Kent D. Chapman

Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that ∼10–20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1–2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic. Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that ∼10–20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1–2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic. Lipid droplets are recognized by their conserved structural organization, which consists of a hydrophobic matrix of neutral lipid covered by a monolayer of phospholipids and associated proteins (1van Meer G. Caveolin, cholesterol, and lipid droplets? J. Cell Biol. 2001; 152: F29-F34Google Scholar). Although traditionally regarded as a simple repository for stored carbon reserves, emerging evidence suggests that droplets function as dynamic organelles with a central role in cellular lipid metabolism, membrane trafficking, and cell signaling (2Beckman M. Cell biology. Great balls of fat. Science. 2006; 311: 1232-1234Google Scholar). Because lipid droplets can be found in bacteria, yeast, plant, and animal cells, over the years they have acquired a variety of names. Recently, we proposed that this diverse collection of names be replaced with the designation adiposome (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar). Thus, an adiposome is an organelle that is specialized for packaging and distributing lipids in cells. In this nomenclature, the droplet is simply the most visible stage in the complex life cycle of an adiposome. During the past few years, a number of reports have focused on the protein composition of lipid droplets isolated from yeast (4Athenstaedt K. Zweytick D. Jandrositz A. Kohlwein S.D. Daum G. Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae. J. Bacteriol. 1999; 181: 6441-6448Google Scholar), plant (5Katavic V. Agrawal G.K. Hajduch M. Harris S.L. Thelen J.J. Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars. Proteomics. 2006; 6: 4586-4598Google Scholar), and animal (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar, 6Brasaemle D.L. Dolios G. Shapiro L. Wang R. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes. J. Biol. Chem. 2004; 279: 46835-46842Google Scholar, 7Fujimoto Y. Itabe H. Sakai J. Makita M. Noda J. Mori M. Higashi Y. Kojima S. Takano T. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7. Biochim. Biophys. Acta. 2004; 1644: 47-59Google Scholar) cells. A consensus view from these studies is that droplets contain structural proteins, proteins involved in the biosynthesis and breakdown of lipids, and proteins that mediate membrane traffic. Thus, the proteome indicates that droplets are actively engaged in membrane traffic, perhaps for the purpose of maintaining the proper lipid composition of different membrane compartments. In contrast to the proteins, surprisingly little is known about the lipid composition of animal cell droplets. Generally, droplets are rich in neutral lipids such as triacylglycerol (TAG) and cholesteryl esters that have a diverse population of esterified fatty acids (8Murphy D.J. The biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog. Lipid Res. 2001; 40: 325-438Google Scholar). Here, we report an analysis of the lipid composition of droplets purified from various types of cultured and tissue cells. We used a combination of NMR spectroscopy and mass spectrometric approaches [including high-throughput, direct infusion electrospray ionization-tandem mass spectrometry (ESI-MS/MS)] to characterize the neutral lipid and phospholipid composition of isolated droplets. We found that droplets are rich not only in TAG and cholesteryl esters esterified with a variety of different fatty acids but also in the ether neutral lipid monoalk(en)yl diacylglycerol (MADAG). Despite representing only 1–2% of the total lipid in the droplet, the phospholipid composition included diverse molecular species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), ether-linked phosphatidylcholine (ePC), and ether-linked phosphatidylethanolamine (ePE) but very little phosphatidylserine (PS) or sphingomyelin (SM). We identified and quantified >160 phospholipid molecular species, which suggests that the simple phospholipid monolayer surrounding each droplet has an amazingly complex lipid composition. The neutral and phospholipid composition of lipid droplets is consistent with adiposomes having a direct role in lipid metabolism and in the intracellular traffic of membrane lipids. FBS and cosmic calf serum were from Hyclone (Logan, UT). DMEM and oleate were from Sigma (St. Louis, MO). Silica gel TLC plates were from Whatman (Brentford, Middlesex, UK). CHO K2 cells were cultured on 150 mm plates with 25 ml of DMEM (high-glucose; 4.5 g/l) containing 10% cosmic calf serum, 40 μg/ml proline, 100 U/ml penicillin, and 100 μg/ml streptomycin. Immortalized human B lymphocytes were cultured in RPMI 1640 containing 10% FBS as described previously (9Michaely P. Li W.P. Anderson R.G. Cohen J.C. Hobbs H.H. The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding and internalization but not for LDL receptor clustering in coated pits. J. Biol. Chem. 2004; 279: 34023-34031Google Scholar). 3T3-L1 cells, primary human fibroblasts, fibroblasts derived from Zellweger patients (No. GM04340; Coriell Cell Repositories, Camden, NJ), NRK cells, and immortalized human fibroblasts (SV589 cells) were maintained in DMEM (low-glucose; 2.5 g/l) and 10% FBS supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Lipid droplets were induced by incubating confluent cells in the presence of 100 μM oleate for NRK cells or 80 μM oleate for other cells for either 48 h for primary human fibroblasts or 16 h for other cells. 3T3-L1 cells were induced to differentiate by incubating them in the presence of 10 μg/ml insulin, 1 μM dexamethasone, and 200 μM isobutylxanthine (Sigma) for 48 h followed by insulin alone for an additional 4 days with a medium change after 2 days. For mass spectrometry and NMR analysis, lipids were extracted into CHCl3 from droplet fractions or whole cells by a modification of the Bligh and Dyer method (10Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (41848) Google Scholar) in which methanol was replaced with 2-propanol (11Chapman K.D. Moore T.S. N-Acylphosphatidylethanolamine synthesis in plants: occurrence, molecular composition, and phospholipid origin. Arch. Biochem. Biophys. 1993; 301: 21-33Google Scholar). Monophasic extracts were partitioned into two phases, and the organic layer was washed three times with 1 M KCl (12Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar). Total lipid mass was estimated gravimetrically after removing solvent under a gentle stream of N2 gas. Lipids were extracted from lipid droplets of tissue cultured cells and mouse liver or from 20 mg of adipose tissue for TLC analysis of unknown neutral lipid using CHCl3/acetone (1:1, v/v). The solvent was removed by N2 gas, and the lipids were dissolved in CHCl3 and separated on TLC plates in hexane-diethyl ether-acetic acid (80:20:1, v/v) for 40 min. Lipid classes were visualized by iodine vapor or charring and semiquantified by densitometric scanning (NIH ImageJ software). Neutral lipid classes were quantified (and identified) according to TLC standards (Nu-Chek Prep, Inc., Elysian, MN) and separated under the same conditions at different amounts to generate standard curves for each major lipid class. In some cases, total lipid extracts were dissolved in CHCl3 for LC-MS analysis or phospholipid profiling. In some cases, TLC was used to separate neutral lipid classes that were then recovered from silica gel in acidified CHCl3 without exposure of lipids to iodine vapor. A nonpolar lipid fraction migrating between TAGs and cholesteryl esters was isolated by TLC and recovered in CHCl3 as described above. Fractions were examined for ionization by direct infusion using ESI and atmospheric pressure chemical ionization (APCI) in both positive and negative ion modes. The strongest ionization was observed with positive ion APCI, which was then coupled with reverse-phase liquid chromatography. An Agilent (Palo Alto, CA) 1100 LC-MS system with a model SL ion trap mass spectrometer was operated with a Zorbax C18 column (2.1 × 150 mm; 5 μm particle size) and isocractic mobile phase of 1:1 methanol-dichloromethane at 0.3 ml/min. Peaks with major ions corresponding to those seen by direct infusion eluted in <10 min. MS conditions were as follows: nebulizer pressure = 60 p.s.i., dry gas flow = 5 l/min, dry gas temperature = 350°C, vaporization temperature = 425°C, corona current = 4,000 nA, and capillary voltage = 3,500 V. Neutral lipid classes from ∼100 mg of adiposome lipids were fractionated by low-pressure flash chromatography on silica gel G60 (63 to ∼200 μm; EM Science, Gibbstown, NJ) and eluted with hexane-diethyl ether (30:1, v/v). Three major fractions corresponding to cholesteryl esters, ether-neutral lipids, and TAGs were collected, dried under high vacuum, and analyzed by NMR and matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrometry. 1H-NMR and 13C-NMR spectra were acquired on Varian 300 MHz or 400 MHz spectrometers. Chemical shifts (δ; ppm) were reported against tetramethylsilane (0 ppm). MALDI-TOF mass spectroscopy was performed on a Voyager-DE PRO biospectrometry workstation (Applied Biosystems, Foster City, CA) using 2,5-dihydroxy benzoic acid as the matrix. Adiposome lipid extracts (2.5 ml) were chromatographed on a 0.3 g silicic acid column packed in chloroform. The column was eluted with 10 ml of chloroform-methanol (98:2, v/v) to produce a neutral lipid fraction. The solvent was evaporated and the sample dissolved in 0.9 ml of chloroform. A 0.1 ml aliquot was diluted into 1.2 ml of chloroform-methanol-300 mM ammonium acetate in water (300:665:35) and introduced in the electrospray source of an Applied Biosystems API 4000 mass spectrometer as described below in Phospholipid Analysis. Q+ spectra were interpreted as TAGs. Product ion spectra of major TAG and MADAG species were determined using a collision energy of 45 V. An automated electrospray ionization-tandem mass spectrometry approach was used, and data acquisition, analysis, and acyl group identification were carried out as described previously (13Wanjie S.W. Welti R. Moreau R.A. Chapman K.D. Identification and quantification of glycerolipids in cotton fibers: reconciliation with metabolic pathway predictions from DNA databases. Lipids. 2005; 40: 773-785Google Scholar, 14Welti R. Wang X. Williams T.D. Electrospray ionization tandem mass spectrometry scan modes for plant chloroplast lipids. Anal. Biochem. 2003; 314: 149-152Google Scholar) with minor modifications. An aliquot of extract (∼0.4 mg of lipid) was taken for mass spectrometry analysis. The lipid extract was combined with solvents and internal standards, such that the ratio of chloroform-methanol-300 mM ammonium acetate in water was 300:665:35 and the final volume was 1 ml. Internal standards, obtained and quantified as described previously (14Welti R. Wang X. Williams T.D. Electrospray ionization tandem mass spectrometry scan modes for plant chloroplast lipids. Anal. Biochem. 2003; 314: 149-152Google Scholar), were 0.66 nmol of di14:0-PC, 0.66 nmol of di24:1-PC, 0.66 nmol of 13:0-lysoPC, 0.66 nmol of 19:0-lysoPC, 0.36 nmol of di14:0-PE, 0.36 nmol of di24:1-PE, 0.36 nmol of 14:0-lysoPE, 0.36 nmol of 18:0-lysoPE, 0.36 nmol of di14:0-phosphatidic acid (PA), 0.36 nmol of di20:0(phytanoyl)-PA, 0.24 nmol of di14:0-PS, 0.24 nmol of di20:0(phytanoyl)-PS, 0.20 nmol of 16:0-18:0-PI, and 0.16 nmol of di18:0-PI. Unfractionated lipid extracts were introduced by continuous infusion into the ESI source on a triple quadrupole MS/MS system (API 4000; Applied Biosystems). Samples were introduced using an autosampler (LC Mini PAL; CTC Analytics AG, Zwingen, Switzerland) fitted with the required injection loop for the acquisition time and presented to the ESI needle at 30 μl/min. The collision gas pressure was set at 2 (arbitrary units). The collision energies, with nitrogen in the collision cell, were 28 V for PE, 40 V for PC and SM, −58 V for PI, −57 V for PA, and −34 V for PS. Declustering potentials were 100 V for PE, SM, and PC and −100 V for PA and PI. Entrance potentials were 15 V for PE, 14 V for PC and SM, and −10 V for PI, PA, and PS. Exit potentials were 11 V for PE, 14 V for PC, −15 V for PI, −14 V for PA, and −13 V for PS. The mass analyzers were adjusted to a resolution of 0.7 amu full width at half height. For each spectrum, 9–150 continuum scans were averaged in multiple channel analyzer mode. The source temperature (heated nebulizer) was 100°C, the interface heater was on, +5.5 kV or −4.5 kV was applied to the electrospray capillary, the curtain gas was set at 20 (arbitrary units), and the two ion source gases were set at 45 (arbitrary units). Lipid species were detected using the scans described previously, including neutral loss of 87 in the negative mode for PS (14Welti R. Wang X. Williams T.D. Electrospray ionization tandem mass spectrometry scan modes for plant chloroplast lipids. Anal. Biochem. 2003; 314: 149-152Google Scholar, 15Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar). Sequential precursor and neutral loss scans of the extracts produce a series of spectra with each spectrum revealing a set of lipid species containing a common head group fragment. SM was determined from the same mass spectrum as PC (precursors of m/z 184 in positive mode) (15Brugger B. Erben G. Sandhoff R. Wieland F.T. Lehmann W.D. Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry. Proc. Natl. Acad. Sci. USA. 1997; 94: 2339-2344Google Scholar, 16Liebisch G. Lieser B. Rathenberg J. Drobnik W. Schmitz G. High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled with isotope correction algorithm. Biochim. Biophys. Acta. 2004; 1686: 108-117Google Scholar) and by comparison with PC internal standards using a molar response factor for SM (in comparison with PC) determined experimentally to be 0.37. The background of each spectrum was subtracted, the data were smoothed, and the peak areas were integrated using a custom script and Applied Biosystems Analyst software. Isotopic overlap corrections were applied, and the lipids in each class were quantified in comparison with the two internal standards of that class using corrected curves determined for the API 4000 mass spectrometer. Lipid droplets were purified from tissue culture cells by the method of Liu et al. (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar). To purify droplets from liver, a whole mouse liver was sliced into small pieces in 20 ml of ice-cold buffer containing 20 mM Tricine, pH 7.8, 250 mM sucrose, and 100 μM PMSF. The sample was homogenized with a glass Dounce homogenizer (20 strokes) on ice and centrifuged at 1,000 g for 10 min at 4°C. The supernatant fraction was transferred to two SW41 tubes, 7 ml each, and overlaid with 4 ml of HEPES buffer (20 mM HEPES, pH 7.4, 100 mM KCl, and 2 mM MgCl2) and centrifuged at 40,000 rpm for 1 h at 4°C. After centrifugation, the white band at the top of the tube was carefully collected and further processed as described (3Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004; 279: 3787-3792Google Scholar). To purify total cell membranes, CHO K2 cells were cultured on 150 mm plates to confluence, collected by scraping in ice-cold PBS with 100 μM PMSF, and homogenized with a nitrogen bomb at 450 p.s.i. for 15 min on ice. The sample was centrifuged at 1,000 g for 10 min at 4°C. The supernatant fraction was recovered and centrifuged at 40,000 rpm in a SW41 tube for 1 h at 4°C to pellet all of the membranes. The lipid composition of the pellet was determined as described. Quantification of lipids on TLC plates was by densitometric scanning of acidified, charred plates using NIH ImageJ software, compared with quantitative TLC standards (Nu-Chek Prep). Initially, we set out to characterize the neutral lipid composition of droplets purified from several different cell types that had been grown in the presence or absence of oleate. The baseline cell type for these studies was the CHO K2 cell, which normally contains numerous droplets (Fig. 1A , B, lane 2). Droplets were purified, and the lipids were extracted and separated by TLC along with standards. These droplets were rich in cholesteryl esters (∼34%), TAG (∼44%), and an unknown neutral lipid (∼20%) that migrated between cholesteryl esters and TAGs. Small amounts of free fatty acids, cholesterol, and phospholipids also were detected. The relative amounts of the major lipid classes changed little when CHO K2 cells were grown overnight in the presence of 80 μM oleate (Fig. 1A, B, lane 3). Moreover, the same relative proportions of neutral lipids were found in droplets isolated from NRK and SV589 cells that had been grown in the presence of oleate (Fig. 1A, B, lanes 4, 5). We used LC-APCI-MS/MS to identify the dominant fatty acid species in the TAG fraction (see supplementary Table I). The acyl composition was consistent with the fatty acids typically found in mammalian cells, including palmitic acid (P), stearic acid (S), oleic acid (O), linoleic acid (L), and linolenic acid (Ln). Based on [M+H]+ ions and diagnostic fragment ions (representing the DAG portion of the molecule), four major TAG species in CHO K2 cell lipid droplets were LLP, LLnO, LLO, and LLS. Mass spectrometric (MALDI-TOF-MS) and NMR (1H-NMR and 13C-NMR) analyses showed that the cholesteryl ester fraction contained at least three species: cholesteryl palmitate, cholesteryl linoleate, and cholesteryl oleate (data not shown). The unusual neutral lipid migrating between cholesteryl esters and TAG yielded [M+H]+ ions by APCI-MS/MS that were distinctly different from TAGs. An accurate mass measurement by MALDI-TOF-MS of one major species in this fraction was 865.7654, which suggested the formula of a large hydrocarbon containing five oxygen atoms. These data, along with key fragmentation ions identified by APCI-MS, were consistent with the possibility that this neutral lipid was a MADAG (see supplementary Table II). To unequivocally identify the unknown neutral lipid, we used NMR spectroscopy and additional mass spectrometry techniques. We purified the neutral lipid by silica gel flash chromatography and determined the 1H-NMR spectrum. The spectrum showed peaks that were characteristic of an esterified glycerol backbone (Fig. 2A , peaks b, c) containing an ether linkage at a terminal carbon (Fig. 2A, peaks a, d). This was supported by two-dimensional 1H-1H correlation spectroscopy (Fig. 2B), in which all peaks could be interpreted by proton signals of an ether lipid containing unsaturated alkyl and acyl chains. ESI-MS/MS analysis of the neutral lipids from adiposomes revealed >100 species of TAGs and MADAGs (Fig. 3A ). The [M+NH4]+ molecular ions represent an identifiable series of TAGs with intervening series of MADAGs in the same total ion chromatogram. Each species differs in mass by one oxygen atom, which represents mixed acyl species consistent with TAG and MADAG species predicted from LC-MS and MALDI-TOF-MS preliminary analyses (see supplementary Tables I, II). A list of TAG and MADAG species with increasing numbers of acyl/alkyl carbons and carbon-carbon double bonds is provided in supplementary Table III. Product ion spectra of TAG and MADAG molecular ions confirmed the nature of the alk(en)yl or acyl groups in the parent ions (Fig. 3B, C). For example, the diacyl moieties derived from the neutral loss of one ammoniated fatty acid revealed the nature of the TAG at nominal mass m/z 900 as a mixture of several species with combinations of different acyl chains totaling 54:4 (Fig. 3B). The acyl groups were consistent with the principal TAGs at this mass being a mixture of 18:1/18:1/18:2, 18:0/18:2/18:2, and 16:0/20:2/18:2 (as well as several less abundant combinations). Similar patterns were seen with product ion scans of the ether neutral lipid species, and Fig. 3C shows a product ion scan derived from the ammoniated e52:3 parent ion. Here, the diacyl or alk(en)yl/acyl products with a neutral loss of ammoniated fatty acid or ammoniated fatty alcohol helped to identify the mixture of species that make up the principal e52:3 MADAG species. There were several possible combinations, but two major species of e52:3 that are consistent with the fragment spectra are e18:1/16:0/18:2 and e16:0/18:1/18:2. The [M+NH4]+ TAGs and MADAGs identified in ESI-MS analyses were consistent with several species tentatively identified by LC-APCI-MS (see supplementary Tables I, II). The TAG shown in Fig. 3B corresponds to the [M+H]+ ion with nominal m/z 883 in supplementary Table I, and the MADAG shown in Fig. 3C corresponds to the [M+Na]+ ion with nominal m/z 865 in supplementary Table II. Finally, we compared the 13C-NMR spectrum of the purified neutral lipid species with the spectrum of pure glycerol trioleate. Two major differences were noted between the two spectra (see supplementary Fig. I). The 13C-NMR spectrum of the purified lipid had two additional peaks between 62 and 73 ppm (see supplementary Fig. IA, B; compare top and bottom spectra), consistent with the presence of an ether linkage in a terminal carbon glycerol backbone. Only one peak was seen in this region of glycerol trioleate (see supplementary Fig. IA, B, top spectra). The purified lipid also contained seven or more peaks between 127 and 131 ppm (see supplementary Fig. IC, bottom spectra). 13C peaks in this region correspond to unsaturated carbon atoms in a double bond. Glyceryl trioleate has only two peaks in this region because all acyl chains in this molecule are oleate, and oleate contains only one double bond (see supplementary Fig. IC, top spectra). The presence of multiple peaks indicated that the fraction contains different alkyl/acyl chains of mixed unsaturation. We conclude that the unknown neutral lipid is a mixture of MADAG (ether neutral lipid) molecular species containing alkyl and acyl chains of various lengths and degrees of unsaturation. In addition to physiologically relevant unsaturated fatty acids, such as 16:1, 18:1, 18:2, 18:3, and 20:4, the mixture may also contain saturated fatty acids, including 14:0, 16:0, and 18:0. This conclusion is supported by ESI-MS analyses of precursor and product ion scans (Fig. 3 and supplementary Table III). MADAG was not present in all cell types. The key step in the synthesis of ether lipids is the conversion of acyl-dihydroxyacetone phosphate (DHAP) to alkyl-DHAP by the peroxisomal enzyme alkyl-DHAP synthase (17van den Bosch H. de Vet E.C. Alkyl-dihydroxyacetonephosphate synthase. Biochim. Biophys. Acta. 1997; 1348: 35-44Crossref PubMed Scopus (24) Google Scholar). Therefore, droplets isolated from cells lacking peroxisomes should contain reduced amounts of MADAG. To determine whether this is correct, we used primary fibroblasts from patients with Zellweger syndrome, which lack functional peroxisomes owing to a defect in peroxisome assembly (18Wanders R.J. Peroxisomes, lipid metabolism, and peroxisomal disorders. Mol. Genet. Metab. 2004; 83: 16-27Crossref PubMed Scopus (161) Google Scholar). CHO K2 cells (Fig. 4A , lane 1) as well as normal (lane 2) and Zellweger (lane 3) human fibroblasts grown for 48 h in the presence of 80 μM oleate were processed to extract lipids from isolated droplets. The MADAG band was present in droplets isolated from normal cells but markedly reduced in Zellweger cells. We also compared the relative amount of MADAG in lipids extracted from adipose tissue (Fig. 4B) and found that MADAG was not detected in either white (lane 2) or brown (lane 3) adipose tissue. Previous studies have shown that during adipocyte differentiation, the level of alkyl-DHAP synthase declines (19Hajra A.K. Larkins L.K. Das A.K. Hemati N. Erickson R.L. MacDougald O.A. Induction of the peroxisomal glycerolipid-synthesizing enzymes during differentiation of 3T3-L1 adipocytes. Role in triacylglycerol synthesis. J. Biol. Chem. 2000; 275: 9441-9446Google Scholar), which may explain the low amounts of MADAG in adipose neutral lipids. Indeed, very little MADAG was found in droplets isolated from differentiated 3T3-L1 cells (Fig. 4B, lane 5), whereas undifferentiated 3T3-L1 cells grown overnight in the presence of oleate had significant amounts of MADAG (Fig. 4B, lane 4). We also detected MADAG in droplets isolated from liver (Fig. 4B, lane 8, arrowhead). Liver droplets were also rich in lipid species that migrate with the relative mobility values of retinoic ester (Fig. 4B, lane 8, asterisk) and free fatty acid (Fig. 4B, lane 8, double asterisk). Finally, we noted that droplets from NRK cells (Fig. 4B, lane 6) as well as NIH 3T3 cells grown in oleate (Fig. 4B, lane 4) contained low amounts of cholesteryl ester but still had considerable amounts of MADAG, which indicates that droplets accumulate these esters independently. These results emphasize two points. First, ether lipid metabolism varies among cell types and, most likely, is dependent on a cooperative interaction between peroxisomes and adiposomes. Second, adiposomes accumulate different types of neutral lipids depending on the specialized function of the cell. Although the proportion of total phospholipids in droplets is small relative to neutral lipids (1–2% of total), phospholipids stabilize the droplet and serve as an interface with other cellular compartments. Recent sensitive mass spectrometric techniques (ESI-MS/MS) have made it possible to identify and quantify phospholipid classes as well as the amount of specific molecular species in each class (20Welti R. Wang X. Lipid species profiling: a high-throughput approach to identify lipid compositional changes and determine the function of genes involved in lipid metabolism and signaling. Curr. Opin. Plant Biol. 2004; 7: 337-344Google Scholar). We used direct infusion ESI followed by MS/MS to identify different classes of polar lipids in the droplet and quantify the amount of the molecular species within each class. The species in each head group class are detected as the head group fragment produced by collision-induced dissociation. Classes are detected by sequential scans during continuous infusion, and quantification is done using internal sta

