Research
Inhibition of RNA and protein synthesis in pollen tube
development of Pinus bungeana by actinomycin D and
cycloheximide
Blackwell Publishing, Ltd.
Huaiqing Hao1, Yiqin Li2, Yuxi Hu1 and Jinxing Lin1
1
Key Laboratory of Photosynthesis and Molecular Environment Physiology, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China;
2
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China
Summary
Author for correspondence:
Jinxing Lin
Tel: +86 10 62836211
Fax: +86 10 62590833
Email: [email protected]
Received: 30 August 2004
Accepted: 19 October 2004
• The effects of actinomycin D and cycloheximide on RNA and protein synthesis
were investigated during pollen tube development of Pinus bungeana.
• RNA and protein contents, protein expression patterns, cell wall components and
ultrastructural changes of pollen tubes were studied using spectrophotometry, SDSPAGE electrophoresis, Fourier transformed infrared (FTIR) microspectroscopy and
transmission electron microscopy (TEM).
• Pollen grains germinated in the presence of actinomycin D, but tube elongation
and RNA synthesis were inhibited. By contrast, cycloheximide inhibited pollen
germination and protein synthesis, induced abnormal tube morphology, and
retarded the tube growth rate. SDS-PAGE analysis showed that protein expression
patterns changed distinctly, with some proteins being specific for each phase. FTIR
microspectroscopy established significant changes in the chemical composition of
pollen tube walls. TEM analysis revealed the inhibitors caused disintegration of
organelles involved in the secretory system.
• These results suggested RNA necessary for pollen germination and early tube
growth were present already in the pollen grains before germination, while the
initiation of germination and the maintenance of pollen tube elongation depended
on continuous protein synthesis.
Key words: actinomycin D, cell wall component, cycloheximide, Pinus bungeana,
pollen tube, protein expression pattern, ultrastructure.
Abbreviations
DMSO, dimethylsulfoxide; FTIR, fourier transformed infrared; CW, cell wall; D,
dictyosome; ER, endoplasmic reticulum; L, lipid droplet; Mi, mitochondria; SV,
secretory vesicle; V, vacuole.
New Phytologist (2005) 165: 721–730
© New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01290.x
Introduction
In higher plants, pollen tubes have the essential task
of delivering the sperm nuclei to the ovules. Pollen tube
elongation proceeds by tip growth, and thus resembles growth
processes in unrelated cell types such as fungal hyphae, root
www.newphytologist.org
hairs, and moss rhizoids (Derksen et al., 1999a; Lennon &
Lord, 2001). Pollen tubes of various species have served as
model systems for studying tip growth (Steer & Steer, 1989;
Cai et al., 1996), intracellular transport (Geitmann et al.,
1996), dynamics of secretion (Ueda et al., 1996; Ibrahim
et al., 2002), the interaction of cells and signal transduction
721
722 Research
(Malho, 1998; Kaothien et al., 2002). However, physiological
and structural differences between pollen tubes of the different
species have received little attention.
Pollen tubes of angiosperms and gymnosperms differ
in many important characteristics (Steer & Steer, 1989;
Derksen et al., 1995a, 1995b, 1999b; Taylor & Hepler, 1997).
Angiosperm pollen tubes grow quickly (Mascarenhas, 1993),
and their cytoplasm shows a zonal distribution of many
organelles (Pierson & Cresti, 1992; Derksen et al., 1999a).
Germination and growth of gymnosperm pollen proceeds
slowly, and the tubes tend to ramify and lack a tip-to-base
zonation of organelles (de Win et al., 1996; Mogami et al.,
1999). In gymnosperms, pollen grains land in the pollination
droplet and then germinate. During the tube growth, the
reproductive cells remain within the pollen grain. After
the tube reaches the egg, there exists a resting period during
which the reproductive cell forms two sperm nuclei. The gap
between pollination and fertilization in gymnosperms is
much longer than that in angiosperm (Singh, 1978; Pettitt,
1985). At present, a significant body of knowledge exists that
has been compiled about various biochemical processes
involved in pollen tube development, especially the synthesis
of polysaccharides (Schlupmann et al., 1993; Li et al., 2002),
RNA (Mascarenhas, 1993) and proteins (Li et al., 1983;
Alena et al., 2001). However, most of this knowledge is based
on studies on angiosperm species, and it remains questionable
whether current ideas are also valid for gymnosperms since
little information from the pollen tube development of
coniferous species is available.
