Journal of Experimental Botany, Vol. 62, No. 5, pp. 1621–1631, 2011
doi:10.1093/jxb/err032
REVIEW PAPER
Cytoplasmic connection of sperm cells to the pollen
vegetative cell nucleus: potential roles of the male germ unit
revisited
Andrea D. McCue1, Mauro Cresti2, José A. Feijó3,4 and R. Keith Slotkin1,*
1
2
3
4
Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA
Dipartimento di Biologia Ambientale, Università di Siena, Via P. A. Mattioli 4, I-53100 Siena, Italy
Instituto Gulbenkian de Ciencia, 2780-156 Oeiras, Portugal
Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Vegetal, C2, 1749-016 Campo Grande, Portugal
* To whom correspondence should be addressed. [email protected]
Received 16 November 2010; Revised 24 January 2011; Accepted 25 January 2011
Abstract
The male germ cells of angiosperm plants are neither free-living nor flagellated and therefore are dependent on the
unique structure of the pollen grain for fertilization. During angiosperm male gametogenesis, an asymmetric mitotic
division produces the generative cell, which is completely enclosed within the cytoplasm of the larger pollen grain
vegetative cell. Mitotic division of the generative cell generates two sperm cells that remain connected by a common
extracellular matrix with potential intercellular connections. In addition, one sperm cell has a cytoplasmic projection
in contact with the vegetative cell nucleus. The shared extracellular matrix of the two sperm cells and the physical
association of one sperm cell to the vegetative cell nucleus forms a linkage of all the genetic material in the pollen
grain, termed the male germ unit. Found in species representing both the monocot and eudicot lineages, the
cytoplasmic projection is formed by vesicle formation and microtubule elongation shortly after the formation of the
generative cell and tethers the male germ unit until just prior to fertilization. The cytoplasmic projection plays
a structural role in linking the male germ unit, but potentially plays other important roles. Recently, it has been
speculated that the cytoplasmic projection and the male germ unit may facilitate communication between
the somatic vegetative cell nucleus and the germinal sperm cells, via RNA and/or protein transport. This review
focuses on the nature of the sperm cell cytoplasmic projection and the potential communicative function of the male
germ unit.
Key words: Cell-to-cell communication, cytoplasmic projection, intercellular transport, male germ unit, pollen, vegetative
nucleus.
Introduction
The presence of mitotic divisions after meiosis but before
fertilization constitutes a fundamental difference in the
process of gametogenesis between the plant and animal
kingdoms. In multicellular plants, the single cell haploid
product of meiosis divides several times to produce
a multicellular gametophyte. On the male side of the
angiosperm reproductive cycle, the meiotic products
(individual microspores) undergo an asymmetric mitotic
division. After this asymmetric division, the smaller generative cell (GC) is completely enclosed within the cytoplasm
of the larger pollen grain vegetative cell (VC; Fig. 1). The
GC divides again to produce the two sperm cells (SCs). The
Abbreviations: ECM, extracellular matrix of the sperm cells and generative cell; GC, pollen generative cell; GCN, pollen generative cell nucleus; GFP, green fluorescent
protein; MGU, male germ unit; SC, sperm cell; SC1, sperm cell 1, closest to the vegetative cell nucleus with a cytoplasmic projection associated with the vegetative
cell nucleus; SC2, sperm cell 2, farther from the vegetative cell nucleus with respect to SC1; SCN, sperm cell nucleus; SEM, scanning electron microscopy; TEM,
transmission electron microscopy; VC, pollen grain vegetative cell; VN, pollen vegetative cell nucleus.
ª The Author [2011]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1622 | McCue et al.
Fig. 1. Development of the SC cytoplasmic projection. Mitotic divisions between the time of meiosis and fertilization distinguish
gametogenesis of the plant kingdom from that of the animal kingdom. In angiosperm pollen development, the first mitotic division after
meiosis is asymmetric, producing a generative cell (GC) within the vegetative pollen grain. Generative cell membrane, shown in orange,
contacts the vegetative nucleus (VN) in the late stages of binucleate pollen. The timing of the second pollen mitotic division varies
between species, occurring either before or after pollen dehiscence and pollen germination. After this division, which produces two
sperm cells (SCs) from the generative cell, only one sperm cell remains connected to the VN. The connection, which persists during
pollen tube growth until just before fertilization, secures the male germ unit.
timing of this second pollen mitosis varies between species,
occurring either before or after anther dehiscence and
pollination.
