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ScienceDirect Roles and regulation of plant cell walls surrounding plasmodesmata J Paul Knox and Yoselin Benitez-Alfonso In plants, the intercellular transport of simple and complex molecules can occur symplastically through plasmodesmata. These are membranous channels embedded in cell walls that connect neighbouring cells. The properties of the cell walls surrounding plasmodesmata determine their transport capacity and permeability. These cell wall micro-domains are enriched in callose and have a characteristic pectin distribution. Cell wall modifications, leading to changes in plasmodesmata structure, have been reported to occur during development and in response to environmental signals. Cell wall remodelling enzymes target plasmodesmata to rapidly control intercellular communication in situ. Here we describe current knowledge on the composition of cell walls at plasmodesmata sites and on the proteins and signals that modify cell walls to regulate plasmodesmata aperture. Addresses Centre for Plant Sciences, School of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom Corresponding author: Benitez-Alfonso, Yoselin ([email protected])
Current Opinion in Plant Biology 2014, 22:93–100 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´
http://dx.doi.org/10.1016/j.pbi.2014.09.009 1369-5266/# 2014 Elsevier Ltd. All right reserved.
Introduction Molecular trafficking between cells is necessary for coordinated development and to regulate responses to the environment [1–5]. Plant cells are delimited by relatively rigid cell walls that act as barriers and restrict intercellular contacts. Small molecules exported to the extracellular spaces can diffuse across cell walls from where they can be imported into neighbouring cells. Intercellular transport in plants can also occur cytoplasm-to-cytoplasm through channels embedded in cell walls named plasmodesmata (PD) [5–7]. These channels are membrane-continuous structures running across the cell walls of neighbouring cells arising, primarily, during cytokinesis (Figure 1) [8,9]. The desmotubule (DT), a rod derived from endoplasmic reticulum (ER), transverses the centre of these membranous structures resulting in thin cytoplasmic spaces or www.sciencedirect.com
cytoplasmic sleeve. This space is not totally free but occupied by cytoskeletal proteins that contribute to channel function. The cytoplasmic sleeve of PD is the main route for intercellular molecular trafficking. The transport of cytoplasmic molecules is determined by their size and shape and/or their association with proteins that modify PD structure/permeability. Transcription factors, RNAs, silencing signals, metabolites and hormones move through PD to regulate plant growth, development and adaptation to the environment [1,2,5,7,10]. Viruses also use this pathway to spread locally between neighbouring cells and systemically to uninfected organs [6,11]. The number of PD found in the interface between two cells, their aperture and architecture modulate the transport of these molecules, and thus determine cell/tissue responses to environmental and developmental cues. Research on the mechanisms that modify PD function highlights the influence of the cell wall on PD biogenesis and structural modification [8,9,12,13]. Changes in cell wall mechanics/composition modify the physical dimensions and dilation properties of PD restricting the space available for the transport of large molecules. The characteristics of cell walls also affect the formation of new PD (which requires targeted cell wall dissolution) and the structural changes (branching and twinning) that PD undergo during development [9]. Hence information on the composition, biogenesis and metabolism of cell wall regions surrounding PD is crucial to understand and manipulate intercellular symplastic transport during plant growth and development. Recent technologies have led to the identification of novel PD structural components [14–17]. In some cases, the function of these proteins at PD has been investigated, as well as the biological process that they regulate. However, there is still ample scope for research on the mechanisms underlying this regulation. Moreover, a large number of proteins contained in the PD proteome have not an assigned PD function [16,18]. These include a range of cell wall remodelling enzymes and signalling proteins. In this review we discuss the current knowledge on the distribution of cell wall components around PD aiming to highlight the differences with other cell wall domains. We comment on recent articles reporting the screening of PD proteins with a particular focus on those involved in cell wall remodelling. We also discuss recent research on the signals and signalling pathways involved in regulating PD Current Opinion in Plant Biology 2014, 22:93–100
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Figure 1
desmotubule (a)
(b) callose
plasma membrane cellulose
CE L
L
W AL L
pectin
middle lamella PD
100 nm Current Opinion in Plant Biology
Plasmodesmata are embedded in the cell wall. (a) Electron tomogram reconstruction of a simple plasmodesmata (PD) in a 6-day-old Arabidopsis root section. (b) Schematic representation of simple plasmodesmata. The desmotubule, plasma membrane and different cell wall components (callose, cellulose, pectin and middle lamella) have been indicated.
in response to developmental and environmental cues. To conclude we highlight areas of cell wall biology in relation to PD that warrant further investigation.
Cell-wall microdomains around plasmodesmata The cell wall surrounding PD physically constrains their shape determining the cytoplasmic aperture of the channel and hence the size of the molecules able to pass through. Despite its importance, the detailed composition and metabolism of these cell wall microdomains remain unknown (Figure 1). Imaging of immuno-labelled tissue using either transmission electron-microscopy (TEM) or three-dimensional structured illumination microscopy (3D-SIM) shows that b-1,3-glucan, callose, accumulates in the form of a collar around the channel neck regions [19,20]. Mutations and conditions that affect the metabolism of callose at PD significantly modify PD transport capacity [13,21,22,23,24,25,26,27,28]. This suggests a role for callose in controlling the channel aperture. Reviews discussing the relevance of this process in plant development and stress responses have recently been published (e.g. [1,5,13]). The importance of PDassociated callose for lateral root formation [21,29], leaf Current Opinion in Plant Biology 2014, 22:93–100
vein development [25,30] and for the generation of an auxin gradient during the tropic response [23] are some of the newest additions to a long list of observations on this topic. Other cell wall components are differentially regulated around PD where they are likely to contribute to maintain PD shape and function. Calcofluor white staining and immunoanalysis of freeze-fractured tobacco trichome cells indicate that, together with callose, there is an enrichment in pectic polysaccharides and a reduction in cellulose content in cell wall regions around PD [9]. High resolution scanning electron microscopy (HRSEM) imaging of PD ultrastructure in the green alga Chara corallina indicated the presence of spoke-like structures linking the PD membrane with the cell wall [14]. Enzymatic digestion suggests that cellulose and pectins form these junctions although the presence of other polysaccharides (such as callose) or proteins cannot be discounted. The presence of these connections in plant species has not been confirmed yet but they could be playing an important role in stabilizing or regulating PD structure. Stabilization and/or regulation might be the reason why pectin composition in the cell walls around www.sciencedirect.com
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the channels differs from other cell wall domains, as shown in earlier work [9,31,32,33]. Pectin comprise a complex set of polysaccharides the major two being homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). These can be interlinked in pectic supramolecules [34]. Pectic composition influences many factors including cell adhesion, porosity of cell wall matrices and stiffness of cell-walls. Immunoassays in different tissues of various plant species indicate that low-esterified HG and (1 ! 5)-a-L-arabinan domains are preferentially localized at pit fields/PD whereas (1 ! 4)-b-galactan is specifically absent [9,31, 32,33]. The arabinan and galactan domains are side chains of RG-I with arabinan-rich forms associated with flexibility and galactan-rich forms with stiffer cell walls [34]. These observations suggest PD-specific RG-I polymers may be inserted around PD to maintain flexibility of movement and to accommodate oscillations of PD apertures. How cell wall microdomains with specific forms of pectic supramolecules are generated is an open question. Interestingly pectin methyl esterase (PME), the enzyme involved in pectic HG de-esterification in muro, has been shown to localize preferentially around PD in flax hypocotyls [35]. Experimental evidence suggests that host PME interacts with the movement protein (MP) of tobacco mosaic virus (TMV) and this interaction is required for virus systemic infection [36,37]. PME also interacts at the cell wall with MPs of turnip vein clearing virus, cauliflower mosaic virus and Chinese wheat mosaic virus [37,38]. Suppressing PME expression or activity in planta affects virus spreading suggesting that this enzyme acts as chaperone or facilitator for MP movement [39,40]. PME-MP interaction might also affect PME activity in HG-deesterification which could lead to changes in the mechanical properties of the cell wall surrounding PD. How changes in pectin de-esterification affects PD transport capacity is not known but potentially this could be a mechanism exploited by viruses to ensure their systemic spreading. In summary, PD are inserted in cell wall microdomains enriched in callose and with a distinct pectin composition (Figure 1). The role of callose in controlling PD size and symplastic permeability has been well defined. By contrast, little is known about the mechanism controlling pectin composition or its importance in modifying the mechanical properties of these cell wall microdomains and the permeability of PD channels.
