The role of the exine coating in pollen stigma interactions in Brassica oleracea L.

Transcription

1 New Phytnl. (1990), 114, The role of the exine coating in pollen stigma interactions in Brassica oleracea L. BY CAROLE J. ELLEMAN AND H. G. DICKINSON School of Plant Sciences, University of Reading, Whiteknights, Reading, RG6 2AS, UK {Received 6 September 1989; accepted 21 October 1989) SUMMARY When the dehydrated pollen grain of Brassica oleracea L. alights on a receptive stigma the pollen coat flows out from the exine to form an appresoria-like ' foot' and, within a matter of some 30 min, gross ultrastructural changes become visible both within the protoplast and in the foot itself. These changes are interpreted as reflecting the limited movement of water, and presumably other materials, from the stigma to the grain. The compatible pollen grain then continues to take up water, whilst undergoing other cytoplasmic changes and eventually producing the pollen tube. The tube grows from the colpus towards the point of contact with the stigma, beneath which the outer layer of the papillar wall has become more loosely packed. The pollen tube enters the wall at this point and, as a consequence of its rapid extension, the grain is frequently lifted away from the papilla. The tube then grows between two layers of the pectocellulosic papillar wall into the stigmatic parenchyma, where it follows an intercellular route. These events are discussed in terms of current views of the relationship between male and female cells at these early stages of the pollen stigma interaction. Key words: Brassica, papillar cell wall, pollen coating, pollen tube. INTRODUCTION The pollen o^ Brassica oleracea L. is shed in a highly dehydrated form. Its water content at dehiscence has been estimated as being c. 20% (Dumas & Gaude, 1982) and, in this condition, it alights on a mature stigma - normally transported thereto by insects. The surface of the Brassica stigma is classified as dry (Heslop-Harrison & Shivanna, 1977), being devoid of any visible stigmatic exudate, and thus hydraulic contact between the stigmatic water and the grain must first be established for water to fiow from the stigma to the grain. Once this has taken place, the water potential of the grain, being lower than that of the stigma, causes water to fiow out from the stigmatic papilla (Heslop-Harrison, 1979). The pollen coat of B. oleracea is mainly lipidic in nature and can be completely removed from the dry grains by rapid washes in organic solvents (Roberts & Dickinson, 1983). However, when the coat is removed from the grains by mechanical means (Dickinson, unpublished data) it appears as a clear, highly viscous fluid. Cytochemical studies have also demonstrated the presence of a range of en^^ymes within the pollen coat (Vithanage & Knox, 1976). Following osmic vapour fixation for electron microscopy the coat appears largely electron-lucent, containing many discrete, roughly spherical particles some 50 nm in diameter. The entire pollen grain, coating and exine is invested by the coating superficial layer (c.s.l), an apparently membranous layer some 10 nm in depth (Dickinson & Elleman, 1985; EUeman & Dickinson, 1986). The pollen coat of all Brassica spp. is applied to the surface of the developing pollen grain prior to dehiscence, and consists of the cellular remains of the degenerating tapetum (Dickinson & Lewis, 1973). In this manner the pollen must carry a range of maternal determinants, some of which may account for the sporophytic control of self-incompatibility observed in this group of plants. Since the pollen coating, and particularly the c.s.l., establish the first contact between the grain and the stigmatic surface, we report here events taking place in this region following pollination. MATERIALS AND METHODS Brassica oleracea var. gemmifera, homozygous for the S alleles S25 and S63, were raised from seed kindly supplied by Dr D. J. Ockendon (IHR, Wellesbourne, Warwick, UK). Pollinations were carried

2 512 Carole y. Elleman and H. G. Dickinson Figure 1. Pollen grain vapour-fixed immediately after dehiscence. Note the spherical particles (s) characteristic of a dry grain and the fragments of plasma membrane (pm). The unconverted pollen coat (c) appears relatively dense but unstructured, x Figure 2. Pollen coat flowing out from between the baculae (b) of a pollen grain to form a 'foot' (f). l^he coat flows before any conversion takes place retaining its dry, translucent, nonstructured form. X Figure 3. Pollen grain coat (c) of a pollen grain showing the converted form. It appears electron opaque and to be filled with membrane-like structures, b, baculae; c.s.l., coating superficial layer, x Figure 4. Partially hydrated pollen grain. The spherical particles of Figure 1 have fused to form the stratified fihrillar layer (sfi). The layer subjacent (v) to the plasma membrane (pm) has become highly

