Articles
Sperm Delivery in Flowering
Plants: The Control of Pollen
Tube Growth
KATHLEEN L. WILSEN AND PETER K. HEPLER
Although most people think of pollen merely as an allergen, its true biological function is to facilitate sexual reproduction in flowering plants. The
angiosperm pollen grain, upon arriving at a receptive stigma, germinates, producing a tube that extends through the style to deliver its cargo to the
ovule, thereby fertilizing the egg, and completing the life cycle of the plant. The pollen tube grows rapidly, exclusively at its tip, and produces a cell
that is highly polarized both in its outward shape and its internal cytoplasmic organization. Recent studies reveal that the growth oscillates in rate.
Many underlying physiological processes, including ionic fluxes and energy levels, also oscillate with the same periodicity as the growth rate, but
usually not with the same phase. Current research focuses on these phase relationships in an attempt to decipher their hierarchical sequence and to
provide a physiological explanation for the factors that govern pollen tube growth.
Keywords: actin cytoskeleton, ion dynamics, molecular switches, oscillatory growth, pollen tube
I
n animals, meiosis produces gametes that fuse to
form a zygote during sexual reproduction. The life cycle of
flowering plants, referred to as an alternation of generations,
is more complex. Here, a multicellular diploid generation,
known as the sporophyte generation, alternates with a multicellular haploid generation, known as the gametophyte generation. Meiosis does not directly produce gametes. Rather,
cells of the sporophyte generation undergo meiosis to produce
haploid spores, which in turn divide mitotically to give rise
to the gamete-producing gametophyte generation. The embryo sac, which bears the ovule and is embedded in the
flower, constitutes the female gametophyte generation. The
pollen grain is the male gametophyte generation produced
from microspores in the anthers of flowering plants, and
consists of a vegetative cell and a generative cell. Either in the
grain or during pollen tube growth, depending on the species,
the generative cell undergoes mitosis to give rise to two sperm
cells (Raven et al. 1999).
Double fertilization is a hallmark of flowering plants
(Dresselhaus 2006). In addition to the fusing of one sperm cell
with an egg cell to give rise to an embryo, the second sperm
cell fuses with the two polar nuclei in the central cell of the
female gametophyte to produce the nutritive triploid tissue
of the endosperm. The embryo and endosperm are packaged
as a seed, which becomes encased in a fruit formed from the
ovary and, in some instances, from additional floral parts. It
will be apparent, therefore, that double fertilization is not only
necessary for sexual reproduction in flowering plants but
also essential for the production of much of the food that we
eat, including nuts, seeds, grains, and fruits.
Because the sperm of flowering plants have no flagella, they
do not depend on water to transport them to the ovule, as
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do the sperm of protists, bryophytes, ferns, and some gymnosperms. Instead, the pollen grain may travel a great distance,
transported by wind or an animal carrier (e.g., bird, bat,
insect), before alighting on a receptive stigma and germinating. The sperm cells then travel in the cytoplasm of the
large vegetative cell of the pollen tube to their target. A
pollen tube is thus the tricellular male gametophyte generation that emerges from a pollen grain to bring about
double fertilization.
Pollen tube growth is fast and highly polarized, with new
material being added at the tip, which is called the apex.
Secretory vesicles inside the pollen tube transport the cellular building blocks required for growth to the apex, where they
are incorporated into the extending pollen tube by exocytosis (Hepler et al. 2006). This process is so efficient that a
pollen tube from Zea mays (corn) can attain growth rates of
up to 1 centimeter (cm) per hour, or approximately 3 micrometers (µm) per second, making it one of the fastest-growing cells known. With the added realization that cell elongation
can be visualized under carefully controlled in vitro conditions,
the pollen tube becomes an excellent model system for studies of plant cell growth. Clearly, it is ideal for enhancing our
understanding of other cells undergoing tip growth, such as
root hairs, fungal hyphae, and fern and moss protonemata,
but the pollen tube may also serve as an effective model for
Kathleen L. Wilsen (e-mail: [email protected]) works in the School
of Biological Sciences at the University of Northern Colorado in Greeley.
Peter K. Hepler (e-mail: [email protected]) works in the Biology and Plant
Biology Graduate Program at the University of Massachusetts in Amherst.
© 2007 American Institute of Biological Sciences.
November 2007 / Vol. 57 No. 10 • BioScience 835
Articles
the many plant cells that exhibit diffuse growth. In defense of
this assertion, we note the similarity in cell wall structure and
composition and membrane trafficking machinery between
pollen tubes and other plant cells.
In this article we describe the structure and physiology of
a growing pollen tube, focusing on several processes believed
to be key in the regulation of growth. We exploit the fact that
pollen tube growth oscillates, as do many underlying physiological processes and structural elements. Through an analysis of the temporal and spatial ordering of these many factors,
we provide insight into the basic regulatory events that control pollen tube growth. Although we include data from
pollen tubes of different species (e.g., Arabidopsis and Nicotiana), we focus in particular on those from the lily family (e.g.,
the Easter lily, Lilium longiflorum), which, because of their
relatively large size and their readiness to germinate in vitro
and grow at in vivo rates, have been the subject of many
insightful studies.
