Genes for glutathione synthesis from tomato: Structure, expression and role in cadmium tolerance

Iulia Alexandra Kovari, Purdue University

Abstract

All organisms must balance the requirement for essential metals with the potential toxicity of these and other non-essential metals. In plants, phytochelatins and metallothioneins may provide mechanisms to keep metals such as zinc, copper and cadmium between beneficial and toxic levels. Phytochelatins are synthesized from glutathione, therefore glutathione biosynthesis is critical for production of these peptides. cDNAs from tomato encoding proteins with homology to $\gamma$-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) were cloned and sequenced. The homology of tomato GSH1 and GSH2 at the protein level with corresponding Arabidopsis genes is 82% and 67%, respectively. The GSH2 protein contains a leucine zipper motif suggesting dimerization of the protein, consistent with observations that eukaryotic GSH2 proteins from dimers. Genomic DNA blot analysis indicated that the tomato GSH1 and GSH2 genes are present in low copy number. Further, RNA blot analysis indicated that the GSH1 and GSH2 2 genes are expressed constitutively in different tissues and are not induced by Cd or other stresses. Hybridization of GSH1 and GSH2 cDNAs to genomic DNA from unselected and selected Cd tolerant cell lines hybridized with the same pattern and intensity of hybridization, indicating that neither gene is amplified in tolerant cells. Amino acid substitutions in the $\gamma$-glutamylcysteine synthetase from wild type and Cd tolerant cell lines might contribute to altered activity of this enzyme in selected cells. Arabidopsis cad2 mutants, which are Cd sensitive and deficient in GSH, were transformed with the tomato GSH1 gene and this partially restored the wild type phenotype. Complementation of the cad2 mutant with tomato GSH1 cDNA under the control of the 35S promoter provides evidence that $\gamma$-GluCys synthetase is critical for Cd tolerance in plants. Cd tolerance, measured by root growth and biomass accumulation, was restored to some variable degree in these transformants. There was a negative correlation between the number of copies of the tomato GSH1 gene inserted in the transformants and the level of Cd tolerance. Overall, these results support the hypothesis that an increased capacity to synthesize glutathione, and therefore phytochelatins, might be used to increase the tolerance of plants to cadmium.

Degree

Ph.D.

Advisors

Goldsbrough, Purdue University.

Subject Area

Molecular biology|Botany|Biochemistry|Agronomy

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