Exine development in Encephalartos altensteinii (Cycadaceae): ultrastructure, substructure and the modes of sporopollenin accumulation

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Review of Palaeobotany and Palynology 132 (2004) 175–193

Exine development in Encephalartos altensteinii (Cycadaceae):

ultrastructure, substructure and the modes

of sporopollenin accumulation

Nina I. Gabarayeva*, Valentina V. Grigorjeva

Komarov Botanical Institute, Popov st., 2, St. Petersburg 197376, Russia

Received 18 February 2003; received in revised form 28 May 2004; accepted 28 May 2004

Abstract

It has been found that in Encephalartos altensteinii all the main characters of the exine appear within the tetrad period. The

framework of the exine—the plasma membrane glycocalyx—appears first and consists of close packed cylinder-like units 120–

160 nm in diameter, oriented perpendicular to the microspore plasma membrane. The main character of the glycocalyx

framework is its dynamism: the units are capable of building up throughout the period of exine development. Sporopollenin

acceptor particles (SAPs) occur distributed alongside the walls of the cylinder-like units, bringing about a sporopollenin

accumulation and the appearance of cylindrical alveolae of the exine pattern. The location of SAPs, which are probably enzyme

molecules, is carried out mainly under control of genome, and only the final step occurs by self-orientation, as a result of a

combination of both strong and weak interactions between glycocalyx macromolecules and enzyme molecules. The initial

accumulation of sporopollenin apparently occurs in the form of microglobules around SAPs with diameter of 30–50 nm.

Subsequent sporopollenin accumulation, which results in the appearance of the mature alongate alveolate pattern of the exine, is

independent of SAPs and can be regarded as secondarily accumulated sporopollenin. We suggest two stages in the exine

development which are realized by self-assembly: (i) the formation of the glycocalyx and (ii) the accumulation of receptor-

independent sporopollenin. The intermediate stage—the initial accumulation of receptor-dependent sporopollenin—is carried

out under the control of SAPs.

D 2004 Elsevier B.V. All rights reserved.

Keywords: exine substructure; sporopollenin acceptor particles (SAPs); initial sporopollenin accumulations; self-assembly; acceptor-dependent

and acceptor-independent sporopollenin; aperture establishment; Encephalartos altensteinii; Cycadales

1. Introduction

Encephalartos altensteinii Lehm. (Lehmann, 1834),

together with other representatives of Cycadales, is

0034-6667/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.revpalbo.2004.05.005

* Corresponding author. Fax: +7-812-234-4512.

E-mail address: [email protected]

(N.I. Gabarayeva).

characterized by some archaic characters and has a

unique long-alveolate type of pollen grain exine. It is

not surprising that Cycadales has attracted the attention

of many investigators, interested in its morphology,

structure, evolution, cytotaxonomy, taxonomic affini-

ties, and pollen ultrastructure (Chamberlain, 1931,

1935; Meyer, 1977; Dehgan and Dehgan, 1988; Ste-

venson, 1990; Moretti, 1990; Nishida, 1994; Osborn

N.I. Gabarayeva, V.V. Grigorjeva / Review of Palae176

and Taylor, 1995; Doyle, 1998). Especially important

contributions to our knowledge of pollen morphology,

ultrastructure and the development of the sporoderm

of cycadalean plants have been made by Audran and

his co-authors (Audran, 1964, 1969, 1970, 1981,

1987; Audran and Masure, 1977).

Our objectives in this study were to trace in detail

at the ultrastructural level the developmental sequence

of events during exine formation. Our aims were: (1)

to reveal the role of the glycocalyx in exine develop-

ment; (2) to clarify the modes of exine deposition; (3)

to try to reveal the initial form of sporopollenin

accumulations and their relations with sporopollenin

acceptor particles (SAPs); (4) to interpret the phases

of the exine development, some of which are probably

carried out by self-assembly.

2. Material and methods

Microsporangia of Encephalartos altensteinii

Lehm. (Lehmann, 1834) were collected from the

glass-houses of the Komarov Botanical Institute (St.

