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.

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.