Engineering and Genetic Approaches to Modulating the Glutathione Network in Plants Spencer Maughana and Christine H

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

REVIEW Engineering and genetic approaches to modulating the glutathione network in plants Spencer Maughana and Christine H. Foyerb,* aInstitute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK bCrop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK

Correspondence Reduced glutathione (GSH) is the most abundant low-molecular weight thiol *Corresponding author, in plant cells. It accumulates to high concentrations, particularly in stress e-mail: [email protected] situations. Because the pathway of GSH synthesis consists of only two

Received 10 October 2005; revised 26 enzymes, manipulation of cellular glutathione contents by genetic interven- November 2005 tion has proved to be relatively straightforward. The discovery of a new bacterial bifunctional enzyme catalysing GSH synthesis but lacking feedback doi: 10.1111/j.1399-3054.2006.00684.x inhibition characteristics offers new prospects of enhancing GSH production and accumulation by plant cells, while the identification of g-glutamyl cysteine and glutathione transporters provides additional possibilities for selective compartment-specific targeting. Such manipulations might also be used to affect plant biology in disparate ways, because GSH and glutathione disulphide (GSSG) have crucial roles in processes as diverse as the regulation of the cell cycle, systemic acquired resistance and xenobiotic detoxification. For example, depletion of the total glutathione pool can be used to manipulate the shoot : root ratio, because GSH is required specifically for the growth of the root meristem. Similarly, chloroplast g-glutamyl cysteine synthetase overexpression could be used to increase the abundance of specific amino acids such as leucine, lysine and tyrosine that are synthesized in the chloroplasts. Here we review the aspects of glutathione biology related to synthesis, compartmentation and transport and related signalling functions that modulate plant growth and development and underpin any assessment of manipulation of GSH homeostasis from the viewpoint of nutritional genomics.

Abbreviations – g-EC, g-glutamyl cysteine; g-ECS, g-glutamyl cysteine synthetase; APR, adenosine 50phosphosulphate reductase; 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 Introduction and defence. Similarly, the analysis of mutants deficient in glutathione has greatly advanced current concepts of The thiol tripepide glutathione [g-Glu-Cys-Gly; reduced how GSH and glutathione disulphide (GSSG) regulate glutathione (GSH)] does not immediately come to mind cell signalling and development. Chemical inhibition of in considerations of human nutrition. However, this GSH synthesis using 1-buthionine-S, R-sulfoximine ubiquitous tripeptide thiol is a vital intracellular and (BSO), a specific inhibitor of g-ECS (Griffith and extracellular protective antioxidant against oxidative/ Meister 1979, Griffith 1982, Maughan and Cobbett nitrosative stresses, which play a key role in the control 2003), has been widely used to study the effects of of many human diseases. GSH is also important in GSH depletion in plants (Hell and Bergman 1990, immunity modulation in animals as well as in remodell- Vernoux et al. 2000). Analysis of Arabidopsis thaliana ing of the extracellular matrix, apoptosis and mitochon- T–DNA insertion and EMS mutants selected by their drial respiration in disease. Mammalian cancer cells ability to grow on BSO concentrations that are inhibi- often accumulate GSH, where there is also a metabolic tory to the wild-type has indicated that different switch to glycolysis rather than TCA cycle as a major mechanisms can confer BSO resistance (Maughan energy source (Pozuelo Rubio et al. 2004). Interactions 2003). of GSH with antiapoptotic factors such as Bcl-2 in can- Since its initial discovery in yeast over 125 years ago, cer cells have been linked to radiation and multidrug glutathione has been shown to have a prodigious number resistance (Ortega et al. 2003). However, because high of critical functions (Fig. 1). GSH is a powerful reducing GSH concentrations are essential for both antioxidant agent and is a key player in the plant redox-state (Noctor and immune defence systems in mammals, tissue GSH and Foyer 1998, Noctor et al. 2002a). Here we will levels can be regulated, particularly in malnourished focus on how manipulation of GSH synthesis and meta- patients through diet and nutrition before therapeutic bolism has advanced current concepts of glutathione treatments (Bray and Taylor 1993). homeostasis and function, concentrating on aspects Plants make abundant amounts of glutathione, and its related to cell signalling, development and defence synthesis and accumulation in either the chloroplast or that may be of importance in any consideration of nutri- cytosolic compartments of the plant cell can be tional genomics involving glutathione. The characteris- enhanced or decreased relatively easily by genetic engi- tics and functional importance of putative thiol-based neering approaches (Strohm et al. 1995, Creissen et al. reactive oxygen species (ROS) sensors and associated 1996, Noctor et al. 1998a, b, Zhu et al. 1999, Xiang redox-signalling cascades have recently been discussed et al. 2001). Ectopic expression of g-glutamyl cysteine synthetase (g-ECS, also called glutamate cysteine ligase) (Noctor et al. 1996) and certain enzymes of sulphur assimilation pathway (Harms et al. 2000) or glutathione Biotic reductase (GR) (Foyer et al. 1991) in transgenic plants Abiotic ROS scavenging results in substantial increases in leaf glutathione. Heavy metal detoxification Systemic Acquired Resistance Glutathione contents have been increased by at least Xenobiotic detoxification Protein thiolation five-fold the values of wild-type plants without negative Salinity protection PCD Oxidative brust effects (Noctor et al. 1998b, 2002a). Only one report to Drought protection Calcium MAPK date has indicated that an enhanced capacity to gener- UV protection signaling cascades ate and accumulate the intermediate g-glutamyl Signaling cysteine (g-EC) can produce negative effects (Creissen et al. 1999). The increased capacity to generate glu- Cell cycle progression Root development tathione and enhance cellular glutathione pools leads to higher rates of sulphur assimilation, modified amino Sulfur storage acid metabolism and enhanced stress tolerance (Noctor Sulfur sensing et al. 1998a, b). Transformed poplar trees with an increased capacity to synthesize and accumulate glu- Carbon - Sulfur - Nitrogen tathione are currently being used in the field for bior- emediation purposes to purify and restore soils polluted Metabolism by human activities (Peuke and Rennenberg, 2005a, b). Genetic engineering approaches have led to an Fig. 1. The functions of glutathione in plants linked by essential signal- improved understanding of how compartment-specific ling functions that integrate plant growth, development and defence alterations in glutathione synthesis affect metabolism processes.

