J. Cell Sci. Si, 109-119 (IQ8I)
Printed in Great Britain © Company of Biologists Limited 1081
MEGASPOROGENESIS IN A HETEROSPOROUS
FERN: FEATURES OF THE ORGANELLES
IN MEIOTIC CELLS AND YOUNG MEGASPORES
P. R. BELL
Department of Botany and Microbiology, University College London,
Gower Street, London WCiE 6BT
SUMMARY
Megasporogenesis in the heterosporous fern Marsilea (Hydropterideae) shows features
intermediate between sporogenesis in homosporous fern3 and that in heterosporous seed
plants. The plastids in the dyads and young spores were associated with tubules 30—35 nm in
diameter, probably a specialized form of endoplasmic reticulum. No consistent differences in
size or cytoplasmic components could be found between the megaspores of a tetrad that might
account for differential survival. The view that megaspore viability within the tetrad is genetically determined is thereby strengthened.
INTRODUCTION
The Hydropterideae are a small group of ferns distinguished by regular heterospory
accompanied by strict segregation of sex. The formation of the female megaspores is
normally accompanied by resorption of 3 of the products of each meiosis. In these
features they resemble seed plants, but differ from all living seed plants in producing
megaspores (as the microspores) in symmetrical tetrahedral tetrads, quite similar to
those characteristic of sporogenesis in ferns generally. Sporogenesis in homosporous
ferns has now been investigated at the ultrastructural level (Sheffield & Bell, 1979)
and the process shows many parallels with microsporogenesis in flowering plants
(e.g. see Dickinson & Heslop Harrison, 1977). Megasporogenesis in flowering plants
is less well known, but the indications are that it is similar in essentials (Dickinson &
Potter, 1978). Although the homosporous ferns and flowering plants represent widely
different levels of evolutionary complexity, their methods of sporogenesis are now
known to have many features in common. The principal differences lie in a regular
loss of distinctness of the plastid envelopes during the first prophase of homosporous
sporogenesis, and the absence of nucleoloids from the cytoplasm of the young spores.
In Marsilea vestita, the subject of the present investigation, 8 tetrads of megaspores
are produced. Three megaspores in each tetrad regress rapidly as the spores separate.
Of the 8 viable megaspores only one, which eventually fills the whole sporangium,
comes to maturity. Previous reports of sporogenesis in Marsilea have all been of light
microscopic studies (Feller, 1953; Boterberg, 1956), and M. vestita itself has not been
investigated before in this respect. The present study was undertaken to discover
whether, when viewed in greater detail, megasporogenesis in Marsilea showed
no
P. R. Bell
features intermediate between sporogenesis in homosporous plants, and mega- and
microsporogenesis in the seed plants. Further, the regularity of the megaspore tetrads
in Marsilea provided an opportunity to assess whether the viability of only one of the
4 megaspores in each instance could be attributed to a conspicuous inequality in the
distribution of cytoplasmic components. There is evidence in Gingko, an archegoniate
seed plant with a single linear tetrad of megaspores, that the innermost viable spore
receives a greater volume of cytoplasm, and correspondingly more organelles, than
those that degenerate (Stewart & Gifford, 1967). Nevertheless, only if such an
inequality were found to be a general feature of megasporogenesis could it be confidently regarded as having causal significance.
The present account takes megasporogenesis in Marsilea as far as the formation of
the megaspore tetrads. The events following the breaking open of the tetrads will be
considered separately.
MATERIALS AND METHODS
Immature sporocarps of M. vestita from a plant of Californian origin, grown in a greenhouse,
were dissected on an agar surface with a microscalpel. Short lengths of receptacle (< 1 mm)
were removed and plunged immediately into 3 % glutaraldehyde (TAAB Laboratories,
Reading, U.K.) in 005 M-phosphate buffer (pH 69) at 10 °C. Tapping was adequate to
remove entrapped air. Fixation was continued for 4 h, and followed by overnight washing in
ice-cold buffer. Osmication (2 % aqueous) was for 2 h at o °C. Dehydration was in acetone and
embedding in Durcupan AR (Fluka AG, Switzerland).
