Molecular Functional and Transcriptome Analysis of Arabidopsis thaliana Overexpression BrBBX21 from Zicaitai (Brassica rapa var. purpuraria)

B-box transcription factors (TFs) in plants are essential for circadian rhythm regulation, abiotic stress responses, hormonal signaling pathways, secondary metabolism, photomorphogenesis, and anthocyanin formation. Here, by blasting the AtBBX21 gene sequence, we identified a total of 18 BBX21 genes from five distinct Brassica species (Arabidopsis thaliana, Brassica rapa, Brassica oleracea, Brassica napus, and Brassica juncea). The BrBBX21-1 gene is most closely linked to the AtBBX21 gene based on phylogeny and protein sequence similarities. The BrBBX21-1 gene, which encodes a polypeptide of 319 amino acids, was identified from Zicaitai (Brassica rapa ssp. purpuraria) and functionally characterized. BrBBX21-1 was localized within the nucleus, and its overexpression in Arabidopsis augmented anthocyanin accumulation in both leaves and seeds. We further performed an RNA-seq analysis between the BrBBX21-OE and WT A. thaliana to identify the key regulators involved in anthocyanin accumulation. In detail, a total of 7583 genes demonstrated differential expression, comprising 4351 that were upregulated and 3232 that were downregulated. Out of 7583 DEGs, 81 F-box protein genes and 9 B-box protein genes were either up- or downregulated. Additionally, 7583 differentially expressed genes (DEGs) were associated with 109 KEGG pathways, notably including plant hormone signal transduction, the biosynthesis of secondary metabolites, metabolic pathways, glutathione metabolism, and starch and sucrose metabolism, which were considerably enriched. A transcriptome analysis led us to identify several structural genes, including DFRA, GSTF12, UGT75C1, FLS1, CHI1, 4CL3, and PAL1, and transcription factors, MYB90, TT8, and HY5, that are regulated by the overexpression of the BrBBX21-1 gene and involved in anthocyanin biosynthesis. Altogether, these findings demonstrate the beneficial regulatory function of BrBBX21-1 in anthocyanin accumulation and offer valuable information about the basis for breeding superior Brassica crops.