Analysis of sulfide signaling in rice highlights specific drought responses.

Hydrogen sulfide regulates essential plant processes, including adaptation responses to stress situations, and the best characterized mechanism of action of sulfide consists of the post-translational modification of persulfidation. In this study, we reveal the first persulfidation proteome described in rice including 3443 different persulfidated proteins that participate in a broad range of biological processes and metabolic pathways. In addition, comparative proteomics revealed specific proteins involved in sulfide signaling during drought responses. Several proteins are involved in the maintenance of cellular redox homeostasis, the tricarboxylic acid cycle and energy-related pathways, and ion transmembrane transport and cellular water homeostasis, with the aquaporin family showing the highest differential levels of persulfidation. We revealed that water transport activity is regulated by sulfide which correlates with an increasing level of persulfidation of aquaporins. Our findings emphasize the impact of persulfidation on total ATP levels, fatty acid composition, levels of reactive oxygen species, antioxidant enzymatic activities, and relative water content. Interestingly, the role of persulfidation in aquaporin transport activity as an adaptation response in rice differs from current knowledge of Arabidopsis, which highlights the distinct role of sulfide in improving rice tolerance to drought.

Persulfidation of ATG18a regulates autophagy under ER stress in Arabidopsis.

Hydrogen sulfide (H2S) is an endogenously generated gaseous signaling molecule, which recently has been implicated in autophagy regulation in both plants and mammals through persulfidation of specific targets. Persulfidation has been suggested as the molecular mechanism through which sulfide regulates autophagy in plant cells. ATG18a is a core autophagy component that is required for bulk autophagy and also for reticulophagy during endoplasmic reticulum (ER) stress. In this research, we revealed the role of sulfide in plant ER stress responses as a negative regulator of autophagy. We demonstrate that sulfide regulates ATG18a phospholipid-binding activity by reversible persulfidation at Cys103, and that this modification activates ATG18a binding capacity to specific phospholipids in a reversible manner. Our findings strongly suggest that persulfidation of ATG18a at C103 regulates autophagy under ER stress, and that the impairment of persulfidation affects both the number and size of autophagosomes.

Persulfidation protects from oxidative stress under nonphotorespiratory conditions in Arabidopsis.

Hydrogen sulfide is a signaling molecule in plants that regulates essential biological processes through protein persulfidation. However, little is known about sulfide-mediated regulation in relation to photorespiration. Here, we performed label-free quantitative proteomic analysis and observed a high impact on protein persulfidation levels when plants grown under nonphotorespiratory conditions were transferred to air, with 98.7% of the identified proteins being more persulfidated under suppressed photorespiration. Interestingly, a higher level of reactive oxygen species (ROS) was detected under nonphotorespiratory conditions. Analysis of the effect of sulfide on aspects associated with non- or photorespiratory growth conditions has demonstrated that it protects plants grown under suppressed photorespiration. Thus, sulfide amends the imbalance of carbon/nitrogen and restores ATP levels to concentrations like those of air-grown plants; balances the high level of ROS in plants under nonphotorespiratory conditions to reach a cellular redox state similar to that in air-grown plants; and regulates stomatal closure, to decrease the high guard cell ROS levels and induce stomatal aperture. In this way, sulfide signals the CO2 -dependent stomata movement, in the opposite direction of the established abscisic acid-dependent movement. Our findings suggest that the high persulfidation level under suppressed photorespiration reveals an essential role of sulfide signaling under these conditions.

Hydroxynitrile lyase defends Arabidopsis against Tetranychus urticae.