Growing pollen tubes are highly organized
Whereas the diameter of a pollen grain is between 10 and 100
µm (lily pollen is about 60 µm), the length of a pollen tube
is much greater, and can reach several centimeters (approximately 10 cm in lily) as it grows through the tissues of the style
to deliver the sperm cells to the embryo sac. The growing lily
pollen tube is around 15 µm in diameter (figure 1) and has
an apical dome that is roughly hemispherical in shape. As the
pollen tube grows, callose plugs are laid down in the shank
in such a manner that the cytoplasm remains in the apical end
of the growing tube. Because of this mechanism, pollen tubes
have been described as moving cells, in that a fixed plug of
cytoplasm containing floating sperm cells moves forward as
the cell wall extends (Sanders and Lord 1992).
Both light and electron microscopy reveal that organelles
have a particular arrangement inside a pollen tube (figures 1,
2; Hepler et al. 2001). Most of the pollen tube is granular in
appearance because of a rich supply of starch-containing
plastids, or amyloplasts (figure 1). However, amyloplasts and
vacuoles are excluded from the extreme apex, creating the
Figure 1. A living lily pollen tube observed using Nomarski differential interference contrast optics. Many
starch-containing amyloplasts give the shank a granular
appearance. However, these amyloplasts are excluded
from the apex, giving rise to the clear zone. All of the cytoplasmic inclusions undergo continuous cytoplasmic
streaming, in which they flow forward along the edge of
the cell and rearward through the central core. This pattern has been called “reverse fountain” streaming, and
gives rise to the funnel-shaped appearance, which is evident at the base of the clear zone. Bar = 10 micrometers.
836 BioScience • November 2007 / Vol. 57 No. 10
so-called clear zone (figure 1). Although lacking certain inclusions, the extreme apex contains numerous secretory vesicles, as well as elements of the endoplasmic reticulum (ER),
mitochondria, and Golgi dictyosomes (figure 2). These vesicles are of particular interest because they contain the cell wall
precursors, which are delivered to the apex of the pollen
tube, where their contents are secreted to the wall, allowing
the cell perimeter to extend. Although mitochondria, Golgi
dictyosomes, and elements of the ER are abundant in the clear
zone, they are not confined to this region; they are present
throughout the pollen-tube shank (Parton et al. 2003, LovyWheeler et al. 2007).
Cytoskeletal elements, including both actin microfilaments
and microtubules, are ubiquitous components of the pollen
tube (Hepler et al. 2001). In lily pollen tubes, microtubules
form a prominent collar at the base of the clear zone (LovyWheeler et al. 2005); their function, however, is not clear. The
depolymerization of microtubules with oryzalin has no effect
on pollen tube growth or organelle positioning in vitro (LovyWheeler et al. 2007). Nevertheless, recent studies show that
some cytoplasmic components, including mitochondria and
Golgi vesicles, move slowly along microtubules, indicating that
these cytoskeletal elements may indeed contribute to the
control of pollen tube growth (Romagnoli et al. 2007).
In contrast to microtubules, a dynamic actin cytoskeleton
is widely acknowledged to be essential for pollen tube growth
(Hepler et al. 2001). Although there is agreement that the
shank of the pollen tube contains longitudinal bundles of actin
parallel to the axis of growth, the organization of these microfilaments in the clear zone has been widely debated.
Lovy-Wheeler and colleagues (2005) resolved this issue by employing an improved method of fixation, followed by labeling with antiactin antibodies, and analysis with confocal
microscopy. This study confirmed that a cortical fringe of actin
is a consistent feature of the clear zone, and that the extreme
apex contains relatively few actin filaments (figure 3; LovyWheeler et al. 2005).
The actin cytoskeleton serves three important functions in
growing pollen tubes. First, actin microfilaments, together with
the motor protein myosin (myosin XI in lily), drive cytoplasmic streaming. This process is thought to transport the
secretory vesicles from their point of origin to the apical end
of the pollen tube, where they ultimately empty their contents
into the expanding cell wall (Yokota and Shimmen 2006). Like
the secretory vesicles, all organelles, including amyloplasts,
dictyosomes, ER, mitochondria, and vacuoles, are propelled
by the actomyosin system and are in constant motion. In
angiosperm pollen tubes, cytoplasmic streaming exhibits a “reverse fountain” pattern in which the lanes move forward
along the edge of the cell and then reverse direction in the clear
zone, creating a massive central retrograde flow.
A second function of the actin cytoskeleton is controlling
the position of organelles within the pollen tube. Under
normal conditions, the polarized distribution of organelles,
including notably the detailed morphology of the clear zone,
is maintained despite the fact that the contents are constantly
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Articles
dome, esterified pectins are enzymatically demethylated, and the cell wall is greatly reinforced when calcium cross-links the acidic residues on the pectin
chains (Bosch and Hepler 2005). Thus, the pollen
tube wall is stronger and more rigid in the shank of
the tube than in the apex. This arrangement means
that only the apical wall is plastic and able to undergo strain deformation in response to internal
turgor pressure (Bosch and Hepler 2005). These
conditions contribute to the polarity of pollen tube
growth.
Calcium ions and protons are
essential for pollen tube growth
For more than 40 years it has been recognized that
calcium ions are required for pollen germination
and pollen tube growth (reviewed in Hepler et al.
