the fate of intine-held antigens on the stigma in compatible and

J. Cell Sci. 9, 239-251 (1971)
Printed in Great Britain
239
POLLEN-WALL PROTEINS: THE FATE OF
INTINE-HELD ANTIGENS ON THE STIGMA
IN COMPATIBLE AND INCOMPATIBLE
POLLINATIONS OF PHALARIS TUBEROSA L.
R. B. KNOX
Department of Botany, Australian National University, Box 4, G.P.O., Canberra A.C.T.,
Australia
AND J. HESLOP-HARRISON
Royal Botanic Gardens, Kew, Richmond, Surrey, England
SUMMARY
By the use of an immunofluorescence technique, the main source of the antigens released by
the pollen grains of Phalaris tuberosa L. (Gramineae) on leaching has been shown to be the
intine. The main concentration is in the thickened zone underlying the germination pore. The
intine is also the site of various hydrolytic enzymes.
The fate of the intine-held antigens has been followed in compatible and incompatible
pollinations. They are lost on to the stigma within 5-10 min, whether or not the pollen grains
germinate. Where germination does occur after a compatible pollination, the antigens remain
spread on the surface of the stigma cells after the tubes have penetrated. There is no indication
that antigenic material of the same type is released during the further growth of the tubes.
The possible roles of the intine-held materials as recognition substances in inter- and intraspecific compatibility reactions are discussed.
INTRODUCTION
The inner stratum of the pollen-grain wall, the cellulosic intine, contains substantial
amounts of protein, including various hydrolytic enzymes (Knox & Heslop-Harrison,
1970). The protein is concentrated principally in the vicinity of the apertures, and is
held in electron-microscopically visible ribbons or leaflets, which are incorporated
during intine growth (Heslop-Harrison, 1963*2,6; Knox, 1971a). Pollen grains are
well known to release proteins, including enzymes, on moistening (Green, 1894;
Stanley & Linskens, 1965; Makinen & Brewbaker, 1967), and it now seems likely that
the bulk of the rapidly emitted material is derived from the wall sites, and not from the
vegetative cell itself. By the use of an immunofluorescence method, we have shown that
much of the antigenic material lost from the pollen of Gladiolusgandavensis and Ambrosia trifida in a soaking period of 24 h comes from the intine (Knox, Heslop-Harrison &
Reed, 1970), and in fact the intine-held antigens of Gladiolus are almost wholly lost
into the bathing medium in a much shorter period of time, of the order of a few minutes
(Knox, 1971a).
The rapidly released intine proteins have been found without exception in every
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R. B. Knox and J. Heslop-Harrison
flowering-plant species examined, so there can be little doubt that they have important
functions. The hydrolytic enzymes are probably concerned in germination, early
pollen-tube nutrition and the penetration of the stigma (Knox & Heslop-Harrison,
1970). However, the enzymes appear to represent only a small fraction of the bulk of
the protein emitted from moistened pollen grains, and they may not contribute much
to the total antigenic activity of leachates. We have suggested that much of the rapidly
released intine material is concerned rather with compatibility reactions (Knox, 1971 a;
Heslop-Harrison, 1971). We refer here to all responses where pollen-borne materials
are concerned with regulating the breeding system, and to such materials, collectively,
we have applied the term recognition substances. It is to be expected that recognition
substances will be heterogeneous, for they are likely to be involved in different kinds
of responses; in inter-specific pollinations, the requirement is to ensure fertilization
of like by like; in the operation of intra-specific incompatibility systems, on the other
hand, the need is for like to reject like to prevent selfing.
In view of what is now known of the pollen wall proteins and what may be postulated
about their functions, it is obviously important to establish their actual fate when a
pollen grain alights on a stigma. It is fortunate that the immunofluorescence method
allows this to be done, for by its use the behaviour of antigenic material from known
wall sites can be followed under various conditions (Knox, 19716). In this paper we
report observations on the early events following the arrival of pollen on the stigmas
of a grass, Phalaris tuberosa L., in compatible and incompatible matings.
