| Hydrogen gas (H2) was recently proposed as a novel antioxidant and signaling molecule in animals. However, the physiological roles of H2in plants are less clear. Ascorbic acid (AsA) was reported to mediate enhancement of plant tolerance against cadmium (Cd) stress. But the involved mechanisms of the effect of AsA were still few studied. To solve the above questions, we studied the topics and acquired results as follows:1. We showed that exposure of alfalfa seedlings to paraquat stress increased endogenous H2production. When supplied with exogenous H2, 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 H2. Further results showed that H2pretreatment could dramatically increase levels of the MsHO1transcript, levels of the protein it encodes, and HO1activity. The above-mentioned H2-mediated responses were specific for HO1, given that the potent HO1inhibitor counteracted the effects of H2. The effects of H2were reversed after the addition of carbon monoxide (CO). We also discovered enhanced tolerance of multiple environmental stresses after plants were pretreated with H2. Taking together, these results suggested that H2might function as an important gaseous molecule that alleviates oxidative stress via HO1signaling.2. We observed that H2production was significantly induced by abscisic acid (ABA) and drought stress. Alfalfa seedlings pretreated with H2in the form of hydrogen-rich water (HRW) were hypersensitive to exogenous ABA in terms of radicle emergence, root elongation, and stomatal closure. Meanwhile, seedlings pretreated with HRW could accumulate higher amounts of hydrogen peroxide (H2O2) in response to exogenous ABA or water deficit than the untreated plants, followed by the enhancement of plant tolerance against drought stress. Further pharmacological data showed that inhibition of NADPH oxidase and chemical scavenging of H2O2reduced HRW-induced drought tolerance. We also found that alfalfa seedlings showed significant increase in the apoplastic pH of leaves in response to HRW treatment or drought stress. Experiments with H+-ATPase inhibitor showed that vanadate could prevent the effect of H2on the alkalinization of the apoplastic sap and stomatal sensitivity to exogenous ABA or water deficit. These responses were interpreted as an effect of H2on sap pH and closure of stomata via an abscisic acid-based mechanism and suggest H2is a novel positive regulator in the alfalfa ABA-dependent drought response.3. Cd stress brought about severe oxidative damage and remarkable decrease in AsA content in alfalfa seedling roots. Exogenous AsA not only increased AsA content in vivo, and strengthened the up-regulation of alfalfa HO1transcript and HO activity triggered by Cd, but also significantly decreased Cd accumulation and oxidative damage, which was confirmed by the histochemical analysis. The responses of AsA were further impaired by the potent inhibitor of HO1, zinc protoporphyrin IX (ZnPP), which were blocked further when50%saturation of CO aqueous solution or bilirubin (BR), two catalytic by-products of HO1, was added, respectively. Molecular evidence illustrated that AsA-triggered the up-regulation of antioxidant enzyme genes, especially Mn-SOD and POD, were sensitive to ZnPP and reversed by CO. In short, above results suggested that cytoprotective roles triggered by AsA might be, at least partially, through HO1-dependent fashion by the induction of antioxidant system and lowering Cd accumulation. |
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