Sexual Plant. Reproduction 9 Springer-Verlag 1993

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1 Sex Plant Reprod (1993) 6:24%256 Sexual Plant Reproduction 9 Springer-Verlag 1993 Pollen-pistil interactions in Populus: fl-galactosidase activity associated with pollen-tube growth in the crosses P. nigra x P. nigra and P. nigra x P. alba M. Villar ~, M. Gaget 2, M. Rougier 3, and C. Dumas 3 i INRA, Station d'am~lioration des Arbres Forestires, F-45160, Ardon, France 2 INRA, Station de recherches fruiti~res m~diterran6ennes, F Montfavet, France 3 RCAP INRA 23879, Universit6 Lyon I, 43, Bd. du 11 Novembre 1918, F Villeurbanne Cedex, France Summary. In order to understand the nature of interspecific barriers in Populus, we have explored pollen/pistil interactions in intra- and interspecific crosses Populus nigra P. nigra and P. nigra x P. alba. The kinetics of pollen-tube growth demonstrated that P. nigra and P. alba pollen tubes have distinct behavioral patterns inside P. nigra pistils. P. alba pollen tubes exhibit an unique S-shaped growth curve and an arrested growth site near the sylodium. P. nigra pollen tubes exhibit two growth phases, in the stigmatic tissues and in the ovarian cavity respectively. P. nigra and P. alba curves diverge 5 h after controlled pollination and could be related to a change in the physiology of the P. nigra pollen tube, which shifts from an autotrophic to a heterotrophic type of nutrition. Protein analysis of pollinated stigmatic extracts (0,6 and 20 h after pollination) revealed qualitative and quantitative differences that are related to the presence of either P. nigra or P. alba pollen tubes inside the stigmatic tissues. Increasing numbers of protein bands were detectable from 0 to 20 h after pollination only in intraspecific cross. Glycoproteins were detected, and the differences observed were dependent of the cross, fl-galactosidase activity was found in pollinated stigmas, but an increase in its activity (one isozyme of phi 4.2) between 6 h and 20 h after pollination was detected only in the intraspecific cross. This enzyme could play a role in heterotrophic pollen-tube nitrition, and its activity could be the final result of a series of interactions started by the initial pollen-stigma dialog. Key words: Incongruity - Pollen-pistil interactions - Populus Pollen-tube growth fl-galactosidase Introduction Among the numerous species of the genus Populus, many interspecific crosses are hindered by barriers that prevent Correspondence to: M. Villar successful hybridization. In attempts to characterize pollen rejection sites, contradictory data have been reported in the literature. The cross Populus deltoides (female) x P. alba (male) is in that sense a very good example. According to Knox et al. (1972a), P. alba pollen tubes strictly fail to penetrate the stigma. While this behavior has also been recorded by Knox et al. (1972b) and Hamilton (1976), they do mention that some tubes penetrate the stigmatic surface. Guries and Stettler (1976) and Gaget et al. (1984) observed pollen-tube arrest in the style. Finally, seedlings were surprisingly obtained from that cross by Stettler et al. (1980). These striking differences that have been reported for just one cross illustrate the deep misunderstandings that exist on interspecific barrier mechanisms in Populus. We have undertaken an hybridization experiment involving Populus nigra and P. alba in an attempt to overcome interspecific barriers and to obtain an understanding of sexual reproduction in these two species. They belong to botanical sections Aigeiros and Leuce, known to be strictly incompatible. The stigma and style, which are involved in the control of prezygotic male-female interactions, have been previously described in Populus sp. by means of cytological, cytochemical and biochemical studies (Gaget et al. 1984; Villar et al. 1987a, b, 1988). The aim of this paper is the comparison of styles of P. nigra after intra- or interspecific pollination both in terms of fluorescence microscopy analysis of pollentube growth and electrophoretic analysis of protein dynamics. Materials and methods Experimental material Populus nigra branches bearing flower buds were collected from trees located in Priay and Neuville/Ain (Ain, France). Male branches of P. alba (clone 5376) were obtained from INRA, Nancy (France) and forced at room temperature. Pollen was collected, and stored in closed vials at - 18 ~ C. Populus nigra female branches

