An update on plant membrane rafts

Available online at www.sciencedirect.com

An update on plant membrane rafts Franc¸oise Simon-Plas1, Artemis Perraki2, Emmanuelle Bayer2, Patricia Gerbeau-Pissot1 and Se´bastien Mongrand2 The dynamic segregation of membrane components within microdomains, such as the sterol-enriched and sphingolipidenriched 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. Addresses 1 UMR Plante-Microbe-Environnement 1088, Institut National de la Recherche Agronomique (INRA)-5184, CNRS-Universite´ de Bourgogne, 21065 Dijon Cedex, France 2 Laboratoire de Biogene`se Membranaire (LBM), UMR 5200 CNRSUniversite´ de Bordeaux, 146 rue Le´o Saignat, 33076 Bordeaux, France Corresponding author: Mongrand, Se´bastien ([email protected])

Current Opinion in Plant Biology 2011, 14:642–649 This review comes from a themed issue on Cell biology Edited by Simon Gilroy and Julia Davies Available online 6th September 2011 1369-5266/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2011.08.003

nanoscale, sterol–sphingolipid-enriched, ordered assemblies of proteins and lipids in which the metastable-raft resting state can be stimulated to coalesce into larger, more stable raft domains by specific lipid–lipid, protein– lipid and protein–protein interactions [3]. Unravelling the molecular composition of rafts has been contingent on the development of methods enabling their isolation. The supposed Lo structure of rafts, preventing the incorporation of detergent molecules, has long led researchers to consider that they could be recovered in membrane fractions insoluble to non-ionic detergents at low temperatures (detergent-insoluble membranes, DIMs). This approach has been validated by the fact that DIMs were significantly enriched in sterols, sphingolipids and specific subsets of proteins, some of which displayed a clustered distribution within the plasma membrane (PM) [3]. However, the correlation between DIMs and membrane rafts has been questioned, and issues rose about the possible artefacts generated by the use of Triton X100 at 4 8C. For example lipid phase behaviour is highly temperature-dependent and the reduction in temperature alone could potentially induce alterations in lipid organisation. Moreover, Triton actually promotes the formation of ordered domains in model bilayers, by reducing further the already low levels of sphingolipids and cholesterol in the ld phase, and so the presence of the detergent may induce segregation of components of the bilayer [4–6]. It is now clear that DIMs should be considered as a tool among others to analyse the PM subcompartmentalisation and that other techniques allowing the in vivo observation of the nanoscale assemblies should be performed to confirm the potential association of a protein or a lipid within rafts, for example [7].

Where do we stand concerning the characterisation of rafts in plants? Lipid composition

Introduction The membrane raft concept was first introduced in 1997 by Simons and Ikonen [1]. It postulated that the sphingolipid/cholesterol/protein assembly into microdomains, characterised by their tight lipid packing – similar to the sterol-dependent, liquid-ordered (Lo) phase observed in model membranes – could organise membrane trafficking and signalling events. The novelty of the raft concept was to give lipids a key role in the subcompartmentalisation of biological membranes, and to provide a clue as to how this lateral heterogeneity could orchestrate membrane bioactivity [2]. Rafts are now defined as dynamic, Current Opinion in Plant Biology 2011, 14:642–649

In plants, most studies performed so far have used DIMs, isolated from pure PM, as a starting material. DIMs have also been purified from the Golgi apparatus but not from the endoplasmic reticulum in plants [8]. Plant PM DIMs represent the 5–10% of total PM proteins and their molecular composition has been characterised in several species [8–12,13]. Lipids can be divided into three classes: sterols, sphingolipids and glycerolipids. The plant PM exhibits a mix of free sterols, representing up to 20% of total PM lipids [14]. In DIMs this amount increases by almost two fold [10,15]. Several studies have established that conjugated sterols (sterylglucosides and acylated www.sciencedirect.com

An update on plant membrane rafts Simon-Plas et al. 643

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Chemical structures of some plant raft lipids. Free sterol: (a) beta-sitosterol. Conjugated sterols: (b) beta-sitosteryl beta-D-glucopyranoside and (c) beta-sitosteryl 60 -O-palmitoyl beta-D-galactopyranoside and sphingolipids: (d) glucosylceramides: Glucosyl beta-Ceramide (d18:1/18:1delta9) (e) glycosylinositol phosphoceramide, GIPC: N-acetylglucosamine (alpha1-4)-glucuronic acid (alpha1-2)-myo-inositol-1-O-phosphoceramide (t18:1/2hydroxy 24:0)

