Plant Physiol. (1999) 120: 183-192
Plastid Sedimentation Kinetics in Roots of Wild-Type and
Starch-Deficient Mutants of Arabidopsis1
Scott A. MacCleery and
John Z. Kiss*
Department of Botany, Miami University, Oxford, Ohio 45056
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ABSTRACT |
Sedimentation and movement of
plastids in columella cells of the root cap were measured in seedlings
of wild-type, a reduced starch mutant, and a starchless mutant of
Arabidopsis. To assay for sedimentation, we used both linear
measurements and the change of angle from the cell center as indices in
vertical and reoriented plants with the aid of computer-assisted image
analysis. Seedlings were fixed at short periods after reorientation,
and plastid sedimentation correlated with starch content in the three
strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the
greatest sedimentation, whereas plastids of the starchless mutant
showed no significant sedimentation in the vertically grown and
reoriented seedlings. Because previous research has shown that a full
complement of starch is needed for full gravitropic sensitivity, this
study correlates increased sensitivity with plastid sedimentation.
However, although plastid sedimentation contributed to
gravisensitivity, it was not required, because the gravitropic
starchless mutant had plastids that did not sediment. This is the first
study, to our knowledge, to measure plastid sedimentation in
Arabidopsis roots after reorientation of seedlings. Taken together, the
results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.
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INTRODUCTION |
Of the many different environmental cues that any organism uses
for orientation, gravity is the most constant and pervasive. All life
has evolved in its presence. Since the emergence of plants from an
aqueous environment, gravity has been an essential factor in plant
development (Barlow, 1995
). Gravitropism can be divided into three
phases, distinct in time and, in higher plants, space: perception,
transduction, and response (Evans et al., 1986; Salisbury, 1993). It
has long been hypothesized that the central columella cells of the root
cap are the graviperceptive cells, i.e. the statocytes, in plant roots
(Volkmann and Sievers, 1979). Direct evidence supporting the role of
columella cells as statocytes includes work by Behrens et al. (1985),
who found that only the columella cells experienced rapid membrane
potential change after reorientation, and work by Blancaflor et al.
(1998), who showed that selective laser ablation of columella cells
reduced gravitropic sensitivity.
When a plant is reoriented with respect to a gravitational force, the
amyloplasts in the columella cells settle on the new "lower" cell
wall. This original observation by Haberlandt (1914) implicated
amyloplasts in graviperception and still is a good indication of a
graviperceptive organ (Volkmann and Sievers, 1979; Sack, 1991;
Salisbury, 1993). On theoretical grounds, Björkman (1988)
calculated that amyloplasts have the mass and mobility to activate a
receptor with enough energy to make it reliable (250 times thermal
noise and 15 times activation energy), whereas other organelles,
including mitochondria and the entire protoplast, do not. But even with
an amyloplast's relatively great mass, some discernible movement is
required for any reliable perception of gravity, according to these
theoretical calculations (Björkman, 1988).
An alternative hypothesis for the perception of gravity that does not
require starch or plastids is the protoplast-pressure model (Wayne et
al., 1990). Despite the large turgor pressure between the protoplasm
and the wall, Wayne and coworkers implicated the total weight of the
protoplast on the cell wall as the susceptor (with integrins as a
possible receptor; see Katembe et al., 1997). This model is supported
by the absence of sedimenting particles in both unicellular gravitactic
organisms such as Euglena gracilis (Häder
et al., 1995; Häder, 1997) and in internodal cells of Chara sp. (Staves et al., 1992; Staves, 1997). Other studies
of the effect of the density of the external medium on
gravity-dependent processes in Chara sp. (Wayne et al.,
1992) and rice (Staves et al., 1997) seem to bolster this hypothesis.
According to Staves (1997), the unidirectional force of gravity can be
perceived through any omnidirectional force of turgor. This perception
could be analogous to thermotropism in maize roots or hydrotropism in
maize and pea roots, in which small signals could be detected through a
much larger background (for discussion, see Staves et al., 1992). According to this view, amyloplasts could be used only as added weight
within the protoplast statolith.
The original theory of Haberlandt (1914) suggests that starch is a
necessary component of graviperception in plants. Plants without starch
would seem to be a good system in which to test this hypothesis.
Studies of starch-depleted wheat coleoptiles (Pickard and Thimann,
1966) found continued gravitropism, whereas studies of cress roots
(Iversen, 1969) and barley pulvini (Song et al., 1988) showed no
response to reorientation. Although it is difficult to reconcile some
of these results, it must be emphasized that the methods used to
destarch plants, especially the columella cells, are harsh and can
affect the plant in many unintended ways (Sack, 1991). The use of
starch-deficient mutants is a more direct means of measuring
differences in graviperception.
