A Novel Gamma-Glutamyl Cyclotransferase Protects Plants from Arsenic Toxicity by Efficiently Recycling Glutamate

Plants detoxify arsenic and other thiol-reactive toxins through a glutathione (GSH)-dependent pathway. GSH homeostasis is maintained by the gamma-glutamyl cycle, which involves GSH synthesis and degradation, and the recycling of component amino acids. The enzyme gamma-glutamyl cyclotransferase (GGCT) is involved in glutamate (Glu) recycling. GGCT activity has been reported in plants, but the underlying gene(s) has not been identified. Here, we report that an Arabidopsis protein named GGCT2;1, functions as a g-glutamyl cyclotransferase. Heterologous expression of GGCT2;1 in yeast strains produced phenotypes that were consistent with decreased GSH content due to the diversion of gamma-glutamyl cysteine  (g-EC) from GSH synthesis to 5-oxoproline (5-OP), and significantly increased the 5-OP content. 5-OP levels were further increased by the addition of arsenite and GSH to the medium, indicating that GGCT2;1 participates in the cellular response to arsenic via GSH degradation. Moreover, Recombinant GGCT2;1, expressed in yeast, converted g-glutamyl alnine to 5-OP in vitro. GGCT2;1 transcripts were upregulated in arsenic-treated Arabidopsis, and ggct2;1 knock-out mutants were more tolerant to arsenic and cadmium, but not to nickel, than the wild type. The constitutive overexpression of GGCT2;1 in Arabidopsis resulted in the accumulation of 5-OP. Isotopic labelling studies on metabilites of the nitrogen assimilation pathway confirmed efficient Glu recycling. Thus, our results suggest that GGCT2;1 ensures sufficient GSH turnover during abiotic stress.