Pistil Factors Controlling Pollination
Ana Maria Sanchez,1 Maurice Bosch,2 Marc Bots, Jeroen Nieuwland,3 Richard Feron, and Celestina Mariani4
Department of Experimental Botany, Graduate School of Experimental Plant Sciences, Faculty of Science,
University of Nijmegen, 6525 ED Nijmegen, The Netherlands
INTRODUCTION
The successful establishment of angiosperms on land is in part
determined by their floral design. Because plants cannot move to
find the ideal mate, they have developed a great variety of flowers
to provide different mechanisms of pollen release, pollen
transfer, and deposition of the pollen from the male to the female
sexual organs, the anther and the pistil, respectively. Pollination
ensures the maintenance of the species, but it is also a means to
increase genetic diversity and, with it, the potential to adapt to
new environments. The position and morphology of the anthers
and the pistil have often coevolved with the mode of pollen
dispersal and pollen receipt, aided either by wind or by animals.
Nevertheless, pollination can fail at various points during these
processes, causing the extinction of rare plants and lower crop
yields (Wilcock and Neiland, 2002).
The pistil, the pollen-accepting organ that occupies the central
position in a flower, is composed of one or more fused carpels
that bear the ovules. Pistil development initiates with the formation
of the carpel primordia, and the floral identity genes of class
C, such as AGAMOUS in Arabidopsis thaliana and PLENA in
Antirrhinum majus, dictate carpel identity (Ng and Yanofsky,
2000). Carpel fusion occurs very early in pistil development. Even
in species with single pistils, fusion of the carpel margins is required
to form a closed carpel. For instance, tobacco (Nicotiana
tabacum) and Arabidopsis pistils are made from the fusion of two
carpels, and that of the cultivated tomato is formed from five
fused carpels (Gasser and Robinson, 1993). Close to the time
of ovary closure, the carpel walls extend vertically to form one
or more hollow cylinders, the styles. This process requires cell
division at first and cell elongation later. The length, number, and
structure of the styles are typical within each species and
variable between them. Stylar extension facilitates pollen
capture, and the wide variety of pistil morphologies reflects the
different pollination mechanisms found among the angiosperms
(Barrett et al., 2000). While the style is elongating, the inner
tissues differentiate to form the specialized secretory zone of the
stigma on the top of the style and the transmitting tissue within
the hollow cylinder of the style. In some species, such as lily, the
style remains hollow, with only one layer of secretory tissue lining
the inner surface of the cylinder (Dickinson et al., 1982).
At flower maturity, when pollination takes place, the pistil is
fully developed and composed of stigma, style, and ovary.
Whether the pollen is transported by the wind or by animal
pollinators, after landing on the stigma the pollen grain hydrates
and germinates a tube. This tube then penetrates the specialized
tissues of the pistil, growing into the stigma and the style to reach
the ovules in the ovary. During this process, numerous cell–cell
interaction events occur between the cells of the sporophyte (the
pistil) and the male gametophyte (the pollen grain and the tube).
Most of the existing knowledge regarding pollen–pistil interaction
was gathered from the study of self-incompatibility in several
species. Nevertheless, considerable data are now available on
the prepollination and postpollination events and molecules that
make the pistil ready to interact with the pollen after compatible
pollinations (Lord and Russell, 2002). Some of these interactions
are discussed in this review.
POLLEN–STIGMA INTERACTIONS
Stigma receptivity to pollen can persist from 1 h to several days in
different species (Heslop-Harrison, 2000) and is influenced by
several factors. Whether it is a dry stigma or a wet stigma,
receptivity is defined as the ability to ‘‘capture’’ pollen by
adhesion, to let it hydrate and consequently germinate a pollen
tube. The appropriate stage of stigma development is crucial for
receptivity. For example, on immature stigmas of pear flowers,
mature pollen can adhere but do not hydrate and germinate.
On a degenerating stigma, pollen can adhere, hydrate, and
germinate, but pollen tube growth arrests abruptly (Sanzol et al.,
2003). A cuticle of varying thickness is deposited on most
stigmas above the epidermis, and compatible pollen deposited
on a heavily cutinized surface never germinates (HeslopHarrison,
2000). At the receptive stage, the cuticle breaks at
some places, aided either by visiting insects or by increasing
turgidity of the stigma, and the activity of some enzymes such as
esterases increases in the stigma of several species (Dafni and
Maues, 1998). The pollen of Brassica napus was shown to carry
an active cutinase to break the cuticle of the papilla (Hiscock
et al., 1994; Edlund et al., 2004).