Proteins attached to PD remodel cell walls to regulate channel aperture In recent years, significant strides have been made in the identification and characterization of proteins bound to PD. Improved methods to isolate PD enriched cell-wall fractions from Arabidopsis cell cultures combined with nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer identified more than www.sciencedirect.com
1000 candidate PD proteins [16]. This list is not by any means a definite demonstration of their presence at PD. A number of contaminants resulting from the purification process were detected highlighting the need of further tests to confirm localization. As a parameter to primarily screen the candidates, only proteins with a predicted secretory signal peptide and either a transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor were considered. These features characterize membrane proteins and were found in PD-located proteins identified in previous proteomic screens [24,41]. However, using alternative approaches, PD targeting of non-membrane predicted proteins has been reported indicating that there is no a priori reason to exclude any of the candidates identified in the proteome. As expected, several cell wall remodelling enzymes appeared in the PD proteome dataset (Table 1). These include enzymes involved in the biosynthesis (callose synthases, CALS) and degradation (b-1,3-glucanases, BG) of callose. Research using mutants, inducible and overexpression lines confirmed the importance of these proteins in regulating PD permeability and the biological processes in which they participate. For example, ATGSL12 (Arabidopsis thaliana glucan synthase-like 12, also named CALS3) was shown to regulate PD-associated callose in the root vasculature affecting vascular patterning and root meristem maintenance [26]. On the other hand, ATGSL8 regulates the non-cell autonomous expression of factors that determine stomata patterning and cell proliferation in leaves, the hypocotyl tropic response and root growth [22,23,42]. Callose-degrading enzymes located at PD sites have also been genetically characterized. A mutant in the PD glucanase ATBG_PAP had increased callose at PD concomitant with a reduction in the systemic spread of the turnip vein clearing virus (TVCV) [28]. During development, a different set of PD-located BG genes (named PdBG1 and PdBG2) regulates callose in the xylem pole pericycle (XPP) in the transition and differentiation zone of the root [21]. pdbg1pdbg2 mutants initiate lateral roots in clusters suggesting that symplastic connectivity is important for lateral root positioning and spatial patterning. The ectopic expression in the XPP of a mutated activated version of CALS3 mimicked the pdbg1pdbg2 phenotype highlighting the importance of controlling callose metabolism at PD during lateral root initiation and patterning. PD-located CALS and BG have also been identified and characterized in different plant species. A recent manuscript reports the identification of the maize TIE-DYED2 gene (TDY2), a callose synthase that regulates symplastic phloem transport and leaf vein development [25]. In Populus two genes encoding PD-located BG were proposed to mediate the movement of Flowering locus T (FT) protein into the bud during dormancy release [43]. As these reports illustrate, the identification of PD-callose Current Opinion in Plant Biology 2014, 22:93–100
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Table 1 Enzymes identified in the PD proteome putatively involved in cell wall remodelling processes. This list is a subset of the full dataset published in [26] and only includes enzymatic activities associated with PD transport in independent studies (see main text for references). Gene ID
Gene name
Cell wall activity
AT2G36850 AT5G13000 AT1G05570 AT2G31960 AT3G07160 AT4G03550 AT5G42100 AT3G55430 AT4G31140 AT5G58090 AT1G64760 AT3G07320 AT1G53840 AT2G47030 AT3G14300 AT4G12390 AT1G65570 AT5G06860 AT5G06870 AT1G02730 AT4G07960 AT4G19900 AT3G46650 AT5G16510 AT3G08900
GLUCAN SYNTHASE-LIKE 8 (GSL8) GLUCAN SYNTHASE-LIKE 12 (CALS3) CALLOSE SYNTHASE 1 GLUCAN SYNTHASE-LIKE 3 GLUCAN SYNTHASE-LIKE 10 GLUCAN SYNTHASE-LIKE 5 b-1,3-GLUCANASE (ATBG_PAP) b-1,3-GLUCANASE b-1,3-GLUCANASE b-1,3-GLUCANASE b-1,3-GLUCANASE b-1,3-GLUCANASE PECTIN METHYLESTERASE 1 PECTINESTERASE (VANGUARD1) PECTIN METHYLESTERASE 26 PECTIN METHYLESTERASE INHIBITOR 1 POLYGALACTURONASE/PECTINASE POLYGALACTURONASE INHIBITING PROTEIN 1 POLYGALACTURONASE INHIBITING PROTEIN 2 GLYCOSYL TRANSFERASE ACTIVITY GLYCOSYL TRANSFERASE ACTIVITY a-1,4-GLYCOSYL TRANSFERASE UDP-GLYCOSYL TRANSFERASE REVERSIBLY GLYCOSYLATED POLYPEPTIDE 5 (RGP5) REVERSIBLY GLYCOSYLATED POLYPEPTIDE 3 (RGP3)
Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Pectin modification Pectin modification Pectin modification Pectin modification Pectin metabolism Pectin metabolism Pectin metabolism Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall
metabolic enzymes may provide new opportunities for crop improvement, as they could be the target to biotechnologically modify important economic and agronomic traits involving developmental events or transitions in which PD participate. Currently, the use of this knowledge is hindered by the lack of a specific motif/domain to distinguish PD proteins from other plasma membrane enzymes. A recent approach to overcome this issue uses phylogeny to identify BG that evolved with PD complexity in land plants [44]. Proteins involved in pectin metabolism were isolated in the PD proteomic screen but there is scarce information (if any) on their role in regulating symplastic intercellular communication (Table 1). As discussed above pectin degradation may affect PD anchorage to cell walls which, presumably, could destabilize the channel structure [14]. Thus, pectinases and polygalacturonases-inhibiting proteins (PGIP) might be important in forming and maintaining PD architecture. Consistent with this hypothesis, pectinase activity was detected at PD, and in the surrounding cell-wall, in tobacco pollen mother cells at the zigotene stage, coincident with the formation of simple and branched PD structures [45]. Uncharacterized members of these protein families were identified in the proteome but localization or function at PD has not been further investigated. Enzymes catalysing the de-methylesterification and de-acetylation of pectins modify the mechanical/physical properties of cell walls, which might Current Opinion in Plant Biology 2014, 22:93–100
polysaccharides polysaccharides polysaccharides polysaccharides polysaccharides polysaccharides
alter the capacity of PD channels to dilate.Pectin methylesterases and their inhibitors are found in the PD proteome but again their localization has not yet been confirmed. The accumulation of PME around PD has been reported in other systems where they play a role in the systemic spreading of viruses [36,37,39,46]. The ectopic expression of the PME inhibitor PMEI reduces tobamovirus systemic movement which support their function in the control of PD transport [40]. Other putative enzymatic activities involved in general cell-wall biosynthesis, dissolution and assembly are represented in the PD proteome[16]. These include glycosyl transferases and reversibly glycosylated polypeptides (RGPs) (Table 1). A role for glycosyl transferases in PD function is supported by the recent characterization of a b-1,6-N-acetylglucosaminyl transferase-like protein in Arabidopsis thaliana [47]. This protein interacts with calreticulin at PD to regulate seed germination and plant growth. How its expression modifies PD structure or function is unknown. Another glycosyl transferase-like protein was identified in a genetic screen for mutants in stomata differentiation [48]. This protein (named KOBITO1, KOB1) was thought to regulate cellulose biosynthesis but a role in pectin, callose and lignin metabolism could not be discarded [49]. In kob1-3 mutant leaves, stomata appear in clusters, concomitant with an increase in GFP diffusion and in the intercellular transport of the transcription factor SPEECHLESS (SPCH). www.sciencedirect.com
