1209
Development 110, 1209-1221 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
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, USA
Department of Botany, University of Maryland, College Park, MD 20742, USA
3
Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA
2
* Author for correspondence
Summary
Fern (Onoclea sensibilis) gametophytes when grown in
the dark form a linear file of cells (one-dimensional)
called a protonema. In the light two-dimensional growth
occurs which results in a heart-shaped prothallus one
cell thick. The objective of this paper is to relate the most
common pattern of cell division observed in developing
gametophytes to the formation of the plasmodesmatal
network. Since the prothalli are only two dimensional,
we can easily determine from thin sections the total
number and the density (number per unit surface area)
of plasmodesmata at each developmental stage. As the
prothallus grows the number of plasmodesmata increases 50-fold in the apical or meristematic cell. This
number eventually reaches a plateau even though the
density continues to increase with each new cell division.
What is particularly striking is that both the number and
density of plasmodesmata between adjacent cells is
precisely determined. Furthermore, the pattern of
plasmodesmata distribution is predictable so that (1) we
can identify the apical meristematic cells by their
plasmodesmata number, or density, as well as by their
size, shape and location, (2) we can predict, again from
plasmodesmata number, the location of a future wall of
the apical cell prior to its actual formation, (3) we can
show that the density of plasmodesmata in the triangular
apical cell of the prothallus (14 plasmodesmata/*m~2) is
comparable to those reported for secretory glands which
are known to have high rates of plasmodesmatal
transport and (4) we can show that once the plasmodesmata have been formed during division, no subsequent change in the number of plasmodesmata occurs
following cell plate formation.
Introduction
into a heart-shaped object, a prothallus. The prothallus
is composed of a flat plate of cells one cell thick. Thus
the one-dimensional pattern of tip growth in the
protonema can be converted to the two-dimensional
planar growth (in the prothallus) by transferring the
gametophyte to the light. The reverse process can also
occur by transferring the prothallus to the dark
(Sobota, 1970).
There are three reasons why the fern gametophyte is
an ideal system for the study of morphogenesis in
plants. First, depending on the form one chooses, a
protonema or a prothallus, one can study either one- or
two-dimensional growth and its regulation without
having to worry about the enormous complexity
produced by a three-dimensional plant. Second, gametophytes grow readily on moist filter paper or agar
medium and do not require complex organic supplements, which may have uncontrolled effects on plant
growth. Third, there is an extensive literature on fern
gametophytes that began at the turn of the century and
Little is known about how cell division, cell expansion
and cell differentiation are related to the generation of
form in plants. Unfortunately most higher plants are
composed of massive three-dimensional organs with
complex intercellular interactions. Thus our observations on cell behavior are usually based on average
values for large populations of heterogeneous cells.
Accordingly, there is a compelling need to exploit a
system in which the behavior of individual cells can be
related to the whole organism as it differentiates.
A system of choice is the fern gametophyte which has
two basic forms (Furuya, 1983; Miller, 1968) (Fig. 1). If
the gametophyte is grown in the dark, it produces a long
filament, the protonema, which consists of a single file
of cells. Protonemal growth occurs by a process of tip
growth whereby cell wall elongation and cell division
are restricted to the apical cell. In contrast, if the spore
is exposed to light, the resulting gametophyte grows
Key words: fern, Onoclea sensibilis, gametophyte,
protonema, prothallus, plasmodesmata.
1210 L. G. Tilney and others
has continued into modern times with numerous studies
on the growth effects of visible light (Charlton, 1938;
Furuya, 1983), X-irradiation (Rottman, 1939) hormones and other growth substances (Smith, 1979),
plasmolysis (Nagai, 1914; Nakazawa, 1963), and microsurgery (Albaum, 1938a; Albaum, 19385; Ito, 1962).
Much background information is also available on the
growth 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). The
literature on fern gametophyte development has been
reviewed by Miller (1968) and Raghavan (1989).
What has not been investigated so far is the possible
morphogenetic role of intercellular communication via
the plasmodesmata, i.e. the cytoplasmic bridges between neighboring cells (for reviews see Gunning and
Overall, 1983; Gunning and Robards, 1976). This is
surprising as there is a large literature available which
documents that the plasmodesmata may somehow play
a key role in regulating fern gametophyte morphogenesis. For example Nagai (1914) and Nakazawa (1963)
demonstrated that a brief exposure to plasmolysis,
sufficient to break the plasmodesmata, induces each cell
in the prothallus to differentiate into a new complete
prothallus. Similar results are achieved with surgical
procedures (Albaum, 19385). All these observations
lead to the same conclusion, namely, that there must be
some signal transmitted via the plasmodesmata from
cell to cell throughout the gametophyte so that the
prothallus behaves as a coordinated unit.
