Journal of Experimental Botany, Vol. 61, No. 7, pp. 2001–2013, 2010
doi:10.1093/jxb/erq065 Advance Access publication 2 April, 2010
REVIEW PAPER
Pollen–pistil interactions and the endomembrane system
Aruna Kumar and Bruce McClure*
Division of Biochemistry, Interdisciplinary Plant Group, 117 Schweitzer Hall, University of Missouri, Columbia, MO 65211-7310, USA
* To whom correspondence should be addressed: E-mail: [email protected]
Received 17 December 2009; Revised 22 February 2010; Accepted 2 March 2010
Abstract
The endomembrane system offers many potential points where plant mating can be effectively controlled. This
results from two basic features of angiosperm reproduction: the requirement for pollen tubes to pass through
sporophytic tissues to gain access to ovules and the physiology of pollen tube growth that provides it with the
capacity to do so. Rapid pollen tube growth requires extravagant exocytosis and endocytosis activity as cell wall
material is deposited and membrane is recovered from the actively growing tip. Moreover, recent results show that
pollen tubes take up a great deal of material from the pistil extracellular matrix. Regarding the stigma and style as
organs specialized for mate selection focuses attention on their complementary roles in secreting material to
support the growth of compatible pollen tubes and discourage the growth of undesirable pollen. Since these
processes also involve regulated activities of the endomembrane system, the potential for regulating mating by
controlling endomembrane events exists in both pollen and pistil.
Key words: Endomembrane, interspecific incompatibility, self-incompatibility, S-RNase.
Introduction
Plants have unique reproductive challenges, in part, because
they are sessile and cannot directly control pollen flow.
Pollination by a closely related plant may lead to inbreeding
depression on the one hand, while wide crosses between
different species or genera may result in aborted or sterile
progeny on the other. Angiosperms have especially welldeveloped systems to control fertilization between the points
when pollen arrives at the stigma and when fertilization
occurs.
The pre-fertilization phase of angiosperm reproduction
begins with the arrival of pollen on the stigma surface.
Reproduction may be controlled from either the pollen side
or the pistil side or by interactions occurring at distinct
stages. For instance, the stigma may or may not provide the
resources needed for hydration and germination, depending
on the interactions between the pollen coat and the stigma.
In other cases, controls may act after pollen germinates and
as the pollen tube grows through the style. As pollen tubes
grow, they interact with several pistil extracellular matrix
(ECM) components, including lipids, carbohydrates, amino
acids, glycoproteins, and polysaccharides. These interactions occur at the plasma membrane and in the pollen
endomembrane system, where internalized ECM components may interact further with pollen proteins. These
processes then signal to the physiological systems supporting pollen tube growth, and the results of this crosstalk are
manifested as either a promotion of pollen tube growth or
as a barrier to fertilization.
Pollen tube growth
A pollen tube is highly dynamic. Growth rates from 0.03–
0.16 cm h 1 in Nicotiana section Alatae (Lee et al., 2008b) to
1 cm h 1 in maize (Valdivia et al., 2007) are known. Pollen,
the male gametophyte, consists of three cells at maturity. The
vegetative cell elaborates the pollen tube and carries two
sperm cells from the stigma to the egg sac, a distance that is
often mm or cm. Growth occurs only at the tip, and
although the total volume of the pollen tube increases
dramatically, cytoplasmic volume is held nearly constant by
periodic deposition of callose plugs (Taylor and Hepler,
1997). Polarized exocytosis at the pollen tube tip supplies
membrane, proteins, and cell wall material for growth. By
one estimate, the amount of membrane delivered to the tip
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2002 | Kumar and McClure
exceeds that needed for tip extension by 79% (Ketelaar et al.,
2008). Endocytosis recovers excess membrane and may also
provide an important route for the internalization of material
from the pistil and thus contribute to signalling between the
pollen and pistil.
The extreme growth rate and the associated endomembrane traffic of exocytic and endocytic vesicles also require
a dynamic cytoskeleton and transport system. Growing pollen
tubes display a cytoplasmic streaming pattern described as
a bidirectional reverse fountain (Cheung and Wu, 2008):
a clear zone consisting of densely packed vesicles occupies the
pollen tube tip, while cytoplasm moves along the tube cortex
to the subapical region and then returns in the central region.
Using refraction-free high-resolution time-lapse differential
interference contrast microscopy in conjunction with pulsechase labelling with styryl FM dyes, Zonia and Munnik
(2008, 2009) provided spatial and temporal details of vesicle
trafficking pathways in growing pollen tubes. Their interpretation is that endocytosis occurs along the shank and at the
tip, and exocytosis occurs in a subapical region. Exocytosis
and endocytosis are, thus, tightly regulated processes vital for
robust pollen tube growth.
Studies of individual genes as well as broader proteomic
and transcriptomic-level studies demonstrate the importance
of endomembrane system regulation for reproduction and
pollen tube growth (Cheung and Wu, 2008). Furthermore,
proteome and transcriptome studies suggest that vesicle
trafficking components are overrepresented in Arabidopsis
pollen (Grobei et al., 2009; Qin et al., 2009; Wang et al.,
2008). Interestingly, Qin et al. (2009) showed that growth
through the stigma and style elicits a novel pollen tube
transcriptome compared with pollen tubes growing in vitro.
They found ;700 additional genes induced in pollen tubes
that have passed through sporophytic tissue when compared
to in vitro-grown pollen tubes or ungerminated pollen. These
additional genes include those involved in endomembrane
processes, such as those encoding GTP-binding proteins and
calcium-binding proteins, and soluble N-ethylmaleimidesensitive factor-attachment protein (SNAP) receptor proteins.
