Tissue-specific regulation of lipid polyester synthesis genes controlling oxygen permeation into Lotus japonicus nodules

Legumes establish endosymbiotic associations with nitrogen-fixing rhizobia, which they host inside root nodules. Here, specific physiological and morphological adaptations, such as the production of oxygen-binding leghemoglobin proteins and the formation of an oxygen diffusion barrier in the nodule periphery, are essential to protect the oxygen-labile bacterial nitrogenase enzyme. The molecular basis of the latter process remains elusive, as the identification of required genes is limited by the epistatic effect of nodule organogenesis over nodule infection and rhizobia accommodation. We overcame this by exploring the phenotypic diversity of Lotus japonicus accessions that uncouple nodule organogenesis from nodule infection when inoculated with a sub-compatible Rhizobium strain. Using comparative transcriptomics, we identified genes with functions associated with oxygen homeostasis and deposition of lipid polyesters on cell walls to be specifically upregulated in infected compared to uninfected nodules. As such hydrophobic modifications on cell walls are pivotal for creating diffusion barriers like the root endodermis, we focused on two Fatty acyl-CoA reductase genes that were specifically activated in the nodule or in the root endodermis. Mutant lines in a Fatty acyl-CoA reductase gene expressed exclusively in the nodule endodermis showed had decreased suberization of this cell layer and increased nodule permeability compared to wild type plants. Oxygen concentrations were significantly increased in the inner cortex of mutant nodules, which correlated with reduced nitrogen fixation rates, and impaired shoot growth. These results provide the first genetic evidence for the formation of the nodule oxygen diffusion barrier, a key adaptation enabling nitrogen fixation in legume nodules. Overall design: Comparative gene expression profiling analysis of RNA-seq data for different Lotus japonicus accessions that were either susceptible to rhizobium infection or not