Yves Jolivet

Prof Arnould

Gilles Chatel-Innocenti

Hélène Vanacker

Raynaud Cécile

Linda De Bont

Jingfang Hao

Shengbin Liu

Marie Dufresne

Sophie Massot

Sophie Blanchet

Severine Domenichini

Holger Ornstrup

Jean-Paul Bares…

Ler Landsberg

Glycerate Glycolate

-Phosphogluconate dehydrogenase ABA Abscisic acid AO Ascorbate oxidase APX Ascorbate peroxidase ASC Reduced ascorbate C Cytosine CAT Catalase Chl Chlorophyll CPR5 CONSTITUTIVE EXPRESSION

Keywords:

I would like to thank the former and the current colleagues in our group. Amna Mhamdi, my first officemate, helped me a lot in learning different techniques and in being familiar with the working environment. The scientific discussion with her has benefited me a lot. Her enthusiasm,

Chi-square sources of ROS within plant cells in the light [START_REF] Foyer | Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria[END_REF].

Increased ROS production can modify intracellular redox state. Because ROS production is influenced by external conditions, these molecules have become recruited by plants as important signals that provide information on environmental conditions relative to the internal status of plant cells. Under non-stressful physiological conditions, intracellular redox homeostasis is maintained by various systems. When the environment becomes more challenging, ROS accumulation can occur, leading to the perturbation of physiological processes that depend on cellular redox state. Depending on its intensity, context and duration, such perturbations can lead to increased resistance in plants through acclimation processes. Alternatively, they may lead to cell death. These responses are well known as outcomes of stress, although it is important to note that even cell death is not necessarily deleterious to plant function and can be used, for instance, as a strategy to resist pathogen invasion.

There is keen interest in understanding plant responses to stress, as they can often determine agricultural yields. The central role of ROS in stress responses explains the explosion of research interest in these molecules over recent decades. While earlier concepts saw ROS as toxic byproducts, they are now considered to be key signaling molecules involved in many physiological processes (Foreman et al., 2003;[START_REF] Wagner | The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana[END_REF]Han et al., 2013a, b;[START_REF] Foyer | Viewing oxidative stress through the lens of oxidative signalling rather than damage[END_REF]. It is important to note that, in the natural and field environments, plants are often exposed to more than one type of stress condition simultaneously. For instance, a plant might have to cope with soil pollutants, excessive salt, and pathogen attack at the same time. Although it is accepted that ROS are key regulators of the outcome of such conditions, the network that controls ROS and plant responses to ROS remains to be elucidated. As explained below, one of the key gaps in our knowledge is the specific role of different enzymes that are involved in controlling accumulation of ROS and cell redox state. This is the context within which the work presented in this thesis has been conducted.

CHAPTER 1 GENERAL INTRODUCTION

3

ROS in plants 1.ROS: definition

The term ROS refers to a group of derivatives of O2 that are more reactive than O2 itself. Since 2.7 billion years ago, when significant amounts of O2 began to accumulate in the earth's atmosphere following the evolution of PSII in O2-evolving cyanobacteria, plants have had to evolve mechanisms to deal with ROS generation within their cells [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]. The production of ROS relies on the distinct molecular characteristics of O2. As a diatomic radical, O2 has two unpaired electrons with the same spin quantum number. Because of the spin restriction, O2 prefers to accept electrons one at a time, which initially produces superoxide. Photodynamic excitation of O2 can also lead, without reduction, to singlet oxygen [START_REF] Krieger-Liszkay | Singlet oxygen production in photosynthesis[END_REF]. Both of these processes lead to the generation of partially reduced or activated derivatives of O2 which are, collectively, known as ROS [START_REF] Halliwell | Oxidative stress and neurodegeneration: where are we now?[END_REF]. As well as the superoxide anion and singlet oxygen, ROS include H2O2 and the very reactive hydroxyl radical (Figure 1.1). Singlet oxygen is a highly reactive form of ROS that is formed in PSII within chloroplasts. It has a short half-time of around 200 ns in cells and can react with various biomolecules such as lipids, proteins and DNA/RNA [START_REF] Gorman | Current perspectives of singlet oxygen detection in biological environments[END_REF]. Singlet oxygen can be generated during photoinhibition, which is caused by excess photosynthetically active radiation [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]; [START_REF] Krieger-Liszkay | Singlet oxygen production in photosynthesis[END_REF]. Photosensitizers like Rose Bengal can also lead to the generation of singlet oxygen [START_REF] Gutiérrez | Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts[END_REF]. Singlet oxygen production and signaling have been well studied in flu mutants in which a defect in a chlorophyll (Chl) biosynthesis regulator leads to ROS accumulation [START_REF] Laloi | Cross-talk between singlet oxygen-and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana[END_REF][START_REF] Lee | EXECUTER1-and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana[END_REF].

3 O 2 1 O 2 O 2 •- O 2 2- H 2 O 2 O 2 3- O -

Krieger

As noted above, superoxide, a short-lived anion radical, is produced as a byproduct of electron transport chains during photosynthesis and respiration. Plasma membrane NADPH oxidases are another source of superoxide anion in plants. Such enzymes translocate electrons from NADPH in the cell across the membrane to O2, which is reduced at the cell surface (Foyer and Noctor, 2000;[START_REF] Mittler | Reactive oxygen gene network of plants[END_REF][START_REF] Li | Oxygen vacancy associated surface fenton chemistry: surface structure dependent hydroxyl radicals generation and substrate dependent reactivity[END_REF]. Because of its high reactivity and toxic characteristic, superoxide anion is then converted to H2O2 and O2 by chemical transformation or by dismutation catalyzed by superoxide dismutase (SOD).

H2O2 has received particular attention as an oxidant and signaling molecule because of its relative stability. As well as being produced secondarily from superoxide (Figure 1.1), H2O2 can be generated at high rates by a two-electron reduction of O2 catalyzed by enzymes such as the photorespiratory glycolate oxidase [START_REF] Foyer | Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria[END_REF]. H2O2 is not a free radical, and it has a relatively long lifespan compared with other ROS. This property allows it to traverse cellular membranes and migrate between different compartments to function in physiological processes. H2O2, however, is highly oxidizing, with a standard redox potential of +1.32 V at pH 7.0 [START_REF] Winterbourn | The biological chemistry of hydrogen peroxide[END_REF]. Despite this potential oxidizing power, it reacts poorly with most biological molecules because of a high activation energy barrier that explains its relative stability. Therefore, the reactions of H2O2 are kinetically rather than thermodynamically driven. This means that they are largely dependent on catalysts such as enzymes. Among various types of enzymes that can metabolize H2O2, catalase (CAT) has received attention because it is highly abundant and active and because it can catalyze the dismutation of H2O2 without the requirement of any reducing co-factor other than H2O2 itself.

The hydroxyl radical is the three-electron reduction product of O2, and can notably be produced by metal-catalyzed reductive cleavage of H2O2 (Figure 1.1). As a free radical, it is short-lived, and is considered the most reactive ROS. It is highly destructive of cellular components, and can attack lipids, proteins, DNA and RNA. Therefore, it is kept at a minimal level compared with the superoxide anion and H2O2, although it is thought to be important in the formation of cell wall polymers. The hydroxyl radical can notably be produced via the Fenton reaction involving suitable transition metals [START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF][START_REF] Li | Oxygen vacancy associated surface fenton chemistry: surface structure dependent hydroxyl radicals generation and substrate dependent reactivity[END_REF]. Plants lack enzymatic mechanisms to scavenge the hydroxyl radical, but its formation can be prevented by metal chelators and rapid removal of H2O2 via a variety of enzyme systems that are discussed below. Together, these mechanisms act to limit Fentoninduced damage through the hydroxyl radical [START_REF] Maurício | Antioxidant activity of caffeic acid against iron-induced free radical generation-a chemical approach[END_REF].

ROS generation

ROS generation and compartmentation

Many developmental processes involve signaling through ROS that are probably generated at lower levels than those associated with stress. It has been well established that reactions associated with photosynthesis, photorespiration and respiration are major sources of ROS. As described above, ROS can also be generated at the extracellular surface by NADPH-dependent oxidases, as well as in the apoplast by peroxidases and oxidases (Figure 1.2; [START_REF] Corpas | Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells[END_REF][START_REF] Foyer | Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria[END_REF][START_REF] Asada | Production and scavenging of reactive oxygen species in chloroplasts and their functions[END_REF][START_REF] Mhamdi | Plant catalases: peroxisomal redox guardians[END_REF][START_REF] Noctor | ROS-related redox regulation and signaling in plants[END_REF] .

The chloroplast was one of the very first sources of ROS to be described in plants [START_REF] Mehler | Studies on reactions of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents[END_REF][START_REF] Asada | Univalent reduction of molecular-oxygen by spinachchloroplasts on illumination[END_REF]. ROS generation occurs during photosynthesis at the sites of both PSI and PSII [START_REF] Asada | Production and scavenging of reactive oxygen species in chloroplasts and their functions[END_REF][START_REF] Foyer | Viewing oxidative stress through the lens of oxidative signalling rather than damage[END_REF]. Specifically, singlet oxygen is produced by the photosensitizer Chl in the reaction center of PSII. When light absorption exceeds the capacity of photosynthetic electron transport, the lifetime of excited triplet states of Chl ( 3 Chl) increases, favoring transfer of the absorbed light energy to ground-state triplet oxygen ( 3 O2) to form singlet oxygen [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][START_REF] Krieger-Liszkay | Singlet oxygen production in photosynthesis[END_REF]. In contrast, PSI is considered to be a less important contributor to singlet oxygen generation in photosynthetic organisms. However, it is the major site for the generation of superoxide anion and H2O2; that is, reduced forms of O2 [START_REF] Mehler | Studies on reactions of illuminated chloroplasts. I. Mechanism of the reduction of oxygen and other Hill reagents[END_REF][START_REF] Asada | The water-water cycle as alternative photon and electron sinks[END_REF]Fryer et al., In animals, the mitochondrial electron transport chain is a major site of ROS generation [START_REF] Liu | Generation of reactive oxygen species by the mitochondrial electron transport chain[END_REF]. Complex I and III are the main sites of ROS production in mitochondria, whereby O2 is reduced to superoxide anion [START_REF] Møller | PLANT MITOCHONDRIA AND OXIDATIVE STRESS : electron transport, NADPH turnover, and metabolism of reactive oxygen species[END_REF]Jezek and Hlavata, 2005). Subsequently, the superoxide anion is converted to H2O2 either by the action of MnSOD or by spontaneous dismutation (Finkel and Holbrook, 2000). Compared to chloroplasts and peroxisomes, the rate of H2O2 generation in plant mitochondria is probably lower [START_REF] Foyer | Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria[END_REF][START_REF] Halliwell | Free Radicals in Biology and Medicine[END_REF]. In the light at least, the mitochondria are not regarded as a major source of ROS in leaves, although this obviously does not exclude an important function for these organelles in ROS-related signaling.

The apoplast is a major site of ROS generation. For example, during plant-pathogen interactions, ROS-producing enzymes are thought to be largely responsible for the apoplastic oxidative burst [START_REF] Qi | Apoplastic ROS signaling in plant immunity[END_REF]. This system consists chiefly of NADPH oxidases, cell wall peroxidases and amine oxidases [START_REF] Petrov | Hydrogen peroxide-a central hub for information flow in plant cells[END_REF][START_REF] Sierla | Apoplastic and chloroplastic redox signaling networks in plant stress responses[END_REF]. NADPH oxidases, also called respiratory burst oxidase homologs (RBOHs), are one of the major ROS-generating enzymes in plants [START_REF] Umezawa | Molecular basis of the core regulatory network in ABA responses: sensing, signaling and transport[END_REF][START_REF] Mittler | ROS signaling: the new wave?[END_REF]. As noted above, they transfer electrons from cytosolic NADPH to apoplastic oxygen, leading to the production of the superoxide anion, which is then converted to H2O2 spontaneously or by SOD. Ten NADPH oxidase genes have been identified in Arabidopsis. Among them, AtrbohD and AtrbohF play important roles in the hypersensitive response (HR; [START_REF] Torres | Reactive oxygen species signaling in response to pathogens[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][START_REF] Kadota | Regulation of the NADPH oxidase RBOHD during plant immunity[END_REF].

Stress and ROS

In optimal physiological conditions, redox homeostasis is maintained and ROS levels are relatively low. However, as sessile organisms, plants often endure environmental challenges that limit growth, reproductive success, yield, quality, or other traits desirable to humans. Collectively, these conditions are defined as stress, and are typically divided into abiotic and biotic stresses. Abiotic stress refers to the negative impact of non-living factors on organisms. Examples are excess salt, heavy metals, heat, chilling, insufficient water or high irradiance. The second is biotic stress that occurs due to damage instigated by other living organisms including bacteria, fungi, viruses, parasites, insects and other native or cultivated plants. A central theme in many stress responses is the accumulation of ROS and ROS-induced changes in cellular redox state. This condition is commonly known as oxidative stress (Foyer and Noctor, 2000;[START_REF] Dat | Catalase-deficient tobacco plants: tools for in planta studies on the role of hydrogen peroxide[END_REF][START_REF] Pastori | Common components, networks, and pathways of cross-tolerance to stress. The central role of "Redox" and abscisic acid-mediated controls[END_REF].

Pollution linked to heavy metals that are redox-active may result in superoxide anion formation because of autoxidation, and subsequently lead to H2O2 production via Fenton-type reactions [START_REF] Li | DNA damage resulting from the oxidation of hydroquinone by copper: role for a Cu(II)/Cu(I) redox cycle and reactive oxygen generation[END_REF]Schutzendübel and Polle, 2002). Drought is another abiotic stress condition in which the roles of ROS have received much attention. In drought conditions, stomatal closure is induced, causing a drop in intracellular CO2 concentrations. This effect tends to favor RuBP oxygenation, thereby increasing glycolate production and accelerating H2O2 production in the peroxisomes [START_REF] Cornic | Partitioning of photosynthetic electron flow between CO2 and O2 reduction in a C3 leaf (Phaseolus vulgaris L.) at different CO2 concentrations and during drought stress[END_REF][START_REF] Noctor | The roles of reactive oxygen metabolism in drought: not so cut and dried[END_REF]. Moreover, drought stress favors electron flux to O2 and restricts NADP + regeneration. The rise in NADPH:NADP + ratio will lead to promotion of the Mehler reaction, and increased photosynthetic control will tend to promote singlet oxygen production. Furthermore, over-reduction of the electron transport chain in chloroplasts and decreased availability of other oxidants for the chain during drought may enhance singlet oxygen, superoxide anion or H2O2 production [START_REF] Asada | Production and scavenging of reactive oxygen species in chloroplasts and their functions[END_REF]Fischer et al., 2013).

The above paragraph describes examples of how abiotic stress conditions can alter ROS production by their effects on basal metabolism. In addition to such effects, rapid programmed production of ROS, known as "oxidative bursts", may also occur. Such processes have received particular attention for their importance in responses to biotic stress [START_REF] Torres | Reactive oxygen species signaling in response to pathogens[END_REF]. Indeed, the first documented "oxidative burst" in plants was described following infection of potato tubers with the pathogenic oomycete, Phytophthora infestans [START_REF] Doke | Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity[END_REF]. Since then numerous studies have shown that ROS production at the cell wall and apoplast is involved in biotic stress responses. Plasma membrane NADPH oxidases and cell wall peroxidases are thought to be the major sources of ROS production during plant-pathogen interactions, although intracellular redox reactions are also involved. NADPH oxidases, especially RBOHD and RBOHF in Arabidopsis, play a key role in apoplastic ROS generation in immune responses [START_REF] Torres | Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response[END_REF][START_REF] Nühse | Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses[END_REF][START_REF] Zhang | A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-induced immunity in plants[END_REF]. In addition, it has been reported that class III apoplastic peroxidases PRX33 and PRX34 are essential for CHAPTER 1 GENERAL INTRODUCTION 9 ROS production in response to flg22 and elf18 in Arabidopsis [START_REF] Daudi | The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity[END_REF].

ROS signaling

A long-held view of the biological role of ROS was that they are toxic by-products of plant metabolism through the processes described above [START_REF] Asada | THE WATER-WATER CYCLE IN CHLOROPLASTS: scavenging of active oxygens and dissipation of excess photons[END_REF]. According to this view, they would have little or no importance in physiological processes, but rather cause damage to lipids, proteins and DNA in the stressed cells, leading to the notion that resistance to stress could be engineered by enhancing antioxidative capacity or by avoiding the production of ROS (Noctor and Foyer, 1998;[START_REF] Asada | THE WATER-WATER CYCLE IN CHLOROPLASTS: scavenging of active oxygens and dissipation of excess photons[END_REF][START_REF] Maxwell | The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells[END_REF][START_REF] Mittler | Oxidative stress, antioxidants and stress tolerance[END_REF]. However, since the beginning of this century, substantial experimental data suggests that ROS are highly controlled signaling molecules. For ROS to be effective as signaling components, living cells have very efficient antioxidant systems to keep them under precise control (Foyer and Noctor, 2005a,b). Secondary messengers activated by ROS can regulate downstream pathways that are physically non-adjacent to the original ROS signaling pathway [START_REF] Nathan | Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling[END_REF]. All these characteristics render ROS able to set specificity as signaling molecules.

ROS function is intimately involved in the activation of gene expression, the induction of specific protein kinases and calcium signatures, the interaction with NADPH oxidases, hormone signal transduction, and the requirement of specific gene modulation for ROS-induced cell death (Figure 1.3; [START_REF] Kovtun | Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants[END_REF][START_REF] Desikan | Regulation of the Arabidopsis transcriptome by oxidative stress[END_REF]Foreman et al., 2003;[START_REF] Kwak | NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis[END_REF][START_REF] Vandenabeele | A comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco[END_REF][START_REF] Wagner | The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana[END_REF][START_REF] Vandenbroucke | Hydrogen peroxide-induced gene expression across kingdoms: A comparative analysis[END_REF][START_REF] Sewelam | Global plant stress signaling: reactive oxygen species at the cross-road[END_REF].

ROS and photosynthesis

Chloroplasts were one of the very earliest sources of ROS to be described in plants. As described above, ROS production in the thylakoid membrane is notably determined by the balance between light capture and the utilisation of light energy by the photosynthetic electron transport chain, which in turn depends on metabolic consumption of ATP and reductant for its continued operation. Any change or imbalance associated with photosynthesis in the chloroplast can affect, directly or indirectly, other functions in plants. It is not surprising that plants have evolved mechanisms to sense photosynthesisassociated ROS accumulation induced by environmental challenges, to enable the ensuing change of redox states in the chloroplast to influence other signaling pathways and lead to appropriate physiological responses [START_REF] Huner | Sensing environmental temperature change through imbalances between energy supply and energy consumption: Redox state of photosystem II[END_REF]. It has been shown that signal transmission from the chloroplast can change nuclear gene expression, which is proposed to act as a retrograde signaling pathway [START_REF] Koussevitzky | Signals from chloroplasts converge to regulate nuclear gene expression[END_REF][START_REF] Pogson | Plastid signalling to the nucleus and beyond[END_REF]Pfannschmidt et al., 2009;[START_REF] Leister | Retrograde signaling in plants: from simple to complex scenarios[END_REF][START_REF] Herz | Shedding light on the chloroplast as a remote control of nuclear gene expression[END_REF][START_REF] Sewelam | Global plant stress signaling: reactive oxygen species at the cross-road[END_REF]. For example, when pharmacological approaches were used to block carotenoid synthesis and hence increase the ability of ROS production in chloroplasts, Arabidopsis seedlings suffered severe photo-oxidative damage. Simultaneously, ROS-responsive marker genes were rapidly up-regulated [START_REF] Kim | 1 O2-mediated and EXECUTER-dependent retrograde plastid-to-nucleus signaling in norflurazon-treated seedlings of Arabidopsis thaliana[END_REF]. Another approach used a genetic system of glycolate oxidase overexpression to perturb H2O2 levels in chloroplasts. The resulting ROS accumulation induced early signaling responses by regulating gene transcription and secondary signaling messengers, providing further evidence for ROS-dependent retrograde signaling pathways [START_REF] Sewelam | Spatial H2O2 signaling specificity: H2O2 from chloroplasts and peroxisomes modulates the plant transcriptome differentially[END_REF]. The redox state of the plastoquinone pool, which plays a key role in electron transport in photosynthesis, was suggested to modulate the expression of cytosolic ascorbate peroxidase (APX) under excess light stress [START_REF] Karpinski | Photosynthetic electron transport regulates the expression of cytosolic ascorbate peroxidase genes in Arabidopsis during excess light stress[END_REF]. Other studies also show that ROS signaling from chloroplast is involved in abiotic and biotic stress responses [START_REF] Fryer | Control of Ascorbate Peroxidase 2 expression by hydrogen peroxide and leaf water status during excess light stress reveals a functional organisation of Arabidopsis leaves[END_REF][START_REF] Agrawal | A dynamic mathematical model to clarify signaling circuitry underlying programmed cell death control in Arabidopsis disease resistance[END_REF][START_REF] Joo | Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone[END_REF][START_REF] Belhaj | The chloroplast protein RPH1 plays a role in the immune response of Arabidopsis to Phytophthora brassicae[END_REF][START_REF] Zurbriggen | Chloroplast-generated reactive oxygen species play a major role in localized cell death during the non[END_REF][START_REF] Serrato | Plastid thioredoxins: a "one-for-all" redox-signaling system in plants[END_REF].

ROS and redox homeostasis

Redox regulation is an essential factor to keep energy conversion and consumption in plants under good control. When suffering environmental challenges, ROS perturbation is perceived and transformed into redox signals, followed by responses at various levels of regulation and in different subcellular compartments [START_REF] Scheibe | Reduction-oxidation network for flexible adjustment of cellular metabolism in photoautotrophic cells[END_REF]. The major soluble non-protein redox couples in plants like glutathione, ascorbate, NAD and NADP, are at the hub of the redox network surrounding ROS generation and control. It was reported that photorespiration-derived H2O2 led to a photoperiodic effect on redox signaling, in which intracellular glutathione state was perturbed by the accumulation of glutathione disulfide (GSSG; Queval et al., 2007). The changes of intracellular redox state could mediate various signaling events through their interaction with many other secondary messengers such as protein kinase and phosphatases, phytohormones and calcium. Much evidence indicates that ROSantioxidant interactions act as a metabolic interface between environmental changes and the ensuing signaling responses (Foyer andNoctor, 2005a, 2012), and this is partly accomplished through modifications of redox state [START_REF] Dietz | Redox signal integration: From stimulus to networks and genes[END_REF]. The plant antioxidative systems that promote redox homeostasis in the face of constant ROS production are discussed further in Section 1.2.

ROS and Mitogen-activated protein kinase (MAPK)

MAPK signaling pathways have been well accepted as a general signal transduction mechanism in eukaryotes. These pathways consist of three functionally linked protein kinases: MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK). By protein phosphorylation cascades from MAPKKK to MAPK, this system links different receptors to their cellular and nuclear targets [START_REF] Tena | Plant mitogen-activated protein kinase signaling cascades[END_REF]. MAPK signaling cascades in Arabidopsis are complex, involving multiple isoforms of each MAPK gene and more than 20 pathways [START_REF] Wrzaczek | Plant MAP kinase pathways : how many and what for?[END_REF][START_REF] Ichimura | Mitogen-activated protein kinase cascades in plants: a new nomenclature[END_REF].

Many environmental constraints were found to stimulate MAPK cascades, like cold, heat, drought, wounding, and pathogens, while these cascades are also involved in phytohormone signaling [START_REF] Bowler | The role of calcium and activated oxygens as signals for controlling crosstolerance[END_REF]. In plants and eukaryotes in general, it has been demonstrated that the transmission of oxidative signals is controlled by protein phosphorylation involving MAPKs [START_REF] Kyriakis | Sounding the alarm: protein kinase cascades activated by stress and inflammation[END_REF][START_REF] Gustin | MAP Kinase pathways in the yeast Saccharomyces cerevisiae[END_REF][START_REF] Pitzschke | Mitogen-activated protein kinases and reactive oxygen species signaling in plants[END_REF][START_REF] Xing | AtMKK1 mediates ABA-induced CAT1 expression and H2O2 production via AtMPK6-coupled signaling in Arabidopsis[END_REF]. It was reported that H2O2 initiated MAPK signaling by activating ANP1, an isoform of MAPKKK. This effect led to the phosphorylation of MPK3 and MPK6 [START_REF] Kovtun | Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants[END_REF]. Oxidative signal inducible 1 (OXI1), a protein kinase, is also involved in the activation of MPK3 and MPK6. Infection by virulent fungal pathogens caused HR in oxi1 null mutant, concomitantly the activation of MPK3 and MPK6 by oxidative stress was compromised [START_REF] Rentel | OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis[END_REF]. Protein phosphorylation was also demonstrated to trigger Ca 2+ -dependent ROS production, which was mediated by NADPH oxidases in Arabidopsis. This study also suggested the existence of a positive feedback regulation of Ca 2+ and ROS [START_REF] Kimura | Protein phosphorylation is a prerequisite for the Ca 2+ -dependent activation of Arabidopsis NADPH oxidases and may function as a trigger for the positive feedback regulation of Ca 2+ and reactive oxygen species[END_REF].

ROS and phytohormones

Plant stress responses are highly integrated with hormone signaling to regulate biological processes. Salicylic acid (SA) signaling is associated with resistance to biotrophic pathogens as well as abiotic stress [START_REF] Vlot | Salicylic Acid, a multifaceted hormone to combat disease[END_REF]. Intracellular and extracellular ROS accumulation can induce SA production, pathogenesis-related (PR) gene expression, and cell death [START_REF] Chamnongpol | Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco[END_REF]Torres et al., CHAPTER 1 GENERAL INTRODUCTION 13 2005;[START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]. All these effects can be reverted in the cat2 mutant by blocking SA synthesis, showing that H2O2-triggered cell death and related responses are not a direct consequence of damage [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]. Intriguingly, the defence responses triggered by increased peroxisomal availability of H2O2 in cat2 occur in a photoperiod-dependent manner: the PR responses such as lesions and SA accumulation appear when plants are grown in long days (LD) but not short days (SD) (Queval et al., 2007;[START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]. ROS-induced SA accumulation can have several effects, notably including enhanced resistance to pathogens through the induction of PR proteins and phytoalexins. In addition, it can also promote stomatal closure, which may also contribute to defence against pathogens that enter plants by this route [START_REF] Khokon | Involvement of extracellular oxidative burst in salicylic acid-induced stomatal closure in Arabidopsis[END_REF]. The application of SA can also induce ROS production via a peroxidase-catalyzed reaction, which also favors stomatal closure [START_REF] Melotto | Plant stomata function in innate immunity against bacterial invasion[END_REF][START_REF] Miura | SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid-induced accumulation of reactive oxygen species in Arabidopsis[END_REF].

Unlike SA, JA and the related compound methyl jasmonate (MeJA) are involved in the response to necrotrophic pathogens and wounding (Devoto and Turner, 2005). Accumulating evidence reveals a strong relationship between ROS and JA signaling. For instance, ROS derived from NADPH oxidases are critical for JA-induced gene expression regulated by MYC2, a transcription factor involved in JAmediated response [START_REF] Maruta | Arabidopsis NADPH oxidases, AtrbohD and AtrbohF, are essential for jasmonic acid-induced expression of genes regulated by MYC2 transcription factor[END_REF]. It was revealed that JA signaling in response to intracellular oxidative stress requires an accompanying accumulation of glutathione (Han et al., 2013b). A complex relationship between SA and JA in physiological processes has been elucidated. Many studies show the opposite effect between JA signaling and SA-dependent pathways [START_REF] Dangl | Plant pathogens and integrated defence responses to infection[END_REF][START_REF] Spoel | NPR1 modulates cross-talk between salicylate-and jasmonate-dependent defense pathways through a novel function in the cytosol[END_REF][START_REF] Takahashi | Antagonistic interactions between the SA and JA signaling pathways in Arabidopsis modulate expression of defense genes and gene-for-gene resistance to cucumber mosaic virus[END_REF]Koorneef et al., 2008). However, the two pathways may interact positively and can also be induced together. For example, it was demonstrated that increased H2O2 levels in cat2 induced both SA and JA signaling pathways, and that both pathways are less induced when glutathione accumulation is genetically blocked by the cad2 mutation (Han et al., 2013b). These observations point to some glutathione-dependent signaling process in the link between H2O2 and induction of phytohormone pathways, an issue that will receive attention in Chapters 4 and 5 of this thesis.

As well as SA and JA, other phytohormones are also involved in stress responses. ABA is known to be induced together with ROS production in different environmental stress responses. It has been reported that ABA-induced stomatal closure during stress is mediated by ROS which is derived from NADPH oxidases [START_REF] Kwak | NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis[END_REF]. Besides, ABA is found to be required for H2O2 production in chloroplasts, mitochondria and peroxisomes under water stress [START_REF] Hu | Abscisic acid is a key inducer of hydrogen peroxide production in leaves of maize plants exposed to water stress[END_REF]. Moreover, ABA has been considered to play a negative role in biotic stress signaling orchestrated by SA, JA and ethylene [START_REF] Coego | An Arabidopsis homeodomain transcription factor, OVEREXPRESSOR OF CATIONIC PEROXIDASE 3, mediates resistance to infection by necrotrophic pathogens[END_REF]. Ethylene is well documented to be a key player in programmed cell death during senescence, ozone stress or pathogen infection (Orzaez and Granell, 1997;[START_REF] Lund | Ethylene regulates the susceptible response to pathogen infection in tomato[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]. Gibberellins (GA), a cyclic diterpene compound with multiple functions in the plant life cycle, are linked with ROS through DELLA proteins which modulate transcript levels of antioxidant enzymes [START_REF] Achard | Plant DELLAs restrain growth and promote survival of adversity by reducing the levels of reactive oxygen species[END_REF].

