Growth inhibition and antioxidative response were investigated in wheat roots cultured in 1/4 Hoagland solution containing zinc (Zn, 500 μM), iron (Fe, 300 μM), and copper (Cu, 300 μM) in combination. Different Zn, Fe, and Cu interactions inhibited seedling growth and increased Zn, Fe, and Cu contents in roots and shoots, with the most significant inhibition due to Zn + Fe + Cu treatment. The elevation of malondialdehyde content and the loss of cell viability resulted from the increases of total and apoplastic hydrogen peroxide (H2O2) and hydroxyl radical (·OH) contents in all treated roots. Except for Zn + Fe stress, root superoxide anion (O2•-) level significantly decreased at other combined treatments. The application of 10 μM diphenylene iodonium suggested that NADPH oxidase activity was lower in Fe + Cu-treated and Zn + Fe + Cu-treated roots than in other roots. Additionally, all combined treatments inhibited superoxide dismutase (SOD) and peroxidase (POD) but stimulated total glutathione reductase (GR) activity in roots. However, in root apoplast, decreased SOD and ascorbate peroxidase activities as well as increased POD, catalase, and GR activities were caused by different Zn, Fe, and Cu interactions. In conclusion, combined Zn, Fe, and Cu stresses exhibited significant inhibition on root growth, with the strongest effect due to Zn + Fe + Cu. Here, it is also indicated that each antioxidantive enzyme including apoplastic enzymes showed specific responses and that the stimulation of some of them played an important protective mechanism against oxidative damage, when wheat roots were treated with different Zn, Fe, and Cu treatments in combination.
Keywords: Antioxidative response; Copper; Iron; Root growth; Wheat; Zinc.