Expansion and Nitrate-Responsive Expression of NRT3 Transport Regulators in Maritime Pine

Nitrate uptake in plants is mediated by coordinated transporter systems, which include NPF, NRT2 and NRT3 proteins. While these families have been extensively studied in angiosperms, their evolution and regulation in conifers are still not well understood. In this work, we examined the NRT3 family in maritime pine (Pinus pinaster) and in representative plant lineages. Phylogenetic analyses of nucleotide sequences and the NRT3 protein revealed a broad expansion in gymnosperms, particularly in conifers, while copy number increases among angiosperms appeared to be more lineage-specific. In addition, we evaluated the expression of nitrate transporter genes in cotyledons, hypocotyls and roots of P. pinaster seedlings exposed to low and high concentrations of nitrate. Several NRT3 genes, particularly PpNRT3.1, PpNRT3.3 and PpNRT3.5, were significantly induced by nitrate, while most NPF and NRT2 genes showed weaker or non-significant transcriptional responses. Correlation analysis revealed distinct expression associations among the NRT3, NRT2 and NPF transporters, including a specific association between PpNRT3.4 and PpNRT2.1, as well as broader correlations among other NRT3 paralogs and NPF genes. These results indicate that the expansion of NRT3 in conifers was accompanied by transcriptional divergence among paralogs and identify potential regulatory relationships for future functional studies.