J. Cell Sci. 25, 125-138 (1977)
Printed in Great Britain
125
DIFFERENTIATION OF THE TAPETUM
IN A VENA
I. THE CELL SURFACE
M. W. STEER
Botany Department, The Queen's University, Belfast, N. Ireland
SUMMARY
The development of the tapetal cell surface and associated structures in Avena has been
followed from cell formation to senescence. Plasmodesmata initially connect the tapetal cells to
each other, the pollen mother cells, and the inner loculus wall cells. These connexions are
subsequently severed, those to the sporogenous cells being broken first at the pollen mother
cell surface during callose wall formation. Loss of cellulose from the tapetal walls was followed
using the decline in the ability of the wall to bind the fluorescent brightener, Calcofluor
White M2R New. Subplasma-membrane microtubules persist after loss of the cellulose wall.
The tapetal plasma membrane facing the meiocytes then develops a series of depressions, or
cups, over its surface, which are later the site of pro-orbicule formation. Sporopollenin is laid
down over the pro-orbicules, to form orbicules, and over other tapetal cell surfaces. No morphological evidence was found for the intracytoplasmic formation of pro-orbicules or polymerized
sporopollenin precursors.
These observations on Avena are compared with those on other plants. The changes in the
cell wall and associated structures, plasmodesmata and microtubules, are considered in detail,
while the general significance of cell wall loss to the water relations of the tissue are assessed.
Proposals that pro-orbicule formation results from non-specific accumulation of lipid at a free
cell surface are rejected, instead this formation is considered to be related to the presence of a
specially modified plasmamembrane surface.
INTRODUCTION
The tapetum is one of the most complex plant cell types, showing a remarkable
specialization of its intracellular and extracellular components. The developmental
sequence of this specialized tissue can be studied in material that is readily accessible,
the anther, and whose growth is easily monitored by reference to the stage of microspore formation. This makes the tapetum a particularly attractive model system for
the study of various aspects of plant cell activity. Of the 2 types of tapetum found
among flowering plants, the secretory type is the more suitable for this type of study
as the cells are static and do not migrate into the anther loculus, as do cells of the
periplasmodial type (Echlin, 1971a).
General descriptions of Avena tapetal and microspore structure are given in Steer
(1974) and Gunning & Steer (1975). This paper will be concerned with establishing
the development of the cell surface and associated structures, a further paper will be
presented on the internal development of the cell.
Echlin & Godwin (1968) first applied improved tissue preparation methods for
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electron microscopy to the study of tapetal development in a dicotyledon, Helleborus,
reporting on many features of the cell surface and laying the groundwork for further
observations and discussions. This was followed by observations from other dicotyledons (Marquardt, Barth & von Rahden, 1968; Risueno, Gimenez-Martin, LopezSaez & Garcia, 1969; Hoefert, 1971; Horner & Lersten, 1971), monocotyledons
(Heslop-Harrison & Dickinson, 1969; De Vries & Ie, 1970; Christensen, Horner &
Lersten, 1972), and agymnosperm (Dickinson and Bell, 1972, 1976a). It is clear from
these observations that the development of the secretory tapetum cell surface is
broadly similar in all these groups. The differences in development between these
major taxonomic groupings that have been reported so far are mainly concerned with
the relative timing of tapetal events compared with the stage of microspore development, and are difficult to assess in view of the limited number of genera examined.
In Avena it has been possible to follow formation of the cellulose wall and associated
plasmodesmata and their loss and replacement by a layer of orbicules that develop in
association with a specialized plasma-membrane surface. The observations reported
here are in very close agreement with those already made on another monocotyledon,
Sorghum (Christensen et al. 1972), and appear similar to those from Zea mays (Skvarla
& Larson, 1966) and wheat (De Vries & Ie, 1970).
All micrographs are arranged so that the pollen mother cell (microspore) lies to the left of
each figure and the tapetal cell to the right.
