Abstract
In the scutellum of maize kernel after imbibition, lipase activity increased rapidly, concomitant with the decrease in storage triacylglycerols. The enzyme activity peaked at day 6, but remained at the same level from day 6-10 when most of the triacylglycerols had been depleted. By in vitro translation with extracted RNAs followed by immunoprecipitation, and by resolving the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, lipase was found to be de novo synthesized in postgermination. The enzyme was synthesized by RNAs extracted from free polyribosomes and not from bound polyribosomes. Both in vitro and in vivo synthesized lipase had the same Mr of 65,000 as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as had the purified authentic enzyme; thus there was no appreciable co- or post-translational processing of the enzyme. Lipase-specific mRNA was present only between day 2-6 after imbibition. At day 6 when lipolysis was most active, more than 60% of the lipase activity was recovered in the lipid body fraction and specifically associated with the organelle membrane. From day 6-10, the lipase activity gradually shifted from the lipid body fraction to other subcellular fractions, including the 10,000 X g pellet, the 120,000 X g pellet, and the 120,000 X g supernatant. Lipase in these subcellular fractions was attributed to represent the enzyme associated with membrane ghosts of the lipid bodies which were fusing with the fragile cell vacuoles; such fusions were observed in situ by electron microscopy.
Highlights
We report experimental results showing that the appearance of lipase activity in postgermination is due to a de novo synthesis of the protein, that thereis noco- or posttranslational processing of the enzyme, and that theenzyme is synthesized on free and not bound polyribosomes
In day 2-6 when lipolysis was most active, more than half of the lipase activity in the homogenate was recoveredin the lipid body fraction (Fig. 1).Earlier findings show that lipase is a hydrophobic protein that binds tightly to themembrane of the lipid bodies [6]
Beyond day 6 when most of the lipid had been depleted, lipase activity shifted from the lipid body fraction to other subcellular fractions, including the 10,000 x g pellet, the 120,000 X g pellet, and the 120,000 X g supernatant
Summary
From the Biology Department, Universityof South Carolina, Columbia, South Carolina 29208. Lipase activity is absent before germination and appears in postgermination concomitant with the disappearance of storage triacylglycerols [5]. This appearance may be due to a de novo synthesis of the enzyme during seedling growth or an activation of an inactive enzyme synthesized during seed maturation. We have been using maize scutella as a model system to study seed lipase and lipid bodies [6,7,8] In this tissue, lipase activity is absent in the maturing andungerminated seed, and appears 2 days after imbibition, concomitant with the disappearance of triacylglycerols. We report experimental results showing that the appearance of lipase activity in postgermination is due to a de novo synthesis of the protein, that thereis noco- or posttranslational processing of the enzyme, and that theenzyme is synthesized on free and not bound polyribosomes. When about 50% of the maximal kernel lipids had been accumulated, the maturing kernels were collected
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