pollen-pistil interactions in interspecific crosses of ... - Semantic Scholar

J. Cell Sci. 72, 173-184 (1984)

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POLLEN-PISTIL INTERACTIONS IN INTERSPECIFIC CROSSES OF POPULUS (SECTIONS AIGEIROS AND LEUCE): POLLEN ADHESION, HYDRATION AND CALLOSE RESPONSES M. GAGET, C. SAID, C. DUMAS Departement de Biologie Vegetale, Universite de Lyon I, Villeurbanne 69622 Cedex, France AND R. B. K N O X Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia

SUMMARY Intersectional crosses between species of sections Aigeiros (Populus deltoides, P. x euramericana or P. nigra) and Leuce (P. alba or P. tremuloides) are known to be reciprocally incompatible. The site of pollen tube arrest is on the stigma surface in pollinations between pistils of section Leuce and pollen of section Aigeiros; tubes failed to penetrate the stigma surface. In reciprocal matings, pollen of section Leuce germinated and tubes penetrated the stigma and style, where arrest occurred. Rejection may be accompanied by swelling of the tube tips, and callose plug formation. In the cross between P. deltoides and P. alba a callose response was detected jn the cell walls of the transmitting tissue, adjacent to the rejected pollen tubes.

INTRODUCTION

Callose, a predominantly l,3-/J-glucan component of plant cell walls, plays a significant role in the reproductive biology of angiosperms. Because of its structure, it may form a physiological barrier sealing off one cell from another, providing for the maintenance of the genetic identity of the meiotic cells (see Knox & Heslop-Harrison, 1970). Callose accumulates in the walls of incompatible pollen grains and tubes and, in certain cases, in stigma cells following mutual rejection (see review by Dumas & Knox, 1983). The number of cases studied is still relatively small and most of the information available is for self-incompatibility systems. In interspecific matings, important for the transfer of desirable genes from wild species into crop plants, data are available only for several Cruciferae, including Brassica (Dickinson & Lewis, 1975; Heslop-Harrison, Knox, Heslop-Harrison & Mattsson, 1975; Kerhoas, Knox & Dumas, 1983), sunflower (Vithanage & Knox, 1977) and recently for Rhododendron (Williams, Knox & Rouse, 1982). Here, we examine interspecific pollinations of several species of Populus belonging to sections Aigeiros and Leuce, between which crosses are reciprocally incompatible (see references quoted by Knox, Willing & Ashford, 1972a; Knox, Willing &Pryor, 19726; review by Zufa, 1975). In this paper, Key words: Populus, pollen adhesion, callose responses.

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M. Gaget, C. Said, C. Dumas and R. B. Knox

the progress of the pollen tubes has been monitored by the decolorized aniline blue fluorescence (ABF) method in cleared whole mounts of pistils. This has enabled us to establish the qualitative patterns and timetable of the progamic phase of fertilization. In a further paper we will examine the cytology of the pathway of pollination, especially the ultrastructure and cytochemistry of pollen-stigma interactions.

MATERIALS

AND

METHODS

Material of P. deltoides and P. alba var. bolleana for one set of crosses was grown at the Australian National University, Canberra, and kindly provided by Professor L. D. Pryor and Mr R. R. Willing; pollen was collected and pollinations were carried out as described by Knox et al. (\972a,b). A minimum of 10 female flowers or a whole inflorescence was employed for each cross, and each experiment was carried out on two or more occasions. Material of P. nigra, P. X euramericana, P. tremidoides and P. alba was obtained from INRA Station des Arbres Forestiers, Orleans and grown at the University de Lyon 1, Villeurbanne, France. Before pollination pollen quality was checked using the FCR (Fluorochromatic Reaction) test (Heslop-Harrison & Heslop-Harrison, 1970; Heslop-Harrison, Heslop-Harrison & Shivanna, 1984); values ranged between 70 and 90% in the Canberra experiments and 30 and 60% in the Lyon experiments. The decolorized aniline blue method for tissue preparation was employed as described by Dumas & Knox (1983), using the methods of Linskens & Esser (1957) and Martin (1959). For scanning electron microscopy, pollinated stigmas were fixed in 3 % glutaraldehyde in 0-05 M-phosphate buffer (pH7-3) and prepared by the critical-point drying method, using equipment manufactured by Balzers, and observed in a Cambridge S600 scanning electron microscope at 15 kV at the C.M.E.A.B.G., University de Lyon. In the present experiments with Populus stigmas, effective pollen adhesion has been monitored qualitatively by scanning electron microscopy, and in fixed and cleared pistils by the ABF method (Dumas & Knox, 1983) using fluorescence microscopy. Stigmas were heavily pollinated, and incubated for 20min or longer before processing.