2006). In addition to calcium being an essential
Figure 2. An electron micrograph of a lily pollen tube, prepared by rapid
component of the surrounding extracellular envifreeze fixation and freeze substitution, shows the clear zone. The apex
ronment, an intracellular calcium gradient and excontains numerous small vesicles; these fuse with the plasma membrane
tracellular fluxes associated with the pollen tube
and contribute material to the expanding cell wall. Mitochondria and
are essential for growth. Like most other cells, plant
elements of the endoplasmic reticulum are also abundant. Note that the
cell wall is thickest at the extreme apex, and that it becomes substantially or animal, the pollen tube experiences a relatively
high external calcium concentration, where it ranges
thinner along the flanks of the dome. It is thought that this transition in
thickness reflects the conversion of the cell wall pectins from methyl esters between 10 micromolar (µM) and 10 millimolar.
However, within the cell the free concentration of
to acids together with calcium cross-linking. Source: Lancelle and Hepler
this ion is substantially lower, with basal con(1992). Bar = 1 micrometer.
centrations from 150 to 300 nanomolar (nM)
in motion. However, this polarized distribution of organelles
(Holdaway-Clarke and Hepler 2003). The underlying reason
is quickly and dramatically disrupted by antimicrofilament
for the low concentration of intracellular calcium may derive
from the physiological incompatibility of millimolar conagents (e.g., latrunculin B; Gibbon et al. 1999, Vidali et al.
centrations of phosphate and calcium, at which point these
2001). By some process that is not well understood, the actin
two ions would react to form an insoluble precipitate of calcytoskeleton appears to create a filter that allows certain orcium phosphate, and destroy phosphate-based energy meganelles such as the vesicles, mitochondria, Golgi dictyosomes,
tabolism.Virtually all living cells thus evolved mechanisms for
and ER to flow into the clear zone while preventing the inreducing the intracellular calcium ion concentration, leading
cursion of amyloplasts and vacuoles.
to the enormous concentration difference between the cytosol
A third function of actin relates to its direct role in the control of tube elongation. Researchers initially assumed that actin
contributed to growth indirectly, through the control of cytoplasmic streaming and the consequent delivery of secretory
vesicles to the apex. However, studies with agents that
control actin polymerization (e.g., latrunculin B, cytochalasin
D, DNAse, profilin) indicate that growth can be inhibited at
concentrations of these agents that are significantly lower
Figure 3. A confocal fluorescence image of actin labeled
than those required to block cytoplasmic streaming (Vidali
with an antiactin antibody. The cell was first subjected to
et al. 2001). These results strongly support the idea that actin
rapid freeze fixation and freeze substitution. It was then
polymerization contributes directly to pollen tube growth.
rehydrated and probed with the antibody. This figure,
Although much more work is needed to completely unravel
which is a projection of several confocal image slices,
the mechanisms of actin’s contribution to cell growth, we
shows the prominent actin fringe that starts about 5
increasingly realize that actin has a profound effect on cell
micrometers (µm) back from the apex and extends
elongation.
basally for another 7 µm. Three-dimensional analysis
The cell wall is another important structural feature of
indicates that the fringe is restricted to the cortex of the
growing pollen tubes (Geitmann and Steer 2006). The freshly
apical dome. Behind the fringe, the actin is evenly dissecreted pollen tube wall in the apex consists of methyltributed throughout the thickness of the pollen tube in
esterified pectins, which are displaced to the shank of the
finely articulated actin bundles. Source: Lovy-Wheeler
pollen tube as the cell grows. On the flanks of the apical
and colleagues (2005). Bar = 10 µm.
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November 2007 / Vol. 57 No. 10 • BioScience 837
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Figure 4. A composite image showing calcium and pH distribution in living lily pollen tubes. These are
selected image pairs from oscillating pollen tubes that show high (top left) and low (top right) calcium, and
high (bottom left) and low (bottom right) pH. Red indicates high calcium concentration and high pH. For
calcium detection, the cell was injected with fura-2-dextran; for pH detection, the cell was injected with
BCECF-dextran. The cells were subjected to ratiometric ion imaging. Source: Hepler and colleagues (2006).
and the extracellular environment. It seems likely that
during the process of evolution, this concentration difference
was exploited for signaling purposes. For example, a primary
messenger (e.g., hormone, light, gravity) relays its presence
to the cell through an interaction that causes the opening of
a calcium-selective pore or channel. Calcium flows into the
cell, generating a large local increase in its intracellular concentration. Calcium becomes the second messenger because
it will now bind to and activate proteins (e.g., calmodulin)
that are poised to carry the signal forward to the next step.
In the pollen tube, there is direct evidence for a localized
domain of elevated calcium (see below), which we presume
behaves as a second messenger and activates growthdependent events, without compromising phosphate-based
energy metabolism.
Several investigators report a region of high intracellular
calcium in the apex of the pollen tube, immediately adjacent
to the plasma membrane, where growth is maximal (figure
4, top row; Holdaway-Clarke and Hepler 2003, Hepler et al.
2006). The concentration of calcium in this localized region
may reach 10 µM (Messerli et al. 2000), and is more than an
order of magnitude greater than that in the shank, where
basal values from 150 to 300 nM are observed (HoldawayClarke and Hepler 2003). This gradient is referred to as
“tip-focused” because the calcium concentration is greatest
in the extreme apex and declines sharply with distance from
the apex. Thus calmodulin, a ubiquitous calcium-binding
protein that binds to and regulates numerous cellular targets,
or key enzymes, such as the calcium-dependent protein
kinases (CDPKs) commonly found in plants, may be saturated
with calcium and maximally active in the apex of growing
pollen tubes but switched off in the region just behind the apex
(Holdaway-Clarke and Hepler 2003). Pollen tube growth
rate and direction are positively correlated with the slope
838 BioScience • November 2007 / Vol. 57 No. 10
and position of the calcium gradient, and growth stops when
the gradient dissipates (Malhó and Trewavas 1996, Pierson et
al. 1996).
