Formation of α-tocopherol hydroperoxide and α-tocopheroxyl radical: relevance for photooxidative stress in Arabidopsis

Tocopherols, lipid-soluble antioxidants play a crucial role in the antioxidant defense system in higher plants. The antioxidant function of alpha -tocopherol has been widely studied; however, experimental data on the formation of its oxidation products is missing. In this study, we attempt to provide spectroscopic evidence on the detection of oxidation products of alpha -tocopherol formed by its interaction with singlet oxygen and lipid peroxyl radical. Singlet oxygen was formed using photosensitizer rose bengal and thylakoid membranes isolated from Arabidopsis thaliana. Singlet oxygen reacts with polyunsaturated fatty acid forming lipid hydroperoxide which is oxidized by ferric iron to lipid peroxyl radical. The addition of singlet oxygen to double bond carbon on the chromanol head of alpha -tocopherol forms alpha -tocopherol hydroperoxide detected using fluorescent probe swallow-tailed perylene derivative. The decomposition of alpha -tocopherol hydroperoxide forms alpha -tocopherol quinone. The hydrogen abstraction from alpha -tocopherol by lipid peroxyl radical forms alpha -tocopheroxyl radical detected by electron paramagnetic resonance. Quantification of lipid and protein hydroperoxide from the wild type and tocopherol deficient (vte1) mutant Arabidopsis leaves using a colorimetric ferrous oxidation-xylenol orange assay reveals that alpha -tocopherol prevents formation of both lipid and protein hydroperoxides at high light. Identification of oxidation products of alpha -tocopherol might contribute to a better understanding of the protective role of alpha -tocopherol in the prevention of oxidative damage in higher plants at high light.