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chanical stimulation in fern protonemal cells. Here we report the discovery of a mechanically induced accu- mulation response of chloroplasts in bryophytes.
Planta (2003) 216: 772–777 DOI 10.1007/s00425-002-0927-x

O R I GI N A L A R T IC L E

Yoshikatsu Sato Æ Masamitsu Wada Æ Akeo Kadota

Accumulation response of chloroplasts induced by mechanical stimulation in bryophyte cells

Received: 20 June 2002 / Accepted: 7 October 2002 / Published online: 30 November 2002 Ó Springer-Verlag 2002

Abstract Chloroplast movement has been studied in many plants but mainly as a model system for light signaling. However, we recently showed that the avoidance response of chloroplasts is also induced by mechanical stimulation in fern protonemal cells. Here we report the discovery of a mechanically induced accumulation response of chloroplasts in bryophytes. When mechanical stimulation was directly applied with a capillary to a part of a cell, chloroplasts moved towards and accumulated at the pressed site within 30 min after the onset of stimulation in all species tested. The accumulation movement of chloroplasts was inhibited by Cremart but not by cytochalasin B in red-light-grown protonemata of Physcomitrella patens (Hedw.) B., S. & G. To determine the contribution of external Ca2+ to the response, we examined the effects on the accumulation movement of gadolinium (Ga3+), an inhibitor of stretch-activated ion channels, and lanthanum (La3+), a potent inhibitor of calcium channels. Mechano-relocation of chloroplasts was abolished by these drugs, but no effects were observed on photo-relocation of chloroplasts, irrespective of light colors and intensity. These results suggest that influx of external Ca2+ through the plasma membrane is essential for the early steps in signaling of mechano-relocation of chloroplasts whose motility system is dependent on microtubules.

Y. Sato Æ M. Wada Æ A. Kadota (&) Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-Osawa 1-1, Hachioji, Tokyo 192-0397, Japan E-mail: [email protected] Fax: +81-426-772559 M. Wada Division of Biological Regulation and Photobiology, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan Present address: Y. Sato Division of Speciation Mechanisms 2, National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan

Keywords Chloroplast movement Æ Mechanical stimulation Æ Light Æ Microtubule Æ Actin filament Æ Physcomitrella Abbreviations BL: blue light. Æ [Ca2+]c: cytosolic Ca2+ concentration. Æ RL: red light

Introduction Precise control of organelle positioning is important for plant responses at the cellular level to environmental stresses such as light or mechanical stimulation (Nagai 1993; Wada et al. 1993; Williamson 1993). Light induction of changes in the intracellular distribution of chloroplasts is a well-known phenomenon, seen across the plant kingdom, that was first comprehensively described by Senn (1908). Depending upon the fluence intensity of the effective wavelength, chloroplasts move towards or away from the illuminated site to the best position for achieving optimum photosynthetic light harvesting. In the fern Adiantum capillus-veneris and the moss Physcomitrella patens, red light (RL) and blue light (BL) are effective wavelengths for induction of chloroplast photo-relocation. These responses are mediated by phytochrome and a BL receptor, respectively (Yatsuhashi et al. 1985; Kadota et al. 2000). On the other hand, we recently discovered that mechanical stimulation also induces the directional movement of chloroplasts in fern protonemal cells, namely, the chloroplast avoidance response away from a stimulation site (Sato et al. 1999). This chloroplast mechano-relocation movement is an excellent experimental system for elucidation of mechano-perception and mechano-transduction at the cellular level because the stimulation site is the mechano-perception site and directional movement is induced relative to this point. However, it is not known whether chloroplast mechano-relocation movement occurs in other plant cells. Bryophytes, including the mosses Ceratodon purpureus and Physcomitrella patens, and a liverwort,

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Marchantia polymorpha, have been used as model systems for studying plant genetics and development because of their simple anatomy (Dyer and Duckett 1984). In these bryophytes, the structure of RL-grown protonemata, which do not form side branches, is similar to that of fern cells (Kadota et al. 2000). We can readily apply mechanical stimulation to part of an intact protonemal cell on a microscope stage and directly observe movement of individual chloroplasts over time. In this study, we examined mechano-induced chloroplast relocation in bryophyte protonemal cells. We found that they indeed exhibit mechano-induced chloroplast relocation movement but, surprisingly, the direction of movement with respect to the site of stimulus was the exact reverse of that seen in fern protonemata. The mechanisms of the chloroplast mechano-stimulated relocation, the accumulation response in bryophytes and the avoidance response in pteridophytes are discussed.

