review engineering and genetic approaches to …...review engineering and genetic approaches to...

REVIEW

Engineering and genetic approaches to modulating theglutathione network in plantsSpencer Maughana and Christine H. Foyerb,*

aInstitute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UKbCrop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK

Correspondence

*Corresponding author,

e-mail: [email protected]

Received 10 October 2005; revised 26

November 2005

doi: 10.1111/j.1399-3054.2006.00684.x

Reduced glutathione (GSH) is the most abundant low-molecular weight thiolin plant cells. It accumulates to high concentrations, particularly in stresssituations. Because the pathway of GSH synthesis consists of only twoenzymes, manipulation of cellular glutathione contents by genetic interven-tion has proved to be relatively straightforward. The discovery of a newbacterial bifunctional enzyme catalysing GSH synthesis but lacking feedbackinhibition characteristics offers new prospects of enhancing GSH productionand accumulation by plant cells, while the identification of g-glutamylcysteine and glutathione transporters provides additional possibilities forselective compartment-specific targeting. Such manipulations might also beused to affect plant biology in disparate ways, because GSH and glutathionedisulphide (GSSG) have crucial roles in processes as diverse as the regulationof the cell cycle, systemic acquired resistance and xenobiotic detoxification.For example, depletion of the total glutathione pool can be used tomanipulate the shoot : root ratio, because GSH is required specifically forthe growth of the root meristem. Similarly, chloroplast g-glutamyl cysteinesynthetase overexpression could be used to increase the abundance ofspecific amino acids such as leucine, lysine and tyrosine that are synthesizedin the chloroplasts. Here we review the aspects of glutathione biology relatedto synthesis, compartmentation and transport and related signalling functionsthat modulate plant growth and development and underpin any assessmentof manipulation of GSH homeostasis from the viewpoint of nutritionalgenomics.

Abbreviations – g-EC, g-glutamyl cysteine; g-ECS, g-glutamyl cysteine synthetase; APR, adenosine 50phosphosulphate reduc-

tase; BS, bundle sheath; BSO, 1-buthionine-SR-sulfoximine; CAT, catalase; CDK, cyclin-dependent kinase; ER, endoplasmic

reticulum; FW, fresh weight; GGT, g-glutamyl transpeptidase; GPX, glutathione peroxidase; GR, glutathione reductase; GRX,

glutaredoxin; GS, glutathione-S; GSH, reduced glutathione; GSH-S, glutathione synthetase; GSNO, S-nitrosoglutathione;

GSSG, glutathione disulphide; GST, glutathione S-transferase; GS-X, glutathione conjugates; Hgt1p, high-affinity glutathione

transporter; JA, jasmonic acid; MAPK, mitogen-activated protein kinase; mBBr, monobromobimane; MRP, multidrug-resistant

transporter; NPR1, non-expressor of PR proteins 1; OPT, oligopeptide transport proteins; PC, phytocheletin; PCD, programmed

cell death; PMF, proton motive force; PR, pathogenesis related; QTL, quantitative trait loci; RNS, reactive nitrogen species;

ROS, reactive oxygen species; SA, salicylic acid; SAR, systemic acquired resistance; TGA, a group of bZIP (basic leucine

repeat)-type transcription factors; TRX, thioredoxin.

382 Physiol. Plant. 126, 2006

Physiologia Plantarum 126: 382–397. 2006 Copyright � Physiologia Plantarum 2006, ISSN 0031-9317

Introduction

The thiol tripepide glutathione [g-Glu-Cys-Gly; reducedglutathione (GSH)] does not immediately come to mindin considerations of human nutrition. However, thisubiquitous tripeptide thiol is a vital intracellular andextracellular protective antioxidant against oxidative/nitrosative stresses, which play a key role in the controlof many human diseases. GSH is also important inimmunity modulation in animals as well as in remodell-ing of the extracellular matrix, apoptosis and mitochon-drial respiration in disease. Mammalian cancer cellsoften accumulate GSH, where there is also a metabolicswitch to glycolysis rather than TCA cycle as a majorenergy source (Pozuelo Rubio et al. 2004). Interactionsof GSH with antiapoptotic factors such as Bcl-2 in can-cer cells have been linked to radiation and multidrugresistance (Ortega et al. 2003). However, because highGSH concentrations are essential for both antioxidantand immune defence systems in mammals, tissue GSHlevels can be regulated, particularly in malnourishedpatients through diet and nutrition before therapeutictreatments (Bray and Taylor 1993).

Plants make abundant amounts of glutathione, and itssynthesis and accumulation in either the chloroplast orcytosolic compartments of the plant cell can beenhanced or decreased relatively easily by genetic engi-neering approaches (Strohm et al. 1995, Creissen et al.1996, Noctor et al. 1998a, b, Zhu et al. 1999, Xianget al. 2001). Ectopic expression of g-glutamyl cysteinesynthetase (g-ECS, also called glutamate cysteine ligase)(Noctor et al. 1996) and certain enzymes of sulphurassimilation pathway (Harms et al. 2000) or glutathionereductase (GR) (Foyer et al. 1991) in transgenic plantsresults in substantial increases in leaf glutathione.Glutathione contents have been increased by at leastfive-fold the values of wild-type plants without negativeeffects (Noctor et al. 1998b, 2002a). Only one report todate has indicated that an enhanced capacity to gener-ate and accumulate the intermediate g-glutamylcysteine (g-EC) can produce negative effects (Creissenet al. 1999). The increased capacity to generate glu-tathione and enhance cellular glutathione pools leadsto higher rates of sulphur assimilation, modified aminoacid metabolism and enhanced stress tolerance (Noctoret al. 1998a, b). Transformed poplar trees with anincreased capacity to synthesize and accumulate glu-tathione are currently being used in the field for bior-emediation purposes to purify and restore soils pollutedby human activities (Peuke and Rennenberg, 2005a, b).Genetic engineering approaches have led to animproved understanding of how compartment-specificalterations in glutathione synthesis affect metabolism

and defence. Similarly, the analysis of mutants deficientin glutathione has greatly advanced current concepts ofhow GSH and glutathione disulphide (GSSG) regulatecell signalling and development. Chemical inhibition ofGSH synthesis using 1-buthionine-S, R-sulfoximine(BSO), a specific inhibitor of g-ECS (Griffith andMeister 1979, Griffith 1982, Maughan and Cobbett2003), has been widely used to study the effects ofGSH depletion in plants (Hell and Bergman 1990,Vernoux et al. 2000). Analysis of Arabidopsis thalianaT–DNA insertion and EMS mutants selected by theirability to grow on BSO concentrations that are inhibi-tory to the wild-type has indicated that differentmechanisms can confer BSO resistance (Maughan2003).

