Characterization Of A Homoglutathione Synthetase In Medicago Truncatula

P. Frendo1, M.J. Hernández Jiménez1, C. Mathieu1, L. Duret2, D. Gallesi1, G. Van de Sype1,

laboratoire de Biologie Végétale et Microbiologie, CNRS FRE 2294, U Nice-Sophia Antipolis, Nice, France

Laboratoire de Biométrie Génétique et Biologie des Populations, CNRS UMR 5558, U Claude Bernard-Lyon 1, Villeurbanne, France

The tripeptide glutathione (y-glutamylcysteinylglycine, GSH) is a low molecular weight thiol which is synthesized in an ATP-dependent two-step reaction. In the first step, catalyzed by y-glutamylcysteine synthetase (y-ECS), y-glutamylcysteine is produced from L-glutamic acid and L-cysteine. In the second step, catalyzed by glutathione synthetase (GSHS), glycine is added to the y-glutamylcysteine to form GSH. In plants, GSH is a storage and transport form of reduced sulfur. It has a crucial role in protecting the plant against xenobiotics, heavy metals and oxidative stress. In addition to GSH, homoglutathione (y-glutamylcysteinyl-P-alanine, hGSH), has been detected in leguminous plants. Homoglutathione synthetase (hGSHS), an enzyme that has a higher affinity for P-alanine than for glycine, catalyzes the second step of hGSH synthesis. We are working on the characterization of GSH and hGSH metabolism in Medicago truncatula, a model plant for the study of legxime-Rhizobium interactions.

Two Medicago truncatula cDNAs (gshsl and gshs2) corresponding to a putative glutathione synthetase (GSHS) and a putative homoglutathione synthetase (hGSHS) were characterized by heterologous expression in an Escherichia coli strain deficient in GSHS activity. The recombinant GSHS1 is a GSHS that does not accept P-alanine as a substrate. The recombinant GSHS2 showed a 10-fold higher affinity for P-alanine than for glycine indicating that GSHS2 is a hGSHS. Leucine-534 and proline-535 present in hGSHS were substituted in GSHS2 by two 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 p-alanine and glycine by hGSHS. Phylogenetic analysis of GSHS2 and GSHS1 indicated that gshs2 and gshsl are the result of a gene duplication that occurred after the divergence between Fabales, Solanales and Brassicales. Analysis of the structure of gshsl and gshs2 genes shows they are both present in a cluster and in the same orientation in the M. truncatula genome, suggesting that the gene duplication occurred via a tandem duplication.

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