Methanotrophy Alleviates Nitrogen Constraint of Carbon Turnover by Rice Root-Associated Microbiomes

The bioavailability of nitrogen constrains primary productivity, and ecosystem stoichiometry implies stimulation of N₂ fixation in association with carbon sequestration in hotspots such as paddy soils. In this study, we show that N₂ fixation was triggered by methane oxidation and the methanotrophs serve as microbial engines driving the turnover of carbon and nitrogen in rice roots. ¹⁵N₂-stable isotope probing showed that N₂-fixing activity was stimulated 160-fold by CH₄ oxidation from 0.27 to 43.3 μmol N g^-1 dry weight root biomass, and approximately 42.5% of the fixed N existed in the form of ¹⁵N-NH₄ ⁺ through microbial mineralization. Nitrate amendment almost completely abolished N₂ fixation. Ecophysiology flux measurement indicated that methane oxidation-induced N₂ fixation contributed only 1.9% of total nitrogen, whereas methanotrophy-primed mineralization accounted for 21.7% of total nitrogen to facilitate root carbon turnover. DNA-based stable isotope probing further indicated that gammaproteobacterial Methylomonas-like methanotrophs dominated N₂ fixation in CH₄-consuming roots, whereas nitrate addition resulted in the shift of the active population to alphaproteobacterial Methylocystis-like methanotrophs. Co-occurring pattern analysis of active microbial community further suggested that a number of keystone taxa could have played a major role in nitrogen acquisition through root decomposition and N₂ fixation to facilitate nutrient cycling while maintaining soil productivity. This study thus highlights the importance of root-associated methanotrophs as both biofilters of greenhouse gas methane and microbial engines of bioavailable nitrogen for rice growth.