Journal of Experimental Botany, Vol. 54, No. 380,
Plant Reproductive Biology Special Issue, pp. 83±92, January 2003
DOI: 10.1093/jxb/erg043
Endo/exocytosis in the pollen tube apex is differentially
regulated by Ca2+ and GTPases
LuõÂsa Camacho and Rui MalhoÂ1
Departamento Biologia Vegetal, Faculdade de CieÃncias de Lisboa, 1749-017 Lisboa, Portugal
Received 12 April 2002; Accepted 19 September 2002
Abstract
Pollen tube growth relies on an extremely fast
delivery of new membrane and wall material to the
apical region where growth takes place. Despite the
obvious meaning of this fact, the mechanisms that
control this process remain very much unknown. It
has previously been shown that apical growth is
regulated by cytosolic free calcium ([Ca2+]c) so it was
decided to test how changes in [Ca2+]c affect endo/
exocytosis in pollen tube growth and reorientation.
The endo/exocytosis was assayed in living cells
using confocal imaging of FM 1-43. It was found that
growing pollen tubes exhibited a higher endo/exocytosis activity in the apical region whereas in nongrowing cells FM 1-43 is uniformly distributed.
During pollen tube reorientation, a spatial redistribution of exocytotic activity was observed with the
highest ¯uorescence in the side to which the cell will
bend. Localized increases in [Ca2+]c induced by
photolysis of caged Ca2+ increased exocytosis. In
order to ®nd if [Ca2+]c changes were modulating
endo/exocytosis directly or through a signalling cascade, tests were conducted to ®nd how changes in
GTP levels and GTPase activity (primary regulators
of the secretory pathway) affect the apical [Ca2+]c
gradient and endo/exocytosis. It was found that
increases in GTP levels could promote exocytosis
(and growth). Interestingly, the increase in [GTP] did
not signi®cantly affect [Ca2+]c distribution, thus suggesting that the apical endo/exocytosis is regulated
in a concerted but differentiated manner by the Ca2+
gradient and the activity of GTPases. Rop GTPases
are likely candidates to mediate the Ca2+/GTP crosstalk as shown by knock-down experiments in
growing pollen tubes.
1
Key words: Antisense, caged probes, calcium, endocytosis,
exocytosis, GTP, pollen tubes, rop GTPase.
Introduction
Pollen tubes are among the most rapidly extending cells,
growing up to 50 mm min±1. These growth rates are
possible due to a highly polarized apical fusion of vesicles,
which transport cell wall components to the growing tip
(Steer and Steer, 1989). It was found some time ago that
the quantity of membrane delivered by exocytosis was in
excess for the pollen tube growth rate, suggesting an
underlying recycling process (Picton and Steer, 1983).
However, little is known about the molecular mechanisms
involved in the regulation of exocytosis and membrane
recycling in growing pollen tubes.
A tip-focused [Ca2+]c gradient is known to play a central
role in the regulation of pollen tube growth and modulation
of the [Ca2+]c concentration results in changes in the rate
and direction of growth. (Pierson et al., 1994; Malho and
Trewavas, 1996). One of the targets of this Ca2+ signalling
pathway was therefore claimed to be the secretory pathway
(Malho et al., 2000). However, up to this moment, only
indirect evidence for this fact has been presented.
Picton and Steer (1985) have suggested that Ca2+ was
also involved in the movement of vesicles towards the tip,
fusion of vesicles with the plasma membrane, and the state
of plasticity of the wall at the tip. Similar claims were
made for GTP-binding proteins (Drgonova et al., 1996; Li
et al., 1999; Lin et al., 1996). GTP-binding proteins act as
molecular switch regulators, cycling between a GDPbound (inactive) state and a GTP-bound (active) state and a
large number of these G-proteins exert important functions
in signalling transduction, namely in different traf®cking
steps on the secretory and endocytic pathways (Bar-Sagi
To whom correspondence should be addressed. Fax: +351 217500069. E-mail: [email protected]
ã Society for Experimental Biology 2003
84 Camacho and MalhoÂ
and Hall, 2000; Ridley, 2001). The Ras superfamily of
small GTPases includes ®ve families: Ras, Rab, Arf, Ran,
and Rho. A Rab2 homologue, necessary for membrane
traf®c between ER and Golgi in mammalian cells (Tisdale
et al., 1992), is present in Arabidopsis pollen grains
(Moore et al., 1997) and was recently shown to be
important for pollen tube growth (Cheung et al., 2002).