Since its initial discovery in yeast over 125 years ago,glutathione has been shown to have a prodigious numberof critical functions (Fig. 1). GSH is a powerful reducingagent and is a key player in the plant redox-state (Noctorand Foyer 1998, Noctor et al. 2002a). Here we willfocus on how manipulation of GSH synthesis and meta-bolism has advanced current concepts of glutathionehomeostasis and function, concentrating on aspectsrelated to cell signalling, development and defencethat may be of importance in any consideration of nutri-tional genomics involving glutathione. The characteris-tics and functional importance of putative thiol-basedreactive oxygen species (ROS) sensors and associatedredox-signalling cascades have recently been discussed

Abiotic Biotic

Heavy metal detoxification

Xenobiotic detoxification

Systemic AcquiredResistance

Oxidative brustMAPK

cascades

Proteinthiolation

PCDCalciumsignaling

Cell cycle progressionRoot development

Sulfur storageSulfur sensing

Carbon - Sulfur - Nitrogen

Metabolism

Signaling

Salinity protection

Drought protection

UV protection

ROS scavenging

Fig. 1. The functions of glutathione in plants linked by essential signal-

ling functions that integrate plant growth, development and defence

processes.

Physiol. Plant. 126, 2006 383

(Foyer and Noctor 2005). In this context, we will con-centrate here on the implications for the plant of mod-ulating glutathione homeostasis.Like ascorbate, glutathione limits the lifetime of ROS

and lipid peroxide signals. Moreover, GSH alters cal-cium signalling in plants (Gomez et al. 2004a) andparticipates in the calcium-dependent pathways ofROS-signal transduction (Rentel and Knight 2004).Through oxidation to GSSG, other signalling cascadescan be initiated through thiol-disulphide exchange andthiolation. Moreover, the interaction between GSH andnitric oxide, catalysed by the enzyme formaldehydedehydrogenase (also known as class III alcohol dehy-drogenase), is also important in signal transduction asS-nitrosoglutathione (GSNO) is thought to be a stableNO transport form (Liu et al. 2001). The formaldehydedehydrogenases, which also catalyse the NAD-dependentformation of S-formylglutathione and S-hydroxymethylglutathione, are regulated by wounding and salicylicacid (SA) (Diaz et al. 2003). Hence, glutathione isdirectly implicated in the coordinated control of sig-nalling by ROS and reactive nitrogen species (RNS)that determines cell fate in situations such as pathogenattack.It is important to note that thioredoxins (TRXs), glu-

tathione and lipid peroxides all target the same proteinresidues, allowing us to envisage a dynamic signallingnetwork where proteins are modulated by these threesignalling systems according to their respective concen-trations and the associated enzymes such as glutaredox-ins (GRXs).

Control of plant growth and development

Glutathione regulation is important in a number of keyprocesses associated with plant growth and develop-ment. These include the following:

The cell cycle and plant development

Current knowledge of how cell division is controlledand how it interacts with different aspects of plantdevelopment such as morphogenesis, architecture andgrowth rate has greatly increased over the last 5 years.Cell-cycle regulation involves components that respondto signals from the external environment as well as intrin-sic developmental programmes, and it ensures that DNAis replicated with high fidelity within the constraints ofprevailing environmental conditions (Dewitte et al. 2003,de Jager et al. 2005). Cyclin-dependent kinases (CDKs)play a central role in cell-cycle regulation, with negative(KRP proteins) and positive (D-type cyclins) regulatorsacting downstream of environmental inputs at the G1

checkpoint (Dewitte and Murray 2003, de Jager et al.2005). The activity of the CDK/cyclin complexes isregulated by a network of regulatory mechanismsincluding transcription, proteolysis, phosphorylation/dephosphorylation, interaction with regulatory proteinsand intracellular trafficking.

In Arabidopsis, the embryo cells of the dry seed arearrested in the G1 phase of the cell cycle. Germinationis initiated by water uptake and the resumption of meta-bolism and cell division. Nitric oxide is a potent regu-lator of germination in dormant seeds such as those ofArabidopsis (Bethke et al. 2004, 2006). Germination isstimulated by the presence of environmental nitratefrom which NO can be synthesized via nitrate reduc-tase. The relatively low abundance of GSH within thedry seed means that GSH has either to be regeneratedfrom oxidized or bound forms upon water uptake or thatit has to be synthesized de novo during germination toachieve the high levels present in seedlings. Low GSHlevels in the dry seed and during germination may pre-vent excessive formation of GSNO, allowing sufficientfree NO to be present to stimulate the germinationprocess. However, it is also possible that NO accumula-tion serves as a trigger for GSH synthesis duringgermination.

While it is well established that the G1 checkpoint isvery sensitive to environmental conditions, it has onlyrecently become clear that this control might be exertedthrough oxidation–reduction (redox) regulation andassociated components, particularly glutathione andascorbate, whose concentrations and redox state influ-ences G1 progression. Glutathione and ascorbate exertindependent controls on the plant cell cycle (Potterset al. 2004).

Numerous interactions between the plant and environ-ment take place at the root/soil interface.Monobromobimane (mBBr) has been widely used todetect GSH in vivo. Application of this GSH-localiza-tion system to Arabidopsis roots shows that GSH accu-mulates in the vascular system and root tip (Fig. 2A).The relatively high concentration of GSH in roottips, where actively dividing cells are found (Fig. 2A),suggests that growing tissues have a requirementfor GSH. Indeed, studies using the GSH-deficientROOTMERISTEMLESS1 mutant (rml1) which has lessthan 2% of the wild-type GSH levels have shown thatwithout GSH root meristem cells arrest in G1 (Chenget al. 1995, Vernoux et al. 2000). This mutation onlyaffects post-embryonic root development, and whileoverall the roots are shorter, the cell files and types arenot aberrant. It is important to note that in contrast to theroot meristem, the shoot meristem is able to produce allthe aboveground organs with development and timing


that are comparable with the wild-type despite very lowGSH levels.

The rml1 phenotype can also be induced chemicallyby treating cells or seedlings with BSO to lower GSHlevels (Fig. 2B–G). This causes the cells in the rootmeristem to stop dividing, and, like the root meristemcells of rml1, they arrest in the G1 phase (Fig. 2F,G).These results suggest that GSH plays a key role inpotentiating signals permitting cell-cycle progressionand hence root growth. Similarly, root nodule formationis prevented when GSH synthesis is blocked by additionof BSO, suggesting that like the root meristem, thenodule meristem is unable to develop in the absence

of GSH (Frendo et al. 2005). While the absolute contentof GSH and the GSH : GSSG ratio clearly influencecell-cycle progression, the targets of glutathione actionare unknown at present. In mammalian systems, thisregulation includes transcription of the retinoblastomagene product as well as its activity, which functiondirectly downstream of G1 activators (Yamauchi andBloom 1997).