3 513 Pollen-Stigma interactions in Brassica out on freshly opened flowers, or on stigmas from buds of various lengths from which the petals and anthers had been removed. Following appropriate time intervals the pistils were fixed in osmium vapour and processed for electron microscopy as described previously (Elleman & Dickinson, 1986). Scanning microscopy of fresh frozen stigmas was carried out in a JEOL 35R electron microscope. Material was frozen in nitrogen slush and gold coated in an EMSCOPE SP2000 cryounit, before transfer to the cold stage of the microscope. The microscope was operated at 15 kv. Cyclohexane treatment of pollinated stigmas was carried out by dipping stigmas momentarily into cyclohexane and allowing the solvent to volatilise for 15 min before vapour fixation. RESULTS Changes to the coating following pollination, and other treatments As described elsewhere (Elleman & Dickinson, 1986), the dry, newly dehisced pollen grain carries an electron-lucent coat closely appressed between the baculae of the exine, and bounded by the c.s.l. Eigure 1 shows the ultrastructure of such a grain; its interior is characterised by a dominant population of spherical particles distributed throughout the protoplast. Not shown in the micrograph, but also present are large elliptical bodies bounded by rough endoplasmic reticulum. These cells also appear to carry an organised plasma membrane. Within minutes of contact between pollen and stigma, and before any internal ultrastructurai changes are apparent, the coat flows out from the baculae to form a ' foot' between the grain and the papilla (see Eig. 2). In this region the coat then assumes a more electron-opaque appearance, with many apparently membranous inclusions becoming visible (Eig. 3). The speed of the reaction is dependent on the genotype of the stigmatic papilla, but the example shown was fixed 30 min after pollination. Either as a consequence of, or an accompaniment to these changes in the coat, the protoplast also undergoes a structural change. The spherical bodies enlarge and coalesce to form a stratified fibrillar layer. The protoplast membrane becomes more distinct and the layer beneath becomes highly vesicular. Vesicles also appear between the protoplast surface and the intine, giving the appearance of a palisade (Eig. 4). These changes have been reported previously (Dickinson & Elleman, 1985; Elleman & Dickinson, 1986) but are repeated here as they provide an indication as to the state of hydration of the pollen grain. We regard the appearance of the stratified fibrillar layer as indicating that the grain has started to take up water. Useful information can also be derived from interspecific pollinations, and Eigure 5 shows a pollen grain of B. oleracea hydrating on the stigma of a species of Hebe. The stratified fibrillar layer is present but the coat has remained translucent. Interestingly, changes do occur in the coat on contact with pure water but, significantly, full conversion only occurs when the coating material is in contact with a B. oleracea stigma. To demonstrate this, pollen was hydrated by placing on filter paper and flooding briefly with water from below. As soon as the grains were hydrated, they were picked off the paper with a compatible stigma. The stigmas were then fixed after a period of 15 min. The results are shown in Eigure 6 ; coating conversion has only taken place where the coat has been in contact with the stigma surface. Significantly the coat does not convert when pollen grains are hydrated in a humid atmosphere, but the cytoplasm reorganizes to the stratified fibrillar stage (Eig. 7). The change in ultrastructure and density of staining of the coat evidently reflects an alteration in its chemical nature. This was further demonstrated by treating pollinated stigmas (30 min post-pollination) with cyclohexane. The solvent completely removed the coat from those areas of the grain that were not in contact with the stigma, while the coat in the foot remained insoluble, even in places where the solvent had direct access (Eig. 8). Emergence of the tube and entry into the stigmatic wall Eollowing a compatible pollination the coat converts and the stratified fibrillar layer is formed. The contents of the grain next become very disorganised in appearance, and the inclusions composing the stratified fibrillar layer move into the tip of the developing tube. No further changes are observed in the pollen coat, but that area of the stigmatic wall lying immediately beneath the foot begins to alter in appearance, even anticipating the production of the pollen tube (Eig. 9). The outer of the two pecto- vesicular. There are also vesicles in the space above the plasma membrane, formiiig a palisade (pi), x Figure 5. Brassica oleracea pollen grain hydrating on the stigma of Ilebe (st). The pollen coat (pc) whilst attaching to the stigma has remained translucent, showing no sign of coat conversion. The interior of the grain has reached the sfl stage but, in this instance, the layer is very dark in appearance. The fused spherical particles are apparently filled with electron opaque materials. The vesicular layer (v) is also visible, x Figure 6. The effect on the pollen coat of rapid hydration in water followed by immediate transfer to the stigma. The coat (pc) is darkening (arrows) where it is in contact with the stigma (st), whilst that area of coat adjacent to the grain remains translucent, x

