Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier protein thioesterase

We have examined production of mediumchain fatty acids by Brassica napus L. plants transformed with a California bay (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (UcFatB1) cDNA under the control of the constitutive cauliflower mosaic virus 35S promoter. These plants were found to accumulate medium-chain fatty acids in seeds but not in leaves or roots. Assay of thioesterase activity in extracts of leaves indicated that lauroyl-ACP thioesterase activity is comparable to oleoyl-ACP thioesterase (EC 3.1.2.14) activity in transformant leaves. Furthermore, leaf lauroyl-ACP thioesterase activity was in excess of that which produced a significant increase in the amount of laurate (12:0) in seed. Studies in which isolated chloroplasts were ¹⁴C-labelled were used to evaluate whether medium-chain fatty acids were produced in transformed leaves. Up to 34% of the fatty acids synthesized in vitro by isolated chloroplasts were 12:0. These results demonstrate that the normally seed-localized lauroyl-ACP thioesterase can be expressed in active form in leaves, imported into chloroplasts and can access acyl-ACP intermediates of leaf de-novo fatty acid synthesis. The most likely explanation for the lack of accumulation of 12:0 in transformed leaves is its rapid degradation by -oxidation. In support of this hypothesis, isocitrate lyase (EC 4.1.3.1) activity was found to be significantly increased in plants transformed with 35S-UcFatB1.Abbreviations ACP acyl carrier protein - CaMV cauliflower mosaic virus - control Brassica napus cultivar 212/86 - event 8 pCGN3831-212/86-8 - event 11 pCGN3831-212/86-11 - FAS fatty acid synthase - IL isocitrate lyase - KAS -keto-acyl ACP synthase - MS malate synthase - OTE oleoyl-ACP thioesterase - TAG triacylglycerol - UcFatB1 California bay medium-chain acyl-ACP thioesterase We are indebted to Calgene's Brossica-transformation, growth-chamber, greenhouse, and lipid-analysis personnel. Maelor Davies conducted the initial tranformant analysis. We thank Laura Olsen for IL and MS Western blot analysis and advice on IL and MS activity assays. This work was supported in part by a grant from the U.S. Department of Energy (No. DE-FG02-87ER12729). Acknowledgement is made to the Michigan Agricultural Experiment Station for its support of this research.     

Induction,purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus)

The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape. Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides. Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatogr aphy, chromatofocusing and gel filtration. Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity. Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related. An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations. Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification. The native molecule has a Mr of 70 kDa indicating a dimeric structure. The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate. The intact enzyme has an N-terminus blocked to protein sequencing. We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions. The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed.     

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene -glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and -amylase inhibitors.     

cDNA cloning and expression of Brassica napus enoyl-acyl carrier protein reductase in Escherichia coli

The onset of storage lipid biosynthesis during seed development in the oilseed crop Brassica napus (rape seed) coincides with a drastic qualitative and quantitative change in fatty acid composition. During this phase of storage lipid biosynthesis, the enzyme activities of the individual components of the fatty acid synthase system increase rapidly. We describe a rapid and simple purification procedure for the plastidlocalized NADH-dependent enoyl-acyl carrier protein reductase from developing B. napus seed, based on its affinity towards the acyl carrier protein (ACP). The purified protein was N-terminally sequenced and used to raise a potent antibody preparation. Immuno-screening of a seed-specific gt11 cDNA expression library resulted in the isolation of enoyl-ACP reductase cDNA clones. DNA sequence analysis of an apparently full-length cDNA clone revealed that the enoyl-ACP reductase mRNA is translated into a precursor protein with a putative 73 amino acid leader sequence which is removed during the translocation of the protein through the plastid membrane. Expression studies in Escherichia coli demonstrated that the full-length cDNA clone encodes the authentic B. napus NADH-dependent enoyl-ACP reductase. Characterization of the enoyl-ACP reductase genes by Southern blotting shows that the allo-tetraploid B. napus contains two pairs of related enoyl-ACP reductase genes derived from the two distinct genes found in both its ancestors, Brassica oleracea and B. campestris. Northern blot analysis of enoyl-ACP reductase mRNA steady-state levels during seed development suggests that the increase in enzyme activity during the phase of storage lipid accumulation is regulated at the level of gene expression.     

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.     