Given the importance of glutathione redox status inregulating cell division, it is perhaps not surprising thatthe GSH : GSSG ratio also regulates embryogenesis inplants (Belmonte and Yeung 2004, Belmonte et al.2005) as it does in animals. Application of GSH in thesecond half of embryo development results in preco-cious germination, while application of GSSG improvedsomatic embryogenesis (Belmonte and Yeung 2004,Belmonte et al. 2005). The redox status of the embryoglutathione pool delineates specific stages of embryodevelopment both in vivo and in vitro. A high GSH/GSSG ratio is essential for cell division and proliferationduring the initial stages of embryo development, butlater in development, the redox status of the glutathionepool decreases (Belmonte et al. 2005). This metabolicshift in cellular glutathione redox status appears to berequired for continual embryonic development, andproper acclimation of storage product deposition is pos-sibly facilitated through interactions with hormonessuch as abscisic acid. Zygotic embryos are held in G1until germination-specific D-type cyclins promote celldivision (Mausubelel et al. 2006). It is thus tempting tosuggest that redox controls may act upstream ofgermination-specific cell-cycle regulation in a fashionsimilar to the regulation of division in the root apicalmeristem.

Because GSH is a modulator of general plant devel-opment, it is not surprising that it has also been impli-cated in senescence processes (Ogawa et al. 2002). Onespecific mode of action affects the activity of the ubi-quitin-dependent 26S proteasome. The activity of thisprotein degradation pathway may be influenced by cel-lular redox status in plants as it is in animals, where highGSH/GSSG ratios impede the binding of target proteins(Hochstrasser 1998, del Pozo et al. 1998, Theriault et al.2000, Sangerman et al. 2001).

Plant defence and systemic acquired resistance

GSH is a transcriptional regulator. It enhances the DNA-binding activity of NFkB, a transcription factor that iscentral to the regulation of the oxidative stress responsein animal systems. Few homologues of major animal ormicrobial redox-sensitive elements and factors havebeen reported in plants to date apart from the activator

A

B D

EC

F G

Fig. 2. The effects of 1-buthionine-S, R-sulfoximine (BSO) on tissue

glutathione and root growth in Arabidopsis thaliana. The root of a 7-day-

old seedling shown in (A) highlights the fact that reduced glutathione

(GSH) is most abundant in the root tip and in the vasculature as

indicated by the blue fluorescence of mBBr. Untreated Arabidopsis

cells (B) were stained for GSH which accumulates in the vacuole (C).

Equivalent BSO-treated (1 mM) cells (D, E) lack any significant staining

indicating the depletion of GSH. In (F), the root apical meristem is

demarcated by the staining of dividing cells by using a fusion of the

GUS enzyme to a mitotic cyclin (CYCB1;1; Colon-Carmona et al. 1999).

Inhibition of GSH synthesis by BSO (1 mM) cell division causes cessation

of root growth (G).


protein-1 (a transcription factor controlling response tooxidative stress) and the antioxidative responsive elementthat is present in the maize catalase (CAT) promoter.However, cytosolic thiol-disulphide status reactionsare crucial in plant innate immune responses. GSSGaccumulation is often observed in close conjunctionwith cell death and systemic acquired resistance (SAR)responses resulting from exposure to pathogens(Vanacker et al. 2000) or abiotic stresses (Gomez et al.2004a). RNS are also important in the plant pathogenresponses, and the strong interaction between GSH andNO in signal transduction process, as GSNO for example,suggests that mutual upregulation of these signallingcomponents may occur in challenged cells.An increase in tissue GSH contents activates the

expression of PR genes through the non-expressor ofPR protein 1 (NPR1) pathway (Mou et al. 2003,Gomez et al. 2004a). The NPR1 protein is part of themechanism through which SA triggers SAR to pathogens.While many aspects of NPR1 function with regard tobinding of the TGA transcription factors and movementto the nucleus to facilitate SAR are not fully understood,this glutathione-modulated ankyrin repeat protein is oneof the key regulators of SA-dependent gene expression(Cao et al. 1994, Delaney et al. 1995). Such studies indi-cate a strong relationship between SA and glutathionehomeostasis in stress signalling. Increased oxidationresulting from exposure to stress stimulates glutathioneaccumulation. SA enhances this response presumably byincreasing oxidation possibly through inhibitory effects onCAT and ascorbate peroxidase.

Cell death and the GSH/GSSG redox couple

The many varied functions of GSH and importance ofthe GSH : GSSG redox couple as a sensitive signal forcellular events necessitates strict GSH homeostasis. Inanimal cells, substantial evidence implicates redoxpotential as an important factor determining cell fate.All aerobic organisms maintain thiols in the reduced(-SH) state. GSH : GSSG ratios of 100:1 are typicalvalues for animal and plant cells. Departures from highcellular GSH : GSSG ratios caused for example byexposure to stress are potentially dangerous, as theyare a cell death trigger; low GSH : GSSG ratios initiat-ing programmed cell death (PCD) (Schafer and Buettner2001). Many stresses cause an initial net oxidation ofthe glutathione pool that is often followed by increasesin total glutathione (Smith et al. 1984, Sen Gupta et al.1991, Willekens et al. 1997, Noctor et al. 2002b,Gomez et al. 2004b). This effect is observed, becauseoxidation of the glutathione pool stimulates of GSHsynthesis to restore cellular GSH : GSSG ratios. This is

a homeostatic mechanism by which [GSH] is increasedin an attempt to offset stress-induced decreases in the[GSH] : [GSSG] ratio. The nature of the link betweenredox-state perturbation and enhanced glutathioneaccumulation is not fully resolved, but it may involvemultiple levels of control. For example, the activity ofadenosine phosphosulphate reductase, a key enzyme insulphate assimilation, is activated by decreases inGSH/GSSG (Bick et al. 2001). In addition, H2O2 and/orlow GSH/GSSG ratios enhance translation of g-ECS(Xiang and Oliver 1998). Significant deviations fromhigh GSH : GSSG ratios result in PCD (reviewed inNoctor et al. 2002a, b). The significance of alterations tothe GSH : GSSG ratio can be quantified using the Nernstequation through which the redox potential of a givenredox couple can be calculated: E 5 Em [–(RT/nF)]ln([Ared]/[Aox]). This allows a quantitative appreciation ofthe impact of changes in total glutathione content on theredox state of the cell. The activity of GR is essential inmaintaining the high cellular GSH : GSSG ratios. Ectopicexpression of bacterial GR in the chloroplast or cytosol oftransgenic plants increased the GSH : GSSG ratio andgave added protection against some stresses (Foyer et al.1991, 1995, Aono et al. 1993, 1995, Creissen et al. 1996).

