Publications by authors named "Françoise Simon-Plas"

40 Publications

Antioxidant Properties of Ergosterol and Its Role in Yeast Resistance to Oxidation.

Antioxidants (Basel) 2021 Jun 25;10(7). Epub 2021 Jun 25.

UMR Procédés Alimentaires et Microbiologiques, University Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, 21000 Dijon, France.

Although the functions and structural roles of sterols have been the subject of numerous studies, the reasons for the diversity of sterols in the different eukaryotic kingdoms remain unclear. It is thought that the specificity of sterols is linked to unidentified supplementary functions that could enable organisms to be better adapted to their environment. Ergosterol is accumulated by late branching fungi that encounter oxidative perturbations in their interfacial habitats. Here, we investigated the antioxidant properties of ergosterol using in vivo, in vitro, and in silico approaches. The results showed that ergosterol is involved in yeast resistance to tert-butyl hydroperoxide and protects lipids against oxidation in liposomes. A computational study based on quantum chemistry revealed that this protection could be related to its antioxidant properties operating through an electron transfer followed by a proton transfer mechanism. This study demonstrates the antioxidant role of ergosterol and proposes knowledge elements to explain the specific accumulation of this sterol in late branching fungi. Ergosterol, as a natural antioxidant molecule, could also play a role in the incompletely understood beneficial effects of some mushrooms on health.
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http://dx.doi.org/10.3390/antiox10071024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8300696PMC
June 2021

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

J Biol Chem 2021 Jan-Jun;296:100602. Epub 2021 Mar 27.

Laboratoire de Biogènese Membranaire, UMR 5200, CNRS, Université de Bordeaux, Villenave d'Ornon Cedex, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbc.2021.100602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099651PMC
March 2021

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

FEBS Lett 2020 11 24;594(22):3719-3738. Epub 2020 Nov 24.

Laboratoire de Biogenèse Membranaire, UMR5200, Université de Bordeaux, CNRS, Villenave d'Ornon, France.

Sphingolipids 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.
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http://dx.doi.org/10.1002/1873-3468.13987DOI Listing
November 2020

Cell stage appears critical for control of plasma membrane order in plant cells.

Plant Signal Behav 2019 25;14(8):1620058. Epub 2019 May 25.

a Agroécologie, AgroSup Dijon, CNRS, INRA , Univ. Bourgogne Franche-Comté, F-21000 , Dijon , France.

Lipids and proteins modulate both the global order of plasma membrane (PM) and its organization in distinct domains. This raises the question of the influence on PM-ordered domain formation of PM composition, which is finely controlled during cell differentiation. Labeling of plant cell PM with an environment-sensitive probe demonstrated that the level of PM order is regulated during anisotropic expansion observed during both cell regeneration from protoplasts and cell differentiation along the growing root. Indeed, PM order progressively decreased during the polarized growth of regenerated tobacco cells, without observed correlation between this parameter and the kinetics of either cell wall regeneration or cell morphology. This suggests that the dynamics of PM formation and renewal could control the PM organization, maybe by involving the secretory pathway.
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http://dx.doi.org/10.1080/15592324.2019.1620058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619956PMC
May 2020

Plant lipids: Key players of plasma membrane organization and function.

Prog Lipid Res 2019 01 19;73:1-27. Epub 2018 Nov 19.

Laboratoire de Biogenèse Membranaire (LBM), CNRS, University of Bordeaux, UMR 5200, F-33882 Villenave d'Ornon, France. Electronic address:

The 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.
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http://dx.doi.org/10.1016/j.plipres.2018.11.002DOI Listing
January 2019

Divide and Rule: Plant Plasma Membrane Organization.

Trends Plant Sci 2018 10 30;23(10):899-917. Epub 2018 Aug 30.