2 O v u l e 200 v 03 C O) cb loo 50 ~ { P, nigraxr alba l I I l I I I l I I I I [ I I I I l I I I I I I I I I I Time after pollination (h) Fig. 1. Kinetics of pollen-tube growth in vivo (20 ~ C). Populus alba pollen tubes exhibit a unique S-shaped growth curve and a cessation of growth near the stylodium. P. nigra pollen tubes exhibit two growth phases in the stigmatic tissues and in the ovarian cavity, respectively. * defines the homogenates realized for the protein analysis of pollinated stigmas. (see Materials and methods) were placed in pots of fresh water and forced; the flowers were matured in growth chambers (20 ~ C, 12 h light/12 h dark, 40% R.H. University Lyon I, France). Pollination and pollen-tube length Scanning electron microscopic (SEM) observations of P. nigra female flowers were realized according to Villar et al. (1987a). Controlled pollinations were carried out, with particular care taken with respect to pollen quality (FCR test, Heslop-Harrison and Heslop-Harrison 1970) and pistil receptivity. The growth kinetics of the pollen tubes was studied in growth chambers (20 ~ C, 12 h light/ 12 h dark, 40% R.H.). Pollen tubes were observed within the stylar tissues by the aniline blue fluorescence method (ABF) in cleared whole mounts of pistils (0.01% aniline blue dissolved in 0.1% K3PO4. 2H20 according to Martin 1959). Six hundred squash preparations were observed by UV fluorescence microscopy (360 nm) using a Reichert Zetopan microscope (excitation filter nr. 11 and barrier filter nr. 3) in order to measure pollen-tube length and to observe callose plug formation. Each point of the curve represents the average length of the 20 longest pollen tubes measured on an average of 6 flowers. The logistic curve was fitted to measurements of P. alba and P. nigra pollen-tube penetration within P. nigra pistil (from 0 to 8 h after pollination). This curve may be given by: a x=xl 4 1 +exp((b-t)/c) where x is pollen-tube penetration (or maximum length) at time t, and xl, a, b and c are parameters. Means, standard errors and fit to the logistic curve were computed on an ECLIPSE S-140 (Data General) computer (Biometry laboratory CNRS , Lyon I University, France). Electrophoresis For the isoelectrofocusing of proteins, homogenates of pollinated stigmas were processed according to the procedure of Villar et al. (1988). Single stigmas were collected at different times after pollination, and the homogeneous distribution of pollen grains on their surfaces was checked under a stereo-microscope. Each type of cross (intra- and interspecific) was characterized by three series of stigmatic extracts. The choice of homogenate was based on the results of the growth kinetics experiments (see * Fig. 1), i.e., homogenates of stigmas immediately after pollination (nrs. 2 and 5, also named 0 h after pollination), 6 h after pollination (nrs. 3 and 6 corresponding to similar phases of growth) and 20 h after pollination (hrs. 4 and 7, corresponding to distinct phases of growth). Unpollinated stigmas were used as a control nr. 1). Gel preparation, focusing conditions and the silver nitrate procedure were according to Villar et al. (1988). Protein isoelectric points were determined by isoelectric point markers (Pharmacia calibration kit). Glycoproteins (Concanavalin A binding proteins) were directly revealed on the gel according to the procedure of Hawkes (1982). Using 5-bromo-4-chloro-3-indoxyl fl-d-galactoside (Sigma, USA) as a substrate we were able to improve the fl-galactosidase visualization protocol of Singh and Knox (1984). Controls were obtained by omitting the substrate and following a 3-min incubation of the extract in boiling water. Results The comparison of these two distinct pollen-tube behaviors (P. nigra and P. alba) is presented on the basis of the growth kinetics data and the electrophoretic analysis of pollinated stigmas. Kinetics of growth Figure I illustrates the set of data points and the corresponding fitted curve. The different parts of the pistil of the P. nigra female flower can be observed in Fig. 2,

