Plasmodesmata and the Control of Symplastic Transport

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Publishing Ltd 2002

26 Original Article

Plant, Cell and Environment (2003) 26, 103–124

Plasmodesmata and the control of symplastic transport

A. G. ROBERTS & K. J. OPARKA

Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

‘There are holes in the sky other cells, some degree of intercellular connection is main- where the rain gets in, tained. This plasmodesmal continuum that potentially but they’re ever so small exists throughout the whole plant is termed the symplast that’s why rain’s so thin’ (Münch 1930). However, the symplast is not the open con- Spike Milligan (1968) tinuum that Münch originally hypothesized, but is divided into functional domains, each tightly regulated by different In 1879 Eduard Tangle discovered cytoplasmic connections forms of plasmodesmata (Erwee & Goodwin 1985; Ehlers between cells in the cotyledons of Strychnos nuxvomica, & Kollmann 2001). Plasmodesmata are now thought of as which he interpreted to be protoplasmic contacts. This led fluid, dynamic structures that can be modified both struc- him to hypothesize that ‘the protoplasmic bodies . . . are turally and functionally to cope with the requirements of united by thin strands passing through connecting ducts in specific cells and tissues. the walls, which put the cells into connection with each other and so unite them to an entity of higher order’ (Carr 1976). This challenged the then current view that cells func- THE STRUCTURE OF PLASMODESMATA tioned as autonomous units. It was after much research in Based on structure, two basic types of plasmodesmata have many other species and cell types that Strasburger, in 1901, been characterized; simple and branched. Simple plas- named these structures plasmodesmata (Carr 1976). modesmata consist of a single pore traversing the cell wall, During the division and differentiation of meristematic whereas branched plasmodesmata have two or more chan- cells, plasmodesmata are formed across each developing nels on either side of the middle lamella, often joined by a cell plate, allowing cytoplasmic and endomembrane conti- central cavity. Electron micrographs of simple plasmodes- nuity to occur between all daughter cells, and ultimately, mata are shown in Fig. 1A and B, and in diagrammatic form between all cells in a developing tissue (Mezitt & Lucas in Fig. 2. Simple plasmodesmata may be grouped together 1996). Those plasmodesmata that form during cell division in primary pit fields and this form has been found to pre- are termed primary plasmodesmata (Jones 1976). Those dominate in immature plant tissues (Oparka et al. 1999). that form de novo across existing cell walls are called sec- Simple plasmodesmata are common in the lower plants ondary plasmodesmata (Ehlers & Kollmann 2001). The for- such as algae and mosses (Franceschi, Ding & Lucas 1994; mation of secondary plasmodesmata allows cells to increase Cook et al. 1997) whereas branched plasmodesmata are their potential for molecular trafficking and allows connec- found extensively in mature tissues and appear to represent tions to be created between cells that are not related more evolutionarily advanced structures (Lucas, Ding & cytokinetically. van der Schoot 1993; Oparka et al. 1999). Both primary and As cells expand and differentiate, their fate determines secondary plasmodesmata are initially simple in structure the extent to which their cytoplasmic connectivity to other but, during tissue development, may intrinsically form cells is maintained (Mezitt & Lucas 1996). Some cell types, branches or fuse with neighbouring plasmodesmata to pro- such as those of the leaf mesophyll, remain closely con- duce complex, branched structures. During the sink–source nected to their neighbours, and may even lay down addi- transition that occurs in leaves, simple plasmodesmata are tional plasmodesmata to increase the continuity (Ding et al. gradually converted to branched plasmodesmata by a 1992a). In other areas of the plant, for instance in vascular mechanism that involves the formation of bridges between tissue, certain cells greatly reduce the number of plas- adjoining simple pores (Fig. 2; see Oparka et al. 1999; modesmata in their adjoining walls (Gamalei 1989). In this Roberts et al. 2001). way, the cytoplasmic continuity can be altered depending Due to the difficulty of isolating intact plasmodesmata on the tissue type (Botha & Evert 1988; Brown et al. 1995). from cell-wall fractions, structural models have tradition- However, although reductions in the number of plasmodes- ally been based on data from transmission electron micro- mata are common, only guard cells surrounding stomata graphs (Robards 1971; Robards & Lucas 1990; Beebe & (Erwee, Goodwin & van Bel 1985; Palevitz & Hepler 1985) Turgeon 1991; Ding, Turgeon & Parthasarathy 1992b; and differentiating xylem elements (Lachaud & Maurous- Botha, Hartley & Cross 1993; Overall & Blackman 1996; set 1996) lose all symplastic connections at maturity. In all Waigmann et al. 1997). Most models depict simple plasmodesmata as linear pores in the cell wall (Figs 1C & D), Plasmodesmata and symplastic transportA. G. Roberts & K. J. Oparka Correspondence: Alison G. Roberts. e–mail: [email protected] each lined by the plasma membrane, which is continuous

104 A. G. Roberts & K. J. Oparka

Figure 1. (A) Longitudinal section through simple plasmodesmata between adjacent vascular parenchyma cells in a mature sugarcane leaf. These plasmodesmata show constricted desmotubules and neck constrictions lacking sphincters. ER, endoplasmic reticulum. (Reproduced with permission from R.F. Evert and Planta; Robinson-Beers & Evert, 1991) Bar = 200 nm. (B) Transverse section through simple plasmodesmata between adjacent vascular parenchyma cells in a mature sugarcane leaf. The inner leaflet of the desmotubule appears as a central, electron-opaque dot. The cytoplasmic sleeve appears mottled due to the presence of electron-opaque, spoke-like extensions that extend from the outer leaflet of the desmotubule toward the inner leaflet of the plasma membrane. (Reproduced with permission from R.F.Evert and Planta; Robinson-Beers & Evert, 1991) Bar = 100 nm. (C) and (D) Diagrammatic representation of the substructure of simple plasmodesmata in longitudinal (C) and transverse (D) sections. ER, endoplasmic reticulum; CW, cell wall; CS, cytoplasmic sleeve; D, desmotubule; CR, central rod; PM, plasma membrane; SP, spoke-like extensions; PMP, plasma membrane-embedded proteins; DP, desmotubule-embedded proteins. (Based on model in Ding et al. 1992b).

between adjacent cells. In the centre of the pore lies a ture with an internal lumen, whereas Tilney et al. (1991) strand of modiﬁed cortical endoplasmic reticulum, the des- speculated that it exists as a solid, proteinaceous rod. The motubule (Robards & Lucas 1990; Ding et al. 1992b; Lucas desmotubule has been found in both an appressed and & Wolf 1993; Epel 1994). Different structural models of the dilated state (Overall, Wolfe & Gunning 1982; Robinson- desmotubule have been postulated. Gunning & Overall Beers & Evert 1991; Waigmann et al. 1997). (1983) showed it to have a cylindrical, membranous struc- It is thought that most endogenous cytoplasmic mole-

Plasmodesmata and symplastic transport 105

Figure 2. Diagrammatic representation of the structural change from simple to branched plasmodesmata. During development, the simple pore develops into a complex, branched structure with a central cavity aligned along the middle lamella of the cell wall. This transformation occurs via an H-shaped intermediate that appears to form by the introduction of a new protoplasmic bridge between neighbouring pairs of simple plasmodesmata. WC, wall collar; CW, cell wall; ML, middle lamella; D, desmotubule; PM, plasma membrane; ER, endoplasmic reticulum; CC, central cavity. (Re-drawn from Oparka et al. 1999)