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The results suggest that KOB1 functions in cell-wall metabolism to establish the correct PD aperture that determines stomata patterning in leaves [48]. Regarding RGPs, members of the class 1 family were identified to bind to PD in independent proteomic studies [50]. These proteins are reversibly glycosylated with UDP-sugars and transfer this substrate to enzymes involved in cell wall (and specifically pectin) biosynthesis. Constitutive expression in tobacco of an Arabidopsis class I RGP (RGP2) increases callose and restricts the transport of photoassimilates into sink tissues leading to aberrant plant phenotypes [27]. Expression of this protein also affects virus spreading [27,51]. RGP3 and RGP5 (identified in the PD proteome) might contribute to the regulation of cell wall modifications and symplastic transport in other tissues and/or stress conditions. The presence in the PD proteome of a large number of proteins involved in cell wall synthesis and remodelling may reflect the need to modify the composition of these cell wall microdomains in a quick and dynamic fashion and independently of other cell wall regions. Recent research highlights the importance of regulating callose at PD for proper plant development and identifies proteins regulating its deposition. The function of other enzymatic activities in regulating PD structure and transport is less clear but the pectic supramolecules would appear to be an important target.
Small signalling molecules induce cell wall modifications to regulate PD transport As membranous structures interfacing the cytoplasm and the cell wall, PD function and development are influenced by changes in the intracellular and the extracellular (apoplast) environment. Research on PD composition indicates that these domains are enriched in receptor and receptor-like proteins involved in signalling pathways regulate cell-to-cell communication that [15,52,53,54]. In some instances, the signals and cues perceived by these receptors have been determined but the downstream signalling components are mostly unknown. These receptor proteins can trigger changes in the composition of cell walls at PD domains as a medium to alter symplastic connectivity. A clear example is the Arabidopsis PD-located protein PDLP5, a receptorlike kinase that negatively regulates symplastic transport during the innate immune response by influencing callose deposition at PD [55]. Research on the signals that regulate PD transport capacity is also gaining momentum. Changes in the concentration of Reactive oxygen species (ROS) and in calcium fluxes are known to induce PD structural modifications likely by altering the activity of callose metabolic enzymes and PD-associated cell wall composition (for a review see [12,56]). Plastidial and mitochondrial proteins (such as THIOREDOXIN-M3 and INCREASED SIZE www.sciencedirect.com
EXCLUSION LIMIT 1 and 2) have been identified to participate in organelle-to-nucleus signalling pathways to regulate the intracellular redox status and maintain PD aperture [57,58]. The mechanism underlying this regulation depends on glutathione homeostasis and involves changes in the expression of genes that control PD architecture and transport capacity [59,60]. ROS generated in the apoplast have been proposed to act as cell wall loosening agents (for a review see Gapper and Dolan, 2006 [61]) which might physically influence how PD form and function. Consistent with this hypothesis, H2O2 and class III peroxidases have been found to accumulate at PD regions in the stem cambial zone of tomato accompanying changes in PD structure [62]. Other molecules that interact with redox signalling pathways are described to modify PD. The most recent example is salicylic acid (SA), which regulates the formation of complex PD and callose biosynthesis to influence symplastic transport [63,64]. Work on the mechanism behind this regulation indicates that SA activates the expression of PDLP5, which modifies the activity of callose metabolic enzymes to regulate symplastic transport and pathogen infection [64]. The phytohormone auxin has also been related with cell wall and PD modifications. The expression of the callosebinding protein PDCB1 was found to be up-regulated in response to auxin treatment in lateral roots [29]. PDCB1 functions in callose deposition at PD [24] and its expression correlates with the symplastic isolation of the lateral root primordia from the overlying tissue during emergence. The expression of PD-located callose degrading enzymes acting during lateral root initiation (PdBG1 and PdBG2) is also induced after treatment with auxin and this regulation is abolished in the dominant auxin signalling mutant solitary root1 (slr1)[21]. These expression data led us to hypothesize a regulatory role for auxin in the control of callose deposition and intercellular transport during lateral root development. Intriguingly, treatment with synthetic auxins does not alter diffusion of fluorescein (a fluorescent dye) in root meristem tissues [65]. Recent experiments shed some light on this apparent contradiction. Han et al. demonstrate that changes in callose perturb the formation of an auxin gradient during the phototropic response [23]. Interestingly they showed that in hypocotyls, the expression of the callose biosynthetic enzyme GSL8 is positively regulated by auxin through a mechanism that involves the auxin response factor 7 (ARF7). These results suggest that auxin can target different callose metabolic genes to regulate symplastic transport but this depends on the tissue/condition analysed and on the auxin-dependent regulation of auxin response factors. Changes in PD aperture in response to auxin might also be associated with modifications in pectin structure/distribution. Recent work demonstrates that auxin accumulation in the shoot apex increases HG-demethyl-esterification Current Opinion in Plant Biology 2014, 22:93–100
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leading to cell wall softening during organ outgrowth [66]. Earlier studies indicate that auxin can increase PME activity which is the enzyme responsible for HG deesterification in the cell wall [67]. PME family members are found at PD [35] and, together with PMEI, they appear in the PD proteome (Table 1). Thus, it is possible that auxin influences on PD transport might be related to its effect on cell wall mechanics through changes in pectin esterification. Other small molecules such as tryptophan [65] and gibberellins (GA) [43] have recently been reported to influence symplastic communication. More research is required to elucidate which are the pathways that these molecules use to modify PD and how they differ from the ones described above.
