Molecular cloning and nucleotide sequence of a gene encoding a cotton palmitoyl-acyl carrier protein thioesterase.

A cotton genomic clone containing a 17.4-kb DNA segment was found to encompass a palmitoyl-acyl carrier protein (ACP) thioesterase (Fat B1) gene. The gene spans 3.6 kb with six exons and five introns, and is apparently the first plant FatB acyl-ACP thioesterase gene to be completely sequenced. The six exons are identical in nucleotide sequence to the open reading frame of the corresponding cDNA, and would encode a preprotein of 413 amino acids. The preprotein can clearly be identified as a FatB acyl-ACP thioesterase from its similarity to the deduced amino acid sequences of other FatB thioesterase preproteins. A 5'-flanking region of 914 bp was sequenced, with the potential TATA basal promoter 324 bp upstream from the ATG initiation codon. The 5'-flanking sequence also has a putative CAAT box and two presumptive basic region helixloop-helix (bHLH) elements with the consensus motif CANNTG (termed an E box), implicated as being a positive regulatory element in seed-specific gene expression.     

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.     

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.     

Deposition of stearate-oleate rich seed fat in Mangifera indica is mediated by a FatA type acyl-ACP thioesterase

Although the mechanism of accumulation of C8-C16 saturated fatty acids in seed oils has been well-studied, the control of stearic (C18:0) acid deposition in high stearate seed fat is still unclear. We investigated the mechanism that regulates high level of stearate and oleate (C18:1) accumulation in mango (Mangifera indica) seeds during its development, and examined the seed plastid extracts for induction of any specialized fatty acyl-ACP thioesterase (Fat) that may control this high level of deposition. Though the specificity of the Fat enzymes does not account directly for the fatty acid composition of mango seeds, our result suggested that an induced synthesis of a FatA type of thioesterase could be responsible for the high content of oleate and stearate in its seed fat. The major thioesterase from developing seed kernel was purified to near homogeneity, and characterized as a heat-labile, dimeric, neutral protein with relative substrate specificity of 100:35:1.8 towards oleoyl-, stearoyl- and palmitoyl-ACP, respectively. This enzyme was confirmed as Mi FatA by mass spectrometric analysis. Additionally, a heat-stable FatB type enzyme (Mi FatB) was also partially purified, with relative substrate specificity for the same substrates as 9:8.5:100, respectively. Mi FatA is an enzyme of great biotechnological interest because of its involvement in the regulation of stearate rich seed fat in mango.     

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.     

Purification of a jojoba embryo wax synthase, cloning of its cDNA, and production of high levels of wax in seeds of transgenic arabidopsis

Wax synthase (WS, fatty acyl-coenzyme A [coA]: fatty alcohol acyltransferase) catalyzes the final step in the synthesis of linear esters (waxes) that accumulate in seeds of jojoba (Simmondsia chinensis). We have characterized and partially purified this enzyme from developing jojoba embryos. A protein whose presence correlated with WS activity during chromatographic fractionation was identified and a cDNA encoding that protein was cloned. Seed-specific expression of the cDNA in transgenic Arabidopsis conferred high levels of WS activity on developing embryos from those plants. The WS sequence has significant homology with several Arabidopsis open reading frames of unknown function. Wax production in jojoba requires, in addition to WS, a fatty acyl-CoA reductase (FAR) and an efficient fatty acid elongase system that forms the substrates preferred by the FAR. We have expressed the jojoba WS cDNA in Arabidopsis in combination with cDNAs encoding the jojoba FAR and a beta-ketoacyl-CoA synthase (a component of fatty acid elongase) from Lunaria annua. (13)C-Nuclear magnetic resonance analysis of pooled whole seeds from transgenic plants indicated that as many as 49% of the oil molecules in the seeds were waxes. Gas chromatography analysis of transmethylated oil from individual seeds suggested that wax levels may represent up to 70% (by weight) of the oil present in those seeds.     

Isolation and Characterization of a cDNA Encoded an Embryogenic Cell Protein 63 Related to Embryogenesis from Carrot

cDNA library with about 6.0 x 108 plaques per μg phage from carrot ( Daucus carota L. cv. Hammaki) torpedo-shaped embryos was constructed by a eDNA cloning system (λgtl0). A fulllength cDNA for embryogenic cell protein 63 (ECP63), an embryogenic cell protein from carrot with a relative molecular weight of 63 000, was isolated from a eDNA library using PCR-amplified DNA as a probe. The nucleotide sequence of ECP63 cDNA was 1 989 bp in length. The cDNA encoded a polypeptide of 569 amino acids, and the calculated molecular weight of this pelypeptide was 62 000. The Northern blot analysis using labeled full-length ECP63 cDNA as a probe showed that the gene for ECP63 was expressed in embryogenic cells, globular, heart-, and torpedo-shaped embryos, but not in seedlings and non-embryogenic cells.     

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.     

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.     

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.     

The cotton GhNHX1 gene encoding a novel putative tonoplast Na(+)/H(+) antiporter plays an important role in salt stress

A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.     

Regulation of lysine catabolism through lysine-ketoglutarate reductase and saccharopine dehydrogenase in Arabidopsis.

In plant and mammalian cells, excess lysine is catabolized by a pathway that is initiated by two enzymes, namely, lysine-ketoglutarate reductase and saccharopine dehydrogenase. In this study, we report the cloning of an Arabidopsis cDNA encoding a bifunctional polypeptide that contains both of these enzyme activities linked to each other. RNA gel blot analysis identified two mRNA bands-a large mRNA containing both lysine-ketoglutarate reductase and saccharopine dehydrogenase sequences and a smaller mRNA containing only the saccharopine dehydrogenase sequence. However, DNA gel blot hybridization using either the lysine-ketoglutarate reductase or the saccharopine dehydrogenase cDNA sequence as a probe suggested that the two mRNA populations apparently are encoded by the same gene. To test whether these two mRNAs are functional, protein extracts from Arabidopsis cells were fractionated by anion exchange chromatography. This fractionation revealed two separate peaks-one containing both coeluted lysine-ketoglutarate reductase and saccharopine dehydrogenase activities and the second containing only saccharopine dehydrogenase activity. RNA gel blot analysis and in situ hybridization showed that the gene encoding lysine-ketoglutarate reductase and saccharopine dehydrogenase is significantly upregulated in floral organs and in embryonic tissues of developing seeds. Our results suggest that lysine catabolism is subject to complex developmental and physiological regulation, which may operate at gene expression as well as post-translational levels.