Functional characterization and structural modelling of Helianthus annuus (sunflower) ketoacyl-CoA synthases and their role in seed oil composition.

MAIN CONCLUSION: The enzymes HaKCS1 and HaKCS2 are expressed in sunflower seeds and contribute to elongation of C18 fatty acids, resulting in the C20-C24 fatty acids in sunflower oil. Most plant fatty acids are produced by plastidial soluble fatty acid synthases that produce fatty acids of up to 18 carbon atoms. However, further acyl chain elongations can take place in the endoplasmic reticulum, catalysed by membrane-bound synthases that act on acyl-CoAs. The condensing enzymes of these complexes are the ketoacyl-CoA synthase (KCSs), responsible for the synthesis of very long chain fatty acids (VLCFAs) and their derivatives in plants, these including waxes and cuticle hydrocarbons, as well as fatty aldehydes. Sunflower seeds accumulate oil that contains around 2-3% of VLCFAs and studies of the fatty acid elongase activity in developing sunflower embryos indicate that two different KCS isoforms drive the synthesis of these fatty acids. Here, two cDNAs encoding distinct KCSs were amplified from RNAs extracted from developing sunflower embryos and named HaKCS1 and HaKCS2. These genes are expressed in developing seeds during the period of oil accumulation and they are clear candidates to condition sunflower oil synthesis. These two KCS cDNAs complement a yeast elongase null mutant and when expressed in yeast, they alter the host's fatty acid profile, proving the encoded KCSs are functional. The structure of these enzymes was modelled and their contribution to the presence of VLCFAs in sunflower oil is discussed based on the results obtained.

New insights in the composition of wax and sterol esters in common and mutant sunflower oils revealed by ESI-MS/MS.

Wax esters (WEs) and steryl esters (SEs) are minor components of sunflower oils formed by the esterification of long chain fatty alcohols and sterols to fatty acids. These compounds have similar carbon numbers and polarities making them difficult to separate using conventional chromatographic methods. In this study, electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) allowed the rapid and accurate profiling of WEs and SEs acyl moieties in total ester fractions of common and mutant sunflower oils with different fatty acid profiles. The acyl composition of both WEs and SEs partially reflected that of the oil and the high oleic background displayed the lowest level of crystallisable waxes. ESI-MS/MS complemented by GC-MS analyses revealed that SEs contain 17-30% of previously unreported methylsterol moieties. We demonstrated that these compounds are overlooked by official sterol analytical methods which may have consequences for quality control and authentication of vegetable oils prior to commercialisation.

Temperature effect on triacylglycerol species in seed oil from high stearic sunflower lines with different genetic backgrounds.

BACKGROUND: This study characterized the influence of temperature during grain filling on the saturated fatty acid distribution in triacylglycerol molecules from high stearic sunflower lines with different genetic backgrounds. Two growth chamber experiments were conducted with day/night temperatures of 16/16, 26/16, 26/26 and 32/26 °C. RESULTS: In all genotypes, independently of the genetic background, higher temperatures increased palmitic and oleic acid and reduced linoleic acid concentrations. Increasing night temperature produced an increase in saturated-unsaturated-saturated species, indicating a more symmetrical distribution of saturated fatty acids. The solid fat index was more affected by temperature during grain filling in lines with high linoleic than high oleic background. Higher variations in symmetry among night temperatures were observed in lines with high oleic background, which are more stable in fatty acid composition. CONCLUSION: The effect of temperature on triacylglycerol composition is not completely explained by its effect on fatty acid composition. Thus night temperature affects oil properties via its effects on fatty acid synthesis and on the distribution of fatty acids in the triacylglycerol molecules. © 2016 Society of Chemical Industry.

Effect of solvents on the fractionation of high oleic-high stearic sunflower oil.

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.

Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

MAIN CONCLUSION: Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of ß-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

Acyl-ACP thioesterases from Camelina sativa: cloning, enzymatic characterization and implication in seed oil fatty acid composition.

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.

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.

Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla) nuts: cloning, characterization and their impact on oil composition.

The mechanisms by which macadamia nuts accumulate the unusual palmitoleic and asclepic acyl moieties, which constitute up to 20% of the fatty acids in some varieties, are still unknown. Acyl-acyl carrier protein (ACP) thioesterases (EC 3.1.2.14) are intraplastidial enzymes that terminate the synthesis of fatty acids in plants and that facilitate the export of the acyl moieties to the endoplasmic reticulum where they can be used in the production of glycerolipids. Here, we have investigated the possible role of acyl-ACP thioesterase activity in the composition of macadamia kernel oil. Accordingly, two acyl-ACP thioesterases were cloned from developing macadamia kernels, one of the FatA type and the other of the FatB type. These enzymes were heterologously expressed in Escherichia coli, and the recombinant thioesterases were purified, characterized kinetically and assayed with a variety of substrates, demonstrating the high specificity of macadamia FatA towards 16:1-ACP. Acyl-ACP thioesterase activity was also characterized in crude extracts from two different varieties of macadamia, Cate and Beaumont, which accumulate different amounts of n-7 fatty acids. The impact of acyl-ACP thioesterase activities on the oil composition of these kernels is discussed in the light of these results.