cules, and plant viruses, utilize the cytoplasm between the (Robinson-Beers & Evert 1991; Waigmann et al. 1997). desmotubule and plasma membrane, a region known as the Very little is currently known about the proteins that con- cytoplasmic sleeve (Esau & Thorsch 1985), to move from tribute to the structure of plasmodesmata, and many, if cell to cell (Lucas & Wolf 1993; Epel 1994; Kragler, Lucas not most, plasmodesmal proteins await isolation and & Monzer 1998a). However, trafficking between cells via functional characterization. the desmotubule has also been reported (Cantrill, Overall & Goodwin 1999). At both ends of the plasmodesma, in TRANSPORT VIA THE CYTOPLASMIC SLEEVE the neck region, the channel is frequently constricted (Fig. 1A), and the cytoplasmic sleeve may be partially Transport of many small molecules through plasmodesmata occluded with globular subunits. The subunits are thought occurs via the cytoplasmic sleeve. Passively loaded fluores- to exist in a helical arrangement that may functionally cent probes (Duckett et al. 1994; Roberts et al. 1997) and divide the space into a number of spiralling channels (Zee microinjected dyes (Goodwin 1983; Erwee et al. 1985; 1969; Robards 1976; Olesen 1979; Overall et al. 1982; Wolf Madore & Lucas 1986; Oparka et al. 1991) utilize this path- et al. 1989; Olesen & Robards 1990; Robards & Lucas 1990; way, and larger molecules, including green fluorescent pro- Ding, Turgeon & Parthasarathy 1991; Robinson-Beers & tein (GFP; see below) may also move via the cytoplasmic Evert 1991; Lucas & Wolf 1993; Lucas et al. 1993; Overall sleeve. Barclay, Peterson & Tyree (1982) showed that dye & Blackman 1996; Waigmann et al. 1997). This arrange- movement was not disrupted when cytoplasmic streaming ment appears to reduce the functional diameter of the was inhibited by treatment with cytochalasin B, although cytoplasmic sleeve, producing microchannels that are plasmodesmal disruption by plasmolysis did cause a much- between 3 and 4 nm in diameter (Lucas et al. 1993; Fisher reduced rate of fluorescein movement. Tucker (1987) also 1999). It is thought that these proteins may be part of an noted that cytoplasmic streaming has no effect on the inter- important regulatory structure that can alter the size exclu- cellular movement of molecules. These data suggest that sion limit (SEL) of plasmodesmata, and which viruses may the movement of small molecules occurs by diffusion, and exploit to modify plasmodesmata in order to move from that movement through plasmodesmata is the rate-limiting cell to cell (Evert, Eschrich & Heyser 1977; Carrington step for intercellular transport (Barclay et al. 1982; Tucker, et al. 1996). Electron micrographs have also shown an elec- Mauzerall & Tucker 1989). tron-opaque ring of material surrounding the neck of the plasmodesma, the wall ‘collar’ or ‘sphincter’, that may TRANSPORT VIA THE ENDOMEMBRANE function to alter the SEL of the pore (Olesen 1979; Olesen SYSTEM & Robards 1990; Beebe & Turgeon 1991; Badelt et al. 1994; Turner, Wells & Roberts 1994). While the structure of most Many electron micrographs show that the cortical endo- plasmodesmata conforms to this general model, a range plasmic reticulum (ER) is closely associated with plas- of substructural variations have been found in different modesmata, and the desmotubule provides a potential plant species and within tissues of the same plant pathway for movement between cells. Different structures

106 A. G. Roberts & K. J. Oparka of the desmotubule have been postulated. Although Tilney Ultimately, the factor that determines the diffusive per- et al. (1991) speculated that the desmotubule might exist as meability of a molecule through plasmodesmata is the a solid proteinaceous rod, most models consider it to be a Stokes radius (RS). Terry & Robards (1987) showed that cylindrical, membranous structure with an internal lumen the rate of diffusion is directly correlated to the radius of (Gunning & Overall 1983). Grabski, de Feijter & Schindler the permeant molecule, and that small changes in the (1993) showed that fluorescent lipid and phospholipid ana- Stokes radius could cause large differences in the mobilities logues could diffuse between cells and redistribute follow- of molecular probes. Based on these experiments, it was ing photobleaching if these lipid-based probes were located proposed that the diameter of individual channels in the in the ER. However, they were unable to move between cytoplasmic sleeve of a plasmodesma is approximately cells when targeted to the plasma membrane. This suggests 3 nm. This is a figure that corresponds well to estimates that at least some lipid-based molecules may diffuse from high resolution electron micrographs (Ding et al. between cells along the membranes that comprise the des- 1992b) and dye-coupling studies (Tucker 1982; Erwee & motubule. Recently, Cantrill et al. (1999) showed that if Goodwin 1983, 1984). However, a more recent recalcula- small dyes were microinjected directly into the cortical ER tion of the Terry & Robards (1987) data has shown that the of a single cell they were capable of movement into adjoin- above figure may be closer to 4 nm, a value that makes a ing cells. Such movement can only occur through the des- considerable difference to the potential SEL of the pore motubule. Trafficking via the ER/desmotubule could (Fisher 1999). For instance, an increase in diameter from 3 potentially occur in three different ways: (1) by passive flow to 4 nm may alter the maximum RS of dextrans that can within the desmotubule lumen; (2) by diffusion along the pass through the plasmodesmal pore from approximately inner desmotubule membranes; or (3) by specific attach- 6 nm to 10 nm, and increase the maximum RS of proteins ment of molecules to the cytoplasmic face of the desmotu- from approximately 4 nm to 14 nm (data derived from Jør- bule followed by facilitated transport through the gensen & Møller 1979; le Maire et al. 1986). cytoplasmic sleeve. This ER-mediated mode of transport may be particularly important at plasmodesmata between REGULATION OF TRANSPORT sieve element and companion cells, as there is virtually no cytoplasm in sieve tubes, but the desmotubule provides a Trafficking of molecules through plasmodesmata may be continuum between the parietal ER of the sieve element non-specific, as described above, or may require a specific and the normal, cortical ER of the companion cell (Black- interaction between a structural motif or sequence-specific man et al. 1998; Oparka & Santa Cruz 2000; van Bel & element of the transported protein with other proteins Knoblauch 2000). close to, or within, plasmodesmata (Itaya et al. 2000; Craw- ford & Zambryski 2000). Both endogenous plant proteins and viral proteins can effect specific trafficking (Mezitt & THE PLASMODESMAL SEL Lucas 1996; Ghoshroy et al. 1997; McLean, Hempel & If molecules are smaller than the basal SEL of the plas- Zambryski 1997). Non-specific trafficking is influenced by modesma, intercellular transport appears to occur by diffu- plant development (Oparka et al. 1999) and also by a num- sion. The basal SEL is defined as being the ‘natural’ SEL ber of cellular factors. For example, GFP can traffic non- of plasmodesmata in mature tissues that have been unmod- specifically through plasmodesmata in sink-leaf tissues of ified by, for example, movement proteins, chemicals or tobacco, where plasmodesmata are predominantly simple environmental stresses that increase the SEL. Sugars, in structure and have a relatively high SEL (Imlau, Truernit metabolites, ions and amino acids are all thought to move & Sauer 1999; Oparka et al. 1999). However, movement of by diffusion through the cytoplasmic sleeve of plasmodes- GFP is restricted in mature source-leaf tissues of tobacco, mata (see Lucas et al. 1993). Molecules larger than the basal where plasmodesmata are branched and have a low SEL SEL require selective transport, in which case conforma- (Oparka et al. 1999; Roberts et al. 2001). A reduction in the tional changes must occur in the plasmodesmal pore. Such SEL of plasmodesmata during maturation of a tissue seems changes are not necessarily permanent structural modifica- to be common. For example, symplastic communication is tions as plasmodesmata are capable of transient increases extensive in young embryos, but this changes with develop- in their SEL (Schulz 1999). It is now apparent that the SEL ment, leading to increased symplastic isolation as cells and varies throughout plant development, within different tis- tissues mature (Rinne & van der Schoot 1998; Gisel et al. sues of an organ, and between different plant species. The 1999). In Arabidopsis roots, undifferentiated epidermal SEL can also be modified by interactions with a number of cells in the meristem and elongation zone are extensively endogenous proteins, viral movement proteins (MP) and connected (as measured by dye coupling), but become less other endogenous molecules. Thus, although many early so as they mature, until in the mature root the epidermis reports suggested that the ‘basal’ SEL of plasmodesmata and root hairs are completely isolated from underlying cell was between 850 and 900 Da (reviewed in Lucas et al. layers (Duckett et al. 1994). Similarly, Cantrill, Overall, & 1993), and some cell types (e.g. epidermal cells) can have a Goodwin (2001) found that the symplastic permeability of

SEL less than 370 Mr (Erwee & Goodwin 1985; Duckett cells in tissue culture was high during early regeneration et al. 1994), it now appears that for many cells the SEL is events, but decreased as shoot formation was initiated. considerably higher than these values. Studies of GFP trafﬁcking have shown that plasmodesmata