RESULTS Morphology of the pistil of Populus T h e female flowers of Populus comprise the pistil held within encircling bracts. T h e b u l b o u s ovary contains many ovules, and is s u r m o u n t e d by a short tubular lower style, and a two or three-branched stylar neck s u r m o u n t e d by m o u n d s of lobe-like stigma

Figs 1-3. Pistil of Populus X euramericana 18 h after self-pollination. Fig. 1. Scanning electron micrograph of female flower; showing one of the two stigma lobes, sg, stigma; ov, ovary; br, bract. Bar, 1 mm. Fig. 2. As Fig. 1, showing detail of stigma surface. Note strong adhesion of pollen (po) and pollen tube (pt) penetrating the stigma surface. Bar, 40jum. Fig. 3. Fluorescence micrograph of a whole cleared pistil; ABF method; pollen grains have germinated (top of picture) and tubes can be seen traversing the stigma towards the style (at base of picture). Bar, 200/lm. Figs 4—6. Scanning electron micrographs of pistils of P. tremuloides pollinated with pollen of P. X euramericana. Abbreviations as in Figs 1-3. Fig. 4. Complete female flower. Bar, 500 fun. Fig. 5. Detail of stigma surface. Note adhesion of pollen is weak. Only a few grains from the many present at pollination remain on stigma, but have produced short coiled pollen tubes (see Fig. 6). Bar, 40/zm. Fig. 6. Detail of germinating pollen grains in Fig. 5, showing tube tip appressed to surface, without apparently having penetrated the stigma surface. X900.

Pollen-pistil interactions in Populus

Figs 1-6

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cells (Fig. 1). The mature stigma cells are generally light green in colour, but are purple in P. tremuloides and have a glistening, flattened and bullate appearance. The stigma is dry and non-papillate in type, following the classification of Heslop-Harrison (1981). Compatible intraspecific pollinations; P. X euramericana (section Aigeiros) and P. tremuloides (section Leuce) After self-compatible pollination in P. X euramericana pollen grains germinated, and tubes penetrated the stigma through the radial walls (Fig. 2). The tubes are generally exposed for about twice the diameter of the grain (i.e. 50/im) before penetration, and are straight or slightly curved in appearance. After 18 h pollen tubes have penetrated through the style (Fig. 3) to the ovary. Stigmas of P. tremuloides are much smaller than those of section Aigeiros, but are basically similar in structure (Fig. 4). Self-pollinations are very similar in appearance to that described for P. X euramericana, with short tubes penetrating directly into the stigma within 18 h. Interspecific incompatibility between P. X euramericana and P. tremuloides In interspecific crosses, pollen of P. tremuloides behaved in a similar way to self (i.e. P. X euramericana) pollen on stigmas of P. X euramericana, except that the tubes were arrested in the style. In contrast, the reaction of P. X euramericana pollen on theP. tremuloides stigmas is quite different. By 18 h and despite heavy pollination, few grains remained on the stigma surface after processing for scanning electron microscopy. Some pollen grains had hydrated and germinated, producing a short, coiling tube that failed to penetrate the stigma surface (Figs 5, 6). These data indicate a general lack of adhesion. Compatible intraspecific pollinations in P. deltoides, P. nigra (section Aigeiros) and P. alba (section Leuce) In self-compatible pollination of P. deltoides and P. nigra after 2-5 h, pollen had germinated and many tubes had penetrated the stigma and grown across to the style (Fig. 7). By 26 h, pollen tubes had reached the ovary (Fig. 8). Germinated pollen of both P. deltoides and P. nigra showed callose accumulation on the side of the grain adjacent to the stigma, and to the pollen tube; it was not uniformly distributed in the Figs 7-10. Squash preparations olPopulus pistils prepared by the ABF method. Stigma surface is at top of photograph; cp, initial callose plug of pollen tube. Bars, 100f*m. Fig. 7. P. deltoides self-pollination fixed after 2-5 h (compatible). Epifluorescence illumination. Fig. 8. P. nigra self-pollination fixed after 26 h (compatible). Transmitted fluorescence illumination. Fig. 9. P. deltoides pistil pollinated with P. alba pollen, fixed after 2-5 h; showing only a few grains adhering, many with an intense ABF fluorescence. Epifluorescence illumination. Fig. 10. As 9, but fixed 20 h after pollination. Arrowheads indicate sites of callosic reaction within stigma. Epifluorescence illumination.