The epidermis of the stigma differentiates to form the specialized
papilla cells. Crucifers, such as Brassica species and
Arabidopsis, have a dry stigma with many large papillae that
interact directly with the pollen. Here, the pollen is accepted if
1 Current address: Laboratory of Plant Genetic Engineering, Instituto de
Technologia Quı´mica e Biologia-Instituto de Biologia Experimental e
Technologia, Quinta do Marqueˆ s, Aptd. 12, 2781-901 Oeiras, Portugal.
2 Current address: Biology Department, University of Massachusetts,
611 North Pleasant St. - Morrill South, Amherst, MA 01003-9297.
3 Current address: Institute of Biotechnology, University of Cambridge,
Tennis Court Road, Cambridge, CB2 1QT, UK.
4 To whom correspondence should be addressed. E-mail mariani@sci.
kun.nl.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.017806.
The Plant Cell, Vol. 16, S98–S106, Supplement 2004, www.plantcell.org ª 2004 American Society of Plant Biologists
compatible, or rejected if incompatible or from a foreign species,
and adheres and hydrates. The best-characterized pollen–pistil
interaction on a dry stigma is the self-incompatibility response
in Brassica. Its components constitute the male determinant
S-locus cystein rich protein, the S-locus glycoprotein female
determinant secreted by the stigma into the cell wall, the S-locus
receptor kinase located in the stigmatic plasma membrane, and
its target arm-repeat-containing protein 1, also produced in the
stigma (Nasrallah, 2000).
However, little is known about the genes required for successful
(compatible) pollen–stigma interaction in crucifers. No
mutants have been identified to date with defects in papillar cell
functions, although cell ablation experiments clearly demonstrated
that papillae are required for pollination. Papillar cell
ablation, in combination with differential display, also was used
to identify genes expressed in the Brassica napus epidermis
(Kang and Nasrallah, 2001). The results of this experiment led to
the identification of the PIS63 gene, which is homologous with an
Arabidopsis gene of unknown function. By a transgenic approach,
it was shown that a reduction of PIS63 expression in the
stigma correlates with reduced pollen germination and seed set,
but pollen adhesion was not affected (Kang and Nasrallah, 2001).
This finding, together with the identification of Arabidopsis pollen
mutants affected in adhesion (Edlund et al., 2004), supports the
idea that pollen adhesion on a dry stigma is a property of
the pollen. The adhesion of pollen on wet stigma is facilitated by
the presence of the exudate, which can be aqueous, as in lily,
or lipidic, as in tobacco and petunia. In addition, proteins and
sugars are present in both types of exudate.
Pollen hydration and tube germination can occur very fast or
may take up to 1 h, depending on the degree of pollen desiccation
at the time of anther dehiscence. Grass pollen, for example,
is never fully dehydrated and is metabolically active when shed
from the anthers. This makes it very vulnerable, but it germinates
within minutes after landing on a stigma. By contrast, pollen of lily
is very dehydrated when released, which enables it to survive
under extreme environmental conditions, but it takes 1 h to
germinate after hydration on the appropriate stigma (HeslopHarrison,
2000). Hydration on the dry stigmas of the crucifers
requires contact with the papillae and is aided by the pollen coat
that flows from the exine to form a contact zone between the two
(Elleman et al., 1992). In particular, lipids and oleosin-like proteins
on the pollen coat were shown to be essential for hydration
in Arabidopsis (Edlund et al., 2004), demonstrating that, as in
pollen adhesion, hydration also is determined by the pollen. We
found no oleosin-like proteins on the coat of tobacco pollen,
suggesting that on this wet stigma hydration may be facilitated
by other factors (Bots and Mariani, 2004).
In tobacco and other solanaceous species, the lipidic exudate
is produced in the cells of the secretory zone of the stigma and is
secreted at pistil maturity. Underneath the exudate, a thin layer of
water (in the form of crystals) surrounds the cells of the secretory
zone. After pollination, pollen grains sink through the exudate
and establish direct contact with the stigma or with each other.