The embedded material was sectioned at 4 /im with a glass knife and searched for megasporangia. These were recognizable by their larger size and their location along the upper edge of
the receptacle. The selected sections were photographed with phase-contrast optics and
remounted by the technique of Woodcock & Bell (1967) for fine-sectioning. Staining was with
saturated aqueous uranyl acetate and lead citrate (Reynolds, 1963).
RESULTS
The developmental changes during sporogenesis are considered in relation to the
condition of the nucleus. This displayed the normal stages of meiosis, although the
synaptonemal complexes at zygotene/pachytene were very indistinct.
Fig. 1. Portion of a section of sporangium showing 2 megaspore mother cells and
tapetal cell (t). a, possible autophagic region, x 25 000.
Insets: top left, thick resin section of sporangium yielding fine sections shown in
figure. The spore mother cells lie grouped at the centre, x 315. Bottom right, common
wall between tapetum (below) and spore mother cell (above) at a slightly later stage.
The position of the middle lamella (arrow) shows that the thickening is now principally on the spore mother cell side. The plasmodesmata are mostly occluded, x 75 000.
In all micrographs: m, mitochondrion ;£, plastid; n, nucleus.
Fig. 2. Portion of spore mother cell at slightly later state than Fig. 1. The boundary
of the plastid is now almost indistinguishable, that of the mitochondrion remains
distinct, r, region rich in ribosomes surrounded by endoplasmic reticulum. x 45 000.
Inset: detail of boundary of plastid (ground cytoplasm, left) showing discontinuity,
x 100000.
Fig. 3. Spore mother cell, early leptotene. Envelope of plastids again distinct. Arrows
indicate small imaginations of innermembraneof envelope into nucleoplasm. x 22 500.
Megasporogenesis in Marsilea
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P. R. Bell
Preleptotene
The megaspore mother cells, 8 in number, lay at the centre of the young sporangium. They were clearly set off as a group from the surrounding tapetum (Fig. i,
upper inset), but each cell had i face in contact with it. It was evident from 4-/*m
sections that the organelles were largely adjacent to these faces, the regions towards
the centre of the cluster being notably vesicular. This asymmetry was presumably a
consequence of the previous mitoses, the organelles remaining in the polar regions of
the preceding spindles. Initially, the face in contact with the tapetum showed open
plasmodesmata (Fig. i), but subsequently the common wall thickened, particularly on
the mother cell side (Fig. i, lower inset), and these plasmodesmata became occluded.
Nodules of wall material, about 1-5 /im across and containing irregular profiles of
membranes, were often seen at the boundaries of the mother cells (Fig. 1, right spore
mother cell). Complex membranous profiles suggestive of autophagic regions were
frequent in the tapetal cells (Fig. 1).
As soon as the mother cells became recognizable a striking change was evident in
the plastid envelopes in the mother cells. Although these remained normal in the
tapetal cells, in the mother cells the envelopes became progressively less distinct
(Fig. 1). The loss of distinctness did not affect the whole envelope uniformly, but
began locally, giving the envelope a discontinuous appearance (Fig. 2, inset). Ultimately the envelope could be distinguished only with difficulty (Fig. 2), and sometimes
not at all. The envelopes of the mitochondria by contrast remained unchanged in
both tapetum and mother cells (Figs. 1, 2).
Regions surrounded by paired membranes, containing high concentrations of
ribosomes (Fig. 2), were occasionally seen at the stage at which the plastid envelopes
were barely detectable.
Prophase
The change in the response of the plastic envelope to fixation and osmication was
found to be transient. By the beginning of leptotene the clarity of the envelopes was
re-established (Fig. 3). The asymmetry in the distribution of the organelles persisted.
Some of the plasmodesmata in the common walls between the spore mother cells
Fig. 4. Portion of nucleus of a spore mother cell, late leptotene, showing extensive
development of vesicular system from inner membrane of nuclear envelope. Arrows
indicate electron-opaque material (probably nucleolar). x 33 500.
Figs. 5, 6. Comparison of representative areas of cytoplasm of spore mother cells at
pre-leptotene (Fig. 5) and late leptotene (Fig. 6), showing fall in frequency of ribosomes. Both, x 50000.