Plant-pest interactions involve multifaceted processes encompassing a complex crosstalk of pathways, molecules, and regulators aimed at overcoming defenses developed by each interacting organism. Among plant defensive compounds against phytophagous arthropods, cyanide-derived products are toxic molecules that directly target pest physiology. Here, we identified the Arabidopsis (Arabidopsis thaliana) gene encoding hydroxynitrile lyase (AtHNL, At5g10300) as one gene induced in response to spider mite (Tetranychus urticae) infestation. AtHNL catalyzes the reversible interconversion between cyanohydrins and derived carbonyl compounds with free cyanide. AtHNL loss- and gain-of-function Arabidopsis plants showed that specific activity of AtHNL using mandelonitrile as substrate was higher in the overexpressing lines than in wild-type (WT) and mutant lines. Concomitantly, mandelonitrile accumulated at higher levels in mutant lines than in WT plants and was significantly reduced in the AtHNL overexpressing lines. After mite infestation, mandelonitrile content increased in WT and overexpressing plants but not in mutant lines, while hydrogen cyanide (HCN) accumulated in the three infested Arabidopsis genotypes. Feeding bioassays demonstrated that the AtHNL gene participated in Arabidopsis defense against T. urticae. The reduced leaf damage detected in the AtHNL overexpressing lines reflected the mite's reduced ability to feed on leaves, which consequently restricted mite fecundity. In turn, mites upregulated TuCAS1 encoding ß-cyanoalanine synthase to avoid the respiratory damage produced by HCN. This detoxification effect was functionally demonstrated by reduced mite fecundity observed when dsRNA-TuCAS-treated mites fed on WT plants and hnl1 mutant lines. These findings add more players in the Arabidopsis-T. urticae interplay to overcome mutual defenses.

Sulfide promotes tolerance to drought through protein persulfidation in Arabidopsis.

Hydrogen sulfide (H2S) is a signaling molecule that regulates essential plant processes. In this study, the role of H2S during drought was analysed, focusing on the underlying mechanism. Pretreatments with H2S before imposing drought on plants substantially improved the characteristic stressed phenotypes under drought and decreased the levels of typical biochemical stress markers such as anthocyanin, proline, and hydrogen peroxide. H2S also regulated drought-responsive genes and amino acid metabolism, and repressed drought-induced bulk autophagy and protein ubiquitination, demonstrating the protective effects of H2S pretreatment. Quantitative proteomic analysis identified 887 significantly different persulfidated proteins between control and drought stress plants. Bioinformatic analyses of the proteins more persulfidated in drought revealed that the most enriched biological processes were cellular response to oxidative stress and hydrogen peroxide catabolism. Protein degradation, abiotic stress responses, and the phenylpropanoid pathway were also highlighted, suggesting the importance of persulfidation in coping with drought-induced stress. Our findings emphasize the role of H2S as a promoter of enhanced tolerance to drought, enabling plants to respond more rapidly and efficiently. Furthermore, the main role of protein persulfidation in alleviating reactive oxygen species accumulation and balancing redox homeostasis under drought stress is highlighted.

Photorespiration: regulation and new insights on the potential role of persulfidation.

Photorespiration has been considered a 'futile' cycle in C3 plants, necessary to detoxify and recycle the metabolites generated by the oxygenating activity of Rubisco. However, several reports indicate that this metabolic route plays a fundamental role in plant metabolism and constitutes a very interesting research topic. Many open questions still remain with regard to photorespiration. One of these questions is how the photorespiratory process is regulated in plants and what factors contribute to this regulation. In this review, we summarize recent advances in the regulation of the photorespiratory pathway with a special focus on the transcriptional and post-translational regulation of photorespiration and the interconnections of this process with nitrogen and sulfur metabolism. Recent findings on sulfide signaling and protein persulfidation are also described.

Persulfidation-based Modification of Cysteine Desulfhydrase and the NADPH Oxidase RBOHD Controls Guard Cell Abscisic Acid Signaling.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that regulates diverse cellular signaling pathways through persulfidation, which involves the post-translational modification of specific Cys residues to form persulfides. However, the mechanisms that underlie this important redox-based modification remain poorly understood in higher plants. We have, therefore, analyzed how protein persulfidation acts as a specific and reversible signaling mechanism during the abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana). Here we show that ABA stimulates the persulfidation of l-CYSTEINE DESULFHYDRASE1, an important endogenous H2S enzyme, at Cys44 and Cys205 in a redox-dependent manner. Moreover, sustainable H2S accumulation drives persulfidation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) at Cys825 and Cys890, enhancing its ability to produce reactive oxygen species. Physiologically, s-persulfidation-induced RBOHD activity is relevant to ABA-induced stomatal closure. Together, these processes form a negative feedback loop that fine-tunes guard cell redox homeostasis and ABA signaling. These findings not only expand our current knowledge of H2S function in the context of guard cell ABA signaling, but also demonstrate the presence of a rapid signal integration mechanism involving specific and reversible redox-based post-translational modifications that occur in response to changing environmental conditions.

Abscisic Acid-Triggered Persulfidation of the Cys Protease ATG4 Mediates Regulation of Autophagy by Sulfide.

