A trade-off between warming responses and ozone sensitivity in nodulating versus non-nodulating soybean rhizosphere nitrogen cycling

Understanding how multiple abiotic stressors interact to shape rhizosphere nitrogen cycling is critical for predicting soil ecological functioning under global change. Biological nitrogen fixation in legumes creates a microbially enriched and highly active rhizosphere, yet it is unclear whether this trait reduces sensitivity or increases vulnerability when warming and oxidative stress occur simultaneously. Here, we compared the rhizosphere nitrogen cycling responses of two soybean genotypes, a nodulating cultivar (Tiefeng-29) and a non-nodulating cultivar (Clark L73-1054), exposed to factorial warming (+2 °C) and elevated ozone (+80 nL L −1 ). Warming consistently increased ammonium and nitrate availability, microbial biomass, and archaeal ammonia oxidation, with the strongest stimulation in the nodulating cultivar. In contrast, elevated ozone reduced microbial biomass, nitrifier gene abundances, urease activity, and nitrate accumulation, with effects markedly stronger in the nodulating cultivar. Elevated ozone significantly reduced grain yield in both cultivars, with a stronger proportional decline in the non-nodulating genotype; however, belowground microbial suppression did not strictly parallel yield reduction patterns. Ozone also increased nirS and nosZ abundances, suggesting enhanced denitrification-related gene abundances, whereas warming promoted nifH and AOA- amoA , indicating stimulation of nitrogen fixation and archaeal nitrification pathways. Redundancy analysis illustrated multivariate separation among warming- and ozone-associated response patterns, with nodulation aligning more closely with warming-related variation than with ozone-associated shifts. Together, these results demonstrate a trait-mediated ecological trade-off: symbiotic nitrogen fixation strengthens rhizosphere functioning under warming but becomes disproportionately sensitive to ozone stress. This has important implications for the stability of rhizosphere processes, nitrogen turnover, and legume-based soil fertility under future climate conditions. • Nodulation reshapes rhizosphere N pools and microbial processes. • Warming strengthens N fixation and archaeal nitrification. • Ozone suppresses nitrifiers and urease, most in nodulating roots. • Ozone increases nirS - nosZ , shifting N cycling toward denitrification. • Warming-ozone trade-off defines nitrogen-fixing soybean responses.