Capsella Embryogenesis: The Synergids Before and After Fertilization
Richardis Schulz; William A. Jensen
American Journal of Botany, Vol. 55, No. 5. (May - Jun., 1968), pp. 541-552.
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‘Thu Jul 13 19:18:06 2006‘Ammer. J. Bot, 85(5): SUL-552, 1968
CAPSELLA EMBRYOGENESIS: THE SYNERGIDS
BEFORE AND AFTER FERTILIZATION:
Sister Ricrarpts Scuuiz? anv WituiaM A. Jensen
Department of Botany, University of California, Berkeley
ABSTRACT
‘The ultrastructure and composition of the synorgids of Capella burea-pasiois were studied
before and after fertilination, The synergide inthe mature embryo sac contain numerous plastids,
rmitochondia, dietyosomes and masses of ER and associated ribosomes. Each synergid contains
‘large chalazal vacuole, a nvclens with a single nucleolus and is surrounded by a wall. This wall
i thiekest at the mieropyle end of tho ell where it proliferates nto the filiform apparatus, At the
‘halazal end of the oallthe wall thins and may be absent for small distanoes. ‘The pollen tube
‘grows into one of the two synergids through the filiform apparatus and extends one-third the
length of the cell before it discharges, Following discharge of the pollen tube, mitochondria and
plastids of the tube can be identified in the synergid as can hundreds of 0:5 polysaccharide
spheres liberated by the tube. The method by which the sperm or sperm nuclei enter the egg
Or central cell s not known although an apparent rupture was found in the wal ofthe egg near
the tip of the pollen tube, The second aynergid changes at the timo the pollen tube enters the
first synergd. These changes result in the disorganization of the nucleus and loss of the chalazal
wall and plasma membrane. Eventually this synergid loses its identity as its cytoplasm merges
swith that of the central col
‘Tum, sywmnows are being assigned an ineress-
ingly important role in. discussions concerning
the entranee and discharge of the pollen tube into
the embryo sae (van der Pluijm, 1964; Jensen,
1965; Van Went and Linskens, 1967; Jensen and
Fisher, 1968). This report deseribes the synergids
of Capsella bursa-pastoris before and after ferti-
Tization. Changes. in ‘ultrastructure and com-
position of these cells are described from thir
ature condition in. the embryo. sae. through
their degeneration after fertilization. The data
shed additional light on the role of the synergids
in the mature megagametophyte and in the
events surrounding pollen tube entrance and
discharge.
Marmarats axp susrions—Plants of Capella
iuraa-pasioris L.. “Medic,” the shepherd's purse,
were grown in the greenhouse from seeds collected
at the Botanieal Garden, University of California,
Berkeley. Whole ovules vere dissected from the
siliques. and fixed, immediately in 2% KMnO,
for 15-25 hr at 4.C. ‘The tips of some of the older
ovules were excised before fixation in order to
Thliate penetration of fratives and embedding
media. Alternately, ovules were placed in 6
lutaraldehyde buffered by 0.06 x phosphate at
DIGS fort hr at 4 C-and post-fixated with 2%
+ Received fr publieation 18 September 1967.
sar pied ty NS grant ON 400 a NTL
grant S-HOT-CA 03650-4,
‘Research ofthe tefor author conducted while an NSF
PMc adgrss: Department of Biology, Rosary
“cent addres: Department. of Biology,
College, River Foret, 1 6098,
mbuifered Os0, containing 4% suerose for 15
hr at 4 C. The tissue was dehydrated in a graded
acetone series. Staining with 1% uranyl nitrate
was done in the 70% acetone, All material was
embedded in. Epon, Sections were stained with
lead citrate (Reynolds, 1963) and observed with
1 Zeiss Electron Microscope EM.9.
‘Material prepared for the histochemical locali-
zation of nucleic acids was fixed in GA-OsOs,
embedded in Epon, sectioned at 2 4 and stained
with Azure B (Fensen, 1962). ‘The periodic acid-
Schiff (PAS) reaction was used for the localization
of insoluble earbyhodrate (Jensen, 1962) and
Aniline Blue Black (1% dye in 7% acetic acid,
stained for 10 min at 60 C, rinsed in 7% acetic
acid) for a, general protein stain (Fisher, un-
published), Tissue for the latter procedures was
fixed in glutaraldehyde, embedded in Epon and
sectioned at 12 u. Permanent mounts were made
of material siainod. with Azure B and PAS with
Zeiss phase-mounting medium 115..‘Tempor
Touts of material stained wth Aniline Blue
Black were made with glycerol to which 5%
acetic ack had been added.