sterylglucosides, see Figure 1) are also enriched in plant DIMs when compared to the PM [8,11,12,15]. Plant sphingolipids have a ceramide backbone structure different from their animal counterparts, with up to eight different long chain bases (LCB), an esterified fatty acid that is often 2-hydroxylated, and a complex polar head that can contain up to 13 sugar moieties. The major plant sphingolipids are glycosylinositolphosphoceramides (GIPCs), which contain mostly 2-hydroxylated saturated or mono-unsaturated very long chain fatty acids, and glucosylceramides see Figure 1 [16]. Total LCB are www.sciencedirect.com

enriched in DIMs when compared to the PM, by a factor of 2–7 depending on the plant species [10,12], probably owing to an overall enrichment of GIPCs. Analyses have shown that the major structural glycerophospholipids are largely depleted from DIMs. Interestingly, specific polyphosphoinositides, a class of minor phospholipids involved in signalling, were found enriched in DIMs [17]. Finally, when considering the ratio of saturated versus unsaturated fatty acyl chains, a significant increase was observed in DIMs compared to the PM, consistent with the putative Lo structure of rafts [8,11]. Current Opinion in Plant Biology 2011, 14:642–649

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Physical properties – organisation

Phytosterols appear as central structural elements of plant membrane rafts. Depletion of sterols induced by methyl-bcyclodextrin resulted in an overall increase in acyl chain disorder and a reduction of the liquid-phase heterogeneity of the isolated PM. The fact that such a treatment also prevented DIM recovery strengthens the link between the sterol-driven lateral heterogeneity of the PM and resistance to solubilisation by non-ionic detergents [18]. These results are in agreement with the ability of phytosterols to induce a Lo phase in ternary mixtures of lipids [19]. Recovery of DIMs also strongly depends on the unsaturation degree of fatty acyl chains. Strong evidence came from the dramatic decrease in the total amount of DIMs, isolated from the Arabidopsis thaliana fad2 and Fad3+ mutants, affected in their fatty acid desaturases [8]. Protein composition

Proteomic analyses of DIMs have been mainly performed on tobacco (Nicotiana tabacum), Arabidopsis and Medicago truncatula [10,13,20]. All studies showed a DIM protein profile distinct from the whole PM. Several hundreds of proteins have been identified, most being involved in signalling and response to biotic/abiotic stresses (particularly the leucine-rich repeat receptor kinase family, LRR), cellular trafficking, and cell wall metabolism [9,10,12,13,21,22]. Interestingly, DIMs extracted from M. truncatula roots, also revealed the presence of a complete PM redox system [12]. As in animals, plant rafts are probably involved in signal transduction processes. Size

It is impossible to define a unique size for membrane rafts given that they can vary from small, short-lived, nanoscale assemblies to more stable membrane domains, depending on the cell type and the physiological conditions [3]. Another hurdle in the estimation of the in vivo membrane raft size, resides in the fact that most conventional microscopic approaches depict a rather low-resolution limit, which is not well adapted for the observation of nanoscale structures. Nevertheless, in plants, the PM-surface distribution of specific proteins and lipids, found enriched in DIMs, was analysed by electron microscopy using immunogold labelling coupled with a pattern-identifying statistical analysis [17,23]. By this approach, the protein remorin (REM) was observed clustering in microdomains of about 70 nm in diameter. REM became randomly redistributed following cyclodextrin treatment, reinforcing the link between the structuring action of phytosterols and the spatial distribution of the protein at the PM [23]. Microdomains of phosphatidylinositol (4,5)-bisphosphate (PIP2) were also visualised in the plane of the PM with a size of 25 nm. These data are consistent with comparable studies performed on animal PM, revealing a clustering of lipids and proteins in domains of 10–70 nm [24–28]. Using the sterol-binding fluorochrome filipin and confocal microscopy, several groups demonstrated the presence of larger Current Opinion in Plant Biology 2011, 14:642–649

sterol-rich PM domains of about 10 to 30 mm. Such domains were visualised in roots [29], in growing tips of pollen tubes [30] or at fungal infection sites [31]. A current challenge consists in reconciling these different observations, using on the same material, similar sets of molecular markers (lipids and proteins) and high-resolution imagery approaches. This should provide key information about the different scales of membrane subcompartmentalisation and their relationships.

