Download Peptide signalling during the pollen tube journey and double

Journal of Experimental Botany, Vol. 66, No. 17 pp. 5139–5150, 2015
doi:10.1093/jxb/erv275 Advance Access publication 11 June 2015
REVIEW PAPER
Peptide signalling during the pollen tube journey and double
fertilization
Li-Jia Qu1,*, Ling Li1, Zijun Lan1 and Thomas Dresselhaus2,*
1 State Key Laboratory for Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences at College of Life Sciences,
Peking University, Beijing 100871, China
2 Cell Biology and Plant Biochemistry, Biochemie-Zentrum Regensburg, University of Regensburg, 93053 Regensburg, Germany
* To whom correspondence should be addressed. E-mail: [email protected] or [email protected]
Received 5 April 2015; Revised 10 May 2015; Accepted 11 May 2015
Editor: Ruediger Simon
Abstract
Flowering seed plants (angiosperms) have evolved unique ways to protect their gametes from pathogen attack and
from drying out. The female gametes (egg and central cell) are deeply embedded in the maternal tissues of the ovule
inside the ovary, while the male gametes (sperm cells) are enclosed in the vegetative pollen tube cell. After germination of the pollen tube at the surface of papilla cells of the stigma the two immobile sperm cells are transported deep
inside the sporophytic maternal tissues to be released inside the ovule for double fertilization. Angiosperms have
evolved a number of hurdles along the pollen tube journey to prevent inbreeding and fertilization by alien sperm cells,
and to maximize reproductive success. These pre-zygotic hybridization barriers require intensive communication
between the male and female reproductive cells and the necessity to distinguish self from non-self interaction partners. General molecules such as nitric oxide (NO) or gamma-aminobutyric acid (GABA) therefore appear to play only
a minor role in these species-specific communication events. The past 20 years have shown that highly polymorphic
peptides play a leading role in all communication steps along the pollen tube pathway and fertilization. Here we review
our current understanding of the role of peptides during reproduction with a focus on peptide signalling during selfincompatibility, pollen tube growth and guidance as well as sperm reception and gamete activation.
Key words: Cysteine rich peptide (CRP), fertilization, pollen tube, receptor-like kinase (RLK), reproductive isolation,
self-incompatibility.
Introduction
Angiosperms are characterized by their unique mode of
sexual reproduction including a double fertilization process,
in which two sperm cells fuse with the egg and central cell,
respectively, to form both embryo and endosperm respectively (Raghavan, 2003). The immobile sperm cells need to
be delivered as cargo toward the deeply embedded female
gametophyte with the help of a pollen tube, representing the
fastest growing plant cell. The entire process consists of a
number of successive steps initiated after pollen deposition
on the stigma, and its adhesion, hydration and germination
to produce the pollen tube. Elongation at the tip is typical
for polar pollen tube growth. Pollen tubes first penetrate the
intercellular space at the stigma and style, and then pave their
way along the nutritious extracellular matrix of transmitting
tract tissues towards the ovary. Transmitting tract tissues
differ significantly between species and may consist of specialized cell files associated with the vascular tissue in plants
like Arabidopsis (Crawford and Yanofsky, 2008) and maize
(Lausser et al., 2010) or hollow walls in plants like the lily
(Lord, 2003).
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
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5140 | Qu et al.
After perceiving signals from the ovule, pollen tubes exit
the transmitting tract, grow on the surface of the placenta
and migrate up the funiculus towards the micropylar opening
of the ovule, where they enter the embryo sac (for review see
Higashiyama and Takeuchi, 2015). This pathway is typical
for many angiosperms including the model plant Arabidopsis.
Due to anatomical differences and the presence of a single
ovule per ovary, this stage of the pollen tube journey from
the transmitting tracts towards the micropyle was named as
ovarial cavity growth in the grasses (Lausser and Dresselhaus,
2010) and likely depends on mechanical rather than on chemotactic cues. Finally, after arrival at the egg apparatus (consisting of the egg cell and accessory synergid cells) the pollen
tube decelerates its speed, grows around the synergid cells
(Leshem et al. 2013) and ultimately bursts into the receptive
synergid cell thereby releasing its cargo for double fertilization. Cell fusions (plasmogamy) take place after both sperm
cells arrive at the junction between egg and central cell where
they are activated (Sprunck et al. 2014). Subsequently the
fertilized egg cell (zygote) develops into an embryo and the
fertilized central cell forms the endosperm, the two major
components of angiosperm seeds (Weterings and Russell,
2004).
In order to achieve and maximize their reproductive success,
flowering plants have evolved complicated signalling mechanisms to assure and regulate every step during the pollen tube
journey and subsequent double fertilization (Hamamura
et al., 2012; Beale and Johnson, 2013; Dresselhaus and
Franklin-Tong, 2013; Bleckmann et al., 2014). Recent studies have revealed that peptide signalling plays a leading role
during short-range signalling along the whole pollen tube
pathway, the regulated release of its cargo and for gamete
activation.
In general, plant peptides can be categorized into two
classes: secreted peptides and non-secreted peptides. Secreted
peptides can be further divided into two major classes:
cysteine-rich peptides (CRPs) and non-CRPs (NCRPs).