Plasmodesmata and symplastic transport 107 are differentially permeable in different tissues or organs long-lived ray and parenchyma cells of tree species and in different plant species. GFP was shown to traffic (Chaffey & Barlow 2001). between cells in Arabidopsis leaf and stem epidermis at all The ER is known to be closely associated with actin developmental stages and yet was unable to move in the filaments (Quader, Hofmann & Schnepf 1987; Boevink epidermis of either tomato or cucumber (Itaya et al. 2000). et al. 1998), and the highly motile cortical ER has been Movement between cell layers also appears to be regulated postulated to move along actin cables (Quader et al. 1987), differently from movement within a cell layer. GFP could possibly using myosin or other motor proteins that link the not pass from the epidermis into the mesophyll of cucum- cytoskeleton to the endomembrane system. Fluorescent ber cotyledons although it could move from the epidermis lipid analogues have been shown to move between cells in into the cortex of hypocotyls (Itaya et al. 2000). During the ER membrane but not in the plasma membrane (Grab- morphogenesis of protoplast-derived calluses, some plas- ski et al. 1993). This suggests that the ER may ‘flow’ modesmata were found to be blocked by a dense, osmio- through plasmodesmata whereas the plasma membrane is philic material that produced numerous symplast domains a more stationary structure. If intrinsic ER proteins can of varying size within the developing tissue (Ehlers, move between cells in this way, it may be possible that Binding & Kollmann 1999. The authors noted that the other molecules could ‘hitch a ride’ on the ER. Molecules blockages were always produced by the surrounding cells inserted into, or temporarily attached to, the ‘flowing’ ER and not within the isolated domain, and suggested that membrane could conceivably move through plasmodes- demarcation of symplastic domains might be a general pre- mata via the desmotubule. In addition, if myosin motors requisite for differential morphogenesis. Plasmodesmal are required for movement along actin cables, this could regulation may therefore use a number of distinct mecha- give rise to polarity of ER-based movement, as all myosins nisms, depending on the developmental stage, tissue and move uni-directionally towards either the pointed (–) or species of plant, the structure of plasmodesmata present, the barbed (+) end of the actin filament (Wells et al. 1999). and the presence of modifying factors such as endogenous Such behaviour could help to explain the apparent direc- or viral MPs. tionality of movement sometimes observed through plasmodesmata. In Nicotiana clevelandii trichome cells, movement of low molecular weight dyes and dextrans THE CYTOSKELETON AND PLASMODESMATA occurred preferentially towards the tip of the trichome Both actin and myosin have been localized to the plas- (Waigmann & Zambryski 1995), although this could be modesmal pore (White et al. 1994; Blackman & Overall due to an increase in simple plasmodesmata towards the 1998; Radford & White 1998). Some authors have sug- trichome tip (Waigmann et al. 1997). In Chara cells, a gested that this distribution may allow constriction and similar linear system, the intercellular transport of relaxation of the entire length of the pore (Zambryski & photo-assimilate also shows directionality (Ding et al. Crawford 2000), whereas others have suggested that these 1991). Tightly controlled and directional movement of cytoskeletal elements are involved in transport per se, transcription factors has also been discovered, even in rather than having a role in regulating the permeability of tissues with a high SEL (see later). the plasmodesma (Blackman & Overall 2001). The latter The presence of both actin and myosin along the length point is supported by the fact that inhibitors of actin and of the pore, possibly as helically arranged ‘spokes’ that myosin do not appear to influence the movement of small connect the desmotubule to the plasma membrane (Over- molecules that pass through plasmodesmata by diffusion, all & Blackman 1996), provides a possible contractile indicating that actin and myosin are not involved in con- mechanism for controlling the aperture of the cytoplasmic trolling the permeability of plasmodesmata, at least at low sleeve (Radford & White 1998; Reichelt et al. 1999). SELs (Tucker 1987). In contrast, complete depolymeriza- Myosin VIII, an unconventional myosin found only in tion of the actin cytoskeleton by cytochalasin resulted in a plants, has also been localized to plasmodesmata (Reichelt widening of the neck region of plasmodesmata in Nephrol- et al. 1999) and its activity could be regulated by calcium epis exaltata (White et al. 1994), and caused an increase in (Knight & Kendrick-Jones 1993). At present, seven puta- the SEL of tobacco plasmodesmata from 1 kDa to over tive myosin VIII proteins have been reported (Baluska 20 kDa (Ding, Kwon & Warnberg 1996). However, when et al. 2001). All of these have a characteristic C-terminal actin microfilaments were stabilized by treatment with structure that includes a probable phosphorylation site for phalloidin, cell–cell transport was inhibited. Additional protein kinases, as well as four calmodulin-binding motifs studies, in which a GFP-talin fusion was biolistically bom- (Reichelt & Kendrick-Jones 2000). Myosin VIII therefore barded into a single cell, showed that the actin microfila- emerges as a good candidate for a plasmodesmal motor ments which became labelled by GFP-talin did not protein whose activity may be regulated by calcium or themselves move between cells (Crawford & Zambryski calmodulin. 2000). These results suggest that actin filaments may form Plasmodesmal regulation may occur along the full length a static scaffold within plasmodesmata, along which mole- of the plasmodesmata (as suggested by the presence of cules move using a myosin-driven mechanism. The cytosk- both actin and myosin along the length of the pore), but eleton has also been hypothesized to be important in regulation could also occur only at the neck regions, pro- maintaining an extensive communication pathway in the ducing the same effect. Centrin, a calcium-binding contrac-

108 A. G. Roberts & K. J. Oparka tile protein, has been localized to the neck region of ever, at lower pressure differentials, dye continued to move plasmodesmata (Blackman, Harper & Overall 1999) and between neighbouring cells. The closure of plasmodesmata could regulate the plasmodesmal aperture. An increase in occurred relatively slowly, over a period of at least 10 min, the concentration of cytoplasmic calcium causes a decrease and the effect lasted for some time, possibly permanently. in the phosphorylation of this protein (Martindale & In the alga, Chara corallina, pressure differences also Salisbury 1990). This dephosphorylation causes the centrin caused plasmodesmata to seal off, preventing further inter- nanofilaments to contract rapidly, potentially closing the cellular exchange (Ding & Tazawa 1989; Reid & Overall plasmodesma (Martindale & Salisbury 1990; Blackman 1992). In contrast, an increase in the turgor of Arabidopsis et al. 1999). Additional data to support this hypothesis root hairs by microinjection of oil droplets did not cause an include evidence that increased levels of calcium lead to alteration in the electrical coupling of cells, apparently plasmodesmal closure (Erwee & Goodwin 1983; Tucker indicating that plasmodesmata did not close completely 1990; Holdaway-Clarke et al. 2000), and the fact that two (Lew 1996). Increasing osmotic tissue stress has also been protein kinases, one calcium-dependent, have been local- shown to cause changes in plasmodesmatal permeability. ized to plant cell walls or plasmodesmata (Citovsky et al. Schulz (1995) showed that increasing mannitol concentra- 1993; Yahalom et al. 1998). In addition, both ATPase activ- tions caused the usually constricted neck regions of plas- ity and calcium-binding sites have been localized to plasmodesmata to widen to approximately the same diameter modesmata in barley roots (Belitser, Zaalishvili & as the central portion of the pore. In parallel to the plas- Sytnianskaja 1982) and the calcium-sequestering protein, modesmal widening, these osmotically stressed root tips calreticulin, has also been localized to plasmodesmata showed increased phloem unloading. In Egeria densa (Baluska et al. 1999, 2001). Calreticulin is normally found leaves, the SEL was also increased for a period of 20 h in the lumen of the ER but has calcium-buffering activity following plasmolysis (Erwee & Goodwin 1984). Together, that could potentially regulate plasmodesmal transport. these results suggest that a sudden pressure differential However, although alterations in cytoplasmic calcium have between adjacent cells, as might occur during wounding, been reported to increase the electrical resistance of plas- will cause isolation of plant cells from their neighbours, modesmata, cytoplasmic acidification with butyric acid whereas an overall drop in tissue turgor, characteristic of doubled the calcium concentration and yet did not affect water stress (hyperosmotic stress), will lead to an increased plasmodesmal resistance (Holdaway-Clarke et al. 2001). SEL. In the latter case, the increase in plasmodesmal Citovsky et al. (1993) showed that a cell-wall-associated permeability may be associated with the plant’s need to kinase was developmentally regulated, and its activity was suddenly increase solute fluxes into organs undergoing correlated with the developmental maturation of plas- osmotic stress. modesmata. This kinase was also able to phosphorylate the Other physiological perturbations can also alter plas- 30 kDa MP of Tobacco mosaic virus (TMV), a protein modesmal SELs. In a study of roots under anaerobic stress, known to be able to interact with, and functionally alter, reduced levels of ATP caused the plasmodesmal SEL to plasmodesmata (Citovsky et al. 1993; see below). Further- increase from less than 1 kDa in controls, to between 5 and more, phosphorylation of this MP led to altered plas- 10 kDa in stressed roots (Cleland, Fujiwara & Lucas 1994). modesmal permeability properties (Waigmann et al. These authors suggested that ATP was required to maintain 2000), and may be a common mechanism to control viral plasmodesmata in a constricted state. However, although movement, as the movement proteins of Potato leafroll hypoxia has been shown to depolarize the membrane virus (PLRV; Sokolova et al. 1997; Tomato mosaic virus potential of wheat root cells, it had little effect on the mem- (Matsushita et al. 2000), and Cucumber mosaic brane resistance or the electrical coupling ratio, which is a virus (CMV; Matsushita et al. 2002b) have all been found measure of plasmodesmal resistance (Zhang & Tyerman to be phosphorylated. The presence of phosphorylation 1997). These authors suggested that their results pointed to sites at, or near, plasmodesmata strongly suggests that the possibility that transport of water and solutes could modification of plasmodesmal-specific, contractile proteins occur via both the cytoplasm and the ER, and that only the by either phosphatases or kinases may play an important ER pathway is interrupted by hypoxia. Furthermore, they role in plasmodesmal control. postulated that hypoxia may cause the desmotubule to con- dense, which would in turn enlarge the cytoplasmic annulus, and might explain the increased SEL found by Cleland TURGOR AND PRESSURE EFFECTS et al. (1994) in anaerobically stressed wheat roots (Zhang Plasmodesmata are known to alter their permeability in & Tyerman 1997. A range of metabolic inhibitors have also response to turgor changes. In contrast to the fine regula- been shown to increase symplastic movement out of the tion of the plasmodesmal SEL imposed by cytoskeletal ele- transport phloem in Arabidopsis roots (Wright & Oparka ments, it seems likely that responses to turgor changes are 1997), whereas increased cytoplasmic concentrations of designed to cope with physiological traumas that arise as a group II ions decreases plasmodesmal permeability (Erwee result of stress or wounding. In Nicotiana clevelandii tri- & Goodwin 1983). Simply changing plant growth condi- chome cells, pressure differentials of more than 200 kPa tions has been reported to alter plasmodesmal permeabil- between adjacent cells prevented the transport of microin- ity; plants grown in a glasshouse exhibited increased jected Lucifer yellow dye (Oparka & Prior 1992). How- movement of GFP compared with plants grown in tissue-

