A global LC-MS2 -based methodology to identify and quantify anionic phospholipids in plant samples

peer reviewedAnionic phospholipids (PS, PA, PI, PIPs) are low-abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at defined spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin-layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive, and accurate quantification of all anionic phospholipid species. Here we developed a method based on high-performance liquid chromatography (HPLC) combined with two-dimensional mass spectrometry (MS 2) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peak, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana. Our work thus paves the way for new studies on how the composition of anionic lipids is finely tuned during plant development and environmental responses

Biosynthesis and functions of very-long-chain fatty acids in the responses of plants to abiotic and biotic stresses

Very-long-chain fatty acids (i.e., fatty acids with more than 18 carbon atoms; VLCFA) are important molecules that play crucial physiological and structural roles in plants. VLCFA are specifically present in several membrane lipids and essential for membrane homeostasis. Their specific accumulation in the sphingolipids of the plasma membrane outer leaflet is of primordial importance for its correct functioning in intercellular communication. VLCFA are found in phospholipids, notably in phosphatidylserine and phosphatidylethanolamine, where they could play a role in membrane domain organization and interleaflet coupling. In epidermal cells, VLCFA are precursors of the cuticular waxes of the plant cuticle, which are of primary importance for many interactions of the plant with its surrounding environment. VLCFA are also major components of the root suberin barrier, which has been shown to be fundamental for nutrient homeostasis and plant adaptation to adverse conditions. Finally, some plants store VLCFA in the triacylglycerols of their seeds so that they later play a pivotal role in seed germination. In this review, taking advantage of the many studies conducted using Arabidopsis thaliana as a model, we present our current knowledge on the biosynthesis and regulation of VLCFA in plants, and on the various functions that VLCFA and their derivatives play in the interactions of plants with their abiotic and biotic environment

Biophysical analysis of the plant-specific GIPC sphingolipids reveals multiple modes of membrane regulation

The plant plasma membrane (PM) is an essential barrier between the cell and the external environment, controlling signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols, and phospholipids. The glycosyl inositol phosphoryl ceramides (GIPCs), representing up to 40% of total sphingolipids, are assumed to be almost exclusively in the outer leaflet of the PM. However, their biological role and properties are poorly defined. In this study, we investigated the role of GIPCs in membrane organization. Because GIPCs are not commercially available, we developed a protocol to extract and isolate GIPC-enriched fractions from eudicots (cauliflower and tobacco) and monocots (leek and rice). Lipidomic analysis confirmed the presence of trihydroxylated long chain bases and 2-hydroxylated very long-chain fatty acids up to 26 carbon atoms. The glycan head groups of the GIPCs from monocots and dicots were analyzed by gas chromatograph–mass spectrometry, revealing different sugar moieties. Multiple biophysics tools, namely Langmuir monolayer, ζ-Potential, light scattering, neutron reflectivity, solid state 2H-NMR, and molecular modeling, were used to investigate the physical properties of the GIPCs, as well as their interaction with free and conjugated phytosterols. We showed that GIPCs increase the thickness and electronegativity of model membranes, interact differentially with the different phytosterols species, and regulate the gel-to-fluid phase transition during temperature variations. These results unveil the multiple roles played by GIPCs in the plant PM.Vers un modèle intégratif de la bicouche lipidique de la membrane plasmique végétaleDéveloppement d’une infrastructure française distribuée pour la métabolomique dédiée à l’innovatio

