POLLEN-STIGMA INTERACTIONS IN BRASSICA OLERACEA

Transcription

1 y. Cell Sci. 66, (1984) 255 Printed in Great Britain The Company of Biologists Limited 1984 POLLEN-STIGMA INTERACTIONS IN BRASSICA OLERACEA II. THE FATE OF STIGMA SURFACE PROTEINS FOLLOWING POLLINATION AND THEIR ROLE IN THE SELF- INCOMPATIBILITY RESPONSE IAN N. ROBERTS*, GILLIAN HARROD AND HUGH G. DICKINSONf Department of Botany, Plant Science Laboratories, The University of Reading, Whiteknights, Reading RG6 2AS, U.K. SUMMARY Mature, self-incompatible stigmas exposed to cycloheximide for 2 h prior to pollination supported identical germination and growth of both cross and self pollen. Treatment of self-pollinated pistils with cycloheximide resulted in the germination of hitherto inactive pollen after some 2 4h. Pollen germination and initial tube growth in an in vitro germination medium were not significantly affected by cycloheximide. A continuous synthesis of stigmatic proteins is therefore essential for the operation of the self-incompatibility (S.I.) system. However, light-microscope autoradiography of stigmas fed with L-[ 3 H]leucine prior to pollination revealed no movement of stigmatic proteins into the pollen, independent of the compatibility of the pollen with respect to the stigma. Further, tunicamycin, when applied in the same way as cycloheximide, had no effect on the S.I. system. These results are discussed in terms of the proposed cycling of proteins in the'papillar cell wall and the involvement of a stigmatic glycoprotein in the S.I. response. INTRODUCTION There is much evidence that proteins and particularly glycoproteins play important roles in mediating self-incompatibility (S.I.) responses in angiosperms (for reviews see de Nettacourt, 1977; Heslop-Harrison, 1978; Shivanna, 1982). Protein constituents of the pistil that specifically inhibit self pollen in vitro have been identified both in species with heterostylic S.I. (Golynskaya, Bashkinova & Tomchuk, 1976; Shivanna, Heslop-Harrison & Heslop-Harrison, 1981) and in species with gametophytic S.I. (Dickinson, Moriarty & Lawson, 1982; Williams et al. 1982). In the sporophytically controlled S.I. system of Brassica oleracea S-allele-specific glycoprotein molecules have been detected in stigma extracts by a number of different methods (reviewed by Dickinson & Roberts, 1983; Nasrallah, Doney & Nasrallah, 1983). The amino acid and carbohydrate compositions of a small number of these glycoproteins have been determined (Ferrari, Bruns & Wallace, 1981; Nishio & Hinata, 1979, 1982), it has been deduced that S-specificity resides in the protein part of the molecule (Hinata, Nishio & Himura, 1982) and one such glycoprotein has been Present address: Department of Cell Biology, John Innes Institute, Colney Lane, Norwich NR4 7UH, U.K. f Author for correspondence.

