Abstract

As the outermost structure of the plant shoot, pubescence is crucial for plant resistance to herbivores. However, the underlying mechanism regulating pubescence density and insect resistance in soybean [Glycine max (L.) Merr.] remains largely unclear. Here, we identified the pubescence density-related gene Glycine soja KNOTTED1-like homeobox 1 (GsKNOX1), a member of the KNOX transcription factor family, in wild soybean [Glycine soja Sieb. & Zucc.] through a genome-wide association study. Overexpression of GsKNOX1 increased soybean pubescence density and resistance to common cutworm (Spodoptera litura Fabricius), reduced leaf size, and altered plant architecture. Furthermore, scanning electron microscopy revealed that GsKNOX1 overexpression decreased leaf epidermal cell area and increased leaf epidermal cell density. RNA sequencing revealed that GsKNOX1 affects the expression levels of several pubescence-related genes, including the dense pubescence (Pd1) gene and the pubescence positive regulator GLABRA2 (GL2), and resistance-related genes such as lipoxygenase (LOX). Metabolomic analysis further demonstrated that GsKNOX1 mediates extensive metabolic reprogramming, leading to the marked accumulation of defense-related metabolites, including flavonoids, terpenoids, and alkaloids. Bimolecular fluorescence complementation and luciferase complementation assays validated the interaction between GsKNOX1 and Pd1. Dual-luciferase transient expression assays revealed that the GsKNOX1–Pd1 protein interaction exhibits stronger activation of glabrous (P1) compared to Pd1 alone. Application evaluation revealed that moderate GsKNOX1 expression does not obviously affect the seed yield per plant in soybean. This study highlights the significant roles of GsKNOX1 in regulating soybean pubescence density and insect resistance and provides a gene target for improving insect resistance in