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Meetings Intercellular communication via plasmodesmata EMBO Workshop on Intercellular Communication in Plant Development and Disease, Bischoffsheim, France, August 2014 Intercellular communication is fundamental to multicellularity. In plants, plasmodesmata (PD) are membrane-lined channels that connect the cytoplasm of adjacent cells, allowing direct molecular exchange (Fig. 1). In growth, development and responses to the external environment, PD act as traffic controllers, allowing or restricting the flux of different types of molecules and the downstream processes which they trigger. The European Molecular Biology Organization (EMBO) workshop on ‘Intercellular communication in plant development and disease’ evolved from the PD conference series to bring together researchers who work on the trafficking of different classes of molecules in a variety of contexts. In doing so, the organizers collated some of the most exciting advances in PD research to date. Because PD are small, membranerich structures embedded in the cell wall, their study presents multiple technological challenges such that, in addition to breakthroughs in knowledge, many presenters outlined novel tools and approaches that are likely to take PD research into new realms and also provide a powerful handle to more general cell biological problems in plants.
characterizing their mode of translocation will unlock transport mechanisms. Away from the microscope, Hanna Rademaker and Alexander Schulz (University of Copenhagen, Denmark) have applied mathematical modelling to test the polymer trap model of sugar transport, in which PD act as filters allowing the transport of sucrose but not raffinose. Despite the very small differences in size of these molecules, Rademaker’s calculations support this model and suggest that bulk flow assists in translocating sugars into sieve elements in the concentrations measured experimentally. Callose deposition in the apoplast surrounding PD induces a restriction in the aperture of the cytoplasmic channel of the PD pore, and thus a restriction in intercellular flux (Maule et al., 2012). Several enzymes involved in callose homeostasis (callose synthases and b-1,3-glucanases) have been identified as PD-resident proteins
‘. . . how does polar transport occur when there are “holes” in the membrane?’
Structure and regulation of PD Analysis of PD function depends upon indirect assays that measure the movement of molecules with specific properties. The capacity of PD to act as channels for the passage of different molecules can be extrapolated from these assays, but is limited in application to flux via nonspecific diffusion. One context in which this kind of assay is particularly powerful is the distribution of photoassimilates. PD connect photosynthetic cells to the phloem and therefore molecules that trace the path of sugars provide a snapshot of tissue connectivity. Karl Oparka (University of Edinburgh, UK) has established a novel chemical screen, screening libraries of fluorescent ‘mobiliphores’ for transport activity. He proposes that 970 New Phytologist (2015) 205: 970–972 www.newphytologist.com
Fig. 1 Schematic representation of plasmodesmata (PD) structure. PD are the tunnel-like structure embedded in cell wall and form symplastic continuum between cells. Endoplasmic reticulum (ER) is continuous between adjacent cells within PD. The cytoplasmic sleeve is the major pathway for cellto-cell movement of micromolecules such as photoassimilates and macromolecules such as proteins and nucleic acids. Specific transcriptional factors (TFs) are passed through PD by an active mechanism which involves protein unfolding. As a regulator of PD connectivity, callose can be accumulated in the apoplast surrounding PD, which restricts the PD connectivity. The turnover of this callose is finely controlled by the balance between activities of callose synthases and b-1,3-glucanases located at PD. Additionally, several receptors located at PD also mediate noncell autonomous signalling through PD following perception of apoplastic signals. The lipid composition of PD plasma membrane (PM) is distinct from that of other regions; complex sphingolipids, sterols and glycerolipids are enriched in PD PM. Such membrane microdomains can act as signalling platform for the receptors. Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust
New Phytologist and associated with the regulation of PD flux in both developmental and disease-related contexts. Callose-mediated PD regulation was a repeated theme throughout the meeting with new contexts identified in which PD are regulated via this mechanism. For example, callose deposition occurs in response to stimuli such as low phosphorous (Steffan Abel, Leibniz Institute of Plant Biochemistry, Halle, Germany), and gibberellins (Christiaan van der Schoot, Norwegian University of Life Sciences, As, Norway). Additionally, new players in the molecular regulation of callose synthases and b-1,3-glucanases at PD have also been identified including PDLP5 (Jung-Youn Lee, University of Delaware, Newark, DE, USA), a PD-located ENOD-like protein (Yoselin Benitez-Alfonso, University of Leeds, UK) and lipid composition of the PD plasma membrane (PM) (Danny Geelen, Ghent University, Belgium; Emmanuelle Bayer, LMB-INRA, Paris, France). The lipid composition of PD and its potential to regulate PD function is an exciting new facet of PD biology. Emmanuelle Bayer has performed analysis of PD membranes and found the PD PM to be enriched in complex sphingolipids, sterols and glycerolipids. Significantly, both Bayer and Geelen observed that disruption of the lipid composition of PD altered the trafficking capacity of PD. Bayer noted that sterols and sphingolipids are defining features of lipid rafts which supports the hypothesis that the PD PM is a specialized membrane domain which contains rafts (Raffaele et al., 2009). Noteworthy is that lipid rafts and membrane microdomains can act as signalling platforms for a variety of receptor proteins and receptor kinases and some of these proteins have specialized localization and/or activity at the PD/PM (see later).