Petersburg), from the plant with inventory number

11830. The material was fixed in a fixative consisting

of 3% glutaraldehyde and 2.5% sucrose in O.1 M

phosphate buffer (pH 7.3, 20 jC, 24 h) and post-fixed

in 2% osmium tetroxide (pH 7.4, 20 jC, 3 h). After

acetone dehydration the samples were embedded in

mixture of Epon and Araldite. Ultrathin sections were

stained with a saturated solution of uranyl acetate in

ethanol and 0.2% lead citrate. Sections were examined

with a Hitachi H-600 TEM.

3. Results

3.1. Early tetrad microspore stage

In the post-meiosis tetrad the microspores are

covered with a thick callose envelope. The topogra-

phy of the plasma membrane is rather flat and lacks

any signs of the glycocalyx (Plate I,1). A large

nucleus with an active nucleolus occupies a consid-

erable volume within the cytoplasm. Plastids with

dark stroma, multimembrane bodies, multivesicular

bodies and cisternae of endoplasmic reticulum (ER)

are seen in the microspore cytoplasm (Plate I,1).

3.2. Young tetrad microspore stage

At the next stage the topography of the plasma

membrane becomes intensively wavy (Plate II,2).

Newly formed portions of the glycocalyx appear in

the invaginations of the plasma membrane. The cyto-

plasm of the microspore is rather dark and contains

many vesicles, ribosomes and plastids with dark

stroma and osmiophylic globules.

3.3. Young tetrad microspore stage in progress

The glycocalyx layer shows at this stage a uniform

thickness (Plate III,3). In some places the outlines of

tufts, especially their binder elements, are seen as

cross lines. The nucleus occupies the considerable

part of the microspore volume.

Later the layer of the glycocalyx is not uniform in

thickness as a result of its successive growth. In a

slightly oblique section of the glycocalyx layer (Plate

III,4) dark contrasted spots are seen surrounded by an

electron transparent halo. These are cross-sectioned

tufts, the basic units of the exine, with electron dense

core subunits and electron lucent binder subunits. The

microspore cytoplasm at this stage is very dense.

3.4. Middle tetrad microspore stage

At this stage the outlines of tufts in the layer of

the glycocalyx are very well pronounced (Plate IV,

5–7). The tufts are cylinder-like and oriented per-

pendicularly to the plasma membrane, their diameter

is about 120–160 nm. Each tuft corresponds to one

alveola of the future ectexine. The stain reversal is

observed: now binder elements are dark contrasted,

whereas core subunits are electron lucent (Plate IV,

5–7). Plate IV,5–6 shows that the walls of tufts bear

particles of intermediate electron density 10–15 nm

in diameter. These are Sporopollenin Acceptor Par-

ticles (SAPs). Later the plasma membrane forms

deep invaginations, and the proximal ends of tufts

have become stretched in (Plate IV,6). SAPs have

enlarged (30–50 nm in diameter) and acquired high

electron density: this change will be discussed below.

The initial tectum appears on the surface of the

proexine (Plate IV,6). Electron dense particles are

sometimes seen around the cylinder-like tufts (Plate

IV,5–7).

obotany and Palynology 132 (2004) 175–193

Palaeobotany and Palynology 132 (2004) 175–193 177

3.5. The transition to late tetrad microspore stage

At this ontogenetic stage many Sporopollenin

Acceptor Particles (SAPs) of tufts become consid-

erably increased in size and electron density (Plate

V,8). The tectum is already well pronounced. Some-

what later the proexine is completed and the long-

N.I. Gabarayeva, V.V. Grigorjeva / Review of

Plate I (see page 179).

1. Early tetrad microspore stage. The plasma membrane (P) of the m

No sign of the glycocalyx is seen on the surface of the plasma m

reticulum; MMB, multimembrane body; Mvb, multivesicular bod

polysomes, SMCE, spore mother cell envelope). Bar = 0.5 Am.

Plate II (see page 180).

2. Young tetrad microspore stage. The plasma membrane of the mic

membrane (IP) the portions of the newly formed glycocalyx (G) a

Bar = 0.2 Am.

Plate III (see page 181).

Later phase of young tetrad microspore stage.