The observation that pollen tubes as well as virtually all
other cells have low basal levels of calcium means that there
are compensatory systems that respond to the ion, sequestering
it or extruding it from the cell. Indeed, within the pollen
tube the region of elevated calcium is restricted to the apical
portion of the clear zone, indicating that the removal system
must be located there. The localized ER and mitochondria are
strong candidates for the sequestering system because they
occur at high density at the base of the clear zone; preliminary
evidence also shows that the ER takes up calcium and transports it basally (Wilsen 2005). Studies directed at the flow of
ER and mitochondria in living pollen tubes reveal that they
continually move into the clear zone, providing a fresh supply of uncharged elements that remove calcium ions and restrict the basal spread of the tip-focused gradient (LovyWheeler et al. 2007). There are also calcium adenosinetriphosphatases (ATPases) on the plasma membrane; these are
membrane-bound enzymes that use the energy of adenosine
triphosphate (ATP) to move calcium against its concentration
gradient (Sze et al. 2006). Taken together, these lines of evidence make it clear that the pollen tube has overlapping
components that respond to elevated calcium and restore the
basal concentration.
Extracellular calcium, as previously noted, is at much
higher concentrations than are concentrations within the
cell. For this reason, an influx of extracellular calcium requires
only the opening of selected pores or channels on the plasma
membrane. Early predictions of an inward flow of calcium ions
into the pollen tube apex from the surrounding environment were confirmed by high-resolution studies using the
calcium-specific vibrating probe. This is an electrode that
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Articles
oscillates between two points close to the pollen tube, measuring the local calcium ion concentration. The difference in
ion concentration between these two points indicates both the
direction and magnitude of flux. Studies on the growing
pollen tube demonstrated that calcium flows in only at its
extreme apex (Kühtreiber and Jaffe 1990, Pierson et al. 1994),
fueling ideas that stretch-activated calcium channels were
responding to the growth-dependent deformation of the
apical plasma membrane. More recently, these stretchactivated calcium channels have been identified in protoplasts from Lilium longiflorum pollen tube tips and from
regions of the pollen grain associated with the site of germination (Dutta and Robinson 2004).
Although it seems clear that calcium ions flow into the cell
from the extracellular environment, it must also be recognized
that a substantial amount of this apparent flow may be
associated with binding to cell wall components. The bulk of
the cell wall material is secreted as methyl-esterified pectins.
As previously noted, these are demethylated by the enzyme
pectin methyl esterase, exposing acidic groups which then
bind calcium in a process that cross-links these pectic residues
and strengthens the cell wall (Bosch and Hepler 2005).
Calculations indicate that more than 90% of the calcium
flux observed with the ion-selective vibrating electrode is
for wall binding, and thus does not represent ions that cross
the plasma membrane (Holdaway-Clarke and Hepler 2003).
Nevertheless, the small amount that does cross the plasma
membrane creates the apical gradient and is essential for
pollen tube growth.
Although receiving much less attention than calcium,
protons are also essential for pollen germination and pollen
tube growth. In experiments with pollen tubes, the culture
medium must be acidic, with growth being optimal at pH 5
to pH 6 (Holdaway-Clarke et al. 2003). However, intracellular pH has proved to be more challenging to gauge than
intracellular calcium because protons, which are considerably
more mobile than calcium ions, are easily perturbed when
probed by an indicator dye. Nonetheless, it has been shown,
using low dye concentrations, that growing pollen tubes exhibit an intracellular pH gradient (figure 4, bottom row). The
apex of the pollen tube is slightly acidic (pH 6.8), whereas the
rest of the clear zone is characterized by the presence of an alkaline band, with a pH of 7.5 (Feijó et al. 1999). In addition
to the intracellular expression of pH noted above, studies with
extracellular ion-selective electrodes reveal a pattern of fluxes
that correlates with the pattern of intracellular pH. These observations indicate that there is an influx of protons at the extreme apex of the tube, precisely at the intracellular acidic
domain. Moreover, there is an efflux along the sides of the clear
zone, which corresponds to the position of the intracellular
alkaline band (Feijó et al. 1999). This pattern of extracellular proton fluxes provides evidence for the presence of a current loop in the apical dome of the pollen tube, which we
presume is driven by a plasma membrane–associated protonATPase. The regulation of pH emerges as a key factor in the
control of pollen tube growth.