There are 432 NCRP genes annotated in the Arabidopsis
genome including post-translationally modified small peptides and some proteins, which are not yet classified (Huang
et al., 2015). Post-translationally modified peptides are generated by proteolytic processing from larger polypeptide precursors with an N-terminal signal peptide. Mature peptides
of this NCRP category are generally less than 20 amino acid
residues in length, typically consist of approximate 10 amino
acid residues, and contain at least one post-translational
modification such as tyrosine sulfation, proline hydroxylation, or hydroxyproline arabinosylation (Matsubayashi, 2014;
Tabata and Sawa, 2014). Most reported post-translationally
modified peptides are involved in plant development and
defence, such as CLEs (CLV3/ESR) (Kiyohara and Sawa,
2012), systemin (Green and Ryan, 1972) and phytosulfokines
(PSK) (Matsubayashi and Sakagami, 1996; see review by
Sauter, 2015). CRPs are significantly larger peptides. They
are derived from precursors that are generally less than 170
amino acid residues in length, and contain 4–16 cysteine
residues (Marshall et al., 2011; Huang et al., 2015). A large
number of CRP-coding genes have been computationally
predicted in plants: 756 were identified, for example, in the
Arabidopsis genome and 598 in the rice genome (Silverstein
et al., 2007; Huang et al., 2015). High-throughput RNA
sequencing (RNA-seq) data has recently shown that 205
CRP-coding genes are expressed in the female gametophyte,
accounting for about 53% of genes specifically expressed or
highly up-regulated in these reproductive cells. Most of the
largest CRP family genes encode defensin-like (DEFL) peptides, lipid-transfer proteins (LTP) and ECA1 (Early Culture
Abundant1) gametogenesis-associated peptides, which have
been reported to be expressed in flower organs before and
after fertilization, and thus likely play roles in reproduction
(Silverstein et al., 2005; Steffen et al., 2007; Murphy et al.,
2012; Tesfaye et al., 2013; review by Gutierrez-Marcos and
Ingram, 2015). The ECA1 subfamily was recently subdivided in EC1 genes, which are predominately expressed in
the egg cell and ECR (EC1-Related) genes, of which many
are expressed in the synergid cells of Arabidopsis (Sprunck
et al., 2014). This specific expression pattern points towards
cell-specific function(s) and the requirement of high peptide
amounts for these yet unknown function(s).
Peptides serving as signalling molecules are usually released
through the classical secretory pathway (Krause et al., 2013).
After secretion they diffuse to neighbouring cells over short
distances (a few cell files) to bind cognate receptors, but longdistance signalling from the root to the shoot has also been
reported (Tabata et al., 2014). A concentration gradient is
generated from the cell where signal molecules are released
to the distance, which adds to the signal sensitivity (Busch
and Benfey, 2010). The affinity of their binding to the cognate receptors is presumably high, as the working concentration is usually in the nM range or even below (Matsubayashi
et al., 2001). After receptor activation, peptide-receptor complexes are often internalized to the cytosol through endocytosis, where the receptor undergoes degradation or recycling
(Geldner and Robatzek, 2008).
In the various male-female interactions during plant reproduction, small peptides secreted either from pollen grains
or tubes (male gametophyte), from embryo sac cells (female
gametophyte) or the sporophytic maternal tissues of the
pistil play critical roles, for example, in self-incompatibility
responses, pollen tube growth support and guidance, pollen
tube burst and gamete activation (Fig. 1). The role of peptides in these reproductive processes will be outlined in more
detail below. An overview about the signalling peptides discussed below is shown in Table 1.
Roles of plant peptides in self-incompatibility
responses
Recognition of self-pollen, pollen from the same or alien species occurs after pollen grains have been deposited at papilla
cells of the stigma. Self-incompatibility (SI) responses are
thereafter activated in many plant species to prevent inbreeding by pollen of the same species. This pre-fertilization reproduction barrier promotes outcrossing and thus avoids the
negative effects associated with inbreeding depression (Losdat
Peptide signalling along the pollen tube journey | 5141
A
Pollen
Peptides
Receptors
SCR/SP11
SRK
PrsS
PrpS
LAT52
LePRKs
Stigma
Style
Transmitting tract
Ovary
Ovule
Embryo sac
Self-incompatibility
response
STIG1
LePRKs
CLE45
SKM1 & SKM2
SCA/LTP5
—
Plantacyanin
—
Chemocyanin
—
—
—
Ovular pollen tube guidance
ZmEA1
—
LUREs
LIP1 & LIP2*
Micropylar pollen tube
guidance
Pollen tube germination
and tip growth
Funiculus
B
Pollen tube
Sperm cells
Synergid cell
Egg cell
Funiculus
Integuments
Central cell
Antipodal cell
Peptides
Receptors
RALFs**
FER/SRN
—
ANX1 & ANX2
ZmES1-4
KZM1***
PMEI1
PME***
EC1
—
Pollen tube reception
Gamete activation
Fig. 1. Schematic representation of secreted peptides (see also Table 1) and their receptors involved in pre-zygotic communication of reproductive cells.
(A) Pollen-pistil interactions in self-incompatibility responses, pollen tube growth and guidance. Please note that LIP1 and LIP2 (one asterisk) are not
direct receptors, but are proposed to represent part of the LURE receptor complex. (B) Enlarged image of an ovule shortly before sperm cell release is
shown [see also highlighted frame in (A)]. The role of peptides and receptors is detailed in the text. The interaction of RALFs (two asterisks) with the FER/
SRN receptor has been shown with root expressed peptides, but not yet with pollen tube expressed RALFs. Please note that peptide targets including
ion channels such as KZM1 and enzymes such as PME (indicated by three asterisks) are not receptors in the ‘classical’ sense.
et al., 2014). In self-incompatibility responses selective inhibition of germination or growth of self-pollen grains (or tubes)
is initiated to prevent self-fertilization and thus contributes
to maintaining the gene pool and genetic variation in plants
(Kitashiba and Nasrallah, 2014).
Self-incompatibility responses are controlled by the multiallelic S-locus in many plant species (Takayama and Isogai,
2005). The S-locus genes determining self-incompatibility in
the Brassicaceae, for example, consist of those encoding the
polymorphic and haplotype-specific cysteine rich protein/Slocus protein 11 (SCR/SP11) and the S-locus receptor kinase
(SRK) (Stein et al., 1991; Schopfer et al., 1999; Takayama
et al., 2000). Stigmas of SRK gain-of-function transformants
reject pollen grains expressing SCR/SP11 of the same haplotypes (Takasaki et al., 2000; Silva et al., 2001). Moreover,
transfer of the SRK and SCR/SP11 genes from one S-locus
of self-incompatible Arabidopsis lyrata to self-fertile A. thaliana is sufficient to cause self-incompatibility in this species
(Nasrallah et al., 2002).
