Genome-wide identification and functional characterization of bHLH transcription factors in Herpetospermum pedunculosum under salt stress

The widely conserved basic helix-loop-helix (bHLH) transcription factors, which constitute a major transcription factor family in plants, play crucial roles in regulating plant growth, development, and environmental stress responses. Despite their importance, the genomic composition and functional roles of bHLH transcription factors in Herpetospermum pedunculosum remain uncharacterized. In this study, we identified 98 HpbHLH genes in H. pedunculosum, which were grouped into 23 subfamilies, each exhibiting distinct conserved motifs, domains, and gene structures. Promoter cis-element analysis revealed that HpbHLH genes are enriched in phytohormone- and stress-response-responsive elements. Transcriptomic analysis of salt-stressed H. pedunculosum highlighted HpbHLH28, HpbHLH45, and HpbHLH51 as candidates for further functional analysis. Subsequent quantitative real-time PCR confirmed their expression patterns under salt stress and demonstrated tissue-specific expression differences. Subcellular localization studies revealed that all three proteins localize to the nucleus. Transcriptional activation assays further demonstrated that HpbHLH28 and HpbHLH51 possess transcriptional activity, whereas HpbHLH45 remains inactive. Additionally, dual-luciferase reporter assays showed that HpbHLH51 binds to and activates the promoter of its downstream target gene HpTIFY6B, which encodes a TIFY family protein. This study presents the first comprehensive characterization of the bHLH gene family in H. pedunculosum, establishing a molecular foundation for elucidating its stress resistance mechanisms and supporting future variety improvement efforts.