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Across the globe, heat waves are getting more intense and frequent. Diatoms are a major group of microalgae at the base of the marine food webs and an important source of long chain polyunsaturated fatty acids that are transferred through the food web. The present study investigates the possible impacts of temperature increase on lipid classes and expression of genes encoding enzymes related to lipid metabolism in Phaeodactylum tricornutum. The heat wave exposure caused an increase in the relative amounts of plastidial lipids such as the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulphoquinovosyldiacylglycerol (SQDG) in parallel with a decrease in the neutral lipid fraction, which includes triacylglycerols. In agreement, gene expression analyses revealed an up-regulation of a gene encoding one MGDG synthase and down-regulation of a diacylglycerol acyltransferase (DGAT), a key enzyme in triacylglycerol synthesis. Our results show that heat waves not only negatively impact the abundance of unsaturated fatty acids such as eicosapentaenoic acid (20:5n-3, EPA) and hexadecatrienoic acid (16:3n-4) as observed by the decrease in their relative abundance in MGDG and neutral lipids, respectively, but also induce changes in the relative amounts of the diverse membrane lipids as well as the proportion of membrane/storage lipids. The expression study of key genes indicates that some of the aforementioned alterations are regulated at the transcription level whereas others appear to be post-transcriptional. The changes observed in plastidial lipids are related to negative impacts on the photosynthesis.

Introduction Jasmonic acid (JA) and its precursors are oxylipins derived from α-linolenic acid (αLA) and hexadecatrienoic acid, and regulate seed development. However, their spatial distribution in the developing Glycine max L. (soybean) seeds has not been elucidated. Objective To investigate the distribution of JA-related compounds in the developing soybean seeds using desorption electrospray ionisation-mass spectrometry imaging (DESI-MSI) and liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) analyses. Methods Cryosections of developing seeds were prepared using adhesive films, and subjected to DESI-MSI analysis. Verification of the DESI-MSI ion images were performed using DESI-tandem MSI (MS/MSI), LC-ESI-MS and tandem MS (MS/MS). Results In the DESI-MSI mass spectrum, peaks matching the chemical formulae of αLA, 12-oxo-phytodienoic acid (OPDA), and 3-oxo-2-(2-(Z)-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0) were detected. These compounds were mainly distributed in the seed coat, especially near the hilum. This was consistent with the quantitative results obtained by LC-ESI-MS. While, DESI-MS/MSI and LC-ESI-MS/MS suggested the presence of isomers for OPDA and OPC-8:0. The effect of isomers on the DESI-MSI ion images was small for OPDA, and considerable for OPC-8:0. Conclusion These results demonstrated that free αLA, OPDA, and OPC-8:0 were the abundant JA-related compounds mainly distributed in the seed coat of the developing soybeans. OPDA and OPC-8:0 might exert a biological role in the seed coat. To the best of my knowledge, this is the first report on the accumulation of OPDA and OPC-8:0 in the seed coat. The combination of DESI-MSI and LC-ESI-MS is a useful tool for distribution analysis of JA-related compounds in the developing seeds.

Diatoms are microscopic biorefineries producing value-added molecules, including unique pigments, triglycerides (TAGs) and long-chain polyunsaturated fatty acids (LC-PUFAs), with potential implications in aquaculture feeding and the food or biofuel industries. These molecules are utilized in vivo for energy harvesting from sunlight to drive photosynthesis and as photosynthetic storage products, respectively. In the present paper, we evaluate the effect of narrow-band spectral illumination on carotenoid, LC-PUFAs and TAG contents in the model diatom Phaeodactylum tricornutum. Shorter wavelengths in the blue spectral range resulted in higher production of total fatty acids, namely saturated TAGs. Longer wavelengths in the red spectral range increased the cell’s content in Hexadecatrienoic acid (HTA) and Eicosapentaenoic acid (EPA). Red wavelengths induced higher production of photoprotective carotenoids, namely fucoxanthin. In combination, the results demonstrate how diatom value-added molecule production can be modulated by spectral light control during the growth. How diatoms could use such mechanisms to regulate efficient light absorption and cell buoyancy in the open ocean is discussed.

A cold-tolerant unicellular green alga was isolated from a meltwater stream on King George Island, Antarctica. Morphological, molecular, and biochemical analyses revealed that the isolate belonged to the species Chlorella vulgaris. We tentatively named this algal strain C.vulgaris KNUA007 and investigated its growth and lipid composition. We found that the strain was able to thrive in a wide range of temperatures, from 5 to 30 &deg, C, however, it did not survive at 35 &deg, C. Ultimate analysis confirmed high gross calorific values only at low temperatures (10 &deg, C), with comparable values to land plants for biomass fuel. Gas chromatography/mass spectrometry analysis revealed that the isolate was rich in nutritionally important polyunsaturated fatty acids (PUFAs). The major fatty acid components were hexadecatrienoic acid (C16:3 &omega, 3, 17.31%), linoleic acid (C18:2 &omega, 6, 8.52%), and &alpha, linolenic acid (C18:3 &omega, 3, 43.35%) at 10 &deg, C. The microalga was tolerant to low temperatures, making it an attractive candidate for the production of biochemicals under cold weather conditions. Therefore, this Antarctic microalga may have potential as an alternative to fish and/or plant oils as a source of omega-3 PUFA. The temperature tolerance and composition of C.vulgaris KNUA007 also make the isolate desirable for commercial applications in the pharmaceutical industry.

