Download Effects of Brefeldin A on Pollen Germination and Tube Growth

Effects of Brefeldin A on Pollen Germination
and Tube Growth. Antagonistic Effects on
Endocytosis and Secretion1[W]
Qinli Wang, Lingan Kong, Huaiqing Hao, Xiaohua Wang, Jinxing Lin*,
Jozef Šamaj, and František Baluška
Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, Chinese
Academy of Sciences, Beijing 100093, China (Q.W., L.K., H.H., X.W., J.L.); Graduate School of the Chinese
Academy of Sciences, Beijing 100049, China (Q.W., X.W.); Institute of Cellular and Molecular Botany,
Department of Plant Cell Biology, Rheinische Friedrich-Wilhelms-University Bonn, D–53115 Bonn,
Germany (J.S., F.B.); Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences,
SK–95007, Nitra, Slovak Republic (J.S); and Institute of Botany, Slovak Academy of Sciences,
SK–84223, Bratislava, Slovak Republic (F.B.)
We assessed the effects of brefeldin A (BFA) on pollen tube development in Picea meyeri using fluorescent marker FM4-64 as
a membrane-inserted endocytic/recycling marker, together with ultrastructural studies and Fourier transform infrared
analysis of cell walls. BFA inhibited pollen germination and pollen tube growth, causing morphological changes in a dosedependent manner, and pollen tube tip growth recovered after transferring into BFA-free medium. FM4-64 labeling showed
typical bright apical staining in normally growing P. meyeri pollen tubes; this apical staining pattern differed from the
V-formation pattern found in angiosperm pollen tubes. Confocal microscopy revealed that exocytosis was greatly inhibited in
the presence of BFA. In contrast, the overall uptake of FM4-64 dye was about 2-fold that in the control after BFA (5 mg mL21)
treatment, revealing that BFA stimulated endocytosis in a manner opposite to the induced changes in exocytosis. Transmission
electron microscopic observation showed that the number of secretory vesicles at the apical zone dramatically decreased,
together with the disappearance of paramural bodies, while the number of vacuoles and other larger organelles increased. An
acid phosphatase assay confirmed that the addition of BFA significantly inhibited secretory pathways. Importantly, Fourier
transform infrared microspectroscopy documented significant changes in the cell wall composition of pollen tubes growing in
the presence of BFA. These results suggest that enhanced endocytosis, together with inhibited secretion, is responsible for the
retarded growth of pollen tubes induced by BFA.
The pollen tube is a highly polarized plant cell with
a rapidly growing tip that is specialized to deliver
genetic material from the site of pollination on the
flower stigma to the site of fertilization at the ovule
(Hepler et al., 2001). Polarized pollen tube growth
results from continued fusion with the plasma membrane by secretory vesicles derived from the Golgi
apparatus. This process provides new plasma membrane and cell wall components, and remodels the cell
1
This work was supported by the National Science Fund of China
for Distinguished Young Scholars (30225005), and by grants from the
European Union Research Training Network TIPNET (project no.
HPRN–CT–2002–00265) obtained from Brussels; from Deutsches
Zentrum für Luft- und Raumfahrt (Bonn); and from Grant Agency
Vega (grant nos. 2/2011/22 and 2031), Bratislava, Slovakia.
* Corresponding author; e-mail [email protected]; fax 0086–10–
62590833.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Jinxing Lin ([email protected]).
[W]
The online version of this article contains Web-only data.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.105.069765.
1692
wall composition (Mascarenhas, 1993). The quantity of
membrane delivered to the cell tip by exocytosis is in
excess of that required for the pollen tube growth rate,
suggesting an underlying recycling process (Parton
et al., 2001; Camacho and Malhó, 2003). Obviously, a
delicate balance between the exocytosis of cell wall
components, cell wall assembly, and endocytosis is
essential for pollen tube growth (Parton et al., 2001).
However, the precise mechanisms involved in the regulation of exocytosis, endocytosis, and vesicle recycling in growing pollen tubes remain speculative.
Exocytosis is a general term used to denote vesicle
fusion at the plasma membrane, and it is the final step
in the secretory pathway that typically begins in the
endoplasmic reticulum (ER), passes through the Golgi
apparatus, and ends at the outside of the cell (Battey
et al., 1999). Endocytosis is postulated to counterbalance membrane secretion (Emans et al., 2002). However,
the growing number of plasma membrane proteins
and cell wall molecules, such as pectins and xyloglucans, accomplish recycling via endocytosis, followed
by exocytosis from secretory endosomes (Baluška et al.,
2002, 2005; Šamaj et al., 2004, 2005). Growing pollen
tubes exhibit higher endocytosis and/or exocytosis
Plant Physiology, December 2005, Vol. 139, pp. 1692–1703, www.plantphysiol.org 2005 American Society of Plant Biologists
Brefeldin A Stimulates Endocytosis and Inhibits Secretion
activity in the apical region (Parton et al., 2001, 2003;
Camacho and Malhó, 2003). Rapid tip growth in
angiosperm pollen tubes has been studied extensively
(Derksen et al., 1995) and is characterized by a particular type of cytoplasmic organization, i.e. the specific
accumulation of secretory vesicles, clathrin, and clathrincoated pits at the tube tip (Derksen et al., 2002). However, pollen tubes of gymnosperms differ from those
of angiosperms in many important characteristics
(Derksen et al., 1995; Wang et al., 2003). Gymnosperm
pollen tubes show natural ramification, and a slow,
mostly Brownian-like, movement of the organelles.
They have little tip-to-base zonation of large organelles. The cytoplasm contains numerous starch grains,
and few microtubules and actin filaments are found in
the cortex. Outside the tip, the mitochondrial density
increases toward the periphery, giving rise to rows of
mitochondria along the tube wall. Unlike in angiosperms, the Golgi does not show any specific zonation
or accumulation in the gymnosperm tube (De Win
et al., 1996). In addition, the initiation of germination
and the maintenance of pollen tube elongation in gymnosperms depend on continuous protein synthesis
(Fernando et al., 2001; Hao et al., 2005). These different physical characteristics may be reflected by another
type of cytoplasmic organization than that known in
angiosperm pollen tubes.
The inhibitor brefeldin A (BFA) is a metabolite produced by fungi that is used for the study of endomembrane vesicle flow in eukaryotic cells (Rojas et al., 1999).
It affects membrane traffic in the animal secretory and
endocytic pathways (Lippincott-Schwartz et al., 1991).
Increasing evidence documents striking and reversible
effects of BFA on secretion from both plant Golgi
apparatus and endosomes, making it a useful tool in
following the secretory pathway in plant cells (Baluška
et al., 2002; Šamaj et al., 2004). In meristematic root
cells, BFA inhibits exocytosis but allows endocytosis
(Baluška et al., 2002, 2005). Using fluorescent marker
FM1-43, Emans et al. (2002) reported that BFA stimulated temperature-dependent endocytosis in BY-2 suspension cells. Moreover, studies on tobacco (Nicotiana
tabacum) pollen tubes established that BFA blocks the
secretion of cell wall material, resulting in growth
arrest (Rutten and Knuiman, 1993). Additionally, BFA
inhibits root hair tip growth, accompanied by the
disappearance of the clear zone, depletion of secretory
vesicles, and the simultaneous relocation of actin and
mitogen-activated protein kinase (Šamaj et al., 2002).
