Development 110, 1209-1221 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

1209

The distribution of plasmodesmata and its relationship to morphogenesis

in fern gametophytes

LEWIS G. TILNEY1'3*, TODD J. COOKE2, PATRICIA S. CONNELLY3 and MARY S. TILNEY1'3

1 Marine Biological Laboratory, Woods Hole, MA 02543, USA2 Department of Botany, University of Maryland, College Park, MD 20742, USA3 Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA

* Author for correspondence

Summary

Fern (Onoclea sensibilis) gametophytes when grown inthe dark form a linear file of cells (one-dimensional)called a protonema. In the light two-dimensional growthoccurs which results in a heart-shaped prothallus onecell thick. The objective of this paper is to relate the mostcommon pattern of cell division observed in developinggametophytes to the formation of the plasmodesmatalnetwork. Since the prothalli are only two dimensional,we can easily determine from thin sections the totalnumber and the density (number per unit surface area)of plasmodesmata at each developmental stage. As theprothallus grows the number of plasmodesmata in-creases 50-fold in the apical or meristematic cell. Thisnumber eventually reaches a plateau even though thedensity continues to increase with each new cell division.What is particularly striking is that both the number anddensity of plasmodesmata between adjacent cells isprecisely determined. Furthermore, the pattern of

plasmodesmata distribution is predictable so that (1) wecan identify the apical meristematic cells by theirplasmodesmata number, or density, as well as by theirsize, shape and location, (2) we can predict, again fromplasmodesmata number, the location of a future wall ofthe apical cell prior to its actual formation, (3) we canshow that the density of plasmodesmata in the triangularapical cell of the prothallus (14 plasmodesmata/*m~2) iscomparable to those reported for secretory glands whichare known to have high rates of plasmodesmataltransport and (4) we can show that once the plasmo-desmata have been formed during division, no sub-sequent change in the number of plasmodesmata occursfollowing cell plate formation.

Key words: fern, Onoclea sensibilis, gametophyte,protonema, prothallus, plasmodesmata.

Introduction

Little is known about how cell division, cell expansionand cell differentiation are related to the generation ofform in plants. Unfortunately most higher plants arecomposed of massive three-dimensional organs withcomplex intercellular interactions. Thus our obser-vations on cell behavior are usually based on averagevalues for large populations of heterogeneous cells.Accordingly, there is a compelling need to exploit asystem in which the behavior of individual cells can berelated to the whole organism as it differentiates.

A system of choice is the fern gametophyte which hastwo basic forms (Furuya, 1983; Miller, 1968) (Fig. 1). Ifthe gametophyte is grown in the dark, it produces a longfilament, the protonema, which consists of a single fileof cells. Protonemal growth occurs by a process of tipgrowth whereby cell wall elongation and cell divisionare restricted to the apical cell. In contrast, if the sporeis exposed to light, the resulting gametophyte grows

into a heart-shaped object, a prothallus. The prothallusis composed of a flat plate of cells one cell thick. Thusthe one-dimensional pattern of tip growth in theprotonema can be converted to the two-dimensionalplanar growth (in the prothallus) by transferring thegametophyte to the light. The reverse process can alsooccur by transferring the prothallus to the dark(Sobota, 1970).

There are three reasons why the fern gametophyte isan ideal system for the study of morphogenesis inplants. First, depending on the form one chooses, aprotonema or a prothallus, one can study either one- ortwo-dimensional growth and its regulation withouthaving to worry about the enormous complexityproduced by a three-dimensional plant. Second, ga-metophytes grow readily on moist filter paper or agarmedium and do not require complex organic sup-plements, which may have uncontrolled effects on plantgrowth. Third, there is an extensive literature on ferngametophytes that began at the turn of the century and

1210 L. G. Tilney and others

has continued into modern times with numerous studieson the growth effects of visible light (Charlton, 1938;Furuya, 1983), X-irradiation (Rottman, 1939) hor-mones and other growth substances (Smith, 1979),plasmolysis (Nagai, 1914; Nakazawa, 1963), and micro-surgery (Albaum, 1938a; Albaum, 19385; Ito, 1962).Much background information is also available on thegrowth patterns of protonemata and prothalli (Dopp,1927; Orth, 1936), RNA and protein synthesis(DeMaggio and Raghavan, 1973) and ionic currents(Cooke and Racusen, 1986; Racusen et al. 1988). Theliterature on fern gametophyte development has beenreviewed by Miller (1968) and Raghavan (1989).