In some angiosperms, one SC may possess a cytoplasmic
projection that is in direct contact with the VC nucleus (VN).
The physical connection of a SC to the VN was first observed
by Jensen and Fisher (1970), but was first described in detail
in Plumbago zeylanica and given a possible structural
significance by Russell and Cass (1981). The existence of this
structural connection between the germinal SCs and the
somatic VN has led to the proposal of a functional unit of
fertilization in angiosperms (Russell and Cass, 1981) and
was coined the ‘male germ unit’ (MGU) by Dumas et al.
(1985). Despite continued controversy about its existence and
prevalence, the MGU has been detected in most species
examined (Table 1). The connection between the SCs and VN
ensures that the VN is associated with the sperm cells as all
three nuclei travel as a unit through the pollen tube (Fig. 1).
However, little is known about the structure of the SC
cytoplasmic projection or the function of the MGU. Some
functional roles have been proposed in the literature, such as
facilitating the movement of the SCs in the pollen tube and
ordering the events of double fertilization (McConchie et al.,
1985; Russell, 1985; Mogensen, 1992; see below). Recent
evidence has suggested direct communication between the VN
and SCs (Slotkin et al., 2009), and the SC cytoplasmic
projection may provide a direct line for this communication.
These new data call for a re-examination of the available
literature on the nature of the SC cytoplasmic projection and
the functional role of the MGU. In this review, what is
known about the molecular nature of the SC cytoplasmic
projection is summarized, with emphasis on its potential role
in aiding communication between the VN and SCs.
Intercellular connections
Sperm cells and the vegetative cell cytoplasm
After pollen mitosis II, the two SCs remain linked until just
prior to fertilization (white arrows, Fig. 2). Both SCs
Potential roles of the male germ unit | 1623
Table 1. Plants examined for the presence of generative cell or
sperm cell cytoplasmic connection to the pollen vegetative nucleus
Plant/Order
Eudicots
Plumbago zeylanica/
Caryophyllales
Spinacia oleracea/
Caryophyllales
Brassica oleracea/
Brassicales
Arabidopsis thaliana/
Brassicales
Petunia hybrida/
Solanales
Nicotiana alata and
Nicotian tabacum/
Solanales
Cyphomandra
betacea/Solanales
Euphorbia dulcis/
Malpighiales
Gerbera jamesonii/
Asterales
Helleborus foetidus/
Ranunculales
Monocots
Zea mays/Poales
Bi- or
tricellular
pollen at
dehiscence?
SC/GC to
Reference
VN
connection
detected?a
Tri
Yes
Tri
Yes
Tri
Yes
Russell and Cass,
1981
Russell, 1984
Wilms and van Aelst,
1983
Dumas et al., 1985
McConchie et al.,
1985
Matthys-Rochon
et al., 1987
Lalanne and Twell,
2002
Wagner and
Mogensen, 1988
Cresti et al., 1985
Tian et al., 1998
Tri
Yes
Bi
Yes
Bi
Yes
Bi
Yes
Hu and Yu, 1988
Tri
No
Tri
No
Murgia and Wilms,
1988
Provoost et al., 1988
Bi
No
Heslop-Harrison
et al., 1986
Tri
Yes
Triticum aestivum/
Poales
Hordeum vulgare/
Poales
Tri
No
McConchie et al.,
1987
Matthys-Rochon
et al., 1987
Mogensen, 1986a
Tri
Yes
Hippeastrum vitatum/
Asparagales
Bi
Yes
Mogensen and
Wagner, 1987
Charzynska et al.,
1988
Mogensen, 1986b
a
Connection detected between either the generative cell (GC) or
sperm cell (SC) and the pollen vegetative nucleus (VN).
possess their own plasma membranes and a thin polysaccharide extracellular matrix (ECM), but lack a rigidly
structured true cell wall comprised of cellulose and callose.
The region between the SCs contains many of what Dumas
et al. (1985) termed ‘trabeculae’, strands of electron-dense
material in transmission electron microscopy (TEM)
images, which potentially link the two SC plasma membranes and cytoplasm (Fig.3f). In addition to these potential channels between the two SCs, the GC and SCs may
also contain connections to the VC cytoplasm. For example, in tobacco (Nicotiana tabacum) Cresti et al. observed
that the GC membrane shares what these authors referred
to as ‘abundant plasmodesmata-like channels’, potentially
linking the GC and VC cytoplasm (Cresti et al., 1987).