However, there is controversy regarding the effects of
BFA on endocytosis (Prydz et al., 1992; Baluška et al.,
2002), and no study has systematically compared differences in the endocytic pathways to changes in the
cell wall components in response to BFA treatment.
Such findings may be important for bridging existing
information among the biochemical, physiological, and
cellular levels.
The goal of this investigation was to evaluate the
effects of BFA on the endocytosis and the abundance
and distribution of secretory vesicles at the apical
region during pollen tube development in the gymnosperm Picea meyeri using several independent methods. In addition, the chemical components of the tube
wall and the ultrastructure of the pollen tubes were
analyzed to gain insight into the structural basis of the
observed effects.
RESULTS
Pollen Germination and Pollen Tube Growth
Much variation was observed in the germination
rate of pollen grains incubated in medium containing
various concentrations of BFA. Pollen began to germinate after 6 h in control culture medium and reached
its maximum germination ratio of 85% after 36 h (Table
I; Fig. 1A). Little difference was observed in the
germination rate with the addition of 1 mg mL21 BFA
to the culture medium. However, with the addition of
5 mg mL21 BFA, the average pollen germination rate
was significantly lower, at 48% (P , 0.05). The pollen
grain germination process was severely retarded
when treated with higher concentrations of BFA (Table
I; Fig. 1B).
When cultured in standard medium, pollen tubes
were long, with uniform diameter and a clear zone at
the apical region (Fig. 1C). The addition of BFA led to
obvious morphological changes, including changes in
growth direction (wavy growth pattern), an increase in
the tube diameter, and variable changes in the tube tip,
such as more pointed tips or tip swelling (Fig. 1, D–F).
Table I. Dose-dependent effects of BFA on P. meyeri pollen germination and pollen tube growth
The germinated pollen grains were counted after 36-h culture, and only those pollen tubes with their length longer than the diameter of pollen grain
were considered germinated.
BFA
Germination Rate
mg mL21
%
0
1
3
5
8
84.5
83.0
68.1
48.3
8.2
6
6
6
6
6
Pollen Tube Length
6h
12 h
18 h
24 h
30 h
36 h
mm
3.4
3.3
2.7
1.9
0.4
Plant Physiol. Vol. 139, 2005
45.0
43.0
39.5
39.2
38.1
6
6
6
6
6
1.8
1.9
1.6
1.8
1.8
91.5
89.8
85.3
78.5
39.5
6
6
6
6
6
3.6
4.5
4.3
3.8
1.8
187.7
185.9
151.3
80.5
45.2
6
6
6
6
6
5.6
5.5
5.5
3.6
2.2
262.5
250.2
175.0
82.3
43.7
6
6
6
6
6
8.4
7.9
7.3
4.0
2.1
300.9
265.7
169.2
83.0
49.0
6
6
6
6
6
9.1
9.0
8.4
4.1
2.4
315.9
265.2
165.1
84.8
48.0
6
6
6
6
6
9.7
9.5
8.2
4.2
2.3
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Wang et al.
Figure 1. Effects of BFA on pollen
germination and pollen tube morphology. A, Healthy P. meyeri pollen tubes
cultured in standard medium for 36 h,
showing good germination and many
long pollen tubes with normal shape.
B, P. meyeri pollen tubes cultured in
medium containing 5 mg mL21 BFA for
36 h, showing poor germination and
a few short tubes with morphological
abnormalities. C, Micrograph of a control P. meyeri pollen tube cultured for
20 h, showing a regularly shaped
pollen tube of constant diameter and
a clear zone at apical region. D to F,
Typical examples of pollen tubes in
the presence of BFA for 20 h. D, Pollen
tube incubated in 1 mg mL21 BFA; stars
indicate changes in growth direction
(wavy pattern). E, Pollen tube incubated in 5 mg mL21 BFA, showing
a swelled tube tip (arrowhead). F,
Most of the pollen grains cultured in
8 mg mL21 BFA, showing aberrant
protrusions, which were not regarded
as germinated. G, Details of cell wall
structure in control pollen tube. The
cell wall was uniform, compact, and
0.4 mm thick. H, Details of cell wall
structure in BFA-treated pollen tube.
The cell wall became more loosely
packed, showing the cell wall thickness from 0.35 to 0.6 mm in the presence of BFA. cz, Clear zone; CW,
cell wall. Bars in A and B 5 100 mm;
in C to F 5 50 mm; and in G and
H 5 0.3 mm.
With increasing incubation durations, the BFA-treated
pollen tubes ruptured, indicating that they were more
fragile. Treatment with 1 mg mL21 BFA decreased the
pollen tube growth rate to 7.8 mm h21. When the BFA
concentration was increased to 5 mg mL21, the average
pollen tube growth rate dropped to 2.5 mm h21, in
comparison with 16.2 mm h21 observed in the control
during 30 h of growth after germination. With increasing BFA concentrations, the inhibitory effect on
pollen tube growth was more pronounced (Table I).
Most of the pollen grains cultured in the presence
of 8 mg mL21 BFA did not germinate. Although the
pollen grains were observed to have short protrusions,
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they cannot be considered as germinated since their
length was shorter than the pollen grain diameter
(Fig. 1F). Thus, BFA inhibited pollen tube growth in
a dose-dependent manner. To determine whether
the effects of BFA were reversible, pollen grains were
first cultured in media supplemented with 5 mg mL21
or 8 mg mL21 BFA for 18 h, and were then transferred into BFA-free medium. Our results showed
that for samples previously treated with 5 mg mL21
or 8 mg mL21 BFA, not only did germination rate
increase but also pollen tubes continued to elongate
when transferred into BFA-free medium (Supplemental Fig. 1).
Plant Physiol. Vol. 139, 2005
Brefeldin A Stimulates Endocytosis and Inhibits Secretion
FM4-64 Staining Distribution Pattern
In median confocal optical sections (Fig. 2A), FM4-64
consistently produced a distinct peripheral and bright
apical staining, which was distributed in the extreme
apical (15–20 mm) region in normally growing P. meyeri
pollen tubes. The corresponding quantitative image
revealed a model of a sharply defined high signal in the
extreme apex; however, beyond this region, FM4-64
staining was generally and significantly weakened
(Fig. 2B). Peripheral staining was shown to be plasma
membrane, and not associated with the cell wall, as
shown by plasmolysis with 100 mM sorbitol in the
presence of FM4-64. Pretreatment of pollen tubes with
500 mM sodium azide impaired dye uptake: FM4-64
fluorescence could only be observed at plasma membrane, but the dye was not internalized over time
(Supplemental Fig. 2).
For pollen tubes cultured in medium containing
5 mg mL21 BFA, the FM4-64 membrane-staining pattern at the apical region was disrupted. Extreme apical
staining was more diffuse and less well localized, no
longer exhibiting the bright region at the apical clear
zone that is characteristic of normally growing pollen
tubes (Fig. 2C). The corresponding quantitative image
showed a broader distribution of FM4-64 staining (Fig.
2D). Furthermore, a typical FM4-64 staining pattern in
a normal pollen tube could be rapidly dispersed by
short-term treatment with BFA (Fig. 3). Within 10 to
15 min, apical FM4-64 staining was largely redistributed from the apex and became continuously scattered
over more subapical regions (Fig. 3, B and C). Ultimately, the apical bright FM4-64 staining pattern
disappeared with increasing time of exposure to BFA
(Fig. 3D).