As starchless mutants became available, some studies of the root
curvature showed a markedly different graviresponse compared with that
seen in WT plants. Although gravity was perceived, the mutant was much
less perceptive than the WT in maize (mutant amylomaize; Hertel et al.,
1969), Arabidopsis (mutant TC7; Kiss et al., 1989), and tobacco (mutant
NS458; Kiss and Sack, 1989, 1990). These observations raised two
important questions: Were the plastids in these starchless mutants
sedimenting in response to gravity? And was the absence of starch
affecting the plants in ways other than by changing the mass of
plastids in the columella cells? The amylomaize mutant had smaller
amyloplasts that sedimented less than the WT amyloplasts (Hertel et
al., 1969), supporting the starch/gravitropism correlation. In initial
tests, the plastidic phosphoglucomutase mutant TC7 showed no
statistically significant difference in vertical plastid positioning
between vertical and inverted roots (Caspar and Pickard, 1989). Caspar
and Pickard concluded that the plastids did not sediment and therefore
could not be the ultimate source of graviperception in these starchless
mutants. Kiss et al. (1989) proposed that because the plastids in the
mutant were the most mobile structure in the columella cells and were
relatively dense, they could still function as statoliths. They
reasoned that at least some starch was needed for full graviperception.
Knowing the positions of the plastids in the early stages of
gravitropism would help in clarifying these issues.
Some new starch-deficient mutants of Arabidopsis have been
characterized and were generated by T-DNA mutagenesis (Kiss et al.,
1996). The ACG 21 mutant is starchless, whereas mutant ACG 20 has 51%
of the WT starch complement. Using measures similar to those tested on
the TC7 mutant (Kiss et al., 1989), Kiss et al. (1996) found the
starchless mutant to be the least responsive to gravity and the reduced
starch mutants to have an intermediate level of graviresponsiveness.
There have been few quantitative studies of plastid movement in
response to gravistimulation by reorientation (in dandelion [Clifford
and Barclay, 1980], in mung bean [Heathcote, 1981], and in corn
[Sack et al., 1985]). However, these were performed on relatively
large organs, and it has been technically difficult to perform these
types of studies with seedlings of small plant species. It is only with
advances in computer-based image analysis that the present study of
(small) Arabidopsis roots has been made possible.
Previous papers have reported on plastid movement in starchless mutants
in a preliminary manner. We (Kiss et al., 1989) performed low-gravity
centrifugation studies to show that the starchless plastids are
relatively dense and moveable. In addition, Caspar and Pickard (1989)
performed some inversion studies that were based on relatively few data
points. The present paper presents a more complete study of plastid
movement in response to reorientation in roots of WT and
starch-deficient mutants of Arabidopsis. The issue of plastid movement
in response to gravity is important in the evaluation of theories of
gravity perception in plants (Salisbury, 1993; Sack, 1997). Thus, the
focus of these experiments is on the early stages of plastid movement
(0.5-60 min), because, according to the starch-statolith hypothesis,
significant movement must take place within this period. The major aims
of this study were to determine if plastid sedimentation is a
requirement for gravitropism and how starch deficiency affects plastid
kinetics in reoriented roots.
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MATERIALS AND METHODS |
Origin of Seed Stock and Plant Propagation
The starch-deficient mutants of Arabidopsis were produced by T-DNA
insertional mutagenesis. Agrobacterium tumefaciens was used
to infect and transform Arabidopsis seeds (T1),
and the seeds produced by infected cells of the
T1 plants were collected and germinated for
mutational determination (Feldmann and Marks, 1987). All seeds,
including the WT (geographic race Wassilewskija), ACG 20, and ACG 21, were propagated from seeds originally obtained from the DuPont culture
collection (Kiss et al., 1996, 1997).
All plants for seed stocks were grown in potting soil (Metro-Mix,
Scotts-Sierra Horticultural Products, Marysville, OH) at room
temperature (21°C) under constant illumination at 80 to 100 µmol
m
2 s1 PAR from 34-W
fluorescent bulbs (Watt Miser, General Electric). They were watered
alternately with tap water and nutrient solution (Haughn and
Somerville, 1986). To control for seed age among the WT and the two
mutants, seeds used in each experiment were grown and harvested
concurrently. After harvest, all seeds were stored at 5°C for 3 to 9 months.
Culture Conditions
Seeds were first surface-sterilized with 30% (v/v) commercial
bleach (sodium hypochlorite) and 0.01% (w/v) Triton X-100 (Sigma) for
10 min and then washed three times with sterile water and Triton X-100.
Using the final wash, the seeds were sown in a pipette onto 1.7% (w/v)
agar medium containing nutrients as described by Haughn and Somerville
(1986) with 1% (w/v) Suc in sterile Petri dishes (60 mm in diameter,
15 mm in depth). The seeds were sown in two rows with 8 to 10 seeds per
row. In reorientation studies, seeds were immediately covered with
0.8% (w/v) agar medium containing nutrients with Suc (temperature
<55°C) to a depth of 3 to 5 mm. All Petri dishes were then sealed
with laboratory film (Parafilm, American National Can, Greenwich, CT).
