Journal of Experimental Botany, Vol. 53, No. 369, pp. 683–687, April 2002
Positional effect of cell inactivation on root gravitropism
using heavy-ion microbeams
Atsushi Tanaka1, Yasuhiko Kobayashi, Yoshihiro Hase and Hiroshi Watanabe
Department of Radiation Research for Environment and Resources, Takasaki Radiation Chemistry Research
Establishment, Japan Atomic Energy Research Institute (JAERI), Watanuki-machi 1233, Takasaki, Gunma
370-1292, Japan
Received 30 June 2001; Accepted 7 November 2001
Abstract
When primary root apical tissues of Arabidopsis
thaliana were irradiated by heavy-ion microbeams
with 120 mm diameter, strong inhibition of root elongation and curvature were observed at the root tip.
Irradiation of the cells that become the lower part
of the root cap after gravistimulation showed strong
inhibition of root curvature, whereas irradiation of
the cells that become the upper part of the root cap
after gravistimulation did not show severe damage in
either root curvature or root growth. Further analysis
using smaller area microbeams with 40 mm diameter
indicated that the greatest inhibition of curvature
occurred at the root tip and the next greatest inhibition occurred in the cells in the lower part of the root
cap. These results indicate not only that the root
tip and columella cells are the most sensitive sites
for root gravity, but also that signalling of root gravity
would go through the lower part of the cap cells after
perception.
Key words: Arabidopsis, heavy ions, microbeam, root
gravitropism, signalling.
Introduction
Root gravitropism, although simple to define, is a
wondrously complex phenomenon in plants, as has been
revealed by physiological and genetical studies (reviewed
in Masson, 1995; Rosen et al., 1999). Many investigations
have proposed that amyloplast sedimentation in the
columella cells is the primary mechanism for gravity
sensing. Recent analyses using starch-deficient mutants
1
of Arabidopsis and unique experiments of flax using
high-gradient magnetic fields also indicate that amyloplasts are important for the induction of root curvature
(Kiss et al., 1997; Kuznetsov and Hasenstein, 1996).
Blancaflor et al. showed that the central cells of storey 2
in the columella cells contributed the most to root
gravitropism using laser ablation (Blancaflor et al., 1998).
On the other hand, the signalling pathway from gravity
sensing to physical change of cell elongation is still
unclear. Ca2q and a Ca2q gradient are probably involved,
and calmodulin may play an important role in its transduction to a local auxin release (Masson, 1995; Sinclair
et al., 1996). As a result, auxin redistribution at the root
tip and asymmetric distribution in the elongation zone
are thought to be the major signalling pathways in root
gravitropism. However, there is no distinct evidence that
auxin transport and auxin distribution across the elongation zone are necessary for root gravitropism in vivo
(Marchant et al., 1999).
To determine the signalling pathway within the root
apex, it is useful to block the function of cell(s) but not
physically to break these cells in order to clarify the cellto-cell interactions. Cell ablation (Day and Irish, 1997)
and UV laser (van den Berg et al., 1995, 1997) techniques
are powerful tools for understanding the fate and function of cells in an organ, but it is difficult to inactivate
a specified tissue or a certain group of cells without
disruption using these techniques.
It is well known that ionizing radiation causes cell
inactivation as a result of DNA damage, without causing
significant damage to the cytoplasm, cell membrane and
so on. Heavy ions have a high relative biological effectiveness (RBE) by means of efficiently producing DNA
double strand breaks and irreparable DNA damage
(reviewed by Blakely, 1992; Lett, 1992). Also in plants,
To whom correspondence should be addressed. Fax: q81 27 346-9688. E-mail: [email protected]
ß Society for Experimental Biology 2002
684
Tanaka et al.
the high efficiency of lethality (Tanaka et al., 1997),
chromosome aberration (Hase et al., 1999) and mutations
(Shikazono et al., 1998, 2001) has been shown. Recently,
a high-energy microbeam apparatus was constructed to
investigate the effect of local irradiation of heavy ions on
biological systems (Kobayashi et al., 2000).
