Analysis of bZIP transcription factors in Rhododendron simsii and functional study of RsbZIP6 in regulating anthocyanin biosynthesis

The basic leucine zipper (bZIP) transcription factors play a critical role in various plant biological processes, including anthocyanin biosynthesis. This study focuses on Rhododendron simsii, a notable ornamental species with insufficiently explored bZIP transcription factors. We identified 66 bZIP transcription factors in the R. simsii genome and conducted comprehensive bioinformatics analyses to determine their gene localization, phylogenetic relationships, grouping, gene/protein structure, duplication events, synteny, and expression profiles. Our analysis identified RsbZIP6, a homolog of HY5 known to influence anthocyanin biosynthesis in many plants, as a potential regulator of this pathway. We cloned the complete coding sequence of RsbZIP6, which encodes a 170amino acid protein spanning 510 bp. Subcellular localization analysis verified the nuclear presence of the RsbZIP6 protein. RT-qPCR analysis revealed the highest expression of RsbZIP6 in petals, which correlated with anthocyanin accumulation. Transgenic experiments indicated that overexpressing RsbZIP6 in Arabidopsis enhanced anthocyanin accumulation by upregulating genes involved in anthocyanin biosynthesis (4CL, CHS, CHI, DFR, F3H, F3'H, ANS and UF3GT). Our findings enhance understanding of the bZIP transcription factor family in R. simsii and underscore the vital role of RsbZIP6 in anthocyanin biosynthesis, providing insights for future genetic enhancement strategies.