The rhizosphere contributes disproportionately to free-living nitrogen fixation in subalpine forest soils

Root activity creates a unique microbial hotspot in the rhizosphere, profoundly regulating free-living nitrogen fixation (FLNF). However, empirical assessments of rhizosphere FLNF and its ecological consequences for nitrogen (N) budgets remain lacking, particularly for different root functional modules. Here, we separately collected rhizosphere soils attached to two root functional modules, absorptive roots and transport roots, and investigated the rates (¹⁵N₂ incorporation) and regulators of rhizosphere FLNF in a N-limited subalpine coniferous forest. The measured rates were further extrapolated to annual fluxes based on the volume of the rhizosphere and the relationship between FLNF rate and temperature. We found that absorptive roots drove significantly higher rhizosphere FLNF rates than did transport roots (12.2 vs. 7.5 ng N g⁻¹ d⁻¹), with both rhizosphere compartments exhibiting rates well exceeding those in the bulk soil (0.9 ng N g⁻¹ d⁻¹). The FLNF rates were positively correlated with soil organic carbon content but showed no significant relationship with the abundance or composition of the diazotrophic community. Moreover, when extrapolated to the ecosystem level the FLNF fluxes were 2-fold greater in the rhizosphere of absorptive roots than in that of transport roots (0.13 vs. 0.04 kg N ha⁻¹ yr⁻¹). Taken together, despite representing only 6.0% of the soil volume, the two rhizosphere compartments contributed as much as 47.2% to the total soil FLNF fluxes. Overall, we provide empirical evidence that despite its limited volume, the rhizosphere contributes disproportionately to the FLNF in subalpine forest soils. Our findings also underscore the critical role of root functional differentiation in regulating rhizosphere FLNF, which is essential for integrating this process into the ecosystem-level N cycle.