Annals of Botany 85 (Supplement A): 39-47, 2000
doi:l10.1006/anbo.1999.1059, available online at http://www.idealibrary.com on IE1
®
Pollen Tube Guidance by the Pistil of a Solanaceous Plant
W. MARY LUSH*, TIMOTHY SPURCK and RUTH JOOSTEN
School of Botany, University of Melbourne, Parkville 3052, Australia
Received: 21 July 1999
Returned for revision: 10 September 1999
Accepted: 15 October 1999
The guidance of pollen tubes into the stigma, through the style and within the ovary of solanaceous plants is
examined. New data based on time-lapse video microscopy of pollen on Nicotiana alata stigmas and in culture, as well
as style squashes and pollinations with pollen bearing the marker gene Gus, are presented. The initial guidance of
pollen tubes as they emerge from grains on the stigma is probably by a gradient in the concentration of water in the
immediate environment of the pollen grain. At later stages of growth on the stigma, the probability that tube tips will
encounter an opening is high because of the tendency of tubes to maintain contact with the surface. Styles support
pollen tube growth either towards or away from the ovary, suggesting that they have no constitutive directionality.
Guidance within solid transmitting tracts is probably by default, i.e. the architecture of the tissue means that pollen
tubes can only grow in the intercellular spaces between the parallel files of cells. Pollen tubes within the ovary of
N. alata do not appear to be precisely guided to the nearest ovule, and further, quantitative studies of growth in this
(()2000 Annals of Botany Company
region are needed.
Key words: Hydrophobin, chemotropism, thigmotropism, Nicotiana atta, tobacco, pollen, Solanaceae, stigma
exudate, TTS protein, GaRSGP.
INTRODUCTION
For at least a century pollination biologists have been intrigued by the ability of pollen tubes to find their way from the
site of pollen hydration and germination, to a remote site
where ovule fertilization occurs. Heavy emphasis has been
given to the possibility that chemotropic pollen tubes
respond to concentration gradients in chemical attractants,
but no guidance cue has yet been unequivocally identified.
It seems unlikely that there is a universal guidance cue,
given the long and unsuccessful history of the search for one
and the structural and chemical variety of pistils. Work on
Nicotiana alata suggests that the main means of pollen tube
guidance may in fact be the interaction of pollen tubes with
their physical environment. Following the usual convention, the style is treated as being made up of three parts,
stigma, style and ovary, and guidance mechanisms are
examined in each part.
THE STIGMA
Nature of the stigma exudate
The stigma is the site at which pollen is 'captured', hydrates
and germinates, and at which tubes start their growth
through female tissues to the ovules. The best descriptions
of pollen hydration and germination involve species with
'dry' stigmas, where pollen is relatively easy to observe (e.g.
Dickinson, 1995). Pollen disappears from view when it
enters the liquid exudate on the surface of 'wet' stigmas
such as those of the Solanaceae, and descriptions of events
* For correspondence. Fax +613 9347 1071, e-mail w.lush@botany.
unimelb.edu.au
0305-7364/00/0A0039 + 09 $35.00/00
within exudates are based on interpretation rather than
direct observation.
Although stigma exudates have long been classified as
either hydrophobic or hydrophilic (Knox, 1984), the
consequences of differences in hydrophobicity tend to be
overlooked. Thus, for example, the description of stigma
exudates as growth media, although accurate in the sense
that the exudate is the medium in which pollen germinates
and tubes start to grow, is misleading if it is interpreted as
meaning that all exudates are reservoirs of the water
required for growth. Some of the chemical and functional
differences between the two major classes of stigma exudate
are discussed below and are summarized in Table 1.
Hydrophilic stigma exudates, for example those of the
Liliaceae, are probably growth media for pollen in the sense
of being reservoirs of both water and nutrients. They have a
continuous water phase, a high content of lipids (which
may occur as drops of lipid in a continuous water phase),
and simple and complex sugars (Labarca and Loewus,
1972). Lily exudate stimulates pollen germination and tube
growth (Rosen, 1971), and studies with radioactively
labelled exudate have shown some transfer of components
to pollen tubes (Kroh, Labarca and Loewus, 1971).