Hydrogen sulfide is a signaling molecule that regulates essential processes in plants, such as autophagy. In Arabidopsis (Arabidopsis thaliana), hydrogen sulfide negatively regulates autophagy independently of reactive oxygen species via an unknown mechanism. Comparative and quantitative proteomic analysis was used to detect abscisic acid-triggered persulfidation that reveals a main role in the control of autophagy mediated by the autophagy-related (ATG) Cys protease AtATG4a. This protease undergoes specific persulfidation of Cys170 that is a part of the characteristic catalytic Cys-His-Asp triad of Cys proteases. Regulation of the ATG4 activity by persulfidation was tested in a heterologous assay using the Chlamydomonas reinhardtii CrATG8 protein as a substrate. Sulfide significantly and reversibly inactivates AtATG4a. The biological significance of the reversible inhibition of the ATG4 by sulfide is supported by the results obtained in Arabidopsis leaves under basal and autophagy-activating conditions. A significant increase in the overall ATG4 proteolytic activity in Arabidopsis was detected under nitrogen starvation and osmotic stress and can be inhibited by sulfide. Therefore, the data strongly suggest that the negative regulation of autophagy by sulfide is mediated by specific persulfidation of the ATG4 protease.

Regulation of V-ATPase by Jasmonic Acid: Possible Role of Persulfidation.

Vacuolar H+-translocating ATPase (V-ATPase) is a proton pump crucial for plant growth and survival. For this reason, its activity is tightly regulated, and various factors, such as signaling molecules and phytohormones, may be involved in this process. The aim of this study was to explain the role of jasmonic acid (JA) in the signaling pathways responsible for the regulation of V-ATPase in cucumber roots and its relationship with other regulators of this pump, i.e., H2S and H2O2. We analyzed several aspects of the JA action on the enzyme, including transcriptional regulation, modulation of protein levels, and persulfidation of selected V-ATPase subunits as an oxidative posttranslational modification induced by H2S. Our results indicated that JA functions as a repressor of V-ATPase, and its action is related to a decrease in the protein amount of the A and B subunits, the induction of oxidative stress, and the downregulation of the E subunit persulfidation. We suggest that both H2S and H2O2 may be downstream components of JA-dependent negative proton pump regulation. The comparison of signaling pathways induced by two negative regulators of the pump, JA and cadmium, revealed that multiple pathways are involved in the V-ATPase downregulation in cucumber roots.

Hydrogen sulfide signaling in plant adaptations to adverse conditions: molecular mechanisms.

Hydrogen sulfide (H2S) is a signaling molecule that regulates critical processes and allows plants to adapt to adverse conditions. The molecular mechanism underlying H2S action relies on its chemical reactivity, and the most-well characterized mechanism is persulfidation, which involves the modification of protein thiol groups, resulting in the formation of persulfide groups. This modification causes a change of protein function, altering catalytic activity or intracellular location and inducing important physiological effects. H2S cannot react directly with thiols but instead can react with oxidized cysteine residues; therefore, H2O2 signaling through sulfenylation is required for persulfidation. A comparative study performed in this review reveals 82% identity between sulfenylome and persulfidome. With regard to abscisic acid (ABA) signaling, widespread evidence shows an interconnection between H2S and ABA in the plant response to environmental stress. Proteomic analyses have revealed persulfidation of several proteins involved in the ABA signaling network and have shown that persulfidation is triggered in response to ABA. In guard cells, a complex interaction of H2S and ABA signaling has also been described, and the persulfidation of specific signaling components seems to be the underlying mechanism.

Mutation in Arabidopsis ß-cyanoalanine synthase overcomes NADPH oxidase action in response to pathogens.

Plant responses to pathogens comprise a complex process, implying a plethora of signals and reactions. Among them, endogenous production of hydrogen cyanide (HCN) has been shown to induce resistance in Arabidopsis to the hemibiotrophic bacterium Pseudomonas syringae pv. tomato (Pst) DC3000. ß-cyanoalanine synthase (CAS-C1) is responsible for the detoxification of HCN in Arabidopsis mitochondria. Here, we show that green fluorescent protein-tagged CAS-C1 is transiently reduced in leaves infected with an avirulent strain of Pst during early interactions and increased in leaves infected with a virulent strain of Pst, supporting previous transcriptional data. Genetic crosses show that mutation in CAS-C1 in Arabidopsis resembles the action of the NADPH oxidase RbohD independently of reactive oxygen species production and that the accumulation of salicylic acid is required for HCN-stimulated resistance to Pst. Finally, we show that the cas-c1 mutation acts on the salicylic acid-dependent response to pathogens by mechanisms other than protein ubiquitination or the increase of monomerization and entry to the nucleus of NPR1, the central regulator of the salicylic acid-mediated response. Considering these results, we propose new mechanisms for modulation of the immune response by HCN.