We have begun to explore intercellular communication in the fern gametophyte with the ultimate aim of
trying to determine how the morphogenesis of this
simple organism is controlled. Using reconstruction
techniques similar to those used to describe the
plasmodesmatal network in Azolla roots (Gunning,
1978), we have characterized the number and the
density [density, in keeping with earlier terminology, is
'number per unit area of cell plate' (Gunning, 1978)] of
plasmodesmata during all stages of gametophyte
development. The favorable geometry of the fern
gametophyte made it practical to perform this exhaustive study of how the plasmodesmatal network is
established in a developing plant. We observed that the
distribution of plasmodesmata between cells relates to
the particular patterns of cell division at each stage of
gametophyte development, the end result being a wellorganized and precisely programmed network of
plasmodesmata that appears to be involved in prothallial development.
Materials and methods
Culture conditions
The culture conditions followed those described by Cooke
and Paolillo (1979) for the preparation of Onoclea sensibilis L.
gametophytes. Briefly, sporophylls were collected from
Thompkins County, New York and stored in polyethylene
bags 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 % agar
made up in Voth's No. 5 medium with common inorganic salts
(Voth, 1943) supplemented with 1% sucrose at pH6.0. The
spores were germinated under cool-white fluorescent lights
with an intensity of 150^Em~ 2 s~' for 24 h, wrapped with
three layers of aluminum foil and stored in the dark for
periods of 10 to 14 days at 25 °C.
Protonemata were obtained directly from the agar medium
after removing the aluminum foil. To obtain prothalli at
different developmental stages, the plates containing the
protonemata were exposed to 150^Em~ 2 s~ 1 of continuous
cool-white fluorescent light for various periods at 25 °C. For
the sake of brevity, protonemata will be referred to as 0 day
gametophytes, gametophytes exposed to light for 2 days as 2
day prothalli, etc. The oldest prothalli examined in this study
were 30 day prothalli which had already produced sexual
organs.
Electron microscopy
At the appropriate time, protonemata or prothalli were
carefully removed from the agar plate with fine forceps and
fixed by immersion in a freshly made fixative solution
containing 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. The
preparation was then rinsed 3 times in distilled water at 4°C
and en bloc stained in 0.5% uranyl acetate for 3h to
overnight, washed and then dehydrated in acetone and
embedded in plastic (Spurr, 1969). The early steps in the
embedding procedure must be done very slowly, from 0 to
10 % plastic over a course of 2 h, in order to avoid shrinkage
artefacts. Gametophytes are flat embedded in small aluminum weighing dishes. In the later stages in this study, we
found that if a glass cover slip is lain over the germinated
spores which were sown on the agar, the protonemata and
prothalli tend to grow as flatter specimens.
Since both the protonema and the prothalli are only one cell
thick, one must cut a frontal section parallel to its upper and
lower surface to see the plasmodesmata in most of the cells of
this flattened object. This requires carefully positioning of the
embedded gametophytes. The thin sections, light purple in
color, were picked up on single hole grids which contained a
thin layer of support film of formvar, covered with a light
carbon coat. The sections were stained with uranyl acetate
and lead citrate and examined in a Philips 200 electron
microscope with which photographs were taken of the whole
gametophyte at 4000x. These plates were enlarged 2.6x and
taped together to form large montages, some of which were 3
by 5 m. Then under a magnifier we counted the number of
plasmodesmata between all the cells in the montage. These
can be easily distinguished on these montages as the section is
enlarged more than 10 000 x. An artist accurately drew the
section with all of its cell walls and recorded on the drawing
the number of plasmodesmata encountered in that section.
Calculations
Reasonable estimates of the density of plasmodesmata, i.e.
the number of plasmodesmata per unit surface area, can be
derived from sectioned views of cell walls perpendicular to the
plane of section as observed in the montages. The density of
plasmodesmata in each rectangular wall of the prothallus can
be calculated as the number of plasmodesmata visualized in
the wall divided by the product of the length of the wall times
the corrected wall thickness which is equal to the sum of
actual section thickness (150 nm) and a correction factor (see
discussion in Robards, 1976). Assuming that the limit of
detectability is one quarter of the outside radius of a
Plasmodesmata and fern morphogenesis 1211
plasmodesma, then the correction factor is equal to 1.5 times
the plasmodesmatal radius (20 nm) and thus the corrected
wall thickness is equal to 180 nm. In protonemata the
transverse walls are typically circular in face view. Thus if the
cross section through the wall is a median one, the observed
wall length equals the wall diameter; then, using analytical
geometry, the surface area of a transverse wall included in the
section, but normal to its plane, can be shown to equal 4 times
the area of a triangle with a height of x and a base of r 2 -* 2 ,
and an arc of a radius of r and an angle of sin"1 -,
r
•or
where x equals one half the corrected section thickness
(90 nm) and r represents one half the wall length. The density
of plasmodesmata in each transverse wall is then calculated as
the number of plasmodesmata observed in the wall divided by
the surface area of the section calculated from the above
equation, and the total number of plasmodesmata per
transverse wall is equivalent to the number of plasmodesmata
per section times the total surface area of the transverse wall
(or itr2) divided by the surface area of the section calculated as
above.