Acyl-acyl carrier protein thioesterase activity from sunflower (Helianthus annuus L.) seeds

During sunflower (Helianthus annuus L.) seed formation there was an active period of lipid biosynthesis between 12 and 28 days after flowering (DAF). The maximum in-vitro acyl-acyl carrier protein (ACP) thioesterase activities (EC 3.1.2.14) were found at 15 DAF, preceding the largest accumulation of lipid in the seed. Data from the apparent kinetic parameters, V _max and K _m, from seeds of 15 and 30 DAF, showed that changes in acyl-ACP thioesterase activity are not only quantitative, but also qualitative, since, although the preferred substrate was always oleoyl-ACP, the affinity for palmitoyl-ACP decreased, whereas that for stearoyl-ACP increased with seed maturation. Bisubstrate assays carried out at 30 DAF seemed to indicate that the total activity found in mature seeds is due to a single enzyme with 100/75/15 affinity for oleoyl-ACP/stearoyl-ACP/palmitoyl-ACP. In contrast, at 15 DAF, enzymatic data together with partial sequences from cDNAs indicated the presence of at least two enzymes with different properties, a FatA-like thioesterase, with a high affinity for oleoyl-ACP, plus a FatB-like enzyme, with preference for long-chain saturated fatty acids, both being expressed during the active lipid biosynthesis period. Competition assays carried out with CAS-5, a mutant with a higher content of palmitic acid in the seed oil, indicated that a modified FatA-type thioesterase is involved in the mutant phenotype. Received: 17 December 1999 / Accepted: 25 February 2000     

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts

Using the expression vector gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.     

Sequence of an oleocin cDNA from Brassica napus

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 pb and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3 untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.     

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.     

Two related,low-temperature-induced genes from Brassica napus are homologous to the human tumour bbc1 (breast basic conserved) gene

In order to identify genes involved in cold acclimation, we have constructed a cDNA library from Brassica napus (cv. Samouraï) cold-acclimated etiolated seedlings. By differential screening, a cDNA clone named pBnC24 (Brassica napus Cold), corresponding to a new cold-inducible plant gene, was isolated. Northern blot hybridizations using total RNA from acclimated and unacclimated seedlings confirmed that BnC24 represents a cold-regulated gene. In contrast with a number of cold-inducible plant genes, BnC24 does not seem to be responsive to abscisic acid (ABA). In addition, further screening of the cold-acclimated cDNA library using pBnC24 cDNA as a probe, allowed the isolation of a second type of homologous cDNA. Sequence analysis showed that the two BnC24 genes encode basic 24 kDa proteins, which are highly hydrophilic and rich in alanine, lysine and arginine. The nucleotide and deduced amino acid sequences of these clones do not show any homology with other previously described cold-induced plants genes. However they have strong homology with a recently discovered human tumour gene, bbc1 (breast basic conserved), which seems to be highly conserved in eukaryotes.     

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.     

Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.

Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C₄ plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane -helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.     

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.     

Characterization and expression of chitinase and 1,3-β-glucanase genes in cotton

We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3--glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3--glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3--glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3--glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.     

Characterization of two acyl-acyl carrier protein thioesterases from developing Cuphea seeds specific for medium-chain- and oleoyl-acyl carrier protein

Two acyl-acyl carrier protein (ACP) thioesterases were partially purified from developing seeds of Cuphea lanceolata Ait., a plant with decanoic acid-rich triacylglycerols. The two enzymes differ markedly in their substrate specificity. One is specific for medium-chain acyl-ACPs, the other one for oleoyl-ACP. In addition, these enzymes are distinct with regard to molecular weight, pH optimum and sensitivity to salt. The thioesterases could be separated by Mono Q chromatography or gel filtration. The medium-chain acyl-ACP thioesterase and oleoyl-ACP thioesterase were purified from a crude extract 29- and 180-fold, respectively. In Cuphea wrightii A. Gray, which predominantly contains decanoic a nd lauric acid in the seeds, two different thioesterases were also found with a similar substrate specificity as in Cuphea lanceolata.     

Isolation and characterization of two safflower oleoyl-acyl carrier protein thioesterase cDNA clones

Oleoyl-acyl carrier protein (18:1-ACP) thioesterase has been partially purified from developing safflower (Carthamus tinctorius) seeds. Protein species with molecular masses of 34 and 40 kD associated with thioesterase activity were identified and partially sequenced. Analysis of amino-terminal and internal cyanogen bromide peptide sequences revealed no differences in the primary structure of the two species. Amino acid sequence was used to design degenerate oligonucleotides for primers in a polymerase chain reaction (PCR) using safflower embryo cDNA as a template. A 380-base pair PCR product was used to isolate two classes of cDNA clones, designated 2-1 and 5-2, from the embryo cDNA library. Clone 2-1 encodes a 389-amino acid protein including a 60-amino acid transit peptide, and contains all of the protein sequence determined from the 34- and 40-kD proteins. Clone 5-2 encodes a 385-amino acid protein with 80% identity to that encoded by 2-1. Expression of the two safflower cDNA clones in Escherichia coli resulted in a 50- to 100-fold increase in the level of 18:1-ACP thioesterase activity. Both thioesterases are most active on 18:1-ACP; however, the enzyme encoded by 5-2 shows less discrimination against saturated 16- and 18-carbon acyl-ACP substrates.     

