INTRODUCTION

Plasmodesmata are cytoplasmic channels that traverse plantcell walls, linking protoplasts into a symplastic continuum (forreviews see Beebe and Turgeon, 1991; Lucas et al., 1993;Oparka, 1993; Robards and Lucas, 1990). Thus they facilitatesymplastic movement of nutrients and metabolites throughoutthe plant. Plasmodesmata are generally considered to offer freepassage to soluble molecules of less than about 1000 Da (Terryand Robards, 1987), but evidence is accumulating for both celltype- and tissue-specific variations in permeability (Kemperset al., 1993), and for the regulation of pore size in response toa variety of stimuli, including pressure differentials (Oparkaand Prior, 1992) and chemical signals such as Ca2+ and inositolphosphates (Erwee and Goodwin, 1983; Tucker, 1988, 1990).The existence of regulated permeability (or ‘gating’) clearlysuggests a potential role for plasmodesmata in controllinggrowth and development, by controlling the traffic of sig-nalling molecules between cells. The propagation of electricalsignals, with a potential role in wound response (Wildon et al.,1992), also occurs via plasmodesmata. In addition, plasmod-esmata are of considerable interest as the routes for cell-cellmovement of plant viruses, in the course of which sizeexclusion limits are increased under the influence of viralmovement proteins (reviewed by Citovsky, 1993; Maule,1991).

The small size of plasmodesmata (typically 40 nm diameterin angiosperms; Robards, 1976) and their inaccessibility withinthe wall has slowed progress in determining details of theirstructure and composition. The currently accepted model of

their structure was established from conventional transmissionelectron microscopy studies (López-Sáez et al., 1966; Overallet al., 1982; reviewed by Robards and Lucas, 1990). In itssimplest form this model describes the plasmodesma as aplasma membrane-lined hole through the cell wall; within thislies an axial rod or tube known as the desmotubule or appressedendoplasmic reticulum (ER), which is an extension from ERon either side of the wall. Between plasma membrane anddesmotubule is a sleeve of cytoplasm through which thepassage of soluble substances is thought to occur. Ill-definedparticles within the sleeve have been reported by severalauthors (e.g. Overall et al., 1982; Thomson and Platt-Aloia,1985; for reviews see Olesen and Robards, 1990; Robards andLucas, 1990).

This model has been refined by Ding et al. (1992), who usedhigh-pressure freezing to preserve fine details of structure intobacco plasmodesmata. This study showed regularly arrangedparticulate structures embedded in both desmotubule andplasma membrane leaflets lining the cytoplasmic sleeve. Theparticles extend into the cytoplasm, partially occluding thechannel. It has been speculated that conformational changes inthese particles might account for gating of the aqueous plas-modesmal channel (Citovsky, 1993; Deom et al., 1992).

Structural specialisation has also been observed outside theplasma membrane, within the cell wall itself. Willison (1976)imaged the surface of the wall using freeze-fracture techniquesand showed the presence of a raised collar region surroundingthe plasmodesmal orifice. Olesen (1979) used tannic acidstaining to highlight regularly spaced particles within the cellwall around plasmodesmata in

Salsola. It was suggested that

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A procedure is described for obtaining clean maize cell wallpreparations that contain embedded plasmodesmata.Negative staining and rotary shadowing have been usedwith transmission electron microscopy to visualise the plas-modesmata in these isolated walls, and to assess the effectsof simple biochemical treatments on plasmodesmal com-ponents. Light protease treatment removes material fromthe exposed ends of plasmodesmata but does not extract theplasmodesmal core, which lies within the cell wall.However, heavy proteolysis occasionally removes thecomplete plasmodesma, including its enclosing collarstructure, from the wall. Extraction with urea has a similareffect. The collar itself appears not to be proteinaceous incomposition, although protein may bind it into the wall.

Callose is localised in the wall around plasmodesmata, butdoes not appear to be a constituent of the collar. Themembrane components of the plasmodesma (plasmamembrane and desmotubule) can be extracted withmembrane-solubilising detergents. This treatment releasesfrom the wall a small number of proteins that are regardedas being potentially of plasmodesmal origin. These resultsshow that plasmodesmata from maize can be dissected bio-chemically and suggest a strategy for the characterisationof individual molecular components.

Key words: plasmodesmata, cell wall, protein, callose, transmissionelectron microscopy

SUMMARY

Plasmodesmata of maize root tips: structure and composition

Adrian Turner, Brian Wells and Keith Roberts

Department of Cell Biology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK

Journal of Cell Science 107, 3351-3361 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

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these might form a sphincter-like structure, largely composedof callose, which could function in regulating the size of theplasmodesmal aperture (Olesen and Robards, 1990). The closespatial association between plasmodesmata and sites of callosedeposition has been demonstrated immunologically (Northcoteet al., 1989), and using the callose-specific aniline blue fluo-rochrome (Currier, 1957; Olesen, 1986).

