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Plant Physiol. 1998 December; 118(4): 1139–1145. | PMCID: PMC34730 |
Root-Growth Behavior of the Arabidopsis Mutant
rgr11
Roles of Gravitropism and Circumnutation in the Waving/Coiling
Phenomenon Jack L. Mullen,* Ed Turk, Karin Johnson, Chris Wolverton, Hideo Ishikawa, Carl Simmons, Deiter Söll, and Michael L. Evans Department of Plant Biology, The Ohio State University, Columbus, Ohio 43210–1293 (J.L.M., E.T., C.W., H.I., M.L.E.) Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 (K.J., D.S.) Pioneer Hi-Bred International, Johnston, Iowa 50131 (C.S.) Received May 15, 1998; Accepted September 11, 1998. In this study we investigated the
kinetics of the gravitropic response of the Arabidopsis
mutant rgr1 (reduced
root gravitropism). Although the rate of
curvature in rgr1, which is allelic to
axr4, was smaller than in the wild type (ecotype
Wassilewskija), curvature was initiated in the same region of the root,
the distal elongation zone. The time lag for the response was
unaffected in the mutant; however, the gravitropic response of
rgr1 contained a feature not found in the wild type:
when roots growing along the surface of an agar plate were
gravistimulated, there was often an upward curvature that initiated in
the central elongation zone. Because this response was dependent on the
tactile environment of the root, it most likely resulted from the
superposition of the waving/coiling phenomenon onto the gravitropic
response. We found that the frequency of the waving pattern and
circumnutation, a cyclic endogenous pattern of root growth, was the
same in rgr1 and in the wild type, so the waving/coiling
phenomenon is likely governed by circumnutation patterns. The
amplitudes of these oscillations may then be selectively amplified by
tactile stimulation to provide a directional preference to the
slanting. Although plant roots appear superficially to be symmetrical, their
growth patterns can be asymmetrical. An example of asymmetrical growth
patterns in a seemingly symmetrical environment is the skewed, wavy
pattern observed in the roots of some ecotypes of Arabidopsis grown on
inclined plates of agar ( Okada and Shimura, 1990, 1992; Simmons et al.,
1995a; also see Fig. A). The waving pattern is caused by a series of
bends in the root that alternate in direction. The skew arises from a
nonrandom preference by the root in the direction of bending relative
to the vertical. There is a strict correlation between the direction of
curvature for each bend of the root and the orientation (left or right)
of the helical spirals formed by epidermal cell files, suggesting that
the pattern is caused by a succession of left- and right-oriented
processes ( Rutherford and Masson, 1996). The pattern of wavy growth has
been suggested to result primarily from an interaction of gravitropism
and thigmotropism ( Okada and Shimura, 1990, 1992) or from an
interaction of gravitropism and circumnutation ( Maher and Martindale,
1980; Simmons et al., 1995b).
In the former model it is postulated that thigmotropism of the root tip
causes a reversal in the direction of rotation of cell files and,
therefore, a reversal in the direction of tip growth. Subsequent
thigmostimulation of the root tip results in another reversal of
cell-file rotation, causing the root to grow in a wavy pattern along
the agar surface. Gravitropic sensitivity provides thigmostimulation by
giving the root a tendency to grow into the agar. According to the circumnutation/gravitropism model, the wavy pattern of
root growth on inclined surfaces results from an endogenous pattern of
root growth (circumnutation) interacting with the gravitropic response,
which causes the roots to grow downward. Because root circumnutation
usually has a chirality favoring the clockwise direction, as viewed
looking downward along the root axis ( Baillaud, 1962; Johnsson, 1997,
and refs. therein), circumnutation may be able to provide the
directional preference for the wavy growth. However, this slanting
preference did not occur in roots embedded in agar, on a soft agar
surface, or on a surface slanted so that gravitropism tended to pull
the roots away from the agar surface ( Rutherford and Masson, 1996; also
see Fig. C). Therefore, asymmetrical mechanical stimulation, which
these treatments minimize, is involved in the slanting response in some
manner. Because gravitropism serves an important role in these models of the
waving pattern of growth, we wanted to investigate the relationships
between this waving phenomenon and gravitropism and other growth
behaviors in the gravitropism-deficient mutant rgr1
( reduced root gravitropism). The
mutant was isolated from the DuPont T-DNA insertional mutagenesis
collection in the Arabidopsis Wassilewskija ecotype by Simmons et al.
