Functional Analysis of SibHLH137 in Regulating Anthocyanin Biosynthesis and Drought Tolerance in Foxtail Millet

Drought is a major abiotic stress constraining the yield of foxtail millet (Setaria italica L.). Anthocyanins serve as natural antioxidants and play important roles in plant stress adaptation; however, their upstream transcriptional regulatory mechanisms remain unclear. In this study, we identified a bHLH transcription factor gene, SibHLH137, which exhibited significantly higher expression levels in various tissues of purple-leaf varieties than in green-leaf varieties. Heterologous over-expression of SibHLH137 in Arabidopsis significantly promoted anthocyanin accumulation and upregulated the expression of late anthocyanin biosynthetic genes (AtDFR, AtANS, AtUGT78D2), and enhanced drought tolerance of transgenic plants by increasing proline content and antioxidant enzyme activities as well as upregulating drought-responsive genes (AtRD29A, AtDREB2A). To validate its function in foxtail millet, we established efficient regeneration and genetic transformation systems and obtained SibHLH137-overexpressing transgenic foxtail millet lines. Compared with wild-type plants, the transgenic lines exhibited darker caryopsis and stem coloration and significantly increased total anthocyanin content, with SiDFR expression upregulated by 30-fold. Under drought stress, the transgenic lines showed increased relative water content and proline content, reduced leaf water loss rate and malondialdehyde accumulation, enhanced antioxidant enzyme activities, and significant upregulation of drought-responsive genes including SiDREB2, SiNCED1, and SiRAB18. Promoter analysis revealed the presence of G-box elements in the promoter regions of target genes. Collectively, SibHLH137 triggers anthocyanin biosynthesis and drought response pathways to improve drought resistance of foxtail millet, which offers valuable genetic resources for drought-resistance molecular breeding.