Triglycerides, however, are the major component of the
continuous oil phase of the hydrophobic exudates of the
Solanaceae (Konar and Linskens, 1966b; Cresti et al.,
1986), and the hydrophobicity of these exudates is probably
critical to their function in pollen hydration and pollen tube
growth. The exudates are not emulsions and to describe
them as lipid-rich, although correct, is to fail to recognize
the fundamental distinction between the two classes of
exudate. The solubility of water in non-polar solvents is
very low and, as might be expected from this fact,
© 2000 Annals of Botany Company
40
Lush et al.-Pollen Tube Guidance
TABLE I. Summary of the properties andfunctions of hydrophilic and hydrophobic stigma exudates discussed in the text
Property or function
Hydrophilic exudates
Hydrophobic exudates
Composition of continuous phase
Other major components
Pollen capture
Pollen adhesion
Water
Lipids, sugars and polysaccharides
Yes
Triglycerides
Yes
Yes
Yes
No
No
Yes
Yes
No
Yes
Yes
Pollen nutrition
Reservoir of water for hydration
Reservoir of nutrients
Establishes hydraulic contact between pollen and stigma
Pollen tube guidance
Source of cue
Provides permissive environment for propagation of cue
Restricts water loss from stigma and pollen
No
9*
Yes
No
*Not known.
solanaceous exudates are not a reservoir of water for pollen
hydration (Konar and Linskens, 1966b). Coverage by
hydrophobic exudates will reduce water loss from stigma
cells and hydrating pollen grains, and restricted water loss
may be essential for full hydration, germination and pollen
tube growth on the stigma (Konar and Linskens, 1966b).
In Table 1 we indicate that the non-aqueous components
of hydrophobic exudates are not known, although there are
reports of substantial amounts of proteins and carbohydrates in solanaceous exudates (e.g. Gane, Clarke and
Bacic, 1995). These reports are puzzling because the
components reported as being present are usually hydrophilic. However, the exudate collection techniques used in
most analyses do not rule out contamination of the exudate
with other components from the stigma, and in some cases
identification of a component in extracts of whole stigmas
(Bacic, Gell and Clarke, 1988) has subsequently been
interpreted as indicating presence in the exudate (Gane
et al., 1995). It is possible, however, that some of the
proteins and carbohydrates reported as present in the
exudate occur in the surface 'skin' (Fig. IA). This skin
reforms at air/exudate and water/exudate boundaries when
exudate is isolated from the stigma, but, to our knowledge,
is not described elsewhere. Its behavioural properties
resemble those of hydrophobins, a class of small (15 kDa),
secreted proteins that self-assemble into 'membrane'
structures at hydrophobic surfaces (Talbot et al., 1996).
Pollen hydration on living stigmas
The stigma of N. alata is papillate, but not as densely so
as the dry stigmas of brassicaceous plants. After N. alata
flowers open, the oily exudate surrounds the base of all
papillae and completely submerges many of them (Fig. A).
Pollen grains that lodge on the tips of papillae above the
surface of the exudate do not hydrate (Lush, Grieser and
Wolters-Arts, 1998). Most pollen grains are completely
submerged in exudate (mature pollen equilibrated with air
at normal relative humidities is hydrophobic), but the
exudate itself is not the reservoir of water for pollen
hydration, because pollen placed in exudate isolated from
the stigma does not hydrate. Konar and Linskens (1966a)
FIG. 1. Surface views of the stigma of N. alata. A, Some papillae
project through stigma exudate which is covered by a surface 'skin'. B,
Small droplets of a liquid that is immiscible in oil occur on the surface
of the stigma beneath the oil layer. This photograph was taken by
flooding an intact sigma with an oil (refined olive oil, which has
properties similar to those of the exudate) and using epi-illumination.
