Bot. J. Linn. SOC., 69: 79-87. With 4 plates
September 1974
Developmental mechanisms in heterospory.
II. Evidence for pinocytosis in the microspores
of Selaginella
J . M. PETTITT, F.L.S.
Department of Palaeontology, British Museum (Natural History),
Cromwell Road, London
Accepted f o r publication July 1974
At a stage in ontogeny while they are still held together in tetrads, but before the formation of
the exine, the microspores of Selaginella brooksii Hieron. show features of the cell surface
which suggest that material is being taken up by pinocytosis. These features, which are confined
to the proximal face of the spore, are: (1) cytoplasmic fringes which arise near, arch over and
enclose membrane-bound particles on the cell surface, (2) invaginations of the plasma
membrane which form smoothsurfaced vesicles, and (3) invaginations of the plasma membrane
to form coated vesicles. The membranes which limit all three kinds of vesicle are asymmetrical.
Sections that cut the surface of the microspore tangentially at or in the vicinity of this surface
activity show a hexagonal lattice which is a surface specialization possibly connected with
pinocytosis. There are indications that the pinocytosed material is digested by lysosomal
enzymes; myelin-like residual bodies are formed which migrate to the periphery of the cell.
These observations are discussed in relation to the nutritional explanation of heterospory in the
pteridoph ytes.
CONTENTS
. . . . . .
Introduction
Materialsandmethods
Observations
. . . . . .
Features of the cell surface
Vesicles in the cytoplasm
Discussion
. . . . . .
Acknowledgements
. . . .
References
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INTRODUCTION
Heterospory in Selaginella is characterized by the presence of spores of two
distinct sizes. In all known cases, germination of the small spores (microspores)
results in the formation of endosporic gametophytes bearing only male
gametangia, while germination of the large spores (megaspores) results in the
formation of endosporic gametophytes bearing only female gametangia. At
maturity, therefore the spores of Selaginella differ both in size and sexual
0
79
80
J . M. PETTITT
expression. But these differences at maturity, in fact, are presaged before
meiosis. In the premeiotic megasporangium there is extensive degeneration of
the sporocytes by a process corresponding to cellular autophagy (Pettitt ,
1971a), leaving only one or a few, which enlarge and divide, while in the
premeiotic microsporangium this phenomenon does not occur, and all the
sporocytes divide. Thus, the developmental commitment of the sporangium
and its contents is established before meiosis; and since there is a difference in
the size of the two kinds of sporocytes, there is also a difference in the size of
the two kinds of spore at their inception. This difference is promoted during
subsequent growth and development of the megaspores; and although
considerable variation exists between species, in all the volume of the ripe
megaspore is appreciably greater than that of the ripe microspore. However, as
Sussex (1966) has pointed out, the differences between microspores and
megaspores extend t o more than those of size and sexual expression. They
differ with regard to type of food reserve, number and appearance of
organelles, nuclear shape and wall construction ; and comparable differences in
other organisms are known to be under genetic control. I t is reasonable to
suppose, however, that these structural differences may be not only a
by-product of distinctive growth and differentiation, but also an instrumental
factor in achieving it. Clearly, since in Selaginella, as in other heterosporous
pteridophytes, divergent development is premeiotic, the distinctions between
the megaspores and microspores cannot be the result of essential genetic
difference. Heterospory in these pteridophytes, therefore, is phenotypic. And
since the genetic constitution is coequal, the proximate cause of the difference
between the spores must be attributable to differences between reaction
systems, that is, to differential response. In other words, cytoplasmic state
plays a discriminating role in respect of genotype-environment interaction
(Lewis & John, 1968).
The belief that the environmental determinant is largely a matter of
nutrition is well established, though never demonstrated. The inference is that
with degeneration of the megasporocytes in the megasporangium the
competition for the same substrate material becomes less, and the enhanced
growth shown by the remaining sporocyte is interpreted as a compensatory
response to this by an increase in synthetic activity. Thus, elimination
determines size; and size is consistently linked with sex. To examine this
postulate, Shattuck (19 10) conducted experiments with Marsilea and found
that by manipulating environmental factors it was possible to induce the
formation of small spores (microspores) in megasporangia and large spores
(megaspores) in microsporangia. When nutritional conditions were unfavourable microspores occurred in presumptive megasporangia. Under favourable
conditions, some of the spores in the developing microsporangia aborted and
the remainder enlarged, and, what is more, the amount of enlargement was
proportional to the degree of abortion. In extreme cases only one large spore
survived to maturity. An equally interesting situation is that in Isoetes pantii
Goswami & Arya, where certain microsporangia contain both microspores and
megaspores. Here the megaspores always occur a t the ligular or proximal end of
the sporangium, and the pattern would seem to be a normal feature of
development in this species, not an anomaly (Goswami & Arya, 1968, 1970).