Actinomycin D and cycloheximide, which inhibit RNA
and protein synthesis, respectively, have been useful tools
in the study of pollen tube development (Mascarenhas,
1975, 1993; Raghavan, 1981; Fernando et al., 2001; Song et al.,
2002; Yang et al., 2003). Roberts et al. (1984) showed that
pollen germination and initial tube growth in vitro were not
significantly affected by cycloheximide. Speranza et al. (2001)
reported that kiwifruit (Actinidia deliciosa var. deliciosa)
pollen germination was completely blocked in the presence of
cycloheximide, and the tube growth was strongly dependent
upon de novo protein synthesis. Fernando et al. (2001) found
that the RNA and protein synthesis during pollen germination and tube elongation in nine coniferous species were
inhibited by actinomycin and cycloheximide, respectively.
However, structural and chemical inhibitor-mediated modifications of pollen tubes, which may be important for integrating results from the biochemical and growth-physiological
levels, have rarely been addressed (de Win et al., 1996; Derksen
et al., 1999a). The purpose of this study was to evaluate the
effects of actinomycin D and cycloheximide on pollen germination and tube growth, and on changes of protein composition
during these processes in pollen of the gymnosperm Pinus
bungeana. In addition, the chemical components of the tube
wall and the ultrastructure of the pollen tube were analyzed to
provide an insight into the structural basis of the effects observed.
New Phytologist (2005) 165: 721–730
Materials and Methods
Plant material
Cones with mature pollen were collected from trees of Pinus
bungeana Zucc. in the Botanical Garden of the Institute of
Botany, Chinese Academy of Sciences, before the beginning
of the pollination season in early May, 2002, and were dried
overnight at room temperature. The dry pollen was stored at
−80°C until use.
In vitro pollen germination
For germination, pollen grains were kept at room temperature
for 30 min, and then suspended in the germination medium
containing 15% sucrose, 0.01% H3BO3, and 0.01% CaCl2
at pH 6.8. Various concentrations of actinomycin D and
cycloheximide (Sigma, St. Louis, MO, USA) dissolved in 1%
dimethylsulfoxide (DMSO) were added to the germination
medium at the beginning of the culture period. Tests with
control media containing 1% DMSO showed no effects on
pollen germination and pollen tube growth. Pollen cultures
were incubated on a shaker (100 rpm) at 27°C in the dark.
Determination of pollen germination percentage and
pollen tube growth
The germination percentage was determined by checking at
least 200 grains per experiment under a light microscope;
pollen grain were considered germinated when the tube
length was greater than the diameter of the grain. To measure
the mean tube length, images of pollen tubes cultured in
different media were taken at 12-h intervals, and the number
of pollen tubes examined was at least 100 for each treatment.
All experiments were performed at room temperature and in
triplicate.
Extraction of total RNA from pollen tubes
To study the effect of actinomycin D on RNA synthesis,
50 mg pollen was cultured in the culture medium and the
total RNA was extracted from pollen tubes using the TRIZOL
reagent (Invitrogen Corp., Carlsbad, CA, USA), according
to the manufacturer’s instructions. The RNA concentration
and purity were analyzed with a spectrophotometer (DU 640
Spectrophotometer, Beckman Instruments, Inc., Fullerton,
CA, USA) by calculating the ratio of optical density at
wavelengths of 260 and 280 nm.
Extraction of proteins and SDS-PAGE gel
electrophoresis
Protein extraction was slightly modified from the method
described by Fernando et al. (2001). For the determination of
www.newphytologist.org © New Phytologist (2005)
Research
Table 1 Effects of actinomycin D on the pollen germination and mean tube length of Pinus bungeana
Pollen tube length (µm)
Actinomycin D
(µg ml−1)
Germination
percentage (%)
2d
3d
4d
5d
0
10−3
10−2
10−1
1
5
93.7 ± 3.2
91.1 ± 2.2
92.0 ± 2.4
91.3 ± 1.2
89.7 ± 3.3
83.4 ± 2.3
60.06 ± 2.14
53.89 ± 3.25
45.56 ± 6.11
42.50 ± 3.91
38.08 ± 4.12
36.59 ± 4.26
99.83 ± 3.25
70.45 ± 3.50
69.90 ± 4.33
60.25 ± 3.41
65.00 ± 4.66
45.64 ± 2.57
114.17 ± 2.55
93.40 ± 3.84
83.32 ± 3.26
80.95 ± 2.92
74.8 ± 5.24
48.7 ± 2.46
132.21 ± 5.46
103.6 ± 6.14
94.56 ± 4.08
89.22 ± 2.23
84.82 ± 3.47
54.12 ± 3.25
Only pollen tubes that were longer than the diameter of pollen grain were measured. Values are means ± SD.
protein, 20 mg pollen in 20 ml medium was used. Frozen
pollen tubes were ground to a fine powder with mortar and
pestle, and resuspended in 65 m Tris-HCl buffer pH 6.8,
1% SDS, 5% glycerol, and 2.5% mercaptoethanol. The
suspension was boiled for 5 min and centrifuged at 14 000 g
for 30 min. The protein concentrations were measured according
to Schaffner & Weismann (1973) with bovine serum albumin
as a standard. The extracted proteins were subjected to
electrophoresis on 12% polyacrylamide gels (1 mm thick) with
SDS in a discontinuous system according to Laemmli (1970).
