Several markers of oxidative stress were measured in 2-to 10-week-old soybean (Glycine max [L.] Merr.) nodules. There were increases in peroxides, protein carbonyls and modi®ed DNA base concentrations with nodule age. The catalytic iron content also increased signi®cantly during nodule ageing. Iron contained in the peribacteroid space was eective in promoting lipid peroxidation and this might contribute to the degradation of the peribacteroid membrane in senescing nodules. The concentration of the oxidized forms of glutathione and homoglutathione increased signi®cantly during nodule development and the concentration of reduced glutathione and homoglutathione decreased during senescence. Taken together, these results are consistent with the development of oxidative stress in senescing nodules. Signi®cant DNA and protein damage also occurred in the ®rst days of nodule development, suggesting that an earlier period of oxidative stress might occur in the period over which the symbiosis becomes established.Abbreviations: CHP = cumene hydroperoxide; GSH = glutathione; hGSH = homoglutathione; MDA = malondialdehyde; PBM = peribacteroid membrane; PBS = peribacteroid space; ROS = reactive oxygen species Correspondence to: A. Puppo;

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Glutathione (GSH) and homo-GSH (hGSH) are the major low-molecular weight thiols synthesized in Medicago truncatula. Two M. truncatula cDNAs (gshs1 and gshs2) corresponding to a putative GSH synthetase (GSHS) and a putative hGSH synthetase (hGSHS) were characterized. Heterologous expression of gshs1 and gshs2 cDNAs in an Escherichia coli strain deficient in GSHS activity showed that GSHS1 and GSHS2 are a GSHS and an hGSHS, respectively. Leucine-534 and proline-535 present in hGSHS were substituted by alanines that are conserved in plant GSHS. These substitutions resulted in a strongly stimulated GSH accumulation in the transformed E. coli strain showing that these residues play a crucial role in the differential recognition of ␤-alanine and glycine by hGSHS. Phylogenetic analysis of GSHS2 and GSHS1 with other eukaryotic GSHS sequences indicated that gshs2 and gshs1 are the result of a gene duplication that occurred after the divergence between Fabales, Solanales, and Brassicales. Analysis of the structure of gshs1 and gshs2 genes shows they are both present in a cluster and in the same orientation in the M. truncatula genome, suggesting that the duplication of gshs1 and gshs2 occurred via a tandem duplication.

SummaryGlutathione (GSH) and homoglutathione (hGSH) were quantified in Medicago truncatula during plant development. hGSH was detectable only 48 h after seed germination whereas GSH was present in the dry seeds, indicating that only GSH is used for sulphur storage in seeds. The hGSH was detectable only in the underground part of mature plants whereas GSH was present in all the organs. γ-EC synthetase (γ-ECS) and GSH synthetase (GSHS) activities were found in roots and leaves whereas hGSH synthetase (hGSHS) was found only in roots. Full-length cDNA encoding γ-ECS and two partial cDNAs (gshs1 and gshs2) showing high identity with GSHS were isolated in M. truncatula. High γ-ECS activity was detected in protein extracts of a γ-ECS-deficient E. coli strain expressing the M. truncatula γ-ECS. Northern blot analysis showed that the γ-ECS gene was similarly expressed in all the mature plant organs tested, whereas gshs1 had a higher expression in leaves and flowers and gshs2 was preferentially expressed in roots and nodules. We hypothesise that gshs1 and gshs2 encode a GSHS and an hGSHS, respectively.