Tung oil is an industrial drying oil containing ca. 90% PUFA. We previously reported on enzymes required for the synthesis of linoleic (6% of FA) and eleostearic (80%) acids and here describe the cloning and functional analysis of an omega-3 FA desaturase (FAD3) required for the synthesis of linolenic acid (1%). The tung FAD3 cDNA was identified by screening a tung seed cDNA library using the polymerase chain reaction and degenerate primers encoding conserved regions of the FAD3 enzyme family. Expression of this cDNA in yeast cells, cultured in the presence of linoleic acid, resulted in the synthesis and accumulation of linolenic acid, which accounted for up to 18% w/w of total cellular FA. Tung FAD3 activity was significantly affected by cultivation temperature, with the greatest amount of linolenic acid accumulating in yeast cells grown at 15°C. The amount of linolenic acid synthesized in yeast cells by tung FAD3 is ca. 10-fold higher than that observed by expression of a rapeseed (Brassica napus) FAD3 in yeast, suggesting that tung FAD3 might be useful for biotechnological production of omega-3 FA in transgenic organisms. EXPRESSION OF TUNG FAD3 IN YEAST CELLS 651 JAOCS, Vol. 81, no. 7 (2004) FIG. 4. Northern blot analysis of tung FAD3 gene expression in tung leaf or seed tissue. (A) Total RNA (15 µg) was isolated from either tung seed or leaf tissue, then size-fractionated on an agarose gel, transferred to membrane, and probed with labeled tung FAD3 under stringent wash conditions. (B) Ethidium bromide-stained gel of total RNA demonstrating equal loading of samples. See Figure 1 for abbreviation.

Ethanolamine kinase, CTP:ethanolaminephosphate cytidylyltransferase (ECT), and ethanolaminephosphotransferase, which sequentially catalyze the primary pathway for phosphatidylethanolamine synthesis, were measured in castor bean (Ricinus communis L. var Hale) endosperm for 6 d after the onset of imbibition. Ethanolamine kinase (EC 2.7.1 3 2 ) activity was cytosolic, increasing slowly during the first 5 d and then declining. Total ECT (EC 2.7.7.14) activity increased until the 4th d, but the endoplasmic reticulum fraction of the activity peaked at d 3, and the mitochondrial activity peaked at d 4. Diacylglycero1:CDPethanolamine ethanolaminephosphotransferase (EC 2.7.8.1) increased during the first 2 d after imbibition began, after which it declined. The lowest activity of ethanolamine kinase during postgermination was more than 5-fold higher than the maximum activity of ECT, and the total activity of diacylglycero1:CDPethanolamine ethanolaminephosphotransferase at d 2 was at least triple that of ECT of the endoplasmic reticulum. We have partially purified ECT from mitochondrial fractions of postgermination castor bean endosperm starting with mitochondria purified by sucrose (SUC) density gradient centrifugation and broken by osmotic shock and homogenization. The membranebound ECT was solubilized with 1.5% 3-[(3-cholamidopropyI) dimethylammonio]-2-hydroxy-l -propanesulfonate and purified approximately 1 18-fold by polyethylene glycol precipitation, chromatography on Sephacryl S-200, and then SUC gradient centrifugation. The continuous presence of both salt (0.5 M NaCI) andhydroxy-1 -propanesulfonate) was necessary to prevent aggregation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final activity peak resulted in a prominent protein band at 35 kD, which correlated with bands from peak ECT activity fractions after both SUC gradient centrifugation and gel filtration on Sephacryl S-200. The activity of this enzyme was enhanced by the addition of several phospholipids.

Lipid-protein complexes were identified in the 104,000 × g supernatant fraction of developing tung seeds. Incubation of this fraction with linoleoyl-CoA promoted an increase of chloroform-extractable lipids in a time-dependent manner. High-performance liquid chromatography analysis indicated that the extracted lipids were similar to mature tung oil triglycerides. Differential extraction using chloroform or chloroform/methanol indicated that linoleoyl-CoA promoted extraction of pre-existing lipids rather than de novo synthesis. An increase in extractable lipids was also observed after incubation with proteinase K. Isolation of lipid-protein complexes by sucrose density centrifugation and analysis of proteins by gel electrophoresis revealed several proteins specifically associated with this lipid fraction. FIG. 1. High-performance liquid chromatography analysis of eleostearoyl lipids extracted after incubation of 104,000 × g supernatant with linoleoyl-CoA. (a) Tung oil standard. (b) Lipids extracted from the 104,000 × g supernatant after 20 min incubation. The three major peaks containing eleostearic acid are labeled 1, 2 and 3. (c) Changes in absorbance of each of the three major peaks observed in (b) throughout the course of the assay.

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