Glycolytic enzymatic activities in developing seeds involved in the differences between standard and low oil content sunflowers (Helianthus annuus L.).

As opposed to other oilseeds, developing sunflower seeds do not accumulate starch initially. They rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Glycolysis is the principal source of carbon skeletons and reducing power for lipid biosynthesis. In this work, glycolytic initial metabolites and enzyme activities from developing seed of two different sunflower lines, of high and low oil content, were compared during storage lipid synthesis. These two lines showed different kinetic lipid accumulation in the developing embryos. Fatty acids levels during the initial and final stage of lipid synthesis were higher in CAS-6 than in ZEN-8. The analysis of the photosynthate and sugars content suggests that, although the hexoses levels were quite similar in both lines, the amount of sucrose produced by the mother plant and available for lipid synthesis was higher in CAS-6. Although, a smaller amount of sucrose is available in the ZEN-8 line, its seeds maintain the levels of intermediate sugars in the initial steps of glycolysis due to an increase in the levels of the invertase, hexokinase and phosphoglucose isomerase activities in ZEN-8, with respect to CAS-6. Also, a readjustment in the final part of this metabolic route took place, with the activities of phosphoglycerate kinase and enolase in CAS-6 being higher, allowing increased synthesis of phosphoenolpiruvate, the intermediate carbon donor for fatty acid synthesis. In addition, recently, it has been shown that Arabidopsis mutants with a lower fat content in their seeds have a higher amount of sucrose. These data together point to these last two enzymatic activities, phosphoglycerate kinase and enolase, as being responsible for the lower fat content in the ZEN-8 line.

Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation.

Acyl-acyl carrier protein (ACP) thioesterases are enzymes that terminate the intraplastidial fatty acid synthesis in plants by hydrolyzing the acyl-ACP intermediates and releasing free fatty acids to be incorporated into glycerolipids. These enzymes are classified in two families, FatA and FatB, which differ in amino acid sequence and substrate specificity. In the present work, both FatA and FatB thioesterases were cloned, sequenced and characterized from castor (Ricinus communis) seeds, a crop of high interest in oleochemistry. Single copies of FatA and FatB were found in castor resulting to be closely related with those of Jatropha curcas. The corresponding mature proteins were heterologously expressed in Escherichia coli for biochemical characterization after purification, resulting in high catalytic efficiency of RcFatA on oleoyl-ACP and palmitoleoyl-ACP and high efficiencies of RcFatB for oleoyl-ACP and palmitoyl-ACP. The expression profile of these genes displayed the highest levels in expanding tissues that typically are very active in lipid biosynthesis such as developing seed endosperm and young expanding leaves. The contribution of these two enzymes to the synthesis of castor oil is discussed.

The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

Characterization and partial purification of acyl-CoA:glycerol 3-phosphate acyltransferase from sunflower (Helianthus annuus L.) developing seeds.

The glycerol 3-phosphate acyltransferase (GPAT, EC 2.3.1.15) from sunflower (Helianthus annuus L.) microsomes has been characterised and partially purified. The in vitro determination of activity was optimized, and the maximum value for GPAT activity identified between 15 and 20 days after flowering. The apparent Michaelis-Menten K(m) for the glycerol 3-phosphate was 354 muM. The preferred substrates were palmitoyl-CoA = linoleoyl-CoA > oleoyl-CoA with the lowest activity using stearoyl-CoA. High solubilisation was achieved using 0.75% Tween80 and the solubilised GPAT was partially purified by ion-exchange chromatography using a Hi-Trap DEAE FF column, followed by gel filtration chromatography using a Superose 12 HR column. The fraction containing the GPAT activity was analysed by SDS-PAGE and contained a major band of 60.1 kDa. Finally, evidence is provided which shows the role of GPAT in the asymmetrical distribution, between positions sn-1 and sn-3, of saturated fatty acids in highly saturated sunflower triacylglycerols. This work provides background information on the sunflower endoplasmic reticulum GPAT which may prove valuable for future modification of oil deposition in this important crop.

Influence of specific fatty acids on the asymmetric distribution of saturated fatty acids in sunflower (Helianthus annuus L.) triacylglycerols.

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 characterization of a high-stearic sunflower mutant displaying a seed stearic acid gradient.