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 103–124 Plasmodesmata and symplastic transport 109 culture containers (Crawford & Zambryski 2000). In short, only in response to aluminium, and not to peroxide appli- plasmodesmata appear to be extremely sensitive to a range cation (Robards & Lucas 1990; Jones et al. 1998), suggest- of physiological perturbations, a fact that underlines their ing that callose deposition at plasmodesmata may be a central importance in regulating exchange between cells survival mechanism for aluminium toxicity in plants and tissues. (Sivaguru et al. 2000). Although some callose is always found in sieve pores as a remnant of sieve-pore genesis, phloem transport can also occur in the presence of large CALLOSE callose deposits across sieve plates (Peterson & Rauser Callose ((1Æ3)-b-glucan) deposition at plasmodesmata has 1979). However, phloem loading in the sucrose export defi- also been postulated to control intercellular exchange, cient (sxd1) maize mutant is prevented by callose deposits although the rates of deposition are generally slow, sug- which specifically block plasmodesmata at the interface gesting that this mechanism may be used more in response between bundle sheath and vascular parenchyma cells to wounding or pathogenesis than as a rapid control mech- (Botha et al. 2000). anism. However, callose plugs at plasmodesmata have During pathogenesis, callose has been found at plas- recently been implicated in the maintenance of dormancy modesmata, suggesting that callose deposition is a ubiqui- by symplastically isolating the meristem from surrounding tous plant response to pathogen spread. Most published tissues (Rinne & van der Schoot 1998; Rinne, Kaikuranta reports have concentrated on viral infections, but callose & van der Schoot 2001). During wounding, callose is has also been found to block plasmodesmata during infec- deposited at plasmodesmata (Hughes & Gunning 1980), tion by the oomycete Phytophthora sojae (Enkerli, Hahn but callose deposition and degradation have been shown to & Mims 1997). Callose has been found in pit fields and be variable both in time and in quantity. During the along the surrounding walls of plants infected with Potato response of oat coleoptiles to plasmolysis, callose was virus X (PVX; Allison & Shalla 1974). In these plants, deposited at plasmodesmata, but most had disappeared 4– callose deposition was extensive within necrotic viral 6 h after full turgor was regained. Recovery of electrical lesions, but could also be detected outside the visible coupling in these cells followed the time course of callose lesion, suggesting that formation of callose collars at plas- removal (Drake, Carr & Anderson 1978), taking several modesmata could be an early defence strategy against this hours to resume. In contrast, in Egeria densa, electrical virus. Callose deposition has also been reported at plas- coupling was re-established within 10 min of deplasmolysis modesmata in plants infected with many other viruses, (Erwee & Goodwin 1984). Callose deposition at the neck including TMV (Wu & Dimitman 1970; Moore & Stone of plasmodesmata has also been linked to alterations in the 1972; Leisner & Turgeon 1993; Beffa et al. 1996), Tomato hormonal balance or the osmotic potential of plant mate- bushy stunt virus (Pennazio et al. 1978), and Maize dwarf rial, and plasmodesmata with callose collars were observed mosaic virus (Choi 1999). In addition, plants deficient in to also contain electron dense material in the cytoplasmic b-1,3-glucanases (callose-degrading enzymes) showed a sleeve which apparently obscured the desmotubule (Botha decreased susceptibility to infection with TMV (Beffa et al. & Cross 2000). Wolf et al. (1991) showed that a reduction 1996; Iglesias & Meins 2000) and to PVX and CMV (Igle- in the SEL, and an inhibition of dye movement in trans- sias & Meins 2000). These plants also showed increased genic tobacco plants expressing the movement protein of callose deposition at plasmodesmata in response to viral tobacco mosaic virus, could be alleviated by treatment of infection, and a reduction in the plasmodesmal SEL. the tissue with an inhibitor of callose synthesis, providing Complementary experiments showed that TMV mutants evidence that callose deposition is involved in regulating that overexpressed the b-1,3-glucanase gene displayed the plasmodesmal SEL. Dye coupling has also been shown increased movement and spread on either wild-type or b- to be reduced in cells adjacent to TMV infection sites, in 1,3-glucanase-deficient plants (Bucher et al. 2001). Moore areas known to have callose deposits at plasmodesmata & Stone (1972) also showed that levels of b-1,3-glucan (Susi 2000; see below). Enhanced callose deposition in a b- hydrolase were increased in leaves infected with a number 1,3-glucanase-deficient mutant also caused a reduction in of other plant viruses. Collectively, these authors have the SEL (Iglesias & Meins 2000) but not an absolute clo- suggested that induction of b-1,3-glucanases might be a sure of the plasmodesmata. It is interesting to note that, strategy used by viruses to enhance their cell-to-cell move- although the effects of callose are generally thought to be ment and spread (Moore & Stone 1972; Beffa et al. 1996; coarser than other control mechanisms, many recent pub- Iglesias & Meins 2000). lications have shown that callose can regulate the SEL, rather than simply constricting or closing the plasmodes- LOSS OR REDUCTION OF PLASMODESMATA mal pore. Additional experiments have shown that callose may be deposited only in response to specific stresses. Cal- In addition to changes in the structure of plasmodesmata, lose deposition is known to be induced by micromolar their frequency can also be developmentally altered in dif- changes in intercellular calcium concentrations (Kauss ferent tissue types throughout the plant. Plants are capable 1987). Induction of stress by application of both aluminium of sealing off, or removing plasmodesmata both tempo- or hydrogen peroxide caused an increase in cytoplasmic rarily and permanently. Erwee & Goodwin (1985) injected calcium concentration and yet, callose deposition occurred a range of differently sized fluorescent dyes into Egeria