Using cryo-scanning electron microscopy, it was evident that at
the contact site between pollen and stigma, the water crystals
gradually disappeared (Wolters-Arts et al., 2002). We produced
transgenic tobacco plants in which the secretory zone of the
stigma was ablated by tissue-specific expression of a Barnase
ribonuclease. Pollen–pistil interaction is arrested in this plant,
resulting in female sterility (Goldman et al., 1994). In particular,
this stigmaless pistil does not produce exudate, and cryoscanning
electron microscopy images confirmed that no water
crystals were present around the dead cells of the ablated stigma
(Wolters-Arts et al., 2002). Yet, upon application of exogenous
tobacco exudate or a mixture of triacylglycerides, pollen still
could hydrate. It is not clear how the water passes through the
dead cells of the stigmaless pistil to the pollen grain. However,
contact alone is not sufficient for hydration, because even the
exertion of a constant mechanical pressure on the pollen grains
did not lead to hydration without exudate or lipids (Wolters-Arts
et al., 2002). Recently, many water channel proteins, collectively
named aquaporins, were described in plants; these indicate the
existence of a very rapid and regulated water transport across
biological membranes (Johanson et al., 2001). Some of these
aquaporins or aquaporin-like genes are expressed in the pistil,
among other plant tissues, of Solanum chacoense (O’Brien et al.,
2002) and Brassica (Marin-Olivier et al., 2000; Dixit et al., 2001).
Although it is attractive to speculate that they play a role in pollen
hydration by the stigma, 35 putative aquaporin genes, classified
on the basis of sequence identity, were found in the genome
of Arabidopsis. With such high redundancy, it may be difficult to
prove the function of aquaporins in the pollen–stigma interaction.
We have produced transgenic tobacco plants in which a class of
PIP2 aquaporins, expressed in flower organs, was silenced by
RNA interference. We found no effects on pollen hydration,
pollen tube growth, and seed set (M. Bots and C. Mariani,
unpublished data).
Once pollen is hydrated and germination occurs, tube growth
is directed within the stigma. In dry stigmas, tube growth occurs
through the papilla cell wall, and pollen tube penetration is
accompanied by cell wall expansion or loosening in the stigma
(Elleman et al., 1992). Tube growth seems to be facilitated by
wall-degrading enzymes produced either by the pollen itself or
by the stigma. This is in agreement with the finding that group I
allergens of grass pollen have expansin activity (Cosgrove et al.,
1997) and that a polygalacturonase was localized in Brassica
germinating pollen (Dearnaley and Daggard, 2001, and references
therein). In tobacco, a species with a wet stigma and a solid
style, pollen tubes grow through the intercellular spaces between
the cells of the secretory zone, within the exudate produced by
these cells. Interestingly, we recently discovered that tobacco
exudate has cell wall–loosening activity (J. Nieuwland, J.
Derksen, C. Hilbers, and C. Mariani, unpublished results). The
pistil pollen allergen-like (PPAL) protein, one of the proteins
secreted in the exudate, is highly similar to b-expansins (Pezzotti
et al., 2002) and obviously was the most suitable candidate for
a cell wall–loosening activity that could facilitate pollen tube
growth in tobacco stigmas. However, we found that PPAL is not
active in cell wall loosening in an in vitro assay using an
expansometer constructed according to Cosgrove (1989).
Moreover, we have determined that another protein is responsible
for cell wall–loosening activity. This protein is a lipid
transfer protein (LTP), the most abundant protein in the exudate
of tobacco (J. Nieuwland J. Derksen, C. Hilbers, and C. Mariani,
unpublished results). LTPs can bind acyl lipids in vitro (Kader,
Pistil Factors Controlling Pollination S99
1996) and constitute the largest group in the catalog of
Arabidopsis genes involved in acyl lipid metabolism (Beisson
et al., 2003). LTPs represent a group of proteins whose
sequences are highly divergent and that may have different
functions in vivo apart from binding lipids. Indeed, another
function for LTP(-like) proteins is that of the SCA protein (stigma/
stylar Cys-rich adhesin) secreted in the aqueous lily exudate
(Lord, 2003). Lily has a hollow stigma and a style with a secretory
epidermis that pollen tubes travel in to the ovary. Therefore, no
tissue penetration is required for pollen tubes to grow from the
stigma into the style. SCA, combined with another small stigma
protein, chemocyanin, induces chemotropic activity on lily pollen
tubes grown in vitro (S. Kim, J.C. Mollet, J. Dong, K. Zhang, S.Y.
Park, and E.M. Lord, unpublished data). Pollen tubes reorient
their growth toward a gradient of chemocyanin, and SCA potentiates
this activity.
Using our stigmaless transgenic tobacco plants (Goldman
et al., 1994), we studied pollen–pistil interaction in this system,
hoping to dissect the components required. The ablated surface
of this stigmaless pistil is dry, and when tobacco pollen is used
for pollination, it fails to hydrate. If humidity is provided, pollen
hydrates but only a few short pollen tubes germinate. This finding
indicates that the defect is only on the stigma side and not in the
pollen. The addition of germination medium containing boron,
calcium, and sugar improved tube germination, but penetration
of the pistil tissues failed. Surprisingly, when we applied exudate
collected from wild-type pistils, pollen tubes germinated and
grew directly in the pistil tissues (Goldman et al., 1994). Currently,
we are investigating the function of the proteins secreted in the
exudate, some of which (LTP and PPAL) have been discussed
above.