SCR/SP11 is a defensin-like CRP with eight conserved
cysteine residues, consisting of about 50 amino acids after
signal peptide cleavage (Takayama et al., 2000; Table 1). In
situ hybridization analyses showed that SCR/SP11 is mainly
expressed in the sporophytic tapetum cell layer, and, in some
species, in the microspores. During pollen maturation, the
peptide is released to the anther locule where it accumulates
on the pollen coats. Upon pollination, SCR/SP11 diffuses
to the papilla cells, where it binds the receptor SRK at the
plasma membrane (Iwano et al., 2003).
SRK is a receptor-like-kinase (RLK) expressed in the
stigma papilla cells prior to flower opening (anthesis). It
forms a homodimer constitutively, which binds SCR/SP11
with high efficiency (Takayama et al., 2001). Nuclear magnetic resonance (NMR) analysis showed that SCR/SP11 folds
into a α/β-sandwich structure containing three anti-parallel
β-sheets and one α-helix, which are connected by loop regions.
The conserved cysteine residues form four disulfide bonds,
which stabilize the peptide structure. The binding-specificity
of SCR/SP11 to its receptor SRK is mainly determined by the
two loop regions. However, the overall binding capacity additionally depends on the whole conformation of the peptide
(Chookajorn et al., 2004). As mentioned above the S-locus
genes harbour high polymorphisms as sequence similarity
between two individuals (haplotypes) is usually less than
5142 | Qu et al.
Table 1. Peptide ligands involved in signalling processes during pollen germination, tube growth and double fertilization
Peptide name
Peptide family
Length (aa)*
Genus
Function
Reference
LAT52
KTI/Ole e I** (CRP)
144
Tomato
Muschietti et al., 1994
STIG1
STIG1 (CRP)
120
Tomato
Pollen hydration and
PT*** growth
Promotes PT growth
STIG1
STIG1 (CRP)
123
SCR/SP11
SCA
DEFL (CRP)
LTP (CRP)
50–77
91
Tobacco/
Petunia
Brassica
Lily
Secretion of pistil
exudates
Pollen SI determinant
Stylar PT adhesion
LTP5
LTP (CRP)
93
Arabidopsis
PT growth
PrsS
S1
Poppy
Chemocyanin
Plantacyanin
CLE45
Phytocyanin
Phytocyanin
CLV3/ESR
96
96
12
Lily
Arabidopsis
Arabidopsis
EA1
EA1-like
49
Maize
LUREs
DEFL (CRP)
62–70
Torenia
Sporophytic SI
determinant
PT reorientation
PT guidance?
Promotes PT growth
at high temperature
PT guidance and
growth arrest
PT attraction
LUREs
RALFs
DEFL (CRP)
RALF (CRP)
71–75
~50
Arabidopsis
Arabidopsis
PT attraction
H+/Ca2+ homeostasis
ES1-4
DEFL (CRP)
Maize
PT burst
PMEI1
EC1
PMEI (CRP)
ECA1 (CRP)
now EC1
Maize
Arabidopsis
PT burst
Sperm cell activation
120
61
~150–160
101–131
Tang et al., 2004;
Huang et al., 2014
Goldman et al., 1994;
Verhoeven et al., 2005
Schopfer et al., 1999
Park et al., 2000;
Mollet et al., 2000
Chae et al., 2010;
Chae and Lord, 2011
Foote et al., 1994
Kim et al., 2003
Dong et al., 2005
Endo et al., 2013
Márton et al., 2005;
Márton et al., 2012
Okuda et al., 2009;
Kanaoka et al., 2011
Takeuchi and Higashiyama, 2012
Haruta et al., 2014****
Murphy and Smet, 2014
Amien et al., 2010;
Woriedh et al., 2015
Woriedh et al., 2013
Sprunck et al., 2012;
Sprunck et al., 2014
* Length of the predicted mature peptides in amino acids (aa); ** KTI/Ole e I, homology to Kunitz-type trypsin inhibitors/Ole e I pollen allergens;
*** PT, pollen tube; **** RALFs are strongly expressed in pollen tubes and bind to the FER/SRN receptor. A signalling role during reproduction
remains to be demonstrated.
50% (Schopfer et al., 1999). Variation is especially high in the
loop 1 region of SCR/SP11, the hypervariable (HV) domain,
which further supports the hypothesis that these sites serve as
decisive regions of a certain haplotype (Mishima et al., 2003).
The S-allele-specific interaction between SCR/SP11 and
SRK triggers SI responses leading to pollen rejection. SRK
undergoes auto-phosphorylation after ligand-receptor binding (Cabrillac et al., 2001; Takayama et al., 2001). Then the
activated kinase domain recruits and works together with an
M-locus protein kinase (MLPK), a receptor-like cytoplasmic
kinase, to phosphorylate Armadillo Repeat-Containing protein 1 (ARC1) (Murase et al., 2004; Kakita et al., 2007). ARC1
acts as an E3 ligase and mediates the ubiquitination and subsequent degradation of stigma membrane protein Exo70A1,
which disturbs pollen grain hydration, and ultimately blocks
successful pollen tube germination (Stone et al., 2003; Samuel
et al., 2009).
The SI response may also occur later during pollen tube
growth inside the transmitting tract. While the above type of
SI was named as sporophytic SI (SSI), the later type is considered as gametophytic SI (GSI) as it is mainly controlled
by the S-alleles of the vegetative pollen tube cell. GSI
involving peptide-mediated communication is best understood in poppy (Papaver rhoeas) (Eaves et al., 2014). Here
the S-determinant (PrsS) encodes a CRP secreted from pistil cells (Foote et al., 1994), which interacts with the pollen
tube S-determinant PrpS and triggers a Ca2+-dependent signalling network in incompatible pollen tubes. This in turn
results in inhibition of pollen tube growth and ultimately
leads to programmed cell death (PCD). PrpS is a small transmembrane protein located at the pollen tube plasma membrane and is involved in Ca2+ influx (Wheeler et al., 2009). In
addition to Ca2+ and K+ influx, acidification of the cytosol
has been reported recently (Wilkins et al., 2015). Altogether
PrsS interaction with its receptor PrpS triggers a number
of downstream events such as depolymerization of F-actin,
phosphorylation and thereby inhibition of sPPases (soluble inorganic pyrophosphatases), activation of p56 MAPK
(mitogen-activated protein kinase), an increase in ROS (reactive oxygen species) and NO as well as the activation of caspase-3-like/DEVDase enzymes, which finally leads to PCD
(for review see Eaves et al., 2014).