In what will probably become a classic study, Qin et al. (2009)
provide direct evidence that female tissues influence pollen
tube gene expression at a level not previously appreciated.
These brief examples of pollen tube and cell biology
studies, as well as those of single candidate genes and broader
proteomic and transcriptomic-level studies, all point toward
a central role for the endomembrane system. The Rop
GTPases, Rab GTPases, and the exocyst complex deserve
special attention because of their roles in transport and
targeting of endomembrane vesicles.
Molecular control of endomembrane
function in pollen tubes
Rop GTPases
Rho family GTPases regulate a variety of processes in
eukaryotes (Bishop and Hall, 2000). Unlike the mammalian
Rho family that includes multiple subfamilies, plant Rho-
like-GTPases belong to the single subfamily, Rops (Yang,
2008). Rop GTPases play a pivotal role in establishing the
robust cell polarity needed for pollen tube tip growth by
acting as a hub for the co-ordination of positive and negative
feedback loops from the actin cytoskeleton, Ca2+ levels, and
vesicular trafficking (Yang, 2008; Yalovsky et al., 2008). In
general, GTP hydrolysis is associated with a conformational
change that switches Rops from an active to an inactive
form. They receive inputs from plasma membrane signalling
proteins, and accessory proteins activate GTPase activity,
facilitate guanine nucleotide exchange, and dissociation of
GDP (GAP, GEF, and GDI factors), respectively (Kost,
2008; Lee and Yang, 2008; Yang, 2008).
The role of Rop proteins in pollen tube growth has been
demonstrated through mutagenesis and expression studies.
The results show that overexpression or constitutively active
mutants of pollen-specific Rop1 GTPase causes depolarization of pollen tube growth (Li et al., 1999; Kost et al., 1999).
Such studies also provide evidence that a Rop1-dependent
pathway directly regulates tip-localized Ca2+ influx (Li et al.,
1999) and F-actin dynamics (Gu et al., 2005). Rop proteins
also interact with lipid signalling systems in the control of
pollen tube tip growth. A Rop-associated lipid kinase activity
seems to be responsible for generating the plasma membranesignalling
lipid,
phosphatidylinositol-4,5-bisphosphate
(PIP2), at the pollen tube tip. PIP2 potentially represents an
effector of activated Rac and may directly effect actin
organization or vesicle trafficking (Kost et al., 1999). PIP2 is
also a precursor of other signalling molecules. Phospholipase
C hydrolyses PIP2 to the signalling molecules inositol 1,4,5trisphophate (IP3) and diacylglycerol, a precursor of phosphatidic acid that is required for tip growth (Monteiro et al.,
2005; Helling et al., 2006). PIP2 and IP3 regulate the tipfocused Ca2+ gradient and apical secretion. Inhibition of
phosphatidic acid production also results in dissipation of the
tip-focused [Ca2+]c gradient and inhibits membrane recycling
(Monteiro et al., 2005). Thus, Rop-mediated polarized
growth intersects several downstream pathways that, in turn,
reinforce establishment of polar cell growth.
A number of factors influence the activity of Rop
proteins in pollen tubes. Rop GTPase activation is modulated by two downstream pathways involving apical F-actin
and Ca2+ (Hwang et al., 2005). GEFs, GDIs, and GAPs
play important roles in spatial restriction of activated Racs/
Rops on the apical membrane. RopGEF1 activates Rop1 in
the control of pollen tube growth (Gu et al., 2006). As the
tip grows, activated Rops are displaced to the flanking
region where they are inactivated by GAPs, thereby
restricting their activity to the apex (Klahre and Kost,
2006; Hwang et al., 2008). Once inactivated, GDI actively
extracts Rac/Rop GTPases from the flanking region back to
the apical region where GDP/GTP exchange takes place to
activate them (Klahre et al., 2006). Studies in tomato
provide evidence that Rop regulation is influenced by
pollen–pistil interactions. A tomato GEF interacts with
a plasma membrane receptor-like kinase and potentially
influences pollen tube growth through the Rop system
(Zhang et al., 2008).
Pollen–pistil endomembranes | 2003
Rab GTPase
Rab GTPases are members of the Ras-related superfamily
of small GTPases that are key regulators of vesicle
trafficking, exocytosis, endocytosis, and membrane recycling. In general, they regulate fusion of sequential steps of
vesicle traffic as components of the endomembrane system
move from, for example, the endoplasmic reticulum (ER) to
the Golgi and then to the plasma membrane (Nielsen et al.,
2008; Stenmark, 2009). The Rab GTPase family is the most
complex subfamily of Ras proteins, and the nomenclature is
confusing (Nielsen et al., 2008). Here, the nomenclature
used by authors of the work being discussed are followed
and reclassification is not attempted.
Like other Ras GTPases, Rab GTPases exist in inactive
GDP-bound and active GTP-bound states. Interconversion
is mediated by factors such as GEFs, GDIs, and GAPs.
