JXB Advance Access originally published online on July 30, 2004
Journal of Experimental Botany 2004 55(404):1851-1859; doi:10.1093/jxb/erh202
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RESEARCH PAPER |
Regulation of calcium signalling and gene expression by glutathione
1Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK
2Institut de Biotechnologie des Plantes, Bâtiment 630, Université Paris XI, F-91405 Orsay cedex, France
3Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
To whom correspondence should be addressed. Fax: +44 (0) 1582 763010. E-mail: christine.foyer{at}bbsrc.ac.uk
Received 4 March 2004; Accepted 17 May 2004
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Abstract |
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The glutathione redox couple is an information-rich redox buffer that interacts with numerous cellular components. To explore the role of glutathione in redox signalling, leaf contents were increased either chemically, by feeding reduced glutathione (GSH), or genetically, by over-expressing the first enzyme of the GSH biosynthetic pathway,
-glutamylcysteine synthetase (-ECS). Leaf discs were also fed glutathione disulphide (GSSG), leading to increases in both GSH and GSSG. The effects of increases in GSH were compared with non-specific changes in leaf thiol status induced by feeding dithiothreitol (DTT) or the monothiol ß-mercaptoethanol (ß-ME). Photosynthesis measurements showed that none of the feeding treatments greatly disrupted leaf physiology. Transgenic plants expressing aequorin were used to analyse calcium signatures during the feeding treatments. Calcium release occurred soon after the onset of GSH or GSSG feeding, but was unaffected by DTT or ß-ME. Pathogenesis-related protein 1 (PR-1) was induced both in the -ECS overexpressors and by feeding GSH, but not GSSG. Feeding DTT also induced PR-1. Key transcripts encoding antioxidative enzymes were much less affected, although glutathione synthetase was suppressed by feeding thiols or GSSG. It is concluded that modulation of glutathione contents transmits information through diverse signalling mechanisms, including (i) the establishment of an appropriate redox potential for thiol/disulphide exchange and (ii) the release of calcium to the cytosol. Key words: Calcium signalling, cytosol, dithiothreitol, gene expression, glutathione, ß-mercaptoethanol, regulation
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Introduction |
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Glutathione has many functions in plant biology, including sulphur metabolism, regulation of growth and development, cell defence, redox signalling, and regulation of gene expression. These functions depend on the concentration and/or redox state of the leaf glutathione pools (Noctor and Foyer, 1998a
; May et al., 1998). Changes in intracellular glutathione status may, therefore, be expected to have important consequences for the cell, through modification of the metabolic functions associated with glutathione-regulated genes. In animal cells, redox regulation of the transcription factor NF
B involves glutathione. This regulation is important for T cell function since glutathione augments the activity of these cells (Suthanthiran et al., 1990). The application of exogenous glutathione can elicit changes in the transcription of genes encoding cytosolic Cu,Zn superoxide dismutase, glutathione reductase, and 2-cys peroxiredoxins (Hérouart et al., 1993; Wingsle and Karpinski, 1996; Baier and Dietz, 1997). Glutathione-inducible hypersensitive elements have been identified in the proximal region of the chalcone synthase (CHS) promoter (Dron et al., 1988). GSH is also a physiological regulator of many thiol-disulphide exchange reactions, including those implicated in chloroplast transcription.
The glutathione redox couple is also considered to be a key player in homeostatic adjustment of the cellular redox potential. In animal cells, substantial evidence implicates redox potential as an important factor determining cell fate, and the glutathione redox couple is the key player (Schafer and Buettner, 2001). Unlike many other redox couples such as ascorbate/dehydroascorbate and NADPH/NADP, the glutathione redox potential depends on both GSH/GSSG and absolute glutathione concentration. The concentration-dependent term of the Nernst equation is second-order with respect to GSH, but first-order with respect to GSSG, hence the accumulation of GSH can offset the change in redox potential caused by decreases in the GSH/GSSG ratio.
It has long been known that defence genes are among those induced by GSH (Wingate et al., 1988; Dron et al., 1988). In particular, thiol-disulphide status appears to be crucial for the expression of pathogenesis-related (PR) proteins, which are induced by salicylic acid (SA) and are involved in systemic acquired resistance (SAR). Increases in SA trigger the reduction of disulphide bonds located on both the regulatory protein NPR1 and on certain TGA transcription factors with which NPR1 interacts (Mou et al., 2003; Després et al., 2003). This reduction stimulates both the translocation of NPR1 from the cytosol to the nucleus and the physical interaction of NPR1–TGA1 that is necessary for the activation of PR gene transcription (Mou et al., 2003; Després et al., 2003). The redox dependence of the pathway suggests that any biotic or abiotic stimulus that can perturb the cellular redox state will up-regulate the same set of defence genes via the NPR1 pathway (Mou et al., 2003). Redox-linked effects explain, for example, PR gene expression in response to UV-B exposure (Green and Fluhr, 1995) or in catalase-deficient mutants (Willekens et al., 1997) where extensive, specific oxidation of the glutathione pool and greatly enhanced glutathione accumulation occurs in certain conditions (Noctor et al., 2000; May and Leaver, 1993). Indeed, it is noteworthy that oxidation of the leaf glutathione pool followed by enhanced glutathione synthesis is also seen in plant–pathogen interactions (Vanacker et al., 2000; Mou et al., 2003). In this situation SAR induction involves an early burst of ROS and a transient or more sustained increase in cellular redox state (more oxidized), followed by a sharp decrease in cellular redox potential (more reduced) as a result of the accumulation of antioxidants such as GSH.
