J. Cell Sci. 36, 1-18 (1979)
Printed in Great Britain © Company of Biologists Limited J979
POLLEN-PISTIL INCOMPATIBILITY IN
PETUNIA HYBRID A: CHANGES IN THE PISTIL
FOLLOWING COMPATIBLE AND
INCOMPATIBLE INTRASPECIFIC CROSSES
M. HERRERO AND H. G. DICKINSON
Department of Botany, Plant Science Laboratories, University of Reading,
Whiteknights, Reading RG6 2AS, England
SUMMARY
The structural events in the stigma and transmitting tissue of Petunia hybrida pistils that
accompany compatible and incompatible intraspecific pollinations have been investigated in
detail, together with the changes in reserve levels that also take place at this time. Many of these
phenomena may be explained in terms of 3 phases of secretion by the cells in the upper regions
of the transmitting tissue. The first, independent of pollination, results in the deposition of an
intercellular matrix, rich in protein and carbohydrate. The second, triggered by pollination,
although independent of the compatibility of the pollen grain, involves synthesis of molecules
believed to be specific to the S(incompatibility)-gene system. The third phase of secretion
occurs only following a compatible mating, and involves the transfer of stylar reserves to
support the growth of the pollen tubes. These observations are discussed in terms of current
models of the incompatibility mechanism operating in Petunia.
INTRODUCTION
In plants with gametophytic control of pollen compatibility with respect to the
style, much attention has been focussed upon the structure and physiology of the
pistil. For example, the cytology of the stigma of Petunia sp. (Konar & Linskens,
1966a) and that of the stylar transmitting tissue in a variety of species (van der Pluijm
& Linskens, 1966; Sassen, 1974; Bell & Hicks, 1976; Cresti, Went, Pacini & Willimse,
1976) has been the subject of recent intensive investigation. Likewise, the stigma and
style of Lilium, a plant with a stylar canal rather than a transmitting tissue per se, have
also been described in detail (Rosen & Thomas, 1970). Physiological studies, however,
have extended into the differences in stylar metabolism following compatible and
incompatible crosses. Bredemeijer (1974) has demonstrated differences in the stylar
peroxidase isozymes following such crosses in Nicotiana, while different levels
of RNA and protein synthesis have been described following crosses of different
compatibility in Petunia by van der Donk (1974).
Structural aspects of these stylar differences have yet to be fully examined.
Certainly, structural changes accompanying cross and self-pollination have been
described in Oenothera (Dickinson & Lawson, 1975I, but here few alterations appear
to occur in the female tissue. In any event, this species possesses neither an organized
transmitting tissue, nor a stylar canal, making localization of the site of the self-
2
M. Herrero and H. G. Dickinson
incompatibility reaction difficult. Likewise, although Lycopersicum has a compact
style with a transmitting tissue, the many elegant ultrastructural investigations into
the differences between compatible and incompatible crosses (e.g. de Nettancourt
et al. 1973) have not considered stylar changes in detail. Our knowledge of the reaction
of compact styles to pollination is thus restricted to studies of the disruption of the
tissue by pollen tube growth in Lychnis (Crang, 1966), and measurement of uptake of
materials from the style by pollen tubes in a number of species (Linskens & Esser,
1959; Kroh, Miki-Hirosige, Rosen & Loewus, 1970; Kroh & Helsper, 1974). Stylar
changes may, however, be of considerable importance since little is known of the
factors that induce relatively slow growth of the incompatible tubes, when compared
with the rate of compatible tube extension. While structural data may be few, models
explaining these events certainly are not. Kroes (1973), for example, suggests that
incompatible tubes lack an enzyme necessary to use the nutrients available in the
style while, in an elegant hypothesis for Petunia, van der Donk (1975) proposes that,
following a pollination-generated stimulus, synthesis of polypeptides takes place in
the style. It is the interaction between these 'pollination-polypeptides' (or a larger
molecular assembly containing them) and the tube that results in the 'compatible' or
'incompatible' style of growth.
In an attempt to align some of these hypotheses with structural events, we describe
here the changes in the stigma and style accompanying compatible and incompatible
intraspecific pollination in Petunia.