Fig. 1. Longitudinal section, 1 /tm thick, of glycol methacrylate-embedded anther
stained with 0 0 1 % Calcofluor White M2R New for 20 min and photographed
using a Zeiss fluorescence microscope. The layer of binucleate tapetal cells lies between
the pollen mother cells, at prophase I, and the loculus wall cells. Note intense
fluorescence of the callose walls and of the cellulose walls in the outer layers of the anther. By comparison there is a complete lack of fluorescence from the tapetal cell walls.
X950.
Fig. 2. As Fig. 1. Longitudinal section tangential to the anther sac showing layer of
binucleate tapetal cells. Fluorescence is absent from the tapetal cell walls, while it is
present from the cellulose around the loculus wall cells, x 1100.
Fig. 3. Early pollen grain development, otherwise as Fig. 1. Exine formation is almost
complete and the 3 layers can. be resolved due to autoftuorescence of sporopollenin in
the ncxine and tcctum. Only the layer of orbicules on. the adjacent tapetal wall shows
evidence of fluorescence, while other walls show a strong fluorescence, x 950.
Fig. 4. The tapetum-pollen mother cell wall (czo) at the time of tapetal cell formation.
The tapetal nucleus (n) had just been reconstituted at the end of mitosis and the cell was
in cytokinesis at the time of fixation. A plasmodesma (pd) interconnects the 2 cell types,
x 9500.
Fig. 5. Two tapetal cells (top and right) abut a pollen mother cell which is at the precallose wall stage. The cell wall has a lightly stained fibrillar appearance and is traversed
by a plasmodesma (pd) which runs out of the plane of section at the top left. Microtubules (nit) occur just below the tapetal plasma membrane and in the adjacent
cytoplasm. They are also present in the pollen mother cell cytoplasm, x 19000.
Avena tapetum. I. Cell surface
9-2
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M. W. Steer
MATERIALS AND METHODS
Seeds of Avena sativa L. var Stormont Sceptre were grown as described previously (Steer,
-75)- Anthers at different stages of development were excised and processed for either light
or electron microscopy. Sections i-/tm thick, from material embedded in glycol methacrylate
(Feder & O'Brien, 1968) were routinely stained with toluidine blue or periodic acid-Schiff's
reaction for examination by transmitted light microscopy. Glycol methacrylate sections were
also stained with o-oi % Calcofluor White M2R New in distilled water (Hughes & McCully
1975) and examined in a Zeiss fluorescence microscope. Thick sections (1 fim) cut from Eponembedded anthers were routinely stained with toluidine blue for light microscopy, while thin
sections from the same anthers were stained with uranyl acetate and lead citrate for electron
microscopy.
IO
OBSERVATIONS
The following observations are based on examination of Avena sativa anthers by
light and electron microscopy. Observations on other oat species have been made
(Gunning & Steer, 1975) and these do not appear significantly different from the
following account. The accompanying figures, representing only a small fraction of
the material available, have been selected to illustrate specific stages of tapetal cell
surface development and concentrate on the earlier stages, since later ones are already
widely reported in the literature. In describing events in the tapetum reference will be
made to the corresponding stage of microspore development, so that comparisons can
be made with tapetum development in other plants.
Fig. 6. Subplasma-membrane microtubules (mt) lie on either side of the cellulose wall
separating a tapetal cell (right) from a pollen mother cell which is laying down callose
at the opposite (innermost) face. There is a noticeable halo around each microtubule
in the tapetal cell, suggesting the presence of a non-staining component, x 57000.
Fig. 7. Early stage of cell wall breakdown at the beginning of prophase I. The tapetal
plasma membrane is slightly withdrawn from the wall which is still distinctly fibrous
on the tapetal side of the middle lamella (ml). Note plasmodesmata (pd). x 15000.
Fig. 8. Callose wall formation has extended around the pollen mother cell to the wall
adjacent to the tapetum. This results in an increased thickness of the wall on the mother
cell side of the middle lamella (ml). The outer leaflet of the tapetal plasma membrane
is heavily stained, x 62300.