What has not been investigated so far is the possiblemorphogenetic role of intercellular communication viathe plasmodesmata, i.e. the cytoplasmic bridges be-tween neighboring cells (for reviews see Gunning andOverall, 1983; Gunning and Robards, 1976). This issurprising as there is a large literature available whichdocuments that the plasmodesmata may somehow playa key role in regulating fern gametophyte morphogen-esis. For example Nagai (1914) and Nakazawa (1963)demonstrated that a brief exposure to plasmolysis,sufficient to break the plasmodesmata, induces each cellin the prothallus to differentiate into a new completeprothallus. Similar results are achieved with surgicalprocedures (Albaum, 19385). All these observationslead to the same conclusion, namely, that there must besome signal transmitted via the plasmodesmata fromcell to cell throughout the gametophyte so that theprothallus behaves as a coordinated unit.

We have begun to explore intercellular communi-cation in the fern gametophyte with the ultimate aim oftrying to determine how the morphogenesis of thissimple organism is controlled. Using reconstructiontechniques similar to those used to describe theplasmodesmatal network in Azolla roots (Gunning,1978), we have characterized the number and thedensity [density, in keeping with earlier terminology, is'number per unit area of cell plate' (Gunning, 1978)] ofplasmodesmata during all stages of gametophytedevelopment. The favorable geometry of the ferngametophyte made it practical to perform this exhaus-tive study of how the plasmodesmatal network isestablished in a developing plant. We observed that thedistribution of plasmodesmata between cells relates tothe particular patterns of cell division at each stage ofgametophyte development, the end result being a well-organized and precisely programmed network ofplasmodesmata that appears to be involved in prothal-lial development.

Materials and methods

Culture conditionsThe culture conditions followed those described by Cookeand Paolillo (1979) for the preparation of Onoclea sensibilis L.gametophytes. Briefly, sporophylls were collected fromThompkins County, New York and stored in polyethylenebags at -20°C. Spores were wetted with 0.1 % Triton X 405(Sigma Chem. Co., St Louis, MO) and then sterilized with

10 % Clorox for 75 s. These spores were plated on 0.8 % agarmade up in Voth's No. 5 medium with common inorganic salts(Voth, 1943) supplemented with 1% sucrose at pH6.0. Thespores were germinated under cool-white fluorescent lightswith an intensity of 150^Em~2s~' for 24 h, wrapped withthree layers of aluminum foil and stored in the dark forperiods of 10 to 14 days at 25 °C.

Protonemata were obtained directly from the agar mediumafter removing the aluminum foil. To obtain prothalli atdifferent developmental stages, the plates containing theprotonemata were exposed to 150^Em~2s~1 of continuouscool-white fluorescent light for various periods at 25 °C. Forthe sake of brevity, protonemata will be referred to as 0 daygametophytes, gametophytes exposed to light for 2 days as 2day prothalli, etc. The oldest prothalli examined in this studywere 30 day prothalli which had already produced sexualorgans.

Electron microscopyAt the appropriate time, protonemata or prothalli werecarefully removed from the agar plate with fine forceps andfixed by immersion in a freshly made fixative solutioncontaining 1% OsO4, 1% glutaraldehyde (from an 8%stock, Electron Microscope Sciences, Fort Washington, PA)and 0.05 M phosphate buffer at pH6.3 at 4°C for 45 min. Thepreparation was then rinsed 3 times in distilled water at 4°Cand en bloc stained in 0.5% uranyl acetate for 3h toovernight, washed and then dehydrated in acetone andembedded in plastic (Spurr, 1969). The early steps in theembedding procedure must be done very slowly, from 0 to10 % plastic over a course of 2 h, in order to avoid shrinkageartefacts. Gametophytes are flat embedded in small alumi-num weighing dishes. In the later stages in this study, wefound that if a glass cover slip is lain over the germinatedspores which were sown on the agar, the protonemata andprothalli tend to grow as flatter specimens.

Since both the protonema and the prothalli are only one cellthick, one must cut a frontal section parallel to its upper andlower surface to see the plasmodesmata in most of the cells ofthis flattened object. This requires carefully positioning of theembedded gametophytes. The thin sections, light purple incolor, were picked up on single hole grids which contained athin layer of support film of formvar, covered with a lightcarbon coat. The sections were stained with uranyl acetateand lead citrate and examined in a Philips 200 electronmicroscope with which photographs were taken of the wholegametophyte at 4000x. These plates were enlarged 2.6x andtaped together to form large montages, some of which were 3by 5 m. Then under a magnifier we counted the number ofplasmodesmata between all the cells in the montage. Thesecan be easily distinguished on these montages as the section isenlarged more than 10 000 x. An artist accurately drew thesection with all of its cell walls and recorded on the drawingthe number of plasmodesmata encountered in that section.

