Figure 17. Representative images of chloroplast mechano- and photo-relocation movement. Mechanical and microbeam light stimulation were continuously applied for 2 h. A-E, Mechano- and photo-relocation movement of chloroplasts in 10 mM MES containing 1mM CaCl2 and 10 mM KCI. F-J,
Mechano- and photo-relocation movement of chloroplasts in 10 mM MES containing 10 mM KCI and 100 JAM Gd3+. A and F, Mechanical stimulation
(Mec); B and G, LRL (1 Wm-2); C and H, LBL (1 Wm-2); D and I, High fluence rate RL (HRL; 600 Wm-2); E and J, HBL (10 Wm-2). Each panel shows the same cell before (upper half) and after (lower half) stimulation.
observed when Gd3+ at 100 µM was applied in the medium wthout CaCI? (Fig.
17F). I examined photo-relocation movement with the latter medium condition , namely, the most unfavorable condition for mechano-relocation (Fig. 17G-J).
All the photo-relocation movements were hardly affected regardless of light quality and of the type of movement, that is, accumulation or avoidance.
Almost the same results were obtained when Lai+ was used instead of Gd3+
(data not shown). These results suggest that influx of external Ca' from extracellular sites is essential for induction of chloroplast mechano-relocation,
but not for the photo-relocation.
Discussion
Chloroplast behavior after mechanical stimulation
In this chapter, I firstly analyzed the behavior of individual chloroplasts during the mechano-relocation using computer-recording systems, which enable us to analyse in time resolution higher than that in the previous study (Chapter 1).
Kinetics of mechano-relocation movement showed similar features to those of photo-relocation, which had previously examined (Kadota and Wada, 1992).
The directional movement of chloroplasts, avoiding the mechanically-stimulated site was clearly demonstrated. Oil drops which flowed on cytoplasmic streaming did not show any avoidance response and always moved much faster than chloroplasts. Thus, mechano-movement of chloroplast is distinct from cytoplamic streaming as is the case of the photo-movement (Kadota and Wada,
-52-1992). Furthermore, the velocity of chloroplast movement during mechano-relocation is close to that of movement during photo-mechano-relocation (Kagawa and Wada, 1996; Kagawa and Wada, 1999). Therefore , common motile system
may be shared in both movements, although the molecular motor responsible for the two responses has not been proven yet.
Mechano-relocation movement is evident in photo-relocated chloroplasts In the previous study, when chloroplast photo-relocation response attained the stationary state, the photo-relocated chloroplasts were surrounded by MFs and their motility was reduced, indicating that they are mechanically anchored to the location by MF cytoskeleton (Kadota and Wada, 1989; Kadota and Wada, 1992).
This suggestion is also supported by the studies that photo-relocated chloroplasts become resistant against centrifugal force in epidermal cells of Vallisneria gigantea (Takagi et al., 1991; Dong et al., 1998). In this study, I clearly showed that the mechano-avoidance response occurred in these anchored chloroplasts under all the photo-relocation conditions tested, suggesting that different signal transduction pathways are activated and regulate chloroplasts in photo- and mechano-relocation. Surprisingly, lag time before avoidance movement was longer in the dark than those in any of the photo-relocation conditions tested whereas no significant difference in the velocity of chloroplast avoidance movement was observed among the conditions.
condition, short lag period in photo-relocated chloroplast may indicate that reactivation of motor apparatus by the mechano-signal leads to chloroplast avoidance on the MF tracks which were already arranged around it.
Independence of mechano-signal transduction from that of light was further supported by the requirement of external Cat+ for mechano- and
photo-relocation of chloroplasts discussed below.