3 251 Fig. 2. Longitudinal section of a female flower of Populus nigra (SEM). sl Stigmatic lobes, st stylodium, ov ovule, x 50. Fig. 3. Fluorescent micrograph of stigmatic lobes of Populus nigra (ABF method). Pollen grains have germinated (top of picture), and tubes can be seen traversing the stigmatic tissues towards the stylodium (base of picture), pt Pollen tubes, st stylodium, x 80. Fig. 4. Fluorescent micrograph of stigmatic lobes of Populus nigra (ABF method). Details of some compatible pollen tubes (pt) 18 h after pollination. Note the large callose plug (cp) near the pollen exine (po). x 200 i.e., the stigmatic lobes, the sylodium, the ovarian cavity and the ovules. Populus nigra pollen tubes exhibit a biphasic type of growth (Fig. 1). These two phases correspond to pollentube growth in the style (stigmatic tissues and stylodium) and in the ovarian cavity, respectively (Fig. 2). The period between 10 h and 15 h after pollination strictly corresponds to the presence of pollen tubes in the stylodium. The overcrowding of pollen tubes that can be detected in this region (Fig. 3) is directly related to narrowness of this passage through which they grow (Fig. 2). Populus alba pollen tubes exhibit a unique S-shaped growth curve (Fig. 1). The growth of these pollen tubes ceases at 11 h after pollination under our experimental conditions. At that time, the tips of the longest pollen tubes are localized close to the stylodium. The values of the parameters for the two curves (Table 1) show the major differences in the growth kinetics of these two types of pollen tubes. From 0 to 8 h after pollination, the growth rates (parameter c) and abscissae Table 1. Values of the parameters used for fitting the logistic curve Populus nigra P. nigra Populus nigra x P. alba SD SD xl a b c a + xl = value of the lag phase; 1/c = growth rate; b = abscissae of inflexion point; SD = standard devistion

4 252 Fig. 5. Comparison of protein patterns in unpollinated and pollinated stigmas (silver nitrate staining, IEF). 1 Non-pollinated stigma, 2 compatible pollinated stigma immediately after pollination, 3 compatible pollinated stigma 6 h after pollination, 4 compatible pollinated stigma 20 h after pollination, 5 incompatible pollinated stigma immediately after pollination, 6 incompatible pollinated stigma 6 h after pollination, 7 incompatible pollinated stigma 20 h after pollination of inflexion points (parameter b) are similar, and since the standard deviations are large, there are not significant differences between estimates computed in the two situations. In other words, theoretical values of P. nigra and P. aiba pollen-tube lengths are similar until the two curves diverge, which occurs approximately 6 h after pollination. The formation of callose plugs has been observed in both situations. The first plug is formed between 4 h and 6 h after pollination in both crosses and is located just beneath the stigmatic surface (Fig. 4). At that time, both compatible and incompatible pollen tubes have already grown through half of the stigmatic lobes (Fig. 1). Electrophoretic analysis of the proteins of pollinated stigmas The choice of extracts (according to the time after pollination, see * in Fig. 1) was determined on the basis of the kinetics of P. nigra and P. atba pollen-tube growth, i.e., divergence of the two curves at 6 h after pollination. From the results of the growth kinetics experiments, we expected major differences in the protein dynamics between the intra- and interspecific cross from 6 to 20 h after pollination. Our electrophoretic technique combined with silver nitrate staining revealed many proteins bands, making the interpretation of the gel difficult. There were, however some qualitative and quantitative differences (Fig. 5). A comparison of the electrophoregrams of extract 1 (non-pollinated stigma) and extract 2 (intraspecific pollination) did not reveal any major differences. On the other hand, a comparison of extract I (non pollinated stigma) and 5 (interspecific pollination) revealed qualitative differences such as band d, which is likely to be related to P. alba pollen. Some interesting protein bands (e.g., protein b) were apparent when pollinated stigmas at 0, 6 and 20 h after intraspecific pollination (electro-

