South Afrtcan Journal of Botany 2001. 67 I • 9 Copynglll tSJ NISC Pty Ltd Prtnted m Sot~II! Afnca -All nghts reserved SOUTH AfRICAN JOURNAL Of BOTANY ISSN 0254-6299

Mini review

Regulation within the supracellular highway - plasmodesma are the key

CEJ Botha'* and RHM Cross2

' Botany Department and 2 Electron Microscopy Unit, Rhodes University, Grahamstown 6140, South Africa * Corresponding author; e-mail: [email protected]

Received 16 October 2000, accepted in revised form 19 October 2000

Plasmodesma! connections are unique, highly dynamic intercellular passages and connections. With time, intercellular structures that are lined by the plas­ these connections have evolved to allow some degree mamembrane. They are believed to be a vital intercellu­ of regulation and traffic control. This paper explores lar communication channel between living cells, linking some of the structure/function relationships in plas­ numbers of living cells into interconnected, highly spe­ modesmata. Attention is focused on the potential role of cialised cellular domains, thus enabling the plant to act the neck region of these remarkable structures and dis­ as an integrated organism. Their evolution in the higher cusses models which may explain the processes plant was inevitable. It is .accepted that cell heterogene­ involved in regulating the movement of substances ity rather than cell divergence pressurised developing from cell to cell. plant systems along a route that led to the formation of

Introduction

Plasmodesma are unique intercellular cytoplasmic commu­ based microinjection techniques have allowed these tech­ nication channels that traverse the walls between living plant niques to be applied in a number of study programmes cells. Within the limiting tubule formed by the plasmamem­ (Schulz, 1999 and references cited) including real-time brane, which traverses the common wall between living observation of injection of fluorescent probes into living cells, the endoplasmic reticulum joins the adjacent cells cells. This new dimension has provided answers to some through the plasmodesma in a highly-modified ER stru cture, important, yet fundamental questions relating to intercellular called the desmotubule. The desmotubule is separated from trafficking (see Botha and Cross 1997, Botha and Cross the inner leaflet of the plasmalemma by a space that is 2000, Botha et at. 2000). Table 1 summarises some of the termed the cytoplasmic sleeve . It is the cytoplasmic sleeve concepts associated with the structure and function of plas­ which is believed to be involved in the direct cell-to-cell traf­ modesma. ficking process. Plasmodesma have diverse functions and it seems that they are capable of trafficking a large variety of Trafficking and transport processes molecules and signalling structures, of widely differing molecular mass. We recognise that substances are able to move through The movement of substances through the transport sys­ adjacent cells, following a pathway that is entirely contained tems in plants has occupied the attention of plant scientists by living interconnected protoplasts, collectively termed the for nearly 150 years. Improvements in technologies such as symplasm. In contrast, the apoplasm, defined as that region microscopy and microinjection have allowed more refined of the plant not bounded by the plasmamembrane, also is observations on cell-to-cell communication processes, and involved with energy-free short- and long-distance transport have allowed us to focus on regulation and control mecha­ in plants. The existence of two transport systems means that nisms within the processes involved. division of labour is possible, but, at the same time, compli­ Given the strong interrelationship between plant structure cates the transport processes within large supracellular and function , It is not surprising that many researchers organisms. It cannot be disputed that the symplasmic path­ remain focussed on the leaf and. in particular, the process­ way (in this context, the phloem) is highly dependent upon es that govern uptake and transport of assimilates and relat­ the apoplasmic pathway (the xylem) for a steady supply of ed materials. These interests remain concentrated at the water, without which little if any symplasmic transport microstructural and physiological levels. Exciting advances through the complex higher plant supracellular organism made in the past decade in light and confocal microscope- would be possible. How are these two contrasting, but com- 2 Bolha and Cross