Physiol. Plant. 126, 2006 383 (Foyer and Noctor 2005). In this context, we will con- checkpoint (Dewitte and Murray 2003, de Jager et al. centrate here on the implications for the plant of mod- 2005). The activity of the CDK/cyclin complexes is ulating glutathione homeostasis. regulated by a network of regulatory mechanisms Like ascorbate, glutathione limits the lifetime of ROS including transcription, proteolysis, phosphorylation/ and lipid peroxide signals. Moreover, GSH alters cal- dephosphorylation, interaction with regulatory proteins cium signalling in plants (Gomez et al. 2004a) and and intracellular trafficking. participates in the calcium-dependent pathways of In Arabidopsis, the embryo cells of the dry seed are ROS-signal transduction (Rentel and Knight 2004). arrested in the G1 phase of the cell cycle. Germination Through oxidation to GSSG, other signalling cascades is initiated by water uptake and the resumption of meta- can be initiated through thiol-disulphide exchange and bolism and cell division. Nitric oxide is a potent regu- thiolation. Moreover, the interaction between GSH and lator of germination in dormant seeds such as those of nitric oxide, catalysed by the enzyme formaldehyde Arabidopsis (Bethke et al. 2004, 2006). Germination is dehydrogenase (also known as class III alcohol dehy- stimulated by the presence of environmental nitrate drogenase), is also important in signal transduction as from which NO can be synthesized via nitrate reduc- S-nitrosoglutathione (GSNO) is thought to be a stable tase. The relatively low abundance of GSH within the NO transport form (Liu et al. 2001). The formaldehyde dry seed means that GSH has either to be regenerated dehydrogenases, which also catalyse the NAD-dependent from oxidized or bound forms upon water uptake or that formation of S-formylglutathione and S-hydroxymethyl it has to be synthesized de novo during germination to glutathione, are regulated by wounding and salicylic achieve the high levels present in seedlings. Low GSH acid (SA) (Diaz et al. 2003). Hence, glutathione is levels in the dry seed and during germination may pre- directly implicated in the coordinated control of sig- vent excessive formation of GSNO, allowing sufficient nalling by ROS and reactive nitrogen species (RNS) free NO to be present to stimulate the germination that determines cell fate in situations such as pathogen process. However, it is also possible that NO accumula- attack. tion serves as a trigger for GSH synthesis during It is important to note that thioredoxins (TRXs), glu- germination. tathione and lipid peroxides all target the same protein While it is well established that the G1 checkpoint is residues, allowing us to envisage a dynamic signalling very sensitive to environmental conditions, it has only network where proteins are modulated by these three recently become clear that this control might be exerted signalling systems according to their respective concen- through oxidation–reduction (redox) regulation and trations and the associated enzymes such as glutaredox- associated components, particularly glutathione and ins (GRXs). ascorbate, whose concentrations and redox state influences G1 progression. Glutathione and ascorbate exert independent controls on the plant cell cycle (Potters Control of plant growth and development et al. 2004). Glutathione regulation is important in a number of key Numerous interactions between the plant and environ- processes associated with plant growth and development take place at the root/soil interface. ment. These include the following: Monobromobimane (mBBr) has been widely used to detect GSH in vivo. Application of this GSH-localization system to Arabidopsis roots shows that GSH accu- The cell cycle and plant development mulates in the vascular system and root tip (Fig. 2A). Current knowledge of how cell division is controlled The relatively high concentration of GSH in root and how it interacts with different aspects of plant tips, where actively dividing cells are found (Fig. 2A), development such as morphogenesis, architecture and suggests that growing tissues have a requirement growth rate has greatly increased over the last 5 years. for GSH. Indeed, studies using the GSH-deficient Cell-cycle regulation involves components that respond ROOTMERISTEMLESS1 mutant (rml1) which has less to signals from the external environment as well as intrin- than 2% of the wild-type GSH levels have shown that sic developmental programmes, and it ensures that DNA without GSH root meristem cells arrest in G1 (Cheng is replicated with high fidelity within the constraints of et al. 1995, Vernoux et al. 2000). This mutation only prevailing environmental conditions (Dewitte et al. 2003, affects post-embryonic root development, and while de Jager et al. 2005). Cyclin-dependent kinases (CDKs) overall the roots are shorter, the cell files and types are play a central role in cell-cycle regulation, with negative not aberrant. It is important to note that in contrast to the (KRP proteins) and positive (D-type cyclins) regulators root meristem, the shoot meristem is able to produce all acting downstream of environmental inputs at the G1 the aboveground organs with development and timing

