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.

Drought stress limits agricultural productivity worldwide. Identifying and characterizing genetic components of drought stress-tolerance networks may improve crop resistance to drought stress. We show that the regulatory module formed by miR166 and its target gene, ATHB14-LIKE, functions in the regulation of drought tolerance in soybean (Glycine max). Drought stress represses the accumulation of miR166, leading to upregulation of its target genes. Optimal knockdown of miR166 in the stable transgenic line GmSTTM166 conferred drought tolerance without affecting yield. Expression of ABA signaling pathway genes was regulated by the miR166-mediated regulatory pathway, and ATHB14-LIKE directly activates some of these genes. There is a feedback regulation between ATHB14-LIKE and MIR166 genes, and ATHB14-LIKE inhibits MIR166 expression. These findings reveal that drought-triggered regulation of the miR166-mediated regulatory pathway increases plants drought resistance, providing new insights into drought stress regulatory network in soybean.