Ángeles Aroca, Cecilia Gotor, Luis C. Romero New insights into protein S-Sulfhydration: A large-scale proteomic study using the Tag Switch Method in Arabidopsis Ángeles Aroca, Cecilia Gotor, Luis C. Romero Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas y Universidad de Sevilla, Sevilla, Spain. ABSTRACT Emerging data in recent years suggest that H2S may function as an important signaling molecule in plant systems (Gotor et al., 2015; Kimura, 2015). With regard to certain stresses, H2S treatment alleviates the inhibitory effect of several abiotic stresses. H2S also plays a role in the regulation of drought stress and has been described as a component of the abscisic acid signaling network in guard cells (Scuffi et al., 2014; Papanatsiou et al., 2015). Moreover, H2S has been shown to modulate photosynthesis through the promotion of chloroplast biogenesis, photosynthetic enzyme expression, and thiol redox modification. Protein S-sulfhydration of the thiol residue of cysteines has been proposed as a mechanism for transforming the sulfide signal into a biological response and recently we have reported the detection of protein S-sulfhydryl modification in plants by using a modification of the biotin switch technique (Aroca et al., 2015). However, we have improved the detection method by using a tag-switch technique that can selectively detect persulfide adducts labelled with a biotin-linked cyanoacetate (CN-biotin) as reporting molecule (Zhang et al., 2014). With this new method, we have increased the number of detected S-sulfhydrated proteins from 106 to 2015 proteins, which are involved in functional processes mainly related with primary metabolism. We have performed a functional analysis of one of these proteins, the cytosolic isoform C of the glyceraldehyde 3-phosphate dehydrogenase enzyme, both in wild type and des1 mutant defective in the generation of sulfide in the cytosol. We have clearly observed that S-sulfhydration alters the cytosolic/nucleic subcellular location of the protein. Figure 1. Schematic illustration of the procedure for identification and quantification of S-sulfhydrated proteins in Arabidopsis leaves isolated by Tag Switch Method. Figure 5. Hydrogen sulfide mediates GAPDH nuclear accumulation. (A) Western blot analysis for the cytosolic GAPDH GapC protein of enriched nuclei and cytosolic extracts isolated from 30-days-old Arabidopsis wild type leaves. (B) Quantitation of the sum of bands from the enriched nucleus extract with respect to Sypro Ruby staining as loading control. Data shown are means ±SD of three independent measures ANOVA was performed and significant differences are indicated by the letter a, b and c (P < 0.05). Figure 2. Gene Ontology classification of the 2018 locus identifiers corresponding to the S-sulfhydrated proteins identified by the CN-Biotin Tag Switch Method. The graphs shows the functional categorization of the loci by annotation for GO Biological Process (A) and Molecular function (B). The last graph shows that only 5% of identified proteins were previously described by other methods (C). Figure 4. Subcellular localization of cytosolic GAPDH isoforms by stable expression of GFP-GAPDH fusion proteins in Arabidopsis wild type and des1 mutant leaves of 30-days old plants. Arrows indicate nuclei. Glycolysis and pentose-phosphate shunt tRNA aminoacylation Abiotic stress response Jasmonic acid biosynthesis Figure 3. Enriched Gene Ontology analysis of the 2018 loci corresponding to the 2015 S-sulfhydrated proteins identified by the CN-Biotin Tag Switch Method. The graph was generated by Singular Enrichment Analysis (SEA) from AgriGO (Du et al, 2010). The gragh displays the GO hieratical image of the GO Biological Process categories containing the statistically significant terms. The right side of the graph shows several enlargements of some highly significant terms. RESULTS   Using an improved tag-switch assay for persulfide detection we have analyzed the protein persulfide content in leaf extract from wild-type Arabidopsis thaliana (Figure 1). A total of 2015 S-sulfhydrated proteins were identified with high confidence (FDR < 1%) and Functional Categorized for their GO Biological Process and Molecular Function (Figure 2A and 2B). Almost the half of proteins identified are involved in metabolic and cellular processes, and the third largest group are proteins involved in response to stress. The 95% of the proteins identified by Tag switch assay are newly described while only 5% have been previously identified by other methods (Aroca et al, 2015) (Figure 2C). Enrichment Gene Ontology analysis have revealed several highly significant terms that include a group of proteins involved in jasmonic acid biosynthesis process, an important plant hormone in pathogen response. An enriched subset of proteins involved in tRNA aminoacylation is also shown highlighting the importance of S-sulfhydration in protein translation. Additionally, S-sulfhydration seems to be over represented in primary metabolic process related with cellular energy production such as glucose catabolism and glycolysis and the pentose phosphate shunt (Figure 3). Functional analysis of S-sulfhydration on the cytosolic GAPDH enzyme was studied in wild type and des1 (L-cysteine desulfhydrase 1) mutant line and demonstrated that S-sulfhydration affects the subcellular cytosolic/nuclear localization of the protein (Figures 4 and 5).