Activity Analysis of Thr 49 Mutants of AtPCS1 and Cadmium Tolerance of AtPCS1 Complementary Transgenic Arabidopsis

Phytochelatin synthase (PCS, EC2.3.2.15) plays important roles in the sequestration of heavy metals in plants. Previous studies demonstrated that phytochelatin synthase (PCS, EC 2.3.2.15) may be regulated at post-translational modifications. By using mutagenesis and E. coli expression system, we had demonstrated that phosphorylation on Thr 49 might play an important role in the formation of the active site through the interaction with Arg183. In order to reveal the physiological role of Thr 49 and Arg 183, we constructed pCAMBIA vectors containing AtPCS1 promoter sequence and its cDNA and selected several transgenic lines that expressed normal or mutated AtPCS1. The expression level and the copy number of AtPCS1 (T49A, T49E) was determined by semi-quantitative RT-PCR, western blotting, and nested IPCR. At modest Cd concentrations, wild type Arabidopsis (ecotype Columbia-0) and AtPCS1 complementary lines were more tolerant to Cd that AtPCS1-T49A and AtPCS1-T49E complementary lines. These data indicated that Thr 49 is important for Arabidopsis to resist the presence of Cd.