Identification of C-terminal Regions in Arabidopsis thaliana Phytochelatin Synthase 1 Specifically Involved in Activation by Arsenite

Phytochelatins (PCs) are major chelators of toxic elements including inorganic arsenic (As) in plant cells. Their synthesis confers tolerance and influences within-plant mobility. Previous studies had shown that various metal/metalloid ions differentially activate PC synthesis. Here we identified C-terminal parts involved in arsenite- [As(III)] dependent activation of AtPCS1, the primary Arabidopsis PC synthase. The T-DNA insertion in the AtPCS1 mutant cad1-6 causes a truncation in the C-terminal regulatory domain that differentially affects activation by cadmium (Cd) and zinc (Zn). Comparisons of cad1-6 with the AtPCS1 null mutant cad1-3 and the double mutant of tonoplast PC transporters abcc1/2 revealed As(III) hypersensitivity of cad1-6 equal to that of cad1-3. Both cad1-6 and cad1-3 showed increased As distribution to shoots compared with Col-0, whereas Zn accumulation in shoots was equally lower in cad1-6 and cad1-3. Supporting these phenotypes of cad1-6, PC accumulation in the As(III)-exposed plants were at trace level in both cad1-6 and cad1-3, suggesting that the truncated AtPCS1 of cad1-6 is defective in PCS activity in response to As(III). Analysis of a C-terminal deletion series of AtPCS1 using the PCS-deficient mutant of fission yeast suggested important regions within the C-terminal domain for As(III)-dependent PC synthesis, which were different from the regions previously suggested for Cd- or Zn-dependent activation. Interestingly, we identified a truncated variant more strongly activated than the wild-type protein. This variant could potentially be used as a tool to better restrict As mobility in plants.