Figs 7-10

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pollen wall. At the site of entry into the stigma a large callose plug is also present in both species (Figs 7, 8). In P. deltoides by 2-5 h only this single plug is evident and not strongly fluorescent. After 26 h, in stigmas of both species the self pollen has differentiated callose plugs at regular intervals along the tubes (see Fig. 8). When P. alba was employed as the female parent in intra-specific pollinations, some germinating grains showed callose accumulation, while others did not. A large callose plug formed at the point of entry and it was often curved in shape. Tubes grew straight towards the style and contained only a few small plugs. By 26 h pollen tubes had reached the ovary and were observed to have penetrated the micropyles of the ovules. Interspecific incompatibility between P. deltoides, P. nigra and P. alba The responses of stigmas of P. deltoides and P. nigra differed in interspecific crosses with P. alba pollen. In P. deltoides after 2-5 h there was little sign of pollen germination, although in a high proportion of grains (about 80%, corresponding to the viability of the pollen) an intense deposition of callose within the grains was present (Fig. 9), indicating that adhesion and hydration had occurred. By 20 h some pollen had germinated. A feature of most of the grains in ABF preparations is that callose appears to occlude the entire pollen grain, not just the wall adjacent to the stigma or pollen tube. Pollen tubes had penetrated the stigma surface (Fig. 10). Most were relatively short and had penetrated only a short distance into the stigma. A few had penetrated almost to the style. In many tubes the tip was swollen, while in others the tip appeared to have burst, judging by the appearance of callose in squash preparations (Fig. 10). This conclusion was not supported when pistils were embedded in plastic resin and semithin sections were examined by the ABF method. In the transmitting tissue of the style the arrested tube walls had heavy deposits of callose. Significantly, adjacent stigma cells also had callose deposits in the walls in contact with the incompatible pollen tubes (Figs 11, 12). These appear to account for the appearance of tube tips in squash preparations. In contrast, compatible P. deltoides self-pollinations showed thin callosic tube walls and no callose reaction was evident in the transmitting tissue cells (Fig. 13). The incompatible pollen tubes were arrested at various sites within the style; most had traversed the funnel-shaped stylar neck and had entered the tubular lower style. In the cross between P. nigra and P. alba tube arrest occurred about one-third of the way to the ovary within the transmitting tissue and was accompanied by swelling of the tips or appearance of a terminal callose plug. Large callose plugs formed at the site of entry into the stigma, as in self-pollinations, and occasional smaller plugs Figs 11-13. Semithin sections of transmitting tissue of P. deltoides, fixed 20h after pollination. ABF method. Bars, 10|lm. Figs 11, 12. Pollination with P. alba. Note callose (arrows) in pollen tubes (pt) and in adjacent cells of transmitting tissue (tt). Fig. 11, epifluorescent illumination; Fig. 12, epifluorescent and transmitted phase-contrast illumination. Fig. 13. Pollination with self pollen (compatible). Note callose in pollen tubes (pt) but absence of callose in transmitting tissue (tt).

