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.

A lipid droplet-targeting fluorescent probe for specific H2S imaging in biosamples and development of smartphone platform.

Hydrogen sulfide (H2S), a significant gas signal molecule, is closely related to various physiological/pathological processes. The monitoring of H2S is crucial in understanding the occurrence and development of diseases such as cancers. Emerging evidence suggests that abnormal regulation of Lipid droplets (LDs) is associated with many human diseases. For example, cancer cells are characterized by the abnormal accumulation of LDs. Therefore, understanding the relationship between LDs and cancer is of great significance for developing therapies against cancer. To address this challenge, we designed and developed a LD-targeting and H2S-activated probe (BTDA-DNB) by engineering a 2,4-dinitrophenyl ether (DNBE) as the H2S reactive site. In the presence of H2S, a strongly fluorescent emitter, 3-(benzo[d]thiazol-2-yl)-N,N-diethyl-2-imino-2H-chromen-7-amine (BTDA) was obtained with the leaving of DNBE group. BTDA-DNB displayed favorable sensitivity, selectivity and functioning well at physiological pH. The probe features excellent LD-targeting specificity and low cellular toxicity. The practical applications of LD-targeting probe BTDA-DNB as H2S probe in living cells, cancer tissues and Arabidopsis seedling have been evaluated. The excellent imaging performance demonstrates a potential ability for cancer diagnosis. Benefitted from the excellent performance on visual recognition H2S, a robust smartphone-integrated platform for H2S analysis was also successfully established.

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.

Functional characterization of the Serine acetyltransferase family genes uncovers the diversification and conservation of cysteine biosynthesis in tomato.

Sulfur-containing compounds are essential for plant development and environmental adaptation, and closely related to the flavor and nutrition of the agricultural products. Cysteine, the first organic sulfur-containing molecule generated in plants, is the precursor for most of these active substances. Serine acetyltransferase (SERAT) catalyzes the rate-limiting step of its formation. However, despite their importance, systematic analyses of these enzymes in individual species, especially in economically important crops, are still limited. Here, The SERAT members (SlSERATs, four in total) were identified and characterized in tomato. Phylogenetically, the four SlSERAT proteins were classified into three subgroups with distinct genomic structures and subcellular localizations. On the function, it was interesting to find that SlSERAT3;1, possessed a high ability to catalyze the formation of OAS, even though it contained a long C-terminus. However, it retained the essential C-terminal Ile, which seems to be a characteristic feature of SERAT3 subfamily members in Solanaceae. Besides, SlSERAT1;1 and SlSERAT2;2 also had high activity levels and their catalyzing abilities were significantly improved by the addition of an OAS-(thiol)-lyase protein. At the transcriptional level, the four SlSERAT genes had distinct expression patterns during tomato plant development. Under abiotic stress conditions, the chloroplast-localized SlSERATs were the main responders, and the SlSERATs adopted different strategies to cope with osmotic, ion toxicity and other stresses. Finally, analyses in the loss-of-function and overexpression lines of SlSERAT1;1 suggested that function redundancy existed in the tomato SERAT members, and the tomato SERAT member was ideal target for S-assimilation manipulating in molecular breeding.

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.

Identification and functional characterization of a cystathionine ß-lyase (CBL) enzyme for H2S production in Arabidopsis thaliana.

Sulfide or sulfur metabolism plays an important role in the growth and development of plants. Cystathionine ß-lyase (CBL) is an important enzyme in methionine synthesis, but a comprehensive understanding of CBL functions is limited. As the third gasotransmitter, hydrogen sulfide (H2S) plays important physiological roles in plants. In this study, we found that the endogenous H2S content in Arabidopsis thaliana cbl mutants was lower than that in the wild type. Under PEG-based osmotic stress conditions, the H2S contents of CBL-overexpression (OE-CBL) plants increased significantly compared with the wild type. Additionally, the OE-CBL plants increased their tolerance to osmotic stress by increasing the transcription levels of drought-related genes and their relative water-loss rates. Compared with cbl and wild type, OE-CBL plants resisted drought stress by significantly closing their stomata, resulting in improved survival rates. Root tip-bending experiments showed that CBL overexpression relieved osmotic, heavy metal and cold stresses in Arabidopsis. The recombinant CBL activity in vitro revealed that CBL produced H2S using L-cysteine as a substrate. Thus, CBL had a very strong cysteine desulfhydrase activity that could produce endogenous H2S using L-cysteine as a substrate, and it played an important role in plant abiotic stress resistance.

