Cell Biology International 1999, Vol. 23, No. 10, 653–661Article No. cbir.1999.0431, available online at http://www.idealibrary.com on

CELL-TO-CELL COMMUNICATION VIA PLANT ENDOMEMBRANES

L. C. CANTRILL1*, R. L. OVERALL2 and P. B. GOODWIN1

1Department of Crop Sciences A20, 2School of Biological Sciences A12, The University of Sydney,NSW 2006, Australia

Received 12 March 1999; accepted 26 May 1999

Cell-to-cell communication was investigated in epidermal cells cut from stem internodal tissue ofNicotiana tabacum and Torenia fournieri. Fluorescently labelled peptides and dextrans weremicroinjected using iontophoresis into the cytoplasm and cortical endomembrane network ofthese cells. The microinjected endomembrane network was similar in location and structure tothe endoplasmic reticulum (ER) as revealed by staining with 3,3�-dihexyloxacarbocyanine iodide(DiOC6). No cell-to-cell movement of dextrans was observed following cytoplasmic injectionsbut injection of dextrans into the endomembrane network resulted in rapid diffusion of theprobes to neighbouring cells. It is proposed that the ER acts as a pathway for intercellularcommunication via the desmotubule through plasmodesmata. � 1999 Academic Press

K: endoplasmic reticulum; plasmodesmata; dextrans; microinjection.

*To whom correspondence should be addressed.

INTRODUCTION

The endoplasmic reticulum (ER) forms a con-tinuous network throughout the cytoplasm ofeukaryotic cells and has the roles of synthesizing,processing and sorting proteins and lipids as well asregulating cytosolic calcium levels (Staehelin,1997). In plants, an ultrastructural relationshipbetween the cortical ER and plasmodesmata hasbeen recognized since the earliest EM images ofplasmodesmata (see Robards, 1976 and referencestherein). The nature of the desmotubule linkbetween the ER of adjacent cells has been thesubject of much speculation. The desmotubule hasbeen modelled as a solid proteinaceous rod (Tilneyet al., 1991), a membrane cylinder (Gunning andOverall, 1983) or as an integration of both features(Ding et al., 1992). The possibility that the desmo-tubule plays a role in intercellular communicationhas also been the topic of much debate (Robards,1976; Robards and Lucas, 1990).

The lipid-packing calculations of Overall et al.(1982) demonstrated the possibility that ER lipidscould be constricted into a tubular strand that

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passes through the centre of plasmodesmata. Withthis model, the desmotubule lumen was reduced tothe diameter of a few water molecules and as aresult the spaces between protein subunits withinthe cytoplasmic sleeve became the preferred routefor cell to cell communication (Lucas et al., 1993;Overall and Blackman, 1996). More recently, newevidence has revived the idea of the desmotubule asa pathway of cell to cell communication. Theintercellular diffusion of ER specific lipids has beendemonstrated (Grabski et al., 1993), and the poten-tial of the ER network to act as a pathway forphototsynthate or water transfer from cell to cell(Gamalei et al., 1994; Zhang and Tyerman, 1997)has also been indicated. Strong evidence also existsfor the cell to cell movement of tobacco mosaicvirus movement protein within the membrane ofthe ER (Reichel and Beachy, 1998).

Evidence is presented in this paper that a micro-injectable subcellular compartment exists in ad-dition to the familiar cytoplasmic, nuclear andvacuolar compartments. This compartment has theappearance of a lacy network. It resembles corticalER in both form and location and is a potentialavenue for cell to cell communication in higherplants.

� 1999 Academic Press

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MATERIALS AND METHODS

Plant material

Explants of Nicotiana tabacum L. cv Wisconsin 38(CSIRO Plant Industry, Canberra, Australia) wereobtained from vegetative stock plants. Stock plantswere maintained under sterile conditions onMurashige and Skoog (MS) medium (Murashigeand Skoog, 1962) supplemented with vitaminsaccording to Linsmaier and Skoog (1965), 30 g/lsucrose, 8 g/l of bacto-agar and with the pHadjusted to 5.7. Light was provided by fluorescenttubes (40W, Crompton, Australia) (26 W/m2) for4-h intervals, each separated by 2 h of darkness.Temperatures during the light and dark periodswere 27�C and 24�C, respectively. Explants of stemepidermis were cut from the second and thirdextended internodes below the apex of 6 to 8 weekold stock plants (modified from Tran Thanh Vanet al., 1974). Each explant was approximately 5 mmlong and 2 mm wide and consisted of one layer ofepidermal cells and 3–7 layers of subepidermalcells. For microinjection, tobacco stem explantswere bathed in liquid MS medium.