Biochemical data that might illuminate details of plasmod-esmal structure and function have been elusive. Labelled viralmovement proteins have been used to probe for plasmodesmalcomponents, but without reported success to date. Conven-tional cell wall preparations were used by Monzer and Kloth(1991) to enrich for potential plasmodesmal proteins fromSolanum nigrum. Proteins of 28 and 43 kDa were extractedfrom these preparations using detergent but could not be iden-tified unambiguously as plasmodesma-associated. Kotlizky etal. (1992) used carefully prepared clean cell walls frometiolated maize mesocotyl as a source for wall-associatedproteins. A small group of proteins ranging from 17 to 80 kDawas enriched in these preparations, and these were consideredto be potentially associated with plasmodesmata.

Antibodies against animal gap junction protein (connexin)have been reported to recognise an endogenous polypeptide inboth maize and Arabidopsis, which in the case of maize islocalised to plasmodesmata (Yahalom et al., 1991). However,recent work indicates that the putative Arabidopsis connexinhomologue (Meiners et al., 1991) is more likely to be a proteinkinase-related protein (Mushegian and Koonin, 1993).

An alternative biochemical approach adopted by Tilney etal. (1991) was to use simple extraction methods for a moleculardissection of plasmodesmata in the fern gametophyte. By usingproteases and membrane-solubilising detergents these authorsfound that plasma membrane and desmotubule could be inde-pendently extracted. Their results suggested that the desmo-tubule is principally composed of protein, and may have acytoskeletal function, determining and stabilising plasmodes-mal morphology.

Here we adopt a similar approach to Tilney and colleaguesto study plasmodesmata of the maize root tip. We also reporta procedure for obtaining cell wall preparations rich in plas-modesmata, and adapt methods of negative staining and rotaryshadowing to study plasmodesmal structure with the electronmicroscope.

MATERIALS AND METHODS

Plant materialSeeds of maize (Zea mays cv Irla; Pioneer Hi-Bred) were surface ster-ilised with 10% ‘Domestos’ bleach and germinated on damp tissues.

Wall preparationRoot apices were excised from emerging roots 2 mm from the tip andplaced in 3 M sorbitol for 10 minutes at 4°C to plasmolyse cells. Tipswere drained, then snap frozen and stored in liquid nitrogen. Collectedtips from about 2000 roots were ground to powder in liquid nitrogenusing a mortar and pestle, then thawed into 30 ml of wall preparationbuffer (100 mM Tris-HCl, pH 8.0, 100 mM KCl, 10% (v/v) glycerol,10 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, and aprotease inhibitor cocktail of 1 mM phenylmethylsulphonyl fluoride(PMSF), 1 mM benzamide, 1

µg/ml leupeptin, 5 mM ε-amino-n-caproic acid and 1 mM p-amino benzamidine). The preparation wasspun at 400

g for 5 minutes and the pellet resuspended in 30 ml wall

preparation buffer without PMSF and benzamide (incomplete buffer).The suspension was passed three times through a French pressure cellusing 13 MPa maximum pressure and minimal outlet aperture.Shearing forces generated by the pressure cell rupture cells thatremain intact and strip cytoplasm from cell walls. The walls were thenwashed clean by sequential centrifugation and resuspension in incom-plete buffer. To ensure that walls were sedimented while subcellularcontaminants were discarded with the supernatants, the spins followeda schedule of 400 g for 5 minutes, 260 g for 3 minutes (twice), then115 g for 3 minutes (twice). Upon resuspension in fresh buffer aftereach spin the material was homogenised for 20 strokes in a glass-on-glass homogeniser. Walls were finally examined by phase-contrastmicroscopy, pelleted at 1100 g, and either processed further (seebelow) or stored at −20°C.

Negative staining for transmission electron microscopy(TEM)A 1-2 µl sample of wall suspension was mixed on a pyroxylin-filmedelectron microscope grid (200 mesh; Agar) with an equal volume of2% (w/v) uranyl acetate. After a few seconds the grid was carefullyblotted to remove all but a thin layer of stain, and allowed to air dry.

Rotary shadowing for TEMPelleted walls were resuspended in 50% (v/v) glycerol andammonium formate, pH 6.8, was added to 135 mM. The suspensionwas mixed thoroughly and a 75 µl sample was sprayed onto a pieceof freshly cleaved mica according to the method of Shotton et al.(1979). The mica was either plunged immediately into nitrogen slushand stored in liquid nitrogen, or else dried overnight under vacuum.Frozen samples were transferred to a Balzers rotary shadowing unitand etched at −100°C under high vacuum for 2-3 hours. They werethen rotary shadowed at a 20° angle with platinum/palladium andcarbon (Heuser, 1981), and replicas were recovered by floating ontowater. Dried samples were rotary shadowed at a 10° angle directly inthe vacuum chamber. All specimens were viewed in a Jeol 1200 EXTEM. Replicas were photographed as stereo pairs (20° tilt) andimages were printed in reverse contrast.

