Cold-responsive interaction between MdRAD23D1 and MdMYB15 confers cold stress tolerance via the CBF pathway in apple (Malus domestica)

Low temperature is a major environmental factor that impairs plant growth and development, posing a significant threat to crop yield and quality. RAD23 (RADIATION SENSITIVE23) proteins belong to the UBL-UBA (Uiquitin-like-ubiquitin associated) family and function as shuttle factors in the UPS (ubiquitin proteasome system). Although UBL-UBA proteins are known regulators of plant stress responses, the function and mechanism of RAD23 in apple under cold stress are poorly understood. Here, we demonstrated that MdRAD23D1 is induced by 4 °C and positively regulates cold tolerance. Silencing MdRAD23D1 impaired cold tolerance in both apple plants and calli. Conversely, its overexpression enhanced cold tolerance in transgenic tobacco, and apple calli and plants. We further demonstrated that MdRAD23D1 interacted with MdMYB15 protein via in vivo and in vitro assays. MdMYB15 functions as a negative regulator of cold stress tolerance. This is evidenced by the enhanced cold tolerance in apple calli and plants in which MdMYB15 expression was silenced, contrasted with the reduced tolerance in materials of overexpressing MdMYB15. Furthermore, yeast one-hybrid (Y1H), dual-luciferase (Dual-LUC), and electrophoretic mobility shift assays (EMSA) showed that MdMYB15 could bind to the promoters of CBF1, CBF2, and CBF3 and inhibit the expressions of the corresponding genes. In addition, MdRAD23D1 promoted MdMYB15 degradation under cold stress, thus enhancing the cold tolerance of apple. In summary, we proposed a mechanism for the response of apple to cold stress that is mediated by the 'MdRAD23D1-MdMYB15-MdCBFs' modula, which enhances our understanding of the regulation of cold tolerance in apple by UBL-UBA protein.