P. tuberosa is a tetraploid species, and in breeding work several self-compatible and
self-incompatible genotypes have been isolated (McWilliam & Latter, 1970). The
detailed genetics of incompatibility in the species have yet to be worked out. In the
diploid species, P. coerulescens, Hayman (1956) has shown that incompatibility is
gametophytically determined, dependent, as in other grasses (Lundquist, 1963)
on 2 major loci, S and Z, with many alleles. The system in P. tuberosa is likely to be
comparable, except in so far as it is modified by tetraploidy. It should be understood
that in this initial study we have not been concerned with tracing the behaviour of
pollen-borne antigens specifically associated with particular incompatibility genotypes,
but with the broader problem of what happens to the total bulk of the wall-held antigens in different matings. The absence of full information concerning the genetics of
the incompatibility system has not been significant, for the comparison to be made
was between the behaviour on selfing in known self-compatible and self-incompatible
genotypes.
MATERIALS AND METHODS
Antigen solutions and antisera were prepared as previously described (Knox et al. 1970).
Four weekly subcutaneous injections of extracts from mixed P. tuberosa pollen were given to
2 rabbits, the first with complete Freund adjuvant, and the others with the incomplete. Pollen
samples for the location of the antigens were freeze-sectioned as described by Knox & HeslopHarrison (1970), and the immunofluorescence technique was that described by Knox eta!. (1970)
as modified by Knox (1971a).
For the study of the behaviour of the pollen-borne antigens on the stigma, 4 P. tuberosa
genotypes were used: 2 self-compatible (nos. 35 and 42) and 2 self-incompatible (nos. 27 and
Intine-held antigens on the stigma of Phalaris
241
30). Inflorescences were collected in the field when the upper florets were just reaching anthesis,
and kept in vials of water until required. Undehisced florets were removed, and the complete
pistil with stigmas carefully dissected out and placed in agar medium (2 % agar, 15-30 % sucrose
and 100 p.p.m. boric acid) to facilitate accurate control of pollination (Lundquist, 1961). Next
morning, fresh pollen from the same inflorescence could be sprinkled on to the stigmas on the
agar plates, or cross-pollinations carried out as required.
Various chemical fixation procedures were tested for stabilizing the emitted proteins, including the methanol-cyanuric chloridefixativeof Goland et al. (1969), but fully satisfactory results
were not obtained. Accordingly the precipitin reaction itself was used as a method of stabilizing
the antigens. The pistils with stigmas bearing the applied pollen were placed directly into antiP. tuberosa serum, maintained at 37 °C to ensure rapid complexing. The pistils were removed
after 1 h, and washed for 10 min in saline without agitation to remove uncomplexed surface
proteins. They were then incubated for 1 h at room temperature in fluorescein isothiocyanate
(FlTC)-labelled goat anti-rabbit IgG serum (Behringwerke, Marburg Lahn, Germany). The
stigmas were then washed for a further 10 min in saline, and mounted for fluorescence
microscopy.
Samplings were made 5-10 min after pollination and again after 40 min. As controls, stigmas
bearing pollen grains were incubated in normal rabbit serum instead of the anti-P. tuberosa
serum. No fluorescent precipitins could be detected at all in control preparations after incubation
in the standard way in the labelled anti-rabbit serum, so the specificity of the reactions observed
is in no doubt.
For fluorescence microscopy, observations were made using a mercury-arc source and an
incident-light illuminator with Zeiss Type 1 excitation filter and a barrier filter excluding
light of wavelength shorter than 500 nm. The preparations could be viewed directly by phase
contrast using transmitted tungsten light.
