New Insights on the Role of ß-Cyanoalanine Synthase CAS-C1 in Root Hair Elongation through Single-Cell Proteomics.

(1) Background: Root hairs are specialized structures involved in water and plant nutrient uptake. They elongate from epidermal cells following a complex developmental program. ß-cyanoalanine synthase (CAS), which is mainly involved in hydrogen cyanide (HCN) detoxification in Arabidopsis thaliana, plays a role in root hair elongation, as evidenced by the fact that cas-c1 mutants show a severe defect in root hair shape. In addition to root hairs, CAS C1 is expressed in the quiescent center and meristem. (2) Methods: To identify its role in root hair formation, we conducted single-cell proteomics analysis by isolating root hair cells using Fluorescence-activated Cell Sorting (FACS) from wild-type and cas-c1 mutants. We also analyzed the presence of S-cyanylation, a protein post-translational modification (PTM) mediated by HCN and affecting cysteine residues and protein activity in proteins of wild type and cas-c1 mutants. (3) Results and Conclusions: We have found that the cas-c1 mutation has no visible effect on quiescent center or meristem root tissue, in both control and nutrient-deprivation conditions. We have identified more than 3900 proteins in root hairs and we have found that several proteins involved in root hair development, related to the receptor kinase FERONIA signaling and DNA methylation, are modified by S-cyanylation.

Persulfidation of plant and bacteroid proteins is involved in legume nodule development and senescence.

Legumes establish symbiosis with rhizobia forming nitrogen-fixing nodules. The central role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in nodule biology has been clearly established. Recently, hydrogen sulfide (H2S) and other reactive sulfur species (RSS) have emerged as novel signaling molecules in animals and plants. A major mechanism by which ROS, RNS, and RSS fulfil their signaling role is the post-translational modification of proteins. To identify possible functions of H2S in nodule development and senescence, we used the tag-switch method to quantify changes in the persulfidation profile of common bean (Phaseolus vulgaris) nodules at different developmental stages. Proteomic analyses indicate that persulfidation plays a regulatory role in plant and bacteroid metabolism and senescence. The effect of a H2S donor on nodule functioning and on several proteins involved in ROS and RNS homeostasis was also investigated. Our results using recombinant proteins and nodulated plants support a crosstalk among H2S, ROS and RNS, a protective function of persulfidation on redox-sensitive enzymes, and a beneficial effect of H2S on symbiotic nitrogen fixation. We conclude that the general decrease of persulfidation levels observed in plant proteins of aging nodules is one of the mechanisms that disrupt redox homeostasis leading to senescence.

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.

Old poisons, new signaling molecules: the case of hydrogen cyanide.

The high phenotypic plasticity developed by plants includes rapid responses and adaptations to aggressive or changing environments. To achieve this, they evolved extremely efficient mechanisms of signaling mediated by a wide range of molecules, including small signal molecules. Among them, hydrogen cyanide (HCN) has been largely ignored due to its toxic characteristics. However, not only is it present in living organisms, but it has been shown that it serves several functions in all kingdoms of life. Research using model plants has changed the traditional point of view, and it has been demonstrated that HCN plays a positive role in the plant response to pathogens independently of its toxicity. Indeed, HCN induces a response aimed at protecting the plant from pathogen attack, and the HCN is provided either exogenously (in vitro or by some cyanogenic bacteria species present in the rhizosphere) or endogenously (in reactions involving ethylene, camalexin, or other cyanide-containing compounds). The contribution of different mechanisms to HCN function, including a new post-translational modification of cysteines in proteins, namely S-cyanylation, is discussed here. This work opens up an expanding 'HCN field' of research related to plants and other organisms.

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.

Proteome Dynamics of Persulfidation in Leaf Tissue under Light/Dark Conditions and Carbon Deprivation.

Hydrogen sulfide (H2S) acts as a signaling molecule in plants, bacteria, and mammals, regulating various physiological and pathological processes. The molecular mechanism by which hydrogen sulfide exerts its action involves the posttranslational modification of cysteine residues to form a persulfidated thiol motif. This research aimed to study the regulation of protein persulfidation. We used a label-free quantitative approach to measure the protein persulfidation profile in leaves under different growth conditions such as light regimen and carbon deprivation. The proteomic analysis identified a total of 4599 differentially persulfidated proteins, of which 1115 were differentially persulfidated between light and dark conditions. The 544 proteins that were more persulfidated in the dark were analyzed, and showed significant enrichment in functions and pathways related to protein folding and processing in the endoplasmic reticulum. Under light conditions, the persulfidation profile changed, and the number of differentially persulfidated proteins increased up to 913, with the proteasome and ubiquitin-dependent and ubiquitin-independent catabolic processes being the most-affected biological processes. Under carbon starvation conditions, a cluster of 1405 proteins was affected by a reduction in their persulfidation, being involved in metabolic processes that provide primary metabolites to essential energy pathways and including enzymes involved in sulfur assimilation and sulfide production.

