A Medicago truncatula homoglutathione synthetase is derived from glutathione synthetase by gene duplication

Glutathione (GSH) and homo-GSH (hGSH) are the major low-molecular weight th iols synthesized in Medicago truncatula. Two M. truncatula cDNAs (gshs1 and gshs2) corresponding to a putative GSH synthetase (GSHS) and a putative hG SH synthetase (hGSHS) were characterized. Heterologous expression of gshs1 and gshs2 cDNAs in an Escherichia coli strain deficient in GSHS activity sh owed that GSHS1 and GSHS2 are a GSHS and an hGSHS, respectively. Leucine-53 4 and proline-535 present in hGSHS were substituted by alanines that are co nserved in plant GSHS. These substitutions resulted in a strongly stimulate d GSH accumulation in th transformed E. coli strain showing that these resi dues play a crucial role in the differential recognition of beta -alanine a nd glycine by hGSHS. Phylogenetic analysis of GSHS2 and GSHS1 with other eu karyotic GSHS sequences indicated that gshs2 and gshs1 are the result of a gene duplication that occurred after the divergence between Fabales, Solana les, and Brassicales. Analysis of the structure of gshs1 and gshs2 genes sh ows 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 occ urred via a tandem duplication.