Microbial Transcriptional and Metabolic Shifts Mediate Rhizosphere Organic Carbon Accumulation in Alpine Grasslands Following Six Years of Continuous Nitrogen Addition

Global nitrogen (N) deposition alters soil organic carbon (SOC) dynamics in alpine grasslands, yet rhizosphere carbon turnover mechanisms remain debated due to a lack of integrated multi-omics insights. This study aimed to systematically investigate the rhizosphere carbon turnover mechanisms and explore the 6-year N addition thresholds in alpine grasslands. Leveraging a six-year in situ N addition experiment, we combined metatranscriptomics and untargeted metabolomics to systematically investigate rhizosphere carbon pathways. The 10 kg N ha−1 yr−1 treatment (N10) alleviated nutrient limitation by increasing microbial biomass nitrogen and dissolved organic nitrogen. This significantly upregulated carbon fixation genes, particularly the reductive tricarboxylic acid (rTCA) cycle, promoting the accumulation of lipids and organic acids, which subsequently increased total SOC, particulate organic carbon (POC), and mineral-associated organic carbon (MAOC). Conversely, when N inputs exceeded this threshold (e.g., the 20 kg N ha−1 yr−1 treatment), the additions induced severe soil acidification, suppressed carbon fixation transcription, and shifted the rhizosphere toward a degradation-dominated state, decreasing key carbonaceous metabolites and reducing all organic carbon fractions. Overall, continuous N input exerts a significant nonlinear threshold effect on rhizosphere carbon sequestration. Sustained N loading beyond the ecological threshold undermines soil carbon pool stability through coupled physicochemical, transcriptional, and metabolic alterations, highlighting the severe ecological risks of continuous N deposition in alpine ecosystems.