Peptide signalling along the pollen tube journey | 5143
Roles of peptides for pollen tube growth
behaviour
During compatible interactions the vegetative cell of compatible pollen grains produces a pollen tube. Signals from both
the pistil and the pollen maintain the polarized growth pattern of pollen tubes by controlling the cytoskeleton dynamics and vesicle trafficking (Chebli et al., 2013; Guan et al.,
2013). In tomato (Solanum lycopersicum), a member of the
Kunitz trypsin inhibitor-like CRPs, LAT52, participates in
pollen tube germination. Pollen grains injected with antisense
LAT52 germinate abnormally (Twell et al., 1989; Muschietti
et al., 1994). LAT52 binds specifically to two pollen receptor
kinases LePRK1 and LePRK2. The 122 amino acids at the
C-terminus of LAT52 are responsible for this receptor-binding activity. Both LAT52 and LePRKs are expressed in mature
pollen grains, while only the expression of LePRKs increases
significantly after germination, consistent with the finding
that the interaction of their encoded proteins is undetectable
when pollen tubes elongate further. There exist two different
forms of LAT52 before and after germination. It is possible
that pollen monitors its germination status by LAT52, since
the binding affinity of LAT52 to LePRKs changes over time,
and the smaller form of LAT52 expressed after germination is
unable to bind LePRKs efficiently (Tang et al., 2002). During
pollen germination LePRK1 and LePRK2 are detected in a
~400 kDa protein complex, which is disrupted in vitro after
germination initiation in the presence of a 3–10 kDa fraction
of style extract. It was therefore hypothesized that a smaller
extracellular peptide may induce complex dissociation after
germination (Wengier et al., 2003). LeSTIG1 (see below)
appears to represent such a candidate peptide.
After germination, pollen tubes penetrate through the style
and transmitting tract and grow towards the ovary. Pollen
tubes grow considerably more slowly in culture medium compared with in vivo growth, indicating that factors derived from
the pistil promote their growth. One example is the tomato
small CRP peptide LeSTIG1, which is secreted from stigma
tissues. Treatment with recombinant LeSTIG1 promotes pollen tube elongation (Tang et al., 2004). LeSTIG1 encodes a
143-amino-acid peptide containing 16 conserved cysteine
residues at its C-terminus. LeSTIG1 is expressed during pistil
maturation, and its expression is restricted to the stigma and
upper section of the style in the mature pistil. After being
processed, a 7 kDa peptide is released and accumulated at
the pollen tube surface where it interacts with LePRK receptors. The tetrapeptide FNYF in the conserved C-terminal
region of LeSTIG1 is the core domain sufficient for binding
to LePRKs. The three pollen tube receptors LePRK1-3 share
similar expression patterns in the flower, but they possess different binding affinities with different peptides (Kim et al.,
2002). For example during pollen germination LePRK2
binds LAT52, while during pollen tube elongation LePRK1
and LePRK2 bind STIG1. By interacting with different peptides, LePRKs regulate pollen tube germination and growth
at different stages (Tang et al., 2002). The cytoplasmic kinase
domain of LePRKs is able to interact with KPP, a plantspecific Rop GTPase exchange factor (GEF) (Kaothien et al.,
2005). It was further reported that ROS inhibits pollen tube
growth in vitro, therefore it is possible that LePRKs regulate
pollen tube growth through induction of downstream signalling events including the regulation of ROS production
(Zhang et al., 2008). Recently LeSTIG1 was reported to be
able to bind phospholipids like PI(3)P. Thus it may indirectly
regulate the cell redox state (Huang et al., 2014). LeSTIG1
homologues in other species appear to have other functions,
for example, STIG1 regulates the release timing of the secretion of exudates from pistils in petunia (Petunia hybrida) and
tobacco (Nicotiana tabacum) (Verhoeven et al., 2005).
Pollen tube growth is also known to be sensitive to fluctuating environmental conditions such as temperature changes.
A post-translationally modified NCRP peptide CLV3/ESRrelated 45 (CLE45) was recently reported to maintain seed
production under high temperature conditions (Endo et al.,
2013). CLE45 is expressed in the stigma and vascular tissues. Upon temperature shift from 22°C to 30°C its distribution expands to the transmitting tract, where it binds two
RLKs (i.e. SKM1 and SKM2) at the surface of pollen tubes.
Synthetic mature CLE45 peptide therefore promotes pollen tube growth in vitro at 30°C but not at 22°C. This signal
thus prolongs the growing period of a pollen tube instead
of affecting elongation rate in vivo, probably through maintaining mitochondrial activity at higher temperature, which
eventually contributes to adequate fertilization (Endo et al.,
2013).
Inside the transmitting tract, pollen tube growth is guided
and supported by many chemical cues. Several members of
the LTP subfamily of CRPs, such as SCA, are expressed in
the pistil to support pollen tube growth (Chae and Lord,
2011). SCA, a 9–10 kDa peptide with a globular structure is
stabilized by four conserved disulfide bonds and secreted from
epidermal cells into the extracellular matrix (ECM) of the
transmitting tract cells. The peptide is believed to participate
in pollen tube adhesion-mediated guidance because it is positively charged, which facilitates binding to negatively-charged
pectin and thus likely results in the adherence of pollen tubes
to the transmitting tract surface for growth support (Lord,
2000). SCA is also reported to bind the tip of growing pollen
tubes in vitro, and appears in endocytic compartments of pollen tubes in vivo (Kim et al., 2006). It is very likely that these
events are caused after ligand-receptor binding, suggesting
that SCA conveys a signal to the pollen tube tip (Kim et al.,
2006). However, until now receptors and downstream signalling pathway(s) have not yet been described. In Arabidopsis,
an SCA-like molecule, LTP5, also possesses a SCA-similar
function (Chae et al., 2010).
