Graham Noctor

Amna Mhamdi

Sejir Chaouch

Y I Han

Jenny Neukermans

Belen Marquez-Garcia

Guillaume Queval

Christine H Foyer

Several members of the laboratory, including myself, contributed to this review paper. Glutathione in plants: an integrated overviewp ce_2400 454..484

Keywords: Antioxidant, detoxification, oxidative stress, pathogens, redox metabolism and signalling, sulphur metabolism, thiols

Plants

cannot survive without glutathione (g-glutamylcysteinylglycine) or g-glutamylcysteinecontaining homologues. The reasons why this small molecule is indispensable are not fully understood, but it can be inferred that glutathione has functions in plant development that cannot be performed by other thiols or antioxidants. The known functions of glutathione include roles in biosynthetic pathways, detoxification, antioxidant biochemistry and redox homeostasis. Glutathione can interact in multiple ways with proteins through thiol-disulphide exchange and related processes. Its strategic position between oxidants such as reactive oxygen species and cellular reductants makes the glutathione system perfectly configured for signalling functions. Recent years have witnessed considerable progress in understanding glutathione synthesis, degradation and transport, particularly in relation to cellular redox homeostasis and related signalling under optimal and stress conditions. Here we outline the key recent advances and discuss how alterations in glutathione status, such as those observed during stress, may participate in signal transduction cascades. The discussion highlights some of the issues surrounding the regulation of glutathione contents, the control of glutathione redox potential, and how the functions of glutathione and other thiols are integrated to fine-tune photorespiratory and respiratory metabolism and to modulate phytohormone signalling pathways through appropriate modification of sensitive protein cysteine residues.

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.

Reactive oxygen species in plants

Molecular oxygen (O 2 ) 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 O 2 -evolving photosynthetic organisms [START_REF] Halliwell | Oxidative stress and neurodegeneration: where are we now?[END_REF]. The O 2 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 O 2 to H 2 O 2 or water by divalent or tetravalent mechanisms, the spin restriction of the O 2 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 O 2 ), superoxide (O 2 -
), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (HO•)). In plants ROS are continuously produced as byproducts of various metabolic pathways localized in different cellular compartments (Figure 1; [START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF]. 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 [START_REF] Mittler | Abiotic Stress, the Field Environment and Stress Combination[END_REF]. 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 [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF].

CHAPTER 1 GENERAL INTRODUCTION

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.

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. Mhamdi et al., 2010b) 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 intermediates [START_REF] Asada | Production and Scavenging of Reactive Oxygen Species in Chloroplasts and Their Functions[END_REF]. O 2 -and H 2 O 2 are formed mainly in PSI, while 1 O 2 is notably produced from the triplet ground state ( 3 O 2 ) by photodynamic transfer of energy from excited triplet state chlorophyll such as the P680 reaction center of PSII [START_REF] Telfer | Isolated photosynthetic reaction center of photosystem II as a sensitizer for the formation of singlet oxygen[END_REF][START_REF] Hideg | Photoinhibition of photosynthesis in vivo results in singlet oxygen production detection via nitroxide-induced fluorescence quenching in broad bean leaves[END_REF].

Figure. 1.1. Major sites of H 2 O 2 production in photosynthetic cells (scheme taken from

As their name implies, peroxisomes are also significant generators of ROS in the form of O 2 -or H 2 O 2 produced by various oxidases [START_REF] Mhamdi | Plant catalases: peroxisomal redox guardians[END_REF]. Indeed, it has been estimated that in some conditions, these organelles may be the fastest H 2 O 2 -generating system in photosynthetic cells (Noctor et al., 2002).

In peroxisomes, H 2 O 2 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 H 2 O 2 is acylCoA oxidase, involved in fatty acid oxidation [START_REF] Nyathi | Plant peroxisomes as a source of signalling molecules[END_REF]. In addition, xanthine oxidase can generate O 2 -, which in turn is converted to H 2 O 2 by Cu,Zn-superoxide dismutase (SOD; [START_REF] Corpas | The Expression of Different Superoxide Dismutase Forms is Cell-type Dependent in Olive (Olea europaea L.) Leaves[END_REF].

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 [START_REF] Møller | PLANT MITOCHONDRIA AND OXIDATIVE STRESS: Electron Transport, NADPH Turnover, and Metabolism of Reactive Oxygen Species[END_REF]. Mitochondrial terminal oxidases such as cytochrome c oxidase (Complex IV) and the alternative oxidase, reduce oxygen via tetravalent mechanisms. H 2 O 2 is formed in mitochondria from O 2 -by Mn-SOD [START_REF] Finkel | Oxidants, oxidative stress and the biology of ageing[END_REF].

Among several types of enzymes which may be required for generating H 2 O 2 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;[START_REF] Torres | ROS signaling in response to pathogens[END_REF]. 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 O 2 to O 2 -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 [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]Bindschedler et al., 2006;[START_REF] Choi | Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens[END_REF]Van Breusegem et al., 2008).

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 [START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF][START_REF] Møller | Elevated frequency of abnormalities in barn swallows from Chernobyl[END_REF].

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;[START_REF] Pastori | Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling[END_REF]Ball et al., 2004;[START_REF] Dowdle | Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling REFERENCES viability[END_REF]Parisy et al., 2007). Both of them are highly reducing and abundant antioxidants found in millimolar (mM) concentrations in plants [START_REF] Foyer | Ascorbate and glutathione: the heart of the redox hub[END_REF]. In addition, both ASC and GSH are reducing co-factors for a range of enzymes that metabolize ROS, as described 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;[START_REF] Ramel | Carotenoid oxidation products are stress signals that mediate gene responses to singlet oxygen in plants[END_REF].

Enzymatic metabolism of ROS

As noted above, superoxide is formed in several cell compartments and the apoplast, and is metabolized to H 2 O 2 and/or O 2 . While direct reduction to H 2 O 2 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) [START_REF] Fink | Molecular evolution and structure-function relationships of thesuperoxide dismutase gene families in Angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases[END_REF].

Whether produced directly from O 2 by oxidases or indirectly from superoxide by SOD, H 2 O 2 is the most stable ROS. It has therefore attracted considerable attention as a signal molecule. Although H 2 O 2 can oxidize some groups such as protein thiols, it can also be cleaved to the highly reactive hydroxyl radical.

Hence, enzyme systems involved in H 2 O 2 metabolism are particularly important to maintain redox homeostasis and avoid uncontrolled cellular oxidation. Numerous types of enzyme can metabolize H 2 O 2 , 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 H 2 O 2 to water and therefore require a reducing co-factor.

Catalases (CAT), exists in almost all organisms, and possess a high capacity for metabolizing H 2 O 2 through the dismutation of H 2 O 2 to water and O 2 . In most organisms, CAT are located mainly in the peroxisomes [START_REF] Zamocky | Evolution of catalases from bacteria to humans[END_REF]. Three CAT genes have been identified in Angiosperm species studied to date, including tobacco, Arabidopsis, maize, pumpkin and rice [START_REF] Willekens | Catalases in plants[END_REF][START_REF] Frugoli | Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh[END_REF][START_REF] Guan | Molecular evolution of maize catalases and their relationship to other eukaryotic and prokaryotic catalases[END_REF][START_REF] Esaka | cDNA cloning and differential gene expression of three catalases in pumpkin[END_REF][START_REF] Iwamoto | Differential diurnal expression of rice catalase genes: the 5'-flanking region of CatA is not sufficient for circadian control[END_REF], and a classification system based on the naming of the tobacco genes was introduced by [START_REF] Willekens | Catalases in plants[END_REF], 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 [START_REF] Frugoli | Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh[END_REF]. 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 [START_REF] Hu | Functional comparison of catalase genes in the elimination of photorespiratory H 2 O 2 using promoter-and 3'-untranslated region exchange experiments in the Arabidopsis cat2 photorespiratory mutant[END_REF].

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;[START_REF] Smykowski | G-Box binding factor1 reduces CATALASE2 expression and regulates the onset of leaf senescence in Arabidopsis[END_REF]. 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 CO 2 ; however, lesions appear when the plant is transferred to air [START_REF] Kendall | The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgare L.)[END_REF]. The appearance of

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 C 3 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 [START_REF] Chamnongpol | Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis-related expression under high light[END_REF](Chamnongpol et al., , 1998;;Willekens et al., 1997;Vandenabeele et al., 2004;[START_REF] Vanderauwera | Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis[END_REF]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). As well as catalases, the best described route for H 2 O 2 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).

APX, ascorbate peroxidase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, glutathione disulphide; MDHAR, monodehydroascorbate reductase. Foyer and Noctor, 2009).

CHAPTER 1 GENERAL INTRODUCTION

In the ascorbate-glutathione pathway, H 2 O 2 is reduced to H 2 O by ascorbate peroxidase (APX), an enzyme with a much higher affinity for H 2 O 2 than CAT, leading to oxidation of ASC to monodehydroascorbate (MDHA; Figure 1.3). As well as APX, which is specific to H 2 O 2 as oxidant, plants also contain several types of other peroxidases that can reduce organic peroxides in addition to H 2 O 2 . Among these, peroxiredoxins (PRX) may have an important function alongside APX in H 2 O 2 removal [START_REF] Dietz | The function of peroxiredoxins in plant organelle redox metabolism[END_REF]. 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;[START_REF] Rouhier | The plant multigenic family of thiol peroxidases. Free Radical[END_REF]Meyer, 2008;[START_REF] Pérez-Ruiz | A proposed reaction mechanism for rice NADPH thioredoxin reductase C, an enzyme with protein disulfide reductase activity[END_REF]. 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 H 2 O 2 was reported to be about half that of soluble APX [START_REF] Dietz | The function of peroxiredoxins in plant organelle redox metabolism[END_REF]. In addition to detoxification of lipid peroxides, glutathione peroxidases (GPXs) are able to remove H 2 O 2 . 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 H 2 O 2 (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.

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

CHAPTER 1 GENERAL INTRODUCTION

of H 2 O 2 -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 [START_REF] Giacomelli | Arabidopsis thaliana deficient in two chloroplast ascorbate peroxidases shows accelerated light-induced necrosis when levels of cellular ascorbate are low[END_REF][START_REF] Miller | Double Mutants Deficient in Cytosolic and Thylakoid Ascorbate Peroxidase Reveal a Complex Mode of Interaction between Reactive Oxygen Species, Plant Development, and Response to Abiotic Stresses[END_REF]Mhamdi et al., 2010a) and exploration of crosstalk between distinct ROS such as 1 O 2 or H 2 O 2 originating in different compartments [START_REF] Laloi | REFERENCES Cross-talk between singlet oxygen-and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana[END_REF][START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 [START_REF] Harir | The ROS signaling network of cells[END_REF].

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 [START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF][START_REF] Møller | Elevated frequency of abnormalities in barn swallows from Chernobyl[END_REF]. 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.

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. et al., 2004).

CHAPTER 1 GENERAL INTRODUCTION 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 [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]. For instance, both HsfA4a and HsfA8 were not induced by enhanced peroxisomal H 2 O 2 in a cat2-deficient line but induced by elevated cytosolic H 2 O 2 in an apx1 knockout mutant [START_REF] Pnueli | Growth suppression, altered stomatal responses, and augmented induction of heat shock proteins in cytosolic ascorbate peroxidase (Apx1)-deficient Arabidopsis plants[END_REF]Davletova et al., 2005;[START_REF] Vanderauwera | Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis[END_REF].

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 [START_REF] Foreman | Reactive oxygen species produced by NADPH oxidase regulate plant cell growth[END_REF][START_REF] Monshausen | Oscillations in extracellular pH and reactive oxygen species modulate tip growth of Arabidopsis root hairs[END_REF][START_REF] Macpherson | NADPH oxidase involvement in cellular integrity[END_REF][START_REF] Potocky | Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth[END_REF]. 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 [START_REF] Liszkay | Production of reactive oxygen intermediates, H 2 O 2 , and OH by maize roots and their role in wall loosening and elongation growth[END_REF], and apoplastic ROS accumulation has also been implicated in the involvement of maize leaf development [START_REF] Rodriguez | Salinity-induced decrease in NADPH oxidase activity in the maize leaf blade elongation zone[END_REF]. Moreover, ROS are involved in abscisic acid (ABA)-mediated stomatal closure, the modulation of cell wall loosening during seed germination, and fruit ripening (Apel andHirt, 2004, Dunand et al., 2007;Müller et al., 2009a, b).

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 H 2 O 2 . Several reports in various plant species suggest that both plasmalemma-located NADPH oxidase and extracellular peroxidases contribute to this rapid increase in ROS [START_REF] Torres | Arabidopsis gp91 phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response[END_REF](Torres et al., , 2005;;Bindschedler et al., 2006;[START_REF] Daudi | The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity[END_REF][START_REF] O'brien | A Peroxidase-Dependent Apoplastic Oxidative Burst in Cultured Arabidopsis Cells Functions in MAMP-Elicited Defense[END_REF]. 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;[START_REF] Volt | Salicylic acid, a multifaceted hormone to combat disease[END_REF][START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 H 2 O 2 -dependent lignification reactions that confine the pathogen to the infection site [START_REF] Durner | Salicylic acid and disease resistance in plants[END_REF]. 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 spectrum of pathogens [START_REF] Fobert | Redox control of systemic acquired resistance[END_REF]. Nonexpressor of pathogenesis-related genes 1 (NPR1)

protein is required for SA signaling and it is a key component of SAR regulation [START_REF] Cao | Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance[END_REF]. 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 [START_REF] Zhao | Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor[END_REF]Chaouch et al., 2010).

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 [START_REF] Lam | Controlled cell death, plant survival and development[END_REF]. 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 [START_REF] Overmyer | Ozone-induced programmed cell death in the Arabidopsis radical-induced cell death1 mutant[END_REF]. 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 [START_REF] Danon | Concurrent activation of cell death-regulating signalling pathways by singlet oxygen in Arabidopsis thaliana[END_REF]Chaouch et al., 2010). Other defence hormones such as jasmonic acid (JA) and ethylene also are involved in ROS-dependent PCD [START_REF] Fath | Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone[END_REF][START_REF] De Jong | A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells[END_REF][START_REF] Overmyer | Ozone-sensitive Arabidopsis rcd1 mutant reveals opposite roles for ethylene and jasmonate signaling pathways in regulating superoxide-dependent cell death[END_REF][START_REF] Moeder | Ethylene synthesis regulated by bi-phasic induction of ACC synthase and ACC oxidase genes is required for H 2 O 2 accumulation and cell death in ozone-exposed tomato[END_REF][START_REF] Danon | Concurrent activation of cell death-regulating signalling pathways by singlet oxygen in Arabidopsis thaliana[END_REF].

Besides phytohormones, nitric oxide (NO) operates in a dose-dependent manner, synergistically with H 2 O 2 , in the modulation of HR-like cell death [START_REF] Delledonne | Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response[END_REF][START_REF] Zago | Nitric oxide-and hydrogen peroxide-responsive gene regulation during cell death induction in tobacco[END_REF]. In addition, lipidic components such as sphingolipids, oxylipins and phospholipids can interact with ROS to modulate PCD [START_REF] Liang | Ceramides modulate programmed cell death in plants[END_REF][START_REF] Loeffler | B1-phytoprostanes trigger plant defense and detoxification responses[END_REF][START_REF] Meijer | Phospholipid-based signaling in plants[END_REF]. Metacaspases and metabolites such as myo-inositol have also been implicated in ROS-induced cell death [START_REF] He | Metacaspase-8 modulates programmed cell death induced by UV and H 2 O 2 in Arabidopsis[END_REF]Chaouch and Noctor, 2010).

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 CHAPTER 1 GENERAL INTRODUCTION [START_REF] Neill | Hydrogen peroxide signalling[END_REF][START_REF] Mittler | Reactive oxygen gene network of plants[END_REF][START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF]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 [START_REF] Dietz | Redox signal integration: from stimulus to networks and genes[END_REF], and such functions could act analogously to the regulation of yAP1 in yeast by peroxidase-dependent oxidation by H 2 O 2 [START_REF] Delaunay | A thiol peroxidase is an H 2 O 2 receptor and redox-transducer in gene activation[END_REF]. 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 [START_REF] Mittler | ROS signalling: the new wave?[END_REF]. 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 [START_REF] Teige | The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis[END_REF][START_REF] Xing | AtMKK1 mediates ABA-induced CAT1 expression and H 2 O 2 production via AtMPK6-coupled signaling in Arabidopsis[END_REF][START_REF] Jammes | MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling[END_REF]. The MEKKI pathway is highly activated during abiotic stress, which was suggested to be specifically linked to H 2 O 2 -mediated activation of MPK4 [START_REF] Nakagami | A Mitogen-activated protein kinase kinase kinase mediates reactive oxygen species homeostasis in Arabidopsis[END_REF][START_REF] Xing | AtMKK1 mediates ABA-induced CAT1 expression and H 2 O 2 production via AtMPK6-coupled signaling in Arabidopsis[END_REF]. 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 [START_REF] Anthony | A protein kinase target of a PDK1 signalling pathway is involved in root hair growth in Arabidopsis[END_REF][START_REF] Rentel | OXI1 kinase is necessary for oxidative burstmediated signalling in Arabidopsis[END_REF]. 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 [START_REF] Jammes | MAP kinases MPK9 and MPK12 are preferentially expressed in guard cells and positively regulate ROS-mediated ABA signaling[END_REF]. 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 [START_REF] Gupta | Redox control of protein tyrosine phosphatases and mitogen-activated protein kinases in plants[END_REF][START_REF] Lee | Arabidopsis MAPK phosphatase 2 (MKP2) positively regulates oxidative stress tolerance and inactivates the MPK3 and MPK6 MAPKs[END_REF]. A MAPK cascade is also implicated in NO signaling, which synergistically functions with ROS signaling in pathogen defence responses such as HR [START_REF] Asai | MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana[END_REF].

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 [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]Davletova et al., 2005;[START_REF] Vanderauwera | Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis[END_REF][START_REF] Gadjev | Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis[END_REF][START_REF] Miller | Reactive oxygen signaling and abiotic stress[END_REF]. These ROS-sensitive TFs can then lead to changes in gene expression [START_REF] Vanderauwera | Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis[END_REF][START_REF] Gadjev | Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis[END_REF].

The intensity, duration and localization of ROS signals are controlled by the complex interaction between ROS generation and ROS scavenging systems (Figure 1.4; [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]. 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 [START_REF] Maxwell | The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells[END_REF][START_REF] Baier | Antisense suppression of 2-Cys peroxiredoxin in Arabidopsis specifically enhanced activities and expression of enzymes associated with ascorbate metabolism but not glutathione metabolism[END_REF][START_REF] Torres | Arabidopsis gp91 phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response[END_REF][START_REF] Epple | Antagonistic control of oxidative stress-induced cell death in Arabidopsis by two related, plant-specific zinc finger proteins[END_REF][START_REF] Foreman | Reactive oxygen species produced by NADPH oxidase regulate plant cell growth[END_REF][START_REF] Rizhsky | The water-water cycle is essential for chloroplast protection in the absence of stress[END_REF][START_REF] Pnueli | Growth suppression, altered stomatal responses, and augmented induction of heat shock proteins in cytosolic ascorbate peroxidase (Apx1)-deficient Arabidopsis plants[END_REF][START_REF] Mittler | Reactive oxygen gene network of plants[END_REF];[START_REF] Sagi | Plant respiratory burst oxidase homologs impinge on wound responsiveness and development in Lycopersicon esculentum[END_REF]Vandenabeele et al., 2004;Mhamdi et al., 2010a;[START_REF] Mittler | ROS signalling: the new wave?[END_REF][START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 CHAPTER 1 GENERAL INTRODUCTION lacking in key ROS network genes can serve as excellent tools to study interactions between different types of ROS generated at different subcellular locations.

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.

Glutathione synthesis

In both animals and plants, GSH is synthesized from glutamate, cysteine and glycine by two ATP-dependent steps (Figure 1.5; [START_REF] Rennenberg | Glutathione metabolism and possible biological roles in higher plants[END_REF]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;[START_REF] Ullman | Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant[END_REF]. 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).

Glycine Cysteine Glutamate

-EC

GSH-S

-EC 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 [START_REF] Shanmugam | ZINC TOLERANCE INDUCED BY IRON 1 reveals the importance of glutathione in the cross-homeostasis between zinc and iron in Arabidopsis thaliana[END_REF]. 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).

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 H 2 O 2 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(Noctor et al., , 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).

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 (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 35 S-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.

CHAPTER 1 GENERAL INTRODUCTION

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 -1 FW. 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 [START_REF] Steinkamp | Degradation of glutathione in plant cells: evidence against the participation of a g-glutamyltranspeptidase[END_REF], 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(Blum et al., , 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;[START_REF] Storozhenko | g-Glutamyl transpeptidase in transgenic tobacco plants. Cellular localization, processing, and biochemical properties[END_REF] 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;[START_REF] Destro | Compensatory expression and substrate inducibility of γ-glutamyl transferase GGT2 isoform in Arabidopsis thaliana[END_REF].

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;[START_REF] Steinkamp | g-Glutamylcyclotransferase in tobacco suspension cultures: catalytic properties and subcellular localization[END_REF].

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.

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. 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 [START_REF] Edwards | Plant glutathione S-transferases: enzymes with multiple functions in sickness and in health[END_REF]Wagner et al., 2002;Dixon andEdwards, 2009, 2010;[START_REF] Geu-Flores | Cytosolic g-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis[END_REF][START_REF] Su | Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF]. 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;[START_REF] Reumann | In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes[END_REF]. 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 [START_REF] Chen | Dehydroascorbate reductase affects leaf growth, development and function[END_REF][START_REF] Yoshida | Cytosolic dehydroascorbate reductase is important for ozone tolerance in Arabidopsis thaliana[END_REF]Tamaoki et al., 2008;Vadassery et al., 2009).

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 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).

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;[START_REF] Sundaram | Transgenic expression of fern Pteris vittata glutaredoxin PvGrx5 in Arabidopsis thaliana increases plant tolerance to high temperature stress and reduces oxidative damage to proteins[END_REF]. 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).

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] n Gly), 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., 2007Blum et al., , 2010;;[START_REF] Peršoh | Multi-tasking phytochelatin synthases[END_REF].

Phytochelatins sequester the metal to form a complex that is then transported into the vacuole (Grill et al., 1987(Grill et al., , 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

CHAPTER 1 GENERAL INTRODUCTION

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 CO 2 or enter C 1 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;[START_REF] Thomma | Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola[END_REF][START_REF] Glawischnig | Camalexin[END_REF] and following treatment with abiotic factors such as UV-B paraquat, and heavy metal ions [START_REF] Zhao | Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor[END_REF][START_REF] Mert-Türk | Camalexin accumulation in Arabidopsis thaliana following abiotic elicitation or inoculation with virulent or avirulent Hyaloperonospora parasitica[END_REF][START_REF] Bouizgarne | Early physiological responses of Arabidopsis thaliana cells to fusaric acid: toxic and signalling effects[END_REF][START_REF] Kishimoto | Analysis of defensive responses activated by volatile allo-ocimene treatment in Arabidopsis thaliana[END_REF]. 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 [START_REF] Su | Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF]. 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 [START_REF] Geu-Flores | Cytosolic g-glutamyl peptidases process glutathione conjugates in the biosynthesis of glucosinolates and camalexin in Arabidopsis[END_REF]. 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 [START_REF] Hopkins | Role of glucosinolates in insect-plant relationships and multitrophic interactions[END_REF]. 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 GGP1 and GGP3 seem to metabolize GSH conjugates in the biosynthesis of glucosinolates in Arabidopsis.

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.

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).

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 H 2 O 2 levels and to oxidation of the glutathione pool [START_REF] Mateo | Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis[END_REF]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 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 [START_REF] Sung | Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants[END_REF]. The ars4 mutation was identified as an allele of phytochrome A (phyA) and caused increased BSO resistance [START_REF] Sung | Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants[END_REF]. 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 [START_REF] Sung | ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis[END_REF].

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;[START_REF] Wingate | Glutathione causes a massive and selective induction of plant defense genes[END_REF]Senda and Ogawa, 2004;Gomez et al., 2004a). Moreover, accumulation of glutathione is triggered by pathogen infection (Edwards et al., 1991;[START_REF] May | An Arabidopsis mutant depleted in glutathione shows unaltered responses to fungal and bacterial pathogens[END_REF].

Similar changes have also been reported following exogenous application of the defence-related hormone SA, or biologically active SA analogs (Mou et al., 2003;[START_REF] Mateo | Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis[END_REF]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 H 2 O 2 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 H 2 O 2 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;[START_REF] Pauly | Reactive oxygen and nitrogen species and glutathione: Key players in the legumerhizobium symbiosis[END_REF]. 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 [START_REF] Wildermuth | Isochorismate synthase is required to synthesize salicylic acid for plant defence[END_REF]. 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

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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF].

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 [START_REF] Browse | Jasmonate passes muster: a receptor and targets for the defense hormone[END_REF]. 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 [START_REF] Spoel | NPR1 modulates cross-talk between salicylate-and jasmonatedependent defense pathways through a novel function in the cytosol[END_REF], 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.,

CHAPTER 1 GENERAL INTRODUCTION

2011).

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.

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).

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(Lindermayr et al., , 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;[START_REF] Leitner | NO signals in the haze: nitric oxide signalling in plant defence[END_REF]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.

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

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 H 2 O 2 production (Smith et al., 1984;Willekens et al., 1997;Rizhsky et al., 2002;Queval et al., 2007Queval et al., , 2009)), and this is a key observation underlying the studies reported in Chapters 2 and 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 [START_REF] Bassham | Plant autophagy-more than a starvation response[END_REF][START_REF] Yang | An overview of themolecular mechanism of autophagy[END_REF][START_REF] Mehrpour | Overview of macroautophagy regulation in mammalian cells[END_REF]. 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;[START_REF] Bassham | Autophagy in development and stress responses of plants[END_REF]. 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 [START_REF] Yoshimoto | Beginning to understand autophagy, an intracellular self-degradation system in plants[END_REF]. 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. In plants, autophagic structures have been observed, largely based on morphological observation by electron microscopy, since the late 1960s [START_REF] Villiers | Cytolysosomes in long-dormant plant embryo cells[END_REF][START_REF] Matile | Vacuolation: origin and development of the lysosomal apparatus in root tip cells[END_REF][START_REF] Matile | Function of lysosomes and lysosomal enzymes in the senescing corolla of the morning glory[END_REF][START_REF] Matile | The lytic compartment of plant cells[END_REF][START_REF] Marty | Cytochemical studies on GERL, provacuoles, and vacuoles in root meristematic cells of Euphorbia[END_REF][START_REF] Van Der Wilden | Protein bodies of mung bean cotyledons as autophagic organelles[END_REF]. 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 [START_REF] Barth | Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p[END_REF][START_REF] Klionsky | A unified nomenclature for yeast autophagy-related genes[END_REF][START_REF] Kanki | Atg32 is a mitochondrial protein that confers selectivity during mitophagy[END_REF][START_REF] Nakatogawa | Dynamics and diversity in autophagy mechanisms: lessons from yeast[END_REF][START_REF] Okamoto | Mitochondria-anchored receptor Atg32 mediates degradation of mitochondria via selective autophagy[END_REF]. 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; [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF][START_REF] Hanaoka | Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene[END_REF], 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;[START_REF] Ghiglione | Autophagy regulated by day length determines the number of fertile florets in wheat[END_REF]Chumg et al., 2009;[START_REF] Shin | OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice[END_REF][START_REF] Xia | Genome-wide identification, classification, and expression analysis of autophagy-associated gene homologues in rice (Oryza sativa L.)[END_REF][START_REF] Kuzuoglu-Ozturk | Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response[END_REF]. 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.

