Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant.
Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) plays an important role in the protection of plants against various types of stress caused by reactive oxygen species, gazeous pollutants, heavy metals and xenobiotics. A cDNA fragment containing the entire coding unit for glutathione synthetase (GSH2) of Arabidopsis thaliana was cloned by complementation of the methylglyoxal sensitivity of a gsh2 mutant of the yeast Saccharomyces cerevisiae. The cDNA encodes a protein of 478 amino acids (deduced Mr: 53783), bearing clear sequence similarities to GSH2 products from frog embryos (Xenopus laevis), rat kidney (Rattus norvegicus) and from the fission yeast (Schizosaccharomyces pombe). A highly conserved glycine-rich domain close to the carboxy-terminus was found in the GSH2 product and appears to be typical for eukaryotic glutathione synthetases. The Mr is similar to those of soluble animal enzymes, 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 and cadmium after transformation with the plasmid bearing the Arabidopsis GSH2 cDNA. Moreover, this increased resistance is correlated to the restoration of GSH content from below detectability in mutants to about 50% of the wild-type levels in transformed cells.
Ullmann
P
P
Départment d'Enzymologie Cellulaire et Moléculaire, Institut de Biologie Moléculaire des Plantes, Strasbourg, France.
Gondet
L
L
Potier
S
S
Bach
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D001483:Base Sequence; D002104:Cadmium / Q000633:toxicity; D003001:Cloning, Molecular; D017343:Genes, Plant; D005816:Genetic Complementation Test; D005978:Glutathione / Q000378:metabolism; D005981:Glutathione Synthase / Q000235:genetics*; D008969:Molecular Sequence Data; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D002104:Cadmium; C000609131:GSH2 protein, Arabidopsis; D005981:Glutathione Synthase; D005978:Glutathione