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 ...

An expressed napin storage protein gene fromBrassica rapa, BcNA1, has been cloned and sequenced. The gene is a member of a family of four to seven napin genes inB. rapaand is highly expressed in developing seeds. An expression cassette containing the DNA flanking the napin coding region of BcNA1 has been engineered and demonstrated to function appropriately, as compared with the gene's endogenous expression, in transgenic rapeseed using the β-glucuronidase reporter gene. TheB. rapaBcNA1 gene and aB. napusnapin gene, gNa, share extremely high nucleotide homology not only throughout their coding regions, but over a DNA locus comprising 4.3 kb. We suggest the gNa gene was contributed by the originalB. rapaprogenitor of the amphidiploidB. napus.

A 715 base pair cDNA clone coding for an acyl carrier protein (ACP) in spinach leaves has been isolated and characterized. The amino acid sequence indicated by the cDNA sequence closely matches the amino acid sequence of the ACP-I isoform. The presence of polyadenylation and DNA sequence coding for a precursor protein with a putative transit peptide, and the absence of hybridization between the cloned DNA and isolated spinach plastid DNA collectively show that the ACP-I gene is nuclear-encoded. The ACP-I cloned DNA did not cross-hybridize with mRNA from spinach tissues in which ACP-II has been found. Cross-hybridization with mRNA from tissues of Brassica campestris was either weak or undetectable. The cloning of an ACP-I gene represents an initial step in the molecular dissection of fatty acid synthetase in plants.

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A genomic clone of an acyl carrier protein gene (Bcg4-4) which is highly expressed in developing embryos of Brassica rapa was isolated and sequenced. The promoter and transcription terminator regions of Bcg4-4 were used to express a beta-glucuronidase reporter gene in transgenic rapeseed. Deletion of repeated domains in the promoter region did not lower beta-glucuronidase expression in seeds.