plementary pathways and specifically, their contents kept are at the heart of cell to cell traffic control and regulation separate from each other? Are there control points and if so (Marchant 1976, Lucas eta/. 1993, Bergmans eta/. 1997, how does the plant maintain a normal functional physiologi­ Jane and Chiang 1993, Eleftheriou 1993, Franceschi el a/. cal state? Clearly, the interactivities between these systems 1994, Vasil'ev 1997). Plasmodesma! permeability is an will result in a balanced and finely tuned transport system active business (Pickard and Beachy 1999) in which mes­ that can be regulated at several points. sengers such as IP3 and ca++ participate in the control of Cell to cell transport is a complexity of processes - each permeability. interacting or acting in solitude to produce a potential cas­ cade controlling mechanism. Water loss and carbohydrate Plasmodesma. meristems, domains and field transfer must be interlinked within the whole plant; other­ boundaries wise, an unorchestrated discordant system will exist. The loading of assimilates must therefore be complex, involving Convergent evolution amongst divergent taxa may well tell a series of cell-to-cell short distance transfer processes via us something about plasmodesma! evolution. Cook and plasmodesma. These are coupled with a freer apoplasmic Graham (1999) suggest that there is a clear correlation water transfer from the xylem to the cell walls and intercel­ between the ability to form plasmodesma and the ability to lular spaces of the mesophyll, as well as into the cytoplasm form meristematic tissue (van der School and Rinne 1999a). of the mesophyll and surrounding tissues. In grasses, part of Previously, Jian eta/. (1997) conducted a series of experi­ the overall ph loem loading controlling mechanism may be ments which demonstrated that there were changes in the found at the junction between the bundle sheath (BS) and ultrastructure, as well as in the subcellular localisation of the mesophyll (MS). It is at the MS- BS interface where ca++ in poplar (Populus deltoides Bartr. ex Marsh) apical many species regulate outward water loss and inward car­ bud cells during the induction of dormancy by short-day (SO) bohydrate uptake. Regulation of the outward loss of water photoperiods. These authors reported an increase in the can be undertaken by the suberin lamella (Botha and Evert number of starch granules, as well as a significant accumu­ 1986 and literature cited), which may force adoption of a lation of vacuolar storage proteins in the apical bud cells. more regulated symplasmic pathway through plasmodesma Coupled with this , they observed constriction and blockage at this interface. Internal to the bundle sheath, three alterna­ of the plasmodesma. These results suggest that under the tive phloem-loading pathways exist - along entirely sym­ influence of SO photoperiods, there are alterations in sub­ plasmic or apoplasmic pathways, or via a mixed mode sym­ cellular Ca-2+ localisation. and changes in ultrastructure of plasmic-apoplasmic pathway. Clearly, there are many plants apical bud cells during the development of dormancy. The where phloem loading occurs via a mixed pathway (refer­ constriction and blockage of plasmodesma it seems, may ences cited in Botha and Cross 1997). cause the cessation of symplastic transport, limit cellular Undoubtedly these important, yet diverse transport super­ communication and signal transduction between adjacent highways remain the subject of significant and directed cells. This in turn, may lead to even ts associated with growth research, and there are now obviously defined focus areas cessation and dormancy development in buds. which are being actively pursued in a number of laboratories Thus, complex cellular interactions, which require the worldwide. The re-focussing of ideas has come about as a exchange of morphogenetic signals, underlie the process of result of the aforementioned applica tion of advances in light morphogenesis within shoot apical meristems (Rinne and and electron micrography, particularly those using fluo­ van der School 1998, 1999b). It has been suggested that all rophores, and coupled with this, the rapid improvements in apical meristem cells are interconnected by plasmodesma confocal microscopy which have contributed a great deal to and thus could be subject to regulation, especially during the rekindling of 'old' interests and. significantly, in some new cell division cycle (Ehlers and Kollmann 1996). Rinne and discoveries (Knoblauch and van Bel 1998). van der School (1998) were able to demonstrate that two Many of the new ideas associated with intercellular traf­ concentric fields exist in the apical meristem of Betula ficking in plants are encompassed in the work published pubescence, which effectively restricts the symplasmic diffu­ recently by van Bel, Gunther and van Kesteren (1999), who sion of small morphogens to the cells within the boundaries state that there is widespread opinion that the evolution of of their specific fields. These authors determined that tran­ the multicellular plant body implicitly requires that corridors sient connections could arise between the fields, thus allow­ of communication have to become established. The authors ing transfer of potential signalling molecules between the argue convincingly that this is necessary in order to regulate fields under these conditions, and potentiating the radial and, in part, possibly control intercellular trafficking of exchange of symplasmically diffusible signalling molecules. metabolites, as well as to co-ordinate and integrate the These exciting results suggest that the apical meristem is a activities of the many hundreds of thousands of cells making compartmentalised structure and that the presence of inter­ up a typical average plant. It is now accepted dogma that cellular communication partitions could involve boundary eel/ heterogeneity rather than cell divergence would have interactions that can be demonstrated electrophysiological­ pressurised the developing plant systems along a route that ly. led to the formation of passages and connections. With time, Interestingly, tannic acid staining and immunolocalisation these connections enabled some degree of regulation and revealed that cell isolation was due to the activation of glu­ traffic control. Cell division is recognised as being an early can synthase complexes (the enzyme responsible for the pressure for the formation of the sub-microscopic intercellu­ synthesis of callose) near or at plasmodesma! orifices. lar communication channels, termed plasmodesma, which Callose plugs formed as a result, and these are associated South African Journal of Botany 2001. 67. 1-9 3