384 Physiol. Plant. 126, 2006 of GSH (Frendo et al. 2005). While the absolute content of GSH and the GSH : GSSG ratio clearly influence cell-cycle progression, the targets of glutathione action are unknown at present. In mammalian systems, this regulation includes transcription of the retinoblastoma gene product as well as its activity, which function BDdirectly downstream of G1 activators (Yamauchi and Bloom 1997). Given the importance of glutathione redox status in regulating cell division, it is perhaps not surprising that the GSH : GSSG ratio also regulates embryogenesis in plants (Belmonte and Yeung 2004, Belmonte et al. 2005) as it does in animals. Application of GSH in the A C E second half of embryo development results in preco- cious germination, while application of GSSG improved somatic embryogenesis (Belmonte and Yeung 2004, Belmonte et al. 2005). The redox status of the embryo glutathione pool delineates specific stages of embryo development both in vivo and in vitro. A high GSH/ GSSG ratio is essential for cell division and proliferation during the initial stages of embryo development, but later in development, the redox status of the glutathione pool decreases (Belmonte et al. 2005). This metabolic shift in cellular glutathione redox status appears to be required for continual embryonic development, and proper acclimation of storage product deposition is possibly facilitated through interactions with hormones FG such as abscisic acid. Zygotic embryos are held in G1 D Fig. 2. The effects of 1-buthionine-S, R-sulfoximine (BSO) on tissue until germination-specific -type cyclins promote cell glutathione and root growth in Arabidopsis thaliana. The root of a 7-day- division (Mausubelel et al. 2006). It is thus tempting to old seedling shown in (A) highlights the fact that reduced glutathione suggest that redox controls may act upstream of (GSH) is most abundant in the root tip and in the vasculature as germination-specific cell-cycle regulation in a fashion indicated by the blue fluorescence of mBBr. Untreated Arabidopsis similar to the regulation of division in the root apical cells (B) were stained for GSH which accumulates in the vacuole (C). meristem. M Equivalent BSO-treated (1 m ) cells (D, E) lack any significant staining Because GSH is a modulator of general plant devel- 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 opment, it is not surprising that it has also been impli- GUS enzyme to a mitotic cyclin (CYCB1;1; Colon-Carmona et al. 1999). cated in senescence processes (Ogawa et al. 2002). One Inhibition of GSH synthesis by BSO (1 mM) cell division causes cessation specific mode of action affects the activity of the ubi- of root growth (G). quitin-dependent 26S proteasome. The activity of this protein degradation pathway may be influenced by cel- that are comparable with the wild-type despite very low lular redox status in plants as it is in animals, where high GSH levels. GSH/GSSG ratios impede the binding of target proteins The rml1 phenotype can also be induced chemically (Hochstrasser 1998, del Pozo et al. 1998, Theriault et al. by treating cells or seedlings with BSO to lower GSH 2000, Sangerman et al. 2001). levels (Fig. 2B–G). This causes the cells in the root meristem to stop dividing, and, like the root meristem Plant defence and systemic acquired resistance cells of rml1, they arrest in the G1 phase (Fig. 2F,G). These results suggest that GSH plays a key role in GSH is a transcriptional regulator. It enhances the DNA- potentiating signals permitting cell-cycle progression binding activity of NFkB, a transcription factor that is and hence root growth. Similarly, root nodule formation central to the regulation of the oxidative stress response is prevented when GSH synthesis is blocked by addition in animal systems. Few homologues of major animal or of BSO, suggesting that like the root meristem, the microbial redox-sensitive elements and factors have nodule meristem is unable to develop in the absence been reported in plants to date apart from the activator

Physiol. Plant. 126, 2006 385 protein-1 (a transcription factor controlling response to a homeostatic mechanism by which [GSH] is increased oxidative stress) and the antioxidative responsive element in an attempt to offset stress-induced decreases in the that is present in the maize catalase (CAT) promoter. [GSH] : [GSSG] ratio. The nature of the link between However, cytosolic thiol-disulphide status reactions redox-state perturbation and enhanced glutathione are crucial in plant innate immune responses. GSSG accumulation is not fully resolved, but it may involve accumulation is often observed in close conjunction multiple levels of control. For example, the activity of with cell death and systemic acquired resistance (SAR) adenosine phosphosulphate reductase, a key enzyme in responses resulting from exposure to pathogens sulphate assimilation, is activated by decreases in

(Vanacker et al. 2000) or abiotic stresses (Gomez et al. GSH/GSSG (Bick et al. 2001). In addition, H2O2 and/or 2004a). RNS are also important in the plant pathogen low GSH/GSSG ratios enhance translation of g-ECS responses, and the strong interaction between GSH and (Xiang and Oliver 1998). Significant deviations from NO in signal transduction process, as GSNO for example, high GSH : GSSG ratios result in PCD (reviewed in suggests that mutual upregulation of these signalling Noctor et al. 2002a, b). The significance of alterations to components may occur in challenged cells. the GSH : GSSG ratio can be quantified using the Nernst An increase in tissue GSH contents activates the equation through which the redox potential of a given 5 expression of PR genes through the non-expressor of redox couple can be calculated: E Em [–(RT/nF)] PR protein 1 (NPR1) pathway (Mou et al. 2003, ln([Ared]/[Aox]). This allows a quantitative appreciation of Gomez et al. 2004a). The NPR1 protein is part of the the impact of changes in total glutathione content on the mechanism through which SA triggers SAR to pathogens. redox state of the cell. The activity of GR is essential in While many aspects of NPR1 function with regard to maintaining the high cellular GSH : GSSG ratios. Ectopic binding of the TGA transcription factors and movement expression of bacterial GR in the chloroplast or cytosol of to the nucleus to facilitate SAR are not fully understood, transgenic plants increased the GSH : GSSG ratio and this glutathione-modulated ankyrin repeat protein is one gave added protection against some stresses (Foyer et al. of the key regulators of SA-dependent gene expression 1991, 1995, Aono et al. 1993, 1995, Creissen et al. 1996). (Cao et al. 1994, Delaney et al. 1995). Such studies indi- Plants that are deficient in CAT activity have also cate a strong relationship between SA and glutathione been used to investigate oxidative signalling resulting homeostasis in stress signalling. Increased oxidation from decreased H2O2 detoxification (Vandenabeele resulting from exposure to stress stimulates glutathione et al. 2002, 2004). CAT-deficient plants are particularly accumulation. SA enhances this response presumably by interesting as overall levels of H2O2 accumulation are increasing oxidation possibly through inhibitory effects on relatively small, but there is a drastic increase in tissue CAT and ascorbate peroxidase. glutathione 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 identify Cell death and the GSH/GSSG redox couple a complete inventory of genes induced or repressed in

The many varied functions of GSH and importance of H2O2-induced PCD, which appears as clusters of dead the GSH : GSSG redox couple as a sensitive signal for palisade parenchyma cells along the leaf veins when cellular events necessitates strict GSH homeostasis. In these plants are exposed to high light (Vandenabeele animal cells, substantial evidence implicates redox et al. 2004). The stimulation of GSH synthesis and mod- potential as an important factor determining cell fate. ified GSH : GSSG ratios are intrinsic features of ozone

