Possible Interraction Mechanisms Of Hydrogen Sulfide-,Hydrogen Peroxide-,and Methane-Induced Lateral Root Formation

The occurrence of lateral root(LR)is an important agronomic trait in plant root morphogenesis.Auxin plays an important role in the process of LR formation.As a new gaseous signalling molecule,hydrogen sulfide(H2S)is involved in auxin-induced LR formation.Hydrogen peroxide(H2O2)is an important component of plant signal transduction systems.Previous studies showd that H2O2 is required for auxin-induced adventitious root and lateral root formation.However,corresponding casual link of H2S and H2O2 governing lateral root(LR)formation is still elusive.In addition,it has been reported that methane(CH4)can reduce myocardial ischemic injury in rats through anti-oxidation,anti-apoptosis and anti-inflammatory effects.In plants,CH4 plays an important role in against unfavorable environmental conditions and adventitious root formation.However,there is no direct report about whether CH4 is involved in auxin-induced LR formation.Meanwhile,the inter-relationship between H2S signaling system and CH4-mediated LR development is largely unclear.Present study showed that:To solve the above questions,we studied the molecular mechanism by pharmacological and genetic evidences,and corresponding results are listed as follows:Using different concentrations of CH4 to treat tomato and Arabidopsis seedlings,it was found that CH4 can participate in auxin-induced LR development in a concentration-dependent manner.Exogenous methane treatment triggered increasing of DES activity and endogenous H2S content by spectrophotography and laser scanning confocal microscope(LSCM)analysis,up-regulation DES1 transcript,and thereafter tomato LR formation.By contrast,above responses achieved by CH4 were sensitive to the H2S scavenger(hypotaurine,HT)and the inhibitor of DES(DL-propargylglycine,PAG).Further molecular evidence revealed that applications of NaHS(H2S donor)and CH4 could regulate cell cycle regulatory genes,miRNAs,and their target genes(ARFs)transcripts,which would be prevented by the co-treatment with HT or PAG.The above results were confirmed in genetic experiments.Compared with Arabidopsis wild-type,the endogenous H2S accumulation and LR occurrence of Arabidopsis DES mutant(atdesl)were significantly inhibited,and the addition of CH4 couldn't reverse the above inhibitory effect.All together,the above results indicated that H2S is a downstream signaling molecule in CH4-induced LR signaling pathway.Similar to hydrogen peroxide(H2O2),hydrogen sulfide(H2S)is a highly reactive molecule involved in diverse physiological functions(root morphogenesis,etc)in plants.However,corresponding casual link governing lateral root(LR)formation is still elusive.Here,we discovered that H2S donor sodium hydrosulfide(NaHS)triggered the accumulation of H2O2,the up-regulation of RBOH1 transcript,and thereafter tomato LR formation.By contrast,above responses achieved by NaHS were sensitive to the H2O2 scavenger(dimethylthiourea;DMTU)and the inhibitor of NADPH oxidase(diphenylene idonium;DPI),showing that the accumulations of H2O2 and increased RBOH1 transcript were respectively prevented.Meanwhile,LR formation was also impaired.Further molecular evidence revealed that H2S-modulated gene expression of cell cycle regulatory genes,including up-regulation of SICYCA2;1,SlCYCA3;1,and SICDKA1,and the down-regulation of SlKRP2,were prevented by the co-treatment with DMTU or DPI.Above mentioned inducing phenotypes were consistent with the changes of LR formation-related miRNA transcripts:up-regulation of miR390 and miR160,and with the opposite tendencies of their target genes(encoding Auxin response factors,ARFs).Contrasting tendencies were observed when DMTU or DPI was added together with NaHS or H2O2.The occurrence of H2S-mediated S-sulfhydration during above responses was discovered.Overall,these results suggested an important role of RBOH1-mediated H2O2 in H2S-elicited tomato LR development,and corresponding H2S-target proteins regulated at transcriptional and post-translational levels.Above results provide insights into H2S signaling in plant development.In summary,the above research results demonstrated that methane as a downstream signal molecule of auxin participates in the LR formation,and H2S is involved in the signaling pathway of CH4-induced LR by regulating the expression of cell cycle regulatory genes and miRNAs.At the same time,the interaction between H2S and H2O2 plays an important role in the LR formation.Therefore,the connection among CH4,H2S,and H2O2 is critical to the occurrence of LR formation and positively affects the growth and development of plants.