From the above observations, it appears that induction of glutathione accumulation has at least two effects. Firstly, it acts as a mechanism to offset stress-initiated oxidation of glutathione and, secondly, it can cause changes in gene transcription either directly or indirectly via interaction with regulatory proteins and/or transcription factors. The aim of the present study was to explore the mechanisms whereby GSH, GSSG, and thiol compounds modulate gene expression in the absence of stress. Photosynthetic CO2 assimilation rates were used as a physiological marker to verify that effects on gene expression and calcium signalling induced by thiol feeding were independent of adverse effects on major leaf functions.
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Materials and methods |
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Plant material and growth conditions
Tobacco seeds (Nicotiana tabacum cv. Samsung) were germinated on moistened filter paper. After 10 d, the seedlings were transferred to compost and grown in the glasshouse at 22 °C. Young fully expanded leaves were harvested from 45-d-old plants and used for thiol treatments, glutathione determinations, calcium signalling, and gene expression analysis.
Tobacco transformation for overexpression of maize -glutamylcysteine synthetase (-ECS)
A maize -ECS cDNA was cloned and sequenced as described in Gomez et al. (2004). The -ECS cDNA was subcloned in the SacI restriction site of pp5ln (derived from pUC19; Pignocchi et al., 2003) by standard recombination techniques. Targeting to the chloroplast was achieved by inserting a RuBisCO small subunit transit peptide from soybean as a XhoI–NcoI fragment upstream of the coding sequence for -ECS, originating the 2x35S--ECS expression cassettes targeted to the cytosol and chloroplast. These expression cassettes were ligated into cj102, a derivative plasmid of pGPTV, as a HindIII–SacI fragment into cj102 in replacement of the 2x35S-ß-glucuronidase cassette (Pignocchi et al., 2003). The cj102--ECS and cj102-transit peptide--ECS constructs obtained were transformed into A. tumefaciens LBA4404 by electroporation.
Sterile cultures of Nicotiana tabacum L. (cv. Petit Havana, mutant SRI) were transformed with the constructs described above by A. tumefaciens leaf disc infection (Gallois and Marinho, 1995). Transgenic and wild-type plants were grown in compost (Petersfield products, Leicester, UK) at 22 °C day/night in controlled environment chambers supplying an irradiance of 250 µmol m–2 s–1 at plant height as a 16 h photoperiod. T1 seeds from primary transformants were germinated in Petri dishes containing 1.5% agar in distilled water (non-selective medium), supplied with 100 mg l–1 of kanamycin (selective medium). T2 seeds were obtained from individual T1 plants and used to generate T2 transgenic progeny that were analysed for glutathione contents and gene expression analysis.
Thiol feeding experiments to intact leaves
Leaves were excised under water from 45-d-old plants and the following solutions were supplied through the petiole for 16 h in darkness: 10 mM 3-(N-morpholino)-propanesulphonic acid buffer (MOPS), pH 6.0 with either 0, 5 mM or 20 mM GSH; 5 mM GSSG; 5 mM ß-ME; or 5 mM DTT. Immediately after the feeding experiments, treated leaves were either used for photosynthesis measurements or RT-PCR as described below or were frozen in liquid N2 for metabolite analysis. Reduced and oxidized glutathione were extracted and assayed as described by Noctor and Foyer (1998b). Spectrophotometric determinations of chlorophyll were performed on 80% (v/v) acetone extracts. The total chlorophyll (a+b) content of leaves (g–1 fresh weight) and leaf discs (m–2) was calculated from the absorbances at 645 nm and 663 nm. The chlorophyll concentration in acetone solution (mg l–1) was calculated as 20.2A645 plus 8.02 A663.