4 514 Carole J. EUeman and H. G. Dickinson pm. loi Figure 7. Pollen grain hydrated for 60 min in a humid atmosphere. The interior of the grain shows the sfl, the vesicular layer (v), an intact pm and the palisade layer (pi). The coat (c) however is dry, translucent, and contains roughly spherical particles some 50 nm in diameter, x Figure 8. A pollen grain and stigma treated with cyclohexane some 15 min after pollination. Note the complete removal of the coat (arrows) from between the baculae (b), except where the pollen grain 'foot' (f) has formed in contact with the stigma (s). X Figure 9. Loosening in the outer wall of the stigmatic papilla beneath the ' foot' of a developing pollen grain, 4 h post-pollination. The inner wall layer (il) is apparently unaffected by the presence of the grain. Several dense vesicles (v) are present in the matrix of the outer layer (ol). The coating (c) in the foot has completely converted and become very electron-opaque, x Figure 10. Pollen tubes (pt) seen in section 4 h post-pollination growing in the 'foot' (0 formed by the converted pollen coat. They appear to be growing towards the area of loosening (arrows) in the outer layer (ol) of the stigmatic cell wall, x

5 Pollen-Stigma interactions in Brassica Figure 11. Another view of Figure 10. pt, pollen tubes; f, foot; ol, outer layer, x Figure 12. Scanning electron micrograph of pollen grains after 16 h following compatible pollination. Note the short tubes (pt) clearly visible between the grain and the point of penetration (arrows), x Figure 13. Compatible pollen grain (g) on a stigmatic papilla (st) at two time intervals (10 min and 2 h) post-pollination. Note the contact point between grain and papilla at time 10 min (arrow), and the fact that it is coincident with the point of entry of the pollen tribe into the stigma wall. The tube is still visible for some short distance once it has penetrated the wall. The picture is two frames from a video-recording of the pollen grain made with the recorded attached to an inverted microscope, x Figure 14. A single frame from a video recording of pollen grains on a compatible stigma showing the grains supported on short tubes (arrows) lifted up and away from the stigmatic surface, x

6 516 Carole jf. Elleman and H. G. Dickinson ol Figure 15. A pollen tuhe (t) penetrating the stigmatic outer wall layer (ol). The delineation between the stigmatic (s) and pollen tube wall (pt) is clear (see arrows), and the inner stigmatic wall layer has become distorted inwards to accommodate the tube, wo, wall opening, x Figure 16. Pollen tube (pt) encased between the two wall layers, outer (ol) and inner (il) in a mature stigma 18 h after pollination. This papilla had a total of five tubes growing down it. x Figure 17. Transverse section of a pollen tube (pt), in an immature stigma, growing hetween the inner wall (iw) and the plasma membrane (pm). x Figure 18. A fluorescence photo-micrograph of pollen tubes stained with aniline blue. The immature stigma (5 mm) was pollinated with compatible pollen. A few tubes grew down to the style but the majority (e.g. pt) make a U turn at the base of the papilla cell, indicating that they were trapped by the inner face of the immature cell wall. x300.

