Identification and immunolocalization of superoxide dismutase isoenzymes of olive pollen

Abstract

Gametophytic tissues of plants are an area largely neglected in the broad literature on free radical processes in plants. In order to study the mechanisms of protection against oxidative stress in pollen, the presence of the key antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) was investigated. Crude extracts of olive tree (Olea europaea L.) pollen were subjected to native PAGE in 10% polyacrylamide gels. The SOD activity staining of gels showed the presence of four isoenzymes. All the SODS were completely inhibited by 2 mM KCN and 5 mM H2O2, and therefore belong to the family of CuZn-SODS. Isoelectric focusing (pH 3.5-7) of crude extracts and further detection of SOD activity allowed determination of isoelectric points for the four isoforms, namely 4.60, 4.78, 5.08 and 5.22. The cross-reactivity of pollen extracts with a polyclonal antibody to cytosolic CuZn-SOD from spinach leaves was assayed by western blotting. After SDS-PAGE and immunoblotting, a major polypeptide band of about 16.5 kDa was detected, which is characteristic of the subunit of most CuZn-SODS. Immunocytochemical studies at TEM level using the same antiserum showed that CuZn-SOD was localized in the cytoplasm of both vegetative and generative cells, and also in material adhered to the pollen wall. The olive pollen CuZn-SODS could function in the protection against oxidative stress during pollen development.