Stock plants of Torenia fournieri Lind. cv ClownMixed (Yates, Australia) were cultured on modi-fied MS media according to Tanimoto and Harada(1979). These were incubated in a controlledenvironment room at a temperature of 30�C undercontinuous illumination (6.8 W/m2) provided byfluorescent tubes (40W, Crompton, Australia).Explants were cut from the second internode belowthe apex of 6 to 8 week old stock plants andwere approximately 5 mm long and 3 mm wide,consisting of the epidermis and many layers ofsubepidermal and cortical cells including some vas-cular tissue (modified from Tanimoto and Harada,1979). For microinjection, Torenia stem explantswere bathed in liquid modified MS medium.

Anchoring explants

Injection slides and anchoring of stem explants wasadapted from Hepler and Callaham (1987). Injec-tion slides were constructed from 1.2-mm thickglass slides (American Scientific McGaw Park, IL,U.S.A.) each drilled with an 18-mm diameter hole.Each hole was covered by a 22�22-mm coverslip(#1 thickness) glued around the edges with dilutenail polish to form a well. The injection slides werewarmed to 37�C on the heated stage of a binoculardissecting microscope prior to anchoring theexplants. Explants were blotted of excess mediumprior to anchoring and were placed one per slide

and epidermis up. A few drops of molten 1%agarose solution (type VII; Sigma) +0.02% TritonX were spread around the sides of the injectionslide well and left to cool to 37�C for 30 s on theheated stage. Agarose was pushed up to the edgesof the explant and allowed to run under it andacross the base of the well. The slide was thenplaced on a freezer brick for 10 s to solidify theagarose. The explants were then immersed oncemore in the appropriate culture medium andallowed to equilibrate for at least 30 min prior tothe first injection.

Probes

F(Glu)3 was synthesized according to the methodof Oparka and Read (1994) by conjugating fluores-cein isothiocyanate isomer I (FITC) (Sigma Chemi-cals, St Louis, MO, U.S.A.) and triglutamic acid[(Glu)3] (Sumac, University of Sydney, Australia).FITC-labelled dextrans (FD 3000, FD 10000 andFD 40000) were obtained from Molecular ProbesInc. (Eugene, OR, U.S.A.). Prior to use, the dex-trans were reprecipitated in 80% ethanol accordingto Derrick et al. (1992) to eliminate unreactedFITC. 6-Carboxyfluorescein (Eastman Chemicals,Rochester, NY, U.S.A.) was initially dissolved in0.3 KOH. All probes were made up to 1-msolutions in millipore filtered (0.45 �m pore size)distilled water and the fluorescent conjugates weretested for low-molecular weight contaminantsusing thin layer chromatography (Kieselgel 60,Merck Darmstadt, Germany) according toGoodwin (1983). Solutions of all fluorochromeconjugates were discarded after 1 day to avoid lowmolecular weight contaminants from microbialdegradation.

Microinjection

Micropipettes were pulled from 1.2-mm boro-silicate glass capillaries with inner filaments (WorldPrecision Instruments (WPI), Sarasota, FL,U.S.A.) on a P87 puller (Sutter InstrumentCompany, Novato, CA, U.S.A.). Tips were filledwith 5 �l of the fluorescent probe, back filled with0.1 m KCl and secured in a microelectrode holder(MEH2SFW12, WPI) attached to the active probeof the electrometer (Intra 767, WPI). A chloridizedsilver wire placed in the bathing solution was usedas a reference electrode and all metal componentsof the set up were electrically grounded. Tip integ-rity of the micropipettes was monitored via thebuilt in electrode resistance test function. Resist-ances of around 1000 M� indicated that the tip

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was undamaged and suitable for microinjection.Injections were performed via an hydraulic micro-manipulator (WR-30, Narishige, Japan) mountedon a coarse manipulator (M-35, Narishige)attached to the microscope stage. Cells wereimpaled at a 45 degree angle in the corner of a cellor close to the nucleus. The micropipette tip waspositioned so as to push gently on the cell wall/membrane and the side of the microscope wastapped to gain entry to the cytoplasm. The mem-brane potential was noted both before and afteriontophoretic injection. Injections where mem-brane potential fluctuated or large changes wereseen after microinjection were rejected. Probes wereinjected by iontophoresis using a current pulse of�1 nA for 15–30 s. The current was generatedas a square wave from a function generator(CFG253, Techtronix, Beaverton, OR, U.S.A.)and was delivered via the breakaway box on theelectrometer.

