Persulfidation proteome reveals the regulation of protein function by hydrogen sulfide in diverse biological processes in Arabidopsis

Abstract

Hydrogen sulfide-mediated signaling pathways regulate many physiological and pathophysiological processes in mammalian and plant systems. The molecular mechanism by which hydrogen sulfide exerts its action involves the post-translational modification of cysteine residues to form a persulfidated thiol motif, a process called protein persulfidation. We have developed a comparative and quantitative proteomic analysis approach for the detection of endogenous persulfidated proteins in wild-type Arabidopsis and L-CYSTEINE DESULFHYDRASE 1 mutant leaves using the tag-switch method. The 2015 identified persulfidated proteins were isolated from plants grown under controlled conditions, and therefore, at least 5% of the entire Arabidopsis proteome may undergo persulfidation under baseline conditions. Bioinformatic analysis revealed that persulfidated cysteines participate in a wide range of biological functions, regulating important processes such as carbon metabolism, plant responses to abiotic and biotic stresses, plant growth and development, and RNA translation. Quantitative analysis in both genetic backgrounds reveals that protein persulfidation is mainly involved in primary metabolic pathways such as the tricarboxylic acid cycle, glycolysis, and the Calvin cycle, suggesting that this protein modification is a new regulatory component in these pathways.

Keywords: Cysteine; hydrogen sulfide; mass spectrometry; persulfidation; post-translational modification; proteomics.

Fig. 1.

Schematic illustration of the procedure to identify and quantify persulfidated proteins in Arabidopsis leaves using the tag-switch method.

Fig. 2.

Functional classification of persulfidated proteins identified by LC-MS/MS in leaf extracts of 30-day-old Arabidopsis wild-type plants. (A) Functional classification of gene ontology (GO) terms categorized by biological processes. (B) Functional classification by subcellular localization.

Fig. 3.

Persulfidated proteins in the plant glycolysis pathway. Red squares represent persulfidated proteins. Cytosolic, GapC1 and GapC2, chloroplastic, GapA1, GapA2, and GapB, and NADP-dependent isoforms of glyceraldehyde-3-phosphate dehydrogenase are highlighted in blue.

Fig. 4.

Functional characterization of proteins quantitatively regulated by persulfidation. (A) Functional distribution of proteins differentially regulated by persulfidation in des1 and wild-type plants. (B) Singular enrichment analysis performed with AgriGO to identify enriched gene ontologies associated with proteins negatively regulated by persulfidation in des1 mutants in comparison with persulfidation patterns in wild-type plants. Box colors indicate levels of statistical significance: yellow, 0.05; orange, e−5; red, e−9.

Fig. 5.

Comparison of S-nitrosylated proteins and persulfidated proteins identified in Arabidopsis plants. (A) Venn diagram of total S-nitrosylated identified proteins in wild-type and gsnor1-3 mutant (Hu et al., 2015) and of total persulfidated identified proteins in wild-type and des1 mutant in this work. (B) Functional classification of gene ontology (GO) terms categorized by biological processes.