Plants that are deficient in CAT activity have alsobeen used to investigate oxidative signalling resultingfrom decreased H2O2 detoxification (Vandenabeeleet al. 2002, 2004). CAT-deficient plants are particularlyinteresting as overall levels of H2O2 accumulation arerelatively small, but there is a drastic increase in tissueglutathione levels (Smith et al. 1984, Smith et al. 1989)together with a low GSH : GSSG ratio (Noctor et al.2002b). CAT-deficient plants have been used to identifya complete inventory of genes induced or repressed inH2O2-induced PCD, which appears as clusters of deadpalisade parenchyma cells along the leaf veins whenthese plants are exposed to high light (Vandenabeeleet al. 2004). The stimulation of GSH synthesis and mod-ified GSH : GSSG ratios are intrinsic features of ozoneand H2O2-dependent signalling. Thus, GSSG and thio-lation may be important in adding specificity to thesignal and/or promoting second messenger signal trans-duction in these pathways (Rentel and Knight 2004,Foyer and Noctor 2005).

Transport systems also fulfil important roles in deter-mining the GSH : GSSG ratios of individual cell com-partments (Foyer et al. 2001). Transport of GSSG intothe vacuole by the MRP transporters or across the plas-malemma (Jamaı et al. 1996) contributes toGSH : GSSG homeostasis in the cytosol. GSH degrada-tion may also be an important factor (Storozhenko et al.2002). However, the g-glutamyl cycle, which contri-butes to GSH metabolism in animals, remains to be


confirmed in plants (Storozhenko et al. 2002).Overexpression of one putative component, g-glutamyltransferase (GGT) in Arabidopsis, did not alter theGSH : GSSG ratio or total tissue glutathione contents(Storozhenko et al. 2002).

Thiol-disulphide exchange reactions are a universalmechanism for perception of redox perturbation. Inaddition, S-glutathionylation or thiolation is a furthercrucial mechanism in perception of altered glutathioneredox state. Protein glutathionylation is a widespreadresponse to oxidizing conditions. It is becoming estab-lished as an important mechanism for reversible mod-ification of protein cysteinyl residues in plants as it is inanimals. While relatively few thiolated proteins havebeen identified to date, those that have been character-ized include important metabolic enzymes such assucrose synthase and acetyl CoA carboxylase anddefence enzymes such as dehydroascorbate reductase(Dixon et al. 2005). Thiolation can have a number ofeffects on proteins, but it primarily protects cysteinylresidues from irreversible oxidation to their sulfinicand sulphonic acid derivatives. The process is reversedby specific GRXs and/or TRXs, but much remains to becharacterized concerning these regulatory systems(Noctor in press). Both types of redox-active proteinsare encoded by quite large gene families in plants(Lemaire 2004). While relatively few plant thiolationtargets have been identified to date, many as yet uni-dentified targets are likely also to be thioredoxin targets,which are becoming increasingly well characterized(Buchanan and Balmer 2005).

Glutathione as a sink for reduced sulphur andregulator of sulphur assimilation

Current evidence suggests that GSH has a role in sul-phur homeostasis. GSH is an abundant low-molecularweight thiol and hence a potentially important sulphursink as well as a predominant transported form ofreduced sulphur (reviewed in Hell 1997). This modelpredicts that excess sulphur will be stored in GSH andwill be released when the cell experiences sulphur defi-ciency or requires increased sulphur availability.Important mechanistic support came from studieswhich show GSH is transported long distances in thephloem and xylem (Rennenberg et al. 1979).Furthermore, GSH is transported to developing tissuesfrom Maize scutella (Rauser et al. 1991). Herschbachet al. (2000) showed that not only is GSH found in thephloem and xylem but also that GSH levels controlsulphate loading into the xylem. Together with cysteine,glutathione forms part of the repertoire of signals thatmodulate sulphate uptake and assimilation (Kopriva and

Rennenberg 2004). GSH accumulation in cells has feed-back effects on the pathway of primary sulphur assim-ilation (Kreuzwieser and Rennenberg 1998). Theaddition of GSH to Arabidopsis roots caused a decreasein adenosine 50 phosphosulphate reductase (APR) activ-ity and transcript abundance (Vauclare et al. 2002) aswell as in ATP sulphurylase activity (Lappartient et al.1999). Long distance GSH transport has also been sug-gested to decrease sulphur assimilation (Lappartient andTouraine 1996) and poplar (Herschbach et al. 2000). Asimple regulatory mechanism has been proposed withregard to the regulation of demand-driven sulphurassimilation by positive signals such as o-acetylserineand negative signals such as GSH (Kopriva andRennenberg 2004).

Effects of manipulation of GSH synthesis onmetabolism and development in mutant andtransgenic plants

Glutathione is synthesized in both photosynthetic andnon-photosynthetic cell types. However, green leavesare often the major organs of GSH production andexport, and chloroplasts contain high (3–5 mM) GSHconcentrations (Foyer and Halliwell 1976, Smith et al.1985). However, certain non-green cell types such astrichomes also have a very high capacity to produce andaccumulate glutathione (Gutierrez Alcala et al. 2002).Variable proportions of leaf glutathione are localized inthe chloroplasts, but values are generally considered tobe between 30 and 40% of the total (Klapheck et al.1987) with 1–4 mM in the cytosol (Noctor et al. 2002a).