The stability of the high-oleic characteristic of tobacco (Nicotiana tabacum) transformants obtained by silencing of microsomal Ω-6 desaturase (FAD2) was investigated when grown at low temperature. Results showed that oleic acid content in total leaf lipids, individual leaf lipids and total seed lipids of transformants decreased drastically at low temperature, with a corresponding increase of polyunsaturated fatty acid contents. The decrease of oleate was caused by neither the restoration of FAD2 transcripts nor the disappearance of small interference RNA. A significant increase of hexadecadienoic acid and hexadecatrienoic acid was observed in MGDG (monogalactosyldiacylglycerol) of transformants grown at lower temperature. Hexadecadienoic and hexadecatrienoic acids, typical fatty acids in MGDG, are produced through the activity of Ω-6 desaturase in plastids (FAD6). The significant increase of Hexadecadienoic and hexadecatrienoic acids suggested the involvement of FAD6 and/or other desaturases in the lipid desaturation of transgenic plants under lower temperature, leading to a significant decrease of oleate in plant cells. These results suggested that the activities of plastidic desaturases must be, at least partially, responsible for the instability of the high-oleic characteristic of FAD2-silenced plants.

showed that the main fatty acids were palmitic acid (29%), linolenic acid (26.5%), linoleic acid (23.5%), hexadecatrienoic acid (11%) and hexadecadienoic acid (8.5%). As it is known that culture conditions greatly influence the composition of microalgae, the experiments were designed to optimise the composition of the medium in order to increase Picochlorum sp. growth from OD680nm = 0.53 to OD680nm = 2.2 and lipid accumulation from 163gkg −1 to 190gkg −1 . The highest lipid contents of 570 and 585gkg −1 were achieved under phosphate starvation and sodium carbonate supplementation, respectively. Under these conditions, the fatty acid profile is dominated by mono-unsaturated and polyunsaturated acids, and is therefore suitable for aqua-culture feeding. However, under high salinity, growth and lipid synthesis are inhibited, and the fatty acids are saturate, and the product is therefore suitable for biodiesel. CONCLUSION:Thishighlipidcontentrichinessentialfattyacids,omega-6andomega-3,endorsesthiswildstrainofPicochlorum sp. as a promising feedstock for aqua-culture and human nutrition or for the production of biodiesel. c � 2013 Society of Chemical Industry

A ω3-fatty acid desaturase gene (maw3) which is involved in biosynthesis of n-3 polyunsaturated fatty acids (PUFAs) was previously isolated from Mortierella alpina 1S-4. In this report, we investigated the products of MAW3 catalyzing reaction with endogenous and exogenous fatty acids in the yeast transformant. Two unusual fatty acids de novo synthesized in the yeast transformant expressing maw3 gene were identified as n-4 hexadecadienoic acid (16:2(9cis, 12cis)) and n-1 hexadecatrienoic acid (16:3(9cis, 12cis, 15)) by GC-MS and (1)H NMR analyses. In addition to the desaturation activity at the ω3-position for 18- and 20-carbon PUFAs, MAW3 in the yeast transformant inserted a double bond at Δ12-position of endogenous palmitoleic acid (16:1(9cis)) and further at Δ15-position of the resulting 16:2(9cis, 12cis) to result in the formation of 16:3(9cis, 12cis, 15) leading to a bifunctional Δ12/Δ15-desaturase for 16-carbon fatty acids. Moreover, we evaluated the activity of MAW3 in the yeast transformant under different temperatures. The MAW3 did not have desaturation activities in M.alpina 1S-4 at 28°C but it had in the yeast transformant for various fatty acids. The MAW3 was demonstrated to be a trifunctional Δ12/Δ15/ω3-desaturase, exhibiting Δ12-desaturation for 16:1(9cis), Δ15-desaturation for 16- and 18-carbon fatty acids that had a preexisting cis-double bond at Δ12 position, and ω3-desaturation for 20-carbon fatty acids having that at Δ14-position. It is the first report that the fatty acid desaturase (MAW3) is shown to have Δ12- and Δ15-desaturation activities for a 16-carbon fatty acid, in addition to its major function, ω3-desaturation activity.

Sorgoleone, produced in root hair cells of sorghum (Sorghum bicolor), is likely responsible for much of the allelopathic properties of sorghum root exudates against broadleaf and grass weeds. Previous studies suggest that the biosynthetic pathway of this compound initiates with the synthesis of an unusual 16:3 fatty acid possessing a terminal double bond. The corresponding fatty acyl-CoA serves as a starter unit for polyketide synthases, resulting in the formation of 5-pentadecatrienyl resorcinol. This resorcinolic intermediate is then methylated by an S-adenosylmethionine-dependent O-methyltransferase and subsequently dihydroxylated, yielding the reduced (hydroquinone) form of sorgoleone. To characterize the corresponding enzymes responsible for the biosynthesis of the 16:3 fatty acyl-CoA precursor, we identified and cloned three putative fatty acid desaturases, designated SbDES1, SbDES2, and SbDES3, from an expressed sequence tag (EST) data base prepared from isolated root hairs. Quantitative real-time RT-PCR analyses revealed that these three genes were preferentially expressed in sorghum root hairs where the 16:2 and 16:3 fatty acids were exclusively localized. Heterologous expression of the cDNAs in Saccharomyces cerevisiae revealed that recombinant SbDES2 converted palmitoleic acid (16:1Δ9) to hexadecadienoic acid (16:2Δ9,12), and that recombinant SbDES3 was capable of converting hexadecadienoic acid into hexadecatrienoic acid (16:3Δ9,12,15). Unlike other desaturases reported to date, the double bond introduced by SbDES3 occurred between carbons 15 and 16 resulting in a terminal double bond aliphatic chain. Collectively, the present results strongly suggest that these fatty acid desaturases represent key enzymes involved in the biosynthesis of the allelochemical sorgoleone.

