Hydrogen Sulfide: A Key Role in Autophagy Regulation from Plants to Mammalians

Autophagy is a degradative conserved process in eukaryotes to recycle unwanted cellular protein aggregates and damaged organelles. Autophagy plays an important role under normal physiological conditions in multiple biological processes, but it is induced under cellular stress. Therefore, it needs to be tightly regulated to respond to different cellular stimuli. In this review, the regulation of autophagy by hydrogen sulfide is described in both animal and plant systems. The underlying mechanism of action of sulfide is deciphered as the persulfidation of specific targets, regulating the pro-or anti-autophagic role of sulfide with a cell survival outcome. This review aims to highlight the importance of sulfide and persulfidation in autophagy regulation comparing the knowledge available in mammals and plants.Ministerio de Ciencia e Innovación 10.13039/ 501100011033, PID2019-109785GB-I00Junta de Andalucía P18-RT-3154, US-1255781Marie Skłodowska-Curie 83412

Hydrogen Sulfide Signaling in Plants: Emerging Roles of Protein Persulfidation

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.Unión Europea European Regional Development Fund BIO2016-76633-

Transient Transcriptional Regulation of the CYS-C1 Gene and Cyanide Accumulation upon Pathogen Infection in the Plant Immune Response

Cyanide is produced concomitantly with ethylene biosynthesis. Arabidopsis (Arabidopsis thaliana) detoxifies cyanide primarily through the enzyme b-cyanoalanine synthase, mainly by the mitochondrial CYS-C1. CYS-C1 loss of function is not toxic for the plant and leads to an increased level of cyanide in cys-c1 mutants as well as a root hairless phenotype. The classification of genes differentially expressed in cys-c1 and wild-type plants reveals that the high endogenous cyanide content of the cys-c1 mutant is correlated with the biotic stress response. Cyanide accumulation and CYS-C1 gene expression are negatively correlated during compatible and incompatible plant-bacteria interactions. In addition, cys-c1 plants present an increased susceptibility to the necrotrophic fungus Botrytis cinerea and an increased tolerance to the biotrophic Pseudomonas syringae pv tomato DC3000 bacterium and Beet curly top virus. The cys-c1 mutation produces a reduction in respiration rate in leaves, an accumulation of reactive oxygen species, and an induction of the alternative oxidase AOX1a and pathogenesis-related PR1 expression.We hypothesize that cyanide, which is transiently accumulated during avirulent bacterial infection and constitutively accumulated in the cys-c1 mutant, uncouples the respiratory electron chain dependent on the cytochrome c oxidase, and this uncoupling induces the alternative oxidase activity and the accumulation of reactive oxygen species, which act by stimulating the salicylic aciddependent signaling pathway of the plant immune systemEspaña MINECO BIO2010-15201Junta de Andalucía grant no. CVI-719

Mitochondrial Sulfide Detoxification Requires a Functional Isoform O-Acetylserine(thiol)lyase C in Arabidopsis thaliana

In non-cyanogenic species, the main source of cyanide derives from ethylene and camalexin biosyntheses. In mitochondria, cyanide is a potent inhibitor of the cytochrome c oxidase and is metabolised by the β-Cyanoalanine synthase CYS-C1, catalysing the conversion of cysteine and cyanide to hydrogen sulfide and β- cyanoalanine. The hydrogen sulfide released also inhibits the cytochrome c oxidase and needs to be detoxified by the O-acetylserine(thiol)lyase mitochondrial isoform, OAS-C, which catalyses the incorporation of sulfide to O-acetylserine to produce cysteine, thus generating a cyclic pathway in the mitochondria. The loss of functional OAS-C isoforms causes phenotypic characteristics very similar to the loss of the CYS-C1 enzyme, showing defects in root hair formation. Genetic complementation with the OAS-C gene rescues the impairment of root hair elongation restoring the wild type phenotype. The mitochondria compromise their capacity to proper detoxify cyanide and the resulting sulfide because the latter cannot re-assimilate into cysteine in the oas-c null mutant. Consequently, we observe an accumulation of sulfide and cyanide and of the alternative oxidase, which is unable to prevent the production of reactive oxygen species probably due to the accumulation of both toxic molecules. Our results allow us to suggest that the significance of OAS-C is related with its role in the proper sulfide and cyanide detoxification in mitochondria.Ministerio de Ciencia e Innovación BIO2010-15201Junta de Andalucía BIO–27

Hydrogen sulfide: From a toxic molecule to a key molecule of cell life

Hydrogen sulfide (H2S) has always been considered toxic, but a huge number of articles published more recently showed the beneficial biochemical properties of its endogenous production throughout all regna. In this review, the participation of H2S in many physiological and pathological processes in animals is described, and its importance as a signaling molecule in plant systems is underlined from an evolutionary point of view. H2S quantification methods are summarized and persulfidation is described as the underlying mechanism of action in plants, animals and bacteria. This review aims to highlight the importance of its crosstalk with other signaling molecules and its fine regulation for the proper function of the cell and its survival.EU Marie Skłodowska-Curie 834120Junta de Andalucía US-125578

