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An understanding of the drivers of hotspot/hot moments of N2O production is required to better constrain the global N2O budget and to plan the mitigation strategies. Hot spots are areas with very high N2O emission rates relative to the surrounding area, while hot moments are short periods of time with very high emission rates. As the decomposition of fresh organic matter is transitory in nature, it may have a strong influence on hotspot and hot moment N2O production. Roots are well known to be hotspots for microbial activity but roots direct contribution to N2O production and emissions in soil remain poorly understood.In this study, we evaluated the role of root decomposition on N2O production and emissions, as a function of soil pore size and water content. We hypothesized that (i) the greatest N2O emissions will be observed from root decomposition in the soil dominated by large (>30 µm Ø) pores due to their high connectivity and (ii) enhanced N2O production by denitrification will be observed due to local anaerobic conditions, generated by O2 consumption by decomposers.To evaluate the role of root decomposition on N2O production we used soil microcosms cultivated with switchgrass (Panicum virgatum L. variety Cave-in-rock). From the same composite soil samples we created two soil materials with contrasting pore architectures, namely soil with prevalence of large pores (≥ 35 μm Ø) and small pores (≤ 10 μm Ø). After four months of growing in a greenhouse, plants were cut and soil microcosms with roots were incubated in the dark at room T for 21 days, at two contrasting soil moisture conditions: 40% and 70% water filled pore space (WFPS). Gas headspace samples were collected at different time points during incubation for N2O and CO2 concentration analysis and isotopic characterization of N2O (δ15Nbulk, site preference (SP), and δ18O).The daily emissions of N2O and CO2 from soil microcosms with grown roots showed the same trend during the incubation period and were significantly higher compared to soil microcosms without roots (control) (p < 0.05). Microcosm with large pores soil had significantly higher N2O flux rates compared to the microcosms with small pore soil for both soil moisture treatments (p < 0.001). The relationship between SP and δ18O (isotope mapping) indicated that heterotrophic bacterial denitrification strongly dominated N2O production between day 1 to 7 of the incubation (≥ 97%) and N2O reduction was higher during this period (40 – 60%) in soil microcosms with both pore size and moisture treatment. Later on, N2O reduction decreased (1 – 35%) while the share of nitrification/fungal sources increased for soil microcosms with large pores.Our results indicated that decomposing roots acted as hotspots enhancing N2O emissions and N2O hotspots occurring during root decomposition are strongly influenced by soil pore architecture. While differences in soil pore architecture did not cause differences in N2O production process at the initial phase of decomposition, it might influence the relative contribution of N2O microbial production pathways in later stage of decomposition.

High temporal and spatial variability of nitrous oxide (N2O) 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 N2O emission in single hotspots is a prerequisite to overcoming this problem.We investigated the decomposing switchgrass roots as N2O hotspots, using isotope dual-labeling (15N and 13C) and zymography. Our main objectives were i) to quantify the contribution of decomposing roots to N2O 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 ‘spatially differentiated’ hotspot enzyme activity indeed related to ‘isotopically differentiated’ hotspot N2O 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 Ø and < 10 mm Ø, referred to as coarse and fine soil), to evaluate how these variables change the contribution of decomposing roots to the N2O production.Our results showed that up to 0.4 % of the root driven N can be emitted as N2O 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 (NH4+) and nitrate (NO3-) during the incubation. Decreasing NH4+ and increasing NO3- suggested nitrification. Surprisingly, both inorganic and organic N content was greater in coarse soil, which likely led to intense hotspots of enzyme activity and N2O 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 N2O emission rate (p< 0.01, R2=0.22), supporting that the microbial hotspot formed near the root is the hotspots of N2O emission.Our study showed that the intensity of root driven N2O 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 N2O emissions in bioenergy systems.