Measurements of CO2 exchange, chlorophyll a fluorescence and metabolite assays
Measurements of CO2 and H2O exchange under steady-state conditions and sampling for metabolite analysis were performed as described in Novitskaya et al. (2002). All experiments were conducted at 22 °C and 50% relative humidity. Irradiance was provided by overhead lamps and adjusted by neutral density sheeting. Gas composition was controlled by a gas mixer supplying CO2 and O2 to the stated partial pressures, with balanced N2. Respiratory CO2 release was monitored for 20 min in the dark, then plants were illuminated until a steady-state rate of CO2 uptake was attained. To analyse the dependence of net CO2 uptake on irradiance, photosynthesis was induced at 360 µbar CO2 at the lowest irradiance (100 µmol m–2 s–1) as shown in Fig. 2 of the Results. Light was then increased step-wise from 100 µmol m–2 s–1 to 1600 µmol m–2 s–1, and measurements were taken on attainment of the steady-state rate at each irradiance. Chlorophyll a fluorescence analysis was conducted using a PAM fluorimeter (Veljovic-Jovanovic et al., 2001).
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Thiol feeding to leaf discs
Leaf discs (2.5 cm diameter) were excised under water from leaves of 45-d-old plants and incubated in Petri dishes containing 10 mM 3-(N-morpholino)-propanesulphonic acid buffer (MOPS), pH 6.0, with either 0, 5 mM, or 20 mM GSH; 5 mM GSSG; 5 mM ß-ME; or 5 mM DTT. Incubations were carried out for 16 h at room temperature in the dark. Discs were then used either for expression analysis or metabolite analysis.
RNA extraction and RT-PCR analysis
Total RNA was extracted from five leaves using Trizol reagent (GibcoBRL) according to the manufacturer's recommendations. DNA contamination was removed from the RNA samples using DNase I (Invitrogen) and checked using actin-specific primers giving differential PCR products in the presence of genomic DNA (Pignocchi et al., 2003). One microgram of total RNA was reverse transcribed using 0.5 µg Oligo(dT)12–18 (GibcoBRL), 0.5 mM dNTP, 10 mM DTT, and 200 U of Superscript II reverse transcriptase (GibcoBRL). PCR conditions were standardized using gene-specific primers for actin content. Linear amplification for semi-quantitative RT-PCR was obtained with 28 cycles and PCR conditions were 5 min at 94 °C, cycle of 40 s at 94 °C, 40 s at 52 °C, and 1 min at 72 °C, followed by 10 min at 72 °C, using 1 µl of the reverse transcriptase reaction. Primer sequences for actin, PR-1, tAPX, sAPX, cAPX, AO, and GLDH were as published in Pignocchi et al. (2003) and GSH-S primers were as published in Gomez et al. (2004).
Calcium imaging
Leaf discs from plants expressing aequorin were excised and incubated in thiols as described above. Aequorin imaging was performed using an intensified CCD camera (model EDC-02), with camera control unit (HRPCS-2) and image acquisition and processing software (IFS216), all from Photek (St Leonards-on-Sea, UK) as described previously (Knight et al., 1999).
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Results |
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Leaf glutathione contents are greatly increased by GSH or GSSG feeding
Tobacco leaf total glutathione contents were increased from below 0.5 µmol mg–1 chlorophyll to about 6.0 µmol and 11.0 µmol mg–1 chlorophyll after feeding with either 5 mM or 20 mM GSH, respectively, for 16 h (Fig. 1A). GSH feeding had less effect on the leaf GSH/GSSG ratios, which were about 11 in control leaves, about 17 in leaves supplied with 5 mM GSH, and about 9 in leaves supplied with 20 mM GSH (Fig. 1B). Feeding with 20 mM GSSG also increased leaf total glutathione contents to 6.5 µmol mg–1 chlorophyll (Fig. 1A), but decreased GSH/GSSG (Fig. 1B). Despite the significant decrease in GSH/GSSG, this ratio remained at around 4, i.e. most of the 12-fold increase in total glutathione induced by feeding GSSG was due to accumulation in the form of GSH (Fig. 1, compare A and B). Feeding 5 mM DTT or ß-ME did not significantly increase leaf glutathione compared with leaves supplied with buffer alone (Fig. 1A) and had relatively small effects on GSH/GSSG (Fig. 1B). A small increase was observed in leaves fed ß-ME (Fig. 1B). In conclusion, total glutathione could be increased by feeding GSH or GSSG, but not DTT or ß-ME. GSH/GSSG was only appreciably affected by supplying GSSG, which caused the ratio to decrease. Similar changes in glutathione to those observed in leaves were found when leaf discs were treated with equivalent solutions (data not shown).
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Feeding GSH or GSSG does not disrupt major leaf function
The light-response curves of photosynthetic CO2 uptake were similar in N. tabacum leaves supplied for 16 h with either GSH (5 mM and 20 mM), GSSG (20 mM), ß-ME (5 mM), or DTT (5 mM), steady-state rates of photosynthesis being comparable on a leaf area basis, at saturating light intensities (Fig. 2A). Thus, leaf glutathione contents were greatly increased by GSH or GSSG feeding (Fig. 1A) without detrimental effects on photosynthetic CO2 assimilation rates (Fig. 2A). The dark-adapted ratio of variable (Fv) to maximal (Fm) chlorophyll a fluorescence was not greatly decreased by feeding 5 mM GSH or GSSG (Fig. 2B). The quantum efficiency of photosystem II (
PSII) decreased with increasing irradiance in a similar manner in leaves supplied with GSH or GSSG to those of controls treated with buffer alone (Fig. 2C). By contrast, leaves supplied with either ß-ME or DTT showed smaller decreases in this parameter with increasing light availability at all but the highest irradiances (Fig. 2C). The slower decrease in PSII at intermediate light intensities caused by these thiols may be related to a decrease in non-photochemical quenching via inhibition of violaxanthin de-epoxidation (Noctor et al., 1991).
