Characterization of acyl‐ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola

Acyl-acyl-carrier protein (ACP) thioesterases are, at least in part, responsible for the fatty acyl chain length composition of seed storage oils. Acyl-ACP thioesterases with specificity for each of the saturated acyl-ACP substrates from 8:0 through 16:0 have been cloned, with the exception of 18:0, and are members of the FatB class of thioesterases. The authors have determined that the tropical tree species mangosteen (Garcinia mangostana) stores 18:0 (stearate) in its seed oil in amounts of up to 56% by weight. Acyl-ACP thioesterase activity as measured in crude mangosteen seed extracts showed a preference for 18:1-ACP substrates, but had significant activity with 18:0 relative to that with 16:0-ACP, suggesting a thioesterase might be involved in the production of stearate. Three distinct acyl-ACP thioesterases were cloned from mangosteen seed cDNA; two representative of the FatA class and one representative of the FatB class. When expressed in vitro, the enzyme encoded by one of the FatAs (Garm FatA1) while preferring 18:1-ACP showed relatively low activity with 16:0-ACP as compared to 18:0-ACP, similar to the substrate preferences shown by the crude seed extract. Expression of Garm FatA1 in Brassica seeds led to the accumulation of stearate up to 22% in seed oil. These results suggest that Garm FatA1 is at least partially responsible for determining the high stearate composition of mangosteen seed oil and that FatA as well FatB thioesterases have evolved for specialized roles.     

Characterization and cloning of a stearoyl/oleoyl specific fatty acyl-acyl carrier protein thioesterase from the seeds of Madhuca longifolia (latifolia)

Deposition of oleate, stearate and palmitate at the later stages of seed development in Mahua (Madhuca longifolia (latifolia)), a tropical non-conventional oil seed plant, has been found to be the characteristic feature of the regulatory mechanism that produces the saturated fatty acid rich Mahua seed fat (commonly known as Mowrah fat). Although, the content of palmitate has been observed to be higher than that of stearate at the initial stages of seed development, it goes down when the stearate and oleate contents consistently rise till maturity. The present study was undertaken in order to identify the kind of acyl-ACP thioesterase(s) that drives the characteristic composition of signature fatty acids (oleate 37%, palmitate 25%, stearate 23%, linoleate 12.5%) in its seed oil at maturity. The relative Fat activities in the crude protein extracts of the matured seeds towards three thioester substrates (oleoyl-, stearoyl- and palmitoyl-ACP) have been found to be present in the following respective ratio 100:31:8. Upon further purification of the crude extract, the search revealed the presence of two partially purified thioesterases: a long-chain oleoyl preferring house-keeping LC-Fat and a novel stearoyl-oleoyl preferring SO-Fat. The characteristic accumulation of oleate and linoleate in the M. latifolia seed fat is believed to be primarily due to the thioesterase activity of the LC-Fat or MlFatA. On the other hand, the SO-Fat showed almost equal substrate specificity towards stearoyl- and oleoyl-ACP, when its activity towards palmitoyl-ACP compared to stearoyl-ACP was only about 12%. An RT-PCR based technique for cloning of a DNA fragment from the mRNA pool of the developing seed followed by nucleotide sequencing resulted in the identification of a FatB type of thioesterase gene (MlFatB). This gene was found to exist as a single copy in the mother plant genome. Ectopic expression of this MlFatB gene product in E. coli strain fadD88 further proved that it induced a higher level of accumulation of both stearic and oleic acids when compared to the negative control line that did not contain this MlFatB gene. It also indicated that SO-Fat indeed is the product of the MlFatB gene present in the maturing seeds of M. latifolia in nature. Additionally, a predicted 3D-structure for MlFatB protein has been developed through use of bioinformatics tools.     

Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli

A cDNA of fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) from developing seed of Madhuca butyracea has been cloned. The deduced amino acid sequence of the cDNA corresponding to the mature polypeptide showed 30-40% and 60-75% identity to the reported FatA and FatB class of plant thioesterases, respectively. This gene, MbFatB, is present as a single copy in M. butyracea genome and the MbFatB protein was detected clearly in seed tissues of this plant but not in that of Indian mustard (Brassica juncea). Heterologous expression of the MbFatB gene driven by different promoters in E. coli wild type and fatty acid beta-oxidation mutant (fadD88) strains resulted production of the recombinant protein with various fusion tags either as biologically inactive (insoluble) or functionally active forms. Expression of functionally active recombinant MbFatB in E. coli affected bacterial growth and cell morphology as well as changed the fatty acid profiles of the membrane lipid and the culture supernatant. Alteration of the fatty acid composition was directed predominantly towards palmitate and to a lesser extent myristate and oleate due to acyl chain termination activity of plant thioesterase in bacteria. Thus, this new MbFatB gene isolated from a non-traditional oil-seed tree can be used in future for transgenic development of oil-seed Brassica, a widely cultivated crop that expresses predominantly oleoyl-ACP thioesterase (FatA) in its seed tissue and has high amount of unwanted erucic acid in edible oil in order to alter the fatty acid profile in a desirable way.     

Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana , designated Ch FatB2 , has been identified, which, when expressed in Escherichia coli , provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1 , another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.     

Cloning,characterization and structural model of a FatA-type thioesterase from sunflower seeds (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

The substrate specificity of acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) determines the fatty acids available for the biosynthesis of storage and membrane lipids in seeds. In order to determine the mechanisms involved in the biosynthesis of fatty acids in sunflower seeds (Helianthus annuus L.), we isolated, cloned and sequenced a cDNA clone of acyl-ACP thioesterase from developing sunflower seeds, HaFatA1. Through the heterologous expression of HaFatA1 in Escherichia coli we have purified and characterized this enzyme, showing that sunflower HaFatA1 cDNA encodes a functional thioesterase with preference for monounsaturated acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/K_m) in catalyzing oleoyl-ACP, both in vivo and in vitro. By comparing this sequence with those obtained from public databases, we constructed a phylogenetic tree that included FatA and FatB thioesterases, as well as related prokaryotic proteins. The phylogenetic relationships support the endosymbiotic theory of the origin of eukaryotic cells and the suggestion that eubacteria from the -subdivision were the guest cells in the symbiosis with archaea. These prokaryotic proteins are more homologous to plant FatB, suggesting that the ancient thioesterases were more similar to FatB. Finally, using the available structure prediction methods, a 3D model of plant acyl-ACP thioesterases is proposed that reflects the combined data from direct mutagenesis and chimera studies. In addition, the model was tested by mutating the residues proposed to interact with the ACP protein in the FatA thioesterase by site-directed mutagenesis. The results indicate that this region is involved in the stabilization of the substrate at the active site.     

Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases

The specificity of plant acyl-acyl carrier protein (ACP) thioesterases is the major determinant of the chain length and level of saturated fatty acids found in most plant tissues. Although these enzymes have been previously characterized from a number of sources, information on kinetic parameters for a wide range of substrates with cloned enzymes is lacking. In the present study the substrate specificity of recombinant FatA thioesterase isoforms from Arabidopsis (AtFatA) and coriander (CsFatA) and FatB from Arabidopsis (AtFatB) have been re-examined with a comprehensive range of substrates including 14:1-ACP and 16:1-ACP. AtFatA displayed the highest catalytic efficiencies (kcat/Km) towards oleoyl-ACP with activities at least 20-fold lower for all other tested substrates and 75-fold lower with palmitoyl-ACP. Both chain length and double bond presence strongly influenced kcat of FatA with minor influence on Km. Arabidopsis FatB substrate specificity was found to differ from previous reports and this difference could be attributed to the influence of ACP structure. FatB activity with palmitoyl-ACP was 2.5-fold higher and the ratio of 16:0-ACP/14:0-ACP hydrolysis was 6.4-fold higher with spinach ACP compared to E. coli ACP. Additionally, the influence of amino acid domains from both AtFatA and AtFatB on their substrate specificity was studied by utilizing a domain-swapping approach. The characterization of the resulting chimeric enzymes pointed to the N-terminus as a determinant of the substrate specificity for both FatA and FatB acyl-ACP thioesterases.     

Acyl-ACP thioesterases from castor (Ricinus communis L.): An enzymatic system appropriate for high rates of oil synthesis and accumulation

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed.     

Improved stearate phenotype in transgenic canola expressing a modified acyl-acyl carrier protein thioesterase.