But we note that these observations on Isoetes, like the experiments with
PINOCYTOSIS IN SELAGLVELLA MICROSPORES
81
Marsilea, relate only to differences in size; it was not determined whether this
was attended by changes in food reserve, organelles and wall construction and a
switch in sex expression.
Although nutrient supply seems to affect the pattern of spore development
in the heterosporous pteridophytes, it is not known whether the spores take up
macromolecules of maternal origin or if the new components which appear in
them are exclusively the products of their own synthetic activity. Clearly, from
what is said above, differential uptake or synthesis might be expected to occur
at particular stages in spore development, and this could be detected. Of
course, such observations would not explain the different morphogenetic
processes, though they would add appreciably to our description of them. A
full explanation would require a full description of how the macromolecules,
intrinsic or extrinsic, are causally related to differential growth and sex
expression and how, in Selaginella at least, the heterosporous condition is
precipitated by the advent of meiosis.
By a very elegant series of experiments, Rowley & Dunbar (1970) have
shown that marker particles are transferred from the sporophyte to the male
gametophyte in angiosperms. When anthers of Populus and Salix were
incubated in colloidal iron, the iron was translocated from the vascular tissue of
the stamen, through the parietal and tapetal cells, to the surface of the
microspore tetrads or the intine of the pollen grains. Some iron was taken into
the cytoplasm of the microspores and pollen grains by, the authors suggest,
pinocytosis. Here, then, in the anthers of flowering plants, there appears to be a
system by which large molecules of maternal origin may selectively cross the
boundary between sporophyte and gametophyte and thus transcend the genetic
confines of the cells. This paper describes a similar occurrence in the
microsporangium of Selaginella.
MATERIALS AND METHODS
Strobili of greenhouse-grown Selaginella brooksii Hieron. and Selaginella
grundis Moore were detached from the plant and immediately immersed in
ice-cold 4.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), and the
sporangia were excised. The sporangia were fixed for 20 h at 4OC, and after
fixation were washed in several changes of buffer and postfixed for 2 h at room
temperature in 1% osmium tetroxide in phosphate buffer (pH 7.3). The
sporangia were washed in buffer again and dehydrated in ethanol (30%absolute), transferred to 1,Zepoxy propane and embedded in Araldite. During
dehydration and infiltration the tubes containing the sporangia were slowly
turned end-over-end in a rotator. Ultrathin sections were cut with diamond
knives and stained with alcoholic uranyl acetate or with uranyl acetate and lead
citrate (Reynolds, 1963).
Strobili of S. brooksii were fixed in 2BD (Darlington & La Cour, 1962),
dehydrated in ethanol, embedded in paraffin wax and sectioned at 10 pm. The
wax was removed from the sections with xylene and the sections were stained
briefly in a saturated solution of orange G in absolute ethanol, then transferred
to xylene and mounted.
82
J. M. PETTITT
OBSERVATIONS
Features of the cell surface
Early in the differentiation of the microspores of S, brooksii, at a stage
before the exine begins to form, fringes or folds of cytoplasm arise from the
proximal surfaces of the spores. Each fringe invariably occurs in close
proximity to membrane-bound particles which lie adjacent to the plasma
membrane (Plate l A , D). I t would appear that when it has reached a certain
elevation the fringe recurves, and at this stage the adjacent particles are seen to
lie within a recess or caveola in the cell surface (Plate l B , C). The recurved
fringe makes contact with the cell surface, thus enclosing the particles within a
vesicle (Plate 2B, C). Two other kinds of vesicle are initiated on the proximal
surface of the microspores in addition t o these. The first, like the cytoplasmic
fringes, are formed in the vicinity of membrane-bound particles on the cell
surface. They arise by invagination of the plasma membrane and appear to
contain minutely particulate or finely fibrous material of low electron density.
