Cloning of the cDNA and genomic clones for glutathione synthetase from Arabidopsis thaliana and complementation of a gsh2 mutant in fission yeast.
Glutathione is essential for protecting plants from a range of environmental stresses, including heavy metals where it acts as a precursor for the synthesis of phytochelatins. A 1658 bp cDNA clone for glutathione synthetase (gsh2) was isolated from Arabidopsis thaliana plants that were actively synthesizing glutathione upon exposure to cadmium. The sequence of the clone revealed a protein with an estimated molecular mass of 53858 Da that was very similar to the protein from higher eukaryotes, was less similar to the gene from the fission yeast, Schizosaccharomyces pombe, and shared only a small region of similarity with the Escherichia coli protein. A 4.3 kb SstI fragment containing the genomic clone for glutathione synthetase was also isolated and sequenced. A comparison of the cDNA and genomic sequences revealed that the gene was composed of twelve exons. When the Arabidopsis cDNA cloned in a special shuttle vector was expressed in a S. pombe mutant deficient in glutathione synthetase activity, the plant cDNA was able to complement the yeast mutation. Glutathione synthetase activity was measurable in wild-type yeast cells, below detectable levels in the gsh2- mutant, and restored to substantial levels by the expression of the Arabidopsis cDNA. The S. pombe mutant expressing the plant cDNA had near wild type levels of total cellular thiols, 109Cd2+ binding activity, and cadmium resistance. Since the Arabidopsis cDNA was under control of a thiamine-repressible promoter, growth of the transformed yeast on thiamine-free medium increased expression of the cDNA resulting in increases in cadmium resistance.
Department of Molecular Biology and Biochemistry, University of Idaho, Moscow 83843, USA.
C L
CL
Wang
D J
DJ
Oliver
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D015723:Gene Library; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D018745:Genome, Plant; D005981:Glutathione Synthase / Q000235:genetics* / Q000378:metabolism; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000096:biosynthesis; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000032:analysis
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D002104:Cadmium; D005981:Glutathione Synthase