A Novel Gamma-Glutamyl Cycle and Its Role in Mediating Oxidative Stress and Glutamate Recycling Via Maintaining Glutathione Homeostasis.

A novel gamma-Glutamyl Cycle and its Role in Mediating Oxidative Stress and Glutamate Recycling via Maintaining Glutathione Homeostasis Om Parkash Dhankher, Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003 Bibin Paulose, Stockbridge School of Agriculture, University of Massachusetts Amherst and Department of Plant Biology, Michigan State University, East Lansing, MI Sudesh Chhikara, Stockbridge School of Agriculture, University of Massachusetts Amherst Glutathione (GSH) is the critical redox buffers in most aerobic cells and plays important roles in protecting cells from oxidative stress caused by exposure to both abiotic and biotic stresses. The g-glutamyl cycle maintains GHS homeostasis, which involves GSH synthesis and degradation, and the recycling of the component amino acids. GSH biosynthesis steps are well characterized in plants and overexpression of GSH synthesis pathway genes increases the levels of GSH and enhanced tolerance to toxic metals. However, GSH degradation pathways are not fully understood in plants. Recently, we have identified a small gene family encoding ?-glutamyl cyclotransferases, GGCTs (GGCT1, GGCT2;1 and GGCT2;2) in Arabidopsis, which are involved in the g-glutamyl cycle required for maintaining GSH homeostasis via recycling Glu, a key nitrogen-storing amino acid. qRT-PCR studies showed the upregulation of GGCT transcripts by heavy metals and other abiotic stressors including ABA, mannitol and salt. Heterologous expression of Arabidopsis GGCT2;1 in the mutant yeast strain DTY, lacking the YCF1 vacuolar transporter and an arsenite efflux pump ACR3, resulted in a significantly higher accumulation of 5-oxoproline (5-OP), a precursor of glutamate (Glu) under arsenite stress. The recombinant GGCT2;1 protein was able to convert ?-glutamyl-Ala and GSH to 5-OP in in vitro conditions as well. Manipulation of GGCT genes in Arabidopsis showed enhanced tolerance to multiple abiotic stresses, produced higher levels of Glu precursor 5-oxoproline (5-OP) as a result of GSH degradation. GGCT2;1 T-DNA mutants become severely sensitive to ABA, salt, and mannitol. Our studies suggest that by efficient recycling of Glu as part of the g-glutamyl cycle, GGCT2;1 decreased the de novo synthesis of Glu, thereby decreasing the nitrogen requirement. Therefore, GSH homeostasis plays important roles in maintaining cellular redox for protection of plants under abiotic stresses and holds great potential for developing ÔClimate Resilient CropsÓ with decreased N requirements.