2.3.1. Enzymatic quantification

The most common spectrophotometric glutathione assay uses 5,5′-dithiobis(2-nitrobenzoic acid), otherwise known as DTNB or Ellman's reagent. Because this compound binds non-specifically to thiol groups, it can be used to measure protein and non-protein thiols alike. In acid extracts, from which proteins have been precipitated, this method measures total non-protein thiols (Figure 2A, bottom). The assay can provide a useful gross estimate of GSH content as this compound is usually the major non-protein thiol in most tissues. However, it should be noted that the method does not specifically measure GSH but all DTNB-reactive thiols present in the extract. It also does not detect GSSG. If NADPH and GR are include in the reaction mix (Figure 2A, middle), DNTB is continuously reduced by electrons provided by NADPH, and glutathione cycles between GSSG and GSH in an intermediate, catalytic role. This technique provides much greater sensitivity and specificity for glutathione than the end-point DTNB assay (Tietze, 1969). Without pre-treatment of extracts, the method measures both GSH and GSSG, ie, total free glutathione. The most common approach to distinguish between the two forms is to treat aliquots with a thiol reagent to remove GSH, then to assay the residual GSSG in the same way as total glutathione. Typical thiol reagents are N-ethylmaleimide and 2-vinylpyridine (Griffith, 1980). These methods can be readily adapted to multi-well assay in plate-readers (Queval and Noctor, 2007).

Figure 2.

Some commonly used methods for measuring glutathione.

(A) Standard in vitro techniques for assaying glutathione and related thiols in protein-free extracts.

(B) Three techniques recently used to measure glutathione in situ. The image in the right middle panel was kindly provided by Bernd Zechmann (University of Graz, Austria). It shows a section through an Arabidopsis wild-type leaf mesophyll cell labelled with an anti-glutathione antibody. The black particles correspond to glutathione immunoprecipitates visible in mitochondria (M), nucleus (N), and plastid (P), and which are much less abundant in the vacuole (V). The scale bar indicates 1 µm. DTNB, 5,5′-dithiobis(2-nitrobenzoic acid). F, fluorescence. GRX, glutaredoxin. GST, glutathione S-transferase. MCB, monochlorobimane.

2.3.2. Liquid chromatography-based assays

The most commonly used HPLC technique is based on fluorescent labelling. Developed during the late 1970's and early 1980's (Fahey et al., 1981), this technique was first used to analyze plant extracts by the Rennenberg group (Schupp and Rennenberg, 1988; Rauser et al., 1991). It involves quantification of thiol-bimane adducts formed by treating extracts with reagents such as monobromobimane (MBB). The advantages of this sensitive technique are that the bimane derivatives have a high fluorescence yield and are quite stable (up to several days in solutions kept in the dark), and that other thiols present in the extract, such as cysteine, can be separated and measured in the same run with glutathione (Figure 2A, top). In the most common application of the technique, aliquots are treated, prior to bimane derivatization, with a reductant such as dithiothreitol (DTT). This pre-treatment converts any GSSG present in the aliquot to GSH, and so only the total glutathione pool is determined, not the redox state. As described above for the spectrophotometric assay, GSSG can be measured by pre-treating extract aliquots with thiol reagents to remove GSH. However, this procedure is somewhat more onerous for the HPLC method because, after GSH is fixed in a complex, GSSG must subsequently be reduced to GSH prior to incubation with MBB. Approximate glutathione redox states can be estimated by simply comparing GS-bimane peak yields in aliquots from the same extract pre-treated or not with reductants like DTT, though the alkaline pH required for reaction with MBB probably causes some artefactual oxidation of extracted GSH if DTT is not present during the derivatization. This method can therefore give somewhat lower reduction states than the spectrophotometric assay (eg, Queval et al., 2011).

Other sensitive HPLC-fluorescence methods can be used based on derivatizing the primary amine group of glutathione. One example is O-phthaldialdehyde (OPA), a reagent that entered widespread use for HPLC analysis of amino acids in the 1980's (Lindroth and Mopper, 1979). Using OPA allows glutathione to be measured along with amino acids (Noctor and Foyer, 1998), though one drawback compared to bimane derivatization is that cysteine is not readily detectable. Other techniques applied to measuring glutathione in plants include LC-MS (Muckenschnabel et al., 2001) and capillary electrophoresis (Mendoza et al., 2008). Compared to targeted thiol analyses, however, these techniques require more specialized equipment and may also be less reproducible and quantitative. Techniques such as LCMS are, however, among the methods of choice for identifying or analyzing GS-conjugates (eg, Brazier-Hicks et al., 2008; Mueller et al., 2008).

2.3.3 Data output

Regardless of the analytical method used to quantify glutathione in extracts, a key factor is sample preparation. The extraction procedure is crucial, and great care has to be taken to ensure that no loss of glutathione or change in redox state occurs. Best results are obtained if samples are quenched and ground in liquid nitrogen and then extracted directly into acid as the material thaws, taking care to homogenize regularly during this procedure. While some neutralization of protein-free acid supernatants is required for most of the techniques, maintaining a slightly acidic pH throughout the sample preparation minimizes GSH oxidation. Once the pH has been adjusted, extracts should be assayed as soon as possible, though from our experience GSH and GSSG are usually quite stable for a few hours in extracts kept on ice. It is not advisable to refreeze extracts, even if kept at acidic pH. For some extracts, this can lead to artefactual oxidation or degradation. As for any metabolite assay, it is advisable to check extraction efficiency, for example, with recovery experiments using known amounts of GSH or GSSG added at the stage of extraction in the presence or absence of the tissue under examination.

Measuring glutathione by enzyme assay or by HPLC-fluorescence with MBB or OPA produces comparable data for tissue contents (Noctor and Foyer, 1998; Queval and Noctor, 2007). Proper application of these methods to analysis of plants grown in controlled conditions gives typical values for leaf glutathione of the order of 200–600 nmol.g¹ fresh weight with the exact value depending on developmental stage and nutritional/light regime (eg, Cobbett et al., 1998; Veljovic-Jovanovic et al., 2001; Hartmann et al., 2003; Queval and Noctor, 2007). Higher values may be obtained for plants grown in less controlled conditions, eg, in crop plants growing in the field.

The overall redox state of glutathione [GSH/(GSH + 2GSSG)] in leaf extracts is around 0.9–0.95. Increases in GSSG relative to GSH are a useful indicator of oxidative stress or ‘disulfide stress’ (Åslund and Beckwith, 1999), and in plants can be observed in response to conditions such as ozone, pathogens, and cold, as well as deficiency in important H₂O₂-metabolizing enzymes such as catalase and ascorbate peroxidase (APX; Smith et al., 1984; Sen Gupta et al., 1991; Edwards et al., 1991; May and Leaver, 1993; May et al., 1996a; Willekens et al., 1997; Vanacker et al., 2000; Kocsy et al., 2000; Riszhky et al., 2002; Gomez et al., 2004a; Queval et al., 2007). A key property of glutathione homeostasis function is that increased oxidation, unless accompanied by appreciable consumption of GSH, is often followed or accompanied by increases in the total pool.

