Characterization, Expression Profiling, and Biochemical Analyses of the Cinnamoyl-CoA Reductase Gene Family for Lignin Synthesis in Alfalfa Plants.

Cinnamoyl-CoA reductase (CCR) is a pivotal enzyme in plant lignin synthesis, which has a role in plant secondary cell wall development and environmental stress defense. Alfalfa is a predominant legume forage with excellent quality, but the lignin content negatively affects fodder digestibility. Currently, there is limited information on CCR characteristics, gene expression, and its role in lignin metabolism in alfalfa. In this study, we identified 30 members in the CCR gene family of Medicago sativa. In addition, gene structure, conserved motif, and evolution analysis suggested MsCCR1-7 presumably functioned as CCR, while the 23 MsCCR-likes fell into three categories. The expression patterns of MsCCRs/MsCCR-likes suggested their role in plant development, response to environmental stresses, and phytohormone treatment. These results were consistent with the cis-elements in their promoters. Histochemical staining showed that lignin accumulation gradually deepened with the development, which was consistent with gene expression results. Furthermore, recombinant MsCCR1 and MsCCR-like1 were purified and the kinetic parameters were tested under four substrates. In addition, three-dimensional structure models of MsCCR1 and MsCCR-like1 proteins showed the difference in the substrate-binding motif H212(X)2K215R263. These results will be useful for further application for legume forage quality modification and biofuels industry engineering in the future.

Transcriptome analysis reveals insight into molecular hydrogen-induced cadmium tolerance in alfalfa: the prominent role of sulfur and (homo)glutathione metabolism.

BACKGROUND: Hydrogen gas (H2) is hypothesised to play a role in plants that are coping with stresses by regulating signal transduction and gene expression. Although the beneficial role of H2 in plant tolerance to cadmium (Cd) has been investigated previously, the corresponding mechanism has not been elucidated. In this report, the transcriptomes of alfalfa seedling roots under Cd and/or hydrogen-rich water (HRW) treatment were first analysed. Then, the sulfur metabolism pathways were focused on and further investigated by pharmacological and genetic approaches. RESULTS: A total of 1968 differentially expressed genes (DEGs) in alfalfa seedling roots under Cd and/or HRW treatment were identified by RNA-Seq. The DEGs were classified into many clusters, including glutathione (GSH) metabolism, oxidative stress, and ATP-binding cassette (ABC) transporters. The results validated by RT-qPCR showed that the levels of relevant genes involved in sulfur metabolism were enhanced by HRW under Cd treatment, especially the genes involved in (homo)glutathione metabolism. Additional experiments carried out with a glutathione synthesis inhibitor and Arabidopsis thaliana cad2-1 mutant plants suggested the prominent role of glutathione in HRW-induced Cd tolerance. These results were in accordance with the effects of HRW on the contents of (homo)glutathione and (homo)phytochelatins and in alleviating oxidative stress under Cd stress. In addition, the HRW-induced alleviation of Cd toxicity might also be caused by a decrease in available Cd in seedling roots, achieved through ABC transporter-mediated secretion. CONCLUSIONS: Taken together, the results of our study indicate that H2 regulated the expression of genes relevant to sulfur and glutathione metabolism and enhanced glutathione metabolism which resulted in Cd tolerance by activating antioxidation and Cd chelation. These results may help to elucidate the mechanism governing H2-induced Cd tolerance in alfalfa.

Methane alleviates alfalfa cadmium toxicity via decreasing cadmium accumulation and reestablishing glutathione homeostasis.

