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

Les Glycosyl Inositol Phosphoryl Céramides (GIPCs), sphingolipides majeurs des plantes : rôle dans la structuration membranaire et dans les relations hôte-pathogènes chez les plantes

Glycosyl Inositol Phosphoryl Ceramides (GIPCs) are the major sphingolipids of the biosphere. They account for up to 40 mol% of the plasma membranes (PM) of plants and fungi. Since their discovery over 50 years ago, GIPCs remained however almost completely ignored. No data are available on their roles in the structure of biological membranes, on their organization in membrane nanodomains and their interactions with other lipids and proteins. Many questions about plant GIPCs remain unanswered, such as the exact chemical structure of the polar as well as the number sugars grafted; their influence on the thickness of the membrane or on the structure of nanodomains; and also their involvement in host-pathogen interactions in plants. The purpose of this project is to purify and characterize the different classes of plant GIPCs to study their structural roles with phytosterols and phospholipids by biophysical and structural biochemistry methods. This multidisciplinary project will enable the emergence of a new theme and will provide an essential database for understanding the structure of plant PM and among others, their roles in the response against pathogens.Les Glycosyl-Inositol Phosphoryl Céramides (GIPCs) sont les sphingolipides majeurs de la biosphère. Ils représentent jusqu’à 40%mol des membranes plasmiques (MP) des plantes et des champignons. Les GIPCs sont cependant restés presque totalement ignorés depuis leur découverte il y a plus de 50 ans. Aucune donnée n’est disponible sur leurs rôles dans la structuration des membranes biologiques, leur organisation en nanodomaines membranaires et leurs interactions avec les autres lipides et les protéines. De nombreuses questions à propos des GIPCs de plantes restent encore sans réponse, telles que la structure chimique exacte de la tête polaire dont le nombre de sucres ; l’influence de ces molécules sur l’épaisseur de la membrane ou encore sur la structuration des nanodomaines ; et enfin l’implication des GIPC dans les relations hôte-pathogène chez les plantes. Le but de ce projet est de purifier et caractériser les différentes classes de GIPCs de plantes, afin d’étudier leurs rôles structurants avec les phytostérols et les phospholipides par des méthodes de biophysique et de biochimie structurale. Ce projet multidisciplinaire permettra l’émergence d’une nouvelle thématique et procurera une base de données indispensable pour comprendre la structure des MP végétales et entre autres, leurs rôles dans la réponse contre les pathogènes

Les Glycosyl Inositol Phosphoryl Céramides (GIPCs), sphingolipides majeurs des plantes : rôle dans la structuration membranaire et dans les relations hôte-pathogènes chez les plantes

Les Glycosyl-Inositol Phosphoryl Céramides (GIPCs) sont les sphingolipides majeurs de la biosphère. Ils représentent jusqu’à 40%mol des membranes plasmiques (MP) des plantes et des champignons. Les GIPCs sont cependant restés presque totalement ignorés depuis leur découverte il y a plus de 50 ans. Aucune donnée n’est disponible sur leurs rôles dans la structuration des membranes biologiques, leur organisation en nanodomaines membranaires et leurs interactions avec les autres lipides et les protéines. De nombreuses questions à propos des GIPCs de plantes restent encore sans réponse, telles que la structure chimique exacte de la tête polaire dont le nombre de sucres ; l’influence de ces molécules sur l’épaisseur de la membrane ou encore sur la structuration des nanodomaines ; et enfin l’implication des GIPC dans les relations hôte-pathogène chez les plantes. Le but de ce projet est de purifier et caractériser les différentes classes de GIPCs de plantes, afin d’étudier leurs rôles structurants avec les phytostérols et les phospholipides par des méthodes de biophysique et de biochimie structurale. Ce projet multidisciplinaire permettra l’émergence d’une nouvelle thématique et procurera une base de données indispensable pour comprendre la structure des MP végétales et entre autres, leurs rôles dans la réponse contre les pathogènes.Glycosyl Inositol Phosphoryl Ceramides (GIPCs) are the major sphingolipids of the biosphere. They account for up to 40 mol% of the plasma membranes (PM) of plants and fungi. Since their discovery over 50 years ago, GIPCs remained however almost completely ignored. No data are available on their roles in the structure of biological membranes, on their organization in membrane nanodomains and their interactions with other lipids and proteins. Many questions about plant GIPCs remain unanswered, such as the exact chemical structure of the polar as well as the number sugars grafted; their influence on the thickness of the membrane or on the structure of nanodomains; and also their involvement in host-pathogen interactions in plants. The purpose of this project is to purify and characterize the different classes of plant GIPCs to study their structural roles with phytosterols and phospholipids by biophysical and structural biochemistry methods. This multidisciplinary project will enable the emergence of a new theme and will provide an essential database for understanding the structure of plant PM and among others, their roles in the response against pathogens

Role of the most abundant plant sphingolipids, Glycosyl Inositol Phosphoryl Ceramides GIPCs, in membrane structure and host/pathogen interactions