Additionally, gradients of peptides like plantacyanins have been described to act as guidance signals for
polarized tip growth of pollen tubes (Dong et al., 2005).
Plantacyanin is a 10 kDa-sized small secreted protein
related to a class of basic copper-containing blue proteins
(Dong et al., 2005). Plantacyanin is expressed in many tissues. During reproduction mature plantacyanin peptide
forms a steep concentration gradient from the stigma
towards the ovule, with a lower concentration at the
stigma end. This distribution pattern is probably regulated
5144 | Qu et al.
by a miRNA pathway, which is important for its function
(Maunoury and Vaucheret, 2011). If this spatial pattern
is disturbed by plantacyanin overexpression, pollen tubes
expand randomly at stigmatic papilla cells showing defective polar growth. Due to its copper-binding motif, it is
predicted to display a high redox potential with a copper
molecule exposed on the surface. This feature reveals a role
of plantacyanin in ROS metabolism and thereby affecting pollen tube growth (Dong et al., 2005). Chemocyanin
in lily (Lilium longiflorum) is a homologue of plantacyanin, acting similarly to chemotropic molecules for pollen
tube growth. However, chemocyanin lacks copper-binding
capability due to a single amino acid substitution at the
binding site, and its specific mechanism regulating pollen
tube remains unclear (Kim et al., 2003).
Roles of peptides for ovular pollen tube
guidance
Based on few eudicotyledonous model species, pollen tube
guidance towards and inside ovules was previously divided
into two steps: funicular guidance and micropylar guidance
(Higashiyama et al., 2003). Due to recent findings the first
step, which is mediated by sporophytic signals, was further
distinguished into two steps namely pre-ovular and ovular
guidance (Higashiyama and Takeuchi, 2015). Micropylar
guidance is mediated by gametophytic signals from the
embryo sac (Márton and Dresselhaus, 2010). In order to
additionally consider the morphology and knowledge from
plant families such as the grasses we suggest distinguishing
between ovular guidance (mediated by signals derived from
sporophytic ovule tissues) and micropylar guidance (guidance mediated by signals from female gametophytic cells).
Until now, little is known about the molecular mechanism
of sporophytic ovular guidance. Recently, two mitogen-activated protein kinases, MPK3 and MPK6, have been discovered to be involved in ovular guidance (Guan et al., 2014). In
vivo pollination assays showed that mpk3 mpk6 pollen tubes
were defective in ovule guidance, but micropylar guidance
in a semi in vivo pollination assay appeared normal (Guan
et al., 2014).
In recent years, elegant studies have elucidated many more
details of the molecular mechanism of micropylar guidance
(Fig. 2A). In 2001, it was demonstrated using a laser ablation assay that a diffusible attraction signal is derived from
the two synergid cells located as part of the egg apparatus
close to the egg cell. Using Torenia foumieri as a model—a
unique plant with a naked embryo sac that protrudes from
the micropyle of the ovule—it was shown that the distance
of pollen tube attraction by the synergid cell is about 100 μm
at most in vitro, indicating that synergid cells produce shortrange attractants (Higashiyama et al., 2001). This implied
for the first time that attractants are more likely larger molecules such as small secreted peptides rather than small molecules like NO or ROS (Higashiyama et al., 1998; Palanivelu
and Preuss, 2006).
The first reported pollen tube attractant was ZmEA1 (Zea
mays EGG APPARATUS1), a peptide exclusively expressed
in the maize egg apparatus before fertilization. Downregulation of ZmEA1 expression resulted in misguidance of
pollen tubes in the micropylar region of the ovule causing
fertilization failure. Therefore, ZmEA1 was considered as a
signalling molecule for short-range pollen tube guidance in
maize (Márton et al., 2005). ZmEA1 encodes a 94 amino acid
hydrophobic precursor protein with a predicted N-terminal
transmembrane domain. Mature ZmEA1 was predicted to be
N-terminally cleaved to generate a 49 amino acid oligopeptide, which is secreted from the egg apparatus and accumulates
in the cell walls of micropylar nucellar cells. If expressed from
Arabidopsis synergid cells, ZmEA1 is capable of guiding maize
pollen tubes in vitro within a distance of 150 μm towards the
micropylar opening of Arabidopsis ovules (Márton et al.,
2012). Recent studies further showed that the ZmEA1 peptide
interacts with the maize pollen tube tip in a species-specific
manner in vitro. After binding, the peptide is immediately
internalized into vesicles and degraded (Uebler et al., 2013).
Fig. 2. Hypothetical model of peptide signalling at the end of the pollen tube journey. (A) Pollen tubes are attracted by LURE and EA1 peptides (blue
dots), respectively, released by constitutive secretion (CS) from synergid cells. Unknown receptors (white) interact with cytoplasmic LIP1/2 (violet)
associated with the LURE receptor complex. Peptides secreted from the pollen tube may include RALF peptides (brown dots), which interact with the
FER/SRN receptor (green) and induce Ca2+ spiking. This activates calcium induced secretion (CIS) of stored secretory vesicles containing ES1-4 (orange
dots) and PMEI peptides (grey dots), respectively. Their release leads to osmotic burst of the pollen tube tip after inactivation of PME (dark green) and
ion influx by channel (pink) opening. (B) Pollen tube burst induces a Ca2+ elevation in the egg cell. Whether CIS leads to release of the sperm activator
peptide EC1 (yellow dots) remains to be determined. The EC1 receptor at the sperm surface is yet unknown.
Peptide signalling along the pollen tube journey | 5145
ZmEA1 belongs to the EA1-like (EAL) family peptides, a
novel class of hydrophobic and polymorphic peptides, which
do not exist in eudicots (see below). Until now only one additional member of this family, EAL1 (EA1-like1), has been
functionally characterized in maize and was shown to be
involved in regulating cell fate determination of female gametophyte cells (Krohn et al., 2012). Homologous sequences are
also present in other grasses such as Brachypodium distachyon
(purple false brome), Sorghum bicolor (sorghum) and Oryza
sativa (rice), but not in A. thaliana or other dicotyledonous
plant species (Dresselhaus et al., 2011), suggesting that EA1like peptides are probably Gramineae-specific.