The Time Course of FM4-64 Internalization
The uptake of FM4-64 into P. meyeri pollen tubes
followed a strict time sequence (Fig. 4, A–F). Staining
associated with the plasma membrane became obvious immediately after dye application to the germination medium (Fig. 4A). This was followed by
internalization of the dye, mainly at the apical region
(Fig. 4, C and D). Within several minutes, the typical
staining pattern of FM4-64 dye was observed, i.e.
bright staining of the entire apical region that extended
to the subapical region with very weak staining (Fig. 4,
E and F), rather than the inverted cone shape typically
observed in angiosperms. The apical fluorescent region corresponds to the so-called clear zone, a region
filled with secretory vesicles but lacking any larger organelles.
In BFA-treated pollen tubes, the FM4-64 internalization continued even more rapidly, and the dye
internalization occurred throughout almost the whole
pollen tube (Fig. 5, A–F). However, the bright apical
FM4-64 staining pattern did not occur. With increasing
time after FM4-64 dye application, only patch-like and
dispersed fluorescence of stained material emerged
over most of the pollen tube (Fig. 5F). To quantify the
effect of BFA on FM4-64 dye internalization, pollen
tubes cultured for 18 h in control medium were
pretreated with BFA, latrunculin B, or sodium azide
for 60 min, and, subsequently, FM4-64 was internalized for 12 min. The images in Figure 6 (A–H) show
that the FM4-64 staining pattern was disrupted by
treatment with BFA (Fig. 6C), latrunculin B (Fig. 6E),
and sodium azide (Fig. 6G) compared to the control
(Fig. 6A). Moreover, quantitative data suggest that
BFA stimulated the total uptake of FM4-64 dye (Fig.
6D). In contrast, latrunculin B inhibited the internalization of FM4-64 dye (Fig. 6F), and sodium azide
blocked FM4-64 internalization almost completely
(Fig. 6H) compared to the control (Fig. 6B). This fluorimetric quantification showed that FM4-64 uptake
was stimulated about 2-fold by BFA and inhibited
about 0.48-fold by latrunculin B, while almost no
dye was internalized after the sodium azide treatment
(Fig. 7).
Ultrastructural Changes in Organelles and the
Production of Secretory Vesicles
A zone filled mainly with secretory vesicles (clear
zone) could be distinguished from the streaming
Figure 2. Confocal images of FM4-64
staining in pollen tubes of P. meyeri.
A, A median focal plane confocal
optical section, showing a typical
FM4-64 staining pattern in a growing
pollen tube; the bright field at onethird size appears as an insert. B, Pixel
values along a central transect through
the fluorescence image in A. C, A
median focal plane confocal optical
section, showing a dispersed and disrupted FM4-64 staining pattern in BFAtreated (5 mg mL21) pollen tube; the
bright field at one-third size appears as
an insert. D, Pixel values along a central transect through the fluorescence
image in C.
Plant Physiol. Vol. 139, 2005
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Wang et al.
Figure 3. Time course of disruption in a normal typical FM4-64
staining induced by 5 mg mL21 BFA. BFA was applied directly to
growing pollen tubes on thin gel layers in 70 mL of 115% to 120%
liquid medium. A, A typical FM4-64 distribution pattern in a normal
growing pollen tube of P. meyeri. B, The changes of FM4-64 staining at
3 min after the addition of BFA, showing the FM4-64 fluorescence
tended to be scattered. C and D, The changed FM4-64 staining
distribution with increasing time, showing FM4-64 fluorescence became more dispersed and almost distributed in the whole pollen tube
after 12 min of BFA treatment. Bar 5 25 mm.
cytoplasm at the very tip of pollen tubes growing in
control medium. In the zones behind the very tip,
many larger organelles such as Golgi stacks, ER, and
mitochondria, as well as many small round-shaped
vacuoles, were well resolved (Fig. 8A). Some of the
secretory vesicles fusing with plasma membrane, and
paramural bodies (PBs), probably resulting from fusion of multivesicular bodies (MVBs) with the plasma
membrane, were observed at higher magnification
(Fig. 8, C and E). Most Golgi stacks in the control pollen
tubes consisted of four to seven flattened cisternae
with a distinct cis-to-trans polarity, and numerous secretory vesicles were present around them (Fig. 8F).
Much variation was observed when pollen grains
were incubated in the medium containing BFA. Treatment with 5 mg mL21 BFA drastically decreased the
number of secretory vesicles and increased the number
of small vacuoles at pollen tube tips, and other larger
organelles such as mitochondria and lipid bodies could
be observed at the very apical region (Fig. 8, B and D).
Upon BFA treatment, Golgi stacks disassembled or
tended to be stacked and curved at the trans face, while
the number of secretory vesicles surrounding them
drastically decreased (Fig. 8, G and H). The ER became
swollen with the detachment of ribosomes from the
rough ER (Fig. 8G). However, no significant differences
were observed between BFA-treated and normal pollen
tubes in terms of mitochondria (data not shown). In
addition, a more loosely packed cell wall, 0.35 to 0.6 mm
thick, was produced (Fig. 1H) in the treated than in the
control pollen tubes, which showed a uniform cell wall
thickness of 0.4 mm (Fig. 1G).
Activity of Secreted Acid Phosphatase
The effects of BFA on the acid phosphatase (acPase)
activity are plotted in Figure 9 and show the average of
three experiments. The acPase activity increased as the
washed pollen grains began to germinate and pollen
tubes elongated in the germination medium (Fig. 9, A,
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a, B, a, and C, a). There were almost parallel increases
in acPase activity and pollen tube length over time
(Fig. 9, A, a, and C, a). When 5 mg mL21 BFA was
added to the medium after 12 h of incubation, the
export of acPase activity (Fig. 9A, b) was immediately
inhibited, along with the inhibition of tube elongation
(Fig. 9C, b) and pollen germination (Fig. 9B, b). Pollen
tube growth and secretion of active acPase were
almost completely inhibited over time with BFA treatment (Fig. 9, A, b, and C, b), whereas pollen germination was less affected (Fig. 9B, b).
Changes in Chemical Components
Fourier transform infrared (FTIR) spectra were analyzed in apical, middle, and basal regions of pollen
tubes. FTIR spectra of pollen tubes growing in control
medium showed similar patterns among the three regions analyzed, although the proportion of proteins to
polysaccharides varied among different regions within
an individual pollen tube. The absorption bands occurred at 1,650 cm21 and 1,550 cm21, corresponding to
amide I and amide II of proteins (McCann et al., 1994),
while a peak at 1,744 cm21 corresponded to saturated
esters (McCann et al., 1994), and polysaccharides absorbed at 1,200 to 900 cm21 (McCann et al., 1994; Fig. 10A).
Treatment with 5 mg mL21 BFA induced displacement of the peaks or changes in absorbance. The
difference spectra generated by digital subtraction of
the spectra of control tube walls from the spectra of
BFA-treated tube walls (Fig. 10B) showed that both the
amide-stretching bands and the carbohydrate bands
Figure 4. FM4-64-uptake time course in a growing P. meyeri pollen
tube. A to F, Median confocal fluorescence images at increasing times
after addition of FM4-64 (2 mM in 115% standard medium). To avoid
osmotic perturbation, pollen tubes were pretreated with 115% medium
before dye application. The rapid uptake suggests an extremely high rate
of endocytosis and membrane traffic; the bright region suggests there is
an accumulation of secretory vesicles in the apical zone. Bar 5 25 mm.
