Protoplasma107, 27--45 (1981) PROTOPLASMA

9 by Springer-Verlag 1981

Pre-Fertilization Ovule Development in Capsella:

Ultrastructure and Ultracytochemical Localization
of Acid Phosphatase in the Meiocyte 1

PATRIClA SCI-IIJLZ 2 * a n d W . A. JENSEN *

2 Department of Biology, University of San Francisco, and 3 Department of Botany,

University of California, Berkeley

Received October 27, 1980

Accepted February 27, 1981

Summary
Pre-meiotic and prophase I ovules of Capsella bursa-pastoris (L.) Medic. (monosporic, Poly-
gonum type of gametophyte development) were fixed routinely or incubated in a modified
Gomori medium containing [3-glycerophosphate as a substrate. Prior to the beginning of
meiosis the potential meiocyte is ultrastructurally similar to the other cells of the nucellus
and is distinguished only by its size and position. At the initiation of prophase I dramatic
ultrastructural and ultracytochemical changes take place in the female meiocyte. These
include the sudden appearance of cytoplasmic structures composed of single and multiple
concentric cisternae, distinctive changes in plastids and mitochondria, and the blebbing of
0.3 ~tm double-membraned vesicles from the nuclear envelope. The concentric cisternae
encapsulate portions of cytoplasm containing ribosomes, plastids, mitochondria, ER fragments
and vesicles. Both single and multiple concentric cisternae localize high levels of acid phos-
phatase and function as autophagic vesicles (AVs) that sequester ribosomes and organelles
for destruction during meiosis. Plastids stop dividing and become more spherical during
prophase I. Some plastids localize acid phosphatase and many show continuities between
the outer membrane and the plastid envelope and acid phosphatase-rich RER cisternae.
Mitochondria appear as dense, contracted spheres or rods. Some mitochondria localize acid
phosphatase but they do not show membrane confluencies with the ER. Some of the plastids
and mitochondria that are segregated into the functional megaspore at meiosis I[ are
destroyed but others apparantly survive meiosis and give rise to the plastid and mitochondrial
populations of the young gametophyte (ScHuLZ and JrNS~N, unpublished). The lateral and
end walls of the meiocyte show patches of intense aniline blue fluorescence and the chalazal
end wall of the ceil is perforated with large numbers of plasmodesmata.
Keywords: Acid phosphatase; Capsella; Female meiocyte; Ovule; Ultrastructure.
Research supported by NSF Grant PCM-79-11018. The authors gratefully acknowledge
the valuable assistance of David Lee Ivans in this project.
* Correspondence and Reprints: Department of Biology, University of San Francisco, San
Francisco, CA 94117, U.S.A.

0 0 3 3 - 1 8 3 X / 8 1 / 0 1 0 7 / 0 0 2 7 / $ 03.80
28 PATRI(~IASCHULZ and W. A. JENSEN

1. Introduction
Female meiosis (megasporogenesis) in flowering plants occurs within a
developing ovule (immature seed) inside the ovary at the base of the pistil.
Capsella has a monosporic, Polygonum pattern of gametophyte development
(MAHeSXVARI1950) in which three of the linearly-arranged meiotic products
(megaspores) degenerate and the fourth (the chalazal megaspore) develops
into the female gametophyte which produces a single egg cell (GuIGNARD
1902, HENRY 1958, SCHULZ and JensEn 1968). Major questions concerning
female meiosis in angiosperms have centered on the problems of 1. the factors
that induce a particular nucellar cell to differentiate into the meiocyte, 2. the
continuity of plastids and mitochondria during meiosis, and 3. the ultra-
structural and molecular mechanisms that effect the turnover of information
during the change from the diploid sporophyte to the haploid gametophyte
generation. Studies on Lilium (tetrasporic development) (RoDKIEWlCZ and
MIKtSLSKA 1965, DICKINSON and HESLOP-HARRISON 1977, DICKINSON and
PoxT~I~ 1978, DICKINSON and ADREWS 1977) and Zea rnays (monosporic
development) (RussELL 1979) have greatly increased our understanding of the
ultrastructural events that accompany female meiosis in angiosperms, but the
answers to these basic questions are still not clearly resolved. In this work
on Capsella ultracytochemical procedures were combined with ultrastructural
studies to gain further insight into the mechanisms involved in cellular dif-
ferentiation during female meiosis.
The paper describes the female meiocyte just prior to and during prophase I
of meiosis. Subsequent papers will describe the products of meiosis I and II
(the dyad and the tetrad), the development of the functional megaspore and
the two-nucleate gametophyte.

2. M a t e r i a l s and M e t h o d s
Plants of Capsella bursa-pastoris (L.) Medic., the shepherd's purse, were collected at the
Botanical Garden, University of California, Berkeley.