Bar = 50 pm. pap, Papilla cell; aq, aqueous droplet.
suggested that Petunia (Solanaceae) pollen derives water
from a thin film that lines the surface of stigma cells beneath
the exudate. However, because pollen beneath the exudate
is not readily visible, there are no detailed descriptions of
hydration on solanaceous stigmas. Here we present
observations from time-lapse sequences made using epiillumination of oil-flooded stigmas on cut styles. As judged
Lush et al.-Pollen Tube Guidance
41
FIG. 2. Pollen germination and tube growth on stigmas of N. alata. A-I, Time-lapse sequences of oil-flooded, intact stigmas. A-D, The initial
direction of growth of pollen tubes from germinating grains is towards the stigma. Grains are displaced when elongating tubes reach the stigma
surface, sometimes resulting in changes in the orientation of pollen tube growth (pollen tube arrowed). The direction of growth changes so that the
tube approaches the stigma. E-G, A partially-hydrated pollen grain in E, completes hydration and germinates to produce a tube that grows
towards the stigma. When the tube (arrow) reaches the stigma the grain is displaced slightly before the tube commences growth along the stigma
surface. H and I, A pollen tube (arrow) following the surface of the stigma grows up a short papilla cell, and subsequently maintains its growth in
the same direction resulting in it leaving the surface of the stigma. Bar in E also applies to H and 1. J, Pollen tube growth on a normal (nonflooded) stigma viewed 12 h after pollination. At least some pollen tubes (arrow) grow along the surface of the stigma. The stigma was excised and
placed in aniline blue just before viewing with epifluorescent illumination. Bars = 50 [tm. Numbers in the right-hand corner are the time (min)
after taking the first frame in the sequence.
by vigorous cytoplasmic streaming in papillae, stigma cells
remained alive for many hours in these conditions.
Drops of an immiscible fluid, presumably aqueous,
occurred on the surface of cells beneath the oil on N. alata
stigmas (Fig. lB), but did not spread to form a film,
indicating that the walls of stigma cells are hydrophobic.
Some of the pollen grains that entered the exudate settled at
the base of papillae and presumably absorbed some water
by direct contact with aqueous drops, but there was no
migration of water droplets towards hydrating pollen
grains. Other pollen grains remained suspended in the
exudate or lodged against the sides of papillae or other
pollen grains (Fig. 2), and were therefore not in direct
contact with free water. Pollen hydration times, measured
as the time for grains to become fully rounded, ranged from
13 to 43 min, with hydration being most rapid in grains
closest to the stigma surface (six grains monitored). The
reservoir of water for hydration of suspended or lodged
grains is thus the stigma cells and free water at the stigma
surface, and it is transferred to pollen through a surrounding oil layer.
Pollen germination and penetration of living stigmas
All the pollen tubes (total of 16) observed emerging from
grains on the flooded stigmas grew towards the stigma
surface (Fig. 2). Typically, pollen tubes became visible after
they made contact with the stigma, when the continuing
extension of the tip into the resistant surface resulted in the
older parts of the tube and the grain being displaced
backwards (Fig. 2A). The backwards displacement was
usually restricted by the presence of other pollen grains
and papillar cells. Neither grains nor tubes appeared to
adhere to the stigma surface. During the displacement of
grains, the original orientation of pollen tube growth
towards the stigma was sometimes disturbed (Fig. 2A and
Lush et al.-Pollen Tube Guidance
42
B), but these pollen tubes reoriented their direction of
growth towards the stigma (Fig. 2C).
Growth towards the stigma was no guarantee of stigma
penetration. Cells at the surface of solanaceous stigmas
separate from each other at maturity, thus bringing the
intercellular spaces of the stigma into direct contact with
external spaces (Cresti et al., 1986). Pollen tubes enter the
stigma through the gaps between cells, which may be
further enlarged by pressure from pollen tubes (Cresti et al.,
1986). Some pollen tubes probably grew directly into
intercellular spaces of N. alata (this could not be observed),
but six of the 16 tubes observed grew along the surface of
the stigma between papillae (Fig. 2E-G) for distances up to
300 Jim before monitoring ceased. One of the six pollen
tubes left the stigma surface. This tube grew up a short
papilla cell in a relatively non-papillate part of the stigma,
and when it reached the apex of the cell, maintained its
direction of growth (Fig. 2H and I).
