Synergistic Regulation Of The Mechanism Of Late Flowering By BpFLC And BpSVP

Flowering is a switch from vegetative growth to reproductive growth, which is an important part of the plant life cycle. Plant flowering regulation is mainly focused on the annual plant, model plant, Arabidopsis thaliana. The flowering regulation of perennial plants is a more complex genetic process, and the molecular mechanism of flowering regulation is still not clear. In view of the huge difference in development process between the herbaceous plants and woody plants, it is significant for the development and utilization of forest resources to extend the growth period of the tree and delay entering into the reproductive stage and the lag phase. In order to explore the regulation mechanism of late flowering of birch, The expression pattern and function of two flowering inhibitor BpFLC and BpSVP genes from birch were studied with molecular biology technique. The main conclusions are as follows:(1) qRT-PCR analysis showed that BpFLC was expressed by all sampled tissues, and the high transcription level was in young shoots and leaves. But their transcription levels decreased rapidly after vernalization. The GUS expression driven by a 2-kb promoter region of BpFLC in Arabidopsis was found in almost any tissues excluding roots, as well as in the sepals, stigma papillae, anthers, filaments, and silique-abscission zones. The promoter was not the main area for vernalization response, a lot of evidence that the key parts of vernalization response was the first intron of the FLC gene.(2) The morphological characteristics of Arabidopsis flc mutant showed early flowering, and the heterologous expression of BpFLC gene can restore the phenotype of flc mutant, the transgenic seedlings showed normal flowering or late flowering phenotype. Overexpression of BpFLC in wild-type Arabidopsis significantly delayed flowering. These transgenic plants produced more luxuriant leaves, larger reproductive organs and prolonged life cycle compared with wild-type Arabidopsis. Ectopic expression of BpFLC failed to affect COOLAIR accumulation during cold exposure.(3) RNA-Seq sequencing was performed with the material of growing points from wild type and overexpressed BpFLC seedlings. There were 237 differentially expressed genes in no-flowering group,51 genes were up-regulated and 186 genes down regulated; There were 852 differentially expressed genes in flowering group,271 genes were up-regulated and 581 genes down regulated, the overlapping genes of the two groups were 140. GO enrichment showed that the differentially expressed genes were mainly distributed in the biological process control, development process, reproductive process, cell composition and other functional groups, moreover, a large number of genes related to reproductive development were screened, such as the formation of pollen wall, pollen cell wall assembly, pollination, sexual reproduction, the process of germ cell, pollen tube development and pollen tube growth.(4) A late flowering regulating genes BpSVP was cloned from birch young leaves cDNA, and the length of open reading frame (ORF) is 678 bp, which belongs to the MIKC type MADS protein family, encoding a predicted polypeptide of 225 amino acids with a molecular weight of 25.25 kDa and a pI of 6.68. Homologous analysis showed that BpSVP was highest homology to Carya cathayensis (92%).(5) qRT-PCR analysis showed that BpSVP was expressed only in vegetative organs, and the expression level in 6-month-old plant was higher than that of the perennial plant. At the same time, the expression level in the young leaves is higher than that of mature leaves and stem tip. pUC-35S::BpSVP-GFP vector was constructed and was transiently transformed into onion epidermal cells. Subcellular localization analysis showed that BpSVP protein is localized in the nuclear. The GUS expression driven by a 2-kb promoter region of BpSVP was found predominantly in all vegetative tissues excluding roots, as well as in the young seedlings, rosette leaves and sepals, suggesting that BpSVP gene is expressed in various stages of plant growth and development.(6) pROKII-BpSVP was constructed and was transformed into Arabidopsis thaliana. BpSVP can be detected in the genome and transcription level of transgenic plants. Compared with the wild type, the transgenic plants showed a late flowering phenotype, which was delayed by about 20 days. At the same time, there were abnormal flowers in the transgenic lines. qRT-PCR results showed that endogenous AtFLC in transgenic plants was up-regulated expression, and the expression level of flowering related genes expression was significantly reduced,such as AtSOCl, AtFD, AtFT, AtLFY, AtAPl. However, the expression level of AtSVP and AtAGL24 was not significant.(7) Yeast two-hybrid assay showed BpFLC can interact with BpSVP protein. Further, BpFLC and BpSVP can respectively interact with themselves and their truncated to form a homodimer. Heterologous interaction between BpFLC and truncated BpSVP proved that the K domain from BpFLC3 and BpSVP3 is the basis for the occurrence the homologous or heterologous interactions between BpFLC and BpSVP. Finally, yeast two hybrid showed that BpFLC could also interact with AtFLC and AtSVP.In summary, this study will greatly increase our understanding of the mechanisms of extending the vegetative phase and enhance genetic improvement of traits in birch and other woody plants, and provide a theoretical basis and related genes for genetic improvement of forest trees.