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.

Detection of protein persulfidation in plants by the dimedone switch method.

Hydrogen sulfide (H2S) is a well-known signaling molecule in both animals and plants, endogenously produced by cells, and involved in a wide variety of biological functions. In plants, H2S regulates a wide range of essential aspects of plant life, including plant responses to numerous stresses and physiological processes as important as abscisic acid (ABA)-dependent stomatal movement, photosynthesis, and autophagy. The best studied molecular mechanism responsible of sulfide signaling is protein persulfidation, a post-translational modification of cysteine residues, where a thiol group (P-SH) is transformed into a persulfide group (P-SSH). In this way, persulfidation has emerged as a new type of cellular redox mechanism that can regulate protein structure and function and interest in this modification has increased exponentially. However, the identification and the development of detection methods have been challenging. Nevertheless, on the basis of the chemical differences between the thiol and the persulfide groups, different methods have been implemented. In plants, different high-throughput proteomic analyzes have been performed using a tag-switch method where in the first step all thiols and persulfides are blocked and then in the second step persulfides are selectively labeled using a specific nucleophile. This chapter outlines a new method, previously described in mammals, that has been applied to detect persulfidation in plants and is based on the same chemical premise but consists of chemoselective persulfide labeling with dimedone-based probes. Here, we provide a detailed workflow of this method that includes procedures for the determination of the persulfidation level of a protein extract visualized and quantified by fluorescence on the gel on one side, and on the other, the labeling and purification of persulfidated proteins for identification by mass spectrometry.

The Combined Partial Knockdown of CBS and MPST Genes Induces Inflammation, Impairs Adipocyte Function-Related Gene Expression and Disrupts Protein Persulfidation in Human Adipocytes.

Recent studies in mice and humans demonstrated the relevance of H2S synthesising enzymes, such as CTH, CBS, and MPST, in the physiology of adipose tissue and the differentiation of preadipocyte into adipocytes. Here, our objective was to investigate the combined role of CTH, CBS, and MPST in the preservation of adipocyte protein persulfidation and adipogenesis. Combined partial CTH, CBS, and MPST gene knockdown was achieved treating fully human adipocytes with siRNAs against these transcripts (siRNA_MIX). Adipocyte protein persulfidation was analyzed using label-free quantitative mass spectrometry coupled with a dimedone-switch method for protein labeling and purification. Proteomic analysis quantified 216 proteins with statistically different levels of persulfidation in KD cells compared to control adipocytes. In fully differentiated adipocytes, CBS and MPST mRNA and protein levels were abundant, while CTH expression was very low. It is noteworthy that siRNA_MIX administration resulted in a significant decrease in CBS and MPST expression, without impacting on CTH. The combined partial knockdown of the CBS and MPST genes resulted in reduced cellular sulfide levels in parallel to decreased expression of relevant genes for adipocyte biology, including adipogenesis, mitochondrial biogenesis, and lipogenesis, but increased proinflammatory- and senescence-related genes. It should be noted that the combined partial knockdown of CBS and MPST genes also led to a significant disruption in the persulfidation pattern of the adipocyte proteins. Although among the less persulfidated proteins, we identified several relevant proteins for adipocyte adipogenesis and function, among the most persulfidated, key mediators of adipocyte inflammation and dysfunction as well as some proteins that might play a positive role in adipogenesis were found. In conclusion, the current study indicates that the combined partial elimination of CBS and MPST (but not CTH) in adipocytes affects the expression of genes related to the maintenance of adipocyte function and promotes inflammation, possibly by altering the pattern of protein persulfidation in these cells, suggesting that these enzymes were required for the functional maintenance of adipocytes.

ß-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified ß-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and ß-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its ß-cyanoalanine synthase activity. Consistent with the ß-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the ß-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite's ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

Persulfidation is the mechanism underlying sulfide-signaling of autophagy.

In this commentary, we highlight the findings described in a recent paper regarding the mechanism of H2S regulation of macroautophagy/autophagy in mammalian cells and discuss the similarities/divergencies with plant cells. The main outcome is that the posttranslational modification of thiol groups of cysteine residues to form persulfides is a conserved molecular mechanism.

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.

Label-Free Quantitative Proteomic Analysis of Nitrogen Starvation in Arabidopsis Root Reveals New Aspects of H2S Signaling by Protein Persulfidation.

Hydrogen sulfide (H2S)-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 posttranslational modification of cysteine residues to form a persulfidated thiol motif. We developed a comparative and label-free quantitative proteomic analysis approach for the detection of endogenous persulfidated proteins in N-starved Arabidopsis thaliana roots by using the tag-switch method. In this work, we identified 5214 unique proteins from root tissue that were persulfidated, 1674 of which were quantitatively analyzed and found to show altered persulfidation levels in vivo under N deprivation. These proteins represented almost 13% of the entire annotated proteome in Arabidopsis. Bioinformatic analysis revealed that persulfidated proteins were involved in a wide range of biological functions, regulating important processes such as primary metabolism, plant responses to stresses, growth and development, RNA translation and protein degradation. Quantitative mass spectrometry analysis allowed us to obtain a comprehensive view of hydrogen sulfide signaling via changes in the persulfidation levels of key protein targets involved in ubiquitin-dependent protein degradation and autophagy, among others.