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 103–124 110 A. G. Roberts & K. J. Oparka densa and showed that the SEL varied between tissues. This plants were also gradually shut off as cells differentiated proved that the symplast is not a cellular continuum with into phloem mother cells (van Bel & van Rijen 1994). Sym- unlimited communication, but is subdivided into functional plastic communication is also reduced during Arabidopsis domains. These domains, and the limits they place on the embryogenesis when a developmental transition reduces movement of signal molecules, are thought to allow co- the plasmodesmal size exclusion limit at the torpedo stage ordinated mitosis, morphogenesis and development in of development (Kim et al. 2002a). Such data suggest that plants (Kragler et al. 1998a; Ehlers & Kollmann 2000; symplastic isolation is a common mechanism to allow reg- Pfluger & Zambryski 2001). ulated and co-ordinated development and differentiation As guard cells differentiate, cell-wall material is depos- in plant tissues. By preventing the movement of signal mol- ited across the pore on the side of the neighbouring epider- ecules, ions and photo-assimilate, plasmodesmal isolation mal or subsidiary cell wall, effectively truncating also affects the turgor potential within a given symplastic plasmodesmata and symplastically isolating the guard cell domain. This aspect of symplastic isolation has long been complex (Wille & Lucas 1984; Palevitz & Hepler 1985). In understood in the function of stomata, but has also recently differentiating xylem tissue of Sorbus torminalis, many been shown to control development of cotton fibres (Ruan, plasmodesmata are found in the pits that connect imma- Llewellyn & Furbank 2001). In these cells, a developmental ture xylem elements to the surrounding mesophyll cells. switch from simple to branched plasmodesmata, concomi- However, during the final stages of programmed cell death tant with a transient closure of plasmodesmata, drives the the pits become sealed off by the deposition of new cell- rapid elongation required in these specialized cells (Pfluger wall material across both ends of the plasmodesmatal & Zambryski 2001; Ruan et al. 2001). pores (Lachaud & Maurousset 1996). The sucrose export At least some plasmodesmal proteins can be targeted for deficient (sxd1) mutant of maize is unable to load sucrose degradation via the ubiquitin pathway, as occurs for cyto- into minor veins due to a blockage of plasmodesmata at solic proteins. In regenerating protoplasts, discontinuous the bundle sheath–vascular parenchyma cell-wall interface plasmodesmata showed high levels of ubiquitin whereas (Russin et al. 1996). This blockage also occurs by deposi- those able to establish secondary contacts between neigh- tion of wall material and/or callose (Russin et al. 1996; bouring cells did not (Ehlers, Schulz & Kollmann 1996). Botha et al. 2000) across the plasmodesmatal pores. It has The accumulation of ubiquitin in these half-plasmodesmata been hypothesized that sxd1 mutants have a defect in the may point to a role for this enzyme in the degradation and signalling mechanism between the chloroplasts and removal of plasmodesmata. nucleus in bundle sheath cells. This defect in the communication pathway affects the differentiation of bundle sheath INTERCELLULAR PROTEIN AND cells during the sink-source transition, leading to deposi- RNA TRAFFICKING tion of callose across plasmodesmata at the bundle sheath–vascular parenchyma cell-wall interface (Mezitt Functional parallels exist between plasmodesmata and Provencher et al. 2001). Since the permanent blockage of nuclear pore complexes, and it seems that common mech- plasmodesmata may share similar underlying mecha- anisms may underlie the transport of mRNA and proteins nisms, the cloning and study of genes such as sxd1 will through both types of pore (Lee, Yoo & Lucas 2000). One hopefully elucidate the molecular events that underlie of the earliest examples of cell–cell signalling was reported plasmodesmatal modifications that lead to loss of sym- by Sussex (1951) who found that the signal that determines plastic continuity. the dorsiventrality of leaves was initiated in the shoot apical During the sink–source transition in tobacco leaves, the meristem (SAM). It is now known that signals that pass numbers of simple plasmodesmata are dramatically between the abaxial and adaxial cell layers of leaves are reduced (Roberts et al. 2001). Some of this loss appears to also required for organ shape (Bowman 2000). During the occur by the conversion of simple plasmodesmata to 1990s, many proteins and RNAs were discovered to move branched plasmodesmata (Oparka et al. 1999). However, it in plants, often displaying non-cell-autonomous functions appears that many simple plasmodesmata are ‘sacrificed’ that had previously been assumed to be the function of during leaf development. Most of these plasmodesmata smaller signal molecules (Hake 2001). The SAM gives rise are lost during the rapid phase of leaf cell expansion when to all aerial parts of the plant and in most species, consists intercellular air spaces are forming in the mesophyll. No of three layers, L1 (the outermost), L2 and L3, which give trace of these plasmodesmata remains in the mature leaf, rise to the epidermis, cortical tissues and inner tissues and they are literally ripped apart during rapid leaf expan- (including the vasculature), respectively. In dicotyledonous sion. It would appear that these simple plasmodesmata are plants, cells in the L1 and L2 layers divide anticlinally, utilized for a brief developmental period during which effectively producing clonal cell layers (Poethig & Sussex the leaf functions as a sink for assimilates (Roberts et al. 1985; Sussex 1989). By using periclinal chimeras, where one 2001). cell layer is not genetically related to the others, it was In the Arabidopsis shoot, communication between cells shown that signals could be transmitted between layers in different areas of the meristem changes during the tran- (Szymkowiak & Sussex 1992, 1993). In tomato periclinal sition from vegetative growth to flowering (Gisel et al. chimeras, signals produced within the L3 layer determine 1999), whereas plasmodesmata in the cambium of Lupinus the number of carpels and size of the floral meristem

Figure 3. Diagrammatic representation of the expression pattern of various transcription factors in apical and floral meristems. Relative quantities of RNA and protein are represented by gradations of colour. In contrast to the unrestrained movement of GFP in meristems (see Fig. 4), a number of RNAs and proteins have been found to move in a tightly regulated manner within the meristem region, or not at all. (a) KNOTTED1 mRNA is found only in the L2 and L3 layers of the meristem whereas KNOTTED1 protein is found in all three layers of the meristem (Jackson et al. 1994). (b) The DEFICIENS mRNA is also present only in the L2 and L3 layers, but the protein shows polar trafficking, being found predominantly in the L1 layer, with lesser quantities present in the L2 and L3 layers (Perbal et al. 1996). (c) Genetic chimeras expressing LEAFY mRNA only in the L1 layer, showed LEAFY protein expression in all layers, most strongly within the L1 layer, but with a gradient of expression extending into inner layers (Sessions et al. 2000). (d) In experiments using genetic chimeras, the APETALA3 protein has been found only in the cell layers in which the APETALA3 RNA is expressed, showing that this protein does not traffic between cells of the meristem (Jenik & Irish 2001). (e) CLAVATA3 mRNA accumulates only in a small zone of cells at the meristem apex in the L1 and L2 layers, plus a few underlying cells in the L3 layer. It is hypothesized that the CLAVATA3 protein is secreted to the extracellular space and then moves through the apoplast into the L3 layer of the meristem and, by acting on the CLAVATA receptor found there, allows co-ordinated development of the meristem (Fletcher et al. 1999).

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 103–124 112 A. G. Roberts & K. J. Oparka although these tissues are formed from cells in the L1 and an effect only on cells in underlying layers, and not on cells L2 layers (Szymkowiak & Sussex 1992). in the same plane (Foster, Veit & Hake 1999). In contrast, The protein KNOTTED1 (KN1) was the first transcrip- other proteins, such as APETALA3 and PISTILATA, also tion factor reported to move between cells (Jackson, Veit found in the SAM, do not move at all (Jenik & Irish 2001). & Hake 1994). The KN1 protein was found in the L1 layer In Arabidopsis, the maintenance of a functional SAM of the SAM although its RNA was restricted to the L2 and requires a gene family called CLAVATA (Fletcher et al. L3 layers (see Fig. 3). The fact that KN1 could move 1999; Brand et al. 2000; Sharma & Fletcher 2002). It is between cells was initially demonstrated by microinjection thought that a small protein encoded by CLA3 binds to of fluorescently labelled KN1 into tobacco leaf cells (Lucas CLA1, a transmembrane serine/threonine kinase, in order et al. 1995) and has since also been shown using GFP- to modulate transcription factors that are required for mer- fusions (Kim et al. 2002b). These experiments showed that istem maintenance. CLA3 RNA is expressed only in a small the protein was not only capable of trafficking between group of stem cells at the centre of the meristem apex but cells in the leaf and the SAM (Kim et al. 2002b), but also the encoded protein is hypothesized to move into deeper increased the plasmodesmal SEL and could specifically layers of the meristem where it activates the CLV stem cell traffic its own RNA between cells in tobacco mesophyll signalling pathway to allow co-ordinated growth (Fletcher (Lucas et al. 1995). However, it is perhaps puzzling that et al. 1999; Rojo et al. 2002). In contrast to the symplastic although KN1 can move into the L1 layer, and specifically pathway thought to be used by other transcription factors, traffic its own mRNA in mesophyll, that the mRNA is recent and elegant work by Rojo et al. (2002) has shown unable to move with the protein into the L1 layer of the that the CLV3 protein is transported through the secretory meristem. This suggests that expression of the mRNA is pathway and localized to the apoplast. The protein is now unnecessary, or perhaps deleterious in the L1 layer and that thought to move through the apoplast into the L3 layer movement of the RNA, even in the presence of the KN1 where it binds with, and activates, the CLV1–CLV2 com- protein is tightly controlled. Several other non-cell- plex at the plasma membrane to co-ordinate stem cell activ- autonomous factors may move through plasmodesmata ity via the transcription factor WUSCHEL (Brand et al. (Mezitt & Lucas 1996) but this may not be the case for all 2000; Rojo et al. 2002). Fluorescent tracer studies by Rinne such signals. KN1 has since been shown to require the & van der Schoot (1998) showed that the SAM of birch activity of a chaperone molecule to unfold the protein, and seedlings maintains symplastic domains to allow co- a receptor molecule to allow movement from cell to cell ordinated morphogenesis. It has been proposed that an (Kragler et al. 1998b). The same receptor apparently plays apoplastic means of transport, as has been shown for a role in the movement of CMV (Kragler et al. 1998b), CLA3, may allow signalling between areas of the SAM that suggesting that a single protein is able to mediate the traf- are not symplastically coupled (Rojo et al. 2002). Figure 3 ficking of both an endogenous protein and viral RNA. Kra- summarizes the reported movement of some known tran- gler et al. (2000) subsequently showed that a peptide scription factors in the SAM. antagonist could interact with a motif involved in plas- A recent publication has highlighted the biological sig- modesmal dilation, preventing the movement of KN1 nificance of the movement of a transcription factor into RNA, but still allowing limited movement of the KN1 pro- cells beyond its site of synthesis. In Arabidopsis, signals tein. Murillo, Cavallarin & San Segundo (1997) found that initiated in older cells and propagated to immature cells are PRms, an 18 kDa maize pathogenesis-related protein that transmitted throughout the root meristem in order to deter- is produced in response to fungal infection, could also move mine the positional fate of cells (van den Berg et al. 1995, independently between cells. This protein was found in 1997). One specific gene has recently been identified that plasmodesmata, and was also detected in both the paren- controls radial patterning of the Arabidopsis root. The chyma cells of the protoxylem and central pith of maize SHORT ROOT (SHR) gene encodes a transcription factor radicles, whereas the PRms RNA was found only in required for formation of the endodermal layer, and parenchyma cells of the protoxylem. mutants that lack this protein do not form an endodermis A number of non-cell-autonomous transcription factors (Helariutta et al. 2000). In situ hybridization has shown that that control flower development have now been described the SHR RNA is expressed only in the vascular tissues, but (see Fig. 3 and Sharma & Fletcher 2002). Transcription fac- the protein product is found in the vascular tissue and in tors that have been found to move symplastically include the neighbouring endodermis (Nakajima et al. 2001). In LEAFY (LFY), in which the RNA is located only in the addition, the localization of SHR in the vascular tissues was L1 layer of the Arabidopsis floral meristem whereas the both nuclear and cytoplasmic, but in the endodermis it was protein can be found in a number of inner cell layers nuclear only. This suggests that the cytoplasmic SHR moves (Sessions, Yanofsky & Weigel 2000), and DEFICIENS one cell away from the vascular origin, presumably through (DEF) which can move within developing Antirrhinum plasmodesmata, into the endodermis where it becomes flowers (Perbal et al. 1996). The DEF protein shows direc- nuclear localized. These authors also showed that when tional movement; it can move from the L2 upwards into the SHR was expressed from either the endodermis-specific L1 layer, but not in the opposite direction (Perbal et al. SCARECROW (SCR) promoter, or the constitutive Cau- 1996). GNARLEY1, another non-cell-autonomous factor liflower mosaic virus (CaMV) 35s promoter, supernumer- from maize leaves also shows directional movement, having ary endodermal cell layers were produced, leading to