DiOC6 staining

DiOC6 (3,3�-dihexyloxacarbocyanine iodide)(Sigma) was dissolved in a small quantity of di-methyl sulfoxide (DMSO; final concentration nomore than 0.1%) and made up into a 5 mg/ml stocksolution with distilled water according to the pro-tocol of Oparka and Read (1994). Internodes ofwhole Torenia and tobacco stems were gentlyabraded with a lint-free tissue to remove cuticle soas to allow DiOC6 to penetrate and stain epidermalcells. This step also removed trichomes from thetobacco epidermis which would have otherwiseinterfered with observations due to fluorescencefrom non specific staining of the trichome cellwalls. Explants were cut from the abraded area andthese were bathed in a staining solution containingthe appropriate MS medium and DiOC6 (5 �g/ml)for 2 min. Stained explants were washed briefly infresh MS medium to remove excess DiOC6 andmounted on slides.

Microscopy

Microinjections were monitored as they occurredwith an Axiophot microscope (Zeiss) equippedwith a �40 water immersion objective. DiOC6staining was observed with a standard �40 and a�63 oil immersion lens. The FITC probes andDiOC6 were observed under blue illumination(excitation 450 to 490 nm). Experiments wererecorded on Fujichrome 400 ASA colour reversalfilm (Fuji Photo Film Co, Tokyo, Japan) andEktachrome 400 or 1600 ASA colour reversal film

(Eastman Kodak). Photographic slides werescanned by a Nikon LS-1000 Super Cool Scan(Nikon Inc., Tokyo Japan). The digitized imageswere printed out on a Codonics NP-1600M dyesublimation printer (Codonics Inc. MiddlebergHeights, OH, U.S.A.).

RESULTS

Small molecule microinjections

The presence of a microinjectable endomembranesystem was first observed when microinjecting CFand F(Glu)3 into tobacco and Torenia explants.Figure 1a shows a typical cytoplasmic injection ofCF into a Torenia internodal epidermal cell andcompares it to an endomembrane injection (Fig.1b). The cytoplasmic injection shows a characteris-tically evenly distributed fluorescence in addition tothe bright nucleus in each of the cells and thiscontrasts with the punctate fluorescence of theendomembrane injection. The higher magnificationimage (Fig. 1c) reveals that the distinct brightpoints which underlie the cell wall of the injectedcell seem to be linked by fluorescent strands. Thesebodies and strands are similar to the strands andlamellae of cortical ER revealed by DiOC6 stainingof Torenia cells (Fig. 1d).

Injections of this type using CF and F(Glu)3were observed at a low frequency in the two celltypes tested (less than 10% of injections; see Table1) and this probably reflects the chance of locatingthe micropipette tip within a structure of smallvolume relative to the rest of the cell. Cytoplasmicinjections of CF and F(Glu)3 moved from cellto cell in the majority of cases although a per-centage of these injections resulted in no cell to cellmovement (see Table 1). In contrast, all casesof endomembrane injection of CF and F(Glu)3resulted in cell to cell movement (see Table 1).Furthermore, these small fluorescent probesseemed to travel more rapidly from cell to cell viaendomembrane injections than via cytoplasmic in-jections, although this was not possible to quantify,as in most cases fluorescence vanished from theendomembrane network within a few seconds ofmicroinjection.