2.3.4. Analyzing glutathione in situ

Several methods have been developed to monitor glutathione in situ. It is important to note that these techniques measure different features of glutathione status, which may be of greater or lesser relevance depending on the information that is sought. Like in vitro analysis of glutathione, all have potential drawbacks and limitations in terms of the information they provide. In some cases, the necessary preparation imposes a stress on the sample, which could modify the factor being measured. Another caveat relates to the use of in vivo techniques under conditions different from those in which the plants are growing (eg, different actinic irradiance in studies where the rate of photosynthesis is important). Nevertheless, the availability of in situ techniques is undoubtably an important development that is adding to the repertoire of tools to analyze the complexities of glutathione homeostasis and related issues of cellular redox metabolism.

Monochlorobimane (MCB) is a less reactive analog of MBB that was used to specifically label the free GSH pool in mammalian cells in situ (Rice et al., 1986). While MBB reacts rapidly and non-specifically with thiol groups in alkaline pH conditions, the chemical reaction of MCB with glutathione is slower and accelerated by endogenous GSTs (Rice et al., 1986). In plants, in situ derivatization of the glutathione pool with MCB was used for fluorescence imaging of epidermal cells (Müller et al., 1999a,b). Subsequent studies in Arabidopsis cells and poplar leaves adapted this technique to quantify extracted GS-bimane in vitro by HPLC (Meyer et al., 2001; Hartmann et al., 2003). This approach has been used to investigate the regulation of glutathione synthesis and breakdown, as well as the compartmentation of glutathione per se (Meyer and Fricker, 2002; Hartman et al., 2003; Grzam et al., 2007; Ohkamu-Ohtsu et al., 2007b; Blum et al., 2010; Queval et al., 2011). Because GSTs are particularly abundant in the cytosol (Dixon et al., 2009), the requirement for these enzymes to obtain rapid labelling may be the most important factor contributing to the potential compartmental specificity of the technique (Hartmann et al., 2003). Based on work in poplar, it was concluded that MCB labels only the cytosolic pool, and that this pool represents about half of total leaf glutathione (Hartmann et al., 2003). In wildtype Arabidopsis leaves, however, the entire leaf glutathione pool appeared to be accessible to MCB labelling within 2h incubation (Queval et al., 2011). Among the many factors that might explain this difference are inter-species specificity in GST compartmentalization or chloroplast glutathione transporters, variation in leaf developmental stage, or the inclusion (or not) of inhibitors during the incubation.

In situ immunolocalization has been used to examine glutathione distribution at the subcellular level by quantitative immunohistochemistry coupled to electron microscopy (Figure 2B). This approach relies on specific antibodies generated against glutaraldehyde-fixed glutathione and on the quantitative evaluation of the obtained signal by stereological methods. Antibodies that detect both GSH and GSSG have been used to label glutathione in several subcellular compartments (Zechmann et al., 2007, 2008; Kolb et al., 2010; Zechmann and Müller, 2010). Interestingly, in both Arabidopsis and tobacco, it was reported that the highest glutathione concentrations were not found in plastids but in mitochondria (Zechmann et al., 2008; Hölleret al., 2010). In wild-type plants, glutathione was found to be negligible or undetectable in the vacuole, in agreement with earlier concepts that glutathione concentrations are usually low in this compartment (Rennenberg, 1982). A leaf glutathione content of 200 nmol.g-¹FW converts to a global concentration of about 0.2 mM. Assuming low concentrations in the vacuole and apoplast, this would give a concentration of around 2 mM in other compartments, in reasonable agreement with reported values in spinach chloroplasts and the cytosol/cytoplasm of Arabidopsis cells (Foyer and Halliwell, 1976; Meyer et al., 2001). Calculations from immunolocalization studies performed in parallel with whole tissue quantification yielded estimated glutathione concentrations of 1 to 6 mM in the plastids, cytosol, and mitochondria of Arabidopsis Col-0 leaf mesophyll cells, with concentrations in the vacuole below 50 µM (Queval et al., 2011). Using non-aqueous fractionation of Arabidopsis leaves, cytosolic, plastid and vacuolar glutathione concentrations were estimated to be 3.5, 3.2 and 0.7 mM, respectively (Krueger et al., 2009). Thus, both these techniques give concentrations of the same order, regardless of potential problems such as cross-contamination between compartments or fractions during sample preparation. Differences in concentrations obtained in independent studies may also reflect differences in plant nutrition (eg, sulfur supply), which can affect the distribution of glutathione (Höller et al., 2010). Oxidative stress is among other factors that affect glutathione concentrations in a compartment-specific manner (Queval et al., 2011).

While the above two methods measure glutathione contents (and, potentially, concentrations) in vivo probes for the redox potential of glutathione have also been developed, based on fluorescent proteins containing oxidizable thiol groups. These include glutathione-specific redox-sensitive protein variants of the yellow (rxYFP) or green (roGFP) fluorescent protein (Hanson et al., 2004; Maulucci et al., 2009). These probes have been adapted for use in plants, particularly to monitor glutathione status in the cytosol (Jiang et al., 2006a; Meyer et al., 2007; Maughan et al., 2010). An important advantage of this method is that the introduced fluorescent proteins are designed to ‘sense’ the glutathione redox potential by equilibration of their thiol-disulfide status with that of glutathione (Figure 2B). Because the glutathione redox potential depends on GSH:GSSG and overall concentration (see section 8.1.2), these probes do not simply measure the glutathione pool size or even the oxidation state. However, as discussed further below, redox potential may be a key factor in glutathione-dependent signaling, and so these tools are providing useful information in studies of mutants or responses to stress (Meyer et al., 2007; Schwarzlander et al., 2008; Marty et al., 2009; Jubany-Mari et al., 2010). Potential constraints of these techniques are related to the limitations of optical microscopy. Most of the information generated to date has been obtained from studies of epidermal cells, and signal intensity may decrease for internal cell layers. However, such problems are less severe for the dual wavelength fluorescence ratio imaging roGFP variants (Schwarzlander et al., 2010). Because these proteins are dependent on glutaredoxin (GRX) activity, it is likely that glutathione redox potential is the major factor contributing to the signal (Meyer et al., 2007). However, recent studies have highlighted interactions between glutathione, GRX, and thioredoxins (TRX; Reichheld et al., 2007; Marty et al., 2009; Gao et al., 2010). It therefore remains to be definitively established that glutathione redox potential is the only component that is sensed by the thiol/disulfide-dependent fluorescent proteins.

