CLONING OF ARABIDOPSIS-THALIANA GLUTATHIONE SYNTHETASE (GSH2) BY FUNCTIONAL COMPLEMENTATION OF A YEAST GSH2 MUTANT

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) plays an import ant role in the protection of plants against various types of stress c aused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for gl utathione synthetase (GSH2) of Arabidopsis thaliana was cloned by comp lementation of the methylglyoxal sensitivity of a gsh2 mutant of the y east Saccharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced M(r): 53783), bearing clear sequence similarities to G SH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus no rvegicus) and from the fission yeast (Schizosaccharomyces pombe). A hi ghly conserved glycine-rich domain close to the carboxy-terminus was f ound in the GSH2 product and appears to be typical for eukaryotic glut athione synthetases. The M(r) is similar to those of soluble animal en zymes, suggesting that the Arabidopsis gene also codes for a cytosolic protein. Genomic DNA-blot analysis indicates the presence of a single GSH2 gene. The yeast gsh2 mutant becomes resistant to methylglyoxal a nd cadmium after transformation with the plasmid bearing the Arabidops is GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectibility in mutants to abo ut 50% of the wild-type levels in transformed cells.