Thiol feeding modulates gene expression
Using marker transcripts (PR-1, AO, GLDH, tAPX, sAPX, cAPX) the role of thiols in co-ordinating gene expression linked to plant–pathogen interactions and crosstalk between the glutathione and ascorbate redox systems was examined. Two approaches were employed. First, transcripts were analysed in leaves supplied with GSH, GSSG, ß-ME, or DTT. Second, transcripts were analysed in tobacco transformed to accumulate GSH.
Feeding leaves or leaf discs with 5 mM or 20 mM GSH resulted in a large increase in the abundance of transcripts encoding PR-1 (Fig. 3). This effect was also observed in transgenic tobacco plants overexpressing the maize -ECS in the chloroplast or cytosol where leaf glutathione was increased by 50–70% (Fig. 4). Unlike DTT, which also increased the level of PR-1 mRNA accumulation, neither GSSG nor ß-ME had any effect compared with the control supplied with buffer alone (Fig. 3). None of the added thiols had any marked effect on the abundance of transcripts encoding the tAPX, sAPX, or cAPX except that cAPX mRNA was lower in leaves supplied with 5 mM GSSG (Fig. 3). GLDH, AO, and GSH-S mRNAs were slightly decreased following feeding with either 20 mM GSH, 5 mM ß-ME, or 5 mM GSSG (Fig. 3). Levels of AO transcripts were also lower in transformed tobacco plants expressing maize -ECS in the chloroplast, but not the cytosol (Fig. 4), whereas GLDH transcript abundance was increased in leaves supplied with GSH (Fig. 3) and in plants expressing maize -ECS in the cytosol (Fig. 4), but similar to controls in transformed tobacco plants expressing maize -ECS in the chloroplast (Fig. 3).
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GSH and GSSG modulate calcium signalling
The level of aequorin luminescence was changed in tobacco leaf discs supplied with GSH or GSSG. Luminescence increased rapidly in leaf discs supplied with 20 mM GSH, reaching a peak within the first 2 h of incubation. Calcium release into the cytoplasm was transient, however, luminescence falling back to a low level within 3 h of the onset of feeding (Fig. 5). A similar transient increase in aequorin luminescence was observed upon GSSG feeding. In this case, however, the peak was reached later and the duration of calcium release was much longer than that observed with 20 mm GSH. Since GSSG feeding led to a marked increase in leaf disc GSH, it is likely that the transient and the kinetic are caused by conversion of added GSSG to internal GSH, which then stimulates calcium release. Moreover, supplying a lower concentration of GSH (5 mM) caused a similar, but somewhat later, transient increase in aequorin luminescence that was also broader than that observed with 20 mm GSH. By contrast, neither ß-ME nor DTT caused any change in the basal level of aequorin luminescence (Fig. 5).
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Discussion |
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Like all other aerobic organisms, plants are able to maintain cytoplasmic thiols in the reduced (-SH) state because of the low thiol-disulphide redox potential imposed by millimolar amounts of glutathione, which acts as a thiol buffer. Although transient disulphide bonds do occur during the catalytic cycle of some enzymes, stable protein disulphide bonds are relatively rare except in quiescent tissues such as seeds. The multiple roles of GSH within the cell, together with the stability of GSSG, may make this redox couple ideally suited to information transduction, and it is becoming increasingly clear that glutathione status is important in signalling. Glutathione strongly interacts with two key signalling factors: H2O2 and, as has been shown here, Ca2+.
Reversible thiol-disulphide exchange has long been recognized as an important mechanism of modulating protein function (Kunert and Foyer, 1993; Demple, 1998). It is only relatively recently, however, that this mechanism has been shown to have a crucial signalling role in plant stress responses (Mou et al., 2003; Després et al., 2003). In addition, reversible protein thiolation protects essential thiol groups on key proteins from irreversible inactivation during oxidative stress and also plays an important regulatory role in controlling metabolism, protein turnover and gene transcription (Foyer and Noctor, 2001).
