Yanjie Xie

Evidence of Arabidopsis salt acclimation induced by up‐regulation of HY1 and the regulatory role of RbohD‐derived reactive oxygen species synthesis

In Arabidopsis thaliana, a family of four genes (HY1, HO2, HO3 and HO4) encode haem oxygenase (HO), and play a major role in phytochrome chromophore biosynthesis. To characterize the contribution of the various haem oxygenase isoforms involved in salt acclimation, the effects of NaCl on seed germination and primary root growth in Arabidopsis wild-type and four HO mutants (hy1-100, ho2, ho3 and ho4) were compared. Among the four HO mutants, hy1-100 displayed maximal sensitivity to salinity and showed no acclimation response, whereas plants over-expressing HY1 (35S:HY1) exhibited tolerance characteristics. Mild salt stress stimulated biphasic increases in RbohD transcripts and production of reactive oxygen species (ROS) (peaks I and II) in wild-type. ROS peak I-mediated HY1 induction and subsequent salt acclimation were observed, but only ROS peak I was seen in the hy1-100 mutant. A subsequent test confirmed the causal relationship of salt acclimation with haemin-induced HY1 expression and RbohD-derived ROS peak II formation. In atrbohD mutants, haemin pre-treatment resulted in induction of HY1 expression, but no similar response was seen in hy1-100, and no ROS peak II or subsequent salt acclimatory responses were observed. Together, the above findings suggest that HY1 plays an important role in salt acclimation signalling, and requires participation of RbohD-derived ROS peak II.

Carbon monoxide enhances salt tolerance by nitric oxide-mediated maintenance of ion homeostasis and up-regulation of antioxidant defence in wheat seedling roots

Salt stress induced an increase in endogenous carbon monoxide (CO) production and the activity of the CO synthetic enzyme haem oxygenase (HO) in wheat seedling roots. In addition, a 50% CO aqueous solution, applied daily, not only resulted in the enhancement of CO release, but led to a significant reversal in dry weight (DW) and water loss caused by 150 mm NaCl treatment, which was mimicked by the application of two nitric oxide (NO) donors sodium nitroprusside (SNP) and diethylenetriamine NO adduct (DETA/NO). Further analyses showed that CO, as well as SNP, apparently up-regulated H(+)-pump and antioxidant enzyme activities or related transcripts, thus resulting in the increase of K/Na ratio and the alleviation of oxidative damage. Whereas, the CO/NO scavenger haemoglobin (Hb), NO scavenger or synthetic inhibitor methylene blue (MB) or N(G)-nitro-l-arginine methyl ester hydrochloride (l-NAME) differentially blocked these effects. Furthermore, CO was able to mimic the effect of SNP by strongly increasing NO release in the root tips, whereas the CO-induced NO signal was quenched by the addition of l-NAME or cPTIO, the specific scavenger of NO. The results suggested that CO might confer an increased tolerance to salinity stress by maintaining ion homeostasis and enhancing antioxidant system parameters in wheat seedling roots, both of which were partially mediated by NO signal.

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.

Haem oxygenase delays programmed cell death in wheat aleurone layers by modulation of hydrogen peroxide metabolism

Haem oxygenase-1 (HO-1) confers protection against a variety of oxidant-induced cell and tissue injury in animals and plants. In this report, it is confirmed that programmed cell death (PCD) in wheat aleurone layers is stimulated by GA and prevented by ABA. Meanwhile, HO activity and HO-1 protein expression exhibited lower levels in GA-treated layers, whereas the hydrogen peroxide (H2O2) content was apparently increased. The pharmacology approach illustrated that scavenging or accumulating H2O2 either delayed or accelerated GA-induced PCD. Furthermore, pretreatment with the HO-1 specific inhibitor, zinc protoporphyrin IX (ZnPPIX), before exposure to GA, not only decreased HO activity but also accelerated GA-induced PCD significantly. The application of the HO-1 inducer, haematin, and the enzymatic reaction product of HO, carbon monoxide (CO) aqueous solution, both of which brought about a noticeable induction of HO expression, substantially prevented GA-induced PCD. These effects were reversed when ZnPPIX was added, suggesting that HO in vivo played a role in delaying PCD. Meanwhile, catalase (CAT) and ascorbate peroxidase (APX) activities or transcripts were enhanced by haematin, CO, or bilirubin (BR), the catalytic by-product of HO. This enhancement resulted in a decrease in H2O2 production and a delay in PCD. In addition, the antioxidants butylated hydroxytoluene (BHT), dithiothreitol (DTT), and ascorbic acid (AsA) were able not only to delay PCD but also to mimic the effects of haematin and CO on HO up-regulation. Overall, the above results suggested that up-regulation of HO expression delays PCD through the down-regulation of H2O2 production.

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis

Hydrogen-mediated stomatal closure in Arabidopsis and drought tolerance involves RbohF-dependent ROS production, subsequent NR-associated NO production, and GORK activation. The signaling role of hydrogen gas (H2) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of H2 in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (ABA) induced stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering intracellular signaling events involving H2, reactive oxygen species (ROS), nitric oxide (NO), and the guard cell outward-rectifying K+ channel (GORK). ABA elicited a rapid and sustained H2 release and production in Arabidopsis. Exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H2 production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that HRW stimulated significant inductions of NO and ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant nitrate reductase1/2 (nia1/2) or the NADPH oxidase-deficient mutant rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the HRW-promoted NO generation is dependent on ROS production. The rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. In addition, both HRW and hydrogen peroxide failed to induce NO production or stomatal closure in the nia1/2 mutant, while HRW-promoted ROS accumulation was not impaired. In the GORK-null mutant, stomatal closure induced by ABA, HRW, NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of H2-regulated stomatal movement involved in the ABA signaling cascade in which RbohF-dependent ROS and nitric reductase-associated NO production, and subsequent GORK activation, were causally involved.