2.1. Electron Microscopy

Whole ovaries and single ovules dissected from ovaries were fixed in 6 % glutaraldehyde (GA)
in 0.1 M sodium cacodylate buffer, p H 7.2, for 4 hours at room temperature. The tissue was
washed for 1 hour with 4 changes of buffer and postfixed with 2 % unbuffered OsO4
containing 4~ sucrose overnight at 4 ~ Acetone was used for dehydration and 1% uranyt
nitrate was added to the 70~ acetone mixture. The material was embedded in Epon and
sectioned with a diamond knife. Sections were stained on grids with lead citrate and observed
with a Zeiss EM 9 A electron microscope.

2.2. Light Microscopy

Tissue was fixed in GA only and embedded in Epon as above. Sections were cut at 1.5 ~tm
and stained with aniline blue black (FISHER 1968).
 Pre-Fertilization Ovule Development in Capsella 29

2.3. Localization of Acid Phosphatase

Whole ovaries or single ovules were prefixed in 3~ GA in 0.05 M sodium cacodylate buffer,
pH 7.0, for 1-2 hours followed by a 24 hour wash with multiple changes of buffer. The
tissues were then incubated in a medium containing sodium ~-glycerophosphate (grade 1,
Sigma) and lead nitrate in a 0.1 M Tris-maleate buffer, p H 5, for 3 89 hours at room tem-
perature following the method of BARIiA and Am)~t~SON (1962). Controls were incubated
without substrate or with substrate plus 0.01 M sodium fluoride, Following incubation the
tissues were washed 1 hour in Tris-maleate buffer, p H 5 , and fixed overnight at the same
30/0 GA solution used for pre-fixation. This was followed by a I hour wash in cacodylate
buffer and postfixation for 4 hours in 20/0 unbuffered OsO4 containing 40/0 sucrose. An
additional postfixation step was carried out using 0.5~ uranyl acetate in veronal acetate
buffer, p H 6.0, for 1 89 hours. Tissues were dehydrated in acetone and embedded in Spurr's
epoxy resin.

2.4. Fluorescence Microscopy

Whole ovaries were fixed for 36 hour in 60/0 GA in 0.1 M sodium cacodylate buffer, p H 7.2,
and then placed in 850/0 lactic acid and heated in boiling water for 1 minute. After a brief
rinse tissues were stained for 10 minutes to 2 hours in 0.005% aniline blue (Fisher) in 0.15 M
phosphate buffer, p H 8.2. Ovaries were then squashed with a coverslip and observed with
a Zeiss fluorescence microscope using a BG 12 exciter filter and barrier filter #53.

3. R e s u l t s
Ovule development in Capsella begins with the outgrowth of the nucellus
from the parietal placenta of the ovary (Fig. 1). At a very e~,rly stage in
development, before the integuments appear, a single hypodermal cell near
the apex of the nucellus enlarges and becomes the potential meiocyte (Figs. 1
and 2).
3.1. The Meiocyte Prior to Prophase I
Prior to the onset of meiosis the potential meiocyte (approximately 7 )< 15 ~m
in median longitudinal section) is distinguishable from the other cells of the
nucellus only by its size and position. It possesses a large, central spherical
nucleus with a dense nucleolus and a complement of organelles similar in
appearance and distribution to those in neighboring nucellar cells. Mito-
chondria are spherical to ovoid in shape (0.5 5< 1.0 tim) (Fig. 2) and contain
ribosomes, intramitochondrial granules and a sparse number of cristae. The
mitochondrial matrix is less dense than the cytosol and contains fibrillar
material presumed to be DNA. Plastids are larger than mitochondria
(0.5 • 1.5 tim) and less numerous (Fig. 2). The plastid matrix contains one
or two unfused thylakoids, osmiophilic droplets, ribosomes and fibrillar
material, but never any starch. Both plastids and mitochondria show
dumbbell-shaped profiles that are interpreted as dividing organelles (Fig. 2).
The potential meiocyte is dense with ribosomes and has short segments of
RER sparsely distributed in the cell (Fig. 2). The cell also contains dictyo-
somes and a few microbodies and small vacuoles. Both the lateral cell walls
and the end walls have plasmodesmata (Fig. 2).
Fig. 1. Ovule development in Capsella begins with the outgrowth of the nucellus from the
parietal placenta. Very early, a single hypodermal cell near the tip of the nucellus (the
potential meiocyte) begins to enlarge. Aniline blue black. • 450
Fig. 2. The potential meiocyte is similar in uhrastructure to the other cells of the nucellus.
Plasmodesmata are present in the lateral and end walls of the cell. Plastid (P), mitochon-
drion (M). • 10,500
Fig. 3. The female meiocyte in pachytene of prophase I shows a dramatic change :in appear-
ance. The cytoplasm contains numberous concentric ER cisternae, spherical plastids (P) and
dense, contracted mitochondria (M). Synaptinemal complexes are present in the nucleus
(arrow) and plasmodesmata occur in the lateral walls and chalazaI end wall of the ceil.
Vacuolate lenticular cells border the meiocyte laterally. • 7,000
32 PATRIC1ASCHULZand W. A. JENSEN

