PsRGL1 negatively regulates chilling- and gibberellin-induced dormancy release by PsF-box1-mediated targeting for proteolytic degradation in tree peony

Tree peony bud endodormancy is a common survival strategy similar to many perennial woody plants in winter, and the activation of the GA signaling pathway is the key to breaking endodormancy. GA signal transduction is involved in many physiological processes. Although the GA-GID1-DELLA regulatory module is conserved in many plants, it has a set of specific components that add complexity to the GA response mechanism. DELLA proteins are key switches in GA signaling. Therefore, there is an urgent need to identify the key DELLA proteins involved in tree peony bud dormancy release. In this study, the prolonged chilling increased the content of endogenously active gibberellins. PsRGL1 among three DELLA proteins was significantly downregulated during chilling- and exogenous GA(3)-induced bud dormancy release by cell-free degradation assay, and a high level of polyubiquitination was detected. Silencing PsRGL1 accelerated bud dormancy release by increasing the expression of the genes associated with dormancy release, including PsCYCD, PsEBB1, PsEBB3, PsBG6, and PsBG9. Three F-box protein family members responded to chilling and GA(3) treatments, resulting in PsF-box1 induction. Yeast two-hybrid and BiFC assays indicated that only PsF-box1 could bind to PsRGL1, and the binding site was in the C-terminal domain. PsF-box1 overexpression promoted dormancy release and upregulated the expression of the dormancy-related genes. In addition, yeast two-hybrid and pull-down assays showed that PsF-box1 also interacted with PsSKP1 to form an E3 ubiquitin ligase. These findings enriched the molecular mechanism of the GA signaling pathway during dormancy release, and enhanced the understanding of tree peony bud endodormancy.