Calredoxin (CRX) is a calcium (Ca2+)-dependent thioredoxin (TRX) in the chloroplast of Chlamydomonas (Chlamydomonas reinhardtii) with a largely unclear physiological role. We elucidated the CRX functionality by performing in-depth quantitative proteomics of wild-type cells compared with a crx insertional mutant (IMcrx), two CRISPR/Cas9 KO mutants, and CRX rescues. 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. Via non-reducing SDS-PAGE assays and mass spectrometry, our data also demonstrated that PRX1 is 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 interconnect 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 ensured. The finding that the absence of CRX under HL conditions severely inhibited light-driven CO2 fixation underpins the importance of CRX for redox tuning, as well as for efficient photosynthesis.

25 ng of labelled and digested NTRC were analysed on an Ultimate 3000 nannoLC (Thermo Fisher Scientific) coupled via a nanospray interface to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Mobile phases consisted of 0.1% formic acid in ultrapure water (A) and 0.1% formic acid/80% acetonitrile in ultrapure water (B). Peptide separation was carried out on a PepsSep 15 reversed-phase column (75 µm x 15cm, 1.9 µm particle size, 100 Å pore size; Bruker). The gradient applied was as follows: 5-40 % B over 30 min, 40-99 % over 2 min, 99% B over 5 min. Two rounds of sample analysis were performed. In the first round, samples were analysed in conventional DDA mode (see above). A spectral library was constructed using BiblioSpec in Skyline 22.2.0.351 (Frewen and MacCoss, 2007; MacLean et al., 2010) by making use of peptide identifications obtained by searching spectra files with MSFragger (Kong et al., 2017) against a concatenated database consisting of the protein sequences of recombinant NTRC and common contaminants (Frankenfield et al., 2022), as well as the E. coli proteome (Uniprot UP000000625). Carbamidomethylation, vinylpyridination and trioxidation of cysteine, oxidation of methionine, and acetylation of protein N-termini were allowed as variable modifications. Using the spectral library, an inclusion list of NTRC peptides was generated, and samples were analysed again, this time in PRM-mode (parallel reaction monitoring) for targeted quantification (120 ms maximum injection time, AGC target 1e5, isolation window 2 m/z, isolation offset 0.5 m/z, resolution 35,000). Raw files acquired in PRM mode were loaded in Skyline for peptide identification and peak integration. By default, a minimum of six fragment ions per peptide were used for quantification. For positive peptide identification, a peptide dotp of >0.9 was required. If minimum requirements for identification and quantification were satisfied in at least one replicate, the presence of at least three fragment ions with a maximum retention time shift of 30 s and a dotp of 0.7 were considered sufficient for quantification. Data normalization was performed using the “global standards” method, i.e., the ratio of the total peak area of a modified peptide and the sum of the total peak areas of two NTRC peptides lacking cysteine (ANLKPVVFEGFR, LVAGQVELDEAGYVK) was calculated.

Recombinant NTRC was incubated alone or with 10 times more recombinant CRX or TRX f in 30 mM MOPS, 100 mM KCl, 2 mM NADPH at a free Ca2+ concentration of 0 µM for 10 min. 50 µg of protein mixtures were labelled with 40 mM chloroacetic acid (CAA) at room temperature for 1 h. After removal of residual CAA by washing four times with 50 mM ammonium bicarbonate (ABC) in centrifugal filter devices (Amicon Ultra 30K, 0.5 ml, Merck), denaturation and reduction of non-labelled cysteines were achieved by adding 10 mM TCEP in 8 mM Urea, 100 mM Tris/HCl, pH 8.5 and incubation for 1 h at 25°C. The same conditions were applied for subsequent cysteine labelling with 4-vinylpyridine (VP, 50 mM) labelling. After four washes with 50 mM ABC to remove excess VP, samples were tryptically digested and prepared for MS analysis following the FASP protocol