Importance of cysteine residues in the C-terminal region of PGRL1 for PSI photoprotection in <i>Chlamydomonas reinhardtii</i>

Photoinhibition is more pronounced in photosystem II (PSII) than in PSI. However, PSI is sensitive to photoinhibition in mutants of PROTON GRADIENT REGULATION5 (PGR5) and PGR5-LIKE1 (PGRL1) in both Arabidopsis thaliana and Chlamydomonas reinhardtii. In this study, we observed severe PSI photoinhibition in the PGRL1-deficient mutant (pgrl1) of C. reinhardtii under high light for 1 h. Because PSI photoinhibition was mitigated in pgrl1-complemented strains, PGRL1 was thought to play a crucial role in PSI photoprotection. To elucidate the mechanism of PGRL1-dependent PSI photoprotection, we introduced cysteine-to-serine (CS) substitutions at the six widely conserved cysteine residues (C63, C167, C256, C259, C284, and C287) of PGRL1 in C. reinhardtii. Among the transformants obtained, no CS variants at C256 and C259 accumulated PGRL1. The C63SC167S variant restored the PSI photosensitive phenotype, whereas the CS variants of the C-terminal region (C284SC287S, C284S, and C287S) exhibited PSI photoinhibition similar to pgrl1 and destabilization of PGRL1 under high light. These variants failed to accumulate PGR5 even under growth light. These results suggest that CS substitutions at the C-terminal region affect the proper conformation of PGRL1, leading to its destabilization and disruption of the PGRL1-PGR5 interaction. Based on these findings, we conclude that PGRL1 C284 and C287 play important roles in regulating cyclic electron flow in C. reinhardtii, and consequently, in PSI photoprotection, by stabilizing PGRL1 and enabling PGR5 accumulation.