Functional And Alternative Splicing Analysis Of FT And FD Homologous Genes In Platanus Acerifolia

Flowering and dormancy regulations have always been the important research fields of plant physiology and plant molecular biology.Recent studies have shown that some of the regulatory elements within the signaling pathway are effective in both flowering and dormancy regulations.Research based on their commonality will be the future development trend.FT and its homoeologous genes have been proved to have an important function in both flower induction and dormancy regulation.The molecular mechanism of FT pathway in flower induction has been intensively studied in many species;and this pathway has also been reported in seed dormancy of Arabidopsis and seasonal dormancy of perennial plants,but it is still very scarce.FD and its homologous genes also play a key role in floral transtion;in addition,other functions(such as flower development or dormancy regulation)have also been reported in a few species.London plane tree(Platanus acerifolia Willd.)is a perennial deciduous tree,and also a significantly important tree in urban landscaping throughout temperate climes.Based on our previous research,this work intends to study on transcriptional and post-transcriptional levels regulation of the FT homologous gene of London plane,including: analysis of annual expression patterns,expression pattern under different photoperiod and temperature treatments,ectopical expression of promoters and the predictions of cis-acting elements,as well as the distribution of alternative splicing regulation signals for PaFT by various mutations.In addition,we carried out research on the expression patterns and functions of FD homologous genes,as well as the interaction between FT and FD protein isoforms.Through this study,we hope to reveal the regulation mechanism and the different biological functions of FT and FD homologous genes,and provide a new theory for comprehensive analysis of the growth rhythm regulation of perennial ornamental plants such as London plane tree,which would be significantly improve their landscape application value.The major results as following:1.Through the analysis of the annual expression patterns of PaFT and PaFTL,it was found that PaFT was predominantly expressed in the dormant stage,while PaFTL showed a high expression level during the periods of floral transition,beginning of inflorescence development and flowering.Through the photoperiod and temperature treatments,it was found that low temperature(LT)could upregulate PaFT,while short-day(SD)enhanced the sensitivity of PaFT to LT;meanwhile,PaFTL was insensitive to both LT and SD.The predictions of promoters partly explained the difference of the expression patterns between PaFT and PaFTL.An attractive hypothesis is that PaFT and PaFTL may be not only important to regulation of floral transitions but have broader functionalities: PaFT probably put particular emphasis on the control of dormancy induced by SD/LT,and PaFTL may focus on the reproductive growth,including flowering and inflorescence development;PaFT and PaFTL are complementary to control the perennial growth in London plane tree.2.Through the analysis of the annual expression of PaFDL1 and PaFDL2,it was found that PaFDLs were predominantly expressed during the periods of floral transition,inflorescence initiation and differentiation.Ectopical expression of PaFDLs in tobacco showed that PaFDLs could induce flowering by promoting the expression of tobacco AP1 homologous gene.In addition,ectopical expression of promoters indicated that PaFDLs were mainly expressed in buds and stems,which partly explained the ability of London plane to form floral meristems in the apical and lateral buds.Protein interaction studies showed that PaFDLs protein isoforms could interact with PaFT-A and PaFTL,and also interact with some PaFT protein isoforms.These results indicated that different PaFDLs could form different complexes with PaFTL,and all or some of the complexes could activate floral meristem identity genes,thereby promoting flowering.Meanwhile,PaFDLs might interact with PaFT and PaFTL to regulate the inflorescence differentiation.3.Through the substitution,deletion and alternative splicing sites mutations of PaFT first intron,it is shown that the first intron of PaFT contains all alternative splicing signals,the regulatory signal of the third alternative splicing site was related to the first alternative splicing site or the nearby region;the regulatory signal of the first alternative splicing site of PaFT second intron was located within the 46-73 sequence of the first intron.The first intron substitution experiment also suggested that the alternative splicing of PaFDL1 may be co-regulated by introns and exons,due to the non-conservative alternative splicing site and/or the degree of intron evolution.4.The yeast one-hybrid libraries of flowering transition stage and dormancy stage were constructed,respectively,and were screened by using the baits of PaFT promoter and 2 introns.The results indicated that we successfully screened some candidate genes that may be involved in flower induction,dormancy regulation or alternative splicing regulation;however,further validation and functional identification are still needed.