A role for raft-driven lateral compartmentalisation in plant cell signalling? Membrane raft ability to temporally and spatially organise protein complexes, thereby allowing regulation of cellular processes, relies on a key concept: dynamic membrane compartmentalisation. Although the underlying molecular basis remains largely undeciphered, two non-exclusive mechanisms might be involved: (1) clustering of nanoscale assemblies into more stable, selective and functional platforms, or (2) association-dissociation of specific proteins to particular domains [2,3]. To date, data obtained in plants point to the latter mechanism with the modification of DIM protein content upon different biological stimuli [13,22,32,33]. In animal cells, evidence has been provided that posttranslational modifications involving the adjunction of saturated lipid-anchors trigger recruitment of both peripheral and transmembrane proteins to rafts, while short, unsaturated, and/or branched hydrocarbon chains prevent raft association [34,35]. In DIMs from BY-2 tobacco cells 16% of the proteins exhibited putative fatty acid modification sites (namely palmitoylation and myristoylation) [21]. These sites were exclusively present in proteins involved in signalling or response to stress, a particularly relevant observation since the reversibility of the palmitoylation/myristoylation processes is well adapted for dynamic signal transduction processes. This hypothesis was confirmed in a very elegant study showing that transient S-acylation of the small Arabidopsis G-protein AtROP6, regulates its association with DIMs. Moreover, acylation-deficient AtROP6 mutants can bind and hydrolyse GTP but display aberrant root hair polar cell growth, endocytosis and reactive oxygen species distribution, indicating that S-acylation regulates signalling processes via dynamic targeting to specialised membrane domains [36]. This is in agreement with quantitative proteomics, revealing that proteins with signalling functions, such as receptor kinases, G-proteins, and calcium signalling proteins, were for the most part identified as fluctuating members of plant rafts, whereas cell wall-related proteins made up a core set of sterol-dependent raft proteins [13].

Membrane rafts as PAMP-associated signalling platforms? Dynamic changes in the distribution of sterol-rich domains at the PM were first observed during early stages www.sciencedirect.com

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of fungus infection at host-cell entry sites [31]. Recently, the putative role of PM rafts in Pathogen-Associated Molecular Patterns PAMPs-induced signalling processes has been addressed by quantitative proteomic approaches. Three models have been studied: Arabidopsis/bacterial flagellin 22 (flg22) [22], tobacco/oomycetal cryptogein [33] and rice/fungal chitin [37]. PAMP receptors are transmembrane proteins containing extracellular LRR domains, and a cytosolic kinase domain. Binding of the elicitor to LRR leads to structural changes that allow oligomerisation of the receptor with

itself or other cofactors such as BAK1 for plant flg22 receptor. On the cytoplasmic side, the kinase domains get into contact and initiate intracellular signalling, probably by transphosphorylation events [38]. At steady state in non-elicited cells (Figure 2A), the Arabidopsis flg22 receptor, FLS2, is mainly found in the non-raft phase of the PM, that is, in the detergent-soluble membranes (DSMs) [22], whereas other signalling components such as the respiratory burst oxidase homolog (RbohD) are associated with DIMs [13,21,37]. Lipase substrates, like polyphosphoinositides, are also enriched in DIMs and found located in nanoscale PM domains [17]. Lipid-using

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Working model of the putative involvement of rafts in the activation of RbohD after elicitation by PAMP. (a) In non-elicited cells, Arabidopsis flg22 receptors are located in non-raft domains. RbohD, PIP2-PLC, DGK and PLD proteins are detected inside rafts, but are inactive probably owing to a lack of activation, or an inhibition, of the enzyme activities. Their substrates, polyphosphoinositides (PIP and PIP2), are also enriched in the DIMs [17]. Typical raft proteins, like REM, locate inside raft domains [23]. (b) Shortly after elicitation, the flg22 receptor, and other RLKs and cofactors, shift from DSM to DIM, and undergo oligomerisation processes. RbohD enzymes are phosphorylated and enzymatically-activated in rafts by the recruitment of regulators of RbohD, such as 14.3.3 and the small G protein RAC. The signalling phospholipid PA, synthesised quickly after elicitation and probably coming from the PLC/DGK and PLD pathways, activates Rboh by binding to the N-terminal domain of the enzymes. www.sciencedirect.com