Embedding and sectioning for TEMWalls were fixed in 2% glutaraldehyde, pH 7, washed with distilledwater and embedded in 0.5% (w/v) low melting point agarose. Thiswas refixed in glutaraldehyde, sliced into 1 mm cubes, washed indistilled water, then incubated in 2% OsO4 for 1-3 hours on ice. Thesamples were rinsed in distilled water, dehydrated through a gradedethanol series on ice and embedded in LR White medium grade resinat room temperature. Resin was heat polymerised overnight at 60°C,sectioned at 0.1-0.2 µm, and stained with 2% uranyl acetate and leadcitrate prior to viewing in the TEM.

For frozen sections, root tips were fixed overnight in 4%paraformaldehyde at pH 6.9, infiltrated with 1.5 M sucrose and 0.5%paraformaldehyde over 3 days, then frozen in liquid ethane andsectioned at 0.1 µm at −110°C.

Detergent extractionWalls were resuspended in 2% (w/v) sodium dodecyl sulphate (SDS),Triton X-100 (TX-100), or 3-[(3-cholamidopropyl) dimethyl-ammonio] 1-propane-sulphonate (CHAPS), and incubated at 4°C withagitation for various times. Walls were then washed by repeatedpelleting and resuspension in distilled water, and prepared for electronmicroscopy or protein extraction as described.

Protease treatmentHeavy proteolysis: walls were resuspended in 1 mg/ml Pronase E(Sigma) and 100 µg/ml proteinase K (Sigma) in 10 mM Tris-HCl, pH7.8, 1 mM CaCl2, 0.5% (w/v) SDS and incubated at 37°C for 4 hourswith agitation, then washed in distilled water as above. Light prote-olysis: walls were resuspended in 50 mM (NH4)2HCO3, pH 8.0, 1 mM

A. Turner, B. Wells and K. Roberts

3353Extraction of plasmodesmata

CaCl2 containing 10 µg/ml trypsin or chymotrypsin (Sigma) andincubated at room temperature for 4 hours. Controls were incubatedin buffer without enzyme. Where required for SDS-polyacrylamidegel electrophoresis (PAGE), supernatants were recovered frompelleted walls, brought to 1× Laemmli (1970) sample buffer (SB) andimmediately boiled for 3 minutes before being loaded onto gels.

Glucanase treatmentWall fragments were incubated in 20 units/ml exo-β(1→3) glucanase(Megazyme, North Rocks, Australia) in 50 mM citrate buffer, pH 4.0,for 5 hours at 10°C with agitation, then washed in distilled water.Controls were incubated in buffer without enzyme.

Immunogold labellingWashed wall fragments were pipetted onto pyroxylin-filmed gold EMgrids and allowed to air dry. The grids were floated on drops ofblocking buffer (phosphate-buffered saline (PBS) containing 10%(v/v) sheep serum and 4% (w/v) bovine serum albumin) for 15minutes, then on drops of PBS containing 1% (v/v) sheep serum(PBSS) and a 1 in 50 dilution of monoclonal mouse anti-β(1→3)glucan antibody (Biosupplies, Parkville, Australia) for 1 hour at roomtemperature. Grids were washed 3× 5 minutes on PBSS, then trans-ferred to PBSS containing a 1 in 30 dilution of 10 nm gold-conju-gated goat anti-mouse antibodies (BioCell) for a further 45 minutes.After 3× 5 minute washes in distilled water the grids were negativestained as described above. Grids that received either no primary orno secondary antibody served as controls.

SDS-PAGEPelleted wall fragments were resuspended in 1× SB and boiled for 10minutes. In some cases sample buffer was used with the addition of10 M urea. Supernatants were boiled for 3 minutes in an equal volumeof 2× SB. SDS-PAGE gels (10% acrylamide) were prepared and runaccording to Laemmli (1970). Gels were stained by the ammoniacalsilver method of Oakley et al. (1980).

RESULTS

Cell wall fragments containing numerous embedded plasmod-esmata can be prepared rapidly from maize root tips by usingthe procedure described in Materials and Methods. Theresulting wall fragments appear to be largely free from conta-minating material. Virtually no cells remain intact as deter-mined by light microscopy (Fig. 1A), while at the electronmicroscope level plasmodesmata are evident embedded in thewall (Fig. 1B). Plasmodesmata appear to have snapped offfrom the plasma membrane and cytoplasm near the wallsurface.

Dried, rotary-shadowed replicas provide a means of viewingplasmodesmata within the cell wall in detail. This techniqueconfirms that maize root tips are a rich source of plasmodes-mata, with frequencies in excess of 20 per µm2 often observedin transverse walls (Fig. 1C); similar values were reported byJuniper and Barlow (1969) using conventional thin-sectionedmaterial. In longitudinal walls, plasmodesmata are lessprevalent with increasing distance from the root tip, but arelocally abundant where gathered together in primary pit fields(Fig. 1D).