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.

Hydrogen sulfide-linked persulfidation of ABI4 controls ABA responses through the transactivation of MAPKKK18 in Arabidopsis.

Hydrogen sulfide (H2S) is a signaling molecule that regulates plant hormone and stress responses. The phytohormone abscisic acid (ABA) plays an important role in plant adaptation to unfavorable environmental conditions and induces the persulfidation of L-CYSTEINE DESULFHYDRASE1 (DES1) and the production of H2S in guard cells. However, it remains largely unclear how H2S and protein persulfidation participate in the relay of ABA signals. In this study, we discovered that ABSCISIC ACID INSENSITIVE 4 (ABI4) acts downstream of DES1 in the control of ABA responses in Arabidopsis. ABI4 undergoes persulfidation at Cys250 that is triggered in a time-dependent manner by ABA, and loss of DES1 function impairs this process. Cys250 and its persulfidation are essential for ABI4 function in the regulation of plant responses to ABA and the H2S donor NaHS during germination, seedling establishment, and stomatal closure, which are abolished in the ABI4Cys250Ala mutated variant. Introduction of the ABI4Cys250Ala variant into the abi4 des1 mutant did not rescue its hyposensitivity to ABA. Cys250 is critical for the binding of ABI4 to its cognate motif in the promoter of Mitogen-Activated Protein Kinase Kinase Kinase 18 (MAPKKK18), which propagates the MAPK signaling cascade induced by ABA. Furthermore, the DES1-mediated persulfidation of ABI4 enhances the transactivation activity of ABI4 toward MAPKKK18, and ABI4 can bind the DES1 promoter, forming a regulatory loop. Taken together, these findings advance our understanding of a post-translational regulatory mechanism and suggest that ABI4 functions as an integrator of ABA and MAPK signals through a process in which DES1-produced H2S persulfidates ABI4 at Cys250.

Activation of Endogenous H2S Biosynthesis or Supplementation with Exogenous H2S Enhances Adipose Tissue Adipogenesis and Preserves Adipocyte Physiology in Humans.

Aims: To investigate the impact of exogenous hydrogen sulfide (H2S) and its endogenous biosynthesis on human adipocytes and adipose tissue in the context of obesity and insulin resistance. Results: Experiments in human adipose tissue explants and in isolated preadipocytes demonstrated that exogenous H2S or the activation of endogenous H2S biosynthesis resulted in increased adipogenesis, insulin action, sirtuin deacetylase, and PPARγ transcriptional activity, whereas chemical inhibition and gene knockdown of each enzyme generating H2S (CTH, CBS, MPST) led to altered adipocyte differentiation, cellular senescence, and increased inflammation. In agreement with these experimental data, visceral and subcutaneous adipose tissue expression of H2S-synthesising enzymes was significantly reduced in morbidly obese subjects in association with attenuated adipogenesis and increased markers of adipose tissue inflammation and senescence. Interestingly, weight-loss interventions (including bariatric surgery or diet/exercise) improved the expression of H2S biosynthesis-related genes. In human preadipocytes, the expression of CTH, CBS, and MPST genes and H2S production were dramatically increased during adipocyte differentiation. More importantly, the adipocyte proteome exhibiting persulfidation was characterized, disclosing that different proteins involved in fatty acid and lipid metabolism, the citrate cycle, insulin signaling, several adipokines, and PPAR, experienced the most dramatic persulfidation (85-98%). Innovation: No previous studies investigated the impact of H2S on human adipose tissue. This study suggests that the potentiation of adipose tissue H2S biosynthesis is a possible therapeutic approach to improve adipose tissue dysfunction in patients with obesity and insulin resistance. Conclusion: Altogether, these data supported the relevance of H2S biosynthesis in the modulation of human adipocyte physiology. Antioxid. Redox Signal. 35, 319-340.

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.

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.

Dataset for proteomic analysis of Chlorella sorokiniana cells under cadmium stress.

Cadmium is one of the most hazardous heavy metal for aquatic environments and one of the most toxic contaminants for phytoplankton. This work provides the dataset associated with the research publication "Effect of cadmium in the microalga Chlorella sorokiniana: a proteomic study" [1]. This dataset describes a proteomic approach, based on the sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS), derived from exposure of Chlorella sorokiniana to 250 µM Cd2+ for 40 h, showing the proteins that are up- or downregulated. The processing of data included the identification of the Chlamydomonas reinhardtii protein sequences equivalent to the corresponding of Chlorella sorokiniana sequences obtained, which made possible to use KEGG Database. MS and MS/MS information, and quantitative data were deposited PRIDE public repository under accession number PXD015932.

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.