3.2. The Meiocyte During Prophase I

At the onset of prophase I the meiocyte, which measures approximately
10 • 24 ~m, shows a dramatic change in ultrastructure (Fig. 3). The nucleus
is displaced toward the micropylar end of the cell leaving the bulk of cyto-
plasm and organlles in the chalazal half. Despite this general polarization of
cytoplasm some plastids, mitochondria and other organelles remain at the
micropylar end of the cell. Synaptonemal complexes, marking the pachytene
stage of prophase I, are visible in the nucleoplasm (Figs. 3 and 4). Vigorous
nucleocytoplasmic interaction is reflected by the blebbing of double-mem-
braned vesicles (0..3 ~tm diameter) that contain dense amorphous and fibrillar
material from the nuclear envelope (Figs. 5-7). The fate of these vesicles,
identified by their possession of nuclear pore complexes (Fig. 7), is not known.
The nuclear envelope is continuous in some places with long RER cisternae
(Fig. 8).
A striking feature of the meiocyte cytoplasm is the appearance of numerous
single and multiple concentric cisternae (Figs. 3 and 9-12). These concentric
cisternae enclose areas of cytoplasm containing primarily ribosomes, but also
some plastids (Fig. 11), mitochondria (Fig. 12), short segments of RER (Fig. 9),
and vesicles (Fig. 10). The single concentric cisternae have ribosomes attached
to the cytoplasmic surfaces of both the inner and the outer membranes of
the cisternum (Figs. 9 and 10), while the multiple concentric cisternae show
ribosomes attached only to the outer membrane of the outermost cisternum
and the inner membrane of the inner most cisternum (Fig. 10). Both single
and multiple concentric cisternae appear to be derived from the RER (Fig. 10)
and both show the specific localization of high levels of acid phosphatase
(Figs. 13 and 14). Control tissues incubated without substrate (Fig. 15) or
with substrate plus sodium fluoride (Fig. 16) do not show enzyme locali-
zation.
The plastids and mitochondria of the meiocyte show distinctive changes in
appearance at the beginning of meiosis. Plastids stop dividing and become
more spherical in shape (1.0 ~m diameter) (Figs. 3, 9, and 17). They lose
much of their internal membrane structure but retain ribosomes, osmiophilic

Fig. 4. Enlarged view of a synaptinemal complex in the nucleoplasm of the meiocyte.

X57,000
Fig. 5. Double-membraned vesicles containing dense material are pinched from the nuclear
envelope of the meiocyte. Nucleus (N). X 50,000
Fig. 6. A further stage in the blebbing of double-membraned vesicles from the nuclear
envelope. Nucleus (N). X40,000
Fig. 7. Double-membraned vesicles released from the nuclear envelope of the meiocyte contain
fibrillar material and show nuclear pore complexes (arrows). Mitochondrion (M). • 66,000
Fig. 8. The nuclear envelope of the meiocyte is continuous with the RER (arrow). Nucleus (N).
X 39,000
 Pre-Fer~ilizadon Ovule Development ~n Capsella 33