In conventionally prepared thin sections of isolated walls, aregion of low electron opacity surrounds the osmium-stainedmembranes of the plasmodesmal core (Fig. 2A). This core isabsent in some instances, suggesting that it has been lost eitherduring extraction, or possibly in the course of wall develop-

ment. (Corresponding replicas are shown in Fig. 2E,G.) Thepale collar region resembles the ‘sphincter’ of amorphousmaterial described by Olesen and Robards (1990), and corre-sponds to the collar seen by Willison (1976) in freeze-fracturepreparations. The present structure appears to traverse the wall,enclosing the plasma membrane tube in a continuous collar,and may project slightly at the ends from the wall proper.

The plasmodesmata shown in Fig. 2A have been sectionedtranversely after osmication and embedding. Negative staining(Brenner and Horne, 1959) was adopted as a quicker methodthat also offers potentially higher resolution of structural detailin wall fragments (Fig. 2B-D). In some cases this techniqueagain revealed the collar as a pale-staining raised lip of about80 nm diameter around a conventional concentric arrangementof plasma membrane, cytoplasmic sleeve and axial component(desmotubule) of 40 nm diameter (Fig. 2B,C). In otherinstances the stain appeared to penetrate within the plasmod-esma to reveal internal structure (Fig. 2D) and emphasise thesleeve-like form of the channel. It is not clear whether theregion of plasmodesma revealed in this way runs right throughthe wall, or is largely the tail region exposed outside the wall.

In Fig. 2D, regularly repeated structures within the cyto-plasmic sleeve are sometimes visible. These structures appearto be associated with the desmotubule, and may correspond toelectron-lucent particles previously reported in thin-sectionedmaterial (Olesen, 1979; Overall et al., 1982). Ding et al. (1992)saw similar features in cryofixed Nicotiana plasmodesmata,although the electron-lucent regions were here interpreted asgaps between electron-opaque particles. It should be empha-sised that, in the present work, the material is dried after thestain is applied, and thus structural features may be distorted.However, the sizes of the different components correspondclosely to those seen in conventionally prepared material (Fig.2A), indicating that structural details are at least broadlyretained. Dried replicas give comparable dimensions (Fig. 2E),although here again drying introduces distortion, as theexposed membranous tail collapses onto the collar and the twobecome indistinguishable.

Replicas of fast-frozen material reveal that the amorphouscollar contains a simple cylindrical structure in the centre, pre-sumably the exposed end of the plasma membrane tube (Fig.2F, absent in Fig. 2G). In frozen preparations the collar doesnot project above the wall surface as seen in dried material,suggesting that in the latter case the drying causes collapse ofthe fibrous wall matrix around the comparatively rigid plas-modesmal collar.

The techniques described above, while illustrating thegeneral structure of plasmodesmata and their integration intothe wall, do not further resolve fine structural organisation,neither do they identify components at the molecular level.Therefore we performed a variety of extraction procedures toattempt a sequential disassembly of the plasmodesma.

Extraction with detergentsWall fragments were incubated in 2% SDS at 4°C for 2 hours.Negative staining of the extracted walls revealed depressionsin the centres of the raised collar regions (Fig. 3A); the con-centric series of membranes seen in Fig. 2B was no longerapparent. The central region at the exposed end of each plas-modesma thus appeared to have been removed by thetreatment. Sectioning and osmium staining of material treated

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with SDS confirmed that membranous plasmodesmal materialwithin the cell wall is successfully extracted by this method.An identical effect occurs if Laemmli sample buffer is usedinstead of SDS (Fig. 3B).

When walls were extracted with 2% Triton X-100 at 4°C,a similar appearance to that seen with cold SDS resulted (Fig.

3C,D). Collar regions remained intact, but internal structurewas removed. Similar results were achieved using CHAPS,another efficient membrane-solubilising detergent. In someinstances a small remnant of material, of uncertain origin,could be observed within the axial region by negativestaining. However, embedding and sectioning of the extracted

A. Turner, B. Wells and K. Roberts

Fig. 1. Preparation of wall fragments containing embedded plasmodesmata. (A) Phase-contrast light micrograph of wall fragments at thecompletion of the preparation procedure. Virtually all cells are broken and cytoplasmic contamination is minimal. Arrows indicate primary pitfields in pieces of longitudinal wall. Bar, 20 µm. (B) Transmission electron micrograph of a fragment of maize cell wall that has beenosmicated, embedded and sectioned. In this relatively thick (~200 nm) section the membrane cores (plasma membrane and desmotubule) of thenumerous plasmodesmata stain darkly, and project from either side of the wall. They are surrounded by electron-lucent collar regions(arrowheads). (C, D) Platinum/palladium replicas of dried wall fragments viewed by TEM. (C) Numerous, randomly spread plasmodesmata arepresent within the wall; a few are indicated by arrows. The random orientation of the cellulose microfibrils indicates that this is a fragment oftransverse wall. (D) Longitudinal wall with oriented cellulose microfibrils in which plasmodesmata are clustered together in primary pit fields.The exposed ends of the plasmodesmata are rather structureless as a result of air drying, so the tubular arrangement of membranes is obscured.Bars (B-D), 200 nm.

