Genome-wide identification of SrbHLH transcription factors highlight its potential role in rebaudioside A (RA) biosynthesis in Stevia rebaudiana

Stevia rebaudiana Bertoni is a valuable medicinal plant and an important source of natural sweetener, steviol glycosides (SGs), with rebaudioside A (RA) being one of the main components of SGs. bHLH genes play a crucial role in plant development and secondary metabolism. In this study, a total of 159 SrbHLH genes were identified from the S. rebaudiana genome, and each gene was named based on its chromosome location. The SrbHLH proteins were then clustered into 18 subfamilies through phylogenetic analysis. The classification of the SrbHLH family was further supported by the analysis of conserved motifs and gene structure. Chromosomal location and gene duplication events of SrbHLH genes were also studied. Moreover, based on the RNA-Seq data of different tissues of S. rebaudiana , 28 SrbHLHs were found to be co-expressed with structural genes involved in RA biosynthesis. The expression pattern of candidate SrbHLH genes was confirmed by qPCR. Finally, dual luciferase reporter assays (DLAs) and subcellular localization analysis verified SrbHLH22, SrbHLH111, SrbHLH126, SrbHLH142, and SrbHLH152 as key regulators of RA biosynthesis. This study provides new insights into the function of SrbHLHs in regulating SGs biosynthesis and lays the foundation for future applications of SrbHLH genes in molecular breeding of S. rebaudiana .