Sterile seedlings of Potamogeton crispus were cultured by tissue culture technique,and used as experimental materials.In this study,different concentrations of Ni²⁺(0,0.05,0.10,0.15 and 0.20 mmol/ L) on contents of hydrogen peroxide (H₂O₂),superoxide anion (O₂^.-) and malondialdehyde (MDA),antioxidant defense system and proline metabolism were investigated.The results indicated that (1) Ni²⁺stress triggered the generation of reactive oxygen species (ROS) as evidenced by a substantial accumulation of H₂O₂ and O₂^.-,accompanied by the increased MDA content with the increasing of Ni²⁺concentration;(2) Activities of superoxide dismutase (SOD),peroxides (POD) and catalase (CAT),as well as contents of reduced glutathione (GSH) and non-protein thiols (NP-SH) increased at first and then decreased,while no outstanding changes were observed in ascorbate (AsA) content,and level of phytochelatins (PCs) increased significantly;(3) Analysis of enzyme activities showed that the accumulation of proline induced by Ni²⁺stress is due to the increases of ornithine-δ-aminotransferase (OAT) at 0.05-0.10 mmol/L and pyrroline-5-carboxylate synthetase (P5CS) activity at 0.15-0.20 mmol/ L,respectively,but there was not a statistically significant difference in ProDH activity.Our results demonstrated that Ni²⁺induced the enhancement of reactive oxygen species level,aggravated lipid peroxidation and interfere with intrinsic balance of antioxidant defense system.In addition,proline accumulation is mediated by increased biosynthesis from ornithine at low concentrations (0.05 and 0.10 mmol/L),while glutamate pathway,instead of the ornithine pathway,plays a vital role when exposed to higher concentrations of Ni²⁺.