Plant Cell Physiol. 42(4): 358–365 (2001) JSPP © 2001 Mechanosensitive Ca2+ Release from Intracellular Stores in Nitella flexilis

Munehiro Kikuyama 1, 3 and Masashi Tazawa 2, 4 1 Biological Laboratory, The University of the Air, 2-11 Wakaba, Mihama-ku, Chiba, 261-8586 Japan 2 Department of Applied Physics and Chemistry, Fukui University of Technology, Gakuen, Fukui, 910-8505 Japan

; We found previously that the cytoplasmic drop isolated cytoplasm that had been squeezed out from an internodal cell from internodal cells of Nitella flexilis releases Ca2+ in of N. flexilis was mixed with a hypotonic solution (Kikuyama response to hypotonic treatment and named the phenome- et al. 1995). Since HICR in the cytoplasmic drop was greatly 2+ non hydration-induced Ca release (HICR). The HICR is inhibited by a water channel inhibitor HgCl2, water channels in assumed to be a result of activation of Ca2+ permeable chan- the membrane of Ca2+ stores are assumed to be involved in nels in the membrane of Ca2+ stores in a stretch-activated HICR (Kikuyama and Tazawa 1998). Thus, a possible mecha- manner. To prove this idea, mechanical stimulus was nism of HICR may be as follows. Rapid hydration of the cyto- applied to the drop by means of shooting isotonic/hypnotic plasm lowers the osmotic pressure of the cytoplasm, which medium or silicon oil into the drop, or compressing the then results in osmotic expansion of putative Ca2+ stores. drop. All these mechanical stimuli induced a rapid increase Osmotic expansion of the stores necessarily stretches the mem- in the Ca2+ concentration of the drop. The chloroplast frac- brane and causes efflux of Ca2+ from the stores. If this is the tion isolated from the cytoplasmic drop released Ca2+ on case, the ion channel in the membrane of the stores would be a compression, while the chloroplast-free cytoplasm did not. stretch-activated one. Suppression of HICR by HgCl2 can be In Chara corallina, the cytoplasmic drop, which shows a accounted for in terms of inhibition of osmotic expansion of very weak HICR, also responded weakly to the mechanical the store membrane (Kikuyama and Tazawa 1998). stimulus, but the chloroplast fraction was inert. When chlo- The present study was undertaken to confirm the above roplasts from Chara were added to the chloroplast-free assumption that mechanical stimulation of the cytoplasmic 2+ cytoplasm of N. flexilis, the cytoplasm recovered the mecha- drop induces Ca release from the stores through activation of noresponse.Starchgrainswereaseffectiveaschloroplasts. mechanosensitive ion channels. The data indicate that Ca2+ permeable channels in the membrane of Ca2+ stores in N. flexilis are really mechano- Materials and Methods sensitive. Plant materials 2+ The materials, N. flexilis and Chara corallina, were cultured in Key words: Aequorin — Ca store — Chloroplast — Hydra- an aquarium filled with tap water under 12 h illumination a day with 2+ tion induced Ca release (HICR) — Nitella flexilis —Mech- two 20 W fluorescent lamps placed about 10 cm above the water sur- anosensitive ion channel. face. Temperature was kept at about 25°C with a thermostat.

Abbreviations: ER, endoplasmic reticulum; EGTA, ethylenegly- Isolation of cytoplasm from internodal cells col bis(2-aminoethylether)-N,N,N¢,N¢-tetraacetic acid; D[Ca2+], increase Isolation of the cytoplasm was carried out in the same manner as in Ca2+ concentration; HICR, hydration-induced calcium release; Osm, described in the previous paper (Kikuyama and Tazawa 1998). Briefly, osmolar; PM, photomultiplier. internodal cells, isolated from neighboring internodal and leaf cells, were kept in an artificial pond water (APW; 0.1 mM each of KCl,

