CpCBF6-Mediated Activation of Antioxidant Defense and Osmotic Adjustment Enhanced Cold Tolerance in Cerasus pseudocerasus Lindl.

Low-temperature stress restricts plant growth, development, and geographical distribution. The C-repeat binding factor (CBF) is a core transcriptional regulator in plant cold stress responses, but its role in the thermophilic "Manaohong" cherry (Cerasus pseudocerasus Lindl.) remains elusive. Here, a cold-induced transcription factor gene, CpCBF6, was isolated and characterized from this species. Phylogenetic analysis showed its protein shares the highest homology with orthologs from Prunus persica and Prunus dulcis in Rosaceae. CpCBF6 localized to the nucleus, and its transactivation function mainly depends on the N-terminal and C-terminal domains. Forward and reverse genetics confirmed that CpCBF6 positively regulates cold tolerance. Heterologous overexpression in Arabidopsis thaliana enhanced cold tolerance, while virus-induced gene silencing (VIGS)-mediated knockdown in "Manaohong" cherry increased cold sensitivity. Physiological and biochemical analyses revealed that CpCBF6 scavenges reactive oxygen species (ROS) by enhancing superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, thereby reducing malondialdehyde (MDA) accumulation. Concurrently, CpCBF6 promoted the synthesis of soluble sugars, soluble proteins, and proline, alleviating oxidative and osmotic stress. Yeast one-hybrid and dual-luciferase assays demonstrated that CpCBF6 directly targets and activates promoters of the cold-responsive genes CpCOR27 and CpCOR47. This study provides the first systematic functional characterization of CpCBF6 in "Manaohong" cherry, elucidating its molecular mechanism in enhancing cold tolerance via activating CpCORs expression, strengthening antioxidant defense, and improving osmotic adjustment, thus offering a key candidate gene and theoretical foundation for cold tolerance breeding in fruit trees.