Nitrogen is an essential nutrient for plants because it represents a major constituent of numerous cellular compounds, including proteins, amino acids, nucleic acids and lipids. While N deprivation is known to have severe consequences for primary carbon metabolism, the effect on chloroplast lipid metabolism has not been analysed in higher plants. Nitrogen limitation in Arabidopsis led to a decrease in the chloroplast galactolipid monogalactosyldiacylglycerol (MGDG) and a concomitant increase in digalactosyldiacylglycerol (DGDG), which correlated with an elevated expression of the DGDG synthase genes DGD1 and DGD2. The amounts of triacylglycerol and free fatty acids increased during N deprivation. Furthermore, phytyl esters accumulated containing medium-chain fatty acids (12:0, 14:0) and a large amount of hexadecatrienoic acid (16:3). Fatty acid phytyl esters were localized to chloroplasts, in particular to thylakoids and plastoglobules. Different polyunsaturated acyl groups were found in phytyl esters accumulating in Arabidopsis lipid mutants and in other plants, including 16:3 and 18:3 species. Therefore N deficiency in higher plants results in a co-ordinated breakdown of galactolipids and chlorophyll with deposition of specific fatty acid phytyl esters in thylakoids and plastoglobules of chloroplasts.

We investigated the characteristics of desaturation in Trichoderma sp. AM076. Although 6,9,12-octadecatrienoic acid (18 : 3ω6) was detected when Trichoderma sp. AM076 was cultivated in the presence of 6,9-octadecadienoic acid (18 : 2ω9), the desaturation products of 6,9,12-octadecatrienoic acid (18 : 3ω6) and 6-octadecenoic acid (18 : 1Δ6) were not detected. These results suggest that the double bonds at the Δ6 position of 18 : 3ω6 and 18 : 1Δ6 disturb their Δ15 and Δ9 desaturation, respectively. This fungus also introduced a double bond at the Δ15 position of 9,12-hexadecadienoic acid (16 : 2ω4), thereby yielding a novel C16 polyunsaturated fatty acid (PUFA) identified as 9,12,15-hexadecatrienoic acid (16 : 3ω1). Further investigations revealed that the mutant having enhanced accumulation of linolenic aid (18 : 3ω3) accumulates 16 : 3ω1 as one of the major PUFAs, together with 9,12-octadecadienoic acid (16 : 2ω4), when grown with palmitoleic acid (16 : 1ω7). These results suggest that, in this strain, the reaction that catalyzes the conversion of linoleic acid to linolenic acid, similar to the conversion of 16 : 2ω4 to 16 : 3ω1, is not ω3 desaturation but Δ15 desaturation.

Algal preparations from Acetabularia crenulata were analyzed for their fatty acid composition to establish the suitability of this alga as a model to study fatty acid oxidation and oxylipin biosynthesis. The work was based on two goals. The first goal of this study was to determine the contribution of fatty acids from contaminating bacteria and how this influenced the total fatty acid composition of cell homogenates of A. crenulata collected in the wild as compared to specimens cultured in sterile conditions. The major fatty acids detected for both specimens were palmitic (C16:0), palmitoleic (C16:1n-7), oleic (C18:1n-9), linoleic (C18:2n-6), linolenic (C18:3n-3), and octadecatetraenoic acid (C18:4n-3). Significant amounts of odd-chain fatty acids common to bacteria were not detected in either sample. Furthermore, branched-chain fatty acids, typical bacterial biomarkers, were not detected in either sample. Data suggest that bacteria do not greatly contribute to the total fatty acid pool of A. crenulata. The second goal was to compare the fatty acid composition of cell homogenates with that of isolated chloroplasts. Comparatively speaking palmitoleic and octadecatetraenoic acid were found at significantly lower concentrations in the chloroplast whereas oleic and linolenic acid were found at significantly higher amounts in this organelle. Furthermore, the amount of hexadecatrienoic acid (C16:3), a fatty acid commonly esterified to monogalactosyldiacylglycerol (MGDG; lipid present at high concentrations inside the chloroplasts of algae), was present at very low concentrations in these plastids (0.7%). Typically green algal follow the “prokaryotic pathway” for MGDG biosynthesis where C18:3 is esterified at the sn-1 position of the glycerol backbone and C18:3 or C16:3 at the sn-2 position, making C16:3 a major fatty acid inside chloroplasts. Interestingly, our results suggest that chloroplasts of A. crenulata appear to follow the “eukaryotic pathway” for MGDG biosynthesis where C18:3 is both at the sn-1 and sn-2 position of MGDG. Taking into account the exceptions noted, the fatty acid composition for A. crenulata is similar to that reported for most chlorophytes.

The effects on mixed ruminal cultures of olive oil (OO) and a hexadecatrienoic acid (HA, cis-C 16:6,9,12 ) extracted from the Hawaiian algae Chaetoceros were studied in 24 h-batch fermentation. HA at 0.5, 1 and 2 ml/l linearly decreased CH 4 production by 25, 47 and 97%, respectively, while OO did not affect it. HA at 0.5, 1 and 2 ml/l increased H 2 accumulation by 2-, 2- and 5-fold, respectively. Release of CO 2 was linearly decreased by HA at 0.5, 1 and 2 ml/l by 10, 32 and 48%, respectively, while OO linearly increased it by 9, 2 and 17%, at the same concentrations. Apparently fermented OM, as estimated through the VFA production stoichiometry, was linearly decreased by HA at 0.5, 1 and 2 ml/l by 9, 19 and 42%, respectively, while OO did not affect it. HA decreased acetate molar percentage, increased propionate, and tended to decrease butyrate. OO tended to decrease acetate molar percentage, and increased propionate and butyrate molar percentages. HA at 0.5, 1 and 2 ml/l linearly decreased NH 4 + concentration by 5, 5 and 21%, respectively. HA was a strong inhibitor of methanogenesis, but decreased fermentation and increased H 2 accumulation. Addition of OO increased propionate production and did not seem to inhibit fermentation.