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enzymes such as phospholipase D (PLD), phospholipase C (PLC), and PIP2-specific PLC are detected in DIMs [9,13,21,22] although their activity is not associated with this fraction in resting cells, probably owing to a lack of enzyme activation [17]. Shortly after elicitation, FLS2 shifted from DSMs to DIMs probably owing to the oligomerisation process, and a significant number of others RLKs displayed similar behaviour. Among others, H+-transporting or Ca2+-transporting ATPase, ATP Binding Cassette (ABC), nitrate, lysine or sugar transporters, vacuolar H+-ATPase, Ca2+dependant kinases and syntaxin also underwent relocalisation into DIMs after flg22 treatment [22]. Elicitation with cryptogein in tobacco cells triggered a decrease in the association with DIMs of four dynamin-related proteins [33], GTPases involved in the pinching off endocytic vesicles from the PM [39]. This is in agreement with the early stimulation of the Reactive Oxygen Species (ROS)-dependent and clathrin-dependent endocytic pathway demonstrated in this model [40]. The functional link between DIMs and endocytosis during plant–microbe signalling events needs to be clarified. An integrative picture of the putative involvement of PM rafts in the early signalling process is currently emerging. In non-elicited cells, a plant raft, with an average size of 30–70 nm, might contain 50–80 lipid molecules and at the most only a dozen membrane proteins [41]. Figure 2 presents a working model of the putative involvement of rafts in the activation of RbohD, summarising what could be inferred from the different studies discussed above. The rapid phosphorylation of RbohD after flg22 treatment [42] and the fact that some of its regulators, such as 14.3.3 [33,43] and small G proteins [37] are recruited in sterol-rich domains, is consistent with a local activation of this enzyme. The discrete accumulation of ROS within patches of 50–100 nm along the PM of cryptogein-elicited cells, fits with a microsegregation and activation of RbohD in nanocale PM domains [44]. Additionally, Zhang et al. [45] showed that the signalling phospholipid phosphatidic acid (PA) activates AtRbohD by binding to the N-terminal domain of the enzyme [45]. Consistently, PA has been shown to accumulate upon elicitation by PAMPs, and a direct application of PA to plants induces pathogen-related gene expression and cell death [46]. Generated by two distinct enzymatic paths, directly by PLD or via the combined action of PLC and diacylglycerol kinase (DGK), the enzymatic origin of elicitor-induced PA is still uncertain. In tomato cells treated with xylanase, PA and its phosphorylated derivative diacylglycerolpyrophosphate, are quickly synthesised after elicitation and appear to produce from both pathways [46]. How all these different proteins, lipidusing enzymes and lipids are organised after elicitation is of outmost importance. For example, whether or not PA is synthesised in the raft domain remains to be elucidated. Current Opinion in Plant Biology 2011, 14:642–649

Role of membrane rafts in legume symbiosis Two groups of plant proteins upregulated during nodulation, namely group 2 remorins (SYMREM) and flotillin (FLOT), were found to be enriched in DIMs purified from the host legume M. truncatula roots infected by Sinorhizobium meliloti and to locate in membrane-associated puncta [47,48]. In addition FLOT4 becomes polarly localised in response to bacterial signals [48]. Moreover, SYMREM-silenced, FLOT2-silenced and FLOT4silenced plants are defective in nodule formation and functioning. These findings highlight the potential role of membrane subcompartments during symbiotic plant microbe interactions.

A functional link between plasmodesmata and membrane raft domains Recent data indicate that a link exists between raft microdomains and the membranous channels plasmodesmata (PD). Remorin, the first membrane raft protein marker identified in plants, also accumulates at the PM lining the PD (PD–PM) in Solanaceae [23], and AtREM1.2 a member of the REM family has been recently identified in the Arabidopsis PD proteome [49]. A role for membrane rafts in PD function has been suggested in the context of viral infection. Variation of REM expression levels in tomato specifically affects the cell-to-cell movement of the virus Potato Virus X (PVX). Moreover, REM directly binds Triple Gene Bock Protein1 (TGBp1) [23], the PVX movement protein that targets PD and promotes viral RNA-PVX cell-to-cell movement [50,51]. The localisation of REM at PD is likely to reflect the presence of raft microdomains in these structures (see also Andrew J Maule, Yoselin BenitezAlfonso and Christine Faulkner ‘‘Plasmodesmata – membrane tunnels with attitude’’, in this issue). REM accumulation at PD may reflect the ‘raft-like’ nature (i.e. an enrichment in sterol and sphingolipids) of the PD–PM, or portion of it. This hypothesis receives support from the fact that several glycosylphosphatidylinositolanchored proteins, a class of proteins known to be preferentially associated with plant rafts [10,13], have already been identified as PD constituents [52,53]. Stable raft-like domains could then participate in the lateral membrane segregation that exists at the PD–PM.

