RESUMO
The rapid increase in lipidomic studies has led to a collaborative effort within the community to establish standards and criteria for producing, documenting, and disseminating data. Creating a dynamic easy-to-use checklist that condenses key information about lipidomic experiments into common terminology will enhance the field's consistency, comparability, and repeatability. Here, we describe the structure and rationale of the established Lipidomics Minimal Reporting Checklist to increase transparency in lipidomics research.
RESUMO
Understanding the pathways involved in chlorophyll breakdown provides a molecular map to the color changes observed in plant life on a global scale each fall. Surprisingly, little is known about the fate of phytol, chlorophyll's 20-carbon branched-chain tail, during this process. A recent study from Gutbrod et al. provides evidence using physiological, genetic, and exquisitely sensitive analytical approaches that phytenal is an intermediate in plant phytol catabolism. These insights and techniques open the door to further investigation of this complicated metabolic system, with implications for plant health and agriculture.
Assuntos
Clorofila/metabolismo , Fitol/metabolismo , Arabidopsis/metabolismo , Folhas de Planta/metabolismoRESUMO
Lipids have been observed attached to lumen-facing surfaces of mature xylem conduits of several plant species, but there has been little research on their functions or effects on water transport, and only one lipidomic study of the xylem apoplast. Therefore, we conducted lipidomic analyses of xylem sap from woody stems of seven plants representing six major angiosperm clades, including basal magnoliids, monocots and eudicots, to characterize and quantify phospholipids, galactolipids and sulfolipids in sap using mass spectrometry. Locations of lipids in vessels of Laurus nobilis were imaged using transmission electron microscopy and confocal microscopy. Xylem sap contained the galactolipids di- and monogalactosyldiacylglycerol, as well as all common plant phospholipids, but only traces of sulfolipids, with total lipid concentrations in extracted sap ranging from 0.18 to 0.63 nmol ml-1 across all seven species. Contamination of extracted sap from lipids in cut living cells was found to be negligible. Lipid composition of sap was compared with wood in two species and was largely similar, suggesting that sap lipids, including galactolipids, originate from cell content of living vessels. Seasonal changes in lipid composition of sap were observed for one species. Lipid layers coated all lumen-facing vessel surfaces of L. nobilis, and lipids were highly concentrated in inter-vessel pits. The findings suggest that apoplastic, amphiphilic xylem lipids are a universal feature of angiosperms. The findings require a reinterpretation of the cohesion-tension theory of water transport to account for the effects of apoplastic lipids on dynamic surface tension and hydraulic conductance in xylem.
Assuntos
Lipídeos/análise , Magnoliopsida/química , Xilema/química , Galactolipídeos/análise , Galactolipídeos/metabolismo , Lipidômica , Magnoliopsida/genética , Magnoliopsida/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Fosfolipídeos/análise , Fosfolipídeos/metabolismo , Filogenia , Estações do Ano , Xilema/metabolismo , Xilema/ultraestruturaRESUMO
Mass-spectrometry-based screening of lipid extracts of wounded and unwounded leaves from a collection of 364 Arabidopsis thaliana T-DNA insertion lines produced lipid profiles that were scored on the number and significance of their differences from the leaf lipid profiles of wild-type plants. The analysis identified Salk_109175C, which displayed alterations in leaf chloroplast glycerolipid composition, including a decreased ratio between two monogalactosyldiacylglycerol (MGDG) molecular species, MGDG(18:3/16:3) and MGDG(18:3/18:3). Salk_109175C has a confirmed insertion in the At5g64790 locus; the insertion did not co-segregate with the recessive lipid phenotype in the F2 generation of a wild-type (Columbia-0) × Salk_109175C cross. The altered lipid compositional phenotype mapped to the At4g30950 locus, which encodes the plastidial ω-6 desaturase FATTY ACID DESATURASE 6 (FAD6). Sequencing revealed a splice-site mutation, leading to the in-frame deletion of 13 amino acids near the C-terminal end of the 448 amino acid protein. Heterologous expression in yeast showed that this deletion eliminates desaturase activity and reduces protein stability. Sequence comparison across species revealed that several amino acids within the deletion are conserved in plants and cyanobacteria. Individual point mutations in four conserved residues resulted in 77-97% reductions in desaturase activity, while a construct with all four alanine substitutions lacked activity. The data suggest that the deleted region of FAD6, which is on the C-terminal side of the four putative transmembrane segments and the histidine boxes putatively involved in catalysis, is critical for FAD6 function.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA Bacteriano , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , LipidômicaRESUMO
Persons with cystic fibrosis (CF) exhibit a unique alteration of fatty acid composition, marked especially among polyunsaturates by relative deficiency of linoleic acid and excess of Mead acid. Relative deficiency of docosahexaenoic acid is variably found. However, the initial development of these abnormalities is not understood. We examined fatty acid composition in young CF ferrets and pigs, finding abnormalities from the day of birth onward including relative deficiency of linoleic acid in both species. Fatty acid composition abnormalities were present in both liver and serum phospholipids of newborn CF piglets even prior to feeding, including reduced linoleic acid and increased Mead acid. Serum fatty acid composition evolved over the first weeks of life in both non-CF and CF ferrets, though differences between CF and non-CF persisted. Although red blood cell phospholipid fatty acid composition was normal in newborn animals, it became perturbed in juvenile CF ferrets including relative deficiencies of linoleic and docosahexaenoic acids and excess of Mead acid. In summary, fatty acid composition abnormalities in CF pigs and ferrets exist from a young age including at birth independent of feeding and overlap extensively with the abnormalities found in humans with CF. That the abnormalities exist prior to feeding implies that dietary measures alone will not address the mechanisms of imbalance.
