Download Female gametophyte-controlled pollen tube guidance

Cell–Cell Communication in Plant Reproduction
Female gametophyte-controlled pollen tube
guidance
Mihaela-Luiza Márton1 and Thomas Dresselhaus
Cell Biology and Plant Biochemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany
Abstract
During the evolution of flowering plants, their sperm cells have lost mobility and are transported from
the stigma to the female gametophyte via the pollen tube to achieve double fertilization. Pollen tube
growth and guidance is largely governed by the maternal sporophytic tissues of the stigma, style and ovule.
However, the last phase of the pollen tube path is under female gametophyte control and is expected to
require extensive cell–cell communication events between both gametophytes. Until recently, little was
known about the molecules produced by the female gametophyte that are involved in this process. In
the present paper, we review the most recent development in this field and focus on the role of secreted
candidate signalling ligands.
Introduction
Double fertilization, a unique and characteristic trait of
flowering plants (angiosperms), is a very subtle and accurately
regulated process. Owing to the fact that the female
gametes (egg and central cell) are deeply embedded into
the maternal tissues of the ovule and ovary respectively,
they are inaccessible to the non-motile male gametes (sperm
cells). Thus angiosperms have acquired a specialized form of
sperm cell delivery and evolved a tricellular male gametophyte
[pollen grain/PT (pollen tube)], consisting of two sperm cells
enclosed by a large vegetative tube cell, which is able to grow
over long distances to deliver its cargo [1]. After hydration
and germination on the stigma surface, pollen grains form
PTs that first penetrate the intercellular spaces between the
papillar cells of the stigma, then grow towards and through
the TT (transmission tract) of the style and ovary. In order
to reach unfertilized ovules, PTs have to exit the TT in
eudicots, emerge on the surface of the placenta and grow
along the epidermis of the funiculus towards the micropylar
entry of the ovule, finally arriving at the hidden FG (female
gametophyte).
The mechanism that precisely directs the PT over such
a long distance through the pistil towards the FG has been
studied for more than a century. It has been shown that,
before entering the ovary, PT guidance requires intensive
communication with the surrounding sporophytic maternal
tissues, and the presence of the FG seems not to be necessary
for the largest part of the PT’s journey [2–4]. However, after
exit from the TT tissue and emergence on the surface of
the placenta, PT guidance is thought to be primarily under
gametophytic control, although the maternal sporophytic
Key words: Arabidopsis, maize, micropyle, pollen tube guidance, signalling, synergid, Torenia.
Abbreviations used: CRP, cysteine-rich protein; FG, female gametophyte; GABA, γ -aminobutyric
acid; pop2, pollen–pistil interaction2; PT, pollen tube; TT, transmitting tract; ZmEA1, Zea mays
EGG APPARATUS1.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2010) 38, 627–630; doi:10.1042/BST0380627
tissues of the ovary might also contribute to guidance cues
towards the ovary [5]. Excellent recent reviews offering
updates in the mechanic and molecular processes of PT
germination and sporophytic guidance have been published
elsewhere [6–9] and we therefore restrict the present review
to PT guidance governed by the FG.
Genetic dissection of gametophytic PT
guidance
To understand the various mechanisms and components of
the FG-controlled last phases of PT growth and guidance,
several approaches have been performed in different plant
species, especially in the Brassicaceae model plant Arabidopsis
thaliana and the Scrophulariaceae species Torenia fournieri.
Genetic approaches were applied in various sporophytic and
gametophytic Arabidopsis mutants to determine whether and
at which steps the PT path may be influenced by the FG.
These studies have shown that PTs are not attracted to
either immature or incompletely formed ovules or to ovules
lacking FGs, and, as a consequence, did not approach the
funiculus [2,10]. In contrast, in mutants displaying FGs with
less severe or later developmental defects, PTs grew along the
funiculus, but did not enter the micropyle [11]. Hence these
results, together with those obtained from the experiments
performed in the in vitro Torenia system [3], suggested that
FG-controlled PT guidance begins when PTs exit the TT and
grow towards the funiculus of each ovule. The last path of PT
attraction controlled by the FG was therefore divided into at
least two steps: (i) guidance from the surface of the placenta
to the funiculus (funicular guidance), and (ii) guidance from
the entrance of the micropyle to the FG or embryo sac
(micropylar guidance) [6,11,12]. Since a funiculus is lacking in
grasses (Poaceae), gametophytic PT attraction involves only
the latter phase [7].
