A Nonaqueous Procedure for Isolating Starch Granules with Associated Metabolites from Maize (Zea mays L.) Endosperm

Abstract

A nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol was developed for the isolation from maize of starch granules with associated metabolites. In this procedure, immature endosperm tissue was quickly frozen at -156 C, freeze-dried, homogenized in cold glycerol, filtered through Miracloth, and centrifuged through a higher density medium of 3-chloro-1,2-propanediol. The procedure was used to isolate starch granules from the endosperm of normal and the mutant amylose-extender dull waxy. Starch and water-soluble polysaccharide recovery was high with low cytoplasmic (RNA) and nuclear (DNA) contamination.Electron microscopic examination of the isolated starch granules failed to demonstrate the presence of the amyloplast's membrane. However, based on an examination of fresh, freeze-dried, and rehydrated freeze-dried normal endosperm, it is suggested that the amyloplast membrane and enclosed stroma metabolites were dried onto the surface of the starch granules during the freeze-drying procedure. Chemical analysis of the glycerol-propanediol isolated granules showed the presence of alcohol-soluble sugars, inorganic phosphate, and phosphate-containing compounds. These soluble metabolites may represent amyloplast stroma metabolites which became bound to the starch granules during freeze-drying. Thus, this isolation procedure should be useful when metabolites closely associated with starch granules in situ are to be evaluated.