Client distribution between Chlamydomonas FDX1 and FDX2 in carbon, nitrogen and sulfur assimilation

Plant-type ferredoxins (Fd) comprise small, soluble protein families that distribute electrons from photosystem I to various client proteins within the chloroplast stroma. In Chlamydomonas reinhardtii, the major, constitutively expressed FDX1/PetF supports Fd-NADP⁺ reductase (FNR) in NADPH production. The highly similar FDX2 is present only when its preferred nitrogen (N) source ammonium is absent, supplying Fd-dependent nitrite reductase (NiR) for nitrate/nitrite assimilation. Surprisingly, despite accumulating to ~10% of FDX1 abundance and preferential interaction with NiR, fdx2 mutants are asymptomatic when grown on nitrate, requiring to additionally deplete FDX1 for growth to be halted. A fdx1 knockout itself appears lethal, severe fdx1 knockdowns have reduced growth rates both in phototrophic and photoheterotrophic conditions, independent of the N source. Transcriptome analyses of fdx1 mutants revealed expression patterns similar to sulfur (S) deficient algae, and fdx1 strains have a reduced total cellular S content. S assimilation requires Fd-dependent sulfite reductase (SiR) activity, an enzyme distantly related to FDX2 client NiR. Expression defects are partially alleviated; growth and S content are less impacted with FDX2 expression. Our mutant analysis shows the two major Fds in Chlamydomonas focus on a specific subset of Fd-dependent metabolism, mostly supplying Fd-dependent enzymes involved in macronutrient assimilation (C/N/S).