Exogenous hydrogen sulphide promotes plant flowering through the Arabidopsis splicing factor AtU2AF65a.

Alternative splicing (AS) is an important regulatory mode at the post-transcriptional level, through which many flowering genes regulate floral transition by producing multiple transcripts, and splicing factors have essential roles in this process. Hydrogen sulphide (H2S) is a newly found gasotransmitter that has critical physiological roles in plants, and one of its potential modes of action is via persulfidation of target proteins at specific cysteine sites. Previously, it has been shown that both the splicing factor AtU2AF65a and H2S are involved in the regulation of plant flowering. This study found that, in Arabidopsis, the promoting effect of H2S on flowering was abolished in atu2af65a-4 mutants. Transcriptome analyses showed that when AtU2AF65a contained mutations, the regulatory function of H2S during the AS of many flowering genes (including SPA1, LUH, LUG and MAF3) was inhibited. The persulfidation assay showed that AtU2AF65a can be persulfidated by H2S, and the RNA immunoprecipitation data indicated that H2S could alter the binding affinity of AtU2AF65a to the precursor messenger RNA of the above-mentioned flowering genes. Overall, our results suggest that H2S may regulate the AS of flowering-related genes through persulfidation of splicing factor AtU2AF65a and thus lead to early flowering in plants.

AtPRMT5-mediated AtLCD methylation improves Cd2+ tolerance via increased H2S production in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5), a highly conserved arginine (Arg) methyltransferase protein, regulates multiple aspects of the growth, development, and environmental stress responses by methylating Arg in histones and some mRNA splicing-related proteins in plants. Hydrogen sulfide (H2S) is a recently characterized gasotransmitter that also regulates various important physiological processes. l-cysteine desulfhydrase (LCD) is a key enzyme of endogenous H2S production. However, our understanding of the upstream regulatory mechanisms of endogenous H2S production is limited in plant cells. Here, we confirmed that AtPRMT5 increases the enzymatic activity of AtLCD through methylation modifications during stress responses. Both atprmt5 and atlcd mutants were sensitive to cadmium (Cd2+), whereas the overexpression (OE) of AtPRMT5 or AtLCD enhanced the Cd2+ tolerance of plants. AtPRMT5 methylated AtLCD at Arg-83, leading to a significant increase in AtLCD enzymatic activity. The Cd2+ sensitivity of atprmt5-2 atlcd double mutants was consistent with that of atlcd plants. When AtPRMT5 was overexpressed in the atlcd mutant, the Cd2+ tolerance of plants was significantly lower than that of AtPRMT5-OE plants in the wild-type background. These results were confirmed in pharmacological experiments. Thus, AtPRMT5 methylation of AtLCD increases its enzymatic activity, thereby strengthening the endogenous H2S signal and ultimately improving plant tolerance to Cd2+ stress. These findings provide further insights into the substrates of AtPRMT5 and increase our understanding of the regulatory mechanism upstream of H2S signals.

Profiling Cystathionine ß/γ-Lyase in Complex Biosamples Using Novel Activatable Fluorogens.

Cystathionine lyase, the key enzyme in transsulfuration and reverse transsulfuration pathways, is involved in a wide array of physiological and pathophysiological processes in both mammals and nonmammals. Though the biological significance of the hydrogen sulfide/cystathionine lyase system in disease states is extensively discussed, the absence of molecular methods for direct monitoring of cystathionine lyase in complex biosamples renders the result unreliable and perplexing. Here, we present the first attempt at designing and developing effective activatable fluorescent probes for cystathionine lyase based on the naphthylamide scaffold. CBLP and CSEP were designed based on the catalytic preference of cystathionine ß-lyase (CBL) and cystathionine γ-lyase (CSE). Briefly, incorporation of cysteine/homocysteine as the recognition moiety and a carbamate ethyl sulfide group as a self-immolated linker proved to be an effective strategy for cystathionine lyase fluorescence reporting. CBLP exhibits high selectivity and sensitivity in vitro in semiquantifying CBL levels in roots of wild-type Arabidopsis thaliana and cbl mutants (cbl knockout: SALK_014740C, overexpressed: OE-CBL). Meanwhile, CSEP successfully detected CSE levels in HCC-LM3 cells, zebrafish models, and upregulated CSE in frozen section slides from the liver tissue of cecal ligation and puncture (CLP)-induced septic rats, which was also validated by Western blotting and immunohistochemical analysis. In summary, the practical design strategy facilitates profiling of cystathionine lyase activity in biological processes. It may pave the way for the development of accurate and efficient methods for the direct estimation of cystathionine lyase.

