Copper-Induced Oxidative Stress and Responses of the Antioxidant System in Roots of Medicago sativa

The effects of various copper (Cu) concentrations on the antioxidative system in the roots of Medicago sativa were explored. The results indicated that the Cu content of the roots reached a value of 854 mu g g(-1) DW at 10 mu M Cu and a value of 4415 mu g g(-1) DW at 100 mu M Cu, suggesting that M. sativa has better ability to tolerate and accumulate Cu than other Cu-bioaccumulators, and is a potential plant for phytoremediation. Treatment with Cu resulted in a significant increment in the levels of H2O2, O-2(center dot-) and OH center dot. The reduced form of ascorbate and glutathione reached a peak at 30 mu M Cu, and was followed by a sharp depletion to a lower level than that of the control. In contrast, the levels of the oxidised forms of ascorbate and glutathione showed a progressive increment with increasing Cu concentrations, suggesting that the antioxidant system was unable to cope with Cu stress at higher Cu levels. Under the Cu concentrations tested, the activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) increased at lower Cu concentrations, and then decreased, reaching a maximum at 30 mu M of Cu for APX and GR, at 10 mu M for CAT, whereas the activities of guaiacol peroxidase (POD, EC 1.11.1.7) were gradually increased with increasing Cu concentrations. PAGE analysis of superoxide dismutase (SOD, EC 1.1.5.1.1) revealed that one band is a Mn-SOD and five bands are identified as Cu, Zn-SOD, whereas Fe-SOD isoforms were not found in the roots of alfalfa. Cu at 10-100 mu M increased the intensity of constitutive isozymes of CAT, APX and POD, whereas it decreased the intensity of isozymes of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) significantly. The activities of lipoxygenases (LOX, EC 1.13.11.12) were gradually augmented with increasing Cu concentrations, demonstrating that LOXs are probably involved in production of lipid hydroperoxides and superoxide anion. There was a continuous and pronounced enhancement in the activity of esterase (EST, EC 3.1.1.1) in roots treated with 10-30 Cu mu M, whereas EST activity in roots exposed to above 30 mu M Cu declined, suggesting that EST plays a protective role under lower Cu concentrations stress.