In nearly all organisms studied to date, GSH is synthe-sized from constituent amino acids in a two-step ATP-dependent reaction sequence (Fig. 3) catalysed byg-ECS and glutathione synthetase (GSH-S) (Hell andBergmann 1988, 1990, Meister 1988). With the excep-tion of a novel enzyme from Streptococcus agalactiaethat catalyses both activities (Janowiak and Griffith2005), g-ECS and GSH-S enzymes have been identifiedas separate proteins and gene products from numerouseukaryotes and gram-negative prokaryotes. The S. aga-lactiae enzyme has several features that are worthy ofnote. This is not only the first bifunctional g-ECS-GSH-Senzyme to be isolated but is also the first GSH synthesisenzyme system to be identified from a gram-positiveorganism (Janowiak and Griffith 2005). The g-ECS-GSH-S sequence encodes an 85 kDA protein, whichwhen purified exhibits GSH-S activity with a similarspecific activity to that of g-ECS. It has similar Km valuesfor cysteine and ATP to the Escherichia. coli g-ECS, butthe streptococcal g-ECS-GSH-S has a lower affinity forglutamate than the E. coli GSH-S. This may be


explained by the fact that gram-positive bacteria main-tain exceptionally high glutamate levels (of up to100 mM). The GSH-S domain has a relatively smallmolecular mass (31 kDaA) with an amino acidsequence more related to D-Ala-ligase than GSH-S(Janowiak and Griffith 2005). It is important to noteGSH inhibits neither the g-ECS nor the GSH-S activityof the streptococcal g-ECS-GSH-S, allowing S. agalac-tiae to maintain much higher GSH concentrations thanfor example E. coli, despite the fact that the overallg-ECS activity is relatively lower. It is interesting to notethat S. agalactiae lack catalase and therefore rely heavilyon GSH-based enzymes for peroxide detoxification(Janowiak and Griffith 2005).The E. coli genes encoding g-ECS and GSH-S have

been used extensively in genetic engineeringapproaches to enhance glutathione contents in plants(Noctor et al. 1998a, Zhu et al. 1999). Targeting of thebacterial g-ECS and GSH-S to either the chloroplastor cytosol has led to marked increases in enzymeactivity. Increases in g-ECS but not GSH-S led to largeconstitutive increases in leaf glutathione (Noctor et al.1996, 1998a, Creissen et al. 1999). Moreover, glu-tathione was also increased in xylem sap, phloem

exudates and roots (Herschbach et al. 2000). Theidentification of a g-ECS-GSH-S enzyme that facilitatesglutathione synthesis in S. agalactiais suggests that similarbifunctional enzymes might occur in other gram-positivebacteria where glutathione synthesis was thought to berelatively uncommon. These bifunctional g-ECS-GSH-Senzymes might be extremely useful in future plantgenetic engineering approaches to manipulateglutathione for nutritional purposes. Similarly, specifichomologues of glutathione could be introduced forexample by ectopic expression of enzymes such ashomoglutathione synthetase.

The endogenous plant g-ECS and GSH-S enzymes dis-play a high degree of sequence homology. In A. thaliana,g-ECS is encoded by a single gene, GSH1, with a plastidtarget signal (May and Leaver 1994). A recent analysisusing a g-ECS-GFP reporter system has indicated thatmost of, if not all, the A. thaliana g-ECS protein is locatedin the chloroplasts (Wachter et al. 2005). Targetinginformation relative to GSH-S suggests that it is presentboth in chloroplasts and the cytosol. The chloroplastand cytosol thus cooperate in de novo GSH synthesis,the chloroplast acting as the source of g-EC forboth chloroplastic and cytosolic GSH production.

Mitochondrion

Cytochromes

Cytoplasm

GSHL-Glycine + ATP

2 GSH

NPR1

Vacuole

(γ-ECn)Gly - Heavy metalsGS-X conjugates

GSSG(GSH)

ER

PMF

GSHGSSGGS-X

GSHGSSGGS-X

Pepides(CysGly)

AAs

Chloroplast

1

O

GSH-Ascorbatecycle

ROS Scavenging(H2O2, 1O2)

OH

O

OOH

HSHN

N H

O

OH

O

O O

OHNH2

HSHN

N H

ATPL-GlutamateGlutathione

(GSH)

L-Glycine+

ATP

L-Cysteine

γ-Glutamylcysteine(γ-EC)2

γ-EC

Nucleus

PR genesRedox

signaling/geneexpression

Reducingpower for

energy chains

Sequestrationof harmfulcompounds

Fig. 3. Glutathione synthesis and compartmentation. Reduced glutathione (GSH) synthesis proceeds by the sequential action of g-glutamyl cysteine

synthetase (g-ECS) (1) localized exclusively in the chloroplast and GSH-S, glutathione synthetase (GSH-S) (2) which is localized in both the chloroplastic

and cytosolic compartments. The many transporters capable of transporting GSH and related metabolites (shown in black) including the tonoplast

ATP-dependent MRP pumps, which can transport GSH, GSSG and GS-X compounds (see also Figs 2B,C), are shown. Other multidrug-resistant

transporters (MRPs) are shown on the plasma membrane. Alongside these are the proton-coupled pumps that can transport GSH, GSSG, GS-X, di/tri-

peptides and amino acids. Significant gaps remain in our current knowledge of GSH transporters, and it is likely that other as yet undescribed

transporters (red) must exist to allow effective GSH homeostasis.


These observations are surprising given that earlier stud-ies localized g-ECS and GSH-S activities in the cystosolas well as the chloroplasts (Klapheck et al. 1987, Helland Bergmann 1988, 1990). Moreover, the studies withtransgenic plants have shown that GSH synthesis can beincreased by targeting the synthetic enzymes to eitherthe chloroplastic or the cytosolic compartments (Noctoret al. 1996, 1998a, Creissen et al. 1999). However,kinetic analysis has revealed that the chloroplast andcytosolic isoforms have similar properties (Noctoret al. 2002a). More work is required to fully rationalizethe molecular genetic evidence with the biochemicalobservations.

The targeting information indicating that g-EC is lar-gely synthesized in the chloroplasts (Wachter et al.2005) has implications for glutathione homeostasis.Firstly, at least some of g-EC produced in the chloroplastmust be exported to the cytosol, as most of the GSH-S islocated in this compartment. Secondly, overexpressionof g-EC in the chloroplast enhanced not only leaf g-ECand glutathione contents, but it also increased the abun-dance of amino acids synthesized in the chloroplastssuch as valine, leucine, isoleucine, lysine and tyrosine(Noctor et al. 1998a). It is interesting to note thatincreases in these amino acids was specific to chloro-plast g-ECS overexpression and that the degree ofenhancement occurred in almost direct proportion toincreases in g-EC and glutathione (Noctor et al. 1998a)

GSH metabolism is more complex in C4 species suchas maize than in C3 species such as Arabidopsis,tobacco and poplar. In maize leaves, cysteine synthesisis exclusively localized in the bundle sheath (BS) tissues(Burgener et al. 1998). g-ECS and GSH-S transcriptsand proteins are similar in abundance in maize leafmesophyll (M) and BS cells, and they were also foundin the epidermis and stomatal guard cells (Gomez et al.2004b). It is important to note however that an earlierstudy found that GSH-S activity primarily in the M cells(Burgener et al. 1998).