Polarisation of sterol-enriched lipid microdomains In animal and yeast cells, rafts are thought to provide platforms for the polar targeting of neosynthesised proteins at the PM, and also mediate localised endocytosis (e.g. [54]). In plants, ABC-transporter/p-glycoprotein (PGP)type auxin efflux carriers, including ABCB19/PGP19, are enriched in DIMs, but significantly less than the polarly localised PINFORMED1 (PIN1) auxin efflux carrier [55]. Endocytosis-defective sterol biosynthetic mutants are affected both in post-cytokinetic acquisition of PIN2 polarity and lateral membrane diffusion of KNOLLE www.sciencedirect.com

An update on plant membrane rafts Simon-Plas et al. 647

[29,56]. Liu et al. [30] reported the involvement of Lo, sterol-rich membrane domains at the tip of Picea meyeri pollen tube growth. Use of a sterol-chelating agent disrupted membrane polarisation, obliterated tip-based ROS formation, dissipated tip Ca2+-gradient and arrested tip growth, establishing a functional link between the steroldriven lateral compartmentalisation of the membrane and polarised growth.

Conclusions The study of raft-based microsegregation at the PM of plants revealed a great diversity in terms of size, composition and function. One future goal will be to decipher this diversity in different biological contexts and tissue types in planta, as well as to determine the topology and dynamics of raft proteins and lipids. In this context development of specific tools allowing the visualisation of specific domains remains one of the most important challenges.

Acknowledgements Membrane raft research in the authors’ laboratory is supported by French program ANR NT09_517917, PANACEA. Thanks go to Alain Glowszak for drawing the figure illustration and Patrick Moreau, Ve´ronique Germain and Jean-Luc Cacas for critical reading. We apologise to all colleagues whose work was not cited owing to space restriction.

References and recommended reading Papers of particular interest published within the period of review have been highlighted as:  of special interest  of outstanding interest

9.

Shahollari B, Peskan Bergho¨fer T, Oelmu¨ller R: Receptor kinases with leucine rich repeats are enriched in Triton X 100 insoluble plasma membrane microdomains from plants. Physiol Plan 2004, 122:397-403.

10. Borner GH, Sherrier DJ, Weimar T, Michaelson LV, Hawkins ND, MacAskill A, Napier JA, Beale MH, Lilley KS, Dupree P: Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts. Plant Physiol 2005, 137:104-116. 11. Mongrand S, Morel J, Laroche J, Claverol S, Carde JP, Hartmann MA, Bonneu M, Simon-Plas F, Lessire R, Bessoule JJ: Lipid rafts in higher plant cells: purification and characterization of Triton X-100-insoluble microdomains from tobacco plasma membrane. J Biol Chem 2004, 279:36277-36286. 12. Lefebvre B, Furt F, Hartmann MA, Michaelson LV, Carde JP, Sargueil-Boiron F, Rossignol M, Napier JA, Cullimore J, Bessoule JJ: Characterization of lipid rafts from Medicago truncatula root plasma membranes: a proteomic study reveals the presence of a raft-associated redox system. Plant Physiol 2007, 144:402-418. 13. Kierszniowska S, Seiwert B, Schulze WX: Definition of  Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-b-cyclodextrin and quantitative proteomics. Mol Cell Proteomics 2009, 8:612-623. This study, performed on Arabidopsis, was the first to challenge the role of phytosterols in the segregation of proteins in the PM, using quantitative proteomics and disruption of sterol-rich membrane domains (DIMs) by methyl-cyclodextrin. Components of plant sterol-rich membrane microdomains were distinguished based on their responsiveness to the drug. Predominantly proteins with signalling functions, such as receptor kinases, G-proteins-, and calcium signalling proteins, were identified as variable members in plant rafts, whereas cell wall-related proteins and specific proteins with unknown functions make up a core set of sterol-dependent plant PM proteins. 14. Hartmann MA: Plant sterols and the membrane environment. Trends Plant Sci 1998, 3:170-175. 15. Furt F, Lefebvre B, Cullimore J, Bessoule JJ, Mongrand S: Plant lipid rafts: fluctuat nec mergitur. Plant Signal Behav 2007, 2:508-511. 16. Pata MO, Hannun YA, Carl KY: Plant sphingolipids: decoding the enigma of the Sphinx. New Phytol 2010, 185:611-630.

1.

Simons K, Ikonen E: Functional rafts in cell membranes. Nature 1997, 387:569-572.

2.

Lingwood D, Simons K: Lipid rafts as a membrane-organizing principle. Science 2009, 327:46-50.

3.

Simons K, Gerl MJ: Revitalizing membrane rafts: new tools and insights. Nat Rev Mol Cell Biol 2010, 11:688-699.

4.

Munro S: Lipid rafts: elusive or illusive? Cell 2003, 115:377-388.

5.

