Research Interests: Genomics, Phylogenetics, Plant Biology, Gene expression, DNA, and 28 morePhylogeny, Plant Genome Project, Fatty acids, Escherichia coli, Jatropha curcas, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Enzyme, Genes, Protein Sequence Analysis, Genomic DNA, Protein Secondary Structure Prediction, Seeds, Sunflower, Substrate Specificity, Fatty Acid, Condensation, Helianthus, Amino Acid Sequence, Genomes, Recombinant Protein, Heterologous Expression, Recombinant Proteins, Leaves, Planta, Helianthus annuus L, Gene expression profiling, Molecular Sequence Data, and Acyl carrier protein
Main conclusion Two sunflower
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Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a... more
Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. β-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two β-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in th...
Research Interests: Plant Biology and Planta
Solvent fractionation of high oleic-high stearic (HOHS) sunflower oil was studied to determine the best solvent to use (hexane or acetone) in terms of the operational parameters and properties of the final stearins. Acetone fractionation... more
Solvent fractionation of high oleic-high stearic (HOHS) sunflower oil was studied to determine the best solvent to use (hexane or acetone) in terms of the operational parameters and properties of the final stearins. Acetone fractionation on two types of HOHS sunflower oils (N17 and N20) was carried out at temperatures from 5 to 10 °C using micelles with different oil/solvent ratios. Acetone was more suitable than hexane as a solvent for HSHO sunflower oil fractionation because it allowed the oil to be fractionated at higher temperatures and at lower supercooling degrees. Likewise, a sunflower soft stearin obtained by dry fractionation of HOHS sunflower oil was also used to produce high-melting point stearins by acetone or hexane fractionation. The fractionation of these stearins could be performed at higher temperatures and gave higher yields. The combination of dry and solvent fractionation to obtain tailor-made stearins is discussed.
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Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free... more
Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo fatty acid biosynthesis in the plastids of higher plants by hydrolyzing the thioester bond between ACP and the fatty acid synthesized. Free fatty acids are then esterified with coenzyme A prior to being incorporated into the glycerolipids synthesized through the eukaryotic pathway. Acyl-ACP thioesterases belong to the TE14 family of thioester-active enzymes and can be classified as FatAs and FatBs, which differ in their amino acid sequence and substrate specificity. Here, the FatA and FatB thioesterases from Camelina sativa seeds, a crop of interest in plant biotechnology, were cloned, sequenced and characterized. The mature proteins encoded by these genes were characterized biochemically after they were heterologously expressed in Escherichia coli and purified. C. sativa contained three different alleles of both the FatA and FatB genes. These genes were expressed most strongly in expanding tissues in which lipids are very actively synthesized, such as developing seed endosperm. The CsFatA enzyme displayed high catalytic efficiency on oleoyl-ACP and CsFatB acted efficiently on palmitoyl-ACP. The contribution of these two enzymes to the synthesis of C. sativa oil was discussed in the light of these results.
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The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus... more
The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyzes the first step of glycerolipid assembly within the stroma of the chloroplast. In the present study, the sunflower (Helianthus annuus, L.) stromal GPAT was cloned, sequenced and characterized. We identified a single ORF of 1344base pairs that encoded a GPAT sharing strong sequence homology with the plastidial GPAT from Arabidopsis thaliana (ATS1, At1g32200). Gene expression studies showed that the highest transcript levels occurred in green tissues in which chloroplasts are abundant. The corresponding mature protein was heterologously overexpressed in Escherichia coli for purification and biochemical characterization. In vitro assays using radiolabelled acyl-ACPs and glycerol-3-phosphate as substrates revealed a strong preference for oleic versus palmitic acid, and weak activity towards stearic acid. The positional fatty acid composition of relevant chloroplast phospholipids from sunflower leaves did not reflect the in vitro GPAT specificity, suggesting a more complex scenario with mixed substrates at different concentrations, competition with other acyl-ACP consuming enzymatic reactions, etc. In summary, this study has confirmed the affinity of this enzyme which would partly explain the resistance to cold temperatures observed in sunflower plants.
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Fats based on stearic acid could be a healthier alternative to existing oils especially hydrogenated fractions of oils or palm, but only a few non-tropical species produce oils with these characteristics. In this regard, newly developed... more
Fats based on stearic acid could be a healthier alternative to existing oils especially hydrogenated fractions of oils or palm, but only a few non-tropical species produce oils with these characteristics. In this regard, newly developed high stearic oil seed crops could be a future source of fats and hard stocks rich in stearic and oleic fatty acids. These oil crops have been obtained either by breeding and mutagenesis or by suppression of desaturases using RNA interference. The present review depicts the molecular and biochemical bases for the accumulation of stearic acid in sunflower. Moreover, aspects limiting the accumulation of stearate in the seeds of this species are reviewed. This included data obtained from the characterization of genes and enzymes related to fatty acid biosynthesis and triacylglycerol assembly. Future improvements and uses of these oils are also discussed.