Conclusions Research indicates that the composition of cell walls surrounding PD significantly differs from other cell wall regions. This is likely to be important to maintain PD structure/architecture and to control the cytoplasmic aperture of the channel. The role of callose in the control of PD transport has been well established. The accumulation of this polysaccharide is regulated by PD-located enzymes and by redox signalling pathways. The expression of callose metabolic enzymes is also regulated by salicylic acid and auxin signalling which provides a mechanism for the tight control of callose (and PD transport) in response to a wide range of developmental and environmental cues. By contrast, little is known of the functional significance of the specific distribution of pectic polysaccharides around PD or about the consequences for PD transport capacity of changes in pectin composition— including both HG and RG-I domains. Interestingly, the PD proteome identified a significant number of candidates implicated in pectin biosynthesis and/or remodelling. Characterization of these proteins (including confirmation of PD association) will provide new clues on the role of pectins in symplastic cell-to-cell communication.
Acknowledgements The authors thank William Nicolas, Emmanuelle Bayer, the Bordeaux imaging centre and the Laboratory Membrane Biogenesis for providing the tomogram reconstructed section of plasmodesmata included in Figure 1.
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23. Han X, Hyun TK, Zhang M, Kumar R, Koh EJ, Kang BH, Lucas WJ, Kim JY: Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling. Dev Cell 2014, 28:132-146. This manuscript provides the first evidences of a role for symplastic transport in the generation and maintenance of the auxin gradient. Through the characterization of a callose synthase (GSL8), the authors propose that the regulation of callose biosynthesis at PD is essential for the hypocotyl tropic response as it affects auxin symplastic transport. 24. 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. 25. Slewinski TL, Baker RF, Stubert A, Braun DM: Tie-dyed2 encodes a callose synthase that functions in vein development and affects symplastic trafficking within the phloem of maize leaves. Plant Physiol 2012, 160:1540-1550. A callose synthase that regulates symplastic transport in the phloem of maize leaves is identified. Mutant in this enzyme is impaired in sucrose export and accumulate starch in leaves indicating defective source-sink distribution. 26. Vaten A, Dettmer J, Wu S, Stierhof YD, Miyashima S, Yadav SR, Roberts CJ, Campilho A, Bulone V, Lichtenberger R et al.: Callose biosynthesis regulates symplastic trafficking during root development. Dev Cell 2011, 21:1144-1155. 27. Zavaliev R, Sagi G, Gera A, Epel BL: The constitutive expression of Arabidopsis plasmodesmal-associated class 1 reversibly glycosylated polypeptide impairs plant development and virus spread. J Exp Bot 2010, 61:131-142. 28. Zavaliev R, Levy A, Gera A, Epel BL: Subcellular dynamics and role of Arabidopsis beta-1,3-glucanases in cell-to-cell movement of tobamoviruses. Mol Plant Microbe Interact 2013, 26:1016-1030. The manuscript reports the characterization of a pathogenesis related beta-glucanase (AtBG2). The gene was highly induced after virus infection but the protein accumulates in the ER and ER-derived bodies. Virus spread and callose deposition was compared in mutants in AtBG2 and in Atbg_pap (a PD-located glucanase). The results suggest that Atbg_pap, but not AtBG2, functions in regulating PD-associated callose and virus movement. 29. Maule J, Gaudioso-Pedraza R, Benitez-Alfonso Y: Callose deposition and symplastic connectivity are regulated prior to lateral root emergence. Commun Integr Biol 2013, 6:e26531. 30. Baker RF, Slewinski TL, Braun DM: The tie-dyed pathway promotes symplastic trafficking in the phloem. Plant Signal Behav 2013, 8:e24540. 31. Giannoutsou E, Sotiriou P, Apostolakos P, Galatis B: Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays. Ann Bot 2013, 112:10671081. In this paper, changes in cell-wall composition and plasmodesmata distribution during the formation of mesophyll cells are described. The data allow to establish correlations between callose deposition and pectin distribution and the appearance of secondary plasmodesmata. 32. Orfila C, Knox JP: Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp. Plant Physiol 2000, 122:775-781. 33. Roy S, Watada AE, Wergin WP: Characterization of the cell wall microdomain surrounding plasmodesmata in apple fruit. Plant Physiol 1997, 114:539-547. 34. Burton RA, Gidley MJ, Fincher GB: Heterogeneity in the chemistry, structure and function of plant cell walls. Nat Chem Biol 2010, 6:724-732. 35. Morvan O, Quentin M, Jauneau A, Mareck A, Morvan C: Immunogold localization of pectin methylesterases in the cortical tissues of flax hypocotyl. Protoplasma 1998, 202:175-184. 36. Dorokhov YL, Makinen K, Frolova OY, Merits A, Saarinen J, Kalkkinen N, Atabekov JG, Saarma M: A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein. FEBS Lett 1999, 461:223-228. www.sciencedirect.com
37. Chen MH, Sheng J, Hind G, Handa AK, Citovsky V: Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-to-cell movement. EMBO J 2000, 19:913-920. 38. Andika IB, Zheng S, Tan Z, Sun L, Kondo H, Zhou X, Chen J: Endoplasmic reticulum export and vesicle formation of the movement protein of Chinese wheat mosaic virus are regulated by two transmembrane domains and depend on the secretory pathway. Virology 2013, 435:493-503. In this manuscript, the authors use bimolecular fluorescent complementation (BIFC) to demonstrate the interaction between pectin methylesterase and movement protein in planta. The results suggest that these proteins interact at the cell wall and reveal the domains require for this interaction. 39. Chen MH, Citovsky V: Systemic movement of a tobamovirus requires host cell pectin methylesterase. Plant J 2003, 35:386-392. 40. Lionetti V, Raiola A, Cervone F, Bellincampi D: Transgenic expression of pectin methylesterase inhibitors limits tobamovirus spread in tobacco and Arabidopsis. Mol Plant Pathol 2014, 15:265-274. 41. Thomas CL, Bayer EM, Ritzenthaler C, Fernandez-Calvino L, Maule AJ: Specific targeting of a plasmodesmal protein affecting cell-to-cell communication. PLoS Biol 2008, 6:e7. 42. De Storme N, De SJ, Van CW, Wewer V, Dormann P, Geelen D: GLUCAN SYNTHASE-LIKE8 and STEROL METHYLTRANSFERASE2 are required for ploidy consistency of the sexual reproduction system in Arabidopsis. Plant Cell 2013, 25:387-403. 43. Rinne PL, Welling A, Vahala J, Ripel L, Ruonala R, Kangasjarvi J, van der Schoot C: Chilling of dormant buds hyperinduces FLOWERING LOCUS T and recruits GA-inducible 1,3-betaglucanases to reopen signal conduits and release dormancy in Populus. Plant Cell 2011, 23:130-146. 44. Gaudioso-Pedraza R, Benitez-Alfonso Y: A phylogenetic approach to study the origin and evolution of plasmodesmatalocalized glycosyl hydrolases family 17. Front Plant Sci 2014, 5:212. 45. Yu C-H, Guo G-Q, Nie X-WNH-W, Zheng G-CCK-C: Cytochemical localization of pectinase activity in pollen mother cells of tobacco during meiotic prophase I and its relation to the formation of secondary plasmodesmata and cytoplasmic channels. Acta Bot Sin 2004, 46:1443-1453. 46. Dorokhov YL, Komarova TV, Petrunia IV, Frolova OY, Pozdyshev DV, Gleba YY: Airborne signals from a wounded leaf facilitate viral spreading and induce antibacterial resistance in neighboring plants. PLoS Pathog 2012, 8:e1002640. 47. Zalepa-King L, Citovsky V: A plasmodesmal glycosyltransferase-like protein. PLOS ONE 2013, 8:e58025. 48. Kong D, Karve R, Willet A, Chen MK, Oden J, Shpak ED: Regulation of plasmodesmatal permeability and stomatal patterning by the glycosyltransferase-like protein KOBITO1. Plant Physiol 2012, 159:156-168. Through the characterization of mutants in the glycosyltransferase-like protein KOBITO1, the authors discovered a role for this cell wall activity in the regulation of symplastic transport in Arabidopsis leaves. The mutant displays defective movement of the transcription factor SPEECHLESS, leading to the formation of stomata clusters. 49. Pagant S, Bichet A, Sugimoto K, Lerouxel O, Desprez T, McCann M, Lerouge P, Vernhettes S, Hofte H: KOBITO1 encodes a novel plasma membrane protein necessary for normal synthesis of cellulose during cell expansion in Arabidopsis. Plant Cell 2002, 14:2001-2013. 50. Sagi G, Katz A, Guenoune-Gelbart D, Epel BL: Class 1 reversibly glycosylated polypeptides are plasmodesmal-associated proteins delivered to plasmodesmata via the golgi apparatus. Plant Cell 2005, 17:1788-1800. 51. Burch-Smith TM, Cui Y, Zambryski PC: Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata. Plant Signal Behav 2012, 7:62-67. Current Opinion in Plant Biology 2014, 22:93–100
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52. Faulkner C, Petutschnig E, Benitez-Alfonso Y, Beck M, Robatzek S, Lipka V, Maule AJ: LYM2-dependent chitin perception limits molecular flux via plasmodesmata. Proc Natl Acad Sci U S A 2013, 110:9166-9170. The authors demonstrate that the chitin-binding protein LYM2 is located preferentially at plasmodesmata where it regulates symplastic transport in response to pathogen attack. This is the first evidence of a role for chitin (and chitin signalling) in the regulation of intercellular symplastic connectivity, which is presumably required for proper plant defense response against fungal pathogens. 53. Jo Y, Cho WK, Rim Y, Moon J, Chen XY, Chu H, Kim CY, Park ZY, Lucas WJ, Kim JY: Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins. Protoplasma 2011, 248:191-203. 54. Stahl Y, Grabowski S, Bleckmann A, Kuhnemuth R, Weidtkamp Peters S, Pinto KG, Kirschner GK, Schmid JB, Wink RH, Hulsewede A et al.: Moderation of Arabidopsis root stemness by CLAVATA1 and ARABIDOPSIS CRINKLY4 receptor kinase complexes. Curr Biol 2013, 23:362-371. This manuscript demonstrates the importance of receptor signalling proteins in the regulation of symplastic intercellular transport during development. The work revealed that the receptor protein CRINKLY4 associates with CLAVATA1 at plasmodesmata in Arabidopsis roots and that this interaction is necessary to maintain root stem cell activity. 55. Lee JY, Wang X, Cui W, Sager R, Modla S, Czymmek K, Zybaliov B, van WK, Zhang C, Lu H, Lakshmanan V: A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis. Plant Cell 2011, 23:3353-3373. 56. Benitez-Alfonso Y, Jackson D, Maule A: Redox regulation of intercellular transport. Protoplasma 2011, 248:131-140. 57. Stonebloom S, Burch-Smith T, Kim I, Meinke D, Mindrinos M, Zambryski P: Loss of the plant DEAD-box protein ISE1 leads to defective mitochondria and increased cell-to-cell transport via plasmodesmata. Proc Natl Acad Sci U S A 2009, 106: 17229-17234. 58. Benitez-Alfonso Y, Cilia M, San RA, Thomas C, Maule A, Hearn S, Jackson D: Control of Arabidopsis meristem development by thioredoxin-dependent regulation of intercellular transport. Proc Natl Acad Sci U S A 2009, 106:3615-3620. 59. Burch-Smith TM, Brunkard JO, Choi YG, Zambryski PC: Organelle-nucleus cross-talk regulates plant intercellular
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communication via plasmodesmata. Proc Natl Acad Sci U S A 2011, 108:E1451-E1460. 60. Stonebloom S, Brunkard JO, Cheung AC, Jiang K, Feldman L, Zambryski P: Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata. Plant Physiol 2012, 158:190-199. Using ro-GFP targeted to the mitochondria or plastids, the authors show that the amount of reduced glutathione in these organelles is modified in plasmodesmata mutants. Chemicals were also used to demonstrate that the origin and/or chemical nature of reactive oxygen species determine how plasmodesmata structure and transport capacity are affected. 61. Gapper C, Dolan L: Control of plant development by reactive oxygen species. Plant Physiol 2006, 141:341-345. 62. Ehlers K, van Bel AJ: Dynamics of plasmodesmal connectivity in successive interfaces of the cambial zone. Planta 2010, 231:371-385. 63. Fitzgibbon J, Beck M, Zhou J, Faulkner C, Robatzek S, Oparka K: A developmental framework for complex plasmodesmata formation revealed by large-scale imaging of the Arabidopsis leaf epidermis. Plant Cell 2013, 25:57-70. This elegant study uses a high-throughput imaging platform to detect the appearance of complex plasmodesmata during the development of leaf epidermal cells. The authors describe the dynamics of complex plasmodesmata formation and the effect, on plasmodesmata number, of treatment with salicylic acid or mannitol. 64. Wang X, Sager R, Cui W, Zhang C, Lu H, Lee JY: Salicylic acid regulates plasmodesmata closure during innate immune responses in Arabidopsis. Plant Cell 2013, 25:2315-2329. The work revealed salicylic acid as a signal to regulate plasmodesmata transport capacity through modulating the activity of the plasmodesmata receptor protein PDLP5 and the synthesis of callose. This mechanism plays a role during Arabidopsis response to virulent bacterial pathogens. 65. Rutschow HL, Baskin TI, Kramer EM: Regulation of solute flux through plasmodesmata in the root meristem. Plant Physiol 2011, 155:1817-1826. 66. Braybrook SA, Peaucelle A: Mechano-chemical aspects of organ formation in Arabidopsis thaliana: the relationship between auxin and pectin. PLOS ONE 2013, 8:e57813. 67. Brian W, Newcomb EH: Stimulation of pectin methylesterase activity of cultured tobacco pith by indole acetic acid. Phys Plant 1954, 7:290-297.
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ScienceDirect Roles and regulation of plant cell walls surrounding plasmodesmata J Paul Knox and Yoselin Benitez-Alfonso In plants, the intercellular transport of simple and complex molecules can occur symplastically through plasmodesmata. These are membranous channels embedded in cell walls that connect neighbouring cells. The properties of the cell walls surrounding plasmodesmata determine their transport capacity and permeability. These cell wall micro-domains are enriched in callose and have a characteristic pectin distribution. Cell wall modifications, leading to changes in plasmodesmata structure, have been reported to occur during development and in response to environmental signals. Cell wall remodelling enzymes target plasmodesmata to rapidly control intercellular communication in situ. Here we describe current knowledge on the composition of cell walls at plasmodesmata sites and on the proteins and signals that modify cell walls to regulate plasmodesmata aperture. Addresses Centre for Plant Sciences, School of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom Corresponding author: Benitez-Alfonso, Yoselin ([email protected])
Current Opinion in Plant Biology 2014, 22:93–100 This review comes from a themed issue on Cell biology Edited by Shaul Yalovsky and Viktor Zˇa´rsky´
http://dx.doi.org/10.1016/j.pbi.2014.09.009 1369-5266/# 2014 Elsevier Ltd. All right reserved.
Introduction Molecular trafficking between cells is necessary for coordinated development and to regulate responses to the environment [1–5]. Plant cells are delimited by relatively rigid cell walls that act as barriers and restrict intercellular contacts. Small molecules exported to the extracellular spaces can diffuse across cell walls from where they can be imported into neighbouring cells. Intercellular transport in plants can also occur cytoplasm-to-cytoplasm through channels embedded in cell walls named plasmodesmata (PD) [5–7]. These channels are membrane-continuous structures running across the cell walls of neighbouring cells arising, primarily, during cytokinesis (Figure 1) [8,9]. The desmotubule (DT), a rod derived from endoplasmic reticulum (ER), transverses the centre of these membranous structures resulting in thin cytoplasmic spaces or www.sciencedirect.com
cytoplasmic sleeve. This space is not totally free but occupied by cytoskeletal proteins that contribute to channel function. The cytoplasmic sleeve of PD is the main route for intercellular molecular trafficking. The transport of cytoplasmic molecules is determined by their size and shape and/or their association with proteins that modify PD structure/permeability. Transcription factors, RNAs, silencing signals, metabolites and hormones move through PD to regulate plant growth, development and adaptation to the environment [1,2,5,7,10]. Viruses also use this pathway to spread locally between neighbouring cells and systemically to uninfected organs [6,11]. The number of PD found in the interface between two cells, their aperture and architecture modulate the transport of these molecules, and thus determine cell/tissue responses to environmental and developmental cues. Research on the mechanisms that modify PD function highlights the influence of the cell wall on PD biogenesis and structural modification [8,9,12,13]. Changes in cell wall mechanics/composition modify the physical dimensions and dilation properties of PD restricting the space available for the transport of large molecules. The characteristics of cell walls also affect the formation of new PD (which requires targeted cell wall dissolution) and the structural changes (branching and twinning) that PD undergo during development [9]. Hence information on the composition, biogenesis and metabolism of cell wall regions surrounding PD is crucial to understand and manipulate intercellular symplastic transport during plant growth and development. Recent technologies have led to the identification of novel PD structural components [14–17]. In some cases, the function of these proteins at PD has been investigated, as well as the biological process that they regulate. However, there is still ample scope for research on the mechanisms underlying this regulation. Moreover, a large number of proteins contained in the PD proteome have not an assigned PD function [16,18]. These include a range of cell wall remodelling enzymes and signalling proteins. In this review we discuss the current knowledge on the distribution of cell wall components around PD aiming to highlight the differences with other cell wall domains. We comment on recent articles reporting the screening of PD proteins with a particular focus on those involved in cell wall remodelling. We also discuss recent research on the signals and signalling pathways involved in regulating PD Current Opinion in Plant Biology 2014, 22:93–100
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Figure 1
desmotubule (a)
(b) callose
plasma membrane cellulose
CE L
L
W AL L
pectin
middle lamella PD
100 nm Current Opinion in Plant Biology
Plasmodesmata are embedded in the cell wall. (a) Electron tomogram reconstruction of a simple plasmodesmata (PD) in a 6-day-old Arabidopsis root section. (b) Schematic representation of simple plasmodesmata. The desmotubule, plasma membrane and different cell wall components (callose, cellulose, pectin and middle lamella) have been indicated.
in response to developmental and environmental cues. To conclude we highlight areas of cell wall biology in relation to PD that warrant further investigation.
Cell-wall microdomains around plasmodesmata The cell wall surrounding PD physically constrains their shape determining the cytoplasmic aperture of the channel and hence the size of the molecules able to pass through. Despite its importance, the detailed composition and metabolism of these cell wall microdomains remain unknown (Figure 1). Imaging of immuno-labelled tissue using either transmission electron-microscopy (TEM) or three-dimensional structured illumination microscopy (3D-SIM) shows that b-1,3-glucan, callose, accumulates in the form of a collar around the channel neck regions [19,20]. Mutations and conditions that affect the metabolism of callose at PD significantly modify PD transport capacity [13,21,22,23,24,25,26,27,28]. This suggests a role for callose in controlling the channel aperture. Reviews discussing the relevance of this process in plant development and stress responses have recently been published (e.g. [1,5,13]). The importance of PDassociated callose for lateral root formation [21,29], leaf Current Opinion in Plant Biology 2014, 22:93–100
vein development [25,30] and for the generation of an auxin gradient during the tropic response [23] are some of the newest additions to a long list of observations on this topic. Other cell wall components are differentially regulated around PD where they are likely to contribute to maintain PD shape and function. Calcofluor white staining and immunoanalysis of freeze-fractured tobacco trichome cells indicate that, together with callose, there is an enrichment in pectic polysaccharides and a reduction in cellulose content in cell wall regions around PD [9]. High resolution scanning electron microscopy (HRSEM) imaging of PD ultrastructure in the green alga Chara corallina indicated the presence of spoke-like structures linking the PD membrane with the cell wall [14]. Enzymatic digestion suggests that cellulose and pectins form these junctions although the presence of other polysaccharides (such as callose) or proteins cannot be discounted. The presence of these connections in plant species has not been confirmed yet but they could be playing an important role in stabilizing or regulating PD structure. Stabilization and/or regulation might be the reason why pectin composition in the cell walls around www.sciencedirect.com
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the channels differs from other cell wall domains, as shown in earlier work [9,31,32,33]. Pectin comprise a complex set of polysaccharides the major two being homogalacturonan (HG) and rhamnogalacturonan-I (RG-I). These can be interlinked in pectic supramolecules [34]. Pectic composition influences many factors including cell adhesion, porosity of cell wall matrices and stiffness of cell-walls. Immunoassays in different tissues of various plant species indicate that low-esterified HG and (1 ! 5)-a-L-arabinan domains are preferentially localized at pit fields/PD whereas (1 ! 4)-b-galactan is specifically absent [9,31, 32,33]. The arabinan and galactan domains are side chains of RG-I with arabinan-rich forms associated with flexibility and galactan-rich forms with stiffer cell walls [34]. These observations suggest PD-specific RG-I polymers may be inserted around PD to maintain flexibility of movement and to accommodate oscillations of PD apertures. How cell wall microdomains with specific forms of pectic supramolecules are generated is an open question. Interestingly pectin methyl esterase (PME), the enzyme involved in pectic HG de-esterification in muro, has been shown to localize preferentially around PD in flax hypocotyls [35]. Experimental evidence suggests that host PME interacts with the movement protein (MP) of tobacco mosaic virus (TMV) and this interaction is required for virus systemic infection [36,37]. PME also interacts at the cell wall with MPs of turnip vein clearing virus, cauliflower mosaic virus and Chinese wheat mosaic virus [37,38]. Suppressing PME expression or activity in planta affects virus spreading suggesting that this enzyme acts as chaperone or facilitator for MP movement [39,40]. PME-MP interaction might also affect PME activity in HG-deesterification which could lead to changes in the mechanical properties of the cell wall surrounding PD. How changes in pectin de-esterification affects PD transport capacity is not known but potentially this could be a mechanism exploited by viruses to ensure their systemic spreading. In summary, PD are inserted in cell wall microdomains enriched in callose and with a distinct pectin composition (Figure 1). The role of callose in controlling PD size and symplastic permeability has been well defined. By contrast, little is known about the mechanism controlling pectin composition or its importance in modifying the mechanical properties of these cell wall microdomains and the permeability of PD channels.
Proteins attached to PD remodel cell walls to regulate channel aperture In recent years, significant strides have been made in the identification and characterization of proteins bound to PD. Improved methods to isolate PD enriched cell-wall fractions from Arabidopsis cell cultures combined with nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer identified more than www.sciencedirect.com
1000 candidate PD proteins [16]. This list is not by any means a definite demonstration of their presence at PD. A number of contaminants resulting from the purification process were detected highlighting the need of further tests to confirm localization. As a parameter to primarily screen the candidates, only proteins with a predicted secretory signal peptide and either a transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor were considered. These features characterize membrane proteins and were found in PD-located proteins identified in previous proteomic screens [24,41]. However, using alternative approaches, PD targeting of non-membrane predicted proteins has been reported indicating that there is no a priori reason to exclude any of the candidates identified in the proteome. As expected, several cell wall remodelling enzymes appeared in the PD proteome dataset (Table 1). These include enzymes involved in the biosynthesis (callose synthases, CALS) and degradation (b-1,3-glucanases, BG) of callose. Research using mutants, inducible and overexpression lines confirmed the importance of these proteins in regulating PD permeability and the biological processes in which they participate. For example, ATGSL12 (Arabidopsis thaliana glucan synthase-like 12, also named CALS3) was shown to regulate PD-associated callose in the root vasculature affecting vascular patterning and root meristem maintenance [26]. On the other hand, ATGSL8 regulates the non-cell autonomous expression of factors that determine stomata patterning and cell proliferation in leaves, the hypocotyl tropic response and root growth [22,23,42]. Callose-degrading enzymes located at PD sites have also been genetically characterized. A mutant in the PD glucanase ATBG_PAP had increased callose at PD concomitant with a reduction in the systemic spread of the turnip vein clearing virus (TVCV) [28]. During development, a different set of PD-located BG genes (named PdBG1 and PdBG2) regulates callose in the xylem pole pericycle (XPP) in the transition and differentiation zone of the root [21]. pdbg1pdbg2 mutants initiate lateral roots in clusters suggesting that symplastic connectivity is important for lateral root positioning and spatial patterning. The ectopic expression in the XPP of a mutated activated version of CALS3 mimicked the pdbg1pdbg2 phenotype highlighting the importance of controlling callose metabolism at PD during lateral root initiation and patterning. PD-located CALS and BG have also been identified and characterized in different plant species. A recent manuscript reports the identification of the maize TIE-DYED2 gene (TDY2), a callose synthase that regulates symplastic phloem transport and leaf vein development [25]. In Populus two genes encoding PD-located BG were proposed to mediate the movement of Flowering locus T (FT) protein into the bud during dormancy release [43]. As these reports illustrate, the identification of PD-callose Current Opinion in Plant Biology 2014, 22:93–100
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Table 1 Enzymes identified in the PD proteome putatively involved in cell wall remodelling processes. This list is a subset of the full dataset published in [26] and only includes enzymatic activities associated with PD transport in independent studies (see main text for references). Gene ID
Gene name
Cell wall activity
AT2G36850 AT5G13000 AT1G05570 AT2G31960 AT3G07160 AT4G03550 AT5G42100 AT3G55430 AT4G31140 AT5G58090 AT1G64760 AT3G07320 AT1G53840 AT2G47030 AT3G14300 AT4G12390 AT1G65570 AT5G06860 AT5G06870 AT1G02730 AT4G07960 AT4G19900 AT3G46650 AT5G16510 AT3G08900
GLUCAN SYNTHASE-LIKE 8 (GSL8) GLUCAN SYNTHASE-LIKE 12 (CALS3) CALLOSE SYNTHASE 1 GLUCAN SYNTHASE-LIKE 3 GLUCAN SYNTHASE-LIKE 10 GLUCAN SYNTHASE-LIKE 5 b-1,3-GLUCANASE (ATBG_PAP) b-1,3-GLUCANASE b-1,3-GLUCANASE b-1,3-GLUCANASE b-1,3-GLUCANASE b-1,3-GLUCANASE PECTIN METHYLESTERASE 1 PECTINESTERASE (VANGUARD1) PECTIN METHYLESTERASE 26 PECTIN METHYLESTERASE INHIBITOR 1 POLYGALACTURONASE/PECTINASE POLYGALACTURONASE INHIBITING PROTEIN 1 POLYGALACTURONASE INHIBITING PROTEIN 2 GLYCOSYL TRANSFERASE ACTIVITY GLYCOSYL TRANSFERASE ACTIVITY a-1,4-GLYCOSYL TRANSFERASE UDP-GLYCOSYL TRANSFERASE REVERSIBLY GLYCOSYLATED POLYPEPTIDE 5 (RGP5) REVERSIBLY GLYCOSYLATED POLYPEPTIDE 3 (RGP3)
Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Callose metabolism Pectin modification Pectin modification Pectin modification Pectin modification Pectin metabolism Pectin metabolism Pectin metabolism Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall Biosynthesis of cell wall
metabolic enzymes may provide new opportunities for crop improvement, as they could be the target to biotechnologically modify important economic and agronomic traits involving developmental events or transitions in which PD participate. Currently, the use of this knowledge is hindered by the lack of a specific motif/domain to distinguish PD proteins from other plasma membrane enzymes. A recent approach to overcome this issue uses phylogeny to identify BG that evolved with PD complexity in land plants [44]. Proteins involved in pectin metabolism were isolated in the PD proteomic screen but there is scarce information (if any) on their role in regulating symplastic intercellular communication (Table 1). As discussed above pectin degradation may affect PD anchorage to cell walls which, presumably, could destabilize the channel structure [14]. Thus, pectinases and polygalacturonases-inhibiting proteins (PGIP) might be important in forming and maintaining PD architecture. Consistent with this hypothesis, pectinase activity was detected at PD, and in the surrounding cell-wall, in tobacco pollen mother cells at the zigotene stage, coincident with the formation of simple and branched PD structures [45]. Uncharacterized members of these protein families were identified in the proteome but localization or function at PD has not been further investigated. Enzymes catalysing the de-methylesterification and de-acetylation of pectins modify the mechanical/physical properties of cell walls, which might Current Opinion in Plant Biology 2014, 22:93–100
polysaccharides polysaccharides polysaccharides polysaccharides polysaccharides polysaccharides
alter the capacity of PD channels to dilate.Pectin methylesterases and their inhibitors are found in the PD proteome but again their localization has not yet been confirmed. The accumulation of PME around PD has been reported in other systems where they play a role in the systemic spreading of viruses [36,37,39,46]. The ectopic expression of the PME inhibitor PMEI reduces tobamovirus systemic movement which support their function in the control of PD transport [40]. Other putative enzymatic activities involved in general cell-wall biosynthesis, dissolution and assembly are represented in the PD proteome[16]. These include glycosyl transferases and reversibly glycosylated polypeptides (RGPs) (Table 1). A role for glycosyl transferases in PD function is supported by the recent characterization of a b-1,6-N-acetylglucosaminyl transferase-like protein in Arabidopsis thaliana [47]. This protein interacts with calreticulin at PD to regulate seed germination and plant growth. How its expression modifies PD structure or function is unknown. Another glycosyl transferase-like protein was identified in a genetic screen for mutants in stomata differentiation [48]. This protein (named KOBITO1, KOB1) was thought to regulate cellulose biosynthesis but a role in pectin, callose and lignin metabolism could not be discarded [49]. In kob1-3 mutant leaves, stomata appear in clusters, concomitant with an increase in GFP diffusion and in the intercellular transport of the transcription factor SPEECHLESS (SPCH). www.sciencedirect.com
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The results suggest that KOB1 functions in cell-wall metabolism to establish the correct PD aperture that determines stomata patterning in leaves [48]. Regarding RGPs, members of the class 1 family were identified to bind to PD in independent proteomic studies [50]. These proteins are reversibly glycosylated with UDP-sugars and transfer this substrate to enzymes involved in cell wall (and specifically pectin) biosynthesis. Constitutive expression in tobacco of an Arabidopsis class I RGP (RGP2) increases callose and restricts the transport of photoassimilates into sink tissues leading to aberrant plant phenotypes [27]. Expression of this protein also affects virus spreading [27,51]. RGP3 and RGP5 (identified in the PD proteome) might contribute to the regulation of cell wall modifications and symplastic transport in other tissues and/or stress conditions. The presence in the PD proteome of a large number of proteins involved in cell wall synthesis and remodelling may reflect the need to modify the composition of these cell wall microdomains in a quick and dynamic fashion and independently of other cell wall regions. Recent research highlights the importance of regulating callose at PD for proper plant development and identifies proteins regulating its deposition. The function of other enzymatic activities in regulating PD structure and transport is less clear but the pectic supramolecules would appear to be an important target.
Small signalling molecules induce cell wall modifications to regulate PD transport As membranous structures interfacing the cytoplasm and the cell wall, PD function and development are influenced by changes in the intracellular and the extracellular (apoplast) environment. Research on PD composition indicates that these domains are enriched in receptor and receptor-like proteins involved in signalling pathways regulate cell-to-cell communication that [15,52,53,54]. In some instances, the signals and cues perceived by these receptors have been determined but the downstream signalling components are mostly unknown. These receptor proteins can trigger changes in the composition of cell walls at PD domains as a medium to alter symplastic connectivity. A clear example is the Arabidopsis PD-located protein PDLP5, a receptorlike kinase that negatively regulates symplastic transport during the innate immune response by influencing callose deposition at PD [55]. Research on the signals that regulate PD transport capacity is also gaining momentum. Changes in the concentration of Reactive oxygen species (ROS) and in calcium fluxes are known to induce PD structural modifications likely by altering the activity of callose metabolic enzymes and PD-associated cell wall composition (for a review see [12,56]). Plastidial and mitochondrial proteins (such as THIOREDOXIN-M3 and INCREASED SIZE www.sciencedirect.com
EXCLUSION LIMIT 1 and 2) have been identified to participate in organelle-to-nucleus signalling pathways to regulate the intracellular redox status and maintain PD aperture [57,58]. The mechanism underlying this regulation depends on glutathione homeostasis and involves changes in the expression of genes that control PD architecture and transport capacity [59,60]. ROS generated in the apoplast have been proposed to act as cell wall loosening agents (for a review see Gapper and Dolan, 2006 [61]) which might physically influence how PD form and function. Consistent with this hypothesis, H2O2 and class III peroxidases have been found to accumulate at PD regions in the stem cambial zone of tomato accompanying changes in PD structure [62]. Other molecules that interact with redox signalling pathways are described to modify PD. The most recent example is salicylic acid (SA), which regulates the formation of complex PD and callose biosynthesis to influence symplastic transport [63,64]. Work on the mechanism behind this regulation indicates that SA activates the expression of PDLP5, which modifies the activity of callose metabolic enzymes to regulate symplastic transport and pathogen infection [64]. The phytohormone auxin has also been related with cell wall and PD modifications. The expression of the callosebinding protein PDCB1 was found to be up-regulated in response to auxin treatment in lateral roots [29]. PDCB1 functions in callose deposition at PD [24] and its expression correlates with the symplastic isolation of the lateral root primordia from the overlying tissue during emergence. The expression of PD-located callose degrading enzymes acting during lateral root initiation (PdBG1 and PdBG2) is also induced after treatment with auxin and this regulation is abolished in the dominant auxin signalling mutant solitary root1 (slr1)[21]. These expression data led us to hypothesize a regulatory role for auxin in the control of callose deposition and intercellular transport during lateral root development. Intriguingly, treatment with synthetic auxins does not alter diffusion of fluorescein (a fluorescent dye) in root meristem tissues [65]. Recent experiments shed some light on this apparent contradiction. Han et al. demonstrate that changes in callose perturb the formation of an auxin gradient during the phototropic response [23]. Interestingly they showed that in hypocotyls, the expression of the callose biosynthetic enzyme GSL8 is positively regulated by auxin through a mechanism that involves the auxin response factor 7 (ARF7). These results suggest that auxin can target different callose metabolic genes to regulate symplastic transport but this depends on the tissue/condition analysed and on the auxin-dependent regulation of auxin response factors. Changes in PD aperture in response to auxin might also be associated with modifications in pectin structure/distribution. Recent work demonstrates that auxin accumulation in the shoot apex increases HG-demethyl-esterification Current Opinion in Plant Biology 2014, 22:93–100
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leading to cell wall softening during organ outgrowth [66]. Earlier studies indicate that auxin can increase PME activity which is the enzyme responsible for HG deesterification in the cell wall [67]. PME family members are found at PD [35] and, together with PMEI, they appear in the PD proteome (Table 1). Thus, it is possible that auxin influences on PD transport might be related to its effect on cell wall mechanics through changes in pectin esterification. Other small molecules such as tryptophan [65] and gibberellins (GA) [43] have recently been reported to influence symplastic communication. More research is required to elucidate which are the pathways that these molecules use to modify PD and how they differ from the ones described above.
Conclusions Research indicates that the composition of cell walls surrounding PD significantly differs from other cell wall regions. This is likely to be important to maintain PD structure/architecture and to control the cytoplasmic aperture of the channel. The role of callose in the control of PD transport has been well established. The accumulation of this polysaccharide is regulated by PD-located enzymes and by redox signalling pathways. The expression of callose metabolic enzymes is also regulated by salicylic acid and auxin signalling which provides a mechanism for the tight control of callose (and PD transport) in response to a wide range of developmental and environmental cues. By contrast, little is known of the functional significance of the specific distribution of pectic polysaccharides around PD or about the consequences for PD transport capacity of changes in pectin composition— including both HG and RG-I domains. Interestingly, the PD proteome identified a significant number of candidates implicated in pectin biosynthesis and/or remodelling. Characterization of these proteins (including confirmation of PD association) will provide new clues on the role of pectins in symplastic cell-to-cell communication.
Acknowledgements The authors thank William Nicolas, Emmanuelle Bayer, the Bordeaux imaging centre and the Laboratory Membrane Biogenesis for providing the tomogram reconstructed section of plasmodesmata included in Figure 1.
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