Symbiotic nitrogen fixation (SNF) is one of the main natural pathways for nitrogen entry in the biosphere and a likely alternative to the overuse of polluting and expensive synthetic fertilizers. The best characterized SNF is the one carried out between legumes and a group of soil bacteria known as rhizobia. Through a complex process these bacteria colonize plant cells of newly developed plant organs, the nodules. There, rhizobia are surrounded by a plant-derived membrane in an organelle-like structure called symbiosome, where rhizobia differentiates into a specialized nitrogen fixing form called bacteroid. This process requires an intense nutrient trafficking between the symbionts, especially of transition metals (copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo) and zinc (Zn)) to satisfy the synthesis of both plant and bacterial metalloenzymes. However, in spite of the importance that metals have on SNF, it remains largely unknown how these micronutrients are delivered to the nodule and to the symbiosome.

Using synchrotron-based X-ray fluorescence (XRF) to study Fe distribution in the nodules of model legume Medicago truncatula, a heterogeneous pattern in different developmental zones of the nodule was observed. At the apical zone, Fe has a spotty, extracellular distribution. In the infection zone, we observed a progressive internalization of the metal in the infected cells, and finally their accumulation in the bacteroids within the nitrogen-fixation zone. Expanding on these results using μXRF and μXANES (X-ray Absorption Near Edge Structure) on frozen hydrated samples, we have observed that the local coordination environment of Fe and Fe oxidation state change longitudinally too. In the apical regions Fe³⁺ is the most abundant species, while Fe²⁺ is more prevalent in the fixation zone. Searching for a more detailed Fe localization we have performed nanoXRF analysis, and nanotomography to assess morphological changes. In future studies we plan to use these techniques to investigate other essential elements for root nodulation such as Cu, Zn and Mo.

Altogether, these snapshots helped us to propose a model of Fe transport in the nodule, which in the long term would support the selection of better symbiotic partners for a sustainable agriculture, as well as design organ-specific fortification programs mimicking mechanisms developed in the nodule.

This work was supported by ERC Starting Grant (ERC-2013-StG-335284), MINECO Grant (AGL-2012-32974; AGL-2015-65866-P), and ESRF proposals EV246, EV323 and EV351 to MGG. I.A is a Juan de la Cierva-Formación Postdoctoral fellow (FJCI-2017-33222).