SummaryThe effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5¢-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, g-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mM L-cysteine, whereas ATP sulphurylase was signi®cantly affected only at 2 mM L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mM and higher concentrations. In the presence of L-buthionine-S, Rsulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mM L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mM GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using 35 SO 4 2± in the presence of 0.5 mM L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the¯ux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the¯ux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the¯ux control coef®cient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the¯ux through the pathway, and that the¯ux control coef®cient of APR for the pathway, including the transport step, is proportionately less, with a signi®cant share of the control exerted by the transport step.

SummaryThe coding sequence of the wild-type, cys-sensitive, cysE gene from Escherichia coli, which encodes an enzyme of the cysteine biosynthetic pathway, namely serine acetyltransferase (SAT, EC 2.3.1.30), was introduced into the genome of potato plants under the control of the cauli¯ower mosaic virus 35S promoter. In order to target the protein into the chloroplast, cysE was translationally fused to the 5¢-signal sequence of rbcS from Arabidopsis thaliana. Transgenic plants showed a high accumulation of the cysE mRNA. The chloroplastic localisation of the E. coli SAT protein was demonstrated by determination of enzymatic activities in enriched organelle fractions. Crude leaf extracts of these plants exhibited up to 20-fold higher SAT activity than those prepared from wild-type plants. The transgenic potato plants expressing the E. coli gene showed not only increased levels of enzyme activity but also exhibited elevated levels of cysteine and glutathione in leaves. Both were up to twofold higher than in control plants. However, the thiol content in tubers of transgenic lines was unaffected. The alterations observed in leaf tissue had no effect on the expression of O-acetylserine(thiol)-lyase, the enzyme which converts O-acetylserine, the product of SAT, to cysteine. Only a minor effect on its enzymatic activity was observed. In conclusion, the results presented here demonstrate the importance of SAT in plant cysteine biosynthesis and show that production of cysteine and related sulfur-containing compounds can be enhanced by metabolic engineering.

Cysteine synthesis from sulfide and O-acetyl-l-serine (OAS) is a reaction interconnecting sulfate, nitrogen, and carbon assimilation. Using Lemna minor, we analyzed the effects of omission of CO 2 from the atmosphere and simultaneous application of alternative carbon sources on adenosine 5Ј-phosphosulfate reductase (APR) and nitrate reductase (NR), the key enzymes of sulfate and nitrate assimilation, respectively. Incubation in air without CO 2 led to severe decrease in APR and NR activities and mRNA levels, but ribulose-1,5-bisphosphate carboxylase/oxygenase was not considerably affected. Simultaneous addition of sucrose (Suc) prevented the reduction in enzyme activities, but not in mRNA levels. OAS, a known regulator of sulfate assimilation, could also attenuate the effect of missing CO 2 on APR, but did not affect NR. When the plants were subjected to normal air after a 24-h pretreatment in air without CO 2 , APR and NR activities and mRNA levels recovered within the next 24 h. The addition of Suc and glucose in air without CO 2 also recovered both enzyme activities, with OAS again influenced only APR. 35 SO 4 2Ϫ feeding showed that treatment in air without CO 2 severely inhibited sulfate uptake and the flux through sulfate assimilation. After a resupply of normal air or the addition of Suc, incorporation of 35 S into proteins and glutathione greatly increased. OAS treatment resulted in high labeling of cysteine; the incorporation of 35 S in proteins and glutathione was much less increased compared with treatment with normal air or Suc. These results corroborate the tight interconnection of sulfate, nitrate, and carbon assimilation.Plants, yeast, and most prokaryotes cover their demand for reduced sulfur, which is essential for function of proteins, oligopeptides, and many coenzymes, by reduction of inorganic sulfate. In the pathway of sulfate assimilation of plants, sulfate is first activated by ATP sulfurylase to adenosine 5Ј-phosphosulfate, which is reduced to sulfite by adenosine 5Ј-phosphosulfate reductase (APR) in a glutathionedependent reaction. Sulfite is further reduced to sulfide by a ferredoxin-dependent sulfite reductase and sulfide incorporated into the amino acid skeleton of O-acetyl-l-Ser (OAS) by OAS (thiol) lyase, forming Cys (Brunold, 1990;Leustek et al., 2000). Cys can further be metabolized to Met or directly incorporated into proteins or glutathione, a tripeptide with important functions as storage and transport form of reduced sulfur, in oxidative stress defense, regulation of sulfur assimilation, etc. (Noctor et al., 1998). Thus, Cys synthesis from OAS and sulfide is a central point of cellular metabolism as this reaction interconnects sulfate, nitrate, and carbon assimilation.Several studies have established regulatory interactions between sulfate and nitrate assimilation in plants (Brunold, 1993;Takahashi and Saito, 1996;Kim et al., 1999;Koprivova et al., 2000). The two assimilatory pathways are well coordinated so that deficiency for one element represses the other pathway. The act...