Assuntos
Fibrose Cística , Humanos , Animais , Suínos , Ácidos Graxos , Furões , Fosfolipídeos , Ácidos Docosa-Hexaenoicos , Ácidos LinoleicosRESUMO
Head group-acylated chloroplast lipids were discovered in the 1960s, but interest was renewed about 15 years ago with the discovery of Arabidopsides E and G, acylated monogalactosyldiacylglycerols with oxidized fatty acyl chains originally identified in Arabidopsis thaliana. Since then, plant biologists have applied the power of mass spectrometry to identify additional oxidized and non-oxidized chloroplast lipids and quantify their levels in response to biotic and abiotic stresses. The enzyme responsible for the head-group acylation of chloroplast lipids was identified as a cytosolic protein closely associated with the chloroplast outer membrane and christened acylated galactolipid-associated phospholipase 1 (AGAP1). Despite many advances, critical questions remain about the biological functions of AGAP1 and its head group-acylated products.
Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/química , Galactolipídeos/química , Lipídeos de Membrana/química , Acilação , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/sangue , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Galactolipídeos/genética , Galactolipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Folhas de Planta/química , Folhas de Planta/genética , Folhas de Planta/metabolismo , Estresse Fisiológico/genéticaRESUMO
Understanding the adaptive changes in wheat pollen lipidome under high temperature (HT) stress is critical to improving seed set and developing HT tolerant wheat varieties. We measured 89 pollen lipid species under optimum and high day and/or night temperatures using electrospray ionization-tandem mass spectrometry in wheat plants. The pollen lipidome had a distinct composition compared with that of leaves. Unlike in leaves, 34:3 and 36:6 species dominated the composition of extraplastidic phospholipids in pollen under optimum and HT conditions. The most HT-responsive lipids were extraplastidic phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol, phosphatidic acid, and phosphatidylserine. The unsaturation levels of the extraplastidic phospholipids decreased through the decreases in the levels of 18:3 and increases in the levels of 16:0, 18:0, 18:1, and 18:2 acyl chains. PC and PE were negatively correlated. Higher PC:PE at HT indicated possible PE-to-PC conversion, lower PE formation, or increased PE degradation, relative to PC. Correlation analysis revealed lipids experiencing coordinated metabolism under HT and confirmed the HT responsiveness of extraplastidic phospholipids. Comparison of the present results on wheat pollen with results of our previous research on wheat leaves suggests that similar lipid changes contribute to HT adaptation in both leaves and pollen, though the lipidomes have inherently distinct compositions.
Assuntos
Metabolismo dos Lipídeos , Pólen/metabolismo , Triticum/metabolismo , Resposta ao Choque Térmico , Temperatura Alta , Ácidos Fosfatídicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Metaboloma/genética , Metabolômica , Arabidopsis/genética , Arabidopsis/metabolismo , Redes e Vias Metabólicas , MutaçãoRESUMO
Identifying lipids that experience coordinated metabolism during heat stress would provide information regarding lipid dynamics under stress conditions and assist in developing heat-tolerant wheat varieties. We hypothesized that co-occurring lipids, which are up-regulated or down-regulated together through time during heat stress, represent groups that can be explained by coordinated metabolism. Wheat plants (Triticum aestivum L.) were subjected to 12 days of high day and/or night temperature stress, followed by a 4-day recovery period. Leaves were sampled at four time points, and 165 lipids were measured by electrospray ionization-tandem mass spectrometry. Correlation analysis of lipid levels in 160 leaf samples from each of two wheat genotypes revealed 13 groups of lipids. Lipids within each group co-occurred through the high day and night temperature stress treatments. The lipid groups can be broadly classified as groups containing extraplastidic phospholipids, plastidic glycerolipids, oxidized glycerolipids, triacylglycerols, acylated sterol glycosides and sterol glycosides. Current knowledge of lipid metabolism suggests that the lipids in each group co-occur because they are regulated by the same enzyme(s). The results suggest that increases in activities of desaturating, oxidizing, glycosylating and acylating enzymes lead to simultaneous changes in levels of multiple lipid species during high day and night temperature stress in wheat.