Although methane (CH4) generation triggered by some environmental stimuli, displays the protective response against oxidative stress in plants, whether and how CH4 regulates plant tolerance against cadmium stress is largely unknown. Here, we discovered that cadmium (Cd) stimulated the production of CH4 in alfalfa root tissues. The pretreatment with exogenous CH4 could alleviate seedling growth inhibition. Less amounts of Cd accumulation was also observed. Consistently, in comparison with Cd stress alone, miR159 transcript was down-regulated by CH4, and expression levels of its target gene ABC transporter was increased. By contrast, miR167 transcript was up-regulated, showing a relatively negative correlation with its target gene Nramp6. Meanwhile, Cd-triggered redox imbalance was improved by CH4, evidenced by the reduced lipid peroxidation and hydrogen peroxide accumulation, as well as the induction of representative antioxidant genes. Further results showed that Cd-triggered decrease of the ratio of reduced/oxidized (homo)glutathione was rescued by CH4. Additionally, CH4-triggered alleviation of seedling growth was sensitive to a selective inhibitor of glutathione biosynthesis. Overall, above results revealed that CH4-alleviated Cd accumulation at least partially, required the modulation of heavy metal transporters via miR159 and miR167. Finally, the role of glutathione homeostasis elicited by CH4 was preliminarily suggested.

Nitric oxide is involved in methane-induced adventitious root formation in cucumber.

Our previous studies revealed that methane (CH4 ) induces adventitious rooting in cucumber. However, the corresponding molecular mechanism is still elusive. In this work, we discovered that CH4 triggered the accumulation of nitric oxide (NO) and thereafter cucumber adventitious rooting, mimicking the inducing effects of sodium nitroprusside (SNP) and NONOate (two NO-releasing compounds). Above mentioned responses were sensitive to NO scavenger(s), showing that the accumulation of NO and adventitious root development were respectively impaired. Inhibitor test and biochemical analysis suggested that endogenous NO mainly produced by mammalian NO synthase-like enzyme and diamine oxidases (DAO), might be required for adventitious root formation elicited by CH4 . Molecular evidence confirmed that CH4 -mediated induction of several marker genes responsible for adventitious root development, including CsDNAJ-1, CsCDPK1, CsCDPK5, cell division-related gene CsCDC6, and two auxin signaling genes, CsAux22D-like and CsAux22B-like, was casually dependent on NO signaling. The possible involvement of S-nitrosylation during the mentioned CH4 responses was preliminarily illustrated. Taken together, through pharmacological, anatomical and molecular approaches, it is suggested that NO might be involved in CH4 -induced cucumber adventitious rooting, and CH4 -eliciated NO-targeted proteins might be partially modulated at transcriptional and post-translational levels. Our work may increase the understanding of the mechanisms underlying CH4 -elicited root organogenesis in higher plants.

Methane alleviates copper-induced seed germination inhibition and oxidative stress in Medicago sativa.

Recent results discovered the protective roles of methane (CH4) against oxidative stress in animals. However, the possible physiological roles of CH4 in plants are still unknown. By using physiological, histochemical and molecular approaches, the beneficial role of CH4 in germinating alfalfa seeds upon copper (Cu) stress was evaluated. Endogenous production of CH4 was significantly increased in Cu-stressed alfalfa seeds, which was mimicked by 0.39 mM CH4. The pretreatment with CH4 significantly alleviated the inhibition of seed germination and seedling growth induced by Cu stress. Cu accumulation was obviously blocked as well. Meanwhile, α/ß amylase activities and sugar contents were increased, all of which were consistent with the alleviation of seed germination inhibition triggered by CH4. The Cu-triggered oxidative stress was also mitigated, which was confirmed by the decrease of lipid peroxidation and reduction of Cu-induced loss of plasma membrane integrity in CH4-pretreated alfalfa seedlings. The results of antioxidant enzymes, including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD) total or isozymatic activities, and corresponding transcripts (APX1/2, Cu/Zn SOD and Mn-SOD), indicated that CH4 reestablished cellular redox homeostasis. Further, Cu-induced proline accumulation was partly impaired by CH4, which was supported by the alternation of proline metabolism. Together, these results indicated that CH4 performs an advantageous effect on the alleviation of seed germination inhibition caused by Cu stress, and reestablishment of redox homeostasis mainly via increasing antioxidant defence.