3355Extraction of plasmodesmata

Fig. 2. Visualisation of plasmodesmata in cell wall fragments. (A) A relatively thick (~200 nm) section through a fragment of osmicated cellwall, showing dark membranous plasmodesmal cores surrounded by electron-lucent collars. The arrow indicates a plasmodesma in which thecore appears to have been lost. (B-D) Negative staining of plasmodesmata. (B,C) Electron-opaque stain accumulates in depressions to highlighta raised outer lip around plasmodesmata (arrowheads), corresponding in position to the collar of sectioned material. Within this, concentricrings correspond to plasma membrane and desmotubule remnants (arrows). (D) The plasmodesmata appear to have snapped off from thecytoplasm a short distance from the wall surface. This has left a ‘tail’ emerging from each embedded plasmodesma. Negative stain haspenetrated inside this fragment to reveal the permeable sleeve between plasma membrane (outside) and desmotubule (inside). The stainingreveals a mixture of face views (long arrows) and longitudinal views (short arrows). In some of the latter, regularly spaced substructures areapparent along the desmotubule (arrowheads). Corresponding structures are just visible in the plasmodesmata revealed in face (transverse)view. (E) Replica of dried wall fragment showing formless plasmodesmal material exposed at the wall surface. One collar appears to have lostits core (arrow). (F, G) Replicas of fast-frozen plasmodesmata. Rapid freezing followed by etching under vacuum preserves better the structureof the plasmodesma in replica, although again the core region may be lost occasionally (G; arrow). This method emphasises the porous natureof the polysaccharide network of the wall. Bar, 200 nm.

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walls again revealed that no membrane remained in plas-modesmata, as indicated by absence of OsO4 staining (notshown).

Detergent extracts from purified wall fragments wereanalysed by SDS-PAGE. Boiling walls in gel sample buffercontaining SDS yielded a moderately complex and highlyreproducible profile of protein bands (Fig. 4, lane 2). Cold SDSalone released a similar set of proteins (not shown). In contrast,TX-100 and CHAPS extracts both contained a relatively smallnumber of bands (Fig. 4, lane 3), all of which were also foundin the SDS extract. Doublets at about 80, 100 and 130 kDa

were seen consistently, and therefore represent candidate plas-modesmal proteins, which are currently under further investi-gation. Bands at 40 kDa (visible in Fig. 4) and 62 kDa (notvisible) were less reproducible and may represent occasionalcontamination with cytoplasmic membrane-associatedproteins.

Treatment with ureaWall fragments were boiled for 10 minutes in sample buffercontaining 10 M urea in an effort to extract further plasmod-esmal material, with mixed results. In many cases, extracted

A. Turner, B. Wells and K. Roberts

Fig. 3. Extraction of plasmodesmata with detergent. (A,B) SDS-extracted plasmodesmata. (A) Walls were incubated with 2% SDS for 2 hoursat 4°C. Negative stain reveals that the detergent has removed material from the core of the plasmodesma, leaving a stain-filled depression in thecentre of each raised collar region (arrows). (B) Sectioned and osmicated plasmodesmata after extraction in SDS sample buffer. Walls wereboiled for 10 minutes in SDS-PAGE sample buffer, washed and prepared for conventional transmission electron microscopy. The absence ofelectron-opaque material within the plasmodesmata indicates that osmiophilic lipid membrane has been extracted by this treament, although theformer sites of plasmodesmata are still apparent as electron-lucent regions (arrows). (C,D) Triton X-100-extracted plasmodesmata. Isolatedwalls were incubated in 2% Triton X-100 for 2 hours at 4°C, washed and negative stained (C) or rotary shadowed (D). Annular structureswithin the plasmodesmata have been removed by the treatment, leaving a depression in the centre of the collar. Bar, 200 nm.

3357Extraction of plasmodesmata

material viewed by electron microscopy appeared similar towalls boiled with sample buffer without urea. However, onsome occasions examples were observed in which the plas-modesmata had disappeared entirely from the wall (Fig. 5A).The membranous core and the surrounding collar or sphincterhad disappeared and a well-defined cylindrical hole was leftin the cell wall, of similar diameter (60-80 nm) to the collarthat surrounds unextracted plasmodesmata. However, PAGEgels of urea extracts revealed no protein bands not alreadyseen in standard protein extracts, and no abnormal propor-

tions of any protein. Thus the substantial collar structure isunlikely to be of proteinaceous composition, a conclusionsupported by its staining properties in conventional TEMpreparations.