MATERIALS AND METHODS
Plant material
Seedlings homozygous with respect to the incompatibility genes were raised by means of
bud pollination of clones Ka3 and T211 (genotypes 22 and 33 respectively), the seeds of which
were originally kindly supplied by Professor H. F. Linskens. Following growth and flowering
in the greenhouse, individual flowers were detached for the pollination studies. These experiments were carried out in a well-lit air-conditioned chamber, maintained at a constant temperature of 25 °C.
Studies of pollen tube growth
Pollen tube growth was measured using material stained with aniline blue examined under a
Leitz epi-UV irradiance system fitted to a Leitz Dialux microscope. Styles were prepared
following a modifications of Linskens & Esser's (1957) technique. Material was first softened
for 1 min at 100 °C in a 5 % aqueous solution of sodium sulphite, and then stained in a O'l %
solution of aniline blue in 01 N K3PO4, for 5 min. The tissues were examined mounted in the
stain.
Determination of starch levels
With the aid of a sharp scalpel short sections were excised from different regions of the style
and, using light pressure, the transmitting tissue contained in the section extruded onto a
clean slide. Difficulties were encountered in extracting the very top of the transmitting tissue,
that which interfaces with the stigma. However, by shaving off the stigmatic papillae with a
microscalpel it was possible to expose, and subsequently express, the top layer of the transmitting tissue.
Starch in these cells was then stained with a saturated solution of iodine in potassium iodide
Self-incompatibility in Petunia
3
for 5 min at room temperature. Following rinsing in 0-3 M Sorensen phosphate buffer, the
transmitting tissue was mounted in glycerol, squashed carefully, and the preparation sealed
with rubber solution. The intensity of the stain was measured using a Vickers M85 scanning
microdensitometer, operating at a wavelength of 500 ran. The method of preparation produced
a monolayer of stained cells, and readings were taken of a constant area of this monolayer,
limited by the field of the densitometer 'mask'. Each field contained between 4 and 6 cells. It
must be emphasized that the readings resulting from this work can in no way be linked stoichiometrically with the starch in the tissue, but they do provide a useful indication of the levels of
this reserve. In some of the results it was considered helpful to express the data as 'amounts
per flower'. To obtain this measure, the average was taken of the readings from each level in
the style.
Preparation for light and electron microscopy
For enzyme cytochemistry, fresh material was sectioned at 5 /im in a Slee cryostat and
stained to reveal the location of acid phosphatase (Knox & Heslop-Harrison, 1970), esterase
(Pearse, i960) and peroxidase (Jensen, 1962).
Tissue for electron microscopy was prepared according to Dickinson & Lawson (1975). To
identify protein in this material, sections were digested with protease (Dickinson & Potter,
1975). Thick sections (1-5 fim) of these Epon blocks were also cut for light microscopy. Carbohydrate was localized in these sections using treatment with periodic acid and Schiff's reagent
(Feder & O'Brien, 1968).
RESULTS
The stigma
At anthesis, degeneration of the stigmatic tissue takes place completely independently of pollination and indeed, the compatibility of that pollination. Prior to this
event, electron micrographs reveal the papillae not only to contain reserves of starch
and saturated lipid, but also plastids (see Figs. 1, 2), dictyosomes, associated vesicles,
microbodies and mitochondria. As degeneration of these commences, large droplets
of lipid become evident inside the cytoplasm of a proportion of the cells (see Fig. 3).
The plasma membranes of these cells then rupture, releasing the lipid to coalesce
with other extracellular droplets (see Figs. 4, 5), and the remaining cytoplasmic
content to form small islets of degenerate cytoplasm that float in the lipid (see Fig. 3).
Cytochemical tests, however, show the stigmatic extract to contain sugars and also acid
phosphatase. This enzyme is not distributed evenly, but apparently contained in droplets in the stigmatic fluid (see Fig. 6). No reaction was observed with the cytochemical
tests for either peroxidase or esterase.
It is into this lipid-rich fluid that the pollen grain alights. Within 30 min it has
germinated, and the pollen tube begun to grow between the papillae (see Figs. 7, 8)
into the stigma.
The cells of the transmitting tissue
The cells of this region present the features of normal somatic meristematic plant
cells, albeit with rather thick walls, some of which may measure up to 0-5 /tm in width.