To confirm that the patterns of growth we observed
on the flooded stigmas were not an artefact of the experimental conditions, we also examined pollen hydration time
and the pattern of tube growth on intact flowers with the
normal coverage of natural exudate. Pollen hydration times
were assessed by observing the shape of pollen grains
washed from the stigma surface (stigmas were immersed in
mineral oil at intervals after pollination). Hydration was
first observed, but at very low frequency (less than I in 1000
grains), 5 min after pollination, and the frequency of
hydrated grains increased the longer the hydration time
(up to 24 h). The pattern of pollen tube growth was
observed by excising the stigma at intervals after pollination, staining with aniline blue and observing the surface of
the stigma with epifluorescent illumination. Pollen tubes
that had meandered along the surface of the stigma were
readily observed (Fig. 2J).
In summary, pollen on the stigma hydrates in a hydrophobic environment. Emerging pollen tubes are not guided
into the stigma with high precision, but their initial growth
towards the stigma and subsequent growth along the stigma
surface must increase (relative to random growth) the
probability of penetration occurring.
IN VITRO RECONSTRUCTIONS OF THE
STIGMA ENVIRONMENT
Experiments in which aspects of the chemical and physical
environment of the stigma were reproduced in culture were
used to expand upon the role of the exudate and of surfaces
in pollen tube guidance.
The role of the exudate
One conceptual view of solanaceous stigmas is that they
are made up of two phases, a hydrophobic phase (exudate)
and a hydrophilic phase (stigma cells and aqueous drops on
the surface). These phases were reproduced in culture, using
exudate (or other oils) collected from the stigma as the
hydrophobic phase, and pollen tube growth media as the
aqueous phase (Lush et al., 1998; Wolters-Arts, Lush and
Mariani, 1998). Pollen at the boundary between phases and
FIG. 3. Progressive hydration and germination of pollen in an oil
(refined olive oil) with similar properties to the stigma exudate. Pollen
grains at the interface between the oil and an aqueous growth medium
germinate to produce tubes that grow into the aqueous phase. Grains
entirely surrounded by oil hydrate and germinate more slowly, and
produce tubes that grow towards the aqueous medium. The interface
between the oil and aqueous phases represents a small resistance to the
growth of pollen tubes, resulting in grains being displaced slightly
before the resistance is overcome and tubes enter the aqueous phase.
Bar = 50 m. Numbers in the lower right-hand corner are the time
(min) since addition of the aqueous medium to the culture. aq,
Aqueous medium; oil, refined olive oil: pg, pollen grain.
thus in direct contact with the aqueous phase, hydrates
within 2 min and pollen tubes grow into the aqueous phase
(Fig. 3A). Pollen surrounded by oil hydrates and germinates
more rapidly the closer it is to the aqueous phase; tubes
emerge from the aperture (one of three) closest to the
aqueous phase and their angle of emergence is towards the
aqueous phase (Fig. 3B). In addition, tubes usually curve
slowly towards the aqueous phase (Fig. 3B). The growth
direction of emerging pollen tubes towards the source of
water in the two-phase culture system is thus strikingly
similar to pollen tube growth towards the source of water
on the stigma. Perhaps the most interesting feature of
directed growth in two-phase cultures is that it does not
require the presence of any style-specific components.
Several oils are effective substitutes for the exudate, and
defined growth media substitute for the stigma (Lush et al.,
1998; Wolters-Arts et al., 1998).
Lush et al.-Pollen Tube Guidance
We concluded, by a process of elimination, that a
gradient of water as a solute in the oil surrounding pollen
grains is the cue that directs pollen tube growth to water (or
the stigma) (Lush et al., 1998). This hypothesis is feasible in
that the guidance chemical (water) is present in stigmas,
and there must be a water gradient between the source of
water and pollen grains (the more rapid hydration of pollen
grains closer to the aqueous phase establishes this).
