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

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.

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

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.

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

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 [33 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.

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

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 ß-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.

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

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.

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

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.

Sterols, pleiotropic players in plant-microbe interactions.

Plant-microbe interactions (PMIs) are regulated through a wide range of mechanisms in which sterols from plants and microbes are involved in numerous ways, including recognition, transduction, communication, and/or exchanges between partners. Phytosterol equilibrium is regulated by PMIs through expression of genes involved in phytosterol biosynthesis, together with their accumulation. As such, PMI outcomes also include plasma membrane (PM) functionalization events, in which phytosterols have a central role, and activation of sterol-interacting proteins involved in cell signaling. In spite (or perhaps because) of such multifaceted abilities, an overall mechanism of sterol contribution is difficult to determine. However, promising approaches exploring sterol diversity, their contribution to PMI outcomes, and their localization would help us to decipher their crucial role in PMIs.

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

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.

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

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.

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

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.

Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts.

Involvement of sterols in membrane structural properties has been extensively studied in model systems but rarely assessed in natural membranes and never investigated for the plant plasma membrane (PM). Here, we address the question of the role of phytosterols in the organization of the plant PM. The sterol composition of tobacco BY-2 cell PM was determined by gas chromatography. The cyclic oligosaccharide methyl-beta-cyclodextrin, commonly used in animal cells to decrease cholesterol levels, caused a drastic reduction (50%) in the PM total free sterol content of the plant material, without modification in amounts of steryl-conjugates. Fluorescence spectroscopy experiments using DPH, TMA-DPH, Laurdan, and di-4-ANEPPDHQ indicated that such a depletion in sterol content increased lipid acyl chain disorder and reduced the overall liquid-phase heterogeneity in correlation with the disruption of phytosterol-rich domains. Methyl-beta-cyclodextrin also prevented isolation of a PM fraction resistant to solubilization by nonionic detergents, previously characterized in tobacco, and induced redistribution of the proteic marker of this fraction, NtrbohD, within the membrane. Altogether, our results support the role of phytosterols in the lateral structuring of the PM of higher plant cells and suggest that they are key compounds for the formation of plant PM microdomains.

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

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.

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

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.

Divide and Rule: Plant Plasma Membrane Organization.

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.

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

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.

Ratiometric Fluorescence Live Imaging Analysis of Membrane Lipid Order in Arabidopsis Mitotic Cells Using a Lipid Order-Sensitive Probe.

Eukaryotic cells contain membranes exhibiting different levels of lipid order mostly related to their relative amount of sterol-rich domains, thought to mediate temporal and spatial organization of cellular processes. We previously provided evidence in Arabidopsis thaliana that sterols are crucial for execution of cytokinesis, the last stage of cell division. Recently, we used di-4-ANEPPDHQ, a fluorescent probe sensitive to order of lipid phases, to quantify the level of membrane order of the cell plate, the membrane structure separating daughter cells during somatic cytokinesis of higher plant cells. By employing quantitative, ratiometric fluorescence microscopy for mapping localized lipid order levels, we revealed that the Arabidopsis cell plate represents a high-lipid-order domain of the plasma membrane. Here, we describe step-by-step protocols and troubleshooting for ratiometric live imaging procedures employing the di-4-ANEPPDHQ fluorescent probe for quantification of membrane lipid order during plant cell division in suspension cell cultures and roots of Arabidopsis thaliana.

Lipids of plant membrane rafts.

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.

An update on plant membrane rafts.

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.