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Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, γ-glutamate cysteine ligase (GSH1), responsible for synthesis of γ-glutamylcysteine (γ-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by γ-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of γ-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by γ-EC in vitro strongly suggests export of γ-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, γ-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1. [ABSTRACT FROM AUTHOR]

Summary: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, γ-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, γ-glutamylcysteine.In vitroenzyme assays showed that thecad2–1mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. TheCAD2locus maps to chromosome 4 and is tightly linked to a gene,GSHA, identified by a previously isolated cDNA. A genomic clone ofGSHAcomplements both the phenotypic and biochemical deficiencies of thecad2–1mutant. 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 theCAD2gene encodes GCS. Thecad2–1mutation 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 thecad2–1mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.. [ABSTRACT FROM AUTHOR]