3. a radial section of a microspore. An even layer of glycocalyx (G) is

with the core and binder elements can be distinguished (arrows).

4. Slightly oblique section of the surface of the microspore. The gly

plasma membrane (P). Electron dense spots in the layer of the glyco

cross-sectioned tufts (arrow) (Ca, callose; Cy, microspore cytopl

envelope). 3: Bar = 0.25 Am; 4: Bar = 0.2 Am.

Plate IV (see page 182).

Middle tetrad microspore stage.

5. the layer of the glycocalyx (G) is evident, consisting of the cylin

the ectexine. Stain reversal is observed: core subunits are electron

of the tufts bear electron dense particles which are sporopollenin a

(IT).

6. developmental stage a little later than that shown in figure 5. The

parts of the tufts are stretched in. Sporopollenin acceptor pa

accumulations of sporopollenin are indicated with arrowheads.

7. a portion of a microspore surface with cylinder-like tufts with SAP

microspore cytoplasm; ER, endoplasmic reticulum; G, glycocalyx

Bar = 0.2 Am; 7: Bar = 0.5 Am.

Plate V (see page 183).

The transition to late tetrad microspore stage.

8. SAPs with accumulated sporopollenin (arrows) are more evident.

9. the alongate alveolate proexine is completed. It is easy to distinguis

one tuft (Ca, callose; G, glycocalyx; IP, invagination of the plasma

SER, smooth endoplasmic reticulum). 8, 9: Bar = 0.1 Am.

alveolate pattern of ectexine is clearly revealed

(Plate V,9). The alveolae have thin sporopollenin

walls. The tectum is thin where sectioned radially

(the right part of Plate V,9), while it seems thick

where sectioned obliquely (the left part of the same

figure). In some places the plasma membrane is still

invaginated.

icrospore is more or less even under the thick callose envelope (Ca).

embrane (Cy, microspore cytoplasm; ER, cisterna of endoplasmic

y; N, nucleus; NE, nuclear envelope; Nu, nucleolus; Pl, plastid; Ps,

rospore has an undulating profile. In the invaginations of the plasma

re visible (Ca, callose; GV, Golgi vesicles; Pl, plastid; R, ribosomes).

seen on the surface of the plasma membrane (P). The outlines of tufts

cocalyx (G) forms an uneven layer on the surface of the undulating

calyx, surrounded by an electron lucent halo, are core elements of the

asm; N, nucleus; NE, nuclear envelope; SMCE, spore mother cell

der-like tuft-units that are progenitors of the future long alveolae of

lucent, but binder subunits have become electron dense. The walls

cceptor particles (SAPs—arrows). The initial tectum is recognisable

plasma membrane is considerably invaginated (IP), and the proximal

rticles are marked by arrows. Some of these SAPs with initial

s which begin to accumulate sporopollenin (arrow) (Ca, callose; Cy,

; GV, Golgi vesicles; N, nucleus; PT, protectum). 5: Bar = 0.1 Am; 6:

The tectum is well pronounced (T).

h the tectum (T) and walls of the alveolae. One alveola corresponds to

membrane; N, nucleus; NE, nuclear envelope; P, plasma membrane,

N.I. Gabarayeva, V.V. Grigorjeva / Review of Palaeobotany and Palynology 132 (2004) 175–193178

3.6. The late tetrad microspore stage

A general view of the late tetrad is shown in Plate

VI,10. The callose envelope has partly disintegrated.

The microspores have acquired typical flattened form.

The distal portion of the microspores has the charac-

teristic alongate alveolate pattern of the ectexine with

a thin tectum. The proximal portion of the micro-

spores has a thick tectum, and the alveolate pattern of

Plate VI (see page 184).

10. Late tetrad microspore stage. A general view of two mi

and continuous, well defined walls of the alveolae. The

part of the alveola walls are lacking (Gap) (CPl, cup-l

microspore distal side; N, nucleus; Nu, nucleolus; Pro

Plate VII (see page 185).

