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1.
J Exp Bot ; 71(18): 5521-5537, 2020 09 19.
Article in English | MEDLINE | ID: mdl-32556164

ABSTRACT

The cuticle is composed of cutin and cuticular waxes, and it is the first protective barrier to abiotic and biotic stresses in fruit. In this study, we analysed the composition of and changes in cuticular waxes during fruit development in nectarine (Prunus persica L. Batsch) cultivars, in parallel with their conductance and their susceptibility to Monilinia laxa. The nectarine waxes were composed of triterpenoids, mostly ursolic and oleanolic acids, phytosterols, and very-long-chain aliphatics. In addition, we detected phenolic compounds that were esterified with sugars or with triterpenoids, which are newly described in cuticular waxes. We quantified 42 compounds and found that they changed markedly during fruit development, with an intense accumulation of triterpenoids during initial fruit growth followed by their decrease at the end of endocarp lignification and a final increase in very-long-chain alkanes and hydroxylated triterpenoids until maturity. The surface conductance and susceptibility to Monilinia decreased sharply at the beginning of endocarp lignification, suggesting that triterpenoid deposition could play a major role in regulating fruit permeability and susceptibility to brown rot. Our results provide new insights into the composition of cuticular waxes of nectarines and their changes during fruit development, opening new avenues of research to explore brown rot resistance factors in stone fruit.


Subject(s)
Ascomycota , Prunus persica , Fruit , Waxes
2.
J Biol Chem ; 289(32): 21984-94, 2014 Aug 08.
Article in English | MEDLINE | ID: mdl-24917677

ABSTRACT

The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1Δ gat2Δ. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.


Subject(s)
Acyltransferases/metabolism , Aldehyde Oxidoreductases/metabolism , Lipids/biosynthesis , Protozoan Proteins/metabolism , Tetrahymena thermophila/metabolism , Acyltransferases/chemistry , Acyltransferases/genetics , Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/genetics , Ethers/metabolism , Gene Fusion , Genes, Protozoan , Genetic Complementation Test , Palmitoyl Coenzyme A/metabolism , Plants, Genetically Modified , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Substrate Specificity , Tetrahymena thermophila/genetics , Nicotiana/genetics , Nicotiana/metabolism
3.
Plant Physiol ; 163(3): 1118-32, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24019425

ABSTRACT

Suberin is found in a variety of tissues, such as root endoderms and periderms, storage tuber periderms, tree cork layer, and seed coats. It acts as a hydrophobic barrier to control the movement of water, gases, and solutes as well as an antimicrobial barrier. Suberin consists of polymerized phenolics, glycerol, and a variety of fatty acid derivatives, including primary fatty alcohols. We have conducted an in-depth analysis of the distribution of the C18:0 to C22:0 fatty alcohols in Arabidopsis (Arabidopsis thaliana) roots and found that only 20% are part of the root suberin polymer, together representing about 5% of its aliphatic monomer composition, while the remaining 80% are found in the nonpolymeric (soluble) fraction. Down-regulation of Arabidopsis FATTY ACYL REDUCTASE1 (FAR1), FAR4, and FAR5, which collectively produce the fatty alcohols found in suberin, reduced their levels by 70% to 80% in (1) the polymeric and nonpolymeric fractions from roots of tissue culture-grown plants, (2) the suberin-associated root waxes from 7-week-old soil-grown plants, and (3) the seed coat suberin polymer. By contrast, the other main monomers of suberin were not altered, indicating that reduced levels of fatty alcohols did not influence the suberin polymerization process. Nevertheless, the 75% reduction in total fatty alcohol and diol loads in the seed coat resulted in increased permeability to tetrazolium salts and a higher sensitivity to abscisic acid. These results suggest that fatty alcohols and diols play an important role in determining the functional properties of the seed coat suberin barrier.