Cystathionine gamma-lyase/H2 S signaling facilitates myogenesis under aging and injury condition.

Hydrogen sulfide (H2 S) can be endogenously produced and belongs to the class of signaling molecules known as gasotransmitters. Cystathionine gamma-lyase (CSE)-derived H2 S is implicated in the regulation of cell differentiation and the aging process, but the involvements of the CSE/H2 S system in myogenesis upon aging and injury have not been explored. In this study, we demonstrated that CSE acts as a major H2 S-generating enzyme in skeletal muscles and is significantly down-regulated in aged skeletal muscles in mice. CSE deficiency exacerbated the age-dependent sarcopenia and cardiotoxin-induced injury/regeneration in mouse skeletal muscle, possibly attributed to inefficient myogenesis. In contrast, supplement of NaHS (an H2 S donor) induced the expressions of myogenic genes and promoted muscle regeneration in mice. In vitro, incubation of myoblast cells (C2C12) with H2 S promoted myogenesis, as evidenced by the inhibition of cell cycle progression and migration, altered expressions of myogenic markers, elongation of myoblasts, and formation of multinucleated myotubes. Myogenesis was also found to upregulate CSE expression, while blockage of CSE/H2 S signaling resulted in a suppression of myogenesis. Mechanically, H2 S significantly induced the heterodimer formation between MEF2c and MRF4 and promoted the binding of MEF2c/MRF4 to myogenin promoter. MEF2c was S-sulfhydrated at both cysteine 361 and 420 in the C-terminal transactivation domain, and blockage of MEF2c S-sulfhydration abolished the stimulatory role of H2 S on MEF2c/MRF4 heterodimer formation. These findings support an essential role for H2 S in maintaining myogenesis, presenting it as a potential candidate for the prevention of age-related sarcopenia and treatment of muscle injury.

H2S aids osmotic stress resistance by S-sulfhydration of melatonin production-related enzymes in Arabidopsis thaliana.

KEY MESSAGE: Hydrogen sulfide closed Arabidopsis thaliana stomata by increasing the transcription of melatonin-producing enzymes and the post-translational modification levels to combat osmotic stress. Hydrogen sulfide (H2S) and melatonin (MEL) reportedly have similar functions in many aspects of plant growth, development and stress response. They regulate stomatal movement and enhance drought resistance. However, their physiological relationship is not well understood. Here, their crosstalk involved in osmotic stress resistance in Arabidopsis thaliana was studied. Exogenous H2S and MEL closed stomata under normal or osmotic stress conditions and increased the relative water contents of plants under osmotic stress conditions. At the same time, exogenous H2S and MEL responded to osmotic stress by increasing the content of proline and soluble sugar, and reducing malondialdehyde (MDA) content and relative conductivity. Using mutants in the MEL-associated production of serotonin N-acetyltransferase (snat), caffeic acid O-methyltransferase (comt1) and N-acetylserotonin methyltransferase (asmt), we determined that H2S was partially dependent on MEL to close stomata. Additionally, the overexpression of ASMT promoted stomatal closure. Exogenous H2S increased the transcription levels of SNAT, ASMT and COMT1. Furthermore, exogenous H2S treatments increased the endogenous MEL content significantly. At the post-translational level, H2S sulfhydrated the SNAT and ASMT, but not COMT1, enzymes associated with MEL production. Thus, H2S appeared to promote stomatal closure in response to osmotic stress by increasing the transcription levels of MEL synthesis-related genes and the sulfhydryl modification of the encoded enzymes. These results increased our understanding of H2S and MEL functions and interactions under osmotic stress conditions.

ATP-Binding Cassette G Transporters SGE1 and MtABCG13 Control Stigma Exsertion.

