0000000000144352
AUTHOR
Françoise Simon-plas
Constitutive expression of clathrin hub hinders elicitor-induced clathrin-mediated endocytosis and defense gene expression in plant cells.
International audience; Endocytosis has been recently implicated in the signaling network associated with the recognition of microbes by plants. In a previous study, we showed that the elicitor cryptogein was able to induce clathrin-mediated endocytosis (CME) in tobacco suspension cells. Herein, we investigate further the induced CME by means of a GFP-tagged clathrin light chain and a CME inhibitor, the hub domain of clathrin heavy chain. Hub constitutive expression does affect neither cell growth nor constitutive endocytosis but abolishes cryptogein-induced CME. Such an inhibition has no impact on early events in the cryptogein signaling pathway but reduces the expression of defense-associ…
Cercospora beticola toxins. Part XVII. The role of the beticolin/Mg2+ complexes in their biological activity Study of plasma membrane H+-ATPase, vacuolar H+-PPase, alkaline and acid phosphatases
Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inh…
Influence of phytosterols on the structural and dynamic properties of plant plasma membrane
National audience; The complex mixture of sterols, namely phytosterols, found in the plasma membrane (PM) of plant cells is an intriguing characteristic. In the present work, we characterized the sterol composition of tobacco BY-2 cell PM, and we adapted a sterol manipulation method to this plant material. Methyl-b-cyclodextrin was found to induce a drastic reduction of the PM total free sterol content, with a low specificity for the different phytosterols, and without effect on steryl-conjugates. The consequences of phytosterols depletion were investigated by means of fluorescence spectroscopy and biochemical detergent resistant membrane (DRM) fraction purification methods. Data obtained w…
Modification of Plasma Membrane Organization in Tobacco Cells Elicited by Cryptogein
Abstract 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 w…
Proteasome comprising a beta1 inducible subunit acts as a negative regulator of NADPH oxidase during elicitation of plant defense reactions.
Elicitation of defense reactions in tobacco by cryptogein, triggered a production of active oxygen species (AOS) via the NADPH oxidase, NtrbohD, and an accumulation of beta1din, a defense induced beta-type subunit of 20S proteasome. The proteasome inhibitor, MG132, stimulated this AOS production. Tobacco cells transformed with sense constructs of beta1din showed an inhibition of the AOS production following elicitin treatment, whereas the antisense transformed cells showed a strongly enhanced AOS production. In cells transformed with sense construct of beta1din, the NtrbohD transcripts failed to be induced by cryptogein as observed in control and antisense transformed cells. Conversely, in …
Sugar transporters in plants and in their interactions with fungi.
International audience; 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 interactio…
Lipid Rafts in Higher Plant Cells
A large body of evidence from the past decade supports the existence of functional microdomains in membranes of animal and yeast cells, which play important roles in protein sorting, signal transduction, or infection by pathogens. They are based on the dynamic clustering of sphingolipids and cholesterol or ergosterol and are characterized by their insolubility, at low temperature, in nonionic detergents. Here we show that similar microdomains also exist in plant plasma membrane isolated from both tobacco leaves and BY2 cells. Tobacco lipid rafts were found to be greatly enriched in a sphingolipid, identified as glycosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methyl…
Active oxygen species production in tobacco cells elicited by cryptogein*
We analyse the relationship between active oxygen species (AOS) production and pH changes induced in tobacco cells by cryptogein, a fungal proteinaceous elicitor of defence mechanisms in plants. When tobacco cells were treated with cryptogein, an intracellular acidification, an alkalinization of the extracellular medium and a transient burst of AOS (H 2 O 2 ) were observed. Treatment of elicited cells with either diphenyleneiodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, or Tiron, which scavenges O2 - , abolished AOS production. These data suggest the involvement of a NADPH oxidase-like enzyme leading to H 2 O 2 production through O 2 - dismutation. Although H 2 O 2 production…
Liquid ordered domains of tobacco cell plasma membrane : in vivo visualization, spatial distribution and modulation upon elicitation
SPE IPM; International audience; The ability of lipids in complex mixtures to form liquid-ordered, sterol-rich phases, together with the association of specific proteins with sterol-enriched biochemical fractions extracted from biological membranes, gave raise to the "lipid rafts" hypothesis. This suggests the presence within biological membranes of small-sized, ordered domains, of particular lipid and protein composition, which could serve as signal transduction platforms, particularly during plant-microorganisms interactions. However, the precise organization of living cell plasma membrane is still poorly described and the spatial distribution of segregating liquid-disordered and liquid-o…
Plasma membrane sterol complexation, generated by filipin, triggers signaling responses in tobacco cells
International audience; 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 …
Antioxidant Properties of Ergosterol and Its Role in Yeast Resistance to Oxidation
International audience; 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 h…
Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells
IF 5.547; International audience; 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 bo…
Regulation of Plant Transporters by Lipids and Microdomains
Transporters in the broad sense, that is, carriers, pumps, and channels, are proteins inserted in a lipid bilayer separating two cellular compartments. This lipid bilayer is not only the physical support of such proteins, but also a powerful way to regulate their activity. This chapter will first summarize the different means by which lipids can regulate the activity of transmembrane proteins (including the physical properties of the bilayer, its dynamic lateral compartmentalization, and the presence of particular lipid species acting as cofactors). It will then illustrate these general rules with examples of such regulations found in plant literature and, as a reference, in animal studies.
