Lipid Body Lipoxygenase Characterized by Protein Fragmentation, cDNA Sequence and very early Expression of the Enzyme during Germination of Cucumber Seeds

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Abstract
Lipid bodies are cellular compartments containing triacylglycerols. They are encompassed by a phospholipid monolayer and decorated with characteristic proteins. In plants, lipid bodies are synthesized during seed formation but acquire new proteins during seed germination. In germinating cucumber (Cucumis sativus) seeds, the set of newly synthesized proteins appearing in the lipid bodies at the early stage of triacylglycerol mobilization comprises a special form of lipoxygenase. We isolated the lipid body lipoxygenase and characterized fragments prepared by limited proteolysis and cleavage with cyanogen bromide. A very early expression of lipid body lipoxygenase was found by studying the rate of de novo synthesis of lipoxygenase forins during germination. This allowed a clear distinction of this enzyme from other lipoxygenase isoforms. Hence, for determining the molecular structure of lipid body lipoxygenase we analyzed a cDNA prepared from mRNA of cotyledons at day 1 of germination. From the cDNA sequence, oligonucleotides were derived that specifically detected lipid body lipoxygenase mRNA on nothern blots. The very early expression of lipid body lipoxygenase was corroborated by this approach. Good agreement was observed between the amino acid sequence deduced from the cDNA sequence and the peptide structures analyzed biochemically. In particular, the cleavage products of cyanogen bromide treatment indicated that we had isolated the lipid body lipoxygenase cDNA. The sequence data show a lipoxygenase form characterized by a molecular mass of 99655 Da, which is significantly higher than the molecular masses of the cytosolic forms. Compared to the cytosolic forms that exhibit a molecular mass of 95 kDa, the lipid body form has an N-terminal extension of 34 amino acid residues. No evidence for a cotranslational or post-translational proteolytic processing was obtained by the size comparison of the in vitro translated lipoxygenase and the lipid body form.