Subcellular Distribution of Four Classes of Sterols in Cotyledons of Soybean Seedlings

Soybean seedlings were grown at 26°C under the light (“green”) or in the dark (“etiolated”) for 5 days, and the cotyledons collected were homogenized with 0.4M sucrose-Tris buffer solution. By differential centrifugation, partly using sucrose stepwise density gradient centrifugation, the homogenates were fractionated into particulate fractions (pellets of 500×g, 1500×g, 15, 000×g (3 sub-fractions) and 105, 000×g (3 to 5 sub-fractions)) and non-particulate fractions (105, 000×g supernatant and lipid layer). The lipid extracts from respective fractions were chromatographed on thin layer plates, and the sterols separated into the four classes, the free form, fatty acid esters, acylated glucosides and non-acylated glucosides, were detected with H2SO4 spray. Either “green” or “etiolated” cotyledons showed that the pattern of sterol distribution in the particulate fractions differed from that in the non-particulate fractions, and the later fractions contained considerably greater ratio of fatty acid esters of sterols. The former fractions as well as the homogenate contained greatest ratio of the free form.