7 Pollen-Stigma interactions in Brassica 517 cellulosic layers, peculiar to the stigma and described in Elleman et al. (1988), is loosened. Frequently moderately electron-opaque vesicles become visible enmeshed within this fibrous matrix. These bodies can appear very dense, especially in immature stigmas. The growing pollen tube extends towards the pollen grain foot, and the area of the stigmatic surface where the changes in the wall are apparent (Figs 10, 11). With the aid of the SFM it was possible to record pollen grains at a range of times after pollination. Figure 12 shows grains, 16 h after pollination, held clear of the stigma surface by their tubes. Time lapse observations, using the video recorder in conjunction with an inverted microscope, were made over a period of 2 h following pollination. The two frames presented, superimposed one on the other (Fig. 13), show the tube entering the papilla at the initial point of contact between the grain and the stigmatic cell surface. This particular grain did not become elevated by the tube, but moved sideways as shown. However, as in Figure 14, grains are more commonly supported on short tubes. a transition from a non-polar to a polar state. It is not clear whether this change is a consequence of water flowing through the coat or if it is an outward sign of the 'water channels' that must be established before water can be pulled into the grain from the stigma. The work with Hebe and other observations on B. oleracea pollen hydrating in a humid atmosphere clearly demonstrates that the grain can hydrate in the absence of any conversion of the coat. The results obtained after transferring hydrated grains from filter paper to stigmas also suggest that the conn'ersion is the result of a specific interaction between the surface of the stigma and the coat, and not merely a consequence of water uptake. It is possible that enzymes, known to be in abundance on the stigma surface, play a role in this reaction. Washing stigmas prior to pollination certainly delays the pollination process, presumably while the surface layers are being replaced (Zuberi & Dickinson, 1985). The function of the converted coat may thus be to enable the pollen grain to draw water from the stigma, from which it is not otherwise readily available. Results obtained with the inverted microscope on individual In T E M section pollen tubes can sometimes be pollen grains have established that water only mo\'es seen entering the papilla. In Figure 1 5 the distinction between female and male surfaces, and not between between male and female tissue is clearh- delinated adjacent grains (Sarker, Flleman & Dickinson, 1988). and the wall of the papillar cell has expanded Further, coat conversion does not occur between dramatically to accommodate the entering tube. As adjacent pollen grains (unpublished observation). It Figure 16 shows, the inner wall of the papilla is never is of course c]uite possible that conversion of the coat breached, under normal circumstances, and the is not associated with hydration, and may be an tubes remain retained between the two distended outward sign of recognition between pollen and wall layers. If the mechanism channelling the tube stigma of the same species, which then results in the between the wall layers fails, as in immature stigmas, changes in the stigmatic wall reported here. the pollen tubes then penetrate the entire wall and are trapped between its inner face and the plasma membrane (Fig. 17). The escape of the tubes from the papillar cell is thus prevented and they frequently can be seen completing a turn of 180 at the base, and growing back ' u p ' the cell (see Fig. 18). Our main interest in studying pollination in B. oleracea is in the mechanism of self-incompatibility. Both self- and cross-pollinations result in the formation of a pollen grain foot, and in conversion of the pollen grain coat. Howe\-er, changes in the stigmatic wall lying beneath the grain ha\-e not been obser\'ed in self-pollinations. This suggests that the initial recognition between pollen and stigma of the lilscussion same, or closely related, species of Brassica spp. It is well established that contact between pollen results in coat conversion which, in compatible grain and stigma results in the adhesion of one to the pollinations, is followed by a loosening of the closely other (Stead, Roberts & Dickinson, 1979), and it packed fibrils of the pectocellulosic cell wall of the thus seems likely that the flowing of the dry pollen stigma. In self-pollinations this process is blocked, coat over the stigmatic surface results in the and we have some evidence which sviggests that after establishment of bonds between the two (for a review- a period of some hours either the incompatible see Gaude & Dumas, 1987). Interestingly, we have pollen grains detach from the stigma, or the 'foot' never noted differences between self- and cross- flows back into the exine. pollinations in the readiness of the pollen coat to In a previous publication we reported that pollen form a foot, using electron microscopy. Stead et al. tubes of compatible B. oleracea pollen grains grow (1979) however, reported that compatible pollen between the two wall layers of the stigma down the grains adhere more readily than incompatible, al- base of the papilla where they enter the middle though eventually both adhere to an equal extent. lamellae, and by which they are guided to the The conversion of the coat from its ' d r y ' electron transmitting tissue (Flleman et al., 1988). This was lucent form to the dense, 'membranous' stage contrary to expectation since, in other pollination associated with pollen hydration evidently reflects a systems, tubes had been reported as growing beneath profound change in its nature, presumably involving the cuticle rather than in the wall (Heslop-Harrison,