Low molecular mass markers (14.4–97 kDa, Amersham
BioSciences, Uppsala, Sweden) were used as standards, and
the gel was silver-stained (Blum et al., 1987). The determinations of RNA and protein are referred to the initial weight of
pollen grains.
Fourier transformed infrared (FTIR) analysis
Pollen tubes cultured in different media for 3 d were collected
and washed three times with deionised water. The samples
were dried at room temperature on a barium fluoride window
(13 mm diameter × 2 mm). Infrared (IR) spectra of the
pollen tube tip were obtained using a MAGNA 750 FTIR
spectrometer (Nicolet Corp., Tokyo, Japan) equipped with
a mercury-cadmium-telluride (MCT) detector. The spectra
were recorded at a resolution of 8 cm−1 with 128 coadded
interferograms and were normalized to obtain the relative
absorbance.
Electron microscopy
For electron microscopy, pollen tubes were fixed for 2 h in
2.5% glutaraldehyde in 100 m phosphate buffer (pH 7.2)
with 2% (w/v) sucrose. Samples were washed with 100 m
phosphate buffer, postfixed in 2% osmium tetroxide for 2 h,
dehydrated in an ethyl-alcohol series, and finally embedded in
Spur resin. Sections were cut using an LKB-V ultromicrotome,
and examined with a JEM-1230 electron microscope ( JEOL
Ltd., Tokyo, Japan).
© New Phytologist (2005) www.newphytologist.org
Results
Effects of actinomycin D and cycloheximide on pollen
germination and pollen tube growth
Under control conditions, pollen started to germinate after
2 d culture in the germination medium and reached its
maximum germination percentage of 93.7% after 108 h.
Actinomycin D at concentrations of up to 5 µg ml−1 did
not delay germination and had little effect on the maximum
germination percentage (Table 1). However, pollen tube
elongation was inhibited substantially by actinomycin D in
a dose-dependent manner. In the presence of 10−3 µg ml−1
actinomycin D, the average growth rate of pollen tubes was
16.6 µm d−1. When actinomycin D concentration increased
to 5 µg ml−1, the average growth rate dropped to 5.8 µm d−1
by comparison with 24.1 µm d−1 in the control during 3-d
growth after germination (Table 1). Actinomycin D led to a
decrease in the content of extant RNA in a dose-dependent
manner. Compared with the control pollen tubes, RNA
content was reduced by 59.3% and 68.9%, respectively, in
the pollen tubes treated with 1 and 5 µg ml−1 actinomycin D
for 5 d (Fig. 1).
By contrast to actinomycin D, cycloheximide reduced the
percentage of pollen germination drastically, and prevented
germination completely when applied at 10−1 µg ml−1 or more
(Table 2). Pollen tube elongation was affected by cycloheximide to a similar extent as pollen germination. In the
presence of 10−2 µg ml−1 cycloheximide, pollen germination
percentage decreased significantly (P < 0.05), pollen tube
elongation was inhibited severely, and pollen tubes stopped
growing after 4 d (Table 2). The mean length of pollen tubes
treated with 10−1 µg ml−1 cycloheximide could not reach the
pollen grain diameter and so the pollen grains were considered
ungerminated. Cycloheximide at 1 and 5 µg ml−1 completely
inhibited the germination of pollen grains, irrespective of the
length of treatment (Table 2). Figure 2 shows that cycloheximide at 10−1 µg ml−1 caused an inhibition of 55.98% in the
protein content. With increasing cycloheximide concentration,
New Phytologist (2005) 165: 721–730
723
724 Research
Table 2 Effects of cycloheximide on pollen germination and tube growth of Pinus bungeana
Pollen tube length (µm)
Cycloheximide
(µg ml−1)
Germination
percentage (%)
2d
3d
4d
5d
0
10−3
10−2
10−1
1
5
93.7 ± 3.2
65.6 ± 4.5
45.2 ± 3.0
0
0
0
60.06 ± 2.14
38.76 ± 4.32
34.07 ± 2.15
0
0
0
99.83 ± 3.25
69.36 ± 3.15
59.6 ± 4.46
0
0
0
114.17 ± 2.55
75.48 ± 3.25
72.52 ± 3.15
0
0
0
132.21 ± 5.46
78.88 ± 2.46
72.99 ± 3.84
0
0
0
Only pollen tubes that were longer than the diameter of pollen grain were measured. Values are means ± SD.
