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Plant Physiol, June 2000, Vol. 123, pp. 665-670
Kinetics of Constant Gravitropic Stimulus Responses in
Arabidopsis Roots Using a Feedback System1,2
Jack L.
Mullen,*
Chris
Wolverton,
Hideo
Ishikawa, and
Michael L.
Evans
Department of Plant Biology, The Ohio State University, Columbus,
Ohio 43210-1293
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ABSTRACT |
The study of gravitropism is hindered by the fact that as a root
responds, the gravitational stimulus changes. Using a feedback system
to connect a rotating stage platform to a video digitizer system, we
were able to maintain a constant angle of gravistimulation to
Arabidopsis roots for long time periods. The rate of curvature approximated the sine rule for angles of stimulation between 20° and
120°. For a given angle of stimulation, the rate of curvature also
remained constant, with no observed diminishment of the response. Although previous reports of Arabidopsis root gravitropism suggest latent periods of approximately 30 min, using a smooth mechanical stage
to reorient the root, we observed a mean time lag of approximately 10 min. This more rapid onset of curvature can, in part, be explained by
reduced mechanical perturbation during the process of gravistimulation. This suggests that mechanical stimulation associated with rapid root
re-orientation may confound investigations of early gravitropic events.
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INTRODUCTION |
Although the gravitropism of plant
roots has been studied for well over 100 years, its mechanisms are
still poorly understood. The use of Arabidopsis, with its plethora of
mutants showing altered gravity response, promises to aid in the
investigation of these mechanisms. Due to the small and fragile nature
of the Arabidopsis root, characterizations of mutants are often
performed with coarse temporal resolution, with the result that the
initiation of the response is unobserved (Hobbie and Estelle, 1995 ;
Kiss et al., 1996; Fukaki et al., 1997; Tian and Reed, 1999). Ishikawa
and Evans (1997) and Mullen et al. (1998b) detailed the kinetics of gravitropic curvature in horizontally stimulated Arabidopsis roots. These studies revealed a time lag between 20 and 45 min before curvature was initiated in the distal elongation zone (DEZ). Yet interpretation of data for later time periods is complicated by the
process of the graviresponse, which changes the stimulation angle at
the root cap, the probable site of gravity perception (for review, see
Sack, 1991). The response also creates a large difference in angle of
orientation along the elongating region of the root and may involve
adaptation to the gravity signal.
Sachs (1882) proposed that the gravitropic response was proportional to
the component of the gravity vector perpendicular to the root axis,
leading to the "sine rule" approximation of the dependence of
response on the stimulation angle. Although the rate of curvature has
been found to be related to the sine of the stimulation angle for roots
(Larsen, 1969; Perbal, 1974) and coleoptiles (Pickard, 1973; Iino et
al., 1996), a simple sine dependence only held for angles of
stimulation less than 90°, and the optimal angle of stimulation was
in some cases greater than 90°. Further, a recent study of maize
roots by Barlow et al. (1993) found no dependence of rate of curvature
on stimulation angle, for angles between 20° and 90°, the range in
which the sine rule approximation should be most valid.
Since prior studies of the relationship between stimulation angle
and response in roots have been equivocal, we have employed a new
technique to investigate this in the roots of Arabidopsis. We used a
computer feedback system to rotate a seedling growing on agar so that
its root tip was constrained to a particular angle from vertical. Thus
we were able to maintain a constant stimulus throughout the
graviresponse. This allowed us to evaluate the dependence of the
response on stimulus angle and to better understand the detailed
kinetics of the gravitropic response.
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RESULTS |
Gravitropism Kinetics
Although experiments measuring the angle of a root tip as a
function of time after gravistimulation provide useful measures of
graviresponse, the strength of the stimulus is time-dependent in such
cases. To better quantitate the kinetics of the graviresponse of
Arabidopsis roots, we used the feedback system to smoothly reorient the
roots from a vertical to a horizontal position. The tips of the roots
were then constrained at an angle of 90° relative to vertical (see
Fig. 1). The roots responded quickly to the gravitational stimulus and
achieved a constant rate of curvature within an hour of stimulation
(Fig. 2). Curvature continued
undiminished well beyond 90°, the amount of curvature that would be
needed for the root tip to attain vertical orientation if it was not
being constrained horizontally. Because the roots responded more
quickly than we expected based on previous studies (Ishikawa and Evans,
1997; Mullen et al., 1998b), we estimated the latent period for the response. To obtain an estimate of the latent period, we looked at the
time interval of  0.5 to 1.5 h (Fig.