Materials and methods Plant material and aseptic culture Protonemata of Physcomitrella patens (Hedw.) B., S. & G. were cultured on BCD medium using the procedure previously described by Sato et al. (2001a), unless otherwise stated. Ceratodon purpureus (Hedw.) Brid. (wild-type strain wt4) was cultured on the following medium: 10 mM KNO3, 0.1 mM CaCl2, 50 lM MgSO4, 1 mM KH2PO4, 8 lM Fe-citrate, 10 mM glucose, 0.5% agar, pH 5.2 (Kagawa et al. 1997). Protonemata of Marchantia polymorpha L. were raised from the spores, which were collected on the campus of Tokyo Metropolitan University (Hachioji, Tokyo) in the summer of 1994 (Nakazato et al. 1999). The same culture medium was used as for C. purpureus. All three species were cultured for 1–2 weeks under continuous RL of about 0.5 W m–2, which was provided by a fluorescent lamp (FL40SD, Toshiba Lighting and Technology Corp., Tokyo, Japan) with a red plastic filter (Shinkolite A, #102; Mitsubishi Rayon Co., Tokyo, Japan). Some experiments were performed using darkadapted protonemata where the RL-grown cells were incubated in the dark for 1 day. Mechanical and light stimulation For mechanical stimulation, each cell was pushed with a capillary until deformation of the cell was observed under the microscope, as was previously done for fern protonemal cells (Sato et al. 1999). For light stimulation, microbeam irradiation was delivered using a custom-made microbeam irradiator (Kadota et al. 2000). Quantitation of the mechano- and photo-relocation of chloroplasts The percentage of chloroplasts that had moved towards or away from the stimulation site was determined to quantitate mechanoand photo-relocation movement of chloroplasts. For quantitation of accumulation responses induced by mechanical and light stimulation, the number of chloroplasts in the area 20–40 lm from the stimulation site was counted before and after stimulation. The percentage of chloroplasts that had moved more than 10 lm towards the stimulation site within 1 h after stimulation was calculated. The avoidance movement induced by strong light stimulation was also quantitated by counting the number of chloroplasts in the irradiation site (20 lm in length) before and 1 h after stimulation.

Inhibitor treatments To determine the nature of the motility system for mechanoaccumulation movement, we used cytochalasin B and Cremart as depolymerization drugs for actin filaments and microtubules, respectively (Sato et al. 2001a). For analysis of the role of external Ca2+ on mechano- and photo-relocation of chloroplasts, lanthanum chloride was used as a calcium-influx inhibitor and gadolinium chloride as a stretch-activated-channel blocker (both were obtained from Sigma, St. Louis, Mo., USA). Calcium-channel blockers were used in 10 mM Mes buffer with 1 mM CaCl2 and 10 mM KCl because in the growth medium, gadolinium (Gd3+) easily forms complexes with other ions, resulting in a white precipitate. Cells were pretreated with each drug for 1 h prior to mechanical or light stimulation. All the experiments described here were repeated at least 3 times on different occasions, and the same results were obtained each time.

Results and discussion The direction of chloroplast movement with respect to the mechanical stimulation site in bryophytes (mosses and liverworts) is the opposite of that in pteridophytes In dark-adapted protonemal cells of the moss P. patens, chloroplasts are normally distributed randomly around the cell periphery. When mechanical stimulation was continuously applied on apposing sides of a cylindrical cell using bent capillaries, an accumulation response of chloroplasts was clearly observed within 30 min after the onset of stimulation (Fig. 1). In the previous study, we reported that the same mechanical treatment in fern protonemata stimulated movement in the opposite direction; that is, the chloroplasts moved away from the stimulus site instead of towards it (Sato et al. 1999, 2001b). The difference in direction of movement between ferns and the moss P. patens is not due to the composition of the medium used, because the direction of movement was unchanged even when the responses were examined in the same growth medium as used for fern cells. Furthermore, the mechano-accumulation response also occurred in the protonemata of another moss, Ceratodon purpureus, and of a liverwort Marchantia polymorpha with similar time course to that in P. patens, suggesting that this phenomenon is general in bryophytes. This is the first demonstration of mechanostimulated accumulation movement of chloroplasts in these plants. Subsequent analyses were performed using P. patens because chloroplast photo-relocation in this species had been examined in detail in earlier studies (Kadota et al. 2000; Sato et al. 2001a). Cytoskeletal tracks responsible for mechano-relocation in the moss P. patens are different from those in the fern A. capillus-veneris Experiments were performed using protonemal cells of P. patens grown under RL without subsequent dark incubation. As a consequence of RL-induced photo-

