Bud dormancy, which serves as a survival mechanism during winter, is crucial for determining the timing and quality of flowering in many perennial woody plants, including tree peony. The gibberellin (GA) signaling pathway participates in breaking bud dormancy in tree peony. Specifically, PsRGL1, a key DELLA protein, is a negative regulator in this process. MADS-box family members participate in plant growth and development regulation. In this study, a MADS-domain transcription factor, AGAMOUS-LIKE 9 (PsAGL9), was identified as a candidate interaction protein of PsRGL1 using a pull-down assay coupled with liquid chromatography–tandem mass spectrometry. PsAGL9 expression was induced by chilling and exogenous GA₃. Yeast two-hybrid (Y2H), pull-down, and luciferase complementation assays (LCAs) confirmed that PsAGL9 interacted with PsRGL1. PsAGL9 overexpression significantly promoted dormancy break and upregulated the expression of marker genes such as PsBG6, PsBG9, PsEBB1, PsEBB3, and PsCYCD, suggesting a potential regulatory function of PsAGL9. Classical and nonclassical CArG motifs were identified in the promoter regions of PsCYCD and PsEBB3, respectively. Yeast one-hybrid (Y1H), electrophoretic mobility shift (EMSA), and dual-luciferase assays confirmed that PsAGL9 directly bound to and activated PsCYCD and PsEBB3 expression, and PsRGL1 abolished the DNA-binding activity of PsAGL9. Furthermore, interaction proteins of PsAGL9 were screened, and MADS-box members PsAGL9, PsAGL6, and PsPI were identified. Y2H, LCA, and pull-down assays confirmed that PsAGL9 formed both homodimers and heterodimers, and heterodimers further promoted target gene expression. This study provides an in-depth exploration of the GA pathway and elucidates a novel pathway, PsRGL1-PsAGL9-PsCYCD, involved in regulating dormancy break in tree peony.

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