Agroécologie, Institut National Supérieur des Sciences Agronomiques, de l'Alimentation, et de l'Environnement (AgroSup) Dijon, CNRS, Institut National de la Recherche Agronomique (INRA), Université Bourgogne Franche-Comté, Dijon, France; These authors contributed equally to this work. Electronic address:

Since the publication of the fluid mosaic as a relevant model for biological membranes, accumulating evidence has revealed the outstanding complexity of the composition and organization of the plant plasma membrane (PM). Powerful new methodologies have uncovered the remarkable multiscale and multicomponent heterogeneity of PM subcompartmentalization, and this is emerging as a general trait with different features and properties. It is now evident that the dynamics of such a complex organization are intrinsically related to signaling pathways that regulate key physiological processes. Listing and linking recent progress in precisely qualifying these heterogeneities will help to draw an integrated picture of the plant PM. Understanding the key principles governing such a complex dynamic organization will contribute to deciphering the crucial role of the PM in cell physiology.
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http://dx.doi.org/10.1016/j.tplants.2018.07.007DOI Listing
October 2018

Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

J Exp Bot 2018 06;69(15):3545-3557

Agroécologie, AgroSup Dijon, CNRS, INRA, Université Bourgogne Franche-Comté, Dijon, France.

The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.
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http://dx.doi.org/10.1093/jxb/ery152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022670PMC
June 2018

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

Biochim Biophys Acta Mol Cell Biol Lipids 2018 Feb 2;1863(2):199-211. Epub 2017 Dec 2.

Centre de Recherche des Cordeliers, INSERM, UMPC Université Paris 6, Université Paris Descartes Paris 5, CNRS, F-75006 Paris, France. Electronic address:

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics.
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http://dx.doi.org/10.1016/j.bbalip.2017.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742559PMC
February 2018

Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains.

Elife 2017 07 31;6. Epub 2017 Jul 31.

Laboratoire de Biogenèse Membranaire (LBM), Unité Mixte de Recherche UMR 5200, CNRS, Université de Bordeaux, Bordeaux, France.

Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.
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http://dx.doi.org/10.7554/eLife.26404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536944PMC
July 2017

Plasma membrane order and fluidity are diversely triggered by elicitors of plant defence.

J Exp Bot 2016 09 18;67(17):5173-85. Epub 2016 Jul 18.

Agroécologie, AgroSup Dijon, CNRS, INRA, Université Bourgogne Franche-Comté, F-21000 Dijon, France

Although plants are exposed to a great number of pathogens, they usually defend themselves by triggering mechanisms able to limit disease development. Alongside signalling events common to most such incompatible interactions, modifications of plasma membrane (PM) physical properties could be new players in the cell transduction cascade. Different pairs of elicitors (cryptogein, oligogalacturonides, and flagellin) and plant cells (tobacco and Arabidopsis) were used to address the issue of possible modifications of plant PM biophysical properties induced by elicitors and their links to other events of the defence signalling cascade. We observed an increase of PM order whatever the elicitor/plant cell pair used, provided that a signalling cascade was induced. Such membrane modification is dependent on the NADPH oxidase-mediated reactive oxygen species production. Moreover, cryptogein, which is the sole elicitor able to trap sterols, is also the only one able to trigger an increase in PM fluidity. The use of cryptogein variants with altered sterol-binding properties confirms the strong correlation between sterol removal from the PM and PM fluidity enhancement. These results propose PM dynamics as a player in early signalling processes triggered by elicitors of plant defence.
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http://dx.doi.org/10.1093/jxb/erw284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5014163PMC
September 2016

Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein.

Plant Cell Environ 2017 Apr 20;40(4):585-598. Epub 2016 Jul 20.

UPMC UnivParis06, UR5, Physiologie Cellulaire et Moléculaire des Plantes, 4 place Jussieu, 75252, Paris cedex 05, France.

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [ P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.
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http://dx.doi.org/10.1111/pce.12771DOI Listing
April 2017

Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids.

Plant Physiol 2016 Jan 30;170(1):367-84. Epub 2015 Oct 30.

