Rafael Garcés

In vitro oleate desaturase in developing sunflower seeds

Abstract The in vitro oleoyl phosphatidylcholine desaturase (ODS) activity in developing seeds of normal and high oleic sunflower lines has been studied. Lysophosphatidylcholine and sorbitol stimulated the enzyme activity, while mg2+ was an absolute requirement. The microsomal fraction was the main site of ODS localization. The oil bodies fraction also retained some activity which could be removed after sonication. In the seeds of the normal genotype the microsomal ODS showed good activity throughout the period of active lipid synthesis. On the contrary, in the high oleic seeds this activity was lost at the beginning of this period. The microsomal ODS was strongly affected by incubation temperature and showed maximal activity at 20°, suggesting that sunflower seeds could be a good model system to study temperature control of ODS enzyme activity.

Sunflower mutant containing high levels of palmitic acid in high oleic background

A new sunflower mutant, CAS-12, was obtained, which has both high palmitic (≈30%) and high oleic acid contents, and also a substantial amount of palmitoleic acid (≈7%). The mutant was selected after X-ray irradiation of dry seeds of the inbred line BSD-2-423, which had normal palmitic (≈3%) and high oleic (≈88%) acid levels. The increase of palmitic and palmitoleic acids occurred at the expense of the oleic acid content, which decreased to around 55% in respect to the original line. Linoleic acid content is always under 5%. Palmitic and palmitoleic acid levels were similar to those of the high palmitic mutant CAS-5 obtained in a previous programme from a low oleic line isogenic to BSD-2-423 using a similar mutagenic treatment. In that previous programme we also selected three high stearic acid mutants using chemical mutagenic treatment on the same sunflower line (RDF-1-532). We attempted to obtain mutants in other lines but were unsuccessful. The isolation of similar mutants in isogenic parental lines illustrates the importance of the genetic background in the development of specific mutants with an altered seed oil fatty acid composition. The oil of this mutant will increase the range of potential uses of sunflower oil.

Temperature regulation of oleate desaturase in sunflower (Helianthus annuus L.) seeds

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.

Characterization of polar and nonpolar seed lipid classes from highly saturated fatty acid sunflower mutants

The seed lipids from five sunflower mutants, two with high palmitic acid contents, one of them in high oleic background, and three with high stearic acid contents, have been characterized. All lipid classes of these mutant seeds have increased saturated fatty acid content although triacylglycerols had the highest levels. The increase in saturated fatty acids was mainly at the expense of oleic acid while linoleic acid levels remained unchanged. No difference between mutants and standard sunflower lines used as controls was found in minor fatty acids: linolenic, arachidic, and behenic. In the high-palmitic mutants palmitoleic acid (16∶1n−7) and some palmitolinoleic acid (16∶2n−7, 16∶2n−4) also appeared. Phosphatidylinositol, the lipid with the highest palmitic acid content in controls, also had the highest content of palmitic or stearic acids, depending on the mutant type, suggesting that saturated fatty acids are needed for its physiological function. Positional analysis showed that mutant oils have very low content of saturated fatty acids in the sn-2 position of triacylglycerols, between the content of olive oil and cocoa butter.

Genetic analysis of the high oleic acid content in cultivated sunflower (Helianthus annuus L.)

SummarySunflower lines breeding true for very high oleic acid content in their oil (average levels higher than 85%) were crossed with standard sunflower lines with mean oleic acid levels of 30%. Analysis of the oil of F1 seeds indicated dominance for high oleic levels and control of the genotype of the embryo. Segregating generations were obtained selfing heterozygous high oleic BCnF1 plants from several generations of a backcrossing program to incorporate the high oleic character to standard inbred lines and testcrossing these plants to low oleic material. Analysis of F2 and testcrossed seeds showed three kind of segregations, in both F2 and testcrossed populations, with different proportions of low, intermediate and high oleic types. Genetic analysis of these data supported the hypothesis, that the high oleic character is controlled by three dominant complementary genes OL1, OL2 and OL3. Additional data showing F1 seeds with intermediate oleic content and segregations for high oleic in progenies of intermediate types, suggest the presence of major factors modifying high oleic acid content.