Table 1: Plasmodesma! origins, classification and transport properties

Origins and definitions Transport, functionality and regulation

Origins: Primary plasmodesma are formed during cytokinesis and Current flow. Many authors have shown that electrical potentials cell plate formation, from the fusing of Golgi-derived vesicles - may be transferred from cell to cell via plasmodesma. Transfer from thought to be a direct product of th e endoplasmic reticulum. These cell to cell is assumed to be via the cytoplasmic sleeve or via the plasmodesma are formed in transverse cell walls in pit fields or desmotubule. singly. Plasmodesma! frequencies within longitudinal walls decline as cell wall expansion is continued, due to sharing between daugh­ Trafficking- Small molecules (< 1kDa) have been shown to traf­ ter cells fic easily along diffusional gradients. These molecules are assumed to be confined to the intrinsic spaces within the cytoplasmic sleeve. Plasmodesma that undergo modification after formation, or those Transport Is diffusional, or may involve pressure now which are formed at the scion during grafting, are called secondary plasmodesma. There is evidence that plant viruses may alter the Macromolecules have been shown to be able to be trafficked structure or the primary plasmodesma. thus forming secondary under certain circumstances. Viral proteins and viral nucleic acids, plasmodesma. If they are formed de novo then they are true sec­ small proteins and mRNA may traffic. ondary plasmodesma Molecular size exclusion limits Plasmodesma may become modified with deposition of additional cell wall material (associated with the primary wall). These plas­ Evidence for plasmodesma! size exclusion limit (SEL) with raffinose modesma are modified primary plasmodesma. series sugars- the 'polymer trap' model (Turgeon and Gowan 1990. Turgeon 1991, 1996). Groups of plants transport raffinose series of Ultrastructural & molecular considerations oligosaccharldes. Low molecular mass sucrose and galactlnol pass into Intermediary cells and are synthesised into raffinose, maintain­ Diameter across outer plasmalemma membrane leaflet varies from ing the diffusion gradient. 70-120nm. Plasmalemma leaflets have been demonstrated to form a helix of Physiological Control electron-dense and electron-lucent structures. These are inter­ spersed with and connected to the central desmoiubule (confusing­ The neck region has been implicated in plasmodesma! gating (see ly referred to as 'appressed ER' in some literature) by fine spoke­ Botha et a/. 1999, and literature cited) which has been suggested to like structures. The 'space' available within the cytoplasmic annulus be ca++ enhanced (Botha et a/. 2000, and literature cited). is from 2-14nm, sufficient to accommodate small molecules. Neck region and exterior of plasmodesma have been associated Molecular size exclusion limits from 0.7 to 4.4kDa (latter by with fimbrin, and/or actin or myosin filaments. These filaments are Kempers et a/. 1993) for pore plasmodesma! units (PPU's) suggested to play a role in plasmodesma! modulation (Overall 1999). Desmotubule concomitant with ER. Regulation through chelating agents that may form soluble com­ The neck region has been demonstrated to be able to be restrict­ plexes with the divalent ca tion. Calcium has been Implicated as a ed or constricted under certain circumstances. Dilation can be messenger for a number of plant responses . it may bind to calmod­ achieved by 2-deoxy-D-glucose, callose synthesis has been shown ulin in the cytosol (Hepler and Wayne 1985). Phenothyazines and lo be enhanced in the presence of 10-40mM ca++, and to be calmidozolium can inhibit, by attaching to the c a++ binding pro­ retarded by the sequestration of ca++ via EDTA. teins.

