Comprehensive genome-wide identification of the NPF gene family and functional characterization of GmNPF6.8 regulating root development in soybean

The nitrate transporter 1/peptide transporter family (NPF) plays a key role in nitrate uptake, transport, and nitrogen use efficiency in plants. Although NPF genes have been widely studied in many species, their genomic organization, evolutionary patterns, and functional roles in soybean remain unclear. Soybean is an important legume with high nitrogen demand and the ability to fix atmospheric nitrogen through symbiosis. In this study, 126 GmNPF genes were identified in the Wm82.a4.v1 genome. These genes were classified into 8 subfamilies and were unevenly distributed across 19 chromosomes. Family expansion was mainly driven by segmental duplication. Ka/Ks analysis indicated strong purifying selection. Promoter analysis revealed cis-regulatory elements associated with light response, phytohormone signaling, and abiotic stress. Expression profiling across tissues showed clear spatial and temporal patterns for 112 GmNPF genes. GmNPF6.8 was predominantly expressed in roots. Under low-nitrogen conditions, many GmNPF genes were differentially expressed. GmNPF5.13, GmNPF5.5, GmNPF7.13, GmNPF7.12, GmNPF7.14, and GmNPF2.11 were significantly upregulated, whereas GmNPF6.8 and GmNPF6.9 were significantly downregulated in soybean roots. Genetic diversity analysis of GmNPF6.8 in 4,068 soybean accessions identified 3 coding-region haplotypes. GmNPF6.8Hap1 showed clear evidence of strong artificial selection. Subcellular localization assays confirmed that GmNPF6.8 is localized to the plasma membrane. Overexpression of GmNPF6.8 in Arabidopsis and soybean hairy roots significantly reduced root length and root density. It also altered the expression of key genes involved in root development. Further analysis showed that GmARF11 directly binds to the promoter of GmNPF6.8 and represses its transcription. This study clarified the genomic and evolutionary features of the GmNPF family and identified GmNPF6.8 as a negative regulator of root development. These findings provide a potential target for improving nitrogen use efficiency in soybean breeding.