Thylakoid-bound and stromal antioxidative enzymes in wheat treated with excess copper

Copper is a catalyst in the formation of reactive free radicals and its toxicity may be due, at least in part, to oxidative damage. The response of thylakoid-bound and stromal antioxidative enzymes against the generation of superoxide radical was investigated in seedlings of wheat (Triticum durum L. cv. Adamello) grown in hydroponic culture for 10 days and subjected to 10 and 50 ?M copper treatments. Electron spin resonance of roots evidenced a spectrum of copper, the intensity of which increased with the treatment, whereas the carbon-centered free radical spectrum detected in the control leaves was not seen anymore in the treated samples. As well as thylakoids, photosystem II (PSII) particles were able to produce the superoxide radical. Increased superoxide production both by thylakoids and PSII was observed in the sample treated with 50 ?M Cu. Induction of thylakoid-bound and stromal antioxidative enzymes, with the exception of dehydroascorbate reductase, was also detected in leaves treated with the highest copper concentration. No Mn-superoxide dismutase (SOD, EC 1.15.1.1) was detected in thylakoids of wheat. Both stromal and thylakoid-bound SOD were CuZn-SOD with 16.2-kDa subunits. Both western blotting and immuno-electron microscopy showed that the SOD subunit was recognized by a polyclonal antibody against glyoxisomal CuZn-SOD from watermelon cotyledon. In the stroma of wheat, ascorbate peroxidase showed at least three well-resolved bands differently induced by copper treatments.