Overexpression of apple MdNRT1.7 enhances low nitrogen tolerance via the regulation of ROS scavenging

Low nitrogen stress significantly limits crop production. The role of NRT1.7 as a nitrate transporter in alleviating low nitrogen stress in apple (Malus domestica) remains unclear. This study investigates how MdNRT1.7 regulates the low nitrogen response in apple using Agrobacterium-mediated transformation of tobacco (Nicotiana benthamiana). The transcription factor (TF) regulating MdNRT1.7 was identified through yeast one-hybrid (Y1H) and dual-luciferase (LUC) reporter assays. MdNRT1.7 was located in the tonoplast and expressed in various growth sites, including leaves, seeds, and young anthers. Overexpressing MdNRT1.7 in tobacco enhanced low nitrogen stress tolerance by exhibiting significantly higher proline content, soluble protein content, superoxide dismutase activity, peroxidase activity, and total nitrogen accumulation compared to wild-type (WT) plants. In contrast, the malondialdehyde and hydrogen peroxide content were significantly lower in transgenic plants than in WT plants. Additionally, the expression of nitrogen metabolism-related genes in transgenic tobacco was significantly higher than in WT tobacco under low nitrogen conditions. Y1H, LUC reporter assays and electrophoretic mobility shift assays (EMSA) demonstrated that a H₂O₂-mediated NAC TF MdJUB1 binds to the MdNRT1.7 promoter and represses its expression. These results suggested that MdNRT1.7, as a crucial nitrate transporter gene, improved the nitrogen metabolism-related enzymes' activity, reduced reactive oxygen species (ROS) accumulation, and thus enhanced low nitrogen stress tolerance.