ANATOMY OF THE UNPOLLINATED AND POLLINATED WATERMELON STIGMA

Transcription

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

2 342 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 ( 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 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 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

3 Unpollinated and pollinated watermelon stigma

4 344 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 C C 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-[6-3l H]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 S4O SS 61 us II IO 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 Fig. 5. Electron micrograph of watermelon stigma papilla cell 5 min after pollination, showing distended ER (er) surrounding clear areas of cytoplasm (c). x 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 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.

5 UnpoUinated and pouinated watermelon stigma 345 CKI. 54

6 346 M. Sedgley a

7 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.

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9 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.

10 35 M. Sedgley

11 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 Fig. 12. Electron micrograph of unpollinated watermelon stigma 24 h after anthesis, showing healthy papilla cells (p) and secretion (s) containing lipid (/). x 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.

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13 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 Exine/ Stigma Stain Specificity Intine pollenkitt secretion Ccomassie brilliant blue PAS Sudan black B Toluidine blueo Aniline blue Autofluorescence t Proteins + Vicinal glycol groups + of carbohydrates Lipids Acidic polysaccharides H Callose H Phenolic or Iigninlike compounds 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 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.

14 354 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. REFERENCES BRONNER, R. (1975). Simultaneous demonstration of lipids and starch in plant tissues. Stain Tecknol. 50, 1-4. COCHRANE, M. P. & DUFFUS, C. M. (1980). The nucellar projection and modified aleurone in the crease region of developing caryopses of barley (Hordeum vulgare L. var. distichum). Protoplasma 103, CURRIER, H. B. (1957). Callose substances in plant cells. Am. J. Bot. 44, DICKINSON, H. G. & LEWIS, D. (1973). Cytochemical and ultrastructural differences between intraspecific compatible and incompatible pollination in Raphanus. Proc. R. Soc. Lond. B, 183, ECHLIN, P. (1971). The role of the tapetum during microsporogenesis of angiosperms. In

15 Unpollinated and pollinated watermelon stigma 355 Pollen Development and Physiology (ed. J. Heslop-Harrison), pp London: Butterworths. EVANS, L. V. & CALLOW, M. E. (1978). Autoradiography. In Electron Microscopy and Cytochemistry of Plant Cells (ed. J. L. Hall), pp Elsevier: North-Holland Biomedical Press. FEDER, N. & O'BRIEN, T. P. (1968). Plant microtechnique: some principles and new methods. Am.jf. Bot. 55, FISHER, D. B. (1968). Protein staining of ribboned epon sections for light microscopy. Histochemie 16, GUNNING, B. E. S. & PATE, J. S. (1974). Transfer cells. In Dynamic Aspects of Plant Ultrastructure (ed. A. W. Robards), pp , U.K.: McGraw-Hill. HBRRBRO, M. & DICKINSON, H. G. (1979). Pollen-pistil incompatibility in Petunia hybrida: changes in the pistil following compatible and incompatible intraspecific crosses. J. Cell Sci. 36, HESLOP-HARRISON, J. (1964). Cell walls, cell membranes and protoplasmic connections during meiosis and pollen development. In Pollen Physiology and Fertilisation (ed. H. F. Linskens), pp Amsterdam: North Holland. HESLOP-HARRISON, J. (1979). Aspects of the structure, cytochemistry and germination of the pollen of rye (Secale cereale L.). Ann. Bot. 44, Suppl. 1, HESLOP-HARRISON, Y. (1977). The pollen-stigma interaction: pollen-tube penetration in Crocus. Ann. Bot. 41, HUGHES, J. E. & GUNNING, B. E. S. (1980). Glutaralderhyde-induced deposition of callose. Can. J. Bot. 58, JENSEN, W. A. & FISHER, D. B. (1969). Cotton embryogenesis: the tissues of the stigma and style and their relation to the pollen tube. Planta 84, KNOX, R. C. (1982). Intercellular Interactions. Encyclopaedia of Plant Physiology (ed. H. F. Linskens & J. Heslop-Harrison). Berlin: Springer-Verlag (In Press). KONAR, R. N. & LINSKENS, H. F. (1966). Physiology and biochemistry of the stigmatic fluid of Petunia hybrida. Planta 71, KOPRIWA, B. M. (1973). A reliable, standardized method for ultrastructural electron microscopic radioautography. Histochemie 37, MATILE, PH. (1974). Lysosomes. In Dynamic Aspects of Plant Ultrastructure (ed. A. W. Robards), pp U.K.: McGraw-Hill. MESQUITA, J. F. (1972). Ultrastructure de formations comparables aux vacuoles autophagiques dans les cellules des racines de VAllium cepa L. et du Lupinus albus L. Cytologia 37, RODKIEWICZ, B. (1973). Callose walls in megaspores in Fuchsia and Epilobium. Carylogia 25 (Suppl), SEDGLEY, M. (1977). Reduced pollen tube growth and the presence of callose in the pistil of the male floral stage of the avocado. Scient. Hort. 7, SEDGLEY, M. (1979). Structural changes in the pollinated and unpollinated avocado stigma and style. J. Cell Sci. 38, SEDGLEY, M. (1981). Ultrastructure and histochemistry of the watermelon stigma. J. Cell Sci. 48, SEDGLEY, M. & SCHOLEFIELD, P. B. (1980). Stigma secretion in the watermelon before and after pollination. Bot. Gaz. 141, SMART, M. G. & O'BRIEN, T. P. (1979). Observations on the scutellum. III. Ferulic acid as a component of the cell wall in wheat and barley. Aust. J. PI. Physiol. 6, SMITH, M. M. & MCCULLY, M. E. (1977). Mild temperature 'stress' and callose synthesis. Planta 136, SMITH, M. M. & MCCULLY, M. E. (1978). A critical evaluation of the specificity of aniline blue induced fluorescence. Protoplasma 95, TRUMP, B. R., SMUCKLER, E. A. & BENDITT, E. P. (1961). A method for staining epoxy sections for light microscopy. J. Ultrastruct. Res. 5, WATERKEYN, L. (1981). Cytochemical localization and function of the 3-linked glucan callose in the developing cotton fibre cell wall. Protoplasma 106, (Received 25 August 1981)

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