Members of the Rho family with high homology to the Rac
subfamilyÐRop (Yang, 2002), have been implicated in
the regulation of pollen tube growth and root hairs,
possibly by interaction with the tip-focused [Ca2+]c
gradient (Lin et al., 1996; Lin and Yang, 1997;
Molendjik et al., 2001) and with the phosphoinositol
signalling pathway (Kost et al., 1999). It was therefore
decided to investigate the effect of GTPase activity and
[Ca2+]c in membrane traf®c in pollen tubes and to what
extent, if any, the two signalling pathways are connected.
For this purpose, the ¯uorescent probe FM 1-43 has
been used. This dye has been found increasingly useful in
exploring the endo- and exocytosis mechanisms in a
variety of biological models (Smith and Betz, 1996). FM
1-43 is a non-toxic, water-soluble dye, virtually non¯uorescent in aqueous medium, and is believed to insert
into the outer lea¯et of the surface membrane where it
becomes ¯uorescent. Since the dye stains membranes in an
activity-dependent manner, it has proved to be useful for
studies of vesicle recycling, exocytosis and endocytosis
(Cochilla et al., 1999). Therefore variations in the apical
¯uorescence of FM 1-43 were monitored while manipulating Ca2+ and GTP levels with caged-probes. It was
found that both activation of GTPase enzymes and increase
in [Ca2+]c stimulate secretion. Similarly, antisense inhibition of Rop GTPases diminish secretion. Furthermore,
evidence was obtained for the existence of a concerted but
differentiated regulation of secretion by GTPase activity
and apical [Ca2+]c.
Materials and methods
Plant material
Pollen of Agapanthus umbellatus L'Her was harvested and stored at
±80 °C. Pollen was germinated in vitro as described previously
(Malho et al., 1994), using a modi®ed version of the Brewbaker and
Kwack (1963) medium: 2.5% sucrose, 0.01% H3BO3, 0.02% MgCl2,
0.02% CaCl2, and 0.02% KCl, pH 6.0 at 27 °C.
FM 1-43 labelling and imaging
After 1 h germination, pollen tubes were labelled with FM 1-43
(Molecular Probes). The dye was added to the germination medium
to a ®nal concentration of 0.2 mM. This value was chosen because it
resulted in high levels of labelling but no toxicity for pollen tubes;
cells grew normally for >2 h in medium with such concentration of
extracellular FM 1-43. Time-course images of FM 1-43 ¯uorescence
were acquired with a Bio-Rad MCR-600 (Microscience Ltd, Hemel
Hempstead, UK) confocal laser scanning microscope (CLSM),
attached to a Olympux BX-51 upright microscope, using the BHS
®lter (excitation: 488 nm; dichroic re¯ector: 510 nm; barrier ®lter:
515 nm). Fluorescence was quanti®ed in terms of average pixel
intensity using the histogram command of COMOS/MPL software
(Bio-Rad).
Modulation of [Ca2+]c and GTP levels
Pollen tubes were loaded by pressure microinjection as described in
Camacho et al. (2000) with one of the following probes (the
concentration refers to the solution injected): (1) caged Ca2+
(NITR-5 tetrasodium, 0.5 mM, Calbiochem) and the guanine
nucleotide analogues (2) GDPbS (1 mM, Sigma-Aldrich) and (3)
[S-(DMNPE)-caged] GTP-g-S (1 mM, Molecular Probes). The
concentration of the solutions was chosen so that pollen tube growth
was not arrested upon microinjection and release. Ca2+ and GTPgS
were released by exposing pollen tubes to a 2 s of UV light pulse
produced by a 100 W mercury lamp via a 400 nm dichroic mirror
and an Iris diaphragm. Controls involved exposing non-loaded tubes
to the same amount of UV light (Malho and Trewavas, 1996).
[Ca2+]c imaging
Pollen tubes were microinjected with the Ca2+-sensitive dye Calcium
Green-1 and the Ca2+-insensitive Rhodamine B, both linked to a
10 kDa dextran (1 mM, Molecular Probes). [Ca2+]c ratio imaging
was performed by CLSM in the dual channel mode (excitation: 488/
568 nm; 520/630 nm double dichroic; barrier ®lter of 522 nm; barrier
®lter 2 of 585 nm). Fluorescence ratio images were calculated with
the TCSM/MPL software (Bio-Rad) as described before (Camacho
et al., 2000) and then quanti®ed in terms of average pixel intensity
(0±255 scale for 8 bit images). For calculation of [Ca2+]c values, an
in vitro calibration was used (Camacho et al., 2000).
Data analysis
Numerical analysis of the ¯uorescence images was performed using
COMOS commands. Growth rates were calculated using ImageProâ Plus 4.0 software (Media Cybernetics).