Guidance is only proposed to occur over distances of
approx. 75 rm, which is within the maximum guidance
range of chemical cues in other biological systems (Lush,
1999). Finally, the hypothesis may apply more generally-a
directional supply of water could also direct pollen tubes on
dry stigmas. At present we doubt that marked differences in
the relative availability of water on opposite sides of a
pollen grain or tube can occur in aqueous environments,
and thus envisage that water ceases to be a cue when tubes
enter the stigma.
If a concentration gradient of water in oil is the cue
directing pollen tubes to the stigma, then the permeability
of the exudate to water could be critical for effective
guidance. The lower limit would be the permeability below
which pollen grains in oil fail to fully hydrate and
germinate. Consistent with this expectation is the finding
that mineral oil, which has an exceptionally low permeability to water, is only partially effective as a substitute
for the exudate (Lush et al., 1998). The upper limit would
be the permeability above which the supply of water to one
side of the grain or tube was not sufficiently different
relative to supply to the other side, for the gradient to be
perceived. Consistent with this view is the finding that
pollen tube guidance is impaired in tricaprylin (a saturated
triglyceride), which is highly permeable to water (for a
triglyceride). High fidelity guidance is restored when the
permeability of tricaprylin is reduced by dilution with
mineral oil (Lush, unpubl. res.). We thus propose that
solanaceous exudates exercise a passive role in pollen tube
guidance, and that their chemical composition is not critical
provided they have the appropriate physico-chemical
properties and are non-toxic to pollen.
The alternative to the hypothesis that the exudate
indirectly guides pollen tubes through its effects on the
concentration of water, is that specific components of
the exudate actively participate in pollen-pistil signalling.
The signalling hypothesis is based on observations that
specific lipids are required for pollen hydration and
directional growth on both wet and dry stigmas (Preuss,
Yen and Davis, 1993; Wolters-Arts et al., 1998; Lolle and
Pruitt, 1999). Unsaturated triglycerides (fatty acid chains
C18) were the only fully-effective substitutes for the exudate
of solanaceous plants, and long-chain lipids (approx. C30)
are critical for pollen hydration on the dry stigmas of
Arabidopsis. The hypothesis is weakened, however, by the
finding that unsaturated triglycerides can rescue pollen of
Arabidopsis mutants with deficiencies in the synthesis of
long-chain lipids (Wolters-Arts et al., 1998), and subsequent findings that mineral oil and tricaprylin are
partially effective substitutes for the exudate.
43
The role of physical surfaces
We compared the response of pollen tubes to contact
with the stigma with the response of pollen tubes grown
in vitro to contact with glass surfaces and other pollen
tubes. Methods were as described in Lush et al. (1997).
Pollen tubes of N. alata tended to maintain their original
direction of growth when touched at the tip (Fig. 4A-C) or
any other part, indicating that in this environment they are
not thigmotropic. Continued tip growth following contact
with surfaces resulted in the surface being displaced, in the
tip sliding along the surface (Fig. 4D and E), or in the older
parts of the tube being displaced backwards (Fig. 4F-I).
Displacement backwards continued until either the angle of
contact of the tube with the surface changed and the tube
started to slide along the surface, or the polarity of the tip
changed. After these adjustments in the direction of tube
growth, tube tips tracked along straight or concave surfaces
(23 tubes observed). They also followed slightly convex
surfaces (18 tubes observed, Fig. 4K), but left these surfaces
when the angle of curvature increased (eight tubes, Fig. 4L).
Pollen tubes could maintain contact with the stigma or
other surfaces by adhering to them (Jauh et al., 1997), or
because touch initiates a thigmotropic response that ensures
that contact between the tube tip and the stigma surface is
maintained (Iwanami, 1959; Hirouchi and Suda, 1975;
Malh6, 1998). However, the ability of in vitro grown pollen
tubes of N. alata to follow surfaces did not require adhesion
to the surface (Fig. 4E) or a thigmotropic response. Instead,
a strong endogenous polarity of tips, combined with
bending of the older parts of tubes, appeared to be all
that was required for tubes to grow along obstructing
surfaces. Only when bending was restricted did changes in
the trajectory of growth require changes in polarity.
