In vivo detection of protein cysteine sulfenylation in plastids.

Protein cysteine thiols are post-translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H 2 O 2 ) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation-sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C-trapping technology to the plastids of light-grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H 2 O 2 pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H 2 O 2 treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle-tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.