Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing ;80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.

The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% ␣-eleostearic acid (18: 3⌬ 9cis,11trans,13trans ), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related ⌬ 12 oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1⌬ 9cis ) into linoleic acid (18:2⌬ 9cis,12cis ) and that FADX converted linoleic acid into ␣-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and ⌬ 12 -desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with ⌬ 12 fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.Conjugated fatty acids are naturally occurring compounds that have specialized uses in nutraceutical and industrial applications. For example, conjugated linoleic acid (CLA) is a potent anticancer compound present in foods derived from ruminant animals (Belury, 2002). This bioactive fatty acid (predominantly the 18:2⌬ 9cis,11trans isomer) is synthesized by rumen bacteria and then absorbed by the animal and concentrated in milk fat or adipose tissue. Rumen bacteria also synthesize 18:1⌬ 11trans , which can be absorbed and then desaturated by an animal stearoyl-CoA desaturase to produce CLA (Corl et al., 2001). Conjugated fatty acids such as ␣-eleostearic acid (18:3⌬ 9cis,11trans,13trans ) have recently shown promise for anticancer applications (Igarashi and Miyazawa, 2000;Kohno et al., 2002), as well as serum lipidlowering effects in mammals (Koba et al., 2002). Oils containing ␣-eleostearic acid may also be used for industrial drying applications. Tung oil, which is derived from seeds of the tung tree (Aleurites fordii Hemsl.), is commonly used in formulations of inks, dyes,...