Several Rab homologues have been identified in different
plant species, indicating a multiplicity of endomembrane
trafficking pathways. Knocking out Rab GTPase genes or
expressing dominant negative or constitutively active
mutants that disturb wild-type protein activity reveals their
functional significance. For example, dominant negative
mutants of NtRab2 GTPase disrupt the transport of pollen
proteins that enter the secretory pathway and suppress
pollen tube elongation (Cheung et al., 2002). Similarly,
Rab11b in pollen tubes regulates vesicle trafficking in
polarized secretion and membrane recycling (de Graaf
et al., 2005). Disruption of AtRabA4d results in bulged
pollen tubes that display a reduced rate of growth in vitro
and altered deposition of cell wall components. The bulged
pollen tube phenotype and interference with polarized tip
growth are similar to that observed in pollen tubes with
overexpressed Rop1 (Li et al., 1999; Szumlanski and
Nielsen, 2009). Rab GTPase (RabA4b)-defined TGN compartments are involved in recruiting phosphoinositide
kinases (PI-4Kb1), which function in the polarized growth
of root hair tips. PI-4Kb1 also interacts with a Ca2+ sensor,
AtCBL1, providing a link between Ca2+ and PIP2 signalling
(Preuss et al., 2006). AtRabA4d also interacts with phosphoinositide kinase (PI-4Kb1) in pollen tubes (Szumlanski
and Nielsen, 2009). Together, these findings provide a set of
examples of regulatory endomembrane proteins that could
be exploited to connect pollen–pistil interactions to pollen
tube physiology.
Exocyst
The exocyst acts as a tethering protein complex that targets
secretory vesicles to specific sites on the plasma membrane.
It functions prior to the docking and fusion events mediated
by SNAREs (Novick et al., 2006; Zarsky et al., 2009). In
yeast, exocyst components interact with Rab and Rho
GTPases to regulate localized exocytosis (Novick et al.,
2006). The exocyst appears to be involved in polarized
exocytosis to the region of the plasma membrane experiencing polarized growth, a phenomenon with obvious parallels
in the pollen tube system. The angiosperm exocyst complex
includes eight subunits (SEC3, SEC5, SEC6, SEC8, SEC10,
SEC15, Exo70, Exo84; Hala et al., 2008), one of which,
SEC3, interacts with a Rop GTPase via the adapter protein
ICR1 (Lavy et al., 2007). Moreover, recent results suggest
that the exocyst is directly involved in pollen–pistil interactions (Samuel et al., 2009).
Pollen–pistil interactions
Pollen–pistil interactions contribute to controlling pollination
during the pre-fertilization stage. In several cases, specific
factors mediating these interactions have been identified. For
example, pollen tube growth is influenced by chemotropic
agents (reviewed by Cheung et al., 2010) as well as a variety
of lipids, ions, proteins, and metabolites produced by the
pistil (Gleeson and Clarke, 1979; Du et al., 1994; Lind et al.,
1994; Wolters-Arts et al., 1998; Park et al., 2000; Wu
et al., 2000; Kim et al., 2003; Palanivelu et al., 2003; Tang
et al., 2004; Juarez-Diaz et al., 2006). The challenge in the
coming years will be to determine how these interactions are
connected to the physiological processes of pollen tube
growth. In many cases, this will probably be a matter of
determining how these interactions signal to endomembrane
system components, such as those just mentioned. Recent
results reviewed in this article bear this out and directly
identify roles for the endomembrane system (Goldraij et al.,
2006; Samuel et al., 2009). Overall, it is now clear that the
endomembrane system provides more than a supporting role
in pollen–pistil interactions: it is actively involved in signalling and in the physiology of pollen rejection.
LePRK activation
An elegant series of experiments show that LePRK2
activation is one instance where a well-defined pollen–pistil
interaction signals directly to proteins involved in the
physiology of pollen tube growth. LePRK2 is a receptor
kinase required for pollen tube growth in tomato (Zhang
et al., 2008). Interestingly, this pollen protein interacts with
both a pollen-specific secreted cysteine-rich protein, LAT52,
as well as with a pistil protein, LeSTIG1 (Tang et al., 2002,
2004). One suggestion is that LeSTIG1 displaces LAT52
upon contact with the stigma and that this switch regulates
pollen tube growth (Tang et al., 2004). Further regulation is
suggested by the observation that LePRK2, which is normally phosphorylated and present in a ; 400 kDa protein
complex with LePRK1, becomes dephosphorylated upon
contact with another style extract component, followed by
dissociation of the LePRK complex (Wengier et al., 2003).
There is evidence that LePRK2 signals to the pollen tube
endomembrane system. The cytoplasmic domain of
LePRK2 interacts with a member of a plant-specific GEF
protein family, known as L. esculentum kinase partner
protein (KPP). Pollen overexpressing KPP shows depolarized tube growth (Kaothien et al., 2005). Moreover,
coexpressing the Arabidopsis homologues, AtRopGEF12
and AtPRK2a, results in ballooned tips, suggesting a link
between Rop-mediated tip growth and pollen receptor
kinases (Zhang and McCormick, 2007). Pollen tubes overexpressing LePRK2 and full-length KPP display widened
2004 | Kumar and McClure
tips (Zhang et al., 2008). Antisense LePRK2 pollen tubes
display abnormally large vacuoles near the tip, suggesting
a role in vacuolar trafficking as well. Together, these results
suggest that LePRK signalling could directly influence
pollen tube growth in the pistil by positively activating the
Rop-mediated polarized tip growth. However, it is yet to be
demonstrated that LePRK recruits both Rop/RacGTPases
and Rop/RacGEFs to a specific plasma membrane domain,
or vice versa. Nevertheless, RopGEFs may be the missing
link between receptor kinases and intracellular signalling.