Lipid characterization in seeds of a high oleic acid sunflower mutant

Abstract The expression of the ‘high oleic’ 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 mutant seeds triacylglycerols contained up to 90% of oleic acid and less than 0.5% of linoleic acid. The content of oleic acid was 2.5 ± 0.7 mg/g of fresh seed all over the developing period.

Evaluation of high oleic-high stearic sunflower hard stearins for cocoa butter equivalent formulation

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.

Reduced expression of FatA thioesterases in Arabidopsis affects the oil content and fatty acid composition of the seeds.

Acyl–acyl carrier protein (ACP) thioesterases are enzymes that control the termination of intraplastidial fatty acid synthesis by hydrolyzing the acyl–ACP complexes. Among the different thioesterase gene families found in plants, the FatA-type fulfills a fundamental role in the export of the C18 fatty acid moieties that will be used to synthesize most plant glycerolipids. A reverse genomic approach has been used to study the FatA thioesterase in seed oil accumulation by screening different mutant collections of Arabidopsis thaliana for FatA knockouts. Two mutants were identified with T-DNA insertions in the promoter region of each of the two copies of FatA present in the Arabidopsis genome, from which a double FatA Arabidopsis mutant was made. The expression of both forms of FatA thioesterases was reduced in this double mutant (fata1 fata2), as was FatA activity. This decrease did not cause any evident morphological changes in the mutant plants, although the partial reduction of this activity affected the oil content and fatty acid composition of the Arabidopsis seeds. Thus, dry mutant seeds had less triacylglycerol content, while other neutral lipids like diacylglycerols were not affected. Furthermore, the metabolic flow of the different glycerolipid species into seed oil in the developing seeds was reduced at different stages of seed formation in the fata1 fata2 line. This diminished metabolic flow induced increases in the proportion of linolenic and erucic fatty acids in the seed oil, in a similar way as previously reported for the wri1 Arabidopsis mutant that accumulates oil poorly. The similarities between these two mutants and the origin of their phenotype are discussed in function of the results.

Comparative study of ozonized olive oil and ozonized sunflower oil

Neste estudo, oleos de oliva e girassol ozonizados foram comparados quimica e microbilogicamente. Estes oleos foram introduzidos em um reator com gas ozonio borbulhante, em banho-maria a temperatura ambiente, ate solidificacao. O teor de peroxido, de iodo e o grau de acidez foram determinados juntamente com a atividade antimicrobiana. Os efeitos da ozonizacao na composicao dos acidos graxos desses oleos foram analisados usando-se a tecnica de Cromatografia Gas-Liquido. Um aumento nos valores de peroxidacao e de acidez foi observado em ambos os oleos, mas foram maiores no oleo de girassol ozonizado. O teor de iodo obtido no azeite de oliva ozonizado foi zero, enquanto no oleo de girassol ozonizado foi de 8,8 g de iodo per 100 g. A atividade antimicrobiana foi similar para os dois oleos ozonizados, com excecao da Concentracao Minima Bactericida de Pseudomona Aruginosa. A composicao dos acidos graxos nos dois oleos ozonizados mostrou um decrescimo gradual de acidos graxos insaturados (C18:1, C18:2), com o aumento gradual da ozonizacao. 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.

Cloning, characterization and structural model of a FatA-type thioesterase from sunflower seeds (Helianthus annuus L.)

The substrate specificity of acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) determines the fatty acids available for the biosynthesis of storage and membrane lipids in seeds. In order to determine the mechanisms involved in the biosynthesis of fatty acids in sunflower seeds (Helianthus annuus L.), we isolated, cloned and sequenced a cDNA clone of acyl-ACP thioesterase from developing sunflower seeds, HaFatA1. Through the heterologous expression of HaFatA1 in Escherichia coli we have purified and characterized this enzyme, showing that sunflower HaFatA1 cDNA encodes a functional thioesterase with preference for monounsaturated acyl-ACPs. The HaFatA1 thioesterase was most efficient (kcat/Km) 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.