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The Plant Cell, Vol. 17, 548–558, February 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
Amyloplasts and Vacuolar Membrane Dynamics in the Living
Graviperceptive Cell of the Arabidopsis Inflorescence Stem
W
Chieko Saito, Miyo T. Morita, Takehide Kato, and Masao Tasaka1
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis)
of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells.
Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these
movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravityoriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting
findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of
amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has
been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation
by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament–disrupted,
and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on
the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts
and (2) the en mass movement of amyloplasts.
INTRODUCTION
Higher plants cannot escape from the place where they germinate and settle, even if the environmental conditions drastically
change. Plants have developed many mechanisms during the
course of evolution to survive in such circumstances by changing
growth direction or architecture of their body shape. Typical of
such growth responses, gravitropism is one of most prominent
phenomena and has been the focus of studies on many plant
species over a long period of time (Knight, 1806; Sack, 1991;
Fukaki et al., 1996b; Chen et al., 1999). In higher plants, shoots
and roots generally show negative and positive gravitropism,
respectively. This response ensures an appropriate positioning
of tissues for efficient photosynthesis, gas exchange, and the
supply of water and minerals.
The gravitropic response in plants can be divided into four
sequential steps: gravity perception, signal formation in the
gravity perceptive cell, intracellular and intercellular signal transduction, and an asymmetric cell elongation between the upper
and lower sides of the responding organs (Tasaka et al., 1999).
Some classical hypotheses have been supported by recent
molecular genetical studies. For example, the starch-statolith
1 To
whom correspondence should be addressed. E-mail m-tasaka@
bs.naist.jp; fax 81-743-72-5489.
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.plantcell.org) is: Masao Tasaka
([email protected]).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.104.026138.
hypothesis is involved in the first step and proposes that the
sedimenting amyloplasts, which contain dense starch granules,
act in sensing the direction of gravity (Sack, 1991; Weise and
Kiss, 1999). However, the mechanism functioning in gravity
perception has not yet been characterized at the molecular level.
A recent molecular approach using Arabidopsis thaliana revealed the graviperceptive cell in an inflorescence stem. We have
isolated several shoot gravitropism (sgr) mutants, which are
defective in the gravitropic response of inflorescence stems. In
the Arabidopsis shoot, the epidermis, cortex, endodermis, and
stele containing vascular tissues are arranged in a radially
symmetrical manner. In the course of the study of sgr1 and
sgr7, which are allelic to scarecrow and short-root, respectively,
the endodermis has been revealed as essential for the gravitropism of the inflorescence stem (Fukaki et al., 1998; Tasaka et al.,
1999). In addition, it is known that amyloplasts differentiate and
sediment to the direction of gravity in the endodermis. These
findings suggest that the endodermis in the inflorescence stem
is essential for the first step of shoot gravitropism, namely the
perception of gravity. The starch-statolith hypothesis was also
supported by these results.
Interestingly, some other sgr mutants, including sgr2, sgr3,
and zig/sgr4, have been shown to still have an endodermis,
although the sedimentation of amyloplasts in those cells is
abnormal (Morita et al., 2002; Yano et al., 2003). These
mutants show a gravitropism defect in the shoot exclusively
(inflorescence stem and hypocotyls) and are normal in root
gravitropism or phototropism. Surprisingly, responsible genes
of these mutants are closely related to the biogenesis or the
integrity of the vacuoles (Kato et al., 2002; Yano et al., 2003).
Moreover, it is revealed that amyloplasts in the wild-type
Subcellular Dynamics of the Graviperceptive Cell
endodermis are almost completely enwrapped by a thin cytoplasmic layer and vacuolar membrane, whereas amyloplasts
are left in the peripheral region of the cytoplasm of the
endodermal cells in the mutants (Morita et al., 2002). When
expressed under the control of the SCARECROW promoter
(ProSCR), which drives expression in the endodermis, the
corresponding wild-type genes restored gravitropism and
amyloplast sedimentation in the mutant (Morita et al., 2002;
Yano et al., 2003). These findings suggest that the early step of
the gravitropism occurring in the endodermis is impaired in the
mutants. They still retain, however, the ability for the latter
steps, signal formation in the gravity perceptive cell, intracellular and intercellular signal transduction, and an asymmetric
cell elongation between the upper and lower sides of the
responding organs for tissue bending. These findings also
suggest that vacuoles in the endodermis may be involved in
the early step of gravitropism to some extent.
In the gravity-perceptive cell of the root (columella cell), several
detailed analyses of the subcellular dynamics have been performed after gravistimulation by reorientation (Legue et al., 1997;
Fasano et al., 2001; Yoder et al., 2001), and it has been shown
that amyloplasts actually sediment to the orientation of gravity
(Yoder et al., 2001). In the shoot, however, only a few attempts
have been made at visualizing the gravity-perceptive cell at
a subcellular level (Johannes et al., 2001), and to our knowledge,
no studies have tried to analyze the movement dynamics of the
vacuolar membrane in the endodermal cell. Here, we describe
amyloplast movement in the living endodermal cell of Arabidopsis inflorescence stems using a vertical stage microscope.
Interestingly, they do not sediment statically but show saltatory
movement. We also visualize the movement dynamics of the
vacuolar membrane in the endodermal cells. Confocal laser
scanning microscopy revealed where amyloplasts pass through.
