Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos

Abstract

Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks. Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids. An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of alpha-eleostearic (18:3Delta(9cis, 11trans,13trans)) and alpha-parinaric (18:4Delta(9cis,11trans, 13trans,15cis)) acids. Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of alpha-eleostearic and alpha-parinaric acids, respectively. From ESTs of both species, a class of cDNAs encoding a diverged form of the Delta(12)-oleic acid desaturase was identified. Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of alpha-eleostearic and alpha-parinaric acids, neither of which is present in untransformed soybean embryos. alpha-Eleostearic and alpha-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX. These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants. These findings also demonstrate a previously uncharacterized activity for Delta(12)-oleic acid desaturase-type enzymes that we have termed "conjugase."

Figure 1

Comparison of the structures of fatty acids with methylene interrupted double bonds (A) vs. those that contain conjugated double bonds (B and C). Shown are α-linolenic acid (18:3Δ^{9cis,12cis,15cis}) (A), α-eleostearic acid (18:3Δ^{9cis,11trans,13trans}) (B), and α-parinaric acid (18:4Δ^{9cis,11trans,13trans,15cis}) (C).

Figure 2

Northern analysis of the expression of genes for a diverged Δ¹²-oleic acid desaturase or Fad2 from Momordica (MomoFadX) and Impatiens (ImpFadX). 32P-labeled probes derived from the MomoFadX or ImpFadX cDNAs were hybridized to 10 μg of total RNA from leaves (L) and developing seeds (S) of M. charantia or from leaves (L) and seed pods (that contain developing seeds) (S) of I. balsamina as indicated in A. The ethidium bromide stained gel corresponding to the Northern blot is shown in B.

Figure 3

Comparison of amino acid sequences of diverged forms of the Fad2 from Impatiens (ImpFadX) and Momordica (MomoFadX) with known Fad2 and Fad2-related polypeptides. The alignment includes the amino acid sequences of the Arabidopsis Fad2 (AraDes), the castor oleic acid hydroxylase (CastorOH), and the Crepis epoxygenase (CrepEpox) and acetylenase (CrepAcet). Colons indicate residues that are identical to those found in the ImpFadX sequence. Gaps in alignments are maintained by dashes (−).

Figure 4

Gas chromatographic analyses of fatty acid methyl esters prepared from an untransformed somatic soybean embryo (A), a transgenic somatic soybean embryo expressing MomoFadX (B), and seeds of M. charantia (C). The asterisks (*) indicate fatty acid methyl esters tentatively identified as cis-trans isomers of methyl α-eleostearic acid. The labeled peaks represent methyl esters of the following fatty acids: 16:0, palmitic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, α-linolenic acid; and 20:1, eicosenoic acid.