A number of GSH-deficient A. thaliana mutants havebeen described in the GSH1 locus. The analysis ofg-ECS-deficient mutants such as rml, cad and rax hasprovided useful information concerning the effects ofglutathione depletion in plants (Cheng et al. 1995,Howden et al. 1995, Vernoux et al. 2000, Ball et al.2004). The mutants have rather different characteristics.For example, the cad2-1, which has 15–30% of wild-type GSH, and the rax 1-1, which has 50% of wild-typeGSH, have different leaf transcriptome profiles (Ballet al. 2004). It is possible that the GSH1 gene productsare themselves involved in signal transduction.

The extensive literature concerning the effects ofmanipulation of g-ECS and GSH-S activities in plants

has already been reviewed (Noctor et al. 1998a, Noctoret al. 2002a). The major factors controlling GSH accu-mulation in plants as in animals are abundance of g-ECSand the availability of cysteine (Noctor et al. 1996,1998a, 1998b, 2002a, Xiang and Oliver 1998, Xiangand Bertrand 2000, Xiang et al. 2001). Overexpressionof enzymes such as g-ECS (Noctor et al. 1996), GR(Foyer et al. 1991, 1995, Aono et al. 1993, 1995) orserine acetyltransferase (Harms et al. 2000) enhancestissue GSH contents. However, there are few attemptsto date to examine the effects of simultaneous expres-sion of these enzymes. Increased tissue glutathione intransformed plants has resulted in largely beneficialeffects (Foyer et al. 1995, Strohm et al. 1995, Noctoret al. 1996, 1998a, Pilon-Smits et al. 1999, Zhu et al.1999). The marked chlorotic phenotype produced bychloroplastic E. coli g-ECS overexpression in trans-formed tobacco is unusual and may result from per-turbed signal transduction rather than direct effects ofGSH or g-EC accumulation per se (Creissen et al. 1999).

Some of the effects of ectopic g-ECS expression areillustrated in Fig. 4 using transformed Arabidopsis seed-lings produced by Cobbett et al. (1998). The high levelof GSH1 transcripts observed in the transformedArabidopsis plants relative to controls (Fig. 4A) isaccompanied by increased total leaf glutathionecontents (Fig. 4B). Moreover, the enhanced capacityfor GSH synthesis arising from the increased expressionof g-ECS results in a much lower effect of BSO in termsof its ability to deplete tissue glutathione (Fig. 4B). Thepronounced positive effect of GSH1 overexpression andhigher tissue GSH availability on Arabidopsis rootgrowth is illustrated in Fig. 4C. It is interesting to notethat in the presence of BSO, the wild-type plants have asimilar phenotype to the rml1 mutant as illustrated bythe absence of a post-embryonic root. However,enhanced g-ECS activity (o/e line) alleviates thisinhibition of root development, presumably due to thehigher capacity for GSH synthesis even in the presenceof BSO, allowing the root to grow.

Sulphur uptake by the roots of transformed bacterialg-EC-expressing poplar trees was enhanced to meet therequirements of increased demand for sulphur causedby enhanced GSH synthesis (Herschbach et al. 2000).However, the glutathione contents of untransformedand transformed poplar leaf discs were increased sub-stantially by incubation with cysteine, particularly in thelight. This suggests that cysteine supply remains a keylimiting factor for glutathione synthesis even wheng-ECS activity is increased (Strohm et al. 1995, Noctoret al. 1996, 1997). The leaf cysteine levels were main-tained at values similar to the wild-type in transgenicpoplars with high g-ECS activities, but the activities of


enzymes involved in sulphate reduction and assimila-tion were not increased (Noctor et al. 1998a,b, Hartmann et al. 2004). Similarly, microarray analysisof plants undergoing sulphate starvation did not revealeffects on g-ECS or GSH-S transcripts suggesting littlecoordination of the pathways of GSH synthesis inresponse to sulphur availability (Hirai et al. 2003,Maruyama-Nakashita et al. 2003). Hence, to removemetabolic limitations in cysteine supply, it will beessential to consider solutions that modulate sulphurassimilation to further enhance the capacity of planttissues to synthesize and accumulate glutathione. Forexample, modulation of oxylipin signalling may beone approach, as methyl jasmonate stimulates transcrip-tion of both sulphur assimilation and GSH synthesisgenes (Harada et al. 2000).

Regulation of glutathione synthesis andaccumulation

There is very little literature information available on thecoordinate regulation of expression of GSH1 and GSH2in plants. It is worth noting the very high abundance ofGSH2 transcripts in the quiescent centre of Arabidopsisroots, compared with surrounding cells (Nawy et al.2005), and it may be that a high level of GSH is requiredin these stem cells for antioxidant defence, becausequiescent centre cells are essentially devoid of reducedascorbate.

GSH1 and GSH2 mRNAs are increased by conditionsthat require enhanced GSH abundance for metabolicfunctions such as heavy metal sequestration. NeitherGSH nor GSSG exercise much control over GSH1 andGSH2 transcription (Xiang and Oliver 1998). Similarly,while H2O2 and CAT inhibitors increase tissue GSH,they did not affect GSH1 or GSH2 transcript abundance(Xiang and Oliver 1998). Exposure to oxidative stressincreased g-ECS activity and glutathione in Arabidopsis,but GSH1 mRNA abundance was unchanged. In con-trast, exposure to heavy metals such as cadmium andcopper increases GSH1 and GSH2 transcripts (Schaferet al. 1998, Xiang and Oliver 1998). While SA does notregulate GSH1 or GSH2 transcripts, jasmonic acid (JA)

20A

B

C

18

16

14

12

10

Rel

ativ

e fo

ld e

xpre

ssio

n of

GSH

1nm

ol G

SH/g

FW

8

6

4

2

0WT GSH1 o/e

WT GSH1 o/e

WT GSH1 o/e

500

400

300

200

100

0

Fig. 4. Effect of g-glutamyl cysteine synthetase (g-ECS) overexpressionin Arabidopsis thaliana. Ten-day-old transgenic A. thaliana (Cobbett

et al. 1998) seedlings were compared with the wild-type. The effects

of constitutive g-ECS expression (g-ECS o/e) are demonstrated by semi-

quantitative RT-PCR (A) tissue reduced glutathione (GSH) contents (B)

and the stimulatory effect of the resultant high GSH levels on seedling

root growth (C). The higher activities of g-ECS activity in the transgenic

plants leads to greater GSH abundance (m) that persists even in the

presence of 0.7 mM 1-buthionine-SR-sulfoximine (M).