Polyunsaturated fatty acid (PUFA) utilization was investigated in skin fibroblasts cultured from a female patient with an inherited abnormality in lipid metabolism. These deficient human skin fibroblasts (DF) converted 85–95% less [1-14C]linoleic acid (18:2n-6) to arachidonic acid (20:4n-6), 95% less [3-14C]tetracosatetraenoic acid (24:4n-6) to docosapentaenoic acid (22:5n-6), and 95% less [1-14C]-linolenic acid (18:3n-3) and [3-14C]tetracosapentaenoic acid (24:5n-3) to docosahexaenoic acid (22:6n-3) than did normal human skin fibroblasts (NF). The only product formed by the DF cultures from [1-14C]tetradecadienoic acid (14:2n-6) was 18:2n-6. However, they produced 50–90% as much 20:4n-6 as the NF cultures from [1-14C]hexadecatrienoic acid (16:3n-6), [1-14C]γ-linolenic acid (18:3n-6), and [1-14C]dihomo-γ-linolenic acid (20:3n-6), PUFA substrates that contain Δ6 double bonds. DF also contained 80% more 18:2n-6 and 25% less 20:4n-6. These results suggested that DF are deficient in Δ6 desaturation. This was confirmed by Northern blots demonstrating an 81–94% decrease in Δ6-desaturase mRNA content in the DF cultures, whereas the Δ5-desaturase mRNA content was reduced by only 14%. This is the first inherited abnormality in human PUFA metabolism shown to be associated with a Δ6-desaturase deficiency. Furthermore, the finding that the 18- and 24-carbon substrates are equally affected suggests that a single enzyme carries out both Δ6 desaturation reactions in human PUFA metabolism. —Williard, D. E., J. O. Nwankwo, T. L. Kaduce, S. D. Harmon, M. Irons, H. W. Moser, G. V. Raymond, and A. A. Spector. Identification of a fatty acid Δ6-desaturase deficiency in human skin fibroblasts. J. Lipid Res. 2001. 42: 501–508.

Human skin fibroblasts converted [5,6,8,9,11,12,14,15- 3 H]arachidonic acid ([ 3 H]20:4) to eicosatrienoic acid (20:3), but appreciable amounts of radiolabeled 20:3 were not detected in corresponding incubations with [1- 14 C]20:4. This indicates that the main pathway for synthesizing 20:3 from arachidonic acid in the fibroblast involves oxidative removal of the carboxyl group of arachidonic acid. Fibroblasts deficient in long-chain acyl coenzyme A dehydrogenase (LCAD) converted [ 3 H]20:4 to [ 3 H]20:3. However, Zellweger fibroblasts that are deficient in peroxisomal fatty acid oxidation did not, indicating that the oxidative removal of the carboxyl group occurs in the peroxisomes. [ 3 H]Hexadecatrienoic acid (16:3) was the main product that accumulated when [ 3 H]20:4 was incubated with normal, LCAD deficient, and very long-chain acyl coenzyme A dehydrogenase (VLCAD) deficient fibroblasts, but Zellweger fibroblasts did not form this product. Normal fibroblasts converted [ 3 H] 16:3 to radiolabeled 20:3 and arachidonic acid. These findings suggest that some of the 16:3 produced from arachidonic acid by peroxisomal β-oxidation can be recycled and that this recycling process constitutes a novel pathway for the conversion of arachidonic acid to 20:3 in human fibroblasts.

Galactolipids represent the predominant membrane lipid class in plants. In general, galactolipids are restricted to plastids, but during phosphate deficiency, they also accumulate in extraplastidial membranes. Two groups of plants can be distinguished based on the presence of a specific fatty acid, hexadecatrienoic acid (16:3), in chloroplast lipids. Plants that contain galactolipids with 16:3 acids are designated "16:3-plants"; the other group of plants which lack 16:3 contain mostly 18:3 in their galactolipids ("18:3-plants"). The methods in this chapter describe the extraction of membrane lipids from whole leaves, or from subcellular fractions, and their analysis via thin-layer chromatography (TLC) with different staining methods. Furthermore, a protocol for membrane lipid quantification is presented starting with the separation via TLC, transmethylation of the isolated lipids to fatty acid methyl esters, and their quantitative analysis via gas chromatography (GC).

285 Pags., with Figs., Tabls., Bibliogr., Objetivos: 1. Regulación de la expresión de las ω-3 desaturasas durante el desarrollo de la hoja en soja. 2. Localización, organización y regulación de las ω-6 y ω-3 desaturasas plastidiales. 3. El ácido graso hexadecatrienoico (16:3) como precursor de la síntesis de jasmonatos.

High-resolution, natural abundance 13C-NMR spectroscopy was used to analyze the positional distribution of fatty acids in the predominant plant glycoglycerolipids. Fatty acid profiles attributed spectroscopically to the sn-1 and sn-2 positions were in very good agreement with the enzymatically measured distribution using a lipase from Rhizopus arrhizus. The 13C-NMR spectroscopy analysis makes use of small shift differences of the aryl carbonyl carbons. As in other lipids, in glycolipids sn-1-bound acyl carbonyls are also shifted further downfield than the sn-2-bound carbonyls. Furthermore, in both positions the carbonyl shift depends on the distance to the nearest double bond resulting in identical series of incremental sequence shifts to higher fields from saturated to Δ9, Δ7 and Δ6 double bonds, respectively. These deductions were enabled by analysis of galactolipids isolated from plants belonging to the Apiaceae and Boraginaceae, which contain high proportions of all-cis-δ7,10,13-hexadecatrienoic and all-cis-Δ6.9,12,15-octadecatetraenoic acid, respectively, in the sn-2 position.