The dishes were placed on the 15-mm edge with seed rows horizontal, and
a line was drawn indicating the gravity vector. The seeds were
germinated and grown under continuous light, as described above, at
18°C to 21°C. The illumination was continuous from side to top to
side, and the clear Petri dishes were placed on white paper to negate
any potential phototropic response. Germination was greater than 95%
in all genotypes. The seedlings were grown for approximately 80 h,
until they were 10 to 15 mm long.
Fixation and Sectioning
Fixation for the nonreoriented seedlings was performed with the
seedlings still adhering to the agar. A row of seedlings was excised
from the Petri dish and placed on glass slides. The glass slides were
placed on the edge (maintaining the original gravity vector) in a
staining dish for 30 min. The transfer process took less than 10 s
for each strip of agar (row of seedlings). The primary fixative, 2%
(v/v) glutaraldehyde in 50 mM sodium cacodylate buffer and
50 mM CaCl2, pH 7.2, was added to the
staining dish, and the dish was kept at 5°C for 2 h. The
staining dish was then washed with buffer three times. The seedlings
that remained on the agar in an upright position were collected, and
their primary roots were cut 10 to 15 mm from the root tip and
postfixed in buffered 2% (w/v) OsO4 at 5°C for
2 h.
For the reoriented seedlings, the Petri dishes containing the seedlings
were turned 90°, a hole was melted in the top with a heated needle,
and fixative was added (the dishes were still sealed with laboratory
film) at the following intervals: 0 (immediately upon reorientation),
0.5, 2, 5, 10, 30, and 60 min after the start of reorientation. Care
was taken when the holes were melted to avoid touching the agar or
seedlings with the hot metal. A nonreoriented control was also fixed.
The fixative consisted of 1% (w/v) p-formaldehyde and 2%
(v/v) glutaraldehyde in 50 mM sodium cacodylate
buffer and 5 mM CaCl2, pH
7.2, at 4°C. The formaldehyde was used to increase the speed of
fixation, because the fixative had to penetrate up to 5 mm of agar.
After the addition of fixative, the samples remained at room
temperature for 30 min and then were kept at 5°C for 120 to 180 min.
After three washings in the cacodylate buffer, the agar-embedded root
tips were excised with a no. 11 scalpel (Fisher Scientific). Each
specimen was left in this agar block (through fixation, dehydration, and embedding) to allow for orientation during sectioning. The agar
block surrounding the specimen was excised from the Petri dish with a
no. 2 cork borer (i.d., 4.5 mm) and marked on the reoriented, lower
side (see Fig. 1). This allowed for
determination of the reorientation direction of the root tip and easier
placement of the specimen in the Quetol resin (Electron
Microscopy Sciences, Fort Washington, PA).
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| Figure 1.
Method used to remove agar-encased seedlings from
the Petri dishes in which they were grown. A cork borer was used to
excise a block of agar that contained the seedling. Processing of the
seedling while it was encased in agar permitted the determination of
the plane of reorientation when the roots were sectioned. The block to
the left, which had already been excised, would be sectioned along the
plane of the page (from front to back).
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Both the specimen containing blocks and the roots from the
nonreoriented group were washed with buffer three times and postfixed in buffered 2% (w/v) OsO4 at 5°C for 2 h.
The tissue was then washed, dehydrated in a graded ethanol series, and
embedded in Quetol resin according to the method of Kushida and Kushida
(1982). Semithin sections (1 µm) were cut with a glass knife and
stained with 0.1% (w/v) toluidine blue.
Digitization, Stereology, and Image Analysis
Toluidine-blue-stained sections were visualized with a research
microscope using bright-field optics (Zeiss) and a CCD (charge-coupled device) camera (Dage-MTI, Michigan City, IN). The images were then
captured using image-analysis hardware and software (Image-1, Universal
Imaging Corp., Media, PA). In some cases, sections were examined and
photographed with bright-field optics using a compound microscope
(BH-2, Olympus) with Technical Pan film (no. 2415, Kodak) at ASA
(American Standards Association) 50.
An exact median longitudinal section of the Wassilewskija WT (see Fig.
2) shows the arrangement of the root-cap columella cells. All of the
mutants used in this study, which were derived from the Wassilewskija
strain, had similar root-cap morphology. The lighter staining of the
cytoplasm and the presence of darkly staining plastids allowed for
fairly easy differentiation of the columella cells.
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| Figure 2.
Light micrographs of 1-µm sections of the root
cap of Arabidopsis WT (A), ACG 20 (51% starch; B), and ACG 21 (0%
starch; C) stained with toluidine blue. Arrowheads indicate plastids in
columella cells. Columella cells are in stories 1 through 3, and
peripheral cells are in story 4. The WT plastids are somewhat larger
than those of the mutants, but otherwise the morphology of the root of
all strains is uniform. The arrow indicates the gravity vector and
equals 25 µm.