In young prothalli where apical cells have reached at least
the 'GG' division (Fig. 2-7) and all older prothalli, the
gametophyte has sufficiently broadened so that the interior
walls are approximately rectangular in shape. In this case,
total number of plasmodesmata per cell wall was obtained as
the number of plasmodesmata per section times the surface
1A
B
area of the total wall (the product of its length and thickness
normal to the plane of sectioning) over the surface area of the
section (the product of the wall length and the thickness of
180 nm). All these numbers were taken directly from the
montages except for wall thicknesses which were estimated by
the following procedure. Median sagittal sections, i.e. the
plane is perpendicular to the flat surface of the gametophyte,
extending from the apical cell to the most basal cell, were cut
from several prothalli of different ages and montages were
constructed by the same method described for frontal
sections. These montages were used to measure the prothallus
thickness as a function of distance from the apical margin of
the prothallus to the basal end. Then the distance from the
midpoint of any wall of interest on a frontal montage was
measured to the most distal part of the apical cell. The
estimate of the wall thickness in that prothallial region could
then be obtained from the sagittal sections. Only estimates for
total plasmodesmatal number per wall could be calculated for
young prothalli that had not attained the 'GG' division
because their walls had a transitional shape between a circle
and a rectangle.
Results
An overview of fern gametophyte development
Fig. 1A illustrates a gametophyte of Onoclea sensibilus
grown in darkness for 30 days following a light
treatment sufficient to induce spore germination. This
gametophyte consists of two cell types with different
X
Fig. 1. (A) Light micrograph of a protonemal thread grown in the dark for 4 weeks. x56 (B) Light micrograph of
prothallus grown in the light for 4 weeks. x!42. Bars, lOjum
1212 L. G. Tilney and others
physiological activities: a colorless, single-celled rhizoid
involved in water uptake and substrate attachment and
those cells comprising the green protonema. The linear
protonema will continue to grow indefinitely in darkness until the nutrient reserves in the original spore
and/or in the culture medium are exhausted. In
contrast, Fig. IB presents a gametophyte of the same
species exposed to 30 days of continuous white light
following protonemal formation. This gametophyte has
developed numerous rhizoids near its base and a heartshaped prothallus, which is composed of a single layer
of photosynthetic cells.
In Fig. 2 we have included a series of drawings
illustrating the most common sequence of cell divisions
in the transition from the protonema to the prothallus
following the transfer from darkness to light. It is
important for what follows to clarify the terminology
used here. In order to identify common walls, it is
useful to designate the walls in the order of their
appearance. Thus, the letters 'AA' or 'aa', indicate the
first wall in a division sequence, 'BB' the second, 'CC
m
J /m
10
Fig. 2. Diagram of representative stages in the early
development of a prothallus from a protonemal thread.
None of the stages are drawn to scale. The heavy line
indicates the most recent division plane and is lettered
appropriately. 1. Protonemal thread (dark grown).
2. Protonemal thread exposed to light for a few hours.
3. Approximately 12h in the light. 4. Approximately 24h
in the light. 5. Approximately 36 h in the light. First
longitudinal division, 'EE', starts two dimensional growth.
6. Approximately 2 1/2 days in the light. 7. 3-4 days in
the light. 'GG' will become part of the surface of a apical
cell. 8. 5 days in the light. 'HH' is the first oblique division
which gives rise to the apical cell indicated by an 'm'. In an
equal number of prothalli, the 'HH' division could have
met the wall of 'GG' obliquely on its left side. 9. 6-7 days
in the light. 'II' is the second oblique division. Notice that
it cuts the former apical cell from the opposite direction,
i.e. the oblique division alternates from the right, then the
left, etc. 10. 7-8 days in the light. 'JJ' is the third oblique
division that alternates from the last cutting to the right.
the third, and so forth. The letters 'ZZ' indicate the last
wall in the division sequence, with 'YY' the next to last,
etc. Lower case letters designate cell divisions that
occurred in protonemata growing in darkness and
upper case letters designate cell divisions that occurred
in light-grown prothalli. The sequence illustrated in
Fig. 2 is derived from our observations as well as from a
classic paper in the field (Dopp, 1927). The pattern in
Fig. 2 is not drawn to scale.