Roles of NIA/NR/NOA1-dependent nitric oxide production and HY1 expression in the modulation of Arabidopsis salt tolerance

Despite substantial evidence on the separate roles of Arabidopsis nitric oxide-associated 1 (NOA1)-associated nitric oxide (NO) production and haem oxygenase 1 (HY1) expression in salt tolerance, their integrative signalling pathway remains largely unknown. To fill this knowledge gap, the interaction network among nitrate reductase (NIA/NR)- and NOA1-dependent NO production and HY1 expression was studied at the genetic and molecular levels. Upon salinity stress, the majority of NO production was attributed to NIA/NR/NOA1. Further evidence confirmed that HY1 mutant hy1-100, nia1/2/noa1, and nia1/2/noa1/hy1-100 mutants exhibited progressive salt hypersensitivity, all of which were significantly rescued by three NO-releasing compounds. The salinity-tolerant phenotype and the stronger NO production in gain-of-function mutant of HY1 were also blocked by the NO synthetic inhibitor and scavenger. Although NO- or HY1-deficient mutants showed a compensatory mode of upregulation of HY1 or slightly increased NO production, respectively, during 2 d of salt treatment, downregulation of ZAT10/12-mediated antioxidant gene expression (cAPX1/2 and FSD1) was observed after 7 d of treatment. The hypersensitive phenotypes and stress-related genes expression profiles were differentially rescued or blocked by the application of NO- (in particular) or carbon monoxide (CO)-releasing compounds, showing a synergistic mode. Similar reciprocal responses were observed in the nia1/2/noa1/hy1-100 quadruple mutant, with the NO-releasing compounds exhibit the maximal rescuing responses. Overall, the findings present the combination of compensatory and synergistic modes, linking NIA/NR/NOA1-dependent NO production and HY1 expression in the modulation of plant salt tolerance.

Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone layers by the modulation of glutathione homeostasis and heme oxygenase-1 expression.

Hydrogen sulfide (H2S) is considered as a cellular signaling intermediate in higher plants, but corresponding molecular mechanisms and signal transduction pathways in plant biology are still limited. In the present study, a combination of pharmacological and biochemical approaches was used to study the effect of H2S on the alleviation of GA-induced programmed cell death (PCD) in wheat aleurone cells. The results showed that in contrast with the responses of ABA, GA brought about a gradual decrease of l-cysteine desulfhydrase (LCD) activity and H2S production, and thereafter PCD occurred. Exogenous H2S donor sodium hydrosulfide (NaHS) not only effectively blocked the decrease of endogenous H2S release, but also alleviated GA-triggered PCD in wheat aleurone cells. These responses were sensitive to hypotaurine (HT), a H2S scavenger, suggesting that this effect of NaHS was in an H2S-dependent fashion. Further experiment confirmed that H2S, rather than other sodium- or sulphur-containing compounds derived from the decomposing of NaHS, was attributed to the rescuing response. Importantly, the reversing effect was associated with glutathione (GSH) because the NaHS triggered increases of endogenous GSH content and the ratio of GSH/oxidized GSH (GSSG) in GA-treated layers, and the NaHS-mediated alleviation of PCD was markedly eliminated by l-buthionine-sulfoximine (BSO, a selective inhibitor of GSH biosynthesis). The inducible effect of NaHS was also ascribed to the modulation of heme oxygenase-1 (HO-1), because the specific inhibitor of HO-1 zinc protoporphyrin IX (ZnPP) significantly suppressed the NaHS-related responses. By contrast, the above inhibitory effects were reversed partially when carbon monoxide (CO) aqueous solution or bilirubin (BR), two of the by-products of HO-1, was added, respectively. NaHS-triggered HO-1 gene expression in GA-treated layers was also confirmed. Together, the above results clearly suggested that the H2S-delayed PCD in GA-treated wheat aleurone cells was associated with the modulation of GSH homeostasis and HO-1 gene expression.

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 alleviates aluminum-induced inhibition of root elongation in alfalfa via decreasing nitric oxide production.

One of the earliest and distinct symptoms of aluminum (Al) toxicity is the inhibition of root elongation. Although hydrogen gas (H2) is recently described as an important bio-regulator in plants, whether and how H2 regulates Al-induced inhibition of root elongation is largely unknown. To address these gaps, hydrogen-rich water (HRW) was used to investigate a physiological role of H2 and its possible molecular mechanism. Individual or simultaneous (in particular) exposure of alfalfa seedlings to Al, or a fresh but not old nitric oxide (NO)-releasing compound sodium nitroprusside (SNP), not only increased NO production, but also led to a significant inhibition of root elongation. Above responses were differentially alleviated by pretreatment with 50% saturation of HRW. The addition of HRW also alleviated the appearance of Al toxicity symptoms, including the improvement of seedling growth and less accumulation of Al. Subsequent results revealed that the removal of NO by the NO scavenger, similar to HRW, could decrease NO production and alleviate Al- or SNP-induced inhibition of root growth. Thus, we proposed that HRW alleviated Al-induced inhibition of alfalfa root elongation by decreasing NO production. Such findings may be applicable to enhance crop yield and improve stress tolerance.

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.