Genome-Wide Analysis of B-Box (BBX) Gene Family in Red Walnut (Juglans regia L.) and JrBBX3 Function in Relation to Anthocyanin Biosynthesis

Walnut is an important nut with a rich nutritional profile and associated health benefits for the human body. B-box (BBX) proteins containing one or two BBX motifs play pivotal roles in plant growth and developmental processes; nevertheless, the functions of JrBBXs in walnut anthocyanin biosynthesis remain inadequately understood. In this study, 39 JrBBXs in red walnut ‘RW-1’ were identified, with phylogenetic analysis suggesting that they were divided into six classes based on the distribution of conserved domains and unevenly distributed on 14 chromosomes. Promoter analysis demonstrated that JrBBX promoters possessed an abundance of light responsiveness elements, ABA responsiveness elements, MYB binding sites and MYC binding sites. The transcriptome analysis results demonstrated that eight JrBBXs were differently expressed in normal green walnut ‘Zhonglin 1’ and red walnut ‘RW-1’ seed coats. Furthermore, qRT-PCR (quantitative real-time polymerase chain reaction) analysis showed that JrBBX3 exhibited lower expression during seed coat development in ‘RW-1’. Y1H (Yeast One-Hybrid) and LUC (dual-luciferase reporter) assays revealed that JrBBX3 directly inhibited the expression of JrUFGT5, considered a key anthocyanin biosynthesis structural gene in research. Subcellular localization analysis indicated both cytoplasmic and nuclear localization of JrBBX3. Transient overexpression of JrBBX3 in walnut leaves resulted in reduced JrUFGT5 expression and anthocyanin accumulation. Collectively, these findings revealed the negative regulation of JrBBX3 in red walnut anthocyanin biosynthesis, and provided a basis to further study the anthocyanin biosynthesis mechanism of red walnut.