Nitrate and flooding induce N2O emissions from soybean nodules

Nitrous oxide (N2O) is one of the three main biogenic greenhouse gases (GHGs) and agriculture represents close to 30 % of the total N2O net emissions. In agricultural soils, N2O is emitted by two main microbial processes, nitrification and denitrification, both of which can convert synthetic nitrogen fertilizer into N2O. Legume-rhizobia symbiosis could be an effective and environmental-friendly alternative to nitrogen fertilization and hence, to mitigate soil N2O emissions. However, legume crops also contribute to N2O emissions. A better understanding of the environmental factors involved in the emission of N2O from nodules would be instrumental for mitigating the release of this GHG gas. In this work, in vivo N2O emissions from nodulated soybean roots in response to nitrate (0, 1, 2 and 4 mM) and flooding have been measured. To investigate the contribution of rhizobial denitrification in N2O emission from nodules, plants were inoculated with B. japonicum USDA110 and napA and nosZ denitrification mutants. The results showed that nitrate was essential for N2O emissions and its concentration enhanced N2O fluxes showing a statistical linear correlation, being the highest N2O fluxes obtained with 4 mM nitrate. When inoculated plants grown with 4 mM nitrate were subjected to flooding, a 150- and 830-fold induction of N2O emission rates from USDA110 and nosZ nodulated roots, respectively, was observed compared to non-flooded plants, especially during long-term flooding. Under these conditions, N2O emissions from detached nodules produced by the napA mutant were significantly lower (p < 0.05) than those produced by the wild-type strain (382 versus 1120 nmol N2O h(-1) g(-1) NFW, respectively). In contrast, nodules from plants inoculated with the nosZ mutant accumulated statistically higher levels of N2O compared to wild-type nodules (2522 versus nmol 1120 N2O h(-1) g(-1) NFW, p < 0.05). These results demonstrate that flooding is an important environmental factor for N2O emissions from soybean nodules and that B. japonicum denitrification is involved in such emission.