One question that arose from the discovery that exudate is
sufficient for pollen tube growth in the style was whether
exudates secreted on the stigmas of other species could
produce a similar effect (Wolters-Arts et al., 1998). Figure 1
shows that exudate of petunia is as good as that of tobacco
in restoring pollen tube penetration, whereas that of lily only
enables pollen hydration and germination. By comparing the
types of exudate and by performing other experiments, we
concluded that lipids might be important for directional pollen
tube growth. One particular class of unsaturated triacylglycerides,
applied on the ablated stigmas of transgenic tobacco, was
revealed to be sufficient for this to occur (Figure 1) (Wolters-Arts
et al., 1998). This conclusion excludes the possibility that the
proteins of the exudate have a direct and decisive function in the
pollen–pistil interaction, at least in tobacco. The cell wall–
loosening activity of the tobacco LTP, for instance, may turn
out to have only a fine-tuning activity on pollen tube growth in
vivo, perhaps to speed up pollen tube growth or to make the
pollen tube wall more permeable to larger pistil compounds. It
also is possible that some functions may be accomplished in
different ways and are redundant in the stigma, to ensure that
pollen tube growth is carried on to deliver the sperm cells to the
embryo sac.
Another function attributed to nonspecific LTPs and to
other proteins secreted in the exudate is that of defense
against pathogens (Garcia-Olmedo et al., 1995). Proteinase
inhibitors (Miller et al., 2000), thaumatin-like proteins, and other
Figure 1. Fluorescence Micrographs of Longitudinal Sections of Stigmaless Pistils at 24 h after Pollination.
(A) After application of petunia exudate, pollen grains on the stigmaless surface become hydrated and germinate and the pollen tubes penetrate the
stylar tissue.
(B) to (D) Effects of the application of lily exudate (B), trilinolein (C), and saturated triacylglycerides (D) are shown.
pg, pollen grain; pt, pollen tube; tt, transmitting tissue; vb, vascular bundle. Bars ¼ 100 mm.
S100 The Plant Cell
defense-related proteins (Kuboyama, 1998, and references
therein) were shown to be expressed and to accumulate in the
stigmas of species of the Solanaceae, possibly to prevent
predator or pathogen attack. However, these functions are not
related directly to pollination.
One conclusion that can be drawn regarding pollen–stigma
interaction is that although pollination on dry stigmas and wet
stigmas appears very different, there are similarities in the two
systems. For example, lipids are required in crucifers and in
solanaceous species for pollen hydration and tube penetration,
and proteins such as expansins have been suggested to aid
pollen tube growth in both plant families. The difference lies only
in where these factors accumulate: the pollen coat in species
with dry stigmas (crucifers) and the exudate in species with wet
stigmas (Solanaceae).
POLLEN TUBE GROWTH IN THE STYLE AND THE OVARY
Based on morphological features, styles can be classified as
open, closed, or semisolid. In open styles, characteristic of many
monocotyledons, such as lily, pollen tubes grow in the canal filled
with mucilage, along the transmitting tract epidermis. Most
dicotyledons, such as Solanaceae species, have a closed style in
which the pollen tubes grow through the transmitting tissue,
a continuum with the stigma secretory zone. The cells of the
transmitting tissue are connected in a file by plasmodesmata on
their transverse walls, whereas their longitudinal walls are separated
by the intercellular matrix (IM) that they secrete. Chemical
analysis has shown that the IM contains free sugars, polysaccharides,
free amino acids, proteins, glycoproteins, proteoglycans,
and phenolic compounds, usually thought to function in
pollen tube nutrition, recognition, and guidance (Cheung, 1996).
In this matrix, in general, pollen tubes grow with considerable
speed. Tobacco pollen tubes, for instance, take 26 to 30 h to
reach the ovary, at a distance of 4 cm from the stigma. This
makes the speed of pollen tube growth 1.7 mm/h. Pollen of
many species can germinate and grow a tube in vitro in a medium
that should contain at least calcium, boron, and an osmoticum.
However, pollen tubes grown in vitro never reach the length they
can attain when growing in the pistil (Lord, 2003). Nevertheless,
the ease with which tube growth can be followed in vitro has been
very valuable in the study of pollen tube cell biology (Hepler et al.,
2001).