All aerobic organisms maintain thiols in the reduced and H2O2-dependent signalling. Thus, GSSG and thio- (-SH) state. GSH : GSSG ratios of 100:1 are typical lation may be important in adding specificity to the values for animal and plant cells. Departures from high signal and/or promoting second messenger signal trans- cellular GSH : GSSG ratios caused for example by duction in these pathways (Rentel and Knight 2004, exposure to stress are potentially dangerous, as they Foyer and Noctor 2005). are a cell death trigger; low GSH : GSSG ratios initiat- Transport systems also fulfil important roles in deter- ing programmed cell death (PCD) (Scha¨fer and Buettner mining the GSH : GSSG ratios of individual cell com- 2001). Many stresses cause an initial net oxidation of partments (Foyer et al. 2001). Transport of GSSG into the glutathione pool that is often followed by increases the vacuole by the MRP transporters or across the plas- in total glutathione (Smith et al. 1984, Sen Gupta et al. malemma (Jamaı¨ et al. 1996) contributes to 1991, Willekens et al. 1997, Noctor et al. 2002b, GSH : GSSG homeostasis in the cytosol. GSH degrada- Gomez et al. 2004b). This effect is observed, because tion may also be an important factor (Storozhenko et al. oxidation of the glutathione pool stimulates of GSH 2002). However, the g-glutamyl cycle, which contri- synthesis to restore cellular GSH : GSSG ratios. This is butes to GSH metabolism in animals, remains to be

386 Physiol. Plant. 126, 2006 confirmed in plants (Storozhenko et al. 2002). Rennenberg 2004). GSH accumulation in cells has feed- Overexpression of one putative component, g-glutamyl back effects on the pathway of primary sulphur assim- transferase (GGT) in Arabidopsis, did not alter the ilation (Kreuzwieser and Rennenberg 1998). The GSH : GSSG ratio or total tissue glutathione contents addition of GSH to Arabidopsis roots caused a decrease (Storozhenko et al. 2002). in adenosine 50 phosphosulphate reductase (APR) activ- Thiol-disulphide exchange reactions are a universal ity and transcript abundance (Vauclare et al. 2002) as mechanism for perception of redox perturbation. In well as in ATP sulphurylase activity (Lappartient et al. addition, S-glutathionylation or thiolation is a further 1999). Long distance GSH transport has also been sug- crucial mechanism in perception of altered glutathione gested to decrease sulphur assimilation (Lappartient and redox state. Protein glutathionylation is a widespread Touraine 1996) and poplar (Herschbach et al. 2000). A response to oxidizing conditions. It is becoming estab- simple regulatory mechanism has been proposed with lished as an important mechanism for reversible mod- regard to the regulation of demand-driven sulphur ification of protein cysteinyl residues in plants as it is in assimilation by positive signals such as o-acetylserine animals. While relatively few thiolated proteins have and negative signals such as GSH (Kopriva and been identified to date, those that have been character- Rennenberg 2004). ized include important metabolic enzymes such as sucrose synthase and acetyl CoA carboxylase and Effects of manipulation of GSH synthesis on defence enzymes such as dehydroascorbate reductase metabolism and development in mutant and (Dixon et al. 2005). Thiolation can have a number of transgenic plants effects on proteins, but it primarily protects cysteinyl residues from irreversible oxidation to their sulfinic Glutathione is synthesized in both photosynthetic and and sulphonic acid derivatives. The process is reversed non-photosynthetic cell types. However, green leaves by specific GRXs and/or TRXs, but much remains to be are often the major organs of GSH production and characterized concerning these regulatory systems export, and chloroplasts contain high (3–5 mM) GSH (Noctor in press). Both types of redox-active proteins concentrations (Foyer and Halliwell 1976, Smith et al. are encoded by quite large gene families in plants 1985). However, certain non-green cell types such as (Lemaire 2004). While relatively few plant thiolation trichomes also have a very high capacity to produce and targets have been identified to date, many as yet uni- accumulate glutathione (Gutie´rrez Alcala´ et al. 2002). dentified targets are likely also to be thioredoxin targets, Variable proportions of leaf glutathione are localized in which are becoming increasingly well characterized the chloroplasts, but values are generally considered to (Buchanan and Balmer 2005). be 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- Glutathione as a sink for reduced sulphur and sized from constituent amino acids in a two-step ATP- regulator of sulphur assimilation dependent reaction sequence (Fig. 3) catalysed by Current evidence suggests that GSH has a role in sul- g-ECS and glutathione synthetase (GSH-S) (Hell and phur homeostasis. GSH is an abundant low-molecular Bergmann 1988, 1990, Meister 1988). With the excep- weight thiol and hence a potentially important sulphur tion of a novel enzyme from Streptococcus agalactiae sink as well as a predominant transported form of that catalyses both activities (Janowiak and Griffith reduced sulphur (reviewed in Hell 1997). This model 2005), g-ECS and GSH-S enzymes have been identified predicts that excess sulphur will be stored in GSH and as separate proteins and gene products from numerous will be released when the cell experiences sulphur defi- eukaryotes and gram-negative prokaryotes. The S. aga- ciency or requires increased sulphur availability. lactiae enzyme has several features that are worthy of Important mechanistic support came from studies note. This is not only the first bifunctional g-ECS-GSH-S which show GSH is transported long distances in the enzyme to be isolated but is also the first GSH synthesis phloem and xylem (Rennenberg et al. 1979). enzyme system to be identified from a gram-positive Furthermore, GSH is transported to developing tissues organism (Janowiak and Griffith 2005). The g-ECS- from Maize scutella (Rauser et al. 1991). Herschbach GSH-S sequence encodes an 85 kDA protein, which et al. (2000) showed that not only is GSH found in the when purified exhibits GSH-S activity with a similar phloem and xylem but also that GSH levels control specific activity to that of g-ECS. It has similar Km values sulphate loading into the xylem. Together with cysteine, for cysteine and ATP to the Escherichia. coli g-ECS, but glutathione forms part of the repertoire of signals that the streptococcal g-ECS-GSH-S has a lower affinity for modulate sulphate uptake and assimilation (Kopriva and glutamate than the E. coli GSH-S. This may be