Subject(s)
Arabidopsis/metabolism , Fatty Alcohols/metabolism , Lipids/analysis , Plant Roots/metabolism , Seeds/metabolism , Aldehyde Oxidoreductases/genetics , Aldehyde Oxidoreductases/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Chromatography, Gas , Down-Regulation , Fatty Acids/metabolism , Gene Expression Regulation, Plant , Lipids/chemistry , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Plant Roots/genetics , Plants, Genetically Modified , Reverse Transcriptase Polymerase Chain Reaction , Seeds/genetics , Waxes/analysis , Waxes/chemistry
4.
Eukaryot Cell ; 12(10): 1326-34, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23748431

ABSTRACT

When they are fed with bacteria, Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs), which are composed of membranous material. It has been proposed that MLBs are a waste disposal system that allows D. discoideum to eliminate undigested bacterial remains. However, the real function of MLBs remains unknown. Determination of the biochemical composition of MLBs, especially lipids, represents a way to gain information about the role of these structures. To allow these analyses, a protocol involving various centrifugation procedures has been developed to purify secreted MLBs from amoeba-bacterium cocultures. The purity of the MLB preparation was confirmed by transmission electron microscopy and by immunofluorescence using H36, an antibody that binds to MLBs. The lipid and fatty acid compositions of pure MLBs were then analyzed by high-performance thin-layer chromatography (HPTLC) and gas chromatography (GC), respectively, and compared to those of amoebae as well as bacteria used as a food source. While the bacteria were devoid of phosphatidylcholine (PC) and phosphatidylinositol (PI), these two polar lipid species were major classes of lipids in MLBs and amoebae. Similarly, the fatty acid composition of MLBs and amoebae was characterized by the presence of polyunsaturated fatty acids, while cyclic fatty acids were found only in bacteria. These results strongly suggest that the lipids constituting the MLBs originate from the amoebal metabolism rather than from undigested bacterial membranes. This opens the possibility that MLBs, instead of being a waste disposal system, have unsuspected roles in D. discoideum physiology.


Subject(s)
Dictyostelium/metabolism , Exocytosis , Fatty Acids/metabolism , Phosphatidylcholines/metabolism , Phosphatidylinositols/metabolism , Secretory Vesicles/metabolism , Dictyostelium/physiology , Dictyostelium/ultrastructure , Phagocytosis , Secretory Vesicles/ultrastructure
5.
Plant J ; 73(5): 733-46, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23384041

ABSTRACT

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl-CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2-like family: CER2, CER26 and CER26-like . Expression pattern analysis showed that the three genes are differentially expressed in an organ- and tissue-specific manner. Using individual T-DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl-CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/metabolism , Fatty Acids/metabolism , Gene Expression Regulation, Plant , Waxes/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Lipid Metabolism , Lipids , Multigene Family , Mutagenesis, Insertional , Organ Specificity , Plant Components, Aerial/cytology , Plant Components, Aerial/genetics , Plant Components, Aerial/metabolism , Plant Epidermis/cytology , Plant Epidermis/genetics , Plant Epidermis/metabolism , Plant Roots/cytology , Plant Roots/genetics , Plant Roots/metabolism , Plants, Genetically Modified , RNA, Plant/genetics , Substrate Specificity , Waxes/chemistry
6.
Plant Cell ; 24(7): 3106-18, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22773744

ABSTRACT

In land plants, very-long-chain (VLC) alkanes are major components of cuticular waxes that cover aerial organs, mainly acting as a waterproof barrier to prevent nonstomatal water loss. Although thoroughly investigated, plant alkane synthesis remains largely undiscovered. The Arabidopsis thaliana ECERIFERUM1 (CER1) protein has been recognized as an essential element of wax alkane synthesis; nevertheless, its function remains elusive. In this study, a screen for CER1 physical interaction partners was performed. The screen revealed that CER1 interacts with the wax-associated protein ECERIFERUM3 (CER3) and endoplasmic reticulum-localized cytochrome b5 isoforms (CYTB5s). The functional relevance of these interactions was assayed through an iterative approach using yeast as a heterologous expression system. In a yeast strain manipulated to produce VLC acyl-CoAs, a strict CER1 and CER3 coexpression resulted in VLC alkane synthesis. The additional presence of CYTB5s was found to enhance CER1/CER3 alkane production. Site-directed mutagenesis showed that CER1 His clusters are essential for alkane synthesis, whereas those of CER3 are not, suggesting that CYTB5s are specific CER1 cofactors. Collectively, our study reports the identification of plant alkane synthesis enzymatic components and supports a new model for alkane production in which CER1 interacts with both CER3 and CYTB5 to catalyze the redox-dependent synthesis of VLC alkanes from VLC acyl-CoAs.