The discrepancy between in vitro and in vivo growth indicates
a strong contribution of the sporophytic tissue of the pistil to
pollen tube growth. A depletion in the amount of reserves within
the transmitting tissue indicates that tube growth occurs at the
expense of the stylar reserves (Herrero and Hormaza, 1996). The
use of exogenous sugars by growing pollen tubes has been
demonstrated for many species, and glycosylated proteins such
as Hyp-rich glycoproteins (HRGPs) are ideal candidates to
provide nutritional support to pollen tubes during growth. HRGPs
are abundant in the pistils of several species, and many of them
are arabinogalactan proteins (AGPs). Up to 90% of their mass
can be contributed by carbohydrates linked by O-glycosylation
to the Hyp and Ser residues of the protein backbone. AGPs in the
pistil have been proposed to act as glue or lubricants and may
guide pollen tubes to the ovary (Cheung and Wu, 1999).
The best-characterized HRGPs that accumulate in the style
are the tobacco transmitting tissue–specific (TTS) glycoproteins.
The mature TTS proteins span a molecular mass range
from 45 to 105 kD in which the backbone peptides are 28 kD,
and the carbohydrate moieties consist mostly of galactose. TTS
glycoproteins do react with Yariv reagent, an AGP binding
b-glucosyl, but are classified as ‘‘nonclassic’’ AGPs (Cheung
and Wu, 1999). A series of elegant experiments has shown
several functions of TTS glycoproteins. They promote pollen
tube elongation in vitro and in vivo (Cheung et al., 1995), they are
deglycosylated by hydrolyzing enzymes, suggesting that they
provide nutrients to pollen tubes (Wu et al., 1995), and they
attract pollen tubes grown in a semi-in vivo culture system
(Cheung et al., 1995). The carbohydrate moiety associated with
the TTS protein is arranged to form a gradient of glycosylation,
increasing toward the bottom end of the style. This gradient may
have a chemotropic effect on growing pollen tubes (Wu et al.,
1995). Recently, the Nicotiana alata homolog of tobacco TTS,
NaTTS, also was shown to stimulate pollen tube growth in vitro
and to attract pollen tubes in the semi-in vivo culture system (Wu
et al., 2000).
Other HRGPs accumulate in the styles of N. alata and tobacco.
They are very similar to TTS in the primary structure of their
backbone but differ in the carbohydrate composition, which can
explain the differences found in pollen tube growth assays. The
galactose-rich style glycoprotein (GaRSGP) and the 120-kD
glycoproteins both were isolated from N. alata (Lind et al., 1994;
Sommer-Knudsen et al., 1996), and pistil-specific extensin-like
protein (PELP) was isolated from tobacco (Bosch et al., 2001).
These proteins do not form a glycosylation gradient, nor are they
deglycosylated after pollination, and they do not attract pollen
tubes in a semi-in vivo assay. Their localization pattern also is
different from that of TTS. Whereas TTS is bound tightly to the IM,
GaRSGP is localized to the cell wall of the transmitting tissue
cells, and the 120-kD glycoprotein is in the IM but translocated
through the pollen tube wall into the tube cytoplasm after
pollination (Lind et al., 1996). Finally, PELPIII accumulates in the
IM and also is translocated in the wall of the pollen tube;
eventually, it ends up in the callose plugs, but it never enters the
tube cell cytoplasm (de Graaf et al., 2003) (Figure 2). It is
noteworthy that such large molecules can get across the pollen
tube wall and in one case across the membrane. Probably the
same or similar translocation mechanism, although not understood,
is used to incorporate the large S-RNase determinant
of the gametophytic self-incompatibility (SI) (Luu et al., 2000; Kao
and Tsukamoto, 2004). This also is in agreement with the finding
that in vitro S-RNases can form protein complexes with TTS and
NaPELPIII glycoproteins (Cruz-Garcia et al., 2003). However, the
function of the three HRGPs reported above is still unknown,
although it is possible that they all contribute to creating an
environment rich in nutrients and lubricants and physically
favorable for pollen tube growth. The transition from tube growth
in the stigma to growth in the style is characterized by a reduction
of the space in which the tubes grow. Competition between
pollen tubes increases during this transition, and only the
fittest tubes reach the ovary. Therefore, it is important that
the pistil provides an optimal environment to support pollen
tube growth.