Fig. 9. Loss of cellulose fibrils from tapetal side of middle lamella and replacement by
a system of granules and fibrils. The pollen mother cells in the loculus of this anther
are at anaphase-telophase I. Microtubules (arrows) are present beneath the tapetal
plasma membrane, x 28500.
Fig. 10. Interdigitating ends of tapetal cells showing plasmodesmatal connexions
between them (arrowed). The tapetal wall inside the middle lamella has almost
disappeared, while the pollen mother cell callose wall (ca) has continued to increase in
thickness, x 20300.
Fig. 11. Several plasmodesmatal connexions (pd) from the tapetum persist after
callose wall formation around the pollen mother cell. The lower one probably passes
out of the plane of section, whilst the upper clearly traverses the middle lamella and
terminates in a slightly swollen end at the callose wall. The plasmodesmata are filled
with a homogenous, stained, material and are lined by a tripartite membrane. Microtubules are present below the tapetal plasma membrane at this tangential wall and the
adjacent radial wall (arrows), x 30000.
Avena tapetum. I. Cell surface
129
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130
M. W. Steer
Tapetal cells are formed when cell division takes place in the cylindrical layer of
cells immediately surrounding the pollen mother cells. The division is slightly
asymmetric, the cell plate being laid down tangentially to the anther cylinder, cutting
off a slightly larger tapetal cell, adjacent to the pollen mother cells, from a smaller
loculus wall cell. Subsequent growth of the tapetal cell increases its volume by a
factor of 10, from about 650 to 6500 /mv3. Most of the increase in cell size is due to
an increase in length of the cell (i.e. parallel to the anther long axis), with smaller
increases occurring in the lengths of the radial and tangential walls.
The newly formed tapetal cell is bounded by a thin cell wall (0-1-0-2/tm thick),
that binds the fluorescent brightener Calcofluor White M2R New (Hughes & McCully,
1975). It contains numerous fine, lightly stained fibrils, and appears to be a typical
primary cellulose wall (Figs. 4, 5). Plasmodesmata connect adjacent tapetal cells to
each other and to the pollen mother cells and the loculus wall cells (Figs. 4, 5, 7, io, 11).
Just beneath the plasma membrane lie a series of microtubules. These are usually
oriented parallel to the cell's (and anther's) long axis on the tangential walls (Fig. 6),
and either in this direction or radially on the radial walls (Figs. 12, 13). Later, just
before mitosis occurs to give binucleate tapetal cells (Figs. 1, 2), aggregates of microtubules occur that are similar to those described as preprophase bands (Fig. 12).
With the extension of the callose walls, from the centre of the pollen mother cell
mass to the outer wall, the tapetal cell walls start to break down. The tangential tapetal
wall adjacent to the pollen mother cells is the first to be affected, followed by changes
to the outer tangential wall and radial walls.
Loss of the wall against the pollen mother cells is accompanied by a pronounced
Fig. 12. Preprophase band microtubules in tapetal cell before mitosis. Other microtubules run parallel to them, and at right angles, along the radial wall separating 2
adjacent tapetal cells. Note the tenuous nature of the intervening cell wall. The outer
membrane of the nuclear envelope is marked by arrows, x 40000.
Fig. 13. Radial wall between 2 tapetal cells at metaphase I in the mciocytes. Only the
middle lamella (ml) remains of the original cellulose wall. Microtubules (mt) run both
alongside the wall and out into the cytoplasm. The outer leaflets of the plasma membranes are darkly stained, x 80000.
Fig. 14. Adjacent tapetal cells (left) have withdrawn from the cellulose wall of the
inner loculus wall cell. Large lipid droplets (arrowed) have been deposited along the
inner face of the loculus wall, especially at the corners of the tapetal cells. The middle
lamella (»//) can be traced through the lipid and along the loculus wall, x 27000.
