SUMMARYIn 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.

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).

Aims and methods The molecular mechanisms and signal transduction pathways of hydrogen sulfide (H 2 S) in plant biology are still unclear. Here, by using pharmacological and biochemical approaches, we report that H 2 S promotes germination and alleviates salinity damage involving nitric oxide (NO) pathway. Results Upon 100 mM NaCl treatment, both H 2 S donor sodium hydrosulfide (NaHS) and NO donor sodium nitroprusside (SNP) at 100 μM could significantly attenuate the inhibition of alfalfa (Medicago sativa) seed germination and thereafter seedling growth inhibition. Meanwhile, the ratio of potassium (K) to sodium (Na) in the root parts was increased.Total, isozymatic activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), or ascorbate peroxidase (APX) were activated differentially, thus resulting in the alleviation of oxidative damage. The above protective roles of NaHS might be related to the induction of endogenous NO, because the addition of the specific scavenger of NO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) reversed above effects. Meanwhile, NaHStriggered NO production was confirmed. Conclusions Our observations indicate that H 2 S enhances plant responses against salinity stress by reducing oxidative damage, which might have a possible interaction with NO.

Hydrogen sulfide (H 2 S) is a gaseous signaling molecule that regulates diverse cellular signaling pathways through persulfidation, which involves the post-translational modification of specific cysteine residues to form persulfides.However, the mechanisms that underlie this important redox-based modification remain poorly understood in higher plants. We have, therefore, analyzed how protein persulfidation acts as a specific and reversible signaling mechanism during the abscisic acid (ABA) response in Arabidopsis thaliana.Here we show that ABA stimulates the persulfidation of L-CYSTEINE DESULFHYDRASE 1 (DES1), an important endogenous H 2 S enzyme, at Cys44 and Cys205 in a redox-dependent manner. Moreover, sustainable H 2 S accumulation drives persulfidation of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG PROTEIN D (RBOHD) at Cys825 and Cys890, enhancing its ability to produce reactive oxygen species. Physiologically, S-persulfidation-induced RBOHD activity is relevant to ABA-induced stomatal closure. Together, these processes form a negative feedback loop that fine-tunes guard cell redox homeostasis and ABA signaling. These findings not only expand our current knowledge of H 2 S function in the context of guard cell ABA signaling, but also demonstrate the presence of a rapid signal integration mechanism involving specific and reversible redox-based post-translational modifications that occur in response to changing environmental conditions.

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 H 2 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.

Summary• Using pharmacological and biochemical approaches, the role of cadmium (Cd)-induced carbon monoxide (CO) release and the relationship between CO and oxidative stress conferred by Cd exposure in the root tissues of alfalfa ( Medicago sativa ) plants were investigated.• Cd treatments showed a dose-dependent enhancement in lipid peroxidation. Both 100 and 200 µ M CdCl 2 treatments caused the increase of CO release, which is consistent with the changes in the activity of the CO synthetic enzyme heme oxygenase (HO) and its HO-1 transcript.• A 100 µ M CdCl 2 exposure enhanced the formation of nonprotein thiols (NPT), and reduced glutathione (GSH) to oxidized glutathione (GSSG), which was potentiated by the pretreatment of CO scavenger hemoglobin (Hb). Plants pretreated for 6 h with 50% CO-saturated aqueous solution, which induced the rapid endogenous CO release followed by a gradual decrease when subsequently exposed to 100 µ M CdCl 2 for 72 h, effectively decreased oxidative damage. Meanwhile, CO pretreatment modulated several enzymes responsible for GSH metabolism, thus resulting in the partial restoration of GSH : GSSG ratio, which was significantly blocked by Hb.• These results are suggestive of a role for CO release as a signal element for the alleviation of Cd-induced oxidative damage by modulating glutathione metabolism.

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BackgroundThe metabolism of hydrogen gas (H2) in bacteria and algae has been extensively studied for the interesting of developing H2-based fuel. Recently, H2 is recognized as a therapeutic antioxidant and activates several signalling pathways in clinical trials. However, underlying physiological roles and mechanisms of H2 in plants as well as its signalling cascade remain unknown.Methodology/Principal FindingsIn this report, histochemical, molecular, immunological and genetic approaches were applied to characterize the participation of H2 in enhancing Arabidopsis salt tolerance. An increase of endogenous H2 release was observed 6 hr after exposure to 150 mM NaCl. Arabidopsis pretreated with 50% H2-saturated liquid medium, mimicking the induction of endogenous H2 release when subsequently exposed to NaCl, effectively decreased salinity-induced growth inhibition. Further results showed that H2 pretreatment modulated genes/proteins of zinc-finger transcription factor ZAT10/12 and related antioxidant defence enzymes, thus significantly counteracting the NaCl-induced reactive oxygen species (ROS) overproduction and lipid peroxidation. Additionally, H2 pretreatment maintained ion homeostasis by regulating the antiporters and H+ pump responsible for Na+ exclusion (in particular) and compartmentation. Genetic evidence suggested that SOS1 and cAPX1 might be the target genes of H2 signalling.ConclusionsOverall, our findings indicate that H2 acts as a novel and cytoprotective regulator in coupling ZAT10/12-mediated antioxidant defence and maintenance of ion homeostasis in the improvement of Arabidopsis salt tolerance.