Laboratoire de Biogenèse Membranaire, Centre National de la Recherche Scientifique-University of Bordeaux, Unité Mixte de Recherche 5200, F-33883 Villenave d'Ornon cedex, France (J.-L.C., Cl.B., J.G., F.F., L.F., V.G., E.B., S.M.);Chimie Biologie des Membranes et Nanoobjets, Unité Mixte de Recherche 5248, Centre de Génomique Fonctionnelle, Université de Bordeaux, F-33076 Bordeaux cedex, France (Co.B., J.-M.S.);Université de Bourgogne, Unité Mixte de Recherche 1347 Agroécologie, Equipes de Recherche Labellisée 6300 Centre National de la Recherche Scientifique, F-21065 Dijon cedex, France (J.-L.C., K.G., P.G.-P.);Institut National de la Recherche Agronomique, Unité Mixte de Recherche 1347 Agroécologie, Equipes de Recherche Labellisée 6300 Centre National de la Recherche Scientifique, F-21065 Dijon cedex, France (J.L., F.R., F.S.-P.);Université de Lille 1, Unité de Glycobiologie Structurale et Fonctionnelle, F-59655 Villeneuve d'Ascq, France (Y.R., E.M.);Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8576, F-59655 Villeneuve d'Ascq, France (Y.R., E.M.);Laboratoire de Biophysique Moléculaire aux Interfaces, Université de Liège, B-5030 Gembloux, Belgium (Cl.B., M.D., L.L.); andInstitut des Sciences de la Vigne et du Vin, Groupe d'Etude des Substances Végétales à Activité Biologique, University of Bordeaux, Equipe Associée 3675, F-33400 Talence, France (S.C.)

The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.
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http://dx.doi.org/10.1104/pp.15.00564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704565PMC
January 2016

Differential effect of plant lipids on membrane organization: specificities of phytosphingolipids and phytosterols.

J Biol Chem 2015 Feb 9;290(9):5810-25. Epub 2015 Jan 9.

From UMR1347 Agroécologie, ERL 6300 CNRS, Université de Bourgogne, 17 Rue Sully, BP 86510, 21065 Dijon Cedex, France,

The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains.
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http://dx.doi.org/10.1074/jbc.M114.598805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342490PMC
February 2015

Long-chain bases and their phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco (Nicotiana tabacum) BY-2 cells.

New Phytol 2015 Feb 10;205(3):1239-1249. Epub 2014 Oct 10.

INRA, UMR 1347 Agroécologie, ERL CNRS 6300, BP 86510, F-21065, Dijon Cedex, France.

The proteinaceous elicitor cryptogein triggers defence reactions in Nicotiana tabacum (tobacco) through a signalling cascade, including the early production of reactive oxygen species (ROS) by the plasma membrane (PM)-located tobacco respiratory burst oxidase homologue D (NtRbohD). Sphingolipid long-chain bases (LCBs) are emerging as potent positive regulators of plant defence-related mechanisms. This led us to question whether both LCBs and their phosphorylated derivatives (LCB-Ps) are involved in the early signalling process triggered by cryptogein in tobacco BY-2 cells. Here, we showed that cryptogein-induced ROS production was inhibited by LCB kinase (LCBK) inhibitors. Additionally, Arabidopsis thaliana sphingosine kinase 1 and exogenously supplied LCB-Ps increased cryptogein-induced ROS production, whereas exogenously supplied LCBs had a strong opposite effect, which was not driven by a reduction in cellular viability. Immunogold-electron microscopy assay also revealed that LCB-Ps are present in the PM, which fits well with the presence of a high LCBK activity associated with this fraction. Our data demonstrate that LCBs and LCB-Ps differentially regulate cryptogein-induced ROS production in tobacco BY-2 cells, and support a model in which a cooperative synergism between LCBK/LCB-Ps and NtRbohD/ROS in the cryptogein signalling pathway is likely at the PM in tobacco BY-2 cells.
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http://dx.doi.org/10.1111/nph.13094DOI Listing
February 2015

Direct purification of detergent-insoluble membranes from Medicago truncatula root microsomes: comparison between floatation and sedimentation.

