Regulation of thylakoid protein phosphorylation during state transition and photosystem II supercomplex mobilization in rice

Abstract

Recent research identified the role of STN8 kinase for phosphorylation of PSII core proteins such as D1, D2, CP43, PsbH, TSP9, CaS and STN7 kinase for not only phosphorylation of LHCII protein but also PSII core proteins and state transition in Arabidopsis. However complete inhibition of PSII core protein phosphorylation was only observed in the Arabidopsis stn7/stn8 double mutant. Hence, the elucidation of specific roles of STN8 and STN7 kinases has been identified by using T-DNA insertional knock-out mutant of the rice (Oryza sativa) STN8 gene, osstn8, STN7 gene, osstn7 and PPH phosphatase gene, ospph. Here, osstn8 and osstn7 mutants has been used to clarify the role of PSII-LHCII protein phosphorylation in regulation of PSII repair cycle, mobilization of PSII supercomplex, state transition. In the osstn8 mutant, PSII core protein phosphorylation (PCPP) was significantly suppressed and the grana were thin and elongated. During high light (HL) illumination, PSII was strongly inactivated in the osstn8 mutants, yet the D1 protein was degraded more slowly than in wild type, and the mobilization of the PSII supercomplex from the grana to stroma lamellae for repair was also suppressed. Taken together, these data show that the absence of STN8 gene is sufficient to abolish PCPP in osstn8 mutants to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8-mediated PCPP in PSII repair. Using both histochemical and fluorescence probes, ROS production, including superoxide and hydrogen peroxide production was increased in osstn8 mutant’s leaves during HL illumination. When superoxide dismutase was inhibited, superoxide production was increased, indicating that superoxide production is the initial event prior to hydrogen peroxide production. However, singlet oxygen production was not different between WT and osstn8. In addition, PSII-driven superoxide production was more in the thylakoid membranes as well as in isolated PSII and PSII-LHCII supercomplex of osstn8. PSII and PSII-LHCII supercomplex of osstn8 leaves showed strong oxidation of proteins under HL illumination. These results suggest that superoxide is the major form of ROS produced in the osstn8 mutant, and that the damaged PSII in the supercomplex is the primary source of superoxide production during HL illumination. Apart from this, the specific kinase and phosphatase for reversible phosphorylation of CP29 (CP29-P) was still unknown. Therefore T-DNA insertion knock-out mutant of STN7 and STN8 kinase as well as PPH phosphatase mutants were used to confirm it. CP29-P was observed in WT, osstn8 complement, osstn7 and ospph mutant but not in osstn8 mutant during high light and light chilling treatment. However dephosphorylation of CP29-P was observed in all genotypes during dark incubation. The phosphorylation and dephosphorylation of LHCII protein was blocked in osstn7 mutant and ospph mutant, as a consequence, state transition was strongly impaired. Mobilization of PSII supercomplex was observed in osstn7 mutant during high light illumination but it was suppressed in ospph mutant. This result proposed that dephosphorylation of LHCII protein was also involved for mobilization of PSII supercomplex. In control condition, the relative change in maximal fluorescence during state transition, ∆F was similar in WT and osstn8 mutant and mobilization of PSII supercomplex was not observed but ∆F become negligible in osstn8 during high light illumination and mobilization of PSII supercomplex was only observed in WT. Upon high light illumination, CP29-P was observed in PSI complex in WT but not in osstn8 mutant. Therefore it is concluded that lack of CP29-P also impaired high light induced state transition in osstn8 mutant of rice.