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Legumes can contribute to emissions of the potent greenhouse gas nitrous oxide (N2O) directly by some rhizobia species that are able to denitrify under free-living conditions and in symbiotic association with the plant. In this study, the capacity of Phaseolus vulgaris-Rhizobium etli symbiosis to emit N2O in response to nitrate (NO3-) has been demonstrated for the first time. We found that bacteroidal assimilatory nitrate reductase (NarB) as well as nitrite reductase (NirK) and nitric oxide reductase (cNor) denitrifying enzymes contribute to nitric oxide (NO) and N2O formation in nodules. We also show that R. etli NarK is involved in NO2- extrusion and links NO3- reduction by NarB in the cytoplasm with NirK and cNor denitrification activities in the periplasm. The knowledge generated in this work will be instrumental for exploring strategies and sustainable practices in agricultural soil management to increase legume crop yield and mitigate greenhouse gas emissions. HIGHLIGHTS Common bean nodules are able to produce N2O in response to nitrate. Nitrate reduction to nitrite in R. etli bacteroids catalyzed by assimilatory nitrate reductase NarB is involved in N2O emissions from common bean nodules. R. etli respiratory nitrite reductase NirK and nitric oxide reductase cNor support N2O production in common bean nodules. R. etli NarB and cNor are involved in NO homeostasis in common bean nodules. R. etli nitrite exporter NarK links nitrate reduction in the cytoplasm with denitrification activities in the periplasm. [ABSTRACT FROM AUTHOR]

Purpose: Alfalfa 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. Methods: Medicago 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. Results: Alfalfa 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. 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. [ABSTRACT FROM AUTHOR]

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c -type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD , and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ–FixK2–NnrR and RegSR–NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD , and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA , a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes. [ABSTRACT FROM AUTHOR]

Nitrous oxide (N2O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N2O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N2, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N2O emissions from agricultural soils. [ABSTRACT FROM AUTHOR]

• Cu addition during growth influenced soybean- Bradyrhizobium diazoefficiens symbiosis. • 20 μM Cu2+ was the stress threshold for soybean plants. • The addition of Cu reduced significantly N 2 O emissions produced by soybean nodules. • Cu accumulation in nodules affected denitrifying enzymes activities of bacteroids. • Nitrate reductase of bacteroids is proposed as a target for mitigation strategies. Agriculture is an important source of the greenhouse gas nitrous oxide (N 2 O) due to the over- or non-synchronised application of nitrogen to crops. Symbiotic nitrogen fixation (SNF) by legume-rhizobia symbiosis can be an effective strategy for N 2 O mitigation, but several environmental factors such as copper (Cu) availability might affect N 2 O emissions derived from legume crops. The aim of this research was to study how Cu can modulate N 2 O emissions by soybean root nodules. Soybean plants inoculated with Bradyrhizobium diazoefficiens USDA110 were grown in the presence of 4 mM KNO 3 and a battery of Cu2+ concentrations added during growth (0, 5, 10, 20, 40, 60 or 100 μM). N 2 O emissions were measured by gas chromatography in both, nodulated roots and detached nodules, after root flooding. Also, 15N isotope dilution was assayed for SNF and N 2 O determinations. Results showed that an excess of added Cu during growth significantly affected plant physiology, nodulation and SNF of the soybean- B.diazoefficiens symbiosis, being 20 μM the threshold that soybean plants can tolerate without suffering Cu stress. Meanwhile, Cu addition reduced statistically N 2 O emissions by soybean nodules. This reduction was correlated with Cu accumulation in nodules, which affected the denitrifying enzymes activities. Cu excess produced a simultaneous decrease of nitrate and nitrite reductase activities, but an increase of nitrous oxide reductase activity. Finally, the modulation of bacteroidal nitrate reductase activity is proposed as an effective target for the strategies for mitigation of N 2 O emissions derived from soybean crops, probably more effective than nitrous oxide reductase activity. [ABSTRACT FROM AUTHOR]