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Functional analysis of glutathione and autophagy inresponse to oxidative stress

Yi Han

To cite this version:Yi Han. Functional analysis of glutathione and autophagy in response to oxidative stress. Agriculturalsciences. Université Paris Sud - Paris XI, 2012. English. �NNT : 2012PA112392�. �tel-01236618�

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UNIVERSITE PARIS‐SUD – UFR des Sciences

ÉCOLE DOCTORALE SCIENCES DU VEGETAL

Thèse Pour obtenir le grade de

DOCTEUR EN SCIENCES DE L’UNIVERSITÉ PARIS SUD

Par

Yi HAN

Functional analysis of glutathione and autophagy in response to

oxidative stress

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

Christine FOYER Professor, University of Leeds, UK Examinateur

Michael HODGES Directeur de Recherche, IBP, Orsay Examinateur

Stéphane LEMAIRE Directeur de Recherche, IBPC, Paris Rapporteur

Graham NOCTOR Professeur, IBP, Orsay Directeur de la thèse

Jean‐Philippe REICHHELD Directeur de Recherche, LGP, Perpignan Rapporteur

ACKNOWLEDGMENTS

It would not have been possible to write this doctoral thesis without the help and support of the kind

people around me. It is a pleasure to thank the many people who made this thesis possible.

First of all, I would like to express my deepest sense of gratitude to my supervisor Prof. Graham Noctor

who offered his continuous advice and encouragement throughout the course of this thesis. I have been

extremely lucky to have a supervisor who cared so much about my work. I thank him for the systematic

guidance and great effort he put into training me in the scientific field.

I also want to express my gratitude to the reviewers of my thesis, Dr. Stéphane Lemaire (Institut de

Biologie Physico-Chimique, FR), Dr. Jean-Philippe Reichheld (Université de Perpignan, FR), Prof.

Christine Foyer (University of Leeds, UK) and Dr. Michael Hodges (Université Paris Sud, FR) for having

accepted to evaluate my PhD work.

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

glutathione.

This thesis would not have been possible without the financial support of China Scholarship Council and

French Agence Nationale de la Recherche project “Vulnoz” as well as the European Union Marie-Curie

project “Chloroplast Signals”.

I am thankful to the former as well as the current colleagues in the lab. My officemate, at the start of my

thesis, Guillaume Queval, for always being prepared to advices and help. Thanks to Sejir Chaouch, for

teaching many biological measurements, and for the scientific discussion. To Jenny Neukermans for the

help and continuous encouragement. To the current colleagues, Amna Mhamdi, a well organized person

for the scientific discussion and laboratory management. I am quite happy to work with you and enjoy all

our discussions about food, cultures and everything in the past four years. To my Chinese colleague

Shengchun Li, and Marie-Sylviane Rahantaniaina for their friendship.

I would like to thank the many people at IBP for their help and support (Patrick Saindrenan, Bertrand

Gakière, Dao-Xiu Zhou, Catherine Bergounioux, Gilles Sante, Gilles Innocenti, Sophie Blanchet,

Caroline Mauve, Françoise Gilard, Pierre Pétriacq, Edouard Boex-fontvieille…). Also, I am indebted to

my many student colleagues at IBP for providing a stimulating and fun environment in which to learn and

grow. I am especially grateful to Linda De-bont, Manon Richard, Yuan Shen, Laure Audonnet, Thomas

Guerinier, and Laure Didierlaurent.

Lastly, and most importantly, I wish to thank my parents and my wife… 感谢我的妈妈，爸爸，岳父和

岳母，还有我可爱的妻子冯峰，这么多年来，正是你们无私的支持和鼓励，我才能顺利完成我的学

业。我将在以后的工作和生活中尽我最大的努力来回报你们长久以来的支持。

Abbreviations

1O2: Singlet oxygen

3PGA: 3-Phosphoglycerate

5-OPase: 5-oxoprolinase

ABA: Abscisic acid

AO: Ascorbate oxidase

APX: Ascorbate peroxidase

ASC: ascorbate

ATG: Autophagy

BSO: Buthionine sulfoximine

CAT: Catalase

CLT: Chloroquinone-like transporter

CO2: Carbon dioxide

DCPIP: 2,6-dichlorophenolindophenol

DHA(R): dehydroascorbate (reductase)

DNA: Deoxyribonucleic acid

DTNB: 5,5’-dithiobis-2-nitrobenzoic acid

Dpi: day post-inoculation

DTT: Dithiothreitol

-ECS: -glutamyl cysteine synthetase

EDTA: Ethylene diamine tetraacetic acid

ER:Endoplasmic reticulum

ETI: Effector-triggered immunity

FDH: Formaldehyde dehydrogenase

G6PDH: Glucose-6-phosphate dehydrogenase

GAPDH: Glyceraldehyde-3-phosphate dehydrogenase

GC-TOF-MS: Gas chromatography-time of flight-mass spectrometry

GDC: Glycine decarboxylase

GGC: -glutamyl-cyclotransferases

GGP: -glutamyl peptidases

GGT: -glutamyl transpeptidase

GO: Glycolate oxidase

GPX: Glutathione peroxidase

GR: Glutathione reductase

GRX: Glutaredoxins

GSH: Reduced glutathione

GSNO(R): S-nitrosylglutathione (reductase)

GSSG: Glutathione disulphide

GST: Glutathione S-transferase

HO•: Hydroxyl radical

HPLC: High performance liquid chromatography

HR: Hypersensitive response

ICS1: Isochorismate synthase 1

JA: Jasmonic acid

MAPK: Mitogen-activated protein kinase

MDHAR: Monodehydroascorbate reductase

MRP: Multidrug resistance-associated protein

MV: Methyl viologen

NO: Nitric oxide

NADP(H): Nicotinamide adenine dinucleotide phosphate (Reduced)

NPR1: Nonexpressor of pathogenesis-related genes 1

NTR: NADPH-thioredoxin reductase

NR: Nitrate reductase

O2: Oxygen

O2−: Superoxide

OPT: Oligopeptide transporter

OXI1: Oxidative signal inducible 1

PB: Protein bodies

PCD: Programmed cell death

PCS: Phytochelatin synthase

PE: Phosphatidylethanolamine

PI3K: Phosphatidylinositol 3-kinase

PR: Pathogenesis-related

RBOHs: Respiratory burst oxidase homologues

PMS: Phenazine methosulfate

RNA: Ribonucleic acid

RT-qPCR: Reverse transcription-quantitative real-time polymerase chain reaction

ROS: Reactive oxygen species

POX: Peroxidase

PRX: Peroxiredoxins

PSVs: Protein storage vacuoles

PTI: Pathogen-associated molecular pattern-triggered immunity

roGFP: Reduction-oxidation sensitive green fluorescent protein

RuBisco: Ribulose 1,5-bisphosphate carboxylase/oxygenase

SA: Salicylic acid

SAR: Systemic acquired resistance

sid2: Salicylic acid induction-deficient 2

SNP: Sodium nitroprusside

SOD: Superoxide dismutase

T-DNA: Transferred deoxyribonucleic acid

TF: Transcription factors

TORC1: Target of rapamycin kinase complex 1

TRX: Thioredoxins

VPD: 2-vinylpyridine

XO: Xanthine oxidase

TABLE OF CONTENTS

CHAPTER 1 GENERAL INTRODUCTION

1.1 Reactive oxygen species in plants 1

1.1.1 ROS generating systems 2

1.1.2 Metabolism of ROS 3

1.1.2.1 Non-enzymatic scavenging mechanisms 3

1.1.2.2 Enzymatic metabolism of ROS 4

1.1.3 Functions of ROS in plants 7

1.1.3.1 The role of ROS in abiotic stress 8

1.1.3.2 Regulation of growth and developmental processes by ROS 8

1.1.3.3 Functions of ROS in plant innate immunity 8

1.1.3.4 ROS in cell death 9

1.1.4 ROS signaling network 9

1.2 Glutathione in plants 11

1.2.1 Glutathione synthesis 11

1.2.1.1 Glutathione deficient mutants 12

1.2.1.2 Regulation and overexpression of the glutathione synthesis pathway 12

1.2.2 Glutathione transport 12

1.2.3 Glutathione degradation 14

1.2.4 Metabolic functions of glutathione in plants 15

1.2.4.1 Glutathione S-transferases 15

1.2.4.2 Glutathione reductase 16

1.2.4.3 Glutaredoxins 17

1.2.4.4 Other glutathione-associated metabolic reactions 18

1.2.5 Glutathione in plant development, growth and environmental responses 20

1.2.5.1 Glutathione in the regulation of plant development and growth 20

1.2.5.2 Light signaling 20

1.2.5.3 Biotic interactions, cell death and defence phytohormones 21

1.2.6 Mechanisms of glutathione dependent redox signaling 24

1.2.6.1 Protein S-glutathionylation 24

1.2.6.2 Protein S-nitrosylation and GSNO reductase 25

1.2.6.3 Glutathione redox potential 25

1.3 Autophagy in plants 26

1.3.1 Functions of autophagy in plants 28

1.3.1.1 Functions of autophagy during abiotic stress 28

1.3.1.2 Functions of autophagy during pathogen infection 29

1.3.1.3 Functions of autophagy during growth and seed development 29

1.3.2 Difficulties of monitoring autophagy in plants 30

1.4 Objectives of the work 31

CHAPTER 2 Functional analysis of Arabidopsis mutants points to novel roles for glutathione in coupling H2O2 to

activation of salicylic acid accumulation and signaling

2.1 Introduction 34

2.2 Results 37

2.2.1 Genetic blocks over H2O2-triggered glutathione accumulation inhibit cell death and associated pathogenesis responses

induced by intracellular oxidative stress

37

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

dependent pathogenesis responses 42

2.2.3 Side-by-side comparison of cat2 cad2 with cat2 npr1 46

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

responses

51

2.3 Discussion 53

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

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

responses

54

2.3.3 Glutathione acts independently of its antioxidant function in transmitting H2O2 signals 55

2.3.4 Glutathione can regulate SA signaling through processes additional to NPR1 56

2.3.5 Multifunctional roles of glutathione status in defence signaling pathways: A model 57

CHAPTER 3 Regulation of basal and oxidative stress-triggered jasmonic acid-related gene expression by glutathione

3.1 Introduction 59

3.2 Results 61

3.2.1 Effects of glutathione content on basal expression of jasmonic acid-associated genes 61

3.2.2 Activation of jasmonic acid signaling by intracellular oxidative stress 63

3.2.3 Modulation of leaf thiol status by the cad2 mutation 63

3.2.4 Comparison of effect of cad2 and npr1 mutations on oxidative stress-triggered JA signalling 65

3.2.5 Effects of complementing mutants with cysteine and glutathione 66

3.3 Discussion 68

3.3.1 Basal expression of the JA pathway is closely correlated with glutathione contents 68

3.3.2 JA-associated gene expression induced by oxidative stress is influenced by glutathione status 68

3.3.3 Glutathione can regulate oxidative stress-triggered activation of JA signaling independent of NPR1 70

3.3.4 Novel roles for glutathione in regulating the JA pathway: a model 71

CHAPTER 4 Analysis of autophagy mutants: interactions with photorespiration and oxidative stress

4.1 Introduction 74

4.2 Results 77

4.2.1 Effects of high CO2 on atg phenotypes and metabolite profiles 78

4.2.2 Impact of oxidative stress on atg-triggered early senescence 80

4.2.3 Impact of atg mutations on cat2-triggered lesions and bacterial resistance 82

4.2.4 Interactions between atg mutations and glutathione 83

4.3 Discussion 84

CHAPTER 5 CONCLUSION AND PERSPECTIVES

5.1 CONCLUSIONS 87

5.1.1 New roles for glutathione in the regulation of defense phytohormone signaling 87

5.1.2 Interactions between autophagy, oxidative stress of peroxisomal origin, and glutathione 88

5.2 PERSPECTIVES 88

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

5.2.1.1 Glutathione oxidation and metabolism in response to H2O2 89

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

5.2.1.3 H2O2, NO, and glutathione 90

5.2.1.4 Glutathione and calcium signaling 91

5.2.1.5 Glutathione and the jasmonic acid pathway 92

5.2.2 Autophagy and oxidative stress 92

CHAPTER 6 MATERIALS AND METHODS

6.1 Plant material and growth conditions 94

6.2 Methods 95

6.2.1 Molecular biology analyses 95

6.2.1.1 DNA extraction and plant genotyping 95

6.2.1.2 RT-qPCR 96

6.2.1.3 CATMA microarray analyses and data-mining 96

6.2.2 Lesion quantification and pathogen tests 97

6.2.3 Cytohistochemical analysis 97

6.2.4 Metabolite analyses 97

6.2.4.1 Assays for ascrobate, glutathione and NADP(H) 97

6.2.4.1.1 Extraction 98

6.2.4.1.2 Quantification of thiols by HPLC 98

6.2.4.1.3 Glutathione measurement by plate reader 98

6.2.4.1.4 Ascorbate 99

6.2.4.1.5 NADP(H) 99

6.2.4.2 Salicylic acid measurement 99

6.2.4.3 Non-targeted GC-TOF-MS analysis 99

6.2.4.4 Peroxide assay 100

6.2.5 Statistical analysis 100

100

REFERENCES

101

ANNEXES

Annex 1A Mhamdi et al., 2010 Plant Physiol.

Annex 1B Confirmation of the genetic interaction between cat2 and gr1 knockout mutations

Annex 2 Noctor et al., 2012 Plant Cell Environ.

CHAPTER 1

GENERAL INTRODUCTION


Unlike animals, plants cannot move to a more comfortable location to escape stresses of external origin.

Instead, plants have developed coping mechanisms that allow them to withstand environmental

fluctuations. Most types of stresses disrupt the metabolic balance of cells, resulting in enhanced

production of reactive oxygen species (ROS). It is clear that, over the course of evolution, ROS in plants

have been recruited to act as a signal in many biological processes such as growth, the cell cycle,

programmed cell death (PCD), and other defence responses to stress. Thus, in addition to their

well-documented toxicity, ROS are important actors in cellular and subcellular signaling. There is a close

relationship between increased ROS availability and glutathione status, and many glutathione-dependent

reactions are involved in oxidative stress-mediated cellular processes. However, while the functions of

glutathione as an antioxidant involved in ROS-scavenging and associated detoxification reactions are

relatively well described, it is much less clear whether ROS-induced adjustments in glutathione status

play important regulatory roles (eg, in ROS signal transmission). One physiological condition in which

regulatory roles of glutathione have been studied in plants is biotic stress. Pathogen challenge clearly

involves ROS signaling but the interactions between ROS and glutathione in determining responses to

biotic stress remain unclear. One important ROS-dependent response to pathogens involves cell death

during the hypersensitive response (HR). Emerging evidence implicates autophagic processes in cell

death, and also in cellular acclimation to abiotic stress as well as resistance to pathogens, but the

relationship between ROS and autophagy is not yet clearly established in plants. As the overall aims of

this thesis were to exploit model study systems to investigate interactions between ROS and glutathione,

and between ROS and autophagy, the general features and concepts surrounding these factors and

processes are described in this first chapter.

1.1 Reactive oxygen species in plants

Molecular oxygen (O2) first began to exist in significant amounts in the earth’s atmosphere over 2.7

billion years ago, an evolutionary event driven largely by the appearance of O2-evolving photosynthetic

organisms (Halliwell, 2006). The O2 molecule is a free radical (triplet biradical in the ground state), as it

has two impaired electrons that have the same spin quantum number. Although several oxidases are able

to catalyze reduction of O2 to H2O2 or water by divalent or tetravalent mechanisms, the spin restriction of

the O2 molecule means that it prefers to accept electrons one at a time, leading to the generation of the

so-called ROS, which are partially reduced or activated derivatives of oxygen (such as singlet oxygen

(1O2), superoxide (O2−), hydrogen peroxide (H2O2), and hydroxyl radical (HO•)). In plants ROS are

continuously produced as byproducts of various metabolic pathways localized in different cellular

compartments (Figure 1; Apel and Hirt, 2004). Adverse environmental conditions lead to an increased

production of ROS, resulting in oxidative stress. The evolution of efficient antioxidant systems has most

likely enabled plant cells to overcome ROS toxicity and to use these reactive species as signal transducers

to regulate diverse physiological processes (Mittler, 2006). These compounds are tightly controlled by a

range of enzymatic and non-enzymatic scavenging mechanisms to maintain the cellular redox state which

will be discussed in more detail in section 1.1.2. In addition, as well as signaling roles, other key

functions for ROS has been identified including the control and regulation of biological processes, such

as growth, the cell cycle, PCD, hormone signaling, biotic and abiotic stress responses and development

(Mittler et al., 2004).

1


1.1.1 ROS generating systems It is well established that cell organelles such as chloroplasts, mitochondria and peroxisomes are major

sources of intracellular ROS production in plant cells through photosynthesis, respiration and

photorespiration. ROS are also generated at the extracellular sites such as plasma membrane

NADPH-dependent oxidases, or peroxidases and oxidases in the apoplast (Figure 1.1). Localized ROS

production in these compartments may trigger different signaling cascades.

GO, glycolate oxidase. 3PGA, 3-phosphoglycerate. POX, peroxidase. RuBisCO, ribulose 1,5-bisphosphate

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

Figure. 1.1. Major sites of H2O2 production in photosynthetic cells (scheme taken from Mhamdi et al., 2010b)

In the chloroplast, the reaction centers of photosystems I and II (PSI and PSII) are the major generation

sites of ROS since they are found in an environment rich in oxygen, reductants and high-energy

intermediates (Asada, 2006). O2−and H2O2 are formed mainly in PSI, while 1O2 is notably produced from

the triplet ground state (3O2) by photodynamic transfer of energy from excited triplet state chlorophyll

such as the P680 reaction center of PSII (Telfer et al., 1994; Hideg et al., 1998).

As their name implies, peroxisomes are also significant generators of ROS in the form of O2− or H2O2

produced by various oxidases (Mhamdi et al., 2012). Indeed, it has been estimated that in some conditions,

these organelles may be the fastest H2O2-generating system in photosynthetic cells (Noctor et al., 2002).

In peroxisomes, H2O2 is notably generated by the enzymatic activity of glycolate oxidase which oxidizes

glycolate to glyoxylate, an important reaction in photorespiration (Figure 1.1). Another enzyme that can

directly produce H2O2 is acylCoA oxidase, involved in fatty acid oxidation (Nyathi and Baker, 2006). In

2


addition, xanthine oxidase can generate O2−, which in turn is converted to H2O2 by Cu,Zn-superoxide

dismutase (SOD; Corpas et al., 2008).

In contrast to mammalian mitochondria, which are well accepted to be a main intracellular source of ROS

in animals, mitochondria in photosynthetic cells probably make a relatively minor contribution to overall

cellular ROS production (Foyer and Noctor, 2003). In mitochondria, the major sites of ROS production

lie in the electron transport chain, especially at the level of Complex I and Complex III (Møller et al.,

2001). Mitochondrial terminal oxidases such as cytochrome c oxidase (Complex IV) and the alternative

oxidase, reduce oxygen via tetravalent mechanisms. H2O2 is formed in mitochondria from O2− by

Mn-SOD (Finkel and Holbrook, 2000).

Among several types of enzymes which may be required for generating H2O2 in the apoplast, plasma

membrane NADPH-dependent oxidases, known as respiratory burst oxidase homologues (RBOHs), have

been the subject of intense investigation (Torres and Dangl, 2005; Torres et al., 2006). In plants,

RBOH-encoding genes belonged to a small multigenic family (e.g. ten members in Arabidopsis and nine

in rice) and contain a multimeric flavocytochrome that is part of a small electron transport chain capable

of reducing extracellular O2 to O2− using NADPH as a cytosolic electron donor. Although much attention

has been given to NADPH oxidases, other ROS-producing systems such as peroxidases and oxidases in

the apoplast are likely to play a role in ROS signaling in response to different stimuli or developmental

signals (Mittler et al., 2004; Bindschedler et al., 2006; Choi et al., 2007; Van Breusegem et al., 2008).

1.1.2 Metabolism of ROS As noted above, the essential reactions of energy transduction and metabolism involve the production of

ROS. Moreover, exposure of plants to adverse environmental conditions such as drought, temperature

extremes, pathogen attack, nutrient deficiency, heavy metal and air pollutants can trigger increased ROS

production. Thus, plants make use of a sophisticated and complex antioxidant machinery, including

enzymatic and non-enzymatic components, to maintain redox homeostasis, and this machinery is likely to

be particularly important in stress conditions (Apel and Hirt, 2004; Møller et al., 2007).

1.1.2.1 Non-enzymatic scavenging mechanisms

Numerous components such as tocopherols, flavonoids, alkaloids and carotenoids have been recognized

as reductants or energy quenchers that can metabolize ROS. While no enzymatic systems has been found

to scavenge singlet oxygen and hydroxyl radical, they are thought to be essentially controlled by

avoidance of their production or by reaction with low molecular weight antioxidants such as glutathione,

ascorbate, tocopherol and carotenoids (Noctor and Foyer, 1998; Asada, 1999). Indeed, although many

metabolites can be oxidized by ROS, the cellular redox buffers ascorbate (ASC) and glutathione (GSH)

are two of the major non-enzymatic antioxidant components, although they also function in many other

biochemical and physiological processes (Cobbett et al., 1998; Vernoux et al., 2000; Pastori et al., 2003;

Ball et al., 2004; Dowdle et al., 2007; Parisy et al., 2007). Both of them are highly reducing and abundant

antioxidants found in millimolar (mM) concentrations in plants (Foyer and Noctor, 2011). In addition,

both ASC and GSH are reducing co-factors for a range of enzymes that metabolize ROS, as described

3


below. Apart from their roles in maintaining redox homeostasis, it has been suggested that some

well-known antioxidant metabolites such as glutathione and carotenoids may be involved in oxidative

stress signaling (Foyer et al., 1997; May et al. 1998a; Ramel et al., 2012).

1.1.2.2 Enzymatic metabolism of ROS

As noted above, superoxide is formed in several cell compartments and the apoplast, and is metabolized

to H2O2 and/or O2. While direct reduction to H2O2 through non-enzymatic systems (e.g., ferredoxin, GSH,

ASC) can occur, dismutation via SOD is thought to the most important factor in superoxide metabolism.

Several isoforms of SOD with different metal co-factors are found in plants, including Mn-SOD

(mitochondria), Fe-SOD (chloroplast) and Cu,Zn-SOD (chloroplast, cytosol, peroxisome, apoplast) (Fink

and Scandalios, 2002).

Whether produced directly from O2 by oxidases or indirectly from superoxide by SOD, H2O2 is the most

stable ROS. It has therefore attracted considerable attention as a signal molecule. Although H2O2 can

oxidize some groups such as protein thiols, it can also be cleaved to the highly reactive hydroxyl radical.

Hence, enzyme systems involved in H2O2 metabolism are particularly important to maintain redox

homeostasis and avoid uncontrolled cellular oxidation. Numerous types of enzyme can metabolize H2O2,

but these can be divided into two broad classes. The first consists of catalases which, like SOD, catalyze a

dismutation reaction and so do not require any other reductant. The second group is more diverse, and is

comprised of various peroxidases which reduce H2O2 to water and therefore require a reducing co-factor.

Catalases (CAT), exists in almost all organisms, and possess a high capacity for metabolizing H2O2

through the dismutation of H2O2 to water and O2. In most organisms, CAT are located mainly in the

peroxisomes (Zamocky et al., 2008). Three CAT genes have been identified in Angiosperm species

studied to date, including tobacco, Arabidopsis, maize, pumpkin and rice (Willekens et al., 1995; Frugoli

et al., 1996; Guan and Scandalios, 1996; Esaka et al., 1997; Iwamoto et al., 2000), and a classification

system based on the naming of the tobacco genes was introduced by Willekens et al. (1995), with class I

CAT designating the major isoform expressed in leaves. According to this grouping, Arabidopsis CAT1,

CAT2, and CAT3 correspond to Class III, Class I, and Class II catalases, respectively (Table 1.1). Two are

located on chromosome 1 (CAT1, CAT3) and one is located on chromosome 4 (CAT2) based on genome

sequencing (Frugoli et al., 1996). The three corresponding amino acid sequences have high similarity. In

Arabidopsis, CAT1 is mainly expressed in pollen and seeds. CAT2 is the major form of leaf catalase

which is highly expressed in photosynthetic tissue, whereas CAT3 is associated with vascular tissues but

also leaves (Hu et al., 2010).

Whereas irreplaceable functions for Arabidopsis CAT1 and CAT3 remain to be described (Mhamdi et al.,

2010b), studies of mutants have demonstrated important physiological roles for CAT2 in photorespiration,

maintenance of leaf redox homeostasis, and modulation of leaf senescence (Queval et al., 2007;

Smykowski et al., 2010). Indeed, the first report of obvious oxidative stress in a catalase-deficient line

came from forward genetics studies of a barley mutant isolated via a photorespiratory screen. The mutant

has about 10% wild-type leaf catalase activity but shows a wild-type phenotype when grown at high CO2;

however, lesions appear when the plant is transferred to air (Kendall et al., 1983). The appearance of

4


lesions in this line is preceded by marked accumulation of oxidized glutathione (Smith et al., 1984). This

effect is also observed in tobacco transformants in which leaf catalase expression has been markedly

decreased by antisense technology (Willekens et al., 1997) or in Arabidopsis cat2 knockouts (Queval et

al., 2007), suggesting that accumulation of oxidized glutathione is a conserved response to catalase

deficiency in the leaves of plants with C3 photosynthesis. Because of the ease of manipulating oxidative

stress symptoms in such lines, they have been used as stress mimic tools to investigate oxidative

stress-related signaling pathways (Chamnongpol et al., 1996, 1998; Willekens et al., 1997; Vandenabeele

et al., 2004; Vanderauwera et al., 2005; Queval et al., 2007; Chaouch et al., 2010; Mhamdi et al., 2010b).

Nevertheless, the functional importance of the changes in glutathione in this signaling have received little

attention. A study of double cat2 gr1 mutants, doubly deficient in catalase and glutathione reductase (GR;

Mhamdi et al., 2010a; annex 1) revealed that dramatic accumulation of oxidized glutathione was

associated with altered phytohormone responses. However, interpretation of these effects is complicated

by the simultaneous loss of function of two antioxidative enzymes, reflected in the extreme phenotype of

the double mutant when grown in air (Mhamdi et al., 2010a).

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

Class I Class II Class III

Tobacco Cat1 Cat2 Cat3

Arabidopsis CAT2 CAT3 CAT1

Maize Cat2 Cat3 Cat1

Pumpkin Cat2 Cat3 Cat1

Rice CatC CatA CatB

As well as catalases, the best described route for H2O2 metabolism is the ascorbate-glutathione pathway

in which GSH (indirectly) and ASC (directly) are involved (Figure 1.2). This pathway exists in several

cell organelles, including chloroplasts, mitochondria and peroxisomes (Jiménez et al., 1997; Chew et al.,

2003).

APX, ascorbate peroxidase; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione;

GSSG, glutathione disulphide; MDHAR, monodehydroascorbate reductase.

Figure 1.2. Two of the major pathways for H2O2 metabolism in plants (scheme taken from Foyer and Noctor, 2009).

5


In the ascorbate-glutathione pathway, H2O2 is reduced to H2O by ascorbate peroxidase (APX), an enzyme

with a much higher affinity for H2O2 than CAT, leading to oxidation of ASC to monodehydroascorbate

(MDHA; Figure 1.3). As well as APX, which is specific to H2O2 as oxidant, plants also contain several

types of other peroxidases that can reduce organic peroxides in addition to H2O2. Among these,

peroxiredoxins (PRX) may have an important function alongside APX in H2O2 removal (Dietz et al.,

2006). The reduced form of PRX is regenerated from the oxidized form by either a thiol or ASC and the

enzymes glutaredoxins (GRX), thioredoxins (TRX) and NADPH-thioredoxin reductase (NTR; Dietz,

2003; Rouhier and Jacquot, 2005; Meyer, 2008; Pérez-Ruiz and Cejudo, 2009). Based on the number of

cysteine residues and the catalytic mechanism, in plants PRXs are divided into four sub-classes: 1-Cys

PRX, 2-Cys PRX, Prx Q and type II PRX (Dietz, 2003; Meyer, 2008). It has been concluded that 2-cys

PRX could play a significant role in the chloroplast, where peroxiredoxin capacity to reduce H2O2 was

reported to be about half that of soluble APX (Dietz et al., 2006). In addition to detoxification of lipid

peroxides, glutathione peroxidases (GPXs) are able to remove H2O2. Despite their name, it is likely that

GPXs function in vivo with TRX rather than GSH (Herbette et al., 2002; Iqbal et al., 2006). Finally,

glutathione S-transferases (GSTs) could contribute to peroxide metabolism. Although the most studied

role of these enzymes is in removal of electrophilic xenobiotics by the formation of GS-conjugates,

several GSTs can also act as GSH-dependent peroxidases and are able to remove H2O2 (Dixon et al., 2009;

Dixon and Edwards, 2010). In plants, GSTs are divided into several groups and some of their possible

functions are further discussed in section 1.2.4.1.

APX, ascorbate peroxidase. ASC, ascorbate. CAT, catalase. DHAR, dehydroascorbate reductase. Fdox, oxidized ferredoxin.

Fdred, reduced ferredoxin. FTR, ferredoxin-thioredoxin reductase. GPX, glutathione(thioredoxin) peroxidase. GR,

glutathione reductase. GRX, glutaredoxin. GSH, glutathione. GSSG, glutathione disulphide. GST, glutathione S-transferase.

MDHA, monodehydroascorbate reductase. NTR, NADPH-thioredoxin reductase. PRX, peroxiredoxin. ROOH, organic

peroxide. TRXox, oxidized thioredoxin. TRXred, reduced thioredoxin

Figure 1.3. Part of the complexity of plant H2O2-metabolizing systems (scheme taken from Mhamdi et al., 2010b).

Unlike catalases, peroxidases require reductant, whether ASC, GSH or other. Maintaining the pools of

these reductants requires several other categories of enzyme such as dehydroascorbate reductase (DHAR),

GR, and NADPH-generating dehydrogenases (Figures 1.2 and 1.3). Given the high number and diversity

6


of H2O2-metabolizing systems, it remains unclear which are the most important. Insight into the

interaction between different ROS and ROS scavenging mechanisms has been obtained by studies of

double or triple mutants that are defective in key ROS-scavenging enzymes found in different subcellular

organelles (Giacomelli et al. 2007; Miller et al. 2007; Mhamdi et al., 2010a) and exploration of crosstalk

between distinct ROS such as 1O2 or H2O2 originating in different compartments (Laloi et al., 2007;

Chaouch et al., 2012). These data have not only revealed much apparent redundancy in the

ROS-scavenging network, but also suggest that different antioxidant enzymes and different ROS in the

same (or different) compartments, mediate specific signals in plant responses to various environmental

stimuli and development (Harir and Mittler, 2009).

1.1.3 Functions of ROS in plants ROS were initially considered as toxic by-products of aerobic metabolism which are tightly regulated by

means of antioxidant and enzymatic components of the ROS scavenging pathways (Apel and Hirt, 2004;

Møller et al., 2007). However, it is now apparent that they are key components in fundamental plant

processes including growth, development, response to biotic and abiotic stresses, as well as PCD. Hence,

ROS are important signaling molecules. A simple view of ROS signaling in plants is shown in the model

in Figure 1.4.

These active oxygens are produced at a number of intercellular and extracellular sites, including mitochondria, chloroplasts,

peroxisomes and apoplast. ROS trigger a series of signal transduction events. The influence of these molecules on cellular

processes is mediated by both the perpetuation of their production and their removal by scavenging enzymes such as

superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT). The location, amplitude, and duration of

production of these molecules determine the specificity and rapidity of the responses they elicit.

Figure 1.4. ROS form in plant cells as a consequence of myriad stimuli ranging from abiotic and biotic stress,

production of hormonal regulators, as well as cell processes such as acclimation and PCD (scheme taken from Mittler

et al., 2004).

7


1.1.3.1 The role of ROS in abiotic stress

Plants are continually exposed to changes in their environment. Abiotic stresses (eg, ozone, excess light,

heavy metal or drought) are perceived by plant cells and induce an acclimatory response through ROS

signaling. The most common abiotic stresses lead to increased ROS production from the different

subcellular sources through down-regulation of scavenging enzymes or up-regulation of generating

systems which dictate the expression pattern of specific sets of genes and the induction of certain

acclimation and defence mechanisms (Mittler et al., 2004). For instance, both HsfA4a and HsfA8 were not

induced by enhanced peroxisomal H2O2 in a cat2-deficient line but induced by elevated cytosolic H2O2 in

an apx1 knockout mutant (Pnueli et al., 2003; Davletova et al., 2005; Vanderauwera et al., 2005).

1.1.3.2 Regulation of growth and developmental processes by ROS

The spatial control of cell growth is a central process in plant development. ROS play an important role in

the regulation of growth through their various effects on cell wall elasticity. Unlike the well-known

signaling roles in pathogen response (see below), the roles of ROS in development are less well

understood. The best studied developmental role for NADPH-derived ROS is in the regulation of tip

growth in root hairs and pollen tubes (Foreman et al., 2003; Monshausen et al., 2007; Macpherson et al.,

2008; Potocky et al., 2007). However, accumulating evidence indicates that ROS may play a more

general role in developmental regulation other than just in tip extension growth. Inhibitor tests indicate

that the promotion of maize root elongation may be regulated by NADPH oxidases (Liszkay et al., 2004),

and apoplastic ROS accumulation has also been implicated in the involvement of maize leaf development

(Rodriguez et al., 2007). Moreover, ROS are involved in abscisic acid (ABA)-mediated stomatal closure,

the modulation of cell wall loosening during seed germination, and fruit ripening (Apel and Hirt, 2004,

Dunand et al., 2007; Müller et al., 2009a, b).

1.1.3.3 Functions of ROS in plant innate immunity

One of the initial responses observed after pathogen attack is the oxidative burst, which involves a rapid

increase in ROS production such as superoxide and H2O2. Several reports in various plant species suggest

that both plasmalemma-located NADPH oxidase and extracellular peroxidases contribute to this rapid

increase in ROS (Torres et al., 2002, 2005; Bindschedler et al., 2006; Daudi et al., 2012; O’Brien et al.,

2012). However, these initial signals at the cell surface/apoplast lead to later downstream adjustments in

intracellular redox state that are notably associated with intracellular ROS generated by chloroplasts,

mitochondria as well as peroxisomes (Edwards et al., 1991; Volt et al., 2009; Chaouch et al., 2012). The

increased ROS may contribute to resistance via several mechanisms. Firstly, invading pathogens may be

directly killed by ROS and ROS may mediate the strengthening of the cell wall through H2O2-dependent

lignification reactions that confine the pathogen to the infection site (Durner et al., 1997). Secondly,

effector-triggered immunity (ETI) is visibly accompanied by the HR in which cell death occurs at the site

of pathogen infection, also acting to limit the pathogen growth. ETI is usually linked with ROS

accumulation. In addition to roles in triggering HR, it has been suggested that ROS act, together with

salicylic acid (SA), to mediate the establishment of systemic acquired resistance (SAR) in the distal

uninoculated systemic tissue of a pathogen-infected plant, conferring improved resistance against a broad

8


spectrum of pathogens (Fobert et al., 2005). Nonexpressor of pathogenesis-related genes 1 (NPR1)

protein is required for SA signaling and it is a key component of SAR regulation (Cao et al., 1994). NPR1

contains an ankyrin-repeat motif and a BTB/POZ domain. The protein can be found in the nucleus, where

it functions in SA-dependent PR gene expression. SA-induced changes in cell redox state trigger the

reduction of NPR1 disulfide bonds, and NPR1 monomers are subsequently translocated from the cytosol

to the nucleus, where they interact with TGA transcription factors to activate the expression of defence

genes such as PATHOGENESIS-RELATED 1 (PR1; Després et al., 2003; Tada et al., 2008; Mou et al.,

2003). It was proposed that the reduction of disulfide bonds of the oligomeric form of NPR1 could be

mediated by TRX (Tada et al., 2008). According to this model, induction of PR gene expression by this

pathway involves monomerization of oligomeric NPR1 driven by SA-induced changes in cellular redox

state, leading to reduction of two NPR1 cysteine residues (Cys82 and Cys216) by, for example, TRX H5

and/or TRX H3. NPR1 monomers are subsequently translocated from the cytosol into the nucleus, where

they induce defence genes by interacting with TGA transcription factors (Després et al., 2003; Mou et al.,

2003).

Finally, camalexin, the major recognized phytoalexin in Arabidopsis thaliana, is a low molecular mass

secondary metabolite with antimicrobial activity. ROS are also generally involved in camalexin

production, as shown by experiments using the oxidative stress-inducing chemical (paraquat) and the

oxidative stress mimic mutant, cat2 (Zhao et al., 1998; Chaouch et al., 2010).

1.1.3.4 ROS in cell death

Plants possess active genetic cell death programs that have become integral parts of their growth,

development, and reactions with the environment (Lam, 2004). This involves various processes starting

as early as embryogenesis. For example, PCD can be observed in the tissues of germinating seeds and

during leaf senescence. PCD can also be observed during HR and ozone stress. ROS closely interplay

with other signaling components in the regulation of PCD (Overmyer et al., 2005). For example,

ROS-induced cell death is influenced by a variety of plant hormones. SA is a key candidate considered to

act in concert with ROS to cause HR-like lesions, and ROS-triggered SA accumulation is sufficient to

cause lesions, even in the absence of pathogen attack (Danon et al., 2005; Chaouch et al., 2010). Other

defence hormones such as jasmonic acid (JA) and ethylene also are involved in ROS-dependent PCD

(Fath et al., 2001; de Jong et al., 2002; Overmyer et al., 2000; Moeder et al., 2002; Danon et al., 2005).

Besides phytohormones, nitric oxide (NO) operates in a dose-dependent manner, synergistically with

H2O2, in the modulation of HR-like cell death (Delledonne et al., 2001; Zago et al., 2006). In addition,

lipidic components such as sphingolipids, oxylipins and phospholipids can interact with ROS to modulate

PCD (Liang et al., 2003; Loeffler et al., 2005; Meijer and Munnik, 2003). Metacaspases and metabolites

such as myo-inositol have also been implicated in ROS-induced cell death (He et al., 2008; Chaouch and

Noctor, 2010).

1.1.4 ROS signaling network

Although specific ROS sensors or receptors has not yet been clearly identified in the plant cell,

redox-sensitive proteins and other as yet unknown factors have been proposed to transduce ROS signals

9


(Neill et al., 2002; Mittler et al., 2004; Apel and Hirt, 2004; Van Breusegem et al., 2008). To date, NPR1

remains the best example of a redox-sensitive protein clearly associated with ROS signaling. As noted

above, TRX may control the NPR1 redox state (Tada et al., 2008). However, recently, other evidence

suggests that redox regulation of this protein also involves S-nitrosylglutathione (GSNO; Lindermayr et

al., 2010). Besides TRX, PRX have been suggested to act as redox sensors in plants (Dietz, 2008), and

such functions could act analogously to the regulation of yAP1 in yeast by peroxidase-dependent

oxidation by H2O2 (Delaunay et al., 2002). Genetic screens for mutants impaired in ROS sensing could

possibly contribute to revealing some of these mechanisms, though surprisingly few have as yet been

identified by such approaches.

Despite the failure to uncover direct “sensors” of ROS in plants, it is apparent that ROS signaling

networks with many different signaling components, eg, protein kinases and calcium/calmodulin (Mittler

et al., 2011). Roles for mitogen-activated protein kinase (MAPK) cascades in ROS signaling are

established. Many different kinases such as MAPKKK MEKK1, MPK3, MPK4 and MPK6 can be

activated following ROS accumulation (Teige et al., 2004; Xing et al., 2008; Jammes et al., 2009). The

MEKKI pathway is highly activated during abiotic stress, which was suggested to be specifically linked

to H2O2-mediated activation of MPK4 (Nakagami et al., 2006; Xing et al., 2008). Two other

ROS-responsive MAP kinases, MPK3 and MPK6, are activated by the serine/threonine protein kinase

OXI1 (OXIDATIVE SIGNAL INDUCIBLE 1), which has been shown to play a role in downstream ROS

signaling (Anthony et al., 2004; Rentel et al., 2004). Recently it has shown that MPK9 and MPK12

function downstream of ROS to regulate ABA-induced stomata closure in guard cells and to activate

anion channels (Jammes et al., 2009). In addition, it has been suggested that some phosphatases such as

the MAPK phosphatase, AtMKP2, and the tyrosine phosphatase, AtPTP1, have negative effects on MPK

activation (Gupta et al., 2003; Lee and Ellis, 2007). A MAPK cascade is also implicated in NO signaling,

which synergistically functions with ROS signaling in pathogen defence responses such as HR (Asai et al.,

2008).

Apart from the activation of the MAPK cascade in ROS signal transduction, several transcription factors

(TF) are affected by ROS availability, including four identified members of the WRKY, Zat, Hsf, and

Myb families (Mittler et al., 2004; Davletova et al., 2005; Vanderauwera et al., 2005; Gadjev et al., 2006;

Miller et al., 2008). These ROS-sensitive TFs can then lead to changes in gene expression (Vanderauwera

et al., 2005; Gadjev et al., 2006).

The intensity, duration and localization of ROS signals are controlled by the complex interaction between

ROS generation and ROS scavenging systems (Figure 1.4; Mittler et al., 2004). Many studies of knockout

and antisense lines for generating and scavenging systems such as CAT2, APX1, chlAOX, mitAOX,

CSD2, 2-cysteine PRX, GR1 and various NADPH oxidases have revealed a strong link between ROS and

processes such as growth, development, stomatal responses and biotic and abiotic stress responses

(Maxwell et al., 1999; Baier et al., 2000; Torres et al., 2002; Epple et al., 2003; Foreman et al., 2003;

Rizhsky et al., 2003; Pnueli et al., 2003; Mittler et al., 2004;; Sagi et al., 2004; Vandenabeele et al., 2004;

Mhamdi et al., 2010a; Vanderauwera et al., 2011; Chaouch et al., 2012). These findings demonstrate the

complex nature of the ROS gene network in plants and its close integration with key biological processes.

Although the redundancy of the ROS network is evidenced by such work, double or triple mutants

10


lacking in key ROS network genes can serve as excellent tools to study interactions between different

types of ROS generated at different subcellular locations.

1.2 Glutathione in plants

The tripeptide glutathione (GSH) is found in most organisms and is an essential metabolite with multiple

functions in plants. Alongside the most widely found form of GSH (-L-glutamyl-L-cysteinyl-glycine)

homologs are found in some groups of plants such as homoglutathione (hGSH;

-L-glutamyl-L-cysteinyl--alanine) and hydroxymethylglutathione -L-glutamyl-L-cysteinyl-L-serine).

In plants, glutathione is the most abundant non-protein thiol, typically accumulating in cells to

concentrations of about 1-5 mM. 1.2.1 Glutathione synthesis In both animals and plants, GSH is synthesized from glutamate, cysteine and glycine by two

ATP-dependent steps (Figure 1.5; Rennenberg, 1980; Meister, 1988; Noctor et al., 2002; Mullineaux and

Rausch, 2005). Each of the synthetic enzymes is encoded by a single gene in Arabidopsis (May and

Leaver, 1994; Ullman et al., 1996). Localization studies in Arabidopsis have demonstrated that the first

enzyme, -glutamylcysteine synthetase (-ECS), encoded by GSH1, is located in plastids, whereas the

second enzyme, glutathione synthetase (GSH-S), encoded by GSH2, is found in both chloroplasts and

cytosol (Wachter et al., 2005).

Glycine

Cysteine Glutamate

-EC

GSH-S

-EC

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

GSH

11


1.2.1.1 Glutathione deficient mutants

While knockout mutations in GSH1 cause lethality at the embryo stage (Cairns et al., 2006), T-DNA

knockouts for GSH2 show a seedling-lethal phenotype (Pasternak et al., 2008). Less severe mutations in

the GSH1 gene, which produce partial decreases in glutathione contents, have contributed appreciably to

the study of the functions of glutathione in plants. Of these, the rml1 mutant, which has less than 5% of

wild-type glutathione contents, shows the most striking phenotype because it fails to develop a root apical

meristem (Vernoux et al., 2000). In other mutants, in which glutathione is decreased to about 25 to 50%

of wild-type contents, developmental phenotypes are weak or absent, but alterations in environmental

responses are observed. In cad2, lower glutathione is associated with enhanced cadmium sensitivity,

whereas rax1 was identified by modified APX2 expression and pad2 shows lower camalexin contents and

enhanced sensitivity to pathogens (Howden et al., 1995; Cobbett et al., 1998; Ball et al., 2004; Parisy et

al., 2007). Very recently, yet another mutant partly deficient in glutathione because of a mutation in GSH1

has been reported. The zir1 mutant has only about 15% wild-type glutathione levels and was isolated in a

screen for mutants that have defects in iron-mediated tolerance to zinc (Shanmugam et al., 2012). As well

as genetic approaches, a pharmacological tool that has frequently been used to analyze the functions of

glutathione is buthionine sulphoximine (BSO), a specific inhibitor of -ECS (Griffith and Meister, 1979).

1.2.1.2 Regulation and overexpression of the glutathione synthesis pathway

The production of glutathione is regulated at multiple levels, including regulation of cysteine synthesis

and GSH1 and GSH2 at the level of transcript accumulation (Xiang and Oliver, 1998; Queval et al., 2009),

post-translational regulation of adenosine phosphosulfate reductase (Bick et al., 2001), and

post-translational regulation of -ECS (May et al., 1998b; Hicks et al., 2007). Glutathione accumulation is

a well known response to enhanced H2O2 availability, and was first described in a barley mutant deficient

in catalase (Smith et al., 1984). This response presumably reflects enhanced rates of glutathione synthesis

and the major drivers in such situations are probably oxidant-induced increases in the limiting amino acid,

cysteine, and post-translational activation of -ECS by oxidation (Queval et al., 2009).

Although the production of glutathione is regulated at multiple levels, plant cells can accommodate

marked increases in overall glutathione. Studies using overexpression of -ECS targeted to either the

chloroplast or the cytosol increase leaf glutathione by up to four-fold in different plant species (Noctor et

al., 1996, 1998; Creissen et al., 1999; Xiang et al., 2001). Recently, considerably greater increases in

glutathione have been achieved by overexpression of a bifunctional -ECS/GSH-S from Streptococcus

(Liedschulte et al., 2010).

1.2.2 Glutathione transport

Glutathione synthesized in the chloroplast and cytosol is transported between cells and organs. One group

of proteins that could be responsible for transport across the plasmalemma is the oligopeptide transporter

12


(OPT) family (Figure 1.6). For example, of the nine annotated Arabidopsis OPT genes, OPT6 may play a

role in long-distance transport, where it may transport GS-conjugates and GS-cadmium as well as GSH

and GSSG (Cagnac et al., 2004).

Several different types of transporter may be important in translocation of glutathione between subcellular

compartments. First, direct analysis of 35S-GSH uptake by wheat chloroplasts suggested the existence of

components able to translocate glutathione across the inner chloroplast envelope (Noctor et al., 2002).

That such transport occurs in vivo is strongly supported by the observation that the wild-type phenotype

of gsh2 knockout mutants, which lack the ability to convert -EC to GSH, can be restored by

transformation with a construct driving GSH-S expression exclusively in the cytosol (Pasternak et al.

2008). Recently, the chloroquinone-like transporter (CLT) family was described. These proteins are

targeted to the plastid envelope and can transport both -EC and glutathione from the chloroplast into the

cytosol (Maughan et al., 2010). These transporters are encoded by three genes in Arabidopsis, called

CLT1, CLT2 and CLT3. Concerning transport into the vacuole, tonoplast multidrug resistance-associated

protein (MRP) transporters of the ATP-binding cassette (ABC) type may act to clear GS-conjugates or

GSSG from the cytosol (Figure 1.6; Martinoia et al., 1993; Rea, 1999; Foyer et al., 2001). While the

anti-apoptotic factor Bcl-2 is considered as a key component to regulate GSH transport into the nucleus

and mitochondria in mammalian tissues (Voehringer et al., 1998), transporters that may regulate the

distribution of glutathione between the nucleus and mitochondria remain to be identified.

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

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

oligopeptide transporter.

13


1.2.3 Glutathione degradation

Rates of glutathione catabolism in Arabidopsis leaves have been estimated to be as high as 30 nmol g-1

FW h-1 (Ohkama-Ohtsu et al., 2008), which can be compared with typical leaf contents of 200-400

nmol.g-1FW. Thus, it is possible that some pools of glutathione could turn over several times in a day. In

plants, several pathways of glutathione turnover have been proposed. Among them, four different types of

enzymes have been described that could initiate glutathione breakdown (Figure 1.7). Some of these

enzymes could use GSH, while others act preferentially on GSSG or other GS-conjugates. Firstly,

glutathione or GS-conjugates could be degraded by carboxypeptidase activity (Steinkamp and

Rennenberg, 1985), which has been detected in barley vacuoles (Wolf et al., 1996). However, specific

genes remain to be indentified.

A second type of enzyme for glutathione catabolism involves the cytosolic enzyme phytochelatin

synthase (PCS). Although this enzyme is most associated with polymerization of GSH-derived -EC units

to form the metal-complexing phytochelatins (see section 1.2.4.4), it could also play a role in

GS-conjugate degradation (Blum et al., 2007, 2010). The third type of enzyme, -glutamyl transpeptidase

(GGT), could cleave glutathione to cysteinylglycine and a -glutamyl-amino acid derivative

(transpeptidase activity) or free glutamate (peptidase activity). New insights have been gained by

analyzing Arabidopsis mutants perturbed in glutathione degradation. In Arabidopsis, GGTs are encoded

by four functional genes. The apoplastic enzyme GGT1 is active against GSH, GSSG and GS-conjugates

and is prominent in all Arabidopsis tissue except seeds and siliques (Martin and Slovin, 2000;

Storozhenko et al., 2002) while another apoplastic GGT2 is mainly expressed in siliques. Unlike the

localization in animal cells, GGT1 and GGT2 are probably bound to the cell wall rather than to the

plasmalemmma (Martin et al., 2007; Ohkama-Ohtsu et al., 2007a). Besides the extracellular GGTs,

intracellular GGT4 (originally called GGT3 in Arabidopsis) is a vacuolar enzyme which is probably

involved mainly in the breakdown of GS-conjugates (Grzam et al., 2007; Martin et al., 2007;

Ohkama-Ohtsu et al. 2007b). The fourth predicted GGT enzyme, now called GGT3, could be detected in

some tissues but is considered unlikely to contribute greatly to GGT activity (Martin et al., 2007; Destro

et al., 2010).

It has been suggested that -glutamyl peptides produced by GGT can be further metabolized to

5-oxoproline by -glutamyl-cyclotransferases (GGC) and subsequently 5-oxoprolinase (5-OPase) to

produce free glutamate (Ohkama-Ohtsu et al., 2008). No gene has yet been identified for GGC in

Arabidopsis but a single gene OXP1 has been identified that likely encodes a cytosolic 5-OPase

(Ohkama-Ohtsu et al., 2008). Based on 5-oxoproline accumulation in single oxp1 mutants, and in triple

oxp1 ggt1 ggt4 mutants that are deficient in the major GGT activities as well as 5-OPase, it was proposed

that the predominant pathway for GSH degradation is cytosolic and initiated by GGC (Figure 1.7), and

not vacuolar or extracellular GGT (Ohkama-Ohtsu et al., 2008). GGC is therefore a fourth type of enzyme

potentially involved in initiating glutathione degradation, although both the rat and tobacco GGC have

been reported to be unable to use GSH (Orlowski and Meister, 1973; Steinkamp et al., 1987).

14


Figure 1.7. Possible pathways of glutathione degradation (scheme from Noctor et al., 2012; annex 2). aa, amino acid; Cpep, carboxypeptidase; GGC, -glutamyl cyclotransferase; GGT, g-glutamyl transpeptidase; 5-OP,

5-oxoproline; 5-OPase, 5-oxoprolinase; PCS, phytochelatin synthase; X, S-conjugated compound. 1.2.4 Metabolic functions of glutathione in plants

GSH is oxidized to glutathione disulfide (GSSG) via not only enzymatic reactions catalyzed by DHAR or

GSH-dependent peroxidases but also non-enzymatic reactions in which GSH is oxidized by ROS or,

chemically, by DHA. The regeneration of GSH mainly is performed by NADPH-dependent GR. In

addition to redox cycling, glutathione is involved in many other types of reaction.

1.2.4.1 Glutathione S-transferases

The plant glutathione S-transferases (GSTs; EC 2.5.1.18) are a large and diverse group of enzymes which

catalyze the conjugation of electrophilic xenobiotics substrates with glutathione. The glutathione

conjugates are then transferred to the vacuole by ATP-dependent GS-X pumps and degraded. In plants,

the activity of most GSTs depends on an active site serine, which stabilizes the GS-thiolate anion (Dixon

and Edwards, 2010). Based on sequence similarity and gene structure of family members, the

monophyletic group of soluble GST super-family is subdivided into evolutionarily distinct classes in

plants, each denoted by a Greek letter. Yet another two classes are DHARs and tetrachlorohydroquinone

dehalogenase-like (TCHQD) enzymes. In all, the Arabidopsis genome contains a total of 55

GST-encoding genes which can be divided into 8 classes (Table 1.2). While specific nomenclatures have

been proposed for these GSTs, much remains to be discovered regarding their functions.

15


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

Class Letter code Number of Arabidopsis gene

Phi F 13

Tau U 28

Theta T 3

Zeta Z 2

Lambda L 3

Dehydroascorbate reductase - 4

Tetrachlorohydroquinone dehalogenase-like - 1

Microsomal - 1

As well as their role in catalysing the conjugation and detoxification of herbicides, other GST-associated

activities have been proposed including intracellular transport of small molecules such as flavonoids,

transient glutathione conjugation to protect reactive metabolites such as porphyrinogens and oxylipins,

introduction of sulfur into secondary metabolites such as glucosinolates and camalaxin, and cis-trans

isomerisation reactions (Edwards et al., 2000; Wagner et al., 2002; Dixon and Edwards, 2009, 2010;

Geu-Flores et al., 2011; Su et al., 2011). Besides these functions, many GSTs have peroxidase or DHAR

activity, and therefore function directly as antioxidant enzymes, as described above in section 1.1.2.2 and

developed further below.

