Functional Characterizations Of EjFT/EjTFL1 On 'Jiefangzhong' Loquat Flowering Time Regulation

Loquat(Eriobotrya japonica Lindl.)is an evergreen fruit tree natived to South China in Maloideae,Rosaceae.Contrary to most Rosaceae plants like Malus,Pyrus and A,mygdalus which flower in spring,the cultivated loquat flowers in fall and fruits mature in summer.However,the mechanism of loquat flowering time shifting remains to be elucidated.FT and TFL1 are two key factors in flowering regulation pathway.FT works as an integration factor which integrates the upstream flowering signals,regulates downstream genes expression to induce SAM differentiation by binding b-ZAP translation factor FD.TFL1 is a key gene for inflorescence meristem maintaining,which combines to FD competing with FT.And it represses the downstream flowering genes like API and LFY expression to extend vegetative growth and delay flowering.In this research,the flowering differentiation period of 'Jiefangzhong' was analyzed using paraffin section,and homologous genes EjFT1,EjFT2,EjTFL1-1,EjTFL1-2 and EjFD were cloned,the sequence phylogenetic analysis were also carried out.Their spatio-temporal expression profiles across different developmental stages and in various tissues were performed by quantitative real-time PCR.Furthermore,functional analysis of EjTFL1-1 and EjTFL1-2 were performed in Arabidopsis.The sub-cellular localizations of EjFTl,EjFT2,EjTFL1-land EjTFL1-2 were detected by tobacco leaves epidemic cells transformation,and BiFC were carried out between EjFTl/EjFT2 and EjFD.The main results were as follow:(1)The visual observation and analysis of morphological anatomy structure about the loquat cultivar 'Jiefangzhong' and 'Zaozhong-6' indicated that the flowering differentiation of them occurs in July.However,the inflorescence duration of this cultivars differs from one another,which may relate to the flowering time delaying of'Jiefangzhong '.(2)EjFTl,EjFT2,EjTFL1-1,EjTFL1-2 and EjFD were cloned and the alignment of these amino acids sequence showed high identity with their homologs in other species,especially the Malus.The important function sites of predicted amino acid were conserved.Phylogenetic analysis suggested close genetic relationships between EjFTl,EjFT2,EjTFL1-1,EjTFL1-2 and their homologs in Malus,to be clustered into narrow features.Besides,EjFD possessed the conserved b-ZIP domain.Promoters of EjTFL1-1 and EjTFL1-2 were cloned and predicted to contain several cis-elements,which suggested that expressions of these genes may under complicated regulation by multiple inner and external factors.(3)The spatial-temporal expressions of the studied genes across different developmental stages of shoot tips,leaves and other tissues of loquat were detected.The results indicated that EjTFL1-1 and EjTFLl-2 were expressed mainly before flowering differentiation,and then significantly reduced.And the quantitative analysis in different tissues showed high expression of EjTFL1-1 and EjTFL1-2 in leaf bud,and EjTFL1-2 was found to be expressed highly in flower and fruit as well,while EjFTl expressed in tissues like fruit,flower bud,leaf bud and leaf.Moreover,the expression of EjFT2 was detected in all tissues except leaf,and especially high in flower and fruit.These results suggested that EjTFL1-1 and EjTFL1-2 may repress floral bud differentiation,and hinted that EjTFL1-2 might regulate flower and fruit development as well,so do EjFT1 and EjFT2 which were also possible to be functional during floral and fruit development.(4)Overexpressions of EjTFL1-1 and EjTFL1-2 in Arabidopsis significantly delayed flowering and altered plant morphological structure,and particularly,the structure of inflorescence was distinctively modified.The qPCR analysis of TFL1 downstream genes API and LFY in transgenic plant showed the significant down-regulation of the expression of API and LFY.The above results indicated the functions of delayed flowering and regulation of inflorescence development of EjTFL1-1 and EjTFL1-2.(5)Tobacco leaves epidemic cells transient transformation suggested that EjFTl,EjFT2,EjTFL1-1 and EjTFLl-2 distributed in the nucleus and cytoplasm.And EjFT1/EjFT2 and EjFD were found to interact with each other in BiFC assay.