Molecular Mechanism Study On The Loquat Flowering Time Regulation

Higher plants is a very complex organism that forms a complex and sophisticated network of regulated flowering transformations.This network can accurately respond to internal and external signals,integrate them to promote flowering during the best time.Different species have formed their own specific flower regulation mechanisms caused by their different evolutionary processes.Loquat(Eriobotrya japonica Lindl.),a woody fruit tree that belonged to Rosaceae,has evolved a unique flowering strategy different from apples,pears,and plums.The difference in flowering time gives the production advantage of loquat compared to other fruit trees,but there have been few reports on the molecular mechanism of flowering regulation of loquat.Although there has been intensive research in the flowering regulation of the pattern plant Arabidopsis,and summarized a variety of regulatory pathways,including photoperiod pathway,gibberellin pathway,vernalization pathway,thermosensory pathway,autonomous pathway and age pathway.However,whether it is applicable in loquat,and what are the main factors that regulate the flowering of loquat still unknown.In this study,we first cloned the homologous genes of key genes related to flowering regulation,and carried out a comprehensive analysis of their spatiotemporal expression and functional verification.In addition,we used exogenous gibberellin and short-day treatment to explore the role of gibberellin pathway and photoperiod pathway in the regulation of flowering.Finally,combined with the production experience,the operability of inflorescence short cut technique in delayed flowering was explored.This study can depth the understand the mechanism of flowering regulation of loquat,make breakthroughs in artificially regulating the flowering,and can provide a reference for the flowering mechanism studies of other woody fruit trees.The main results of this study are as follows:1.The flower bud differentiation occurred was determined from late June to early July in Guangzhou by paraffin section and q PCR,and the obvious inflorescence was observed in early September.At the same time,it was confirmed that Ej AP1-1,Ej AP1-2,Ej LFY-1,and Ej LFY-2 could be used as marker genes to identify the flower bud differentiation time.2.Two FT homologous genes were cloned,Ej FT1 and Ej FT2,and the expression patterns of these two genes were different in loquat.Through our research,we found that Ej FT1 regulates the growth of loquat and Ej FT2 regulates the flower bud differentiation of loquat.Moreover,Ej FT2 is regulated by the photoperiod and GA₃,and it shows an obvious circadian rhythm.Ej FT1 is located in the cytoplasm and nucleus like Ej FT2,and Ej FD is localized in the nucleus.In addition,we cloned the Ej FT1 and Ej FT2 promoter regions and found that the Ej FT2 promoter region contains 4 CO protein specific binding elements CCAAT-motif and 3 photo-sensing elements Tbox,but not in the Ej FT2 promoter region.element.Moreover,Ej CO can significantly activate the Ej FT2 promoter activity,and Ej FT2can significantly activate the Ej AP1s promoter activity,the Ej FD-Ej FT2 complex enhances the activation of the Ej AP1-2 promoter by Ej FT2.Thus,the regulation mechanism of Ej FT2in loquat was summarized:Ej CO activates the expression of Ej FT2 under long-term conditions,and then Ej FT2 can directly bind to the Ej AP1s promoter to activate its expression,while Ej FT2-Ej FD complex bind to the Ej AP1-2 promoter to strongly activate its expression,then the flower buds began to differentiate.Finally,we overexpressed two Ej FTs into Arabidopsis Col-0 and found that both promote Arabidopsis flowering.Finally,we determined that the two Ej FTs perform different functions in the loquat,and the specific reason may be caused by their difference promoter areas.3.The expression levels of the two Ej TFL1 decreased sharply during the flower-forming process,and the expression trends of the two Ej AP1s were inconsistent,showing the flowering inhibitory factor of loquat.Moreover,after GA₃ and short-day treatment,there was an upward trend in the expression of flower bud differentiation,which was determined to be the flower-forming inhibitor of loquat.At the same time,we found that Ej TFL1s localization is consistent with Ej FTs in the cytoplasm and nucleus,and can also interact with Ej FD,but its function is opposite,Ej FT2 is a flower activating factor,while Ej TFL1s is a flowering inhibitor.4.The two EjSOC1 cloned from loquat are Arabidopsis At SOC1 homologous genes,which are closely related to other plants of Rosaceae;Both EjSOC1s are located in the nucleus;EjSOC1s are regulated by GA₃ and photoperiod in loquat,GA₃ and short-day treatment inhibited the transcription of EjSOC1s;It was determined that EjSOC1s regulates flower bud differentiation through the gibberellin pathway and photoperiod pathway;Finally,we overexpressed two EjSOC1s into Arabidopsis Col-0 or soc1-2 mutant and found that both promote Arabidopsis flowering.5.EjSVPs in loquat are orthologs of At SVP,but their functions in loquat are different from those in Arabidopsis.EjSVP-1 may regulate cell proliferation in loquat,and EjSVP-2may be involved in cell expansion.6.After the short cut the inflorescences,buts can sprout from the vicinity of the cross-section.The new sprout buds include leaf buds,flower buds,and leaf buds combined with flower buds.According to statistics,the number of flower buds and the inflorescence of the secondary flowering buds after the short cut treatment were significantly reduced.Comparing the experiment results,it is found that the optimal operation method for the secondary flowering induced by short-cutting treatment is the upper short section,and it is more suitable for the orange-pulp loquat variety during the full flowering period.The method of the lower short-cut treatment and the white-pulp loquat variety are not applicable.These new inflorescences induced by short cut treatment can produce normal flowers,and fruit ripening can be delayed by 2-4 months later than the fruit of normal flowering inflorescences.In a word,this short cut method can effectively delay the flowering and fruit-setting of loquat.