Fig. 1 The inhibitory effect of actinomycin D on RNA synthesis. RNA
was extracted and the concentration was determined after 5 d
cultured in various concentrations of actinomycin D.
Fig. 2 The inhibitory effect of cycloheximide on protein synthesis.
Total protein was extracted and determined after 5 d cultured in
different concentrations of cycloheximide.
the inhibitory effect on protein content increased. It is of
interest to note that in the presence of the inhibitors only a
lower percentage of branching of pollen tubes was observed,
similar to the case detected in the normal pollen tubes.
in pollen grains treated with 1 µg ml−1 cycloheximide. Several
proteins increased after germination; some of them (34 kDa,
73 kDa, and 78 kDa) accumulated gradually as the pollen
tubes developed, while another protein (37 kDa) decreased in
staining intensity as the pollen tube elongated. At least two
proteins (18 kDa and 27 kDa) increased in intensity during
the early stages of pollen tube elongation, but became less
intense 5 d after germination.
Protein profiles of pollen grains and pollen tubes
Proteins extracted from pollen tubes of different stages of
elongation were separated by SDS-PAGE (Fig. 3). Although
the protein patterns at different stages of development shared
similarities, some characteristic differences were evident.
Most conspicuously, a 39 kDa protein was present in the
pollen grains, but it gradually disappeared as the pollen tubes
elongated. The intensity of three proteins (54 kDa, 59 kDa
and 83 kDa) declined after germination and then increased
again during the later stages of pollen tube growth. A 36 kDa
protein was found at the later stages of pollen tube growth
but not in other stages. In addition, a protein of 45 kDa was
present in pollen tubes of all stages, but seemed not to be
expressed at all in the ungerminated mature pollen grains and
New Phytologist (2005) 165: 721–730
Fourier transformed infrared (FTIR) spectra from pollen
tubes
FTIR spectra obtained from the tip region of pollen tubes
growing in control medium showed the absorption maxima
of amide I and amide II at 1657 cm−1 and 1547 cm−1
(Sutherland, 1952; McCann et al., 1994), while a peak at
1743 cm−1 corresponded to saturated esters (Morikawa et al.,
1978; McCann et al., 1994) (Fig. 4 CK). Treatment with 10−2
µg ml−1 of either actinomycin D or cycloheximide induced
displacements of the peaks or changes of absorbance (Fig. 4
www.newphytologist.org © New Phytologist (2005)
Research
Fig. 3 SDS gel electrophoresis of proteins.
The proteins were extracted from
ungerminated Pinus bungeana pollen grains
(CK), pollen tubes cultured in the normal
medium for 2, 3, 4, 5 d (2 d–5 d) and pollen
grains treated with 1 µg ml−1 cycloheximide
(CH). Arrows indicate 45 kDa and 36 kDa
protein, respectively. M: Protein marker.
Fig. 4 FTIR spectrum obtained from the tip regions of normal Pinus
bungeana pollen tubes (CK), pollen tubes treated with 10−2 µg ml−1
actinomycin D (ActD) and 10−2 µg ml−1 cycloheximide (CHX).
ActD, CHX). The difference spectra (Fig. 5a) showed that
actinomycin D led to decreases of the amide I and II peaks
(1643 cm−1 and 1535 cm−1, respectively), indicating that the
protein content of tube walls was reduced. Furthermore, the
saturated ester peak (1743 cm−1) decreased and the carboxylic
acids peak (1597 cm−1) increased (McCann et al., 1994),
suggesting that actinomycin D induced a reduction in the
content of saturated ester and an increase in synthesis of
© New Phytologist (2005) www.newphytologist.org
Fig. 5 FTIR spectral differences generated by digital subtraction of
the spectra CK from ActD (a) and that of the spectra CK from CHX
(b). Both of the spectra show that there were less protein and
saturated ester, but more carboxylic acid in the inhibitor-treated Pinus
bungeana pollen tubes.
carboxylic acids. Cycloheximide evoked qualitatively identical
effects. Figure 5(b) reveals the difference spectrum generated
by digital subtraction of spectrum (CK) from spectrum (CHX),
showing the decrease in the amide-stretching bands at
1659 cm−1 and 1535 cm−1, saturated ester band at 1743 cm−1,
and the increase in the carboxylic acid stretches at 1597 cm−1.
New Phytologist (2005) 165: 721–730
725
726 Research
Fig. 6 Ultrastructure of pollen tubes of Pinus
bungeana cultured in different media. Bars,
60 µm (a,b), 3.4 µm (c,d), 0.5 µm (e,f).
(a) Pollen tubes cultured in normal medium
for 3 d, showing normal shape and long tube.
(b) Pollen tubes treated with 10−2 µg ml−1
cycloheximide for 3 d, showing swelling of
the pollen tube tip. (c) Tip region of normal
pollen tube tip, showing secretory vesicles
(arrow head) and organelles at the tip.