3.4.1. Glutathione regulation of sulfur assimilation

As a major storage and transport form of reduced sulfur and non-protein cysteine, glutathione has been implicated in the regulation of sulfur metabolism in Arabidopsis, as in other plants (Kopriva and Rennenberg, 2004). Several components involved in sulfur assimilation are regulated by glutathione. Inhibition of sulfate uptake by GSH occurs in tobacco (Herschbach and Rennenberg, 1994). Similarly, glutathione has been reported to repress sulfur assimilation and uptake activities of sulfate transporters in a number of species (Buchner et al., 2004). In Arabidopsis, GSH inhibits the expression of AST68, a sulfate transporter, and ATP sulfurylase 1 (APS1), accompanied by a decrease of sulfate influx and APS activity in the roots (Lappartient et al., 1999). All three isoforms of the second enzyme of the sulfate assimilation pathway, adenosine 5′-phosphosulfate reductase (APR), seem to be particularly sensitive to inhibition by glutathione at the levels of transcripts and activity in Arabidopsis roots (Vauclare et al., 2002).

Available evidence suggests that GSH is the most important reductant for APR (Leustek, 2002). This enzyme contains a carboxyl domain with homology to TRX that appears to act as a functional GRX, as it uses GSH to reduce adenosine 5′-phosphosulfate to sulfite with apparent KMGSH values of 0.6 to 1 mM (Bick et al., 1998). Post-translational redox control of APR (particularly APR1) marks it out as a significant control point in response to glutathione oxidation (Leustek, 2002).

3.4.2. Manipulation of sulfur assimilation and effects on glutathione

Sulfur availability limits GSH accumulation in plants (Nikiforova et al., 2003). An Arabidopsis mutant defective in the Sultr1;2 gene, encoding a high affinity sulphate transporter, has decreased levels of glutathione (Maruyama-Nakashita et al., 2003) while constitutive expression of a bacterial APR in Arabidopsis increases the contents of both cysteine and glutathione (Tsakraklides et al., 2002). Overexpression of the cysteine synthesis pathway enhances cysteine and glutathione contents in Arabidopsis and tobacco (Harms et al, 2000; Noji and Saito, 2002; Wirtz and Hell, 2007). Environmental stresses such as ozone concomitantly increase cysteine and glutathione levels, associated with the posttranslational activation of APR1 (Bick et al., 2001). Genes encoding all three APRs are also induced by intracellular H₂O₂, as are cysteine, γ-EC, and glutathione (Queval et al., 2009).

4.1.1. γ-ECS mutants

Knockout mutations in GSH1 produce an embryo-lethal phenotype (Cairns et al., 2006). Less severe mutations in this gene, which produce partial decreases in glutathione contents, have been extremely useful in elucidating the functions of glutathione in plants. The rml1 (rootmeristemless1) mutant has less than 5% of wild-type glutathione contents. It fails to develop a root apical meristem because the cells arrest at the G1 phase of the cell cycle (Vernoux et al., 2000). This mutant is much less affected in its shoot phenotype. By combining the rml1 mutation with mutations in the two genes encoding dual-addressed cytosolic/mitochondrial NADPH-thioredoxin reductases (NTRA, At2g17420; NTRB At4g35460), it was shown that there is functional redundancy between glutathione and TRX systems in the control of shoot apical meristem function (Reichheld et al., 2007). Moreover, the combination of the cad2 mutation with ntra and ntrb resulted in perturbed auxin transport and metabolism, together with a loss of apical dominance and reduced secondary root production (Bashandy et al., 2010).

Several mutations in GSH1 decrease glutathione to a residual but still significant level (about 25 to 50% of wild-type). In themselves, these decreases do not markedly affect Arabidopsis development. They do, however, cause alterations in responses to stress and the environment. Of these mutants, cad2 was identified by its enhanced sensitivity to cadmium, rax1 by altered expression of APX2 (At3g09640) and pad2 by decreased camalexin contents and enhanced sensitivity to pathogens (Howden et al., 1995; Cobbett et al., 1998; Ball et al., 2004; Parisy et al., 2006).

4.1.2. Glutathione synthetase

As stated above, GSH-S is encoded by a single gene, GSH2, but alternative splicing results in localization of the gene product in both the chloroplast and cytosol (Wachter et al., 2005). While GSH1 knockouts are embryo-lethal, GSH2 knockout mutants are seedling-lethal, probably reflecting partial replacement of GSH functions by γ-EC, which accumulates to very high levels in these plants (Pasternak et al., 2008). The wild-type phenotype can be restored in gsh2 by complementing the mutant with targeted expression of the enzyme to the cytosol alone (Pasternak et al., 2008). This provides further evidence that GSH can be imported from the cytosol into the plastid, consistent with radiolabelling studies of isolated wheat chloroplasts (Noctor et al., 2002).

Figure 3.

Pathway of glutathione biosynthesis and principal methods of regulation.

(A) Scheme showing production of glutathione from constituent amino acids and cysteine synthesis enzymes that are likely important in regulating glutathione concentration. Important regulatory mechanisms are shown as red lines (feedback inhibition) or green lines (up-regulation in response to oxidation or other factors). Green dotted lines indicate likely transcriptional activation mechanisms while solid lines indicate post-translational processes. The role of GSSG and the exact mechanisms involved in the activation of γ-ECS by thiol oxidation remain to be established. For simplicity, not all enzymes involved in sulfate reduction are shown. APR, adenosine phosphosulfate reductase. γ-ECS, γ-glutamylcysteine synthetase. GRX, glutaredoxin. GSH-S, glutathione synthetase. JA, jasmonic acid. OAS, O-acetylserine thiol lyase. SAT, serine acetyl transferase.

(B) Expression of GSH1 and GSH2 in response to a range of environmental stresses ( www.genevestigator.com; Hruz et al., 2008). Examples of significant responses are highlighted. Botrytis, rosettes infected with B.cinerea or B.graminis versus uninfected rosettes. Drought, leaves from draughted plants versus watered plants. Light, light-grown seedlings compared to dark-grown seedlings. MeJa, methyl jasmonate-treated cell cultures versus solvent-treated cell cultures.