Apart from the conventional cell contents, the cytoplasm of these cells possess reserves
of lipid and starch, considerable numbers of dictyosomes, mitochondria, and a large
endoplasmic reticulum (see Fig. 9). Frequently, these cells contain microbodies. Close
M. Herrero and H. G. Dickinson
Self-incompatibility in Petunia
5
examination of the transmitting tissue with the electron microscope reveals it to be
differentiated into 2 distinct regions, each with a characteristic cell morphology. The
tissue in the 'neck' of the style contains large spherical cells (see Fig. 10), possessing
characteristic ridges in their walls (see Fig. 11) which cause them to 'key' into an
adjacent cell. In the remainder of the transmitting tissue, the cells are elongate, more
loosely packed and do not have superficial ridges. The organelle content of both types
of cell is similar, but the 'neck' cells often appear to contain more endoplasmic
reticulum and ribosomes. The endoplasmic reticulum, be it in the neck cells or elsewhere in the transmitting tissue, is regularly associated with vesicles (see Fig. 12).
These vesicles are also frequently observed subjacent to, or apparently merging with
the plasma membrane.
Particularly striking are the large intercellular spaces characteristic of the transmitting tissue, which appear to contain an electron-opaque matrix (see Fig. 10).
Again differences occur between cells in the neck of the style and those in the remainder
of the tissue, for the content of the spaces formed between the 'neck' cells is particularly sensitive to protease digestion (see Fig. n ) , while that between cells elsewhere in the transmitting tissue remains unaffected, even following long periods of
digestion. Cytochemical investigation of the intercellular spaces also reveals differences
between the 'neck' and lower regions of the tissue, for, while acid phosphatase (see
Fig. 13), peroxidase, and carbohydrate (see Fig. 14) are common to spaces throughout
the tissue, the neck cells are surrounded by spaces containing esterase, in addition to
generally larger amounts of these other constituents.
While all the preceding events take place in virgin styles, other changes do overcome
the 'neck' cells of the transmitting tissue on pollination, independent of the compatibility of the mating. The first indications of this stylar reaction to pollination is
a rise in the number of polyribosomes in these cells (see Fig. 15). This takes place
between 0-5 and 2 h after pollination and is accompanied by large numbers of singlemembraned cytoplasmic inclusions becoming associated with the plasma membrane
to form a characteristic 'embayment' (see Fig. 16). These embayments may measure
up to 0-2 /im in the maximum dimension and contain a grey fibrillar matrix, which
merges with that of the wall. This activity at the plasma membrane is comparatively
Figs. 1, 2. Young 8tigmatic papillar cells of Petunia hybrida containing a full complement of organelles, including mitochondria (»/) and starch-containing plastids (p).
Droplets of saturated lipid (i) are also present in this cytoplasm. Fig. i, x 7540; Fig. 2,
x 5256.
Fig. 3. Degenerating stigmatic papillae showing accumulations of unsaturated lipid (/)
in intact protoplasts (p). Other cells (c) have completely degenerated to form lipid
droplets and vesicles (arrows) containing fibrogranular material, x 3078.
Fig. 4. Scanning electron micrograph of stigmatic surface. Individual papillae (s) protrude through the stigmatic fluid. The spheres visible (arrows) may represent the
droplets of unsaturated lipid visible in Fig. 5. x 770.
Fig. 5. Transmission electron micrograph of material depicted in Fig. 4. A proportion
of the papillae (s) remain intact and are invested by the lipidic residues of the degenerate cells, x 3348.
M. Herrero and H. G. Dickinson
Self-incompatibility in Petunia
y
short-lived and, within about 4 h, this membrane has returned to its original
aspect.
All these events occur before the arrival of the pollen tubes, which are at this time
growing at a rate of about 150/tm/h through stigmatic tissue. Close to these extending
pollen tubes many of the 'neck' cells of the transmitting tissue appear necrotic,
containing dark granular cytoplasm, large numbers of vesicles, and disorganized
plastids (see Fig. 17). In addition, cytoplasmic fragments may frequently be observed
in the intercellular spaces dividing these cells (see Fig. 18). These necrotic cells, if
tested some 24 h after pollination, react most strikingly with protease, appearing to
be almost totally sensitive to the enzyme (see Fig. 19).
The stylar cells following self- and cross-pollinations
Although the cells of all regions of the transmitting tissue appear to react independently of the compatibility of the pollen tube approaching them, their behaviour
following its passage differs considerably according to the nature of the mating.