NaCl and CaCl2) for a day, then immersed in 10 mM KCl for several hours or more to make cells inexcitable to mechanical stimulation. The D 2+ KCl treatment was essential to avoid excitation-induced [Ca ]c that D 2+ Introduction interferes with measurement of [Ca ]c caused by HICR (Hayama and Tazawa 1978, Hayama et al. 1979). In order to remove Ca2+ from the vacuole, the vacuolar sap was In internodal cells of Nitella flexilis, transcellular osmosis replaced with the isotonic perfusion medium composed of 5 mM induces a transient increase in the concentration of cytoplas- EGTA, 10 mM PIPES (pH 7 with KOH), 6 mM MgCl2 and 300 mM 2+ 2+ 2+ mic free Ca ([Ca ]c) on the endosmosis side (Tazawa et al. sorbitol (345 mOsm). With this procedure, possible release of Ca 2+ D 2+ from fragments of the central vacuole is completely neglected, even if 1994, Tazawa et al. 1995). The increase in [Ca ]c ( [Ca ]c) was assumed to be a result of Ca2+ release from intracellular they were formed during the isolating procedure and remained in the cytoplasmic drop. Then the cell content was squeezed out onto a sheet stores that is evoked by a rapid hydration of the cytoplasm on of Parafilm (American National Can, Greenwich, CT, U.S.A.) by using the endosmosis side (Tazawa et al. 1995). The hydration- forceps (Fig. 1). The isolated drop will be called simply cytoplasmic induced calcium release (HICR) was also observed when the drop hereafter.

3 Present address: Department of Biology, Faculty of Science, Niigata University, Niigata, 950-2181 Japan. 4 Corresponding author: E-mail, [email protected]; Fax, +81-77-524-9221.

358 Mechanosensitive Ca2+ Release in Nitella 359

Fig. 1 Method for isolating cytoplasmic drop. (1) Vacuolar per- fusion. An internodal cell (cell) was placed on a Plexiglas bench (B) Fig. 2 A method for inducing HICR and/or mechanosensitive and the vacuolar sap was removed and replaced with isotonic per- response in cytoplasmic drop (Drop) placed in silicone oil (oil). (a) fusion medium (PM) by vacuolar perfusion. (2) Squeeze out. Cyto- Injection of perfusion medium. (b) Shooting of a drop of silicone oil. plasm concomitant with the perfusion medium was squeezed out onto A silicone oil dorp was shot into the cytoplasmic drop using a pump a sheet of Parafilm (PF) using forceps (F). (3) Put in silicone oil and (Piezo pump). (c) Compression of cytoplasmic drop. Compression and add aequorin. Two ml of the cytoplasmic drop (drop) was placed in sil- decompression were performed by horizontal movements of a Plex- icone oil and added with aequorin by using a glass micropipette (p). iglas rod connected to a piezoelectric device (Piezo Actuator). The specimen containing aequorin was placed just over the photomul- tiplier tube, and light emission from aequorin was measured.

fch-aequorin, 150 mM KCl, 6 mM MgCl2, 0.15 mM EGTA, 0.5 mM PIPES (pH 7.0 with KOH) was added to the specimen (Fig. 1). Preparation of chloroplast fraction and chloroplast-free fraction Although the amount of the aequorin solution added was in the order The isolated cytoplasmic drop from two internodal cells was cen- of 100 pl, the actual amount was significantly changed in each meas- trifuged at 125´g for 5 s using a centrifuge (5415C, Eppendorf, Ham- urement partly because we had an impression that the cellular sensitiv- burg, Germany). The supernatant was isolated from the precipitate. ity of Ca2+ release fluctuated seasonally. This is a major reason why The supernatant was a mixture of cytoplasm and the isotonic per- the measured photomultiplier current are largely different in each fig- fusion medium and contained no chloroplast. It will be named simply ure. as chloroplast-free fraction hereafter. The precipitate (about 2 ml) containing mostly chloroplasts was Measurement of ,[Ca2+] upon hypotonic treatment and mechanical resuspended in 20 ml perfusion medium, centrifuged (125´g,30s)and stimuli the supernatant was discarded. After two rinses, precipitated chloro- The cuvette with specimen was placed in a dark box equipped plasts were dispersed in 4 ml perfusion medium. The suspension will with a photomultiplier tube (PM) (R1924P, Hamamatsu Photonics, be named simply as chloroplast fraction. Hamamatsu, Japan) in the same manner as described in the previous Increase in the Ca2+ concentration (D[Ca2+]) in response to vari- paper (Kikuyama and Tazawa 1998). ous stimuli was measured in isolated cytoplasmic drops, chloroplast- The HICR of each specimen was induced by applying a hypot- free fractions and chloroplast fractions as increases in light emission onic medium (the same ionic composition as the isotonic perfusion from aequorin added in each fraction. Two ml of specimen was placed medium but lacking sorbitol; about 45 mOsm) from a microsyringe in a cuvette and covered with silicone oil (10 cs; KF-96-10CS, Shin- (1705, Hamilton, U.S.A.) which was driven by a motor (CYLINOID, Etsu Chemical, Tokyo, Japan). Then aequorin solution (0.5 mg ml–1 CA-2, Kamaden, Tokyo) (Fig. 2a). 360 Mechanosensitive Ca2+ Release in Nitella