Macromolecule microinjections

Injections of FD 3000 into the cytoplasm oftobacco cells resulted in no cell to cell movement ofthis dextran probe (see Table 1; Fig. 2a,b). Figure2a is focused at the level of the external cell wall of

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Fig. 1. Fluorescence images of internodal epidermal cells of Torenia fournieri. (a) A microinjection of CF showing thecharacteristic cytoplasmic distribution of the fluorescent probe. Fluorescence is observed in the cell nuclei (arrows) and is presentin an even band inside the cell wall. Bar=50 �m. (b) A CF injection showing the punctate fluorescence of an endomembranenetwork. Bar=50 �m. (c) At a higher magnification, the small bodies are more evident (arrows, �) and in places these seem tobe linked by small fluorescent strands (darts, �). Bar=25 �m. (d) DiOC6 staining of ER in an internodal epidermal cell ofTorenia fournieri. Fluorescent bodies (arrows) joined by strands (darts) are similar to those observed in 1c. Bar=25 �m.

Table 1.Frequency of intercellular movement observed during endomembrane and cytoplasmic injections

into tobacco and Torenia explants

Explant type Tobacco Torenia

Probe F(Glu)3 FD 3000 FD 10000 CF F(Glu)3 FD 3000 FD 10000Molecular weight 799 �3000 �10,000 376 799 �3000 �10,000Total number of injections 14 18 8 64 67 6 33Cytoplasmic injections 13 17 8 61 64 6 30Intercellular movement observed 11 0 0 55 46 0 0

(84.6)* (90.1)* (71.9)*Endomembrane injections 1 1 0 3 3 0 3Intercellular movement observed 1 1 0 3 3 0 3

(100)† (100)† (100)† (100)† (100)†

*% of cytoplasmic injections where intercellular movement observed.†% of endomembrane injections where intercellular movement observed.

a tobacco epidermal cell and FITC fluorescence isconcentrated in the cytoplasm which is seen verybrightly in the cell periphery but also exists just

below the cell wall in view and which appears as afluorescent haze all over the central part of the cell.This fluorescence is uniform and is not located in

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small bodies just below the cell wall. Observing thesame cell at a lower focal plane (Fig. 2b) provides asimilar picture, with the nucleus in focus this timeand the peripheral cytoplasm surrounding a darkcentral area (the vacuole) which contains littleor no fluorescence. However, Fig. 2c presents atobacco cell in which FD 3000 was injected into acompartment that was different from the usualcytoplasmic injections. A lacy intracellular networkappeared to be located just below the cell wall inthe cell cortex. Not only was there a distinctlocalization of fluorescence to this network butthere was also a lack of the normal all-over fluor-escence seen at similar focal planes in cytoplasmicinjections (Figs 1a and 2a). Dye was distributedwithin a network of small fluorescent bodies whichappeared to be interlinked by fine strands. At a midplane focus, this network is shown to be confinedto the area just inside the cell wall (Fig. 2d). Figure2e reveals the most striking feature of this injection;a large fluorescent body in each of the neighbour-ing cells. These were the nuclei of the neighbouringcells and the presence of fluorescence within themindicated that FD 3000 had moved from cell to cell.The similarity in form and location between corti-cal ER in a tobacco cell stained with DiOC6 (Fig.2f and g) and the microinjected endomembranenetwork (Fig. 2c and d) suggests that they areidentical structures.

Similar observations were made with injectionsof FD 10000 into freshly prepared Torenia explantsand as with the other probes already discussed,only a small proportion (

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endomembrane injection might also indicate thatthere is gating or control over the molecular sizeexclusion limit in this pathway.

With epifluorescence it was difficult to determineif the dye was in the nuclear envelope or nucleo-plasm of neighbouring cells. If dye was in thenucleoplasm after an endomembrane injection, itcould mean that a previously unobserved pathwayfor transport exists between the ER/nuclear en-velope lumen and the nucleoplasm. Indeed, Singhet al. (1998) have described a trans-nucleoplasmicreticulum that significantly increases the surfacearea of the nuclear envelope by invaginating deeplyinto the nucleoplasm and which is continuous withthe endomembrane system.

Intercellular movement through subcellularmembrane networks has been observed or pro-posed in other systems. Shepherd et al. (1993a,b)reported that a system of tubules and vacuolesmoved intercellularly through the dolipore in thewall between the terminal and penultimate cells offungal hyphae. Lazzaro and Thomson (1996)reported an intercellular vacuolar system withinsecretory trichomes of chickpea that transportedLucifer yellow hydrazide from the basal to the tipcells via plasmodesmata. These latter authorsspeculated on a possible relationship between thechickpea vacuoles and the ER network seen inother plant cells and the possibility that the ERlumen might also provide an avenue for cell to celltransport. Recent evidence suggests that vacuolesare derived from specialized regions of the ER(Staehelin, 1997 and references therein) and thusthe relationship could be very close. Waigmannet al. (1997) have also reported ‘open’ desmotu-bules in EM images of trichome cells of Nicotianaclevlandii and that these cells possessed a basal sizeexclusion limit in the macromolecular range(Waigmann and Zambryski, 1995). However, theseauthors speculated that in trichome cells, the

desmotubule might be open to the cytoplasm ratherthan being continuous with the ER.

Viral movement in several systems has beenproposed to involve ER membranes. Geminiviruses recruit ER to form specialized tubules con-taining movement protein that enable cell to cellmovement of the virus particle via the tubule lumen(Ward et al., 1997). Tobacco mosaic virus move-ment protein has also been localized to ER mem-branes suggesting that it and the viral RNA mightbe transported from cell to cell via the movement ofER through plasmodesmata (Reichel and Beachy,1998). Indeed, the ER also appears to be readymade for many of the active macromolecule traf-ficking functions that are now attributed to plas-modesmata. ER undergoes continual movementand reorganization (Knebel et al., 1990) and isclosely association with cytoskeletal elementsincluding actin and myosin (Liebe and Quader,1994; Reuzeau et al., 1997) which have also beenlocalized to plasmodesmata (White et al., 1994;Blackman and Overall, 1998; Radford and White,1998). Furthermore, the protein targeting (Bar-Peled et al., 1996) and protein folding functionsattributed to ER (Boston et al., 1996) make theseendomembranes very attractive sites for furtherstudies of macromolecular cell-to-cell communi-cation. It is clear that the technique reported hereof microinjection into plant endomembrane sys-tems has reinforced this evidence of the ER playingan active role in cell to cell communication throughplasmodesmata.

Fig. 2. Fluorescence images of internodal epidermal cells of tobacco. (a) Dye distribution of a cytoplasmic injection of FD 3000into an internodal epidermal cell of tobacco seen at the focal plane of the external wall. The yellow haze observed across the cellshows the presence of an even layer of cytoplasm just below the cell wall. The nucleus is indicated (arrow). Bar=25 �m. (b) Thesame cell focused at the level of the nucleus (arrow) shows that most of the cell appears clear due to the presence of the largecentral vacuole. A narrow line of fluorescence around the cell is the probe contained within the thin layer of peripheralcytoplasm. No neighbouring cells contain fluorescent nuclei or cytoplasm indicating that this probe does not move from cell tocell in this tissue type. Bar=25 �m. (c) An endomembrane injection of FD 3000 into an internodal epidermal cell of tobacco. Inthe cell wall focal plane shown here, the cell appears to contain a lacy network of fluorescent bodies linked by fluorescent strands.Bar=25 �m. (d) The same cell focused at the level of the nucleus (arrow). Some fluorescent bodies are visible at the edges of thecell but the central area is free of fluorescence indicating that the network is likely to be located in the cell cortex. Bar=25 �m.(e) A wider field view of the cell in (c) and (d). Note the fluorescent nuclei in neighbouring cells (arrows) indicating theintercellular movement of FD 3000 after an endomembrane microinjection. Bar=25 �m. (f) DiOC6 staining of tobaccointernodal epidermal cells showing the presence of interlinked fluorescent bodies of the ER in the cell cortex. These are similarto the fluorescent bodies in Fig. 2c. Bar=25 �m. (g) The same cell at a lower focal plane showing the absence of ER in the centralregion of the cell. Bar=25 �m.

ACKNOWLEDGEMENTS

L. C. Cantrill acknowledges support by theChristian Rowe Thornett Postgraduate Scholar-ship. This study was supported through grants to

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P. B. Goodwin and R. L. Overall from theAustralian Research Council.

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CELL-TO-CELL COMMUNICATION VIA PLANT ENDOMEMBRANESINTRODUCTIONMATERIALS AND METHODSPlant materialAnchoring explantsProbesMicroinjectionDiOC6 stainingMicroscopy

RESULTSSmall molecule microinjectionsMacromolecule microinjectionsFig. 1Table 1

DISCUSSIONFig. 2

ACKNOWLEDGEMENTSREFERENCES