Transport through the desmotubule would require conformational Plasmodesma! permeability has been demonstrated to be an active changes. process (Pickard and Beachy 1999, Tucker 1988). Control is thought to be via ca++ and IP3 messengers. A gating structure would narrow down the plasmodesma! orifice. This would form a bottleneck and could act as a rate-controlling Gating can effect non synchronous cell division in apical cells, step (Schulz 1999). Neck diameters when throttled back usually 20 and separation of cell domains or fields. -40nm (Overall el a/. 1982, Botha eta/. 1993, Schulz 1995).

Cytosolic gateway would be advantageous for symplasmlc solutes, as no membrane translocation step will be involved. with morphogenetic deactivation. Clearly, domain or sym­ boundaries could be set up as a gradient of effect in the ini­ plasmic field formation, and the isolation of these fields or tial cells (Rinne and van der Schoot 1998). domains within the corpus, must lead to specific cellular dif­ ferentiation steps. The involvement of callose in the activity Plasmodesma - regulated entry to the functional of sphincters in a controlled deposition of callose supports intercellular transport pathway? the evidence presented recently by Botha and Cross (2000a), in which these au thors concern themselves with Plasmodesma have been described as highly specialised the role of callose in regulating cell to cell communication gatable trans-wall channels that interconnect con tiguous and transport in plants. It follows that the mechanism con­ cells and function in direct cytoplasm-to-cytoplasm intercel­ trolling plasmodesma! narrowing or closure at the field lular transport (Epel 1994). Analysis of computer-enhanced 4 Bolha and Cross

digital electron micrographs has provided new information major enzyme under calmodulin control, namely ca++_ATP­ on plasmodesma! fine structure (Botha et at. 1993 and liter­ ase, which is concerned with pumping ca++ from the ature cited). It is now becoming clear that plasmodesma are cytosol into cell walls. dynamic quasi-organelles whose conductivity can be regu­ lated by environmental and developmental signals. New Gating, and the interaction between calcium and findings suggest that signalling and controlling mechanisms calmodulin - closing plasmodesma without cellu­ exist which allow the plasmodesmata! pore to dilate to allow lar perturbation or damage macromolecular transport. Schulz (1999) states that it is possible to envisage a control mechanism, which involves Many workers accept that the process involved in gating is the dilation of the plasmodesma. Many authors have studied considered one that allows for a degree of selectivity. The the regulation, control and functional state of plasmodesma rate-controlling step must be associated with the outer, or (Botha and Cross 2000, Botha et at. 2000 and references neck regions, of individual plasmodesma, but can be cited) and others (Holdaway-Clarka 1996, 2000, and refer­ extended to include the mid regions of plasmodesma tra­ ences cited) have been concerned with the controlling versing interfaces delimited by suberin lamellae. Here plas­ mechanisms. Transport through these microchannels and, modesma are constricted by the suberin lamella such as particularly, the regulation of pore aperture size, must influ­ that which exists at the bundle sheath-vascular parenchyma ence the transport capacity on a per plasmodesma, per cell interface in grasses (Botha et at. 1993). and whole organ basis. Schulz ( 1999) envisages this gating Tucker (1988) demonstrated that 0-myo-inositol 1,4 bis­ process to be similar to that of a lock in which, through a phosphate (IP2) and 0-myo-inositol 1,4,5 trisphosphate selective state of 'openness' or 'closedness', the passage of (IP3) could play an important role in regulatory mechanisms variously sized molecules will either be allowed or denied. in plants. Tucker (1988) co-injected IP2 with buffered 5(6) This gating model envisages plasmodesma as the regulator carboxyfluorescein. Tucker (1988) reports that IP3 is hydrol­ of intercellular molecular size exclusion (SEL) limits. This is ysed to inositol 1 ,4,5 trisphosphate and diacylgycerol (IP3- an important concept, as many researchers believe that 0G) by the enzyme phosphodiesterase. The IP3-0G second conformational changes (such as those that virons have to messenger system (Berridge and Irvine 1984, Tucker 1988) make) are necessary to allow unhindered passage of large seems to occur in many plants. Significantly, IP3-0G stimu­ proteins and virons with or without pilot molecules or chap­ lates a release of calcium, most li~ely from the ER. erones. Oiacylgycerol has also been shown to elicit a response from Olesen (1980) suggested that there was a structure akin protein kinase C. According to Tucker (1988) protein phos­ to a sphincter associated with the neck region of plasmod­ phorylation by these two kinases, evokes a cellular esma. This region has been linked to putative glucan syn­ response. The lack of CF transport between cells and other thase complexes at the neck region (Olesen 1980, Olesen evidence such as inhibition of cytoplasmic streaming (Allen and Robards 1990, Rinnie and van der School 1998), which 1987) and the stimulation of a ca++ release from vacuolar were thought to act as a crude form of sphincter. Clearly, membrane vesicles (Shumaker and Sze 1987) are strong plasmodesma play an important role in intercellular traffic evidence for an elicited cellular response from IP2 and IP3. control. Yahalom et at. (1998) recently suggested that plasmodes­ ma! conductivity may also be regulated by a phosphorylation Wounding responses - the role of calcium and mechanism. In a maize mesocotyl cell wall fraction, a ca++ other messengers? -dependent protein kinase (COPK) is present that phospho­ rylates approximately 8 of 20 wall-associated proteins. The In 1995 Apitius and Lehmann showed that cells of the liver­ kinase is membrane-associated and is not easily extracted wort Riel/a could be separated from the thallus in two differ­ using routine methodologies. Two polypeptides in the cell ent ways; firstly, by killing the surrounding cells very careful­ wall fraction, with apparent molecular masses of 51 and ly with a glass needle, without touching the isolated cells, 56kD, cross-react with an Arabidopsis COPK anti-serum and and secondly, by touching the cells with a scalpel while undergo in situ ca++ -dependent autophosphorylation on simultaneous killing the neighbouring cells. In the first case, nitrocellulose. These authors demonstrated that the molecu­ the only wound stimulation observed was the destruction of lar masses of the COPKs extracted from the cell wall fraction plasmodesma and a change in cell turgor. A thin layer of new are different from those extracted from the cell membrane wall material was seen to become attached to the old wall fraction, suggesting that wall-associated COPK is unique to within 24 hours as a wound reaction, which, within six days the cell wall fraction. Using immunoflu orescence of wounding produced a thicker new cellulosic wall. When microscopy, Yahalom et at. (1998) were able to localize the thallus was more severely damaged, callose was formed COPK to discrete punctate loci in isolated cell wall material. in the cell wall adjacent to the dead cells within 10 minutes Isolated plasmodesma challenged with COPK anti-serum of wounding. Most of the damaged cells on the other side of show a pattern of cross-reactivity similar to the cell wall frac­ the callose plugs died within hours of the appearance of the tion. This exciting data is evidence for the cell wall-associat­ relatively large callose deposits. Significantly Apitius and ed COPK being a putative plasmodesmal-associated mem­ Lehmann (1995) demonstrated that callose formation was brane protein, which may be involved in regulating plas­ dependent upon the presence of calcium ions. The require­ modesma! conductivity. The question that remains unan­ ment of this secondary signal molecule implicates theca++ swered is where exactly these COPK molecules are located. binding protein, calmodulin, which, in turn, involves the South African Journal of Botany 2001 . 67. 1-9 5