The present data suggest that glutathione-associated signalling may be complex and diverse, and this could reflect the various parameters associated with the glutathione redox couple. The glutathione redox potential is related to [GSH]2/GSSG, and so depends on both GSH/GSSG and the total concentration of the glutathione pool in any given compartment (Schafer and Buettner, 2001). Thus, accumulation of glutathione can make the redox potential more negative even if GSH/GSSG does not change. Conversely, GSSG can accumulate without changes in the redox potential, if there is a compensatory increase in the total concentration. It is therefore interesting to examine which parameters are most important in controlling glutathione-associated gene expression and calcium signalling.
Diverse pathways of glutathione-regulated gene expression
Table 1 shows a simple summary of the principal data presented in this manuscript. Increasing leaf GSH contents either by direct feeding to leaves or leaf discs or by constitutive overexpression of -ECS led to the accumulation of PR-1 transcripts, as did supplying DTT. Significantly, ß-ME was without effect, as was GSSG, even though the latter treatment caused GSH to increase (Fig. 1, compare A and B). Although it is worth noting that glutathionylation between GSH and reduced protein thiols can occur by enzymatic mechanisms (Starke et al., 2003), the failure of GSSG feeding to induce PR-1 transcripts suggests that GSSG-dependent glutathionylation is not involved in the regulation of PR-1 expression. The data are consistent with recent findings that the reduction of disulphides on NPR1 and interacting transcription factors is crucial in the link between SA and PR gene expression (Mou et al., 2003; Després et al., 2003). Thus, GSH and DTT are able to mimic this part of the pathogen response, presumably by facilitating the disulphide reduction of NPR1 and TGA, which is a process downstream of salicylic acid. This would be coherent with the notion that reduction of NPR1 and TGA is accomplished by protein disulphide reductases (thioredoxins or glutaredoxins). The active sites of these proteins can be reduced efficiently by DTT but much less effectively by the monothiol ß-ME. The effect of the monothiol GSH on PR-1 expression may reflect the establishment of redox potentials sufficiently negative to increase the reduction status of key thioredoxins or glutaredoxins. When GSSG is supplied, the resulting decrease in GSH/GSSG means that such high redox potentials are not reached, despite the increase in overall glutathione concentration, and this may explain the differential effects of the two forms of glutathione (Table 1). Interestingly, recent data with Arabidopsis shows that a specific cytosolic thioredoxin is co-expressed with PR-1 in response to stress, including pathogen attack (Laloi et al., 2004).
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Pathogen-induced increases in the intracellular GSH concentration and GSH-dependent induction of phenylalanine ammonia lyase and chalcone synthase (CHS) have been demonstrated previously (Dron et al., 1988
; Wingate et al., 1988; Vanacker et al., 2000). However, using an artificial precursor of GSH biosynthesis, L-oxothiazolidine-4-carboxylate, to increase intracellular thiol concentrations, Edwards et al. (1991) showed that enhanced intracellular GSH concentrations were not sufficient to induce PAL induction and phytoalexin synthesis. They concluded that changes in the intracellular glutathione concentration in response to pathogen attack were too slow to be consistent with the initiation of the elicitation response. However, it has recently been suggested that the simultaneous presence of pro- and anti-oxidants may be important in the NPR1/TGA interaction (Mou et al., 2003; Després et al., 2003). Indeed, a complex response of the glutathione couple is well documented in plant–pathogen interactions, where an oxidative burst and rapid induction of glutathione accumulation precede the maximal induction of transcripts encoding phenylpropanoid metabolism enzymes (Zhang et al., 1997; Vanacker et al., 2000).
GSH feeding induced PR-1 expression without any inhibition of photosynthesis or visible changes in leaf phenotype. Moreover, the transformed plants expressing maize -ECS in either the cytosol or chloroplasts were morphologically comparable to controls (data not shown). A similar type of thiol-disulphide regulation may control the activities of the two abscisic acid-signalling cascade phosphatases, ABI1 and ABI2, since oxidation of component protein cysteines inactivates these two stress signalling components (Meinhard and Grill, 2001; Meinhard et al., 2002). The signalling cascade leading to GSH-S, APX, AO, and GLDH expression is not known, but may involve modulation of these protein phosphatases.
Glutathione triggers calcium release
The results presented here (Fig. 5) demonstrate that feeding GSH rapidly and specifically triggers calcium release. Unlike PR-1 induction, calcium release is triggered by both GSH and GSSG (Table 1). Measurements of the leaf GSH/GSSG ratio suggest that most of the GSSG absorbed by the leaf is converted to GSH. The more rapid induction of calcium release by 5 mM GSSG than 5 mM GSH can be explained by the higher affinity of the plasma-membrane glutathione transporter for GSSG than GSH (Foyer et al., 2001). Moreover, the rapidity of the response to calcium is more or less correlated with total GSH supplied to the leaf (Fig. 5), 5 mM GSSG being potentially equivalent to 10 mM GSH. Thus, it is possible that calcium release responds to the total GSH concentration, rather than glutathione redox potential, and this could explain the different effects of GSSG on PR-1 expression and on calcium signatures. Mou et al. (2003) suggest that a GSH/GSSG ratio of about 15 is required for transfer of NPR1 from the cytosol to the nucleus. While, this value may vary according to experimental conditions, the data presented here clearly show that when the GSH/GSSG ratio falls to a low value (below 5), as occurs upon GSSG feeding (Fig. 1), PR-1 is not expressed, even though total glutathione is much enhanced and calcium release occurs (Table 1).