Fig. 4 Transient increase in PM current of aequorin light emission in Fig. 3 Transient increase in PM current of aequorin light emission in response to a shot of silicone oil. (a) A silicone oil drop was shot at response to a jet of isotonic (a) or hypotonic medium (b). In (b) the time zero. The resulted PM current shows a single peak. (b) First, a sil- first spike is followed by a slower and long-lasting one. icone oil drop was shot at time zero and then a jet of hypotonic medium was applied at a time of 8.3 s (arrow). The increase in PM current is composed of two processes, the first rapid increase and the second slower one. Three types of mechanical stimuli were used. (1) Injection of perfusion medium. About 4 ml of either isotonic or hypotonic per- fusion medium was injected into the cytoplasmic drop within 2 s from Results a microsyringe (Fig. 2a). (2) Shooting of a drop of silicone oil. A drop m of silicone oil (about 0.3 l) was shot into the cytoplasmic drop by Injection of perfusion medium into cytoplasmic drop using a pump (Piezo pump; NS-02D, Nippon Keiki Works Ltd., Tokyo, Japan) driven by a piezoelectric device (Fig. 2b). (3) Compres- A jet of the isotonic medium into the cytoplasmic drop sion of cytoplasmic drop. The cytoplasmic drop was compressed verti- caused a single large spike of the PM current (= a spike of cally with a Plexiglas rod connected to a piezoelectric device (Piezo aequorin light emission), indicating an abrupt D[Ca2+](Fig.3a). Actuator; P2-50, Denso, Kariya, Japan) (Fig. 2c). The extent of defor- In this case, no osmotic expansion of Ca2+ stores was induced. mation was about 30 mm except noted otherwise. By contrast, application of the hypotonic medium caused Preparation of starch grains increase in the PM current composed of two phases; the first Starch grains from potato, commercial name Katakuriko, was spike was very similar to that induced by isotonic injection and purchased from a market. Before use, the powder was rinsed three the second one, beginning at the recovery phase of the first 2+ times with the isotonic perfusion medium to remove Ca . spike, was a slower increase of the PM current followed by a gradual recovery (Fig. 3b). In this case, the Ca2+ stores were assumed to be subjected to two stimuli, mechanical and osmotic. The first spike in Fig. 3b may correspond to Ca2+ release from putative Ca2+ stores in response to mechanical Mechanosensitive Ca2+ Release in Nitella 361

Fig. 5 Increase in PM current caused by compression of cytoplasmic drop. Cytoplasmic drop was intermittently compressed for 5 s as indi- cated with the upper trace in the figure. Spikes were observed at the moment of compression and/or decompression.

Fig. 7 HICR in chloroplast-free fraction (a) and in chloroplast frac- tion (b). Hypotonic medium was added at time zero.