Methane enhances aluminum resistance in alfalfa seedlings by reducing aluminum accumulation and reestablishing redox homeostasis.

Methane (CH4) is emerging as a candidate of signal molecule recently. However, whether or how CH4 enhances plant adaptation to aluminum (Al)-contaminated environment is still unknown. In this report, the physiological roles and possible molecular mechanisms of CH4 in the modulation of Al toxicity in alfalfa seedlings were characterized. Our results showed that, CH4 pretreatment could alleviate Al-induced seedling growth inhibition and redox imbalance. The defensive effects of CH4 against Al toxicity including the remission of Al-induced root elongation inhibition, nutrient disorder, and relative electrolyte leakage. Moreover, contents of organic acids, including citrate, malate, and oxalate, were increased by CH4. These results were paralleled by the findings of CH4 regulated organic acids metabolism and transport genes, citrate synthase, malate dehydrogenase, aluminum-activated malate transporter, and aluminum activated citrate transporter. Consistently, Al accumulation in seedling roots was decreased after CH4 treatment. In addition, Al-induced oxidative stress was also alleviated by CH4, through the regulation of the activities of anti-oxidative enzymes, such as ascorbate peroxidase, superoxide dismutase, and peroxidase, as well as their corresponding transcripts. Our data clearly suggested that CH4 alleviates Al toxicity by reducing Al accumulation in organic acid-dependent fashion, and reestablishing redox homeostasis.

Hydrogen Gas Is Involved in Auxin-Induced Lateral Root Formation by Modulating Nitric Oxide Synthesis.

Metabolism of molecular hydrogen (H2) in bacteria and algae has been widely studied, and it has attracted increasing attention in the context of animals and plants. However, the role of endogenous H2 in lateral root (LR) formation is still unclear. Here, our results showed that H2-induced lateral root formation is a universal event. Naphthalene-1-acetic acid (NAA; the auxin analog) was able to trigger endogenous H2 production in tomato seedlings, and a contrasting response was observed in the presence of N-1-naphthyphthalamic acid (NPA), an auxin transport inhibitor. NPA-triggered the inhibition of H2 production and thereafter lateral root development was rescued by exogenously applied H2. Detection of endogenous nitric oxide (NO) by the specific probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) and electron paramagnetic resonance (EPR) analyses revealed that the NO level was increased in both NAA- and H2-treated tomato seedlings. Furthermore, NO production and thereafter LR formation induced by auxin and H2 were prevented by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO; a specific scavenger of NO) and the inhibitor of nitrate reductase (NR; an important NO synthetic enzyme). Molecular evidence confirmed that some representative NO-targeted cell cycle regulatory genes were also induced by H2, but was impaired by the removal of endogenous NO. Genetic evidence suggested that in the presence of H2, Arabidopsis mutants nia2 (in particular) and nia1 (two nitrate reductases (NR)-defective mutants) exhibited defects in lateral root length. Together, these results demonstrated that auxin-induced H2 production was associated with lateral root formation, at least partially via a NR-dependent NO synthesis.

Heme-heme oxygenase 1 system is involved in ammonium tolerance by regulating antioxidant defence in Oryza sativa.