In our experiments, the protonemata were grown in
complete darkness for 10 to 14 days until the apical cell
had typically undergone two cell divisions ('aa' and 'bb'
in Fig. 2-1). After the protonemata are transferred
from darkness to our standard light conditions, within
2h the apical cell starts to swell in a lateral direction
(Fig. 2-2). Subsequently in 12 to 14 h it undergoes one
transverse division ('CC' in Fig. 2-3) and then a second
transverse division 12 to 18 h. later ('DD' in Fig. 2-4).
The third division ('EE' in Fig. 2-5), which is typically
longitudinal to the protonemal axis, marks the initiation
of two-dimensional growth. The young prothallus
begins to broaden as a planar structure without any
increase in its thickness. The next divisions can occur in
several cells, but we have depicted a common sequence
of apical cell divisions (a transverse division, 'FF' in
Fig. 2-6 and a longitudinal division, 'GG' in Fig. 2-7).
These divisions produce 3 cells at the apical end of the
young prothallus. What follows is an oblique division in
the central cell of the 3 apical cells with the new cell wall
contacting the most recent longitudinal cell wall ('HH'
in Fig. 2-8). This division marks the formation of the
triangular apical cell which will divide by an alternating
series of oblique divisions; e.g. the right-sided 'II' in
Fig. 2-9. and the left-sided 'JJ' in Fig. 2-10. Of interest
to our discussion later is the fact that the new cell wall
contacts approximately the middle of the preceding
wall.
The distribution of plasmodesmata
Prothalli were examined at sufficient magnification to
accurately and unambiguously count the plasmodesmata (Fig. 3). Fig. 3B is illustrated at twice the
magnification we used in our montages. We doubled the
magnification to make up for any loss in resolution upon
reproduction in the journal. It should be obvious from
this figure that we can accurately count the number of
plasmodesmata in a thin section.
Initially we were concerned that in a single thin
section we were not looking at the true distribution of
plasmodesmata because they might not be randomly
distributed in the cell wall, but clustered. We alleviated
these fears by comparing the counts on the number of
plasmodesmata from a number of sections through the
cell wall. The counts were remarkably consistent from
section to section as seen in Figs 4D and 9. We also
sectioned several different prothalli at the same stage
(Fig. 9) and again found the pattern consistent from
prothallus to prothallus.
A total of 35 montages of gametophytes of different
ages (and serial sections of the same age) were
constructed as described in the Materials and methods.
Plasmodesmata and fern morphogenesis 1213
3A
Fig. 3. (A) Thin section through a triangular apical cell from a prothallus placed in the light for 6 days. The whole
prothallus is illustrated in Fig. 6, albeit a mirror image. The nucleus of the apical cell is indicated by an n. The rectangle
outlined is illustrated at higher magnification in B. X5400. Of particular interest are the plasmodesmata, a few of which are
indicated by the arrows.The magnification here is twice that used to count the plasmodesmata number in the following
figures. X21100. Bars,
We have included a representative sample in Figs 4-7
to illustrate how we arrived at the numbers used in the
graphs (Figs 9 and 11) and the summary illustration
(Fig. 8).
In dark-grown protonemata, the number of plasmodesmata present in our thin sections of cell walls
between protonemal cells varied from 0-9 (Fig. 4A)
with an average of 2. These low numbers are consistent
with earlier observations on the protonemata of
Polypodium vulgare (Fraser and Smith, 1974) and
Dryopteris pseudo-mas (Cran, 1979). Interestingly, the
average number of plasmodesmata between protonemal cells and adjacent rhizoids was a significantly
higher value of 12 per section. When the protonemata
are placed in the light, the apical cell swells and then
divides transversely twice ('CC and 'DD' of Fig. 2-2
and 2-3; Fig. 4B). The plasmodesmatal number in a
thin section of the first transverse division ('CC') is 1-5
with an average of 3 (see Fig. 4B) and 4-14 with an
average of 8 for the second transverse division, 'DD'.
There are 6 in Fig. 4B for division 'DD'. The third
division, 'EE', which is longitudinal, has a range of
5-21 with an average of 13.
The next two divisions in the apical end of the
prothallus, 'FF' and 'GG' result in the formation of
three apical cells. There is a dramatic increase in the
number of plasmodesmata in the 'GG' division relative
to 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 cell
wall indicates the number of plasmodesmata present. The scale on each drawing indicates the dimensions in microns. In D
are serial sections of the same prothallus. Note that in A and C the cell wall between the two cells at the bottom of each
protonema, which will give rise to a rhizoid, has more plasmodesmata than the subsequent cells. The letters on B-D
correspond to the sequence of divisions illustrated in Fig. 2. Note that the plasmodesmatal number in 'GG' in C and D is
much, much greater than that of earlier divisions, e.g. 'EE' or 'FF'.
range of 25-37 with an average number of 30. The next
division, 'HH' (Fig. 2-8) will be an oblique division that
will produce the triangular apical cell. Although this
division in principle could extend from the apical end of
the prothallus and contact the lateral surface of either
the 'EE' or 'GG' wall, both of which are of comparable
length, (see Fig. 2), in fact it always contacts the 'GG'
wall, distinguishable by its large number of plasmodesmata relative to the 'EE' wall or a portion of it. The
differences in numbers here are 6-8 for the portion of
the 'EE' wall available for contact with the newly
forming 'HH' wall versus 25-37 for the 'GG' wall.