3). A sixth-order polynomial was fit to
data in this interval by non-linear regression, and the second
derivative with respect to time was taken. The local maximum of the
resulting equation, which represents the time of greatest change in the
rate of curvature, was used as the time of commencement of the
response. Using this method, we calculated a latent period of 10.5 ± 2.4 min (mean ± SE, n = 12). To
better compare this estimate of the latent period with previous studies, we repeated the experiment, but we gravistimulated the root by
removing the Petri dish from the vertical stage, manually rotating the
root to a horizontal position, and reattaching the dish to the stage
(Fig. 3). The average latent period for manually stimulated roots
was 19.6 ± 2.6 min (n = 9), a significant
difference (P = 0.02, t test). Thus, it
seems that the feedback system provided a reduction in mechanical
stresses, which allowed a quicker response.
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Figure 1.
Feedback system for constraining root
orientation. A, After an image of the root is captured, the image is
divided into segments, which are a fixed distance from the root tip.
The angles of orientation for the segments are then determined and
compared with the desired angle of constraint. The vertical stage is
then rotated by the amount necessary to achieve the desired angle of
orientation. B, Time-lapse images of an Arabidopsis root showing the
development of gravitropic curvature while the root tip remains
horizontal.
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Figure 2.
Kinetics of the gravitropic response of an
Arabidopsis root. The root tip was constrained at 90° relative to
vertical at 0 h. The measured rotation was the rotation of the
vertical stage necessary to keep the root tip horizontal.
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Figure 3.
Early time-course for the gravitropic response of
typical Arabidopsis roots. The stage-rotated root was reoriented to a
horizontal position by means of the rotating vertical stage. The
manually rotated root was removed from the stage, reoriented by hand,
and reattached to the vertical stage. The fitted lines are sixth-order
polynomials from which latent periods were calculated
(r2 = 0.995 and 0.976 for the
stage-rotated and manually-rotated samples, respectively).
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Dependence on Stimulation Angle
Because the curvature of the root as a function of time is linear
over a long time interval (Fig. 2), the average rate of curvature could
be determined by linear regression. This value is a useful measure of
the strength of the gravitropic response for a given angle of
stimulation. To find the relation between the strength of the response
and the dose of the gravity signal perpendicular to the root axis, we
measured the mean rate of curvature for roots with tips constrained at
different angles of stimulation. The rate of curvature increased with
angle of stimulation for angles less than 90° (Fig.
4). The relation can be approximated by a
sinusoidal function of the form:
where y is the rate of curvature,
x is the stimulation angle, and a,  , and
yo are parameters calculated by non-linear
regression to be 15.9, 14.7, and 7.9, respectively. A physical
interpretation of this equation is that the threshold angle for
gravitropic response is 15°, since at this angle the sinusoidal term
of the function is nil.
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Figure 4.
The dependence of the rate of root curvature on
the stimulation angle. Error bars indicate SE
(n = 10-12). The curve is a sinusoidal function fitted
to the data by non-linear regression.
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Threshold Angle for Gravitropic Response
Since the previous data suggested that roots must be reoriented at
least 15° from vertical before a response is elicited, we made
measurements of gravitropic responses at low stimulation angles to test
this prediction. Because the gravitropic response is characteristic in
its rapid onset and constant rate of curvature (Fig. 2), we were able
to measure the proportion of roots, at a given angle of constraint,
which responded to the gravitational stimulus. Figure
5 shows the proportion of roots
responding as a function of stimulation angle. The angle at which 50%
of the population responds gravitropically is approximately 15°, in
good agreement with the prediction from the rate of curvature data.
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Figure 5.
Threshold of gravitropic response in Arabidopsis
roots. Roots were scored as responders if the root began curving within
an hour of stimulation and continued curving throughout the course of
the experiment (n = 10).
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Localized Contributions
Following gravistimulation, a root enters a phase of curvature
development; but, after several hours, curvature in the elongating region, both within the apical region of the elongation zone (240-480 µm from the tip) and the middle of the central elongation zone (CEZ,
480-720 µm from the tip) reaches a steady state (Fig.