Cloning of a Rop gene fragment
Two degenerated oligonucleotides (R1- 5¢-GTI GGN GAC/T GGI
GCI GTI GGN AAA/G AC-3¢ and R2- 5¢-TA A/GTC C/TTC C/TTG
ICC IGC NGT A/GTC-3¢, in which I is inosine and N represents all
four nucleotides) were used as PCR primers (Yang and Watson,
1993). Total RNA was isolated from germinated pollen by the
phenol/SDS method and reverse transcribed using primer R1. The
single-stranded cDNA was used as the template for PCR and
ampli®ed with primers R1 and R2. The ampli®cation product
(around 165 bp long) was cloned using the pT7Blue3-Perfectly
BluntÔ Cloning Kit (Novagen) and sequenced for both strands using
the ABI PRISMÔ dRhodamine Terminator Cycle Sequencing
Ready Reaction Kit (PE Applied Biosystems). cDNA sequences
were analysed using BLASTN software (Altschul et al., 1997).
The 3¢ end of Rop gene was obtained by 3¢RACE (Frohman et al.,
1988). The ®rst strand of cDNA was synthesized using primer QT
(5¢- CCA GTG AGC AGA GTG ACG AGG ACT CGA GCT CAA
GC (dT)17-3¢) (Bespalova et al., 1998) and used as template for PCR
ampli®cation. Nested PCR was performed. The ®rst PCR used the
primers R1 and QO (5¢-CCA GTG AGC AGA GTG ACG AG-3¢)
and the second ampli®cation the primers RacF (5¢-TCG TAC ACC
AGC AAC ACT TTT -3¢) and QI (5¢-ACG AGG ACT CGA GCT
CAA GC-3¢). Cloning, sequencing and analysis of the ampli®cation
product were performed as described before.
Knock-down of a Rop gene expression
The expression of the Rop gene was reduced by loading pollen with
antisense oligonucleotides (AS-ODN) complementary to the cloned
AuRop cDNA fragment. The Rop2-AS antisense primer, that
yielded the best results, was 16 bp long and modi®ed in the last
four bases of both the 5¢ and 3¢ terminus with phosphorothioate
Ca2+ and GTP modulate endo/exocytosis in pollen tubes
bonds, as indicated in lowercase: 5¢-tga gCA TGC AGG tct t-3¢.
Delivery of the AS-ODN to pollen was accomplished by combining
the AS-ODN with the cationic lipid Cytofectin GS 3815 (Glen
Research, Sterling, VA, USA). The cytofectin vesicles were
prepared according to supplier's instructions and mixed with ASODN to a ®nal concentration of 15 mg ml±1 lipid and 30 mM ASODN (Moutinho et al., 2001). After 15 min incubation at room
temperature, the cytofectin/AS-ODN complex was diluted with 45 ml
of growth medium and pollen was germinated in this supplemented
medium. The controls performed were the absence of the cytofectin
and/or of the AS-ODN, as well as transfection with cytofectin and
sense ODN complex (Moutinho et al., 2001). Transmitted light
images were captured through the CSLM ®bre optic device. Length,
diameter and growth rates were calculated for 20 pollen tubes per
assay using Image-Proâ Plus 4.0 software (Media Cybernetics).
Results
FM 1-43 dye is a valid marker for studying
endo/exocytosis
In order to understand how the endo/exocytosis is regulated in growing pollen tubes, the use of FM 1-43 as a
marker to study membrane recycling in Agapanthus
umbellatus was optimized. An external dye concentration
of 0.2 mM was chosen, as it did not affect growth rate (data
not shown) and yielded a good ¯uorescent signal.
FM 1-43 is highly ¯uorescent in a membrane environment. However, its permanent charge prevents the dye
from passively crossing the membrane and so the loading
into the cell is limited by an uptake from endocytic vesicles
(Cochilla et al., 1999). Staining of the pollen tubes plasma
membrane started immediately after the addition of FM 143 dye to the germination medium. This was followed by
internalization of the dye, mainly in the tip region, and
within a few minutes the typical staining pattern of the FM
1-43 dye was observed: a bright staining in all the apical
region that extends to the sub-apical region with an
inverted cone shape (Fig. 1A). The most ¯uorescent region
corresponds to the so-called clear zone, a region of large
organelle exclusion ®lled with secretory vesicles (Lancelle
and Hepler, 1992). The staining pattern was smooth
without large hotspots indicating that, mostly, near- and
sub-resolution organelles (e.g. vesicles) were labelled. In
non-growing pollen tubes, the ¯uorescence hotspot in the
apical region was absent and ¯uorescence was almost all
con®ned to the plasma membrane without being internalized (Fig. 1B).