These potential connections occur where the GC wall and
VN are in close proximity (Cresti et al., 1987; Fig. 3A, B).
However, the appearance of what these authors termed
‘plasmodesmata-like’ connections are transient, as they are
lost upon tobacco pollen germination and movement of the
GC into the pollen tube (Cresti et al., 1987). It has been
argued that GC and SC viability is dependent on these
connections to the VC, as angiosperm SCs are small, often
lack plastids, and possess very little cytoplasm (reviewed in
Boavida et al., 2005). However, isolated SCs remain viable
and can participate in in vitro fertilization (Faure et al.,
1994), and the GC can remain viable inside of a dead VC
for days (Twell, 1995), demonstrating that, to some extent,
SCs are autonomous.
Direct connection of one SC to the VN
In addition to the apparent connection of the SCs to the VC
cytoplasm, most angiosperms display a direct connection of
one SC directly to the VN (Table 1; Fig. 1; red arrows in
Fig. 2). The SC that contacts the VN is termed SC1 and, in
species with dimorphic SC size (such as Plumbago), SC1 is
larger than SC2 (Russell, 1984, refers to SC1 and SC2 as
SVN and SUA). SC1 is connected to the VN by a cytoplasmic
projection of GC origin (Russell and Cass, 1981). The
projection can be detected by light microscopy (Fig. 2A, B),
SEM (Fig. 2C), fluorescence microscopy with the appropriate fluorophore marker (Fig. 2E, D–F), and TEM (Fig. 3E).
Of note, the distal SC2 also has a smaller cytoplasmic tail or
projection region, which does not extend toward the VN
(Fig. 2C; Russell and Cass, 1981).
The size of the cytoplasmic extension varies among
species and changes during pollen development, germination, and tube growth. The cytoplasmic projection often
appears short in the ungerminated pollen grain and
elongated in the pollen tube, as it seems to tether the SCs
to the VN (Fig. 1; Yu et al., 1992). The most well-studied
SC cytoplasmic projection is from Plumbago. Its projection
to the VN in the rapidly elongating pollen tube may extend
in excess of 30 lm long and less than 1 lm in width (Russell
and Cass, 1981).
Development of the SC cytoplasmic
projection
Formation of the cytoplasmic projection
Whether a plant species displays bicellular or tricellular
pollen at dehiscence, the timing of the SC1 cytoplasmic
projection formation is the same. The formation of the
cytoplasmic projection occurs after pollen mitosis I, as the
GC is formed and migrates to central a position within the
VC cytoplasm (Fig. 1). In Plumbago, the GC becomes
polarized with microtubule extension toward the VN, and
this polarized organization of the GC leads to dimorphic
1624 | McCue et al.
Fig. 2. Different microscopy methods used to detect the pollen cytoplasmic projection from the published literature. The cytoplasmic
projection is detected using phase contrast microscopy (A, B), scanning electron microscopy (C), and fluorescence microscopy (D, E, F).
In all images a red arrow points to the cytoplasmic projection connecting the SC and VN, while a white arrow points to the
interconnection of the two SCs. Scale bars are defined for each image. (A) The MGU isolated from the pollen tube of tobacco (from Tian
et al., 1998, and reproduced by kind permission of Springer). Black scale bar¼10 lm. (B) The isolated MGU from Brassica oleracea
pollen (from Matthys-Rochon et al., 1987, and reproduce by kind permission of The American Society of Plant Biologists). Black scale
bar¼10 lm. (C) The connection between the two SCs and the VN in tobacco pollen tubes (from Tian et al., 1998, and reproduced by
kind permission of Springer). White scale bar¼2 lm. (D) The SC cytoplasmic projection illuminated with DAPI fluorescence in the
tobacco pollen tube (from Yu et al., 1989, and reproduced by kind permission of Springer). The image is 31280 magnification. (E) Twophoton fluorescence microscopy image of an Arabidopsis pollen grain with a SC-specific promoter driving the expression of GFP (shown
in black) from McCormick (2004) and reproduced by kind permission of The American Society of Plant Biologists. (F) Fluorescence
microscopy image of an Arabidopsis pollen grain with a GFP-labelled SC plasma membrane (green) in the same pollen grain as a RFPlabelled VN (red). White scale bar¼10 lm.
