Biochar-driven redistribution of soil nitrogen pools enhances nitrogen uptake in wheat under elevated CO2

• Biochar increases wheat N uptake under e CO 2 by improving both fertilizer-N and native soil-N use. • Biochar shifts soil N to labile pools and retains it within micro-aggregates. • Biochar mitigates elevated CO 2 -induced NO 3 - -N accumulation. Under accelerating climate change, elevated atmospheric CO 2 ( e CO 2 ) presents a substantial challenge to nitrogen (N) cycling in agricultural systems. This study elucidated the mechanistic role of biochar in regulating soil N transformations and plant N acquisition under e CO 2 conditions through a three-year pot experiment (2019-2022) with wheat. Using 15 N isotope tracing combined with metagenomic sequencing, we examined the interactions between two CO 2 concentrations ( a CO 2 400 μmol mol −1 vs . e CO 2 600 μmol mol −1 ) and 2% (w/w) biochar amendment. Our results demonstrated that under e CO 2 , biochar application reduced the incorporation of fertilizer-derived N into the recalcitrant heavy fraction organic N (HFON) by 39.4%, while enhancing the content of native soil-derived N in the light fraction N (LFON) by 34.0%. Concurrently, biochar promoted the formation of micro-aggregates (<0.25 mm) and particulate organic N (PON) by 37.3 and 13.2%, respectively. Metagenomic analysis revealed that biochar under e CO 2 suppressed the relative abundance of key N-cycling genes (involved in assimilation, nitrification, and nitrate reduction) that were upregulated under a CO 2 condition. These physicochemical processes, coupled with microbial modulation, resulted in a 52.6% reduction in soil NO 3 - -N accumulation and a significant increase in aboveground N uptake. Structural equation modeling indicated that biochar counteracted the adverse effects of e CO 2 on micro-aggregate stability and N-cycling gene abundance. Synergistically, biochar enhanced the uptake of fertilizer-N and native soil-N by 30.8% and 111.4%, respectively, under e CO 2 , leading to a 55.4% increase in grain N accumulation. Our findings demonstrate that biochar is an effective amendment for mitigating e CO 2 -induced N limitation by redistributing N from recalcitrant to labile pools, enhancing N bioavailability, and ultimately supporting crop productivity in future CO 2 -enriched agroecosystems.