Pretreatment of pollen tubes with 100 mM sodium azide
impaired dye uptake. At this concentration growth stops,
but cytoplasmic streaming remains active. FM 1-43
associated with the plasma membrane, but was not
internalized (Fig. 1C). This control excludes passive
diffusion of dye into the cell (Parton et al., 2001)
con®rming the involvement of an endocytic mechanism
in the internalization of FM 1-43. Dye release by
exocytosis also took place as revealed by washout
experiments. If FM 1-43 was removed from the medium,
85
Fig. 1. FM 1-43 staining distribution in (A) a growing pollen tube of
Agapanthus umbellatus (B) a non-growing pollen tube and (C) a
pollen tube pretreated with sodium azide (100 mM). The apical
inverted cone-shaped ¯uorescence, corresponding to the secretory
vesicles in the clear zone, is observed only in growing cells. In nongrowing cells, the plasma membrane is stained, but almost no dye is
internalized. Inhibition of FM 1-43 uptake by sodium azide con®rms
that the dye is loaded by an endocytic pathway. All images correspond
to median focal planes and are displayed with the same intensity
settings. Bar=10 mm.
Fig. 2. FM 1-43 labelling in a pollen tube growing in an
isodiametrical way is observed all around the apical region (A). Here
some hotspots are visible and probably correspond to small vacuoles
or clusters of vesicles. The diagram (B) illustrates the hypothesized
distribution of the secretory vesicles in the apex of such a pollen tube.
Bar=10 mm.
the labelling pattern was maintained, but ¯uorescence in
the cell slowly decreased; the rate of this process varies
from cell to cell with ¯uorescence persisting from 30 min
to >90 min (data not shown).
FM 1-43 as a marker of polar growth
FM 1-43 was tested for its ability to act as a marker for the
prediction of a new site of polarization. In pollen tubes
with isodiametrical growth (e.g. induced by a series of
electrical pulses; Malho et al., 2000), FM 1-43 ¯uorescence was observed around all the apical region (Fig. 2).
However, in pollen tubes that stopped and later recovered
apical growth, a ¯uorescence hotspot became visible in the
area where the new growing tip was about to emerge
(Fig. 3A±D); when growth fully resumed, the typical
inverted cone shaped apical staining of FM 1-43 was again
observed (Fig. 3E, F). This ¯uorescence redistribution is
likely to be a combination of new internalized dye and
movement of previously stained vesicles.
A similar result was found when the pollen tubes
reorientated their growth. Just prior to reorientation, FM 143 apical staining became asymmetric with the highest
86 Camacho and MalhoÂ
Fig. 3. FM 1-43 staining distribution in a pollen tube that stopped and
recovers its polarity. (A) Apex shortly after growth arrest; previous
direction of growth (left side) is still the most ¯uorescent region. (B)
Apex swells and a new growing apex starts to form; a ¯uorescence
hotspot appears where the new growing tip is about to emerge
(indicated by arrow) while ¯uorescence in the former apex decreases.
(C) The polarized fusion events continue and (D) give rise to a new
growing tip. (E, F) The new tip exhibits the typical inverted cone
shape. Bar=10 mm.
¯uorescence associated to the side of the dome to which
the tube will bend (Fig. 4B, C, F, G). When the tube ceases
to curve, the apical hotspot was again re-centred in the
growing tip (Fig. 4D±H).
Cytosolic calcium modulates endo/exocytosis
To understand the role of [Ca2+]c in the processes of endo/
exocytosis, apical FM 1-43 ¯uorescence was monitored
while manipulating [Ca2+]c levels. The [Ca2+]c levels were
increased in the tube cytoplasm by releasing caged Ca2+
(NITR-5). This allowed the monitoring of changes in the
secretory process just before and after the increase in
[Ca2+]c.
The increase of [Ca2+]c upon release did not alter
signi®cantly the growth rates (n=9; Table 1). One-third of
the tubes slowed down the growth slightly (to about 97%
of the original growth rate), while the remaining two-thirds
increased the growth rate by 3% (results not shown).
Despite the almost stable growth rate, apical FM 1-43
¯uorescence was affected by the increase in [Ca2+]c. 75%
of the pollen tubes had a decrease in apical ¯uorescence
intensity (Fig. 5A). This reduction averaged 22% relative
to the ¯uorescence value recorded prior to release; in the
control cells, FM 1-43 ¯uorescence remained constant
before and after the UV pulse (data not shown). These data
indicate that changes in [Ca2+]c which do not signi®cantly
affect growth rates may still affect the endo/exocytosis
mechanism suggesting the existence of a different growth
regulator mechanism.