Hexadecatrienoic acid (C16:3), octadecatetraenoic acid (C18:4), eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6) were purified in two steps by counter-current chromatography (CCC). The selected liquid phases were heptane-acetonitrile-water for the first step and heptane-methanol-water for the second. The upper non-polar phase was kept stationary in the coil of the instrument by a rotational force field. The separation was followed by UV detection at 210 nm and by analysing the eluted fractions by gas chromatography after esterification. The purity of the fatty acids therefore purified was checked by capillary gas chromatograpy. CCC may be an alternative to HPLC for the preparative-scale purification of such compounds.

The effect of increasing NaCl concentrations up to 170 mM on the lipid composition of leaf membranes was studied on rape (Brassica napus L. var. oleifera Metzg) in hydroponic culture for one month. At NaCl concentrations lower than 102 mM, the polar glycerolipid biosynthesis was stimulated. NaCl concentrations higher than 102 mM reduced the contents in total lipids, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phospholipids. In contrast, the neutral lipid contents increased with increasing NaCl concentration. An increase in NaCl induced a decrease in linolenic and hexadecatrienoic acid contents and an increase in linoleic and palmitic acid contents in all lipid groups. Key words: rape, lipids, sodium chloride.

The present study was undertaken in order to characterize then to purify fatty acids from marine phytoplankton. From a crude mixture of fatty acid methyl esters it was possible to isolate by countercurrent chromatography a mixture of four polyunsaturated fatty acid methyl ester identified as being hexadecatrienoic acid methyl ester, octadecatetraenoic acid methyl ester, eicosapentaenoic acid methyl ester and docosahexaenoic acid methyl ester by gas chromatography coupled with mass spectrometry in electron impact and in positive-ion chemical ionization mode. The four polyunsaturated fatty acids are in different ratios in mixtures from the two microorganisms:Skeletonema costatum andIsochrysis galbana.

Caenorhabditis elegans FAT-2 has been characterized as fatty acid Δ12-desaturase able to desaturate C16 and C18 fatty acids. However, in this report we show that when expressed in yeast cells this enzyme can also catalyze Δ15 desaturation. This results in the production of both linoleic acid (ω6 C18:2Δ9,12) and linolenic acid (ω3 C18:3Δ9,12,15) from oleic acid (C18:1Δ9) substrate, and hexadecadienoic acid (ω4 C16:2Δ9,12) and hexadecatrienoic acid (ω1 C16:3Δ9,12,15) from palmitoleic acid (C16:1Δ9) substrate. In addition, this enzyme can also produce C14:2Δ9,12, C15:2Δ9,12, C17:2Δ9,12, and C18:4Δ6,9,12,15 when C14:1Δ9, C15:1Δ9, C17:1Δ9, and C18:3Δ6,9,12 substrates are available in yeast cells. Mass spectrometry analysis of 2,4-dimethyloxazoline modification of fatty acid methyl esters confirms the positions of all newly formed double bonds. These results indicate that when expressed in yeast the C. elegans Δ12-desaturase CeFAT-2 shows a characteristic of a bifunctional Δ12/Δ15-desaturase and has a great deal of elasticity with respect to fatty acid chain length in being able to accept fatty acids ranging from C14 to C18. Interestingly, despite possessing a bifunctional Δ12/Δ15 desaturation activity, phylogenetic analysis suggests that C. elegans Δ12-desaturase CeFAT-2 might have arisen independently from other reported dual Δ12/Δ15-desaturases from fungi and protozoa.

The fatty acid compositions of algae show characteristic patterns accoding to species. This paper is the first to identify and compare the main fatty acids of the two endemic Codium species: C. dimorphum and C. fernandezianum found in Chile. In addition the fatty acids of the introduced species C. fragile were investigated. Palmitic acid was shown to be the main fatty acid, comprising between 28 to 54%, followed by oleic and linolenic acids. We also confirm, for the first time, the presence of hexadecatrienoic acid (C16:3) in all three species, which is suggested to be a characteristic fatty acid for Codium genus, so this can be used in chemotaxonomy for these species around the world

The fatty acid compositions of algae show characteristic patterns accoding to species. This paper is the first to identify and compare the main fatty acids of the two endemic Codium species: C. dimorphum and C. fernandezianum found in Chile. In addition the fatty acids of the introduced species C. fragile were investigated. Palmitic acid was shown to be the main fatty acid, comprising between 28 to 54%, followed by oleic and linolenic acids. We also confirm, for the first time, the presence of hexadecatrienoic acid (C16:3) in all three species, which is suggested to be a characteristic fatty acid for Codium genus, so this can be used in chemotaxonomy for these species around the world.

7,10,13-Hexadecatrienoic acid (16:3) is abundant in many plant species. However, its metabolism through the lipoxygenase pathway is not sufficiently understood. The goal of present work was to investigate the oxygenation of 16:3 by different plant lipoxygenases and to study the occurrence of oxygenated derivatives of 16:3 in plant seedlings. The recombinant maize 9-lipoxygenase specifically converted 16:3 into (7S)-hydroperoxide. Identification of this novel oxylipin was substantiated by data of GC-MS, LC-MS/MS, 1H-NMR, and 2D-COSY as well as by deuterium labeling from [(2)H(6)]16:3. Soybean lipoxygenase 1 produced 91% (11S)-hydroperoxide and 6% racemic 14-hydroperoxide. Recombinant soybean lipoxygenase 2 (specifically oxidizing linoleate into 13-hydroperoxide) lacked any specificity towards 16:3. Lipoxygenase 2 produced 7-, 8-, 10-, 11-, 13-, and 14-hydroperoxides of 16:3, as well as a significant amount of bis-allylic 9-hydroperoxide. Seedlings of several examined plant species possessed free hydroxy derivatives of 16:3 (HHTs), as well as their ethyl esters. Interestingly, HHTs occur not only in "16:3 plants", but also in typical "18:3 plants" like pea and soybean seedlings.