Hydrogen sulfide inhibits the abscission of tomato pedicel through reconstruction of a basipetal auxin gradient.

Abscission is an important developmental process and an essential agricultural trait. Auxin and ethylene are two phytohormones with important roles in the complex, but still elusive signaling network of abscission. Here, we found that hydrogen sulfide (H2S), a newly identified gasotransmitter, inhibits the initiation of tomato pedicel abscission. The underlying mechanism was explored through transcriptome profile analysis in various pedicel tissues with or without H2S treatment in the early abscission stage. The data suggested that H2S strongly influences the global transcription of pedicel tissues, exerts differential expression regulation along the pedicel, and markedly influences both the auxin and ethylene signaling pathways. Computational analysis revealed that H2S reconstructs a basipetal auxin gradient along the pedicel at 4 h after treatment; this finding was further substantiated by the GUS-staining results of DR5::GUS pedicels. The inhibitory effect of H2S to the ethylene signaling pathway might be an indirect action. Moreover, the subtilisin-like proteinase family members involved in the release of peptide signal molecules are critical components of the abscission signaling network downstream of auxin and ethylene.

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.

Effect of hydrogen sulfide on glycolysis-based energy production in mouse erythrocytes.

Hydrogen sulfide (H2 S) is a gasotransmitter that regulates both physiological and pathophysiological processes in mammalian cells. Recent studies have demonstrated that H2 S promotes aerobic energy production in the mitochondria in response to hypoxia, but its effect on anaerobic energy production has yet to be established. Glycolysis is the anaerobic process by which ATP is produced through the metabolism of glucose. Mammalian red blood cells (RBCs) extrude mitochondria and nucleus during erythropoiesis. These cells would serve as a unique model to observe the effect of H2 S on glycolysis-mediated energy production. The purpose of this study was to determine the effect of H2 S on glycolysis-mediated energy production in mitochondria-free mouse RBCs. Western blot analysis showed that the only H2 S-generating enzyme expressed in mouse RBCs is 3-mercaptopyruvate sulfurtransferase (MST). Supplement of the substrate for MST stimulated, but the inhibition of the same suppressed, the endogenous production of H2 S. Both exogenously administered H2 S salt and MST-derived endogenous H2 S stimulated glycolysis-mediated ATP production. The effect of NaHS on ATP levels was not affected by oxygenation status. On the contrary, hypoxia increased intracellular H2 S levels and MST activity in mouse RBCs. The mitochondria-targeted H2 S donor, AP39, did not affect ATP levels of mouse RBCs. NaHS at low concentrations (3-100 µM) increased ATP levels and decreased cell viability after 3 days of incubation in vitro. Higher NaHS concentrations (300-1000 µM) lowered ATP levels, but prolonged cell viability. H2 S may offer a cytoprotective effect in mammalian RBCs to maintain oxygen-independent energy production.

H2S Persulfidated and Increased Kinase Activity of MPK4 to Response Cold Stress in Arabidopsis.

Hydrogen sulfide (H2S) is a gasotransmitter along with nitric oxide and carbon oxide, which is involved in plant growth and development as well as biotic and abiotic stress resistance. In a previous study, we reported that mitogen-activated protein kinases, especially MPK4, are important downstream components of H2S involved in alleviating cold stress; however the underlying mechanism is unclear. In this study, we determined that the ability of H2S to alleviate cold stress is impaired in mpk4 mutants, but not in the upstream mek2 and crlk1 mutants. MPK4 was basically persulfidated, and NaHS (H2S donor) further increased the persulfidation level of MPK4. MEK2 was not persulfidated by H2S. NaHS treatments increased the MPK4 activity level nearly tenfold. The persulfidation signal of MPK4 did not disappear after eight cystein residues in MPK4 were site-mutated, respectively. Above all, our results suggested that H2S alleviates cold stress directly by persulfidating MPK4 and increasing the MPK4 kinase activity.

Hydrogen sulfide promotes flowering in heading Chinese cabbage by S-sulfhydration of BraFLCs.