Changes in GTPase activity lead to alterations in
endo/exocytosis
In yeast and animal cells, several GTPases have been
shown to modulate both endo- and/or exocytosis and it was
Fig. 4. FM 1-43 staining distribution in a pollen tube changing
growth direction. When the pollen tube is growing straight (A, E), FM
1-43 ¯uorescence is almost symmetrical, within the apical dome.
Upon reorientation, a larger recruitment of vesicles to one side of the
apical region occurs (B, C, F, G) and new cell wall and membrane is
added in an asymmetrical way. Once a new axis of polarity has been
established, the typical FM 1-43 apical staining is recovered (D, H).
(I) The magni®ed apex of (G) (circled region) showing detail of the
asymmetric distribution. The diagrams (J±L) illustrate the
hypothesized distribution of the secretory vesicles in the apex of a
reorienting pollen tube. Bar=10 mm.
decided to test if they play a similar role in pollen tubes.
One way to modulate GTPase activity is by bounding the
GTPase enzymes to the guanine nucleotide analogues
GTPgS and GDPbS. The non-hydrolysable analogue of
GTP (GTPgS) keeps G proteins (both ras-like monomeric
and heteromeric) in a permanently active conformation,
while the analogue of GDP (GDPbS) renders the enzymes
inactive. Therefore, the guanine nucleotide analogues were
microinjected in pollen tubes preloaded with FM 1-43.
The caged release of GTPgS in the cytoplasm led to a
signi®cant increase in pollen tube growth rate with an
average increase of 18% (n=19, Table 1). By contrast, a
microinjection of the inactivator GDPbS led to a decrease
in growth rate of about 29% (n=5; Table 1).
Photoactivation of caged GTPgS led, in 88% of the
cases, to a decrease in FM 1-43 apical ¯uorescence
(Table 1). The decrease in ¯uorescence occurred in a
sustained but sometimes (40% of the cases) quite abrupt
way (Fig. 5B). The effect of GDPbS could not be tested in
this respect because a caged version was not available.
These results strongly suggested that the process of
Ca2+ and GTP modulate endo/exocytosis in pollen tubes
87
Table 1. Effect of NITR-5 (n=9), GDPbS (n=5) and GTPgS (n=19) in pollen tube growth rate and on the average apical
¯uorescence of FM 1-43 dye (0±255 scale)
In brackets is the percentage of variation relative to the initial value. Standard deviation values for the FM 1-43 ¯uorescence are not shown
since these are independent.
Treatment
Growth rate (mm s±1)
FM 1-43 apical ¯uorescence
Before
After
Before
After
NITR-5
(Caged Ca2+)
Caged GTPgS
0.3660.05
134.55
GDPbS
0.3560.05
0.3660.05
(0%)
0.3960.07
(+18.18%)
0.2560.06
(±29.38%)
104.81
(±22.10%)
91.32
(±20.75%)
±
0.3360.07
115.23
±
Fig. 5. Correlation between pollen tube growth and FM 1-43 apical staining before and after release of (A) caged Ca2+ and (B) caged GTPgS; the
UV pulse corresponding to the caged photolysis is indicated by a vertical bar. (A) Though the average growth rate is kept approximately
unchanged, the increase in [Ca2+]c results in a signi®cant decrease of apical ¯uorescence. (B) The release of GTPgS leads to an increase of the
growth rate and decrease of the FM 1-43 ¯uorescence. Dotted line: growth rate (mm s±1); solid line: FM 1-43 ¯uorescence (average intensity in a
0±255 scale).
endo/exocytosis in growing pollen tubes was controlled by
GTPases.
[Ca2+]c and GTPases cross-talk
From the results described above, it was concluded that
both GTPases and [Ca2+]c are involved in the control of
growth and membrane recycling in pollen tubes. In an
attempt to unravel if both GTPases and [Ca2+]c interact
physiologically, caged GTPgS was released in cells
preloaded with the Ca2+-sensitive dye Calcium Green-1
and the Ca2+-insensitive dye Rhodamine B, both linked to
a 10 kDa dextran molecule. This dye combination for
[Ca2+]c imaging has been previously assessed (Camacho
et al., 2000) and found to be the one which produced more
consistent results. Along with an increase in growth rate,
release of GTPgS induced an apical [Ca2+]c decrease in
78% of the pollen tubes (n=16; Fig. 6).