Fig. 7. Telophase of first meiotic division. The mitochondria and plastids are congregated at the equatorial plate. The nuclear envelope of the daughter nuclei has not
reformed, x 12500.
Fig. 8. Plastids in a dyad associated with tubules in longitudinal section (upper arrow)
and transverse (lower arrow) x 37 500.
Inset: an array of tubules in the vesicular area of the cytoplasm, a few above cut
transversely, x 60000.
Megasporogenesis in Marsilea
P. R. Bell
13
Megasporogenesis in Marsilea
115
enlarged to form cytomictic channels about 25 nm wide, the remainder disappeared.
The channels were themselves not seen following leptotene.
Striking changes occurred at the nuclear envelope during prophase. At the beginning
of leptotene the inner membrane of the envelope began to invaginate into the nucleoplasm (Fig. 3, arrows) and these imaginations became more conspicuous as prophase
proceeded (Fig. 4). Membrane profiles also appeared within the imaginations (Fig. 4).
indicating a complex folding of their surfaces as the invaginations extended throughout
the nucleus. By diplotene/diakinesis, connections between the vesicular formations,
now occupying much of the nucleus, and the inner membranes of the envelope were
rarely seen, suggesting that structures beginning as invaginations were now free in
the nucleoplasm. No single nucleolus could be recognized at any time during meiosisi
but aggregates of electron-opaque material, probably nucleolar, about o-i fim in
diameter, were scattered amongst the invaginations (Fig. 4, arrows). The synaptonemal complexes were similarly distributed.
By the end of prophase the cytoplasm was markedly less dense. Although there
were insufficient areas of ground cytoplasm clear of membrane to make meaningful
counts, sections of pre-leptotene cells and those at the end of prophase showed a clear
fall in the frequency of ribosomes (Figs. 5, 6). This was not accompanied by any
substantial change in the volume of the cells.
Dyads
At telophase of the first division of meiosis the plastids and mitochondria, despite
their asymmetrical distribution in prophase, were congregated in the region of the
equatorial plate (Fig. 7). The cytoplasm in the polar regions was again largely vesicular.
A conspicuous new feature in the dyads was the association of the plastids, now often
containing prominent starch, with tubules 30-35 nm in diameter. These lay very close
to the outer membrane of the envelope and sometimes appeared continuous with it
(Fig. 8, upper arrow). Arrays of these tubules were occasionally seen elsewhere in the
cytoplasm (Fig. 8, inset). Sometimes single tubules could be seen continous with
profiles of endoplasmic reticulum (Fig. 9); but the width of the tubules (here about
47 nm) was always less than the external dimension of the sheet (about 60 nm). The
boundary of the tubules frequently had a clear unit membrane profile (Fig. 8). This
was about 13 nm in width, slightly wider than the reticular membrane with which (in
Fig. 8) it was continuous.
Fig. 9. Profile of endoplasmic reticulum in dyad cytoplasm continuous with tubule.
x 75 coo.
Fig. 10. Section of tetrahedral tetrad passing through median plane of 3 spores. The
surrounding tapetum is beginning to lose its cellular structure. The arrow indicates
the strongly osmiophilic plasmalemma of the young spore, x 4625.
Fig. 11. Plastid in young spore associated with tubules. Arrows indicate where
tubules are possibly fusing with the envelope, x 100000.
Fig. 12. Possibly autophagic area in cytoplasm of young spore in tetrad, x 22 750.
Fig. 13. Possible nucleoloids in cytoplasm of young spore, x 60000.
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P.R. Bell
Tetrads
The final division of meiosis, despite much searching, was never encountered. It
appeared to take place in the spore mother cells of one sporangium more or less
simultaneously, since all of the tetrads encountered in sectioning were at closely
similar stages of development. The tetrads were uniformly tetrahedral and there was
no evidence that the tetrads were oriented in any particular way in relation to the
longitudinal axis of the sporangium. The youngest tetrad found is shown in Fig. 10.
The plasmalemma of the young spores was already notably osmiophilic, possibly
indicating the beginning of the secretion of exine.