In this study, microbeam irradiation was first used
for botanical research with Arabidopsis plants, and
the positional effects of cell inactivation caused by
microbeams on root gravitropism was analysed.
Materials and methods
Plant material and growth condition
Seedlings of Arabidopsis thaliana (L.) Heynh. ecotype Columbia
were used in this study. Seeds were surface-sterilized using 70%
ethanol for 1 min and 5% bleachu0.05% Tween 20 for 10 min,
and then rinsing in sterile, distilled water. The seeds were
planted on a nutritive agar plate (0.1% Hyponex, 1.5% agar)
and vertically incubated for 3 d under fluorescent lights
(c. 25 mmol m 2 s 1) at 23 8C.
For microbeam irradiation, 6–8 uniformly-grown plants were
carefully chosen from 30–40 plants incubated and were put on a
micro slide glass (76 3 26 mm), then they were dripped with a
few drops of nutrient-sterile water (0.1% Hyponex). Roots and
hypocotyls were covered with a small piece of microwave wrap
(Saran wrap, Asahi Chemical Co. Ltd, c. 50 3 20 mm) to prevent
drying of the seedlings.
Microbeam irradiation
The heavy-ion microbeam used in this study has been described
previously (Kobayashi et al., 1997). In this study, 220 MeV C5q
ions were used for microbeam irradiation. The physical
properties of 220 MeV C5q ions are as follows: incident
energy is 18.3 MeVuu, linear energy transfer (LET) in a root
is 110 keVumm as water equivalent, and the range of ions is
c. 1.0 mm. Before irradiation, the laser beam which is installed
upstream of the collimators, is used for the establishment of the
irradiation position. Plants on the glass plate were positioned
by micropositioning the X–Y stage, and then irradiated. The
intensity and the energy of the ion beams on the target micropositioning stage or after the beam has penetrated the target
are measured with a plastic scintillator, CR-39 track detector
and solid-state detector (SSD) in the air. During irradiation, the
target can be observed with an optical microscope system.
Measurement of growth and curvature
After irradiation, irradiated plants were removed from the
glass plate and incubated vertically on a fresh nutrient agarcontaining plastic plate. For a mock control, seedlings grown on
the same plate were also transferred to fresh medium without
microbeam irradiation. In order to measure the horizontal
and vertical length of root elongation after irradiation and
gravistimulation, plotting paper (10 3 5 mm) was put on top of
each root and the position of the root-tip end was checked.
Then, irradiated plants were gravistimulated by rotating 908
from the vertical for another 3 d in the dark to prevent
phototropic effects. The horizontal length and vertical length
of root elongation were measured under an optical microscope
with a micrometer eyepiece. Root curvature in degrees were
calculated as tan 1 (vertical lengthuhorizontal length).
Results
The root apical tissues were irradiated with 220 MeV
carbon-ion beams with diameters of 40, 120 and 250 mm,
and then gravistimulated by incubation with a 908
rotation (Fig. 1). Root apical tissues including the distal
elongation zone (i.e. the sites of gravitropic perception
and signalling) were irradiated with carbon ions in a
250 mm diameter beam to determine the dose needed to
inactivate these cells (Figs 1A, 2). Root growth was
completely inhibited by a dose of 100 Gy. The dose–
response curve of vertical root elongation showed a small
shoulder up to about 10 Gy, followed by an exponential
decrease until 75 Gy. On the other hand, the dose–
response curve of the horizontal root elongation has a
shoulder at around 30 Gy and an exponential decrease
until 100 Gy. Thus, the effect of ion beams on gravistimulated root growth was more effective on vertical
elongation than horizontal elongation. It is concluded
that 75 Gy is a suitable dose for detecting sites that are
sensitive to gravitropism.