CalculationsReasonable estimates of the density of plasmodesmata, i.e.the number of plasmodesmata per unit surface area, can bederived from sectioned views of cell walls perpendicular to theplane of section as observed in the montages. The density ofplasmodesmata in each rectangular wall of the prothallus canbe calculated as the number of plasmodesmata visualized inthe wall divided by the product of the length of the wall timesthe corrected wall thickness which is equal to the sum ofactual section thickness (150 nm) and a correction factor (seediscussion in Robards, 1976). Assuming that the limit ofdetectability is one quarter of the outside radius of a

Plasmodesmata and fern morphogenesis 1211

plasmodesma, then the correction factor is equal to 1.5 timesthe plasmodesmatal radius (20 nm) and thus the correctedwall thickness is equal to 180 nm. In protonemata thetransverse walls are typically circular in face view. Thus if thecross section through the wall is a median one, the observedwall length equals the wall diameter; then, using analyticalgeometry, the surface area of a transverse wall included in thesection, but normal to its plane, can be shown to equal 4 timesthe area of a triangle with a height of x and a base of r2-*2,

and an arc of a radius of r and an angle of sin"1 -,r

•orwhere x equals one half the corrected section thickness(90 nm) and r represents one half the wall length. The densityof plasmodesmata in each transverse wall is then calculated asthe number of plasmodesmata observed in the wall divided bythe surface area of the section calculated from the aboveequation, and the total number of plasmodesmata pertransverse wall is equivalent to the number of plasmodesmataper section times the total surface area of the transverse wall(or itr2) divided by the surface area of the section calculated asabove.

In young prothalli where apical cells have reached at leastthe 'GG' division (Fig. 2-7) and all older prothalli, thegametophyte has sufficiently broadened so that the interiorwalls are approximately rectangular in shape. In this case,total number of plasmodesmata per cell wall was obtained asthe number of plasmodesmata per section times the surface

area of the total wall (the product of its length and thicknessnormal to the plane of sectioning) over the surface area of thesection (the product of the wall length and the thickness of180 nm). All these numbers were taken directly from themontages except for wall thicknesses which were estimated bythe following procedure. Median sagittal sections, i.e. theplane is perpendicular to the flat surface of the gametophyte,extending from the apical cell to the most basal cell, were cutfrom several prothalli of different ages and montages wereconstructed by the same method described for frontalsections. These montages were used to measure the prothallusthickness as a function of distance from the apical margin ofthe prothallus to the basal end. Then the distance from themidpoint of any wall of interest on a frontal montage wasmeasured to the most distal part of the apical cell. Theestimate of the wall thickness in that prothallial region couldthen be obtained from the sagittal sections. Only estimates fortotal plasmodesmatal number per wall could be calculated foryoung prothalli that had not attained the 'GG' divisionbecause their walls had a transitional shape between a circleand a rectangle.

Results

An overview of fern gametophyte developmentFig. 1A illustrates a gametophyte of Onoclea sensibilusgrown in darkness for 30 days following a lighttreatment sufficient to induce spore germination. Thisgametophyte consists of two cell types with different

1A BX

Fig. 1. (A) Light micrograph of a protonemal thread grown in the dark for 4 weeks. x56 (B) Light micrograph ofprothallus grown in the light for 4 weeks. x!42. Bars, lOjum

1212 L. G. Tilney and others

physiological activities: a colorless, single-celled rhizoidinvolved in water uptake and substrate attachment andthose cells comprising the green protonema. The linearprotonema will continue to grow indefinitely in dark-ness until the nutrient reserves in the original sporeand/or in the culture medium are exhausted. Incontrast, Fig. IB presents a gametophyte of the samespecies exposed to 30 days of continuous white lightfollowing protonemal formation. This gametophyte hasdeveloped numerous rhizoids near its base and a heart-shaped prothallus, which is composed of a single layerof photosynthetic cells.

In Fig. 2 we have included a series of drawingsillustrating the most common sequence of cell divisionsin the transition from the protonema to the prothallusfollowing the transfer from darkness to light. It isimportant for what follows to clarify the terminologyused here. In order to identify common walls, it isuseful to designate the walls in the order of theirappearance. Thus, the letters 'AA' or 'aa', indicate thefirst wall in a division sequence, 'BB' the second, 'CC

mJ / m

10

Fig. 2. Diagram of representative stages in the earlydevelopment of a prothallus from a protonemal thread.None of the stages are drawn to scale. The heavy lineindicates the most recent division plane and is letteredappropriately. 1. Protonemal thread (dark grown).2. Protonemal thread exposed to light for a few hours.3. Approximately 12h in the light. 4. Approximately 24hin the light. 5. Approximately 36 h in the light. Firstlongitudinal division, 'EE', starts two dimensional growth.6. Approximately 2 1/2 days in the light. 7. 3-4 days inthe light. 'GG' will become part of the surface of a apicalcell. 8. 5 days in the light. 'HH' is the first oblique divisionwhich gives rise to the apical cell indicated by an 'm'. In anequal number of prothalli, the 'HH' division could havemet the wall of 'GG' obliquely on its left side. 9. 6-7 daysin the light. 'II' is the second oblique division. Notice thatit cuts the former apical cell from the opposite direction,i.e. the oblique division alternates from the right, then theleft, etc. 10. 7-8 days in the light. 'JJ' is the third obliquedivision that alternates from the last cutting to the right.