Glutathione biosynthesis in Arabidopsis trichome cells

In Arabidopsis thaliana, trichome cells are specialized unicellular structures with uncertain functions. Based on earlier observations that one of the genes involved in cysteine biosynthesis (Atcys-3A) is highly expressed in trichomes, we have extended our studies in trichome cells to determine their capacity for glutathione (GSH) biosynthesis. First, we have analyzed by in situ hybridization the tissue-specific expression of the genes Atcys-3A and sat5, which encode O-acetylserine(thio)lyase (OASTL) and serine acetyltransferase (SAT), respectively, as well as gsh1 and gsh2, which encode γ-glutamylcysteine synthetase and glutathione synthetase, respectively. The four genes are highly expressed in leaf trichomes of Arabidopsis, and their mRNA accumulate to high levels. Second, we have directly measured cytoplasmic GSH concentration in intact cells by laser-scanning microscopy after labeling with monochlorobimane as a GSH-specific probe. From these measurements, cytosolic GSH concentrations of 238 ± 25, 80 ± 2, and 144 ± 19 μM were estimated for trichome, basement, and epidermal cells, respectively. Taking into account the volume of the cells measured using stereological techniques, the trichomes have a total GSH content more than 300-fold higher than the basement and epidermal cells. Third, after NaCl treatment, GSH biosynthesis is markedly decreased in trichomes. Atcys-3A, sat5, gsh1, and gsh2 mRNA levels show a decrease in transcript abundance, and [GSH](cyt) is reduced to 47 ± 5 μM. These results suggest the important physiological significance of trichome cells related to GSH biosynthesis and their possible role as a sink during detoxification processes.Dirección General de Investigación Científica y Técnica PB96-1367Junta de Andalucía CVI011

Cysteine and cysteine-related signaling pathways in arabidopsis thaliana

Cysteine occupies a central position in plant metabolism because it is a reduced sulfur donor molecule involved in the synthesis of essential biomolecules and defense compounds. Moreover, cysteine per se and its derivative molecules play roles in the redox signaling of processes occurring in various cellular compartments. Cysteine is synthesized during the sulfate assimilation pathway via the incorporation of sulfide to O-acetylserine, catalyzed by O-acetylserine(thiol)lyase (OASTL). Plant cells contain OASTLs in the mitochondria, chloroplasts, and cytosol, resulting in a complex array of isoforms and subcellular cysteine pools. In recent years, significant progress has been made in Arabidopsis, in determining the specific roles of the OASTLs and the metabolites produced by them. Thus, the discovery of novel enzymatic activities of the less-abundant, like DES1 with L-cysteine desulfhydrase activity and SCS with S-sulfocysteine synthase activity, has provided new perspectives on their roles, besides their metabolic functions. Thereby, the research has been demonstrated that cytosolic sulfide and chloroplastic S-sulfocysteine act as signaling molecules regulating autophagy and protecting the photosystems, respectively. In the cytosol, cysteine plays an essential role in plant immunity; in the mitochondria, this molecule plays a central role in the detoxification of cyanide, which is essential for root hair development and plant responses to pathogens.Ministerio de Economía y Competitividad BIO2010-15201Junta de Andalucía CVI-719

Signaling in the plant cytosol: cysteine or sulfide?

Cysteine (Cys) is the first organic compound containing reduced sulfur that is synthesized in the last stage of plant photosynthetic assimilation of sulfate. It is a very important metabolite not only because it is crucial for the structure, function and regulation of proteins but also because it is the precursor molecule of an enormous number of sulfur-containing metabolites essential for plant health and development. The biosynthesis of Cys is accomplished by the sequential reaction of serine acetyltransferase (SAT) and O-acetylserine(thiol)synthase (OASTL). In Arabidopsis thaliana, the analysis of specific mutants of members of the SAT and OASTL families has demonstrated that the cytosol is the compartment where the bulk of Cys synthesis takes place and that the cytosolic OASTL enzyme OAS-A1 is the responsible enzyme. Another member of the OASTL family is DES1, a novel L-cysteine desulfhydrase that catalyzes the desulfuration of Cys to produce sulfide, thus acting in a manner opposite to that of OAS-A1. Detailed studies of the oas-a1 and des1 null mutants have revealed the involvement of the DES1 and OAS-A1 proteins in coordinate regulation of Cys homeostasis and the generation of sulfide in the cytosol for signaling purposes. Thus, the levels of Cys in the cytosol strongly affect plant responses to both abiotic and biotic stress conditions, while sulfide specifically generated from the degradation of Cys negatively regulates autophagy induced in different situations. In conclusion, modulation of the levels of Cys and sulfide is likely critical for plant performance

Hydrogen sulfide action in the regulation of plant autophagy

Hydrogen sulfide is a signalling molecule with a well-established impact on both plant and animal physiology. Intense investigation into the regulation of autophagy by sulfide in Arabidopsis thaliana has revealed that the post-translational modification of persulfidation/S-sulfhydration plays a key role. In this review focused on plants, we discuss the nature of the sulfide molecule involved in the regulation of autophagy, the final outcome of this modification and the persulfidated autophagy proteins identified so far. A detailed outline of the actual knowledge of the regulation mechanism of the autophagy-related proteins ATG4a and ATG18a from Arabidopsis by sulfide is also included. This information will be instrumental for furthering research on the regulation of autophagy by sulfide.Ministerio de Ciencia e Innovación PID2019-109785GB-I00Junta de Andalucía 18-RT-3154, US-1255781European Union 83412