Identi®cation of a Rop homologue and knock-down by
antisense assay
In order to identify a GTPase involved in apical growth,
PCR cloning and 3¢RACE techniques were used to identify
cDNAs encoding putative Rop proteins from germinated
pollen. From the isolated clones, a fragment containing
Fig. 6. Correlation between pollen tube growth and apical [Ca2+]c
before and after release of caged GTPgS; the UV pulse corresponding
to the caged photolysis is indicated by a vertical bar. The increase of
GTPase activity induced by GTPgS leads to an increase in the growth
rate and a slight decrease of [Ca2+]c. Dotted line: growth rate (mm s±1);
solid line: [Ca2+]c ¯uorescence(average ratio intensity in a 0±255
scale).
~800 bp encoding an open reading frame of 187 aa
including the stop codon was chosen. Analysis of the
sequences using BLASTN program revealed the cDNA
was 92% identical to the Rac subfamily of Rho GTPases
from other monocot plants (barley, rice and maize). It has
the three typical GTP binding sites, an effector region and
88 Camacho and MalhoÂ
the typical C-terminal CXXL motif, required for geranylation (boxes, Fig. 7).
Based on the cloned cDNA sequences, antisense
oligonucleotides were designed transiently to inhibit the
expression of the Rop protein and to investigate the effect
of its knockdown in the growth of pollen tubes (Moutinho
et al., 2001). During the ®rst hour of germination,
antisense-treated pollen tubes were similar to controls
(absence of the cytofectin and/or of the AS-ODN;
transfection with cytofectin and sense ODN complex),
presenting an average growth rate of 0.3760.03 mm s±1 and
a tube diameter of 10.460.43 mm. However, after that
period, the antisense effect started to become visible in
cells treated with RopAS2 oligonucleotides. Pollen tubes
slowed down growth rate (0.1060.01 mm s±1) and
increased tip diameter (16.463.0 mm) in contrast to the
normal phenotype exhibited in the controls performed
(Fig. 8A, B).
Antisense-treated pollen tubes also took up FM 1-43 dye
from the germination medium, but at a much slower rate
when compared to control cells (~10 min instead of a few
seconds). Although ¯uorescence was still higher at the tip,
the hotspot occupied the entire abnormally extended
apex and was more con®ned to the membrane region
(Fig. 8C, D).
In agreement with previous observations, the antisensetreated, but still growing, pollen tubes, revealed a highly
localized tip-focused [Ca2+]c gradient (Fig. 8E) which was
also more con®ned to the membrane region than normal
cells (Camacho et al., 2000). In pollen tubes more severely
affected by the antisense treatment (e.g. totally lost apical
growth), this [Ca2+]c gradient could no longer be observed.
Thus, the spatial de®nition of the [Ca2+]c gradient seems
also to depend on Rop protein expression.
Discussion
The use of FM 1-43 to study membrane traf®cking
FM 1-43 proved to be a valid probe for monitoring the
processes of endo/exocytosis in A. umbellatus pollen tubes.
The rapid uptake suggests an extremely high rate of
endocytosis and membrane traf®c, as expected in such
fast-growing cells. The dye uptake was inhibited by the
metabolic inhibitor sodium azide con®rming that FM 1-43
does not accumulate into the cytoplasm by diffusion. The
staining pattern also agrees with an endocytic uptake of the
dye as revealed by the higher ¯uorescence intensity at the
tip of the growing tube. When growth is arrested and the
clear cap dissipated, an indication that vesicles no longer
accumulate in the apex (Lancelle and Hepler, 1992), the
¯uorescence pattern changes to a uniform distribution
which again suggests that the dye is loaded (and released)
into vesicles. The apical accumulation is in agreement with
previous studies that show that both endo- and exocytosis
occur in this region of the cell (Steer and Steer, 1989;
O'Driscoll et al., 1993; Derksen et al., 1995; Parton et al.,
2001).
FM 1-43 was also found to be a marker for polar growth.
In pollen tubes exhibiting isodiametrical growth, ¯uorescence was approximately uniform all over the apical
region. However, if apical growth was recovered, the
region of the new growing tip showed a ¯uorescence
hotspot. A similar observation was made when a pollen
tube changed its growth direction. It has been hypothesized
that this event results in relocation of the secretory vesicles
to one side of the dome (Malho et al., 2000). Indeed, it was
observed that the side of the dome to which the cell bent
became more ¯uorescent, thus suggesting vesicle relocation and an asymmetric distribution of the fusion events.
Fig. 7. Nucleotide and deduced aminoacid sequence of the cDNA clone AuRop. Numbers on the right refer to the nucleotide sequence. Numbers
on the left refer to the amino acid sequence. Amino acids are numbered following the Rop protein complete sequence. The DNA sequences
corresponding to the degenerated PCR primers are underlined with dashed lines and to the 3¢RACE primer is underlined with a dash±dot line. The
antisense oligonucleotide DNA sequence is in bold.