While ROS or DHA can react chemically with glutathione at significant rates, DHARs, members of the

GST superfamily, provide an enzymatic link between GSH and DHA, thus promoting maintenance of

ASC at the expense of the glutathione pool. Unlike most other GSTs, DHARs have an active site cysteine

instead of serine/tyrosine, so rather than stabilising the thiolate anion of GSH, this residue instead forms a

mixed disulfide with GSH as part of the catalytic mechanism (Dixon et al., 2002b). Also, at the protein

level, DHARs behave differently from most other GSTs in being expressed as monomers rather than as

dimers (Dixon et al., 2002a). They are found in peroxisomes, mitochondria, cytosol, and chloroplast

(Chew et al., 2003; Reumann et al., 2009). Arabidopsis has 5 DHAR-like genes of which 3 are transcribed

and encode functional proteins, namely DHAR1, DHAR2, and DHAR3. DHAR4 appears to be a

pseudogene encoding a full-length but inactive enzyme, while the fifth DHAR corresponds to an

untranscribed region encoding an N-terminally truncated, hence inactive enzyme. Several genetic studies

show that DHARs play an important role in plants, particularly in conditions of oxidative stress such as

ozone, aluminium stress, or pathogen inoculation (Chen and Gallie, 2006; Yoshida et al., 2006; Tamaoki

et al., 2008; Vadassery et al., 2009).

1.2.4.2 Glutathione reductase

In most living organisms, NADPH-dependent GR maintains a high cellular GSH:GSSG ratio. GR is a

NADPH-dependent dimeric flavoprotein belonging to the pyridine nucleotide-disulfide oxidoreductase

group. Although NTR can also reduce GSSG in a TRX-dependent manner (Marty et al., 2009), GR is

more efficient than the NTR-TRX system. Two genes encode GR in Arabidopsis. GR2 encodes an

16


enzyme that is dual-addressed to plastids and mitochondria, while GR1 encodes a protein that is found in

the cytosol and peroxisome (Chew et al., 2003; Kataya and Reumann, 2010).

Arabidopsis knockout mutants for the chloroplast/mitochondrial GR2 are embryo-lethal (Tzafrir et al.,

2004), demonstrating the importance of maintaining an appropriate glutathione reduction state in plastids,

mitochondria, or both. In contrast, gr1 knockout mutants do not show phenotypic effects, despite a

30-60% reduction in extractable enzyme activity (Marty et al., 2009; Mhamdi et al., 2010a). Genetic

analyses have shown that the aphenotypic nature of gr1 mutants results from partial replacement of GSSG

regeneration by the cytosol-located NTR-TRX system, though this is not sufficient to prevent significant

accumulation of GSSG in the mutant (Marty et al., 2009).

1.2.4.3 Glutaredoxins

GRXs are small oxidoreductases structurally related to thioredoxins and generally considered to be

reduced by glutathione. In Arabidopsis, over 30 GRX genes are annotated to encode GRX. They have two

major glutathione-dependent biochemical properties: the reduction of disulphide bonds and the binding of

iron-sulphur clusters. Normally, GRXs reduce a mixed disulfide between a protein and glutathione or a

disulfide bridge via two catalytic mechanisms including monothiol and dithiol dependent catalytic

mechanisms (Figure 1.8). In land plants, GRXs can be classified into three distinct subgroups based on

amino acid motifs in the active site (Lemaire, 2004; Rouhier, 2010). The activity of the first CYPC-type

class, which contain GRXs with C[P/G/S][Y/F][C/S] motifs, include thiol-disulphide exchange,

regeneration of PRX and methionine sulphoxide reductase (Rouhier et al., 2002; Rouhier et al., 2006;

Zaffagnini et al., 2008; Tarrago et al., 2009; Gao et al., 2010). The second CGFS-type class, which

possesses a conserved CGFS active site sequence, could catalyze protein deglutathionylation and are

involved in iron–sulphur cluster assembly. While some of these GRX have been characterized

biochemically, their in vivo roles remain unclear. In Arabidopsis, GRXs can interact with ion channels and

may play a role in response to oxidative stress (Cheng and Hirschi, 2003; Cheng et al., 2006; Guo et al.,

2010; Sundaram and Rathinasabapathi, 2010). The third CC-type class, which regroups proteins with

CC[M/L][C/S/G/A/I] active sites, is specific to land plants, because of the lack of related sequences in

genomes of lower photosynthetic organisms, bacteria and mammals (Lemaire, 2004). Although little

information about the biochemistry of the plant specific CC-type GRXs is available, some member of this

class are the best studied GRXs in terms of physiological function. Several studies in Arabidopsis have

revealed that three GRX forms are involved in plant development and phytohormone responses through

interaction with TGA transcription factors (Ndamukong et al., 2007; Li et al., 2009). Notably, GRX480

overexpression was shown to repress the JA signaling marker gene, PDF1.2 (Ndamukong et al., 2007),

while ROXY1 and ROXY2 are involved in petal and anther development (Li et al., 2009). In addition,

overexpression of ROXY1 in Arabidopsis increased susceptibility to Botrytis cinerea (Wang et al., 2009c).

17


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

1.2.4.4 Other glutathione-associated metabolic reactions

As well as the above processses, other glutathione-associated metabolic reactions include phytochelatin

synthesis, glyoxylase and formaldehyde metabolism, camalexin production and glucosinolate synthesis.

Glutathione is the precursor of phytochelatins ([-Glu-Cys]nGly), which are required for responses to

cadmium and other heavy metals. These compounds are produced from glutathione by PCS, a cytosolic

enzyme. In Arabidopsis, there are two genes encoding PCS, of which PCS1 appears to be the most

important. As noted in section 1.2.3, PCS1 may play a role in degradation of GS-conjugates as well as

phytochelatin synthesis (Rea et al., 2004; Blum et al., 2007, 2010; Clemens and Peršoh, 2009).

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

1987, 1989; Cobbett and Goldsbrough, 2002; Rea et al., 2004). The importance of sufficient amounts of

GSH to confer heavy metal resistance is evidenced by the identification of the glutathione-deficient cad2

mutant which is sensitive to cadmium (Howden et al., 1995; Cobbett et al., 1998). This is in agreement

with increased heavy metal resistance by transgenic up-regulation of glutathione synthesis (Zhu et al.,

1999a,b). As well as a precursor role in phytochelatin synthesis, glutathione could be involved in heavy

metal resistance via its antioxidant function.

Oxo-aldehydes such as methylglyoxal are reactive compounds that may interfere with sensitive cellular

compounds. Metabolism of such oxo-aldehydes through the glyoxylase system involves conjugation to

GSH, catalyzed by glyoxalase 1 and then the hydrolytic reaction catalysed by glyoxalase 2 liberates the

hydroxyacid and free GSH. In several plant species, overexpression of these enzymes increases tolerance

to exogenous methylglyoxal and/or salt (Singla-Pareek et al., 2003, 2008; Deb Roy et al., 2008). Apart

from its involvement in the catabolism of oxo-aldehydes, GSH may also act in detoxification of

formaldehyde via formaldehyde dehydrogenase (FDH) and S-formylglutathione hydrolase. Formaldehyde

18


can enter plants through stomata or be produced by endogenous metabolism (Haslam et al., 2002). Both

of these enzymes act to oxidize formaldehyde to formic acid, which may then be converted to CO2 or

enter C1 metabolism. Genes for both enzymes have been identified in Arabidopsis (Martínez et al., 1996;

Haslam et al., 2002; Achkor et al., 2003). However, it seems that one FDH also encodes an enzyme with

GSNO reductase activity (GSNOR; Sakamoto et al., 2002; Díaz et al., 2003).

Glutathione has long been implicated in reactions linked to metabolite synthesis (Dron et al., 1988;

Edwards et al., 1991). Camalexin (3-thiazol-2-yl-indole) is the major phytoalexin with antimicrobial

activity that accumulates in Arabidopsis after infection with a range of biotrophic and necrotrophic

pathogens (Tsuji et al., 1992; Thomma et al., 1999; Glawischnig, 2007) and following treatment with

abiotic factors such as UV-B paraquat, and heavy metal ions (Zhao et al., 1998; Mert-Türk et al., 2003;

Bouizgarne et al., 2006; Kishimoto et al., 2006). It is an indole phytoalexin that contains one S atom per

molecule and whose thiazole ring is derived partly from cysteine (Glazebrook and Ausubel, 1994). The

Arabidopsis pad2 mutant was first identified as deficient in camalexin, with accumulation in the pad2

mutant decreased by 90% compared with wild-type plants following infection with Pseudomonas

syringae pv. maculicola strain ES4326 (Glazebrook and Ausubel, 1994). Subsequent genetic studies

identified the affected gene in pad2 as GSH1 and the mutation results in GSH contents that during growth

in optimal conditions are decreased about 75% relative to wild-type (Parisy et al., 2007). Recent data

support the idea that GSH is the sulfur donor required for camalexin biosynthesis, in which GSTF6

catalyzes the conjugation of GSH with indole-3-acetonitrile (IAN), which is then hydrolyzed to Cys-IAN

in a process in which the GGT1 and GGT2 as well as the PCS1 have been proposed to be involved (Su et

al., 2011). However, Geu-Flores et al. (2011) have argued against a role for GGTs and have presented

evidence of the involvement of -glutamyl peptidases 1 and 3 (GGP1 and GGP3) in metabolizing

GSH(IAN) conjugation.

In addition to camalexin, the glucosinolates are well-studied sulfur-containing secondary metabolites

found in almost all plants of the order Brassicales, and the sulfur donor in their biosynthesis was recently

shown to be GSH (Geu-Flores et al., 2011). The first step in the biosynthesis of indole glucosinolates is

shared with that of tryptophan (Trp)-derived camalexin. Together with the enzyme myrosinase,

glucosinolates are hydrolyzed into various active substances in the presence of water, which serve as

defence against generalist-feeding insects (Hopkins et al., 2009). Indeed, the first indication of the

involvement of GSH in the biosynthesis of glucosinolates came from the analysis of pad2 mutant plants.

The pad2 mutant shows decreased resistance to feeding of the generalist insect Spodoptera littoralis, an

effect that is linked to decreased accumulation of glucosinolates (Schlaeppi et al., 2008). This effect is

specific to GSH, was not complemented by treatment of pad2 with the strong reducing agent

dithiothreitol (DTT). Analysis of the ascorbate-deficient mutant vtc1-1 showed that the effect was not

dependent on the ascorbate level, suggesting that it is linked to a requirement for GSH in biosyntheses

rather than mediated via generalized changes in antioxidant capacity (Schlaeppi et al., 2008). An

indication of the involvement of GSH as direct sulfur donor has come from the de novo engineering of

benzylglucosinolate into Nicotiana benthamiana (Geu-Flores et al., 2009). Finally, Geu-Flores et al.

(2011) have provided direct genetic evidence of the involvement of GSH as sulfur donor in the

biosynthesis of glucosinolates by showing that an Arabidopsis double ggp1 ggp3 mutant has altered

glucosinolate levels and accumulates up to 10 related GSH conjugates. Thus, the cytosolic enzymes

19


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

Arabidopsis.

1.2.5 Glutathione in plant development, growth and environmental responses It has long been recognized that GSH is oxidized by ROS as part of the antioxidant barrier that

contributes to preventing excessive oxidation of sensitive cellular components. As noted above, however,

glutathione is an essential metabolite with multiple functions in plants. Against the background of this

complexity, current work is generating new information on the roles of glutathione in influencing the

plant developmental processes and their interaction with the environment.

1.2.5.1 Glutathione in the regulation of plant development and growth

Accumulating evidence demonstrate that glutathione status is a key regulator in plant development and

growth. The most intriguing data has come from analysis of the phenotypes of glutathione-deficient

Arabidopsis mutants, which has shown that glutathione has critical functions in embryo and meristem

development (Vernoux et al., 2000; Cairns et al., 2006; Reichheld et al., 2007; Frottin et al., 2009;

Bashandy et al., 2010). Analysis of several triple mutants such as ntra nrtb rml1 and ntra ntrb cad2 have

established redundancy between glutathione and other thiol systems such as TRX in the regulation of

meristem development and growth related-auxin metabolism (Reichheld et al., 2007; Bashandy et al.,

2010). Additionally, glutathione synthesis is also required for pollen germination and pollen tube growth

in vitro (Zechmann et al., 2011). It has been shown that glutathione depletion by a specific inhibitor BSO

in pollen grains causes disturbances in auxin metabolism which lead to inhibition of pollen germination

(Zechmann et al., 2011). While the roles of glutathione in the plant cell cycle remain unclear, evidence

has been presented that the recruitment of GSH into the nucleus in the G1 phase of the plant cell cycle has

a profound effect on the redox state of the cytoplasm and the expression of redox-related genes (Pellny et

al., 2009; Diaz-Vivancos et al., 2010a,b). A subsequent increase in the total cellular GSH pool above the

level present at G1 is essential for the cells to progress from the G1 to the S phase of the cycle

(Diaz-Vivancos et al., 2010a,b).

1.2.5.2 Light signaling

Evidence for the role of glutathione in irradiance-dependent signaling came from the identification of the

Arabidopsis rax1 mutant. This line contains less than 50% wild-type glutathione contents, and shows

enhanced constitutive expression of the high light-induced gene, APX2 (Ball et al., 2004). Glutathione has

also been implicated in the signaling pathways that facilitate acclimation of chloroplast processes to high

light (Ball et al., 2004). Exposure to photoinhibitory light can lead to increases in leaf H2O2 levels and to

oxidation of the glutathione pool (Mateo et al., 2006; Muhlenbock et al., 2008). The abundance of

glutathione or its homologs in leaves does not vary greatly over the day/night cycle. However, young

poplar trees show more variation throughout development, with a gradient from young to old leaves

(Arisi et al., 1997). The effect of stresses such as drought can also influence the relative abundance of

glutathione/homoglutathione in different leaf ranks. Several studies in Arabidopsis and other species point

20


to a potential link between growth daylength, light signaling and glutathione status (Becker et al., 2006;

Queval et al., 2007; Bartoli et al., 2009). Relationships between glutathione and photoreceptor signaling

have been suggested in studies on the arsenic tolerant mutants, ars4 and ars5 (Sung et al., 2007). The

ars4 mutation was identified as an allele of phytochrome A (phyA) and caused increased BSO resistance

(Sung et al., 2007). The ars5 mutant, which is affected in a 26S proteasome component, had increased

levels of glutathione when exposed to arsenic, accompanied by increased GSH1 and GSH2 transcripts

(Sung et al., 2009).

1.2.5.3 Biotic interactions, cell death and defence phytohormones

As well as serving as a source of sulfur donor in the biosynthesis of secondary metabolites upon pathogen

and insect challenge, it is likely that glutathione is involved in signaling processes during biotic challenge.

Exogenous GSH can mimic certain pathogens in activating the expression of defence-related genes such

as PR1 (Dron et al., 1988; Wingate, et al., 1988; Senda and Ogawa, 2004; Gomez et al., 2004a). Moreover,

accumulation of glutathione is triggered by pathogen infection (Edwards et al., 1991; May et al., 1996).

Similar changes have also been reported following exogenous application of the defence-related hormone

SA, or biologically active SA analogs (Mou et al., 2003; Mateo et al., 2006; Koornneef et al., 2008).

Further evidence that glutathione is involved in defence against biotic stress came from the studies of

Arabidopsis mutants altered in GSH synthesis or metabolism. Resistance to fungal and bacteria pathogens

is compromised in the glutathione-deficient Arabidopsis mutants pad2, cad2 and rax1, while

overexpressing Lycopersicon esculentum -ECS in tobacco enhances resistance to Pseudomonas syringae

pv. tabaci (Table 1.3). These observations suggest that there is a critical glutathione status below which

the accumulation of pathogen-defence related molecules and consequently disease resistance, is impaired.

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

Analysis of catalase-deficient tobacco and Arabidopsis have indicated that H2O2 triggered changes in

glutathione status accompany the activation of pathogen responses (Willekens et al., 1997; Chamnongpol

et al., 1998; Chaouch et al. 2010). Glutathione oxidation and accumulation triggered by increased H2O2

availability in catalase-deficient cat2 is associated with HR-like lesions formation, induced PR genes,

camalexin production and increased bacterial resistance as part of a wide spectrum of defence responses

(Chaouch et al. 2010). Glutathione is also required for the production of nodules in the association

between legumes and nitrogen-fixing rhizobia. These organs contain high levels of both GSH and

homoglutathione, and deficiency in these molecules inhibits nodule formation (Frendo et al., 2005; Pauly

et al., 2006).

21


Table 1.3. Disease resistance phenotypes in glutathione synthesis mutants

(- Decreased resistance, + Increased resistance, = No difference relative to Col-0, OE Overexpression)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The phytohormone SA has been implicated as an important signal for the activation of both

effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered

immunity (PTI). SA accumulated in response to pathogen infection is synthesized from chorismate, with

the key step being catalyzed by isochorismate synthase (ICS1), the enzyme that is inactive in the salicylic

acid induction-deficient mutant, sid2 (Wildermuth et al., 2001). Signaling downstream from SA is largely

regulated via NPR1. Most of the attention paid to roles of glutathione in pathogenesis responses has

focused on NPR1, because this protein is known to be influenced by thiol-disulfide status and

SA-dependent PR gene expression can be induced by exogenous GSH (see section 1.1.3.3). The notion

that NPR1 function might be linked to glutathione redox state via GRX activity (Mou et al., 2003) has

been superseded by a model based on activation by TRX h (Tada et al., 2008), some of which are known

to be inducible by oxidative stress and pathogens (Laloi et al., 2004). However, glutathione could be

indirectly involved through GSNO-dependent nitrosylation of NPR1 monomers, stimulating

reoligomerization, which was postulated to be necessary to prevent protein depletion (Tada et al., 2008).

The identification of NPR1 as a target of S-nitrosylation is another example of the involvement of GSH in

plant disease resistance, in which protein S-nitrosylation is considered to be a key post-translational

modification. The Atgsnor1 mutant for the enzyme GSNO reductase has compromised resistance to both

virulent and avirulent pathogens (Feechan et al., 2005). However, redox regulation of NPR1 and the

transcription factors with which NPR1 interacts is turning out to be complex. Four cysteine residues on

TGA1 underwent S-nitrosylation and S-glutathionylation after GSNO treatment of the purified protein,

while GSNO treatment of Arabidopsis protoplasts caused translocation of an NPR1-GFP fusion protein

into the nucleus (Lindermayr et al., 2010). The importance of S-nitrosylation in regulating NPR1 function

is in line with an important role for this modification in plant disease resistance.

Although activation of NPR1 involves a reductive change, the initial signal for the events leading to PR

gene expression generally involves ROS accumulation. Oxidative bursts can also trigger the HR, in which

22


necrotic lesions develop at the site(s) of pathogen infection in plants. While redox changes involved in

HR have been most closely associated with events triggered by extracellular ROS production, oxidative

stress of intracellular origin can also trigger such effects. It has been proposed that the glutathione redox

state is a key determinant of cell death and dormancy (Kranner et al., 2006). According to this concept, an

increase in GSSG accumulation or increase in redox potential above a threshold reducing value

necessarily causes death. For instance, increases in the total glutathione contents of leaves produced by

ectopic -ECS overexpression in the tobacco chloroplast caused accumulation of GSSG and this was

associated with lesion formation and enhanced expression of PR genes (Creissen et al., 1999). In addition,

the HR-like lesions triggered by intracellular oxidative stress are associated with GSSG accumulation

(Smith et al., 1984; Willekens et al., 1997; Chamnongpol et al., 1998; Chaouch et al., 2010). However,

based on current understanding on work in catalase-deficient cat2 and related double mutants, changes in

glutathione redox potential or GSSG accumulation do not seem to be sufficient to cause cell death. Firstly,

HR-like lesions can be abolished by genetically blocking SA synthesis even though this effect is

associated with unchanged or greater GSSG accumulation (Chaouch et al., 2010). Secondly, the

hyper-accumulation of GSSG in cat2 gr1 does not cause HR-like lesions but seems to repress the SA

pathway relative to the single cat2 mutant (Mhamdi et al. 2010a). Thirdly, analyses of double cat2

atrbohF Arabidopsis mutants, which lack both the major catalase and one of the major leaf NADPH

oxidase activities, demonstrate that glutathione is much less perturbed than in the cat2 single mutant, yet

this is associated with unchanged or even increased lesion formation (Chaouch et al., 2012).

While glutathione status is implicated in SA signaling, glutathione may also modulate signaling through

the JA pathway, which is involved in the regulation of development and in responses to necrotrophic

pathogens and herbivores and is regulated antagonistically to the SA pathway. Accumulation of JA is

triggered by wounding or various types of pathogens, and occurs from linolenic acid in the chloroplast

and subsequent synthesis in the peroxisome (Browse, 2009). JA induces genes for glutathione synthesis

enzymes and GR (Xiang and Oliver, 1998). Subsequent work has shown that other antioxidative genes

can also be activated by JA (Sasaki-Sekimoto et al., 2005). In gr1 mutants lacking the

cytosolic/peroxisomal GR, several JA-associated genes are repressed (Mhamdi et al. 2010a). Among the

components that could link JA signaling and glutathione are GRXs and GSTs. Overexpression of the

SA-inducible protein GRX480 represses JA-triggered induction of the defensin, PDF1.2 (Ndamukong et

al., 2007). At the same time as relaying part of the SA-dependent signaling, NPR1 antagonizes JA

signaling (Spoel et al., 2003), and this antagonism was abolished by treatments with a glutathione

biosynthesis inhibitor, BSO, suggesting that SA-JA interactions may be mediated by transient changes in

the redox status (Koornneef et al., 2008). Based on these data, glutathione could act to repress JA

signaling through SA-dependent induction of NPR1 and GRX480, both of which interact with TGA

transcription factors. Other possible effects of glutathione could occur through the regulation of JA

synthesis or excess accumulation of intermediates like 12-oxophytodienoic acid (OPDA). Several GSTs

may catalyze formation of GS-oxylipin conjugates based on JA- or oxylipin-dependent gene expression

patterns (Yan et al., 2007; Mueller et al., 2008) and the peroxidatic or conjugation activities of GSTs

against electrophilic metabolites (Wagner et al., 2002; Davoine et al., 2006). Roles for GS-conjugates in

JA metabolism receive support from the observation that ggt4 mutants lacking an enzyme likely to be

important in GS-conjugate degradation (section 1.2.3) were found to accumulate a GS-conjugate of the

JA synthesis precursor, OPDA, during incompatible interactions with bacteria (Ohkama-Ohtsu et al.,

23


2011).

1.2.6 Mechanisms of glutathione dependent redox signaling

Thiol-disulfide exchange reactions are a fundamental mechanism of regulation enzyme activity in plants

(Buchanan and Balmer, 2005). While thiol-disulfide redox signaling through the chloroplast TRX system

was well established by the end of the 1970s, attention has been focused on glutathione-linked redox

signaling in plants only more recently (Foyer et al., 1997; May et al., 1998a). The following discussion

will outline factors that could link glutathione to modification of target protein activity.

1.2.6.1 Protein S-glutathionylation

Glutathione is involved in a post-translational modification called glutathionylation. Protein

S-glutathionylation involves the formation of a stable mixed disulphide bond between glutathione and a

protein cysteine residue (Figure 1.9). Described protein targets include enzymes involved in primary

metabolism such as TRXf, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycine

decarboxylase (GDC; Michelet et al., 2005; Zaffagnini et al., 2007; Palmieri et al., 2010). TRX of the f

type is glutathionylated on a cysteine residue found outside the active site, decreasing its activity against

target proteins such as plastidial GAPDH, while another TRX-independent isoform of GAPDH is

inhibited by glutathionylation (Michelet et al., 2005; Zaffagnini et al., 2007). In the mitochondria, GDC

has been shown to be sensitive to oxidative stress (Taylor et al., 2002), and is inhibited by thiol

modifications, including S-glutathionylation (Palmieri et al., 2010). However, the in vivo mechanisms

underlying S-glutathionylation remain to be established. The mechanisms of S-glutathionylation may

involve GRX-catalysed thiol-disulphide exchange between protein thiol groups and GSSG, conversion of

thiol groups to thiyl radicals or sulphenic acids, or be GSNO mediated (Dixon et al., 2005; Holtgrefe et al.,

2008; Gao et al., 2009; Palmieri et al., 2010). The reverse reaction, deglutathionylation, could be

mediated by certain class I and class II GRX. From a signaling perspective, GRXs activities could

regulate the status of protein thiols through reactions involving disulfides or mixed disulfides (Figure

1.9).

Figure 1.9. Some possible glutathione linked

signaling mechanisms (scheme taken from

Foyer and Noctor, 2011).

GR, Glutathione reductase; GRX, Glutaredoxin;

GS-R, Glutathione S-conjugate; GSNOR,

GSNO reductase; GST, glutathione

S-transferase; NO; Nitric oxide; R, small

organic molecule; SH, sulfhydryl (thiol) group;

SSG, glutathionylated protein Cys residue, TRX,

Thioredoxin.

24


1.2.6.2 Protein S-nitrosylation and GSNO reductase

As well as S-glutathionylation, S-nitrosylation of protein Cys residues may be mediated by GSNO in

plants (Lindermayr et al., 2005, 2010; Romero-Puertas et al., 2008; Lindermayr and Durner, 2009).

S-nitrosylation is a post-translational modification involving the reversible covalent binding of NO to the

cysteine residue of proteins to form S-nitrosocysteine (SNO). GSNO is thought to be the physiological

NO molecule that mediates direct protein S-nitrosylation. Inducible NO-producing enzymes in plants

remain to be clearly described, but GSNOR may be an important enzyme that can regulate SNO

homeostasis via the breakdown of GSNO into GSH and ammonia (Sakamoto et al., 2002; Barroso et al.,

2006; Díaz et al., 2003). This enzyme has also been implicated in the regulation of cell death, pathogen

resistance and other functions such as thermotolerance (Feechan et al., 2005; Lee et al., 2008; Chen et al.,

2009). It is not yet clear whether changes in glutathione status can affect GSNO availability or the

probability of either protein S-glutathionylation and S-nitrosylation. In Arabidopsis many potential protein

candidates carrying cysteine residues have already been reported such as NPR1, SA-binding protein 3,

GAPDH, GDC and type II metacaspase 9 (Tada et al., 2008; Wang et al., 2009a; Leitner et al., 2009;

Palmieri et al., 2010). S-nitrosylation of type II PRX has been proposed to play a role in ROS signaling

(Romero-Puertas et al., 2007). As described above, S-nitrosylation is thought to act in opposition to

disulphide reduction by TRX in the regulation of NPR1 (Tada et al., 2008). While the monomerisation

reaction of NPR1 was shown to be catalysed TRX, oligomerisation is facilitated by GSNO (Tada et al.,

2008). However, the accumulation of NPR1-GFP fusion protein in the nucleus was significantly increased

when Arabidopsis mesophyll protoplasts were treated with the physiological NO donor, GSNO

(Lindermayr et al., 2010). In the nucleus, NPR1 interacts with the transcription factor TGA1 and binds to

the promoter region to active PR gene expression (Després et al., 2003). In vitro studies using GSNO

have shown that S-nitrosylation of both NPR1 and TGA1 favoured DNA binding and TGA1 stability

(Lindermayr et al., 2010). Thus, S-nitrosylation may play a crucial role in regulating SA mediated plant

innate immunity.

1.2.6.3 Glutathione redox potential

A key factor governing interactions between glutathione and protein targets could be redox potential.

Genetic support for the physiological importance of glutathione-dependent disulphide reduction comes

from the observations that the NADPH-TRX and glutathione systems play overlapping roles in

development (Reichheld et al., 2007; Marty et al. 2009; Bashandy et al., 2010). It is possible that in some

conditions changes in glutathione redox potential could influence the mechanisms that contribute to

changes in the TRX redox potential and thus the biological activity of TRX targets. The actual glutathione

redox potential is related to [GSH]2:GSSG. Thus, unlike many other redox couples (e.g.

NADP+/NADPH), the glutathione redox potential depends on and can be influenced by absolute

concentration as well as by changes in GSSG relative to GSH (Mullineaux and Rausch, 2005; Meyer,

2008). Even if the GSH:GSSG ratio remains unchanged, decreases in glutathione concentration alone will

lead to an increase in redox potential (less reducing). It has been experimentally confirmed that the

cytosolic redox potential as detected by reduction-oxidation sensitive green fluorescent protein (roGFP)

was more positive in cad2 in which glutathione is decreased to about 30% of wild-type contents (Meyer

et al., 2007). Moreover, increases in the cytosolic but not plastidial redox potential in clt1 clt2 clt3

25


mutants, lacking the chloroplast envelope glutathione transporters, are also consistent with a depleted

glutathione pool in the cytosol but not the chloroplast (Maughan et al., 2010). In addition, physiological

and environmental factors might cause shifts in glutathione redox potential in vivo. For example, the

cytosolic glutathione redox potential has been shown to increase in response to wounding (Meyer et al.

2007). Whole tissue GSH:GSSG ratios can be markedly affected in catalase deficient mutants in

conditions favouring high rates of H2O2 production (Smith et al., 1984; Willekens et al., 1997; Rizhsky et

al., 2002; Queval et al., 2007, 2009), and this is a key observation underlying the studies reported in

Chapters 2 and 3.

1.3 Autophagy in plants

As sessile organisms, intracellular recycling is an important mechanism that is critical for the responses of

plants to adverse environmental conditions. A key intracellular degradation process during intracellular

remodeling, senescence and nutrient starvation is vacuolar autophagy, whereby cytoplasmic components

are degraded in the vacuole to provide raw materials and energy for growth, and also to eliminate

damaged or toxic components, for the maintenance of essential cellular functions. The term ‘autophagy’

comes from the Greek words ‘phagy’ meaning eat, and ‘auto’ meaning self. The basic autophagy process

is conserved among eukaryotes from yeast to animals and plants (Bassham, 2007; Yang and Klionsky,

2009; Mehrpour et al., 2010). While several of types of autophagy have been described in many species,

two types of autophagy, called microautophagy and macroautophagy, are found in plants (Figure 1.10;

Bassham et al., 2006). Microautophagy involves the formation of a small intravacuolar vesicle called an

autophagic body by invagination of the tonoplast, thus engulfing cytoplasmic components. On the other

hand, macroautophagy is the most extensively studied in plants and is mediated by a special organelle

termed the autophagosome (Yoshimoto, 2012). The core marker for autophagy is the double

membrane-bound autophagosome in plants into which, during macroautophagy, bulk cytosolic

constituents and organelles are sequestered. The outer membrane of the autophagosome then fuses with

the vacuolar membrane and thus delivers the inner membrane structure and its cargo, namely the

autophagic body, inside the vacuole for degradation by vacuolar hydrolases (Figure 1.10), and the

products are exported from the vacuole to the cytoplasm for reuse. The discussion here will focus on the

plant macroautophagy pathway, which hereafter is referred to as autophagy.

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

26


In plants, autophagic structures have been observed, largely based on morphological observation by

electron microscopy, since the late 1960s (Villiers, 1967; Matile and Moor, 1968; Matile and Winkenbach,

1971; Matile, 1975; Marty, 1978; Van der Wilden et al., 1980). However, the underlying molecular

mechanisms of autophagy have only recently been begun to be identified. Progress in understanding the

mechanistic basis of autophagy has been greatly facilitated by studies of yeast autophagy. The initial

studies of most genes functioning in autophagy pathways were done via mutagenesis studies in the yeast,

Saccharomyces cerevisiae. Hence, the identification of the ATG genes in yeast led to molecular analysis

of autophagy in higher eukaryotes. Since the first autophagy (ATG) genes were identified in yeast in 1993

(Tsukada and Ohsumi, 1993), the number of identified yeast autophagy-related genes has increased to 32

(Barth et al., 2001; Klionsky et al., 2003; Kanki et al., 2009; Nakatogawa et al., 2009; Okamoto et al.,

2009). These genes can be divided into several functional groups: the ATG1 protein kinase complex; the

phosphatidylinositol 3-kinase (PI3K) complex specific for autophagy; the ATG9 complex; and two

ubiquitin-like conjugation systems, leading to AG8 lipidation and ATG12 protein conjugation (Table 1.4).

The knowledge gained from yeast research has greatly facilitated the identification of homologous genes

that are required for autophagy in plants. Indeed, the genes essential for the central autophagic machinery

consist of 18 ATG genes in yeast. Although 4 homologues of the 18 yeast ATG genes have not yet been

identified in plants, approximately 30 Arabidopsis ATG genes have been identified on the basis of their

sequences (Table 1.4; Doelling et al., 2002, Hanaoka et al., 2002, Xiong et al., 2005). It has thus been

suggested that Arabidopsis as well as other crop species such as rice and maize may share the core

autophagic machinery with yeast (Su et al., 2006; Ghiglione et al., 2008; Chumg et al., 2009; Shin et al.,

2009; Xia et al., 2011; Kuzuoglu-Ozturk et al., 2012).

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

a T-DNA insertional knock-out mutants of these genes have been published.

b Unpublished data.

TORC1, TOR kinase complex 1; PI(3)P, phosphatidylinositol 3-phosphate; PE, phosphatidylethanolamine.

27


1.3.1 Functions of autophagy in plants Although morphological studies provided initial insights into the structural components and

characteristics of the process in plants several decades ago, these results provided only static images of

autophagy, and the physiological roles of plant autophagy remain largely unknown. The recent

identification of Arabidopsis homologs of most of the core ATG genes has allowed studies of many

autophagy-defective plants with impaired ATG genes (Bassham et al., 2006; Hayward et al., 2009). As

with other systems such as yeast and animals, autophagy in plants has essential roles not only during

starvation, but also during development, senescence, and in response to different abiotic stresses as well

as pathogens (Bassham, 2007; Bassham, 2009; Hayward and Dinesh-Kumar, 2011). Even under favorable

environments, basal autophagy has an important housekeeping function by clearing damaged or unwanted

cytoplasmic components for recycling and to provide a source of amino acids, lipids and sugars for basic

cellular processes.

1.3.1.1 Functions of autophagy during abiotic stress

Autophagy is induced under nutrient starvation such as carbon or nitrogen deprivation, which is one of

most studied abiotic stresses (Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005; Xiong et

al., 2005; Phillips et al., 2008). During nutrient starvation, the breakdown of damaged or unwanted cell

materials such as bulk protein, carbohydrates and lipids through autophagy degradative pathways

becomes crucial to replenish the supply of carbon and nitrogen needed for survival and new development.

The early morphological studies demonstrated that autophagic structures are often observed under

nutrient-limiting conditions in plant cells, suggesting a nutrient recycling function for plant autophagy.

Indeed, when Atatg mutant plants are grown under nitrogen- and carbon-depleted or limiting conditions,

autophagy defective plants display accelerated starvation-induced chlorosis and stunted growth, most

likely because autophagy is required for nutrient remobilization to allow temporal adaptation of plant

cells to nutrient starved conditions (Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005;

Xiong et al., 2005; Phillips et al., 2008). It has been recently confirmed that autophagy is required for

nitrogen remobilization (Guiboileau et al., 2012). It is well established that the target of rapamycin (TOR)

complex integrates nutrient signaling and associates with the ATG1 complex to inhibit the activity of

autophagy in yeast and mammals (He and Klionsky, 2009). As well as the interaction between TOR and

ATG1 as the primary molecular switch for starvation-induced autophagy in yeast and mammals, it has

also been shown that in plants TOR may function as a negative regulator of autophagy (Liu and Bassham,

2010). In addition, autophagy has been shown to be a rather general response to a variety of abiotic

stresses (Bassham, 2007). When treatment of plants with H2O2 or methyl viologen (MV) results in severe

oxidative stress, autophagy is quickly induced. The autophagy-defective AtATG18a knockdown

transgenic plants are hypersensitive to MV treatment and accumulate higher levels of oxidized proteins,

implying that the defence mechanism of autophagy is to degrade oxidized components (Xiong et al.,

2007). Similar effects have also been observed in the rice Osatg10 mutant (Shin et al., 2009). Moreover,

autophagy is essential for plant tolerance to drought and salt stresses (Liu et al., 2009; Kuzuoglu-Ozturk

et al., 2012). These results suggest a protective role for autophagy in transporting oxidized proteins or

organelles into the vacuole for degradation in plant cells. However, it remains unknown whether this

transport is selective. Additionally, AtTSPO, an ABA-induced protein, has been shown to be degraded by

28


the autophagy pathway, implying the involvement of autophagy in responses to ABA in plants (Vanhee et

al., 2011).

1.3.1.2 Functions of autophagy during pathogen infection

Conflicting results have been reported on the relationship between autophagy and cell death in plants. It

has been reported that autophagy can either restrict or promote cell death upon pathogen infection

(Hayward and Dinesh-Kumar, 2011; Hofius et al., 2009; Yoshimoto et al., 2009; Lenz et al., 2011a,b; Lai

et al., 2011; Wang et al., 2011). The roles of autophagy may vary depending on the type of pathogen and

the age of the plant. Plant pathogens are often divided into two types based on their lifestyles:

necrotrophic and biotrophic. Necrotrophic pathogens derive nutrients from dead or dying cells, whereas

biotrophic pathogens feed on living host tissue for survival (Glazebrook, 2005). In Arabidopsis, infection

with the necrotrophic fungal pathogen Botrytis cinerea induces the expression of autophagy genes and

formation of autophagosomes (Lai et al., 2011). Autophagy defective mutants exhibit susceptibility to the

necrotrophic fungal pathogen Botrytis cinerea and Alternaria brassicicola (Lai et al., 2011; Lenz et al.,

2011b). These results suggest that autophagy functions in a pro-survival role during necrotrophic

pathogen infection. However, the role of autophagy upon biotrophic pathogen infection is open to debate.

When plants are infected with tobacco mosaic virus (TMV) pathogen, the execution of HR cell death is

not compromised, but spreads to throughout the infected leaves and even into the healthy uninfected

tissue of the BECLIN1/ATG6-silenced tobacco plants (Liu et al., 2005). Similar results have been also

confirmed in Arabidopsis ATG6 RNAi lines and atg5 mutants using the avirulent pathogen Pseudomonas

syringae pv. tomato (Pst) DC3000 (avrRPM1; Patel and Dinesh-Kumar, 2008; Yoshimoto et al., 2009).

These results suggest that autophagy functions to restrict the spread of HR cell death, thus serving a

pro-survival role. By contrast, it was found that cell death is suppressed and resistance is increased in atg7

and atg9 knockout mutants when challenged with the avirulent pathogen Pst DC3000(avrRps4; Hofius et

al., 2009). Furthermore, several atg knockout mutants (atg2, atg5, atg7 and atg10) which are defective in

autophagy exhibited enhanced resistance to the powdery mildew Golovinomyces cichoracearum (Wang et

al., 2011). Other biotrophic pathogens including the virulent bacterial strain PstDC3000 and avirulent

Hyaloperonospora arabidopsidis have recently been used in resistance tests in ATG-defective mutant

plants (Hofius et al., 2009; Lenz et al., 2011b). The ATG-defective plants all showed enhanced resistance

to both of these biotrophic pathogens (Hofius et al., 2009; Lenz et al., 2011b). These different results

indicate that autophagy may have both a pro-death role and pro-survival functions upon challenge with

biotrophic pathogens. It has been proposed that the contrasting results observed during biotrophic

pathogen challenge could be due to the differences of plant age and growth conditions (Hayward and

Dinesh-Kumar, 2011; Liu and Bassham, 2012), and the exact roles of autophagy in plant innate immune

response remain to be elucidated.

1.3.1.3 Functions of autophagy during growth and seed development

Although a lot of autophagy studies have been focused on stress related conditions, the functions of

autophagy under normal growth conditions have also been investigated (Slavikova et al., 2005; Inoue et

al., 2006; Yano et al., 2007; Yoshimoto et al., 2009). Here, it is important to note that plants may maintain

a basal level of housekeeping autophagy to eliminate damaged materials, which are continually generated

29


even under favorable growth conditions (Bassham et al., 2006). As well as slower growth, almost all of

the autophagy-defective mutants have an early senescence phenotype under nutrient-rich conditions,

suggesting that autophagy has a pro-survival function in development (Doelling et al., 2002; Hanaoka et

al., 2002; Patel and ; Thompson et al., 2005; Xiong et al., 2005; Qin et al., 2007; Phillips et al., 2008;

Dinesh-Kumar, 2008; Yoshimoto et al., 2009). It is interesting that premature senescence is genetically

blocked by inactivation of the SA synthesis or signaling pathways in atg5 sid2 and atg5 npr1 double

mutants, and that this is accompanied by decreased H2O2 (Yoshimoto et al., 2009). This raises an

interesting question regarding the relationship between autophagy and H2O2 during SA related leaf

senescence. As discussed above, autophagy could be involved in degrading oxidized proteins under

oxidative stress conditions in Arabidopsis (Xiong et al., 2007). It is also possible that autophagy may

function selectively to remove specific H2O2-generating organelles such as chloroplasts and peroxisomes.

However, the underlying mechanisms remain to be fully investigated, and the relationship between

autophagy and H2O2 generated intracellularly is a focus of Chapter 4 of this study.

During senescence, plants could recycle nutrients from senescing leaves to newly forming organs such as

developing seeds. Because autophagy is involved in recycling cytoplasmic materials, it is understandable

that it functions during seed development, which is a process of extensive nutrient remobilization.

Although no obvious defect in seed formation was observed in Arabidopsis atg mutants in under normal

growth conditions, several ATG genes are up regulated during seed maturation and desiccation

(Angelovici et al., 2009). More recently it has been shown that autophagy is required for seed filling

(Guiboileau et al., 2012). Seed storage proteins are synthesized in the endoplasmic reticulum (ER) and

form protein bodies (PB) during seed development, and accumulate in protein storage vacuoles (PSVs)

which are degraded to support the growth of newly forming organs in plants (Ibl and Stoger, 2012).

Indeed, it has been reported that intact PB is transported to the vacuoles by autophagy pathway in wheat

(Levanomy et al., 1992). Furthermore, after being assembled in the ER, zeins, the prolamin storage

protein in maize, were recently found to be delivered from ER to aleurone PSVs in atypical prevacuolar

compartments by an atypical autophagy pathway (Reyes et al., 2011).

1.3.2 Difficulties of monitoring autophagy in plants As mentioned above, autophagic processes were identified by electron microscopy in early plant studies.

More recently, a more reliable marker used to monitor plant autophagy has been developed, ie,

monitoring of the ATG8 protein. ATG8 is integrated into the phagophore during the early stages of

autophagosome formation. ATG8 is covalently attached to the lipid phosphatidylethanolamine (PE) to

produce ATG8-PE that is bound to autophagic membranes via its lipid moiety. Hence, plants engineered

to express ATG8-GFP reporter constructs have been particularly informative. Autophagic vesicles can be

easily visualized in plant cells using ATG8-GFP by fluorescence confocal microscopy (Yoshimoto et al.,

2004; Thompson et al., 2005; Chung et al., 2010). Some of these studies have revealed that structures

observed in plants sometimes seem to be protein aggregates other than autophagosomes. Such

aggregation often occurs when proteins are overexpressed in plant cells and it has therefore been

considered that autophagic flux should be confirmed using biochemical approaches (Chung et al., 2010).

ATG8-GFP fusions are transported into the vacuole as a consequence of autophagy, and accumulation

of free GFP released from the GFP-ATG8-PE conjugate during breakdown of autophagic bodies in the

30


vacuole can be monitored biochemically by immunoblotting with an anti-GFP antibody (Chung et al.,

2010; Suttangkakul et al., 2011). This accumulation is almost completely blocked in autophagy-deficient

mutants. Furthermore, concanamycin A or protease inhibitors such as E64D have also been used to

monitor the autophagic process in combination with the tagging of ATG8 in plant cells. Because

autophagic bodies are short lived in the vacuole of plant cells, their stability and thus detection is greatly

improved by prior incubation of plants with inhibitors of vacuolar degradation (Yoshimoto et al., 2004;

Thompson et al., 2005; Ishida et al., 2008). In addition, LysoTracker red or monodansylcadaverine has

also been used to monitor acidic autophagosomes in plants as well as in animals (Comtento et al., 2005;

Liu et al., 2005; Patel et al., 2008). However, these dyes should be used cautiously because of their

potential to cross identify other acidic bodies in the tissue (Yoshimoto, 2012).

1.4 Objectives of the work

In our laboratory, a conditional photorespiratory mutant cat2 has been used as a stress mimic tool to

assess the importance of components that could be important in oxidative stress signaling. Oxidative

stress occurring in cat2 is derived from enhanced H2O2 availability through photorespiration and can be

prevented by growth at high CO2 or low light (Queval et al., 2007). During growth of cat2 in air in long

days (but not short days), intracellular oxidative stress triggers SA-dependent responses including SA

accumulation, PR gene expression, HR-like cell death and increased resistance to pathogens, effects that

are all accompanied by the oxidation and accumulation of glutathione (Chaouch et al., 2010). However, it

remains unclear whether glutathione oxidation and accumulation is required for intracellular oxidative

stress-mediated SA-dependent pathogenesis responses and, more generally, there is little direct in vivo

evidence that glutathione plays a signaling role downstream of H2O2. To analyze these questions, novel

and double triple mutants were constructed, and the results obtained are presented in Chapter 2.

A second aim of this work arose from the observation that JA-associated gene expression was repressed

in gr1, an effect that was reinforced in cat2 gr1, which has a strongly perturbed leaf glutathione pool.

These effects point to possible links between the JA pathway and glutathione status (Mhamdi et al., 2010a;

annex 1). However, the repression of JA signaling gene expression is complicated by the extreme

phenotype of cat2 gr1, which has severe oxidative stress. Hence, it is unknown whether the previously

described repression of JA signaling is linked to glutathione status rather than GR activity, especially in

an oxidative stress context. In Chapter 3, several of the mutants reported in Chapter 2 are exploited to

analyze this specific question and the role of glutathione in interplay between SA and JA pathways.

Leaf senescence has been considered to be linked to enhanced oxidative stress. Indeed, senescence can be

induced by a range of stresses such as salt, drought and pathogens (Dwidedi et al., 1979; Dhindsa et al.,

1981; Bohnert et al., 1995; Balazadeh et al., 2010). One signaling molecule that could be important in the

regulation of senescence is H2O2. The roles of antioxidant enzymes such as CAT and APX, which

metabolize H2O2, are therefore of particular interest, and it has been reported that the onset of leaf

senescence is associated with lower expression and activity of CAT2 (Smykowski et al., 2010).

Furthermore, it has been proposed that autophagy may function in degrading oxidized proteins to confer

tolerance to oxidative stress (Xiong et al., 2007). Indeed, several autophagy mutants such as atg2 and

31


32

atg5 exhibit an early senescence phenotype which is associated with enhanced oxidative stress as well as

the activation of SA pathway (Yoshimoto et al., 2009). Chapter 4 reports an analysis of possible interplay

between photorespiration, oxidative stress and autophagy in the regulation of senescence and

SA-dependent lesion formation by phenotypic and other analyses of double mutants including cat2 atg2,

cat2 atg5 and cat2 atg18a.

CHAPTER 2

Functional analysis of Arabidopsis mutants points to novel roles for

glutathione in coupling H2O2 to activation of salicylic acid

accumulation and signaling

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

original paper:

Han Y., Chaouch S., Mhamdi A., Queval G., Zechmann B. and Noctor G. (2012). Functional analysis

of Arabidopsis mutants points to novel roles for glutathione in coupling H2O2 to activation of salicylic

acid accumulation and signaling. Antioxidants & Redox Signaling

CHAPTER 2

Summary Through its interaction with H2O2, glutathione is a candidate for transmission of signals in plant responses

to pathogens but identification of signaling roles is complicated by its antioxidant function. Using a

genetic approach based on a conditional catalase-deficient Arabidopsis mutant, cat2, this study aimed to

establish if glutathione plays an important functional role in the transmission of signals downstream of

H2O2. For this, glutathione content or antioxidative capacity was independently modified in an H2O2

signaling background. Introducing the cad2 or allelic mutations in the glutathione synthesis pathway into

cat2 blocked H2O2-triggered glutathione oxidation and accumulation. While no effects on NADP(H) or

ascorbate were observed, and H2O2-induced decreases in growth were maintained, blocking glutathione

modulation antagonized salicylic acid (SA) accumulation and SA-dependent responses. Other novel

double and triple mutants were produced and compared with cat2 cad2 at the levels of phenotype,

expression of marker genes, non-targeted metabolite profiling, accumulation of SA, and bacterial

resistance. The effects of the cad2 mutation on H2O2-triggered responses were distinct from those

produced by mutations for GLUTATHIONE REDUCTASE1 (GR1) or NONEXPRESSOR OF

PATHOGENESIS-RELATED GENES 1 (NPR1), and were linked to compromised induction of

ISOCHORISMATE SYNTHASE1 (ICS1) and ICS1-dependent SA accumulation. This study of new

double and triple mutants allowed us to infer previously undescribed regulatory roles for glutathione, in

which this compound acts in parallel to its antioxidant role to allow increased intracellular H2O2 to

activate SA signaling, a key defence response in plants.

Résumé

Grâce à ses interactions avec le H2O2, le glutathion se présente comme un candidat pour jouer des rôles

dans la transmission de signaux lors des interactions plantes-pathogènes. Cependant, l’identification de

tels rôles est compliquée par la fonction antioxydante de glutathion. En utilisant une approche génétique

basée sur cat2, un mutant d’Arabidopsis conditionnel déficient en catalase, cette étude visait à établir si le

glutathion joue des rôles de signalisation en aval du H2O2. Pour ce faire, la teneur en glutathion ou sa

capacité antioxydante ont été modifiées, d’une manière indépendante, dans le fond génétique cat2.

L’introduction de la mutation cad2 ou d’autres mutations alléliques pour la voie de synthèse de glutathion

produit un blocage sur l’oxydation et l’accumulation de glutathion H2O2-dépendante qui sont autrement

observées chez le mutant cat2. Alors que les pools de NADP(H) et d’ascorbate restent inaffectés, et la

diminution de la croissance causée par le H2O2 sont maintenues au niveau cat2 chez cat2 cad2, le blocage

de la réponse du glutathion produit un effet antagoniste sur l’accumulation de l’acide salicylique (SA) et

des effets SA-dépendants. D’autres nouveau doubles et triples mutants ont été générés et comparés avec

cat2 cad2 au niveau de leurs phénotypes, de l’expression de gènes marqueurs, de profils métaboliques

non-ciblés, de l’accumulation du SA, et de la résistance aux bactéries. Ces analyses ont révélé que les

effets de la mutation cad2 chez cat2 sont distincts de ceux produits par des mutations pour la

GLUTATHION REDUCTASE1 (GR1) ou la NONEXPRESSOR OF PATHOGENESIS-RELATED

GENES 1 (NPR1), et sont liés à une induction attenuée de l’ISOCHORISMATE SYNTHASE 1 (ICS1) et

l’accumulation de SA ICS1-dépendante. L’étude de ces doubles et triples mutants nous permet de

conclure à des rôles nouveaux pour le glutathion, qui semble agir en parallèle à son rôle antioxydant pour

coupler le H2O2 intracellulaire à la signalisation par le SA, une réponse de defénse clé chez les plantes.

33

CHAPTER 2

2.1 Introduction

As potent signal molecules, reactive oxygen species (ROS) play important roles in transmitting

information during responses of both plant and animal cells to the environment. Since the seminal studies

that established key roles for ROS in plant-pathogen interactions (Doke, 1983; Apostol et al., 1989;

Levine et al., 1994), primary redox-linked events have been considered to be extracellular or

plasmalemma-located, mediated primarily by NADPH oxidases, peroxidases, or other enzymes (Torres et

al., 2002, 2005; Bindschedler et al., 2006; Daudi et al., 2012; O’Brien et al., 2012). However, these initial

signals at the cell surface/apoplast lead to later downstream adjustments in intracellular redox state

(Edwards et al., 1991; Vanacker et al., 2000; Volt et al., 2009) that are notably associated with

thiol-dependent activation of the cytosolic protein, NON EXPRESSOR OF PATHOGENESIS RELATED

GENES1 (NPR1). Reduction of NPR1 is required to link part of the salicylic acid (SA) signaling pathway

to induction of genes such as PATHOGENESIS-RELATED1 (PR1; Mou et al., 2003).

One of the key players governing intracellular redox state is the thiol/disulfide compound, glutathione.

Several reports have used single mutants or transformants deficient in glutathione in studies of

pathogenesis responses (May et al., 1996; Ball et al., 2004; Parisy et al., 2007; Höller et al., 2010;

Dubreuil-Maurizi et al., 2011; Baldacci-Cresp et al., 2012). While some of these have described effects of

altered glutathione status, it remains unknown where glutathione acts in the signaling chain and how it

interacts with ROS-dependent events. Interpretation of the role of glutathione is complicated by its

antioxidant function which depends on regeneration of the thiol form, GSH, from the disulfide form,

GSSG, by glutathione reductase (GR). The predominant concept of glutathione function is as a negative

regulator that acts to oppose or limit H2O2 signals. In terms of thiol-dependent signaling functions in

pathogenesis responses, the NPR1 pathway remains by far the best studied (Koornneef et al., 2008;

Ghanta et al., 2011; Maughan et al., 2010). However, although exogenous glutathione can induce PR1

expression, NPR1 reduction has been reported to be mediated through thioredoxin-dependent systems

(Tada et al., 2008). Adding to these uncertainties are recent studies that have revealed the complexity both

of NPR1 redox regulation (Lindermayr et al., 2010) and of potential functional overlap between cytosolic

thiol/-disulfide systems (Reichheld et al., 2007; Marty et al., 2009; Bashandy et al., 2010).