Despite substantial evidence showing the ammonium-altered redox homeostasis in plants, the involvement and molecular mechanism of heme-heme oxygenase 1 (heme-HO1), a novel antioxidant system, in the regulation of ammonium tolerance remain elusive. To fill in these gaps, the biological function of rice HO1 (OsSE5) was investigated. Results showed that NH4 Cl up-regulated rice OsSE5 expression. Oxidative stress and subsequent growth inhibition induced by excess NH4 Cl was partly mitigated by pretreatment with carbon monoxide (CO, a by-product of HO1 activity) or intensified by zinc protoporphyrin (ZnPP, a potent inhibitor of HO1 activity). Pretreatment with HO1 inducer hemin, not only up-regulated OsSE5 expression and HO activity, but also rescued the down-regulation of antioxidant transcripts, total and related isozymatic activities, thus significantly counteracting the excess NH4 Cl-triggered reactive oxygen species overproduction, lipid peroxidation and growth inhibition. OsSE5 RNAi transgenic rice plants revealed NH4 Cl-hypersensitive phenotype with impaired antioxidant defence, both of which could be rescued by CO but not hemin. Transgenic Arabidopsis plants over-expressing OsSE5 also exhibited enhanced tolerance to NH4 Cl, which might be attributed to the up-regulation of several antioxidant transcripts. Altogether, these results illustrated the involvement of heme-HO1 system in ammonium tolerance by enhancing antioxidant defence, which may improve plant tolerance to excess ammonium fertilizer.

Methane-rich water induces cucumber adventitious rooting through heme oxygenase1/carbon monoxide and Ca(2+) pathways.

KEY MESSAGE: Methane-rich water triggered adventitious rooting by regulating heme oxygenase1/carbon monoxide and calcium pathways in cucumber explants. Heme oxygenase1/carbon monoxide (HO1/CO) and calcium (Ca(2+)) were reported as the downstream signals in auxin-induced cucumber adventitious root (AR) formation. Here, we observed that application of methane-rich water (MRW; 80% saturation) obviously induced AR formation in IAA-depleted cucumber explants. To address the universality, we checked adventitious rooting in soybean and mung bean explants, and found that MRW (50 and 10% saturation, respectively) exhibited the similar inducing results. To further determine if the HO1/CO system participated in MRW-induced adventitious rooting, MRW, HO1 inducer hemin, its activity inhibitor zinc protoporphyrin IX (ZnPP), and its catalytic by-products CO, bilirubin, and Fe(2+) were used to detect their effects on cucumber adventitious rooting in IAA-depleted explants. Subsequent results showed that MRW-induced adventitious rooting was blocked by ZnPP and further reversed by 20% saturation CO aqueous solution. However, the other two by-products of HO1, bilirubin and Fe(2+), failed to induce AR formation. Above responses were consistent with the MRW-induced increases of HO1 transcript and corresponding protein level. Further molecular evidence indicted that expression of marker genes, including auxin signaling-related genes and cell cycle regulatory genes, were modulated by MRW alone but blocked by the cotreatment with ZnPP, the latter of which could be significantly rescued by the addition of CO. By using the Ca(2+)-channel blocker and Ca(2+) chelator, the involvement of Ca(2+) pathway in MRW-induced adventitious rooting was also suggested. Together, our results indicate that MRW might serve as a stimulator of adventitious rooting, which was partially mediated by HO1/CO and Ca(2+) pathways.

Hydrogen-rich water confers plant tolerance to mercury toxicity in alfalfa seedlings.

In this report, the effect of hydrogen-rich water (HRW), which was used to investigate the physiological roles of hydrogen gas (H2) in plants recently, on the regulation of plant adaptation to mercury (Hg) toxicity was studied. Firstly, we observed that the exposure of alfalfa seedlings to HgCl2 triggered production of reactive oxygen species (ROS), growth stunt and increased lipid peroxidation. However, such effects could be obviously blocked by HRW. Meanwhile, significant decreases in the relative ion leakage and Hg accumulation were observed. Hg-induced increases in total and isozymatic activities of superoxide dismutase (SOD) were significantly reversed by HRW. Further results suggested that HRW-induced the activities of guaiacol peroxidase (POD) and ascorbate peroxidase (APX), two hydrogen peroxide-scavenging enzymes, was at transcriptional levels. Meanwhile, obvious increases of the ratios of reduced/oxidized glutathione (GSH), homoglutathione (hGSH), and ascorbic acid (AsA) and corresponding gene expression were consistent with the decreased oxidative damage in seedling roots. In summary, the results of this investigation indicated that HRW was able to alleviate Hg toxicity in alfalfa seedlings by (i) alleviating growth stunt and reducing Hg accumulation, and (ii) avoidance of oxidative stress and reestablishment of redox homeostasis.