The triangular apical cell is easy to identify in 6 day
and older prothalli (Fig. 5, indicated by M). Its external
surface is continuous with the apical edge of the
prothallus and the other 2 walls extend from the
external surface towards each other at an oblique angle.
It is characteristic of cell divisions in fern prothalli for a
younger wall to contact an older wall near its midpoint
and thus any new wall tends to bisect the cell in which it
occurs (Dopp, 1927 and our observations). (The only
exceptions to these general rules are the asymmetric
divisions that result in specialized structures such as
hairs, rhizoids, antheridia and archegonia.) Thus we
can easily map out the last 5 divisions of the apical cell
in this prothallus. The last division is 'ZZ', the next to
last division 'YY', etc. These are indicated in Fig. 5. Of
interest is that the apical cell in Fig. 5 has a total of 83
plasmodesmata and its most recent precursor cell,
defined by walls 'YY' and most of 'XX' has 77. Given
the smaller internal wall area of the apical cell, this
means that the apical cell has the greatest density of
plasmodesmata per unit wall length; a point to which we
will return later.
By 9 days the prothalli are just beginning to become
heart shaped with the apical cell in the exact center of
the developing heart (Fig. 6). As is the case with 6 day
prothalli, the walls of the apical cell and its most recent
derivative display the highest number of plasmodesmata per section: 174 and 168 in Fig. 6. Again the
Plasmodesmata and fern morphogenesis 1215
lOjum
B
Fig. 5. Drawing of a serial section (A) through a
prothallus 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 to
last, 'XX', etc.
density of plasmodesmata per unit wall length is highest
in the apical cell.
The 30 day prothalli are composed of large numbers
of cells, but the apical cell can still be identified by its
triangular shape, by its central position, and by the high
number of plasmodesmata (Fig. 7). All the observations made in reference to the 9 day prothalli also
apply to these older prothalli. (1) The apical cell has the
highest density of plasmodesmata in the prothallus,
(2) The density of plasmodesmata in the walls ('ZZ',
'YY', 'XX', etc) derived from the apical cell declines as
one proceeds away from the apical cell.
The data included so far, plus other montages not
presented are summarized in Fig. 8.
Quantitation of the distribution of plasmodesmata in
the apical cells at different stages of development
The number of plasmodesmata counted in the walls of
the apical cells in thin sections through individual
gametophytes is plotted as a function of age in Fig. 9.
As expected from the discussion in the previous
section, the number of plasmodesmata in the apical cell
walls depends on the particular stage of gametophyte
development, with a minimum of 2 or less in 0 day
protonemata to a maximum of around 170 in 9 day and
older prothalli.
However, the number of plasmodesmata does not
provide any measure of the potential fluxes across these
walls, which must instead depend on the density of
plasmodesmata, or the number per unit surface area of
cell wall. Since our serial sections indicate a random
distribution of plasmodesmata, their density can easily
be calculated from a single section of known thickness.
Fig. 9 shows that the density of plasmodesmata in the
apical cells increases as the gametophyte develops from
the dark-grown protonema (<1 plasmodesmata ,um~2)
to the 9 day prothallus (an average of 10 plasmodesmata ^m^ 2 ). The density continues to increase after
9 day to 30 day prothalli (15 plasmodesmata,um~2),
even though the number of plasmodesmata levels off
between 9 and 30 day prothalli (Fig. 9). This apparent
paradox can be rationalized by the fact that the apical
cell is becoming progressively smaller in older prothalli.
In those gametophytes whose internal walls present a
known geometry, i.e. the walls in protonemata and
older prothalli approach circular and rectangular
shapes, respectively, it is also possible to estimate the
total number of plasmodesmata in each wall from the
number of plasmodesmata per section, the corrected
section thickness and an estimate of the thickness of the
prothallus. The last estimate was obtained by median
sagittal, sections (Fig. 10) from the apical cell to the
most basal cells of prothalli (montages not shown). The
total number of plasmodesmata is expressed in Fig. 9C
as a function of gametophyte age. Significantly, the
total plasmodesmata number exhibits a linear rise from
less than 600 in dark-grown protonemata to 3xl0 4 or
more in 9 and 30 day prothalli, representing a more
than 50-fold increase. If the average outside diameter of
a plasmodesma is assumed to be 40 nm, this means that
plasmodesmata constitute an average of almost 2 % of
the total internal wall surface of the apical cell in 30 day
prothalli.
