Molecular study of assembly proteins required for photosystem biogenesis in Chlamydomonas reinhardtii

Abstract

Photosystem is a multi-protein complex where absorbs light energy and transports electron for photosynthesis. The polypeptides of this complex are accurately transported and assembled to the thylakoid membranes via various nuclear-encoded assembly proteins identified in higher-plants. However, a large part of the transport and assembly for photosystem is still unrevealed in microalgae. In this study, to reveal in vivo function of proteins involved in the transport and assembly of photosystem in green microalgae Chlamydomonas reinhardtii, specific gene knockout mutants were generated by both forward and reverse genetic approaches (random insertional mutagenesis and CRISPR-Cas9 method), and their phenotypic characteristics were analyzed. First, to investigate proteins taking part in the transport and assembly of light-harvesting chlorophyll- and carotenoid-binding protein (LHCP) composing of light-harvesting antenna complex in photosystem, two putative components in chloroplast signal recognition particle (CpSRP) pathway, CpSRP54 and LTD, were targeted. As already reported in A. thaliana, both CpSRP54 knockout mutant tla4 and LTD knockout mutant Crltd showed reduced LHCP contents, indicating CpSRP54 and LTD functions in LHCP assembly in C. reinhardtii. Despite this similarity, a structural difference showed in the 3D modeling of CpSRP54 between A. thaliana and C. reinhardtii and a specific reduction of LHCI in Crltd mutant give us clues that both CpSRP54 and LTD proteins may have a limited role in microalgae. Next, the single and double knockout mutants for LPA2 and LPA3 (Crlpa2, Crlpa3 and Crlpa2/3) were generated to elucidate in vivo functions of putative LPA2 and LPA3 proteins, which were predicted to be involved in assembly of CP43 into photosystem II (PSII) core complex in C. reinhardtii. All mutants displayed a significant reduction in PSII core complex, particularly CP43. This means that LPA2 and LPA3 proteins are involved in CP43 assembly in C. reinhardtii as in A. thaliana. Some pleiotropic effects (increase of total chlorophyll and LHCI levels) were observed in the C. reinhardtii mutants, raising the possibility that a PSII reduction may lead the accumulation of other photosynthetic apparatus in C. reinhardtii. In conclusion, CpSRP54, LTD, LPA2, and LPA3 proteins of C. reinhardtii play a conserved role in the transport and assembly of photosystem. However, the detailed phenotypes of C. reinhardtii knockout mutants exhibited several differences from those in higher-plants, supporting that microalgae can establish a unique mechanism originated from the ancestor of the green lineage to adapt for their specific habitat.