Self-incompatibility and interspecificincompatibility
Self-incompatibility (SI) systems are relatively well-understood
mechanisms that prevent pollination by self-pollen and pollen
from close relatives. Interspecific pollen is also rejected, but
the mechanisms underlying this system are not as well understood. Some mechanisms have already been shown to
intersect with the pollen tube endomembrane system, and
more connections are likely to be discovered as the mechanisms become better described.
SI provides for genetically controlled recognition and
rejection of self-pollen and pollen from close relatives.
Usually, compatibility is controlled by a single locus, called
the S-locus. Two types of genetic control are recognized:
gametophytic (GSI) and sporophytic (SSI) (de Nettancourt,
1977, 2001). GSI is the more phylogenetically widespread SI
system found in the Solanaceae, Papaveraceae, Ranunculaceae, Rosaceae, Poaceae, Scrophularaceae, Leguminosae,
and Onagraceae families (Igic and Kohn, 2001). In GSI,
compatibility is determined by the haploid genotype of the
male gametophyte; pollen is rejected when its S-haplotype
matches either of the two S-haplotypes present in the diploid
pistil. In SSI, compatibility is determined by the S-haplotypes
of the diploid sporophyte acting as the pollen parent. In
simple SSI systems, rejection occurs when either S-haplotype
of the pollen parent matches either S-haplotype in the
pistilate parent (Rea and Nasrallah, 2008).
At the molecular level, the Brassica S-locus encodes
separate proteins expressed in the stigma and the pollen
that determine pollination specificity (see Rea et al., 2010;
Chapman and Goring, 2010, in this issue). As shown in Fig.
1, S-locus receptor kinase (SRK) proteins are expressed in
the stigma papilla cell and localized in the plasma membrane. S-locus cysteine-rich proteins (SCR, also designated
SP11) (Schopfer et al., 1999; Takayama et al., 2000) are
expressed in anthers and deposited in the pollen coat. SRK
is a transmembrane protein. When the extracellular domain
of SRK binds a cognate SCR protein, an inhibitory
thioredoxin is displaced from the SRK cytosolic domain
and autophosphorylation occurs (Cabrillac et al., 2001;
Kachroo et al., 2001; Takayama et al., 2001).
Endomembrane events that connect SRK–SCR signalling
to the physiology of pollen rejection in the papillar cell are
beginning to be defined. These events include changes in
papillar cell vacuoles, targeting resources to the site of pollen
attachment, and internalization of SRK itself. Important
factors include the armadillo repeat containing (ARC1) E3
ligase (Gu et al., 1998), M-locus protein kinase (MLPK)
(Murase et al., 2004), and Exo70A1 (Samuel et al., 2009).
MLPK is a plasma membrane anchored serine-threonine
kinase that acts with SRK to transduce SI signalling (Murase
et al., 2004; Kakita et al., 2007). ARC1 interacts with SRK in
vitro and is also an SRK substrate (Gu et al., 1998). ARC1 is
a positive regulator of self-incompatibility and proteasomalmediated degradation (Stone et al., 2003).
Recent results focus attention on SRK–SCR signalling
and the endomembrane system. Using ultra-high-voltage
Sporophytic self-incompatibility and the endomembrane
system
Many of the details of the SSI response have been defined in
the Brassicaceae (see Rea et al., 2010; Chapman and
Goring, 2010, in this issue). Species that display SSI
typically have a dry stigma, and the SI response operates at
the level of interaction between a pollen grain and a papillar
cell on the stigma surface. SSI is initiated in stigmatic
papillar cells in response to proteins present in the pollen
coat. It is very rapid and highly localized; a single papillar
cell can respond to two opposite stimuli, accepting crosspollen and rejecting self-pollen grains placed near each
other (Sarker et al., 1988). At the biological level, incompatible pollen often fails to adhere, hydrate, and
germinate. Thus, the response in the papillar cell probably
involves the highly localized release of water and nutrients
that facilitate these early events in the pollen.
Fig. 1. Possible role of papillar cell secretion—SSI in Brassica. A
papillar cell (SaSe) is shown with compatible (SbSc) and incompatible (SaSd) pollen. SRK (S-locus Receptor Kinase) is present in the
papillar cell plasma membrane and SCR (S-locus Cysteine-Rich
protein) are presented in the pollen exine. Localized secretion of
stigmatic resources occurs in compatible crosses, a process that
requires the exocyst and Exo70A1. The incompatible SRKa–SCRa
interaction leads to displacement of an inhibitory thioredoxin (THX)
from SRK, autophosphorylation, activation of ARC1 (Armadillo
Repeat Containing E3 ligase), and subsequent down-regulation of
Exo70A1. This model is based on the work of Samuel et al. (2009).
Pollen–pistil endomembranes | 2005
electron microscopy (HVEM), Iwano et al. (2007) observed
actin dynamics and the three-dimensional structure of the
B. rapa papillar cell vacuolar system after self- and non-self
pollinations. Before pollination, prominent tubular vacuoles
are connected to the central vacuole and also form
branching structures. One hour after cross (i.e. compatible)
pollination, tubular vacuoles coexist with large vacuoles and
are similar to the papilla cells prior to the pollination.
However, a network of elongated and large vacuoles are
directed to the plasma membrane below the cross-pollen
grain attachment site. With incompatible self pollinations, in
contrast, pollen hydration and germination can not be
detected 1 h post-pollination. Ultra-HVEM tomography of
papillar cells following self-pollination revealed a vacuolar
network with a different structure. Few elongated vacuoles
are seen near the plasma membrane, and the apical vacuoles
appear fragmented. These results indicate that changes in
vacuolar structure, rather than maintenance of a pre-existing
structure, in the papilla cell is linked to pollen rejection.