(1995a) and is allelic to the axr4 mutant isolated by Hobbie
and Estelle (1995). Primary roots of these mutants are characterized by
reduced gravitropism, as indicated by a slower rate of gravitropic
curvature ( Hobbie and Estelle, 1995) and more random orientation about
the vertical after gravitropic response ( Simmons et al., 1995a). This
confirms the importance of gravitropism in the waving phenomenon,
because the roots of rgr1 seedlings grown on inclined
surfaces do not form wavy patterns, but instead form circular coils
that have the same directional preference as the wild-type slanting
preference ( Simmons et al., 1995b; Fig. D). Roots of rgr1
seedlings also exhibit resistance to growth inhibition by auxin, which
may explain their reduced gravitropic sensitivity. We have developed an automated video-digitizer system for detailed
measurement of the growth and curvature of roots of Arabidopsis. We
used this new system to compare the kinetics of gravitropism and other
growth behaviors between the roots of rgr1 and wild-type
seedlings in a series of different tactile environments. These
comparisons provide insight into the basis of the waving/coiling growth
phenomenon. Plant Material Seeds of Arabidopsis of either the wild type (ecotype
Wassilewskija) or the mutant rgr1 were surface-sterilized by
agitation in a 5.25% (v/v) NaOCl solution for 5 min, followed by
several rinses in distilled water. Seeds were sown in a row (three to
five seeds per row) on sterile agar (1%, w/v) in Petri dishes (60 mm
in diameter, 15 mm in height) sealed with laboratory film (Parafilm,
American Can, Greenwich, CT). The agar medium contained 1% (w/v) Suc,
one-half-strength Murashige-Skoog medium ( Murashige and Skoog, 1962),
and 1 m m Mes, pH 5.8. The rows of seeds were placed on the
agar surface perpendicular to the cylindrical axis of the dish so that
the roots would grow along the surface or on a surface parallel to the
axis, so that the roots would grow downward through the agar. The Petri
dishes were either placed immediately in a culture room under
continuous white light from fluorescent lamps (F30T8-CW, Sylvania) with
a fluence rate of approximately 47 μm m −2
s −1 at a temperature of 23°C, or they were
refrigerated at 4°C for 1 to 6 d before being transferred to the
culture room. The Petri dishes were placed vertically or at the
indicated angle of tilt and used for experimentation when the seedlings
were 4 to 5 d old. Video-Digitizer System The seedlings were viewed by a CCD (charge-coupled device) camera
(Marshall Electronics, Culver City, CA) connected to a computer via a
frame-grabber circuit board (ImageNation, Beaverton, OR). The roots
were illuminated from behind with a fiber-optic illuminator (Fiber
Lite, Leica) or with an IR light-emitting diode (Radio Shack, Fort
Worth, TX). No difference in growth or response was observed between
the two light conditions. The growth behavior of the roots was analyzed using a modified version
of the Multi-ADAPT software described by Ishikawa and Evans (1997). The
new version of ADAPT measures the rate of elongation of the opposite
flanks of the root as well as the angles (relative to vertical) of
different segments of the root, defined by their distance from the root
tip. The software determines total elongation on opposite sides of the
root by tracing the root edges from the calculated root-tip position to
the position of fixed reference points in the nonelongating region.
Regression of the changes in length of a side gives its elongation
rate. ADAPT defines segments of the root by searching along arcs of
fixed radius for the edges of the root (Fig.
). A particular segment can then be
described by a line segment connecting the midpoints of the arcs
defining the boundaries of the segment. The angle of this line segment
from the vertical is then calculated as a function of time.
Interaction of Gravitropism and Tactile Stimulation For the gravitropism experiments, root growth was first analyzed
by the digitizer system with the seedling oriented vertically. The
Petri dish containing the seedling was then rotated 90° in either a
clockwise or a counterclockwise direction, and data collection was
resumed. To determine the extent to which tactile stimulation of the
root as a result of contact with the agar surface influenced the
gravitropic response, gravitropism was measured both with the root
growing along the surface of the agar and with the root growing through
a solid block of agar. To minimize tactile stimulation, we also
examined growth and gravitropism of roots growing in a liquid medium or
in humid air. To create these conditions of minimal stimulation, we cut
a small cube out of the agar block, just below the tip of the root.
