Functional Analysis Of Flavonoid Biosynthesis Related Transcription Factors CpbHLH1 And CpMYB111 From Chimonanthus Praecox

Wintersweet(Chimonanthus praecox)is a traditional Chinese flowering shrub.Its flower color is mainly yellow,and the inner tepals of some varieties are covered with red stripes.Its flower color is relatively monotonous.Flower color is an important trait in ornamental plants,and therefore flower color breeding is the main aim of wintersweet breeding.The research on the mechanism underlying wintersweet flower color formation can lay a solid foundation for its breeding.The pigment components in wintersweet flowers are mainly flavonoids,including anthocyanins and flavonols.The biosynthesis of anthocyanins and flavonols is controlled by a series of transcription factors and structural genes.Some important structural genes have been identified in our laboratory though the transcriptome library and expression profile of wintersweet flowers.However,in addition to CpMYB2,other transcription factors that regulate the biosynthesis of anthocyanins and flavonols have not been identified in wintersweet.In this study,the b HLH transcription factor CpbHLH1 that may regulate the biosynthesis of anthocyanins and the MYB transcription factor CpMYB111 that may regulate the biosynthesis of flavanols were screened by using the transcriptome and expression profile data,and then the functional analysis of them were performed.The main results are as follows:1.CpbHLH1 is the first subgroup IIIf b HLH repressor of anthocyanin biosynthesis identified in dicotyledons.CpbHLH1 encodes a subgroup IIIf b HLH protein localized in the nucleus.The analysis of spatiotemporal expression of CpbHLH1 in wintersweet suggested a negative correlation between the expression level of CpbHLH1 and the expression level of Cp ANS1 during the wintersweet flower developmental stages.Phenotypic identification and quantitative RT-PCR(q RT-PCR)analysis of35S::CpbHLH1 Arabidopsis and tobacco plants showed that CpbHLH1 inhibited the accumulation of anthocyanin and the expression of late biosynthesis genes in the flavonoid pathway.Transcriptional activity analysis showed that CpbHLH1 is a transcriptional repressor and the repressive motif(s)of CpbHLH1 is located in the region between the MYB-interacting region(MIR)and the basic Helix-Loop-Helix(b HLH)domain.Yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(Bi FC)assays revealed that CpbHLH1 can interact with the MYB activators of anthocyanin biosynthesis.Dual luciferase analysis suggested that CpbHLH1 could not directly inhibit the expression of the genes related to anthocyanin biosynthesis,but it could be achieved by inhibiting the activity of MYB-b HLH complex.2.CpbHLH1 promotes the initiation of trichomes.The number of trichomes onthe leaves and stems of Arabidopsis overexpressing CpbHLH1 increased significantly.The GUS activity analysis of the promoter of CpbHLH1 in Arabidopsis showed that the promoter of CpbHLH1 had higher activity at the site where the trichomes were produced in the aboveground part of Arabidopsis.The q RT-PCR analysis of Arabidopsis overexpressing CpbHLH1 showed that CpbHLH1 inhibited the expression of trichome development-related genes GL2,TTG2,CPC and ETC1.Y2 H analysis revealed that CpbHLH1 can interact with key transcription factors GL1 and TTG1 that regulate trichome development in Arabidopsis.These results suggest that CpbHLH1 is a transcription factor that promotes trichome initiation,but the detailed mechanism is still waiting to be investigated.3.CpMYB111 promotes the biosynthesis of flavonol by directly activating the expression of the structural genes related to flavonol biosynthesis.CpMYB111 encodes a R2R3-MYB protein localized in the nucleus.The analysis of spatiotemporal expression in wintersweet suggested that the expression of CpMYB111 was positively correlated with flavonol biosynthesis.By using the plant transformation system mediated by Agrobacterium tumefaciens GV3101,CpMYB111 was overexpressed in Arabidopsis and early-flowering tobacco.The high performance liquid chomatography(HPLC)analysis and q RT-PCR analysis of 35S::CpMYB111 Arabidopsis and tobacco plants showed that CpMYB111 promoted the accumulation of flavonol and the expression levels of the structural genes of flavonol biosynthesis.Transcriptional activity analysis showed that CpMYB111 is a transcriptional activator.Dual luciferase analysis suggested that CpMYB111 could activate the promoter of the structural genes involved in flavonol biosynthesis.These results indicate that CpMYB111 is a transcription factor that promotes the biosynthesis of flavonol by directly activating the expression of the structural genes.In this study,the functions of the transcription factors CpbHLH1 and CpMYB111 were identified,and their mechanisms in regulating the biosynthesis of flavonoids were analyzed,which provided a theoretical foundation for the flower color breeding of wintersweet.