The SRK–SCR interaction also appears to signal to the
exocyst. As mentioned, the SRK–SCR interaction results in
ARC1 protein phosphorylation. ARC1 is known to interact
with Exo70A1 (Samuel et al., 2009), a subunit of the
exocyst complex that functions in regulated or targeted
vesicle trafficking to the plasma membrane in yeast and
animal systems (Novick et al., 2006; Zarsky et al., 2009).
Samuel et al. (2009) propose that Exo70A1 functions in the
polarized delivery of vesicles containing factors that facilitate pollen hydration, germination, and growth (Fig. 1) and
that the self-SRK–SCR interaction interferes by targeting
Exo70A1 for degradation through the action of ARC1.
Results favouring this interpretation include fluorescent
fusion protein studies in an Arabidopsis model showing the
redistribution of ARC1 and AtExo70A1 to punctate
structures where proteasomal degradation could occur.
Furthermore, loss of Exo70A1 in Brassica or Arabidopsis
stigmas results in reduced compatibility, and overexpressing
BnExo70A1 in self-incompatible Brassica partially overcomes SSI (Samuel et al., 2009).
Ivanov and Gaude (2009) suggest that SRK–SCR signalling occurs in specific plasma membrane domains and is
followed by endocytosis of the receptor. They observed
a patchy distribution of SRK in the papillar cell plasma
membrane and proposed that the distribution corresponds
to ‘ready-to-be-activated’ regions in intimate communication with underlying endosomes. This arrangement, they
argue, may prevent the localized activation of SRK from
spreading and may also help explain the highly localized
response in the papillar cell. Upon ligand recognition, SRK
is directed to sorting endosomes where THL1 causes signal
attenuation and is destined for degradation.
Gametophytic self-incompatibility and the
endomembrane system
Two distinct GSI mechanisms have been studied at the
molecular and cellular levels (McClure and Franklin-Tong,
2006). SI in Papaver rhoeas is controlled by interactions
between a small stigmatic protein and a newly identified
pollen receptor (Wheeler et al., 2009). In vitro studies have
elucidated connections between this interaction and the
physiology of pollen tube growth. SI species in Solanaceae,
Scrophulariaceae, and Rosaceae display S-RNase-based SI,
in which pollen rejection is controlled by S-RNase proteins
in the pistil and S-locus F-box proteins (SLF/SFB) in the
pollen (McClure et al., 1989; Huang et al., 1994; Murfett
et al., 1994; Ushijima et al., 2003; Qiao et al., 2004a).
Gametophytic self-incompatibility in Papaver
Physiological studies of SI are more advanced in P. rhoeas than
in any other SI system. In GSI systems, self-incompatibility
factors from the pistil initiate a cell-autonomous pollen response that results in the inhibition of pollen tube growth
when the pollen S-haplotype is matched by the pistil. In
P. rhoeas, a single stigma protein, PrsS, induces the
incompatibility response, and no other pistil-side factors
are required (Franklin-Tong et al., 1988; Foote et al., 1994).
The pollen-side specificity protein, PrpS, has recently been
identified as a low molecular weight membrane protein in
the pollen tube (Wheeler et al., 2009). The ability to
faithfully reconstruct the SI responses in vitro by using
recombinant PrsS (Foote et al., 1994) has enabled detailed
physiological studies. As illustrated in Fig. 2, the incompatible PrsSa–PrpSa interaction causes a very rapid calcium
influx, which, in turn, acts as a second messenger signal to
a number of physiological subsystems contributing to pollen
tube growth (Franklin-Tong et al., 1993). Pollen responds
to incompatible PrsS with a rapid inhibition of growth as
well as a longer term permanent response. The very rapid
responses include the activation of a phospholipase C activity that possibly signals the further release of calcium from
internal and external sources (Franklin-Tong et al., 1996),
Fig. 2. GSI processes in Papaver. An incompatible Sa-pollen tube
expressing PrpSa (Papaver rhoeas pollen S) is shown growing in
the presence of PrsSa (P. rhoeas stigma S) and PrsSb proteins.
The incompatible PrpSa–PrsSa interaction triggers rapid Ca2+
influx. Downstream signalling leads to rapid growth inhibition that
is reversible and longer term irreversible responses that ultimately
lead to PCD (programmed cell death).
2006 | Kumar and McClure
effects on the actin cytoskeleton that would impact vesicle
trafficking (Snowman et al., 2002), and inhibition of
inorganic pyrophosphatase activity (de Graaf et al., 2006).
Morphological changes in the endomembrane system affecting Golgi, mitochondria, and endoplasmic reticulum are
observed within 1 h of pollination (Geitmann et al., 2004).
Long-term responses over a few hours contribute to
programmed cell death (i.e. permanent growth inhibition).
These responses include activation of mitogen-activated
protein kinase (MAPK) activity, mitochondrial cytochrome
c release, activation of caspase activity, and DNA fragmentation (Bosch and Franklin-Tong, 2008). Although much
remains to be learned about Papaver SI, it is clear that the
system rapidly affects intracellular calcium and the actin
cytoskeleton. These responses would be expected to impact
membrane traffic directly to and from the pollen tube tip
that are needed for continued growth.