the third, and so forth. The letters 'ZZ' indicate the lastwall in the division sequence, with 'YY' the next to last,etc. Lower case letters designate cell divisions thatoccurred in protonemata growing in darkness andupper case letters designate cell divisions that occurredin light-grown prothalli. The sequence illustrated inFig. 2 is derived from our observations as well as from aclassic paper in the field (Dopp, 1927). The pattern inFig. 2 is not drawn to scale.

In our experiments, the protonemata were grown incomplete darkness for 10 to 14 days until the apical cellhad typically undergone two cell divisions ('aa' and 'bb'in Fig. 2-1). After the protonemata are transferredfrom darkness to our standard light conditions, within2h the apical cell starts to swell in a lateral direction(Fig. 2-2). Subsequently in 12 to 14 h it undergoes onetransverse division ('CC' in Fig. 2-3) and then a secondtransverse division 12 to 18 h. later ('DD' in Fig. 2-4).The third division ('EE' in Fig. 2-5), which is typicallylongitudinal to the protonemal axis, marks the initiationof two-dimensional growth. The young prothallusbegins to broaden as a planar structure without anyincrease in its thickness. The next divisions can occur inseveral cells, but we have depicted a common sequenceof apical cell divisions (a transverse division, 'FF' inFig. 2-6 and a longitudinal division, 'GG' in Fig. 2-7).These divisions produce 3 cells at the apical end of theyoung prothallus. What follows is an oblique division inthe central cell of the 3 apical cells with the new cell wallcontacting the most recent longitudinal cell wall ('HH'in Fig. 2-8). This division marks the formation of thetriangular apical cell which will divide by an alternatingseries of oblique divisions; e.g. the right-sided 'II' inFig. 2-9. and the left-sided 'JJ' in Fig. 2-10. Of interestto our discussion later is the fact that the new cell wallcontacts approximately the middle of the precedingwall.

The distribution of plasmodesmataProthalli were examined at sufficient magnification toaccurately and unambiguously count the plasmo-desmata (Fig. 3). Fig. 3B is illustrated at twice themagnification we used in our montages. We doubled themagnification to make up for any loss in resolution uponreproduction in the journal. It should be obvious fromthis figure that we can accurately count the number ofplasmodesmata in a thin section.

Initially we were concerned that in a single thinsection we were not looking at the true distribution ofplasmodesmata because they might not be randomlydistributed in the cell wall, but clustered. We alleviatedthese fears by comparing the counts on the number ofplasmodesmata from a number of sections through thecell wall. The counts were remarkably consistent fromsection to section as seen in Figs 4D and 9. We alsosectioned several different prothalli at the same stage(Fig. 9) and again found the pattern consistent fromprothallus to prothallus.

A total of 35 montages of gametophytes of differentages (and serial sections of the same age) wereconstructed as described in the Materials and methods.

Plasmodesmata and fern morphogenesis 1213

3AFig. 3. (A) Thin section through a triangular apical cell from a prothallus placed in the light for 6 days. The wholeprothallus is illustrated in Fig. 6, albeit a mirror image. The nucleus of the apical cell is indicated by an n. The rectangleoutlined is illustrated at higher magnification in B. X5400. Of particular interest are the plasmodesmata, a few of which areindicated by the arrows.The magnification here is twice that used to count the plasmodesmata number in the followingfigures. X21100. Bars,

We have included a representative sample in Figs 4-7to illustrate how we arrived at the numbers used in thegraphs (Figs 9 and 11) and the summary illustration(Fig. 8).

In dark-grown protonemata, the number of plasmo-desmata present in our thin sections of cell wallsbetween protonemal cells varied from 0-9 (Fig. 4A)with an average of 2. These low numbers are consistentwith earlier observations on the protonemata ofPolypodium vulgare (Fraser and Smith, 1974) andDryopteris pseudo-mas (Cran, 1979). Interestingly, theaverage number of plasmodesmata between proto-nemal cells and adjacent rhizoids was a significantlyhigher value of 12 per section. When the protonemata

are placed in the light, the apical cell swells and thendivides transversely twice ('CC and 'DD' of Fig. 2-2and 2-3; Fig. 4B). The plasmodesmatal number in athin section of the first transverse division ('CC') is 1-5with an average of 3 (see Fig. 4B) and 4-14 with anaverage of 8 for the second transverse division, 'DD'.There are 6 in Fig. 4B for division 'DD'. The thirddivision, 'EE', which is longitudinal, has a range of5-21 with an average of 13.