BMC Plant Biol 2014 Sep 30;14:255. Epub 2014 Sep 30.

UMR1347 INRA/Agrosup/Université de Bourgogne Agroécologie, Pôle Interactions Plantes-Microorganismes - ERL 6300 CNRS, 17 Rue Sully, Dijon Cedex, BP 86510, F-21065, France.

Background: Membrane microdomains are defined as highly dynamic, sterol- and sphingolipid-enriched domains that resist to solubilization by non-ionic detergents. In plants, these so-called Detergent Insoluble Membrane (DIM) fractions have been isolated from plasma membrane by using conventional ultracentrifugation on density gradient (G). In animals, a rapid (R) protocol, based on sedimentation at low speed, which avoids the time-consuming sucrose gradient, has also been developed to recover DIMs from microsomes as starting material. In the current study, we sought to compare the ability of the Rapid protocol versus the Gradient one for isolating DIMs directly from microsomes of M. truncatula roots. For that purpose, Triton X-100 detergent-insoluble fractions recovered with the two methods were analyzed and compared for their sterol/sphingolipid content and proteome profiles.

Results: Inferred from sterol enrichment, presence of typical sphingolipid long-chain bases from plants and canonical DIM protein markers, the possibility to prepare DIMs from M. truncatula root microsomes was confirmed both for the Rapid and Gradient protocols. Contrary to sphingolipids, the sterol and protein profiles of DIMs were found to depend on the method used. Namely, DIM fractions were differentially enriched in spinasterol and only shared 39% of common proteins as assessed by GeLC-MS/MS profiling. Quantitative analysis of protein indicated that each purification procedure generated a specific subset of DIM-enriched proteins from Medicago root microsomes. Remarkably, these two proteomes were found to display specific cellular localizations and biological functions. In silico analysis of membrane-associative features within R- and G-enriched proteins, relative to microsomes, showed that the most noticeable difference between the two proteomes corresponded to an increase in the proportion of predicted signal peptide-containing proteins after sedimentation (R) compared to its decrease after floatation (G), suggesting that secreted proteins likely contribute to the specificity of the R-DIM proteome.

Conclusions: Even though microsomes were used as initial material, we showed that the protein composition of the G-DIM fraction still mostly mirrored that of plasmalemma-originating DIMs conventionally retrieved by floatation. In parallel, the possibility to isolate by low speed sedimentation DIM fractions that seem to target the late secretory pathway supports the existence of plant microdomains in other organelles.
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http://dx.doi.org/10.1186/s12870-014-0255-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4193990PMC
September 2014

Different tobacco retrotransposons are specifically modulated by the elicitor cryptogein and reactive oxygen species.

J Plant Physiol 2014 Oct 23;171(16):1533-40. Epub 2014 Jul 23.

INRA, UMR 1347 Agroécologie, ERL CNRS 6300, 17 Rue Sully, BP 86510, 21065 Dijon Cedex, France. Electronic address:

Interactions of plant retrotransposons with different steps of biotic and abiotic stress-associated signaling cascades are still poorly understood. We perform here a finely tuned comparison of four tobacco retrotransposons (Tnt1, Tnt2, Queenti, and Tto1) responses to the plant elicitor cryptogein. We demonstrate that basal transcript levels in cell suspensions and plant leaves as well as the activation during the steps of defense signaling events are specific to each retrotransposon. Using antisense NtrbohD lines, we show that NtrbohD-dependent reactive oxygen species (ROS) production might act as negative regulator of retrotransposon activation.
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http://dx.doi.org/10.1016/j.jplph.2014.07.003DOI Listing
October 2014

Dynamic changes in the subcellular distribution of the tobacco ROS-producing enzyme RBOHD in response to the oomycete elicitor cryptogein.

J Exp Bot 2014 Sep 1;65(17):5011-22. Epub 2014 Jul 1.

Université de Bourgogne, UMR1347 Agroécologie, ERL CNRS 6300, BP 86510, F-21065 Dijon Cedex, France

Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), have been identified as a major source of reactive oxygen species (ROS) during plant-microbe interactions. The subcellular localization of the tobacco (Nicotiana tabacum) ROS-producing enzyme RBOHD was examined in Bright Yellow-2 cells before and after elicitation with the oomycete protein cryptogein using electron and confocal microscopy. The plasma membrane (PM) localization of RBOHD was confirmed and immuno-electron microscopy on purified PM vesicles revealed its distribution in clusters. The presence of the protein fused to GFP was also seen in intracellular compartments, mainly Golgi cisternae. Cryptogein induced, within 1h, a 1.5-fold increase in RBOHD abundance at the PM and a concomitant decrease in the internal compartments. Use of cycloheximide revealed that most of the proteins targeted to the PM upon elicitation were not newly synthesized but may originate from the Golgi pool. ROS accumulation preceded RBOHD transcript- and protein-upregulation, indicating that ROS resulted from the activation of a PM-resident pool of enzymes, and that enzymes newly addressed to the PM were inactive. Taken together, the results indicate that control of RBOH abundance and subcellular localization may play a fundamental role in the mechanism of ROS production.
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http://dx.doi.org/10.1093/jxb/eru265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144778PMC
September 2014

Interplays between nitric oxide and reactive oxygen species in cryptogein signalling.

Plant Cell Environ 2015 Feb 12;38(2):331-48. Epub 2014 Mar 12.

INRA, UMR 1347 Agroécologie, Pôle Mécanisme et Gestion des Interactions Plantes-Microorganismes - ERL CNRS 6300, 17 rue Sully, BP 86510, 21065, Dijon cédex, France.

Nitric oxide (NO) has many functions in plants. Here, we investigated its interplays with reactive oxygen species (ROS) in the defence responses triggered by the elicitin cryptogein. The production of NO induced by cryptogein in tobacco cells was partly regulated through a ROS-dependent pathway involving the NADPH oxidase NtRBOHD. In turn, NO down-regulated the level of H2O2. Both NO and ROS synthesis appeared to be under the control of type-2 histone deacetylases acting as negative regulators of cell death. Occurrence of an interplay between NO and ROS was further supported by the finding that cryptogein triggered a production of peroxynitrite (ONOO(-)). Next, we showed that ROS, but not NO, negatively regulate the intensity of activity of the cryptogein-induced protein kinase NtOSAK. Furthermore, using a DNA microarray approach, we identified 15 genes early induced by cryptogein via NO. A part of these genes was also modulated by ROS and encoded proteins showing sequence identity to ubiquitin ligases. Their expression appeared to be negatively regulated by ONOO(-), suggesting that ONOO(-) mitigates the effects of NO and ROS. Finally, we provided evidence that NO required NtRBOHD activity for inducing cell death, thus confirming previous assumption that ROS channel NO through cell death pathways.
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http://dx.doi.org/10.1111/pce.12295DOI Listing
February 2015

Modification of plasma membrane organization in tobacco cells elicited by cryptogein.

Plant Physiol 2014 Jan 14;164(1):273-86. Epub 2013 Nov 14.

Université de Bourgogne , Unité Mixte de Recherche 1347 Agroécologie, Equipe de Recherche Labelisée 6300 Centre National de la Recherche Scientifique, BP 86510, F-21000 Dijon, France.