Effect of cadmium in the microalga Chlorella sorokiniana: A proteomic study.

Cadmium is one of the most common heavy metals in contaminated aquatic environments and one of the most toxic contaminants for phytoplankton. Nevertheless, there are not enough studies focused on the effect of this metal in algae. Through a proteomic approach, this work shows how Cd can alter the growth, cell morphology and metabolism of the microalga Chlorella sorokiniana. Using the sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS), we concluded that exposure of Chlorella sorokiniana to 250 µM Cd2+ for 40 h caused downregulation of different metabolic pathways, such as photosynthesis, oxidative phosphorylation, glycolysis, TCA cycle and ribosomal proteins biosynthesis. However, photorespiration, antioxidant enzymes, gluconeogenesis, starch catabolism, and biosynthesis of glutamate, cysteine, glycine and serine were upregulated, under the same conditions. Finally, exposure to Cd also led to changes in the metabolism of carotenoids and lipids. In addition, the high tolerance of Chlorella sorokiniana to Cd points to this microalga as a potential microorganism to be used in bioremediation processes.

Hydrogen sulfide, a signaling molecule in plant stress responses.

Gaseous molecules, such as hydrogen sulfide (H2 S) and nitric oxide (NO), are crucial players in cellular and (patho)physiological processes in biological systems. The biological functions of these gaseous molecules, which were first discovered and identified as gasotransmitters in animals, have received unprecedented attention from plant scientists in recent decades. Researchers have arrived at the consensus that H2 S is synthesized endogenously and serves as a signaling molecule throughout the plant life cycle. However, the mechanisms of H2 S action in redox biology is still largely unexplored. This review highlights what we currently know about the characteristics and biosynthesis of H2 S in plants. Additionally, we summarize the role of H2 S in plant resistance to abiotic stress. Moreover, we propose and discuss possible redox-dependent mechanisms by which H2 S regulates plant physiology.

HCN Regulates Cellular Processes through Posttranslational Modification of Proteins by S-cyanylation.

Hydrogen cyanide (HCN) is coproduced with ethylene in plant cells and is primarily enzymatically detoxified by the mitochondrial ß-CYANOALANINE SYNTHASE (CAS-C1). Permanent or transient depletion of CAS-C1 activity in Arabidopsis (Arabidopsis thaliana) results in physiological alterations in the plant that suggest that HCN acts as a gasotransmitter molecule. Label-free quantitative proteomic analysis of mitochondrially enriched samples isolated from the wild type and cas-c1 mutant revealed significant changes in protein content, identifying 451 proteins that are absent or less abundant in cas-c1 and 353 proteins that are only present or more abundant in cas-c1 Gene ontology classification of these proteins identified proteomic changes that explain the root hairless phenotype and the altered immune response observed in the cas-c1 mutant. The mechanism of action of cyanide as a signaling molecule was addressed using two proteomic approaches aimed at identifying the S-cyanylation of Cys as a posttranslational modification of proteins. Both the 2-imino-thiazolidine chemical method and the direct untargeted analysis of proteins using liquid chromatography-tandem mass spectrometry identified a set of 163 proteins susceptible to S-cyanylation that included SEDOHEPTULOSE 1,7-BISPHOSPHATASE (SBPase), the PEPTIDYL-PROLYL CIS-TRANS ISOMERASE 20-3 (CYP20-3), and ENOLASE2 (ENO2). In vitro analysis of these enzymes showed that S-cyanylation of SBPase Cys74, CYP20-3 Cys259, and ENO2 Cys346 residues affected their enzymatic activity. Gene Ontology classification and protein-protein interaction cluster analysis showed that S-cyanylation is involved in the regulation of primary metabolic pathways, such as glycolysis, and the Calvin and S-adenosyl-Met cycles.

Abscisic acid-triggered guard cell l-cysteine desulfhydrase function and in situ hydrogen sulfide production contributes to heme oxygenase-modulated stomatal closure.