Other components involved in ROS signaling

In addition to the aspects mentioned above, there are also some other second messengers such as GTPbinding proteins (G proteins) and Ca 2+ which mediate the ROS signals. The role of G proteins in stress responses, especially in plant-pathogen interactions, has been extensively reported [START_REF] Assmann | G protein regulation of disease resistance during infection of rice with rice blast fungus[END_REF][START_REF] Trusov | Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene-and abscisic acid-mediated defense signaling[END_REF][START_REF] Maruta | Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis[END_REF]. It was revealed that under ozone stress, the first biphasic oxidative burst is greatly attenuated or absent in mutants lacking Gα protein or Gβ protein [START_REF] Joo | Different signaling and cell death roles of heterotrimeric G protein α and β subunits in the Arabidopsis oxidative stress response to ozone[END_REF]. AtRbohD and AtRbohF were suggested to receive initial signals from G proteins to mediate ozone responses in guard cells [START_REF] Suharsono | The heterotrimeric G protein α subunit acts upstream of the small GTPase Rac in disease resistance of rice[END_REF]. Calcium signaling is involved in many signal transduction pathways. Elevations in cytosolic Ca 2+ represent an early response to many different biotic and abiotic stresses [START_REF] Mcainsh | Shaping the calcium signature[END_REF][START_REF] Dodd | The language of calcium signaling[END_REF]. ABA and H2O2 treatments are among those that can increase cytosolic Ca 2+ concentrations. Moreover, Ca 2+ has been reported to work both upstream and downstream of ROS production in signaling pathways [START_REF] Bowler | The role of calcium and activated oxygens as signals for controlling crosstolerance[END_REF][START_REF] Abuharbeid | Cytotoxicity of the novel anti-cancer drug rViscumin depends on HER-2 levels in SKOV-3 cells[END_REF][START_REF] Monshausen | Ca 2+ regulates reactive oxygen species production and pH during mechanosensing in Arabidopsis roots[END_REF][START_REF] Sewelam | Ethylene response factor 6 is a regulator of reactive oxygen species signaling in Arabidopsis[END_REF].

ROS processing

The above discussion underlines the close integration of ROS-dependent redox signaling in numerous physiological processes, while also revealing that our knowledge of the details of redox signaling remains incomplete. Although many downstream responses to ROS have been described, the key redox events that lie at the heart of ROS-triggered signaling remain to be elucidated. A key player in enabling and regulating ROS signals is the plant antioxidative system. This system is complex and includes efficient enzymatic and non-enzymatic mechanisms that have been developed during evolution. Enzymatic systems consist of CAT, APX, SOD, various types of peroxiredoxins (PRX), glutathione/thioredoxin peroxidases (GPX) and glutathione S-transferases (GST). Non-enzymatic components include glutathione and ascorbate, which can be considered major components of a redox hub, but also tocopherol, carotenoids and phenolic compounds. Through the network of these mechanisms, plants regulate ROS accumulation and maintain redox homeostasis (Figure 1.4;Noctor et al., 2017).

Catalase

CAT was the first discovered antioxidative enzyme. All known CAT forms in eukaryotes are haemdependent. Among them two main types have been characterized, which are monofunctional CATs (also known as typical CATs) and bifunctional CAT-peroxidases [START_REF] Zamocky | Evolution of catalases from bacteria to humans[END_REF]. They can be distinguished by the affinity for H2O2 and the sensitivity to the inhibitor 3-amino-1,2,4-triazole (3-AT) [START_REF] Margoliash | Irreversible reaction of 3-amino-1:2:4-triazole and related inhibitors with the protein of catalase[END_REF][START_REF] Regelsberger | Occurrence and biochemistry of hydroperoxidases in oxygenic phototrophic prokaryotes (cyanobacteria)[END_REF]. Unlike monofunctional CATs, which exist in diverse organisms, the CAT-peroxidases are only found in some fungi and prokaryotes [START_REF] Mutsuda | The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli[END_REF][START_REF] Regelsberger | Occurrence and biochemistry of hydroperoxidases in oxygenic phototrophic prokaryotes (cyanobacteria)[END_REF].

The typical CAT reaction is the dismutation of H2O2. The reaction is initiated by splitting the O-O bond of H2O2 to produce a molecule of H2O, as well as an oxy-ferryl enzyme intermediate (compound I) and a porphyrin cation radical. Then a second H2O2 is oxidized to O2. Simultaneously, compound I is reduced back to the initial state by releasing the bound O which is involved in the formation of the second molecule of water [START_REF] Regelsberger | The role of distal tryptophan in the bifunctional activity of catalase-peroxidases[END_REF][START_REF] Alfonso-Prieto | The molecular mechanism of the catalase reaction[END_REF]. Intriguingly, monofunctional CATs can also catalyze some H2O2-dependent peroxidation of reducing substrates, making the functional division between the two types of CAT indistinct. During this process, the reduction of compound I is performed by the interaction with small compounds like ethanol instead of the second H2O2 [START_REF] Zamocky | Evolution of catalases from bacteria to humans[END_REF]. CAT-associated peroxidation has been reported in both mammalian and plants [START_REF] Havir | Enhanced-peroxidatic activity in specific catalase isozymes of tobacco, barley, and maize[END_REF][START_REF] Kirkman | Mammalian catalase: a venerable enzyme with new mysteries[END_REF], but its physiological significance remains as yet unclear.

Regenerators of reduced forms

MDAR

GR

CAT has high specificity for H2O2. So far three CAT genes have been identified in angiosperm species such as Arabidopsis, rice, tobacco, maize and pumpkin [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]Esaka et al., 1997;[START_REF] Iwamoto | Differential diurnal expression of rice catalase genes: the 5'flanking region of CatA is not sufficient for circadian control[END_REF]. In Arabidopsis, CAT1 and CAT3 are located contiguously on chromosome 1 while CAT2 is located on chromosome 4 [START_REF] Frugoli | Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh[END_REF]. According to the classification system based on tobacco genes and first proposed by [START_REF] Willekens | Catalases in plants[END_REF], Class I CATs are mainly expressed in photosynthetic tissues, Class II CATs are associated with vascular tissues, while Class III CATs are notably expressed in seeds and reproductive tissues.

For Arabidopsis, CAT1, CAT2 and CAT3 correspond to Class III, Class I and Class II, respectively (Table 1.1). The CAT1 gene is mainly expressed in pollens and seeds and at very low levels in leaves, CAT2 is expressed in photosynthetic tissues but also in roots and seeds, while CAT3 is associated with vascular tissues. It has been reported that compared to CAT1, the transcript levels of CAT2 and CAT3 are much higher in mature Arabidopsis rosettes [START_REF] Frugoli | Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh[END_REF][START_REF] Mcclung | Regulation of catalases in Arabidopsis[END_REF]. In addition,

CAT2 transcripts show a day-night rhythm with a peak expression at the night/day transition, which is opposite to that of CAT3 [START_REF] Zhong | Interactions between light and the circadian clock in the regulation of CAT2 expression in Arabidopsis[END_REF]Mhamdi et al., 2010a). Studies on subcellular localization showed that CATs exist mainly in peroxisomes in plants. Evidence supporting this conclusion includes the high CAT activity in peroxisomes and the identification of an import mechanism due to a Peroxisomal Targeting Sequence 1 (PTS1) pathway [START_REF] Mullen | Identification of the peroxisomal targeting signal for cottonseed catalase[END_REF]. Previous studies on gene-specific T-DNA insertion mutants showed that CAT2 encodes the major leaf CAT isoform and this enzyme makes the major contribution to leaf CAT activity (Queval et al., 2007;Mhamdi et al., 2010a). In cat2 knock-out mutants, the CAT activity in rosettes is decreased by around 90% compared with that in wild type, and root CAT activity is also attenuated, though less severely [START_REF] Bueso | The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene[END_REF]. In cat3, there is around 20% activity lost. In contrast, the effect of the cat1 mutation on leaf CAT activity is negligible. This is consistent with its relatively low transcript levels in leaves. To date, therefore, it seems that CAT2 and CAT3 may play the major roles in Arabidopsis rosette tissue. However, the relative contribution of these CAT isoforms changes according to the developmental stage of the plants [START_REF] Zimmermann | Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh[END_REF]. For instance, as a senescence-associated gene, CAT3 transcripts increase with leaf age.

Because of the close association with photorespiration, Arabidopsis CAT2-deficient mutants (cat2)

have been extensively used in various studies of plant development and physiology. Under conditions where photorespiratory H2O2 production is highly active, CAT function deficiency leads to increased availability of endogenous H2O2, with effects on cell redox state. The intracellular H2O2 signal can be modulated by changing photorespiration-related growth conditions, such as CO2 concentration and irradiance (Queval et al., 2007). When grown in air (400 µL CO2 L -1 ) with a moderate irradiance of 200 μmol m -2 s -1 at the surface of leaves, cat2 shows a dwarf phenotype accompanied by redox perturbation, evidenced by the decreases in the GSH:GSSG ratio and increases in total glutathione. By contrast, it has a wild-type phenotype when grown in high CO2 (hCO2; 3000 µL L -1 ) or if the irradiance is lower than 50 μmol m -2 s -1 , both conditions in which photorespiration is rather inactive.

Among the most obvious phenotypes of cat2 grown in air at moderate light intensity are necrotic lesions. This phenotype is photoperiod-dependent. When grown in a 16 h photoperiod (LD), cat2

shows spreading necrotic lesions. By contrast, no lesions are observed in plants grown in an 8 h photoperiod (SD), even though the dwarf phenotype and redox perturbation are as marked as in LD (Queval et al., 2007[START_REF] Queval | H2O2activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast[END_REF]. The lesion appearance in cat2 bears a striking resemblance to the HR, and includes SA accumulation, PR genes induction (e.g. PR1 and PR2), activation of camalexin and its synthesis pathway, and induced resistance to bacterial challenge. As well as necrotic lesions, all these effects are absent in SD. By the combination of pharmacological and genetic approaches, it is revealed that exogenous SA treatment could induce these responses in SD and revert it in LD when SA synthesis is genetically blocked. All these observations show that SA is involved in the peroxisomal H2O2-triggered HR-like lesion formation in cat2 grown in LD air condition [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF].

Because of their influence on cell redox state, peroxisomal CATs act as regulators to fine-tune redox signaling, which is also a function of the carbon flux through photorespiration. This makes cat2 mutants an interesting model system to investigate the possible relationship between photorespiration and other metabolic, transcriptional and physiological processes, especially those possessing a redox component. Reductive pathways for H2O2 processing appear to compensate quite rapidly when CAT is deficient. Even though glutathione redox status is perturbed within hours after the onset of photorespiratory H2O2 production in cat2, little or no change is found in the redox states of glutathione-associated redox compounds, like ascorbate/dehydroascorbate or NADPH/NADP + (Queval et al., 2007;Mhamdi et al., 2010b,c). Glutathione status appears to play an active part in the H2O2triggered signal transduction provoked by CAT deficiency. For example, the GR knockout line gr1, which shows qualitatively similar changes in glutathione to those observed in cat2, has a wild-type phenotype, but nevertheless shows gene expression patterns that partly recapitulate those observed in cat2. Further, when the gr1 mutation is introduced into the cat2 background, H2O2-associated transcript profiles are significantly affected, suggesting that glutathione plays a role in transmitting H2O2-induced signals (Mhamdi et al., 2010b). Further evidence for this conclusion was obtained by analysis of cat2 lines in which glutathione accumulation was genetically impaired (Han et al., 2013a,b).

Extensive evidence reveals that phytohormones are important in determining cat2 phenotypes.

Transcriptome analysis of cat2 roots points to the modulation of ethylene and auxin signaling [START_REF] Bueso | The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene[END_REF]. Another study showed that the decreased CAT activity in cat2 induced effects on ABA signaling [START_REF] Jannat | The roles of CATALASE2 in abscisic acid signaling in Arabidopsis guard cells[END_REF]. Glutathione was shown to be a key player in linking H2O2 to SA signaling (Han et al., 2013a). It was also reported that CAT2 participates in SA-mediated repression of auxin accumulation and JA biosynthesis during pathogen infection [START_REF] Yuan | CATALASE2 coordinates SA-mediated repression of both Auxin accumulation and JA biosynthesis in plant defenses[END_REF]. Another study reported that CAT2 and APX1 work in a coordinated way during the nuclear DNA damage response (DDR). Compared with the cat2 single mutant, the apx1 cat2 double mutant is more tolerant to oxidative stress imposed by high light, heat and paraquat application, and genome-wide 20 transcriptome analysis shows an activation of typical DDR hallmarks [START_REF] Vanderauwera | Extranuclear protection of chromosomal DNA from oxidative stress[END_REF].

Ascorbate and glutathione in ROS metabolism

Many compounds function as effective antioxidants in cells by regulating ROS accumulation. Among the best studied players are the cellular redox buffers, ascorbate and glutathione. They are well accepted to be key in controlling concentrations of ROS, although both metabolites are involved in multiple physiological processes [START_REF] Cobbett | The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in γ-glutamylcysteine synthetase[END_REF][START_REF] Vernoux | The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development[END_REF]Dowdle et al., 2007).

Ascorbate and glutathione are distinguished from most other antioxidant small molecules by (1) specific enzyme systems (peroxidases) that couple them to H2O2 metabolism; (2) the relative stability of their oxidized forms;

(3) recycling of oxidized forms to reduced compounds by high-capacity reductases and associated systems that depend on the key electron carrier, NAD(P)H. Based on these features, ascorbate and glutathione can effectively regulate cellular redox state by repeated redox cycling (Foyer and Noctor, 2011). The pools of these two metabolites are generally highly reduced (over 95%) within the cytosol, chloroplasts and mitochondria, while the oxidized forms accumulate in compartments lacking efficient redox-recycling mechanisms like the vacuole and the apoplast [START_REF] Schwarzländer | Confocal imaging of glutathione redox potential in living plant cells[END_REF][START_REF] Queval | Increased intracellular H2O2 availability preferentially drives glutathione accumulation in vacuoles and chloroplasts[END_REF][START_REF] Noctor | Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation[END_REF].

Glutathione in plants

Glutathione (γ-glutamylcysteinylglycine) is the principal low-molecular-weight thiol in most cells. It is an essential metabolite with multiple functions in plant development, biosynthetic pathways, detoxification, antioxidant biochemistry, and redox homeostasis. The fundamental function of glutathione is in thiol-disulfide interactions, in which the interconversion of reduced glutathione (GSH) and GSSG allows an appropriate cell redox state to be achieved. The concentration of cellular glutathione is high, as is its reduction state in the absence of stress. Under optimal physiological conditions the average ratio of GSH:GSSG in tissues such as leaves is at least 20:1 (Mhamdi et al., 2010a;Han et al., 2013a). However, this value may be higher (e.g. cytosol) or lower (e.g. vacuole) in specific subcellular compartments [START_REF] Meyer | Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the redox potential of the cellular glutathione redox buffer[END_REF][START_REF] Queval | Increased intracellular H2O2 availability preferentially drives glutathione accumulation in vacuoles and chloroplasts[END_REF].

CHAPTER 1 GENERAL INTRODUCTION 21

In plants glutathione is synthesized by two ATP-dependent steps which rely on the activity of γ-EC synthetase (γ-ECS) and glutathione synthetase (GSH-S) [START_REF] Rennenberg | Glutathione metabolism and possible biological roles in higher plants[END_REF][START_REF] Meister | Glutathione metabolism and its selective modification[END_REF][START_REF] Noctor | Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling[END_REF][START_REF] Mullineaux | Glutathione, photosynthesis and the redox regulation of stressresponsive gene expression[END_REF]. Each synthetic enzyme is encoded by a single gene [START_REF] May | Arabidopsis thaliana gamma-glutamylcysteine synthetase is structurally unrelated to mammalian, yeast, and Escherichia coli homologs[END_REF]Ullman et al., 1996), and both are indispensable to plant growth and development.

Knocking out GSH1, the gene encoding γ-ECS, leads to lethality at the embryo stage [START_REF] Cairns | Maturation of Arabidopsis seeds is dependent on glutathione biosynthesis within the embryo[END_REF] while knocking out GSH2, the gene encoding GSH-S, causes a seedling-lethal phenotype [START_REF] Pasternak | Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development[END_REF]. In Arabidopsis γ-ECS is located in plastids [START_REF] Wachter | Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae[END_REF], while GSH-S is found in both chloroplasts and cytosol, with the latter compartment considered to contain the major form of GSH-S because of the higher abundance of the corresponding transcript [START_REF] Wachter | Differential targeting of GSH1 and GSH2 is achieved by multiple transcription initiation: implications for the compartmentation of glutathione biosynthesis in the Brassicaceae[END_REF]. Glutathione synthesis, which responds to factors such as oxidative stress or heavy metals, can be modulated by regulating γ-ECS expression and activity, or by changing cysteine availability [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;[START_REF] Creissen | Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress[END_REF][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]. Both GSH1 and GSH2 transcripts respond to phytohormone and heavy metals [START_REF] Xiang | Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis[END_REF][START_REF] Sung | ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis[END_REF], as well as to light, drought and pathogens.

While glutathione synthesis is stress-responsive, degradation is also important to achieve appropriate levels. In Arabidopsis there are four different types of enzyme which have been implicated in glutathione degradation. These are phytochelatin synthase (PCS), γ-glutamyl transpeptidase (GGT), γglutamyl cyclotransferase (GGC), and 5-oxoprolinase (5-OPase). Most attention has thus far focused on GGT, which is considered to degrade mainly GSSG located in the vacuole or the apoplast [START_REF] Masi | Localization of gammaglutamyl transferase activity and protein in Zea mays organs and tissues[END_REF]Ferretti et al., 2009;[START_REF] Ohkama-Ohtsu | 12-Oxo-phytodienoic acid-glutathione conjugate is transported into the vacuole in Arabidopsis[END_REF][START_REF] Su | Glutathione-Indole-3-Acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana[END_REF].

Ascorbate in plants

Ascorbate, also known as vitamin C, is a highly abundant metabolite and a water-soluble antioxidant in both animals and plants. It occurs in a reduced form (ascorbic acid) and two oxidized forms, which are the immediate product of one-electron oxidation (monodehydroascorbate, or MDHA) and dehydroascorbate (DHA), which has lost two electrons compared to ascorbate and is largely the result of MDHA dismutation. Homeostasis between reduced and oxidized ascorbate is essential for plants to cope with environmental stress. Ascorbate is found in different subcellular compartments like chloroplasts, mitochondria, peroxisomes, and vacuoles [START_REF] Jiménez | Evidence for the presence of the ascorbateglutathione cycle in mitochondria and peroxisomes of pea leaves[END_REF][START_REF] Jiménez | Role of the ascorbateglutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves[END_REF][START_REF] Noctor | Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling[END_REF][START_REF] Takahama | Oxidation of vacuolar and apoplastic phenolic substrates by peroxidase: physiological significance of the oxidation reactions[END_REF][START_REF] Zechmann | Subcellular distribution of ascorbate in plants[END_REF]. It has been well documented that ascorbate can be synthesized through the L-galactose (L-Gal) pathway starting from glucose [START_REF] Linster | L-Ascorbate biosynthesis in higher plants : the role of VTC2[END_REF][START_REF] Wheeler | The biosynthetic pathway of vitamin C in higher plants[END_REF][START_REF] Wheeler | Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes[END_REF][START_REF] Bulley | The regulation of ascorbate biosynthesis[END_REF]. It was shown that vtc1 and vtc2 mutants, deficient in the enzymes associated with this pathway, contain only 25-30% and 10-20% of wild type ascorbate levels, respectively. Furthermore, the loss of ascorbate biosynthetic capacity in the vtc2 vtc5

mutant cannot be compensated by other identified ascorbate biosynthetic pathways (Dowdle et al., 2007). Hence, the L-Gal pathway is suggested to be the primary pathway that generates ascorbate in most plant organs. Nevertheless, alternative ascorbate biosynthetic pathways through galacturonate and glucuronate have also been proposed (Li et al., 2010). Ascorbate has been considered of great importance in plants with key functions in growth development, metabolism and stress responses (Noctor and Foyer, 1998;[START_REF] Smirnoff | Ascorbate biosynthesis and function in photoprotection[END_REF][START_REF] Pastori | Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling[END_REF][START_REF] Anjum | Metal/metalloid stress tolerance in plants: role of ascorbate, its redox couple, and associated enzymes[END_REF]. The finding that near-total ascorbate depletion in the vtc2 vtc5 mutant is associated with seedling lethality (Dowdle et al., 2007) suggests that, like glutathione, ascorbate is an indispensable player during at least some stages of plant development.

Ascorbate-glutathione pathway

While ascorbate and glutathione can react chemically with ROS, and also have other functions as enzyme co-factors and in defense, their roles in the ascorbate-glutathione pathway continue to be a major research focus. The pathway is involved in ROS metabolism in plant cells by removal of H2O2 [START_REF] Polle | Dissecting the superoxide dismutase-ascorbate-glutathione-pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis[END_REF][START_REF] Smirnoff | Vitamin C: The metabolism and functions of ascorbic acid in plants[END_REF][START_REF] Gest | Light-dependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol[END_REF][START_REF] Johnston | Monodehydroascorbate reductase mediates TNT toxicity in plants[END_REF][START_REF] Noctor | Lighting the fuse on toxic TNT[END_REF]. Consumption of GSH in this pathways leads to GSSG, which can be reduced back to GSH by the action of glutathione reductase (GR). This reaction needs electrons provided by NADPH. During this sequence of reactions, ascorbate and glutathione are not consumed: instead they are involved in a cyclic transfer of reducing equivalents by the action of four enzymes, which permits the reduction of H2O2 to H2O using electrons derived from NAD(P)H (Foyer and Halliwell, 1976;Noctor and Foyer, 1998;[START_REF] Donahue | Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications[END_REF]. Both ascorbate and glutathione are abundant and stable antioxidants which may be maintained at thermodynamic equilibrium with each other under optimal conditions. Within the highly reducing intracellular context, ROS accumulation can lead to the transient or sustained adjustment of this system, and such adjustments can be sensed by signaling pathways, leading to downstream responses.

For glutathione, there is a close correlation between its status and the expected intracellular ROS availability. For example, it has been extensively reported that pharmacologically or genetically blocking CAT activities causes glutathione to become both more oxidized and more abundant [START_REF] Smith | The regulation of the biosynthesis of glutathione in leaves of barley (Hordeum vulgare L.)[END_REF]May andLeaver 1993., Willekens et al., 1997;[START_REF] Queval | H2O2activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast[END_REF]Mhamdi et al., 2010a).

Work going back over two decades has pointed to novel outcomes of modulating parts of the ascorbate-glutathione pathway, suggesting that the pathway does not function simply as an antioxidative system but may also act in redox signaling. For example, cytosolic DHARs are involved in the regulation of glutathione status triggered when H2O2 metabolism shifts from CAT to reductive pathways. Genetically blocking these enzymes weakens, rather than reinforces, cell death and defense responses induced by CAT deficiency [START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF]. Another report showed that MDHAR can act as a pro-oxidant rather than an antioxidant by conferring sensitivity to soil-borne pollutant 2,4,6-trinitrotoluene (TNT) on Arabidopsis [START_REF] Johnston | Monodehydroascorbate reductase mediates TNT toxicity in plants[END_REF][START_REF] Noctor | Lighting the fuse on toxic TNT[END_REF].

One current view is that perturbation of the status of the ascorbate-glutathione pathway may be important in signaling, i.e., the pathway has a dual antioxidant-signaling role in which some downstream responses are triggered by increased H2O2 metabolism through the pathway, rather or in addition to increased H2O2 concentrations [START_REF] Foyer | Stress-triggered redox signalling: what's in pROSpect?[END_REF]. In such a scenario, glutathione status seems likely to be an influential player, based on possible interactions with protein thioldisulfide groups. In contrast to glutathione, the ratio of ascorbate to DHA is less likely to be involved in the transmission of ROS signals. Instead, ascorbate limits the lifetime of ROS signals by scavenging ROS chemically and enzymatically. Ascorbate contents are sensitive to factors such as irradiance [START_REF] Gatzek | Antisense suppression of l-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l-galactose synthesis[END_REF][START_REF] Smirnoff | Vitamin C: The metabolism and functions of ascorbic acid in plants[END_REF], and this response may be partly related to functions in tocopherol regeneration and xanthophyll cycle.

Other pathways of ROS processing

In addition to CATs and the ascorbate-glutathione pathway, plants also contain other antioxidative systems to cope with H2O2 accumulation. Among them, PRX has been well documented to function in maintaining appropriate H2O2 levels, at least in chloroplasts [START_REF] Awad | 2cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions[END_REF]. PRX is a thioldependent peroxidase existing in almost all organisms. Depending on the isoform, PRX regeneration from the oxidized form to the reduced state can be accomplished by glutaredoxin (GRX), thioredoxin (TRX), NADPH-thioredoxin reductase (NTR), glutathione or ascorbate [START_REF] Dietz | Plant peroxiredoxins[END_REF][START_REF] Rouhier | The plant multigenic family of thiol peroxidases[END_REF][START_REF] Meyer | The integration of glutathione homeostasis and redox signaling[END_REF][START_REF] Pérez-Ruiz | A proposed reaction mechanism for rice NADPH thioredoxin reductase C, an enzyme with protein disulfide reductase activity[END_REF][START_REF] Liebthal | Peroxiredoxins and redox signaling in plants[END_REF]. GPX is also able to remove H2O2 but these enzymes are likely to function with TRX instead of GSH [START_REF] Herbette | Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities[END_REF][START_REF] Iqbal | Hydroperoxide reduction by thioredoxin-specific glutathione peroxidase isoenzymes of Arabidopsis thaliana[END_REF]. GST could also play a role as GSH-dependent peroxidases to remove H2O2 [START_REF] Dixon | Enzyme activities and subcellular localization of members of the Arabidopsis glutathione transferase superfamily[END_REF], although their exact importance in this respect is still not clear.

All the enzymes discussed above are found in the aqueous cell phases. Within the membrane phase, carotenoids and tocopherols are well known as important compounds to control the accumulation of ROS and derived molecules. Carotenoids can act either to avoid ROS production (e.g. by quenching excited Chl states) or react with existing ROS by quenching singlet oxygen (Fischer et al., 2013). Most evidence available so far suggests that the functions of tocopherols (vitamin E) are more essential in seeds than in leaves. Tocopherol-deficient mutants of Arabidopsis show clear effects associated with enhanced lipid peroxidation and the activation of defense pathways during germination [START_REF] Sattler | Nonenzymatic lipid peroxidation reprograms gene expression and activates defense markers in Arabidopsis tocopherol-deficient mutants[END_REF].

NADPH-linked reaction in plants

NADPH is a key redox carrier in the aqueous phase of many cells and plays a crucial role in physiological processes like cell growth, proliferation, detoxification, redox homeostasis and biosynthesis. It is the reductant required for sugar-phosphate production in the photosynthetic Calvin-Benson cycle and is also required for other key anabolic pathways such as lipid and amino acid synthesis. Alongside its roles in biosynthetic pathways, NADPH functions in defence reactions and the regulation of cellular redox status. For example, in the ascorbate-glutathione pathway, NADPH is required for the reactions catalyzed by GR and MDHAR (Figure 1.5; [START_REF] Noctor | Metabolic signalling in defence and stress: the central roles of soluble redox couples[END_REF] while TRX needs NADPH to protect against oxidative damage [START_REF] Nordman | Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase[END_REF]. The functions of NADPH are not limited to providing electrons for biosynthetic reactions and antioxidative enzymes. Indeed, NADPH may have a pro-oxidant function, for example, in providing reductant for plasma membrane NADPH oxidase that produce suyperoxide and, subsequently, H2O2 (Foreman et al., 2003;[START_REF] Torres | Reactive oxygen species signaling in response to pathogens[END_REF].

NADPH is also a cofactor for the generation of nitric oxide (NO) from arginine, catalyzed by animaltype NO synthases [START_REF] Corpas | Evidence supporting the existence of Larginine-dependent nitric oxide synthase activity in plants[END_REF], although the existence of such enzymes in plants is controversial.

The regeneration of NADPH in plants is accomplished by several systems. The primary source in the chloroplast is ferredoxin-NADP reductase (FNR). This enzyme mediates electron transfer from ferredoxin to NADP + in a light-dependent manner [START_REF] Arakaki | Plant-type ferredoxin-NADP + reductases: a basal structural framework and a multiplicity of functions[END_REF] may also contribute to the production of NADPH (Figure 1.6; [START_REF] Donahue | Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications[END_REF]. While the primary function of FNR is considered to be in photosynthesis, the physiological roles of other NADPH-generating enzymes remain unclear. The following sections present a concise overview of some of these NADPH-producing systems in plants.

