Identification of the PsEXP Gene Family and Functional Analysis of PsEXPA4-1 During Flower Opening Process in Tree Peony (Paeonia suffruticosa)

Background/Objectives: Tree peony (Paeonia suffruticosa) has a short flowering period, and its regulatory mechanisms remain poorly understood. These limitations have severely constrained its industrial development. Expansins (EXPs) are key proteins that mediate cell wall loosening associated with petal expansion, yet they remain uncharacterized in tree peony. Methods: This study utilized gene family identification, key gene screening and functional research, as well as regulatory network analysis to reveal the role of the EXP family in the regulation of flower opening. Results: This study presents the first genome-wide identification of 36PsEXPgenes in tree peony, classifying them into four evolutionarily conserved subfamilies (PsEXPA, PsEXPB, PsEXLA, and PsEXLB). Promoter analysis revealed that 28 out of 36PsEXPpromoters contain gibberellin (GA)-responsive elements. Exogenous GA3treatment significantly promoted flower opening and upregulated eightPsEXPs, withPsEXPA4-1showing the highest expression level and promoter GA-responsive element abundance. Subcellular localization confirmed that PsEXPA4-1 was targeted to the cell wall. Overexpression ofPsEXPA4-1inArabidopsisled to early flowering and enlarged petals, indicating thatPsEXPA4-1had the potential to promote cell expansion, consistent with its proposed role in tree peony flower opening. Mechanistically, we identified a bHLH transcription factor, PsbHLH25, whose expression was induced by GA. Y1H and dual-luciferase assays indicated that PsbHLH25 can bind to thePsEXPA4-1promoter. Conclusions: This study systematically characterized the EXP gene family in tree peony, revealedPsEXPA4-1as a key effector downstream of GA promoting flower opening, and discoveredPsbHLH25as a transcriptional activator linking GA signaling toPsEXPA4-1. These findings provided important insights into GA-mediated floral opening and genetic resources for understanding the molecular mechanisms and enabling precise flowering time regulation in tree peony.