5 253 Fig. 6. Comparison of glycoprotein patterns in unpollinated and pollinated stigmas (ConA/peroxidase reaction, IEF). For explanation of lanes 1-7, see Fig. 5 Fig. 7. Comparison of/~-galactosidase activity in unpollinated and pollinated stigmas (IEF). For explanation of lanes 1 7, see Fig. 5 phoregrams 2, 3, and 4) were compared while other proteins were no more apparent 6 h after pollination (protein a). The comparison between pollinated stigmas at 0, 6 and 20 h after interspecific pollination (electrophoregrams 5, 6 and 7) revealed a general decrease in the number of protein bands in pollinated stigmas, especially between 6 h and 20 h after pollination. Some proteins were no more discernible 20 h after pollination (proteins c, e and f). The Concanavalin A/peroxidase reaction allowed the visualization of 15 stigmatic and pollinic glycoproteins (Fig. 6). According to the type of cross, differences in banding patterns were observed. Some glycoproteins were specifically linked to P. nigra or P. alba pollen (a, e in extracts 3 and 4 and b, c in extracts 6 and 7), while other glycoproteins gradually increased their activity, whatever the type of cross (e.g., glycoprorein d that presents the same increase in activity from 6 h to 20 h after pollination in both the intra- and interspecific cross). Lastly, the activity of I distinct glycoprotein (e), not detectable in interspecific pollinated stigmas gradually increased in the intraspecific cross between 6 h and 20 h after pollination. The zymogram of the gel stained for/~-galactosidase revealed one isoenzyme (a in extracts 3 and 4) of phi 4.2 detected in the intraspecific cross (Fig. 7); non-pollinated stigmas did not show any /%galactosidase activity. Moreover, this band was more intense 20 h after pollination. (On the other hand, a very feeble band,of phi 4.4 (b) was discernable on the original gel 6 h after interspecific pollination. Unfortunately, this band cannot be seen on the photograph. This isoenzyme was not detectable 20 h after pollination). Controls did not show any /3-galactosidas activity.

6 254 Discussion Growth dynamics between 0 and 6 h after pollination The values of the growth kinetics of P. nigra and P. alba pollen tubes in the stigmatic tissues between 0 and 6 h after pollination are not significantly different. Our electrophoretic study of pollinated stigmas reveals the presence of protein bands that are related to P. alba and P. nigra pollen and pollen tubes. However, these data do not reveal any major differences in protein dynamics between 0 and 6 h after pollination, suggesting that these pollen tubes exhibit similar physiological behavior. Our hypothesis is that both compatible and incompatible pollen tubes have an initial autotrophic nutrition. Moreover, P. nigra and P. alba pollen easily germinate in vitro without any carbon source, suggesting that endogenous substances are mobilized in the pollen during its germination (Villar 1987). These results correlate with the cytochemical observation of numerous amyloplasts in the cytoplasm of P. alba and P. nigra pollen (Gaget et al. 1989b). Growth divergence The lengths of P. nigra tubes diverge from those of P. alba tubes 6 h after pollination. This precise timing corresponds strictly to the deposit of the first callose plug in both pollen tubes. This plug is formed just beneath the stigmatic surface, isolating the long pollen tube from the remaining pollen grain exine on the surface. Such a deposit could be related to an important change in the physiology of the pollen tube, i.e., indicating that a new physiological phenomenon has relayed the autotrophic type of nutrition in the intraspecific situation. in Plumbago zeylanica (Russell 1986). The stylodium in P. nigra could represent the final site of intense pollenpollen competition, which is known to occur in the style for sexual selection of faster growing pollen tubes (Snow and Spira 1991). A typical growth curve of the P. nigra pollen tube is observed in the ovarian cavity. Electrophoretic data demonstrate a general increase in protein dynamics. Moreover, the increase in a particular glycoprotein is clearly observed only in the intra-specific cross. Such a phenomenon represents the first clear biochemical evidence of separate distinct behavior patterns for P. nigra and P. alba pollen tubes in vivo. Such an increase in activity has been recorded in Malus domestica, but under in vitro germination conditions (Calzoni and Speranza 1989). The enzymatic nature of these glycoproteins has been