Cercospora beticola toxins. Use of fluorescent cyanine dye to study their effects on tobacco cell suspensions
Abstract The fluorescent dye 3,3′-diethylthiadicarbocyanine iodide [diS-C 2 -(5)] was used to observe plasmalemma transmembrane potential variations of tobacco cells treated with uncoupler (FCCP), respiratory inhibitors (azide and cyanide), and H + -ATPase inhibitors (DCCD and a carbanilate derivative). These chemicals induced an increase in fluorescence, indicating a dissipation of the transmembrane potential. The [diS-C 2 -(5)] was also used to study the effects of two Cercospora beticola toxins on tobacco cells. Changes in fluorescence of [diS-C 2 -(5)] suggested that these two toxins caused a dissipation of the transmembrane potential with a different magnitude whereas kinetics of their…
Interplays between nitric oxide and reactive oxygen species in cryptogein signalling
The cellular messenger nitric oxide (NO) has many functions in plants. In this study, we investigated its interplays with Reactive Oxygen Species (ROS) in the defense responses triggered by the elicitin cryptogein produced by the oomycete Phytophthora cryptogea. The production of NO induced by cryptogein in tobacco cell suspensions was partly regulated through a ROS-dependent pathway involving the NADPH oxidase NtRBOHD. In turn, NO down-regulated the level of H2O2 derived from NtRBOHD activity. Both NO and ROS synthesis appeared to be under the control of two redundant isoforms of histone deacetylases of type 2 acting as negative regulators of cell death. Occurrence of an interplay between …
Quantitative proteomics reveals a dynamic association of proteins to detergent-resistant membranes upon elicitor signaling in tobacco.
International audience; 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, w…
NADPH Oxidase-Mediated Reactive Oxygen Species Production: Subcellular Localization and Reassessment of Its Role in Plant Defense
International audience; 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 membran…
Dynamic changes in the subcellular distribution of the tobacco ROS-producing enzyme RBOHD in response to the oomycete elicitor cryptogein.
Highlight text The oomycete elicitor cryptogein triggers the relocation of RBOHD from intracellular compartments to the plasma membrane in tobacco cells. This suggests that intracellular trafficking is a potential determinant of RBOHD activity.
Cercospora beticola toxins. IX. Relationship between structure of beticolins, inhibition of plasma membrane H+ -ATPase and partition in lipid membranes
Beticolins are yellow toxins produced by the fungus Cercospora beticola. The effect of one of them, beticolin-1, has been investigated on corn root plasma membrane H + -ATPase (EC 3.6.1.35) at different purification levels (plasma membrane fraction. partially, or highly purified enzyme). The results obtained demonstrated that (1) the purified proton pump was inhibited directly by low amounts of the toxin (I 50 =1.62 ± 0.18 μM), (2) the biological effects of beticolin-1 were similar to those of CBT (Cercospora beticola toxin). Furthermore, it was established that the efficiency of the different beticolins was clearly related to their ability to interact with the lipid bilayers, determined by…
Regulation of plant NADPH oxidase.
Addendum to: Regulation of Reactive Oxygen Species Production by a 14-3-3 Protein in Elicited Tobacco Cells. T. Elmayan, J. Fromentin, C. Riondet, G. Alcaraz, J. Blein and F. Simon-Plas. Plant Cell Environ 2007; 30:722–32; International audience; The production of Reactive Oxygen Species (ROS) is one of the key events occurring during the response of plants to environmental changes, and contributing to establish adaptive signaling pathways. A plasma membrane bound NADPH oxidase enzyme has been evidenced as the ROS producing system in various plant‑microorganisms interactions. We very recently reported, that a protein of the 14‑3‑3 family was able to interact directly with the C‑terminus par…
Plant lipids: Key players of plasma membrane organization and function
International audience; The Plasma Membrane (PM) is a key structure protecting the cell, regulating nutrient exchanges and acting as a control tower allowing the cell to perceive signals. Plasma comes from the greek πλάσμα meaning "which molds", meaning that the PM takes the shape of the cell by delimitating it. The PM harbors the appropriate signaling cascades allowing adaptive responses ensuring proper cell functions in a continuously fluctuating environment, crucial for cell survival. To address this challenge, the PM needs to be both stable and robust yet incredibly fluid and adaptable. This amazing combination of long-term stability and short-term dynamics in order to adapt to signals …
Rôle des microdomaines du plasmalemme dans la réponse de la cellule végétale au stress biotique
National audience
The Plant Defense Elicitor Cryptogein Stimulates Clathrin-Mediated Endocytosis Correlated with Reactive Oxygen Species Production in Bright Yellow-2 Tobacco Cells
Abstract The plant defense elicitor cryptogein triggers well-known biochemical events of early signal transduction at the plasma membrane of tobacco (Nicotiana tabacum) cells, but microscopic observations of cell responses related to these early events were lacking. We determined that internalization of the lipophilic dye FM4-64, which is a marker of endocytosis, is stimulated a few minutes after addition of cryptogein to tobacco Bright Yellow-2 (BY-2) cells. This stimulation is specific to the signal transduction pathway elicited by cryptogein because a lipid transfer protein, which binds to the same receptor as cryptogein but without triggering signaling, does not increase endocytosis. To…
The fungal elicitor cryptogein induces cell wall modifications on tobacco cell suspension
Upon addition of the fungal elicitor cryptogein, suspension cells of tobacco (Nicotiana tabacum cv. Xanthi) aggregated in clusters. Cytochemical experiments indicated that elicited cells displayed fibrillar expansions of pectin along the primary cell wall. Immunocytochemical detection of pectin epitopes indicated that the fibrillar material surrounding the treated cells was mostly composed of low methylated galacturonan sequences, but the use of the cationic probe did not reveal the presence of negatively charged carboxyl groups: the presence of important amounts of calcium ions in these pectic fibrillar expansions accounts for these observations. These data indicate that tobacco cells trea…
The plasma membrane oxidase NtrbohD is responsible for AOS production in elicited tobacco cells
Summary A cDNA encoding a protein, NtrbohD, located on the plasma membrane and homologue to the flavocytochrome of the neutrophil NADPH oxidase, was cloned in tobacco. The corresponding mRNA was accumulated when tobacco leaves and cells were treated with the fungal elicitor cryptogein. After elicitation with cryptogein, tobacco cells transformed with antisense constructs of NtrbohD showed the same extracellular alkalinization as the control, but no longer produced active oxygen species (AOS). This work represents the first demonstration of the function of a homologue of gp91–phox in AOS production in elicited tobacco cells.
Differential Effect of Plant Lipids on Membrane Organization
SPE IPM; International audience; 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…
Rôle de la dynamique de la membrane plasmique dans la mise en place des réactions de défense chez le tabac
National audience; Lors des interactions entre les plantes et les microorganismes, des molécules spécifiques élicitent les réponses de défense des cellules végétales. La cryptogéine, une élicitine produite par l’oomycète Phytophtora cryptogea, induit une réponse hypersensible (HR) et une résistance non systémique chez le tabac (1). Aussi bien sur plante entière que sur suspension cellulaire, la reconnaissance au niveau de la membrane plasmique et la fixation de cet éliciteur par un récepteur non identifié déclenche une cascade d’événements au niveau de la membrane plasmique comme un influx rapide de calcium, des efflux d’ions, une alcalinisation extracellulaire. De plus, une NADPH oxydase, …
Plant lipid rafts : from proteic composition to physiological roles
International audience; A large body of evidence 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 (1). We demonstrated the presence, in plants, of detergent resistant fractions isolated from plasma membrane, with a lipidic composition similar to animal microdomains (2). Electrophoresis experiments indicated that these fractions were able to recruit a specific set of plasma membrane proteins and exclude others. We used mass spectrometry to give an extensive description of a tobacco plasma membrane microdomains. This led to the identification of 145 proteins wh…
Lipids of the Plant Plasma Membrane
The plasma membrane (PM) is arguably the most diverse membrane of the plant cell. Furthermore, the protein and lipid composition of the PM varies with cell type, developmental stage, and environment. Physical properties of lipids and associate proteins allow the formation of a barrier that is selectively permeable to macromolecules and solutes. As the plasma membrane delineates the interface between the cell and the environment, it is the primary part of signal recognition and transduction into intracellular responses for nutritional uptake/distribution, environmental responses, and developmental signaling. Many essential PM functions are carried out by proteinaceous components. However, PM…
Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes.
International audience; We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5 kbar at 30 °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-β-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 hig…
Asymétrie de la membrane plasmique végétale : caractérisation et rôle dans la signalisation immunitaire des plantes
National audience; La membrane plasmique (MP) forme une barrière sélective entre la cellule et le milieu extérieur.C’est un senseur des modifications environnementales et une plateforme orchestrant latransduction du signal permettant de déployer une réponse adaptée. Elle joue donc un rôleessentiel dans la physiologie cellulaire. Décrypter les bases moléculaires de l’organisation dela MP apparaît donc nécessaire pour comprendre comment les plantes s’adaptent auxmodifications environnementales. La MP est une bicouche de lipides à laquelle des protéinessont associées, et les données accumulées sur ce modèle ont révélé son organisation complexeet dynamique et le rôle essentiel joué par les lipi…
Purification, characterization and influence on membrane properties of the plant-specific sphingolipids GIPC
AbstractThe plant plasma membrane (PM) is an essential barrier between the cell and the external environment. The PM is crucial for signal perception and transmission. It consists of an asymmetrical lipid bilayer made up of three different lipid classes: sphingolipids, sterols and phospholipids. The most abundant sphingolipids in the plant PM are the Glycosyl Inositol Phosphoryl Ceramides (GIPCs), representing up to 40% of total sphingolipids, assumed to be almost exclusively in the outer leaflet of the PM. In this study, we investigated the structure of GIPCs and their role in membrane organization. Since GIPCs are not commercially available, we developed a protocol to extract and isolate …
Plasma membrane microdomains in plants: for which physiological roles?
International audience; A large body of evidence 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 (1). We demonstrated the presence, in plants, of detergent resistant fractions isolated from plasma membrane, with a lipidic composition similar to animal microdomains (2). Electrophoresis experiments indicated that these fractions were able to recruit a specific set of plasma membrane proteins and exclude others. We used mass spectrometry to give an extensive description of a tobacco plasma membrane microdomains. This led to the identification of 145 proteins wh…
Rac regulation of NtrbohD, the oxidase responsible for the oxidative burst in elicited tobacco cell
Five cDNAs encoding Rac protein homologues to the Rho-related proteins from plants (Rop) were isolated in tobacco, and the function of one of them, Ntrac5, was studied. The Ntrac5 mRNA is repressed when tobacco leaves and cells are treated with the fungal elicitor cryptogein. Tobacco cells were transformed with sense constructs of Ntrac5 or Ntrac5V15, encoding the native GTP/GDP-bound form of this Rac protein homologue or the constitutively active mutant in its GTP-bound form, respectively. Immunological studies indicate that the corresponding protein is continuously located on the plasma membrane (PM). Both types of transformed cells show the same extra-cellular alkalinization as the contr…
Revisiting Plant Plasma Membrane Lipids in Tobacco: A Focus on Sphingolipids
International audience; 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 …
Tobacco cells contain a protein, immunologically related to the neutrophil small G protein Rac2 and involved in elicitor-induced oxidative burst.
Abstract Suspension-cultured cells of Nicotiana tabacum generated active oxygen species (AOS) when they were treated with the proteinaceous elicitor, cryptogein. This response was blocked by diphenylene iodonium, an inhibitor of the neutrophil NADPH oxidase. When microsomal extracts of tobacco cells were probed with an antibody directed against the human small G protein Rac2, two immunoreactive proteins were detected at 18.5 and 20.5 kDa. The same experiment performed with cytosolic extracts of tobacco cells led to the observation of a strong immunoreactive protein at 21.5 kDa only in the cryptogein-treated cells. The appearance of this cytosolic protein was related to the production of AOS…
Regulation of reactive oxygen species production by a 14-3-3 protein in elicited tobacco cells.
International audience; The regulation of the system responsible for the production of reactive oxygen species (ROS) during plant–microorganism interaction is still largely unknown. The protein NtrbohD has been recently demonstrated as the plasma membrane oxidase responsible for ROS production in elicited tobacco cells. Here, its C-terminus part was used as a bait in a two-hybrid screen in order to identify putative regulators of this system. This led to the isolation of a cDNA coding for a member of the 14-3-3 protein family. The corresponding transcript was induced after infiltration of tobacco leaves with the fungal elicitor cryptogein. Tobacco cells transformed with an antisense constru…