Brown DA: Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda) 2006, 21:430-439.

18. Roche Y, Gerbeau-Pissot P, Buhot B, Thomas D, Bonneau L, Gresti J, Mongrand S, Perrier-Cornet JM, Simon-Plas F: Depletion of phytosterols from the plant plasma membrane provides evidence for disruption of lipid rafts. FASEB J 2008, 22:3980-3991.

6.

Tanner W, Malinsky J, Opekarova´ M: In plant and animal cells, Detergent-Resistant Membranes do not define functional membrane rafts. Plant Cell 2011, 23:1191-1193.

19. Beck JG, Mathieu D, Loudet C, Buchoux S, Dufourc EJ: Plant sterols in ‘‘rafts’’: a better way to regulate membrane thermal shocks. FASEB J 2007, 21:1714-1723.

7. 

Eggeling C, Ringemann C, Medda R, Schwarzmann G, Sandhoff K, Polyakova S, Belov VN, Hein B, Von Middendorff C, Scho¨nle A: Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 2008, 457:1159-1162. In this pioneering work, the authors demonstrate the ability of stimulatedemission depletion far-field fluorescence nanoscopy to detect single diffusing (lipid) molecules in nanosized areas of the plasma membrane of living cells. Tuning of the probed area to spot sizes 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently trapped in cholesterol-mediated molecular complexes dwelling within 20-nm diameter areas. This ability to combine temporal and spatial resolution in a non-invasive way is a powerful new approach to study the dynamics of biomolecules in living cells. 8.

Laloi M, Perret AM, Chatre L, Melser S, Cantrel C, Vaultier MN, Zachowski A, Bathany K, Schmitter JM, Vallet M: Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells. Plant Physiol 2007, 143:461.

www.sciencedirect.com

17. Furt F, Ko¨nig S, Bessoule JJ, Sargueil F, Zallot R, Stanislas T, Noirot E, Lherminier J, Simon-Plas F, Heilmann I: Polyphosphoinositides are enriched in plant membrane rafts and form microdomains in the plasma membrane. Plant Physiol 2010, 152:2173-2187.

20. Peskan T, Westermann M, Oelmu¨ller R: Identification of lowdensity Triton X-100-insoluble plasma membrane microdomains in higher plants. Eur J Biochem 2000, 267:6989-6995. 21. Morel J, Claverol S, Mongrand S, Furt F, Fromentin J, Bessoule JJ, Blein JP, Simon-Plas F: Proteomics of plant detergent-resistant membranes. Mol Cell Proteomics 2006, 5:1396-1411. 22. Keinath NF, Kierszniowska S, Lorek J, Bourdais G, Kessler SA,  Shimosato-Asano H, Grossniklaus U, Schulze WX, Robatzek S, Panstruga R: PAMP (pathogen-associated molecular pattern)induced changes in plasma membrane compartmentalization reveal novel components of plant immunity. J Biol Chem 2010, 285:39140-39149. In this study, quantitative proteomics was used to monitor the variation of protein amount in DIMs after short-term flagelin elicitation in Arabidopsis cells. A set of proteins was found significantly enriched in this fraction: among them proton pump ATPases and receptor-like kinases, including the flagellin receptor FLS2. The role of some of these proteins Current Opinion in Plant Biology 2011, 14:642–649

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in flg22 responses was further analysed by reverse genetics, namely DET3, AHA1 and FER. No proteins were found excluded from DIM after elicitation.

34. Levental I, Lingwood D, Grzybek M, Coskun U¨, Simons K: Palmitoylation regulates raft affinity for the majority of integral raft proteins. Proc Natl Acad Sci USA 2010, 107:22050-22054.

23. Raffaele S, Bayer E, Lafarge D, Cluzet S, Retana SG, Boubekeur T,  Leborgne-Castel N, Carde JP, Lherminier J, Noirot E: Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement. Plant Cell 2009, 21:1541-1555. This study identifies the first biochemical marker of PM rafts in plant, and demonstrates the existence of microsegregation of REM protein in nanodomains of 70 nm in a sterol-dependent way, thus making a link between the association of a protein to DIMs and its lateral segregation within particular membrane domains. In addition, it proposes the first physiological role of REM protein as a putative receptor for the movement protein TGBP1 of PVX virus. This study finally suggests that the PM-lining the PD exhibit raft properties.

35. Levental I, Grzybek M, Simons K: Greasing their way: lipid modifications determine protein association with membrane rafts. Biochemistry 2010, 49:6305-6316.

