Molecular Mechanism Of Methane-induced Cucumber Adventitious Root Formation

Adventitious root development is partly determined by several endogenous and environmental factors,particularly including auxin,wounding,light,and gravity.Methane(CH4)is usually used as a type of promising fuel and is also regarded as a greenhouse gas for the atmosphere;however,its biological functions were discovered recently.Previous results reported that CH4 induce cucumber adventitious rooting by using methane-rich water(a safe and easily available way in comparison with exogenously applied methane gas).However,the molecular mechanisms are not well understood.In addition,although CH4 production was reported to be increased in response to abiotic stress,the source(s)where CH4 come from in plant remain elusive.To answer the above questions,we studied the cross-talk between CH4 and three other signaling molecules(auxin,H2O2 and H2S),which were reported to be useful for cucumber adventitious rooting,through pharmacological and anatomical as well as molecular genetic approaches.Proteomic analysis was also used to further discover the molecular mechanisms of adventitious rooting.Acquired results are listed as follows:First,we observed that indole acetic acid(IAA)triggered the accumulation of CH4 in cucumber explants.Subsequent results showed that the CH4 accumulation and adventitious rooting elicited by IAA were sensitive to dimethylthiourea(DMTU;a scavenger of H2O2),showing that the accumulation of CH4 and adventitious root development were partly impaired.Further molecular evidence indicated that expression of marker genes for adventitious rooting,including CsDNAJ-1,CsCDPK1,CsCDPK5,cell division-related gene CsCDC6,and two auxin signaling genes,CsAux22D-like and CsAux22B-like,were promoted by IAA and H2O2 alone,but partly or completely blocked by the co-treatment with DMTU.Genetic evidence showed that the adventitious root formation was impaired in the NADPH oxidase deficient mutant rbohF of Arabidopsis thaliana.Moreover,the application of CH4 and H2O2 alone significantly promote the adventitious root number,while IAA treatment showed obviously reduced sensitivity for adventitious rooting in this mutant line.These results indicated that IAA triggers cucumber adventitious rooting through increasing endogenous CH4 production,and H2O2 mediates IAA-induced CH4 accumulation and adventitious rooting in cucumber.Second,we observed that the activities of DES,endogenous H2S production,and thereafter adventitious root development were induced by CH4 and NaHS(an H2S donor).Some responses were sensitive to hypotaurine(HT;a scavenger of H2S),showing that endogenous H2S production and adventitious rooting were obviously blocked.The development of adventitious root primordia was also impaired.Further molecular evidence revealed that CH4-induced gene expression of CsDNAJ-1,CsCDPK1,CsCDPK5,CsCDC6,CsAux22D-like and CsAux22B-like were blocked by the co-treatment with HT.The occurrence of CH4-elicited S-sulfhydration during above responses was also sensitive to the removal of endogenous H2S,suggesting the requirement of H2S.These results revealed a vital role of endogenous H2S in CH4-triggered cucumber adventitious root development,and thus provided a direct demonstration window of the complex signaling transduction pathway in CH4-mediated root organogenesis.We further used the label-free proteomics to explore the molecular mechanisms for adventitious rooting.The results showed that a total of 1660 proteins were identified from CH4-,IAA-,NaHS-,and distilled water(Con)-treated samples.A total of 934 significantly differential proteins were identified in CH4-,IAA-,and NaHS-treated samples(compared with control).Furthermore,40 from the 934 proteins were selected for further bioinformatic analysis.The results indicated that they fall into five categories:sulfur compound metabolic process,amino acid and protein metabolic process,carbohydrate and energy metabolic process,secondary metabolic process,and metal ion homeostasis.In addition,the protein expression patterns were consistent with the results of increased non-protein thiols abundance and iron and zinc contents.We also discovered and preliminarily confirmed that glutathione(GSH)could induce the adventitious rooting by pharmacological approach.In summary,this study revealed the possible molecular mechanisms of CH4-induced cucumber adventitious rooting,and described the cross-talk between CH4 and other signaling molecules related to adventitious rooting in detail.