Figs. 4-8
;3 Protoplasma 107/1--~
34 PATRIelASCHULZand W. A. JENSEN

droplets and fibrillar material in a dense matrix (Figs. 9 and 17). An unusual
feature of meiocyte plastids is that they frequently show membrane continu-
ities between the outer membrane of the plastid envelope and the membranes
of the RER (Fig. 17). Plastid, RER membrane continuities, which are com-
mon in the meiocyte during prophase I, are not observed at earlier or later
stages of development. Some plastids show acid phosphatase localization
(Figs. 14 and 18) and these appear to have direct membrane continuities with
acid-phosphatase-rich (Fig. I8). Although starch may be present in the plastids
of the nucellar cells at this time, it never accumulates in the plastids of the
meiocyte.
During prophase I mitochondria appear as dense, contracted spheres (0.3 ~tm
diameter) or rods (0.2 X 1.0 ~m) which are strikingly different in appearance
from the mitochondria in the neighboring nucellar cells (Figs. 3, 9, and 19)
and the mitochondria of the potential meiocyte (Fig. 2). The mitochondrial
matrix contains ribosomes, dense granules and fibrillar material and is
penetrated by long, slender cristae (Fig. 19). Some mitochondria show acid
phosphatase localization (Fig. 14) but continuities between the mitochondrial
membranes and the RER are never seen.
The meiocyte contains active dictyosomes (Figs. 10 and 17) and a few micro-
bodies (Fig. 21), small vacuoles and lipid droplets (Fig. 3). The cytoplasm
is filled with ribosomes (Figs. 10 and I7) and microtubules are abundant
(Figs. 6, 17, and 19).
The plasmalemma of the meiocyte is the site of vigorous exocytotic activity.
Numerous double-membraned vesicles (0.3 pm diameter) with electron trans-
lucent contents fuse with the cell membrane and release single-membraned
vesicle into the cell wall (Figs. 20 and 21). The origin of the double-mem-
braned vesicles is not known but they may be related to the deposition of
aniline blue fluorescent substances into the cell walls of the meiocyte (Figs. 22
and 23). Plasmodesmata are present in small numbers in the lateral walls
and in large numbers in the chalazal end wall, but they are completely ex-
cluded from the micropylar end wall of the cell (Figs. 3 and 24). The
chalazal end wall, which is richly perforated with plasmodesmata, has a close
spatial relationship with SER membranes in both the meiocyte and the
adjacent nucellar cell (Fig. 24). Surface views suggest that SER membranes
may be continuous with the plasmalemma bordering the chalazal end wall in
both cells (Fig. 24).
Fig. 9. Concentric ER cisternae (AVs) in the meiocyte envelope ribosomes and short segments
of RER. Note the differences in mitochondrial (M) morphology in the meiocyte (bottom)
and adjacent nucellar cell (top). Plastid (P). •
Fig. 10. Numerous single and multiple concentric ER cisternae (AVs) in the meiocyte
envelope portions of cytoplasm containing ribosomes and small vesicles. • 47,300
Fig. 11. A meiocyte plastid trapped inside of an AV. •
Fig. 12. A meiocyte mitochondrion trapped inside of an AV. X 39,000
 Pre-Fertilization Ovule Development ~ri Capselta 35

Figs. 9-12
3*
36 PaT~mm SCHVLZet al.: Pre-Fertilization Ovule Development in CapseIla

3.3. The Nucellus Surrounding the Meiocyte

The nucellus enveloping the meiocyte has a specific organization not described
in light microscope studies of pre-fertilization ovule development in Capsella
(GuIGNARD 1902, HENleY1958).
A single layer of cuboidal epidermal cells (5 • 5 ~tm) delimits the surface of
the ovule (Fig. 1). A layer of lenticular-shaped cells (6 • 14 ~tm) immediately
envelops the meiocyte on all sides (Figs. 1 and 3), and files of nucellar cells
extend basipetally from the chalazal ends of the meiocyte and the lenticular
cells (Figs. 1 and 3). The ultrastructure of the nucellar cells (which will not
be described here) is distinctly different from that of the meiocyte (Fig. 3)
and specific changes occur in these cells as the meiocyte goes through meiosis I
and II. Of special note here is the development of large vacuoles in the
lenticular nucellar cells that border the meiocyte laterally (Fig. 3). The
vacuolation of these ceils may exert pressure on the meiocyte that could in-
fluence the pattern of meiotic cell divisions and the subsequent destruction
of the non-functional megaspores.
3.4. The Integuments
During prophase I the inner and outer integuments are initiated from the
base of the ovule (Fig. 22).

4. D i s c u s s i o n

Dramatic ultrastructural and cytochemical changes occur in the cytoplasm of

the female meiocyte of Capsella during prophase I of meiosis. One of the
most striking changes is the sudden appearance of structures composed of
single and multiple concentric cisternae. Somewhat similar structures have
been well documented in the female meiocytes of Lilium candidum (RoDKIE-
WlCZ and MIKULSI(A1965) and Lilium longifiorum (DICKINSONand ANDREWS
1977, nI~ BOER-DE JEu 1978), which have a tetrasporic pattern of embryo
sac development (MAHESHWARI 1950). This study of Capsella is the first
report of the presence of such concentric membrane structures in the female
meiocyte of a monosporic species. DICKINSONand ANDREWS(1977) interpret
the concentric membranes as "membrane inclusions" that protect functional
ribosomes enclosed within them from hydrolytic enzymes that degrade the
rest of the ribosomes in the cell. RODKIEWICZand co-workers observed the
disappearance of the contents enclosed within the concentric membranes
(RODXlEWlCZ and MmULStCA1965) and demonstrated acid phosphatase locali-
zation at the light microscope level in the same region of the embryo sac that
contains the concentric membranes (RoDK~EXVICZand KXrIATKOWSKA1965).
They conjectured that the structures hold reserve substances that are used up
during gametophyte formation. In Capsella the concentric membrane-bound
Fig. 13. Single concentric ER cisternae (AVs) in the meiocyte show acid phosphatase locali-
zation. X 32,400
Fig. 14. Multiple concentric ER cisternae (AVs) in the meiocyte show acid phosphatase
localization. Some plastids (P) and mitochondria (M) also localize acid phosphatase. X36,600
38 PArt~ici~_ScmrLz et al.: Pre-Fertilization Ovule Developmentin Capselia