Study of plasma membrane lipids of Arabidopsis thaliana

Chez tous les organismes, la membrane plasmique (PM) forme une barrière sélective entre la cellule et le milieu extracellulaire et joue de multiples rôles, notamment dans l'homéostasie, la régulation de l'état nutritionnel, la signalisation et la réponse au stress. Chez les plantes, la compréhension de l'organisation de la PM ainsi que sa dynamique sont au centre de diverses études. En effet, si les lipides présents dans la PM des plantes ont déjà été déterminés, la quantité précise de chaque espèce lipidique dans cette membrane est encore manquante. Dans ce contexte, le but de ce travail était de d’étudier le lipidome de la PM ainsi que ça distribution chez Arabidopsis thaliana, une des espèces les plus étudiées en biologie végétale.À cette fin, la PM a été purifié à partir de suspensions cellulaires d'Arabidopsis thaliana à l'aide d'une méthode de purification par partition à deux phases (PEG/Dextran). La combinaison de trois méthodes analytiques différentes a été utilisé pour l'identification et la quantification des lipides. Tout d'abord, l'hydrolyse des lipides de la PM et leur analyse ultérieure par chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS) ont permis de quantifier les stérols, les acides gras à chaîne longue et les acides gras à chaîne très longue. Sur la base de cette analyse, la proportion des trois principales classes de lipides (stérols, glycérolipides et sphingolipides, respectivement) dans la PM végétale a été déterminée. Ensuite, une approche plus approfondie a été utilisée pour déterminer les familles de lipides précises dans chaque classe de lipides. Plus précisément, les familles de lipides ont été analysées par chromatographie sur couche mince. Enfin, l'identification précise des espèces moléculaires de chaque lipide dans la PM ont été déterminées par chromatographie liquide couplée à la spectrométrie de masse (LC-MS).Les résultats de l’étude ont permis de déterminer le lipidome PM d'A. thaliana. Tout d'abord, nous avons confirmé la proportion de stérols, de phospholipides et de sphingolipides dans la PM de la plante. A l'intérieur de chaque classe de lipides, la quantité précise de chaque famille de lipides et la proportion relative de chaque espèce moléculaire ont ensuite été déterminées, ce qui nous permet d’établir le lipidome complet de la PM d'A. thaliana. Les résultats obtenus sont de grand intérêt et ouvrent la voie à la distribution asymétrique des lipides dans les deux feuillets de la membrane plasmiqueIn all organisms, the plasma membrane (PM) forms a selective barrier between the cell and the extracellular environment and plays multiple roles, including homeostasis, regulation of nutritional status, signaling and stress response. In plants, the understanding of the organization of the MP as well as its dynamics is the focus of various studies. Indeed, if the lipids present in the PM of plants have already been determined, the precise amount of each lipid species in this membrane is still missing. In this context, the aim of this work was to study the lipidome of the PM and its distribution in Arabidopsis thaliana, one of the most studied species in plant biology.To this end, PM was purified from Arabidopsis thaliana cell suspensions using a two-phase partitioning purification method (PEG/Dextran). The combination of three different analytical methods was used for lipid identification and quantification. First, hydrolysis of the MP lipids and their subsequent analysis by gas chromatography-mass spectrometry (GC-MS) allowed the quantification of sterols, long-chain fatty acids, and very long-chain fatty acids. Based on this analysis, the proportion of the three main lipid classes (sterols, glycerolipids, and sphingolipids, respectively) in plant PM was determined. Next, a more in-depth approach was used to determine the specific lipid families within each lipid class. Specifically, the lipid families were analyzed by thin layer chromatography. Finally, the precise identification of the molecular species of each lipid in the MP were determined by liquid chromatography coupled with mass spectrometry (LC-MS).The results of the study were used to determine the PM lipidome of A. thaliana. First, we confirmed the proportion of sterols, phospholipids, and sphingolipids in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were then determined, allowing us to establish the complete lipidome of the PM of A. thaliana. The results obtained are of great interest and open the way to the asymmetric distribution of lipids in the two sheets of the plasma membrane