CHAPTER 1 GENERAL INTRODUCTION

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 [START_REF] Bassham | Autophagy in development and stress responses of plants[END_REF][START_REF] Hayward | Autophagy and plant innate immunity: Defense through degradation[END_REF]. 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 [START_REF] Bassham | Plant autophagy-more than a starvation response[END_REF][START_REF] Bassham | Function and regulation of macroautophagy in plants[END_REF][START_REF] Hayward | What can plant autophagy do for an innate immune response?[END_REF]. 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.

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 [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF][START_REF] Hanaoka | Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene[END_REF][START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF]Xiong et al., 2005;[START_REF] Phillips | The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana[END_REF]. 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 [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF][START_REF] Hanaoka | Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene[END_REF][START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF][START_REF] Xiong | AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana[END_REF][START_REF] Phillips | The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana[END_REF]. It has been recently confirmed that autophagy is required for nitrogen remobilization [START_REF] Guiboileau | Autophagy machinery controls nitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis[END_REF]. 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 [START_REF] He | Regulation mechanisms and signaling pathways of autophagy[END_REF]. 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 [START_REF] Liu | TOR is a negative regulator of autophagy in Arabidopsis thaliana[END_REF]. In addition, autophagy has been shown to be a rather general response to a variety of abiotic stresses [START_REF] Bassham | Plant autophagy-more than a starvation response[END_REF]. When treatment of plants with H 2 O 2 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 [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF]. Similar effects have also been observed in the rice Osatg10 mutant [START_REF] Shin | OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice[END_REF]. Moreover, autophagy is essential for plant tolerance to drought and salt stresses [START_REF] Liu | Autophagy is required for tolerance of drought and salt stress in plants[END_REF][START_REF] Kuzuoglu-Ozturk | Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response[END_REF]. 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

CHAPTER 1 GENERAL INTRODUCTION

the autophagy pathway, implying the involvement of autophagy in responses to ABA in plants [START_REF] Vanhee | A TSPO-related protein localizes to the early secretory pathway in Arabidopsis, but is targeted to mitochondria when expressed in yeast[END_REF].

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 [START_REF] Hayward | What can plant autophagy do for an innate immune response?[END_REF][START_REF] Hofius | Autophagic components contribute to hypersensitive cell death in Arabidopsis[END_REF][START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]Lenz et al., 2011a,b;[START_REF] Lai | A critical role of autophagy in plant resistance to necrotrophic fungal pathogens[END_REF][START_REF] Wang | ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis[END_REF]. 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 [START_REF] Lai | A critical role of autophagy in plant resistance to necrotrophic fungal pathogens[END_REF]. Autophagy defective mutants exhibit susceptibility to the necrotrophic fungal pathogen Botrytis cinerea and Alternaria brassicicola [START_REF] Lai | A critical role of autophagy in plant resistance to necrotrophic fungal pathogens[END_REF]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 [START_REF] Liu | Autophagy regulates programmed cell death during the plant innate immune response[END_REF]. 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; [START_REF] Patel | Arabidopsis ATG6 is required to limit the pathogen-associated cell death response[END_REF][START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. 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; [START_REF] Hofius | Autophagic components contribute to hypersensitive cell death in Arabidopsis[END_REF]. 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 [START_REF] Hofius | Autophagic components contribute to hypersensitive cell death in Arabidopsis[END_REF]Lenz et al., 2011b). The ATG-defective plants all showed enhanced resistance to both of these biotrophic pathogens [START_REF] Hofius | Autophagic components contribute to hypersensitive cell death in Arabidopsis[END_REF]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 [START_REF] Hayward | What can plant autophagy do for an innate immune response?[END_REF][START_REF] Liu | Autophagy: pathways for self-eating in plant cells[END_REF], and the exact roles of autophagy in plant innate immune response remain to be elucidated.

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 [START_REF] Slavikova | The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants[END_REF]Inoue et al., 2006;[START_REF] Yano | A novel type of autophagy occurs together with vacuole genesis in miniprotoplasts prepared from tobacco culture cells[END_REF][START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. Here, it is important to note that plants may maintain a basal level of housekeeping autophagy to eliminate damaged materials, which are continually generated even under favorable growth conditions [START_REF] Bassham | Autophagy in development and stress responses of plants[END_REF]. 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 [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF][START_REF] Hanaoka | Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene[END_REF][START_REF] Patel | Arabidopsis ATG6 is required to limit the pathogen-associated cell death response[END_REF][START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF][START_REF] Xiong | AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana[END_REF][START_REF] Qin | Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development[END_REF][START_REF] Phillips | The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana[END_REF][START_REF] Patel | Arabidopsis ATG6 is required to limit the pathogen-associated cell death response[END_REF][START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. 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 H 2 O 2 [START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. This raises an interesting question regarding the relationship between autophagy and H 2 O 2 during SA related leaf senescence. As discussed above, autophagy could be involved in degrading oxidized proteins under oxidative stress conditions in Arabidopsis [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF]. It is also possible that autophagy may function selectively to remove specific H 2 O 2 -generating organelles such as chloroplasts and peroxisomes.

However, the underlying mechanisms remain to be fully investigated, and the relationship between autophagy and H 2 O 2 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 [START_REF] Angelovici | Deciphering transcriptional and metabolic networks associated with lysine metabolism during Arabidopsis seed development[END_REF]. More recently it has been shown that autophagy is required for seed filling [START_REF] Guiboileau | Autophagy machinery controls nitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis[END_REF]. 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 [START_REF] Ibl | The formation, function and fate of protein storage compartments in seeds[END_REF].

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 [START_REF] Reyes | Delivery of prolamins to the protein storage vacuole in maize aleurone cells[END_REF].

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 [START_REF] Yoshimoto | Processing of ATG8s, ubiquitinlike proteins, and their deconjugation by ATG4s are essential for plant autophagy[END_REF][START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF][START_REF] Chung | ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci[END_REF]. 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 [START_REF] Chung | ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci[END_REF].

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 vacuole can be monitored biochemically by immunoblotting with an anti-GFP antibody [START_REF] Chung | ATG8 lipidation and ATG8-mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci[END_REF]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 [START_REF] Yoshimoto | Processing of ATG8s, ubiquitinlike proteins, and their deconjugation by ATG4s are essential for plant autophagy[END_REF][START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF][START_REF] Ishida | Mobilization of Rubisco and stroma-localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process[END_REF]. 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;[START_REF] Liu | Autophagy regulates programmed cell death during the plant innate immune response[END_REF][START_REF] Patel | Arabidopsis ATG6 is required to limit the pathogen-associated cell death response[END_REF]. However, these dyes should be used cautiously because of their potential to cross identify other acidic bodies in the tissue [START_REF] Yoshimoto | Beginning to understand autophagy, an intracellular self-degradation system in plants[END_REF].

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 H 2 O 2 availability through photorespiration and can be prevented by growth at high CO 2 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 H 2 O 2 . 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 [START_REF] Dwidedi | Biochemical changes in excised leaves of Oryza sativa subjected to water stress[END_REF][START_REF] Dhindsa | Leaf senescence: Correlated with increased levels membrane permeability and lipid peroxidation and decreased levels of SOD and CAT[END_REF][START_REF] Bohnert | Adaption to environmental stresses[END_REF][START_REF] Balazadeh | A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence[END_REF]. One signaling molecule that could be important in the regulation of senescence is H 2 O 2 . The roles of antioxidant enzymes such as CAT and APX, which metabolize H 2 O 2 , 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 [START_REF] Smykowski | G-Box binding factor1 reduces CATALASE2 expression and regulates the onset of leaf senescence in Arabidopsis[END_REF].

Furthermore, it has been proposed that autophagy may function in degrading oxidized proteins to confer tolerance to oxidative stress [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF]. Indeed, several autophagy mutants such as atg2 and atg5 exhibit an early senescence phenotype which is associated with enhanced oxidative stress as well as the activation of SA pathway [START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF] CHAPTER 2

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 [START_REF] Doke | Involvement of superoxide anion generation in the hypersensitive response of potato tuber tissues to infection with an incompatible race of Phytophthora infestans and to the hyphal wall components[END_REF][START_REF] Apostol | Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: Role in defence and signal transduction[END_REF]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 [START_REF] Torres | Arabidopsis gp91 phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response[END_REF](Torres et al., , 2005;;Bindschedler et al., 2006;[START_REF] Daudi | The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity[END_REF][START_REF] O'brien | A Peroxidase-Dependent Apoplastic Oxidative Burst in Cultured Arabidopsis Cells Functions in MAMP-Elicited Defense[END_REF]. However, these initial signals at the cell surface/apoplast lead to later downstream adjustments in intracellular redox state (Edwards et al., 1991;[START_REF] Vanacker | Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hypersensitive response in the barley-powdery mildew interaction[END_REF][START_REF] Volt | Salicylic acid, a multifaceted hormone to combat disease[END_REF] 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 [START_REF] May | An Arabidopsis mutant depleted in glutathione shows unaltered responses to fungal and bacterial pathogens[END_REF]Ball et al., 2004;Parisy et al., 2007;Höller et al., 2010;[START_REF] Dubreuil-Maurizi | Glutathione deficiency of the Arabidopsis mutant pad2-1 affects oxidative stress-related events, defense gene expression, and the hypersensitive response[END_REF][START_REF] Baldacci-Cresp | Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula[END_REF]. 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 H 2 O 2 signals. In terms of thiol-dependent signaling functions in pathogenesis responses, the NPR1 pathway remains by far the best studied (Koornneef et al., 2008;[START_REF] Ghanta | Nicotiana REFERENCES tabacum overexpressing γ-ECS exhibits biotic stress tolerance likely through NPR1-dependent salicylic acid-mediated pathway[END_REF]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;[START_REF] May | An Arabidopsis mutant depleted in glutathione shows unaltered responses to fungal and bacterial pathogens[END_REF][START_REF] Vanacker | Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hypersensitive response in the barley-powdery mildew interaction[END_REF]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 H 2 O 2 signaling to be studied specifically and controllably, because the endogenous signal can be manipulated easily by external conditions [START_REF] Dat | Catalase-deficient tobacco plants: tools for in planta studies on the role of hydrogen peroxide[END_REF]Mhamdi et al., 2010b). When conditions allow active photorespiration, increased H 2 O 2 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 H 2 O 2 to downstream defence responses or are rather an CHAPTER 2

accompanying, passive response of the cell to enhanced H 2 O 2 .

Increases in H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF].

The study presented in this chapter uses the cat2 mutant as the basis for a targeted analysis of the role of CHAPTER 2 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 CO 2 .

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 [START_REF] Nawrath | Salicylic acid induction deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation[END_REF]. Allowed ICS1 to be identified as major source of pathogen-induced salicylic acid production in Arabidopsis [START_REF] Wildermuth | Isochorismate synthase is required to synthesize salicylic acid for plant defence[END_REF]. 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 [START_REF] Cao | Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance[END_REF]. 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.

____________________________________________________________________________________ CHAPTER 2

Results

Plants deficient in the major leaf catalase show accumulation of glutathione that is conditional on H 2 O 2 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 H 2 O 2 -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 [START_REF] Wildermuth | Isochorismate synthase is required to synthesize salicylic acid for plant defence[END_REF]. During growth at high CO 2 , 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 CO 2 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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 CO 2 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 CO 2 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 CO 2 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.

Genetic blocks over H 2 O 2 -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 CO 2 ), leaf glutathione remained at wild-type levels and reduction state (about 95% reduced). Within 4h after transfer to air from high CO 2 , 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 H 2 O 2 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 H 2 O 2 availability (Queval et al., 2007;Mhamdi et al., 2010b). It has (B) Leaf peroxide contents in Col-0, cat2, cat2 cad2 and cat2 gr1.

Plants were grown at high CO 2 , 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.

CHAPTER 2

been previously reported that H 2 O 2 itself (measured as in situ staining or extractable H 2 O 2 ) is minor or undetectable in cat2, even when plants are clearly undergoing oxidative stress (Chaouch et al., 2010[START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]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 CO 2 to air, no difference was apparent between any of the lines, suggesting that any excess H 2 O 2 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 H 2 O 2 that is transient and relatively minor, suggesting that other, reductive systems are able to replace the major leaf catalase in efficiently metabolizing photorespiratory H 2 O 2 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 H 2 O 2 -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;[START_REF] Zechmann | Subcellular compartmentation of glutathione in dicotyledonous plants[END_REF]. 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). The effect of genetically blocking glutathione accumulation on H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 -triggered lesion extent, none of the secondary mutations reverted CHAPTER 2 the decreased rosette size in cat2 (Figure 2.4). Thus, blocking H 2 O 2 -triggered up-regulation of glutathione deficiency led to down-regulation of lesion spread without producing general effects on the cat2 oxidative (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 H 2 O 2 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? The rax1 and pad2 mutations (Ball et al., 2004;Parisy et al., 2007)

______________________________________________________________________

Contrasting responses in cat2 cad2 and cat2 gr1 reveal a non-antioxidant role for glutathione in coupling H 2 O 2 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 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).

cat2 sid2 cat2 cad2 sid2

Col-0 cad2 cat2 cat2 cad2

Lesion formation (%) 21 9 0 0 0 0

Lesion formation (%) 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 CO 2 , 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 CO 2 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 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. Leaf glutathione content (µmol g -1 FW)

Col

cad2 r1 cad2 gr1 d2 sid2 d2 1 sid2 Glutathione GSSG GSSG GSH GSH cat2 gr1 sid2

CHAPTER 2

Several recent publications have implicated myo-inositol in SA-dependent responses [START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF]Chaouch and Noctor, 2010;[START_REF] Donahue | The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppression of cell death[END_REF]. 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 CO 2 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 H 2 O 2 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. Samples were taken 4d after transfer of plants grown at high CO 2 to air, and metabolites were measured by GC-MS (means ± SE of three biological repeats). Different letters show significant difference at P < 0.05.

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

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, H 2 O 2 -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. (C) Growth of virulent P.syringae DC3000 in all seven genotypes. Plants were grown at high CO 2 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. (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

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

A

Col-0 cat2 cad2 cat2 cad2 npr1 cat2 npr1 npr1 effect cad2 effec -1.5 +1.5 A Col-0 cat2 cad2 cat2 cad2 npr1 cat2 npr1
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.

CHAPTER 2

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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF] 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 [START_REF] Shirano | A gain-of-function mutation in an Arabidopsis Toll Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat type R gene triggers defense responses and results in enhanced disease resistance[END_REF][START_REF] Zhang | A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1[END_REF]. In marked contrast to the effect of npr1, the cad2 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 [START_REF] Wildermuth | Isochorismate synthase is required to synthesize salicylic acid for plant defence[END_REF]

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 CO 2 following prior growth in air confirmed that both were significantly less accumulated in cat2 cad2 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 [START_REF] Rate | The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth[END_REF][START_REF] Shah | A recessive mutation in the Arabidopsis SSI2 gene confers SA-and NPR1-independent expression of PR genes and resistance against bacterial and oomycete pathogens[END_REF]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 Plants were grown at high CO 2 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. 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.

Col-0 cat2 cat2 cad2 cat2 cad2 sid2 Col-0 cat2 cad2 cat2 cad2 sid2 Col -0 cat2 cad2 a b e f f cd cd cd c cat2 cad2 sid2 f Mock GSH GSSG 0.0 0.6 1.2 1.8 Col -0 cat2 cat2 cad2 Col-0 cat2 cad2 Col -
+ - + - + - + - + - + - + - Col-0 npr1 cad2 cat2 cat2 cad2 npr1 cat2 npr1 cat2 cad2

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 H 2 O 2 signaling by seeking to genetically abrogate oxidation-triggered accumulation of glutathione.

Partial impairment of -ECS function confers a genetic block on H 2 O 2 -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 H 2 O 2 -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 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 H 2 O 2 -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 H 2 O 2 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.

An essential role for glutathione in activation of H 2 O 2 -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 H 2 O 2 in cat2 show considerable similarity with responses to pathogenic bacteria [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. Thus, intracellular H 2 O 2 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;[START_REF] Volt | Salicylic acid, a multifaceted hormone to combat disease[END_REF] while the analysis of glycolate oxidase mutants provides further indications of roles for peroxisomally produced H 2 O 2 in pathogenesis responses [START_REF] Rojas | Glycolate oxidase modulates reactive oxygen species-mediated signal transduction during nonhost resistance in Nicotiana benthamiana and Arabidopsis[END_REF]. 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[START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. The present study provides direct evidence that glutathione is a key player linking increased H 2 O 2 to downstream phytohormone signaling. Blocking up-regulation of glutathione in cat2 antagonizes H 2 O 2 -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 H 2 O 2 -triggered signaling through the ICS1-dependent pathway. This conclusion is supported by the attenuated induction of the ICS1 gene in cat2 cad2

CHAPTER 2 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 H 2 O 2 . The intermediates in this signaling pathway remain to be identified. 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.

Glutathione

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 H 2 O 2 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 H 2 O 2 -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 [START_REF] Mittler | ROS signalling: the new wave?[END_REF].

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 (H 2 O 2 in this study), intracellular oxidative stress can drive several distinct phenotypic outcomes.

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 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 H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 signaling at the level of induction of SA itself.

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

The observations reported in this study reveal that H 2 O 2 -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 H 2 O 2 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 [START_REF] Vanacker | Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hypersensitive response in the barley-powdery mildew interaction[END_REF]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 Oxidation by H 2 O 2 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;[START_REF] Király | Sulfate supply influences compartment specific glutathione metabolism and confers enhanced resistance to Tobacco mosaic virus during a hypersensitive response[END_REF]. 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 [START_REF] Noctor | Metabolic signalling in defence and stress: the central roles of soluble redox couples[END_REF]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.

CHAPTER 3

Regulation of basal and oxidative stress-triggered jasmonic CHAPTER 3

acid-related gene expression by glutathione

Introduction

As a major determinant of cell redox status, glutathione is a key player in stress responses [START_REF] Noctor | Glutathione in plants: an integrated overview[END_REF]. 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 [START_REF] Rouhier | The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation[END_REF]Schlaeppi et al., 2008;[START_REF] Su | Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF].

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;[START_REF] Vanacker | Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hypersensitive response in the barley-powdery mildew interaction[END_REF]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 [START_REF] May | An Arabidopsis mutant depleted in glutathione shows unaltered responses to fungal and bacterial pathogens[END_REF]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 H 2 O 2 -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- [START_REF] Leon-Reyes | Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway[END_REF]. 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 [START_REF] Spoel | NPR1 modulates cross-talk between salicylate-and jasmonatedependent defense pathways through a novel function in the cytosol[END_REF]Koornneef et al., 2008). Because of the role of glutathione in linking H 2 O 2 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.

CHAPTER 3 Since glutathione status impacts on H 2 O 2 -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.

H

CHAPTER 3

Results

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). 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

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).

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, H 2 O 2 (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 H 2 O 2 -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 CO 2 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).

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, 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). The well-documented antagonism between SA and JA pathways is considered partly to depend on NPR1 function [START_REF] Spoel | NPR1 modulates cross-talk between salicylate-and jasmonatedependent defense pathways through a novel function in the cytosol[END_REF]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 [START_REF] Spoel | NPR1 modulates cross-talk between salicylate-and jasmonatedependent defense pathways through a novel function in the cytosol[END_REF][START_REF] Leon-Reyes | Salicylate-mediated suppression of jasmonate-responsive gene expression in Arabidopsis is targeted downstream of the jasmonate biosynthesis pathway[END_REF]. 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). 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.

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.

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). CHAPTER 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 [START_REF] Foyer | Ascorbate and glutathione: the heart of the redox hub[END_REF]. 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;[START_REF] Noctor | Glutathione in plants: an integrated overview[END_REF]. 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.

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).

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 CO 2 to air at moderate irradiance shows that changes in intracellular redox state driven by enhanced availability of photorespiratory H 2 O 2 can trigger activation of multiple defence hormone pathways in parallel. The potential relevance of redox-triggered events in cat2 is underscored by our CHAPTER 3

previous reports on metabolite profiles and the effects of introducing mutations for recognized players in pathogenesis into the cat2 background (Chaouch et al., 2010[START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 H 2 O 2 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(Queval et al., , 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.

CHAPTER 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 [START_REF] Brosché | Low antioxidant concentrations impact on multiple signaling pathways in Arabidopsis thaliana partly through NPR1[END_REF]. 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 [START_REF] Cao | Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance[END_REF] 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, [START_REF] Rate | The gain-of-function Arabidopsis acd6 mutant reveals novel regulation and function of the salicylic acid signaling pathway in controlling cell death, defenses, and cell growth[END_REF]. 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 [START_REF] Rouhier | The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation[END_REF]. 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 CHAPTER 3

Figure 3.2), where SA signaling is only weakly or not activated and GRX480 expression is decreased. Oxidation by H 2 O 2 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 H 2 O 2 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).

Novel roles for glutathione in

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.

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 CO 2 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 CO 2 in atg2 cad2 double mutants with decreased glutathione. Taken together, these results may indicate that oxidative stress originating from excess peroxisomal H 2 O 2 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

Introduction

Autophagy is an intracelluar macromolecule degradation process that delivers unwanted cell materials into the vacuole/lysosome for recycling of needed nutrients [START_REF] Liu | Autophagy: pathways for self-eating in plant cells[END_REF][START_REF] Pérez-Pérez | Reactive oxygen species and autophagy in plants and algae[END_REF]. 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;[START_REF] Barth | Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p[END_REF]. 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 [START_REF] Kamada | Tor directly controls the Atg1 kinase complex to regulate autophagy[END_REF]. 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 [START_REF] Nakatogawa | Dynamics and diversity in autophagy mechanisms: lessons from yeast[END_REF][START_REF] Kamada | Tor directly controls the Atg1 kinase complex to regulate autophagy[END_REF]. 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 [START_REF] Bassham | Autophagy in development and stress responses of plants[END_REF].

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 [START_REF] Doelling | The APG8/12-activating enzyme APG7 is required for proper nutrient recycling and senescence in Arabidopsis thaliana[END_REF][START_REF] Hanaoka | Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene[END_REF][START_REF] Thompson | Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways[END_REF][START_REF] Xiong | AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana[END_REF][START_REF] Phillips | The ATG12-conjugating enzyme ATG10 is essential for autophagic vesicle formation in Arabidopsis thaliana[END_REF], Arabidopsis atg mutants are also hypersensitive to salt and drought stresses [START_REF] Liu | Autophagy is required for tolerance of drought and salt stress in plants[END_REF]. Moreover, oxidative stress triggered by exogenous H 2 O 2 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 [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF].

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 [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF]. Although autophagy has been suggested to be involved in resistance to oxidative stress in plants [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF][START_REF] Liu | Autophagy is required for tolerance of drought and salt stress in plants[END_REF], 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 [START_REF] Liu | Autophagy regulates programmed cell death during the plant innate immune response[END_REF][START_REF] Hofius | Autophagic components contribute to hypersensitive cell death in Arabidopsis[END_REF][START_REF] Hayward | What can plant autophagy do for an innate immune response?[END_REF]. 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 CHAPTER 4 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 [START_REF] Liu | Autophagy: pathways for self-eating in plant cells[END_REF][START_REF] Fujiki | An Arabidopsis homolog of yeast ATG6/VPS30 is essential for pollen germination[END_REF][START_REF] Harrison-Lowe | Autophagy protein 6 (ATG6) is required for pollen germination in Arabidopsis thaliana[END_REF]. 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 [START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. 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 [START_REF] Sakai | Pexophagy: autophagic degradation of peroxisomes[END_REF][START_REF] Kanki | Atg32 is a mitochondrial protein that confers selectivity during mitophagy[END_REF][START_REF] Pérez-Pérez | Reactive oxygen species and autophagy in plants and algae[END_REF]. 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 H 2 O 2 availability [START_REF] Smykowski | G-Box binding factor1 reduces CATALASE2 expression and regulates the onset of leaf senescence in Arabidopsis[END_REF][START_REF] Wu | JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis[END_REF], which could be a signal involved in the regulation of senescence. As discussed in Chapter 1, plants have developed numerous antioxidant systems to metabolize H 2 O 2 (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 [START_REF] Volk | Photoinactivation of catalase at low temperature and its relevance to photosynthetic and peroxide metabolism in leaves[END_REF][START_REF] Hertwig | Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions[END_REF][START_REF] Dorey | Tobacco Class I and II catalases are differentially expressed during elicitor-induced hypersensitive cell death and localized acquired resistance[END_REF]. Recently, it has been suggested that decreased CAT2 expression and activity is linked to the onset of leaf senescence, via regulation of the intracellular H 2 O 2 content [START_REF] Smykowski | G-Box binding factor1 reduces CATALASE2 expression and regulates the onset of leaf senescence in Arabidopsis[END_REF]. Precocious senescence and a higher level of H 2 O 2 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 H 2 O 2 signaling in plant function [START_REF] Mhamdi | Plant catalases: peroxisomal redox guardians[END_REF]. To analyze interactions between photorespiration and autophagy, I also examined whether CO 2 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 CO 2 , the effects of induced oxidative stress can be studied by transferring mutants grown CHAPTER 4 initially at high CO 2 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.

Long days, air

CHAPTER 4 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 [START_REF] Inoue | AtATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells[END_REF][START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]Lenz et al., 2011b). RT-PCR analysis of atg18a showed that transcripts of ATG18a were undetectable in this line

(Figure 4.2B). 1 2 1 2 1 2 Col-0 atg2 cat2 atg2 1 2 1 2 1 2 Col-0 atg5 cat2 atg5 1 2 Col-0 cat2 atg2 1 2 1 2 1 2 1 2 cat2 atg5 cat2 cat2 atg18a 1 2 1 2 1 2 Col-0 atg18a cat2 atg18a A ATG18a ACTIN2 Col-0 Col-0 atg18a
Col-0 atg18a atg18a Primer combinations are given in Table 6.2 (Chapter 6) CHAPTER 4

RT-PCR B

Effects of high CO 2 on atg phenotypes and metabolite profiles

As a first step to analyzing interacttions between photorespiration and autophagy, single atg mutants were grown in short days (8h light/16h dark), either at high CO 2 (3000 µL.L -1 ) 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 [START_REF] Xiong | AtATG18a is required for the formation of autophagosomes during nutrient stress and senescence in Arabidopsis thaliana[END_REF][START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF], 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 CO 2 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 CO 2 . The early senescence phenotype was most evident for atg2, and was correlated with decreased rosette size (Figure 4.3C). CHAPTER 4

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 CO 2 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 CO 2 . A large sub-cluster of compounds showed higher contents in these two mutants specifically at high CO 2 (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 CO 2 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 CO 2 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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]

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 CO 2 , 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 CO 2 . 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 CO 2 , 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 (

CHAPTER 4 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 Leaf glutathione (µmol.g -1 FW)

Interactions between atg mutations and glutathione

Leaf ascorbate (µmol.g -1 FW)

A B

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 CO 2 and also in atg2 grown in air. Thus, there seems to be an overall correlation between early senescence phenotypes (

Discussion

Cytoplasmic components are recycled and remobilized by autophagy process [START_REF] Guiboileau | Autophagy machinery controls nitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis[END_REF].

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 CO 2 .