Control of cellular glutathione status
The increases in glutathione obtained by overexpressing the maize -ECS in tobacco confirm numerous earlier studies that this enzyme is a limiting factor in glutathione accumulation in plants (Rüegsegger and Brunold, 1992; Noctor et al., 1996; Cobbett et al., 1998; Xiang et al., 2001). Overexpression of an E. coli -ECS, but not GSH-S, in poplar or tobacco substantially increases leaf glutathione contents (Noctor et al., 1996, 1998; Creissen et al., 1999), as does homologous overexpression of the Arabidopsis -ECS (Xiang et al., 2001). The marked chlorotic phenotype produced by chloroplastic E. coli -ECS overexpression in tobacco was not observed when maize -ECS was expressed in tobacco, but leaf GSH contents were increased and PR-1 protein expression was observed in both cases (Fig. 4; Creissen et al., 1999). The results presented here (Fig. 3) show that supplying leaves with either 20 mM GSH or 5 mM GSSG decreases the abundance of GSH-S transcripts in tobacco leaves, perhaps implying a type of feedback control on GSH-S expression or GSH-S transcript stability via the total glutathione pool size. Unfortunately, -ECS transcripts were below the level of detection in wild-type tobacco plants under the conditions used in Fig. 3.
In addition to -ECS activity, the most important factor controlling plant glutathione is the availability of cysteine (Noctor et al., 1997), and these two factors are likely co-ordinated. In vivo activity of -ECS is determined by control at multiple levels. The 5' untranslated region (5' UTR) of the gsh1 gene encoding -ECS was found to interact with a repressor-binding protein that was released upon the addition of H2O2 or decreases in the GSH/GSSG ratio (Xiang and Bertrand, 2000). H2O2 increases tissue glutathione contents, whereas jasmonic acid increases the transcript abundance of the enzymes of GSH synthesis but does not affect GSH concentration (Xiang and Oliver, 1998). A redox-sensitive 5' UTR-binding complex is thus suggested to control -ECS mRNA translation and hence GSH synthesis in A. thaliana (Xiang and Bertrand, 2000). The pro-oxidative events linked to NPR1 activation may, therefore, not only be required for monomerization of the NPR1 protein as suggested by Mou et al. (2003) but may also be required for activation of -ECS mRNA translation. Consistent with this notion is the observation that oxidation of the glutathione pool precedes glutathione accumulation in catalase mutants (Noctor et al., 2000), in maize subject to cold stress (Gomez et al., 2004), and in barley undergoing a pathogen-induced oxidative burst (Vanacker et al., 2000). The nature of the link between redox state perturbation and enhanced glutathione accumulation is therefore complex and remains unresolved. However, current literature data suggest at least two possibilities with regard to effects on GSH synthesis. Firstly, the activity of adenylylsulphate reductase, a key enzyme in sulphate assimilation, may be activated by decreases in GSH/GSSG (Bick et al., 2001). Secondly, as noted above, H2O2 or associated decreases in GSH/GSSG ratios kickstart -ECS translation (Xiang and Oliver, 1998).
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Conclusions |
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The glutathione redox couple interacts with numerous cellular components. Glutathione status is probably involved in the transmission of oxidative stress signals, for example, through GSSG-driven or enzyme-catalysed protein glutathionylation, reactions that may be reversed (and therefore, regulated) by specific enzymes such as glutaredoxins (Johansson et al., 2004
; Lemaire, 2004). On the other hand, decreases in glutathione redox potential (‘overreduction’), linked to an enhanced GSH/GSSG ratio, increases in total glutathione, or both, may be an obligatory event in the modulation of redox potential necessary to allow the efficient reduction of disulphides on regulatory proteins. Lastly, glutathione acts to stimulate calcium release into the cytosol, an effect that appears to depend on total glutathione concentration. As previously discussed in depth (Noctor and Foyer, 1998a), the accumulation of glutathione occurs in response to various stresses, and the attendant release of calcium reported here may mean that glutathione accumulation is equally as important in signal transduction as in chemical defence against reactive oxygen. By triggering the activation of calcium-dependent protein kinases, increases in glutathione concentration may participate in early signal transduction events and function in the integration of multiple abiotic and biotic stimuli. |
Acknowledgements |
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The authors thank Emmanuelle Issakidis-Bourguet for discussion. Leonardo Gomez is grateful to the Royal Society (UK) for a Research Fellowship.
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Footnotes |
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* Present address: Centre for Novel Agricultural Products, Department of Biology, University of York, PO Box 373, York YO10 5 YW, UK.
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References |
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Baier M, Dietz KJ. 1997. The plant 2-cys peroxiredoxin BAS1 is a nuclear-encoded chloroplast protein: its expressional regulation, phylogenetic origin, and implications for its specific physiological function in plants. The Plant Journal 12, 179–190.[CrossRef][Web of Science][Medline]
Bick JA, Setterdahl AT, Knaff DB, Chen Y, Pitcher LH, Zilinskas BA, Leustek T. 2001. Regulation of the plant-type 5'-adenylyl sulfate reductase by oxidative stress. Biochemistry 40, 9040–9048.[CrossRef][Medline]
Cobbett CS, May MJ, Howden R, Rolls B. 1998. The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase. The Plant Journal 16, 73–78.[CrossRef][Web of Science][Medline]
Creissen G, Firmin J, Fryer M, et al. 1999. Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress. The Plant Cell 11, 1277–1291.
Demple B. 1998. A bridge to control. Science 279, 1655–1656.
Després C, Chubak C, Rochon A, Clark R, Bethune T, Desveaux D, Fobert PR. 2003. The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1. The Plant Cell 15, 2181–2191.
Dron M, Clouse SD, Dixon RA, Lawton MA, Lamb CJ. 1988. Glutathione and fungal elicitor regulation of a plant defense gene promoter in electroporated protoplasts. Proceedings of the National Academy of Sciences, USA 85, 6738–6742.
Edwards R, Blount JW, Dixon RA. 1991. Glutathione and elicitation of the phytaolexin response in legume cultures. Planta 184, 403–409.[Web of Science]
Foyer CH, Noctor G. 2001. The molecular biology and metabolism of glutathione. In: Grill D, Tausz M, De KoK L, eds. Significance of glutathione in plant adaptation to the environment. The Netherlands: Kluwer Academic Publishers, 27–57.
Foyer CH, Theodoulou FL, Delrot S. 2001. The functions of intercellular and intracellular glutathione transport systems. Trends in Plant Science 6, 486–492.[CrossRef][Web of Science][Medline]
Gallois P, Marinho P. 1995. Leaf disk transformation using Agrobacterium tumefaciens-expression of heterologous genes in tobacco. In: Jones H, ed. Methods in molecular biology, Vol. 49. Plant gene transfer and expression protocols. Totowa, NJ: Humana Press, 39–48.
Gomez L, Vanacker H, Buchner P, Noctor G, Foyer CH. 2004. Intercellular distribution of glutathione synthesis in maize leaves and its response to short-term chilling. Plant Physiology 134, 1662–1671.
Green R, Fluhr R. 1995. UV-B-induced PR-1 accumulation is mediated by active oxygen species. The Plant Cell 7, 203–212.[Abstract]
Herouart D, Van Montagu M, Inzé D. 1993. Redox-activated expression of the cytosolic copper/zinc superoxide dismutase gene in Nicotiana. Proceedings of the National Academy of Sciences, USA 90, 3108–3112.
Johansson C, Lillig CH, Holmgren A. 2004. Human mitochondrial glutaredoxin reduces S-glutathionylated proteins with high affinity accepting electrons from either glutathione or thioredoxin reductase. Journal of Biological Chemistry 279, 7537–7543.
Knight H, Veale EL, Warren GJ, Knight MR. 1999. The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif. The Plant Cell 11, 875–886.
Kunert KJ, Foyer, CH 1993. Thiol/disulphide exchange in plants. In: De Kok LJ, Stulen I, Rennenberg H, Brunold C, Rauser W, eds. Sulfur nutrition and assimilation in higher plants, regulatory, agricultural and environmental aspects. The Hague: SPB Academic Publishers, 139–151.
Laloi C, Mestres-Ortega D, Marco Y, Meyer Y, Reichheld JP. 2004. The Arabidopsis cytosolic thioredoxin h5 gene induction by oxidative stress and its W-Box-mediated response to pathogen elicitor. Plant Physiology 134, 1006–1016.
Lemaire SD. 2004. The glutaredoxin family in oxygenic photosynthetic organisms. Photosynthesis Research 79, 305–318.[CrossRef][Web of Science][Medline]
May MJ, Leaver CJ. 1993. Oxidative stimulation of glutathione synthesis in Arabidopsis thaliana suspension cultures. Plant Physiology 130, 621–627.
May MJ, Vernoux T, Leaver C, van Montagu M, Inzé D. 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. Journal of Experimental Botany 49, 649–667.
Meinhard M, Grill E. 2001. Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis. FEBS Letters 508, 443–446.[CrossRef][Web of Science][Medline]
Meinhard M, Rodriguez PL, Grill E. 2002. The sensitivity of ABI2 to hydrogen peroxide links the abscisic acid-response regulator to redox signalling. Planta 214, 775–782.[CrossRef][Web of Science][Medline]
Mou Z, Fan W, Dong X. 2003. Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell 27, 935–944.[Web of Science]
Noctor G, Arisi ACM, Jouanin L, Foyer CH. 1998. Manipulation of glutathione and amino acid biosynthesis in the chloroplast. Plant Physiology 118, 471–482.