Enhanced oxidation of glutathione, accompanied by increases in the total pool, is a well-documented

response in plants subjected to treatments such as ozone, cold, pathogens, or SA (Edwards et al., 1991;

Sen Gupta et al., 1991; May et al., 1996; Vanacker et al., 2000; Bick et al., 2001; Gomez et al., 2004b;

Koornneef et al., 2008). Photorespiratory catalase-deficient plants are model systems in which such

changes are particularly evident (Smith et al., 1984; Willekens et al., 1997; Rizhsky et al., 2002; Queval et

al., 2007). The interest of these systems is that they allow the role of H2O2 signaling to be studied

specifically and controllably, because the endogenous signal can be manipulated easily by external

conditions (Dat et al., 2001; Mhamdi et al., 2010b). When conditions allow active photorespiration,

increased H2O2 availability in the Arabidopsis knockout cat2 mutant triggers accumulation of glutathione

followed by induction of pathogenesis responses in the absence of pathogen challenge (Queval et al.,

2007; Chaouch et al., 2010). The well-defined changes in glutathione in catalase-deficient plants are

useful as a readout of altered redox state, but their functional impact is unclear. It remains to be

established whether they play any role in coupling H2O2 to downstream defence responses or are rather an

34

CHAPTER 2

accompanying, passive response of the cell to enhanced H2O2.

Increases in H2O2 in catalase-deficient plants are much less apparent and reproducible than those

observed in glutathione (Mhamdi et al., 2010b), raising the possibility that some of the signals

downstream of intracellular H2O2 may be mediated by changes in glutathione status. Altered glutathione

status in catalase-deficient plants or in response to stress presumably reflects an increased load on

reductant-requiring antioxidant pathways. This is underscored by analysis of Arabidopsis gr1 knockout

mutants for one of the two GR-encoding genes. While GR1 deficiency does not in itself lead to an

oxidative stress phenotype, the gr1 mutation greatly enhances stress in the cat2 background, showing that

glutathione-dependent antioxidative pathways are increasingly solicited when catalase activity is

compromised (Mhamdi et al., 2010a).

By exploiting cat2 as a model H2O2 signaling background, evidence was recently reported that

intracellular oxidative stress interacts with specific NADPH oxidases to determine activation of salicylic

acid (SA) accumulation and SA-dependent pathways. The atrbohF mutant lacking expression of a

specific NADPH oxidase shows compromised SA accumulation and resistance to virulent bacteria, and

the atrbohF mutation also attenuates cat2-triggered SA accumulation and induced resistance (Chaouch et

al., 2012). Intriguingly, the clearest indicator of redox interactions between the cat2 and atrbohF

mutations was not H2O2 itself but the status of glutathione: attenuation of SA contents and SA-dependent

responses was correlated with decreased accumulation of glutathione in cat2 atrbohF compared to cat2

(Chaouch et al., 2012).

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

glutathione status in transmitting H2O2 responses. This mutant was chosen because it is a well-defined

conditional redox signaling system in which H2O2 provokes oxidative modulation of the glutathione pool

accompanied by activation of SA signaling and associated pathogenesis-related responses. Relationships

between enhanced H2O2 production, the response of glutathione, and the activation of the SA pathway

were explored to answer the following specific questions. (1) Does glutathione play a specific role in

H2O2 signaling, independent of other potentially redundant thiol systems? (2) To what extent is any such

role dependent on glutathione status, rather than glutathione redox turnover in an antioxidant function? (3)

What is the relationship between glutathione status and NPR1 function in transmitting signals

downstream of H2O2? To examine these questions, mutant lines available in Arabidopsis that have

decreased glutathione or salicylic acid, or loss of GR1 or NPR1 function, were used (Table 2.1). By

functional analysis of the effects of these specific mutations in the cat2 H2O2 signaling background,

previously unidentified roles for glutathione in controlling oxidative stress-triggered SA signaling are

inferred.

35

CHAPTER 2

Table 2.1. Details of mutant lines used in this study. The principal characteristics of the lines are given, as well as the reference(s)

in which the mutant and/or mutation was first reported or characterized.

_____________________________________________________________________________________

cat2 T-DNA knockout for CATALASE2.

Conditional photorespiratory oxidative stress mutant (Queval et al., 2007). Constitutive activation of salicylic acid

pathway under oxidative stress conditions (Chaouch et al., 2010). Wild-type phenotype when grown under high CO2.

gr1 T-DNA knockout for GLUTATHIONE REDUCTASE1.

Wild-type phenotype in optimal conditions due to back-up NADPH-thioredoxin system for GSSG reduction (Marty et al.

2009), but gr1 shows altered responses to oxidative stress (Mhamdi et al., 2010a).

cad2 Deletion mutation in GSH1 gene leading to loss of two amino acids in -GLUTAMYLCYSTEINE SYNTHETASE.

Identified in a screen for enhanced cadmium sensitivity, cad2 has about 30% wild-type leaf glutathione contents

(Howden et al., 1995; Cobbett et al., 1998). Wild-type phenotype in optimal growth conditions.

rax1 Point mutation in GSH1 gene leading to an amino acid substitution in -GLUTAMYLCYSTEINE SYNTHETASE.

Identified in a screen for altered high light responses, rax1 has about 30-40% wild-type leaf glutathione contents (Ball et

al., 2004). Wild-type phenotype in optimal growth conditions.

pad2 Point mutation in GSH1 gene leading to an amino acid substitution in -GLUTAMYLCYSTEINE SYNTHETASE.

Identified in a screen for decreased phytoalexin (camalexin) contents, pad2 has about 20-25% wild-type leaf glutathione

contents (Parisy et al. ,2007). Wild-type phenotype in optimal growth conditions.

sid2 Point mutation in ISOCHORISMATE SYNTHASE 1.

Identified in a screen for decreased induction of salicylic acid (Nawrath and Métraux 1999). Allowed ICS1 to be

identified as major source of pathogen-induced salicylic acid production in Arabidopsis (Wildermuth et al., 2001). In

response to bacterial challenge, salicylic acid stays close to basal uninduced Col-0 levels or below. Wild-type phenotype

in optimal growth conditions.

npr1 Point mutation in NONEXPRESSOR OF PATHOGENESIS RELATED GENES 1.

Identified in a screen for mutants that fail to induce pathogenesis-related PR genes (Cao et al., 1994). The NPR1 protein

was subsequently shown to be thiol-regulated, with reduction allowing the protein to move into the nucleus from the

cytosol to induce PR genes (Mou et al., 2003). Wild-type phenotype in optimal growth conditions.

____________________________________________________________________________________

36

CHAPTER 2

2.2 Results

Plants deficient in the major leaf catalase show accumulation of glutathione that is conditional on H2O2

production through photorespiration (Queval et al., 2007). When cat2 is grown in air, glutathione

accumulation accompanies the appearance of lesions which initially develop after about two weeks

growth then spread to cover about 15% total rosette area within the following week. This is associated

with induction of SA-dependent PR genes and enhanced resistance to bacteria. All these H2O2-triggered

effects in cat2, except glutathione accumulation, can be reverted by the sid2 mutation (Chaouch et al.,

2010), which blocks SA synthesis through the ICS1-dependent pathway (Wildermuth et al., 2001). During

growth at high CO2, where photorespiratory glycolate oxidase activity is negligible, cat2 shows no signs

of oxidative stress and is phenotypically indistinguishable from the wild-type, Col-0. However, when

plants grown first at high CO2 are transferred to air, glutathione in cat2 accumulates to similar levels to

those observed in cat2 grown in air from seed. This effect is followed by initiation of pathogenesis-related

responses about five days after transfer to air and their continued development during subsequent days

(Chaouch et al., 2012). These features allow cat2 to be used as a conditional model in which the roles of

glutathione in defence signaling triggered by intracellular oxidative stress can be examined either during

growth in air from seed or by the induced stress that follows the transfer of plants from high CO2 to air.

As reported in this study, both conditions allow activation of the SA pathway in the cat2 mutant. However,

studying responses after transfer from high CO2 to air is particularly useful for double mutants in which

the interpretation is complicated by their extreme phenotypes when they are grown from seed in air. Thus,

for some experiments, transfer from high CO2 condition was preferred as a protocol for inducing

oxidative stress. However, the principal effects of glutathione on the SA pathway here were observed

using both experimental protocols.

2.2.1 Genetic blocks over H2O2-triggered glutathione accumulation inhibit cell death and associated

pathogenesis responses induced by intracellular oxidative stress

When cat2 plants were grown in non-stress conditions (at high CO2), leaf glutathione remained at

wild-type levels and reduction state (about 95% reduced). Within 4h after transfer to air from high CO2,

the reduction state in cat2 fell from 95% to 61% reduced (Figure 2.1A). Glutathione oxidation continued

in cat2 on subsequent days, and the pool was more than 50% oxidized 4d after transfer. This oxidation

was accompanied by a more than 2-fold increase in the total pool. The cat2-triggered oxidation and

accumulation of glutathione were blocked by the cad2 mutation: in cat2 cad2, glutathione remained about

90% reduced, and was only slightly increased above cad2 levels after 4d exposure to air (Figure 2.1A). In

contrast to the effects of the cat2 and cad2 mutations on glutathione, two major intracellular redox pools

that glutathione donates electrons to (ascorbate) or receives electrons from (NADPH) were much less

affected. Ascorbate reduction status was above 80% in all lines while NADP(H) pools were constant at

between 50 and 60% reduced. Thus, when plants are grown in conditions permissive for photorespiratory

H2O2 production, the cat2 mutation triggers a response of the glutathione pool that is quite specific and

introduction of the cad2 mutation produces a specific block on this response.

Studies of plants deficient in catalase grown in different conditions suggest that the glutathione pool is in

close correspondence to the predicted H2O2 availability (Queval et al., 2007; Mhamdi et al., 2010b). It has

37

CHAPTER 2

A

B

0

250

500

750

1000

0

250

500

750

1000

cat2

ca

d2

Co

l-0

cat2

cat2

gr1

4h 4d

bc

b

Per

oxi

de

cont

ent

(nm

ol.g

-1F

W)

aab

a a a a

cat2

ca

d2

Co

l-0

cat2

cat2

gr1

0

250

500

750

1000

0

250

500

750

1000

cat2

ca

d2

Co

l-0

cat2

cat2

gr1

cat2

ca

d2

Co

l-0

cat2

cat2

gr1

4h 4d

bc

b

Per

oxi

de

cont

ent

(nm

ol.g

-1F

W)

aab

a a a a

cat2

ca

d2

Co

l-0

cat2

cat2

gr1

cat2

ca

d2

Co

l-0

cat2

cat2

gr1

0

10

20

30

40

0

10

20

30

40

Lea

f NA

DP

(H)

(nm

ol.g

-1F

W) a a a a a a a a

0

10

20

30

40

0

10

20

30

40

Lea

f NA

DP

(H)

(nm

ol.g

-1F

W) a a a a a a a aa a a a a a a a

0

2

4

6

8

0

2

4

6

8

Lea

f asc

orba

te(µ

mol

.g-1

FW

) a a a a a a a a

0

2

4

6

8

0

2

4

6

8

Lea

f asc

orba

te(µ

mol

.g-1

FW

) a a a a a a a aa a a a a a a a

cat2

cad

2

Col

-0

cat2

cad

2

cat2

cad

2

Col

-0

cat2

cad

2

cat2

cad2

Co

l-0

cat2

cad

2

cat2

cad2

Co

l-0

cat2

cad

2

0

400

800

1200

0

400

800

1200b d a c b d a c

Leaf

glu

tath

ione

(n

mol

.g-1

FW)

0

400

800

1200

0

400

800

1200b d a c b d a c

Leaf

glu

tath

ione

(n

mol

.g-1

FW)

4h 4d

Figure 2.1. Oxidant and antioxidant capacity in plant lines with modulated glutathione contents.

(A) Leaf contents of NADP(H), ascorbate, and glutathione in Col-0, cad2, cat2 and cat2 cad2. White bars, reduced forms. Black

bars, oxidized forms.

(B) Leaf peroxide contents in Col-0, cat2, cat2 cad2 and cat2 gr1.

Plants were grown at high CO2, where the cat2 mutation is silent, then transferred to air to initiate oxidative stress in the cat2

backgrounds. Samples were taken 4h and 4d after transfer. Data are means ± SE of at least three biological repeats. Different letters

indicate significant difference at P<0.05 for leaf contents. Note that x-axis legends to parts A and B are different.

38

CHAPTER 2

been previously reported that H2O2 itself (measured as in situ staining or extractable H2O2) is minor or

undetectable in cat2, even when plants are clearly undergoing oxidative stress (Chaouch et al., 2010, 2012;

Mhamdi et al., 2010a). In the present study, extractable peroxides in cat2 were re-examined and compared

with levels found in cat2 cad2 at two time-points after transfer to oxidative stress conditions. For

comparison, the cat2 gr1 double mutant was included, in which loss of GR1 function exacerbates

oxidative stress compared to cat2 (Mhamdi et al., 2010a). At 4d after transfer from high CO2 to air, no

difference was apparent between any of the lines, suggesting that any excess H2O2 had been efficiently

metabolized at this point (Figure 2.1B). At 4h after transfer, however, a 60% increase in peroxides relative

to Col-0 was detected in cat2 and this increase was slightly more pronounced in the cat2 gr1 double

mutant (Figure 1B). In cat2 cad2 at this early time-point, peroxides were intermediate between Col-0 and

cat2. Together, these findings show that when placed in air, the cat2 background causes an accumulation

of H2O2 that is transient and relatively minor, suggesting that other, reductive systems are able to replace

the major leaf catalase in efficiently metabolizing photorespiratory H2O2 when CAT2 function is lost. Its

marked, progressive in cat2 implicates glutathione as a significant component in these reductive systems,

and the cat2 cad2 mutant offers an interesting system to establish the functional significance of

H2O2-triggered adjustments in glutathione.

To examine how the cad2 mutation affects the subcellular distribution of glutathione in cat2 in oxidative

stress conditions, a specific antibody was exploited to quantify glutathione pools in different

compartments (Zechmann et al., 2007; Zechmann and Müller, 2010). In agreement with the increase in

total tissue glutathione (Figure 2.1A), immunogold labelling was significantly increased in cat2 compared

to Col-0 (Figure 2.2A). This increase was mainly due to enhanced signal in chloroplasts, vacuoles, and

the cytosol (Figure 2.2B). All these effects were annulled by the cad2 mutation. In cat2 cad2, glutathione

was decreased in all measured compartments especially the chloroplast, cytosol, nucleus, and

peroxisomes (Figure 2.2). From multiple images of photosynthetic mesophyll cells, subcellular

glutathione concentrations were estimated in the three lines (see Materials and methods for details). They

ranged from 1-10 mM in Col-0 and cat2, whereas concentrations in all compartments except the

mitochondria did not greatly exceed 1 mM in cat2 cad2 (Table 2.2).

______________________________________________________________________

Table 2.2. Subcellular glutathione concentrations in Col-0, cat2, and cat2 cad2.

Plants were grown as described for Figure 2.1. Values are in mM and were calculated from relative gold labelling densities as

described in Materials and methods based on mean values of multiple counts of different cells (n>20 for peroxisomes and vacuoles,

and n>60 for all other cell compartments).

__________________________________________________________________________________________________________

Col-0 cat2 cat2 cad2

Cytosol 4.8 7.4 0.8

Nuclei 7.5 9.0 1.2

Chloroplasts 1.1 2.7 0.1

Mitochondria 10.3 10.1 7.5

Peroxisomes 5.4 6.0 0.8

Vacuole 0.04 0.5 0.03

__________________________________________________________________________________________________________

39

CHAPTER 2

-1

0

1

2

3

4 cat2

** *

*

-4

-3

-2

-1

0

1

cat2 cad2

*

* * * *

*

B

Fo

ld-c

hang

e in

glu

tath

ione

lab

elin

g

rela

tive

to C

ol-0

(lo

g2

mu

tan

t/Co

l-0)

M C N Cy VPx

A Col-0

cat2

cat2 cad2

Figure 2.2. Effects of the cad2 mutation on

subcellular distribution of glutathione in response to

oxidative stress.

(A) Transmission electron micrographs showing

subcellular distribution of gold-labelled glutathione in

photosynthetic mesophyll cells of Col-0, cat2 and cat2

cad2. The bars indicate 1 µm.

(B) Relative quantitation of glutathione labelling in

different subcellular compartments. Plants were grown

as described for Figure 1. Values show log fold change

compared to Col-0. C, chloroplast. CW, cell wall. Cy,

cytosol. M, mitochondria. N, nucleus. Px, peroxisome.

St, starch. V, vacuole. *Significant difference from Col-0

at P<0.05.

The effect of genetically blocking glutathione accumulation on H2O2-triggered pathogenesis responses

was first examined by growing plants in air from seed. This analysis revealed that the cad2 mutation

markedly affected the lesion formation that is spontaneously triggered in cat2 in the absence of pathogen

challenge and the associated induced resistance to subsequent bacterial challenge (Figure 2.3). PR gene

expression, which is spontaneously activated alongside lesion formation in cat2, was also decreased in

cat2 cad2, and the cad2 mutation decreased cat2-triggered resistance to bacteria to wild-type levels

(Figure 2.3). An effective block over H2O2-induced glutathione accumulation and decreased lesion extent

were also observed in double mutants carrying allelic mutations in the same glutathione synthesis gene as

cad2 (Figure 2.4). The decrease in lesion extent was in good agreement with the severity in glutathione

deficiency, with rax1, the weakest allele, producing a slightly weaker effect than cad2 or pad2 (Figure

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

40

CHAPTER 2

the decreased rosette size in cat2 (Figure 2.4). Thus, blocking H2O2-triggered up-regulation of glutathione

deficiency led to down-regulation of lesion spread without producing general effects on the cat2 oxidative

Figure 2.3. Effect of the secondary cad2 mutation on cat2-induced phenotype, PR gene expression, and bacterial resistance.

(A) Phenotype of plants grown in air from seed. Photographs and samples were taken 21 days after sowing. Bars indicate 1 cm.

(B) Lesion quantification in the different genotypes as a percentage of the total rosette area. ND, not detected. Values are means ±

SE of at least 12 plants as in (A).

(C) and (D) PR transcripts quantified by qPCR in the four genotypes, plants as in (A).

(E) Growth of virulent P.syringae DC3000 in the four genotypes.

stress phenotype. Of the three alleles, cad2 is the best characterized and is intermediate in its effects on

glutathione contents (Parisy et al., 2007; Figure 2.4). Because of this, the cat2 cad2 line was chosen for a

detailed examination of the processes underlying the role of glutathione in linking intracellular H2O2 to

induction of pathogenesis responses, with the aim of answering the following specific questions: How is

the effect of blocking glutathione accumulation related to an antioxidant function? And how is it related

to the SA pathway and/or NPR1 function?

0

13

26

Col-0 cad2

cat2 cat2 cad2

Les

ions

(% r

ose

tte

are

a)

Col

-0

cad

2

cat2

cat2

cad

2

a

b

ND ND

c c

PR

1tr

ansc

ript

(rel

. A

CT

IN2

)s

C A

0

2

4

6

a

bc c

0

2

4

6

PR

2tr

ansc

ripts

(rel

. A

CT

IN2

)c c

a

b

D

B

3

6

9

Ba

cte

rialg

row

th )

E

(l og 1

0 c

fucm

-2

Co

l-0

cad2

cat2

cat2

cad

2

a a ab

41

CHAPTER 2

0

300

600

900

1200

Les

ion

s(%

ros

ette

are

a)Le

af g

luta

thio

ne

(nm

ol.g

-1F

W)

Col

-0

pad2

cad

2

rax1

cat2

cat2

pad

2

cat2

cad

2

cat2

rax

1

0

6

12

18

24

ND ND ND ND

a

b

cc

dddd ddd

a

b

cdefgh

0

200

400

600

Fre

sh w

eig

ht(m

g)

a a aa

b b b b

`

Col-0 pad2

cad2 rax1

cat2 cat2 pad2

cat2 cad2 cat2 rax1

Figure 2.4. Confirmation of effects of genetic deficiency in glutathione on H2O2-triggered lesion formation using allelic

mutants.

The rax1 and pad2 mutations (Ball et al., 2004; Parisy et al., 2007) were introduced into the cat2 background by crossing and

compared with cat2 cad2. Bars in photographs indicate 1 cm. For fresh weight, values are means ±SE of ten to twenty-eight plants.

Values are means ± SE of three biological repeats. For lesion formation, values are means ± SE of fifteen plants. For glutathione,

white bars indicate GSH and black bars, GSSG. Letters indicate significant difference at P<0.05.

2.2.2 Contrasting responses in cat2 cad2 and cat2 gr1 reveal a non-antioxidant role for glutathione

in coupling H2O2 to SA-dependent pathogenesis responses

Spreading lesions and associated responses in cat2 can be abolished by introducing the sid2 mutation

(Chaouch et al., 2010). To check whether the decreased lesion extent in cat2 cad2 occurs through the

42

CHAPTER 2

same ICS1-dependent pathway, a cat2 cad2 sid2 triple mutant was produced. Growth of cat2

backgrounds under conditions promoting lesion formation in cat2 (continuous oxidative stress in air)

revealed that, like cat2 sid2, cat2 cad2 sid2 showed no detectable lesion formation (Figure 2.5).

cat2 sid2 cat2 cad2 sid2

Col-0 cad2 cat2

cat2 cad2

Lesion formation (%)21

9 0 0

0 0

Lesion formation (%)

Figure 2.5. The cat2 cad2 sid2 triple mutant shows that residual H2O2-induced lesions in cat2 cad2 are dependent on the ICS1

pathway.

Photographs show plants after 21 d growth from seed in air. The bar indicates 1 cm.

Lesion quantification was performed on 12 plants for each genotype.

Because partial glutathione deficiency does not affect growth or biomass production in the cat2 mutant,

effects of the cad2 mutation on cat2 responses may not be related to changes in oxidative stress intensity.

This hypothesis was tested by comparing cat2 cad2 phenotypes and SA dependency with those observed

in cat2 gr1, in which loss of GR1 function exacerbates oxidative stress in cat2, as reflected by an extreme

dwarf phenotype and dramatic accumulation of oxidized glutathione (Mhamdi et al., 2010a). To analyze

the dependence of responses on SA and glutathione accumulation, the sid2 and cad2 mutations were

introduced into cat2 gr1.

To enable a meaningful comparison of the interactions between the different mutations, plants were

initially grown at high CO2, where the cat2 mutation is silent. In this condition, cat2-dependent

phenotypes and, in particular, the extreme phenotype of cat2 gr1, are annulled. Accordingly, in high CO2

growth conditions, no phenotypic difference was observed between any of the lines (data not shown).

After transferring plants to air, the phenotypes of sid2, gr1 and cad2 were indistinguishable from Col-0

and none of these four genotypes presented lesions on the leaves (Figure 2.6). In contrast, the cat2

mutation induced lesions that first became visible after 5 to 6d, spreading to become very apparent after

10d in air (Figure 2.7A). Similar to its effects on cat2 grown in air from seed, the cad2 mutation restricted

lesion spread while the sid2 mutation abolished it completely so that no lesions were visible in either cat2

sid2 or cad2 cad2 sid2 (Figure 2.7A). All lines in which both cat2 and gr1 were present showed rapid

43

CHAPTER 2

onset of leaf bleaching, which was severe after 4d exposure to air, irrespective of whether or not the sid2

or cad2 mutations were additionally present (Figure 2.7A). Unlike the more slowly forming, persistent

lesions observed in cat2 and, residually, in cat2 cad2, the phenotypes of cat2 gr1 backgrounds were

transient and the bleaching had largely disappeared within 10 days after transfer to air (Figure 2.7A).

Although cat2 gr1 and cat2 gr1 sid2 dramatically accumulated oxidized glutathione, the phenotype

induced by the combination of cat2 and gr1 mutations did not require this accumulation: introduction of

the cad2 mutation into cat2 gr1 restricted glutathione contents well below cat2 levels yet reversible leaf

bleaching was still observed (Figure 2.7A and 2.7B). Thus, the rapid-onset bleaching associated with loss

of glutathione recycling capacity appears to be associated with an antioxidant function rather than

glutathione status per se and does not require ICS1-dependent SA accumulation. This phenotypic

response is therefore quite distinct from that produced by modulating glutathione status through the cad2

mutation.

Col-0

sid2

gr1

cad2

Col-0

sid2

gr1

cad2

4 days 10 days

Transfer to air from high CO2

0.0

0.4

0.8

1.2

Col

-0 gr1

sid

2

cad2

µm

ol g

-1 F

W

Glutathione

Figure 2.6. Phenotypes and glutathione contents in

Col-0, sid2, gr1 and cad2 mutants following transfer

to air after three weeks growth from seed at high

CO2.

Bars in the photographs indicate 1 cm. For glutathione,

white bars show GSH, black bars GSSG. Leaf

glutathione contents in the different lines. White bars,

GSH. Black bars, GSSG. Values are means ± SE of 3

plants, sampled 4d after transfer to air.

44

CHAPTER 2

Figure 2.7. Blocking glutathione accumulation produces distinct effects on H2O2-triggered phenotypes to those produced by

decreasing glutathione-dependent antioxidative capacity.

(A) Phenotypes in cat2 cad2 and cat2 gr1 induced by onset of oxidative stress in cat2 and comparison with lines additionally

carrying the sid2 mutation in the SA synthesis pathway. Plants were grown for 3 weeks at high CO2 then transferred to air to induce

oxidative stress in the cat2 backgrounds. Photographs were taken 4d and 10d after transfer of plants to air. Scale bars indicate 1 cm.

Bleaching in gr1 genotypes is indicated by arrowheads.

(B) Leaf glutathione contents in the different lines. White bars, GSH. Black bars, GSSG. Values are means ± SE of 3 plants, sampled

4d after transfer to air.

0

1

4

5

0

1

4

5

0

1

4

5

0

1

4

5

0

1

4

5

0

1

4

5

0

1

4

5

A

cat2 cat2

4 days

cat2 cad2 cat2 cad2cat2 cad2 cat2 cad2

cat2 gr1 cat2 gr1cat2 gr1 cat2 gr1

cat2 sid2cat2 sid2 cat2 sid2

cat2 cad2 gr1 cat2 cad2 gr1cat2 cad2 gr1 cat2 cad2 gr1

cat2 gr1 sid2cat2 gr1 sid2

cat2 cad2 sid2 cat2 cad2 sid2cat2 cad2 sid2 cat2 cad2 sid2

10 days

Time after transfer to air from high CO2

B

cat2

cat2 g

cat2

cat2 ca

cat2

cat2 si

cat2 gr

Le

afg

luta

thion

eco

nten

t (µm

ol g

-1 F

W)

cad2

r1

cad2 gr1

d2 sid2

d2

1 sid2

Glutathione

GSSGGSSGGSHGSH

cat2 gr1 sid2

45

CHAPTER 2

Several recent publications have implicated myo-inositol in SA-dependent responses (Meng et al., 2009;

Chaouch and Noctor, 2010; Donahue et al., 2010). In cat2, decreases in myo-inositol occur prior to SA

accumulation and are necessary for induction of SA-dependent responses as these can be prevented

simply by treatment with exogenous myo-inositol (Chaouch and Noctor, 2010). In both cat2 and cat2

cad2, 4d after transfer from high CO2 to air, myo-inositol was decreased and this was associated with

decreases in galactinol, which is a product of myo-inositol (Figure 2.8). In contrast, the bleaching

phenotype of cat2 gr1 and cat2 gr1 cad2 was associated with much less marked effects on these

compounds. These observations provide further evidence (1) that myo-inositol metabolism is important in

linking intracellular H2O2 to downstream SA responses, and (2) that loss of glutathione antioxidant

recycling function entrains an SA-independent stress response that is distinct from the SA-dependent

pathway operating in cat2 and cat2 cad2.

0.0

0.1

0.2

0.3

0.0

0.4

0.8

Re

lativ

e u

nits myo-InositolGalactinol

aab ab

b b

cd

aabc bc b

cdd

Col

-0

cad2

cat2

gr1

cat2

cad

2

cat2

gr1

cat2

cad

2 gr

1

Co

l-0

cad

2

cat2

gr1

cat2

ca

d2

cat2

gr1

cat2

ca

d2 g

r1

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

Samples were taken 4d after transfer of plants grown at high CO2 to air, and metabolites were measured by GC-MS (means ± SE

of three biological repeats). Different letters show significant difference at P < 0.05.

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

To establish whether the effect of glutathione deficiency on pathogenesis responses in cat2 is related to

impaired NPR1 function, double cat2 npr1 mutants were produced and analyzed in parallel with cat2

cad2. The npr1 mutation did not affect glutathione status in either Col-0 or cat2 backgrounds (Figure.

2.9A). It did, however, decrease lesion formation in cat2, though less markedly than did cad2 (Figure

2.9B). The triple cat2 cad2 npr1 line showed similar glutathione contents and lesion formation to those

observed in cat2 cad2 (Figure 2.9). Thus, H2O2-induced alterations in glutathione status were independent

of the presence or absence of functional NPR1 in both cat2 and cat2 cad2 backgrounds. None of the

mutations affected bacterial growth, when samples were taken immediately after inoculation (data not

shown) but clear differences were observed in growth in samples taken at two days post inoculation (Fig.

2.9C). Like cad2, npr1 annulled cat2-induced resistance: both cat2 cad2 and cat2 npr1 showed Col-0

resistance levels (Fig. 2.9C). However, cat2 npr1 still showed higher resistance than npr1.

46

CHAPTER 2

3

6

9

0

5

10

15

20

Lesi

ons

(% r

ose

tte a

rea)

NDND ND

B a

b

c cd dd

0

5

10

15

20

Lesi

ons

(% r

ose

tte a

rea)

NDND ND

B a

b

c cd dd

Col

-0

npr

1

cad2

cat2

cat2

np

r1

cat2

cad

2

cat2

ca

d2 n

pr1

Col

-0

npr

1

cad2

cat2

cat2

np

r1

cat2

cad

2

cat2

ca

d2 n

pr1

0

300

600

900

1200

Lea

f glu

tath

ione

(n

mol

.g-1

FW

)

A a a

b b

c de0

300

600

900

1200

Lea

f glu

tath

ione

(n

mol

.g-1

FW

)

A a a

b b

c de

C

Bac

teria

lgro

wth

(log

10

cfu

cm-2

)

a ab b b b

c

Figure 2.9. Comparison of lesions, glutathione contents, and bacterial resistance in cat2 cad2 and cat2 npr1.

(A) Leaf glutathione contents. White bars, GSH. Black bars, GSSG. Values are means ± SE of 3 plants.

(B) Lesion quantification as a percentage of the total rosette area. ND, not detected. Values are means ± SE of at least 12 plants.

(C) Growth of virulent P.syringae DC3000 in all seven genotypes. Plants were grown at high CO2 for 3 weeks to prevent any cat2

phenotype, and inoculations were performed on plants 7d after transfer to air to induce oxidative stress in cat2 backgrounds. No

difference in bacterial growth was observed between the genotypes at 0 hpi.

47

CHAPTER 2

A

Col

-0

cat2

cad2

cat2

cad

2

npr1

cat2

npr

1

npr1 effect

cad2effec

-1.5 +1.5

A

Col

-0

cat2

cad2

cat2

cad

2

npr1

cat2

npr

1

0

1

2

3

0

1

2

B

736cat2 sid2 cat2 cad29

337 cat2 npr18cat2 sid2

914 cat2 npr12cat2 cad2

Tryp

toph

an

Thre

onin

e

Thre

itol

SA-g

luco

pyra

nosi

de

Rha

mno

se

Putr

esci

ne

Isol

euci

ne

Rib

ose

Glu

coni

c ac

id

Mal

ic a

cid

Laur

ic a

cid

Serin

e

Thre

onin

e

Arg

inin

e

α-k

etog

luta

ric a

cid

Thre

onin

e

Ala

nine

Glu

coni

c ac

id

Glu

coni

c ac

id

Rat

io r

ela

tive

to c

at2

Ra

tio r

elat

ive

to c

at2

Ra

tio r

elat

ive

to c

at2

0

1

2

n.d. n.d.

Signal in cat2

Signal in cat2

Signal in cat2

9 common metabolites



t

npr1 effect

cad2effec

-1.5 +1.5

Col

-0

cat2

cad2

cat2

cad

2

npr1

cat2

npr

1

t

npr1 effect

cad2effec

-1.5 +1.5

t

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

(A) Heatmap showing hierarchical clustering of all detected metabolites. Values were centered-reduced prior to clustering analysis.

(B) Comparison of metabolite profiles in cat2 cad2 and cat2 npr1 with those previously reported for cat2 sid2 (Chaouch et al.,

2010). The values in the Venn diagrams indicate the number of metabolites that were significantly different in each double mutant

relative to cat2. Overlapping sections indicate the number of metabolites that showed same-direction significant effects in cat2 sid2

and cat2 cad2 (top), cat2 sid2 and cat2 npr1 (middle), and cat2 cad2 and cat2 npr1 (bottom). For each comparison, the graphs show

values (normalized to cat2) plotted for common metabolites in each comparison using the same color coding as in the Venn

diagrams. Red type is used for the only two metabolites affected similarly by all three secondary mutations.

Plants were grown from seed in air in long days and sampled 23 days after sowing. Three biological repeats were analyzed for each

genotype.

48

CHAPTER 2

In a first approach to analyze whether the effects of blocking glutathione up-regulation were linked to

impaired NPR1 function, comparative GC-TOF-MS analysis of the two double mutants was performed.

By generating information on about 100 different compounds, this technique produces a metabolic

signature for intracellular oxidative stress in cat2 which shows substantial overlap with that induced by

bacterial pathogens (Chaouch et al., 2012). A key feature of the cat2 metabolic signature is accumulation

of a wide range of metabolites in an SA-dependent fashion, ie, most of the signature is annulled in cat2

sid2 double mutants (Chaouch et al., 2010).

Both cad2 and npr1 mutations affected cat2-triggered metabolite profiles, but in very different ways

(Figure 2.10A). Comparison of the profiles shown in Figure 2.10A with those previously described for

cat2 sid2 sampled in the same conditions revealed that cat2 npr1 and cat2 cad2 recapitulated different

parts of the cat2 sid2 profile (Figure. 2.10B). For this comparative analysis, only metabolites that were

detected both in the earlier study of cat2 sid2 (Chaouch et al., 2010) and the present one were included.

For example, 17 metabolites were significantly different from cat2 in cat2 npr1 but only 11 of these were

also detected in the previous study. Of these 11, the sid2 and npr1 mutations significantly affected eight in

the same-direction (Figure 2.10B, middle). Using the same approach, only two metabolites that were

detected in this study and the earlier one (gluconic acid and threonine) were affected similarly by the cad2

and npr1 mutations in the cat2 background. These two compounds are among those strongly induced in

cat2 and in response to bacterial infection (Chaouch et al., 2012) and the induction of both was

significantly decreased by sid2, cad2 or npr1 mutations (Figure 2.10B). Six other metabolites were

affected similarly in cat2 sid2 and cat2 npr1 (Figure 2.10B, middle) whereas the response of seven

metabolites in cat2 was unaffected by npr1 but decreased by both cad2 and sid2 mutations (Figure 2.10B,

top). These seven compounds included several metabolites associated with pathogenesis responses,

including tryptophan, putrescine, and the glucosylated form of SA.

Targeted quantification of free and total SA by HPLC showed that both accumulated in cat2 and that their

accumulation was markedly enhanced by the npr1 mutation (Figure 2.11A). In cat2 npr1, total SA

accumulated to three times the values in cat2. This hyper-accumulation of SA is consistent with effects of

the npr1 mutation in other mutants showing constitutive induction of SA-dependent pathogenesis

responses (Shirano et al., 2002; Zhang et al., 2003). In marked contrast to the effect of npr1, the cad2

mutation substantially decreased SA accumulation (Figure 2.11A). SA levels in the triple cat2 cad2 npr1

mutant were similar to the cat2 single mutant (Figure 2.11A). Thus, the cad2 mutation decreased SA

accumulation in both the cat2 and cat2 npr1 backgrounds, providing further evidence that the principal

effect of glutathione deficiency on H2O2-triggered pathogenesis responses is mediated via a different

route than impairment of NPR1 function.

49

CHAPTER 2

A

B

6 8 10 12 14 16 18 20 22 24 260.0

0.1

0.2

0.3

0.4

ICS1

Tra

nsc

ript

abu

ndan

cere

lativ

e to

AC

TIN

2

Time (days)

Fre

e sa

licyl

icac

id(µ

g g

-1 F

W)

0

20

40

90

120

Col

-0

npr

1

cad2

cat2

cat2

npr

1

a

b b

cd d d

cat2

cad

2 n

pr1

cat2

ca

d2

To

tal s

alic

ylic

acid

(µg

g-1

FW

)

0

1

2

5

6

a

b

cde e e

Figure 2.11. Analysis of salicylic acid (SA) and ICS1 transcripts in double and triple mutants.

(A) Free and total SA in cat2, cad2, npr1 and double and triple mutants. Different letters indicate significant difference at P<0.05.

Plants were grown in air and samples taken 21 days after sowing.

(B) Time course of ICS1 transcript levels in Col-0 (black circles), cat2 (white circles) and cat2 cad2 (triangles). Plants were grown

as in (A). Time indicates days after sowing.

All values are means ± SE of three biological replicates.

Because of the decreased SA accumulation in cat2 cad2, the expression of ICS1, the key enzyme in the

production of SA in response to pathogens (Wildermuth et al., 2001) was analysed, during continuous

growth of plants in air from seed. ICS1 expression showed a marked transient increase in cat2 (Figure

2.11B), with the initial increase correlating with the onset of lesions, which begin to be visible after about

50

CHAPTER 2

16 days growth. The induction of ICS1 was strongly damped in cat2 cad2 (Figure 2.11B), in agreement

with the decreased lesions (Figure 2.3) and SA contents (Figure 2.11A).

2.2.4 Complementation experiments reveal non-redundant roles for glutathione and NPR1 in the

activation of SA-dependent responses

Experiments in which SA content and ICS1 and PR1 transcripts were quantified after transfer to high CO2

following prior growth in air confirmed that both were significantly less accumulated in cat2 cad2

compared to cat2 (Figures 2.12A and 2.12B). To examine whether SA accumulation in cat2 cad2 could be

restored by glutathione, plants were treated with GSH or GSSG. Both forms induced SA above basal

levels in Col-0, with GSSG being the more effective of the two (Figure 2.12A). In cat2 cad2, in which the

cat2 mutation should activate H2O2 signaling, GSH was most effective, restoring total SA to about 60% of

cat2 levels (Figure 2.12A). This effect was not observed in cat2 cad2 sid2, showing that it was dependent

on functional ICS1. We therefore investigated the effects of glutathione supplementation on ICS1

expression. Restoration of SA accumulation in cat2 cad2 by added GSH was associated with enhanced

induction of both ICS1 and PR1 genes (Figure 2.12B). Further, GSSG effectively induced ICS1, even in

the absence of an H2O2 signal (Col-0), although GSSG was much less effective than GSH in inducing

PR1. The glutathione treatments also promoted lesion formation in cat2 cad2.

To establish whether down-regulation of SA-dependent responses in cat2 cad2 was linked to attenuated

SA accumulation or impaired NPR1 function, the effects of SA supplementation on PR1 expression were

examined in the different lines. In Col-0, npr1, and cad2, PR1 expression was very low in the absence of

added SA (Figure 2.13). SA treatment strongly induced PR1 in Col-0 but not significantly in npr1. In

cad2, SA was able to induce PR1 but to lower levels than in Col-0 (Figure 2.13). SA treatment of cat2

showed that PR1, already strongly induced in the absence of added SA, was induced a further 2-fold

(Figure 2.13). In cat2 npr1, PR1 was induced in the absence of SA, though to a lower level than in cat2

(Figure 2.13). This suggests that an NPR1-independent pathway of PR gene induction can operate in the

cat2 npr1 background, as previously reported during SA-dependent interactions with certain pathogens

(Rate et al., 1999; Shah et al., 2001; Liu et al., 2010a). While SA was unable to further induce PR1 in cat2

npr1, PR1 was induced by SA about 8-fold in cat2 cad2, so that transcripts were intermediate between

cat2 –SA and cat2 + SA (Figure 2.13).

51

CHAPTER 2

0

10

20

30

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

Plants were grown at high CO2 for 3 weeks to prevent any cat2 phenotype, then transferred to air to induce oxidative stress in cat2

backgrounds. From the first day after transfer to air, rosettes were treated once daily by spraying with water, 1 mM GSH, or 1 mM

GSSG. Samples were taken after 9 days.

(A) Free and total SA contents in Col-0, cat2, cat2 cad2, and cat2 cad2 sid2.

(B) ICS1 and PR1 expression in Col-0, cat2, and cat2 cad2.

Values are means ± SE of 3 biological replicates. Different letters indicate significant difference at P<0.05.

Col

-0

cat2

cat2

ca

d2

cat2

cad

2 si

d2

Col

-0

cat2

ca

d2

cat2

cad

2 si

d2

Col

-0

cat2

ca

d2

a

b

e f fcd cd

cdc

cat2

cad

2 si

d2

f

Mock GSH GSSG

0.0

0.6

1.2

1.8

Col

-0

cat2

cat2

cad

2

Col

-0

cat2

cad

2

Col

-0

aa

b

b

cc

PR1

Tra

nscr

ipt

abun

danc

ere

lativ

e to

AC

TIN

2

A

Fre

e sa

licyl

icac

id(µ

g g

-1 F

W)

To

tal s

alic

ylic

acid

(µg

g-1

FW

)

cat2

cad

2

b

0.0

0.5

1.0

1.5

ab

c cd

b

a

d

aba

d

0.00

0.04

0.08

0.12

aa

ab

ab

b

cc

ICS1

Tra

nscr

ipt

abun

danc

ere

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AC

TIN

2

BMock GSH GSSG

52

CHAPTER 2

0

3

6

9

0

3

6

9

0

3

6

9

PR

1tr

ansc

ripta

bun

danc

ere

lativ

e to

AC

TIN

2

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

cad2npr1

cat2npr1

cat2cad2

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

Plants were grown in air from seed for 20 days after sowing and then sprayed either (-) with water (mock) or (+) with 0.5 mM SA

and samples were taken 24h later. Values are means ± SE of 3 biological replicates.

2.3 Discussion

A substantial body of work shows that glutathione is a multifunctional metabolite with diverse functions

in plant defence and antioxidative metabolism. Because signaling roles of glutathione might be mediated

by several types of reactions, many of which could be dependent on functionally redundant proteins

encoded by quite large gene families, establishing the role of specific glutathione-dependent components

is a formidable task. This study investigated the role of glutathione-dependent processes in H2O2

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

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

of glutathione

The cad2, rax1 and pad2 mutations in the single gene encoding the first committed enzyme of glutathione

synthesis (-ECS) produce constitutive partial decreases in glutathione (Cobbett et al., 1998; Ball et al.,

2004; Parisy et al., 2007). Here, we show that these mutations also block H2O2-triggered up-regulation of

glutathione. In Arabidopsis, the -ECS protein is located in the chloroplast (Wachter et al., 2005). There is

little evidence that accumulation of glutathione in cat2 is linked to enhanced synthesis of -ECS protein

(Queval et al., 2009). Based on current knowledge, the major player is post-translational activation of

-ECS by disulfide bond formation (Hicks et al., 2007; Gromes et al., 2008). However, the factors that

mediate oxidation of -ECS thiols in vivo remain to be identified. Unlike the wild-type enzyme present in

cat2, the mutant -ECS does not appear to be oxidatively activated in cat2 cad2. However, whereas in

cat2 cad2 glutathione remained close to cad2 values, in cat2 gr1 glutathione accumulated to much higher

levels than in cat2 (Figure 2.7). Thus, glutathione stayed highly reduced and below Col-0 levels in cat2

cad2 while a highly oxidized glutathione pool in cat2 gr1 was associated with dramatic accumulation.

These observations are consistent with a model for -ECS regulation in which GSSG produced from GSH

53

CHAPTER 2

oxidation allows activation of the enzyme to up-regulate glutathione synthesis. This receives support from

glutathione contents in cat2 cad2 gr1, which were intermediate between cat2 cad2 and cat2 (Figure 2.7).

Indeed, the chloroplast was among the cellular compartments that showed the highest H2O2-triggered

glutathione accumulation in cat2 (Table 2.2). Since the accumulated glutathione in cat2 is almost all in

the disulfide form (Figures 2.1 and 2.9), it is highly likely that in cat2 the chloroplast is enriched in GSSG

but that this enrichment does not occur in cat2 cad2.

In all three double mutants blocked in glutathione accumulation, including cat2 rax1 carrying the weakest

allele, the overall leaf glutathione pool remains highly reduced in conditions that permit increased H2O2

availability (Figure 2.4). In cat2 cad2 gr1, a limited accumulation of glutathione was accompanied by

significant oxidation relative to cat2 cad2 (Figure 2.7). From a cellular point of view, these striking

observations suggest that the plant cell glutathione redox system is configured so that not only does

oxidation drive enhanced accumulation of total glutathione but also decreased contents inhibit glutathione

oxidation. This two-way interaction may be important in setting appropriate conditions for cellular

signaling. The underlying factors remain unclear but could include, for example, affinities of the enzymes

that oxidize GSH to GSSG or the differences in glutathione compartmentation in cat2 and cat2 cad2.

Whatever the underlying causes, glutathione contents are known to change during development (Queval

et al., 2007) and in response to factors such as sulfur nutrition (Nikiforova et al., 2003). A strong interplay

between concentration and redox state could influence the outcome of redox-dependent responses to

external stresses in different circumstances.

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

related pathogenesis responses

Alongside up-regulation of glutathione, the cat2 mutation induces SA and a wide range of SA-dependent

responses in an ICS1-dependent manner (Chaouch et al., 2010). Using non-targeted metabolite profiling

and targeted analysis of recognized defence compounds, it was recently reported that SA-dependent

responses triggered by intracellular H2O2 in cat2 show considerable similarity with responses to

pathogenic bacteria (Chaouch et al., 2012). Thus, intracellular H2O2 generated by the peroxisome-located

photorespiratory glycolate oxidase closely mimics redox processes involved in biotic stress. Several

studies suggest that down-regulation of catalase plays some role in pathogenesis responses (Mhamdi et al.,

2010b; Volt et al., 2009) while the analysis of glycolate oxidase mutants provides further indications of

roles for peroxisomally produced H2O2 in pathogenesis responses (Rojas et al., 2012). The potential

relevance of redox-triggered events in cat2 is underscored by effects observed in double mutants in which

cat2 responses are modulated by loss of function of recognized players in pathogenesis (Chaouch et al.,

2010, 2012). The present study provides direct evidence that glutathione is a key player linking increased

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

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

bacteria.

The residual SA-linked responses in cat2 cad2 are completely annulled in cat2 cad2 sid2, suggesting that

glutathione status modulates the efficiency of H2O2-triggered signaling through the ICS1-dependent

pathway. This conclusion is supported by the attenuated induction of the ICS1 gene in cat2 cad2

54

CHAPTER 2

compared to cat2 (Figure 2.11). Like -ECS, ICS1 is a chloroplast enzyme that could potentially be

redox-regulated. However, post-translational control of ICS1 by thiol-disulfide status has been discounted

on the basis of the protein’s structural features (Strawn et al., 2007). Our data reveal that the expression of

ICS1 may be under redox regulation through glutathione-dependent processes downstream of H2O2. The

intermediates in this signaling pathway remain to be identified.

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

A central premise of this study was that H2O2-triggered modulation of glutathione (as a result of the

antioxidant function of glutathione) might be perceived by the cell as a signal. To test this hypothesis, the

aim was to manipulate glutathione status in an H2O2 signaling system, without affecting overall cellular

antioxidant capacity. That the block over glutathione up-regulation in cat2 cad2 and allelic lines achieves

this objective is evidenced by the following observations. First, introduction of pad2, cad2, or rax1

mutations does not affect the oxidative stress-dependent decreased growth phenotype of cat2. Second, the

block over H2O2-triggered glutathione accumulation in cat2 cad2 is quite specific, and not accompanied

by increased oxidation of ascorbate or NADPH, or by enhanced accumulation of peroxides. Third,

blocking the glutathione synthesis pathway decreases rather than increases cat2-dependent lesion spread.

Fourth, and most crucially, the ICS1-linked effects observed in cat2 cad2 are quite distinct from the

responses to exacerbated oxidative stress produced by knocking out both CAT2 and GR1.

In addition to the ICS1-independent phenotypes of cat2 gr1, the contrasting effects of cad2 and gr1

mutations on the cat2 response are underscored by analysis of metabolite markers for the SA-dependent

pathway. Entrainment of SA-dependent responses in cat2 requires decreased myo-inositol (Chaouch and

Noctor, 2010). In cat2 cad2, as in cat2, this compound and the related metabolite galactinol were

decreased (Figure 2.8), even though the cat2-triggered SA response was attenuated in the double mutant.

This indicates that glutathione status exerts its effects on the SA pathway downstream of myo-inositol.

Together with previous analyses of cat2, it suggests a model in which H2O2 decreases myo-inositol

concentrations, an effect that then allows SA accumulation through glutathione-dependent processes.

Despite the enhanced oxidative stress in cat2 gr1 and cat2 cad2 gr1, both these compounds remained at

levels close to wild-type, suggesting that decreases in myo-inositol are not simply related to oxidative

stress intensity.

Together, these data further illustrate that SA-dependent lesions in cat2 are not a simple consequence of

oxidative stress intensity. Rather, they are the result of an H2O2-initiated programmed response whose

intensity is modulated by glutathione. Elucidation of events underlying the intriguing reversibility of the

SA-independent phenotypes in cat2 gr1 backgrounds will require further study, although it is perhaps

worth noting that Arabidopsis lines deficient in both CAT2 and APX1 show induction of novel protective

mechanisms that are not observed in lines deficient in only CAT2 or APX1 (Vanderauwera et al., 2011).

Failure of cat2 gr1 to induce the SA-dependent phenotype observed in cat2 perhaps suggests that an

optimal level of oxidation is required to entrain effective induction of pathogenesis responses. Beyond a

certain threshold intensity, SA-dependent pathogenesis programme are no longer activated. Thus, even

when triggered by a single type of ROS (H2O2 in this study), intracellular oxidative stress can drive

several distinct phenotypic outcomes.

55

CHAPTER 2

2.3.4 Glutathione can regulate SA signaling through processes additional to NPR1

NPR1 is by far the best characterized thiol-dependent protein involved in pathogenesis responses, and can

be activated by glutathione addition to cells or leaves (Mou et al., 2003; Gomez et al., 2004a). Pathogens

and SA can both cause adjustments in leaf glutathione pools (Edwards et al., 1991; May et al., 1996;

Vanacker et al., 2000; Mateo et al., 2006; Koornneef et al., 2008). However, the precise nature of the

redox factors controlling NPR1 in vivo is still open to debate. While h-type thioredoxins, which are

known to be induced during pathogenesis responses (Laloi et al., 2004), were shown to perform the

reductive activating step (Tada et al., 2008), a recent study has revealed the complexity of NPR1

regulation (Lindermayr et al., 2010).

As well as abolishing H2O2-triggered increases in chloroplast glutathione, the cad2 mutation prevented

increases in cytosolic and nuclear concentrations (Figure 2.2; Table 2.2). Based on current knowledge,

this effect might be predicted to compromise NPR1 function. Indeed, the NPR1 pathway is clearly

functional in cat2, as the npr1 mutation produced the following effects in cat2 npr1: impaired lesion

spread relative to cat2, partial loss of PR gene expression, a complete loss of PR1 inducibility by

exogenous SA, and a characteristic metabolite signature, including hyper-accumulation of SA. Consistent

with previous studies of plants with altered cytosolic glutathione status (Maughan et al., 2010), exogenous

SA induced PR1 less effectively in cad2 than in Col-0. However, if the effect of the cad2 mutation on

cat2 responses occurred exclusively through partial or complete loss of NPR1 function, it would be

predicted that cat2 cad2 should show same-direction responses to cat2 npr1. In fact, of the cat2 npr1

features listed above, the only one shared by cat2 cad2 was decreased lesion spread relative to cat2. PR1

expression was even lower in cat2 cad2 than in cat2 npr1 and was inducible by exogenous SA. Crucially,

blocking glutathione accumulation in cat2 cad2 produced a metabolite signature that was distinct from

that observed in cat2 npr1. These distinct effects were most striking for SA contents: if effects of the cad2

mutation were purely explainable in terms of loss of NPR1 function, cat2 cad2 should be expected to

have higher SA contents than cat2 (as in cat2 npr1). In fact, the opposite is observed. Thus, it seems that

glutathione plays a regulatory role in SA signaling additional to NPR1. Indeed, the similar glutathione

status in cat2 and cat2 npr1 provides little evidence for feedback between NPR1 and glutathione.

Hyper-accumulation of SA in cat2 npr1 was associated with H2O2-triggered NPR1-independent PR1

expression and bacterial resistance. The operation of this NPR1-independent pathway seems itself to be

dependent on glutathione, because cat2 cad2 npr1 showed similarly low resistance (Figure 2.9C) and PR1

expression to npr1 (Figure 2.13), showing that the cad2 and npr1 mutations act additively to annul part of

the cat2-induced resistance responses. Again, this suggests that blocking up-regulation of glutathione

impairs H2O2-triggered pathogenesis responses via effects that are distinct from those of the npr1

mutation. A key difference between cat2 npr1 and cat2 npr1 cad2 is that the hyper-accumulation of SA in

the former is not observed in the latter. Together with differences in SA accumulation in cat2 and cat2

cad2, this suggests that glutathione plays an important role in H2O2 signaling at the level of induction of

SA itself.

