Control of Enzyme Activities in Cotton Cotyledons during Maturation and Germination

Abstract

Microbodies were isolated by zonal-rotor sucrose density gradient centrifugation from cotton (cv. DP 61) seeds at two distinct stages of embryogenesis (38 and 50 days after anthesis) and after 48 hours postgerminative growth. In all cases, beta-oxidation activity (palmitoyl-coenzyme A (CoA)-dependent reduction of acetylpyridine adenine dinucleotide or production of acetyl-CoA) and activities of the enzymes palmitate:CoA ligase, acyl-CoA oxidase, enoyl hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, plus catalase, were localized exclusively in the microbody fractions, i.e. none of the activities were associated with mitochondria. Acyl-CoA dehydrogenase activity could not be detected in any of the gradient fractions or in homogenates.Glyoxysomes isolated from cotyledons of 48-hour-germinated seeds were capable of beta-oxidation of acyl-CoAs of various chain lengths and degrees of unsaturation and were the sole site of 3-cis-2-trans enoyl-CoA isomerase activity. Direct measurement of the isomerase is the first demonstration of an enzyme required for unsaturated fatty acid catabolism in a higher plant. Palmitoyl-carnitine was not oxidized by any of the organelle fractions.Subfractionation of glyoxysomes by osmotic shock revealed that none of the beta-oxidation enzymes were tightly membrane-associated.