Optimizing soil profile nitrogen distribution alleviates nutrient limitations in wheat fields by influencing the...

Optimizing the spatial distribution of nitrogen (N) fertilizer is critical for improving nutrient use efficiency and mitigating soil resource limitations in agroecosystems. We conducted a four-year field experiment in Northwest China to compare conventional surface application (N8; all fertilizer placed at 0-8 cm) with layered fertilization (N1-2-1; fertilizer placed in a 1:2:1 ratio at 0-8, 8-16, and 16-24 cm). Soil physicochemical properties, extracellular enzyme stoichiometry, high-throughput sequencing, and cross-kingdom microbial network analysis were combined to assess nutrient availability, microbial community assembly, and their links to wheat N uptake. N1-2-1 decreased NO₃^--N accumulation in 0-24 cm soil while increasing available phosphorus and microbial biomass in the rhizosphere. Enzyme stoichiometry and vector analysis showed that N1-2-1 alleviated microbial N limitation and shifted metabolic investment toward C and P acquisition. Microbial diversity responded differently across soil compartments, with N1-2-1 enriching Proteobacteria and Basidiomycota in the rhizosphere. Cross-kingdom network analysis indicated that N1-2-1 enhanced network complexity, positive interactions, and natural connectivity, especially in the rhizosphere. Partial least squares path modeling further demonstrated that soil properties and key microbial taxa indirectly promoted wheat N accumulation by stimulating extracellular enzyme activities. Overall, layered fertilization reshaped soil nutrient-microbe interactions, mitigated microbial N limitation, and strengthened microbial network stability, thereby improving nutrient uptake and yield while reducing topsoil nutrient losses. These findings offer a sustainable N management strategy for intensive wheat production systems.