Figure 3 and Table 1 show typical results of the
effect of irradiation with a 120 mm diameter microbeam.
Irradiation at position a (Fig. 1B), which was in the root
tip, strongly inhibited root elongation and curvature.
Root elongation and curvature were weakly inhibited by
irradiation at position d (which becomes the upper part
of the root cap and columella cells after gravistimulation),
whereas they were strongly inhibited by irradiation at
position b (which becomes the lower part after gravistimulation). Irradiation of position c often resulted in
upward root curvature (negative gravitropism) after
gravistimulation. Out of a total of 30 plants, nine showed
negative gravitropism. On the other hand, irradiation at
position e, which is thought to be in the distal elongation
(DE) zone, or at position f, which is thought to be in the
main elongation (ME) zone, had hardly any effect on
either root elongation or curvature (Fig. 3).
To identify more accurately the site that is responsive
for gravitropism, an additional experiment was conducted
with a finer (40 mm diameter) microbeam. The position
of the beam is shown in Fig. 1C and the results are shown
in Table 2. As was found with the 120 mm diameter beam,
the position that showed the greatest sensitivity to irradiation with respect to vertical elongation and curvature
was position a at the tip of the root. However, negative
gravitropism was not observed in the case of the 40 mm
diameter irradiation. On the other hand, vertical elongation and curvature were severely inhibited by irradiation
at position b, and moderately inhibited by irradiation
at position e. Irradiation at position c had no effect.
Irradiation at position d, that becomes the upper site after
Effect of microbeams on root gravitropism 685
Fig. 1. Irradiation positions used in this study. Vertically grown Arabidopsis primary roots were irradiated by microbeams with diameters of 250 mm
(A), 120 mm (B) and 40 mm (C). After irradiation, plants were gravistimulated by rotation at 908 from the vertical and incubated in the dark.
EZ, elongation zone; M, meristematic; RC, root cap; ME, main elongation zone; DE, distal elongation zone; QC, quiescent centre; C, columella.
Discussion
Fig. 2. Dose–response curves of vertical and horizontal root elongation
after microbeam irradiation. Apical root tissues were irradiated with
a 250 mm diameter beam (Fig. 1A), then gravistimulated by rotation at
908 and incubated in the dark. Data ("SE) are the average of 6–9 plants
(see Materials and methods).
gravistimulation, had a small effect compared with that at
position b. One of the central sites, position x, although it
contains most of the columella and meristem cells, is less
sensitive than position b, which becomes the lower side
after gravistimulation.
Of the areas tested, the root tip was the most sensitive
to microbeams with respect to both root growth and
curvature after gravistimulation. Several investigations
have shown that columella cells have a role in root gravity
sensing because they contain amyloplasts, which are the
primary machinery for the perception of gravity. This
study showed that the root tip area, especially the a or
a position in Fig. 2, is the most sensitive area for root
curvature. The a or a position consists of root cap cells
and outer columella cells. Blancaflor et al. showed by
means of the laser ablation technique that ablation of
root cap and tip cells did not alter root curvature, but
ablation of the two innermost columella stories (storeys
1 and 2) caused the strongest inhibitory effect without
affecting root growth rates (Blancaflor et al., 1998). These
differences from data of this study are likely to be as a
result of the number of different cell types being affected
in this study whereas specific cell types were ablated in the
Blancaflor et al. study. This concern is highlighted by the
results presented in Tables 1 and 2 where considerably
greater effects were seen in roots treated with 120 mm
beams compared to the 40 mm beams, such as position
a versus position a. It is plausible that the columella is
the most sensitive, and therefore, important tissue for
graviresponsiveness, but a number of additional cell types
might also be strongly related to root gravitropism.
After the tip cells, cells that become the lower side
after gravistimulation, such as the cells in the b position
686
Tanaka et al.
Fig. 3. Root bending after irradiation with a 120 mm diameter microbeam. Alphabets indicate the irradiation positions drawn in Fig. 1B.