has a marked stimulatory effect (Xiang and Oliver1998). Methyl jasmonate caused a rapid transientincrease in transcripts encoding sulphur assimilationenzymes and GSH1 and GSH2 mRNA levels inArabidopsis (Harada et al. 2000). Although transcriptabundance was increased by heavy metals and JA, oxi-dative stress was required for the translation of the tran-scripts, implicating regulation at the post-transcriptionallevel and a possible role for factors such as H2O2 ormodified GSH/GSSG ratios in de-repressing translationof the existing mRNA (Xiang and Oliver 1998). Stress-induced increases in glutathione, such as thoseobserved in plants deficient in catalase, have shownthat glutathione accumulation is preceded or accompan-ied by a marked decrease in the reduction state of thepool (Smith et al. 1984, Willekens et al. 1997). A similarresponse was elicited by exposing poplar leaves toozone (Sen Gupta et al. 1991). The 50untranslatedregion (50UTR) of the GSH1 gene was found to interactwith a repressor-binding protein that was released uponaddition of H2O2 or changes in the GSH/GSSG ratio(Xiang and Bertrand 2000). A redox-sensitive 50UTR-binding complex is thus suggested to control g-ECSmRNA translation in A. thaliana (Xiang and Bertrand2000).

It has long been accepted that post-translational reg-ulation of g-ECS through end-product inhibition by GSHmay control tissue GSH concentrations (Hell andBergmann 1990, Schneider and Bergmann 1995, Jezet al. 2004). Moreover, g-ECS activity is inhibited bythiols in plant extracts (Noctor et al. 2002a) as well asin the purified recombinant enzyme (Jez et al. 2004).Such feedback control of g-ECS activity by GSH con-centrations is easy to understand in terms of the exclu-sive localization of this enzyme in the chloroplast,where localized GSH accumulation in the limitedspace of the organelle would cause a specific feedbacklimitation on g-ECS activity, until excess GSH could beexported to the cytosol.

Post-translational control of g-ECS activity remains apossibility (May et al. 1998), but protein phosphoryl-ation similar to that reported in animals (Sun et al. 1996)has not yet been found in plants. In animals, a smallerregulatory subunit increases the catalytic potential ofthe larger catalytic subunit by alleviating the effects offeedback control (Huang et al. 1993, Mulcahy et al.1995). A recent analysis in human cells has shownthat a number of enzymes affecting glutathione metab-olism and breakdown are targets for of 14-3-3 proteins(Pozuelo Rubio et al. 2004), and this may be also thecase in plants. Because the precise pathway of glu-tathione degradation in plants remains poorly resolved,and many important plant enzymes are controlled by

interactions with 14-3-3 proteins (DeLille et al. 2001),this type of regulation of GSH synthesis and/or degrad-ation is a possibility that merits further investigation.

GSH homeostasis-transport

The physical separation of the rate-limiting step of GSHsynthesis catalysed by g-ECS from the second reactionstep suggests that g-EC and GSH transport have funda-mental roles in glutathione homeostasis. Recent datahas implicated oligopeptide transport proteins (OPTs)in GSH transport. However, none of the nine AtOPTshas a high affinity for GSH transport, and the cellularlocalizations of these proteins have not been resolved.Chloroplastic transporters are particularly important, asthey are essential for the export of g-EC and GSH pro-duced within the chloroplast. However, none of theknown transporters that have been implicated in GSH/GSSG transport have been shown to be localized in thechloroplast despite the fact that recent data suggests thatthe chloroplast may be the major site of g-EC produc-tion. One or more high-affinity g-EC/GSH/GSSG trans-porters targeted to the chloroplast may be expected, asillustrated in Fig. 3. Preliminary biochemical experi-mentation has shown that GSH is transported into iso-lated wheat chloroplasts. Noctor et al. (2002a) observedthat 35S-labelled GSH was taken up by chloroplasts in atime-dependent linear fashion. There appeared to betwo independent systems for uptake with at least oneof those being an active transport system. This wasinferred by observing linear uptake of labelled GSH upto 20–30 mM GSH showing saturation at a concentra-tion of 100–200 mM, which was followed by anincrease in the rate of uptake until a concentration of1 mM was reached. From these initial experiments,there is evidently great potential for modulating GSHconcentration by transport in response to an altereddemand for GSH caused by either the enzymes of thecytosol or ascorbate–glutathione cycle in the chloroplast.

GSH transport has not only been observed at thechloroplast membrane. Early experiments with hetero-trophic tobacco cells suggested that GSH could betransported across plasma membranes of plants(Schneider et al. 1992). In this study, both high-affinity(Km 18 mM) and low-affinity (Km 780 mM) GSH trans-port systems were identified. Other biochemical studiesindicated that some transporters had preferences forspecific GSH metabolites. For example, the bean plasma-lemma transporter exhibited a significantly greater affi-nity for GSSG compared with GSH (Jamaı et al. 1996).Taken together, the biochemical data from chloroplastsand plasma membrane preparations suggest that there isdiversity in the GSH transport mechanisms.


To date, the best-characterized glutathione transpor-ters are all members of the peptide transporter super-family. Early research in yeast identified the YeastCadmium Factor (YCF1) protein as a glutathione-S-con-jugate (GS-conjugate) transporter (Szczypka et al. 1994;Falcon-Perez et al. 1999). YCF1 is a vacuolar membraneprotein that transports Cd2þ.GSH2 and consequently isessential for Cd2þ tolerance in yeast. YCF1 is a memberof the ABC-type transporters similar to the multipledrug-resistance transporters (MRPs) in humans(Decottignies and Goffeau 1997). A common feature ofmany MRP proteins is the ability to transport GS-con-jugates and, in some cases, cotransport of GSH anddrugs (Rappa et al. 1997). MRP homologues are alsofound in plants (Kolukisaoglu et al. 2002). InArabidopsis, there are 14 genes (and one putative pseu-dogene) encoding MRP transporters (Kolukisaoglu et al.2002, Martinoia et al. 2002). Among these, onlyAtMRP1, AtMRP2, AtMRP3 and AtMRP5 have beencharacterized. Transport of GS conjugates was observedfor AtMRP1, AtMRP2 and AtMRP3 and is consistentwith the phenotype observed in an AtMRP5 insertionmutant (Lu et al. 1997, Lu et al. 1998, Tommasini et al.1998, Gaedeke et al. 2001, Liu et al. 2001). Morerecently, AtMRP3 has been implicated in Cd detoxifica-tion, as it is upregulated by Cd treatment which suggeststhat it may be transporting GSH-Cd or PC-Cd complexes(Bovet et al. 2005). However, the ability of the MRPproteins to transport GSH and GSSG is variable. Forexample, while only AtMRP2 and AtMRP4 exhibitedcompetitive inhibition of GS-conjugate transport byGSH, all four showed competitive inhibition in the pre-sence of oxidized glutathione (GSSG). Because GSSGcan be considered a GS-conjugate, this is consistentwith the MRP proteins being predominantly GS-conju-gate transporters. However, the observed GSSG trans-port may also suggest that they play an important role inremoving oxidized glutathione as a mechanism to helpmaintain the redox poise of the cell (Foyer et al. 2001).A second peptide transporter belonging to a different