Is it at the neck region or is it in the plasmalemma cell wall an antibody against calmodulin did not label plasmodesma. complex, surrounding the neck region? Blackman et at. (1999)· and Harper eta/. (1996) suggested Lew (1994) used a voltage clamp to measure the voltage that centrin is a component of calcium-sensitive contractile dependence of cell-to-cell coupling via plasmodesma nanofilaments in the neck region of plasmodesma and between root hairs of Arabidopsis thaliana (L.) Heynh.). In somehow facilitates calcium-induced regulation of intercellu­ addition, Lew (1994) used iontophoresis to introduce a vari­ lar transport. ety of ions (Ca++ inositol-Iris phosphate, u+, K+, Mg++, eth­ Clearly, the lack of plasmodesma! association with the ylene glycol-bis(beta-aminoethyl ether)-N,N,N' ,N'-tetra­ antibody to calmodulin is strong evidence that the ca++ acetic acid (EGTA), 1 ,2-bis(2-aminophenoxy)ethane­ binding protein is not directly associated with the plasmod­ N,N,N',N'-tetraacetic acid (BAPTA), H+, and OW) to exam­ esma, and may be spatially separated from them. ine whether they regulate cell-to-cell coupling. Electrical Visualisation of immunofluorescence labelling of coupling showed high variability in this single cell type at the Arabidopsis roots has previously been limited to single cell same developmental stage; the coupling ratio ranged from layers. Using confocal microscopy, Harper eta/. (1996) were near 0% to about 90% (mean of 32%). The coupling ratio able to visualise the whole root, after antibody penetration appeared to be voltage-independent for intracellular into several cell layers. Confocal optical sectioning allowed transplasmodesmatal voltage gradients of -163 to 212mV. visualisation of the cellular arrangement of microtubules, While ca++ stimulated closure of the plasmodesma! con­ callose, calmodulin and a phosphoprotein epitope. nections (at concentrations higher than those causing ces­ Subsequently, Hold away-Clarke et at. (2000) conducted a sation of cytoplasmic streaming), inositol-trisphosphate and series of elegant experiments which persuasively showed lithium were shown to be without effect. Apparently, inositol­ that increased cytosolic ca++ levels resulted in transient trisphosphate may not cause increased concentrations of plasmodesma! closure, and that ca++cyt was capable of cytosolic ca++ to appear in root hairs. Alkalinisation by OH­ rapid change, which is in line with earlier experiments which iontophoresis caused a modest decline in cell-to-cell cou­ demonstrated that membrane depolarisation was induced pling. as did acidification by H+ iontophoresis, to an extent by cold treatment in corn coleoptiles (Nelles and Laske causing the cell fo become flaccid. Interestingly, increases in 1982) and in cucumber seedlings (Minorsky and Spanswick cytosolic K+, Mg++. and the calcium chelator, BAPTA intro­ 1989). Cold shock induces an increase in extracellular ca++ duced by iontophoresis, had no effect on cell-to-cell cou­ influx into maize roots (Zocchi and Hanson 1982) as well as pling. The regulation (and lack thereof) reported here for in alfalfa (Monroy and Dhindsa 1995). K+ (Zocchi and plant plasmodesma is quite similar to that of gap junctions. Hanson 1982) and ca++ influx across the plasmalemma It should be clear from the above discussion that the liter­ has been shown to be a major component of the cold shock ature unequivocally supports the notion that plasmodesma! response In tobacco as well as in Arabidopsis (see Knight et closure is triggered by an increase in cytosolic calcium a/. 1996). Calcium has been identified as a major factor in (Erwee and Goodwin 1993, Baron Epel eta/. 1988, Tucker the regulation of intercellular communication via plasmodes­ 1988, 1990, Tucker and Boss 1996, Holdaway-Clarka, ma Beebe and Turgeon (1991 ). Holdaway-Ciark et a!. Walker, Hepler and Overall 2000). It becomes tempting to (2000) state that although cold produced on average, a two­ suggest that cytosolic calcium concentration, its various fold increase in cytosolic ca++(expressed as [Ca++l Cy1 ) in interactions with calmodulin and ca++ATP-ase, and related corn suspension culture cells, ca++ concentration could be to this calmodulin activity, (Harper et a/. 1996, Botha et a/. influenced by leakage across the plasmamembrane. It is 2000 and references cited) and I P3, must have an important interesting to speculate that here perhaps is a reversal role role in selective gating capacity of this important cytoplasmic for the recognised role of ca++ ATP-ase, actively pumping gateway. ca++ into the cytosol. Interestingly, Holdaway-Ciark et at. Whilst there are many papers that deal with the interaction (2000) also suggest that increases in [Ca++l cy1 at either of calcium with calmodulin in the literature, there are less physiological or non-physiological levels, can influence than 30 that deal with the interrelationship of plasmodesma opening or closing of the plasmodesma in the corn suspen­ with calcium in plant species, and only two relate cytosolic sion cultures. calcium concentration to calmodulin and plasmodesma! gat­ Actin, myosin and centrin have been localised to higher ing directly (Blackman et at. 1999, Harper et a/. 1996). plant plasmodesma (see White et at. 1994, Radford and Blackman eta/. (1999) demonstrated that antibodies against White 1998, Blackman et at. 1999). The actomyacin interac­ centrin, the ubiquitous calcium-binding contractile protein, tion and contraction of centrin nanofilaments have been recognised a 17k0a protein in extracts of onion root tips and shown to be c a++_dependent (Martindale and Salisbury cauliflower florets. Anti-centrin antibodies have been 1990). However, Holdaway-Clarka et at. (2000) are quite localised to the developing cell plate of onion and cauliflower convinced that the fast response times recorded during their root tip cells using immunofluorescence microscopy. In cau­ experiments (recorded as changes in membrane potential liflower florets, these antibodies localised to the walls in a (PD) to injected ca++) means that callose is not involved in punctate manner, consistent with the distribution of plas­ effecting closure of plasmodesma, as the process is, accord­ modesma, as shown by colocalisation with callose. Anti-cen­ ing to these authors. one which may take hours rather than trin antibodies were also localised to plasmodesma of onion seconds (Drake et a/. 1978). This observation contrasts root tips and cauliflower florets with immunogold electron strongly with a supportive statement by Overall {1999) who, microscopy, where this label was shown to be concentrated citing Olesen and Robards (1990) stated that callose could around the neck regions of the plasmodesma. In contrast, 6 Botha and Cross