Examination of 6 tetrads, including that shown in Fig. 10 in which the section lay
close to the median plane of 3 of the spores, gave no indication of convincing inequality
of volume or of frequency of plastids and mitochondria in the visible spores. All the
spores appeared equally well formed. It seems unlikely, in view of the irregular
arrangement of the tetrads, that the fourth spore out of the plane of the section would
in each of these 6 instances have been strikingly different in size and contents. There
was no evidence of elimination or degeneration of organelles so long as the spores
remained adhering to each other within the boundary of the original spore mother cell.
The plastids in the young spore continued to be closely associated with tubules
(Fig. 11), and some profiles again indicated actual fusion with the envelope (e.g. Fig.
11, above). Regions of convoluted membrane enclosing cytoplasm (Fig. 12) were
occasionally seen towards the distal part of the young spore. A feature of particular
interest was the presence of electron-opaque bodies 0-3-0-6 ftm in diameter, resembling
nucleolar material (Fig. 13), in both nucleus and cytoplasm. They resembled, but
were larger than, the bodies seen only in the nuclei during prophase. The cytoplasm
of the young spores (Fig. 10) began to increase in density and resemble that of the
pre-leptotene spore mother cells.
DISCUSSION
There are close similarities between megasporogenesis in Marsilea and sporogenesis
in the homosporous fern Pteridium (Sheffield & Bell, 1979). These includetheocclusion
of the plasmodesmata between the spore mother cells and their replacement by
cytomictic channels, also subsequently eliminated, and the presence in the mother
cells of regions of cytoplasm surrounded by membranes and rich in ribosomes. The
occurrence of nodules of wall material with membranous inclusions, the temporary
absence from the plastids of well defined envelopes, and the progressive fall in
ribosome frequency during prophase are also all features of sporogenesis in Pteridium.
There are however undoubted differences in the extent of these features and their
timing. In Pteridium the cytomictic channels persist into late prophase, in Marsilea
they were not seen following leptotene. The aggregates of ribosomes found in the
pre-leptotene spore mother cells, although resembling the pseudo-nucleoloids of
Sheffield & Bell (1979), were not so conspicuous, and there was no evidence that they
were involved in replenishing the ribosome population at a later stage of sporogenesis.
Megasporogenesis in Marsilea
117
The alteration in the nature of the plastid envelope was also more transient; in
Ptertdium it persisted into prophase but in Marsilea the envelopes were fully restored
in early leptotene, similar to the situation during microsporogenesis in Pinus (Dickinson
& Bell, 1976). The possible nature of this striking change in the envelope has been
discussed (Sheffield & Bell, 1979). The several instances of discontinuous envelopes
seen in the present work show that the entire envelope was not affected simultaneously.
Although ultimately the whole was uniformly indistinct, Marsilea may indicate the
beginning of the weakening of this effect. In megasporogenesis in Lilium, for example,
the plastid envelopes diminish in distinctness at diplotene, but nevertheless remain
readily detectable (Dickinson & Potter, 1978).
The formation of the vesicular nuclear inclusions during prophase was closely
similar in Marsilea and Pteridium. This phenomenon is now known to accompany
meiosis in several plants and animals (Sheffield, Cawood, Bell & Dickinson, 1979;
Fouquet & Dang, 1980), but its significance is not yet known.
