Control of assimilatory sulphate reduction

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Control of S flux from sulphate to cysteine appears to be controlled in several ways (Figure 4). The first concerns the transcriptional control over the sulphate transporting membrane proteins. As discussed before,, expression of these proteins is linked to low concentrations of cysteine, glutathione and sulphate though there are reasons for suspecting that glutathione serves as the long distance signal and that cysteine acts as the principal direct sensor for initiating transcription. Conversely, high concentrations of OAS appear to enhance the expression of the sulphate transporting proteins. The activities of various enzymes of the sulphate assimilation pathway are also influenced by factors that affect glutathione synthesis. Cultured tobacco cells and plants exhibit large increases in the extractable activity of ATP sul-phurylase when exposed to S stress (for a review, see Brunold 1990). S dep rivation influences the expression of ATP sulphurylase and APS-kinase indicating that control over sulphate assimilation occurs, at least in part, at the level of transcription (see Hell 1997). However, of the enzymes of sulphate assimilation, APS sulphotransferase seems to be especially responsive. For example, APS sulphotransferase activity increases 2-fold in response to S deprivation (Brunold et al. 1987) and up to 20-fold in plants exposed to herbicide safeners (Farago and Brunold 1990); conversely, the activity decreases on exposure to S02 or H2S (Tschanz et al. 1986) and to exogenous cysteine (Jenni et al. 1980). These responses provide conditions for enhancing S flux under conditions where there is an increased demand for glu-tathione synthesis and suggest that control of APS sulphotransferase activity is important in assimilatory sulphate reduction. The activity of ATP sul-phurylase does not appear to be especially sensitive to the demand for S (Brunold et al. 1987), consistent with the very high activity of this enzyme in plant tissues. However, ATP sulphurylase activity appears the linked to the rate of N assimilation.

Since glutathione is important in many aspects of plant function (Figure 3), Bergmann and Rennenberg (1993) have suggested that it is a likely regulatory factor in glutathione synthesis. May et al. (1998) have proposed a model for the control of sulphate assimilation by glutathione in which glu-tathione blocks the transduction into the nucleus of an unidentified factor which controls the expression of the proteins of the sulphate assimilation pathway. Perhaps this model might also be applicable to regulation of the sulphate transporter.

Control of glutathione synthesis from cysteine

In theory, the synthesis of glutathione can be regulated by the availability of one or more of the constituent amino acids, cysteine, glutamate and glycine, and/or the activity of one or more of the enzymes involved in coupling them (Brunold and Rennenberg 1997). Under normal conditions, glutamate and glycine do not appear to be limiting. Rather, the endogenous concentration of cysteine and the activity of y-glutamylcysteine synthetase (and hence the concentration of y-glutamylcysteine) appear to be critically important (Brunold and Rennenberg 1997). A significant feature is that both the K.m(cysteine) of y-glutamylcysteine synthetase and the intracellular concentration of cysteine are very low thereby making the synthesis of y-glutamylcysteine especially sensitive to the endogenous concentration of cysteine. In illuminated plants addition of exogenous cysteine consistently enhances the internal concentration of glutathione (Buwalda et al. 1990,

Farago and Brunold 1994, Schneider and Bergmann 1995, Noctor et al. 1996, 1997) implying that the production of glutathione is limited by the availability of y-glutamylcysteine which in turn is limited by the internal concentration of cysteine. Other evidence is consistent with this view. In the leaves of poplar plants in which the limited production of is overcome by overexpression of y-glutamylcysteine synthetase, glutathione synthesis is limited by glutamate rather than cysteine (Noctor et al. 1996). This implies that the synthesis of cysteine from sulphate is responsive to increased demand for cysteine for glutathione synthesis. Studies of y-glutamylcysteine synthetase activity in relation to glutathione and PC synthesis in plants and cell cultures treated with Cd are consistent with the proposals set out above.

Studies with poplar plants that overexpress glutathione synthetase indicate that the activity of this enzyme does not normally limit glutathione (Strohm et al. 1995, Brunold and Rennenberg 1997). The endogenous glu-tathione concentration of transgenic plants was similar to that of the wild type and addition of cysteine enhanced the endogenous glutathione concentration in both plants. However, upon addition of cysteine the concentration of glutathione did not increase with time indefinitely, either in the wild type or transgenic plants, even though they accumulated cysteine. This implies that some other factor limits the synthesis of glutathione from cysteine. This appears to involve y-glutamylcysteine synthetase activity since addition of y-resulted in much higher concentrations of glutathione in the plants, which overexpressed glutathione synthetase (Strohm et al. 1995). Since glutathione strongly inhibits y-glutamylcysteine synthetase in vitro (Hell et al. 1990), other observations with Cd-treated cells and plants have been interpreted in terms of a model involving negative post-transcriptional control of y-glutamylcysteine synthetase by glutathione (Ruegsegger and Brunold 1992, Bergmann and Rennenberg 1993). However, feedback control of synthetase by glutathione was not evident in trans formed poplar plants which overexpressed synthetase even though the concentration of glutathione in these plants was 3-fold higher than in the wild type. Another relevant factor here is that, under some conditions, glutathione accumulates in response to various external factors (Table 1) indicative of lack of feedback inhibition. However, it is not known whether these concentrations apply at the subcellular sites where the regulatory mechanisms of glutathione synthesis are located.