56

CHAPTER 2

57

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

The observations reported in this study reveal that H2O2-triggered changes in glutathione status are not

merely a passive response to oxidative stress. Rather, they suggest that modulation of glutathione status is

required to link increases in intracellular H2O2 production to activation of the ICS1-dependent SA

pathway. This function would operate within the context of dynamic modulation of glutathione that

involves initial oxidation leading to downstream reduction during some pathogenesis responses (Vanacker

et al., 2000; Mou et al., 2003; Koornneef et al., 2008). While a potential role for glutathione in the

reductive phase has been described at the level of NPR1, results presented in this chapter provide the first

direct evidence that initial oxidative events necessary for SA accumulation require changes in glutathione

status. The down-regulation of the SA pathway in cat2 cad2, in which glutathione stays highly reduced,

suggests that these oxidative events involve a decrease in the GSH:GSSG ratio (Figure 2.14). Failure to

produce an appropriately oxidized glutathione status would then inhibit initiation of the pathway,

explaining our observation that introducing the cad2 mutation antagonizes SA accumulation, PR1

induction and induced resistance in both cat2 and cat2 npr1 (Figure 2.14). When glutathione oxidation is

accompanied by excessively severe oxidative stress, as in cat2 gr1 backgrounds, an alternative response

to the SA pathway is entrained.

H2O2

SA accumulation

NPR1GSH/GSSG

Thioredoxins/Glutathione

Initiation and control of oxidative signaling

Downstream reductive signaling

SA-dependent gene expression

Cell death

Resistance

ICS1

NPR1-independent

pathway

Signal readout

H2O2

SA accumulation

NPR1GSH/GSSG

Thioredoxins/Glutathione

Initiation and control of oxidative signaling

Downstream reductive signaling

SA-dependent gene expression

Cell death

Resistance

ICS1

NPR1-independent

pathway

Signal readout

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

Oxidation by H2O2 modulates glutathione status. This oxidative modulation is part of the signal network required for optimal

ICS1-dependent SA accumulation that then leads to activation of NPR1 function through reductive processes (Mou et al., 2003), to

which glutathione may also contribute.

Both pathogenesis responses and glutathione status are influenced by sulfur nutrition (Höller et al., 2010;

Király et al., 2012). More generally, photoautotrophism means that redox modulation is a central player in

linking energy and nutritional status to appropriate stress outcomes within the complex metabolic network

and plastic developmental program of plants (Noctor, 2006; Foyer and Noctor, 2009). Glutathione status

may be an important factor to take into account in attempts to understand and optimize pathogenesis

responses in plants growing in natural environments with variable nutrition.

CHAPTER 3

Regulation of basal and oxidative stress-triggered jasmonic

acid-related gene expression by glutathione

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

following authors:

Han Y., Mhamdi A., Chaouch S. and Noctor G. Regulation of basal and oxidative stress-triggered

jasmonic acid-related gene expression by glutathione. Submitted to Plant, Cell & Environment

CHAPTER 3

Summary

As one of the components that determine protein thiol-disulfide status, emerging concepts view

glutathione as potentially important in cellular signaling. While several studies have indicated interactions

between phytohormone signaling and glutathione in biotic stress responses, effects of glutathione could

reflect several processes. In chapter 2, the results showed that glutathione accumulation may play an

important role in linking oxidative stress to salicylic acid (SA) dependent signaling. Antagonistic

interactions between SA and jasmonic acid (JA) are well described in plants. Most attention has been

focused on the thiol-regulated protein NPR1. This chapter reports evidence for potentially important

NPR1-independent roles for glutathione in setting signaling strength through the jasmonic acid (JA)

pathway. First, it is shown that basal expression of JA-associated genes in the absence of stress is

correlated with leaf glutathione content. Second, analyses of an oxidative stress mutant, cat2, reveal that

up-regulation of the JA pathway triggered by intracellular H2O2 signals requires accompanying

glutathione accumulation. Genetically blocking this accumulation largely annuls H2O2-induced

expression of JA-linked genes, and this effect can be rescued by exogenously supplying glutathione.

Through a comparison of JA-linked gene expression in double mutants in which oxidative stress occurs in

the absence of either glutathione accumulation or NPR1 function, evidence is provided that the regulation

of H2O2-triggered JA signaling by glutathione can be mediated by factors additional to NPR1. Hence, this

study provides new information that implicates glutathione as a factor determining basal JA gene

expression and points to novel glutathione-dependent control-points that regulate JA signaling in response

to oxidative stress.

Résumé

Vu son role en tant que composante determinant le statut thiol-disulfure, le glutathion est potentiellement

important dans la signalisation cellulaire. Plusieurs études ont indiqué l’existence d’interactions entre la

signalisation phytohormonale et le glutathion lors des réponses des plantes aux stress biotiques, mais ces

interactions pourrait avoir lieu grâce à plusieurs processus dont les détails restent à élucider. Dans le

chapitre 2, nous avons vu que l’accumulation de glutathion pourrait jouer un rôle pour relier le stress

oxydatif à la signalisation dépendant de l’acide salicylique (SA). Les interactions antagonistes entre SA et

acide jasmonique (JA) sont bien documentées chez les plantes, notamment au niveau de la protéine

régulatrice, NPR1. Ce chapitre met en évidence des rôles NPR1-indépendants pour le glutathion dans la

détermination de l’intensité de signlisation JA. D’abord, l’expression basale de gènes associés à la voie

JA est corrélée avec les teneurs en glutathion. Ensuite, des analyses du cat2, un mutant « stress oxydant »,

revèlent que l’induction de la voie JA déclenché par le H2O2 nécessite une accumulation de glutathion.

Introduisant un blocage génétique de cette accumulation chez cat2 diminue significativement l’induction

H2O2-dépendante des gènes associés au JA, un effet qui peut être surmonté en fournissant du glutathion

éxogène à la plante. Une comparaison de l’expression génique associée au JA dans des doubles mutants

chez lesquels le stress oxydant a lieu en absence de l’accumulation du glutathion ou bien en absence de la

fonction NPR1, suggère que la régulation de la signalisation JA déclenchée par le glutathion pourrait être

médiée par d’autres facteurs que NPR1. Donc, les résultats impliquent le glutathion comme facteur

déterminant l’expression basale de la voie JA et indiquent l’existence de nouveaux points de régulation

glutathion-dépendants qui régulent la signalisation JA en réponse au stress oxydant.

58

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3.1 Introduction

As a major determinant of cell redox status, glutathione is a key player in stress responses (Noctor et al.,

2012). In addition to its accepted antioxidant function mediated by the thiol form, GSH, it influences

defence, secondary metabolism and regulation of protein activity through conjugation reactions to

metabolites or to protein cysteine residues (Rouhier et al., 2008; Schlaeppi et al., 2008; Su et al., 2011).

Glutathione contents are influenced by light and sulphur nutrition, and adjustments in glutathione status

can be triggered by a variety of stresses (Bick et al., 2001; Gomez et al., 2004b; Höller et al., 2010). As

discussed in the preceding chapters, attention has been paid to roles of glutathione in biotic stress, for

several reasons. First, such stresses are known to modulate glutathione status (Edwards et al., 1991;

Vanacker et al., 2000; Mou et al., 2003; Koornneef et al., 2008). Second, alterations in responses to

pathogens have been described in some studies of mutants with modified glutathione contents or

reduction (May et al., 1996; Ball et al., 2004; Parisy et al., 2007; Mhamdi et al., 2010a; Dubrueil et al.,

2011). Third, NONEXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), a major player in

signal transmission linked to some pathogenesis responses, is known to be regulated by modulation of

thiol status (Mou et al., 2003; Tada et al., 2008; Lindermayr et al., 2010). However, the exact role of

glutathione status in regulating NPR1 is not clearly established, because pathogen-induced thioredoxins

(TRX) have also been implicated (Laloi et al., 2004, Tada et al., 2008). Other important

glutathione-sensitive regulatory proteins may await description, and, generally, much remains to be

elucidated on how glutathione impacts on pathogenesis-related signaling.

As described in Chapter 2, activation of pathogenesis responses in cat2 is correlated with perturbations in

leaf glutathione, is followed by activation of SA-dependent processes and pathogen resistance, and the

H2O2-triggered up-regulation of the SA pathway is glutathione-dependent. Jasmonic acid (JA) is another

important defence hormone and its signaling strength is known to be negatively controlled by SA

(Leon-Reyes et al., 2010). There is some evidence that glutathione also interacts with the JA pathway.

Genes encoding glutathione synthesis enzymes and glutathione reductase (GR) are induced by JA (Xiang

and Oliver, 1998) and gr1 mutants with decreased leaf GR capacity show repression of the JA pathway in

both basal and oxidative stress contexts (Mhamdi et al., 2010a). Glutathione has also been implicated in

the interplay between SA and JA pathways at the level of NPR1, which acts to relay part of the negative

control of JA signaling by SA (Spoel et al., 2003; Koornneef et al., 2008). Because of the role of

glutathione in linking H2O2 to activation of the SA pathway (Chapter 2), and the known antagonism of SA

and JA pathways, the aim of the work presented in this chapter was to explore whether blocking

cat2-triggered glutathione accumulation has any impact on JA signaling and, if so, whether any such

impact is mediated by NPR1 (Figure 3.1). By analysis of the glutathione-deficient cad2, the oxidative

stress signaling system cat2 and the double mutants cat2 cad2 and cat2 npr1, evidence is provided that

glutathione status is a determinant of the abundance of transcripts associated with JA synthesis, signaling

and downstream processes, and that this effect appears to occur through novel components additional to

NPR1.

59

CHAPTER 3

H2O2

SA accumulati

NPR

?GSH/GSSG

Oxidation and induction of glutathione

on

1

JA-dependegene expres

ICS1

ntsion

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

Since glutathione status impacts on H2O2-triggered SA signaling, and SA is known to repress JA signaling, key questions addressed

in this chapter are whether glutathione status has any effect on the JA pathway and, if so, whether these can be explained purely in

terms of glutathione-dependent effects on SA signaling via ICS1 and/or NPR1. ICS1, Isochorismate synthase. NPR1, Nonexpressor

of pathogenesis-related genes 1.

60

CHAPTER 3

3.2 Results

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

To investigate effects of partial glutathione deficiency on phytohormone-linked pathways, microarray

analysis of the cad2 mutant was performed. The data revealed that multiple genes associated with

phytohormone signaling were differentially expressed in cad2 compared to Col-0. Of these genes, most

are annotated as being associated with JA and/or wounding. A suite of genes down-regulated in cad2

included genes involved in JA synthesis and activation (LOX3, LOX4, OPR3, JAR1), seven members of

the jasmonate/ZIM domain family of JA-induced JA signaling repressors (JAZ1, JAZ2, JAZ3, JAZ6, JAZ7,

JAZ9, JAZ10), and transcription factors and other genes known to be induced by wounding (Table 3.1).

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

Locus Description cad2/Col JA/Wounding

At1g01060 Late elongated hypocotyl 1 (LHY1) -1.03 JA At1g01470 LATE EMBRYOGENESIS ABUNDANT 14 (LEA14) -1.14 Wounding At1g17420 Lipoxygenase_ lipoxygenase 3 (LOX3) -2.35 JA At1g18710 Myb family transcription factor (MYB47) -1.59 JA At1g19180 Jasmonate-zim-Domain Protein 1 (JAZ1) -1.67 JA At1g28480 Glutaredoxin family protein (GRX480) -1.13 JA At1g43160 Related to AP 2.6 (RAP2.6) -2.11 JA At1g52890 Arabidopsis NAC Domain Containing Protein 19 (ANAC019) -1.72 JA At1g70700 Jasmonate ZIM-Domain protein 9 (JAZ9) -1.65 JA At1g72450 Jasmonate ZIM-Domain protein 6 (JAZ6) -1.62 JA At1g72520 Lipoxygenase 4 (LOX4) -2.16 JA At1g74430/40 Arabidopsis thaliana MYB domain protein 95 (MYB95) -1.58 JA At1g74950 Jasmonate ZIM-Domain protein 2 (JAZ2) -1.55 JA At1g80840 Pathogen induced-transcription factor (WRKY40) -1.29 JA At2g06050 12-oxophytodienoate reductase (OPR3) -2.09 JA At2g27690 Oxygen binding/fatty acid (omega-1)-hydroxylase_CYP94C1 -1.78 JA At2g29440 glutathione S-transferas 24 (GSTU6) -1.30 JA At2g34600 Jasmonate ZIM-Domain protein 7 (JAZ7) -2.04 JA At2g46370 Jasmonate resistant 1 (JAR1) -2.32 JA At2g46510 ABA-inducible BHLH-type transcription factor (AIB) -1.11 JA At3g10985 Arabidopsis wound-induced protein 12 -1.05 Wounding At3t17860 Jasmonate ZIM-Domain protein 3 (JAZ3) -1.17 JA At3g23250 MYB Domain protein 15 (MYB15) -1.51 JA At3g50260 Cooperatively regulated by ethylene and jasmonate 1 -1.20 JA At4g37260/70 MYB Domain protein 73 (MYB73); -1.02 JA At5g05730 Jasmonated-induced defective lateral root 1 (JDL1) -0.87 JA At5g06870 Polygalacturonase inhibiting protein 2 (PGIP2) -1.08 JA At5g13220 Jasmonate ZIM-Domain protein 10 (JAZ10) -2.52 JA At5g47220 Ethylene-responsive element-binding factor 2 (ERF2) -2.18 JA At5g47240 Arabidopsis thaliana nudix hydrolase homolog 8 (ATNUDT8) -1.19 Wounding At5g53750 CBS domain-containing protein -1.49 JA At5g54170 Polyketide cyclase/dehydrase and lipid transport family protein -1.70 JA At5g60890 Myb-like transcription factor (MYB34) -1.08 JA At5g67300/10 Myb domain protein 44 (MYB44); Member of CYP81G -1.48 JA

Microarray analyses and data processing were performed as in Mhamdi et al. (2010a). Only genes that were significantly repressed in independent biological replicates of cad2 RNA dye-swapped with Col-0 RNA are included. The full data set is available at http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=256

61

http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=256

CHAPTER 3

To further investigate the relationship between glutathione and JA, we examined how the levels of

transcripts of two JA signaling marker genes (PDF1.2, VSP2) responded to glutathione deficiency in two

available allelic mutants (cad2, rax1) or in Col-0 treated with the glutathione synthesis inhibitor,

buthionine sulfoximine (BSO). Irrespective of the genetic background, the results revealed a close

correlation between leaf glutathione content and expression of the two JA marker genes (Figure 3.2).

Figure 3.2. The basal level of jasmonic acid-associated gene expression is closely correlated with glutathione contents.

(A) Total glutathione contents. (B) PDF1.2a transcripts. (C) VSP2 transcripts.

Col-0 and the two allelic glutathione-deficient lines, cad2 and rax1, were grown on agar for two weeks. The glutathione synthesis

inhibitor, buthionine sulfoximine (BSO), was supplied to Col-0 plants in the medium at the indicated concentration. All values are

means ± SE of three biological repeats. Different letters indicate significant differences at P<0.05.

0.0000

0.0002

0.0004

0.0006

0.0008

0.00

0.04

0.08

0.12

B

C

Tra

nscr

ipt

abu

ndan

cere

lativ

e to

AC

TIN

2

PDF1.

VSP2

Col-0 0.2 0.5

2a

cad2 rax1

BSO (mM)

a

a

a

b

Tra

nscr

ipt

abu

ndan

cere

lativ

e to

AC

TIN

2

b b b

bc

c c

0

100

200

300

400A

Lea

fco

nte

nt

(nm

olg

-1 F

W)

Glutathi

b

a

d

one

cb

62

CHAPTER 3

3.2.2 Activation of jasmonic acid signaling by intracellular oxidative stress

Because of its role as an antioxidant, glutathione status is likely to become particularly important during

enhanced production of ROS, in particular, H2O2 (Mhamdi et al., 2010b). To explore relationships

between glutathione, intracellular oxidative stress, and JA signaling, the cat2 mutant was used. This line

is a conditional oxidative stress signaling mutant which, when exposed for several days to air and

moderate light, shows H2O2-activated oxidation and accumulation of glutathione, predominantly as GSSG

(Queval et al., 2007; Mhamdi et al., 2010a). To establish whether intracellular oxidative stress in cat2

activates the JA pathway, four genes were selected for qRT-PCR analysis in cat2 and Col-0. Two of the

genes encode enzymes involved in JA synthesis (LOX3, OPR3), one encodes the JA signaling protein

JAZ10, and the last is a JA/wounding marker gene (VSP2). Inducing oxidative stress by transferring cat2

from high CO2 to air caused accumulation of transcripts of all four genes in a time-dependent manner:

while transcript abundance remained at Col-0 levels 4d after transfer to air, induction of several-fold was

observed 8d after transfer (Figure 3.3).

0.00

0.05

0.10

0.20

0.30

0.00

0.02

0.04

0.06

Col-0 cat2

Tra

nsc

ript

abu

ndan

cere

lativ

e to

A

CT

IN2

*OPR3

*

VSP2

*

*

Col-0 cat2

0.00

0.01

0.08

0.10

0.000

0.005

0.010

0.015LOX3 *

*

JAZ10 *

*

Tra

nsc

rip

tab

und

ance

rela

tive

to

AC

TIN

2

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

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

3.2.3 Modulation of leaf thiol status by the cad2 mutation

To establish the functional importance of glutathione modulation in oxidative stress-triggered expression

of JA-associated genes, the the cat2 cad2 double mutant described in Chapter 2 was analysed. As

shown in Chapter 2, the cad2-linked block on cat2-triggered glutathione accumulation prevented the

oxidative up-regulation of glutathione that was otherwise observed in cat2. To explore this effect further,

63

CHAPTER 3

leaf thiols were determined by HPLC. Profiling of soluble thiols showed that, in agreement with previous

studies (Queval et al., 2009), glutathione and its precursors, cysteine and -glutamylcysteine (-EC), were

all accumulated in cat2 to about two- to three-fold Col-0 levels (Figure 3.4). In cad2, carrying a mutation

in the enzyme that converts cysteine to -EC, glutathione and -EC levels were lower than in Col-0 while

cysteine contents were increased. These effects are consistent with previous studies of cad2 (Cobbett et

al., 1998). Introducing the cad2 mutation into cat2 showed that the cad2 mutation overrode the main

features of the cat2 effect on thiol profiles. These were essentially similar in cat2 cad2 and cad2, except

that cysteine contents were increased in cat2 cad2 to even higher levels than in cat2, and reached about

5-fold Col-0 levels (Figure 3.4). Cys-gly, which is a product of glutathione degradation by -glutamyl

transpeptidases, was detected at low levels in Col-0 but accumulated to significantly higher levels in cat2

(Figure 3.4). In line with the low glutathione levels in cad2 and cat2 cad2, cys-gly was below the limit of

detection in these lines (Figure 3.4).

Figure 3.4. Profiling of free thiols in cad2, cat2, and cat2 cad2.

The scheme on the right indicates a simplified version of the glutathione synthesis pathway and the first step of glutathione

breakdown. -ECS, -glutamylcysteine synthetase. GGT, -glutamyl transpeptidase. GSH-S, glutathione synthetase. Different letters

indicate significant difference at P < 0.05. ND, not detected.

Lea

f cy

ste

ine

(nm

ol.g

-1F

W)

0

25

50

75 a

bb

a

Lea

f gl

utat

hion

e

(nm

ol.g

-1F

W) a

d

b

c

0

7

14

21 a

b

c cLea

f -

EC

(n

mo

l.g-1

FW

)

0

300

600

900

1200

0

5

10

15

Lea

f cy

s-g

ly

(nm

ol.g

-1F

W) a

b cND ND

c

cat2

cat2

ca

d2

cad

2

Col

-0

cat2

cat2

ca

d2

cad

2

Col

-0

Cys-Gly

Glutathione

-Glu-Cys

Cys

GGTGGT

GSH-SGSH-S

-ECS-ECS cad2

cat2

Sulfate

+

+

64

CHAPTER 3

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

The well-documented antagonism between SA and JA pathways is considered partly to depend on NPR1

function (Spoel et al., 2003; Koornneef et al., 2008). The data in Figure 3.3 in this chapter and Figure 2.3

in the previous chapter show that both pathways can be activated in parallel following the glutathione

oxidation and accumulation that occurs in cat2 within a few days of transferring to oxidative stress

conditions. Based on current concepts, disabling NPR1 function in cat2 npr1 should allow oxidative

stress to hyper-induce JA-associated genes. Similarly, if any effects of blocking glutathione accumulation

on the oxidative stress-induced JA pathway are mediated by down-regulation of SA accumulation or

defective NPR1 function (or both), the cad2 mutation should act in a qualitatively similar manner to npr1,

ie, the effects of the two secondary mutation on JA-associated genes should be in the same direction.

qRT-PCR analysis showed that the npr1 mutation did not significantly affect stress-induction of the two

genes involved in JA synthesis but that JAZ10 and, particularly, VSP2, were more induced than in cat2

(Figure 3.5). The response of JAZ10 and VSP2 in cat2 npr1 is consistent with previous studies

implicating NPR1 in SA-mediated repression of the JA pathway (Spoel et al., 2003; Leon-Reyes et al.,

2010). In stark contrast to effects observed in cat2 npr1, the cad2 mutation did not cause enhanced

induction of JA-associated genes. Rather, expression of all four genes remained close to Col-0 levels in

cat2 cad2 (Figure 3.5). Hence, both the SA and JA pathways were down-regulated in parallel in cat2 cad2

relative to cat2 (Figure 3.5; Chapter 2).

0.0

0.2

0.8

1.0

1.2

*

LOX3cat2cat2

0

1

2

3

*

*JAZ10

cat2cat2

Fo

ldch

an

ge r

ela

tive

to

cat2

0.0

0.4

0.8

1.2

*

OPR3cat2cat2

0

1

10

15

20

*

*

VSP2

cat2cat2

Col-0 cat2

npr1cat2 cad2

Col-0 cat2 npr1

cat2 cad2

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

mutations.

Effects of cad2 and npr1 mutations on gene expression were examined at 8d after transfer. The dotted line shows expression level in

cat2, which is set to a value of 1 and indicated by arrows. *Significant difference of double mutants from cat2 at P<0.05.

65

CHAPTER 3

3.2.5 Effects of complementing mutants with cysteine and glutathione

Because the cad2 mutation not only prevents cat2-triggered accumulation of glutathione but also further

promotes cysteine accumulation, effects of exogenous supplying either cysteine or glutathione on the cat2

phenotype and expression of the JA pathway were examined. The aim of these experiments was to

establish whether increased cysteine (as occurs in cat2 cad2) contributes to the effects of the cad2

mutation on cat2-triggered expression of the JA-associated genes. In Col-0, neither cysteine nor

glutathione treatments affected rosette phenotype but both produced some induction of the JA-associated

genes (Figure 3.6). Treating cat2 with cysteine or glutathione did not markedly affect lesion spread or

expression of the JA pathway (Figure 3.6). Thus, these observations provide no evidence that the effects

of the cad2 mutation observed in cat2 cad2 are linked to increased cysteine.

To test the second possibility – that the effects of the cad2 mutation are caused by the block on

glutathione accumulation – we examined whether induction of the four genes could be rescued by

supplying glutathione exogenously to cat2 cad2. As shown in Figure 3.7, the antagonistic effect of the

cad2 mutation on cat2 responses could largely be overcome by supplying glutathione. Exogenous

glutathione, particular in the form of GSH, allowed all four transcripts in cat2 cad2 to approach cat2

levels. GSH or GSSG also stimulated expression in Col-0, albeit to a lesser extent (Figure 3.7).

0.00

0.06

0.12

0.18

0.00

0.25

0.50

0.00

0.25

0.50

LOX

3tr

ansc

ripts

(rel

. A

CT

IN2)

OP

R3

tran

scrip

ts(r

el.

AC

TIN

2)V

SP

2tr

ansc

ripts

(rel

. AC

TIN

2)

0.000

0.013

0.026

JAZ

10tr

ansc

ripts

(rel

. AC

TIN

2)

Col

-0

cat2

Col

-0

cat2

Col

-0

cat2

Mock Cys GSH

a a

a

bb

c

aa

a

bc

d

aa

a

b bc

a

a

a

bbc

Mock Cys GSH

Col-0 Col-0 Col-0

cat2 cat2 cat2

Figure 3.6. Effects of externally supplied cysteine or

glutathione on rosette phenotype and jasmonic

acid-related gene expression in Col-0 and cat2.

Plants were grown at high CO2 for three weeks to

prevent any cat2 phenotype, then transferred to air to

induce oxidative stress in cat2 backgrounds. From the

first day after transfer to air, rosettes were treated once

daily by spraying with water, 1 mM cysteine, or 1 mM

GSH. Photographs show plants eight days after transfer

to air. Bars indicate 1 cm. Transcripts were measured at

the same point after spraying leaves with water, cysteine,

or glutathione. Different letters indicate significant

difference at P < 0.05.

66

CHAPTER 3

0.00

0.04

0.08

0.12

0.0

0.1

0.2

0.00

0.01

0.02

0.0

0.4

0.8

1.2

Mock GSH GSSG

Co

l-0

cat2

cat2

cad

2

Co

l-0

cat2

cad

2

Co

l-0

cat2

cad

2

Tra

nsc

ripta

bund

ance

rela

tive

to A

CT

IN2

LOX3

OPR3

JAZ10

VSP2

a

ababb

ccdd

aa

a a a

b b

a

bc c

c

c

dd

a a

bc b

c

d d

Figure 3.7. Complementation of jasmonic acid-related gene expression in cat2 cad2 by glutathione.

Plants were grown and treated as described for Figure 3.6. GSSG was supplied at 1 mM. Different letters indicate significant

difference at P < 0.05.

67

CHAPTER 3

3.3 Discussion

Glutathione has long been recognized as a key player in oxidative stress metabolism in plants. It can act

as a general reductant, notably in direct metabolism of ROS or to maintain the reduced form of ascorbate

(Foyer and Noctor, 2011). Because this role involves oxidation of GSH to GSSG, and because glutathione

can interact with protein cysteines, several authors have proposed that changes in glutathione status could

be part of oxidative signaling networks (Foyer et al., 1997; May et al., 1998a; Meyer et al., 2007; Noctor

et al., 2012). According to such hypotheses, ROS-triggered changes in glutathione would be important in

activating downstream signaling pathways. However, direct evidence that glutathione plays such a role is

scarce. It was shown in Chapter 2 that genetic modulation of glutathione status antagonizes activation of

the SA pathway by intracellular oxidative stress.. Because of the close interplay between SA and JA

signaling, present study used cat2 cad2 and other genetic tools to examine possible roles for glutathione

in regulating JA signaling.

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

Previous studies of mutants deficient in glutathione point to roles in responses to pathogens and

wounding, although the underlying processes remain to be defined (Ball et al., 2004; Parisy et al., 2007;

Schlaeppi et al., 2008). Data included in one of these studies pointed to slight repression of wounding

genes in the pad2 mutant (Schlaeppi et al., 2008), though this effect was much less clear than those shown

here in Table 3.1 and Figure 3.2. In the light of subsequent annotation of the Arabidopsis genome,

examination of a previous study of cad2 and rax1 based on a more limited analysis of the transcriptome

(about 8500 genes; Ball et al., 2004) also reveals that some JA- or wounding-related genes were among

defence genes affected by glutathione deficiency. It is important to note that growth conditions such as

irradiance or photoperiod could influence the extent to which glutathione affects JA signaling. The effects

observed here were found to be dependent on day length: when the cad2 transcriptome was analyzed in

short day conditions, repression of JA-linked genes was much less evident (data not shown). The data

point to a role for glutathione-dependent processes in regulating basal expression of the JA pathway in the

standard conditions used throughout this study (growth in long day conditions). This is evident both from

the high number of JA genes down-regulated in cad2 (Table 3.1) and the close correlation between JA

marker gene expression and glutathione content (Figure 3.2). Thus, glutathione status, which can be

influenced by several abiotic factors (eg, irradiance, mineral nutrition, heavy metal stress), may be

influential in setting signal strength through the JA pathway in response to biotic stress. Further work is

required to elucidate the mechanisms, although it is intriguing that a glutaredoxin (GRX480) and a

glutathione S-transferase (GSTU6), both of which have been previously implicated in JA signaling, were

among the genes down-regulated in response to partial glutathione deficiency (Table 3.1).

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

Induction of both SA and JA signaling by the cat2 mutation in a time-dependent manner following

transfer from high CO2 to air at moderate irradiance shows that changes in intracellular redox state driven

by enhanced availability of photorespiratory H2O2 can trigger activation of multiple defence hormone

pathways in parallel. The potential relevance of redox-triggered events in cat2 is underscored by our

68

CHAPTER 3

previous reports on metabolite profiles and the effects of introducing mutations for recognized players in

pathogenesis into the cat2 background (Chaouch et al., 2010, 2012). A well characterized response to

catalase deficiency is accumulation of glutathione, mainly as GSSG (Chapter 2). Blocking this response

through the cad2 mutation largely prevents the up-regulation of JA-associated genes that is observed in

cat2 (Figue 3.5), indicating that glutathione-dependent processes link intracellular oxidative stress to

activation of the JA pathway.

Thiol profiling of cat2 cad2 shows that the cad2 mutation produces an effective block in

oxidation-triggered glutathione accumulation at the level of -ECS, the first dedicated enzyme of

glutathione synthesis (Figure 3.4). Thus, under conditions of active photorespiration, the cat2 mutation

drives accumulation of thiols associated with glutathione metabolic pathways, ie, cysteine, -EC,

glutathione, and cys-gly. This suggests that enhanced availability of intracellular H2O2 activates both

cysteine and glutathione synthesis, and that the accumulation of glutathione also activates glutathione

turnover through enzymes such as GGTs. Transcript data suggests that up-regulation of cysteine and

glutathione synthesis in cat2 is closely associated with the chloroplast, while immunolabelling analyses

revealed that the ensuing enrichment of glutathione occurs in most subcellular compartments (Queval et

al., 2009, 2011). Production of cys-gly in cat2 could occur though the action of GGTs located in the

vacuole or apoplast (Grzam et al., 2007; Ohkamu-Ohtsu et al., 2007a,b). Based on marked accumulation

of GSSG in the vacuole (Queval et al., 2011), our working hypothesis is that this compartment could be

an important site of glutathione turnover in cat2.

In cat2 cad2, the block over cat2-triggered glutathione accumulation is reflected in decreased glutathione

is almost all measured subcellular compartments (Table 2.2 in Chapter 2). The present analysis shows that

the secondary cad2 mutation also decreases -EC and cys-gly in cat2 cad2 to cad2 levels, observations

that are consistent with restraint at the level of -ECS and thus limitation of glutathione synthesis and

subsequent turnover. However, it seems that oxidative activation of cysteine synthesis may still occur in

cat2 cad2 because cysteine accumulates to at least two-fold cat2 levels, ie, about five times the level in

Col-0. As discussed in Chapter 2, it seems unlikely that effects of blocking glutathione accumulation

could be ascribed simply to possible changes in oxidative stress intensity because (1) leaf peroxides,

NADP(H), ascorbate, and rosette growth are largely unaffected compared to cat2, and (2) the phenotype

of cat2 cad2 is quite distinct from cat2 gr1 double mutants, in which oxidative stress is clearly

exacerbated compared to cat2. However, the possibility remains that differences in gene expression

between cat2 and cat2 cad2 could reflect accumulated cysteine in the latter, rather than decreased

glutathione. The complementation experiments provide little evidence for this. First, providing additional

cysteine to cat2 (to mimic the high cysteine contents of cat2 cad2) did not decrease JA-associated gene

expression (Figure 3.6). Second, restoration of cat2-triggered JA-associated gene expression by supplying

glutathione to cat2 cad2 (to mimic conditions in cat2; Figure 3.7) suggests that the factors underlying the

differences in JA transcripts between cat2 and cat2 cad2 are associated with glutathione rather than

cysteine. Nevertheless, we cannot definitively rule out some influence of differences in cysteine, or the

intriguing possibility that the cell may possess mechanisms that sense changes in the glutathione

synthesis/metabolism pathways rather than glutathione status per se or glutathione status alone.

69

CHAPTER 3

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

NPR1

NPR1 is by far the best characterized thiol-dependent protein involved in pathogenesis responses. A

recent study indicates that NPR1 can interact with leaf concentrations of both glutathione and ascorbate in

the regulation of ozone-dependent induction of both SA and JA pathways (Brosché and Kangasjärvi,

2012). The NPR1 protein appears to be involved in responses observed in cat2, because previously

documented NPR1-dependent responses are impaired in cat2 npr1, notably decreased induction of

SA-dependent PR genes (Cao et al., 1994) and hyper-accumulation of SA (Figure 2.11 in Chapter 2), an

effect that is known to occur in the single npr1 mutant challenged with certain pathogens (eg, Rate et al.,

1999). However, as described in the previous chapter, comparisons of effects of secondary npr1 and cad2

mutations on cat2-triggered SA signaling suggest that glutathione-dependent processes play regulatory

roles additional to NPR1 in regulating SA accumulation (Chapter 2). This is consistent with the decreased

SA accumulation in cat2 cad2, an effect that is opposite to the very marked accumulation of SA in cat2

npr1.

In conditions where both SA and JA signaling are induced by oxidative stress, as in cat2, the decreased

accumulation of SA produced by the cad2 mutation should mitigate SA-dependent restraint of JA

signaling. Similarly, if the cad2 mutation negatively affects activation of NPR1, the intensity of this

restraint would also be predicted to decrease. Based on these notions, any effects of the cad2 mutation on

cat2-triggered induction of JA genes would be expected to be positive. In fact, the opposite is observed

when glutathione accumulation is blocked: the cad2 mutation down-regulates JA-associated gene

expression at the same time as down-regulating the SA pathway. Indeed, the contrast between effects of

cad2 and npr1 mutations on cat2 responses is striking (Figure 3.5). Disabling NPR1 function had no

effect on two JA synthesis genes while allowing markedly increased expression of JAZ10 and VSP2. That

all four genes were down-regulated in cat2 cad2 relative to cat2 and, even more so (for JAZ10 and VSP2)

relative to cat2 npr1, is not consistent with an action of glutathione at the level of NPR1. This does not

rule out roles for glutathione in mediating repression of JA signaling through this previously described

mechanism, but it does imply the existence of other glutathione-dependent processes in the regulation of

the JA pathway.

Previous studies have drawn attention to roles of GRX480 in mediating certain aspects of the interaction

between SA and JA pathways (Ndamukong et al., 2007). GRX480 encodes a member of the largest

sub-class of plant glutaredoxins, proteins that are likely important in linking glutathione and protein

cysteine status (Rouhier et al., 2008). This class of glutaredoxins is specific to plants (Lemaire, 2004).

Their biochemical activities remain to be characterized, although at least some members are known to

interact with transcription factors (Ndamukong et al., 2007; Wang et al., 2009c). GRX480 is inducible by

SA and its overexpression represses the JA marker gene, PDF1.2, although knockout mutants do not

show altered PDF1.2 expression, possibly because of gene redundancy (Ndamukong et al., 2007). While

GRX480 and potentially redundant similar genes could play some role in the effects we describe here,

other factors are probably at work because (1) cat2 cad2 shows repression of SA signaling as well as JA

signaling; (2) our observations suggest a generalized effect of glutathione on JA-associated genes, not just

PDF1.2; and (3) this effect appears to be apparent even in basal, non-induced conditions (Table 3.1 and

70

CHAPTER 3

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

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

Based on the above discussion, it is possible that novel glutathione-dependent components involved in JA

signaling exist (Figure 3.8). Two possibilities could explain the dual effect of glutathione on signaling

through the SA and JA pathways in response to oxidative stress. First, it is possible that glutathione status

acts to modulate a master switch that governs the oxidative activation of both pathways (Figure 3.8,

dotted line). A second scenario would involve regulation of both pathways in parallel (Figure 3.8, dashed

lines). Our analysis of the SA pathway implicates glutathione in regulating SA accumulation at the level

of ICS1 expression (Chapter 2; Figure 3.8). Whatever the glutathione-dependent process(es) involved in

the initial activation of the JA pathway, interplay between the SA and JA pathways mediated by other

redox-linked factors such as thiol-regulation of NPR1 or induction of GRX480 could act downstream to

determine the relative signaling strength of these two key defence hormones.

H2O2

Oxidation and induction of glutathione

GSH/GSSG

ICS1

JA-dependent gene expression accu

SA mulation

NPR1

ICS1?

?

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

expression.

Oxidation by H2O2 modulates glutathione status. This oxidative modulation is part of the signal network required for optimal

ICS1-dependent SA accumulation that then leads to activation of NPR1 function through reductive processes (Mou et al., 2003), to

which glutathione may contribute. Through a pathway that is parallel to SA activation (dashed lines) or simultaneously induced by a

master switch (dotted lines), glutathione modulation also links H2O2 to induction of JA signaling. The impact of glutathione on

JA-related gene expression, which is also apparent in non-induced (basal) conditions, operates alongside restraint of JA signaling by

the SA pathway, eg, through reductive activation of NPR1 (Koornneef et al., 2008).

71

CHAPTER 3

72

It is possible that the regulatory influence of glutathione is mediated by direct control of protein

thiol-disulfide status through redox potential (Meyer et al., 2007). This notion receives some support from

the observation that plants lacking expression of GR1 also show repression of the JA pathway (Mhamdi et

al., 2010a), albeit less strongly than in cad2. Both gr1 and cad2 should have an increased glutathione

redox potential compared to Col-0, ie, glutathione should be less reducing (Meyer et al., 2007; Marty et

al., 2009). In cat2 gr1 double mutants, JA-associated genes are strongly repressed, as in cat2 cad2

(Mhamdi et al., 2010a). Although the interpretation of effects in cat2 gr1 is complicated by a much more

extreme phenotype than in cat2 cad2, it is possible that repression of the JA pathway reflects mechanisms

that are common to the two double mutants. In cat2 gr1, the absence of GR1 function and the consequent,

marked accumulation of GSSG are expected to increase the glutathione redox potential relative to cat2. It

is more difficult to predict differences in glutathione redox potential between cat2 cad2 and cat2 because

the double mutant has a more reduced leaf glutathione pool than cat2 (favouring a lower redox potential)

but the total glutathione pool is lower (favouring a higher redox potential). Direct measurements using

redox-sensitive GFP could be informative here, though such technologies are currently difficult to apply

to photosynthetic leaf mesophyll cells, in which cat2 effects are expected to predominate.

As well as effects on signaling proteins that could be mediated by glutathione redox potential or

S-glutathionylation reactions, other possible mechanisms are related to glutathione S-transferase activities.

Conjugation reactions have been implicated in JA metabolism (Ohkamu-Ohtsu et al., 2011). Because of

the large number of genes encoding proteins such as GRXs and GSTs, and possible gene redundancy,

further work is required to elucidate the mechanistic details of what seem to be multiple roles of

glutathione in modulating defence hormone-linked responses. Understanding these mechanisms may be

relevant to understanding and optimizing the biotic stress responses of plants growing in a variable

environment that can lead to marked changes in tissue glutathione status.

CHAPTER 4

Analysis of autophagy mutants: interactions with photorespiration and

oxidative stress

CHAPTER 4

Summary

Autophagy involves degradation of cytoplasmic materials and may be important in plants during nutrient

starvation, senescence, pathogenesis responses or under oxidative stress conditions. While much remains

to be elucidated, over 30 genes have been implicated in autophagic processes in plants. Autophagy has

been considered to play a positive role in nutrient recycling under conditions of carbon or nitrogen

limitation, but an early senescence phenotype is still observed in atg mutants under optimal conditions.

The aim of this study was to use previously identified atg mutants to analyze possible interactions

between autophagy, photorespiration and oxidative stress using the cat2 and cad2 lines described in

previous chapters. Growth of atg2, atg5 and atg18a mutants at high CO2 showed that the

accelerated-senescence of these lines was enhanced compared to growth in air. Data on metabolic

profiling and glutathione indicated that this effect was related to intracellular oxidative stress. However,

introducing the cat2 mutation into atg mutants delayed the atg-triggered senescence, suggesting that other

ROS-generating pathways contribute to this process. On the other hand, induced pathogen resistance in

cat2 seems to be compromised by atg2 and atg18a mutations, while SA-dependent lesion formation was

not different in cat2 compared to any of the three double cat2 atg mutants. Additionally, preliminary data

are presented on a defect in the development of young leaves in high CO2 in atg2 cad2 double mutants

with decreased glutathione. Taken together, these results may indicate that oxidative stress originating

from excess peroxisomal H2O2 can act antagonistically to the programs induced in atg mutants and that

oxidative stress in these mutants is caused by other cellular sources of ROS.

Résumé

L’autophagie a été impliquée dans la dégradation de composantes cytoplasmiques. Ce processus pourrait

jouer des rôles importants chez les plantes dans des conditions comme une carence nutritive, la

sénescence, les réponses aux pathogènes, ou encore les stress oyxdants. Alors que nos connaissances

restent limitées, plus de 30 gènes ont été impliqués dans les processus d’autophagie chez les plantes.

L’autophagie jouerait un rôle positif lors du recyclage nutritif dans des conditions de limitation en

carbone ou en azote. Cependant, un phénotype de sénescence précoce est observé dans des mutants atg

cultivés dans des conditions optimales. Cette étude avait pour but d’utiliser des mutants atg déjà identifiés

afin d’analyser des interactions éventuelles entre autophagie, photorespiration et stress oxydant grâce aux

lignées cat2 et cad2 décrites dans les chapitres précédents. La culture de mutants atg2, atg5 et atg18a à

fort CO2 a montré que leurs phénotypes de sénescence accélérée sont plus marqués par rapport à ceux

observés lors de la culture dans l’air. Des profilages métaboliques et des données sur le glutathion

indiquent que ces effets sont associés à une condition de stress oxydant. Cependant, la mutation cat2

retarde la sénescence atg-dépendante, suggérant que d’autres voies de production de ROS contribuent à

ce processus. En revanche, la résistance aux pathogènes induite chez cat2 semble être compromise par les

mutations atg2 et atg18a, alors que la formation de lésions SA-dépendantes est essentiellement similaire

chez cat2 et les trois doubles mutants cat2 atg. De plus, des données préliminaires içi présentées

indiquent un défaut de développement de jeunes feuilles dans les doubles mutants atg2 cad2 cultivées à

fort CO2. Dans leur ensemble, ces résultats indiqueraient qu’un stress oxydant déclenché par un excès de

H2O2 peroxysomal pourrait agir d’une manière antagoniste aux programmes induits dans les mutants atg

et que le stress oxydant dans ces mutants serait causé par des ROS provenant d’autres sources cellulaires.

73

CHAPTER 4

4.1 Introduction Autophagy is an intracelluar macromolecule degradation process that delivers unwanted cell materials

into the vacuole/lysosome for recycling of needed nutrients (Liu and Bassham, 2012; Pérez-Pérez et al.,

2012). Upon the induction of autophagy, cytoplasmic constituents and organelles are engulfed into

double-membraned autophagosomes, which deliver this cargo into the vacuole for further degradation.

Although there have been reports of autophagy-like processes in plants since the early 1960s, the

molecular mechanism is best described via mutagenesis studies in yeast (Tsukada and Ohsumi, 1993;

Thumm et al., 1994; Barth et al., 2001). However, most of the autophagy-related (ATG) genes are well

conserved in plants (see Chapter 1), suggesting that the core molecular basis of autophagy is similar

between plants and yeast. For instance, the ATG1/13 kinase complex initiates autophagosome formation

in response to nutrient demands sensed by an assemblage of upstream regulators in yeast. Under

nutrient-rich conditions, activation of the Target Of Rapamycin (TOR) complex represses the

ATG1/ATG13 kinase by the hyperphosphorylation of the ATG13 subunit which in turn prevents complex

assembly (Kamada et al., 2010). During starvation, TOR is inhibited and, consequently, ATG13 is no

longer phosphorylated. ATG1 can associate with the ATG13 subcomplex, subsequently leading to

autophagy induction through several steps (Nakatogawa et al., 2009; Kamada et al., 2010). It has been

shown that the ATG1/ATG13 kinase complex in plants plays a key role in inducing autophagy under

nutrient-limiting conditons, as in yeast (Suttangkakul et al., 2011). Furthermore, the defect in autophagy

was also observed in many Arabidopsis atg knockout lines (Bassham et al., 2006).

Over the past decade, our physiological understanding of autophagy and its molecular mechanisms in

plants has greatly increased. While most attention has focused on the functions of autophagy in nutrient

starvation in plants (Doelling et al., 2002; Hanaoka et al., 2002; Thompson et al., 2005; Xiong et al., 2005;

Phillips et al., 2008), Arabidopsis atg mutants are also hypersensitive to salt and drought stresses (Liu et

al., 2009). Moreover, oxidative stress triggered by exogenous H2O2 or methyl viologen (MV) treatment

can induce autophagy and ATG18a RNAi lines are more sensitive to oxidative stress, a sensitivity that is

accompanied by enhanced accumulation of oxidized proteins relative to wild-type (Xiong et al., 2007).

Based on this study, plant autophagy has been proposed to play an important role in the degradation of

oxidized proteins as well as nutrient recycling under stress conditions (Xiong et al., 2007). Although

autophagy has been suggested to be involved in resistance to oxidative stress in plants (Xiong et al., 2007;

Liu et al., 2009), the precise function of autophagy and whether plant cells also use autophagy as a

defence against oxidative stress triggered by specific source of ROS in different organelles remains

unclear. Besides the impact of autophagy in abiotic stresses, accumulating evidence points to a key role of

autophagy in the regulation of plant immune responses (Liu et al., 2005; Hofius et al., 2009; Hayward and

Dinesh-Kumar, 2011). For instance, atg5 or atg18a mutations not only result in an increased resistance to

the virulent bacterium to P. syringae DC3000, but also increase susceptibility to infection with the fungus

Alternaria brassicicola (Lenz et al., 2011b). This suggests that autophagy could play differential roles in

responses to invasion of biotropic and necrotrophic pathogens.

Besides studies of the physiological function of autophagy in stress conditions, autophagy could also play

a housekeeping role to remove unwanted cellular compounds generated by basal metabolism or to

remobilize nutrients, even under optimal growth conditions. For example, almost all Arabidopsis

74

CHAPTER 4

autophagy mutants are able to grow from seed to seed except for ATG6 T-DNA lines (which are lethal)

but many of them such as atg2, atg5, and atg10 display slower growth phenotype under nutrient-rich

conditions (Liu and Bassham, 2012; Fujiki et al., 2007; Harrison-Lowe and Olsen, 2008). Several atg

mutants such as atg2 and atg5 have an early senescence phenotype even under favorable conditions, and

this is linked to the NPR1-dependent SA signaling pathway (Yoshimoto et al., 2009). As for the

mechanism of how autophagy negatively regulates premature senescence, one possibility is that

autophagy may be selectively capable of removing specific ROS-generating organelles such as

mitochondrion and peroxisomes in plants as observed in yeast (Sakai et al., 2006; Kanki et al., 2009;

Pérez-Pérez et al., 2012). However, the underlying mechanism of organelle-specific autophagy remains to

be identified in plants. It is also possible that the induction of oxidative stress and related senescence

could be caused by failure to recycle oxidized proteins and lipids, leading to the accumulation of damaged

and toxic compounds.

While ROS have been implicated in senescence, the relationships between plant senescence and oxidative

stress are not yet clear. Indeed, leaf senescence has been particularly associated with enhanced H2O2

availability (Smykowski et al., 2010; Wu et al., 2012), which could be a signal involved in the regulation

of senescence. As discussed in Chapter 1, plants have developed numerous antioxidant systems to

metabolize H2O2 (Mhamdi et al., 2010b), and changes in the potency of some of these systems may be

influential in the regulation of senescence. While there are three CAT genes in Arabidopsis, CAT2 is

highly expressed in leaf peroxisomes and is the major form of the enzyme in photosynthetic tissues

(Queval et al., 2007). It has been shown that leaf catalase activity can be decreased by a variety of stresses

including salt, heat, cold and biotic stress (Volk and Feierabend, 1989; Hertwig et al., 1992; Dorey et al.,

1998). Recently, it has been suggested that decreased CAT2 expression and activity is linked to the onset

of leaf senescence, via regulation of the intracellular H2O2 content (Smykowski et al., 2010). Precocious

senescence and a higher level of H2O2 are no longer observed in a gbf1 mutant that was reported to lack

senescence-associated down-regulation of CAT2.

In the second half of my thesis studies, I undertook to begin an analysis of interactions between

peroxisomal-triggered oxidative stress and autophagy. For this, three published autophagy mutants were

selected (atg2, atg5, and atg18a) and crossed with cat2 which, as we have seen in Chapter 2 and 3, is a

useful system to study the importance of photorespiratory H2O2 signaling in plant function (Mhamdi et al.,

2012). To analyze interactions between photorespiration and autophagy, I also examined whether CO2

level affects responses in the three atg mutants. In addition, because the cat2 mutant activates

SA-dependent signaling (Chaouch et al., 2010; Chapter 2), and some of the atg mutants have also been

implicated in regulating this pathway, I wanted to analyze whether cat2-dependent lesion formation was

affected by secondary atg mutations. The phenotypic analysis of interactions between cat2 and atg was

performed in several growth conditions, because the cat2 mutant is highly conditional. When grown in air

in long days (as for the studies reported in Chapters 2 and 3), the cat2 mutation triggers SA-dependent

responses and lesion formation. These responses are absent when cat2 is grown in short days, even

though oxidative stress is still apparent. This property allows cat2 to be used as a system to analyze

effects of oxidative stress in the presence or absence of induction of the SA pathway (Figure 4.1).

Because the oxidative stress observed in cat2 in air is absent when photorespiration is repressed by

growth at high CO2, the effects of induced oxidative stress can be studied by transferring mutants grown

75

CHAPTER 4

initially at high CO2 to air, which also allows induction of the SA pathway specifically after transfer to air

in long days but without the decreased growth in cat2 grown in air from germination (Figure 4.1). Finally,

because of the roles of glutathione reported in the two previous chapters, I introduced the cad2 mutation

into atg2 (the mutant that shows the most striking phenotype of the three atg selected for study). These

double mutants were obtained in the final year of the thesis and their functional analysis is as yet

preliminary. This chapter reports some of the results already obtained through phenotyping and metabolite

profiling.

Long days, airHigh CO2Short days, air

No oxidative stressWild-type phenotype

Oxidative stressDecreased growtSA accumulation

Lesions

Oxidative stressDecreased growth

No lesions

h

Induced oxidative stress

WITHOUT decreased growth

(no lesions)

Induced oxidativstress

SA accumulationLesions

WITHOUT decreased growth

e

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

and on growth daylength.

76

CHAPTER 4

4.2 Results T-DNA mutants for atg2, atg5, and atg18a obtained from seed banks were genotyped to identify plants

homozygous for the mutations (Figure 4.2A). Homozygotes were crossed with cat2 and double

homozygotes identified in the F2 generation (Figure 4.2A). The atg2 and atg5 mutations cause knockout

of the expression of the corresponding genes (Inoue et al., 2006; Yoshimoto et al., 2009; Lenz et al.,

2011b). RT-PCR analysis of atg18a showed that transcripts of ATG18a were undetectable in this line

(Figure 4.2B).

1 2 1 2 1 2

Col‐0

atg2

cat2 atg2

1 2 1 2 1 2

Col‐0

atg5

cat2 atg5

1 2

Col‐0

cat2 atg2

1 2 1 2 1 2 1 2

cat2 atg5

cat2

cat2 atg18a

1 2 1 2 1 2

Col‐0

atg18a

cat2 atg18a

A

ATG18a

ACTIN2

Col‐0

Col‐0

atg18a

Col‐0

atg18a

atg18a

RT‐PCRB

Figure 4.2. Genotyping and expression analysis of atg

mutants.

(A) PCR analysis of homozygous single atg2, atg5, and

atg18a T-DNA mutants and after crossing into cat2 to

produce double mutants. For each gene, lane 1 indicates

primer combinations to amplify the wild-type gene and

lane 2 primer combinations to amplify gene-specific

fragments using a T-DNA-specific primer.

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

transcript in atg18a.

Primer combinations are given in Table 6.2 (Chapter 6)

77

CHAPTER 4

4.2.1 Effects of high CO2 on atg phenotypes and metabolite profiles

As a first step to analyzing interacttions between photorespiration and autophagy, single atg mutants were

grown in short days (8h light/16h dark), either at high CO2 (3000 µL.L-1) or in air. Short day conditions

were chosen because this greatly retards flowering in Arabidopsis, enabling effects on leaf senescence to

be studied without this complicating process. In agreement with previous reports (Xiong et al., 2005;

Yoshimoto et al., 2009), phenotypic analysis revealed that all three atg mutants showed a phenotype of

accelerated senescence on older leaves when they were grown in air. This phenotype was first observed

after about 6 weeks growth in air (Figure 4.3A), a stage at which no senescence was observed in Col-0.

However, appearance of senescent leaves occurred significantly earlier in atg mutants grown in high CO2

such that this phenotype was already apparent after 5 weeks growth in this condition (Figure 4.3A and

4.3B). Similar to growth in air, no phenotypic symptoms of senescence were observed in Col-0 at high

CO2. The early senescence phenotype was most evident for atg2, and was correlated with decreased

rosette size (Figure 4.3C).

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

(A) Development of senescent yellowing in mutants grown in high CO2 (black circles) or in air (white circles) for six weeks in short

days. *Significant difference at P<0.05 (n = 6-10 different plants of each line).

(B) Photographs of plants after five weeks growth in the two conditions.

(C) Rosette diameter (left) and fresh mass (right) after five weeks growth in the two conditions. *Significant difference at P<0.05 (n

= 12 or more different plants of each line).