Hydrogen gas acts as a novel bioactive molecule in enhancing plant tolerance to paraquat-induced oxidative stress via the modulation of heme oxygenase-1 signalling system.

Hydrogen gas (H2) was recently proposed as a novel antioxidant and signalling molecule in animals. However, the physiological roles of H2 in plants are less clear. Here, we showed that exposure of alfalfa seedlings to paraquat stress increased endogenous H2 production. When supplied with exogenous H2 or the heme oxygenase-1 (HO-1)-inducer hemin, alfalfa plants displayed enhanced tolerance to oxidative stress induced by paraquat. This was evidenced by alleviation of the inhibition of root growth, reduced lipid peroxidation and the decreased hydrogen peroxide and superoxide anion radical levels. The activities and transcripts of representative antioxidant enzymes were induced after exposure to either H2 or hemin. Further results showed that H2 pretreatment could dramatically increase levels of the MsHO-1 transcript, levels of the protein it encodes and HO-1 activity. The previously mentioned H2-mediated responses were specific for HO-1, given that the potent HO-1-inhibitor counteracted the effects of H2. The effects of H2 were reversed after the addition of an aqueous solution of 50% carbon monoxide (CO). We also discovered enhanced tolerance of multiple environmental stresses after plants were pretreated with H2 . Together, these results suggested that H2 might function as an important gaseous molecule that alleviates oxidative stress via HO-1 signalling.

RNAi knockdown of rice SE5 gene is sensitive to the herbicide methyl viologen by the down-regulation of antioxidant defense.

Plant heme oxygenase (HO) catalyzes the oxygenation of heme to biliverdin, carbon monoxide (CO), and free iron (Fe(2+))-and Arabidopsis and rice (Oryza sativa) HOs are involved in light signaling. Here, we identified that the rice PHOTOPERIOD SENSITIVITY 5 (SE5) gene, which encoded a putative HO with high similarity to HO-1 from Arabidopsis (HY1), exhibited HO activity, and localized in the chloroplasts. Rice RNAi mutants silenced for SE5 were generated and displayed early flowering under long-day conditions, consistent with phenotypes of the null mutation in SE5 gene reported previously (se5 and s73). The herbicide methyl viologen (MV), which produces reactive oxygen species (ROS), was applied to determine whether SE5 regulates oxidative stress response. Compared with wild-type, SE5 RNAi transgenic plants aggravated seedling growth inhibition, chlorophyll loss and ROS overproduction, and decreased the transcripts of some representative antioxidative genes. By contrast, administration of exogenous CO partially rescued corresponding MV hypersensitivity in the SE5 RNAi plants. Alleviation of seed germination inhibition, chlorophyll loss and ROS overproduction, as well as the induction of antioxidant defense were further observed when SE5 or HY1 was overexpressed in transgenic Arabidopsis plants, indicating that SE5 may be useful for molecular breeding designed to improve plant tolerance to oxidative stress.

Mutation of Arabidopsis HY1 causes UV-C hypersensitivity by impairing carotenoid and flavonoid biosynthesis and the down-regulation of antioxidant defence.