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Figures 1-4: Aspects of the structure of Tradescantia pa/lida stamina! hair cells and associated plasmodesma! fields between concomitant hair filament cells; Figures 1-2: Epifluorescence micrographs, after staimng in 0.05% aniline blue and viewed using an epinuorescence micro­ scope fitted with a Chroma #11 003 Blue/Violet filter set (Chroma Technology Corp., Brattleboro VT USA); Figure 1: Basal attachment point of one stamina! hair. Note the brightly-fluorescing cross wall (arrows); Figure 2: Higher magnification view showing two cross walls in a stami­ na! hair filament. Regions of bright punctate fluorescence are individual plasmodesma; Figures 3-4: Aspects of the ultrastructure of plas­ modesma between concomitant cross walls In a single stamina! hair. Note evidence of some damage (in cell above, Figure 3, and in cell at right, Figure 4). Individual plasmodesma are either simple or branched. Electron-dense material co-exists withER and other globular materi­ al. Note globular material associated with the neck region of the plasmodesma (arrows) shown at high magnification in Figure 3. Scale bars: 1 =500 JJm, 2 =250 JJm . 3 =20 0nm, 4 = 100nm act as a crude sphincter mechanism and that callose was could result in the prevention of normal cell wall separation deposited very rapidly but takes hours to disappear. in regions containing plasmodesma by protecting the sur­ Van Bel et a/. (1999) suggested that there could be sever­ rounding wall surrounding the plasmodesma from hydrolytic al forms of gating involved, and that plasmodesma! closure enzyme breakdown during ripening (Roy et at. 1997). may be a strongly energetic process and that it would be Similar wall structures have been observed in barley aleu­ acceptable to regard the sealing of plasmodesma as a rone plasmodesma (Taiz and Jones 1973). However, Overall potential wounding response reaction. which is effected to (1999) states that it remains to be seen just how the pectin prevent the loss of cytoplasmic material. sleeve either modifies, or plays a role in altering the func­ In addition to callose, pectins seem to be implicated in tional dimensions of plasmodesma. some way to callose deposition at plasmodesma, having So we have a number of intriguing possibilities associated been involved in plasmodesma in tomato pericarp cells with plasmodesma! function. Unequivocal evidence exists (Cassero and Knox 1995). Low esterified pectin has been for a central role for the secondary messenger ca++ and located around the plasmodesma in ripening apples but is coupled to this, calmodulin. Many supportive role players apparently not involved in calcium cross-bridging. Pectins like actin, centrin, fimbrin IP3 and myosin clearly are South Afncan Journal or Botany 2001, 67 1·9 7