24. Prior IA, Muncke C, Parton RG, Hancock JF: Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J Cell Biol 2003, 160:165-170. 25. Parton RG, Hancock JF: Lipid rafts and plasma membrane microorganization: insights from Ras. Trends Cell Biol 2004, 14:141-147. 26. Fujita A, Cheng J, Hirakawa M, Furukawa K, Kusunoki S, Fujimoto T: Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling. Mol Biol Cell 2007, 18:2112-2122. 27. Lillemeier BF, Pfeiffer JR, Surviladze Z, Wilson BS, Davis MM: Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc Natl Acad Sci USA 2006, 103:18992-18997. 28. Plowman SJ, Muncke C, Parton RG, Hancock JF: H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton. Proc Natl Acad Sci USA 2005, 102:15500-15505. 29. Men S, Boutte´ Y, Ikeda Y, Li X, Palme K, Stierhof YD, Hartmann MA, Moritz T, Grebe M: Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity. Nat Cell Biol 2008, 10:237-244. 30. Liu P, Li RL, Zhang L, Wang QL, Niehaus K, Balusˇka F, Sˇamaj J,  Lin JX: Lipid microdomain polarization is required for NADPH oxidase dependent ROS signaling in Picea meyeri pollen tube tip growth. Plant J 2009, 60:303-313. Using di-4-ANEPPDHQ, a fluorescent probe whose emission properties are sensitive to membrane organisation, the authors showed that the growing tip of pollen tube corresponded to a sterol-enriched ordered phase. Moreover, sterol sequestration disrupted this membrane polarisation and inhibited several signalling processes, thereby arrested tip growth. Finally they demonstrated that the NADPH oxidase responsible for growth-related ROS production clustered at the growing tip, within the ordered membrane domains, was partially associated with DIMs, and that this localisation was important for its enzymatic activity. These results suggest a strong link between the presence of the NADPH oxidase within lipid microdomains of the apical PM, and the growth of the pollen tube in a highly polarised manner. 31. Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R: Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc Natl Acad Sci USA 2005, 102:3135-3140. 32. Minami A, Fujiwara M, Furuto A, Fukao Y, Yamashita T, Kamo M, Kawamura Y, Uemura M: Alterations in detergent-resistant plasma membrane microdomains in Arabidopsis thaliana during cold acclimation. Plant Cell Physiol 2009, 50:341-359. 33. Stanislas T, Bouyssie D, Rossignol M, Vesa S, Fromentin J,  Morel J, Pichereaux C, Monsarrat B, Simon-Plas F: Quantitative proteomics reveals a dynamic association of proteins to detergent-resistant membranes upon elicitor signaling in tobacco. Mol Cell Proteomics 2009, 8:2186-2198. Quantitative proteomics using metabolic labelling was used for the first time to demonstrate the variation of protein content of DIMs in a defencesignalling context. After short-term elicitation of BY-2 tobacco cells by the proteinaceous elicitor cryptogein, four dynamin-related proteins were less abundant in the DIM, whereas a 14-3-3 protein was enriched. Current Opinion in Plant Biology 2011, 14:642–649