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 [START_REF] Liu | Autophagy is required for tolerance of drought and salt stress in plants[END_REF][START_REF] Shin | OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice[END_REF]Péréz-Péréz et al., 2012). Indeed, after treatment with methyl viologen, which produces CHAPTER 4

ROS in chloroplasts or mitochondria in plants, ATG18a RNAi lines display a more chlorotic phenotype relative to Col-0 [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF]. In addition, atg2 and atg5 mutants were reported to suffer oxidative stress, even in the absence of external stress [START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. Inversely, exogenous H 2 O 2 and MV could rapidly induce autophagy [START_REF] Xiong | Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis[END_REF]. 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 CO 2 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 [START_REF] Zimmermann | Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh[END_REF][START_REF] Smykowski | G-Box binding factor1 reduces CATALASE2 expression and regulates the onset of leaf senescence in Arabidopsis[END_REF], 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 CO 2 to air, loss of CAT2 function seems to oppose the spread of senescence. This observation could point to negative interactions between photorespiratory H 2 O 2 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 [START_REF] Hofius | Autophagic components contribute to hypersensitive cell death in Arabidopsis[END_REF][START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]Lenz et al., 2011b;[START_REF] Wang | ATG2, an autophagy-related protein, negatively affects powdery mildew resistance and mildew-induced cell death in Arabidopsis[END_REF]. 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 [START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. 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 CO 2 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 CO 2 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 [START_REF] Noctor | Glutathione in plants: an integrated overview[END_REF] 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 CO 2 (Figure 4.9). While it is not clear why this effect should be observed at high CO 2 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 CO 2 -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.

CHAPTER 5 CONCLUSION AND PERSPECTIVES

CHAPTER 5 CONCLUSION AND PERSPECTIVES

CONCLUSIONS

Numerous studies have demonstrated that H 2 O 2 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 H 2 O 2 -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 H 2 O 2 metabolism. In this work, we adopted a reverse genetics approach to studying the interaction of glutathione and autophagy with increased H 2 O 2 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 H 2 O 2 production occurs in peroxisomes through a physiologically relevant pathway (photorespiration) and it can be easily controlled by growth conditions such as ambient CO 2 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 H 2 O 2 in cat2 arbohF double mutants are associated with altered glutathione status [START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF], it remained unclear how important this altered statusis in linking phytohormone signaling to oxidative stress.

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 H 2 O 2 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 H 2 O 2 -triggered JA signaling by 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.

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 H 2 O 2 [START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. In addition, CAT2 expression is down-regulated at the onset of leaf senescence [START_REF] Zimmermann | Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh[END_REF]. 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 H 2 O 2 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 CO 2 . 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 H 2 O 2 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.

PERSPECTIVES

Potential mechanisms on the effects of blocking glutathione accumulation in response to enhanced H 2 O 2 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 H 2 O 2 , 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 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.

Glutathione oxidation and metabolism in response to H 2 O 2

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 [START_REF] Foyer | Ascorbate and glutathione: the heart of the redox hub[END_REF]. When catalase is deficient, APX may be the major enzyme that is engaged to deal with the extra H 2 O 2 ; 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;[START_REF] Queval | Day length is a key regulator of transcriptomic responses to both CO 2 and H 2 O 2 in Arabidopsis[END_REF]. 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.

Glutathione, H 2 O 2 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 [START_REF] Wagner | The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana[END_REF], classical genetic screens have failed to identify "H 2 O 2 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 "H 2 O 2 receptors" is that H 2 O 2 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 H 2 O 2 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).

H 2 O 2 , NO, and glutathione

Although the individual roles of NO and H 2 O 2 in plant defence against pathogens is well established, the two molecules interact to activate the HR and pathogen resistance [START_REF] Delledonne | Nitric oxide functions as a signal in plant disease resistance[END_REF][START_REF] Delledonne | Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response[END_REF]. Indeed, the NO donor sodium nitroprusside (SNP) provokes cell death in catalase-deficient tobacco leaves exposed to moderate light stress [START_REF] Zago | Nitric oxide-and hydrogen peroxide-responsive gene regulation during cell death induction in tobacco[END_REF]. 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 H 2 O 2 -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 (NOS) has been identified in the green algae Ostreococcus tauri, the exact source of NO in higher plants

has not yet been elucidated [START_REF] Foresi | Characterization of a nitric oxide synthase from the plant kingdom: NO generation from the green alga Ostreococcus tauri is light irradiance and growth phase dependent[END_REF].

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 H 2 O 2 , 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 [START_REF] Flores-Pérez | A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids[END_REF][START_REF] Gas | Hunting for plant nitric oxide synthase provides new evidence of a central role for plastids in nitric oxide metabolism[END_REF]. 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 [START_REF] Liu | OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function[END_REF]. 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.

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 [START_REF] Ma | Ca 2+ , cAMP, and transduction of non-self perception during plant immune responses[END_REF].

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 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 [START_REF] Qi | Calcium entry mediated by GLR3.3, an Arabidopsis glutamate receptor with a broad agonist profile[END_REF]. Additionally, catalase activity may be activated by calmodulin binding in the presence of calcium [START_REF] Yang | Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin[END_REF]. 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 H 2 O 2 signaling to changes in calcium concentrations.

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) (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 H 2 O 2 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 [START_REF] Kendall | The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgare L.)[END_REF][START_REF] Lin | Nitric oxide and protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice[END_REF]. 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.

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 accelerated in high CO 2 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 [START_REF] Yoshimoto | Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis[END_REF]. 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 H 2 O 2 -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 [START_REF] Mittler | ROS signalling: the new wave?[END_REF][START_REF] Chaouch | AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis[END_REF]. 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 CO 2 . 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.

CHAPTER 6 MATERIALS AND METHODS

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) 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). 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 CO 2 concentration was maintained at 400 μL L -1 (air) or 3000 μL L -1 (high CO 2 ). 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 CO 2 as described above. On transfer from high CO 2 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.

Methods

Molecular biology analyses

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 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 CO 2 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.

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 10 5 colony-forming units mL -1 . Leaf discs (0.5 cm 2 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 H 2 O, plating appropriate dilutions on soild LB medium containing rifampicin (100 μg/mL) and kanamycin (25 μg/mL), and counting colony numbers after 3 days.

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 [START_REF] Zechmann | Subcellular compartmentation of glutathione in dicotyledonous plants[END_REF]. 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 -1 FW [START_REF] Winter | Subcellular volumes and metabolite concentrations in spinach leaves[END_REF].

Metabolite analyses

Assays for ascorbate, glutathione and NADP(H)

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.

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.

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.

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 [START_REF] Tietze | Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues[END_REF]. Specific assay of GSSG was done by pre-treatment of extract aliquots with 2 μL of 2-vinylpyridine (VPD), which efficiently complexes GSH [START_REF] Griffith | Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine[END_REF]. To measure total glutathione, the mixture contained 100 μL of 0.2 M NaH 2 PO 4 , 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 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. for 30 min at room temperature. Both of above assays were done in triplicate for each extract. According to a standard extinction coefficient of 14 mM -1 cm -1 , absorbance changes in A 265 were converted to quantities of ascorbate (Queval and Noctor, 2007).

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.

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.

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 [START_REF] Noctor | A comparative study of amino acid analysis in leaf extracts by gas chromatography-time of flight-mass spectrometry and high performance liquid chromatography with fluorescence detection[END_REF], except that a mix of alkanes was 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.

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 NH 3 (pH 9.5), and 0.05 mL of 0.5 mM luminol (prepared in 0.2 M NH 3 , pH 9.5), vortexed rapidly, and injected into a luminometer. The reaction was started by adding 100 mL of 0.5 mM K 3 Fe(CN) 6 (in NH 3 ), and luminol chemiluminescence was monitored for 2 s. To check the linearity of this assay, a standard curve of H 2 O 2 was done for each experiment, and assay standards and extracts were assayed in triplicate.

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. Foyer et al., 1997;May et al., 1998;[START_REF] Foyer | Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses[END_REF]. The glutathione/GR system is involved in H 2 O 2 metabolism by reducing dehydroascorbate generated following the (per)oxidation of ascorbate (Asada, 1999). This pathway is one way in which H 2 O 2 reduction could be coupled to NADPH oxidation, with the first reaction catalyzed by ascorbate peroxidase (APX) and the last by GR, although ascorbate regeneration can occur independently of reduced glutathione (GSH) through NAD(P)H-dependent or (in the chloroplast) ferredoxin-dependent reduction of monodehydroascorbate (MDAR; Asada, 1999). Additional complexity of the plant antioxidative system has been highlighted by identification of several other classes of antioxidative peroxidases that could reduce H 2 O 2 to water. These include the TRX fusion protein CDSP32 [START_REF] Rey | Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses[END_REF] and several types of peroxiredoxin, many of which are themselves TRX dependent (Dietz, 2003). Plants lack animaltype selenocysteine-dependent glutathione peroxidase (GPX), instead containing Cys-dependent GPX (Eshdat et al., 1997;Rodriguez Milla et al., 2003). Despite their annotations as GPX, these enzymes are now thought to use TRX rather than GSH (Iqbal et al., 2006). However, H 2 O 2 could still oxidize GSH via peroxidatic glutathione S-transferases (GSTs) and/or glutaredoxin (GRX)-dependent peroxiredoxin (Wagner et al., 2002;[START_REF] Jacquot | The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation[END_REF]. Metabolism of H 2 O 2 may occur through APX or through ascorbate-independent GSH peroxidation, both of which potentially depend on GR activity, as well as through TRX-dependent pathways.

The relative importance of these pathways remains unclear.

The major sites of intracellular H 2 O 2 production in most photosynthetic plant cells are the chloroplasts and peroxisomes (Noctor et al., 2002). Despite this, Davletova et al. (2005) demonstrated a crucial role for cytosolic APX1 in redox homeostasis in Arabidopsis. This finding implies that the cytosolic ascorbateglutathione pathway is important in metabolizing H 2 O 2 originating in other organelles, thus marking out this compartment as a key site in which redox signals are integrated to drive appropriate responses. Cytosolic metabolism of H 2 O 2 could be key in setting appropriate conditions for thiol-disulfide regulation through cytosolic/nuclear signal transmitters such as NPR1 and TGA transcription factors [START_REF] Despre ´s | The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1[END_REF]Mou et al., 2003;Rochon et al., 2006;Tada et al., 2008). Because peroxisomal reactions can be a major producer of H 2 O 2 , a related issue that remains unresolved is to what extent reductive H 2 O 2 metabolism overlaps with metabolism through catalase, which is confined to peroxisomes and homologous organelles. Intriguingly, double antisense tobacco (Nicotiana tabacum) lines in which both catalase and cytosolic APX were down-regulated showed a less marked phenotype than single antisense lines deficient in either enzyme alone (Rizhsky et al., 2002). This observation implies that if the major role of the GR-glutathione system is indeed to support reduction of dehydroascorbate to ascorbate, down-regulation of cytosolic GR capacity should produce a similar effect to APX deficiency.

Two genes are annotated to encode GR in plants. Pea (Pisum sativum) chloroplast GR was the first plastidial protein shown also to be targeted to the mitochondria (Creissen et al., 1995). Dual targeting of this protein also occurs in Arabidopsis (Chew et al., 2003), where the plastidic/mitochondrial isoform is named GR2. The second gene, GR1, is predicted to encode a cytosolic enzyme. In pea, cytosolic GR has been well characterized at the biochemical level (Edwards et al., 1990;Stevens et al., 2000), but the functional significance of the enzyme remains unclear. Underexpression or overexpression studies in tobacco and poplar (Populus species) have reported significant effects of modifying chloroplast GR capacity (Aono et al., 1993;Broadbent et al., 1995;Foyer et al., 1995;Ding et al., 2009). Less evidence is available supporting an important role for cytosolic GR. In insects, GSSG reduction can also be catalyzed by NADPH-TRX reductases (NTRs; [START_REF] Kanzok | Substitution of the thioredoxin system for glutathione reductase in Drosophila melanogaster[END_REF], and it has recently been shown that Arabidopsis cytosolic NTR can functionally replace GR1 (Marty et al., 2009).

Therefore, key outstanding issues in the study of redox homeostasis and signaling in plants are (1) the importance of GR/glutathione in H 2 O 2 metabolism and/or H 2 O 2 signal transmission and (2) the specificity of GSH and TRX systems in H 2 O 2 responses. In this study, we sought to address these questions by a genetically based approach in which the effects of modified H 2 O 2 and glutathione were first analyzed in parallel in single mutants and then directly through the production of double mutants. This was achieved using gr1 insertion mutants and a catalase-deficient Arabidopsis line, cat2, in which intracellular H 2 O 2 drives the accumulation of GSSG in a conditional manner (Queval et al., 2007). Our study provides in vivo evidence for a specific irreplaceable role for GR1 in H 2 O 2 metabolism and signaling.

RESULTS

Characterization of gr T-DNA Mutants

While T-DNA insertions in the coding sequence of dual-targeted chloroplast/mitochondrial GR2 are embryo lethal (Tzafrir et al., 2004), homozygous gr1 mutants were readily obtained. Reverse transcription (RT)-PCR confirmed the absence of GR1 transcript, while total extractable GR activity was decreased by 40% relative to ecotype Columbia (Col-0; Supplemental Fig. S1). Despite these effects, repeated observations over a period of 5 years showed that the mutation produced no difference in rosette growth rates from Col-0 in either short days (SD) or long days (LD) or in flowering timing and leaf number (data not shown). Thus, our analysis is in agreement with the report of Marty et al. (2009) that absence of cytosolic GR activity caused a measurable decrease in leaf GR activity but that this did not cause phenotypic effects.

Comparative Analysis of Glutathione-and H 2 O 2 -Dependent Changes in Gene Expression

Because gr1 mutants are aphenotypic in typical growth conditions, this study analyzed the potential role of the enzyme in H 2 O 2 metabolism and signaling. First, we performed parallel microarray analysis of GSSG-accumulating gr1 and the catalase-deficient mutant cat2, which conditionally accumulates GSSG triggered by photorespiratory H 2 O 2 production (Queval et al., 2007). To compare effects on transcript abundance while minimizing possible interference of longterm developmental effects due to oxidative stress in cat2 grown from seed in air, plants were sampled following initial growth at high CO 2 , where the cat2 mutation is silent (Queval et al., 2007). This experimental design was chosen to allow comparison of the effects of modified glutathione status in gr1 with similar H 2 O 2 -induced effects triggered in cat2 after transfer from high CO 2 to air. Our rationale was that while H 2 O 2 may modify gene expression through a number of signaling mechanisms, any effects medi-ated via glutathione should also be observed in gr1, even in the absence of the oxidative stress that drives GSSG accumulation in cat2 (Fig. 1A).

As previously reported, cat2 showed a wild-type phenotype at high CO 2 . The consequences of the cat2 mutation for gene expression and phenotype in air are strongly influenced by growth photoperiod (Queval et al., 2007). Thus, measurements of transcripts and antioxidant were performed after transfer of gr1 and cat2 from high CO 2 to air in both SD (8-h photoperiod) and LD (16-h photoperiod). In both photoperiods (and at high CO 2 ), the glutathione reduction state was lower in gr1 than in Col-0 (Fig. 1B). In cat2, however, this factor was only lower than in Col-0 after transfer to air, where it was decreased below the values observed in gr1. In both mutants, leaf ascorbate contents were much less affected than glutathione. The only significant difference was observed in ascorbate reduction state in cat2 in LD (Supplemental Fig. S2).

Analysis of microarray data was performed by considering as being differentially expressed genes with a Bonferroni P # 0.05, as described by Gagnot 2008). An additional selection criterion was introduced by considering only genes that showed statistically significant same-direction change in both biological replicates. The analysis showed that H 2 O 2 -regulated gene expression (in cat2) was highly daylength dependent (Fig. 1C). More genes were significantly induced in cat2 transferred to air in SD than in LD, whereas the reverse was true for significantly repressed genes. Fewer genes were affected in gr1, but effects were also daylength conditioned. Of genes induced in gr1 in SD, only three were also induced in LD, together with another four LD-specific genes. The number of repressed genes in gr1 in LD was similar to that in SD (Fig. 1C), but of these, only two were common (Supplemental Table S2). Thus, as for genes regulated by an H 2 O 2 signal, gene expression dependent on a drop in glutathione reduction state was strongly determined by photoperiod context. Of the 58 genes showing significantly modified expression in response to a mild perturbation of glutathione redox state in gr1, most showed significant samedirection changes in at least one cat2 replicate (Supplemental Table S2). It should be noted that Figure 1C provides a conservative estimate of overlap between gr1 and cat2 transcriptomes because only genes that showed statistically significant same-direction changes in all four dye-swap repeats (two gr1/Col-0 and two cat2/Col-0) are considered.

Several genes whose expression was modified in gr1 encoded stress-related proteins, notably cadmium-, salt-, and cold-responsive genes, as well as two GSTs (GSTU7 and GSTU6) and three multidrug resistanceassociated proteins (Supplemental Table S2). This is consistent with the roles of glutathione in redox homeostasis, heavy metal resistance, cold acclimation, and metabolite conjugation and transport (Cobbett et al., 1998;May et al., 1998;[START_REF] Kocsy | Inhibition of glutathione synthesis reduces chilling tolerance in maize[END_REF]Wagner et al., 2002;Gomez et al., 2004).

Sixteen of the 58 gr1-sensitive genes are annotated as phytohormone associated. In all cases, induction or repression of these genes was dependent on daylength context (Table I). A putative monoxygenase with similarity to salicylic acid (SA)-degrading enzymes was among the induced genes, whereas other differentially expressed genes in gr1 were associated with auxin, gibberellin, abscisic acid, and ethylene function (Table I). The most striking impact of the gr1 mutation was on the expression of genes involved in jasmonic acid (JA) synthesis and signaling. Of the 18 genes repressed in gr1 in LD, eight have been shown to be early JA-responsive genes (Yan et al., 2007). These genes encoded, among others, LIPOXYGENASE3 (LOX3), MYB95, GSTU6, and two of the 12 JASMONATE/ ZIM DOMAIN (JAZ) proteins recently characterized as JA-inducible repressors of JA signaling [START_REF] Staswick | JAZing up jasmonate signaling[END_REF][START_REF] Browse | Jasmonate passes muster: a receptor and targets for the defense hormone[END_REF]. All but one of the JA-dependent genes repressed in gr1 in LD showed the same response in the GSSG-accumulating cat2 mutant (Table I).

Two induced stress-associated genes and four JAdependent genes that showed similar responses in gr1 and cat2 were selected for quantitative PCR analysis. This confirmed the induction in gr1 of the cold-regulated COR78 in SD and of the cadmium-responsive At3g14990 in both daylength conditions (Fig. 2). For the JAassociated genes, quantitative PCR revealed that in addition to repression in gr1 and cat2 in LD, three of the four genes (LOX3, MYB95, and JAZ10) were also induced in SD (Fig. 2). Thus, GR1-dependent glutathione status influences the expression of genes involved in JA synthesis and signaling in a daylength-dependent manner.

Genetic Analysis of GR1 Function in Responses to H 2 O 2

To directly explore the role of GR1 and glutathione status in the H 2 O 2 response, the gr1 mutation was crossed into the cat2 background. Preliminary analysis 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 CO 2 , where, as noted above, the cat2 mutation is phenotypically silent because photorespiratory H 2 O 2 production is largely shut down. Genotyping of these seeds identified several plants with cat2/cat2 gr1/gr1 genotypes, suggesting selection against cat2 gr1 double homozygotes in the presence of photorespiratory H 2 O 2 production (air) but not in its absence (high CO 2 ). When F4 seeds obtained from F3 cat2 gr1 double homozygotes identified at high CO 2 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 CO 2 (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 increased intracellular H 2 O 2 availability. To further investigate this point, we analyzed the phenotypic responses of cat2 gr1 grown at high CO 2 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 H 2 O 2 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 CO 2 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 CO 2 showed any apparent phenotype (data not shown). Of 93 F2 plants transferred from high CO 2 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).

Microarray Analysis of the H 2 O 2 -GR1 Interaction

To identify H 2 O 2 -regulated genes whose expression is dependent on GR1, transcript profiling of cat2 and cat2 gr1 was performed after transfer of plants grown at high CO 2 to air. In view of the above phenotypic observations and the daylength-dependent transcript profiles for cat2 and gr1 single mutants (Fig. 1; Supplemental Table S2), the analysis was performed after transfer to both SD and LD. A full list of significantly different genes and signal intensities is given in Supplemental Table S3.

Clustering analysis of significantly different transcripts delineated two major patterns of H 2 O 2 -induced gene expression (Fig. 5). The first was largely composed of genes that were induced by photorespiratory H 2 O 2 in cat2 and cat2 gr1 (cluster 1), while the second comprised genes that responded in a daylengthdependent manner (cluster 2). The most striking impact of the gr1 mutation in the cat2 background was to annul or impair the induction of genes by photorespiratory H 2 O 2 in SD, an effect exemplified by a subcluster of genes in cluster 2. Of 62 genes in this subcluster, 33 are annotated as involved in JA-or wounding-dependent responses [START_REF] Taki | 12-Oxo-phytodienoic acid triggers expression of a distinct set of genes and plays a role in wound-induced gene expression in Arabidopsis[END_REF]Yan et al., 2007; http://www.arabidopsis.org) and are highlighted yellow in Figure 5. Data mining of all significantly different genes using the above data sets revealed that, in all, 47 genes inducible by JA or wounding, or encoding proteins involved in JA synthesis, conjugation, or signaling, were differentially expressed in cat2 and/or cat2 gr1 (Supplemental Table S4). Twenty-five of these were among 35 genes recently shown to be induced by wounding in a JA-dependent manner (Yan et al., 2007), while among the others were established JA-associated genes such as LOX2, LOX3, OPR3, JAZ1, JAZ3, JAZ7, JAZ9, JAR1, and JMT1.

In the cat2 single mutant, the predominant effect of H 2 O 2 on genes involved in JA synthesis and signaling was induction in SD and/or repression in LD. This daylength-dependent regulation was strongly modulated by the absence of GR1 function in cat2 gr1. Of the 47 JA-associated transcripts whose abundance was significantly modified in cat2 and/or cat2 gr1 relative to Col-0, only 10 did not show differential expression between the two lines in at least one photoperiod (Supplemental Table S4). The overwhelmingly predominant effect of the gr1 mutation in the cat2 background was to cause or enhance repression (e.g. LOX2, JMT1, JAZ3), to oppose induction in SD (e.g. RAP2.6, JAZ10, AOS), or both (LOX3, TAT3, OPR3).

Profiling of Glutathione and Associated Gene Expression in cat2 gr1

Consistent with the absence of phenotype at high CO 2 (Fig. 4A), cat2 glutathione status was similar to the wild type (95% GSH), whereas in cat2 gr1 the glutathione pool was only about 70% reduced (i.e. similar to the gr1 single mutant in air or at high CO 2 ; Fig. 6A; Supplemental Fig. S2). These observations are consistent with the silent nature of the cat2 mutation at high CO 2 and with a constitutive oxidation of glutathione in the gr1 genotype. The impact of the gr1 mutation on glutathione in conditions of increased H 2 O 2 was assessed under the same conditions as for the microarray analysis. A time course following transfer to air revealed that leaf glutathione (1) became progressively oxidized and accumulated in cat2; (2) became much more rapidly oxidized and accumulated more strongly in cat2 gr1; and (3) was most oxidized and most strongly accumulated in cat2 gr1 in SD. The changes in glutathione involved a fall in the GSH to GSSG ratio to below 1 in cat2 and cat2 gr1, with the lowest ratio observed in cat2 gr1 in SD (Fig. 6A).

Mining of the transcriptome data for glutathioneassociated genes showed that modified glutathione status in cat2 and cat2 gr1 was associated with the induction of genes encoding three GSTs of the tau class (GSTU7, GSTU8, GSTU19) and two of the phi class (GSTF2, GSTF8), two multidrug resistanceassociated proteins (MRP2, MRP14), a glutathione/ thioredoxin peroxidase (GPX6), a glyoxylase I family protein, two members of the GRX family, and a GRXlike expressed protein (Fig. 6B). These genes were equally or more strongly induced in cat2 gr1 compared with cat2, although the effect depended on daylength. In no case did the gr1 mutation oppose induction of these genes in cat2, which were all found to be grouped in cluster 1 on the heat map shown in Figure 5.

Five glutathione-associated genes showed a different expression pattern (Fig. 6B, bottom) and were all found within cluster 2 shown in Figure 5. While one GRX (At5g18600) showed a tendency to repression in all conditions, repression was only significant in cat2 gr1 in SD. GSTU5 and GSTU6 were both induced in cat2 but not in cat2 gr1 in SD and were repressed in both genotypes in LD (Fig. 6B). The gr1 mutation in the cat2 background also produced repression of GGT1 and GRX480, specifically in LD. Thus, the expression patterns of these five genes were similar to those of JAdependent genes (Fig. 5; Supplemental Table S4). In contrast to the differential expression of these glutathionedependent genes, the expression of cytosolic TRXs and associated genes involved in thiol-disulfide exchange reactions was little or not affected in either the cat2 or cat2 gr1 genotype (Supplemental Table S5).

Analysis of Oxidative Stress and the Ascorbate-Glutathione Pathway

Catalase-independent pathways of H 2 O 2 metabolism depend on ascorbate, which is considered to be partly regenerated by glutathione, while NADPH is required for reduction of GSSG. Despite this, the effects of gr1 and cat2 mutations on glutathione pools were not associated with marked perturbation of leaf ascorbate pools, which remained more than 80% reduced in all plants. In LD conditions, ascorbate was decreased in cat2 and cat2 gr1, although this small effect was only statistically significant for cat2 (Fig. 7A). Like ascorbate, the NADP reduction state was not greatly affected in any of the samples (Fig. 7B). The only significant effects were observed in cat2 gr1, which in SD had more NADPH than Col-0 and in LD less NADP + . Because of these effects, the most reduced NADP(H) pools were observed in cat2 gr1. Assay of lipid peroxidation products using thiobarbituric acid also showed no significant effect between any of the samples in either condition (Fig. 7C).

Potential problems that must be taken into account in measuring H 2 O 2 include low assay specificity, interference, and extraction efficiency [START_REF] Wardman | Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects[END_REF]Queval et al., 2008). In this study, we used three different techniques to assess H 2 O 2 or reactive oxygen species (ROS) levels in cat2 and cat2 gr1. In situ staining using 3,39-diaminobenzidine did not produce evidence for generalized accumulation of H 2 O 2 in gr1, cat2, or cat2 gr1 leaves in SD or LD (Supplemental Fig. S5A). Likewise, assays of extractable peroxides using luminol chemiluminescence did not find increased contents in cat2 or cat2 gr1 in either daylength condition (Supplemental Fig. S5B). Semiquantitative ROS visualization within the mesophyll cells in vivo also revealed that the cat2 mutation caused only minor increases in dichlorofluorescein fluorescence (Fig. 8). The cat2 gr1 double mutant showed an appreciably stronger fluorescence signal, but this effect was specific to plants in SD (Fig. 8).

To further assess the impact of the mutations on the ROS-antioxidant interaction, we measured the extractable activities and expression of APX, catalase, GR, and dehydroascorbate reductase (DHAR), the enzyme linking glutathione and ascorbate pools. The only one of these enzyme activities that was significantly different between Col-0 and gr1 was GR (Fig. 9A). Similarly, the gr1 mutation in the Col-0 background produced no significant change in transcripts other than GR1 (Fig. 9C). Decreased catalase activity in cat2 was associated with an increase in APX activity in both conditions (Fig. 9A). Increased APX activity was accompanied by induction of transcripts for APX1, encoding the cytosolic isoform (Davletova et al., 2005). Of eight APX transcripts present on the chip, only APX1 was significantly increased in cat2, and as for APX activity, this effect was similar in both SD and LD (Fig. 9C). Induction of APX1 in cat2 was accompanied by induction of a cytosolic DHAR (DHAR2), although this effect was stronger in SD than in LD and was not associated with a marked increase in the overall activity of this enzyme. Compared with Col-0, effects in cat2 gr1 were similar to those observed in cat2 (i.e. induction of APX1 in both SD and LD, an increase in extractable APX in the two conditions, and induction of DHAR2 transcripts, particularly in SD; Fig. 9). Overall, the presence of the gr1 mutation in Col-0 or cat2 backgrounds did not greatly affect the responses of these antioxidative systems relative to the respective controls. Despite the oxidation of the glutathione pool Figure 6. Glutathione contents and glutathione-related gene expression in cat2 and the cat2 gr1 double mutant. A, Time course of changes in percentage glutathione reduction and total glutathione in the different lines following transfer from high CO 2 to air. Black circles, Col-0; white circles, cat2; white triangles, cat2 gr1. Top, 100 3 GSH/total glutathione (the numbers indicate GSH to GSSG ratio for the final time points). Bottom, total glutathione (GSH + 2 GSSG). Error bars that are not apparent are contained within the symbols. FW, Fresh weight. B, Expression values of significantly different 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.

in cat2 and its very marked oxidation in cat2 gr1 (Fig. 6A), neither genotype showed compensatory induction of GR2 transcripts, encoding the chloroplast/ mitochondrial isoform (Fig. 9C).