11–12. The end of the tetrad period. 11—the equatorial portion

proximal surfaces of the microspore the tectum become

the first of them is the outermost primordial lamella

elements (arrow). The second stain reversal: core

microspore. The tectum is very thin. The lamellated

ectexine (Ect). Tuft substructure is seen in the form t

arrow) (AE, alveolate ectexine; Cy, microspore cy

membrane). 11: Bar = 0.2 Am; 12: Bar = 0.25 Am.

Plate VIII (see page 186).

13–14. The end of the tetrad period. Oblique sections through

elements of tufts (arrows). The binder subunits are sh

them sporopollenin has accumulated (black–white as

lamellae; T, the area of tectum). 13: Bar = 0.2 Am; 14

Plate IX (see page 187).

15–16. The tapetum at the end of the tetrad period. 15—A par

layer of the glycocalyx (G) is seen on the surface of

invaginations of the plasma membrane of the tapeta

membrane; M, mitochondrion; P, plasma membrane; R

Plate X (see page 188).

17–18. The end of the tetrad period. Proximal portions of a m

are modified (only their proximal part is present leavin

Cy, microspore cytoplasm; End, lamellated endexin

68,000�); 18: Bar = 0.1 Am.

the ectexine is disturbed: a vast space (gap) has been

formed between the tectum and the alveolae.

3.7. The end of the tetrad period

Most of the callose envelope has disintegrated, but

microspores are still held in tetrads because the

mother cell wall remains. Equatorial portion of the

microspore is shown in Plate VII,11; distal portion is

crospores of a tetrad. The microspore distal face shows a thin tectum

microspore proximal face shows a thick tectum (T), and the outermost

ike plastid; Cr, remnants of callose; Cy, microspore cytoplasm; Dist,

x, microspore proximal side). Bar = 1 Am (ca. 11,000�).

of a microspore. At the transition regions between the equatorial and

s thicker (T). Several lamellae of the endexine have appeared (End),

(PL). Tuft substructure is seen in the form of loops of the binder

subunits are electron dense again. 12—the distal portion of the

endexine with white lines is well-distinct (End), as is the alveolate

he core elements (small arrows) and a binder element loops (large

toplasm; D, dictyosome; ER, endoplasmic reticulum; P, plasma

the exine. Electron dense spots in the centre of the alveolae are core

own with arrowheads. On the surface of the alveolae and between

terisks) (Cy, microspore cytoplasm; End, the area of the endexine

: Bar = 0.1 Am.

t of a tapetal cell bordering the cavity of microsporangium. A sparse

the tapetal cell. 16—complex orbicules (CO) are observed in the

l cell (ER, endoplasmic reticulum; IP, invagination of the plasma

, ribosomes; Ta, tapetum). 15: Bar = 0.1 Am; 16: Bar = 0.25 Am.

icrospore. The tectum (T) is very thick, and the walls of the alveolae

g a hollow space (Gap) beneath the tectum) (AE, alveolate ectexine;

e; G, glycocalyx; P, plasma membrane). 17: Bar = 0.1 Am (ca.

Plate I (Caption on page 177).

N.I. Gabarayeva, V.V. Grigorjeva / Review of Palaeobotany and Palynology 132 (2004) 175–193 179

Plate II (Caption on page 177).


Plate III (Caption on page 177).


Plate IV (Caption on page 177).


Plate V (Caption on page 177).


Plate VI (Caption on page 177).


Plate VII (Caption on page 178).


Plate VIII (Caption on page 178).


Plate IX (Caption on page 178).


Plate X (Caption on page 178).



seen in Plate VII,12. At this stage the lamellae of the

endexine with white lines appear. The outermost

lamella appears first (primordial lamella). The central

part of the microspore is occupied by a large nucleus.

In the peripheral cytoplasm many dyctiosomes and

their vesicles, cisternae of smooth endoplasmic retic-

ulum, plastids and ribosomes are seen. The tufts inside

the alveolae show binder and core elements, core

elements are again electron dense (the second stain

reversal—Plate VII,11–12). These core and binder

elements are also distinct in the subtangential sections

through the exine (Plate VIII,13–14).

