Nitrate Has Stronger Copper Detoxification Ability by Reducing Copper Translocation, Enhancing Cell Wall Immobilization, and Modulating Transcriptional Regulation in Malus Rootstock

An appropriate nitrogen (N) supply alleviates copper (Cu) toxicity, but the underlying mechanisms of different N forms remain unclear. This study investigated the regulatory mechanisms of nitrate (NO₃⁻-N) and ammonium (NH₄⁺-N) on Cu uptake, translocation, and tolerance in Malus plants under Cu exposure conditions. Under excess Cu, NO₃⁻-N reduced root and leaf Cu concentrations and root-to-shoot translocation but increased Cu sequestration in root cell walls, accompanied by elevated pectin, hemicellulose (HC1 and HC2) contents, and pectin methylesterase (PME) activity, and decreased polygalacturonase activity relative to NH₄⁺-N. In contrast, NH₄⁺-N increased water-soluble Cu, bioconcentration/translocation factors and subcellular Cu in sensitive fractions, indicating enhanced Cu mobility and phytotoxicity. NO₃⁻-N lowered reactive oxygen species (ROS) and enhanced nonenzymatic and enzymatic antioxidants. RNA sequencing showed that NO₃⁻-N upregulated cell wall metabolism genes, whereas NH₄⁺-N activated Cu uptake/transport genes. Weighted gene coexpression network analysis identified WRKY51 with HUB genes EXPA and BXL. These findings establish physiological and molecular foundations for NO₃⁻-N-mediated Cu stress mitigation.