Tag

6. LPAT (lysophosphatidic acid acyltransferase)

7. PAP (phosphatidic acid phosphatase)

8. DGAT (diacylglycerol acyltransferase)

9. PDAT (phospholipid:diacylglycerol acyltransferase)

FIGURE 2.1 Schematic diagram of a-eleostearic acid and triacylglycerol biosynthesis in developing tung (Vernicia fordii) seeds. Eleostearic acid is synthesized from linoleic acid attached to endoplasmic reticulum (ER) phospholipids, primarily phosphatidylcholine (PC), then removed from the phospholipid pool and channeled into triacylglycerols through any of several coenzyme A (CoA)-dependent or -independent acyltransferase reactions.

efficient channeling of novel fatty acids into storage TAG. We have focused primarily on the diacylglycerol acyltransferases (DGATs), the enzyme family that catalyzes the final acylation reaction of the Kennedy pathway, converting acyl-CoA and diacylglycerol (DAG) to TAG. At least three unrelated classes of DGAT enzymes exist in higher plants; two are targeted to the membrane of the endoplasmic reticulum (ER) (DGAT1 and DGAT2) [20,21], and the other is the soluble DGAT3 class [22]. Our work has compared the properties of the DGAT1 and DGAT2 enzymes from castor and tung and to the orthologous enzymes from yeast and Arabidopsis, both of which do not synthesize hydroxylated or conjugated fatty acids. Tung DGAT1 and castor DGAT1 showed either no preference or only slight preference for substrates containing the appropriate novel fatty acid, and the expression patterns of both genes compared rather poorly to the time course of seed TAG synthesis [23-26], although the protein expression profile for castor DGAT1 does match the oil synthetic timeline much better [27].

However, both tung and castor DGAT2 enzymes showed strong preferences for their respective novel fatty acids. When expressed in yeast fed tung oil fatty acids, tung DGAT2 synthesized substantially more trieleostearin than did tung DGAT1, yeast DGAT, or either Arabidopsis DGAT1 or DGAT2 [24]. These data suggested that tung DGAT2 may play a more important role than tung DGAT1 in trieleostearin synthesis in developing tung seeds. Besides the apparent preference of tung DGAT2 for substrates containing conjugated fatty acids, cell biological analysis of the tung DGATs also suggested that each enzyme might play a different role: each is localized to discreet subdomains within the ER membrane [24]. These experiments were among the first to definitively demonstrate compartmentalization of enzymes within discreet, nonoverlapping regions of the ER membrane and support long-standing hypotheses that suggested colocalization of sets of enzymes dedicated to the same function [28]. We are currently analyzing other tung acyltransferases with regard to their targeting within plant cells to extend the depth of our understanding of this fascinating process.

Castor DGAT2 has also proven very useful in manipulating fatty acid content. It was initially characterized with respect to its ability to increase the levels of HFAs present in the seed oils of transgenic Arabidopsis expressing the castor hydroxylase FAH12. Singular expression of FAH12 in Arabidopsis results in HFA levels of 17%-18%, and repeated efforts to find better FAH12 plant lines have consistently failed [11,12,29]. Coexpression of castor DGAT2, however, resulted in multiple independent lines that produced up to 30% HFA, by far the highest level of HFA yet reported for a transgenic system [23]. As shown in Figure 2.2, detailed analysis of the TAG species produced by the FAH12/RcDGAT2 (Ricinus communis DGAT2) double-transgenic lines showed that castor DGAT2 coexpression dramatically shifted the TAG fatty acid profile toward lipid species containing two or more HFAs; the FAH12/RcDGAT2 double-transgenic plants contain higher levels of all eight major TAG species present in native castor oil (including triricindein) than do the FAH12 single transgenics.

The preference of castor DGAT2 for ricinoleic acid-containing substrates was demonstrated directly by enzyme assay after overexpression in yeast. The castor enzyme exhibited a 10-fold preference for diricinolein as acyl acceptor over two

TAG Species

FIGURE 2.2 Quantitative analysis of individual triacylglycerol (TAG) species from FAH12-and FAH12/RcDGAT2-transgenic Arabidopsis seeds. Relative amounts of eight individual TAGs, corresponding to the eight TAG species that make up more than 97% of castor oil, were identified and quantified in the seed oils of CL7 and line 544 by liquid chromatography tandem mass spectrometry (LC/MS/MS; see ref. 19 for details). Data are means plus or minus the standard deviation of three independent measurements. P, palmitate (16:0); S, stearate (18:0); O, oleate (18:1); L, linoleate (18:2); Ln, linolenate (18:3); and R, ricinoleate (18:1, 12-OH).

TAG Species

FIGURE 2.2 Quantitative analysis of individual triacylglycerol (TAG) species from FAH12-and FAH12/RcDGAT2-transgenic Arabidopsis seeds. Relative amounts of eight individual TAGs, corresponding to the eight TAG species that make up more than 97% of castor oil, were identified and quantified in the seed oils of CL7 and line 544 by liquid chromatography tandem mass spectrometry (LC/MS/MS; see ref. 19 for details). Data are means plus or minus the standard deviation of three independent measurements. P, palmitate (16:0); S, stearate (18:0); O, oleate (18:1); L, linoleate (18:2); Ln, linolenate (18:3); and R, ricinoleate (18:1, 12-OH).

other traditional DAG substrates [23]. These findings were extended even further by Kroon et al. [25], who demonstrated that RcDGAT2 could synthesize triricinolein, the primary TAG species in castor oil. Collectively, these data strongly suggest that endogenous Arabidopsis DGAT activities are poorly suited to accommodate HFA-containing substrates, and that RcDGAT2 is a major determinant of castor oil fatty acid composition.

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