structures appear to originate from the RER, are rich in acid phosphatase and
encapsulate ribosomes, plastids, mitochondria, short segments of RER and
other cytoplasmic substances. As meiosis proceeds the multiple-concentric-
membrane-bound structures that segregate into the functional megaspore are
ultimately absorbed into the large vacuoles of the functional megaspore and
the two-nucleate gametophyte (SCHULZand JENSEN 1978).
The single-concentric-cisternum-bound structures on the other hand appear to
be transformed into small vacuoles in the young gametophyte by the digestion
of the sequestered cytoplasm and the inner membrane of the cisternum
(SCHtJLZ and JENSEN, unpublished). We interpret both types of concentric-
membrane-bound structure as autophagic vacuoles (AVs) (BuvAr 1971) that
function in the destruction of ribosomes, some plastids and mitochondria and
other cytoplasmic substances that control the expression of the sporophyte
generation during the changeover to the gametophyte phase of the life cycle.
The ribosomal population destroyed inside of AVs is restored by the massive
synthesis and outpouring of new ribosomal subunits from the huge nucleolus
in the nucleus of the surviving megaspore (SCHULZ and JENSEN, un-
published).
Despite numerous studies on the subject, the precise fate of plastids and
mitochondria during female meiosis in angiosperms is difficult to interpret.
An early paper by ISRAEL and SAGAWA (1965) reported that the plastids and
mitochondria of the female meiocyte of the orchid Dendrobiurn degenerate
during prophase I. In contrast, the plastids and mitochondria of Myosurus
minirnus (WooDCOCK and BELL 1968) and Lilium Iongiflorum (DICKINSON
and HESLoV-HARRISON 1977) are reported to go through stages of dedifferen-
tiation and redifferentiation during female meiosis. Another report on
Lilfurn, however, suggests that a portion of the mitochondrial and plastid
populations may actually be eliminated (DICKINSON and POTTER 1978).
RUSSELL (I979) observed that the plastids and mitochondria in Zea rnays
become structurally simplified during meiosis, increase in complexity in the
functional megaspore and two-nucleate gametophyte and then degenerate in
the four-nucleate gametophyte. In Capsella the plastids of the female meiocyte
stop dividing, become more spherical and lose much of their internal mem-

Fig. 15. Acid phosphatasecontrol tissue incubated without substrate. X 23,400

Fig. 16. Acid phosphatase control tissue incubated with substrate plus 0.01 M sodium fluoride.
X33,750
Fig. 17. Meiocyte plastids (P) become spherical and lose most of their internal membrane
structure, but ribosomes and fibrillar material remain in the matrix. Many meiocyte plastids
show direct continuities between the outer membrane of the plastid envelope and the
membranes of RER cisternae (arrow). Mitochondrion(M). • 40,000
Fig. 18. Some meiocyte plastids show acid phosphatase localization and show direct membrane
connectionswith acid-phosphatase-richRER. > 61,250
Figs. 15-18
40 PATRmIASCHULZand W. A. JENSEN

Fig. 19. Meiocyte mitochondria have a characteristic dense, contracted appearance. Long,
slender cristae penetrate the dense matrix which contains ribosomes, mitochondrial granules
and fibrillar material Microtubules are abundant in the cytoplasm. X 60,700
Fig. 20. Double-membraned vesicles (arrow) with electron translucent contents fuse with
the plasmalemma (PL) of the meiocyte. Cell wall (CW). X 50,000
Fig. 21. Double-membraned vesicles (arrow) that fuse with the plasmalemma of the meiocyte
release single-membraned vesicles into the cell wall (CW). Microbody (MB). >(40,000

brahe structure. A portion of the plastid population is sequestered within

AVs (Fig. 11). Some of the AV-encased plastids segregate into the functional
megaspore at meiosis II and are absorbed into the large vacuoles of the func-
tional megaspore and two-nucleate gametophyte where digestion is completed
(ScHurz and J~NSEN, unpublished). Of the meiocyte plastids not encased in
 Pre-Fertilization Ovule Development in Capsella 41

Fig. 22. Whole ovule containing a meiocyte in prophase I, viewed with phase contrast micros-
copy. Note the initiation of the inner and outer integuments from the base. of the ovule
(arrows). X340
Fig. 23. The same field as shown in Fig. 22 but viewed with UV fluorescence microscopy.
Pat&es of aniline blue-induced fluorescent material are present in the lateral and end wails
of the meiocyte. • 340
Fig. 24. A view of the chalazal end wall of the meiocyte (MC) showing many plasmodesmata
(PL) in cross section. SER membranes show a close relationship and possible continuity with
the plasmalemmas bordering the wall in both the meiocyte and the adjacent nucellar cell (NU)
(arrows). X 35,700
42 PATRIelA SCHULZ and W. A. JENSEN