Triacylglycerols from plants, familiar to most people as vegetable oils, supply 25% of dietary calories to the developed world and are increasingly a source for renewable biomaterials and fuels. Demand for vegetable oils will double by 2030, which can be met only by increased oil production. Triacylglycerol synthesis is accomplished through the coordinate action of multiple pathways in multiple subcellular compartments. Recent information has revealed an underappreciated complexity in pathways for synthesis and accumulation of this important energy-rich class of molecules.

The compartmentation of neutral lipids in plants is mostly associated with seed tissues, where triacylglycerols (TAGs) stored within lipid droplets (LDs) serve as an essential physiological energy and carbon reserve during postgerminative growth. However, some nonseed tissues, such as leaves, flowers and fruits, also synthesize and store TAGs, yet relatively little is known about the formation or function of LDs in these tissues. Characterization of LD-associated proteins, such as oleosins, caleosins, and sterol dehydrogenases (steroleosins), has revealed surprising features of LD function in plants, including stress responses, hormone signaling pathways, and various aspects of plant growth and development. Although oleosin and caleosin proteins are specific to plants, LD-associated sterol dehydrogenases also are present in mammals, and in both plants and mammals these enzymes have been shown to be important in (steroid) hormone metabolism and signaling. In addition, several other proteins known to be important in LD biogenesis in yeasts and mammals are conserved in plants, suggesting that at least some aspects of LD biogenesis and/or function are evolutionarily conserved.

Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid-cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid beta oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies ("gnarls"), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

Lipins are phosphatidate phosphatases that generate diacylglycerol (DAG). In this study, we report that yeast lipin, Pah1p, controls the formation of cytosolic lipid droplets. Disruption of PAH1 resulted in a 63% decrease in droplet number, although total neutral lipid levels did not change. This was accompanied by an accumulation of neutral lipids in the endoplasmic reticulum (ER). The droplet biogenesis defect was not a result of alterations in neutral lipid ratios. No droplets were visible in the absence of both PAH1 and steryl acyltransferases when grown in glucose medium, even though the strain produces as much triacylglycerol as wild type. The requirement of PAH1 for normal droplet formation can be bypassed by a knockout of DGK1. Nem1p, the activator of Pah1p, localizes to a single punctum per cell on the ER that is usually next to a droplet, suggesting that it is a site of droplet assembly. Overall, this study provides strong evidence that DAG generated by Pah1p is important for droplet biogenesis.

Summary High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol ( TAG ). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in N icotiana tabacum (tobacco) leaves by the co‐expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild‐type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum . When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil‐processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

Lipid droplets, also known as oil bodies or lipid bodies, are plant organelles that compartmentalize neutral lipids as a hydrophobic matrix covered by proteins embedded in a phospholipid monolayer. Some of these proteins have been known for decades, such as oleosins, caleosins, and steroleosins, whereas a host of others have been discovered more recently with various levels of abundance on lipid droplets, depending on the tissue and developmental stage. In addition to a growing inventory of lipid droplet proteins, the subcellular machinery that contributes to the biogenesis and degradation of lipid droplets is being identified and attention is turning to more mechanistic questions regarding lipid droplet dynamics. While lipid droplets are mostly regarded as storage deposits for carbon and energy in lipid-rich plant tissues such as seeds, these organelles are present in essentially all plant cells, where they display additional functions in signaling, membrane remodeling, and the compartmentalization of a variety of hydrophobic components. Remarkable metabolic engineering efforts have demonstrated the plasticity of vegetative tissues such as leaves to synthesize and package large amounts of storage lipids, which enable future applications in bioenergy and the engineering of high-value lipophilic compounds. Here, we review the growing body of knowledge about lipid droplets in plant cells, describe the evolutionary similarity and divergence in their associated subcellular machinery, and point to gaps that deserve future attention.

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets.

CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf-specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in β-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of crop plants.

Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth.

Direct visualization of plant tissues by matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) has revealed key insights into the localization of metabolites in situ. Recent efforts have determined the spatial distribution of primary and secondary metabolites in plant tissues and cells. Strategies have been applied in many areas of metabolism including isotope flux analyses, plant interactions, and transcriptional regulation of metabolite accumulation. Technological advances have pushed achievable spatial resolution to subcellular levels and increased instrument sensitivity by several orders of magnitude. It is anticipated that MALDI-MSI and other MSI approaches will bring a new level of understanding to metabolomics as scientists will be encouraged to consider spatial heterogeneity of metabolites in descriptions of metabolic pathway regulation.

Lipid droplets (LDs) in plants have long been viewed as storage depots for neutral lipids that serve as sources of carbon, energy, and lipids for membrane biosynthesis. While much of our knowledge of LD function in plants comes from studies of oilseeds, a recent surge in research on LDs in non-seed cell types has led to an array of new discoveries. It is now clear that both evolutionarily conserved and kingdom-specific mechanisms underlie the biogenesis of LDs in eukaryotes, and proteomics and homology-based approaches have identified new protein players. This review highlights some of these recent discoveries and other new areas of plant LD research, including their role in stress responses and as targets of metabolic engineering strategies aimed at increasing oil content in bioenergy crops.

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ionization-MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry.

Recent pressures to obtain energy from plant biomass have encouraged new metabolic engineering strategies that focus on accumulating lipids in vegetative tissues at the expense of lignin, cellulose and/or carbohydrates. There are at least three important factors that support this rationale. (i) Lipids are more reduced than carbohydrates and so they have more energy per unit of mass. (ii) Lipids are hydrophobic and thus take up less volume than hydrated carbohydrates on a mass basis for storage in tissues. (iii) Lipids are more easily extracted and converted into useable biofuels than cellulosic-derived fuels, which require extensive fractionation, degradation of lignocellulose and fermentation of plant tissues. However, while vegetative organs such as leaves are the majority of harvestable biomass and would be ideal for accumulation of lipids, they have evolved as "source" tissues that are highly specialized for carbohydrate synthesis and export and do not have a propensity to accumulate lipid. Metabolism in leaves is directed mostly toward the synthesis and export of sucrose, and engineering strategies have been devised to divert the flow of photosynthetic carbon from sucrose, starch, lignocellulose, etc. toward the accumulation of triacylglycerols in non-seed, vegetative tissues for bioenergy applications.

There is considerable interest in the de novo production of omega-3 long chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), not least of all given the importance of these fatty acids in both aquaculture and human nutrition. Previously we have demonstrated the feasibility of using metabolic engineering in transgenic plants (Camelina sativa) to modify the seed oil composition to now include EPA and/or DHA. In this study, we further tailored the seed oil profile to reduce the omega-6 content, and evaluated the performance of such GM plants under field conditions (i.e. environmental releases), in terms of agronomic performance and also the lipidomic profile of seed oil. We used MALDI- mass spectrometry imaging to identify discrete tissue-types in the seed in which these non-native fatty acids preferentially accumulated. Collectively, these data provide new insights into the complexity of plant lipid metabolism and the challenges associated with predictive manipulation of these pathways. However, this study identified the likely dispensable nature of a Δ12-desturase activity in our omega-3 metabolic engineering rationales for Camelina.

