Effect of in vivo iron supplementation on oxygen radical production by soybean roots

Both total iron content and the in vitro rate of iron reduction were higher in roots grown in the presence of exogenously added iron (up to 500 microM) than in roots grown in absence of supplemented iron. In vivo generation of reactive O2 species by intact roots was assessed employing 2',7'-dichlorofluorescein diacetate oxidation to a fluorescent product. Roots developed in the presence of 500 microM Fe-EDTA, showed significantly increased fluorescence (64%), as compared to roots developed in absence of added iron, suggesting that iron supplementation led to oxidative stress in vivo. At the subcellular level, iron content and Fe-EDTA reduction rate were significantly increased in microsomes isolated from roots developed in the presence of exogenously added iron, as compared to microsomes from roots grown in the absence of iron. Microsomes from Fe supplemented plants exhibited a 51% increase in 2',7'-dichlorofluorescein diacetate oxidation rate, a 55% increase in superoxide anion generation, and a four-fold increase in hydroxyl radical production as compared to controls. Iron supplementation did not affect the activity of antioxidant enzymes or the content of total thiols, however alpha-tocopherol content was significantly decreased in the homogenates and the microsomes isolated from roots supplemented with iron, as compared with values in roots developed in absence of iron. These data suggest that in vivo iron supplementation increased oxygen radical generation.