OBSERVATIONS
Localization of the antigens in ungerminated pollen
An earlier study of the pollen of 3 other grass species, Alopecurus pratensis, Coix
lachryma-jobi and Lolium temulentum, showed that protein was present in the intine,
where acid phosphatase and ribonuclease activity could be detected cytochemically
(Knox & Heslop-Harrison, 1970). In Phalaris tuberosa, most of the antigenic material
is undoubtedly held in the same site. The loss from fully mature pollen prepared for
freeze-sectioning in gelatine slurries is very rapid, so that observations on sections of
such material did not reveal the full content of the intine, much appearing in the
surrounding medium. However, it was found that the antigens leaked much less
readily from late vacuolate grains still held within the anther loculi (compare
Knox, 1971a, b). In preparations made of pollen at this stage, the localization of
antigens in the intine was dramatically precise, and the concentration in the vicinity
of the germination pore could readily be seen (Fig. 1). For comparison, the localization
of acid phosphatase, detected by the method of Barka & Anderson (1962) using anaphthyl acid phosphate as substrate, is shown in Fig. 2. Again the concentration in
the intine, particularly at the pore, is quite evident.
From this evidence and that given elsewhere for other pollens (Knox & HeslopHarrison, 1970; Knox et al. 1970; Knox, 1971a, b), it may be accepted that antigenic
material detected by the immunofluorescence method outside of the grain after
moistening is derived from mainly the wall sites. We have no indication from any of
our observations of massive loss of antigens from the vegetative cell, although proteins from this source must be present in long-term leachates made in saline solution.
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Reactions of the unpollinated stigma
The results of attempts to localize pollen-borne antigens on the stigma would be
meaningless were cross-reacting proteins present in, or on the surface of, the stigma
cells. In fact, cross-reacting materials were either absent, or present in such low concentrations as to be essentially undetectable against the faint nativefluorescenceof the
stigma cell walls and cell contents. The point may be verified from Figs. 3-8.
Release of the pollen-borne antigens
Compatible pollinations. In P. tuberosa, as shown by Hayman (1956) for P. coeru-
lescens, germination takes place in compatible pollinations within a few minutes of the
pollen reaching the stigma. In the compatible selfing 42 x 42, the intine antigens were
found to be released very rapidly, and they were readily detected, spread over the
surface of the stigma cells, in 5-10 min preparations, whether or not a pollen tube
had emerged (Fig. 3). Where several grains had alighted on the same stigma filament,
the entire surface was coated with the fluorescent precipitins, with a marked concentration immediately around the grains. The same distribution appeared in the compatible crossing, 42 x 27 (Fig. 6). In some preparations, the precipitin complex was
noticeably granular (Fig. 5); it is, of course, impossible to say whether this is characteristic of the distribution of the antigens, or results from the antigen-antibody reaction
itself.
After 40 min many more grains had germinated, and the tubes had penetrated
considerably farther between the cells of the stigma. However, the antigens were
distributed just as in the 5-10 min preparations, and it was quite evident that no more
antigenic material of the same type as that released from the grain immediately after
landing was being emitted by the growing tubes.
Incompatible pollinations. With both of the self-incompatible genotypes tested, there
was no germination within 5-10 min after selfing. Nevertheless, the intine-held antigens were freely discharged. Perhaps because of the absence of tubes, the grains were
mostly lost from the stigmas during the washing after antibody complexing, but the
sites of their arrival were clearly marked by localized depositions of intensely fluorescent precipitins (Fig. 7). Apart from the disturbance caused by the detachment of the
grains, there was no indication of any difference in the distribution of the antigens in
compatible and incompatible pollinations.
In preparations made after 40 min, some grains were found adhering to the stigmas,
suggesting that tubes had penetrated a short distance. However, it was often apparent
by phase microscopy that the tubes had simply coiled around the grains or a neighbouring stigma process, without true penetration. Hayman (1956) noted that pollen
tubes did penetrate a short distance into the stigma after 20 h in incompatible pollinations of P. coerulescens.
Intine-held antigens on the stigma of Phalaris
243
DISCUSSION
These results show that the intine-held antigens leave individual pollen grains and
spread out on the stigma surface very soon after contact is made with the stigma,
certainly within 5-10 min. Furthermore, they suggest there is no difference between
compatible and incompatible pollination in the behaviour of the discharged materials.
Doubtless the substances responsible for the incompatibility reaction will be among
the emitted antigens. While no direct evidence is yet available for antigens specifically
associated with particular incompatibility genotypes in P. tuberosa, this may be deduced
from the fact that the control over tube growth is exerted also within 5-10 min of
pollination, particularly in the light of the results of the elegant experiments of Lewis,
Burrage & Walls (1967) on Oenothera organensis. In the experiments on Oenothera,
it was shown that the antigen associated with a particular S allele can be detected by a
precipitin reaction in a halo around an individual grain when it is placed on agar
containing an antiserum for an extract of pollen of the same genotype.
