Ensifer meliloti denitrification is involved in infection effectiveness and N2O emissions by alfalfa root nodules

Abstract

Abstract Purpose: Ensifer meliloti, the endosymbiont of alfalfa, contains all the denitrification genes but the capacity of alfalfa root nodules to produce N2O is not known. In this work, N2O emissions as well as the influence of bacteroidal denitrification on nodulation competitiveness and N2O release from alfalfa nodules has been investigated. Methods: Medicago sativa cv. Victoria plants were inoculated with E. meliloti 1021 and napA-, nap+ and nosZ- mutants. Plants were grown in the presence of different nitrate and copper treatments and subjected to flooding during one week before harvesting. MV+-NR and MV+-NIR enzymatic activities were measured in isolated bacteroids by analysing the capacity of the cells to produce or consume nitrite, respectively. Bacteroidal nitrous reductase (N2OR) activity was determined by measuring N2O consumption capacity. N2O was analysed by using a gas cromatograph. Results: Alfalfa root nodules are able to produce N2O in response to nitrate and flooding. Overexpression of the periplasmic nitrate reductase (Nap) improved nodulation competitiveness and induced N2O emissions. The addition of Cu to the plant nutrient is required for an effective symbiosis as well as triggered a reduction of N2O production by alfalfa nodules due to the induction of the N2OR and a reduction of NIR activities in the bacteroids. Conclusion: Alfalfa root nodules emit N2O. Nap is involved in nodulation competitiveness and in N2O emissions by the nodules. Bacteroidal N2OR and NIR activities are modulated by Cu and may be considered as effective targets for the mitigation strategies of N2O emissions derived from alfalfa crops.