Fig. 6 Relation between the amplitude of PM current and that of compression of the cytoplasmic drop. Compression of 10 Hz was applied for 1 s as indicated with the lower trace. The amplitude of suc- course of D[Ca2+] was very similar to that shown in Fig. 3a. In cessive deformation was 30 (first), 24 (2nd), 18 (3d), 12 (4th), 18 (5th), 24 (6th) and 30 (7th) mm, respectively. The spike was signifi- Fig. 4b, two kinds of stimuli were sequentially applied to the cant only when the deformation was 24 and 30 mm. same cytoplasmic drop, shooting of silicone oil at time zero and then shooting of the hypotonic medium (shown by an arrow) at 8.3 s. Again, D[Ca2+] in response to the first and the second stimulation were essentially the same as those shown in stimulation and the second slow spike may represent HICR Fig. 4a and Fig. 3b, respectively. from the Ca2+ stores. Compression of cytoplasmic drop Silicone oil shooting Mechanical stimulus by means of oil shooting cannot be Addition of the isotonic medium into the cytoplasmic drop applied uniformly onto the cytoplasmic drop. Also it is diffi- necessarily changes the composition of the drop. In order to cult to control the strength of the stimulus and to repeat it at an apply a pure mechanical stimulus onto the cytoplasmic drop arbitrary time interval. To overcome these defects of oil shoot- without causing any change of its composition, we first shot a ing, we designed another method named the compression small drop of silicone oil (about 0.3 ml) into the cytoplasmic method (Fig. 2c). drop using the Piezo pump. In Fig. 4a, a drop of silicone oil In Fig. 5, a cytoplasmic drop was compressed for 5 s, was shot into the cytoplasmic drop at time zero. Clearly, the repeatedly at the time interval of 10–15 s as indicated with the mechanical stimulation induced a significant D[Ca2+]. The time upper trace of the figure. Again, this mechanical stimulation 362 Mechanosensitive Ca2+ Release in Nitella

Fig. 9 Increase in PM current in response to compression of chloro- plast-free fraction of N. flexilis to which Chara chloroplasts (a) and Fig. 8 Response of PM current to compression in chloroplast-free starch grains (b) were added. fraction (a) and chloroplast fraction (b). The increase in PM current indicated by an arrow in (a) may not be a response to mechanical stim- ulation because it took place independently of compression. In (b) increase in PM current was observed on each mechanical stimulation. fractions, the chloroplast-free fraction and the chloroplast frac- tion. Both fractions showed HICR in a similar manner (Fig. 7a, b), indicating that not only the chloroplast-free fraction but also caused a significant D[Ca2+]. Furthermore, the spike indicating the chloroplast fraction act as Ca2+ stores. a quick D[Ca2+] was observed steadily at the moment of com- pression and/or decompression. The rate of compression or Responses of chloroplast-free and chloroplast fractions to –1 decompression was estimated to be about 23 mm s from the compression m amplitude (30 m) and the time (1.3 ms) needed. Mechanical compression was applied to the chloroplast- In Fig. 6, compressions with various amplitudes were free and chloroplast fractions. The D[Ca2+] was not observed in applied with a frequency of 10 Hz for 1 s. The D[Ca2+]wassig- all chloroplast-free fractions tested (0/5, Fig. 8a) but observed nificant for compressions of 24 mmand30mm but insignifi- m in all chloroplast fractions (3/3, Fig. 8b). Based on these facts, cant for those of 18 m or less. Since the time for the compres- 2+ sion was constant (1.3 ms), this fact indicates that D[Ca2+] two alternatives are possible to explain the Ca release from depends on the rate of compression. In other words, the Ca2+ whole cytoplasmic drops on compression (Fig. 5). One is that 2+ release from Ca2+ stores depends on the rate of deformation of only chloroplasts are Ca stores sensitive to mechanical stimu- the cytoplasmic drop. lation, and the other is that the presence of chloroplasts in the cytoplasmic drop is essential for mechanical stimulation to HICR in chloroplast-free fraction and chloroplast fraction Ca2+ stores other than chloroplasts. This was examined by the The isolated cytoplasmic drop was separated into two following experiments. Mechanosensitive Ca2+ Release in Nitella 363

