Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and are the precursors of many lipid-derived signaling molecules. Here, pathways for PUFA synthesis are described that do not require desaturation and elongation of saturated fatty acids. These pathways are catalyzed by polyketide synthases (PKSs) that are distinct from previously recognized PKSs in both structure and mechanism. Generation of cis double bonds probably involves position-specific isomerases; such enzymes might be useful in the production of new families of antibiotics. It is likely that PUFA synthesis in cold marine ecosystems is accomplished in part by these PKS enzymes.

Population growth, arable land and fresh water limits, and climate change have profound implications for the ability of agriculture to meet this century’s demands for food, feed, fiber, and fuel while reducing the environmental impact of their production. Success depends on the acceptance and use of contemporary molecular techniques, as well as the increasing development of farming systems that use saline water and integrate nutrient flows.

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Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rpa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs ofB. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.Canola and other temperate vegetable oils are composed predominantly of unsaturated 18-carbon fatty acids: the monounsaturated oleic (18:1) and polyunsaturated linoleic (18:2) and linolenic (18:3) acids. In addition to these fatty acids, most oils also contain small but significant amounts of the saturated palmitic (16:0) and stearic (18:0) acids (1). The plastid-localized enzyme stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the initial desaturation reaction in fatty acid biosynthesis (Fig. 1A) and thus plays a key role in determining the ratio oftotal saturated to unsaturated fatty acids in plants (2,4,5).Specialized fatty acid compositions desired for edible and industrial purposes have been produced in oilseed crops through traditional breeding and selection alone or in combination with mutagenesis programs (6-9). Although the molecular basis for the changes is largely unknown, examples such as the removal of erucic acid from rapeseed oil to create canola (10), reduction of linolenic acid content in flax seed (11), and increases in stearate content of up to six times the wild-type level in safflower (up to 12% stearate) (12) and soybean (up to 30%o stearate) oil (13,14) demonstrate the plasticity of fatty acid composition in seed oil. It should also be possible to modify seed oil composition by the use of genetic engineering techniques (15-17). Antisense RNA technology has proven to be an effective means of reducing the level of specific enzymes in plants (18-21). Because fatty acid biosynthesis is an essential metabolic pathway in all tissues ofthe plant, modification of seed oil biosynthesis may require tissue-specific control of antisense RNA expression. Reduction of stearoyl-ACP desaturase in seeds should alter the ratio of saturated to unsaturated fatty acids and lead to the production of a novel storage oil without compromising the integrity of membrane lipids in leaf and other plant tissues.We report the isolation of a Brassica rapa (syn. Brassica campestris, turnip rape) stearoyl-ACP desaturase cDNAt and expression of antisense stearoyl-ACP desaturase constructs in seeds of B. rapa and Brassica napus. The activity and amount of stearoyl-ACP desaturase...

BackgroundWheat (Triticum spp.) is an important source of food worldwide and the focus of considerable efforts to identify new combinations of genetic diversity for crop improvement. In particular, wheat starch composition is a major target for changes that could benefit human health. Starches with increased levels of amylose are of interest because of the correlation between higher amylose content and elevated levels of resistant starch, which has been shown to have beneficial effects on health for combating obesity and diabetes. TILLING (Targeting Induced Local Lesions in Genomes) is a means to identify novel genetic variation without the need for direct selection of phenotypes.ResultsUsing TILLING to identify novel genetic variation in each of the A and B genomes in tetraploid durum wheat and the A, B and D genomes in hexaploid bread wheat, we have identified mutations in the form of single nucleotide polymorphisms (SNPs) in starch branching enzyme IIa genes (SBEIIa). Combining these new alleles of SBEIIa through breeding resulted in the development of high amylose durum and bread wheat varieties containing 47-55% amylose and having elevated resistant starch levels compared to wild-type wheat. High amylose lines also had reduced expression of SBEIIa RNA, changes in starch granule morphology and altered starch granule protein profiles as evaluated by mass spectrometry.ConclusionsWe report the use of TILLING to develop new traits in crops with complex genomes without the use of transgenic modifications. Combined mutations in SBEIIa in durum and bread wheat varieties resulted in lines with significantly increased amylose and resistant starch contents.

Fatty acid hydroperoxide lyase (HPL) is a novel P-450 enzyme that cleaves fatty acid hydroperoxides to form short-chain aldehydes and oxo-acids. In cucumber seedlings, the activities of both fatty acid 9HPL and 13HPL could be detected. High 9HPL activity was especially evident in hypocotyls. Using a polymerase chain reaction-based cloning strategy, we isolated two HPL-related cDNAs from cucumber hypocotyls. One of them, C17, had a frameshift and it was apparently expressed from a pseudogene. After repairing the frameshift, the cDNA was successfully expressed in Escherichia coli as an active HPL with specificity for 13-hydroperoxides. The other clone, C15, showed higher sequence similarity to allene oxide synthase (AOS). This cDNA was also expressed in E. coli, and the recombinant enzyme was shown to act both on 9-and 13-hydroperoxides, with a preference for the former. By extensive product analyses, it was determined that the recombinant C15 enzyme has only HPL activity and no AOS activity, in spite of its higher sequence similarity to AOS. ß

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Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils. Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined. The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA. The primary structure of the protein, as deduced from the nucleotide sequence of the eDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme. Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition. However, active enzyme was detected when assayed in vitro with added spinach ferredoxin. The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E. coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme.Membrane fluidity and function are greatly influenced by the ratios of saturated to unsaturated fatty acids in the membrane lipids. In plants (1) and bacteria (2), the saturated fatty acids are synthesized in two-carbon increments as acyl thioesters of acyl carrier protein (ACP). In enteric bacteria such as Escherichia coli, the primary unsaturated fatty acids are cis-All C18:1 (vaccenic acid) and cis-A9 C16:1 (palmitoleic acid). Vaccenic and palmitoleic acids are synthesized by elongation of precursor monounsaturated acyl-ACPs; the saturated 16-and 18-carbon fatty acids (palmitic and stearic acids) are synthesized from precursor saturated acyl-ACPs. In higher plants, however, the unsaturated 16-carbon transhexadec-9-enoic acid and 18-carbon oleic acid (cis-A9 C18:1) are formed directly from palmitic and stearic acids esterified to specific glycerol lipids or to ACP (3). These reactions take place in the chloroplast (or proplastid in nonphotosynthetic tissues). Further double bonds are introduced into the monounsaturated acyl-lipids, typically at the 12 position followed by desaturation at the 15 or 6 positions of the diunsaturated species; saturated acyl groups generally do not serve as substrates for desaturation at the 6, 12, or 15 position in the carbon chain. Thus in higher plants, the ratio of saturated fatty acids to unsaturated fatty acids in membrane lipids is directly regulated by the enzymes that catalyze the conversion of saturated species to monounsaturated ones.Our interests lie in the regulation of the enzymatic steps in higher plants that determine the relative amounts of specific saturated and unsaturated fatty acids in neutral storage lipids. Unsaturated fatty acids in seed oils are predominantly 18 carbons or more in length and are derived by a series of enzymatic steps following the conversion of stearoyl-ACP to oleoyl-ACP. Stearoyl-ACP desaturase (EC 1.14.99.6) is therefore a necessary enzyme ...