Glucose-6-phosphate dehydrogenase

G6PDH is the first and rate-limiting enzyme in the OPPP,which is a source of NADPH in nonphotosynthetic tissues as well as in leaves in the dark (Emes and Neuhaus, 1997;[START_REF] Thom | Sweet pepper plastids: enzymic equipment, characterisation of the plastidic oxidative pentose-phosphate pathway, and transport of phosphorylated intermediates across the envelope membrane[END_REF]. It catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconolactone accompanied by the conversion of NADP + to NADPH. The NADPH thus produced can subsequently be used for the various NADPH-requiring reactions discussed above. Many studies have shown that G6PDH is associated with various environmental stress responses. G6PDH activity can be stimulated by exogenous application of H2O2 and NaCl, as well as drought and pathogen attack [START_REF] Wang | Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress[END_REF][START_REF] Scharte | Isoenzyme replacement of glucose-6-phosphate dehydrogenase in the cytosol improves stress tolerance in plants[END_REF][START_REF] Li | Glucose-6-phosphate dehydrogenasedependent hydrogen peroxide production is involved in the regulation of plasma membrane H + -ATPase and Na + /H + antiporter protein in salt-stressed callus from Carex moorcroftii[END_REF][START_REF] Dal Santo | Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in Arabidopsis[END_REF][START_REF] Corpas | NADPH-generating dehydrogenases: their role in the mechanism of protection against nitro-oxidative stress induced by adverse environmental conditions[END_REF]. It has been reported that drought stress activated the expression of G6PDH as well as its activity [START_REF] Liu | Glucose-6-phosphate dehydrogenase plays a pivotal role in tolerance to drought stress in soybean roots[END_REF], and overexpression of G6PDH-encoding genes could enhance the resistance to drought stress [START_REF] Scharte | Isoenzyme replacement of glucose-6-phosphate dehydrogenase in the cytosol improves stress tolerance in plants[END_REF]. Although many of the studies have been correlative in nature, attention has focused on relationship between G6PDH and the ascorbate-glutathione pathway [START_REF] Wang | Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress[END_REF].

In plants, 6 G6PD genes have been identified. They encode four plastidic isoforms (G6PD1-4) and two cytosolic isoforms (G6PD5 and G6PD6). Moreover, the plastidic isoforms are separated into classes P1 and P2, which can be distinguished by specific antibodies, regulatory mechanisms, and expression patterns [START_REF] Wendt | Evidence for functional convergence of redox regulation in G6PDH isoforms of cyanobacteria and higher plants[END_REF][START_REF] Wakao | Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis[END_REF]. In Arabidopsis G6PD1 and G6PD2 may play the major roles in the plastid because T-DNA insertions in the G6PD3 gene do not affect the activity in zymograms and G6PD4 activity and mRNA abundance are very low. In terms of cytosolic isoforms, G6PD5 is highly expressed in leaves but at lower levels in other tissues. By contrast, G6PD6 is extensively expressed in all tissues. As well as expression, kinetic characteristics may determine the influence of each isoform. For instance, the KM NADP + of G6PD6 is considerably higher than that of G6PD5 [START_REF] Wakao | Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis[END_REF]. Seeds of a g6pd5 g6pd6 double mutant showed higher oil content and weight compared to the wild type, suggesting that deficiency of cytosolic G6PDH may modulate the metabolism of developing seeds [START_REF] Wakao | Functional analyses of cytosolic glucose-6-phosphate dehydrogenases and their contribution to seed oil accumulation in Arabidopsis[END_REF]. Moreover, cytosolic G6PDH-produced NADPH has been described as a factor that influences tobacco responses to pathogen attack, an effect the authors explained in terms of reductant for NADPH oxidases [START_REF] Scharte | Isoenzyme replacement of glucose-6-phosphate dehydrogenase in the cytosol improves stress tolerance in plants[END_REF]. It has also been reported that the activity of G6PDH can be stimulated by glycogen synthase kinase 3 (GSK3) during salt stress, leading to the alteration of redox status and favoring ROS metabolism in stress conditions [START_REF] Dal Santo | Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in Arabidopsis[END_REF].

Isocitrate dehydrogenase

ICDHs mediate the oxidative decarboxylation of isocitrate to 2-oxoglutarate (2-OG), accompanied by the production of NADPH and CO2. As a carbon skeleton, 2-OG is required for ammonia assimilation through the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway [START_REF] Hodges | Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation[END_REF]. In Arabidopsis there are three genes encoding different ICDH isoforms located in the cytosol, chloroplasts/mitochondria and peroxisomes. Among them cytosolic ICDH (cICDH) is the major isoform in leaves, accounting for more than 90% of the NADP + -dependent activity in leaf extracts [START_REF] Kruse | Antisense inhibition of cytosolic NADPdependent isocitrate dehydrogenase in transgenic potato plants[END_REF][START_REF] Hodges | Enzyme redundancy and the importance of 2-oxoglutarate in plant ammonium assimilation[END_REF]Mhamdi et al., 2010c). ICDH has been shown to provide NADPH for the promotion of redox signaling or homeostasis in response to oxidative stress [START_REF] Møller | The role of NADP in the mitochondrial matrix[END_REF][START_REF] Hodges | Higher plant NADP + -dependent isocitrate dehydrogenases, ammonium assimilation and NADPH production[END_REF]. Deficiency of cICDH in the cat2 mutant exacerbates glutathione oxidation and lesion formation, and also enhances PR transcripts and bacterial resistance, pointing to some role for cICDH in redox signaling linked to pathogenesis responses (Mhamdi et al., 2010c). Moreover, ICDH activity is found to be stimulated by O3 treatment in a day length-dependent manner [START_REF] Dghim | Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod-influenced responses to ozone using Arabidopsis knockout mutants[END_REF].

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase

npGAPDH mediates the irreversible oxidation of glyceraldehyde-3-phosphate (Ga3P) to 3phosphoglycerate (3-PGA), accompanied by the production of NADPH [START_REF] Arnon | A new glyceraldehyde phosphate dehydrogenase from photosynthetic tissues[END_REF][START_REF] Iglesias | On the metabolism of triose-phosphates in photosynthetic cells. Their involvement on the traffic of ATP and NADPH[END_REF]. This enzyme is a conserved cytosolic protein in higher plants [START_REF] Arnon | A new glyceraldehyde phosphate dehydrogenase from photosynthetic tissues[END_REF][START_REF] Mateos | Occurrence of phosphorylating and non-phosphorylating NADP +dependent glyceraldehyde-3-phosphate dehydrogenases in photosynthetic organisms[END_REF][START_REF] Gao | NADPH supply and mannitol biosynthesis. Characterization, cloning, and regulation of the non-reversible glyceraldehyde-3-phosphate dehydrogenase in celery leaves[END_REF], and is encoded by a single gene [START_REF] Bustos | Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is post-translationally phosphorylated in heterotrophic cells of wheat (Triticum aestivum)[END_REF][START_REF] Rius | Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase[END_REF]. It is also found in green algae and some specialized bacteria [START_REF] Mateos | Occurrence of phosphorylating and non-phosphorylating NADP +dependent glyceraldehyde-3-phosphate dehydrogenases in photosynthetic organisms[END_REF][START_REF] Brunner | Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase from Thermoproteus tenax[END_REF][START_REF] Iddar | Widespread occurrence of nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase among gram-positive bacteria[END_REF]. Its function provides an alternative route for Ga3P oxidation in glycolysis, as a bypass to oxidation by NAD-dependent GAPDH and 3-PGA kinase [START_REF] Iglesias | On the metabolism of triose-phosphates in photosynthetic cells. Their involvement on the traffic of ATP and NADPH[END_REF][START_REF] Plaxton | The organization and regulation of plant glycolysis[END_REF].

CHAPTER 1 GENERAL INTRODUCTION 29

The function of npGAPDH in plants remains unclear. In photosynthetic cells, it was suggested to be involved in a shuttle system which exports photosynthetic NADPH from the chloroplast to the cytosol [START_REF] Kelly | A mechanism for the indirect transfer of photosynthetically reduced nicotinamide adenine dinucleotide phosphate from chloroplasts to the cytoplasm[END_REF]. Reduction of sugars to polyols in plants may also require NADPH derived from npGAPDH [START_REF] Rumpho | A pathway for photosynthetic carbon flow to mannitol in celery leaves : activity and localization of key enzymes[END_REF][START_REF] Gao | NADPH supply and mannitol biosynthesis. Characterization, cloning, and regulation of the non-reversible glyceraldehyde-3-phosphate dehydrogenase in celery leaves[END_REF]. In non-photosynthetic cells, npGAPDH was found to be a target for posttranslational phosphorylation [START_REF] Bustos | Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase is post-translationally phosphorylated in heterotrophic cells of wheat (Triticum aestivum)[END_REF].

It can be phosphorylated by the interaction with 14-3-3 proteins in heterotrophic tissues. This leads to a lower activity and enhanced sensitivity to regulation by adenylates and inorganic pyrophosphate [START_REF] Bustos | Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from heterotrophic cells of wheat interacts with 14-3-3 proteins[END_REF]. It was also recently reported that npGAPDH influences PR responses triggered by hCO2 [START_REF] Mhamdi | High CO2 primes plant biotic stress defences through redox-linked pathways[END_REF].

NADP-malic enzyme

NADP-malic enzyme (NADP-ME) mediates the reversible oxidative decarboxylation of malate to produce pyruvate and CO2, simultaneously reducing NADP + to NADPH [START_REF] Chang | Structure and function of malic enzymes, a new class of oxidative decarboxylases[END_REF]. Its substrate malate is a crucial tricarboxylic acid cycle intermediate and plays different roles in plant physiological processes, like defense responses, stomatal movement and metabolite signaling [START_REF] Lee | The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2[END_REF][START_REF] Parker | Metabolomic analysis reveals a common pattern of metabolic re-programming during invasion of three host plant species by Magnaporthe grisea[END_REF]Finkemeier et al., 2013). One of the products of the NADP-ME reaction, pyruvate, is involved in multiple pathways. It is notably the final product of glycolysis, where it is produced from phosphoenolpyruvate (PEP) in the cytosol, before being translocated to the mitochondria for metabolism through the TCA cycle. Apart from oxidative decarboxylation, NADP-ME can also catalyze the reductive carboxylation of pyruvate and the decarboxylation of oxaloacetate [START_REF] Wheeler | Arabidopsis thaliana NADP-malic enzyme isoforms: high degree of identity but clearly distinct properties[END_REF].

NADP-ME is involved in many kinds of biological processes. It not only takes part in the photosynthetic pathway in some C4 and CAM species, but also functions as a non-photosynthetic isoforms in many plants (Drincovich et al., 2001). To date four NADP-ME genes encoding specific isoforms have been identified in Arabidopsis. Among them NADP-ME 1-3 are localized in the cytosol and NADP-ME 4 is plastidic [START_REF] Wheeler | A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis[END_REF]. These isoforms exhibit different kinetic, regulatory and structural properties [START_REF] Wheeler | A comprehensive analysis of the NADP-malic enzyme gene family of Arabidopsis[END_REF], 2008;[START_REF] Maurino | Redundancy is sometimes seen only by the uncritical: does Arabidopsis need six malic enzyme isoforms?[END_REF]. Genetic analysis indicates that NADP-ME2 is responsible for most of the activity analyzed in mature plant tissues. Recent studies show that the loss of function of NADP-ME2 alters metabolic profiles [START_REF] Brown | C4 acid decarboxylases required for C4 photosynthesis are active in the mid-vein of the C3 species Arabidopsis thaliana, and are important in sugar and amino acid metabolism[END_REF][START_REF] Voll | Loss of cytosolic NADP-malic enzyme 2 in Arabidopsis thaliana is associated with enhanced susceptibility to Colletotrichum higginsianum[END_REF]. This enzyme is activated in response to various environmental challenges [START_REF] Voll | Loss of cytosolic NADP-malic enzyme 2 in Arabidopsis thaliana is associated with enhanced susceptibility to Colletotrichum higginsianum[END_REF][START_REF] Li | Analysis of knockout mutants suggests that Arabidopsis NADP-MALIC ENZYME2 does not play an essential role in responses to oxidative stress of intracellular or extracellular origin[END_REF]. The overexpression of a cytosolic NADP-ME of rice in Arabidopsis enhances the tolerance to osmotic and salt stress [START_REF] Cheng | A cytosolic NADP-malic enzyme gene from rice (Oryza sativa L.) confers salt tolerance in transgenic Arabidopsis[END_REF][START_REF] Liu | Expression of an NADP-malic enzyme gene in rice (Oryza sativa L) is induced by environmental stresses; overexpression of the gene in Arabidopsis confers salt and osmotic stress tolerance[END_REF].

Lesion-mimic mutants in plants

One outcome of excess ROS availability is induced cell death. Current concepts suggest that ROSdependent cell death is not just due to cell collapse caused by damage but rather is under genetic control. Indeed, components required for ROS-induced cell death in plants (EXECUTOR proteins) have been identified by genetic studies [START_REF] Wagner | The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana[END_REF]. As described in detail above, the extensive studies of cat2 reveal that loss of this antioxidative enzyme leads to an HR-like phenotype in which plants present lesions on the leaves linked to SA accumulation. Many other mutations have been described that lead to similar, spontaneous lesion formation. To date, more than 40 lesion-mimic mutants have been identified in Arabidopsis [START_REF] Bruggeman | To die or not to die? Lessons from lesion mimic mutants[END_REF]. The appearance of lesions in lesion-mimic mutants is often accompanied by phenomena associated with pathogen attack, like SA induction, defense marker gene expression, and altered resistance to biotic challenge [START_REF] Lorrain | Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants?[END_REF].

The first lesion-mimic mutant described was lsd1 [START_REF] Dietrich | Arabidopsis mutants simulating disease resistance response[END_REF]. This mutant presents lesions on the leaves in a day length-dependent manner. When plants are grown in LD or transferred from SD to LD, lesions form and gradually spread until consuming the entire leaf. LSD1 codes for a zinc finger protein which may act as a negative regulator of a pro-death signal. By binding to the transcription factor AtbZIP10, LSD1 stops it from entering the nucleus and thereby suppresses cell death [START_REF] Dietrich | A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death[END_REF][START_REF] Kaminaka | bZIP10-LSD1 antagonism modulates basal defense and cell death in Arabidopsis following infection[END_REF]. A second mutant that shows a similar phenotype is mips1, a mutant defective in the synthesis of myo-Inositol [START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF]. This mutant exhibits spontaneous cell death which is affected by both the amount of light and day length. Studies strongly suggest that myo-Inositol plays important roles in regulating cell death formation and this regulation is closely linked to phytohormones like SA [START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF]Donahue et al., 2010).

Arabidopsis: a model to aid quick progress in understanding plant function

Arabidopsis has been well accepted as the most important model plant system by the scientific community. It has a relatively small size, as well as a small genome, compared with other plant species. Since the sequencing of its whole genome in 2000, lots of genetic tools have become available. Nowadays both scientific information and biological materials are easily accessible through various databases and biological stock centers, and it is relatively easy to produce genetic resources.

Indeed, Arabidopsis is an excellent model for both classical (forward) genetics studies and analyses using reverse genetics. These two approaches can both be powerful and are distinguished as follows.

Forward genetics aims to identify the sequence change that underlies a specific mutant phenotype, by which we can find genetic determinants of a specific physiological process or a signaling pathway. A big advantage of forward genetics is that this approach does not depend on prior assumptions. Instead, all the genes are considered, in an unbiased fashion, to find mutations that affect the phenotype of interest. Chemical mutagenesis by agents such as ethyl methanesulfonate (EMS) are most popular in forward genetics, although screens can also be conducted on other collections, such as T-DNA insertion mutants [START_REF] Page | The art and design of genetic screens: Arabidopsis thaliana[END_REF][START_REF] Peters | Forward genetics and map-based cloning approaches[END_REF][START_REF] Qu | Generation and identification of Arabidopsis EMS mutants[END_REF]. Reverse genetics, in contrast to forward genetics, begins with a clearly identified gene of interest. Plants carrying a mutation in this gene are then analyzed to determine if it is associated with any interesting phenotype, with the advantage relative to forward genetics that the phenotypic analysis can be more [START_REF] Mccallum | Targeted screening for induced mutations[END_REF][START_REF] Greene | Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis[END_REF]

Aims of the project

The overall objectives of this thesis were to continue the group's work on enzyme systems involved in metabolizing H2O2 in plants, with a particular focus on CATs and enzymes involved in or related to the ascorbate-glutathione pathway. Most available functional analysis of CAT has been focused on CAT2 functions in leaves. As the major leaf isoform, CAT2 accounts for most of the extractable CAT activity

and it has been demonstrated to play an important role in oxidative stress signaling. When grown in LD, but not SD, cat2 shows spreading necrotic lesions, SA accumulation, PR gene induction, accumulation of camalexin and its synthesis pathway, and enhanced resistance to bacterial challenge [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]. Although the SA pathway and related genes are only strongly induced in LD, many other genes, including numerous oxidative stress marker genes, are induced more strongly in SD [START_REF] Queval | Daylength is a key regulator of transcriptomic responses to both CO2 and H2O2 in Arabidopsis[END_REF]. Irrespective of the growth day length, all of the stress responses triggered by loss of CAT2 function can be prevented by growth at hCO2 or low light (Queval et al., 2007). These characteristics make cat2 a useful stress mimic tool to investigate the importance of components involved in oxidative signaling. One issue is whether the influence of day length on oxidative signaling is related to differences in stress intensity or not. A second issue bears on the specific functions of CAT1 or CAT3, for which there is as yet little information. To explore these questions, single and double CAT mutants were investigated and the results obtained are presented in Chapter 2.

As described above, NADPH is a crucial component in cell redox homeostasis. The production of NADPH relies on several different enzymes. G6PDH is one of the major NADPH-producing enzymes that could play important roles in oxidative stress conditions, when the enzyme would traditionally be considered likely to play an antioxidative role. However, according to unpublished work in our group, a knockout for a specific isoform (G6PD5) was found to abolish, rather than exacerbate, the cat2 lesion phenotype. Moreover, the abolition of cat2-induced lesions by G6PD5 deficiency appears to be linked to inhibition of cat2-triggered activation of the SA pathway. To investigate the processes that might be involved, we carried out a forward genetic screen on cat2 g6pd5, based on identifying mutants that allow lesions to be observed in this genetic background. This work is described in Chapter 3.

One of the pathways for which NADPH is required is the ascorbate-glutathione pathway, which becomes particularly important in stress conditions. In total, four enzymes are involved in the ascorbate-glutathione pathway. Unlike the other three (APX, DHAR, GR), little genetically based information is available on the role of specific MDHAR isoforms. As discussed above, ascorbate can be regenerated from MDHA or DHA by enzymatic and non-enzymatic processes, some of which require GSH and/or NADPH. Recent work in our group showed that the loss of all three DHAR functions does not affect leaf ascorbate pools in either optimal growth conditions or oxidative stress [START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF]. The ability to maintain a largely reduced leaf ascorbate pool when DHAR activity is negligible may be explained by other routes, such as MDHAR. To explore the roles of specific isoforms of MDHAR in the ascorbate-glutathione pathway, mutants for three MDHARencoding genes were obtained and analyzed in both Col-0 and cat2 oxidative stress backgrounds. The functional analysis of these lines is presented in Chapter 4.

CHAPTER 2 The function of specific catalases in plants

A modified version of this chapter has been submitted for publication by the following authors:

Yang Z, Mhamdi A and Noctor G. Analysis of single and double catalase mutants in Arabidopsis underscores the essential role of CATALASE2 for plant growth and day length-dependent oxidative signalling in leaves.

Summary

Three genes encode CAT in Arabidopsis. While the role of CAT2 in photorespiration is well established, the importance of the different CATs in other processes is less clear. In this work, we examined CAT functions by using T-DNA mutants for cat1, cat2, cat3, as well as cat1 cat2 and cat2 cat3 double mutants. In roots and in leaves, the cat2 mutants showed the largest effect on activity. Like rosette growth, root growth was inhibited in all cat2-containing lines. Root growth inhibition was prevented by growth at hCO2, suggesting that it is an indirect effect of oxidative stress in the leaves.

Marked phenotypes were not identified in single cat1 or cat3 mutants, but analysis of double mutants suggested some overlap between CAT2 and CAT3 in leaves and CAT1 and CAT2 in seeds. The response of H2O2 marker genes to equal-time exposure of cat2 to oxidative stress was highly influenced by growth day length context. Similar responses in cat2 and cat2 cat3 suggest that this is not related to day length effects on CAT3 expression. The non-specific CAT inhibitor, 3-aminotriazole (3-AT), produced qualitatively similar day length-dependent effects to those observed in cat2.

Together, our data (1) underline the importance of CAT2 in Arabidopsis;

(2) suggest that CAT1 and CAT3 are mainly "back-up" enzymes; and (3) establish that day length-dependent responses to CAT deficiency are independent of the duration of oxidative stress.

Introduction

Among the many enzymes that plants may use to remove H2O2, CAT is the most rapid and is strongly expressed [START_REF] Tuzet | Analyzing the function of catalase and the ascorbateglutathione pathway in H2O2 processing: Insights from an experimentally constrained kinetic model[END_REF]. Accordingly, under standard assay conditions, CAT shows a very high extractable activity [START_REF] Noctor | Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation[END_REF]. Enzyme capacity is particularly high in photosynthetic tissues but activities are also considerable in other plant parts [START_REF] Mhamdi | Plant catalases: peroxisomal redox guardians[END_REF]. Most plants so far examined contain three genes encoding CAT [START_REF] Frugoli | Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh[END_REF][START_REF] Dat | Catalase-deficient tobacco plants: tools for in planta studies on the role of hydrogen peroxide[END_REF]Mhamdi et al., 2010a). One of these (CAT2 in Arabidopsis) is essential for optimal plant growth in C3 plants growing in air, but the roles of the other two (CAT1 and CAT3 in Arabidopsis) remain much less clear.

The expression patterns of the Arabidopsis CATs suggested that CAT2 encodes the photorespiratory CAT [START_REF] Frugoli | Catalase is encoded by a multigene family in Arabidopsis thaliana (L.) Heynh[END_REF] that genetic studies in barley and tobacco showed to be important in leaves [START_REF] Kendall | The isolation and characterisation of a catalase-deficient mutant of barley (Hordeum vulgare L.)[END_REF][START_REF] Willekens | Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants[END_REF]. An RNAi construct based on the CAT2 sequence produced Arabidopsis plants that were smaller, more sensitive to high light, and that showed induction of oxidative stress-responsive genes [START_REF] Vandenabeele | Catalase deficiency drastically affects gene expression induced by high light in Arabidopsis thaliana[END_REF][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]. Analysis of gene-specific T-DNA knockouts confirmed that CAT2 is responsible for a large part of the leaf activity in Arabidopsis and identified the encoded enzyme as an important player in photorespiration (Queval et al., 2007).

While the importance of CAT2 in photorespiration is clear, less is known about other possible functions of this gene. In addition to loss of most of the leaf CAT activity, the cat2 mutant was previously reported to produce a 50% decrease in root activity [START_REF] Bueso | The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene[END_REF]. The mutant also shows decreased post-germinative growth that, like phenotypes observed in mature leaves, was linked to auxin signaling [START_REF] Gao | Mutation of Arabidopsis CATALASE2 results in hyponastic leaves by changes of auxin levels[END_REF][START_REF] Liu | CATALASE2 functions for seedling post-germinative growth by scavenging H2O2 and stimulating ACX2/3 activity in Arabidopsis[END_REF]. Auxin is one of a number of phytohormones whose contents or signaling pathways are modified by the oxidative stress caused by loss of CAT2 function [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]Mhamdi et al., 2010b;[START_REF] Tognetti | Perturbation of indole-3-butyric acid homeostasis by the UDP-glucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance[END_REF]Han et al., 2013a, b).

Interestingly, the effect of cat2-induced oxidative stress in leaves has been linked to day length. Loss of CAT2 function causes lesions when plants are grown in LD but not in SD, and this effect seems to be under genetic control (Queval et al., 2007;[START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF][START_REF] Li | The protein phosphatase subunit PP2A-B'γ is required to suppress day length-dependent pathogenesis responses triggered by intracellular oxidative stress[END_REF]. Transcriptomes elicited by the cat2 mutation are also different in LD compared to SD (Queval et al., 2007[START_REF] Queval | Daylength is a key regulator of transcriptomic responses to both CO2 and H2O2 in Arabidopsis[END_REF], but the significance of this observation with respect to photoperiod-dependent signaling is potentially obscured by the differences in the duration of exposure to photorespiration (and, therefore, oxidative stress).

Available evidence does not suggest that there is a great difference in the biochemical properties between the three CATs. Gene sequences have high similarity and promoter-swap studies revealed that CAT3 under the control of the CAT2 promoter, but not CAT2 under the control of the CAT1 or CAT3

promoter, can fully rescue cat2 phenotypes [START_REF] Hu | Functional comparison of catalase genes in the elimination of photorespiratory H2O2 using promoter-and 3'-untranslated region exchange experiments in the Arabidopsis cat2 photorespiratory mutant[END_REF]. This suggests that any specificity of function may be largely controlled by differences in expression. For example, CAT3 protein increases relative to CAT2 as leaves age [START_REF] Zimmermann | Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh[END_REF] and CAT1 expression is closely associated with reproductive tissues (Mhamdi et al., 2010a). However, the roles of CAT1 and CAT3 remain largely unknown and, while mutants for these genes have been briefly described (Mhamdi et al., 2010a), they have not been the focus of any in-depth study.

Since the CAT2 gene is located on a different chromosome from the CAT1 and CAT3 genes, double mutants for cat1 cat2 and cat2 cat3 are relatively straightforward to obtain. In contrast, it is difficult to obtain the cat1 cat3 combination by simple crossing because the two genes are located adjacently on chromosome 1. A very recent study using CRISPR technology reported a triple loss-of-function line but the plants were not analyzed in depth [START_REF] Su | The Arabidopsis catalase triple mutant reveals important roles of catalases and peroxisome derived signaling in plant development[END_REF]. In the present study, we used gene-specific mutants to analyze the roles of the three CAT genes in Arabidopsis. Our aims were (1) to examine potential roles for CAT1 and CAT3 in plant growth and development;

(2) to assess the importance of CAT2 in organs other than leaves;

(3) to establish whether day length-dependent oxidative signaling in cat2 leaves is independent of stress duration and whether it is influenced by CAT3.

Material and methods

Plant material

The T-DNA CAT mutants were obtained from Nottingham Arabidopsis Stock Centre (NASC), Nottingham University, UK. While cat2 and cat3 are from the SALK collection and are in the Col-0 background, cat1 is in the Col-3 background. The polymorphisms were SAIL_311_d07 for cat1, SALK_076998 for cat2 (cat2-1, [START_REF] Queval | H2O2activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast[END_REF], and SALK_092911 for cat3. Genotyping was performed by PCR using the primers listed in Supporting Information Table S1. Double mutants were generated by crossing cat2 with cat1 or cat3. After verification that mutations were heterozygous in F1 generation by PCR, double homozygotes were identified in F2 by the same way. F3 seeds were used for experiments.

Growth conditions and sampling

For all experiments, plants were grown at a temperature of 20°C (day)/18°C (night) at an irradiance of 200 µmol.m -2 s -1 at the leaf surface (16h light: 8h dark). For analysis of effects on roots, plants were grown on 0.5 Murashige-Skoog medium for up to two weeks in the presence or absence of added sucrose (1%), either in air (400 µL.L -1 ) or 3000 µL.L -1 CO2 (hCO2), which was used to decrease photorespiration to very low levels.

For experiments investigating processes in leaves or rosettes, plants were grown in soil in a controlled environment growth chamber and given nutrient solution twice a week, as in Queval et al. (2007). In these conditions, the day length was either 8h (SD) or 16h (LD) and the CO2 concentration either air or hCO2. Unless otherwise stated, plants were grown to the age of 19-21d (LD) or 26-28d (SD), at which point samples were taken. In experiments to analyze the effect of day length on oxidative stress marker gene expression, plants were grown at hCO2 in either short or LD and then transferred to air for 8h, and samples were taken before and after the transfer. All samples were rapidly frozen in liquid nitrogen and stored at -80°C.

CHAPTER 2 39

Enzyme activities

Enzyme assays were performed as described in detail in [START_REF] Noctor | Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation[END_REF]. About 100 mg root or shoot tissue was ground in liquid nitrogen then extracted into phosphate buffer (pH 7.5), 1 mM EDTA.

Centrifuged extracts were desalted on a NAP-5 column, and immediately used for spectrophotometric assay. CAT was measured as the disappearance of H2O2 at 240 nm, APX as the H2O2-dependent disappearance of ascorbate at 290 nm, DHAR as the GSH-and extract-dependent formation of ascorbate at 265 nm, and GR as the GSSG-dependent oxidation of NADPH at 340 nm.

Measurements of transcript abundance

Total RNA was extracted with Trizol following the manufacturer's protocols, and qPCR procedures were performed as in Queval et al. (2007), except that transcripts were normalized to two independent constitutive genes, which were ACTIN2 and RCE1. Gene-specific primer sequences used in this study are listed in Supporting Information Table S1.

Ascorbate, glutathione and ROS assays

Ascorbate and glutathione were measured by plate-reader spectrophotometry following the procedures described by Queval and Noctor (2007). ROS visualization was performed by dichlorodihydrofluorescein-diacetate (DCFH2-DA) staining of the primary roots of ten individual plants of each genotype in each condition, using a method adapted from Mhamdi et al. (2010b). 10 day-old seedlings were incubated in 50 µM staining solution at room temperature for 20 min in dark.

Then seedlings were washed three times with phosphate buffer for 5 min in dark. Fluorescence was observed with a Zeiss Axio Imager 2 fluorescence microscope.

Results

CAT expression and activity in roots

We first analyzed transcript levels of all three CATs in the roots of seedlings grown in the presence or absence of added sucrose (Figure 2.1A). The cat1, cat2 and cat3 mutations decreased respective transcripts to low levels, although low transcript signals and variability in roots meant the differences were not always significant relative to the wild-type. Both CAT2 and CAT3 tended to be higher in plants grown on sucrose, with a significant increase in CAT2 in cat3 and in CAT3 in cat2 (P < 0.05).

Compensatory changes in the expression of other CATs were also observed for some of the mutants in both growth conditions (Figure 2.1A). CAT1 transcripts were increased in the two cat3 backgrounds while CAT3 transcripts were higher in cat2 backgrounds other than cat2 cat3, although the increase in CAT3 was not significant in cat1 cat2 grown with added sucrose.