Distribution of plasmodesmata in the last 5 cell walls
formed from the apical cell - evidence that there is no
net formation of plasmodesmata following cell
division
In Fig. 11 we have plotted the distribution of plasmodesmata in the last five walls derived from apical cell
divisions ('VV to 'ZZ') for 3 to 30 day prothalli. These
divisions are indicated in Figs 5 to 7. The data plotted in
these graphs represent average values computed from
those prothalli of the same age (plus their serial
sections) which had reached at least the 'GG' division as
depicted in Fig. 2-7. The number of plasmodesmata per
section, the density of plasmodesmata, and the total
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 of
an indentation that will be the center of the heart-shaped prothallus can be seen. Notice in this region one can identify the
triangular-shaped apical cell with its striking number of plasmodesmata. In B we have diagrammed the last 5 consecutive
divisions of this prothallus.
number of plasmodesmata are estimated by the same
procedures as above.
What we would like to determine is whether
additional plasmodesmata appear in existing cell walls
or is the number fixed at the time of cell wall formation.
Two sets of observations, summarized in Fig. 11, bear
directly on this question. First, if one proceeds from the
most 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 obvious
that the number of plasmodesmata per section is
increasing with each successive apical cell division
('VV to 'ZZ'). Thus, younger cell walls have more
plasmodesmata than older ones, e.g. compare 'ZZ' to
'WW'. It follows from this simple observation that there
can be no significant secondary formation of plasmodesmata following the formation of the initial cell plate,
unless formation of new plasmodesmata is balanced by
the loss of existing ones. Second, in 30 day prothalli the
number 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 synthesis
of plasmodesmata occurs following the initial appearance of the cell wall.
Since additional plasmodesmata are not added to cell
walls that have already formed, yet existing cell walls
expand as the prothallus grows, it must be true that the
density of plasmodesmata must fall as each cell 'moves'
basally from the apical notch. This fact is easily
observed in Fig. 11B.
Discussion
Since we are ultimately interested in how intercellular
communication regulates the morphogenesis of a plant,
the obvious initial step was to describe the plasmodesmatal network. The fern gametophyte offers a most
favorable geometry for this task because a single frontal
section of any gametophyte provides sufficient information, to deduce the sequence of recent cell divisions at
that particular developmental stage. In addition, since
the gametophyte grows as a two-dimensional structure,
one cell thick, one can easily use that frontal section to
calculate the density of plasmodesmata, i.e. the number
per unit surface area of a particular cell wall, as well as
the total number of plasmodesmata inserted into that
cell. Such calculations are much, much more difficult in
the three-dimensional structures of higher plants or
even the sporophytic structures of lower plants such as
mosses and ferns. Furthermore, if one compares the
frontal sections of different stages from dark-grown
protonemata to mature prothalli, it becomes possible to
reconstruct the complete formation of the plasmodesmatal network between individual cells throughout
gametophyte development. It is absolutely crucial to
follow the plasmodesmatal network on the basis of
individual cells, because fern gametophytes, like many
other plant structures, exhibit reproducible growth
patterns that arise from the activity of apical cells
(Dopp, 1927).
The picture that emerges from our electron micro-
Plasmodesmata and fern morphogenesis 1217
lOjum
Fig. 7. Drawing of a section through the apical end of a 30 day old prothallus. The apical cell, M, is readily recognized by
its shape, location and number of plasmodesmata. In the insert we have diagrammed the last 7 consecutive divisions of this
prothallus.
graphs is that the distribution of plasmodesmata is
tightly regulated in the apical cell and its derivatives at
every stage of fern gametophyte development. Furthermore, there is a 50-fold increase in plasmodesmata in
the walls of the apical cell from the protonemata to the
mature prothallus, but once the initial cell plate forms,
no new plasmodesmata appear.
Previous descriptions of plasmodesmatal networks
have been restricted to small specialized structures such
as secretory glands (Eleftheriou and Hall, 1983;
Gunning and Hughes, 1976) or to certain developmental stages of isolated organs such as roots (Juniper and
Barlow, 1969; Gunning, 1978). Gunning's monumental
study on Azolla roots is certainly worthy of considerable discussion. Using the precise organization of cell
lineages which are derived from 55 or so divisions of the
single apical cell, Gunning (1978) was able to characterize the distribution of plasmodesmata in the subapical
to basal regions of entire Azolla roots ranging in age
from a young root whose apical cell had undergone its
24th division to older roots whose apical cell had just
completed its 55th division. This work led to several
important conclusions with respect to the plasmodesmatal network during steady-state and senescent
growth: (1) plasmodesmatal number is precisely regu-
lated according to the position of the new cell wall, (2)
no secondary formation of plasmodesmata is seen in
older walls, and (3) the last subapical cells formed from
the senescent apical cell have fewer plasmodesmata
inserted in their walls. A subsequent study, which
examined many roots whose apical cells had undergone
between 20 and 55 divisions, demonstrated that the
striking decrease in plasmodesmatal number of the
senescent apical cell is accompanied by a corresponding
decrease in the electrical coupling to its most recent
derivatives (Overall and Gunning, 1982). Our study
complements Gunning's work on Azolla roots (1978) as
it provides developmental information on the plasmodesmatal network both during the initiation of the
apical cell and as the apical cell differentiates.