Covering the inner (cytoplasmic) surface of the limiting membrane is a coat
consisting of radiating fibrils or “bristles”, a feature which characterizes them
as coated vesicles (Roth & Porter, 1964; Plate 3A, D). The second kind are
initiated as small flask-shaped invaginations in the cell surface and are evidently
pinched off into the cytoplasm as the sides of the invagination come together.
These vesicles have no detectable contents and are somewhat larger than the
coated variety (Plates 2D and 3B, C). The components of the membranes which
limit all these vesicles are differentially stained ; the outer (luminal) leaflet is
more electron-dense than the inner (cytoplasmic) one and the structure
therefore appears asymmetrical (Plates 1C and 3C, D).
At this early stage in development each microspore is enclosed within a coat
composed of acid mucopolysaccharide (Pettitt & Jermy, in press), which has
been rendered pictorially indistinct by the methods of preparation employed in
this investigation. However, in some sections random fibrils of the coat are
discernible in the area bordered by the proximal faces of the spores in the
tetrad and within the surface caveolae (Plate 2A). A layer of more densely
staining coat material is sometimes seen bridging the opening of the caveola
when the recurved cytoplasmic fringe is near to the cell surface (Plate 2A).
Since this increase in density is both localized and temporal, it presumably
signifies a change in the organization of the components of the coat coincident
with closure of the caveola. Sections which cut the surface of the microspore
tangentially at or in the vicinity of a cytoplasmic fringe show a hexagonal
lattice (Plate l D , E). That this lattice is a feature of the cell surface and is
intimately associated with the plasma membrane is certain; but what is not
certain is whether it is a feature of the mucopolysaccharide surface coat, of a
submembranal specialization or of the laminae of the membrane itself. These
possibilities are discussed later.
Vesicles in the cytoplasm
The microspore cytoplasm contains a variety of membrane-limited vesicles.
Some of them contain membrane-bound particles identical with those in the
PINOCYTOSIS IN SELAGINELLA MICROSPORES
83
surface caveolae and these vesicles would seem to represent an early stage in the
transferrence of the particles into the cell (Plate 3G). Other cytoplasmic
vesicles contain granular material together with small, somewhat indistinct
membrane-bound particles (Plate 3 E), while yet others contain granular
material together with membranous wefts or arrays (Plate 3F). Quite frequently, smaller vesicles with circular profiles and grey structureless contents
occur juxtaposed to these structures (Plate 3H).
Microspores of S. grundis a t a stage in development judged somewhat more
advanced than that just described for S. brooksii (using the size of the
sporangium as an indication of age), but before the establishment of a
continuous exine layer, contain numerous cytoplasmic vesicles enclosing whorls
of parallel membranes some of which have a myelin-like form (Plate 4A, B).
Large masses of this myelin-like material also occur at the periphery of the
spore (Plate 4A). Microspores of S. brooksii at an equivalent ontogenetic stage
were not encountered in the series processed for electron microscopy, but they
did occur in the strobili fixed for optical microscopy. Plate 4C, D shows
peripheral osmophilic and birefringent myelin-like masses in the microspores in
this species.
DISCUSSION
The difficulties of interpreting sequential activity from thin sections
notwithstanding, the observations presented here provide considerable evidence
that at a certain stage in their development the microspores of Selaginella
brooksii take up material by pinocytosis. But, clearly, since other interpretations can be proposed, a basis for preference must exist, and that is afforded by
a comparison with pinocytosis in animal cells. All in all such comparisons seem
to favour an explanation that the process in Selaginella is one of uptake rather
than liberation.
Pinocytosis is a highly selective physiological process which utilizes the
capacities of the cell surface for specific binding and configurational change
(Bennett, 1969b). The specificity is conferred on the process by an acid
polysaccharide-protein component on the surface of the plasma membrane
which Bennett (1963, 1969a) has named the glycocalyx. As mentioned above,
each microspore in Seluginella is enclosed by a strongly acidic fibrillar coat
which, both in position and composition, fulfills the requirements of a
glycocalyx (Pettitt & Jermy, in press). Bennett (1956) postulated that
pinocytosis is activated by the binding of an inducer to the cell surface and
Marshall & Nachmias (1965) showed this t o be correct by demonstrating that
pinocytosis in an amoeba occurs when a cationic inducer is bound to the
glycocalyx. Since this circumstance is believed to hold for all pinocytic events
(Bennett, 1969b), it is reasonable to suppose that the mechanism in Selaginella
is activated by the attachment of the membrane-bound particles and perhaps
other inconspicuous substances to the glycocalyx. The source of the particles is
uncertain. However, some cells of the tapetum show signs of necrosis and are
releasing their contents into the microsporangium loculus at this stage, and it is
probable that the particles originate in these.