Physiol. Plant. 126, 2006 387 γ ( -ECn)Gly - Heavy metals Nucleus Sequestration GS-X conjugates Cytochromes of harmful GSSG (GSH) NPR1 PR genes Mitochondrion compounds Reducing Redox power for Vacuole signaling/gene energy chains expression L-Glycine + ATP GSH γ-EC GSH ER 2 GSH Cytoplasm GSSG GS-X Chloroplast PMF Pepides L-Cysteine (CysGly) AAs γ-Glutamylcysteine γ 1 2 ( -EC) GSH HS GSSG L-Glycine OH H O ATP Glutathione N OH L-Glutamate GS-X (GSH) + O N ATP O H HS OH H OO N O N OH ROS Scavenging H NH 1 O 2 (H2O2, O2) GSH-Ascorbate cycle

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. explained by the fact that gram-positive bacteria main- exudates and roots (Herschbach et al. 2000). The tain exceptionally high glutamate levels (of up to identification of a g-ECS-GSH-S enzyme that facilitates 100 mM). The GSH-S domain has a relatively small glutathione synthesis in S. agalactiais suggests that similar molecular mass (31 kDaA) with an amino acid bifunctional enzymes might occur in other gram-positive sequence more related to D-Ala-ligase than GSH-S bacteria where glutathione synthesis was thought to be (Janowiak and Griffith 2005). It is important to note relatively uncommon. These bifunctional g-ECS-GSH-S GSH inhibits neither the g-ECS nor the GSH-S activity enzymes might be extremely useful in future plant of the streptococcal g-ECS-GSH-S, allowing S. agalac- genetic engineering approaches to manipulate tiae to maintain much higher GSH concentrations than glutathione for nutritional purposes. Similarly, specific for example E. coli, despite the fact that the overall homologues of glutathione could be introduced for g-ECS activity is relatively lower. It is interesting to note example by ectopic expression of enzymes such as that S. agalactiae lack catalase and therefore rely heavily homoglutathione synthetase. on GSH-based enzymes for peroxide detoxification The endogenous plant g-ECS and GSH-S enzymes dis- (Janowiak and Griffith 2005). play a high degree of sequence homology. In A. thaliana, The E. coli genes encoding g-ECS and GSH-S have g-ECS is encoded by a single gene, GSH1, with a plastid been used extensively in genetic engineering target signal (May and Leaver 1994). A recent analysis approaches to enhance glutathione contents in plants using a g-ECS-GFP reporter system has indicated that (Noctor et al. 1998a, Zhu et al. 1999). Targeting of the most of, if not all, the A. thaliana g-ECS protein is located bacterial g-ECS and GSH-S to either the chloroplast in the chloroplasts (Wachter et al. 2005). Targeting or cytosol has led to marked increases in enzyme information relative to GSH-S suggests that it is present activity. Increases in g-ECS but not GSH-S led to large both in chloroplasts and the cytosol. The chloroplast constitutive increases in leaf glutathione (Noctor et al. and cytosol thus cooperate in de novo GSH synthesis, 1996, 1998a, Creissen et al. 1999). Moreover, glu- the chloroplast acting as the source of g-EC for tathione was also increased in xylem sap, phloem both chloroplastic and cytosolic GSH production.