Lipid mixtures within artificial membranes undergo a separation into liquid-disordered and liquid-ordered phases. However, the existence of this segregation into microscopic liquid-ordered phases has been difficult to prove in living cells, and the precise organization of the plasma membrane into such phases has not been elucidated in plant cells. We developed a multispectral confocal microscopy approach to generate ratiometric images of the plasma membrane surface of Bright Yellow 2 tobacco (Nicotiana tabacum) suspension cells labeled with an environment sensitive fluorescent probe. This allowed the in vivo characterization of the global level of order of this membrane, by which we could demonstrate that an increase in its proportion of ordered phases transiently occurred in the early steps of the signaling triggered by cryptogein and flagellin, two elicitors of plant defense reactions. The use of fluorescence recovery after photobleaching revealed an increase in plasma membrane fluidity induced by cryptogein, but not by flagellin. Moreover, we characterized the spatial distribution of liquid-ordered phases on the membrane of living plant cells and monitored their variations induced by cryptogein elicitation. We analyze these results in the context of plant defense signaling, discuss their meaning within the framework of the "membrane raft" hypothesis, and propose a new mechanism of signaling platform formation in response to elicitor treatment.
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http://dx.doi.org/10.1104/pp.113.225755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3875808PMC
January 2014

Lipids of plant membrane rafts.

Prog Lipid Res 2012 Jul 30;51(3):272-99. Epub 2012 Apr 30.

Laboratoire de Biogenèse Membranaire, UMR 5200 CNRS, Université de Bordeaux, 146 Rue Léo Saignat, 33076 Bordeaux, France.

Lipids tend to organize in mono or bilayer phases in a hydrophilic environment. While they have long been thought to be incapable of coherent lateral segregation, it is now clear that spontaneous assembly of these compounds can confer microdomain organization beyond spontaneous fluidity. Membrane raft microdomains have the ability to influence spatiotemporal organization of protein complexes, thereby allowing regulation of cellular processes. In this review, we aim at summarizing briefly: (i) the history of raft discovery in animals and plants, (ii) the main findings about structural and signalling plant lipids involved in raft segregation, (iii) imaging of plant membrane domains, and their biochemical purification through detergent-insoluble membranes, as well as the existing debate on the topic. We also discuss the potential involvement of rafts in the regulation of plant physiological processes, and further discuss the prospects of future research into plant membrane rafts.
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http://dx.doi.org/10.1016/j.plipres.2012.04.001DOI Listing
July 2012

Sugar transporters in plants and in their interactions with fungi.

Trends Plant Sci 2012 Jul 17;17(7):413-22. Epub 2012 Apr 17.

UMR INRA 1347, Agrosup, Université de Bourgogne, Agroécologie, Pôle Interactions Plantes Microorganismes ERL CNRS 6300, BP 86510, 21065 Dijon Cedex, France.

Sucrose and monosaccharide transporters mediate long distance transport of sugar from source to sink organs and constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Even if numerous families of plant sugar transporters are defined; efflux capacities, subcellular localization and association to membrane rafts have only been recently reported. On the fungal side, the investigation of sugar transport mechanisms in mutualistic and pathogenic interactions is now emerging. Here, we review the essential role of sugar transporters for distribution of carbohydrates inside plant cells, as well as for plant-fungal interaction functioning. Altogether these data highlight the need for a better comprehension of the mechanisms underlying sugar exchanges between fungi and their host plants.
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http://dx.doi.org/10.1016/j.tplants.2012.03.009DOI Listing
July 2012

An update on plant membrane rafts.

Curr Opin Plant Biol 2011 Dec 6;14(6):642-9. Epub 2011 Sep 6.

UMR Plante-Microbe-Environnement 1088, Institut National de la Recherche Agronomique-5184, CNRS-Université de Bourgogne, 21065 Dijon Cedex, France.