Consistent with previous reports for activities measured in leaves (Mhamdi et al., 2010a), the cat1 mutant showed wild-type levels of CAT activity (Figure 2.1 B). This was observed when cat1 was compared to either Col-0 or Col-3, which showed very similar root CAT activities (Supporting Information Figure. S1). As in leaves (Queval et al., 2007), loss of CAT2 function caused a substantial decrease in root activity (Figure 2.1B). Like cat1, the cat3 mutation alone had no significant effect on activity. However, in cat2 cat3 grown without added sucrose, the activity was decreased compared to cat2, although this effect was not apparent when plants were grown on sucrose (Figure 2.1B).

Root and seed phenotypes in CAT mutants

Growth analysis revealed that cat2 had a substantial impact on root length in plants grown without sucrose in air (Figure 2.2A, left). A slight effect of cat3 was observed in the cat2 cat3 double mutant.

The cat1 mutant showed no decrease in root length ( To explore root responses to increased ROS signals in the mutants, we analyzed APX activity and APX1 transcripts, as well as two markers for oxidative stress. All these factors have been well

documented to be strongly induced in cat2 leaves (Queval et al., 2007;Mhamdi et al., 2010a,b;[START_REF] Noctor | Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation[END_REF] shown).

To investigate the functional impact of the three CATs in reproductive tissues, seed number and seed formation were analyzed in the different genotypes. A slight but significant decrease in the number of seeds per silique was observed in cat1 and cat3, and this was slightly stronger when these mutations were combined with cat2 (Supporting Information Figure . S4, top). The CAT mutations also had some impact on the number of aborted seeds per silique: averaged over at least 100 siliques, the % aborted seeds rose from below 1% in the wild-type to over 4% in cat1 cat2, with slight but significant increases also observed in cat2 and cat3 (Supporting Information Figure . S4, bottom).

CAT2 and CAT3 function in leaves

As shown above, CAT2 is essential for optimal root growth, adding to observations that expression of this gene is required for rosette development (Queval et al., 2007;[START_REF] Gao | Mutation of Arabidopsis CATALASE2 results in hyponastic leaves by changes of auxin levels[END_REF]Mhamdi et al., 2010a). CAT1 does not seem to make a significant contribution to leaf CAT activity, with CAT3 responsible for most of the CAT2-independent activity (Mhamdi et al., 2010a). We therefore focused on the respective roles of CAT2 and CAT3 in ensuring optimal rosette growth. When plants were grown in air, where photorespiration is relatively rapid, cat3 rosettes were similar to the wild-type, in contrast to cat2, in which the rosette was much smaller (Figure 2.4, right). The additional presence of the cat3 mutation in cat2 cat3 did not exacerbate the effect of cat2. At hCO2, both cat2 and cat3

showed wild-type rosette characteristics, but a significant decrease in rosette size and mass was observed in cat2 cat3 (Figure 2.4, left). Further, the cat2 cat3 mutation increased time to flowering significantly compared to both Col-0 and cat2 in LD (Supporting Information Figure . S5).

The effects on rosette size were specific to growth in hCO2 in LD: all three mutant lines showed wildtype rosette size when grown at hCO2 in SD (Supporting Information Figure . S6). The day lengthdependent effect was not obviously correlated to differences in CAT activity, which was affected similarly by mutations in both growth conditions. Leaf activity was decreased by about 75% in cat2, 15-25% in cat3 and about 90% in cat2 cat3 (Figure 2.5). When measured in plants growing at hCO2, other antioxidative enzymes showed either no effect of the CAT mutations or day length (GR, DHAR)

or slight increases linked to the cat2 mutation (APX1), although this was only statistically significant in cat2 cat3 (Supporting Information Figure. S7).

The above observations in plants grown at hCO2 point to some photorespiration-independent function of CAT3 that becomes apparent when CAT2 function is lost. Moreover, it suggests that such an effect may be conditional on growth day length. We have previously described an interaction between oxidative signaling in cat2 and growth day length, in that both cat2 phenotypes and transcriptomes are markedly different in LD and SD (Queval et al., 2007[START_REF] Queval | Daylength is a key regulator of transcriptomic responses to both CO2 and H2O2 in Arabidopsis[END_REF][START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]Mhamdi et al., In all cases, CAT activities at the end of the experiment were similar in the two day length conditions (Figure 2.6B). Exposure to air for several days after growth at hCO2 increases CAT2-dependent CAT activity (Queval et al., 2007), but such effects were minor in the shorter-term (8h) exposure used here Despite this stability of key members of core antioxidative systems over the course of the experiment, 8h exposure of cat2 to air was sufficient to strongly induce two oxidative stress marker genes (Figure 2.7A, middle panels). Strikingly, however, induction of both genes was strongly dependent on the day length in which the plants had been growing prior to transfer to air. Induction of GSTU24 in LD plants was modest while UGT73b3 was not induced in cat2 (Figure 2.7A). By contrast, in SD plants, GSTU24 and UGT73b3 were induced about 100-fold and 10-fold respectively (Figure 2.7B, note the log scale). Equally strikingly, the influence of day length was also apparent for 3-AT treatment, with both genes being much more strongly induced after treatment of SD-grown plants than LD-grown plants. It is also noteworthy that 3-AT treatment of the wild-type produced an effect that was about 10 times stronger than the cat2 mutation for both genes (Figure 2.7).

In a second experiment, we included cat3 and cat2 cat3 genotypes. Similar effects of growth day length were apparent for cat2 and the 3-AT treatment, although this time 3-AT induced the marker genes only about 2-3 times more strongly than cat2 in SD (Figure 2.8). The single cat3 mutation did not cause a significant induction, while induction in cat2 cat3 showed a similar pattern to that observed in cat2 (Figure 2.8). Finally, we examined whether the stronger gene induction in SD plants was linked to photorespiration by analyzing effects in plants kept at hCO2 for the 8h test period. Under these conditions, none of the mutant lines showed a marked induction of the two genes (Figure 2.9). Both genes showed some induction by 3-AT treatment, although less than in plants exposed to air (cf. Figures 2.7 and 2.8). In contrast with the effects of the cat2 mutation and 3-AT treatment when plants were exposed to air, there was no evidence that 3-AT treatment in hCO2 induced the two genes more strongly in plants grown in SD.

Discussion

Our aim in this study was to conduct an in-depth comparative analysis of the roles of the three CATs in growth and development of Arabidopsis. To this end, we exploited a cat2 mutant line that has been well-studied at the leaf level (Queval et al., 2007) and cat1 and cat3 mutants that have been previously reported but which have not been investigated in detail (Mhamdi et al., 2010a). Below, we discuss the major issues coming out of this work.

Decreased root growth is specific to cat2 and is a secondary effect

It was reported previously that the cat2 mutation causes a decrease of about 80% in extractable leaf CAT activity in leaves and about 50% in roots [START_REF] Bueso | The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene[END_REF]Queval et al., 2007). Our present data are largely consistent with these earlier reports, although we found a greater decrease in roots than did [START_REF] Bueso | The lithium tolerance of the Arabidopsis cat2 mutant reveals a cross-talk between oxidative stress and ethylene[END_REF]. This is possibly due to differences in root age (three weeks in the earlier study compared to two weeks here). The remaining root activity when CAT2 function is lost appears to be attributable to CAT3, since when plants were grown without sucrose, the cat2 cat3 double mutant had very low activity whereas cat1 cat2 had similar activity to cat2 (Figure 2.1). Interestingly, when sucrose was included in the medium, cat2 cat3 and cat2 activities were similar. This suggests possible induction of CAT1 by sucrose, although we note that CAT1 transcripts were similar in plants grown in the presence and absence of sucrose.

Analysis of leaf responses in cat2 has established that this mutation causes alterations in leaf redox state, reconfiguration of transcriptomes, and induction of enzymes of the ascorbate-glutathione Col-0

LD SD cat2 LD SD Col-0 + 3AT LD SD cat3 LD SD cat2 cat3 LD SD GSTU24 UGT73b3
CHAPTER 2 53 pathway (Queval et al., 2007[START_REF] Queval | H2O2activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast[END_REF]Mhamdi et al., 2010b;[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]. By contrast, increased ROS signals are not so easy to detect (Mhamdi et al., 2010b, Chaouch et al., 2012), possibly due to technical difficulties or due to rapid removal of H2O2 by alternative enzymes [START_REF] Noctor | Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation[END_REF][START_REF] Tuzet | Analyzing the function of catalase and the ascorbateglutathione pathway in H2O2 processing: Insights from an experimentally constrained kinetic model[END_REF]. ROS analysis with the DCFH2-DA stain, which is not easy to use in leaves, revealed a clear increase in root signals in cat2 genotypes and, to a lesser extent, in cat3. More intense ROS signals were accompanied by decreased root growth in all cat2 genotypes grown in the absence of sugar (Figure 2.2).

A recent analysis of 5-day old seedlings also described decreased root growth in cat2, an effect that was completely annulled by growth on sucrose [START_REF] Liu | CATALASE2 functions for seedling post-germinative growth by scavenging H2O2 and stimulating ACX2/3 activity in Arabidopsis[END_REF]. While we also found that the cat2dependent inhibition was sensitive to sucrose, this effect was only partial (Figure 2.2). Moreover, [START_REF] Liu | CATALASE2 functions for seedling post-germinative growth by scavenging H2O2 and stimulating ACX2/3 activity in Arabidopsis[END_REF] reported that sucrose decreased DAB staining in cotyledons, but we found little evidence that root ROS signals were decreased when root growth was favored. This is in line with a changing perception of ROS. Long considered to be potentially damaging molecules, their positive role in plant growth is now being increasingly recognized (Foyer & Noctor, 2005;[START_REF] Dietz | Recent progress in understanding the role of reactive oxygen species in plant cell signaling[END_REF][START_REF] Foyer | Viewing oxidative stress through the lens of oxidative signalling rather than damage[END_REF][START_REF] Mittler | ROS are good[END_REF]. [START_REF] Liu | CATALASE2 functions for seedling post-germinative growth by scavenging H2O2 and stimulating ACX2/3 activity in Arabidopsis[END_REF] reported that CAT activity was decreased by growing wild-type seedlings on sucrose, perhaps due to a less developed capacity for photosynthesis (and, hence, photorespiration). Our root-specific analyses found no evidence that sucrose decreased the CAT activity in genotypes other than cat2 cat3, in which the activity was stimulated by added sugar.

Further, CAT2 transcripts were more abundant in the roots of plants grown with sugar (Figure 2.1).

The lack of a simple correlation between root growth, root CAT activity and root ROS signals perhaps

suggests that any effect of the cat2 mutation on root development is an indirect consequence of effects in the shoot. Direct evidence that this is the case comes from the almost complete reversion of the cat2 root phenotype by growing the plants at hCO2 (Figure 2.2). While we cannot exclude an effect of hCO2 on biochemical pathways in the root, this observation probably reflects the inhibition of photorespiration, which poses a stress on cat2 leaves and leads to an indirect effect on root growth.

Further evidence in support of this conclusion comes from an analysis of antioxidative enzymes and H2O2 marker transcripts in roots. Both of these factors respond strongly to stress in cat2 leaves but we found little evidence of a concerted oxidative stress response in cat2 roots (Figure 2.3). Interestingly, however, the increase in root ROS signals in cat2 was accompanied by a slight induction of APX1.

Since root ROS signals were decreased to wild-type levels in all CAT mutants grown at hCO2 (Figure 2.2), this raises the intriguing possibility that ROS may be produced in the roots as a secondary response to oxidative stress in cat2 shoots. While we cannot discount direct translocation of ROS, a more likely explanation is activation of cell-to-cell propagation of ROS production through components such as NADPH oxidases [START_REF] Mittler | ROS signaling: the new wave?[END_REF] which have been implicated in systemic signaling in cat2 leaves [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 enigmatic roles of non-photorespiratory CATs in Arabidopsis

While our data add to previous studies that attest to the essential function of CAT2 in Arabidopsis, they do not provide substantial evidence for important biological roles for CAT1 and CAT3. Although CAT3 appears to encode most of the CAT2-independent CAT activity in leaves and roots (Figures 2.1 and 2.5), the cat3 mutation did not produce a significant effect on growth, despite some evidence of increased ROS signals (Figure 2.2). In the double cat2 cat3 mutant grown in air, loss of CAT3 function did not reinforce the cat2-dependent effect on growth. However, the cat2 mutation in itself is sufficient to produce a marked dwarf phenotype. To circumvent this effect, and to analyze possible roles of CAT3 in metabolizing H2O2 produced through processes other than photorespiration, we examined effects of cat3 in plants grown at hCO2, where CAT2 is not required for optimal growth. In this condition, rosette size in cat2 cat3 was slightly but significantly decreased compared to Col-0, cat2, and cat3 (Figure 2.4). This suggests that when CAT2 is absent, CAT3 plays some role in the processing of either residual photorespiratory H2O2 or photorespiration-independent H2O2 in leaves. Interestingly, this effect was not observed in cat2 cat3 grown in SD at hCO2, pointing to a day length-dependent role for CAT3. CAT3 expression increases in the older leaves of Arabidopsis grown in LD [START_REF] Zimmermann | Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh[END_REF]. Indeed, CAT3 is annotated as a senescence-associated gene. Perhaps as an indication of this role, we found that cat2 cat3 was significantly slower to produce a flowering stem compared to Col-0 and cat2, although the cat3 single mutation had no effect (Supporting Information Figure . S5).

CHAPTER 2 55

Our data suggests that when other CATs are functional, CAT1 makes at most a minor contribution to overall activity in roots and leaves (Figure 2.1; Mhamdi et al., 2010a). CAT1 expression is strongest in reproductive tissues, including stamens, pollen and seeds (Mhamdi et al., 2010a). It is interesting that the cat1 mutation did produce some effect on silique viability when introduced into the cat2 background (Supporting Information Figure . S4). Nevertheless, this effect points to overlapping roles of CAT1 and CAT2 these organs, since the cat1 mutation alone had no significant effect, and the effect of cat2 alone was minor and similar to that cat3. Despite the apparently low CAT1-linked activity, evidence for functional overlap with CAT2 in roots perhaps comes from the experiments in which plants were grown at hCO2, where growth in the cat1 cat2 double mutant showed the biggest difference from wild-type (Figure 2.2).

As well as their expression patterns in different organs, the different CATs show cell-and tissuespecificity within organs [START_REF] Zimmermann | Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh[END_REF]. Despite such specificity, our data identify a number of compensatory responses in specific CAT transcripts in response to loss of other CAT functions. This is most obvious for CAT3 transcripts in the cat2 background and CAT1 transcripts in cat3 and cat2 cat3 lines (Figure 2.1; Supporting Information Figure . S8). Taken together, our data perhaps suggest that CAT1 and CAT3 may be mainly back-up players to CAT3 and CAT2, respectively. Triple CAT mutants that have very recently been reported [START_REF] Su | The Arabidopsis catalase triple mutant reveals important roles of catalases and peroxisome derived signaling in plant development[END_REF] may enable a functional analysis of overlap between CAT1 and CAT3 functions.

Growth day length affects oxidative signaling independent of oxidative stress duration

While previous reports have documented day length-specific outcomes of the cat2 mutations at the level of phenotypes and gene expression, they have not established that this effect is independent of stress intensity. The transcripts of many oxidative stress-sensitive genes accumulate more strongly in cat2 grown in SD than in LD (Queval et al., 2007[START_REF] Queval | Daylength is a key regulator of transcriptomic responses to both CO2 and H2O2 in Arabidopsis[END_REF]), but it is not clear whether this simply reflects attenuation of the response after a certain duration of oxidative stress. Here, we sought to establish, first, whether or not this is the case and, second, whether day length-dependent effects on oxidative stress transcriptomes can be explained by differences in cat2-independent CAT activity in LD compared to SD.

Data shown in Figures 2.7 and 2.8 suggest that the impact of day length on cat2-triggered gene expression is not the result of differences in the duration of oxidative stress. When the responses of two H2O2 marker transcripts to equal-time exposure to oxidative stress were examined, plants grown in SD clearly showed much stronger induction in response to CAT deficiency than those grown in LD.

Interestingly, this was the case whether CAT deficiency was provoked by the cat2 mutation or by treatment with 3-AT, a general inhibitor of CAT activity. Moreover, 3-AT produced a stronger effect on gene expression than did the cat2 mutation. The similarity between gene induction in cat2 and cat2 cat3 suggest that this was not due to additional inhibition of CAT3, even though extractable CAT activity after 3-AT treatment was very similar to that in the double mutant (Figure 2.6). While 3-AT is an inhibitor of histidine biosynthesis, it has been commonly used as an inhibitor of CAT (Ferguson & Dunning, 1986;[START_REF] May | Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures[END_REF]. It is effective in knocking down CAT activities within hours after spraying (Figure 2.6) without affecting the extractable activities of several other enzymes involved in the ascorbate-glutathione pathway (Supporting Information Figures S7 andS9).

It is interesting that when 3-AT treatment was performed at hCO2, the two marker genes were not more strongly induced in SD than in LD. This perhaps suggests that the interaction between day length and oxidative signaling is linked somehow to photorespiration, a process that is tightly embedded into photosynthetic and respiratory metabolism in C3 plants [START_REF] Donahue | Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications[END_REF]. Our observations add to the growing awareness of the impact of nutritional and environmental conditions on defence signalling [START_REF] Kangasjärvi | Photosynthesis, photorespiration, and light signalling in defence responses[END_REF]. They are consistent with previous studies showing that responses of plants to ozone exposure are influenced by the day length context [START_REF] Vollsnes | Visible foliar injury and infrared imaging show that daylength affects short-term recovery after ozone stress in Trifolium subterraneum[END_REF][START_REF] Dghim | Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod-influenced responses to ozone using Arabidopsis knockout mutants[END_REF].

For instance, ozone-triggered lesions in Arabidopsis are more easily induced by equal-time exposure in LD compared to SD [START_REF] Dghim | Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod-influenced responses to ozone using Arabidopsis knockout mutants[END_REF], similar to LD-dependent lesion formation in cat2 [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]. Such effects may be related to circadian clock rhythms, which have been reported to interact with oxidative stress and related phytohormone signaling [START_REF] Nitschke | Circadian stress regimes affect the circadian clock and cause jasmonic acid-dependent cell death in cytokinin-deficient Arabidopsis plants[END_REF].

Here, by using a short-term exposure to oxidative stress of intracellular origin, we have shown that CHAPTER 2 57 gene expression responses in leaves are strongly dependent on the history of the plant, and that this cannot be explained by day length-dependent differences in CAT3 expression. Although we cannot exclude a stressful effect of 3-AT that is additional to CAT inhibition, it is possible that the stronger effect of 3-AT compared to cat2 and cat2 cat3 may be another reflection of the redundancy between the three enzymes.

CHAPTER 3

Effect of a specific isoform of glucose-6-phosphate dehydrogenase on H2O2-induced SA signaling: a genetic screen for revertant lines

Introduction

Pathogen attack is one of the common constraints during plant growth and development. The perception of an invading pathogen commonly stimulates an oxidative burst in infected tissues, with accumulation of H2O2. Although there are several sources of pathogen-induced H2O2 accumulation, NADPH oxidases have received particular attention (Torres and Dangl, 2005). The high level of H2O2 triggers rapid death of plant cells directly in contact with or close to the pathogen. This effect is termed the hypersensitive response (HR; [START_REF] Mur | The hypersensitive response; the centenary is upon us but how much do we know?[END_REF]. HR is a widespread phenomenon which contributes to the activation and establishment of plant immunity to disease. As one of the most efficient and immediate resistance reactions, it plays a crucial role in preventing the infection from spreading by sacrificing both infected and adjacent cells. It can also lead to systemic acquired resistance (SAR), a mechanism with enhanced resistance against a wide range of pathogens following an earlier localized attack by a pathogen [START_REF] Dietrich | Arabidopsis mutants simulating disease resistance response[END_REF][START_REF] Torres | Reactive oxygen species signaling in response to pathogens[END_REF][START_REF] Van Breusegem | Reactive oxygen species in plant cell death[END_REF]. Accompanied by lesion formation and SAR, the interaction of plants and pathogen also cause PR genes induction and SA accumulation [START_REF] Takahashi | Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels[END_REF][START_REF] Chamnongpol | Defense activation and enhanced pathogen tolerance induced by H2O2 in transgenic tobacco[END_REF][START_REF] Mittler | Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection[END_REF].

The cat2 mutant has been well described as a lesion-mimic mutant because it shows spontaneous localized lesions similar to those observed in response to bacterial pathogens. As mentioned in the last chapter, oxidative stress responses in cat2 leaves, including lesion formation, are day lengthdependent. Previous work in our group revealed that cat2 plants grown in LD, but not SD, show obvious lesions (Queval et al., 2007). The lesion phenotype in cat2 is associated with accumulation of SA and activation of pathogenesis response pathways. The sid2 mutant is defective in ISOCHORISMATE SYNTHASE1 (ICS1), an enzyme associated with SA synthesis in Arabidopsis challenged with pathogens. When the sid2 mutation was introduced into the cat2 background, the lesion phenotype observed in LD condition was reverted, and this effect was accompanied by inhibition of oxidative stress-induced SA accumulation and induction of PR genes (Figure 3.1).

Furthermore, treatment of rosette leaves with SA restored the lesion phenotype in cat2 sid2 in LD and also triggered lesion formation in cat2 in SD. These results demonstrate that H2O2-induced lesion and CHAPTER 3 75 pathogenesis associated response are dependent on SA through the isochorismate pathway (Chaouch et al., 2010). NADPH is a crucial component required for ROS metabolism by the ascorbate/glutathione pathway and TRX pathway, as well as ROS production by NADPH oxidases. The production of NADPH relies on several different enzymes [START_REF] Donahue | Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications[END_REF]. As noted in Chapter 1, G6PDH is a potentially important source of intracellular NADPH and may be involved in various environmental stress responses. In Arabidopsis, the different isoforms are distinguished by their separate localization and function. When I arrived in the laboratory, it was known from the unpublished work of Amna Mhamdi that knocking out a specific cytosolic isoform of G6PDH (G6PD5) abolished the cat2 lesion phenotype (Figure 3.2A). Further, transcriptome analysis showed that most of the genes induced in cat2 were much less induced when G6PD5 function is lost (Figure 3.2B). Strikingly, the abolition of cat2-induced lesions by G6PD5 deficiency is very similar to that caused by the ICS1 mutation.

Microarray analyses revealed a significant overlap between the cat2 g6pd5 and cat2 sid2 transcriptomes. For instance, for the top 50 genes that are highly induced in cat2 (relative to Col-0), With the completion of genome sequencing programmes, emphasis in genomics has shifted from sequence analysis to gene function investigation. For example, with a sequenced reference genome from the Columbia (Col-0) accession (The Arabidopsis Genome Initiative, 2000), Arabidopsis has been used extensively as a model organism to understand plant development, physiology, and

Col-0 cat2 cat2 g6pd5 A Col-0 cat2 cat2 g6pd5 B C
CHAPTER 3 77 metabolism. Over the past few decades, many techniques of forward and reverse genetics have been developed to identify causative mutations in Arabidopsis thaliana, like chemical, irradiation and insertional mutagenesis [START_REF] Page | The art and design of genetic screens: Arabidopsis thaliana[END_REF][START_REF] Peters | Forward genetics and map-based cloning approaches[END_REF][START_REF] Henikoff | Single-nucleotide mutations for plant functional genomics[END_REF].

Forward genetics has been proven to be a powerful tool for identifying the components involved in a specific biological process or a signaling pathway. In comparison to reverse genetics approaches such as that described in Chapter 2, this strategy is much less reliant on prior knowledge, and can therefore uncover novel functions for genes through an unbiased approach.

Among all methods in forward genetics, T-DNA insertion and chemical mutagenesis such as EMS are the most widely used. The insertional mutations tend to result in complete knockouts of the gene, making it difficult to link a phenotype other than death with essential genes. Therefore the mutational spectrum with effects on gene function can be limited. In addition, plants mutagenized with T-DNA typically have only one to three insertions per line. So the size of a "saturated population", ie, one in which at least one insertion mutation is randomly generated for most genes, is typically extremely high. Screening so many plants can be prohibitive if the mutant screen being performed is laborious or slow. As a complementary approach, chemically induced mutants have been shown to provide an efficient alternative. Firstly, chemical mutagenesis may produce promoter or missense mutations in the coding region, resulting in a non-lethal knock-down rather than a potentially lethal knockout of a protein function. Consequently, it is easier to understand the functions of essential genes by generating weak non-lethal alleles. Because each individual line can bear single point missense and nonsense substitutions in hundreds of genes, an allelic series of induced mutations with different effects on a given gene's function can be isolated by screening a few thousand mutagenized plants. An additional potential advantage of chemical mutagenesis is that it can be used not only to identify the function of a given gene but also to understand the role of specific amino acids in protein function, thanks to the production of short deletions or point mutations.

As a chemical mutagen, EMS may preferentially induce alkylation of guanine (G), forming O6ethylguanine which pairs with thymine (T) instead of cytosine (C). Thereafter the single-base change leads to the conversion of C/G pairs to T/A during the subsequent DNA repair. This substitution occurs at a high frequency during the mutagenesis, even though EMS also generates G/C to C/G or G/C to T/A transversions by 7-ethylguanine hydrolysis or A/T to G/C transition by 3-ethyladenine pairing errors. The efficiency of this mutation effect may be dose-dependent. In general, the higher the EMS concentration used to treat seeds, the larger the number of mutations induced in each individual [START_REF] Martín | A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis[END_REF]. In addition, some genotypes may be more susceptible to EMS treatment than others. Other factors that affect mutation frequency are the gene's position in the genome and the treatment condition during mutagenesis. For example, effects such as embryo-lethality and mutations that cause decreases in Chl are directly related to the length of exposure to a fixed concentration of EMS [START_REF] Koornneef | EMS-and radiation-induced mutation frequencies at individual loci in Arabidopsis thaliana (L.) Heynh[END_REF].

Identification of the genetic basis of a mutant phenotype has become much easier and faster within the past few years. Until recently, map-based cloning or positional cloning was still required, which are relatively lengthy and onerous [START_REF] Arondel | Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis[END_REF][START_REF] Meyer | A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana[END_REF]. This strategy is usually based on outcrossing the mutant plant to a divergent Arabidopsis accession, often involving crosses between Col-0 and Landsberg erecta (Ler). In the F2 generation, the mutant phenotype is scored and molecular markers are then used to provide an approximate location of the mutated gene's physical position.

Finally, plants with intra-chromosomal recombination events are used to narrow down the genetic interval. The recombination frequency has been shown to vary across the genome with low recombination frequencies hindering fine mapping [START_REF] Lynn | Covariation of synaptonemal complex length and mammalian meiotic exchange rates[END_REF]Drouaud et al., 2006). Further, the whole mapping processes can be difficult if the mutant phenotype is subtle and if assaying the phenotype is labor-intensive. To avoid this time-consuming effect, traditional map-based cloning has progressively been replaced by direct identification through sequencing. Initially, a pool of DNA isolated from bulked segregants was sequenced and used for simultaneous mapping and mutant identification [START_REF] Schneeberger | SHOREmap: simultaneous mapping and mutation identification by deep sequencing[END_REF][START_REF] Cuperus | Identification of MIR390a precursor processing-defective mutants in Arabidopsis by direct genome sequencing[END_REF][START_REF] Austin | Next-generation mapping of Arabidopsis genes[END_REF]. Although these approaches are fast and straightforward, the considerable phenotypic variation in F2 populations from crosses between different accessions impairs the recognition of mutants with subtle phenotypic alterations. In addition, if genetic screens involve modifiers of a preexisting mutant, the mapping depends on the availability of the primary mutant in another suitable accession, the introgression of the mutation in such a background, or the laborious additional genotyping for the presence of the first-site CHAPTER 3 79 mutation. However, these limitations have largely disappeared with the advent of very rapid genome sequencing technology. This allows direct identification of mutations, meaning that mapping is no longer necessary. Currently, a simpler approach of backcrossing mutants to their non-mutagenized progenitor can be used [START_REF] Abe | Genome sequencing reveals agronomically important loci in rice using MutMap[END_REF][START_REF] Hartwig | Fast isogenic mapping-bysequencing of ethyl methanesulfonate-induced mutant bulks[END_REF][START_REF] Thole | Next-generation sequencing as a tool to quickly identify causative EMS-generated mutations[END_REF]. Combined with bulk segregation analysis and comparative whole genome sequencing, the number of causal candidates identified in the genome of the mutated plant can be reduced to an acceptable level.

In this part of the work, an EMS mutagenesis of cat2 g6pd5 was performed and a genetic screen for revertants that present lesions was conducted. Our aim was to further investigate the underlying mechanisms by which G6PD5 is required for lesion formation and, thereby, to define novel components that might link G6PD5 to the activation of SA-associated pathways.

Results

The evaluation of mutagenesis efficiency

To produce a population of chemically induced mutant lines for the forward genetics study, we treated around 2000 seeds of cat2 g6pd5 homozygotes (M0) with 0.3% EMS [START_REF] Qu | Generation and identification of Arabidopsis EMS mutants[END_REF] for 15 h with gentle rotation. Subsequently, these mutagenized seeds (M1) were sown in square pots (nine seeds per pot). Plants were self-pollinated and seeds of the first generation (M2) were harvested in pools. To estimate the efficiency of the mutagenesis, we quantified embryo lethality in siliques of M1 plants as an indicator [START_REF] Till | Large-scale discovery of induced point mutations with highthroughput TILLING[END_REF][START_REF] Martín | A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis[END_REF]. This is performed by estimating the proportion of fully or nearly fully sterile fruits (class A) and the proportion of semi sterile or normal fertile fruits (class B and C). We dissected ten siliques from each of five individual plants selected randomly. As is shown in Table 3.1, among all fifty siliques dissected, only one class B and one class C fruit were observed. Therefore the frequency of class (B+C) is 4%, which is in accordance with the expected range between 2% and 35% [START_REF] Martín | A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis[END_REF]. The efficiency of the EMS mutagenesis was towards the high end of this range, but was considered adequate to continue the genetic screen.

