<p>High temporal and spatial variability of nitrous oxide (N<sub>2</sub>O) emission from soils has been a challenge for the systematic prediction of global climate change. It is attributed to multiple hotspots occurring simultaneously and affecting the N dynamics cumulatively on an ecosystem scale. Understanding the mechanisms and contributing factors of N<sub>2</sub>O emission in single hotspots is a prerequisite to overcoming this problem.</p><p>We investigated the decomposing switchgrass roots as N<sub>2</sub>O hotspots, using isotope dual-labeling (<sup>15</sup>N and <sup>13</sup>C) and zymography. Our main objectives were i) to quantify the contribution of decomposing roots to N<sub>2</sub>O emission along with the N contents in the soil (total, organic, and inorganic N) and microbial pools, and ii) to differentiate the extracellular enzyme activity in decomposing roots from the bulk soil, and test if the &#8216;spatially differentiated&#8217; hotspot enzyme activity indeed related to &#8216;isotopically differentiated&#8217; hotspot N<sub>2</sub>O emissions. We treated the soils of the same origin to have different moisture contents (40% and 70% water-filled pore space, WFPS) and pore size distributions (dominant pores of >30 &#216; and < 10 mm &#216;, referred to as coarse and fine soil), to evaluate how these variables change the contribution of decomposing roots to the N<sub>2</sub>O production.</p><p>Our results showed that up to 0.4 % of the root driven N can be emitted as N<sub>2</sub>O gas, only within 21 days of the decomposition. Approximately 21 ~35% of root N was transformed to dissolved organic N, while less than 1 % of the root N remained as ammonium (NH<sub>4</sub><sup>+</sup>) and nitrate (NO<sub>3</sub><sup>-</sup>) during the incubation. Decreasing NH<sub>4</sub><sup>+</sup> and increasing NO<sub>3</sub><sup>-</sup> suggested nitrification. Surprisingly, both inorganic and organic N content was greater in coarse soil, which likely led to intense hotspots of enzyme activity and N<sub>2</sub>O emission. However, there was no difference in microbial biomass between the soil materials. Higher chitinase activity and relatively large pores in coarse soils suggest that the fungal activity was higher in coarse soils compared to the fine soils. Root chitinase activity was positively correlated with the root driven N<sub>2</sub>O emission rate (p< 0.01, R<sup>2</sup>=0.22), supporting that the microbial hotspot formed near the root is the hotspots of N<sub>2</sub>O emission.</p><p>Our study showed that the intensity of root driven N<sub>2</sub>O hotspots can highly depend on the soil physical characteristics, being mediated by decomposed substances, and enzyme activity. Tracking the fate of N during the plant root decomposition can provide a new perspective on the strategies to minimize N<sub>2</sub>O emissions in bioenergy systems.</p>