Resvuns—Before Sertilization—At the micro
pylar end of the embryo sac the mature egg
flanked by two synengids in triangular artange-
‘ment termed the egg apparatus. The synergids
are the same length as the xg (254) but
ases penetrate deep into the mieropylar apex of
the embryo sac whe the base of the ea al
tached at the side of the embryo sae approxi-
mately 10) back from the tip (Fig. 1). This
aL[Vol. 35
AN JOURNAL OF BOTANY‘May-June, 1968]
Fig. 2,
SCHULZ AND JENSEN—CAPSELLA EMBRYOGENESIS as
—Fig. 2. Longitudinal soction through the two synergids showing the intense staining of the nucleus, nu-
leolti, and eytopiasm for protein. The filiform apparatus (PA) and the synergid wall (double arrow) remain unstained.
‘The fused polar nuclei (PN) ean also be seen. Aniline Bl
Black, x 2,000-—Fig. 8. Longitudinal rection through the
‘sro synergide (SY) showing the PAS-positive reaction of the wall double arrows) and filiform apparatus for insoluble
‘carbohydrate, The plastids (P) stain intensely for protein, Aniline Blue Black and PAS, X 2,000.
causes the chalazal end of the egg to extend
beyond the synergids making the exe appear
longer. The synergids possess a centrally located
nucleus and a large chalazal vacuole (Wig. 1).
The micropylar end of both cells is occupied by
the complex striated filiform apparatus (Haber-
mann, 1906); (Fig. 2, 3). ‘The bulk of the
eytoplasm lies between the chalazal vacuole and
filiform apparatus. ‘The synergids arise at_ the
third mitotic division of megagametogenesis
which yields the nucleate embryo sac. They
are shortlived cells and become disorganized
and disappear soon after fertilization of the egw.
‘A cell wall surrounds each synergid. ‘This
wall is thickest toward the micropylar end of
the cell where wall synthesis began but becomes
thin and irregular over the chalazal end of the
cell where it borders the central cell. Often the
plasma membranes of the synergid and central
cell lie in close contact (Fig. 4), while at other
places the two membranes are clearly separated
by an clectron-translueent space which contains
small patehes of electron-dense material (Fig.
‘The region between the two membranes is PAS.
positive (Fig. 3) and is presumed to contain wall
material. The synergid wall becomes continuous
with the embryo sae wall in the mid-region of
the cell. Each synergid also shares. common
wall with the egg (Fig, 1) and with its sister
synengid (Fig. 2, 3). Plasmodesmata are -seen
in all parts of the wail except where it is contin-
uous with the embryo sae wall (Fig. 5).
At the mieropylar end of each. synergid ‘the
wall becomes thickened and folded into the long
finger-like projections of the filiform apparatus
extending deep into the cytoplasm (Wig. 2, 3, 6).
The filiform apparatus appears to consist of
‘two structural phases: a central electron-dense
core surrounded by more electron-translucent
phase (Fig. 6, 7). The core consists of a. tightly
Fig. 1. The micropylar end ofthe embryo sue showing the structure and relative positions of the ogg, synergid (SY),
and central ell (CC). GA-0s04, X50.AMERICAN JOURNAL OF BOTANY {Vol. 55‘May-June, 1963}
packed mass of microfibrils while the peripheral
Tegion contains fewer loosely organized, micro-
fibrils set in a translucent: matrix (Pig. 7). ‘The
filiform apparatus. gives a strong PAS-positive
reaction for insoluble earbohydrate (Fig. 8) but
is negative for nucleic acids and protein (Fig. 2).
‘The plasma membrane follows the contours of
the filiform apparatus which greatly increases its
surface area in this region (Fig. 7). ‘The plasma
rmerene and th Bare oquently eon
in the region of the filiform apparatus (Fig. 6) an
ae parallel inthe chalaal Tlf of the cell (ig
4). This parallel relationship gives the plasma-
Jesama the sopearance of being » double mem.
rane synergid has a large (0 X 11 1)
vacuole in the chalazal half of the eell and may
contain several smaller randomly distributed
vacuoles (1 ») (Fig, 1).
“Phe central nucieus is $-6 2 in diam and has
single, prominent nucleolus (3.5 a) consisting
of a mass of densely packed granules with a few
small clear areas in it, Small dense masses of
chromatin are frequently seen near the nuclear
‘membrane. The synergid cytoplasm is filled with
ribosomes and stains intensely for protein (Fig. 2)
and RNA. Characteristic of synergd eytopltam
are long profiles of ER frequently appearing in
groups of two to four strands oriented parallel
to the long axis of the cell (Fig. 5). In general,
the cistemal phase of the ER’ is narrow and
“uniform except at the tips where it may be swollen
(Fig. 8). An exception to this is the ER closely
paralleling the plasma membrane which has a
‘vide cisternal phase. At the chalazal end of the
synergid the ER often encircles plastids and
mitochondria. (Fig. 1)
“Phere are relatively few plastids in the synergid
compared with their numbers in the egg and
central cell (Fig. 1). Most of the plastids are
located in. the eytoplasm between the nucleus
and the filiform apparatus although a few may
‘ccur at the chalazsl end of the cell.
‘Many cup-shaped plastids are present (Fig. 6).
The appearance’ of the eytoplasm within’ the
invaginations is altered and contains fewer
ribosomes than the neighboring eytoplasm. This
form of plastid i also common in the central
cell, egg, zygote, and young embryo.
‘The plastids ‘are surromded by a double
membrane, and each contains several single
lamellae which are invaginations of the inner
membrane (Fig. §). Some lamellae are quite
vesiculate in appearance and only rarely are two
Big. 4, 5.-Fig. 4. The jt
SCHULE AND JENSEN—CAPSELLA EMBRYOGENESIS
BAS
fused. The plastid matrix is very electron-dense
and iain intnaly for prota allowing ito be
easily seen in light-microscope sections (Fig. 3
‘There are a few diffuse, light areas in the matric
late alo staan of.
(ig. 5). The
dense, spherical (700A diam) droplets (Fi
and ribosome-like partiles (150 A). They cont
ery litle starch, even, when stare
in the egg and central cell of the same mega-
snmetophyte,
Mitochondria are much more numerous than
plastids. They oceur throughout. the eell but are
‘more concentrated near the, filiform apparatus
(Fig. 1, 6). They are rod-shaped (1X 244 u)
and contain many short vesicular eristae. Clear
‘areas which contain tiny fibrils (25 A) sometimes
‘oceur in the center of the mitochondria between
the eristae (Fig. 6). The mitochondria (Fig. 6)
also contain intramitochondrial granules (300 A)
(Peachey, 1962) and ribosome-like particles
(050 A).
‘The uniform dictyosomes are ubiquitous but
fare present in higher numbers in the region
between the filiform apparatus and the nucleus
(Fig. 1, 5). They have 4-6 flat cisternae (0.7-
0.9.4) which produce vesicles at their tips.
‘There are a few single membrane-bound
organelles (0.3 ») randomly distributed in the cell
which are spherical or rod-shaped and have
dense granular contents (Fig. 6). Crystals are
sometimes seen in the BR cisternae. ‘They are
long and narrow (0.5 X 0.04) and appear
white in GA-Os0, black in KMnO,, They are
Believed to be, inorganic beeause they aro pre-
served with KMnOi which destroys protein
crystals (Jensen, 1965). Microtubules (Ledbetter
and Porter, 1963) have also been observed in the
synengid cytoplasm,
After friization—The pollen tube enters the
embryo sae through the flform apparatus. After
entering. the ‘synergid_eytoplaam. it continues
{fo grov in a chalagal direction close to the com-
ron egg-synergid wall until it has traveled one-
third the length of the cell. At this point the tp
of the pollen tube opens and discharge occurs
‘Thereafter both synergids undergo characteristic
changes. "The most immediate and apparent
henge oer in the eerie i penetra
by the pollen tube (degenerating synergid). ‘The
other synergid. (persistent synergid) remains
sotive fora short time after fertilization and then
‘becomes disorganized in a manner different from,
tion ofthe egg, synergid (SY), and conta ell (CC) showing the close parallel relationship
‘of the ER with the plasma membrane of the synergd (arrows). Gaps which contain a dense material (DM) separate the
‘lasina membranes of the synergid and central eall, but in some areas the two membranes are very close and appear to
have little oF no intervening material between them (double arrow). GA-OsO,, x 25,600.—Fig. 6. View of the synergid
(SY) and a portion of the egg at the level of the synergid nucleus (N) showing the relative abundance of ER, mito-
chondria (M), and dictycsomes (D). Plasmodesmata (PD) are eommon in the wall between the synengid and the exg.
Plaatids (P) are relatively undifferentiated and contain dense droplets. GA-OsO,, X 25,600,546 AMERICAN JOURNAL OF BOTANY [Vol. 55‘May-June, 1968]
‘the degenerating synengid. ‘The contents of both
synergids are ultimately reabsorbed by the
growing embryo sac.
Degenerating synergid—'There are _several
ryt paso the daca yer
fid. and pollen tube oytoplasing. ‘The large
chalazal vacuole of the synergid disappears and
the upper part of the ell colapecs against the
zygote. The cytoplasm is very dark but several
structures are still discernible. The nucleus and
Icleolus are somewhat flattened and the nuclear
membrane has disappeared (Fig. 8). Organelle
structure is disorganized and in GA-OsO, material
‘these structures are unrecognizable except where
the occasional persistence of a starch grain
identifies a plastid (Fig. 8). In KMnO, material
some mitochondria can be recognized in the
synergid and pollen tube by the presence of long
parallel tae (Fig. 10). This type of mito-
chondrion is not seen in the synergids before
fertlzaton. Dense ribosomes remain inthe
synergid and pollen tube. Many are randomly
Oren ut ota ar nour i li
Gig. 9). The plasma membrane has also dis-
{pyar rom che sutfacr of tie syntgié Cg 8)
‘pollen tube liberates hundreds of spherical
parte (0102 ) phi. spread dougie
id above. the,naintoF rupture (Fig.
3 0 Tn GAa-Os0, these particles have a dense,
ular center and are surrounded by a thin
Fran of lear material (Pig. 4). In KMnO, they
nye‘ coar eutars ant ace. gartouped bys
dark band which may be a unit membrane (Fig.
What appears to be a rupture i the wall of
the egg is seen in Fig. 11. Such structures were
repeatedly seen in the egg wall near the tip of the
discharged pollen tube
“Tho eytgplasm of tho degenerated. synereid
stains much more intensely with Aniline Blue
Black, Azure B, and PAS than it did before the
entrance of the pollen tube.
Persistent synergid—After a brief period of
normal activity following fertilization the per-
sistent synergid begins to show signs of di
organization different from those of the do-
generating synergid. One of the most notable
changes occurs in the nucleus (Fig. 12, 13). ‘The
ER becomes oriented in several layers around the
nucleus and is continuous. with. the nucl
membrane. In some places it is difficult to dis-
SCHULZ AND JENSEN—CAPSELLA EMBRYOGENESIS
27
tinguish the nuclear envelope from the ER.
‘The inner nuclear membrane invaginates. to
produce small vesicles and several short strands
of ER (with ribosomes attached) appear in the
center of the nucleus (Fig. 12). The nucleolus
breaks up into several large particulate maises
which migrate to the periphery of the nucleus
(Fig. 12). Small. groups of polyribosomes, pre-
sumably originating from the larger mastes of
nucleolar material, are seen throughout the
nucleus (Fig. 12, 18). These nuclear ribosomes are
the same size as the ribosomes in the neighboring
cytoplasm. ‘The nucleus gradually takes on the
appearance of the. eytoplasm (Fig. 12) and
eventually loses its identity.
Parts of the wall and plasma membrane at the
chalazal end of the synergid disappear and a
large vacuole of the central eell makes. contact
h the chalazal vacuole of the synengid (Fig. 14).
Eventually” the chalazal wall” and’ membrane
completely disappear and there is a gradual
mixing of the synergid and central cell eytoplasms.
As early as the first division of the zygote the
large, well-developed plastids of the central cell
ean be seen less than 14 from the filiform ap-
paratus of the persistent synergid.
Discussrox—The fine structural image of th
synergids before fertilization is one of metabol
cally ‘active cells. ‘The activity of the synergid
with its large numbers of dictyosomes and mito-
chondria. and abundance of ER is sharply con-
trasted with the image of the egg which reflects
relatively quiescent eytoplasm. ‘This study sup-
ports the conclusion that the synergids function
in the absorption. and transport of compounds
from the surrounding integuments to the exg and
possibly the central cell (Jensen, 1965).
‘The filiform apparatus is a modification of the
cell wall which greatly increases the surface ares
of the plasma membrane. The position of the
filiform apparatus is also important. because it
ecupies the micropylar tip of the embryo. sue
and comes into intimate contact with metabolite-
Jaden. integuments. The proximity of _mito-
chondria, offers an available energy supply for
active transport across the plasma membrane,
and the closely associated ER provides a direct
channel to metabolic sites deep in the evtoplasm
and ultimately regions bordering,
tho gt and contra cals Tho bes parle ap
sociation of the ER and plasma membrane in
these regions could facilitate the transfer of sub-
_ Big. 6,7,
in the region
traversed by tiny
‘The plastid P)
—Fig. 6. View of the filiform apparatus (PA) and associated cytoplasm. There are many mitochondria (M)
‘of the filiform apparatus which have short vesicular erstae, intramitochondtial granules, and clear areas
fibrils (F). The ER. is closely associated with the plasma membrane of the flform apparatus.
in the upper right corner is wrapped around a portion of the eytoplasm which appears to he inside the
organelle, GA-O:0,, X 32,800.—Fig. 7. Enlarged view of the fllform apparatus showing its microuibrillar strusture and
the close relationship of the plasma membrane. GA.OsO,, X 45,500.AMERICAN JOURNAL OF BOTANY
BAS.‘May-June, 1963} SCHULZ AND JENSEN—CAPSELEA RMBRYOGENESIS 9
Fig. 10, 11—Fig, 10, The open polle tube (PT) in the degenerating synergid (D SY). Mitochondria with long
tubular evistao are frequently seen in the eytoplasm of both the degenersting synergid and the pollen tube, Small
spherical particles resembling wall material are lerated from the open end of the pollen tube KMnO,, X 25,600.
Fig. 11. View of the open pollen tube (PT) inthe degenerating aynergid and the ruptured sygote (2) wall (W). GA-Os0,,
25,600,
Fig. 8. ‘The degenerating synengid containing the pollen tube (PT) which is open at one end, Starch gras (ST) mark
the presence of plastids. Hundreds of small particles are liberated from the pollen tube, ‘The mileus (N) and
filiform apparstus (PA) of the degenerating synergid can be seen. GA-Os0, X 9,200.