36. Sorek N, Segev O, Gutman O, Bar E, Richter S, Poraty L, Hirsch JA,  Henis YI, Lewinsohn E, Ju¨rgens G: An S-acylation switch of conserved G domain cysteines is required for polarity signaling by ROP GTPases. Curr Biol 2010, 20:914-920. In this very elegant study, the authors demonstrate that the transient Sacylation of a small G protein of the RAC family (AtROP6) takes place on two conserved cysteine residues of the G domain, and that this process regulates both the membrane-association dynamics of the protein and its association to the DIMs. Moreover, using site-specific acylation-deficient AtROP6 mutants, they showed that although the ability of the protein to hydrolyse GTP was maintained, its ability to regulate several signalling events was affected, suggesting that S-acylation could activate ROP signalling by enhancing ROP partitioning into lipid rafts and stabilising ROP membrane interactions. 37. Fujiwara M, Hamada S, Hiratsuka M, Fukao Y, Kawasaki T, Shimamoto K: Proteome analysis of detergent-resistant membranes (DRMs) associated with OsRac1-mediated innate immunity in rice. Plant Cell Physiol 2009, 50:1191-1200. 38. Boller T, Felix G: A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 2009, 60:379-406. 39. Saalbach G, Erik P, Wienkoop S: Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes. Proteomics 2002, 2:325-337. 40. Leborgne-Castel N, Lherminier J, Der C, Fromentin J, Houot V, Simon-Plas F: The plant defense elicitor cryptogein stimulates clathrin-mediated endocytosis correlated with reactive oxygen species production in bright yellow-2 tobacco cells. Plant Physiol 2008, 146:1255-1266. 41. Mongrand S, Stanislas T, Bayer EM, Lherminier J, Simon-Plas F: Membrane rafts in plant cells. Trends Plant Sci 2010, 15:656-663. 42. de la Fuente van Bentem S, Hirt H: Using phosphoproteomics to reveal signalling dynamics in plants. Trends Plant Sci 2007, 12:404-411. 43. Elmayan T, Fromentin J, Riondet C, Alcaraz G, Blein JP, SimonPlas F: Regulation of reactive oxygen species production by a 14-3-3 protein in elicited tobacco cells. Plant Cell Environ 2007, 30:722-732. 44. Lherminier J, Elmayan T, Fromentin J, Elaraqui KT, Vesa S, Morel J, Verrier JL, Cailleteau B, Blein JP, Simon-Plas F: NADPH oxidasemediated reactive oxygen species production: subcellular localization and reassessment of its role in plant defense. Mol Plant Microbe Interact 2009, 22:868-881. 45. Zhang Y, Zhu H, Zhang Q, Li M, Yan M, Wang R, Wang L, Welti R,  Zhang W, Wang X: Phospholipase dalpha1 and phosphatidic acid regulate NADPH oxidase activity and production of reactive oxygen species in ABA-mediated stomatal closure in Arabidopsis. Plant Cell 2009, 21:2357-2377. This very nice study aimed at determining the role of phospholipase D alpha1 and its lipid product PA in abscisic acid (ABA)-induced production of ROS in Arabidopsis thaliana guard cells. The pld alpha1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pld alpha1 cells, whereas PA-stimulated the NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidases RbohD and RbohF. The PA binding motifs were identified, and punctual mutation of corresponding aminoacids triggered the loss of PA binding and activation of RbohD. Moreover, a mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure, demonstrating that PA is as a central lipid signalling molecule in the ABA signalling network in guard cells. 46. Testerink C, Munnik T: Molecular, cellular, and physiological responses to phosphatidic acid formation in plants. J Exp Bot 2011, 62:2349-2361. www.sciencedirect.com

An update on plant membrane rafts Simon-Plas et al. 649

47. Lefebvre B, Timmers T, Mbengue M, Moreau S, Herve´ C, To´th K, Bittencourt-Silvestre J, Klaus D, Deslandes L, Godiard L: A remorin protein interacts with symbiotic receptors and regulates bacterial infection. Proc Natl Acad Sci USA 2010, 107:2343-2348. 48. Haney CH, Long SR: Plant flotillins are required for infection by nitrogen-fixing bacteria. Proc Natl Acad Sci USA 2010, 107:478-483. 49. Fernandez-Calvino L, Faulkner C, Walshaw J, Saalbach G, Bayer E, Benitez-Alfonso Y, Maule A: Arabidopsis plasmodesmal proteome. PLoS ONE 2011, 6:e18880.

52. Levy A, Erlanger M, Rosenthal M, Epel BL: A plasmodesmataassociated beta-1,3-glucanase in Arabidopsis. Plant J 2007, 49:669-682. 53. Simpson C, Thomas C, Findlay K, Bayer E, Maule AJ: An Arabidopsis GPI-anchor plasmodesmal neck protein with callose binding activity and potential to regulate cell-to-cell trafficking. Plant Cell 2009, 21:581-594. 54. Hanzal-Bayer MF, Hancock JF: Lipid rafts and membrane traffic. FEBS Lett 2007, 581:2098-2104.

50. Verchot-Lubicz J, Ye CM, Bamunusinghe D: Molecular biology of potexviruses: recent advances. J Gen Virol 2007, 88:1643-1655.

55. Titapiwatanakun B, Blakeslee JJ, Bandyopadhyay A, Yang H, Mravec J, Sauer M, Cheng Y, Adamec J, Nagashima A, Geisler M: ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis. Plant J 2009, 57:27-44.

51. Howard AR, Heppler ML, Ju HJ, Krishnamurthy K, Payton ME, Verchot-Lubicz J: Potato virus X TGBp1 induces plasmodesmata gating and moves between cells in several host species whereas CP moves only in N. benthamiana leaves. Virology 2004, 328:185-197.

56. Boutte´ Y, Frescatada-Rosa M, Men S, Chow CM, Ebine K, Gustavsson A, Johansson L, Ueda T, Moore I, Ju¨rgens G: Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis. EMBO J 2009, 29:546-558.

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Current Opinion in Plant Biology 2011, 14:642–649