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Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 ω3-desaturase showed low activity in contrast to similar expression of microsomal FAD3... more
Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterologous expression in yeast of the Helianthus annuus FAD7 ω3-desaturase showed low activity in contrast to similar expression of microsomal FAD3 ω3-desaturases. However, the removal of the plastidial transit peptide and the incorporation of a KKNL motif to the C-terminus of HaFAD7 increased the activity by 10-fold compared to the native protein. N-terminal fusion of transmembrane-domains from either the yeast microsomal ELO3, (a type III signal anchor domain), or FAE1, an endoplasmic reticulum membrane anchoring domain, resulted in moderate increases in enzyme activity (5- and 7-fold, respectively), suggesting that the first, most hydrophobic transmembrane domain of HaFAD7 is sufficient to direct targeting to, and insertion into, the endoplasmic reticulum membrane. Furthermore, fusing a hemagglutinin (HA) epitope tag upstream of an endogenous C-terminal KEK motif resulted in a significant loss of activity compared to the un-tagged construct, indicating that the endogenous KEK C-terminal di-lysine motif is capable of directing in yeast the ER-retention of this normally plastidial-located protein. Western blotting analysis of constructs with internal HA epitope revealed that in whole cell extracts, with the exception of the one bound to C-terminal, it did not display a reduced level of protein accumulation. Whilst ferredoxin was shown to be required for HaFAD7 activity in yeast, it appears not necessary for protein stability and accumulation of this plastidial desaturase in the endoplasmic reticulum.Metabolic engineering is focus in the production of a huge diversity of polyunsaturated fatty acids with an economical interest. In this study, we characterized factors contributing to improve the activity of sunflower plastidial ω3-desaturase, HaFAD7, expressed in yeast.
Research Interests: Phytochemistry, Protein Stability, Signal Transduction, Protein Targeting, Biological Sciences, and 13 moreSaccharomyces cerevisiae, Omega-3 fatty acids, Endoplasmic Reticulum, CHEMICAL SCIENCES, Sunflower, Western blot, Fatty Acid, Enzyme activity, Helianthus, Amino Acid Sequence, Heterologous Expression, Helianthus Annuus, and Molecular Sequence Data
The 1,3-random-2-random theory was proposed several years ago to explain the fatty acid distribution in vegetable oil triacylglycerols. However, by demonstrating an asymmetry between positions sn-1 and sn-3 in olive oil, cocoa butter,... more
The 1,3-random-2-random theory was proposed several years ago to explain the fatty acid distribution in vegetable oil triacylglycerols. However, by demonstrating an asymmetry between positions sn-1 and sn-3 in olive oil, cocoa butter, sunflower oil, etc., a number of studies have shown that this theory does not hold true for some oils and fatty acids. Accordingly, the distribution of fatty acids in sunflower triacylglycerols has been studied, calculating the alpha coefficient of asymmetry in several combinations of standard linoleic, high-oleic, and high-stearic sunflower oils. The results obtained from the oils of these lines and from single seed oil samples indicate that the asymmetry for saturated fatty acids is greater in high-oleic than in standard linoleic backgrounds. Hence, the distribution of the fatty acids within the triacylglycerol molecule appears to depend not only on the fatty acid under study but also on the other fatty acids in the oil. Thus, it is demonstrated for the first time that certain fatty acids can influence the distribution of other fatty acids within triacylglycerols.
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Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of... more
Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 μmol min−1 mg−1 and 1,011 μmol min−1 mg−1 protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.
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A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted... more
A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation.
Research Interests: Metabolism, Plant Biology, Plant Physiology, Sequence alignment, Escherichia coli, and 27 morePlant, Temperature, Enzyme, Molecular cloning, Storage, Seeds, Specific Activity, Sunflower, Enzyme activity, Seed storage, Lb, Helianthus, Amino Acid Sequence, Base Sequence, Heterologous Expression, Recombinant Proteins, SDS PAGE, Level, Hydrogen-Ion Concentration, Size Exclusion Chromatography, Time Course, Helianthus Annuus, Full Length Movies, Helianthus annuus L, Structural model, Seed storage proteins, and Molecular Sequence Data
Fractionation of fats and oils makes it possible to generate products with specific properties from natural fats that contain a variety of triacylglycerol (TAG) species. High-oleic high-stearic (HOHS) sunflower oils contain high levels of... more
Fractionation of fats and oils makes it possible to generate products with specific properties from natural fats that contain a variety of triacylglycerol (TAG) species. High-oleic high-stearic (HOHS) sunflower oils contain high levels of saturated fatty acids, mainly stearate, on a high-oleic background. Accordingly, HOHS oils could be a source of disaturated TAGs appropriate for cocoa butter equivalent formulations. We examined the kinetics of HOHS oil crystallization, paying special attention to the influence of crystal seeding and temperature on the process and the composition of the final fractions. This oil was fractionated at 18 °C, and seeding increased the amount of disaturated TAGs recovered in the precipitate from 23% to up to 30%. The experimental data collected were fitted using the models of Gompertz and Avrami to study how well these models fitted the data and their utility in predicting the progress of crystallization. At seeding additions above 0.25% there was a change in the crystallization mechanism that improved the process of fractionation. The effect of temperature was also studied, showing important increases in the maximum rates of crystallization when fractionations were carried out at lower temperatures. Finally, the melting profiles of the fractions enriched in saturated fatty acids were studied, showing amounts of solids intermediate between the initial oil and cocoa butter.