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C.sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine.

Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady-state maintenance and turnover of plant LDs, particularly in non-seed tissues, are relatively unknown. Previously, we showed that the LD-associated proteins (LDAPs) are a family of plant-specific, LD surface-associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two-hybrid library using the Arabidopsis LDAP3 isoform as 'bait' in an effort to identify other novel LD protein constituents. One of the candidate LDAP3-interacting proteins was Arabidopsis At5g16550, which is a plant-specific protein of unknown function that we termed LDIP (LDAP-interacting protein). Using a combination of biochemical and cellular approaches, we show that LDIP targets specifically to the LD surface, contains a discrete amphipathic α-helical targeting sequence, and participates in both homotypic and heterotypic associations with itself and LDAP3, respectively. Analysis of LDIP T-DNA knockdown and knockout mutants showed a decrease in LD abundance and an increase in variability of LD size in leaves, with concomitant increases in total neutral lipid content. Similar phenotypes were observed in plant seeds, which showed enlarged LDs and increases in total amounts of seed oil. Collectively, these data identify LDIP as a new player in LD biology that modulates both LD size and cellular neutral lipid homeostasis in both leaves and seeds.

Summary Despite the importance of oilseeds to worldwide human nutrition, and more recently to the production of bio‐based diesel fuels, the detailed mechanisms regulating seed oil biosynthesis remain only partly understood, especially from a tissue‐specific perspective. Here, we investigated the spatial distributions of lipid metabolites and transcripts involved in oil biosynthesis from seeds of two low‐erucic acid genotypes of Brassica napus with high and low seed‐oil content. Integrated results from matrix‐assisted laser desorption/ionization‐mass spectrometry imaging ( MALDI ‐ MSI ) of lipids in situ , lipidome profiling of extracts from seed tissues, and tissue‐specific transcriptome analysis revealed complex spatial distribution patterns of lipids and transcripts. In general, it appeared that many triacylglycerol and phosphatidylcholine species distributed heterogeneously throughout the embryos. Tissue‐specific transcriptome analysis identified key genes involved in de novo fatty acid biosynthesis in plastid, triacylglycerols assembly and lipid droplet packaging in the endoplasmic reticulum ( ER ) that may contribute to the high or low oil phenotype and heterogeneity of lipid distribution. Our results imply that transcriptional regulation represents an important means of impacting lipid compartmentalization in oil seeds. While much information remains to be learned about the intricacies of seed oil accumulation and distribution, these studies highlight the advances that come from evaluating lipid metabolism within a spatial context and with multiple omics level datasets.

Lipid droplets (LDs) are organelles that compartmentalize nonbilayer-forming lipids in the aqueous cytoplasm of cells. They are ubiquitous in most organisms, including in animals, protists, plants and microorganisms. In eukaryotes, LDs are believed to be derived by a budding and scission process from the surface of the endoplasmic reticulum, and this occurs concomitantly with the accumulation of neutral lipids, most often triacylglycerols and steryl esters. Overall, the mechanisms underlying LD biogenesis are difficult to generalize, in part because of the involvement of different sets of both evolutionarily conserved and organism-specific LD-packaging proteins. Here, we briefly compare and contrast these proteins and the allied processes responsible for LD biogenesis in cells of animals, yeasts and plants.

Plant oils represent an energy-rich and carbon-dense group of hydrophobic compounds. These oils are not only of economic interest, but also play important, fundamental roles in plant and algal growth and development. The subcellular storage compartments of plant lipids, referred to as lipid droplets (LDs), have long been considered relatively inert oil vessels. However, research in the last decade has revealed that LDs play far more dynamic roles in plant biology than previously appreciated, including transient neutral lipid storage, membrane remodeling, lipid signaling, and stress responses. Here we discuss recent developments in the understanding of LD formation, turnover and function in land plants and algae.

Seeds of the desert shrub, jojoba (Simmondsia chinensis), are an abundant, renewable source of liquid wax esters, which are valued additives in cosmetic products and industrial lubricants. Jojoba is relegated to its own taxonomic family, and there is little genetic information available to elucidate its phylogeny. Here, we report the high-quality, 887-Mb genome of jojoba assembled into 26 chromosomes with 23,490 protein-coding genes. The jojoba genome has only the whole-genome triplication (γ) shared among eudicots and no recent duplications. These genomic resources coupled with extensive transcriptome, proteome, and lipidome data helped to define heterogeneous pathways and machinery for lipid synthesis and storage, provided missing evolutionary history information for this taxonomically segregated dioecious plant species, and will support efforts to improve the agronomic properties of jojoba.

Phospholipid catabolism is essential to cell function and encompases a variety of processes including metabolic channeling of unusual fatty acids, membrane reorganization and degradation, and the production of secondary messengers. Phospholipases are grouped into classes depending upon their general site of cleavage, and multiple isoforms of two phospholipases have been identified. Recent advances have improved our understanding of these different activities and their physiological consequences in the context of plant development and in response to environmental stress.

We used spontaneously active neuronal networks derived from dissociated embryonic murine spinal cord and auditory cortex and grown on substrate-integrated thin-film microelectrodes to determine characteristic responses to the cannabinoid agonists anandamide (AN) and methanandamide (MA). AN and MA reversibly inhibited spike and burst production in both tissue types. Responses of 21 cultures ranging in age from 23 to 111 days in vitro (d.i.v.) showed high intra- and inter-culture reproducibility at all ages. However, responses were tissue and substance-dependent. AN and MA were equipotent in cortical cultures and terminated bursting and spiking at 2.5 +/- 0.9 microM (n = 10). Spinal cultures were shut-off by 1.3 +/- 0.7 microM (n = 15) AN, but required 5.8 +/- 1.2 microM MA for activity cessation. MA, but not AN, demonstrated a biphasic influence: excitation at 0.25-3.5 microM and suppression at 4-7.1 microM. Palmitoylethanolamide, a related lipophilic molecule with no reported binding to the CBI receptor (to which AN and MA bind in the central nervous system), did not affect network activity at concentrations up to 6.5 microM. Irreversible cessation of activity was observed after 30 min applications of AN or MA at > 7 microM.

Three classes of phospholipase D (PLD), designated PLDα, -β, and -γ, have been cloned from plants, but their substrate selectivities have not been established. Using active PLDs expressed from their cDNAs inEscherichia coli,we compared the hydrolytic activities of these three PLDs toward various phospholipids and the influence of substrate composition on their substrate selectivities. When single-class phospholipid vesicles of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP2),N-acylphosphatidylethanolamine (NAPE), and cardiolipin (CL) were examined, PLDα hydrolyzed PC, PE, and PG but PLDβ and -γ showed no activity toward any of these lipids. When PIP2was included in mixed vesicles with the phospholipids above, PLDα showed the same PC-, PE-, and PG-hydrolyzing ability, whereas PLDβ and -γ were able to hydrolyze both PE and PS. When both PE and PIP2were included in substrate vesicles, PLDβ and PLDγ hydrolyzed PC, PG, and NAPE, showing that both PE and PIP2are required for PC, PG, and NAPE hydrolysis by PLDβ and -γ. The PE activation of PLDβ and -γ required lipid vesicles made of mostly PE, suggesting that PE may affect the substrate presentation rather than serve as a cofactor of these PLDs. Under equivalent reaction conditions, PLDβ displayed a similar preference for PC and NAPE, whereas PLDγ preferred NAPE to PC by nearly three times. None of the three PLDs used PI, CL, or PIP2as substrates. These results have identified PS- and NAPE-hydrolyzing PLDs and have indicated an important role for lipid composition in regulating the substrate selectivity of PLDβ and -γ.

N-Acylethanolamines (NAEs) are fatty acid amides that are derived from an N-acylated phoshatidylethanolamine presursor, a minor membrane lipid constituent of plant and animal cells. Historically, the formation of N-acylethanolamines was associated with cellular stress and tissue damage in mammals, but more recently has been shown to be part of the endocannabinoid signaling system that regulates a variety of normal physiological functions, including neurotransmission, immune responses, vasodilation, embryo development and implantation, feeding behavior, cell proliferation, etc. The widespread regulation of vertebrate physiology by this class of lipid mediators and the conservation of the mechanisms for NAE formation, perception and degradation in higher plants raises the possibility that the metabolism of NAEs represents an evolutionarily conserved lipid signaling pathway that regulates an array of physiological processes in multicellular eukaryotes. Here the recent information on NAEs in plants is reviewed in the context of the occurrence, metabolism and functions of this bioactive class of lipid mediators.

An expanding appreciation for the varied functions of neutral lipids in cellular organisms relies on a more detailed understanding of the mechanisms of lipid production and packaging into cytosolic lipid droplets (LDs). Conventional lipid profiling procedures involve the analysis of tissue extracts and consequently lack cellular or subcellular resolution. Here, we report an approach that combines the visualization of individual LDs, microphase extraction of lipid components from droplets, and the direct identification of lipid composition by nanospray mass spectrometry, even to the level of a single LD. The triacylglycerol (TAG) composition of LDs from several plant sources (mature cotton (Gossypium hirsutum) embryos, roots of cotton seedlings, and Arabidopsis thaliana seeds and leaves) were examined by direct organelle mass spectrometry and revealed the heterogeneity of LDs derived from different plant tissue sources. The analysis of individual LDs makes possible organellar resolution of molecular compositions and will facilitate new studies of LD biogenesis and functions, especially in combination with analysis of morphological and metabolic mutants. Furthermore, direct organelle mass spectrometry could be applied to the molecular analysis of other subcellular compartments and macromolecules.

Changes in ambient temperature represent a major physiological challenge to membranes of poikilothermic organisms. In plants, the endoplasmic reticulum (ER)-localized omega-3 fatty-acid desaturases (Fad3) increase the production of polyunsaturated fatty acids at cooler temperatures, but the <i>FAD3</i> genes themselves are typically not up-regulated during this adaptive response. Here, we expressed two closely related plant <i>FAD3</i> genes in yeast cells and found that their enzymes produced significantly different amounts of omega-3 fatty acids and that these differences correlated to differences in rates of protein turnover. Domain-swapping and mutagenesis experiments revealed that each protein contained a degradation signal in its N terminus and that the charge density of a PEST-like sequence within this region was largely responsible for the differences in rates of protein turnover. The half-life of each Fad3 protein was increased at cooler temperatures, and protein degradation required specific components of the ER-associated degradation pathway including the Cdc48 adaptor proteins Doa1, Shp1, and Ufd2. Expression of the Fad3 proteins in tobacco cells incubated with the proteasomal inhibitor MG132 further confirmed that they were degraded via the proteasomal pathway in plants. Collectively, these findings indicate that Fad3 protein abundance is regulated by a combination of <i>cis</i>-acting degradation signals and the ubiquitin-proteasome pathway and that modulation of Fad3 protein amounts in response to temperature may represent one mechanism of homeoviscous adaptation in plants.

COMPARATIVE GENE IDENTIFICATION-58 (CGI-58) is a key regulator of lipid metabolism and signaling in mammals, but its underlying mechanisms are unclear. Disruption of CGI-58 in either mammals or plants results in a significant increase in triacylglycerol (TAG), suggesting that CGI-58 activity is evolutionarily conserved. However, plants lack proteins that are important for CGI-58 activity in mammals. Here, we demonstrate that CGI-58 functions by interacting with the PEROXISOMAL ABC-TRANSPORTER1 (PXA1), a protein that transports a variety of substrates into peroxisomes for their subsequent metabolism by β-oxidation, including fatty acids and lipophilic hormone precursors of the jasmonate and auxin biosynthetic pathways. We also show that mutant cgi-58 plants display changes in jasmonate biosynthesis, auxin signaling, and lipid metabolism consistent with reduced PXA1 activity in planta and that, based on the double mutant cgi-58 pxa1, PXA1 is epistatic to CGI-58 in all of these processes. However, CGI-58 was not required for the PXA1-dependent breakdown of TAG in germinated seeds. Collectively, the results reveal that CGI-58 positively regulates many aspects of PXA1 activity in plants and that these two proteins function to coregulate lipid metabolism and signaling, particularly in nonseed vegetative tissues. Similarities and differences of CGI-58 activity in plants versus animals are discussed.

The emergence of ‘omics’ technologies (i.e. genomics, proteomics, metabolomics, etc.) have revealed new avenues for exploring plant metabolism through data-rich experimentation and integration of complementary methodologies. Over the past decade, the lipidomics field has benefited from advances in instrumentation, especially mass spectrometry (MS)-based approaches that are well-suited for detailed lipid analysis. The broad classification of what constitutes a lipid lends itself to a structurally diverse range of molecules that contribute to a variety of biological processes in plants including membrane structure and transport, primary and secondary metabolism, abiotic and biotic stress tolerances, extracellular and intracellular signaling, and energy-rich storage of carbon. Progress in these research areas has been advanced in part through approaches analyzing chemical compositions of lipids in extracts from cells, tissues and/or whole organisms (e.g. shotgun lipidomics), and through visualization approaches primarily through microscopy-based methodologies (e.g. fluorescence, bright field, electron microscopy, etc.). While these techniques on their own provide rich biochemical and biological information, coordinated analyses of the complexity of lipid composition with the localization of these lipids at a high spatial resolution will help to develop a new level of understanding of lipid metabolism within the context of tissue/cellular compartmentation. This review will elaborate on recent advances of one such approach – mass spectrometry imaging (MSI) – that integrates in situ visualization with chemical-based lipidomics. We will illustrate, with an emphasis on oilseed lipid metabolism, how MS imaging can provide new insights and questions related to the spatial compartmentation of lipid metabolism in plants. Further it will be apparent that this MS imaging approach has broad application in plant metabolic research well beyond that of triacylglycerol biosynthesis in oilseeds.

The regulation of lipid synthesis in oil seeds is still not fully understood. Oilseed rape (Brassica napus) is the third most productive vegetable oil crop on the global market; therefore, increasing our understanding of lipid accumulation in oilseed rape seeds is of great economic, as well as intellectual, importance. Matrix-assisted laser/desorption ionization-mass spectrometry imaging (MALDI-MSI) is a technique that allows the mapping of metabolites directly onto intact biological tissues, giving a spatial context to metabolism. We have used MALDI-MSI to study the spatial distribution of two major lipid species, triacylglycerols and phosphatidylcholines. A dramatic, heterogenous landscape of molecular species was revealed, demonstrating significantly different lipid compositions between the various tissue types within the seed. The embryonic axis was found to be particularly enriched in palmitic acid, while the seed coat/aleurone layer accumulated vaccenic, linoleic, and α-linoleic acids. Furthermore, the lipid composition of the inner and outer cotyledons differed from each other, a remarkable discovery given the supposed identical functionality of these two tissues. Triacylglycerol and phosphatidylcholine molecular species distribution was analyzed through a developmental time series covering early seed lipid accumulation to seed maturity. The spatial patterning of lipid molecular species did not vary significantly during seed development. Data gathered using MALDI-MSI was verified through gas chromatography analysis of dissected seeds. The distinct lipid distribution profiles observed imply differential regulation of lipid metabolism between the different tissue types of the seed. Further understanding of this differential regulation will enhance efforts to improve oilseed rape productivity and quality.