(d) Tip region of pollen tube treated with
10−2 µg ml−1 cycloheximide; organelles were
damaged, number of vacuole increased and
vesicles almost disappeared. (e) Normal
pollen tube tip, showing typical morphology
of dictyosomes and endoplamic reticulum.
(f ) Dictyosomes in pollen tube after
10−2 µg ml−1 cycloheximide treatment for 3 d;
the diameter of dictyosomes increased, and
the cisternae of dictyosomes began to disrupt.
Effects of actinomycin D and cycloheximide on the
ultrastructure of the pollen tubes
Pollen tubes growing in control medium were long with
uniform diameter and a clear zone at the tip (Fig. 6a). The tip
cytoplasm contained large amounts of endoplasmic reticulum
(ER), dictyosomes, and mitochondria, as well as various
kinds of vesicles (Fig. 6c). Most of the dictyosomes had six to
eight cisternae and they did not show any specific zonation.
Numerous secretory vesicles and coated vesicles were present
around the dictyosomes (Fig. 6e).
Much variation was observed when pollen grains were
incubated in the medium containing cycloheximide. Cycloheximide caused swelling of the pollen tube tips (Fig. 6b).
Treatment with 10−2 µg ml−1 cycloheximide drastically decreased
the number of secretory vesicles and increased the number of
New Phytologist (2005) 165: 721–730
vacuoles in the pollen tube tips (Fig. 6d). The ER membrane
became irregular and even fragmented (Fig. 7a). The mitochondria appeared swollen or disrupted, and showed a reduced
number of cristae (Fig. 7c). The diameter of dictyosome
seemed to increase (Fig. 6f ), and eventually the dictyosome
disintegrated (Fig. 7a,b). Actinomycin D at 10−2 µg ml−1 caused
ER swelling and the detachment of the ribosomes from the
rER (Fig. 7d). The membranes of the mitochondria appeared
damaged and the number of cristae reduced. At 5 µg ml−1,
actinomycin D caused an accumulation of secretory vesicles
around dictyosomes, while the number of secretory vesicles in
the very tip decreased (Fig. 7e). Finally, the dictyosomes and
the mitochondria disorganized completely (Fig. 7f ). These
results suggested that cycloheximide and actinomycin D
damaged the structure of the main organelles and interfered
with the secretory system at the pollen tube tip.
www.newphytologist.org © New Phytologist (2005)
Research
Fig. 7 Ultrastructure of pollen tube tip of
Pinus bungeana in the presence of
cycloheximide or actinomcin D. Bars, 0.5 µm.
(a–c). Pollen tubes treated with 10−2 µg ml−1
cycloheximide for 4 d. (a) The cisternae of
dictyosomes ruptured into vesicular structure.
(b) Disaggregation of dictyosomes. (c) The
mitochondria membrane swelled and
disrupted (arrow). (d) Pollen tubes treated
with 10−2 µg ml−1 actinomcin D for 3 d.
Endoplasmic reticulum swelled, ribosomes fell
off the rER and dictyosome was damaged.
(e–f) Pollen tubes treated with 5 µg ml−1
actinomcin D for 3 d and 4 d, respectively.
(e) Vesicles accumulated around the
dictyosomes. (f) Dictyosomes disrupted.
Discussion
When the pollen grain arrives on the surface of a compatible
stigma, it hydrates and then germinates to extrude a pollen
tube. It has been suggested that pollen germination and/or
tube growth require active protein synthesis (Capkova et al.,
1988; Franklin-Tong, 1999). However, the dependence of
pollen tube generation on RNA and protein synthesis varies
with the plant species (Mascarenhas, 1975, 1993). In the
present study, we found that pollen germination of Pinus
bungeana was not affected by actinomycin D, while pollen
tube elongation and RNA synthesis were inhibited. From
these results, which are consistent with previous reports
(Frankis, 1990; Fernando et al., 2001), it appears that RNA
necessary for pollen germination and early tube growth were
present already in the pollen grains before germination, while
continuous RNA synthesis seems required for sustained tube
© New Phytologist (2005) www.newphytologist.org
elongation at normal rates. Taken together, the consequences
for pollen development of inhibiting RNA synthesis appear to
be similar in conifers and angiosperms (Mascarenhas, 1993).