Plant Physiol. Vol. 139, 2005
Brefeldin A Stimulates Endocytosis and Inhibits Secretion
concentrations of BFA used in this study are within
a physiological range (Supplemental Fig. 1). The results
presented here indicated that the pollen tube growth
was closely linked to the cellular process of the secretory pathway, the disruption of which led to the pollen
tube growth arrest. However, it should be noted that,
for P. meyeri, only concentrations of BFA greater than
1 mg mL21 exerted a significant effect. This concentration
is much higher than that reported for angiosperms, e.g.
0.1 mg mL21 of BFA in the pollen tubes of tobacco
(Rutten and Knuiman, 1993). This large discrepancy
may be due to differences in the plant species examined,
and may also reflect alterations in the activity of the Golgi
apparatus.
Figure 5. Time course of changes in dye distribution of FM4-64-loaded
P. meyeri pollen tubes treated with 5 mg mL21 BFA. A to F, Confocal
fluorescence images of a BFA-treated pollen tube at different times,
showing BFA treatment did not inhibit the internalization of FM4-64
dye through endocytosis but the dispersed FM4-64 distributing pattern
indicating BFA inhibited secretory vesicle accumulation at tube tip.
Bar 5 25 mm.
were dramatically reduced after BFA treatment in all
three regions analyzed. In contrast to a small reduction
in protein content, there was a marked decrease in
polysaccharide content among the three regions. Further, the reduction of polysaccharides in the newly
formed tip region was far more obvious than in the
already formed middle and basal regions.
DISCUSSION
BFA Retarded Pollen Tube Development
The fungal metabolite BFA, which has been shown
to interfere with protein transfer through the endomembrane system, is an inhibitor of the secretory
pathway of intracellular protein transfer and mainly
induces dysfunction of the Golgi stacks (Nebenführ
et al., 2002). Lanubile et al. (1997) reported that BFA
affects the synthesis and transport of cell wall polysaccharides and proteins in pea (Pisum sativum) root
seedlings. Additionally, the comparison of FM1-43 uptake in normal and BFA-treated tobacco suspension
cells showed that BFA can stimulate endocytosis (Emans
et al., 2002). However, the reliability of FM1-43 as an
endocytic tracer for plant cells has not been confirmed
(Meckel et al., 2004; Šamaj et al., 2005).
Little information about the effects of BFA on the
secretory pathway during pollen germination and
pollen tube growth has been reported. Rutten and
Knuiman (1993) reported that the secretion and pollen
tube growth were two separate processes. In this study,
BFA not only reduced pollen germination rates (Table I;
Fig. 1B) and retarded pollen tube growth (Table I), but
also resulted in abnormalities in pollen tube shape (Fig.
1, D–F). The recovery experiments indicated that the
effects of BFA were reversible, clearly documenting the
Plant Physiol. Vol. 139, 2005
BFA Disrupted Secretory Vesicle Accumulation at the
Pollen Tube Apex
Regulated secretory vesicle delivery, distribution,
vesicle fusion, and rapid membrane recycling can be
tracked in living cells using FM4-64 dye because this
dye is nontoxic and water soluble (Šamaj et al., 2005).
For studies of plant and fungal cells, FM4-64 is usually
preferred to FM1-43 because of its superior brightness,
greater contrast, and higher photostability, and, more
importantly, unlike FM1-43, the dye is not retained in
cell walls (Bolte et al., 2004). Because the dye stains
membranes in an activity-dependent manner, it has
been found increasingly useful in exploring the endocytosis and secretory mechanisms in a variety of
biological models (Smith and Betz, 1996). The present
experiment of plasmolysis with sorbitol and the effect
of sodium azide on FM4-64 uptake (Supplemental Fig.
2) confirmed earlier reports that FM4-64 staining was
plasma membrane rather than cell wall associated,
and its internalization occurred via an endocytic
mechanism instead of passive diffusion (Parton et al.,
2001, 2003; Camacho and Malhó, 2003). The median
confocal optical images of FM4-64 fluorescence in
normally growing P. meyeri tubes consistently revealed
bright peripheral and apical staining (Fig. 2A). The
FM4-64 staining pattern detected in P. meyeri was
different from the V-shaped apical staining reported
in angiosperm species (Parton et al., 2001, 2003). In
addition to the staining pattern, the proportion of the
apical region stained by FM4-64 to the whole pollen
tube in P. meyeri was much lower than that in lily
(Lilium longiflorum) pollen tubes. In P. meyeri, the ratio
of the length of the FM4-64-labeled apex region (15–
20 mm) to the pollen tube diameter (35–40 mm) was
about 1:2 in comparison with the corresponding parameter (1:1) in lily pollen tubes, in which both the length
of the FM4-64 staining region and the pollen tube
diameter were about 15 to 20 mm (Parton et al., 2001,
2003). Because FM4-64 staining corresponds strikingly
to the location and distribution of secretory vesicles at
the apical region (Lancelle and Hepler, 1992; Derksen
et al., 1995), we may conclude that the slow growth
rate of P. meyeri pollen tubes was largely caused by the
smaller region of secretory vesicles at the tip.
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Wang et al.
Figure 6. Confocal images showing uptake of 2 mM FM4-64 into control and inhibitor-treated pollen tubes of P. meyeri within
12 min. All the pixel values did not include the peripheral region. A, A median focal plane confocal optical section of a control
pollen tube; the bright field at one-third size appears as an insert. B, Pixel values along a central transect through the fluorescence
image in A. C, A median focal plane confocal optical section of a pollen tube pretreated with 5 mg mL21 BFA for 60 min; the
bright field at one-third size appears as an insert. D, Pixel values along a central transect through the fluorescence image in C,
showing that BFA stimulated the internalization of FM4-64 dye. E, A median focal plane confocal optical section of a pollen tube
pretreated with 1 mM latrunculin B for 60 min; the bright field at one-third size appears as an insert. F, Pixel values along a central
transect through the fluorescence image in E, showing latrunculin B inhibited the internalization of FM4-64 dye. G, A median
focal plane confocal optical section of a pollen tube pretreated with 500 mM sodium azide for 60 min; the bright field at one-third
size appears as an insert. H, Pixel values along a central transect through the fluorescence image in G, showing sodium azide
blocked internalization of FM4-64 dye almost entirely. Bar 5 25 mm.
Previous studies have established that treatment
with BFA can result in a particular reorganization of
the cytoplasm at the pollen tube apex (Parton et al.,
2003). Our experiment with FM4-64 indicated that the
typical bright FM4-64 staining pattern that appeared at
the apical region in normal pollen tubes is lost in BFAtreated pollen tubes, and is replaced by a scattered and
dispersed distribution of the dye (Fig. 2, C and D). This
implies that the distribution and number of secretory
vesicles at the tip region varied after BFA treatment.
The time course of dissociation and dissipation of
a normal apical FM4-64 staining pattern after the
addition of BFA in P. meyeri revealed a dynamic process; BFA can rapidly disperse and reduce the abundance of secretory vesicles located in the tube apex
(Fig. 3). The decreased number and dispersed distribution of secretory vesicles in the apical region indicated that vesicle secretion became less active after
BFA treatment, which likely resulted from the effect of
BFA on the production of secretory vesicles and/or
vesicle delivery to the apical region. Vesicle trafficking
1698
in plants is dependent on the actin cytoskeleton, and
previous reports have shown that BFA affects both
actin organization and actin-dependent endosomal
motility within the growing tips of root hairs (Šamaj
et al., 2002; Voigt et al., 2005). The phenotype of wavy
pollen tubes with increased diameter produced upon
BFA treatment (Fig. 1, D–F) that was observed in our
experiment was possibly caused by the improper
delivery to and depletion of secretory vesicles from
the clear zone of the pollen tubes. Nevertheless, we did
not detect BFA-induced vesicular aggregation, as was
found in BFA-treated lily pollen tubes (Parton et al.,
2003), probably because of the different type of cytoplasmic streaming found in conifer and angiosperm
pollen tubes (De Win et al., 1996; Lazzaro et al., 2003).
