Characterization of Ser352 phosphorylation site in Arabidopsis phytochelatin synthase

• Main Authors: Yan-Hung Chen, 陳彥宏
• Other Authors: 莊榮輝
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/69842648528242915374

碩士 === 國立臺灣大學 === 生化科技學系 === 103 === Phytochelatin synthase (PCS, EC2.3.2.15) plays important roles in the sequestration of heavy metals in Arabidopsis thaliana. It uses two substrates, GSH and Cd-GS2, to synthesize phytochelatin (PC) via ping-pong mechanism. PC chelates heavy metals and forms a high molecular complex, which is then transferred to vacuoles to prevent from damaging the cells. PCS composes of a catalytic domain at N-terminus and a Cys-rich C-terminal domain which is responsive to cadmium (Cd). Many studies show that PCS is constitutively expressed in cytosol but could be only activated under heavy metal stress. The phenomenon indicates that PCS may be regulated by post-translational modification. Our previous data have demonstrated that recombinant AtPCS1 could be activated by in vitro phosphorylation. Furthermore, phosphorylation at Ser352 of recombinant AtPCS1 might affect the catalysis of the enzyme. Ser352 is close to Cys358Cys359, which is a heavy-metal binding site of the C-terminal domain. This study showed that mutation at Ser352 decreased the catalytic activity of PCS while increased the Cd binding ratio of the enzyme. Moreover, Recombinant AtPCS1 with a mutation at Ser352 showed a lower catalytic activity because of the significantly reduced Vmax along with the higher affinities to both substrates, which suggested the role of Ser352 in activation of PCS. On the other hand, this study selected 6 Arabidopsis transgenic lines that express AtPCS1 and AtPCS1-S352A in a PC-deficient mutant, cad1-3. Although S352A has a low catalytic activity in vitro, AtPCS1-S352A transgenic lines showed no significant changes in Cd tolerance comparing to the wild type Arabidopsis. Thus, we speculated that partially-activated PCS could synthesize enough PC to rescue the Cd tolerance of the transgenic lines, and the PCS might also act as a chelator to bind heavy metals in cytosol.