Associations of maize protein bodies with cytoskeleton, membranes, and ribosomes in the endosperm of wild type and opaque-2 mutant

Maize endosperm was homogenized in a cytoskeleton-stabilizing buffer, filtered and layered on gradients of 20–80% sucrose and analyzed by monitoring their UV absorbance. A major peak of UV-light absorbing material was detected on the gradient, at about 60–65% sucrose (density of approximately 1.3 g·ml⁻¹). Biochemical, fluorescence microscopic, and immunoblot analyses of this peak showed that it consisted of protein bodies associated with actin, membranes, and RNA (ribosomes). Seeds of wild type and opaque-2 mutant were then homogenized, the homogenate was modified using detergents and/or cytoskeleton-disrupting agents, and centrifuged on sucrose gradients. In wild type maize endosperm, detergent treatment caused the major peak (protein bodies) to increase in density so that they sediment further down the gradient. However, in opaque-2 the protein bodies formed a broader, but smaller peak which, upon treatment with detergent, generated protein bodies which pelleted to the bottom of the gradient. Analysis of gradient fractions by gel electrophoresis and immuno-blotting showed that both the wild type and the mutant had cytoskeleton proteins in the upper regions (soluble, non-polymerized microfilaments and microtubules) as well as in the peak regions. Comparisons of both the UV-absorbance profiles and the immunoblot data suggest that the protein bodies from the two maize types associate differently with the membranes and the cytoskeleton.