Class category Number of siliques

A (aborted﹥20 %) 48 B (20 % ≥ aborted 6.7 %) 1 C (aborted﹤6.7%) 1

(B+C)% 4

The revertant screen

All M2 seeds were pooled together and used for the screen. As is shown in Figure 3.3A, the strategy was based on a screen for lesion phenotypes. Any plants that showed lesions were analyzed by PCR at the CAT2 and G6PD5 loci to confirm their homozygosity for the T-DNA insertions, and confirmed cat2 g6pd5 homozygotes were self-fertilized and individually harvested. After further confirmation of the phenotype and genotype in the next generation, they were backcrossed with cat2 g6pd5.

Subsequently, segregants showing cell death were pooled for sequencing to find the candidates for the causal mutation.

Specifically, the screen was carried out with plants sown and grown in soil. Nine seeds were sown in each square pot and more than 1600 seeds were planted in total for each batch of screened individuals.

As negative and positive controls for phenotyping and genotyping, Col-0, cat2 and cat2 g6pd5 were always grown simultaneously alongside mutated plants. After two days in a cold room at 4 °C in dark, plants were transferred to a controlled-environment growth chamber room in LD (16 h light/8 h dark).

In cat2, lesions initially develop after about two weeks growth and then spread clearly on the rosette area within the following week. Hence, the lesion phenotype screen was performed on plants at 21-25 d of age to make sure that any revertants to the cat2 phenotype would be visible. Plants showing clear cell death were screened and first genotyped by PCR to check that they were cat2 g6pd5 homozygotes. To date, six M2 plants that show a cat2-like lesion phenotype have been identified using this strategy (Figure 3.3B). It should be noted that while these plants are all cat2 g6pd5 genotypes that CHAPTER 3 81 show cat2-like lesions, they do present some differences in leaf shape and rosette size. In some lines, the rosettes have a similar size to cat2 while others are much smaller. These screened plants were conserved and seeds of M3 were harvested for confirmation of the lesion phenotype and cat2 g6pd5 homozygous genotype in M3 plants.

Backcross to cat2 g6pd5 and segregation analysis

Because EMS mutagenesis induces multiple point mutations in each plant, the high rate of mutagenesis makes it possible that relatively few plants in the screen will be enough to find those with the phenotype of interest. On the other hand, a high mutation density implies that undesired background mutations in selected mutant lines need to be cleaned up to identify the causal mutation. This is partly accomplished by backcrossing the newly identified plants with their parental lines [START_REF] Weigel | Arabidopsis: A Laboratory Manual[END_REF]. An example is shown in Figure 3.4 for the first identified line, 9.11.

The M3 plants of line 9.11, all of which showed clear lesions, were first backcrossed with their parental line cat2 g6pd5. The principle behind this approach is that the causal mutation of line 9.11 introduced by EMS mutagenesis is homozygous in M3. In contrast, other introduced mutations unlinked to the lesion phenotype are likely to be random in zygosity. Backcross with cat2 g6pd5 does not change the homozygous genotype at the CAT2 and G6PD5 loci but the causal mutation should be heterozygous in the F1 backcross. Therefore, the phenotype of F1 plants provides a first indicator to distinguish if the causal mutation is dominant or recessive. Specifically, if F1 plants do not show lesions, the mutation should be recessive; otherwise it is likely to be dominant. This information is also an important reference for the following phenotype segregation. By now the six lines we have obtained are all recessive mutations according to the phenotype of the F1 after backcross (Table 3.2). From the self-fertilized F1 plants, F2 seeds were harvested from several individual plants and the phenotype segregation was checked for each plant. According to the number of plants showing lesions or not in F2, we can obtain the phenotype segregation ratio for each F1 plant. These data provide an indicator to infer the likely number of causal mutations involved in the phenotype effect and to confirm whether causal mutations are dominant or recessive. For instance, if one quarter of the plants shows lesions, the simplest explanation would be a single causal mutation that is recessive. If three quarters show lesions, this would be consistent with the causal mutation being single and dominant.

For more complicated cases, e.g. if two unlinked recessive mutations are involved, the segregation ratio should be 1/16. After re-confirming the cat2 g6pd5 homozygote genotype, plants with lesions were used for the second backcross and segregation was checked once again. Details of the backcross and segregation in F2 generation were shown in Tables 3.2 and 3.3 respectively. To evaluate how well After at least one backcross event, multiple leaf samples of F2 plants displaying a lesion phenotype were pooled together and the bulked DNA was subjected to whole-genome sequencing along with non-mutated parental cat2 g6pd5 DNA as the reference. For each line we collected the sample from at least 50 plants that presented lesions. This is because, during the backcross, the phenotype should segregate with the causal mutation, while non-causal mutations should be distributed more evenly in F2 (unless they are closely linked genetically to the causal mutation). As shown schematically in

Identification of causal mutations

During whole-genome sequencing of both novel mutants and the parental line cat2 g6pd5, CHAPTER 3 85 SHOREmap was used to identify the causal mutations. The causal mutation should show the highest frequency among all EMS-induced changes [START_REF] Ossowski | Sequencing of natural strains of Arabidopsis thaliana with short reads[END_REF][START_REF] Schneeberger | SHOREmap: simultaneous mapping and mutation identification by deep sequencing[END_REF]; Non-causal mutations X,Y,Z distribute evenly in F2 [START_REF] Hartwig | Fast isogenic mapping-bysequencing of ethyl methanesulfonate-induced mutant bulks[END_REF]. To date, the putative causal mutations in four of the six lines have been tentatively identified by sequencing. In three lines, the top candidate is MIPS1, a gene encoding myo-Inositol phosphate synthase, while CPR5, encoding CONSTITUTIVE EXPRESSION OF PR GENES5 is the most probable candidate in another line (Table 3.4). As discussed below, mutations in both of these genes have previously been reported to be involved in lesion formation. In the other two lines, several novel candidates for the causal mutation were identified (Table 3.4), but further work is required to establish which of them is responsible for lesion formation in the cat2 g6pd5 background.

Discussion

NADPH is an essential component required in plant metabolism and cellular redox homeostasis [START_REF] Noctor | Metabolic signalling in defence and stress: the central roles of soluble redox couples[END_REF][START_REF] Donahue | Redox regulation in photosynthetic organisms: signaling, acclimation, and practical implications[END_REF]. Within the context of biotic stress and SA-related signaling, potentially important functions are providing reductant for NADPH oxidases, but also reduction of ascorbate, glutathione, and cytosolic TRXs, all of which have been implicated in PR signaling [START_REF] Pastori | Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling[END_REF][START_REF] Torres | Reactive oxygen species signaling in response to pathogens[END_REF][START_REF] Parisy | Identification of PAD2 as a γ-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis[END_REF][START_REF] Tada | Plant immunity requires conformational changes of NPR1 via S-nitrosylation and thioredoxins[END_REF]. It has been well established that NADPH can be generated by several enzymatic approaches. One of the major pathways in the chloroplast is the FNR that provides NADPH for CO2 assimilation and related reactions [START_REF] Arakaki | Plant-type ferredoxin-NADP + reductases: a basal structural framework and a multiplicity of functions[END_REF]. In the cytosol, where many signaling processes occur, NADPH is generated by at least four major NADP-dependent dehydrogenases (Emes and Neuhaus, 1997;[START_REF] Thom | Sweet pepper plastids: enzymic equipment, characterisation of the plastidic oxidative pentose-phosphate pathway, and transport of phosphorylated intermediates across the envelope membrane[END_REF][START_REF] Kruse | Antisense inhibition of cytosolic NADPdependent isocitrate dehydrogenase in transgenic potato plants[END_REF][START_REF] Hodges | Higher plant NADP + -dependent isocitrate dehydrogenases, ammonium assimilation and NADPH production[END_REF][START_REF] Arnon | A new glyceraldehyde phosphate dehydrogenase from photosynthetic tissues[END_REF][START_REF] Iglesias | On the metabolism of triose-phosphates in photosynthetic cells. Their involvement on the traffic of ATP and NADPH[END_REF][START_REF] Chang | Structure and function of malic enzymes, a new class of oxidative decarboxylases[END_REF]. Even though in the cytosol. These similarities suggest it is worthwhile investigating the relationships between these two enzymes, even though G6PD5 participates in the catalysis of 6-phosphogluconate synthesis, a biological reaction different from that catalyzed by MIPS1.

The second gene we identified by sequencing revertant lines was CPR5. Like mips1, the cpr5 mutant has previously been reported to be a member of the lesion-mimic mutant family. Lesion formation in cpr5 is accompanied by induction of PR gene expression, SA accumulation and enhanced resistance to pathogens. Unlike cat2, the cpr5 lesion phenotype is not dependent on environmental conditions such as photoperiod [START_REF] Bowling | The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance[END_REF]. In addition to spontaneous lesion formation, it also exhibits the characteristic of accelerated leaf senescence, indicating that CPR5 controls multiple physiological processes in Arabidopsis [START_REF] Jing | Arabidopsis onset of leaf death mutants identify a regulatory pathway controlling leaf senescence[END_REF][START_REF] Jing | Arabidopsis CPR5 is a senescenceregulatory gene with pleiotropic functions as predicted by the evolutionary theory of senescence[END_REF][START_REF] Jing | Early leaf senescence is associated with an altered cellular redox balance in Arabidopsis cpr5/old1 mutants[END_REF][START_REF] Yoshida | Identification of a novel gene HYS1/CPR5 that has a repressive role in the induction of leaf senescence and pathogen-defence responses in Arabidopsis thaliana[END_REF][START_REF] Trotta | Regulatory subunit B'gamma of protein phosphatase 2A prevents unnecessary defense reactions under low light in Arabidopsis[END_REF].

EMS mutagenesis introduces multiple mutations in the genome. Even if non-causal mutations can be cleaned by backcrossing, it is difficult to remove their influence completely without a series of multiple, time-consuming backcrosses. The principle of identifying the causal mutation is based on its occurring at the highest frequency among all modifications induced by the mutagenesis. To confirm the phenotype effect of these identified genes, one of the approaches will be to obtain T-DNA insertion mutants and to cross them into the cat2 g6pd5 background. Hence, T-DNA mutants of mips1 and cpr5 will be crossed with cat2 g6pd5 to get triple homozygotes, and the resulting plants will be analyzed to confirm that the candidate genes are indeed responsible for the observed reversion. PR gene expression and SA content can be analyzed and compared with cat2 g6pd5 to establish the pathways underlying the lesion phenotype.

A key remaining question is whether the mutations identified in the revertant screen are dependent on the presence of the cat2 mutation and, therefore, on oxidative stress induced by intracellular H2O2. Our main aim in conducting this screen was to shed light on the mechanisms by which loss of G6PD5 function modulates the cat2-triggered response. To this end, we focused on revertants that show a similar phenotype to that observed in cat2. Several other potential revertants were observed that showed symptoms such as yellowing or early senescence but such plants were disregarded because CHAPTER 3 89 these phenotypes do not correspond to those observed in the cat2 single mutant. To date, we cannot judge to what extent the lesion phenotypes linked to the mutations identified in the different revertant lines are dependent on the cat2 mutation. Indeed, the fact that mips1 and cpr5 have already been identified as lesion mimic mutants (i.e., their mutation alone is sufficient to induce lesions) suggests that some of their effects might be independent of the presence of cat2. This question could be elucidated by comparing the different genotypes under our growth conditions. For example, a comparison of mips1, cat2 mips1, g6pd5 mips1 and cat2 g6pd5 mips1 could shed some light on this issue. A second approach would be to transform the cat2 g6pd5 revertant with the wild-type CAT2 sequence. However, as described in the next chapter, the use of a targeted reverse genetics approach to analyze an NADPH-requiring enzyme involved in the ascorbate-glutathione pathway has identified an interesting avenue of inquiry concerning the biochemical mechanisms underlying the effect of the g6pd5 mutation on responses in cat2.

CHAPTER 4

Analysis of the roles of monodehydroascorbate reductases in H2O2 metabolism using gene-specific mutants

Introduction

As noted previously in this thesis, plants are often exposed to various stress conditions in the field. A central theme in most stress responses is the accumulation of ROS, such as H2O2, and ROS-induced perturbation on intracellular redox state (Queval et al., 2007). Plants have evolved several enzymatic antioxidative systems to escape from oxidative damage [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]. As one of the major enzymes to metabolize ROS, CATs can initiate a dismutation reaction in which H2O2 is decomposed into water and O2. Alongside CAT, the ascorbate-glutathione pathway is also considered to be an essential system to protect plants from oxidative stress by consuming H2O2. In this pathway, H2O2 can obtain electrons from ascorbate and can thereby be converted to water. This reaction is catalyzed by APX and accompanied by the production of MDHA. As a short-lived radical, MDHA can be recycled immediately to reduced ascorbate (ASC) by MDHAR via electron transfer from NAD(P)H.

Alternatively, it disproportionates spontaneously to ascorbate and DHA. DHA can be reduced back to ascorbate, which is catalyzed by glutathione-dependent DHAR (Foyer and Halliwell, 1977;[START_REF] Nakano | Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts[END_REF]. The product of glutathione oxidation, GSSG, is reduced by GR using NADPH as an electron donor. Among all these enzymes, DHAR and MDHAR may be important for maintaining ascorbate in its reduced state. Interestingly, although the degradation of H2O2 in plant peroxisomes is primarily attributed to CATs, the affinity of CAT for H2O2 is much lower than that of APX. This leads to the suggestion that APX/MDHAR system might have a much higher efficiency to scavenge H2O2 when its concentration is lower [START_REF] Bunkelmann | Ascorbate peroxidase. A prominent membrane protein in oilseed glyoxysomes[END_REF][START_REF] Mullen | Biogenesis and membrane properties of peroxisomes: does the boundary membrane serve and protect?[END_REF].

Nevertheless, modelling analyses suggest that both CAT and APX can function together to maintain H2O2 at low levels (of the order of 1 µM: [START_REF] Tuzet | Analyzing the function of catalase and the ascorbateglutathione pathway in H2O2 processing: Insights from an experimentally constrained kinetic model[END_REF].

Although the ascorbate-glutathione pathway is considered to be a key player in metabolizing H2O2, the relative importance of specific MDHAR isoforms is not clearly established. Activity of MDHAR has been reported in various plant cell compartments such as chloroplasts [START_REF] Hossain | Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide[END_REF], the cytosol [START_REF] Dalton | Subcellular localization of oxygen defense enzymes in soybean (Glycine max [L.] Merr.) root nodules[END_REF], mitochondria and peroxisomes [START_REF] Jiménez | Evidence for the presence of the ascorbateglutathione cycle in mitochondria and peroxisomes of pea leaves[END_REF]. Cytosolic MDHAR has been purified from cucumber fruits [START_REF] Hossain | Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme[END_REF], soybean root nodules [START_REF] Dalton | Purification and characterization of monodehydroascorbate reductase from soybean root nodules[END_REF] and potato tubers [START_REF] Borraccino | Purification and properties of ascorbate free-radical reductase from potato tubers[END_REF]. A mitochondrial MDHAR has been CHAPTER 4 93 purified from potato tubers (De Leonardi S et al., 1995) and a chloroplast MDHAR from spinach leaves [START_REF] Sano | Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach[END_REF]. Analysis of the Arabidopsis genome has revealed that five genes encode MDHAR (Table 4.1). The encoded proteins are approximately 50 kDa in mass, and are targeted to various subcellular compartments (Table 4.1). Among them, MDHAR1 and MDHAR4 are targeted to the peroxisomal matrix and membrane, respectively. MDHAR2 and MDHAR3 are targeted to the cytosol [START_REF] Lisenbee | Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase[END_REF] while MDHAR5/6 is targeted to mitochondria or chloroplasts, depending on the different transcription initiation sites [START_REF] Obara | The use of multiple transcription starts causes the dual targeting of Arabidopsis putative monodehydroascorbate reductase to both mitochondria and chloroplasts[END_REF]. As one of the enzymes in the ascorbate-glutathione cycle, MDHAR may play an important role in various physiological processes. Overexpression of MDHAR has been reported to enhance tolerance to temperature and methyl viologen in tomato (Li et al., 2010). MDHAR genes are involved in the response to abiotic stresses through maintaining redox status in acerola (Eltelib et al., 2011). As well as possible roles in abiotic stresses, MDHAR genes were found to play a negative role in pathogen recognition in wheat (Feng et al., 2014a(Feng et al., , 2014b;;[START_REF] Abou-Attia | TaMDAR6 acts as a negative regulator of plant cell death and participates indirectly in stomatal regulation during the wheat stripe rust-fungus interaction[END_REF]. In Arabidopsis, MDHAR1

and MDHAR4 were proposed to be important for ascorbate recycling in the peroxisomes [START_REF] Lisenbee | Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase[END_REF]. MDHAR genes may also be involved in the resistance to ozone, salt and polyethylene glycol (Eltayeb et al., 2007). Fungi such as Piriformospora indica can cause accumulation of MDHAR transcripts [START_REF] Vadassery | Monodehydroascorbate reductase 2 and dehydroascorbate reductase 5 are crucial for a mutualistic interaction between Piriformospora indica and Arabidopsis[END_REF]. MDHAR4 was found to influence seed storage oil hydrolysis and seedling growth (Eastmond, 2007). Recently, evidence was presented for a new pro-oxidant effect of MDHAR6 in mediating TNT toxicity by promoting reactive oxygen production [START_REF] Johnston | Monodehydroascorbate reductase mediates TNT toxicity in plants[END_REF][START_REF] Noctor | Lighting the fuse on toxic TNT[END_REF]. Further work is required to elucidate the precise roles of the different isoforms.

Very recent work in our group showed that dhar mutations did not affect leaf ascorbate pools in optimal and oxidative stress conditions, even though the cat2-induced glutathione oxidation and total glutathione accumulation is decreased by multiple dhar mutations [START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF]. The negligible effect of DHAR mutation on ascorbate status leads us to investigate the potential importance of other routes reducing DHA or else avoiding its formation by consuming MDHA. Are MDHAR isoforms important in maintaining the ascorbate redox state by catalyzing the reduction of MDHA? To answer this question, we generated loss-of-function mutants for different MDHAR genes in Arabidopsis. We also introduced these mutants into a CAT-deficient background (cat2) to obtain double mutant lines. Because CAT2 encodes the irreplaceable photorespiratory CAT enzyme, cat2

itself has been extensively used as a system in which H2O2 signals can be switched on and off by transferring plants between photorespiratory and non-photorespiratory conditions (Queval et al., 2007). Decreased CAT activity forces H2O2 metabolism through alternative systems that are dependent on cellular reductants, notably the ascorbate-glutathione pathway (see Figure 1.4). For example, it has notably been shown that a specific isoform of GR that is mainly located in the cytosol is essential to allow glutathione regeneration when CAT function is lost (Mhamdi et al., 2010b). Here, we used a similar strategy to analyze the roles of genes encoding MDHAR in Arabidopsis, with a focus on MDHAR1, encoding a peroxisomal enzyme, and MDHAR2 and MDHAR3, which encode cytosolic enzymes. For this, a series of mdhar and cat2 mdhar mutants were analyzed that had been obtained and partially characterized in the laboratory by Gilles Chatel-Innocenti and Hélène Vanacker.

Results

Identification of mdhar single mutants

To analyze the specific roles of different MDHAR genes, we obtained at least one line of T-DNA insertion mutants for MDHAR1, MDHAR2, MDHAR3 and MDHAR4 (Table 4.1). In a first analysis, a single line for each gene was chosen for functional analysis. Among them, mdhar1, mdhar2 and mdhar3 are mutants with T-DNA insertions in the corresponding coding sequence, while for mdhar4

the T-DNA is located in the promoter (Figure 4.1). All four mutants were identified to be homozygotes CHAPTER 4

95 The results of reverse transcription-polymerase chain reaction (RT-PCR) suggest that mdhar1, mdhar2 and mdhar3 are true knockouts, which is consistent with the location of insertions in the coding sequences. In contrast, mdhar4 is not affected by the T-DNA insertion at the level of transcript (Figure 4.2B), suggesting that the insertion in the promoter did not significantly modify expression of the gene. To study the functional impact of single mdhar mutations, plants were grown in standard conditions. Little difference in rosette phenotype was found when compared with Col-0 after three weeks' growth in LD, even though the mdhar4 mutation caused a slight but significant decrease in fresh weight (Figure 4.2C and D). This suggests functional redundancy among MDHAR isoforms or with other systems when plants are grown in non-stressful conditions. Because expression of the corresponding gene did not seem to be altered in mdhar4, we focused on the mdhar1, mdhar2, and mdhar3 mutants in the rest of the study described below.

MDHAR transcripts in response to intracellular oxidative stress

Based on current concepts of the ascorbate-glutathione pathway as an important player in the plant antioxidative system, we explored the importance of the three MDHAR genes in oxidative stress conditions. Because of its enhanced intracellular H2O2 availability, cat2 has been well established as a system to mimic oxidative stress (Queval et al., 2007;Mhamdi et al., 2010bc). Hence, we crossed each mdhar mutant into the cat2 background to produce double mutants. F1 plants were identified as heterozygotes at both the CAT2 and MDHAR loci. Subsequently, homozygotes were identified by PCR genotyping in the F2 generation (Figure 4.3A). RT-PCR confirmed the absence of CAT2 and MDHAR transcripts (Figure 4.3B). F3 seeds were used for functional analysis.

qRT-PCR analysis confirmed that the mutations in mdhar1 and mdhar2 decreased the respective transcripts to low levels. The mdhar3 mutation also decreased the MDHAR3 transcript to very low levels, although the difference to the wild-type is not statistically significant because of some variability between the Col-0 replicates (Figure 4.4). Interestingly, loss of CAT2 function induced the expression of MDHAR2 and MDHAR3 but not MDHAR1. Moreover, the induction of MDHAR3 was CHAPTER 4 97 weakened significantly in cat2 mdhar2. These results provide a first indication that MDHAR2 and MDHAR3 may be involved in the response to oxidative stress occurring in cat2.

Impact of mdhar mutations on cat2-triggered lesion formation and phytohormone signaling

To study the phenotypic impact of the mdhar mutations in an oxidative stress background, plants were grown in standard conditions in LD. Like cat2, all three double mutants were significantly smaller than the wild-type. While cat2 mdhar1 was somewhat smaller than cat2, cat2 mdhar2 and cat2 mdhar3 showed a similar rosette mass to cat2 (data not shown). The most striking phenotypic effect was on cat2-triggered lesion formation. While cat2-triggered HR-like lesions were clearly visible on cat2 mdhar1 and cat2 mdhar3 leaves, they were largely abolished by the loss of function of MDHAR2 (Figure 4.5A and B). As described in Chapter 3, previous studies revealed that the lesion formation in cat2 is associated with SA signaling (Queval et al., 2007;[START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]. Hence, we analyzed the effects of each mdhar mutation on transcript levels for SA-associated genes. PR1, PR2 and PR5 are marker genes in SA-dependent PR responses, and ICS1 is a gene involved in SA synthesis. None of the single MDHAR mutations affected these transcripts in the wild-type background in standard conditions, with all the markers maintained at the low basal level observed in the wild type (data not shown). In cat2, all four genes were markedly induced by the intracellular oxidative stress: while mdhar1 did not affect this response, the mdhar3 mutation tended to reinforce it, although this effect was only statistically significant for PR1 (Figure 4.5C). The most dramatic effect was that of the mdhar2 mutation, which strongly inhibited the induction of SA marker gene expression triggered by cat2. In cat2 mdhar2, transcripts for all four genes were similar to the basal levels observed in the wild-type, Col-0 (Figure 4.5C).

In view of the effects of the mdhar2 mutation on lesions and SA marker transcripts, SA contents were measured by HPLC. Although some variability meant that differences were not always statistically significant, this analysis revealed that cat2 mdhar2 showed similar SA levels to the wild-type while SA contents in cat2 mdhar1 and cat2 mdhar3 were similar to cat2 (Figure 4.6). Therefore, loss of MDHAR2 function appears to suppress SA contents, induction of SA-dependent genes and, to a large extent, lesion formation triggered by intracellular oxidative stress. C. SA marker transcripts analysis in Col-0, cat2, and double mutants. Data are means ± SE of four biological replicates. * Indicates significant differences of mutants to Col-0 at P < 0.05; + indicates significant differences of double mutants compared with cat2 at P < 0.05. At present, an allelic mutant for MDHAR2 is not available (Table 4.1). A backcross strategy was therefore used to test the link between the mdhar2 mutation and the phenotype by crossing the cat2 mdhar2 line with cat2. Phenotype and genotype segregation were quantified and evaluated in F2 (Table 4.2). Data analysis showed that the lesion phenotype segregation ratio is close to 3:1, and most of the plants showing less or no lesions are cat2 mdhar2 homozygotes. The semi-quantitative nature of the phenotype analysis meant that the genotype-phenotype link was not absolute. Nevertheless, the analysis revealed that about 90% of F2 segregants with one or two wild-type MDHAR2 alleles showed phenotypes similar to cat2. In contrast, this figure was around 30% in cat2 mdhar2 double homozygotes. These results suggest that MDHAR2 is important in linking H2O2 to lesion formation and SA signaling induction.

CHAPTER 4 101

As well as SA, JA is also a phytohormone that is closely linked to oxidative stress (Devoto and Turner, 2005;[START_REF] Maruta | Arabidopsis NADPH oxidases, AtrbohD and AtrbohF, are essential for jasmonic acid-induced expression of genes regulated by MYC2 transcription factor[END_REF]. A complex relationship between SA and JA signaling pathway has been reported [START_REF] Dangl | Plant pathogens and integrated defence responses to infection[END_REF][START_REF] Spoel | NPR1 modulates cross-talk between salicylate-and jasmonate-dependent defense pathways through a novel function in the cytosol[END_REF][START_REF] Takahashi | Antagonistic interactions between the SA and JA signaling pathways in Arabidopsis modulate expression of defense genes and gene-for-gene resistance to cucumber mosaic virus[END_REF]Koorneef et al., 2008). Therefore, we analyzed levels of several marker transcripts involved in JA synthesis and signaling (Figure 4.7). PDF1.2 and JAZ10 are JA signaling marker genes, while LOX3 encodes an enzyme involved in JA synthesis and MYB95 is a transcription factor which responds to JA signaling. As observed for the SA markers, the single mdhar mutations did not affect the basal level of the JA markers in the wild-type background (data not shown). Also similar to the response of the SA marker genes, the cat2 mutation induced all four genes, and this effect was not obviously impacted by loss of function of MDHAR1 or MDHAR3. However, the MDHAR2 mutation inhibited the transcript of PDF1.2 in cat2 mdhar2 to a basal level close to Col-0. It also slightly but significantly alleviated the induction of LOX3 and MYB95 (Figure 4.7). It is interesting that PDF1.2 was the most strongly impacted, as this gene is also ethylene-responsive. These results suggest that MDHAR2 may play roles in linking oxidative stress to signaling through several phytohormones, although its function seems to be particularly important for activation of the SA signaling pathway.

Impact of the loss of MDHAR functions on leaf redox status

To explore the functional impact of mdhar mutations in oxidative stress responses, several major antioxidative enzyme activities were analyzed in the different mutants. The single mdhar mutations did not affect any of the enzyme activities in the Col-0 background. As shown in Chapter 2, the cat2 105 mutation decreased leaf CAT activity to around 20% of that in the wild type. This effect was not substantially changed by the mdhar mutations although cat2 mdhar2 showed slightly lower CAT activity than in cat2. To assess the effect of the mutations on the capacity of the ascorbate-glutathione system, the activities of APX, GR and DHAR were measured. All of them were increased by the cat2 mutation, but none of the mdhar mutations had an obvious impact except cat2 mdhar2, which presented significantly lower APX activity than that in cat2 (Figure 4.8).

As mentioned in Chapter 2, ROS signals are not easy to measure directly, especially in leaves, but transcripts known to be H2O2-responsive can serve as a useful proxy. Consequently, transcripts for the oxidative markers GSTU24, UGT73b3 and APX1 were quantified in both single and double mutants.

The single mdhar mutations did not impact the expression of these marker genes in the Col-0 background, but the cat2 mutation caused marked induction of all three (Figure 4.9). The upregulation of GSTU24 in response to the cat2 mutation was not changed by blocking the function of MDHAR1, MDHAR2 or MDHAR3. However, the induction of UGT73b3 and APX1 was partly reversed by the loss of function of MDHAR2 (Figure 4.9).

Perturbation of redox homeostasis triggered by the cat2 mutation notably involves increased oxidation of glutathione and total glutathione accumulation (Queval et al., 2007[START_REF] Queval | H2O2activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast[END_REF]Mhamdi et al., 2010b).