SA Signaling and Pathogen Reponses

Given the observed effects on JA-associated genes (Fig. 5; Table I; Supplemental Table S4) and the opposition between JA signaling and SA-dependent pathways [START_REF] Dangl | Plant pathogens and integrated defence responses to infection[END_REF][START_REF] Koorneef | Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation[END_REF], we analyzed SA and expression of SA marker genes in gr1, cat2, and cat2 gr1 in both daylength conditions. SA-dependent PR genes (PR1, PR2) were both significantly induced in cat2 in LD but less strongly or not at all in SD (Fig. 10A). Induction of SA-dependent PR genes in cat2 in LD was antagonized by the gr1 mutation, producing significantly lower expression in cat2 gr1. Leaf SA contents were similar in all samples in SD, although slightly decreased in cat2 gr1, whereas in LD H 2 O 2 -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 redoxmodulated 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 overlap between glutathione and TRX systems (Reichheld et al., 2007;Marty et al., 2009). A key question, therefore, concerns the specificity of cytosolic GRglutathione and NTR-TRX functions. Glutathione has been proposed to play a role in H 2 O 2 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.

GR1 Plays a Nonredundant Role in H 2 O 2 Metabolism and Signaling

In this study, the cat2 mutant was used both as a reference GSSG-accumulating H 2 O 2 signaling system and as a genetic background in which to directly explore the GR1-H 2 O 2 interaction. Because daylengthdependent effects have been described for redox signaling during pathogen and oxidative stress responses [START_REF] Dietrich | Arabidopsis mutants simulating disease resistance response[END_REF][START_REF] Karpinski | Light perception in plant disease defence signalling[END_REF]Queval et al., 2007;[START_REF] Vollsnes | Visible foliar injury and infrared imaging show that daylength affects short-term recovery after ozone stress in Trifolium subterraneum[END_REF], we analyzed the importance of GR1 in Col-0 and cat2 backgrounds in two growth daylengths.

Our analysis of gr1 in the Col-0 background confirm the results of Marty et al. (2009) that GR1 is not required for growth in optimal conditions. However, GR1 plays an important role when intracellular H 2 O 2 production is increased and during pathogen challenge. The effect of gr1 on the cat2 phenotype contrasts with results in which plants were exposed to exogenous H 2 O 2 present in agar, where no difference was found between gr1 and Col-0 phenotypes, even at highly challenging H 2 O 2 concentrations (Marty et al., 2009). This suggests that the site of H 2 O 2 production is crucial. Enhanced H 2 O 2 availability in cat2 leaves placed in air occurs intracellularly through the photorespiratory enzyme, glycolate oxidase, which is located in peroxisomes. In these conditions, redox perturbation in cat2 produces similar effects to those observed in stress conditions, notably oxidation and accumulation of glutathione and changes in the abun- dance of many stress-related transcripts. GR activity has been detected in pea leaf peroxisomes, and it is likely that GR1 is found in Arabidopsis peroxisomes as well as in the cytosol [START_REF] Jime ´nez | Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves[END_REF][START_REF] Kaur | Peroxisome biogenesis and function[END_REF]. Therefore, it is possible that GR1 is important in peroxisomal ascorbate-and/or glutathione-dependent H 2 O 2 metabolism. At least one (APX3) and possibly as many as three (APX3, APX4, APX5) Arabidopsis APXs are targeted to peroxisomes or the peroxisomal membrane [START_REF] Narendra | The Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development[END_REF], but the corresponding transcripts were not significantly induced in gr1, cat2, or cat2 gr1 (Fig. 9). Most of the antioxidative genes that were induced in cat2 and cat2 gr1 encoded cytosolic enzymes, including APX1, DHAR2, and MDAR2 (Supplemental Table S3). Our analysis thus points to tight coupling between H 2 O 2 produced in the peroxisomes and cytosolic antioxidative systems.

The dramatic accumulation of GSSG in cat2 gr1 placed in air shows that GSH regeneration through alternative pathways is rapidly exceeded under conditions of enhanced intracellular H 2 O 2 availability. To our knowledge, the accumulation of GSSG in cat2 gr1 (up to 2 mmol g 21 fresh weight; Fig. 6A) exceeds previously reported values for this compound in leaf tissues. Further work is required to identify the compartments in which such marked accumulation of GSSG occurs. Analyses of the gr1 single mutant in air and the cat2 gr1 double mutant at high CO 2 show that mild perturbation of the glutathione pool is not sufficient to produce marked phenotypic effects, while effects produced by the more severe perturbation of glutathione in cat2 gr1 in air were dependent on growth daylength. Lesions were not observed in cat2 gr1 in SD, even though these plants showed the most dramatic GSSG accumulation and the lowest GSH to GSSG ratios. Oxidized glutathione pools are considered to be among the cellular factors underlying dormancy and cell death (Kranner et al., 2002(Kranner et al., , 2006)). However, our data show that although growth arrest was linked to glutathione perturbation in both daylengths, leaf cells can tolerate extreme perturbation of intracellular thiol-disulfide state without undergoing bleaching (Fig. 5). Analysis of ascorbate, thiobarbituric acid-reactive substances, and ROS also provided little evidence that oxidative stress was stronger in LD compared with SD.

The dramatic perturbation of glutathione pools in cat2 gr1 compared with cat2 provides direct evidence for the role of GR1 in intracellular H 2 O 2 metabolism. Analysis of ROS suggests that H 2 O 2 accumulation in cat2 is localized and/or minimized by metabolism through ascorbate-and/or glutathione-dependent pathways. Little or no detectable increase in H 2 O 2 in cat2 compared with the wild type is consistent with previous studies of catalase-deficient tobacco and barley (Hordeum vulgare) lines (Willekens et al., 1997;Noctor et al., 2002;Rizhsky et al., 2002). Although certain antioxidative enzymes and transcripts were up-regulated in cat2 and cat2 gr1, these effects were at least as marked in SD as in LD. ROS signal intensity was increased in cat2 gr1 in SD, but increases were less evident in this genotype in LD (Fig. 8), conditions in which bleaching occurred (Fig. 5). One explanation of the requirement of LD conditions for leaf bleaching is that other daylength-linked signals are required in addition to oxidative stress intensity.

The phenotype of cat2 gr1 in LD contrasts with observations of tobacco plants deficient in both cytosolic APX and catalase, which showed an ameliorated phenotype compared with parent lines (Rizhsky et al., 2002). One explanation of the different effects of GR and APX deficiency in catalase-deficient plants is that some proportion of H 2 O 2 is metabolized through GR-dependent but ascorbate-independent pathways. Hence, as well as GSH oxidation by dehydroascorbate, glutathione perturbation in cat2 may occur through enzyme-catalyzed peroxidation of GSH (e.g. catalyzed by certain GSTs, as discussed further below). Although increases in APX activities in cat2 and cat2 gr1 were associated with induction of APX1 (Fig. 9), several glutathione-associated genes with potential peroxidative functions were also induced in these lines (Fig. 6). Although there was no evidence of oxidative stress or phenotypic effects in the gr1 single mutant, transcriptomes in this line partly overlapped with that of the H 2 O 2 signaling reference system, cat2. Both transcriptomes were found to be highly dependent on daylength. This observation suggests that modulation of redox-linked gene expression by growth daylength is not restricted to oxidative stress or is a secondary consequence of stress-induced phenotypes.

Fewer genes were affected in gr1 than in cat2. Genes affected in cat2 but not gr1 could reflect H 2 O 2 signaling that occurs independently of changes in glutathione (Fig. 1A). The difference could also be related to the stronger perturbation of the glutathione pool in cat2 (Fig. 1B). If the difference can be partly explained by weaker changes in glutathione in gr1, then the overlap in transcriptomes we report could provide a minimum estimate of the importance of glutathione in H 2 O 2 signaling Many TRX-linked components play roles in oxidative stress (Dietz, 2003;[START_REF] Rey | Analysis of the proteins targeted by CDSP32, a plastidic thioredoxin participating in oxidative stress responses[END_REF][START_REF] Pe ´rez-Ruiz | NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage[END_REF], but none of these transcripts was induced 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 cat2 gr1 (4). Units are mmol mg 21 protein min 21 (catalase) and nmol mg 21 protein min 21 (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 catalaseand ascorbate-dependent H 2 O 2 metabolism. C, Abundance (log 2 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. by increased H 2 O 2 availability in cat2 or cat2 gr1. A lack of response was also observed for chloroplast ferredoxin-TRX reductase subunit genes and for the two cytosolic NTRs and cytosolic TRXs (Supplemental Table S5). The only TRX-linked gene that was significantly induced by H 2 O 2 was GPX6, and this effect was largely independent of both daylength and the gr1 mutation (Fig. 6).

In contrast to the lack of response of TRX systems, H 2 O 2 -or gr1-driven perturbation of glutathione triggered changes in the abundance of transcripts of several glutathione-associated genes, notably GSTs and GRXs. Analysis of GST functions is complicated by the number of genes in plants, notably due to the presence of large plant-specific phi and tau subclasses, as well as substrate overlap between the different enzymes. Our transcript profiling analysis showed that H 2 O 2 modified transcript levels for two phi class (GSTF2, GSTF8) and three tau class (GSTU7, GSTU8, GSTU19) enzymes and that in all cases, these effects were further modulated by daylength and/or the gr1 mutation. Type F GSTs exhibit both conjugase and peroxidase activities, with GSTF8 showing considerable activity against cumene hydroperoxide (Wagner et al., 2002;Dixon et al., 2009). As well as GSTFs, some GSTUs, including GSTU8, show quite high peroxidase activity (Dixon et al., 2009).

Analysis of GRX function is also complicated by the number of genes, notably due to a large subclass that is specific to higher plants. At least 30 GRX genes have been described in Arabidopsis (Lemaire, 2004;Meyer et al., 2008). Based on active-site sequences, these are subclassified into CPYC, monothiol CGFS, and CCtype GRX, with the last subclass being specific to land plants (Lemaire, 2004). Although at least one CGFS GRX is implicated in oxidative stress (Cheng et al., 2006), no CPYC or CGFS GRXs showed differential expression in gr1, cat2, or cat2 gr1 in either photoperiod condition (Supplemental Table S5). All differentially expressed GRX genes observed in our analysis belong to the CC-type subclass, consisting of 20 identified members that are thought to encode cytosolic proteins. The functions of most CC-type GRXs remain obscure, but two have been implicated in the regulation of gene expression by interacting with TGA transcription factors. GRX480 overexpression represses the JA marker gene, PDF1.2, while ROXY1 is required for petal development (Ndamukong et al., 2007;Li et al., 2009). The predicted protein encoded by the GRX-like sequence (Fig. 6B) has an active-site CCMS motif that is shared with nine annotated GRX proteins, and BLAST analysis revealed that the protein has 58% to 60% amino acid identity with five of these CCMS GRXs. The H 2 O 2 -and/or gr1-dependent changes in Figure 10. Pathogen-associated responses in GRdeficient lines. A, PR gene expression and SA contents in Col-0, gr1, cat2, and cat2 gr1 in two photoperiods after transfer from high CO 2 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 between 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).

CC-type GRXs suggest that the encoded proteins may play roles in the redox regulation of gene expression.

GR1 Is Required for SA Responses and for Expression of Genes Involved in JA Signaling

The cat2 mutation causes accumulation of SA and induction of SA responses in LD but not SD (Fig. 10). GR1 function appears to be required for optimal SA production, as this was compromised in the gr1 single mutant relative to Col-0 and in cat2 gr1 relative to cat2 (Fig. 10). Furthermore, gr1 showed decreased resistance to bacteria, while induction of PR genes in cat2 gr1 was compromised compared with cat2. As well as effects on SA production itself, the gr1 mutation could interfere with SA-dependent gene expression by altered redox regulation of NPR1 (Mou et al., 2003;Tada et al., 2008). Because loss of GR1 function in an H 2 O 2 signaling context produces accelerated lesion formation, the enzyme appears to be required both to restrict leaf bleaching and to enable the induction of SA and PR genes.

Several antioxidative genes can be induced by JA, including genes encoding enzymes of glutathione synthesis (Xiang and Oliver, 1998;Sasaki-Sekimoto et al., 2005). Recent reports implicate glutathioneassociated components in JA signaling (Ndamukong et al., 2007;[START_REF] Koorneef | Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation[END_REF]Tamaoki et al., 2008). In both Col-0 and cat2 backgrounds, the gr1 mutation affected a suite of JA-associated genes in a daylength-dependent manner. We observed induction of JA genes in cat2 and gr1 single mutants in SD but repression in all mutant genotypes in LD. This may indicate (1) an optimum glutathione redox status for induction of JA genes and (2) daylength-dependent signals that operate to modulate the impact of changes in glutathione status on JA signaling. The daylengthand gr1-modulated expression of several GRX and GSTs suggests that these components may functionally link glutathione and JA signaling. Such components could include GRX480 (Ndamukong et al., 2007), GSTU8-dependent formation of glutathione-oxylipin conjugates (Mueller et al., 2008), and GSTU6, which is among the JA-dependent genes that are rapidly induced by wounding (Yan et al., 2007). In our analysis, similar expression patterns were observed for these glutathione-associated genes and for a suite of JAdependent genes. Other genes that link glutathione and JA may include GSTU7, GSTU19, and MRP2, which were induced most strongly in cat2 gr1 in SD and less strongly in cat2 and cat2 gr1 in LD. These genes are up-regulated by phytoprostane or 12-oxophytodienoic acid treatment (Mueller et al., 2008). Several of the glutathione-associated genes shown in Figure 6 are rapidly induced by methyl jasmonate, 12-oxo-phytodienoic acid, phytoprostane, or wounding (Supplemental Fig. S6). Based on JA-or oxylipindependent expression patterns, the activities of GSTs against electrophilic metabolites, and interactions of CC-type GRX with transcription factors (Wagner et al., 2002;Ndamukong et al., 2007;Yan et al., 2007;Mueller et al., 2008;Li et al., 2009), it is possible that several GSTs and CC-type GRX could be involved in linking glutathione to JA signaling.

Interestingly, the glutathione-deficient pad2 mutant is compromised in insect resistance (Schlaeppi et al., 2008). Glutathione status is modulated by various stresses, notably biotic challenge, while sulfur deficiency, which can induce JA synthesis genes [START_REF] Hirai | Global expression profiling of sulfur-starved Arabidopsis by DNA macroarray reveals the role of O-acetyl-L-serine as a general regulator of gene expression in response to sulfur nutrition[END_REF], is an important determinant of glutathione concentration. As well as the influence of glutathione status, our data also underline the potential importance of daylength context in modulating redox-triggered signaling through the JA pathway.

CONCLUSION

While TRX-and glutathione-dependent pathways have overlapping functions in plants (Reichheld et al., 2007;Marty et al., 2009), this study shows that GR1 plays specific roles in intracellular H 2 O 2 metabolism, in daylength-linked control of phytohormone gene expression, and in certain responses to biotic stress. The transcriptomic patterns we report also point to glutathione-and TRX-specific pathways in redox signaling, with relatively little cross talk between the two pathways at the level of gene expression.

Two nonexclusive hypotheses could explain how the GR-glutathione system influences H 2 O 2 -linked gene expression. In the first, glutathione turnover would influence H 2 O 2 concentration through its antioxidative function. Most of our observations do not suggest that this is the main cause of the gr1-linked changes in gene expression, although in situ visualization of ROS suggests that it could contribute to differences between cat2 and cat2 gr1, particularly in SD. In the second hypothesis, glutathione status would be a key part of H 2 O 2 -triggered signal transduction. This hypothesis receives support from several of our observations, notably the partial overlap between cat2 and gr1 transcriptomes, but further work is required to confirm the role of glutathione status per se.

MATERIALS AND METHODS

Plant Material

Arabidopsis (Arabidopsis thaliana) mutant lines carrying T-DNA insertions in the GR1 gene (At3g24170) were identified using insertion mutant information obtained from the SIGnAL Web site (http://signal.salk.edu), and seeds were obtained from the Nottingham Arabidopsis Stock Centre (http://nasc. nott.ac.uk). The default gr1 line was gr1-2 (SALK_060425). A second gr1 insertion mutant (SALK_105794), named gr1-1 by Marty et al. (2009), was used to confirm the phenotypic effects of the cat2 gr1 interaction. After identification of homozygotes, all further analyses were performed on plants grown from T3 seeds. The cat2 line was cat2-2 (Queval et al., 2007), now renamed cat2-1.

Identification of Homozygous gr1 Insertion Mutants

Leaf DNA was amplified by PCR (30 s at 94°C, 30 s at 60°C, 1 min at 72°C, 30 cycles) using primers specific for left T-DNA borders and the GR1 gene (Supplemental Table S1). Fragments obtained were sequenced to confirm the insertion site. Zygosity was analyzed by PCR amplification of leaf DNA, and RT-PCR analysis of GR1 transcripts was performed using gene-specific primers (Supplemental Table S1).

Plant Growth and Sampling

Seeds were incubated for 2 d at 4°C and then sown either on agar or in soil in 7-cm pots. Plants were grown in a controlled-environment growth chamber at the specified photoperiod and an irradiance of 200 mmol m 22 s 21 at leaf level, 20°C/18°C, 65% humidity, and given nutrient solution twice per week. The CO 2 concentration was maintained at 400 mL L 21 (air) or 3,000 mL L 21 (high CO 2 ). Samples were rapidly frozen in liquid nitrogen and stored at 280°C until analysis. Unless otherwise stated, data are means 6 SE of three independent samples from different plants, and significant differences are expressed using t test at P , 0.1, P , 0.05, and P , 0.01.

Pathogen Tests

The virulent Pseudomonas syringae pv tomato strain DC3000 was used for resistance tests in a medium titer of 5 3 10 5 colony-forming units mL 21 . Whole leaves of 3-week-old plants grown in a 16-h photoperiod were infiltrated using a 1-mL syringe without a needle. Leaf discs of 0.5 cm 2 were harvested from inoculated leaves at the appropriate time points. For each time point, four samples were made by pooling two leaf discs from different treated plants. Bacterial growth was assessed by homogenizing leaf discs in 400 mL of water, plating appropriate dilutions on solid King B medium containing rifampicin and kanamycin, and quantifying colony numbers after 3 d.

Microarray Analysis

Microarray analysis was performed using the CATMA arrays containing 24,576 gene-specific tags 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++). This array resource has been used in 40 publications over the last 5 years using a protocol based on technical repeats of two independent biological replicates produced from pooled material from independent plants [START_REF] Achard | Plant DELLAs restrain growth and promote survival of adversity by reducing the levels of reactive oxygen species[END_REF][START_REF] Besson-Bard | Nitric oxide contributes to cadmium toxicity in Arabidopsis by promoting cadmium accumulation in roots and by up-regulating genes related to iron uptake[END_REF][START_REF] Krinke | Phospholipase D activation is an early component of the salicylic acid signaling pathway in Arabidopsis thaliana cell suspensions[END_REF], and this approach was adopted in this study. For each biological repeat and each point, RNA samples were obtained by pooling leaf material from independent sets, each of two plants. Samples of approximately 200 mg fresh weight were collected from plants after 3.5 weeks of growth in the conditions specified above. Total RNA was extracted using Nucleospin RNAII kits (Macherey-Nagel) according to the supplier's instructions. For each comparison, one technical replication with fluorochrome reversal was performed for each biological replicate (i.e. four hybridizations per comparison). The labeling of complementary RNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science Products), the hybridization to the slides, and the scanning were performed as described by [START_REF] Lurin | Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis[END_REF].

Statistical Analysis of Microarray Data

Experiments were designed with the statistics group of the Unite ´de Recherche en Ge ´nomique Ve ´ge ´tale. Normalization and statistical analysis were based on two dye swaps (i.e. four arrays, each containing 24,576 GSTs and 384 controls) as described by Gagnot et al. (2008). To determine differentially 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 familywise 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). We considered as being differentially expressed the genes with a Bonferroni P # 0.05, as described by Gagnot et al. (2008). Only genes that showed samedirection statistically significant change in both biological replicates of at least one sample type were considered as statistically significant.

Quantitative RT-PCR Analysis

RNA was extracted using the NucleoSpin RNA plant kit (Macherey-Nagel) and reverse transcribed with the SuperScript III First-Strand Synthesis System (Invitrogen). ACTIN2 transcripts were measured as a control. cDNAs were amplified using the conditions described above for analysis of genomic DNA except that the number of cycles was 50. Quantitative RT-PCR was performed according to Queval et al. (2007). Gene-specific primers are listed in Supplemental Table S1.

Enzyme Assays, Metabolite, and ROS Analysis

Extractable enzyme activities were measured as described previously [START_REF] Veljovic-Jovanovic | Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system[END_REF]. Oxidized and reduced forms of glutathione, ascorbate, and NADP were measured by plate-reader assay as described by Queval and Noctor (2007). SA was measured according to the protocol of Langlois-Meurinne et al. (2005). Thiobarbituric acid-reactive substances were assayed and calculated as described by Yang et al. (2009). In situ visualization of ROS was performed using dichlorodihydrofluorescein-diacetate (DCFH 2 -DA) by a protocol modified from [START_REF] Oracz | The mechanisms involved in seed dormancy alleviation by hydrogen cyanide unravel the role of reactive oxygen species as key factors of cellular signaling during germination[END_REF]. Leaves were vacuum infiltrated twice for 15 min at room temperature with DCFH 2 -DA, carefully rinsed, and kept in the dark. DCF fluorescence at 510 to 550 nm was visualized on a confocal microscope, simultaneously with red chlorophyll autofluorescence, using argon laser excitation at 488 nm.

Microarray data from this article will be deposited at the Gene Expression Omnibus and can be consulted at CATdb (http://urgv.evry.inra.fr/cgi-bin/ projects/CATdb/consult_expce.pl?experiment_id=256). [START_REF] Meister | Glutathione-ascorbic acid system in animals[END_REF]. In plant cells, where reductive H2O2 metabolism has been linked to ascorbate since the 1970s, the absolutely irreplaceable role of glutathione was less apparent. However, glutathione depletion in Arabidopsis knockouts lacking the first enzyme of the committed pathway of GSH synthesis causes embryo lethality (Cairns et al. 2006). Thus, both plant and mammalian cells rely on at least some of the multifunctional properties of glutathione for their vigour and survival. Glutathione is the principal low-molecular-weight thiol in most cells. However, there are some intriguing variations in organisms such as halobacteria, in which GSH can be replaced by other sulphur compounds like gglutamylcysteine (g-EC) and thiosulphate [START_REF] Newton | g-Glutamylcysteine and thiosulfate are the major low-molecular-weight thiols in halobacteria[END_REF]. Similarly, in some parasitic protozoa, trypanothione [N 1 ,N 8 -bis(glutathionyl)spermidine] can substitute for glutathione [START_REF] Fairlamb | Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids[END_REF]. Some plant taxa contain glutathione homologues, in which the C-terminal residue is an amino acid other than glycine [START_REF] Rennenberg | Glutathione metabolism and possible biological roles in higher plants[END_REF][START_REF] Klapheck | Homoglutathione: isolation, quantification and occurrence in legumes[END_REF][START_REF] Klapheck | g-Glutamylcysteinylserine -a new homologue of glutathione in plants of the family Poaceae[END_REF][START_REF] Meuwly | g-Glutamylcysteinylglutamic acid -a new homologue of glutathione in maize seedlings exposed to cadmium[END_REF]. These compounds include homoglutathione (g-Glu-Cys-b-Ala), which is found alongside GSH in many legumes [START_REF] Macnicol | Homoglutathione and glutathione synthetases of legume seedlings: partial purification and substrate specificity[END_REF][START_REF] Klapheck | Homoglutathione: isolation, quantification and occurrence in legumes[END_REF]. Interestingly, gene duplication during evolution has resulted in the coexistence of different synthetases that produce GSH or homoglutathione [START_REF] Frendo | A Medicago trunculata homoglutathione synthetase is derived from glutathione synthetase by gene duplication[END_REF]. Cereals produce another GSH variant (hydroxymethylGSH; g-Glu-Cys-Ser) through reactions that remain to be fully elucidated but which likely involve modification of GSH rather than alternative synthesis pathways [START_REF] Klapheck | g-Glutamylcysteinylserine -a new homologue of glutathione in plants of the family Poaceae[END_REF][START_REF] Okumura | Synthesis of hydroxymethylglutathione from glutathione and L-serine catalyzed by carboxypeptidase Y[END_REF]Skipsey, Davis & Edwards 2005a). Disulphide forms of these homologues are reducible by GR [START_REF] Klapheck | Homoglutathione: isolation, quantification and occurrence in legumes[END_REF][START_REF] Klapheck | g-Glutamylcysteinylserine -a new homologue of glutathione in plants of the family Poaceae[END_REF][START_REF] Oven | Homo-phytochelatins are synthesized in response to cadmium in azuki beans[END_REF]). Therefore, current information suggests that they do not require alternative reductive systems. Novel homologues may remain to be discovered (Skipsey et al. 2005a). For example, highperformance liquid chromatography (HPLC) analysis of thiols in poplar overexpressing a bacterial form of the second enzyme of glutathione synthesis revealed two novel Correspondence: G. Noctor. e-mail: graham.noctor@u-psud.fr GSH is used here to indicate the thiol (reduced) form of glutathione while GSSG denotes the disulphide form. The term 'glutathione' is used where no distinction is drawn or both forms may be concerned. Like other thiols, glutathione can undergo numerous redox reactions. As well as GSSG, oxidized forms notably include the formation of 'mixed disulphides' with proteins and other thiol molecules. Other oxidized forms include thiyl radicals, sulphenic acid (SOH) and, possibly, sulphinic (SO2H) or sulphonic (SO3H) acids on the cysteine moiety of glutathione, similar to their formation on protein cysteine residues. Moreover, a wide array of glutathione conjugates can be formed with endogenous and xenobiotic electrophilic species [START_REF] Ballatori | Endogenous glutathione conjugates: occurrence and biological functions[END_REF]Dixon & Edwards 2010) while interactions with the nitric oxide (NO) system via formation of S-nitrosoglutathione (GSNO) broaden the scope of glutathione as a reservoir of signalling potential (Lindermayr, Saalbach & Dürner 2005).

It has long been recognized that GSH is oxidized by reactive oxygen species (ROS) as part of the antioxidant barrier that prevents excessive oxidation of sensitive cellular components. Unlike the oxidized forms of many other primary and secondary metabolites that can also react with ROS, GSSG is rapidly recycled by the GRs in key organelles and the cytosol [START_REF] Halliwell | Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography[END_REF][START_REF] Smith | Properties and functions of glutathione reductase in plants[END_REF]Edwards, Rawsthorne & Mullineaux 1990;Jiménez et al. 1997;Chew, Whelan & Millar 2003;Kataya & Reumann 2010). A characteristic feature of glutathione is its high concentration in relation to other cellular thiols. In general, glutathione accumulates to millimolar concentrations, with tissue contents well in excess of free cysteine. A second key characteristic of the cellular glutathione pool is its high reduction state. In the absence of stress, tissues such as leaves typically maintain measurable GSH: GSSG ratios of at least 20:1 (e.g. Mhamdi et al. 2010a). It is important to note that this is an average value across tissues, and that ratios may be higher (e.g. cytosol) or lower (e.g. vacuole) in specific subcellular compartments (Meyer et al. 2007;Queval et al. 2011).