Subject(s)
Alkanes/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cytochromes b5/metabolism , Nuclear Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Alkanes/chemistry , Amino Acid Motifs , Arabidopsis/chemistry , Arabidopsis/genetics , Arabidopsis/ultrastructure , Arabidopsis Proteins/genetics , Carbon-Carbon Lyases , Cytochromes b5/genetics , Gene Expression Regulation, Plant , Models, Molecular , Mutagenesis, Site-Directed , Nuclear Proteins/genetics , Oxidation-Reduction , Plant Epidermis/chemistry , Plant Epidermis/genetics , Plant Epidermis/metabolism , Plant Epidermis/ultrastructure , Plant Stems/chemistry , Plant Stems/genetics , Plant Stems/metabolism , Plant Stems/ultrastructure , Plants, Genetically Modified , Protein Interaction Mapping , Protein Isoforms/genetics , Protein Isoforms/metabolism , Recombinant Fusion Proteins , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Seedlings/chemistry , Seedlings/genetics , Seedlings/metabolism , Seedlings/ultrastructure , Transgenes
7.
PLoS One ; 7(1): e30686, 2012.
Article in English | MEDLINE | ID: mdl-22295104

ABSTRACT

CD36 is a ubiquitous membrane glycoprotein that binds long-chain fatty acids. The presence of a functional CD36 is required for the induction of satiety by a lipid load and its role as a lipid receptor driving cellular signal has recently been demonstrated. Our project aimed to further explore the role of intestinal CD36 in the regulation of food intake. Duodenal infusions of vehicle or sulfo-N-succinimidyl-oleate (SSO) was performed prior to acute infusions of saline or Intralipid (IL) in mice. Infusion of minute quantities of IL induced a decrease in food intake (FI) compared to saline. Infusion of SSO had the same effect but no additive inhibitory effect was observed in presence of IL. No IL- or SSO-mediated satiety occurred in CD36-null mice. To determine whether the CD36-mediated hypophagic effect of lipids was maintained in animals fed a satietogen diet, mice were subjected to a High-Protein diet (HPD). Concomitantly with the satiety effect, a rise in intestinal CD36 gene expression was observed. No satiety effect occurred in CD36-null mice. HPD-fed WT mice showed a diminished FI compared to control mice, after saline duodenal infusion. But there was no further decrease after lipid infusion. The lipid-induced decrease in FI observed on control mice was accompanied by a rise in jejunal oleylethanolamide (OEA). Its level was higher in HPD-fed mice than in controls after saline infusion and was not changed by lipids. Overall, we demonstrate that lipid binding to intestinal CD36 is sufficient to produce a satiety effect. Moreover, it could participate in the satiety effect induced by HPD. Intestine can modulate FI by several mechanisms including an increase in OEA production and CD36 gene expression. Furthermore, intestine of mice adapted to HPD have a diminished capacity to modulate their food intake in response to dietary lipids.


Subject(s)
CD36 Antigens/metabolism , Diet , Intestinal Mucosa/metabolism , Satiety Response , Animals , CD36 Antigens/genetics , Eating/drug effects , Gene Expression Regulation/drug effects , Intestines/drug effects , Ligands , Male , Mice , Mice, Inbred C57BL , Oleic Acids/biosynthesis , Oleic Acids/metabolism , Oleic Acids/pharmacology , Protein Binding , Satiety Response/drug effects , Succinimides/metabolism , Succinimides/pharmacology , Time Factors
8.
Mol Biol Cell ; 23(2): 233-46, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22090344

ABSTRACT

For many years, lipid droplets (LDs) were considered to be an inert store of lipids. However, recent data showed that LDs are dynamic organelles playing an important role in storage and mobilization of neutral lipids. In this paper, we report the characterization of LOA1 (alias VPS66, alias YPR139c), a yeast member of the glycerolipid acyltransferase family. LOA1 mutants show abnormalities in LD morphology. As previously reported, cells lacking LOA1 contain more LDs. Conversely, we showed that overexpression results in fewer LDs. We then compared the lipidome of loa1Δ mutant and wild-type strains. Steady-state metabolic labeling of loa1Δ revealed a significant reduction in triacylglycerol content, while phospholipid (PL) composition remained unchanged. Interestingly, lipidomic analysis indicates that both PLs and glycerolipids are qualitatively affected by the mutation, suggesting that Loa1p is a lysophosphatidic acid acyltransferase (LPA AT) with a preference for oleoyl-CoA. This hypothesis was tested by in vitro assays using both membranes of Escherichia coli cells expressing LOA1 and purified proteins as enzyme sources. Our results from purification of subcellular compartments and proteomic studies show that Loa1p is associated with LD and active in this compartment. Loa1p is therefore a novel LPA AT and plays a role in LD formation.


