Sulfenome mining in Arabidopsis thaliana

Abstract

Reactive oxygen species (ROS) have been shown to be potent signaling molecules. Today, oxidation of cysteine residues is a well-recognized posttranslational protein modification, but the signaling processes steered by such oxidations are poorly understood. To gain insight into the cysteine thiol-dependent ROS signaling in Arabidopsis thaliana, we identified the hydrogen peroxide (H2O2)-dependent sulfenome: that is, proteins with at least one cysteine thiol oxidized to a sulfenic acid. By means of a genetic construct consisting of a fusion between the C-terminal domain of the yeast (Saccharomyces cerevisiae) AP-1-like (YAP1) transcription factor and a tandem affinity purification tag, we detected ∼ 100 sulfenylated proteins in Arabidopsis cell suspensions exposed to H2O2 stress. The in vivo YAP1-based trapping of sulfenylated proteins was validated by a targeted in vitro analysis of dehydroascorbate reductase2 (DHAR2). In DHAR2, the active site nucleophilic cysteine is regulated through a sulfenic acid-dependent switch, leading to S-glutathionylation, a protein modification that protects the protein against oxidative damage.

Keywords: cysteine oxidation; oxidative stress; redox regulation.

Fig. 1.

Dose- and time-dependent formation of YAP1C-involving complexes. (A) Cell cultures overproducing the YAP1C/YAP1A-GS probe treated with 0, 0.1, 1, 5, 10, and 20 mM H₂O₂ for 1 h. Complexes (marked with an arrow) are visualized with the PAP antibody complex. The H₂O₂ concentration and the signal intensity clearly correlate. Treatment of protein samples with 50 mM tris(2-carboxyethyl)phosphine led to reduction of the complexes. (B) Cell cultures treated with 1 mM H₂O₂. The time course was taken after 0, 5, 10, 30, 60, and 120 min. The initial signal intensity peak returns to a near basal level after 120 min of treatment. The asterisk denotes an unknown protein recognized by the antiserum. (C and D) Schematic comparison of datasets identified after treatment of cultures with 0, 1, and 20 mM H₂O₂ for 1 h (C) and 1 mM H₂O₂ for 10 min (early response) and 1 h (late response) (D).

Fig. 2.

Experimental set-up for in vivo identification of the Arabidopsis sulfenome. (A) Cell cultures overproducing the YAP1C/YAP1A probes treated with H₂O₂ as described (see main text). (B and C) Proteins isolated and subjected to a two-step purification procedure based on IgG-protein G and SBP affinity. Numbers indicate the sequence of elution steps. (D) LC-MS/MS analysis of proteins after elution. Comparison of interactors between the negative control probes of YAP1C and YAP1A potentially undergoing cysteine sulfenic acid (-SOH) formation under oxidative stress.

Fig. 3.

Requirement of DHAR2 cysteines and GSH protection against overoxidation. (A) Mean initial velocities ± SD of three independent measurements of DHAR2 determined on progress curves under different conditions. The reaction was started under V_max conditions. (B) Dimedone labeling of DHAR2-SOH in vitro. DHAR2 (20 µM) nontreated or treated with 1 mM GSH either not or incubated with 100 µM H₂O₂ in the presence or absence of dimedone (1 mM). DHAR2-SOH formation was analyzed by immunoblot with an anticysteine sulfenic acid antibody. (C) Identification of the dimedone modification Cys20 of DHAR2. The LC-MS/MS spectrum shows data obtained from a +2 parent ion with m/z 935.5. The cysteine residue corresponds to a dimedone-modified sulfenic acid, which produces a +138-Da mass increment.