Incompatibility in Populus: Structural and cytochemical characteristics of the receptive stigmas of Populus alba and P. nigra

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1 Incompatibility in Populus: Structural and cytochemical characteristics of the receptive stigmas of Populus alba and P. nigra M. VILLAR', M. GAGET 2, C. SAID 2, R. B. KNOX 3 and C. DUMAS 2 ^Station d'ainelioration des Arbres Forestiers, lnra Ardon Olivet, France 2 Recotmaissatice Cellulaire et Amelioration des Plantes, De'partement de Biologie Yegetale, Universite de Lyon I, Vdleurbanne Cedes France and UM CNRS , France 3 Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia Summary Structural and cytochemical characteristics of stigmas of Populus alba (section Leuce) and P. nigra (section Aigeiros) have been studied to provide an understanding of the cell biology of the interspecific incompatibility reactions between these two species. In addition to specific morphological characteristics, stigma surfaces differ in the presence of an exudate studied by ultrastructural and stigma print techniques. P. nigra shows features of a dry-type stigma covered by a pellicle, whereas P. alba has a copious exudate typical of a wet-type stigma that contains unsaturated lipids. Key words: Populus, stigma, exudate, interspecific incompatibility. Introduction Interspecific incompatibility in Populus has been extensively studied with the aim of obtaining hybrids for forest tree breeding programs (Knoxetal. 1972a,b; Willing & Pryor, 1976; Stettler & Guries, 1976; Stettler et al. 1980). Hybrids are readily obtained between species within a section, but not between sections, except by manipulative techniques. Pollen mixtures (Mentor pollen technique, Stettler, 1968) or solvent treatment of the stigma surface (Willing & Pryor, 1976) have been used to overcome incompatibility barriers. In attempts to understand the nature of interspecific incompatibility in Populus, we have explored the interactions between the male and female partners during incompatible crosses. Structural and biochemical characteristics of the pollen and pistil have provided the first data on the informational molecules involved (Knox et al. \911a; Ashford & Knox, 1980). A key feature of the stigma surface is the pellicle, the initial membrane-like layer that receives pollen, and may regulate hydration in Brassica (Mattsson et al. 1974). Observations of poplar pollen tube behaviour have shown that stigma organization is a major factor in the control of incompatibility (Knox et al ; Gaget et Journal of Cell Science 87, (1987) Printed in Great Britain The Company of Biologists Limited 1987 al. 1984). A comparison is made here of some cytological and cytochemical characteristics of receptive stigmas of Populus nigra (section Aigeiros) and P. alba (section Leuce). Major differences in these features are reported between these two species. Materials and methods Populus nigra and P. alba shoots from female trees were obtained from the INRA Station des Arbres Forestiers, Orleans and grown in a greenhouse at the University of Lyon, Villeurbanne. For scanning electron microscopy (SEM), stigmas were fixed in 3 % glutaraldehyde in 0-1 M-sodium cacodylate buffer (ph 7-4) and prepared by the critical-point drying method using equipment manufactured by Balzers (Liechtenstein) and observed in a JEOL CF 35 SEM at 15kV at the CMEABG, University of Lyon. Uncoated fresh pistil samples were observed directly after rapid liquid nitrogen freezing (Dumas & Lecocq, 1975), under 15 kv on a Cambridge S 600 SEM. For cytochemistry, pistil samples were fixed in 3 % glutaraldehyde in 0-1 M-sodium cacodylate buffer (ph 7-4) for 1 h, post-fixed in 1 % osmium tetroxide and embedded in Epon resin, according to the method of Dumas et al. (1978). Semithin sections of 1 ^m were obtained and stained for polysaccharides (PAS, Jensen, 1962), for lignin and phenolic components (Toluidine Blue, Trump et al. 1961), for 483

