Poster: Heavy Metals

Abs # 109: Coordinate overexpression of enzymes involved in biosynthesis of phytochelatins significantly enhance resistance to arsenic and mercury but hypersensitive to cadmium

Presenter:	Li, Yujing , yujingli@arches.uga.edu
Authors	Li, Yujing  (A)   Dhankher, Om P (A)   Balish, Rebecca S (A)   Meagher, Richard B (A)
Affiliations:	(A): The University of Georgia

Three enzymes are required to catalyze three successive reactions for biosynthesis of phytochelatins which are believed to play very important roles in resistance to thiol-reactive heavy metals/metalloids. These enzymes include gamma-glutamylcysteine synthetase (gamma-ECS), glutathione synthetase (GS), and phytochelatin synthase (PCS). Engineered plants overexpressing gamma-ECS or GS or PCS alone perform enhanced resistance to arsenic and mercury and hypersensitivity to cadmium. However, manipulation of single gene could play limited role for the metabolic engineering. Therefore, coordinate overexpression of multiple proteins is indispensable in metabolic engineering. Using classic genetic approach, three enzymes were coordinately overexpressed in Arabidopsis using a constitutive actin2 promoter (ACT2p) and a light regulated soybean rubisco small subunit promoter (SRS1p). The resistance to arsenic and mercury was significantly improved in the engineered plants coordinately expressing the three enzymes as compared to WT and the transgenic lines that overexpress gamma-ECS or GS or PCS alone. Obviously, the classic genetic approach has some disadvantages such as time-consuming, , negative impact of position effect in the genome, and gene silencing. Theoretically, mRNA polycistrons were an ideal tool to deal with the shortcomings. In this study, some internal ribosome entry sites (IRESs) previously identified in genomes of virus animal and humans were used to construct polycistrons with PCS gene in the first, gamma-ECS in the second and GS in the third cistronic position. Characterization of Arabidopsis plants engineered with these constructs, including the protein levels, quantification of non-protein thiols and metals/metalloids resistance assays, are in process.