Following a compatible cross, for example, cells are characterized by large vacuoles
invested by a thin peripheral layer of cytoplasm. This cytoplasm is no longer rich in
reserves, but instead contains a nucleus, few mitochondria and scattered dictyosomes
(see Figs. 20, 21). The transmitting tissue cells of self-pollinated flowers, on the other
hand, resemble those of unpollinated flowers. Little depletion of reserves is indicated
by the micrographs (see Fig. 22), and the cytoplasm: vacuole ratio remains largely
unchanged.
Starch metabolism during pollination
While the changes above may be easily observed qualitatively, a quantitative measure
of the events is not so simply obtained. Nevertheless, microdensitometry of preparations
in which the starch has been stained (see Fig. 23) provides a quantitative representation
of levels of this reserve.
From such results, it is clear that in unpollinated flowers no change occurs in the
Fig. 6. Light-microscopic preparation treated to reveal acid phosphatase. The enzyme
is localized in small droplets which occur both on the papillar surfaces (s) and in the
large spaces between the loosely packed cells of the stigma, x 1176.
Fig. 7. Scanning electron micrograph of a pollen grain germinating on the stigma.
The pollen tube (t) has penetrated the stigmatic surface (arrow). A papilla (s) is also
visible, as is a tube (ij) from another grain, x 1534.
Fig. 8. Transmission electron micrograph of material shown in Fig. 7. The pollen
tube (t) is growing down between stigmatic papillae (s) invested by droplets (arrows)
of the lipidic remains of degenerated cells, x 6016.
Fig. 9. Transmitting tissue cell of Petunia showing a starch-containing plastid (/>),
mitochondria (m), a dictyosome (d) and elements of the endoplasmic reticulum
(arrows), x 18165.
Fig. 10. The ' neck' region of the transmitting tissue. The cells of this region are spherical and possess unusual 'key' junctions (arrows) with their neighbours (shown better
in Fig. 11). Note also the electron-opaque intercellular spaces, x i960.
M. Herrero and H. G. Dickinson
Self-incompatibility in Petunia
9
starch content of the transmitting tissue until the final degeneration of the style. After
a cross-pollination, however, a massive decrease in starch levels takes place, while,
following a self-mating, there is only a slight depletion of the reserve (see Table 1).
In order to investigate a possible relationship between the passage of the pollen
tubes and depletion of the starch, the number of pollen tubes at different levels in the
styles was expressed as a percentage of the number of pollen grains present, and this
percentage considered in terms of the starch levels in the entire pistil. When these
results are plotted (see Fig. 24) a clear relationship may be detected in compatible
crosses, the starch of the stylar cells decreasing with the passage of the pollen tubes
through the adjacent intercellular spaces. Following incompatible crosses, this inverse
relationship is not nearly as marked.
Table 1. Levels of IKI-stainable material, expressed as arbitrary
microdensitometer units per flower. {The standard errors are shown in parentheses.)
24 h
48 h
Unpollinated
Self-pollinated
Cross-pollinated
20-7910-38
20-9711-31
180110-83
II-6I±O-O8
I6-57±I-66
1-8610-46
This difference in starch metabolism between tubes of differing compatibility is
particularly well underlined by a series of readings taken at the neck of the transmitting tissue, subjacent to the stigma, when pollen tube distribution is almost
identical (see Fig. 25 A, E). Starch levels in the selfed styles are far in excess of those
found following cross-pollination.
In addition to these effects of pollination, measurement of starch content at 4 levels
at 24 and 48 h after pollination indicates that in styles pollinated with grains of
Fig. 11. Protease-digested material similar to that shown in Fig. 10. The loss of electron
opacity of the intercellular spaces is striking. The 'key' junctions between cells
(arrows) are also conspicuous in this micrograph, x 4504.
Fig. 12. Portion of cell in ' neck' region of the transmitting tissue. Elements of the endoplasmic reticulum are evident («) as are associated vesicles (arrow), x 25500.
Fig. 13. Light-microscopic preparation of 'neck' region of transmitting tissue, treated
to reveal acid phosphatase. Note the presence of this enzyme in the intercellular
spaces, x 1178.
Fig. 14. As Fig. 13, but material reacted to show the location of carbohydrate. Starch
(arrows) is visible within the cells, but the intercellular matrix is also PAS-positive.
x 761.
Fig. 15. Cells of the 'neck' region of the transmitting tissue 2 h after pollination.