Treatment with proteaseIn view of the above, it is perhaps surprising that heavy pro-teolysis of wall fragments also led to the extraction of completeplasmodesmata together with their associated collars from aproportion of the wall fragments imaged (Fig. 5B). In suchcases, which included both randomly spread and clusteredplasmodesmata, a clean and well-defined hole of approxi-mately 60-80 nm diameter resulted, which appeared to runthrough the width of the wall. This was most obvious in occa-sional examples of negative staining where the wall appearedto be partially detached from the plastic film of the TEM grid;here stain did not accumulate in the holes, which thereforeappeared pale (Fig. 5C).

In cases where the collar was not removed by heavy prote-olysis, both exposed and internal membranes of the core weregenerally absent (not shown), due to the presence of SDS inthe extraction buffer.

Limited proteolysis using low concentrations of eithertrypsin or chymotrypsin (without SDS), on the other hand, didnot remove the entire plasmodesma, but did extract materialfrom the region of the orifice, as determined using negativestaining (not shown). Thin sections of this material, however,showed no significant differences from untreated controls inthe core of the plasmodesma. Similarly, plasmodesmata infrozen sections of whole tissue appeared unaltered after directexposure to the proteases. The restriction of proteolytic effects

Fig. 4. Detergent-extractablewall-associated proteins. SDS-PAGE analysis of proteinsextracted from whole purifiedwall preparations using SDS(lane 2) or CHAPS (lane 3).Lane 1 contains proteins fromthe non-wall fraction. The 10%acrylamide gel was silverstained. Numbers on the leftindicate molecular mass in kDa.

kDa

Fig. 5. Extraction of plasmodesmata with urea and protease. (A) Replica of a primary pit field from a wall fragment that has been boiled inbuffer containing 10 M urea. (B) Replica of wall after incubation in protease solution (Pronase E and proteinase K). In both (A) and (B) entireplasmodesmal structures, including their surrounding collars, have been removed. (C) Negative staining of a protease-treated wall fragment.Straight-sided holes left by extracted plasmodesmata are unstained and appear to run right through the wall (arrows). Bar, 200 nm.

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to the orifice region may reflect the presence there of protease-sensitive cytoplasmic remnants associated with the plasmod-esmal tails. Lack of any discernible effect in the plasmodesmal

core argues against the presence of major proteinaceous struc-tures in this region.

Supernatants harvested from the wall digests were analysed

A. Turner, B. Wells and K. Roberts

Fig. 6. Collar composition. (A) Negative-stained wall after boiling in SDS sample buffer. The raised, pale-staining collars are still in evidence(arrows), but are formless and somewhat swollen. Neither annular membrane structures nor central depressions are present. (B) Replica of wallthat has been boiled in water. This treatment is sufficient to cause the collars to swell and distort (arrows). (C) Replica of wall after treatmentwith exo-β(1→3) glucanase. The plasmodesmal collars are still present (arrows). (D,E) Wall fragments labelled with anti-β(1→3) glucanantibodies conjugated to 10 nm gold particles. Walls were previously extracted with SDS sample buffer to fully expose the collars. The labelcolocalises with plasmodesmata, but appears to be specific for wall around and between the plasmodesmata, and does not lie directly on theexposed collars. (D) Transverse wall; (E) two primary pit fields in a longitudinal wall. (F) Thin-sectioned material labelled with the anti-β(1→3) glucan antibodies. With this method of sample preparation, callose (indicated by gold particles) appears to be more directly associatedwith plasmodesmata (arrows). A-E Bar, 200 nm; F bar, 200 nm.

3359Extraction of plasmodesmata

by SDS-PAGE in an effort to obtain peptide fragments ofputative plasmodesmal proteins. However, no candidatepeptides were detectable by this method.

Collar compositionWhen cell walls were boiled for 10 minutes in Laemmli samplebuffer containing SDS, plasmodesmata showed no sign of thecentral depression that was produced by extraction with coldSDS. Rather, negative staining showed pale, raised mounds atthe sites of plasmodesmata (Fig. 6A), confirmed in replicaswhere amorphous lumps could be seen at the plasmodesmalorifice (not shown). Walls boiled in the absence of detergentwere found to have the same form (Fig. 6B). It appears,therefore, that boiling causes swelling of material at the plas-modesmal opening, presumably of the collar itself.

Collars are not obvious around all plasmodesmata, but theirwidespread appearance might be expected if they arecomposed of callose, synthesised as a response to plasmo-lysis and wounding (Currier, 1957). However, immunogoldlabelling of wall fragments with anti-β(1→3) glucan anti-bodies, while clearly showing co-localisation with plasmodes-mata, appeared to decorate regions of wall between the collarsrather than the collars themselves (Fig. 6D,E). Incubation ofwall fragments in β(1→3) glucanase eliminated labelling withthe anti-β(1→3) glucan antibodies (not shown), but did notremove the collars (Fig. 6C). While immunogold label clearlyassociates with plasmodesmata in conventional thin-sectionedmaterial (Fig. 6F), such images are difficult to interpret. Onlythe surface of a 100 nm thick section is accessible to theantibody, and precise identification of the labelled material inside views of essentially intact plasmodesmata is impossible,even with stereo pairs. Labelling of negative stained wholewall fragments (Fig. 6D,E) is thus likely to give a truer indi-cation of callose distribution.

