The miR166–ATHB14-LIKE module regulates flavonoid biosynthesis in soybean

Flavonoids are crucial secondary metabolites widely distributed in plants, playing vital roles in diverse biological processes. Although the flavonoid biosynthesis pathway has been extensively characterized, the transcriptional regulatory mechanisms remain poorly understood. In this study, we identify the miR166–ATHB14-LIKE module comprising the miR166 and its target gene ATHB14-LIKE as a key regulator of flavonoid biosynthesis in soybean (Glycine max). Knockdown of miR166 or overexpression of ATHB14-LIKE upregulated multiple flavonoid biosynthesis genes, leading to increased flavonoid accumulation. Conversely, miR166 overexpression suppressed these genes and reduced flavonoid levels. We further show that ATHB14-LIKE directly activates specific flavonoid biosynthesis genes by binding to their promoters. Additionally, ATHB14-LIKE forms homodimers and heterodimers with homologous proteins to regulate downstream flavonoid biosynthesis genes. Together, our findings demonstrate that the miR166–ATHB14-LIKE module controls soybean flavonoid content by coordinating the expression of key biosynthetic genes.