Large numbers of polyribosomes (arrows) are present and the embayments (e) are
also visible, x 27675.
Fig. 16. As Fig. 15. The embayments (e) of the plasma membrane are particularly
striking, as is their fibrillar content. X 14287.
Fig. 17. Pollen tube (i) growing through the 'neck' region of the transmitting tissue.
Near the tube some of the cells («) appear disorganized and necrotic, while others (c)
display a normal aspect, x 5771.
M. Herrero and H. G. Dickinson
Self-incompatibility in Petunia
i1
either compatability, a detectable 'wave' of starch synthesis precedes the tube tip in
its passage through the transmitting tissue (see Fig. 25 c, D).
DISCUSSION
The maturing stigma and first events after pollination
The final stages in stigmatic maturation which appear to involve the 'degeneration'
of the papillae (Dumas, 1975) are clearly independent of pollination and, indeed, the
compatibility of pollination. Konar & Linskens (19666) have proposed that the stigma
acts purely as a location for the germination of pollen and is not involved in its
nutrition per se, and there is little from the present investigation to indicate otherwise.
However, while the pollen grains are germinating in this stigmatic exudate, striking
changes appear to be induced in the subjacent regions of the transmitting tissue.
These start with a marked increase in polyribosome number in the cells, and continue
with the formation of the characteristic 'embayments'. While conclusive evidence is,
of course, unavailable, these profiles do indicate a transfer of cytoplasmically synthesized material into the carbohydrate and pectin rich intercellular spaces (Kroh,
1973; Kroh & van Bakel, 1973; Sassen, 1974). The nature of the stimulus that travels
to these cells of the stylar neck and triggers these changes is not clear. Linskens &
Spanjers (1973) have recorded a difference in the electrical resistance of the pistil
after pollination, but it remains equally possible that these changes are induced by a
flow of chemical messenger. These events must also be accompanied by biochemical
changes, such as the activation of the glutamic dehydrogenase described by Roggen
(1967).
Biochemical studies (van der Donk, 1975) have indicated pollination to stimulate
the production of 'recognition' polypeptides, specific to the incompatibility (S)
genotype of the plant. It is striking that this aggregation of ribosomes into polysomes
and the apparent secretion of materials by the cells in the neck of the transmitting
Fig. 18. 'Neck' region of transmitting tissue during passage of pollen tubes. Intact
cells (c) are present, but also visible is a degenerate protoplast (n) and cytoplasmic
fragments (/). x 5769.
Fig. 19. Degenerate protoplast (n) as shown in Fig. 18, following digestion with
protease. This cell is almost totally sensitive to the enzyme, whereas intact cells (c)
are not. x 7185.
Fig. 20. Tangential section of a transmitting tissue cell following the passage of
compatible pollen tubes. Note the absence of reserves from this protoplast, x 9585.
Fig. 21. As Fig. 20, but median section revealing the large vacuoles (v) of these cells,
x 6140.
Fig. 22. Transmitting tissue cell following the passage of incompatible pollen tubes.
Note the presence of starch (si) in the plastids and droplets of lipid ([) free in the
cytoplasm, x 9060.
Fig. 23. Light-microscopic preparation of lower region of the transmitting tissue
treated with PAS to reveal the presence of starch. Although the cell walls react and
the intercellular regions are slightly sensitive, the main staining is in the starch grains
of the stylar cells, x 934.
M. Herrero and H. G. Dickinson
12
100
20-
Stigma
Style
100 -
- 25
80-
-20
r
60-
-15 3
:
40-
- 10 j j
20-
- 5
_c
u
Stigma
0-25
0-5
0-75
Style
Fig. 24. The number of pollen tubes and levels of starch at different points in the
style 24 h after pollination following compatible (A), and incompatible (B) pollinations.
The pollen tube number (•) is expressed as a percentage of the grains on the stigma
surface. The levels of starch ( • ) are in arbitrary microdensitometer units.
Self-incompatibility in Petunia
171]
Fig. 25. Levels of IKI-stained starch, expressed in arbitrary microdensitometer
units, in unpollinated (D), selfed (^), and crossed (3§) flowers at 4 levels in the
pistil, 24 (A,B,C,D) and 48 (E,F,G,H) h after pollination. The pollen tube numbers are
given in brackets under the lower axes. The regions of the pistil from which measurements were taken are as follows: A, E, stigma; B, F, top quarter of style; c, G,
second quarter; and D, H, third quarter.
