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...

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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 ...

The pyridoxal phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14), the key enzyme in ethylene biosynthesis, is inactivated by its substrate S-adenosylmethionine (AdoMet). Apple ACC synthase was purified with an immunoaffinity gel, and its active site was probed with NaB3H4 or Ado[(4C]Met. HPLC separation of the trypsin digest yielded a single radioactive peptide. Peptide sequencing of both 3H-and 14C-labeled peptides revealed a common dodecapeptide of Ser-Leu-Ser-Xaa-Asp-Leu-Gly-LeuPro-Gly-Phe-Arg, where Xaa was the modified, radioactive residue in each case. Acid hydrolysis of the 3H-labeled enzyme released radioactive N-pyridoxyllysine, indicating that the active-site peptide contained lysine at position 4. Mass spectrometry of the "4C-labeled peptide indicated a protonated molecular ion at m/z 1390.6, from which the mass of Xaa was calculated to be 229, a number that is equivalent to the mass of a lysine residue alkylated by the 2-aminobutyrate portion of AdoMet, as we previously proposed. These results indicate that the same active-site lysine binds the PLP and convalently links to the 2-aminobutyrate portion of AdoMet during inactivation. The active site of tomato ACC synthase was probed in the same manner with Ado [14C]Met. Sequencing of the tomato active-site peptide revealed two highly conserved dodecapeptides; the minor peptide possessed a sequence identical to that of the apple enzyme, whereas the major peptide differed from the minor peptide in that methionine replaced leucine at position 6.The plant hormone ethylene regulates many aspects of plant growth and development. Adams and Yang (1) have established that ethylene is biosynthesized via the following se-

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