Our current view of guidance on the stigma is as follows.
The initial polarity of the tips emerging from pollen grains
is set by a gradient of water, and results in pollen tubes
encountering the stigma surface soon after their emergence
from the grain. Some tubes, by chance, grow directly
through intercellular spaces and into the stigma. Others
follow the stigma surface in a direction determined by their
angle of contact. Tubes following the surface may eventually penetrate the stigma in a manner similar to that
illustrated in Fig. 4K-M. Here, a pollen tube following the
slightly convex surface of another pollen tube, left that
surface when the angle of curvature increased. However, the
tube's forward growth was limited by contact with another
surface, resulting in the tip being directed into the
'intercellular space' between restricting surfaces.
THE STYLE
In the work just described, a pollen tube entered a parallel
array of cells and its growth was subsequently directed
through the spaces between those cells (Fig. 4K-M). Pollen
tubes in the solid styles of solanaceous plants also grow
through intercellular spaces between parallel files of cells
(transmitting tract cells). Provided that transmitting tract
cells are suitably rigid, guidance through the style may
44
Lush et al.-Pollen Tube Guidance
FIG. 4. Pollen tubes in culture tend to follow surfaces that the tip encounters because older parts of the tube bend. A-C, The tip of a micropipette
is advanced to make contact with the tip of a pollen tube. The polarity of the tip does not change, but the direction of tube growth alters because
the continued growth of the tip results in backwards displacement and bending of older parts of the tube until the tip can slide past the pipette.
D and E, A pollen tube makes contact with a glass surface. The tip slides along the surface in the direction determined by the angle of contact, and
the direction of growth changes because the tube bends. There is little change in the polarity of the tip and no adhesion between the tube and the
glass surface. F-J, The initial contact angle between a tube and a glass surface is approx. 90° . Initially the tube slides to the right, increasing the
curvature of older parts of the tube. Older parts of the tube are also displaced backwards (tube moves out of focus). Ultimately the tube follows
the surface by growing towards the left. This redirection seems to require both bending of older parts of the tube and a change in polarity of the
tip. K-M, Pollen tubes tend to follow other pollen tubes, sometimes resulting in parallel files of pollen tubes. A pollen tube (arrow) follows the
slightly convex surface of another pollen tube until the curvature of the surface increases sharply. At this point the pollen tube leaves the surface
and continues forward growth until it encounters another surface. The tip advances through the space between surfaces. Bar = 50 tm. Numbers
in the lower right-hand corner are time (min) since taking the first frame in the series.
therefore be by default, i.e. growth is only possible within
and along the channels formed by intercellular spaces.
One of the key differences between the hypothesis that
guidance through the style is physical guidance-by-default,
and the hypothesis that chemotropic pollen tubes follow a
chemical gradient, is that the direction of growth is determined only by the direction of growth at the point of entry.
Chemical guidance, by contrast, requires the style to have a
constitutive directionality towards the ovary. Experimentation has failed to reveal any convincing evidence that
constitutive gradients exist within styles (Heslop-Harrison,
1987). Thus, pollen introduced at the middle or base of
both solid and hollow styles produces some tubes that grow
towards the stigma (Buchholz, Doak and Blakeslee, 1932;
Iwanami, 1959; Hecht, 1964; Mulcahy and Mulcahy, 1987).
Furthermore, pollen tubes grow out of cut styles and into
artificial growth media (e.g. Cheung, Wang and Wu, 1995;
Higashiyama et al., 1998), despite the presence of putative
attractants in the style and their absence in the media.
Perhaps the only evidence of constitutive directionality
comes from work in which pollen was applied to the
middle of N. alata styles (Mulcahy and Mulcahy, 1987).