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Signaling molecules are essential
for pollen tube growth
Pollen tube growth is also regulated by a complex network of
signaling molecules and pathways. Two categories of molecules and their associated enzymes and cofactors have been
localized to the apex of the pollen tube and appear to play
central roles in regulating growth; these include the phosphoinositides and the small G-proteins, which are guanosine
triphosphate (GTP)-binding proteins, such as ROP (Rho
family GTPases of plants). The phosphoinositides are membrane lipids of which phosphatidyl inositol 4,5 bisphosphase
(PIP2) has received the most attention. It is a multifaceted molecule that associates with certain actin-binding proteins, including actin depolymerizing factor (ADF), profilin, and
villin (Martin 1998), which are common in pollen tubes. In
addition, PIP2 is enzymatically cleaved by phospholipase C
(PLC) to produce two more signaling agents, diacylglycerol
and inositol 1,4,5 trisphosphate. The former remains in the
plasma membrane, and can be converted to phosphatidic acid,
which itself is a regulatory molecule that can affect actin dynamics (Žárský et al. 2006). The latter diffuses in the cytoplasm
and can release calcium from internal stores (Franklin-Tong
et al. 1996). It is noteworthy, therefore, that PIP2 localizes to
the apical plasma membrane of the pollen tube (figure 5; Kost
et al. 1999).
Observations support the importance of these phosphoinositides to pollen tube growth. For example, an increase in
PIP2 at the apex is associated with an increase in pollen tube
growth (Dowd et al. 2006). Further studies on PLC reveal that
modulation of its activity also modulates growth; thus inhibition of PLC activity markedly blocks pollen tube growth
(Helling et al. 2006). There is evidence that PLC activity may
impinge most directly on the actin cytoskeleton, because
cells compromised with an inactive form of the enzyme can
be partially rescued with the actin polymerization inhibitor
latruculin B (Dowd et al. 2006). Both of these recent studies
support the idea that PLC cleaves PIP2 that might migrate away
from the apex. PLC activity thus restricts PIP2 to the extreme
apex of the tube and in so doing contributes centrally to the
control of pollen tube growth polarity (Dowd et al. 2006,
Helling et al. 2006).
The small G-proteins are also potential central regulators
of pollen tube growth (Hwang and Yang 2006). ROPs constitute a subfamily of molecular switches, unique to plants,
that function in developmental and signaling events. In the
cell, ROP toggles between an active GTP-bound form and
an inactive guanosine diphosphate–bound form, under the
influence of guanine nucleotide exchange factors, GTPaseactivating proteins, and guanine nucleotide dissociation
inhibitors. It has been shown that certain ROPs localize to the
apical region of the plasma membrane of pollen tubes, the site
of growth (figure 6; Kost et al. 1999, Hwang and Yang 2006).
As with PIP2, the structural studies on ROPs reveal the presence of regulating factors that inactivate these agents if they
migrate from the extreme apex to the flank of the apical
dome (Gu et al. 2006, Klahre and Kost 2006, Klahre et al. 2006).
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Figure 5. Phosphatidyl inositol 4,5, bisphosphate (PIP2)
localizes to the apical plasma membrane. PIP2 has been
identified through its binding to the pleckstrin homology
domain of phospholipase C, which is fused to green fluorescent protein. This medial confocal image clearly shows
that PIP2 resides in the apical plasma membrane. Source:
Kost and colleagues (1999). Bar = 10 micrometers.
These conditions ensure that the active species will be
confined to the extreme apex of the tube, where they may
participate in the control of pollen tube growth polarity. It
seems important that both ROPs and PIP2 localize to the
same extreme apical domain and may act together in a
common pathway (Kost et al. 1999).
Pollen tube growth oscillates
An exciting finding that has emerged in the last 15 years is that
pollen tubes do not extend uniformly but exhibit pronounced
oscillations in their growth rate. For example, in lily pollen
tubes the growth rate oscillates between 100 and 500 nanometers per second, with a periodicity of about 20 to 50
seconds (Holdaway-Clarke and Hepler 2003). Inevitably the
question arises as to why pollen tube growth oscillates.
Although the answer cannot be stated with certainty, there are
some important considerations that help us understand the
matter. First, many vital processes exhibit oscillatory behavior
(Goldbeter 1996, Feijó et al. 2001). It has been known for many
years that cultures of yeast cells generate oscillations in reduced
nicotinamide adenine dinucleotide phosphate (NAD[P]H),
a coenzyme essential in numerous energetic reactions within
the cell (Goldbeter 1996). To the extent that pollen tubes
produce and use NAD(P)H, we can expect similar oscillations
in those cells. But beyond these general considerations there
are issues that pertain more specifically to pollen tube growth.
In our studies of lily, we note that when the pollen grains first
germinate, the emerging tubes exhibit fluctuations in their
growth rate but not regular oscillations. However, once they
reach a length of about 700 µm, they begin to exhibit regular oscillations, and at this point their overall growth speeds
up (Pierson et al. 1996). Oscillatory growth thus is more
rapid than nonoscillatory growth. Because only one pollen
tube will fertilize each egg cell and thereby perpetuate its
DNA, speed of growth is a trait that enjoys a selective advantage (Snow and Spira 1991).
As the rate of growth oscillates, so do several underlying
physiological processes, typically with the same periodicity as
growth, but usually not with the same phase. To identify how
key processes are connected to pollen tube growth, investigators determine the phase relationships between oscillating
physiological events and pollen tube growth. To do this, we
have used cross-correlation analysis, which is a mathematical procedure that provides a detailed comparison of the related oscillatory data and makes it possible to infer whether
a physiological process precedes or follows growth (Hepler et
al. 2006).