Summary Twenty years ago, N ‐acylethanolamines ( NAE s) were considered by many lipid chemists to be biological ‘artifacts’ of tissue damage, and were, at best, thought to be minor lipohilic constituents of various organisms. However, that changed dramatically in 1993, when anandamide, an NAE of arachidonic acid ( N ‐arachidonylethanolamine), was shown to bind to the human cannabinoid receptor ( CB 1) and activate intracellular signal cascades in mammalian neurons. Now NAE s of various types have been identified in diverse multicellular organisms, in which they display profound biological effects. Although targets of NAE s are still being uncovered, and probably vary among eukaryotic species, there appears to be remarkable conservation of the machinery that metabolizes these bioactive fatty acid conjugates of ethanolamine. This review focuses on the metabolism and functions of NAE s in higher plants, with specific reference to the formation, hydrolysis and oxidation of these potent lipid mediators. The discussion centers mostly on early seedling growth and development, for which NAE metabolism has received the most attention, but also considers other areas of plant development in which NAE metabolism has been implicated. Where appropriate, we indicate cross‐kingdom conservation in NAE metabolic pathways and metabolites, and suggest areas where opportunities for further investigation appear most pressing.

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

Abstract In this glossary of plant cell structures, we asked experts to summarize a present-day view of plant organelles and structures, including a discussion of outstanding questions. In the following short reviews, the authors discuss the complexities of the plant cell endomembrane system, exciting connections between organelles, novel insights into peroxisome structure and function, dynamics of mitochondria, and the mysteries that need to be unlocked from the plant cell wall. These discussions are focused through a lens of new microscopy techniques. Advanced imaging has uncovered unexpected shapes, dynamics, and intricate membrane formations. With a continued focus in the next decade, these imaging modalities coupled with functional studies are sure to begin to unravel mysteries of the plant cell.

Pennycress ( Thlaspi arvense L.) is being domesticated as an oilseed cash cover crop to be grown in the off-season throughout temperate regions of the world. With its diploid genome and ease of directed mutagenesis using molecular approaches, pennycress seed oil composition can be rapidly tailored for a plethora of food, feed, oleochemical and fuel uses. Here, we utilized Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to produce knockout mutations in the FATTY ACID DESATURASE2 ( FAD2 ) and REDUCED OLEATE DESATURATION1 ( ROD1 ) genes to increase oleic acid content. High oleic acid (18:1) oil is valued for its oxidative stability that is superior to the polyunsaturated fatty acids (PUFAs) linoleic (18:2) and linolenic (18:3), and better cold flow properties than the very long chain fatty acid (VLCFA) erucic (22:1). When combined with a FATTY ACID ELONGATION1 ( fae1 ) knockout mutation, fad2 fae1 and rod1 fae1 double mutants produced ∼90% and ∼60% oleic acid in seed oil, respectively, with PUFAs in fad2 fae1 as well as fad2 single mutants reduced to less than 5%. MALDI-MS spatial imaging analyses of phosphatidylcholine (PC) and triacylglycerol (TAG) molecular species in wild-type pennycress embryo sections from mature seeds revealed that erucic acid is highly enriched in cotyledons which serve as storage organs, suggestive of a role in providing energy for the germinating seedling. In contrast, PUFA-containing TAGs are enriched in the embryonic axis, which may be utilized for cellular membrane expansion during seed germination and seedling emergence. Under standard growth chamber conditions, rod1 fae1 plants grew like wild type whereas fad2 single and fad2 fae1 double mutant plants exhibited delayed growth and overall reduced heights and seed yields, suggesting that reducing PUFAs below a threshold in pennycress had negative physiological effects. Taken together, our results suggest that combinatorial knockout of ROD1 and FAE1 may be a viable route to commercially increase oleic acid content in pennycress seed oil whereas mutations in FAD2 will likely require at least partial function to avoid fitness trade-offs.

Imaging has long supported our ability to understand the inner life of plants, their development, and response to a dynamic environment. While optical microscopy remains the core tool for imaging, a suite of novel technologies is now beginning to make a significant contribution to visualize plant metabolism. The purpose of this review was to provide the scientific community with an overview of current imaging methods, which rely variously on either nuclear magnetic resonance (NMR), mass spectrometry (MS) or infrared (IR) spectroscopy, and to present some examples of their application in order to illustrate their utility. In addition to providing a description of the basic principles underlying these technologies, the review discusses their various advantages and limitations, reveals the current state of the art, and suggests their potential application to experimental practice. Finally, a view is presented as to how the technologies will likely develop, how these developments may encourage the formulation of novel experimental strategies, and how the enormous potential of these technologies can contribute to progress in plant science.

The activation of N-acylphosphatidylethanolamine (NAPE) metabolism in plants appears to be associated mostly with cellular stresses. In response to pathogen elicitors, NAPE is hydrolzyed by phospholipase-D (PLD), and corresponding medium-chain, saturated N-acylethanolamines (NAEs) are released by plant cells where they act as lipid mediators to modulate ion flux and activate defense gene expression. In desiccated seeds of higher plants, long-chain, saturated and unsaturated NAEs are prevalent, but are rapidly metabolized during the first few hours of imbibition, a period of substantial osmotic stress. NAPE synthesis is increased in seeds during this same period of rapid rehydration. A membrane-bound enzyme designated NAPE synthase has been purified from imbibed cottonseeds and its unusual biochemical properties suggest that it may scavenge free fatty acids in vivo. This feature of NAPE metabolism may be unique to higher plants a may be a mechanism for the rapid recycling of fatty acids back into membrane-associated NAPE. Altogether, increasing evidence indicates that NAPE metabolism in plants shares functional similarities with NAPE metabolism in animal systems, including signal transduction and cellular protection. In particular, the emerging role of released NAEs as lipid mediators in plant defense signaling represents an intriguing parallel to ‘endocannabinoid signaling’ in several mammalian cell types.

Glyoxysomes in cotyledons of cotton (Gossypium hirsutum, L.) seedlings enlarge dramatically within 48 h after seed imbibition (Kunce, C.M., R.N. Trelease, and D.C. Doman. 1984. Planta (Berl.). 161:156-164) to effect mobilization of stored cotton-seed oil. We discovered that the membranes of enlarging glyoxysomes at all stages examined contained a large percentage (36-62% by weight) of nonpolar lipid, nearly all of which were triacylglycerols (TAGs) and TAG metabolites. Free fatty acids comprised the largest percentage of these nonpolar lipids. Six uncommon (and as yet unidentified) fatty acids constituted the majority (51%) of both the free fatty acids and the fatty acids in TAGs of glyoxysome membranes; the same six uncommon fatty acids were less than 7% of the acyl constituents in TAGs extracted from cotton-seed storage lipid bodies. TAGs of lipid bodies primarily were composed of palmitic, oleic, and linoleic acids (together 70%). Together, these three major storage fatty acids were less than 10% of both the free fatty acids and fatty acids in TAGs of glyoxysome membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constituted a major portion of glyoxysome membrane phospholipids (together 61% by weight). Pulse-chase radiolabeling experiments in vivo clearly demonstrated that 14C-PC and 14C-PE were synthesized from 14C-choline and 14C-ethanolamine, respectively, in ER of cotyledons, and then transported to mitochondria; however, these lipids were not transported to enlarging glyoxysomes. The lack of ER involvement in glyoxysome membrane phospholipid synthesis, and the similarities in lipid compositions between lipid bodies and membranes of glyoxysomes, led us to formulate and test a new hypothesis whereby lipid bodies serve as the dynamic source of nonpolar lipids and phospholipids for membrane expansion of enlarging glyoxysomes. In a cell-free system, 3H-triolein (TO) and 3H-PC were indeed transferred from lipid bodies to glyoxysomes. 3H-PC, but not 3H-TO, also was transferred to mitochondria in vitro. The amount of lipid transferred increased linearly with respect to time and amount of acceptor organelle protein, and transfer occurred only when lipid body membrane proteins were associated with the donor lipid bodies. 3H-TO was transferred to and incorporated into glyoxysome membranes, and then hydrolyzed to free fatty acids. 3H-PC was transferred to and incorporated into glyoxysome and mitochondria membranes without subsequent hydrolysis. Our data are inconsistent with the hypothesis that ER contributes membrane lipids to glyoxysomes during postgerminative seedling growth.(ABSTRACT TRUNCATED AT 400 WORDS)

In vertebrates, the endocannabinoid signaling pathway is an important lipid regulatory pathway that modulates a variety of physiological and behavioral processes. N -Acylethanolamines (NAEs) comprise a group of fatty acid derivatives that function within this pathway, and their signaling activity is terminated by an enzyme called fatty acid amide hydrolase (FAAH), which hydrolyzes NAEs to ethanolamine and their corresponding free fatty acids. Bioinformatic approaches led to the identification of plant homologues of FAAH that are capable of hydrolyzing NAEs in vitro . To better understand the role of NAEs in plants, we identified T-DNA knockouts to Arabidopsis FAAH ( AtFAAH ; At5g64440) and generated plants overexpressing AtFAAH . Here we show that seeds of AtFAAH knockouts had elevated levels of endogenous NAEs, and seedling growth was hypersensitive to exogenously applied NAE. On the other hand, seeds and seedlings of AtFAAH overexpressors had lower endogenous NAE content, and seedlings were less sensitive to exogenous NAE. Moreover, AtFAAH overexpressors displayed enhanced seedling growth and increased cell size. AtFAAH expression and FAAH catalytic activity increased during seed germination and seedling growth, consistent with the timing of NAE depletion during seedling establishment. Collectively, our results show that AtFAAH is one, but not the only, modulator of endogenous NAE levels in plants, and that NAE depletion likely participates in the regulation of plant growth.

A cotton (Gossypium hirsutum L.) genomic clone encompassing a 17.9-kb DNA fragment was found to contain a delta-12 fatty acid desaturase gene (designated FAD2-4). The FAD2-4 open reading frame has 1,155bp and is uninterrupted, encoding a conceptual FAD2-4 polypeptide of 384 amino acids that has 98% identity with the cotton FAD2-3 polypeptide. The FAD2-4 gene has a single intron of 2,780 bp in its 5'-untranslated region (5'-UTR). The 3'-flanking regions and 5'-UTR introns in the FAD2-4 and FAD2-3 genes are quite different, indicating that the genes might be paralogs in the cotton genome. Reverse transcriptase (RT)-PCR analysis indicated that the FAD2-4 and FAD2-3 genes were expressed in all tissues examined, including seeds, seedling tissues, young and mature leaves, roots, stems, developing flower buds, and ovule fibers. These constitutive patterns of expression were notably different from that of the FAD2-1 gene, which was restricted to seeds and developing flower buds, or to the expression of a newly-identified FAD2-2 gene isoform, which was barely detectable in roots, hypocotyls, stems, and fibers, but was expressed in all other tissues. The FAD2-4 coding region was expressed in yeast and shown to encode a functional delta-12 desaturase, converting oleic acid (C18:1) to linoleic acid (C18:2) in recombinant yeast cells. In addition, both the FAD2-4 and the FAD2-3 genes were transferred into the Arabidopsis thaliana fad2-1 mutant background where they effectively restored wild type fatty acid composition and growth characteristics. Finally, the cotton FAD2-4 green fluorescent protein (GFP) fusion polypeptide appeared to be localized in the endomembrane system of transgenic Arabidopsis plants in the complemented fad2-1 mutant background, supporting a functional ER location for the cotton FAD2-4 polypeptide in this heterologous plant system. Thus, a new functional member of the FAD2 gene family in cotton has been characterized, indicating a complex regulation of membrane lipid desaturation in this important fiber/oilseed crop.

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. In plants, NAEs are generally elevated in desiccated seeds, suggesting that they may play a role in seed physiology. NAE and abscisic acid (ABA) levels were depleted during seed germination, and both metabolites inhibited the growth of Arabidopsis thaliana seedlings within a similar developmental window. Combined application of low levels of ABA and NAE produced a more dramatic reduction in germination and growth than either compound alone. Transcript profiling and gene expression studies in NAE-treated seedlings revealed elevated transcripts for a number of ABA-responsive genes and genes typically enriched in desiccated seeds. The levels of ABI3 transcripts were inversely associated with NAE-modulated growth. Overexpression of the Arabidopsis NAE degrading enzyme fatty acid amide hydrolase resulted in seedlings that were hypersensitive to ABA, whereas the ABA-insensitive mutants, abi1-1, abi2-1, and abi3-1, exhibited reduced sensitivity to NAE. Collectively, our data indicate that an intact ABA signaling pathway is required for NAE action and that NAE may intersect the ABA pathway downstream from ABA. We propose that NAE metabolism interacts with ABA in the negative regulation of seedling development and that normal seedling establishment depends on the reduction of the endogenous levels of both metabolites.

Engineering compositional changes in oilseeds is typically accomplished by introducing new enzymatic step(s) and/or by blocking or enhancing an existing enzymatic step(s) in a seed-specific manner. However, in practice, the amounts of lipid species that accumulate in seeds are often different from what one would predict from enzyme expression levels, and these incongruences may be rooted in an incomplete understanding of the regulation of seed lipid metabolism at the cellular/tissue level. Here we show by mass spectrometry imaging approaches that triacylglycerols and their phospholipid precursors are distributed differently within cotyledons and the hypocotyl/radicle axis in embryos of the oilseed crop Camelina sativa, indicating tissue-specific heterogeneity in triacylglycerol metabolism. Phosphatidylcholines and triacylglycerols enriched in linoleic acid (C18:2) were preferentially localized to the axis tissues, whereas lipid classes enriched in gadoleic acid (C20:1) were preferentially localized to the cotyledons. Manipulation of seed lipid compositions by heterologous over-expression of an acyl-acyl carrier protein thioesterase, or by suppression of fatty acid desaturases and elongases, resulted in new overall seed storage lipid compositions with altered patterns of distribution of phospholipid and triacylglycerol in transgenic embryos. Our results reveal previously unknown differences in acyl lipid distribution in Camelina embryos, and suggest that this spatial heterogeneity may or may not be able to be changed effectively in transgenic seeds depending upon the targeted enzyme(s)/pathway(s). Further, these studies point to the importance of resolving the location of metabolites in addition to their quantities within plant tissues.

Plant oils represent one of the most energyrich sources of renewable fuels available in Nature. Most of these oils occur in the form of triacylglycerols (TAGs) that can be transformed into biodiesel by conversion of their acyl chains into fatty acid methyl esters. In 2009, 14 billion litres of biodiesel were produced worldwide from plant oils (largely in the EU). This compares with 70 billion litres of ethanol (largely from Brazil and the USA). Both of these fuels now depend on land and crops (e.g. oil seeds, palm trees, maize and sugar cane) that are also used for foods. To meet growing demand and avoid competition with food, major expansion of biofuel production and development of new sources of biofuel are required. In this article, we outline how plants synthesize oils and describe some ways in which supplies of oils from plants could be increased to provide a larger contribution to renewable energy supplies.

Summary Mass spectrometry (MS) advances in recent years have revolutionized the biochemical analysis of lipids in plants, and made possible new theories about the structural diversity and functional complexity of lipids in plant cells. Approaches have been developed to profile the lipidome of plants with increasing chemical and spatial resolution. Here we highlight a variety of methods for lipidomics analysis at the tissue, cellular and subcellular levels. These procedures allow the simultaneous identification and quantification of hundreds of lipids species in tissue extracts by direct‐infusion MS, localization of lipids in tissues and cells by laser desorption/ionization MS, and even profiling of lipids in individual subcellular compartments by direct‐organelle MS. Applications of these approaches to achieve improved understanding of plant lipid metabolism, compartmentation and function are discussed.

While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, lipid droplets are delineated by a half-unit membrane whose protein constituents are poorly understood, except in the specialized case of oleosins, which are associated with seed lipid droplets. Recently, we identified a new class of lipid-droplet associated proteins called LDAPs that localize specifically to the lipid droplet surface within plant cells and share extensive sequence similarity with the small rubber particle proteins (SRPPs) found in rubber-accumulating plants. Here, we provide additional evidence for a role of LDAPs in lipid accumulation in oil-rich fruit tissues, and further explore the functional relationships between LDAPs and SRPPs. In addition, we propose that the larger LDAP/SRPP protein family plays important roles in the compartmentalization of lipophilic compounds, including triacylglycerols and polyisoprenoids, into lipid droplets within plant cells. Potential roles in lipid droplet biogenesis and function of these proteins also are discussed.