CEL 36
14
M. Herrero and H. G. Dickinson
tissue is so closely synchronized with the proposed synthesis of the s-specific polypeptides, all these events occurring in the style ahead of the advancing pollen tubes.
Once the pollen tubes reach the transmitting tissue, there is little doubt that some
cellular degeneration takes place. This has been reported in Gossypium (Jensen &
Fisher, 1969) and would appear to be a general feature of pollen tube growth, irrespective of its compatibility. Although results have yet to be analysed statistically,
more transmitting tissue cells appear to degenerate in the vicinity of the growing
pollen tubes than elsewhere in the tissue. The fate of the cytoplasm released into the
intercellular space by the rupture of these protoplasts is not yet clear, but it may often
be seen to invest the pollen tubes.
The metabolism of starch over the course of pollination
There is little doubt that a slight increase in stylar starch synthesis is stimulated by
pollination itself. As with the previous cytoplasmic effects, it is not yet clear how the
stimulus for this process is transmitted from the stigma. Other changes in the carbohydrate metabolism of the pistil also occur at this point, for Tupy (1961), working
on Nicotiana, has reported an increase in glucose and fructose levels at the ovary 1 day
after pollination, combined with a decline in the stigmatic sugars.
The pronounced drop in starch levels accompanying the growth of compatible pollen
tubes is not unexpected. Starch is clearly an energy reserve, and if the tube is growing
heterotrophically, depletion of such reserves should certainly occur. These observations
are consistent with those of Linskens (1955) who reported free sugars to decrease to
below half their pre-pollination levels following the passage of the pollen tubes, and
of Roggen (1967) who demonstrated induction of enzymes for carbohydrate metabolism
in the vicinity of the growing pollen tubes. The uses to which these extracellular
carbohydrates may be put are many. O'Kelley (1955) describes their utilization in
respiration, while Kessler, Feingold & Hassid (i960) have, in studies in vitro, described
their employment in the synthesis of sucrose, callose and starch. In an investigation
into the fate of the intracellular pectic substances, Kroh et al. (1970) and Kroh &
Helsper (1974), reported their incorporation into the pollen tube wall.
The lack of starch mobilization following the passage of incompatible tubes is not
so easily understood. This does not simply result from fewer tubes generally growing
in such circumstances, for areas at the top of the transmitting tissue containing
equivalent numbers of pollen tubes exhibit differences in starch metabolism dependent upon the compatibility of the cross. Again, biochemical investigations tend to
support these observations; Linskens (1953, 1955) has reported a decrease in respiratory rate of pistils 12 h following self-pollination, and also differences between
selfed and crossed styles in their endogenous levels of 'glucan-hydrolases' (Linskens
et al. 1969). Taken in toto this evidence indicates that, in incompatible tubes, major
pathways of carbohydrate metabolism are either blocked, or alternatively not activated.
Self-incompatibility in Petunia
15
The structural differences in the transmitting tissue following compatible or incompatible
pollinations
Since the difference in pollen tube number normally encountered between compatible and incompatible crosses might cause effects that would be incorrectly
interpreted, results are only discussed from the upper regions of the transmitting
tissue where numbers of pollen tubes are equal irrespective of their compatibility
and true stylar changes are thus most conspicuous.
The very different aspects displayed by transmitting tissue permeated by compatible
tubes, and that containing incompatible tubes may almost be fully explained in terms
of the utilization of reserves. The tissue with incompatible tubes looks very similar
to that of pollinated flowers prior to the passage of the pollen tubes, while that in
contact with compatible tubes is deficient in reserves in the form of starch and lipid,
and contains far fewer microbodies.
Such a 'degeneration' of stylar tissue following the passage of compatible pollen
tubes was reported by Crang (1966) to occur in Lychnis. In another species, Lilium,
Crang (1969) found degeneration to occur in the parenchymatous cells investing the
stylar canal cells, although these latter cells seemed not to be affected. While this
work did not indicate clearly the cause of this degeneration, Crang (1969) proposed
it to result from either enzymic secretion by the pollen tubes, or the stimulation by
the pollen tube of 'autolytic bodies' in the stylar cells.