Because oscillating processes that lag growth are unlikely
to initiate it, the emphasis has been on identifying oscillating
processes that peak before growth does. However, the significance of events that follow growth should not be overlooked,
because they may be involved in terminating growth. Thus,
the events preceding growth may be viewed as the accelerator and those following growth as the brakes: as with a speeding car, to quickly negotiate a tortuous path, brakes together
with an accelerator are essential components of the regulatory machinery. Ultimately, we would like to determine what
sets the oscillations in motion, and how these oscillations
translate into growth of the pollen tube.
Because oscillatory growth is known almost entirely from
studies of pollen tubes grown in vitro, questions are frequently raised about whether this process occurs in vivo. The
answer is yes; Iwano and colleagues (2004) have demonstrated growth oscillations in Arabidopsis pollen tubes growing in styles. Interestingly, the in vivo pollen tubes grew faster
and oscillated more quickly than their in vitro counterparts.
In addition, using transgenic Arabidopsis plants expressing the
yellow cameleon calcium indicator, they demonstrated oscillations in the tip-focused calcium gradient similar to those
observed in pollen tubes grown in vitro (Iwano et al. 2004).
Setting the pace for growth:
Available energy oscillates
Figure 6. Rac/ROP localizes to the apical plasma membrane. The small G-protein Rac2, which is a member of
ROP family, has been identified as the fusion construct:
GFP-At-Rac2. Although more broadly spread along the
apical plasma membrane than PIP2 (phosphatidyl inositol 4,5, biphosphate), the distribution of Rac2 nevertheless is maximally centered at the apex of the tube. Source:
Kost and colleagues (1999). Bar = 10 micrometers.
840 BioScience • November 2007 / Vol. 57 No. 10
Given the rapidity of cell elongation and the necessary synthesis, transport, and secretion of cell wall components, it can
be appreciated that an abundant supply of energy is essential
for pollen-tube growth. ATP is the fuel for key events, such
as actin polymerization, proton pumping, exocytosis, and
kinase activity. From these brief considerations, it follows
that ATP plays a key role in pollen tube growth.
Cárdenas and colleagues (2006) recently demonstrated
fluctuations in NAD(P)H fluorescence in growing pollen
tubes. This essential coenzyme drives many biosynthetic
processes, including ATP production in mitochondria. The results show that the dominant signal colocalizes with mitowww.biosciencemag.org
Articles
chondria at the base of the clear zone, and further that
NAD(P)H fluorescence oscillates with the same periodicity
as growth rate, with peaks following growth by approximately 95° (i.e., by 95 out of 360, or 26.4%, of a growth
oscillation) and troughs anticipating growth by approximately –80°. Cárdenas and colleagues (2006) focus on the
troughs because they anticipate growth and also because
they show as strong a correlation with growth as do the
peaks. The suggestion is made that the changes in fluorescence
represent the periodic oxidation of NAD(P)H to form
NAD(P)+, with the concomitant production of ATP. Oxidation of NAD(P)H may therefore precede and regulate pollen
tube growth by fueling ATP production. If ATP output does
indeed oscillate, the activity of many ATP-dependent processes
will also oscillate.
Accelerating growth: Alkalinity oscillates
Sequential examination of appropriately stained pollen tubes
reveals that pH oscillates with the same period as growth rate
(figure 7). Determination of the phase relationship reveals that
the alkaline band is most alkaline approximately –95° before
a growth surge, whereas acidity peaks approximately 70°
after growth (Lovy-Wheeler et al. 2006). Phase analysis of the
extracellular proton influx shows that it too lags growth, and
by a magnitude similar to the intracellular peak in acidity (approximately 100°; Messerli et al. 1999). The results further indicate that the formation of the alkaline band and the increase
in acidity at the apex are driven by two independent events.
It seems likely that the alkaline band derives from activity of
a proton-ATPase on the plasma membrane that pumps
protons out of the cell (figure 8; Feijó et al. 1999). These
conclusions are consistent with recent observations by Lefebvre and colleagues (2005) showing a proton-ATPase that is located along the shank of the tube, but not at the extreme apex.
By contrast, the acidic domain at the apex may arise through
the growth-dependent opening of stretch-activated channels at the apex, in a manner similar to that shown for calcium
(Dutta and Robinson 2004).
The observation that the pH of the alkaline band peaks
before growth does (figure 7; Lovy-Wheeler et al. 2006)
means that alkalinity becomes a candidate for an event or
process that might serve as a growth initiator. One important
entity that could be regulated by pH is the formation and
activity of the actin cytoskeleton. Given that the actin fringe
colocalizes with the alkaline band, it is possible that the band
helps organize actin in this region (Lovy-Wheeler et al. 2006).
ADF and pollen-specific actin-interacting protein also
localize to the actin fringe (Lovy-Wheeler et al. 2006) and are
known to enhance depolymerization of F-actin at alkaline pH
(reviewed in Hepler et al. 2006). Although it may seem that
ADF would only degrade actin microfilaments, it is important to emphasize that during the process of F-actin
fragmentation, free plus (fast-growing) ends of actin microfilaments are exposed. These can then serve as templates
upon which new actin microfilaments can grow. ADF thus
promotes the turnover of F-actin, which may translate into
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faster pollen tube growth. However, when protons flow into
the apex, the pH in this region is lowered, which results in
decreased actin turnover and slower growth. Thus, the oscillations in growth rate may be a direct response to the
oscillations in pH in the pollen tube apex, which in turn
cause their effect by modulating the actin cytoskeleton.