A standardized experiment was conducted during 2009 and 2010 at 20 location‐years across U.S. cotton ( Gossypium hirsutum L.)‐producing states to compare the N use requirement of contemporary cotton cultivars based on their planting seed size. Treatments consisted of three cotton varieties with planting seed of different numbers of seed per kg and N rates of 0, 45, 90, and 134 kg ha –1 . Soil at each trial location was sampled and tested for nitrate presence. High levels of soil nitrate (&gt;91 N‐NO 3 – kg ha –1 ) were found in Arizona and western Texas, and soil nitrate in the range of 45 to 73 kg N‐NO 3 – ha –1 was found at locations in the central United States. Cotton lint yield responded to applied N at 11 of 20 locations. Considering only sites that responded to applied N, highest lint yields were achieved with 112 to 224 kg ha –1 of applied plus pre‐plant residual soil NO 3 —translating to an optimal N requirement of 23 kg ha –1 per 218 kg bale of lint produced. Among the varieties tested those with medium‐sized seed produced higher yields in response to N than did larger and smaller seeded varieties. Varieties with larger seed had longer and stronger fibers, higher fiber length uniformity than small seeded varieties and decreased micronaire. Seed protein and oil increased and decreased slightly in response to increasing amounts of soil nitrate plus applied N, respectively.

Seed mass and oil content of the quiescent cotton seed are positively associated with seedling vigor. In contrast, seed size has been negatively associated with lint yield due to selection for cultivars with greater lint percent. The current study addressed the hypothesis that planting seed mass and total oil + protein calorie content of the quiescent cotton seed would be strongly predictive of seedling vigor across most field conditions and that the impact of seed traits on yield would be dependent upon yield environment. When considered in each yield environment, seedling vigor was positively associated with seed size and the total oil + protein calorie content per seed in four out five environments tested. Regression analysis of cultivar mean oil + protein kcal content per seed versus seedling vigor across all environments indicated a strong, positive relationship between the two parameters (r2 = 0.65). Although lint percent was positively correlated with lint yield in every environment, planting seed mass and calorie content were not correlated with lint yield in four of the five environments tested or when cultivar means for lint yield and seed characteristics were averaged across all environments. Thus, it is concluded that individual planting seed mass and total energy content for oil + protein are strong predictors of early seedling vigor. Furthermore, selecting commercially available cultivars with characteristics indicative of seedling vigor does not appear to limit lint yield in most environments tested.

SEIPIN proteins are localized to endoplasmic reticulum (ER)-lipid droplet (LD) junctions where they mediate the directional formation of LDs into the cytoplasm in eukaryotic cells. Unlike in animal and yeast cells, which have single SEIPIN genes, plants have three distinct SEIPIN isoforms encoded by separate genes. The mechanism of SEIPIN action remains poorly understood, and here we demonstrate that part of the function of two SEIPIN isoforms in Arabidopsis (Arabidopsis thaliana), AtSEIPIN2 and AtSEIPIN3, may depend on their interaction with the vesicle-associated membrane protein (VAMP)-associated protein (VAP) family member AtVAP27-1. VAPs have well-established roles in the formation of membrane contact sites and lipid transfer between the ER and other organelles, and here, we used a combination of biochemical, cell biology, and genetics approaches to show that AtVAP27-1 interacts with the N termini of AtSEIPIN2 and AtSEIPIN3 and likely supports the normal formation of LDs. This insight indicates that the ER membrane tethering machinery in plant cells could play a role with select SEIPIN isoforms in LD biogenesis at the ER, and additional experimental evidence in Saccharomyces cerevisiae supports the possibility that this interaction may be important in other eukaryotic systems.

Abstract Design of environmentally friendly lubricants derived from renewable resources is highly desirable for many practical applications. Here, Orychophragmus violaceus (Ov) seed oil is found to have superior lubrication properties, and this is based on the unusual structural features of the major lipid species—triacylglycerol (TAG) estolides. Ov TAG estolides contain two non-hydroxylated, glycerol-bound fatty acids (FAs) and one dihydroxylated FA with an estolide branch. Estolide branch chains vary in composition and length, leading to their thermal stability and functional properties. Using this concept, nature-guided estolides of castor oil were synthesized. As predicted, they showed improved lubrication properties similar to Ov seed oil. Our results demonstrate a structure-based design of novel lubricants inspired by natural materials.

Abstract Thirty years ago, the discovery of a cannabinoid (CB) receptor that interacts with the psychoactive compound in Cannabis led to the identification of anandamide, an endogenous receptor ligand or endocannabinoid. Research on endocannabinoids has since exploded, and additional receptors along with their lipid mediators and signaling pathways continue to be revealed. Specifically, in humans, the release of endocannabinoids from membrane lipids occurs on demand and the signaling process is rapidly attenuated by the breakdown of the ligand suggesting a tight regulation of the endocannabinoid system (ECS). Additionally, the varying distribution of CB receptors between the central nervous system and other tissues allows for the ECS to participate in a wide range of cognitive and physiological processes. Select plant-derived ‘phyto’cannabinoids such as Δ-9-tetrahydrocannabinol (Δ9-THC) bind to the CB receptors and trigger the ECS, and in the case of Δ9-THC, while it has therapeutic value, can also produce detrimental effects. Current research is aimed at the identification of additional phytocannabinoids with minimal psychotropic effects with potential for therapeutic development. Although decades of research on the ECS and its components have expanded our understanding of the mechanisms and implications of endocannabinoid signaling in mammals, it continues to evolve. Here, we provide a brief overview of the ECS and its overlap with other related lipid-mediated signaling pathways.

Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially fatal disorder caused by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) deficiency. Prompt identification/exclusion of TTP can thus be facilitated by rapid ADAMTS13 testing. The most commonly utilized (enzyme-linked immunosorbent assay [ELISA]-based) assay takes several hours to perform and so does not generally permit rapid testing.To evaluate the utility of a new automated test for ADAMTS13 activity, the HemosIL AcuStar ADAMTS13 Activity assay, based on chemiluminescence and able to be performed on an ACL AcuStar instrument within 33 minutes.This multicenter (n = 8) assessment included testing of more than 700 test samples, with similar numbers of prospective (n = 348) and retrospective (n = 385) samples. The main comparator was the Technozym ADAMTS13 Activity ELISA. We also assessed comparative performance for detection of ADAMTS13 inhibitors using a Bethesda assay.Overall, the chemiluminescent assay yielded similar results to the comparator ELISA, albeit with slight negative bias. ADAMTS13 inhibitor detection was also comparable, albeit with slight positive bias with the AcuStar assay. Assay precision was similar with both assays, and we also verified assay normal reference ranges.The HemosIL AcuStar ADAMTS13 Activity assay provided results rapidly, which were largely comparable with the Technozym ADAMTS13 Activity ELISA assay, albeit lower on average. Conversely, inhibitor levels tended to be identified at a higher level on average. Thus, the HemosIL AcuStar ADAMTS13 Activity assay provides a fast and accurate means to quantitate plasma levels of ADAMTS13 for TTP/ADAMTS13 identification/exclusion, and potentially also for other applications.

Thrombotic thrombocytopenic purpura (TTP) is a prothrombotic condition caused by a deficiency of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In turn, ADAMTS13 (also called von Willebrand factor (VWF) cleaving protease (VWFCP)) acts to cleave VWF multimers and thus reduce plasma VWF activity. In the absence of ADAMTS13 (i.e., in TTP), plasma VWF accumulates, in particular as "ultra-large" VWF multimers, and this leads to thrombosis. In most patients with confirmed TTP, ADAMTS13 deficiency is an acquired disorder due to the development of antibodies against ADAMTS13, which either promote clearance of ADAMTS13 from circulation or cause inhibition of ADAMTS13 activity. The current report describes a protocol for assessment of ADAMTS13 inhibitors, being antibodies that inhibit ADAMTS13 activity. The protocol reflects the technical steps that help identify inhibitors to ADAMTS13, whereby mixtures of patient plasma and normal plasma are then tested for residual ADAMTS13 activity in a Bethesda-like assay. The residual ADAMTS13 activity can be assessed by a variety of assays, with a rapid test able to be performed within 35 minutes on the AcuStar instrument (Werfen/Instrumentation Laboratory) used as an example in this protocol.

Two overlapping genomic clones spanning 16.5 kb of cotton DNA were found to encompass a Delta-12 fatty acid desaturase (FAD2-3) gene. A partial FAD2-3 cDNA clone was also analyzed. The FAD2-3 gene has one large intron of 2967 bp entirely within its 5'-untranslated region, only 12 bp upstream from the ATG initiation codon. Several potential promoter elements, including several light-responsive motifs, occur in the 5'-flanking region. The continuous FAD2-3 coding region is 1155 bp and would encode a protein of 384 amino acids. The polypeptide has four putative membrane-spanning helices, indicative of an integral membrane protein, and is most likely localized in the endoplasmic reticulum. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.

Abstract Cottonseed typically contains about 15% oleic acid. Here we report the development of transgenic cotton plants with higher seed oleic acid levels. Plants were generated by Agrobacterium ‐mediated transformation. A binary vector was designed to suppress expression of the endogenous cottonseed †‐12 desaturase ( fad2 ) by subcloning a mutant allele of a rapeseed fad2 gene downstream from a heterologous, seedspecific promoter (phaseolin). Fatty acid profiles of total seed lipids from 43 independent transgenic lines were analyzed by gas chromatography. Increased seed oleic acid content ranged from 21 to 30% (by weight) of total fatty acid content in 22 of the primary transformants. The increase in oleic acid content was at the expense of linoleic acid, consistent with reduced activity of cottonseed FAD2. Progeny of some lines yielded oleic acid content as high as 47% (three times that of standard cottonseed oil). Molecular analyses of nuclear DNA from transgenics confirmed the integration of the canola transgene into the cotton genome. Collectively, our results extend the metabolic engineering of vegetable oils to cottonseed and should provide the basis for the development of a family of novel cottonseed oils.

We have identified a microsomal phospholipid as N-acylphosphatidylethanolamine (NAPE) that was radiolabeled following incubation of 1-day-old cotyledons of cotton seedlings with [14C]ethanolamine. Radiolabeled NAPE comigrated with commercially available L-alpha-dipalmitoyl phosphatidyl(N-palmitoyl)ethanolamine (std-NAPE) in one- and two-dimensional TLC. This NAPE comprised 7.2 +/- 1.0% (by weight) of microsomal phospholipids when hot isopropanol was used to inactivate endogenous phospholipases prior to extraction of lipids. In vitro degradation of putative cottonseed radiolabeled NAPE by Streptomyces chromofuscus phospholipase D resulted in production of a ninhydrin-reactive, radiolabeled lipid which comigrated with N-acylethanolamine (NAE) that was produced from a similar enzymatic cleavage of std-NAPE. Transmethylation of cottonseed radiolabeled NAE yielded radiolabeled ethanolamine and fatty acid methyl esters, nearly all of which were saturated (myristate, palmitate, and stearate together were 92% of the acyl components of cottonseed NAE). Positional analysis and relative abundance of the O-acyl groups of cottonseed microsomal NAPE were determined following a double enzymatic cleavage with Trimeresurus flavoviridis venom (phospholipase A2 activity) and S. chromofuscus phospholipase D. We substantiated our identification of cottonseed NAPE by 1H NMR spectroscopy and by mass spectrometry (fast-atom-bombardment ionization and tandem MS, FAB-MS/MS). Radiolabeled NAPE was synthesized in vivo in varying amounts from [14C]ethanolamine applied to maturing seeds of cotton and soybean, cotyledons of dark-grown cotton and soybean seedlings, cotyledons of light-grown okra, cotton and soybean seedlings, endosperm tissue of castor bean, and suspension cell cultures of rice. In pulse-chase radiolabeling experiments in cotyledons of 1-day-old cotton seedlings, radiolabeled NAPE increased and radiolabeled phosphatidylethanolamine (PE) decreased over a 12-h chase period (in the dark or light), suggesting that NAPE was synthesized from PE in vivo. In vitro, the synthesis of NAPE from PE (radiolabeled in vivo) proceeded in a linear fashion in microsomes of cotton cotyledons with or without 1 mM EGTA and with or without 1 mM CaCl2 for 90 min. NAPE was synthesized in vitro from PE synthesized by the exchange pathway (microsomes preincubated with [14C]++e+thanolamine) and from PE synthesized by the nucleotide pathway (microsomes preincubated with [14C]CDPethanolamine). Collectively, our data indicate that (a) NAPE is a widespread, natural phospholipid component of plants, (b) NAPE is synthesized in vivo under normal physiological growth conditions in cotyledons of cotton seedlings, (c) NAPE is localized and synthesized in cottonseed microsomes, and (d) NAPE is likely synthesized by a direct acylation of PE.

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120-126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [14C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [14C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133-142) raises the possibility that a similar mechanism may operate in plant cells.

N-Acylethanolamines (NAEs) were quantified in seeds of several plant species and several cultivated varieties of a single species (cotton [Gossypium hirstutum]) by gas chromatography-mass spectroscopy. The total NAE content of dry seeds ranged from 490 +/- 89 ng g(-1) fresh weight in pea (Pisum sativum cv early Alaska) to 1,608 +/- 309 ng g(-1) fresh weight in cotton (cv Stoneville 7A glandless). Molecular species of NAEs in all seeds contained predominantly 16C and 18C fatty acids, with N-linoleoylethanolamine (NAE18:2) being the most abundant (approaching 1,000 ng g(-1) fresh weight in cottonseeds). Total NAE levels dropped drastically following 4 h of imbibition in seeds of pea, cotton, and peanut (Arachis hypogea cv Virginia), and this decline was most pronounced for NAE18:2. A novel enzyme activity was identified in cytosolic fractions of imbibed cottonseeds that hydrolyzed NAE18:2 in vitro. NAE degradation was optimal at 35 degrees C in 50 mM MES buffer, pH 6.5, and was inhibited by phenylmethylsulfonyl fluoride and 5, 5'-dithio-bis(2-nitrobenzoic acid), which is typical of other amide hydrolases. Amidohydrolase activity in cytosolic fractions exhibited saturation kinetics toward the NAE18:2 substrate, with an apparent K(m) of 65 &mgr;M and a V(max) of 83 nmol min(-1) mg(-1) protein. Total NAE amidohydrolase activity increased during seed imbibition, with the highest levels (about four times that in dry seeds) measured 2 h after commencing hydration. NAEs belong to the family of "endocannabinoids," which have been identified as potent lipid mediators in other types of eukaryotic cells. This raises the possibility that their imbibition-induced metabolism in plants is involved in the regulation of seed germination.

Abstract N-Acylethanolamines (NAEs) are endogenous lipids in plants produced from the phospholipid precursor,N-acylphosphatidylethanolamine, by phospholipase D (PLD). Here, we show that seven types of plant NAEs differing in acyl chain length and degree of unsaturation were potent inhibitors of the well-characterized, plant-specific isoform of PLD—PLDα. It is notable that PLDα, unlike other PLD isoforms, has been shown not to catalyze the formation of NAEs fromN-acylphosphatidylethanolamine. In general, inhibition of PLDα activity by NAEs increased with decreasing acyl chain length and decreasing degree of unsaturation, such thatN-lauroylethanolamine andN-myristoylethanolamine were most potent with IC50s at submicromolar concentrations for the recombinant castor bean (Ricinus communis) PLDα expressed inEscherichia coli and for partially purified cabbage (Brassica oleracea) PLDα. NAEs did not inhibit PLD from Streptomyces chromofuscus, and exhibited only moderate, mixed effects for two other recombinant plant PLD isoforms. Consistent with the inhibitory biochemical effects on PLDα in vitro,N-lauroylethanolamine, but not lauric acid, selectively inhibited abscisic acid-induced closure of stomata in epidermal peels of tobacco (Nicotiana tabacum cv Xanthi) andCommelina communis at low micromolar concentrations. Together, these results provide a new class of biochemical inhibitors to assist in the evaluation of PLDα physiological function(s), and they suggest a novel, lipid mediator role for endogenously produced NAEs in plant cells.