Bennett (1969b) suggests that the consequence of adsorption of an inducer
is a changed chemical configuration at the cell surface, to which the cell
a4
J. M. PETTITT
responds by invaginating the area of the membrane to which the particles are
bound. There would seem to be no observable differences between the
formation of cytoplasmic folds which envelop the surface bound particles in
Selaginella and the process of pinocytosis in animal cells, c)r the formation of
vesicles by invagination of the plasma membrane and the process of
micropinocytosis (Fawcett (1965) and Threadgold (1967) give full descriptions). Indeed, comparisons extend even to the asymmetry of the membranes
limiting the vesicles (Slautterback, 1967; Deams, Wisse & Brederoo, 1969). The
significance of this particular feature is by no means clear, but Brandt (1958)
cites experimental data from work with an amoeba which suggests that the
permeability of the plasma membrane is altered after its incorporation into the
pinocytic vesicle. Thus, one possibility is that the observed change in
membrane symmetry denotes a change in permeability. Sufficient evidence can
be advanced in support of the claim that micropinocytic vesicles of the coated
variety are concerned in the uptake of proteins (Roth & Porter, 1962, 1964;
Fawcett, 1965; Friend & Farquhar, 1967). Further, studies on the formation of
these vesicles provide clear evidence that the coating on the inner (cytoplasmic)
surface is actually a series of hexagons and pentagons rather than a palisade of
hair-like projections (Bowers, 1964; Friend & Farquhar, 1967; Kanaseki &
Kadota, 1969). In some cells this same geometrical pattern is also discernible in
areas of specialization immediately beneath the plasma membrane; areas which
when invaginated will adorn the vesicle with its characteristic coat (Kanaseki &
Kadota, 1969). There is a striking similarity between the geometry of the
material coating these vesicles and the hexagonal lattice seen in those sections
which have cut the surface of the Selaginella microspores tangentially at or near
a point of pinocytosis. One conclusion to be drawn from this finding,
therefore, is that the lattice in the microspores represents a localized
submembranal specialization. But an alternative exists. Slautterback (1967)
believes that the hexagonal lattice which he detected on the micropinocytic
vesicles in Hydra is due to the disposition of the subunits in the coat which
lines the luminal surface of the vesicles-that is, the glycocalyx. Slautterback
suggests that this disposition might impose a similar order on the substructure
in that leaflet of the membrane to which the coat is intimately attached, and it
is interesting to note that this leaflet is the denser of the two. Whatever the
details in Selaginella, it seems that the lattice could correspond to those regions
of the cell surface to which Bennett (1969b) alludes as ones in which the
molecular architecture is adapted in such a way that pinocytosis would be an
obligatory response to specific binding.
Many investigations have cytochemically demonstrated that the material
taken up by pinocytosis in animal cells is digested by hydrolytic enzymes
originating in lysosomes; indeed, this process would seem to account for all
instances (see de Duve & Wattiaux, 1966; Straus, 1967; Deams, Wisse &
Brederoo, 1969 for reviews). There are indications that such a process also
occurs in the microspores of Seluginellu. These indications are the occurrence
of cytoplasmic vesicles containing indistinct membrane-bound particles,
granular material and membranous arrays, which closely resemble secondary
lysosomes (de Duve & Wattiaux, 1966). Further, if, as would seem, these
represent various stages in the intravesicular digestion of the pinocytosed
material, they would correspond to heterolysosomes (de Durve & Wattiaux,
PINOCYTOSIS IN SELAGINELLA MICROSPORES
85
1966; Straus, 1967). Furthermore, the masses of myelin-like material which are
a common feature of the cytoplasm in S. grandis at a somewhat later stage in
spore development conform to typical lysosomal residual bodies (Deams, Wisse
& Brederoo, 1969). But the demonstration of the lysosomal nature of a cell
particle rests on the detection of the distinctive hydrolytic enzymes, and until
these can be shown to be present or absent from the vesicles in Selaginella their
identity remains conjectural. Nevertheless, the masses of myelin-like membrane
migrate to the periphery of the microspore in S. brooksii and S. grandis and are
possibly extruded from the cell. This last point is virtually impossible to
establish, however, because of the difficulty in discriminating the plasma
membrane from the membrane of the residual body when the two are in
contact. There is evidence which suggests that in these two species, and in
SelaginelZa selaginoides (L.) Link (Robert, 1971), the membrane masses are in
some way implicated in the establishment of the microspore exine. The nature
and extent of their participation in this process is not yet clear, but in this
connection it would be interesting to know whether, in view of their probable
lysosomal ancestry, the myelin-like masses possess any enzymic activity.
