Involvement Of Reduced Glutathione,Hydrogen Peroxide,and Nitric Oxide In β-cyclodextrin-hemin-induced Tamato Lateral Root Formation

Lateral root(LR)formation is not only an important agronomic trait for vegetables,but also a research model for lateral root.Recent results showed that animal-sourced hemin,a heme oxygenase-1(HO-1)inducer,is a novel functioned compound as a plant growth inducer responsible for lateral tomato root formation.However,the disadvantage of poor water-solubility and high photosensitivity seriously restricted its application in agricultures.Our recent study results also showed that β-cyclodextrin-hemin complex(β-CDH)could overcome above shortcomings,and efficiently induce tomato lateral root development.However,the molecular mechanism underlying β-CDH-induced tomato lateral root formation is not fully elucidated.In this research,the involvement of reduced glutathione(GSH),hydrogen peroxide(H2O2)and nitric oxide(NO)in β-CDH-inducing tomato lateral root formation is investigated.Our study showed that:1.Similar to the responses of β-CDH,exogenously applied with 100 μM GSH not only increased endogenous GSH content determined by spectrophotography and the monochlorobimane(MCB)-dependent fluorescent analysis,but also induced thereafter LR formation.Meanwhile,both P-CDH-and GSH-induced lateral root primordia(LRP)exhibited a similar accelerated anatomic structure.Above inducible responses were blocked significantly when the L-buthionine-(S,R)-sulfoximine(BSO),the GSH synthetic inhibitor,was separately applied.Exogenously applied with β-CDH could rescue N-1-naphthylphthalamic acid(NPA;IAA depletion)-triggered inhibition of LR formation.Further molecular evidence revealed that both P-CDH and GSH modulated gene expression of cell cycle regulatory genes(CYCA2;1,CYCA3;1,CYCD3;1 and CDKA1)and auxin signaling genes(ARF7 and RSI-1),six marker genes responsible for LR formation.By contrast,above changes were sensitive to the cotreatment with BSO.All together,these results suggest a novel role for GSH in the regulation of tomato LR development triggered by β-CDH.2.H2O2 might be involved in β-CDH-induced lateral root formation in tomato.P-CDH induced H2O2 content by analyzing the fluorescence intensity of H2O2,thereafter lateral root formate.Exogenous β-CDH and H2O2 not only induced the production of H2O2,but also induced lateral root formation in tomato.While,the H2O2 scavenger dimethylthiourea(DMTU),ascorbic acid(AsA),and its synthesis inhibitor diphenyleneiodonium(DPI)impaired these tendencies.β-CDH and H2O2 induced lateral root formation by regulating the expression of auxin response genes(ARF7,RSI-1,and IAA14)and auxin transport genes(LAX3,PIN3,and PIN7),indicating that auxin signaling played an important role inβ-CDH-induced lateral root formation.Identifing possible target protein in β-CDH-induced lateral root formation by proteomic analysi was also carried out.These results suggested that H2O2 acts as a signaling molecular in β-CDH-induced lateral root formation.3.β-CDH-induced tomato lateral root formation could be reversed by a NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide(PTIO),while NO contents were decreased.Thus,these results suggested that NO might be involved in β-CDH-induced lateral root formation.In summary,this research demonstrated GSH,H2O2 and NO might be involvement inβ-CDH-induced tomato lateral root formation by using pharmacological and molecular approaches.