We analysed chloroplast lipids of Nicotiana tabacum var. John William's Broadleaf, cultivated under an increased Pco2 of 700 p.p.m. Glycolipids and phospholipids remain constant under these conditions, whereas the carotenoid content undergoes a quantitative change. The saturation degree of fatty acids increases due to an increase in palmitic acid and decreases in hexadecatrienoic acid and linolenic acid.

The effect of recalcitrant hydrocarbons on the fatty acid profile from leaf, basal corm, and roots of Cyperus laxus plants cultivated in greenhouse phytoremediation systems of soils from aged oil spill-impacted sites containing from 16 to 340 g/Kg total hydrocarbons (THC) was assessed to investigate if this is a C18:3 species and if the hydrocarbon removal during the phytoremediation process has a relationship with the fatty acid profile of this plant. The fatty acid profile was specific to each vegetative organ and was strongly affected by the hydrocarbons level in the impacted sites. Leaf extracts of plants from uncontaminated soil produced palmitic acid (C16), octadecanoic acid (C18:0), unsaturated oleic acids (C18:1-C18:3), and unsaturated eichosanoic (C20:2-C20:3) acids with a noticeable absence of the unsaturated hexadecatrienoic acid (C16:3); this finding demonstrates, for the first time, that C. laxus is a C18:3 plant. In plants from the phytoremediation systems, the total fatty acid contents in the leaf and the corm were negatively affected by the hydrocarbons presence; however, the effect was positive in root. Interestingly, under contaminated conditions, unusual fatty acids such as odd numbered carbons (C15, C17, C21, and C23) and uncommon unsaturated chains (C20:3n6 and C20:4) were produced together with a remarkable quantity of C22:2 and C24:0 chains in the corm and the leaf. These results demonstrate that weathered hydrocarbons may drastically affect the lipidic composition of C. laxus at the fatty acid level, suggesting that this species adjusts the cover lipid composition in its vegetative organs, mainly in roots, in response to the weathered hydrocarbon presence and uptake during the phytoremediation process.

Intermediates of the aldehyde biosynthesis in Thalassiosira rotula are investigated. Use of labeled precursors and cell preparations proves production of 2 E ,4 Z -octadienal ( 1a ) from 6 Z ,9 Z ,12 Z -hexadecatrienoic acid (C16:3 ω − 4) through the lipoxygenase-dependent intermediate (9 S )-9-hydroperoxyhexadeca-6,10,12-trienoic acid. On the contrary, synthesis of 2 E ,4 Z ,7 Z -decatrienal ( 2a ) involves mainly EPA (C20:5 ω − 3) by a 11 R -lipoxygenase, as suggested by identification of chiral 11 R -HEPE (12% e.e.) in the diatom extracts. Consistently with the necessity to have a rapid transport and metabolization of the intermediate hydroperoxides, we show that lipoxygenase and lyase activities are both found in the same subcellular fraction of the microalga.

The free-living soil protozoon Acanthamoeba castellanii synthesizes a range of polyunsaturated fatty acids, the balance of which can be altered by environmental changes. We have isolated and functionally characterized in yeast a microsomal desaturase from A. castellanii, which catalyzes the sequential conversion of C(16) and C(18) Delta9-monounsaturated fatty acids to di- and tri-unsaturated forms. In the case of C(16) substrates, this bifunctional A. castellanii Delta12,Delta15-desaturase generated a highly unusual fatty acid, hexadecatrienoic acid (16:3Delta(9,12,15)(n-1)). The identification of a desaturase, which can catalyze the insertion of a double bond between the terminal two carbons of a fatty acid represents a new addition to desaturase functionality and plasticity. We have also co-expressed in yeast the A. castellanii bifunctional Delta12,Delta15-desaturase with a microsomal Delta6-desaturase, resulting in the synthesis of the highly unsaturated C(16) fatty acid hexadecatetraenoic acid (16:4Delta(6,9,12,15)(n-1)), previously only reported in marine microorganisms. Our work therefore demonstrates the feasibility of the heterologous synthesis of polyunsaturated fatty acids of the n-1 series. The presence of a bifunctional Delta12,Delta15-desaturase in A. castellanii is also considered with reference to the evolution of desaturases and the lineage of this protist.

Diatoms are unicellular plants broadly present in freshwater and marine ecosystems, where they play a primary role in sustaining the marine food chain. In the last 10 years, there has been accumulating evidence that diatoms may have deleterious effects on the hatching success of zooplankton crustaceans such as copepods, thus affecting dynamics of planktonic populations and limiting secondary production. At the molecular level, failure to hatch is ascribed to the presence of a family of inhibitory oxylipins, which we propose to collectively name polyunsaturated short-chain aldehydes (abbreviated here as PUSCAs). Here we describe the origin of PUSCAs produced by the marine diatom Skeletonema costatum via a lipoxygenase-mediated pathways involving non-esterified polyunsaturated fatty acids (PUFA). Experiments with complex lipids proved the pivotal role of chloroplast-derived glycolipids, especially monogalactosyldiacylglycerol (MGDG), in providing hexadecatrienoic acid (C16:3 omega-4), hexadecatetraenoic acid (C16:4 omega-1) and eicosapentaenoic acid (C20:5 omega-3) to the downstream process leading to 2E,4Z-octadienal (C8:2 omega-4), 2E,4Z,7-octatrienal (C8:3 omega-1) and 2E,4Z-heptadienal (C7:2 omega-3), respectively. Under physiological conditions, the hydrolytic process is associated to galactolipid hydrolyzing enzyme capable of removing fatty acids from both sn positions of glycerol.