AVs, a portion of these localize acid phosphatase and some show direct
continuities of the outer membrane of the plastid envelope with the RER
(Figs. 17 and 18). The occurrence of plastid-RER membrane continuities,
which have also been observed in the dividing microsporocytes of Gingko
(WO~NIAt~ 1976) and in fern gametophyte tissue (CRoTTY and Lt~DBETTER
1973), suggests a mechanism for the entry, of acid phosphatase from the RER
directly into the plastid and may also reflect an early stage in the encasement
of the plastid in an AV by the subsequent wrapping of the attached RER
around the organelle. Some of the plastids that segregate into the functional
megaspore appear to survive meiosis and give rise to the plastids of the
gametophyte generation (SCHuLz and J~NSEN 1978). There is no evidence
from this study of Capsella to suggest that all of the plastids are destroyed
or that the plastids of the gametophyte are formed de novo.
The mitochondria of the Capsella meiocyte have a dense, contracted ap-
pearance characteristic of the mitochondria of the female meiocyte of Liliurn
(DICKINSON and POTTER 1978). In Capsella, a small number of meiocyte
mitochondria are encapsulated and destroyed inside of AVs (Fig. 12). Of the
mitochondria not encased in AVs, some localize acid phosphatase (Fig. 14)
but none show membrane continuities with the RER. Some of the meiocyte
mitochondria not associated with AVs that segregate into the functional
megaspore are destroyed during meiosis and young gametophyte development,
but others appear to survive meiosis and give rise to the mitochondria of the
new generation (SCHULZ and J~NS~N, unpublished). The presence of high
levels of acid phosphatase in some mitochondria (and plastids) not encased in
AVs may signal the immanent destruction of these organelles or may reflect
some form of molecular reorganization occurring within organelles destined
to survive. HILL (1977) observed high levels of acid phosphatase in plastids
and mitochondria of the synergids of cotton (cells that are destined to
degenerate) a few hours after anthesis.
A major unanswered question concerning the mitochondria of the female
meiocyte is the origin of the dense, contracted organelles which are conspicu-
ously different in morphology when compared with the mitochondria of the
potential meiocyte (Fig. 2) and the mitochondria of the cells of the nucellus
that surround the meiocyte (Figs. 3 and 9). Do the meiocyte mitochondria
develop directly from the pre-existing mitochondria of the potential meiocyte,
possibly by a conformational change (HAcKENm~OCKet al. 1971) reflecting
metabolic changes in the cell or do they arise de novo? The best known
examples of strong nucleocytoplasmic interactions suggesting possible organelle
generation in the reproductive ceils of plants occur in the maturing egg cells
of ferns which produce conspicuous sac-like evaginations of the nuclear
envelope (B~LL 1972, 1974). Similar nuclear evaginations or blebs have not
been observed in the egg cells of flowering plants (SCHULZand JENSEN 1968)
but do appear at earlier stages in development, i.e., during sporogenesis
 Pre-Fertilization Ovule Development in Capsella 43

(DICKINSON1971) and during the growth of the megaspore (CoccuccI 1969).

