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 GA3-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 GA3 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.

Dual functions of PsmiR172b-PsTOE3 module in dormancy release and flowering in tree peony (Paeonia suffruticosa).

MicroRNAs (miRNAs) are non-coding RNAs that interact with target genes and are involved in many physiological processes in plants. miR172-AP2 mainly plays a role in the regulation of flowering time and floral organ differentiation. Bud dormancy release is necessary for forcing culture of tree peony in winter, but the mechanism of dormancy regulation is unclear. In this study, we found that a miR172 family member, PsmiR172b, was downregulated during chilling-induced bud dormancy release in tree peony, exhibiting a trend opposite to that of PsTOE3. RNA ligase-mediated (RLM) 5'-RACE (rapid amplification of cDNA ends) confirmed that miR172b targeted PsTOE3, and the cleavage site was between bases 12 (T) and 13 (C) within the complementary site to miR172b. The functions of miR172b and PsTOE3 were detected by virus-induced gene silencing (VIGS) and their overexpression in tree peony buds. PsmiR172b negatively regulated bud dormancy release, but PsTOE3 promoted bud dormancy release, and the genes associated with bud dormancy release, including PsEBB1, PsEBB3, PsCYCD, and PsBG6, were upregulated. Further analysis indicated that PsTOE3 directly regulated PsEBB1 by binding to its promoter, and the specific binding site was a C-repeat (ACCGAC). Ectopic expression in Arabidopsis revealed that the PsmiR172b-PsTOE3 module displayed conservative function in regulating flowering. In conclusion, our results provided a novel insight into the functions of PsmiR172-PsTOE3 and possible molecular mechanism underlying bud dormancy release in tree peony.