In contrast to 16:3 plants like rapeseed (Brassica napus), which contain α-linolenic acid (18:3Δ 9,12,15) and hexadecatrienoic acid (16:3Δ 7,10,13) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5Δ 5,8,11,14,17) and a different isomer of hexadecatrienoic acid (16:3Δ 6,9,12). In this report, we describe the characterization of two cDNAs having sequence homology to Δ12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Δ12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) andSynechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1Δ 9), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the ω9-fatty acids 18:1Δ 9 and 16:1Δ 7, in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1Δ 9. Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum(16:3Δ 6,9,12) is of plastidial origin.

Sitosterol was the principal sterol in Arabidopsis thaliana with campesterol being second most abundant. Small amounts of cholesterol, stigmasterol, campestanol, brassicasterol, stigmastanol, fucosterol and isofucosterol were identified by GC-MS. Free sterol made up 88% of the total sterol, but both esters and glycosides were detected. No significant changes in sterol composition were noted in Arabidopsis thaliana or in mutant PM-11 in response to chilling temperatures. Phytyl esters accumulated in mutant PM-11 after two or more days of chilling temperature, but did not accumulate in the wild type under chilling conditions. The fatty acids in these phytyl esters are atypical of the total fatty acids of Arabidopsis and are dominated by short chain, saturated fatty acids and hexadecatrienoic acid (16:3). The accumulation of these phytyl esters would be consistent with chlorophyll degradation, inhibition of late stages in fatty acid biosynthesis and degradation of the chloroplast.

Numerous Δ12-, Δ15- and multifunctional membrane fatty acid desaturases (FADs) have been identified in fungi, revealing great variability in the enzymatic specificities of FADs involved in biosynthesis of polyunsaturated fatty acids (PUFAs). Here, we report gene isolation and characterization of novel Δ12/Δ15- and Δ15-FADs named CpFad2 and CpFad3, respectively, from the opportunistic pathogenic yeast Candida parapsilosis. Overexpression of CpFad3 in Saccharomyces cerevisiae strains supplemented with linoleic acid (Δ9,Δ12-18:2) and hexadecadienoic acid (Δ9,Δ12-16:2) leads to accumulation of Δ15-PUFAs, i.e., α-linolenic acid (Δ9,Δ12,Δ15-18:3) and hexadecatrienoic acid with an unusual terminal double bond (Δ9,Δ12,Δ15-16:3). CpFad2 produces a range of Δ12- and Δ15-PUFAs. The major products of CpFad2 are linoleic and hexadecadienoic acid (Δ9,Δ12-16:2), accompanied by α-linolenic acid and hexadecatrienoic acid (Δ9,Δ12,Δ15-16:3). Using GC/MS analysis of trimethylsilyl derivatives, we identified ricinoleic acid (12-hydroxy-9-octadecenoic acid) as an additional product of CpFad2. These results demonstrate that CpFAD2 is a multifunctional FAD and indicate that detailed analysis of fatty acid derivatives might uncover a range of enzymatic selectivities in other Δ12-FADs from budding yeasts (Ascomycota: Saccharomycotina).

Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways. The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid. Here we describe a new class of mutants, designatedgly1, with reduced levels of hexadecatrienoic acid. Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway. Genetic and biochemical approaches revealed that thegly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway. The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves. Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype. These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast. The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P.

Trienoic fatty acids, namely α-linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by ω-3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid ω-3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid ω-3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.

A new radiolabeled metabolite was released into the extracellular fluid by normal human skin fibroblasts that were labeled with [5,6,8,9,11,12,14,15-3H] arachidonic acid. This product continued to accumulate during a 24 h incubation, and its formation was not saturated at arachidonic acid concentrations up to 15 mumol/L. The compound, identified as hexadecatrienoic acid, was not produced by Zellweger fibroblasts which are deficient in peroxisomal fatty acid beta-oxidation. By contrast, radiolabeled hexadecatrienoic acid was produced by mutant fibroblasts having other peroxisomal defects, including X-linked adrenoleukodystrophy, adult Refsum's disease, and rhizomelic chondrodysplasia punctata. This radiolabeled metabolite also was produced by mutant fibroblasts that cannot oxidize long-chain fatty acids in the mitochondria. These results indicate that hexadecatrienoic acid is synthesized from arachidonic acid by peroxisomal beta-oxidation. The absence of this pathway may account for some of the biochemical and functional abnormalities that occur in Zellweger's syndrome.

Twenty macroalgae belonging to the Chlorophyta, Phaeophyta and Rhodophyta were collected from the Hadramout coast, and samples were subsequently analysed for their lipids. All samples of algae had broadly similar lipid compositions, but with quantitative differences. The predominant saturated fatty acid in all the algae was palmitic acid (16:0). The Phaeophyta and Rhodophyta were, as a rule, richer than the Chlorophyta in eicosatetraenoic (20:4) and eicosapentaenoic (20:5) acids, but poorer in linolenic (18:3) acid. These polyunsaturated fatty acids occurred at high levels in monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) in the Phaeophyta and the Rhodophyta, but not in those of the Chlorophyta where the MGDG and DGDG were rich in linolenic (18:3) and hexadecatrienoic acid (16:3)

Twelve macroalgae belonging to the Chlorophyta, Phaeophyta and Rhodophyta were collected from the Arabian Gulf. Field samples and samples that were first incubated at 5° C and 24° C in the light for 1 week were analysed for lipids and fatty acids. The lipid contents varied according to the macroalga and, within the Chlorophyta and Phaeophyta, some representatives accumulated more lipids at 5° C and others at 24° C. All samples of algae had similar lipid composition with only quantitative differences. The temperature did not have a common effect on the lipid composition of representative algae, although changes in the relative concentration of specific classes were recorded. The Phaeophyta and Rhodophyta were as a rule richer than the Chlorophyta in eicosatetranoic (20:4) and eicosapentaenoic (20:5) but poorer in linolenic (18:3) acids. In most of the algae, incubation at 5° C was associated with lowering the proportion of palmitic acid (16:0) in the total lipids, and, but only in the Phaeophyta and Rhodophyta, increasing the concentration of 20:4 and 20:5. These polyunsaturated fatty acids occurred in high levels in monogalactosyldiacylglycerols (MGDG) and digalactosyldiacylglycerols (DGDG) of the Phaeophyta and Rhodophyta but not the Chlorophyta, the MGDG and DGDG of which were rich in 18:3 and hexadecatrienoic acid (16:3).