Previous pharmacological results confirmed that haem oxygenase-1 (HO-1) is involved in protection of cells against ultraviolet (UV)-induced oxidative damage in soybean [Glycine max (L.) Merr.] seedlings, but there remains a lack of genetic evidence. In this study, the link between Arabidopsis thaliana HO-1 (HY1) and UV-C tolerance was investigated at the genetic and molecular levels. The maximum inducible expression of HY1 in wild-type Arabidopsis was observed following UV-C irradiation. UV-C sensitivity was not observed in ho2, ho3, and ho4 single and double mutants. However, the HY1 mutant exhibited UV-C hypersensitivity, consistent with the observed decreases in chlorophyll content, and carotenoid and flavonoid metabolism, as well as the down-regulation of antioxidant defences, thereby resulting in severe oxidative damage. The addition of the carbon monoxide donor carbon monoxide-releasing molecule-2 (CORM-2), in particular, and bilirubin (BR), two catalytic by-products of HY1, partially rescued the UV-C hypersensitivity, and other responses appeared in the hy1 mutant. Transcription factors involved in the synthesis of flavonoid or UV responses were induced by UV-C, but reduced in the hy1 mutant. Overall, the findings showed that mutation of HY1 triggered UV-C hypersensitivity, by impairing carotenoid and flavonoid synthesis and antioxidant defences.

Haem oxygenase-1 is involved in salicylic acid-induced alleviation of oxidative stress due to cadmium stress in Medicago sativa.

This work examines the involvement of haem oxygenase-1 (HO-1) in salicylic acid (SA)-induced alleviation of oxidative stress as a result of cadmium (Cd) stress in alfalfa (Medicago sativa L.) seedling roots. CdCl(2) exposure caused severe growth inhibition and Cd accumulation, which were potentiated by pre-treatment with zinc protoporphyrin (ZnPPIX), a potent HO-1 inhibitor. Pre-treatment of plants with the HO-1 inducer haemin or SA, both of which could induce MsHO1 gene expression, significantly reduced the inhibition of growth and Cd accumulation. The alleviation effects were also evidenced by a decreased content of thiobarbituric acid-reactive substances (TBARS). The antioxidant behaviour was confirmed by histochemical staining for the detection of lipid peroxidation and the loss of plasma membrane integrity. Furthermore, haemin and SA pre-treatment modulated the activities of ascorbate peroxidase (APX), superoxide dismutase (SOD), and guaiacol peroxidase (POD), or their corresponding transcripts. Significant enhancement of the ratios of reduced/oxidized homoglutathione (hGSH), ascorbic acid (ASA)/dehydroascorbate (DHA), and NAD(P)H/NAD(P)(+), and expression of their metabolism genes was observed, consistent with a decreased reactive oxygen species (ROS) distribution in the root tips. These effects are specific for HO-1, since ZnPPIX blocked the above actions, and the aggravated effects triggered by SA plus ZnPPIX were differentially reversed when carbon monoxide (CO) or bilirubin (BR), two catalytic by-products of HO-1, was added. Together, the results suggest that HO-1 is involved in the SA-induced alleviation of Cd-triggered oxidative stress by re-establishing redox homeostasis.

An unexpected discovery toward argon-rich water amelioration of cadmium toxicity in Medicago sativa L.

Argon has organ-protective effects on animals. However, whether or how argon influences plant responses remains elusive. In this study, we discovered that the growth inhibition of hydroponically cultured alfalfa seedlings under 100 µM CdCl2 condition was significantly ameliorated by 100 % saturated argon-rich water (ARW). Less Cd uptake and accumulation were also observed in both root and shoot parts, which could be explained by the modified root cell walls, including the increased cell wall thickness, lignin content, and demethylation degree of covalently bound and ion-bound pectin, as well as the down-regulated expression of natural-resistance-associated-macrophage protein1 (Nramp1) encoding a heavy metal ion transporter in root tissue. The hindered Cd translocation from root to shoot achieved by ARW addition was validated by the decreased expression of heavy metal ATPase 2/4 (HMA2/4) in roots and decreased Cd content in xylem saps. The reestablished glutathione (GSH) homeostasis and redox balance, two important indicators of plant defense against Cd poisoning, were also observed. Further greenhouse experiments demonstrated that the phenotypic and physiological performances of alfalfa plants cultured in Cd-contaminated soil were significantly improved by irrigating with ARW. Above results implied that ARW confers plants tolerance against cadmium toxicity by impairing Cd uptake and accumulation and restoring GSH and redox homeostasis. These findings might open a new window for understanding argon biology in higher plants.