2 proteins (Coomassie Blue, Fisher, 1968) and lipids (Sudan Black B, Bronner, 1975). A method for microscopic examination of the surface components of stigmas was developed. Stigma prints were obtained by pressing fresh stigmas between two glass sides, then fixed with glutaraldehyde or osmium tetroxide vapour and stained by different cytochemical techniques (Said et al. 1985). For SEM observations, stigma prints were first fixed and gold coated before observation. For enzyme cytochemical studies with transmission electron microscopy (TEM), stigmas from receptive flowers were treated with the reaction mixture for non-specific esterase (Mattsson et al. 1974) using (r-naphthyl acetate as substrate and hexazotized pararosanilin as coupling agent. Treated stigmas were post-fixed in glutaraldehyde as described above, thin sections cut and observed at 80 kv with Hitachi HU 12A and Philips EM 300 microscopes at CMEABG, University of Lyon. Ultrathin sections were post-stained with uranyl acetate (lomin) and lead citrate (8min) following Reynolds' (1963) procedures. Additional cytochemical tests of the stigma surface included the use of lanthanum nitrate to test for surface membrane permeability, and cationized ferritin for surface polyanions (methods described by Gaude & Dumas, 1986). Results The receptive stigma surface The stigmas of P. nigra (Fig. 1) and P. alba (Fig. 2) show striking differences in morphological characteristics (Table 1). In spite of the smaller stigmatic area of P. alba, the receptive surfaces in both P. nigra and P. alba inflorescences are equivalent, because of the larger number of flowers per catkin in P. alba. On the other hand, pollination of P. alba stigmas is partly obstructed by numerous bract hairs borne near the base of the pedicel. In both species, the stigma papillae are rounded and bullate (Figs 3, 4), covered with small spherical surface wax deposits. Semithin sections of the receptive stigmas of P. nigra and P. alba showed that the receptive cells are characterized by the presence of large polyphenol vacuoles that stain green with Toluidine Blue. Stigma cells are generally much larger than their non-receptive counterparts. Comparison is rendered difficult by the thickness of the papillar cell walls of both species. On the outer surface a positively stained surface film is apparent in sections stained with Coomassie Blue for Table 1. Morphological characteristics of the female catkins of P. alba and P. nigra Characteristics P. alba P. nigra No. of flowers per catkin Size (mm) of stigmatic lobe (shape) (filiform, (triangular, erect) procumbent) No. of lobes 4 4 Table 2. Cytochemistry of fresh stigma prints of P. alba and P. nigra Stains Specificity P. nigra P. alba Coomassie Blue PAS Esterase activity Sudan Black B NT, not tested. Proteins Polysaccharides Substrate: c-naphthyl acetate Lipids proteins. This surface layer presumably represents both cuticle and pellicle, since it is positively stained with the PAS reaction for polysaccharides and Sudan Black B for lipids (including cutin). The wall of the receptive cells is multi-layered, and the major part of the wall is stained red with PAS, especially the inner layer adjacent to the cytoplasm. Cytochemistry of stigma prints P. alba stigma prints (see Table 2) are obtained from stigmatic compounds mainly localized in the clefts of the stigma surface. Each print comprises a distinct exudate when detected by SEM (Fig. 5), and shows positive staining with Coomassie Blue (Fig. 6) and esterase activity (Fig. 7). On the other hand P. nigra prints represent the wax droplets of the receptive cell surface (Fig. 8). There is no staining for polysaccharides and proteins (Table 2). Ultrastructure of the receptive stigma cells The receptive surface of P. nigra stigmas is covered by a diffuse electron-dense layer of irregular thickness (approximately 30 nm) comprising the surface pellicle (Fig. 9). Immediately beneath this layer, and largely outside the cuticle, are wax platelets. These surface layers contain cytochemically detectable reaction product for esterase (not illustrated). The underlying cuticle is a thin layer abutting directly against the cell wall surface (Fig. 9). The wall of the receptive cell appears bilayered and heterogeneous: a reticulate outer zone containing spherical electron-opaque droplets, and a fibrillar inner zone (Fig. 9). Further resolution of the stigma surface is provided by using two cytochemical probes: lanthanum nitrate treatment gives deposits in the pellicle and cell wall (Fig. 10); cationized ferritin-labelled preparations show deposits in the pellicle only (Figs 11, 12), which is clearly differentiated from the underlying cuticle. The mature stigma cell contains a large central vacuole that displaces that cytoplasm to the periphery NT 484 M. Villar et al.

