Heme Oxygenase-1/Carbon Monoxide Signaling System Is Involved In Hydrogen Sulfide, Beta-CD-Hemin And Hydrogen-Induced Cucumber Adventitious Root Formation

The previous result suggested that heme oxygenase-1/carbon monoxide (HO-1/CO) acts as a downstream signaling system in the auxin-induced pathway leading to cucumber (Cucumis sativus) adventitious root formation (Xuan et al.2008).Hydrogen sulphide (H2S) has been proposed as a new endogenous gaseous signaling molecule. The objective of this study is to test whether HO-1is also involved in hydrogen sulfide (H2S)-induced adventitious root formation. Cucumber explants were treated with HO-1inducer hemin, H2S donor sodium hydrosulfide (NaHS) in combination with the specific inhibitor of HO-1zinc protoporphyrin IX (ZnPP) and the by-products of HO-1(CO, BR and Fe2+). The results showed that NaHS brought about the inducible responses which were similar to hemin, and the further experiment verified that H2S or HS-rather than other sulfur-containing components derived from NaHS was ascribed to the stimulation respons. The exogenous H2S could up-regulate cucumber HO-1transcripts (CsHO1), its protein levels and target genes responsible for HO-1/CO-induced adventitious root formation, including CsDNAJ-1and CsCDPKl/5, and thereafter induce adventitious root formation. The inducible effect was significantly reduced by treatment with ZnPP, and the inhibitory effects were reversed partially when30%CO-saturated aqueous solution was added. However, hypotaurine (HT), the H2S scavenger, could not influence the hemin-and CO-induced adventitious rooting in IAA-depleted cucumber explants. Together, above results suggested that HO-1was involved in the H2S-induced cucumber adventitious root formation.The previous result showed that β-cyclodextrin-hemin complex (CDH) exhibited a vital protective role against cadmium-induced oxidative damage and toxicity in alfalfa seedling roots by the regulation of heme oxygenase-1(HO-1) gene expression. In this report, we further test whether CDH exhibited the hormonal-like response. The application of CDH and an inducer of HO-1, hemin, were able to induce the up-regulation of cucumber HO-1gene (CsHO1) expression and thereafter the promotion of adventitious rooting in cucumber explants. The effect is specific for HO-1since the HO-1inhibitor ZnPP blocked the above responses triggered by CDH, and the inhibitory effects were reversed further when30%saturation of CO aqueous solution was added together. Further molecular evidence showed that CDH-triggered the increases of the HO-1-mediated target genes responsible for adventitious rooting, including CsDNAJ-1and CsCDPKl/5, were inhibited by ZnPP and reversed by CO. The calcium (Ca2+) chelator ethylene glycol-bis (2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) and the Ca2+channel blocker lanthanum chloride (LaCl3not only compromised the induction of adventitious rooting induced by CDH, but also decreased the transcripts of above three target genes.However, the application of ascorbic acid (AsA), a well-known antioxidant in plants, failed to exhibit similar inducible effect on adventitious root formation. In short, above results illustrated that the response of CDH in the induction of cucumber adventitious rooting might be through HO-1-dependent mechanism and calcium signaling.In recent years, a lot of results showed that hydrogen (H2) is a therapeutic anti-oxidant in the animal models and clinic trails. However, the underlying physiological roles and mechanisms of H2in plants remain unknown. In this report, we adopted pharmacological and molecular approaches to test the role of HO-1/CO signaling in the H2-induced responses. Similar to the responses of hemin,50%saturation of H2aqueous solution could induce adventitious root formation and also up-regulate CsHO1, its protein levels and target genes including CsDNAJ-1and CsCDPK1/5in IAA-depleted cucumber explants.The inducible effect was significantly reduced by treatment with ZnPP, and the inhibitory effects were reversed partially when30%CO-saturated aqueous solution was added. Together, above results suggested that the possible response of H2in the induction of cucumber adventitious rooting might be through HO-1/CO signaling system.