0

4

8

12

Col-0 atg2 atg5 atg18a

Col-0 atg2

Hig

h C

O2

Air

atg5 atg18a

0

300

600

900

Fre

sh w

eigh

t (m

g)

Ros

ette

dia

met

er(c

m)

* * * * *

** * *

A

C

B

0

10

20

30

0

10

20

30

0

10

20

30

Sen

esce

nt a

reas

(% r

oset

te a

rea)

Weeks after sowing3 4 5 63 4 5 6 3 4 5 63 4 5 6 3 4 5 63 4 5 6

Weeks after sowing Weeks after sowi

atg2 atg5 atg18a*

*

*

*

*

*

**

Col

-0at

g2

atg

5at

g18a

ng

High CO2 Air

Col

-0at

g2

atg5

atg1

8a

Col

-0at

g2

atg

5at

g18a

Col

-0at

g2

atg

5at

g18a

High CO2 Air

Col

-0at

g2

atg5

atg1

8a

Col

-0at

g2

atg5

atg1

8a

Col

-0at

g2

atg5

atg1

8a

High CO2 Air

Col

-0at

g2

atg5

atg1

8a

Col

-0at

g2

atg5

atg1

8a

Col

-0at

g2

atg5

atg1

8a

High CO2 Air

Col

-0at

g2

atg5

atg1

8a

Col

-0at

g2

atg5

atg1

8a

78

CHAPTER 4

To gain further insight into the underlying metabolic features of this phenotype, non-targeted metabolite

profiling was performed using GC-TOF-MS (Figure 4.4). Hierchical clustering of all detected compounds

showed that different metabolic patterns were obtained in high CO2 and air for the wild-type, Col-0

(Figure 4.4A). Whereas the atg mutations did not radically alter profiles in air, atg2 and atg18a strongly

impacted on the metabolite profiles observed at high CO2. A large sub-cluster of compounds showed

higher contents in these two mutants specifically at high CO2 (Figure 4.4A, bottom half of heatmap).

Metabolites that were significantly different from Col-0 were identified (Figure 4.4B). In agreement with

the phenotype (Figure 4.3), the highest number of significantly different metabolites was detected in atg2

and then atg18a (Figure 4.4B). A clear interaction with CO2 was observed, because the number of

signficant metabolites was lower in air (Figure 4.4B). This pattern was exemplified by isoleucine (Figure

4.4C), which was one of many amino acids that were increased in abundance in atg2 in high CO2 but less

so in air. Comparison with metabolite profiles for cat2 (Chapter 2) indicated substantial overlap with the

profile observed in atg2. Of 26 compounds that were significantly different between Col-0 and atg2,

almost two-thirds also showed the same significant difference between cat2 and Col-0 (Figure 4.4D).

These metabolites included gluconic acid, which is the most markedly accumulatedcompound in cat2

(Chaouch et al., 2012). Indeed, of the 17 common metabolites that were induced in cat2 and atg2 (Table

4.1), seven were among the most strongly accumulated compounds in response to both oxidative stress

and bacterial challenge (Chaouch et al., 2012).

-2.0 +2.5

Col

-0at

g2

atg5

atg1

8a

Col

-0at

g2

atg5

atg1

8a

High CO2 AirA

Figure 4.4. Metabolite profiling of Col-0 and atg mutants grown at high CO2 and air. (A) Heatmap display of all data after

center-reduction. (B) Significantly increased (red) or decreased (green) metabolites in each mutant grown in air or at high CO2 after

t tests against Col-0 in the same condition (P<0.05). (C) Graph to illustrate typical high CO2-specific response in atg2. The position

of the selected metabolite on the heatmap is indicated in (A). (D) Overlap between atg2 metabolite profile and oxidative

stress-triggered profiles described for the catalase-deficient mutant, cat2 (Chapter 2). Note that the analysis only considers

metabolites that were detected in both series of experiments.

2,4

2‐

Al

Am

Ar

b‐

b‐

Cit

Et

Fr

GA

Ga

Ga

Ga

Gl

Gl

Ho

Iso

Ly

Ma

M

O‐

Ph

Ph

Pu

Ra

Rh

Ri

Se

Sh

Su

Te

Th

Th

Ty

Ty

Ur

Va

Xy

‐OHbutanoic acid

hydroxyglutaric acid

anine

inoadipic acid

abinose

alanine

sitosterol

ramalic acid

hanolamine

uctose

BA

lactono‐1,4‐lactone

lactinol

lactose

uconic acid

ycerate

moserine

leucine

sine

late

annose

acetylserine

enylalanine

osphate

trescine

ffinose

amnose

bose

rine

ikimic acid

crose

tradecanoic acid

reonine

reonic acid

rosine

ramine

acil

line

lose

High CO2 Air

2 5 18a2 5 18a 2 5 18a2 5 18a

B

C

Metabolite

0.000

0.002

0.004

0.006

*

Rel

ativ

e p

ea

kin

tens

ity

Co

l-0

atg

2

Col

-0

atg2

CO2 Air

0.000

0.002

0.004

0.006

*

Rel

ativ

e p

ea

kin

tens

ity

Co

l-0

atg

2

Col

-0

atg2

CO2 Air

Isoleucine

Isoleucine Datg2 c

179

at2

32

79

CHAPTER 4

Table 4.1. Overlapping metabolites induced by atg2 and cat2 mutations. Metabolites were identified by t test at P<0.05 (n = 3

biologically independent samples) and numbers give mean fold-change relative to Col-0 sampled in the same conditions. *Among

metabolites that most strongly accumulate in response to both oxidative stress and bacterial challenge (Chaouch et al., 2012).

atg2/Col-0 cat2/Col-0

Gluconic acid * 12.2 159.9

Threonic acid 4.2 2.8

Isoleucine * 4.2 2.6

2-Hydroxyglutaric acid * 3.5 8.8

2,4-Dihydroxybutanoic acid 3.5 4.2

Ribose * 3.1 3.6

Phenylalanine * 2.6 2.0

Malic acid 2.4 3.1

Valine 2.3 2.4

Shikimic acid 2.1 1.6

Putrescine * 2.0 2.8

Lysine 1.9 1.8

Rhamnose 1.8 2.3

β-alanine 1.8 1.8

Glyceric acid 1.4 2.0

Serine 1.3 3.5

Threonine * 1.3 2.3

4.2.2 Impact of oxidative stress on atg-triggered early senescence

To analyze possible effects of oxidative stress on the atg phenotype, all three mutants were crossed with

cat2. First, as for the experiments described above, the double mutants were analyzed in short-day

conditions. Under this growth regime, cat2 shows oxidative stress in air but not at high CO2, and

oxidative stress that occurs in air is not associated with activation of the SA pathway and SA-dependent

responses (Figure 4.1; Queval et al., 2007; Chaouch et al., 2010). No obvious effect of the cat2 mutation

on the early senescence phenotype was observed when the plants were grown at high CO2. This is

unsurprising because the cat2 mutant is phenotypically silent in this condition. When plants were grown

to an age of six weeks in air, at which point early senescence is apparent in atg single mutants (Figure

4.3), no senescence was observed in cat2 atg double mutants (Figure 4.5B). However, this observation is

somewhat difficult to interpret because of the general effects of the cat2 mutation on rosette growth when

plants are grown in air from seed (Figure 4.1). Single atg2 and double cat2 atg2 mutants were therefore

grown for four weeks in high CO2, at which point early senescence is initiated in atg2 (Figure 4.3), and

then transferred to air to induce oxidative stress in cat2 genotypes and grown for a further week. This

analysis revealed that whereas the atg2 single mutant develops obvious senescence on the outer leaves,

this phenotype was less apparent in cat2 atg2 (Figure 4.5C).

80

CHAPTER 4


cat2 cat2 atg2 cat2 atg5 cat2 atg18a

Col-0


atg2 atg5 atg18a

Col-0

cat2 cat2 atg2

atg2

0

10

20

Co

l-0

cat2

atg2

cat2

atg

2

Sen

esce

nt a

reas

(% r

oset

te a

rea)

ND ND

*

A

C

B

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

high CO2 and air.

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

(B) Growth in air for six weeks (oxidative stress in cat2)

(C) Growth in high CO2 for four weeks then in air for one week (one week of oxidative stress in cat2). *Significant difference

between atg2 and cat2 atg2 at P<0.05 (n = 8 different plants).

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CHAPTER 4

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

Previous studies of cat2 have established that the lesion formation is SA-dependent (Chapter 2) and does

not occur in short day growth conditions (Figure 4.1; Queval et al., 2007). Introduction of atg mutations

into cat2 does not promote lesions in short day conditions (Figure 4.5). We therefore compared the impact

of atg mutations on cat2 phenotypes in long days, conditions that are permissive for cat2-triggered SA

accumulation and spreading SA-dependent lesions. This analysis revealed little effect of any of the atg

mutations on lesion formation in cat2, whether lesions were induced during growth from seed in air

(Figure 4.6A) or following transfer to air after initial growth at high CO2 (Figure 4.6B). Because the cat2

mutation induces enhanced resistance to bacteria, the influence of the atg mutations on this response was

tested. While the phenotypes of atg5 and atg18a were associated with a similar level of induced

resistance to that observed in cat2, the atg2 and atg18a mutations both annulled the cat2-triggered

resistance (Figure 4.7).

0

10

20

30



A



B

0

10

20

30

cat2

cat2

atg

2

cat2

atg

5

cat2

atg

18a

cat2

cat2

atg

2

cat2

atg

5

cat2

atg

18a

Lesions(% rosette area)

Lesions(% rosette area)

Figure 4.6. Effect of secondary atg mutations on cat2-triggered formation of salicylic acid-dependent lesions.

(A) Growth for three weeks in air in long days to produce constitutive induction of pathogenesis responses in cat2

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

82

CHAPTER 4

3

4

5

6

7

3

4

5

6

7B

acte

rialg

row

th(lo

g 10c

fucm

-2) (l og

10cfu

cm-2)

Co

l-0

atg

2

atg

5

atg

18a

cat2

cat

2 a

tg2

cat

2 at

g5

cat2

atg

18a

Co

l-0

atg

2

atg

5

atg

18a

cat

2

cat

2 at

g2

cat

2 at

g5

cat2

atg

18a

0 dpi 2 dpi

a a a a a a a a

a a a ab b b b

Bacte

rialgrow

th

Figure 4.7. Effect of secondary atg mutations on Pst resistance induced by the cat2 mutation.

Plants were grown as for Figure 4.6B to induce salicylic acid accumulation and Pst resistance in cat2. Different letters indicate

significant difference at P<0.05.

4.2.4 Interactions between atg mutations and glutathione

0.0

0.2

0.4

0.6

0.8

0

1

2

3

4

** *

*

Col

-0

atg

2

atg

5

atg1

8a

Col

-0

atg

2

atg5

atg1

8a

Col

-0a

tg2

atg5

atg1

8a

Col

-0

atg2

atg

5at

g18a

High CO2 High CO2Air Air

Leaf

glu

tath

ione

(µ

mo

l.g-1

FW

)

Leaf

asc

orb

ate

(µm

ol.g

-1F

W)

A B

Assay of ascorbate and glutathione as redox markers showed that none of the atg mutations affected

ascorbate contents but that glutathione was increased slightly in all mutants grown at high CO2 and also in

atg2 grown in air. Thus, there seems to be an overall correlation between early senescence phenotypes

(Figure 4.3) and leaf glutathione content (Figure 4.8). To test the possible influence of glutathione, the

cad2 mutation was crossed into atg2 (Figure 4.9). As in the cat2 background (Chapters 2 and 3), the cad2

mutation effectively decreased glutathione in cat2 atg2 (Figure 4.9C) but did not greatly affect rosette

mass (Figure 4.9B). The cad2 mutation also seemed to have little effect on the early senescence

phenotype observed in atg2 (Figure 4.9A). However, interpreting this result is complicated because the

interaction between atg2 and cad2 seemed to produce a defect in leaf development, which was observed

in the double mutant grown at high CO2 but not in air (Figure 4.9A).

Figure 4.8. Ascorbate and glutathione in atg mutants grown in high CO2 or air.

(A) Ascorbate. White bars, reduced form. Black bars, dehydroascorbate.

(B) Glutathione. White bars, GSH. Black bars, GSSG.

*Significant difference at P<0.05 (n = 3 biological repeats).

83

CHAPTER 4

0

500

1000

Col-0 cad2 atg2 atg2 cad2F

resh

we

igh

t(m

g)

Col-0 cad2 atg2 atg2 cad2

Hig

h C

O2

Air

Col

-0

atg2

atg2

cad

2

High CO2 Air

**

* *

0.0

0.4

0.8Le

af g

luta

thio

ne

(µm

ol.g

-1F

W)

High CO2 Air

cad2

Col

-0

atg2

atg2

cad

2

cad2

Col

-0

atg2

atg2

cad

2

cad2

Col

-0

atg2

atg2

cad

2

cad2

A

B C

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

(A) Phenotypes of plants grown for five weeks in short days at high CO2 or in air.

(B) Fresh weight after five weeks growth. *Significant difference at P<0.05 (n = 10 different plants).

(C) Leaf glutathione. White bars, GSH. Black bars, GSSG.

4.3 Discussion

Cytoplasmic components are recycled and remobilized by autophagy process (Guiboileau et al., 2012).

It is therefore clear that autophagy functions in nutrient remobilization during senescence. If autophagy is

the only intracellular process for degradation in senescing leaves, a delayed senescence phenotype should

be predicted when the autophagic process is defective. Intriguingly, however, loss of ATG functions

causes an early senescence phenotype, indicating that activation of autophagy-independent pathway(s)

may function in recycling cytoplasmic materials from old leaves to remobilize nutrients when autophagic

process is impaired. Indeed, many amino acid and sugar related compounds are significantly increased in

atg2 and atg18a mutants relative to Col-0, and the analyses reported here show that this effect, together

with the early senescence phenotype, is much stronger when plant are grown at high CO2.

It has been reported that autophagy can confer tolerance against oxidative stress in plants such as salt and

drought, suggesting that autophagy plays some role in the regulation of ROS metabolism (Liu et al., 2009;

Shin et al., 2009; Péréz-Péréz et al., 2012). Indeed, after treatment with methyl viologen, which produces

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CHAPTER 4

ROS in chloroplasts or mitochondria in plants, ATG18a RNAi lines display a more chlorotic phenotype

relative to Col-0 (Xiong et al., 2007). In addition, atg2 and atg5 mutants were reported to suffer oxidative

stress, even in the absence of external stress (Yoshimoto et al., 2009). Inversely, exogenous H2O2 and MV

could rapidly induce autophagy (Xiong et al., 2007). Moreover, tolerance of oxidative stress is tightly

related with completion of life cycle. Several aspects of the present results suggest that the early

senescence occurring in atg2 in high CO2 may be associated with intracellular oxidative stress. First, most

of the significantly accumulating metabolites identified in atg2 were also observed to respond similarly in

cat2, which triggers intracellular oxidative stress through the photorespiratory pathway (Figure 4.4D).

Second, glutathione accumulation was observed in atg mutants in high CO2 (Figure 4.8). These

observations indicate that the atg2 mutation is indeed associated with intracellular oxidative stress but

that this is unlikely to be linked to enhanced availability of photorespiratory H2O2 generated in the

peroxisomes, because both the phenotypic and metabolomics responses were stronger at high CO2. Based

on this logic, we propose that other ROS-generating systems are associated with intracellular oxidative

stress in atg2. While it has been proposed that down-regulation of CAT2, leading to enhanced availability

of ROS, is a contributor to leaf senescence (Zimmermann et al., 2004; Smykowski et al., 2010), other

ROS-producing compartments such as chloroplasts and mitochondria contribute to the intracellular

oxidative stress signaling network. Furthermore, transcriptomic profiles of cat2 provide relatively little

evidence that senescence programs are activated by loss of catalase function (Queval et al., 2007;

Mhamdi et al., 2010b). Indeed, the cat2 mutation did not accelerate early senescence phenotypes of atg

mutants; in fact, in atg2 transferred from high CO2 to air, loss of CAT2 function seems to oppose the

spread of senescence. This observation could point to negative interactions between photorespiratory

H2O2 and senescence, and maybe suggests that ROS of different origins have differential effects on

autophagy-regulated senescence.

Autophagy has been proposed to be involved in the regulation of pathogenesis responses as well as

SA-dependent senescence (Hofius et al., 2009; Yoshimoto et al., 2009; Lenz et al., 2011b; Wang et al.,

2011). Both atg5 and atg18a mutants show increased resistance to virulent bacterial PstDC3000 and

increased SA during this bacterial challenge (Lenz et al., 2011b). As in cat2 (Chaouch et al., 2010), the

atg5 mutation was also able to trigger some SA accumulation in the absence of pathogen challenge

(Yoshimoto et al., 2009). However, phenotypic observation revealed little effect of atg mutations on cat2

triggered SA-dependent lesion formation (Figure 4.6). In fact, the atg2 and atg18a mutations

compromised cat2-triggered induced resistance to PstDC3000 when plants were inoculated at 7 days after

transfer from high CO2 to air (Figure 4.7). One possibility is that the potential activation of senescence

programs in atg2 and atg18a after 3 weeks growth at high CO2 has an antagonistic effect on

cat2-triggered enhanced pathogen resistance. Further experiments to determine pathogen resistance and

SA levels using different inoculation times, as well as different pathogens and growth conditions (Figure

4.1), could be informative.

One developmental process in which autophagy could be particularly important is recycling materials

from old leaves to allow the development of young leaves. Glutathione is involved in the regulation of

sulphur metabolism (Noctor et al., 2012) and also in meristem control (Vernoux et al., 2000; Reichheld et

al., 2007; Bashandy et al., 2010). It is therefore intriguing that the atg2 cad2 double mutants exhibit a

defect in the development of young leaves in high CO2 (Figure 4.9). While it is not clear why this effect

85

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86

should be observed at high CO2 but not in air, it could indicate that glutathione may be degraded by

autophagy-independent pathways to provide amino acids for biosynthesis, or act through a regulatory

function involving redox control or roles as a sulfur donor in key metabolic pathways. The high

CO2-specific interaction between cad2 and atg2 was observed in two independent experiments, but will

require further study both to confirm the effect and to investigate the underlying causes.

CHAPTER 5

CONCLUSION AND PERSPECTIVES


5.1 CONCLUSIONS

Numerous studies have demonstrated that H2O2 plays an important role in plant pathogen responses and

cell death as a signal, but much remains to be elucidated concerning the details of the underlying

processes. In particular, the importance of H2O2-driven changes in cell thiol-disulfide remains to be

clearly described. While the glutathione pool is an important component of cell thiol-disulfude status, and

is crucial as an antioxidant, its physiological function(s) in oxidative signaling remain(s) largely unknown.

Autophagy has also been implicated in processes such as PCD and senescence partly through the

modulation of H2O2 metabolism. In this work, we adopted a reverse genetics approach to studying the

interaction of glutathione and autophagy with increased H2O2 availability in the regulation of

pathogenesis-linked responses (Chapters 2 and 3) and senescence (Chapter 4) in Arabidopsis thaliana. A

useful genetic system to test above interactions is the catalase-deficient Arabidopsis mutant, cat2 (Queval

et al., 2007). This line is of interest because intracellular H2O2 production occurs in peroxisomes through

a physiologically relevant pathway (photorespiration) and it can be easily controlled by growth conditions

such as ambient CO2 level and light intensity. Previous work of the laboratory showed that glutathione

oxidation and accumulation in the cat2 mutant precedes or accompanies the activation of SA-dependent

pathogenesis-associated responses and bacterial resistance (Chaouch et al., 2010). While other work from

the laboratory recently reported that down-regulation of SA responses triggered by intracellular H2O2 in

cat2 arbohF double mutants are associated with altered glutathione status (Chaouch et al., 2012), it

remained unclear how important this altered statusis in linking phytohormone signaling to oxidative

stress.

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

In Chapter 2 of this thesis, through a direct genetics-based approach in which -ECS activity is

compromised, it was shown that blocking glutathione oxidation and accumulation largely annulled

cat2-triggered SA responses. We suggested that these effects are difficult to explain in terms of potentially

altered oxidative stress intensity when the response of glutathione is blocked because (1) leaf peroxides,

NADP(H), ascorbate, and rosette growth are largely unaffected compared to cat2, and (2) the phenotype

of cat2 cad2 is quite distinct from cat2 gr1 double mutants, in which oxidative stress is clearly

exacerbated compared to cat2. Furthermore, side by side comparison of cat2 cad2 with cat2 npr1, in

which thiol-regulated NPR1 function is disabled, revealed little overlap between responses in the two

double mutants, and indicates that glutathione oxidation and accumulation in cat2 play a signaling role in

coupling enhanced H2O2 availability to SA-dependent responses through the activation of ICS1-derived

SA accumulation upstream of NPR1.

JA is another defence phytohormone which is negatively controlled by SA and this antagonistic

interaction is considered to be at least partly mediated by NPR1. In Chapter 3, by further analysis of the

genetic tools reported in Chapter 2, it was shown that, while the cat2 mutation triggers up-regulation of

JA-associated gene expression in parallel to activation of SA pathway, genetically blocking glutathione

accumulation attenuates JA-associated gene expression triggered by cat2 mutation, decreasing expression

of the JA pathway to close to Col-0 levels. Contrasting responses in the transcript abundance of JA

associated genes in cat2 cad2 and cat2 npr1 reveal that the regulation of H2O2-triggered JA signaling by

87


glutathione can be mediated by NPR1-independent pathway(s). Overall, the work presented in the

Chapters 2 and 3 of this thesis points to new roles for glutathione-dependent processes in linking

oxidative stress to downstream SA and JA hormone signaling and the perspective arising from the main

parts of the study are discussed below, in section 5.2.

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

As noted in Chapter 4, it has been shown that the function of autophagy is associated with the regulation

of senescence associated cell death. Early senescence phenotype occurs in several autophagy-deficient

mutants and is dependent on SA signaling, which is associated with increased H2O2 (Yoshimoto et al.,

2009). In addition, CAT2 expression is down-regulated at the onset of leaf senescence (Zimmermann et al.,

2004). However, given the close association of leaf CAT2 with photorespiration, decreased expression of

this enzyme is in line with an overall increase in photosynthetic competence as leaves undergo a

transition from classical carbon-producing source organs to become a source of remobilized nutrients for

reproductive organs. The specific importance of peroxisomally-sourced H2O2 and CAT2 expression in

regulating senescence therefore remains to be established.

The analysis reported in Chapter 4 confirmed the early senescence phenotypes described by other groups,

but also reveal that the early-senescence phenotypes of atg mutants are enhanced when plants are grown

at high CO2. A comparison of metabolite profiles with those observed in cat2 suggests that the senescence

phenotypes may involve some of the same processes as those elicited by intracellular oxidative stress.

However, the phenotypes observed when atg mutants are crossed with cat2 provide little evidence that

enhanced oxidative stress of peroxisomal origin promotes these senescence processes; in fact, it seems

that the cat2 mutation has an antagonistic effect, acting rather to delay the development of the atg

phenotypes. The lack of induction of a generalized senescence programme in cat2 has been previously

discussed (Queval et al., 2007; Mhamdi et al., 2010b). While the cat2 mutation can clearly induce

SA-dependent lesions (Chapter 2), it is also possible that in certain conditions or genotypes, ROS act as a

pro-life signal or that SA-dependent HR-like cell death caused by excess intracellular H2O2 is in

opposition to senescence programmes. A study of cat2 atg double mutants in conditions promoting

SA-dependent HR-like lesions (long days) also generated little evidence that the corresponding ATG

genes play an important non-redundant role in controlling this response. Clearly, the results of Chapter 4

will need to be extended by further analyses, and some perspectives are discussed at the end of this

chapter.

5.2 PERSPECTIVES

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

enhanced H2O2 availability

While the genetic analysis reported in Chapters 2 and 3 provide clear evidence for potential roles of

glutathione status in transmitting signals downstream of H2O2, the details of the underlying mechanisms

remain to be described. The double and triple mutants described in these chapters could be very useful

tools for further analyses based on cellular and biochemical approaches, twinned with the production of

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other mutants to genetically test the importance of specific proteins involved in glutathione metabolism or

glutathione-dependent reactions. Some of these possibilities are now discussed.

5.2.1.1 Glutathione oxidation and metabolism in response to H2O2

Glutathione in cat2 is about 2-3 fold higher than in Col-0, and accumulates essentially in the form of

GSSG (Chapters 2 and 3). Exogenous GSSG treatment has some positive effects both on SA and JA

associated gene expression, indicating that GSSG produced from H2O2-triggered GSH oxidation may act

as an indirect signal of H2O2 levels to transmit information to activate downstream phytohormone

responses. A role for GSSG is possible based on the fact that the cad2 and allelic mutations not only block

glutathione accumulation in cat2 but also cause the glutathione pool to stay close to the highly reduced

values observed in Col-0. The reasons underlying this surprising observation are not yet clear. A key

uncertainty relates to the mechanism of GSSG production in cat2, an issue related to the interdependence

of ascorbate and glutathione pools in reductive metabolism of H2O2 (Foyer and Noctor, 2011). When

catalase is deficient, APX may be the major enzyme that is engaged to deal with the extra H2O2; in this

case, GSH oxidation to GSSG could occur through DHA reduction, either chemically or catalyzed by

DHAR. However, several other types of enzyme could catalyze GSSG production from GSH,

independent of the ascorbate pool. These questions are currently being analyzed by production of cat2

double mutants in the laboratory.

Cys-gly, which is a product of glutathione degradation by -glutamyl transpeptidases (GGT), accumulated

to significantly higher levels in cat2 relative to Col-0 (Chapter 3), suggesting that the marked

accumulation of glutathione in cat2 stimulates not only glutathione neosynthesis but also turnover. In

plants, GGT1 is probably the major apoplastic enzyme for glutathione degradation (Martin et al., 2007;

Ohkama-Ohtsu et al., 2007a). Moreover, analyses of compartmentation of glutathione were performed

using in situ immunolabelling (Chapter 2), which can be used to detect glutathione in different organelles

of Arabidopsis leaf and root cells (Zechmann et al., 2008). Glutathione accumulation in cat2 occurs in

most organelles, but the increase relative to Col-0 is strongest in the vacuoles (Queval et al., 2011;

Chapter 2), where another GGT (GGT4) is specifically located. To explore the influence of these

pathways, mutations for GGT1 and GGT4 have been crossed with cat2 and the characterization of double

mutants is underway in the laboratory. Concerning glutathione accumulation in the vacuole, multi-drug

resistance associated proteins (MRPs) are the primary candidates to perform transport from the cytosol

(Lu et al., 1998). Indeed, several mrp genes were reported to be up-regulated in microarray studies of cat2

(Mhamdi et al., 2010a; Queval et al., 2012). To analyze whether any of the encoded proteins play an

important role in glutathione homeostasis during oxidative stress, several double cat2 mrp mutants have

been produced and these lines are currently being studied.

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

Lesion formation and assaociated pathogenesis responses in cat2 do not seem to be caused simply by

widespread, indiscriminate, irreversible cellular damage, because they can be prevented simply by

blocking SA synthesis (Chaouch et al. 2010). Nevertheless, with the notable exceptions of two genes

identified in the singlet oxygen-accumulating flu mutant (Wagner et al., 2004), classical genetic screens

89


have failed to identify “H2O2 sensors”. This may reflect chance or limitations in experimental design. In

this respect, secondary mutagenesis of the cat2 mutant could produce interesting results, and a search for

cell death revertants in chemically mutagenized cat2 is currently in progress in the laboratory of Frank

Van Breusegem (Ghent, Belgium). An alternative possibility to explain unidentified “H2O2 receptors” is

that H2O2 signaling is mediated by multiple reactions operating in parallel. If so, the data shown here in

Chapters 2 and 3 suggest that these reactions are influenced by glutathione: the down-regulation of both

SA and JA signaling when glutathione synthesis is blocked point to new thiol-sensitive signals, in

addition to the previously described NPR1 protein. Given the possible involvement of glutathione in

influencing protein thiol-disulfide status, such reactions could involve oxidation of sensitive protein thiol

groups that form intra- or intermolecular disulfides or mixed disulfides with glutathione as the redox

potential of the free glutathione pool becomes more positive. Hence, it would be interesting to examine

the response in cat2 and cat2 cad2 of green fluorescent proteins that are sensitive to the glutathione redox

potential (roGFP; Jiang et al., 2006; Meyer et al., 2007). While reliable data of this type would be

informative, they would not demonstrate that the glutathione redox potential is the factor mediating the

signaling. Applying this approach in cat2 and derived mutants is complicated by the need to measure the

signal with accuracy in underlying leaf mesophyll cells, where the initial H2O2 trigger is expected to

occur in CAT2-deficient lines. Because of technical difficulties, most studies to date have used roGFP in

non-photosynthetic epidermal cells. The stomatal guard cells, epidermal cells that are photosynthetic,

could be an interesting compromise to obtain interesting data here. A comparison of glutathionylated

proteins in cat2 and cat2 cad2 could identify possible candidates involved in the signaling cascade. Here,

it is important to consider quantitative aspects as the effects of glutathionylation on the biological activity

of a protein may be progressive and gradual, rather than all or nothing. Such an approach is envisaged in

collaboration with S Lemaire (Paris, France).

5.2.1.3 H2O2, NO, and glutathione

Although the individual roles of NO and H2O2 in plant defence against pathogens is well established, the

two molecules interact to activate the HR and pathogen resistance (Delledonne et al., 1998, 2001). Indeed,

the NO donor sodium nitroprusside (SNP) provokes cell death in catalase-deficient tobacco leaves

exposed to moderate light stress (Zago et al., 2006). Work over recent years has established

S-nitrosoglutathione (GSNO) as an important reservoir of NO that is likely a physiologically important

reagent involved in modifying protein thiol groups through either S-nitrosylation or S-glutathionylation

(eg, Lindermayr et al., 2010). During the first year of my PhD study, I performed experiments seeking to

examine the effect of NO on responses in the cat2 mutant. However, treatment with exogenous SNP

under standard growth conditions did not accelerate or enhance lesions in cat2. One explanation is that

increased H2O2-triggered intracellular NO induction is sufficient for execution of HR-like responses or

that exogenously supplied SNP may not adequately mimic endogenous NO production. The response of

endogenous NO to oxidative stress in cat2 remains to be established. Because of technical difficulties

with the most commonly used techniques for NO detection, I did not attempt to resolve this question.

Despite their widespread use for over a decade in plants, the limitations of in vivo studies using

NO-generating agents and NO detection are becoming increasingly recognized. Unfortunately,

well-defined genetic resources for studies of NO also remain limited. Although a nitric oxide synthase

90


(NOS) has been identified in the green algae Ostreococcus tauri, the exact source of NO in higher plants

has not yet been elucidated (Foresi et al., 2010).

Despite doubt over the existence of a specific NOS in plants, three proteins potentially involved in NO

production or metabolism have been described, including nitrate reductase (NR), GSNO reductase

(GSNOR), and nitric oxide associated 1 (NOA1). With the aim of exploring interactions between H2O2,

glutathione and NO, I introduced mutations for some of these systems into cat2. So far, functional

analysis remains preliminary, although it seems that the cat2 noa1 double mutant shows much decreased

lesion formation compared to cat2 (Y. Han, unpublished results). However, like the noa1 single mutant,

cat2 noa1 shows a pale green phenotype (compared to Col-0 and cat2). Indeed, other authors have

concluded that impaired NO production in noa1 is indirectly due to the impact of normal chloroplast

function (Flores-Pérez et al., 2008; Gas et al., 2009). Furthermore, it has been reported from quantitative

proteomic analyses that several photorespiratory enzymes were down-regulated in noa1 RNAi mutants,

and that these lines also show decreased chlorophyll content and photosynthetic capacity (Liu et al.,

2010b). Additionally, treating the double cat2 noa1 mutant with SNP did not restore the cat2-triggered

lesion phenotype (Y. Han, unpublished results). Hence, we cannot discount the possibility that decreased

lesion formation in cat2 noa1 results from the attenuation of oxidative stress intensity through decreased

photorespiratory flux rather than the function of NO signaling per se.

The cytosolic enzyme, NR, which is required to catalyze the first step of nitrate assimilation, is encoded

by two genes (NIA1 and NIA2) in Arabidopsis. This enzyme can also contribute to the reduction of nitrite

to NO. Thus, the triple mutant cat2 nia1 nia2 is presently under construction although, like the noa1

mutation, possible effects on plant nutritional or energy status could complicate the interpretation of any

observed effects. Mutants for GSNOR would seem to be particularly interesting to further explore roles

for NO-linked processes in the observations reported in Chapters 2 and 3. Given our results in cat2 cad2,

it would be interesting to establish whether the effects of blocking glutathione accumulation are mediated

by impaired GSNO levels and associated S-nitrosylation processes. Moreover, GSNOR is a key player in

regulating S-nitrosylation, because the enzyme catalyzes the NADH-dependent reduction of GSNO to

S-aminoglutathione, which then decomposes to free GSH and ammonia or other compounds (Sakamoto et

al., 2002; Barroso et al., 2006; Díaz et al., 2003). In Arabidopsis, GSNOR appears to be a cytosolic

protein encoded by a single gene, GSNOR1 (Martínez et al., 1996). A double mutant cat2 gsnor1 is

currently being produced and characterized in the laboratory by Dr. A Mhamdi.

5.2.1.4 Glutathione and calcium signaling

One of the earliest steps in the plant response to many pathogens involves an increase in cytosolic

calcium levels. Close interaction have been described between intracellular oxidative stress and cytosolic

calcium in response to pathogen attack. Indeed, increases in cytosolic calcium can be accompanied by

activation of SA-dependent responses as well as the production of ROS (Ma et al., 2009).

Calcium-dependent calmodulin binding to the CBP60g protein has been shown to be essential for its

function in SA signaling (Wang et al., 2009a). It has been also reported that cytosolic calcium

concentrations and SA-dependent PR1 gene expression can both be modulated by glutathione content,

suggesting a possible interaction between calcium signaling and glutathione in the regulation of

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http://www.ncbi.nlm.nih.gov/pubmed/8944774


SA-dependent responses (Gomez et al., 2004a). Interestingly, at least one glutamate receptor identified in

plants is sensitive to glutathione and may be involved in linking glutathione contents to calcium flux into

the cytosol (Qi et al., 2006). Additionally, catalase activity may be activated by calmodulin binding in the

presence of calcium (Yang and Poovaiah, 2002). Finally, at least some glutaredoxins have been identified

that may interact with ion channels (Cheng and Hirschi, 2003). In view of these elements, and the data

presented in Chapters 2 and 3, it would be interesting to study the effects of crossing cat2 with mutants

for candidate genes involved in linking glutathione status to ion channel activity or for transporters that

could link H2O2 signaling to changes in calcium concentrations.

5.2.1.5 Glutathione and the jasmonic acid pathway

SA generally antagonizes JA signaling in plants by processes that are at least partly dependent on NPR1.

In addition, SA antagonism of JA signaling was abolished by the glutathione biosynthesis inhibitor, BSO,

suggesting that glutathione status plays a key role in the crosstalk between SA and JA signaling

(Koornneef et al., 2008). Results shown in Chapter 3 strongly suggest the existence of novel

glutathione-dependent processes that can regulate JA signaling independently of NPR1. While the data

shown in Chapter 2 indicate that glutathione is somehow involved in determining the accumulation of SA

itself in response to oxidative stress, we do not yet know whether effects on the JA pathway are linked to

changes in JA accumulation. This question is currently under investigation. Inversely, it could be

interesting to analyze glutathione contents and glutathione-associated gene expression when JA synthesis

mutants are introduced into the cat2 background. Futhermore, it has been reported that overexpression of

GRX480 completely blocked the induction of the JA marker gene PDF1.2 by MeJA in an

NPR1-independent manner (Ndamukong et al., 2007). Interestingly, in our microarray analysis of cad2,

expression of GRX480 was modulated in cad2 in a similar way to other genes associated with JA function.

Hence, genetic and physiological characterizations of the effect of the GRX480 mutation in the cat2

background will possibly give more information on the interaction of glutathione with hormone signaling.

As well as GRX480, potentially redundant genes encoding this type of glutaredoxin could play some role

in the effects. On the other hand, recent studies have pointed to functional overlap between glutathione

and cytosolic NADPH-dependent thioredoxin reductase (NTR) system in the control of developmental

processes (Reichheld et al., 2007; Marty et al., 2009; Bashandy et al., 2010). It would be interesting to

investigate interactions between glutathione and NTR systems in H2O2 signaling (e.g. by collaborations

with JP Reichheld, Perpignan). Finally, it could also be interesting to examine whether some of the effects

observed in the Chapters 2 and 3 are relevant to agriculturally important species such as barley and rice in

which catalase deficient mutants are available (Kendall et al., 1983; Lin et al., 2012). For example, work

in our laboratory has confirmed that the barley catalase mutant shows visible lesion formation and

accumulation and oxidation of glutathione that is very similar to effects observed in cat2 (J. Neukermans,

unpublished results). Analysis of such plants could be of interest, for example, to test effects of blocking

glutathione accumulation using pharmacological approaches.

5.2.2 Autophagy and oxidative stress

The data shown in Chapter 4 remain somewhat preliminary, and several obvious complementary analyses

are apparent. Because the onset of the early senescence phenotype in the atg mutants seems to be

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93

accelerated in high CO2 compared to air, it would be useful to investigate the underlying processes more

closely using molecular markers such as selected senescence associated genes (eg. SAG12) in the two

conditions. It has been reported that early senescence phenotypes in atg mutants are dependent on the SA

pathway (Yoshimoto et al., 2009). Therefore, it would be interesting to understand whether the delayed

senescence phenotype in double mutants of cat2 atg in short days is linked to compromised SA levels. As

well as analysis of SA levels, this could include crosses with the sid2 mutant (Chapter 2). Additionally,

although peroxisomal H2O2-triggered oxidative stress seems to have a delaying effect on the atg

senescence phenotypes, other potential ROS-generating systems could interact with ATG functions to

determine this response, perhaps in antagonistic ways to the cat2 mutation. For instance, ascorbate

peroxidase 1 (APX1) and AtRbohF could be players in oxidative stress-associated leaf senescence, and

have been suggested as key actors in the regulation of intracellular oxidative stress signaling

(Vanderauwera et al., 2011; Chaouch et al., 2012). It would be interesting to produce double mutants such

as atg2 apx1 and atg2 atrbohF to further investigate possible interactions of ROS with atg-triggered

senescence and the location and source of oxidative stress with which ATG interact.

It should be noted that although the early senescence phenotypes of atg mutants have been linked to SA,

they are morphologically quite distinct from the SA-dependent lesions triggered by the cat2 mutation in

long days, which are very similar to HR-like processes triggered by certain pathogens. Although the cat2

HR-like phenotype was not affected by the atg mutations, decreased resistance to PstDC3000 in cat2 atg2

and cat2 at18a relative to cat2 was observed, suggesting that some of SA-dependent pathogenesis

responses activated in cat2 could be regulated by the autophagic process. Hence, SA levels and

SA-dependent marker genes should be examined in these conditions. Moreover, time course analyses of

resistance to other pathogens as well as PstDC3000 in atg single and double mutants will be useful to

extend our understanding of the role of autophagy in oxidative stress-triggered pathogen resistance.

Very preliminary results indicate that combining atg2 and cad2 double mutants affects leaf development

at an early stage, when plants are grown at high CO2. One possibility is that this reflects a role for

glutathione in the regulation of nutrient remobilization. Indeed, glutathione is well known to be a form of

intercellular transport of organic sulfur in plants. Further investigation of this interaction would require

genetic confirmation by crossing atg2 with other allelic glutathione-deficient mutants such as pad2 and

rax1.

CHAPTER 6

MATERIALS AND METHODS


6.1 Plant material and growth conditions

The Arabidopsis mutant lines used in this study were all in the Columbia genetic background and are

listed in Table 6.1. Double and triple mutants were produced by crossing. After verification of double

heterozygosity in F1 plants, F2 individuals were genotyped to identify double homozygotes. Genotyping

of the new mutants produced for the analyses described in Chapters 2 and 3 is depicted in Figure 6.1.

Functional analysis was performed on F3 plants.

200 bp

400 bp

600 bp

WP MP

cat2cat2

WP MP WP MP 200 bp

400 bp

600 bp

123 bp

362 bp

507 bp

624 bp

Genotyping at CAT2 locus Genotyping at CAD2 (GSH1) locus

sensCAT2 + revCAT2

sensCAT2 + LB1

sensGR1 + revGR1

sensGR1 + LB1

sensSID2 + revSID2+

Digestion

sensCAD2 + revCAD2+

Digestion

Col

-0

cat2

cad

2 g

r1

cat2

cad

2 s

id2

cat2

gr1

sid

2

CAT2cat2

CAT2CAT2

CAD2cad2

CAD2CAD2

cad2cad2

B

Co

l-0

npr1

cat2

npr

1

cat2

cad

2 n

pr1

Genotyping at NPR1 locus

250 bp500 bp

C

A

Figure 6.1. Genotyping of double and triple mutants.

(A) Genotyping of cat2 cad2 mutant. MP, mutant primer pair. WP, wild-type primer pair.

(B) Genotyping at the NPR1 locus to obtain cat2 npr1 and cat2 cad2 npr1 mutants.

(C) Genotyping of other triple mutants by crossing cat2 cad2 with cat2 sid2 (Chaouch et al., 2010) and cat2 gr1 (Mhamdi et al.,

2010a).

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

Line AGI code Origin

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

Unless mentioned otherwise, seeds were directly sown on soil in square 7 cm pots and incubated for 2 d

at 4ºC in the dark. Plants were grown in a controlled-environment growth chamber in a 16h (long days) or

8h (short days) photoperiod and an irradiance of 200 μmol m-2 s-1 at leaf level, 20ºC day/18ºC night

temperature, 65% humidity, and given nutrient solution twice per week. The CO2 concentration was

maintained at 400 μL L-1 (air) or 3000 μL L-1 (high CO2). For buthionine sulfoximine (BSO) treatment

Col-0 and the two allelic glutathione-deficient lines, cad2 and rax1, were grown on agar in 0.5x

Murashige-Skoog medium without sugar for two weeks at an irradiance of 40 μmol m-2 s-1. BSO was

supplied to Col-0 plants in the medium at the indicated concentration. Samples were rapidly frozen in

liquid nitrogen and stored at -80ºC until analysis. For complementation experiments, plants were grown

on soil at high CO2 as described above. On transfer from high CO2 to air, rosettes were sprayed once daily

with 1 mM cysteine, 1 mM GSH or 1 mM GSSG. Unless otherwise stated, data are means ± SE of at least

three independent samples from different plants.

6.2 Methods

6.2.1 Molecular biology analyses 6.2.1.1 DNA extraction and plant genotyping

Leaf DNA was extracted in 400 μL extraction buffer (200 mM Tris pH7.5, 250 mM NaCl, 25 mM EDTA

and 0.5%SDS) using a bead shaker. The obtained solution was centrifuged for 10 min at 10 000g at 4ºC.

200 μL supernatant was mixed well with 200 μL isopropanol and centrifuged for 10 min at room

temperature. The pellet was washed with 1 mL 70% ethanol (v/v), dried and then redissolved in 100 μL

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TE buffer (10 mM Tris pH 8.0, 1mM EDTA). For T-DNA insertion lines, leaf DNA was amplified by

PCR using primers specific for left T-DNA borders (Table 6.2). Zygosity was analyzed by PCR

amplification of leaf DNA. Homozygote of the point/deletion mutations was established using restriction

length polymorphism (Table 6.2).

6.2.1.2 RT-qPCR

Total RNA was extracted with TRIzol (Invitrogen) following the manufacturer’s instructions. RNA

quality and concentration were determined by gel electrophoresis and estimated using a nanodrop

spectrophotometer at 260 nm respectively. Reverse transcription and first-strand cDNA synthesis were

performed using the SuperScript III First-Strand Synthesis System (Invitrogen). qPCR was performed

according to Queval et al. (2007). Primer sequences are listed in Table 6.2.

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

N° Oligonucleotide Sequence Restriction enzyme Technique

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

6.2.1.3 CATMA microarray analyses and data-mining

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

96


corresponding to 22,089 genes from Arabidopsis (Crowe et al., 2003; Hilson et al., 2004) plus 1,217

probes for microRNA genes and putative small RNA precursors (information available at http://urgv.

evry.inra.fr/projects/FLAGdb++). Samples of approximately 200 mg fresh weight were collected from

plants after 4 days transfer when plant were initially grown at high CO2 3 weeks. To determine

differentially expressed genes, a paired t test was performed on the log ratios, assuming that the variance

of the log ratios was the same for all genes. The raw P values were adjusted by the Bonferroni method,

which controls the family wise error rate (with a type I error equal to 5%) in order to keep a strong control

of the false positives in a multiple comparison context (Ge et al., 2003). Differentially expressed genes

were considered to be those with a Bonferroni P ≤ 0.05, as described by Gagnot et al. (2008). Only

genes that showed same direction statistically significant changes in biologically independent replicates

were considered as statistically significant. The full dataset is available at

http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=256.

6.2.2 Lesion quantification and pathogen tests Percentages of lesion areas were quantified using IQmaterials software. Growth of virulent Pseudomonas

syringae pv. tomato strain DC3000 was assessed 48 h post-inoculation according to Chaouch et al. (2010).

Three of the middle leaves from 5 to 7 different plants of each genotype were inoculated using a 1-mL

syringe without a needle with virulent Pst DC3000 in a medium titer of 5 x 105 colony-forming units

mL-1. Leaf discs (0.5 cm2 each) were taken for analysis either immediately (0h) or 48 h later. Four to six

samples were made by pooling two leaf discs from different inoculated leaves. Bacterial growth was

assessed by homogenizing leaf discs in 400 μL of H2O, plating appropriate dilutions on soild LB medium

containing rifampicin (100 μg/mL) and kanamycin (25 μg/mL), and counting colony numbers after 3

days.

6.2.3 Cytohistochemical analysis Sample preparation for cytohistochemical investigations was performed as previously described in detail

(Zechmann et al., 2007; Queval et al., 2011). Immunolocalization of glutathione on samples prepared in

Orsay was performed by Dr Bernd Zechmann (University of Graz, Austria) according to Zechmann and

Müller (2010). Subcellular concentrations of glutathione from immunogold labelling densities were

estimated according to Queval et al. (2011). Concentrations for each genotype were based on global leaf

GSH + GSSG contents measured in Col-0, cat2, and cat2 cad2. The amount of glutathione in each

compartment (nmol g-1 FW) was obtained by multiplying the leaf glutathione contents by the measured

fractional contribution of each compartment to the overall gold label. From these values, concentrations

were calculated using sub-cellular volumes estimated in leaf sections of each genotype. Measurements of

the percentage volume for each compartment were estimated according to Queval et al. (2011).

Subcellular volumes for each cell compartment were finally calculated per g fresh weight from the

percentage volumes based on a mesophyll volume per leaf mass of 773 μL g-1FW (Winter et al., 1994).

6.2.4 Metabolite analyses 6.2.4.1 Assays for ascorbate, glutathione and NADP(H)

97

http://urgv.evry.inra.fr/cgi-bin/projects/CATdb/consult_expce.pl?experiment_id=256


Oxidized and reduced forms of ascorbate, glutathione and NADP(H) were assayed using a plate-reader

method previously described in Queval and Noctor (2007), as described in detail below. Total glutathione

(GSH+GSSG) was also measured in acid extracts, along with other non-protein thiols, by

HPLC-fluorescence.

6.2.4.1.1 Extraction

Ascorbate, dehydroascorbate (DHA), reduced glutathione (GSH), glutathione disulfide (GSSG) and

NADP+ were extracted into 0.2 mM HCl while NADPH was extracted in 0.2 M NaOH. To measure

ascorbate, glutathione, thiols and NADP+, approximately 100 mg of leaf tissue was ground in liquid

nitrogen with a pestle and mortar and then extracted into 1 mL of 0.2 mM HCl. The homogenate was

centrifuged at 16 000g for 10 min at 4ºC. An aliquot of 0.5 mL of the supernatant was neutralized to pH

4.5-5 for assaying thiols, glutathione and ascorbate. Another 0.2 mL of the same supernatant was

incubated in boiling water for 1 min, rapidly cooled and neutralized to pH 6.5-7 for assaying NADP+. To

measure NADPH, parallel leaf material was extracted as for NADP+ except that the extraction solution

was 0.2 M NaOH and the heated supernatant aliquot was neutralized with 0.2 mM HCl to a final pH 7-8.

6.2.4.1.2 Quantification of thiols by HPLC

Thiols were measured by HPLC separation of bimane derivatives followed by fluorescence detection, as

described in Queval and Noctor (2007). Following neutralization, 0.2 mL extract supernatant was added

to 0.1 ml of 0.5 M Ches (pH 8.5) and 20 µL of 10 mM DTT. After incubation at room temperature for 30

min to reduce disulfide forms, all thiols were derivatized by the addition of 20 µL of 30 mM

monobromobimane. The mixture was incubated in the dark at room temperature for 15 min, and then the

reaction was stopped by the addition of 0.66 mL of 10% (v/v) acetic acid. The derivatized mix was

centrifuged at 10,000g for 10 min, and 0.9 mL supernatant was filtered through 0.2 µm mesh into

autosampler vials. Vials were loaded into the HPLC, and 50 µL from each vial was injected onto the

column by the HPLC autosampler. Bimane derivatives were separated by isocratic elution with 10%

methanol and 0.25% acetic acid (pH 4.3) at a flow rate of 0.8 mL/min and a column temperature of 40 °C.

Bimane derivatives of Cys, Cys-Gly, -EC and glutathione can be identified and quantified according to

this protocol. Peaks were identified by reference to standards and were quantified according to solutions

of mixed standard using quadratic curve-fitting available within the Waters Empower software.

6.2.4.1.3 Glutathione measurement by plate reader

This method relies on GR-dependent reduction of DNTB by GSH monitored at 412 nm and is used to

measure either total glutathione (GSH + GSSG) or GSSG (Tietze, 1969). Specific assay of GSSG was

done by pre-treatment of extract aliquots with 2 μL of 2-vinylpyridine (VPD), which efficiently

complexes GSH (Griffith, 1980). To measure total glutathione, the mixture contained 100 μL of 0.2 M

NaH2PO4, 10 mM EDTA (pH 7.5), 10 μL 10 mM NADPH, 10 μL 12 mM DTNB, 10 μL of neutralized

extract and 60 μL water. After shaking, the reaction was started by addition of 10 μL GR. GSSG

measurement was based on the same principle except that extract aliquots were incubated with VPD for

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30 min at room temperature and were centrifuged twice for 15 min at 4ºC before assay. The increase in

absorbance at 412 nm was monitored for 5 min and all assays were done in triplicate for each extract.

Rates were converted to quantities of glutathione using standard curves generated by assay of known

concentration in the same plate.

6.2.4.1.4 Ascorbate

Ascorbate was measured by the absorbance at 265 nm that is specifically removed by ascorbate oxidase

(AO). The reduced form was measured in untreated acid extract aliquots, while total ascorbate (DHA +

ascorbate) were measured following pre-incubation of aliquots with DTT to reduce DHA to ascorbate. To

assay ascorbate, the buffer contained 100 μL 0.2 M NaH2PO4 (pH 5.6), 40 μL extract and 55 μL H2O. The

absorbance at 265 nm was read before and 5 min after adding 0.2 U of AO. Total ascorbate was measured

after incubation of 100 μL neutralized extract with 140 μL 0.12 M NaH2PO4 (pH7.5), 10 μL 25 mM DTT

for 30 min at room temperature. Both of above assays were done in triplicate for each extract. According

to a standard extinction coefficient of 14 mM-1 cm-1, absorbance changes in A265 were converted to

quantities of ascorbate (Queval and Noctor, 2007).

6.2.4.1.5 NADP(H)

The assay involves the phenazine methosulfate (PMS)-catalyzed reduction of dichlorophenolindophenol

(DCPIP) in the presence of glucose-6-phosphate (G6P) and glucose-6-phosphate dehydrogenase

(G6PDH). NADP+ and NADPH are distinguished by preferential dectruction in acid or base. To measure

NADP+ and NADPH, triplicate 20 μL aliquots of neutralized extract (acid extraction for NADP+ and

basic extraction for NADPH) were added to plates which contained 100 μL HEPES, 2 mM EDTA (pH

7.5), 20 μL 1.2 mM DCPIP, 10 μL 20 mM PMS, 10 μL 10 mM G6P, and 30 μL water. After shaking, the

reaction was started by addition of 10 μL G6PDH. The decrease in absorbance at 600 nm was monitored

for 5 min. Pyridine nucleotide contents were calculated using standard curves generated simultaneously

by assay of standard solutions on the same plate.

6.2.4.2 Salicylic acid measurement

Salicylic acid (SA) was extracted as described in Langlois-Meurinne et al. (2005). Radio-labelled SA was

added to each sample as an internal standard. After extraction, samples were dried in a Speed-Vac. One

series of dried samples were used to analyze free SA while another series of dried samples were subjected

to acid hydrolysis to assay total SA. SA was assayed using fluorescence detection by High Performance

Liquid Chromatography (HPLC). Identification and quantification was performed by comparison of

peaks with SA standards. Extraction yields of radioactively marked SA were estimated using a

scintillation counter.

6.2.4.3 Non-targeted GC-TOF-MS analysis

Non-targeted metabolite profiling was performed by gas chromatography-time of flight spectrometry

(GC-TOF-MS) on triplicate biological repeats as in Noctor et al. (2007), except that a mix of alkanes was

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100

included during derivatization to aid metabolite identification. Compounds identified by retention index

were confirmed by reference to mass spectra libraries. Peak area was quantified based on specific

fragments and was corrected on the basis of an internal standard (ribitol) and sample fresh weight. Data

display was performed using TMEV4 software. Peak areas were mean-centered and reduced by dividing

by the standard deviation across all samples before hierarchical clustering and production of “heat-maps”.

To identify significantly different metabolites, peak areas normalized to ribitol and fresh weight were

subject to ANOVA (P < 0.05) in TMEV4 or pair-wise t tests in Microsoft Excel.

6.2.4.4 Peroxide assay

Peroxides were measured by luminol luminescence according to Queval et al. (2008). Tissue samples of

50 mg fresh weight were ground to a fine powder in liquid nitrogen and then extracted into 1 mL of 0.2 N

HCl. The homogenate was centrifuged for 10 min at 10,000g. The extract was neutralized to pH 5.6 with

0.2 M NaOH. To avoid interference from ascorbate, an aliquot (50 mL) was treated with ascorbate

oxidase, and the treated aliquot was added to 0.5 mL of 0.2 M NH3 (pH 9.5), and 0.05 mL of 0.5 mM

luminol (prepared in 0.2 M NH3, pH 9.5), vortexed rapidly, and injected into a luminometer. The reaction

was started by adding 100 mL of 0.5 mM K3Fe(CN)6 (in NH3), and luminol chemiluminescence was

monitored for 2 s. To check the linearity of this assay, a standard curve of H2O2 was done for each

experiment, and assay standards and extracts were assayed in triplicate.