The next two divisions in the apical end of theprothallus, 'FF' and 'GG' result in the formation ofthree apical cells. There is a dramatic increase in thenumber of plasmodesmata in the 'GG' division relativeto earlier divisions (Figs 4C and 4D). Here we find a

1214 L. G. Tilney and others

D

Fig. 4. Drawings of protonemata (A) and prothalli grown in the light for 2-4 days (B-D). The number placed on each cellwall indicates the number of plasmodesmata present. The scale on each drawing indicates the dimensions in microns. In Dare serial sections of the same prothallus. Note that in A and C the cell wall between the two cells at the bottom of eachprotonema, which will give rise to a rhizoid, has more plasmodesmata than the subsequent cells. The letters on B-Dcorrespond to the sequence of divisions illustrated in Fig. 2. Note that the plasmodesmatal number in 'GG' in C and D ismuch, much greater than that of earlier divisions, e.g. 'EE' or 'FF'.

range of 25-37 with an average number of 30. The nextdivision, 'HH' (Fig. 2-8) will be an oblique division thatwill produce the triangular apical cell. Although thisdivision in principle could extend from the apical end ofthe prothallus and contact the lateral surface of eitherthe 'EE' or 'GG' wall, both of which are of comparablelength, (see Fig. 2), in fact it always contacts the 'GG'wall, distinguishable by its large number of plasmo-desmata relative to the 'EE' wall or a portion of it. Thedifferences in numbers here are 6-8 for the portion ofthe 'EE' wall available for contact with the newlyforming 'HH' wall versus 25-37 for the 'GG' wall.

The triangular apical cell is easy to identify in 6 dayand older prothalli (Fig. 5, indicated by M). Its externalsurface is continuous with the apical edge of theprothallus and the other 2 walls extend from theexternal surface towards each other at an oblique angle.It is characteristic of cell divisions in fern prothalli for ayounger wall to contact an older wall near its midpointand thus any new wall tends to bisect the cell in which it

occurs (Dopp, 1927 and our observations). (The onlyexceptions to these general rules are the asymmetricdivisions that result in specialized structures such ashairs, rhizoids, antheridia and archegonia.) Thus wecan easily map out the last 5 divisions of the apical cellin this prothallus. The last division is 'ZZ', the next tolast division 'YY', etc. These are indicated in Fig. 5. Ofinterest is that the apical cell in Fig. 5 has a total of 83plasmodesmata and its most recent precursor cell,defined by walls 'YY' and most of 'XX' has 77. Giventhe smaller internal wall area of the apical cell, thismeans that the apical cell has the greatest density ofplasmodesmata per unit wall length; a point to which wewill return later.

By 9 days the prothalli are just beginning to becomeheart shaped with the apical cell in the exact center ofthe developing heart (Fig. 6). As is the case with 6 dayprothalli, the walls of the apical cell and its most recentderivative display the highest number of plasmo-desmata per section: 174 and 168 in Fig. 6. Again the

Plasmodesmata and fern morphogenesis 1215

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BFig. 5. Drawing of a serial section (A) through aprothallus grown in the light for 5 days. The apical cell, M,is indicated. In B we have indicated the last cell division,'ZZ', the next to last, 'YY', and the next to the next tolast, 'XX', etc.

density of plasmodesmata per unit wall length is highestin the apical cell.

The 30 day prothalli are composed of large numbersof cells, but the apical cell can still be identified by itstriangular shape, by its central position, and by the highnumber of plasmodesmata (Fig. 7). All the obser-vations made in reference to the 9 day prothalli alsoapply to these older prothalli. (1) The apical cell has thehighest density of plasmodesmata in the prothallus,(2) The density of plasmodesmata in the walls ('ZZ','YY', 'XX', etc) derived from the apical cell declines asone proceeds away from the apical cell.

The data included so far, plus other montages notpresented are summarized in Fig. 8.

Quantitation of the distribution of plasmodesmata inthe apical cells at different stages of developmentThe number of plasmodesmata counted in the walls of

the apical cells in thin sections through individualgametophytes is plotted as a function of age in Fig. 9.

As expected from the discussion in the previoussection, the number of plasmodesmata in the apical cellwalls depends on the particular stage of gametophytedevelopment, with a minimum of 2 or less in 0 dayprotonemata to a maximum of around 170 in 9 day andolder prothalli.

