RNA Interference-Mediated Change in Protein Body Morphology and Seed Opacity through Loss of Different Zein Proteins

Opaque or nonvitreous phenotypes relate to the seed architecture of maize (Zea mays) and are linked to loci that control the accumulation and proper deposition of storage proteins, called zeins, into specialized organelles in the endosperm, called protein bodies. However, in the absence of null mutants of each type of zein (i.e. α, β, γ, and δ), the molecular contribution of these proteins to seed architecture remains unclear. Here, a double null mutant for the δ-zeins, the 22-kD α-zein, the β-zein, and the γ-zein RNA interference (RNAi; designated as z1CRNAi, βRNAi, and γRNAi, respectively) and their combinations have been examined. While the δ-zein double null mutant had negligible effects on protein body formation, the βRNAi and γRNAi alone only cause slight changes. Substantial loss of the 22-kD α-zeins by z1CRNAi resulted in protein body budding structures, indicating that a sufficient amount of the 22-kD zeins is necessary for maintenance of a normal protein body shape. Among different mutant combinations, only the combined βRNAi and γRNAi resulted in drastic morphological changes, while other combinations did not. Overexpression of α-kafirins, the homologues of the maize 22-kD α-zeins in sorghum (Sorghum bicolor), in the β/γRNAi mutant failed to offset the morphological alterations, indicating that β- and γ-zeins have redundant and unique functions in the stabilization of protein bodies. Indeed, opacity of the β/γRNAi mutant was caused by incomplete embedding of the starch granules rather than by reducing the vitreous zone.