6.2.5 Statistical analysis Unless stated otherwise, the statistical analysis of data was based on Student’s t-tests. Calculations were

performed on a minimum of three independent data sets, assuming two sample equal variance and a

two-tailed distribution. Unless otherwise indicated, significant difference is expressed using t-test at P <

0.05.

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ANNEX 1A

Mhamdi et al., 2010 Plant Physiol.

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

This article first reported the cat2 gr1 mutant that was used in Chapter 2 of this study. My

main contribution to results shown in this paper was the production and characterization of an

allelic double mutant to confirm the genetic interaction between cat2 and gr1 knockout

mutations (data shown in Annex 1b).

Arabidopsis GLUTATHIONE REDUCTASE1 Plays aCrucial Role in Leaf Responses to Intracellular HydrogenPeroxide and in Ensuring Appropriate Gene Expressionthrough Both Salicylic Acid and Jasmonic AcidSignaling Pathways1[C][W][OA]

Amna Mhamdi, Jutta Hager, Sejir Chaouch, Guillaume Queval2, Yi Han, Ludivine Taconnat,Patrick Saindrenan, Houda Gouia, Emmanuelle Issakidis-Bourguet, Jean-Pierre Renou, and Graham Noctor*

Institut de Biologie des Plantes, UMR8618 CNRS, Universite de Paris Sud, 91405 Orsay cedex, France (A.M.,J.H., S.C., G.Q., Y.H., P.S., E.I.-B., G.N.); Departement de Biologie, Faculte des Sciences de Tunis, Universite deTunis El Manar, 2092 Tunis, Tunisia (A.M., H.G.); and Plant Genomics Research Unit, Unite de Recherche enGenomique Vegetale, 91057 Evry cedex, France (L.T., J.-P.R.)

Glutathione is a major cellular thiol that is maintained in the reduced state by glutathione reductase (GR), which is encoded bytwo genes in Arabidopsis (Arabidopsis thaliana; GR1 and GR2). This study addressed the role of GR1 in hydrogen peroxide(H2O2) responses through a combined genetic, transcriptomic, and redox profiling approach. To identify the potential role ofchanges in glutathione status in H2O2 signaling, gr1 mutants, which show a constitutive increase in oxidized glutathione(GSSG), were compared with a catalase-deficient background (cat2), in which GSSG accumulation is conditionally driven byH2O2. Parallel transcriptomics analysis of gr1 and cat2 identified overlapping gene expression profiles that in both lines weredependent on growth daylength. Overlapping genes included phytohormone-associated genes, in particular implicatingglutathione oxidation state in the regulation of jasmonic acid signaling. Direct analysis of H2O2-glutathione interactions in cat2gr1 double mutants established that GR1-dependent glutathione status is required for multiple responses to increased H2O2availability, including limitation of lesion formation, accumulation of salicylic acid, induction of pathogenesis-related genes,and signaling through jasmonic acid pathways. Modulation of these responses in cat2 gr1 was linked to dramatic GSSGaccumulation and modified expression of specific glutaredoxins and glutathione S-transferases, but there is little or noevidence of generalized oxidative stress or changes in thioredoxin-associated gene expression. We conclude that GR1 plays acrucial role in daylength-dependent redox signaling and that this function cannot be replaced by the second Arabidopsis GRgene or by thiol systems such as the thioredoxin system.

Thiol-disulfide exchange plays crucial roles in pro-tein structure, the regulation of enzymatic activity, and

redox signaling, and it is principally mediated bythioredoxin (TRX) and glutathione reductase (GR)/glutathione systems (Buchanan and Balmer, 2005;Jacquot et al., 2008; Meyer et al., 2008). Arabidopsis(Arabidopsis thaliana) lines identified in independentscreens for alterations in heavy metal tolerance, mer-istem function, light signaling, and pathogen resis-tance have been shown to harbor mutations in thegene encoding the first enzyme of glutathione synthe-sis (Cobbett et al., 1998; Vernoux et al., 2000; Ball et al.,2004; Parisy et al., 2007). Studies on the rml1 mutant,which is severely deficient in glutathione synthesis,define a specific role for glutathione in root meristemfunction (Vernoux et al., 2000). However, shoot meri-stem function is regulated in a redundant manner bycytosolic glutathione and TRX (Reichheld et al., 2007),providing a first indication for functional overlapbetween these thiol-disulfide systems in plant devel-opment.

Modifications of cellular thiol-disulfide status maybe important in transmitting environmental changesthat favor the production of oxidants such as hydrogen

1 This work was supported by the French Agence Nationale dela Recherche-GENOPLANTE program Redoxome, by the TunisianMinistry of Higher Education and Scientific Research and theUniversite de Paris Sud XI (to A.M.), by the French Centre Nationalde la Recherche Scientifique (to S.C.), and by the Chinese ScholarshipCouncil (to Y.H.).

2 Present address: Department of Plant Systems Biology, FlandersInstitute for Biotechnology, and Department of Plant Biotechnologyand Genetics, Ghent University, 9052 Gent, Belgium.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Graham Noctor ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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

1144 Plant Physiology�, July 2010, Vol. 153, pp. 1144–1160, www.plantphysiol.org � 2010 American Society of Plant Biologists

peroxide (H2O2; Foyer et al., 1997; May et al., 1998;Foyer and Noctor, 2005). The glutathione/GR systemis involved in H2O2 metabolism by reducing dehy-droascorbate generated following the (per)oxidationof ascorbate (Asada, 1999). This pathway is one way inwhich H2O2 reduction could be coupled to NADPHoxidation, with the first reaction catalyzed by ascor-bate peroxidase (APX) and the last by GR, althoughascorbate regeneration can occur independently of re-duced glutathione (GSH) throughNAD(P)H-dependentor (in the chloroplast) ferredoxin-dependent reduc-tion of monodehydroascorbate (MDAR; Asada, 1999).Additional complexity of the plant antioxidativesystem has been highlighted by identification of sev-eral other classes of antioxidative peroxidases thatcould reduce H2O2 to water. These include the TRXfusion protein CDSP32 (Rey et al., 2005) and severaltypes of peroxiredoxin, many of which are themselvesTRX dependent (Dietz, 2003). Plants lack animal-type selenocysteine-dependent glutathione peroxi-dase (GPX), instead containing Cys-dependent GPX(Eshdat et al., 1997; Rodriguez Milla et al., 2003). De-spite their annotations as GPX, these enzymes are nowthought to use TRX rather than GSH (Iqbal et al., 2006).However, H2O2 could still oxidize GSH via peroxidaticglutathione S-transferases (GSTs) and/or glutaredoxin(GRX)-dependent peroxiredoxin (Wagner et al., 2002;Jacquot et al., 2008). Metabolism of H2O2 may occurthrough APX or through ascorbate-independent GSHperoxidation, both of which potentially depend on GRactivity, as well as through TRX-dependent pathways.The relative importance of these pathways remainsunclear.The major sites of intracellular H2O2 production in

most photosynthetic plant cells are the chloroplastsand peroxisomes (Noctor et al., 2002). Despite this,Davletova et al. (2005) demonstrated a crucial role forcytosolic APX1 in redox homeostasis in Arabidopsis.This finding implies that the cytosolic ascorbate-glutathione pathway is important in metabolizing H2O2originating in other organelles, thus marking out thiscompartment as a key site in which redox signals areintegrated to drive appropriate responses. Cytosolicmetabolism of H2O2 could be key in setting appropri-ate conditions for thiol-disulfide regulation throughcytosolic/nuclear signal transmitters such as NPR1and TGA transcription factors (Despres et al., 2003;Mou et al., 2003; Rochon et al., 2006; Tada et al., 2008).Because peroxisomal reactions can be a major pro-ducer of H2O2, a related issue that remains unresolvedis to what extent reductive H2O2 metabolism overlapswith metabolism through catalase, which is confinedto peroxisomes and homologous organelles. Intrigu-ingly, double antisense tobacco (Nicotiana tabacum)lines in which both catalase and cytosolic APX weredown-regulated showed a less marked phenotypethan single antisense lines deficient in either enzymealone (Rizhsky et al., 2002). This observation impliesthat if the major role of the GR-glutathione system isindeed to support reduction of dehydroascorbate to

ascorbate, down-regulation of cytosolic GR capacityshould produce a similar effect to APX deficiency.

Two genes are annotated to encode GR in plants. Pea(Pisum sativum) chloroplast GR was the first plastidialprotein shown also to be targeted to the mitochondria(Creissen et al., 1995). Dual targeting of this proteinalso occurs in Arabidopsis (Chew et al., 2003), wherethe plastidic/mitochondrial isoform is named GR2.The second gene, GR1, is predicted to encode a cyto-solic enzyme. In pea, cytosolic GR has been well cha-racterized at the biochemical level (Edwards et al.,1990; Stevens et al., 2000), but the functional signifi-cance of the enzyme remains unclear. Underexpres-sion or overexpression studies in tobacco and poplar(Populus species) have reported significant effects ofmodifying chloroplast GR capacity (Aono et al., 1993;Broadbent et al., 1995; Foyer et al., 1995; Ding et al.,2009). Less evidence is available supporting an impor-tant role for cytosolic GR. In insects, GSSG reductioncan also be catalyzed by NADPH-TRX reductases(NTRs; Kanzok et al., 2001), and it has recently beenshown that Arabidopsis cytosolic NTR can function-ally replace GR1 (Marty et al., 2009).

Therefore, key outstanding issues in the study ofredox homeostasis and signaling in plants are (1) theimportance of GR/glutathione in H2O2 metabolismand/or H2O2 signal transmission and (2) the specific-ity of GSH and TRX systems in H2O2 responses. In thisstudy, we sought to address these questions by agenetically based approach in which the effects ofmodified H2O2 and glutathione were first analyzed inparallel in single mutants and then directly throughthe production of double mutants. This was achievedusing gr1 insertion mutants and a catalase-deficientArabidopsis line, cat2, in which intracellular H2O2drives the accumulation of GSSG in a conditionalmanner (Queval et al., 2007). Our study provides invivo evidence for a specific irreplaceable role for GR1in H2O2 metabolism and signaling.

RESULTS

Characterization of gr T-DNA Mutants

While T-DNA insertions in the coding sequence ofdual-targeted chloroplast/mitochondrial GR2 areembryo lethal (Tzafrir et al., 2004), homozygous gr1mutants were readily obtained. Reverse transcription(RT)-PCR confirmed the absence of GR1 transcript,while total extractable GR activity was decreased by40% relative to ecotype Columbia (Col-0; Supplemen-tal Fig. S1). Despite these effects, repeated observa-tions over a period of 5 years showed that themutationproduced no difference in rosette growth rates fromCol-0 in either short days (SD) or long days (LD) or inflowering timing and leaf number (data not shown).Thus, our analysis is in agreement with the report ofMarty et al. (2009) that absence of cytosolic GR activitycaused a measurable decrease in leaf GR activity butthat this did not cause phenotypic effects.

Glutathione Reductase in H2O2 Responses

Plant Physiol. Vol. 153, 2010 1145

Comparative Analysis of Glutathione- and

H2O2-Dependent Changes in Gene Expression

Because gr1 mutants are aphenotypic in typicalgrowth conditions, this study analyzed the potentialrole of the enzyme in H2O2 metabolism and signaling.First, we performed parallel microarray analysis ofGSSG-accumulating gr1 and the catalase-deficient mu-tant cat2, which conditionally accumulates GSSG trig-gered by photorespiratory H2O2 production (Quevalet al., 2007). To compare effects on transcript abun-dance while minimizing possible interference of long-term developmental effects due to oxidative stress incat2 grown from seed in air, plants were sampledfollowing initial growth at high CO2, where the cat2mutation is silent (Queval et al., 2007). This experi-mental design was chosen to allow comparison ofthe effects of modified glutathione status in gr1 withsimilar H2O2-induced effects triggered in cat2 aftertransfer from high CO2 to air. Our rationale was thatwhile H2O2 may modify gene expression through anumber of signaling mechanisms, any effects medi-

ated via glutathione should also be observed in gr1,even in the absence of the oxidative stress that drivesGSSG accumulation in cat2 (Fig. 1A).

As previously reported, cat2 showed a wild-typephenotype at high CO2. The consequences of the cat2mutation for gene expression and phenotype in air arestrongly influenced by growth photoperiod (Quevalet al., 2007). Thus, measurements of transcripts andantioxidant were performed after transfer of gr1 andcat2 from high CO2 to air in both SD (8-h photoperiod)and LD (16-h photoperiod). In both photoperiods (andat high CO2), the glutathione reduction state was lowerin gr1 than in Col-0 (Fig. 1B). In cat2, however, thisfactor was only lower than in Col-0 after transfer to air,where it was decreased below the values observedin gr1. In both mutants, leaf ascorbate contents weremuch less affected than glutathione. The only signif-icant difference was observed in ascorbate reductionstate in cat2 in LD (Supplemental Fig. S2).

Analysis of microarray data was performed byconsidering as being differentially expressed geneswith a Bonferroni P # 0.05, as described by Gagnot

Figure 1. Comparison of H2O2- and glutathione-regulated changes in gene expression. A, Schemedepicting experimental strategy. B, Glutathionereduction states (100 GSH/total glutathione) inCol-0, gr1, and cat2 in high CO2 or in air in 8-h(SD) or 16-h (LD) growth photoperiods. Differentletters indicate significant difference betweengenotypes at P , 0.05. The numbers show theGSH to GSSG ratios. Data are means 6 SE of fiveto seven independent extracts. C, Overlap insignificantly induced or repressed genes in cat2or gr1 following induction of glutathione oxida-tion in cat2 after transfer from high CO2 to air.[See online article for color version of this figure.]

Mhamdi et al.

1146 Plant Physiol. Vol. 153, 2010

et al. (2008). An additional selection criterion wasintroduced by considering only genes that showedstatistically significant same-direction change inboth biological replicates. The analysis showed thatH2O2-regulated gene expression (in cat2) was highlydaylength dependent (Fig. 1C). More genes were sig-nificantly induced in cat2 transferred to air in SD thanin LD, whereas the reverse was true for significantlyrepressed genes. Fewer genes were affected in gr1, buteffects were also daylength conditioned. Of genesinduced in gr1 in SD, only three were also inducedin LD, together with another four LD-specific genes.The number of repressed genes in gr1 in LD wassimilar to that in SD (Fig. 1C), but of these, only twowere common (Supplemental Table S2). Thus, as forgenes regulated by an H2O2 signal, gene expressiondependent on a drop in glutathione reduction statewas strongly determined by photoperiod context. Ofthe 58 genes showing significantly modified expres-sion in response to a mild perturbation of glutathioneredox state in gr1, most showed significant same-direction changes in at least one cat2 replicate (Sup-plemental Table S2). It should be noted that Figure 1Cprovides a conservative estimate of overlap betweengr1 and cat2 transcriptomes because only genes thatshowed statistically significant same-direction changesin all four dye-swap repeats (two gr1/Col-0 and twocat2/Col-0) are considered.Several genes whose expression was modified in gr1

encoded stress-related proteins, notably cadmium-,salt-, and cold-responsive genes, as well as two GSTs(GSTU7 and GSTU6) and three multidrug resistance-associated proteins (Supplemental Table S2). This isconsistent with the roles of glutathione in redox ho-meostasis, heavy metal resistance, cold acclimation,and metabolite conjugation and transport (Cobbettet al., 1998; May et al., 1998; Kocsy et al., 2000; Wagneret al., 2002; Gomez et al., 2004).

Sixteen of the 58 gr1-sensitive genes are annotated asphytohormone associated. In all cases, induction orrepression of these genes was dependent on daylengthcontext (Table I). A putative monoxygenase with sim-ilarity to salicylic acid (SA)-degrading enzymes wasamong the induced genes, whereas other differentiallyexpressed genes in gr1 were associated with auxin,gibberellin, abscisic acid, and ethylene function (TableI). The most striking impact of the gr1mutation was onthe expression of genes involved in jasmonic acid (JA)synthesis and signaling. Of the 18 genes repressedin gr1 in LD, eight have been shown to be earlyJA-responsive genes (Yan et al., 2007). These genes en-coded, among others, LIPOXYGENASE3 (LOX3),MYB95, GSTU6, and two of the 12 JASMONATE/ZIM DOMAIN (JAZ) proteins recently characterizedas JA-inducible repressors of JA signaling (Staswick,2008; Browse, 2009). All but one of the JA-dependentgenes repressed in gr1 in LD showed the same responsein the GSSG-accumulating cat2 mutant (Table I).

Two induced stress-associated genes and four JA-dependent genes that showed similar responses in gr1and cat2 were selected for quantitative PCR analysis.This confirmed the induction in gr1 of the cold-regulatedCOR78 in SD and of the cadmium-responsiveAt3g14990in both daylength conditions (Fig. 2). For the JA-associated genes, quantitative PCR revealed that in addi-tion to repression in gr1 and cat2 in LD, three of the fourgenes (LOX3, MYB95, and JAZ10) were also induced inSD (Fig. 2). Thus, GR1-dependent glutathione statusinfluences the expression of genes involved in JA synthe-sis and signaling in a daylength-dependent manner.

Genetic Analysis of GR1 Function in Responses to H2O2

To directly explore the role of GR1 and glutathionestatus in the H2O2 response, the gr1 mutation wascrossed into the cat2 background. Preliminary analysis

Table I. Annotated phytohormone-associated genes showing significantly modified transcript levelsin gr1

Gene Identifier Name 8-h Days 16-h Days Hormone

At3g50970a XERO2 Induced – Abscisic acidAt4g15760 MO1 Induced – SAAt4g23600a CORI3 (JR2) Induced – JAAt5g05730 ASA1 Induced – EthyleneAt1g74670a Unknown protein Repressed – GAAt5g54490 PBP1 Repressed – AuxinAt5g61590 ERF B3 member Repressed – EthyleneAt5g61600 ERF B3 member Repressed – EthyleneAt1g17420a LOX3 – Repressed JAAt1g74430/40a MYB95 – Repressed JAAt2g24850 TAT3 – Repressed JAAt2g29440a GSTU6 – Repressed JAAt2g34600a JAZ7 – Repressed JAAt2g38240a 2OGOR – Repressed JAAt4g15440a HPL1 – Repressed JAAt5g13220a JAZ10 – Repressed JA

aSignificant same-direction response in cat2 in the same condition.



of F2 seeds grown in air failed to identify double cat2gr1 homozygotes. Thus, F3 seeds from a cat2/cat2 GR1/gr1 genotype were germinated and grown at high CO2,where, as noted above, the cat2 mutation is phenotyp-ically silent because photorespiratory H2O2 produc-tion is largely shut down. Genotyping of these seedsidentified several plants with cat2/cat2 gr1/gr1 geno-types, suggesting selection against cat2 gr1 doublehomozygotes in the presence of photorespiratory H2O2production (air) but not in its absence (high CO2).

When F4 seeds obtained from F3 cat2 gr1 doublehomozygotes identified at high CO2 were sown onagar plates in air, germination was similar to Col-0,gr1, and cat2, but growth was severely compromisedin all cat2 gr1 plants from the cotyledon stage onward(Fig. 3A). Although cat2 gr1was able to grow on soil atmoderate irradiance, all plants with this genotypeshowed a dwarf rosette phenotype that was muchmore severe than that observed in cat2 (Fig. 3B).

The cat2 gr1 phenotype was completely revertedby growth at high CO2 (Fig. 4A). Together with theaphenotypic nature of the single gr1 mutant, thisobservation indicates that the functional importanceof GR1 is highly correlated with conditions of in-creased intracellular H2O2 availability. To further in-vestigate this point, we analyzed the phenotypicresponses of cat2 gr1 grown at high CO2 after transferto air in either SD or LD. Lesions developed in cat2 at5 to 6 d after transfer to LD but did not develop in SD(data not shown). Four days after the transfer to LD,few or no lesions were apparent on cat2 leaves (Fig.4B). By contrast, extensive bleaching was observed oncat2 gr1 leaves as early as 2 d after the transfer to air inLD (Fig. 4B). In both photoperiods, photorespiratoryH2O2 induced slower growth in cat2, and this effectwas exacerbated by the absence of GR1 function (Fig.4C). Remarkably, however, bleaching was limited orabsent in cat2 gr1 transferred to air in SD.

To verify the conditional genetic interaction betweenthe cat2 and gr1 mutations, a second allelic gr1 T-DNAline was obtained and crossed with cat2. F2 plantsfrom this cross were grown at high CO2 and thentransferred to air in LD, and lesions were quantifiedand plants were genotyped (Supplemental Fig. S3).Neither F1 CAT2/cat2 GR1/gr1 double heterozygotesgrown in air nor any F2 plants grown at high CO2showed any apparent phenotype (data not shown). Of93 F2 plants transferred from high CO2 to air in LD,seven displayed readily visible lesions within 4 d(Supplemental Fig. S3). Genotyping confirmed that allplants showing substantial lesions were cat2/cat2 gr1/gr1 double homozygotes, and lesion quantificationand rosette fresh mass distinguished these plants fromthe eight other genotypes (Supplemental Fig. S3).

Microarray Analysis of the H2O2-GR1 Interaction

To identify H2O2-regulated genes whose expression isdependent on GR1, transcript profiling of cat2 and cat2gr1was performed after transfer of plants grown at high

Figure 2. Verification of selected genes repressed or induced in gr1 byquantitative RT-PCR. Data are means6 SE of two independent extracts.* P , 0.1, ** P , 0.05, *** P , 0.01.

Mhamdi et al.


CO2 to air. In view of the above phenotypic observationsand the daylength-dependent transcript profiles for cat2and gr1 single mutants (Fig. 1; Supplemental Table S2),the analysis was performed after transfer to both SD andLD. A full list of significantly different genes and signalintensities is given in Supplemental Table S3.Clustering analysis of significantly different tran-

scripts delineated two major patterns of H2O2-inducedgene expression (Fig. 5). The first was largely com-posed of genes that were induced by photorespiratoryH2O2 in cat2 and cat2 gr1 (cluster 1), while the secondcomprised genes that responded in a daylength-dependent manner (cluster 2). The most striking im-pact of the gr1 mutation in the cat2 background wasto annul or impair the induction of genes by photo-respiratory H2O2 in SD, an effect exemplified by asubcluster of genes in cluster 2. Of 62 genes in thissubcluster, 33 are annotated as involved in JA- orwounding-dependent responses (Taki et al., 2005; Yanet al., 2007; http://www.arabidopsis.org) and arehighlighted yellow in Figure 5. Data mining of allsignificantly different genes using the above data setsrevealed that, in all, 47 genes inducible by JA orwounding, or encoding proteins involved in JA syn-thesis, conjugation, or signaling, were differentiallyexpressed in cat2 and/or cat2 gr1 (Supplemental TableS4). Twenty-five of these were among 35 genes recentlyshown to be induced by wounding in a JA-dependentmanner (Yan et al., 2007), while among the others wereestablished JA-associated genes such as LOX2, LOX3,OPR3, JAZ1, JAZ3, JAZ7, JAZ9, JAR1, and JMT1.

In the cat2 single mutant, the predominant effect ofH2O2 on genes involved in JA synthesis and signalingwas induction in SD and/or repression in LD. Thisdaylength-dependent regulation was strongly modu-lated by the absence of GR1 function in cat2 gr1. Of the47 JA-associated transcripts whose abundance wassignificantly modified in cat2 and/or cat2 gr1 relativeto Col-0, only 10 did not show differential expressionbetween the two lines in at least one photoperiod(Supplemental Table S4). The overwhelmingly pre-dominant effect of the gr1 mutation in the cat2 back-ground was to cause or enhance repression (e.g. LOX2,JMT1, JAZ3), to oppose induction in SD (e.g. RAP2.6,JAZ10, AOS), or both (LOX3, TAT3, OPR3).

Profiling of Glutathione and Associated Gene Expressionin cat2 gr1

Consistent with the absence of phenotype at highCO2 (Fig. 4A), cat2 glutathione status was similar to thewild type (95% GSH), whereas in cat2 gr1 the gluta-thione pool was only about 70% reduced (i.e. similar tothe gr1 single mutant in air or at high CO2; Fig. 6A;Supplemental Fig. S2). These observations are consis-tent with the silent nature of the cat2 mutation at highCO2 and with a constitutive oxidation of glutathionein the gr1 genotype. The impact of the gr1 mutationon glutathione in conditions of increased H2O2 wasassessed under the same conditions as for the micro-array analysis. A time course following transfer to airrevealed that leaf glutathione (1) became progressively

Figure 3. Germination and growthphenotype of cat2 gr1 double mutants.A, Germination and growth on agar. B,Plants grown on soil from germinationin a 16-h/8-h day/night regime. Aster-isks indicate significant differencesbetween mutants and Col-0, whilepluses indicate significant differencesbetween cat2 and cat2 gr1. FW, Freshweight. [See online article for colorversion of this figure.]



oxidized and accumulated in cat2; (2) became muchmore rapidly oxidized and accumulatedmore stronglyin cat2 gr1; and (3) was most oxidized and moststrongly accumulated in cat2 gr1 in SD. The changesin glutathione involved a fall in the GSH to GSSG ratioto below 1 in cat2 and cat2 gr1, with the lowest ratioobserved in cat2 gr1 in SD (Fig. 6A).

Mining of the transcriptome data for glutathione-associated genes showed that modified glutathionestatus in cat2 and cat2 gr1 was associated with theinduction of genes encoding three GSTs of the tauclass (GSTU7, GSTU8, GSTU19) and two of the phiclass (GSTF2, GSTF8), two multidrug resistance-associated proteins (MRP2, MRP14), a glutathione/thioredoxin peroxidase (GPX6), a glyoxylase I familyprotein, two members of the GRX family, and a GRX-like expressed protein (Fig. 6B). These genes wereequally or more strongly induced in cat2 gr1 com-pared with cat2, although the effect depended ondaylength. In no case did the gr1 mutation opposeinduction of these genes in cat2, which were all foundto be grouped in cluster 1 on the heat map shown inFigure 5.

Five glutathione-associated genes showed a differentexpression pattern (Fig. 6B, bottom) and were all foundwithin cluster 2 shown in Figure 5. While one GRX(At5g18600) showed a tendency to repression in allconditions, repression was only significant in cat2 gr1 inSD. GSTU5 and GSTU6 were both induced in cat2 butnot in cat2 gr1 in SD and were repressed in both ge-notypes in LD (Fig. 6B). The gr1 mutation in the cat2background also produced repression of GGT1 andGRX480, specifically in LD. Thus, the expression pat-terns of these five genes were similar to those of JA-dependent genes (Fig. 5; Supplemental Table S4). Incontrast to the differential expression of these glutathione-dependent genes, the expression of cytosolic TRXs andassociated genes involved in thiol-disulfide exchangereactions was little or not affected in either the cat2 orcat2 gr1 genotype (Supplemental Table S5).

Analysis of Oxidative Stress and theAscorbate-Glutathione Pathway

Catalase-independent pathways of H2O2 metabo-lism depend on ascorbate, which is considered to be

Figure 4. Rescue of a wild-type phe-notype in cat2 gr1 at high CO2, andrapid daylength-dependent inductionof leaf bleaching following transfer toair. A, Plants were germinated andgrown for 25 d at high CO2 (3,000 mLL21). FW, Fresh weight. B, Plants trans-ferred from high CO2 to air in either SDor LD conditions. False-color imagingof lesions (red) are shown under eachphotograph. C, Rosette fresh weights ofplants transferred to air in either SD orLD. Histograms show means 6 SE of atleast six plants. Black asterisks indicatesignificant differences between mutantand Col-0, and red asterisks indicatesignificant differences between cat2and cat2 gr1: ** P , 0.05, *** P ,0.01.

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partly regenerated by glutathione, while NADPH isrequired for reduction of GSSG. Despite this, theeffects of gr1 and cat2 mutations on glutathione poolswere not associated with marked perturbation of leafascorbate pools, which remained more than 80% re-duced in all plants. In LD conditions, ascorbate wasdecreased in cat2 and cat2 gr1, although this smalleffect was only statistically significant for cat2 (Fig.7A). Like ascorbate, the NADP reduction state was notgreatly affected in any of the samples (Fig. 7B). Theonly significant effects were observed in cat2 gr1,which in SD had more NADPH than Col-0 and inLD less NADP+. Because of these effects, the mostreduced NADP(H) pools were observed in cat2 gr1.Assay of lipid peroxidation products using thiobarbi-turic acid also showed no significant effect betweenany of the samples in either condition (Fig. 7C).Potential problems that must be taken into account

in measuring H2O2 include low assay specificity, in-terference, and extraction efficiency (Wardman, 2007;

Queval et al., 2008). In this study, we used threedifferent techniques to assess H2O2 or reactive oxygenspecies (ROS) levels in cat2 and cat2 gr1. In situstaining using 3,39-diaminobenzidine did not produceevidence for generalized accumulation of H2O2 in gr1,cat2, or cat2 gr1 leaves in SD or LD (Supplemental Fig.S5A). Likewise, assays of extractable peroxides usingluminol chemiluminescence did not find increasedcontents in cat2 or cat2 gr1 in either daylength condi-tion (Supplemental Fig. S5B). Semiquantitative ROSvisualization within the mesophyll cells in vivo alsorevealed that the cat2 mutation caused only minorincreases in dichlorofluorescein fluorescence (Fig. 8).The cat2 gr1 double mutant showed an appreciablystronger fluorescence signal, but this effect was spe-cific to plants in SD (Fig. 8).

To further assess the impact of the mutations on theROS-antioxidant interaction, we measured the extract-able activities and expression of APX, catalase, GR,and dehydroascorbate reductase (DHAR), the enzyme

Figure 5. Comparison of gene expres-sion in cat2 and cat2 gr1 followingtransfer from high CO2 to air in either8-h (SD) or 16-h (LD) growth condi-tions. Hierarchical clustering was per-formed using MultiExperimentViewersoftware. The exploded heat map at theright shows a subcluster of 62 genesthat includes 33 JA- or wounding-associated genes (highlighted yellow).



linking glutathione and ascorbate pools. The onlyone of these enzyme activities that was significantlydifferent between Col-0 and gr1 was GR (Fig. 9A).Similarly, the gr1 mutation in the Col-0 backgroundproduced no significant change in transcripts otherthan GR1 (Fig. 9C). Decreased catalase activity in cat2was associated with an increase in APX activity in bothconditions (Fig. 9A). Increased APX activity was ac-companied by induction of transcripts for APX1, en-coding the cytosolic isoform (Davletova et al., 2005). Ofeight APX transcripts present on the chip, only APX1was significantly increased in cat2, and as for APXactivity, this effect was similar in both SD and LD (Fig.

9C). Induction of APX1 in cat2 was accompanied byinduction of a cytosolic DHAR (DHAR2), although thiseffect was stronger in SD than in LD and was notassociated with a marked increase in the overall ac-tivity of this enzyme. Compared with Col-0, effects incat2 gr1 were similar to those observed in cat2 (i.e.induction of APX1 in both SD and LD, an increase inextractable APX in the two conditions, and inductionof DHAR2 transcripts, particularly in SD; Fig. 9).Overall, the presence of the gr1 mutation in Col-0 orcat2 backgrounds did not greatly affect the responsesof these antioxidative systems relative to the respectivecontrols. Despite the oxidation of the glutathione pool

Figure 6. Glutathione contents andglutathione-related gene expression incat2 and the cat2 gr1 double mutant.A, Time course of changes in percent-age glutathione reduction and totalglutathione in the different lines fol-lowing transfer from high CO2 to air.Black circles, Col-0; white circles,cat2; white triangles, cat2 gr1. Top,1003GSH/total glutathione (the num-bers indicate GSH to GSSG ratio for thefinal time points). Bottom, total gluta-thione (GSH + 2 GSSG). Error bars thatare not apparent are contained withinthe symbols. FW, Fresh weight. B, Ex-pression values of significantly differ-ent glutathione metabolism transcriptsin cat2 and cat2 gr1 relative to Col-0.Black bars, cat2; white bars, cat2 gr1.The top two panels show genes thatwere induced, while the bottom panelshows genes that were significantlyrepressed in at least one sample type.

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

SA Signaling and Pathogen Reponses

Given the observed effects on JA-associated genes(Fig. 5; Table I; Supplemental Table S4) and the oppo-sition between JA signaling and SA-dependent path-ways (Dangl and Jones, 2001; Koorneef et al., 2008), weanalyzed SA and expression of SA marker genes ingr1, cat2, and cat2 gr1 in both daylength conditions.SA-dependent PR genes (PR1, PR2) were both signif-icantly induced in cat2 in LD but less strongly or notat all in SD (Fig. 10A). Induction of SA-dependentPR genes in cat2 in LD was antagonized by the gr1mutation, producing significantly lower expression

in cat2 gr1. Leaf SA contents were similar in allsamples in SD, although slightly decreased in cat2gr1, whereas in LD H2O2-induced accumulation wasobserved in cat2 but not in cat2 gr1 (Fig. 10A). The gr1mutation also decreased basal SA levels in the Col-0background in LD (Fig. 10A).

We investigated the response of gr1 to infection withthe virulent bacterium Pseudomonas syringae pv tomatostrain DC3000 (Fig. 10B). Bacterial growth in gr1 leaveswas intermediate between that observed in Col-0and the npr1-1 mutant, in which cytosolic redox-modulated NPR1 function is lacking (Mou et al.,2003). In agreement with the data of Figure 10A,increased sensitivity to bacteria in gr1 was associatedwith lower total SA levels than in Col-0 (Fig. 10B).

DISCUSSION

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

GR1 Plays a Nonredundant Role in H2O2 Metabolismand Signaling

In this study, the cat2 mutant was used both as areference GSSG-accumulating H2O2 signaling systemand as a genetic background in which to directlyexplore the GR1-H2O2 interaction. Because daylength-dependent effects have been described for redox sig-naling during pathogen and oxidative stress responses(Dietrich et al., 1994; Karpinski et al., 2003; Quevalet al., 2007; Vollsnes et al., 2009), we analyzed theimportance of GR1 in Col-0 and cat2 backgrounds intwo growth daylengths.

Our analysis of gr1 in the Col-0 background confirmthe results of Marty et al. (2009) that GR1 is notrequired for growth in optimal conditions. However,GR1 plays an important role when intracellular H2O2production is increased and during pathogen chal-lenge. The effect of gr1 on the cat2 phenotype contrastswith results in which plants were exposed to exoge-nous H2O2 present in agar, where no difference wasfound between gr1 and Col-0 phenotypes, even athighly challenging H2O2 concentrations (Marty et al.,2009). This suggests that the site of H2O2 production iscrucial. Enhanced H2O2 availability in cat2 leavesplaced in air occurs intracellularly through the pho-torespiratory enzyme, glycolate oxidase, which islocated in peroxisomes. In these conditions, redoxperturbation in cat2 produces similar effects to thoseobserved in stress conditions, notably oxidation andaccumulation of glutathione and changes in the abun-

Figure 7. Leaf ascorbate, NADP(H), and thiobarbituric acid-reactivesubstances (TBARS) in the four genotypes placed in SD (left) and LD(right) in air following growth at high CO2. A, Ascorbate (white bars)and dehydroascorbate (black bars). B, NADP+ (white bars) and NADPH(black bars). C, TBARS. For A and B, numbers above each bar indicatepercentage reduction states [100 ascorbate/(ascorbate + dehydroascor-bate) in A and 100 NADPH/(NADP+ + NADPH) in B]. Samples weretaken 4 d after transfer to air. All data are means 6 SE of threeindependent leaf extracts. Asterisks within blocks indicate significantdifferences from Col-0 values in the same condition: * P , 0.1. FW,Fresh weight.



dance of many stress-related transcripts. GR activityhas been detected in pea leaf peroxisomes, and it islikely that GR1 is found in Arabidopsis peroxisomes aswell as in the cytosol (Jimenez et al., 1997; Kaur et al.,2009). Therefore, it is possible that GR1 is important inperoxisomal ascorbate- and/or glutathione-dependentH2O2 metabolism. At least one (APX3) and possibly asmany as three (APX3, APX4, APX5) Arabidopsis APXsare targeted to peroxisomes or the peroxisomal mem-brane (Narendra et al., 2006), but the correspondingtranscripts were not significantly induced in gr1, cat2,or cat2 gr1 (Fig. 9). Most of the antioxidative genes thatwere induced in cat2 and cat2 gr1 encoded cytosolicenzymes, including APX1, DHAR2, and MDAR2(Supplemental Table S3). Our analysis thus points totight coupling between H2O2 produced in the perox-isomes and cytosolic antioxidative systems.

The dramatic accumulation of GSSG in cat2 gr1placed in air shows that GSH regeneration throughalternative pathways is rapidly exceeded under con-ditions of enhanced intracellular H2O2 availability. Toour knowledge, the accumulation of GSSG in cat2 gr1(up to 2 mmol g21 fresh weight; Fig. 6A) exceedspreviously reported values for this compound in leaftissues. Further work is required to identify the com-partments in which such marked accumulation ofGSSG occurs. Analyses of the gr1 single mutant inair and the cat2 gr1 double mutant at high CO2 showthat mild perturbation of the glutathione pool is notsufficient to produce marked phenotypic effects, whileeffects produced by the more severe perturbation ofglutathione in cat2 gr1 in air were dependent ongrowth daylength. Lesions were not observed in cat2gr1 in SD, even though these plants showed the mostdramatic GSSG accumulation and the lowest GSH toGSSG ratios. Oxidized glutathione pools are consid-ered to be among the cellular factors underlyingdormancy and cell death (Kranner et al., 2002, 2006).

However, our data show that although growth arrestwas linked to glutathione perturbation in both day-lengths, leaf cells can tolerate extreme perturbation ofintracellular thiol-disulfide state without undergoingbleaching (Fig. 5). Analysis of ascorbate, thiobarbituricacid-reactive substances, and ROS also provided littleevidence that oxidative stress was stronger in LDcompared with SD.

The dramatic perturbation of glutathione pools incat2 gr1 compared with cat2 provides direct evidencefor the role of GR1 in intracellular H2O2 metabolism.Analysis of ROS suggests that H2O2 accumulation incat2 is localized and/or minimized by metabolismthrough ascorbate- and/or glutathione-dependentpathways. Little or no detectable increase in H2O2 incat2 compared with the wild type is consistent withprevious studies of catalase-deficient tobacco andbarley (Hordeum vulgare) lines (Willekens et al., 1997;Noctor et al., 2002; Rizhsky et al., 2002). Althoughcertain antioxidative enzymes and transcripts wereup-regulated in cat2 and cat2 gr1, these effects were atleast as marked in SD as in LD. ROS signal intensitywas increased in cat2 gr1 in SD, but increases were lessevident in this genotype in LD (Fig. 8), conditions inwhich bleaching occurred (Fig. 5). One explanation ofthe requirement of LD conditions for leaf bleaching isthat other daylength-linked signals are required inaddition to oxidative stress intensity.

The phenotype of cat2 gr1 in LD contrasts withobservations of tobacco plants deficient in both cyto-solic APX and catalase, which showed an amelioratedphenotype compared with parent lines (Rizhsky et al.,2002). One explanation of the different effects of GRand APX deficiency in catalase-deficient plants is thatsome proportion of H2O2 is metabolized throughGR-dependent but ascorbate-independent pathways.Hence, as well as GSH oxidation by dehydroascorbate,glutathione perturbation in cat2 may occur through

Figure 8. In vivo visualization of ROS usingdichlorofluorescein fluorescence. Leaf mesophyllcells of plants grown at high CO2 and thentransferred to air for 4 d in SD or LD were imagedin intact tissue by confocal microscopy. Each pairof images shows the same area measured forgreen dichlorofluorescein fluorescence (left) andred chlorophyll autofluorescence (right). Repre-sentative examples from three independent ex-periments are shown. Magnification was the samefor all samples. [See online article for colorversion of this figure.]

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enzyme-catalyzed peroxidation of GSH (e.g. catalyzedby certain GSTs, as discussed further below). Althoughincreases in APX activities in cat2 and cat2 gr1 wereassociated with induction of APX1 (Fig. 9), severalglutathione-associated genes with potential peroxida-tive functions were also induced in these lines (Fig. 6).

H2O2 and Glutathione Modulate the Expression ofSpecific Glutathione-Associated Genes in aDaylength-Dependent Manner

Although there was no evidence of oxidative stressor phenotypic effects in the gr1 single mutant, tran-scriptomes in this line partly overlapped with that ofthe H2O2 signaling reference system, cat2. Both tran-scriptomes were found to be highly dependent on

daylength. This observation suggests that modulationof redox-linked gene expression by growth daylengthis not restricted to oxidative stress or is a secondaryconsequence of stress-induced phenotypes.

Fewer genes were affected in gr1 than in cat2. Genesaffected in cat2 but not gr1 could reflect H2O2 signalingthat occurs independently of changes in glutathione (Fig.1A). The difference could also be related to the strongerperturbation of the glutathione pool in cat2 (Fig. 1B). Ifthe difference can be partly explained by weakerchanges in glutathione in gr1, then the overlap in tran-scriptomes we report could provide a minimum esti-mate of the importance of glutathione in H2O2 signaling

Many TRX-linked components play roles in oxida-tive stress (Dietz, 2003; Rey et al., 2005; Perez-Ruizet al., 2006), but none of these transcripts was induced

Figure 9. Major antioxidative enzyme activities and transcript levels in Col-0, gr1, cat2, and cat2 gr1 after transfer to air in SD(gray backgrounds) or LD (white backgrounds). A, Enzyme activities. The x axis numbers refer to Col-0 (1), gr1 (2), cat2 (3), andcat2 gr1 (4). Units are mmol mg21 protein min21 (catalase) and nmol mg21 protein min21 (other enzymes), and values are meansof three independent extracts. Asterisks indicate significant differences from Col-0 at P, 0.05. B, Simplified scheme of catalase-and ascorbate-dependent H2O2 metabolism. C, Abundance (log2 scale, relative to Col-0) of corresponding transcripts for whichprobes were present on the CATMA array. Asterisks indicate transcripts that showed significant same-direction differences fromcontrols in both biological replicates. Where no bar is apparent, transcripts were almost identical in abundance in the mutantand the corresponding Col-0 control.



by increased H2O2 availability in cat2 or cat2 gr1. Alack of response was also observed for chloroplastferredoxin-TRX reductase subunit genes and for thetwo cytosolic NTRs and cytosolic TRXs (SupplementalTable S5). The only TRX-linked gene that was signif-icantly induced by H2O2 was GPX6, and this effect waslargely independent of both daylength and the gr1mutation (Fig. 6).

In contrast to the lack of response of TRX systems,H2O2- or gr1-driven perturbation of glutathione triggeredchanges in the abundance of transcripts of severalglutathione-associated genes, notably GSTs and GRXs.Analysis of GST functions is complicated by the num-ber of genes in plants, notably due to the presence oflarge plant-specific phi and tau subclasses, as well assubstrate overlap between the different enzymes. Ourtranscript profiling analysis showed that H2O2 modi-fied transcript levels for two phi class (GSTF2, GSTF8)and three tau class (GSTU7, GSTU8, GSTU19) en-zymes and that in all cases, these effects were furthermodulated by daylength and/or the gr1 mutation.Type F GSTs exhibit both conjugase and peroxidaseactivities, with GSTF8 showing considerable activityagainst cumene hydroperoxide (Wagner et al., 2002;Dixon et al., 2009). As well as GSTFs, some GSTUs,including GSTU8, show quite high peroxidase activity(Dixon et al., 2009).

Analysis of GRX function is also complicated by thenumber of genes, notably due to a large subclass that isspecific to higher plants. At least 30 GRX genes havebeen described in Arabidopsis (Lemaire, 2004; Meyeret al., 2008). Based on active-site sequences, these aresubclassified into CPYC, monothiol CGFS, and CC-type GRX, with the last subclass being specific to landplants (Lemaire, 2004). Although at least one CGFSGRX is implicated in oxidative stress (Cheng et al.,2006), no CPYC or CGFS GRXs showed differentialexpression in gr1, cat2, or cat2 gr1 in either photoperiodcondition (Supplemental Table S5). All differentiallyexpressed GRX genes observed in our analysis belongto the CC-type subclass, consisting of 20 identifiedmembers that are thought to encode cytosolic proteins.The functions of most CC-type GRXs remain obscure,but two have been implicated in the regulation ofgene expression by interacting with TGA transcriptionfactors. GRX480 overexpression represses the JAmarker gene, PDF1.2, while ROXY1 is required forpetal development (Ndamukong et al., 2007; Li et al.,2009). The predicted protein encoded by the GRX-likesequence (Fig. 6B) has an active-site CCMS motif thatis shared with nine annotated GRX proteins, andBLAST analysis revealed that the protein has 58% to60% amino acid identity with five of these CCMSGRXs. The H2O2- and/or gr1-dependent changes in

Figure 10. Pathogen-associated responses in GR-deficient lines. A, PR gene expression and SAcontents in Col-0, gr1, cat2, and cat2 gr1 in twophotoperiods after transfer from high CO2 to air.Top, 8-h days; bottom, 16-h days. Data are means6SE of two to three biological samples. FW, Freshweight. B, Bacterial resistance and SA contents inCol-0 and gr1 grown under standard conditions inair. Asterisks indicate significant differences be-tween mutants and Col-0 (* P , 0.1, ** P , 0.05,*** P , 0.01), while pluses indicate significantdifferences between cat2 and cat2 gr1 (+ P , 0.1,++ P , 0.05, +++ P , 0.01).

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

GR1 Is Required for SA Responses and for Expression

of Genes Involved in JA Signaling

The cat2 mutation causes accumulation of SA andinduction of SA responses in LD but not SD (Fig. 10).GR1 function appears to be required for optimal SAproduction, as this was compromised in the gr1 singlemutant relative to Col-0 and in cat2 gr1 relative to cat2(Fig. 10). Furthermore, gr1 showed decreased resis-tance to bacteria, while induction of PR genes in cat2gr1 was compromised compared with cat2. As well aseffects on SA production itself, the gr1 mutation couldinterfere with SA-dependent gene expression by al-tered redox regulation of NPR1 (Mou et al., 2003; Tadaet al., 2008). Because loss of GR1 function in an H2O2signaling context produces accelerated lesion forma-tion, the enzyme appears to be required both to restrictleaf bleaching and to enable the induction of SA andPR genes.Several antioxidative genes can be induced by JA,

including genes encoding enzymes of glutathionesynthesis (Xiang and Oliver, 1998; Sasaki-Sekimotoet al., 2005). Recent reports implicate glutathione-associated components in JA signaling (Ndamukonget al., 2007; Koorneef et al., 2008; Tamaoki et al.,2008). In both Col-0 and cat2 backgrounds, the gr1mutation affected a suite of JA-associated genes in adaylength-dependent manner. We observed inductionof JA genes in cat2 and gr1 single mutants in SD butrepression in all mutant genotypes in LD. This mayindicate (1) an optimum glutathione redox status forinduction of JA genes and (2) daylength-dependentsignals that operate to modulate the impact of changesin glutathione status on JA signaling. The daylength-and gr1-modulated expression of several GRX andGSTs suggests that these components may functionallylink glutathione and JA signaling. Such componentscould include GRX480 (Ndamukong et al., 2007),GSTU8-dependent formation of glutathione-oxylipinconjugates (Mueller et al., 2008), and GSTU6, which isamong the JA-dependent genes that are rapidly in-duced by wounding (Yan et al., 2007). In our analysis,similar expression patterns were observed for theseglutathione-associated genes and for a suite of JA-dependent genes. Other genes that link glutathioneand JA may include GSTU7, GSTU19, and MRP2,which were induced most strongly in cat2 gr1 in SDand less strongly in cat2 and cat2 gr1 in LD. Thesegenes are up-regulated by phytoprostane or 12-oxo-phytodienoic acid treatment (Mueller et al., 2008).Several of the glutathione-associated genes shownin Figure 6 are rapidly induced by methyl jasmonate,12-oxo-phytodienoic acid, phytoprostane, or wound-ing (Supplemental Fig. S6). Based on JA- or oxylipin-dependent expression patterns, the activities of GSTsagainst electrophilic metabolites, and interactions ofCC-type GRX with transcription factors (Wagner et al.,

2002; Ndamukong et al., 2007; Yan et al., 2007; Muelleret al., 2008; Li et al., 2009), it is possible that severalGSTs and CC-type GRX could be involved in linkingglutathione to JA signaling.

Interestingly, the glutathione-deficient pad2 mutantis compromised in insect resistance (Schlaeppi et al.,2008). Glutathione status is modulated by variousstresses, notably biotic challenge, while sulfur defi-ciency, which can induce JA synthesis genes (Hiraiet al., 2003), is an important determinant of glutathioneconcentration. As well as the influence of glutathionestatus, our data also underline the potential importanceof daylength context in modulating redox-triggeredsignaling through the JA pathway.

CONCLUSION

While TRX- and glutathione-dependent pathwayshave overlapping functions in plants (Reichheld et al.,2007; Marty et al., 2009), this study shows that GR1plays specific roles in intracellular H2O2 metabolism,in daylength-linked control of phytohormone geneexpression, and in certain responses to biotic stress.The transcriptomic patterns we report also point toglutathione- and TRX-specific pathways in redox sig-naling, with relatively little cross talk between the twopathways at the level of gene expression.

Two nonexclusive hypotheses could explain howthe GR-glutathione system influences H2O2-linkedgene expression. In the first, glutathione turnoverwould influence H2O2 concentration through its anti-oxidative function. Most of our observations do notsuggest that this is the main cause of the gr1-linkedchanges in gene expression, although in situ visuali-zation of ROS suggests that it could contribute todifferences between cat2 and cat2 gr1, particularly inSD. In the second hypothesis, glutathione statuswould be a key part of H2O2-triggered signal trans-duction. This hypothesis receives support from severalof our observations, notably the partial overlap be-tween cat2 and gr1 transcriptomes, but further work isrequired to confirm the role of glutathione status per se.

MATERIALS AND METHODS

Plant Material

Arabidopsis (Arabidopsis thaliana) mutant lines carrying T-DNA insertions

in the GR1 gene (At3g24170) were identified using insertion mutant informa-

tion obtained from the SIGnAL Web site (http://signal.salk.edu), and seeds

were obtained from the Nottingham Arabidopsis Stock Centre (http://nasc.

nott.ac.uk). The default gr1 line was gr1-2 (SALK_060425). A second gr1

insertion mutant (SALK_105794), named gr1-1 byMarty et al. (2009), was used

to confirm the phenotypic effects of the cat2 gr1 interaction. After identifica-

tion of homozygotes, all further analyseswere performed on plants grown from

T3 seeds. The cat2 line was cat2-2 (Queval et al., 2007), now renamed cat2-1.

Identification of Homozygous gr1 Insertion Mutants

Leaf DNAwas amplified by PCR (30 s at 94�C, 30 s at 60�C, 1 min at 72�C,30 cycles) using primers specific for left T-DNA borders and the GR1 gene



(Supplemental Table S1). Fragments obtained were sequenced to confirm the

insertion site. Zygosity was analyzed by PCR amplification of leaf DNA, and

RT-PCR analysis of GR1 transcripts was performed using gene-specific

primers (Supplemental Table S1).

Plant Growth and Sampling

Seeds were incubated for 2 d at 4�C and then sown either on agar or in soil

in 7-cm pots. Plants were grown in a controlled-environment growth chamber

at the specified photoperiod and an irradiance of 200 mmol m22 s21 at leaf

level, 20�C/18�C, 65% humidity, and given nutrient solution twice per week.

The CO2 concentration was maintained at 400 mL L21 (air) or 3,000 mL L21

(high CO2). Samples were rapidly frozen in liquid nitrogen and stored at

280�C until analysis. Unless otherwise stated, data are means 6 SE of three

independent samples from different plants, and significant differences are

expressed using t test at P , 0.1, P , 0.05, and P , 0.01.

Pathogen Tests

The virulent Pseudomonas syringae pv tomato strain DC3000 was used for

resistance tests in a medium titer of 53 105 colony-forming units mL21. Whole

leaves of 3-week-old plants grown in a 16-h photoperiod were infiltrated

using a 1-mL syringe without a needle. Leaf discs of 0.5 cm2 were harvested

from inoculated leaves at the appropriate time points. For each time point,

four samples were made by pooling two leaf discs from different treated

plants. Bacterial growth was assessed by homogenizing leaf discs in 400 mL of

water, plating appropriate dilutions on solid King B medium containing

rifampicin and kanamycin, and quantifying colony numbers after 3 d.

Microarray Analysis

Microarray analysis was performed using the CATMA arrays containing

24,576 gene-specific tags corresponding to 22,089 genes from Arabidopsis

(Crowe et al., 2003; Hilson et al., 2004) plus 1,217 probes for microRNA genes

and putative small RNA precursors (information available at http://urgv.

evry.inra.fr/projects/FLAGdb++). This array resource has been used in 40

publications over the last 5 years using a protocol based on technical repeats of

two independent biological replicates produced from pooled material from

independent plants (Achard et al., 2008; Besson-Bard et al., 2009; Krinke et al.,

2009), and this approach was adopted in this study. For each biological repeat

and each point, RNA samples were obtained by pooling leaf material from

independent sets, each of two plants. Samples of approximately 200 mg fresh

weight were collected from plants after 3.5 weeks of growth in the conditions

specified above. Total RNA was extracted using Nucleospin RNAII kits

(Macherey-Nagel) according to the supplier’s instructions. For each compar-

ison, one technical replication with fluorochrome reversal was performed for

each biological replicate (i.e. four hybridizations per comparison). The label-

ing of complementary RNAs with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-

NEN Life Science Products), the hybridization to the slides, and the scanning

were performed as described by Lurin et al. (2004).