Heading Chinese cabbage (Brassica rapa L. syn. B. campestris L. ssp. chinensis Makino var. pekinensis (Rupr.) J. Cao et Sh. Cao) is a cruciferous Brassica vegetable that has a triplicate genome, owing to an ancient genome duplication event. It is unclear whether the duplicated homologs have conserved or diversified functions. Hydrogen sulfide (H2S) is a plant gasotransmitter that plays important physiological roles in growth, development, and responses to environmental stresses. The modification of cysteines through S-sulfhydration is an important mechanism of H2S, which regulates protein functions. H2S promotes flowering in Arabidopsis and heading Chinese cabbage. Here we investigated the molecular mechanisms of H2S used to promote flowering in the latter. Four, five, and four BraFLC, BraSOC I, and BraFT homologs were identified in heading Chinese cabbage. Different BraFLC proteins were bound to different CArG boxes in the promoter regions of the BraSOC I and BraFT homologs, producing different binding patterns. Thus, there may be functionally diverse BraFLC homologs in heading Chinese cabbage. Exogenous H2S at 100 µmol L-1 significantly promoted flowering by compensating for insufficient vernalization. BraFLC 1 and BraFLC 3 underwent S-sulfhydration by H2S, after which their abilities to bind most BraSOC I or BraFT promoter probes weakened or even disappeared. These changes in binding ability were consistent with the expression pattern of the BraFT and BraSOC I homologs in seedlings treated with H2S. These results indicated that H2S signaling regulates flowering time. In summary, H2S signaling promoted plant flowering by weakening or eliminating the binding abilities of BraFLCs to downstream promoters through S-sulfhydration.

H2S-stimulated bioenergetics in chicken erythrocytes and the underlying mechanism.

The production of H2S and its effect on bioenergetics in mammalian cells may be evolutionarily preserved. Erythrocytes of birds, but not those of mammals, have a nucleus and mitochondria. In the present study, we report the endogenous production of H2S in chicken erythrocytes, which was mainly catalyzed by 3-mercaptopyruvate sulfur transferase (MST). ATP content of erythrocytes was increased by MST-generated endogenous H2S under normoxic, but not hypoxic, conditions. NaHS, a H2S salt, increased ATP content under normoxic, but not hypoxic, conditions. ATP contents in the absence or presence of NaHS were eliminated by different inhibitors for mitochondrial electron transport chain in chicken erythrocytes. Succinate and glutamine, but not glucose, increased ATP content. NaHS treatment similarly increased ATP content in the presence of glucose, glutamine, or succinate, respectively. Furthermore, the expression and activity of sulfide:quinone oxidoreductase were enhanced by NaHS. The structural integrity of chicken erythrocytes was largely maintained during 2-wk NaHS treatment in vitro, whereas most of the erythrocytes without NaHS treatment were lysed. In conclusion, H2S may regulate cellular bioenergetics as well as cell survival of chicken erythrocytes, in which the functionality of the electron transport chain is involved. H2S may have different regulatory roles and mechanisms in bioenergetics of mammalian and bird cells.

[Prokaryotic expression, purification and functional identification of epidermal pattern factors in Arabidopsis thaliana].

Stomatal density is important for crop yield. In this paper, we studied the epidermal pattern factors (EPFs) related to stomatal development. Prokaryotic expression vectors were constructed to obtain EPFs. Then the relationship between EPFs and hydrogen sulfide (H2S) was established. First, AtEPF1, AtEPF2 and AtEPFL9 were cloned and constructed to pET28a vectors. Then recombinant plasmids pET28a-AtEPF1, pET28a-AtEPF2 and pET28a-AtEPFL9 were digested and sequenced, showing successful construction. Finally, they were transformed into E. coli BL21(DE3) separately and induced to express by isopropyl ß-D-galactoside (IPTG). The optimized expression conditions including IPTG concentration (0.5, 0.3 and 0.05 mmol/L), temperature (28 °C, 28 °C and 16 °C) and induction time (16 h, 16 h and 20 h) were obtained. The bands of purified proteins were about 18 kDa, 19 kDa and 14.5 kDa, respectively. In order to identify their function, the purified AtEPF2 and AtEPFL9 were presented to Arabidopsis thaliana seedlings. Interestingly, the H2S production rate decreased or increased compared with the control, showing significant differences. That is, EPFs affected the production of endogenous H2S in plants. These results provide a foundation for further study of the relationship between H2S and EPFs on stomatal development, but also a possible way to increase the yield or enhance the stress resistance.