investigated by different osidases. Among them,/~-galactosidase, which is known to be involved in the regulation of pollen-tube growth (Singh and Knox 1986). The activation of the/3-galactosidase detected in our experiments could be associated with the biphasic type of growth of the compatible pollen tube. Hydrolase activities have been detected in Petunia hybrida in both pollen and style (Linskens et al. 1969). According to these authors, the increase in activity would be induced by the association of style and pollinic isozymes, which leads to the formation of hybrid heteropolymers more active than the initial isozymes. In P. nigra, our results tend to support the idea of the pollinic isozyme because no activity was detected in the unpollinated stigma and because/%galactosidase isozymes have been detected in crude extracts of pollen (Gaget 1988). Moreover, the two isozymes of phi 4.2 and 4.4 correspond closely to pollinic isozymes found in the pollen extracts of P. nigra and P. alba, respectively (Gaget 1988). Role of ~-galactosidase in pollen-tube nitrition Growth dynamics between 6 h and 20 h after pollination The P. alba pollen tube seems to be unable to effect the physiological change in growth pattern. Its monophasic S-shaped growth curve is not modified, and pollen-tube growth is arrested 11 h after pollination. This arrest of growth parallels the general decrese in protein dynamics observed in extracts of interspecific pollinated stigmas. The P. nigra pollen tubes exhibits the physiological change described above but shows a slowing of growth between 10 h and 15 h after pollination that could be explained by the presence of a large number of pollentube tips in the stylodium area. Our hypothesis is that this overcrowding of pollen tubes in the stylodium area ("bottle-neck") could be responsible for the decreased mean pollen-tube length, as has been mentioned in Nicotiana glauca (Cruzan 1986). This slowing phase is likely to be due to a structural change in the transmitting tissue. Such a phenomenon, independent from the physiology of the pollen tube, has been previously recorded /~-Galactosidase is involved in cellular growth and is especially responsible for the hydrolysis of polysaccharides that occurs during autolysis and for the loosening of the cell wall that takes place during extension growth (for review, Dopico et al. 1989). Singh and Knox (1985) found/~-galactosidase activity in pollen and postulated that it possibly has a hydrolytic action on stylar arabinogalactans, thereby providing on exogenous carbon source for pollen-tube growth. This enzyme has been located in the cytoplasm and in the walls of pollen and pollen tubes of many species (Table 2). No significant change in the activity of pollinic hydrolases has been reported during in vitro germination (Table 2). On the other hand, the in vivo activity of this hydrolase in Petunia hybrida (self-incompatible species), which is constant after self pollination, is greatly enhanced (5 times) in the compatible situation (Linskens et al. 1969), suggesting a key role for this enzyme in pollen-tube nutrition. Therefore, our present results support the notion that /%galactosidase is likely to be one of the enzymes involved in heterotrophic pollen-tube growth.

7 255 Table 2. Evolution of pollen hydrolases during the in vitro germination (% compared to initial activity) Enzyme Species Evolution during Cytological Reference in vitro germination localization N-acetyl-~-glucosaminidase Petunia + 40% Linskens et al. (1969) /~-Galactosidase hybrida + 70% c~-mannosidase + 40% ~-Galactosidase No change Phosphatase Pyrus No change in Present in the Rosenfield and e-glucosidase communis activity pollen-tube wall Matile (1979) /~-Glucosidase c~-galactosidase ]%Galactosidase c~-mannosidase Callase Pectinase Cellulase c~-glucosidase ~-Galactosidase /?