The inner, locular sides of the tapetal cells at this

time are strongly invaginated, and the surface is

covered with the sparse layer of the glycocalyx (Plate

IX,15–16). In the invaginations of the tapetal cells

complex orbicules are located (Plate IX,16). The

tapetal cytoplasm is packed with mitochondria, cis-

ternae of SER and ribosomes.

The details of the proximal portions of microspores

are shown in Plate X,17,18. The tectum is very thick,

and the alveolate pattern of the ectexine is interrupted.

The base parts of the alveola walls are well defined,

but the distal parts are missing: instead, a vast hollow

space (gap) is formed under the tectum.

4. Discussion

4.1. The appearance and growth of the microspore

glycocalyx

The invaginated character of the plasma membrane

in the young stages of the tetrad microspore develop-

ment of Encephalartos altensteinii is typical actually

for all species under ontogenetic study (Gabarayeva,

2000). This universal feature is probably connected

with the mode of the glycocalyx growth. The glyco-

calyx units are built up continuously in their proximal

part, from the side of the plasma membrane, up to and

including the late tetrad stage, when sporopollenin has

already started to accumulate. The involvement of the

plasma membrane movements (micromovements) in

the process of building-up the glycocalyx framework

is evident (Gabarayeva, 2000). In Stangeria eriopus,

during mid-tetrad stage, some portions of the plasma-

lemma are considerably invaginated, which results in

widening of the periplasmic space and stretching out

of the glycocalyx units (Gabarayeva and Grigorjeva,

2002). Oscillatory movements of the plasmalemma

were confirmed by our stereometric study in S.

eriopus (Gabarayeva and Grigorjeva, 2002). As pre-

viously proposed (Gabarayeva, 1990, 1993, 2000),

short stretching and detachment of the glycocalyx

from the plasma membrane could be needed for radial

growth of the glycocalyx units by self-assembly.

The microspore glycocalyx in Encephalartos, as in

other species, consists of radially oriented exine units,

named tufts by Rowley and his co-authors (Rowley et

al., 1981, 1995; Rowley, 1990). At the middle tetrad

stage, before sporopollenin accumulation has been

initiated, it is already possible to observe the future

pattern of the long-alveolate ectexine. One tuft corre-

sponds to one future alveola. At this time the

microspore surface coating—glycocalyx—is well de-

veloped. In radial sections it shows a pattern of closely

packed cylinder-like units which are especially evident

in Plate IV,5 and confirmed by cross-sections in Plate

VIII,13–14. The image seen in Plate IV,5 is very

similar to that shown by Audran (1981) at the

corresponding stage during microspore development

in Ceratozamia mexicana and to that shown for micro-

spore development in Stangeria eriopus in Plate VIII,

17 (Gabarayeva and Grigorjeva, 2002).

In Encephalartos we have observed a stain reversal

between binder and core elements of the young exine

units, as was observed in other species (Betula—

Dunbar and Rowley, 1984; Epilobium—Rowley and

Claugher, 1996; Borago—Gabarayeva et al., 1998)

and hypothetically explained by Rowley (1987–88,

1995). Rowley’s interpretation is that the staining is

strong because the coils of core subunits are located

within the core, and the contrast is weak when the

coils of core subunits are interdigitated with binder

coils; in other words, core subunits are mobile. In

Encephalartos a stain reversal between binder and

core elements occurs twice: at the beginning of the

middle tetrad stage (Plate IV,5–6) and partly in the

end of the tetrad period (Plate VII,11–12).

4.2. Sporopollenin receptors

The most important character of the mid-tetrad

glycocalyx is the presence of dark roundish particles

seen in the walls of cylinder-like units (Plate IV,5–6).

