# Identification and Expression Analysis of the bHLH Gene Family in Rhododendron × pulchrum Sweet with Different Flower Colors

## TL;DR

This study identifies and analyzes bHLH genes in Rhododendron × pulchrum, linking them to flower development and coloration.

## Contribution

The study identifies 109 bHLH genes in Rhododendron × pulchrum and links them to flower coloration and development.

## Key findings

- 109 bHLH genes were identified and grouped into 24 subfamilies in Rhododendron × pulchrum.
- Gene duplication events were found to contribute to functional diversification of RpbHLH genes.
- 25 RpbHLH genes are enriched in the flavonoid biosynthetic pathway, suggesting roles in pigment synthesis.

## Abstract

Basic helix–loop–helix (bHLH) transcription factors play significant roles in plant growth and 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 Sweet was identified and characterized using whole-genome data. A total of 109 bHLH family genes (RpbHLHs) were identified in R. pulchrum, and their expression levels were analyzed in flowers of different colors and developmental stages. The results showed that the RpbHLH family is divided into 24 subfamilies. Chromosomal localization and collinearity analyses 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 the 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 development and pigment synthesis. A Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of the target RpbHLHs revealed that 25 genes are enriched in the flavonoid biosynthetic pathway. Potential RpbHLHs related to flower development and pigment synthesis were identified through a transcriptome analysis and validated through quantitative reverse transcription PCR (qRT-PCR). This study will enhance our understanding of RpbHLH functions and provide a reference for the study of flower development and coloration in R. pulchrum.