<i>N</i>-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information is available for this enzyme in plants. Here we report the identification, the heterologous expression (in <i>Escherichia coli</i>), and the biochemical characterization of an <i>Arabidopsis thaliana</i> FAAH homologue. Candidate <i>Arabidopsis</i> DNA sequences containing a characteristic amidase signature sequence (PS00571) were identified in plant genome data bases, and a cDNA was isolated by reverse transcriptase-PCR using <i>Arabidopsis</i> genome sequences to develop appropriate oligonucleotide primers. The cDNA was sequenced and predicted to encode a protein of 607 amino acids with 37% identity to rat FAAH within the amidase signature domain (18% over the entire length). Residues determined to be important for FAAH catalysis were conserved between the <i>Arabidopsis</i> and rat protein sequences. In addition, a single transmembrane domain near the N terminus was predicted in the <i>Arabidopsis</i> protein sequence, similar to that of the rat FAAH protein. The putative plant FAAH cDNA was expressed as an epitope/His-tagged fusion protein in <i>E. coli</i> and solubilized from cell lysates in the nonionic detergent, dodecyl maltoside. Affinity-purified recombinant protein was indeed active in hydrolyzing a variety of naturally occurring <i>N</i>-acylethanolamine types. Kinetic parameters and inhibition data for the recombinant <i>Arabidopsis</i> protein were consistent with these properties of the enzyme activity characterized previously in plant and animal systems. Collectively these data now provide support at the molecular level for a conserved mechanism between plants and animals for the metabolism of NAEs.

Abstract Two cDNAs encoding functional carbonic anhydrase (CA) enzymes were recently isolated from a non-photosynthetic, cotyledon library of cotton (Gossypium hirsutum) seedlings with putative plastid-targeting sequences (GenBank accession nos. AF132854 andAF132855). Relative CA transcript abundance and enzyme activity increased 9 and 15 times, respectively, in cotton embryos during the maximum period of reserve oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [14C]acetate incorporation into total lipids in cotton embryos in vivo, and in embryo plastids in vitro, suggesting a role for CA in plastid lipid biosynthesis. CA inhibitors did not affect acetyl-coenzyme A carboxylase activity or total storage protein synthesis. Similar results were obtained for two other plant systems: cell suspensions (and isolated plastids therefrom) of tobacco (Nicotiana tabacum), and chloroplasts isolated from leaves of transgenic CA antisense-suppressed tobacco plants (5% of wild-type CA activity). In addition, tobacco cell suspensions treated with the CA inhibitor ethoxyzolamide showed a substantial loss of CO2 compared with controls. The rate of [14C]acetate incorporation into lipid in cell suspensions was reduced by limiting external [CO2] (scrubbed air), and this rate was further reduced in the presence of ethoxyzolamide. Together, these results indicate that a reduction of CA activity (biochemical or molecular inhibition) impacts the rate of plant lipid biosynthesis from acetate, perhaps by impairing the ability of CA to efficiently “trap” inorganic carbon inside plastids for utilization by acetyl-coenzyme A carboxylase and the fatty acid synthesis machinery.

Abstract While cannabinoids are secondary metabolites synthesized by just a few plant species, N ‐acylethanolamines (NAEs) are distributed widely in the plant kingdom, and are recovered in measurable, bioactive quantities in many plant‐derived products. NAEs in higher plants are ethanolamides of fatty acids with acyl‐chain lenghts of C 12 C 18 and zero to three CC bonds. Generally, the most‐abundant NAEs found in plants and vertebrates are similar, including NAE 16 : 0, 18 : 1, 18 : 2, and 18 : 3. Like in animal systems, NAEs are formed in plants from N ‐acylphosphatidylethanolamines (NAPEs), and they are hydrolyzed by an amidase to yield ethanolamine and free fatty acids (FFA). Recently, a homologue of the mammalian fatty acid amide hydrolase (FAAH‐1) was identified in Arabidopsis thaliana and several other plant species. Overexpression of Arabidopsis FAAH (AtFAAH) resulted in plants that grew faster, but were more sensitive to biotic and abiotic insults, suggesting that the metabolism of NAEs in plants resides at the balance between growth and responses to environmental stresses. Similar to animal systems, exogenously applied NAEs have potent and varied effects on plant cells. Recent pharmacological approaches combined with molecular‐genetic experiments revealed that NAEs may act in certain plant tissues via specific membrane‐associated proteins or by interacting with phospholipase D‐ α , although other, direct targets for NAE action in plants are likely to be discovered. Polyunsaturated NAEs can be oxidized via the lipoxygenase pathway in plants, producing an array of oxylipin products that have received little attention so far. Overall, the conservation of NAE occurrence and metabolic machinery in plants, coupled with the profound physiological effects of elevating NAE content or perturbing endogenous NAE metabolism, suggest that an NAE‐mediated regulatory pathway, sharing similarities with the mammalian endocannabinoid pathway, indeed exists.

N-acylethanolamines are a group of lipid mediators that accumulate under a variety of neurological and pathological conditions in mammals. N-acylethanolamine signaling is terminated by the action of diverse hydrolases, among which fatty acid amide hydrolase (FAAH) has been well characterized. Here, we show that transgenic Arabidopsis lines overexpressing an AtFAAH are more susceptible to the bacterial pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. AtFAAH overexpressors also were highly susceptible to non-host pathogens P. syringae pv. syringae and P. syringae pv. tabaci. AtFAAH overexpressors had lower amounts of jasmonic acid, abscisic acid and both free and conjugated salicylic acid (SA), compared with the wild-type. Gene expression studies revealed that transcripts of a number of plant defense genes, as well as genes involved in SA biosynthesis and signaling, were lower in AtFAAH overexpressors than wild-type plants. Our data suggest that FAAH overexpression alters phytohormone accumulation and signaling which in turn compromises innate immunity to bacterial pathogens.

Summary. Background: von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF ‘activity’ assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen‐binding (VWF:CB) and VWF mAb‐based (VWF activity [VWF:Act]) assays are used by some laboratories.Objective:To perform a cross‐laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B.Methods:A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays.Results:The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within‐method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers.Conclusions:We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura. Summary. Background: von Willebrand disease (VWD), the most common inherited bleeding disorder, is caused by deficiencies and/or defects in von Willebrand factor (VWF). An effective diagnostic and VWD typing strategy requires plasma testing for factor VIII, and VWF antigen plus one or more VWF ‘activity’ assays. VWF activity is classically assessed by using VWF ristocetin cofactor activity (VWF:RCo), although VWF collagen‐binding (VWF:CB) and VWF mAb‐based (VWF activity [VWF:Act]) assays are used by some laboratories. To perform a cross‐laboratory study to specifically evaluate these three VWF activity assays for comparative sensitivity to loss of high molecular weight (HMW) VWF, representing the form of VWF that is most functionally active and that is absent in some types of VWD, namely 2A and 2B. A set of eight samples, including six selectively representing stepwise reduction in HMW VWF, were tested by 51 different laboratories using a variety of assays. The combined data showed that the VWF:CB and VWF:RCo assays had higher sensitivity to the loss of HMW VWF than did the VWF:Act assay. Moreover, within‐method analysis identified better HMW VWF sensitivity of some VWF:CB assays than of others, with all VWF:CB assays still showing better sensitivity than the VWF:Act assay. Differences were also identified between VWF:RCo methodologies on the basis of either platelet aggregometry or as performed on automated analyzers. We believe that these results have significant clinical implications for the diagnosis of VWD and monitoring of its therapy, as well as for the future diagnosis and therapy monitoring of thrombotic thrombocytopenic purpura.

Thrombotic microangiopathy (TMA) is a term used to describe a group of disorders characterized by hemolytic anemia (with prominent red blood cell fragmentation), thrombocytopenia, and thrombosis in the microvasculature. It may be used when describing patients with thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome, atypical hemolytic uremic syndrome, as well as a myriad of other disorders in which the TMA may be secondary to another disease or disorder. While limited information exists as to the exact cause of microthrombosis in many TMA, recent advances have been made in the understanding of TTP and its pathophysiology. This progress can be attributed to discovery of the von Willebrand factor cleaving protease ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), whose absence in TTP has given the disorder a distinct molecular identity. The discovery of this metalloprotease has prompted a significant amount of research relating to its role in TTP as well as its general function in hemostasis. The exact mechanisms by which this metalloprotease achieves its role are slowly being understood and these now provide other avenues by which TMA may occur.

The majority of commercial cotton varieties planted worldwide are derived from Gossypium hirsutum, which is a naturally occurring allotetraploid produced by interspecific hybridization of A- and D-genome diploid progenitor species. While most cotton species are adapted to warm, semi-arid tropical and subtropical regions, and thus perform well in these geographical areas, cotton seedlings are sensitive to cold temperature, which can significantly reduce crop yields. One of the common biochemical responses of plants to cold temperatures is an increase in omega-3 fatty acids, which protects cellular function by maintaining membrane integrity. The purpose of our study was to identify and characterize the omega-3 fatty acid desaturase (FAD) gene family in G. hirsutum, with an emphasis on identifying omega-3 FADs involved in cold temperature adaptation.Eleven omega-3 FAD genes were identified in G. hirsutum, and characterization of the gene family in extant A and D diploid species (G. herbaceum and G. raimondii, respectively) allowed for unambiguous genome assignment of all homoeologs in tetraploid G. hirsutum. The omega-3 FAD family of cotton includes five distinct genes, two of which encode endoplasmic reticulum-type enzymes (FAD3-1 and FAD3-2) and three that encode chloroplast-type enzymes (FAD7/8-1, FAD7/8-2, and FAD7/8-3). The FAD3-2 gene was duplicated in the A genome progenitor species after the evolutionary split from the D progenitor, but before the interspecific hybridization event that gave rise to modern tetraploid cotton. RNA-seq analysis revealed conserved, gene-specific expression patterns in various organs and cell types and semi-quantitative RT-PCR further revealed that FAD7/8-1 was specifically induced during cold temperature treatment of G. hirsutum seedlings.The omega-3 FAD gene family in cotton was characterized at the genome-wide level in three species, showing relatively ancient establishment of the gene family prior to the split of A and D diploid progenitor species. The FAD genes are differentially expressed in various organs and cell types, including fiber, and expression of the FAD7/8-1 gene was induced by cold temperature. Collectively, these data define the genetic and functional genomic properties of this important gene family in cotton and provide a foundation for future efforts to improve cotton abiotic stress tolerance through molecular breeding approaches.

Arabidopsis thaliana has been widely used as a model plant to study acyl lipid metabolism. Seeds of A. thaliana are quite small (approximately 500×300μm and weigh ~20μg), making lipid compositional analyses of single seeds difficult to achieve. Here we have used matrix assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to map and visualize the three-dimensional spatial distributions of two common membrane phospholipid classes, phosphatidylcholine (PC) and phosphatidylinositol (PI), in single A. thaliana seeds. The 3D images revealed distinct differences in distribution of several molecular species of both phospholipids among different seed tissues. Using data from these 3D reconstructions, the PC and PI mol% lipid profiles were calculated for the embryonic axis, cotyledons, and peripheral endosperm, and these data agreed well with overall quantification of these lipids in bulk seed extracts analyzed by conventional electrospray ionization-mass spectrometry (ESI-MS). In addition, MALDI-MSI was used to profile PC and PI molecular species in seeds of wild type, fad2-1, fad3-2, fad6-1, and fae1-1 acyl lipid mutants. The resulting distributions revealed previously unobserved changes in spatial distribution of several lipid molecular species, and were used to suggest new insights into biochemical heterogeneity of seed lipid metabolism. These studies highlight the value of mass spectrometry imaging to provide unprecedented spatial and chemical resolution of metabolites directly in samples even as small as a single A. thaliana seeds, and allow for expanded imaging of plant metabolites to improve our understanding of plant lipid metabolism from a spatial perspective.

Modified fatty acids (mFA) have diverse uses; for example, cyclopropane fatty acids (CPA) are feedstocks for producing coatings, lubricants, plastics and cosmetics. The expression of mFA-producing enzymes in crop and model plants generally results in lower levels of mFA accumulation than in their natural-occurring source plants. Thus, to further our understanding of metabolic bottlenecks that limit mFA accumulation, we generated transgenic Camelina sativa lines co-expressing Escherichia coli cyclopropane synthase (EcCPS) and Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT). In contrast to transgenic CPA-accumulating Arabidopsis, CPA accumulation in camelina caused only minor changes in seed weight, germination rate, oil accumulation and seedling development. CPA accumulated to much higher levels in membrane than storage lipids, comprising more than 60% of total fatty acid in both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) versus 26% in diacylglycerol (DAG) and 12% in triacylglycerol (TAG) indicating bottlenecks in the transfer of CPA from PC to DAG and from DAG to TAG. Upon co-expression of SfLPAT with EcCPS, di-CPA-PC increased by ~50% relative to lines expressing EcCPS alone with the di-CPA-PC primarily observed in the embryonic axis and mono-CPA-PC primarily in cotyledon tissue. EcCPS-SfLPAT lines revealed a redistribution of CPA from the sn-1 to sn-2 positions within PC and PE that was associated with a doubling of CPA accumulation in both DAG and TAG. The identification of metabolic bottlenecks in acyl transfer between site of synthesis (phospholipids) and deposition in storage oils (TAGs) lays the foundation for the optimizing CPA accumulation through directed engineering of oil synthesis in target crops.

Heparin induced thrombocytopenia (HIT) is a rare but potentially fatal complication of heparin therapy. In some patients, HIT causes platelet activation and thrombosis (sometimes abbreviated HITT), which leads to adverse clinical sequalae ('pathological HIT'). The likelihood of HIT is initially assessed clinically, typically using a scoring system, of which the 4T score is that most utilised. Subsequent laboratory testing to confirm or exclude HIT facilitates exclusion or diagnosis and management. The current investigation comprises a multicentre (n=9) assessment of contemporary laboratory testing for HIT, as performed over the past 1-3 years in each site and comprising testing of over 1200 samples. The primary laboratory test used by study participants (n=8) comprised a chemiluminescence procedure (HIT-IgG(PF4-H)) performed on an AcuStar instrument. Additional immunological testing performed by study sites included lateral flow (STiC, Stago), enzyme linked immunosorbent assay (ELISA), Asserachrom (HPIA IgG), PaGIA (BioRad), plus functional assays, primarily serotonin release assay (SRA) or platelet aggregation methods. The chemiluminescence procedure yielded a highly sensitive screening method for identifying functional HIT, given high area under the curve (AUC, generally ≥0.9) in a receiver operator characteristic (ROC) analysis against SRA as gold standard. ELISA testing resulted in lower ROC AUC scores (<0.8) and higher levels of false positives. Although there is clear association with the likelihood of HIT, the 4T score had less utility than literature suggests, and was comparable to a previous study reported by some of the authors.

The number of described contact sites between different subcellular compartments and structures in eukaryotic cells has increased dramatically in recent years and, as such, has substantially reinforced the well-known premise that these kinds of connections are essential for overall cellular organization and the proper functioning of cellular metabolic and signaling pathways. Here, we discuss contact sites involving plant lipid droplets (LDs), including LD-endoplasmic reticulum (ER) connections that mediate the biogenesis of new LDs at the ER, LD-peroxisome connections, that facilitate the degradation of LD-stored triacylglycerols (TAGs), and the more recently discovered LD-plasma membrane connections, which involve at least three novel proteins, but have a yet unknown physiological function(s).

