Identification and Expression Analysis of the bHLH Gene Fam-ily in Rhododendron pulchrum: Insights into Flower Color and Developmental Stages

Basic Helix-Loop-Helix (bHLH) transcription factors play significant roles in plant growth, organ development, and diverse biochemical processes. However, the function of bHLH transcription factors in woody plants is not fully understood. In this study, the bHLH gene family in Rhododendron pulchrum (R. pulchrum) was identified and charac-terized using whole-genome data. A total of 109 bHLH family genes (RpbHLH) in R. pulchrum were identified, and their expression levels were analyzed in flower with dif-ferent colors and developmental stages. The results showed that the RpbHLH gene family is divided into 24 subfamilies. Chromosomal localization and collinearity anal-yses revealed numerous duplication events during evolution, which is one of the main reasons for the diversification of gene functions. The bHLH domains showed relative conservation of RpbHLH proteins. In the promoter regions of RpbHLHs, various cis-regulatory elements involved in light response, gibberellic acid (GA) response, and abscisic acid (ABA) response were identified. These elements may regulate flower de-velopment and pigment synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of RpbHLH target genes revealed that 25 genes are en-riched in the flavonoid biosynthetic pathway. Potential RpbHLH genes related to flow-er development and pigment synthesis were identified through transcriptome analysis and validated by quantitative reverse transcription PCR (qRT-PCR). This study will enhance the understanding of RpbHLHs functions and provide references for the study of flower development and coloration in R. pulchrum.