CBSX2 is required for the efficient oxidation of chloroplast redox-regulated enzymes in darkness.

Thiol/disulfide-based redox regulation in plant chloroplasts is essential for controlling the activity of target proteins in response to light signals. One of the examples of such a role in chloroplasts is the activity of the chloroplast ATP synthase (CF o CF 1 ), which is regulated by the redox state of the CF 1 γ subunit and involves two cysteines in its central domain. To investigate the mechanism underlying the oxidation of CF 1 γ and other chloroplast redox-regulated enzymes in the dark, we characterized the Arabidopsis cbsx2 mutant, which was isolated based on its altered NPQ (non-photochemical quenching) induction upon illumination. Whereas in dark-adapted WT plants CF 1 γ was completely oxidized, a small amount of CF 1 γ remained in the reduced state in cbsx2 under the same conditions. In this mutant, reduction of CF 1 γ was not affected in the light, but its oxidation was less efficient during a transition from light to darkness. The redox states of the Calvin cycle enzymes FBPase and SBPase in cbsx2 were similar to those of CF 1 γ during light/dark transitions. Affinity purification and subsequent analysis by mass spectrometry showed that the components of the ferredoxin-thioredoxin reductase/thioredoxin (FTR-Trx) and NADPH-dependent thioredoxin reductase (NTRC) systems as well as several 2-Cys peroxiredoxins (Prxs) can be co-purified with CBSX2. In addition to the thioredoxins, yeast two-hybrid analysis showed that CBSX2 also interacts with NTRC. Taken together, our results suggest that CBSX2 participates in the oxidation of the chloroplast redox-regulated enzymes in darkness, probably through regulation of the activity of chloroplast redox systems in vivo.