6, segments 2 and 3, respectively). This
steady state arises from a balance between continuing curvature
formation and the migration of curvature out of the elongation zone as
the root grows. A comparison of the deviation of steady-state angles
for different regions of the elongation zone from the angle of the root
tip can be used as an index of relative contributions to the
gravitropic response. Comparing the deviation of the apical region of
the elongation zone to that of the middle of the CEZ (for elongation
zone analysis, see Mullen et al., 1998a), we found that the apical
region contributed the majority of the curvature when the root was
reoriented to large angles (Fig. 7).
However, at smaller angles of stimulation, the more basal region of the
elongation zone had significantly greater importance in the response
than it had at larger stimulation angles (P = 0.01).
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Figure 6.
Localized changes in Arabidopsis root orientation
upon gravistimulation. The data show the angles of orientation of
different regions of a representative root when the tip-most segment is
constrained at 120°. Inset is an image of the root, illustrating the
division of the apical region into segments, numbered 1 to 3, corresponding to the angle data.
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Figure 7.
Relative local contributions to gravitropic
curvature. The deviations in the orientation of the root tip from the
central portion of the elongation zone and from the apical region
of the elongation zone are compared to provide an index of the
relative contributions of these regions to the gravitropic curvature.
The ratio of these deviations was determined both for low angles of
stimulation (n = 10) and high angles (n = 15). Error bars indicate SE. A large ratio
indicates a relatively greater contribution to curvature by the central
portion of the elongation zone, whereas a small ratio indicates a
relatively greater contribution by the apical region of the elongation
zone.
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DISCUSSION |
By measuring the kinetics of Arabidopsis root gravitropism with a
feedback system, we observed a rapid onset of curvature, averaging
approximately 10 min. Therefore, the perceived stimulus must be
transduced to a response in the DEZ and CEZ more rapidly than
previously expected. The sedimentation time of amyloplasts has been
measured to be approximately 5 min (MacCleery and Kiss, 1999). The
presentation time for Arabidopsis roots has been estimated to be
between 20 s and 5 min (Kiss et al., 1989, 1996). It has previously been suggested that harsh gravistimulations may affect the
sensing process (Barlow et al., 1993). Although we did not attempt to
measure presentation time using the rotating stage device, we expect
that any changes in the presentation time will be reflected in the
shorter latent period. Although potential electrical (Ishikawa and
Evans, 1990; Weisenseel et al., 1992) and chemical (Perera et al.,
1999) signals have been detected within this time interval, caution
must be exercised when assessing components of the early phases of the
gravitropic response. The manner in which roots were gravistimulated
affected the observed latent period. Roots gravistimulated smoothly by
the feedback system exhibited a reduced gravitropic latent period. This
suggests that mechanical stress associated with rapid reorientation may alter the response and could confound the identification of true gravitropic signal transduction components.
Once the roots commenced bending, the rate of curvature remained
constant over long time periods. This rate of curvature varied with the
angle of stimulation and followed a sinusoidal dependence with a phase
shift of 15°. This contrasts with observations in maize roots (Barlow
et al., 1993), which found no relation between the rate of curvature
and stimulation angle. These results also differ from older reports
(Audus, 1964; Perbal, 1974) suggesting a "modified sine rule" with
maximal curvature at angles of 120° to 135°. However, we were able
to make observations at a constant root tip orientation for long
periods of time. Since the root cap is the major site of perception of
the gravity signal (Sack, 1991; Blancaflor et al., 1998), allowing
changes in the orientation of the root tip during gravitropic response
can confound results. It seems likely that the large curvature observed
at high angles of stimulation in other studies (Audus, 1964) was caused
by the root tip spending more time oriented at large angles rather than a larger magnitude response at these high angles.
The changes in the magnitude of the gravitropic response with
stimulation angle were accompanied by changes in the location of the
response (Fig. 6). At small reorientation angles, much of the curvature
occurred in the CEZ. However, as the stimulation angle increased,
differential growth in the DEZ and the apical portion of the CEZ
increased more than in the central CEZ, shifting the region of greatest
curvature acropetally. This type of response is consistent with a model
of gravitropism in which the signal originates in the root cap and
dampens with distance from the cap only if one assumes that the central
CEZ is more sensitive to the signal than the more apical region
of the elongation zone. To accommodate the observation that the region
of maximal curvature shifts acropetally when the root is stimulated at
higher angles (Fig. 7), one may speculate that the apical region,
because of its proximity to the signal source and the higher strength
of the signal at higher stimulation angles, now receives a signal sufficiently above its response threshold to account for a large contribution to overall curvature. Alternatively, the apical region of
the elongation zone might be capable of directly perceiving a
gravistimulus for sufficiently large angles of stimulation, though this
is a model which is difficult to reconcile with the observation that
the gravitropic response is lost when the columella cells of the root
cap are destroyed (Blancaflor et al., 1998).
