A 20% nitrogen reduction threshold optimizes soil carbon stability and microbial functional resilience under long-term straw return in a rice-wheat rotation.

Reducing synthetic nitrogen (N) fertilizer while maintaining soil fertility and crop productivity is a major challenge for sustainable rice production. Straw return has been widely adopted to enhance soil organic carbon (SOC) sequestration; however, the mechanisms by which reduced N input interacts with straw return to regulate SOC stabilization remain poorly understood. Here we evaluated graded N reduction (0, 20, and 30%) under continuous straw return in a nine-year rice-wheat double-cropping experiment in central China. Soil carbon pools, aggregate stability, organic carbon functional groups, and microbial community structure and function were assessed using carbon fractionation, wet-sieving, FTIR spectroscopy, and metagenomic analyses. We found that 20% N reduction (80%NR) maintained grain yield comparable to conventional N fertilization while significantly increasing readily oxidizable organic carbon (ROOC, +18-26%), water-soluble organic carbon (WSOC, +12-21%), microbial biomass carbon (MBC, +15-24%), and carbon management index (CMI, +42-58% relative to NR). Moderate N reduction also enhanced macroaggregate-associated SOC and the relative abundance of aliphatic and aromatic carbon functional groups. Metagenomic analyses revealed that 80%NR enriched Firmicutes (+26%), Actinobacteria (+50%), and Nitrospira, reduced methanogenic archaea, and triggered a metabolic shift from CO oxidation (coxL/cutL, -15 to -22%) toward reductive carbon fixation via the Wood-Ljungdahl pathway (hdrA2, +6 to +19%). In contrast, 30% N reduction (70%NR) undermined subsurface aggregate stability and eroded microbial functional diversity, identifying 20% N reduction as an optimal threshold within the tested gradients. These findings provide a mechanistic basis for optimizing nitrogen management in sustainable rice production systems.