SC size after pollen mitosis II (Russell et al., 1996). The
extension of the polarized GC becomes associated with
a pre-formed groove on the surface of the VN (Russell et al.,
1996). Growth and expansion of the cytoplasmic projection
is fuelled by microtubule elongation and is associated with
vesicle formation at the tip of the projection (Yu et al.,
1992). Yu et al. (1992) noted that this process of vesicle
formation in tobacco appears similar to the production of
enucleated cytoplasmic bodies and vesicle-containing bodies
that shed mass of the GC and SCs as they become smaller.
In fact, production of vesicle-containing bodies and reduction of GC and SC size occurs throughout pollen
development, as even species that are assumed to have
isomorphic SC size display dimorphism in late maturation
Potential roles of the male germ unit | 1625
Fig. 3. TEM images of pollen grains and tubes. All material was cryo-fixed and cryo-substituted as described elsewhere (Van Aelst et al.,
1993). In each image the VN is coloured red and the GC nucleus (GCN) is coloured yellow. Scale bar¼1 lm in all panels. (A) Crosssection of a hydrated pollen grain of Papaver rhoeas. In this species the GC shows a typical convoluted shape, which increases the
surface area in contact with the surrounding VC. The GCN occupies practically all the volume of the GC, and the VN is crescent-shaped,
surrounding most of the GC. (B) Enlargement of the dashed line rectangle in panel A (rotated counter-clockwise). The polysaccharide
nature of the GC ECM is highlighted by the darkened contrast given by the PATAg test. Using this contrast, the highly perforated SC
ECM is visible (arrows). Cresti et al (1987) described these channels as ’plasmodesmata-like’ and speculated that they effectively
connect the cytoplasm of the VC and GC. (C) A germinating pollen grain of tobacco, showing GC deformation just before entry into the
pollen tube. This deformation is highly suggestive of a mechanical pulling force from the VN at the site where the VN and GC wall are in
very close vicinity (dashed line rectangle). (D) Enlargement of the dashed line rectangle in (C). The tangential section of the GC wall (GCW)
highlights the existence of microtubules inside the GC (arrow). (E) Mid-section through a dehydrated mature pollen grain of Arabidopsis.
A convoluted and often perforated VN is typical of the final stages of pollen dehydration. The adjacent SC is attached at one end via its
cytoplasmic projection to the outer envelope of the VN (arrow). The SC nucleus (SCN) is shown coloured in blue. (F) Cross-section of an
Arabidopsis pollen tube, showing the close relationship between the two SCs (arrow), with the respective ECMs forming a jigsaw-like
pattern.
1626 | McCue et al.
Post-pollination
Fig. 4. Integrated model of the SC cytoplasmic projection
connected with the VN. The cartoon is adapted from Fig. 1 of
McConchie et al. (1985) and is reproduced by kind permission of
Springer. The objects in the image are not drawn to scale.
ER¼endoplasmic reticulum.
(Tian et al., 2001). The same timing of cytoplasmic projection formation likely occurs in the tricellular pollen of
Arabidopsis thaliana, as the GC and VN are still associated
in duo1 pollen, a mutant that lacks pollen mitosis II
(Rotman et al., 2005).
Connection to VN
At the time of the cytoplasmic projection formation, the
surface of the VN is highly lobed, possessing many
indentations and numerous nuclear pores (Russell and Cass,
1981; Fig. 3E). The SC cytoplasmic projection wraps
around the VN and attaches to the VN at the sites of these
indentations, referred to as ‘clasping grooves’ by Russell
(1984) on the VN surface (Fig. 4). The cytoplasmic projection has been shown on occasion to penetrate the VN
through a convoluted passage, terminating within its
enclaves at the far side of VN entry (Figs 2F, 4; Russell,
1984; McConchie et al., 1985; Kaul et al., 1987). Within the
‘clasping grooves’ of the VN, rough endoplasmic reticulum
(rough ER) forms membrane arrays. In addition, laminar
and smooth ER is enriched on the VN membrane adjacent
to the SCs (Dumas et al., 1985; Cresti et al., 1987; Russell
and Cass, 1981). Rough and smooth ER are also observed
in portions of the cytoplasmic projection itself (Russell and
Cass, 1981; Yu et al., 1989). The connection between the
VN and SC is made through this ER-dense material
surrounding the VN (Fig. 4; Yamamoto et al., 2003). These
regions rich in ER are probably the site of protein and lipid
synthesis from the VN, perhaps localized to this region due
to higher levels of nuclear export. However, direct functional evidence of ER accumulation at these sites is lacking.