Subject(s)
Acyltransferases/metabolism , Homeostasis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Triglycerides/metabolism , Acyltransferases/genetics , Endoplasmic Reticulum/enzymology , Gene Knockdown Techniques , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
9.
Plant Physiol ; 156(1): 29-45, 2011 May.
Article in English | MEDLINE | ID: mdl-21386033

ABSTRACT

Land plant aerial organs are covered by a hydrophobic layer called the cuticle that serves as a waterproof barrier protecting plants against desiccation, ultraviolet radiation, and pathogens. Cuticle consists of a cutin matrix as well as cuticular waxes in which very-long-chain (VLC) alkanes are the major components, representing up to 70% of the total wax content in Arabidopsis (Arabidopsis thaliana) leaves. However, despite its major involvement in cuticle formation, the alkane-forming pathway is still largely unknown. To address this deficiency, we report here the characterization of the Arabidopsis ECERIFERUM1 (CER1) gene predicted to encode an enzyme involved in alkane biosynthesis. Analysis of CER1 expression showed that CER1 is specifically expressed in the epidermis of aerial organs and coexpressed with other genes of the alkane-forming pathway. Modification of CER1 expression in transgenic plants specifically affects VLC alkane biosynthesis: waxes of TDNA insertional mutant alleles are devoid of VLC alkanes and derivatives, whereas CER1 overexpression dramatically increases the production of the odd-carbon-numbered alkanes together with a substantial accumulation of iso-branched alkanes. We also showed that CER1 expression is induced by osmotic stresses and regulated by abscisic acid. Furthermore, CER1-overexpressing plants showed reduced cuticle permeability together with reduced soil water deficit susceptibility. However, CER1 overexpression increased susceptibility to bacterial and fungal pathogens. Taken together, these results demonstrate that CER1 controls alkane biosynthesis and is highly linked to responses to biotic and abiotic stresses.


Subject(s)
Alkanes/metabolism , Arabidopsis Proteins/genetics , Arabidopsis/enzymology , Plant Diseases/immunology , Stress, Physiological , Waxes/metabolism , Arabidopsis/genetics , Arabidopsis/microbiology , Arabidopsis/physiology , Arabidopsis Proteins/metabolism , Ascomycota/physiology , Biosynthetic Pathways , Disease Susceptibility , Gene Expression , Gene Expression Regulation, Plant , Mutagenesis, Insertional , Organ Specificity , Phenotype , Plant Components, Aerial/enzymology , Plant Components, Aerial/genetics , Plant Components, Aerial/microbiology , Plant Components, Aerial/physiology , Plant Diseases/microbiology , Plant Epidermis/enzymology , Plant Epidermis/genetics , Plant Epidermis/microbiology , Plant Epidermis/physiology , Plants, Genetically Modified , Pseudomonas syringae/physiology , Seedlings/enzymology , Seedlings/genetics , Seedlings/microbiology , Seedlings/physiology
10.
Plant Physiol ; 153(4): 1539-54, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20571114

ABSTRACT

Suberin is a protective hydrophobic barrier consisting of phenolics, glycerol, and a variety of fatty acid derivatives, including C18:0-C22:0 primary fatty alcohols. An eight-member gene family encoding alcohol-forming fatty acyl-coenzyme A reductases (FARs) has been identified in Arabidopsis (Arabidopsis thaliana). Promoter-driven expression of the beta-glucuronidase reporter gene indicated that three of these genes, FAR1(At5g22500), FAR4(At3g44540), and FAR5(At3g44550), are expressed in root endodermal cells. The three genes were transcriptionally induced by wounding and salt stress. These patterns of gene expression coincide with known sites of suberin deposition. We then characterized a set of mutants with T-DNA insertions in FAR1, FAR4, or FAR5 and found that the suberin compositions of roots and seed coats were modified in each far mutant. Specifically, C18:0-OH was reduced in far5-1, C20:0-OH was reduced in far4-1, and C22:0-OH was reduced in far1-1. We also analyzed the composition of polymer-bound lipids of leaves before and after wounding and found that the basal levels of C18:0-C22:0 primary alcohols in wild-type leaves were increased by wounding. In contrast, C18:0-OH and C22:0-OH were not increased by wounding in far5-1 and far1-1 mutants, respectively. Heterologous expression of FAR1, FAR4, and FAR5 in yeast confirmed that they are indeed active alcohol-forming FARs with distinct, but overlapping, chain length specificities ranging from C18:0 to C24:0. Altogether, these results indicate that Arabidopsis FAR1, FAR4, and FAR5 generate the fatty alcohols found in root, seed coat, and wound-induced leaf tissue.