The engineering of crops for selected fatty acid production is one of the major goals of plant biotechnology. The Garm FatA1, an acyl-acyl carrier protein (ACP) thioesterase isolated from Garcinia mangostana, generates an elevated stearate (18:0) phenotype in transgenic Brassica plants. By site-directed mutagenesis, we generated seven mutants that showed up to a 13-fold increase in specific enzyme activity toward 18:0-ACP in vitro. The seed-specific expression of mutant S111A/V193A in Brassica plants results in transgenic plants that accumulate 55-68% more stearate than plants expressing the wild-type enzyme. Our results demonstrate that a thioesterase can be engineered to increase specific activity and that its improved function demonstrated in vitro is retained in vivo.     

Cloning and characterization of cDNAs encoding for long-chain saturated acyl-ACP thioesterases from the developing seeds of Brassica juncea

Four types of cDNAs corresponding to the fatty acyl-acyl carrier protein (ACP) thioesterase (Fat) enzyme were isolated from the developing seeds of Brassica juncea, a widely cultivated species amongst the oil-seed crops. The mature polypeptides deduced from the cDNAs showed sequence identity with the FatB class of plant thioesterases. Southern hybridization revealed the presence of at least four copies of BjFatB gene in the genome of this amphidiploid species. Western blot and RT-PCR analyses showed that the BjFatB class thioesterase is expressed poorly in flowers and leaves, but significantly in seeds at the mid-maturation stage. The enzymatic activities of different BjFatB isoforms were established upon heterologous expression of the four BjFatB CDSs in Escherichia coli K27fadD88, a mutant strain of fatty acid β-oxidation pathway. The substrate specificity of each BjFatB isoform was determined in vivo by fatty acid profile analyses of the culture supernatant and membrane lipid of the recombinant K27fadD88 and E. coli DH10B (fadD⁺) clones, respectively. The BjFatB1 and BjFatB3 were predominantly active on C18:0-ACP substrate, whereas BjFatB2 and BjFatB4 were specific towards C18:0-ACP as well as C16:0-ACP. These novel FatB genes may find potential application in metabolic engineering of crop plants through their over-expression in seed tissues to generate stearate-rich vegetable fats/oils of commercial importance.     

Mapping minor QTL for increased stearic acid content in sunflower seed oil

Increased stearic acid (C18:0) content in the seed oil of sunflower would improve the oil quality for some edible uses. The sunflower line CAS-20 (C18:0 genotype Es1Es1es2es2), developed from the high C18:0 mutant line CAS-3 (C18:0 genotype es1es1es2es2; 25% C18:0), shows increased C18:0 levels in its seed oil (8.6%). The objective of this research was to map quantitative trait loci (QTL) conferring increased C18:0 content in CAS-20 in an F₂ mapping population developed from crosses between HA-89 (wild type Es1Es1Es2Es2; low C18:0) and CAS-20, which segregates independently of the macromutation Es1 controlling high C18:0 content in CAS-3. Seed oil fatty acid composition was measured in the F₂ population by gas-liquid chromatography. A genetic linkage map of 17 linkage groups (LGs) comprising 80 RFLP and 19 SSR marker loci from this population was used to identify QTL controlling fatty acid composition. Three QTL affecting C18:0 content were identified on LG3, LG11, and LG13, with all alleles for increased C18:0 content inherited from CAS-20. In total, these QTL explained 43.6% of the C18:0 phenotypic variation. Additionally, four candidate genes (two stearate desaturase genes, SAD6 and SAD17, and a FatA and a FatB thioesterase gene) were placed on the QTL map. On the basis of positional information, QTL on LG11 was suggested to be a SAD6 locus. The results presented show that increased C18:0 content in sunflower seed oil is not a simple trait, and the markers flanking these QTL constitute a powerful tool for plant breeding programs.     

Efficient free fatty acid production in Escherichia coli using plant acyl-ACP thioesterases