Before discussing the possible developmental roles of
the plasmodesmatal network, we should emphasize the
following 4 points. First, an increase in plasmodesmatal
number comparable to what we observed in the
developing gametophyte has never been documented
for the apical cell(s) of any other system. Second, apical
cells are traditionally identified by their distinctive
shapes and strategic positions; but the present study
shows that these apical cells are also characterized by
having the highest density of plasmodesmata relative to
1218 L. G. Tilney and others
200
160
120
•S 80
40
30
(38)
46
51
/ *
0
*
2
4
6
8
10
20
30
Gametophyte age (d)
B
16
jo
12
Fig. 8. In this diagram, which is the same used in Fig. 2,
the letters indicating the successive divisions leading to an
apical cell are eliminated. On the walls that are thicker,
indicating the most recent cell plate formation, we have
placed the number of plasmodesmata that we would find
on these walls. This number is an average number derived
from either several sections of the same prothallus or
sections of several prothalli of the same age. One number
(38) enclosed by parentheses, is the number we expect to
find at this stage. We unfortunately do not have an
example of this.
•a
o
c
'o
'% 0
0
2
4
6
8
10
30
20
Gametophyte age (d)
2 5xlO4
C
'Si
any other cells in the developing prothallus. Third, it
seems that the elaboration of the plasmodesmatal
network does not happen as a passive feature of overall
prothallial development but the network may instead
contribute to the construction of the triangular apical
cell itself. This tentative interpretation comes from the
observation that an abrupt increase in plasmodesmatal
number occurs in the cell wall that is destined to
construct one side of the future apical cell before that
apical cell appears. Fourth, in fern prothalli the
formation of all plasmodesmata occurs only during new
cell wall formation. This conclusion is consistent with
the observations in certain other systems (Gunning,
1978), although secondary formation of plasmodesmata
in mature walls is seen in unusual circumstances, which
include graft unions and parasitic haustoria (see
Gunning and Steer, 1975; Binding et al. 1987; Kollman
and Glockmann, 1985; Kollmann et al. 1985).
Plasmodesmatal densities in apical cells are
comparable to the maximum densities present in
secretory tissues
In many plants there is circumstantial evidence to
suggest that plasmodesmata act to convey small
molecules throughout the plant. All plant cell walls
examined to date with the notable exceptions of those
walls between the reproductive cells (spores and
gametes) and adjacent vegetative tissue (Carr, 1976) are
observed to contain plasmodesmata. Dye injection
•§ 4X10 4
E
2xlO 4
H
lxlO4
0
2
4
6
8
10
20
Gametophyte age (d)
Fig. 9. (A) Graph expressing the total number of
plasmodesmata encountered in a section through the walls
of the apical cell as a function of the length of time the
prothallus was exposed to light (gametophyte age in days).
(B) Graph depicting the density of plasmodesmata or
number of plasmodesmata per /im2 of cell wall of the
apical cell as a function of age of the prothallus
(gametophyte age in days). (C) Graph depicting the total
number of plasmodesmata present attached to the apical
cell walls as a function of age of the prothallus
(gametophyte age in days).
studies have shown that the plasmodesmata in most
plant structures can readily transport water soluble
molecules of 800 Mt or less between adjacent cells
(Barclay et al. 1982; Goodwin, 1983; Tucker, 1982;
Tucker, 1987). These observations are entirely consist-
30
Plasmodesmata and fern morphogenesis 1219
80
I 70
.£. 6d Longitudinal section
60
50
v
30d Longitudinal section
340
30
0
100
200
300 400 500 600 700
Distance from apex (/an)
800
900
Fig. 10. Graph expressing the thickness of the prothallus in
lim as determined by a mid sagittal section through 6 and
30 day prothalli as a function of the distance from the apex
or apical notch towards the basal end of the prothallus.
ent with electrophysiological measurements of intercellular coupling which demonstrate that the plasmodesmata represent a low resistance pathway relative to
alternative routes across the plasma membranes (Drake
etal. 1978; Overall and Gunning, 1982; Racusen, 1976;
Spanswick, 1972). With these observations in mind, one
would suspect that specialized cells such as secretory
cells and sieve elements known to transport metabolites
at high rates would be characterized by high densities
and/or enlarged diameters of their plasmodesmata
(Gunning and Steer, 1975; Ledbetter and Porter, 1970).