Stigma exsertion is an important agricultural trait that facilitates the application of heterosis in crop breeding. Although several quantitative trait loci associated with stigma exsertion have been fine-mapped or cloned, the underlying genetic basis, particularly in legumes, remains unclear. In this study, we identified and characterized the exserted stigma mutant stigma exsertion1 (sge1) in the model legume Medicago truncatula The exserted stigma phenotype of sge1 is mainly caused by physical interaction between floral organs, in which normal petal and stamen elongation are inhibited due to flower cuticle defects. SGE1 encodes an ATP-binding cassette G (ABCG) transporter that plays a critical role in regulating floral cutin and wax secretion in M. truncatula SGE1 physically interacts with another half-size transporter, MtABCG13, to form a functional heterodimer. Mutation of MtABCG13 results in flower cuticle defects similar to those in sge1 as well as stigma exsertion, indicating that SGE1 and MtABCG13 are indispensable for flower cuticle secretion and collaboratively control stigma exsertion in M. truncatula Our findings reveal novel functions for ABCG transporters in determining stigma exsertion by affecting the physical interactions of floral organs, providing insight into the molecular mechanism underlying stigma exsertion in leguminous plants with complex zygomorphic flowers.

SMALL LEAF AND BUSHY1 controls organ size and lateral branching by modulating the stability of BIG SEEDS1 in Medicago truncatula.

Organ size is a major agronomic trait that determines grain yield and biomass production in crops. However, the molecular mechanisms controlling organ size, especially in legumes, are poorly understood. Using forward genetic approaches in a Tnt1 insertion mutant population of the model legume Medicago truncatula, we identified SMALL LEAF AND BUSHY1 (SLB1), which is required for the control of organ size and lateral branching. Loss of function of SLB1 led to reduced leaf and flower size but increased lateral branch formation in M. truncatula. SLB1 encodes an F-box protein, an orthologue of Arabidopsis thaliana STERILE APETALA (SAP), that forms part of an SKP1/Cullin/F-box E3 ubiquitin ligase complex. Biochemical and genetic analyses revealed that SLB1 controls M. truncatula organ growth and lateral branching by modulating the stability of BIG SEEDS1 (BS1). Moreover, the overexpression of SLB1 increased seed and leaf size in both M. truncatula and soybean (Glycine max), indicating functional conservation. Our findings revealed a novel mechanism by which SLB1 targets BS1 for degradation to regulate M. truncatula organ size and shoot branching, providing a new genetic tool for increasing seed yield and biomass production in crop and forage legumes.

Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

KEY MESSAGE: This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L. Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a ß-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

The role of H2S in low temperature-induced cucurbitacin C increases in cucumber.

KEY MESSAGE: In this study, we first linked the signal molecule H2S with cucurbitacin C, which can cause the bitter taste of cucumber leaves and fruit, and specifically discuss its molecular mechanism. Cucurbitacin C (CuC), a triterpenoid secondary metabolite, enhances the resistance of cucumber plants to pathogenic bacteria and insect herbivores, but results in bitter-tasting fruits. CuC can be induced in some varieties of cucumber on exposure to plant stressors. The gasotransmitter hydrogen sulfide (H2S) participates in multiple physiological processes relating to plant stress resistance. This study focused on the effect of H2S on low temperature-induced CuC synthesis in cucumber. The results showed that treatment of cucumber leaves at 4 °C for 12 h enhanced the content and production rate of H2S and increased the expression of genes encoding enzymes involved in H2S generation, Csa2G034800.1 (CsaLCD), Csa1G574800.1 (CsaDES1), and Csa1G574810.1 (CsaDES2). In addition, treatment at 4 °C or with exogenous H2S upregulated the expression of CuC synthetase-encoding genes and the resulting CuC content in cucumber leaves, whereas pretreatment with hypotaurine (HT, a H2S scavenger) before treatment at 4 °C offset these effects. In vitro, H2S could increase the S-sulfhydration level of His-Csa5G156220 and His-Csa5G157230 (both bHLH transcription factors), as well as their binding activity to the promoter of Csa6G088690, which encodes the key synthetase for CuC generation. H2S pretreatment enhanced the cucumber leaves resistance to the Phytophthora melonis. Together, these results demonstrated that H2S acts as a positive regulator of CuC synthesis as a result of the modification of proteins by S-sulfhydration, also providing indirect evidence for the role of H2S in improving the resistance of plants to abiotic stresses and biotic stresses by regulating the synthesis of secondary metabolites.

