J. Cell Sri. 54, 341-355 (1982)
Printed in Great Britain © Company of Biologists Limited 1982
341
ANATOMY OF THE UNPOLLINATED AND
POLLINATED WATERMELON STIGMA
M. SEDGLEY
CSIRO, Division of Horticultural Research, G.P.O. Box 350,
Adelaide, S.A. 5001, Australia
SUMMARY
The structure of the watermelon stigma before and after pollination was studied using light
and electron microscopy, freeze-fracture and autoradiography.
The wall thickenings of the papilla transfer cells contained callose and their presence prior
to pollination was confirmed using EM-autoradiography, freeze-fracture and fixation. No
further callose thickenings were produced following pollination.
Pollination resulted in a rapid increase in aqueous stigma secretion and localized disruption
of the cuticle, which appeared to remain on the surface of the secretion. Autorysis of the papilla
cells, which had commenced prior to pollination, was accelerated and appeared to take place
via cup-shaped vacuoles developed from distended endoplasmic reticulum. The reaction was
localized to the papilla cells adjacent to the pollen tube only.
Both pollen-grain wall and stigma secretion contained proteins, carbohydrates, acidic polysaccharides, lipids and phenolics.
INTRODUCTION
The pollen-stigma interaction is one of the most important processes in the life of
the flowering plant because the production of the future generation is dependent
upon its successful operation. It is not surprising, therefore, that pollen germination
and early tube growth involve a complex series of events (Heslop-Harrison, 1979),
many of which are poorly understood. Moreover, the diversity of flower type in the
angiosperms is matched by variation in pollen and stigma structure, and in the
breeding system in the species studied to date (Knox, 1982).
In the watermelon the stigma papillae are transfer cells (Sedgley, 1981) that have
the capacity to produce large amounts of exudate in response to pollination (Sedgley
& Scholefield, 1980). In this paper the anatomy of pollen-stigma interaction in the
watermelon is investigated further. Evidence is presented to show that the wall
thickenings of the papilla cells are aniline-blue-positive. Such material (callose) is
not normally found in situations where free passage across the cell wall would be
expected, but its existence in the papilla cells is shown by a number of methods. The
possible mode of papilla cell death in response to pollination is also described.
342
M. Sedgley
MATERIALS AND METHODS
Plant material
Watermelon (Citrullut lanatus (Thunb.) Matsum and Nakai, cv. ' Early Yates') plants were
grown in 150 mm diameter pots in a growth cabinet with a day/night temperature regime of
either 30/25 °C or 25/20 °C, a 14 h photoperiod and a photon flux density of 640 /iEinsteins
m~' s"1 (400-700 nm). Plants were also grown outside in the ground in an area close to a
commercial watermelon-producing region. The mean maximum and minimum temperatures
during flowering Were 25-9 and 15-6 °C, respectively.
Stigma tissue was sampled unpollinated at anthesis and at 24 h following anthesis. Female
flowers were pollinated by hand with a small paint-brush. Tissue was sampled at 1, 5, io, 15
and 30 min and at 1, 2, 8 and 24 h after pollination. Anther tissue was sampled at
anthesis.
Transmission electron microscopy
Tissue wasfixedin 3 % glutaraldehyde in 0-025 M-phosphate buffer (pH 7) for 18 h, followed
by postfixation in 1 % osmium tetroxide in the same buffer. In some cases 5 % glucose, sucrose
or a combination of the two was included in the buffer when it was found that these sugars
were present in the stigma secretion (J. S. Hawker, personal communication). Tissue was
dehydrated in an ethanol series, through propylene oxide and embedded in Araldite. Sections
mounted on grids were stained with uranyl acetate and lead citrate.
Electron microscopic autoradiography
Unpollinated stigmas at anthesis were submerged in o-i ml D-[6-'H]glucose (100/*Ci) in
aqueous solution (sp. act. 22-5 Ci/mmol, batch 27, Amersham) by application of the undiluted
precursor in vivo. The precursor was held in the cup formed by the petals. After labelling for
30 min the precursor was removed and the stigmas were washed thoroughly with distilled
water. After a period of 1 h, to allow metabolism of free label, the stigmas were fixed and
processed as described above with the addition of five 30 min washes between fixation and
post-fixation to remove any remaining unmetabolized label. Autoradiography was carried out
according to the method of Kopriwa (1973). Autoradiographs were analysed by comparing
the number of labelled components with the total number of components falling within circles
in a quadratic array (Evans & Callow, 1978).
Light microscopy and histochemistry
Glutaraldehyde-fixed tissue was embedded in glycol methacrylate (GMA) (Feder & O'Brien,
1968). Sections were cut at 1 /*m and stained with periodic acid-Schiff's reagent (PAS) (Feder
& O'Brien, 1968), Coomassie brilliant blue (Fisher, 1968), aniline blue (Currier, 1957) or left
unstained for autofluorescence (Smart & O'Brien, 1979). Araldite-embedded tissue was sectioned
at 1 Jim and stained with Sudan black B (Bronner, 1975) or toluidine blue O (Trump, Smuckler
& Benditt, 1961).
Stigma tissue was also frozen, while still attached to the plant, by immersing in melting
Fig. 1. Light micrograph of unpollinated watermelon stigma papilla cells (p) at
anthesis, showing wall thickenings (tot) stained with PAS. x 500.
Fig. 2. Fluorescence micrograph of unpollinated watermelon stigma papilla cells (p)
at anthesis, showing fluorescent wall thickening (tot), but not cell wall (to), stained
with aniline blue. Adjacent section to that in Fig. 1. x 500.
Fig. 3. Electron microscopic autoradiograph of unpollinated watermelon stigma
papilla cell at anthesis, showing labelled wall thickening (tct), golgi (g) and secretory
vesicles (v). x 27500.
Unpollinated and pollinated watermelon stigma
344
M. Sedgley
Freon 22 for 10 s. The stigma/style was severed from the plant and transferred to liquid
nitrogen for 5 min and then to 95 % ethanol/acetic acid (3:1) fixative at —20 CC for 24 h.
Tissue was embedded in GMA and sections stained with aniline blue.
Fresh hand-cut sections were observed with Nomarski interference optics or stained with
aniline blue.
Freeze-fracture
Glutaraldehyde-fixed tissue was placed in 23 % aqueous glycerol for 24 h and frozen in
25 % glycerol on a gold specimen disc in melting Freon 22. Fresh tissue was frozen in 100 %
glycerol. Freeze-fracture replicas were cleaned in 80% sulphuric acid followed by sodium
hypochlorite.
RESULTS
Stigma anatomy
Unpollinated stigma papilla cells have wall thickenings that stain with PAS (Fig. 1).
Serial 1 /im sections showed that these wall thickenings, but not the papilla cell wall,
are also aniline-blue-positive (Figs. 1, 2). As aniline-blue-positive material (callose)
can be induced in response to wounding (Currier, 1957), temperature stress (Smith &
McCully, 1977) and glutaraldehyde fixation (Hughes & Gunning, 1980), the possibility that the wall thickenings are artefacts was investigated further. Callose wall
thickenings were present in stigmas of plants grown under all conditions tested
Table 1. Labelling with D-[6-3lH]glucose of cellular and extracellular
components of watermelon stigma papilla cells at anthesis
Component
Endoplasmic reticulum
Cell wall
Wall thickenings
Golgi and secretory vesicles
Secretion
Cytoplasm, nucleus and
mitochondria
Plastids
Vacuole
Number of
circles
Number of
grains
Activity
relative to vacuole
332
506
61
6-1
us
7-5
181
56
IO-2
327
176
17-8
974
9
03
S4O
48
29
161
II
10SS
32
2-3
i-o
Fig. 4. Freeze-fracture electron micrograph of unpollinated watermelon stigma
papilla cell at anthesis, showing wall thickening (tot), and secretion (s) with similar
hydration to the cytoplasm (cy) but greater than the cell wall (to), x 14500.
Fig. 5. Electron micrograph of watermelon stigma papilla cell 5 min after pollination,
showing distended ER (er) surrounding clear areas of cytoplasm (c). x 21500.
Fig. 6. Electron micrograph of watermelon stigma papilla cell 5 min after pollination,
showing curved vacuolar profile (cv) and distended ER (er) adjacent to clear area of
cytoplasm (c). x 23000.
Fig. 7. Electron micrograph of watermelon stigma papilla cell 5 min after pollination,
showing curved vacuolar profile (cv) and clear area of cytoplasm (c), both with dense
areas, x 7500.
UnpoUinated and poUinated watermelon stigma
345
CKI. 54
346
a
M. Sedgley
Unpollinated and pollinated watermelon stigma
347
at a daytime temperature of 30 or 25 °C, and in pots or in the ground. Following
labelling of stigmas with tritiated glucose for 30 min, followed by a 1 h chase period,
most of the label was present in the Golgi and secretory vesicles but a large proportion
was also present in both cell wall and wall thickenings (Fig. 3, Table 1). Wall thickenings were present in both fresh and fixed freeze-fractured stigma tissue (Fig. 4) and
in frozen tissue that had been freeze-substituted with ethahol fixative. Fresh hand-cut
sections also showed wall thickenings with Nomarski interference optics and these
fluoresced with aniline blue. The wall thickenings did not show autofluorescence.
At 1 min following pollination the cytoplasm of the papilla cell adjacent to a pollen
grain contained many vesicles, apparently of Golgi origin, with a range of sizes
(Fig. 9), and the secretion had lost its pre-pollination fibrillar appearance (cf. Figs.
8, 9). Cup-shaped vacuolar profiles subtending clear areas of cytoplasm were prominent. These profiles, which often appeared curved in section, were present before
pollination (Fig. 8) and appeared to form from distended smooth endoplasmic reticulum (ER) (Figs. 5, 6). Dense areas were sometimes present in either the profile, the
cytoplasm or both (Figs. 7, 8). By 15 min following pollination large clear areas of
cytoplasm were present in the papilla cell adjacent to the pollen grain and pollen
tube (Fig. 10). The cell was deformed by the pollen tube, and the remaining ground
cytoplasm was dark and contained many vesicles and vacuoles. This effect was very
localized, as only the cell immediately adjacent to the pollen tube degenerated; the
next cell appearing relatively unchanged (Fig. 10). The vesicles and vacuoles gradually
disappeared until by 24 h following pollination the cytoplasm of the papilla cell
adjacent to the pollen tube had shrunk against the cell wall (Fig. 11). The cell adjacent
to the degenerated papilla still appeared healthy, as did all papillae of the unpollinated
stigma at 24 h following anthesis (Fig. 12).
Pollen tube growth did not result in the development of further wall thickenings
in the papilla cells (Figs. 13, 14), even when the papilla cell was deformed (Fig. 14).
However, callose was deposited on the walls of the cells of the transmitting tissue
below the stigma papillae by 24 b following pollination (Fig. 15), by which time
callose was absent from the stigma papilla cells.
The presence of the pollen grain on the stigma resulted in rapid disruption of the
cuticle (Fig. 16), and the secretion lost its characteristic chambered appearance.
However, following pollination the cuticle appeared beyond the germinated pollen
grain on the surface of the secretion (Fig. 17). The disrupted cuticle appeared to be
carried beyond the pollen grain by the increasing volume of secretion. At 15 min
Fig. 8. Electron micrograph of unpollinated watermelon stigma papilla cell at anthesis,
showing curved vacuolar profiles (cv) adjacent to clear areas of cytoplasm (c) some with
dense areas. Also note Golgi (g) and fibrillar appearance of secretion (1) with lipid
(/). X7500.
Fig. 9. Electron micrograph of watermelon stigma papilla cell 1 min after pollination,
showing vesicles (y), vacuoles (va) and curved vacuolar profiles (cv) with clear areas of
cytoplasm (c). Also note Golgi (g) and loss of fibrillar appearance and lipid in secretion
(s). The papilla cell is adjacent to a pollen grain (not shown), x 7500.
348
M. Sedgley
V.7
Unpollinated and pollinated watermelon stigma
349
following pollination the freeze-fractured secretion showed similar hydration to the
vacuole of the papilla cell (Fig. 18) and greater hydration than the secretion before
pollination (Fig. 4), as judged by the comparative extent of ice-crystal nucleation.
Pollen anatomy
The pollen-grain wall consisted of an inner intine and an outer exine closely associated with pollenkitt (Figs. 10, 17). External to the intine was a z-layer or endexine
(Fig. 17). The ektexine was composed of a nexine layer thickened adjacent to the
aperture (Fig. 10) and a sexine consisting of baculae with an incomplete tectum (Figs,
io, 17). The staining properties of the pollen-grain wall components and the stigma
secretion are shown in Table 2. Some components of the pollen-grain wall were
positive to all stains tested, indicating that protein, carbohydrate, acidic polysaccharide, lipid, phenolic compounds and callose were present. The stigma secretion
was positive to all stains except aniline blue.
The pollen tube appeared at 10 min following pollination and the wall of the
germination aperture was left attached to the pollen grain, displaced to one side of
the tube (Fig. 10). The pollen grain and pollen tube cytoplasm was rich in lipid and
starch (Fig. 10). The starch grains in the pollen tube appeared more dispersed than
in the pollen grain (Fig. 13) and the wall was aniline-blue-negative at 15 min following
pollination (Fig. 14). Deposition of the inner callose layer commenced between 15 and
30 min following pollination, and by 24 h following pollination the callose layer of the
pollen tube wall was very thick (Fig. 11), callose plugs were present in the tube
(Fig. 15) and there were few organelles (Fig. 11).
The inclusion of sugars in the fixative buffers improved the preservation of the
secretion and developing pollen tube.
DISCUSSION
Watermelon stigma papilla cells are transfer cells with callose wall thickenings.
Callose has been reported to be induced by wounding (Currier, 1957) and adverse
temperatures during growth (Smith & McCully, 1977), and may also be an artefact
of glutaraldehyde fixation (Hughes & Gunning, 1980). It is generally considered to
form a barrier to further cell damage in wounded tissue (Currier, 1957) and to
parental molecules, which may affect the genetic autonomy of developing gametophytes, both male (Heslop-Harrison, 1964) and female (Rodkiewicz, 1973). Moreover,
it is commonly deposited following incompatible pollinations, either in the stigma
Fig. 10. Electron micrograph of watermelon stigma 15 min after pollination, showing
degenerating papilla cell (dp) with large clear areas of cytoplasm (c) and adjacent healthy
papilla cell (ftp). The pollen grain (pg) wall consists of intine (1), z-layer (z), nexine (n)
and baculae (b) associated with lipidic pollenkitt (pk). The intine, z-layer and exine (e)
of the germination aperture (a) are pushed aside by the germinating pollen tube (pi).
The cytoplasm of both pollen grain and pollen tube contain lipid (/) and starch (si).
Note the presence of lipid (/) in the stigma secretion (s). The section passes through cell
wall (to) of the deformed degenerating papilla cell, x 4000.
35°
M. Sedgley
Unpollinated and pollinated watermelon stigma
351
papillae or in the pollen grain and tube (see Knox, 1982), and it has been associated
with the reduced fertility of the male-stage flower in the avocado (Sedgley, 1977). For
these reasons the existence of callose in the wall thickenings of watermelon papilla
cells seemed unlikely, as rapid passage of molecules is expected where transfer cells
occur (Gunning & Pate, 1974), and this has been shown to be so for the watermelon
stigma (Sedgley & Scholefield, 1980). All the methods employed to investigate this
problem indicated that the wall thickenings were present in vivo and that they contained callose. Electron-microscopic autoradiography indicated that the wall thickenings were normal components of the cell wall structure, as they contained proportions
of grains similar to those in the cell wall following labelling with tritiated glucose.
The experiment does not rule out the possibility that the wall thickenings are artefactual, but freezing in melting Freon 22 would be expected to immobilize the tissue
before wound callose synthesis could occur, and ethanol fixation, observation of fresh
tissue and growing the plants under a range of conditions eliminated some of the other
possible causes of the callose. Cochrane & Duffus (1980) have also reported callose
wall thickenings in the developing caryopses of barley, where rapid passage across
the wall would also be expected. It has been suggested that callose areas of cell wall
may have a more open network of wall construction than that of other wall regions,
and may merely represent recent wall deposition (Smith & McCully, 1978;
Waterkeyn, 1981). This could well explain their occurrence in transfer cells, and
also explains why the wall thickenings of watermelon papilla cells are no longer
aniline-blue-positive by 24 h after anthesis.
Various authors have reported that the papilla cells degenerate, either before or
after pollination (Jensen & Fisher, 1969; Dickinson & Lewis, 1973; Heslop-Harrison,
1977; Herrero & Dickinson, 1979; Segley, 1979), but no explanation of the mode of
degeneration has been given. The profiles of curved and dilated ER described here
are similar to those observed in onion and lupin root cells during autophagocytosis
Fig. 11. Electron micrograph of watermelon stigma 24 h after pollination, showing
shrunken cytoplasm of degenerated papilla cell (dp) and normal cytoplasm of the
adjacent healthy papilla cell (hp). The pollen tube {pi) wall has an outer fibrillar layer
(/) and a thick inner callose layer (ca). The pollen tube lumen (lit) contains few organelles. Note that the secretion (s) has dried down around the pollen tube, leaving a thick
layer of lipid (/)• x 4000.
Fig. 12. Electron micrograph of unpollinated watermelon stigma 24 h after anthesis,
showing healthy papilla cells (p) and secretion (s) containing lipid (/). x 5500.
Fig. 13. Light micrograph of watermelon stigma 30 min after pollination stained with
PAS, showing pollen tube (pt) with starch (si) more dispersed than in the pollen grain
(pg). Wall thickenings (zot) in the papilla cells (p) do not appear to be produced in
response to the presence of the pollen tube. Note heavy staining of intine (»') of pollengrain wall, x 550.
Fig. 14. Fluorescence micrograph of watermelon stigma 15 min after pollination,
stained with aniline blue, showing pollen tube (pt) with unstained wall. Wall thickenings (tet) in the papilla cells (p) do not appear to be produced in response to the
presence of the pollen tube even in the deformed cells (d). Note the staining of both the
intine (1) and exine (e) of the pollen grain (pg) wall, x 400.
352
M. Sedgley
Unpollinated and pollinated watermelon stigma
353
(Mesquita, 1972). As these profiles, which are associated with clear areas of cytoplasm,
are present before pollination, it would appear that the watermelon papilla cells have
commenced autolysis. This is certainly the case in cotton, where the papillae have
autolysed completely prior to pollination (Jensen & Fisher, 1969). However, the
autolysis in watermelon proceeds no further until the stigma is pollinated, as the
ultrastructure of the unpollinated stigma is unchanged at 24 h following anthesis,
when the petals have closed. The papilla cells appear to maintain some metabolic
Table 2. Staining properties of the pollen-grain wall and stigma secretion
Pollen wall layer
Stain
Specificity
Ccomassie
brilliant blue
PAS
Sudan black B
Toluidine
blueO
Aniline blue
Autofluorescence
Intine
Proteins
+
Vicinal glycol groups
of carbohydrates
Lipids
Acidic polysaccharides
+
Exine/
pollenkitt
Stigma
secretion
H
Callose
H
Phenolic or Iigninlike compounds
— t No staining.
+, Some staining.
+ +, Strong staining.
activity despite the commencement of autolysis. They can synthesize cell wall
material as shown by autoradiography, and the loss of callose staining from the wall
thickenings at 24 h after anthesis also suggests further cell wall metabolism. It appears
that the trigger to continue autolysis comes from the pollen, as degeneration is both
rapid and localized following pollination. Final loss of cell contents proceeds via a
progressive reduction of vacuoles, presumably due to rupture of the tonoplast and
loss of cell compartmentation (Matile, 1974). Degeneration of the papilla cells may
supply reserves for the growing pollen tube (Herrero & Dickinson, 1979).
Fig. 15. Fluorescence micrograph of watermelon stigma 24 h after pollination stained
with aniline blue, showing thick callose (ca) wall and callose plugs (pi) of the pollen
tube {pi). Note also the callose (ca) deposited in the germinated but not the ungerminated (ug) pollen grains, and the callose walls of the transmitting tissue (tt). x 150.
Fig. 16. Light micrograph of watermelon stigma 15 min after pollination stained with
Sudan black B, showing the disrupted cuticle (cu) adjacent to the pollen grains (pg)
and pollen tubes (pi). Note also the heavy staining of the pollenkitt (pk). x 400.
Fig. 17. Electron micrograph of watermelon stigma 30 min after pollination, showing
disrupted cuticle (cu) and lipid droplets (/) beyond the pollen grain (pg). The pollengrain wall consists of intine (i), z-layer (z), nexine (n), baculae (b) and tectum (t), with
lipidic pollenkitt (pk). x 6000.
Fig. 18. Freeze-fracture electron micrograph of watermelon stigma 15 min after
pollination, showing secretion (1) with similar hydration to the vacuole (va) of the
papilla cell (p) and greater hydration than the cell wall (w) and cytoplasm (cy). x IOOOO.
354
M. Sedgley
Pollination results in a rapid increase in vesicles, apparently produced by the Golgi
apparatus in the cytoplasm of the adjacent papilla, and in the volume of extracellular
secretion. Thus it is likely that the vesicles are contributing to the secretion, which
loses its pre-pollination fibrillar appearance. The apparent hydration of the secretion
following pollination is greater than that before pollination. The comparison of icecrystal nucleation can give only an indication of hydration but suggests that the
secretion following pollination is more aqueous than that before. Thus the reaction
is largely due to an outflow of water containing 5-10% sucrose (J. S. Hawker,
personal communication). Localized breakdown of the cuticle is rapid following
pollination, and the lipid droplets and internal lipid lamellae become dispersed.
However, lipid still appears to be present on the surface of the secretion following
pollination, as has also been described in Petunia by Konar & Linskens (1966). This
would be possible in the watermelon, as the lipid droplets and internal lamellae
present before pollination would provide sufficient lipid to create an external barrier
for the increased volume of aqueous secretion by an oil-on-water effect. This explanation is considered particularly likely as the freshly pollinated stigma does not cause
loss of vacuum in the scanning electron microscope, as occurs when an aqueous
surface is present. The appearance of the surface of the secretion is also very similar
before and after pollination (Sedgley & Scholefield, 1980).
The features of pollen structure and pollen germination are generally similar to
those described in other species (Heslop-Harrison, 1979; Knox, 1982). Proteins,
carbohydrates, acidic polysaccharides, lipids and possibly phenolics are all present in
both the pollen-grain wall and stigma secretion, and may all be involved in the early
processes leading to recognition, germination and tube growth. Watermelon pollen
is transferred by insects, which may explain the lipid-rich pollenkitt associated with
the pollen exine (Echlin, 1971). Proteins and carbohydrates are at present generally
considered to be the molecules responsible for initial pollen-stigma recognition (Knox,
1982), but much further work is required on this and on the numerous other important
early reactions, including the trigger for increased secretion and for papilla cell
degeneration.
Thanks to Nathalie Chaly for advice with the autoradiography, to Meredith Blesing,
Christine Annells and Cheryl Mares for assistance and to Brian Loughman of the Department
of Agricultural Science, University of Oxford, for valuable discussion.
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