Here, we assess several potential plant-based lubricants to reveal their functional characteristics in comparison to the traditionally used synthetic lubricant polyalphaolefin 4 and 10 oils. Our results indicate that jojoba and castor oils demonstrate the best oxidation resistance at elevated temperatures. The low erucic acid (LE) oils demonstrated a decrease in the lubrication efficiency already at 150 °C. This performance was inferior to that of canola oil which also has low erucic acid content, possibly due to the LE pennycress oil having relatively higher polyunsaturated fatty acid content. These results provide a comprehensive overview of plant-based oil candidates, including some with very long chain fatty acids and/or hydroxylated fatty acids, and suggest new concepts for lubrication efficiency improvement.

Thrombotic thrombocytopenic purpura (TTP) is a prothrombotic condition caused by a significant deficiency of the enzyme, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In the absence of adequate levels of ADAMTS13 (i.e., in TTP), plasma VWF accumulates, in particular as "ultra-large" VWF multimers, and this leads to pathological platelet aggregation and thrombosis. In addition to TTP, ADAMTS13 may be mildly to moderately reduced in a range of other conditions, including secondary thrombotic microangiopathies (TMA) such as those caused by infections (e.g., hemolytic uremic syndrome (HUS)), liver disease, disseminated intravascular coagulation (DIC), and sepsis, during acute/chronic inflammatory conditions, and sometimes also in COVID-19 (coronavirus disease 2019)). ADAMTS13 can be detected by a variety of techniques, including ELISA (enzyme-linked immunosorbent assay), FRET (fluorescence resonance energy transfer) and by chemiluminescence immunoassay (CLIA). The current report describes a protocol for assessment of ADAMTS13 by CLIA. This protocol reflects a rapid test able to be performed within 35 min on the AcuStar instrument (Werfen/Instrumentation Laboratory), although certain regional approvals may also permit this testing to be performed on a BioFlash instrument from the same manufacturer.

In a recent study of N-acylphosphatidylethanolamine (NAPE) metabolism in elicitor-treated tobacco (Nicotiana tabacum L.) cells, we identified a rapid release and accumulation of medium-chain N-acylethanolamines (NAEs) (e.g. N-myristoylethanolamine or NAE 14:0) and a compensatory decrease in cellular NAPE (K.D. Chapman, S. Tripathy, B. Venables, A.D. Desouza [1998] Plant Physiol 116: 1163-1168). In the present study, we extend this observation and report a 10- to 50-fold increase in NAE 14:0 content in leaves of tobacco (cv Xanthi) plants treated with xylanase or cryptogein elicitors. Exogenously supplied synthetic NAE species affected characteristic elicitor-induced and short- and long-term defense responses in cell suspensions of tobacco and long-term defense responses in leaves of intact tobacco plants. In general, synthetic NAEs inhibited elicitor-induced medium alkalinization by tobacco cells in a time- and concentration-dependent manner. Exogenous NAE 14:0 induced expression of phenylalanine ammonia lyase in a manner similar to fungal elicitors in both cell suspensions and leaves of tobacco. NAE 14:0, but not myristic acid, activated phenylalanine ammonia lyase expression at submicromolar concentrations, well within the range of NAE 14:0 levels measured in elicitor-treated plants. Collectively, these results suggest that NAPE metabolism, specifically, the accumulation of NAE 14:0, are part of a signal transduction pathway that modulates cellular defense responses following the perception of fungal elicitors.

Seven molecular species of N-acylethanolamines were quantified in seeds from selected members of the legume family. Total concentrations for the 14 taxa studied ranged over approximately three orders of magnitude with no consistent overall relationship to phylogeny. Elevated concentrations observed in some species make them good candidates for natural sources of these compounds which are of increasing therapeutic interest in the modulation of the mammalian endocannabinoid system.

The lipid profiles of cotton fiber cells were determined from total lipid extracts of elongating and maturing cotton fiber cells to see whether the membrane lipid composition changed during the phases of rapid cell elongation or secondary cell wall thickening. Total FA content was highest or increased during elongation and was lower or decreased thereafter, likely reflecting the assembly of the expanding cell membranes during elongation and the shift to membrane maintenance (and increase in secondary cell wall content) in maturing fibers. Analysis of lipid extracts by electrospray ionization and tandem MS (ESI-MS/MS) revealed that in elongating fiber cells (7-10 d post-anthesis), the polar lipids-PC, PE, PI, PA, phosphatidylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylglycerol-were most abundant. These same glycerolipids were found in similar proportions in maturing fiber cells (21 dpa). Detailed molecular species profiles were determined by ESI-MS/MS for all glycerolipid classes, and ESI-MS/MS results were consistent with lipid profiles determined by HPLC and ELSD. The predominant molecular species of PC, PE, PI, and PA was 34:3 (16:0, 18:3), but 36:6 (18:3,18:3) also was prevalent. Total FA analysis of cotton lipids confirmed that indeed linolenic (18:3) and palmitic (16:0) acids were the most abundant FA in these cell types. Bioinformatics data were mined from cotton fiber expressed sequence tag databases in an attempt to reconcile expression of lipid metabolic enzymes with lipid metabolite data. Together, these data form a foundation for future studies of the functional contribution of lipid metabolism to the development of this unusual and economically important cell type.

Fatty acid amides are a group of nitrogen-containing, lipid-soluble fatty acid derivatives, many of which display potent biological activities at very low concentrations. This group of compounds includes the family of N-acylethanolamines (NAE), minor lipid constituents naturally present in a variety of organisms from fungi to plants to mammals. Most of the work on NAE thus far has been in mammalian systems where these lipids exert diverse physiological, behavioral and neurological roles as part of the endocannabinoid signaling system. Corresponding studies on the function of NAE in plants have appeared only recently and much remains to be understood regarding the role of these fatty acid amides in plant physiology. The potent effects of NAE on plants, and the recent discovery of plant proteins that bind to, and catalyze the synthesis and degradation of NAE, point to the possibility that a signaling pathway similar to the endocannabinoid system occurs in plants. However, recent evidence suggests that the plant fatty acid amide hydrolase (FAAH), an enzyme involved predominantly in NAE catabolism, may have other functions that are independent of its catalytic activity, particularly in regard to salicylic acid (SA) and abscisic acid (ABA) signaling. Furthermore, the recent identification of a plant N-acylphosphatidylethanolamine (NAPE) synthase, the enzyme that catalyzes the formation of NAPE, the immediate lipid precursor of NAE, represents a significant breakthrough not only for plant NAE research but also for studies on the endocannabinoid system in animals. Although plants, like animals, possess the cellular machinery for metabolizing and responding to NAE, research in plants points to intriguing aspects of NAE signaling that have not been uncovered in studies with animals. Additional examples of bioactive plant fatty acid amides such as alkamides, N-acyl-homoserines, and amino acid-conjugated fatty acids (e.g., N-jasmonyl-isoleucine) may interact with this endogenous NAE metabolic machinery and modulate various phytohormone signaling pathways.

EDITORIAL article Front. Plant Sci., 27 June 2013Sec. Plant Physiology https://doi.org/10.3389/fpls.2013.00216

Abstract Modification of cottonseed quality traits is likely to be achieved through a combination of genetic modification, manipulation of nutrient allocation, and selective breeding. Oil and protein stores account for the majority of mass of cottonseed embryos. A more comprehensive understanding of the relationship between lint quality, lint yield, and embryo reserve accumulation will assist breeders in their efforts to improve seed value. Here we report the development of a rapid, nondestructive, simultaneous method for quantifying oil and protein levels within cottonseed by low‐field 1 H time‐domain nuclear magnetic resonance (TD‐NMR). This approach is suitable for a minimal amount of seed and represents an accurate (oil R 2 = 0.998, protein R 2 = 0.95), noninvasive alternative to conventional, time‐consuming chemical extractions. We demonstrate the value of this approach by surveying seed reserve content, identifying extremes of either protein and/or oil, in two sets of diverse germplasm.

N-Acylethanolamines (NAEs) are fatty-acid derivatives with potent biological activities in a wide range of eukaryotic organisms. Polyunsaturated NAEs are among the most abundant NAE types in seeds of Arabidopsis thaliana, and they can be metabolized by either fatty acid amide hydrolase (FAAH) or by lipoxygenase (LOX) to low levels during seedling establishment. Here, we identify and quantify endogenous oxylipin metabolites of N-linolenoylethanolamine (NAE 18:3) in Arabidopsis seedlings and show that their levels were higher in faah knockout seedlings. Quantification of oxylipin metabolites in lox mutants demonstrated altered partitioning of NAE 18:3 into 9- or 13-LOX pathways, and this was especially exaggerated when exogenous NAE was added to seedlings. When maintained at micromolar concentrations, NAE 18:3 specifically induced cotyledon bleaching of light-grown seedlings within a restricted stage of development. Comprehensive oxylipin profiling together with genetic and pharmacological interference with LOX activity suggested that both 9-hydroxy and 13-hydroxy linolenoylethanolamides, but not corresponding free fatty-acid metabolites, contributed to the reversible disruption of thylakoid membranes in chloroplasts of seedling cotyledons. We suggest that NAE oxylipins of linolenic acid represent a newly identified, endogenous set of bioactive compounds that may act in opposition to progression of normal seedling development and must be depleted for successful establishment.

Von Willebrand disease (VWD) is reportedly the most common inherited bleeding disorder and can also arise as an acquired syndrome (AVWS). These disorders develop due to defects and/or deficiency of the plasma protein von Willebrand factor (VWF). Laboratory testing for the VWF-related disorders requires assessment of both VWF level and VWF activity, the latter requiring multiple assays because of the many functions carried out by VWF to help prevent bleeding. As an additional step, an evaluation of VWF structural features by multimer analysis is useful in selective investigations. The current paper therefore describes a protocol for assessment of VWF multimers by gel electrophoresis, thus enabling identification of protein bands that represent differently sized multimers. The sample protocol described in this chapter is the methodology developed by Sebia.

Summary Fat storage‐inducing transmembrane protein 2 ( FIT 2) is an endoplasmic reticulum ( ER )‐localized protein that plays an important role in lipid droplet ( LD ) formation in animal cells. However, no obvious homologue of FIT 2 is found in plants. Here, we tested the function of FIT 2 in plant cells by ectopically expressing mouse ( Mus musculus ) FIT 2 in Nicotiana tabacum suspension‐cultured cells, Nicotiana benthamiana leaves and Arabidopsis thaliana plants. Confocal microscopy indicated that the expression of FIT 2 dramatically increased the number and size of LD s in leaves of N. benthamiana and Arabidopsis, and lipidomics analysis and mass spectrometry imaging confirmed the accumulation of neutral lipids in leaves. FIT 2 also increased seed oil content by ~13% in some stable, overexpressing lines of Arabidopsis . When expressed transiently in leaves of N. benthamiana or suspension cells of N. tabacum , FIT 2 localized specifically to the ER and was often concentrated at certain regions of the ER that resembled ER ‐ LD junction sites. FIT 2 also colocalized at the ER with other proteins known to be involved in triacylglycerol biosynthesis or LD formation in plants, but not with ER resident proteins involved in electron transfer or ER ‐vesicle exit sites. Collectively, these results demonstrate that mouse FIT 2 promotes LD accumulation in plants, a surprising functional conservation in the context of a plant cell given the apparent lack of FIT 2 homologues in higher plants. These results suggest also that FIT 2 expression represents an effective synthetic biology strategy for elaborating neutral lipid compartments in plant tissues for potential biofuel or bioproduct purposes.

The seeds of many nondomesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered seeds remains low and is often hampered by their inefficient exclusion from phospholipids. Recent studies have established the feasibility of increasing triacylglycerol content in plant leaves, which provides a novel approach for increasing energy density of biomass crops. Here, we determined whether the fatty acid composition of leaf oil could be engineered to accumulate unusual fatty acids. Eleostearic acid (ESA) is a conjugated fatty acid produced in seeds of the tung tree (Vernicia fordii) and has both industrial and nutritional end-uses. Arabidopsis thaliana lines with elevated leaf oil were first generated by transforming wild-type, cgi-58 or pxa1 mutants (the latter two of which contain mutations disrupting fatty acid breakdown) with the diacylglycerol acyltransferases (DGAT1 or DGAT2) and/or oleosin genes from tung. High-leaf-oil plant lines were then transformed with tung FADX, which encodes the fatty acid desaturase/conjugase responsible for ESA synthesis. Analysis of lipids in leaves revealed that ESA was efficiently excluded from phospholipids, and co-expression of tung FADX and DGAT2 promoted a synergistic increase in leaf oil content and ESA accumulation. Taken together, these results provide a new approach for increasing leaf oil content that is coupled with accumulation of unusual fatty acids. Implications for production of biofuels, bioproducts, and plant-pest interactions are discussed.

Fatty acid amide hydrolase (FAAH) is the signal-terminating enzyme of the N-acylethanolamine signaling pathway with an established role in seedling development. The crystal structure of Arabidopsis thaliana FAAH was recently solved, revealing for the first time the structural features of FAAH from plants and explaining the enzyme’s promiscuity toward N-acyl amide substrates. A second group of FAAH enzymes in angiosperms has been identified with conserved substitutions in the substrate-binding pocket altering the size, shape, and physicochemical properties for substrate recognition. Fatty acid amide hydrolase (FAAH) is an enzyme that belongs to the amidase signature (AS) superfamily and is widely distributed in multicellular eukaryotes. FAAH hydrolyzes lipid signaling molecules – namely, N-acylethanolamines (NAEs) – which terminates their actions. Recently, the crystal structure of Arabidopsis thaliana FAAH was solved and key residues were identified for substrate-specific interactions. Here, focusing on residues surrounding the substrate-binding pocket, a comprehensive analysis of FAAH sequences from angiosperms reveals a distinctly different family of FAAH-like enzymes. We hypothesize that FAAH, in addition to its role in seedling development, also acts in an N-acyl amide communication axis to facilitate plant–microbe interactions and that structural diversity provides for the flexible use of a wide range of small lipophilic signaling molecules. Fatty acid amide hydrolase (FAAH) is an enzyme that belongs to the amidase signature (AS) superfamily and is widely distributed in multicellular eukaryotes. FAAH hydrolyzes lipid signaling molecules – namely, N-acylethanolamines (NAEs) – which terminates their actions. Recently, the crystal structure of Arabidopsis thaliana FAAH was solved and key residues were identified for substrate-specific interactions. Here, focusing on residues surrounding the substrate-binding pocket, a comprehensive analysis of FAAH sequences from angiosperms reveals a distinctly different family of FAAH-like enzymes. We hypothesize that FAAH, in addition to its role in seedling development, also acts in an N-acyl amide communication axis to facilitate plant–microbe interactions and that structural diversity provides for the flexible use of a wide range of small lipophilic signaling molecules. a group of HLs that are produced by some photosynthetic bacteria and contain an aromatic tail instead of the ‘canonical’ aliphatic acyl chains in AHLs. enzymes that are best known for their role in terminating NAE signaling pathways in multicellular eukaryotes. group of amino acid residues located at the N terminus of the arabidopsis FAAH, where they define the opening of the MAC and were shown to undergo conformational changes and close the MAC of AtFAAH on ligand binding. a family of functionally diverse signaling lipids. They comprise a fatty acid linked by an amide bond to ethanolamine and are classified based on the chain length and degree of unsaturation of their acyl chain. a class of QS signals produced by Gram-negative bacteria to coordinate their group activity based on the population density. They comprise an acyl chain linked by an amide bond to a homoserine lactone head group. the NAE abbreviation includes two numbers x:y, where x indicates the number of carbons and y indicates the number of double bonds in the corresponding acyl chain. For example, NAE 16:0 is an NAE with a 16-carbon acyl chain that has no double bonds, while NAE 18:2 is an NAE with an 18-carbon acyl chain that has two double bonds. bioactive lipid metabolites that are derived from polyunsaturated fatty acids or polyunsaturated NAEs (e.g., NAE 18:2, NAE 18:3) via the actions of a family of dioxygenases named lipoxygenases (9-LOX and 13-LOX), which introduce hydro(pero)xy functional groups at the C9 or C13 position of the acyl chain.