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Ozonization of theobroma oil at different applied ozone dosages was carried out with measurement of peroxide index values, oxygen percentage content and fatty acids composition. The comparison of peroxide values with percentage content of... more
Ozonization of theobroma oil at different applied ozone dosages was carried out with measurement of peroxide index values, oxygen percentage content and fatty acids composition. The comparison of peroxide values with percentage content of oxygen at different applied ozone dosages showed good correlation (r=0.9923). Unsaturated fatty acids and triacylglycerols decrease with ozone applied dosage due to ozone reaction with double bonds. Small amounts of oleic acid were consumed with applied ozone dosage at 35 mg/g, which demonstrated that peroxide values and oxygen content were not principally increased by the ozone attack on the double bonds, but other mechanisms could be involved in the reaction system.
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Ozonation of sunflower oils with genetic modification High Oleic and High Oleic-Palmitic (AO and PO respectively) and without modification, High Linoleic (AL) at different applied ozone dosages was carried out with measurement of peroxide... more
Ozonation of sunflower oils with genetic modification High Oleic and High Oleic-Palmitic (AO and PO respectively) and without modification, High Linoleic (AL) at different applied ozone dosages was carried out with measurement of peroxide and acidity indexes values, fatty acids composition, oxygen percentage content and antimicrobial activity. The comparison of peroxides indexes and oxygen content at different applied ozone dosages in each oil showed good correlation (r = 0,99). Higher amount of oleic acid was consumed at higher applied ozone dosage in PO oil than AO oil, which can be related to the increase of acidity index. The antimicrobial activity was better for AL and PO ozonized oils.
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We have obtained a simulation of the final steps of de novo fatty acid biosynthesis in sunflower control line RHA-274. For this simulation, we have used data from the evolution of fatty acids during seed formation and from the biochemical... more
We have obtained a simulation of the final steps of de novo fatty acid biosynthesis in sunflower control line RHA-274. For this simulation, we have used data from the evolution of fatty acids during seed formation and from the biochemical characterization of beta-keto-acyl-ACP synthetase II (FASII), stearoyl-ACP desaturase (SAD) and acyl-ACP thioesterase activities and the program GEPASI (based on the metabolic control-analysis theory). When physiological data from high- and medium-stearic acid mutants seed development were used with this model the predicted changes in SAD and TE were very similar to those actually found in the biochemical characterization of these mutants. However, the model had to be modified when results from high-palmitic mutants, accumulating unusual fatty acids like palmitoleic, asclepic and palmitolinoleic acids, were used. The emerging model, that fits all of our results, predicts the existence of a dynamic channelling between the FASII complex and SAD, that channelling being responsible for the alternative pathway starting with the desaturation of palmitic acid by the stearoyl-ACP desaturase. This channelling is consistent with our previous results. For instance, the determination of SAD activity on sunflower seed crude extracts only rendered oleic acid when the stearic acid used as a substrate was obtained from a KASII assay, but not when the stearic acid came from in vitro synthesis using acyl-ACP synthetase from Escherichia coli. This theoretical approximation will be very useful in predicting the evolution of the system when introducing new or modified activities; similar approximations in other oil-seed crops could be of great interest.
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Two sunflower (Helianthus annuus L.) mutants with high concentrations of saturated fatty acids in their seed oil have been identified and studied extensively. The mutant line CAS-5 has high concentrations of palmitic acid (C16:0) (>25%... more
Two sunflower (Helianthus annuus L.) mutants with high concentrations of saturated fatty acids in their seed oil have been identified and studied extensively. The mutant line CAS-5 has high concentrations of palmitic acid (C16:0) (>25% compared with 7% in standard sunflower seed oil) and low-C18:0 values (3%). CAS-3 is characterized by its high levels of stearic acid (C18:0) (>22% compared with 4% in standard sunflower seed oil) and a low-C16:0 content (5%). CAS-5 also possesses elevated levels of palmitoleic acid (C16:1) (>5%), which is absent in standard sunflower seed oil. The objective of this study was to determine the relationships between the loci controlling the high-C16:0 and the high-C18:0 traits in these mutants. Plants of both mutants were reciprocally crossed. Gas chromatographic analyses of fatty acids from the seed oil of F1, F2, F3 and the BC1F1 to CAS-5 generations indicated that the loci controlling the high-C16:0 trait exerted an epistatic effect over the loci responsible for the high-C18:0 character. As a result, the phenotypic combination containing both the high-C16:0 levels of CAS-5 and the high-C18:0 levels of CAS-3 was not possible. However, phenotypes with a saturated fatty acid content of 44% (34.5% C16:0+9.5% C18:0) were identified in the F3 generation. These are the highest saturated (C16:0 and C18:0) levels reported so far in sunflower seed oil. When F3 C16:0 segregating generations in both a high- and a low-C18:0 background were compared, the high-C16:1 levels were not expressed as expected in the high-C18:0 background (CAS-3 background). In this case, the C16:1 content decreased to values below 1.5%, compared with >5% in a low-C18:0 background. As the stearoyl-ACP desaturase has been reported to catalyze the desaturation from C16:0-ACP to C16:1-ACP, these results suggested that a decrease in its activity was involved in the accumulation of C18:0 in the high-C18:0 mutant CAS-3.