involved. Time will tell which are the important ones and and other related questions can lead to significant answers which are not. to what could otherwise be described as a purely academic study. Plasmodesma! studies can provide more information Plasmodesma in action about the as yet, poorly understood phenomenon of zone regulation which was highlighted recently by van der School We have chosen to illustrate this review with one example, and Rinne (1999a,b) who demonstrated that plasmodesma Tradescantia pal/Ida (Rose) DR Hunt, which we are current­ can create regulating zones within the shoot apex, for exam­ ly using to model the effects of changing the plasmodesma! ple. Plasmodesma! gateability has become an area of great microenvironment (e.g. changes in the [Ca++ cytl : osmolari­ interest, as it is the relative degree of openness or closed­ ty, effect of adding chelating agents and studying the result­ ness that governs intercellular trafficking. It is the trafficked ant intercellular transport), using electrophysiological and signals which allow the formation of domains within the reverse current microiontophoresis. Our work also involves shoot and root apical meristems and which control synchro­ examination of the structural characteristics and appearance nous and asynchronous cell division within these complex of plasmodesma. regions of the vegetative plant. Whilst is clear that these Figures 1-4 show aspects of the functionality (Figures 1-2) minute but complex structures are under the control of many and ultrastructure of Tradescantia pal/ida stamina! hair cells, interactives. such as the secondary messengers ca++ and and their associated plasmodesma! fields. Figure 1 shows IP3, the elicited responses result in gating, regulation and part of an attached basal cell and the lowermost stamina! control of intercellular transport. Clearly, the complexity of hair filament cells, stained in 0.05% (w/v) aniline blue in form, and the functional parameters associated with this water, and viewed under blue light, using an epifluorescence form, and the interactions of these components, must be microscope. The brig ht fluorescence band is associated with studied to provide answers about regulation and functionali­ large plasmodesma! fields. At higher magnification (Figure ty. Until the questions listed above are answered, plasmod­ 2) the callose reaction is localised as punctate spots, each esma will remain incompletely understood structures. of which is an Individual aniline bl ue-induced callose region. Perhaps when we understand these structures, we will be When viewed with the TEM (Figures 3-4), these plasmodes­ able to apply some of the answers in a more practical way. ma show a variety of form - some are simple and Pickard and Beachy (1999) so succinctly summed this up by unbranched and others are bra nched. The cell to the left in sta ting that by understanding plasmodesma! form and func­ Figure 4 (and at higher magnification, below, in Figure 3) tion, we may get new insights into the control of viral move­ shows a relatively unperturbed state compared with the cell ment and secondly, if we can control the flow of nitrogen-rich to the right in Figure 4 (and at higher magnification, above in compounds, we co uld effect a significant increase in yield. Figure 3) where large aggregations of ER are interspersed Clearly, the future of plasmodesma! research will continue with membranous and fibrillar material. Some plasmodesma to demand microscopy techniques, coupled with microinjec­ (arrows, Figure 3) are associated with electron-dense mate­ tion and, specifically, investigation of in situ experiments rial, which seems to form a collar surrounding the plasmod­ showing the effects of additions to, or manipulations of, the esma. cytosol. These can only be accomplished and visualised with real-time digital fluorescence and confocal microscopy. Conclusions Understanding plasmodesma! function remains the key.

