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 tox icity may be due, at least in part, to oxidative damage. The response of th ylakoid-bound and stromal antioxidative enzymes against the generation of s uperoxide 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 mu M copper treatments. Electron spin resonance of roots evidenced a spectrum of copper, the intensify of which increased with the treatment, w hereas the carbon-centered free radical spectrum detected in the control le aves was not seen anymore in the treated samples. As well as thylakoids, ph otosystem 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 mu M Cu. Induction of thylakoid-bound and strom al 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 o f wheat. Both stromal and thylakoid-bound SOD were CuZn-SOD with 16.2-kDa s ubunits. Both western blotting and immune-electron microscopy showed that t he SOD subunit was recognized by a polyclonal antibody against glyoxisomal CuZn-SOD from watermelon cotyledon. In the stroma of wheat, ascorbate perox idase showed at least three well-resolved bands differently induced by copp er treatments.