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

PurposeAlfalfa is one of the most valuable forage crops in temperate climate zones. Ensifer meliloti, the endosymbiont of alfalfa, contains all the denitrification genes but the capacity of alfalfa root nodules to produce nitrous oxide (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.MethodsMedicago sativa cv. Victoria plants were inoculated with E. meliloti 1021, a periplasmic nitrate reductase (Nap) defective mutant, a Nap overexpressing strain and a nitrous oxide reductase defective mutant. Plants were grown in the presence of different nitrate and copper treatments and subjected to flooding during one week before harvesting. N2O production by the nodules was analysed by using gas chromatography. Methyl viologen-dependent nitrate reductase (MV+-NR), nitrite reductase (MV+-NIR) and nitrous oxide reductase (N2OR) enzymatic activities were measured in isolated bacteroids.ResultsAlfalfa root nodules produce N2O in response to nitrate and flooding. Overexpression of Nap improved nodulation competitiveness and induced N2O emissions from nodules. Copper is required for an effective symbiosis as well as triggered a reduction of N2O production due to the induction of the N2OR and a reduction of NIR activities in the bacteroids.ConclusionAlfalfa 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.