In most angiosperms, cycloheximide has no effect on pollen
germination (Mascarenhas, 1993). By contrast, pollen germination and early tube growth were severely or completely
inhibited in P. bungeana by cycloheximide (Table 2). Cycloheximide also induced abnormal morphologies in the pollen tubes
and inhibition of protein synthesis. Thus, the initiation of
germination and the maintenance of pollen tube elongation
depend on continuous protein synthesis in P. bungeana. Notably,
pollen grains of P. bungeana took as long as 2 d to germinate,
and the pollen tube growth rate was only c. 24 µm d−1, much
slower than in angiosperms. We infer that the processes of pollen
tube development are possibly limited by the availability of
specific proteins in P. bungeana, giving rise to delayed germinations and low growth rates. These facts, however, fit well into
New Phytologist (2005) 165: 721–730
727
728 Research
gymnosperm reproductive biology, where the period between
pollination and fertilization can be up to 2 yr (Singh, 1978).
Protein synthesis during pollen germination and tube
growth has been studied extensively in angiosperm species
(Mascarenhas, 1993). Some proteins are synthesized preferentially during pollen tube development, such as the 65-kDa
and 69-kDa proteins, which play important roles in the
formation of the pollen tube wall in Nicotiana tabacum
(Capkova et al., 1994). The protein expression pattern in
pollen of P. bungeana (Fig. 3) differed from those reported
from angiosperms. Three protein bands of 54, 59 and 83 kDa
decreased in intensity 2 d after germination, implying that
they may function in pollen germination and early tube
growth. In addition, we found a protein (45 kDa) that seemed
to be specific for pollen tube of P. bungeana. We are currently
analyzing the variation in proteins expressed during pollen
development in P. bungeana by two-dimensional gel electrophoresis, to elucidate protein function at different stages.
In addition to polysaccharides, structural proteins are inserted
into the growing pollen tube cell wall (Rae et al., 1985;
Mascarenhas, 1993). FTIR microspectroscopy provides a fast
and nondestructive assay for cell wall components of different
types of cells (McCann et al., 1992, 1994; Wang et al., 2003;
Wu et al., 2003). Using FTIR analysis, we confirmed that
proteins were present in the pollen tube walls of P. bungeana,
as has been previously reported from other species (Li et al.,
1983; Capkova et al., 1987). It was further found that the
protein content of the tube walls decreased dramatically
when treated with actinomycin D or cycloheximide. This
result indirectly proved the inhibitory effect of actinomycin D
or cycloheximide on pollen tube growth. In addition, actinomycin D or cycloheximide also caused changes in the content
of carboxylic acids and saturated ester. These results demonstrate that actinomycin D and cycloheximide induce modifications in the composition of the pollen tube walls, probably
by affecting the deposition of proteins and polysaccharides.
Tip growth of pollen tubes requires the integrity of the
secretory system (Moscatelli & Cresti, 2001). During tube
growth, secretory vesicles derived from the dictyosomes transport polysaccharides to the sites of wall construction (Li et al.,
1997; Taylor & Hepler, 1997). In the presence of actinomycin
D, the secretory vesicles vanished from the very tip, while
their number increased around the dictyosomes (Fig. 7e),
suggesting that the toxin affected the transport of secretory
vesicles to the tube tip rather than influenced the fusion
of vesicles with the plasma membrane (Mascarenhas, 1993).
Similarly, cycloheximide caused a structural disintegration of
the organelles of the secretory system (Figs 6 and 7). As the
concentrations of two inhibitors increased, the dictyosomes
became disorganized, indicating that a complete inhibition
of the secretory system resulted in the change in proteins
detected in the pollen tubes and the decrease in tube wall proteins. In summary, our results suggest that actinomycin D and
cycloheximide affected pollen tube growth and by inhibiting
New Phytologist (2005) 165: 721–730
the synthesis of RNA and protein, respectively. These inhibitions led to modified compositions of newly synthesized cell
wall materials; the assembly of these materials to form new cell
wall was further impeded by the structural breakdown of the
secretory system.
Acknowledgements
This work was supported by the National Science Fund of
China for Distinguished Young Scholars (30225005). The
authors thank Dr Winfried Peters (University of Giessen,
Germany) for his expert advice and patient correction for the
first draft of manuscript and Dr Shi-Fu Wen (Peking University)
for his technical assistance with FTIR microspectroscopy.
References
Alena F, Petr S, Jaroslav T, Vera C. 2001. Glycoproteins 66 and 69 kDa of
pollen tube wall: properties and distribution in angiosperms. Journal of
Plant Physiology 158: 1367–1374.
Blum H, Beier H, Gross HJ. 1987. Improved silver staining of plant
proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:
93–99.
Cai G, Moscatelli A, Del Casino C, Cresti M. 1996. Cytoplasmic motors
and pollen tube growth. Sex Plant Reproduction 9: 59–64.
Capkova V, Hrabetova E, Tupy J. 1987. Protein changes in tobacco pollen
culture: a newly synthesized protein related to pollen tube growth. Journal
of Plant Physiology 130: 307–314.
Capkova V, Hrabetova E, Tupy J. 1988. Protein synthesis in pollen tubes:
preferential formation of new species independent of transcription. Sex
Plant Reproduction 1: 150–155.