Der Effekt von Licht (500 Lux, 20 000 Lux), Temperatur (5°C, 15°C, 25°C] und Stickstoffernahrung (2 mval N, 4 mval N, 16 mval N) auf die Fettsaure-Zusammensetzung von Galaktolipiden aus alten und jungen Rapsblattern (Brassica napus L.) wurde untersucht. Der Linolensauregehalt (18:3) von Lipiden junger Blatter wurde hauptsachlich durch Licht und Temperatur beeinflust. Alte Blatter reagierten dagegen im wesentlichen auf Licht und Stickstoffernahrung. Bei jungen Blattern trat eine signifikante Licht/Temperatur-Interaktion auf. Der hochste Linolensauregehalt (72%) wurde bei 5°C/500 Lux beobachtet. Bei den alten Blattern wiesen die Anderungen im Fettsauremuster auf eine verzogerte Alterung mit zunehmender Stickstoffversorgung hin. Die 16 mval/20 000 Lux-Behandlung bewirkte die hochsten Gehalte an ungesattigten Fettsauren, In Lipiden junger Blatter stiegen Linolensaure und Hexadecatriensaure [16:3) gemeinsam an. Dagegen wurde der Anstieg des Linolensauregehaltes mit zunehmender Stickstoffversorgung durch einen Abfall im Hexadecatriensauregehalt begleitet. The Variability of the Fatty Acid Composition of Galactolipids from Leaves of Oil-Seed Rape as Affected by Environmental Factors The influences of light (500 Lux, 20 000 Lux), temperature (5°C, 15°C, 25°C) and nitrogen-nutrition (2 mval N, 4 mval N, 16 mval N) treatments on the fatty acid composition of galactolipids from old and young leaves of oil-seed rape (Brassica napus) were tested. The linolenic acid (18:3) content of lipids from young leaves mainly was effected by light and temperature. However, older leaves principally reacted on light and nitrogen-nutrition. With young leaves a significant light/temperature interaction was obvious. The highest level of linolenic acid (72%) was observed at 5°C/500 Lux. In relation to older leaves the changes in the fatty acid composition pointed at a retarded senescence by an increased nitrogen supply. The 16 mval N/20 000 Lux treatment resulted in the highest percentages of unsaturated fatty acids. In lipids from young leaves linolenic and hexadecatrienoic (16:3) acids increased together. In older leaves, however, the high level of linolenic acid caused by increased nitrogen-nutrition was accompanied by a decrease of hexadecatrienoic acid.

Summary The positional distribution of palmitic acid in digalactosyl diglycerides from 29 plants selected from algae to flowering plants is not as exclusive as in some phospholipids or triglycerides and varies with the taxonomic position of the plant. The tendency was observed that the lower the position in the plant system the higher the proportion of palmitic acid at C-2. In angiosperms substantial amounts of palmitic acid were found at C-2 in those plants, which contain hexadecatrienoic acid in monogalactosyl diglycerides. Fatty acid pairings were investigated in unhydrogenated compounds by gas-liquid chromatography and mass spectrometry. In all vascular plants 18:3/18:3- and 18:3/16:0-combinations are predominant. More impressively than analysis of overall fatty acid composition, analysis by mass spectrometry demonstrates the great differences in the diglyceride parts of monogalactosyl-, digalactosyl- and sulfoquinovosyl diglycerides.

The fatty acid composition of total lipids was determined in edible portions and seeds of 15 commonly used vegetables from the families Brassicaceae, Apiaceae and Asteraceae. Tabulated data showed that vegetative parts of Brassicaceae (leaves, flowers, sprouts, tubers) contained no erucic acid, 40-60% linolenic acid and more cis-vaccenic (cis-11-octadecenoic) than oleic (cis-9-octadecenoic) acid, while the opposite ratio was observed in seed lipids. Hexadecatrienoic acid (16:3) was present in the vegetative part of most of the vegetables at 0.3-11.8% especially those rich in linolenic acid, but not in seed oils. Only 4 vegetables contained high % of linoleic acid (44-66%) in the edible portion (black salsify, carrot, turnip-rooted parsley, Florence fennel). Seed lipids of the Brassicaceae contained 31-47% erucic acid, and seed oils of the Apiaceae (carrots, parsley, fennel) contained 65-80% petroselenic acid.

Changes in the content of polar lipids and in their fatty acid composition in spinach leaves during storage were studied in order to clarify the mechanism of leaf-yellowing.The total glycolipids, particularly monogalactosyldiglyceride (MGDG) and diga-lactosyldiglyceride (DGDG), decreased markedly during storage at 25°C. The total phospholipids, however, decreased moderately during storage at 25°C, the decrease in phosphatidylglycerol (PG) being the most notable.In the chloroplast lipids (MGDG, DGDG and PG), the relative amounts of hexadecatrienoic acid in MGDG, and palmitoleic acid and linolenic acid in PG decreased during storage at 25°C. Conversely, the relative amount of linoleic acid in MGDG, DGDG and PG increased. The percentage of unsaturated fatty acids in all the polar lipids, however, showed hardly any change.From these results, it is inferred that the degradation of chlorophylls may be caused by the hydroperoxides of free fatty acids which have been formed by the degradation of polar lipids such as MGDG, DGDG and PG.