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The distribution of amyloplasts within the columella cells was
quantified using stereology to allow the best control over cell-to-cell
comparisons. First, the cells were grouped into three stories (as
described by Sack and Kiss, 1989), with the first story (S1) being made
up of the youngest cells, which were just emerging from the meristem,
and the third story (S3) being made up of the oldest cells, which were
about to become peripheral cells. In these studies, a maximum of three
micrographs per cell was used. To avoid counting the same plastid
twice, if a cell was used more then once we determined that the
positions of the measured plastids did not coincide. To allow the best
stereological control over plastid positioning and comparisons, we used
cell micrographs only if the nucleus was visible and none of the walls was sectioned tangentially. These three criteria helped to standardize the data.
The Image-1 computer program, with a Trinitron monitor (Sony
Electronics Inc., San Jose, CA), was used to visualize and capture the
digitized micrographs and make all of the measurements. After each
image was calibrated, the following data points were determined: each
cell's corners (four), the bottom of the plastid, the center of the
plastid, and the bottom of the cell wall directly below the plastid
(see Fig. 3).
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| Figure 3.
Central columella cell (inside-"right")
after reorientation of the root. As the diagram suggests, the distal
and proximal cell walls were not perpendicular to the root axis. The
plastid angle relative to gravity, the plastid angle relative to the
cell corner, and the sedimentation measurements are indicated (the
original "up" vector being 0°). Pl, Plastid; Nu, nucleus. Each
shape represents a measured point. Black circle, Center of cell
(calculated); open circles, measured points for each plastid; gray
squares, corners of cell.
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Seedlings were reoriented 90° in the second group, and plastid
movement relative to the new, lower cell wall was measured. This
implies movement in two dimensions, not just a linear sedimentation. Because root-cap columella cells in Arabidopsis are not exactly rectangular, as some studies seem to suggest, measurement of the radial
movement of plastids around the cell permits a more complete picture of
plastid motion (Sack et al., 1985). By using the centroid as a relative
center of each cell, an angle for each plastid was determined at all
times (vertical and 0, 0.5, 2, 5, 10, 30, and 60 min). The centroid was
the origin, whereas a line to each plastid and a line from the centroid
up the root, parallel with the original gravity (as the plant was
grown), made the sides (see Fig. 3). The following data points were
recorded for each plastid: (a) the angle from the axis of the root, (b)
the distance from the origin, (c) the distance to the distal cell wall,
(d) the distance to the new bottom cell side, and (e) the absolute
Cartesian coordinates of all points.
From this point in the center of the cell, two fixed reference lines,
relative to general cell morphology, were used to determine angular
plastid movement. One line was relative to the gravity vector, and the
other was relative to the cell corner (by which plastids must pass if
they are to sediment to a new lower wall). The former measurement
relates the movement of the plastids to gravity and provides an
indication of where the plastids might move if there were no
obstructions and no other forces acting on them. This measurement also
reveals whether the calculated centroid is a valid point on which
measurements can be based, because before reorientation, plastids
should be along the gravity vector "below" the centroid
(approximately 180°). The second measurement, relative to the cell
corner between the original and the new, lower cell walls, is important
because it accounts for the fact that the columella cells are not
rectangular (see Fig. 2). These cells are trapezoidal, but the corners
are almost never 90°. Measurement relative to a corner helps correct
for the true morphology of the cell and affords yet another measure of
movement (Sack et al., 1985). With this measurement, one can determine
if and when a plastid ever moves past the corner.
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RESULTS |
Root-Cap and Columella Cell Morphology
Bright-field microscopy of roots from 4-d-old (vertically grown)
Arabidopsis seedlings demonstrated that the root cap consists of four
horizontal stories and four vertical files of cells (Fig. 2). The stories are labeled 1 to 4 from
the meristem to the root tip according to the method of Sack and Kiss
(1989), and usually the first three stories are considered columella
cells. Beyond story 3, the columella cells usually lose polarity and
develop enlarged vacuoles, and thus become peripheral cells, before
being shed from the tip. Cells were determined to be columella cells if
they were polarized, had a small vacuolar area, and stained much
lighter than the surrounding cells (peripheral cells and the
meristematic region). In vertically grown seedlings, a typical columella cell has a polarized morphology, with the nucleus always at
or near the proximal end and the plastids near the distal end (Fig. 2).
Even in the starchless mutant, the plastids were not near the "top"
of the cell, close to the nucleus.
Although only columella cells from stories 2 and 3 were used for this
study, there is still variation in size and shape within this cell
type. There are two inner and two outer cells in each story that differ
in shape. These are referred to as the central and flank columella
cells, respectively, according to Blancaflor et al. (1998). As shown in
Figure 2, the cells on one side of the center line are mirror images of
the cells on the other side. Even though they still have longitudinal
running sides, the proximal and distal cell walls are sloped toward the
center. An approximately equal number of cells from each side of the
center line was used to normalize the data. Also, it was assumed that
this morphology also occurs in the Z direction (to and away from
the observer). This was compensated for by specifying a certain cell
morphology for all cells in which plastid position was analyzed: The
nucleus must be sectioned in approximately the middle third, the
longitudinal walls must be parallel, and the cytoplasm must be light,
continuous, and demarcated abruptly at the cell wall.
