Genome-Wide Identification and Expression Analysis of the PEBP Gene Family in Cymbidium sinense Reveals CsFTL3 as a Floral Inhibitor

This study comprehensively characterizes the PEBP gene family in <i>Cymbidium sinense</i>, an orchid with a prolonged vegetative phase that limits its industrial production. Genome-wide analysis identified six CsPEBPs, classified into <i>FT</i>-like, <i>TFL1</i>-like, and <i>MFT</i>-like subfamilies. Evolutionary, gene structure, and collinearity analyses revealed both conservation and lineage-specific diversification of these genes. <i>CsFTL3</i>, a distinctive FT-like member, displayed notably high expression during the bud undifferentiated stage, followed by a sharp downregulation upon floral initiation. Functional studies identified <i>CsFTL3</i> as a key floral repressor. Heterologous overexpression in <i>Arabidopsis</i> delayed flowering time from 32.0 days (wild-type) to 63.0-75.3 days (transgenic) and increased rosette leaf number from 12.6 to 33.0-34.5, while its knockdown via virus-induced gene silencing (VIGS) in <i>C. sinense</i> accelerated floral bud development and upregulated flowering-promoter genes. Phylogenetically, CsFTL3 falls within the flowering repressor FT-I clade, and multiple sequence alignment identified critical amino acid substitutions (Y134S, W138L, Q140E) that likely underpin its functional divergence from typical flowering promoters. Furthermore, promoter analysis revealed an enrichment of light-, hormone-, and stress-responsive cis-elements, and its expression was modulated by gibberellin (GA), abscisic acid (ABA), and low-temperature treatments. Predicted protein-protein interaction and transcriptional regulatory networks provide preliminary insights into its complex regulation. We conclude that <i>CsFTL3</i> acts as a crucial floral inhibitor, integrating environmental and endogenous cues to repress flowering. These findings offer fundamental insights into the molecular mechanisms of flowering in orchids and provide a valuable genetic resource for molecular breeding programs aimed at achieving precise flowering time control.