BFA Stimulated Endocytosis
There have been reports of endocytosis associated
with tip growth in the development of pollen tubes,
root hairs, and fungal hyphae (Derksen et al., 2002;
Plant Physiol. Vol. 139, 2005
Brefeldin A Stimulates Endocytosis and Inhibits Secretion
endosomes (Geldner et al., 2003; Murphy et al., 2005)
transport components needed for cell wall expansion
(Camacho and Malhó, 2003). In this experiment, we
observed that secretory vesicles dominated the apex in
P. meyeri pollen tubes, and their distribution corresponded strikingly to the FM4-64 staining pattern in
Figure 7. FM4-64 internalization in control and inhibitor-treated pollen
tubes showing that dye uptake is stimulated by BFA (5 mg mL21) inhibited by latrunculin B (1 mM) and sodium azide (500 mM). Cell-associated
FM4-64 fluorescence was quantified after a 12-min internalization of the
dye and normalized to control pollen tubes labeling. Data shown are
means 6 SD and are representative of three experiments, each containing
three individual measurements. CK, Control; LTB, latrunculin B; SAD,
sodium azide.
Read and Kalkman, 2003; Voigt et al., 2005). Studies on
yeast (Saccharomyces cerevisiae), tobacco BY-2 cells, and
lily pollen tubes have indicated that FM dyes were
taken up by the endocytic pathway (Parton et al., 2001;
Emans et al., 2002). In this study, we found that FM4-64
dye was internalized to induce a typical bright staining pattern at the apical region (Fig. 4) within 30 min,
indicating that endocytosis occurred during normal
pollen tube growth in P. meyeri, similar to reports from
other species (Parton et al., 2001; Camacho and Malhó,
2003). In BFA-treated pollen tubes, FM4-64 dye uptake
was more rapid, probably because the dye internalization occurred at broader range, in spite of dispersed
staining (Fig. 5), implying that BFA treatment stimulated endocytosis in contrast to untreated pollen tubes.
Furthermore, latrunculin B and sodium azide inhibited and blocked FM4-64 dye internalization, respectively, while BFA obviously stimulated endocytosis
(Fig. 6, A–H). This conclusion was also strongly supported by a fluorimetric quantitative analysis (Fig. 7).
Because endocytosis is postulated to counterbalance
membrane secretion (Emans et al., 2002), we conclude
that decreased tube growth is likely a direct consequence of increased endocytosis and inhibited secretion from both Golgi stacks and endosomes. Stimulated
apical endocytosis after BFA treatment, through its
action on an ADP ribosylation-guanine nucleotide exchange factor involved in endocytosis, was indeed
reported earlier in tobacco BY-2 suspension cells
exposed to FM1-43 (Emans et al., 2002).
BFA Disassembled the Golgi Apparatus
Tip growth in pollen tubes requires the integrity of
the secretory system (Moscatelli and Cresti, 2001).
During tube growth, secretory vesicles derived from
the Golgi apparatus and/or from the early/recycling
Plant Physiol. Vol. 139, 2005
Figure 8. Electron micrographs in control and BFA-treated (5 mg mL21)
pollen tubes. A, Tip region of a control pollen tube, showing the apical
clear zone. B, Tip region of a BFA-treated pollen tube, showing the
apical clear zone was occupied by many organelles, e.g. mitochondria
and vacuoles. C, Magnified picture of a control pollen tube tip region,
from where many vesicles could be observed; some of them were
fusing with, or releasing from, the plasma membrane. D, Tip region of
a BFA-treated pollen tube. No PBs and secretory vesicles could be
observed, but many other types of vesicles and large organelles, such as
mitochondria, vacuoles, and lipid bodies, appeared. E, Another apical
zone in control pollen tube, indicating a typical PB (arrow). F, Golgi
apparatus in control pollen tubes, showing the Golgi stacks contain
four flattened cisternae with a distinct cis-trans polarity with numerous
vesicles attached to them. G, Abnormal arrangements of the Golgi
stacks in BFA-treated pollen tubes, indicating the disassembly of the
Golgi apparatus accompanied by vesiculation of trans-side (arrow), less
Golgi-derived vesicles, as well as the ER dilations (arrow). H, Another
abnormal arrangement of Golgi apparatus induced by BFA treatment.
Golgi cisternae appeared abnormally stacked and curved, engulfing
some large vesicles. Bar 5 2 mm in A and B; 1 mm in C and D; 0.5 mm in
E; and 0.2 mm in F, G, and H. SV, Secretory vesicles; M, mitochondrion;
V, vacuole; L, lipid body; G, Golgi apparatus; PB, paramural body.
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Wang et al.
other large organelles are detected in the apical region
of the pollen tube (Hao et al., 2005). Deducing from the
facts that the pollen tube growth rate in P. meyeri
(16 mm h21) ranks between that of the lily (390 mm h21)
and other gymnosperm species (1 mm h21; De Win et al.,
1996; Yang et al., 1999; Hao et al., 2005), we speculate
that the pollen tube growth rate mainly depends on
the particular cellular organization of the apical region. In the presence of BFA (5 mg mL21), the secretory
vesicles rapidly vanished from the very tip, while
many small vacuoles and mitochondria, as well as some
lipid bodies and various types of vesicles, appeared in
the apical region (Fig. 8, B and D). Furthermore, the
number of secretory vesicles at the trans-Golgi side
decreased because of the structural disintegration of
the Golgi apparatus (Fig. 8, G and H), suggesting that
BFA inhibited the production of secretory vesicles by
disorganizing the Golgi apparatus and trans-Golgi
network. The ER also dilated after BFA treatment
(Fig. 8G). Because the pollen tube growth rate depends
on the supply of new material to the cell walls
Figure 9. A typical inhibitor experiment showing the effects of BFA on
acPase activity (A), germination frequency (B), and mean tube length
(C). Pollen grains were incubated in germination medium. After 12 h,
BFA (5 mg mL21) was added to one-half of the pollen culture (:),
whereas the other half served as a control (¤). All three parameters
were determined every 6 h. The data points represent means with SDs of
three independent experiments.
both location and distribution, as described previously. Because many of the secretory vesicles received
the internalized endocytic tracer FM4-64 within a few
minutes of exposure, we propose that many of these
secretory vesicles are derived from the early/recycling
endosomes, as reported for the apices of tip-growing
root hairs (Voigt et al., 2005). However, it is important
to note that the vesicle distribution at the extreme
apical region shows a lower density (Fig. 8C) than that
found in the V-shaped vesicle accumulation in angiosperm pollen tubes (Parton et al., 2001, 2003) and also
differed from other coniferous species, in which many
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Figure 10. FTIR spectra obtained from the apical region, middle
region, and basal region of P. meyeri pollen tubes cultured for 20 h.