Pistil Factors Controlling Pollination S101
Besides nutrition, it is proposed that the style provides guidance
cues to the pollen tubes (Lord, 2003). An opposite theory,
however, says that chemical cues guide the tube only on the
stigma and in the ovary, but that growth in the style is constrained
by physical structures (Lush et al., 2000). Strong evidence for
pollen tube guidance in the style, in addition to the chemotropic
response to the glycosylation gradient of TTS, comes from the
contact-stimulated guidance of the extracellular matrix in lily
styles (Lord, 2003). SCA, an LTP-like protein, was isolated from
the style of lily in combination with another stylar molecule, later
identified as a low-esterified pectic polysaccharide (Mollet et al.,
2000; Park et al., 2000). The SCA/pectin combination was
identified using a functional adhesion assay in which an artificial
stylar matrix was made by immobilizing crude stylar extract on
a nitrocellulose membrane (Jauh et al., 1997). When germinated,
lily pollen were incubated with the artificial matrix in ‘‘in vitro’’
germination medium and pollen tubes adhered and grew on the
matrix as they do on the cells of the transmitting tract epidermis.
Recently, it was demonstrated that a recombinant SCA protein
produced in Escherichia coli is as functional in the adhesion
assay as the native SCA (Park and Lord, 2003). The SCA and
pectin bind each other and provide an adhesive matrix that acts
as a contact-stimulated, or haptotactic, guidance mechanism for
pollen tubes. It has been demonstrated that no chemotropic
gradients exist in the lily style (Iwanami, 1959). When pollen is
applied to an open style, pollen tubes grow toward both the
stigma and the ovary.
A different mechanism of pollen tube guidance was discovered
recently. In this case, a gradient of g-aminobutyric acid
(GABA) was detected in the Arabidopsis pistil (Palanivelu et al.,
2003; Edlund et al., 2004). This important discovery was made
while studying the function of the pollen pistil 2 (POP2) gene of
Arabidopsis, which was shown to encode a GABA transaminase.
This finding also was in agreement with the finding that wild-type
unpollinated pistils accumulate low levels of GABA in the stigma,
an increasing amount in the style, and even more in the ovary
locule, providing a gradient of GABA to the growing pollen tubes.
In the pop2 mutant, GABA is not degraded and accumulates at
much higher levels than in the wild type, but it is distributed more
equally through the whole pistil. Pollen tubes of the pop2 mutant
fail to grow in the mutant pistil, perhaps because they cannot
degrade the excess GABA. By contrast, wild-type pollen tubes,
which carry the functional allele of POP2, can grow in the
presence of a higher concentration of GABA, creating a small
gradient around the tip of the tubes. Further studies with pollen
tube growth in vitro have shown that wild-type pollen tubes were
stimulated to grow in the presence of 1 to 10 mM GABA, but
higher levels were inhibitory (Palanivelu et al., 2003; Edlund et al.,
2004). However, pollen tubes do not respond chemotropically to
a GABA gradient in vitro, so other molecules must be involved in
this system.
By far, the best signal transduction mechanism described in
the pollen–pistil interaction is the sporophytic SI response in
Brassica. However, new, very interesting signaling systems
are emerging in which stylar molecules play a crucial role.
McCormick and co-workers have demonstrated that the two
tomato pollen-specific receptor-like kinases LePRK1 and
LePRK2 coimmunoprecipitate from pollen and yeast when
Figure 2. Localization of PELPIII in Style Sections Containing Pollen Tubes after 16 or 24 h of Pollination.
PELPIII is distributed mainly over the pollen tube callose walls (cw), whereas the detection of PELPIII in the intercellular matrix (IM) is lost. This PELPIII
distribution in the tube walls was found in transverse (A) and longitudinal (B) sections of pollinated style parts. cp, callose plug; pt, pollen tube; TT,
transmitting tissue.
S102 The Plant Cell
expressed together. The 400-kD complex persists in pollen after
pollen tube germination in vitro, but it dissociates if stylar extract
is added to the germination medium. Dissociation of the complex
is caused by the dephosphorylation of LePRK2. A model for
signal transduction in vivo would predict that a small stylar
ligand (the dissociation factor resides in a 3- to 10-kD protein
fraction) binds to LePRK2 and dephosphorylates it, and the
complex dissociates in the pollen tube (Wengier et al., 2003;
McCormick, 2004). The significance of this signal route in pollen
tube growth is still unknown.
Upon arrival at the placenta, the path for the growing pollen
tube is much less defined. In some cases, pollen tubes wander
around on the placenta surface covered by secretion produced
and released by specialized structures, such as the obturators
(Herrero, 2001). These structures appear as little bumps along
the placenta, facing the ovule entrance, and seem to control
pollen tube growth at that point. In some species, pollen tubes
arrest if the obturator is not active in the production of secretion
(Herrero, 2001). The final attraction of the pollen tube to the ovule
was shown recently to depend largely on the female gametophyte.