Recent studies have demonstrated that hydrogen sulfide (H2 S) produced through the activity of l-cysteine desulfhydrase (DES1) is an important gaseous signaling molecule in plants that could participate in abscisic acid (ABA)-induced stomatal closure. However, the coupling of the DES1/H2 S signaling pathways to guard cell movement has not been thoroughly elucidated. The results presented here provide genetic evidence for a physiologically relevant signaling pathway that governs guard cell in situ DES1/H2 S function in stomatal closure. We discovered that ABA-activated DES1 produces H2 S in guard cells. The impaired guard cell ABA phenotype of the des1 mutant can be fully complemented when DES1/H2 S function has been specifically rescued in guard cells and epidermal cells, but not mesophyll cells. This research further characterized DES1/H2 S function in the regulation of LONG HYPOCOTYL1 (HY1, a member of the heme oxygenase family) signaling. ABA-induced DES1 expression and H2 S production are hyper-activated in the hy1 mutant, both of which can be fully abolished by the addition of H2 S scavenger. Impaired guard cell ABA phenotype of des1/hy1 can be restored by H2 S donors. Taken together, this research indicated that guard cell in situ DES1 function is involved in ABA-induced stomatal closure, which also acts as a pivotal hub in regulating HY1 signaling.

Cadmium induces reactive oxygen species-dependent pexophagy in Arabidopsis leaves.

Cadmium treatment induces transient peroxisome proliferation in Arabidopsis leaves. To determine whether this process is regulated by pexophagy and to identify the mechanisms involved, we analysed time course-dependent changes in ATG8, an autophagy marker, and the accumulation of peroxisomal marker PEX14a. After 3 hr of Cd exposure, the transcript levels of ATG8h, ATG8c, a, and i were slightly up-regulated and then returned to normal. ATG8 protein levels also increased after 3 hr of Cd treatment, although an opposite pattern was observed in PEX14. Arabidopsis lines expressing GFP-ATG8a and CFP-SKL enabled us to demonstrate the presence of pexophagic processes in leaves. The Cd-dependent induction of pexophagy was demonstrated by the accumulation of peroxisomes in autophagy gene (ATG)-related Arabidopsis knockout mutants atg5 and atg7. We show that ATG8a colocalizes with catalase and NBR1 in the electron-dense peroxisomal core, thus suggesting that NBR1 may be an autophagic receptor for peroxisomes, with catalase being possibly involved in targeting pexophagy. Protein carbonylation and peroxisomal redox state suggest that protein oxidation may trigger pexophagy. Cathepsine B, legumain, and caspase 6 may also be involved in the regulation of pexophagy. Our results suggest that pexophagy could be an important step in rapid cell responses to cadmium.

Signaling by hydrogen sulfide and cyanide through post-translational modification.

Two cysteine metabolism-related molecules, hydrogen sulfide and hydrogen cyanide, which are considered toxic, have now been considered as signaling molecules. Hydrogen sulfide is produced in chloroplasts through the activity of sulfite reductase and in the cytosol and mitochondria by the action of sulfide-generating enzymes, and regulates/affects essential plant processes such as plant adaptation, development, photosynthesis, autophagy, and stomatal movement, where interplay with other signaling molecules occurs. The mechanism of action of sulfide, which modifies protein cysteine thiols to form persulfides, is related to its chemical features. This post-translational modification, called persulfidation, could play a protective role for thiols against oxidative damage. Hydrogen cyanide is produced during the biosynthesis of ethylene and camalexin in non-cyanogenic plants, and is detoxified by the action of sulfur-related enzymes. Cyanide functions include the breaking of seed dormancy, modifying the plant responses to biotic stress, and inhibition of root hair elongation. The mode of action of cyanide is under investigation, although it has recently been demonstrated to perform post-translational modification of protein cysteine thiols to form thiocyanate, a process called S-cyanylation. Therefore, the signaling roles of sulfide and most probably of cyanide are performed through the modification of specific cysteine residues, altering protein functions.

Multilevel Regulation of Peroxisomal Proteome by Post-Translational Modifications.