GLUTATHIONE ACCUMULATION, TURNOVER AND TRANSPORT

GSH biosynthetic pathway

As in animals, GSH is synthesized in plants from its constituent amino acids by two ATP-dependent steps [START_REF] Rennenberg | Glutathione metabolism and possible biological roles in higher plants[END_REF]Meister 1988;Noctor et al. 2002a;Mullineaux & Rausch 2005). Each of the synthetic enzymes is encoded by a single gene (May & Leaver 1994;[START_REF] Ullman | Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant[END_REF], and Arabidopsis knockout lines for either have lethal phenotypes. While knocking out expression of GSH1, encoding g-EC synthetase (g-ECS), causes lethality at the embryo stage (Cairns et al. 2006), knockouts for GSH2, encoding glutathione synthetase (GSH-S), show a seedling-lethal phenotype (Pasternak et al. 2008). Using forward genetics approaches, several mutants have been identified in which decreased GSH contents are caused by less severe mutations in the GSH1 gene. Of these, the rml1 (rootmeristem-less1) 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 decreased camalexin contents and enhanced sensitivity to pathogens (Howden et al. 1995;Cobbett et al. 1998;Ball et al. 2004;[START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]. In addition to genetic approaches, a pharmacological tool that has frequently been used to deplete glutathione is buthionine sulphoximine (BSO), a specific inhibitor of g-ECS (Griffith & Meister 1979).

The activity of g-ECS is strongly associated with chloroplasts in wheat (Noctor et al. 2002a). Localization studies in Arabidopsis have demonstrated that the enzyme is restricted to plastids in this species (Wachter et al. 2005). The Arabidopsis GSH-S is found in both chloroplasts and cytosol. Of the two transcripts encoded by the GSH2 gene, the most abundant one is the shorter form, which is translated to produce a cytosolic GSH-S (Wachter et al. 2005). Thus, the first step of glutathione synthesis is plastidic while the second step is probably predominantly located in the cytosol.

Regulation of biosynthesis

Many factors affect the synthesis of glutathione, but the most important are considered to be g-ECS activity and cysteine availability. Accordingly, constitutive increases in glutathione can be produced either by overexpression of the first enzyme of the committed glutathione synthesis pathway or of enzymes involved in cysteine synthesis [START_REF] Strohm | Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula x P.alba) overexpressing glutathione synthetase[END_REF]Noctor et al. 1996Noctor et al. , 1998;;Creissen et al. 1999;[START_REF] Harms | Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione[END_REF][START_REF] Noji | Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants[END_REF][START_REF] Wirtz | Dominant-negative modification reveals the regulatory function of the multimeric cysteine synthase protein complex in transgenic tobacco[END_REF]. Other factors that may affect GSH contents in certain conditions include glycine and ATP [START_REF] Buwalda | Cysteine, g-glutamylcysteine and glutathione contents of spinach leaves as affected by darkness and application of excess sulfur. II. Glutathione accumulation in detached leaves exposed to H2S in the absence of light is stimulated by the supply of glycine to the petiole[END_REF][START_REF] Noctor | The role of glycine in determining the rate of glutathione synthesis in poplars. Possible implications for glutathione production during stress[END_REF][START_REF] Ogawa | Level of glutathione is regulated by ATP-dependent ligation of glutamate and cysteine through photosynthesis in Arabidopsis thaliana: mechanism of strong interaction of light intensity with flowering[END_REF]). Increased g-ECS activity may result from transcriptional or posttranscriptional changes (May et al. 1998), but so far relatively few conditions have been shown to cause marked induction of GSH1 or GSH2 transcripts. Both genes are induced by jasmonic acid (JA) and heavy metals (Xiang & Oliver 1998;[START_REF] Sung | ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis[END_REF]) and also respond to light and some stress conditions such as drought and certain pathogens. However, neither externally applied H2O2 nor intracellularly generated H2O2 leads to increased abundance of GSH1 or GSH2 transcripts in Arabidopsis, despite the well-described increases in glutathione in these conditions (Smith et al. 1984;May & Leaver 1993;Willekens et al. 1997;[START_REF] Sánchez-Fernández | Cloning and expression analyses of AtMRP4, a novel MRP-like gene from Arabidopsis thaliana[END_REF]Xiang & Oliver 1998;Queval et al. 2009). While some evidence has been presented that production of the g-ECS protein is regulated at the level of translation [START_REF] Xiang | Glutathione synthesis in Arabidopsis: multilevel controls coordinate responses to stress[END_REF], more attention has focused on post-translational redox controls. A partially purified enzyme preparation from tobacco was shown to be sensitive to inhibition by dithiols [START_REF] Hell | g-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization[END_REF], and similar effects were subsequently reported in other species (Noctor et al. 2002a;[START_REF] Jez | Arabidopsis thaliana glutamate-cysteine ligase. Functional properties, kinetic mechanism, and regulation of activity[END_REF]). Recently, it has been shown that the plant g-ECS forms a homodimer linked by two disulphide bonds [START_REF] Hothorn | Structural basis for the redox control of plant glutamate cysteine ligase[END_REF], one of which is involved in redox regulation (Hicks et al. 2007;Gromes et al. 2008). This is likely an important factor in the well-known up-regulation of glutathione synthesis in response to oxidative stress. Another mode of regulation that is likely to be important in glutathione homeostasis is feedback inhibition of g-ECS by GSH. First reported for the animal enzyme [START_REF] Richman | Regulation of g-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione[END_REF], this regulatory mechanism also occurs in plants [START_REF] Hell | g-Glutamylcysteine synthetase in higher plants: catalytic properties and subcellular localization[END_REF]Noctor et al. 2002a). Alleviation of feedback inhibition is likely to be an important mechanism driving accelerated rates of synthesis under conditions in which glutathione is being consumed (e.g. in the synthesis of phytochelatins). The mechanistic links between feedback inhibition and thiol/disulphide redox regulation of g-ECS remain to be elucidated.

Integration of glutathione synthesis with sulphur assimilation

In accordance with the observation that enhanced cysteine supply favours glutathione accumulation, increases in GSH synthesis are associated with up-regulation of the cysteine synthesis pathway. For example, glutathione accumulation triggered by oxidative stress causes accumulation of transcripts encoding adenosine 5'-phosphosulphate reductase (APR) and serine acetyltransferase (SAT; Queval et al. 2009). While all three Arabidopsis APR genes encode plastidial enzymes, only one of the five SAT gene produces an isoform located in this compartment [START_REF] Kawashima | Characterization and expression analysis of a serine acetyltransferase gene family involved in a key step of the sulfur assimilation pathway in Arabidopsis[END_REF]. Although the mitochondrial SAT makes the major contribution to cysteine synthesis under standard conditions [START_REF] Haas | Mitochondrial serine acetyltransferase functions as pacemaker of cysteine synthesis in plant cells[END_REF][START_REF] Watanabe | Comparative genomics and reverse genetics analysis reveal indispensable functions of the serine acetyltransferase gene family in Arabidopsis[END_REF], the chloroplast SAT was the most strongly induced during H2O2-triggered accumulation of glutathione (Queval et al. 2009). As well as up-regulation at the transcript level, ozone exposure activates at least one APR at the post-translational level (Bick et al. 2001). As in the case of post-translational activation of g-ECS, the exact mechanisms regulating APR activation state remain unclear. One appealing possibility is that oxidation-triggered decreases in GSH:GSSG activate glutathione synthesis by increasing the chloroplast glutathione redox potential and allowing glutaredoxin (GRX)mediated activation of both enzymes. Consistent with this model, accumulation of glutathione in catalase-deficient barley and Arabidopsis is associated with markedly increased chloroplast GSSG content [START_REF] Smith | The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.)[END_REF]Queval et al. 2011). However, regulation could also be linked to thioredoxin (TRX) activity. Whatever the details of the underlying regulatory mechanisms, intracellular oxidative stress can drive glutathione accumulation to severalfold basal levels (Smith et al. 1984;Willekens et al. 1997). It has been estimated that a fivefold accumulation of glutathione in Arabidopsis cat2 mutants means that the amount of sulphur in the glutathione cysteine residue approaches that which is found in protein cysteine and methionine residues combined (Queval et al. 2009). As well as the changes in transcripts and extractable activities previously mentioned, H2O2-triggered glutathione accumulation in barley is accompanied by increased uptake of labelled sulphate [START_REF] Smith | The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.)[END_REF]. Accordingly, marked accumulation of glutathione achieved by transgenic enhancement using a bacterial enzyme with both g-ECS and GSH-S activities was dependent on sufficient sulphur supply (Liedschulte et al. 2010).

Overexpression of glutathione biosynthesis

Regardless of the controls over synthesis, and the signalling roles discussed later, tissue glutathione contents can be markedly enriched in plants. Whereas overexpression of Escherichia coli GSH-S in poplar produced little effect on glutathione contents in optimal conditions (Foyer et al. 1995;[START_REF] Strohm | Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula x P.alba) overexpressing glutathione synthetase[END_REF], introduction of the E. coli g-ECS caused a two-to fourfold increase in leaf glutathione, and this was observed whether the bacterial g-ECS was targeted to the cytosol or the chloroplast (Noctor et al. 1996(Noctor et al. , 1998;;Arisi et al. 1997). Expression of the same g-ECS in the tobacco chloroplast also produced substantial increases in leaf glutathione (Creissen et al. 1999) whereas homologous overexpression of g-ECS in Arabidopsis resulted in about twofold glutathione enrichment (Xiang et al. 2001). More recently, considerably greater increases in glutathione have been achieved by overexpression of a bifunctional g-ECS/ GSH-S from Streptococcus (Liedschulte et al. 2010).

There is still some debate about the impact of increasing glutathione contents in plants. Whereas increased glutathione triggered by chloroplastic overexpression of g-ECS in tobacco was accompanied by oxidation and lesion formation (Creissen et al. 1999), studies with the same E. coli construct introduced into other species did not report marked phenotypic effects (Noctor et al. 1998;Zhu et al. 1999a). More recently, it has been shown that one of the chloroplast lines with multiple insertions shows symptoms of early leaf senescence [START_REF] Herschbach | Over-expression of bacterial g-glutamylcysteine synthetase (GSH1) in plastids affects photosynthesis, growth and sulphur metabolism in poplar (Populus tremula ¥ Populus alba) dependent on the resulting g-glutamylcysteine and glutathione levels[END_REF]. Another study of a single cytosolic overexpressor grown for 3 years in the field reported several effects, including lower biomass and photosynthesis [START_REF] Ivanova | Overexpression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on contaminated soil[END_REF]. In terms of interpretation of phenotypes, a clear limitation of studies in poplar is the difficulty of genetic studies in this species. This necessitates analysis of several independent lines to be certain that the observed effects are linked to increases in glutathione. To date no marked deleterious effects have been reported in the tobacco overexpressors with very high glutathione (Liedschulte et al. 2010), though these lines are clearly interesting systems in which to analyse the impact of high glutathione concentrations on plant function. The reasons for the apparent discrepancy between the studies of Creissen et al. (1999) and Liedschulte et al. (2010) remain to be elucidated. An important factor could be differences in the introduced proteins. Unlike the E. coli g-ECS, the Streptococcus protein has both GSH-S and g-ECS activities.

Several studies have shown the benefits of elevating glutathione through overexpression of g-ECS. These include enhanced resistance to heavy metals and certain herbicides (Zhu et al. 1999a;[START_REF] Gullner | Enhanced tolerance of transgenic poplar plants overexpressing g-glutamylcysteine synthetase towards chloroacetanilide herbicides[END_REF][START_REF] Ivanova | Overexpression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on contaminated soil[END_REF]. Intriguingly, increased glutathione produced by overexpression of g-ECS in the chloroplast was associated with higher leaf contents of several free amino acids, including tyrosine, leucine, isoleucine and valine (Noctor et al. 1998). This could bear some relation to redox regulation of the enzymes of amino acid metabolism in the chloroplast, several of which are potential TRX targets [START_REF] Montrichard | Thioredoxin targets in plants: the first 30 years[END_REF].

Although overexpression of GSH-S alone has less marked effects on tissue glutathione contents than boosting g-ECS capacity (Foyer et al. 1995;[START_REF] Strohm | Regulation of glutathione synthesis in leaves of transgenic poplar (Populus tremula x P.alba) overexpressing glutathione synthetase[END_REF]Noctor et al. 1998), the effects could be condition dependent if GSH-S becomes limiting when g-EC supply is increased, for example, during exposure to cadmium (Zhu et al. 1999b). Similarly, expression of a soybean homoGSH-S in tobacco was successfully used to confer tolerance to the herbicide, fomesafen (Skipsey et al. 2005b). Interestingly, in this study, the homoGSH-S was expressed together with a homoGSHpreferring soybean GST (Skipsey et al. 2005b).

Turnover and degradation

Biochemical studies of glutathione degradation in tobacco conducted by the Rennenberg group [START_REF] Rennenberg | 5-Oxoprolinase in Nicotiana tabacum: catalytic properties and subcellular localization[END_REF][START_REF] Steinkamp | g-Glutamyltranspeptidase in tobacco suspension cultures: catalytic properties and subcellular localization[END_REF], 1985;[START_REF] Steinkamp | g-Glutamylcyclotransferase in tobacco suspension cultures: catalytic properties and subcellular localization[END_REF] have been significantly extended over the last decade, notably by genetically based studies in Arabidopsis. Four different types of enzymes have been described that could initiate glutathione breakdown (Fig. 2). 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 [START_REF] Steinkamp | Degradation of glutathione in plant cells: evidence against the participation of a g-glutamyltranspeptidase[END_REF], which has been detected in barley vacuoles [START_REF] Wolf | Degradation of glutathione S-conjugates by a carboxypeptidase in the plant vacuole[END_REF]. A second type of enzyme that has been implicated in GS-conjugate breakdown is the cytosolic enzyme phytochelatin synthase (PCS; Blum et al. 2007Blum et al. , 2010)). Because GS-conjugates are usually rapidly transported into the vacuole, the extent to which they accumulate in the cytosol remains unclear. However, some data obtained from work on Arabidopsis suggest that PCS may play some role in certain cell types or when the enzyme is activated by heavy metals [START_REF] Grzam | Vacuolar sequestration of glutathione S-conjugates outcompetes a possible degradation of the glutathione moiety by phytochelatin synthase[END_REF]Blum et al. 2007;[START_REF] Brazier-Hicks | Catabolism of glutathione-conjugates in Arabidopsis thaliana. Role in metabolic reactivation of the herbicide safener fenchlorim[END_REF].

The third type of enzyme, g-glutamyl transpeptidase (GGT), has been the focus of several research groups in recent years. These enzymes act in the mammalian g-glutamyl cycle (Meister 1988), and catalyse the hydrolysis or transpeptidation of GSH at the plasma membrane. The resulting g-glutamyl amino acid derivatives are further processed by g-glutamyl cyclotransferases (GGC) and 5-oxoprolinase (5-OPase) to produce free glutamate. In Arabidopsis, GGTs are encoded by at least three functional genes. Two of these (GGT1 and GGT2) encode apoplastic enzymes with activity against GSH, GSSG and GS-conjugates (Martin & Slovin 2000;[START_REF] Storozhenko | g-Glutamyl transpeptidase in transgenic tobacco plants. Cellular localization, processing, and biochemical properties[END_REF]. Extracellular GGT has also been described in barley and maize [START_REF] Masi | Localization of gamma-glutamyl transferase activity and protein in Zea mays organs and tissues[END_REF][START_REF] Ferretti | Gamma-glutamyl transferase in the cell wall participates in extracellular glutathione salvage from the root apoplast[END_REF]. Unlike their counterparts 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). Based on phenotypes of mutants and other studies, these enzymes may be important in countering oxidative stress or in salvaging excreted GSSG (Ohkama-Ohtsu et al. 2007a;[START_REF] Ferretti | Gamma-glutamyl transferase in the cell wall participates in extracellular glutathione salvage from the root apoplast[END_REF][START_REF] Destro | Compensatory expression and substrate inducibility of g-glutamyl transferase GGT2 isoform in Arabidopsis thaliana[END_REF]. Besides the extracellular GGTs, Arabidopsis has at least one vacuolar GGT, which is probably involved in the breakdown of GS-conjugates (Grzam et al. 2007;Martin et al. 2007;Ohkama-Ohtsu et al. 2007b). Along with PCS, GGTs may be important in metabolizing GS-conjugates that are formed during the synthesis of certain secondary metabolites (Ohkama-Ohtsu et al. 2011;[START_REF] Su | Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF].

In animals, g-glutamyl peptides produced by GGT are further metabolized by GGC.An Arabidopsis gene (OXP1) has been identified that likely encodes 5-OPase. This enzyme catalyses the hydrolysis of 5-oxoproline, which is the product of GGC activity (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 (Fig. 2), 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 & Meister 1973;[START_REF] Steinkamp | g-Glutamylcyclotransferase in tobacco suspension cultures: catalytic properties and subcellular localization[END_REF]. The first gene encoding GGC was identified in humans recently, but no obviously homologous sequences exist in plants [START_REF] Oakley | The identification and structural characterization of C7orf24 as g-glutamyl cyclotransferase. An essential enzyme in the g-glutamyl cycle[END_REF]). Yet other proteins may contribute to some extent to glutathione turnover, for example, GGP1, which has been implicated in the removal of the Glu residue from a GS-conjugate during glucosinolate synthesis (Geu-Flores et al. 2009). Because of this complexity, several questions remain on glutathione degradation in plants. These include cellular/tissue specificities, activities against the different forms of glutathione and, in some cases, the gene identities.

Despite the plethora of possible routes of glutathione catabolism, the extent of glutathione turnover and resynthesis remains unclear. 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). Extractable Arabidopsis leaf GSH-S activities can reach 10 nmol g -1 FW min -1 , while the extractable activity of the rate-limiting enzyme, g-ECS, is much lower, only about 0.5 nmol g -1 FW min -1 (Queval et al. 2009). This rate is therefore very close to the rates of turnover estimated by Ohkama-Ohtsu et al. (2008). However, this close correspondence could be coincidental. Extractable g-ECS activities should theoretically be higher than true in vivo fluxes, because substrates, notably cysteine, may be less limiting and feedback inhibition by glutathione negligible in assays of desalted extracts. It should be noted, however, that redox regulation and the absence of other factors may make it difficult to recover true in vivo capacities of g-ECS in in vitro assays. Indeed, assuming that accumulation of glutathione reflects neosynthesis and that there are no alternative routes awaiting description, our recent data strongly suggest that at least under some conditions, g-ECS can work considerably faster in vivo than the measured extractable value. Glutathione oxidation in cat2 gr1 double mutants triggers accumulation of total glutathione to more than 10-fold basal levels within 4 d (Mhamdi et al. 2010a). Within the first 4 h after the beginning of excess H2O2 production (a consequence of the cat2 background), about 800 nmol glutathione g -1 FW was newly accumulated in cat2 gr1, that is, a mean minimum synthesis rate of around 200 nmol g -1 FW h -1 .

While the interplay between degradation and neosynthesis remains to be elucidated, typical leaf glutathione contents are 300 nmol g -1 FW. Thus, degradation rates of 30 nmol g -1 FW h -1 would mean that some glutathione pools must turn over and be resynthesized within a few hours. Another question concerns the possible role of glutathione degradation during oxidative stress. In this regard, it is interesting that GGT1 was among glutathioneassociated genes that were significantly down-regulated in GSSG-accumulating cat2 gr1 mutants (Mhamdi et al. 2010a). By virtue of their activities in cleaving GSconjugates, some enzymes implicated in glutathione degradation may also be important in biosynthesis pathways (e.g. [START_REF] Su | Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF]; see below).

Compartmentation and transport

Glutathione is one of the major forms of organic sulphur translocated in the phloem [START_REF] Herschbach | Influence of glutathione (GSH) on net uptake of sulfate and sulfate transport in tobacco plants[END_REF], 1995;[START_REF] Bourgis | S-methylmethionine plays a major role in phloem sulfur transport and is synthesized by a novel type of methyltransferase[END_REF][START_REF] Mendoza-Cózatl | Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation[END_REF], and must therefore move between cells, either apoplastically, symplastically or both. Glutathione can be detected in apoplastic extracts but at much lower levels than in whole tissue extracts [START_REF] Vanacker | Pathogeninduced changes in the antioxidant status of the apoplast in barley leaves[END_REF]. This is in line with immunolocalization studies that have detected weak or no labelling in the cell wall and apoplast (Zechmann et al. 2008). The apoplastic pool is also likely more oxidized than many intracellular pools. Current concepts suggest that both the apoplast and vacuole have low glutathione concentrations and GSH:GSSG ratios, and that most of the glutathione is concentrated in other compartments. While immunolocalization studies point to particularly high concentrations in the mitochondria (Zechmann et al. 2008), because of their greater volume the cytosol and chloroplasts have been estimated to account for about 50 and 30%, respectively, of total glutathione in Arabidopsis leaf mesophyll cells (Queval et al. 2011).

Uptake of both GSH and GSSG has been documented in both cells and protoplasts [START_REF] Schneider | Reduced glutathione (GSH) transport in cultured tobacco cells[END_REF][START_REF] Jamaï | Characterization of glutathione uptake in broad bean leaf protoplasts[END_REF]. Transport across the plasmalemma could be accomplished by the oligopeptide transporter (OPT) family. Within this family, the Brassica uncea and rice genes, BjGT1 and OsGT1, are homologous to the yeast HGT1 transporter, which can transport GSH, GSSG and GS-conjugates, but also other small peptides [START_REF] Bourbouloux | Hgt1p, a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae[END_REF][START_REF] Bogs | Functional chacterization and expression analysis of a glutathione transporter, BjGT1, from Brassica juncea: evidence for regulation by heavy metal exposure[END_REF][START_REF] Zhang | A novel family of transporters mediating the transport of glutathione derivatives in plants[END_REF]. Of the nine annotated Arabidopsis OPT genes [START_REF] Koh | An oligopeptide transporter gene family in Arabidopsis[END_REF], OPT6 may play a role in long-distance transport (Cagnac et al. 2004). The OPT6 gene is highly expressed in the vasculature, where it may transport GS-conjugates and GS-cadmium complexes as well as GSH and GSSG (Cagnac et al. 2004). Another study reported that GSH, but not GSSG, was transported by OPT6 [START_REF] Pike | Arabidopsis OPT6 is an oligopeptide transporter with exceptionally broad substrate specificity[END_REF]. Knockout opt6 mutants are aphenotypic, suggesting gene redundancy, and the transporter may be important in translocating other peptides as well as glutathione [START_REF] Pike | Arabidopsis OPT6 is an oligopeptide transporter with exceptionally broad substrate specificity[END_REF].

Several different types of transporter may be important in translocation of glutathione between subcellular compartments. Inner chloroplast envelope transporters have recently been described that likely act to link plastidic g-ECS and cytosolic GSH-S via g-EC export across the chloroplast envelope (Maughan et al. 2010). These transporters are encoded by three genes in Arabidopsis, called CLT1, CLT2 and CLT3. Further, the wild-type phenotype of gsh2 knockout mutants can be restored by transformation with a construct driving GSH-S expression exclusively in the cytosol (Pasternak et al. 2008). This observation provides further evidence that GSH can be imported from the cytosol into the plastid, consistent with radiolabelling studies of isolated wheat chloroplasts (Noctor et al. 2002a). Thus, current concepts suggest that g-EC is synthesised exclusively in the chloroplast, then either converted to GSH in this compartment or transported to the cytosol, where part of any GSH formed can be transported into the chloroplast (Fig. 3).

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 (Martinoia et al. 1993;Rea 1999;Foyer, Theodoulou & Delrot 2001). Indeed, certain Arabidopsis MRPs are competent in GSSG as well as GS-conjugate transport (Lu et al. 1998). Barley vacuoles can take up GSSG much more rapidly than GSH, and the removal of cytosolic GSSG may play a role in maintaining glutathione redox status in this compartment [START_REF] Tommasini | Transport of oxidized glutathione into barley vacuoles: evidence for the involvement of the glutathione-S-conjugate ATPase[END_REF]. Although glutathione concentrations in the vacuoles of unstressed plants have long been considered to be low or negligible [START_REF] Rennenberg | Glutathione metabolism and possible biological roles in higher plants[END_REF]Zechmann et al. 2008), accumulation of GSSG in this compartment could be a physiologically important part of oxidative stress responses (Queval et al. 2011). Further, induction of specific MRPs occurs in response to oxidising agents [START_REF] Sánchez-Fernández | Cloning and expression analyses of AtMRP4, a novel MRP-like gene from Arabidopsis thaliana[END_REF]) and also accompanies GSSG accumulation in Arabidopsis (Mhamdi et al. 2010a).

Immunolocalization studies detect nuclear glutathione concentrations that are similar to those in the cytosol in cells in the undividing G0 state (Zechmann et al. 2008). Other recent studies suggest that nuclear/cytosol distribution of glutathione may be dynamic, with glutathione being recruited into the nucleus early in the cell cycle in both mammalian and plant cells [START_REF] Markovic | Glutathione is recruited into the nucleus in early phases of cell proliferation[END_REF]Diaz-Vivancos et al. 2010a). A nuclear redox cycle within the cell cycle has been proposed, according to which GSH moves into the nucleus during the G1 phase: this in turn promotes oxidation of the cytosol and, consequently, enhanced glutathione accumulation (Diaz-Vivancos et al. 2010b). Oxidation of the cytosol at G1 is accompanied by enhanced levels of ROS and lowering of the oxidative defense shield (Diaz-Vivancos et al. 2010a). The accumulated GSH is divided between the daughter cells, in which the process begins again. These observations suggest the presence of proteins in plants that are able to alter the permeability of nuclear pores, facilitating GSH sequestration in the nucleus. Such proteins remain to be identified in plants. However, the anti-apoptotic factor Bcl-2 is thought to be a crucial component regulating GSH transport into the nucleus in mammalian tissues, as it is in mitochondria (Voehringer et al. 1998). Despite the lack of evidence for GSH synthesis in the mitochondria, high glutathione concentrations have been detected in this organelle (Zechmann et al. 2008). It is possible that pore-regulating proteins are involved in regulating mitochondrial and nuclear GSH concentrations (Fig. 3).

Like mitochondria, peroxisomes contain glutathione and GR (Jiménez et al. 1997) while apparently lacking the enzymes of glutathione synthesis. Immunolocalization studies suggest that the peroxisomal glutathione concentration is similar to the cytosolic concentration (Zechmann et al. 2008), and it has been estimated in leaf mesophyll cells to be around 3-4 mm (Queval et al. 2011). Presumably, this pool results from import across the single peroxisomal membrane, but transporters responsible for this activity remain to be characterized.

METABOLIC FUNCTIONS OF GLUTATHIONE

Regulation of sulphur assimilation

As a significant non-protein sink for reduced sulphur, glutathione contents are influenced by sulphur supply. Indeed, glutathione may be one of the S-containing compounds that link changes in sulphur nutrition to resistance to some pathogens, a phenomenon termed sulphur-induced resistance (SIR; [START_REF] Gullner | Elevation of glutathione level of glutathione-related enzymes affect virus infection in tobacco[END_REF][START_REF] Bloem | Facts and fiction about sulfur metabolism in relation to plant-pathogen interactions[END_REF]Zechmann et al. 2007;Höller et al. 2010). Stresses that involve an oxidative component also cause up-regulation of the S assimilation pathway. For example, exposure to ozone increases cysteine and glutathione levels, effects linked to posttranslational activation of APR1 (Bick et al. 2001). In catalase mutants, increased intracellular H2O2 triggers accumulation of glutathione and precursors, and this is accompanied by accumulation of transcripts for all three APRs (Queval et al. 2009).

