Flavonoid biosynthesis mediated by GmF3Hs contributes to drought tolerance in soybean.
Flavonoids are central to abiotic stress responses, yet the specific signaling roles and evolutionary dynamics of flavonoid biosynthetic intermediates in crop drought adaptation remain elusive. Here, we demonstrate that dihydrokaempferol (DHK) and dihydroquercetin (DHQ), specific intermediate products of the soybean flavanone 3-hydroxylases GmF3H1/2, function as potent signaling molecules that mitigate drought stress. Exogenous DHK/DHQ promoted abscisic acid-dependent stomatal closure and enhanced drought tolerance across diverse dicot species, including soybean and tobacco, highlighting a broadly conserved stress-mitigating signaling mechanism. CRISPR/Cas9-generated gmf3hs double mutants exhibited severe drought hypersensitivity due to compromised redox homeostasis and defective stomatal regulation, which could be specifically rescued by DHK/DHQ application. Furthermore, the loss of GmF3H triggered a distinct reproductive trade-off under stress, leading to increased pod initiation but severe filling defects. Multiomics network analysis revealed extensive rewiring of broader stress-responsive pathways and identified upstream transcription factors, among which GmPHL11 directly binds to and activates the GmF3H1 promoter; overexpression of GmPHL11 promoted DHK accumulation and enhanced drought stress tolerance in soybean hairy roots. Finally, population genomic analyses demonstrated that the GmF3H1H1 haplotype, which confers superior enzymatic activity and robust root growth under drought stress, might have undergone positive selection during soybean domestication. Collectively, our findings redefine the role of GmF3H-derived specific intermediates as potent signaling molecules, providing comprehensive mechanistic and evolutionary insights into flavonoid-mediated drought resilience, developmental trade-offs, and molecular breeding in crops.