S-RNase-based gametophytic self-incompatibility
The cytotoxic model is widely accepted as the basis of SRNase-based pollen rejection (McClure, 2009). S-RNases
function as pistil-side specificity determinants and also
directly inhibit growth of incompatible pollen by functioning as cytotoxins that target pollen RNA (McClure et al.,
1989, 1990; Gray et al., 1991; Lee et al., 1994; Murfett et al.,
1994). Several models have been presented to account for
the resistance to S-RNase cytotoxicity in compatible
pollinations (McClure, 2006, 2008; Hua et al., 2008; Zhang
et al., 2009; Chen et al., 2010). In all models, the pollen-side
and pistil-side specificity determinants interact to determine
compatibility. The pollen-side specificity determinants, SLF/
SFB genes, were identified by sequencing regions around SRNase genes in Antirrhinum, Prunus, and Petunia (Lai et al.,
2002; Entani et al., 2003; Ushijima et al., 2003, 2004; Ikeda
et al., 2004; Qiao et al., 2004a, b; Sijacic et al., 2004;
Sonneveld et al., 2005). SLF/SFB genes encode F-box
proteins, best known for their role in ubiquitin-mediated
protein degradation (Vierstra, 2009). Several pollen proteins
are known to form complexes with SLF/SFB, and SCFSLF/
SFB
-like complexes are likely to be important (Hua and
Kao, 2006; Huang et al., 2006; Qiao et al., 2004a). Still, it
remains unclear how the interaction between S-RNase and
SLF/SFB determines compatibility.
One complication is that additional pistil-side factors are
required for S-RNase-based SI in some families. HT-B and
the 120 kDa glycoprotein (120K), are two such factors
(McClure et al., 1999, 2000; O’Brien et al., 2002; Hancock
et al., 2005). HT-B was identified in a differential screen to
identify sequences expressed in SI Nicotiana alata but not in
self-compatible (SC) N. plumbaginifolia (McClure et al.,
1999). Antisense suppression of HT-B protein levels prevents pollen rejection in some systems (McClure et al., 1999;
O’Brien et al., 2002). It is, therefore, significant that HT-B
protein is degraded in compatible pollen tubes in Nicotiana
(Goldraij et al., 2006). 120K and similar arabinogalactan
proteins (AGPs) are abundant in the pistil ECM. 120K
binds to S-RNase in vitro (Cruz-Garcia et al., 2005), and
RNAi experiments suggest that it is required for efficient
pollen rejection (Hancock et al., 2005). Other AGPs,
including NaTTS (N. alata transmitting tract specific)
and NaPELPIII (N. alata pistil extensin-like protein III),
Goldman et al., 1992), also bind to S-RNases (Cruz-Garcia
et al., 2005). Although the exact roles of HT-B and 120K
proteins are not known, both these pistil proteins enter
pollen tubes, and neither is required for S-RNase uptake
(Goldraij et al., 2006). Thus, S-RNase is not capable of
causing pollen rejection without these additional factors,
even when it is inside the pollen tube. These factors must,
therefore, be required for a step in pollen rejection that
occurs late in incompatibility. Further studies of how pistil
proteins move through pollen tubes are needed.
S-RNase uptake
The biology of GSI implies a cell-autonomous effect on
pollen tubes. In Papaver, this is likely to be a result of
a PrsS–PrpS interaction on the plasma membrane that
signals to pollen tube growth processes (Fig. 2). However,
growing evidence suggests that signalling occurs inside the
pollen tube in S-RNase-based SI. There are clear parallels
to other plant signalling systems where processes once
assumed to be localized to the plasma membrane are now
thought to take place in a dynamic plasma membraneendosome system (Geldner and Robatzek, 2008). Luu et al.
(2000) were the first to use immunolocalization to demonstrate non-S-specific uptake of S-RNase in Solanum chacoense. Goldraij et al. (2006) later showed that both SRNase and 120K are taken up by compatible as well as
incompatible pollen tubes. Interestingly, 120K protein
marks the boundary of a vacuolar compartment that
contains internalized S-RNase. S-RNase appears to remain
stably compartmentalized in compatible pollen tubes. The
vacuole breaks down in incompatible pollen tubes, and the
concomitant release of S-RNase could obviously cause
pollen rejection. It is noteworthy that pollen rejection in SRNase-based SI is not a sudden phenomenon. Rather,
pollen tubes probably slow their growth progressively and
eventually cease growth altogether. Further studies of the
time-course of rejection are needed, but it is likely that the
endomembrane system gradually loses its integrity with
progressively more dire consequences.
Further experiments showed that S-RNases are taken up
normally in the absence of factors such as HT-B and 120K.
Since pollen rejection does not occur in these plants, compartmentalization of S-RNase appears to be a sufficient mechanism
to evade its cytotoxicity. However, it is not clear how S-RNase
in the lumen could interact with SLF/SFB. Immunolocalization of SLF/SFB in Antirrhinum showed that the protein is
located in the cytoplasm and near the ER (Wang and Xue,
2005). Perhaps, a portion of the S-RNase that enters the
pollen tube passes to the ER by retrograde transport, much as
cytotoxins like ricin are known to do (McClure, 2006). If this
occurs, the topological problem may disappear because
mechanisms for the transport of proteins from the ER lumen
to the cytoplasm are known (Roberts and Smith, 2004).