In order to reach the FG and to execute fertilization,
PTs entering from the funiculus in Arabidopsis first have
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to find their way to the micropylar opening formed by
the outer integuments. In this channel-like structure, PTs
can easily reach the naked micropylar region of the FG
consisting of the egg cell and two glandular-like cells, the
synergids [13,14]. In the case of T. fournieri, the other
prime model plant for PT attraction studies, its naked FG
is even more exposed, protruding through the micropylar
opening of the integuments [3]. After arrival at the micropyle,
one PT enters one of the two synergid cells (the receptive
synergid) directly via the filiform apparatus, a thickened
area of secondary cell wall material generated via plasma
membrane invaginations of both synergids [15–17]. In the
Poaceae, the FG is additionally embedded by a few layers
of stack-like nucellus cells, representing an additional hurdle
before PTs can finally enter the embryo sac (Figures 1A and
1B) [18,19]. Similar to the eudicot model plants described
above, one PT in grasses first targets the filiform apparatus
and then releases its contents explosively into the receptive
synergid cell [14,20]. Second PTs were rarely observed in
the micropyle of wild-type ovules of several plant species
[18,21,22], indicating either that attractant(s) are no longer
secreted, degraded or inactivated, or that additional PTs are
actively repulsed.
Signalling in gametophytic PT guidance
A complex signalling network seems to be required for PT
guidance by the FG. However, the molecular mechanism
of funicular guidance still awaits to be discovered, and it
is still unclear whether there is a diffusible signal emitted
directly from the developing and mature FG or an indirect
signal that causes a change in the ECM (extracellular matrix)
of sporophytic cells [12]. On the basis of genetic, cell
ablation and in vitro investigations with excised Arabidopsis
and Torenia ovules, micropylar or short-range PT guidance
appears to be governed by species-specific chemotactic signals
secreted by the synergid cells of the FG. The maximum
distance of attraction in vitro is in the range of approx.
200 μm in T. fournieri [6], whereas approx. 100 μm has been
observed in Arabidopsis and maize [7,15,18,21]. Laser-assisted
cell ablation experiments with Torenia ovules containing
naked FGs have shown further that the synergid cells are
necessary and sufficient to attract PTs and that neither the
egg cell nor the central cell seems to be involved in this
process and are capable of generating sufficient amounts of
guidance molecule(s). However, at least in maize, the egg
cell seems to contribute to the production of the attraction
signal in addition to the synergid cells [18]. It has been
shown that, in Arabidopsis, even the central cell might play
a role in micropylar PT guidance [23], although it might be
possible that the corresponding central cell guidance (CCG)
gene encoding a potential transcriptional regulator might
act rather indirectly and be required, for example, for FG
maintenance or maturation. This hypothesis is supported by
the observation that once the central cell was destroyed by
laser cell ablation in Torenia ovules, most of the FGs were
no longer able to maintain their structure and deteriorated.
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Authors Journal compilation Furthermore, it was observed that this resulted in an alteration
of PT guidance [22].
Ca2+ has long been considered a potential attractant
because it plays an important role for PT tip growth, and
high concentrations have been detected in the synergid cells
[24]. However, in vitro pollination assays using the in vitro
Torenia system have indicated that it either does not function
as an attractant at all or it belongs to an attractant cocktail of
molecules [25]. A role in micropylar PT attraction was also
proposed for the GABA (γ -aminobutyric acid) molecule,
because it forms a gradient in the pistil with the highest
concentration at the inner integument of the ovule [5].
PTs were sensitive to higher concentrations of GABA and
guidance was impaired after self-pollination in the pollen–
pistil interaction2 (pop2) Arabidopsis mutant. The POP2
gene encodes a transaminase that degrades GABA [5,26].
However, until now, a chemoattractant role for the GABA
molecule has not been demonstrated, and PT guidance in a
pistil with low GABA levels has not been shown. Thus it
is more likely that gametophytic PT attraction and sperm
release depends on species-specific factors rather than on
general molecules such as Ca2+ or GABA.
The first gene encoding a candidate extracellular ligand
involved in PT attraction controlled by the FG was identified
in maize. Zea mays EGG APPARATUS1 (ZmEA1), which
is specifically expressed in egg and synergids cells, encodes
a polymorphic precursor protein shown to be secreted to
the cell walls of micropylar nucellus cells. PTs arrived at the
micropyle of ZmEA1-knockdown plants without penetrating
the intercellular space of micropylar nucellus cells, suggesting
a role for ZmEA1 in short-range gametophytic PT guidance
[18] (Figures 1B and 1C). An N-terminally cleaved predicted
mature ZmEA1 protein of 49 amino acids is capable of
attracting maize PTs directly at a low concentration (<10 μM)
in vitro (Figures 1D–1F). Moreover, Arabidopsis ovules
expressing ZmEA1–GFP (green fluorescent protein) fusion
protein driven by the synergid-cell-specific MYB98 promoter
are capable of attracting maize PTs in vitro (M.-L. Márton,
A. Fastner and T. Dresselhaus, unpublished work), indicating
that it is possible to overcome wide crossing barriers
combining genetic engineering with the tools currently being
developed in plant reproduction research.
