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Protein persulfidation in the Arabidopsis proteome is widespread and is the molecular mechanism by which hydrogen sulfide performs its signaling role.

Cysteine (Cys) occupies a central position in plant metabolism due to its biochemical functions. Arabidopsis (Arabidopsis thaliana) cells contain different O-acetylserine(thiol)lyase (OASTL) enzymes that catalyze the biosynthesis of Cys. Because they are localized in the cytosol, plastids, and mitochondria, this results in multiple subcellular Cys pools. Much progress has been made on the most abundant OASTL enzymes; however, information on the less abundant OASTL-like proteins has been scarce. To unequivocally establish the enzymatic reaction catalyzed by the minor cytosolic OASTL isoform CS-LIKE (for Cys synthase-like; At5g28030), we expressed this enzyme in bacteria and characterized the purified recombinant protein. Our results demonstrate that CS-LIKE catalyzes the desulfuration of L-Cys to sulfide plus ammonia and pyruvate. Thus, CS-LIKE is a novel L-Cys desulfhydrase (EC 4.4.1.1), and we propose to designate it DES1. The impact and functionality of DES1 in Cys metabolism was revealed by the phenotype of the T-DNA insertion mutants des1-1 and des1-2. Mutation of the DES1 gene leads to premature leaf senescence, as demonstrated by the increased expression of senescence-associated genes and transcription factors. Also, the absence of DES1 significantly reduces the total Cys desulfuration activity in leaves, and there is a concomitant increase in the total Cys content. As a consequence, the expression levels of sulfur-responsive genes are deregulated, and the mutant plants show enhanced antioxidant defenses and tolerance to conditions that promote oxidative stress. Our results suggest that DES1 from Arabidopsis is an L-Cys desulfhydrase involved in maintaining Cys homeostasis, mainly at late developmental stages or under environmental perturbations.

Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems.

Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H 2 S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H 2 S is enzymatically produced in the cytosol through the activity of L-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H 2 S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H 2 S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H 2 S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H 2 S, suggesting that H 2 S required a functional ABI1. Further studies to unravel the cross talk between H 2 S and NO indicate that (1) H 2 S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H 2 S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.Abscisic acid (ABA) regulates diverse physiological and developmental processes in plants, among which seed dormancy and stomatal movement are the most studied. Stomata are pores bordered by pairs of specialized cells named guard cells located in the epidermis of the aerial part of most land plants. Due to the waxy cuticle of plants, stomatal pores regulate approximately 90% of all the gas exchange (i.e. the uptake of CO 2 required for photosynthesis and the loss of water vapor during transpiration) between the plant and the environment (Hetherington and Woodward, 2003). Thus, stomatal movement is a key process for the regulation of plant water status and biomass production. In guard cells, ABA induces an increase of intracellular calcium concentrations, which, in turn, induces an efflux of anions that causes membrane depolarization. In this voltage milieu, ion uptake is blocked through the inactivation of inward-rectifying potassium channels, and ion efflux is induced through activation of outward-rectifying potassium channels. This solute relocation drives water out of the guard cells and closes the stomatal pore as a result of a reduction in guard cell turgor (Blatt, 2000;Kim et al...

Hydrogen sulfide (H2S) has been largely referred as a toxic gas and environmental hazard, but recent years, it has emerged as an important gas-signaling molecule with effects on multiple physiological processes in both animal and plant systems. The regulatory functions of H2S in plants are involved in important processes such as the modulation of defense responses, plant growth and development, and the regulation of senescence and maturation. The main signaling pathway involving sulfide has been proven to be through protein persulfidation (alternatively called S-sulfhydration), in which the thiol group of cysteine (-SH) in proteins is modified into a persulfide group (-SSH). This modification may cause functional changes in protein activities, structures, and subcellular localizations of the target proteins. New shotgun proteomic approaches and bioinformatic analyses have revealed that persulfidated cysteines regulate important biological processes, highlighting their importance in cell signaling, since about one in 20 proteins in Arabidopsis is persulfidated. During oxidative stress, an increased persulfidation has been reported and speculated that persulfidation is the protective mechanism for protein oxidative damage. Nevertheless, cysteine residues are also oxidized to different post-translational modifications such S-nitrosylation or S-sulfenylation, which seems to be interconvertible. Thus, it must imply a tight cysteine redox regulation essential for cell survival. This review is aimed to focus on the current knowledge of protein persulfidation and addresses the regulation mechanisms that are disclosed based on the knowledge from other cysteine modifications.

In Arabidopsis thaliana, DES1 is the only identified L-Cysteine desulfhydrase located in the cytosol, and it is involved in the degradation of cysteine and the concomitant production of H 2 S in this cell compartment. Detailed characterization of the T-DNA insertion mutants des1-1 and des1-2 has provided insight into the role of sulfide metabolically generated in the cytosol as a signaling molecule. Mutations of L-CYS DESULFHYDRASE 1 (DES1) impede H 2 S generation in the Arabidopsis cytosol and strongly affect plant metabolism. Senescence-associated vacuoles are detected in mesophyll protoplasts of des1 mutants. Additionally, DES1 deficiency promotes the accumulation and lipidation of the ATG8 protein, which is associated with the process of autophagy. The transcriptional profile of the des1-1 mutant corresponds to its premature senescence and autophagy-induction phenotypes, and restoring H 2 S generation has been shown to eliminate the phenotypic defects of des1 mutants. Moreover, sulfide is able to reverse ATG8 accumulation and lipidation, even in wild-type plants when autophagy is induced by carbon starvation, suggesting a general effect of sulfide on autophagy regulation that is unrelated to sulfur or nitrogen limitation stress. Our results suggest that cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile of Arabidopsis.