DISCUSSION

While advances in ultrastructural techniques are providing evermore detailed insights into the fine structure of plasmodesmata(Ding et al., 1992), significant progress in understanding plas-modesmal form and function awaits the isolation and charac-

terisation of specific biochemical components. We have usedhere a range of simple extraction procedures to attempt a struc-tural and biochemical dissection of plasmodesmata in isolatedmaize root cell walls.

As the first step, purified cell walls were prepared using asimple and reliable procedure, which should be useful for thestudy of other wall components besides plasmodesmata. Asimilar procedure has been described by Kotlizky et al. (1992)for obtaining clean wall preparations from maize mesocotyl.This method employed a nitrogen pressure bomb to disruptcells, followed by filtering of cell debris to isolate walls. Plas-molysis was not utilised in this procedure. Protein profilesobtained with both methods are not directly comparable asdifferent tissues were used. However, a polypeptide doublet ofabout 80 kDa is enriched in both methods, as well as a bandat about 42 kDa. A prominent 26 kDa plasmodesmal associ-ated protein of mesocotyl (PAP 26; Yahalom et al., 1991) isabsent from roots. Wall-associated bands obtained in thepresent work at around 90, 100 and 120-130 kDa are onlyfaintly visible or absent in the extracts of Kotlizky et al. (1992).These differences may reflect tissue-specific protein comple-ments, or differences in relative proportions of the proteins thatare retained in walls prepared by the different methods.

The isolated maize root tip cell walls contain embeddedplasmodesmata that, before further extraction, consist of asimple tube of plasma membrane and an enclosed desmotubuletraversing the wall. Extending on either side from these com-ponents are short remnants of membrane that originallyconnected them to the protoplast in the intact cell. A cylinderof amorphous material, the collar, ensheaths this core complexand is itself embedded within the wall matrix. Extraction withmembrane-solubilising detergents removes both the plasmamembrane tube and the desmotubule from the plasmodesma,leaving the cell wall collar undisturbed. With light proteasetreatment there is little evidence of structural alteration in theplasmodesmal core. Occasionally after heavy proteasetreatment the amorphous collar and all the structure inside itare completely removed. Urea may also extract the collar,again not reproducibly. The effects of these extractions aresummarised diagrammatically in Fig. 7.

The removal of plasma membrane from plasmodesmata inmaize walls by membrane-solubilising detergents (in the

Fig. 7. Diagrammaticrepresentation of the suggestedcourse of plasmodesmalextraction. In (A) the intactplasmodesma is shown,including a prominent collar andplasma membrane tubesurrounding a desmotubule.Wall fragment preparation (B)leaves an intact plasmodesmalcore within the wall, with tailsof membrane attached on eitherside; (C) shows the effect ofdetergent extraction inremoving plasma membraneand desmotubule. After protease

or urea treatment (D) all plasmodesmalstructure may be absent, leaving a holein the wall of 60-80 nm diameter.

3360

present work TX-100, CHAPS and SDS) resembles the effectof TX-100 on fern gametophyte plasmodesmata as observedby Tilney et al. (1991). However, these workers found that thedesmotubule was resistant to this extraction, and could be seensubsequently in electron micrographs to lie in a void regionwithin the cell wall, where the plasma membrane had been. Inthe present work, however, there was also loss of structure inthe region of the desmotubule after treatment with detergent.This suggests that the maize desmotubule is composed largelyof membrane, in contrast to the situation in the fern gameto-phyte (Tilney et al., 1991).

TX-100 and CHAPS both release a relatively small com-plement of protein types, which clearly are of interest aspossible plasmodesmal components. Among these are twopairs of proteins at around 100 and 130 kDa. These are of lowabundance and may be membrane-associated. A third doubletof about 80 kDa is abundant and will leach from the wall inthe absence of detergent. Interestingly, Monzer and Kloth(1991) reported that TX-100 releases a 43 kDa protein fromclean Solanum cell walls. Maize mesocotyl walls also yieldeda protein of this approximate mass (Kotlizky et al., 1992), asdid maize root tips in the present work, although not repro-ducibly.

Tilney et al. (1991) found that protease extracted the desmo-tubule of fern gametophyte plasmodesmata, leaving the sur-rounding plasma membrane intact. In the present work a strongprotease treatment occasionally extracted the entire plasmod-esmal structure, including the surrounding collar. It was notpossible to determine whether the desmotubule had beenspecifically degraded in this case. As it appears unlikely thatthe entire collar is proteinaceous, it is possible that the effectof protease treatment, and also of urea, is to disrupt the con-nections responsible for binding the collar and its enclosedplasmodesma into the wall. This might simply reflect aloosening of the wall matrix after extraction, through degrada-tion of structurally important wall proteins. The lack of repro-ducibility of this effect might be due to variations in exposureto the extractive agent, or to variations in the composition ofthe plasmodesmata or walls themselves.