Statistical Analysis of Microarray Data

Experiments were designed with the statistics group of the Unite de

Recherche en Genomique Vegetale. Normalization and statistical analysis

were based on two dye swaps (i.e. four arrays, each containing 24,576 GSTs

and 384 controls) as described by Gagnot et al. (2008). To determine differ-

entially expressed genes, we performed a paired t test on the log ratios,

assuming that the variance of the log ratios was the same for all genes. Spots

displaying extreme variance (too small or too large) were excluded. The raw P

values were adjusted by the Bonferroni method, which controls the family-

wise error rate (with a type I error equal to 5%) in order to keep a strong

control of the false positives in a multiple comparison context (Ge et al., 2003).

We considered as being differentially expressed the genes with a Bonferroni

P # 0.05, as described by Gagnot et al. (2008). Only genes that showed same-

direction statistically significant change in both biological replicates of at least

one sample type were considered as statistically significant.

Quantitative RT-PCR Analysis

RNAwas extracted using the NucleoSpin RNA plant kit (Macherey-Nagel)

and reverse transcribed with the SuperScript III First-Strand Synthesis System

(Invitrogen). ACTIN2 transcripts were measured as a control. cDNAs were

amplified using the conditions described above for analysis of genomic DNA

except that the number of cycles was 50. Quantitative RT-PCR was performed

according to Queval et al. (2007). Gene-specific primers are listed in Supple-

mental Table S1.

Enzyme Assays, Metabolite, and ROS Analysis

Extractable enzyme activities were measured as described previously

(Veljovic-Jovanovic et al., 2001). Oxidized and reduced forms of glutathione,

ascorbate, and NADP were measured by plate-reader assay as described by

Queval and Noctor (2007). SA was measured according to the protocol of

Langlois-Meurinne et al. (2005). Thiobarbituric acid-reactive substances were

assayed and calculated as described by Yang et al. (2009). In situ visualization

of ROS was performed using dichlorodihydrofluorescein-diacetate (DCFH2-

DA) by a protocol modified from Oracz et al. (2009). Leaves were vacuum

infiltrated twice for 15 min at room temperature with DCFH2-DA, carefully

rinsed, and kept in the dark. DCF fluorescence at 510 to 550 nmwas visualized

on a confocal microscope, simultaneously with red chlorophyll autofluores-

cence, using argon laser excitation at 488 nm.

Microarray data from this article will be deposited at the Gene Expression

Omnibus and can be consulted at CATdb (http://urgv.evry.inra.fr/cgi-bin/

projects/CATdb/consult_expce.pl?experiment_id=256).

Supplemental Data

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

Supplemental Figure S1. Characterization of the gr1 mutant.

Supplemental Figure S2. Glutathione and ascorbate status in gr1 and cat2

mutants grown at high CO2 and then transferred to air in 8-h or 16-h

days.

Supplemental Figure S3. Analysis of the conditional genetic interaction

between cat2 and gr1 in an allelic double cat2 gr1 mutant.

Supplemental Figure S4. Quantitative PCR confirmation of changes in

JA-linked genes in cat2 and cat2 gr1.

Supplemental Figure S5. H2O2 visualization in leaves of Col-0, gr1, cat2,

and cat2 gr1 in SD and LD conditions.

Supplemental Figure S6. Genevestigator analysis (Hruz et al., 2008) of

responses of glutathione-associated genes showing significantly differ-

ent expression in cat2 and/or cat2 gr1 (Fig. 6).

Supplemental Table S1. PCR primers used in this study.

Supplemental Table S2. Summary of modified daylength-dependent gene

expression in gr1 and overlap with cat2 transcriptomes.

Supplemental Table S3. Complete data set of significantly modified genes

in gr1, cat2, and cat2 gr1 in two daylength regimes.

Supplemental Table S4. List of JA-associated genes showing differential

expression in cat2 and cat2 gr1 and relative expression values.

Supplemental Table S5. Summary of expression of GRXs, cytosolic TRXs,

and related genes in cat2 and cat2 gr1.

ACKNOWLEDGMENTS

We thank the Salk Institute Genomic Analysis Laboratory for providing

the sequence-indexed Arabidopsis T-DNA insertion mutants, the Notting-

ham Arabidopsis Stock Centre for supply of gr1 seed stocks, and Gaelle

Cassin (Institut de Biologie des Plantes) for help with preliminary character-

ization of gr1. We are also grateful to Marie-Noelle Soler and Spencer Brown

(Imagif, Centre National de la Recherche Scientifique) for advice on confocal

microscopy imaging and to Eddy Blondet (Unite de Recherche en Genomique

Vegetale) for help with microarray analyses.

Received January 24, 2010; accepted May 19, 2010; published May 20, 2010.

Mhamdi et al.


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Mhamdi et al.


ANNEX 1B

Confirmation of the genetic interaction between cat2 and gr1 knockout

mutations

ANNEX 1B. Confirmation of the genetic interaction between cat2 and gr1 knockout

mutations, presented as Supplemental Figure 3 in Mhamdi et al. (2010).

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

A

069gr1/gr1CAT2/CAT247

0.769GR1/GR1cat2/cat246

0108GR1/GR1CAT2/cat245

15.229gr1/gr1cat2/cat244

070GR1/gr1CAT2/CAT243




075gr1/gr1CAT2/cat239

052GR1/GR1CAT2/CAT238

050GR1/gr1CAT2/cat237



1.281GR1/gr1CAT2/CAT234



059GR1/GR1cat2/cat231








2.265GR1/gr1cat2/cat223












051GR1/gr1cat2/cat211
























1.281GR1/gr1CAT2/CAT234






























































































































Plantnumber

Genotype

CAT2 GR1

RosetteFW (mg)

Lesions(%)

Plantnumber

Genotype

CAT2 GR1

RosetteFW (mg)

Lesions(%)

B

***

Ros

ette

FW

(m

g)

0

0

0

Lesi

ons

(% r

oset

te a

rea)

***

***

CA

T2/

CA

T2

GR

1/G

R1

0

60

120

0

10

20

CA

T2/

CA

T2

gr1

/gr1

CA

T2/

CA

T2

GR

1/g

r1

cat2

/ca

t2 G

R1

/GR

1

cat2

/ca

t2 G

R1

/gr1

cat2

/ca

t2 g

r1/g

r1

CA

T2/

cat2

GR

1/G

R1

CA

T2/

cat2

gr1

/gr1

CA

T2/

cat2

GR

1/gr

1

Number of plants

Theoretical (n = 93)

Observed

5.8

10

5.8

3

11.6

11

5.8

8

5.8

7

11.6

10

11.6

6

11.6

7

23.3

31

C

Supplemental Figure 3. Analysis of the conditional genetic interaction between cat2 and gr1 in an allelic double cat2 gr1 mutant. F2 seeds from F1 double cat2 gr1-1 heterozygotes were germinated and grown at high CO2 for three weeks. 93 plants were transferred to air in 16h days and four days later plants were photographed, genotyped, and rosette fresh weight and lesions quantified. A, Photographs of plants four days after transfer to air with cat2 gr1 double homozygotes boxed in red. Enlarged photographs of two of the seven cat2 gr1 plants are shown below the main figure. B, List of genotypes, rosette masses and lesion areas with cat2 gr1 plants boxed in red. C, Histograms of rosette fresh weight and lesions in the different genotypes. Asterisks indicate significant difference from CAT2/CAT2 GR1/GR1 segregants.

ANNEX 2

Noctor et al., 2012 Plant Cell Environ.

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

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

Glutathione in plants: an integrated overviewpce_2400 454..484

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

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

ABSTRACT

Plants cannot survive without glutathione(g-glutamylcysteinylglycine) or g-glutamylcysteine-containing homologues. The reasons why this small mol-ecule is indispensable are not fully understood, but it can beinferred that glutathione has functions in plant develop-ment that cannot be performed by other thiols or antioxi-dants. The known functions of glutathione include rolesin biosynthetic pathways, detoxification, antioxidant bio-chemistry and redox homeostasis. Glutathione can interactin multiple ways with proteins through thiol-disulphideexchange and related processes. Its strategic positionbetween oxidants such as reactive oxygen species and cel-lular reductants makes the glutathione system perfectlyconfigured for signalling functions. Recent years have wit-nessed considerable progress in understanding glutathionesynthesis, degradation and transport, particularly in relationto cellular redox homeostasis and related signalling underoptimal and stress conditions. Here we outline the keyrecent advances and discuss how alterations in glutathionestatus, such as those observed during stress, may participatein signal transduction cascades. The discussion highlightssome of the issues surrounding the regulation of glu-tathione contents, the control of glutathione redox poten-tial, and how the functions of glutathione and other thiolsare integrated to fine-tune photorespiratory and respiratorymetabolism and to modulate phytohormone signallingpathways through appropriate modification of sensitiveprotein cysteine residues.

Key-words: Antioxidant; detoxification; oxidative stress;pathogens; redox metabolism and signalling; sulphurmetabolism; thiols.

INTRODUCTION

Glutathione (or a functionally homologous thiol) is anessential metabolite with multiple functions in plants(Fig. 1). The fundamental and earliest recognized functionof glutathione is in thiol-disulphide interactions, in whichreduced glutathione (GSH) is continuously oxidized to a

disulphide form (GSSG) that is recycled to GSH byNADPH-dependent glutathione reductase (GR). Thecentral role of glutathione in defence metabolism inanimals was established long ago, largely because selenium-dependent glutathione peroxidase (GPX) is a central pillarof animal antioxidant metabolism. Pharmacologicallyinduced GSH deficiency in newborn mammals such as ratsand guinea pigs leads to rapid multi-organ failure and deathwithin a few days (Meister 1994). In plant cells, wherereductive H2O2 metabolism has been linked to ascorbatesince the 1970s, the absolutely irreplaceable role of glu-tathione was less apparent. However, glutathione depletionin Arabidopsis knockouts lacking the first enzyme of thecommitted pathway of GSH synthesis causes embryolethality (Cairns et al. 2006). Thus, both plant and mamma-lian cells rely on at least some of the multifunctional prop-erties of glutathione for their vigour and survival.

Glutathione is the principal low-molecular-weight thiolin most cells. However, there are some intriguing varia-tions in organisms such as halobacteria, in which GSHcan be replaced by other sulphur compounds like g-glutamylcysteine (g-EC) and thiosulphate (Newton & Javor1985). Similarly, in some parasitic protozoa, trypanothione[N1,N8-bis(glutathionyl)spermidine] can substitute for glu-tathione (Fairlamb et al. 1985). Some plant taxa containglutathione homologues, in which the C-terminal residueis an amino acid other than glycine (Rennenberg 1980;Klapheck 1988; Klapheck et al. 1992; Meuwly, Thibault &Rauser 1993). These compounds include homoglutathione(g-Glu-Cys-b-Ala), which is found alongside GSH in manylegumes (MacNicol 1987; Klapheck 1988). Interestingly,gene duplication during evolution has resulted in the coex-istence of different synthetases that produce GSH or homo-glutathione (Frendo et al. 1999). Cereals produce anotherGSH variant (hydroxymethylGSH; g-Glu-Cys-Ser) throughreactions that remain to be fully elucidated but which likelyinvolve modification of GSH rather than alternative syn-thesis pathways (Klapheck et al. 1992; Okumura, Koizumi &Sekiya 2003; Skipsey, Davis & Edwards 2005a). Disulphideforms of these homologues are reducible by GR (Klapheck1988; Klapheck et al. 1992; Oven et al. 2001). Therefore,current information suggests that they do not require alter-native reductive systems. Novel homologues may remain tobe discovered (Skipsey et al. 2005a). For example, high-performance liquid chromatography (HPLC) analysis ofthiols in poplar overexpressing a bacterial form of thesecond enzyme of glutathione synthesis revealed two novel

Correspondence: G. Noctor. e-mail: [email protected]

GSH is used here to indicate the thiol (reduced) form of glu-tathione while GSSG denotes the disulphide form. The term ‘glu-tathione’ is used where no distinction is drawn or both forms maybe concerned.

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

© 2011 Blackwell Publishing Ltd454

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

Like other thiols, glutathione can undergo numerousredox reactions. As well as GSSG, oxidized forms notablyinclude the formation of ‘mixed disulphides’ with proteinsand other thiol molecules. Other oxidized forms includethiyl radicals, sulphenic acid (SOH) and, possibly, sulphinic(SO2H) or sulphonic (SO3H) acids on the cysteine moiety ofglutathione, similar to their formation on protein cysteineresidues. Moreover, a wide array of glutathione conjugatescan be formed with endogenous and xenobiotic electro-philic species (Wang & Ballatori 1998; Dixon & Edwards2010) while interactions with the nitric oxide (NO) systemvia formation of S-nitrosoglutathione (GSNO) broaden thescope of glutathione as a reservoir of signalling potential(Lindermayr, Saalbach & Dürner 2005).

It has long been recognized that GSH is oxidized byreactive oxygen species (ROS) as part of the antioxidantbarrier that prevents excessive oxidation of sensitive cellu-lar components. Unlike the oxidized forms of many otherprimary and secondary metabolites that can also react withROS, GSSG is rapidly recycled by the GRs in keyorganelles and the cytosol (Halliwell & Foyer 1978; Smith,Vierheller & Thorne 1989; Edwards, Rawsthorne & Mul-lineaux 1990; Jiménez et al. 1997; Chew, Whelan & Millar2003; Kataya & Reumann 2010). A characteristic feature ofglutathione is its high concentration in relation to othercellular thiols. In general, glutathione accumulates to milli-molar concentrations, with tissue contents well in excess offree cysteine. A second key characteristic of the cellularglutathione pool is its high reduction state. In the absence of

stress, tissues such as leaves typically maintain measurableGSH: GSSG ratios of at least 20:1 (e.g. Mhamdi et al.2010a). It is important to note that this is an average valueacross tissues, and that ratios may be higher (e.g. cytosol) orlower (e.g. vacuole) in specific subcellular compartments(Meyer et al. 2007; Queval et al. 2011).

GLUTATHIONE ACCUMULATION, TURNOVERAND TRANSPORT

GSH biosynthetic pathway

As in animals, GSH is synthesized in plants from its con-stituent amino acids by two ATP-dependent steps (Rennen-berg 1980; Meister 1988; Noctor et al. 2002a; Mullineaux &Rausch 2005). Each of the synthetic enzymes is encoded bya single gene (May & Leaver 1994; Ullman et al. 1996), andArabidopsis knockout lines for either have lethal pheno-types. While knocking out expression of GSH1, encodingg-EC synthetase (g-ECS), causes lethality at the embryostage (Cairns et al. 2006), knockouts for GSH2, encodingglutathione synthetase (GSH-S), show a seedling-lethalphenotype (Pasternak et al. 2008). Using forward geneticsapproaches, several mutants have been identified in whichdecreased GSH contents are caused by less severe muta-tions in the GSH1 gene. Of these, the rml1 (rootmeristem-less1) mutant, which has less than 5% of wild-typeglutathione contents, shows the most striking phenotypebecause it fails to develop a root apical meristem (Vernouxet al. 2000). In other mutants, in which glutathione isdecreased to about 25 to 50% of wild-type contents, devel-opmental phenotypes are weak or absent, but alterationsin environmental responses are observed. In cad2, lower

Defence/Primary

C and N metabolism Secondary metabolism

Xenobioticdetoxification

Defence/

Detoxificationmetabolism

γ-EC GSHCys

Glu Gly

S metabolism

GS-conjugates Heavy metal

detoxification

Phytochelatinsy

NADPH

NADP+

Pyridine nucleotide

ROS GSNO RNS

signalling

GR

GSSGNADPHy

synthesis and reduction ROS

signalling

Redox signalling

Figure 1. General overview of some of the most important glutathione functions (synthesis, redox turnover, metabolism, signalling). Cys,cysteine; g-EC, g-glutamylcysteine; GS-conjugates, glutathione S-conjugates; GSNO, S-nitrosoglutathione; Glu, glutamate; Gly, glycine;RNS, reactive nitrogen species; ROS, reactive oxygen species.

Glutathione status and functions 455

© 2011 Blackwell Publishing Ltd, Plant, Cell and Environment, 35, 454–484

glutathione is associated with enhanced cadmium sensitiv-ity, whereas rax1 was identified by modified APX2 expres-sion and pad2 shows decreased camalexin contents andenhanced sensitivity to pathogens (Howden et al. 1995;Cobbett et al. 1998; Ball et al. 2004; Parisy et al. 2006). Inaddition to genetic approaches, a pharmacological tool thathas frequently been used to deplete glutathione is buthion-ine sulphoximine (BSO), a specific inhibitor of g-ECS (Grif-fith & Meister 1979).

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

Regulation of biosynthesis

Many factors affect the synthesis of glutathione, but themost important are considered to be g-ECS activity andcysteine availability. Accordingly, constitutive increases inglutathione can be produced either by overexpression ofthe first enzyme of the committed glutathione synthesispathway or of enzymes involved in cysteine synthesis(Strohm et al. 1995; Noctor et al. 1996, 1998; Creissen et al.1999; Harms et al. 2000; Noji & Saito 2002; Wirtz & Hell2007). Other factors that may affect GSH contents incertain conditions include glycine and ATP (Buwalda et al.1990; Noctor et al. 1997; Ogawa et al. 2004). Increasedg-ECS activity may result from transcriptional or post-transcriptional changes (May et al. 1998), but so far rela-tively few conditions have been shown to cause markedinduction of GSH1 or GSH2 transcripts. Both genes areinduced by jasmonic acid (JA) and heavy metals (Xiang &Oliver 1998; Sung et al. 2009) and also respond to light andsome stress conditions such as drought and certain patho-gens. However, neither externally applied H2O2 nor intrac-ellularly generated H2O2 leads to increased abundanceof GSH1 or GSH2 transcripts in Arabidopsis, despite thewell-described increases in glutathione in these conditions(Smith et al. 1984; May & Leaver 1993;Willekens et al. 1997;Sánchez-Fernández et al. 1998; Xiang & Oliver 1998;Queval et al. 2009). While some evidence has been pre-sented that production of the g-ECS protein is regulated atthe level of translation (Xiang & Bertrand 2000), moreattention has focused on post-translational redox controls.A partially purified enzyme preparation from tobacco wasshown to be sensitive to inhibition by dithiols (Hell & Berg-mann 1990), and similar effects were subsequently reportedin other species (Noctor et al. 2002a; Jez, Cahoon & Chen2004). Recently, it has been shown that the plant g-ECSforms a homodimer linked by two disulphide bonds(Hothorn et al. 2006), one of which is involved in redox

regulation (Hicks et al. 2007; Gromes et al. 2008). This islikely an important factor in the well-known up-regulationof glutathione synthesis in response to oxidative stress.Another mode of regulation that is likely to be important inglutathione homeostasis is feedback inhibition of g-ECS byGSH. First reported for the animal enzyme (Richman &Meister 1975), this regulatory mechanism also occurs inplants (Hell & Bergmann 1990; Noctor et al. 2002a). Alle-viation of feedback inhibition is likely to be an importantmechanism driving accelerated rates of synthesis underconditions in which glutathione is being consumed (e.g.in the synthesis of phytochelatins). The mechanistic linksbetween feedback inhibition and thiol/disulphide redoxregulation of g-ECS remain to be elucidated.

Integration of glutathione synthesis withsulphur assimilation

In accordance with the observation that enhanced cysteinesupply favours glutathione accumulation, increases in GSHsynthesis are associated with up-regulation of the cysteinesynthesis pathway. For example, glutathione accumulationtriggered by oxidative stress causes accumulation of tran-scripts encoding adenosine 5’-phosphosulphate reductase(APR) and serine acetyltransferase (SAT; Queval et al.2009). While all three Arabidopsis APR genes encode plas-tidial enzymes, only one of the five SAT gene produces anisoform located in this compartment (Kawashima et al.2005). Although the mitochondrial SAT makes the majorcontribution to cysteine synthesis under standard condi-tions (Haas et al. 2008;Watanabe et al. 2008), the chloroplastSAT was the most strongly induced during H2O2-triggeredaccumulation of glutathione (Queval et al. 2009). As well asup-regulation at the transcript level, ozone exposure acti-vates at least one APR at the post-translational level (Bicket al. 2001). As in the case of post-translational activation ofg-ECS, the exact mechanisms regulating APR activationstate remain unclear. One appealing possibility is thatoxidation-triggered decreases in GSH:GSSG activate glu-tathione synthesis by increasing the chloroplast glutathioneredox potential and allowing glutaredoxin (GRX)-mediated activation of both enzymes. Consistent with thismodel, accumulation of glutathione in catalase-deficientbarley and Arabidopsis is associated with markedlyincreased chloroplast GSSG content (Smith et al. 1985;Queval et al. 2011). However, regulation could also belinked to thioredoxin (TRX) activity. Whatever the detailsof the underlying regulatory mechanisms, intracellular oxi-dative stress can drive glutathione accumulation to several-fold basal levels (Smith et al. 1984; Willekens et al. 1997).It has been estimated that a fivefold accumulation ofglutathione in Arabidopsis cat2 mutants means that theamount of sulphur in the glutathione cysteine residueapproaches that which is found in protein cysteine andmethionine residues combined (Queval et al. 2009). As wellas the changes in transcripts and extractable activities pre-viously mentioned, H2O2-triggered glutathione accumula-tion in barley is accompanied by increased uptake of

456 G. Noctor et al.


labelled sulphate (Smith et al. 1985). Accordingly, markedaccumulation of glutathione achieved by transgenicenhancement using a bacterial enzyme with both g-ECS andGSH-S activities was dependent on sufficient sulphursupply (Liedschulte et al. 2010).

Overexpression of glutathione biosynthesis

Regardless of the controls over synthesis, and the signallingroles discussed later, tissue glutathione contents can bemarkedly enriched in plants. Whereas overexpression ofEscherichia coli GSH-S in poplar produced little effect onglutathione contents in optimal conditions (Foyer et al.1995; Strohm et al. 1995), introduction of the E. coli g-ECScaused a two- to fourfold increase in leaf glutathione, andthis was observed whether the bacterial g-ECS was targetedto the cytosol or the chloroplast (Noctor et al. 1996, 1998;Arisi et al. 1997). Expression of the same g-ECS in thetobacco chloroplast also produced substantial increases inleaf glutathione (Creissen et al. 1999) whereas homologousoverexpression of g-ECS in Arabidopsis resulted in abouttwofold glutathione enrichment (Xiang et al. 2001). Morerecently, considerably greater increases in glutathione havebeen achieved by overexpression of a bifunctional g-ECS/GSH-S from Streptococcus (Liedschulte et al. 2010).

There is still some debate about the impact of increasingglutathione contents in plants. Whereas increased glu-tathione triggered by chloroplastic overexpression of g-ECSin tobacco was accompanied by oxidation and lesion forma-tion (Creissen et al. 1999), studies with the same E. coliconstruct introduced into other species did not reportmarked phenotypic effects (Noctor et al. 1998; Zhu et al.1999a). More recently, it has been shown that one of thechloroplast lines with multiple insertions shows symptomsof early leaf senescence (Herschbach et al. 2009). Anotherstudy of a single cytosolic overexpressor grown for 3 yearsin the field reported several effects, including lower biomassand photosynthesis (Ivanova et al. 2011). In terms of inter-pretation of phenotypes, a clear limitation of studies inpoplar is the difficulty of genetic studies in this species. Thisnecessitates analysis of several independent lines to becertain that the observed effects are linked to increases inglutathione. To date no marked deleterious effects havebeen reported in the tobacco overexpressors with very highglutathione (Liedschulte et al. 2010), though these lines areclearly interesting systems in which to analyse the impactof high glutathione concentrations on plant function. Thereasons for the apparent discrepancy between the studies ofCreissen et al. (1999) and Liedschulte et al. (2010) remain tobe elucidated. An important factor could be differences inthe introduced proteins. Unlike the E. coli g-ECS, the Strep-tococcus protein has both GSH-S and g-ECS activities.

Several studies have shown the benefits of elevating glu-tathione through overexpression of g-ECS. These includeenhanced resistance to heavy metals and certain herbicides(Zhu et al. 1999a; Gullner, Komives & Rennenberg 2001;Ivanova et al. 2011). Intriguingly, increased glutathione pro-duced by overexpression of g-ECS in the chloroplast was

associated with higher leaf contents of several free aminoacids, including tyrosine, leucine, isoleucine and valine(Noctor et al. 1998). This could bear some relation to redoxregulation of the enzymes of amino acid metabolism in thechloroplast, several of which are potential TRX targets(Montrichard et al. 2009).

Although overexpression of GSH-S alone has lessmarked effects on tissue glutathione contents than boostingg-ECS capacity (Foyer et al. 1995; Strohm et al. 1995; Noctoret al. 1998), the effects could be condition dependent ifGSH-S becomes limiting when g-EC supply is increased, forexample, during exposure to cadmium (Zhu et al. 1999b).Similarly, expression of a soybean homoGSH-S in tobaccowas successfully used to confer tolerance to the herbicide,fomesafen (Skipsey et al. 2005b). Interestingly, in this study,the homoGSH-S was expressed together with a homoGSH-preferring soybean GST (Skipsey et al. 2005b).

Turnover and degradation

Biochemical studies of glutathione degradation in tobaccoconducted by the Rennenberg group (Rennenberg,Steinkamp & Kesselmeier 1981; Steinkamp & Rennenberg1984, 1985; Steinkamp, Schweihofen & Rennenberg 1987)have been significantly extended over the last decade,notably by genetically based studies in Arabidopsis. Fourdifferent types of enzymes have been described that couldinitiate glutathione breakdown (Fig. 2). Some of theseenzymes could use GSH, while others act preferentially onGSSG or other GS-conjugates. Firstly, glutathione orGS-conjugates could be degraded by carboxypeptidaseactivity (Steinkamp & Rennenberg 1985), which has beendetected in barley vacuoles (Wolf, Dietz & Schröder 1996).A second type of enzyme that has been implicated inGS-conjugate breakdown is the cytosolic enzyme phytoch-elatin synthase (PCS; Blum et al. 2007, 2010). BecauseGS-conjugates are usually rapidly transported into thevacuole, the extent to which they accumulate in the cytosolremains unclear. However, some data obtained from workon Arabidopsis suggest that PCS may play some role incertain cell types or when the enzyme is activated by heavymetals (Grzam et al. 2006; Blum et al. 2007; Brazier-Hickset al. 2008).

The third type of enzyme, g-glutamyl transpeptidase(GGT), has been the focus of several research groupsin recent years. These enzymes act in the mammaliang-glutamyl cycle (Meister 1988), and catalyse the hydrolysisor transpeptidation of GSH at the plasma membrane. Theresulting g-glutamyl amino acid derivatives are furtherprocessed by g-glutamyl cyclotransferases (GGC) and5-oxoprolinase (5-OPase) to produce free glutamate. InArabidopsis, GGTs are encoded by at least three func-tional genes. Two of these (GGT1 and GGT2) encodeapoplastic enzymes with activity against GSH, GSSG andGS-conjugates (Martin & Slovin 2000; Storozhenko et al.2002). Extracellular GGT has also been described in barleyand maize (Masi et al. 2007; Ferretti et al. 2009). Unlike theircounterparts in animal cells, GGT1 and GGT2 are probably



bound to the cell wall rather than to the plasmalemmma(Martin et al. 2007; Ohkama-Ohtsu et al. 2007a). Based onphenotypes of mutants and other studies, these enzymesmay be important in countering oxidative stress or in sal-vaging excreted GSSG (Ohkama-Ohtsu et al. 2007a; Fer-retti et al. 2009; Destro et al. 2011). Besides the extracellularGGTs, Arabidopsis has at least one vacuolar GGT, which isprobably involved in the breakdown of GS-conjugates(Grzam et al. 2007; Martin et al. 2007; Ohkama-Ohtsu et al.2007b). Along with PCS, GGTs may be important inmetabolizing GS-conjugates that are formed during thesynthesis of certain secondary metabolites (Ohkama-Ohtsuet al. 2011; Su et al. 2011).

In animals, g-glutamyl peptides produced by GGT arefurther metabolized by GGC.An Arabidopsis gene (OXP1)has been identified that likely encodes 5-OPase. Thisenzyme catalyses the hydrolysis of 5-oxoproline, whichis the product of GGC activity (Ohkama-Ohtsu et al.2008). Based on 5-oxoproline accumulation in single oxp1mutants, and in triple oxp1 ggt1 ggt4 mutants that are defi-cient in the major GGT activities as well as 5-OPase, it wasproposed that the predominant pathway for GSH degrada-tion is cytosolic and initiated by GGC (Fig. 2), and not

vacuolar or extracellular GGT (Ohkama-Ohtsu et al. 2008).GGC is therefore a fourth type of enzyme potentiallyinvolved in initiating glutathione degradation, althoughboth the rat and tobacco GGC have been reported to beunable to use GSH (Orlowski & Meister 1973; Steinkampet al. 1987). The first gene encoding GGC was identified inhumans recently, but no obviously homologous sequencesexist in plants (Oakley et al. 2008). Yet other proteins maycontribute to some extent to glutathione turnover, forexample, GGP1, which has been implicated in the removalof the Glu residue from a GS-conjugate during glucosino-late synthesis (Geu-Flores et al. 2009). Because of thiscomplexity, several questions remain on glutathione degra-dation in plants. These include cellular/tissue specificities,activities against the different forms of glutathione and, insome cases, the gene identities.

Despite the plethora of possible routes of glutathionecatabolism, the extent of glutathione turnover and resyn-thesis remains unclear. Rates of glutathione catabolism inArabidopsis leaves have been estimated to be as high as30 nmol g-1 FW h-1 (Ohkama-Ohtsu et al. 2008). Extract-able Arabidopsis leaf GSH-S activities can reach10 nmol g-1 FW min-1, while the extractable activity of the

ApoplastGGT

GSSG γ-Glu-aa + cys-gly

GGT

γ-Glu-aa+ cys-gly

GSSG

GSSG

GGT

Cpep

γ-Glu-aa

GGC

γ-EC+ gly

GSHGS X

VacuoleGGC?

5OPase5-OP+ aa

or cys-gly?

GGC

GS-X

GS-X

or cys-gly?

Glu

PCS

PCS

γ-EC + gly Cpep

GGT

γ-Glu-aa+ cys(X)-gly

Cytosolγ-EC-X + gly

γ-EC-X+ gly

Figure 2. Possible pathways of glutathione degradation. For simplicity, not all possible downstream reactions are shown, for example,metabolism of g-EC by GGC. Disulphide forms of GSSG breakdown products are omitted for the same reason. As well astranspeptidation, GGT could also catalyse hydrolysis of GSH to Glu and Cys-Gly. aa, amino acid; Cpep, carboxypeptidase; GGC,g-glutamyl cyclotransferase; GGT, g-glutamyl transpeptidase; 5-OP, 5-oxoproline; 5-OPase, 5-oxoprolinase; PCS, phytochelatin synthase;X, S-conjugated compound.



rate-limiting enzyme, g-ECS, is much lower, only about0.5 nmol g-1 FW min-1 (Queval et al. 2009). This rate istherefore very close to the rates of turnover estimated byOhkama-Ohtsu et al. (2008). However, this close correspon-dence could be coincidental. Extractable g-ECS activitiesshould theoretically be higher than true in vivo fluxes,because substrates, notably cysteine, may be less limitingand feedback inhibition by glutathione negligible in assaysof desalted extracts. It should be noted, however, that redoxregulation and the absence of other factors may make itdifficult to recover true in vivo capacities of g-ECS in invitro assays. Indeed, assuming that accumulation of glu-tathione reflects neosynthesis and that there are no alter-native routes awaiting description, our recent data stronglysuggest that at least under some conditions, g-ECS can workconsiderably faster in vivo than the measured extractablevalue. Glutathione oxidation in cat2 gr1 double mutantstriggers accumulation of total glutathione to more than10-fold basal levels within 4 d (Mhamdi et al. 2010a).Withinthe first 4 h after the beginning of excess H2O2 production(a consequence of the cat2 background), about 800 nmolglutathione g-1FW was newly accumulated in cat2 gr1,that is, a mean minimum synthesis rate of around200 nmol g-1 FW h-1.

While the interplay between degradation and neosynthe-sis remains to be elucidated, typical leaf glutathione con-tents are 300 nmol g-1 FW. Thus, degradation rates of30 nmol g-1 FW h-1 would mean that some glutathionepools must turn over and be resynthesized within a fewhours. Another question concerns the possible role of glu-tathione degradation during oxidative stress. In this regard,it is interesting that GGT1 was among glutathione-associated genes that were significantly down-regulatedin GSSG-accumulating cat2 gr1 mutants (Mhamdi et al.2010a). By virtue of their activities in cleaving GS-conjugates, some enzymes implicated in glutathione degra-dation may also be important in biosynthesis pathways (e.g.Su et al. 2011; see below).

Compartmentation and transport

Glutathione is one of the major forms of organic sulphurtranslocated in the phloem (Herschbach & Rennenberg1994, 1995; Bourgis et al. 1999; Mendoza-Cózatl et al. 2008),and must therefore move between cells, either apoplasti-cally, symplastically or both. Glutathione can be detected inapoplastic extracts but at much lower levels than in wholetissue extracts (Vanacker, Carver & Foyer 1998). This is inline with immunolocalization studies that have detectedweak or no labelling in the cell wall and apoplast (Zech-mann et al. 2008). The apoplastic pool is also likely moreoxidized than many intracellular pools. Current conceptssuggest that both the apoplast and vacuole have low glu-tathione concentrations and GSH:GSSG ratios, and thatmost of the glutathione is concentrated in other compart-ments. While immunolocalization studies point to particu-larly high concentrations in the mitochondria (Zechmannet al. 2008), because of their greater volume the cytosol and

chloroplasts have been estimated to account for about 50and 30%, respectively, of total glutathione in Arabidopsisleaf mesophyll cells (Queval et al. 2011).

Uptake of both GSH and GSSG has been documented inboth cells and protoplasts (Schneider, Martini & Rennen-berg 1992; Jamaï et al. 1996). Transport across the plas-malemma could be accomplished by the oligopeptidetransporter (OPT) family. Within this family, the Brassicauncea and rice genes, BjGT1 and OsGT1, are homologousto the yeast HGT1 transporter, which can transport GSH,GSSG and GS-conjugates, but also other small peptides(Bourbouloux et al. 2000; Bogs et al. 2003; Zhang et al.2004). Of the nine annotated Arabidopsis OPT genes (Kohet al. 2002), OPT6 may play a role in long-distance transport(Cagnac et al. 2004). The OPT6 gene is highly expressed inthe vasculature, where it may transport GS-conjugates andGS-cadmium complexes as well as GSH and GSSG(Cagnac et al. 2004). Another study reported that GSH, butnot GSSG, was transported by OPT6 (Pike et al. 2009).Knockout opt6 mutants are aphenotypic, suggesting generedundancy, and the transporter may be important in trans-locating other peptides as well as glutathione (Pike et al.2009).

Several different types of transporter may be importantin translocation of glutathione between subcellular com-partments. Inner chloroplast envelope transporters haverecently been described that likely act to link plastidicg-ECS and cytosolic GSH-S via g-EC export across thechloroplast envelope (Maughan et al. 2010). These trans-porters are encoded by three genes in Arabidopsis, calledCLT1, CLT2 and CLT3. Further, the wild-type phenotypeof gsh2 knockout mutants can be restored by transforma-tion with a construct driving GSH-S expression exclusivelyin the cytosol (Pasternak et al. 2008). This observation pro-vides further evidence that GSH can be imported from thecytosol into the plastid, consistent with radiolabellingstudies of isolated wheat chloroplasts (Noctor et al. 2002a).Thus, current concepts suggest that g-EC is synthesisedexclusively in the chloroplast, then either converted toGSH in this compartment or transported to the cytosol,where part of any GSH formed can be transported into thechloroplast (Fig. 3).

Tonoplast multidrug resistance-associated protein(MRP) transporters of the ATP-binding cassette (ABC)type may act to clear GS-conjugates or GSSG from thecytosol (Martinoia et al. 1993; Rea 1999; Foyer, Theodoulou& Delrot 2001). Indeed, certain Arabidopsis MRPs arecompetent in GSSG as well as GS-conjugate transport (Luet al. 1998). Barley vacuoles can take up GSSG much morerapidly than GSH, and the removal of cytosolic GSSG mayplay a role in maintaining glutathione redox status inthis compartment (Tommasini et al. 1993). Although glu-tathione concentrations in the vacuoles of unstressed plantshave long been considered to be low or negligible (Rennen-berg 1980; Zechmann et al. 2008), accumulation of GSSG inthis compartment could be a physiologically important partof oxidative stress responses (Queval et al. 2011). Further,induction of specific MRPs occurs in response to oxidising



agents (Sánchez-Fernández et al. 1998) and also accompa-nies GSSG accumulation in Arabidopsis (Mhamdi et al.2010a).

Immunolocalization studies detect nuclear glutathioneconcentrations that are similar to those in the cytosol incells in the undividing G0 state (Zechmann et al. 2008).Other recent studies suggest that nuclear/cytosol distribu-tion of glutathione may be dynamic, with glutathione beingrecruited into the nucleus early in the cell cycle in bothmammalian and plant cells (Markovic et al. 2007; Diaz-Vivancos et al. 2010a). A nuclear redox cycle within the cellcycle has been proposed, according to which GSH movesinto the nucleus during the G1 phase: this in turn promotesoxidation of the cytosol and, consequently, enhanced glu-tathione accumulation (Diaz-Vivancos et al. 2010b). Oxida-tion of the cytosol at G1 is accompanied by enhanced levelsof ROS and lowering of the oxidative defense shield (Diaz-Vivancos et al. 2010a). The accumulated GSH is dividedbetween the daughter cells, in which the process beginsagain. These observations suggest the presence of proteinsin plants that are able to alter the permeability of nuclearpores, facilitating GSH sequestration in the nucleus. Suchproteins remain to be identified in plants. However, theanti-apoptotic factor Bcl-2 is thought to be a crucial

component regulating GSH transport into the nucleus inmammalian tissues, as it is in mitochondria (Voehringeret al. 1998). Despite the lack of evidence for GSH synthesisin the mitochondria, high glutathione concentrations havebeen detected in this organelle (Zechmann et al. 2008). It ispossible that pore-regulating proteins are involved in regu-lating mitochondrial and nuclear GSH concentrations(Fig. 3).

Like mitochondria, peroxisomes contain glutathioneand GR (Jiménez et al. 1997) while apparently lackingthe enzymes of glutathione synthesis. Immunolocalizationstudies suggest that the peroxisomal glutathione concentra-tion is similar to the cytosolic concentration (Zechmannet al. 2008), and it has been estimated in leaf mesophyll cellsto be around 3–4 mm (Queval et al. 2011). Presumably, thispool results from import across the single peroxisomalmembrane, but transporters responsible for this activityremain to be characterized.

METABOLIC FUNCTIONS OF GLUTATHIONE

Regulation of sulphur assimilation

As a significant non-protein sink for reduced sulphur, glu-tathione contents are influenced by sulphur supply. Indeed,

OPT

GSSGGSH

Apoplast

C

OPT

Vacuole MITCYT

Cys

Glu

γ-ECGSH

GSSG

MRPGSSG

GR2γ-ECS

γ-EC

Gly

GSH

CLT

Gly

GSH-SGSH-S

GSH

GSH

?GR2

GR1

GSH GSHChloroplast PERNucleus

GR1

Figure 3. Compartmentation of glutathione synthesis, reduction and transport. g-EC(S), g-glutamylcysteine (synthetase); BAG,Bcl2-associated anathogene; CYT, cytosol. PER, peroxisome; GSH-S, glutathione synthetase; GR, glutathione reductase; GSSG,glutathione disulphide; CLT, chloroquinone resistance transporter-like transporter; MIT, mitochondrion; MRP, multidrugresistance-associated protein; OPT, oligopeptide transporter.



glutathione may be one of the S-containing compounds thatlink changes in sulphur nutrition to resistance to somepathogens, a phenomenon termed sulphur-induced resis-tance (SIR; Gullner et al. 1999; Bloem et al. 2007; Zechmannet al. 2007; Höller et al. 2010). Stresses that involve an oxi-dative component also cause up-regulation of the S assimi-lation pathway. For example, exposure to ozone increasescysteine and glutathione levels, effects linked to post-translational activation of APR1 (Bick et al. 2001). Incatalase mutants, increased intracellular H2O2 triggers accu-mulation of glutathione and precursors, and this is accom-panied by accumulation of transcripts for all three APRs(Queval et al. 2009).

Glutathione is an important form of translocated organicS and may thus act as an internal ‘barometer’ of plant Sstatus (Kopriva & Rennenberg 2004). Glutathione regu-lates several steps involved in S assimilation. These includeGSH inhibition of sulphate uptake and assimilationthrough effects on specific transporters and enzymes suchas ATP sulphurylase 1 (APS1) and APR (Herschbach &Rennenberg 1994; Lappartient et al. 1999; Vauclare et al.2002; Buchner et al. 2004). However, the role of glutathionein S assimilation is complex, because as well as these repres-sive effects, GSH is required by APR as reductant toproduce sulphite (Leustek 2002). This occurs through adomain of the APR enzyme that allows it to act as a GRX,using GSH with an apparent KM value of about 1 mm (Bicket al. 1998). Further, thiol-disulphide status also acts tocontrol certain APR isoforms post-translationally throughactivation in response to increased glutathione oxidation(Leustek 2002).

The influence of sulphur assimilation activity on glu-tathione contents also underlines the close relationship

between glutathione and S status (Nikiforova et al. 2003).Arabidopsis mutants defective in certain sulphate trans-porters show decreased leaf glutathione accumulation(Maruyama-Nakashita et al. 2003). By contrast, expressionof a bacterial APR in Arabidopsis enriches tissue cysteineand glutathione contents (Tsakraklides et al. 2002). Quali-tatively similar effects are produced by overexpression ofenzymes of the cysteine synthesis pathway (Harms et al.2000; Noji & Saito 2002; Wirtz & Hell 2007).

Glutathione S-transferases

The classical reaction catalysed by GSTs is the formation ofa covalent bond between the sulphur atom of glutathioneand an electrophilic compound (Fig. 4). In plants, the activ-ity of most GSTs depends on an active site serine, whichstabilizes the GS-thiolate anion (Dixon & Edwards 2010),though in some GSTs such as the dehydroascorbate reduc-tases (DHARs) this residue is replaced by a cysteine. Thischange confers the capacity for reversible disulphide bondformation with glutathione that is part of the DHAR cata-lytic mechanism (Dixon, Davis & Edwards 2002). Longconsidered as cytosolic enzymes, several GSTs may alsolocalize at least partly to other compartments, including thechloroplast, peroxisome and nucleus (Thatcher et al. 2007;Dixon et al. 2009).

The GST family in plants is notable for its structural andfunctional diversity, and the biochemical and physiologicalfunctions of specific members remain to be elucidated. Aswell as or instead of catalysing conjugase reactions, someGSTs may have antioxidative functions.The DHAR type ofGST is one example, while several subclasses of GST haveperoxidase activity (Wagner et al. 2002; Dixon et al. 2009).

GS-X

GST

GSHCO

CHOH

COO-

CHOH HOC SG

HCHO

HCOO

GSH

GST

X

GSHCHO

CO

CH3

CHOH

CO

GSGS

GSHCHOH

CH3

CHOH

CH3

HOC-SG HCOO-

FDH FGHGLYIGLYII

Me

PCS

CO

CH3

(γ-Glu-Cys)n-Gly

MeMe

Figure 4. Detoxification pathways involving glutathione S-conjugate formation and metabolism. The glyoxalase pathway showsmethylglyoxal metabolism, but other oxo-aldehydes might also be metabolized through this route. FDH, formaldehyde dehydrogenase;FGH, S-formylglutathione hydrolase; GLYI, glyoxalase I; GLYII, glyoxalase II; GST, glutathione S-transferase; Me, heavy metal; PCS,phytochelatin synthase; X, electrophilic compound.



GSTs are able to reduce organic peroxides though somespecificity is observed between different peroxides (Wagneret al. 2002; Dixon et al. 2009; Dixon & Edwards 2010). SomeGSTs are strongly inducible by H2O2 (Levine et al. 1994;Willekens et al. 1997; Wagner et al. 2002). For example,certain GST transcripts can be considered useful markersfor increased intracellular availability of H2O2 (Vanderau-wera et al. 2005; Queval et al. 2007, 2009; Chaouch et al.2010), though several are also inducible by salicylic acid(SA; Sappl et al. 2009). The same or other GSTs may havefunctions in the detoxification of electrophilic xenobiotics,but also be important in biosynthetic or catabolic pathways.Recently described examples of biosynthetic pathwaysinvolving GSTs are the production of glucosinolates andcamalexin, discussed further later. For a more detailedrecent discussion of GSTs, we refer the reader to Dixon &Edwards (2010).

Glyoxalase and formaldehyde metabolism

Oxo-aldehydes such as glyoxal are reactive compounds thatmay interfere with sensitive cellular compounds.A commontoxic oxo-aldehyde is methylglyoxal, which can be pro-duced from triose phosphate as an intermediate in thetriose-phosphate isomerase reaction (Maiti et al. 1997;Marasinghe et al. 2005). The glyoxalase system acts toconvert these compounds to non-toxic hydroxyacids such aslactate, and consists of two enzymes acting in concert(Fig. 4). Glyoxalase I isomerizes the spontaneously gener-ated GS-adduct while the hydrolytic reaction catalysed byglyoxalase II liberates the hydroxyacid and free GSH. Inseveral plant species, overexpression of these enzymesincreases tolerance to exogenous methylglyoxal and/or salt(Singla-Pareek, Reddy & Sopory 2003; Deb Roy et al. 2008;Singla-Pareek et al. 2008).

In addition to acting as a substrate for GSTs and glyox-alases, GSH may also act in detoxification reactions viaformaldehyde dehydrogenase. Formaldehyde can enterplants through stomata or be produced by endogenousmetabolism (Haslam et al. 2002). Two enzymes, formalde-hyde dehydrogenase (FDH) and S-formylglutathionehydrolase (FGH), act to oxidize formaldehyde to formicacid, which may then be converted to CO2 or enter C1

metabolism (Fig. 4). Genes for both enzymes have beenidentified in Arabidopsis (Martínez et al. 1996; Haslam et al.2002; Achkor et al. 2003). An important feature of theencoded FDH is that it is able to act as a GSNO reductase(GSNOR; Sakamoto, Ueda & Morikawa 2002; Díaz et al.2003).

Phytochelatin synthesis

A conditionally important role of GSH is in the responseto excessive levels of heavy metals (Fig. 4). Glutathione isthe precursor of phytochelatins ([g-Glu-Cys]nGly), com-pounds that are synthesized in response to cadmium andother heavy metals. Phytochelatins sequester the metal toform a complex that is then transported into the vacuole

(Grill, Winnacker & Zenk 1987; Grill et al. 1989; Cobbett &Goldsbrough 2002; Rea, Vatamaniuk & Rigden 2004).These compounds are produced from glutathione orhomologues by PCS, a cytosolic enzyme. In Arabidopsis,there are two genes encoding PCS, one of which (PCS1) isthe gene affected by the cad1 mutation that exacerbatescadmium sensitivity (Howden & Cobbett 1992; Ha et al.1999; Cazalé & Clemens 2001). As noted previously, it ispossible that PCS has other biochemical roles, in additionto phytochelatin synthesis, for example, turnover ofGS-conjugates (Rea et al. 2004; Blum et al. 2007, 2010;Clemens & Peršoh 2009).

The importance of sufficient amounts of GSH to supportphytochelatin synthesis is evidenced by the identification ofthe cad2 mutant as affected in g-ECS, causing a decrease inleaf GSH to about 20% wild-type levels (Howden et al.1995; Cobbett et al. 1998). As well as a precursor role inphytochelatin synthesis, glutathione could be involved inheavy metal resistance via an antioxidant function: manyheavy metals are considered to provoke perturbation ofcellular redox homeostasis through several mechanisms, forexample, displacement of redox-active metals from boundsites. Increased heavy metal tolerance has been observed intransgenic lines with enhanced glutathione synthesis (Zhuet al. 1999a,b; Lee et al. 2003), contrasting with effects ofoverexpressing PCS itself, which has generally yielded lessclear-cut results (Peterson & Oliver 2006; Picault et al.2006). While both shoots and roots may make a contribu-tion to heavy metal detoxification, it is interesting that phy-tochelatin and GS-cadmium concentrations have beenreported to be much higher in the phloem than in the xylemduring exposure of B. napus to cadmium (Mendoza-Cózatlet al. 2008).

Glutathione metabolic reactions in defenceagainst biotic stress

Glutathione has long been implicated in reactions linked tosecondary metabolism and pathogen responses (Dron et al.1988; Edwards, Blount & Dixon 1991). While at least someof the effects of glutathione during interactions with patho-gens probably involve a signalling role, it now appears thatothers are linked to hormone and secondary metabolitesynthesis. Crucial information had been generated by theanalysis of glutathione-deficient mutants. The first iden-tified glutathione-deficient Arabidopsis mutant, cad2, wasreported to show unchanged resistance to virulent andavirulent strains of the oomycete Peronospora parasitica aswell as to the bacterium Pseudomonas syringae (May et al.1996b). However, a later study of cad2 and rax1-1 reportedincreased susceptibility to avirulent P. syringae (Ball et al.2004). Mutants defective in the CLT glutathione chloroplastenvelope transporters showed decreased expression of PR1and also lower resistance to the oomycete Phytophthorabrassicae (Maughan et al. 2010). Decreased PR1 expressionlinked to lower SA accumulation was also observed in gr1mutants lacking cytosolic/peroxisomal GR (Mhamdi et al.2010a).



The Arabidopsis pad2 mutant was first identified as defi-cient in camalexin, an indole phytoalexin that contains oneS atom per molecule and whose thiazole ring is derivedpartly from cysteine (Glazebrook & Ausubel 1994). Thisline shows enhanced susceptibility to various bacterial,fungal and oomycete pathogens (Ferrari et al. 2003; Parisyet al. 2006). The affected gene in pad2 is GSH1 and themutation results in GSH contents that are slightly lowerthan those in cad2 (Parisy et al. 2006). Of cad2, rax1 andpad2 mutants in GSH1, rax1 has the highest leaf glutathionecontents, pad2 the lowest while cad2 is intermediate. Theglutathione contents of these lines correlate inversely withtheir resistance to P. brassicae, though only pad2 wasreported to be markedly affected in camalexin contents andits response to P. syringae (Parisy et al. 2006). Thus, thereappears to be a certain level of glutathione required for thesynthesis of pathogen defense-related molecules anddisease resistance. This level could vary according to condi-tions and pathogen specificity. In the case of camalexinsynthesis, recent data show that GSH is required as a pre-cursor of the thiazole ring (Fig. 5a; Böttcher et al. 2009; Suet al. 2011).

As well as effects on camalexin and resistance to micro-organisms, pad2 shows decreased resistance to feeding ofinsect larvae, an effect that is linked to decreased accumu-lation of glucosinolates (Schlaeppi et al. 2008). This effectcan be rescued by supplementation with GSH but not thegeneral disulphide reductant, dithiothreitol (Schlaeppi et al.2008). Using a dedicated microarray of more than 200 geneswhose expression is associated with insect feeding, it wasshown that unlike the JA signalling mutant, coi1, the pad2mutant did not show significant differences in the expres-sion of these genes, including glucosinolate synthesis genes,in response to feeding (Schlaeppi et al. 2008). This observa-tion is consistent with a requirement for a certain level ofGSH to support glucosinolate synthesis as a sulphur source,rather than as a signalling or regulatory molecule. Indeed, ithas recently been reported that formation of the glucosino-late thioglucose moiety involves a GS-conjugate intermedi-ate and that this compound is metabolized by a g-glutamylpeptidase, GGP (Geu-Flores et al. 2009). Formation andmetabolism of such GS-conjugates parallels stages inthe camalexin synthesis pathway (Fig. 5), and the lack ofGSH for the respective GST activities may explain both

Camalexin

(b)(a)

NS

GlucosinolateS

Glc

Cys

S

??

CYPsN

SH

R C N OH

R C N O SO3

-

γ-Glu-Cys

S

Cys-Gly

S

Indole-CHCN??

CSL

Cys-Gly

S

γ-Glu-Cys-Gly

S

Indole-CHCN Indole-CHCN

GGTPCS γ-Glu-Cys-Gly

GGP

R C N OH

I d l CH CN

Indole-CHCN

S

GSTF6

Nit il id

S

R C N OH

GST ?

Tryptophan

Indole-CH2CN

CYPs

Amino acid

Nitrile oxide

CYPs

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



constitutive deficiency of camalexin and lower induction ofglucosinolates in pad2 (Parisy et al. 2006; Schlaeppi et al.2008). Both pathways shown in Fig. 5 involve formationof a GS-conjugate followed by further metabolism byGS-conjugate degrading enzymes. Studies of mutants impli-cated GGT1, GGT2 and, to a lesser extent, PCS1, in thecontrol of Botrytis-induced camalexin accumulation (Suet al. 2011), whereas ggt4 mutants were found to accumulatea GS-conjugate of the JA synthesis precursor, 12-oxo-phytodienoic acid (OPDA), during incompatible interac-tions with P. syringae (Ohkama-Ohtsu et al. 2011).

Metabolism of ROS and ascorbate

Glutathione can react chemically with ROS and also withdehydroascorbate (DHA), the relatively stable oxidisedform of ascorbate generated by dismutation of monodehy-droascorbate (MDHA). In particular, there is a closerelationship between increased availability of H2O2 and glu-tathione status. This is most evident from studies in whichH2O2-metabolizing enzymes have been genetically or phar-macologically inhibited (Smith et al. 1984; May & Leaver1993;Willekens et al. 1997; Noctor et al. 2002b; Rizhsky et al.2002; Queval et al. 2007, 2009; Chaouch et al. 2010). Time-course analyses in conditional catalase mutants have shownthat an initial conversion of GSH to GSSG, measurablewithin hours of exposure to the onset of H2O2 production, isfollowed by a several-fold induction of the total glutathionepool over the subsequent period of 3–4 d (Smith et al. 1984;Queval et al. 2009). At moderate rates of endogenous H2O2

production, this response involves a decrease in the whole-leaf GSH : GSSG ratio from above 20 to close to one(Mhamdi et al. 2010a). Such effects make glutathione statusa useful marker for oxidative stress triggered by increasedintracellular H2O2 production.