Pollen–pistil endomembranes | 2007
Pollen proteins bind pistil ECM factors
The fate of pistil ECM components after endocytic uptake
must be controlled by interactions with pollen proteins. The
three most abundant S-RNase binding proteins in Nicotiana
(NaTTS, NaPELPIII, 120K) are AGPs that share a conserved
cysteine-rich C-terminal domain (CTD) (Cruz-Garcia et al.,
2005). Only 120K is known to be required for SI, but all
three AGPs interact with pollen in some way (Hancock et al.,
2005). The CTD from NaTTS and 120K were used as yeast
two-hybrid baits to identify three interacting pollen proteins:
an S-RNase-Binding Protein (NaSBP1), previously identified
in Petunia and Solanum (Sims and Ordanic, 2001; O’Brien
et al., 2004); a putative cysteine protease; and a pollenspecific C2 domain-containing protein (NaPCCP) (Lee et al.,
2008a). Like the Petunia and Solanum SBP1 proteins,
NaSBP1 has a RING domain identifying it with E3 ubiquitin
ligases. SBP1 was first identified in P. hybrida as an S-RNasebinding protein (Sims and Ordanic, 2001), and more recent
studies in P. inflata revealed interactions with PiSLF, SRNases, PiCul1-G, and an E2 conjugating enzyme (Hua and
Kao, 2006). However, SBP1 proteins are expressed in all
organs tested and interact with proteins not involved in
pollination (Ben-Naim et al., 2006). Thus, SBP1 is more
likely to fulfil a function carried on in all cell types than
a function unique to pollination. The function of the cysteine
protease interaction with AGP-CTD, if any, is unknown.
NaPCCP has features consistent with a role in intracellular trafficking of pistil proteins in the pollen tube endomembrane system. NaPCCP has a C2 domain, a modular and
lipid-binding domain found in proteins that function in
vesicular transport, GTPase regulation, lipid modification,
protein phosphorylation, and ubiquitinylation. NaPCCP
interacts specifically with phosphatidylinositol-3-phosphate
(PI3P) in a Ca2+-independent manner (Lee et al., 2009).
PI3P has roles in the assembly of protein complexes needed
for merging, sorting, and recycling of endocytic vesicles
(Czech, 2003). A separate NaPCCP domain allows for interaction with the CTD of NaTTS and 120K (Lee et al.,
2009). Thus, NaPCCP is bifunctional; it binds to components of the pistil extracellular matrix and to PI3P, a component of the pollen tube endomembrane system. It could,
therefore, function in sorting pistil ECM components inside
pollen tubes.
Biochemical, immunolocalization, and live imaging studies confirm that NaPCCP is associated with the pollen tube
endomembrane system. About half the NaPCCP in pollen
tube extracts pellets with the membrane fraction, while half
the protein remains with a soluble marker (Lee et al., 2009).
NaPCCP::FLAG and NaPCCP::GFP constructs were
expressed in pollen tubes for immunolocalization and liveimaging studies. The results showed the association of
NaPCCP with the plasma membrane and pollen tube
endomembrane system, which is consistent with a role in
endocytosis (Lee et al., 2009). FM4-64 labelling experiments
showed that NaPCCP vesicles include plasma membranederived material. However, little or no overlap was seen
with the anti-RabF2a, a marker that labels endosomes
delivered to the vacuole through the multivesicular body
pathway. Although it has not been possible to determine
whether NaPCCP and 120K are co-localized in pollen
tubes, the available data are consistent with a model in
which NaPCCP is somehow involved in the intracellular
transport of pistil ECM proteins. Since NaPCCP does not
appear to be on the pathway directed to the vacuole, it may
be involved in transport to another pathway, such as
retrograde transport to the Golgi and ER.
What is the basis for compatibility in
S-RNase-based SI?
There are currently two broad models to explain S-RNasebased GSI. The role of S-RNases as cytotoxic molecules is
clear, and, by definition, the S-RNase-SLF/SFB interaction
determines the specificity of pollination. Still, how these
phenomena are connected is yet to be determined. Identification of SLF, which is an F-box protein, as the pollen Slocus factor was a critical discovery. Since F-box proteins
often function in ubiquitin-mediated protein degradation,
one model proposes that non-self-S-RNase is degraded by
action of an SCFSLF/SFB complex and the 26S proteasome
(Zhang et al., 2009). There is evidence that SCF-like
complexes do indeed form, and variations of the S-RNasedegradation model have been proposed (Hua et al., 2008;
Zhang et al., 2009). This is a plausible mechanism for pollen
tubes to acquire resistance to S-RNase in a compatible
pollination. S-RNase degradation models propose that selfS-RNase is stable and that its expressed cytotoxicity causes
pollen tube growth inhibition in incompatible pollinations.
Although S-RNase degradation models are appealing, they
do not easily accommodate or explain observations of large
amounts of S-RNase inside compatible pollen tubes or the
requirement for pistil factors such as HT-B and 120K.
Observations of S-RNase uptake suggest that compartmentalization of S-RNase in the endomembrane system
is important in SI, but much remains to be discovered.
Figure 3 illustrates some of the processes that are likely to be
involved. Since S-RNase has been observed in pollen
vacuoles, it is possible that uptake occurs by endocytosis,
perhaps in the subapical region described by Zonia et al.
(2008). The S-RNase-binding AGP 120K also ends up in
pollen vacuoles, and it is possible that uptake occurs in the
form of S-RNase-AGP complexes (Cruz-Garcia et al., 2003).