Bar indicates 200 mm.
Table 1. Growth rates and curvature of Arabidopsis root (I)
Table 2. Growth rates and curvature of Arabidopsis root (II)
Values represent mean"SE of two independent experiments (nG12).
Values represent the mean"SE of results from between 8 and 14 plants.
Position in Fig. 2B
Position in Fig. 2C
Control
a
b
c
d
Percentage length
Vertical
Horizontal
100.0"10.9
5.3"2.2
25.8"6.9
3.3"13.2a
47.3"8.0
100.0"13.9
20.9"6.6
51.7"9.9
36.8"9.9
68.5"7.3
Curvature (degree)
56.5"4.5
7.5"3.2
29.0"7.0
4.9"12.8a
39.1"6.4
Percentage length
Vertical
Horizontal
100.0"11.3
41.9"9.6
47.6"6.7
74.2"6.5
70.4"7.1
62.8"10.9
68.5"10.9
100.9"10.3
100.0"15.1
86.6"15.4
75.8"16.2
102.5"13.8
75.8"12.1
96.4"16.0
82.5"20.1
103.4"15.8
Curvature (degree)
a
Vertical length and curvature of root bending toward upper part
against gravity was measured as a minus number.
Control
a
b
x
d
e
W
c
69.5"2.5
41.6"7.2
52.8"8.2
60.4"4.8
65.9"4.5
55.0"6.4
57.8"8.5
68.0"3.3
in Fig. 1B, were the next most sensitive site for root
gravitropism. Among this latter group of cells, irradiation
of the lower part of the quiescent centre and innermost
columella cells (the b position in Fig. 1C) caused the
strongest inhibition, whereas irradiation of the opposite
site (d position) caused little effect. This result suggests
that there is some signalling from the tip cells to the
elongation zone, and that the signal transduction occurs
in the lower part of the root apical tissues. Auxin
redistribution at the tip cells and asymmetric distribution
in the elongation zone are believed to be a main signalling
pathway, but it is still unknown how the graviperception
signal is transduced and transported to the elongation
zone (reviewed by Rosen et al., 1999). Based on the
present results, it is possible to hypothesize that auxin
or another signal is transported from the root tip to the
elongation zone through cells on the lower side after
gravistimulation, to become accumulated in the elongation zone and inhibiting cell elongation at the lower side
leading to root bending. However further work is
required to confirm this hypothesis.
Negative gravitropism was caused by irradiating
region c in Fig. 1B. The c position includes the a position, but irradiation of the a position only inhibited root
gravitropism, whereas irradiation of the c position caused
upward bending of the root. The root bending mechanism
may have been disrupted because irradiating the c position with a 120 mm diameter ion beam damaged not only
gravity perception (in the a position) but also signalling
in adjacent cells. Therefore, irradiation of only a part of
the c position with a 40 mm diameter microbeam would
not result in negative gravitropism.
As the predominant effect of ionizing radiation on
the cell is DNA damage, dividing cells are more sensitive
to microbeams than non-dividing cells. However, the
meristem regions such as the x or w positions in Fig. 1C
do not seem to be more sensitive to ionizing radiation
than the other regions. The most sensitive regions are
the root tip cells and the cells that become the lower part
after gravistimulation, such as the cells in the a, b and e
positions in Fig. 1C. These cells were not undergoing cell
Effect of microbeams on root gravitropism 687
division during the present experiments. Thus, the root
meristem is not important for root gravity sensing.
Acknowledgements
We thank Professor Shigemitsu Tano, Dr Yutaka Oono,
Dr Naoya Shikazono, and Dr Ayako Sakamoto for their
helpful comments and discussions. We are also grateful to the
staff of the Takasaki Ion Accelerators for Advanced Radiation
Application (TIARA) of the Japan Atomic Energy Research
Institute (JAERI) for their assistance with the heavy-ion
microbeam irradiation.
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