Induction of cystathionine gamma-lyase expression and metallothionein-1 S-sulfhydration alleviate cadmium-induced cell death in myoblast cells.

Hydrogen sulfide (H2S), a multifunctional gasotransmitter, participates in a wide range of cellular signal transduction and pathophysiological processes. Cystathionine gamma-lyase (CSE) acts as a major H2S-generating enzyme in peripheral organs and tissues. As a cysteine-rich and heavy metal-binding protein, metallothionein-1 (MT-1) is known to protect cells from various environmental stresses. Here we demonstrated that exposure of cadmium (Cd) induced oxidative stress, depleted intracellular thiols, and stimulated apoptotic cell death in mouse myoblast cells. CSE expression and H2S production were significantly enhanced by Cd treatment. NaHS, a well-known H2S donor, at physiologically relevant concentration significantly alleviated Cd-induced damage in both myoblasts and mouse skeletal muscles. In contrast, down-regulation of CSE/H2S system deteriorated Cd-stimulated oxidative stress and cell death. Exposure of the cells to Cd lead to increased expressions of metal regulatory transcription factor 1 and MT-1, while siRNA-mediated MT-1 knockdown alleviated Cd-induced CSE expression and caused more oxidative stress and cell death. In addition, H2S post-translationally modified MT-1 by S-sulfhydration and stabilized zinc-protein complex. Taken together, these data suggest that CSE/H2S system would protect myoblasts and skeletal muscles from Cd-induced damage by S-sufhydrating MT-1.

AGAMOUS AND TERMINAL FLOWER controls floral organ identity and inflorescence development in Medicago truncatula.

Angiosperms integrate a multitude of endogenous and environmental signals to control floral development, thereby ensuring reproductive success. Here, we report the identification of AGAMOUS AND TERMINAL FLOWER (AGTFL), a novel regulator of floral development in Medicago truncatula. Mutation of AGTFL led to the transformation of carpels and stamens into numerous sepals and petals and altered primary inflorescence identity. AGTFL encodes a nucleus-localized protein containing a putative Myb/SANT-like DNA-binding domain and a PKc kinase domain. Molecular and genetic analyses revealed that AGTFL regulates the transcription of MtAGs and MtTFL1 to control floral organ identity and inflorescence development.

The Ca2+ /calmodulin2-binding transcription factor TGA3 elevates LCD expression and H2 S production to bolster Cr6+ tolerance in Arabidopsis.

Heavy metal (HM) contamination on agricultural land not only reduces crop yield but also causes human health concerns. As a plant gasotransmitter, hydrogen sulfide (H2 S) can trigger various defense responses and help reduce accumulation of HMs in plants; however, little is known about the regulatory mechanisms of H2 S signaling. Here, we provide evidence to answer the long-standing question about how H2 S production is elevated in the defense of plants against HM stress. During the response of Arabidopsis to chromium (Cr6+ ) stress, the transcription of L-cysteine desulfhydrase (LCD), the key enzyme for H2 S production, was enhanced through a calcium (Ca2+ )/calmodulin2 (CaM2)-mediated pathway. Biochemistry and molecular biology studies demonstrated that Ca2+ /CaM2 physically interacts with the bZIP transcription factor TGA3, a member of the 'TGACG'-binding factor family, to enhance binding of TGA3 to the LCD promoter and increase LCD transcription, which then promotes the generation of H2 S. Consistent with the roles of TGA3 and CaM2 in activating LCD expression, both cam2 and tga3 loss-of-function mutants have reduced LCD abundance and exhibit increased sensitivity to Cr6+ stress. Accordingly, this study proposes a regulatory pathway for endogenous H2 S generation, indicating that plants respond to Cr6+ stress by adjusting the binding affinity of TGA3 to the LCD promoter, which increases LCD expression and promotes H2 S production. This suggests that manipulation of the endogenous H2 S level through genetic engineering could improve the tolerance of grains to HM stress and increase agricultural production on soil contaminated with HMs.

