Immunohistochemical localization of alpha-amylase in barley aleurone cells

Alpha-Amylase was localized in aleurone cells of barley using immunohistochemical methods. Anti-alpha-amylase antibody was produced by rabbits immunized with enzyme purified from malt diastase and Himalaya variety barley seeds. Immunoelectrophoresis showed that the antibodies to both antigens were immunologically similar, therefore, they were used interchangeably in the localization of alpha-amylase. Fluorescence of 8-10 mum sections of freeze-substituted and paraffin embedded, gibberellic acid (GA)-treated aleurone tissue incubated with rabbit anti-alpha-amylase IgG and rhodamine-conjugated goat-anti-rabbit IgG is localized in the cytoplasm, the nuclear region and the innermost portion of the cell wall. Cytoplasmic immunofluorescence is not associated with a specific organelle but rather is diffusely distributed. The fluorescence of the nuclear region, however, is intense and in thinner (4-5 mum) sections is associated not with the nucleoplasm but with the nuclear envelope and perinuclear region of the cytoplasm. Fluorescence of the cell wall is confined to the inner boundary of the wall corresponding to the resistant wall layer. The immunofluorescent properties of non-GA-treated cells are quantitatively different; fluorescence of these sections is low and diffuse and is particularly reduced in the nuclear region. Electron microscopy shows that GA-treatment results in the proliferation of endoplasmic reticulum (ER) in the perinuclear region of the cell. We suggest that the alpha-amylase localized by immunofluorescence in the perinuclear region of the cell is localized in this ER produced in response to GA treatment. Immunohistochemical localization of alpha-amylase in cells zonated by centrifugation also suggests that the enzyme is intimately associated with the perinuclear area.