Collectively, the available data indicate that glutathione synthetase is not an important regulatory site and that the reaction catalysed by this enzyme is close to thermodynamic equilibrium (Noctor et al. 1998). Conversely, the reaction catalysed by y-glutamylcysteine synthetase is far removed from chemical equilibrium and appears to be regulated in several different ways, both pre-translationally and post-translationally. In addition to in vitro evidence of feedback control by GSH (Hell et al. 1990), post-transcriptional control of this enzyme is supported by the poor expression of Arabidopsis y-glutamylcysteine synthetase in transformed E. coli, leading to the suggestion that post-transcriptional factors, absent in the bacteria, are necessary for full expression of the plant gene (May et al. 1998). It appears therefore that y-glutamylcysteine is the primary point of regulation of glutathione synthesis. It is controlled by both the availability of cysteine and by the activity of y-

synthetase. As plants that overexpress synthetase appear to adjust their rate of S assimilation to support y-giutamylcysteine (and hence glutathione) synthesis, then the availability of y-glutamylcysteine might represent a secondary regulatory point in the pathway of glutathione synthesis. This would also prevent the concentration of glutathione exceeding a certain limit, perhaps for physiologically relevant reasons.

Under special conditions plants can produce abnormally high concentrations of cysteine. This can be achieved by fumigating plants with H2S which becomes assimilated into cysteine (thereby acting as a detoxification mechanism) and simultaneously placing the plants in the dark to inhibit photorespi-ratory production of glycine. Under these conditions, the production of glutathione can be limited by the availability of glycine (Buwalda et al. 1988, 1990).

As noted before, glutathione synthesis occurs in both the chloroplast and the cytosol. This raises the question of the availability of the constituent amino acids and the permeability of the intracellular membranes to cysteine, glycine and glutamate. Since it is likely that the amino acids used in glu-tathione synthesis in chloroplasts and the cytosol have different origins, perhaps glutathione synthesis could be directly or indirectly regulated by intra-cellular amino acid transport (e.g. transport of cysteine from the primary site of synthesis in the chloroplast for use in glutathione synthesis in the cytosol, Rennenberg 1997).

Regulatory responses associated with exposure to cadmium and herbicide fasteners

Cd promotes the synthesis of PCs from glutathione. Sustained production of PCs implies increased flux of S from sulphate via cysteine into glutathione. According to the scheme given in Figure 4, the short-term decrease in the concentration of glutathione following the administration of Cd should lead to increased flux of S from sulphate into glutathione. This has been confirmed by experiment. For example, Nussbaum et al. (1988) and Ruegsegger and Brunold (1992) reported that Cd enhanced the rate of sulphate assimilation in the root and increased the turnover of cysteine, y-glutamylcysteine and glutathione, consistent with the proposed role of glu-tathione as a direct or indirect signal for controlling the flux of S from sulphate into glutathione. Cd enhanced the activities of several enzymes of the sulphate assimilation pathway and glutathione synthesis, including ATP sulphurylase, APS sulphotransferase, y-glutamylcysteine synthetase and glutathione synthetase (Nussbaum et al. 1988, Ruegsegger et al. 1990, Rueg-segger and Brunold 1992). Presumably this also involved regulation of the sulphate transporters in response to the increased flux of S from sulphate into PCs.

In Arabidopsis, Cd and Cu, which promote the production of PCs, also promote expression of the mRNAs for y-glutamylcysteine synthetase and glutathione synthetase (Xiang and Oliver 1998) implying control at the tran-scriptional level. Heavy metals that do not induce PC synthesis do not affect transcription of these genes. Supplying exogenous gSh or GSSG to the plants also did not promote transcription. Xiang and Oliver (1998) proposed that, in Arabidopsis, a signal transduction pathway that does not involve GSH or GSSG elicits the transcriptional response to heavy metals, perhaps involving jasmonic acid. Transcriptional regulation of the enzymes involved in the synthesis of glutathione from cysteine is consistent with the more sluggish restoration of the intracellular glutathione pool compared with the very rapid drop following the application of Cd which is thought to involve post-translational activation of PC synthase by Cd (Grill et al. 1989, Loeffler et al. 1989).

Some forms of environmental stress, unlike Cd and Cu, cause glutathione to accumulate in plant tissues (Table 1) implying that the rate of glutathione synthesis exceeds the rate of demand. In these instances, accumulation of glutathione appears to be an intrinsic part of the plant's response to stress and implies that the postulated regulatory mechanism involving inhibition of y-glutamylcysteine synthetase by glutathione must be countermanded. Thus, Farago and Brunold (1990) demonstrated that two herbicide safeners enhanced accumulation of glutathione, sulphate assimilation, and cysteine and glutathione synthesis in maize roots. The safeners also increased the activities of both ATP sulphurylase and APS sulphotransferase. One of the safeners also enhanced sulphate uptake. Although these data were obtained 6 d after addition of the safeners, the increased rate of sulphate assimilation and the association of high enzyme activities with high glutathione concentrations is inconsistent with repression of sulphate assimilation by glutathione via the mechanisms shown in Figure 4. Moreover, the high glutathione levels found in the presence of the safeners indicate that the proposed feedback inhibition of y-glutamylcysteine synthetase by glutathione must have been overcome by another factor(s). One possibility is compartmentation: if glutathione is spatially separated from the site of y-glutamy lcysteine synthetase then feedback inhibition of this enzyme would be effectively eliminated. Also, the increased gene expression of y-glutamylcysteine synthetase induced by the safeners could more than compensate for the post-translational inhibition by glutathione. Consistent with this, transformed poplar plants, which overexpress y-glutamylcysteine synthetase activity, contain higher concentrations of glutathione.

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