In dicotyledonous plants, another group of peptides was
identified as pollen tube attractants that act in a speciespreferential manner. CRPs of the DEFL subclass named as
LUREs (LURE1 and LURE2) were first identified as attractant molecules in Torenia fournieri involved in micropylar
pollen tube guidance. Predicted mature LUREs in Torenia
species consist of 62–70 amino acid residues (Table 1) and
contain six cysteine residues. Down-regulation of LURE
genes by injection of morpholino antisense oligomers into the
embryo sac cells of T. fournieri strongly decreases the attraction frequency of pollen tubes by the ovule (Okuda et al.,
2009). Recombinant LURE1 and LURE2 proteins expressed
in Escherichia coli exhibit similar pollen tube attraction activity in vitro. 40 pM of LUREs (∼1000 molecules) are sufficient
to attract pollen tubes. LUREs belong to the most strongly
expressed genes in synergid cells. The encoded peptides are
secreted towards the filiform apparatus and likely generate
the necessary concentration gradient around the micropyle,
which is important for pollen tube attraction and guidance
towards the egg apparatus (Okuda et al., 2009). Related
TcCRP1 from T. concolor, which differs from LURE1 of
T. fournieri by eight amino acids, also showed species-preferential activity (Kanaoka et al., 2011) indicating that this step
of the pollen tube journey is strongly regulated and represents
a major pre-zygotic hybridization crossing barrier in plants.
Further support for this hypothesis comes from research in
A. thaliana, where five CRPs (AtLURE1.1–AtLURE1.5;
predicted mature peptides are 71–75 amino acids in length),
were identified as attractants controlling pollen tube guidance into the micropyle of Arabidopsis ovules. AtLUREs
are also secreted from the synergid cells and spread around
the funiculus. With the exception of AtLURE1.5, which
lacks one conserved cysteine, all AtLUREs are capable of
attracting pollen tubes (Takeuchi and Higashiyama, 2012).
Moreover, it was shown that AtLUREs attracted pollen tubes
of A. thaliana better than tubes of A. lyrata indicating that
they act in a species-preferential manner. The comparison
of LUREs from Torenia and Arabidopsis species shows little
homology (Higashiyama and Takeuchi, 2015), but based on
the number and arrangement of cysteine residues, they are all
grouped into the defensin-like (DEFL) subclass of CRPs. In
flowering plants DEFL genes form the largest CRP subfamily, with 317 DEFL genes in Arabidopsis. They partly appear
to have evolved by tandem and segmental duplication events
(Takeuchi and Higashiyama, 2012). We assume that this type
of CRP peptide is also used by other eudicotyledonous plant
species to attract pollen tubes into the ovule. However, the
low sequence conservation between Torenia and Arabidopsis
LUREs indicates that it will be difficult to predict these
from the large number of DEFL genes in other species.
Experimental pollen tube guidance assays have to be applied
to test candidates identified by bioinformatic approaches.
Receptors perceiving LURE signals are not yet identified
in any plant species. However, two Arabidopsis receptor-like
cytoplasmic kinases (RLCKs), called Lost In Pollen Tube
Guidance 1 (LIP1) and 2 (LIP2), were recently identified to
be involved in AtLURE1-mediated micropylar pollen tube
guidance (Liu et al., 2013). LIP1 and LIP2 are predominately
expressed in pollen tubes and anchored to the inner plasma
membrane in the pollen tube tip region via palmitoylation.
This localization is essential for their function in pollen tube
guidance control. In lip1 lip2 double mutants the attraction
of pollen tubes towards AtLURE1 was significantly reduced.
The observation that loss of LIP1/2 function resulted in
more than 60% reduction of male transmission in vivo
while retaining 70% of the in vitro response activity towards
AtLURE1 suggests that there might be other RLCKs participating in AtLURE1-mediated pollen tube guidance signalling, or that LIP1 and LIP2 are likely to simultaneously
participate in other peptide-mediated signalling pathways
during pollen tube guidance (Liu et al., 2013). Because LIP1
and LIP2 lack extracellular domains to bind AtLUREs
directly, it is more likely that they function as components
of a receptor complex (Fig. 2A). A receptor complex often
consists of two RLKs, a receptor containing an extracellular
domain for ligand interaction and a co-receptor RLK with
a shorter extracellular domain, and a RLCK lacking extracellular domains. Two examples are the BRI1-BAK1-BSK1
receptor complex in BR signalling and FLS2-BAK1-BIK1
receptor complex in innate immunity signalling (Kim and
Wang, 2010; Lu et al., 2010). The identification of LIP1 and
LIP2 will now facilitate identifcation and characterization of
the other components of the receptor complex in pollen tube
guidance in Arabidopsis.
Finally, it is important to note that loss of function of
MYB98, a transcription factor specifically expressed in synergid cells of Arabidopsis, resulted in more severe defects in
pollen tube guidance compared to AtLURE1 RNAi lines
(Takeuchi and Higashiyama, 2012), indicating that there
might be additional pollen tube attractants expressed in
synergid cells in eudicots, which need to be identified in the
future. In contrast it was shown that maize ovules lacking
embryo sacs showed the same pollen tube attraction phenotype as ZmEA1-RNAi lines (Lausser et al., 2010) suggesting
that the conserved ZmEA1 peptide likely represents the sole
micropylar attractant in the grasses.
Peptides involved in pollen tube reception
After reaching the micropyle, growth of the pollen tube
ceases, and the pollen tube enters the female gametophyte
where it grows beyond the filiform apparatus to enter the synergid cell at a more distant site, where it ruptures to release its
contents, including the two sperm cells (Leshem et al., 2013).
5146 | Qu et al.
This process is called pollen tube reception (Kasahara et al.,
2005).
