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.

-EJB 93 0737/1 cDNA clones encoding the precursor of the T protein of the glycine decarboxylase complex have been isolated from a pea leaf cDNA library in 2 gtl 1. The longest cDNA insert of 1430 bp encodes a polypeptide of 408 amino acid residues of which 30 residues constitute an N-terminal cleavable presequence and 378 residues make up the mature protein. Several results confirmed the identity of the cDNA and the exactness of the predicted primary structure. Firstly, we purified the T protein to homogeneity and its mass was measured by mass spectrometry. The mass obtained (40966 i 5 Da) was the value predicted from the cDNA (40961 Da). Secondly, the purified T protein was chemically cleaved with cyanogen bromide and the peptide fragments were analysed by high-performance liquid chromatography/electrospray ionization mass spectrometry and/or fast-atom-bombardment mass spectrometry. The mass values of all the peptides generated by chemical cleavage and measured by these techniques were very close to the values calculated from the prcdictcd primary structure. Thirdly, microsequencing of some of these peptides, which represent 35% of the total protein, fits perfectly with the primary structure deduced from the cDNA. In the present HPLC/electrospray ionization MS studies we never detected the presence of covalently bound tetrahydropteroylpolyglutamate (H,PteGlu,,), either in the native T protein or in thc different pcptide fragments generalcd by the chemical cleavage. The absence of H,PteGlu,, bound to the T protein in our experimental conditions demonstrates that H,PteGlu, is not covalently linked to the T protein. Northern blot analysis showed that the steady-state level of the mRNA corresponding to the T protein was high in green leaves compared to the level in etiolated leaves (= 8-10-fold higher). Surprisingly, a non-negligible amount of mRNA corresponding to the T protein was present in roots whereas the mRNA encoding the H protein was not detectable. Westcrn blot analysis showed that the P, L and T proteins of the glycine decarboxylase complex were present in roots whereas the H protein was no1 detectable. Southern hybridization to pea genomic DNA indicated the presence of a single gene encoding the T protein of the glycine decarboxylase complex in the haploid genome.

Glyoxylate accumulation within cells is highly toxic. In humans, it is associated with hyperoxaluria type 2 (PH2) leading to renal failure. The glyoxylate content within cells is regulated by the NADPH/NADH dependent glyoxylate/hydroxypyruvate reductases (GRHPR). These are highly conserved enzymes with a dual activity as they are able to reduce glyoxylate to glycolate and to convert hydroxypyruvate into D-glycerate. Despite the determination of high-resolution X-ray structures, the substrate recognition mode of this class of enzymes remains unclear. We determined the structure at 2.0 Å resolution of a thermostable GRHPR from Archaea as a ternary complex in the presence of D-glycerate and NADPH. This shows a binding mode conserved between human and archeal enzymes. We also determined the first structure of GRHPR in presence of glyoxylate at 1.40 Å resolution. This revealed the pivotal role of Leu53 and Trp138 in substrate trafficking. These residues act as gatekeepers at the entrance of a tunnel connecting the active site to protein surface. Taken together, these results allowed us to propose a general model for GRHPR mode of action.

Halobacterium salinarum is an extreme halophile archaeon with an absolute requirement for a multimolar salt environment. It accumulates molar concentrations of KCl in the cytosol to counterbalance the external osmotic pressure imposed by the molar NaCl. As a consequence, cytosolic proteins are permanently exposed to low water activity and highly ionic conditions. In non-adapted systems, such conditions would promote protein aggregation, precipitation, and denaturation. In contrast, in vitro studies showed that proteins from extreme halophilic cells are themselves obligate halophiles. In this paper, adaptation via dynamics to low-salt stress in H. salinarum cells was measured by neutron scattering experiments coupled with microbiological characterization. The molecular dynamic properties of a proteome represent a good indicator for environmental adaptation and the neutron/microbiology approach has been shown to be well tailored to characterize these modifications. In their natural setting, halophilic organisms often have to face important variations in environmental salt concentration. The results showed deleterious effects already occur in the H. salinarum proteome, even when the external salt concentration is still relatively high, suggesting the onset of survival mechanisms quite early when the environmental salt concentration decreases.

Bradyrhizobium japonicum is a symbiotic nitrogen-fixing soil bacteria that induce root nodules formation in legume soybean (Glycine max.). Using (13)C- and (31)P-nuclear magnetic resonance (NMR) spectroscopy, we have analysed the metabolite profiles of cultivated B. japonicum cells and bacteroids isolated from soybean nodules. Our results revealed some quantitative and qualitative differences between the metabolite profiles of bacteroids and their vegetative state. This includes in bacteroids a huge accumulation of soluble carbohydrates such as trehalose, glutamate, myo-inositol and homospermidine as well as Pi, nucleotide pools and intermediates of the primary carbon metabolism. Using this novel approach, these data show that most of the compounds detected in bacteroids reflect the metabolic adaptation of rhizobia to the surrounding microenvironment with its host plant cells.

In vivo molecular dynamics in Halobacterium salinarum cells under stress conditions was measured by neutron scattering experiments coupled with microbiological characterization. Molecular dynamics alterations were detected with respect to unstressed cells, reflecting a softening of protein structures consistent with denaturation. The experiments indicated that the neutron scattering method provides a promising tool to study molecular dynamics modifications in the proteome of living cells induced by factors altering protein folds.