Fig. 15. Tapetal cell plasma-membrane with a series of cups (arrows). The cell wall
between this membrane and the middle lamella consists of a series of granules interspersed with fibrils. On the other side of the middle lamella is the meiocyte callose wall
enclosing a cell at anaphase II meiosis. x 60000.
Fig. 16. Pro-orbicule (po) formation on the tapetal surface. The lipid droplets appear
in the plasma membrane cups without any indication of similar droplets in the cytoplasm. Note profiles of the endoplasmic reticulum (er) containing a stained matrix;
some of these (unlabelled arrows) approach the plasma membrane and are bifacial
(lacking ribosomes on one face). Part of the granular tapetal cell wall can be seen here,
elsewhere in this section are newly formed microspores at the end of meiosis II.
x 48 000.
Avena tapetum. I. Cell surface
igr
132
M.W. Steer
swelling and loss of staining properties, including the ability to bind Calcofluor
(Figs. 1-3) and to stain with periodic acid-Schiff's reaction. By late meiosis I, the
fine ordered fibrils of the primary wall are replaced with a loose meshwork of coarse
fibres and granules which lies adjacent to the callose wall that surrounds the meiocytes
(Fig. 9). Microtubules are still present beneath the tapetal plasma membrane, even
when all remnants of the original wall have disappeared (Figs. 9-11). The plasmodesmatal connexions to the pollen mother cell are broken by the formation of the intervening callose walls, leaving plasmodesmata projecting from the surface of the
tapetal cell (Fig. 11). The lumen of these plasmodesmata is filled with a substance
that stains uniformly. Later they disappear completely, either due to absorption by
the cell or to becoming detached from this suface and lost in the loculus cavity.
At the inner loculus wall the tapetal plasma membrane becomes withdrawn,
cutting off the plasmodesmata. Lipoidal material accumulates along this wall and
frequently forms large droplets appressed to the wall and running round the corners
and up the radial walls between adjacent tapetal cells (Fig. 14). The radial walls
become more tenuous and loose their fine fibrils as the plasma membrane on each
side shrinks away from the wall. Again plasmodesmatal connexions are broken,
though some plasma membrane connexions have been found stretched between
adjacent tapetal cells at maturity. The plasma membrane of the radial walls (Fig. 13)
and the lipid lining of the loculus wall (Figs. 14, 20) become coated with a thin,
dark-staining layer which may be a thin lamella of sporopollenin (Dickinson &
Bell, 1972).
The most prominent feature of the tapetum at maturity is the carpet of orbicules
that coat the cell surface of the inner tangential wall. Each orbicule consists of a
globular lipid core (about 150 nm diameter) surmounted by a decorated sporopollenin
coat (about 200 nm thick). The orbicules develop on the cell surface following a
series of discrete changes in the plasma membrane.
Fig. 17. Dense plaques of sporopollenin forming around pro-orbicules on the tapetal
cell surface (arrows). The wall between this cell and the microspore is distinctly
bipartite, with the callose layer becoming more diffuse (see Fig. 15). x 51000.
Fig. 18. Accretion of sporopollenin at distinct nodes over the pro-orbicule surface
initiates development of the orbicule spines. The adjacent cell wall layers are almost
completely disintegrated leaving the orbicules in close proximity to the developing
microspore exine. x 5500.
Fig. 19. Development of the characteristic channelling in the layer of sporopollenin
encrusting the pro-orbicules can be seen. Note the microbodies in the adjacent tapetal
cytoplasm (arrows), x 36500.
Fig. 20. Nodules of sporopollenin accumulating along the inner loculus wall (s). They
appear to be attached to a thin, dark-staining layer (arrows), probably also of sporopollenin. x 39000.
Fig. 21. Orbicule formation almost complete at the time of exine completion and pollen
grain mitosis, x 39000.
