Proteomic characteriaztion of the high light response of Calredoxin mutants (C. reinhardtii)

Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas reinhardtii. To further elucidate the function of CRX, we performed in-depth quantitative proteomics taking advantage of a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, CRX rescues and wild type strains. These analyses revealed that the chloroplast NADPH-dependent TRX reductase (NTRC) is co-regulated with CRX. Electron transfer measurements revealed that CRX inhibits NADPH-dependent reduction of oxidized chloroplast 2-Cys peroxiredoxin (PRX1) via NTRC and that the function of the NADPH-NTRC complex is under strict control of CRX. Our data also demonstrated via non-reducing SDS-PAGE assays and redox proteomics that PRX1, Transketolase (TRK1), ATPC and Proton Gradient Related like 1 (PGRL1) are more oxidized under high light (HL) conditions in the absence of CRX. The redox tuning of PRX1 and control of the NADPH-NTRC complex via CRX interconnects redox control with active photosynthetic electron transport and metabolism as well as Ca2+ signaling. In this way, an economic use of NADPH for PRX1 reduction is assured. The finding, that in the absence of CRX, light-driven CO2 fixation is severely inhibited under HL conditions underpins the importance of CRX for redox tuning as well as for efficient photosynthesis.