Ca2+ and GTP modulate endo/exocytosis in pollen tubes
89
ionophore, induced thickening of the wall at the tip by the
accretion of the precursor bodies.
However, these data also suggest that cell growth is not
strictly dependent on a Ca2+-mediated stimulation of
exocytosis. This is in agreement with the results of Roy
et al. (1999) using the Yariv reagent, thus suggesting that a
Ca2+-dependent exocytosis serves mainly to secrete cell
wall components. This explains why rises in apical Ca2+
alone led to augmented secretion, reorientation of the
growth axis, but not increase in growth rates. A tight
connection between [Ca2+]c and cell wall material have
also been suggested by Holdaway-Clarke et al. (1997).
These authors found that during oscillatory growth, peaks
of fast growth were not coincident with peaks of high
[Ca2+]c. In addition, they proposed a model where the
intracellular [Ca2+]c gradient determines the rate of
deposition of wall material.
Fig. 8. Effect of AuRop antisense oligonucleotides in growing pollen
tubes. (A) Control pollen tube (transfection with no antisense ODN)
showing a normal phenotype. (B±E) The pollen tubes treated with the
cytofectin/ODN Rop2AS complex, exhibit a pronounced increase in
tube diameter after 1 h and abnormal apical growth; (C, D)
transmitted light image and FM 1-43 ¯uorescence staining in an
antisense treated tube; (E) [Ca2+]c distribution in an antisense treated,
but still growing, pollen tube. Although disturbed, a gradient is still
observed. The colour code shown next refers to an in vitro calibration
(Camacho et al., 2000). Bar=10 mm.
The cargo, which is released upon vesicle fusion, provides
the material for the construction of the new cell wall
needed for growth (not simply elongation of existing wall).
Cytosolic calcium controls secretion of cell wall
material?
The results of FM 1-43 ¯uorescence upon reorientation of
the pollen tube growth axis strongly resembled those
obtained by the authors when [Ca2+]c was mapped during
this process (Malho and Trewavas, 1996). This suggests
that [Ca2+]c is a regulator of the coupling between growth
and endo/exocytosis. Indirect support for this statement
can be found in older literature. Reiss and Herth (1978)
found that chlortetracycline disturbed the apical cytoplasmic gradient in pollen tubes, and induced the formation of
abnormal wall thickenings composed of aggregates of the
dictyosome-derived wall precursor bodies. These authors
attribute the effect to the chelating of Ca2+ and the
consequent disruption of exocytosis. But the same authors
(Reiss and Herth, 1979) also found that A23187, a calcium
GTPases control growth and membrane retrieval?
Modulation of GTP levels was shown to have a strong
effect on pollen tube growth. The non-hydrolysable
analogues of guanine nucleotide interfere in biological
processes by modulating the activity of GTPase enzymes
and it was observed that GDPbS decreased growth rate,
while GTPgS had the opposite effect. Similar results were
obtained by Ma et al. (1999) in lily pollen tubes and
con®rm the importance of GTPases for apical growth (Lin
et al., 1996; Lin and Yang, 1997).
GTP levels were also shown to affect endo/exocytosis
making this signalling pathway a likely candidate to
mediate secretion and growth. The release of GTPgS, thus
an increase in GTPase activity, caused FM 1-43 ¯uorescence to decrease. This can be due to a decrease in
endocytosis rate (lower dye uptake) or an increase in
exocytosis (higher dye release). Given the rapid rate of
¯uorescence decrease (when compared to the dye washout
experiments), the latter is a more likely explanation.
Exocytosis seems to be more dependent on the active state
of GTPases rather then on the cycling between the GTP±
GDP bond state (Zheng and Yang, 2000). Therefore, while
GDPbS has a negative effect on exocytosis by rendering
GTPases inactive, GTPgS has a positive effect.
Selective exposure to elevated Ca2+ alone is not
suf®cient to explain the selectivity of membrane retrieval
(Smith et al., 2000). It is therefore possible that GTPases
play an active role in exocytosis by coupling the actin
cytoskeleton to the sequential steps underlying membrane
traf®cking at the site of exocytosis. This suggestion ®nds
support in the results of Fu et al. (2001) who reported that
F-actin dynamics in the tip of pollen tubes is GTPasedependent. GTPases were reported to modulate not only
the actin cytoskeleton, but also phosphoinositol metabolism (Kost et al., 1999) and protein kinase activity (Nagata
and Hall, 1996), both known to affect apical growth
(MalhoÂ, 1998; Moutinho et al., 1998).
