Study On Sucrose And Cmoant1.2 Response In Graft Union Development In The Heterograft Of Cucumber Onto Pumpkin

Grafting efficiency depends on rate of graft union healing.To clarify the mechanism of graft formation it not only would provide of important theoretical value for the research on communication between scion and rootstock,tissue model reconstruction and vascular bundle tissue regeneration,but also produce a technological guide for stock screening and production of grafted seedlings.Cucumber(Cucumis sativus L.)is an important protected cultivation crop,and usually grafted onto pumpkin(Cucurbita moschata Duch.)to improve tolerance for soil-pathogen and abiotic stress in production.Additionally,cucumber and pumpkin have very developed vascular bundles,and they are suitable materials for study of graft union formation.In our study,cucumber is grafted onto pumpkin rootstock with splicing grafting,the sucrose response during graft union formation in cucumber/pumpkin was studied.CmoANT1.2,which belongs to the AINTEGUMENTA-like(AIL)transcription factor gene family,was only significantly changed in the rootstock during graft union formation.The AINTEGUMENTA-like(AIL)transcription factor gene family in pumpkin(Cucurbita moschata Duch.)was analysis using bioinformation tool,and CmoANT1.2 response in graft union healing of arabisopsis was also studied.The main results are as follows:(1)Sugar metabolism exhibited significantly changed during graft union development in the heterograft of cucumber onto pumpkin,including the sugar responsive genes(ApL3,STP1,DIN6),sugar content(glucose,fructose,sucrose,stachyose,raffinose,and starch),and the enzyme activity of sucrose metabolic enzymes(S-AI,NI,SPS,and SS in the synthesis and degrading directions),RFO metabolic enzymes(GAL,STS)and starch metabolic enzymes(SBE,SSS).Compared with the grafted WT seedlings,the grafted etiolated cucumber seedlings which have lower sugar levels,grew slowly,both phloem and xylem connections were delayed,and graft efficency and biomass accumulacation was decreased.But these biological processes were improved by the presence of exogenous glucose,including graft efficiency and biomass,and xylem connection,and the grafted normal seedlings were markebly promoted by exogenous glucose.The sugar signal genes(TOR?SnRK1?,SnRK2.1,SnRK2.2,S6K2,E2Fa)were trigged distinct responses between rootstock and scion,and these genes expression were significantly affected by exogenous glucose.Graft efficiency was not affected by the spraying of TOR inhibitor AZD-8055,whereas vascular bundle reconnection was delayed and the growth of grafted plants was inhibited by AZD-8055.Moreover,the energy charge was lower at the graft junction after inhibitor treatment during the healing process,and this could be partially complemented by the spraying of exogenous glucose.Interestingly,the expression of CsTOR and CsS6K2 was higher under TOR inhibitors with/without exogenous glucose treatments,whereas CmTOR and CmS6K2 were markedly downregulated under these treatments.The expression of E2Fa was significantly inhibited by AZD-8055 in both the scion and rootstock;in contrast,SnRK1? was triggered at the graft junction,and the upregulation of SnRK1? was slightly relieved by exogenous sugars.(2)We identified 12 CmoAIL genes in the pumpkin genome encoding proteins predicted to be localized in the nucleus.Phylogenetic analysis showed that the CmoAIL gene family could be classified into six major subfamilies,with each member encoding two AP2/ERF domains separated by a linker region.CmoAIL genes were expressed at varying levels in the examined tissues,including root,stem,leaf,tip,and tendril,axillary bud from the top of the lateral branch,female buds,and male buds at the 10th stage of flower development,female and male at anthesis,purp and seeds from the mature fruits.CmoANT genes(CmoANT1.1,CmoANT1.2,CmoANT1.3,CmoANT1.4)showed different expression patterns under auxin(IAA),1-naphthylphthalamic acid(NPA),and abscisic acid(ABA)treatments.Ectopic overexpression of CmoANT1.2 in Arabidopsis increased organ size and promoted growth of grafted plants by accelerating graft union formation.However,there was no significant difference at the graft junction for WT/WT and WT/ANT under IAA or NPA treatments.