Long-distance Transport Of Citrus Florigen FT And Its Response Mechanism To Environmental Stress

Citrus,like most fruit trees,needs a long period of time from the seedling to the flowering,its called juvenile period.It generally takes 6-8 years to go through the juvenile period,which seriously hinders the process of citrus breeding.Therefore,the research on flowering mechanism is of great importence to fruit trees.In recent years,the studies from model plants have suggested that the protein encoded by FT is synthesized in the leaves,long-distance transported through phloem,and finally reach apical meristem to regulate the formation of flower buds.It has been proved in Arabidopsis,rice and other model plants that FT can be transported between rootstock and scions by grafting.Grafting is an important technique in fruit tree production.If FT can be transferred from rootstocks with strong flowering ability to scions by grafting in woody fruit trees,it will greatly shorten the juvenile of scion and improve breeding efficiency.Therefore,to reveal the function,regulation mode and movement mechanism of FT is of great significance in the flowering process.In this study,to study the role in flower formation and their performance during graft migration,we cloned compared the differences in FT sequences and protein structures between arabidopsis and trifoliate orange,and transformed both FT genes into tomato and trifoliate orange,respectively.Meantime,the elements of FT and its promoter were analyzed.The upstream and downstream regulatory genes were also screened through yeast one and two hybrid screening.Finally,the screened genes were also transformed in model plants for functional verification.1.We used bioinformatics to compare the sequences and protein structure between AtFT and PtFT,found both of them showed some differences in nucleotide sequence and predicted protein structure.Therefore,we constructed the over-expression vector of this two genes and genetically transformed in tomato and trifoliate orange,respectively.By observing the phenotype,we found that both genes had the function of promoting flower formation in tomato and trifoliate orange.Real-time PCR results showed that the expression levels of FT and its downstream genes were significantly up-regulated in transgenic plants.Then,we grafted wild-type tomato scions to the rockstock plants overexpressing the FT genes to confirm the mobility of FT.The flowering time of grafted scion was significantly earlier than that of the control,indicating that the FT in the rootstock can move into the scion and cause the early flowering.Western Blot experiments showed that both AtFT and PtFT proteins could be detected in the meristem at the top of the grafted scion,but the movement of their mRNA level can not be detected in RT-PCR.It indicated that AtFT and PtFT proteins can move after graftting and caused early flowering of the scion in tomato.In the citrus grafting experiment,we grafted wild-type trifoliate orange on the positive callus and observed the GFP signal in the upper scion,but no GFP signal was found in the control.Moreover,the results from real-time PCR results showed that the expression of FT in scions with positive callus had no significant change from that in control.Similarly,in the outdoor grafting experiment with AtFT and PtFT overexpression as the rootstock and WT as the scion,the GFP signal from the lower rootstock can be clearly detected in the upper scion,but the RT-PCR result did not detected the mRNA movement,which also indicated that the signal of movement through grafting was FT protein rather than mRNA.2.Transgenic plants over-expressing AtFT and PtFT could not only bloom in tubes,but also bloom in succession within six months after transplanting to the greenhouse.Therefore,we selected two lines of AtFT and PtFT transgenic plants(A1:tube flowering line from A tFT transgenic plants,A7:early flowering line from A tFT transgenic plants,P2:tube flowering line from PtFT transgenic plants,P4:early flowering line from PtFT transgenic plants)and wild-type control to perform comparative transcriptome sequencing analysis.A total of 3541 differentially expressed genes(DEGs)were found in the A1 vs WT combination,1813 were up-regulated and 1728 were down-regulated.A7 vs WT combination produced 1049 DEGs,and 623 up-regulated and 456 down-regulated.P2 vs WT combination produced 2770 DEGs,including 1716 up-regulated genes and 1054 down-regulated genes;Total 3037 DEGs were found in the P4 vs WT combination,including 1853 up-regulated genes and 1184 down-regulated genes.A1 and A7 combination contain 693 DEGs compared with WT;P2 and P4 contain 1867 DEGs with WT,respectively.There were 153 common genes in the four combinations,of which 81 were up-regulated and 72 were down-regulated.To gain insight into these DEGs,we performed KEGG enrichment analysis.153 DEGs were mainly involved in flavonoid biosynthesis,RNA degradation,fatty acid degradation,fatty acid metabolism,tryptophan metabolism,plant circadian rhythm,amino sugar and nucleotide sugar metabolism,glycerol Metabolism,vitamin B6 metabolism.3.The PtFT promoter was analyzed by the website,and found that it contains various elements,incuding heat shock factors elements;hormones-related elements such as ABA response,auxin response,jasmonate response,and brassinolide response,etc;elements related to abiotic stress such as salt/drought response;and binding sites for transcription factors such as WRKY,NAC,Zinc Finger,MYB,MADS.Then we treated the promoter of PtFT with different stress and found that it responded to low temperature,mannitol,salt stress,and ABA,but the specific mechanism and regulatory network of its response to resistance are still unclear.Therefore,we constructed the pAbAi vector linked with PtFT promoter and carried on the yeast one-hybrid analysis.Finally,a zinc finger protein SAP1 was obtained.The qRT-PCR results showed that both PtFT and PtSAP1 were significantly increased under salt stress.To further study the molecular mechanism of PtSAP1 under salt stress,we used Agrobacterium-mediated genetic transformation to obtain tobacco overexpression lines.The overexpressing PtSAP1 lines growed much better than wild-type after salt stress,as well as the relative electrical conductivity was lower than that of wild-type.In contrast,the chlorophyll content was significantly higher compared with the wild-type plants.These results indicate that PtSAP1 plays a positive regulatory role in citrus salt stress.At the same time,we also screened the PtFT interaction protein by the yeast two-hybrid,a protein TIFY8 related to abiotic stress was obtained.The yeast two-hybrid,BiFC,and pull-down experiments had verified the interaction between these PtFT and TIFY8 proteins.Finally,TIFY8 was overexpressed in Arabidopsis and the phenotype showed significantly earlier flowering compared to the wild type.This study laid a foundation for further understanding the molecular mechanism of FT regulating citrus flowering under stress.