In the seeds of the high-stearic sunflower mutant CAS-14 a gradient of increasing stearic acid exists from the embryo to the terminal extreme of the cotyledon. This gradient modifies the fatty acid composition of the total lipids, triglycerides, and phospholipids, which can best be appreciated in the triglycerides that pass from 16% stearic acid content in the embryo to 37.1% in the other extreme. This increase in the triglycerides occurs principally at the cost of the oleic acid content. The stearic content at position sn-2 of triglycerides is low, rising from 1.3% in the embryo to 3.4%, whereas at positions sn-1 +3 the stearic content is high and augments from 25.2% in the embryo to 41.0% at the other extreme. The molecular species of triglycerides are also modified; the disaturated triglycerides increase from 15.5 to 51.7%. Furthermore, for the first time in sunflower seeds, it is demonstrated that trisaturated triglycerides are present, arising probably due to a modification in the acyltransferase system that synthesizes the triglycerides.

Lipid characterization of seed oils from high-palmitic, low-palmitoleic, and very high-stearic acid sunflower lines.

Information obtained in recent years regarding the enzymes involved in FA synthesis can now be applied to develop novel sunflower lines by incorporating enzymes with specific characteristics into lines with a defined background. We have generated three highly saturated mutant lines in this way and characterized their FA content. The new high-palmitic, low-palmitoleic lines CAS-18 and CAS-25, the latter on a high-oleic background, have been selected from the high-stearic mutant CAS-3 by introducing a deficient stearic acid desaturase in a high-palmitic background from the previously developed mutant lines CAS-5 and CAS-12, respectively. As such, the desaturation of palmitic acid and the synthesis of palmitoleic acid and its derivatives (asclepic and palmitolinoleic acids) were reduced in these high-palmitic lines, increasing the stearic acid content. Likewise, introducing a FA thioesterase from a high-palmitic line (e.g., CAS-5) into the high-stearic CAS-3 increased the stearic acid content from 27 to 32% in the new high-stearic line CAS-31. As previously described in high-palmitic lines, high growth temperatures did not reduce the linoleic acid content of the oil. Furthermore, the FA composition of TAG, DAG, and phospholipids was modified in these lines. Besides a high degree of saturation, the TAG from these new vegetable oils have a low content of saturated FA in the sn-2 position. The alpha asymmetric coefficient obtained also indicates that the saturated FA are asymmetrically distributed within the TAG molecules. Indeed, the disaturated TAG content rose from 31.8 to 48.2%. These values of disaturated TAG are the highest to date in a temperate oilseed.

Oils from improved high stearic acid sunflower seeds.

Seed oils from new recombinant high-stearic sunflower lines (Helianthus annuus L.) have been characterized. These new lines were generated by crossing high stearic acid lines between themselves or by crossing them with standard and high-oleic sunflower lines. Of the novel lines generated, the lines CAS-29 and CAS-30 are on a standard background and contain up to 34.5% of stearic acid. In contrast, CAS-15 and CAS-33 are on a high oleic acid background and contain only 24.9 and 17.4% of stearic acid, respectively. The stearic acid contents of lines CAS-19 and CAS-20 are 10.0 and 21.5%, respectively, and they have only one of the two genes that control the high stearic acid trait. In accordance with their vegetable origin, these lines have a low percentage of stearic acid in the sn-2 position of the TAGs, from 0.6 to 2.1%. The amount of disaturated TAGs increases with the stearic acid content, from 1.8% in the standard line to between 5.1% in CAS-20 and 38.5% in CAS-29. There was also a concomitant reduction in triunsaturated TAGs, which were reduced to levels as low as 8.4% in CAS-29, as opposed to the 67.9% that they constitute in the standard line RHA-274. The asymmetrical distribution of the saturated fatty acids between the sn-1 and sn-3 TAG positions ranges from 0.26 to 0.36, being lower in those lines with higher oleic acid content.

Sequential one-step extraction and analysis of triacylglycerols and fatty acids in plant tissues.

A method for plant tissue digestion and triacylglycerol (TAG) extraction followed by transmethylation of TAGs to produce the fatty acid methyl esters (FAMEs) from small storage tissue samples is presented. The method allows the analysis of both TAGs and FAMEs from the same sample. Several reagent mixtures and different experimental conditions were tested on sliced sunflower seeds. The best results were obtained using a mixture that was 33.3% a solution of NaCl (0.17 M) in methanol and 66.6% heptane by volume. The TAGs in the heptane solution were transmethylated with a mixture containing methanol:toluene:dimethoxypropane:H(4)SO(2) (39:20:5:2, by vol). The method was also tested on other oil seed storage tissue (soybean) and fruit tissues from olive and acorn. In all cases, sunflower, soybean, olive, and acorn, the TAGs and FAMEs composition data obtained by this method were quite similar to data from a standard analysis method. In samples with high protein content, such as soybean and sunflower seeds, the TAG extraction was incomplete. The water content of fruit samples did not interfere with TAG extraction obtained by this method.