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 36 PsEXP genes in tree peony, classifying them into four evolutionarily conserved subfamilies (PsEXPA, PsEXPB, PsEXLA, and PsEXLB). Promoter analysis revealed that 28 out of 36 PsEXP promoters contain gibberellin (GA)-responsive elements. Exogenous GA3 treatment significantly promoted flower opening and upregulated eight PsEXPs, with PsEXPA4-1 showing the highest expression level and promoter GA-responsive element abundance. Subcellular localization confirmed that PsEXPA4-1 was targeted to the cell wall. Overexpression of PsEXPA4-1 in Arabidopsis led to early flowering and enlarged petals, indicating that PsEXPA4-1 had 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 the PsEXPA4-1 promoter. Conclusions: This study systematically characterized the EXP gene family in tree peony, revealed PsEXPA4-1 as a key effector downstream of GA promoting flower opening, and discovered PsbHLH25 as a transcriptional activator linking GA signaling to PsEXPA4-1. 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.