Mechanosensivive Ca2+ stores in chloroplast-free fraction from putative Ca2+ stores that are disrupted by mechanical The cytoplasmic drop of C. corallina also released Ca2+ in stimuli. Although we cannot completely deny this possibility, response to compression/decompression of the drop, although we assume the major D[Ca2+]asaCa2+ release through stretch- the response was much smaller than that of N. flexilis.The activated Ca2+ channels as follows. In other Characeae plants chloroplast fraction of Chara was proved to be almost insensi- such as N. axilliformis and C. corallina, rapid injection of tive to mechanical stimulation (data not shown). Then Chara hypotonic medium into cytoplasmic drop never caused rapid chloroplasts were mixed with the chloroplast-free fraction D[Ca2+] but only a slow and a very small D[Ca2+](Shimadaet (4 ml) of N. flexilis. When a portion of the mixture (2 ml) was al. 1996) and this was also confirmed in the present study. This subjected to compression, a spike showing D[Ca2+]was is strong evidence showing that disruption of Ca2+ stores rarely observed on each mechanical stimulation (Fig. 9a). In another takes place, if any, at the moment of medium injection. experiment, starch grains from potato (grain size between 10 Namely, disruption of Ca2+ stores should induce significantly and 80 mm) were mixed with the chloroplast-free fraction of N. large D[Ca2+] also in these Characeae cells because cytoplasmic flexilis. The mixture contained about 20,000 starch grains in content of calcium in these Characeae cells is about half to that 2 ml. Fig. 9b shows that starch grains are as effective as chloro- in N. flexilis (Tazawa et al. 2001) as discussed later in detail. plasts in inducing D[Ca2+]. The fact that the chloroplast-free Furthermore, we have no evidence showing that the mem- fraction of N. flexilis regained the sensitivity to mechanical branes of Ca2+ stores of N. flexilis are mechanically much stimulation through introduction of inert Chara chloroplasts or weaker than those in other Characeae cells. starch grains is a decisive evidence for the presence of Ca2+ The compression method was developed to control the stores in the chloroplast-free fraction. strength of the mechanical stimulus and to give stimulus repeatedly at arbitrary intervals. The D[Ca2+] was induced Discussion repeatedly without being damped upon repeated stimulation (Fig. 5). It should be noted that the D[Ca2+] was induced when Evidence for the mechanosensitive Ca2+ release from Ca2+ the deformation rate of the cytoplasmic drop was maximal, stores namely either at the moment of compression and/or decom- Mechanical signaling in plant cells is mediated by pression. The amplitude of D[Ca2+] was dependent on the cytosolic calcium. Touch and wind signals applied to young amplitude of deformation (Fig. 6). These results suggest that D 2+ D 2+ 2+ seedlings of tobacco induced a transient [Ca ]c.The the [Ca ] is a result of mechanosensitive activation of Ca increased calcium was assumed to originate from intracellular permeable channels in the membrane of Ca2+ stores. stores (Knight et al. 1991, Knight et al. 1992). Increase in 2+ [Ca ]c in response to mechanical stimuli was reported in vari- Responses of chloroplast-free and chloroplast fractions to ous plants by many researchers (Bush 1993, Haley et al. 1995). mechanical and osmotic stimuli As briefly discussed in the introduction, HICR observed The present study has revealed that the membrane of Ca2+ in the cytoplasmic drop of N. flexilis was assumed to be a stores in the cytoplasm of N. flexilis has Ca2+ permeable chan- response of the Ca2+ storing organelles (Ca2+ stores) to stretch- nels that are directly activated by either mechanical distortion ing of the membrane induced by osmotic swelling. This or osmotic swelling of the membrane. The former stimulus assumption was examined by several means. Both a jet of isot- occurs faster compared with the latter stimulus, as indicated by onic medium (Fig. 3a) and a shot of silicone oil (Fig. 4a, b) the discrete time separation of the two types of aequorin light caused a significant D[Ca2+]. In these cases, the PM