Subject(s)
Aldehyde Oxidoreductases/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Fatty Alcohols/metabolism , Lipids/biosynthesis , Nuclear Proteins/metabolism , Aldehyde Oxidoreductases/genetics , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Gene Expression Profiling , Gene Expression Regulation, Plant , Mutagenesis, Insertional , Mutation , Nuclear Proteins/genetics , Plant Roots/enzymology , RNA, Plant/genetics , Seeds/enzymology , Stress, Physiological
11.
Plant Physiol ; 152(2): 670-84, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19965969

ABSTRACT

In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.


Subject(s)
Acyltransferases/metabolism , Brassica rapa/enzymology , Plant Oils/metabolism , Plant Proteins/metabolism , Seeds/metabolism , Acyltransferases/genetics , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Brassica rapa/genetics , Cloning, Molecular , DNA, Plant/genetics , Gene Expression Regulation, Plant , Gene Library , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Promoter Regions, Genetic , Seeds/genetics , Sequence Alignment , Triglycerides/biosynthesis
12.
FEBS J ; 276(21): 6412-24, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19796168

ABSTRACT

In yeast, both phosphatidylinositol and phosphatidylserine are synthesized from cytidine diphosphate-diacylglycerol. Because, as in other eukaryotes, phosphatidylinositol contains more saturated fatty acids than phosphatidylserine (and other phospholipids), it has been hypothesized that either phosphatidylinositol is synthesized from distinct cytidine diphosphate-diacylglycerol molecules, or that, after its synthesis, it is modified by a hypothetical acyltransferase that incorporates saturated fatty acid into neo-synthesized molecules of phosphatidylinositol. We used database search methods to identify an acyltransferase that could catalyze such an activity. Among the various proteins that we studied, we found that Psi1p (phosphatidylinositol stearoyl incorporating 1 protein) is required for the incorporation of stearate into phosphatidylinositol because GC and MS analyses of psi1Delta lipids revealed an almost complete disappearance of stearic (but not of palmitic acid) at the sn-1 position of this phospholipid. Moreover, it was found that, whereas glycerol 3-phosphate, lysophosphatidic acid and 1-acyl lysophosphatidylinositol acyltransferase activities were similar in microsomal membranes isolated from wild-type and psi1Delta cells, microsomal membranes isolated from psi1Delta cells are devoid of the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity that is present in microsomal membranes isolated from wild-type cells. Moreover, after the expression of PSI1 in transgenic psi1Delta cells, the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity was recovered, and was accompanied by a strong increase in the stearic acid content of lysophosphatidylinositol. As previously suggested for phosphatidylinositol from animal cells (which contains almost exclusively stearic acid as the saturated fatty acid), the results obtained in the present study demonstrate that the existence of phosphatidylinositol species containing stearic acid in yeast results from a remodeling of neo-synthesized molecules of phosphatidylinositol.


Subject(s)
Phosphatidylinositols/analysis , Phosphatidylinositols/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Stearic Acids/analysis , Acyltransferases/metabolism , Microsomes/chemistry
13.
J Biol Chem ; 284(28): 18734-41, 2009 Jul 10.
Article in English | MEDLINE | ID: mdl-19447891

ABSTRACT

N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D beta/gamma isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. beta-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.


Subject(s)
Acyltransferases/chemistry , Arabidopsis/enzymology , Acyltransferases/physiology , Amino Acid Sequence , Bacterial Proteins/chemistry , Chromatography, Thin Layer/methods , Escherichia coli/enzymology , Gene Expression Regulation, Plant , Hydrolysis , Lipids/chemistry , Luminescent Proteins/chemistry , Molecular Sequence Data , Phospholipase D/chemistry , Plant Roots/metabolism , Plants/metabolism , Protein Isoforms , Sequence Homology, Amino Acid
15.
Plant Mol Biol ; 67(5): 547-66, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18465198

