Transcriptional Regulation Of ?-farnesene Synthesis By MdMYC2 And MdERF3 In Apple Fruit

?-farnesene is a sesquiterpene volatile compound in leaves,flowers and fruits of many plants,and plays an important role in plant defense.It is known to be associated with plant cold tolerance,insect attraction and physiological disorder-superficial scald of apple or pear fruit during cold storage.farnesene is the precursor of biofuel farnesane,which has broad market value,it has attracted extensive attention of the society in recent years.Previous studies related to ?-farnesene mainly focused on gene cloning,biological function and metabolic engineering of key enzymes in metabolic pathway,but the mechanism of transcription factors regulating apple ?-farnesene biosynthesis has not been clarified.The results of this study revealed that the transcription factors MdMYC2 and MdERF3 can bind to the promoter region of MdAFS,which is the terminal enzyme gene in the ?-farnesene synthesis pathway,activate the expression of MdAFS gene,and promote the accumulation the ?-farnesene.The main conclusions are as follows:(1)Analysis of hormones-inducing the expression of MdAFS in White Winter Pearmain apple leaves by qRT-PCR indicated that MdAFS was regulated by jasmonic acid(JA),ethylene(ETH),Abscisic acid(ABA)and salicylic acid(SA).Under ambient and low temperature storage conditions,JA and ETH treatments could promote the synthesis of ?-farnesene in apple fruits,while 1-MCP treatment inhibited the synthesis of ?-farnesene.(2)Using the PlantCARE database to analyze and predict the MdAFS promoter,it was found that there were multiple cis-acting elements in response to phytohormones(ETH,JA,ABA)and abiotic stress in the promoter region,as well as MYB and MYC binding sites.According to the analyses of binding sites,we selected MdMYC2 and MdERF3,which are important transcription factors in the pathway of jasmonic acid and ethylene,to study the regulation mechanism of ?-farnesene synthesis.The results of qRT-PCR showed that the up-regulated expressions of MdMYC2 and MdERF3 were induced by ethylene and jasmonic acid treatments,and the MdAFS gene was also up-regulated,which indicated that the expression of MdMYC2,MdERF3 and MdAFS was correlated.(3)It was found that MdMYC2 and MdERF3 could activate MdAFS promoter by a firefly luciferase(Luc)complementation imaging assay.Yeast one-hybrid assay analysis indicated that MdMYC2 and MdERF3 could bind to the MdAFS promoter in vitro.EMSA further confirmed that MdERF3 could directly bind to the MdAFS gene promoter.MdMYC2 and MdERF3 were overexpressed in apple callis,and the expression of MdAFS gene was increased.The expression level of MdAFS was markedly lower in MdMYC2-supressed calli than in the control.(4)A more rapid and efficient transient overexpression assay was chosen to examine the function of MdMYC2 and MdERF3 in vivo.The results of GC-MS indicated that MdMYC2 and MdERF3 promoted ?-farnesene synthesis in the peel of White Winter Pearmain apple fruit.(5)MdMYC2 and MdERF3 also affected the expression of other key enzymes HMGR and FPPS genes in the synthesis pathway of ?-farnesene,which indicated that MdMYC2 and MdERF3 could effectively improve the synthesis of ?-farnesene by activating the co-expression of multiple genes in the ?-farnesene metabolic pathway.