2 256 /. N. Roberts, G. Harrod and H. G. Dickinson shown to cause inhibition in vivo of otherwise compatible pollen when applied to the pollen surface (Ferrari et al. 1981). However, the manner in which these molecules of the stigma interact with their counterparts in the pollen remains unclear. Information is lacking in two major areas. Firstly, the pollen recognition molecules have not conclusively been identified; the techniques used to detect S-specific molecules in stigma extracts have largely failed when applied to similar extracts of the pollen. Apart from early reports of S-specific antigens in pollen of Oenothera (Makinen & Lewis, 1962) and Petunia (Linskens, 1960), and callose eliciting S- specific proteins in Brassica (Heslop-Harrison, Knox & Heslop-Harrison, 1974), little progress has been made in this area. Pollen must nevertheless carry determinants of S.I. or the discriminatory reactions that result in the acceptance of cross-pollen, and rejection of self-pollen could not occur. Secondly, although a r61e for stigmatic proteins in recognition events was suggested some years ago (Lewis, 1965), surprisingly little is known of their behaviour during the pollen-stigma interaction. Of crucial importance to any hypothesis to explain the molecular mechanism of S.I. is whether stigmatic proteins actually enter the pollen in order to effect self-rejection directly, or whether self-recognition by these molecules stimulates a secondary rejection response. It is also important to determine whether the proposed cycling of proteins between papillar cell wall and cytoplasm (Roberts, Harrod & Dickinson, 19836) is of any significance to the operation of the S.I. system. In this paper we present evidence that this cycling may well be essential for the expression of S.I. in Brassica and we discuss further the roles of stigmatic proteins and glycoproteins in the S.I. response. MATERIALS AND METHODS Plant material Plant material was prepared as described by Roberts et al. (19836). Studies on the effect of cycloheximide Excised pistils were maintained in small pots containing modified White's medium as described previously (Roberts et al ). At various times before and after cross and self-pollination cycloheximide (Sigma, U.K.) was added to the medium to a final concentration of 2 X 10~ 4 M. Stigmas were removed from pistils thus treated at 2-h intervals, squashed in decolourized aniline blue and observed under ultraviolet illumination in a Leitz DiaJux light microscope (Roberts et al. 1979). Control stigmas receiving identical treatments but omitting cycloheximide were also examined. The percentage of pollen grains germinating was scored and the average pollen tube length was calculated. Cycloheximide (2 X 10~ 4 M) was also added to in vitro germination medium (Roberts, Gaude, Harrod & Dickinson, 1983a) and its effect on germination and tube growth was studied. Studies on the effect of tunicamycin Excised pistils were prepared, treated and examined exactly as described above, but tunicamycin (Sigma, U.K.) was added in place of cycloheximide, to a final concentration of 1 /Jg/ml. Light microscope autoradiography Pistils were supplied with L-[ 3 H]leucine as described previously (Roberts et al ), chased in cold L-leucine for 16 h, cross and self-pollinated, and left for periods of 1, 2 and 3 h. The stigmas

3 Self-incompatibility in B. oleracea 257 were then removed, fixed, and embedded in Epon, also as described by Robertsef al. (19836). Thick sections (2 nm) were cut onto water, dried down on a slide warmer at Cfor 1-2 h, and coated with Ilford GS nuclear emulsion using the loop technique described by Caro & van Tubergen (1962). Autoradiographs were exposed for 2 weeks at 4 C, developed in Kodak D19, and observed in a Leitz Ortholux microscope under phase-contrast optics. RESULTS The effects of cycloheximide and tunicamyctn When cycloheximide was added to the medium 2h prior to pollination no differences between the behaviour of cross and self pollen were detected (Table 1). This was in striking contrast with controls, where no germination of self pollen was observed, but normal germination and tube growth of cross pollen occurred. Tunicamycin, however, had no effect on the operation of the S.I. system when added in the same way as cycloheximide. Stigmas that were pollinated and placed immediately in medium in the presence and absence of cycloheximide gave the results presented in Table 2. The germination and tube growth of self pollen was found to proceed in much the same way as that of cross pollen but the onset of germination and each subsequent stage of tube growth was delayed by some 2-4 h. Self pollen was effectively inhibited until 2h after cycloheximide treatment of the stigma, whereupon restrictions on the development of this pollen apparently ceased. Tubes greater than 150 ptvc\ were not observed in any of the stigmas treated with cycloheximide even when left for 24 h, after which time untreated cross-pollen tubes had penetrated considerable distances down the style. Similarly, pollen grown in vitro in the presence of cycloheximide achieved average tube lengths of only ^im compared to 350 jum in the control (Table 3). Use of polyethylene glycol as osmoticum Table 1. The effect on pollen germination and tube growth of treating stigmas urith cycloheximide and tunicamycin for 2 h prior to pollination Time after pollination (h) + Cycloheximide Cross Self + Tunicamycin Cross Self Key: + = 0-20% germination. + + =20-40% germination =40-60% germination = % germination = % germination. - = Av. tube length of 0-30/im. = Av. tube length of 30 60^m. = Av. tube length of \km. = Av. tube length of ^m = Av. tube length of ^m