Cadmium-induced heme oxygenase-1 gene expression is associated with the depletion of glutathione in the roots of Medicago sativa.

Following previous findings that cadmium (Cd) induces heme oxygenase-1 (HO1) gene expression in alfalfa seedling roots, we now show that the decreased glutathione (GSH) and ascorbic acid (AsA) contents, induction of HO-1 gene expression and its protein level by Cd was mimicked by a GSH depletor diethylmaleate (DEM). Meanwhile, above Cd- or DEM-induced decreased GSH content followed by HO-1 up-regulation could be strengthened or reversed differentially by the application of a selective inhibitor of GSH biosynthesis L: -buthionine-sulfoximine (BSO), or exogenous GSH and AsA, respectively. The antioxidative behavior of HO-1 induction was further confirmed by histochemical staining for the detection of loss of membrane integrity in a short period of treatment time. Additionally, the induction of HO-1 transcript was inhibited by the transcriptional inhibitor actinomycin D (ActD) or protein synthesis inhibitor cycloheximide (CX, especially). In contrast, the level of HO-2 transcript did not change upon various treatments. Together, above results suggested that Cd-induced up-regulation of HO-1 gene expression is associated with GSH depletion, which is at least existing transcriptional regulation level, thus leading to enhanced antioxidative capability transiently.

Heme oxygenase-1 is associated with wheat salinity acclimation by modulating reactive oxygen species homeostasis.

Heme oxygenase-1 (HO-1) has been recently identified as an endogenous signaling system in animals. In this study, HO-1 upregulation and its role in acquired salt tolerance (salinity acclimation) were investigated in wheat plants. We discovered that pretreatment with a low concentration of NaCl (25 mmol/L) not only led to the induction of HO-1 protein and gene expression, as well as enhanced HO activity, but also to a salinity acclimatory response thereafter. The effect is specific for HO-1, since the potent HO-1 inhibitor zinc protoporphyrin IX blocks the above cytoprotective actions, and the cytotoxic responses conferred by 200 mmol/L NaCl are reversed partially when HO-1 inducer hemin is added. Heme oxygenase catalytic product, carbon monoxide (CO) aqueous solution pretreatment, mimicked the salinity acclimatory responses. Meanwhile, the CO-triggered re-establishment of reactive oxygen species (ROS) homeostasis was mainly guaranteed by the induction of total and isozymatic activities, or corresponding transcripts of superoxide dismutase, ascorbate peroxidase, and cytosolic peroxidase (POD), as well as the downregulation of NADPH oxidase expression and cell-wall POD activity. A requirement of hydrogen peroxide homeostasis for HO-1-mediated salinity acclimation was also discovered. Taken together, the above results suggest that the upregulation of HO-1 expression was responsible for the observed salinity acclimation through the regulation of ROS homeostasis.

The Importance of Nitric Oxide as the Molecular Basis of the Hydrogen Gas Fumigation-Induced Alleviation of Cd Stress on Ganoderma lucidum.

Whether or not hydrogen gas (H2) can reduce cadmium (Cd) toxicity in Ganoderma lucidum has remained largely unknown. Here, we report that Cd-induced growth inhibition in G. lucidum was significantly alleviated by H2 fumigation or hydrogen-rich water (HRW), evaluated by lower oxidative damage and Cd accumulation. Moreover, the amelioration effects of H2 fumigation were better than of HRW in an optimum concentration of H2 under our experimental conditions. Further results showed that H2-alleviated growth inhibition in G. lucidum was accompanied by increased nitric oxide (NO) level and nitrate reductase (NR) activity under Cd stress. On the other hand, the mitigation effects were reversed after removing endogenous NO with its scavenger cPTIO or inhibiting H2-induced NR activity with sodium tungstate. The role of NO in H2-alleviated growth inhibition under Cd stress was proved to be achieved through a restoration of redox balance, an increase in cysteine and proline contents, and a reduction in Cd accumulation. In summary, these results clearly revealed that NR-dependent NO might be involved in the H2-alleviated Cd toxicity in G. lucidum through rebuilding redox homeostasis, increasing cysteine and proline levels, and reducing Cd accumulation. These findings may open a new window for H2 application in Cd-stressed economically important fungi.