Particles of similar size and electron density were


observed earlier during exine formation in Poinciana

(Skvarla and Rowley, 1987), Asimina (Waha, 1987),

Anaxagorea (Gabarayeva, 1995), Nuphar (Rowley et

al., 1995), Borago (Gabarayeva et al., 1998) and

Stangeria (Gabarayeva and Grigorjeva, 2002) and

were referred to as sporopollenin accepter particles

(SAPs: Rowley et al., 1999). The proteinaceous nature

of these particles was suggested based on results from

oxidative experiments on a variety of species using 4-

methylmorpholine N-oxide monohydrate, followed

by treatment with phosphotungstic acid in acetone

(Rowley et al., 2001) and with potassium permanga-

nate (Gabarayeva et al., 2003a—Cabomba; Gabar-

ayeva et al., 2003b—Lavatera arborea, Stangeria

eriopus, Oenothera speciosa). It has been suggested

that sporopollenin accumulation was carried out by

enzyme molecules, distributed through the glycocalyx

(Gabarayeva, 1990, 1993). It is very probable that

SAPs correspond to enzyme molecules. Indeed, in

proexine of Encephalartos and Stangeria sporopollen-

in accumulation initiates exactly in areas of clustering

of these particles (the walls of cylinder tuft-units),

resulting in the establishment of the long-alveolate

ectexine.

4.3. Microglobules—the initial form of sporopollenin

accumulation

As seen in Plate IV,6–7 and especially in Plate V,

8, the walls of the long alveolae at the middle tetrad

stage include small osmiophilic globules with diame-

ter about 30–50 nm. We suggest that these micro-

globules are initial sporopollenin accumulations

around SAPs. Gabarayeva (1990, 1993, 2000)

hypothesised that the glycocalyx is formed by self-

assembly. Sporopollenin receptor molecules are

bounded covalently with macromolecules of the gly-

cocalyx and occupy the definite places through the

glycocalyx units. The final step of the insertion of

asymmetrical enzyme molecules into spiral macro-

molecules of the glycocalyx occurs by the process of

self-orientation, as a result of a combination of both

strong and weak interactions between molecules (en-

zyme molecules turn through some angle). Because

the chemical composition of a glycocalyx of a species

is under control of genome and is species-specific, the

distribution of enzyme molecules is generally under

control of genome, but its final step occurs by self-

orientation. The next step is accumulation of sporo-

pollenin around the receptor molecules in the form of

small droplets—emulsion units, which are the initial

sporopollenin particles (Gabarayeva, 1995). Later

these initial particles fuse with one another leading

to the formation of the initial sporopollenin pattern—

the proexine (primexine).

Self-assembly and an important role of physical–

chemical interactions in the process of exine formation

have been suggested by several authors (Heslop-

Harrison, 1972; Gerasimova-Navashina, 1973; Roland

and Vian, 1979; Dickinson and Sheldon, 1986; Van

Uffelen, 1991). These ideas were developed and con-

firmed experimentally mainly by Hemsley and his co-

authors (Hemsley et al., 1992, 1994, 1996; 1998,

2000; Collinson et al., 1993). These authors argue that

sporopollenin is formed as an emulsion in situ, and that

exine formation is carried out by simple physical and

chemical interactions. Our hypothesis is similar, but

there is also a difference. We consider that the exine

framework—the glycocalyx with its receptor sites—is

formed by self-assembly, and subsequent sporopollen-

in accumulation is determined by sporopollenin recep-

tors, whereas Hemsley and coauthors suggested that

exines are sporopollenin colloidal crystal-like struc-

tures, formed lagely by self-assembly.

We suggest that the initial microglobular sporopol-

lenin accumulation is determined by sporopollenin

acceptor particles (SAPs) of the glycocalyx units.

Further sporopollenin accumulations (‘‘receptor-inde-

pendent sporopollenin’’, or ‘‘secondarily accumulated

sporopollenin’’—Rowley and Claugher, 1991, 1996)

probably are self-assembled from sporopollenin emul-

sion. It looks like the main reason why this second-

arily accumulated sporopollenin is not resistant to

various physical- and chemical-degradation experi-

ments (Rowley and Claugher, 1996; Rowley et al.,

2001; Gabarayeva et al., 2003a,b) is the mode of its

accumulation.

4.4. Foot layer and endexine

The endexine in Enchephalartos altensteinii con-

sists of several lamellae. It is thin, but quite distinct.

The endexine appears at the end of tetrad period,

when the callose envelope of the tetrads is almost

disintegrated, and the microspores are kept in tetrads

only by virtue of the mother cell envelope. Every


lamella has a central white line. The outer part of

the first (the outermost, primordial) lamella is the

foot layer. This accords with the opinion of Nabli

(1975) that a foot layer is an outer half-part of the

primordial lamella, with a white line as a border

between ect- and endexine. However, Lugardon

(1995) considers that in Gymnosperms (sensu lato)

the whole outmost lamella belongs, as the rest of

lamellae, to endexine. It is interesting that in some

gymnosperms the endexine is established earlier

than the ectexine (Chamaecyparis lawsoniana,

Lugardon (1995)). This is an example of hetero-

chrony (Blackmore, 1986; Blackmore and Barnes,

1990).

4.5. The aperture

Our study on the exine development in Stangeria

eriopus has shown that the pollen grains of this

species have a proximal rather than distal aperture

(Gabarayeva and Grigorjeva, 2002). Indeed, the prox-

imal portion of the Stangeria pollen grain has much

thinner sporoderm than the distal portion. In Ence-

phalartos microspores the proximal portion of the

exine has a space, where alveolus walls are lacking

beneath a very thick tectum. The exit of the pollen

tube from the proximal pole seems unlikely. There-

fore, we think that Encephalartos pollen grains, unlike

Stangeria, have a distal aperture.

5. Conclusions

(1) The first step in exine development is the

appearance of the framework of the exine—the gly-

cocalyx. This glycocalyx consists of separate radially

oriented units (tufts) 120–160 nm in diameter. The

formation of the glycocalyx is always accompanied by

a considerable invagination of the plasma membrane.

Oscillatory movements of the plasma membrane are

evidently necessary for building up the tuft units from

the side of the plasma membrane. One tuft in Enche-

phalartos corresponds to one future alveola. It is

proposed that the glycocalyx is formed by the process

of self-assembly.

(2) The most prominent character of the middle

tetrad stage is the appearance of electron dense

particles alongside the walls of tufts. We consider

these particles to be sporopollenin acceptor particles

(SAPs—enzyme molecules) which are distributed

through the glycocalyx mainly under control of ge-

nome and finally self-oriented. SAPs always appear at

those very places where sporopollenin accumulation

is going to initiate (the walls of cylinder tuft units in

Encephalartos, bringing about a formation of the

alongate alveolate structure).

(3) Small osmiophilic globules with diameter about

30–50 nm appear around sporopollenin acceptor

particles at the next developmental stage. These

microglobules are evidently initial sporopollenin or

sporopollenin precursor accumulations around sporo-

pollenin acceptor particles (SAP), the progenitor form

of sporopollenin accumulation. Hence, at this stage

the initial sporopollenin accumulation is determined

by sporopollenin receptors.

(4) Subsequent sporopollenin accumulation, which

results in the appearance of the mature alongate

alveolate pattern of the exine, is independent of SAPs

and can be regarded as secondary accumulated spo-

ropollenin. The latter is probably accumulated by self-

assembly. Thus we suggest that two stages in exine

development are realized by self-assembly: the for-

mation of the glycocalyx and the accumulation of

receptor-independent sporopollenin. The intermediate

stage—the initial accumulation of receptor-dependent

sporopollenin—is carried out mainly under control of

genome by SAPs.

(5) The thin foot layer and the endexine, consisting

of several lamellae, appear in the end of the tetrad

period.

(6) In the proximal portion of the mature exine the

alveolate pattern is modified, only the distal walls of

the alveoli are present with a vast space (gap) under-

lying the very thick tectum. The distal aperture site is

regularly alongate alveolate and is covered with a thin

tectum.

Acknowledgements

We are very grateful to N.N. Arnautov (Komarov

Botanical Institute) for the possibility of using living

materials from the greenhouses for this study, and to

our electron microscope engineer Peter Zinnman. We

also greatly appreciate all the facilities presented by

J.R. Rowley and B. Walles (Stockholm University).


This research was supported by the Russian

Foundation of Fundamental Investigations (RFFI)

No. 03-04-49108.

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Documents

Exine development in Encephalartos altensteinii (Cycadaceae): ultrastructure, substructure and the modes of sporopollenin accumulation