Glutathione is an important form of translocated organic S and may thus act as an internal 'barometer' of plant S status [START_REF] Kopriva | Control of sulphate assimilation and glutathione synthesis, interaction with N and C metabolism[END_REF]. Glutathione regulates several steps involved in S assimilation. These include GSH inhibition of sulphate uptake and assimilation through effects on specific transporters and enzymes such as ATP sulphurylase 1 (APS1) and APR [START_REF] Herschbach | Influence of glutathione (GSH) on net uptake of sulfate and sulfate transport in tobacco plants[END_REF][START_REF] Lappartient | Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem-translocated compound[END_REF][START_REF] Vauclare | Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5′-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols[END_REF][START_REF] Buchner | Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H2S and pedospheric sulfate nutrition[END_REF]). However, the role of glutathione in S assimilation is complex, because as well as these repressive effects, GSH is required by APR as reductant to produce sulphite [START_REF] Leustek | Sulfate metabolism[END_REF]. This occurs through a domain of the APR enzyme that allows it to act as a GRX, using GSH with an apparent KM value of about 1 mm [START_REF] Bick | Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5′-adenylylsulfate reductase[END_REF]. Further, thiol-disulphide status also acts to control certain APR isoforms post-translationally through activation in response to increased glutathione oxidation [START_REF] Leustek | Sulfate metabolism[END_REF].

The influence of sulphur assimilation activity on glutathione contents also underlines the close relationship between glutathione and S status (Nikiforova et al. 2003). Arabidopsis mutants defective in certain sulphate transporters show decreased leaf glutathione accumulation [START_REF] Maruyama-Nakashita | Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global effects of sulfur nutrition on multiple metabolic pathways[END_REF]. By contrast, expression of a bacterial APR in Arabidopsis enriches tissue cysteine and glutathione contents [START_REF] Tsakraklides | Sulfate reduction is increased in transgenic Arabidopsis thaliana expressing 5′-adenylylsulfate reductase from Pseudomonas aeruginosa[END_REF]. Qualitatively similar effects are produced by overexpression of enzymes of the cysteine synthesis pathway [START_REF] Harms | Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione[END_REF][START_REF] Noji | Molecular and biochemical analysis of serine acetyltransferase and cysteine synthase towards sulfur metabolic engineering in plants[END_REF][START_REF] Wirtz | Dominant-negative modification reveals the regulatory function of the multimeric cysteine synthase protein complex in transgenic tobacco[END_REF].

Glutathione S-transferases

The classical reaction catalysed by GSTs is the formation of a covalent bond between the sulphur atom of glutathione and an electrophilic compound (Fig. 4). In plants, the activity of most GSTs depends on an active site serine, which stabilizes the GS-thiolate anion (Dixon & Edwards 2010), though in some GSTs such as the dehydroascorbate reductases (DHARs) this residue is replaced by a cysteine. This change confers the capacity for reversible disulphide bond formation with glutathione that is part of the DHAR catalytic mechanism (Dixon, Davis & Edwards 2002). Long considered as cytosolic enzymes, several GSTs may also localize at least partly to other compartments, including the chloroplast, peroxisome and nucleus [START_REF] Thatcher | Differential gene expression and subcellular targeting of Arabidopsis glutathione-S-transferase F8 is achieved through alternative transcription start sites[END_REF]Dixon et al. 2009).

The GST family in plants is notable for its structural and functional diversity, and the biochemical and physiological functions of specific members remain to be elucidated. As well as or instead of catalysing conjugase reactions, some GSTs may have antioxidative functions. The DHAR type of GST is one example, while several subclasses of GST have peroxidase activity (Wagner et al. 2002;Dixon et al. 2009). GSTs are able to reduce organic peroxides though some specificity is observed between different peroxides (Wagner et al. 2002;Dixon et al. 2009;Dixon & Edwards 2010). Some GSTs are strongly inducible by H2O2 (Levine et al. 1994;Willekens et al. 1997;Wagner et al. 2002). For example, certain GST transcripts can be considered useful markers for increased intracellular availability of H2O2 [START_REF] Vanderauwera | Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis[END_REF]Queval et al. 2007Queval et al. , 2009;;Chaouch et al. 2010), though several are also inducible by salicylic acid (SA; [START_REF] Sappl | The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing of multiple genes results in altered metabolic sensitivity to oxidative stress[END_REF]). The same or other GSTs may have functions in the detoxification of electrophilic xenobiotics, but also be important in biosynthetic or catabolic pathways.

GS-X

GST

GSH

Recently described examples of biosynthetic pathways involving GSTs are the production of glucosinolates and camalexin, discussed further later. For a more detailed recent discussion of GSTs, we refer the reader to Dixon & Edwards (2010).

Glyoxalase and formaldehyde metabolism

Oxo-aldehydes such as glyoxal are reactive compounds that may interfere with sensitive cellular compounds.A common toxic oxo-aldehyde is methylglyoxal, which can be produced from triose phosphate as an intermediate in the triose-phosphate isomerase reaction [START_REF] Maiti | Molecular characterization of glyoxalase II from Arabidopsis thaliana[END_REF][START_REF] Marasinghe | Structural studies on a mitochondrial glyoxalase II[END_REF]). The glyoxalase system acts to convert these compounds to non-toxic hydroxyacids such as lactate, and consists of two enzymes acting in concert (Fig. 4). Glyoxalase I isomerizes the spontaneously generated GS-adduct while the hydrolytic reaction catalysed by glyoxalase II liberates the hydroxyacid and free GSH. In several plant species, overexpression of these enzymes increases tolerance to exogenous methylglyoxal and/or salt (Singla-Pareek, Reddy & Sopory 2003;Deb Roy et al. 2008;Singla-Pareek et al. 2008).

In addition to acting as a substrate for GSTs and glyoxalases, GSH may also act in detoxification reactions via formaldehyde dehydrogenase. Formaldehyde can enter plants through stomata or be produced by endogenous metabolism (Haslam et al. 2002). Two enzymes, formaldehyde dehydrogenase (FDH) and S-formylglutathione hydrolase (FGH), act to oxidize formaldehyde to formic acid, which may then be converted to CO2 or enter C1 metabolism (Fig. 4). Genes for both enzymes have been identified in Arabidopsis (Martínez et al. 1996;Haslam et al. 2002;Achkor et al. 2003). An important feature of the encoded FDH is that it is able to act as a GSNO reductase (GSNOR; Sakamoto, Ueda & Morikawa 2002;Díaz et al. 2003).

Phytochelatin synthesis

A conditionally important role of GSH is in the response to excessive levels of heavy metals (Fig. 4). Glutathione is the precursor of phytochelatins ([g-Glu-Cys]nGly), compounds that are synthesized in response to cadmium and other heavy metals. Phytochelatins sequester the metal to form a complex that is then transported into the vacuole (Grill, Winnacker & Zenk 1987;Grill et al. 1989;Cobbett & Goldsbrough 2002;Rea, Vatamaniuk & Rigden 2004). These compounds are produced from glutathione or homologues by PCS, a cytosolic enzyme. In Arabidopsis, there are two genes encoding PCS, one of which (PCS1) is the gene affected by the cad1 mutation that exacerbates cadmium sensitivity [START_REF] Howden | Cadmium-sensitive mutants of Arabidopsis thaliana[END_REF][START_REF] Ha | Phytochelatin synthase genes from Arabidopsis and Schizosaccharomyces pombe[END_REF][START_REF] Cazalé | Arabidopsis thaliana expresses a second functional phytochelatin synthase[END_REF]. As noted previously, it is possible that PCS has other biochemical roles, in addition to phytochelatin synthesis, for example, turnover of GS-conjugates (Rea et al. 2004;Blum et al. 2007Blum et al. , 2010;;[START_REF] Peršoh | Multi-tasking phytochelatin synthases[END_REF].

The importance of sufficient amounts of GSH to support phytochelatin synthesis is evidenced by the identification of the cad2 mutant as affected in g-ECS, causing a decrease in leaf GSH to about 20% wild-type levels (Howden et al. 1995;Cobbett et al. 1998). As well as a precursor role in phytochelatin synthesis, glutathione could be involved in heavy metal resistance via an antioxidant function: many heavy metals are considered to provoke perturbation of cellular redox homeostasis through several mechanisms, for example, displacement of redox-active metals from bound sites. Increased heavy metal tolerance has been observed in transgenic lines with enhanced glutathione synthesis (Zhu et al. 1999a,b;[START_REF] Lee | Higher levels of ectopic expression of Arabidopsis phytochelatin synthase do not lead to increased cadmium tolerance and accumulation[END_REF], contrasting with effects of overexpressing PCS itself, which has generally yielded less clear-cut results [START_REF] Peterson | Leaf-targeted phytochelatin synthase in Arabidopsis thaliana[END_REF][START_REF] Picault | Chloroplast targeting of phytochelatin synthase in Arabidopsis: effects on heavy metal tolerance and accumulation[END_REF]. While both shoots and roots may make a contribution to heavy metal detoxification, it is interesting that phytochelatin and GS-cadmium concentrations have been reported to be much higher in the phloem than in the xylem during exposure of B. napus to cadmium [START_REF] Mendoza-Cózatl | Identification of high levels of phytochelatins, glutathione and cadmium in the phloem sap of Brassica napus. A role for thiol-peptides in the long-distance transport of cadmium and the effect of cadmium on iron translocation[END_REF].

Glutathione metabolic reactions in defence against biotic stress

Glutathione has long been implicated in reactions linked to secondary metabolism and pathogen responses (Dron et al. 1988;Edwards, Blount & Dixon 1991). While at least some of the effects of glutathione during interactions with pathogens probably involve a signalling role, it now appears that others are linked to hormone and secondary metabolite synthesis. Crucial information had been generated by the analysis of glutathione-deficient mutants. The first identified glutathione-deficient Arabidopsis mutant, cad2, was reported to show unchanged resistance to virulent and avirulent strains of the oomycete Peronospora parasitica as well as to the bacterium Pseudomonas syringae (May et al. 1996b). However, a later study of cad2 and rax1-1 reported increased susceptibility to avirulent P. syringae (Ball et al. 2004). Mutants defective in the CLT glutathione chloroplast envelope transporters showed decreased expression of PR1 and also lower resistance to the oomycete Phytophthora brassicae (Maughan et al. 2010). Decreased PR1 expression linked to lower SA accumulation was also observed in gr1 mutants lacking cytosolic/peroxisomal GR (Mhamdi et al. 2010a).

The Arabidopsis pad2 mutant was first identified as deficient in camalexin, an indole phytoalexin that contains one S atom per molecule and whose thiazole ring is derived partly from cysteine (Glazebrook & Ausubel 1994). This line shows enhanced susceptibility to various bacterial, fungal and oomycete pathogens (Ferrari et al. 2003;[START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]. The affected gene in pad2 is GSH1 and the mutation results in GSH contents that are slightly lower than those in cad2 [START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]. Of cad2, rax1 and pad2 mutants in GSH1, rax1 has the highest leaf glutathione contents, pad2 the lowest while cad2 is intermediate. The glutathione contents of these lines correlate inversely with their resistance to P. brassicae, though only pad2 was reported to be markedly affected in camalexin contents and its response to P. syringae [START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]. Thus, there appears to be a certain level of glutathione required for the synthesis of pathogen defense-related molecules and disease resistance. This level could vary according to conditions and pathogen specificity. In the case of camalexin synthesis, recent data show that GSH is required as a precursor of the thiazole ring (Fig. 5a; [START_REF] Böttcher | The multifunctional enzyme CYP71B15 (PHYTOALEXIN DEFICIENT3) converts cysteine-indole-3acetonitrile to camalexin in the indole-3-acetonitrile metabolic network of Arabidopsis thaliana[END_REF][START_REF] Su | Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF].

As well as effects on camalexin and resistance to microorganisms, pad2 shows decreased resistance to feeding of insect larvae, an effect that is linked to decreased accumulation of glucosinolates (Schlaeppi et al. 2008). This effect can be rescued by supplementation with GSH but not the general disulphide reductant, dithiothreitol (Schlaeppi et al. 2008). Using a dedicated microarray of more than 200 genes whose expression is associated with insect feeding, it was shown that unlike the JA signalling mutant, coi1, the pad2 mutant did not show significant differences in the expression of these genes, including glucosinolate synthesis genes, in response to feeding (Schlaeppi et al. 2008). This observation is consistent with a requirement for a certain level of GSH to support glucosinolate synthesis as a sulphur source, rather than as a signalling or regulatory molecule. Indeed, it has recently been reported that formation of the glucosinolate thioglucose moiety involves a GS-conjugate intermediate and that this compound is metabolized by a g-glutamyl peptidase, GGP (Geu-Flores et al. 2009). Formation and metabolism of such GS-conjugates parallels stages in the camalexin synthesis pathway (Fig. 5), and the lack of GSH for the respective GST activities may explain both constitutive deficiency of camalexin and lower induction of glucosinolates in pad2 [START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]Schlaeppi et al. 2008). Both pathways shown in Fig. 5 involve formation of a GS-conjugate followed by further metabolism by GS-conjugate degrading enzymes. Studies of mutants implicated GGT1, GGT2 and, to a lesser extent, PCS1, in the control of Botrytis-induced camalexin accumulation [START_REF] Su | Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF], whereas ggt4 mutants were found to accumulate a GS-conjugate of the JA synthesis precursor, 12-oxophytodienoic acid (OPDA), during incompatible interactions with P. syringae (Ohkama-Ohtsu et al. 2011).

Metabolism of ROS and ascorbate

Glutathione can react chemically with ROS and also with dehydroascorbate (DHA), the relatively stable oxidised form of ascorbate generated by dismutation of monodehydroascorbate (MDHA). In particular, there is a close relationship between increased availability of H2O2 and glutathione status. This is most evident from studies in which H2O2-metabolizing enzymes have been genetically or pharmacologically inhibited (Smith et al. 1984;May & Leaver 1993;Willekens et al. 1997;Noctor et al. 2002b;Rizhsky et al. 2002;Queval et al. 2007Queval et al. , 2009;;Chaouch et al. 2010). Timecourse analyses in conditional catalase mutants have shown 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 production, this response involves a decrease in the wholeleaf GSH : GSSG ratio from above 20 to close to one (Mhamdi et al. 2010a). Such effects make glutathione status a useful marker for oxidative stress triggered by increased intracellular H2O2 production. Several pathways may be involved in GSH-dependent H2O2 metabolism, while H2O2 may be metabolized by GSHindependent pathways (Fig. 6). The chemical reaction of GSH with H2O2 is slow, but three distinct types of peroxidases appear as the principal candidates to link peroxide reduction to GSH oxidation (Table 1). These are ascorbate peroxidase (APX), certain types of peroxiredoxin (PRX) and GSTs. Haem-based peroxidases are divided into two super-families, one of which (non-animal peroxidases) contains plant enzymes and is divided into three classes [START_REF] Welinder | Superfamily of plant, fungal and bacterial peroxidases[END_REF]. While class II haem peroxidases are found in fungi and notably include secreted enzymes involved in lignin degradation, haem peroxidases in plants are found in classes I or III [START_REF] Zámocky | Evolution of structure and function of Class I peroxidases[END_REF]. Class III enzymes are found only in plants. Also known as 'guaiacol-type' peroxidases, they are encoded by numerous genes and are located in the apoplast or vacuole. Their functions are not clearly established, but some are known to be involved in biosynthetic processes and/or in ROS production (Bindschedler et al. 2006;[START_REF] Cosio | Specific functions of individual class III peroxidase genes[END_REF]. In contrast, class I haem peroxidases include the intracellular antioxidative enzyme, APX. Like catalase, APX is relatively specific to H2O2 and does not metabolize other peroxides at high rates. It can participate in the 'ascorbate-glutathione' pathway in which H2O2 reduction is ultimately linked to NAD(P)H oxidation via ascorbate and glutathione pools. Alternatively APX activity could be coupled to NAD(P)H oxidation independently of glutathione via MDHAR activity (Fig. 6). While GSH can chemically reduce DHA at significant rates (though slower at pH 7 than at pH 8), an enzymatic link beween ascorbate and glutathione pools is provided by DHAR. Overexpression of this enzyme underlines the importance of GSH-dependent ascorbate pools in physiological processes like the regulation of stomatal opening [START_REF] Chen | Increasing vitamin C content of plants through enhanced ascorbate recycling[END_REF], but the role of DHAR in the in vivo metabolism of peroxides still remains assumed rather than demonstrated.

In contrast to haem-based enzymes, thiol peroxidases such as PRX are less specific to H2O2, and can also reduce other organic peroxides, though some may have preference for H2O2 or relatively small peroxides (Dietz 2003;[START_REF] Tripathi | Peroxiredoxins: a less studied component of hydrogen peroxide detoxification in photosynthetic organisms[END_REF].There are four types of PRX described in plants, and three of these use either TRX or similar components such as NADPH-thioredoxin reductase (NTR) C (Dietz 2003[START_REF] Pulido | Functional analysis of the pathways for 2-Cys peroxiredoxin reduction in Arabidopsis thaliana chloroplasts[END_REF]. One type, PRX II, can use GSH as a reductant via GRX action (Rouhier, Gelhaye & Jacquot 2002). Peroxiredoxins also include GPXs. Originally identified on the basis of their homology to animal GPX (Eshdat et al. 1997), and subsequently implicated in plant stress responses and signalling (Rodriguez Milla et al. 2003;[START_REF] Miao | An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought responses[END_REF][START_REF] Chang | Arabidopsis chloroplastic glutathione peroxidases play a role in cross talk between photooxidative stress and immune responses[END_REF]), these enzymes are now considered to act as TRX-dependent peroxiredoxins (Herbette et al. 2002;Iqbal et al. 2006;[START_REF] Navrot | Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stress[END_REF] and are, therefore, misleadingly named. Unlike the animal enzyme, where the reduction of peroxides involves formation of a mixed disulphide between a first GSH and the selenocysteine SeOH group, the GPXs found in plants generate a disulphide bridge from the initial sulphenic acid, with the disulphide intermediate being reduced back to the dithiol form by TRX. They are therefore unlikely to be involved in peroxide-mediated glutathione oxidation (Fig. 6). By contrast, peroxidation of GSH could be catalysed by GSTs (Wagner et al. 2002;Dixon et al. 2009). Other enzymes such as methionine sulphoxide reductase (MSR) may also contribute to ROS-triggered glutathione oxidation (Tarrago et al. 2009). Finally, although these studies, relying predominantly on in vitro analyses, have established the relative specificities in reducing substrates, it should be noted that the glutathione and TRX systems may be linked to some extent during in vivo ROS metabolism (Michelet et al. 2005;Reichheld et al. 2007;Marty et al. 2009).

In conclusion, GSH could be linked to H2O2 and/or peroxide reduction by at least two ascorbate-independent routes as well as the ascorbate-glutathione pathway (Fig. 6). Among these, only GSTs appear to act as direct GPXs (Table 1), all other enzymes requiring at least one additional protein to link peroxide reduction to GSH oxidation. The evidence from gene expression makes it clear that certain APX, GPX and GST genes are induced in response to oxidative stress (Willekens et al. 1997;Wagner et al. 2002;Levine et al. 1994;[START_REF] Sappl | The Arabidopsis glutathione transferase gene family displays complex stress regulation and co-silencing of multiple genes results in altered metabolic sensitivity to oxidative stress[END_REF]. Data from transcriptomics and enzyme and metabolite assays of plants deficient in catalase and/or GR suggest that the ascorbateglutathione pathway and enzymes such as GSTs may act in concert to remove excess H2O2 and/or other peroxides (Mhamdi et al. 2010a).

Glutathione reductase

The GSSG produced by GSH oxidation is reduced by GR. While NTR can also reduce GSSG in a TRX-dependent manner (Marty et al. 2009), this enzyme is less efficient than GR, which is encoded by two genes in plants studied so far.

Chloroplast and mitochondrial GR is encoded by GR2, while GR1 encodes a protein that is found in the cytosol and peroxisome (Creissen et al. 1995;Chew et al. 2003;Kataya & Reumann 2010). The dual targeting of these two genes is therefore sufficient to explain biochemical data on GR localization in all of these compartments (Edwards et al. 1990;[START_REF] Rasmusson | NADP-utilizing enzymes in the matrix of plant mitochondria[END_REF]Jiménez et al. 1997;Stevens, Creissen & Mullineaux 2000;[START_REF] Romero-Puertas | Glutathione reductase from pea leaves: response to abiotic stress and characterization of the peroxisomal isozyme[END_REF]. Despite the long-standing association of GR and glutathione with resistance to various stresses [START_REF] Esterbauer | Seasonal variation of glutathione and glutathione reductase in needles of Picea abies[END_REF][START_REF] Tausz | The glutathione system as a stress marker in plant ecophysiology: is a stress-response concept valid[END_REF]), overexpression of GR in several plant species has not in itself been reported to lead to marked increases in stress resistance [START_REF] Foyer | Effects of elevated cytosolic glutathione reductase activity on the cellular glutathione pool and photosynthesis in leaves under normal and stress conditions[END_REF](Foyer et al. , 1995;;Aono et al. 1993;Broadbent et al. 1995;[START_REF] Kornyeyev | Field-grown cotton plants with elevated activity of chloroplastic glutathione reductase exhibit no significant alteration of diurnal and seasonal patterns of excitation energy partitioning and CO2 fixation[END_REF]Ding et al. 2009). However, increases in the reduction state of the ascorbate pool in plants overexpressing GR are consistent with efficient coupling of the reactions of the ascorbate-glutathione pathway (Foyer et al. 1995).

While Arabidopsis T-DNA mutants for the chloroplast/ mitochondrial GR2 are embryo-lethal (Tzafrir et al. 2004), 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 (Marty et al. 2009), though this is not sufficient to prevent significant accumulation of GSSG in the mutant. Accumulation of GSSG is massively increased compared with the parent lines in cat2 gr1 double mutants deficient in both the major leaf catalase and GR1 (Mhamdi et al. 2010a).This effect is associated with a much exacerbated phenotype compared with cat2, showing that the NTR-TRX system cannot replace GR1 under conditions where H2O2 production is increased. Further, the gr1 mutation alters Arabidopsis responses to pathogens and expression of genes involved in defence hormone signalling, notably JA-associated genes (Mhamdi et al. 2010a).

Although transcriptomic patterns documented in this study point to at most limited overlap between glutathione and TRX systems in oxidative stress functions, the observation of Marty et al. (2009) shows that interplay is possible at the biochemical level. For example, changes in glutathione and TRX redox states could act mutualistically to reinforce each other during plant interactions with pathogens. This issue, which is important to defining the frequently used but vague term 'cellular redox state' more clearly, remains an outstanding issue in understanding redox signalling in plants.

GLUTATHIONE IN PLANT DEVELOPMENT, GROWTH AND ENVIRONMENTAL RESPONSES

It has long been known that certain cell types, for example, the root quiescent centre and cells in organs such as seeds, maintain a highly oxidized intracellular state [START_REF] Kranner | Significance of thiol-disulfide exchange in resting stages of plant development[END_REF]Kranner et al. 2002Kranner et al. , 2006)).Auxin accumulation in the root stem cell niche is dependent on the oxidized status of the cells (Jiang & Feldman 2010). Treatment of Arabidopsis root tips with BSO led to disappearance of the auxin maximum in the root tips and altered expression of quiescent centre markers [START_REF] Koprivova | Arabidopsis root growth dependence on glutathione is linked to auxin transport[END_REF]).

The glutathione redox potential of such cell types is relatively high (i.e. positive or oxidizing). Even in cells where the global glutathione pool is highly reduced, compartments that lack GR or that are deficient in NADPH may contain low GSH:GSSG ratios (e.g. the vacuole or endoplasmic reticulum; [START_REF] Hwang | Oxidized redox state of glutathione in the endoplasmic reticulum[END_REF][START_REF] Enyedi | Redox state of the endoplasmic reticulum is controlled by Ero1L-alpha and intraluminal calcium[END_REF]. While an increase in the redox potential above the threshold-reducing value of more redox-sensitive compartments has been linked to growth arrest and/or death (Kranner et al. 2006), cell identity has a profound influence on the processes that govern cell fate (Jiang et al. 2006a,b) and associated responses to abiotic stress [START_REF] Dinneny | Cell identity mediates the response of Arabidopsis roots to abiotic stress[END_REF].

Plant growth and auxin

The analysis of the phenotypes of glutathione-deficient Arabidopsis mutants has demonstrated that GSH is required for plant development fulfilling 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). The rml1 mutant, which has less than 5% of the glutathione present in the wild type, has a strong developmental phenotype that is characterized by a non-functional root meristem while the shoot meristem is largely unaffected (Vernoux et al. 2000). Crossing rml1 with ntra, ntrb double mutants produced an additive shoot meristemless phenotype (Reichheld et al. 2007). However, when the ntra ntrb double mutants were crossed with cad2 (which has about 30% of the glutathione present in the wild type), the resultant triple mutants developed normally at the rosette stage and underwent the floral transition but they produced almost naked flowering stems (Bashandy et al. 2010). The perturbation of the floral meristem in the ntra ntrb, cad2 triple mutants was linked to altered levels and transport of auxin, which plays an important role in the integration of meristem development (Bashandy et al. 2010). Glutathione synthesis is also required for pollen germination and pollen tube growth (Zechmann, Koffler & Russell 2011). In this study, glutathione depletion was shown to result from disturbances in auxin metabolism and transport (Zechmann et al. 2011). The auxin-resistant axr1 and axr3 mutants were found to be less sensitive to BSO than the wild-type Arabidopsis plants and treatment of the tips of primary roots with BSO altered auxin transport [START_REF] Koprivova | Arabidopsis root growth dependence on glutathione is linked to auxin transport[END_REF]. However, in these experiments, the effects of GSH on root growth could be partially reversed by dithiothreitol, suggesting that an as yet unidentified posttranscriptional redox mechanism is involved in the regulation of the expression of PIN proteins and hence auxin transport in roots [START_REF] Koprivova | Arabidopsis root growth dependence on glutathione is linked to auxin transport[END_REF]. The stunted phenotype of mutants such as cat2 and derived lines, which accumulate GSSG when grown from seed in air, may be related to such thiol regulation of plant growth, as could the effects of GSH on the regeneration efficiency of somatic embryos [START_REF] Belmonte | Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca)[END_REF].

Cell cycle regulation

Concepts of the regulation of mitosis in animals incorporate an intrinsic redox cycle in which transient oxidations serve to regulate progression through the cell cycle [START_REF] Menon | A redox cycle within the cell cycle: ring in the old with the new[END_REF][START_REF] Burhans | The cell cycle is a redox cycle: linking phase-specific targets to cell fate[END_REF]. While very few studies have been conducted to establish whether similar processes operate in the control of the plant cell cycle, 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). As the movement of GSH into the nucleus can be visualized in the dividing cells of the developing lateral root meristem (Diaz-Vivancos et al. 2010a), it will be intriguing to see if this process underlies the post-transcriptional redox regulation of the PIN proteins and auxin transport in roots [START_REF] Koprivova | Arabidopsis root growth dependence on glutathione is linked to auxin transport[END_REF].

