Identification, evolution, and expression analysis of the PEBP gene family in tobacco (Nicotiana tabacum) and its role in flowering regulation via interaction with 14-3-3 proteins

The phosphatidylethanolamine-binding protein (PEBP) gene family plays a crucial role in regulating key biological processes in plants, such as flowering time, morphological architecture, and seed germination. However, the diversity, expression patterns, and functions of PEBP genes in Nicotiana tabacum remain largely unexplored. In this study, 32 PEBP gene family members were identified in Nicotiana tabacum and systematically classified into four subfamilies: FT-like (17 members), TFL1-like (9 members), MFT-like (2 members), and YbhB-like (4 members). Conserved motifs DPDxP and GxHR, essential for anion-binding activity, were identified in NtPEBPs. Promoter cis-acting element analysis indicated that NtPEBPs are extensively involved in light response, hormone regulation, stress response, and growth and development in tobacco. Transcriptome data revealed that some genes exhibit tissue-specific expression patterns, with certain NtBFT and NtTSF members showing high expression levels in leaves and flowers, while some members of the MFT-like and YbhB-like subfamilies were significantly expressed in dry capsules. Subcellular localization confirmed that NtATC4, NtBFT1, and NtBFT4 are localized in the cytoplasm, while NtFT7 is localized in both the nucleus and cytoplasm. Yeast two-hybrid and bimolecular fluorescence complementation assays further demonstrated that NtATC4, NtFT7, NtBFT1, and NtBFT4 can all interact with the Nt14-3-3 protein, suggesting their potential involvement in the tobacco flowering process through the formation of flowering regulatory complexes. Virus-induced gene silencing (VIGS) assays revealed that silencing of NtATC4 delays flowering and promotes vegetative growth. In NtATC4-silenced plants, the expression of flowering-related genes NbAP1 and NbSVP was upregulated, whereas NbSOC1 expression was downregulated. This study provides a comprehensive gene inventory and interaction dataset for further research on NtPEBPs and identifies candidate targets for molecular breeding in tobacco.