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Ferredoxins are proteins that participate in photosynthesis and in other processes that require reducing equivalents, such as the reduction of nitrogen or fatty acid desaturation. Two classes of ferredoxins have been described in plants:... more
Ferredoxins are proteins that participate in photosynthesis and in other processes that require reducing equivalents, such as the reduction of nitrogen or fatty acid desaturation. Two classes of ferredoxins have been described in plants: light-regulated photosynthetic ferredoxins and heterotrophic ferredoxins whose activity is not influenced by light. Genes encoding the two forms of ferredoxin have been cloned and characterized in developing sunflower cotyledons. Here, these genes were overexpressed in Escherichia coli and they were purified by ion exchange and size exclusion chromatography to study their capacity to supply electrons to two different sunflower desaturases: soluble stearoyl-ACP desaturase from sunflower cotyledons, and membrane bound desaturase FAD7 expressed in yeast. In both cases photosynthetic ferredoxin was the form that promoted the strongest desaturase activity.
Research Interests: Photosynthesis, Plant Biology, Saccharomyces cerevisiae, Plant Physiology, Escherichia coli, and 18 moreGenetic transformation, Nitrogen, HETEROTROPHIC, Soil sciences, Genes, Plant Physiology and Biochemistry, Membrane, Photosynthetic, Sunflower, Fatty Acid, Heterotrophy, Ion Exchange, Helianthus, Oxidation-Reduction, Size Exclusion Chromatography, Helianthus Annuus, Linoleic Acid, and Alpha-linolenic acid
Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length... more
Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of ferredoxins. Using degenerate oligonucleotides designed from known photosynthetic and heterotrophic plant ferredoxin sequences, two full-length ferredoxin cDNAs were cloned from sunflower (Helianthus annuus L.) leaves and developing seeds, HaFd1 and HaFd2, homologous to photosynthetic and non-photosynthetic ferredoxins, respectively. Based on these cDNAs, the respective genomic sequences were obtained and the presence of DNA polymorphisms was investigated. Complete sequencing of the HaFd1 and HaFd2 genes in different lines indicated the presence of two haplotypes for HaFd2 and their alignment showed that sequence polymorphisms are restricted to the 5′-NTR intron. In addition, specific DNA markers for the HaFd1 and HaFd2 genes were developed that enabled the genes to be mapped. Accordingly, the HaFd1 locus maps to linkage group 10 of the public sunflower map, while the HaFd2 locus maps to linkage group 11. Both ferredoxins display different spatial-temporal patterns of expression. While HaFd2 is expressed at similar levels in all tissues tested (leaves, stem, roots, cotyledons and developing seeds), HaFd1 is more strongly expressed in green tissues than in all the other tissues tested. Both photosynthetic- and heterotrophic-ferredoxins are present in sunflower seeds and may contribute to fatty acid desaturation during oil accumulation. Nevertheless, the levels of HaFd2 expression during seed formation are distinct in lines that only varied in the HaFd2 haplotypes they expressed.
Research Interests: Genetics, Technology, Genomics, Polymorphism, Photosynthesis, and 33 moreAgriculture, Molecular Genetics, Gene expression, Biological Sciences, DNA, Phylogeny, Gene Mapping, Sequence alignment, Haplotypes, Linkage, Gene, Genes, Molecular cloning, Gen, Sunflower, Degeneration, Fatty Acid, Linkage Group, Clones, Genome sequence, Helianthus, Amino Acid Sequence, Leaves, PLANT PROTEINS, Genetic Markers, Oxidation-Reduction, Helianthus Annuus, DNA marker, Full Length Movies, Helianthus annuus L, DNA polymorphism, Chloroplasts, and Molecular Sequence Data
D type phospholipases (PLD) are enzymes that hydrolyze the head group of phospholipids to produce phosphatidic acid. This activity is ubiquitous in plant tissues, and has been isolated and characterized from different species and organs.... more
D type phospholipases (PLD) are enzymes that hydrolyze the head group of phospholipids to produce phosphatidic acid. This activity is ubiquitous in plant tissues, and has been isolated and characterized from different species and organs. Several families of these proteins have been described in plants on the basis of their gene sequences (PLD α, β, γ, δ, ζ and ε). They have been shown to be involved in many metabolic events, such as response to abiotic stress, signal transduction, and membrane lipid turnover and degradation. In the present study, PLD activity was measured in the soluble fractions isolated from different organs of this plant. A PLD of α type was cloned from leaf cDNA that was responsible for most of this activity. The gene encoding this 810 aa protein was heterologously expressed in E. coli. This protein was not lethal for the eukaryotic host, although it altered its phospholipid profile. PLDα was purified to almost homogeneity by His-tag affinity chromatography, displaying an optimum pH of 6.5 and strong dependence on the presence of Ca2+ and SDS in the assay medium. The enzyme was active towards phosphatidylcholine, Phosphatidylethanolamine and phosphatidylglycerol. Furthermore, the HaPLDα gene was found to be expressed at high levels in leaf and stem tissues.