Immuno-electrophoretic studies on the anti-P. tuberosa serum will be described in
another paper, but here it may be noted that such work has shown the intine antigens
to be heterogeneous, as are those of Gladiolus (Knox, 1971 a, b) and other species tested.
The enzymes from the wall sites may be among the antigens, but as already suggested
most may be recognition substances, expected themselves to be heterogeneous. The
proportion of the total concerned in the self-incompatibility reaction remains a matter
for speculation. The work of Makinen & Lewis (1962) suggests that in Oenothera
organensis the S protein must form a substantial part of the total mobile protein of the
pollen grain. This is also indicated by the results of Lewis et al. (1967). The fact that
these workers were able to detect S gene segregation in diffusion tests using antisera
against total pollen extracts can only mean that the S protein forms a major part of
the total, otherwise the segregation would be lost against the background of reactions
involving non-specific proteins - including, of course, the enzymes.
It will be informative in further work to examine the responses in close interspecific pollinations, where a stigma rejects foreign pollen. While inter-specific
incompatibility control could depend upon other factors than emitted proteins, the
idea that these are concerned is attractive, because such a mechanism might allow a
higher degree of gene-determined specificity than other possibilities that can be
envisaged. Hybrids of Populus alba and P. deltoides have recently been obtained using
killed pollen of the compatible type mixed with fresh, incompatible pollen (Knox,
Willing & Pryor, in preparation). One interpretation of this result could be that the
incompatible pollen is here 'borrowing' recognition material from the compatible.
Similar work with Phalaris species is planned.
We are indebted to Dr Ian D. Marshall of the John Curtin School of Medical Research
of the Australian National University for help in preparing the anti-Phalaris serum, to Dr
J. R. McWilliam of CSIRO, Division of Plant Industry, Canberra, for providing the Phalaris
plants, and to Miss E. Evans and Mrs L. Tompkins for their valued technical assistance.
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T. & ANDERSON, P. J. (1962). Histochemical methods for acid phosphatase using hexazonium pararosanalin as coupler. J. Histochein. Cytocliem. 10, 741-753.
GOLAND, P., GRAND, N. G. & BOOKER, B. F. (1969). Immunofluorescence microscopy of
cyanurated tissues. Stain Technol. 44, 227-233.
GREEN, J. R. (1894). On the germination of the pollen grain and the nutrition of the pollen tube.
Aim. Bot. 8, 225-228.
HAYMAN, D. L. (1956). The genetical control of incompatibility in Plialaris coerulescevs Desf.
Aust.J. biol. Sci. 9, 321-331.
HESLOP-HARRISON, J. (1963a). Ultrastructural aspects of differentiation in sporogenous tissue.
Symp. Soc. exp. Biol. 17, 315-340.
HESLOP-HARRISON, J. (19636). An ultrastructural study of pollen wall ontogeny in Silene
pendula. Grana palynol. 4, 7-24.
HESLOP-HARRISON, J. (1971). Sporopollenin in the biological context. In Sporopollenin (Proceedings of an International Symposium, Sept. 1970), ed. M. Muir. London: Academic Press.
(In the Press.)
KNOX, R. B. (1971a). Pollen-wall proteins: localization, enzymic and antigenic activity during
development in Gladiolus (Iridaceae). J. Cell Sci. 9, 209-237.
KNOX, R. B. (19716). Localization of proteins in plant cells by immunofluorescence. Zeiss
Information (Australia). (In the Press).
KNOX, R. B. & HESLOP-HARRISON, J. (1970). Pollen-wall proteins: localization and enzymic
activity. J. Cell Sci. 6, 1-27.
KNOX, R. B., HESLOP-HARRISON, J. & REED, C. (1970). Localisation of antigens associated with
the pollen grain wall by immunofluorescence. Nature, Land. 225, 1066-1068.