Treatment with a microfilament-disrupting drug severely affected the movement of amyloplasts before gravistimulation by
reorientation, although the gravity-perceptive ability of the inflorescence stem segment still remained. In zig-1/sgr4-1 mutants, the movement of amyloplasts was severely affected, and
their gravitropism was almost completely defective as shown
previously. We will discuss the possible role of the gravityperceptive system in the dynamics of amyloplasts and the close
interaction with the vacuolar membrane. This study opens the
possibility of a new concept for gravity perception in the inflorescence stem.
RESULTS
Methods for the Observation of Organelle Dynamics and
Subcellular Events in a Living Endodermal Cell
A transgenic plant containing green fluorescent protein (GFP)marked amyloplasts in its endodermis (pspt3-6) was generated
by transforming into wild type an expression construct carrying
a GFP sequence fused to a plastid-targeted transit peptide,
under the control of ProSCR (Figures 1A and 1B). Amyloplasts
were still detectable using autofluorescence even in the nontransgenic plant (wild type); however, a clearer image of amylo-
549
Figure 1. Visualization of Organelle Dynamics in the Endodermis.
(A) Construction for the endodermal-specific expression of plastid-GFP
(top, for pspt3-6 line) and tonoplast-GFP (bottom, for psgt4-1 line)
markers.
(B) Endodermal-specific expression in the inflorescence stem observed
in the longitudinal section. Left, blue light–excited fluorescence image;
right, Bright-field image. En, endodermal cell files. Bar ¼ 20 mm.
(C) Epifluorescence image of endodermal cells fixed, embedded, sectioned (1 mm thick), and stained with DiOC6. Bar ¼ 5 mm.
(D) Transmission electron micrograph of an ultrathin section of endodermal cells. Bar ¼ 2 mm.
(E) Epifluorescence image of living endodermal cells of the pspt3-6 lines
observed under blue light excitation with a wide-range emission filter set.
Amyloplasts were visualized by the expression of plastid-targeted GFP.
Chloroplasts in cortex cells or stele were also visualized by their
autofluorescence. Co, cortex cell file; En, endodermal cell file; St, stele.
Bar ¼ 5 mm.
(F) Epifluorescence image of living endodermal cells of the psgt4-1 line
under blue light excitation with a band-path filter set. Vacuolar membrane in the endodermal cells was visualized by GFP-gTIP expression
under ProSCR. Bar ¼ 5 mm.
plasts was obtained in the transgenic line (Figure 1E). The images
are comparable to those made from a fixed sample section
(Figures 1C and 1D). ProSCR and GFP were further applied for the
visualization of the vacuolar membrane. GFP and g-TIP (tonoplast intrinsic protein g), a marker of the lytic vacuole (Maurel
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et al., 1993), were fused and expressed in wild-type plants under
the control of ProSCR. In the transgenic plant (psgt4-1), the
vacuolar membrane is visualized within the endodermis specifically with the band-path filter set (Figure 1F; NIBA; Olympus,
Tokyo, Japan). As far as we examined, no significant leak of
the marker molecules to other layers was detected in the
inflorescence stem, and both pspt3-6 and psgt4-1 lines showed
a normal gravitropic response (data not shown).
Dynamics of Amyloplasts in Endodermal Cells of
the Inflorescence Stem before and after
Gravistimulation by Reorientation
First, we wanted to examine the dynamics of amyloplasts using
time-lapse imaging. Sequential images were acquired at 10-s
intervals for 5 to 20 min. Amyloplasts did not sediment statically but moved within the endodermal cell dynamically, as
Figure 2. Dynamics of the Amyloplasts in a Wild-Type Endodermal Cell before and after Gravistimulation by Reorientation.
(A) Left, time-lapse images of amyloplasts at 1-min intervals before gravistimulation by reorientation. Right, trace of amyloplasts. Each color represents
the trace of an individual amyloplast. Bars ¼ 5 mm.
(B) Top, time-lapse images of amyloplasts at 1-min intervals after gravistimulation by reorientation. Bottom, trace of amyloplasts. Each color represents
the trace of an individual amyloplast. Bars ¼ 5 mm.
(C) Definition of the axis for the quantitative analysis of amyloplast dynamics.
(D) Histograms showing the frequency of the speed of amyloplasts tracks, extracted to Dx and Dy components in the wild type. Braces represent
increased gravity-oriented movements of the amyloplasts. Asterisks show the populations of the positive Dy components with large absolute values
(>3 mm/10 s). Total number subjected to the analysis before gravistimulation by reorientation (tracks n ¼ 825, cells n ¼ 3) and after gravistimulation
by reorientation (tracks n ¼ 443, n ¼ 3).
Subcellular Dynamics of the Graviperceptive Cell
shown by a time-lapse movie (see Supplemental Movie 2
online, x100 speed) and the tracing of movement (Figure 2A,
right). They moved not only along the peripheral area, but also
through the central region of the endodermal cell very frequently. These dynamic movements were observed continuously even after 20 min (up to 40 min maximum) after
preparation of samples.
After gravistimulation by reorientation, the amyloplasts
changed their localization pattern drastically and some of them
reached the lower end of the cell (Figure 2B, 3 min). They did not
stay at the basal end but continued to move around (see
Supplemental Movie 3 online). All of the movements of amyloplasts were not directly toward the gravity orientation during this
short time period. Some amyloplasts appeared to actually move
along a newly applied gravity orientation and others not, as
shown by the trace of each amyloplast, after gravistimulation by
reorientation (Figure 2B, bottom).