388 Physiol. Plant. 126, 2006 These observations are surprising given that earlier stud- has already been reviewed (Noctor et al. 1998a, Noctor ies localized g-ECS and GSH-S activities in the cystosol et al. 2002a). The major factors controlling GSH accu- as well as the chloroplasts (Klapheck et al. 1987, Hell mulation in plants as in animals are abundance of g-ECS and Bergmann 1988, 1990). Moreover, the studies with and the availability of cysteine (Noctor et al. 1996, transgenic plants have shown that GSH synthesis can be 1998a, 1998b, 2002a, Xiang and Oliver 1998, Xiang increased by targeting the synthetic enzymes to either and Bertrand 2000, Xiang et al. 2001). Overexpression the chloroplastic or the cytosolic compartments (Noctor of enzymes such as g-ECS (Noctor et al. 1996), GR et al. 1996, 1998a, Creissen et al. 1999). However, (Foyer et al. 1991, 1995, Aono et al. 1993, 1995) or kinetic analysis has revealed that the chloroplast and serine acetyltransferase (Harms et al. 2000) enhances cytosolic isoforms have similar properties (Noctor tissue GSH contents. However, there are few attempts et al. 2002a). More work is required to fully rationalize to date to examine the effects of simultaneous expres- the molecular genetic evidence with the biochemical sion of these enzymes. Increased tissue glutathione in observations. transformed plants has resulted in largely beneficial The targeting information indicating that g-EC is lar- effects (Foyer et al. 1995, Strohm et al. 1995, Noctor gely synthesized in the chloroplasts (Wachter et al. et al. 1996, 1998a, Pilon-Smits et al. 1999, Zhu et al. 2005) has implications for glutathione homeostasis. 1999). The marked chlorotic phenotype produced by Firstly, at least some of g-EC produced in the chloroplast chloroplastic E. coli g-ECS overexpression in trans- must be exported to the cytosol, as most of the GSH-S is formed tobacco is unusual and may result from per- located in this compartment. Secondly, overexpression turbed signal transduction rather than direct effects of of g-EC in the chloroplast enhanced not only leaf g-EC GSH or g-EC accumulation per se (Creissen et al. 1999). and glutathione contents, but it also increased the abun- Some of the effects of ectopic g-ECS expression are dance of amino acids synthesized in the chloroplasts illustrated in Fig. 4 using transformed Arabidopsis seed- such as valine, leucine, isoleucine, lysine and tyrosine lings produced by Cobbett et al. (1998). The high level (Noctor et al. 1998a). It is interesting to note that of GSH1 transcripts observed in the transformed increases in these amino acids was specific to chloro- Arabidopsis plants relative to controls (Fig. 4A) is plast g-ECS overexpression and that the degree of accompanied by increased total leaf glutathione enhancement occurred in almost direct proportion to contents (Fig. 4B). Moreover, the enhanced capacity increases in g-EC and glutathione (Noctor et al. 1998a) for GSH synthesis arising from the increased expression GSH metabolism is more complex in C4 species such of g-ECS results in a much lower effect of BSO in terms as maize than in C3 species such as Arabidopsis, of its ability to deplete tissue glutathione (Fig. 4B). The tobacco and poplar. In maize leaves, cysteine synthesis pronounced positive effect of GSH1 overexpression and is exclusively localized in the bundle sheath (BS) tissues higher tissue GSH availability on Arabidopsis root (Burgener et al. 1998). g-ECS and GSH-S transcripts growth is illustrated in Fig. 4C. It is interesting to note and proteins are similar in abundance in maize leaf that in the presence of BSO, the wild-type plants have a mesophyll (M) and BS cells, and they were also found similar phenotype to the rml1 mutant as illustrated by in the epidermis and stomatal guard cells (Gomez et al. the absence of a post-embryonic root. However, 2004b). It is important to note however that an earlier enhanced g-ECS activity (o/e line) alleviates this study found that GSH-S activity primarily in the M cells inhibition of root development, presumably due to the (Burgener et al. 1998). higher capacity for GSH synthesis even in the presence A number of GSH-deficient A. thaliana mutants have of BSO, allowing the root to grow. been described in the GSH1 locus. The analysis of Sulphur uptake by the roots of transformed bacterial g-ECS-deficient mutants such as rml, cad and rax has g-EC-expressing poplar trees was enhanced to meet the provided useful information concerning the effects of requirements of increased demand for sulphur caused glutathione depletion in plants (Cheng et al. 1995, by enhanced GSH synthesis (Herschbach et al. 2000). Howden et al. 1995, Vernoux et al. 2000, Ball et al. However, the glutathione contents of untransformed 2004). The mutants have rather different characteristics. and transformed poplar leaf discs were increased sub- For example, the cad2-1, which has 15–30% of wild- stantially by incubation with cysteine, particularly in the type GSH, and the rax 1-1, which has 50% of wild-type light. This suggests that cysteine supply remains a key GSH, have different leaf transcriptome profiles (Ball limiting factor for glutathione synthesis even when et al. 2004). It is possible that the GSH1 gene products g-ECS activity is increased (Strohm et al. 1995, Noctor are themselves involved in signal transduction. et al. 1996, 1997). The leaf cysteine levels were main- The extensive literature concerning the effects of tained at values similar to the wild-type in transgenic manipulation of g-ECS and GSH-S activities in plants poplars with high g-ECS activities, but the activities of

Physiol. Plant. 126, 2006 389 enzymes involved in sulphate reduction and assimila- 20 A tion were not increased (Noctor et al. 1998a, 18 b, Hartmann et al. 2004). Similarly, microarray analysis 16 of plants undergoing sulphate starvation did not reveal effects on g-ECS or GSH-S transcripts suggesting little 14 coordination of the pathways of GSH synthesis in 12 response to sulphur availability (Hirai et al. 2003, 10 Maruyama-Nakashita et al. 2003). Hence, to remove metabolic limitations in cysteine supply, it will be 8 essential to consider solutions that modulate sulphur 6 assimilation to further enhance the capacity of plant 4 tissues to synthesize and accumulate glutathione. For Relative fold expression of GSH1 example, modulation of oxylipin signalling may be 2 one approach, as methyl jasmonate stimulates transcrip- 0 tion of both sulphur assimilation and GSH synthesis WT GSH1 o/e genes (Harada et al. 2000).

B 500 Regulation of glutathione synthesis and accumulation

400 There is very little literature information available on the coordinate regulation of expression of GSH1 and GSH2 in plants. It is worth noting the very high abundance of 300 GSH2 transcripts in the quiescent centre of Arabidopsis roots, compared with surrounding cells (Nawy et al. 2005), and it may be that a high level of GSH is required in these stem cells for antioxidant defence, because 200 quiescent centre cells are essentially devoid of reduced nmol GSH/g FW ascorbate. GSH1 and GSH2 mRNAs are increased by conditions 100 that require enhanced GSH abundance for metabolic functions such as heavy metal sequestration. Neither GSH nor GSSG exercise much control over GSH1 and 0 WT GSH1 o/e GSH2 transcription (Xiang and Oliver 1998). Similarly, while H2O2 and CAT inhibitors increase tissue GSH, they did not affect GSH1 or GSH2 transcript abundance C (Xiang and Oliver 1998). Exposure to oxidative stress increased g-ECS activity and glutathione in Arabidopsis, but GSH1 mRNA abundance was unchanged. In contrast, exposure to heavy metals such as cadmium and copper increases GSH1 and GSH2 transcripts (Scha¨fer et al. 1998, Xiang and Oliver 1998). While SA does not regulate GSH1 or GSH2 transcripts, jasmonic acid (JA)

Fig. 4. Effect of g-glutamyl cysteine synthetase (g-ECS) overexpression in 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 WT GSH1 o/e presence of 0.7 mM 1-buthionine-SR-sulfoximine (M).