family of peptide transporters from yeast has providedfurther insights into GSH transport. A high-affinity glu-tathione transporter (Hgt1p) was identified using thehgt1 mutant of Saccharomyces cerevisiae which wasunable to transport GSH (Bourbouloux et al. 2000).The Hgt1p protein is a member of the oligopeptidetransporter (OPT) family (Stacey et al. 2002). InArabidopsis, there are nine OPT orthologues of theyeast OPT proteins (Hgt1p and Opt2p) (Koh et al.2002). All of the OPT proteins are believed to be integ-ral membrane proteins transporting small peptides (3–5amino acids) (Koh et al. 2002). Heterologous expressionof the Arabidopsis OPT genes in S. cerevisiae has

confirmed that most are able to transport small peptides,while only some exhibit glutathione transport capabil-ities and none exhibit high-affinity transport similar toHgt1p (Foyer et al. 2001, Koh et al. 2002). Thus,although glutathione transport has been demonstratedin plant cells, no GSH-specific transporters, like Hgt1p,have been identified. Thus, while considerable move-ment of GSH/GSSG has been observed in biochemicalstudies, little is know about the factors responsible forthis transport.

Conclusions and perspectives: prospects formanipulation of glutathione metabolism forimproved nutritional quality

The ubiquity of GSH and its critical functions regulatingplant growth and as an important sulphur sink makeGSH an attractive target for manipulation to improvecrops for enhanced nutritional value as well asimproved crop growth, sustainability, predictabilityand vigour. Fortunately, plant biology now has anextensive toolbox available to help achieve this aim.Many of the genes found in model plant systems suchas Arabidopsis have counterparts in important crop spe-cies, and hence it is expected that much of the knowl-edge on GSH gained in laboratory systems will bereadily transferable to important crop varieties. In addi-tion, plant cell cultures could be used as a cheaper cellsynthetic factory for the production of g-EC, GSH, GSSGand related homologues than the yeast system that iscurrently used for GSH production. Glutathione haspotential for use in the food industry, for example as anatural food additive with flavour-enhancing properties.In whole plants, there is likely to be a ceiling on theenhanced glutathione concentrations that can beachieved, set by the level of feedback inhibition onGSH synthesis within the chloroplast. However, thisceiling GSH content may vary between plant species,because it is known that some alpine plants hyperaccu-mulate other antioxidants particularly ascorbate, andthe same may be true of glutathione. There are essen-tially no metabolic arguments suggesting that GSHhyperaccumulation in plants is impossible, becauseGSH accumulation is simply a matter of compartmenta-tion, such that the site of GSH accumulation is separatefrom that of synthesis, preventing feedback inhibition.This separation could easily be achieved in plant cellculture systems where GSH synthesis and export wereenhanced such that GSH and related peptides weretransported into the culture medium from which theycould be continually harvested. Moreover, because thebifunctional streptococcal g-ECS-GSH-S shows no feed-back inhibition by GSH, plant cells transformed to


express this enzyme may be able to maintain muchhigher GSH concentrations than those expressing theplant or E. coli enzymes.

Manipulating GSH should affect three main pheno-typic aspects (1) enhanced crop resilience and growth; (2)improved innate immune responses and basal resistanceto pathogen attack and (3) increasing sulphur/cysteinecontent in crop plants. A number of studies havedemonstrated the feasibility of enhancing plant glu-tathione synthesis and accumulation. Such plants havebeen used successfully for phytoremediation purposesand to reclaim arable land. All studies to date haveshown that increasing the GSH content of plants con-comitantly increases the total organic sulphur content.Comparatively, the manipulation of GSH partitioning,both throughout the plant and between cellular com-partments, has lagged behind, because no high-affinityGSH transporters have been identified to date. Theidentification of these will be a key step in combiningand interpreting current results in GSH research.Moreover, while GSH transport mutants will allow fora more detailed dissection of signalling and stress path-ways, they will also provide for future targeted nutriomicsapproaches where glutathione might be enriched inspecific cell types or in discrete compartments of theplant cell.

The fully sequenced genomes of species such asArabidopsis and rice together with new breeding tech-nologies and large scale ‘omics’, systems biologyapproaches will allow increasingly rapid advances inunderstanding and applications. For example, predict-ability of crop yield and hence income to the farmer andreliable sources for the food industry are severely ham-pered by environmental factors. Collectively, the envir-onmental abiotic stresses restrict plant vigour and createa ‘yield gap’. This is the difference between the theore-tical maximum or yield potential of the crop and theactual yield achieved by the farmer. Over the last 50years, plant breeders have improved the yield potential,but the yield gap remains, and it is forecast to increaseas the world climate becomes less predictable. Inmeasurements made under field conditions, maize leafglutathione contents correlate well with chilling resistanceand yield. One factor determining the sensitivity ofmaize to both short-term and long-term chilling is therestriction over glutathione reduction and cyclingbetween the maize leaf BS and M cell types (Gomezet al. 2004b). By understanding how glutathione is usedby the plant in acclimation responses to environmentalcues at the molecular level, we can use this informationto widen the range in which crop plants grow andproduce crops that perform closer to their theoreticalmaximum. The quantitative trait loci (QTL) analysis in

plants such as maize is essential to determine chromo-some regions carrying genes which control GSH accu-mulation and stress tolerance and other important traitsin crops. Mapping experiments at field sites haveallowed us to identify two major QTL for the totalamount of glutathione in maize, with a confidenceinterval of about 30 cM. To date, little information onQTL for glutathione has appeared in the literature.Detailed fine maps of QTL regulating glutathione accu-mulation under field conditions in different plant spe-cies will be useful not only to the scientific communitybut also to plant breeders and associated industries.

Acknowledgements – Rothamsted Research and the

Institute of Biotechnology receive grant-aided support from

the Biotechnology and Biological Sciences Research Council

of the UK [BB/C51508X/1, (C.F) and BB/C515047/1 (SM)].

Spencer Maughan is a Marie Curie International Incoming

Fellow of the EU (509962). We thank Dr Jeroen Nieuwland

for help with the mBBr-staining techniques and confocal

microscopy.

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