A constitutively active cortical nitrate-uptake cell state underlies nitrogen-use efficiency in perennial ryegrass

Perennial ryegrass (Lolium perenne) is the most widely sown forage grass of temperate agriculture and a major sink for fertiliser nitrogen, yet the cellular basis of its nitrogen-use efficiency (NUE) is unknown. We built a single-nucleus atlas of 76,684 root nuclei from a high- and a low-NUE genotype under matched nitrogen resupply. High-affinity nitrate uptake is concentrated in one cortical cell state that co-expresses nine NRT2 transporters and all three NAR2 partner proteins, a state that whole-root RNA-seq of the same tissue cannot resolve. Under reduced nitrogen the low-NUE genotype induces this state, whereas the high-NUE genotype does not; it expresses NRT2.3 at a higher baseline and uses different NRT2 paralogs, with one array member nearly silent. Across 44 annual and perennial Pooideae genomes, the same gene families show no life-history difference in copy number, evolutionary rate or selection intensity. Their regulation, however, varies in the same direction as between the two genotypes: the perennial Brachypodium sylvaticum expresses the NRT2.3 ortholog at the level that the annual B. distachyon reaches only under nitrogen deprivation, and an N-efficient barley genotype does not induce the transporters that an N-sensitive genotype induces. NUE differences are therefore associated with constitutive versus inducible deployment of a conserved toolkit, and the expression and presence/absence variants we identify are candidate breeding markers. The atlas, the first for a forage grass, is released as an open web resource.