Cystathionine gamma-lyase/hydrogen sulfide system is essential for adipogenesis and fat mass accumulation in mice.

Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter analogous to nitric oxide and carbon monoxide. Cystathionine gamma-lyase (CSE)-derived H2S is implicated in the regulation of insulin resistance and glucose metabolism, but the involvement of CSE/H2S system in energy homeostasis and fat mass has not been extensively explored. In this study, a potential functional role of the CSE/H2S system in in vitro adipocyte differentiation and in vivo adipogenesis and the underlying mechanism was investigated. CSE expression and H2S production were increased during adipocyte differentiation, and that the pattern of CSE mRNA expression was similar to that of CCAAT/enhancer-binding protein (C/EBP) ß and δ, two key regulators for adipogenesis. C/EBPß and γ bind to the CCAAT box in CSE promoter and stimulate CSE gene transcription. H2S induced PPARγ transactivation activity by S-sulfhydrating all the cysteine residues in the DNA binding domain and stimulated adipogenesis. High fat diet-induced fat mass was lost in CSE deficient mice, and exogenously applied H2S promoted fat mass accumulation in fruit flies. In conclusion, CSE/H2S system is essential for adipogenesis and fat mass accumulation through enhancement of PPARγ function in adipocytes. This study suggests that the CSE/H2S system is involved in the pathogenesis of obesity in mice.

Reversal of Sp1 transactivation and TGFß1/SMAD1 signaling by H2S prevent nickel-induced fibroblast activation.

Nickel as a heavy metal is known to bring threat to human health, and nickel exposure is associated with changes in fibroblast activation which may contribute to its fibrotic properties. H2S has recently emerged as an important gasotransmitter involved in numerous cellular signal transduction and pathophysiological responses. Interaction of nickel and H2S on fibroblast cell activation has not been studied so far. Here, we showed that a lower dose of nickel (200 µM) induced the activation of human fibroblast cells, as evidenced by increased cell growth, migration and higher expressions of α-smooth muscle actin (αSMA) and fibronectin, while high dose of nickel (1 mM) inhibited cell viability. Nickel reduced intracellular thiol contents and stimulated oxidative stress. Nickel also repressed the mRNA and protein expression of cystathionine gamma-lyase (CSE, a H2S-generating gene) and blocked the endogenous production of H2S. Exogenously applied NaHS (a H2S donor) had no effect on nickel-induced cell viability but significantly attenuated nickel-stimulated cell migration and the expression of αSMA and fibronectin. In contrast, CSE deficiency worsened nickel-induced αSMA expression. Moreover, H2S incubation reversed nickel-stimulated TGFß1/SMAD1 signal and blocked TGFß1-initiated expressions of αSMA and fibronectin. Nickel inhibited the interaction of Sp1 with CSE promoter but strengthened the binding of Sp1 with TGFß1 promoter, which was reversed by exogenously applied NaHS. These data reveal that H2S protects from nickel-stimulated fibroblast activation and CSE/H2S system can be a potential target for the treatment of tissue fibrosis induced by nickel.

H2S protects lipopolysaccharide-induced inflammation by blocking NFκB transactivation in endothelial cells.

Hydrogen sulfide (H2S) is a novel gasotransmitter and acts as a multifunctional regulator in various cellular functions. Past studies have demonstrated a significant role of H2S and its generating enzyme cystathionine gamma-lyase (CSE) in the cardiovascular system. Lipopolysaccharide (LPS), a major pathogenic factor, is known to initiate the inflammatory immune response. The cross talk between LPS-induced inflammation and the CSE/H2S system in vascular cells has not yet been elucidated in detail. Here we showed that LPS decreased CSE mRNA and protein expression in human endothelial cells and blocked H2S production in mouse aorta tissues. Transfection of the cells with TLR4-specific siRNA knockdown TLR4 mRNA expression and abolished the inhibitory role of LPS on CSE expression. Higher dose of LPS (100µg/ml) decreased cell viability, which was reversed by exogenously applied H2S at physiologically relevant concentration (30µM). Lower dose of LPS (10µg/ml) had no effect on cell viability, but significantly induced inflammation gene expressions and cytokines secretion and stimulated cell hyper-permeability. H2S treatment prevented LPS-induced inflammation and hyper-permeability. Lower VE-cadherin expression in LPS-incubated cells would contribute to cell hyper-permeability, which was reversed by H2S co-incubation. In addition, H2S treatment blocked LPS-induced NFκB transactivation. We further validated that LPS-induced hyper-permeability was reversed by CSE overexpression but further deteriorated by CRISPR/Cas9-mediated knockout of CSE. In vivo, deficiency of CSE sensitized the mice to LPS-induced inflammation in vascular tissues. Take together, these data suggest that CSE/H2S system protects LPS-induced inflammation and cell hyper-permeability by blocking NFκB transactivation.