Compatible pollen tubes grew at similar rates towards the
stigma and towards the ovary, unless the stigma was
compatibly pollinated at the same time. When the stigma
was pollinated too, growth of pollen tubes towards the
stigma from mid-style pollinations was 'impeded'. In the
Mulcahys' experiment it was not possible to distinguish
between pollen tubes originating from the stigma and those
that originated from the middle of the style, except by
restricting the experiment to short times so that the pollen
tubes did not meet. We extended the Mulcahys' experiment
by making compatible, mid-style pollinations with pollen
bearing the marker gene Gus, and compatible, stigma
pollinations with wild-type pollen (Fig. 5). In our experiment, small numbers of pollen tubes (typically about ten
tubes) grew up and down the style from mid-style pollinations, even when the stigma was pollinated simultaneously.
There were many pollen tubes in the transmitting tract
midway between the two pollination positions, most of
which were Gus-negative (derived from the stigma) and
some Gus-positive (derived from mid-style pollinations).
Thus pollen tubes can grow in different directions through
the same part of the style.
Lush et al.-Pollen Tube Guidance
FIG. 5.Bidirectional pollen tube growth in the style of N. alata. A small
amount (approx. 500 grains) of compatible, wild-type pollen was
applied to the stigma at the same time as compatible pollen bearing the
Gus marker gene was applied to an incision 15 mm below the stigma.
Intact styles of cut flowers were held at high humidity for 12 h before
the transmitting tract was dissected from sections of the style, stained
for the presence of Gus protein, and counterstained for callose with
aniline blue. The style section (7 mm below the stigma) contained many
fluorescent pollen tubes, some of which were derived from the mid-style
pollination as shown by blue, Gus-positive staining in bright field
illumination of the same section.
A valid criticism of all the experiments on the directionality of styles is that the techniques used are invasive and
may disrupt guidance gradients. The controls in our work
with N. alata (flowers subjected to the same treatment but
in which styles were not harvested), however, set seeds,
showing that no gradients that were critical for reproduction were disrupted. The weight of evidence at present
favours the hypothesis that the transmitting tract is a nondirectional pathway, but the alternative hypothesis, that the
transmitting tract is directional, has not been completely
rejected.
Two chemical guidance models are currently being evaluated (Cheung, Wang and Wu, 1995; Jauh et al., 1997). Of
particular relevance to guidance in solanaceous plants is the
suggestion that a transmitting tract specific protein (TTS
protein) of N. tabacum guides chemotropic pollen tubes
(Cheung et al., 1995). The hypothesis is based upon the
apparent attraction of pollen tubes grown in vitro to a
source of TTS protein. TTS protein, however, is also a
growth stimulant in this assay and the possibility that these
experiments reveal differential growth stimulation rather
than effects on the direction of growth are not ruled out. No
concentration gradient of TTS protein has been demonstrated within styles, but there appears to be a gradient in
the degree of glycosylation of TTS protein, and on this
basis it was proposed that pollen tubes are attracted to the
most highly glycosylated forms which occur at the base of
the style.
The major problem with this and other models of guidance is the length of the style (about 50 mm in N. tabacum).
The maximum guidance range for diffusible chemical
attractants in other systems is about 1mm (reviewed in
Lush, 1999). The maximum distance over which chemical
morphogens influence development is also estimated to be
about I mm (Crick, 1970). Attraction over even 1 mm
requires a 10000-fold difference in concentration at each
45
end of the pathway, but the gradient in glycosylation of
TTS protein from the top to the bottom of the style is only
about four-fold (Wu, Wang and Cheung, 1995).
The role of TTS protein in styles remains unclear. A
homologue from N. alata (GaRSGP) has quite different
biological properties to TTS protein, and is probably
located in the cell wall not the intercellular space (SommerKnudsen et al., 1998). GBGP, another homologue of TTS
protein, is located in the walls of cultured N. tabacum cells
(Takeichi et al., 1998). Concentration gradients in bound
chemicals have a longer guidance range than diffusible
chemicals, but the styles of N. alata are still far too long for
GaRSGP to function as an attractant even if a concentration gradient could be demonstrated. GaRSGP is a
metal-binding protein (Sommer-Knudsen, pers. comm.),
and its function in the style may be associated with this
property.