Exposure of wall fragments to light proteolysis with trypsinor chymotrypsin, on the other hand, consistently had limitedeffects on plasmodesmal structure. While negative stainingindicated some degradation of the exposed tail region, in thinsections the plasmodesmata appeared to be substantially intact.This held true even when plasmodesmal cores were exposedto the protease directly by use of frozen-sectioned whole tissuerather than wall fragments. There was no obvious preferentialdegradation of the desmotubule as was seen by Tilney et al.(1991). These authors concluded that the desmotubule is sub-stantially proteinaceous in composition, while Ding et al.(1992) suggested that prominent electron-opaque particlesassociated with the desmotubule in Nicotiana are probablycomposed of protein. In the present work, subtle effects of pro-teolysis on such structures might have occurred but been unde-tectable. The absence of major effects, however, does supportthe contention that the desmotubule in maize root tips is prin-cipally a membranous structure.

In the present work, confirmation of the effects of extractionon the desmotubule and other internal structure required thatmaterial be embedded and sectioned for conventional TEM.However, negative staining and rotary shadowing techniques

were mainly adopted here, as they provided means of rapidlyassessing the effects of a given treatment on surface structure,while giving some indication of internal effects. Sophisticatedexamination of fine internal structure requires the use of morerefined techniques such as cryofixation and freeze-substitution(Ding et al., 1992). The model of gross structure proposed inthe present work serves to emphasise that plasmodesmalstructure is closely integrated with that of the cell wall. Thevalue of considering both the membranous core and the wall-located components of the plasmodesma in models of plas-modesmal function should not be overlooked.

The presence of a collar region around the plasmodesma isa conspicuous feature not only of maize root tip cell walls, butof plasmodesmata from a variety of species and tissues,observed in a variety of ways (see, for example, Overall et al.,1982; Thomson and Platt-Aloia, 1985). However, not allpublished electron micrographs of plasmodesmata exhibit thisfeature prominently (for example, the cryofixed and freeze-substituted material of Ding et al., 1992). Indeed, the possibil-ity exists that the collar reflects a wound reponse to tissuefixation or to the plasmolysis used in producing wallfragments. However, walls prepared in the present study fromunplasmolysed, snap-frozen root tips still displayed prominentcollars. Apparent differences among published images of plas-modesmata might reflect differences in sample preparation orstaining. Nevertheless, it is quite likely that the presence orabsence of the collar reflects or indeed determines differentfunctional states of a plasmodesma. Stimuli such as woundingor stress might induce the formation of collars, just as theyinduce the deposition of callose (Currier, 1957; Smith andMcCully, 1977).

Localised synthesis of the collar region around the plas-modesmal neck suggests the presence of localised synthase(s),possibly within the plasmodesmal plasma membrane itself.Knowledge of what the collar is composed of might thus givea hint as to the identity of one biochemical component withinthe plasmodesma proper. Callose is known to co-localise withplasmodesmata (Northcote et al., 1989; Olesen, 1986), and aputative callose synthase subunit has been immunolocalised tothe same sites (Delmer et al., 1993). It has been assumed thatthe collar is composed at least in part of callose or some relatedpolysaccharide containing β(1→3)-linked glucans (Olesen,1986). However, immunogold localisation of callose near butnot on collars, and the resistance of the collars to callose-degrading enzyme suggest that other molecules must beinvolved. The cell wall around the plasmodesmal core thusappears to be structurally subdivided, with an inner amorphouscollar surrounded by a peripheral zone in which callose is inter-spersed with the fibrillar wall material. Such a configurationmay still allow callose deposition to restrict the plasmodesmalorifice as suggested by Olesen (1986) and the experiments ofWolf et al. (1991), as a constricting force exerted on the collarcould still be transmitted to the plasmodesmal core within.Indeed, a semi-rigid collar ensheathing the core may allowplasmodesmal permeability to be subtly altered by such anexternal force while preventing collapse of the membranouscylinder of the core.

Identification of the molecular components of the collarshould allow a clearer view of its function. Similarly,molecular characterisation of components in the core of theplasmodesma - the plasma membrane tube, the cytoplasmic

A. Turner, B. Wells and K. Roberts

3361Extraction of plasmodesmata

sleeve, and the desmotubule - will illuminate their function.Further work along the lines described above, combined withimmunological and molecular techniques, may assist towardsthis end.

We thank Maureen McCann, Graham Hills and Robyn Overall forhelpful advice, and members of this Department for technical assis-tance and stimulating discussions. Thanks to Sue Bunnewell for helpwith photography, and to Simon Preece (Pioneer Hi-Bred) forsupplying seed. A.T. was supported by EEC BRIDGE grant no. BIOTCT-900156.

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(Received 28 April 1994 - Accepted, in revised form,18 August 1994)