Comparative Genomics Reveals the Evolutionary Expansion and Diversification of the NPF Gene Family in Grasses

Background/Objectives: NPF proteins are important transporters that mediate nitrate uptake, nutrient allocation, and abiotic stress responses in plants. However, the evolutionary patterns of the NPF gene family in grasses remain largely unclear. This study aimed to clarify the evolutionary expansion and stress response characteristics of NPF genes in Poaceae. Methods: A comprehensive comparative genomic analysis was conducted across nine representative Poaceae species and Arabidopsis thaliana. Multiple analytical approaches were used, including gene family identification, phylogenetic classification, collinearity analysis, Ka/Ks calculation, cis-element prediction, protein interaction analysis, and RNA-seq expression verification. Results: A total of 1109 NPF genes were identified with substantial variation in gene copy number among species, particularly the remarkable expansion observed in hexaploid Triticum aestivum. Phylogenetic analysis classified grass NPF proteins into eight major subfamilies, while collinearity analyses revealed that whole-genome duplication and segmental duplication were the primary drivers of NPF expansion. Most duplicated gene pairs exhibited Ka/Ks values below 1, indicating strong purifying selection during evolution. Promoter analyses identified abundant stress- and hormone-responsive cis-elements, especially in Triticeae species. In addition, protein-protein interaction and RNA-seq analyses suggested potential functional associations among NPF genes and revealed expression variation under low-temperature treatments in rice and wheat. Conclusions: Collectively, this study objectively clarifies the evolutionary expansion, functional conservation, and potential stress-responsive diversification of the NPF gene family in grasses. These findings provide straightforward and reliable insights for further evolutionary and functional research on the NPF gene family in Poaceae.