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.

The expression of the genes encoding the four proteins (P, H, T, and 1) of glycine decarboxylase, a multienzymatic complex involved in the mitochondrial step of the photorespiration pathway, was examined during pea (fisum sativum) leaf development in comparison with ribulose-l,5-bisphosphate carboxylase/oxygenase. Mitochondria from the primary leaf were isolated at severa1 welldefined stages of development. Their capacity to oxidize glycine was negligible during the earlier stages but increased dramatically once the leaflet opened. This was correlated with the accumulation of the glycine decarboxylase complex (CDC) proteins, which was shown to occur in preexisting mitochondria, producing an increase in their density. The transcription of the CDC genes was coordinated and occurred early, with a peak at 7 d, a stage at which mitochondria are unable to oxidize glycine. This implies the existente of posttranscriptional control of gene expression. The comparison of the expression patterns of the genes encoding specific proteins of CDC with that of rbcS genes suggests a common regulation scheme that is related to light induction. However, ribulose-1,5-bisphosphate carboxylase/oxygenase is present in the chloroplast well before CDC fills the mitochondria, suggesting that the setup of photorespiration occurs in cells already engaged in active photosynthesis.

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using (13)C- and (31)P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N(2) fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.