Gravity-Oriented Movement of Amyloplasts Occurs during
the First Three Minutes after Reorientation
Attempts were made to analyze the movements of amyloplasts
quantitatively and statistically. Tracks of some amyloplasts
were traced continuously at 10-s intervals using time-lapse
images (Figures 2A, right, and 2B, bottom). To extract gravityoriented movement, each movement at every 10-s interval was
dissected into two components, one horizontally (Dx) and the
other vertically (Dy), as shown in Figure 2C. Taking time
dependency of the movement into account, the time period
was divided into 0 to 1 min, 1 to 2 min, 2 to 3 min, 3 to 4 min,
and 4 to 5 min after application of gravistimuli. Figure 2D
shows histograms of the frequency of Dx and Dy components.
Before gravistimulation by reorientation, histograms of both Dx
and Dy showed a Gaussian distribution–like shape and were
symmetric to the axis of Dx ¼ 0 and Dy ¼ 0, respectively. After
gravistimulation by reorientation, in the time period of 0 to
1 min, 1 to 2 min, and 2 to 3 min, clear asymmetric distributions
551
of the Dy components were detected (Figure 2D, braces). The
mean values of Dy during this period are statistically significantly increased compared with the mean value of Dy before
gravistimulation by reorientation (Welch’s t test, one-tailed, P <
0.05). During the time period of 1 to 2 min and 2 to 3 min, small
but certain populations of the positive Dy components with
large absolute values were also detected (asterisks, >3 mm/
10 s). This population was 0.9, 2.3, 2.6, 0, and 1.37% in 0 to
1 min, 1 to 2 min, 2 to 3 min, 3 to 4 min, and 4 to 5 min,
respectively (before reorientation, 1.5%). The average value of
Dy components from the same data set of the tracks is shown
in Table 1. During 0 to 3 min, the averaged speed to the gravity
is 1.05 mm/min to 1.42 mm/min. These quantitative results
indicate that the gravity-oriented movements of amyloplasts
(both the mass translocation to gravity and the positive Dy
component with large absolute value) actually occurred after
gravistimulation by reorientation. The mean values of Dx in the
time periods of 2 to 3 min and 3 to 4 min are also statistically
significantly increased compared with the mean value of Dx
before gravistimulation by reorientation (Welch’s t test, onetailed, P < 0.05).
Amyloplasts Are in the Transvacuolar Strand, Both before
and after Gravistimulation by Reorientation
It has been revealed using electron microscopy that the
amyloplasts in the endodermal cells are surrounded almost
completely by a thin layer of cytoplasm and the vacuolar
membrane (Morita et al., 2002). The narrow areas of cytoplasm
enclosed by the vacuolar membrane and continuous to the
peripheral cytoplasm have been referred to as ‘‘transvacuolar
strands.’’ It was expected that amyloplasts move through these
transvacuolar strands before or after gravistimulation by reorientation. Vacuolar membranes in the endodermal cell are
visualized well by expressing the GFP-g-TIP fusion molecule
driven under ProSCR. As shown in Figure 1F, the vacuolar
membrane in the endodermal cell emitted fluorescence in the
psgt4-1 line, but the strand-like structure was not observed so
Table 1. Averaged Speed of Amyloplasts (mm/min) Extracted to Dx and Dy Components
þLatB
Wild Type
Before
After applying gravistimuli
0
1
2
3
4
to
to
to
to
to
1
2
3
4
5
min
min
min
min
min
zig-1/sgr4-1
Dx
Dy
n
Dx
Dy
n
Dx
Dy
n
0.1 6 5.9
0.2 6 6.7
825
0.12 6 2.1
0 6 2.2
404
0.1 6 3.4
0.4 6 4.3
869
0.03
0.1
1.05*
1.21*
1.2
1.05*
1.42*
1.11*
0.4
0.1
117
87
76
90
73
0.72*
0.40
0.44
0.41*
0.31
1.72*
0.94*
0.76*
0.86*
0.11
125
115
97
93
90
0.3
0.1
0.14
0.03
0.25
0.46
0.19
0.4
0.49
0.25
102
102
99
96
91
6
6
6
6
6
5.5
4.6
3.6
5.6
4.8
6
6
6
6
6
5.4
6.1
5.4
4.5
5.3
6
6
6
6
6
2.6
3.4
2.9
3.1
1.9
6
6
6
6
6
3.2
5.0
3.7
3.1
2.4
6
6
6
6
6
2.5
3.1
3.0
3.8
2.0
6
6
6
6
6
2.2
2.0
2.8
3.2
1.6
The averaged speed was calculated from the same data set that was used in the histograms shown in Figures 2D, 4E, and 5D (cell number, n ¼ 3, total
tracks used in the calculation was represented in column n). The axis was defined as shown in Figure 2C. For the direction of movements along Dx and
Dy, positive values mean right or down, and negative values indicate left or up, respectively. 6, standard deviations. Asterisks show the time period of
the mean values of Dx or Dy statistically significantly increased compared with the mean value of Dx or Dy before gravistimulation by reorientation
(Welch’s t test, one-tailed, P < 0.05). LatB, latrunculin B.