Les Glycosyl-Inositol Phosphoryl Céramides (GIPCs) sont les sphingolipides majeurs de la biosphère. Ils représentent jusqu’à 40%mol des membranes plasmiques (MP) des plantes et des champignons. Les GIPCs sont cependant restés presque totalement ignorés depuis leur découverte il y a plus de 50 ans. Aucune donnée n’est disponible sur leurs rôles dans la structuration des membranes biologiques, leur organisation en nanodomaines membranaires et leurs interactions avec les autres lipides et les protéines. De nombreuses questions à propos des GIPCs de plantes restent encore sans réponse, telles que la structure chimique exacte de la tête polaire dont le nombre de sucres ; l’influence de ces molécules sur l’épaisseur de la membrane ou encore sur la structuration des nanodomaines ; et enfin l’implication des GIPC dans les relations hôte-pathogène chez les plantes. Le but de ce projet est de purifier et caractériser les différentes classes de GIPCs de plantes, afin d’étudier leurs rôles structurants avec les phytostérols et les phospholipides par des méthodes de biophysique et de biochimie structurale. Ce projet multidisciplinaire permettra l’émergence d’une nouvelle thématique et procurera une base de données indispensable pour comprendre la structure des MP végétales et entre autres, leurs rôles dans la réponse contre les pathogènes.Glycosyl Inositol Phosphoryl Ceramides (GIPCs) are the major sphingolipids of the biosphere. They account for up to 40 mol% of the plasma membranes (PM) of plants and fungi. Since their discovery over 50 years ago, GIPCs remained however almost completely ignored. No data are available on their roles in the structure of biological membranes, on their organization in membrane nanodomains and their interactions with other lipids and proteins. Many questions about plant GIPCs remain unanswered, such as the exact chemical structure of the polar as well as the number sugars grafted; their influence on the thickness of the membrane or on the structure of nanodomains; and also their involvement in host-pathogen interactions in plants. The purpose of this project is to purify and characterize the different classes of plant GIPCs to study their structural roles with phytosterols and phospholipids by biophysical and structural biochemistry methods. This multidisciplinary project will enable the emergence of a new theme and will provide an essential database for understanding the structure of plant PM and among others, their roles in the response against pathogens

Sphingolipids in plants: a guidebook on their function in membrane architecture, cellular processes, and environmental or developmental responses

National audienceSphingolipids are fundamental lipids involved in various cellular, developmental and stress-response processes. As such, they orchestrate not only vital molecular mechanisms of living cells but also act in diseases, thus qualifying as potential pharmaceutical targets. Sphingolipids are universal to eukaryotes and are also present in some prokaryotes. Some sphingolipid structures are conserved between animals, plants and fungi, whereas others are found only in plants and fungi. In plants, the structural diversity of sphingolipids, as well as their downstream effectors and molecular and cellular mechanisms of action, are of tremendous interest to both basic and applied researchers, as about half of all small molecules in clinical use originate from plants. Here, we review recent advances towards a better understanding of the biosynthesis of sphingolipids, the diversity in their structures as well as their functional roles in membrane architecture, cellular processes such as membrane trafficking and cell polarity, and cell responses to environmental or developmental signals

Plant lipids: Key players of plasma membrane organization and function

International audienceThe plasma membrane (PM) is the biological membrane that separates the interior of all cells from the outside. The PM is constituted of a huge diversity of proteins and lipids. In this review, we will update the diversity of molecular species of lipids found in plant PM. We will further discuss how lipids govern global properties of the plant PM, explaining that plant lipids are unevenly distributed and are able to organize PM in domains. From that observation, it emerges a complex picture showing a spatial and multiscale segregation of PM components. Finally, we will discuss how lipids are key players in the function of PM in plants, with a particular focus on plant-microbe interaction, transport and hormone signaling, abiotic stress responses, plasmodesmata function. The last chapter is dedicated to the methods that the plant membrane biology community needs to develop to get a comprehensive understanding of membrane organization in plants

A nanodomain-anchored scaffolding complex is required for the function and localization of phosphatidylinositol 4-kinase alpha in plants

Phosphoinositides are low-abundant lipids that participate in the acquisition of membrane identity through their spatiotemporal enrichment in specific compartments. Phosphatidylinositol 4-phosphate (PI4P) accumulates at the plant plasma membrane driving its high electrostatic potential, and thereby facilitating interactions with polybasic regions of proteins. PI4Kα1 has been suggested to produce PI4P at the plasma membrane, but how it is recruited to this compartment is unknown. Here, we pin-point the mechanism that tethers Arabidopsis thaliana phosphatidylinositol 4-kinase alpha1 (PI4Kα1) to the plasma membrane via a nanodomain-anchored scaffolding complex. We established that PI4Kα1 is part of a complex composed of proteins from the NO-POLLEN-GERMINATION, EFR3-OF-PLANTS, and HYCCIN-CONTAINING families. Comprehensive knockout and knockdown strategies revealed that subunits of the PI4Kα1 complex are essential for pollen, embryonic, and post-embryonic development. We further found that the PI4Kα1 complex is immobilized in plasma membrane nanodomains. Using synthetic mis-targeting strategies, we demonstrate that a combination of lipid anchoring and scaffolding localizes PI4Kα1 to the plasma membrane, which is essential for its function. Together, this work opens perspectives on the mechanisms and function of plasma membrane nanopatterning by lipid kinases

Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins

Necrosis and ethylene-inducing peptide 1-like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity