J. Cell Sri. 54, 341-355 (1982) 341Printed in Great Britain © Company of Biologists Limited 1982

ANATOMY OF THE UNPOLLINATED AND

POLLINATED WATERMELON STIGMA

M. SEDGLEYCSIRO, 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 lightand electron microscopy, freeze-fracture and autoradiography.

The wall thickenings of the papilla transfer cells contained callose and their presence priorto pollination was confirmed using EM-autoradiography, freeze-fracture and fixation. Nofurther callose thickenings were produced following pollination.

Pollination resulted in a rapid increase in aqueous stigma secretion and localized disruptionof the cuticle, which appeared to remain on the surface of the secretion. Autorysis of the papillacells, which had commenced prior to pollination, was accelerated and appeared to take placevia cup-shaped vacuoles developed from distended endoplasmic reticulum. The reaction waslocalized to the papilla cells adjacent to the pollen tube only.

Both pollen-grain wall and stigma secretion contained proteins, carbohydrates, acidic poly-saccharides, lipids and phenolics.

INTRODUCTION

The pollen-stigma interaction is one of the most important processes in the life ofthe flowering plant because the production of the future generation is dependentupon its successful operation. It is not surprising, therefore, that pollen germinationand 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 theangiosperms is matched by variation in pollen and stigma structure, and in thebreeding system in the species studied to date (Knox, 1982).

In the watermelon the stigma papillae are transfer cells (Sedgley, 1981) that havethe capacity to produce large amounts of exudate in response to pollination (Sedgley& Scholefield, 1980). In this paper the anatomy of pollen-stigma interaction in thewatermelon is investigated further. Evidence is presented to show that the wallthickenings of the papilla cells are aniline-blue-positive. Such material (callose) isnot normally found in situations where free passage across the cell wall would beexpected, but its existence in the papilla cells is shown by a number of methods. Thepossible 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 weregrown in 150 mm diameter pots in a growth cabinet with a day/night temperature regime ofeither 30/25 °C or 25/20 °C, a 14 h photoperiod and a photon flux density of 640 /iEinsteinsm~' s"1 (400-700 nm). Plants were also grown outside in the ground in an area close to acommercial watermelon-producing region. The mean maximum and minimum temperaturesduring flowering Were 25-9 and 15-6 °C, respectively.

Stigma tissue was sampled unpollinated at anthesis and at 24 h following anthesis. Femaleflowers were pollinated by hand with a small paint-brush. Tissue was sampled at 1, 5, io, 15and 30 min and at 1, 2, 8 and 24 h after pollination. Anther tissue was sampled atanthesis.

Transmission electron microscopy

Tissue was fixed in 3 % glutaraldehyde in 0-025 M-phosphate buffer (pH 7) for 18 h, followedby postfixation in 1 % osmium tetroxide in the same buffer. In some cases 5 % glucose, sucroseor a combination of the two was included in the buffer when it was found that these sugarswere present in the stigma secretion (J. S. Hawker, personal communication). Tissue wasdehydrated in an ethanol series, through propylene oxide and embedded in Araldite. Sectionsmounted 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) inaqueous solution (sp. act. 22-5 Ci/mmol, batch 27, Amersham) by application of the undilutedprecursor in vivo. The precursor was held in the cup formed by the petals. After labelling for30 min the precursor was removed and the stigmas were washed thoroughly with distilledwater. After a period of 1 h, to allow metabolism of free label, the stigmas were fixed andprocessed as described above with the addition of five 30 min washes between fixation andpost-fixation to remove any remaining unmetabolized label. Autoradiography was carried outaccording to the method of Kopriwa (1973). Autoradiographs were analysed by comparingthe number of labelled components with the total number of components falling within circlesin 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 leftunstained for autofluorescence (Smart & O'Brien, 1979). Araldite-embedded tissue was sectionedat 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) atanthesis, 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), stainedwith aniline blue. Adjacent section to that in Fig. 1. x 500.Fig. 3. Electron microscopic autoradiograph of unpollinated watermelon stigmapapilla cell at anthesis, showing labelled wall thickening (tct), golgi (g) and secretoryvesicles (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 liquidnitrogen 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 withaniline blue.

Freeze-fracture

Glutaraldehyde-fixed tissue was placed in 23 % aqueous glycerol for 24 h and frozen in25 % 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 sodiumhypochlorite.

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 possi-bility that the wall thickenings are artefacts was investigated further. Callose wallthickenings were present in stigmas of plants grown under all conditions tested

Table 1. Labelling with D-[6-3lH]glucose of cellular and extracellularcomponents of watermelon stigma papilla cells at anthesis

Component

Endoplasmic reticulumCell wallWall thickeningsGolgi and secretory vesiclesSecretionCytoplasm, nucleus andmitochondria

PlastidsVacuole

Number ofcircles

332506181

3279 7 4

S4O

161

10SS

Number ofgrains

61

u s56

1769

48

I I

32

Activityrelative to vacuole

6-1

7-5IO-217-8

0 3

2 9

2-3i - o

Fig. 4. Freeze-fracture electron micrograph of unpollinated watermelon stigmapapilla cell at anthesis, showing wall thickening (tot), and secretion (s) with similarhydration 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 ofcytoplasm (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 denseareas, x 7500.

UnpoUinated and poUinated watermelon stigma 345

CKI. 54

346 M. Sedgley

a

Unpollinated and pollinated watermelon stigma 347

at a daytime temperature of 30 or 25 °C, and in pots or in the ground. Followinglabelling 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 proportionwas also present in both cell wall and wall thickenings (Fig. 3, Table 1). Wall thicken-ings were present in both fresh and fixed freeze-fractured stigma tissue (Fig. 4) andin frozen tissue that had been freeze-substituted with ethahol fixative. Fresh hand-cutsections also showed wall thickenings with Nomarski interference optics and thesefluoresced with aniline blue. The wall thickenings did not show autofluorescence.

At 1 min following pollination the cytoplasm of the papilla cell adjacent to a pollengrain 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 pro-minent. These profiles, which often appeared curved in section, were present beforepollination (Fig. 8) and appeared to form from distended smooth endoplasmic reti-culum (ER) (Figs. 5, 6). Dense areas were sometimes present in either the profile, thecytoplasm or both (Figs. 7, 8). By 15 min following pollination large clear areas ofcytoplasm were present in the papilla cell adjacent to the pollen grain and pollentube (Fig. 10). The cell was deformed by the pollen tube, and the remaining groundcytoplasm was dark and contained many vesicles and vacuoles. This effect was verylocalized, as only the cell immediately adjacent to the pollen tube degenerated; thenext cell appearing relatively unchanged (Fig. 10). The vesicles and vacuoles graduallydisappeared until by 24 h following pollination the cytoplasm of the papilla celladjacent to the pollen tube had shrunk against the cell wall (Fig. 11). The cell adjacentto the degenerated papilla still appeared healthy, as did all papillae of the unpollinatedstigma at 24 h following anthesis (Fig. 12).

Pollen tube growth did not result in the development of further wall thickeningsin 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 tissuebelow the stigma papillae by 24 b following pollination (Fig. 15), by which timecallose was absent from the stigma papilla cells.

The presence of the pollen grain on the stigma resulted in rapid disruption of thecuticle (Fig. 16), and the secretion lost its characteristic chambered appearance.However, following pollination the cuticle appeared beyond the germinated pollengrain on the surface of the secretion (Fig. 17). The disrupted cuticle appeared to becarried 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 withdense 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 ofcytoplasm (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 thevacuole of the papilla cell (Fig. 18) and greater hydration than the secretion beforepollination (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 associ-ated 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 theaperture (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 stigmasecretion are shown in Table 2. Some components of the pollen-grain wall werepositive to all stains tested, indicating that protein, carbohydrate, acidic polysac-charide, lipid, phenolic compounds and callose were present. The stigma secretionwas positive to all stains except aniline blue.

The pollen tube appeared at 10 min following pollination and the wall of thegermination aperture was left attached to the pollen grain, displaced to one side ofthe tube (Fig. 10). The pollen grain and pollen tube cytoplasm was rich in lipid andstarch (Fig. 10). The starch grains in the pollen tube appeared more dispersed thanin the pollen grain (Fig. 13) and the wall was aniline-blue-negative at 15 min followingpollination (Fig. 14). Deposition of the inner callose layer commenced between 15 and30 min following pollination, and by 24 h following pollination the callose layer of thepollen 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 thesecretion 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 adversetemperatures during growth (Smith & McCully, 1977), and may also be an artefactof glutaraldehyde fixation (Hughes & Gunning, 1980). It is generally considered toform a barrier to further cell damage in wounded tissue (Currier, 1957) and toparental molecules, which may affect the genetic autonomy of developing gameto-phytes, 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, showingdegenerating papilla cell (dp) with large clear areas of cytoplasm (c) and adjacent healthypapilla 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 cellwall (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 associatedwith the reduced fertility of the male-stage flower in the avocado (Sedgley, 1977). Forthese reasons the existence of callose in the wall thickenings of watermelon papillacells seemed unlikely, as rapid passage of molecules is expected where transfer cellsoccur (Gunning & Pate, 1974), and this has been shown to be so for the watermelonstigma (Sedgley & Scholefield, 1980). All the methods employed to investigate thisproblem indicated that the wall thickenings were present in vivo and that they con-tained callose. Electron-microscopic autoradiography indicated that the wall thicken-ings were normal components of the cell wall structure, as they contained proportionsof 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 arte-factual, but freezing in melting Freon 22 would be expected to immobilize the tissuebefore wound callose synthesis could occur, and ethanol fixation, observation of freshtissue and growing the plants under a range of conditions eliminated some of the otherpossible causes of the callose. Cochrane & Duffus (1980) have also reported callosewall thickenings in the developing caryopses of barley, where rapid passage acrossthe wall would also be expected. It has been suggested that callose areas of cell wallmay 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, andalso explains why the wall thickenings of watermelon papilla cells are no longeraniline-blue-positive by 24 h after anthesis.

Various authors have reported that the papilla cells degenerate, either before orafter pollination (Jensen & Fisher, 1969; Dickinson & Lewis, 1973; Heslop-Harrison,1977; Herrero & Dickinson, 1979; Segley, 1979), but no explanation of the mode ofdegeneration has been given. The profiles of curved and dilated ER described hereare similar to those observed in onion and lupin root cells during autophagocytosis

Fig. 11. Electron micrograph of watermelon stigma 24 h after pollination, showingshrunken cytoplasm of degenerated papilla cell (dp) and normal cytoplasm of theadjacent 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 organ-elles. Note that the secretion (s) has dried down around the pollen tube, leaving a thicklayer 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 withPAS, 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 inresponse to the presence of the pollen tube. Note heavy staining of intine (»') of pollen-grain 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 thicken-ings (tet) in the papilla cells (p) do not appear to be produced in response to thepresence of the pollen tube even in the deformed cells (d). Note the staining of both theintine (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 havecommenced autolysis. This is certainly the case in cotton, where the papillae haveautolysed completely prior to pollination (Jensen & Fisher, 1969). However, theautolysis in watermelon proceeds no further until the stigma is pollinated, as theultrastructure 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

Exine/ StigmaStain Specificity Intine pollenkitt secretion

Ccomassiebrilliant blue

PAS

Sudan black BToluidineblueO

Aniline blueAutofluorescence

— t

Proteins +

Vicinal glycol groups +of carbohydrates

LipidsAcidic polysaccharides H

Callose HPhenolic or Iignin-

like compoundsNo staining. +, Some staining. + +, Strong staining.

activity despite the commencement of autolysis. They can synthesize cell wallmaterial as shown by autoradiography, and the loss of callose staining from the wallthickenings at 24 h after anthesis also suggests further cell wall metabolism. It appearsthat the trigger to continue autolysis comes from the pollen, as degeneration is bothrapid and localized following pollination. Final loss of cell contents proceeds via aprogressive reduction of vacuoles, presumably due to rupture of the tonoplast andloss of cell compartmentation (Matile, 1974). Degeneration of the papilla cells maysupply reserves for the growing pollen tube (Herrero & Dickinson, 1979).

Fig. 15. Fluorescence micrograph of watermelon stigma 24 h after pollination stainedwith aniline blue, showing thick callose (ca) wall and callose plugs (pi) of the pollentube {pi). Note also the callose (ca) deposited in the germinated but not the ungermi-nated (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 withSudan 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, showingdisrupted cuticle (cu) and lipid droplets (/) beyond the pollen grain (pg). The pollen-grain wall consists of intine (i), z-layer (z), nexine (n), baculae (b) and tectum (t), withlipidic pollenkitt (pk). x 6000.Fig. 18. Freeze-fracture electron micrograph of watermelon stigma 15 min afterpollination, showing secretion (1) with similar hydration to the vacuole (va) of thepapilla 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 Golgiapparatus in the cytoplasm of the adjacent papilla, and in the volume of extracellularsecretion. Thus it is likely that the vesicles are contributing to the secretion, whichloses its pre-pollination fibrillar appearance. The apparent hydration of the secretionfollowing pollination is greater than that before pollination. The comparison of ice-crystal nucleation can give only an indication of hydration but suggests that thesecretion following pollination is more aqueous than that before. Thus the reactionis largely due to an outflow of water containing 5-10% sucrose (J. S. Hawker,personal communication). Localized breakdown of the cuticle is rapid followingpollination, and the lipid droplets and internal lipid lamellae become dispersed.However, lipid still appears to be present on the surface of the secretion followingpollination, as has also been described in Petunia by Konar & Linskens (1966). Thiswould be possible in the watermelon, as the lipid droplets and internal lamellaepresent before pollination would provide sufficient lipid to create an external barrierfor the increased volume of aqueous secretion by an oil-on-water effect. This expla-nation is considered particularly likely as the freshly pollinated stigma does not causeloss of vacuum in the scanning electron microscope, as occurs when an aqueoussurface is present. The appearance of the surface of the secretion is also very similarbefore and after pollination (Sedgley & Scholefield, 1980).

The features of pollen structure and pollen germination are generally similar tothose described in other species (Heslop-Harrison, 1979; Knox, 1982). Proteins,carbohydrates, acidic polysaccharides, lipids and possibly phenolics are all present inboth the pollen-grain wall and stigma secretion, and may all be involved in the earlyprocesses leading to recognition, germination and tube growth. Watermelon pollenis transferred by insects, which may explain the lipid-rich pollenkitt associated withthe pollen exine (Echlin, 1971). Proteins and carbohydrates are at present generallyconsidered 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 importantearly reactions, including the trigger for increased secretion and for papilla celldegeneration.

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 Departmentof Agricultural Science, University of Oxford, for valuable discussion.

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(Received 25 August 1981)