Although Rosen & Thomas (1970) confirmed that no ultrastructural changes overcame the stylar canal cells of Lilium on pollination, they reported an increase in
secretory activity by these cells. Likewise, Yamada (1956) described the loss of cytoplasmic organization by the parenchymatous cells after pollination in Lilium, and
pointed to the coincident arrival of a mucilagenous substance on the surface of the
canal cells. The fact that starch and lipid were the first components of the cytoplasm
to disappear from the cells of Lychnis and Lilium led Crang (1969) to propose that
these reserves were utilized in the nutrition of the pollen tubes. In Gossypium, on the
other hand, the position appears to differ, for Jensen & Fisher (1969) report the
maintenance of stylar starch and lipid levels over the course of pollination. Results
from the present investigation, however, concur well with those of Crang (1969),
Yamada (1965), Rosen & Thomas (1970) and indicate that compatible pollen tubes
stimulate the mobilization of stylar reserves, and subsequently utilize these products
in the course of their metabolism.
Whilst this difference in metabolism of reserves between compatible and incompatible tubes is doubtless of significance, it is perhaps the events immediately prior
to, and following pollination that are possibly most important to our understanding
of the incompatibility system in Petunia. In an autoradiographic investigation,
Labarca & Loewus (1973) reported that secretion by cells of the transmitting tissue
into the intercellular space was independent of pollination, results supported by the
electron-microscopic work of Sassen (1974) which indicated a release of intercellular
substances throughout maturation of the style, reaching a maximum prior to anthesis.
In a more recent investigation Cresti et al. (1976) have examined the formation of
16
M. Herrero and H. G. Dickinson
the intercellular matrix in Lycopersicum, and report that first secreted are pectic
substances followed, after conspicuous activity of the endoplasmic reticulum and
associated polyribosomes, by the formation of a small amount of protein.
While our results agree with this in part, there is little doubt that there are at least
3 phases of synthesis, one massive and prior to pollination, one stimulated by pollination and involving the 'embayments', and a final phase of secretion when mobilized
stylar reserves are transferred to the pollen tube. The second phase occurs prior to
the passage of the pollen tubes and is independent of compatibility, while the third
occurs during pollen tube growth, and depends upon compatibility. These events may
clearly be explained in terms of the model of van der Donk (1975) in which the S gene
acts as a ' master-gene', switching on a battery of stylar genes that result in the support
of pollen tube growth. In this connexion, it is noteworthy that little starch and lipid
appears to be mobilized in transmitting tissue cells on self-pollination.
Alternatively, the differences observed between compatible and incompatible tubes
may result from heterotrophic and autotrophic styles of growth. This concept is not
new, for Rosen & Gawlick (1966) suggested, from work on Lilium, that changes they
observed in incompatible tubes could be explained in terms of an inability to switch
from an autotrophic to heterotrophic form of growth. Although this proposal has
received much consideration in the literature (Kroes, 1973; Vasil, 1974), it has little
support from the autoradiographic work of Kroh et al. (1970), where incompatible
tubes were shown to take up more labelled precursor from the style than compatible
tubes. This work, though far from conclusive, and the results of the study presented
here would perhaps indicate that a metabolic deficiency lies within the incompatible
tube itself.
Whatever model of pollen tube metabolism is finally adopted, the fact remains that
incompatible pollen tubes do germinate, stimulate stylar metabolism and must utilize
a portion, albeit very small, of the stylar reserves. At first sight this might appear to
to be an inefficient use of stylar reserves, but under poor cross-pollination conditions
such a mechanism enables self-pollination to ' prime' the style for the growth of the
few cross-pollen tubes available.
The precise point of recognition presumably occurs when the pollen tube makes
contact with S-specific polypeptides, or a larger molecular assembly containing them.
The nature of these molecules and, indeed, their receptors in the pollen tube are not
known. It is probably in species in which the contents of the stylar canal may be
extracted without physiological damage to the surrounding cells, that the search for
these recognition molecules may most profitably be carried out.
Thanks are due to the ARC and OECD for financial support, to the Royal Society for
the provision of photomicrographic equipment, and to Ursula Potter for help with the
illustrations.
REFERENCES
J. & HICKS, G. (1976). Transmitting tissue in the pistil of Tobacco. Light and electron
microscopic observations. Planta 131, 187-200.
BELL,
Self-incompatibility in Petunia
17
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{Received 27 July 1978)