Nitrate is the primary form of nitrogen uptake in plants, mainly transported by nitrate transporters (NRTs), including NPF (NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY), NRT2 and NRT3. In this study, we identified a total of 78 NPF, seven NRT2, and two NRT3 genes in maize. Phylogenetic analysis divided the NPF family into eight subgroups (NPF1-NPF8), consistent with the results in Arabidopsis thaliana and rice. The NRT2 family appears to have evolved more conservatively than the NPF family, as NRT2 genes contain fewer introns. The promoters of all NRTs are rich in cis-acting elements responding to biotic and abiotic stresses. The expression of NRTs varies in different tissues and developmental stages, with some NRTs only expressed in specific tissues or developmental stages. RNA-seq analysis using Xu178 revealed differential expression of NRTs in response to nitrogen starvation and nitrate resupply. Moreover, the expression patterns of six key NRTs genes (NPF6.6, NPF6.8, NRT2.1, NRT2.5 and NRT3.1A/B) varied in response to alterations in nitrogen levels across distinct maize inbred lines with different nitrogen uptake rates. This work enhances our understanding of the structure and expression of NRTs genes, and their roles in nitrate response, paving the way for improving maize nitrogen efficiency through molecular breeding.

Nitrogen (N) fertilizer sustains modern crop production, yet less than half of applied reactive N is recovered in harvested products, with the remainder lost through volatilization, denitrification, leaching, and runoff. Although hundreds of genes regulating N uptake, assimilation, and remobilization have been identified, their translation into stable field-level improvements remains limited. This gap reflects the fact that nitrogen use efficiency (NUE) is not a single-gene trait, but an emergent property of the integrated plant–soil–microbe system. Here, we organize the genetic determinants of NUE into four functional layers: hardware, comprising transporters and assimilatory enzymes that execute N fluxes; software, encompassing signaling and transcriptional networks that regulate these components; balance, referring to carbon-nitrogen coordination that supplies the energy and carbon skeletons required for assimilation; and the plant-associated microbiome as a second genome that expands plant nitrogen-acquisition capacity. Unlike previous NUE reviews organized by gene family or by physiological process, we tier each target by evidence strength, the supporting endpoint (yield at a given N rate or grain N recovery at maintained yield), and whether its benefit depends on N supply, and we retain failure cases and model-only paradigms alongside successes. Across these layers, effective NUE engineering should prioritize repair of steps shown to constrain flux and mitigation of associated trade-offs rather than indiscriminate enhancement of N flux. Key strategies include spatially constrained expression of high-activity modules, release of transcriptional brakes, carbon–nitrogen coordination through regulatory hubs, and breeding of stable host–microbe partnerships. We further propose a translational roadmap centered on cis-regulatory engineering and combinatorial testing of mechanistically complementary alleles. Treating NUE as an engineering problem of the integrated plant–soil–microbe system is essential for bridging the laboratory-to-field translation gap in nitrogen-efficient crop breeding.