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1992; Sato et al. 1999) while they travel along actin filaments for slow movement and along microtubules for rapid movement in P. patens (Sato et al. 2001a). To determine which tracks are responsible for mechanoaccumulation of chloroplasts, we examined the effects of cytoskeletal drugs. We used 0.1 mM cytochalasin B and 10 lM Cremart, which are sufficient concentrations to induce depolymerization of actin filaments or microtubules within 1 h, respectively (Sato et al. 2001a). Mechanical stimulation was applied to cells that had been pre-incubated in each drug for 1 h. The accumulation response was completely inhibited by 10 lM Cremart, but not by 0.1 mM cytochalasin B (Fig. 2B, C). These results suggest that mechano-accumulation movement of chloroplasts in P. patens is dependent on the microtubule system. This conclusion is also supported by the chloroplast velocity during mechano-relocation estimated from the slope in Fig. 2B (ca. 2.5 lm min–1), which is closer to that of a microtubule-based photomovement (ca. 2.4 lm min–1) than that of an actin-based movement (ca. 0.5 lm min–1) in the BL response (Sato et al. 2001a). The different usage of cytoskeletal tracks could be linked to the opposing directional movement of chloroplasts in fern and moss. External Ca2+ is essential for chloroplast mechanorelocation but not for photo-relocation

Fig. 1 Mechano-accumulation response of chloroplasts in protonemal cells of bryophytes. Mechanical stimulation was applied by bent capillaries or a capillary with a rounded tip designed not to penetrate a cell. Bar = 20 lm

relocation movement, most chloroplasts gathered at the septa, which divide adjacent cells, facing the incident RL (Fig. 2A). When mechanical stimulation was applied to the center of a cylindrical cell, we could attract chloroplasts to the stimulated site from both sides of the cell (Fig. 2A). Time courses of the movement show that chloroplasts accumulated at the stimulation site within 30 min after the onset of the stimulation (Fig. 2B). This type of response was hardly observed in cells that had not received mechanical stimulation (Fig. 2C). The time required to reach a maximum level of this accumulation response (30 min) is apparently shorter than that of the avoidance response in the fern A. capillus-veneris (1–2 h; Sato et al. 1999), suggesting that different motility systems are responsible for these opposing movements. Indeed, chloroplast motility was found to be regulated differently in these plants: chloroplasts only move along actin filaments in A. capillus-veneris (Kadota and Wada

It is well known that in many organisms the direct mechanical deformation of a cell causes changes in the ion permeability of the plasma membrane via stretch-activated ion channels (Morris 1990). In the previous study, we showed that the mechano-avoidance response in fern required Ca2+ influx through the plasma membrane (Sato et al. 2001b). Here, we examined the contribution of external Ca2+ to mechano-accumulation movement in a moss. At first, we tested the effect of external Ca2+ by changing the concentration of CaCl2 in the bathing medium. Even the omission of Ca2+ from the medium had little effect on the mechano-relocation of chloroplasts (data not shown). However, the mechano-accumulation response was abolished when cells were treated with 1 mM lanthanum (La3+), a potent inhibitor of Ca2+ influx through the plasma membrane, or 100 lM gadolinium (Gd3+), an inhibitor of stretch-activated channels (Fig. 3A). In contrast, when we investigated the effects of these inhibitors on chloroplast photorelocation movement induced by partial irradiation of a cell, neither photo-accumulation nor the photo-avoidance response was affected at all. We confirmed these observations in the same cells by sequential application of mechano- and light-stimulation (Fig. 3B). Cells in which chloroplasts were prevented from moving towards the mechanical stimulation site by 100 lM Gd3+ were irradiated with low- and high-intensity BL or RL. Both the accumulation and avoidance responses, i.e. chloroplast movement towards or away from the light spot, were clearly induced regardless of wavelength of light

775 Fig. 2A–C Effects of cytoskeletal inhibitors on the mechanoaccumulation response of chloroplasts in protonemal cells of P. patens. A Representative images of cells before and after mechanical stimulation for 1 h. Bar = 20 lm B Time course of chloroplast accumulation movement in each drug-treated cell shown in A. C Quantitative analysis of the accumulation response under each drug treatment, showing the number of chloroplasts that had moved more than 10 lm towards the stimulation site within 1 h after stimulation. Each bar represents the mean ± SE

used. Essentially the same responses were obtained from cells treated with 1 mM La3+ (data not shown). These results strongly suggest that external Ca2+ is a major determinant of chloroplast mechano-relocation but not photo-relocation. This study also means that influx of external Ca2+ is commonly essential as a mechanospecific signaling for the regulation of chloroplast movement, although downstream of the signal, the system for chloroplast movement in the fern A. capillusveneris is different from that in the moss P. patens. The failure of omission of CaCl2 from the medium to block the response would be explained if the Ca2+ pooled in the cell wall were sufficient to act as the external source for the induction of the response (Russell et al. 1998). Elevation of the cytosolic Ca2+ concentration, [Ca2+]c, induced by mechanical and light stimulation has been studied in P. patens transformed with an aequorin gene (Haley et al. 1995; Russell et al. 1998). Haley et al. (1995) concluded from their inhibitor studies that internal Ca2+ stores rather than extracellular Ca2+ make the major contribution to the mechano-induced elevation of [Ca2+]c. However, their data also show that [Ca2+]c elevation is reduced to a small degree by the drugs that inhibit entry of external Ca2+. Thus, even the influx of external Ca2+ makes a small contribution to