current emission (Fig. 3b, 4b). increased rapidly without time lag, reached the peak within a The mechanical stimulation induced D[Ca2+] not in the second and declined rapidly to the original level. By contrast, a chloroplast-free fraction (Fig. 8a) but in the chloroplast frac- jet of hypotonic solution caused two phases of D[Ca2+](Fig. tion (Fig. 8b), while the osmotic stimulation did so in both 3b); the first one was a rapid spike quite similar to that fractions (Fig. 7a, b). Kikuyama et al. (1995) already observed observed when the drop was stimulated by a jet of isotonic occurrence of HICR in both chloroplast-free and chloroplast medium (Fig. 3a) or by a shot of oil drop (Fig. 4a, b), and the fractions. Since there was no significant difference in the inten- second one is a slower D[Ca2+] followed by a slower decline sity of PM current between both fractions, they concluded that (Fig. 3b, 2nd D[Ca2+] in Fig. 4b). Since a jet of the hypotonic chloroplasts may play a minor role in HICR. In our previous medium contains two kinds of stimuli, mechanical and hypoos- paper cited above, the precipitate after removal of the superna- motic, the initial D[Ca2+] may correspond to the mechanical tant was not rinsed and subjected directly to hypotonic treat- shock which is transmitted without time lag and the second one ment. It may be possible that a significant amount of to the osmotic swelling of putative Ca2+ stores which proceeds chloroplast-free fraction remained in the precipitate. In the slowly via diffusion of solutes and movement of water across present experiment, however, we rinsed the precipitate two the membrane. times with the isotonic perfusion medium. The chloroplast It may be possible, however, that the rapid D[Ca2+]in fraction thus prepared is expected to be mostly composed of response to mechanical stimuli simply reflects outflows of Ca2+ chloroplasts. The chloroplast fraction (Fig. 7b) responded to 364 Mechanosensitive Ca2+ Release in Nitella the hypotonic treatment to the same extent as the chloroplast- stores, poor distribution of Ca2+ permeable channels in the free fraction (Fig. 7a). This indicated that both fractions con- membrane of Ca2+ stores and a low Ca2+ content in the Ca2+ tribute to HICR of whole cytoplasmic drop (Fig. 3b). Further- stores. Recently, we found a big difference in the Ca2+ content more, the present study strongly suggests that Ca2+ channels, in the cytoplasm between N. flexilis and C. corallina, while no which are activated by mechanical stimuli including osmotic significant difference exists in the Mg2+ content (Tazawa et al. expansion of the membrane, are present not only in the mem- 2001). N. flexilis contains about 4.5 times more Ca2+ than that brane of cytoplasmic organelles (ER?) but also in the chloro- of C. corallina (21.0 mM vs. 4.7 mM). As for the chloroplast- plast membrane (Fig. 7, 8). However, the contribution of the free cytoplasm, N. flexilis contains 2.3 times more Ca2+ than C. chloroplasts to HICR may be minor, since the amount of chlo- corallina. As for the chloroplast the former contains 6.8 times roplasts in the cytoplasmic drop is much smaller than in chloro- more Ca2+ than the latter. It is clear that most of Ca2+ in cell plast fraction. organelles are bound with Ca2+-binding proteins (Meldolesi and Pozzan 1998, Ettinger et al. 1999). Therefore the differ- Recovery of mechanosensitive Ca2+ release in chloroplast-free ence between the two species of Characeae may reflect the dif- fraction ference in bound Ca2+ in the stores. The cytoplasmic drop was insensitive to mechanical stim- Assuming that bound Ca2+ is released quickly with lower- uli after removal of chloroplasts (Fig. 8a). However, it recov- ing of the free Ca2+ concentration, bound Ca2+ may also be ered the ability to release Ca2+ when particles insensitive to mobilized for the Ca2+ release from the stores as is the case in mechanical stimulation were introduced into the chloroplast- sarcoplasmic reticulum in muscle contraction. A big difference free fraction (Fig. 9a, b). The fact that even starch grains can in the Ca2+ content of Ca2+ stores may possibly be a factor replace chloroplasts