Noctor G, Arisi ACM, Jouanin L, Valadier MH, Roux Y, Foyer CH. 1997. The role of glycine in determining the rate of glutathione synthesis in poplar. Possible implications for glutathione production during stress. Physiologia Plantarum 100, 255–263.[CrossRef]
Noctor G, Foyer CH. 1998a. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279.[CrossRef][Web of Science][Medline]
Noctor G, Foyer CH. 1998b. Simultaneous measurement of foliar glutathione, -glutamylcysteine and amino acids by high-performance liquid chromatography: comparison with two other assay methods for glutathione. Analytical Biochemistry 264, 98–110.[CrossRef][Web of Science][Medline]
Noctor G, Rees D, Young A, Horton P. 1991. The relationship between zeaxanthin, energy-dependent quenching of chlorophyll fluorescence, and transthylakoid pH gradient in isolated chloroplasts. Biochimica et Biophysica Acta 1057, 320–330.
Noctor G, Strohm M, Jouanin L, Kunert KJ, Foyer CH, Rennenberg H. 1996. Synthesis of glutathione in leaves of transgenic poplar (Populus tremulaxP. alba) overexpressing -glutamylcysteine synthetase. Plant Physiology 112, 1071–1078.[Abstract]
Noctor G, Veljovic-Jovanovic S, Foyer CH. 2000. Peroxide processing in photosynthesis: antioxidant coupling and redox signalling. Proceedings of the Royal Society of London, B 355, 1465–1475.
Novitskaya L, Trevanion S, Driscoll S, Foyer CH, Noctor G. 2002. How does photorespiration modulate leaf amino acids? A dual approach through modelling and metabolite analysis. Plant, Cell and Environment 25, 821–836.[CrossRef]
Pignocchi C, Fletcher JM, Wilkinson JE, Barnes JD, Foyer CH. 2003. The function of ascorbate oxidase in tobacco. Plant Physiology 132, 1631–1641.
Ruegsegger A, Brunold C. 1992. Effect of cadmium on -glutamylcysteine synthesis in maize seedlings. Plant Physiology 99, 428–423.
Schafer FQ, Buettner GR. 2001. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radicals in Biology and Medicine 30, 1191–212.[CrossRef]
Starke DW, Chock PB, Mieyal JJ. 2003. Glutathione thiyl radical scavenging and transferase properties of human glutaredoxin (thioltransferase). Potential role in redox signal transduction. Journal of Biological Chemistry 278, 14607–14613.
Suthanthiran M, Anderson ME, Sharma VK, Meister A. 1990. Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens. Proceedings of the National Academy of Sciences, USA 87, 3343–3347.
Vanacker H, Carver TLW, Foyer CH. 2000. Early H2O2 accumulation in mesophyll cells leads to induction of glutathione during the hypersensitive response in the barley-powdery mildew interaction. Plant Physiology 123, 1289–1300.
Veljovic-Jovanovic S, Pignocchi C, Noctor G, Foyer CH. 2001. Low vitamin C in the vtc1 mutant of Arabidopsis thaliana is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiology 127, 426–435.
Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels C, Van Montagu M, Inzé D, Van Camp W. 1997. Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants. EMBO Journal 16, 4806–4816.[CrossRef][Web of Science][Medline]
Wingate VPM, Lawton MA, Lamb CJ. 1988. Glutathione causes a massive and selective induction of plant defense genes. Plant Physiology 87, 206–210.
Wingsle G, Karpinski S. 1996. Differential redox regulation by glutathione of glutathione reductase and CuZn-superoxide dismutase gene expression in Pinus sylvestris L. needles. Planta 198, 151–157.[Web of Science][Medline]
Xiang C, Bertrand D. 2000. Glutathione synthesis in Arabidopsis: multilevel controls coordinate responses to stress. In: Brunold C, Rennenberg H, De Kok LJ, Stulen I, Davidian JC, eds. Sulfur nutrition and sulphur assimilation in higher plants. Bern, Switzerland: Paul Haupt, 409–412.
Xiang C, Oliver DJ. 1998. Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in arabidopsis. The Plant Cell 10, 1539–1550.
Xiang C, Werner BL, Christensen EM, Oliver DJ. 2001. The biological functions of glutathione revisited in Arabidopsis transgenic plants with altered glutathione levels. Plant Physiology 126, 564–574.
Zhang K, Yang EB, Tang WY, Wong KP, Mack P. 1997. Inhibition of glutathione reductase by plant polyphenols. Biochemical Pharmacology 54, 1047–1053.[CrossRef][Web of Science][Medline]
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