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;

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.

Soybean (Glycine max cv. Stevens) suspension cells were used to investigate the expression of the alternative oxidase (Aox) multigene family. Suspension cells displayed very high rates of cyanide-insensitive respiration, but Aox3 was the only isoform detected in untreated cells. Incubation with antimycin A, citrate, salicylic acid or at low temperature (10 degrees C) specifically induced the accumulation of the Aox1 isoform. Aox2 was not observed under any conditions in the cells. Increases in Aox1 protein correlated with increases in Aox1 mRNA. Treatment of soybean cotyledons with norflurazon also induced expression of Aox1. Reactive oxygen species (ROS) were detected upon incubation of cells with antimycin, salicylic acid or at low temperature, but not during incubation with citrate. Aox1 induction by citrate, but not by antimycin, was prevented by including the protein kinase inhibitor staurosporine in the medium. The results suggest that multiple pathways exist in soybean to regulate expression of Aox genes and that Aox1 specifically is induced by a variety of stress and metabolic conditions via at least two independent signal transduction pathways.

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