PD and plant development PD function is finely regulated to control cell-to-cell communication for normal growth and development. Dynamic alteration of PD function regulates cell-to-cell diffusion of developmental signals upon the basis of their molecular size. It is in this context that callose-mediated regulation of PD functions to restrict PD aperture. At this workshop the regulation of PD callose turnover by auxin was presented (Jae-Yean Kim, Gyeongsang National University, Jinju, South Korea). This work addressed a
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considerable conundrum in PD biology – how does polar transport occur when there are ‘holes’ in the membrane? Kim suggested that auxin triggers PD closure, stopping nonpolar diffusion of auxin through PD. This exciting finding sparked some discussion as it remains to be resolved how small molecules, including sugars, are transported when PD are closed. This is a consideration that must be taken into account during any transient or prolonged closure of PD. In contrast to passive diffusion, specific transcription factors (TFs) are trafficked via PD and transported into their target cells by an active mechanism. This contributes to cell fate determination in meristems, embryos and root ground tissues. Dave Jackson (Cold Spring Harbor Laboratory, NY, USA) reported that chaperonins partially unfold TFs following translocation through PD, identifying the first mechanistic components of active TF translocation. Yvonne Stahl and R€ udiger Simon (Heinrich-Heine University, D€usseldorf, Germany) also identified that PD-located receptor kinases ACR4 and CLV1 regulate the transduction of an as yet unidentified signal that specifies cell stemness in the root meristem. Simon proposed that an ACR4/CLV1 complex may specifically phosphorylate a signal such that it can either pass or not through the PD, echoing previous models of virus movement (Lee & Lucas, 2001).
Intercellular transport of RNA In addition to TFs, the role of small noncoding RNAs as mobile signals that regulate gene expression is significant to development and defence. Mobile microRNAs (miRNA) provide positional information to regulate cell stemness in the shoot meristem (Thomas Laux, Albert Ludwigs University, Freiburg, Germany) and to establish adaxial–abaxial polarity in the leaf (Marja Timmermans, Cold Spring Harbor Laboratory). This short range mobility contrasts with the long range mobility and signalling capability of siRNAs. Two new players in the spread of siRNA mediated silencing were presented: Olivier Voinnet (ETH, Zurich, Switzerland) and Patrice Dunoyer (IBMP-CNRS, France) identified a specific receptor-kinase with PD association and Rosemary White (CSIRO Plant Industry, Clayton South, Australia) identified apoplastic peroxidases. The mechanism by which small RNAs
Fig. 2 Photoassimilates and RNAs are translocated in the phloem vascular tissue throughout the plant. Cells outside the vasculature are symplasmically connected by plasmodesmata (PD), plasma membranelined channels with a diameter of c. 50 nm. PD allow for transport of photoassimilates and signalling molecules via the cytoplasmic sleeve, connecting cells in different tissues. Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust
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move between cells has been a longstanding question, particularly with respect to the differences in range exhibited between miRNAs and siRNAs – these presentations suggest an answer will soon be within reach. The role of phloem as a conduit for long distance transport of molecular information (primarily RNA) was also a point of discussion (Fig. 2). In addition to siRNAs and sugars, macromolecules such as proteins and mRNAs are able to move over long distances via the phloem. Indeed, Friedrich Kragler (Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany) has determined that the phloem transports > 3000 mRNAs. David Hannapel (Iowa State University, Ames, IA, USA) detailed that leaf-to-stolon translocation of mRNA of the TF StBEL5 regulates tuberization in Solanum tuberosum. He has identified that BEL5 mobility is mediated by an untranslated region (UTR), which is able to confer translocation capacity to related, immobile BEL mRNAs. Again, these presentations suggest the mechanism of symplastic macromolecular transport is within reach.
