Function Analysis Of Three CmMYBs Transcription Factors In Regulation Of Floral Coloration In Chrysanthemum

Chrysanthemum(Chrysanthemum morifolium)is one of the ten traditional flowers in China as well as four famous cut flowers in the world,with high ornamental and economic values.Floral color is one of the most important traits of chrysanthemum.Although there are many kinds of colors in chrysanthemum,the mechanism of coloration remains to be clarified.Flavonoids are important pigments responsible for flowral color.Thus,it’s meaningful to understand floral coloration in chrysanthemum by investigating regulatory networks controlling flavonoid biosynthesis.The mutants with color variation and wild type of chrysanthemum,provide plant materials with nearly identical genetic backgrounds,and could be used for selecting key genes in regulation of floral color.In this study,analysis of pigment components and transcriptomes in chrysanthemum cultivars ‘Anastasia Pink’with pink color and its white mutant PM,as well as ‘Anastasia Dark Green’ with green color and its pink mutant GM was performed to select key differentially expressed genes.Then,the part mechanism of regulation in flavonoid biosynthesis were clarified by investigating the function of three CmMYBs which were differentially expressed.The main contents and conclusions in this research are as follows:1.Analysis of pigments in petals of ‘Anastasia Pink’ and its mutant PM at five flowering stages,as well as RNA sequencing of three key stages for coloration was performed.The results showed that down-regulation of EGBs(CmCHS1,Cm F3 H and Cm F3’H1)and LGBs(CmDFR,Cm ANS,Cm UF3 GT,Cm2Ma T1 and Cm2 Ma T2)in the mutant petals from uncolored stage to fully uncolored stage resulted in the reduced anthocyanins and turned flowral color from pink into wihte.Multiple regulatory genes which differentially expressed were detected.Among these genes,down-regultaion of CmMYB6 and up-regulation of CmMYB21 were presumably involved in suppression of anthocyanin biosynthesis in the mutant.2.Analysis of pigments in petals of ‘Anastasia Dark Green’ and its mutant GM at five flowering stages,and RNA sequencing in three coloration stages were performed.The results showed that decreased chlorophylls and increased anthocyanins in the mutant comparing with those in wild type.EGBs(CmCHS1 and Cm F3H)and LGBs(CmDFR,Cm ANS,Cm UF3 GT,Cm2Ma T1 and Cm2 Ma T2)were up-regulated in the mutant during all flowering stages.It was speculated that down-regulation of CmCHS2 and CmCHI in mutant petals resulted in fewer content of total flavonoids;largely up-regulation of CmMYB6 and CmbHLH2 was involved in anthocyanin accumulation in mutant petals.Analysis of differentially expressed genes in ‘Anastasia Pink’ and its mutant,as well as ‘Anastasia Dark Green’ and its mutant suggested that MYB transcription factors including CmMYB9 a,CmMYB11,CmMYB12 and CmMYB21,and bHLH transcription factors including CmbHLH16 may be involved in regulation of flavonoid biosynthesis.3.An R2R3-MYB transcription factor,CmMYB9 a was cloned.Analysis of phylogenetic tree and protein sequences showed that CmMYB9 a belonging to SG7 subfamily,harbored an SG7 motif with variation in C-terminal.The spatio-temporal expression analysis showed that CmMYB9 a specially expressed in the petals of ‘Anastasia Pink’ from uncolored stage to initially colored stage.CmMYB9 a was localized in the nucleus.CmMYB9 a exhibited transcriptional activation activity at the C-terminal.Over-expression of CmMYB9 a in tobacco promoted the anthocyanin accumulation and suppressed the flavonols accumulation in petals.Transient over-expression of CmMYB9 a in petals of ‘Anastasia Pink’ promoted the the accumulation of anthocyanins and flavones.Yeast one-hybrid assay and luciferase assay showed that CmMYB9 a activates CmCHS2,CmDFR,Cm ANS and Cm FNS,and suppress Cm FLS by binding to their promoters.Yeast two-hybrid assay showed that CmMYB9 a interacts with CmbHLH16,rather than CmMYB6 or CmbHLH2.Those results suggested that CmMYB9 a promotes floral coloration by activating biosynthesis of anthocyanins and flavones,and suppressing flavonol biosynthesis;CmMYB9 a plays a role in floral coloration of chrysanthemum.4.An R2R3-MYB transcription factor,CmMYB11 was cloned.Analysis of phylogenetic tree and protein sequences showed that CmMYB11 belonging to SG7 subfamily,harbored a relatively conservative SG7 motif in C-terminal.The spatio-temporal expression analysis showed that CmMYB11 specially expressed in the petals of ‘Anastasia Pink’ from uncolored stage to fully colored stage.CmMYB11 was localized in the nucleus.CmMYB11 exhibited transcriptional activation activity at the C-terminal.Over-expression of CmMYB11 in tobacco promoted the accumulation of total flavonoids,including anthocyanins in petals.Yeast one-hybrid assay and luciferase assay showed that CmMYB11 activates promoters of CmCHS2,CmCHI,CmDFR,Cm ANS,Cm FNS and Cm FLS.The expression patterns of CmMYB11 in six ‘Anastasia’ cultivars with different colors,correlated to the total flavonoid contents as well as expression patterns of CmCHS2 and CmCHI.Those results suggested that CmMYB11 promotes total flavonoid biosynthesis in chrysanthemum,and down-regulation of CmMYB11 presumably resulted in decreased total flavonoids in the mutant of ‘Anastasia Dark Green’.5.An R2R3-MYB transcription factor,CmMYB21 was cloned.Analysis of phylogenetic tree and protein sequences showed that CmMYB21 belonging to SG19 subfamily,was most closely related to At MYB21 in Arabidopsis.The spatio-temporal expression analysis showed that CmMYB21 specially expressed in the petals of ‘Anastasia Pink’after the full coloration.CmMYB21 was localized in the nucleus.CmMYB21 exhibited no transcriptional activation activity.Over-expression of CmMYB21 in tobacco resulted reduction of anthocyanins and decoloration in petals.Yeast one-hybrid assay and EMSA showed that CmMYB21 binds to the CAACCA element in promoter of CmDFR.Luciferase assay showed that CmMYB21 suppresses the expression of CmDFR.The RT-qPCRanalysis showed that the expression levels of CmMYB21 and CmDFR in cultivars including ‘Baifeiwu’,‘Fenluoli’,and ‘Zisongyue’ with color mutation,had opposite trends.Those results suggested that CmMYB21 inhibits anthocyanin biosynthesis by suppressing CmDFR,and up-regulation of CmMYB21 may be involved in decreased anthocyanins in the mutant of ‘Anastasia Pink’.