Wenbiao Shen

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.

The Heme Oxygenase/Carbon Monoxide System Is Involved in the Auxin-Induced Cucumber Adventitious Rooting Process

Indole acetic acid (IAA) is an important regulator of adventitious rooting via the activation of complex signaling cascades. In animals, carbon monoxide (CO), mainly generated by heme oxygenases (HOs), is a significant modulator of inflammatory reactions, affecting cell proliferation and the production of growth factors. In this report, we show that treatment with the auxin transport inhibitor naphthylphthalamic acid prevented auxin-mediated induction of adventitious rooting and also decreased the activity of HO and its by-product CO content. The application of IAA, HO-1 activator/CO donor hematin, or CO aqueous solution was able to alleviate the IAA depletion-induced inhibition of adventitious root formation. Meanwhile, IAA or hematin treatment rapidly activated HO activity or HO-1 protein expression, and CO content was also enhanced. The application of the HO-1-specific inhibitor zinc protoporphyrin IX (ZnPPIX) could inhibit the above IAA and hematin responses. CO aqueous solution treatment was able to ameliorate the ZnPPIX-induced inhibition of adventitious rooting. Molecular evidence further showed that ZnPPIX mimicked the effects of naphthylphthalamic acid on the inhibition of adventitious rooting, the down-regulation of one DnaJ-like gene (CSDNAJ-1), and two calcium-dependent protein kinase genes (CSCDPK1 and CSCDPK5). Application of CO aqueous solution not only dose-dependently blocked IAA depletion-induced inhibition of adventitious rooting but also enhanced endogenous CO content and up-regulated CSDNAJ-1 and CSCDPK1/5 transcripts. Together, we provided pharmacological, physiological, and molecular evidence that auxin rapidly activates HO activity and that the product of HO action, CO, then triggers the signal transduction events that lead to the auxin responses of adventitious root formation in cucumber (Cucumis sativus).

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.

Roles of hydrogen sulfide and nitric oxide in the alleviation of cadmium-induced oxidative damage in alfalfa seedling roots

Despite hydrogen sulfide (H2S) and nitric oxide (NO) are important endogenous signals or bioregulators involved in many vital aspects of plant growth and responses against abiotic stresses, little information was known about their interaction. In the present study, we evaluated the effects of H2S and NO on alfalfa (Medicago sativa L.) plants exposed to cadmium (Cd) stress. Pretreatment with an H2S donor sodium hydrosulfide (NaHS) and well-known NO donor sodium nitroprusside (SNP) decreased the Cd toxicity. This conclusion was supported by the decreases of lipid peroxidation as well as the amelioration of seedling growth inhibition and Cd accumulation, in comparison with the Cd-stressed alone plants. Total activities and corresponding transcripts of antioxidant enzymes, including superoxide dismutase, peroxidase and ascorbate peroxidase were modulated differentially, thus leading to the alleviation of oxidative damage. Effects of H2S above were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the specific scavenger of NO. By using laser confocal scanning microscope combined with Greiss reagent method, further results showed that NO production increased significantly after the NaHS pretreatment regardless of whether Cd was applied or not, all of which were obviously inhibited by cPTIO. These decreases of NO production were consistent with the exaggerated syndromes associated with Cd toxicity. Together, above results suggested that NO was involved in the NaHS-induced alleviation of Cd toxicity in alfalfa seedlings, and also indicated that there exists a cross-talk between H2S and NO responsible for the increased abiotic stress tolerance.

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.

Heme oxygenase/carbon monoxide system participates in regulating wheat seed germination under osmotic stress involving the nitric oxide pathway.

To investigate the mechanism and signaling pathway of carbon monoxide (CO) and hematin in alleviating seed germination inhibition and lipid peroxidation, polyethylene glycol-6000 (PEG) was used to mimic osmotic stress in a series of experiments. The results showed that wheat seeds pretreated with a lower dose of PEG (12.5%) showed higher tolerance against osmotic stress as well as the up-regulation of heme oxygenase (HO, EC 1.14.99.3) and decreased lipid peroxidation during recuperation, compared to those with a higher dose of PEG (50%). Exposure of wheat seeds to 25% PEG, HO-1 inhibitor or specific scavenger of nitric oxide (NO) alone differentially led to seed germination inhibition. The PEG-induced inhibitory effects on seed germination were ameliorated by the HO-1 inducer hematin, CO or NO donor. Additionally, hematin was able to markedly boost the HO/CO system. However, the addition of the HO-1 inhibitor or the specific scavenger of NO not only reversed the protective effects conferred by hematin, but also blocked the up-regulation of HO/CO. In addition, hematin-driven NO production in wheat seeds under osmotic stress was confirmed. Based on these results, we conclude that the endogenous HO/CO signal system is required for the alleviation of osmotic stress-induced wheat seed germination inhibition and lipid peroxidation, which might have a possible interaction with NO.

Haem Oxygenase-1 is Involved in Hydrogen Sulfide-induced Cucumber Adventitious Root Formation

Results from our previous study suggested that haem oxygenase-1/carbon monoxide (HO-1/CO) acts as a downstream signal system in the auxin-induced pathway leading to cucumber (Cucumis sativus) adventitious root formation. The objective of this study was to test whether HO-1 is also involved in hydrogen sulfide (H2S)-induced adventitious root formation. Cucumber explants were treated with HO-1 inducer haemin and H2S donor sodium hydrosulfide (NaHS) in combination with the specific inhibitor of HO-1 zinc protoporphyrin IX (ZnPPIX), and their effects on cucumber adventitious root development in IAA-depleted explants were compared. The results showed that similar to inducible responses of haemin, NaHS brought about the induction of cucumber HO-1 transcripts (CsHO-1) and its protein levels, and thereafter adventitious root formation. A further experiment verified that H2S or HS- rather than other sulfur-containing components derived from NaHS was ascribed to the stimulation response. The inducible effect is specific for CsHO-1 because ZnPPIX significantly suppressed the above responses, and the inhibitory effects were reversed partially when 30% CO-saturated aqueous solution was added. Molecular evidence further suggested that the NaHS-triggered upregulation of target genes responsible for HO-1/CO-induced adventitious root formation, including CsDNAJ-1 and CsCDPK1/5, was inhibited significantly by ZnPPIX. These decreases were reversed obviously by the addition of CO aqueous solution. However, hypotaurine (HT), the H2S scavenger, could not influence the haemin- and CO-induced adventitious rooting in IAA-depleted cucumber explants. Together, the above results suggested that HO-1 was involved in H2S-induced cucumber adventitious root formation.

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.