4.1.3. Buthionine sulfoximine

As well as mutants, one tool that has proved very useful in functional analysis of glutathione in plants is buthionine sulfoximine (BSO), a specific inhibitor that binds to the γ-ECS active site (Griffith and Meister, 1979). This compound is structurally homologous to methionine sulfoximine (MSO), which inhibits both γ-ECS and glutamine synthetase. The reactions catalyzed by both enzymes involve ATP-dependent ligation of an amine group (ammonia or the amino group of cysteine) to the γ-carboxy group of glutamate. Unlike MSO, BSO does not inhibit glutamine synthetase and so can be used in studies specifically focused on the role of glutathione synthesis and concentration in plants (eg, Cobbett et al., 1998; Kocsy et al., 2000; Meyer et al., 2007; Reichheld et al., 2007). Recently, a screen for BSO-insensitive Arabidopsis mutants revealed the identity of the chloroplast γ-EC and GSH transporter, called chloroquinone-like transporter (CLT; Maughan et al., 2010). These transporters are encoded by three genes: CLT1 (At5g19380), CLT2 (At4g24460), and CLT3 (At5g12160/12170). This finding, taken with the available information on γ-ECS and GSH-S localization, leads to the view that the synthesis of γ-EC is restricted to the plastid but that GSH synthesis can also occur in the cytosol following γ-EC export across the plastid envelope (Wachter et al., 2005; Maughan et al., 2010).

4.2.1. Expression of glutathione synthesis genes

Relatively few conditions cause very marked induction of GSH1 or GSH2 transcripts. These genes are induced by JA and heavy metals (Xiang and Oliver, 1998; Sung et al., 2009) and also respond to light and some stress conditions such as drought and certain pathogens (Figure 3B). Increases in H₂O₂ availability are known to trigger accumulation of glutathione in several plant species (Smith et al., 1984; May and Leaver, 1993; Willekens et al., 1997; Queval et al., 2009). However, neither exogenous H₂O₂ nor intracellularly generated H₂O₂ cause increased abundance of GSH1 or GSH2 transcripts in Arabidopsis (Xiang and Oliver, 1998; Sánchez-Fernández et al., 1998; Queval et al., 2009). Some evidence has been presented that production of the γ-ECS protein is regulated at the level of translation (Xiang and Bertrand, 2000).

4.2.2. Post-translational regulation of γ-ECS

Feedback inhibition of enzyme activity by GSH has long been established in animals (Richman and Meister, 1975) and also occurs in plants (Hell and Bergmann, 1990; Noctor et al., 2002). Other types of regulation were proposed based on the failure of Arabidopsis γ-ECS to fully restore wild-type GSH levels in yeast mutants (May et al., 1998b). The tobacco enzyme was shown to be inhibited by dithiols (Hell and Bergmann, 1990), and this effect has also been described for the wheat and Arabidopsis enzymes (Noctor et al., 2002; Jez et al., 2004). The plant enzyme forms a homodimer linked by two disulfide bonds (Hothorn et al., 2006), one of which is involved in redox regulation (Hicks et al., 2007; Gromes et al., 2008). Though the exact mechanistic details are still subject to debate, activation through disulfide formation very probably contributes to the well-known up-regulation of glutathione synthesis in response to oxidative stress conditions. It remains unclear whether this mode of regulation interacts mechanistically with feedback inhibition.

4.2.3. Cysteine availability

Up-regulation of the cysteine synthesis pathway occurs concomitantly with that of GSH synthesis. Two levels of co-regulation have been described under conditions in which oxidative stress triggers accumulation of glutathione in Arabidopsis. In catalase-deficient mutants, accumulation of transcripts encoding APR and serine acetyltransferase (SAT) was observed (Queval et al., 2009). Like γ-ECS, APRs are localized in plastids, whereas the two enzymes of cysteine synthesis are found in several compartments (Droux, 2004; Kopriva and Rennenberg, 2004; Martin et al., 2005; Kawashima et al., 2005). Recent studies show that the mitochondrial SAT (encoded by SERAT2.2; At3g13110) makes the major contribution to cysteine synthesis under standard conditions, and that knocking out this enzyme affects flux into glutathione and leaf glutathione contents (Haas et al., 2008; Watanabe et al., 2008). However, transcripts for the chloroplast SAT (SERAT2.1; At1g55920) were the most strongly induced during H₂O₂-triggered up-regulation of glutathione in Arabidopsis (Queval et al., 2009).

Exposure of Arabidopsis to ozone activates at least one APR at the post-translational level through formation of a disulfide bond (Bick et al., 2001). Although much more is known about thiol-disulfide regulation mediated by TRX (Buchanan and Ballmer, 2005), GRX are also important players in redox regulation (see section 8.3). One simple explanation of the link between decreased GSH:GSSG and enhanced glutathione synthesis is that increases in the chloroplast glutathione redox potential allow GRX-mediated oxidative activation of both APR and γ-ECS (Figure 3A). This would be consistent with a model for APR regulation in which the enzyme is maintained in a reduced inactive form by TRX and activated by GSSG (Leustek, 2002). Accordingly, H₂O₂-triggered accumulation of glutathione in barley is associated with appreciable increases in chloroplast GSSG content (Smith et al., 1985), and in situ analysis of glutathione in leaf mesophyll cells strongly points to a similar effect in Arabidopsis (Queval et al., 2011).

4.2.4. Overexpression of the glutathione synthesis pathway

Despite the multiplicity of controls over glutathione synthesis, plant cells can accommodate marked increases in overall glutathione content. Studies using expression of E.coli γ-ECS targeted to the poplar chloroplast or cytosol or to the tobacco chloroplast produced increases in leaf glutathione of up to four-fold (Noctor et al., 1996, 1998a; Creissen et al., 1999). Even higher contents were achieved by combined overexpression of γ-ECS and GSH-S (Creissen et al., 1999). Overexpression of γ-ECS in Arabidopsis also enhanced GSH, though less markedly (Xiang et al., 2001). Initial studies of several independent lines of poplar overexpressing γ-ECS showed no phenotypic effects (Noctor et al., 1998a). One of these lines has been reported to show symptoms of early leaf senescence (Herschbach et al., 2009). Clear effects of γ-ECS overexpression in tobacco included lesion formation and high levels of GSSG accumulation (Creissen et al., 1999), whereas marked negative phenotypic effects were absent in poplar and Brassica juncea expressing the same gene (Noctor et al., 1998a; Zhu et al., 1999a). Recently, expression of a bifunctional feedback-insensitive γ-ECS/GSH-S enzyme from the bacterium Streptococcus thermophilus in tobacco has shown that the glutathione pool can be boosted by up to 20-fold without apparent marked effects on plant growth and development (Liedschulte et al., 2010). In view of the information from several recent studies (see section 8.6), these plants present a very interesting system in which to analyze responses to biotic and other stresses. Increased glutathione in plants overexpressing γ-ECS is associated with enhanced resistance to heavy metals and certain herbicides (Zhu et al., 1999a; Gullner et al., 2001). Overexpression of γ-ECS in the poplar chloroplast increased not only glutathione but also levels of several free amino acids, such as leucine, isoleucine, leucine, tyrosine, and lysine (Noctor et al., 1998a). The causes underlying these effects remain unclear, though it is interesting to note that several enzymes involved in synthesis or metabolism of some of these amino acids are among those that have been identified as potential TRX targets through redox protemics approaches (Montrichard et al., 2009).