in conferring mechanosensitivity on the responsible for the difference in the mechano- and osmo-sensi- chloroplast-free fraction suggests that collision of cell tive Ca2+ release between two species of Characeae. organelles (Ca2+ stores) with particles is essential for the mech- Intracellular mobilization of Ca2+ in response to mechani- anoresponse of the cytoplasm. In the absence of particles, cal stimulus was suggested in epidermal cells of Nicotiana movement of cell organelles caused by mechanical stimulus plumbaginifolia and protoplasts isolated from leaves (Haley et may be smooth without collision with particles. Then al. 1995). In tendrils of Bryonia dioica presence of mechano- organelles hardly suffer distortion of the membrane. It is to be sensitive Ca2+ channels was suggested in the ER membrane noted that not only collision between cell organelles and chlo- (Klüsener et al. 1995). Our data demonstrate also the presence roplasts but also intercollision between chloroplasts are effec- of a mechanosensitive Ca2+ release mechanism in the cyto- tive for releasing Ca2+. Since the chloroplast fraction contained plasm of N. flexilis. The present study is the first demonstra- a high density of chloroplasts, the chance of intercollision tion of Ca2+ release from cytoplasm in vitro in response to should have been very high. On the other hand, the cytoplasmic mechanical stimulation. The method of mechanical stimulation drop contained a low density of chloroplasts, since it was the including compression and mixing inert particles such as starch mixture of the cytoplasm and the perfusion medium. Then, the grains may serve as a tool for studying mechanosensitivity of chance for the intercollision of chloroplasts should have been cytoplasm and cell organelles in plant cells in general. low. Thus chloroplasts may contribute little to D[Ca2+]ofthe cytoplasmic drop. Acknowledgements We started our study under the assumption that HICR may be a result of activation of mechanosensitive Ca2+ permeable We thank Dr. Osamu Shimomura of the Marine Biological Labo- channels in the membrane of putative Ca2+ stores by osmotic ratory at Woods Hole, MA, U.S.A., Prof. Yoshito Kishi of Department swelling. Occurrence of Ca2+ signals in response to direct of Chemistry, Harvard University, Cambridge, MA, U.S.A. and Dr. mechanical stimuli supports the above assumption. Involve- Satoshi Inouye of Yokohama Research Center, Chisso Corporation, 2+ Yokohama, Japan for their generous gift of recombinant semi-synthetic ment of Ca -permeable stretch-activated channels in the vol- aequorins. This work was supported in part by a Grant from the Uni- ume regulation was reported in epithelial intestine cells sub- versity of the Air to M.K. and by a Grant in Aid for Scientific jected to hypotonic treatment (Okada et al. 1990). Research (C) from the ministry of Education, Science, Sports and Cul- In Characeae, HICR is most significant in N. flexilis.The ture to M.T. (No. 11640658). D[Ca2+] induced by transcellular osmosis in three other species of Characeae (N. axilliformis, C. corallina, Nitellopsis obtusa) References waslessthan1/10thatofN. flexilis (Shimada et al. 1996). The cytoplasmic drop of C. corallina showed a very weak response Bush, D.S. (1993) Regulation of cytosolic calcium in plants. Plant Physiol. 103: 7–13. to mechanical stimulus. Then, a very weak HICR in the cyto- Ettinger, W.F., Clear, A.M., Fanning, K.J. and Peck, M.L. (1999) Identification plasmic drop of C. corallina is partly due to a very weak of a Ca2+/H+ antiport in the plant chloroplast thylakoid membrane. Plant mechano-sensitivity of the membrane of the Ca2+ stores of Physiol. 119: 1379–1385. Haley, A., Russell, A.J., Wood, N., Allan, A.C., Knight, M., Cambell, A.K. and Chara. Several factors may be responsible for the weak Trewavas, A.J. (1995) Effects of mechanical signaling on plant cell cytosolic 2+ mechano-sensitivity of Chara cytoplasm; low density of Ca - calcium. Proc. Natl. Acad. Sci. USA 92: 4124–4128. Mechanosensitive Ca2+ Release in Nitella 365

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(Received November 17, 2000; Accepted January 16, 2001)