Peroxisomes, which are ubiquitous organelles in all eukaryotes, are highly dynamic organelles that are essential for development and stress responses. Plant peroxisomes are involved in major metabolic pathways, such as fatty acid ß-oxidation, photorespiration, ureide and polyamine metabolism, in the biosynthesis of jasmonic, indolacetic, and salicylic acid hormones, as well as in signaling molecules such as reactive oxygen and nitrogen species (ROS/RNS). Peroxisomes are involved in the perception of environmental changes, which is a complex process involving the regulation of gene expression and protein functionality by protein post-translational modifications (PTMs). Although there has been a growing interest in individual PTMs in peroxisomes over the last ten years, their role and cross-talk in the whole peroxisomal proteome remain unclear. This review provides up-to-date information on the function and crosstalk of the main peroxisomal PTMs. Analysis of whole peroxisomal proteomes shows that a very large number of peroxisomal proteins are targeted by multiple PTMs, which affect redox balance, photorespiration, the glyoxylate cycle, and lipid metabolism. This multilevel PTM regulation could boost the plasticity of peroxisomes and their capacity to regulate metabolism in response to environmental changes.

ß-Cyanoalanine Synthase Action in Root Hair Elongation is Exerted at Early Steps of the Root Hair Elongation Pathway and is Independent of Direct Cyanide Inactivation of NADPH Oxidase.

In Arabidopsis thaliana, cyanide is produced concomitantly with ethylene biosynthesis and is mainly detoxified by the ß-cyanoalanine synthase CAS-C1. In roots, CAS-C1 activity is essential to maintain a low level of cyanide for proper root hair development. Root hair elongation relies on polarized cell expansion at the growing tip, and we have observed that CAS-C1 locates in mitochondria and accumulates in root hair tips during root hair elongation, as shown by observing the fluorescence in plants transformed with the translational construct ProC1:CASC1-GFP, containing the complete CAS-C1 gene fused to green fluorescent protein (GFP). Mutants in the SUPERCENTIPEDE (SCN1) gene, that regulate the NADPH oxidase gene ROOT HAIR DEFECTIVE 2 (RHD2)/AtrbohC, are affected at the very early steps of the development of root hair that do not elongate and do not show a preferential localization of the GFP accumulation in the tips of the root hair primordia. Root hairs of mutants in CAS-C1 or RHD2/AtrbohC, whose protein product catalyzes the generation of ROS and the Ca2+ gradient, start to grow out correctly, but they do not elongate. Genetic crosses between the cas-c1 mutant and scn1 or rhd2 mutants were performed, and the detailed phenotypic and molecular characterization of the double mutants demonstrates that scn1 mutation is epistatic to cas-c1 and cas-c1 is epistatic to rhd2 mutation, indicating that CAS-C1 acts in early steps of the root hair development process. Moreover, our results show that the role of CAS-C1 in root hair elongation is independent of H2O2 production and of a direct NADPH oxidase inhibition by cyanide.

Hydrogen Sulfide Regulates the Cytosolic/Nuclear Partitioning of Glyceraldehyde-3-Phosphate Dehydrogenase by Enhancing its Nuclear Localization.

Hydrogen sulfide is an important signaling molecule comparable with nitric oxide and hydrogen peroxide in plants. The underlying mechanism of its action is unknown, although it has been proposed to be S-sulfhydration. This post-translational modification converts the thiol groups of cysteines within proteins to persulfides, resulting in functional changes of the proteins. In Arabidopsis thaliana, S-sulfhydrated proteins have been identified, including the cytosolic isoforms of glyceraldehyde-3-phosphate dehydrogenase GapC1 and GapC2. In this work, we studied the regulation of sulfide on the subcellular localization of these proteins using two different approaches. We generated GapC1-green fluorescent protein (GFP) and GapC2-GFP transgenic plants in both the wild type and the des1 mutant defective in the l-cysteine desulfhydrase DES1, responsible for the generation of sulfide in the cytosol. The GFP signal was detected in the cytoplasm and the nucleus of epidermal cells, although with reduced nuclear localization in des1 compared with the wild type, and exogenous sulfide treatment resulted in similar signals in nuclei in both backgrounds. The second approach consisted of the immunoblot analysis of the GapC endogenous proteins in enriched nuclear and cytosolic protein extracts, and similar results were obtained. A significant reduction in the total amount of GapC in des1 in comparison with the wild type was determined and exogenous sulfide significantly increased the protein levels in the nuclei in both plants, with a stronger response in the wild type. Moreover, the presence of an S-sulfhydrated cysteine residue on GapC1 was demonstrated by mass spectrometry. We conclude that sulfide enhances the nuclear localization of glyceraldehyde-3-phosphate dehydrogenase.