90 Camacho and MalhoÂ
A Rop protein affects endo/exocytosis and apical
growth
[Ca2+]c and GTP control endo/exocytosis in a
concerted but differential way
A large number of members of the Rho family of small
GTPases ®t the model described above, as they behave as
simple on±off switches: when bound to GTP, they transmit
extracellular signals to downstream effectors, whereas in
the GDP state, signal propagation does not occur (Symons
and Settleman, 2000). Possible candidates in pollen tubes
are rac-like proteins (Rops). Rac proteins were shown to
regulate a late step in exocytosis in neurons (Doussau et al.,
2000), chromaf®n (Gasman et al., 1999) and mast cells
(Hong-Geller and Cerione, 2000). In pollen, expression of
dominant negative Rac inhibited tube growth (Kost et al.,
1999), whereas expression of constitutive active Rop
induced ectopic growth (Li et al., 1999) suggesting that it
is the active protein that promotes apical growth. These
data using antisense oligonucleotides to reduce Rop
expression support this assumption. These experiments
revealed (1) that perturbation of polar growth can be
accomplished by reducing the expression of Rop proteins;
(2) provided that growth is not totally disrupted, a [Ca2+]c
gradient and, therefore, Ca2+ in¯ux, can be maintained; (3)
that Rop inhibition results in a substantial decrease in the
rate of FM 1-43 uptake, but not signi®cant changes in the
¯uorescence pattern. Taken together these data suggest
that the Rop protein is involved in endo/exocytosis and
membrane retrieval, but may not be crucial for secretory
vesicle targetting.
One striking feature that emerged from recent data on
GTPases is the extensive cross-talk and co-operation that
exists between GTPase-regulated signal transduction
pathways (Bar-Sagi and Hall, 2000). Using microinjection
of speci®c antibodies, Li et al. (1999) previously reported
an interaction between Ca2+ and the expression of a Rop 1
protein. These authors demonstrated that inhibition of
Rop1At proteins modi®es Ca2+ in¯ux, but could not
determine inhibition of the Ca2+ in¯ux altogether (which
would require electrophysiological methods). It was actually found that growth inhibition was reversed by higher
extracellular Ca2+, which means that in¯ux was still
possible. The apparent discrepancy between those data and
that of the present study may simply be a matter of
technique and interpretation. It is unlikely that any Rop
protein will have the same effect on Ca2+ dynamics and
growth rates. Furthermore, inhibition (or knock-out) of any
protein crucial for tip growth will certainly affect the Ca2+
gradient either directly or through a signalling loop. This
study's results support a model in which Ca2+ and Rop
GTPases act differentially but in a concerted form in the
sequential regulation of pollen tube secretion and membrane retrieval. Although there may be several different
interpretations to these data, it is suggested that the most
likely explanation is that Ca2+ plays a major role in the
secretion of cell wall components while Rop GTPases
appear to play a key role in fusion of docked vesicles and
endocytosis. It is clear, though, that the exact position of
these elements in the signalling cascade controlling pollen
tube growth and orientation is still uncertain and requires
further investigation.
Rapid endocytosis in pollen tubes?
Endocytosis has been generally associated with clathrin
coats, but electron microscope studies have shown that
clathrin coated vesicles are not abundant in the pollen tube
apex (Lancelle and Hepler, 1992). Recent data, however,
has revealed the existence of a different form of
endocytosis which do not require clathrin coats (Nichols
and Lippincott-Schwartz, 2001).
The results obtained in pollen tubes favours the
existence of a rapid endocytosis mechanism. This type of
endocytosis is a Ca2+-dependent process, coupled to
exocytosis, that requires GTP hydrolysis and dynamin
but not clathrin. Dynamin is a GTPase involved in the
pulling and detachment of the endocytic vesicle from the
plasma membrane. This protein, already identi®ed in plant
cells (Jin et al., 2001), is thought to be involved both in
constitutive and rapid endocytosis and to require the
hydrolysis of GTP to accomplish its function. Along with
this rapid endocytosis, a second mechanism where endoand exocytosis are uncoupled should be present. This
mode is favoured if Ca2+ is lowered and/or GTP increases.
According to this hypothesis, it is proposed that secretion
is modulated by Ca2+ and GTPase activity to attain optimal
conditions for coupled exocytosis and endocytosis according to intra and extracellular stimuli.
Acknowledgements
This work was supported by an HFSP grant and FundacËaÄo CieÃncia e
Tecnologia, Portugal (BCI/32605/99 and BD/19642/99)
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