However, the number of plasmodesmata does notprovide any measure of the potential fluxes across thesewalls, which must instead depend on the density ofplasmodesmata, or the number per unit surface area ofcell wall. Since our serial sections indicate a randomdistribution of plasmodesmata, their density can easilybe calculated from a single section of known thickness.Fig. 9 shows that the density of plasmodesmata in theapical cells increases as the gametophyte develops fromthe dark-grown protonema (

1216 L. G. Tilney and others

Fig. 6. Drawing of a section through the apical end of a 9 day prothallus. By this stage in most prothalli the beginning ofan indentation that will be the center of the heart-shaped prothallus can be seen. Notice in this region one can identify thetriangular-shaped apical cell with its striking number of plasmodesmata. In B we have diagrammed the last 5 consecutivedivisions of this prothallus.

number of plasmodesmata are estimated by the sameprocedures as above.

What we would like to determine is whetheradditional plasmodesmata appear in existing cell wallsor is the number fixed at the time of cell wall formation.Two sets of observations, summarized in Fig. 11, beardirectly on this question. First, if one proceeds from themost recent wall ('ZZ') to earlier walls ('YY', 'XX','WW, and finally 'VV') from apical cell divisions in 3,4, 6, and 9 day prothalli (Fig. 11A or C), it is obviousthat the number of plasmodesmata per section isincreasing with each successive apical cell division('VV to 'ZZ'). Thus, younger cell walls have moreplasmodesmata than older ones, e.g. compare 'ZZ' to'WW'. It follows from this simple observation that therecan be no significant secondary formation of plasmo-desmata following the formation of the initial cell plate,unless formation of new plasmodesmata is balanced bythe loss of existing ones. Second, in 30 day prothalli thenumber of plasmodesmata in the most recent ('ZZ')and in earlier cell walls ('YY', 'XX', 'WW, and ' W )remains constant (Fig. 11A or C). Thus no net synthesisof plasmodesmata occurs following the initial appear-ance of the cell wall.

Since additional plasmodesmata are not added to cellwalls that have already formed, yet existing cell wallsexpand as the prothallus grows, it must be true that thedensity of plasmodesmata must fall as each cell 'moves'basally from the apical notch. This fact is easilyobserved in Fig. 11B.

Discussion

Since we are ultimately interested in how intercellularcommunication regulates the morphogenesis of a plant,the obvious initial step was to describe the plasmo-desmatal network. The fern gametophyte offers a mostfavorable geometry for this task because a single frontalsection of any gametophyte provides sufficient infor-mation, to deduce the sequence of recent cell divisions atthat particular developmental stage. In addition, sincethe gametophyte grows as a two-dimensional structure,one cell thick, one can easily use that frontal section tocalculate the density of plasmodesmata, i.e. the numberper unit surface area of a particular cell wall, as well asthe total number of plasmodesmata inserted into thatcell. Such calculations are much, much more difficult inthe three-dimensional structures of higher plants oreven the sporophytic structures of lower plants such asmosses and ferns. Furthermore, if one compares thefrontal sections of different stages from dark-grownprotonemata to mature prothalli, it becomes possible toreconstruct the complete formation of the plasmodes-matal network between individual cells throughoutgametophyte development. It is absolutely crucial tofollow the plasmodesmatal network on the basis ofindividual cells, because fern gametophytes, like manyother plant structures, exhibit reproducible growthpatterns that arise from the activity of apical cells(Dopp, 1927).

The picture that emerges from our electron micro-

Plasmodesmata and fern morphogenesis 1217

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Fig. 7. Drawing of a section through the apical end of a 30 day old prothallus. The apical cell, M, is readily recognized byits shape, location and number of plasmodesmata. In the insert we have diagrammed the last 7 consecutive divisions of thisprothallus.

graphs is that the distribution of plasmodesmata istightly regulated in the apical cell and its derivatives atevery stage of fern gametophyte development. Further-more, there is a 50-fold increase in plasmodesmata inthe walls of the apical cell from the protonemata to themature prothallus, but once the initial cell plate forms,no new plasmodesmata appear.

