Regulation And Functional Analysis Of Populus Tomentosa PtSEP3 Gene In Floral Induction And Secondary Growth

In previous studies, PtSEP3 protein was isolated from the regeneration system of the SVS in P.tomentosa and PtSEP3 was found to play important roles in flower induction and secondary growth. SEP3 protein participate in flower development in Arabidopsis, but there is no evidence that SEP3 plays important roles in secondary growth. Therefore, it is meaningful to study the mechanism that PtSEP3 has function not only in flower induction but also in secondary growth.In this study, we firstly collected wild type tobacco internodes in rooting stage(WRT), stem bolting stage(WSB), rosette stage(WRS), strengthly growing stage(WSG) and flower budding stage(WFB) and collected 35S::Pt SEP3 transgenic tobacco in rooting stage(TRT), stem bolting stage(TSB) and flower budding stage(TFB) for De novo Transcriptome Assembly. On this basis, we used RACE technique to clone 9 flower induction related genes and 15 secondary growth related genes. 8 RNA-Seqs were applied using next-generation sequencing technology in the 5 developmental growth stages of wild type tobacco and 3 developmental growth stages of 35S::PtSEP3 transgenic tobacco. By conduct a comparison between the 8 RNA-Seqs, we found that secondary growth related genes in rooting stage, stem bolting stage and flower budding stage of 35S::PtSEP3 transgenic tobacco have similar expression patterns with rosette stage, strengtrly growing stage and flower budding stage of wild type tobacco. We made expression analysis with flower induction related genes and secondary growth related genes in every internode in the 5 developmental growth stages of wild type tobacco and the 3 developmental growth stages of transgenic tobacco. Analyzing with the results of 8 RNA-Seqs and the expression, we found that promoting factor genes in flowering were up-regulated and repressing factor genes in flowering were down-regualted. But the secondary growth related genes have comprehensive expression pattern. In every internode and in every stage, NtCRE1 have higher expression level in transgenic tobacco than that in wild type tobacco.For understanding of the comprehensive function of PtSEP3, we generated 35S::Pt SEP3 transgenic Arabidopsis plants and the phenotypes were as blew: early flowering; flower without sepal appeared; leaves changed small, shrink, curly; short lifetime; short siliques, less number of seeds with some seeds deletion and abnormal; vigorous root growth. We made analysis of expression level and found that promoting factor genes in flowering were up-regulated and repressing factor genes in flowering were down-regualted. The expression levels of CRE1 and SEP3 were up-regulated. It suggests that PtSEP3 can not only promote early flowering, but also regualte vascular development.In this sense, we made expression analysis in cre1–12 ahk2–2tk ahk3–3, cre1–12 and 35S::PtSEP3/cre1–12 and we found that the expression level of CRE1 in 35S::Pt SEP3/cre1–12 is less than that in cre1–12 ahk2–2tk ahk3–3 and cre1–12 and the SEP3 expression level increased by 4 times. The expression of CRE1 and FT were up-regulated in 35S::PtSEP3 transgenic Arabidopsis, while the expression level of FT in cre1–12, cre1–12 ahk2–2tk ahk3–3 and 35S::PtSEP3/cre1–12 was half of that in WT. It suggests that PtSEP3 activated CRE1 and CRE1 activated FT.In order to study the functions of PtSEP3 in vscular tissue, we chose 3 arabidopsis vascular tissue tissue-specific promoter to construct 3 expression vectors(ProVND6::PtSEP3, ProNAC012::PtSEP3, ProNAC073::PtSEP3). Then, we generated transgenic tobacco using infection. Expression analysis was performed and we found that the expression of NtCRE1, NtCO and NtFT were up-regulated. While, the expression of other flowering time related genes were down-regulated. The change of the expression of NtSEP3 was not obvious. The expression of NtCRE1, NtSEP3 and all the flowering time related genes were up-regulated. It suggests that PtSEP3 upregulated NtCRE1, NtCO and NtFT1 in vascular tissue. So it means the differentiation of vascular tissue is closely related to flowering.In order to determine the relationship between the secondary growth and flowering time, we do graft experiments between two plants with different genetic background. The plants grafted using stock or scion from older plant flowered earlier than control. In other words, the secondary growth in stem has relationship with the flowering time.We conclude the interaction pathway based on the expressin analysis in transgenic tobacco and transgenic Arabidopsis: PtSEP3 upregulated CO, CRE1 and SEP3 firstly; CO and CRE1 activate other downstream flowering genes.