4 oo oooooo 258 /. A r. Roberts, G. Harrod and H. C. Dickinson Table 2. The effect on pollen germination and tube growth of treating stigmas with cycloheximide immediately following pollination Time after pollination (h) + Cycloheximide A i \ Cross Self Cross Control Self 2 (a few tube initials only) Key: as for Table 1. Table 3. The effect on pollination and tube growth in vitro of adding cycloheximide to the medum (scored after 24 h) Cycloheximide added Osmoticum + Cycloheximide after 1-2 min Control Sucrose ( /Jm) PEG ( jun) Key: as for Table 1. PEG, polyethylene glucol. in place of sucrose increased the average tube length slightly but control tube lengths also increased. The effect of cycloheximide in limiting tube growth was the same whether added prior to the pollen or after 1-2 min. Cycloheximide also caused a reduction in germination of up to 20 % relative to controls. The fate of L-[ 3 H]leucine-labelled stigmatic proteins following pollination Light-microscope autoradiography of labelled stigmas 1, 2 and 3 h after cross and self-pollination revealed no movement of proteins into the pollen (Fig. 1A, B). The numbers of silver grains associated with pollen coating and pollen cytoplasm were not significantly different from background levels, independent of the time after pollination or the compatibility of pollination. DISCUSSION The sporophytically controlled S.I. system of Brassica provides an interesting

5 Self-incompatibility in B. oleracea 259 V. % 1A Fig. 1. A. Light microscope autoradiograph of stigma previously fed with L-[ 3 H]leucine and pollinated with unlabelled pollen. Considerable incorporation of label is apparent. X773. B. As A, showing no evidence of movement of labelled stigma proteins into pollen despite high levels of radioactivity in the papillae. X4261. model system not only for studies of angiosperm pollen-stigma interactions, but also for more general investigations into the role of the plant cell surface in short-range intercellular communication. It is becoming increasingly clear that sporophytic S.I. systems exhibit a number of features more closely akin to host pathogen interactions and to somatic cell surface responses than to other less-highly evolved angiosperm S.I. systems. For example, self-recognition in Brassica results in weak pollen adhesion and inadequate rehydration, in addition to inhibition of tube emergence and growth (Stead et al. 1979; Roberts, Stead, Ockenden & Dickinson, 1979). In gametophytic S.I. systems only tube growth is affected by self-recognition (Heslop-Harrison, 1983), whereas adhesion and hydration responses have also been shown to be important in the host pathogen interaction. Furthermore, a number of in vitro bioassays for S.I. have been developed for gametophytic systems but simulation of incompatibility in vitro in sporophytic systems has proved elusive (Ferrari et al. 1981) and remains so despite the advent of media supporting satisfactory pollen tube growth (Roberts et al. 1983a). An explanation of this may be provided by the observed effects of cycloheximide; possibly a continuous synthesis of stigmatic molecules is essential

6 260 /. N. Roberts, G. Harrod and H. G. Dickinson for the operation of the S.I. system, although it is also possible that the structural organization of the stigma is equally important for the expression of S.I. (Roberts et al ). In any event, the dynamic role of the cell wall in recognition and rejection responses is another feature common to sporophytic S.I. systems and the hostpathogen interaction (Albersheim et al. 1981). Complex molecules moving between wall and cytoplasm have been shown to be required for phytoalexin elicitation, and stigmatic glycoproteins may well play a similar role in S.I. in Brassica. Whether gametophytic systems are equally dynamic has yet to be determined but current evidence points to a simple inhibition of the self-pollen tube tip by pre-synthesized stylar glycoproteins (Heslop-Harrison & Heslop-Harrison, 1982), although the possibility that continued synthesis post-pollination exerts some effect cannot be ignored. It is also important to note here that the reversibility of gametophytic systems is difficult to determine. Early work onnicotiana and other genera by East (1934) and Sears (1937) showed that in an incompatible pollination tubes are inhibited in only certain parts of the style, and that those tubes that do traverse this resume the normal rate of growth. Interestingly, this type of inhibitory response can be regarded as involving a kind of specific adhesion (Heslop-Harrison, 1983), but whether the tubes that do not succeed in traversing the critical parts of the style may still be viable after, say, 24 h has not been satisfactorily determined. The frequently observed distortion and bursting of such tubes suggests that they would not. In both Brassica S.I. and host pathogen interactions removal of pollen or pathogen from the inhibitory regime results in a return to viability; that is to say the inhibition is biostatic in nature (Albersheim et al. 1981; Roberts et al. 1983a). This represents yet another shared feature. The light microscope autoradiography reveals that stigmatic proteins do not pass into the pollen and therefore one might assume that a 'secondary messenger' is responsible for the inhibition. However, we are still unable to exclude the more simplistic hypothesis that a structural barrier to water flow is produced in the cell wall by a glycoprotein gelation and forms the basis of the S.I. system (Dickinson & Roberts, 1983). There is much evidence tending to favour this latter possibility, particularly the effect of raised atmospheric humidity in overcoming the S.I. response (Carter, Williams & McNeilly, 1975, 1976), the fact that pollen incubated on a self stigma will germinate readily when added to in vitro germination medium with no apparent lagphase (Roberts et al. 1983a), and the observation that water is the sole activator of germination (Ferrari et al. 1981). On the other hand, we have been unable to find evidence of any physical barrier to water flow. No differences in rate of transpiration or rate of plasmolysis are detected after self-pollination (Roberts et al ), nor is any accumulation of labelled protein potentially capable of forming a water-trapping gel found at the site of contact of self pollen. Our more recent results therefore lead us to form a hypothesis that a continuous cycling of recognition glycoproteins produces, on self-recognition, a secondary messenger that inhibits pollen, probably by an effect on the membrane that prevents normal rehydration. The inhibition must be very mild, overcome by atmospheric humidity, and readily reversible. This suggests that the inhibitor must be rapidly metabolized by the pollen, and therefore

