Production of γ-ECS antibody and homeostasis of GSH in Arabidopsis

Abstract

Glutathione (GSH) plays a crucial role in plant sulfur metabolism and antioxidant defenses. The physiological functions of the tripeptide, [Gamma]- glutamylcysteinylglycine, include maintaining the ascorbate pool, detoxifying many organic contaminants, and protecting plants from oxidative stress. GSH is synthesized by a two-step ATP-dependent reaction. The first reaction forms [Gamma]- glutamylcysteine ([Gamma]-EC) by the enzyme [Gamma]-EC synthetase ([Gamma]-ECS) and this reaction is the rate-limiting step. The second reaction is catalyzed by GSH synthetase. Previous works done by Dr. Chengbin Xiang showed that glutathione metabolic genes coordinately responded to heavy metals and jasmonic acid in Arabidopsis. However, H202 treatment raised the GSH concentration but did not increase the level of mRNAs for GSH synthesis. This suggests that an additional translational control mechanism was functioning. Antibody against [Gamma]-ECS was prepared to detect the actual [Gamma]-ECS protein level in response to heavy metal, JA, and H202. Dr. Xiang's work also showed that 5' untranslated region of the gene encoding [Gamma]ECS, gshl, might be involved in regulating [Gamma]-ECS expression, so a construct was made with the 5'-UTR of gsh1 placed upstream GUS gene. Arabidopsis was transformed with this plasmid and homozygous lines were obtained. By looking at the GUS response to JA/H2O2/heavy metal, we hope to unveil the function of 5'-UTR.