Biotic interactions, cell death and defence phytohormones

In addition to serving as a source of reduced S during synthesis of secondary metabolites, glutathione is involved in signalling processes. Exogenous GSH can mimic fungal elicitors in activating the expression of defence-related genes (Dron et al. 1988;Wingate, Lawton & Lamb 1988) including PATHOGENESIS-RELATED1 (PR1; Senda & Ogawa 2004;Gomez et al. 2004a). Moreover, accumulation of glutathione is triggered by pathogen infection (Edwards et al. 1991;May, Hammond-Kosack & Jones 1996a), and this can involve characteristic transient changes in glutathione redox state [START_REF] Vanacker | Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hypersensitive response in the barley-powdery mildew interaction[END_REF][START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]. Similar changes have also been reported following exogenous application of the defence-related hormone salicylic acid (SA), or biologically active SA analogs (Mou, Fan & Dong 2003;[START_REF] Mateo | Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis[END_REF]Koornneef et al. 2008). Glutathione perturbation in catalase-deficient cat2 is linked to hypersensitive response (HR)-like lesions as part of a wide spectrum of defence responses that are conditionally induced in this line (Chaouch et al. 2010). Glutathione is also required for the development of symbiotic N2-fixing nodules between legumes and rhizobia. Both GSH and the legume homologue, homoGSH, are found at high concentrations in N2-fixing nodules formed during symbiotic interactions with rhizobia: deficiency in these compounds inhibits nodule formation (Frendo et al. 2005;[START_REF] Pauly | Reactive oxygen and nitrogen species and glutathione: Key players in the legumerhizobium symbiosis[END_REF].

Thiol-disulphide status is clearly involved in the regulation of the SA-dependent NONEXPRESSOROFPAT HOGENESISRELATEDGENES 1 (NPR1) pathway (Després et al. 2003;Mou et al. 2003;Rochon et al. 2006;Tada et al. 2008). Induction of PR gene expression by this pathway involves monomerization of the oligomeric cytosolic protein NPR1, which unmasks a nuclear localization signal motif that allows the protein to relocalize to the nucleus where it interacts with TGA transcription factors, themselves redox-sensitive (Després et al. 2003;Mou et al. 2003). The notion that this might be linked to glutathione redox state via GRX activity (Mou et al. 2003) has been superseded by a model based on activation by TRXh (Tada et al. 2008), some of which are known to be inducible by oxidative stress and pathogens (Laloi et al. 2004). If this model is correct, the well-documented ability of genetically or chemically increased GSH to induce PR gene expression could possibly be explained by redox interactions between glutathione and TRX.

Although activation of NPR1 involves a reductive change, the initial trigger for the events leading to PR gene expression involves oxidation. Excessive oxidation can also trigger HR, which is the most studied instance of environmentally induced cell death in plants. While redox changes involved in HR have been most closely associated with events triggered by apoplastic ROS production, oxidative stress of intracellular origin can also trigger such effects. Several observations are suggestive of a role for glutathione redox potential (or GSSG accumulation) in the regulation of genetically programmed cell suicide pathways. For example, increases in the total glutathione contents of leaves produced by ectopic g-ECS overexpression in the tobacco chloroplast caused accumulation of GSSG and this was associated with lesion formation and enhanced expression of pathogenesis-related (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). Alterations in pathogen responses have been described in mutants that are partly deficient in glutathione (Ball et al. 2004;[START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]. However, some of our own recent work in catalase-deficient cat2 and derived Arabidopsis lines suggest that there is unlikely to be a simple relationship between glutathione redox potential and engagement of cell death. Firstly, HR-like lesions in cat2 are under daylength control and this control does not correlate with the degree of glutathione oxidation (Queval et al. 2007). Secondly, daylength control is linked to SA synthesis: HR-like lesions can be prevented by blocking SA synthesis even though this effect is associated with a more oxidized glutathione status (Chaouch & Noctor 2010;Chaouch et al. 2010). Thirdly, in cat2 gr1 mutants, GSSG accumulates to a greater extent than in cat2 single mutants but this does not cause HR-like lesions (Mhamdi et al. 2010a). Despite these observations, however, we cannot exclude the possibility that values for whole-leaf glutathione status may not precisely reflect events in specific intracellular compartments (Queval et al. 2011). Even if this is the case, tissue glutathione status appears to be a poor marker for the engagement of cell suicide pathways. Nevertheless, analysis of H2O2 signalling in glutathione-deficient mutants demonstrates that some factor related to glutathione status is an important modulator of cell death triggered by oxidative stress (authors' unpublished results). Further, analyses of double cat2 atrboh Arabidopsis mutants, which lack both the major catalase and NADPH oxidase activities, point to a crucial role for AtrbohF in permitting accumulation of oxidized glutathione in response to intracellular H2O2. In double cat2 atrbohF (but not cat2 atrbohD) mutants, glutathione is much less perturbed than in the cat2 single mutant and this is associated with much lower accumulation of SA and related defence molecules (Chaouch, Queval & Noctor, unpublished results).

As well as possible roles in SA signalling, glutathione may modulate signalling through the JA pathway, which is involved in the regulation of development and in responses to necrotrophic pathogens and herbivores. JA induces GSH1,GSH2 and GR (Xiang & Oliver 1998),as well as other antioxidative genes (Sasaki-Sekimoto et al. 2005). In gr1 mutants lacking the cytosolic/peroxisomal GR, a suite of JA genes are repressed while introduction of the gr1 mutation into the cat2 background modulates H2O2-triggered expression of these and other JA-associated genes (Mhamdi et al. 2010a). Consistent with these interactions between JA and glutathione, prior wounding was shown to induce resistance to Botrytis cinerea, an effect that was partly or wholly suppressed in glutathione-deficient mutants [START_REF] Chassot | Wounding of Arabidiopsis leaves causes a powerful but transient protection against Botrytis infection[END_REF]. This effect was associated, at least in part, with impaired induction of camalexin [START_REF] Chassot | Wounding of Arabidiopsis leaves causes a powerful but transient protection against Botrytis infection[END_REF]). Among the components that could link JA signalling and glutathione are GRXs and GSTs. The SA-inducible protein, GRX480, represses up-regulation of certain JA-induced genes (Ndamukong et al. 2007). As well as acting in the activation of the SA pathway, NPR1 represses JA pathway [START_REF] Spoel | NPR1 modulates cross-talk between salicylate-and jasmonatedependent defense pathways through a novel function in the cytosol[END_REF], and BSO treatment was shown to anatagonize this repressive effect (Koornneef et al. 2008). Together, these observations suggest that glutathione may act to repress JA signalling through SA-dependent induction of NPR1 and GRX480, both of which interact with TGA transcription factors. However, gene expression profiles and SA contents suggest that the more oxidized glutathione status in gr1 is associated with repression of both SA and JA pathways, and therefore that the roles of glutathione may be complex and not limited to regulating antagonism between the two hormones (Mhamdi et al. 2010a). Other possible effects of glutathione could occur through the regulation of JA synthesis or excess accumulation of intermediates like OPDA. Several GSTs are among early JA-induced genes or may catalyse formation of GS-oxylipin conjugates (Davoine et al. 2006;Yan et al. 2007;Mueller et al. 2008). Indeed, enhanced accumulation of a GS-OPDA conjugate in ggt4 mutants, which are expected to be unable to degrade vacuolar GS-conjugates, was recently reported. However, loss of GGT4 function did not affect contents of JA or derivatives such as the hormonally active JA-isoleucine conjugate compared with wild-type plants (Ohkama-Ohtsu et al. 2011). In addition to these interactions with glutathione, JA and wounding were also shown to decrease GSNOR transcripts (Díaz et al. 2003).

Light signalling

The abundance of glutathione or its homologues in leaves does not vary greatly over the day/night cycle. Similarly, in soybean, all leaves except those approaching senescence contain similar total amounts of glutathione/ homoglutathione, as illustrated in Fig. 7. However, young poplar trees show more variation, 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 (Fig. 7). Shade conditions that involve a decrease in the red/far red ratio of the light environment favour a much lower leaf glutathione pool relative to conditions where the red/far red ratios are similar (Bartoli et al. 2009). While leaf glutathione contents are lower in plants grown under shade conditions, leaf GSH:GSSG ratios are relatively unaffected by light quality (Bartoli et al. 2009). Moreover, the adjustments in leaf glutathione pool in response to different red/ far red ratios were very slow in comparison with the leaf ascorbate pool (Bartoli et al. 2009). Regardless of the relatively slow responses of the leaf glutathione pool to changes in light intensity and light quality, some potential links between daylength, light signalling and glutathione status have been reported (Becker et al. 2006;Queval et al. 2007). Relationships between glutathione and photoreceptor signalling have been suggested in studies on the arsenictolerant mutants, ars4 and ars5 [START_REF] Sung | Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants[END_REF]). The ars4 mutation was identified as an allele of phytochrome A (phyA) and caused increased BSO resistance [START_REF] Sung | Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants[END_REF]). 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 [START_REF] Sung | ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis[END_REF] The redox states of the plastoquinone and TRX pools are considered to be important components of the redox regulation model for the control of gene expression that adjusts energetic and metabolic demands to light-induced changes of the photosynthetic apparatus structure [START_REF] Bräutigam | Hypothesis: a binary redox control mode as universal regulator of photosynthetic light acclimation[END_REF]. Glutathione has also been implicated in the signalling 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 [START_REF] Mateo | Controlled levels of salicylic acid are required for optimal photosynthesis and redox homeostasis[END_REF]Muhlenbock et al. 2008).A light shift that favoured excitation of photosystem II (PSII) relative to photosystem I (PSI) slightly increased the amounts of glutathione in leaves, whereas glutathione levels were slightly decreased following a light shift that favoured excitation of PSI relative to PSII [START_REF] Bräutigam | Dynamic plastid redox signals integrate gene expression and metabolism to induce distinct metabolic states in photosynthetic acclimation in Arabidopsis[END_REF][START_REF] Bräutigam | Hypothesis: a binary redox control mode as universal regulator of photosynthetic light acclimation[END_REF].According to the model of [START_REF] Bräutigam | Hypothesis: a binary redox control mode as universal regulator of photosynthetic light acclimation[END_REF], higher g-ECS activities would be favoured by exposure to light wavelengths that predominantly drive PSII. However, this model remains to be substantiated.

GLUTATHIONE-LINKED SIGNALLING MECHANISMS

Redox regulation is inherent to all energy exchange processes. It is required to balance supply and demand between energy-producing and energy-utilizing processes, as these are driven by redox changes. Of the mechanistic controls that achieve this homeostasis, thiol-disulphide reactions are the best characterized and probably among the most important (Buchanan & Balmer 2005). Key thiol components in enzyme regulation or ROS metabolism include TRX, GRX, glutathione, GPX and PRX (Dietz 2003;Lemaire 2004;Meyer et al. 2008;Rouhier 2010). The roles of TRX in redox signalling are well established. By comparison, the study of glutathione-dependent signalling is still in its infancy, although it is interesting to note that interest in the roles of glutathione in cellular regulation dates back many years [START_REF] Wolosiuk | Thioredoxin and glutathione regulate photosynthesis in chloroplasts[END_REF]. The following discussion first outlines factors that could link glutathione to modification of target protein activity and then analyses some of the issues surrounding the regulation and impact of altered glutathione status in cellular signalling.

Glutaredoxins

Glutaredoxins are also known as thiol transferases. The classical GRX reaction encompasses the reduction of a protein disulphide bond to two thiols with conversion of 2 GSH to GSSG. Some of these enzymes can also catalyse protein S-glutathionylation or de-glutathionylation. Land plants contain a large GRX family subdivided into three classes that can be distinguished on the basis of amino acid motifs in the active site (Lemaire 2004;Rouhier 2010). Most class I GRXs have a TRX-like active site consisting of two cysteines separated by two intervening amino acids. They can catalyse thiol-disulphide exchange but also other reactions such as regeneration of PRX and methionine sulphoxide reductase (MSR; Rouhier et al. 2002;Rouhier, Couturier & Jacquot 2006;Zaffagnini et al. 2008;Tarrago et al. 2009;Gao et al. 2010). Class II GRXs have only a single cysteine at the active site and are therefore sometimes called 'monothiol' GRX, though this term is potentially misleading as in at least some cases this cysteine may form a disulphide bridge with another cysteine located distally in the same protein or on another GRX of the same type (Gao et al. 2009a(Gao et al. , 2010)). Both class I and class II GRX play roles in assembly of iron-sulphur clusters [START_REF] Rouhier | Functional, structural, and spectroscopic characterization of a glutathioneligated [2Fe-2S] cluster in poplar glutaredoxin C1[END_REF][START_REF] Bandyopadhay | Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe-S] clusters[END_REF]Rouhier 2010). They can also catalyse protein de-glutathionylation, though some class II GRX do this in a dithiol-rather than GSH-dependent manner, and in vivo may depend on the TRX system for their regeneration (Zaffagnini et al. 2008;Gao et al. 2009aGao et al. , 2010)). While some of these GRX have been characterized biochemically, their in vivo roles remain unclear. Arabidopsis class II GRXs can interact with ion channels and have been implicated in responses to oxidative stress (Cheng & Hirschi 2003;Cheng et al. 2006;Guo et al. 2010;[START_REF] Sundaram | Transgenic expression of fern Pteris vittata glutaredoxin PvGrx5 in Arabidopsis thaliana increases plant tolerance to high temperature stress and reduces oxidative damage to proteins[END_REF].

GRXs in the third class contain two adjacent cysteines at their active site and form the largest class in land plants. Reverse genetics approaches in Arabidopsis have revealed that three GRX forms are involved in plant development and phytohormone responses through interaction with TGA transcription factors. These are GRX480, implicated in SA-JA interactions, and ROXY1 and ROXY2, which function in petal and flower development (Ndamukong et al. 2007;Li et al. 2009). Overexpression and complementation studies point to biochemical redundancy between these proteins (Li et al. 2009;Wang et al. 2009). Based on these observations, the specificity of class III GRX function may be determined by regulation of their expression patterns (Ziemann, Bhave & Zachgo 2009). This notion receives some support from microarray analysis of GRX gene expression in mutants with perturbations in leaf glutathione redox state, in which specific members of class III were the only GRX that were found to respond (Mhamdi et al. 2010a). Among these four genes was GRX480, which showed expression patterns similar to many other JA-associated genes. In contrast, transcripts for ROXY1 or ROXY2 remained at wild-type abundance levels.

Protein S-glutathionylation

Protein S-glutathionylation involves the formation of a stable mixed disulphide bond between glutathione and a protein cysteine residue. This reaction could modify the conformation, stability or activity of the target protein.

The existence of S-glutathionylated plant proteins has been known for some time [START_REF] Butt | Acyl carrier protein is conjugated to glutathione in spinach seed[END_REF]. However, the nature and role of this phenomenon has only been subject to thorough investigation relatively recently. Reversible S-glutathionylation is part of the catalytic cycle of glutathione-dependent enzymes such as GR, DHAR, MSRB1, as well as some GRXs and PRXs [START_REF] Arscott | Mixed disulfide with glutathione as an intermediate in the reaction catalyzed by glutathione reductase from yeast and as a major form of the enzyme in the cell[END_REF]Dixon et al. 2002;Tarrago et al. 2009;Rouhier 2010). Several techniques have been developed to identify other target proteins, allowing an inventory of potential target proteins to be established [START_REF] Ito | The sugar-metabolic enzymes aldolase and triose-phosphate isomerase are targets of glutathionylation in Arabidopsis thaliana: detection using biotinylated glutathione[END_REF]Dixon et al. 2005;Michelet et al. 2005[START_REF] Michelet | In vivo targets of S-thiolation in Chlamydomonas reinhardtii[END_REF]Zaffagnini et al. 2007;Holtgrefe et al. 2008;Gao et al. 2009a). Despite these advances, major gaps in our current knowledge remain, for example concerning quantification (i.e. the fraction of a given protein that is S-glutathionylated at a given time) and the in vivo significance of the modification. An additional uncertainty is the specificity of the modification, that is, whether the modified cysteine residue is glutathionylated in vivo, rather than nitrosylated or a target of TRX (or some combination of these modifications).

Most targets of glutathionylation identified to date are relatively abundant proteins. These notably 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). Glutathionylation of a TRXf cysteine found outside the active site lowers the protein's efficacy in activating chloroplast NADP-GAPDH, while another TRX-independent isoform of GAPDH is inhibited by glutathionylation (Zaffagnini et al. 2007). In the mitochondria, GDC has been shown to be inhibited by oxidative stress (Taylor, Day & Millar 2002), and is subject to thiol modifications, including S-glutathionylation (Palmieri et al. 2010). This enzyme is a major producer of NADH during conditions of active photorespiration. Mitochondrial redox homeostasis in these conditions may depend on appropriate activation of the alternative oxidase (Igamberdiev, Bykova & Gardeström 1997), which has been shown to be redox regulated through the TRX system [START_REF] Vanlerberghe | Alternative oxidase activity in tobacco leaf mitochondria. Dependence on tricarboxylic acid cycle-mediated redox regulation and pyruvate activation[END_REF][START_REF] Gelhaye | A specific form of thioredoxin h occurs in plant mitochondria and regulates the alternative oxidase[END_REF]. The glycolytic NADdependent GAPDH, a cytosolic enzyme found not only in the cytosol but also in the mitochondrion and nucleus [START_REF] Giegé | Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells[END_REF][START_REF] Anderson | Cytosolic glyceraldehyde-3-P dehydrogenase and the B subunit of the chloroplast enzyme are present in the pea leaf nucleus[END_REF], is sensitive to oxidation and has been identified as a glutathionylated protein (Dixon et al. 2005;[START_REF] Hancock | Doing the unexpected: proteins involved in hydrogen peroxide perception[END_REF]Holtgrefe et al. 2008).

Taken together, the current evidence suggests that glutathione and TRX systems work closely together to finetune photosynthetic and respiratory metabolism through appropriate modification of sensitive protein cysteine residues. 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. 2009b;Palmieri et al. 2010). The reverse reaction, deglutathionylation, could be mediated by certain class I and class II GRX. Current evidence suggests that some class II GRX catalyse this reaction in a TRX reductase-dependent manner (Zaffagnini et al. 2008;Gao et al. 2009aGao et al. , 2010)). Despite the potential physiological importance of S-glutathionylation, and the proposed involvement of class I and class II GRX in either glutathionylation or de-glutathionylation, no GRX of either type responded at the transcript level in Arabidopsis mutants with increased H2O2 and GSSG (Mhamdi et al. 2010a).

Protein S-nitrosylation and GSNO reductase

GSNO can cause S-nitrosylation and S-glutathionylation of protein cysteine residues (Lindermayr et al. 2005(Lindermayr et al. , 2010;;Romero-Puertas et al. 2008;Lindermayr & Durner 2009). Enzymes undergoing both modifications include GAPDH and GDC, with both effects leading to decreased activity [START_REF] Lindermayr | Differential inhibition of Arabidopsis methionine adenosyltransferases by protein S-nitrosylation[END_REF]Holtgrefe et al. 2008;Palmieri et al. 2010). Oxidative inhibition of GAPDH may act to regulate the oxidative pentose pathway relative to glycolysis in stress conditions (Holtgrefe et al. 2008). Inhibition of GDC by modifications of thiols may also have physiological significance, as photorespiratory metabolism involves high-flux pathways capable of modifying intracellular redox states (Foyer et al. 2009). S-nitrosylation of type II PRX has been proposed to play a role in ROS signalling (Romero-Puertas et al. 2007).

It was reported that S-nitrosylation acts in opposition to disulphide reduction by TRX in the regulation of NPR1. While monomerization of the NPR1 protein to the active form was TRX dependent, GSNO-dependent nitrosylation of NPR1 monomers was required for reoligomerization, possibly to inhibit depletion of the protein (Tada et al. 2008). 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. Production of the plant hormone ethylene, which regulates developmental responses like senescence as well as responses to certain pathogens, is known to be inhibited by NO. This effect may be mediated by inhibition of several enzymes involved in ethylene synthesis, including GSNO-triggered S-nitrosylation of a specific methionine adenosyltransferase [START_REF] Lindermayr | Differential inhibition of Arabidopsis methionine adenosyltransferases by protein S-nitrosylation[END_REF].

Inducible NO-producing enzymes in plants remain to be clearly described, and most attention has focused on the importance of a secondary reaction of nitrate reductase. However, studies in animals and plants have revealed that GSNOR is a key player in regulating S-nitrosylation. These enzymes catalyse 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). Originally identified as a formaldehyde dehydrogenase, the Arabidopsis GSNOR appears to play a key role in biotic stress responses, and Atgsnor1 mutants show decreased resistance to virulent and avirulent pathogens (Feechan et al. 2005). This enzyme has also been implicated in the regulation of cell death and other functions (Lee et al. 2008;Chen et al. 2009). It is also interesting to note that carbon monoxide (CO) is now considered to be a gaseous signalling molecule in animals and plants. It is possible that glutathione is involved in CO signalling in Medicago sativa [START_REF] Han | Carbon monoxide alleviates cadmium-induced oxidative damage by modulating glutathione metabolism in the roots of Medicago sativa[END_REF].

Factors affecting the glutathione redox potential

Despite the advances described previously, it remains unclear to what extent glutathione-mediated changes in protein thiol-disulphide status are important in signalling.

Regulation of thiol-disuphide status has traditionally been associated with TRX (Buchanan & Balmer 2005). However, 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;Bashandy et al. 2010). A key factor governing interactions between glutathione and protein targets is redox potential, which depends on the relative rates of glutathione oxidation and its NADPH-dependent reduction. 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 & 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, that is, the potential will become more positive and thus less reducing. This is in accordance with measurements of cytosolic GRXdependent thiol/disulphide redox potential as detected by redox-sensitive roGFP in the glutathione-deficient cad2 mutant compared with the wild type (Meyer et al. 2007). Increases in the cytosolic but not plastidial redox potential in clt1 clt2 clt3 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).

Linking glutathione redox potential to target proteins

No components have yet been identified that directly link glutathione redox potential to modifications in target proteins. However, based on information from the much better studied TRX-dependent changes in protein thiol-disulphide status, we can infer that a change in glutathione redox potential of approximately 50 mV is likely to be biologically significant. Such changes are sufficient to alter the balance between oxidized and reduced forms of TRX-regulated proteins [START_REF] Setterdahl | Effect of pH on the oxidation-reduction properties of thioredoxins[END_REF]. For example, an increase in TRX redox potential from -350 to -300 mV converts chloroplast glucose-6-phosphate dehydrogenase from almost completely inactive to active [START_REF] Née | Redox regulation of chloroplastic glucose-6-phosphate dehydrogenase: a new role for f-type thioredoxin[END_REF]. Indeed, in vitro redox titration of the thiol/disulphide-dependent roGFP in the presence of GRX at different glutathione concentrations revealed that interconversion of the oxidized and reduced forms of the sensor were associated with a calculated redox potential span of about 50 mV (Meyer et al. 2007). Despite this, calibrated quantification of in vivo changes in cytosolic redox potential using these probes has thus far revealed shifts of only about 20 mV in glutathionedeficient mutants compared with wild-type or in stress versus optimal conditions (Meyer et al. 2007;[START_REF] Jubany-Mari | Use of a redox-sensing GFP (c-roGFP1) for real-time monitoring of cytosol redox status in Arabidopsis thaliana water-stressed plants[END_REF]. Nevertheless, the physiological importance of such a shift cannot be discounted because systems that are able to sense glutathione redox potential with high sensitivity may await discovery. Furthermore, based on recent reports of interactions between cytysolic glutathione and TRX systems (Marty et al. 2009), 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.

Physiological and environmental factors that might cause shifts in glutathione redox potential in vivo

As previously discussed, the whole leaf and chloroplast GSH:GSSG ratios are not markedly influenced by variations in light intensity or light-dark transitions. However, oxidant production and oxidative stress can exert a strong influence over glutathione status. Oxidant production is governed by the rates of photosynthesis, photorespiration and respiration, as well as NADPH oxidases and other ROS-producing systems (Foyer & Noctor 2003). Presumably by changing the rate of ROS production through these processes or by affecting the rate of ROS removal, various stresses such as cold, drought, pollution and pathogens can modify whole leaf glutathione redox state (Sen [START_REF] Gupta | Response of photosynthesis and cellular antioxidants to ozone in Populus leaves[END_REF][START_REF] Vanacker | Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hypersensitive response in the barley-powdery mildew interaction[END_REF]Bick et al. 2001;Gomez et al. 2004b). Indeed, the cytosolic glutathione redox potential has been shown to increase in response to wounding (Meyer et al. 2007). Numerous studies on plants deficient in H2O2-metabolizing enzymes have documented marked changes in GSH:GSSG ratios (Smith et al. 1984;Willekens et al. 1997;Rizhsky et al. 2002;Queval et al. 2007Queval et al. , 2009)). These are most evident for catalase-deficient plants exposed to light under photorespiratory conditions, and are accompanied by several-fold changes in the total glutathione pool. It remains to be established to what extent these changes in whole tissue GSH:GSSG ratios involve changes in glutathione redox potential in specific compartments.

The importance of changes in absolute glutathione concentration compared with changes in GSH:GSSG

Accumulation of GSSG is often followed by an increase in the total glutathione pool size (Fig. 8). This response has been documented in plants exposed to ozone, is particularly evident in response to increased intracellular H2O2 and also occurs during pathogen responses. In such circumstances, changes in the total glutathione pool size occur predominantly through oxidant-induced changes in the expression and post-translational activities of enzymes involved in both cysteine and glutathione synthesis. This presumably acts as a homeostatic mechanism to offset what would otherwise be more severe increases in glutathione redox potential. If so, this response in itself suggests that the glutathione redox potential is important for at least some cellular functions. Further circumstantial evidence in favour of this notion comes from the observation that accumulation of GSSG is compartment specific, and relative increases are most marked in the vacuole (Queval et al. 2011). This is in line with the documented capacity of vacuoles to important GGSG more efficiently than GSH [START_REF] Tommasini | Transport of oxidized glutathione into barley vacuoles: evidence for the involvement of the glutathione-S-conjugate ATPase[END_REF]. Thus, mechanisms appear to have evolved to stabilize cytosolic glutathione redox potential during increased ROS production, and these include increases in total glutathione and accumulation of GSSG in compartments that may be less sensitive to redox perturbation (Fig. 8).

Although studies of mutants have shown that relatively small changes in glutathione concentration can modulate gene expression as well as environmental and developmental processes in plants (Ball et al. 2004;[START_REF] Parisy | Identification of PAD2 as a g-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF]Bashandy et al. 2010), it is not yet established that such effects are caused by altered glutathione redox potential. A decrease in total glutathione from 2.5 mm to 50 mm would cause the redox potential to become 50 mV more positive (Meyer et al. 2007). This depletion is rather extreme, and corresponds to the situation in root tips in seedlings of the rml1 mutant, which contain less than 5% wild-type glutathione (Cairns et al. 2006;Meyer et al. 2007). Thus, while an increased glutathione redox potential could explain the observations reported in rml1 and ntra ntrb rml1 mutants (Vernoux et al. 2000;Reichheld et al. 2007), it is less clear whether it can account for effects observed in less severely affected backgrounds. If changes in glutathione redox potential are indeed an important part of glutathionedependent oxidative signalling in wild-type plants, it seems that GSSG accumulation is likely to be a critical factor. Accumulation of GSSG could be relatively small (in absolute terms) if, as discussed further in the next section, the 'resting state' concentration in unstressed conditions is as low as some recent measurements imply.

As increased GSSG generally drives glutathione accumulation as part of what is assumed to be a homeostatic mechanism, decreased glutathione concentrations might be expected to cause a compensatory increase in GSH:GSSG. This should happen if the NADP(H) redox potential does not change and the two couples are in equilibrium. However, if GSSG concentrations are indeed in the nanomolar range, it is possible that kinetic limitations over GR (KMGSSG 10-50 mm;[START_REF] Smith | Properties and functions of glutathione reductase in plants[END_REF]Edwards et al. 1990) become increasingly severe as glutathione decreases, thus explaining the failure to maintain the glutathione redox potential in equilibrium with NADP(H), even when the decrease in concentration is relatively modest.