Fig. 22. Senescence of tapetal cell (right) results in the complete loss of cell contents
(compare Fig. 21). The orbicules, which have lost their lipid cores, remain abutting the
adjacent microspore exine (lower left). Sporopollenin deposits are clearly seen along
the radial wall between adjacent tapetal cells (arrows), x 35000.
Avena tapetum. I. Cell surface
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M. W. Steer
Following loss of the cell wall the inner facing tapetal cell surface is seen as a
relatively smooth expanse of plasma membrane. During the early stages of meiosis II
this smooth membrane develops a series of small depressions, or cups, 50 nm deep
and 100 nm across, at intervals over the cell surface (Fig. 15). At the end of meiosis a
lipid droplet, or pro-orbicule, is formed in each pocket which is initially 50-75 nm in
diameter (Fig. 16). These grow in size until they reach 150 nm across, the same size as
the lipid cores in the mature orbicules. The whole process is strictly synchronized
over the cell surface, all pro-orbicules appearing at the same time. The origin of the
pro-orbicules is not clear. There are no comparable lipid droplets in the cytoplasm at
this time, nor are any of the cell organelles specifically associated with the cell surface
at this stage more than at preceding or subsequent stages.
The completed pro-orbicule is coated with sporopollenin at the time of microspore
separation from the tetrads and exine formation. First discrete plaques of sporopollenin are laid on the lipid surface (Fig. 17), which then enlarge by accretion of
more material (Fig. 18), and coalesce to form a complete coating (Figs. 19, 21).
Sporopollenin is also laid down in small quantities along the radial walls (Fig. 22)
and in larger accumulations on the lipid lining of the inner loculus wall (Fig. 20).
At pollen mitosis the tapetal cell cytoplasm starts to senesce, so that by the time the
generative cells are formed the tapetal cells are devoid of contents and are only
outlined by the orbicules on the tangential wall and other sporopollenin deposits
along the radial walls (Fig. 22).
DISCUSSION
This account of the Avena tapetum cell surface can be compared with those previously published on other monocotyledons and with accounts of secretory tapetum
development in various dicotyledons. The basic developmental sequence is broadly
similar in all these accounts. However, there are variations in the timing of tapetal cell
surface changes when related to the stage of microspore development, for example in
Beta loss of the inner tangential wall occurs at the tetrad stage (Hoefert, 1971), while
it is much earlier in Avena. Further, in Helleborus, pro-orbicule formation does not
occur until after exine formation has commenced (Echlin and Godwin, 1968), but
in Avena it occurs at the end of meiosis. Detailed comparisons between this and the
other accounts of tapetum development reveal a number of similarities and differences,
both in the observations and their interpretation, which will be discussed further.
The early stages of tapetal wall development in Helleborus have been described by
Echlin & Godwin (1968). They interpreted the increased thickness of the inner
tapetal tangential wall (abutting the pollen mother cell callose wall) as being due to the
deposition of cellulose. However, in Avena the cellulose component is very rapidly lost
from this wall as judged by fluorescence microscopy and the increase in wall thickness
appears to be accompanied by a net loss of material visible in electron micrographs.
The overall sequence of tapetal wall loss found in Avena has also been reported from
Beta (Hoefert, 1971), Sorghum (Christensen et al. 1972) and Pinus (Dickinson & Bell,
1972). The formation of granules and coarse fibrils as an intermediate stage in the
Avena tapetum. I. Cell surface
135
breakdown of cellulose walls has also been seen in cotyledons of germinating seeds
(Smith, 1974, and personal communication). The granules may be protein, as they do
not stain for carbohydrates, in which case they could be extracellular enzymes.
Presumably the tapetal cells' physiology changes when the cellulose wall is lost.