A, FTIR spectra obtained from the tip, middle, and basal regions of
pollen tubes cultured in standard medium (CK) or in medium containing 5 mg mL21 BFA (BFA) revealed that BFA treatment induced
displacements of the peaks or changes of absorbance. B, Difference
spectra generated by digital subtraction of spectra CK from spectra BFA,
showing that the content of proteins and polysaccharides decreased
after BFA treatment and that the reduction in polysaccharide was most
obvious in apical region.
Plant Physiol. Vol. 139, 2005
Brefeldin A Stimulates Endocytosis and Inhibits Secretion
(Derksen et al., 1995), the disorder inflicted on the secretory system (ER, Golgi apparatus, trans-Golgi network) by treatment with BFA inevitably retarded the
elongation of the pollen tubes, as discussed previously.
The PB, a general term used to describe all membrane systems associated with the plasma membrane,
comprising a group of membranous elements situated
between the plant cell wall and the plasma membrane,
is involved in exocytosis and cell wall deposition
(Marchant and Robards, 1968). Because PBs possessed
vesicles similar to the internal vesicles of MVBs, they
may eventually originate from fusions of MVBs, representing endosomes with apical plasma membranes
(Tse et al., 2004). In this study, we noted that many PBs
appeared in the normal growing pollen tube apex (Fig.
8E), suggesting that active exocytosis of MVBs occurred during normal pollen tube growth. However,
in the BFA-treated pollen tube apical region, no PBs
were observed (Fig. 10D), providing strong evidence
that BFA disrupted the secretory activity of the MVBs,
which may contribute to pollen tube wall deposition
during pollen tube elongation (Derksen et al., 1995).
BFA Lowered AcPase Activity
In plant cells, secretion can be measured by monitoring the release of secretory products in cell cultures
(Ibrahim et al., 2002). For example, the growth of yeast
is accompanied by the local secretion of acPase at the
growing site of the cell (Field and Schekman, 1980).
Based on a study of lily pollen tube development,
Ibrahim et al. (2002) suggested that the secreted acPase
activity released via the secretory pathway may serve
as a useful indicator of exocytotic activity in terms of
the conspicuous correlation between acPase secretion
and tube growth. The present experiment showed that
secreted acPase activity was strongly correlated with
pollen grain germination and tube growth in P. meyeri.
With the development of normal pollen tubes, the acPase
activity increased (Fig. 9), suggesting that acPase was
secreted during P. meyeri pollen tube growth, and its
secretory activity increased with the elongation of pollen
tubes. In the presence of BFA, the export of acPase activity (Fig. 9A, b) was dramatically inhibited, along with the
inhibition of pollen germination (Fig. 9B, b) and pollen
tube growth (Fig. 9C, b), suggesting that the cellular
process leading to secretion was disrupted by BFA. Because acPase was probably secreted together with the
cell wall material at the tube tip from where it diffuses
into the medium (Ibrahim et al., 2002), it seems reasonable to conclude that the inhibition of production of
secretory vesicles induced by BFA led to the suppression
of acPase secretion.
BFA Altered the Tube Cell Wall Composition
and Structure
Pollen tube elongation requires the insertion of
many polysaccharides and structural proteins into
the tube walls (Mascarenhas, 1993). Cell wall proteins,
Plant Physiol. Vol. 139, 2005
polysaccharides, and other components of the wall
matrix are synthesized in the ER-Golgi system and
transported into the apoplastic space via secretory
vesicles (Moore et al., 1991). Interference with the
production and transport of secretory vesicles results
in the modified secretion of cell wall components
(Cheung et al., 2002). FTIR microspectroscopy is a reliable and highly reproducible assay used to study cell
wall composition (McCann et al., 1994) and pollen
tube walls cultured in different media (Yang et al.,
1999; Wang et al., 2003). Using FITR analysis, we
confirmed that proteins were present in the pollen
tube walls of P. meyeri (Fig. 10A), as has been reported
for other species (Yang et al., 1999). The difference
spectra (Fig. 10B) showed that the protein and polysaccharide contents along the whole tube walls were
reduced when treated with BFA, directly demonstrating the inhibitory effect of BFA on tube wall deposition
during pollen tube growth. Furthermore, the polysaccharide content decreased more obviously than
that of protein, particularly at the apical region (Fig.
10B). This explains the differential sensitivity to BFA
treatment, at the Golgi apparatus, toward the synthesis of cell wall polysaccharides and the machinery of
vesicular transport of proteins to the wall. The BFAinduced modifications in the composition of pollen
tube walls were also reflected by the noncompact and
disordered wall structure (Fig. 1H).
It has been reported that cell walls at the apical
region are highly enriched with pectin (Li et al., 2002).
The FTIR analysis described here showed that polysaccharides decreased more obviously in the newly
formed tip region than in the already formed regions
after treatment with BFA. Because most of the bands in
the polysaccharide region between 1,200 and 900 cm21
are the vibrations associated with the sugar monomers of pectin (Chen et al., 1997), we conclude that the
prominent decrease in polysaccharide content at the
apical region was caused by a decrease in pectin content. Cell wall pectins cross-linked with boron and
calcium are internalized into plant cells (Baluška et al.,
2002, 2005) and apparently recycled via early/recycling endosomes (Baluška et al., 2002; Šamaj et al., 2004).
Thus, we propose that BFA-induced aberrant cell walls
are the result not only of inhibited secretion but also of
enhanced endocytosis of cell wall pectins in the tip region. Further studies should test this scenario.
In summary, our results clearly showed that BFA
retarded pollen tube development; the effects of BFA
were reversible because pollen tubes can recover their
tip growth after the removal of BFA. BFA treatment
inhibited exocytosis by disrupting the distribution and
reducing the abundance of secretory vesicles at the
apical region through disorganizing the Golgi apparatus, trans-Golgi network, and early/recycling endosomes. Importantly, endocytosis was stimulated, as
revealed by confocal microscopy and fluorimetry measurements, in a manner opposite to the inhibition
of exocytosis in the presence of BFA. The results
also demonstrated that the distribution pattern and
1701
Wang et al.
abundance of secretory vesicles found at the pollen
tube apical region in P. meyeri differ from the V-formation
of vesicle accumulation in angiosperms, using both
ultrastructural examination and fluorescent marker
FM4-64 labeling. FTIR analysis further demonstrated
that the changes in the cell wall composition could
clearly be attributed not only to inhibited exocytosis
but also to the enhanced uptake of cell wall components (especially pectins) through increased endocytosis. Based on these results, we conclude that enhanced
endocytosis, together with inhibited secretion, is responsible for the abnormal morphology and growth
inhibition of pollen tubes induced by BFA, a phenomenon that has not been reported previously.
MATERIALS AND METHODS
aliquot served as a control. After 60-min incubation, all of these aliquots were
supplemented with 2 mM FM4-64 for 12 min. Then, the aliquots were washed
three times with control medium by centrifugation (at 1,000g for 5 min). Cellassociated fluorescence was quantitated by fluorimetry using an F-4500
fluorospectrometer (Japan). FM4-64 was excited at 514 nm (5-nm bandpass),
and emission was detected at 580 nm (5-nm bandpass). Cell-associated
fluorescence was normalized to the cell-associated fluorescence of control
pollen tube labeling and the results of three measurements were averaged.