3 Abbreviations used in figures: st-l, stigmatic lobe; sty, stylodium; br, bract; pa, papillae; wa, waxes; w, wall; cy, cytoplasm; la, tannins; pe, pellicle; v, vacuole; cu, cuticle; ex, exudate. Figs 1-4. Scanning electron micrographs of P. alba and P. nigra flowers. Fig. 1. P. nigra female flower, showing four stigmatic lobes hiding the stylodium. The ovary is protected by a bract. X40. Fig. 2. P. alba is also four-branched. Site of contract between stigma and ovary, the stylodium is visible. X70. Fig. 3. P. nigra stigmatic receptive cells are bullate and covered by deposits of waxes. X400. Fig. 4. P. alba stigmatic receptive cells. X800. (Fig. 9) and contains osmiophilic tannins. The cytoplasm contains numerous mitochondria, plastids and an extensive rough endoplasmic reticulum (ER). In contrast, the stigma of P. alba bears a surface exudate. This secretion is relatively free-flowing, and present in the clefts between the papillae (Fig. 13) and over their surfaces (Fig. 15). Osmiophilic droplets, resembling the exudate are observed in the cytoplasm near the plasmalemma (Fig. 15). These droplets may be secretory products. The stigma pellicle, readily detected by its esterase activity (Fig. 13), is dispersed when the massive secretion is released over the surface (Fig. 15). In the cytochemical control for esterase activity (Fig. 14), the pellicle is of low electron opacity. Discussion Stigmatic prints and ultrastructural analyses demonstrate marked differences in stigma surface properties between P. nigra and P. alba. P. nigra presents a typical dry stigma, having at maturity a hydrated proteinaceous extracuticular layer, the pellicle, first described in Raphanus by Mattson et al. (1974). The stigma surfaces of both species possess wax structures Incompatibility in Populus 485

4 Table 3. Characteristics of selected self- and interspecific incompatibility systems for comparison with data presented in this paper concerning interspecific incompatibility in Populus Species Stigma type Arrest site Pollen type Reference Gamctophytic self-incompatible systems Lycopersicon penivianum Pmnus avium Wet Wet Style Style Bicellular Bicellular Dumas el al. (1978) Rait el al. (1983) Sporophytic self-incompatible systems Brassica oleracea Raphanus sativus Dry Dry Stigma surface Stigma surface Tricellular Tricellular Knox el al. (1975); Gaude & Dumas (1986) Dickinson & Lewis (1973) Interspecific incompatible systems P. alba P. nigra RJiododendmn 3pp. Wet Dry Wet Style Style Stigma, style or ovary ±Bicellular ±Bicellular Bicellular Gaget et al. (1984); this paper Gaget el al. (1984); this paper Williams el al. (1982) s. ; *. - * ; -.;.. :. ' : *.. ;.. Figs 5 8. Cytochemistry of stigma prints. Fig. 5. SEM of P. alba stigmatic prints. X800. Fig. 6. P. alba stigmatic prints stained with Coomassie Blue. X1400. Fig. 7. P. alba stigmatic prints stained for esterase activity. X240. Fig. 8. Optical micrograph of P. nigra stigmatic pnnts stained with Sudan Black B. X M. Villar et al.