This removes the mechanical constraint to cell size and shape, and might be thought to
upset the cells' osmoregulatory system. However, loss of the wall is accompanied by
loss of the cell vacuoles, thus removing one solute reservoir that would normally
contribute to the cells' water potential. The surrounding tissues, although capable of
imposing mechanical constraint on the tapetal cells, are not in contact with its plasma
membrane. Hence the solute concentration of the loculus must balance the internal
solute concentration of the tapetum, presumably the loculus solute concentration is
increased by the accumulation of tapetal cell wall and callose wall breakdown products. Movement of water from the apoplast of the surrounding anther tissue into the
loculus may be controlled by the lipoidal coating of the inner loculus wall.
The subject of plasmodesmatal connexions between the various tissues of the
microsporangium has given rise to numerous comments in other publications. In
some of these confusion has been caused by failure to qualify statements about their
occurrence with the corresponding stage of development. For example in a recent
review on the occurrence of plasmodesmata in reproductive structures of plants it is
asserted that they are absent from the tapetum - male meiocyte cell wall (Carr, 1976),
a similar statement can also be found in Robards (1975). Both these references presumably refer to later stages of development, since plasmodesmata have been found
in this situation by Heslop-Harrison (1966), Horner & Lersten (1971), Christensen
et al. (1972) and, more recently, by Dickinson & Bell (1976a). Their presence and
loss in Avena have been reported here and it is of interest that the connexions are
first broken at the pollen mother cell surface at callose formation, leaving the plasma
membrane-lined structure projecting from the tapetal cell surface.
Echlin & Godwin (1968) reported on the occurrence of microtubules beneath the
tapetal plasma membrane and their persistence until the tetrad stage. Similarly in
Avena they persist until long after the loss of the cellulose cell wall. These observations
raise questions about their function in the cell at this time. Traditionally subplasmamembrane microtubules have been associated with orientation of cellulose fibrils as
they are formed in the adjacent plant cell wall (for a review see Hepler & Palevitz,
1974), however, cellulose production only occurs at the earliest stages of tapetal cell
development. Microtubules seen at later stages may only be relics of the earlier
activity, although it seems possible that they might contribute to the maintenance of
tapetal cell shape, as in many animal cells (Tilney, 1968).
The origin of the lipid cores of the orbicules, first termed pro-orbicules by HeslopHarrison & Dickinson (1969), has attracted a great deal of attention. Echlin & Godwin
(1968) were the first to attempt an analysis of pro-orbicule formation. In their material,
Helleborus, conspicuous lipid droplets are present in the tapetal cytoplasm and it was
concluded that these are extruded through the plasma membrane on to the tapetal
cell surface forming the pro-orbicules. However, these authors reported 2 observations which cast doubt on this interpretation, they noted that the lipid bodies in the
136
M.W. Steer
cytoplasm were larger than the pro-orbicules and that they were present at all stages
of development, even after orbicule formation had ceased. A subsequent report by
Heslop-Harrison & Dickinson (1969) supported this model for pro-orbicule formation,
but in later work by Horner & Lersten (1971) and Dickinson & Bell (1972) no firm
evidence was found for the formation of pro-orbicules by this method. Risueno et al.
(1969) presented evidence for the formation of pro-orbicules in elements of the
endoplasmic reticulum and their subsequent movement to the surface, however,
their micrograph (fig. 4 in the above paper) illustrating this latter event seems to show
parts of three different cells (see Fig. 10 of this paper for a similar view, and Fig. 13
for a view of the radial wall which could be confused with an intracytoplasmic channel).
In Avena no evidence was found for the formation of lipid cores in the tapetal cytoplasm. The strict synchrony found in development of Avena pro-orbicules, also
found in Pinns (Dickinson & Bell, 19766), contrasts with the continuous production
of these bodies reported for Helleborus (Echlin & Godwin, 1968) and Beta (Hoefert,
1971).