Assuntos
Resposta ao Choque Térmico , Metabolismo dos Lipídeos , Lipídeos/análise , Folhas de Planta/metabolismo , Estresse Fisiológico , Triticum/metabolismo , Diglicerídeos/metabolismo , Glicolipídeos/metabolismo , Glicosídeos/metabolismo , Redes e Vias Metabólicas , Oxirredução , Fosfolipídeos/metabolismo , Filogenia , Esteróis/metabolismo , Triglicerídeos/metabolismo , Triticum/genéticaRESUMO
Understanding how wheat (Triticum aestivum L.) plants under high temperature (HT) regulate lipid composition is critical to developing climate-resilient varieties. We measured 165 glycerolipids and sterol derivatives under optimum and high day and night temperatures in wheat leaves using electrospray ionization-tandem mass spectrometry. Levels of polar lipid fatty acyl chain unsaturation were lower in both heat-tolerant genotype Ventnor and susceptible genotype Karl 92 under HT, compared with optimum temperature. The lower unsaturation was predominantly because of lower levels of 18:3 acyl chains and higher levels of 18:1 and 16:0 acyl chains. Levels of 18:3-containing triacylglycerols increased threefold/more under HT, consistent with their possible role in sequestering fatty acids during membrane lipid remodelling. Phospholipids containing odd-numbered or oxidized acyl chains accumulated in leaves under HT. Sterol glycosides (SG) and 16:0-acylated sterol glycosides (ASG) were higher under HT than optimum temperatures. Ventnor had lower amounts of phospholipids with oxidized acyl chains under HT and higher amounts of SG and 16:0-ASG than Karl 92. Taken together, the data demonstrate that wheat leaf lipid composition is altered by HT, in which some lipids are particularly responsive to HT, and that two wheat genotypes, chosen for their differing physiological responses to HT, differ in lipid profile under HT.
Assuntos
Temperatura Alta , Lipídeos/química , Folhas de Planta/fisiologia , Estresse Fisiológico , Triticum/fisiologia , Genótipo , Glicosídeos/metabolismo , Fosfolipídeos/metabolismo , Plastídeos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Esteróis/metabolismo , Triticum/genéticaRESUMO
Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin-like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.
Assuntos
Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Triglicerídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Mutação , Óleos de Plantas/análise , Óleos de Plantas/metabolismo , Plantas Geneticamente Modificadas , Sementes/genética , Sementes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Fatores de Tempo , Triglicerídeos/análiseRESUMO
A direct-infusion electrospray ionization triple-quadrupole mass spectrometry method with multiple reaction monitoring (MRM) was employed to measure 264 lipid analytes extracted from leaves of Arabidopsis thaliana subjected to mechanical wounding. The method provided precise measurements with an average coefficient of variation of 6.1%. Lipid classes analyzed comprised galactolipids and phospholipids (including monoacyl molecular species, molecular species with oxidized acyl chains, phosphatidic acids (PAs)), tri- and tetra-galactosyldiacylglycerols (TrGDGs and TeGDGs), head-group-acylated galactolipids, and head-group-acylated phosphatidylglycerol (acPG), sulfoquinovosyldiacylglycerols (SQDGs), sphingolipids, di- and tri-acylglycerols (DAGs and TAGs), and sterol derivatives. Of the 264 lipid analytes, 254 changed significantly in response to wounding. In general, levels of structural lipids decreased, whereas monoacyl molecular species, galactolipids and phosphatidylglycerols (PGs) with oxidized fatty acyl chains, PAs, TrGDGs, TeGDGs, TAGs, head-group-acylated galactolipids, acPG, and some sterol derivatives increased, many transiently. The observed changes are consistent with activation of lipid oxidizing, hydrolyzing, glycosylating, and acylating activities in the wounding response. Correlation analysis of the levels of lipid analytes across individual control and treated plants was used to construct a lipid dendrogram and to define clusters and sub-clusters of lipid analytes, each composed of a group of lipids which occurred in a coordinated manner. Current knowledge of metabolism supports the notion that observed sub-clusters comprise lipids generated by a common enzyme and/or metabolically downstream of a common enzyme. This work demonstrates that co-occurrence analysis, based on correlation of lipid levels among plants, is a powerful approach to defining lipids generated in vivo by a common enzymatic pathway.
Assuntos
Arabidopsis/metabolismo , Lipídeos/análise , Lipídeos/química , Folhas de Planta/metabolismo , Galactolipídeos/análise , Galactolipídeos/metabolismo , Ácidos Fosfatídicos/análise , Ácidos Fosfatídicos/metabolismo , Fosfolipídeos/análise , Folhas de Planta/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Phospholipids with a choline head group are abundant components of all biological membranes, performing critical functions in cellular structure, metabolism, and signaling. In spite of their importance, our ability to visualize choline phospholipids in vivo remains very limited. We present a simple and robust chemical strategy to image choline phospholipids, based on the metabolic incorporation of azidocholine analogues, that accurately reflects the normal biosynthetic incorporation of choline into cellular phospholipids. Azidocholine-labeled phospholipids can be imaged in cells with high sensitivity and resolution, following derivatization with fluorophores, by bio-orthogonal chemical reactions compatible with live-cell imaging. We used this method to visualize the subcellular localization of choline phospholipids. We also demonstrate that double metabolic labeling with azidocholine and propargylcholine allows sensitive two-color imaging of choline phospholipids. Our method represents a powerful approach to directly image phospholipids, and to study their dynamics in cells and tissues.
Assuntos
Química Click , Imagem Molecular/métodos , Fosfolipídeos/análise , Fosfolipídeos/química , Animais , Azidas/química , Colina/análogos & derivados , Colina/química , Corantes Fluorescentes/química , Camundongos , Estrutura Molecular , Células NIH 3T3/metabolismo , Fosfatidilcolinas/análise , Fosfatidilcolinas/química , Fosfolipídeos/metabolismo , Sensibilidade e EspecificidadeRESUMO
Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.
Assuntos
Proteínas Hedgehog/metabolismo , Imagem Óptica , Transdução de Sinais , Esteróis/análise , Alcinos/química , Animais , Azidas/química , Colesterol/análogos & derivados , Química Click , Cobre/química , Reação de Cicloadição , Humanos , Camundongos , Microscopia/métodos , Sondas Moleculares/química , Células NIH 3T3 , Imagem Óptica/métodos , Esteróis/metabolismoRESUMO
ABCA12 mutations disrupt the skin barrier and cause harlequin ichthyosis. We previously showed Abca12(-/-) skin has increased glucosylceramide (GlcCer) and correspondingly lower amounts of ceramide (Cer). To examine why loss of ABCA12 leads to accumulation of GlcCer, de novo sphingolipid synthesis was assayed using [(14)C]serine labeling in ex vivo skin cultures. A defect was found in ß-glucocerebrosidase (GCase) processing of newly synthesized GlcCer species. This was not due to a decline in GCase function. Abca12(-/-) epidermis had 5-fold more GCase protein (n = 4, P < 0.01), and a 5-fold increase in GCase activity (n = 3, P < 0.05). As with Abca12(+/+) epidermis, immunostaining in null skin showed a typical interstitial distribution of the GCase protein in the Abca12(-/-) stratum corneum. Hence, we tested whether the block in GlcCer conversion could be circumvented by topically providing GlcCer. This approach restored up to 15% of the lost Cer products of GCase activity in the Abca12(-/-) epidermis. However, this level of barrier ceramide replacement did not significantly reduce trans-epidermal water loss function. Our results indicate loss of ABCA12 function results in a failure of precursor GlcCer substrate to productively interact with an intact GCase enzyme, and they support a model of ABCA12 function that is critical for transporting GlcCer into lamellar bodies.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Epiderme/metabolismo , Glucosilceramidase/metabolismo , Glucosilceramidas/metabolismo , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ceramidas/análise , Ceramidas/metabolismo , Cromatografia em Camada Fina , Epiderme/efeitos dos fármacos , Epiderme/embriologia , Glucosilceramidas/administração & dosagem , Glucosilceramidas/farmacologia , Células HEK293 , Humanos , Immunoblotting , Imuno-Histoquímica , Lipídeos/análise , Lipídeos/química , Camundongos , Camundongos Knockout , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/efeitos dos fármacos , Pele/embriologia , Pele/metabolismoRESUMO
ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [P, <0.000309], rs312462 [P, <0.028499], rs6502998 [P, <0.029794], and rs434473 [P, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12. In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Toxoplasmose Congênita/genética , Alelos , Araquidonato 12-Lipoxigenase/química , Araquidonato 12-Lipoxigenase/genética , Ácido Araquidônico/metabolismo , Caspase 1/genética , Caspase 1/metabolismo , Linhagem Celular , Estudos de Coortes , Citocinas/genética , Citocinas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Variação Genética , Humanos , Masculino , Monócitos/metabolismo , Monócitos/parasitologia , Plasmídeos/genética , Interferência de RNA , RNA Interferente Pequeno , Toxoplasmose Congênita/imunologia , Toxoplasmose Congênita/parasitologiaRESUMO
The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, ß, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ácidos Graxos/metabolismo , Fosfolipases A/metabolismo , Fosfolipídeos/metabolismo , Óleos de Plantas/metabolismo , Sementes/enzimologia , Acil Coenzima A/análise , Acil Coenzima A/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Ácidos Graxos/análise , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Mutação , Especificidade de Órgãos , Fosfatidilcolinas/metabolismo , Fosfolipases A/genética , Fosfolipases A/isolamento & purificação , Óleos de Plantas/análise , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA de Plantas/genética , Sementes/citologia , Sementes/genética , Triglicerídeos/análise , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
The release of fatty acids from membrane lipids has been implicated in various plant processes, and the patatin-related phospholipases (pPLAs) constitute a major enzyme family that catalyzes fatty acid release. The Arabidopsis thaliana pPLA family has 10 members that are classified into three groups. Group 3 pPLAIII has four members but lacks the canonical lipase/esterase consensus catalytic sequences, and their enzymatic activity and cellular functions have not been delineated. Here, we show that pPLAIIIß hydrolyzes phospholipids and galactolipids and additionally has acyl-CoA thioesterase activity. Alterations of pPLAIIIß result in changes in lipid levels and composition. pPLAIIIß-KO plants have longer leaves, petioles, hypocotyls, primary roots, and root hairs than wild-type plants, whereas pPLAIIIß-OE plants exhibit the opposite phenotype. In addition, pPLAIIIß-OE plants have significantly lower cellulose content and mechanical strength than wild-type plants. Root growth of pPLAIIIß-KO plants is less sensitive to treatment with free fatty acids, the enzymatic products of pPLAIIIß, than wild-type plants; root growth of pPLAIIIß-OE plants is more sensitive. These data suggest that alteration of pPLAIIIß expression and the resulting lipid changes alter cellulose content and cell elongation in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Crescimento Celular , Celulose/análise , Metabolismo dos Lipídeos , Fosfolipases A/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Ácidos Graxos não Esterificados/análise , Ácidos Graxos não Esterificados/farmacologia , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fosfolipases A/genética , Fosfolipídeos/metabolismo , Epiderme Vegetal/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimentoRESUMO
Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG (galactose-acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.
Assuntos
Arabidopsis/química , Galactolipídeos/química , Galactose/química , Folhas de Planta/química , Solanum lycopersicum/química , Triticum/química , Acilação , Arabidopsis/microbiologia , Esterificação , Ácidos Graxos/química , Congelamento , Interações Hospedeiro-Patógeno , Espectrometria de Massas , Estrutura Molecular , Oxirredução , Folhas de Planta/microbiologia , Pseudomonas syringae/fisiologia , Especificidade da Espécie , Estresse MecânicoRESUMO
Food-derived exosome-like nanoparticles pass through the intestinal tract throughout our lives, but little is known about their impact or function. Here, as a proof of concept, we show that the cells targeted by grape exosome-like nanoparticles (GELNs) are intestinal stem cells whose responses underlie the GELN-mediated intestinal tissue remodeling and protection against dextran sulfate sodium (DSS)-induced colitis. This finding is further supported by the fact that coculturing of crypt or sorted Lgr5⺠stem cells with GELNs markedly improved organoid formation. GELN lipids play a role in induction of Lgr5⺠stem cells, and the liposome-like nanoparticles (LLNs) assembled with lipids from GELNs are required for in vivo targeting of intestinal stem cells. Blocking ß-catenin-mediated signaling pathways of GELN recipient cells attenuates the production of Lgr5⺠stem cells. Thus, GELNs not only modulate intestinal tissue renewal processes, but can participate in the remodeling of it in response to pathological triggers.