From these observations it would appear that these mechanisms of uptake
and intracellular digestion are normal features of development in the
microsporangium of Selaginella brooksii. I t would appear too that the duration
of the uptake process, certainly at the magnitude described here, is short and
that after this, during subsequent stages of development, the cell surface is
relatively quiescent. However, evidence from these later stages in a number of
species suggests that appearances may not reveal the truth. In these species the
structure of the exine on the proximal face of the microspore is markedly
different from that which covers the lateral and distal faces. This is exemplified
by Selaginella sulcata (Dew.) Spring, where the proximal exine is interrupted
by numerous channels while the lateral and distal exine is uniform and
continuous (Plate 4E). One possible and perhaps valid conclusion to be drawn
from this difference is that the organization of the proximal exine is influenced
by continued surface activity of the proximal face of the spore. Moreover, in
descriptions given earlier it was shown that the microspore exine in Selaginella
forms within the glycocalyx (Pettitt, 1971b).
The significance of these observations with regard t o the nutritional
explanation of heterospory in the pteridophytes is clear. Microspores of
Seluginella take up material, probably of maternal origin, by the highly
selective process of pinocytosis, and it appears the ingested material is degraded
within the cell by a vacuolar system which allows some of the products of
digestion to pass out into the cytoplasm (Deams, Wisse & Brederoo, 1969).
There remains, of course, the question of how all this compares with the female
side. Experiments are in progress to examine this question in the light of
Bennett’s (1969b) hypothesis that the differing appetites of differing pinocytic
cells can be explained, in part at least, in terms of variations in their respective
cell surfaces.
ACKNOWLEDGEMENTS
I should like t o thank the Director of the Royal Botanic Gardens, Kew, for
permission to work on the living fern collections and Mr H. J. Bruty, who until
86
PINOCYTOSIS IN SELAGINELLA MICROSPORES
his recent retirement was Foreman Gardener in charge of the fern collections at
the Royal Botanical Gardens, for his willing cooperation. I should also like to
thank Mr C. H. Shute for technical assistance.
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active transport and ion pumping. J. biophys. biochem. Cytol., 2(4),Suppl. 2. 99-103.
BENNETT, H. S., 1963.Morphological aspects of extracellular polysaccharides. J. Histochem. Cytochem..
1 1 : 14-23.
BENNETT, H. S., 1969a. The cell surface: components and configurations. In A. Limade-Faria (Ed.).
Handbook of molecular cytology: 1261-Y 3 . Amsterdam & London: North Holland Pub. Co.
BENNETT, H. S.. 1969b. The cell surface: movements and recombinations. In A. Lima-de-Faria (Ed.),
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BOWERS, B., 1964. Coated vesicles in the pericardial cells of the aphid (Myzus persicae Sulz.).
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BRANDT, P. W., 1958.A study of the mechanism of pinocytosis. Expl Cell Res., 15: 300-13.
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Unwin btd.
DEAMS, W. Th., WISSE, E. & BREDEROO. P., 1969. Electron microscopy of the vacuolar apparatus. In
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de DUVE, C. & WATTIAUX, R., 1966.Functions of lysosomes. A . Rev. Physiol.. 28: 435-92.
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GOSWAMI, H. K. & ARYA, B. S., 1968.Heterosporous sporangia in Isoetes. Br. Fern Gaz., 10: 39-40.
GOSWAMI, H. K. & ARYA. B. S.. 1970.A new species of Isoetes from Narasinghgarh. Madhya Pradesh.
