Molecular Mechanism Of Hydrogen Peroxide And Glutathione Functioning In Methane And Hydrogen Sulfide Induced Lateral Roots

Lateral roots(LR)is an important agronomic trait that plays an important role in the growth and development of plants.Tomato and Arabidopsis thaliana are the model plants for studying LR formation.Previous studies have shown that methane(CH4)can be directly produced and emited by terrestrial plants under aerobic conditions.Further study found that CH4 can induce the formation of tomato lateral roots.CH4 may be a new gas signal molecule like hydrogen sulfide which participate in many physiological responses of plants,including lateral roots information.Redox signals hydrogen peroxide(H2O2)and reduced glutathione(GSH)as important components of plant signal transduction system play vital roles in lateral root formation.And threre are studies have been reported that H2O2 and GSH can mediate the formation of lateral and adventitious roots in plants.However,whether redox signals(H2O2 and GSH)were invoved in CH4 and H2S induced lateral root formation and the related molecular mechanisms are unclear.In this study,the tomato(Solanum lycopersicum L.)and Arabidopsis thaliana(col-0)were used as experimental materials.By using pharmacological,genetics,and molecular biology approaches,the molecular mechanism of redox signals(H2O2 and GSH)in the development of lateral roots induced by CH4 and H2S was mainly detected in this study.The specific research results are as follows:1.CH4 gas fumigation can promote the formation of tomato lateral roots,while argon and nitrogen fumigation can not affect the formation of lateral roots.CH4 promoted the development of tomato lateral root primordium(LRP)and lateral root(LR),and accompanied by an increase in endogenous H2O2 content.In order to further prove that H2O2 is involved in CH4-induced tomato lateral root development,we used H2O2,H2O2 scavenger dimethylthiourea(DMTU),and NADPH oxidase inhibitor diphenylene idonium(DPI;related to H2O2 synthesis)for our study.The results showed that both H2O2 and CH4 can promote the formation of tomato lateral roots,CH4 can promote endogenous H2O2,and H2O2 has little effect on CH4 content;DMTU and DPI can block H2O2 and CH4-induced tomato lateral root formation,and endogenous H2O2 content corresponds to phenotype;the detection of NADPH oxidase activities indicate that H2O2 is derived from NADPH oxidase.At the same time,CH4 can also regulate the expression of genes related to tomato lateral root development,including four cell cycle circulating genes(SICDKA1,SICYCA2;1,SICYCA3;1 and SlKRP2)and two miRNAs(miR390a and miR160)and their target genes(SIARF4 and SlARF16),whose effects can be reversed by DMTU and DPI,and correspond to the phenotype and H2O2 content.Therefore,pharmacological and molecular biological experiments have proved that NADPH oxidase-mediated H2O2 participates in the CH4 induced lateral roots formation.2.Lateral root of tomato can be induced by sodium hydrosulfide(NaHS;H2S donor)and glutathione(GSH).H2S-induced lateral roots formationis accompanied by an increase in GSH content,but there is no significant difference in endogenous H2S content of GSH-induced lateral root formation,so it is preliminarily determined that GSH as a downstream signal molecule of H2S in tomato lateral roots formation.Meanwhile,we also used H2S synthesis inhibitor DL-propargylglycine(PAG)and GSH synthesis inhibitor L-buthionine-sulfoximine(BSO)for pharmacological experiments.The results showed that PAG can block H2S-induced lateral roots formation,but it can not block GSH-induced lateral roots;BSO can block H2S and GSH-induced lateral roots;The endogenous GSH content corresponds to the phenotype,and GSH is derived from y-ECS.Quantitative PCR results showed that,exogenous NaHS induced cell cycle genes(SlCDKA1,SlCYCA2;1,SICYCA3;1 and SlKRP2),auxin-related genes(S1PIN1,S1PIN2 and SlARF7)and miRNA(SlmiR160 and SlmiR390a)and its target genes(SlARF16 and SlARF4)was repressed by BSO.To further verify the results,Arabidopsis genetic mutant material Atcad 2(Arabidopsis thaliana mutant with y-ECS defect)was also used in our study.Compared with wild type(WT),endogenous GSH content,lateral root formation and related gene transcription level in Atcad 2 were severely inhibited,and exogenous NaHS could not reverse it.Above results were also proved that endogenous GSH mediated H2S-induced lateral root formation.In summary,H2O2 and GSH can be induced by CH4 and H2S in the formation of LR.The related cell cycle regulating genes,auxin related genes,miRNA and their target genes were invoved in this process.Above results could provide a preliminary research basis for further in-depth study of the signal network in CH4 and H2S induced LR information.