H(2)S signaling in redox regulation of cellular functions.

Hydrogen sulfide (H(2)S) is traditionally recognized as a toxic gas with a rotten-egg smell. In just the last few decades, H(2)S has been found to be one of a family of gasotransmitters, together with nitric oxide and carbon monoxide, and various physiologic effects of H(2)S have been reported. Among the most acknowledged molecular mechanisms for the cellular effects of H(2)S is the regulation of intracellular redox homeostasis and post-translational modification of proteins through S-sulfhydration. On the one side, H(2)S can promote an antioxidant effect and is cytoprotective; on the other side, H(2)S stimulates oxidative stress and is cytotoxic. This review summarizes our current knowledge of the antioxidant versus pro-oxidant effects of H(2)S in mammalian cells and describes the Janus-faced properties of this novel gasotransmitter. The redox regulation for the cellular effects of H(2)S through S-sulfhydration and the role of H(2)S in glutathione generation is also recapitulated. A better understanding of H(2)S-regualted redox homeostasis will pave the way for future design of novel pharmacological and therapeutic interventions for various diseases.

Arginine methylation mediated by the Arabidopsis homolog of PRMT5 is essential for proper pre-mRNA splicing.

Protein arginine methylation, one of the most abundant and important posttranslational modifications, is involved in a multitude of biological processes in eukaryotes, such as transcriptional regulation and RNA processing. Symmetric arginine dimethylation is required for snRNP biogenesis and is assumed to be essential for pre-mRNA splicing; however, except for in vitro evidence, whether it affects splicing in vivo remains elusive. Mutation in an Arabidopsis symmetric arginine dimethyltransferase, AtPRMT5, causes pleiotropic developmental defects, including late flowering, but the underlying molecular mechanism is largely unknown. Here we show that AtPRMT5 methylates a wide spectrum of substrates, including some RNA binding or processing factors and U snRNP AtSmD1, D3, and AtLSm4 proteins, which are involved in RNA metabolism. RNA-seq analyses reveal that AtPRMT5 deficiency causes splicing defects in hundreds of genes involved in multiple biological processes. The splicing defects are identified in transcripts of several RNA processing factors involved in regulating flowering time. In particular, splicing defects at the flowering regulator flowering locus KH domain (FLK) in atprmt5 mutants reduce its functional transcript and protein levels, resulting in the up-regulation of a flowering repressor flowering locus C (FLC) and consequently late flowering. Taken together, our findings uncover an essential role for arginine methylation in proper pre-mRNA splicing that impacts diverse developmental processes.

Transcriptomic analysis reveals the functions of H2S as a gasotransmitter independently of Cys in Arabidopsis.

