Phytoremediation potential of Linum usitatissimum for copper-contaminated soil: Role of nitrogen to improve phytoextraction through altering physio-biochemical and molecular process

Flax ( Linum usitatissimun ) is a promising candidate for copper (Cu) phytoextraction from agricultural soils. However, the effects of nitrogen (N) fertilizer driving the efficiency of Cu phytoextraction remain poorly understood. This study aimed to evaluate the dynamics of different levels of N (0, 125, 250, 375 mg kg −1 soil) on flax growth and Cu uptake with Cu-contaminated soil (2137 mg kg −1 Cu) and in mixtures of Cu-contaminated soil and natural soil (1189 mg kg −1 Cu). Our results demonstrated that the application of N (250 mg kg −1 soil) alleviated Cu toxicity by increasing plant growth and biomass, including dry weight, plant height, stem length, number of pods and number of branches per plant by 153.7 %, 43.1 %, 52.2 %, and 220.8 %, respectively, compared to control. N application at Cu 0.5 N 2 significantly reduced malondialdehyde, proline, and peroxidase activity by 26 %, 66.8 %, and 60.1 %, respectively, while superoxide dismutase activity was maximally enhanced by 365.6 % under Cu 0.5 N 3 treatment. N application progressively modulated antioxidants response, indicating that N plays a protective role against Cu-induced oxidative stress. Furthermore, transcriptome analysis identified differentially expressed genes (DEGs) associated with signal transduction and the phenylpropanoid biosynthesis pathway, which play key roles in N-mediated Cu detoxification. The involvement of phytohormones, including auxin (Aux1, LAX, and SAUR), gibberellic acid (DELLA), and salicylic acid (PR1, TGA), suggests that N improves heavy metals (HMs) tolerance via hormone signal transduction. Notably, N fertilization activated the expression of genes encoding peroxidase (E1.11.1.7) and respiratory burst oxidase homolog (RBOH) in the phenylpropanoid biosynthesis and MAPK signaling pathways, respectively, thereby alleviating Cu-induced damage. These findings indicate that varying N levels enhance Cu accumulation while maintaining ionic balance, facilitated by the activity of key transporters such as ZIP, CTR1, and AMT. These findings provide deeper insights into the mechanisms through which N alleviates Cu phytotoxicity, laying the foundation for optimizing phytoremediation strategies. • Varying N levels influence physio-biochemical, and gene expression processes under Cu stress. • N mitigates Cu-induced growth inhibition in flax plants. • The signal transduction pathways are crucial in N-mediated alleviation of Cu toxicity. • N enhances Cu uptake by regulating key transporters.