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The Plant Cell
clearly using epifluorescence microscopy. However, using
a high-speed confocal scanner unit (CSU-10; Yokogawa,
Tokyo, Japan), a complex network of the transvacuolar strands
and some dilated areas, where amyloplasts are expected to
localize, were observed both before (Figure 3A) and after
(Figure 3B) gravistimulation by reorientation. From time-lapse
confocal laser scanning images, vacuolar membranes were
also shown to be quite dynamic even before gravistimulation
by reorientation (see Supplemental Movies 4 and 5 online). A
silhouette of the vacuolar membrane often showed the same
size, shape, and number of amyloplasts in the endodermal
cell. These images strongly suggest that amyloplasts move
within the transvacuolar strands both before and after gravistimulation by reorientation.
To confirm that amyloplasts translocate through transvacuolar strands, we visualized both amyloplasts autofluorescing
and the vacuolar membrane (by GFP-gTIP expression) in the
same cell of the psgt4-1 line with a very short time difference by
changing the filter set. Figure 3C shows time-lapse images of
the vacuolar membrane and amyloplasts observed alternately at
10-s intervals. Most of the GFP-gTIP signals were detected very
close to the autofluorescence of amyloplasts. These images
clearly indicate that amyloplasts move within the cell wrapped by
the vacuolar membrane.
The Saltatory Movement of Amyloplasts Is
Microfilament Dependent
Figure 3. Vacuolar Membrane Dynamics in a Wild-Type Endodermal
Cell.
(A) and (B) Time-lapse images of the vacuolar membrane visualized in
the psgt4-1 line and captured by confocal laser scanning microscope
before ([A], 10-s intervals) and after ([B], 5-s intervals) applying gravistimuli. Bars ¼ 5 mm.
(C) Epifluorescence micrographs of amyloplasts and the vacuolar
membrane in an endodermal cell captured very shortly after gravistimulation by reorientation. Left, amyloplasts observed by their autofluorescence with a wide-range filter set (WIB; Olympus); right, vacuolar
membrane visualized by GFP-gTIP expression and observed under
a band-path filter set (NIBA; Olympus). Bar ¼ 5 mm.
To address the molecular basis of the movement of amyloplasts
and to examine their association with gravitropism, the effect of
the disruption of actin cytoskeleton was analyzed.
After a 10-min treatment with the antimicrofilament drugs
cytochalasin B and latrunculin B, the saltatory movements of
amyloplasts were severely attenuated (data not shown). As
reported previously (Yamamoto and Kiss, 2002), latrunculin
B–treated inflorescence stems are able to respond to gravity.
These findings were confirmed in this study after 14 h of
treatment with latrunculin B (Figures 4A and 4B). It was also
shown that the network of actin filaments was completely
disrupted, as detected by Pro35S GFP talin (Figure 4B, right).
The motility of amyloplasts was considerably reduced, and
almost all of them were located at the basal region of the
endodermal cell (Figure 4C; see Supplemental Movie 6 online).
After gravistimulation by reorientation, however, some populations of amyloplasts actually sedimented, whereas others
remained static (Figure 4D; see Supplemental Movie 7 online).
Statistical analysis showed that the distribution of Dy components became steeper, indicating that the movements of
amyloplasts were inhibited before gravistimulation by reorientation (Figures 2B and 4E). Positive Dy components showing
large absolute values were detected (asterisks, >3 mm/10 s),
and this population was 0.8, 2.5, 1.0, 0, and 0% in 0 to 1 min,
1 to 2 min, 2 to 3 min, 3 to 4 min, and 4 to 5 min, respectively
(before reorientation, 0%). The average values of Dy components were comparable to those of the wild type (Table 1; 1.72
mm/min, 0.94 mm/min, 0.76 mm/min, and 0.86 mm/min in 0 to
1 min, 1 to 2 min, 2 to 3 min, and 3 to 4 min, respectively). The
mean values of Dy during this period are statistically significantly increased compared with the mean value of Dy before
gravistimulation by reorientation (Welch’s t test, one-tailed, P <
0.05). The mean values of Dx in the time periods of 0 to 1 min
and 3 to 4 min are also statistically significantly increased
Subcellular Dynamics of the Graviperceptive Cell
553
Figure 4. The Disruption of the Actin Cytoskeleton and Saltatory Movement of Amyloplasts and Retention of Gravitropism after Treatment with
Latrunculin B.
(A) and (B) Gravitropism assay of stem segment and F-actin filament in an endodermal cell visualized by GFP-tallin without (A) or with (B) latrunculin B
treatment. Left top, 0 min after gravistimulation by reorientation; left bottom, 150 min after gravistimulation by reorientation; right, actin microfilament
visualized by Pro35S-driven GFP-talin expression. Bars ¼ 5 mm.
(C) Left, time-lapse images of amyloplasts in the endodermal cell of latrunculin B–treated stem segment at 1-min intervals before gravistimulation by
reorientation. Right, trace of amyloplasts. Each color represents the trace of an individual amyloplast. Bars ¼ 5 mm.
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The Plant Cell
compared with the mean value of Dx before gravistimulation by
reorientation (Welch’s t test, one-tailed, P < 0.05). Amyloplasts
were still covered almost entirely by the vacuolar membrane
(Figure 4F). The effect of a microtubule-disrupting drug (amiprophosmethyl) on the movements of amyloplasts was further
examined. The saltatory movements of amyloplasts were not
affected by the treatment with amiprophosmethyl, and the
stem showed normal gravitropic response (data not shown).
components were also calculated (Table 1), and from this the
value in zig-1/sgr4-1 was found to be noticeably reduced
(0.46 mm/min, 0.19 mm/min, and 0.4 mm/min in 0 to 1 min, 1 to
2 min, and 2 to 3 min, respectively). The occurrence of the Dy
positive components with large absolute values was not detected
in zig-1/sgr4-1.
