Glutathione in plants: an integrated overview

transcript

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: graham.noctor@u-psud.fr

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

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

Pyridine nucleotide

ROS GSNO RNS

signalling

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

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

γ-Glu-aa+ cys-gly

γ-Glu-aa

γ-EC+ gly

GSHGS X

VacuoleGGC?

5OPase5-OP+ aa

or cys-gly?

γ-EC + gly Cpep

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,

GSSGGSH

Apoplast

Vacuole MITCYT

γ-ECGSH

MRPGSSG

GR2γ-ECS

GSH-SGSH-S

GSH GSHChloroplast PERNucleus

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).

CHOH HOC SG

GSHCHO

GSHCHOH

HOC-SG HCOO-

FDH FGHGLYIGLYII

(γ-Glu-Cys)n-Gly

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)

GlucosinolateS

R C N OH

R C N O SO3

γ-Glu-Cys

Cys-Gly

Indole-CHCN??

Cys-Gly

γ-Glu-Cys-Gly

Indole-CHCN Indole-CHCN

GGTPCS γ-Glu-Cys-Gly

R C N OH

I d l CH CN

Indole-CHCN

Nit il id

R C N OH

Tryptophan

Indole-CH2CN

Amino acid

Nitrile oxide

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

GR NTR

Glutathione TRX

Ascorbate

GPXGST

GRX-PRX TRX-PRX

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

oxidants

GSH GSH

GSH GSH Redox-sensitive

Glutathione-independent ROS signalling

Activation of

de novosynthesis

Increased engagement

of GSH in oxidant

metabolism

Subcellular

redistribution

Redox sensitive

compartment

Basal state

positive

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

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

Glutathione in plants: an integrated overview

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