Recently, secreted CRPs (cysteine-rich proteins) named
LUREs have been identified as the FG-derived attractants
in T. fournieri [27]. These polymorphic proteins belong to
a subgroup of the DEFL (defensin-like) gene superfamily
of CRPs and are synthesized and secreted by the synergid
cells. Their recombinant mature proteins have been shown to
attract competent PTs in vitro in a species-specific manner.
Moreover, microinjection of LURE antisense oligomers into
mature embryo sacs resulted in a significantly impairment
in PT attraction, providing additional evidence that LUREs
1 and 2 are the micropylar PT attractants derived from the
synergid cells of T. fournieri.
A number of additional genes and proteins have been
postulated to play a role in funicular and micropylar PT
guidance in Arabidopsis [28–33]. However, the majority of the
Cell–Cell Communication in Plant Reproduction
Figure 1 PT guidance by ZmEA1 signalling in maize
(A) Depiction of PT attraction by the FG and sperm discharge inside the receptive synergid cell (RSY). Visible in the PT
(dark blue) is the nucleus of the vegetative tube cell. The two sperm cells (violet) are already released into the RSY. Outer
and inner integuments are shown in pink and orange respectively, and nucellus cells are shown in yellow. The persistent
synergid cell is shown in green, the egg cell in red, central cell in white and antipodals in grey. (B and C) Monitoring of PT
growth and sperm cell discharge 24 h after pollination in ovules of wild-type (wt) plants and ZmEA1-RNAi (RNA interference)
plants (EA1-RNAi) using transgenic ActinP::GUS (β-glucuronidase) maize pollen. Modified from [18] with permission from
AAAS. (B) PT releases its contents into the wild-type receptive synergid cell. (C) PT grew normally at the surface of the
inner integument, but did not enter the micropyle and continued growth at random directions in approx. 50 % ovaries of
ZmEA1-RNAi lines. The arrows label the micropylar opening of the ovule. (D–F) In vitro PT-guidance assay using an N-terminal
cleaved predicted mature ZmEA1 protein of 49 amino acids. (D) ZmEA1 was released using a microcapillary at a 50 μm
distance from an active maize PT. (E) At 22 min after application, PT tip growth was reoriented towards the region where the
ZmEA1 droplet was placed (*). (F) Once the PT finally reached its target, it stopped growing. Abbreviations: AP, antipodals;
CC, central cell; EA, egg apparatus; MC, microcapillary; PG, pollen grain; RSY, receptive synergid cell. Scale bars, 50 μm.
genes seem to be required for PT growth, and FG maturation
thus plays a more indirect or no role for PT guidance.
Various studies have clearly indicated that only fully mature
FGs are capable of producing/secreting the micropylar
attractants [16,21]. In magatama (maa) mutants, for example,
which contain FGs exhibiting late developmental defects,
PTs grew along the funiculus, but not inside the micropyle
[11,32]. Mutations in an Arabidopsis synergid-expressed
gene encoding the transcription factor MYB98 showed an
immature and less organized filiform apparatus that, as a
consequence, affected micropylar PT guidance [15].
Concluding remarks
Despite the fact that the PT path through the pistil towards
the FG has been studied for more than a century, we are
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only now beginning to understand the underlying molecular
mechanisms preceding gamete fusion in flowering plants.
The last few years have seen tremendous efforts in finding
the molecules derived from the FG that are involved in
PT guidance, but only ZmEA1 in maize and LURE1 and
LURE2 in T. fournieri have been identified to date that
accomplish the properties of a true attractant. However,
owing to the development of the necessary tools and methods
as well as the identification of the first FG-derived attractants as highly polymorphic small proteins, further progress
in this field is expected soon, and the identification of the
respective Arabidopsis attractants and probably other plant
species seems to be close. A major task now represents the
identification of the attractant-specific receptors at the PT tip
surface as well as the associated signal transduction cascades
responsible to reorient PT growth direction. It will be exciting
to find out whether small GTPases and ion channels that play
a key role in animal sperm chemotaxis [34,35] play a similar
role in PT growth direction in plants, or whether plants have
evolved different mechanisms.
Funding
Our work is funded by the German Research Foundation (DFG) [grant
number MA 3976/1-1].
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Received 2 October 2009
doi:10.1042/BST0380627