Adenosine 5-phosphosulfate (APS) sulfotransferase and APS reductase have been described as key enzymes of assimilatory sulfate reduction of plants catalyzing the reduction of APS to bound and free sulfite, respectively. APS sulfotransferase was purified to homogeneity from Lemna minor and compared with APS reductase previously obtained by functional complementation of a mutant strain of Escherichia coli with an Arabidopsis thaliana cDNA library. APS sulfotransferase was a homodimer with a monomer M r of 43,000. Its amino acid sequence was 73% identical with APS reductase. APS sulfotransferase purified from Lemna as well as the recombinant enzyme were yellow proteins, indicating the presence of a cofactor. Like recombinant APS reductase, recombinant APS sulfotransferase used APS (K m ‫؍‬ 6.5 M) and not adenosine 3-phosphate 5-phosphosulfate as sulfonyl donor. The V max of recombinant Lemna APS sulfotransferase (40 mol min ؊1 mg protein ؊1 ) was about 10 times higher than the previously published V max of APS reductase. The product of APS sulfotransferase from APS and GSH was almost exclusively SO 3 2؊ . Bound sulfite in the form of S-sulfoglutathione was only appreciably formed when oxidized glutathione was added to the incubation mixture. Because SO 3 2؊ was the first reaction product of APS sulfotransferase, this enzyme should be renamed APS reductase.Higher plants and many microorganisms growing with sulfate as sulfur source reduce it to the level of sulfide for the synthesis of cysteine, methionine, coenzymes, and iron-sulfur clusters of enzymes (1-5). The reaction sequence from sulfate to sulfide is called assimilatory sulfate reduction as opposed to dissimilatory sulfate reduction, which occurs in certain anaerobic organisms such as Desulfovibrio and Desulfotomaculum, where sulfate functions as an electron acceptor during oxidation of organic substrates and where reduced forms of sulfur are excreted into the surroundings (3).The first step of assimilatory sulfate reduction is an activation of sulfate catalyzed by ATP sulfurylase (EC 2.7.7.4). The adenosine 5Ј-phosphosulfate (APS) 1 is the substrate for APS kinase (EC 2.7.1.2.5), which forms adenosine 3Ј-phosphate 5Ј-phosphosulfate (PAPS) in a second activation step (1-5). The subsequent reduction sequence starting from PAPS is well established in bacteria (4) and fungi (6), where a PAPS reductase (EC 1.8.99) reacts first with reduced thioredoxin then with PAPS to form SO 3 2Ϫ , oxidized thioredoxin, and adenosine 3Ј-phosphate 5Ј-phosphate (PAP) (4, 7, 8). The SO 3 2Ϫ is reduced to sulfide by sulfite reductase (EC 1.8.7.1). Sulfide is finally incorporated into O-acetyl-L-serine via O-acetyl-L-serine thiollyase (EC 4.2.99.8), thus forming cysteine (1-4). All these enzymes were detected in plants (1-5, 7-11), indicating that the identical reaction sequence is operative. In two early reports it was demonstrated, however, that plants and algae use APS rather than PAPS as sulfonyl donor for the first reduction step (12, 13). APS-dependent enzymes were partially p...

With the aim of analysing the relative importance of sugar supply and nitrogen nutrition for the regulation of sulphate assimilation, the regulation of adenosine 5'-phosphosulphate reductase (APR), a key enzyme of sulphate reduction in plants, was studied. Glucose feeding experiments with Arabidopsis thaliana cultivated with and without a nitrogen source were performed. After a 38 h dark period, APR mRNA, protein, and enzymatic activity levels decreased dramatically in roots. The addition of 0.5% (w/v) glucose to the culture medium resulted in an increase of APR levels in roots (mRNA, protein and activity), comparable to those of plants kept under normal light conditions. Treatment of roots with d-sorbitol or d-mannitol did not increase APR activity, indicating that osmotic stress was not involved in APR regulation. The addition of O-acetyl-l-serine (OAS) also quickly and transiently increased APR levels (mRNA, protein, and activity). Feeding plants with a combination of glucose and OAS resulted in a more than additive induction of APR activity. Contrary to nitrate reductase, APR was also increased by glucose in N-deficient plants, indicating that this effect was independent of nitrate assimilation. [35S]-sulphate feeding experiments showed that the addition of glucose to dark-treated roots resulted in an increased incorporation of [35S] into thiols and proteins, which corresponded to the increased levels of APR activity. Under N-deficient conditions, glucose also increased thiol labelling, but did not increase the incorporation of label into proteins. These results demonstrate that (i) exogenously supplied glucose can replace the function of photoassimilates in roots; (ii) APR is subject to co-ordinated metabolic control by carbon metabolism; (iii) positive sugar signalling overrides negative signalling from nitrate assimilation in APR regulation. Furthermore, signals originating from nitrogen and carbon metabolism regulate APR synergistically.