390 Physiol. Plant. 126, 2006 has a marked stimulatory effect (Xiang and Oliver interactions with 14-3-3 proteins (DeLille et al. 2001), 1998). Methyl jasmonate caused a rapid transient this type of regulation of GSH synthesis and/or degrad- increase in transcripts encoding sulphur assimilation ation is a possibility that merits further investigation. enzymes and GSH1 and GSH2 mRNA levels in Arabidopsis (Harada et al. 2000). Although transcript GSH homeostasis-transport abundance was increased by heavy metals and JA, oxidative stress was required for the translation of the tran- The physical separation of the rate-limiting step of GSH scripts, implicating regulation at the post-transcriptional synthesis catalysed by g-ECS from the second reaction level and a possible role for factors such as H2O2 or step suggests that g-EC and GSH transport have funda- modified GSH/GSSG ratios in de-repressing translation mental roles in glutathione homeostasis. Recent data of the existing mRNA (Xiang and Oliver 1998). Stress- has implicated oligopeptide transport proteins (OPTs) induced increases in glutathione, such as those in GSH transport. However, none of the nine AtOPTs observed in plants deficient in catalase, have shown has a high affinity for GSH transport, and the cellular that glutathione accumulation is preceded or accompan- localizations of these proteins have not been resolved. ied by a marked decrease in the reduction state of the Chloroplastic transporters are particularly important, as pool (Smith et al. 1984, Willekens et al. 1997). A similar they are essential for the export of g-EC and GSH pro- response was elicited by exposing poplar leaves to duced within the chloroplast. However, none of the ozone (Sen Gupta et al. 1991). The 50untranslated known transporters that have been implicated in GSH/ region (50UTR) of the GSH1 gene was found to interact GSSG transport have been shown to be localized in the with a repressor-binding protein that was released upon chloroplast despite the fact that recent data suggests that addition of H2O2 or changes in the GSH/GSSG ratio the chloroplast may be the major site of g-EC produc- (Xiang and Bertrand 2000). A redox-sensitive 50UTR- tion. One or more high-affinity g-EC/GSH/GSSG trans- binding complex is thus suggested to control g-ECS porters targeted to the chloroplast may be expected, as mRNA translation in A. thaliana (Xiang and Bertrand illustrated in Fig. 3. Preliminary biochemical experi- 2000). mentation has shown that GSH is transported into iso- It has long been accepted that post-translational reg- lated wheat chloroplasts. Noctor et al. (2002a) observed ulation of g-ECS through end-product inhibition by GSH that 35S-labelled GSH was taken up by chloroplasts in a may control tissue GSH concentrations (Hell and time-dependent linear fashion. There appeared to be Bergmann 1990, Schneider and Bergmann 1995, Jez two independent systems for uptake with at least one et al. 2004). Moreover, g-ECS activity is inhibited by of those being an active transport system. This was thiols in plant extracts (Noctor et al. 2002a) as well as inferred by observing linear uptake of labelled GSH up in the purified recombinant enzyme (Jez et al. 2004). to 20–30 mM GSH showing saturation at a concentra- Such feedback control of g-ECS activity by GSH con- tion of 100–200 mM, which was followed by an centrations is easy to understand in terms of the exclu- increase in the rate of uptake until a concentration of sive localization of this enzyme in the chloroplast, 1mM was reached. From these initial experiments, where localized GSH accumulation in the limited there is evidently great potential for modulating GSH space of the organelle would cause a specific feedback concentration by transport in response to an altered limitation on g-ECS activity, until excess GSH could be demand for GSH caused by either the enzymes of the exported to the cytosol. cytosol or ascorbate–glutathione cycle in the chloroplast. Post-translational control of g-ECS activity remains a GSH transport has not only been observed at the possibility (May et al. 1998), but protein phosphoryl- chloroplast membrane. Early experiments with hetero- ation similar to that reported in animals (Sun et al. 1996) trophic tobacco cells suggested that GSH could be has not yet been found in plants. In animals, a smaller transported across plasma membranes of plants regulatory subunit increases the catalytic potential of (Schneider et al. 1992). In this study, both high-affinity the larger catalytic subunit by alleviating the effects of (Km 18 mM) and low-affinity (Km 780 mM) GSH trans- feedback control (Huang et al. 1993, Mulcahy et al. port systems were identified. Other biochemical studies 1995). A recent analysis in human cells has shown indicated that some transporters had preferences for that a number of enzymes affecting glutathione metab- specific GSH metabolites. For example, the bean plasma- olism and breakdown are targets for of 14-3-3 proteins lemma transporter exhibited a significantly greater affi- (Pozuelo Rubio et al. 2004), and this may be also the nity for GSSG compared with GSH (Jamaı¨ et al. 1996). case in plants. Because the precise pathway of glu- Taken together, the biochemical data from chloroplasts tathione degradation in plants remains poorly resolved, and plasma membrane preparations suggest that there is and many important plant enzymes are controlled by diversity in the GSH transport mechanisms.