The tubules adjacent to the plastid envelopes in the dyads, and subsequently in
young spores, have not been observed before in sporogenesis. They were consistently
larger than microtubules, and there was no evidence that their walls had a similar
subunit structure. Further, although microtubules may have lateral connections with
membranes (Cronshaw, 1967; Bell, 1978), there are no reports of microtubules having
direct continuity with membrane profiles. Despite their arising from endoplasmic
reticulum, the Marsilea tubules are clearly different from the tubules described by
Jensen (1968) in cotton, by Quan, Chi & Caplin (1974) in cultured broccoli and by
Hoefert (1975) in Thlaspi, since in all these instances the tubules were never observed
outside endoplasmic reticulum cisternae. The Marsilea tubules much more closely
resemble tubules arising from the endoplasmic reticulum in the leaf gland of Phaseolus
(Steer & Newcomb, 1969). Nevertheless they are not identical. The diameter of the
Marsilea tubules falls between that of the 'small' (29 nm) and 'large' (56-66 nm)
tubules in Phaseolus. The boundary of the Phaseolus tubules also shows no clear unitmembrane profile, and the tendency of the small tubules to disaggregate, and give rise
to the large, finds no parallel in Marsilea. Although the Phaseolus tubules arise from
the endoplasmic reticulum, the absence of any clear unit-membrane profile in the
wall, and the ability of the wall to disaggregate, suggests that any lipid present is in
discrete micelles, and not as a bi-molecular leaflet, now generally accepted as the
normal conformation in cell membranes. The wall of the Marsilea tubule, by contrast,
retains a clear membrane profile. The function of the tubules can at present only be
speculative, but it is conceivable that they are a specialized form of endoplasmic
reticulum, facilitating a particularly active metabolic interchange between plastids,
interlamellar space and cisternae of the normal reticulum at this stage of spore development. It is striking that the tubules show no similar association with the mitochondria.
The electron-opaque bodies in the young megaspores of M. vestita are probably
identical with those staining densely with haematoxylin in the nucleus and cytoplasm
at a similar stage of megasporogenesis in M. diffusa (Boterberg, 1956). They closely
resemble the nucleoloids described in micro- and megasporogenesis in Lillium
(Dickinson & Heslop Harrison, 1977; Dickinson & Potter, 1978) and are believed to
n8
P. R. Bell
be the source of many of the ribosomes appearing in the young spores. These nucleoloids are regarded as being produced in the nucleus and left in the cytoplasm at
telophase. This may be true of the similar bodies in the young megaspore of Marsilea,
but electron-opaque aggregates visually identical to nucleoli can also appear in the
cytoplasm without rupture of the nuclear envelope (e.g. in egg cells of the fern
Lygodium; Hutchinson & Bell, unpublished). Since there is no evidence that the few
pseudo-nucleoloids seen in prophase in Marsilea persist, a nucleoloid mechanism for
replenishing the ribosomes of the young spores is very probable. The membranous
regions in young spores (Fig. 9) do not resemble the dispersing pseudo-nucleoloids of
Pteridium (Sheffield & Bell, 1979; Plate 4B) and may be sites of local autophagy.
The evolution of regular, sexually differentiated heterospory may therefore have been
accompanied by direct passage of nucleolar material into the cytoplasm of the forming
spores, with the concurrent loss of the pseudo-nucleoloids characteristic of homospory.
The early stages of megasporogenesis have given no explanation as to why only one
megaspore in each tetrad survives. Dyads always appeared equally matched and no
consistent differences between the 4 spores of the tetrad could be established. There
was thus no evidence that the polarized distribution of the organelles in the megaspore
mother cells (also detected by Pettitt (1970) in an un-named species of Marsilea)
persisted, and to regard it as effective in determining megaspore viability is unwarranted. The same doubt can be cast on Pettitt's (1977) interpretation of the cytoplasmic
gradient in the megaspore mother cells of SelagineUa sulcata. Here, however, megasporogenesis does not lead to the regular 1 :3 ratio of M. vestita, and the situation in
the lycopods generally may not be directly comparable to that in Marsilea and the
seed plants. Gradients have also been held responsible for the failure of all but one of
the microspores in each tetrad to develop in certain species of the Epacridaceae
(Ford, 1972 a, b). In this instance the explanation is more satisfactory since there is no
clear formation of dyads, and the gradient in organelle frequency in the pollen mother
cell persists during meiosis. Three of the haploid nuclei move into the less-dense
cytoplasm and are resorbed. The formation of monad pollen in the Cyperaceae has been
ascribed to spatial and physiological constraints arising from the radial packing of the
wedge-shaped microspore mother cells (Strandhede, 1973). These considerations also
seem inappropriate in Marsilea since the 8 tetrads of megaspores, separated by the
intrusive tapetum, are arranged in no recognizable order, and the position of the viable
spore in each is unrelated to the axis of the sporangium. As far as megaspore survival
in the tetrad is concerned, the present observations are in line with the argument
developed elsewhere (Bell, 1979) that differential viability expressed asa 1:3 ratio
has a Mendelian basis.
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