Both the absolute and the fractional cell data were quantified to
characterize the position and movement of plastids in the columella
cells of Arabidopsis. Whereas many studies have measured absolute or
fractional cell plastid positional information, almost none has made
direct measurements of both parameters.
Vertically Oriented Seedlings
The extent of sedimentation of the plastids was determined in
roots of vertically oriented seedlings, and this analysis showed that
the cell fractional sedimentation differed significantly (Table
I), as determined by ANOVA with Tukey's
posttest (P < 0.05). The sedimentation of the WT plastids was
significantly different and greater in magnitude than that of plastids
from both starch-deficient mutants. In addition, plastids of the
starchless mutant were approximately one-half as sedimented as the WT
plastids.
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Table I.
Plastid sedimentation by cell fraction in the
root columella
Seedlings were fixed vertically at 4 d. The sedimentation and
total cell height were measured from 1-µm sections stained with
toluidine blue. Sedimentation was defined as the ratio of the distance
from the center of the plastid to the distal cell wall. The values
indicated with different letters are significantly different as
determined by ANOVA/Tukey's method (P < 0.05). n, No.
of plastids.
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Reoriented Seedlings
Linear Plastid Sedimentation
Because the plastid-statolith hypothesis suggests that it is the
settling of plastids upon some receptor that allows graviperception, an
important measurement of plastid movement is linear movement parallel
to gravity. Presumably, it is this movement toward the new, lower wall
that allows graviperception (e.g. interaction with the ER or the
cytoskeleton). In measurements relative to the new, lower cell wall
(both absolute distance in micrometers and fraction of cell height),
the WT plastids sedimented more than the plastids of the two
starch-deficient mutants (Figs. 3 and
4). The positions of plastids of the WT
and starch-deficient seedlings became statistically significantly
different compared with the positions of the plastids of the starchless
mutant at 0.5 and 10 min, respectively (ANOVA with Tukey's posttest,
P < 0.05; Fig. 4). Once their positions diverged from those of
the starchless plastids, the plastids of the other two strains
continued to sediment, and the difference (from the starchless mutant)
increased.
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| Figure 4.
Time-course studies of linear plastid
sedimentation in the root-cap columella cells of WT and mutant
Arabidopsis. The seedlings were reoriented 90°, and the vertical
distance was measured relative to the calculated cell center (A and B)
or the new cell bottom (C and D) at the times indicated. Plastids of
both starch-deficient mutants sedimented less than those of the WT.
Stars and crosses indicate when the WT and 51% starch-deficient
mutant, respectively, became statistically significantly different from
the starchless mutant (ANOVA with Tukey's posttest, P < 0.05).
A, Mean vertical plastid position relative to the cell center measured
as a fraction of the cell size. Because the seedlings were reoriented
90°, the mean plastid position was almost zero ("middle" of the
cell) at time 0. B, Mean vertical plastid position relative to the cell
center as absolute distance (µm). The average distance across the
cell was 10 µm. C, Mean vertical plastid position relative to the
new, lower cell wall measured as a fraction of the cell size. D, Mean
vertical plastid position relative to the new, lower cell wall as
absolute distance (µm). Values in parentheses indicate the amount of
starch relative to the WT, and error bars represent SE.
Each data point represents a mean of 25 to 40 plastids.  , WT;  ,
ACG 20;  , ACG 21. Time zero (*) is added to the logarithm time scale
for comparison.
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The WT and reduced-starch plastids sedimented at approximately the same
rate, whereas the sedimentation of the starchless mutant was not
significantly different (ANOVA with Tukey's posttest, P > 0.05).
At the WT presentation time of 5.3 min (Kiss et al., 1996), the
plastids had sedimented 68% to 87% of the final distance (relative to
the 30- and 60-min points).
Angular Data
Because roots were rotated 90° in the reorientation study,
movement in two dimensions, not just linear sedimentation, was measured
in these studies. Considering that root-cap columella cells in
Arabidopsis are not exactly rectangular, as some studies seem to imply,
measurement of the radial movement of plastids around the cell permits
a more complete picture of plastid motion (Sack et al., 1985). An angle
was determined by first finding the "center" of the cell, which was
done by manually tracing the cell and having the software determine the
center of area (see ``Materials and Methods'').
From the center of the cell, two fixed reference lines, compared
with general cell morphology, were used to determine angular plastid movement: (a) relative to the gravity vector, and (b) relative to the cell corner by which the plastids must pass if they are
to sediment to a new, lower wall (see Fig. 3). The former measurement
gives some indication of where the plastids might move if there was
nothing in their way and no other forces acting on them.