-Glucosidase /~-Galactosidase Amaryllis vitata No change in activity Not present in the Sharma et pollen-tube wall. Enzyme release via al. (1981) the tube tip Conclusion Both our physiological and biochemical results argue for the succession of an autotrophic and heterotrophic type of pollen nutrition in the compatible situation in P. nigra. This phenomenon has been described in Prunus avium (Raft and Knox 1983), Petunia hybrida and Lycopersicon esculentum (Mulcahy and Mulcahy 1983) and in Nicotiana glauca (Cruzan 1986). The arrest in the growth of the P. alba pollen tube in the P. nigra pistil is located in the stylar tissue, i.e., rejection of the gametophytic phenotype. Physiological, biochemical and cytochemical data argue for the hypothesis of the passive arrest of growth of the interspecific pollen tube at the end of the autotrophic phase: 1) The growth curve of the P. alba pollen tube is typically S-shaped and is similar to the growth curve obtained in vitro without any carbon source (Villar 1987). Moreover, this classical curve does not show any modification in its curvature, contrary to the compatible growth curve. 2) A general qualitative and quantitative decrease in proteins was found in interspecific pollinated stigma, particularly between 6 h and 20 h after pollination. 3) The callose response from the stigmatic cells and at the tip of the pollen tube, which is related to the rejection phenomenon (for review, Dumas and Knox, 1983), was not detected in our interspecific cross. In that sense interspecific rejection in P. nigra would be due to the impossibility of the P. alba pollen tube to gain access to stylar nutrients. In other words, the style would leave the incompatible tube to its own fate. Such interspecific rejection after the exhaustion of pollen-tube nutrients has been suggested in LiIium sp. by Rosen (1971) and Ascher (review in 1986). In P. nigra, these interspecific barriers seems to be very different from the active model of interspecific incompatibility (for review, Fritz and Hanneman 1989). This final rejection of P. alba pollen tubes could be related to a case of incongruity as defined by Hogenboom (1984). Such reproductive barriers have been reported in interspecific crosses in Salicaceae, in the genus Populus (Stettler et al. 1980) and in Salix (Mosseler 1989). Is the shift from an autotrophic to a heterotrophic type of nutrition in the compatible pollen tube dependent on specific compounds localized in the male and female partners? In other words, is glycan hydrolases' activity a response to recognition phenomena? We believe that a male-female dialog exists in poplars. Informational molecules, in the form of proteins and glycoproteins have been shown to diffuse from pollen and the pollen tube in vitro (Knox et al. 1972a; Asford and Knox 1980; Villar 1987; Gaget 1988). Moreover, disorganization of the pellicle (stigmatic read-out system, Gaude and Dumas 1987) with a detergent prevents the normal hydration of pollen grains (Villar 1987). Rougier et al. (1988) have also mentioned recognition mechanisms on the stigmatic surface. These authors have demonstrated the implication of adenylate cyclase activity in pollen adhesion, hydration and germination on the stigmatic surface in both an intra- (P. deltoides x P. deltoides) and interspecific cross (P. deltoides x P. alba). Moreover, they have also demonstrated that such activity in stylar tissues while present in the intraspecific cross, is no longer apparent in the interspecific situation. These data argue for the absence of successful dialog between the incompatible pollen tube and the stylar tissues. This disappearance of adenylate cyclase activity could be another argument in agreement with our hypothesis of the passive arrest of growth of the P. alba pollen tube. Our results have demonstrated that compatibility in P. nigra could be characterized by the association of a biphasic growth curve and activation of a pollinic enzymatic system. The same isozyme of phi 4.2 has been detected in pollinic extracts of P. deltoides by Gaget

8 256 (1988). According to her results and our hypothesis on the relation between p-galactosidase and biphasic growth curve, P. deltoides pollen tubes should reach the embryo sac of P. nigra. This is precisely the case: fertilization does occur in such a cross (Melchior and Seitz 1968). This particular cross is also characterized by postzygotic rejection, demonstrating that other physiological and biochemical barriers exist in interspecific crosses in poplars. Acknowledgements. We are grateful to D. Debouzie (Biometry Laboratory, Lyon I University, France) for valued statistical assistance. References Ascher PD (1986) Incompatibility and incongruity: two mechanisms preventing gene transfer between taxa. In: Mulcahy DL, Mulcahy GB, Ottaviano