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,...

Diacylglycerol acyltransferases (DGAT) catalyze the final and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT genes have been identified in numerous organisms. Multiple isoforms of DGAT are present in eukaryotes. We previously cloned DGAT1 and DGAT2 genes of tung tree (Vernicia fordii), whose novel seed TAGs are useful in a wide range of industrial applications. The objective of this study was to understand the developmental regulation of DGAT family gene expression in tung tree. To this end, we first cloned a tung tree gene encoding DGAT3, a putatively soluble form of DGAT that possesses 11 completely conserved amino acid residues shared among 27 DGAT3s from 19 plant species. Unlike DGAT1 and DGAT2 subfamilies, DGAT3 is absent from animals. We then used TaqMan and SYBR Green quantitative real-time PCR, along with northern and western blotting, to study the expression patterns of the three DGAT genes in tung tree tissues. Expression results demonstrate that 1) all three isoforms of DGAT genes are expressed in developing seeds, leaves and flowers; 2) DGAT2 is the major DGAT mRNA in tung seeds, whose expression profile is well-coordinated with the oil profile in developing tung seeds; and 3) DGAT3 is the major form of DGAT mRNA in tung leaves, flowers and immature seeds prior to active tung oil biosynthesis. These results suggest that DGAT2 is probably the major TAG biosynthetic isoform in tung seeds and that DGAT3 gene likely plays a significant role in TAG metabolism in other tissues. Therefore, DGAT2 should be a primary target for tung oil engineering in transgenic organisms.

No abstract

For better understanding of the adhesive properties of different fractions of cottonseed protein, cottonseed meals from both glanded and glandless cotton varieties were separated into several fractions. Each meal was sequentially extracted with water and 1 M NaCl solution, or with phosphate buffer and NaCl solution. Adhesives were prepared from the recovered fractions and hot-pressed onto maple veneer strips and tested for their properties. The adhesive strength of the water-and buffer-washed solid fractions (i.e., the un-extractable residues of the meals) from the glanded seed ranged from 1.32 to 1.62 MPa and were unchanged or increased compared with the adhesive strength of the original meal that varied from 0.98 and 1.49 MPa. Soaking the wood specimens bonded at 80°C revealed that the water resistance of these water-and buffer-washed adhesives was significantly improved in that they exhibited no delamination during soaking compared with the meal adhesive that showed some delamination (20-30 % of the samples). Furthermore, the water resistance of these fractions with wet shear strength around 1.5 MPa was comparable to that of cottonseed protein isolate ([90 % protein) when the joints were bonded at 100°C. The preparations from glandless cottonseed meals showed similar adhesive performances. Additional extraction of the meals with NaCl solution reduced adhesive performance. The results suggest that water-or buffer-washed cottonseed meal fractions can be used as wood adhesives and would be less costly to prepare than cottonseed protein isolates.

No abstract

No abstract

Chinese melon (Momordica charantia L.), also known as bitter gourd, is a tropical crop, grown throughout Asian countries for use as food and medicinals. In 1993, four cultivars of Chinese melon were grown in Mississippi and the seeds were collected. Oil contents of the seeds ranged from 41 to 45% and the oils contained 63-68% eleostearic acid and 22-27% stearic acid. Industrially important tung oil, a "fast-drying oil" used in paints and varnishes, contains 90% eleostearic and 2-3% stearic acid. The ratio of stearic to eleostearic in Chinese melon seed oil is ten times greater than that in tung oil. The higher ratio should reduce the rate of drying and crosslinking and could be advantageous in the paint industry. The defatted meals contained 52-61% protein and would be a good source of methionine.