the total increase in [Ca2+]c, it is likely that the limited entry of Ca2+ from external pools is crucial in mechanosignaling for regulation of chloroplast movement. On the other hand, the BL-induced increase in [Ca2+]c was shown to be completely abolished by La3+ at a concentration which failed to affect chloroplast photorelocation movement in this study (Russell et al. 1998). These data may indicate that a transient increase in [Ca2+]c transmits the BL signal for phenomena other than chloroplast photo-relocation. Alternatively, it is possible that the sensitivity of the detection system for [Ca2+]c is not sufficient to observe the small change in [Ca2+]c required for photo-relocation movement. In conclusion, we have clearly shown the occurrence of chloroplast mechano-accumulation movement in bryophyte protonemata. In P. patens, localized deformation of the cell is suggested to induce influx of Ca2+ through stretch-activated channels, leading to alterations in microtubule-dependent chloroplast movement. Differences in mechano-response between P. patens and A. capillus-veneris are summarized in Fig. 4. In the previous study, we speculated that the avoidance response in pteridophytes is conducted for shade avoidance of chloroplasts, as mechanical stress in natural environment usually means the ‘‘shading’’ of a cell by an

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Fig. 4 Scheme for the mechanism of chloroplast mechano-relocation in P. patens and A. capillus-veneris. External Ca2+ is commonly essential for chloroplast mechano-relocation in both plants. However, chloroplasts move towards the mechanically stimulated site using microtubule (MT) tracks in P. patens whearas they move away from the site using actin microfilaments (MFs) in A. capillus-veneris

(Puchta 1998; Reski 1998, 1999), provides an opportunity to analyze the molecular mechanism of mechanoperception and mechano-transduction in plants at the cellular level. The significant differences in the role of the cytoskeleton and Ca2+ between the mechano- and lightsignaling pathways described here could be crucial in elucidating the transduction of mechanical and light stimuli.

Fig. 3A, B Effects of Gd3+ and La3+ on mechano- and photorelocation of chloroplasts of P. patens. A Quantitative analyses of mechano- and photo-relocation of chloroplasts. Chloroplast photo-relocation was induced by irradiation with polarized light. Mec Mechanical stimulation, LRL low fluence rate of RL (1 W m–2), LBL low fluence rate of BL (1 W m–2), HRL high fluence rate of RL (10 W m–2), HBL high fluence rate of BL (100 W m–2). Each bar represents the mean ± SE. B Effects of Gd3+ on mechano- and photo-relocation. After treatment with 100 lM Gd3+, which prevented mechano-relocation, the same cell was sequentially irradiated as indicated. Note that photo-relocation movements can be seen in cells that failed to show mechano-relocation. Beam size = 20 lm

Acknowledgements We are grateful to Dr. Jane Silverthorne (University of California, Santa Cruz) for critical reading of the manuscript. We thank Prof. E. Hartmann and Dr. T. Lamparter of Berlin Free University for the gift of the wild-type wt4 line of Ceratodon purpureus. This work was carried out under the NIBB Cooperative Research Program (1-120) and was partly supported by Grant-in-Aid for Scientific Research (C) (Grant No 13640655) to A.K. from the Japan Society for the Promotion of Science; Grant-in-Aid for Scientific Research (B) (Grant No 09440270) and PROBRAIN (Program for Promotion of Basic Research Activities for Innovative Biosciences) to M.W.; and also by a grant from Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (Grant No 12740202) and the Sasagawa Scientific Research Grant from the Japan Science Society to Y.S.

References object (Sato et al. 1999). This explanation may not be appropriate in bryophytes. A coherent explanation for the physiological and ecological meaning of chloroplast mechano-relocation movement has not so far been given. However, the opposite directionality of chloroplast mechano-relocation in pteridophytes and bryophytes could be ascribed to a different choice of cytoskeletal tracks for movement. Further analyses, such as measurement of [Ca2+]c changes after mechanical stimulation, are urgently required to discover the mechanism by which the direction of chloroplast movement is determined. The discovery of a new physiological phenomenon in response to mechanical stimulation in P. patens, where powerful genetic and molecular approaches are available

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