Microbial biosynthesis of fatty acid-like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Free fatty acids can be produced by introducing an acyl–acyl carrier protein thioesterase gene into Escherichia coli. The presence of the acyl-ACP thioesterase will break the fatty acid elongation cycle and release free fatty acid. Depending on their sequence similarity and substrate specificity, class FatA thioesterase is active on unsaturated acyl-ACPs and class FatB prefers saturated acyl group. Different acyl-ACP thioesterases have different degrees of chain length specificity. Although some of these enzymes have been characterized from a number of sources, information on their ability to produce free fatty acid in microbial cells has not been extensively examined until recently. In this study, we examined the effect of the overexpression of acyl-ACP thioesterase genes from Diploknema butyracea, Gossypium hirsutum, Ricinus communis and Jatropha curcas on free fatty acid production. In particular, we are interested in studying the effect of different acyl-ACP thioesterase on the quantities and compositions of free fatty acid produced by an E. coli strain ML103 carrying these constructs. It is shown that the accumulation of free fatty acid depends on the acyl-ACP thioesterase used. The strain carrying the acyl-ACP thioesterase gene from D. butyracea produced approximately 0.2 g/L of free fatty acid while the strains carrying the acyl-ACP thioesterase genes from R. communis and J. curcas produced the most free fatty acid at a high level of more than 2.0 g/L at 48 h. These two strains accumulated three major straight chain free fatty acids, C14, C16:1 and C16 at levels about 40%, 35% and 20%, respectively.     

Effects of genomic position and copy number of Acyl-ACP thioesterase transgenes on the level of the target fatty acids in Brassica napus L.

The effects of genomic position and copy number of acyl-acyl carrier protein (ACP) thioesterase (TE) transgenes on the major target fatty acid, either lauric acid (C12:0) or palmitic acid (C16:0) depending on the TE, in transgenic Brassica napus seed oil were investigated. Four transgenic parental lines, transformed individually with the bay-TE (Uc FatB1), elm-TE (Ua FatB1), nutmeg-TE (Mf FatB1) and Cuphea-TE (Ch FatB1) transgenes, were crossed with the non-transgenic recipient genotypes '212/86' or 'QO4'. Bay-TE and Cuphea-TE F₁ seeds, which carry half the number of the construct copies compared to the self-pollinated seeds of the transgenic parents, showed significantly lower levels of the target fatty acid. Doubled haploid (DH) lines were developed through microspore culture from F₁ hybrids with the elm-TE or the Cuphea-TE transgenes. DH lines carrying one to five copies of the Cuphea-TE transgene displayed a positive correlation between transgene copy number and the target fatty acid C16:0 level (r = 0.77**). DH lines with elm-TE transgene copies at four different loci showed different C16:0 levels, with one of the loci (E-II) leading to significantly higher C16:0 levels. This study supports the importance of the selection of high transgene copy number and/or the optimum genomic integration site in order to achieve maximum expression levels of the target fatty acid in transgenic oil quality modification.     

Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds.

In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains. We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively. In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths. Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates. Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP. After expression in B. napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0. The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme. These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented.     

Purification and properties of a thioesterase from lactating rat mammary gland which modifies the product specificity of fatty acid synthetase

An acyl coenzyme A hydrolase (thioesterase II) has been purified to near homogeneity from lactating rat mammary gland. The enzyme is a monomer of molecular weight 33,000 and contains a single active site residue. The enzyme is specific for acyl groups, as acyl-CoA thioesters, containing eight or more carbon atoms and can also hydrolyze oxygen esters. Thioesterase II is capable of shifting the product specificity of rat mammary gland fatty acid synthetase from predominately long chain fatty acids (C14, C16, and C18) to mainly medium chain fatty acids (C8, C10, and C12). Thioesterase II can restore the capacity for fatty acid synthesis to fatty acid synthetase in which the thioesterase component (thioesterase I) has been inactivated with phenylmethanesulfonyl fluoride or removed by trypsinization. No evidence was found of significant levels of thioesterase II in lactating rat liver. The presence of thioesterase II in the lactating mammary gland and the ability of the enzyme to hydrolyze acyl-fatty acid synthetase thioesters of intermediate chain length, are indicative of a major role for this enzyme in the synthesis of the medium chain fatty acids characteristic of milk fat.     

Redirection of metabolic flux for high levels of omega‐7 monounsaturated fatty acid accumulation in camelina seeds

Seed oils enriched in omega‐7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis‐vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. We demonstrate a strategy for enhanced production of omega‐7 monounsaturated fatty acids in camelina (Camelina sativa) and soybean (Glycine max) that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)‐acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)‐acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed‐specific co‐expression of a mutant ?9‐acyl‐ACP and an acyl‐CoA desaturase with high specificity for 16:0‐ACP and CoA substrates, respectively. This strategy was most effective in camelina where seed oils with ~17% omega‐7 monounsaturated fatty acids were obtained. Further increases in omega‐7 fatty acid accumulation to 60–65% of the total fatty acids in camelina seeds were achieved by inclusion of seed‐specific suppression of 3‐keto‐acyl‐ACP synthase II and the FatB 16:0‐ACP thioesterase genes to increase substrate pool sizes of 16:0‐ACP for the ?9‐acyl‐ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications.     