ABSTRACT

As precursors of wax compounds, very long chain fatty acids participate in the limitation of non-stomatal water loss and the prevention of pathogen attacks. They also serve as energy storage in seeds and as membrane building blocks. Their biosynthesis is catalyzed by the acyl-CoA elongase, a membrane-bound enzymatic complex containing four distinct enzymes (KCS, KCR, HCD and ECR). Twenty-one 3-ketoacyl-CoA synthase (KCS) genes have been identified in Arabidopsis thaliana genome. In this paper we present an overview of the acyl-CoA elongase genes in Arabidopsis focusing on the entire KCS family. We show that the KCS family is made up of 8 distinct subclasses, according to their phylogeny, duplication history, genomic organization, protein topology and 3D modelling. The analysis of the subcellular localization in tobacco cells of the different subunits of the acyl-CoA elongase shows that all these proteins are localized in the endoplasmic reticulum demonstrating that VLCFA production occurs in this compartment. The expression patterns in Arabidopsis of the acyl-CoA elongase genes suggest several levels of regulations at the tissular or organ level but also under stress conditions suggesting a complex organization of this multigenic family.


Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis/enzymology , Coenzyme A Ligases/chemistry , Coenzyme A Ligases/genetics , Gene Expression Profiling , Arabidopsis/genetics , Arabidopsis Proteins/classification , Coenzyme A Ligases/classification , Endoplasmic Reticulum/enzymology , Genes, Plant , Phylogeny , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic
16.
Plant Physiol ; 143(1): 461-72, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17114270

ABSTRACT

The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual Delta(5)-sterols are replaced by 9beta,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that Delta(5)-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.


Subject(s)
Arabidopsis/metabolism , Cell Membrane/metabolism , Membrane Lipids/physiology , Membrane Microdomains/metabolism , Onions/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Biological Transport/physiology , Cell Membrane/drug effects , Cells, Cultured , Membrane Lipids/metabolism , Microsomes/metabolism , Morpholines/pharmacology , Mutation , Onions/drug effects , Phospholipids/metabolism , Seedlings/drug effects , Seedlings/metabolism , Steroid Isomerases/antagonists & inhibitors , Sterols/metabolism , Subcellular Fractions
17.
Plant Physiol Biochem ; 43(10-11): 938-46, 2005.
Article in English | MEDLINE | ID: mdl-16325411

ABSTRACT

We investigated phosphatidylcholine (PC) and phosphatidylethanolamine (PE) labelling in shoots and roots from leek plantlets, maize seedlings and Arabidopsis thaliana through the incorporation of radiolabelled acetate. Regardless of the pathway followed in shoots, PC labelling was always higher than PE labelling. However, we obtained an opposite situation in leek and A. thaliana roots since PC labelling was much lower than PE labelling. Several hypotheses to explain the origin(s) of these discrepancies between roots and shoots were tested. Among them, neither the level of the respective AAPT activities, nor specific regulations of PC biosynthesis through the mRNA levels of several enzymes (choline citidylyltransferase (CCT), ethanolamine citidylyltransferase (ECT), phosphoethanolamine methyltransferase (PEAMT)), nor the fatty acyl chain composition of PC, PE, and diacylglycerol, were responsible for the differences observed between PC and PE metabolism in roots and shoots. Finally, we investigated the acylation of PC and PE in vitro in both shoots and roots of A. thaliana seedlings, and demonstrated that some specific remodelling of PC and PE by acylation was responsible for the differences in labelling observed in vivo.


Subject(s)
Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Plant Roots/metabolism , Plant Shoots/metabolism , Acylation , Allium/metabolism , Arabidopsis/metabolism , Base Sequence , Fatty Acids/metabolism , Molecular Sequence Data , RNA, Messenger/genetics , Zea mays/metabolism
18.
Biochim Biophys Acta ; 1734(3): 247-58, 2005 Jun 01.
Article in English | MEDLINE | ID: mdl-15914083

ABSTRACT

Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic compounds. We found in the available database 282 candidate genes that may play a role in wax synthesis, regulation and transport. To identify the most interesting candidates, we measured the level of expression of 204 genes in the aerial parts of 15-day-old Arabidopsis seedlings by performing microarray experiments. We showed that only 25% of the putative candidates were expressed to significant levels in our samples, thus significantly reducing the number of genes which will be worth studying using reverse genetics to demonstrate their involvement in wax accumulation. We identified a beta-keto acyl-CoA synthase gene, At5g43760, which is co-regulated with the wax gene CER6 in a number of conditions and organs. By contrast, we showed that neither the fatty acyl-CoA reductase genes nor the wax synthase genes were expressed in 15-day-old leaves and stems, raising questions about the identity of the enzymes involved in the acyl-reduction pathway that accounts for 20% of the total wax amount.