Lipid droplets (LDs) are neutral-lipid-containing organelles found in all kingdoms of life and are coated with proteins that carry out a vast array of functions. Compared to mammals and yeast, relatively few LD proteins have been identified in plants, particularly those associated with LDs in vegetative (non-seed) cell types. Thus, to better understand the cellular roles of LDs in plants, a more comprehensive inventory and characterization of LD proteins is required. Here, we performed a proteomics analysis of LDs isolated from drought-stressed Arabidopsis leaves and identified EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) as a putative LD protein. mCherry-tagged ERD7 localized to both LDs and the cytosol when ectopically expressed in plant cells, and the protein’s C-terminal senescence domain (SD) was both necessary and sufficient for LD targeting. Phylogenetic analysis revealed that ERD7 belongs to a six-member family in Arabidopsis that, along with homologs in other plant species, is separated into two distinct subfamilies. Notably, the SDs of proteins from each subfamily conferred targeting to either LDs or mitochondria. Further, the SD from the ERD7 homolog in humans, spartin, localized to LDs in plant cells, similar to its localization in mammals; although, in mammalian cells, spartin also conditionally localizes to other subcellular compartments, including mitochondria. Disruption of ERD7 gene expression in Arabidopsis revealed no obvious changes in LD numbers or morphology under normal growth conditions, although this does not preclude a role for ERD7 in stress-induced LD dynamics. Consistent with this possibility, a yeast two-hybrid screen using ERD7 as bait identified numerous proteins involved in stress responses, including some that have been identified in other LD proteomes. Collectively, these observations provide new insight to ERD7 and the SD-containing family of proteins in plants and suggest that ERD7 may be involved in functional aspects of plant stress response that also include localization to the LD surface.

Plant-derived oils are a major agricultural product that exist in both ubiquitous forms such as common vegetable oils and in specialized forms such as castor oil and coconut oil. These specialized oils are the result of lineage-specific metabolic pathways that create oils rich in unusual fatty acids. Considerable progress has been made toward understanding the enzymes that mediate fatty acid biosynthesis, triacylglycerol assembly, and oil storage. However, efforts to translate this knowledge into renewable bioproducts via engineered oil-producing plants and algae have had limited success. Here, we review recent evidence that protein-protein interactions in each of the three major phases of oil formation appear to have profound effects on specialized oil accumulation. We suggest that furthering our knowledge of the noncatalytic attributes of enzymes and other proteins involved in oil formation will be a critical step toward creating renewable bioproducts derived from high performing, engineered oilseeds.

Abstract Introduction Lupus anticoagulant (LA) testing is commonly performed within hemostasis laboratories, and the ACL TOP 50 family of instruments represent a new “single platform” of hemostasis instrumentation. Our aim was to evaluate these instruments and manufacturer reagents or alternatives for utility in LA testing. Methods Comparative evaluations of LA testing using newly installed ACL TOPs 550 and 750 as well as comparative assessments with existing “reference,” predominantly Stago, instrumentation, and reagents. Evaluations comprised both dilute Russell viper venom time (dRVVT) and activated partial thromboplastin time (APTT)‐based assays. Establishment of normal reference ranges (NRR). Results The HemosIL dRVVT‐based assays showed good comparability with the existing Stago reference method (R &gt; 0.9) and could be considered as verified as fit for purpose. A variety of APTT assays was additionally evaluated for LA utility, and we identified from the assessment good utility of a non‐Werfen solution in Hyphen BioMed Cephen reagents. NRR were established based on ≥120 normal individual plasma samples. Conclusion This evaluation of LA reagents on ACL TOP 50 Family instruments identified overall acceptable performance of both dRVVT (Werfen solution) and APTT (non‐Werfen solution) to enable harmonization of LA testing in our large network.

N-Acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), was recently shown to be synthesized from PE and free fatty acids in cotyledons of cotton (Gossypium hirsutum L.) seedlings (K.D. Chapman, T.S. Moore [1993] Plant Physiol 102: 761-769). Here we report that NAPE is present in dry seeds of cotton and increases with time of imbibition from 2.31 nmol/seed in dry seeds to 4.26 nmol/seed in 4-h-soaked seeds. Total phospholipid/seed also increased such that the relative percentage of NAPE was similar in dry and soaked seeds (2.3 mol% compared to 2.6 mol%, respectively). The major molecular species of NAPE were identified in both dry and soaked seeds by fast atom bombardment mass spectrometry and collisionally activated dissociation tandem mass spectrometry as 16:0/18:2-PE(N-palmitoyl), 16:0/18:2-PE(N-linoleoyl), and 18:2/18:2-PE(N-palmitoyl). The specific activity of NAPE synthase in seed extracts increased with increasing time of imbibition from 35 pmol h-1 mg-1 protein in dry seeds to 129 pmol h-1 mg-1 protein in 4-h-soaked seeds. Collectively, our results indicate that NAPE is present in dry cottonseeds and synthesized during imbibition. The biosynthesis of NAPE provides a mechanism for maintaining membrane integrity during seed rehydration and may indicate that NAPE plays a protective role in intracellular membranes of plant tissues, as has been suggested for intracellular membranes of animal tissues.

Saturated and unsaturated N-acylethanolamines (NAEs) occur in desiccated seeds primarily as 16C and 18C species with N-palmitoylethanolamine and N-linoleoylethanolamine (NAE 18:2) being most abundant. Here, we examined the metabolic fate of NAEs in vitro and in vivo in imbibed cotton (Gossypium hirsutum) seeds. When synthetic [1-(14)C]N-palmitoylethanolamine was used as a substrate, free fatty acids (FFA) were produced by extracts of imbibed cottonseeds. When synthetic [1-(14)C]NAE 18:2 was used as a substrate, FFA and an additional lipid product(s) were formed. On the basis of polarity, we presumed that the unidentified lipid was a product of the lipoxygenase (LOX) pathway and that inclusion of the characteristic LOX inhibitors nordihydroguaiaretic acid and eicosatetraynoic acid reduced its formation in vitro and in vivo. The conversion of NAE 18:2 in imbibed cottonseed extracts to 12-oxo-13-hydroxy-N-(9Z)-octadecanoylethanolamine was confirmed by gas chromatography-mass spectrometry, indicating the presence of 13-LOX and 13-allene oxide synthase, which metabolized NAE 18:2. Cell fractionation studies showed that the NAE amidohydrolase, responsible for FFA production, was associated mostly with microsomes, whereas LOX, responsible for NAE 18:2-oxylipin production, was distributed in cytosol-enriched fractions and microsomes. The highest activity toward NAE by amidohydrolase was observed 4 to 8 h after imbibition and by LOX 8 h after imbibition. Our results collectively indicate that two pathways exist for NAE metabolism during seed imbibition: one to hydrolyze NAEs in a manner similar to the inactivation of endocannabinoid mediators in animal systems and the other to form novel NAE-derived oxylipins. The rapid depletion of NAEs by these pathways continues to point to a role for NAE metabolites in seed germination.

The role of the anticholinergic drug, ipratropium bromide, in maintenance antiasthmatic therapy was evaluated in a double-blind crossover trial of three bronchodilator regimens: (1) inhaled ipratropium, placebo, and oral oxtriphylline; (2) inhaled fenoterol, placebo, and oral oxtriphylline; and (3) both inhaled ipratropium and fenoterol plus oral oxtriphylline. Twenty-two asthmatics were treated with all three regimens, each for 1 mo, allocated in random sequence. On the first and last treatment days of each month, spirometric measurements were performed before and 0.5, 1, 2, 3, 4, and 6 hr after administration of the test drugs. On the first treatment day of each month, all regimens produced significant bronchodilatation at 30 min after dose, an improvement that declined between 3 and 6 hr after dose. After continuous administration for 1 mo the two combinations employing fenoterol showed a decline in bronchodilator responsiveness from the initial treatment day, measured as the level of response (V50) or duration of response (FEV1, VC). Ipratropium plus oxtriphylline showed no such decline, suggesting the development of tolerance to long-term administration of fenoterol. Overall benefit at the end of 1 mo, measured as the area under the curves of FEV1, VC, or V50 vs time after dose, was greatest for the triple drug regimen. There were no differences in heart rate, blood pressure response, or side effects among the three treatments. It is concluded that when the anticholinergic drug ipratropium is administered concurrently with an inhaled beta 2 agonist and an oral theophylline derivative, increased bronchodilatation occurs with no detectable additional side effects.

N-acylethanolamines (NAEs) are lipids upregulated in response to cell and tissue injury and are involved in cytoprotection. Arachidonylethanolamide (AEA) is a well characterized NAE that is an endogenous ligand at cannabinoid and vanilloid receptors, but it exists in small quantities relative to other NAE types. The abundance of other NAE species, such as palmitoylethanolamine (PEA), together with their largely unknown function and receptors, has prompted us to examine the neuroprotective properties and mechanism of action of PEA. We hypothesized that PEA protects HT22 cells from oxidative stress and activates neuroprotective kinase signaling pathways.Indeed PEA protected HT22 cells from oxidative stress in part by mediating an increase in phosphorylated Akt (pAkt) and ERK1/2 immunoreactivity as well as pAkt nuclear translocation. These changes take place within a time frame consistent with neuroprotection. Furthermore, we determined that changes in pAkt immunoreactivity elicited by PEA were not mediated by activation of cannabinoid receptor type 2 (CB2), thus indicating a novel mechanism of action. These results establish a role for PEA as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.The results from this study reveal that PEA protects HT22 cells from oxidative stress and alters the localization and expression levels of kinases known to be involved in neuroprotection by a novel mechanism. Overall, these results identify PEA as a neuroprotectant with potential as a possible therapeutic agent in neurodegenerative diseases involving oxidative stress.

N-acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the "endocannabinoid" signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives that occur in all eukaryotes. In plants, NAEs have potent negative growth-regulating properties, and fatty acid amide hydrolase (FAAH)-mediated hydrolysis is a primary catabolic pathway that operates during seedling establishment to deplete these compounds. Alternatively, polyunsaturated (PU)-NAEs may serve as substrates for lipid oxidation. In Arabidopsis, PU-NAEs (NAE 18:2 and NAE 18:3) were the most abundant NAE species in seeds, and their levels were depleted during seedling growth even in FAAH tDNA knock-out plants. Therefore, we hypothesized that lipoxygenase (LOX) participated in the metabolism of PU-NAEs through the formation of NAE-oxylipins. Comprehensive chromatographic and mass spectrometric methods were developed to identify NAE hydroperoxides and -hydroxides. Recombinant Arabidopsis LOX enzymes expressed in Escherichia coli utilized NAE 18:2 and NAE 18:3 as substrates with AtLOX1 and AtLOX5 exhibiting 9-LOX activity and AtLOX2, AtLOX3, AtLOX4, and AtLOX6 showing predominantly 13-LOX activity. Feeding experiments with exogenous PU-NAEs showed they were converted to hydroxide metabolites indicating that indeed Arabidopsis seedlings had the capacity for LOX-mediated metabolism of PU-NAEs in planta. Detectable levels of endogenous NAE-oxylipin metabolites were identified in FAAH fatty acid amide hydrolase seedlings but not in wild-type or FAAH overexpressors, suggesting that NAE hydroxide pools normally do not accumulate unless flux through hydrolysis is substantially reduced. These data suggest that Arabidopsis LOXs indeed compete with FAAH to metabolize PU-NAEs during seedling establishment. Identification of endogenous amide-conjugated oxylipins suggests potential significance of these metabolites in vivo, and FAAH mutants may offer opportunities to address this in the future. N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives that occur in all eukaryotes. In plants, NAEs have potent negative growth-regulating properties, and fatty acid amide hydrolase (FAAH)-mediated hydrolysis is a primary catabolic pathway that operates during seedling establishment to deplete these compounds. Alternatively, polyunsaturated (PU)-NAEs may serve as substrates for lipid oxidation. In Arabidopsis, PU-NAEs (NAE 18:2 and NAE 18:3) were the most abundant NAE species in seeds, and their levels were depleted during seedling growth even in FAAH tDNA knock-out plants. Therefore, we hypothesized that lipoxygenase (LOX) participated in the metabolism of PU-NAEs through the formation of NAE-oxylipins. Comprehensive chromatographic and mass spectrometric methods were developed to identify NAE hydroperoxides and -hydroxides. Recombinant Arabidopsis LOX enzymes expressed in Escherichia coli utilized NAE 18:2 and NAE 18:3 as substrates with AtLOX1 and AtLOX5 exhibiting 9-LOX activity and AtLOX2, AtLOX3, AtLOX4, and AtLOX6 showing predominantly 13-LOX activity. Feeding experiments with exogenous PU-NAEs showed they were converted to hydroxide metabolites indicating that indeed Arabidopsis seedlings had the capacity for LOX-mediated metabolism of PU-NAEs in planta. Detectable levels of endogenous NAE-oxylipin metabolites were identified in FAAH fatty acid amide hydrolase seedlings but not in wild-type or FAAH overexpressors, suggesting that NAE hydroxide pools normally do not accumulate unless flux through hydrolysis is substantially reduced. These data suggest that Arabidopsis LOXs indeed compete with FAAH to metabolize PU-NAEs during seedling establishment. Identification of endogenous amide-conjugated oxylipins suggests potential significance of these metabolites in vivo, and FAAH mutants may offer opportunities to address this in the future.

Targeted increases in monounsaturated (oleic acid) fatty acid content of refined cottonseed oil could support improved human nutrition and cardiovascular health. Genetic modifications of cottonseed fatty acid composition have been accomplished using several different molecular strategies. Modification of oleic acid content in cottonseed embryos using a dominant-negative protein approach, while successful in effecting change in the desired fatty acid composition, resulted in reduced oil content and seed viability. Here these changes in fatty acid composition were associated with changes in dominant molecular species of triacylglycerols (TAGs) and their spatial distributions within embryo tissues. A combination of mass spectrometry (MS)-based lipidomics approaches, including MS imaging of seed cryo-sections, revealed that cotton embryos expressing a non-functional allele of a Brassica napus delta-12 desaturase showed altered accumulation of TAG species, especially within cotyledonary tissues. While lipid analysis of seed extracts could demonstrate detailed quantitative changes in TAG species in transgenics, the spatial contribution of metabolite compartmentation could only be visualized by MS imaging. Our results suggest tissue-specific differences in TAG biosynthetic pathways within cotton embryos, and indicate the importance of considering the location of metabolites in tissues in addition to their identification and quantification when developing a detailed view of cellular metabolism.

ABSTRACT Recent studies have suggested that cottonseed ( Gossypium spp.) has the potential to contribute to the effort against world hunger, particularly by providing a high‐quality protein source. This report analyzed the diversity in protein content and other seed quality factors in the U.S. National Cotton Germplasm Collection. Nine genomes (one tetraploid and eight diploid) and 33 species (five tetraploid and 28 diploid) were surveyed in this examination of 2256 accessions. A novel nondestructive nuclear magnetic resonance technique was applied to measure oil and protein content, seed indices were calculated, and these data were associated with molecular marker information. Oil content ranged from 8 to 27%, protein values ranged from 10 to 36%, and seed index was lowest at 1 g per 100 seeds and extended up to 18 g per 100 seeds. Most of the range in values for these traits resided within G. hirsutum L. and G. barbadense L.; thus implying that variability for cottonseed quality can be introduced with relative ease in current breeding programs. The diploid genomes generally had extremely low values for oil, protein, and seed index. Molecular marker information indicated that chromosome 21 was likely associated with oil content. Understanding how these seed quality factors vary independently and in relation to each other will allow us to better select parents for breeding programs, and identifying associations with molecular markers may help us enhance progress through marker‐assisted selection approaches.