No abstract

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.

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.

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.

Hydrogen sulfide (H 2 S) is a gaseous signaling molecule that regulates diverse cellular signaling pathways through persulfidation, which involves the post-translational modification of specific cysteine 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 thaliana.Here we show that ABA stimulates the persulfidation of L-CYSTEINE DESULFHYDRASE 1 (DES1), an important endogenous H 2 S enzyme, at Cys44 and Cys205 in a redox-dependent manner. Moreover, sustainable H 2 S 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 H 2 S 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.

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, gamma-glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine-synthase complex.