ig. 0. Enlarged view of tho eytoplasm of the dogoncrsting synergid. Particles liberated from the pollen tube have &
srantiar appearance, Chains of ubosomes and. spherical bodies which may be disorganized mitochondria (MD are fre=
tently seen, GA-OsO,, % 59,000.AMERICAN JOURNAL OF BOTANY‘May-June, 1968] SCHULZ AND JENSEN:
A EMBRYOGENESIS
Fig. 14. The persistent synergid (PSY) and a portion of the zygote (Z) showing the gap in the chalazal wall (W)
fad membrane of the synergid and the cloce relationship hetween the vacuoles of the synergid and central call (CC),
GA.Oe04, % 25,00.
stances. Numerous plasmodesmata in the walls
connecting the synergid with the egg and eéntral
cell provide an additional route for the free flow
‘of metabolites. The presence of many active
dietyosomes is characteristic of cells engaged in
secretion (Mollenhauer and Morré, 1966).
The egg and central ecll store metabolites in
the form of starch and lipid which are used in
the early stages of development, following ferti-
lization. The actual amounts of these compounds
stored depends in part on the general state of
nutrition and metabolism of the plant, but the
egg and central coll always have more starch
and lipid than the synergids. In contrast. to
Capsella the synergids of cotton store large
amounts of starch (Jensen, 1965), but this differ
ence may be related, in part, to the slower rate
of development of the cotton egg.
‘There has been much discussion about the role
of the synergids in directing the growth and en-
trance of the pollen tube into the embryo. sac
(ean der Phun, 1964; Diboll and Larson, 1966;
Jensen, 1965; Van Went and Linskens,” 19673
Jensen’ and Pisher, 1968). ‘The pollen tube. in
Capella was found to enter and discharge only
into a synergid, ‘The pollen tube was never seen
to enter the egg or grow through to the central
cell. Moreover; the discharge of the pollen tube
twas found only’ in the synergid. Thus Capsella
fits the pattem established for all the species
examined thus far with the electron microscope
Torenia fournieri (van der Pluijm, 1964), petunia
(Van Went and Linskens, 1967), and cotton
Gensen and Fisher, 1968).’‘The data: emphasize
the importance of the degenerating synergid in
the terminal growth of the pollen tube and pollen
tube discharge
Ultrastructural studies on the synergids of
cotton (Jensen, 1965), Torenia (van der Pluijm,
1964), and maize (Diboll” and Larson, 1966)
Fig. 12, 18.—Fig. 12, The disorganization of the persistent synergid nucleus. Polysomes and rough ER (arrow) appear
inside the nucleus (N). ‘Tho nuclear mombrane js difieult to distinguish from the surrounding ER and the nucleolus is
dispersed into clumps (NU). GA-sO,, X 16,250.—Fig. 18. Hnlarged view of polysomes inside the persistent synergid
nutes which have: probabl
‘come from the nucleolus (NU). GA-Os0,, X 59,000.552
show that a wall is absent at the chalazal end of
the cell. ‘The absence of a wall in this position
‘would presumably facilitate the movement of
the sperm nuclei into the egg and the central cell.
‘The synergids of Capsella, then, seem unique in
possessing’ chalazal wall. In Capsella, how do
the two sperm nuclei reach the egg and ‘the polar
nuclei once they are discharged from the pollen
tube? This is a controversial question because
these events happen so quickly that it is difficult
tovobserve the material at crtial stages. The
present data suggest that the nucleus which
fertilizes the exg moves through a rupture in the
common egg-synergid wall near the open end of
the pollen tube (Fig. 13). This rupture is similar
in size to the male gamete and has been seen in
the same relation to the pollen tube in material
prepared for light and electron microscopy.
ire untaual hehavor ofthe persistent synergid
in the method by which it becomes disorganized
had ‘heen umexpected and stands in sharp con-
trast to the way in which the degenerating syner-
id is destroyed. This is a good example of how
different, mechanisms of destruction may, be
employed by cells of the same organism. ‘The
sequence of events suggests that before the cyto-
plasm of the persistent synergid can mix with
that of the central ell the nucleus must. be
removed from the eell. The persistent synergidl
cotton forms a thiek ‘wall around itself before it
finally, undergoes disorganization (Jensen, un-
published)
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