Study On Molecular Mechanisms Of Chrysanthemum Morifolium CmARF3 And CmTCP7 In Regulating Flowering Time

Chrysanthemum(Chrysanthemum morifolium),a perennial plant of high ornamental and economic values,belongs to the tenth traditional Chinese famous flowers and one of the fourth cut flowers worldwide.Appropriate flowering time is important for the year-round production and profits.At present,the flowering time of chrysanthemum is usually reuglated by lighting or shading and temperature,which increase cost of production to a great extend.Therefore,the breeding of varieties with different flowering time is essential for year-round supply of chrysanthemum.The molecular mechanism analysis of flowering time will do aid to molecular breeding of flowering time of chrysanthemum.Based on the previous studies,ARF3 is linked to flowering time,however,the function of CmARF3 in regulating flowering time in chrysanthemum remained unclear.Here,we showed that CmARF3 directly transcriptionally inhibiting the expression of CmTCP7,thereby relieves CmTCP7’s antagonism on CmFTL3-CmFDL1 complex induced expression of CmAP1L1,consequently inducing flowering in C.morifolium ’Jinba’.The main findings are as follows:1.e firstly identified the function of the ARF3 homologous gene CmARF3 in chrysanthemum.CmARF3 gene is widely expressed in the roots,stems,leaves and apex of chrysanthemum ’Jinba’ at the vegetative period and flowering transition period,and the expression level is highest in apex;the expression pattern of CmARF3 showed no obvious rhythm,however,CmARF3 expression level were induced in leaves and apex after short-day(SD)treatment,suggest that CmARF3 is probably involved in the flowering,but not rely on the circadian rhythm patterns.Under the SD induction,the budding time of CmARF3 overexpressing lines OECmARF3 were about 8-9 days earlier than wild type(WT)plants,and full blooming time of OECmARF3 lines were 7-8 days earlier than WT plants.And the budding time of amiRCmARF3 lines were 2-3 days later than WT,and full blooming time were 1-2 days delayed in amiRCmARF3 lines compared with that of WT plants.In a conclusion,CmARF3 promotes the flowering time of chrysanthemum.2.In order to explore the molecular mechanism of CmARF3 in promoting flowering,the transcriptome sequencing analyses were performed using CmARF3 transgenic plants.The differentially expressed gene CmTCP7,a flowering inhibitor,was found.The expression level of CmTCP7 is repressed by CmARF3 under SD conditions.Three AuxREs(5’TGTCTC-3’)cis-elements were found in the promoter sequence of CmTCP7.The EMSA assays showed that CmARF3 protein could bind the sequence contains first two palindrome AuxREs elements(from-789 bp to-669 bp in CmTCP7 promoter)in vitro.Then,we used ChIP-qPCR to verify the binding sites of CmARF3 protein to the Cm TCP7 promoter in vivo.The results showed a significant enrichment at the AuxRE ranging from-871 bp to-767 bp in CmTCP7 promoter.The dual-luciferase assays via chrysanthemum protoplast transient transformation showed that the expression of luciferase gene driven by the CmTCP7 promoter was significantly supressed by CmARF3,indicating that CmARF3 repress the expression of CmTCP7 in vivo.In a summary,CmARF3 could directly bind to the promoter of Cm TCP7 and inhibit its expression.3.In order to illustrate the molecular network of CmARF3-CmTCP7 transcription cascade in regulating the flowering time of chrysanthemum,if the CmTCP7 is involved in the CmFTL3-CmFDL1 flowering pathway was testfied.The BiFC and CoIP assays showed that CmTCP7 could interact with CmFDLl and CmFTL3,respectively.The dual-luciferase assays via chrysanthemum protoplast transient transformation showed that CmFTL3CmFDL1 inducing CmAP1L1 expression in chrysanthemum ’Jinba’.CmTCP7 could partially inhibit the expression of CmAP1L1 which was induced by the CmFTL3-CmFDL1 complex.Thus,the CmARF3-CmTCP7 transcription cascade fine regulates the expression level of floral meristem identity gene CmAP1L1 through CmFTL3-CmFDLl module,thereby promote flowering of chrysanthemum.