Étude des lipides de la membrane plasmique d’Arabidopsis thaliana

In all organisms, the plasma membrane (PM) forms a selective barrier between the cell and the extracellular environment and plays multiple roles, including homeostasis, regulation of nutritional status, signaling and stress response. In plants, the understanding of the organization of the MP as well as its dynamics is the focus of various studies. Indeed, if the lipids present in the PM of plants have already been determined, the precise amount of each lipid species in this membrane is still missing. In this context, the aim of this work was to study the lipidome of the PM and its distribution in Arabidopsis thaliana, one of the most studied species in plant biology.To this end, PM was purified from Arabidopsis thaliana cell suspensions using a two-phase partitioning purification method (PEG/Dextran). The combination of three different analytical methods was used for lipid identification and quantification. First, hydrolysis of the MP lipids and their subsequent analysis by gas chromatography-mass spectrometry (GC-MS) allowed the quantification of sterols, long-chain fatty acids, and very long-chain fatty acids. Based on this analysis, the proportion of the three main lipid classes (sterols, glycerolipids, and sphingolipids, respectively) in plant PM was determined. Next, a more in-depth approach was used to determine the specific lipid families within each lipid class. Specifically, the lipid families were analyzed by thin layer chromatography. Finally, the precise identification of the molecular species of each lipid in the MP were determined by liquid chromatography coupled with mass spectrometry (LC-MS).The results of the study were used to determine the PM lipidome of A. thaliana. First, we confirmed the proportion of sterols, phospholipids, and sphingolipids in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were then determined, allowing us to establish the complete lipidome of the PM of A. thaliana. The results obtained are of great interest and open the way to the asymmetric distribution of lipids in the two sheets of the plasma membrane.Chez tous les organismes, la membrane plasmique (PM) forme une barrière sélective entre la cellule et le milieu extracellulaire et joue de multiples rôles, notamment dans l'homéostasie, la régulation de l'état nutritionnel, la signalisation et la réponse au stress. Chez les plantes, la compréhension de l'organisation de la PM ainsi que sa dynamique sont au centre de diverses études. En effet, si les lipides présents dans la PM des plantes ont déjà été déterminés, la quantité précise de chaque espèce lipidique dans cette membrane est encore manquante. Dans ce contexte, le but de ce travail était de d’étudier le lipidome de la PM ainsi que ça distribution chez Arabidopsis thaliana, une des espèces les plus étudiées en biologie végétale.À cette fin, la PM a été purifié à partir de suspensions cellulaires d'Arabidopsis thaliana à l'aide d'une méthode de purification par partition à deux phases (PEG/Dextran). La combinaison de trois méthodes analytiques différentes a été utilisé pour l'identification et la quantification des lipides. Tout d'abord, l'hydrolyse des lipides de la PM et leur analyse ultérieure par chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS) ont permis de quantifier les stérols, les acides gras à chaîne longue et les acides gras à chaîne très longue. Sur la base de cette analyse, la proportion des trois principales classes de lipides (stérols, glycérolipides et sphingolipides, respectivement) dans la PM végétale a été déterminée. Ensuite, une approche plus approfondie a été utilisée pour déterminer les familles de lipides précises dans chaque classe de lipides. Plus précisément, les familles de lipides ont été analysées par chromatographie sur couche mince. Enfin, l'identification précise des espèces moléculaires de chaque lipide dans la PM ont été déterminées par chromatographie liquide couplée à la spectrométrie de masse (LC-MS).Les résultats de l’étude ont permis de déterminer le lipidome PM d'A. thaliana. Tout d'abord, nous avons confirmé la proportion de stérols, de phospholipides et de sphingolipides dans la PM de la plante. A l'intérieur de chaque classe de lipides, la quantité précise de chaque famille de lipides et la proportion relative de chaque espèce moléculaire ont ensuite été déterminées, ce qui nous permet d’établir le lipidome complet de la PM d'A. thaliana. Les résultats obtenus sont de grand intérêt et ouvrent la voie à la distribution asymétrique des lipides dans les deux feuillets de la membrane plasmiqu

Study of plasma membrane lipids of Arabidopsis thaliana

Chez tous les organismes, la membrane plasmique (PM) forme une barrière sélective entre la cellule et le milieu extracellulaire et joue de multiples rôles, notamment dans l'homéostasie, la régulation de l'état nutritionnel, la signalisation et la réponse au stress. Chez les plantes, la compréhension de l'organisation de la PM ainsi que sa dynamique sont au centre de diverses études. En effet, si les lipides présents dans la PM des plantes ont déjà été déterminés, la quantité précise de chaque espèce lipidique dans cette membrane est encore manquante. Dans ce contexte, le but de ce travail était de d’étudier le lipidome de la PM ainsi que ça distribution chez Arabidopsis thaliana, une des espèces les plus étudiées en biologie végétale.À cette fin, la PM a été purifié à partir de suspensions cellulaires d'Arabidopsis thaliana à l'aide d'une méthode de purification par partition à deux phases (PEG/Dextran). La combinaison de trois méthodes analytiques différentes a été utilisé pour l'identification et la quantification des lipides. Tout d'abord, l'hydrolyse des lipides de la PM et leur analyse ultérieure par chromatographie en phase gazeuse couplée à la spectrométrie de masse (GC-MS) ont permis de quantifier les stérols, les acides gras à chaîne longue et les acides gras à chaîne très longue. Sur la base de cette analyse, la proportion des trois principales classes de lipides (stérols, glycérolipides et sphingolipides, respectivement) dans la PM végétale a été déterminée. Ensuite, une approche plus approfondie a été utilisée pour déterminer les familles de lipides précises dans chaque classe de lipides. Plus précisément, les familles de lipides ont été analysées par chromatographie sur couche mince. Enfin, l'identification précise des espèces moléculaires de chaque lipide dans la PM ont été déterminées par chromatographie liquide couplée à la spectrométrie de masse (LC-MS).Les résultats de l’étude ont permis de déterminer le lipidome PM d'A. thaliana. Tout d'abord, nous avons confirmé la proportion de stérols, de phospholipides et de sphingolipides dans la PM de la plante. A l'intérieur de chaque classe de lipides, la quantité précise de chaque famille de lipides et la proportion relative de chaque espèce moléculaire ont ensuite été déterminées, ce qui nous permet d’établir le lipidome complet de la PM d'A. thaliana. Les résultats obtenus sont de grand intérêt et ouvrent la voie à la distribution asymétrique des lipides dans les deux feuillets de la membrane plasmiqueIn all organisms, the plasma membrane (PM) forms a selective barrier between the cell and the extracellular environment and plays multiple roles, including homeostasis, regulation of nutritional status, signaling and stress response. In plants, the understanding of the organization of the MP as well as its dynamics is the focus of various studies. Indeed, if the lipids present in the PM of plants have already been determined, the precise amount of each lipid species in this membrane is still missing. In this context, the aim of this work was to study the lipidome of the PM and its distribution in Arabidopsis thaliana, one of the most studied species in plant biology.To this end, PM was purified from Arabidopsis thaliana cell suspensions using a two-phase partitioning purification method (PEG/Dextran). The combination of three different analytical methods was used for lipid identification and quantification. First, hydrolysis of the MP lipids and their subsequent analysis by gas chromatography-mass spectrometry (GC-MS) allowed the quantification of sterols, long-chain fatty acids, and very long-chain fatty acids. Based on this analysis, the proportion of the three main lipid classes (sterols, glycerolipids, and sphingolipids, respectively) in plant PM was determined. Next, a more in-depth approach was used to determine the specific lipid families within each lipid class. Specifically, the lipid families were analyzed by thin layer chromatography. Finally, the precise identification of the molecular species of each lipid in the MP were determined by liquid chromatography coupled with mass spectrometry (LC-MS).The results of the study were used to determine the PM lipidome of A. thaliana. First, we confirmed the proportion of sterols, phospholipids, and sphingolipids in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were then determined, allowing us to establish the complete lipidome of the PM of A. thaliana. The results obtained are of great interest and open the way to the asymmetric distribution of lipids in the two sheets of the plasma membrane

