Changes Of Endogenous Hormones During Graft Healing Process In Carya Cathayensis And Functional Analysis Of CcABCB19 In Grafting

The mechanism of graft healing of Carya cathayensis is still unclear,and lack of systematic endogenous hormone changes and molecular mechanism related researches.In this study,the hormones and its component contents in rootstock and scion at 0,7 and 14 days after grafting were analyzed by LC-MS/MS.Changes of endogenous hormones and related gene expression were comprehensively studied based on the hormones contents and corresponding transcriptome data during the early period of graft healing.The auxin efflux transporter gene CcABCB19,a key gene involved in graft healing,previous studies suggest that CcABCB19 may play an important role in graft healing,was cloned from Carya cathayensis.We analyzed the structure,subcellular localization,tissue-specific expression patterns of CcABCB19,in addition,the effect of overexpressing CcABCB19 on graft healing in Arabidopsis thaliana was also investigated.The main findings are as follows:（1）Dynamic changes of endogenous hormones during graft healing in Carya cathayensis and synergistic regulation of healing.The key genes IPTs,ACS1,ACO1,and ACO5 related to cytokinin and ethylene synthesis were up-regulated,and corresponded with the contents of t Z and ACC.In addition,the expression of cytokinin signal transduction related genes RR3,ARR4,ZFP5 and ethylene signal transduction related genes MKK9,ESE1 and ESE3 were up-regulated during the early stage of graft healing.On the contrary,auxin,jasmonic acid,abscisic acid,and strigolactone synthesis-related genes TAR2,AOC4,JMT and signal transduction-related genes COV1,ETA3,NAC,WRKY,SMAX1 were down-regulated,corresponded with the sharp decrease in the contents of IAA,JA-ILE,ABA and 5DS.This result indicated that synergistic regulation of endogenous hormones promote the callus formation of grafted callus of Carya cathayensis.（2）Expression pattern analysis showed that CcABCB19 was predominantly expressed in flowers,followed by leaves and roots.In addition,CcABCB19 was up-regulated in both rootstock and scion during the graft healing of Carya cathayensis.Promoter element analysis found that GA₃-and ETH-related cis-acting elements were abundantly distributed on the promoter of CcABCB19.Meanwhile,hormone treatment experiment confirmed the expression of CcABCB19 was regulated by hormones,specifically,it was induced by NAA,GA₃ and ETH,and inhibited by NPA.The subcellular localization of CcABCB19showed that it was localized to the cell membrane.Grafting experiments revealed that the graft survival rates of wild-type Arabidopsis and atabcb19 mutants were 89.83±1.97%and 76.31±3.00%,respectively,while the graft survival rate of CcABCB19-overexpressing Arabidopsis lines was 93.75±3.61%.This indicated that CcABCB19 could promote graft healing and improve graft survival rate.In summary,that dynamic changes and synergistic regulation of endogenous hormones promote graft healing of Carya cathayensis.The auxin efflux transporter CcABCB19 could positively regulate the graft survival of Carya cathayensis.