Several pathways may be involved in GSH-dependentH2O2 metabolism, while H2O2 may be metabolized by GSH-independent pathways (Fig. 6). The chemical reaction ofGSH with H2O2 is slow, but three distinct types of peroxi-dases appear as the principal candidates to link peroxidereduction to GSH oxidation (Table 1). These are ascorbateperoxidase (APX), certain types of peroxiredoxin (PRX)and GSTs. Haem-based peroxidases are divided into twosuper-families, one of which (non-animal peroxidases)contains plant enzymes and is divided into three classes(Welinder 1992).While class II haem peroxidases are foundin fungi and notably include secreted enzymes involved inlignin degradation, haem peroxidases in plants are found inclasses I or III (Zámocky, Furtmüller & Obinger 2010).Class III enzymes are found only in plants. Also known as‘guaiacol-type’ peroxidases, they are encoded by numerousgenes and are located in the apoplast or vacuole. Theirfunctions are not clearly established, but some are known tobe involved in biosynthetic processes and/or in ROS pro-duction (Bindschedler et al. 2006; Cosio & Dunand 2009). Incontrast, class I haem peroxidases include the intracellularantioxidative enzyme,APX. Like catalase,APX is relativelyspecific to H2O2 and does not metabolize other peroxides at

high rates. It can participate in the ‘ascorbate-glutathione’pathway in which H2O2 reduction is ultimately linked toNAD(P)H oxidation via ascorbate and glutathione pools.Alternatively APX activity could be coupled to NAD(P)Hoxidation independently of glutathione via MDHAR activ-ity (Fig. 6). While GSH can chemically reduce DHA at sig-nificant rates (though slower at pH 7 than at pH 8), anenzymatic link beween ascorbate and glutathione pools isprovided by DHAR. Overexpression of this enzyme under-lines the importance of GSH-dependent ascorbate pools inphysiological processes like the regulation of stomatalopening (Chen et al. 2003), but the role of DHAR in the invivo metabolism of peroxides still remains assumed ratherthan demonstrated.

In contrast to haem-based enzymes, thiol peroxidasessuch as PRX are less specific to H2O2, and can also reduceother organic peroxides, though some may have preference

NADPH

GR NTR

Glutathione TRX

MDHAR

DHAR

Ascorbate

GPXGST

GRX-PRX TRX-PRX

ROOH

APX

CAT

Figure 6. Glutathione function within major pathways forperoxide metabolism. APX, ascorbate peroxidase; CAT,catalase; DHA(R), dehydroascorbate (reductase); GPX,glutathione/thioredoxin peroxidase; GR, glutathione reductase;GRX, glutaredoxin; GST, glutathione S-transferase; MDHA(R),monodehydroascorbate (reductase); PRX, peroxiredoxin;ROOH, H2O2 or organic peroxide; TRX, thioredoxin.Ferredoxin-dependent pathways may be important in TRX andMDHA reduction in the chloroplast.



for H2O2 or relatively small peroxides (Dietz 2003; Tripathi,Bhatt & Dietz 2009).There are four types of PRX describedin plants, and three of these use either TRX or similarcomponents such as NADPH-thioredoxin reductase (NTR)C (Dietz 2003, Pulido et al. 2010). One type, PRX II, can useGSH as a reductant via GRX action (Rouhier, Gelhaye &Jacquot 2002). Peroxiredoxins also include GPXs. Origi-nally identified on the basis of their homology to animalGPX (Eshdat et al. 1997), and subsequently implicated inplant stress responses and signalling (Rodriguez Milla et al.2003; Miao et al. 2006; Chang et al. 2009), these enzymes arenow considered to act as TRX-dependent peroxiredoxins(Herbette et al. 2002; Iqbal et al. 2006; Navrot et al. 2006)and are, therefore, misleadingly named. Unlike the animalenzyme, where the reduction of peroxides involves forma-tion of a mixed disulphide between a first GSH and theselenocysteine SeOH group, the GPXs found in plants gen-erate a disulphide bridge from the initial sulphenic acid,with the disulphide intermediate being reduced back tothe dithiol form by TRX. They are therefore unlikely tobe involved in peroxide-mediated glutathione oxidation(Fig. 6). By contrast, peroxidation of GSH could be cataly-sed by GSTs (Wagner et al. 2002; Dixon et al. 2009). Otherenzymes such as methionine sulphoxide reductase (MSR)may also contribute to ROS-triggered glutathione oxida-tion (Tarrago et al. 2009). Finally, although these studies,relying predominantly on in vitro analyses, have establishedthe relative specificities in reducing substrates, it should benoted that the glutathione and TRX systems may be linkedto some extent during in vivo ROS metabolism (Micheletet al. 2005; Reichheld et al. 2007; Marty et al. 2009).

In conclusion, GSH could be linked to H2O2 and/or per-oxide reduction by at least two ascorbate-independentroutes as well as the ascorbate-glutathione pathway (Fig. 6).Among these, only GSTs appear to act as direct GPXs(Table 1), all other enzymes requiring at least one addi-tional protein to link peroxide reduction to GSH oxidation.The evidence from gene expression makes it clear thatcertain APX, GPX and GST genes are induced in responseto oxidative stress (Willekens et al. 1997; Wagner et al.2002; Levine et al. 1994; Sappl et al. 2009). Data from

transcriptomics and enzyme and metabolite assays of plantsdeficient in catalase and/or GR suggest that the ascorbate-glutathione pathway and enzymes such as GSTs may act inconcert to remove excess H2O2 and/or other peroxides(Mhamdi et al. 2010a).

Glutathione reductase

The GSSG produced by GSH oxidation is reduced by GR.While NTR can also reduce GSSG in a TRX-dependentmanner (Marty et al. 2009), this enzyme is less efficient thanGR, which is encoded by two genes in plants studied so far.Chloroplast and mitochondrial GR is encoded by GR2,while GR1 encodes a protein that is found in the cytosol andperoxisome (Creissen et al. 1995; Chew et al. 2003; Kataya &Reumann 2010). The dual targeting of these two genes istherefore sufficient to explain biochemical data on GRlocalization in all of these compartments (Edwards et al.1990; Rasmusson & Møller 1990; Jiménez et al. 1997;Stevens, Creissen & Mullineaux 2000; Romero-Puertaset al. 2006). Despite the long-standing association of GRand glutathione with resistance to various stresses (Ester-bauer & Grill 1978;Tausz, Sircelj & Grill 2004), overexpres-sion of GR in several plant species has not in itself beenreported to lead to marked increases in stress resistance(Foyer et al. 1991, 1995; Aono et al. 1993; Broadbent et al.1995; Kornyeyev et al. 2005; Ding et al. 2009). However,increases in the reduction state of the ascorbate pool inplants overexpressing GR are consistent with efficient cou-pling of the reactions of the ascorbate-glutathione pathway(Foyer et al. 1995).

While Arabidopsis T-DNA mutants for the chloroplast/mitochondrial GR2 are embryo-lethal (Tzafrir et al. 2004),gr1 knockout mutants do not show phenotypic effects,despite a 30–60% reduction in extractable enzyme activity(Marty et al. 2009; Mhamdi et al. 2010a). Genetic analyseshave shown that the aphenotypic nature of gr1 mutantsresults from partial replacement of GSSG regeneration bythe cytosol-located NTR-TRX system (Marty et al. 2009),though this is not sufficient to prevent significant accumula-tion of GSSG in the mutant. Accumulation of GSSG is

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

Enzyme ClassProsthetic orcatalytic group Oxidant Reductant Functional link to glutathione

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

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



massively increased compared with the parent lines in cat2gr1 double mutants deficient in both the major leaf catalaseand GR1 (Mhamdi et al. 2010a).This effect is associated witha much exacerbated phenotype compared with cat2, showingthat the NTR-TRX system cannot replace GR1 under con-ditions where H2O2 production is increased. Further, the gr1mutation alters Arabidopsis responses to pathogens andexpression of genes involved in defence hormone signall-ing, notably JA-associated genes (Mhamdi et al. 2010a).Although transcriptomic patterns documented in this studypoint to at most limited overlap between glutathione andTRX systems in oxidative stress functions, the observation ofMarty et al. (2009) shows that interplay is possible at thebiochemical level. For example, changes in glutathione andTRX redox states could act mutualistically to reinforce eachother during plant interactions with pathogens. This issue,which is important to defining the frequently used but vagueterm‘cellular redox state’ more clearly, remains an outstand-ing issue in understanding redox signalling in plants.

GLUTATHIONE IN PLANT DEVELOPMENT,GROWTH AND ENVIRONMENTAL RESPONSES

It has long been known that certain cell types, for example,the root quiescent centre and cells in organs such as seeds,maintain a highly oxidized intracellular state (Kranner &Grill 1996; Kranner et al. 2002, 2006).Auxin accumulation inthe root stem cell niche is dependent on the oxidized statusof the cells (Jiang & Feldman 2010). Treatment of Arabi-dopsis root tips with BSO led to disappearance of the auxinmaximum in the root tips and altered expression of quies-cent centre markers (Koprivova, Mugford & Kopriva 2010).The glutathione redox potential of such cell types is rela-tively high (i.e. positive or oxidizing). Even in cells wherethe global glutathione pool is highly reduced, compart-ments that lack GR or that are deficient in NADPH maycontain low GSH:GSSG ratios (e.g. the vacuole or endo-plasmic reticulum; Hwang, Sinskey & Lodish 1992; Enyedi,Várnai & Geiszt 2010). While an increase in the redoxpotential above the threshold-reducing value of moreredox-sensitive compartments has been linked to growtharrest and/or death (Kranner et al. 2006), cell identity has aprofound influence on the processes that govern cell fate(Jiang et al. 2006a,b) and associated responses to abioticstress (Dinneny et al. 2008).

Plant growth and auxin

The analysis of the phenotypes of glutathione-deficient Ara-bidopsis mutants has demonstrated that GSH is required forplant development fulfilling critical functions in embryo andmeristem development (Vernoux et al. 2000; Cairns et al.2006;Reichheld et al. 2007;Frottin et al. 2009;Bashandy et al.2010). The rml1 mutant, which has less than 5% of theglutathione present in the wild type, has a strong develop-mental phenotype that is characterized by a non-functionalroot meristem while the shoot meristem is largely unaffected(Vernoux et al. 2000). Crossing rml1 with ntra, ntrb double

mutants produced an additive shoot meristemless pheno-type (Reichheld et al. 2007). However, when the ntra ntrbdouble mutants were crossed with cad2 (which has about30% of the glutathione present in the wild type), the result-ant triple mutants developed normally at the rosette stageand underwent the floral transition but they producedalmost naked flowering stems (Bashandy et al. 2010). Theperturbation of the floral meristem in the ntra ntrb, cad2triple mutants was linked to altered levels and transport ofauxin, which plays an important role in the integration ofmeristem development (Bashandy et al. 2010).

Glutathione synthesis is also required for pollen germi-nation and pollen tube growth (Zechmann, Koffler &Russell 2011). In this study, glutathione depletion wasshown to result from disturbances in auxin metabolism andtransport (Zechmann et al. 2011). The auxin-resistant axr1and axr3 mutants were found to be less sensitive to BSOthan the wild-type Arabidopsis plants and treatment of thetips of primary roots with BSO altered auxin transport(Koprivova et al. 2010). However, in these experiments, theeffects of GSH on root growth could be partially reversedby dithiothreitol, suggesting that an as yet unidentified post-transcriptional redox mechanism is involved in the regula-tion of the expression of PIN proteins and hence auxintransport in roots (Koprivova et al. 2010). The stunted phe-notype of mutants such as cat2 and derived lines, whichaccumulate GSSG when grown from seed in air, may berelated to such thiol regulation of plant growth, as could theeffects of GSH on the regeneration efficiency of somaticembryos (Belmonte et al. 2005).

Cell cycle regulation

Concepts of the regulation of mitosis in animals incorporatean intrinsic redox cycle in which transient oxidations serveto regulate progression through the cell cycle (Menon &Goswami 2007; Burhans & Heintz 2009). While very fewstudies have been conducted to establish whether similarprocesses operate in the control of the plant cell cycle, therecruitment of GSH into the nucleus in the G1 phase of theplant cell cycle has a profound effect on the redox state ofthe cytoplasm and the expression of redox-related genes(Pellny et al. 2009; Diaz-Vivancos et al. 2010a,b). A subse-quent increase in the total cellular GSH pool above thelevel present at G1 is essential for the cells to progress fromthe G1 to the S phase of the cycle (Diaz-Vivancos et al.2010a,b). As the movement of GSH into the nucleus can bevisualized in the dividing cells of the developing lateral rootmeristem (Diaz-Vivancos et al. 2010a), it will be intriguingto see if this process underlies the post-transcriptionalredox regulation of the PIN proteins and auxin transport inroots (Koprivova et al. 2010).

Biotic interactions, cell death anddefence phytohormones

In addition to serving as a source of reduced S during syn-thesis of secondary metabolites, glutathione is involved in



signalling processes. Exogenous GSH can mimic fungalelicitors in activating the expression of defence-relatedgenes (Dron et al. 1988; Wingate, Lawton & Lamb 1988)including PATHOGENESIS-RELATED1 (PR1; Senda &Ogawa 2004; Gomez et al. 2004a). Moreover, accumulationof glutathione is triggered by pathogen infection (Edwardset al. 1991; May, Hammond-Kosack & Jones 1996a), and thiscan involve characteristic transient changes in glutathioneredox state (Vanacker, Carver & Foyer 2000; Parisy et al.2006). Similar changes have also been reported followingexogenous application of the defence-related hormone sali-cylic acid (SA), or biologically active SA analogs (Mou, Fan& Dong 2003; Mateo et al. 2006; Koornneef et al. 2008).Glutathione perturbation in catalase-deficient cat2 is linkedto hypersensitive response (HR)-like lesions as part of awide spectrum of defence responses that are conditionallyinduced in this line (Chaouch et al. 2010). Glutathione isalso required for the development of symbiotic N2-fixingnodules between legumes and rhizobia. Both GSH and thelegume homologue, homoGSH, are found at high concen-trations in N2-fixing nodules formed during symbioticinteractions with rhizobia: deficiency in these compoundsinhibits nodule formation (Frendo et al. 2005; Pauly et al.2006).

Thiol-disulphide status is clearly involved in the regula-tion of the SA-dependent NONEXPRESSOROFPATHOGENESISRELATEDGENES 1 (NPR1) pathway(Després et al. 2003; Mou et al. 2003; Rochon et al. 2006;Tada et al. 2008). Induction of PR gene expression by thispathway involves monomerization of the oligomeric cyto-solic protein NPR1, which unmasks a nuclear localizationsignal motif that allows the protein to relocalize to thenucleus where it interacts with TGA transcription factors,themselves redox-sensitive (Després et al. 2003; Mou et al.2003). The notion that this might be linked to glutathioneredox state via GRX activity (Mou et al. 2003) has beensuperseded by a model based on activation by TRXh (Tadaet al. 2008), some of which are known to be inducible byoxidative stress and pathogens (Laloi et al. 2004). If thismodel is correct, the well-documented ability of geneticallyor chemically increased GSH to induce PR gene expressioncould possibly be explained by redox interactions betweenglutathione and TRX.

Although activation of NPR1 involves a reductivechange, the initial trigger for the events leading to PR geneexpression involves oxidation. Excessive oxidation can alsotrigger HR, which is the most studied instance of environ-mentally induced cell death in plants. While redox changesinvolved in HR have been most closely associated withevents triggered by apoplastic ROS production, oxidativestress of intracellular origin can also trigger such effects.Several observations are suggestive of a role for glutathioneredox potential (or GSSG accumulation) in the regulationof genetically programmed cell suicide pathways. Forexample, increases in the total glutathione contents ofleaves produced by ectopic g-ECS overexpression in thetobacco chloroplast caused accumulation of GSSG andthis was associated with lesion formation and enhanced

expression of pathogenesis-related (PR) genes (Creissenet al. 1999). In addition, the HR-like lesions triggered byintracellular oxidative stress are associated with GSSGaccumulation (Smith et al. 1984; Willekens et al. 1997;Chamnongpol et al. 1998; Chaouch et al. 2010). Alterationsin pathogen responses have been described in mutants thatare partly deficient in glutathione (Ball et al. 2004; Parisyet al. 2006). However, some of our own recent work incatalase-deficient cat2 and derived Arabidopsis linessuggest that there is unlikely to be a simple relationshipbetween glutathione redox potential and engagement ofcell death. Firstly, HR-like lesions in cat2 are under day-length control and this control does not correlate with thedegree of glutathione oxidation (Queval et al. 2007). Sec-ondly, daylength control is linked to SA synthesis: HR-likelesions can be prevented by blocking SA synthesis eventhough this effect is associated with a more oxidized glu-tathione status (Chaouch & Noctor 2010; Chaouch et al.2010). Thirdly, in cat2 gr1 mutants, GSSG accumulates to agreater extent than in cat2 single mutants but this does notcause HR-like lesions (Mhamdi et al. 2010a). Despite theseobservations, however, we cannot exclude the possibilitythat values for whole-leaf glutathione status may not pre-cisely reflect events in specific intracellular compartments(Queval et al. 2011). Even if this is the case, tissue glu-tathione status appears to be a poor marker for the engage-ment of cell suicide pathways. Nevertheless, analysis ofH2O2 signalling in glutathione-deficient mutants demon-strates that some factor related to glutathione status is animportant modulator of cell death triggered by oxidativestress (authors’ unpublished results). Further, analyses ofdouble cat2 atrboh Arabidopsis mutants, which lack boththe major catalase and NADPH oxidase activities, point toa crucial role for AtrbohF in permitting accumulation ofoxidized glutathione in response to intracellular H2O2. Indouble cat2 atrbohF (but not cat2 atrbohD) mutants, glu-tathione is much less perturbed than in the cat2 singlemutant and this is associated with much lower accumulationof SA and related defence molecules (Chaouch, Queval &Noctor, unpublished results).

As well as possible roles in SA signalling, glutathionemay modulate signalling through the JA pathway, which isinvolved in the regulation of development and in responsesto necrotrophic pathogens and herbivores. JA inducesGSH1,GSH2 and GR (Xiang & Oliver 1998),as well as otherantioxidative genes (Sasaki-Sekimoto et al. 2005). In gr1mutants lacking the cytosolic/peroxisomal GR, a suite of JAgenes are repressed while introduction of the gr1 mutationinto the cat2 background modulates H2O2-triggered expres-sion of these and other JA-associated genes (Mhamdi et al.2010a). Consistent with these interactions between JA andglutathione, prior wounding was shown to induce resistanceto Botrytis cinerea, an effect that was partly or whollysuppressed in glutathione-deficient mutants (Chassot et al.2008). This effect was associated, at least in part, withimpaired induction of camalexin (Chassot et al. 2008).Among the components that could link JA signalling andglutathione are GRXs and GSTs.The SA-inducible protein,



GRX480, represses up-regulation of certain JA-inducedgenes (Ndamukong et al. 2007). As well as acting in theactivation of the SA pathway, NPR1 represses JA pathway(Spoel et al. 2003), and BSO treatment was shown toanatagonize this repressive effect (Koornneef et al. 2008).Together, these observations suggest that glutathione mayact to repress JA signalling through SA-dependent inductionof NPR1 and GRX480, both of which interact with TGAtranscription factors. However, gene expression profiles andSA contents suggest that the more oxidized glutathionestatus in gr1 is associated with repression of both SA and JApathways, and therefore that the roles of glutathione may becomplex and not limited to regulating antagonism betweenthe two hormones (Mhamdi et al. 2010a). Other possibleeffects of glutathione could occur through the regulation ofJA synthesis or excess accumulation of intermediates likeOPDA. Several GSTs are among early JA-induced genes ormay catalyse formation of GS-oxylipin conjugates (Davoineet al. 2006; Yan et al. 2007; Mueller et al. 2008). Indeed,enhanced accumulation of a GS-OPDA conjugate in ggt4mutants, which are expected to be unable to degrade vacu-olar GS-conjugates, was recently reported. However, loss ofGGT4 function did not affect contents of JA or derivativessuch as the hormonally active JA-isoleucine conjugate com-pared with wild-type plants (Ohkama-Ohtsu et al. 2011).In addition to these interactions with glutathione, JA andwounding were also shown to decrease GSNOR transcripts(Díaz et al. 2003).

Light signalling

The abundance of glutathione or its homologues in leavesdoes not vary greatly over the day/night cycle. Similarly,in soybean, all leaves except those approaching sene-scence contain similar total amounts of glutathione/homoglutathione, as illustrated in Fig. 7. However, youngpoplar trees show more variation, with a gradient fromyoung to old leaves (Arisi et al. 1997). The effect of stressessuch as drought can also influence the relative abundanceof glutathione/homoglutathione in different leaf ranks(Fig. 7). Shade conditions that involve a decrease in thered/far red ratio of the light environment favour a muchlower leaf glutathione pool relative to conditions where thered/far red ratios are similar (Bartoli et al. 2009). While leafglutathione contents are lower in plants grown under shadeconditions, leaf GSH:GSSG ratios are relatively unaffectedby light quality (Bartoli et al. 2009). Moreover, the adjust-ments in leaf glutathione pool in response to different red/far red ratios were very slow in comparison with the leafascorbate pool (Bartoli et al. 2009). Regardless of the rela-tively slow responses of the leaf glutathione pool to changesin light intensity and light quality, some potential linksbetween daylength, light signalling and glutathione statushave been reported (Becker et al. 2006; Queval et al. 2007).Relationships between glutathione and photoreceptor sig-nalling have been suggested in studies on the arsenic-tolerant mutants, ars4 and ars5 (Sung et al. 2007). The ars4mutation was identified as an allele of phytochrome A

(phyA) and caused increased BSO resistance (Sung et al.2007). The ars5 mutant, which is affected in a 26S protea-some component, had increased levels of glutathione whenexposed to arsenic, accompanied by increased GSH1 andGSH2 transcripts (Sung et al. 2009)

The redox states of the plastoquinone and TRX pools areconsidered to be important components of the redox regu-lation model for the control of gene expression that adjustsenergetic and metabolic demands to light-induced changesof the photosynthetic apparatus structure (Bräutigam,Dietzel & Pfannschmidt 2010). Glutathione has also beenimplicated in the signalling pathways that facilitate acclima-tion of chloroplast processes to high light (Ball et al. 2004).Exposure to photoinhibitory light can lead to increases inleaf H2O2 levels and to oxidation of the glutathione pool(Mateo et al. 2006; Muhlenbock et al. 2008).A light shift thatfavoured excitation of photosystem II (PSII) relative tophotosystem I (PSI) slightly increased the amounts of glu-tathione in leaves, whereas glutathione levels were slightlydecreased following a light shift that favoured excitation ofPSI relative to PSII (Bräutigam et al. 2009, 2010).Accordingto the model of Bräutigam et al. (2010), higher g-ECS activi-ties would be favoured by exposure to light wavelengthsthat predominantly drive PSII. However, this modelremains to be substantiated.

GLUTATHIONE-LINKEDSIGNALLING MECHANISMS

Redox regulation is inherent to all energy exchange pro-cesses. It is required to balance supply and demand betweenenergy-producing and energy-utilizing processes, as theseare driven by redox changes. Of the mechanistic controlsthat achieve this homeostasis, thiol-disulphide reactions arethe best characterized and probably among the most impor-tant (Buchanan & Balmer 2005). Key thiol components inenzyme regulation or ROS metabolism include TRX, GRX,glutathione, GPX and PRX (Dietz 2003; Lemaire 2004;Meyer et al. 2008; Rouhier 2010).The roles of TRX in redoxsignalling are well established. By comparison, the study ofglutathione-dependent signalling is still in its infancy,although it is interesting to note that interest in the roles ofglutathione in cellular regulation dates back many years(Wolosiuk & Buchanan 1977).The following discussion firstoutlines factors that could link glutathione to modificationof target protein activity and then analyses some of theissues surrounding the regulation and impact of alteredglutathione status in cellular signalling.

Glutaredoxins

Glutaredoxins are also known as thiol transferases. Theclassical GRX reaction encompasses the reduction of aprotein disulphide bond to two thiols with conversion of 2GSH to GSSG. Some of these enzymes can also catalyseprotein S-glutathionylation or de-glutathionylation. Landplants contain a large GRX family subdivided into threeclasses that can be distinguished on the basis of amino acid



motifs in the active site (Lemaire 2004; Rouhier 2010). Mostclass I GRXs have a TRX-like active site consisting of twocysteines separated by two intervening amino acids. Theycan catalyse thiol-disulphide exchange but also other reac-tions such as regeneration of PRX and methionine sul-phoxide reductase (MSR; Rouhier et al. 2002; Rouhier,Couturier & Jacquot 2006; Zaffagnini et al. 2008; Tarragoet al. 2009; Gao et al. 2010). Class II GRXs have only a singlecysteine at the active site and are therefore sometimes

called ‘monothiol’ GRX, though this term is potentiallymisleading as in at least some cases this cysteine may forma disulphide bridge with another cysteine located distally inthe same protein or on another GRX of the same type (Gaoet al. 2009a, 2010). Both class I and class II GRX play rolesin assembly of iron-sulphur clusters (Rouhier et al. 2007;Bandyopadhay et al. 2008; Rouhier 2010). They can alsocatalyse protein de-glutathionylation, though some class IIGRX do this in a dithiol- rather than GSH-dependent

Figure 7. The effects of ontogeny and drought stress on total tissue contents of homoglutathione plus glutathione (a) and reductionstate (b) in 5-week-old soybean (Glycine max cv Williams 82) plants. For the drought study, plants were deprived of water for 15 d untilthe soil water in the drought treatment had fallen to about half that measured under the optimal watering regime (76.97 � 2.93). Datashow the mean � SE (n = 3) and are expressed in relative units. L, leaf; N, nodule; TF, trifoliate leaf.



manner, and in vivo may depend on the TRX system fortheir regeneration (Zaffagnini et al. 2008; Gao et al. 2009a,2010). While some of these GRX have been characterizedbiochemically, their in vivo roles remain unclear. Arabidop-sis class II GRXs can interact with ion channels and havebeen implicated in responses to oxidative stress (Cheng &Hirschi 2003; Cheng et al. 2006; Guo et al. 2010; Sundaram &Rathinasabapathi 2010).

GRXs in the third class contain two adjacent cysteinesat their active site and form the largest class in landplants. Reverse genetics approaches in Arabidopsis haverevealed that three GRX forms are involved in plantdevelopment and phytohormone responses throughinteraction with TGA transcription factors. These areGRX480, implicated in SA-JA interactions, and ROXY1and ROXY2, which function in petal and flower develop-ment (Ndamukong et al. 2007; Li et al. 2009). Overexpres-sion and complementation studies point to biochemicalredundancy between these proteins (Li et al. 2009; Wanget al. 2009). Based on these observations, the specificity ofclass III GRX function may be determined by regulationof their expression patterns (Ziemann, Bhave & Zachgo2009). This notion receives some support from microarrayanalysis of GRX gene expression in mutants with pertur-bations in leaf glutathione redox state, in which specificmembers of class III were the only GRX that werefound to respond (Mhamdi et al. 2010a). Among these fourgenes was GRX480, which showed expression patternssimilar to many other JA-associated genes. In contrast,transcripts for ROXY1 or ROXY2 remained at wild-typeabundance levels.

Protein S-glutathionylation

Protein S-glutathionylation involves the formation of astable mixed disulphide bond between glutathione and aprotein cysteine residue. This reaction could modify theconformation, stability or activity of the target protein.The existence of S-glutathionylated plant proteins hasbeen known for some time (Butt & Ohlrogge 1991).However, the nature and role of this phenomenon hasonly been subject to thorough investigation relativelyrecently. Reversible S-glutathionylation is part of the cata-lytic cycle of glutathione-dependent enzymes such as GR,DHAR, MSRB1, as well as some GRXs and PRXs(Arscott, Veine & Williams 2000; Dixon et al. 2002; Tarragoet al. 2009; Rouhier 2010). Several techniques have beendeveloped to identify other target proteins, allowing aninventory of potential target proteins to be established(Ito, Iwabuchi & Ogawa 2003; Dixon et al. 2005; Micheletet al. 2005, 2008; Zaffagnini et al. 2007; Holtgrefe et al.2008; Gao et al. 2009a). Despite these advances, major gapsin our current knowledge remain, for example concerningquantification (i.e. the fraction of a given protein that isS-glutathionylated at a given time) and the in vivo signifi-cance of the modification. An additional uncertainty is thespecificity of the modification, that is, whether the modi-fied cysteine residue is glutathionylated in vivo, rather

than nitrosylated or a target of TRX (or some combina-tion of these modifications).

Most targets of glutathionylation identified to date arerelatively abundant proteins. These notably includeenzymes involved in primary metabolism such as TRXf,glyceraldehyde-3-phosphate dehydrogenase (GAPDH)and glycine decarboxylase (GDC; Michelet et al. 2005; Zaf-fagnini et al. 2007; Palmieri et al. 2010). Glutathionylation ofa TRXf cysteine found outside the active site lowers theprotein’s efficacy in activating chloroplast NADP-GAPDH,while another TRX-independent isoform of GAPDH isinhibited by glutathionylation (Zaffagnini et al. 2007). Inthe mitochondria, GDC has been shown to be inhibited byoxidative stress (Taylor, Day & Millar 2002), and is subjectto thiol modifications, including S-glutathionylation (Palm-ieri et al. 2010). This enzyme is a major producer of NADHduring conditions of active photorespiration. Mitochondrialredox homeostasis in these conditions may depend onappropriate activation of the alternative oxidase (Igamber-diev, Bykova & Gardeström 1997), which has been shown tobe redox regulated through the TRX system (Vanlerbergheet al. 1995; Gelhaye et al. 2005). The glycolytic NAD-dependent GAPDH, a cytosolic enzyme found not only inthe cytosol but also in the mitochondrion and nucleus(Giegé et al. 2003; Anderson, Ringenberg & Carol 2004), issensitive to oxidation and has been identified as a glutathio-nylated protein (Dixon et al. 2005; Hancock et al. 2006;Holtgrefe et al. 2008).

Taken together, the current evidence suggests that glu-tathione and TRX systems work closely together to fine-tune photosynthetic and respiratory metabolism throughappropriate modification of sensitive protein cysteine resi-dues. The mechanisms of S-glutathionylation may involveGRX-catalysed thiol-disulphide exchange between proteinthiol groups and GSSG, conversion of thiol groups to thiylradicals or sulphenic acids, or be GSNO mediated (Dixonet al. 2005; Holtgrefe et al. 2008; Gao et al. 2009b; Palmieriet al. 2010).The reverse reaction, deglutathionylation, couldbe mediated by certain class I and class II GRX. Currentevidence suggests that some class II GRX catalyse this reac-tion in a TRX reductase-dependent manner (Zaffagniniet al. 2008; Gao et al. 2009a, 2010). Despite the potentialphysiological importance of S-glutathionylation, and theproposed involvement of class I and class II GRX in eitherglutathionylation or de-glutathionylation, no GRX of eithertype responded at the transcript level in Arabidopsismutants with increased H2O2 and GSSG (Mhamdi et al.2010a).

Protein S-nitrosylation and GSNO reductase

GSNO can cause S-nitrosylation and S-glutathionylation ofprotein cysteine residues (Lindermayr et al. 2005, 2010;Romero-Puertas et al. 2008; Lindermayr & Durner 2009).Enzymes undergoing both modifications include GAPDHand GDC, with both effects leading to decreased activity(Lindermayr et al. 2006; Holtgrefe et al. 2008; Palmieri et al.2010). Oxidative inhibition of GAPDH may act to regulate



the oxidative pentose pathway relative to glycolysis in stressconditions (Holtgrefe et al. 2008). Inhibition of GDC bymodifications of thiols may also have physiological signifi-cance, as photorespiratory metabolism involves high-fluxpathways capable of modifying intracellular redox states(Foyer et al. 2009). S-nitrosylation of type II PRX has beenproposed to play a role in ROS signalling (Romero-Puertaset al. 2007).

It was reported that S-nitrosylation acts in opposition todisulphide reduction by TRX in the regulation of NPR1.While monomerization of the NPR1 protein to the activeform was TRX dependent, GSNO-dependent nitrosylationof NPR1 monomers was required for reoligomerization,possibly to inhibit depletion of the protein (Tada et al.2008). However, redox regulation of NPR1 and the tran-scription factors with which NPR1 interacts is turning out tobe complex. Four cysteine residues on TGA1 underwentS-nitrosylation and S-glutathionylation after GSNO treat-ment of the purified protein, while GSNO treatment ofArabidopsis protoplasts caused translocation of an NPR1-GFP fusion protein into the nucleus (Lindermayr et al.2010). The importance of S-nitrosylation in regulatingNPR1 function is in line with an important role for thismodification in plant disease resistance. Production of theplant hormone ethylene, which regulates developmentalresponses like senescence as well as responses to certainpathogens, is known to be inhibited by NO. This effectmay be mediated by inhibition of several enzymes in-volved in ethylene synthesis, including GSNO-triggeredS-nitrosylation of a specific methionine adenosyltransferase(Lindermayr et al. 2006).

Inducible NO-producing enzymes in plants remain to beclearly described, and most attention has focused on theimportance of a secondary reaction of nitrate reductase.However, studies in animals and plants have revealed thatGSNOR is a key player in regulating S-nitrosylation.These enzymes catalyse the NADH-dependent reduction ofGSNO to S-aminoglutathione, which then decomposes tofree GSH and ammonia or other compounds (Sakamotoet al. 2002; Barroso et al. 2006; Díaz et al. 2003). Originallyidentified as a formaldehyde dehydrogenase, the Arabidop-sis GSNOR appears to play a key role in biotic stressresponses, and Atgsnor1 mutants show decreased resistanceto virulent and avirulent pathogens (Feechan et al. 2005).This enzyme has also been implicated in the regulation ofcell death and other functions (Lee et al. 2008; Chen et al.2009). It is also interesting to note that carbon monoxide(CO) is now considered to be a gaseous signalling moleculein animals and plants. It is possible that glutathione isinvolved in CO signalling in Medicago sativa (Han et al.2008).

Factors affecting the glutathioneredox potential

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

Regulation of thiol-disuphide status has traditionally beenassociated with TRX (Buchanan & Balmer 2005). However,genetic support for the physiological importance ofglutathione-dependent disulphide reduction comes fromthe observations that the NADPH-TRX and glutathionesystems play overlapping roles in development (Reichheldet al. 2007; Bashandy et al. 2010). A key factor governinginteractions between glutathione and protein targets isredox potential, which depends on the relative rates of glu-tathione oxidation and its NADPH-dependent reduction.The actual glutathione redox potential is related to[GSH]2:GSSG. Thus, unlike many other redox couples (e.g.NADP+/NADPH), the glutathione redox potential dependson and can be influenced by absolute concentration as wellas by changes in GSSG relative to GSH (Mullineaux &Rausch 2005; Meyer 2008). Even if the GSH:GSSG ratioremains unchanged, decreases in glutathione concentrationalone will lead to an increase in redox potential, that is, thepotential will become more positive and thus less reducing.This is in accordance with measurements of cytosolic GRX-dependent thiol/disulphide redox potential as detected byredox-sensitive roGFP in the glutathione-deficient cad2mutant compared with the wild type (Meyer et al. 2007).Increases in the cytosolic but not plastidial redox potentialin clt1 clt2 clt3 mutants, lacking the chloroplast envelopeglutathione transporters, are also consistent with a depletedglutathione pool in the cytosol but not the chloroplast(Maughan et al. 2010).

Linking glutathione redox potential totarget proteins

No components have yet been identified that directly linkglutathione redox potential to modifications in target pro-teins. However, based on information from the much betterstudied TRX-dependent changes in protein thiol-disulphidestatus, we can infer that a change in glutathione redoxpotential of approximately 50 mV is likely to be biologicallysignificant. Such changes are sufficient to alter the balancebetween oxidized and reduced forms of TRX-regulatedproteins (Setterdahl et al. 2003). For example, an increase inTRX redox potential from -350 to -300 mV converts chlo-roplast glucose-6-phosphate dehydrogenase from almostcompletely inactive to active (Née et al. 2009). Indeed, invitro redox titration of the thiol/disulphide-dependentroGFP in the presence of GRX at different glutathioneconcentrations revealed that interconversion of the oxi-dized and reduced forms of the sensor were associated witha calculated redox potential span of about 50 mV (Meyeret al. 2007). Despite this, calibrated quantification of in vivochanges in cytosolic redox potential using these probes hasthus far revealed shifts of only about 20 mV in glutathione-deficient mutants compared with wild-type or in stressversus optimal conditions (Meyer et al. 2007; Jubany-Mariet al. 2010). Nevertheless, the physiological importance ofsuch a shift cannot be discounted because systems that areable to sense glutathione redox potential with high sensi-tivity may await discovery. Furthermore, based on recent



reports of interactions between cytysolic glutathione andTRX systems (Marty et al. 2009), it is possible that in someconditions changes in glutathione redox potential couldinfluence the mechanisms that contribute to changes in theTRX redox potential and thus the biological activity ofTRX targets.

Physiological and environmental factors thatmight cause shifts in glutathione redoxpotential in vivo

As previously discussed, the whole leaf and chloroplastGSH:GSSG ratios are not markedly influenced by varia-tions in light intensity or light-dark transitions. However,oxidant production and oxidative stress can exert a stronginfluence over glutathione status. Oxidant production isgoverned by the rates of photosynthesis, photorespirationand respiration, as well as NADPH oxidases and otherROS-producing systems (Foyer & Noctor 2003). Presum-ably by changing the rate of ROS production through theseprocesses or by affecting the rate of ROS removal, variousstresses such as cold, drought, pollution and pathogens canmodify whole leaf glutathione redox state (Sen Gupta,Alscher & McCune 1991; Vanacker et al. 2000; Bick et al.2001; Gomez et al. 2004b). Indeed, the cytosolic glutathioneredox potential has been shown to increase in response towounding (Meyer et al. 2007). Numerous studies on plantsdeficient in H2O2-metabolizing enzymes have documented

marked changes in GSH:GSSG ratios (Smith et al. 1984;Willekens et al. 1997; Rizhsky et al. 2002; Queval et al. 2007,2009). These are most evident for catalase-deficient plantsexposed to light under photorespiratory conditions, andare accompanied by several-fold changes in the totalglutathione pool. It remains to be established to whatextent these changes in whole tissue GSH:GSSG ratiosinvolve changes in glutathione redox potential in specificcompartments.

The importance of changes in absoluteglutathione concentration compared withchanges in GSH:GSSG

Accumulation of GSSG is often followed by an increase inthe total glutathione pool size (Fig. 8). This response hasbeen documented in plants exposed to ozone, is particularlyevident in response to increased intracellular H2O2 and alsooccurs during pathogen responses. In such circumstances,changes in the total glutathione pool size occur predomi-nantly through oxidant-induced changes in the expressionand post-translational activities of enzymes involved inboth cysteine and glutathione synthesis. This presumablyacts as a homeostatic mechanism to offset what would oth-erwise be more severe increases in glutathione redox poten-tial. If so, this response in itself suggests that the glutathioneredox potential is important for at least some cellular func-tions. Further circumstantial evidence in favour of this

Increased

H2O2 or

other

oxidants

GSH GSH

GSH GSH

GSH GSH Redox-sensitive

Glutathione-independent ROS signalling

Activation of

de novosynthesis

Increased engagement

of GSH in oxidant

metabolism

GSSG

Subcellular

redistribution

Redox sensitive

compartment

Basal state

More

positive

redox

potential

Offset increases in redox potential in sensitive

compartments caused by ongoing conversion

of GSH to GSSG

GSSGGSSG GSSG GSSG

Redox-insensitive

compartment

Increased

engagement Increased p

Homeostatic signal damping

in oxidant metabolismoxidation of

TRX by GSSG?

Thiol-disulfide signalling

ofreducedTRX

TRX

Figure 8. Hypothetical scheme showing some glutathione-linked responses in oxidative stress signalling. Increased engagement of GSHin metabolism of H2O2 or other oxidants causes accumulation of GSSG and thus a more positive glutathione redox potential. Signallinginitiated by this change includes activation of glutathione neosynthesis and transport, both of which act to offset increases in redoxpotential. Modified glutathione redox potential could contribute to signalling through components such as glutaredoxins or proteinS-glutathionylation or, more indirectly, by oxidation of thioredoxins (TRX). Both thiol components may contribute to thiol-disulphidesignalling, which is depicted as one part of a larger reactive oxygen species (ROS) signalling network.



notion comes from the observation that accumulation ofGSSG is compartment specific, and relative increases aremost marked in the vacuole (Queval et al. 2011). This is inline with the documented capacity of vacuoles to importantGGSG more efficiently than GSH (Tommasini et al. 1993).Thus, mechanisms appear to have evolved to stabilize cyto-solic glutathione redox potential during increased ROSproduction, and these include increases in total glutathioneand accumulation of GSSG in compartments that may beless sensitive to redox perturbation (Fig. 8).

Although studies of mutants have shown that relativelysmall changes in glutathione concentration can modulategene expression as well as environmental and developmen-tal processes in plants (Ball et al. 2004; Parisy et al. 2006;Bashandy et al. 2010), it is not yet established that sucheffects are caused by altered glutathione redox potential. Adecrease in total glutathione from 2.5 mm to 50 mm wouldcause the redox potential to become 50 mV more positive(Meyer et al. 2007). This depletion is rather extreme, andcorresponds to the situation in root tips in seedlings of therml1 mutant, which contain less than 5% wild-type glu-tathione (Cairns et al. 2006; Meyer et al. 2007). Thus, whilean increased glutathione redox potential could explain theobservations reported in rml1 and ntra ntrb rml1 mutants(Vernoux et al. 2000; Reichheld et al. 2007), it is less clearwhether it can account for effects observed in less severelyaffected backgrounds. If changes in glutathione redoxpotential are indeed an important part of glutathione-dependent oxidative signalling in wild-type plants, it seemsthat GSSG accumulation is likely to be a critical factor.Accumulation of GSSG could be relatively small (in abso-lute terms) if, as discussed further in the next section, the‘resting state’ concentration in unstressed conditions is aslow as some recent measurements imply.

As increased GSSG generally drives glutathione accumu-lation as part of what is assumed to be a homeostaticmechanism, decreased glutathione concentrations might beexpected to cause a compensatory increase in GSH:GSSG.This should happen if the NADP(H) redox potential doesnot change and the two couples are in equilibrium.However, if GSSG concentrations are indeed in the nano-molar range, it is possible that kinetic limitations over GR(KMGSSG 10–50 mm; Smith et al. 1989; Edwards et al. 1990)become increasingly severe as glutathione decreases, thusexplaining the failure to maintain the glutathione redoxpotential in equilibrium with NADP(H), even when thedecrease in concentration is relatively modest.

Relationships between the glutathione andNADP(H) redox couples in signalling

The midpoint redox potential of NADP(H) at pH 7 isapproximately -320 mV while that of glutathione is -230to -240 mV. But what is the actual redox potential of glu-tathione in vivo? Many researchers assume that it is quiteclose to the midpoint potential and that differencesbetween this value and the TRX redox potential, whichfor most TRX is quite similar to NADP(H), is one factor

that potentially explains in vivo preference of proteintargets for glutathione or TRX. However, as discussedpreviously, some roGFP studies suggest that the glu-tathione redox potential in wild-type Arabidopsis in theabsence of stress is lower than -300 mV in the cytosol andin other compartments, that is, it approaches values similarto the TRX redox potential (Meyer et al. 2007; Schwar-zländer et al. 2008; Jubany-Mari et al. 2010). Given thatthe cytosolic NADPH:NADP+ ratio is not far removedfrom one (Igamberdiev & Gardeström 2003), these redoxpotential values suggest that glutathione and NADP(H)are close to redox equilibrium in this compartment. This isa key unresolved issue in assessing the potential signifi-cance of redox signalling through glutathione-mediateddisulphide bond formation. If sensitive thiol proteins arepresent, maintenance of a very low glutathione redoxpotential under optimal conditions could allow oxidativesignalling to be initiated by protein disulphide bond trig-gered by relatively minor accumulation of GSSG. Glu-tathione redox potential values below -300 mV wouldrequire the GSH:GSSG ratio to be in the range of 105 to106, whereas global ratios are much lower in plant tissues,even in the absence of stress, where they are typicallyabout 20–30 (Queval & Noctor 2007; Mhamdi et al.2010a). It is likely that, while they provide a useful indi-cator of redox status, particularly the presence of oxidativestress, global cellular or tissue glutathione data give only arelative measure of GSH:GSSG ratios in intracellularcompartments (Queval et al. 2011).

There is little evidence as yet that ROS-triggered changesin glutathione are accompanied by marked changes inglobal tissue NADP(H) status (Mhamdi et al. 2010a,b). Theextent to which cytosolic NADP pools turn over in optimaland stress conditions remains uncertain. Further, it is notclear how important the demand of GR is on the cytosolicNADPH pool, and whether there is any functional associa-tion between different NADPH-requiring and -generatingenzymes. GR has a KM for NADPH below 10 mm (Smithet al. 1989; Edwards et al. 1990), while total cytosolicNADPH concentrations are above 100 mm (Igamberdiev &Gardeström 2003). Although a large proportion of pyridinenucleotide pools is bound to proteins at any one time, andthus available ‘free’ concentrations are lower than the total,it is generally considered that changes in NADPH are notlikely to impact the glutathione redox potential throughkinetic effects on GR. However, shifts in NADPH:NADP+

could perhaps impact the glutathione redox potential ther-modynamically, as the reaction catalysed by GR is revers-ible. As previously discussed, kinetic limitations may occurthrough GSSG, whose resting concentration could be sub-stantially below the GR KM value. Finally, it is interestingthat the measurable capacity of GR in many plant systemsis often somewhat lower than other enzymes of theascorbate-glutathione pathway. No doubt this could reflectthe presence of other pathways that can regenerate glu-tathione independent of ascorbate (Fig. 6). However, as wehave emphasized in this review, glutathione may also belinked to ROS metabolism independently of ascorbate.



Comparatively low GR activity may be a feature of plantredox systems that allows appropriate changes in GSSG tomediate signalling functions.

CONCLUSION AND PERSPECTIVES

A large body of literature is now available that illustratesthe complex and integrated regulation of glutathione statusby nutritional and environmental factors. Glutathione maybe seen as a central or ‘hub’ molecule in cellular meta-bolism and redox signalling (Fig. 9). Even though manyglutathione-dependent reactions are involved in oxidativestress-mediated cellular processes, the simple vision of glu-tathione as a ROS-scavenging antioxidant is inaccurate andmisleading. For example, at least three types of mechanismcan be defined that link glutathione to the regulation ofbiotic stress reactions: (1) redox signalling, mediated bycomponents such as GRX or GSNO; (2) the synthesisof S-containing or secondary metabolites; and (3) GS-conjugate formation and metabolism to regulate thebiological activity of metabolic intermediates or activeproducts.

Key questions related to the antioxidant roles of glu-tathione concern the importance of different glutathione-dependent enzymes and the influence of changes inglutathione status within cell signalling pathways. Thedegree of interplay between TRX and glutathione systemsin physiological conditions remains to be fully described.The emerging picture is that changes in the ‘general redoxstate’ (i.e. redox potential) of these two componentswork together with altered activity or abundance of specific

redox signalling components (e.g. GSNOR, GRX).Throughtheir coordinated operation, such effects can reinforce sig-nalling through a given pathway. Alternatively, antagonisticchanges could act to restrain activation of signalling path-ways, a control that is important within a cellular networkreceiving multiple evironmental inputs. This conceptreceives support from the operation of multiple interdepen-dent redox modifications reported for the bacterial H2O2

sensor, OxyR (Kim et al. 2002). A similar picture is emerg-ing in plants, where the notion of relatively simple thiol-disuphide regulation of NPR1 (Mou et al. 2003) has givenway to recognition that control is more complex (Tada et al.2008; Lindermayr et al. 2010). By operating in conjunctionwith other regulatory mechanisms, such nuanced, flexiblecontrol integrates multiple inputs into a variable and appro-priate response output. This is likely to be particularlyimportant in plants, which must constantly deal with envi-ronmental changes of varying intensity and predictability.Thus, the challenge for the future is not only to characterizeglutathione-linked redox modifications and to assess theirinteractions with other types of redox regulation, but also toevaluate the significance of these changes within the widerhorizon of the cellular network.

ACKNOWLEDGMENTS

We thank Bernd Zechmann (University of Graz, Austria)for discussions on glutathione concentrations in differentsubcellular compartments and Stéphane Lemaire (Institutde Biologie Physico-Chimique, Paris, France) for discus-sions on glutaredoxins and protein glutathionylation reac-tions. The authors acknowledge funding from the FrenchAgence National de la Recherche project ‘Vulnoz’ (Orsay)and the European Union Marie-Curie Initial TrainingNetwork ‘COSI’ project (Orsay, Leeds). Y.H. is a recipientof a Chinese Scholarship Council fellowship. B.M.G.thanks Subprograma Estancias de Movilidad posdoctoralen centros extranjeros (2009), Ministerio de Educación(Spain) for a fellowship.

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Received 22 April 2011; received in revised form 14 June 2011;accepted for publication 4 July 2011



Summary H2O2 is a recognized signal in activation of defence mechanisms in response to various stresses, and its accumulation is thus tightly controlled by plant antioxidant systems. Because H2O2 signals may be transmitted by thiol-dependent processes, glutathione status could play an important role. While the antioxidant role of this compound is long established, the importance of glutathione in signaling remains unclear. To study this question, this work exploited a stress mimic mutant, cat2, which has a defect in metabolism of peroxisomal H2O2 that conditionally leads to oxidation and accumulation of glutathione. In cat2, changes in glutathione are accompanied by activation of both salicylic acid (SA)-dependent responses and jasmonic acid (JA)-associated genes in a time-dependent manner. This up-regulation of both phytohormone signaling pathway by intracellular oxidative stress can be largely prevented by genetically blocking glutathione accumulation in a double mutant, cat2 cad2, that additionally carries a mutation in the pathway of glutathione synthesis. Contrasting phenotypes between cat2 cad2 and cat2 gr1, in which loss of GR1 activity exacerbates oxidative stress, suggest that glutathione-dependent processes couple H2O2 to activation of SA-dependent pathogenesis responses through an effect that is additional to glutathione antioxidant functions. Direct comparison of cat2 cad2 and cat2 npr1 double mutants suggests that the effects of blocking glutathione accumulation on cat2-triggered up-regulation of both SA and JA pathways are not mediated by defective NPR1 function. Autophagy has been implicated in processes such as senescence, and may interact with oxidative stress and SA signaling. To explore relationships between autophagy and oxidative stress, selected atg mutants were crossed with cat2. Phenotypic analysis revealed that SA-dependent lesion spread observed in cat2 grown in long days is similar in three cat2 atg double mutants, whereas increased peroxisomal H2O2 availability in cat2 delays an oxidative stress related-senescence triggered by atg in short days. Overall, the work suggests that (1) novel glutathione-dependent functions are important to couple intracellular H2O2 availability to the activation of both SA and JA signaling pathways and (2) H2O2 produced through photorespiration may play an antagonistic role in the early senescence phenotype observed in atg mutants. Résumé Le H2O2 est reconnu comme un signal dans l’activation des mécanismes de défense en réponse à divers stress, et son accumulation est donc régulée étroitement par le système antioxydant des plantes. Puisque la signalisation par le H2O2 peut être transmise par des processus thiol-dépendants, le statut du glutathion pourrait jouer un rôle important. Le rôle de ce composé en tant que molécule antioxydante est bien établi; cependant, son importance en tant que signal reste à élucider. Afin d’étudier cette question, ce travail a utilisé un mutant, cat2, ayant un défaut dans son métabolisme du H2O2 peroxysomal qui engendre, d’une manière conditionnelle, une oxydation et une accumulation du glutathion. Les modifications du glutathion dans cat2 sont accompagnées par l’activation à la fois de réponses dépendantes de l’acide salicylique (SA) ainsi que l’expression de gènes associés à l’acide jasmonique (JA). L’activation des deux voies phytohormonales par le stress oxydant intracellulaire est largement empêchée en bloquant génétiquement l’accumulation du glutathion dans un double mutant, cat2 cad2, qui porte une mutation additionnelle dans la voie de synthèse du glutathion. Les phénotypes contrastants de cat2 cad2 et cat2 gr1, dans lequel la perte de l’activité GR1 aggrave le stress oxydant, suggèrent que des processus glutathion-dépendants relient le H2O2 et l’activation des réponses de pathogenèse SA-dépendantes par un effet qui est additionnel aux fonctions antioxydantes du glutathion. Des comparaisons directes de cat2 cad2 et cat2 npr1 indiquent que les effets de bloquer l’accumulation du glutathion sur l’induction des voies SA et JA chez cat2 ne sont pas causés par une déficience dans la fonction de la NPR1. L’autophagie a été impliquée dans des processus comme la sénescence, et interagirait à la fois avec le stress oxydant et avec la signalisation par le SA. Afin d’explorer des relations entre autophagie et stress oxydant, des mutants atg ont été sélectionnés et croisés avec le cat2. Des analyses phénotypiques ont révélé que l’étendue de lésions SA-dépendantes observée chez cat2 cultivé en jours longs est similaire chez trois double mutants cat2 atg, alors que l’augmentation de la disponibilité en H2O2 peroxysomal liée à la mutation cat2 retarde la sénescence précoce observée chez les mutants atg. Dans son ensemble, le travail suggère que (1) des nouvelles fonctions glutathion-dépendantes sont importantes pour relier la disponibilité en H2O2 intracellulaire et activation des voies de signalisation SA et JA, et (2) que le H2O2 produit par la photorespiration pourrait jouer un rôle antagoniste dans les phénotypes de sénescence précoce observée chez les mutants atg.

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Functional analysis of glutathione and autophagy in