LEWIS, D., BURRAGE, S. & WALLS, D. (1967). Immunological reactions of single pollen grains,
electrophoresis and enzymology of pollen protein exudate. J. exp. Bot. 18, 374-378.
LUNDQUIST, A. (1961). A rapid method for the analysis of incompatibility in grasses. Hereditas
47, 7O5-7O7LUNDQUIST, A. (1963). The genetics of incompatibility. In Genetics Today (Proc. XI Int. Conf.
Genet. 1963), vol. 3 (ed. S. J. Geerts), pp. 637-647. New York: Pergamon.
MAKINEN, Y. & BREWBAKER, J. L. (1967). Isoenzyme polymorphism infloweringplants. I. Diffusion of enzymes out of intact pollen grains. Physiologia PI. 20, 477-482.
MAKINEN, Y. & LEWIS, D. (1962). Immunological analysis of incompatibility (S) proteins and of
cross-reacting material in a self-compatible mutant of OenoUiera organensis. Genet. Res., Camb.
3- 352-363MCWILLIAM, J. R. Quantitative genetic analysis in Plialaris and its breeding implications.
Theoret. appl. Genet. 40, 63-72.
STANLEY, R. G. & LINSKENS, H. F. (1965). Protein diffusing from germinating pollen. Physiologia PI. 18, 47-53.
[Received 28 February 1971)
BARKA,
Fig. 1. Freeze-sectioned somewhat immature pollen of Phalaris tuberosa, immunofluorescence technique, A, with anti-P. tuberosa serum; B, control with normal rabbit
serum. The antigens are localized in the intine, with a concentration at the pores
(arrows). The pollen grain exines are visible because of the nativefluorescenceof sporopollenin. x 780 approx.
Fig. 2. Section from the same anther as that of Fig. 1. A, a-naphthyl acid phosphatepararosanalin reaction for acid phosphatase, showing enzyme activity at the pore
(arrow); B, control without substrate, phase contrast; the arrow indicates a pore, x 780
approx.
Intine-held antigens on the stigma of Phalaris
2A
2B
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R. B. Knox and J. Heslop-Harrison
Figs. 3-8. Plialaris tuberosa. Complete mounts of stigmas and attached pollen grains and
antigens; immunofluorescence technique using anti-P. tuberosa serum. All x 500
approx.
Fig. 3. Compatible pollination, no. 42 x no. 42, 5-10 min. A,fluorescenceimage;
B, phase contrast of the same field. The pollen tube has emerged (arrow) and is penetrating the stigma; the antigens are spreading on the stigma surface.
Fig. 4. As Fig. 3. A, fluorescence image; B, phase contrast of the same field. A large
mass of antigens, probably derived from several grains, isflowingover the surface of the
stigma processes.
Intine-held antigens on the stigma of Phalaris
4B ..
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R. B. Knox and J. Heslop-Harrison
Fig. 5. As Fig. 3. A, fluorescence image; B, phase contrast. The pollen tube has
grown well down into the stigma, leaving the mass of antigens on the surface; marked
granularity can be seen at the arrow.
Fig. 6. Compatible pollination, no. 27 x no. 42, 5-10 min. A, fluorescence image;
B, phase contrast of the same field. In this compatible pollination between a selfcompatible and a self-incompatible genotype, the pollen tubes have emerged and are
growing vigorously into the stigma, and the antigens are once more left at the surface.
Intine-held antigens on the stigma of Phalans
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R. B. Knox and J. Heslop-Harrison
Fig. 7. Incompatible pollination, no. 27 x no. 27, 5-10 min. A, fluorescence image;
B, phase contrast of the same field. No germination has taken place, and the pollen
grains have all become detached. The sites can, however, be distinguished by the antigens spread out in the vicinity.
Fig. 8. Incompatible pollination, no. 27 x no. 27, 40 min. A, fluorescence image; B,
phase contrast of the same field. After this time, some grains have been retained, suggesting that the tubes have emerged and are penetrating slightly. Antigens retained at
the surface.
Intine-held antigens on the stigma of Phalaris