Compared to boosting γ-ECS capacity, overexpression of GSH-S has less marked effects on glutathione contents. No effect of substantial increases in GSH-S activity was observed in poplar (Foyer et al., 1995; Noctor et al., 1998a), though enhanced synthesis of GSH was induced when γ-EC was supplied to leaf tissue from the transformants (Strohm et al., 1995). Thus, GSH-S may become limiting when γ-EC supply is increased. Brassica juncea lines overexpressing GSH-S showed improved resistance to cadmium (Zhu et al., 1999b). Expression of a soybean homoGSH-S was used as part of an interesting strategy to confer herbicide resistance in tobacco, which is sensitive to the herbicide, fomesafen (Skipsey et al., 2005b). When expressed together with a homoGSH-preferring GST from soybean, which is fomesafen-tolerant, enhanced resistance was observed in the transformed tobacco lines (Skipsey et al., 2005b).

7.3.1. Genes and Subcellular Localization

The high cellular GSH:GSSG ratio is maintained by GRs, which are flavoproteins with high affinity for both GSSG and NADPH (Halliwell and Foyer, 1978; Edwards et al., 1990). Classical fractionation studies described high GR activities in chloroplasts (Foyer and Halliwell, 1976) but GR has also been detected in the cytosol, mitochondria, and peroxisomes (Edwards et al., 1990; Rasmusson and Møller, 1990; Jiménez et al., 1997; Stevens et al., 2000; Romero--Puertas et al., 2006). Two genes encode GR in Arabidopsis. The first, GR2 (At3g54660) encodes an enzyme corresponding to the pea GR that was the first protein shown to be dual-addressed to plastids and mitochondria (Creissen et al., 1995). Dual localization of the gene product in these two compartments has also been demonstrated in Arabidopsis (Chew et al., 2003). Cytosolic GR activity is encoded by GR1 (At3g24170) but proteomic analysis suggested that this gene also encodes peroxisomal GR activity (Kaur et al., 2009). It has recently been shown that import of GR1 into the peroxisomes occurs through the Peroxisomal Targeting Sequence 1 (PTS1) pathway, though targeting efficiency was considered to be relatively weak (Kataya and Reumann, 2010). From results obtained in pea, it seems that the peroxisomal enzyme represents a relatively minor part of the GR1 gene product (Romero-Puertas et al., 2006), which overall accounts for 30 to 60% of the total leaf enzyme activity in Arabidopsis (Marty et al., 2009; Mhamdi et al., 2010a). Available information therefore suggests that GR1 is mainly targeted to the cytosol, though an intriguing question concerns the physiological significance of its localization in the peroxisomes, which may be important sources of ROS and related signals (Foyer and Noctor, 2003; del Río et al., 2006; Nyathi and Baker, 2006; Mhamdi et al., 2010c).

7.3.2. Overexpression Studies

Glutathione reductase has been overexpressed in a number of plant species. In all cases the reported effects of enhanced GR activity in the chloroplast or cytosol are relatively modest, indicating that under many conditions, activity is sufficient to maintain glutathione pools and function (Aono et al., 1993; Broadbent et al., 1995; Foyer et al., 1991, 1995; Korneyev et al., 2005; Ding et al., 2009). However, increases in GR activity do increase the reduction state of the ascorbate pool (Foyer et al., 1995). These observations are entirely consistent with efficient coupling of the reactions of the ascorbate-glutathione pathway. Nevertheless, as noted above, there are specific situations such as increased availability of H₂O₂ where the glutathione pool becomes preferentially oxidized, suggesting that GR may become limiting for GSSG reduction. This could be a key property of pathway regulation, allowing altered glutathione status to transmit signaling information, and is discussed further below (section 8.1.1).

7.3.3. Mutagenesis Studies in Arabidopsis

Arabidopsis T-DNA mutants for GR2 are embryo-lethal (Tzafrir et al., 2004), presumably reflecting the crucial importance of maintaining appropriate glutathione reduction states in plastids, mitochondria, or both, at least during certain developmental stages. In contrast, gr1 knockout mutants are aphenotypic, despite a significant decrease in both total extractable GR activity and GSH:GSSG ratios (Marty et al., 2009; Mhamdi et al., 2010a). The aphenotypic nature of gr1 mutants is due to the ‘back-up’ GSSG regeneration activity by the cytosol-located NTR-TRX system (Marty et al., 2009). The TRX system also replaces GR in insects and yeast (Kanzok et al., 2001 ; Meyer et al., 2008). Triple mutants in which both NTRs and GR1 are knocked out show a male-sterile phenotype, demonstrating the requirement for at least one of the systems for plant survival (Marty et al., 2009). Further, it was reported that gr1 mutants were little affected in abiotic stress responses, and no increase in sensitivity to exogenously supplied H₂O₂ was observed (Marty et al., 2009). Based on this study, the specificity of GR1 or NTR-TRX function with regard to glutathione reduction remained unclear. However, when the gr1 mutation is introduced into the cat2 background, in which intracellular H₂O₂ places an increased load on regeneration of GSH, the resulting double mutants show a much exacerbated phenotype compared to cat2 (Mhamdi et al., 2010a). This effect is linked to dramatic oxidation and accumulation of glutathione in cat2 gr1, while the gr1 mutation is associated with compromised responses to pathogens and altered expression of genes involved in defense hormone signaling (Mhamdi et al., 2010a). These observations strongly suggest that, although not required for growth in controlled environment conditions, GR1 plays a specific role in stress, particularly biotic stress. They also underline the importance of the compartmentation of H₂O₂ production in the analysis of potential roles of specific components such as GR1 in redox homeostasis and stress responses. Further, transcriptomic patterns suggest that GR-GRX systems may function largely independently of TRXs in oxidative stress signaling, at least at the level of transcript abundance (Mhamdi et al., 2010a). However, the results of Marty et al. (2009) suggest that, under conditions in which GSSG accumulates, the resulting engagement of the NTR-TRX system could be one mechanism potentially contributing to TRX oxidation and signaling.

8.1.1. Insights from studies with redox-sensitive GFP

In vivo roGFP studies suggest that the glutathione redox potential in wild-type Arabidopsis in the absence of stress is lower than -300 mV in the cytosol and in other compartments (Meyer et al., 2007; Schwarzlander et al., 2008; Jubany-Mari et al., 2010). Given that the cytosolic NADPH:NADP⁺ ratio is not far removed from one (Igamberdiev and Gardeström, 2003), these redox potential values suggest that the two couples are close to redox equilibrium in this compartment. Such a situation could confer high sensitivity on the signaling function of glutathione redox potential mediated through GRX-dependent changes in protein thiol-disulfide status. A change in redox potential of about 50 mV is sufficient to significantly alter the balance between oxidized and reduced forms of TRX-regulated proteins (Setterdahl et al., 2002). For example, an increase in redox potential from -350 to -300 mV converts the TRX-regulated chloroplast glucose-6-phosphate dehydrogenase 1 (At5g35790) from almost completely inactive to active (Née et al., 2009).

The relationship between glutathione redox potential and redox state is shown in Figure 7. Knockout mutants for cytosolic NADP-isocitrate dehydrogenase (At1g65930) do not show a significant difference in whole leaf NADP⁺:NADPH ratios, but the absence of this enzyme does modify the GSH:GSSG ratio under conditions of increased H₂O₂ availability (Mhamdi et al., 2010b). This is consistent with the notion that changes in NADP reduction state that are undetectable in whole tissues may be sufficient in vivo to allow significant changes in glutathione redox potential, and hence signal amplification. Such effects may be of physiological significance, especially if localized to specific cells in certain conditions, eg, during plant responses to pathogens. Given the low KMof GR for NADPH (Smith et al., 1989; Edwards et al., 1990) compared to likely cytosolic NADPH concentrations (around 150 µM: Igamberdiev and Gardeström, 2003), this could occur through adjustment of reactant concentrations in the NADP-glutathione equilibrium rather than kinetic limitation of GR activity by NADPH.

The above discussion suggests that glutathione oxidation during stress is unlikely to be caused by kinetic limitation of GR by reductant but that decreases in NADPH:NADP⁺ could impact thermodynamically on the glutathione pool. Moreover, a substantial body of literature data shows that the extractable activity of GR is the lowest of the classical ascorbate-glutathione pathway enzymes. Given that enzymes other than DHAR may also contribute to GSH oxidation under oxidative stress conditions (Figure 6), the relatively low capacity of GR may be important to allow GSSG accumulation to fulfil signaling functions. This may involve gradual uncoupling of ascorbate and glutathione reduction states as flux through the pathway accelerates. This notion is consistent with the behaviour of the two pools observed in several studies of catalase-deficient plants.

8.1.2. The significance of glutathione concentration

A potentially important factor in redox signaling is glutathione concentration. This is because the actual glutathione redox potential is related to [GSH]²:GSSG (Mullineaux and Rausch, 2005; Meyer, 2008). Thus, if the GSH:GSSG ratio remains unchanged, decreases in glutathione concentration in themselves cause the redox potential to increase, ie, become more positive (Figure 7). The cytosolic thiol/disulfide redox potential as detected by roGFP was more positive in both cad2 and gr1 mutants (Meyer et al., 2007; Marty et al., 2009), underscoring the contributions of concentration (decreased in cad2) and GSH:GSSG (decreased in gr1) to the measured redox potential. Quantification of the glutathione redox potential in triple clt1 clt2 clt3 mutants, perturbed in chloroplast envelope glutathione transport, revealed an increase of about 20 mV in the cytosol while the plastidial potential was not affected (Maughan et al., 2010). Decreased reduction state of GRXs caused by a more positive redox potential could explain observations in plants that both lack NTR and are deficient in glutathione (Reichheld et al., 2007; Bashandy et al., 2010).

Given that glutathione concentration can clearly increase in response to decreases in GSH:GSSG, it is unclear why the GSH:GSSG ratio does not seem to increase to compensate for decreases in glutathione concentration. One possibility is that the GSH:GSSG ratio is already extremely high in some compartments, as discussed above, and so cannot be increased further in response to decreases in glutathione concentration. Because the KMGSSG values of GRs have been determined to be in the 10–50 µM range (Smith et al., 1989; Edwards et al., 1990), this may partly reflect a limitation over redox equilibrium with NADPH at very low GSSG concentrations.

8.3.1. Class I and Class II Glutaredoxins

The activities of class I GRXs include thiol-disulfide exchange, (de)glutathionylation, and regeneration of PRX and methionine sulfoxide reductase (MSR; Rouhier et al., 2002, 2006; Zaffagnini et al., 2008; Tarrago et al., 2009; Gao et al., 2010). A DHAR activity has long been known for GRX (Wells et al., 1990) and appears to be specific to this type, but its physiological importance remains unclear. Members of both classes of GRX have been shown to play roles in assembly of iron-sulfur clusters (Rouhier et al., 2007; Bandyopadhyay et al., 2008), though this activity is most strongly associated with class II GRX (Rouhier, 2010).

Arabidopsis class II GRXs have been identified as interacting with ion channels or as involved in oxidative stress responses (Cheng and Hirschi, 2003; Cheng et al., 2006). The plastidic glutaredoxin CXIP1 (AtGRXcp1; At3g54900) is able to rescue the sensitivity of yeast GRX5 mutants to H₂O₂ and Arabidopsis mutants for this GRX have slightly increased sensitivity to exogenous H₂O₂ (Cheng et al., 2006). Manipulation of the expression of a tomato GRX with a CGFS active site in both Arabidopsis and tomato also modulates the antioxidative system and alters stress tolerance (Guo et al., 2010). Expression of a GRX from an arsenic-hyperaccumulating fern was also reported to increase thermotol-erance in Arabidopsis (Sundaram and Rathinasabapathi, 2010).

As discussed further below, both class I and class II GRX have been shown to be competent in protein deglutathionylation in vitro (Gao et al., 2009a). Although members of the CGFS (class II) sub-family are sometimes referred to as ‘monothiol’ GRX, in at least some cases catalytic activity involves formation of an intramolecular disulfide bond with a second cysteine located distally from the GRX active site motif. These types of GRX may be dependent on TRX reductase rather than GSH for their reduction (Zaffagnini et al., 2008; Gao et al., 2009a).

8.3.2. Class III Glutaredoxins

Compared to the other GRX, relatively little is known about the biochemistry of the plant-specific class III GRX (CC-type or ROXY GRX). However, certain members of this class are the best characterized GRX in terms of physiological function, with three members implicated in plant development and defense. GRX480/ROXY19 (At1g28480) overexpression represses the JA marker gene, PDF1.2 (Ndamukong et al., 2007) while ROXY1 (At3g02000) and ROXY2 (At5g14070) are involved in petal and anther development (Li et al., 2009). Both of these functions involve interaction with TGA transcription factors. Complementation experiments showed that the rice orthologs of ROXY1 or ROXY2 can rescue the defect in petal development in Arabidopsis roxy1 mutants, as can GRX480 (Li et al., 2009; Wang et al., 2009). Overexpression of Arabidopsis or rice ROXY1 in Arabidopsis also increased susceptibility to Botrytis cinerea (Wang et al., 2009). This biochemical redundancy suggests that the key factor that determines the specificity of Class III GRX function may be temporal and/or spatial expression patterns (Ziemann et al., 2009). Both this notion and the proposed roles of this type of GRX in ROS signaling receive support from the analysis of plants with perturbations in glutathione status (Mhamdi et al., 2010a). H₂O₂-triggered alteration of glutathione status in cat2 and cat2 gr1 mutants modifies the abundance of transcripts encoding four class III GRXs but not other GRX types. These genes included GRX480 as part of a generalized effect on JA-associated genes, while ROXY1 or ROXY2 expression were not affected (Mhamdi et al., 2010a).

8.6.1. Light Signaling

Evidence for the role of glutathione in irradiance-dependent signaling came from the identification of the Arabidopsis rax1 mutant. This line contains less than 50% wild-type glutathione contents, and shows enhanced constitutive expression of the high light-induced gene, APX2 (Ball et al., 2004). Interestingly, overexpression of a tomato CGFS-type (class II) GRX in Arabidopsis also enhances expression of APX2 (Guo et al., 2010). Several studies in Arabidopsis and other species point to a potential link between growth daylength, light signaling and glutathione status (Becker et al., 2006; Queval et al., 2007; Bartoli et al., 2008). Genetic evidence of a link between glutathione and photoreceptor signaling has been provided by identification of arsenic-tolerant mutants, called ars4 and ars5 (Sung et al., 2007). While ars4 was shown to be a phytochrome A (phyA) mutant (Sung et al., 2007), ars5 is affected in a component of the 26S proteasome (Sung et al., 2009). Mutants for phyA showed increased resistance to BSO as well as to arsenic (Sung et al., 2007), and ars5 had enhanced levels of glutathione when exposed to arsenic, accompanied by increased GSH1 and GSH2 transcripts (Sung et al., 2009).

8.6.2. Biotic Interactions, Salicylic Acid, and Phytoalexin Synthesis

It is well established that exogenous GSH acts similarly to fungal elicitors in activating the expression of defense-related genes (Wingate et al., 1988; Dron et al., 1990) including PATHOGENESIS-RELATED1 (PR1, Gomez et al., 2004b; Senda and Ogawa, 2004). Moreover, accumulation of glutathione is triggered by pathogen infection (Edwards et al., 1991; May et al., 1996a), and this can involve characteristic transient changes in glutathione redox state (Vanacker et al., 2000; Parisy et al., 2006). Similar changes have also been reported following exogenous application of the defense-related hormone salicylic acid (SA), or biologically active SA analogs (Mou et al., 2003; Mateo et al., 2006; Koornneef et al., 2008). Glutathione perturbation in catalasedeficient cat2 is also linked to hypersensitive response (HR)-like lesions as part of a wide spectrum of defense responses that are conditionally induced in this line (Chaouch et al., 2010).

Thiol-disulfide status is clearly involved in the regulation of the SA-dependent NPR1 pathway (Déspres et al., 2003; Mou et al., 2003; Rochon et al., 2006; Tada et al., 2008). It was proposed that SA-dependent PR1 induction requires reduction of disulfide bonds of the oligomeric form of NPR1 that could be mediated by TRX or GRX in vivo (Mou et al., 2003) A more recent study reported that monomerization of the NPR1 protein to the active form was catalyzed by TRX but that glutathione is involved through GSNO-dependent nitrosylation of NPR1 monomers (Tada et al., 2008). The reoligomerization was postulated to be necessary to prevent protein depletion (Tada et al., 2008). The identification of NPR1 as a target of S-nitrosylation underpins the involvement of GSH in plant disease resistance, in which protein S-nitrosylation is considered to be a key post-translational modification. The Atgsnor1 mutant for the enzyme GSNO reductase has compromised resistance to both virulent and avirulent pathogens (Feechan et al., 2005).

Further evidence that glutathione is involved in defense against biotic stress came from the studies of Arabidopsis mutants altered in GSH synthesis or metabolism. Studies on the effect of GSH deficiency have reported conflicting results. Analysis of responses of cad2 to virulent and avirulent strains of the oomycete Peronospora parasitica and the bacterium Pseudomonas syringae did not reveal compromised resistance (May et al., 1996b) but subsequent studies of cad2 and rax1-1 reported increased susceptibility to avirulent strains of P. syringae (Ball et al., 2004). Triple mutants defective in glutathione transport across the chloroplast envelope showed decreased expression of PR1 and resistance to the oomycete Phytophthora brassicae (Maughan et al., 2010). The pad2 mutant is deficient in the Arabidopsis phytoalexin camalexin (Glazebrook and Ausubel, 1994) and shows enhanced susceptibility to various bacterial, fungal and oomycete pathogens as well as to insects (Ferrari et al., 2003; Parisy et al., 2006; Schlaeppi et al., 2008). The affected gene in pad2 was identified as GSH1 and the line was shown to have GSH contents that were lower than those in cad2 (Parisy et al., 2006). A comparative study of the allelic mutants rax1, cad2, and pad2 revealed an inverse correlation between resistance to P. brassicae and glutathione contents, though only pad2 was markedly affected in camalexin contents and its response to P. syringae (Parisy et al., 2006). These observations suggest that there is a critical glutathione status below which the accumulation of pathogen-defense related molecules and consequently disease resistance, is impaired. This level could vary according to conditions and pathogen specificity. In the case of camalexin synthesis, it is not yet clear whether GSH acts as a regulatory component or as a precursor of the thiazole ring, or both (Parisy et al., 2006; Glawischnig, 2007; Nafisi et al., 2007). However, knockout mutants for GR1, which are not glutathione-deficient but which have a decreased GSH:GSSG ratio, accumulate less SA than wild-type and show increased sensitivity to virulent P. syringae (Mhamdi et al., 2010a). This observation suggests that glutathione redox potential could be one factor that determines some of the effects reported in glutathione-deficient mutants.

The components that link changes in glutathione status to pathogen responses remain for the most part unidentified. As well as GRX, GSNO, and S-glutathionylation reactions, GSTs may play important roles (Lieberherr et al., 2003; Sappl et al., 2004; Davoine et al., 2006). Induction of GSTs by SA and other hormones has been systematically studied (Sappl et al., 2009). An active process occuring at the pathogen entry site consists of conjugation of GSH to insect-deterrent indole glucosinolate hydrolysis products to mediate the accumulation of other antifungal glucosinolate compounds (Bednarek et al., 2009). An activation of both GST transcripts and protein activity was also observed in cryptogein-induced tobacco HR together with the conjugation of oxylipins to GSH. This was suggested to be an HR-linked event with a signaling role during plant defense (Davoine et al., 2006).