A discrete number of Arabidopsis mutants defective in
embryo sac development or function also were found to be
unable to guide, attract, and accept pollen tubes (Hulskamp
et al., 1995; Ray et al., 1997; Shimizu and Okada, 2000; Huck
et al., 2003; Skinner et al., 2004). Furthermore, an elegant study
using laser cell ablation on ovules of Torenia has definitively
shown that the cue for pollen attraction is a diffusible signal
emitted by the synergid cells surrounding the egg cells
(Higashiyama et al., 2001; Skinner et al., 2004).
Together, these data leave no doubt that the pistil produces
molecules for nutrition and cues for pollen tube growth.
However, many of these molecules seem to be redundant and
present in the pistil before pollination occurs, implying that the
pistil is preset to support pollen tube growth. This notion also is
supported by the fact that, to date, only a few genes have been
found to be induced strictly by pollination, such as 1-aminocyclopropane-1-carboxylic
acid (ACC) synthase (ACS) in orchids
(Bui and O’Neill, 1998), although there is enough evidence for
gene expression modulated by this event. Many nonprotein
factors also are produced in the style and were implicated in
pollen tube growth. However, these often are isolated examples
in one particular species, which makes it difficult to discuss
them all.
INTERSPECIES POLLEN REJECTION
To this point, only events occurring in self-pollinated species
have been considered here. In nature, however, many different
types of pollen are present simultaneously and may be transported
by wind or insect on a pistil that is not of the same species.
To prevent the ‘‘wrong’’ cross, many plants have developed
barriers that operate in the pistil either before fertilization, inhibiting
pollen tube growth, or after fertilization, causing abortion
of the illegitimate embryo. The pistil, therefore, plays an important
role in the prevention of gene flow between species
and in the maintenance of the species. In a 2001 publication,
de Nettancourt extensively examines the two mechanisms of
intraspecies and interspecies barriers. Lewis and Crowe (1958)
studied the compatibility relationship between different selfincompatible
(SI) and self-compatible (SC) species of several
angiosperm families. They observed that most of the crosses
were compatible only in one direction, when the SC species
was used as the female parent in the cross. By contrast, if
the SI species was used as the female parent, the SI pistil would
reject the SC pollen. These results gave origin to the SI 3 SC rule,
and interspecies incompatibility was named ‘‘unilateral incompatibility’’
(UI).
To date, several examples of UI governed by the SI locus have
been described, and the elegant work of Murfett et al. (1996)
demonstrated the implication of S-RNases as barriers in some
interspecies crosses within the genus Nicotiana. Furthermore, by
genetic mapping of a quantitative trait locus associated with UI in
Lycopersicon, Bernacchi and Tanksley (1997) showed that the SI
locus was a major determinant in UI, although two additional
quantitative trait loci were involved in enhancing UI. However,
there are exceptions to the SI 3 SC rule (de Nettancourt, 2001).
For example, interspecies barriers also act in crosses involving
two SC species. Liedl et al. (1996) showed that in a cross of
Lycopersicon species, the timing, location, and physiology of
pollen tube arrest were distinct from those in self-incompatibility.
The barriers in interspecies crosses between SC species
are mostly referred to as ‘‘incongruity’’ (Hogenboom, 1975),
indicating the incompleteness of a relationship caused by
evolutionary divergence of two species. Incongruity is an event
determined by many genes unrelated to the SI locus. An interesting
study of incongruity was performed by Kuboyama et al.
(1994). They used the SC tobacco as the female parent in distinct
crosses with three other SC species, N. repanda, N. rustica, and
N. trygonofilla. Pollen tubes of these latter species arrested in
three different places in the mature pistils of tobacco. Figure 3
shows pollen tubes of N. rustica arrested halfway in the style of
tobacco at 20 h after pollination. Pollen tubes also curl and
wind and in some cases bend back toward the stigma (A.M.
