Functionnal Characterization Of NnFTIP1 Of Nelumbo Nucifera And Its Mechanism Of Florigen Transport

Lotus(Nelumbo Nucifera Gaertn.),a perennial aquatic dicotyledonous plant in the family Nelumbonaceae.It belongs to the basal group of true dicotyledonous plants.Lotus is orginated from China.With abundant germplasm resources,China is also the distribution and cultivation center of locus in the world.Lotus is an excellent aquatic ornamental plant in summer.Flowering time is one of the most important characters affecting its ornamental value.‘China Antique',a temperate lotus,is a type of long-day plant that usually blooms from June to August and withers in September and October in China's mainland.The temporary blooming of locus could not satisfy the lotus lovers and significantly affect its economic benefits.So it is crucial to study the molecular mechanisms of flowering time control in lotus and then apply novel genetic engineering approaches to modify flowering traits.The Nelumbo necifera cultivar ‘China Antique' was used for this study.We analysed the evolutionary relationship and structural domains of the NnFTIP1 protein as well as the expression levels of NnFTIP1 and its homologous genes in different organs of lotus.We characterized the NnFTIP1 and NnFT1 and their expression patterns in Arabidopsis.We detected the physical interaction between NnFTIP1 and NnFT1 by yeast two-hybrid,firefly luciferase complementation imaging,pull down and Coimmunoprecipitation assays.The results revealed that NnFTIP1 interacts with NnFT1 and affects NnFT1 transport from leaves to the SAM.In addition,an MYB transcription factor,Nn UOF8,was screened to act as the direct regulator of NnFTIP1.The main research results are listed as follows.1.In higher plants,the evolution of FTIP1 is relatively conserved.We could not attain the ho conservative mology in lower plants or animals.Plants may conservatively select the FTIP1-mediated florigen transport pathway during the long evolutionary process.2.NnFTIPs consist of the N-terminal C2 domains and the C-terminal transmembrane regions.We used the Arabidopsis FTIP1 sequence to blast the NnFTIPs in lotus and found a total of seven FTIP1-like FTIPs.Except NnFTIP1 and NnFTIP3 contain three contain three C2 domains,the other five NnFTIPs contain four C2 domains.3.The spatial expression of NnFTIP1 and NnFT1 was analyzed by q RT-PCR and GUS staining assay in various lotus tissues,including the roots,stems,leaves,petals,and seeds.The results showed that both NnFTIP1 and NnFT1 were ubiquitously expressed in all tissues detected,and the highest expression occurred in vascular tissues of lotus leaves.The expression levels of seven NnFTIPs were relatively high in leaves.The expression levels of NnFTIPs were also higher in roots except NnFTIP5.4.NnFTIP1 and NnFT1 could promote flowering when they were overexpressed in Arabidopsis,respectively,by activating the expression of AP1 and LFY.Moreover,NnFT1 could interact with FD to modulate the flowering time of Arabidopsis.5.To check the subcellular localization of NnFTIP1 and NnFT1,we monitored the transient expression or coexpression signal of green fluorescent protein(GFP)fused with NnFTIP1 and red fluorescent protein(RFP)with the NnFT1 in Nicotiana benthamiana leaf epidermal cells.The results revealed that NnFTIP1 was localized to endoplasmic reticulum and NnFT1 was localized to endoplasmic reticulum and nuclei.Both of them were colocalized to the endoplasmic reticulum.6.The interaction between NnFTIP1 and NnFT1 was examined by various approaches,including yeast two-hybrid assay,firefly luciferase complementation imaging,pull down and Co-immunoprecipitation experiment.The yeast two-hybrid results also revealed that the truncated proteins of NnFTIP1 strongly interact with NnFT1 when both the first and third C2 domains were present.In addition,NnFTIP1,NnFTIP5 and NnFTIP7 could interact with FT in Arabidopsis,but the interaction between NnFTIP1 and FT was the strongest.7.We created ftip1 SUC2:NnFT1-3FLAG and ftip1 SUC2:NnFTIP1 transgenic Arabidopsis and then hybridized to get ftip1 SUC2:NnFT1-3FLAG SUC2:NnFTIP1 transgenic Arabidopsis.Gentic and protein assay showed that NnFTIP1 could affect the transport of NnFT1 from leaves to shoot apical meristem,and thus affect plant flowering.8.There are a large number of binding sites for MYB transcription factors in the promoter region of NnFTIP1.We performed a yeast one-hybrid screen with the NnFTIP1 promoter sequence using a c DNA library prepared from the leaves of the lotus.And an MYB transcription factor Nn UOF8 was improved could directly activate the expression of NnFTIP1 by yeast one-hybrid and dual fluorescence reporter assay.In summary,our results revealed that NnFTIP1 and NnFT1 promote flowering in Arabidopsis,demonstrated that the functions of FTIP1 and FT are conserved in evolution in flowering plants and confirmed Nn UOF8 as an upstream regulator of NnFTIP1.The identification of key flowering regulators such as NnFT1 and NnFTIP1 will contribute to improving lotus flowering traits through either classical breeding or novel genetic engineering approaches.