In most angiosperm species the VN precedes the SCs in the
growing pollen tube, while the connection between the VN
and SCs in the pollen tube remains intact (Fig. 2A–D). In
the pollen tube, the SCs of some species remain very close
to the VN, whereas the SCs of other species, such as
tobacco, remain connected to the VN by the cytoplasmic
extension at a distance behind the VN (Fig. 2D). This
distance between the VN and the distal SC can reach up to
100 lm (Yu et al., 1989). Whether they lag far behind the
VN or remain closely associated with the VN, the SCs and
the VN travel as a linked MGU in the pollen tube. The
cytoplasmic projection becomes reduced near the end of the
pollen tube growth and is severed following release of the
SCs. In Plumbago, which lacks synergid cells, the cytoplasmic projection is fractured into pieces upon entry into the
embryo sac and is later isolated in membrane-bound bodies
near the site of gamete fusion between the zygote and
endosperm (Russell, 1984). The fragmented cytoplasmic
projection can remain in enucleated bodies for days postfertilization before completely degenerating (Russell, 1982,
1983, 1985).
Molecular nature of the cytoplasmic
projection
Very little is known about the structural components of the
cytoplasmic projection. The projection is thought to possess
a microtubule-based cytoskeletal framework (Fig. 4) that
may provide structural support to the cytoplasmic projection. The overall organization of microtubules in the SCs
is fundamentally different compared with somatic cells of
the plant, as arrays do not emanate from the SC nuclear
envelope (McConchie et al., 1985; Southworth and Cresti,
1997). In tobacco, a GFP fluorescent a-tubulin6 reporter
protein highlights the cytoplasmic extension from the GC to
the VN, demonstrating that microtubules are present within
the cytoplasmic projection in living cells (Oh et al., 2010).
During formation of the projection, two microtubule ridges
form and fuse into a single microtubule array at the tip of
the cytoplasmic projection where it enters the VN (Fig. 4;
McConchie et al., 1985; Palevitz and Cresti, 1988). In
Tradescantia SCs, thick bundles of microtubules branch to
thinner strands arranged longitudinally to the cell axis,
surrounding the SC nucleus (Palevitz and Cresti, 1988).
McConchie et al. (1985) described this arrangement as
‘cage-like’ and suggested that this organization dictates SC
shape, imparts increased rigidity to the SCs and MGU, and
could potentially play a role in the maintenance of the
cytoplasmic projection’s contact with the VN. Supporting
the potential role of microtubules in influencing cell shape,
microtubule bundles disappeared when cell shape was
altered upon purification of the GC (Theunis et al., 1992).
However, it was later argued that microtubules are only
partially responsible for maintaining SC shape (Southworth
and Cresti, 1997). Breakdown of the cytoskeletal network
Potential roles of the male germ unit | 1627
may not only cause SCs to assume a spherical shape but
may also influence MGU disassociation (Ge et al., 2011). In
mutants of the gem1 microtubule-associated protein of
tobacco and Arabidopsis, tubulin arrays are not established
correctly, resulting in both a lack of polarization of the
uninucleate pollen grain and proper germ cell identity (Oh
et al., 2010).
The most extensive genetic analysis of the cytoplasmic
projection and the MGU linkage was performed as
a DAPI-stained, microscopy-based forward mutant pollen
screen of EMS-mutagenized plants (Lalanne and Twell,
2002). A class of mutants named gum (germ unit malformed)
displayed a displaced VN, separated from the SCs. In these
mutants, after pollen mitosis II, the VN migrates to the
pollen cell wall, disrupting normal compaction of the
MGU. Although the VN and SC are separated at a larger
than normal distance, long projections of the SC1 to the
VN were occasionally observed. This demonstrates that
although the components of the MGU are physically
separated in gum mutants, they can still be linked by the
cytoplasmic projection. Importantly, gum mutants affected
the transmission of the mutant allele through pollen,
suggesting that the association and spacing of the MGU is
critical for successful fertilization.
The GEX2 protein of Arabidopsis is a transmembrane
domain protein that is specifically localized to the SC
plasma membrane. When the GEX2 promoter region
is used to drive GFP, the exterior of the SCs and the cytoplasmic projection fluoresce (Engel et al., 2005; Fig. 2E, F).
This demonstrates that the plasma membrane material of
the cytoplasmic projection is of SC origin, confirming that
the SC plasma membrane extends to contact the VN
(Yu et al., 1992).