Sanchez and C. Mariani, unpublished results). In the reciprocal
cross, using N. rustica as the female parent, tobacco pollen
tubes easily reach the ovary. Interestingly, when the three types
of incongruous pollinations were performed on immature
tobacco pistils, all pollen tubes reached the ovary (Kuboyama
et al., 1994). This finding suggests that, if the arresting factor is
determined by the pistil, it is most likely produced during pistil
maturation. It could be argued that the arrest of pollen tubes is
caused by the longer distance they have to grow in the tobacco
pistil compared with their own pistil (i.e., 3.6-cm tobacco style
versus 1-cm N. rustica style). We used N. rustica pollen on the
pistil of N. sylvestris and the tubes grew 3.2 cm, which is
approximately the length of the tobacco style (A.M. Sanchez and
C. Mariani, unpublished results). This finding indicates that N.
rustica pollen tubes are capable of growing longer than the length
of their own styles. Other physiological factors have been ruled
out (Sanchez, 2001), but the mechanism of pollen tube arrest in
these incongruous crosses is not yet understood. In some cases,
incongruous pollen tubes grow down to the ovary and even
fertilize the embryo sac. This is the case when petunia pollen is
used to pollinate a tobacco pistil. In this case, not only are seeds
produced, although they abort, but also proteins of the tobacco
Pistil Factors Controlling Pollination S103
transmitting tissue are translocated in the growing incongruous
pollen tube, indicating an intimate pollen–pistil interaction typical
of congruous pollinations (B. de Graaf, B. Knuiman, E. Pierson,
G. van de Weerden, and C. Mariani, unpublished results).
Using a molecular approach, we set out to compare transcript
profiles after congruous and incongruous pollinations on immature
and mature tobacco pistils. Neither differential display
reverse transcriptase–mediated PCR nor cDNA amplified fragment
length polymorphism revealed transcripts specifically
expressed in incongruous pollinations. Putative differentially
expressed transcripts appeared to correlate with the developmental
stage of the pistil and with the nature of the pollen used
(A.M. Sanchez and R. Feron, unpublished results). If new
transcripts are not detectable or are not made in incongruous
pollinations, it still can be the case that well-characterized genes
expressed in unpollinated and pollinated pistils play a role in
incongruity. In most species, pollination is accompanied by an
increase of ethylene production in the pistil and by the
expression of genes that encode enzymes necessary for the
hormone biosynthesis (O’Neill, 1997; Bui and O’Neill, 1998).
Therefore, we followed the expression of ACS and ACC oxidase
(ACO) after congruous and incongruous pollinations in tobacco.
Their expression indeed correlated very well with the growth of
different pollen tube types. The level of ACS expression was
higher in self-pollinated tobacco pistils and lowest in the same
pistils when pollinated with N. maritima, whose pollen tubes
arrest around the stigma. Similar results were obtained for the
expression of ACO (Sanchez and Mariani, 2002). In conclusion,
the expression of these genes in the pistil of tobacco is
modulated differently depending on the type of pollen used
and possibly on the length the respective pollen tubes achieve.
Because of the paucity of experiments performed on incongruity,
it is difficult to conclude which factors other than the SI locus
contribute to the arrest of foreign pollen tubes in a pistil.
CONCLUDING REMARKS
There is no doubt that the pistil plays a key role in plant
reproduction and that it has multiple functions: it can accept or
reject the proper pollen, it sustains pollen tube growth, and it
produces and protects the female gametophyte in the ovary. The
pistil is essential for flowering plant reproduction, even when
asexual mechanisms of reproduction are available in some cases
(Bicknell and Koltunow, 2004). The pistil must maintain the right
balance between two important functions: on the one hand, it is
a guarantee that pollen carries the sperm cells to the embryo sac,
but on the other hand, it should select the fittest pollen tube to
ensure the production of vigorous progeny. Therefore, it should
not be surprising that many checkpoints exist in pollen tube
growth in the pistil that are regulated by finely tuned mechanisms.
Examples of these mechanisms are the formation of
gradients in the style to attract pollen tubes, such as the gradient
of glycosylation of TTS and the gradient of GABA, and the
production of adhesion molecules that may function in a similar
manner. Also, the redundancy of some proteins, such as HRGPs,
probably is a safeguard to provide a good growth environment
for pollen tubes. The fact that many genes are expressed in the
pistil at some level before pollination takes place also suggests
that the pistil is poised to accept pollen (or reject it if it is
incompatible or incongruous) when pollination commences.
Figure 3. N. rustica and Tobacco Are Incongruous.
(A) and (B) N. rustica pollen grains germinate normally on the tobacco stigma (A), and initial pollen tube growth is normal (B).
(C) and (D) At half the length of the tobacco style, N. rustica pollen tubes curl and wind, and no further growth is observed.
(E) Sometimes pollen tubes can be seen growing backward.
(F) Division of the N. rustica generative cells inside the tobacco style takes place at 6 h after pollination.
(G) Tobacco pollen tubes arrive easily at the ovary of N. rustica.
In (A) and (B), bars ¼ 250 mm; in (C) to (G), bars ¼ 25 mm.
S104 The Plant Cell
Although this may seem an anthropomorphic view of pollination,
the pistil surely guarantees the continuation of plant life.
Received September 26, 2003; accepted December 26, 2003.
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