Comprehensive analysis of the alfalfa FTIP family reveals MsFTIP1 as a positive regulator of early flowering

Background Flowering time is a pivotal agronomic trait that determines forage yield, quality, and environmental adaptability in alfalfa ( Medicago sativa ). The FT-INTERACTING PROTEIN (FTIP) family, a subgroup of multiple C2 domain and transmembrane region proteins (MCTPs), plays an essential role in florigen transport and flowering control across plant species. However, comprehensive characterization of FTIP family genes in alfalfa ( MsFTIPs ) has not been reported to date. Results In this study, 23 MsFTIPs from the alfalfa genome were identified and systematically analyzed their physicochemical properties, phylogenetic relationships, chromosomal localization, gene structures, conserved motifs, cis-acting regulatory elements, and expression patterns. We further performed functional characterization of MsFTIP1 , a homolog of AtFTIP1 . Subcellular localization assays demonstrated that MsFTIP1 was targeted to the endoplasmic reticulum (ER). Heterologous overexpression of MsFTIP1 in Arabidopsis resulted in significant early flowering under long-day conditions. Real-time quantitative PCR (RT-qPCR) further revealed that transcript levels of the key flowering-promoting genes AtSOC1 , AtAP1 , AtFT and AtLFY were significantly upregulated in transgenic lines. Yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), and luciferase complementation imaging (LCI) assays confirmed the direct physical interaction between MsFTIP1 and the florigen protein MsFT. Conclusion In our study, we identified 23 distinct FTIP genes in alfalfa, which were classified into six clades. We systematically characterized multiple gene-level features, encompassing physiochemical properties, phylogenetic phylogeny, exon-intron organization, conserved protein motifs, chromosomal distribution, gene duplication, cis-acting regulatory elements, protein tertiary structures, tissue-specific expression profiling, and transcriptional responses to drought, salinity and cold stresses. Furthermore, we had revealed the conserved function of MsFTIP1 in facilitating flowering by interacting with MsFT. Our findings provide a solid foundation for elucidating the molecular mechanisms underlying flowering-time regulation and supporting molecular breeding in alfalfa.