Evolution of the SC cytoplasmic projection
The cytoplasmic projection connecting the SC1 to the VN
has been detected in many angiosperms, independent of
whether the species is a monocot or eudicot, or whether
the species displays bi- or tricellular pollen at dehiscence
(Table 1). The cytoplasmic connection has even been found
in the pollen of grasses such as maize, in which the
connection was originally thought to be absent (McConchie
et al., 1987). The maize MGU connection is detected only
after pollen hydration and activation (Matthys-Rochon
et al., 1987; McConchie et al., 1987). Due to the transient
nature of the MGU and cytoplasmic projection, the projection may exist but remain undetected in many organisms
that have been reported to lack the connection altogether
(Mogensen, 1992).
Unlike seedless plants, the SCs of angiosperms do not
have flagella or self-generated autonomous mobility.
Rather, movement is generated by growth of the pollen
tube and cytoskeletal motors associated with the SC ECM
and other organelles. The SCs of cycads and Ginkgo biloba
have proper flagella, while the SCs of other gymnosperms
do not (Southworth and Cresti, 1997). Because the SCs
have a unique microtubule organization that is distinct from
that of other somatic plant cells, it has been speculated that
the angiosperm SC cytoplasmic projection is a flagellar
remnant (reviewed in Palevitz and Tiezzi, 1992). This
angiosperm SC microtubule organization is comparable
(although greatly reduced) to the complex multi-layered
microtubule structures that form the base of the flagella in
Ginkgo and cycad sperm. It is interesting to speculate
further that both the cytoplasmic projection and the process
of double fertilization could only evolve after the loss of
sperm flagella; thus the MGU or pollen cytoplasm could
potentially co-ordinate one sperm to the egg cell and one to
the polar nuclei, rather than having both self-motile sperm
competing to fertilize the same egg cell.
Potential roles of the SC cytoplasmic
projection
Structural stabilization of the MGU
The most obvious potential role of the cytoplasmic projection is the physical connection and stabilization of the
MGU in the pollen tube. Recent data have demonstrated
that MGU linkage is necessary to direct the movement of
the MGU in the growing pollen tube (Ge et al., 2011).
Using live cell imaging, Ge et al. found that as SCs enter the
pollen tube, their movement can be somewhat random, as
the cytoplasmic projection can be in front of or behind the
SC in the developing tube. As the tube elongates, polarity is
established, and the cytoplasmic projection precedes the
SCs in the pollen tube. However, even at the peak of SC
movement in the pollen tube, the cytoplasmic projection
displays dynamic and independent movements and changes
in length. During pollen tube growth, destabilizing the
MGU by exposure to heat can result in disassociation of
the SCs, the loss of SC movement, or the failure of SCs to
enter the growing pollen tube (Ge et al., 2011). These data
suggest that the cytoplasmic projection and MGU linkage
are essential for the movement of SCs within the growing
pollen tube.
The mechanism of how the MGU moves in the pollen
tube remains largely unknown. The overall movement of
the MGU is consistently forward in the pollen tube,
punctuated by more minor advances and retreats (Kliwer
and Dresselhaus, 2010; Ge et al., 2011). The MGU seems to
move independently of the organellar and cytoplasmic
cycling occurring in the growing pollen tube (Cai and
Cresti, 2009). Myosin I is present on the surfaces of the VN
and GC (Heslop-Harrison and Heslop-Harrison, 1989b;
Miller et al., 1995), and the growing pollen tube contains
an actin microfilament cytoskeleton (Heslop-Harrison and
Heslop-Harrison, 1989b; Cardenas et al., 2008). The movement of the VN in the pollen tube is dependent on these
microfilaments, contorting the VN as it enters and moves in
the pollen tube (Heslop-Harrison and Heslop-Harrison,
1989a).
In addition to actin filaments, longitudinal microtubule
tracks form during pollen germination, creating a network
around the VN and GC. Both the migration and the relative
1628 | McCue et al.