Pollen—pistil interactions in Populus

Figs 11-13

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Table 1. Selected list of intersectionalmatings in Populus, showing site of interspecific incompatibility Site of pollen tube arrest Pollen Mating (FXM) Section Leuce pistils: P. alba X P. nigra P. tremuloides X P. X euramericana P. tremuloides X P. tremuloides P. tremuloides X P. trichocarpa Section Aigeiros pistils: P. deltoides X P. alba

Adhesion

Hydration

Stigma surface

Style

+ +

NAf NA

Reference

None +•

This paper This paper This paper

NA

Stettler & Guries (1976); Stettler etal. (1980)

This paper; Knox

etal. (\972a,b) P. X euramericana X P. tremuloides P. deltoides X P. tremuloides P. deltoides X P. trichocarpa P. nigra X P. alba P. nigra X P. tremuloides P. nigra X P. trichocarpa Section Tacamahaca pistils: P. trichocarpa X P. tremuloides

This paper Stettler et al. (1980) Stettler et al. (1980) This paper Stettler et al. (1980) and this paper Stettler et al. (1980)

Stettler & Guries (1976); Stettler etal. (1980)

The extent of pollen germination and tube growth in styles was assessed by the ABF method; + indicates most grains showed response; ± indicates only a small proportion of grains showed response; — indicates no grains showed response. * Long, twisted tubes present on stigma surface. fNA, not applicable.

appeared during growth. There were no callose deposits in the transmitting tissue adjacent to the rejected tubes. In the cross between P. alba and P. nigra pollen, even after 26 h no pollen grains remained on the stigma surface after processing the pistils by the ABF method, indicating that pollen grains had not adhered to the stigma surface and had presumably not been hydrated.


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Sites of pollen and pollen tube arrest The sites where pollen grains or tubes were observed to be arrested following the interspecific matings described above are presented in Table 1. A striking finding is that in both cases where section Leuce pistils were employed the section Aigeiros pollen was arrested on the stigma surface with virtually no pollen adhesion or hydration. In contrast, section Leuce pollen is able to germinate on the stigmas of section Aigeiros, and pollen tube arrest occurs within the style. In most cases arrest is within the lower style, but in P. deltoides X P. alba the site of arrest was variable, some tubes ceasing to grow while within the funnel-shaped stylar neck. DISCUSSION

The cytological evaluation of interspecific incompatibility in Populus spp. belonging to sections Aigeiros and Leuce has revealed that the response is effectively unilateral. In crosses with Leuce pistils, i.e. P. alba or P. tremuloides, the Aigeiros pollen grains are arrested on the surface of the stigma. In reciprocal crosses with Aigeiros pistils, i.e. P. deltoides, P. X euramericana or P. nigra, the Leuce pollen grains germinated and their tubes grew at least one third of the way down the style before arrest. One explanation of these striking differences is in terms of the biocommunication between the stigma and pollen (see review by Dumas, Knox & Gaude, 1984). In the case of the Leuce stigmas most of the Aigeiros pollen grains failed to adhere and hydrate. This response is characteristic of foreign pollination. In the reciprocal crosses with Leuce pollen germination and tube growth proceeded as in compatible matings, but tubes were arrested in the style. In one case (P. alba) there was variability in the site of arrest on P. deltoides stigmas. We may conclude that the Aigeiros stigmas are able to accept a wider spectrum of pollen from Populus species. A similar unilateral response to intersectional matings has been reported by Stettler & Guries (1976) and Stettler, Koster & Steenackers (1980) for P. tremuloides X P. trichocarpa (see Table 1). Here, the P. trichocarpa pollen tubes were mainly long and twisted, unable to penetrate the stigma surface. In P. nigra X P. alba crosses three types of pollen tubes occurred: short or long surface-located tubes, or short-penetrating tubes (M. Villar, personal communication, 1984), similar to those described for other interspecific crosses by Stettler et al. (1980). Adhesion is currently considered as one of the first interactions in pollen-stigma recognition. Part of the adhesion process is non-specific (chemical adhesion or electrical contact) and part is specific, involving putative S-gene products (see review by Dumas et al. 1984). The present experiments with Populus demonstrate the importance of adhesion in pistil interactions. Unfortunately, in both assays non-adhering grains were lost during processing. Adhesion is a difficult parameter to estimate quantitatively, although an assay has been developed experimentally for Brassica pollen (Stead, Roberts & Dickinson, 1979, 1980). Hydration can presumably take place, given a high relative humidity in the atmosphere during pollination. The

