Comprehensive Identification and Unveiling Key Nitrate-Transporting Proteins in Oat (Avena sativa L.)

Nitrate transporter NRT1/PTR family (NPF) proteins are crucial for plant nitrogen uptake and utilization. As an important hexaploid crop for grain and forage, oat (Avena sativa L.) requires substantial levels of nitrogen. However, the oat nitrate transporter 1 (NRT1) family remains uncharacterized. In this study, the oat NRT1 subfamily members were identified through the Hmm and Pfam databases. Bioinformatics analysis was performed using the MEGA 11 and TBtools software to elucidate the physicochemical properties, evolutionary relationships, chromosomal localization, and gene structures. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) analysis and the green fluorescent protein (GFP) fusion expression vector were utilized to investigate the candidate oat NRT1s. Phylogenetic classification categorized oat NRT1s into eight subfamilies, with the most abundant being the NPF5 subfamily. Physicochemical property analysis revealed that the number of amino acids in the proteins encoded by these genes ranged from 235 to 673, with their molecular weights (MWs) ranging from 26 kDa to 74 kDa. Chromosomal localization revealed that these genes were unevenly distributed across all 12 oat chromosomes. Promoter analysis revealed that light-responsive elements appeared most frequently in the promoters of these genes (39.3%), followed by abscisic acid (ABA)-responsive elements (13.5%) and methyl jasmonate (MeJA)-responsive elements (9.4%). qRT-PCR analysis revealed that most of the genes exhibited tissue-specific expression patterns. Among them, AsNPF2.6 was highly expressed in the leaves at 1 h post-low nitrogen (LN) treatment, while AsNPF4.5 was highly expressed in the leaves at 12 h. Both these genes exhibited low expression levels in the roots. However, AsNPF7.16 and AsNPF7.19 were both highly expressed in the roots at 9 h post-LN treatment but exhibited low expression in the leaves. Subcellular localization revealed that all five proteins (AsNPF2.6, AsNPF4.5, AsNPF7.16, AsNPF6.8, and AsNPF7.19) were localized to the cytoplasm and cell membrane. Our results demostrate the involvement of AsNRT1 family members in nitrogen transport in oat, providing theoretical support for further investigation into the functions and molecular mechanisms of action of oat NRT1s in nitrogen transport.