Relationships between the glutathione and NADP(H) redox couples in signalling

The midpoint redox potential of NADP(H) at pH 7 is approximately -320 mV while that of glutathione is -230 to -240 mV. But what is the actual redox potential of glutathione in vivo? Many researchers assume that it is quite close to the midpoint potential and that differences between this value and the TRX redox potential, which for most TRX is quite similar to NADP(H), is one factor that potentially explains in vivo preference of protein targets for glutathione or TRX. However, as discussed previously, some roGFP studies suggest that the glutathione redox potential in wild-type Arabidopsis in the absence of stress is lower than -300 mV in the cytosol and in other compartments, that is, it approaches values similar to the TRX redox potential (Meyer et al. 2007;[START_REF] Schwarzländer | Confocal imaging of glutathione redox potential in living plant cells[END_REF][START_REF] Jubany-Mari | Use of a redox-sensing GFP (c-roGFP1) for real-time monitoring of cytosol redox status in Arabidopsis thaliana water-stressed plants[END_REF]. Given that the cytosolic NADPH:NADP + ratio is not far removed from one [START_REF] Igamberdiev | Regulation of NADand NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves[END_REF], these redox potential values suggest that glutathione and NADP(H) are close to redox equilibrium in this compartment. This is a key unresolved issue in assessing the potential significance of redox signalling through glutathione-mediated disulphide bond formation. If sensitive thiol proteins are present, maintenance of a very low glutathione redox potential under optimal conditions could allow oxidative signalling to be initiated by protein disulphide bond triggered by relatively minor accumulation of GSSG. Glutathione redox potential values below -300 mV would require the GSH:GSSG ratio to be in the range of 10 5 to 10 6 , whereas global ratios are much lower in plant tissues, even in the absence of stress, where they are typically about 20-30 (Queval & Noctor 2007;Mhamdi et al. 2010a). It is likely that, while they provide a useful indicator of redox status, particularly the presence of oxidative stress, global cellular or tissue glutathione data give only a relative measure of GSH:GSSG ratios in intracellular compartments (Queval et al. 2011).

There is little evidence as yet that ROS-triggered changes in glutathione are accompanied by marked changes in global tissue NADP(H) status (Mhamdi et al. 2010a,b). The extent to which cytosolic NADP pools turn over in optimal and stress conditions remains uncertain. Further, it is not clear how important the demand of GR is on the cytosolic NADPH pool, and whether there is any functional association between different NADPH-requiring and -generating enzymes. GR has a KM for NADPH below 10 mm [START_REF] Smith | Properties and functions of glutathione reductase in plants[END_REF]Edwards et al. 1990), while total cytosolic NADPH concentrations are above 100 mm [START_REF] Igamberdiev | Regulation of NADand NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves[END_REF]. Although a large proportion of pyridine nucleotide pools is bound to proteins at any one time, and thus available 'free' concentrations are lower than the total, it is generally considered that changes in NADPH are not likely to impact the glutathione redox potential through kinetic effects on GR. However, shifts in NADPH:NADP + could perhaps impact the glutathione redox potential thermodynamically, as the reaction catalysed by GR is reversible. As previously discussed, kinetic limitations may occur through GSSG, whose resting concentration could be substantially below the GR KM value. Finally, it is interesting that the measurable capacity of GR in many plant systems is often somewhat lower than other enzymes of the ascorbate-glutathione pathway. No doubt this could reflect the presence of other pathways that can regenerate glutathione independent of ascorbate (Fig. 6). However, as we have emphasized in this review, glutathione may also be linked to ROS metabolism independently of ascorbate.

Comparatively low GR activity may be a feature of plant redox systems that allows appropriate changes in GSSG to mediate signalling functions.

CONCLUSION AND PERSPECTIVES

A large body of literature is now available that illustrates the complex and integrated regulation of glutathione status by nutritional and environmental factors. Glutathione may be seen as a central or 'hub' molecule in cellular metabolism and redox signalling (Fig. 9). Even though many glutathione-dependent reactions are involved in oxidative stress-mediated cellular processes, the simple vision of glutathione as a ROS-scavenging antioxidant is inaccurate and misleading. For example, at least three types of mechanism can be defined that link glutathione to the regulation of biotic stress reactions: (1) redox signalling, mediated by components such as GRX or GSNO; (2) the synthesis of S-containing or secondary metabolites; and (3) GSconjugate formation and metabolism to regulate the biological activity of metabolic intermediates or active products.

Key questions related to the antioxidant roles of glutathione concern the importance of different glutathionedependent enzymes and the influence of changes in glutathione status within cell signalling pathways. The degree of interplay between TRX and glutathione systems in physiological conditions remains to be fully described. The emerging picture is that changes in the 'general redox state' (i.e. redox potential) of these two components work together with altered activity or abundance of specific redox signalling components (e.g. GSNOR, GRX). Through their coordinated operation, such effects can reinforce signalling through a given pathway. Alternatively, antagonistic changes could act to restrain activation of signalling pathways, a control that is important within a cellular network receiving multiple evironmental inputs. This concept receives support from the operation of multiple interdependent redox modifications reported for the bacterial H2O2 sensor, OxyR [START_REF] Kim | OxyR: a molecular code for redox-related signaling[END_REF]. A similar picture is emerging in plants, where the notion of relatively simple thioldisuphide regulation of NPR1 (Mou et al. 2003) has given way to recognition that control is more complex (Tada et al. 2008;Lindermayr et al. 2010). By operating in conjunction with other regulatory mechanisms, such nuanced, flexible control integrates multiple inputs into a variable and appropriate response output. This is likely to be particularly important in plants, which must constantly deal with environmental changes of varying intensity and predictability. Thus, the challenge for the future is not only to characterize glutathione-linked redox modifications and to assess their interactions with other types of redox regulation, but also to evaluate the significance of these changes within the wider horizon of the cellular network.

Heavy metal resistance

Herbicide resistance

Figure 1 . 2 .

 12 Figure 1.2. Two of the major pathways for H 2 O 2 metabolism in plants (scheme taken from Foyer and Noctor, 2009).

Figure 1 . 3 .

 13 Figure 1.3. Part of the complexity of plant H 2 O 2 -metabolizing systems (scheme taken from Mhamdi et al., 2010b).

Figure 1 . 4 .

 14 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

Figure 1 . 5 .

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

Figure 1 . 6 .

 16 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.

Figure 1 . 7 .

 17 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.

Figure 1 . 8 .

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

Figure

  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.

Figure 1 . 10 .

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

Figure 2 . 1 .

 21 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.

  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 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.

Figure 2 . 3 .

 23 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).

  Figure 2.4. Confirmation of effects of genetic deficiency in glutathione on H 2 O 2 -triggered lesion formation using allelic mutants.

  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.

Figure 2 . 5 .

 25 Figure 2.5. The cat2 cad2 sid2 triple mutant shows that residual H 2 O 2 -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.

  Figure 2.6. Phenotypes and glutathione contents in Col-0, sid2, gr1 and cad2 mutants following transfer to air after three weeks growth from seed at high CO 2 . 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 plants, sampled 4d after transfer to air.

Figure 2 . 8 .

 28 Figure 2.8. Effects of cat2 and secondary cad2 and gr1 mutations on leaf myo-inositol and galactinol contents.

  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.

Figure 2 . 10 .

 210 Figure 2.10. GC-TOF-MS analysis of the impact of cad2 and npr1 mutations on H 2 O 2 -dependent metabolite profiles. (A) Heatmap showing hierarchical clustering of all detected metabolites. Values were centered-reduced prior to clustering analysis.

  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.

Figure 2 . 11 .

 211 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.

  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 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).

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

Figure 2 .

 2 Figure 2.13. Comparison of PR1 inducibility by salicylic acid (SA) in npr1, cad2, cat2, and double and triple mutants.

Figure 2

 2 Figure 2.14. Glutathione status is a key player linking intracellular H 2 O 2 to activation of the salicylic acid pathway.

  Figure 3.1. Possible hypothesis for the interaction among glutathione, SA and JA in the H 2 O 2 signaling background.

Figure 3 . 2 .

 32 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.

Figure 3

 3 Figure 3.3. Time-dependent induction of jasmonic acid-associated gene expression in the cat2 mutant. White bars, 4d after transfer from high CO 2 to air. Black bars, 8d after transfer. *Significant cat2/Col-0 difference at P<0.05.

Figure 3 . 4 .

 34 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.

Figure 3 . 5 .

 35 Figure 3.5. Differential regulation of cat2-triggered induction of jasmonic acid-associated gene expression by cad2 and npr1 mutations.

Figure 3 . 7 .

 37 Figure 3.6. Effects of externally supplied cysteine or glutathione on rosette phenotype and jasmonic acid-related gene expression in Col-0 and cat2. Plants were grown at high CO 2 for three 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 cysteine, or 1 mM GSH. Photographs show plants eight days after transfer to air. Bars indicate 1 cm. Transcripts were measured at the same point after spraying leaves with water, cysteine, or glutathione. Different letters indicate significant difference at P < 0.05.

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

  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 CO 2 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 CO 2 . Dans leur ensemble, ces résultats indiqueraient qu'un stress oxydant déclenché par un excès de H 2 O 2 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.

Figure 4 . 1 .

 41 Figure 4.1. Summary of the conditional nature of the cat2 mutant, showing its dependence on CO 2 level and on growth daylength.

Figure

  Figure 4.2. Genotyping and expression analysis of atg mutants. (A) PCR analysis of homozygous single atg2, atg5, and atg18a T-DNA mutants and after crossing into cat2 to 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 transcript in atg18a.

Figure 4 . 3 .

 43 Figure 4.3. Phenotypes of atg2, atg5 and atg18a grown in high CO 2 and air for five weeks in short day conditions. (A) Development of senescent yellowing in mutants grown in high CO 2 (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).

  Figure 4.4. Metabolite profiling of Col-0 and atg mutants grown at high CO 2 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 CO 2 after t tests against Col-0 in the same condition (P<0.05). (C) Graph to illustrate typical high CO 2 -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.

Figure

  Figure 4.5. Interactions between peroxisome-generated oxidative stress and atg mutations during growth in short days in high CO 2 and air. (A) Growth in high CO 2 for five weeks (no oxidative stress in cat2). (B) Growth in air for six weeks (oxidative stress in cat2) (C) Growth in high CO 2 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).

(Figure 4 . 7 .

 47 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 CO 2 in short days, then eight days in air in long days to trigger pathogenesis responses in cat2

  Figure4.3) and leaf glutathione content(Figure 4.8). To test the possible influence of glutathione, the cad2 mutation was crossed into atg2 (Figure4.9). As in the cat2 background (Chapters 2 and 3), the cad2 mutation effectively decreased glutathione in cat2 atg2 (Figure4.9C) but did not greatly affect rosette mass (Figure4.9B). The cad2 mutation also seemed to have little effect on the early senescence phenotype observed in atg2 (Figure4.9A). 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 CO 2 but not in air (Figure4.9A).

Figure 4 . 8 .

 48 Figure 4.8. Ascorbate and glutathione in atg mutants grown in high CO 2 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).

Figure 4 . 9 .

 49 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 CO 2 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.

  Figure 6.1. Genotyping of double and triple mutants.

Figure 1 .

 1 Figure 1. Comparison of H 2 O 2 -and glutathioneregulated 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 CO 2 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 oxidation in cat2 after transfer from high CO 2 to air. [See online article for color version of this figure.]

Figure 2 .

 2 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.

Figure 3 .

 3 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. Asterisks 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.]

Figure 4 .

 4 Figure 4. Rescue of a wild-type phenotype in cat2 gr1 at high CO 2 , and rapid daylength-dependent induction of leaf bleaching following transfer to air. A, Plants were germinated and grown for 25 d at high CO 2 (3,000 mL L 21 ). FW, Fresh weight. B, Plants transferred from high CO 2 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.

Figure 5 .

 5 Figure5. Comparison of gene expression in cat2 and cat2 gr1 following transfer from high CO 2 to air in either 8-h (SD) or 16-h (LD) growth conditions. Hierarchical clustering was performed using MultiExperimentViewer software. The exploded heat map at the right shows a subcluster of 62 genes that includes 33 JA-or woundingassociated genes (highlighted yellow).

Figure 7 .

 7 Figure 7. Leaf ascorbate, NADP(H), and thiobarbituric acid-reactive substances (TBARS) in the four genotypes placed in SD (left) and LD (right) in air following growth at high CO 2 . A, Ascorbate (white bars) and dehydroascorbate (black bars). B, NADP + (white bars) and NADPH (black bars). C, TBARS. For A and B, numbers above each bar indicate percentage reduction states [100 ascorbate/(ascorbate + dehydroascorbate) in A and 100 NADPH/(NADP + + NADPH) in B]. Samples were taken 4 d after transfer to air. All data are means 6 SE of three independent leaf extracts. Asterisks within blocks indicate significant differences from Col-0 values in the same condition: * P , 0.1. FW, Fresh weight.

Figure 8 .

 8 Figure 8. In vivo visualization of ROS using dichlorofluorescein fluorescence. Leaf mesophyll cells of plants grown at high CO 2 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). Representative examples from three independent experiments are shown. Magnification was the same for all samples. [See online article for color version of this figure.]

  H 2 O 2 and Glutathione Modulate the Expression of Specific Glutathione-Associated Genes in a Daylength-Dependent Manner

  peaks, in addition to GSH. The peaks were particularly abundant in conditions in which leaf glycine contents are depleted such as darkness (G. Noctor, A.C.M. Arisi, L. Jouanin, C.H. Foyer, unpublished data).

Figure 1 .

 1 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.

Figure 2 .

 2 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.

Figure 3 .

 3 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.

Figure 5 .

 5 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.

Figure 7 .

 7 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.

Figure 8 .

 8 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.

Figure 9 .

 9 Figure 9. Physiological importance of glutathione. The outer green circle shows examples of physiological processes affected by glutathione status or by glutathione-dependent components. The inner pink circle gives examples of major biochemical functions of glutathione. The darker pink spokes indicate components that are involved in these functions.

Table 1 .1. Probable classification of the three catalases found in different plant species

 1 (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

Table 1 .2. Nomenclature of the GST super-family in Arabidopsis

 1 (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

Table 1 .3. Disease resistance phenotypes in glutathione synthesis mutants

 1 (-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

Table 1 .4. Arabidopsis Atg homologs (from

 1

[START_REF] Yoshimoto | Beginning to understand autophagy, an intracellular self-degradation system in plants[END_REF]

.

CHAPTER 2 Functional analysis of Arabidopsis mutants points to novel roles for glutathione in coupling H 2 O 2 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

	CHAPTER 2
	Summary
	. Chapter 4 reports an analysis of possible interplay Through its interaction with H 2 O 2 , glutathione is a candidate for transmission of signals in plant responses
	between photorespiration, oxidative stress and autophagy in the regulation of senescence and to pathogens but identification of signaling roles is complicated by its antioxidant function. Using a
	SA-dependent lesion formation by phenotypic and other analyses of double mutants including cat2 atg2, genetic approach based on a conditional catalase-deficient Arabidopsis mutant, cat2, this study aimed to
	cat2 atg5 and cat2 atg18a. establish if glutathione plays an important functional role in the transmission of signals downstream of
	H 2 O 2 . For this, glutathione content or antioxidative capacity was independently modified in an H 2 O 2
	signaling background. Introducing the cad2 or allelic mutations in the glutathione synthesis pathway into
	cat2 blocked H 2 O 2 -triggered glutathione oxidation and accumulation. While no effects on NADP(H) or
	ascorbate were observed, and H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 to
	activate SA signaling, a key defence response in plants.
	Résumé
	Grâce à ses interactions avec le H 2 O 2 , 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
	of Arabidopsis mutants points to novel roles for glutathione in coupling H 2 O 2 to activation of salicylic basée sur cat2, un mutant d'Arabidopsis conditionnel déficient en catalase, cette étude visait à établir si le
	acid accumulation and signaling. Antioxidants & Redox Signaling glutathion joue des rôles de signalisation en aval du H 2 O 2 . 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 H 2 O 2 -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 H 2 O 2 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 H 2 O 2 intracellulaire à la signalisation par le SA, une réponse de defénse clé chez les plantes.

Table 2 .1. Details of mutant lines used in this study. The

 2 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.

Table 2 .2. Subcellular glutathione concentrations in Col-0, cat2, and cat2 cad2. Plants

 2

	__________________________________________________________________________________________________________
		C o l -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
	__________________________________________________________________________________________________________

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).

cat2 cad2 gr1 cat2 cad2 gr1 cat2 cad2 gr1 cat2 cad2 gr1 cat2 gr1 sid2 cat2 gr1 sid2 cat2 cad2 sid2 cat2 cad2 sid2 cat2 cad2 sid2 cat2 cad2 sid2 10

		B
	4 days
		cat2
		cat2
	cat2 cad2 cat2 cad2	cat2 cad2 cat2 cad2
		cat2 si
	cat2 sid2 cat2 sid2	cat2 sid2
		cat2 ca
		cat2 g
	cat2 gr1 cat2 gr1	cat2 gr1 cat2 gr1
		cat2
		cat2 gr

days Time after transfer to air from high CO 2

  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 (Figure2.12A). In cat2 cad2, in which the cat2 mutation should activate H 2 O 2 signaling, GSH was most effective, restoring total SA to about 60% of cat2 levels (Figure2.12A). 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 H 2 O 2 signal (Col-0), although GSSG was much less effective than GSH in inducing

acts independently of its antioxidant function in transmitting H 2 O 2 signals A

  central premise of this study was that H 2 O 2 -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 H 2 O 2 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 H 2 O 2 -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.

  well as abolishing H 2 O 2 -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

  .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.

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 H 2 O 2 signals requires accompanying glutathione accumulation. Genetically blocking this accumulation largely annuls H 2 O 2 -induced expression of JA-linked genes, and this effect can be rescued by exogenously supplying glutathione.

	Introduisant un blocage génétique de cette accumulation chez cat2 diminue significativement l'induction
	H 2 O 2 -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

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 H 2 O 2 -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 H 2 O 2 nécessite une accumulation de glutathion. glutathion-dépendants qui régulent la signalisation JA en réponse au stress oxydant.

Table 3 .1. Genes associated with JA/wounding showing significantly repressed transcript levels in cad2.

 3

	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

Table 4 .1. Overlapping metabolites induced by atg2 and cat2 mutations.

 4 Metabolites were identified by t test at P<0.05 (n = 3

	B
	High CO 2	Air
	2 5 18a 2 5 18a	2 5 18a 2 5 18a
		2,4 -OHbutanoic acid
		2-	hydroxyglutaric acid
		Al	anine
		Am inoadipic acid
		Ar abinose
		b-	alanine
		b-	sitosterol
		Cit ramalic acid
		Et	hanolamine
		Fr uctose
		GA	BA
		Ga lactono-1,4-lactone
		Ga lactinol
		Ga lactose
		Gl	uconic acid
		Gl	ycerate
		Ho	moserine
		Iso	leucine
		Ly	sine
		Ma late
		M annose
		O-	acetylserine
		Ph	enylalanine
		Ph	osphate
		Pu	trescine
		Ra ffinose
		Rh	amnose
		Ri	bose
		Se	rine
		Sh	ikimic acid
		Su	crose
		Te	tradecanoic acid
		Th	reonine
		Th	reonic acid
		Ty	rosine
		Ty	ramine
		Ur acil
		Va line
		Xy	lose

* *

  .

		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

  .4D). Second, glutathione accumulation was observed in atg mutants in high CO 2(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 H 2 O 2 generated in the peroxisomes, because both the phenotypic and metabolomics responses were stronger at high CO 2 . 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

  associated gene expression, indicating that GSSG produced from H 2 O 2 -triggered GSH oxidation may act as an indirect signal of H 2 O 2 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 H 2 O 2

Table 6 .

 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

  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 NaH 2 PO 4 (pH 5.6), 40 μL extract and 55 μL H 2 O. 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 NaH 2 PO 4 (pH7.5), 10 μL 25 mM DTT

	6.2.4.1.4 Ascorbate
	Ascorbate was measured by the absorbance at 265 nm that is specifically removed by ascorbate oxidase
	(AO).

  (1999b). Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiol. 119: 73-79

	peroxide (H 2 O 2 ;

Zhu, Y.L., Pilon-Smits, E.A.H., Jouanin, L., and Terry, N.

Table I .

 I Annotated phytohormone-associated genes showing significantly modified transcript levels in gr1

	Gene Identifier	Name	8-h Days	16-h Days	Hormone
	At3g50970 a	XERO2	Induced	-	Abscisic acid
	At4g15760	MO1	Induced	-	SA
	At4g23600 a	CORI3 (JR2)	Induced	-	JA
	At5g05730	ASA1	Induced	-	Ethylene
	At1g74670 a	Unknown protein	Repressed	-	GA
	At5g54490	PBP1	Repressed	-	Auxin
	At5g61590	ERF B3 member	Repressed	-	Ethylene
	At5g61600	ERF B3 member	Repressed	-	Ethylene
	At1g17420 a	LOX3	-	Repressed	JA
	At1g74430/40 a	MYB95	-	Repressed	JA
	At2g24850	TAT3	-	Repressed	JA
	At2g29440 a	GSTU6	-	Repressed	JA
	At2g34600 a	JAZ7	-	Repressed	JA
	At2g38240 a	2OGOR	-	Repressed	JA
	At4g15440 a	HPL1	-	Repressed	JA
	At5g13220 a	JAZ10	-	Repressed	JA

a Significant same-direction response in cat2 in the same condition.

  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 CO 2 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.

	C
							disulphide form (GSSG) that is recycled to GSH by
							NADPH-dependent glutathione reductase (GR). The
							central role of glutathione in defence metabolism in
							animals was established long ago, largely because selenium-
							dependent glutathione peroxidase (GPX) is a central pillar
							of animal antioxidant metabolism. Pharmacologically
							induced GSH deficiency in newborn mammals such as rats
							and guinea pigs leads to rapid multi-organ failure and death
							within a few days
			CAT2/CAT2 gr1/gr1	CAT2/CAT2 GR1/gr1	cat2/cat2 GR1/GR1	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. INTRODUCTION
	Glutathione (or a functionally homologous thiol) is an
	essential metabolite with multiple functions in plants
	(Fig. 1). The fundamental and earliest recognized function
	of glutathione is in thiol-disulphide interactions, in which
	reduced glutathione (GSH) is continuously oxidized to a

  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.

								GST
		GSH						CHO CO	X			GSH
								CH 3	HCHO
	COO -CHOH CHOH CH 3	GLYII	GS	CO CHOH CHOH CH 3	GLYI	GS	CHOH CO CO CH 3	GSH	FDH	HOC SG HOC-SG	FGH	HCOO HCOO -
								Me
									PCS
								(γ-Glu-Cys) n -Gly
								Me Me
	Figure 4.

Table 1 .

 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.

  SummaryH 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 availability in cat2 delays an oxidative stress relatedsenescence triggered by atg in short days. Overall, the work suggests that (1) novel glutathione-dependent functions are important to couple intracellular H 2 O 2 availability to the activation of both SA and JA signaling pathways and (2) H 2 O 2 produced through photorespiration may play an antagonistic role in the early senescence phenotype observed in atg mutants.RésuméLe H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 intracellulaire et activation des voies de signalisation SA et JA, et (2) que le H 2 O 2 produit par la photorespiration pourrait jouer un rôle antagoniste dans les phénotypes de sénescence précoce observée chez les mutants atg.

Plant Physiology Ò , July 2010, Vol. 153, pp. 1144-1160, www.plantphysiol.org Ó 2010 American Society of Plant Biologists

Plant Physiol.Vol. 153, 2010

Plant Physiol. Vol. 153, 2010

© 2011 Blackwell Publishing Ltd

© 2011 Blackwell Publishing Ltd, Plant,Cell and Environment, 35,

ACKNOWLEDGMENTS

We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, the Nottingham Arabidopsis Stock Centre for supply of gr1 seed stocks, and Gae ¨lle Cassin (Institut de Biologie des Plantes) for help with preliminary characterization 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 microscopy imaging and to Eddy Blondet (Unite ´de Recherche en Ge ´nomique Ve ´ge ´tale) for help with microarray analyses. Received January 24, 2010; accepted May 19, 2010; published May 20, 2010.

ACKNOWLEDGMENTS We thank Bernd Zechmann (University of Graz, Austria) for discussions on glutathione concentrations in different subcellular compartments and Stéphane Lemaire (Institut de Biologie Physico-Chimique, Paris, France) for discussions on glutaredoxins and protein glutathionylation reactions. 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 of a Chinese Scholarship Council fellowship. B.M.G. thanks Subprograma Estancias de Movilidad posdoctoral en centros extranjeros (2009), Ministerio de Educación (Spain) for a fellowship.

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 genetic interaction between cat2 and gr1 knockout mutations (data shown in Annex 1b).

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).

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. RevCAD2 5'-TCCTTGTCAGTGTCTGTCC-3' Genotyping 7 ForGR1 5'-TTCTGGGGAAACATTCTCCTCTTGC-3' Genotyping 8

RevGR1 5'-GAGGTAACTAGTGCCTGCAATATGACACAC-3' Genotyping 9

ForSID2 5'-GCTCTGCAGCTTCAATGC-3'

CATMA microarray analyses and data-mining

Microarray analysis was performed using the CATMA arrays containing 24,576 gene-specific tags 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 protein structure, the regulation of enzymatic activity, and redox signaling, and it is principally mediated by thioredoxin (TRX) and glutathione reductase (GR)/ glutathione systems (Buchanan and Balmer, 2005;[START_REF] Jacquot | The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation[END_REF]Meyer et al., 2008). Arabidopsis (Arabidopsis thaliana) lines identified in independent screens for alterations in heavy metal tolerance, meristem function, light signaling, and pathogen resistance have been shown to harbor mutations in the gene encoding the first enzyme of glutathione synthesis (Cobbett et al., 1998;Vernoux et al., 2000;Ball et al., 2004;Parisy et al., 2007). Studies on the rml1 mutant, which is severely deficient in glutathione synthesis, define a specific role for glutathione in root meristem function (Vernoux et al., 2000). However, shoot meristem function is regulated in a redundant manner by cytosolic glutathione and TRX (Reichheld et al., 2007), providing a first indication for functional overlap between these thiol-disulfide systems in plant development.

Modifications of cellular thiol-disulfide status may be important in transmitting environmental changes that favor the production of oxidants such as hydrogen * Corresponding author; e-mail graham.noctor@u-psud.fr. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Graham Noctor (graham.noctor@u-psud.fr).

[C] Some figures in this article are displayed in color online but in black and white in the print edition. [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.110.153767

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Characterization of the gr1 mutant.

Supplemental Figure S2. Glutathione and ascorbate status in gr1 and cat2 mutants grown at high CO 2 and then transferred to air in 8-h or 16-h days.

Supplemental Figure S3. Analysis of the conditional genetic interaction between cat2 and gr1 in an allelic double cat2 gr1 mutant.

Supplemental Figure S4. Quantitative PCR confirmation of changes in JA-linked genes in cat2 and cat2 gr1.

Supplemental Figure S5. H 2 O 2 visualization in leaves of Col-0, gr1, cat2, and cat2 gr1 in SD and LD conditions.

Supplemental Figure S6. Genevestigator analysis [START_REF] Hruz | Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes[END_REF] of responses of glutathione-associated genes showing significantly different expression in cat2 and/or cat2 gr1 (Fig. 6).

Supplemental Table S1. PCR primers used in this study.

Supplemental Table S2. Summary of modified daylength-dependent gene expression in gr1 and overlap with cat2 transcriptomes.

Supplemental Table S3. Complete data set of significantly modified genes in gr1, cat2, and cat2 gr1 in two daylength regimes.

Supplemental Table S4. List of JA-associated genes showing differential expression in cat2 and cat2 gr1 and relative expression values.

Supplemental Table S5. Summary of expression of GRXs, cytosolic TRXs, and related genes in cat2 and cat2 gr1.

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