The Mechanisms Of CmMYB#7 And CmbHLH2 Regulating Anthocyanin Metabolism In Chrysanthemum

Chrysanthemum(Chrysanthemum morifolium Ramat)is one of the most important ornamental plants.Flower color is a key trait for its commercial value.Anthocyanins are the main pigment produced by red chrysanthemum cultivars,which regulated by the external environment and their development.From previous study,MYB and bHLH are key factors that involved in the regulation of anthocyanins metabolism in many plant species.However,the identification of key transcription factors in chrysanthemum is comparatively lagging behind.In this paper,on the basis of previous identifying anthocyanin activator CmMYB6,we used different chrysanthemum cultivars including'Z1'(dark red flower),'Z2'(pink flower),'Z3'(yellow flower),'white Jimba'(WJ)and 'Turning red Jimba'(TRJ)as plant materials.isolated and identified a bHLH,CmbHLH2,involved in anthocyanin metabolism and a MYB,CmMYB#7,played an negative role in the transcriptional regulation of anthocyanin metabolism in TRJ.The main results are as follows:1.Identification of CmbHLH2 involved in anthocyanin metabolism of chrysanthemum.CmbHLH2 was isolated from EST database by evolutionary analysis and protein sequence alignment.Its expression was positively correlated with anthocyanin contents and anthocyanin biosynthetic genes expression in three different cultivars 'Z1'.,'Z2'and'Z3'.Promoter induction analysis showed that CmbHLH2 could co-activate CmDFR promoter with the reported anthocyanin activator CmMYB6.Yeast one-hybrid showed both CmMYB6 and CmbHLH2 could bind to CmDFR promoter,and CmMYB6 could interact with CmbHLH2 to form a complex according to yeast two-hybrid.Transient over-expression of CmMYB6 together with CmbHLH2 into tobacco leaves could induce anthocyanin accumulation.2.Screening anthocyanin related MYBs from chrysanthemum.Using chrysanthemum RNAseq database.we isolated 91 MYBs expressed in 'Jimba'flower petals.The different expressed MYBs in WJ and TRJ were analyzed and expressed fold more than 4 were screen out for candidate.Among them,the expression of CmMYB#17,CmMYB#16,CmMYB#39 and CmMYB#85 were up-regulated in TRJ than WJ.CmMYB#7 and CmMYB#15 were down-regulated in TRJ than WJ.The expression of CmMYB#17,CmMYB#16 and CmMYB#85 in three different colored petals of TRJ were positively correlated with anthocyanin contents except CmMYB#39,and the expression of CmAYB#7,CmMYB#15 were negatively correlated with anthocyanin contents.Therefore,those five MYBs are considered as important candidates for anthocyanin metabolism regulation.3.Elucidate the molecular mechanism of R3-type MYB repressor CmMYB#7 regulating anthocyanin metabolism of chrysanthemum.The promoter induction effects of 5 candidate MYBs including CmMYB#17,CmMYB#16,CmMYB#85.CmMYB#7 and CmMYB#15 were analyzed.The results showed that none of them could activate or inhibit the activation of structural genes CmDFR and CmUFGT promoter alone,but when mixed with CmMYB6 and CmbHLH2,CmMYB#7 could inhibit the activation effect of CmDFR and CmUFGT that actived by CmMYB6-CmbHLH2 complex.Further Yeast-two hybrid assays,site-directed mutagenesis assays and Firefly Luciferase Complement Image assays showed that CmMYB#7 could compete for binding CmbHLH2 to inhibit the transcription activation effect of CmMYB6-CmbHLH2 complex on anthocyanin structural genesIn conclusion,we found an anthocyanin related bHLH transcription factor CmbHLH2,which could interact with CmMYB6,a reported anthocyanin activator,to enhance the transcriptional activity of anthocyanin pathway gene CmDFR promoter.In addition,we identified an R3-type MYB anthocyanin repressor,CmMYB#7.It competed with CmMYB6 for binding CmbHLH2 to inhibit the transcriptional activity of anthocyanin biosynthetic genes CmDFR and CmUFGT promoters of chrysanthemum and participated in the transcriptional regulation of TRJ anthocyanin metabolism.