Subject(s)
Arabidopsis/genetics , Arabidopsis/metabolism , Genes, Plant , Plant Proteins/genetics , Starch Synthase/genetics , Acyltransferases/biosynthesis , Acyltransferases/genetics , Aldehyde Oxidoreductases/biosynthesis , Aldehyde Oxidoreductases/genetics , Arabidopsis/enzymology , Arabidopsis Proteins/biosynthesis , Arabidopsis Proteins/genetics , Carbon-Carbon Lyases , Gene Expression Profiling , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Plant Leaves/metabolism , Plant Proteins/biosynthesis , Plant Proteins/metabolism , Seedlings/metabolism , Starch Synthase/biosynthesis , Starch Synthase/metabolism , Time Factors
19.
Biochim Biophys Acta ; 1687(1-3): 152-63, 2005 Feb 21.
Article in English | MEDLINE | ID: mdl-15708363

ABSTRACT

The membrane-bound acyl-CoA elongase complex is a key enzyme responsible for erucoyl-CoA synthesis. Among the four putative genes encoding the four moieties of this complex in Brassica napus seeds, only one has been characterized, the Bn-fae1 gene, which encodes the 3-ketoacyl-CoA synthase. The genes encoding the other enzymes (3-ketoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase and trans-2,3-enoyl-CoA reductase) have not been identified. We cloned two 3-ketoacyl-CoA reductase cDNA isoforms, Bn-kcr1 and Bn-kcr2, from B. napus seeds. Their function was identified by heterologous complementation in yeast by restoring elongase activities. The comparison of Bn-kcr mRNA expression in different B. napus tissues showed that the genes were preferentially expressed in seeds and roots. We also investigated the regulation of gene expression in High Erucic Acid Rapeseed (HEAR) and in Low Erucic Acid Rapeseed (LEAR) cultivars during seed development. The co-expression of Bn-fae1 and Bn-kcr observed in HEAR cultivar during seed development was different in LEAR cultivar, suggesting that expression of both genes was directly or indirectly linked.


Subject(s)
Alcohol Oxidoreductases/metabolism , Brassica napus , Erucic Acids/metabolism , Gene Expression Regulation, Enzymologic , Seeds/enzymology , Seeds/growth & development , 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase , Alcohol Oxidoreductases/genetics , Amino Acid Sequence , Brassica napus/anatomy & histology , Brassica napus/chemistry , Brassica napus/enzymology , Brassica napus/genetics , Cloning, Molecular , Fatty Acids/biosynthesis , Gene Expression Regulation, Plant , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Sequence Data , Plant Roots/enzymology , Sequence Alignment
20.
Phytochemistry ; 66(2): 131-8, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15652569

ABSTRACT

Chemical analysis of 4-day-old corn (Zea mays L.) root cell walls revealed that the lipophilic biopolymer suberin forms an important constituent of rhizodermal and hypodermal cell walls. Identified aliphatic monomers had chain lengths ranging from C16 to C26 and they belonged to 5 substance classes (omega-hydroxycarboxylic acids, 1,omega-dicarboxylic acids, 2-hydroxycarboxylic acids, carboxylic acids and alcohols) by which suberin is characterized. Biochemical experiments proved the occurrence of elongase activities in corn roots. Highest enzymatic activities were found in corn root microsomes, and major products synthesized by root elongases were elongated fatty acids with chain lengths ranging from C20 to C24. Preferred substrates of root elongases were acyl-CoAs of the chain length C18 and C20, whereas monounsaturated acyl-CoAs (C16:1 and C18:1) and acyl-CoAs of lower (C12-C16) and higher chain lengths (C22-C24) were rarely elongated. Elongase activities significantly decreased over the length (40 cm) of 10-day-old corn roots going from the young tip to the older base of the root. Thus, results presented here show the presence and activity of elongases in roots of plants.


Subject(s)
Acetyltransferases/metabolism , Plant Roots/enzymology , Zea mays/enzymology , Fatty Acid Elongases , Lipids , Membrane Lipids/biosynthesis , Plant Roots/ultrastructure , Zea mays/ultrastructure
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