Untargeted LC–HRMS profiling followed by targeted fractionation to discover new taste-active compounds in spirits

International audienceTaste is a key driver of food and beverage acceptability due to its role in consumers’ pleasure. The great interest that natural food and beverages now arouse lies notably in the complexity of their taste, which in turn is related to a wide range of taste-active compounds. Going beyond the classic divide between targeted and untargeted strategies, an integrative methodology to spirits was applied. Untargeted profiling of several cognac spirits was implemented by LC–HRMS to identify compounds of interest among hundreds of ions. A targeted fractionation protocol was then developed. By using HRMS and NMR, dihydrodehydrodiconiferyl alcohol was identified and described for the first time in spirits and oak wood. It was characterized as sweet at 2 mg/L in two matrices and was quantified in spirits up to 4 mg/L. These findings demonstrated how this methodology is relevant and effective to discover new taste-active compounds

A global LC-MS2-based methodology to identify and quantify anionic phospholipids in plant samples

Anionic phospholipids (PS, PA, PI, PIPs) are low abundant phospholipids with impactful functions in cell signaling, membrane trafficking and cell differentiation processes. They can be quickly metabolized and can transiently accumulate at define spots within the cell or an organ to respond to physiological or environmental stimuli. As even a small change in their composition profile will produce a significant effect on biological processes, it is crucial to develop a sensitive and optimized analytical method to accurately detect and quantify them. While thin layer chromatography (TLC) separation coupled with gas chromatography (GC) detection methods already exist, they do not allow for precise, sensitive and accurate quantification of all anionic phospholipid species. Here we developed a method based on high performance liquid chromatography (HPLC) combined with two-dimensional mass spectrometry (MS2) by MRM mode to detect and quantify all molecular species and classes of anionic phospholipids in one-shot. This method is based on a derivatization step by methylation that greatly enhances the ionization, the separation of each peaks, the peak resolution as well as the limit of detection and quantification for each individual molecular species, and more particularly for PA and PS. Our method universally works in various plant samples. Remarkably, we identified that PS is enriched with very long chain fatty acids in the roots but not in aerial organs of Arabidopsis thaliana. Our work thus paves the way to new studies on how the composition of anionic lipids is finely tuned during plant development and environmental responses

Cytotoxic activity of Nep1-like proteins on monocots

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are found throughout several plant-associated microbial taxa and are typically considered to possess cytolytic activity exclusively on dicot plant species. However, cytolytic NLPs are also produced by pathogens of monocot plants such as the onion (Allium cepa) pathogen Botrytis squamosa. We determined the cytotoxic activity of B. squamosa BsNep1, as well as other previously characterized NLPs, on various monocot plant species and assessed the plant plasma membrane components required for NLP sensitivity. Leaf infiltration of NLPs showed that onion cultivars are differentially sensitive to NLPs, and analysis of their sphingolipid content revealed that the GIPC series A : series B ratio did not correlate to NLP sensitivity. A tri-hybrid population derived from a cross between onion and two wild relatives showed variation in NLP sensitivity within the population. We identified a quantitative trait locus (QTL) for NLP insensitivity that colocalized with a previously identified QTL for B. squamosa resistance and the segregating trait of NLP insensitivity correlated with the sphingolipid content. Our results demonstrate the cytotoxic activity of NLPs on several monocot plant species and legitimize their presence in monocot-specific plant pathogens