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PETTITT, J. M. & JERMY, A. C.,1974.The surface coats o n spores. Biol. J. Linn. SOC.,6: 245-57.
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ROBERT, D., 1971, Etude, en microscopie Clectronique, des modalitis d’kdification des parois
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B0t.J. Linn. Soc., 69 (1'974)
J. M. PE'I'TITT
Plate 1
(Facing p. 86)
Bot. J. Linn. Soc., 69 (1974)
J. n1. PETTIT'I'
Plate 2
Bot.J. Linn. S'oc., 69 (1974)
]. M. PETTITT
Plate 3
Bot.]. Linn. Soc., 69 (llJ74)
]. lVI. PElTITT
Plate 4
J. M. PETTITT
EXPLANATION OF PLATES
PLATE 1
Selaginella brooksii. Sections in A, Band D stained with uranyl acetate and lead citrate; section
in C stained with uranyl acetate alone.
A. A cytoplasmic fringe arising from the proximal surface of a microspore in close proximity
to membrane-bound particles on the cell surface. x 12,500.
B. A recurved cytoplasmic fringe: the particles lie in a surface caveola. x 37,500.
C. A surface caveola similar to that in B and beneath it a vesicle formed by the fusion of a
fringe with the cell surface. The leaflets of the membrane limiting this vesicle are differentially
stained (arrow). x 37,500.
D. This section has cut the proximal surface of a micros pore tangentially at the point of origin
of a cytoplasmic fringe (arrow) which was recurving to surround the particles seen on the right
of the picture. There is a hexagonal lattice associated with the plasma membrane. x 40,000.
E. Detail of the lattice in D. x 75,000.
PLATE 2
Selaginella brooksii. Sections in A, C and D stained with uranyl acetate and lead citrate; section
in B stained with uranyl acetate alone.
A. Recurved cytoplasmic fringes on the proximal surface of a spore Fibrils of the surface coat
are discernible between proximal faces of adjacent spores and within the surface caveolae
(arrows). A layer of more densely staining coat material bridges the gap between a recurved
fringe and the cell surface (arrow head). Fibrillar material is also seen within a cytoplasmic
vesicle. x 25,000.
B, C. Fringes have made contact with the cell surface and enclosed particulate material. B,
X 37,500; C, X 40,000.
D. Stages in the formation of micropinocytic vesicles at the proximal surface of a microspore.
X 60,000.
PLATE 3
Selaginella brooksii. All sections stained with uranyl acetate and lead citrate ..
A. Coated vesicles at the proximal surface of a microspore. The characteristic coating on the
inner (cytoplasmic) surface of the limiting membrane is clearly seen (arrow). x 75,000.
B. Formation of a micropinocytic vesicle by invagination of the plasma membrane at the
proximal surface of a microspore. The cytoplasmic vesicle in the adjacent spore (upper part of
the picture) probably arose in this manner. x 62,000.
C. Closure of a micropinocytic vesicle. The outer (luminal) leaflet of the membrane limiting
the vesicle (arrow) is more electron dense than the inner (cytoplasmic) leaflet. x 105,000.
D. Formation of a coated vesicle by invagination of the plasma membrane at the proximal face
of a micros pore. The vesicle is forming beneath particles which lie on the surface of the spore.
X 50,000.
E, F, H .. Cytoplasmic vesicles containing membrane-bound particles, granular material and
membranous arrays. E, x 52,500; F, x 15,000; H, x 30,000.
G. Cytoplasmic vesicles containing membrane-bound particles identical with those in the
surface caveolae (see 1B). The leaflets of the membranes which limit these vesicles are
differentially stained. x 45,000.
PLATE 4
A, B, Selaginella grandis; C, D, Selaginella brooksii; E, Selaginella sulcata. Sections in A, B and
E stained with uranyl acetate and lead citrate.
A. Masses of myelin-like material in the cytoplasm and at the periphery of a microspore.
X 60,000.
B. Cytoplasmic vesicles enclosing whorls of parallel membranes. x 37,500.
C, D. Peripheral myelin-like material in the microspores. C, bright field x 750; D, polarized
light X 750.
E. The structure of the proximal exine (arrow) in this microspore is interrupted by numerous
channels while the lateral and distal exine is uniform and continuous. x 4500.
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