Previous descriptions of plasmodesmatal networkshave been restricted to small specialized structures suchas secretory glands (Eleftheriou and Hall, 1983;Gunning and Hughes, 1976) or to certain developmen-tal stages of isolated organs such as roots (Juniper andBarlow, 1969; Gunning, 1978). Gunning's monumentalstudy on Azolla roots is certainly worthy of consider-able discussion. Using the precise organization of celllineages which are derived from 55 or so divisions of thesingle apical cell, Gunning (1978) was able to character-ize the distribution of plasmodesmata in the subapicalto basal regions of entire Azolla roots ranging in agefrom a young root whose apical cell had undergone its24th division to older roots whose apical cell had justcompleted its 55th division. This work led to severalimportant conclusions with respect to the plasmo-desmatal network during steady-state and senescentgrowth: (1) plasmodesmatal number is precisely regu-

lated according to the position of the new cell wall, (2)no secondary formation of plasmodesmata is seen inolder walls, and (3) the last subapical cells formed fromthe senescent apical cell have fewer plasmodesmatainserted in their walls. A subsequent study, whichexamined many roots whose apical cells had undergonebetween 20 and 55 divisions, demonstrated that thestriking decrease in plasmodesmatal number of thesenescent apical cell is accompanied by a correspondingdecrease in the electrical coupling to its most recentderivatives (Overall and Gunning, 1982). Our studycomplements Gunning's work on Azolla roots (1978) asit provides developmental information on the plasmo-desmatal network both during the initiation of theapical cell and as the apical cell differentiates.

Before discussing the possible developmental roles ofthe plasmodesmatal network, we should emphasize thefollowing 4 points. First, an increase in plasmodesmatalnumber comparable to what we observed in thedeveloping gametophyte has never been documentedfor the apical cell(s) of any other system. Second, apicalcells are traditionally identified by their distinctiveshapes and strategic positions; but the present studyshows that these apical cells are also characterized byhaving the highest density of plasmodesmata relative to

1218 L. G. Tilney and others

30 (38) 46 51

Fig. 8. In this diagram, which is the same used in Fig. 2,the letters indicating the successive divisions leading to anapical cell are eliminated. On the walls that are thicker,indicating the most recent cell plate formation, we haveplaced the number of plasmodesmata that we would findon these walls. This number is an average number derivedfrom either several sections of the same prothallus orsections of several prothalli of the same age. One number(38) enclosed by parentheses, is the number we expect tofind at this stage. We unfortunately do not have anexample of this.

any other cells in the developing prothallus. Third, itseems that the elaboration of the plasmodesmatalnetwork does not happen as a passive feature of overallprothallial development but the network may insteadcontribute to the construction of the triangular apicalcell itself. This tentative interpretation comes from theobservation that an abrupt increase in plasmodesmatalnumber occurs in the cell wall that is destined toconstruct one side of the future apical cell before thatapical cell appears. Fourth, in fern prothalli theformation of all plasmodesmata occurs only during newcell wall formation. This conclusion is consistent withthe observations in certain other systems (Gunning,1978), although secondary formation of plasmodesmatain mature walls is seen in unusual circumstances, whichinclude graft unions and parasitic haustoria (seeGunning and Steer, 1975; Binding et al. 1987; Kollmanand Glockmann, 1985; Kollmann et al. 1985).

Plasmodesmatal densities in apical cells arecomparable to the maximum densities present insecretory tissuesIn many plants there is circumstantial evidence tosuggest that plasmodesmata act to convey smallmolecules throughout the plant. All plant cell wallsexamined to date with the notable exceptions of thosewalls between the reproductive cells (spores andgametes) and adjacent vegetative tissue (Carr, 1976) areobserved to contain plasmodesmata. Dye injection

200

160

120

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Gametophyte age (d)

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Gametophyte age (d)

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0 2 4 6 8 10 20Gametophyte age (d)

30

Fig. 9. (A) Graph expressing the total number ofplasmodesmata encountered in a section through the wallsof the apical cell as a function of the length of time theprothallus was exposed to light (gametophyte age in days).(B) Graph depicting the density of plasmodesmata ornumber of plasmodesmata per /im2 of cell wall of theapical cell as a function of age of the prothallus(gametophyte age in days). (C) Graph depicting the totalnumber of plasmodesmata present attached to the apicalcell walls as a function of age of the prothallus(gametophyte age in days).

studies have shown that the plasmodesmata in mostplant structures can readily transport water solublemolecules of 800 Mt or less between adjacent cells(Barclay et al. 1982; Goodwin, 1983; Tucker, 1982;Tucker, 1987). These observations are entirely consist-

Plasmodesmata and fern morphogenesis 1219

80

I 70

60

50

3 4 0

30

.£. 6d Longitudinal section

v 30d Longitudinal section

0 100 200 300 400 500 600 700 800 900Distance from apex (/an)

Fig. 10. Graph expressing the thickness of the prothallus inlim as determined by a mid sagittal section through 6 and30 day prothalli as a function of the distance from the apexor apical notch towards the basal end of the prothallus.

