Phylogenetic, Molecular, and Functional Characterization of PpyCBF Proteins in Asian Pears (Pyrus pyrifolia)

Abstract

C-repeat binding factor/dehydration-responsive element (CBF/DRE) transcription factors (TFs) participate in a variety of adaptive mechanisms, and are involved in molecular signaling and abiotic stress tolerance in plants. In pear (Pyrus pyrifolia) and other rosaceous crops, the independent evolution of CBF subfamily members requires investigation to understand the possible divergent functions of these proteins. In this study, phylogenetic analysis divided six PpyCBFs from the Asian pear genome into three clades/subtypes, and collinearity and phylogenetic analyses suggested that PpyCBF3 was the mother CBF. All PpyCBFs were found to be highly expressed in response to low temperature, salt, drought, and abscisic acid (ABA) as well as bud endodormancy, similar to PpyCORs (PpyCOR47, PpyCOR15A, PpyRD29A, and PpyKIN). Transcript levels of clade II PpyCBFs during low temperature and ABA treatments were higher than those of clades I and III. Ectopic expression of PpyCBF2 and PpyCBF3 in Arabidopsis enhanced its tolerance against abiotic stresses, especially to low temperature in the first case and salt and drought stresses in the latter, and resulted in lower reactive oxygen species (ROS) and antioxidant gene activities compared with the wild type. The increased expression of endogenous ABA-dependent and -independent genes during normal conditions in PpyCBF2- and PpyCBF3-overexpressing Arabidopsis lines suggested that PpyCBFs were involved in both ABA-dependent and -independent pathways. All PpyCBFs, especially the mother CBF, had high transactivation activities with 6XCCGAC binding elements. Luciferase and Y1H assays revealed the existence of phylogenetically and promoter-dependent conserved CBF–COR cascades in the pear. The presence of a previously identified CCGA binding site, combined with the results of mutagenesis of the CGACA binding site of the PpyCOR15A promoter, indicated that CGA was a core binding element of PpyCBFs. In conclusion, PpyCBF TFs might operate redundantly via both ABA-dependent and -independent pathways, and are strongly linked to abiotic stress signaling and responses in the Asian pear.