Research Interests: Plant Biology, Abiotic Stress, Gene expression, Signal Transduction, Plant Physiology, and 20 moreEscherichia coli, Degradation, Gene Dosage, Affinity chromatography, Pi, Enzyme, Molecular cloning, Sunflower, Phospholipase D, Helianthus, Amino Acid Sequence, Heterologous Expression, PLD, Recombinant Proteins, Pip, PLANT PROTEINS, Phosphatidic acid, Helianthus Annuus, Plant Leaves, and Molecular Sequence Data
CAS-12 is a sunflower mutant with increased levels of palmitic (C16: 0 = 30%) and oleic (C18: 1 = 55%) acids in its seed oil, hence it has a reduced linoleic acid content (C18: 2 < 5%). This study was conducted to determine the... more
CAS-12 is a sunflower mutant with increased levels of palmitic (C16: 0 = 30%) and oleic (C18: 1 = 55%) acids in its seed oil, hence it has a reduced linoleic acid content (C18: 2 < 5%). This study was conducted to determine the inheritance of high C16: 0 content and its relationship with high C18: 1 content in CAS-12. Reciprocal crosses involving CAS-12, CAS-5 (high C16: 0 content), HAOL-9 (high C18: 1 content) and HA-89 (standard fatty acid profile) were made. The F1, F2 and BC1F1 generations were obtained. The genetic control of the high C16: 0 trait in CAS-12 was partially recessive and gametophytic. In all cases, this character segregated in the ratio 19: 38: 7 (low: intermediate: high C16: 0 content) in the F2 generation. These results, together with the lack of segregation for C16: 0 content in crosses between CAS-12 and CAS-5, indicated that the genetic control of the high C16: 0 trait in CAS-12 was similar to that in CAS-5 in being controlled by partially recessive alleles (p1, p2, and p3) at three loci. Crosses between HA-89 and CAS-12, and HAOL-9 and CAS-5 (segregating for C16: 0 and C18: 1) demonstrated that the high C16: 0 and the high C18: 1 traits were independently inherited. However, C18: 1 segregation in these crosses exhibited reversal of dominance. Apparently, the low C18: 1 parental lines carried modifier genes causing the deviation.
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L. A, GARCÍA-ZAPATEIRO, MA DELGADO, JM FRANCO, С. VALENCIA, MV RUIZ-MÉNDEZ, R. GARCES, AND С. GALLEGOS between 10 and 120 °C, are listed in Table 4. As can be observed, increments ¡n estolide kinematic viscosities of around 1000% and 500%... more
L. A, GARCÍA-ZAPATEIRO, MA DELGADO, JM FRANCO, С. VALENCIA, MV RUIZ-MÉNDEZ, R. GARCES, AND С. GALLEGOS between 10 and 120 °C, are listed in Table 4. As can be observed, increments ¡n estolide kinematic viscosities of around 1000% and 500% at 40 ...
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Fatty acid desaturases (FAD) play an important role in plant lipid metabolism and they can be found in several subcellular compartments such as the plastids and endoplasmic reticulum. Lipids are critical components of the cell membrane... more
Fatty acid desaturases (FAD) play an important role in plant lipid metabolism and they can be found in several subcellular compartments such as the plastids and endoplasmic reticulum. Lipids are critical components of the cell membrane and, as a consequence, they are fundamental for the proper growth and development of all living organisms. We have used sequences from the conserved regions of known omega-3-desaturases to design degenerated oligonucleotides and clone a cDNA encoding a plastidial omega-3 desaturase from sunflower (HaFAD7). From its presumed full-length sequence, we predict that Hafad7 encodes a protein of 443 amino acids with a molecular mass of 50.8 kDa, and that it contains a putative chloroplast transit peptide of 51 amino acids. The predicted hydrophobicity of the protein identifies four potential membrane-spanning regions and, according to the TargetP algorithm, the protein should be targeted to the plastid/chloroplast membrane. RT-PCR analysis of its expression shows the transcript is preferentially expressed in photosynthetically active tissues. Heterologous expression of this protein in the unicellular cyanobacterium Synechocystis sp. PCC 6803 confirmed that the protein produced from this cDNA has omega-3 desaturase activity.
Research Interests: Plant Biology, Cyanobacteria, Race, Phylogeny, Growth and development, and 21 moreEndoplasmic Reticulum, Lipid metabolism, Soil sciences, Plant Physiology and Biochemistry, Molecular cloning, Liquid Chromatography / Electrospray Ionization Mass Spectrometry, Sunflower, Degeneration, Fatty Acid, Amino Acid Profile, Molecular Mass, Helianthus, Amino Acid Sequence, Chloroplast, Heterologous Expression, Helianthus Annuus, Full Length Movies, Das, Fas, Molecular Sequence Data, and Cell Membrane
Abstract The sunflower (Helianthus annuus L.) line CAS-4 obtained by mutagenesis has an increased stearic acid (C18: 0) content in its seed oil (130 g kg− 1), which is desired for some food products. The objectives of this study were to... more
Abstract The sunflower (Helianthus annuus L.) line CAS-4 obtained by mutagenesis has an increased stearic acid (C18: 0) content in its seed oil (130 g kg− 1), which is desired for some food products. The objectives of this study were to determine (i) the inheritance of ...
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Ozonized theobroma fat is used as raw material in the manufacture of pessaries and cosmetic creams. Ozonization of theobroma fat with water was carried out at different applied ozone dosages, and the resultant PV, acid value, iodine... more
Ozonized theobroma fat is used as raw material in the manufacture of pessaries and cosmetic creams. Ozonization of theobroma fat with water was carried out at different applied ozone dosages, and the resultant PV, acid value, iodine value, total hydroperoxide content, and FA content were determined. PV and total hydroperoxide content showed a notable increase with applied ozone dosage up to 35.7 mg/g. Acid value varied slightly from 4.1 to 9.9 mg KOH/g, and the iodine value fell to zero. PV and total hydroperoxide content increased slightly with a higher applied ozone dosage. The comparison of total hydroperoxide measurement using ferrous oxidation in xylenol orange assay and traditional iodometric assay for PV determination showed a significant linear correlation. Small amounts of oleic acid were found in ozonized theobroma fat samples with iodine value equaling zero, which demonstrated that iodine value determination is an inexact assay. During ozonization of theobroma fat, an increase in acid value of 18.9-fold with respect to the initial value was observed owing to decomposition of peroxide.
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To further characterize the stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) and the acyl-ACP thioesterase FatB (EC 3.1.2.14) activities from sunflower seeds, we cloned, sequenced and expressed the recombinant genes in... more
To further characterize the stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) and the acyl-ACP thioesterase FatB (EC 3.1.2.14) activities from sunflower seeds, we cloned, sequenced and expressed the recombinant genes in Escherichia coli. We obtained two partially purified proteins, His-SAD and His-FATB, each of about 45 000 Da. The expression of either proteins produced changes in the E. coli fatty acid profile indicating the functionality of the recombinant proteins. While the expression of His-SAD produced an effect similar to that produced by overexpression of the fabA gene, responsible for the fatty acid desaturation in E. coli, the expression of His-FATB gave rise to an unbalance between unsaturated fatty acids and a toxic effect in E. coli.
Research Interests: Analytical Chemistry, Fatty acids, Escherichia coli, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Enzyme, and 12 moreMolecular cloning, Seeds, Sunflower, Fatty Acid, Helianthus, Amino Acid Sequence, Base Sequence, Recombinant Protein, Recombinant Proteins, Helianthus annuus L, Biochemistry and cell biology, and Acyl carrier protein
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... more
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 δ-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.
Research Interests: Protein Structure Prediction, Plant Biology, Gene expression, Phylogeny, Sequence alignment, and 19 moreEscherichia coli, Enzyme, Molecular cloning, Active site, Sunflower, Substrate Specificity, Fatty Acid, Phylogenetic Tree, Protein Conformation, Helianthus, Amino Acid Sequence, Heterologous Expression, Site-directed Mutagenesis, Planta, Helianthus Annuus, Helianthus annuus L, Structural model, Molecular Sequence Data, and Acyl carrier protein
Sunflower genotypes with increased levels of palmitic acid (C16 : 0) in the seed oil could be useful for food and industrial applications. The objective of the present study was to determine the inheritance of the high C16 : 0 content in... more
Sunflower genotypes with increased levels of palmitic acid (C16 : 0) in the seed oil could be useful for food and industrial applications. The objective of the present study was to determine the inheritance of the high C16 : 0 content in the sunflower mutant line CAS-5 (>25% of the total oil fatty acids). This mutant was reciprocally crossed with the lines HA-89 (5.7% C16 : 0) and BSD-2-691 (5.4% C16 : 0), the latter being the parental line from which CAS-5 was isolated. No maternal effect for the C16 : 0 content was observed from the analysis of F1 seeds in any of the crosses. The inheritance study of the C16 : 0 content in F1, F2 and BC1F1 seeds from the crosses of CAS-5 with its parental line BSD-2-691 indicated that the segregation fitted a model of two alleles at one locus with partial dominance for the low content. The analysis of the fatty acid composition in the F2 populations from the crosses with HA-89 revealed a segregation fitting a ratio 19 : 38 : 7 for low (<7.5%), middle (7.5–15%), and high (>25%) C16 : 0 content, respectively. This segregation was explained on the basis of three loci (P1, P2, P3) each having two alleles showing partial dominance for low content. The genotypes with a high C16 : 0 content were homozygous for the recessive allele p1 and for at least one of the other two recessive alleles, p2 or p3. This model was further confirmed with the analysis of the F3 and the BC1F1 generations. It was concluded that both the recessive alleles p2 and p3 were already present in the BSD-2-691 line, the allele p1 being the result of a mutation from P1. This genetic study will facilitate breeding strategies associated with the incorporation of the high C16 : 0 trait into agronomically acceptable sunflower hybrids.
