Mechanism Of Glutathione And Hydrogen Peroxide Involved In Hydrogen Gas-induced Lateral Roots Formation

Both glutathione（GSH）and hydrogen peroxide（H₂O₂）play the key roles in maintaining redox homeostasis,besides,they are both involved in signal transduction.In recent years,hydrogen gas（H₂）has been proven as a new type of gas signal molecule.H₂ participate in the growth and development of plants and improve plant resistance.In order to further study the molecular mechanism of the botanical effects of H₂,this study took lateral roots（LRs）as a typical biological phenotype,explored the molecular mechanism of GSH and H₂O₂involved in the H₂-induced lateral roots formation.The main results presented here:1.Compared with Con,exogenous H₂（0.39 m M）induced the accumulation of GSH intomato（Solanum lycopersicum L.）roots,the inhibitor of rate-limiting enzyme（γ-ECS）in GSH synthesis,_DL-buthionine-sulfoximine（BSO）,could block above effect.Real-time quantitative PCR（RT-q PCR）results showed that H₂ up-regulate the expression Sl GSH1,which encode tomatoγ-ECS.The result suggest that H₂ may regulateγ-ECS at the transcriptional level,thereby stimulating GSH synthesis.Further LR phenotype results showed that,similar to GSH,exogenous application of H₂ can induce tomato lateral roots formation.Combined with BSO treatment,the above phenomenon was suppressed.It is speculated that GSH may be the downstream signal in H₂ induced LR development.RT-q PCR analysis revealed that H₂ and GSH could induce LR formation may be relatedto the transcripts of cell cycle regulation genes（Sl CDKA1,Sl CYCD3;1,Sl CYCA2;1 and Sl CYCA3;1）and auxin signal transduction genes（Sl ARF7 and Sl RSI-1）.Further genetic evidence showed that,unlike Arabidopsis thaliana wild-type（WT）,the cad2-1 mutant（γ-ECS inactivation）were not sensitive to H₂-induced GSH accumulation,transcripts of LR formation related genes,and LR development.In summary,GSH dependent onγ-ECS acts as a downstream signaling compound governing H₂-induced LR formation by regulating auxin signaling-related genes and cell cycle regulatory genes.2.Application of exogenous H₂ and H₂O₂ significantly induced the accumulation of H₂O₂in tomato roots.The effection can be removed by the H₂O₂ membrane permeable scavenger diphenyleneiodonium（DMTU）.According to the analysis on the LR phenotype,it was found that both H₂ and H₂O₂ could induce LR formation in tomato.Above phenomenon was reversed by DMTU.It was speculated that H₂-induced tomato lateral roots formation depend on the accumulation of H₂O₂.RT-q PCR was used to explore the mechanism of H₂-and H₂O₂-induced tomato lateral roots formation.It was found that H₂ and H₂O₂ regulate cell cycle,auxin transport and signal transduction at the transcriptional level,which is manifested as an increase（Sl PIN1,Sl ARF7,Sl CYCA2;1,Sl CYCA3;1 and Sl CDKA1）in the expression of related genes or down（Sl PIN3,Sl IAA14 and Sl KRP2）.Above effect caused by H₂ and H₂O₂ were blocked by DMTU.Diphenyleneiodonium（DPI）,an inhibitor of respiratory burst oxidase homolog（RBOH）,was used for pharmacological exploration.It was found that similar to DMTU,DPI could inhibit the accumulation of H₂O₂,LR formation,and the changes in the transcripts of target gens induced by H₂.The result suggest that H₂ controls the H₂O₂ content by RBOH,subsequently promotes the LR development.Further exploration was conducted with Arabidopsis WT and Cr HYD1 transgenic lines（mimicking endogenous H₂ production by overexpressing hydrogenase1 gene Cr HYD1 from Chlamydomonas reinhardtii）.Compared with WT,Cr HYD1 transgenic lines induced H₂O₂accumulation,LR development,and accompanied by changes in the transcripts of related genes.The above-mentioned differences between the transgenic lines and WT could be eliminated by DMTU and DPI.The above results indicate that H₂-induced LR formation by the regulation of cell cycle,auxin transport and signal transduction,during which,H₂O₂ produced by RBOH was required.Above all,this study reveals that GSH and H₂O₂ may act as downstream signal molecules during H₂-induced LR formation.The new branch of H₂-triggered LR formation has been expanded.