The functional characterization of LjNRT2.4 indicates a novel, positive role of nitrate for an efficient nodule N2 -fixation activity.

Atmospheric nitrogen (N2) -fixing nodules are formed on the roots of legume plants as result of the symbiotic interaction with rhizobia. Nodule functioning requires high amounts of carbon and energy, and therefore legumes have developed finely tuned mechanisms to cope with changing external environmental conditions, including nutrient availability and flooding. The investigation of the role of nitrate as regulator of the symbiotic N2 fixation has been limited to the inhibitory effects exerted by high external concentrations on nodule formation, development and functioning. We describe a nitrate-dependent route acting at low external concentrations that become crucial in hydroponic conditions to ensure an efficient nodule functionality. Combined genetic, biochemical and molecular studies are used to unravel the novel function of the LjNRT2.4 gene. Two independent null mutants are affected by the nitrate content of nodules, consistent with LjNRT2.4 temporal and spatial profiles of expression. The reduced nodular nitrate content is associated to a strong reduction of nitrogenase activity and a severe N-starvation phenotype observed under hydroponic conditions. We also report the effects of the mutations on the nodular nitric oxide (NO) production and content. We discuss the involvement of LjNRT2.4 in a nitrate-NO respiratory chain taking place in the N2 -fixing nodules.