The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L.

The composition and positional distribution of lipids in developing and mature transgenic Brassica napus seeds accumulating up to 7 mol% of caprylate (8:0), 29 mol% caprate (10:0) or 63 mol% of laurate (12:0) were examined. The accumulation of 8:0 and 10:0 resulted from over-expression of the medium-chain-specific thioesterase (Ch FatB2) alone or together with the respective chain-length-specific condensing enzyme (Ch KASIV). Seeds containing high levels of 12:0 were obtained from plants expressing bay thioesterase (BTE) alone or crossed with a line over-expressing the coconut lysophosphatidic acid acyltransferase (LPAAT), an enzyme responsible for the increase in acylation of 12:0 at the sn-2 position. In all instances, 10:0 and 12:0 fatty acids were present in substantial amounts in phosphatidylcholine during seed development with a drastic decrease of 80–90% in mature seeds. At all stages of seed development however, 8:0 was barely detectable in this membrane lipid. Altogether, these results indicate that these transgenic seeds exclude and/or remove the medium-chain fatty acids from their membrane and that this mechanism(s) is more effective with the shorter-chain fatty acids. Furthermore, seeds of 8:0- and 10:0-producing lines had only negligible levels of these fatty acids present in the sn-2 position of the triacylglycerols. In contrast, all 12:0-producing seeds had a substantial amount of this fatty acid in the sn-2 position of the triacylglycerols, suggesting that the endogenous LPAAT is able to acylate 12:0 if no other acyl-CoA species are available. Received: 11 February 2000 / Accepted: 2 May 2000     

Acyl-CoA Synthetase in Maturing Safflower Seeds

Acyl-CoA synthetase in maturing seeds of safflower (Carthamustinctorius) was membranebound, and the highest specific activitywas associated with microsomes. Activity absolutely dependedon the concentrations of fatty acid, CoA, ATP and Mg²⁺. Theapparent K_m values were 4.2 µM for oleate, 24 µMfor CoA, and 250 µM for ATP. The optimum pH of the reactionwas 7.5. Triacsin C, a potent inhibitor of the animal and bacterialacyl-CoA synthetase, was ineffective for the safflower enzyme.The enzyme utilized C₁₆ and C₁₈ long-chain fatty acids preferentially,while medium-chain and very-long-chain fatty acids were poorsubstrates. The order of specificity for native fatty acidswas linoleate > oleate=palmitate > stearate. Althoughactivity per seed varied during seed maturation, it was enoughto account for the rate of triacylglycerol synthesis in vivo. (Received February 2, 1993; Accepted March 3, 1993)     

Cuphea wrightii thioesterases have unexpected broad specificities on saturated fatty acids

Cuphea wrightii A. Gray is an herbaceous annual that accumulates 30% caprate (10:0) and 54% laurate (12:0) in seed storage lipids. We investigated the role of acyl-acyl carrier protein (ACP) thioesterases (TE) in acyl chain-length regulation in C. wrightii. Two embryo-derived cDNAs, encoding the TEs Cw FatB1 and Cw FatB2, were isolated. Both proteins were detected in developing embryos and mature seeds but not in other tissues, suggesting involvement in seed oil synthesis. Although expected to be 10:0/12:0-ACP-specific, these genes produced a broad range of fatty acids (12:0, 14:0, and 16:0) in transgenic Arabidopsis with the greatest accumulation at 14:0. Cw FatB2 transformants also accumulated small amounts of 10:0. Because C. wrightii accumulates only ca. 5% 14:0 and ca. 2% 16:0, we tested the possibility that gene dosage effects might significantly alter the overall kinetics of the pathway. Phenotypic comparisons of progeny segregating for the transgenes individually and in a hybrid population demonstrated that increased enzyme pools in vivo had a minor effect on diverting fatty acid production to shorter chains. We propose that Cw FatB1 and Cw FatB2 may be necessary but not sufficient determinants of the C. wrightii phenotype.