Capkova V, Zbrozek J, Tupy J. 1994. Protein synthesis in tobacco pollen
tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins.
Sex Plant Reproduction 7: 57–66.
Derksen J, Li YQ, Knuiman B, Geurts H. 1999b. The wall of Pinus sylvestris
L. pollen tubes. Protoplasma 208: 26–36.
Derksen J, Rutten T, Lichtscheidl IK, de Win AHN, Pierson ES,
Rongen G. 1995b. Quantitative analysis of the distribution of organelles
in tobacco pollen tubes: implication for exocytosis and endocytosis.
Protoplasma 188: 267–276.
Derksen J, Rutten T, van Amstel A, de Win AHN, Doris F, Steer MW.
1995a. Regulation of pollen tube growth. Acta Botany Neerl 44:
93–119.
Derksen J, van Amstel ANM, Rutten ALM, Knuiman B, Li YQ,
Pierson ES. 1999a. Pollen tubes: cellular organization and control of
growth. In: Clement C, Pacini E, Audran J-C, eds. Anther and Pollen.
Berlin Heidelberg New York Tokyo: Springer, 161–174.
Fernando DD, Owens JNYuXS, Ekramoddoullah AKM. 2001. RNA and
protein synthesis during in vitro pollen germination and tube elongation
in Pinus monticola and other conifers. Sex Plant Reproduction 13: 259–264.
Frankis RC Jr. 1990. RNA and protein synthesis in germinating pine pollen.
Journal of Experimental Botany 41: 1469–1473.
Franklin-Tong VE. 1999. Signaling and the modulation of pollen tube
growth. Plant Cell 11: 727–738.
Geitmann A, Wojciechowicz K, Cresti M 1996. Inhibition of intracellular
pectin transport in pollen tubes by monensin, brefeldin A and cytochalasin
D. Botanica Acta 109: 373–381.
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.
www.newphytologist.org © New Phytologist (2005)
Research
Kaothien P, Ok SH, Kelley D, Cotter R, McCormick S. 2002. Signaling via
receptor kinases during pollen tube growth: The downstream components.
Molecular Bio Cell 13: 257a –258a.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227: 680 – 685.
Lennon KA, Lord EM. 2001. In vivo pollen tube cell of Arabidopsis thaliana
I. Tube cell cytoplasm and wall. Protoplasma 214: 45 –56.
Li YQ, Croes AF, Linskens HF. 1983. Cell-wall proteins in pollen and roots
of Lilium longiflorum: extraction and partial characterization. Planta 158:
422–427.
Li YQ, Mareck A, Faleri C, £Faleri C, Moscatelli A, Liu Q, Cresti M. 2002.
Detection and localiazation of pectin methylesterase isoforms in pollen
tubes of Nicotiana tabacum L. Planta 214: 734 –740.
Li YQ, Moscatelli A, Cai G, Cresti M. 1997. Functional interactions among
cytoskeleton, membranes and cell wall in pollen tubes of flowering plants.
International Review of Cytology 176: 133 –199.
Malho R. 1998. Role of 1,4,5-inositol triphosphate-induced Ca2+ release in
pollen tube orientation. Sexl Plant Reproduction 11: 231– 235.
Mascarenhas JP. 1975. The biochemistry of angiosperm pollen
development. Botany Review 41: 259 –314.
Mascarenhas JP. 1993. Molecular mechanisms of pollen tube growth and
differentiation. Plant Cell 5: 303 –314.
McCann MC, Hammouri M, Wilson R, Belton P, Roberts K. 1992.
Fourier transform infrared microspectroscopy is a new way to look at plant
cell walls. Plant Physiology 100: 1940 –1947.
McCann MC, Shi J, Roberts K, Carpita NC. 1994. Changes in pectin
structure and localization during the growth of unadapted and NaCladapted tobacco cells. Journal of Plant 5: 773 –785.
Mogami N, Nakamura S, Nakamura N. 1999. Immunolocalization of the
cell wall components in Pinus densiflora pollen. Protoplasma 206: 1–10.
Morikawa H, Hayashi R, Senda M. 1978. Infrared analysis of pea stem cell
walls and oriented structure of matrix polysaccharides in them. Plant Cell
Physiology 19: 1151–1159.
Moscatelli A, Cresti M. 2001. Pollen germination and pollen tube growth.
In: Bhoiwani SS, Soh WY, eds. Current Trends in the Embryology of
Angiosperms. Dordrecht, The Netherlands: Kluwer Academic Publishers,
33–65.
Pettitt JM. 1985. Pollen tube development and characteristics of the protein
emission in conifers. Annals of Botany 56: 379 – 397.
