Journal of Experimental Botany, Vol. 53, No. 369, pp. 683-687,
April 1, 2002
© 2002 Oxford University Press
Original Papers |
Positional effect of cell inactivation on root gravitropism using heavy-ion microbeams
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
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Abstract |
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When primary root apical tissues of Arabidopsis thaliana were irradiated by heavy-ion microbeams with 120 µm 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 µm 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.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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 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. Ca2+ and a Ca2+ 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 cell-to-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, 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.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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% bleach/0.05% 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 µmol m-2 s-1) at 23 °C.
For microbeam irradiation, 6–8 uniformly-grown plants were carefully chosen from 30–40 plants incubated and were put on a micro slide glass (76x26 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. 50x20 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 C5+ ions were used for microbeam irradiation. The physical properties of 220 MeV C5+ ions are as follows: incident energy is 18.3 MeV/u, linear energy transfer (LET) in a root is 110 keV/µm 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 agar-containing 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 (10x5 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 90° 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 length/horizontal length).
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Results |
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The root apical tissues were irradiated with 220 MeV carbon-ion beams with diameters of 40, 120 and 250 µm, and then gravistimulated by incubation with a 90° 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 µm 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.
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Figure 3
and Table 1 show typical results of the effect of irradiation with a 120 µm 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).
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12).To identify more accurately the site that is responsive for gravitropism, an additional experiment was conducted with a finer (40 µm 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 µm diameter beam, the position that showed the greatest sensitivity to irradiation with respect to vertical elongation and curvature was position 
at the tip of the root. However, negative gravitropism was not observed in the case of the 40 µm diameter irradiation. On the other hand, vertical elongation and curvature were severely inhibited by irradiation at position ß, and moderately inhibited by irradiation at position 
. Irradiation at position 
had no effect. Irradiation at position 
, that becomes the upper site after gravistimulation, had a small effect compared with that at position ß. One of the central sites, position 
, although it contains most of the columella and meristem cells, is less sensitive than position ß, which becomes the lower side after gravistimulation.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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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
position in Fig. 2, is the most sensitive area for root curvature. The a or 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 µm beams compared to the 40 µm beams, such as position a versus position . 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 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 ß position in Fig. 1C) caused the strongest inhibition, whereas irradiation of the opposite site ( 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 µm 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 µm 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 or 
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 , ß and positions in Fig. 1C. These cells were not undergoing cell division during the present experiments. Thus, the root meristem is not important for root gravity sensing.
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Acknowledgments |
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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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Notes |
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1 To whom correspondence should be addressed. Fax: +8127346-9688. E-mail: atanaka{at}taka.jaeri.go.jp
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References |
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