Identification and Functional Characterization of the PEBP Gene Family in Regulating Flowering Time in Saccharum spontaneum and Saccharum officinarum

The phosphatidylethanolamine-binding protein (PEBP) family serves as pivotal regulators of plant flowering, orchestrating floral transition through the coordination of photoperiodic and hormonal signaling pathways. This study systematically characterized PEBP gene family members in the ancestral Saccharum species S. spontaneum and S. officinarum, elucidating their structural architecture, evolutionary trajectories, and expression profiles to unravel the molecular mechanisms governing flowering regulation and facilitate molecular breeding strategies.

The PEBP gene family was identified by performing sequence alignment of Arabidopsis and rice PEBP protein sequences against the genomes of S. spontaneum and S. officinarum, supplemented with screening using the hidden Markov model profile PF01161. Phylogenetic reconstruction, gene structure analysis, conserved motif identification, and synteny evaluation were performed using MEGA-X and TBtools. Putative cis-regulatory elements within promoter regions were predicted via the PlantCARE database. Transcriptomic profiling coupled with qRT-PCR validation delineated expression dynamics, while functional characterization of FT3 and TFL1 genes was achieved through Arabidopsis transformation.

Our analysis identified 23 and 20 PEBP genes in S. spontaneum and S. officinarum, respectively, classified into three subfamilies: FT-like, TFL1-like, and MFT-like. Protein characterization revealed greater variability in S. spontaneum PEBPs (151-364 aa; 17.1-40.4 kDa) compared to S. officinarum (170-191 aa; 19.2-20.8 kDa), though both predominantly encoded hydrophilic alkaline proteins. Conserved motif analysis demonstrated stringent conservation in TFL1-like subfamily members, while MFT-like proteins exhibited remarkable structural plasticity, exemplified by elongated PEBP domains in SspMFT1.1/1.2/1.3. Gene architecture analysis showed FT-like members possessed the highest exon variability (2-5), contrasting with TFL1-like (3-4) and MFT-like (fixed 4-exon) subfamilies. Evolutionary analysis revealed whole-genome and segmental duplications as primary expansion mechanisms, with tandem duplication frequency substantially higher in S. officinarum (8%) than S. spontaneum (1%). Syntenic analysis uncovered species-specific duplication events (e.g., SspFT5.1/SspFT5.2 and SoFT12.1/SoFT12.2) and stronger conservation between S. spontaneum and sorghum. Promoter analysis identified abundant light- and hormone-responsive elements, particularly jasmonic acid and abscisic acid response motifs. Expression profiling identified two key genes, FT3 and TFL1, with contrasting expression dynamics. FT3 displayed a sustained, photoperiod-sensitive upregulation, whereas TFL1 showed an initial decrease followed by an increase and was independent of photoperiod regulation. Transgenic validation confirmed FT3 orthologs consistently accelerated flowering (~19 days early), whereas TFL1 genes functioned as floral repressors.

Genomic analysis revealed 23 and 20 PEBP genes in S. spontaneum and S. officinarum, respectively, phylogenetically clustered into three well-defined subfamilies (FT-like, TFL1-like, and MFT-like) that exhibited substantial structural and functional divergence. FT3 and TFL1 demonstrated opposite expression patterns during floral induction, with FT3 showing marked photoperiod responsiveness and significantly elevated transcript abundance in S. spontaneum relative to S. officinarum. Transgenic functional analysis confirmed that FT3 typically promotes flowering, whereas the TFL1 gene suppresses it.