Plasmodesma clearly are important structures that we now Acknowledgements - Research in the Authors laboratory is sup­ understand to be involved in many more fun ctions than sim­ ported by a grant from The National Research Foundation (NRF ply providing the channels for intercellular symplasmic traf­ GUN2034568) and the Rhodes University Joint research ficking of sugars and other energy sources. Clearly. th ey are Committee. involved in intercellular communication, and are implicated in the communication of incompletely understood signalling References agen ts. Whilst some would argue that we already know enough about the role of plasmodesma in cellular regulation Allen NS (1987) Intercellular particle movements and cytoplasmic streaming in Acetabularia cells and cytoplasts. Plant Physiology and intercellular trafficking processes, it is clear to us th at supplement 105 (abstract) not all the questions have been answered yet. There remain Apitius A, Lehmann H (1995) The deposition of different wall mate­ many unanswered questions pertaining to the functional rials as a wound reaction in the liveiWort Riel/a helicophyl/a. state of plasmodesma, and how the functional state of plas­ Cryptogamic Botany 4: 351 -359 modesma is maintained. Whilst there is evidence for a role Baron-Epel 0. Hernandez D. Jiang LW, Meiners S, Schindler M for ca+\yt and its interaction with calmodulin, there is no (1998) Dynamic continuity of cytoplasmic and membrane com­ direct evidence of the actual plasmodesma I gating stimulant. partments between plant cells. Journal of Cell Biology 106: 715- If it is ca++ then what levels of cytosolic calcium [Ca++]cyt 721 are required? How do ca++ amplification second messen­ Beebe DU, Turgeon R {1991) Current perspectives on plasmodes­ gers effect their roles in callose synthesis? Where are the mata structure and function. Physlologia Plantarum 83: 194-199 Bergmans ACJ, De Boer AD, Derksen JWM, van der School Cl effectors or locators for CDPK's situated? What is the role of (1997) The symplasmlc coupling of 1-2-cells diminishes in early changing osmotic potential? floral development of iris. Planta 203: 245-252 We have chosen to highlight a few questions and have Berridge MJ. Irvine RF (1984) Inositol-triphosphate, a novel mes­ reviewed only part of the literature, but. we believe, these senger In cellular signal transduction. Nature 312: 315-320 8 Bolha and Cross

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