The Role Of NtMYC Transcription Factors In The Regulation Of Nicotine Biosynthesis By Ethylene

Tobacco is an important economic crop in China.Nicotine is not only an important quality index of tobacco,but also an important raw material in the fields of industry,agriculture,medicine and chemical industry.Nicotine content is affected by many factors which can regulate nicotine biosynthesis in varying degrees,such as genetics,drought,topping,pruning,mechanical damage,diseases and insect pests.Among all plant hormones involved in the regulation of nicotine synthesis and metabolism,jasmonic acid(JA)is a positive regulator,and ethylene is a negative regulator.The expression of genes related to nicotine biosynthesis in tobacco is mostly regulated by the jasmonic acid pathway.However,ethylene can inhibit the biosynthesis of nicotine induced by jasmonic acid.Tobacco topping can promote the synthesis of JA,and MYC2 is the key transcription factor in the JA signal-mediated nicotine synthesis.Our laboratory also found that two kind of MYC1 transcription factors(MYC1a and MYC1b)responding to tobacco topping can affect nicotine synthesis by directly regulating PMT gene expression.Therefore,MYC1 transcription factor plays an important role in nicotine synthesis.So,whether MYC transcription factor joins in the regulation of nicotine synthesis by ethylene has not been reported so far.This study focuses on the role of MYC transcription factor in the regulation of nicotine synthesis by ethylene.The main results are as follows:1.In order to explore the regulatory role of ethylene in nicotine synthesis,the effects of ethylene on the expression of genes related to nicotine synthesis in tobacco roots and the nicotine content in tobacco leaves were detected.The results showed that the expression of genes related to nicotine synthesis(PMT1?PMT2?A662?ADC?ODC?QPT and MPO)in roots were down-regulated,and the nicotine content in tobacco leaves was significantly decreased,indicating that ethylene could inhibit the biosynthesis of nicotine.2.In order to explore the regulatory effect of ethylene on the JA signal-mediated nicotine synthesis pathway,the promoter sequence of MYC transcription factor was analyzed,and it was found that it contains JA response elements and ERE ethylene answer elements,indicating that MYC transcription factor may be regulated by ethylene and JA signal.After tobacco plants were treated with the exogenous ethephon,qPCR results showed that the expressions of genes related to JA synthesis(DAD1?AOC?AOS and OPR3),Nt MYC1 and Nt MYC2 transcription factor genes were significantly decreased,and the JA content in roots is also significantly reduced,suggesting that ethylene can not only reduce root JA content,but also down-regulate the expression of Nt MYC transcription factor.The results of yeast two-hybrid showed that Nt MYC1 could interact with Nt JAZ1,which indicated that Nt MYC1 transcription factor could also participate in the JA-mediated nicotine synthesis via interacting with JAZ1 protein.Therefore,during the process of ethylene reducing nicotine synthesis,it can not only reduce the release of MYC transcription factor through decreasing synthesis of JA but also directly decrease the expression of MYC transcription factor.3.In order to explore the regulatory effects of ERF transcription factors on nicotine synthesis,tobacco plants were treated with exogenous ethephon.The qPCR results showed that the expression of ERF32?ERF189 and ERF142 were down-regulated by exogenous ethephon,and the down-regulation of ERF32 and ERF189 was more than 2times.The analysis of PMT2 gene promoter sequence showed that it contained the GCC site which ERF transcription factor can specifically bind,indicating that ERF transcription factors may regulate the expression of PMT2 gene.Yeast one-hybrid results showed that ERF32 and ERF189 transcription factors could interact with PMT2 gene promoter,suggesting that these two transcription factors could regulate the expression of PMT2 gene.Therefore,ethylene can influence the expression of PMT through directly regulating the expression of NtERF32 and NtERF189 transcription factors,then reduce nicotine synthesis.4.In order to explore the regulatory effect of MYC transcription factor on NtERF189 gene,the promoter sequence of NtERF189 gene was analyzed.It was found that the promoter contained G-box sequence that MYC transcription factor can specifically recognize and bind.The qPCR results showed that the expressions of NtERF32,NtERF142 and NtERF189 in transgenic tobacco plants with overexpression of MYC1 were higher than wild-type tobacco plants,indicating that MYC transcription factor may regulate the expression of these ERF genes.The results of yeast one-hybrid showed that MYC1 transcription factor could interact with NtERF189 gene promoter,suggesting that MYC1 transcription factor could regulate expression of NtERF189 gene.Therefore,MYC transcription factors can regulate the expression of PMT through both directly binding its promoter and regulating the expression of ERF transcription factors,thus regulating nicotine synthesis.