Identification, Classification, and Evolutionary Profile of the NPF Gene Family in Sorghum bicolor

Nitrate transporter 1/peptide transporter family (NPF) proteins play pivotal roles in nitrogen uptake, translocation, and stress adaptation in plants. To comprehensively investigate this gene family in sorghum (Sorghum bicolor), we conducted the genome-wide identification and characterization of SbNPF genes. A total of 88 SbNPF members were identified and classified into eight subfamilies based on phylogenetic analysis, displaying diverse gene structures, conserved motifs, and evolutionary relationships. Gene duplication analysis revealed that tandem duplication was the primary driver of SbNPF family expansion, with most duplicated pairs undergoing purifying selection. Synteny analysis showed extensive collinearity between sorghum and rice, but limited conservation with Arabidopsis, highlighting the evolutionary divergence between monocots and dicots. Cis-regulatory element prediction suggested that SbNPF genes are widely involved in abiotic stress responses, hormone signaling, and light responsiveness. Expression profiling using RNA-seq data revealed distinct tissue-specific expression patterns, indicating functional specialization across roots, stems, leaves, and reproductive tissues. Furthermore, RT-qPCR analysis under low-nitrogen (LN) conditions demonstrated that several SbNPF genes, including SbNPF1.1, SbNPF2.6, SbNPF2.7, and SbNPF4.5, were significantly upregulated in shoots, whereas SbNPF1.2, SbNPF2.7, and SbNPF3.1 were downregulated in roots, suggesting differential regulatory roles in nitrogen acquisition and utilization under nutrient-limiting environments. Collectively, these findings provide novel insights into the evolutionary dynamics and potential functions of the SbNPF gene family and establish a foundation for future functional studies and molecular breeding aimed at improving nitrogen use efficiency in sorghum.