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

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

Traffic

Cell polarity is achieved by regulators such as small G proteins, exocyst members and phosphoinositides, with the latter playing a key role when bound to the exocyst proteins Sec3p and Exo70p, and Rho GTPases. This ensures asymmetric growth via the routing of proteins and lipids to the cell surface using actin cables. Previously, using a yeast mutant for a lysophosphatidylinositol acyl transferase encoded by the PSI1 gene, we demonstrated the role of stearic acid in the acyl chain of phosphoinositides in cytoskeletal organization and secretion. Here, we use a genetic approach to characterize the effect on late steps of the secretory pathway. The constitutive overexpression of PSI1 in mutants affecting kinases involved in the phosphoinositide pathway demonstrated the role of molecular species containing stearic acid in bypassing a lack of phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane, which is essential for the function of the Cdc42p module. Decreasing the levels of stearic acid-containing phosphoinositides modifies the environment of the actors involved in the control of late steps in the secretory pathway. This leads to decreased interactions between Exo70p and Sec3p, with Cdc42p, Rho1p and Rho3p, because of disruption of the GTP/GDP ratio of at least Rho1p and Rho3p GTPases, thereby preventing activation of the exocyst

The receptor kinase FERONIA regulates phosphatidylserine localization at the cell surface to modulate ROP signaling

Cells maintain a constant dialog between the extracellular matrix and their plasma membrane to fine tune signal transduction processes. We found that the receptor kinase FERONIA (FER), which is a proposed cell wall sensor, modulates phosphatidylserine plasma membrane accumulation and nano-organization, a key regulator of Rho GTPase signaling in Arabidopsis. We demonstrate that FER is required for both Rho-of-Plant 6 (ROP6) nano-partitioning at the membrane and downstream production of reactive oxygen species upon hyperosmotic stimulus. Genetic and pharmacological rescue experiments indicate that phosphatidylserine is required for a subset of, but not all, FER functions. Furthermore, application of FER ligand shows that its signaling controls both phosphatidylserine membrane localization and nanodomains formation, which, in turn, tunes ROP6 signaling. Together, we propose that a cell wall-sensing pathway controls via the regulation of membrane phospholipid content, the nano-organization of the plasma membrane, which is an essential cell acclimation to environmental perturbations

Homodimerization of the Death-Associated Protein Kinase Catalytic Domain: Development of a New Small Molecule Fluorescent Reporter

Agence Nationale de Recherche (ANR); Centre National de la Recherche Scientifique (CNRS); University of StrasbourgBackground: Death-Associated Protein Kinase (DAPK) is a member of the Ca(2+)/calmodulin regulated serine/threonine protein kinases. Its biological function has been associated with induced cell death, and in vivo use of selective small molecule inhibitors of DAPK catalytic activity has demonstrated that it is a potential therapeutic target for treatment of brain injuries and neurodegenerative diseases. Methodology/Principal Findings: In the in vitro study presented here, we describe the homodimerization of DAPK catalytic domain and the crucial role played by its basic loop structure that is part of the molecular fingerprint of death protein kinases. Nanoelectrospray ionization mass spectrometry of DAPK catalytic domain and a basic loop mutant DAPK protein performed under a variety of conditions was used to detect the monomer-dimer interchange. A chemical biological approach was used to find a fluorescent probe that allowed us to follow the oligomerization state of the protein in solution. Conclusions/Significance: The use of this combined biophysical and chemical biology approach facilitated the elucidation of a monomer-dimer equilibrium in which the basic loop plays a key role, as well as an apparent allosteric conformational change reported by the fluorescent probe that is independent of the basic loop structure

The odd one out: Arabidopsis reticulon 20 does not bend ER membranes but has a role in lipid regulation

Reticulons are integral ER membrane proteins characterised by a reticulon homology domain comprising four transmembrane domains which results in the proteins sitting in the membrane in a W-topology. Here we report on a novel subgroup of reticulons with an extended N-terminal domain and in particular on arabidopsis reticulon 20. Using high resolution confocal microscopy we show that reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue reticulon 19 labels the whole ER. Other than demonstrated for the other members of the reticulon protein family RTN20 and 19 do not display ER constriction phenotypes on over expression. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in roots indicating a role in lipid regulation. A third homologue in this family -3BETAHSD/D1- is unexpectedly localised to ER exit sites resulting in an intriguing location difference for the three proteins

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

Universalité des méthodologies protéomiques pour l'identification et la caractérisation des protéines

La grande diversité des problèmes biologiques abordés ces dernières années par l'analyse protéomique a montré que celle-ci devait être encore développée en fonction du type de question posée mais aussi en fonction des contraintes techniques imposées par les échantillons. Les méthodologies protéomiques sont composées de plusieurs étapes mettant en jeu la préparation d'échantillon, la séparation des protéines et peptides et enfin l'analyse des protéines et peptides par spectrométrie de masse suivie de l'interprétation des données générées. Les études présentées ont nécessité d'adapter ces différentes étapes pour répondre au mieux aux diverses questions biologiques abordés au cours de cette thèse : L'identification des protéines impliquées dans des complexes protéine/protéine et ARN/protéine : une stratégie protéomique impliquant une purification du complexe d'intérêt et des analyses par nanoLC-MS/MS a pu être développée. La caractérisation fine des protéines phosphorylées purifiées : une méthodologie adaptée, en deux étapes, a été mise en place pour la détermination de l'état de phosphorylation des protéines entières et la localisation des sites exacts de phosphorylation sur le squelette peptidique. L'identification et la quantification relative des protéines plasmatiques exprimées chez le manchot Adélie lors du jeûne prolongé à l'aide d'une stratégie de quantification par analyse d'image et d'une stratégie d'identification par des approches d'interprétation des données MS/MS. Ces travaux de thèse montrent l'universalité des approches protéomiques, qui devront toujours être adaptées très finement pour garder leur potentiel pour l'analyse d'échantillons provenant d'origines diverses et de complexités variables.The high diversity of biological questions treated by proteomics shows that adaptation of proteomic strategies is needed to answer each specific question and cope with technical constraints imposed by biological samples. Proteomic methodologies consist in several steps, as sample preparation, proteins and peptides separation and finally proteins and peptides analyses by mass spectrometry followed by interpretation of generated data. The different steps were needed to be adapted to answer at most to the numerous biological questions, which are explored in this PhD thesis: Identification of proteins involved in protein/protein and RNA/protein complexes: a proteomic strategy involving the purification of complex of interest and nanoLC-MS/MS analyses has been developed. Characterization of phosphorylated protein: a two-step adapted methodology has been developed to determine entire protein phosphorylation state and phosphorylation sites localization. Identification and relative quantification of plasmatic proteins expressed during the prolonged fasting of Adélie penguin with a double strategy of quantification by image analyses and identification by MS/MS data interpretation approach. Those PhD studies show the universality of proteomic methodologies, which needed to be cleverly adapt to prove their potentiality in analyses of samples coming from diverse origins and showing different complexities