As is shown in Figure 4.10, when grown in standard conditions, glutathione is kept in a highly reduced state in the wild type (>90% GSH). The mdhar single mutations did not in themselves affect glutathione status. In cat2, glutathione becomes much more oxidized and accumulates, almost entirely due to GSSG accumulation (Figure 4.10, upper panel). Loss of function of MDHAR2 significantly exacerbated the oxidation of glutathione in cat2. While glutathione was almost 50% reduced in cat2, the pool fell to about 20% reduced in cat2 mdhar2 (Figure 4.10). In contrast, the mdhar3 mutation produced a slight but significant decrease in glutathione oxidation in the cat2 background. In contrast to glutathione, ascorbate is less affected in cat2 (Mhamdi et al., 2010b,c). No difference in the reduction state of ascorbate was found in any of the mutants compared with Col-0, but the total ascorbate pool was significantly decreased in cat2 mdhar2 (Figure 4.10, lower panel). A slight effect of this type was also observed in the single mdhar3 mutant. Taken together, these results suggest that

Discussion

The ascorbate-glutathione pathway has long been considered to be among the major components of plant antioxidative systems (Noctor and Foyer, 1998;Foyer and Noctor, 2013). While the enzymes and small molecules in this pathway have been well described, only limited information is available for the quantitatively estimated using a modeling approach [START_REF] Tuzet | Analyzing the function of catalase and the ascorbateglutathione pathway in H2O2 processing: Insights from an experimentally constrained kinetic model[END_REF]. Gene-specific studies have revealed an important role for cytosolic isoforms of APX, GR, and DHAR during oxidative stress conditions (Mhamdi et al., 2010b;[START_REF] Vanderauwera | Extranuclear protection of chromosomal DNA from oxidative stress[END_REF][START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF]. In addition to these enzymes, the cytosolic ascorbate-glutathione pathway is dependent on NAD(P)H that may be produced by several enzymes. As described in Chapter 3, a gene encoding a cytosolic G6PDH seems to be particularly important to link oxidative stress in cat2 to downstream phytohormone signaling and lesion formation. The data in this chapter provide evidence that as specific cytosolic MDHAR isoform is also important in this respect.

MDHAR has been found to be induced by different external stresses at the levels of gene expression and/or enzyme activity [START_REF] Kayıhan | Excess boron responsive regulations of antioxidative mechanism at physio-biochemical and molecular levels in Arabidopsis thaliana[END_REF][START_REF] Sudan | Oxidative stress induced expression of monodehydroascorbate reductase gene in Eleusine coracana[END_REF]. Genetic studies by Eastmond (2007) found that MDHAR4 is required for storage oil hydrolysis during Arabidopsis seed germination and that this effect is linked to removal of H2O2 derived from β-oxidation of fatty acids. During postgermination development of photosynthetic tissues, however, photorespiration becomes more important than fatty acid β-oxidation in generating large quantities of H2O2. Our phenotypic analyses of single mdhar mutants in standard conditions (LD, moderate light intensity) did not reveal a marked impact of loss of MDHAR1, MDHAR2 or MDHAR3 functions. The slightly decreased rosette size of mdhar4 might be consistent with previous analyses on Arabidopsis seedlings (Eastmond, 2007) but since this mutant did not show decreased MDHAR4 transcripts, the biological relevance of this observation is unclear. Nevertheless, some evidence that peroxisomal isoforms might be important in supporting H2O2 metabolism in leaves comes from the smaller size of cat2 mdhar1 compared to cat2 (Figure 4.5). Taken together, however, our data suggest that none of MDHAR1, MDHAR2 or MDHAR3 is critical for plant growth and development in standard conditions, suggesting there may be functional redundancy among them or with other systems. Interestingly, none of the mdhar mutations affected leaf ascorbate pools to a great extent (Figure 4.10). Along with DHAR, MDHAR is considered one of the enzymes responsible for regenerating the reduced form of ascorbate after its oxidation by reactions such as that catalyzed by APX. However, none of the mutant combinations for the three DHARs in Arabidopsis, including a triple dhar1 dhar2 dhar3 mutant, affected the leaf ascorbate pool in either optimal or oxidative stress conditions [START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF]. This may reflect the existence of multiple routes for ascorbate recycling. As well as enzymatic reduction by MDHAR and DHAR, non-enzymatic reactions may also contribute. Nevertheless, it will be interesting to analyze ascorbate pools in double or triple mdhar mutants generated by crossing. This work is currently underway.

A key outstanding question is the effect of the specific mdhar mutations on extractable MDHAR activity. To what extent does each gene contribute to the total leaf MDHAR capacity? Unfortunately, this enzyme is not so easy to measure, notably because the substrate, MDHA, is an unstable free radical that must be generated in the assay. I have not been able to resolve this question within the timescale of the thesis, but it will be addressed soon by developing appropriate procedures to enable an accurate assay of MDHAR activities in the different lines.

Irrespective of their relative contributions to total leaf MDHAR activity, the data provide further evidence that cytosolic isoform of enzymes involved in the ascorbate-glutathione pathway play key roles in responses to oxidative stress. First, analysis of different MDHAR transcripts showed that MDHAR2 expression is obviously induced in response to cat2-induced oxidative stress (Figure 4.4).

The second gene encoding a cytosolic isoform, MDHAR3, was also significantly induced in the cat2 background. In addition to database evidence that these genes are induced by oxidative stress in Arabidopsis, reports have described induction of certain MDHAR transcripts in other plants such as Brassica campestris [START_REF] Yoon | Molecular cloning of the monodehydroascorbate reductase gene from Brassica campestris and analysis of its mRNA level in response to oxidative stress[END_REF]. Our analyses of double mutants suggest that cytosolic MDHAR isoforms play a role in responses to intracellular oxidative stress. Based on the analysis of cat2 mdhar mutants, MDHAR3 may be less important than MDHAR2 because the block of MDHAR3 expression had little effect on the cat2 phenotype whereas the mdhar2 mutation largely abolished lesions and the induction of the SA pathway (Figure 4.5A-C). While the backcross analysis suggests that this effect was not all-or-nothing, it is remarkable because it shows that knocking out an additional antioxidative enzyme can down-regulate rather than up-regulate cat2-triggered cell death. It should be CHAPTER 4 111 noted, however, that this observation is consistent with previous reports on cat2 apx1 and cat2 gr1 lines (Mhamdi et al., 2010b;[START_REF] Vanderauwera | Extranuclear protection of chromosomal DNA from oxidative stress[END_REF]. It offers a further indication that oxidative stress-induced lesions are not simply the result of accumulated damage, but occur rather through a process of signaling linked to cell redox state.

As well as SA, JA is also an important defence hormone whose signaling is closely linked to ROS [START_REF] Maruta | Arabidopsis NADPH oxidases, AtrbohD and AtrbohF, are essential for jasmonic acid-induced expression of genes regulated by MYC2 transcription factor[END_REF]Han et al., 2013b). According to the marker transcripts analysis (Figure 4.7), JA signaling is activated by the cat2 mutation, consistent with a previous report of our group (Han et al., 2013b). Among all the four genes analyzed, PDF1.2 induction was abolished by loss of MDHAR2 function (Figure 4.6), but this effect was slight for other genes, suggesting that MDHAR2 may influence cat2-induced JA signaling rather than JA synthesis. Further analyses will be required to establish whether the effect on JA signaling is a secondary effect of the marked impact of mdhar2 on oxidative stress-induced SA signaling.

As a lesion-mimic mutant, cat2-induced lesion formation has been shown to share many features with the HR response, including induction of PR genes, accumulation of SA and phytoalexins, and enhanced resistance to pathogens [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]. The present results on SA signaling in the cat2 mutant are therefore consistent with previous reports. Strikingly, even though neither the mdhar1 nor the mdhar3 mutation markedly affected gene expression in cat2, the specific loss of MDHAR2 function almost completely abolished the cat2-induced accumulation of SA and SA-associated transcripts. Since the cat2 lesion phenotype has been shown to be dependent on SA synthesis through ICS1 [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF]Chapter 3), the effect of mdhar2 on this phenotype likely occurs by suppression of the SA pathway during oxidative stress.

What could be the link between MDHAR2 and uncoupling of the SA pathway from oxidative stress?

Oxidative stress in cat2 is accompanied by the induction of multiple oxidative marker transcripts and antioxidative enzyme activities [START_REF] Noctor | Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation[END_REF]. Our analysis revealed that the up-regulation of APX, one of the major antioxidative enzymes, is partly antagonized by the mdhar2 mutation when plants are exposed to intracellular oxidative stress caused by the cat2 mutation. This effect is also observed at the level of APX transcripts, with a decreased induction of the gene encoding cytosolic APX1 in cat2 mdhar2 (Figures 4.8 and 4.9). These effects may be a compensatory response to perturbed regeneration of ascorbate in conditions that impose an accelerated oxidation of this molecule. This would be consistent with the decreased ascorbate pools in cat2 mdhar2 (Figure 4.10), possibly caused by an imbalance between ascorbate and MDHA. The plant might be configured to respond by measures that decrease the efficiency of ascorbate oxidation to slow down the flux through the pathway. Further evidence that redox state is perturbed when MDHAR2 function is lost in the cat2 background comes from the decreased induction of UGT73B3 transcripts and decreased CAT activity.

All these observations reveal that MDHAR2 plays some role in regulating plant redox state in response to intracellular oxidative stress caused by enhanced H2O2 availability.

Whether or not flux through the ascorbate-glutathione pathway is indeed decreased in response to loss of MDHAR2 function, a striking effect is enhanced oxidation of glutathione in cat2 mdhar2 (Figure 4.10). As previously described, the cat2 mutation alone causes a marked oxidation of glutathione, and this effect is somehow required for activation of the SA pathway by oxidative stress (Mhamdi et al., 2010b;Han et al., 2013a). The enhanced oxidation in cat2 mdhar2 suggests that MDHAR2 is required for maintaining glutathione redox state in oxidative stress conditions, even though glutathione is not a substrate or a product of the MDHAR reaction. The simplest explanation is that decreased capacity to regenerate ascorbate from MDHA leads to enhanced dismutation of MDHA to DHA, leading in turn to faster rates of DHA-dependent glutathione oxidation. Indeed, this explanation receives support from kinetic modelling analyses that suggest there is an inverse relationship between MDHAR capacity and glutathione oxidation in oxidative stress conditions [START_REF] Tuzet | Analyzing the function of catalase and the ascorbateglutathione pathway in H2O2 processing: Insights from an experimentally constrained kinetic model[END_REF].

As well as documenting an important role for MDHAR2 in oxidative stress responses, the data presented in this chapter offer an intriguing explanation of the effects of loss of G6PD5 function discussed in Chapter 3. As outlined in Chapter 3, the g6pd5 mutation abolishes the cat2 lesion phenotype and modifies cat2 transcriptomes in a similar manner to sid2. While work by our group and others has shown that various loss-of-function mutants for reductive pathways of H2O2 metabolism (ascorbate-glutathione-NADPH systems) can impact cat2 responses, the mdhar2 and g6pd5 mutations CHAPTER 4

113 are those we have so far identified that produce the most striking effects on the SA pathway and lesion formation. Both genes encode cytosolic proteins, one that produces NADPH (G6PD5) and one that requires NADPH (MDHAR2). One possibility, therefore, is that at least part of the effect of the g6pd5 mutation is due to compromised production of NADPH for MDHAR2 activity. Since glutathione status is a critical factor for induction of the SA pathway by oxidative stress (Han et al., 2013a;[START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF], the hyper-oxidation of glutathione linked to compromised MDHAR activity may be key in suppressing induction of the SA pathway in cat2. This could occur either by direct genetic loss of MDHAR2 function or sub-optimal production of NADPH for MDHAR2 when G6PD5 function is lost. Evidence that this is an interesting avenue to pursue comes from data showing that g6pd5 produces a similar effect on cat2 glutathione status to mdhar2 (Amna Mhamdi, unpublished results).

In conclusion, the results in this chapter suggest that a specific isoform of MDHAR is replaceable in unchallenging conditions but is particularly important in response to oxidative stress. In oxidative stress conditions, the mdhar2 mutation mimics the effects of the sid2 and g6pd5 mutations described in Chapter 3. This observation, and some of the perspectives it raises, will be discussed further in the next chapter.

CHAPTER 5 GENERAL CONCLUSIONS AND PERSPECTIVES

CHAPTER 5 GENERAL CONCLUSIONS AND PERSPECTIVES 115

Conclusions

Numerous studies have revealed that H2O2 plays an important role in response to various environmental constraints and that this mainly occurs through its effect as a signaling molecule rather than as a toxic byproduct. Although a lot of attention has focused on H2O2 accumulation, metabolism and its role in signal transduction, many of the underlying mechanisms that convert redox changes to phenotypic and functional outputs still remain to be elucidated.

A primary factor in governing oxidative signaling is the rate of H2O2 processing systems by various systems existing in plants. Among these systems, CATs and the ascorbate-glutathione pathways are recognized as key players. In Arabidopsis, CAT2 has been demonstrated to be the major isoform of CAT in leaves and its function has been shown to be closely linked to photorespiration. In contrast, the contribution of CAT1 and CAT3 to physiological processes is less well established. NADPH is one of the components involved in both ROS metabolism and production. At the outset of my thesis, work in our group had found that loss of function of a specific enzyme catalyzing NADPH production, G6PD5, abolished H2O2-triggered lesion formation in cat2. However, the underlying mechanism was unknown. The role of the ascorbate-glutathione pathway in processing H2O2 largely depends on NADPH, as this co-factor is the source of reductant for MDHAR and GR. When I began this work, little information was available on the importance of specific isoforms of MDHAR in response to oxidative stress. The following sections outline how my work has provided new information on these different aspects of H2O2 metabolism and its consequences.

The function of specific CAT isoforms

Most studies on CAT have focused on roles of this enzyme in leaves. In the work reported in Chapter 2, the role of CAT in roots was also investigated. Functional analysis by using different CAT single and double mutants showed that CAT2 is the most important isoform in roots as well as in leaves. The decreased root length caused by the cat2 mutation was partly rescued in the presence of sugar.

Moreover, the hCO2 growth condition rescued it almost completely. As is well established, the hCO2 condition blocks photorespiration in photosynthetic cells, so the cat2 root phenotype is probably a secondary nutritional effect of insufficient CAT activity in leaves. The data from CAT activities and plant growth suggest that, compared to CAT2, the other two CATs do not play a major role in plant development in non-stressful conditions.

Previous work in our laboratory showed a day length-dependent effect of the cat2 mutation in that the transcripts of rapidly induced oxidative stress marker genes accumulated more strongly in SD than in LD (Queval et al., 2007[START_REF] Queval | Daylength is a key regulator of transcriptomic responses to both CO2 and H2O2 in Arabidopsis[END_REF]. However, it was still unclear whether this effect of day length was simply caused by the duration of oxidative stress or some other factor related to photoperiod. In this work we avoided the differences in the duration of exposure to photorespiration by keeping plants grown in LD or SD hCO2 to a similar development stage before inducing oxidative stress.

Consequently, the analysis of H2O2 marker gene expression after an equal-time exposure to air revealed that the day length-dependent effect is not simply due to the differences in the duration of stress.

Effect of G6PD5 on the H2O2-induced SA signaling pathway

Based on the crucial role of CAT2 in consuming H2O2 produced in photorespiration, a T-DNA knockout cat2 mutant has been extensively used as a tool to mimic oxidative stress. Unpublished work in our laboratory reveals that the loss of function of G6PD5, a gene involved in NADPH production, abolished the lesion phenotype as well as a large part of the cat2 transcriptome. Interestingly, these effects are quite similar to those caused by the sid2 mutation. With the aim of identifying the underlying mechanisms via an unbiased approach, we used a forward genetics strategy to try to find revertants of the cat2 g6pd5 phenotype. As described in Chapter 3 of the thesis, two genes have so far been identified with some confidence. In addition, novel candidate genes were identified in two other revertants but further work is required to identify exactly which genes are regulating lesion formation in cat2 g6pd5 in these lines.

The first two genes that we have identified are MIPS1 and CPR5. Mutants for both of these genes have previously been found to stimulate lesion formation in Arabidopsis [START_REF] Bowling | The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance[END_REF][START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF]Donahue et al., 2010). MIPS1 catalyzes the conversion of glucose-6-phospate to myo-Inositol phosphate, which shares the same substrate with G6PD5 in the cytosol. In addition, the lesion formation in mips1 is linked to day length or light regime and is regulated by SA, features that are quite similar to those observed for cat2-triggered lesions. In contrast to mips1, cpr5 displays a day length-independent lesion phenotype, even though it is linked with HR response [START_REF] Bowling | The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance[END_REF]. Further work is required to establish the linkage between these genes and the cat2 mutation in determining lesion formation. Possible approaches will be discussed in the following perspectives section.

Functions of specific MDHAR isoforms in response to oxidative stress

As one of the enzymes in the ascorbate-glutathione pathway, the function of MDHAR requires NADPH as an electron donor. These enzymes are considered to function in maintenance of the reduced state of ascorbate, together with DHAR. However the roles of specific MDHAR isoforms are still unclear. As noted in Chapter 4, recent work in our laboratory showed that the loss of DHAR function did not affect the leaf ascorbate pools in either optimal or oxidative stress conditions [START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF]. Thus, we explored the potential importance of MDHAR in the function of the NADPH-dependent ascorbate-glutathione pathway.

The functional analysis in Chapter 4 showed that single mdhar1, mdhar2 and mdhar3 mutations did not affect the phenotype markedly in standard conditions, suggesting functional redundancy among these genes or with other systems. This is further confirmed by similar redox states compared with the wild-type, as indicated by analysis of ascorbate, glutathione, and oxidative marker transcripts. The mutants also showed similar levels of PR gene expression and SA content to the wild type.

Interestingly, transcripts analysis in the cat2 background suggested that the cytosolic isoforms MDHAR2 and MDHAR3 are involved in responses to intracellular oxidative stress. Direct analysis of double mutants to test this hypothesis showed that MDHAR2 is important for lesion formation and induction of the SA pathway in cat2. Subsequently, SA content analysis confirmed the effect of MDHAR2 on cat2-induced SA signaling in that the loss of MDHAR2 function largely abolished the cat2-induced SA accumulation. Activation of JA signaling by the cat2 mutation was also affected by the mdhar2 mutation, albeit to a lesser extent than up-regulation of the SA pathway.

In addition to the effect on lesion formation and phytohormone pathways, MDHAR2 is involved in the up-regulation of antioxidative marker transcripts and enzyme activities in cat2. Our analysis showed that both accumulation of APX1 transcripts and induction of extractable APX activity, which are observed in cat2, were partly antagonized by the loss of MDHAR2 function. One explanation of this effect could be that the plant responds to decreased efficiency of ascorbate regeneration by decreasing the capacity for H2O2-dependent ascorbate oxidation by APX. Some evidence that ascorbate pools were affected by loss of MDHAR2 function is apparent from decreased ascorbate in cat2 mdhar2.

Intriguingly, however, redox analysis also revealed an effect of the mdhar2 mutation on cat2-induced glutathione oxidation, an effect that is discussed further below. Together, these observations suggest that MDHAR2 is required for maintaining redox homeostasis in oxidative stress conditions.

Perspectives

Specificity of CAT functions and interactions between oxidative signaling and day length

The work reported in Chapter 2 underscored the crucial role of CAT2 in plant growth and development. In addition to the well described effect on leaf CAT activity, loss of CAT2 function markedly decreased the root CAT activity and this was accompanied by a decrease in root length. By contrast, loss of either CAT1 or CAT3 function did not produce a substantial effect on plant growth.

One possibility is that there is functional redundancy between these two genes. Because of the contiguous position of the two genes, it is very difficult to test this hypothesis by simple crossing.

Although the cat2 mutation strongly affects root and shoot growth, linked to an 80% decrease in extractable CAT activity, the mutation is not lethal under standard growth conditions. This raises the question of whether plants can survive without any CAT at all. Analysis of this question would require production of triple loss-of-function lines, in which all three genes are inactivated. Recently, a preliminary analysis of such lines was reported, by using triple mutants by CRISPR/Cas9 technology to inactivate CAT1 expression in the cat2 cat3 double mutant [START_REF] Su | The Arabidopsis catalase triple mutant reveals important roles of catalases and peroxisome derived signaling in plant development[END_REF]. While the authors reported that the plants showed a somewhat exacerbated phenotype compared to cat2 cat3, no lethality was observed [START_REF] Su | The Arabidopsis catalase triple mutant reveals important roles of catalases and peroxisome derived signaling in plant development[END_REF].

Data shown in Chapter 2 provide evidence that the effect of day length on oxidative signaling in cat2

is not simply the result of differences in oxidative stress. One possibility is that the effect of day length is somehow linked to differences in photosynthesis. Alternatively, some light-dependent signaling pathway that is differentially active in SD and LD growth conditions might modulate the response to oxidative stress. Further analysis of photosynthetic characteristics in LD and SD could throw some light on the first possibility. One way to test a signaling role of day length would be to cross cat2 with photoreceptor mutants and analyze whether the effect of day length on marker transcripts is altered.

Photoreceptors include phytochromes and cryptochromes, both of which have been implicated in some stress responses as well as in light signaling [START_REF] Kangasjärvi | Photosynthesis, photorespiration, and light signalling in defence responses[END_REF]. Such genetic resources are currently being characterized in the laboratory.

Further analysis of revertant mutations that allow lesion formation in cat2 g6pd5

As noted in Chapter 3, two genes have been identified in the revertant screen. Genome sequencing results suggest that mutations in MIPS1 and CPR5 were responsible for the reversion of the lesion phenotype in four lines of the mutated cat2 g6pd5 plants. Given that three MIPS1 alleles have been identified, it seems highly likely that inactivation of this gene function is responsible for reversion of the cat2 g6pd5 phenotype in these lines. Like mips1, cpr5, which was the top hit in another revertant line, is known to promote lesion formation and PR responses. As recognized lesion-mimic mutants, both mips1 and cpr5 show spontaneous lesion formation [START_REF] Bowling | The cpr5 mutant of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance[END_REF][START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF]Donahue et al., 2010), albeit with some phenotypic differences and a different dependence on growth conditions. Further work is envisaged to establish the extent to which these genes are involved in mechanisms by which g6pd5 mutation prevents cat2-triggered lesion formation and SA signaling.

Phenotype analysis of the mdhar single mutants showed no difference when compared with the wild type, suggesting functional redundancy among MDHAR isoforms or with other systems in standard conditions. Therefore it would be interesting to produce the mutants with loss of function of multiple MDHAR isoforms. Based on the location of subcellular MDHARs, crosses between MDHAR1 and MDHAR4, as well as between MDHAR2 and MDHAR3 seem to be most interesting, because the first two genes are targeted to the peroxisome and the second two are targeted to the cytosol. Furthermore, according to the effects of different MDHAR genes, especially MDHAR2, in response to oxidative stress as reported in Chapter 4, the study of multiple MDHAR mutations in oxidative stress could help us to understand more about the function and relationship of different MDHAR isoforms. With this objective, it would be interesting to produce triple mutants of cat2 mdhar1 mdhar4 and cat2 mdhar2 mdhar3. . The production of such double and triple mutants is in progress.

In Chapter 4 of the thesis, by a direct genetic approach, it was shown that MDHAR2 is required for cat2-triggered lesion formation and SA signaling pathway. As the laboratory had previously established for the g6pd5 mutation, the loss of MDHAR2 function largely abolished PR gene expression and SA accumulation induced by cat2. As noted in the introduction, SA signaling is involved in resistance to certain types of pathogen, and is also involved in localized cell death that occurs to restrict pathogen spread within the plant (HR response; [START_REF] Mur | The hypersensitive response; the centenary is upon us but how much do we know?[END_REF]. In cat2, SA accumulation occurs alongside HR-like lesion formation, and is accompanied by increased resistance to 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]Han et al., 2013a). Thus, it seems that the mdhar2 mutation, like that for G6PD5, abolishes PR responses that are triggered by perturbation of intracellular redox state.

Hence, it would be interesting to do pathogen tests on cat2 mdhar2 mutants, to confirm that the effect on SA-dependent responses is accompanied by a loss of cat2-induced resistance.

At present, we do not have an allelic line of mdhar2 to confirm the effect of MDHAR2 in oxidative stress responses. Even though the backcross of cat2 mdhar2 with cat2 showed a phenotype segregation ratio close to 3:1, the semi-quantitative nature of the phenotype analysis does not provide an absolute confirmation of the link between mdhar2 genotype and phenotype. Hence, we cannot discount a possible influence of other unknown T-DNA-related mutations in the single line of mdhar2 and cat2 mdhar2. In the absence of an allelic mutant line, it would therefore be very interesting to complement the mdhar2 and cat2 mdhar2 lines with the wild-type MDHAR2. One possibility would be to use a construct to express MDHAR2-GFP, as this would enable the subcellular compartmentation of MDHAR2 to be confirmed as well as functional analysis to evaluate if the complementation restores the phenotype and SA responses in cat2 mdhar2.

A key point is the contribution of the specific MDHAR isoforms to total activity in leaves. Thus far, I

have not been able to do these measurements. One difficulty is that the unstable nature of the substrate, MDHA, which is a free radical, makes the assay somewhat more difficult than those of the other antioxidative enzymes I measured during this work. Nevertheless, such experiments are important, and likely to add useful information to clarify the contribution of specific MDHAR isoforms to overall activity. The technique will be developed soon, with the view of analyzing the impact of the different mdhar mutations on enzyme activity in Col-0 and cat2 backgrounds.

A functional link between G6PD5 and MDHAR2 in H2O2 signaling?

One intriguing avenue of enquiry generated by this work is the possibility that specific isoforms of G6PDH and MDHAR are critical in determining signaling outcomes of intracellular oxidative stress.

As noted in Chapter 4, the loss of function of MDHAR2 largely abolished the lesion phenotype and activation of SA signaling induced by cat2. The analyses conducted so far suggest that these effects are quite similar to those produced by the mutation of g6pd5 (Chapter 3). G6PDH is one of the sources of NADPH for the function of MDHAR in the ascorbate-glutathione pathway. Moreover, both G6PD5

and MDHAR2 are targeted to the cytosol. These characteristics suggest that the effect of the g6pd5 mutation in the cat2 background could be due to compromised production of NADPH for MDHAR2 activity. Another result that supports this interpretation is the hyper-oxidation of glutathione linked to the mdhar2 mutation. As shown in Chapter 4, cat2 mdhar2 shows a significantly more oxidized glutathione status compared to cat2. This effect is consistent with modelling analyses that suggest that MDHAR is a key fulcrum that determines the degree of glutathione oxidation in response to increased H2O2 availability [START_REF] Tuzet | Analyzing the function of catalase and the ascorbateglutathione pathway in H2O2 processing: Insights from an experimentally constrained kinetic model[END_REF]. Intriguingly, a similar hyper-oxidation of glutathione has also

CHAPTER 5 GENERAL CONCLUSIONS AND PERSPECTIVES

123 been found in cat2 g6pd5 (Amna Mhamdi, unpublished results). Previous reports point to a crucial role for glutathione status in linking SA signaling pathway with intracellular oxidative stress (Han et al., 2013a;[START_REF] Rahantaniaina | Cytosolic and chloroplastic DHARs cooperate in oxidative stress-driven activation of the salicylic acid pathway[END_REF].

A scheme that summarizes the possible interactions is shown in Figure 5.1. Down-regulation of MDHAR may compromise MDHA reduction to ascorbate, allowing more MDHA to dismutate to DHA, which engages the glutathione pool and leads to increased glutathione oxidation (Figure 5.1).

Exacerbated oxidation of glutathione caused by the mdhar2 mutation may lead to the inhibition of SA pathway induction in cat2. All these effects could also occur by indirect perturbation of MDHAR function when NADPH availability is compromised in cat2 g6pd5 (Figure 5.1). To provide a first confirmation of this hypothesis, it would be interesting to do a side-by-side analysis of glutathione and ascorbate in cat2 mdhar2 and cat2 g6pd5. Alongside the direct transcriptomic comparison of cat2 g6pd5 and cat2 mdhar2, it could also be interesting to identify whether there is overlap between MDHAR2 and G6PD5 interactants at the protein level. This can be achieved by analysis of protein interactions. For example, pulldown experiments can identify unknown proteins interacting with a protein of interest, using tags such as GFP fused to the protein of interest. Pulldown experiments using a G6PD5-GFP construct have already identified a number of possible in planta protein partners for G6PD5 (Amna Mhamdi, unpublished results). The MDHAR2-GFP constructs which will be produced for transformation of cat2 mdhar2 (Section 5.2.3) could also be applied in pulldown experiments to find possible proteins interacting with MDHAR2. Thereafter, the protein partners of G6PD5 and MDHAR2 can be compared to see how much overlap there is between them. This will help us to further understand the relationship between G6PD5 and MDHAR2 in H2O2 signaling.

CHAPTER 6 MATERIALS AND METHODS

CHAPTER 6 MATERIALS AND METHODS 125 6.1 Plant materials and growth conditions

Plant materials

The T-DNA mutants were obtained from Nottingham Arabidopsis Stock Centre (NASC), Nottingham University, UK. While cat2, cat3, mdhar1, mdhar2 and mdhar4 are from the SALK collection and are in the Col-0 background, cat1 and mdhar3 are in the Col-3 background. The polymorphisms were SAIL_311_d07 for cat1, SALK_076998 for cat2 (cat2-1, [START_REF] Queval | H2O2activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast[END_REF], SALK_092911 for cat3, SALK_034893 for mdhar1, SALK_028874 for mdhar2, SAIL_31_A09 for mdhar3, SALK_015596 for mdhar4. Genotyping was performed by PCR using the primers listed in Table 6.1. Double mutants were generated by crossing cat2 with the other single mutants. After heterozygote verification for both genes in the F1 generation by PCR, double homozygotes were identified in F2 in the same way. Thee F3 seeds produced were used for experiments. This work was accomplished with the collaboration of my colleagues in the group.

Growth and sampling

For plants grown in soil or on agar, seeds were first placed at 4 °C for 2 d in the dark and then transferred to a controlled-environment growth chamber in LD (16 h light/8 h dark) or SD (8 h light/16 h dark), an irradiance of 200 µmol m -2 s -1 at the leaf surface, 20°C/18°C, 65% humidity, and given nutrient solution twice per week. CO2 concentration was maintained at 400 µL L -1 (air) or 3000 µL L -1 (hCO2). For chemical treatment, the surface of the leaf was sprayed with 2 mM 3-AT and samples were collected 8 h later. Once collected at the stated developmental stage, samples were immediately frozen in liquid nitrogen and stored at -80 °C. Unless otherwise stated, data are means ± SE of three independent experiments with samples from different plants.