The bifunctional NaPCCP may assist sorting of these
proteins by binding to both PI3P and the AGP CTD. The
fact that S-RNase accumulates in pollen tube vacuoles in
compatible pollen tubes, as well as in otherwise incompatible
pollen tubes when HT-B and 120K are absent, suggests that
transport to the vacuole represents a default pathway. One
speculation is that, in the absence of these factors, S-RNaseSLF/SFB recognition does not occur and pollen tubes are
resistant to S-RNase cytotoxicity because the S-RNase does
not have access to the pollen tube cytoplasm. A further
speculation is that another transport pathway exists that
takes S-RNase to the ER. While there is, as yet, no direct
evidence for this in SI, other cytotoxins gain access to the
2008 | Kumar and McClure
Fig. 3. Possible roles of the endomembrane system in S-RNase (S-locus ribonuclease)-based SI. An incompatible SLFa (S-locus F-box
protein) expressing pollen tube is shown growing in the presence of Sa- and Sb-RNases. S-RNases are taken up into the pollen tube
endomembrane system along with other pistil proteins such as 120K and HT-B. A major pathway sends S-RNases to the pollen tube
vacuole. A hypothetical alternative pathway sends S-RNase to the ER by retrograde transport, possibly with the involvement of NaPCCP
(Nicotiana alata pollen-specific C2 domain-containing protein). In a compatible interaction, symbolized for convenience by an SLFa-nonself-S-RNase interaction, HT-B protein is degraded, S-RNase remains compartmentalized, and pollen tube growth proceeds normally.
Stable compartmentalization of S-RNase and degradation of HT-B are viewed as default phenomena and do not depend on a non-selfS-RNase-SLF interaction. An incompatible interaction ultimately leads to release of S-RNase, pollen RNA degradation and the inhibition
of pollen tube growth.
cytoplasm by this route (Roberts and Smith, 2004). Moreover, SLF has been observed in association with the ER. By
whatever mechanism, some S-RNase must escape the lumen
and interact with SLF/SFB to initiate downstream events,
such as HT-B degradation or the degradation of pollen RNA
in compatible or incompatible pollinations, respectively.
Interspecific pollen rejection
Interspecific and intergeneric pollen rejection systems are
not as well understood as SI, but they must also feed into
physiological processes necessary for pollen tubes to penetrate to the ovary—such as those that inhibit pollen
adhesion, germination, or pollen tube growth. For example,
although ultrastructural studies have revealed differences
between SI and interspecific pollen rejection, similarities
such as tip swelling and altered callose deposition are
known to be common (de Nettancourt et al., 1974; Covey
et al., unpublished data).
Interspecific pollen rejection is related to SI in some cases,
while in other cases the responses are clearly distinct.
Interspecific crosses are frequently governed by the SI3SC
rule—where the SI species rejects pollen from SC relatives,
but the reciprocal cross is compatible (de Nettancourt,
1977)—also known as unilateral incompatibility (UI). There
is genetic evidence that the S-locus contributes to UI in the
tomato clade on both the pollen and pistil sides (Chetelat
and DeVerna, 1991; Bernacchi and Tanksley, 1997). Plant
transformation studies have directly demonstrated that SRNase from N. alata causes rejection of both SC N.
plumbaginifolia and SC N. tabacum pollen; however, SC N.
tabacum pollen is also sensitive to an S-RNase independent
rejection pathway (Murfett et al., 1996). Clear cases also
exist of S-RNase-independent UI in the tomato clade,
where SC accessions of otherwise SI species that do not
express S-RNase nevertheless reject pollen from SC cultivated tomato (Covey et al., unpublished data).
Further studies of interspecific pollen rejection are needed
to determine how these systems signal to the physiological
processes of pollen tube growth. It is possible that the
endomembrane system will be intimately involved. In one early
ultrastructural study, de Nettancourt et al. (1974) highlighted
alterations of the pollen tube endomembrane system in
interspecific pollen rejection. It will be interesting to determine
whether there is a role for compartmentalization in evading
S-RNase cytotoxicity in interspecific systems, as pollen from
some SC species completely lack an S-locus and, therefore,
SLF/SFB proteins cannot play a role. Until recently, it has
not been possible to observe endomembrane system dynamics
in interspecific crosses directly. However, advances in fluorescent protein imaging and confocal microscopy should make
this feasible. Many endomembrane compartments have been
marked with GFP and its fluorescent derivatives (Cheung and
Wu, 2008). In principle, these markers should allow imaging
of the endomembrane system in live pollen tubes growing in
the pistil. Figure 4 shows how this can be applied to
interspecific pollination. In this example, SC N. tabacum
pollen expressing the vacuolar marker d-TIP::GFP and SC N.
plumbaginifolia pollen expressing both d-TIP::GFP and
a cytosolic red fluorescent protein (tdTomato, coloured bluein Fig. 4) are growing side-by-side in an N. tabacum pistil. In
this example, both pollen from species are compatible, and
the vacuoles are normal in appearance. The figure illustrates
how multi-colour live imaging can be used to observe pollen
tube processes directly in the pistil, the most biologically
relevant context for such observations. Such live imaging
studies will add considerably to our understanding of
endomembrane system dynamics during pollination.
Conclusion
There is growing appreciation for the role the endomembrane system plays in pollen–pistil interactions. It has long
Pollen–pistil endomembranes | 2009
tracellular transport. Overall, physiological studies of pollen
tube growth and biochemistry and molecular biology studies
of pollen–pistil signalling are converging on the endomembrane system.
Acknowledgements
We thank Melody Kroll for editorial assistance and
preparation of figures. Professor Felipe Cruz-Garcia, Department of Biochemistry, National Autonomous University of Mexico, assisted with Fig. 4. The authors are
supported by funding from the US National Science
Foundation (IOB 09614962 and DBI 0605200).
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