Rice Root Iron Plaque as a Mediator to Stimulate Methanotrophic Nitrogen Fixation

Iron plaque (IP) on rice root surfaces has been extensively documented as a natural barrier that effectively reduces contaminant bioavailability and accumulation. However, its regulatory mechanisms in rhizospheric methane oxidation and biological nitrogen fixation (BNF) remain elusive. This study reveals a previously unrecognized function of IP: mediating methanotrophic nitrogen fixation through coupled aerobic methane oxidation and IP reduction (Fe-MOX). Using a hydroponic coculture system integrating methane-oxidizing bacteria and rice seedlings, we demonstrated that IP enhanced microbial methane oxidation by 46.8% and significantly stimulated BNF rate by 33.6%, with methane-derived carbon accounting for 89.1% of the BNF energy source. Notably, dissolved iron removal did not diminish the BNF enhancement, excluding mediation by soluble iron species. Intriguingly, ferrihydrite supplementation at equivalent iron concentrations failed to replicate the BNF stimulation observed with IP, suggesting the indispensability of root-associated iron redox cycling. Mechanistic analyses identified that <i>Methylosinus</i>/<i>Methylocystis</i> species mediated Fe(III) reduction, synergistically collaborating with specific rhizobial strains to execute Fe-MOX-dependent BNF. These findings uncover a previously overlooked yet pronounced contribution of IP to BNF, providing novel insights for developing dual-strategy approaches to mitigate methane emissions and reduce nitrogen fertilizer dependency in paddy ecosystems.