The PavbHLH162-Like-PavbHLH106 Module Positively Regulates Cold Tolerance in Sweet Cherry (Prunus avium L.)

bHLH transcription factors serve as crucial regulators of plant growth, development, and responses to abiotic stress. However, their specific functions in the cold resistance of sweet cherry remain elusive. In this study, we cloned PavbHLH162-like and performed gain- and loss-of-function assays to explore its role under cold stress. PavbHLH162-like was upregulated by cold stress, and its encoded protein was localized in the nucleus and possessed transcriptional activation activity. Overexpression of PavbHLH162-like in tobacco significantly enhanced cold tolerance, as evidenced by reduced malondialdehyde (MDA) content and reactive oxygen species (ROS) accumulation, along with increased levels of soluble sugars, soluble proteins, and antioxidant enzyme activities compared to the wild-type (WT) plants. Conversely, silencing PavbHLH162-like increased cold sensitivity, leading to elevated MDA and ROS levels, and decreased contents of soluble compounds and antioxidant enzyme activities. Additionally, overexpression of PavbHLH162-like enhances seed germination and root growth in tobacco. Both overexpression and silencing affected the expression of cold-related genes. Yeast two-hybrid and luciferase assays demonstrated that PavbHLH162-like interacts with PavbHLH106. Furthermore, PavbHLH162-like binds to the promoter of PavNAC29-like and activates its transcription. This activation was strengthened by the PavbHLH162-like-PavbHLH106 complex. Collectively, our findings indicate that PavbHLH162-like acts as a positive regulator of cold tolerance in sweet cherry, which provides insights into the molecular mechanisms of cold resistance in this species.