Molecular cloning of three cDNAs encoding putative larval cuticle protein expressed differentially after larval ecdysis from the mulberry longicorn beetle, Apriona germari

Three cDNAs encoding putative larval cuticle protein (LCP) were cloned from the mulberry longicorn beetle, Apriona germari. The three cDNA sequences were 309 bp, 396 bp and 408 bp in length, encoding 103, 132 and 136 amino acid residues, respectively. The predicted molecular masses for these LCPs were approximately 9.2 kDa (AgLCP9.2), 12.3 kDa (AgLCP12.3) and 12.6 kDa (AgLCP12.6). Pairwise identity among AgLCP9.2, AgLCP12.3 and AgLCP12.6 were relatively low. Each AgLCP contained a type-specific consensus sequence identifiable in other insect cuticle proteins. The deduced amino acid sequence of AgLCP9.2 is most similar to Bombyx mori LCP18 and those of AgLCP12.3 and AgLCP12.6 are both most similar to B. mori LCP17. Northern blot analysis revealed that the three AgLCPs showed epidermis-specific expression. The expression profile of AgLCPs after larval ecdysis revealed by Northern blot analysis that the high-level mRNA expression of AgLCPs was detected on the first day of larval ecdysis for AgLCP9.2, on the fifth day for AgLCP12.3 and from the first day of larval ecdysis to the fifth day after larval ecdysis for AgLCP12.6, demonstrating that AgLCP mRNAs are differentially expressed in epidermis after larval ecdysis.     

Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences

A cDNA library was derived from the poly(A)⁺ RNA of young tomato leaves. The library was cloned in a gt11 system and screened by synthetic oligonucleotide probes having sequences that match the codes of conserved regions of amino acid sequences of Cu,Zn superoxide dismutase (SOD) proteins from a wide range of eukaryotic organisms. Two cDNAs were isolated, cloned and sequenced. One of the cDNAs, P31, had a full-size open reading frame of 456 bp with a deduced amino acid sequence having an 80% homology with the deduced amino acid sequence of the cytosolic SOD-2 cDNA of maize. The other cDNA, T10 (extended by T1), had a 651 bp open reading frame that revealed, upon computer translation, 90% homology to the amino acid sequence of mature spinach chloroplast SOD. The 5 end of the reading frame seems to code for a putative transit peptide. This work thus suggests for the first time an amino acid sequence for the transit peptide of chloroplast SOD. Northern hybridizations indicated that each of the P31 and T10 clones hybridized to a blotted poly(A)⁺ RNA species. These two species are differentially expressed in the plant organs: e.g., the species having the T10 sequence was detected in the leaves but not in roots, while the one with the P31 sequence was expressed in both leaves and roots. The cDNA clones P31 and T10 were also hybridized to Southern blots of endonuclease fragmented tomato DNA. The clones hybridized to specific fragments and no cross hybridization between the two clones was revealed under stringent hybridization conditions; the hybridization pattern indicated that, most probably, only one locus is coding for each of the two mRNA species.     

Cloning of Brassica napus CTP:phosphocholine cytidylyltransferase cDNAs by complementation in a yeast cct mutant

CTP: phosphocholine cytidylyltransferase is a rate-limiting enzyme in biosynthesis of phosphatidylcholine in plant cells. We have isolated four cDNAs for the cytidylyltransferase from a root cDNA library of Brassica napus by complementation in a yeast cct mutant. The deduced amino-acid sequences of the B. napus enzymes resembled rat and yeast enzymes in the central domain. Although all cytidylyltransferases ever cloned from B. napus and other organisms were predicted to be structurally hydrophilic, the yeast cct mutant transformed with one of the B. napus cDNA clones restored the cytidylyltransferase activity in the microsomal fraction as well as in the soluble fraction. These results are consistent with a recent view that yeast cells contained a machinery for targeting the yeast cytidylyltransferase to membranes, and may indicate that the B. napus enzyme was compatible with the machinery.