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A sunflower mutant, CAS-3, with about 25% stearic acid (C18:0) in the seed oil was recently isolated after a chemical-mutagen treatment of RDF-1-532 seeds (8% C18:0). To study the inheritance of the high C18:0 content, CAS-3 was... more
A sunflower mutant, CAS-3, with about 25% stearic acid (C18:0) in the seed oil was recently isolated after a chemical-mutagen treatment of RDF-1-532 seeds (8% C18:0). To study the inheritance of the high C18:0 content, CAS-3 was reciprocally crossed to RDF-1–532 and HA-89 (5% C18:0). Significant reciprocal-cross differences were found in one of the two crosses, indicating possible maternal effects. In the CAS-3 and RDF-1–532 crosses, the segregation patterns of the F1, BC1, and F2 populations fitted a one-locus (designated Es1) model with two alleles (Es1, es1) and with partial dominance of low over high C18:0 content. Segregation patterns in the CAS-3 and HA-89 crosses indicated the presence of a second independent locus (designated Es2) with two alleles (Es2, es2), also with partial dominance of low over high C18:0 content. From these results, the proposed genotypes (C18:0 content) of each parent were as follows: CAS-3 (25.0% C18:0) =es1es1es2es2; RDF-1–532 (8.0% C18:0) =Es1Es1es2es2; and HA-89 (4.6% C18:0) =Es1Es1Es2Es2. The relationship between the proposed genotypes and their C18:0 content indicates that the Es1 locus has a greater effect on the C18:0 content than the Es2 locus. Apparently, the mutagenic treatment caused a mutation of Es1 to es1 in RDF-1–532.
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For the first time, an active fatty-acid metabolism is indicated for triacylglycerols (TAG) of developing sunflower (Helianthus annuus L.) seeds. When the developing seeds were transferred to low temperature, the total amount of oleate... more
For the first time, an active fatty-acid metabolism is indicated for triacylglycerols (TAG) of developing sunflower (Helianthus annuus L.) seeds. When the developing seeds were transferred to low temperature, the total amount of oleate found in TAG decreased as that of linoleate increased, while the contents of total lipids and TAG remained unchanged. These results suggest that oleate from TAG was used for desaturation. This occurred first in microsomal TAG, but after a long cold period it was observed mainly in the oil-body fraction. Thesn-2 position of TAG was preferentially enriched in linoleate. Apparently, more linoleate than necesary for the maintenance of membrane fluidity was synthesized at the expense of TAG oleate.
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The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase... more
The effect of temperature on oleate desaturation in developing sunflower (Helianthus annuus L.) seeds has been examined. When seeds from plants grown at low (20/10° C, day/night) temperature were transferred for 24 h to 10° C, an increase in the linoleate/oleate ratio in phosphatidylcholine and triacylglycerol was observed, but not when transfer was to 20 or 30° C. The same effect was observed in triacylglycerol, phosphatidylcholine and phosphatidylethanolamine in the newly synthesized lipids after in-vivo incubation with [1-14C]oleate at 10° C. The microsomal oleoyl phosphatidylcholine desaturase (ODS) activity of the seeds maintained at 10† C was also enhanced. The stimulation was observed after only 3 h in plants grown at high temperature (30/20° C). This effect was inhibited by cycloheximide, implying that the low-temperature stimulation of the ODS activity was caused by the synthesis of new enzyme. As a consequence, seeds from plants grown at low temperature had higher ODS activities and linoleate contents than those grown at high temperature. The microsomal ODS activity of seeds from plants grown at low temperature was dependent on incubation temperature and showed a maximum at 20° C. By contrast, this activity was almost temperature-insensitive in seeds from plants grown at high temperature. These results could explain how temperature regulates the fatty-acid composition in sunflower-seed lipids.
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... Mutation ; Mutagenesis ; High content ; Screening ; Helianthus annuus ; Saturated fatty acid ;Sunflower oil ; Compositae ; Dicotyledones ; Angiospermae ; Spermatophyta ; Genetic improvement ; Oil plant(vegetal) ; Mots-clés français /... more
... Mutation ; Mutagenesis ; High content ; Screening ; Helianthus annuus ; Saturated fatty acid ;Sunflower oil ; Compositae ; Dicotyledones ; Angiospermae ; Spermatophyta ; Genetic improvement ; Oil plant(vegetal) ; Mots-clés français / French Keywords. ...