E (eds) Biotechnology and ecology of pollen. Springer, Berlin Heidelberg New York, pp Ashford AE, Knox RB (1980) Characteristics of pollen diffusates and pollen wall cytochemistry in poplars. J Cell Sci 44:1-17 Calzoni LC, Speranza A (1989) Glycoproteins in germinating apple pollen. Plant Physiol Biochem 27: Cruzan MB (1986) Pollen tube distribution in Nicotiana glauca: evidence for density dependant growth. Am J Bot 73 : Dopico B, Nicolas G, Labrador E (1989) Partial purification of cell wall/~-galactosidase from Cicer arietinum epicotyls. Relationships with cell wall autolytic processes. Physiol Plant 75: Dumas C, Knox RB (1983) Callose and determination of pistil viability and incompatibility. Theor Appl Genet 67:1-10 Fritz NK, Hanneman RE (1989) Interspecific incompatibility due to stylar barriers in tuber-bearing and closely related non-tuberbearing Solanums. Sex Plant Reprod 2: Gaget M (1988) Incompatibilit6 intersp6cifique chez Populus: Effet mentor. PhD thesis, Universit6 Lyon I, France Gaget M, Said C, Dumas C, Knox RB (1984) Pollen-pistil interactions in interspecific crosses of Populus (sections Aigeiros and Leuce): pollen adhesion, hydration and callose response. J Cell Sci 72 : Gaget M, Villar M, Dumas C (1989b) Sexual reproduction in Populus. II. Informational molecules of the pollen grain. Ann Sci For 46 [Suppl] : Gaude T, Dumas C (1987) Molecular and cellular events of selfincompatibility. Int Rev Cytol 107: Guries RP, Stettler RF (1976) Prefertilization barriers to hybridization in poplars. Silvae Genet 25 : Hamilton D (1976) Intersectional imcompatibility in Populus. PhD thesis, Australian University, Canberra, Australia Hawkes R (1982) Identification of Concanavalin A binding proteins after sodium dodecyl sulfate gel electrophoresis and protein blotting. Anal Biochem 123 : Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence, intracellular hydrolysis of fluorescein diacetate. Stain Technol 45: Hogenboom NG (1984) Incongruity: non-functioning of intercellular and intracellular partner relationships through non-matching information. In: Linskens HF, Heslop-Harrison J (eds) Cel- lular interactions. (Encyclopedia of plant physiology, new series vol 17). Springer, Berlin Heidelberg New York, pp Knox RB, Willing RR, Ashford AE (1972a) Role of pollen wall proteins as recognition substances in interspecific incompatibility in poplars. Nature 237: Knox RB, Willing RR, Pryor LD (1972b) Interspecific hybridization in poplars using recognition pollen. Silvae Genet 21 : Linskens HF, Havez R, Lindner R, Saldem M, Randoux A, Laniez D, Coustaut D (1969) Etude des glycanne-hydrolases au cours de la croissance du pollen chez Petunia habrida auto-incompatible. CR Acad Sci Paris 269: Martin FW (1959) Staining and observing pollen tubes in the style by means of fluorescence. Stain Technol 34: Melchior GH, Seitz FW (1968) Interspezifische Kreuzungsterilit/it innerhalb der Pappelsketion Aigeiros. Silvae Genet 17:88-93 Mosseler A (1989) Interspecific pollen-pistil incongruity in Salix. Can J For Res 19: Mulcahy DL, Mulcahy GB (1983) A comparison of pollen tube growth in bi- and trinucleate pollen. In: Mulcahy DL, Ottaviano E (eds) Pollen: biology and implications for plant breeding. Elsevier Biomedical, New York, pp Raft J, Knox RB (1983) Pollen tube growth in Prunus avium. In: Williams EG, Knox RB, Gilbert P, Bernhardt P (eds) Pollination 82. School of Botany, University of Melbourne, Australia, pp Rosen WG (1971) Pistil-pollen interactions in LiBum. In: Heslop- Harrison J (ed) Pollen, development and physiology. Butterworths, London, pp Rosenfield CL, Matile PH (1979) Glycosidases in pear pollen tube development. 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Theor Appl Genet 58: Villar M (1987) Incompatibilit6 intersp6cifique chez Populus: Approches physiologique et biochimique. PhD thesis, Universit6 Lyon I, France Villar M, Gaget M, Dumas C (1987a) The route of pollen tube from stigma to ovule in Populus nigra: a new look. Ann Sci For 44: Villar M, Gaget M, Said C, Knox RB, Dumas C (1987b) Incompatibility in Populus: structural and cytochemical characteristics of the receptive stigmas of Populus alba and Populus nigra. J Cell Sci 87:483~490 Villar M, Gaget M, Dumas C (1988) Micro-isoelectrofocusing of proteins from single stigmas of Populus. Can J For Res 18: