Weiti Cui

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.

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. CdCl2 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.

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.

Cadmium-induced hydrogen sulfide synthesis is involved in cadmium tolerance in Medicago sativa by reestablishment of reduced (homo)glutathione and reactive oxygen species homeostases.

Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H2S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H2S production as well as the activation of two H2S-synthetic enzymes activities, including L-cysteine desulfhydrase (LCD) and D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H2S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H2S production triggered by Cd, but also alleviated Cd toxicity in a H2S-dependent fashion. By contrast, the inhibition of H2S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H2S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.

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.

Melatonin confers plant tolerance against cadmium stress via the decrease of cadmium accumulation and reestablishment of microRNA-mediated redox homeostasis

Although melatonin-alleviated cadmium (Cd) toxicity both in animals and plants have been well studied, little is known about its regulatory mechanisms in plants. Here, we discovered that Cd stress stimulated the production of endogenous melatonin in alfalfa seedling root tissues. The pretreatment with exogenous melatonin not only increased melatonin content, but also alleviated Cd-induced seedling growth inhibition. The melatonin-rich transgenic Arabidopsis plants overexpressing alfalfa SNAT (a melatonin synthetic gene) exhibited more tolerance than wild-type plants under Cd conditions. Cd content was also reduced in root tissues. In comparison with Cd stress alone, ABC transporter and PCR2 transcripts in alfalfa seedlings, PDR8 and HMA4 in Arabidopsis, were up-regulated by melatonin. By contrast, Nramp6 transcripts were down-regulated. Changes in above transporters were correlated with the less accumulation of Cd. Additionally Cd-triggered redox imbalance was improved by melatonin. These could be supported by the changes of the Cu/Zn Superoxide Dismutase gene regulated by miR398a and miR398b. Histochemical staining, laser scanning confocal microscope, and H2O2 contents analyses showed the similar tendencies. Taking together, we clearly suggested that melatonin enhanced Cd tolerance via decreasing cadmium accumulation and reestablishing the microRNAs-mediated redox homeostasis.

The AtrbohF-dependent regulation of ROS signaling is required for melatonin-induced salinity tolerance in Arabidopsis

Abstract Although several literatures confirmed the beneficial roles of exogenous melatonin in the enhancement of salinity tolerance in plants, whether or how endogenous melatonin confers plant salinity tolerance is still elusive. In the report, we observed impaired melatonin level and salinity hypersensitivity in atsnat, the Arabidopsis melatonin synthesis mutant. Above hypersensitivity was rescued by melatonin or hydrogen peroxide. Meanwhile, melatonin‐mediated salt tolerance in wild‐type was abolished by an NADPH oxidase inhibitor, suggesting the possible role of NADPH oxidase‐dependent reactive oxygen species (ROS). Genetic evidence further showed that the rapid stimulated RbohF transcripts and production of ROS elicited by melatonin in stressed wild‐type plants were largely abolished by the mutation of AtrbohF. Meanwhile, salinity sensitivity of atrbohF mutant was not altered by melatonin, which was consistent with the higher Na+ content and the resulting greater Na+/K+ ratio, compared with those in wild‐type plants. Further changes of SOS1, SOS2, and SOS3 transcripts suggested that the melatonin‐triggered SOS‐mediated Na+ efflux might be mediated by AtrbohF‐dependent ROS. The addition of melatonin could intensify the increased antioxidant defence in stressed wild‐type but not in atrbohF mutant, both of which were confirmed by the histochemical staining for ROS production and lipid peroxidation during the later period of stress. Collectively, our genetic and molecular evidence revealed that the AtrbohF‐dependent ROS signaling is required for melatonin‐induced salinity tolerance via the reestablishment of ion and redox homeostasis. Graphical abstract Figure. No Caption available. HighlightsSalt hypersensitivity of atsnat mutant is rescued by melatonin and H2O2.Melatonin‐mediated salt tolerance is sensitive to DPI, an inhibitor of NADPH oxidase.AtrbohF is required for melatonin‐induced salinity tolerance.Ion homeostasis and redox homeostasis are reestablished by melatonin via AtrbohF.

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.

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.

Hydrogen-Modulated Stomatal Sensitivity to Abscisic Acid and Drought Tolerance Via the Regulation of Apoplastic pH in Medicago sativa

Hydrogen gas (H2) was recently proposed as a novel gaseous signaling molecule. In our previous study, H2-mediated enhancement of plant tolerance to drought stress was preliminarily suggested. However, the detailed mechanisms of the action of H2 have not been fully explored. In this study, we observed that H2 production and hydrogenase activity were significantly induced by abscisic acid (ABA) and drought stress. Alfalfa seedlings pretreated with hydrogen-rich water (HRW) were hypersensitive to exogenous ABA. In response to ABA or water deficit, HRW-pretreated seedlings rapidly accumulated higher amounts of hydrogen peroxide (H2O2), and exhibited more tolerance to drought stress. By contrast, the inhibition or scavenging of H2O2 reduced HRW-induced drought tolerance. Further results showed that the apoplastic pH of leaves was significantly increased by HRW and/or drought stress. Cotreatment with the H+-ATPase inhibitor, however, could prevent the effects of H2 on the alkalinization of the apoplastic sap and stomatal sensitivity to exogenous ABA or water deficit. These responses were interpreted as an effect of H2 on sap pH and closure of stomata in alfalfa via an ABA-based mechanism. Overall, these results suggested a novel regulating mechanism of H2 in plant drought response.