Purification and Characterization of Amyloplasts from Etiolated Epicotyls of Pisum Sativum

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Etiolated epicotyls of Pisum sativum L. cv. Alaska contain two distinct classes of amyloplasts: 1) small, unigranular amyloplasts (UGA) found primarily in the cortex; and 2) larger, multigranular amyloplasts (MGA) found exclusively in the starch sheath surrounding the central vascular cylinder. Only the MGA are displaced in a 1 × g field and are thus presumably involved in gravity perception. To help understand the graviperception mechanism, we have purified and partly characterized these statolithic amyloplasts. Brief mechanical homogenization followed by filtration and centrifugation at 120 × g for 3 min yields an enriched amyloplast fraction (ca. 2 : 1 ratio of MGA's : UGA's). Further purification by isopycnic centrifugation in step gradients of the iodinated compound urografin separated free starch grains (density = 1.44 g/ml) from intact amyloplasts (density = 1.41-1.43 g/ml). Partial resolution of MGA's and UGA's has been achieved with this system. Gradient purified amyloplasts are substantially free of contamination from other organellar fractions, as determined by marker enzyme distribution. Isolated amyloplasts have been judged intact by three criteria: 1) light microscopy; 2) osmotic sensitivity; and 3) the presence of a N, N'-dicyclohexyl carbodiimide-insensitive Mg++-dependent ATPase apparently associated with the amyloplast envelope. The gradient-purified amyloplasts contain significant amounts of DNA and RNA.

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