This created an open cavity, which was then covered with a layer of
agar. For some experiments the cavity was filled with a liquid medium
(one-half-strength Murashige-Skoog medium plus 1 mm Mes, pH
5.8). Circumnutation and Calculation of Curvature To observe the direction of circumnutation in a horizontal plane,
root growth was measured in two perpendicular planes by placing two
cameras at right angles to one another. The direction of curvature in
the horizontal plane was then found by plotting the angle of the root
tip in one plane in relation to the angle in the other. The curvature
(the reciprocal of the radius of curvature) of points along this curve
was calculated using seven-point quadratic numerical differentiation
formulae, as described by Silk and Erickson (1978). Conventions of Terminology Angle of Plate Orientation For purposes of this report, plates oriented vertically with the
roots growing downward were assigned an angle of 0; plates tilted so
that the gravity vector pointed from the root toward the agar surface
were assigned positive values of tilt; and plates inclined so that the
gravity vector was away from the surface were assigned negative values. Direction of Asymmetric Growth and Plate Rotation For this report we assumed that the viewer's perspective is
looking at the seedling through the Petri dish cover with the seedling
in front of the agar surface. From this perspective the directions of
rotation are consistent with those in previous reports of root coiling
( Mirza, 1987; Simmons et al., 1995b). It is from this same frame of
reference that we refer to the directional preference of the wavy
growth pattern as slanting to the left. Gravitropism and the Waving Response The wavy growth pattern of a wild-type root growing along an
inclined plane has a directional preference to the left ( Okada and
Shimura, 1990), which shifts the root's direction of growth away from
the gravity vector; therefore, gravitropism should play a role in the
wavy growth pattern. Indeed, the magnitude of waving and of directional
preference are dependent on the angle of incline (Fig. , A–C). They
are also angle dependent in rgr1, but the “waves” change
to coils at positive angles (Fig. , D–F). To investigate the
interactions of gravitropism and waving/coiling, we analyzed the
kinetics of gravitropism after seedlings were reoriented to a
horizontal position with a clockwise or a counterclockwise rotation. For roots of wild-type seedlings, the gravitropic responses after
counterclockwise rotation (gravitropism and slanting are in the same
direction; Fig. A) and clockwise
rotation (responses have opposing directional preferences; Fig. B)
were similar. In both cases, downward curvature was initiated in a
region 200 to 300 μm from the root tip. It is in this region that
roots show a characteristic divergence of orientation angle after
gravistimulation (segments shown by arrows in Fig. ). Segments apical
to this region show a change in angle attributable to the change of
their basal neighbor, whereas segments basal to this region do not show
curvature until growth causes the curvature to migrate back into these
segments. Because we were able to localize the region of initial
downward curvature, we calculated the time lag until curvature
initiation as the time until the difference in angle between a segment
apical to and a segment basal to the region of curvature reached 5% of
the maximal difference minus the initial difference in angle. The
difference in direction of gravistimulation did not cause a
statistically significant difference in the time lag (Table
I), which averaged 27 min. As a measure
of the magnitude of the growth response, we also calculated the
average rate of curvature for the 2 h immediately after the time
lag. The mean rate of curvature was 18° h −1,
with no significant difference between clockwise and counterclockwise
treatments (Table I). Therefore, the directional preference of the
waving was not strong enough to interfere with the wild-type
gravitropic response.
| Table IComparative gravitropism kinetics for roots of
wild-type and rgr1 seedlings growing on the surface of vertically
oriented agar plates |
The kinetics of the gravitropic response for the roots of
rgr1, however, did depend on the direction of rotation (Fig.
). Seedlings rotated counterclockwise
bent downward because of the initial curvature in the region 200 to 300
μm from the tip (Fig. A). This response is similar to that of the
wild type, except for the reduced rate of curvature (Table I). However,
for seedlings rotated clockwise, 63% of the roots showed upward
curvature (Fig. B). This upward bending originates in the central
elongation zone, farther back from the tip than the site of major
downward curvature, which was found in the same location as in the wild
type. Because of this upward curvature in roots rotated clockwise,
there was a difference in the mean rates of curvature for the clockwise
and counterclockwise treatments (Table I). The time lags for the
gravitropic response were unaffected in rgr1 (Table I).