External Cat+ is crucial for mechano-relocation of chloroplasts
Many pharmacological studies have been done to investigate the role of Ca' on chloroplast movement induced exclusively by light (Haupt and Scheuerlein,
1990; Nagai, 1993; Wada et al., 1993 and references therein). The presence of Cat+ is found to be essential for chloroplast photomovement, but whether they function in the light signaling processes remains obscure because of the following reasons. One is that the specificity of the drugs which block influx of Ca' from extracellular sites such as EGTA, is influenced by their concentration and their incubation periods, because they also modify Cat+ release from the
internal pools through the shift of equilibrium maintained by Cat+ cycling between internal and external stores (Gilroy et al., 1991; Tlalka and Gabrys, 1993; Tlalka and Fricker, 1999). Thus, examination with various periods of incubation in different concentrations of drugs would be necessary to interprete the effects of these drugs. Another point is that perturbation of internal Cat+
level affects the nature of movement itself; that is, these drugs also affect the
-54-actomyosin motile system which is not related to the early signaling processes (Kadota and Wada, 1992).
Experimental system of fern protonemal cell has an advantage that MF-based directional movement of chloroplast can be induced with different stimuli.
Inhibition of mechano-response but not of photo-relocation was found in the medium without Ca'. Partial inhibition of the mechano-relocation may suggest that substantial Ca' are stored in the cell wall. I also demonstrated that chloroplast mechano-relocation was completely abolished with low concentrations of La' and Gd3+ while the same conditions failed to affect the photo-relocation movement. Thus the entry of external Cat+ through the plasma membrane seems essential for chloroplast directional movement in an early step of mechano-signaling, possibly through the stretch-activated channel.
Stretch-activated Ca' channels has been characterised in onion epidermal cells (Ding and Pickard, 1993). Recently, gene encoding stretch-activated channel, which is sensitive to Gd3+, was idetified in the yeast Saccharomyces cerevisiae (Kanzaki et al., 1999).
Results that external Cat+ plays important roles on mechano-response in A. capillus-veneris do not support the indication by the previous studies at whole plants or tissue level in in the trasgenic plants that express apoaequorin;
Ca' influx from apoplastic sites is not essential for mechano-response (Knight et al., 1992; Haley et al., 1995). The differences may be interpreted that because
specialized cells is hidden behind the responses in the other cells and we can
not detect the response of the minor cells in the analysis of whole plant or of cell
masses. Another reason may be ascribed to the different ways in application of
mechanical stimulation; direct touch on a cell which caused localized
deformation of the cell was applied in this study, while wind or application of
isotonic medium was used as mechanical stimulation in the studies of the
transgenic plants.
External Ca2+ has no major contribution for signaling in chloroplast photo-relocation
Results from the pharmacological studies strongly suggest that external Ca2+ is not crucial for photo-relocation of chloroplasts irrespective of the wavelength and fluence rate of stimuli. Recently, BL-induced elevation of [Cal; was observed in Physcomitrella patens, Arabidopsis thaliana, and Nicotiana plumbaginifolia transformed with aequorin gene (Russell et al., 1998; Baum et al., 1999). The elevation of [Ca2+]; in the level of the tissue or whole-seedling was completely abolished by La3+ at concentrations close to that which had no effects on chloroplast photo-relocation in the present study. This suggests that [Cal;
elevation is not involved in an early signaling of the photo-relocation or increase of [Ca2+]; in signaling for photo-relocation of chloroplasts was too small to detect in the tissue level analysis. The experimental system would be necessary, in which spacial information on [Ca2+]; at the subcellular level can be detected.
-56-Conclusion
I clearly demonstrated the different contribution of external Ca' as the signal transducer for mechano- and photo-relocation of chloroplasts even the responses show the same in direction with respect to stimuli . I propose that localized deformation of a cell stimulates the influx of external Ca' through the stretch-activated channel, allowing the chloroplast to show the avoidance movement away from the site of mechanical stress. Since Ca' mobility in the cytoplasm is much lower than that in free solution, results that only chloroplasts near the stimulation site respond, may indicate that localized stimulus establishes a localized [Ca2+]; gradient in the cell.