DISCUSSION
Dynamic Movement of Amyloplasts Is Severely Restricted
in sgr2, sgr3, and zig/sgr4 Mutants Both before and
after Gravistimuli
Among sgr mutants, sgr2, sgr3, and zig/sgr4 were selected to
observe the dynamics of amyloplasts in the endodermis of the
inflorescence stem because these genes are likely to function in
the biogenesis or the integrity of the vacuolar membrane (Kato
et al., 2002; Yano et al., 2003). Electron microscopy studies have
revealed that amyloplasts in the endodermis of the inflorescence
stem of these sgr mutants did not sediment normally, nor were
they wrapped by a vacuolar membrane (Morita et al., 2002; Yano
et al., 2003). The movement of amyloplasts in these mutants is
severely restricted in the endodermal cells of the inflorescence
stem. Figures 5A and 5B show the behavior of amyloplasts in the
endodermal cell of zig-1/sgr4-1 as a representative result from
these mutants (see also Supplemental Movies 8 and 9 online).
The amyloplasts in the endodermal cell of the mutants never
moved through the central region of the cell, although they
occasionally moved a very short distance in the peripheral area of
the cell. To examine the defects of the vacuolar membrane in the
endodermal cell, the GFP-g-TIP fusion construct was expressed
in zig-1/sgr4-1 and the stems were subjected to microscopic
observation. As a result, transvacuolar strand-like structures
were not observed in the endodermal cell. In addition, many
abnormal vesicle-like structures accumulated in these cells
(Figure 5C), which occasionally showed very dynamic movement
(see Supplemental Movie 10 online). Statistical analysis of the
movement of amyloplasts in zig-1/sgr4-1 showed that before
gravistimulation by reorientation, the distribution of Dy and Dx
components became steeper, indicating that the movements of
amyloplasts were inhibited (Figures 2D and 5D). After gravistimulation by reorientation, a small shift of Dy components to the
positive occurred, but asymmetry of the distribution to the axis of
x ¼ 0 was not significant. Neither mean values of Dx nor Dy in all
time periods are statistically significantly increased compared
with the mean values before gravistimulation by reorientation
(Welch’s t test, one-tailed, P < 0.05). Average values of the Dy
Subcellular Dynamics of the Endodermis in the
Arabidopsis Inflorescence Stem
In this study, the subcellular dynamics of the gravity-perceptive
cell in the inflorescence stem of Arabidopsis were observed
using fine time and image resolution, with several intriguing
findings being noted. First, saltatory movement of amyloplasts
was shown both before and after gravistimulation by reorientation in the living graviperceptive cell. Quantitative analysis of
a large data set obtained from fine time-lapse imaging revealed
that the amyloplasts actually moved toward the direction of
gravity 0 to 3 min after gravistimulation by reorientation in the
wild-type cell (Figure 2, Table 1). Saltatory movements of
amyloplasts in the living graviperceptive cell have also been
reported in the dandelion (Taraxacum officinale) flower stalks
(Clifford and Barclay, 1980), mung bean (Vigna radiate) hypocotyls (Heathcote, 1981), and the maize (Zea mays) coleoptile (Sack
and Leopold, 1985). Rapid movements of amyloplasts toward
the gravity were detected (>3 mm/10 s, ;20 mm/min) as previously reported in the living graviperceptive cell; however,
amyloplasts undergoing such movements constituted only
a fraction of the overall population. Averaging the data sets of
the amyloplast movement shows that gravity-oriented movement is attenuated by the upward movement component,
although the movement can still be detected at ;1 mm/min. It
is plausible that both kinds of gravity-oriented movement are
actually occurring 0 to 3 min after gravistimulation by reorientation (Table 1, Figure 2D). These findings are consistent with the
presentation time previously reported (Fukaki et al., 1996a).
The vacuolar membrane was visualized and revealed that the
translocation path of the amyloplasts occurs in transvacuolar
strands, thin cytoplasmic areas that penetrate through the central
vacuole. Sack and Leopold (1985) suggested that amyloplasts
may move through transvacuolar strands. This study showed
using confocal laser scanning microscopy that amyloplasts in the
peripheral area of the cytoplasm were almost entirely covered
with a vacuolar membrane. The vacuolar membrane itself seems
to be dynamic, but it is continuously bent and deformed by
Figure 4. (continued).
(D) Top, time-lapse images of amyloplasts in the endodermal cell of latrunculin B–treated stem segment at 10-s intervals after gravistimulation by
reorientation. Bottom, traces of amyloplasts. Each color represents the trace of an individual amyloplast. Bars ¼ 5 mm.
(E) Histograms showing the frequency of the speed of amyloplasts tracks, extracted to Dx and Dy components in a wild-type stem segment treated with
latrunculin B for 14 h. Total number subjected to the analysis before gravistimulation by reorientation (tracks n ¼ 404, cells n ¼ 3) and after
gravistimulation by reorientation (tracks ¼ 520, cells n ¼ 4).