One RLK, FERONIA/SIRENE (FER/SRN), which
localizes predominately at the surface of synergid cells,
plays an essential role in growth arrest of the pollen tube
(Escobar-Restrepo et al., 2007). The extracellular domain of
FER/SRN is variable in different plant species, consistent
with species-specificity observed in this pollen tube reception
event. It was previously suggested that FER/SRN may interact with an unidentified ligand on the surface of the pollen
tubes or with a signalling ligand derived from the pollen tube
(Rotman et al., 2003; Huck et al., 2003). Due to recent findings (see below) we speculate that CRPs of the RALF family may represent these ligands (Fig. 2A). While FER/SRN
is localized to the female gametophyte and lacks in pollen
tubes, ANXUR1 (ANX1) and ANXUR2 (ANX2), two close
homologues of FER/SRN in the same RLK subfamily, are
expressed in pollen and were reported to be involved in maintaining pollen tube integrity during growth. Simultaneous
inactivation of ANX1 and ANX2 results in growth arrest
and burst of pollen tubes in vitro, and in failure of pollen
tubes to reach the female gametophyte in vivo (BoissonDernier et al., 2009; Miyazaki et al., 2009). These findings
further indicate that the release of sperm cells is controlled
by signalling events occurring between both female and male
gametophytes. Therefore the identification of the ligands of
ANX1 and ANX2 as well as FER/SRN is another goal to
be accomplished in the near future. Recently a small and
secreted peptide of the RALF (Rapid Alkalinization Factor)
subclass of CRPs was reported to bind FER/SRN in roots
and induce the suppression of cell elongation in the primary
root. RALF-FER/SRN interaction causes inhibition of
the plasma membrane H+-ATPase thereby mediating a pH
change in the cytoplasm (Haruta et al., 2014). In addition,
RALF induces the increase of Ca2+ concentration in the cytosol (Pearce et al., 2001; Haruta and Constabel, 2003; Haruta
et al., 2008), which may also affect pollen tube growth and
burst. Whether RALFs are also capable of interacting with
ANX1 and ANX2 remains to be shown. In maize RALF
encoding genes belong to the strongest expressed genes in
pollen tubes (M. Woriedh and T. Dresselhaus, unpublished)
pointing to the possibility of both ANX1 and ANX2 as
well as FER/SRN interaction with RALF peptides during
reproduction.
ZmES1–4 (Zea mays Embryo Sac1–4) were the first peptide genes showing a specific expression pattern in reproductive cells. They encode members of the DEFL subclass
of CRPs and were shown to be specifically expressed in
the whole female gametophyte with strongest expression in
the synergid cells (Cordts et al., 2001). Later it was demonstrated that they induce pollen tube burst in a species-specific
manner (Amien et al., 2010). Application of ZmES4 led to
quick depolarization of the pollen tube plasma membrane,
induced potassium channel KZM1 opening and thus K+ flux.
Whether the activity of other channels is changed due to
membrane depolarization is unknown. However, K+ flux and
influx of other ions such as Na+ and Ca2+ and sugars likely
leads to water uptake, osmotic changes and cytoskeleton
disassembly. This ultimately results in pollen tube rupture at
the very tip, the weakest point of the pollen tube due to the
lack of callose-containing cell wall material at this region.
Recently, it was reported that another small apoplastic protein of the PMEI (pectin methyl esterase inhibitor) subgroup
of CRPs is strongly expressed in both gametophytes of maize.
ZmPMEI1 inhibits PMEs (pectin methyl esterase) whose
activity is required to remove methyl and demethoxy groups
from homogalacturonan (a major class of pectin and thus of
the pollen tube cell wall) and thereby disturbs the integrity
and stiffness of the elongating pollen tube apex. Application
of recombinant ZmPMEI1 to growing pollen tubes leads to
burst at the subapical region. The current model is that the
CRPs ZmES1–4 and ZmPMEI1 act in concert to induce pollen tube rupture upon arrival of the pollen tube at the synergid cell (Woriedh et al., 2013; see also Fig. 2A). Whether
secretion of these peptides/small proteins is induced by the
FER/SRN signalling pathway has to be shown in further
experimentation.
Peptides involved in gamete activation
After release, one sperm cell fuses with the egg cell to form
the embryo and the other one fuses with the central cell to
initiate the development of the triploid endosperm. This
process, double fertilization, represents a (the) major characteristic of flowering plants and is at the same time the last
possibility to control compatibility of gametes and thus may
represent a final pre-zygotic hybridization barrier. Many signalling events are involved, including cell recognition, gamete
activation, plasma membrane adhesion and fusion as well
as nuclear fusion (karyogamy). Until now, only one peptide
class has been identified playing an essential signalling role
in this process (Fig. 2B). Five Arabidopsis peptides, named
Egg Cell1 (EC1), are secreted by the egg cell after sperm cell
release (Sprunck et al., 2012). EC1s belong to the EC1 subgroup of ECA1 (Early Culture Abundant 1) gametogenesisrelated CRPs (Sprunck et al., 2014) and consist of ~100–130
amino acids after signal peptide removal. They contain six
conserved cysteine residues and two conserved signature
sequence motifs (Sprunck et al., 2012). All five Arabidopsis
EC1 genes (EC1.1–EC1.5) function redundantly to activate
sperm cells, thereby enabling them to fuse with the two female
gametes. Simultaneous inactivation of all five EC1 genes
leads to inhibition of gamete fusion and reduced seed set.
Multiple pollen tubes are attracted in the quintuple mutant
and to some extent sperm cells acquire late competence for
fusion 2–3 days after pollination (Rademacher and Sprunck,
2013).
Upon sperm cell arrival, EC1s are released from the egg
cell and trigger the re-localization of HAP2/GCS1 (Sprunck
et al., 2012), a sperm-specific integral membrane protein,
which is required for gamete fusion. From hap2/gcs1 mutant
pollen tubes sperm cells are properly released, but fail to fuse
with female gametes (Mori et al., 2006; von Besser et al.,
2006). Exogenous application of EC1 triggers HAP2/GCS1
redistribution from the endomembrane system to the sperm
Peptide signalling along the pollen tube journey | 5147
cell surface and thus activates sperm cells to fuse with the two
female gametes (Sprunck et al., 2012).