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.

Hydrogen Sulfide Regulates Energy Production to Delay Leaf Senescence Induced by Drought Stress in Arabidopsis.

Hydrogen sulfide (H2S) is a novel gasotransmitter in both mammals and plants. H2S plays important roles in various plant developmental processes and stress responses. Leaf senescence is the last developmental stage and is a sequential degradation process that eventually leads to leaf death. A mutation of the H2S-producing enzyme-encoding gene L-cysteine desulfhydrase1 (DES1) leads to premature leaf senescence but the underlying mechanisms are not clear. In this present study, wild-type, DES1 defective mutant (des1) and over-expression (OE-DES1) Arabidopsis plants were used to investigate the underlying mechanism of H2S signaling in energy production and leaf senescence under drought stress. The des1 mutant was more sensitive to drought stress and displayed accelerated leaf senescence, while the leaves of OE-DES1 contained adequate chlorophyll levels, accompanied by significantly increased drought resistance. Under drought stress, the expression levels of ATPß-1, -2, and -3 were significantly downregulated in des1 and significantly upregulated in OE-DES1, and ATPε showed the opposite trend. Senescence-associated gene (SAG) 12 correlated with age-dependent senescence and participated in the drought resistance of OE-DES1. SAG13, which was induced by environmental factors, responded positively to drought stress in des1 plants, while there was no significant difference in the SAG29 expression between des1 and OE-DES1. Using transmission electron microscopy, the mitochondria of des1 were severely damaged and bubbled in older leaves, while OE-DES1 had complete mitochondrial structures and a homogeneous matrix. Additionally, mitochondria isolated from OE-DES1 increased the H2S production rate, H2S content and ATPase activity level, as well as reduced swelling and lowered the ATP content in contrast with wild-type and des1 significantly. Therefore, at subcellular levels, H2S appeared to determine the ability of mitochondria to regulate energy production and protect against cellular aging, which subsequently delayed leaf senescence under drought-stress conditions in plants.

Hydrogen sulfide alleviates the cold stress through MPK4 in Arabidopsis thaliana.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that mediates physiological processes in animals and plants. In this study, we investigated the relationship of H2S and mitogen activated protein kinase (MAPK) under cold stress in Arabidopsis. H2S up-regulated MAPK expression levels and was involved in the cold stress-related upregulation of MAPK genes expression. We then chose MPK4 whose expression level was influenced the most by H2S as a target and found that H2S's ability to alleviate cold stress required MPK4. Both H2S and MPK4 regulated the expression levels of the cold response genes inducer of CBF expression 1 (ICE1), C-repeat-binding factors (CBF3), cold responsive 15A (COR15A) and cold responsive 15B (COR15B). H2S inhibited the opening of stomata under cold stress, which required the participation of MPK4. In conclusion, MPK4 is a downstream component of H2S-related cold-stress resistance, and H2S and MPK4 both regulated the cold response genes and stomatal movement to response the cold stress.

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.

Role of hydrogen sulfide in the methyl jasmonate response to cadmium stress in foxtail millet.

Methyl jasmonate (MeJA), a vital cellular regulator, mediates diverse developmental processes and defense responses against environmental stresse. Recently, a novel gasotransmitter, hydrogen sulfide (H2S), was found to have similar functions, but the interactions between H2S and MeJA in the acquisition of cadmium (Cd) tolerance have not been reported. Treating foxtail millet with 1 microM MeJA not only enhanced Cd tolerance and alleviated growth inhibitions but also decreased the contents of hydrogen peroxide, malondialdehyde and Cd in seedlings under 200 microM of Cd stress. Exogenous application of MeJA inhibited the transcript levels of the Natural Resistance-Associated Macrophage Protein (NRAMP1 and NRAMP6) and intensified Cd-induced expression of the homeostasis-related genes (MTP1, MTP12, CAX2 and ZIP4, besides HMA3). In addition, treatment with MeJA induced the production of endogenous H2S. Fumigation with sodium hydrosulfide (H2S donor) significantly enhanced MeJA-induced Cd tolerance, but this ability was weakened when H2S biosynthesis was inhibited with hydroxylamine. These results suggest that pretreatment with MeJA alleviated Cd stress and that this improvement was mediated by H2S in foxtail millet.