The roots of rgr1 showed a growth feature not found in the
wild type: upward curvature in the central elongation zone. This
feature has a clockwise directional preference, which is the same
directionality found in the coiling response of rgr1. To
determine whether the upward curvature was a true component of the
gravitropic response or if it was part of the root-coiling pattern
superimposed on the gravitropic response, we measured gravitropic
kinetics under different tactile environments. Upward Curvature and Altered Tactile and Light Environments To test the behavior of rgr1 under a more uniform
tactile environment, we measured the gravitropic response of roots
growing through solid blocks of agar. Figure
shows the time course for the
gravitropic response for such a root after clockwise reorientation.
There was no initial upward curvature in the central elongation zone of
roots growing through solid agar. However, downward curvature occurred
just as it did in roots growing along the surface (initiating 200–300
μm from the root tip). This suggests that an asymmetrical tactile
environment is necessary for the upward-curvature component of the
growth pattern. It is also consistent with the upward curvature being
part of the root-coiling phenomenon, which also disappears when the
root is embedded in agar ( Rutherford and Masson, 1996), rather than
being a part of the gravitropic response.
To minimize the overall tactile stimulation, we also observed root
behavior while the roots were growing in a liquid medium or in a
chamber of humid air. The pattern of growth after clockwise stimulation
was similar to that of roots growing in solid agar, with no upward
curvature (E. Turk, unpublished data). To rule out light as a signal
for upward curvature, we compared the behavior of a root growing along
the agar surface after gravistimulation when the light source was in
front of the plate and when it was behind it. We also measured the
kinetics of gravitropism when the seedlings were exposed to no light
during the experiment (except IR radiation for imaging by the camera).
The upward bending pattern does not depend on the type of light
treatment provided (E. Turk, unpublished data). Circumnutation One model of the wavy growth phenomenon suggests that endogenous
oscillations in the growth direction, called circumnutation, drive the
pattern. Selective amplification of the pattern in an asymmetrical
tactile environment could then create a directional preference.
Therefore, we investigated roots growing vertically through a solid
volume of agar for periodic oscillations in the root-tip direction.
Roots of both wild type and rgr1 showed periodic
oscillations in tip angle, with similar periods of 11.9 ±
1.1 h (mean ± se; n = 10) and
11.0 ± 1.7 h (n = 5), respectively (Fig.
). Although the amplitude of the
circumnutation seems variable, there was no obvious difference in the
amplitude of the responses between rgr1 and wild-type
seedlings. Therefore, the magnitude of the circumnutation was not
dependent on the strength of the root's gravitropic response. There
was no long-term directional preference evident in roots growing
through agar.
This pattern of circumnutation in a uniform tactile environment (Fig.
) is very similar to the kinetics of the wavy growth pattern of a root
growing along the agar surface (Fig. ).
We measured the periodicity of this pattern as 11.1 ± 2.1 h
(n = 7) and 12.0 ± 2.2 h (n
= 5) for rgr1 and wild type, respectively, which is not
significantly different from the circumnutation period. There was,
however, a long-term directional preference for the root tip to stay to
the left of vertical in roots growing along the surface, which can be
seen by the shift in the angle of the axis of oscillations away from 0
(Fig. ).
To determine if the circumnutation behavior had any directional
preference, we used two perpendicular cameras to measure the
three-dimensional growth of rgr1 roots through solid agar.
Although both clockwise and counterclockwise growth was observed, most
root growth was in a counterclockwise direction, as viewed from above.
If there was no directional preference to circumnutation, we would
expect no mean curvature. However, the observed mean curvature was
1.2 ± 0.4 milliradians−1 (mean ±
se; n = 10) and 1.8 ± 0. 7
milliradians−1 (n = 7) for rgr1 and
wild type, respectively, a significant difference from the theoretical
mean (P < 0.05). Therefore, we found chirality to circumnutation,
even in a homogeneous tactile environment. These investigations of root behavior illustrate the complexities
in growth kinetics that can occur as a result of the interactions of
separate growth responses. Roots of rgr1 seedlings tended to
slant to the left when growing on vertical plates. This slanting
contributed to the observed gravitropism kinetics in a manner dependent
on the direction of rotation (Fig. ), suggesting that the relative
magnitudes of the early curvature responses in rgr1 and the
wild type may not have accurately reflected the difference in the
strengths of the true gravitropic response. This was made more evident
by the fact that the kinetics of the overall response of wild-type
roots were not dependent on the direction of gravistimulation.
Therefore, the magnitude of the gravitropic response in the wild type
varied with the direction of rotation and offset the slanting
preference of the root. The interaction between slanting and gravitropism also illustrates the
difficulty in calculating time lags based solely on changes in the
angle of the root tip. Because we were able to localize the initial
downward curvature to the region 200 to 300 μm from the tip, we could
calculate time lags based on changes in angle occurring in this region
only, excluding from consideration curvature arising farther back in
the root, where the slanting phenomenon arises. The region initiating
downward curvature corresponds to the location of the distal elongation
zone ( Mullen et al., 1998), showing that rgr1 roots have a
functional distal elongation zone. The upward curvature in
gravistimulated rgr1 roots occurred in the central
elongation zone, which is consistent with the location of major bending
in waving/coiling growth ( Okada and Shimura, 1990). The lack of upward curvature after clockwise gravistimulation of
rgr1 roots growing in more uniform tactile environments
(Fig. ) provides further evidence that the upward curvature is not a
true component of gravitropism, but is a part of the wavy growth
pattern. These results provide further evidence that an asymmetrical
tactile environment is necessary for the establishment of a directional
preference to the waving/coiling phenomenon. The similarity in the frequencies of the wavy growth pattern and the
circumnutation pattern of roots growing in uniform tactile environments
(Figs. and ) suggests that circumnutation may be the driving force
behind the induction of tactile stimulation. Differences in growth rate
also had no direct correlation with the period of the oscillations,
which is in agreement with previous observations ( Johnsson, 1979). The
frequency of waving remained the same despite differences in growth
rate between roots growing on the surface of agar and those growing in
the agar, or between rgr1 and wild-type roots growing in
agar (J.L. Mullen and E. Turk, unpublished data). An alternative to the model that circumnutation drives the waving
pattern is the idea that a series of gravitropic and thigmotropic
responses are sufficient to create the pattern. However, it seems
unlikely that this scenario could explain the similarity in the
frequency of the waving pattern in rgr1 and wild type, given
their difference in gravitropic response. It is also difficult to
explain the directional preference with such a model. Therefore, to
account for all aspects of the waving/coiling phenomenon, both
circumnutation and thigmostimulation are necessary components. In this
combined model of the waving/coiling phenomenon, it is circumnutation
that drives the pattern of wavy growth. Leftward slanting arose when
the counterclockwise chirality of circumnutation caused differential
thigmostimulation in an asymmetric tactile environment. Because of the
chirality, the root was growing to the left at the point of
greatest tactile stimulation. This thigmostimulation can manifest
itself as an alteration in circumnutational amplitude. Further
investigation into the nature of the three-dimensional circumnutation
process may aid in the understanding of the tactile forces involved.
The genetic analysis of mutants may also provide clues to the basis
of the chirality. For example, sku mutants of Arabidopsis
show increased magnitude of directional preference ( Rutherford and
Masson, 1996), and Marinelli et al. (1997) found mutants with inverted
chirality. Whether the growth pattern takes the form of waving or coiling is based
on whether the amount of bending in the clockwise direction during a
half-wavelength of the pattern is equal to the amount in a
counterclockwise direction during the successive half-wavelength. For
waving to occur, the sum of the gravitropic response and the
thigmoresponse must be the same during clockwise and counterclockwise
bending. Because the tactile stimulation is direction-dependent, the
gravitropic response must be modulated to maintain a constant overall
response, as seen for wild-type roots in Figure . The reduced
gravitropic response of rgr1 is unable to counter the
differential thigmoresponse, so the amount of clockwise curvature is
greater than the amount in the counterclockwise direction and the
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