THE OVARY
The ovary of N. alata has two locules, each containing
about 400 ovules (based on the number of seeds produced),
separated by a central septum. Pollen tubes enter the locules
of N. tabacum by growing along a groove at the top of the
ovary before dispersing over the placental surface (Chandra
Sekhar and Heij, 1995), but details of their passage to
ovules are not known. Whatever the guidance mechanism is
in N. alata, it does not appear to result in an orderly pattern
of ovule fertilization. When pollen bearing the Gus marker
protein reaches the ovary, Gus-staining ovules start to
appear almost randomly over the placental surface, and
some pollen tubes leave the placental surface, by-pass
micropyles and emerge on the external surface of the ovules
where they grow in a disorganized manner (Fig. 6).
Numerical analysis is required to determine whether pollen
tube growth in N. alata ovaries is random or simply
imprecise.
Imprecise guidance in multi-ovule ovaries is not necessarily inconsistent with chemical guidance. Because the
concentration of a diffusible guidance cue originating at the
micropyle of ovules will be, at any particular point, the sum
of the amounts diffusing from all ovules, the steepest
gradients will not necessarily lead to a micropyle (Fig. 7).
The problem of pollen tube guidance in multi-ovule ovaries
is analogous to the problem of yeast cells locating a mating
partner from one of several nearby sources of a diffusible
mating factor. Usually a tip-growing outgrowth called a
'shmoo' grows towards the highest concentration of the
mating factor (Jackson and Hartwell, 1990), but a theoretical analysis suggests that the steepest gradient does not
necessarily lead to another cell (Barkai, Rose and Wingreen, 1998). Yeast cells may deal with this problem by
destroying the cue, effectively decreasing the cue's guidance
range but increasing its focus (Barkai et al., 1998). In
ovaries, an imprecisely focused gradient might decrease the
accuracy of guidance.
In Arabidopsis, contact between pollen tubes and surfaces
within the locule appears to be important for effective
guidance, and non-contact mutants of Arabidopsis are
interpreted as lacking components required for adhesion
Lush et al.-Pollen Tube Guidance
46
the ovary. Physical guidance tends to be associated with
thigmotropism or adhesion, but in fact all that is required
for pollen tubes to follow surfaces is that the pollen tube
should have a strong endogenous polarity, be capable of
bending, and that the surface should not be sharply convex.
The importance of chemotropism remains unclear. It is
possible to construct a conceptual model to account for
almost any pattern of pollen tube growth in terms of
chemical guidance, as we have done above for the stigma
and the ovary, and as has been done by others throughout
the 1900s. Not one of these models is yet proven. To
establish what, if any, role chemical attractants or repellents
play in guidance, requires further critical, and quantitative,
experimentation.
FIG. 6. Pollen tube growth in ovaries of N. alata, harvested 35 d after
pollination of wild-type pistils with compatible pollen bearing the Gus
marker gene. A, Blue ovules (Gus-positive) occur at various positions
across the placental surface. B, Some pollen tubes (arrows) grow to the
external surface of the ovules where they meander in a disorganized
manner.
ACKNOWLEDGEMENTS
We thank P. Gerola, J. Pickett-Heaps, C. O'Brien,
B. McGinness, C. Schultz and the Australian Research
Council for their contributions.
LITERATURE CITED
FIG. 7. Conceptual model of concentration gradients resulting from the
secretion of a diffusible pollen tube attractant from the micropyle of
two ovules. The pollen tube entering the chemical field on the left is
guided along the steepest gradient to a micropyle. The pollen tube
entering in a region of overlap between the two chemical fields is also
guided along the steepest gradient, but this gradient does not lead to a
micropyle. The pollen tube subsequently may be chemically restricted
to the high concentration regions of the chemical field, within which it
will grow randomly and may, by chance, encounter a micropyle.
(Preuss et al., 1993). The ability of pollen tubes to follow
surfaces that they do not adhere to, as discussed earlier and
also shown by Hirouchi and Suda (1975), suggests, however, that non-contact mutants may also occur whenever
the endogenous polarity of pollen tubes is weakened by
internal or external factors.
CONCLUSION
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