Pierson ES, Cresti M. 1992. Cytoskeleton and cytoplasmic organization of
pollen and pollen tubes. International Review of Cytology 140: 73–125.
Rae AL, Harris PJ, Basic A, Clarke AE. 1985. Composition of the cell walls
of Nicotiana alata Link et Otto pollen tubes. Planta 166: 128 –133.
Raghavan V. 1981. Distribution of poly (A)-containing RNA during
normal pollen development and during induced pollen embryogenesis in
Hyoscyamus niger. Journal of Cell Biology 89: 593 – 606.
© New Phytologist (2005) www.newphytologist.org
Roberts IN, Harrod G, Dickinson HG. 1984. Pollen–stigma interactions in
Brassica oleracea. II. The fate of stigma surface proteins following
pollination and their role in the self-incompatibility response. Journal of
Cell Science 66: 255–264.
Schaffner WC, Weismann C. 1973. A rapid, sensitive and specific method
for the determination of protein in dilute solution. Analytical Biochemistry
56: 502–514.
Schlupmann H, Basic A, Read SM. 1993. A novel callose synthase from
pollen tubes of Nicotiana. Planta 191: 470–481.
Singh H. 1978. Embryology of gymnosperms. In: Zimmermann W,
Carlquist S, Ozenda P, Wuff HD, eds. Handbuch der Pflanzenanatomie,
10. Berlin, Germany: Gebruder Borntraeger.
Song JJ, Nada K, Tachibana S. 2002. Suppression of S-adenosylmethionine
decarboxylase activity is a major cause for high-temperature inhibition of
pollen germination and tube growth in tomato (Lycopersicon esculentum
Mill.). Plant Cell Physiology 43: 619–627.
Speranza A, Scoccianti V, Crinelli R, Calzoni GL, Magnani M. 2001.
Inhibition of proteasome activity strongly affects kiwifruit pollen
germination. Involvement of the ubiquitin/proteasome pathway as a
major regulator. Plant Physiology 126: 1150–1161.
Steer MW, Steer JM. 1989. Pollen tube tip growth. New Phytology 111:
323–325.
Sutherland GBBM. 1952. Infrared analysis of the structure of amino
acids, polypeptides and proteins. Biochemistry et Biophysica Acta 6:
291–318.
Taylor LP, Hepler PK. 1997. Pollen germination and tube growth. Annual
Review of Plant Physiological Plant Molecular Biology 48: 461–491.
Ueda T, Tsukaya H, Anai T, Hirata A, Hasegawa K, Uchimiya H. 1996.
Brefeldin A induces the accumulation of unusual membrane structures in
elongating pollen tubes of Nicotiana tabacum L. Journal of Plant Physiology
149: 683–389.
Wang QL, Lu LD, Wu XQ, Li YQ, Lin JX. 2003. Boron influences pollen
germination and pollen tube growth in Picea meyeri. Tree Physiology 23:
345–351.
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 Reproduction 9: 93–101.
Wu XQ, Lin JX, Zhu JM, Hu YX, Hartmann K, Schreiber L. 2003.
Casparian strips in needles of Pinus bungeana: isolation and chemical
characterization. Physiological Plantarum 117: 421–424.
Yang HQ, Jie YL, Liu LX, Tang WY, Yu MG. 2003. Regulation of abscisic
acid and its biosynthesis inhibitors on pomegranate pollen germination
and tube growth. In: Lee JM, Zhang DL, eds. XXVI International
Horticultural Congress: Asian Plants with Unique Horticultural Potential:
Genetic Resources, Cultural Practices and Utilization. International Society
for Horticultural Science, Leuven, Belgium, 209–213.
New Phytologist (2005) 165: 721–730
729
730 Research
About New Phytologist
• New Phytologist is owned by a non-profit-making charitable trust dedicated to the promotion of plant science, facilitating projects
from symposia to open access for our Tansley reviews. Complete information is available at www.newphytologist.org.
• Regular papers, Letters, Research reviews, Rapid reports and Methods papers are encouraged. We are committed to rapid
processing, from online submission through to publication ‘as-ready’ via OnlineEarly – the 2003 average submission to decision time
was just 35 days. Online-only colour is free, and essential print colour costs will be met if necessary. We also provide 25 offprints
as well as a PDF for each article.
• For online summaries and ToC alerts, go to the website and click on ‘Journal online’. You can take out a personal subscription to
the journal for a fraction of the institutional price. Rates start at £109 in Europe/$202 in the USA & Canada for the online edition
(click on ‘Subscribe’ at the website).
• If you have any questions, do get in touch with Central Office ([email protected]; tel +44 1524 592918) or, for a local
contact in North America, the USA Office ([email protected]; tel 865 576 5261).
New Phytologist (2005) 165: 721–730
www.newphytologist.org © New Phytologist (2005)