Thus, when the plastid angle relative to gravity was examined, it
became apparent that the plastids of all three strains started at
approximately the same mean point (
180° along the gravity vector),
but WT plastids had the greatest angular displacement (Fig.
5A). At 2 min and later, the WT plastids
had a statistically significantly greater angle than both the
starchless and the reduced-starch mutants (P < 0.05). In
addition, WT plastid displacement was at a faster rate than plastid
displacement in the reduced-starch and starchless mutants. The WT
plastids had their greatest angular velocity between 2 and 10 min and
slowed markedly after 30 min. By 60 min, the WT plastids had a less
random positioning, as shown by the relatively small SE.
The plastids of both starch-deficient mutants were displaced at about
the same rate, which was less than the rate of the WT.
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| Figure 5.
Time-course studies of angular plastid
sedimentation in the root-cap columella cells of WT and mutant
Arabidopsis. The seedlings were reoriented 90°, and the plastid angle
from the calculated cell center was measured relative to the cell
corner or the root axis (gravity). By 2 min, WT plastids sedimented
significantly more than plastids in both starch-deficient mutants
(P < 0.05). A, Mean angle of plastids relative to the root axis
(gravity). B, Mean angle of plastids relative to the cell corner.
Values in parentheses indicate the amount of starch relative to the WT.
Each data point represents a mean of 25 to 40 plastids, and error bars
represent SE. , WT; , ACG 20; , ACG 21. Time zero
(*) is added to the logarithm time scale for comparison.
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The second angular measurement (Fig. 5B), plastid angle relative to
cell corner (i.e. relative to the cell corner between the original and
the new, lower cell walls), is important because it accounts for the
fact that the columella cells are not rectangular (see Fig. 2).
Although the central cells are approximately rectangular, the flanking
cells deviate from this shape. Thus, measurement relative to a corner
helps correct for the true morphology of the cell and affords another
important measure of movement (Sack et al., 1985). With this
measurement, one can determine if and when the plastids ever move past
the corner. If, as proposed by many researchers (for review, see Sack,
1991), plastids allow perception by falling onto a sensitive surface,
then it is only after they pass the corner that they are able to fully
apply pressure to such a surface.
Although plastids of all three strains started at different angles
relative to the cell wall corner, WT plastids had the greatest angular
displacement (Fig. 5B). Despite the fact that the WT plastids were
larger than the plastids of either of the mutants, they moved farther
around the cell. The WT amyloplasts, in fact, were the only plastids to
pass the corner, after which their movement slowed to a great
degree.
A series of light micrographs qualitatively illustrate the
plastid-sedimentation patterns (Fig. 6)
that were documented in the quantitative time-course studies (Figs. 4
and 5). Amyloplasts in columella cells in roots of WT seedlings showed
the most obvious sedimentation (Fig. 6A), whereas plastids in the
reduced-starch mutant also appeared to sediment (Fig. 6B). In contrast,
plastids in columella cells of the starchless mutant lacked
obvious sedimentation (Fig. 6C).
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| Figure 6.
Time-course photomicrographs of root caps of WT
and mutant Arabidopsis seedlings after gravistimulation by
reorientation. The seedlings were grown for 4 d, reoriented 90°,
and then fixed at the times shown. Row A shows WT, row B shows
ACG 20 (51% starch), and row C shows ACG 21 (0% starch) at 0, 5, and
60 min after reorientation. Arrowheads indicate plastids in columella
cells. WT plastids sedimented in response to reorientation, whereas
starchless mutant plastids did not appear to sediment. In the
starchless mutant, a plastid can be seen in the proximal end of the
cell above the nucleus (bottom arrowhead). This was not common, and it
was never observed in the WT. The direction of gravity is toward the
bottom of the figure, as indicated by the arrow. Bar = 25 µm.
|
|
| |
DISCUSSION |
Comparison with Other Studies of Plastid Sedimentation
Uncertainties about definite receptors, the original asymmetrical
signal, and even the signal transmitted from the root cap to the
elongation zone have compounded the problem of finding the original
gravity susceptor(s). The aim of this research was to analyze plastid
movement from many different and biologically important perspectives.
This study continues the preliminary observations of plastid movement
in WT and starchless Arabidopsis roots reported by Caspar and Pickard
(1989) and Kiss et al. (1989). Thus, a more complete study of plastid
movement in response to reorientation in roots of WT and
starch-deficient mutants of Arabidopsis is presented here. Furthermore,
an accurate study of plastid kinetics in the extremely small roots of
Arabidopsis was made possible only by recent advances in computer-based
image analysis.
