Maize root growth, oxygen and N availability drive formation of N2O hotspots in soil

• Rhizoboxes equipped with planar O 2 optodes to monitor root growth and O 2 in soil. • Maize roots shaped depth gradients of nutrients and denitrification gene abundances. • Microsensors revealed distinct patterns with highest N 2 O concentrations around roots. • Understanding root-soil-microbe interactions crucial to reduce N 2 O emissions. Plant roots can modify all major controls of denitrification in soils, particularly the availability of the main substrates (NO 3 − and C org ), soil moisture, soil O 2 content, and root-associated microbial communities, and thus play an important role in N 2 O formation. Direct in-situ measurements of N 2 O concentrations in the rhizosphere are lacking, yet are crucial to better understand how rhizosphere denitrification contributes to overall N 2 O emissions from soil. We equipped rhizoboxes with O 2 -sensitive planar optodes to simultaneously monitor root growth and rhizosphere/soil O 2 concentrations. We measured soil surface N 2 O fluxes and linked them to root growth, soil moisture, and root/soil O 2 concentrations. Based on root growth and O 2 concentrations, we identified regions of interest (ROI) and sampled small soil volumes, which were analyzed for C and N content, and abundance of genes indicative of microbial denitrifiers ( nirK , nirS ) and N 2 O reducers ( nosZ I, nosZ II), and soil N 2 O concentrations. Plant roots determined depth gradients of nutrients and denitrification gene abundances in the soil of the rhizoboxes with higher resource availability (NO 3 – , DOC) and lower soil moisture in the upper soil layers, which also had higher abundances of total bacteria, nirK and nosZ II. These findings indicate that the uppermost soil layers largely contributed to N 2 O formation. Our study provides the first direct evidence of roots creating distinct O 2 and N gradients controlling N 2 O production at the process scale leading to high in-situ N 2 O concentrations.