5 similar to the waxy substances observed on Brassica stigma papillae (Roggen, 1972) and detected in P. deltoides stigmas by Hamilton (1976). On the other hand, the presence of a surface exudate on P. alba receptive cells, demonstrates that this species possesses a stigma of the wet-type, as defined by Heslop- Harrison & Shivanna (1977). The transport of secretory products through cytoplasm and cell wall to the stigmatic surface, suggest the occurrence of eccrine secretion in P. alba. This secretion resembles the lipophilic stigmatic secretion of Lycopersicum peruvianum (Dumas et al. 1978). Thus Populus appears as a heterogeneous genus in terms of stigma type. Such striking differences have also been observed in the two morphs (pin and thrum) of LJnum grandiflorum (Ghosh & Shivanna, 1980) and Primula obconica (Schou, 1984). Populus has been considered to have a surface (sporophytic) type of interspecific incompatibility system (Hamilton, 1976), but recently Gagetef al. (1984) demonstrated that pollen tube arrest in P. nigra X P. alba occurs in the style, and that the stigmatic surface 12 Figs Ultrastructure of the receptive stigma surface of P. nigra. Fig. 9. Presence of bilayered cell wall, cuticle and pellicle, with mitochondria-rich peripheral cytoplasm. Uranyl acetate/lead citrate staining. X Fig. 10. Lanthanum nitrate as probe of surface, showing deposits in pellicle and cell wall. X Figs 11, 12. Cationized ferritin as probe of surface, showing deposits in the pellicle. Fig. 11, X52 500; Fig. 12, X Incompatibility in Populus 487

6 does not represent a strong sexual barrier even for intergeneric crosses (Villar et al. 1986). In addition, Populus pollen is mainly bicellular, but both species show a constant percentage of tricellular pollen at anthesis, less than 10% according to our unpublished data. Such a low percentage is in contrast with the 80-90% of tricellular grains observed by Hamilton & Langridge (1976). On the basis of these new data, an attempt to classify P. nigra and P. alba is proposed in Table 3. Both species resemble the gametophytic \ pe 15 Figs Ultrastmcture and cytochemistry of the receptive stigma surface of P. alba. Fig. 13. Cytochemical reaction for esterase showing electron-opaque deposits in the pellicle and other surface layers. X5400. Fig. 14. Control without substrate for Fig. 13, showing reduced electron opacity of pellicle. X5400. Fig. 15. As Fig. 13, but showing older stigma with heavier surface secretion and dispersed pellicle. X4200. Fig. 16. Thin section stained with uranyl acetate and lead citrate to show dispersal of pellicle, presence of surface exudate and ultrastructure of stigma cells. X M. Villar et al.

7 incompatibility system (pollen tube arrest in the style), even though P. nigra possess a stigma of the dry-type, and bicellular pollen grains. These differences in classification of Populus species in terms of the classical incompatibility systems may provide an explanation for interspecific incompatibility. Moreover, the significant differences between the stigmas of these two species could explain the unilateral hybridization phenomena reported in Populus (Ronald, 1982). In the literature, (see Willing & Pryor, 1976) only hybrids between a P. alba female partner and P. nigra have been reported. The reciprocal crosses have not succeeded. A similar situation exists for P. deltoides. Natural hybrids between P. deltoides X P. nigra produced P. euramericana but P. nigra X P. deltoides has never been obtained. Interspecific incompatibility is strongly expressed. Even breeding techniques such as the Mentor effect have failed to overcome this incompatibility in crosses between P. nigra and P. alba (M. Gaget et ai, unpublished data). In this paper, we have demonstrated significant differences in the nature of the receptive stigma surface of these two species. The read-out system for pollen tubes may differ in the two species, thus accounting for pollen tube failure in the style. Alternatively, the specific pollen tube signal may interact with stylar gene-products, that act to suppress foreign pollen tube growth in the style, but not self species tubes. This study presents the cytochemical features of P. nigra and P. alba stigmas, sites of pollen-pistil interactions. It represents a step towards the understanding of interspecific incompatibility in Populus. Biochemical investigations are in progress to complement this cytochemical study. References ASHFORD, A. E. & KNOX, R. B. (1980). 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