7 Self-incompatibility in B. oleracea 261 continuous recognition and release of this secondary messenger is essential for the expression of S.I. Little is known of the role of secondary messengers in plant cell biology; camp is not found in plant cells, and as a rule Ca 2+ acts as a mediator of cellular communication. It is not easy to see how Ca 2+ could bring about S.I., although chelation of calcium would inhibit tube growth, if not grain hydration. Polyamines may also be involved as regulators of tube growth (Roberts et al. 1983a). Perhaps the most likely candidate for a secondary messenger, should one exist, would be a phytoalexin-like molecule. Some evidence that such molecules are present in the stigma oibrassica already exists (Hodgkin & Lyon, 1983; Roberts, unpublished) but the case for their release as a consequence of self-pollination is far from proven. However, it is clear from the present study that the levels of protein in the cell wall are critical for the maintenance of the S.I. system; these levels fall by 33% after cycloheximide treatment (Roberts et al ), thus allowing the growth of self pollen. The build-up of levels of stigmatic glycoprotein during stigma maturation (Roberts et al. 1979; Nasrallah et al. 1983) and the amount of S-antigen (Sedgely, 1974) have also been shown to affect the strength of the S.I. system. The lack of movement of stigmatic protein into the pollen is therefore somewhat surprising, particularly as pollen proteins rapidly penetrate the stigma both in Raphanus (Dickinson, unpublished) and in the Gramineae (Vithanage & Heslop-Harrison, 1979). Nevertheless, stigma proteins were not found to enter the pollen of Raphanus either (Dickinson, unpublished). This suggests that under conditions of natural hydration on the stigma the grain achieves good control of movement across the plasma membrane. It is, of course, possible that small, undetectable amounts of stigma proteins enter the pollen and we have some preliminary data suggesting movement of stigma proteins into the pollen coat after 2-3 h and into the cross pollen tube after 3 h. This would be too late to affect the S.I. system and it still appears that in the first hours of pollination a contra-flow system exists, with water moving from stigma to pollen and protein moving vice versa. The effects of cycloheximide in vitro and tunicamycin in vivo require some further explanation. It seems unlikely that a pollen tube of 150 [J.m could grow in vitro without concomitant protein synthesis and the results obtained are more probably correctly interpreted as a function of the penetration kinetics of cycloheximide, the dictyosome turnover time, and the secretory vesicle residence time (Picton & Steer, 1983). A similar interpretation could be applied to the results of Ferrari & Wallace (1976), although we were not able to repeat the inhibitory effect of cycloheximide when added after 1 2min in our somewhat different medium. Further, the effect of cycloheximide in overcoming S.I. when added to the pollen (Ferrari & Wallace, 1977) could also be attributable to the effect on stigma metabolism that we have described, rather than to an effect on activatory and inhibitory regimes of pollen metabolism as discussed by these authors. Cycloheximide-coated pollen may well have been effective in transferring the drug to the stigma, whereas the control used to eliminate this possibility, involving brushing the stigma with 'dry' cycloheximide, may not have been so effective. The lack of inhibition by tunicamycin is surprising, considering the central role of stigma glycoproteins in the pollen-stigma interaction. Possibly, the

8 262 /. N. Roberts, G. Harrod and H. G. Dickinson linkage between carbohydrate and protein is of a type not affected by tunicamycin, but it is perhaps more likely that the penetration kinetics of tunicamycin into the pistil are slower than those of cycloheximide. More work on the effect of tunicamycin is needed before firm conclusions may be drawn. Ultimately, the elucidation of the mechanism of S.I. (and indeed all other plant cellular communication systems of the type involving short-range cell-surface interactions) will depend on the determination of the manner by which interaction of cognitive macromolecules can result in a secondary response effectively altering cell metabolism. In the S.I. system of Brassica, at least, it is clear that the dynamic nature of the papillar cell wall, its adaptations to regulating the movement of water and proteins, and its ability to interpret biological signals and direct cell metabolism accordingly, all result in a finely tuned outbreeding system conferring considerable advantages to the species in terms of evolutionary potential. Self pollen tubes rarely enter the stigma and self pollen is not irreversibly damaged by rejection responses, so no flower or indeed papillar cell or pollen grain is necessarily 'wasted'. Furthermore, a simple reduction of S-glycoprotein concentration in the cell wall would appear to be all that is required for a change to self-compatibility should circumstances dictate. Such changes to self-compatibility are often detected at the end of thefloweringseason or in isolated populations at the edge of the plants' range. The authors thank the Agricultural Research Council forfinancialsupport during this work. REFERENCES ALBERSHEIM, P., DARVILL, A. G., MCNEIL, M., VALENT, B. S., HAHN, M. G., LYON, G., SHARP, J. K., DESJARDINS, A. E., SPELLMAN, M. W., ROSS, L. M., ROBERTSEN, B. K., AMAN, P. & FRANZEN, L.-E. (1981). Structure and function of complex carbohydrates active in regulating plant-microbe interactions. Pure appl. Chem. S3, CARO, L. G. & VANTUBERGEN, R. P. (1962). High resolution autoradiography. I. Methods. J. Cell Biol. 15, CARTER, A. L., WILLIAMS, S. T. &MCNEILLY, T. (1975). Effects of increased humidity on pollen growth and seed set following self-pollination in Brussels sprout (Brassica oleracea var. 'gemmifera'). Euphytica 24, CARTER, A. L., WILLIAMS, S. T. & MCNEILLY, T. (1976). Increased atmospheric humidity postpollination : a possible aid to the production of inbred line seed from mature flowers in the Brussels sprout (Brassica oleracea var. 'gemmifera'). Euphytica 25, DE NETTANCOURT, D. (1977). Incompatibility in Angiosperms. Monographs on Theoretical and Applied Genetics, 3. Berlin, Heidelberg, New York: Springer-Verlag. DICKINSON, H. G., MORIARTY, J. F. & LAWSON, J. R. (1982). Pollen-pistil interaction in Lilium longiflorum: the r61e of the pistil in controlling pollen tube growth following cross- and selfpollinations. Proc. R. Soc. Land. B, 215, DICKINSON, H. G. & ROBERTS, I. N. (1983). Cell surface receptors in the pollen-stigma interaction of Brassica oleracea. In Receptors in Plants and Slime Moulds (ed. D. R. Garrod & D. M. Chadwick). New York: Marcell Dekker (in press). EAST, E. M. (1934). The reaction of the stigmatic tissue against pollen-tube growth in selfed selfsterile plants. Proc. natn. Acad. Sci. U.SA. 20, FERRARI, T. E., BRUNS, D. & WALLACE, D. H. (1981). Isolation of a plant glycoprotein involved with control of intercellular recognition. PI. Physiol. 67, FERRARI, T. E. & WALLACE, D. H. (1976). Pollen protein synthesis and control of incompatibility in Brassica. Theor. appl. Genet. 48,

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10 264 /. N. Roberts, G. Harrod and H. G. Dickinson STEAD, A. D., ROBERTS, I. N. & DICKINSON, H. G. (1979). Pollen-pistil interactions in Brassica oleracea: Events prior to pollen germination. Planta 146, VITHANAGE, H. I. M. V. & HESLOP-HARRISON, J. (1979). The pollen-stigma interaction: Fate of fluorescent-labelled pollen-wall proteins on the stigma surface in Rye {Secale cereale). Ann. Bot. 43, WILLIAMS, E. G., RAMM-ANDERSON, S., DUMAS, C, MAU, S. L. & CLARKE, A. E. (1982). The effect of isolated components of Prunus avium L. styles in in vitro growth of pollen tubes. Planta 156, (Received 4 July 1983-Accepted 12 September 1983)