title: The glutathione-deficient, cadmium-sensitive mutant, cad2-1, of Arabidopsis thaliana is deficient in gamma-glutamylcysteine synthetase.
abstract: This paper reports that the glutathione (GSH)-deficient mutant, cad2-1, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, gamma-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, gamma-glutamylcysteine. In vitro enzyme assays showed that the cad2-1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA, identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2-1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2-1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2-1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.
authors.affiliation: Department of Genetics, University of Melbourne, Parkville, Australia. c.cobbett@genetics.unimelb.edu.au
authors.forename: C S
authors.initials: CS
authors.lastname: Cobbett
authors.forename: M J
authors.initials: MJ
authors.lastname: May
authors.forename: R
authors.initials: R
authors.lastname: Howden
authors.forename: B
authors.initials: B
authors.lastname: Rolls
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology*; D002874:Chromosome Mapping; D004151:Dipeptides / Q000378:metabolism; D017343:Genes, Plant; D005721:Glutamate-Cysteine Ligase / Q000172:deficiency* / Q000235:genetics*; D005978:Glutathione / Q000172:deficiency*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length
chemical_list: D004151:Dipeptides; D010940:Plant Proteins; D002104:Cadmium; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; C017341:gamma-glutamylcysteine