Electron Microscopy
For electron microscopy, pollen tubes incubated for 20 h in media
containing 0 or 5 mg mL21 BFA were fixed in 2.5% glutaraldehyde in 100 mM
phosphate buffer, pH 7.2, for 2 h at room temperature. Pollen tubes were
washed three times with this buffer, post-fixed with 1% OsO4 for 3 h in 100 mM
phosphate buffer, pH 7.2. After washing with this buffer three times, pollen
tubes were dehydrated in ethanol series and finally embedded in Spurr resin.
Ultrathin sections of pollen tubes were mounted on Formvar coated grids and
stained with 2% aqueous uranyl acetate and lead citrate, and observed with an
electron microscope (JEOL 1210) at 80 kV.
Plant Materials
Secreted AcPase Assay
Cones with mature pollen were collected from trees of Picea meyeri Rehd. et
Wils. growing in the Botanical Garden of the Institute of Botany, Chinese
Academy of Sciences, prior to the beginning of the pollination season at the
middle of April 2003, and were dried overnight at room temperature. The dry
pollen was stored at 220C until use.
Pollen grains of 50 mg were suspended in 50 mL of germination medium
and gently shaken for 1 min. Pollen grains were washed five times by
subsequently pelleting the grains, resuspending in fresh medium, and shaking
for 1 min to remove cell wall-bound acPase. After this washing procedure, the
pollen suspension was pipetted into petri dishes for germination. AcPase
secretion was monitored by sampling aliquots every 6 h and analyzing them for
acPase activity. After 30 h the pollen grains and tubes were pelleted and aliquots
of the supernatant containing the secreted acPase activity were frozen and
stored at 220C. The activity of the acPase was measured according to Ibrahim
et al. (2002) by measuring the release of p-nitrophenol from p-nitrophenyl
phosphate. Samples of 100 mL were incubated with 400 mL of reaction buffer
containing 100 mM acetic acid, pH 5.0, 5 mM p-nitrophenyl phosphate for 45 min
at 30C. The reaction was stopped by the addition of 200 mL of 500 mM NaOH,
pH 9.8, and the concentration of p-nitrophenol was determined at 405-nm
wavelength (U3000; Hitachi). All assays were performed as triplicates.
Pollen Culture
Stored pollen was equilibrated at room temperature for 30 min and carefully suspended in 12% (w/v) Suc medium containing 0, 1, 3, 5, 8, or 10 mg mL21
BFA, respectively. The pH of the media was adjusted to 6.4 with phosphatebuffered saline. The cultures were incubated on a shaker (100 rpm) at 25C. For
recovery experiments, samples exposed to the drug were gently centrifuged and
transferred to drug-free medium. For confocal images, pollen grains were
transferred to thin layers of 0.2% agar solidified culture medium after hydration
for 30 min in liquid media. Thin gel layers were prepared according to Parton
et al. (2001). Thin gel layers were sown with pollen suspension and incubated in
a dark humid environment at room temperature.
Determination of Pollen Germination and
Pollen Tube Growth
The germination rate was determined by checking at least 500 grains under
a Nikon microscope. Pollen grains were considered as germinated when the
pollen tube length was greater than the diameter of the pollen grain (Wang
et al., 2003). To measure the mean tube length, images of pollen tubes cultured
in the above media were taken at 6-h intervals, and the number of pollen tubes
examined was at least 200 for each treatment. All experiments were performed
at room temperature and in triplicate.
Confocal Microscopy
Loading cells with 2 mM FM4-64 dye was generally achieved by application
during the imbibition of pollen grains by direct addition of dye solutions in 115%
liquid medium to growing tubes on thin gel layers. BFA (5 mg mL21), latrunculin
B (1 mM), and sodium azide (500 mM) were applied directly to growing pollen
tubes on thin gel layers in 70 mL of 115% to 120% liquid medium. Fluorescence
from FM4-64 staining was detected using a Bio-Rad MRC 600 laser confocal
scanning unit attached to an Optiphot microscope (Nikon). The samples were
excited at 514 nm with a 25-mW argon ion laser operated at full power at an
intensity of 3%, achieved by means of neutral-density filters, with a nearly closed
pinhole and the gain adjusted to below level 7.00.
Fluorimetry Measurements
After 18-h culture, the pollen suspension of 20 mL was divided into four
equal aliquots. Three of these aliquots were incubated with BFA (5 mg mL21),
latrunculin B (1 mM), or sodium azide (500 mM) for 60 min, whereas another
1702
FTIR Analysis
Pollen tubes cultured either in the absence of BFA or supplemented with
5 mg mL21 BFA for 20 h were collected and washed with deionized water three
times. The samples were dried at room temperature on a barium fluoride
window (13 mm diameter 3 2 mm). Infrared spectra were obtained from the
apical region, middle region, and basal region of pollen tubes, respectively, using
a MAGNA 750 FTIR spectrometer (Nicolet) equipped with a mercury-cadmiumtelluride detector. The spectra were recorded at a resolution of 8 cm21 with 128 coadded interferograms and were normalized to obtain the relative absorbance.
ACKNOWLEDGMENTS
We thank Drs. Mathew Benson and Shi-fu Wen for valuable discussions at
the early stages of these experiments and technical assistance with FTIR
microspectroscopy. We also thank Dr. Richard Turner for his patient correction of the draft of the manuscript. This is journal paper number 0503 of the
Institute of Botany, Chinese Academy of Sciences.
Received August 10, 2005; revised August 10, 2005; accepted September 2,
2005; published November 18, 2005.
LITERATURE CITED
Baluška F, Hlavacka A, Šamaj J, Palme K, Robinson DG, Matoh T,
McCurdy DW, Menzel D, Volkmann D (2002) F-actin-dependent
endocytosis of cell wall pectins in meristematic root cells. Insights
from Brefeldin A-induced compartments. Plant Physiol 130: 422–431
Baluška F, Liners F, Hlavacka A, Schlicht M, Van Cutsem P, McCurdy D,
Menzel D (2005) Cell wall pectins and xyloglucans are internalized into
Plant Physiol. Vol. 139, 2005
Brefeldin A Stimulates Endocytosis and Inhibits Secretion
dividing root cells and accumulate within cell plates during cytokinesis.
Protoplasma 225: 141–155
Battey NH, James NC, Greenland AJ, Brownlee C (1999) Exocytosis and
endocytosis. Plant Cell 11: 643–659
Bolte S, Talbot C, Boutte Y, Catrice O, Read ND, Satiat-Jeunemaitre B
(2004) FM-dyes as experimental probes for dissecting vesicle trafficking
in living plant cells. J Microsc 214: 159–173
Camacho L, Malhó R (2003) Endo/exocytosis in the pollen tube apex is
differentially regulated by Ca21 and GTPases. J Exp Bot 54: 83–92
Chen L, Wilson RH, McCann MC (1997) Infra-red microspectroscopy of
hydrated biological systems: design and construction of a new cell with
atmospheric control for the study of plant cell walls. J Microsc 188: 62–71
Cheung AY, Chen YH, Glaven RH, de Graaf BHJ, Vidali L, Hepler PK, Wu
HM (2002) Rab2 GTPase regulates vesicle trafficking between the
endoplasmic reticulum and the Golgi bodies and is important to pollen
tube growth. Plant Cell 14: 945–962
Derksen J, Knuiman B, Hoedemaekers K, Guyon A, Bonhomme S,
Pierson ES (2002) Growth and cellular organization of Arabidopsis
pollen tubes in vitro. Sex Plant Reprod 15: 133–139
Derksen J, Rutten T, Lichtscheidl IK, Dewin AHN, Pierson ES, Rongen G
(1995) Quantitative analysis of the distribution of organelles in tobacco
pollen tubes: implications for exocytosis and endocytosis. Protoplasma
188: 267–276
De Win AHN, Knuiman B, Pierson ES, Geurts H, Kengen HMP, Derksen J
(1996) Development and cellular organization of Pinus sylvestris pollen
tubes. Sex Plant Reprod 9: 93–101
Emans N, Zimmermann S, Fischer R (2002) Uptake of a fluorescent marker
in plant cells is sensitive to brefeldin A and wortmannin. Plant Cell 14:
71–86
Fernando DD, Owens JN, Yu XS, Ekramoddoullah AKM (2001) RNA and
protein synthesis during in vitro pollen germination and tube elongation in Pinus monticola and other conifers. Sex Plant Reprod 13: 259–264
Field C, Schekman R (1980) Localized secretion of acid phosphatase
reflects the pattern of cell surface growth in Saccharomyces cerevisiae.
