Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzym... 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...
Solvent fractionation of high oleic-high stearic (HOHS) sunflower oil was studied to determine th... 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.
Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo ... 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.
The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) c... 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.
Fats based on stearic acid could be a healthier alternative to existing oils especially hydrogena... 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.
Cocoa butter equivalents High oleic-high stearic sunflower oil Shea stearin Isosolid diagram Phas... more Cocoa butter equivalents High oleic-high stearic sunflower oil Shea stearin Isosolid diagram Phase behaviour a b s t r a c t Cocoa butter equivalents (CBEs) are produced from vegetable fats by blending palm mid fraction (PMF) and tropical butters coming from shea, mango kernel or kokum fat. In this regard, high oleic-high stearic (HOHS) sunflower hard stearins from solvent fractionation can be used in CBE production since their compositions and physical properties are similar to those found in the above-mentioned tropical butters. In this work, three sunflower hard stearins (SHS) ranging from 65% to 95% of disaturated triacylglycerols and a shea stearin (used as reference) were blended with PMF to evaluate their potential use in CBEs formulation. Isosolid phase diagrams of mixtures of PMF/SHS showed eutectic formation for SHS 65 and SHS 80, but monotectic behaviour with softening effect for SHS 95. Three CBEs from SHS and shea stearin were formulated according to phase behaviour diagrams and solid fat content data at 25°C. Isosolid phase diagrams of mixtures of these CBEs with cocoa butter showed no eutectic behaviour. Therefore, CBEs elaborated from SHS exhibited full compatibility with cocoa butter.
Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterolog... 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.
The 1,3-random-2-random theory was proposed several years ago to explain the fatty acid distribut... 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.
Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. ... 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.
A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. dev... 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.
Fractionation of fats and oils makes it possible to generate products with specific properties fr... 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.
Ozonization of theobroma oil at different applied ozone dosages was carried out with measurement ... 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.
Ozonation of sunflower oils with genetic modification High Oleic and High Oleic-Palmitic (AO and ... 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.
We have obtained a simulation of the final steps of de novo fatty acid biosynthesis in sunflower ... 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.
Two sunflower (Helianthus annuus L.) mutants with high concentrations of saturated fatty acids in... 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.
Ferredoxins are proteins that participate in photosynthesis and in other processes that require r... 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.
Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of fe... 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.
Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzym... 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...
Solvent fractionation of high oleic-high stearic (HOHS) sunflower oil was studied to determine th... 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.
Acyl-acyl carrier protein (ACP) thioesterases are intraplastidial enzymes that terminate de novo ... 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.
The acyl-[acyl carrier protein]:sn-1-glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) c... 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.
Fats based on stearic acid could be a healthier alternative to existing oils especially hydrogena... 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.
Cocoa butter equivalents High oleic-high stearic sunflower oil Shea stearin Isosolid diagram Phas... more Cocoa butter equivalents High oleic-high stearic sunflower oil Shea stearin Isosolid diagram Phase behaviour a b s t r a c t Cocoa butter equivalents (CBEs) are produced from vegetable fats by blending palm mid fraction (PMF) and tropical butters coming from shea, mango kernel or kokum fat. In this regard, high oleic-high stearic (HOHS) sunflower hard stearins from solvent fractionation can be used in CBE production since their compositions and physical properties are similar to those found in the above-mentioned tropical butters. In this work, three sunflower hard stearins (SHS) ranging from 65% to 95% of disaturated triacylglycerols and a shea stearin (used as reference) were blended with PMF to evaluate their potential use in CBEs formulation. Isosolid phase diagrams of mixtures of PMF/SHS showed eutectic formation for SHS 65 and SHS 80, but monotectic behaviour with softening effect for SHS 95. Three CBEs from SHS and shea stearin were formulated according to phase behaviour diagrams and solid fat content data at 25°C. Isosolid phase diagrams of mixtures of these CBEs with cocoa butter showed no eutectic behaviour. Therefore, CBEs elaborated from SHS exhibited full compatibility with cocoa butter.
Although plant plastidial ω3-desaturases are closely related to microsomal desaturases, heterolog... 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.
The 1,3-random-2-random theory was proposed several years ago to explain the fatty acid distribut... 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.
Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. ... 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.
A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. dev... 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.
Fractionation of fats and oils makes it possible to generate products with specific properties fr... 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.
Ozonization of theobroma oil at different applied ozone dosages was carried out with measurement ... 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.
Ozonation of sunflower oils with genetic modification High Oleic and High Oleic-Palmitic (AO and ... 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.
We have obtained a simulation of the final steps of de novo fatty acid biosynthesis in sunflower ... 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.
Two sunflower (Helianthus annuus L.) mutants with high concentrations of saturated fatty acids in... 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.
Ferredoxins are proteins that participate in photosynthesis and in other processes that require r... 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.
Fatty acid desaturation in plastids and chloroplasts depends on the electron-donor activity of fe... 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.
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