This pattern of response is also consistent with the observed
similarity between circumnutation and low-angle gravistimulation (Barlow et al., 1993) since circumnutation primarily occurs in the CEZ
(Okada and Shimura, 1990; Mullen et al., 1998b). Study of the growth
patterns of roots constrained to angles below the gravitropic threshold
may shed light on the relationship between circumnutation and
gravitropism. The ability of the feedback system to smoothly rotate the
seedling in order to maintain a constant angle of orientation for a
specific region of the root, such as the root cap, provides a means for
measuring curvature development while keeping the gravitropic stimulus
constant. This ability should prove useful in understanding the
influence of gravity on other growth responses.
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MATERIALS AND METHODS |
Plant Material
Seeds of Arabidopsis (ecotype Columbia) were surface-sterilized
by agitation in 5.25% (v/v) NaOCl solution for 5 min, followed by
several rinses in sterile distilled water. Seeds were sown in a row
(three seeds per row) on a sterile agar (1% w/v) medium in Petri
dishes (60-mm diameter, 15 mm high) sealed with Parafilm (American Can
Co., Greenwich, CT). The agar medium contained 1% (w/v) Suc,
one-half-strength Murashige-Skoog medium (Murashige and Skoog, 1962),
and 1 mM 2-(N-morpholino) ethanesulfonic
acid (pH 5.8). The Petri dishes were either immediately placed
vertically in a culture room under continuous white light from
fluorescent lamps (Sylvania, Danvers, MA; F30T8-CW, fluence rate
approximately 60 µm m 2 s1) at 24°C or
refrigerated for 1 to 5 d before being transferred to the culture
room. The seedlings were used for experimentation at age 4 to 5 d,
when the roots had a length of 10 to 20 mm.
Feedback System
The seedling to be observed was first repositioned so that its
root tip was at the center of the Petri dish (center of rotation). This
minimized the translational movement of the root during the stimulation
process. Repositioning was accomplished by placing forceps under the
hypocotyl, lifting slightly, and sliding the plant along the agar. The
dish containing the seedling was then attached to a vertical stage, and
the seedling was allowed to recover for at least 1.5 h, during
which time the growth rate of the root returned to normal (J.L. Mullen
and C. Wolverton, unpublished data). The feedback system was then
started, so that the root tip was constrained at the desired angle
relative to vertical. The feedback system consisted of a CCD camera
(Marshall Electronics, Culver City, CA) focused on a seedling root,
illuminated by an infrared light-emitting diode (Radio Shack, Fort
Worth, TX). The camera was connected to a computer via a frame grabber circuit board (Imagenation, Beaverton, OR). A rotatable vertical stage
(Optec Ltd., Tokyo) was also connected to the computer and controlled
by custom software. This software utilized the algorithm described by
Mullen et al. (1998b) to calculate the angle of the root tip, as well
as that of other root segments. The software constrained the
user-defined segment of the root to the desired angle, making
corrections as frequently as every 20 s. Changes in the angle of
the constrained segment caused a stepper motor to rotate the vertical
stage in the necessary direction, correcting the angle of that segment
(Fig. 1). The custom software recorded the angle of the root segments and the rotation of the vertical stage.
The root could be reoriented to a horizontal position (90°) within
approximately 60 s by means of individual steps of the motor
corresponding to 0.08°.
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FOOTNOTES |
Received October 22, 1999; accepted February 9, 2000.
1
This work was supported by the National
Aeronautics and Space Administration (NASA; grant nos. NAG5-6385 and
NAG2-1190), by the NASA/National Science Foundation Joint Program in
Plant Biology, Network for Research on Plant Sensory Systems (grant no.
IBN-9421856), and by the Institute of Space and Astronautical Science (Japan).
2
This paper is dedicated to the memory of Paul B. Green
whose exceptional creativity in the design of novel equipment for plant growth studies and whose exemplary approaches to quantitative plant
biology have been an inspiration to the authors.
*
Corresponding author; e-mail mullen.37{at}osu.edu; fax
614-292-6345.
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TOP
ABSTRACT
INTRODUCTION