Methods

Phenotypic analysis and lesion quantification

Rosette diameter, leaf number and fresh weight from at least 18 plants of each genotype were quantified for phenotypic comparison. Photographs were also taken at the same time and then analyzed with IQ materials software to estimate the percentage of the rosette area that showed lesions.

DNA extraction and plant genotyping

Tissue samples were pre-cooled together with beads in 2 ml eppendorf tubes by liquid nitrogen. Then fine powder was obtained using a bead shaker. After adding 400 µl extraction buffer (200 mM Tris pH 7.5, 250 mM NaCl, 25 mM EDTA and 0.5% SDS), the solution was thoroughly vortexed and centrifuged at 11,000 rpm for 10 min at 4°C. 200 µl supernatant was mixed well with 200 µl isopropanol by vortex and centrifuged at 11,000 rpm for 10 min at room temperature. The pellet was washed with 1 ml 70% ethanol (v/v) and then dried in a fume hood. The pellet was redissolved in 100 µl sterile water. For T-DNA insertion lines, leaf DNA was amplified by PCR using primers specific for left T-DNA borders and the respective gene coding sequences (See Table 6.1).

RNA extraction and transcripts analysis

Total RNA was extracted with Trizol following the manufacturer's protocols. Around 100 mg leaf sample was thoroughly ground using a bead shaker following pre-cooling with beads in a 2 ml eppendorf tube in liquid nitrogen. For qPCR, the samples were shaken twice. Then 1 ml Trizol was added and the tube vortexed. For qPCR, the homogenate was incubated for 5 min at room temperature. 200 µl chloroform was added and mixed well by gentle shaking for 15 s. After incubation for 2-3 min at room temperature, the solution was centrifuged at 11,000 rpm for 15 min at 4°C. The upper aqueous phase was transferred into 200 µl isopropanol and vortexed. The solution was incubated at room temperature for 10 min and then centrifuged at 11,000 rpm for 10 min at 4 °C. The precipitate was CHAPTER 6 MATERIALS AND METHODS 127 washed with 1 ml 70% ethanol (v/v) and centrifuged for another 5 min. The remaining liquid was removed and the pellet was dried at room temperature for 10 min. Then 40 µl RNase-free water was added, and the pellet resuspended by 10 min incubation at 65°C. The RNA solution was stored at -80°C.

The RNA concentration was estimated using a NanoDrop spectrophotometer. Then 4 µg RNA was reverse-transcribed using imPromll reverse transcriptase (Promega). For semi-quantitative RT-PCR, ACTIN2 transcripts were used as a constitutive control. Quantitative real-time RT-PCR was performed with the LightCycler® 480 SYBR Green I Master (Roche) according to [START_REF] Queval | H2O2activated up-regulation of glutathione in Arabidopsis involves induction of genes encoding enzymes involved in cysteine synthesis in the chloroplast[END_REF]. For qPCR, both ACTIN2 and RCE1 transcripts were used as controls. Gene-specific primer sequences used in this study are listed in Table 6.1. For CAT, GR and DHAR activity analysis, around 150 mg leaves or 100 mg roots from different plants was ground in liquid nitrogen with 50-100 mg PVP. Following grinding, 1.5 ml 0.1 M NaH2PO4 (pH 7.5) containing 1 mM EDTA was added immediately. If APX activity was involved in the analysis, 1 mM L-ascorbic acid was included in the buffer. Once thawed, the homogenate was transferred into a 2 ml Eppendorf tube, and centrifuged at 14,000 rpm for 10 min at 4°C. 1.1 ml supernatant was transferred into a fresh tube, then 0.5 ml was desalted on a NAP-5 column that had been previously washed with 10 ml sterile water and pre-equilibrated with 10 ml 0.1 M NaH2PO4 containing 1 mM EDTA (pH 7.5). After desalting, the elution was performed with 1 ml of the same buffer. To prepare proteins for analysis of APX activity, pre-equilibration and elution were carried out separately with 0.1 M NaH2PO4 containing 1 mM EDTA and 1 mM L-ascorbic acid (pH 7.5).

Activity assay

Extractable enzyme activities were measured as described by [START_REF] Noctor | Oxidative stress and antioxidative systems: Recipes for successful data collection and interpretation[END_REF]. CAT activity was measured by H2O2 consumption at 240 nm. 20 µl H2O2 was added to 880-930 µl 0.1 M NaH2PO4, 1 mM EDTA (pH 7.5). The reaction was started by the addition of 50-100 µl desalted extract. The absorbance decrease at 240 nm was monitored for 1-2 min, and the linear part of the curve (first 30-60 s) was used for calculation. APX activity was measured as ascorbate oxidation at 290 nm. 50 µl 10 mM L-ascorbic acid was added to 890 µl 0.1 M NaH2PO4, 1 mM EDTA (pH 7.5), followed by the addition of 50 µl desalted extract. Then, the absorbance decrease at 290 nm was monitored for 2 min after adding 10 µl 20 mM H2O2. GR activity was measured as the absorbance decrease at 340 nm caused by NADPH oxidation. 10 µl 10 mM NADPH was added to 880 µl 0.1 M NaH2PO4, 1 mM EDTA (pH 7.5), followed by 100 µl desalted extract. Then the reaction was started by adding 10 µl 50 mM GSSG and the decrease in 340 nm was monitored for 2 min. DHAR activity was monitored as the increase in 265 nm due to DHA reduction to ascorbate. 50 µl 4 mM DHA and 25 µl 100 mM GSH were added to 905 µl 0.1 M NaH2PO4, 1 mM EDTA (pH 7.0). Then the absorbance increase at 265 nm was monitored for 2 min after the addition of 20 µl desalted extract.

Ascorbate analysis

Ascorbate measurements were done by monitoring the A265 which is specifically removable by ascorbate oxidase (AO). For ASC, the reaction system contained 100 µl 0.2 M NaH2PO4 (pH 5.6), 55 µl H2O and 40 µl neutralized extract. Following programmed shaking (three times), the A265 was read.

Then, 5µl AO (40 U/ml) was added and A265 was recorded again after 5 min. For total ascorbate, 100 µl neutralized extract was incubated with 140 µl 0.12 M NaH2PO4 (pH 7.5) and 10 µl 25 mM DTT for 30 min at room temperature. This treatment converts DHA to ASC. Then, the assay was performed as described for ASC. Triplicate assays were done for each extract in the same plate (Queval and Noctor, 2007).

Total SA assay by High Performance Liquid Chromatography (HPLC)

Total SA was measured following a protocol described in [START_REF] Langlois-Meurinne | Pathogen-responsive expression of glycosyltransferase genes UGT73B3 and UGT73B5 is necessary for resistance to Pseudomonas syringae pv tomato in Arabidopsis[END_REF].

Approximately 200 mg leaf sample was ground in liquid nitrogen and homogenized with 1.5 ml 90% methanol. Extracts were vortexed and centrifuged at 16000 g for 15 min at 4 ˚C. The supernatant was transferred to a new tube and set aside, and the precipitate was re-suspended with 0.5 ml 100% methanol by vortex. The supernatant obtained by centrifuging for 15 min at 16000 g was then mixed

with the first supernatant. Subsequently, the combined supernatant was recentrifuged to produce the final supernatant, which was dried in a speed-vac for at least two hours. The dried extracts were subjected to acid hydrolysis by adding 600 µl 3 N HCl and incubating for 45 mins at 80 ˚C. 1 ml diethylether was added to the extracts. After vortexing for 20 mins, the preparation was centrifuged for 1 min at 16000 g, and the organic phase was transferred to a new tube. The remaining liquid was reextracted by adding another 1 ml diethylether and the organic phase was mixed with the first one. The combined organic phase was evaporated to dryness in the fume hood. Finally, the dried extracts were dissolved in 200 µl resuspension buffer for HPLC analysis. Identification and quantification were performed by comparison of peaks with SA standards.

ROS visualization in roots

ROS visualization was performed using DCFH2-DA staining, following a method adapted from Mhamdi et al. (2010b). 10 d-old seedlings were incubated in 50 µM staining solution at room temperature for 20 min in dark. Excess staining solution was removed by washing seedlings three times in phosphate buffer for 5 min in the dark. The fluorescence was observed with a Zeiss Axio Imager 2 fluorescence microscope.

EMS screen

EMS mutagenesis

Two 50 ml plastic tubes were prepared, each containing approximately 1000 seeds of cat2 g6pd5, and 25 ml 0.3% EMS (300 µl EMS stock solution/100 ml distilled water) was added. The tubes were incubated for 15 h at room temperature with gentle rotating. Because of the hazardous nature of EMS, a face-mask and gloves were worn, and all procedures were carried out in a fume hood. After the incubation with EMS, the M1 seeds were rinsed eight to nine times with 50 ml sterilized water. The waste washing solution was poured into a special container and deactivated with 0.1 N NaOH solution for 12 h in a fume hood. For the final rinse, seeds were kept in water and rotated for at least 1 h. Waste water was then poured off and the seeds were suspended with 0.1% agarose (0.1 g agarose powder/100 ml TBE).

The seeds were pipetted one by one into soil (9 seeds per square pot) immediately after mutagenesis.

In total, approximately 1700 seeds were sown in the experiment. After vernalization for 2 d in the cold room, plants were transferred into a controlled-environment growth chamber in LD (16 h light/8 h dark), an irradiance of 200 µmol m -2 s -1 at leaf level, 20°C/18°C. To assess the EMS effect and the efficiency of the mutagenesis treatment, we quantified embryo lethality in M1 plants. Five plants were randomly chosen and 10 mature siliques from each plant were dissected under a stereomicroscope.

Fruits were categorized into three classes based on the visual determination of the number of normal and aborted seeds in each silique. Class A has a proportion of 3:1 or smaller (aborted seeds﹥20 %), class B has a proportion between 4:1 and 13:1 (20 % ≥ aborted seeds﹥6.7 %), and class C has a proportion of 14:1 or larger (aborted seeds﹤6.7%). A frequency of fertile fruits (B+C) between 2% and 35% is regarded as an acceptable range to continue the work [START_REF] Martín | A high-density collection of EMS-induced mutations for TILLING in Landsberg erecta genetic background of Arabidopsis[END_REF].

Phenotype screen

M2 seeds were harvested from all plants in bulk. The screen was carried out in a photoperiod of 16 h light/8 h dark with irradiance of 200 µmol m -2 s -1 at leaf level. Nine seeds were sown in each square pot and approximately 1600 plants were screened in each batch. Plants showing clear lesion phenotypes on the leaf surface were selected and genotyped by PCR to confirm that they were cat2 g6pd5 homozygotes.

Backcross with cat2 g6pd5

For identified cat2 g6pd5 M2 mutants that showed lesions, seeds of the next generation were grown to confirm the phenotype and genotype. At least twelve M3 plants of each potential mutant line were grown to confirm the stability of the previously recorded phenotype. For those lines in which all M3 plants showed clear lesions and which were reconfirmed as cat2 g6pd5 homozygotes, plants were backcrossed with the non-mutagenized parental line cat2 g6pd5. Theoretically, the first filial generation (F1) plants obtained by backcross should be heterozygotes for the causal mutation. The F1 phenotype gives an important indication of whether the mutation is dominant or recessive. A lesion phenotype in all F1 plants indicates that the mutation is dominant; if none show lesions, the causal mutation is likely to be recessive.

F2 progeny were obtained by self-pollination of F1 plants. Seeds were collected from single plants and more than 60 seeds from four F1 plants were sown for phenotype segregation analysis. Those showing obvious lesions were identified, the genotype was confirmed again by PCR, and the segregation ratio was calculated. This ratio provides an indicator of the number of potential causal mutations in a given with 0.5 ml 70% ethanol, then dried in air for at least 15 min before being resuspended in 55 µl

DNAse-free water. The DNA concentration was determined with a NanoDrop spectrophotometer.

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 ttest at P < 0.05. réponses au stress explique l'explosion de l'intérêt porté à la recherche sur ces molécules au cours des dernières décennies. Alors que les concepts antérieurs considéraient les ROS comme des sous-produits toxiques, ils sont désormais considérés comme des molécules de signalisation clés impliquées dans de nombreux processus physiologiques (Foreman et al., 2003;[START_REF] Wagner | The genetic basis of singlet oxygen-induced stress responses of Arabidopsis thaliana[END_REF]Han et al., 2013a, b;[START_REF] Foyer | Viewing oxidative stress through the lens of oxidative signalling rather than damage[END_REF] Title : Functional analysis of catalase mutants and their application to the analysis of NADPH-linked pathways in oxidative signaling in Arabidopsis thaliana Keywords : catalase, NADPH, oxidative stress, H2O2, signaling Abstract: Stress conditions lead to modified redox states and signaling linked to reactive oxygen species (ROS), whose cellular concentrations are regulated by complex antioxidative systems. While the importance of ROS in cell signaling remains very fragmentary. This work used forward and reverse genetics to analyze the roles of catalases and the NADPH-glutathione-ascorbate systems in H2O2 metabolism and related signaling in Arabidopsis thaliana. An analysis of T-DNA mutants revealed that cat2, but not cat1 or cat3, substantially impacted plant growth and development. These effects were annulled by growth at high CO2, suggesting that they were caused, directly or indirectly, by compromised capacity to metabolize photorespiratory H2O2. An analysis conducted in cat2 rosettes following growth in different photoperiods revealed that oxidative signaling is strongly influenced by day length in a manner that is independent of stress intensity. When cat2 is grown in long days, oxidative stress induces the salicylic acid (SA) pathway, leading to visible lesions on the leaves. This response to oxidative stress is annulled in cat2 g6pd5, which has additionally lost the function of a specific glucose-6phosphate dehydrogenase (G6PDH). A forward genetics approach identified several genes that may be involved in regulating SA-dependent lesion formation in this double mutant.

To explore the roles of specific monodehydroascorbate reductases (MDHAR) in optimal and stress conditions, insertion mutants for several MDHAR-encoding genes were obtained. One of them markedly altered induction of the SA pathway by oxidative stress when introduced into the cat2 background. Together, the results underline the importance of CAT2 and point to functional coupling between specific NADPH-producing G6PDH and NADPH-requiring MDHAR in oxidative stress signaling pathways in Arabidopsis.

Figure 1 . 1 .

 11 Figure 1.1. Generation of different ROS forms.

Figure 1

 1 Figure 1.3. A scheme explaining how ROS function at the cross-roads of various key signaling events. ROS work upstream and downstream of the other signaling components, e.g. membranes, NADPH oxidases, G-proteins, calcium, redox homeostasis, photosynthesis, MAPKs, plant hormones [such as salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), and ethylene] and transcription factors. Solid arrows for direct ROS interactions with other signaling components, dashed arrows for expected indirect interactions. Figure taken from Sewelam et al. (2016).

Figure 1 . 4 .

 14 Figure 1.4. Plant antioxidative systems and their localization within the cell. APX, ascorbate peroxidase. CAT, catalase. DHAR, dehydroascorbate reductase. GR, glutathione reductase. GRX, glutaredoxin. MDHAR, monodehydroascorbate reductase. NTR, NADPH-thioredoxin reductase. PRX, peroxiredoxin. SOD, superoxide dismutase. TRX, thioredoxin. Figure taken from Noctor et al. (2017).

Figure 1 . 5 .

 15 Figure 1.5. Two of the major pathways for H2O2 metabolism in plants. APX, ascorbate peroxidase; MDHA, monodehydroascorbate; MDHAR, monodehydroascorbate reductase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GSH, reduced glutathione; GSSG, glutathione disulfide; GR, glutathione reductase. Figure taken from Foyer and Noctor (2009).

  extensive and conducted at different levels(visible, molecular, biochemical). Key resources for reverse genetics are T-DNA and transposon-tagged mutant collections, in which expression of given genes are modified. More recently, TILLING (Targeting Induced Local Lesions in Genomes) has also become a commonly used reverse genetic strategy, in which random chemical mutagenesis is combined with PCR-based screening to identify point mutations in genes of interest. This technology allows a time and cost-saving discovery of induced point mutations in a big population of mutagenized individuals

  Figure 2.2A, left). The effects on growth were broadly correlated with ROS signals estimated using DCFH2-DA (Figure 2.2B and Supporting Information Figure. S2), with the strongest ROS signals observed in cat2 genotypes, although some

Figure 2 . 1 .

 21 Figure 2.1. Catalase transcript levels (A) and extractable catalase activities (B) in the roots of single and double catalase-deficient mutants. Plants were grown on 1/2MS ± sucrose for 14 d in LD air. Then approximately 100 mg root samples were collected for analysis. Left, no sucrose. Right, 1% sucrose. For transcript analysis, data are means ± SE of three (cat1, cat1 cat2) or six (other genotypes) biological replicates. For activities, all data are means ± SE of three biological replicates. * indicates significant difference from wild type at P < 0.05; + indicates significant difference between double mutants and cat2 at P < 0.05.

Figure 2 . 3 .

 23 Figure 2.3. Oxidative stress markers in the roots of single and double catalase-deficient mutants. Plants were grown in the presence and absence of sucrose. Left, no sucrose. Right, 1% sucrose. A, extractable ascorbate peroxidase activity. B, three oxidative stress marker transcripts (APX1, GSTU24, UGT73b3). Data are means ± SE of three biological replicates. * indicates significant difference from wild type at P < 0.05; + indicates significant difference between double mutants and cat2 at P < 0.05.

Figure 2 . 4 .Figure 2 . 5 .Figure 2 . 6 .49Figure 2 .

 2425262 Figure 2.4. Comparison of rosette phenotype in Col-0, cat2, cat3 and cat2 cat3. Plants were grown in LD hCO2 or LD air for 20 d. Data are means ± SE of 24 plants. Significant differences to Col-0 at P<0.05 are indicated by *. + indicates significant difference between cat2 cat3 and cat2 at P < 0.05.

(

  Figure 2.6B). Accordingly, CAT2 and CAT3 transcripts were not markedly different between plants kept at hCO2 or transferred to air for 8h (Supporting Information Figure. S8). As reported for roots (Figure 2.1), the cat3 mutation was associated with some induction of CAT1 transcripts, although this was independent of the CO2 regime (Supporting Information Figure. S8). Similarly, little evidence of marked up-regulation of other antioxidative enzymes was apparent within the short exposure to air (Supporting Information Figure. S9). It is also noteworthy that 3-AT did not affect the activity of these enzymes (Supporting Information Figure. S9).

Figure 2 . 7 .Figure 2

 272 Figure 2.7. Analysis of light-dependent induction of oxidative marker gene transcripts in Col-0 and cat2 after growth at different photoperiod regimes. Left, GSTU24. Right, UGT73b3. Plants were grown as described in Figure 2.6A in LD hCO2 (light grey column) or SD hCO2 (dark grey column). A set of Col-0 plants were sprayed with 3-AT and untreated and treated Col-0 plants were transferred to air alongside a group of cat2 plants. Samples were taken either immediately before (0h) or after the treatment/transfer (8h). A, Transcript abundance relative to constitutive genes. Top, Col-0. Middle, cat2. Bottom, Col-0+3-AT. Note the different y-axis scale for the bottom two graphs. Data are means ± SE of 3 biological replicates. *Indicates significant difference between LD and SD at P < 0.05. B, Logarithmic plots of transcript induction by transfer to air following growth at hCO2 in LD or SD. Data are means ± SE of 3 biological replicates. Mean values at 8h were divided by mean values at 0h. Significant difference for the three biological replicates at 0h and 8h is indicated at *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 2 . 9 .

 29 Figure 2.9. Analysis of effect of growth day length on oxidative marker gene transcripts in plants kept at hCO2. Plants were grown in LD hCO2 or SD hCO2 as for Figure 2.8 except that plants were kept at hCO2 instead of being transferred to air. Samples were taken for RNA extraction and analysis of oxidative stress marker transcripts. Data are means ± SE of 3 biological replicates. *Indicates significant increase at P < 0.05 compared to Col-0 in the same condition.

  Photos sequence

Figure 3 . 1 .

 31 Figure 3.1. Effects of sid2 mutation on cat2-induced lesion and SA signaling. Figures taken from Chaouch et al. (2010) A. Phenotype of rosette of plants grown in LD air for 25 d. B. PR1 transcript relative to ACTIN2 transcripts. C. SA accumulation in different genotypes. Results derived from metabolite profiling by GC-MS analysis.

  g6pd5 and sid2 secondary mutations produced a very similar effect and repressed many of these transcripts to the Col-0 level (Figure3.2C). These results demonstrate that g6pd5 mutation mimics the sid2 mutation in abolishing transcriptome responses triggered by intracellular H2O2. In order to get mechanistic insights on the function of G6PD5 in transducing H2O2 signals, a first strategy that the group took before the start of my PhD, was to analyze links between the production of NADPH by G6PDH and the function of NADPH-dependent RBOHs. These and other approaches are ongoing. In this chapter, I describe work that introduced a forward genetics approach to elucidate the role of G6PD5 in H2O2-triggered lesion formation.

Figure 3 . 2 .

 32 Figure 3.2. Effects of the secondary g6pd5 mutation on cat2 phenotype and genes expression. Figures taken from Mhamdi et al. (unpublished) A. Phenotypes of plants grown in LD air for 3 weeks from seeds. B. Transcriptome comparison among Col-0, cat2 and cat2 g6pd5. C. cat2 g6pd5 transcriptome overlap with cat2 sid2. The figure includes the top 50 transcripts highly induced in cat2 relative to Col-0.

Figure 3 . 3 .

 33 Figure 3.3. Screening strategy (A) and photographs of revertant plants (B). Photographs were taken after grown in LD air for three weeks.

Figure 3 . 4 .

 34 Figure 3.4. Example of backcross for line 9.11. This plant was found to show cell death during the original screen. After the phenotype and the cat2 g6pd5 homozygous genotype were confirmed in the next generation (M3), it was backcrossed to cat2 g6pd5. The cat2 g6pd5 genotype was confirmed for all the following generations. F1 plants did not show lesions (B1F1).Segregants showing lesions in the next generation (B1F2) were backcrossed to cat2 g6pd5 again. Then F2 plants with lesions (B2F2) were pooled for sequencing.

Figure 3

 3 Figure 3.5, after self-fertilization, line 9.11 showed phenotype segregation in F2. Plants showing lesions are homozygotes for the causal mutation, but those that are wild-type or heterozygous at the affected locus do not have a lesion phenotype. The more plants included in the pooled sample for DNA extraction, the higher the frequency of the causal mutation should be relative to non-causal mutations. Following the recommendations of our collaborators in Ghent, we used 50 lesion-bearing plants to obtain the pooled DNA (Figure 3.5).

Figure 3 . 5 .

 35 Figure 3.5. Principle of pooled F2 plants for DNA sequencing. After a cross between the revertant M9.11 and the parental line, F1 plants are heterozygous for the causal mutation and show no lesions. In F2, plants that show lesions should be homozygous for the causal mutation. Non-causal mutations present in the original revertant, defined as mutations that do not promote lesion formation, should distribute evenly and be less well represented in the sequencing data.

Figure 4 . 1

 41 Figure 4.1 Schematic diagram of T-DNA insertion in Arabidopsis mdhar mutants. Exons and introns are depicted to scale by boxes and lines, respectively. The coding region of the gene is shown as black boxes. The position of the T-DNA is indicated by the triangle and the arrow indicates the direction of the T-DNA insertion. Seeds of Arabidopsis (Arabidopsis thaliana) mutants in the ecotype Columbia (Col-0) background were obtained from the Nottingham Arabidopsis Stock Center (http://nasc.nott.ac.uk).

Figure 4 . 2

 42 Figure 4.2 Genotyping and transcripts (RT-PCR) of all mdhar single mutants (mdhar1, mdhar2, mdhar3, mdhar4) and phenotype. A. PCR analysis of homozygous single mdhar T-DNA insertion mutants. B. RT-PCR analysis of different mdhar single mutants. C. Photographs of plants grown in LD air for 3 weeks. D. Fresh weight of rosettes after grown in LD air for 3 weeks. Data are means of at least 10 plants. * Indicates significant differences between mdhar mutants and Col-0 at P < 0.05.

Figure 4 . 3 .

 43 Figure 4.3. Genotyping and transcripts analysis of cat2 mdhar1, cat2 mdhar2 and cat2 mdhar3. A. PCR analysis of all double mutants. B. Transcripts analysis by semi-quantitative RT-PCR.

Figure 4 . 4 .

 44 Figure 4.4. MDHAR genes expression in different mdhar mutants in Col-0 and cat2 background. Data are means ± SE of four biological replicates. * Indicates significant differences of mutants to Col-0 at P < 0.05; + indicates significant differences of double mutants compared with cat2 at P < 0.05.

Figure 4 . 5 .

 45 Figure 4.5. Effects of mdhar mutations on the cat2 phenotype and SA marker transcripts. A. Photographs of plants grown in LD air for 3 weeks. B. Effects of different mdhar mutations on cat2-triggered lesions. Data are means ± SE of 12 plants. * Indicates significant differences in percentage of lesions on cat2 background mutant leaves compared with cat2.

  Figure 4.6. Total leaf salicylic acid (SA) contents in the different mutants. Data are means ± SE of three biological replicates. * Indicates significant differences of mutants to Col-0 at P < 0.05; + indicates significant differences of double mutants compared with cat2 at P < 0.05.

Figure 4 . 7 .

 47 Figure 4.7. Effects of different mdhar mutations on cat2-induced JA-associated transcripts. Data are means ± SE of four biological replicates. * Indicates significant differences of mutants to Col-0 at P < 0.05; + indicates significant differences of double mutants compared with cat2 at P < 0.05.

Figure 4 . 8 .

 48 Figure 4.8. Major antioxidative enzyme activities in different mdhar mutants in Col-0 and cat2 background. Data are means ± SE of three biological replicates. * Indicates significant differences of mutants to Col-0 at P < 0.05; + indicates significant differences of double mutants compared with cat2 at P < 0.05.

Figure 4 . 9 .Figure 4

 494 Figure 4.9. Oxidative marker transcripts in different mdhar mutants in Col-0 and cat2 background. Data are means ± SE of four biological replicates. * Indicates significant differences of mutants to Col-0 at P < 0.05; + indicates significant differences of double mutants compared with cat2 at P < 0.05.

  enzyme isoforms in the response to oxidative stress. Previous studies from our and other laboratories have used CAT-deficient mutants to analyze this question, because decreased CAT capacity forces enhanced flux through the ascorbate-glutathione pathway, as has recently been

Figure 5 . 1 .

 51 Figure 5.1. Scheme showing possible interplay between G6PD5 and MDHAR2 in activation of the SA pathway through their effects on cytosolic glutathione status. G6PD5-supported MDHAR2 activity is required to allow efficient regeneration of ascorbate from MDHA and to avoid excessive production of DHA that could over-oxidize the glutathione pool and inhibit the activation of signaling responses such as the SA pathway.

  Chapter 3, the g6pd5 mutation largely abolishes the transcriptomic response in cat2, thereby mimicking the sid2 mutation. This result underscores the role of G6PD5 in linking SA signaling pathway to intracellular oxidative stress. Given the similar effect of mdhar2 reported in this thesis, it would be interesting to analyze how loss of MDHAR2 function affects the transcriptome response in cat2. A direct transcriptomic comparison of cat2 mdhar2, cat2 g6pd5 and cat2 sid2 can help us to understand if the mdhar2 mutation indeed modifies the transcriptome response induced by cat2 in a similar manner to that of the g6pd5 and sid2 mutations. This comparison could shed further light on the possible links between G6PD5 and MDHAR2 in regulating the SA signaling pathway in response to intracellular oxidative stress.

Ferretti M ,

 , Destro T, Tosatto SC, La Rocca N, Rascio N, Masi A (2009) Gamma-glutamyl transferase in the cell wall participates in extracellular glutathione salvage from the root apoplast. New Phytol 181: 115-126 Finkel T, Holbrook NJ (2000) Oxidants, oxidative stress and the biology of ageing. Nature 408: 239-247 Finkemeier I, Kӧnig AC, Heard W, Nunes-Nesi A, Pham PA, Leister D, Fernie AR, Sweetlove LJ (2013) Transcriptomic analysis of the role of carboxylic acids in metabolite signaling in Arabidopsis leaves. Plant Physiol 162: 239-253 Fischer BB, Hideg É, Krieger-Liszkay A (2013) Production, detection, and signaling of singlet oxygen in photosynthetic organisms. Antioxid Redox Signal 18: 2145-2162 Foreman J, Demidchik V, Bothwell JH, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JD, et al (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422: 442-446 Foyer CH, Bloom AJ, Queval G, Noctor G (2009) Photorespiratory metabolism: genes, mutants, energetics, and redox signaling. Annu Rev Plant Biol 60: 455-484 Foyer CH, Halliwell B (1977) Purification and properties of dehydroascorbate reductase from spinach leaves. Phytochemistry 9: 1347-1350 Foyer CH, Halliwell B (1976) The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133: 21-25 Foyer CH, Neukermans J, Queval G, Noctor G, Harbinson J (2012) Photosynthetic control of electron transport and the regulation of gene expression. J Exp Bot 63: 1637-1661 Foyer CH, Noctor G (2011) Ascorbate and glutathione: the heart of the redox hub. Plant Physiol 155: 2-18 Foyer CH, Noctor G (2012) Managing the cellular redox hub in photosynthetic organisms. Plant Cell Environ 35: 199-201 Foyer CH, Noctor G (2005a) Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28: 1056-1071 Foyer CH, Noctor G (2000) Oxygen processing in photosynthesis: regulation and signalling. New Phytol 146: 359-388 Foyer CH, Noctor G (2005b) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17: 1866-1875 Résumé Les plantes sont des organismes sessiles et ne peuvent donc pas s'éloigner de conditions défavorables. Parce qu'elles sont constamment mises au défi par les changements environnementaux tels que l'infection par des agents pathogènes, les fluctuations de température et d'intensité lumineuse, la disponibilité de l'eau et la pollution chimique, les plantes ont mis au point de vastes systèmes physiologiques pour faire face à divers stimuli et maintenir une croissance et un développement normaux. Il existe un vif intérêt pour comprendre les réactions des plantes au stress, car elles peuvent souvent déterminer les rendements agricoles. Le rôle central des ROS dans les

  longs, le stress oxydant induit la voie de l'acide salicylique (SA), provoquant des lésions visibles sur les feuilles. Cette réponse au stress oxydant est annulée dans un double mutant cat2 g6pd5, chez lequel l'expression d'une forme spécifique de la glucose-6-phosphate déshydrogénase (G6PDH) a également été inactivée. Une approche de génétique classique a permis d'identifier plusieurs gènes susceptibles d'être impliqués dans la régulation de la formation de lésions SA-dépendante dans ce double mutant.Afin d'explorer les rôles des monodéshydroascorbate réductases (MDHAR) spécifiques dans des conditions optimales et de stress, des mutants d'insertion pour plusieurs gènes codant la MDHAR ont été obtenus. A la suite de son introduction dans le fond cat2, l'un d'entre eux a fortement modifié l'induction de la voie SA par le stress oxydant. Pris dans leur ensemble, les résultats soulignent l'importance de CAT2 et permettent de dessiner un lien fonctionnel entre des G6PDH et MDHAR spécifiques dans les voies de signalisation oxydative chez Arabidopsis, lien qui pourrait s'expliquer par la production de NADPH par la G6PDH et son utilisation par la MDHAR.