The dynamic segregation of membrane components within microdomains, such as the sterol-enriched and sphingolipid-enriched membrane rafts, emerges as a central regulatory mechanism governing physiological responses in various organisms. Over the past five years, plasma membrane located raft-like domains have been described in several plant species. The protein and lipid compositions of detergent-insoluble membranes, supposed to contain these domains, have been extensively characterised. Imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level at the plant plasma membrane, correlating detergent insolubility and membrane-domain localisation of presumptive raft proteins. Finally, the dynamic association of specific proteins with detergent-insoluble membranes upon environmental stress has been reported, confirming a possible role for plant rafts as signal transduction platforms, particularly during biotic interactions.
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http://dx.doi.org/10.1016/j.pbi.2011.08.003DOI Listing
December 2011

Membrane rafts in plant cells.

Trends Plant Sci 2010 Dec 8;15(12):656-63. Epub 2010 Oct 8.

Laboratoire de Biogenèse Membranaire, Unité Mixte de Recherche 5200 (UMR 5200) Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux, France.

Over the past five years, the structure, composition and possible functions of membrane raft-like domains on plant plasma membranes (PM) have been described. Proteomic analyses have indicated that a high proportion of proteins associated with detergent-insoluble membranes (DIMs), supposed to contain raft-like domains isolated from the PM, might be involved in signalling pathways. Recently, the dynamic association of specific proteins with the DIM fraction upon environmental stress has been reported. Innovative imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level in the plant PM, correlating detergent insolubility and membrane-domain localization of presumptive raft proteins. These data suggest a role for plant rafts as signal transduction platforms, similar to those documented for mammalian cells.
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http://dx.doi.org/10.1016/j.tplants.2010.09.003DOI Listing
December 2010

Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells.

Biochim Biophys Acta 2010 Nov 30;1798(11):2150-9. Epub 2010 Jul 30.

Universite de Bourgogne, Dijon Cedex, France.

The effects of changes in plasma membrane (PM) sterol lateral organization and availability on the control of signaling pathways have been reported in various animal systems, but rarely assessed in plant cells. In the present study, the pentaene macrolide antibiotic filipin III, commonly used in animal systems as a sterol sequestrating agent, was applied to tobacco cells. We show that filipin can be used at a non-lethal concentration that still allows an homogeneous labeling of the plasma membrane and the formation of filipin-sterol complexes at the ultrastructural level. This filipin concentration triggers a rapid and transient NADPH oxidase-dependent production of reactive oxygen species, together with an increase in both medium alkalinization and conductivity. Pharmacological inhibition studies suggest that these signaling events may be regulated by phosphorylations and free calcium. By conducting FRAP experiments using the di-4-ANEPPDHQ probe and spectrofluorimetry using the Laurdan probe, we provide evidence for a filipin-induced increase in PM viscosity that is also regulated by phosphorylations. We conclude that filipin triggers ligand-independent signaling responses in plant cells. The present findings strongly suggest that changes in PM sterol availability could act as a sensor of the modifications of cell environment in plants leading to adaptive cell responses through regulated signaling processes.
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http://dx.doi.org/10.1016/j.bbamem.2010.07.026DOI Listing
November 2010

Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes.

Biochim Biophys Acta 2010 Aug 8;1798(8):1601-7. Epub 2010 Apr 8.

Laboratoire Plantes-Microbe-Environnement, UMR INRA 1088/CNRS 5184/Université de Bourgogne, Dijon, France.

We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5kbar at 30 degrees C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-beta-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5 kbar but was much less important at higher pressures (above 2 kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5 kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5-3.5 kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.
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http://dx.doi.org/10.1016/j.bbamem.2010.03.024DOI Listing
August 2010

Polyphosphoinositides are enriched in plant membrane rafts and form microdomains in the plasma membrane.

Plant Physiol 2010 Apr 24;152(4):2173-87. Epub 2010 Feb 24.

Laboratoire de Biogenèse Membranaire, UMR 5200, CNRS-Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France.

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-beta-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.
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http://dx.doi.org/10.1104/pp.109.149823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2850013PMC
April 2010

Quantitative proteomics reveals a dynamic association of proteins to detergent-resistant membranes upon elicitor signaling in tobacco.