mal components that interact with transcription factors remains a challenge for the future.

LONG-DISTANCE MOVEMENT OF PROTEINS AND RNA IN PLANTS A number of proteins have been found to traffic within phloem tissues but these are described elsewhere in this issue (see review by van Bel, pages 125–149 in this issue). However, many of the proteins known to move between cells in the phloem also traffic over long distances through the phloem to subsequently unload in sink tissues. In heterografts between members of the Cucurbitceae, rootstock- specific structural phloem proteins and their precursors were found to be present in scion tissues (Golecki et al. 1998), having moved extensively through the phloem (Golecki, Schulz & Thompson 1999). It should be noted however, that although a number of proteins have now been found to pass through the plasmodesmata that connect sieve elements and companion cells of the phloem, and to unload in sink tissues, that no common structural or sequence-specific motifs have been identified among this group of proteins. This fact suggests that protein trafficking Figure 4. Emerging lateral root of a transgenic Arabidopsis plant in the phloem must occur in one of two ways. Transport may expressing GFP from the AtSUC2 companion cell-specific probe very highly specialized, with specific interactions moter. GFP unloads from the phloem and moves freely within the between each protein and plasmodesmal receptors. How- developing tissues of the root tip, including the meristem. GFP (green) is present in the cytoplasm of all cell layers. The cell walls ever, it seems unlikely that such a wide array of receptors are stained red with propidium iodide. (Image courtesy of K. could exist at the plasmodesmal pore, and even more Wright, SCRI). Bar = 50 mm. unlikely that receptors would exist for non-plant proteins such as GFP. The alternative, and more likely scenario, is that trafficking within and through the phloem is malformed roots and showing that expression of a gene in relatively unrestricted and does not rely on specific the same tissues as the required protein product can actu- interactions with plasmodesmal proteins. ally be detrimental to plant development (Nakajima et al. In addition to proteins, plant RNAs are also trafficked a 2001). These results highlight the fact that the controlled long distance through the phloem, in some cases in conjunc- movement of signal molecules and gene products is essen- tion with endogenous proteins, and the RNA can subse- tial for accurate development, and may go some way to quently exert effects in sink tissues. The CmPP16 phloem explaining why, for instance, the KNOTTED mRNA is not protein from Cucurbita maxima allows the transport of found in the L1 layer of the meristem. both sense and antisense RNA in the phloem, and can When considering the movement of transcription factors move from cell to cell, increasing the plasmodesmal SEL in meristems, an apparent paradox emerges. The SAM is during the process (Xoconostle-Cázares et al. 1999). These known to have a high SEL (Ormenese et al. 2000), a char- characteristics may allow this protein, and other similar acteristic of immature plant tissues in which simple plas- host proteins, to facilitate the long-distance delivery of modesmata predominate (Oparka et al. 1999). Similarly the RNAs through the phloem to allow the regulation of trans- root meristem shows extensive cell-to-cell trafficking of lational events in developing tissues of sink organs (Jor- phloem-unloaded GFP (K.M. Wright et al. unpubl. results; gensen et al. 1998; Xoconostle-Cázares et al. 1999). Another Fig. 4). Given the apparent high basal SEL of root and endogenous phloem protein, PP2, a dimeric lectin and the shoot meristems, what is surprising is not that transcription most abundant component of Cucurbita spp. phloem exu- factors move from cell to cell, but that they do not move date, has also been shown to interact with RNA (Owens, more extensively away from their site of cellular synthesis Blackburn & Ding 2001). This protein can interact with and throughout tissues (Fig. 3). Clearly, a tightly regulated Hop stunt viroid. Viroids are small, circular, unencapsi- movement of transcription factors occurs, allowing this dated RNA pathogens that lack mRNA activity. PP2 is able class of proteins to modify the plasmodesmata leading into to move from cell to cell through plasmodesmata (Bal- neighbouring cells, but at the same time preventing their achandran et al. 1997) and can itself traffic through the further spread into cells beyond. Such highly specific move- phloem (Golecki et al. 1999), suggesting that, in addition to ment points to a complex and orchestrated interaction viroids, other RNAs may be moved through the phloem by between the transcription factor and the plasmodesmata interactions with chaperone proteins (Owens et al. 2001). present in meristematic tissues. Identifying the plasmodes- Viral proteins can also act in a non-specific way to improve

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 103–124 114 A. G. Roberts & K. J. Oparka phloem transport. Groundnut rosette virus (GRV) is otides; Hamilton & Baulcombe 1999), which can enter the unusual in that it does not encode for a coat protein, nor- phloem and subsequently unload in sink tissues (Voinnet mally required to protect the viral RNA during systemic et al. 1998) in a manner that resembles the unloading pat- movement. The protein encoded by GRV ORF3 has been terns of photo-assimilate or viruses (Oparka & Santa Cruz shown to act as a trans-acting long-distance movement fac- 2000). This PTGS mechanism is used as a defence against tor that can facilitate the movement of unrelated viral a number of plant viruses. A recent report suggests that the RNAs through the phloem (Ryabov, Robinson & Taliansky short RNAs are not required for methylation of the trans- 1999). gene, a state required to allow and maintain gene silencing, Endogenous Cucurbita maxima CmNACP mRNA has and are unnecessary for the production and transmission of been found to move through the phloem and unload in the silencing signal (Mallory et al. 2001). These results sug- apical tissues of heterografts. CmNACP, part of the NAC gest that alternative or additional signals are involved, and domain gene family which may be involved in apical mer- that gene silencing, and symplastic RNA signalling in gen- istem development, was found to move over long distances eral, is likely to be far more complex than we currently and accumulate in vegetative, root and floral meristems. understand. These results proved the existence of a transport system for specific mRNA transcripts into developing apices, provid- VIRUS MOVEMENT ing evidence of a novel mechanism that could integrate developmental and physiological processes throughout the Until relatively recently, plant viruses were the only agents plant (Ruiz-Medrano, Xoconostle-Cázares & Lucas 1999). known in which a single protein was able to interact with A further example of specific mRNA trafficking has proven plasmodesmata to allow the transfer of proteins and nucleic that developmental regulation is possible using this sys- acids from one cell to another. As such, many studies on temic RNA signalling mechanism. Kim et al. (2001) showed intercellular protein movement in plants were carried out that RNA transcripts of the dominant leaf mutant from using recombinant viral MPs that were microinjected into tomato, Mouse ears (Me), could move a long distance plant cells along with a fluorescent reporter molecule (Fuji- through the phloem and cause a change in leaf morphology. wara et al. 1993; Noueiry, Lucas & Gilbertson 1994; Waig- The Me mutant transcripts were graft transmissible, local- mann et al. 1994; Ding et al. 1995). The fact that facilitated izing to the same areas of the normal scion apex as in non- protein trafficking can occur in the absence of a viral infec- grafted Me plants, and proving that the pattern of transcript tion (see above) showed that these mechanisms were likely accumulation was due to controlled trafficking of the Me to be of general relevance to plant physiology. This led to transcripts (Kim et al. 2001). This study does not confirm a debate as to whether viral MPs may have originally been that the altered morphology seen in scion tissue of the plant proteins that were ‘trapped’ by viral genomes and grafted plants was due directly to the trafficked RNA; a used to facilitate viral movement (Lucas & Gilbertson secondary signal, such as a growth regulator or hormone, 1994; see also reviews by Maule 1994 and Mezitt & Lucas might cause the morphological change, as pointed out by 1996). Although essential for plant development and func- Jackson (2001). However, this elegant study does illustrate tion, plasmodesmata represent an ‘Achilles heel’ that can the involvement of RNA trafficking in plant development. be exploited and manipulated by viruses to allow them to It now seems highly likely that plants routinely use endog- spread throughout plant tissues (Oparka & Roberts 2001). enous RNAs as long-distance signals in the phloem to inte- Local virus movement occurs via plasmodesmata, grate and regulate plant development (Jorgensen et al. whereas long-distance virus spread uses the vascular tissues 1998; Ruiz-Medrano, Xoconostle-Cázares & Lucas 2001; (Samuel 1934; Hull 1989; Maule 1991; Leisner & Turgeon Wu, Weigel & Wigge 2002). 1993; Lucas & Gilbertson 1994; Carrington et al. 1996). Post-transcriptional gene silencing (PTGS) in plants is a Most viruses utilize the phloem to spread systemically. Evi- response to foreign nucleic acids; either inserted transgenes dence for this has come from a variety of experiments or viral nucleic acids (Vaucheret et al. 1998; Voinnet, Pinto showing that movement is influenced by the flow of metab- & Baulcombe 1999). Plant gene silencing signals are also olites (Roberts 1952; Leisner & Turgeon 1993) and that thought to move through plasmodesmata; for example, when stems are ‘ringed’ or steamed to kill the phloem, virus they can enter guard cells of immature leaves, but are spread is prevented or delayed (Roberts 1952). Virus par- excluded from these symplastically isolated cells in mature ticles have also been seen in electron micrographs of tissue (Voinnet et al. 1998). A silencing response that is phloem (Price 1966; Esau, Cronshaw & Hoefert 1967). generated by introduction of ectopic DNA into a few cells However, very few studies have examined the entire path- can spread, via plasmodesmata, into neighbouring cells and way taken from the infection site to the phloem, or how subsequently the phloem, allowing systemic spread of the viruses are transported into and out of the vascular tissue signal (Voinnet et al. 1998). Palauqui & Vaucheret 1998) (Roberts et al. 1997; Nelson & van Bel 1998). showed that PTGS can spread to cells that do not express Some viruses move through plasmodesmata as intact vir- the source transgene, causing the silencing of genes that ions, causing permanent modification to plasmodesmal have homology with the short RNAs which act as signals structure. Viruses such as CaMV, Tomato spotted wilt virus (Hamilton & Baulcombe 1999; Di Serio et al. 2001). The and Cowpea mosaic virus use protein tubules, encoded by signals are thought to be short RNA species (25 nucle- viral proteins, to line the plasmodesma; allowing the virions

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 103–124 Plasmodesmata and symplastic transport 115 to pass through the tubule (Hull 1992; Storms et al. 1995). Others viruses, such as PVX, Dahlia mosaic virus and Tobacco etch virus (TEV) do not use tubules, but pass through the pore as intact virions (Kitajima & Lauritis 1969; Weintraub, Ragetli & Leung 1976; Santa Cruz et al. 1998). Another group of plant viruses, typified by TMV, cause more subtle and often transient alterations to plasmodesmata, allowing the viral genome to move as a ribonucleoprotein complex, and encode for viral MPs which modify plasmodesmata (Deom, Lapidot & Beachy 1992; Citovsky & Zambryski 1993; McLean et al. 1993; Lucas & Gilbertson 1994). The plethora of viral movement mechanisms suggest that a unified ‘strategy’ for viral movement is unlikely to occur, given the variety of viral proteins and genome organizations involved. Viral genomes can consist of either single- or double- stranded DNA or RNA. For those viruses that move as ribonucleoprotein complexes, it has been postulated that movement occurs using single-stranded genomes (Citovsky et al. 1992), which would be narrower than double-stranded complexes, therefore requiring less plasmodesmal modification. In support of this hypothesis, it was found that the MP of CaMV, a double-stranded DNA virus which replicates via an RNA intermediate, can selectively bind single-stranded nucleic acids, and binds RNA with greater efficiency than single-stranded DNA (Citovsky, Knorr & Zambryski 1991; Thomas & Maule 1995). However, more Figure 5. (A) Confocal image showing epidermal cells on a evidence will be required to validate this model and take source leaf of transgenic Nicotiana tabacum plants expressing the into account larger, circular genomes such as those pos- TMV movement protein-GFP fusion. The movement protein-GFP sessed by Geminiviruses. fusion is localized to branched plasmodesmata (green) in the walls Some viral MPs have been shown to be antigenically between the epidermal cells (imaged in false transmission; red). Bar 10 m. (B) When the TMV 30K-GFP fusion protein is related to endogenous plant proteins (Xoconostle-Cázares = m expressed in transgenic tobacco plants under the AtSUC2 compan- et al. 1999), and have provided essential tools for the study ion cell-specific promoter, the protein is only found in plasmodes- of intracellular transport. The most studied viral MP is the mata within phloem tissues. Confocal micrograph showing the 30 kDa MP of TMV. Some time ago, this protein was shown movement protein localized to plasmodesmata in the longitudinal to modify the basal SEL of plasmodesmata in infected cells walls between phloem elements (running left to right). to permit virus movement from cell to cell (Deom, Oliver Bar = 10 mm. & Beachy 1987; Wolf et al. 1989; Derrick, Barker & Oparka 1990; Waigmann et al. 1994). The TMV MP has been shown to localize to plasmodesmata (Fig. 5A; Tomenius, Clapham et al. 2000b). A second, contrasting study involving a differ- & Meshi 1987; Ding et al. 1992a) and, in common with the ent MP mutant showed that this mutant could localize to CMV 3a MP, and probably the PLRV (Prüfer et al. 1997) microtubules, but was unable to target plasmodesmata or MP, accumulates only in branched plasmodesmata (Ding move between cells, and interfered with wild-type MP et al. 1992a; Itaya et al. 1998; Hofius et al. 2001; Roberts targeting to microtubules (Kotlizky et al. 2001). However, et al. 2001). The TMV MP binds single-stranded nucleic a recent study has shown that TMV is able to replicate acids (Citovsky et al. 1990, 1992), and interacts with the ER, and move in the absence of microtubules, forcing a re- actin and microtubules (Heinlein et al. 1995; McLean, evaluation of current models for TMV movement Zupan & Zambryski 1995; Mas & Beachy 1999, 2000; (Gillespie et al. 2002). The cytoskeleton is involved in some Boyko et al. 2000a; Gillespie et al. 2002). The MP appears stages of the viral infection process, as is the ER, but to to form a complex with the viral genomic RNA, and many date, the exact mechanism for TMV movement remains to models of TMV movement implicate microtubules as the be finalized. In animal cells, actin and myosin have been cytoskeletal element responsible for transporting the MP implicated in the transport and anchoring of mRNAs to to the plasmodesmal pore (Heinlein et al. 1995; Boyko et al. specific sites within the cell, suggesting that the cytoskele- 2000b; Mas & Beachy 2000). The TMV MP has a conserved ton could also play a role in endogenous mRNA trafficking microtubule-binding motif, and mutants with an altered (Bassel & Singer 1997; Sundell & Singer 1991), and similar motif showed no interaction with microtubules, which pre- data has recently been published for the unicellular alga vented both viral infectivity and movement, although the Acetabularia acetabulum (Vogel, Grieninger & Zetsche, mutant MP continued to target plasmodesmata. (Boyko 2002).

CMV also requires the ER for infection. CMV moves as found to interact strongly with the CaMV MP, and dis- a ribonucleoprotein complex that includes a MP and viral played fluorescence resonance energy transfer (FRET) in coat protein (Canto et al. 1997; Blackman et al. 1998). Once situ. MPI7 was widely expressed throughout different tis- the complex enters sieve elements, the MP associates with sues of Arabidopsis and was localized to punctate spots at the parietal ER, and viral assembly occurs in membrane- the cytoplasmic periphery, which may be plasmodesmata. bound vesicles derived from the ER (Blackman et al. 1998). Sequence data suggests that the protein may be akin to a However, there are viruses that move without any interac- mammalian rab acceptor protein (Huang et al. 2001), a tion between the ER or cytoskeletal elements (Satoh et al. family of proteins involved in protein transport through the 2000) and viral MPs that, although essential for movement endomembrane system, and possibly associated with the do not localize to plasmodesmata (Solovyev et al. 2000). ER. More detailed studies are required to confirm whether Yeast two-hybrid and far-western screens of cDNA librar- MPI7 is indeed associated with plasmodesmata. ies have recently shown that viral MPs can also interact Biochemical purification of plasmodesmal proteins from with a number of host proteins such as putative transcrip- wall preparations has proved difficult due to the small tional co-activators that may modulate host gene expres- amounts of protein present in plasmodesmata, and the dif- sion during pathogenesis (Matsushita et al. 2001, 2002a), ficulty in obtaining pure plasmodesmal extracts that are and also proteins with homologies to myosin, kinesin and free of wall contaminants (Epel 1994; Epel et al. 1995). DnaJ-like chaperones, again suggesting an involvement of Antibodies raised to such extracts have been immunolocal- the cytoskeleton during viral infection (von Bargen et al. ized to plasmodesmata, but none of the genes encoding the 2001). proteins have yet been isolated or characterized (Epel 1994; Waigmann et al. 1997). One of the problems in identifying plasmodesmal genes with whole-plant genetic INTERACTION BETWEEN PLASMODESMAL screens is likely to be that any mutants arising will have a PROTEINS AND VIRAL MPS high chance of lethality, making screens at the embryonic The continued study of viral MPs is beginning to offer clues stage necessary. Many plasmodesmal proteins and struc- about plasmodesmal proteins that may be involved in viral tural components are probably essential for plant function intercellular transport. The TMV MP interacts with pectin and development. A potential plasmodesmal regulatory methylesterase (Dorokhov et al. 1999; Chen et al. 2000), an protein, encoded by the gene increased size exclusion 1 (ise- enzyme that modifies the pectin in plant cell walls. When 1), increased the movement of molecules between cells but this enzyme was screened for interactions with other viral was indeed lethal at the embryonic stage (Zambryski & MPs, both Turnip vein clearing virus and CaMV MPs bound Crawford 2000). to pectin methylesterase (Chen et al. 2000). A pectin methylesterase-binding domain has been identified on the PLASMODESMATA IN AND AROUND TMV MP, and deletion of this domain prevents cell–cell THE PHLOEM movement of the virus (Chen et al. 2000). Pectin methylesterase has been localized to plasmodesmata (Morvan Plasmodesmata in phloem tissue are particularly difficult to et al. 1998), and has also been shown to have RNA-binding study as they are deeply embedded within the plant. The properties (Dorokhov et al. 1999), suggesting that it is a unique pore-plasmdesma units (PPUs) that connect sieve good candidate for a MP receptor in plasmodesmata. elements and companion cells are described elsewhere in Additional genes involved in the viral movement process this issue (see review by Aart van Bel, pages 125–149 in this are being discovered in plants resistant to viral infection. issue) and will not be dealt with here. For many viruses, For instance, the Arabidopsis mutation vsm1 restricts the there is a fundamental difference between movement systemic movement of TMV and the related Tobamovirus, through plasmodesmata in mesophyll tissues and move- Turnip vein clearing virus (Lartney, Ghoshroy & Citovsky ment into and through the vascular tissue (Gilbertson & 1998), and at least three genes, RTM1, RTM2 and RTM3 Lucas 1996). There may be a control point for virus entry in Arabidopsis restrict the movement of TEV, preventing into the phloem at the junction between the vascular paren- systemic spread (Mahajan et al. 1998; Chisholm et al. 2000, chyma cells and the companion cell (Leisner & Turgeon 2001). RTM1 and RTM2 are located only in sieve elements 1993; Oparka & Turgeon 1999; Santa Cruz 1999), but our and their interaction is specific to TEV, and does not understanding of viral entry into the phloem is limited. involve a hypersensitive response or induction of systemic Once viruses enter the phloem, they are carried with the acquired resistance (Mahajan et al. 1998; Whitham et al. flow of photo-assimilate and unload in sink tissues, appar- 2000). Further analysis of such mutants may reveal if these ently without the same level of control that accompanied genes operate by preventing movement of the viral genome their entry (Roberts et al. 1997; Santa Cruz 1999). With only through plasmodesmata in the phloem. a few exceptions (Petty & Jackson 1990; Ryabov et al. A potential plasmodesmal protein was discovered 1999), additional viral proteins (most commonly the coat recently using the yeast two-hybrid system to screen for protein) are required to facilitate a systemic infection Arabidopsis proteins that interacted with the CaMV MP, a (Scholthof et al. 1995). In one recent study, domains within protein known to be necessary for movement through plas- the coat protein of African cassava mosaic geminivirus were modesmata (Huang et al. 2001). One protein, MPI7, was found to mediate nuclear import and export, as well as

© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 103–124 Plasmodesmata and symplastic transport 117 targeting GFP-fusions to the cell periphery, presumably to plasmodesmal composition, not every protein that regu- plasmodesmata (Unseld et al. 2001). Viral MPs can also lates plasmodesmal function will be discovered through this differ in their interactions with mesophyll plasmodesmata route. Some genes/proteins may interact only transiently and phloem plasmodesmata. Neither the TMV MP, nor with plasmodesmata to exert their influence, whereas oth- antibodies raised to the MP, localize to phloem plasmodes- ers may exert effects on plasmodesmal function without mata in transgenic plants expressing the MP under a con- ever locating to plasmodesmata. A case in hand is the sxd1 stitutive promoter (Ding et al. 1992a), although this MP mutant of maize (Russin et al. 1996; Botha et al. 2000) does target phloem plasmodesmata when expressed from a whose mutation leads to a downstream effect on plas- companion cell-specific promoter (K.M. Wright, personal modesmal development that is not intuitive, given its pri- comm.; Fig. 5B). It has been suggested that the TMV MP mary role in chloroplast-to-nucleus signalling (Mezitt is not required in the phloem (Blackman & Overall 2001) Provencher et al. 2001). Unravelling the pleiotropic effects but we do know that, even though plasmodesmata are not of different genes on plasmodesmal structure/function targeted by this MP in sink leaves, it is essential for viral remains a major challenge for the future. movement in sink leaves (Roberts et al. 2001). In contrast, With the onset of the functional genomics era, imagina- the MP of CMV does bind to all plasmodesmata in the tive whole-plant or tissue screens, such as that developed phloem, including those that connect sieve elements and for the ise mutants (Zambryski & Crawford 2000; Kim et al. companion cells (Blackman et al. 1998). Similarly, the 2002a), will be required to characterize the plethora of PLRV MP has been found within phloem plasmodesmata genes and proteins arising from genomics databases. The in both minor and major veins (Hofius et al. 2001). challenge for the future is not to provide further descriptive analyses of plasmodesmal structure and function, but to provide future researchers in this rapidly moving field with THE FUTURE the tools necessary to initiate the molecular dissection of Previous views of plasmodesmata as static pores in the plasmodesmata. walls between cells are no longer tenable. The current pic- ture that emerges is one in which plasmodesmata function ACKNOWLEDGMENTS as highly flexible structures that can exert considerable control of the molecules that pass through them. Although The authors are supported by grants awarded by the Scot- many published models depict simple plasmodesmata such tish Executive Environment and Rural Affairs Department as those shown in Fig. 1, it is clear that there are fundamen- (SEERAD). The authors would like to thank Kath Wright tal differences in architecture between plasmodesmata in for providing Fig. 4, and Don Fisher for discussions regard- different tissues, and at different interfaces within a single ing the calculation of Stokes radii. tissue. In the same way that extensive families of solute transporters have been discovered in recent years (for REFERENCES example over 20 types of sucrose transporter have now Allison A.V. & Shalla T.A. 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