Plastids in the Vertically Oriented Starch-Deficient Seedlings
Sediment Less Than WT Plastids
One standard measure of graviresponsiveness is the orientation of
seedlings around the gravity vector (Sack, 1991; Kiss et al., 1996,
1997). Although the gravity vector is unchanged, Arabidopsis starch-deficient mutants are not as oriented around gravity as the WT
(Kiss et al., 1996). This presumably is a function of the smaller,
buoyant mass of the plastids, but to our knowledge, until this study it
was not known if there was less sedimentation of plastids. Based on
comparison of micrographs, it appears that the cell morphology is
unchanged except for the positions and sizes of the plastids. In
vertically grown seedlings, the plastids within the columella cells
were at different positions relative to the distal cell wall (which
here is the cell bottom with respect to gravity) and differed in their
arrangement. Plastids of the starchless mutant (ACG 21) were only about
one-half as sedimented as WT plastids.
The word "sedimented" is used in a general sense in this
discussion, relative to the whole cell, as it has been used by workers in the field (e.g. Sack et al., 1985, 1986). It is important to note
that sedimentation is not an absolute measure. Depending on how, and
relative to what, sedimentation is defined (available space for
sedimentation, relative to which cellular apparatus), many different
views can be supported.
The positioning of the starch-deficient plastids also was less regular
than that of the WT amyloplasts, as indicated by the greater
SE. The plastid positioning in nonreoriented roots agrees with data from inverted studies of the TC7 starchless mutant (Caspar and Pickard, 1989) and qualitative observations of TC7 (Saether and
Iversen, 1991) as well as data from the starch-deficient mutant of maize (Hertel et al., 1969). The similar results at the plastid level, along with studies of the kinetics of gravitropic curvature (Kiss et al., 1996), are particularly significant, because these two
starchless mutants were isolated from different ecotypes and by
different mutational methods (chemical and T-DNA insertion).
Starchless plastids are less than one-half as large as the WT plastids
and are more irregularly shaped, as measured by light microscopy. The
large size of the WT amyloplasts theoretically would prevent them from
packing as tightly as the starch-deficient plastids. Thus, the
difference in sedimentation detected here could be somewhat
underestimated. However, it was observed that the WT plastids were more
tightly packed within a cell (in the reoriented seedlings as well). One
simple explanation for the rather regular arrangement of the WT
plastids is that they sediment and come to some equilibrium point near
the bottom of the cell. The large size and tight grouping of these WT
plastids would subdue movement compared with the starch-deficient
plastids. It is important to note that the plastids in all genotypes
are not at "rest" and are most likely continually moving in one
direction or another by limited cytoplasmic streaming and saltations
(Sack et al., 1986). These saltations, if of equal strength in the
mutants, would likely cause a less concentrated plastid group in the
reduced-starch mutants, because the plastids presumably have a smaller
mass.
It is also interesting to note the sedimentation when the volume
displaced by other organs is taken into account. All columella cells in
Arabidopsis have a large (about one-quarter of the cell length),
proximally located nucleus (Kiss et al., 1989). Thus, if one were to
subtract the proximal vertical distance of the cell that the nucleus
occupies, the starchless mutant plastids would be found at the vertical
midpoint of the cell.
WT Plastids Sediment Faster and to a Greater Magnitude When the
Root Is Reoriented Compared with Plastids of the Starch-Deficient
Mutants
Linear measurement in the reoriented seedlings showed the
sedimentation of the plastids to the new, lower cell wall. Presumably, it is this movement toward or settling onto the new, lower wall that
allows graviperception (interaction with the ER or the cytoskeleton). This traditional measurement of sedimentation shows that the plastids with more starch have a greater degree of sedimentation. Although a
seemingly obvious observation, this is an important consideration in
the evaluation of possible models of perception. At the WT presentation
time of 5.3 min, 68% to 87% of the plastids had sedimented.
It is important to note that only the WT plastids pass the cell corner.
If, as claimed by many researchers in the field (for review, see Sack,
1991), plastids allow perception by falling onto a sensitive surface
(presumably below the plastids), then it is only here (after they pass
the corner) that they first are able to apply full pressure to any
supposed sensitive surface.
For the plastid-reorientation study, precision in sectioning is
important in both the radial (around the root) and longitudinal (along
the axis of the root) directions (Fig.
7). The longitudinal precision was
controlled by close observation of the morphology of the cells. The
radial imprecision could not be observed and corrected. Although the
roots remained encased in agar during the fixation and embedding
procedure, some twisting could still take place, causing the section to
be out of radial alignment (about one-tenth of the original seedlings
were discarded when twisting was observed after root excision but
before infiltration of resin). A section not in the correct radial
plane would result in measurement of a less sedimented, more random
plastid position, because the section would not be 90° from the
plastid movement. Therefore, we cannot conclude that the sedimentation
of the plastids shown in the reoriented seedlings is a realistic
maximum. Rather, the data measured here can be viewed as a slightly low
approximation, and, because all genotypes were sectioned identically,
the differences observed presumably would still hold.
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| Figure 7.