8 518 Carole jf. Elleman and H. G. Dickinson \911; Clarke et al., 1977). Some early workers had, however, reported Brassica pollen tubes apparently ACKNOWLEDGEMENTS growing in the wall, but these observations did not This work was supported by a research grant from the UK clearly indicate the subsequent route taken by these Agricultural and Food Research Council under its cell tubes (Kroh, 1964). The reaction in the stigmatic signalling initiative. The authors wish to thank Mrs H. wall beneatb the compatible pollen grain, and the Slade for her technical assistance. Figure 14 was kindly eventual location of the tube within the wall are provided by Dr R. H. Sarker. pertinent to the debate on the site of the incompatibility mechanism. The cuticle, the pecto- REFERENCES cellulosic cell wall and the availability of water have A. E., CONSIDINE, ]. A., WARD, R. & KNOX, R. B. (1977). all been proposed as barriers to the incompatible CLARKE, Mechanism of pollination in Gladiolus: roles of the stigma and tube. However, in B. oleracea, self-incompatible pollen tube guide. Annals of Botany 41, pollen grains may either show no development at all DICKINSON, H. G. (1989) Self-incompatibility in Howering plants. In Bioessays (in the press). or produce short tubes, depending upon tbe par- DICKINSON, H. G. & ELLEMAN, C. J. (1985)..Structural changes in ticular S-allele possessed by the plant, and on the the pollen grain of Brassica oleracea during dehydration in the anther and development on the stigma as revealed by anhydrous genetic background in which it is expressed. Since fixation techniques. Micron and Microscopica Acta 16 (4), incompatible tubes have been observed penetrating the cuticle it is unlikely that the cuticle specifically DICKINSON, II. G. & LEWIS, D. (1973). Cytochemical and ultrastructurai differences between intraspecific compatible and inhibits the incompatible pollen tube (Dickinson & incompatible pollinations in Raphanus. Proceedings of the Royal Lewis, 1973). Society of London B183, The changes occurring in the cell wall beneath the Dumas, D. & Gaude, T. (1982). Stigma-pollen recognition and pollen hydration. Phytomorphology 31, compatible pollen grain foot strongly suggest that ELLEMAN, C. J. & DICKINSON, H. G. (1986). Pollen-stigma the outer wall layer is a barrier to incompatible tubes interactions in Brassica. IV. Structural reorganisation in the on the occasions that they are formed; not because it pollen grains during hydration. Journal of Cell Science 80, specifically resists entry by the incompatible tube, C. J., WILLSON, C. E., SARKER, R. H. & DICKINSON, H. but ratber because the wall is not loosened by tbe ELLEMAN, G. (1988). Interaction between the pollen tube and stigmatic adjacent development of a compatible pollen grain. wall following pollination in Brassica oleracea. New Phytologist 109, In conclusion, the pollen coat appears to have GAUDE, T. & DUMAS, C. (1987). Molecular and cellular events of several functions; the first is presumably in aiding in self-incompatibility. International Review of Cytology 107, the dispersal of the grain, which has not been considered here. Secondly, it is responsible for the HESLOP-HARRISON, J. (1979). Aspects of the structure, cytochemistry and germination of rye (Secale cereale). Suppl. 1. attachment of the grain to the stigmatic surface while Annals of Botany 44, hydration and germination take place. The changes HESLOP-HARRISON, Y. (1977). The pollen stigma interaction: pollen tube penetration in Crocus. Annals of Bot any Al, occurring in the coat as the grain hydrates suggest HESLOI'-HARRISON, Y. & SHIVANNA, K. R. (1977). The receptive that the coat may play an active role in establishing surface of the angiosperm stigma. Annals of Botany 42, a flow of water from the stigma to the grain. Some of our observations lead us to propose that once the KROH, M. (1964). An electron microscopic study of the behaviour of Cruciferae pollen after pollination. In : Pollen Physiology and tube is produced, the direction in which it grows is Fertilisation (ed. by H. F. Linskens), pp , North dependent on the 'hydration gradient'. Under Holland Publishing Co. normal circumstances the most hydrated area of the ROBERTS, I. N. & DICKIN.SON, H. G. (1983). Intraspecific incompatibility on the stipma of Brassica. Phytomorphology 31, coat is the 'foot', and it is towards this and that the tube grows. The tube is thus guided toward the SARKER, R. H., ELLEMAN, C. J. & DICKINSON, H. G. (1988). Control of pollen hydration in Brassica requires continued point of contact between the grain and the papilla. protein synthesis and glycosylation is necessary for intraspecific Einally, the coat appears to be involved in the incompatibility. Proceedings of National Academy of Sciences recognition processes between the grain and the USA 85, stigma. It has long been considered that the male STEAD, A. D., ROBERTS, I. N. & DICKINSON, H. G. (1979). Pollen-pistil interaction in Brassica oleracea: events prior to self-incompatibility determinants are carried in tbe germination. Planta 146, coating, and we would now suggest that certain ViTHANAGE, H. I. M. V. & KNOX, R. B. (1976). Pollen-wall 'compatibility' factors are also contained in this proteins : quantitative cytochemistry of the origins of intine and exine enzymes in Brassica oleracea. Journal of Cell Science 21, layer, which are essential for the normal germination and penetration of a B. oleracea stigma by B. oleracea ZuBERi, M. I. & DICKINSON, H. G. (1985). Modification of the pollen. The putative dual role of the S-linked and Spollen-stigma interaction in Brassica oleracea by water. Annals of Botany 56, related glycoproteins, some of wbich are apparently held in the coating, is discussed in detail elsewhere (Dickinson, 1989).

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