(F) A confocal laser scanning microscopic image of the vacuolar membrane in the endodermal cell of a latrunculin B–treated stem segment. Bar ¼ 5 mm.
Subcellular Dynamics of the Graviperceptive Cell
555
Figure 5. Impaired Dynamics of Both Amyloplasts and the Vacuolar Membrane in the zig-1/sgr4-1 Mutant Endodermal Cell.
(A) Left, time-lapse images of amyloplasts at 1-min intervals before gravistimulation by reorientation. Right, trace of representative amyloplasts. Each
color represents the trace of an individual amyloplast. Bars ¼ 5 mm.
(B) Left, time-lapse images of amyloplasts at 1-min intervals after gravistimulation by reorientation. Right, trace of representative amyloplasts. Each
color represents the trace of an individual amyloplasts. Bars ¼ 5 mm.
(C) Time-lapse images of vacuolar membrane at 10-s intervals before gravistimulation by reorientation. Bar ¼ 5 mm.
(D) Histograms showing the frequency of the speed of amyloplast tracks, extracted to Dx and Dy components in a zig-1/sgr4-1 stem segment. Total
number subjected to the analysis before gravistimulation by reorientation (tracks n ¼ 869, cells n ¼ 3) and after gravistimulation by reorientation
(tracks ¼ 490, n ¼ 3).
the amyloplasts as well. As shown in Figures 1F and 3C, the
amyloplasts are always closely attached and enwrapped by the
vacuolar membrane. This interaction between vacuolar membrane and amyloplasts may represent one of the most striking
differences between the subcellular organization of the root and
shoot graviperceptive cells. In root collumera cells, amyloplasts
are not enwrapped by the vacuolar membrane and are thought to
move within the cytoplasm with little or no interaction with the
vacuole. These findings are consistent with the observation that
sgr2, sgr3, and zig/sgr4 mutants still have normal gravitropism in
the root, in spite of their shoot gravitropism defects.
Amyloplast Movement for Gravity Perception Is Likely to
Be Actin Cytoskeleton Independent
The actin cytoskeleton network has been characterized well in
many plant species, with the thick actin cable often observed
within the transvacuolar strands (Traas et al., 1987; McCurdy
et al., 1988; Tominaga et al., 2000). This study also clearly
showed the actin cytoskeleton in the transvacuolar strands and
its involvement with the movement of amyloplasts. Findings from
the actin disruption experiment suggest that the saltatory movement of amyloplasts through transvacuolar strands is dependent
upon the actin cytoskeleton.
556
The Plant Cell
The actin cytoskeleton has been proposed as a major player in
plant gravitropism (Yoder et al., 2001; Blancaflor, 2002; Hou et al.,
2003). Recent studies, however, showed that an intact actin
cytoskeleton is not required for gravity perception in inflorescence stems and roots (Yamamoto and Kiss, 2002; Hou et al.,
2003) and likely acts to downregulate gravitropism by continuously resetting the gravitropic-signaling system (Hou et al.,
2004). The former finding was confirmed in this study, and in
addition, the movement of amyloplasts was shown to correlate
well with gravity-perceptive ability. In the actin disrupted cell,
saltatory movements decline considerably both before and after
gravistimulation by reorientation. After gravistimulation by reorientation, translocation of amyloplasts toward the gravity occurs,
as shown by both time-lapse imaging and quantitative analysis
(Figure 4, Table 1; see Supplemental Movies 6 and 7 online). A
few of the amyloplasts can show dramatic and significant
movement, even though most of the plastids do not move after
reorientation. Although we cannot conclude from these observations that the movement of the amyloplasts was enhanced
after actin disruption, as suggested by Yamamoto and Kiss
(2002), we can say that the mass translocation of amyloplasts in
the stem is comparable to the wild type. These results suggest
that the movement of amyloplasts for graviperception is not actin
dependent and that the saltatory movement of amyloplasts is
actin dependent and not essential for graviperception.
What Is the Importance of Movement of Amyloplasts
for Graviperception?
In this study, amyloplast dynamics were compared using three
types of inflorescence stem: drug-untreated wild type, latrunculin B–treated wild type, and zig-1/sgr4-1 mutant. The former two
are graviperceptive, and the latter is a nongraviperceptive stem.
The results are summarized in Figure 6. These findings suggest
two kinds of movement showing good correlation between
gravity-perceptive ability. The first kind is the mass translocation
of amyloplasts toward gravity. The average speed of the gravityoriented movement of amyloplasts in the latrunculin B–treated
sample is comparable to that of the wild-type sample, whereas
that of zig-1/sgr4-1 is greatly reduced (Table 1). These findings
may indicate that mass translocation of amyloplasts toward the
lower end of the cell induces gravity signal transduction and
therefore is important for graviperception. The second kind of
movement is displayed by a population of amyloplasts that
sediment at high velocity (>3 mm/10 s; Figures 2 and 4, asterisks).
That population is very small, a few percent, but they may retain
a large amount of kinetic energy, taking into account that the
kinetic energy is proportional to the square of the velocity, and
this large amount of kinetic energy may be enough to overcome
the threshold to elicit the signal for a downstream response. This
kind of movement may correspond with the sedimentation of
amyloplast ‘‘faster and to a greater magnitude,’’ as discussed by
MacCleery and Kiss (1999) or ‘‘the leading amyloplasts’’ (Sack
et al., 1984). The mean values of Dx direction in some time
periods (in wild type, 2 to 3 and 3 to 4 min, and in the latrunculin
B–treated samples 0 to 1 min and 3 to 4 min are reorientation) are
also statistically significantly increased compared with the mean
values before reorientation. The possible explanation for this
Figure 6. Summary of This Work.
observation is that amyloplasts tend to move toward the previous upper side of the cell because of the larger area than the
previous bottom side of the cell.