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experiments to date have used greenhouse conditions in which neither temperature nor relative humidity are precisely controlled. Both these factors have profound effects on self-incompatibility reactions (see Dumas & Gaude, 1983; Knox, 1984), but their influence on interspecific incompatibility in Populus remains to be determined. An apparent unilateral effect on seed set in Leuce X Aigeiros hybridizations has been reported by Ronald (1982). He obtained significant levels of hybrid seed set using Leuce pistils rather than the Aigeiros pistils used in previous studies. While these results are apparently at variance with the responses reported here, it has to be noted that the effects obtained in Ronald's (1982) experiments depended on the use of pollen from excised branches forced precociously into dehiscence, which may have affected pollen quality. Also branches of female trees were forced into prematureflowering— conditions that may prevent the differentiation of normal pistil receptivity. The timetable of events in the progamic phase of fertilization also differed between self-compatible intraspecies crosses and the interspecific-incompatible matings. The behaviour of Leuce pollen on Aigeiros stigmas showed an initial surface retardation. At 2-5 h after pollination pollen grains of P. alba had not germinated, although callose deposition had apparently increased. In Populus pollen, callose is present in the intine of some but not all mature pollen grains (Ashford & Knox, 1980). By 18-20 h, the grains had germinated and pollen tubes penetrated the stylar neck and even into the lower style, where inhibition occurred. One explanation for this late pollen tube arrest is that the interaction occurs between pollen tube and stylar components, as in the classical gametophytic self-incompatible systems (see review by Heslop-Harrison, 1983). In section Leuce, the phenotype of the incompatibility responses is typical of a sporophytically controlled system occurring at the stigma surface. In contrast, in the other two sections surveyed in Table 1, Aigeiros and Tacamahaca, the responses are typical of a gametophytically controlled system, occurring mainly in the style. In view of the finding by Hamilton & Langridge (1975) of both bicellular and tricellular pollen types in P. deltoides it is possible that the response of the pollen may be correlated with the incompatibility reaction (see review by Heslop-Harrison, 1975). This possibility is currently being investigated. A striking finding now reported is the occurrence of callose in the cell walls of the stylar transmitting tissue of P. deltoides adjacent to incompatible/1, alba pollen tubes. Compatible self-pollinations, while also showing callose plugs in the pollen tubes, elicited no response in the stylar cells. This reaction in the transmitting tissue of P. deltoides appears to be a specific rejection response, analogous to those reported in various Cruciferae and Compositae (see review by Knox, 1984). One possible explanation is that the stylar cells in contact with the rejected pollen tube(s) seal themselves from the inhibitory factors likely to be secreted, by means of adcrustations of callose (see review by Dumas & Knox, 1983). We thank Dr Eric Teissier du Cros (INRA, Orleans) for making facilities and material available and for valued discussion; Dr Anne Ashford for providing plastic sections of stigmas; A.F.M.E. for financial support to M.G., INRA-CNRS for financial support for the research in France and the Australian Research Grants Scheme for research support in Australia; and Anne-Marie Thierry for helpful technical assistance.


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(Received 25 April 1984 -Accepted 13 June 1984)