Identification and characterization of chloroplast protein phosphatases involved in regulation of photosynthesis in Chlamydomonas reinhardtii

Abstract

The aim of this thesis was to provide novel information about the regulation of photosynthetic electron transfer in the green alga Chlamydomonas reinhardtii, with a focus on the regulation of thylakoid protein phosphorylation. In Arabidopsis, two antagonist pairs of protein kinases and phosphates are known. The kinase STN7 and the phosphatase PPH1 act on light-harvesting antennae proteins in regulating state transitions, while the kinase STN8 and the phosphatase PBCP act on PSII core subunits. In this project, the thylakoid kinase/phosphatase network in Chlamydomonas is explored. As yet, in Chlamydomonas only the STN7 homologue, STT7, was known. Within this work three chloroplast phosphatases regulating the photosynthetic electron transfer chain are newly identified and characterized: CrPPH1, CrPBCP and CrPBCP-LIKE. The thesis begins with a general introduction on the photosynthetic electron chain and its regulation, with a particular focus on the role of thylakoid protein phosphorylation and the known kinases and phosphatases involved. Phosphorylation of the light-harvesting antenna regulates the allocation of light energy to the two photosystems, in a process known as state transitions. Phosphorylation of the light-harvesting antenna favours its binding to PSI (state 2) while its de-phosphorylation favours its association with PSII (state 1). In the first chapter of the results the roles of CrPPH1 and CrPBCP are investigated, specifically during state transitions. Like its homologue in Arabidospis, CrPPH1 is required for efficient de-phosphorylation of the light-harvesting antenna and transition from state 2 to state 1. However CrPBCP is also involved in these process, unlike PBCP in Arabidopsis. In Chlamydomonas the two phosphatases have partly redundant roles, reflected in the partial overlap of their substrate specificities for the subunits of the light-harvesting antenna. The second chapter of the results presents the characterization of CrPBCP-LIKE, specifically under high light. This phosphatase, which is closely related to CrPBCP, is not required for state transitions, but plays a role in acclimation to high light and photoprotection. The third chapter of the results begins with a review of the biochemical approaches used to study the structural reorganization of the thylakoid membrane protein complexes during light acclimation processes, with a discussion of some technical issues. An optimized protocol is presented, to isolate thylakoid membranes from Chlamydomonas while preserving the phosphorylation state of thylakoid proteins and to study thylakoid supercomplex organization. Our results indicate that the roles of PPH1 and particularly PBCP homologs in Chlamydomonas are different from those of the plant counterparts, and a different scenario for the regulation of LHCII phosphorylation and the state transition mechanism appear in the green algae. The thesis ends with a general discussion and all the results are put together in a wider perspective.