Indeed, some of the highest reliable values for
plasmodesmatal densities for cell walls located in
photosynthetic tissues are found in secretory cells: 12.6
plasmodesmata per ,um2 in Abutilon nectary hairs
(Gunning and Hughes, 1976), 7 to 35 per ^m2 in
Utricularia trap hairs (Fineran and Lee, 1975), 6 to 10
per (Um2 in Limonium salt glands (Faraday et al. 1986),
and 9.1 to 16.6 per fim2 in Gossypium secretory hairs
100
/
A
\
\
"y
16
9d
30 d
f9d
V
J5
12
•S
8
1 60
•a
o
o
40
20
VV
WW
XX
YY
zz
Cell wall
3xlO 4
WW
XX
YY
ZZ
Cell wall
C
2xlO4
s
v
/
'
./ / \
\ ^ . . -
V
'^30d
. -*6d
.Q
f
VV
lxlO4
VV
WW
XX
Cell wall
YY
ZZ
Fig. 11. (A) Graph expressing the number of plasmodesmata
per section in the cell walls that resulted from the last 5
divisions of the apical cell. The last division is 'ZZ', the next to
last 'YY', and so forth. Information on the last 5 divisions of
prothalli exposed to light for 3, 4, 6, 9 and 30 days are shown
in the graph. (B) Graph expressing the density of
plasmodesmata or the number of plasmodesmata per /m\2 in the
cell walls that resulted from the last 5 divisions of the apical
cell. As in A, the last division is 'ZZ', the next to last 'YY',
and so forth. Information on the last 5 divisions of the apical
cell of prothalli exposed to light for 3, 4, 6, 9 and 30 days are
included. (C) Graph expressing the total number of
plasmodesmata in the cell walls that resulted from the last 5
divisions of the apical cell. The last division is 'ZZ', the next to
last 'YY', and so forth. Information on the last 5 divisions of
the apical cell of prothalli exposed to light for 3, 4, 6, 9 and 30
days are shown on this graph.
1220 L. G. Tilney and others
(Eleftheriou and Hall, 1983). Of interest is that the
densities just mentioned are comparable to the density
of plasmodesmata in the triangular apical cell of the 30
day prothallus. This suggests that the apical cell may be
capable of metabolic transport at rates comparable to
those measured in secretory structures. Recent studies
have already demonstrated very rapid fluorescent dye
movement between prothallial cells (Tucker and
Cooke, 1990).
What might be the developmental consequences of this
patterned distribution of plasmodesmata?
Nagai (1914) was the first to demonstrate that temporary plasmolysis is sufficient to completely disrupt
prothallial development. He observed that almost every
cell in a prothallus returned to normal osmotic
conditions will subsequently divide perpendicular to the
surface of the prothallus to form a rhizoid initial and a
protonemal initial in a manner that mimics the first
division in a germinating spore. The protonemal initial
will then develop into a mature prothallus of normal
appearance. These original observations have been
repeated with enough other species to confirm that this
response to plasmolysis is a general feature of fern
gametophytes (see reviews of Miller, 1968; Raghavan,
1989). Equally revealing was an experiment of Ito
(1962). Using a microneedle to ablate all the surrounding cells, he showed that isolated cells that maintain
their turgor pressure are also able to regenerate entire
prothalli. Moreover, he observed a definite pattern in
the timing of regeneration: the closer the cell is to the
apical cell, the longer the interval between plasmolysis
and regrowth. Therefore the initiation of new prothalli
from mature cells must necessarily depend on the
disruption of intercellular communication via the
plasmodesmata. It is unlikely that it can be attributed to
unknown side effects of the different treatments
because each employs a unique method to disrupt the
plasmodesmata.
In short, from the evidence in the literature the
plasmodesmata must be transporting a substance or
substances that disciplines all the cells in the prothallus
to behave in a coordinated fashion. Furthermore, since
the microsurgical removal of the apical half will also
induce certain cells in the basal half to produce new
secondary prothalli (Albaum, I938a,b), it stands to
reason that the triangular apical cell and/or the entire
apical region must be exporting this 'disciplinary
substance(s)' at a prodigious rate. Such activity will
necessarily require a high density of plasmodesmata,
which is true for the triangular apical cell and its most
recent derivatives.
Given the favorable geometry of the fern gametophyte and the sensitivity of its cells, it may be possible
to actually identify the intercellular signal being
transported in the plasmodesmata.
We would like to thank Scott Poethig and Ed Tucker for
animated 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 the
montages for this paper. Supported by a grant from NIH, HD
144-74.
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{Accepted 31 August, 1990)