What Is the Role of the Complex Vacuolar Configuration—For
Making Paths for Amyloplasts or Other Functions?
The complex feature of the vacuole in the endodermal cell is
presumed to have a function in gravitropism of the inflorescence
stem. Because this close interaction of vacuolar membrane with
amyloplasts was also reported in cress hypocotyls (Volkmann
et al., 1993), this feature may be common in the shoot (in
hypocotyls and inflorescence stems). Two possibilities of the
function of vacuolar membrane in graviperception are proposed.
First, it is proposed that the transvacuolar strands may facilitate
smooth translocation of amyloplasts. In the wild type, amyloplasts are translocated within transvacuolar strands; however, in
the zig/sgr4 mutant, amyloplasts cannot pass through the transvacuolar strands. In the latrunculin B–treated sample, the movement through transvacuolar strands is not clearly captured, but
amyloplasts are still almost entirely enwrapped by a vacuolar
membrane and therefore should pass thorough transvacuolar
strands. The second proposal is that the vacuolar membrane
itself may receive the signal directly from the gravity-oriented
movement of amyloplasts, for example, through the change of
tension in the membrane as a result of gliding amyloplasts.
However, using this hypothesis, it is difficult to explain two
factors. How is the noisy signal eliminated from continuously
moving amyloplasts, and how do we know whether the signal
coming from the vacuolar membrane within the cell is upward
and/or downward. Because, at present, the vacuolar membrane
itself seems not to have any particular polarity. Further careful
study is required to address these questions.
Subcellular Dynamics of the Graviperceptive Cell
METHODS
Sources and Constructions of GFP Marker Lines
The GFP (S65T) with plastid transit peptide (Pt-GFP) of RBCS1A (Chiu
et al., 1996; Niwa et al., 1999) was provided by Yasuo Niwa (Niwa et al.,
1999; University of Shizuoka, Japan). A BamHI recognition sequence was
inserted at 38 of RBCS1A. The BamHI-EcoRI fragment containing PtGFP and Nos terminator was then inserted into the BamHI and EcoRI sites
of pBI121del_pSCR (Morita et al., 2002) downstream of ProSCR. The GFP
(S65T) fused with g-tonoplast intrinsic protein (GFP-g-TIP) was provided
by Hiroshi Abe (RIKEN, Wako, Saitama, Japan). The XbaI-EcoRI fragment
containing GFP-g-TIP and Nos terminator was inserted into the XbaI and
EcoRI sites of pBI121del_pSCR (Morita et al., 2002) downstream of
ProSCR. The Pro35S:GFP-talin fusion construct was provided by Nam-Hai
Chua (Kost et al., 1998; The Rockefeller University, New York, NY).
The constructs were transformed into Agrobacterium tumefaciens
strain MP90 by electroporation using the Gene pulser (Bio-Rad, Hercules,
CA) and then introduced into wild-type Columbia plants using the floral
dipping method (Clough and Bent, 1998). The T1 plants were selected
with resistance to kanamycin. The construction of the transgene in these
plants was tested using PCR. Segregation of the transgene in the T2
generation was confirmed. The representative lines were selected from
T3 homozygous lines by checking the GFP fluorescence in the endodermis of the inflorescence stems. The transgenes were transferred to sgr
mutants by transfection (GFP-g-TIP in zig-1/sgr4-1) or crossing (Pt-GFP
in sgr2, sgr3, and zig-1/sgr4-1).
Plant Materials and Growth Conditions
In this study, pspt3-6 and psgt4-1 were used as a ProSCR:Pt-GFP and
a ProSCR:GFP-g-TIP expressing line, respectively. Seeds were imbibed,
sawed on MS medium (13 MS salt mixture, 1% [w/v] sucrose, 0.01% [w/
v] myoinositol, and 0.5% [w/v] gellan gum, pH 5.8) vernalized in the dark at
48C for 1 to 2 d, and then grown at 238C under continuous light. Seedlings
were then transplanted to soil to grow inflorescence stems 7 to 14 d after
germination. Only the first inflorescence stem was used in the experiments.
Imaging System for Vertical Stage Microscope
An epifluorescence microscope (BX-50; Olympus) equipped with rotary
stage was mounted on its back so that the rotary stage was oriented
vertically (Legue et al., 1997). Each endodermal cell in the inflorescence
stem was observed while it was vertical, and its gravitropic response was
elicited by rotating the stage through 908 until the cell layer was horizontal.
The endodermal cell was observed before and after gravistimulation. GFP
fluorescence or autofluorescence of amyloplasts was acquired with
a cooled CCD camera (COOL SNAP cf; Photometrics, Nippon Roper,
Tokyo, Japan) and processed using IPLab software (Scanalytics, Fairtax,
VA). For confocal laser scanning microscopic observation, a confocal
scanner unit (CSU-10; Yokogawa) was mounted to the vertical stage
microscope. Images were captured by a CCD camera (ORCA-ER;
Hamamatsu Photonics, Hamamatsu, Japan) and processed with Metamorph (Universal Imaging, Downingtown, PA) and IPLab.