Physiol. Plant. 126, 2006 391 To date, the best-characterized glutathione transpor- confirmed that most are able to transport small peptides, ters are all members of the peptide transporter super- while only some exhibit glutathione transport capabil- family. Early research in yeast identified the Yeast ities and none exhibit high-affinity transport similar to Cadmium Factor (YCF1) protein as a glutathione-S-con- Hgt1p (Foyer et al. 2001, Koh et al. 2002). Thus, jugate (GS-conjugate) transporter (Szczypka et al. 1994; although glutathione transport has been demonstrated Falcon-Perez et al. 1999). YCF1 is a vacuolar membrane in plant cells, no GSH-specific transporters, like Hgt1p, 2þ protein that transports Cd .GSH2 and consequently is have been identified. Thus, while considerable move- þ essential for Cd2 tolerance in yeast. YCF1 is a member ment of GSH/GSSG has been observed in biochemical of the ABC-type transporters similar to the multiple studies, little is know about the factors responsible for drug-resistance transporters (MRPs) in humans this transport. (Decottignies and Goffeau 1997). A common feature of many MRP proteins is the ability to transport GS-con- Conclusions and perspectives: prospects for jugates and, in some cases, cotransport of GSH and manipulation of glutathione metabolism for drugs (Rappa et al. 1997). MRP homologues are also improved nutritional quality found in plants (Kolukisaoglu et al. 2002). In Arabidopsis, there are 14 genes (and one putative pseu- The ubiquity of GSH and its critical functions regulating dogene) encoding MRP transporters (Kolukisaoglu et al. plant growth and as an important sulphur sink make 2002, Martinoia et al. 2002). Among these, only GSH an attractive target for manipulation to improve AtMRP1, AtMRP2, AtMRP3 and AtMRP5 have been crops for enhanced nutritional value as well as characterized. Transport of GS conjugates was observed improved crop growth, sustainability, predictability for AtMRP1, AtMRP2 and AtMRP3 and is consistent and vigour. Fortunately, plant biology now has an with the phenotype observed in an AtMRP5 insertion extensive toolbox available to help achieve this aim. mutant (Lu et al. 1997, Lu et al. 1998, Tommasini et al. Many of the genes found in model plant systems such 1998, Gaedeke et al. 2001, Liu et al. 2001). More as Arabidopsis have counterparts in important crop spe- recently, AtMRP3 has been implicated in Cd detoxifica- cies, and hence it is expected that much of the knowl- tion, as it is upregulated by Cd treatment which suggests edge on GSH gained in laboratory systems will be that it may be transporting GSH-Cd or PC-Cd complexes readily transferable to important crop varieties. In addi- (Bovet et al. 2005). However, the ability of the MRP tion, plant cell cultures could be used as a cheaper cell proteins to transport GSH and GSSG is variable. For synthetic factory for the production of g-EC, GSH, GSSG example, while only AtMRP2 and AtMRP4 exhibited and related homologues than the yeast system that is competitive inhibition of GS-conjugate transport by currently used for GSH production. Glutathione has GSH, all four showed competitive inhibition in the pre- potential for use in the food industry, for example as a sence of oxidized glutathione (GSSG). Because GSSG natural food additive with flavour-enhancing properties. can be considered a GS-conjugate, this is consistent In whole plants, there is likely to be a ceiling on the with the MRP proteins being predominantly GS-conju- enhanced glutathione concentrations that can be gate transporters. However, the observed GSSG trans- achieved, set by the level of feedback inhibition on port may also suggest that they play an important role in GSH synthesis within the chloroplast. However, this removing oxidized glutathione as a mechanism to help ceiling GSH content may vary between plant species, maintain the redox poise of the cell (Foyer et al. 2001). because it is known that some alpine plants hyperaccu- A second peptide transporter belonging to a different mulate other antioxidants particularly ascorbate, and family of peptide transporters from yeast has provided the same may be true of glutathione. There are essen- further insights into GSH transport. A high-affinity glu- tially no metabolic arguments suggesting that GSH tathione transporter (Hgt1p) was identified using the hyperaccumulation in plants is impossible, because hgt1 mutant of Saccharomyces cerevisiae which was GSH accumulation is simply a matter of compartmenta- unable to transport GSH (Bourbouloux et al. 2000). tion, such that the site of GSH accumulation is separate The Hgt1p protein is a member of the oligopeptide from that of synthesis, preventing feedback inhibition. transporter (OPT) family (Stacey et al. 2002). In This separation could easily be achieved in plant cell Arabidopsis, there are nine OPT orthologues of the culture systems where GSH synthesis and export were yeast OPT proteins (Hgt1p and Opt2p) (Koh et al. enhanced such that GSH and related peptides were 2002). All of the OPT proteins are believed to be integ- transported into the culture medium from which they ral membrane proteins transporting small peptides (3–5 could be continually harvested. Moreover, because the amino acids) (Koh et al. 2002). Heterologous expression bifunctional streptococcal g-ECS-GSH-S shows no feed- of the Arabidopsis OPT genes in S. cerevisiae has back inhibition by GSH, plant cells transformed to

392 Physiol. Plant. 126, 2006 express this enzyme may be able to maintain much plants such as maize is essential to determine chromo- higher GSH concentrations than those expressing the some regions carrying genes which control GSH accu- plant or E. coli enzymes. mulation and stress tolerance and other important traits Manipulating GSH should affect three main pheno- in crops. Mapping experiments at field sites have typic aspects (1) enhanced crop resilience and growth; (2) allowed us to identify two major QTL for the total improved innate immune responses and basal resistance amount of glutathione in maize, with a confidence to pathogen attack and (3) increasing sulphur/cysteine interval of about 30 cM. To date, little information on content in crop plants. A number of studies have QTL for glutathione has appeared in the literature. demonstrated the feasibility of enhancing plant glu- Detailed fine maps of QTL regulating glutathione accu- tathione synthesis and accumulation. Such plants have mulation under field conditions in different plant spe- been used successfully for phytoremediation purposes cies will be useful not only to the scientific community and to reclaim arable land. All studies to date have but also to plant breeders and associated industries. shown that increasing the GSH content of plants con- comitantly increases the total organic sulphur content. Acknowledgements – Rothamsted Research and the Comparatively, the manipulation of GSH partitioning, Institute of Biotechnology receive grant-aided support from the Biotechnology and Biological Sciences Research Council both throughout the plant and between cellular com- of the UK [BB/C51508X/1, (C.F) and BB/C515047/1 (SM)]. partments, has lagged behind, because no high-affinity Spencer Maughan is a Marie Curie International Incoming GSH transporters have been identified to date. The Fellow of the EU (509962). We thank Dr Jeroen Nieuwland identification of these will be a key step in combining for help with the mBBr-staining techniques and confocal and interpreting current results in GSH research. microscopy. Moreover, while GSH transport mutants will allow for a more detailed dissection of signalling and stress pathways, they will also provide for future targeted nutriomics References approaches where glutathione might be enriched in Aono M, Kubo A, Saji H, Tanaka K, Kondo N (1993) specific cell types or in discrete compartments of the Enhanced tolerance to photooxidative stress of transgenic plant cell. Nicotiana tabacum with high chloroplast glutathione The fully sequenced genomes of species such as reductase activity. 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