Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge

Abstract

AbstractRedox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as proteinS-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyseS-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processesin vivois still unknown. We generate knockout lines ofGRXC5as the only chloroplast class I GRX of the mossPhyscomitrium patens.While we find that class I PpGRXC5 has high activities in glutathione-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 (roGFP2) as substratesin vitro, Δgrxc5plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromalEGSH(Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys oxidation and decreased reduction rates after oxidative challenge in Δgrxc5plantsin vivo, indicating kinetic uncoupling of the protein Cys redox state from glutathione redox potential. Protein Cys disulfide andS-glutathionylation formation rates after H2O2treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation.Our observations suggest specific roles for class I GRX in the efficient redox equilibration betweenEGSHand protein Cys in the plastid stroma as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function as efficient kinetic couplers of protein Cys redox state to the dynamic stromalEGSHand highlight the importance of identifyingin vivotarget proteins of GRXC5.One sentence summaryRemoval of class I GRX activity in the chloroplast stroma ofP. patenskinetically uncouples GRX-dependent cysteine redox changes from the local glutathione redox potential, without an effect on NPQ or photosynthetic carbon reactions.