Accelerating growth: ROPs oscillate
Recent studies on ROP1 reveal that it forms an apical gradient in growing pollen tubes and that its activity oscillates with
the same periodicity as growth rate (Hwang et al. 2005). The
observation that ROP1 activity leads growth by approximately –90° supports the idea that it also plays a role in
promoting pollen tube growth. It has been argued that
ROPs control pollen tube growth through their regulation of
both calcium and actin cytoskeletal dynamics (Hwang and
Yang 2006). More specifically, Gu and colleagues (2005)
suggest that ROP1 brings about its effects on pollen tube
growth by altering the activity of RICs (ROP-interactive
CRIB-motif-containing proteins). With a focus on RIC3 and
RIC4, Gu and colleagues (2005) report that overexpression
of RIC4 results in the formation of a dense network of actin
in the apex of the pollen tube. Thus, through an interaction
with RIC4, ROPs may be responsible for establishing the
correct configuration of actin in the apex of growing pollen.
By contrast, overexpression of RIC3 results in an elevated
intracellular calcium concentration, together with the inhibition of growth and the formation of a swollen apical domain.
Figure 7. A graph showing the oscillation in growth rate
(dark blue) together with the oscillation in pH (pink) for
the same lily pollen tube. It is clear that the pH oscillations exhibit the same periodicity as those for growth;
however, from visual inspection it is not possible to
determine the phase relationship. Application of crosscorrelation analysis reveals that the alkaline domain
increases in pH in anticipation of an increase in growth
rate, whereas the decline in pH at the apex follows the
increase in growth rate. Source: Lovy-Wheeler and
colleagues (2006). Abbreviations: µm, micrometer; sec,
second.
November 2007 / Vol. 57 No. 10 • BioScience 841
Articles
Because the effects of RIC3 are suppressed in the presence of
a calcium channel blocker, it has been suggested that the
normal function of RIC3 is to regulate the influx of calcium,
which stimulates F-actin disassembly (Gu et al. 2005).
signal and stimulate processes that would promote growth.
The results show instead that it is the growth process that
controls the calcium response. As the pollen tube surges
forward, calcium rushes in through stretch-activated channels. Elevated calcium may then retard growth.
Applying the brakes: Calcium oscillates
Calcium is thought to bring about its effects through
Apical calcium ion concentration is yet another component
intermediary proteins, including calmodulin and CDPKs.
that oscillates in growing pollen tubes; changes in concenAlthough calmodulin is evenly distributed throughout the
tration from 0.75 µM to more than 3 µM during a single
pollen tube, its activity is greatly enhanced in the apex (Rato
growth oscillation have been reported (Pierson et al. 1996).
et al. 2004). A CDPK with elevated activity in the pollen tube
Once again, although these oscillations show the same period
apex was found to be redistributed prior to changes in growth
as the growth rate, they do not show the same phase. Analyorientation (Moutinho et al. 1998). More recently two different
sis of the phase relationship between intracellular calcium and
CDPK isoforms have been identified in Petunia pollen tubes;
growth rate using cross-correlation analysis has yielded an
one (PiCDPK1) affects pollen tube polarity, whereas the
unexpected result, namely, that the intracellular calcium
other (PiCDPK2) affects the overall tube growth (Yoon et al.
gradient follows, rather than leads, the peak in growth rate by
2006). Whereas calmodulin is known to interact with myosin
38° (Messerli et al. 2000). This conclusion has come as a surand villin, and possibly also regulate ACA9, a plasma memprise because it had been thought that calcium, as an intrabrane calcium ATPase (Sze et al. 2006), the downstream
targets of CDPKs in pollen tubes are unknown.
cellular signaling molecule, would appear as an anticipatory
Several structures and related processes are modulated by calcium, which may have a profound
effect on oscillatory pollen tube growth. The cytoskeleton is a prime example. Actin-binding proteins
that are found commonly in pollen tubes include
profilin, villin, and gelsolin-like protein; they all respond to calcium or to calcium plus calmodulin,
and either prevent actin polymerization or fragment
existing bundles of F-actin (Yokota et al. 2005,
Yokota and Shimmen 2006). Another important
cellular process that is regulated by calcium is cytoplasmic streaming (Yokota and Shimmen 2006).
Myosin XI, a motor protein involved in actin-based
motility, is commonly found in pollen tubes and is
blocked by elevated levels of calcium (Yokota and
Shimmen 2006), accounting for the observations
that cytoplasmic streaming is inhibited in the tube
apex, but is prominent in the shank, where calcium is present at basal levels. A conclusion from
these studies is that the local calcium concentration
Figure 8. Summary diagram of a pollen-tube apex showing structural ele- plays a key role in determining the structure and
ments and physiological processes that participate in pollen-tube growth.
activity of the actin cytoskeleton. In oscillatory cell
At the apex of the tube, calcium (Ca2+) and protons (H+) enter the cell.
elongation, the decrease in actin polymerization and
The local high calcium concentration facilitates secretion of the vesicles
increase in F-actin fragmentation brought about by
(V). The excess calcium is taken up by the endoplasmic reticulum (ER),
elevated calcium prevent the surge in growth caused
and also most likely extruded to the outside (not shown). Mitochondria
by actin polymerization (Gibbon et al. 1999, Vidali
(Mito) metabolize sugars, which have been obtained from starch (ST)et al. 2001).
