PpCBFs selectively regulate PpDAMs and contribute to the pear bud endodormancy process.

KEY MESSAGE: PpCBF2 directly binds to the promoters of PpCBF3 and PpCBF4 to activate their expressions and selectively regulates PpDAMs during the leaf bud endodormancy process of 'Wonhwang' pear (Pyrus pyrifolia). Endodormancy is critical for temperate plant survival under freezing winter conditions, and low temperature is a vital environmental factor in endodormancy regulation. A C-repeat binding factor (CBF) has been found to regulate important DAM transcription factors during endodormancy in pear (Pyrus pyrifolia). In this study, we analyzed the regulation of pear DAM genes by CBFs in further detail. Four CBF and three DAM genes were identified in the pear cultivar 'Wonhwang'. Under natural conditions, PpDAM1 expression decreased from the start of chilling accumulation, while the other two DAM and three CBF genes peaked during endodormancy release. Under chilling treatment, the expressions of PpDAM1, PpDAM2 and PpCBF1 genes were similar to those under natural conditions. Different biochemical methods revealed that PpCBF2/4 can bind to the promoter of PpDAM1 and activate its expression and that PpCBF1/4 can activate PpDAM3. Interestingly, we found that PpCBF2 can activate PpCBF3/4 transcription by directly binding to their promoters. The ICE-CBF regulon is conserved in some plants; three ICE genes were identified in pear, but their expressions did not obviously change under natural and artificial chilling conditions. On the contrary, the selective transcriptional induction of PpCBFs by PpICE1s was observed in a dual-luciferase assay. Considering all these results, we propose that the PpCBF1-PpDAM2 regulon mainly responds to low temperature during endodormancy regulation, with further post-translational regulation by PpICE3. Our results provide basic information on CBF genes functional redundancy and differentiation and demonstrate that the CBF-DAM signaling pathway is involved in the pear bud endodormancy process.