Another plant-microbe interaction in which GSH plays a significant role is symbiosis between legumes and nitrogen-fixing rhizobia, which involves the production of nodules. These organs contain high levels of both GSH and homoglutathione, and deficiency in these molecules inhibits nodule formation (Frendo et al., 2005; Pauly et al., 2006).

8.6.3. Glutathione and Cell Death

It has been proposed that the glutathione redox state is a key determinant of cell death and dormancy (Kranner et al., 2006). According to this model, an increase in redox potential above a threshold reducing value necessarily causes death and/or growth arrest. Increased glutathione leaf contents triggered by γ-ECS overexpression in the tobacco chloroplast triggered lesion formation and induced PR gene expression (Creissen et al., 1999). In tobacco deficient in catalase, lesions also appear under conditions that cause accumulation of GSSG (Chamnongpol et al., 1996; Willekens et al., 1997). Double Arabidopsis knockouts for catalase and GR1 (cat2 gr1) show dramatic oxidation of glutathione (more than 2 µmol GSSG g-¹ FW, representing 95% of the leaf glutathione pool). While cat2 gr1 undergoes leaf bleaching when grown in long days, this effect is associated with repression rather than stimulation of SA-dependent pathogen responses (Mhamdi et al., 2010a), even though a wide range of these responses is associated with the less marked glutathione oxidation observed in cat2 single mutants (Chaouch et al., 2010). Moreover, H₂O₂-triggered lesions on the leaves of cat2 can be reverted by blocking SA synthesis through the isochorismate pathway or by treating leaves with myo-inositol, but in neither case does the suppression of cell death correlate with less oxidized tissue glutathione status (Chaouch and Noctor, 2010; Chaouch et al., 2010). Glutathione perturbation is equally or more marked in cat2 gr1 in short day as in long day growth conditions, but neither bleaching nor necrotic lesions are observed in the double mutant in short days (Mhamdi et al., 2010a). These observations suggest that perturbation of glutathione status is not in itself sufficient to cause bleaching or cell death. Accumulation of GSSG in cat2 grown from seed in air is associated with a stunted phenotype, possibly related to the roles of thiols in the plant cell cycle, growth and development (Reichheld et al., 1999, 2007; Vernoux et al., 2000). However, significant GSSG accumulation is also observed in gr1 knockout mutants, which are aphenotypic (Marty et al., 2009; Mhamdi et al., 2010a). Together with the recent study of Liedschulte et al. (2010), the available data suggest that high levels of glutathione (as GSH or GSSG) can be tolerated by plants and are not in themselves sufficient to trigger cell death pathways. As well as cellular tolerance of shifts in glutathione status, subcellular compartmentation could be a key issue.

One explanation of the difference between the effects of overexpressing monofunctional γ-ECS and bifunctional γ-ECS/ GSH-S in the tobacco chloroplast is that lesions are caused by γ-EC accumulation. This metabolite can accumulate to high levels when γ-ECS is overexpressed alone, either in the cytosol or the chloroplast (Noctor et al., 1996, 1998a; Creissen et al., 1999). Support for this idea comes from the observation that tobacco overexpressing both γ-ECS and GSH-S had an ameliorated phenotype compared to tobacco expressing the first enzyme alone (Creissen et al., 1999). Differences in γ-EC accumulation may therefore explain the differences between the studies of Creissen et al. (1999) and Liedschulte et al. (2010). However, the extent of γ-EC accumulation in tobacco overexpressing the bifunctional bacterial enzyme has not yet been documented, and there are several lines of evidence that argue against this interpretation. The first is that γ-EC was increased rather than decreased in tobacco overexpressing both enzymes of glutathione synthesis (Creissen et al., 1999). Second, abolition of photorespiratory glycine production by high CO₂ enhances γ-EC accumulation in poplar expressing E.coli γ-ECS in the cytosol or chloroplast. In this condition, γ-EC can accumulate to more than 1 µmol.g-¹ FW without triggering lesions (Noctor et al., 1997, 1999). Third, H₂O₂activated glutathione accumulation in Arabidopsis cat2 plants is accompanied by increased γ-EC contents and this does not in itself promote lesions (Queval et al., 2009). Currently available information suggests that neither total glutathione nor GSSG accumulation necessarily induces cell death in tobacco, poplar or Arabidopsis, and that further work is required to establish the significance of γ-EC accumulation in lesion formation. Despite this, there is a clear though apparently complex link between glutathione status and SA-dependent cell death.

8.6.4. Jasmonic Acid and Responses to Herbivores

While glutathione status is clearly implicated in SA signaling, several strands of evidence point to an equally influential role in JA signaling. The first indication of a link was provided by Xiang and Oliver (1998), who reported JA induction of transcripts encoding enzymes of GSH synthesis and GR. Subsequent work has shown that other antioxidative genes can be induced by JA (Sasaki-Sekimoto et al., 2005) while this hormone has been implicated, together with thiol status, in resistance to selenite (Tamaoki et al., 2008). Overexpression of GRX480 represses JA-triggered induction of the defensin, PDF1.2 (Ndamukong et al., 2007) and SA-dependent repression of this marker gene is correlated with changes in glutathione status and opposed by inhibition of glutathione synthesis (Koornneef et al., 2008).

Analysis of cat2-deficient plants provided some evidence that H₂O₂ may oppose JA-regulated gene expression (Vanderauwera et al., 2005). A comparative analysis of cat2 and gr1 knockouts suggests that the H₂O₂-JA interaction is daylength-conditioned and may be at least partly mediated by glutathione status (Mhamdi et al., 2010a). Based on JA- or oxylipin-dependent expression patterns (Yan et al., 2007; Mueller et al., 2008), the peroxidatic or conjugation activities of GSTs against electrophilic metabolites (Wagner et al., 2002; Davoine et al., 2006), and the known interaction of CC-type GRX with transcription factors (Ndamukong et al., 2007; Li et al., 2009), glutathione may influence JA signaling through several GSTs and GRX. Specific genes within the GST family and the class III GRX subfamily show modified expression in response to changes in glutathione status in caf2 and gr1 mutants (Mhamdi et al., 2010a). Interestingly, GSNO reductase transcripts are decreased by both wounding and JA (Diaz et al., 2003).

Resistance to some insects is compromised in the glutathione-deficient pad2 mutant and this is linked to decreased glucosinolates (Schlaeppi et al., 2008). The effect could be reverted by GSH treatment but not DTT, possibly suggesting that it is linked to a requirement for GSH in biosyntheses rather than mediated via redox changes (Schlaeppi et al., 2008). This is consistent with studies in tobacco engineered to express the Brassica glucosinolate pathway (Geu-Flores et al., 2009). It therefore seems that changes in glutathione status could impact functionally on JA-dependent stress responses through both a redox signaling effect, possibly mediated by GRX, and a requirement for GSH in glucosolinate synthesis.