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A procedure for the digestion of fresh tissue, transmethylation of lipids, and the extraction of fatty acid methyl esters (FAMES) in one step is described. Fresh tissues or isolated lipids are heated with a reagent containing... more
A procedure for the digestion of fresh tissue, transmethylation of lipids, and the extraction of fatty acid methyl esters (FAMES) in one step is described. Fresh tissues or isolated lipids are heated with a reagent containing methanol:heptane:benzene:2,2-dimethoxypropane:H2SO4 (37:36:20:5:2, by vol), methanol:heptane:toluene:2,2-dimethoxypropane:H2SO4 (39:34:20:5:2, by vol), or methanol:heptane:tetrahydrofuran:2,2-dimethoxypropane:H2SO4 (31:42:20:5:2; by vol). At 80°C the simultaneous digestion and lipid transmethylation takes place in a single phase. After cooling at room temperature two phases are formed; the upper one contains the FAMES ready for GLC analysis. The procedure allows the determination of the fatty acid composition of lipids from tissues containing high proportions of triacylglycerols (oil seeds), water (leaves), or both (oil fruits). Due to the complete extraction of the lipids from the tissue, their content can be determined by adding an internal standard to the sample.
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Page 1. Abstract. In-vivo experiments with developing sunflow-er (Helianthus annuus L.) seeds demonstrated that oleate desaturase activity was stimulated by low temperature (10 °C), repressed by high temperature (30 °C) and ...
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The expression of the &#x27;high oleic&#x27; character in a sunflower mutant takes place exclusively in the developing seeds, during the synthesis of reserve lipids. The fatty acid composition of different lipid classes was... more
The expression of the &#x27;high oleic&#x27; character in a sunflower mutant takes place exclusively in the developing seeds, during the synthesis of reserve lipids. The fatty acid composition of different lipid classes was similar in each genotype and depended on growth temperature. In the ...
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In vivo oleate incorporation and desaturation in developing seeds of normal and the high oleic acid mutant of sunflower have been studied. In seeds less than 15 days after flowering (DAF) of both genotypes, incorporation and desaturation... more
In vivo oleate incorporation and desaturation in developing seeds of normal and the high oleic acid mutant of sunflower have been studied. In seeds less than 15 days after flowering (DAF) of both genotypes, incorporation and desaturation was similar and took place mainly in ...
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Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids... more
Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids because of its neutral effect on serum cholesterol lipoproteins. Selection of a very high stearic acid sunflower (Helianthus annuus L.) line (CAS-14), with up to a 37.3% of stearic acid in the seed oil, and the relationship between the expression of this character and the growth temperature are presented. The mutant was selected from the M(2) progeny of 3000 mutagenized seeds (4 mM sodium azide mutagenesis treatment) by analysing the fatty acid composition of half-seed by gas liquid chromatography. In order to genetically fix the mutant character, plants were grown at high day/night temperatures during seed formation. We found that temperatures higher than 30/20 degrees C are required for good expression of the phenotype, the maximum stearic acid content being obtained at 39/24 degrees C. This behaviour is totally opposed to that observed in normal and previously isolated high-stearic acid sunflower lines that contain more stearic acid at low temperature. Thus, a new type of temperature regulation on the stearate desaturation must occur. This line is the sunflower mutant with the highest stearic acid content reported so far.
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Research Interests:
In this study the ozonized olive and sunflower oils are chemical and microbiologically compared. These oils were introduced into a reactor with bubbling ozone gas in a water bath at room temperature until they were solidified. The... more
In this study the ozonized olive and sunflower oils are chemical and microbiologically compared. These oils were introduced into a reactor with bubbling ozone gas in a water bath at room temperature until they were solidified. The peroxide, acidity and iodine values along with antimicrobial activity were determined. Ozonization effects on the fatty acid composition of these oils were analyzed using Gas-Liquid Chromatographic Technique. An increase in peroxidation and acidity values was observed in both oils but they were higher in ozonized sunflower oil. Iodine value was zero in ozonized olive oil whereas in ozonized sunflower was 8.8 g Iodine per 100 g. The antimicrobial activity was similar for both ozonized oils except for Minimum Bactericidal Concentrations of Pseudomona aeruginosa. Composition of fatty acids in both ozonized oils showed gradual decrease in unsaturated fatty acids (C18:1, C18:2) with gradual increase in ozone doses.
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Research Interests:
The triacylglycerol (TAG) composition of oils from new high-saturated sunflower lines has been studied by means of GLC. The TAG profiles have been compared with the TAG reconstruction made after lipase hydrolysis (according to the... more
The triacylglycerol (TAG) composition of oils from new high-saturated sunflower lines has been studied by means of GLC. The TAG profiles have been compared with the TAG reconstruction made after lipase hydrolysis (according to the 2-random 1,3-random theory). New TAG species with asclepic (cis,Delta11-octadecenoic acid, isomer of oleic acid), araquidic, or behenic acids have been synthesized and identified in oils from mutant lines. The TAG molecular species that contain asclepic acid instead of oleic acid have a longer retention time. Because each mutant oil has a specific TAG GLC pattern, this method could be used for a more precise validation of oil type than current fatty acid methyl ester analysis. The comparison of the results obtained by GLC with the reconstruction after pancreatic lipase hydrolysis shows, in general, a good agreement between both methods. However, results shown in this paper show that this is not always the case. TAG species containing two molecules of linoleic acid show a higher presence of palmitic or stearic acid than could be expected from a random distribution. The abundance of SLL increased in proportion to the stearic acid content of the oil, and the amount of TAG species with three unsaturated fatty acids (LLL or OLL) was therefore reduced.