Numerous studies have revealed the gasotransmitter functions of hydrogen sulfide (H2S) in various biological processes. However, the involvement of H2S in sulfur metabolism and/or Cys synthesis makes its role as a signaling molecule ambiguous. The generation of endogenous H2S in plants is closely related to the metabolism of Cys, which play roles in a variety of signaling pathway occurring in various cellular processes. Here, we found that exogenous H2S fumigation and Cys treatment modulated the production rate and content of endogenous H2S and Cys to various degrees. Furthermore, we provided comprehensive transcriptomic analysis to support the gasotransmitter role of H2S besides as a substrate for Cys synthesis. Comparison of the differentially expressed genes (DEGs) between H2S and Cys treated seedlings indicated that H2S fumigation and Cys treatment caused different influences on gene profiles during seedlings development. A total of 261 genes were identified to respond to H2S fumigation, among which 72 genes were co-regulated by Cys treatment. GO and KEGG enrichment analysis of the 189 genes, H2S but not Cys regulated DEGs, indicated that these genes mainly involved in plant hormone signal transduction, plant-pathogen interaction, phenylpropanoid biosynthesis, and MAPK signaling pathway. Most of these genes encoded proteins having DNA binding and transcription factor activities that play roles in a variety of plant developmental and environmental responses. Many stress-responsive genes and some Ca2+ signal associated genes were also included. Consequently, H2S regulated gene expression through its role as a gasotransmitter, rather than just as a substrate for Cys biogenesis, and these 189 genes were far more likely to function in H2S signal transduction independently of Cys. Our data will provide insights for revealing and enriching H2S signaling networks.

H2S-mediated balance regulation of stomatal and non-stomatal factors responding to drought stress in Chinese cabbage.

Increased evidence has shown that hydrogen sulfide (H2S), a novel gasotransmitter, could enhance drought resistance in plants by inducing stomatal closure, with concurrent enhancement of photosynthetic efficiency, but little is known about the mechanism behind this contradictory phenomenon. This study examined the regulating mechanism of H2S in response to drought stress from stomatal and non-stomatal factors in Chinese cabbage. The results showed that exogenous H2S could increase the accumulation of photosynthetic pigments and alleviate the damage caused by drought stress. It also regulated the expression in transcriptional level and the activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (BrRuBisCO) under drought stress. The large subunit of BrRuBisCO was found to be modified by S-sulfhydration, which might be the reason for its increased enzyme activity. The fluxes of Cl-, K+, and H+ in the guard cells were detected by non-invasive micro-test techniques while under drought stress. The results indicated that H2S signaling induced a transmembrane Cl- and H+ efflux and inhibited K+ influx, and the Cl- channel was the main responders for H2S-regulated stomatal movement. In conclusion, H2S signal not only activated the ion channel proteins located in the guard cell membrane to induce stomatal closure, but also regulated the transcriptional expression and the activity of RuBisCO, a non-stomatal factor to enhance the photosynthetic efficiency of leaves. There is therefore a beneficial balance between the regulation of H2S signaling on stomatal factors and non-stomatal factors due to drought stress, which needs to be better understood to apply it practically to increase crop yields.

Specificity protein-1 as a critical regulator of human cystathionine gamma-lyase in smooth muscle cells.

Cystathionine γ-lyase (CSE) is the major enzyme in vascular smooth muscle cells (SMCs) that catalyzes the endogenous production of H(2)S. Phenotypic switching of SMCs is affected by endogenous H(2)S level and alterations of this switching may result in vascular disorders. To date, the mechanisms underlying the alteration of CSE expression and H(2)S production in vascular proliferative diseases have been unclear. In the present study, we found that serum deprivation induced SMC differentiation marker gene expressions and increased CSE expression and H(2)S production in cultured human aorta SMCs (HASMCs). Carotid artery ligation in mice resulted in enhanced neointima formation and down-regulation of CSE expression, suggesting an important role of CSE in SMC differentiation. Transient transfection of HASMCs with human CSE (hCSE) promoter/luciferase reporter revealed that the region between -226 to +140 base pair contains the core promoter for the hCSE gene. Deletion and mutation analysis demonstrated that two specificity protein-1 (Sp1) consensus binding sites were present in the core promoter region of the hCSE gene. Incubation of HASMCs with Sp1 binding inhibitor mithramycin inhibited CSE mRNA expression in a dose-dependent manner. Overexpression of Sp1 alone was sufficient to increase the activity of the hCSE core promoter and CSE protein expression. Chromatin immunoprecipitation assay showed that the binding of Sp1 to the hCSE promoter was increased in differentiated HASMCs compared with that in proliferated HASMCs. Exogenously applied H(2)S at 100 µM stimulated SMC differentiation, which was reversed by p38 MAPK inhibitor SB203580. These results suggest that transcript factor Sp1 is a critical regulator of the hCSE expression during SMC differentiation, and CSE/H(2)S system is essential for maintenance of SMC phenotype.