The JrCSN5a-JrBBX31-JrNCED5/JrPRX72 module regulates low-temperature tolerance in walnut

B-box (BBX) proteins represent a critical class of transcription factors that regulate plant growth, development, and stress responses. However, the functional characterization of BBX genes remains insufficiently studied in walnut ( Juglans regia ), an economically important oil-bearing tree species. In this study, we demonstrate that the cold-induced BBX transcription factor JrBBX31 enhances cold tolerance in transgenic Arabidopsis thaliana and walnut. Mechanistic analyses, including DAP-Seq, EMSA, and dual-luciferase reporter assays, reveal that JrBBX31 directly binds to the promoter of the 9- cis -epoxycarotenoid dioxygenase-encoding gene JrNCED5 and activates its transcription, thereby elevating endogenous ABA levels. Concurrently, JrBBX31 directly activates the expression of the class III peroxidase-encoding gene JrPRX72 , promoting lignin accumulation. Protein interaction studies via Y2H, Pull-down, BiFC, and Co-IP confirm the physical interaction between JrBBX31 and the COP9 signalosome subunit JrCSN5a. In vivo and in vitro degradation assays further establish that JrCSN5a destabilizes JrBBX31, interfering with its transcriptional activation of JrNCED5 and JrPRX72 , leading to the attenuation of JrBBX31 -mediated cold tolerance. Collectively, this study provides the mechanistic insight into the cold-stress response in walnut. Our findings elucidate the JrCSN5a-JrBBX31-JrNCED5/JrPRX72 signaling axis as a critical regulator of ABA and lignin accumulation, which regulates cold tolerance in walnut.