Layered nitrogen placement increases wheat yield by transcriptionally regulating root functional strategies and reshaping soil enzymatic hotspots

The objective of this study was to identify the soil–root regulatory mechanisms by which nitrogen (N) application at varying soil depths increases nutrient use efficiency and grain yield in wheat. In a two-year field experiment on the Loess Plateau, 240 kg N ha −1 applied in a 1:2:1 ratio to the 8-, 16-, and 24-cm soil layers redistributed NO 3 − –N, soil organic carbon, available P, and enzyme activities relative to application only at the 8-cm depth. These changes were associated with enhanced root growth in deeper soil, including greater specific root length and area, higher nitrate reductase activity, and increased root N and P concentrations. Root functional analyses indicated a shift toward more acquisitive strategies, supported by the upregulation of genes related to lateral root formation and root hair development. The 8–16 cm soil layer contributed most to yield formation, explaining 43.0% of yield variation. Layered N placement increased subsoil nutrient availability, root functional differentiation, and depth-specific transcriptional activation, promoting nutrient accumulation and increasing grain yield.