100

1 60•ao

40

20

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ent with electrophysiological measurements of inter-cellular coupling which demonstrate that the plasmo-desmata represent a low resistance pathway relative toalternative routes across the plasma membranes (Drakeetal. 1978; Overall and Gunning, 1982; Racusen, 1976;Spanswick, 1972). With these observations in mind, onewould suspect that specialized cells such as secretorycells and sieve elements known to transport metabolitesat high rates would be characterized by high densitiesand/or enlarged diameters of their plasmodesmata(Gunning and Steer, 1975; Ledbetter and Porter, 1970).Indeed, some of the highest reliable values forplasmodesmatal densities for cell walls located inphotosynthetic tissues are found in secretory cells: 12.6plasmodesmata per ,um2 in Abutilon nectary hairs(Gunning and Hughes, 1976), 7 to 35 per ^m2 inUtricularia trap hairs (Fineran and Lee, 1975), 6 to 10per (Um2 in Limonium salt glands (Faraday et al. 1986),and 9.1 to 16.6 per fim2 in Gossypium secretory hairs

16

J5 12

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o

30 d

f9d

VV WW XX YY ZZCell wall

Fig. 11. (A) Graph expressing the number of plasmodesmataper section in the cell walls that resulted from the last 5divisions of the apical cell. The last division is 'ZZ', the next tolast 'YY', and so forth. Information on the last 5 divisions ofprothalli exposed to light for 3, 4, 6, 9 and 30 days are shownin the graph. (B) Graph expressing the density ofplasmodesmata or the number of plasmodesmata per /m\2 in thecell walls that resulted from the last 5 divisions of the apicalcell. As in A, the last division is 'ZZ', the next to last 'YY',and so forth. Information on the last 5 divisions of the apicalcell of prothalli exposed to light for 3, 4, 6, 9 and 30 days areincluded. (C) Graph expressing the total number ofplasmodesmata in the cell walls that resulted from the last 5divisions of the apical cell. The last division is 'ZZ', the next tolast 'YY', and so forth. Information on the last 5 divisions ofthe apical cell of prothalli exposed to light for 3, 4, 6, 9 and 30days are shown on this graph.

1220 L. G. Tilney and others

(Eleftheriou and Hall, 1983). Of interest is that thedensities just mentioned are comparable to the densityof plasmodesmata in the triangular apical cell of the 30day prothallus. This suggests that the apical cell may becapable of metabolic transport at rates comparable tothose measured in secretory structures. Recent studieshave already demonstrated very rapid fluorescent dyemovement between prothallial cells (Tucker andCooke, 1990).

What might be the developmental consequences of thispatterned distribution of plasmodesmata?Nagai (1914) was the first to demonstrate that tempor-ary plasmolysis is sufficient to completely disruptprothallial development. He observed that almost everycell in a prothallus returned to normal osmoticconditions will subsequently divide perpendicular to thesurface of the prothallus to form a rhizoid initial and aprotonemal initial in a manner that mimics the firstdivision in a germinating spore. The protonemal initialwill then develop into a mature prothallus of normalappearance. These original observations have beenrepeated with enough other species to confirm that thisresponse to plasmolysis is a general feature of ferngametophytes (see reviews of Miller, 1968; Raghavan,1989). Equally revealing was an experiment of Ito(1962). Using a microneedle to ablate all the surround-ing cells, he showed that isolated cells that maintaintheir turgor pressure are also able to regenerate entireprothalli. Moreover, he observed a definite pattern inthe timing of regeneration: the closer the cell is to theapical cell, the longer the interval between plasmolysisand regrowth. Therefore the initiation of new prothallifrom mature cells must necessarily depend on thedisruption of intercellular communication via theplasmodesmata. It is unlikely that it can be attributed tounknown side effects of the different treatmentsbecause each employs a unique method to disrupt theplasmodesmata.

In short, from the evidence in the literature theplasmodesmata must be transporting a substance orsubstances that disciplines all the cells in the prothallusto behave in a coordinated fashion. Furthermore, sincethe microsurgical removal of the apical half will alsoinduce certain cells in the basal half to produce newsecondary prothalli (Albaum, I938a,b), it stands toreason that the triangular apical cell and/or the entireapical region must be exporting this 'disciplinarysubstance(s)' at a prodigious rate. Such activity willnecessarily require a high density of plasmodesmata,which is true for the triangular apical cell and its mostrecent derivatives.

Given the favorable geometry of the fern gameto-phyte and the sensitivity of its cells, it may be possibleto actually identify the intercellular signal beingtransported in the plasmodesmata.

We would like to thank Scott Poethig and Ed Tucker foranimated discussions of this work as new results appeared.We wish to thank Lisa Ireland, Bob Golder and especially

Doug Rugh, who did the lion's share, for drawing themontages for this paper. Supported by a grant from NIH, HD144-74.

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{Accepted 31 August, 1990)