positioning of the MGU in the pollen tube are dependent
on the presence of microtubules, and studies from in vitro
motility assays suggest that microtubules also play a regulatory role in the trafficking of some organelles (Romagnoli
et al., 2007; reviewed in Cai and Cresti, 2011). However,
the movement of the MGU is seemingly independent from
the movement of the VC organelles. Furthermore, the
MGU movement through the pollen tube relies more on
microtubules than does the cycling movement of the VC
organelles. Treatment of pollen tubes with microtubule
inhibitors does not halt pollen tube growth or organelle
movement, but Åström et al. (1995) observed restricted
movement of the tobacco VN and GC within the pollen
tube. Treatment using another microtubule inhibitor
resulted in increased spacing between the VN and GC
compared with an untreated control (Heslop-Harrison
et al., 1988). An interesting but speculative conclusion from
both of these studies was that, at intermediate levels of
microtubule inhibitors, the GC moved into the pollen tube
before the VN (if the VN entered at all) more often than in
control pollen (Heslop-Harrison et al., 1988; Åström et al.,
1995). At higher concentrations of microtubule inhibitors,
neither the GC nor VN entered the pollen tube. Based on
these findings, both sets of authors of these studies
suggested that the VC microtubules are more susceptible to
inhibition compared with the GC microtubules, potentially
due to the additional membrane layers of the GC. Second,
the authors noted that the GC is able to move into the
pollen tube without movement of the VN and with apparent
disassociation of the MGU, arguing against the requirement
of microtubules and the MGU for initial GC and SC
movement.
Ordered arrival and preferential fertilization
The cytoplasmic projection may play a role in dictating the
ordered arrival of each SC to the embryo sac, thereby
determining which SC fertilizes the egg. In Plumbago, SC
cytoplasm and organelles are transferred to the zygote and
endosperm (Russell, 1983, 1985). Based on the SC plastid
and mitochondria morphology and subsequent content of
the Plumbago embryo, it was found that SC1 preferentially
fuses with the polar nuclei, generating the endosperm.
Likewise, SC2 in Plumbago preferentially fertilizes the egg
cell (Russell, 1985). These studies in Plumbago suggest that
either SC dimorphism and/or the direct connection to the
VN order the events of fertilization. However, the embryo
sac of Plumbago does not have synergid cells and may
represent an exception to the general trend. In addition to
the example of Plumbago, classic studies on B chromosomes
in maize indicated that B chromosomes preferentially
segregate into one of the two SCs, and this SC was more
often used for fertilization of the egg cell (Roman, 1947,
1948). However, it has subsequently been determined that
although there is preferential fertilization, either SC is
capable of fertilizing the egg cell in vitro (Faure et al.,
1994), independent of the presence or absence of B
chromosomes (Faure et al., 2003). As the experimental
conditions of in vitro fertilization may alter the properties of
the SCs, these results remain open to debate.
Research in Arabidopsis has provided contrasting results,
with some studies supporting a SC preference for fertilizing
the egg cell (Iwakawa et al., 2006; Nowack et al., 2006), and
others showing no fertilization preference (Chen et al., 2008;
Ingouff et al., 2009). The most recent evidence used a pollen
mutant for the cyclin dependent kinase A1 gene, in which
some pollen has only one SC, and demonstrated that the
single SC has equal ability to fertilize the egg cell or polar
nuclei (Aw et al., 2010). Therefore, aside from the clear
exception of Plumbago (and possibly maize SCs with
B chromosomes), the current consensus is that there is no
predetermination or preference for an individual SC to
fertilize the egg cell. Given the explosive discharge of SCs
into the synergid (Rotman et al., 2003), little is known
about how each SC is directed to its respective target cell.
During fertilization, the MGU may co-ordinate the simultaneous deposition of the SCs into the embryo sac, providing the SCs with access to the egg cell and polar nuclei at
the same time, leading to non-preferential fertilization.
A potential new role for the cytoplasmic projection in
cell-to-cell communication
In addition to the possible structural roles of the cytoplasmic projection and MGU, Mogensen (1992) proposed
a potential new function for this structure. He envisioned
a role for the connection between SC1 and the VN
facilitating the transfer of material or information between
these two entities. Although no transfer of RNA or protein
through the cytoplasmic projection has been observed
directly, the cell non-autonomous impact of transcriptional
products from the VN has been recently documented. The
production of an artificial microRNA transcript from the
VN is able to reduce the fluorescence of its GFP reporter
target transcript in SCs of Arabidopsis (Slotkin et al., 2009).