Table 1 .

 1

1. Probable classification of the three CATs found in different plant species (taken from

Mhamdi et al., 2010a)

Figure 1.6. Some NADPH-producing and consuming reactions in the cytosol. G6PDH

	, glucose-6-phosphate
	dehydrogenase; npGAPDH, nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase; ICDH, isocitrate
	dehydrogenase; ME, malic enzyme; NADHK, NADH kinase; NTR, NADPH-thioredoxin reductase; RBOH, respiratory burst
	oxidative homolog; TRXox/TRXred, oxidized/reduced thioredoxin. Figure taken from Foyer and Noctor (2009).

G6PDH ICDH npGAPDH ME NADP + NADPH GR GSSG GSH NTR TRX ox TRX red RBOH O 2 O 2 - MDHAR Asc MDHA DHAR NADHK NADH DHA

Table S1 .

 S1 Oligonucleotide sequences used in this study

	Supporting Information N° Oligonucleotide	Sequence	Technique
	ForCAT1	TCCAGGTCAATTACTTCCCTTCA	Genotyping
	RevCAT1	AGGCTCCGAAAGCGCTTCAA	Genotyping
	ForCAT2	CCCAGAGGTACCTCTTCTTCTCCCATG	Genotyping
	RevCAT2	TCAGGGAACTTCATCCCATCGC	Genotyping
	ForCAT3	TCGATTGTTTAGACGTCCGAC	Genotyping
	RevCAT3	AGGTTTGAAATGTCATGGGTG	Genotyping
	Lb1	TGGACCGCTTGCTGCAACTCT	Genotyping
	LBSail	GAAATGGATAAATAGCCTTGCTTCC	Genotyping
	ForACTIN2	CTGTACGGTAACATTGTGCTCAG	qRT-PCR
	RevACTIN2	CCGATCCAGACACTGTACTTCC	qRT-PCR
	ForRCE1	CTGTTCACGGAACCCAATTC	qRT-PCR
	RevRCE1	GGAAAAAGGTCTGACCGACA	qRT-PCR
	ForCAT1	AAGTGCTTCATCGGGAAGG	qRT-PCR
	RevCAT1	CTCCGAAAGCGCTTCAAC	qRT-PCR
	ForCAT2	TGCTGGAAACTACCCTGAATGG	qRT-PCR
	RevCAT2	TCAACACCATACGTCCAACAGG	qRT-PCR
	ForCAT3	CCACTTGATGTGACCAAGATCTG	qRT-PCR
	RevCAT3	GTAGATTCCAGGAACCACAAGACC	qRT-PCR
	ForGSTU24	TCCATAGCTGGTTTGCAGTG	qRT-PCR
	RevGSTU24	TAGCGACGCTCTCTCTCTCC	qRT-PCR
	ForUGT73B3	CCTCACCACACCTCTCAACTC	qRT-PCR
	RevUGT73B3	TCTGGTAACCCGAGATCCAC	qRT-PCR
	ForAPX1	GCACTATTGGACGACCCTGT	qRT-PCR
	RevAPX1	GCAAACCCAAGCTCAGAAAG	qRT-PCR

Table 3 .

 3 1. Embryo lethality degree in the siliques of M1 plants. Ten siliques from each of five M1 Plants (50 in total) were examined for aborted seeds. Siliques were categorized into three classes based on the visual determination of normal and aborted seeds number. Class A: aborted seeds﹥20 %; class B: 20 % ≥ aborted seeds ≥ 6.7 %; class C: aborted seeds﹤6.7%.

Table 3 . 3 .

 33 Phenotype segregation ratios for individual plants. For each line, segregation ratios were determined in an F2 population for at least two to five F1 plants. Ratios of plants showing lesions to the total number of viable plants are presented. * Indicates classes with segregation data fitting the hypothesis of 3:1 ratio according to Chi-square test. df = 1, χ 2 0.999 = 10.827 (P < 0.001).

	line		1 st backcross (plants with lesions/total plants)		2 nd backcross (plants with lesions/total plants)
		F1	F1	F1	F1	F1	F1	F1	F1	F1
		plant1	plant2	plant3	plant4	plant5	plant1	plant2	plant3	plant4
	9.11	8/44*	7/49*	6/52*	6/48*	--	12/58*	3/59	7/60*	5/53*
	13.27	4/64	0/64	5/64*	4/64	8/64*	9/95	15/96*	12/89*	6/87
	17.21	7/69*	2/58	16/77*	7/51*	--	20/137* 22/128*	0/119	15/109*
	19.17 33/204* 39/173*	--	--	--	--	--	--	--
	21.14 11/68*	13/85*	22/84*	8/96	--	--	--	--	--
	26.19 10/43*	14/76*	--	--	--	--	--	--	--

Table 3 .

 3 4. Identification of mutations. For each line, at least 50 plants of the segregants showing lesions were pooled for DNA extraction and whole-genome sequencing. In parallel, their parental line cat2 g6pd5 was also sequenced to distinguish between the novel mutations and the genetic background. At the end, all EMS-induced mutations were selected for SHOREmap to identify the causative changes with the highest frequency.

	Line	Candidate gene
	9.11	MIPS1
	13.27	MIPS1
	17.21	Several novel candidates
	19.17	CPR5
	21.14	MIPS1
	26.19	Several novel candidates

Table 4 .

 4 1. Summary of different MDHARs and T-DNA mutants available in the laboratory

	List of genes	Localization	Protein molecular mass	T-DNA mutants available
	MDHAR1	Peroxisome	50.2 KDa	mdhar1-1, mdhar1-2
	MDHAR2	Cytosol	47.5 KDa	mdhar2-1
	MDHAR3	Cytosol	48.3 KDa	mdhar3-1, mdhar3-2,
				mdhar3-3
	MDHAR4	Peroxisome	53.5 KDa	mdhar4-1, mdhar4-2
	MDHAR5/6	Chloroplast/Mitochondrion	53.3 KDa	--

Table 4 .

 4 2. Backcross of cat2 mdhar2 with cat2. At least four plants of cat2 mdhar2 were crossed with cat2. Seeds were collected and pooled. F2 seeds were collected from two F1 plants, and lesion phenotypes were compared with genotypes for 90 (plant 1) and 86 (plant 2) F2 individuals.

Table 6 . 1 .

 61 Oligonucleotide sequences used in this study

		N Oligonucleotide	Sequence	Technique
	°
	1	ForCAT1	TCCAGGTCAATTACTTCCCTTCA	Genotyping
	2	RevCAT1	AGGCTCCGAAAGCGCTTCAA	Genotyping
	3	ForCAT2	CCCAGAGGTACCTCTTCTTCTCCCATG	Genotyping
	4	RevCAT2	TCAGGGAACTTCATCCCATCGC	Genotyping
	5	ForCAT3	TCGATTGTTTAGACGTCCGAC	Genotyping
	6	RevCAT3	AGGTTTGAAATGTCATGGGTG	Genotyping
	7	ForG6PD5	CTTGTCGCCATGGAGAAACC	Genotyping
	8	RevG6PD5	ATTTGGGAAGGTTTGATTTGG	Genotyping
	9	ForMDHAR1	TGTTGGCGGAGGTTACATTG	Genotyping
	10 RevMDHAR1	TCGTCAAACGAAACCAAACC	Genotyping
	11 ForMDHAR2	TGGTTTATCCAGAACCTTGG	Genotyping
	12 RevMDHAR2	GTCTACCACCGACACCCACT	Genotyping
	13 ForMDHAR3	TTGCAACCGGTTCTACTGTG	Genotyping
	14 RevMDHAR3	TTGATTCCCTTGTTGGCATAG	Genotyping
	15 ForMDHAR4	CAAGTCGTGCAAGCAAAGAG	Genotyping
	16 RevMDHAR4	TTACGGGTTCTTCGGAAATG	Genotyping

.4 Antioxidative enzyme activity measurements 6.2.4.1 Extraction

  . Il est important de noter que, dans les environnements naturels et sur le terrain, les plantes sont souvent exposées simultanément à plusieurs types de conditions de stress. Par exemple, une plante peut être confrontée à la fois à des polluants du sol, à un excès de sel et à une attaque d'agents pathogènes. Bien qu'il soit accepté que les ROS soient des régulateurs clés du résultat de telles conditions, le réseau qui contrôle les ROS et les réponses des usines à ces ROS reste à élucider. L'une des principales lacunes de nos connaissances concerne le rôle spécifique de différentes enzymes impliquées dans le contrôle de l'accumulation de ROS et de l'état d'oxydo-réduction cellulaire. C'est dans ce contexte que les travaux présentés dans cette thèse ont été conduits.Trois gènes encodent CAT chez Arabidopsis. Bien que le rôle de CAT2 dans la photorespiration soit bien établi, l'importance des différents CAT dans d'autres processus est moins claire. Dans ce travail, nous avons examiné les fonctions de la CAT en utilisant des mutants d'ADN-T pour cat1, cat2, cat3 ainsi que les doubles mutants cat1 cat2 et cat2 cat3. Dans les racines et les feuilles, les mutants cat2 ont montré le plus grand effet sur l'activité. Comme la croissance de la rosette, la croissance des racines était inhibée dans toutes les lignées contenant cat2. L'inhibition de la croissance des racines a été empêchée par la croissance à hCO2, ce qui suggère qu'il s'agit d'un effet indirect du stress oxydatif sur les feuilles. Les phénotypes marqués n'ont pas été identifiés chez les mutants simples cat1 ou cat3, mais l'analyse des mutants doubles a suggéré un certain chevauchement entre CAT2 et CAT3 dans les feuilles et CAT1 et CAT2 dans les semences. La réponse des gènes marqueurs H2O2 à une exposition égale de la cat2 au stress oxydatif était fortement influencée par le contexte de la longueur de la journée de croissance. Des réponses similaires dans cat2 et cat2 cat3 suggèrent que cela n'est pas lié aux effets de la durée du jour sur l'expression de CAT3. Le 3-aminotriazole (3-AT), un inhibiteur non spécifique de la CAT, a produit des effets dépendants de la longueur du jour qualitativement similaires à ceux observés chez cat2. Ensemble, nos données (1) soulignent l'importance de CAT2 chez Arabidopsis; (2) suggèrent que CAT1 et CAT3 sont principalement des enzymes de «sauvegarde»; et (3) établir que les réponses au déficit en CAT dépendant de la longueur du jour sont indépendantes de la durée du stress oxydatif.La formation de lésions et la signalisation associée à l'acide salicylique (SA) déclenchée par la mutation cat2 peuvent être supprimées en inactivant la voie de synthèse de la SA par la mutation sid2. Il est intéressant de noter que la neutralisation d'une isoforme spécifique de la glucose-6-phosphate déshydrogénase (G6PD5) dans l'arrièreplan cat2 produit un effet très similaire, mais les raisons restent à élucider. Pour identifier les gènes impliqués dans la liaison de H2O2 intracellulaire et de G6PD5 dans la signalisation SA, un criblage génétique classique (en avant) a été utilisé. Des graines de cat2 g6pd5 ont été mutagénisées avec du méthanesulfonate d'éthyle et la population M2 a été examinée visuellement pour rechercher des individus présentant un phénotype similaire à cat2 (lésions sur les feuilles). Six révertants candidats présentant ce phénotype ont été obtenus. Après au moins un rétrocroisement avec cat2 g6pd5, l'ADN a été isolé d'individus F2 regroupés qui présentaient des lésions et les gènes candidats ont été identifiés par séquençage du génome. Les données montrent que quatre de ces six lignées sont mutées dans des gènes connus pour être impliqués dans la régulation de la mort cellulaire liée à la voie SA.Sur ces quatre, trois portent des mutations dans le gène codant pour la myo-INOSITOL PHOSPHATE SYNTHASE 1 (MIPS1), tandis que l'un est muté dans CONSTITUTIVE EXPRESSOR OF PR GENES 5 (CPR5). Dans les deux autres lignées, les mutations identifiées ne concernaient pas des acteurs reconnus impliqués dans la formation de lésions, offrant des perspectives intéressantes pour des travaux ultérieurs visant à caractériser les rôles des gènes impliqués.Avec la catalase, la voie de l'ascorbate-glutathion est considérée comme un acteur clé dans le métabolisme de H2O2 produit à l'intérieur de la cellule. Les données de la littérature suggèrent que le flux par la voie ascorbateglutathion est accéléré pendant le stress et dans les systèmes génétiques mimant le stress, tels que le mutant cat2.Quatre types d'enzymes différents fonctionnent dans la voie ascorbate-glutathion et sont codés par plus d'un gène. Une de ces enzymes est la monodéhydroascorbate réductase (MDHAR) dépendante de NAD (P) H, pour laquelle cinq gènes sont annotés dans Arabidopsis. Pour analyser l'importance des isoformes spécifiques du MDHAR, des mutants spécifiques du gène ont été obtenus pour un isoforme peroxisomal (MDHAR1) et deux isoformes cytosoliques (MDHAR2, MDHAR3). Les conséquences de la perte de ces fonctions MDHAR ont été analysées dans un contexte de type sauvage et déficient en catalase. Bien qu'aucune des mutations de mdhar n'ait eu d'effet marqué sur la croissance ou le phénotype de rosette dans le contexte de type sauvage, mdhar2 a eu plusieurs effets saisissants sur les modifications de l'état redox déclenchées par cat2 et l'activation de la voie SA.Comparé à cat2, un double mutant cat2 mdhar2 a montré une formation de lésion beaucoup plus réduite accompagnée d'une activation plus faible de la voie SA. Ces effets étaient liés à une perturbation de la voie ascorbate-glutathion et, plus particulièrement, à une oxydation accrue du glutathion par rapport à cat2. Ensemble, les résultats suggèrent qu'un MDHAR cytosolique spécifique joue un rôle important dans la signalisation des réponses au H2O2 intracellulaire. Analyse fonctionnelle des mutants de la catalase et de leur application à l'analyse des voies liées au NADPH dans signalisation oxydative chez Arabidopsis thaliana Mots clés : catalase, NADPH, stress oxydant, H2O2, signalisation Résumé : Les conditions contraignantes provoquent la modification de l'état redox et la signalisation liée aux formes actives de l'oxygène (ROS), dont les concentrations sont régulées par des systèmes antioxydant complexes. Mais nous avons toujours des connaissances fragmentaires quant à l'importance des ROS pour la signalisation cellulaire chez les plantes. Cette étude a utilisé des approches de génétique classique et inverse chez Arabidopsis thaliana dans le but d'élucider les rôles des catalases et des systèmes NADPH-glutathion-ascorbate dans le métabolisme du H2O2 et la signalisation qui en dépend. Une analyse de mutants ADN-T a révélé que la mutation cat2, à la différence de cat1 et de cat3, a fortement affecté la croissance et le développement de la plante. Mais ces effets étaient absents lors de la culture des plantes sous un taux de CO2 élevé, suggérant que la taille diminuée est causée, directement ou indirectement, par une capacité compromise de métaboliser le H2O2 produit par la photorespiration. Une étude de cat2 cultivé dans des photopériodes différentes a mis en évidence une forte influence de la période d'illumination sur la signalisation oxydative et ceci d'une manière qui est indépendante de l'intensité du stress. Lorsque cat2 est cultivé en jours

Université Paris-Saclay

Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre :

22 107 175

host interaction between tobacco and Xanthomonas campestris pv. vesicatoria. Plant J 60: 962-973

ACKNOWLEDGMENTS

ACKNOWLEDGMENTS Z.Y. thanks the China Scholarship Council for the award of a PhD grant. This work received financial support from the French Agence Nationale de la Recherche grants ANR12BSV60011 (Cynthiol) to G.N.

This thesis would not have been possible without the financial support of China Scholarship Council and French Agence Nationale de la Recherche grants ANR12BSV60011 (Cynthiol).

Finally, I would like to take the opportunity to thank my parents and brother for your understanding and support.

Summary

Lesion formation and associated salicylic acid (SA) signaling triggered by the cat2 mutation can be abolished by inactivating the SA synthesis pathway through the sid2 mutation. Interestingly, knocking out a specific isoform of glucose-6-phosphate dehydrogenase (G6PD5) in the cat2 background produces a very similar effect, but the reasons remain to be elucidated. To identify genes involved in linking intracellular H2O2 and G6PD5 in SA signaling, a classical (forward) genetics screen was employed. Seeds of cat2 g6pd5 were mutagenized with ethyl methanesulfonate and the M2 population was visually screened for individuals that show a similar phenotype to cat2 (lesions on the leaves). Six candidate revertants with this phenotype were obtained. Following at least one backcross to cat2 g6pd5, DNA was isolated from pooled F2 individuals that showed lesions and candidate genes were identified by genome sequencing. The data show that four of these six lines are mutated in genes known to be involved in cell death regulation linked to the SA pathway. Of these four, three carry mutations in the gene encoding myo-INOSITOL PHOSPHATE SYNTHASE 1 (MIPS1) while one is mutated in CONSTITUTIVE EXPRESSOR OF PR GENES 5 (CPR5). In the other two lines, the identified mutations were not in recognized players involved in lesion formation, providing interesting perspectives for further work to characterize the roles of the genes involved. the segregation data fit the predicted segregation ratio of 3:1, we performed Chi-square (χ 2 ) statistical test (Table 3.3). In this study we only focused on lesion phenotype, so two classes are involved: lesions and no-lesions. Therefore, the degree of freedom (df) is 1. For each segregation result, the calculated values of χ 2 were compared with χ 2 0.999 which is 10.827 [START_REF] Weigel | Arabidopsis: A Laboratory Manual[END_REF].

CHAPTER 3 83

Those ratios lower than 10.827 were considered acceptable for 3:1 segregation, which is consistent with the phenotype being caused by a single recessive mutation. As shown in Table 3.3, the majority of the segregation ratios are lower than 1/4 and higher than 1/16. The skewed ratios may be an effect of several factors, such as the segregation of multiple unrelated mutations in a mutagenized background, partial lethality in the male or female gametes, or growth conditions [START_REF] Weigel | Arabidopsis: A Laboratory Manual[END_REF].

We also cannot exclude a partly lethal effect of the causal mutation during embryonic and postembryonic growth, which would be a simple explanation of lesion phenotype ratios below 1/4.

CHAPTER 3

87 some conclusions have been drawn based on pharmacological and genetic studies [START_REF] Valderrama | The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants[END_REF][START_REF] Dizengremel | Metabolic-dependent changes in plant cell redox power after ozone exposure[END_REF]Mhamdi et al., 2010c;[START_REF] Piattoni | Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate[END_REF][START_REF] Dghim | Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod-influenced responses to ozone using Arabidopsis knockout mutants[END_REF][START_REF] Li | Analysis of knockout mutants suggests that Arabidopsis NADP-MALIC ENZYME2 does not play an essential role in responses to oxidative stress of intracellular or extracellular origin[END_REF][START_REF] Wang | Glucose-6-phosphate dehydrogenase plays a central role in modulating reduced glutathione levels in reed callus under salt stress[END_REF], the relative importance of different NADPH-generating enzymes remains unclear. Unpublished work in our group over the last several years has revealed that the loss of function of G6PD5, encoding a specific isoform of G6PDH, abolishes the cat2 lesion phenotype, as well as cat2-induced transcriptomes (Figure 3.2). Because these effects on cat2 are quite similar to those caused by the sid2 mutation (Figure 3.1), this suggests some role for G6PD5 in regulating SA signaling and lesion formation in cat2. The present study aimed to explore components that might be involved in this process by genetically screening revertants of the cat2 g6pd5 phenotype.

The first revertant line we identified carries a mutation in MIPS1. MIPS catalyzes the conversion of glucose-6-phospate to myo-inositol phosphate, which is the rate limiting step for myo-inositol biosynthesis [START_REF] Loewus | Myo-inositol: its biosynthesis and metabolism[END_REF][START_REF] Loewus | Myo-Inositol metabolism in plants[END_REF]. myo-Inositol is a ubiquitous compound with diverse biological functions in plants, such as a possible precursor for ascorbate synthesis, and roles in signal transduction and stress tolerance [START_REF] Loewus | Myo-Inositol metabolism in plants[END_REF]. mips1, a mutant defective in MIPS, has previously been identified as a lesion-mimic mutant [START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF]Donahue et al., 2010). Interestingly, like lesions in cat2, the lesion phenotype in mips1 was initially linked with day length. When grown in SD it is indistinguishable from the wild type in rosette phenotype, but transferring plants from SD to LD leads to spontaneous cell death formation within several days. However, this phenomenon is more closely linked with the higher quantity of light received in LD [START_REF] Meng | Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis[END_REF]. As in cat2 and several other lesion mimic mutants, the lesion formation in mips1 is regulated by SA. Previous work in our laboratory showed that the isochorismate pathway of SA synthesis is involved in the lesion formation and associated defense responses in cat2 [START_REF] Chaouch | Peroxisomal hydrogen peroxide is coupled to biotic defense responses by ISOCHORISMATE SYNTHASE1 in a daylength-related manner[END_REF].

Although the cat2-induced lesions are light-and day length-dependent, the characteristics seem to somewhat different from those of mips1. Cell death in cat2 was only observed in LD, but not SD, even if higher irradiance is used in SD (Queval et al., 2007). Nevertheless, it is worth mentioning that the functions of both MIPS1 and G6PD5 require glucose-6-phospate as the substrate, and both are located

Summary

Together with catalase, the ascorbate-glutathione pathway is considered to be a key player in metabolizing H2O2 produced inside the cell. Literature data suggest that flux through the ascorbateglutathione pathway is accelerated during stress and in genetic systems that mimic stress, such as the Confirmation with T-DNA insertion mutants is one of the possible approaches. Such mutants can be crossed to cat2, g6pd5 and cat2 g6pd5. Functional analysis of double and triple mutants will establish whether the mutations identified in the revertant screen are indeed causal in allowing lesion formation.

If so, loss of function of the identified genes in the T-DNA lines should revert the phenotype in cat2 g6pd5, and allow lesions to appear. Furthermore, based on the fact that the g6pd5 mutation mimics the sid2 mutation in abolishing transcriptome responses triggered by cat2, it will be worthwhile to check if the SA signaling pathway is affected when introducing the identified mutation into cat2 g6pd5, e.g. at the levels of PR gene expression, SA contents, and pathogen resistance. Currently, a T-DNA mutant for mips1 has been crossed into cat2 g6pd5 by Amna Mhamdi (Ghent university). Crosses between other candidates and cat2 g6pd5 are underway.

MDHAR isoforms in responses to H2O2

The work presented in Chapter 4 reported that the functional analysis of mdhar1, mdhar2 and mdhar3

mutants revealed an irreplaceable role of MDHAR2 in regulating lesion formation and SA signaling pathway in response to oxidative stress. MDHAR1 may play some role in H2O2 metabolism because the loss of MDHAR1 function slightly decreased the rosette size of cat2. MDHAR3 may also be involved to some extent in the oxidative stress responses, since this gene was significantly induced in cat2. However, introduction of the mdhar3 mutation into the cat2 background did not cause substantial changes in the cat2 phenotype or associated SA signaling. One interesting perspective would be to extend this analysis to include mutants for MDHAR4 or MDHAR5/6. Previous reports suggest that MDHAR4 is involved in ascorbate recycling in the peroxisomes [START_REF] Lisenbee | Arabidopsis peroxisomes possess functionally redundant membrane and matrix isoforms of monodehydroascorbate reductase[END_REF], while loss of function of MDHAR4 also affected seed oil hydrolysis and seedling development (Eastmond, 2007).

New evidence revealed a pro-oxidant effect of MDHAR6 in mediating TNT toxicity by promoting reactive oxygen production [START_REF] Johnston | Monodehydroascorbate reductase mediates TNT toxicity in plants[END_REF][START_REF] Noctor | Lighting the fuse on toxic TNT[END_REF]. It would be interesting to do the functional analysis of mdhar4 and mdhar6 in optimal growth condition and in oxidative stress, e.g. by introducing mutations for these two genes into the cat2 background. Analyses of all mdhar mutants in other stress conditions (e.g. pathogens, salt, heavy metals) is also an interesting perspective. This work is currently underway in the laboratory.

Metabolite analysis

6.2.5.1 Glutathione and ascorbate assay by plate reader

Extraction

Oxidized and reduced forms of ascorbate and glutathione analysis were performed following a platereader method which was described in Queval and Noctor (2007). Around 100 mg leaves was ground to a fine powder in liquid nitrogen with a pestle and mortar. 1 ml 0.2 mM HCl was added immediately and the homogenate was ground again. After centrifuging at 14000 rpm for 10 min at 4 ˚C, 500 µl supernatant was transferred to a fresh tube and mixed with 50 µl of 0.2 M NaH2PO4 (pH 5.6). Then the solution was neutralized to pH 4.5-5 with 0.2 M NaOH for the following assay.

Glutathione analysis

Glutathione measurements were performed by monitoring GR-dependent reduction of 5,5'-dithiobis

(2-nitro-benzoic acid) (DTNB, Ellman's reagent) by GSH at 412 nm [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]. For total glutathione, the reaction system contained 0.1 ml of 0.2 M NaH2PO4, 10 mM EDTA (pH 7.5), 60 µl H2O, 10 µl of 12 mM DTNB, 10 µl of 10 mM NADPH and 10 µl neutralized extract. The components were mixed three times by programmed shaking, and then 10 µl GR (20 U/ml) was added to start the reaction. The absorbance increase at 412 nm was monitored for 5 min and rates were calculated over the first 90 s. For GSSG, the measurement was based on the same principle except that an additional incubation step was performed to complex GSH in extract aliquots before reaction. For this, 200 µl neutralized extract was incubated with 2 µl 2-vinylpyridine (VPD) for 30 min at room temperature [START_REF] Griffith | Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine[END_REF]. Excess VPD in the extract was then removed by centrifugation twice at 14000 rpm for 15 min at 4 ˚C. Finally 20 µl supernatant was added to the reaction system. Rates were converted to quantities of glutathione. Triplicate assays were performed for each extract as well as standards in the same plate.

line. In theory, if the segregation ratio of plants without lesions to those showing lesions is 3:1, it indicates that a single recessive mutation is probably responsible for the phenotype. Two mutations could be causal if the ratio is 15:1. The screened F2 plants with lesions were backcrossed to cat2 g6pd5 for a second time, and genotypes and phenotypes analyzed in the resulting F2 generation. For a given line, F2 segregation ratios should be similar for the first and second backcrosses.

Sample collection for sequencing

Leaves from at least 50 F2 individuals that showed lesions following the first or second backcross were pooled and the bulked genomic DNA was sequenced. In parallel, the parental line cat2 g6pd5

was also sequenced as the reference genome.

Nuclear DNA isolation for sequencing

Rosette material from approximately fifty plants was ground in liquid nitrogen then reground in 15 ml HBM buffer (25 mM Tris-HCl, pH 7.5, 440 mM sucrose, 10 mM MgCl2, 0.1% Triton X-100, 10 mM mercaptoethanol) with continued grinding during thawing. The suspension was vortexed, kept on ice for 20 min, then filtered through Miracloth into 50 ml Falcon tubes. The filtrate was centrifuged at 3000 g for 10 min at 4°C, then the supernatant was discarded and the pellet resuspended in 0.5 ml NIB buffer (20 mM Tris-HCl, pH 7.5, 250 mM sucrose, 5 mM MgCl2, 5 mM KCl, 0.1% Triton X-100, 10 mM mercaptoethanol). The resuspended pellet was carefully layered onto a 15-50% Percoll gradient in NIB buffer and centrifuged at 3000 rpm for 10 min at 4°C. Pelleted nuclei were washed and recentrifuged at 3000 rpm for 10 min at 4°C. The pellet was then resuspended in 0.5 mL pre-heated CTAB solution and incubated at 60°C for 30 min with frequent agitation. The suspension was clarified by centrifugation at 12000 g for 5 min and then combined with 0.5 ml chloroform:isoamyl alcohol (24:1) and gently mixed for about 1 min. After centrifugation for 5 min at 7000 g (4°C), the upper phase was transferred to a fresh Eppendorf tube and treated with RNAse for 30 min at 37°C. Then, 350 µl isopropanol was added with gentle mixing. Following overnight incubation at -20°C, the suspension was centrifuged at 13000 g for 6 min at 4°C.