The formation of pro-orbicules in cup-shaped depressions of the plasma membrane
was first observed in Sorghum by Christensen et al. (1972). The presence of these
cups has probably been responsible for some of the confusion over the intracytoplasmic origin of pro-orbicules (Echlin & Godwin, 1968) and they have been interpreted as discharging dictyosome vesicles (Horner & Lersten, 1971). Cup formation
appears to be a specialization of the plasma membrane at one particular tapetal
surface. In animal cells the formation of such plasma membrane configurations is often
associated with systems of microfilaments below the cell surface (Reaven & Axline,
1973) but these cannot be positively identified in the micrographs from Avena. In
Sorghum a much clearer association between the cups and subsurface endoplasmic
reticulum was found than in the present work. In both Sorghum and Avena a granular
staining material is present in the endoplasmic reticulum that could be involved in
orbicule formation (Fig. 16). However, other studies indicate a quite different fate
for these intracisternal contents (Steer, 1974; Gunning & Steer, 1975 and unpublished
work). Lipid synthesis is known to occur in the endoplasmic reticulum of plants
(Moore, Lord, Kagawa & Beevers, 1973) as well as animals (Jungalwala & Dawson,
1970) and the possibility that such lipid ultimately finds its way to the pro-orbicules
is not excluded by the present work. Once on the surface of the tapetal cell the lipid
accumulates as discrete spheres within the cups, rather than as a continuous layer.
Christensen et al. (1972) suggested that this is due to the absence of mechanical
restraint on lipid accumulation around the tapetum, contrasted with the presence of a
callose wall around the microspore, which they believe restricts such accumulation to
a thin continuous layer. However, in Avena a structurally similar primexine develops
in the absence of a callose wall, which disappears after meiosis II.
Christensen et al. (1972) have considered the possible evolutionary origins for the
tapetum's ability to form orbicules and favour the view that it represents a vestigial
capacity of a tissue that was once sporogenous. This may be true of the ability to lay
down sporopollenin on external surfaces and could explain the general accumulation
of sporopollenin around the tangential and radial walls of the tapetum. But it is
Avena tapetum. I. Cell surface
137
inadequate to account for either the specialization of one face of the plasma membrane,
to form a series of cup-shaped depressions, or the specific accumulation of lipid
droplets within them. Surely pro-orbicule formation is an example of evolutionary
specialization producing a specific product that forms the base for sporopollenin
deposition and orbicule formation, not a vestigial capacity to produce a pollen
grain wall. The problem lies in trying to elucidate the function of orbicules so that the
nature of the evolutionary pressure can be understood (for a review see Echlin, 1971 a).
Suggesting that the sporopollenin provides a non-wetting surface so that pollen
grains may be more easily dispersed (Heslop-Harrison & Dickinson, 1969) ignores
the fact that the radial walls are so covered without recourse to orbicule formation.
The answer may lie in the particular physical properties of the three-dimensional
surface generated by the orbicules.
The discovery that sporopollenin contains polymerized carotenoids (Brooks, 1971)
initiated a search for the cytoplasmic source of these precursors. Early stages of
carotene polymerization to form sporopollenin have been claimed to occur in the
endoplasmic reticulum in Allium (Risueno et al. 1969) and in the endoplasmic
reticulum and associated vesicles in Pinus (Dickinson & Bell, 19766). There is no
evidence for such intracellular formation of sporopollenin in the tapetum of Avena,
supporting the view expressed by Echlin (19716) that such polymerization is always
extracellular. In particular there are no deposits in the cytoplasmic organelles cited
above that resemble either carotenoid pigment globules or sporopollenin. Doubt has
already been expressed in this section over the interpretation of the micrographs in
Risueno et al. (1969). Dickinson & Bell (19766) provide only one example of their
intracytoplasmic vesicles containing precursors of sporopollenin with no evidence for
its movement 'intact' into the loculus. Clearly electron microscopy alone cannot
provide an understanding of these dynamic events.
The expert technical assistance of Mr D. Kernoghan and Mr G. McCartney is gratefully
acknowledged. Part of this work was supported by a grant from the U.K. Science Research
Council.
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{Received 15 October 1976)