Sample Preparation for the Vertical Stage Microscope
Pieces of cover glass (;170 mm thick; Matsunami, Tokyo, Japan) were
cut into ;5-mm-wide strips. These strips were stuck to a glass slide as
a spacer using a waterproof adhesive agent (ARON ALPHA; TOAGOSEI,
Tokyo, Japan). Double-sided adhesive tape was prepared using two
557
sheets of an adhesive tape stuck together by ARON ALPHA. The doublesided adhesive tape was cut very thinly and stuck onto the spacer.
Another piece of the double-sided adhesive tape was stuck inside the
spacer, and an inflorescence stem segment (<1 cm in length, the region of
1 to 2 cm from the top of the inflorescence stem) was excised and stuck to
the tape. The segment was cut longitudinally using a razor blade held with
forceps. Immediately after cutting, a small amount of GM liquid medium
(13 MS salt mixture and 1% sucrose) with 0.1% agar was added. The
space inside the well was covered with a cover slip and sealed with
double-sided adhesive tape (see Supplemental Figure 1 online).
Electron Microscopy
The samples of inflorescence stem were embedded in Spurr’s resin
(Nisshin EM, Tokyo, Japan) following the methods of Morita et al. (2002).
Ultrathin sections were cut (70 to 90 nm thick), stained with uranil acetate
and lead nitrate, and examined with a transmission electron microscope
(H-7100; Hitachi, Tokyo, Japan).
Statistical Analysis of the Amyloplast Movement
The movement of amyloplasts was subjected to quantitative analysis
using time-lapse images. Amyloplasts that could be traced for a substantial period (approximately more than five frames) were selected.
Each coordinate of the amyloplast in the image frame was recorded,
and Dx and Dy was calculated at 10-s intervals. The axis of the coordinate was defined as shown in Figure 2. Pseudomovement caused by
the directional and negligible stage drifting was corrected using the
coordinate of an immobile marker (for example, the corner of the cell) in
the first and last frames. Traces of the tracks were drawn using
a representative example of each condition (before or after gravistimulation by reorientation in the wild type, zig-1/sgr4, and latrunculin
B–treated stem segment). The frequency of Dx and Dy at each 10 s
interval was presented in a histogram.
Drug Treatment
Stock solutions of latrunculin B (Calbiochem, La Jolla, CA; 20 mM) and
amiprophosmethyl (Nihon Bayer Agrochem, Tokyo, Japan; 200 mM)
were prepared in dimethyl sulfoxide. A stock solution of Cytochalasin B
(Wako, Tokyo, Japan; 20 mM) was prepared in distilled water. First, drugs
were added directly into the GM liquid medium. Latrunculin B and
cytochalasin B effectively inhibited the saltatory movement of amyloplasts in the endodermal cell in 2 and 0.2 mM, respectively. The drug
incorporation experiment was performed according to the methods of
Fukaki et al. (1996a), with some modification. Distal 4-cm stem segments
of primary inflorescence stems with total lengths between 4 and 12 cm
were used for gravitropic response assays. The distal 4-cm parts were cut
from the primary inflorescence stems with a razor blade, and their basal
ends were inserted into a 1.5-mL microfuge tube positioned vertically on
a tube rack. Several stem segments were soaked together in one tube so
that they stood in a vertical position. They were preincubated for 14 to
16 h in white light at 238C in GM liquid medium without sucrose. After
incubation, basal 5-mm pieces of these stem segments were put into GM
gellan gum blocks that were fixed onto one face of plastic plates (Fukaki
et al., 1996a). To maintain a high humidity in the plates, a wet paper towel
was placed inside the plate. After the stem segments were incubated in
a vertical orientation for 1 h under white light at 238C in the plates,
gravistimulation was initiated by rotating the plates through 908 in
darkness at 238C. Images were taken at the start point and after
150 min using the COOL SNAP cf camera.
558
The Plant Cell
ACKNOWLEDGMENTS
We thank Yasuo Niwa of the University of Shizuoka for the plantoptimized GFP clone, Hiroshi Abe of RIKEN for the GFP-gTIP fusion
construct, Nam-Hai Chua for the GFP-talin fusion construct, and the
members of the Tasaka laboratory for technical help and valuable
comments on the manuscript. Especially, we would like to thank Seiko
Ishihara for her excellent experimental assistance and Hidehiro Fukaki
for his precious advice. We also thank Dean DellaPenna for enabling
C.S. to accomplish part of this work in his laboratory. This work was
supported by Grants-in-Aid from the Ministry of Education, Science,
Sports, and Culture of Japan, by a Special Coordination Fund for
Promotion of Science and Technology from the Science and Technology Agency of Japan, by a Research Fellowship of the Japan Society for
the Promotion of Science for Young Scientists (C.S.; 03419), and by
a grant from the Bioarchitect Project of RIKEN (M.T.M.).
Received July 28, 2004; accepted November 19, 2004.
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Amyloplasts and Vacuolar Membrane Dynamics in the Living Graviperceptive Cell of the
Arabidopsis Inflorescence Stem
Chieko Saito, Miyo T. Morita, Takehide Kato and Masao Tasaka
Plant Cell 2005;17;548-558
DOI 10.1105/tpc.104.026138
This information is current as of June 18, 2017
Supplemental Data
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This article cites 33 articles, 13 of which can be accessed free at:
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