As a control, the same VC-specific promoter was used to
drive the expression of separate artificial microRNA
that does not target GFP, which displayed no effect on
SC GFP fluorescence. MicroRNAs are known to act cellautonomously in the plant body (reviewed in Voinnet,
2005); therefore, this ability of a VN-transcribed microRNA
to repress SC GFP suggests that the cellular connection
between the VC and SCs is intimate, similar to cells of the
meristem or phloem. This experiment suggests that at least
one direction of communication exists in the mature pollen
grain: from the VN to the SCs. Although communication
was detected, the developmental timing of this event is
unknown. Communication could potentially occur in bicellular pollen, between the VC and the GC, ensuring
that both SCs receive the same information. The repression
of GFP may even initiate in the unicellular microspore,
as, although GFP expression is not present using this
SC-specific GFP transgene at this stage (Engel et al., 2005),
the expression of the VC-specific promoter driving the
artificial microRNA has been observed in late unicellular
microspores (Eady et al., 1994). Therefore, the microRNAs
Potential roles of the male germ unit | 1629
could carry forward from the unicellular microspore to the
GC and SCs, and the lack of GFP expression in mature SCs
would then merely be reflective of this previous repression
event. However, in separate experiments, GFP encoded by
the same VC-specific promoter used to drive the artificial
microRNAs does not accumulate in the GC (Chen et al.,
2009), suggesting that the GC does not directly inherit RNA
transcripts present in the unicellular microspore. Though
the developmental timing of the above events is unclear,
these experiments suggest the cell non-autonomy of VC
small RNAs, giving credence to Mogensen’s (1992) proposed communicative function of the SC cytoplasmic
projection.
The study of microRNA movement in pollen was an
artificial system to model small RNA movement in the
pollen grain (Slotkin et al., 2009). Of primary interest was
the detection of transposable element transcripts produced
from the VN. The enzymatic mechanisms required to
suppress transposable element expression and mobilization
are absent in the VN (Slotkin et al., 2009), while components of this machinery necessary to epigenetically silence
transposable elements are specifically expressed in the SCs
(Pina et al., 2005; Borges et al., 2008; Chen et al., 2009;
Grant-Downton et al., 2009; Schoft et al., 2009; Slotkin
et al., 2009). In addition to transposable element expression,
high levels of some transposable element small RNAs
accumulate in SCs, and these authors speculated that
transposable element small RNAs were communicating
epigenetic information from the VN to the SCs in a manner
similar to the proposed microRNA cell non-autonomy.
Communication between the VC and SCs could potentially move directly from the VN to the SC via the
cytoplasmic projection or indirectly from the VC cytoplasm
into the SC. In support of the theory that the cytoplasmic
projection aids the communication between the two pollen
cell types, nuclear pores of the VC are organized in a polar
manner, seemingly facilitating transfer of information to
the connected SC. In Medicago sativa (alfalfa), pores
are uniformly distributed on the nuclear envelope before
the attachment of the GC to the VN. However, after the
establishment of the connection between the two cells,
there is a 69% higher pore density on the envelope surface
adjacent to the cytoplasmic connection, compared with the
opposite side of the same vegetative nuclear envelope (Shi
et al., 1991). In the pollen of Hippeastrum vitatum and
Clivia miniata, ATPase activity peaks at the interface of the
VN and GC, suggestive of the ATP hydrolysis required for
nuclear trafficking (Tang, 1988). These two studies of the
VC’s nuclear envelope suggest that the cytoplasmic projection may serve to increase the surface area and interaction between the connected SC and VN (Yu et al.,
1992). This physiological relationship between the VN and
its associated SC may potentially aid the interaction,
movement, and cell non-autonomy of RNA and/or proteins
in the pollen grain. In conclusion, great potential exists for
the transfer of information from the somatic VC to the
germinal SCs.
Impact of this new potential role of the MGU
The morphological data reviewed here demonstrate a direct
physical connection between the VN and SCs in angiosperms. The MGU concept has remained polemic to some
extent and without a clear functional significance. Recent
molecular data have now suggested that biological information can be communicated from the VN to the SCs. Due to
this correlation, we believe that there is a direct communicative role for the MGU, and the SC cytoplasmic projection
may, both physically and functionally, link the MGU. This
would constitute a re-examination of the MGU concept, the
implications of which are barely beginning to be understood. It is envisioned that this transfer of information to
the gametes could play a role in parental programming of
the next diploid generation, ultimately affecting the development of the endosperm and embryo.
Acknowledgements
The authors thank David Twell and Scott Russell for
critically reviewing this manuscript and Scott Russell for
sharing data prepublication. ADM is supported by The
Ohio State Graduate School’s University Fellowship. JAF
acknowledges funding from FCT grants PTDC/BIA-BCM/
108044/2008 and PTDC/QUI/64339/06. RKS’s laboratory is
funded by the National Science Foundation research grant
MCB-1020499.
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