Molecular Mechanism Of Glutathione And Cyclic Guanosine Monophosphate-mediated Methane Triggered Adventitious Root Formation

Adventitious root(AR)is an important part of plant roots,and its formation is a prerequisite for vegetative reproduction of many crops,while adventitious roots are also regulated by many signaling molecules.It is known that cell-permeable cyclic guanosine monophosphate(cGMP)acts as a downstream target signal for nitric oxide to induce the formation of cucumber AR.Although a number of previous studies have reported that glutathione(GSH)plays a key regulatory role in antioxidant defense,redox signaling,and cell division and differentiation,recent studies suggest that GSH is also an indispensable signal for plant root formation.Component,Our laboratory research has found that methane(CH4)can induce the occurrence of cucumber AR,but it is not clear about its downstream signaling molecules.In response to the above problems,this experiment used morphological,pharmacological,anatomical,molecular biology and genetics to explore the possibility of GSH and cGMP as downstream signaling molecules involved in CH4-induced AR and related molecular mechanisms.The specific experimental results are as follows:1)Although the key role of methane(CH4)in the induction of cucumber adventitious rooting has been observed previously,the target molecules downstream of the CH4 action are yet to be fully elucidated.Here,we reported that exogenous glutathione(GSH)induced cucumber adventitious root formation;while L-buthionine-sulfoximine(BSO)treatment inhibited it.BSO is a known inhibitor of y-glutamyl cysteine synthetase(y-ECS),an enzyme involved in GSH biosynthesis.Further investigations showed that endogenous GSH content was rapidly increased by CH4 application,which was correlated with the increased CsGSH1 transcript and y-ECS activity.Mimicking the responses of GSH,CH4 could up-regulate cell cycle regulatory genes(CsCDC6,CsCDPK1,CsCDPK5and CsDNAJ-1),and auxin-response genes(CsAux22D-like and CsAux22B-like).Meanwhile,adventitious rooting-related CsmiR160 and CsmiR167 were increased or decreased,respectively,and contrasting tendencies were observed in the changes of their target genes,that included CsARF17 and CsARF8.The responses above were impaired by the removal of endogenous GSH with BSO,indicating that CH4-triggered adventitious rooting was GSH-dependent.Genetic evidence revealed that in the presence of CH4,Arabidopsis mutants cad2(a y-ECS-defective mutant)exhibited,not only the decreased GSH content in vivo,but also the defects in adventitious root formation,both of which were rescued by GSH administration other than CH4.Together,it can be concluded that y-ECS-dependent GSH homeostasis might be required for CH4-induced adventitious root formation.2)Exogenous cGMP could also induced the occurrence of cucumber AR.Further experimental results showed that CH4 treatment could increased the content of endogenous cGMP and the activity of guanylate cyclase activity(GC),compared with the control;CH4 and cGMP alone or combination treatment could accelerated the five stages of AR formation,and improve the expression of cell cycle genes and auxin response genes,by the ways of toluene blue staining and qPCR.The above induction phenomena were all inhibited by 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalin-l-one(ODQ;GC inhibitor)and 6-anilino-5,8-quinolinedione(LY83583;GC inhibitor),suggesting that cGMP might be involved in CH4-induced AR formation.Genetic results showed that the MtMCR transgenic lines(overexpression mutants of methane)had an increasing trend in cGMP content and GC activity,and also induced the occurrence of AR and the transcription of related genes expression,compared with wild-type(WT)Arabidopsis;exogenous ODQ and LY83583 can inhibited the above-mentioned induction phenomenon.The above responses results shown that cGMP might be involved in the induction of adventitious roots induced by CH4.Collectively,our results revealed that both GSH and cGMP might act as two downsteam molecules involved in CH4-triggered AR formation,and these responses might be related with the modulation of some AR-related genes,cell cycle regulatory genes,and auxin-response genes.