Mol Cell Proteomics 2009 Sep 13;8(9):2186-98. Epub 2009 Jun 13.

Institut National de Recherche Agronomique, Unité Mixte de Recherche Plante Microbe Environnement 1088/CNRS 5184/Université de Bourgogne, 17 Rue Sully, BP 86510 F-21000 Dijon, France.

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains playing important roles in protein sorting, signal transduction, or infection by pathogens. In plants, as previously observed for animal microdomains, detergent-resistant fractions, enriched in sphingolipids and sterols, were isolated from plasma membrane. A characterization of their proteic content revealed their enrichment in proteins involved in signaling and response to biotic and abiotic stress and cell trafficking suggesting that these domains were likely to be involved in such physiological processes. In the present study, we used (14)N/(15)N metabolic labeling to compare, using a global quantitative proteomics approach, the content of tobacco detergent-resistant membranes extracted from cells treated or not with cryptogein, an elicitor of defense reaction. To analyze the data, we developed a software allowing an automatic quantification of the proteins identified. The results obtained indicate that, although the association to detergent-resistant membranes of most proteins remained unchanged upon cryptogein treatment, five proteins had their relative abundance modified. Four proteins related to cell trafficking (four dynamins) were less abundant in the detergent-resistant membrane fraction after cryptogein treatment, whereas one signaling protein (a 14-3-3 protein) was enriched. This analysis indicates that plant microdomains could, like their animal counterpart, play a role in the early signaling process underlying the setup of defense reaction. Furthermore proteins identified as differentially associated to tobacco detergent-resistant membranes after cryptogein challenge are involved in signaling and vesicular trafficking as already observed in similar studies performed in animal cells upon biological stimuli. This suggests that the ways by which the dynamic association of proteins to microdomains could participate in the regulation of the signaling process may be conserved between plant and animals.
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http://dx.doi.org/10.1074/mcp.M900090-MCP200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742443PMC
September 2009

NADPH oxidase-mediated reactive oxygen species production: subcellular localization and reassessment of its role in plant defense.

Mol Plant Microbe Interact 2009 Jul;22(7):868-81

UMR PME INRA 1088/CNRS 5184/Université de Bourgogne, Dijon-cedex, France.

Chemiluminescence detection of reactive oxygen species (ROS) triggered in tobacco BY-2 cells by the fungal elicitor cryptogein was previously demonstrated to be abolished in cells transformed with an antisense construct of the plasma membrane NADPH oxidase, NtrbohD. Here, using electron microscopy, it has been confirmed that the first hydrogen peroxide production occurring a few minutes after challenge of tobacco cells with cryptogein is plasma membrane located and NtrbohD mediated. Furthermore, the presence of NtrbohD in detergent-resistant membrane fractions could be associated with the presence of NtrbohD-mediated hydrogen peroxide patches along the plasma membrane. Comparison of the subcellular localization of ROS in wild-type tobacco and in plants transformed with antisense constructs of NtrbohD revealed that this enzyme is also responsible for the hydrogen peroxide production occurring at the plasma membrane after infiltration of tobacco leaves with cryptogein. Finally, the reactivity of wild-type and transformed plants to the elicitor and their resistance against the pathogenic oomycete Phytophthora parasitica were examined. NtrbohD-mediated hydrogen peroxide production does not seem determinant for either hypersensitive response development or the establishment of acquired resistance but it is most likely involved in the signaling pathways associated with the protection of the plant cell.
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http://dx.doi.org/10.1094/MPMI-22-7-0868DOI Listing
July 2009

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Plant Cell 2009 May 26;21(5):1541-55. Epub 2009 May 26.

Laboratoire de Biogenèse Membranaire, Unité Mixte de Recherche 5200, Centre National de la Recherche Scientifique-University of Bordeaux, Bordeaux 33076, France.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.
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http://dx.doi.org/10.1105/tpc.108.064279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700541PMC
May 2009
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