Hydrogen sulfide acts downstream of methane to induce cucumber adventitious root development.

Previous results have shown that hydrogen sulfide (H2S), mainly catalyzed by l-cysteine desulfhydrase (DES) in plants, triggers adventitious rooting. The objective of this study was to test whether H2S is involved in methane (CH4)-induced adventitious root development in cucumber explants. First, we observed that the activities of DES, endogenous H2S production, and thereafter adventitious root development were induced by CH4 and NaHS (an H2S donor). Some responses were sensitive to hypotaurine (HT; a scavenger of H2S), showing that endogenous H2S production and adventitious rooting were obviously blocked. The development of adventitious root primordia was also impaired. Further molecular evidence revealed that CH4-induced gene expression of CsDNAJ-1, CsCDPK1, CsCDPK5, CsCDC6 (a cell-division-related gene), CsAux22D-like, and CsAux22B-like (two auxin-signaling genes), several molecular markers responsible for adventitious rooting, were blocked by the co-treatment with HT. The occurrence of CH4-elicited S-sulfhydration during the above responses was also sensitive to the removal of endogenous H2S, suggesting the requirement of H2S. Taken together, our results reveal a vital role of endogenous H2S in CH4-triggered cucumber adventitious root development, and thus provide a comprehensive window into the complex signaling transduction pathway in CH4-mediated root organogenesis.

Proteomic analysis provides insights into the molecular bases of hydrogen gas-induced cadmium resistance in Medicago sativa.

Recently, molecular hydrogen (H2) has emerged as a bio-regulator both in animals and plants. Normally, functions of endogenous generated H2 could be mimicked by exogenously applied hydrogen-rich water (HRW) or hydrogen-rich saline (particularly in animals). Although alfalfa seedlings showed more cadmium (Cd) resistance after the administration with HRW, corresponding molecular mechanism is still elusive. To address this gap, iTRAQ-based quantitative proteomics was used. The results showed that a total of 2377 proteins were identified with <1% FDR, and 1254 protein abundance perturbations were confidently assessed. Total of 248 significant differential proteins were identified in Cd- and/or HRW-treated samples. Furthermore, 92 proteins from the 248 proteins were selected for further bioinformatics analysis. Interestingly, results indicated that they were classified into seven categories: defense and response to stress, sulfur compound metabolic process, amino acid and protein metabolic process, carbohydrate and energy metabolic process, secondary metabolic process, oxidation-reduction process, and metal ion homeostasis. In addition, the protein expression patterns were consistent with the results of decreased lipid peroxidation, increased non-protein thiols abundance, as well as iron and zinc content. These suggest that HRW alleviates Cd toxicity mainly by decreasing oxidative damage, enhancing sulfur compound metabolic process, and maintaining nutrient element homeostasis. BIOLOGICAL SIGNIFICANCE: Contamination of soils by Cd has become a potential concern to crops. Medicago sativa is a widely used forage around the world. Recently, hydrogen gas (H2) was suggested as a candidate of signal molecule, and found to effectively attenuate Cd-induced damage in alfalfa seedlings. However, the underlying molecular mechanism still needs to be further elucidated. In the present work, an iTRAQ-based quantitative proteomics was firstly carried out, and the results revealed the main molecular targets and metabolic processes associated with Cd resistance conferred by H2. This study may expand our understanding of hydrogen gas-medicated heavy metal tolerance in plants.