Some investigators interpret these nuclear evaginations to represent the
de novo generation of mitochondria and plastids (BELL 1972, COCCUCCI1969),
a view that has not gained widespread popularity. In support of this view,
however, is a recent paper by BELL (1979) that shows the presence of small
patches of reaction product for succinic dehydrogenase deposited in the
envelopes of mitochondria-like nuclear evaginations in fern egg ceils.
The fate of pre-meiotic mitochondria has not been traced in detail in the
potential meiocyte of Capsella, nor has the fate of the double-membraned
vesicles pinched from the nuclear envelope of the meiocyte been determined
(Figs. 5-7). At present there is no direct evidence to suggest that nuclear-
evolved vesicles differentiate into mitochondria. An alternative view is that
nuclear blebbing represents the transfer of large quantities of macromolecular
substances to the cytoplasm to direct the ensuing growth and differentiation
of the meiocyte. Such mechanisms for the transfer of nuclear material have
been commonly observed in animal cells (SZOLLOSI1965, HAY 1968).
Equally elusive is the answer to the question of the factors that induce the
differentiation of the meiocyte. Early electron microscope studies indicated
that plasmodesmata were entirely absent from the cell walls of the potential
female meiocyte (ISRAEL and SAGAWA 1965). Later observations of aniline
blue-induced fluorescence in the cell walls of the female meiocytes of several
species suggested that these walls were rich in callose (a complex carbohydrate
consisting primarily of ~-1,3 glucans) (RoOKIEWICZ 1968). Further studies
showed that callose-enveloped microspores have reduced permeability to
radioactive precursors (HEsLoP-HARKISON and MACKENZIE 1967) and led
some investigators to speculate that the young meiocyte was somewhat
chemically isolated from the other cells of the nucellus and that this isolation
triggered the development of the gametophyte (HESLoP-HAI~RISON 1964,
RODKIEWlCZ 1970). More recently RODKIEWlCZ and his co-workers have
shown that callose-free plasmodesmata are present in large numbers in the
chalazal end walls of the female meiocytes of several orchids with mono-
sporic Polygonum type of development, and that the chalazal end wall of
some species possesses transfer wall ingrowths (RoDKIEWICZ 1975, RODKIEWICZ
and BEDNARA1976). Plasmodesmata have also been observed in the chalazal
end wall of the megasporocyte of Zea mays (RusSELL 1979).
The present study shows that large number of plasmodesmata also occur in
the chalazal end wall of the meiocyte of Capsella and that some, but many
fewer, are present in the lateral walls. In addition, there is an intimate
relationship suggesting possible continuity of SER membranes with the
plasmalemma bordering the chalazal end wall in both the meiocyte and the
neighboring nucellar cell. These observations support the emerging concept
that the female meiocyte of monosporic Polygonum type species is not an
isolated cell but is in close communication with the nucellus in the region of
44 PATRIeIASCHULZand W. A, JENSEN

the chalazal end wall which appears to be the p r i m a r y site for the transfer
of metabolites and other substances into the cell. The polarized transport of
substances into the meiocyte may, in part, influence the pattern of meiotic
cell divisions and the survival of the chalazal megaspore of the tetrad. In
monosporic Oenothera type ovules where the m i c r o p y l a r megaspore develops
into the gametophyte, there is a reduction in aniline blue fluorescence
(RoDKIEWlCZ 1970) and evidence of plasmodesmata in the m i c r o p y l a r end
wall of the meiocyte (JALoUZOT 1978). Recent studies b y SMITH and McCuLLY
(1978) have cast doubt on the specificity of aniline blue-induced fluorescence
for the presence of [3-1,3 glucans (callose). This w o r k reopens the question
of the identification of aniline blue fluorescent substances in the meiocyte
cell wall and their function during meiosis.
The factors that trigger the differentiation of the meiocyte are p r o b a b l y more
subtle and m a y be related to the position of the cell with respect to vascular
supply, centers of hormone production and oxygen tension. Stress forces
imposed b y the surrounding tissues of the ovule m a y also p l a y an i m p o r t a n t
role in determining the position of the developing megagametophyte
(LINTILHAC1974).
References
BARKA, T., ANDERSON, P. J., 1962: Histochemical methods for acid phosphatase using
hexonium pararosanilin as coupler. J. Histochem. Cytochem. 10, 741--753.
BELL, P. R., 1972: NucIeocytoplasmic interaction in the eggs of Pteridium aquilinum maturing
in the presence of thiouracil. J. Cell Sci. 11, 739--755.
- - 1974: Nuclear sheets in the egg of a fern, Dryopteris filix-mas. J. Cell Sci. 14, 69--83.
-- 1979: Demonstration of succinic dehydrogenase in mitochondria of fern egg cells at
electron microscope level. Histochem. 62, 85--91.
DE BO~R-Dr JEU, M. J., 1978: Ultrastructural aspects of megasporogenesis and initiation of
megagametogenesis in Liliurn. Bull. Soc. bot. Fr., Actualit6s botaniques 125, 175--181.
BUVAT, R., 1971: Origin and continuity of ceil vacuoles. In: Origin and continuity of cell
organelles (REINERT,J., URSPRUNG,H., eds.). Berlin-Heidelberg-New York: Springer.
Cor162162A. E., 1969: Embriologia de orquideas: La megaspora de Epidendrurn scutella.
Kurtziana 5, 7--21.
CROTTY, W. J., LEDB~TT~R,M. C., 1973: Membrane continuities involving chloroplasts and
other organelles in plant cells. Science 182, 839--841.
DICKINSON, H. G., 1971: Nucleocytoplasmic interaction following meiosis in the young
microspores of Lilium Iongiflorum. Events at the nuclear envelope. Grana palynol. 11,
107--127.
- - ANDREWS,L., 1977: The role of membrane-bound cytoplasmic inclusions during gameto-
genesis in Lilium Iongiflorurn Thunb. Planta 134, 229--240.
--HESLoV-HARRISON, J., 1977: Ribosomes and organelles during meiosis in angiosperms.
Phil. Trans. R. Soc. Lond. B 277, 327--342.
-- POTTrR, U., 1978: Cytoplasmic changes accompanying the female meiosis in Lilium Iongi-
florum Thunb. J. Cell Sci. 29, 147--169.
FISH~R, D. B., 1968: Protein staining of ribboned Epon sections for light microscopy. Histo-
chem. 16, 92--96.
GUIGNARD,M. L., 1902: La double f&ondation chez les crucif~res. J. Bot. Paris 16, 361--368.
 Pre-Fertilization Ovule Development in Capsella 45