Root-cap cells in Arabidopsis. The four stories
(S1-S4) and four files (central and flank) are labeled. The arrows
indicate the possible orientational errors when sectioning. Although
the left/right and up/down of the root tip could be well accounted for,
twisting (a possibility when cutting the seedlings out of the agar) of
the root could introduce errors in the absolute measurements. This is
one reason why multiple methods to measure plastid sedimentation were
used in this study.
|
|
A Greater Degree of Plastid Sedimentation Is Required for a
Full Gravitropic Response
As measured by the three methods used in this study (i.e. vertical
orientation, angular reorientation, and linear reorientation), WT
plastids sedimented faster and to a greater degree than the starchless
plastids, and the reduced-starch plastids sedimented an intermediate
amount. This demonstrates a correlation of starch content with the rate
and magnitude of plastid sedimentation, and because previous research
(Kiss et al., 1996, 1997, 1998) has shown that starch content is
positively correlated with increased gravisensitivity, the present and
previous studies demonstrate that graviresponse is correlated with the
rate and magnitude of plastid sedimentation. Although not necessarily a
cause, sedimentation does seem to be closely involved with perception.
However, this does not explain the lack of significant sedimentation in
starchless plants that are still able to perceive gravity. This finding
seems to indicate that there is a different, less sensitive mechanism that allows the starchless mutant to perceive gravity, possibly a
protoplast- based system. Redundancy at various levels seems to
be common in the evolution of plant perceptual systems (Barlow, 1995).
Alternatively, it is possible that the plastids still perceive gravity
but that any gross movement is not required (Nick et al., 1997).
Presentation time, a measure that includes the perception and
transduction/transmission steps, and perception time, which includes
the perception and perhaps the intracellular statocyte asymmetry
transduction, have been used traditionally as measures of relative
graviperception (Volkmann and Sievers, 1979). For WT roots (Kiss et
al., 1996), the calculated presentation time is 5.3 min, and the
present study demonstrates that there is significant movement of
plastids toward the new, lower wall after gravistimulation by
reorientation.
Perception times can be approximated by using intermittent stimulation,
alternating with clinostat rotation. In this manner, stimulation times
can be summed to produce an effect (a single application would not
cause a response). Kiss et al. (1996) found that with 6:4 intermittent
stimulation, the reduced starch mutant had 64% of the WT curvature,
whereas the starchless mutant had 20% of the WT curvature. With these
estimated measurements, relative gravisensitivity was determined. In
the present study, calculations of the percentage of WT plastid
sedimentation (Fig. 4) showed an approximate correlation to these
values. Thus, the reduced starch mutant had 55% of WT plastid
sedimentation and the starchless mutant had 16% of the sedimentation
of the WT.
Sedimentation of Plastids Can Play a Role in Graviperception but Is
Not Required for Perception
When a columella cell is reoriented with respect to gravity, the
plastids sediment to the new, lower cell wall. This is the original
observation that implicated plastids in graviperception (Haberlandt,
1914). Even in recent studies mapping the functional role of columella
cells, those with the most readily sedimentable plastids were the ones
that seemed to contribute the most to graviperception (Blancaflor et
al., 1998).
The protoplast model proposes that amyloplasts are used only as added
weight within the protoplast statolith (Staves et al., 1992; Staves,
1997). However, it seems probable that these models are not mutually
exclusive (Barlow, 1995). Multiple systems could be working at
different levels. When starch is present, the sensitive plastid-statolith mechanism would predominate. Yet when the seedling is
starch free and the plastids in the columella cells do not sediment
appreciably, the protoplast mechanism would still allow a minimum level
of graviperception.
One important shortcoming of the starch-statolith theory is the lack of
a definitive receptor. This greatly affects any interpretation of
plastid positioning and movement. Nevertheless, in this study we
examined plastid sedimentation in many different and presumably biologically important ways. The starch-deficient mutants, which are
less gravitropic than the WT, have plastids that sediment less than the
WT amyloplasts. However, plastids in starchless plants, which can
perceive gravity, do not show statistically significant sedimentation,
and this observation implies that there is a different, parallel system
capable of detecting gravity. Taken together, the results of this study
are consistent with the classic plastid-based and protoplast-based
models of graviperception and suggest that multiple systems of
perception exist in plant cells (Barlow, 1995).
| |
FOOTNOTES |
1
This research was supported by the National
Aeronautics and Space Administration (grant no. NAG 2-1017).
*
Corresponding author; e-mail kissjz{at}muohio.edu; fax
1-513-529-4243.
Received November 9, 1998;
accepted January 22, 1999.
| |
ABBREVIATIONS |
Abbreviations:
ANOVA, analysis of variance.
WT, wild type.
| |
ACKNOWLEDGMENTS |
We thank Dr. Roger Meicenheimer (Miami University) for the use
of image-analysis facilities in his laboratory and Dr. Tim Caspar
(DuPont) for providing the original seed stock used in this study.
| |
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Top
Abstract
Introduction