containing amyloplasts (AP), and generate adenosine triphosphate
The oscillating calcium gradient may have a
(ATP). ATP, through its coupled hydrolysis to adenosine diphosphate
profound effect on the activity of the proton(ADP), powers many key processes of pollen tube growth. Some of these
ATPase in the apical domain. As Feijó and colinclude the H+-ATPase at the cell surface, which creates the alkaline band
leagues (1999) noted, high calcium inhibits
(OH–), and the extracellular proton (H+) flux, polymerization of actin
proton-ATPase activity in other plant cell systems.
and transport along microfilaments (Actin), the uptake of calcium by
If this holds for the pollen tube, the elevated calcium
the ER, and the trafficking and fusion of vesicles in the apex. Certain key
in the apex may explain why this enzyme is not
signaling molecules, including, notably, Rho family GTPases of plants
observed in the extreme apical domain. In addition,
(ROP) and phosphatidyl inositol 4,5 bisphosphase (PIP2), are localized
oscillatory increases in calcium together with those
to the apical plasma membrane and contribute to cell polarity.
in acidity may spread sufficiently and block proton842 BioScience • November 2007 / Vol. 57 No. 10
www.biosciencemag.org
Articles
ATPase activity on the plasma membrane bordering the clear
zone. This would cause a decrease in the alkaline band and
in the promotive effect that it would have on growth.
Membrane trafficking, including exocytosis (secretion)
and endocytosis, is another process that is modulated by
high intracellular calcium concentrations (Battey et al. 1999).
Thus, the apical calcium gradient promotes fusion of secretory vesicles, which is necessary for growth and which accounts
for the close correlation between the location of the gradient
and the site of maximal growth. However, it remains a question whether the oscillation in the calcium gradient generates
an equivalent oscillation in secretion. Preliminary data, summarized in a recent review (Holdaway-Clarke and Hepler
2003), fail to note a similar phase relationship between the increase in calcium and the increase in secretion (wall thickness).
The explanation may derive from the observation that the
tip-focused calcium gradient, even at its low point, is close to
or above the threshold for stimulation of secretion. Sutter and
colleagues (2000) have shown in corn protoplasts that
secretion saturates at 1.5 µM and is half maximal at 0.9 µM.
Oscillations from 0.75 to above 3 µM have been reported for
pollen tubes (Pierson et al. 1996). However, it is important to
realize that these values are probably underestimates for the
true concentration of calcium at the membrane surface,
where secretion occurs. Where the ion enters the cell, the
local concentration will be very high but restricted to an extremely small volume of cytoplasm, which is below the limit
of microscopic resolution. The diffusing indicator dye does
not accurately sample this tiny volume; rather, its response is
dominated by nearby domains where the ion concentration
is much reduced. It is possible, therefore, that the local calcium
concentration is always above the threshold for secretion,
and that oscillatory increases in the ion do not further stimulate this process.
Summary
Much has been discovered about the structure and physiology of the pollen tube, and considerable insight has been
gained concerning the characterization and interaction of the
many processes that control tube growth. However, several
issues remain to be elucidated, including, notably, the identity of the central pacemaker and the pathways through
which it generates oscillatory growth. Figure 8 is a summary
diagram of the apical domain of the pollen tube that attempts to show the major components and where they participate. It is clear that calcium and protons play essential roles
in pollen tube growth. The high level of calcium in the apex
inhibits cytoplasmic streaming and fragments the actin cytoskeleton, whereas the zone of high pH in the region of the actin
fringe is thought to promote actin turnover, and thereby
serves as an initiator of pollen tube growth. The discovery that
certain key signaling factors, including PIP2 and ROPs, localize
on the plasma membrane at the apex of the growing pollen
tube has focused our attention on these agents as central
switches in pollen tube growth. The ROPs, with their anticipatory increase in activity during oscillatory growth, emerge
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as candidate agents that serve a primary role in controlling
growth initiation. However, it is still unclear what the pertinent pathways are, both upstream and down, through which
these small G-proteins bring about and coordinate the myriad processes required for pollen tube growth.
Energy is fundamental to biological processes, and we
anticipate that ATP synthesis occupies a position very close
to the central pacemaker of pollen tube growth. Because
mitochondrial metabolism possesses the properties of an
endogenous oscillator in other cells, notably yeast cells (Goldbeter 1996), it seems plausible that similar events take place
in the pollen tube. Thus through the endogenous oscillation
of the pollen mitochondrial electron transport chain, the
ATP concentration oscillates, and this underpins all other
oscillatory events. For example, through nucleoside diphosphate kinase, ATP drives the synthesis of GTP, which is essential for all G-proteins, including ROPs discussed above.
Although key processes such as an oscillatory production of
ATP have not been shown, there are experimental procedures available with which these fundamental questions can
be probed. We are encouraged, therefore, that the knowledge gained from the examination of oscillatory processes will
lead to new studies that will provide an even greater insight
into the basic mechanism of pollen tube growth.
Acknowledgments
We thank Benedikt Kost for providing figures 5 and 6. We also
thank our colleagues Colleen Parris, Alex Bannigan, Alenka
Lovy-Wheeler, and Sylvester McKenna for help in the preparation of the figures. Our research has been supported by a
grant from the National Science Foundation (MCB-0516852).
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