EC1 proteins are found in all angiosperm species investigated, including the basal angiosperm Amborella trichopoda
(Sprunck et al., 2012). It will now be interesting to find out
whether Arabidopsis EC1 is capable of activating sperm cells
of other plant species and vice versa. Moreover, the central
cell also contains CRPs (Schmid et al., 2012) and it will now
be interesting to find out to what extent it contributes to
sperm cell activation and the precise signalling during double fertilization. First studies point in this direction; in the
female gametophyte mutant glc (glauce) one sperm cell successfully fuses with the egg cell, while the second sperm cell
fails to fuse with the central cell. glc mutants lack a central
cell-expressed BAHD acyltransferase, which is involved in
secondary metabolism and possibly required to generate a
central cell-derived signalling molecule (Leshem et al., 2012).
Conclusions
In the past few years, an increasing knowledge of peptide
signalling has contributed significantly to the understanding
of the molecular mechanisms regulating plant reproduction,
especially along the pollen tube journey and during double
fertilization. A number of secreted peptides mainly of various
CRP subclasses were demonstrated to participate in pollen
grain recognition at the stigma, in pollen tube growth support
and guidance, in attracting the tube to enter the embryo sac
and in mediating gamete interaction (Figs 1, 2).
Peptides involved in double fertilization appear to belong
to large gene families with multiple members. Most members show similar expression patterns and function redundantly, as revealed by the studies of LUREs (Takeuchi and
Higashiyama, 2012), ES1–4 (Amien et al., 2010; Woriedh
et al. 2015) and EC1s (Sprunck et al., 2012). CRP peptide
subfamilies like LTPs and RALFs are strongly expressed
along the pollen tube journey, but precise roles are not yet
known. Other peptide families appear to play a predominant
role after fertilization such as the MEG1 CRPs and AE peptides, whose diverse roles are highlighted elsewhere in this
special issue (Gutierrez-Marcos and Ingram, 2015). However,
considering that 756 CRP and 432 non-CRP genes were
annotated in the Arabidopsis genome, of which the majority
is expressed specifically during reproduction and seed development (Huang et al., 2015), it can be expected that many
more functions about peptide signalling during these important biological processes will be elucidated in the near future.
As ligands, apoplastic peptides are supposed to be perceived
by one or more cell surface receptors. However for the majority of peptides functioning or being expressed along the pollen
tube pathway and in double fertilization, the corresponding
receptors have not been identified. RLKs, with more than 630
members, comprise the largest receptor family in plants (Shiu
and Bleecker, 2001), and most peptide receptors identified so
far belong especially to the Leucine-Rich-Repeat (LRR)-type
RLKs (Shiu and Bleecker, 2003). Many RLKs are expressed
in plant gametophytes (Wang et al., 2014; Rutley and Twell,
2015; see also cited references in these articles) implying that
they may play important roles in reproduction, probably by
acting as peptide receptors during male-female communication. Until now few peptide ligand-receptor pairs involved
in plant reproduction have been identified (Fig. 1). Novel
and high-throughput biochemical methods (Uebler and
Dresselhaus, 2014) combined with highly sensitive mass spectrometric instruments will now help to identify more receptors of peptide ligands in the near future.
A further task for the future will be to elucidate how peptide signals are transduced into cellular responses. Many wellstudied peptides are reported to bind extracellular domains of
receptor oligomers, for example SCR binds an SRK homodimer (Takayama et al., 2001), which causes autophosphorylation of the RLK kinase domain and subsequently activates
phosphorelay cascades (Guo et al., 2013). Downstream
responses then include fast responses including protein modification (Takayama et al., 2001), protein degradation (Stone
et al., 2003), Ca2+ fluctuation (Haruta et al., 2008), changes
of redox state (Zhang et al., 2008), alteration of cytoskeleton
dynamics (Guan et al., 2013) etc. or delayed responses associated with gene expression changes, which have been well
studied, for example, during stem cell homeostasis in plants
(Richards et al., 2015), but not during reproduction.
In conclusion, our current knowledge about the roles of
peptides during pollen tube germination and growth as well
as during double fertilization is still very limited. In addition
to the induction of signalling pathways they probably have
roles such as binding lipids, regulating vesicular trafficking,
cell wall integrity and cytoskeleton dynamics, as is the case,
for example, for STIG1, which appears to possess multiple
roles (see above). How signalling peptides themselves are
regulated, both transcriptionally and post-translationally, to
generate or maintain specific spatiotemporal expression patterns, also needs to be investigated in more detail. Moreover,
exploring the function of the numerous uncharacterized
peptides expressed during reproduction (Huang et al., 2015)
represents a major task for the plant research community to
obtain a more comprehensive understanding of the processes
that lead to successful double fertilization or species isolation
in plants.
Integrated methods from various fields including mass
spectrophotometric imaging and structure biology as well
as more advanced live cell and single molecule imaging technologies are now necessary and will allow the study of the
dynamics of peptide-ligand interactions and the activated
signalling pathway(s) in more detail. Moreover, at present
our understanding of peptide signalling during reproduction has been derived from the analysis of few peptides in
many plant taxa such as Arabidopsis, Brassica, lily, maize,
Petunia, poppy, tobacco, tomato and Torenia (Table 1). It is
yet unclear whether the mechanisms described above are conserved among plant species or whether plants species/genera
have developed independent peptide signalling mechanisms
during reproduction. However, the understanding of peptide
based male-female communication likely represents a key
to elucidate speciation mechanisms in plants. The various
hybridization barriers along the pollen tube journey require
5148 | Qu et al.
precise and compatible peptide-receptor interactions. Thus
this knowledge has great potential for future exploitation in
plant breeding to overcome crossing barriers between plant
species or even genera that cannot be crossed today. We look
forward to more exciting studies about peptide signalling during plant reproduction in the near future.
Acknowledgements
This work was supported by the Natural Science Foundation of China
(31370344 and 31230006 to LJQ), the National Basic Research Program
of China (2011CB915402 and 2012CB944800) and the German Research
Council (via DFG collaborative research centre SFB924 to TD).
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