J Cell Biol 86: 123–128
Geldner N, Anders N, Wolters H, Keicher J, Kornberger W, Muller P,
Delbarre A, Ueda T, Nakano A, Jürgens G (2003) The Arabidopsis
GNOM ARF-GEF mediates endosomal recycling, auxin transport, and
auxin-dependent plant growth. Cell 112: 219–230
Hao HQ, Li YQ, Hu YX, Lin JX (2005) Inhibition of RNA and protein
synthesis in pollen tube development of Pinus bungeana by actinomycin
D and cycloheximide. New Phytol 165: 721–730
Hepler PK, Vidali L, Cheung AY (2001) Polarized cell growth in higher
plants. Annu Rev Cell Dev Biol 17: 159–187
Ibrahim H, Pertl H, Pittertschatscher K, Fadl-Allah E, El-Shahed A,
Bentrup FW, Obermeyer G (2002) Release of an acid phosphatase
activity during lily pollen tube growth involves components of the
secretory pathway. Protoplasma 219: 176–183
Lancelle SA, Hepler PH (1992) Ultrastructure of freeze-substituted pollen
tubes of Lilium longiflorum. Protoplasma 167: 215–230
Lanubile R, Piro G, Dalessandro G (1997) Effect of Brefeldin A on the
synthesis and transport of cell wall polysaccharides and proteins in pea
root seedlings. J Exp Bot 48: 1925–1933
Lazzaro MD, Donohue JM, Soodavar FM (2003) Distribution of cellulose
synthesis by isoxaben causes tip swelling and disorganizes cortical
microtubules in elongating conifer pollen tubes. Protoplasma 220:
201–207
Li YQ, Mareck A, Faleri C, Moscatelli A, Liu Q, Cresti M (2002) Detection
and localization of pectin methylesterase isoforms in pollen tubes of
Nicotiana tabacum L. Planta 214: 734–740
Lippincott-Schwartz J, Yuan LC, Tipper M, Amherdt LO, Klausner RD
(1991) Brefeldin A’s effects on endosomes, lysosomes, and the TGN
suggest a general mechanism for regulating organelle structure and
membrane traffic. Cell 67: 601–616
Plant Physiol. Vol. 139, 2005
Marchant R, Robards AW (1968) Membrane systems associated with the
plasmalemma of plant cells. Ann Bot 32: 457–471
Mascarenhas JP (1993) Molecular mechanisms of pollen tube growth and
differentiation. Plant Cell 5: 303–314
McCann MC, Roberts JSK, Carpita NC (1994) Changes in pectin structure
and localization during the growth of unadapted and NaCl-adapted
tobacco cells. Plant J 5: 773–785
Meckel T, Hurst AC, Thiel G, Homann U (2004) Endocytosis against high
turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP-tagged K1-channel
KAT1. Plant J 39: 182–193
Moore PJ, Sword KMM, Lynch MA, Staehelin LA (1991) Spatial
organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants. J Cell Biol 112:
589–602
Moscatelli A, Cresti M (2001) Pollen germination and pollen tube growth.
In SS Bhoiwani, WY Soh, eds, Current Trends in the Embryology of
Angiosperms. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 33–65
Murphy AS, Bandyopadhyay A, Holstein SE, Peer WA (2005) Endocytotic
cycling of PM proteins. Annu Rev Plant Biol 56: 221–251
Nebenführ A, Ritzenthaler C, Robinson DG (2002) Brefeldin A:
deciphering an enigmatic inhibitor of secretion. Plant Physiol 130:
1102–1108
Parton RM, Fischer-Parton S, Trewavas AJ, Watahiki MK (2003) Pollen
tubes exhibit regular periodic membrane trafficking events in the
absence of apical extension. J Cell Sci 116: 2707–2719
Parton RM, Fischer-Parton S, Watahiki MK, Trewavas AJ (2001) Dynamics
of the apical vesicle accumulation and the rate of growth are related in
individual pollen tubes. J Cell Sci 114: 2685–2695
Prydz K, Hansen SH, Sandvig K, van Deurs B (1992) Effects of brefeldin A
on endocytosis, transcytosis and transport to the Golgi complex in
polarized MDCK cells. J Cell Biol 119: 259–272
Read ND, Kalkman ER (2003) Does endocytosis occur in fungal hyphae?
Fungal Genet Biol 39: 199–203
Rojas M, Owen TP Jr, Kathleen NL (1999) Brefeldin A inhibits secondary
cell wall synthesis in developing tracheary elements of Zinnia elegans.
Int J Plant Sci 160: 683–690
Rutten TLM, Knuiman B (1993) Brefeldin A effects on tobacco pollen
tubes. Eur J Cell Biol 61: 247–255
Šamaj J, Baluška F, Voigt B, Schlicht M, Volkmann D, Menzel D (2004)
Endocytosis, actin cytoskeleton, and signaling. Plant Physiol 135:
1150–1161
Šamaj J, Ovecka M, Hlavacka A, Lecourieux F, Meskiene I, Lichtscheidl I,
Lenart P, Salaj J, Volkmann D, Bögre L, et al (2002) Involvement of the
mitogen-activated protein kinase SIMK in regulation of root hair tip
growth. EMBO J 21: 3296–3306
Šamaj J, Read ND, Volkmann D, Menzel D, Baluška F (2005) The
endocytic network in plants. Trends Cell Biol 15: 425–433
Smith CB, Betz WJ (1996) Simultaneous independent measurement of
endocytosis and exocytosis. Nature 380: 531–534
Tse YC, Mo B, Hillmer S, Zhao M, Lo SW, Robinson DG, Jiang LW (2004)
Identification of multivesicular bodies as prevacuolar compartments in
Nicotiana tabacum BY-2 cells. Plant Cell 16: 672–693
Voigt B, Timmers ACJ, Šamaj J, Hlavacka A, Ueda T, Preuss M, Nielsen E,
Mathur J, Emans N, Stenmark H, et al (2005) Actin-propelled motility
of endosomes is tightly linked to polar tip-growth of root hairs. Eur J
Cell Biol 84: 609–621
Wang QL, Lu LD, Wu XQ, Li YQ, Lin JX (2003) Boron influences pollen
germination and pollen tube growth in Picea meyeri. Tree Physiol 23:
345–351
Yang XD, Sun SQ, Li YQ (1999) Boron deficiency causes changes in the
distribution of major polysaccharides of pollen tube wall. Acta Bot Sin
41: 1169–1176
1703