HACK~NBROCK, C. R., R~HN, T. G., WEINI3ACH,E. C., LAMASTERS, J. J., 1971: Oxidative
phosphorylation and ultrastructural transformation in mitochondria in the intact ascites
tumor cell. J. Cell Biol. 51, 123--137.
HAY, E. D., 1968: Structure and function of the nucleolus in developing cells. In: The
nucleus (DALTON,A. J., HAGUUNAU,F., eds.), pp. 1--79. New York: Academic Press.
HrNRY, A., 1958: Formation du gam&ophyte femelle chez le Capsella bursa-pastoris. Bull.
Soc. Bot. Fr. 105, 20--25.
HESLOP-HARI~ISON,J., 1964: Cell walls, cell membranes and protoplasmic connections during
meiosis and pollen development. In: Pollen physiology and fertilization (LINSK~NS,H. F.,
ed.). Amsterdam: North-Holland.
- - MACKENZIE,A., 1967: Autoradiography of soluble (2J4C)-thymidine derivatives during
meiosis and microsporogenesis in Lilium anthers. J. Cell Sci. 2, 387--400.
HILL, R. A., 1977: The ultrastructure of the synergids of Gossypium hirsutum (cotton): From
anthesis through pollen tube discharge. Ph.D. dissertation, University of California,
Berkeley.
ISRAEL, H. W., SAGAWA, Y., 1965: Post-pollination ovule development in Dendrobiurn
orchids. III. Fine structure of meiotic prophase I. Caryologia 18, 15--34.
JALOUZOT, M.-F., 1978: Differentiation des 616ments de la t&rade femelle chez Oenothera
erythrosepala. Bull. Soc. Bot. Fr., Actualit6s botaniques 125, 167--170.
LINTILHAC, P. M., 1974: Differentiation, organogenesis and the tectonics of cell wall orien-
tation. II. Separation of stress in a two-dimensional model. Amer. J. Bot. 61, 135--140.
MAH;SHWA~I, P., 1950: An introduction to the embryology of angiosperms. New York:
McGraw-Hill.
RoI3KIEwIcz, B., 1968: Differences in the distribution pattern of callose in cell walls during
megasporogenesis in some species of flowering plants. Bull. Acad. Polon. C1. V, 16,
663--665.
- 1970: Callose in cell walls during megasporogenesis in angiosperms. Planta 93;, 39--47.
-

- - 1975: Sieve-like distribution of callose in meiocyte chalazal wall in ovules of orchid

Epipactis. Bull. Acad. Polon. Sci. C1. II S6r. Sci. biol. 23, 707--711.
- B~DNAitA, J., 1976: Cell wall ingrowths and callose distribution in megasporogenesis in
-

some orchidaceae. Phytomorph. 26, 276--281.

- - KWIATKOWSKA,M., 1965: Enzymy hydrolityczne w rozwijajacym fie woreczku zalazkowyn
lilii. Acta Soc. Bot. Poloniae 34, 235--242.
- MIKULSKA,E., 1965: The development of cytoplasmic structures in the embryo sac of
-

Lilium candidum, as observed with the electron microscope. Planta 67, 297--304.
RUSSrLL, S. D., 1979: Fine structure of megagametophyte development in Zea mays. Canad.
J. Bot. 57, 1093--1110.
SCMULZ,P., J~NS~N, W. A., 1968: Capsella embryogenesis: the egg, zygote and young embryo.
Amer. J. Bot. 55, 807~819.
1978: Ultrastructural localization of acid phosphatase during female meiosis in
Capsella. J. Cell Biol. 79, 25 a.
SMITH, M. M., McCuLLY, M. E., 1978: A critical evaluation of the specificity of aniline blue
induced fluorescence. Protoplasma 95, 229--254.
SzoI.LosI, D., 1965: Extrusion of nucleoli from pronuclei of the rat. J. Cell Biol. 25, 545--562.
WOLNIAK, S., 1976: Organelle distribution and apportionment during meiosis in the micro-
sporocyte of Ginkgo biloba L. Amer. J. Bot. 63, 251--258.
WOODCOCK, C. L. F., B~LL, P. R., 1968: Features of the ultrastructure of the female
gametophyte of Myosurus minimus. J. Ultrastruct. Res. 22, 546--563.