PD and disease Until recently, research into the role of PD in disease centred on the ability of viral movement proteins to manipulate PD. This workshop has indicated that research has now expanded to encompass a range of molecular interactions between hosts and pathogens in what appears to be a battle to access and control PD. At this meeting two models for virus movement via PD have emerged: one involving the translocation of viral replication complexes (VRCs); the other involving the movement of encapsidated vRNA. In support of the first model, Manfred Heinlein (IBMP-CNRS) outlined data that suggest Tobacco mosaic virus (TMV) VRCs form intracellularly via interaction with endoplasmic reticulum–microtubule junctions en route to PD. The second model is supported by Jens Tilsner’s (University of St Andrews, UK) observations that the Potato virus X (PVX) TGB2/3 proteins cap either side of the PD and that viral replication occurs at PD, in a process tightly linked to intercellular PVX movement. Significantly, viruses are no longer the only pathogens known to manipulate PD with preliminary evidence presented that other biotic pathogens such as bacteria and fungi also regulate PD function. Effectors from bacterial (Joe Aung, Michigan State University, East Lansing, MI, USA) and fungal (Lingxue Cao, University of Amsterdam, the Netherlands) pathogens were also shown to localize to PD albeit for as yet unspecified functions. Furthermore, identification of a PD-resident chitin receptor (Christine Faulkner, John Innes Centre, Norwich, UK) and
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PDLPs (Jung-Youn Lee, Christine Faulkner) that respond to pathogen invasion further suggest that hosts do not surrender PD without a battle.
The future of PD biology In hosting research from fields that had not previously interacted, this workshop has established novel avenues of research that are sure to propel the study of cell-to-cell communication in new directions. The regulation of PD biology by lipids, callose and receptor kinases, and new opportunities to dissect PD function in TF and RNA movement are just some of the key research directions that emerged at this meeting. Manfred Heinlein, Christophe Ritzenthaler and Veronique Ziegler-Graff (IBMP-CNRS) are to be congratulated on the exceptional programme and organization of this exciting workshop.
Acknowledgements The workshop was financially supported by EMBO. The authors are winners of poster prizes awarded at the workshop and thank Christine Faulkner (John Innes Centre) and Olivier Voinnet (ETH) for comments on the report. Munenori Kitagawa1*, Danae Paultre2 and Hanna Rademaker3 1
RIKEN Centre for Sustainable Resource Science, Yokohama, Japan; 2 Institute of Molecular Plant Sciences, University of Edinburgh, Edinburgh, UK; 3 Department of Physics, Technical University of Denmark, Lyngby, Denmark (*Author for correspondence: tel +81 45 503 9576; email
[email protected])
References Lee JY, Lucas WJ. 2001. Phosphorylation of viral movement proteins–regulation of cell-to-cell trafficking. Trends in Microbiology 9: 5–8; discussion 8. Maule A, Faulkner C, Benitez-Alfonso Y. 2012. Plasmodesmata “in communicado”. Frontiers in Plant Science 3: 30. Raffaele S, Bayer E, Lafarge D, Cluzet S, Retana SG, Boubekeur T, Leborgne-Castel N, Carde J-P, Lherminie J, Noirot E et al. 2009. Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus x movement. Plant Cell 21: 1541–1555. Key words: cell-to-cell communication, lipid raft, phloem transport, plant defence, plant development, plasmodesmata, small RNA.
Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust
