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Journal of Experimental Botany, Vol. 60, No. 12, pp. 3301–3310, 2009 doi:10.1093/jxb/erp193 Advance Access publication 21 June, 2009 DARWIN REVIEW Chloroplast anchoring: its implications for the regulation of intracellular chloroplast distribution Shingo Takagi*, Hideyasu Takamatsu and Nami Sakurai-Ozato† Department of Biological Sciences, Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan Received 8 December 2008; Revised 18 May 2009; Accepted 19 May 2009 Abstract The intracellular distribution of organelles plays a pivotal role in the maintenance and adaptation of a wide spectrum of cellular activities in plants. Chloroplasts are a special type of organelle able to photosynthesize, capturing light energy to fix atmospheric CO2. Consequently, the intracellular positioning of chloroplasts is crucial for plant growth and development. Knowledge of the photoreceptors and cellular apparatus responsible for chloroplast movement has gradually accumulated over time, yet recent advances have allowed improved understanding. In this article, several aspects of research progress into the mechanisms for maintaining the specific intracellular distribution patterns of chloroplasts, namely, chloroplast anchoring, are summarized, together with a brief consideration of the future prospects of this subject. Our discussion covers developmental, physiological, ecophysiological, and recent cell biological research areas. Key words: Actin cytoskeleton, chloroplast anchoring, chloroplast movement, chloroplast positioning. What is chloroplast anchoring? Light-dependent chloroplast movement allows the adaptation of cellular activities to fluctuating environmental light conditions. Under dim light, chloroplasts move in order to maximize light interception, while under strong light, chloroplasts move to minimize light interception. It is generally accepted that the physiological significance of these movements is to increase photosynthetic activity under dim light (Zurzycki, 1955), while to evade photodamage caused by excess light (Zurzycki, 1957; Park et al., 1996; Kasahara et al., 2002). Chloroplasts exhibit species-specific patterns of movement, for example, the rotation of single ribbon-shaped chloroplasts in the cylindrical cells of green algae (Wagner and Klein, 1981), face and profile positioning of multiple chloroplasts in cylindrical cells of algae, moss, and ferns, or the noted accumulation and avoidance responses of chloroplasts observed in leaf mesophyll cells of various angiosperms (Haupt and Scheuerlein, 1990). Conversely, under non-fluctuating light conditions favourable for photosynthesis, chloroplasts actively maintain their intracellular distribution. Otherwise cytoplasmic streaming would not allow the chloroplasts to maintain their position within the cell, resulting in decreased light use efficiency. As described later, the concept of ‘chloroplast anchoring’ has arisen from careful analysis of chloroplast behaviour in various types of plant cells at different developmental stages and under different physiological conditions. This article aims to summarize several aspects of chloroplast anchoring and discuss possible mechanisms underlying this intriguing phenomenon. Since comprehensive reviews on chloroplast movement and its motile apparatuses have already been published (Wada et al., 2003; Lee and Liu, 2004; Shimmen and Yokota, 2004; Wada and Suetsugu, 2004), this article does not cover those subjects. Developmental view Intracellular movement and distribution of chloroplasts have been extensively investigated in C3 plants, whereas our * To whom correspondence should be addressed: E-mail: [email protected] y Present address: Department of Anatomy and Molecular Cell Biology, Graduate School of Medicine, Nagoya University, Tsurumai 65, Showa, Nagoya 466-8550, Japan. ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: [email protected] 3302 | Takagi et al. knowledge on C4 plants is rather limited. In C4 leaf tissue, mesophyll cells are the site for the primary fixation of CO2 by phosphoenolpyruvate (PEP) carboxylase into the fourcarbon oxaloacetate, while decarboxylation of malate, formed from oxaloacetate, and the refixation of CO2 are carried out in bundle sheath cells (Hatch and Slack, 1970). Chloroplasts in C4 plant mesophyll cells are more or less randomly distributed, whereas the distribution pattern of chloroplasts in bundle sheath cells is unique; chloroplasts are distributed either centripetally, close to the vascular tissue, or centrifugally, around the periphery of the bundle sheath. Different distribution patterns of chloroplasts in bundle sheath cells are probably advantageous for the efficient exchange of metabolites between the mesophyll and the bundle sheath cells, the minimization of CO2 leakage, and so on. In the NAD-malic-enzyme type C4 plant finger millet (Eleusine coracana), Miyake and Yamamoto (1987) demonstrated that the distribution pattern of chloroplasts in bundle sheath cells was established during leaf development, independent of the light environment, with etioplasts exhibiting the centripetal chloroplast distribution in mature leaves developing in the dark. Miyake and Nakamura (1993) further examined possible factors involved in the establishment of the centripetal distribution of chloroplasts using various chemicals. Auxin, cycloheximide, and the actin-depolymerizing reagent cytochalasin B exhibited substantial inhibitory effects, while other hormones and colchicine did not. Auxin seemed to affect the early processes in the establishment of centripetal distribution, suggesting a role in modulating cell elongation and/or developmental stage, while cycloheximide and cytochalasin B appeared to exert more direct effects on the migration of chloroplasts to the centripetal regions. The inhibitory effects of cytochalasin B, but not of colchicine, strongly suggest the involvement of the actin cytoskeleton in chloroplast migration and anchoring. Recently, Kobayashi et al. (2009) demonstrated that actin filaments, intimately associated with the surface of chloroplasts in bundle sheath cells, may play a central role in the re-establishment of the characteristic distribution pattern of chloroplasts after displacement caused by centrifugation. However, the maintenance of a specific distribution pattern of chloroplasts did not seem to be dependent upon the actin cytoskeleton. Physiological view Chloroplast movement in C3 angiosperms is regulated by blue light. In Arabidopsis thaliana, the blue-light receptors phototropin1 (phot1) and phototropin2 (phot2) have been identified as being responsible for the accumulation response of chloroplasts, while phot2 alone mediates the avoidance response (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). On the other hand, both blue and red light are effective in inducing chloroplast movement in several species of cryptogam plants (Wada et al., 2003). The effects of light in the red region are mediated by the plantspecific photoreceptor phytochrome (Wada and Kadota, 1989). Although molecular species of functional phytochrome for chloroplast movement have not yet been identified in many cases, in the fern Adiantum capillusveneris, neochrome1, which is a chimeric receptor consisting of the chromophore-binding domain of phytochrome and the full-length phototropin (Nozue et al., 1998), is responsible for red-light-induced chloroplast movement (Kawai et al., 2003). In protonemal cells of the fern Dryopteris sparsa, Yatsuhashi and Kobayashi (1993) examined the effects of red light on light-induced chloroplast movement. After the completion of the photo-relocation movement of chloroplasts induced by red light, when protonemata were illuminated with far-red light to convert phytochrome from its far-red-light-absorbing form (Pfr) back to its red-lightabsorbing form (Pr), the induced specific distribution pattern of chloroplasts decayed much faster compared with dark-incubated controls (Yatsuhashi and Kobayashi, 1993). It appears that phytochrome in this fern is involved not only in the induction of the photo-relocation movement of chloroplasts, but also in the maintenance of their specific distribution pattern, although mechanisms for both processes remain to be elucidated. Whether such roles of phytochrome are specific to this fern is also an open question. In the aquatic hydrocharitaceae species Elodea canadensis and Vallisneria spiralis, Seitz (1980) extensively investigated the nature of light-dependent chloroplast movement, developing the concept of ‘chloroplast centrifugability’, namely, how easily chloroplasts in living cells are displaced by centrifugal force as an estimate for the motile activity of cytoplasm. This makes the implicit assumption that chloroplast movement is passive and solely a result of movements of the cytoplasmic matrix. Putative components involved in the centrifugability of chloroplasts include the viscosity of cytoplasm (passive component), the motility of cytoplasm (active component), the magnitude of adhesion of chloroplasts to the cortical cytoplasm, and various kinds of interactions with other organelles such as mitochondria and the vacuole. Even now it is difficult to distinguish and specify the relative importance of each of these components. Still, in carefully designed experiments, centrifugation methods provide useful information on intracellular microconditions associated with organelles, including the mechanical properties of the cytoplasmic matrix (Hiramoto and Kamitsubo, 1995). In leaf epidermal cells of V. spiralis, Seitz (1971) noted dose-dependent effects of illumination; low-fluence-rate illumination resulted in a decrease in the centrifugability of chloroplasts, whereas high-fluence-rate illumination increased it. Seitz concluded that reductions in chloroplast centrifugability reflected lowered cytoplasmic motility under low-fluence-rates, while it was increased when exposed to high-fluence-rates. Based on differential wavelength dependence on the effects of blue light of low- and high-fluencerates, Seitz (1979) tentatively concluded that photosynthetic pigments and flavin function as the photoreceptor systems under low- and high-fluence-rates, respectively. More Chloroplast anchoring | 3303 importantly, the effects of blue light of different fluence rates exhibited a contrasting sensitivity to metabolic inhibitors (Seitz, 1979, 1980). While the inhibitors for photosynthetic electron transport suppressed the blue-light-induced decrease in chloroplast centrifugability, those for oxidative phosphorylation suppressed the increase in centrifugability. This led Seitz (1980) to propose that the cytoplasmic ATP level, supplied by photosynthesis and respiration, may change in relation to light intensity, and that a difference in ATP levels between the periclinal and anticlinal regions is the first cause for the different distribution patterns of chloroplasts induced by light of different intensities. Specifically, chloroplasts tend to migrate out of highly motile cytoplasmic regions with high levels of ATP and accumulate in quiescent cytoplasmic regions containing less ATP. However, such a mode of regulation by ATP seems unlikely, as the cytoplasmic ATP level is now known to be maintained at a more or less constant concentration, irrespective of irradiance (Keifer and Spanswick, 1979; Kikuyama et al., 1979), at concentrations sufficiently high to account for the observed motile activity of actomyosin interactions (Shimmen, 1978), which seems to be important in the movement of cytoplasm in higher plant cells (Shimmen and Yokota, 2004). More recent hypotheses for chloroplast anchoring based on the roles of the cytoskeleton will be discussed later. Ecophysiological view CO2 diffusion through leaf tissues may be one of the most important physiological factors influencing chloroplast movement and positioning (Walczak and Gabryś, 1981). The area of the chloroplast surface facing the intercellular airspace (Sc) is important in determining the conductance of CO2 in leaf tissues. To maximize the diffusion of CO2 from the intercellular airspace to the site of CO2 fixation, the chloroplast stroma, chloroplasts tend to be located contiguous with the plasma membrane (PM) (Evans and von Caemmerer, 1996; Terashima et al., 2006). Using photoacoustic analysis, Gorton et al. (2003) examined whether chloroplast movement had any effect on CO2 diffusion in the leaves of Alocasia brisbanensis. No supporting result was obtained that was favourable for the role of the avoidance response of chloroplasts in modulating internal CO2 diffusion in leaves nor the distance between chloroplasts and the intercellular air space. Rather, a liming effect of the avoidance response of chloroplasts on photosynthetic activity was reported recently. Tholen et al. (2008) analysed the relationship between the distribution patterns of chloroplasts and Sc in Arabidopsis thaliana. After induction of the avoidance response of chloroplasts to high-fluence-rate light, which tends to orientate chloroplasts along cell–cell boundaries from cell–airspace boundaries, Sc was decreased significantly together with CO2 conductance. The changes in these two parameters were not noted in the phot2 mutant, which is deficient in the avoidance response of chloroplasts, and the chup1 mutant, which exhibits no chloroplast movement. Tholen et al. (2008) concluded that the avoidance response of chloroplasts may represent a limiting factor for photosynthetic activity under some conditions. Under very high light, such responses may be advantageous in protecting the photosynthetic machinery from photodamage. Consequently, there is no doubt that chloroplast anchoring is not only an attractive subject in terms of developmental and cell biology, but is also important in understanding plant ecophysiology. In the following sections, the possible mechanisms underlying chloroplast anchoring investigated to date, in which the actin cytoskeleton has been suggested to play central roles, will be discussed. Cytoskeletal involvement The actin cytoskeleton has been postulated to play an important role in determining the intracellular distribution of chloroplasts. Close association of chloroplasts with actin filaments has been demonstrated in vascular plants as well as algae (Takagi, 2003). A possible involvement of actin filaments in chloroplast anchoring was suggested from the effects of actin-depolymerizing reagents. In A. thaliana mesophyll cells, disruption of actin filaments in a basketlike configuration surrounding each chloroplast by the actin-depolymerizing reagents produced a disordered arrangement of chloroplasts (Kandasamy and Meagher, 1999). A similar treatment of leaf epidermal cells of Vallisneria gigantea rendered the chloroplasts less resistant to centrifugal force (Dong et al., 1998), although this was not the case for chloroplasts in the bundle sheath cells of finger millet as already described (Kobayashi et al., 2009). Although there are several reports on microtubuledependent chloroplast movement in algae (Mizukami and Wada, 1981; Maekawa et al., 1986; Menzel and Schliwa, 1986) and a moss (Sato et al., 2001), evidence for the involvement of microtubules in chloroplast anchoring is scarce. In the Chenopodiaceae, a unique cell type, in which the entire C4 photosynthesis cycle is completed within single chlorenchyma cells, has been reported (Edwards et al., 2004). In those cells, chloroplasts and other organelles exhibit specific distribution patterns. Essentially, chloroplasts of two different types occupy distinct intracellular positions in the single chlorenchyma cell to achieve their respective roles, namely, primary CO2 fixation by PEP carboxylase and its subsequent liberation and refixation by Rubisco. Using cytoskeletal inhibitors and immunofluorescence microscopy, Chuong et al. (2006) suggested that microtubules play an important role in the maintenance of chloroplast positioning. Whether the role of microtubules is specific to chloroplasts or more general in maintaining the cytoplasmic architecture remains to be investigated. A role for microtubules was also suggested in the anchorage of non-green plastids in the hypocotyl epidermis of dark-grown tobacco (Kwok and Hanson, 2003). Although the motility of plastids is under the control of the actin cytoskeleton, treatment of the cells with 3304 | Takagi et al. microtubule-disrupting reagents induced an acceleration of plastid motility. Chloroplast motility and actin cytoskeleton There have been a couple of reports examining a possible causal relationship between actin organization and the motility and/or positioning of chloroplasts (Takagi, 2000). In protonemal cells of a fern, Adiantum capillus-veneris, circular arrays of actin filaments associated with each chloroplast appeared after the termination of photorelocation movement and disappeared before the chloroplasts retrieved their motility in darkness (Kadota and Wada, 1992). The characteristic circular arrays of actin filaments may function to maintain the specific distribution pattern of chloroplasts. A brief illumination of leaf epidermal cells of V. gigantea with low-fluence-rate red light increased chloroplast motility within a few minutes (Izutani et al., 1990; Dong et al., 1996). Thereafter, the motility drastically declined and the chloroplasts were almost immobile in less than an hour. This loss of chloroplast motility appears to be an important first step in the accumulation response of chloroplasts along the outer periclinal walls of the cells. Photobiological dissection of these processes revealed that the acceleration of chloroplast motility was initiated by the formation of Pfr, whereas the subsequent deceleration was regulated by photosynthesis (Dong et al., 1995, 1996). With the deceleration of chloroplast motility, the configuration of actin filaments along the outer periclinal walls changed dynamically from a loose network array to a honeycomb array, in which individual chloroplasts are tightly surrounded by actin filaments (Dong et al., 1996, 1998). On the other hand, during the avoidance response of chloroplasts, immobile chloroplasts must become mobile before the start of the relocation movement. In the epidermal cells of V. gigantea, strong blue light initially induces randomly oriented, short-range movement of chloroplasts within a few minutes (Sakurai et al., 2005). Such chloroplasts become easily displaced by centrifugation, suggesting their release from an anchored state (Sakai and Takagi, 2005). Thereafter the pattern of chloroplast movement becomes increasingly unidirectional. Concomitant with these changes in the mode of chloroplast movement, the configuration of actin filaments along the outer periclinal walls changes from being a tightly packed basket-like array around the chloroplasts to linearly aggregated bundles. By contrast, along the anticlinal walls, the chloroplasts migrating from the outer periclinal region became surrounded by actin filaments in a basket-like array (Sakai and Takagi, 2005). These chloroplasts regained their resistance to centrifugal force. These processes may contribute to an efficient redistribution of chloroplasts in the epidermal cells exposed to high-fluence-rate illumination. Importantly, in the presence of an inhibitor of photosynthetic electron transport, neither low-fluence-rate red light (Dong et al., 1996) nor high-fluence-rate blue light (Sakai and Takagi, 2005) produced any detectable changes in the configuration of actin filaments along the outer periclinal walls or anticlinal walls, respectively. Under these conditions, chloroplasts exhibited a high level of motility and easily obeyed centrifugal force. Although there was a contradictory report concerning the role of photosynthesis (Ślesak and Gabryś, 1996), Seitz (1979, 1980) repeatedly argued that photosynthesis is involved in light-induced decreases in chloroplast centrifugability. The results obtained in V. gigantea may support this proposal and, moreover, strongly suggest the critical involvement of the actin cytoskeleton in the regulation of light-induced chloroplast anchoring. In the case of A. thaliana mesophyll cells, in which typical light-dependent accumulation and avoidance responses of chloroplasts are observed (Trojan and Gabryś, 1996; Kagawa and Wada, 2000), Krzeszowiec et al. (2007) showed that blue light never induced any specific reorganization in the actin cytoskeleton regardless of its fluence rate. However, since the samples appeared to be illuminated with actinic light immediately after incision into small pieces of the leaf, the possible effects of physical injury on the cytoskeleton should be considered. Nevertheless, there is a possibility that actin reorganization in a very localized, tiny region is functional in the regulation of chloroplast movement and positioning in A. thaliana. Interaction of chloroplasts with the cortical cytoplasm/plasma membrane For proper chloroplast anchoring, interaction between chloroplasts and the cortical cytoplasm and/or PM may be indispensable. Oikawa et al. (2003) identified a unique mutant, chloroplast unusual positioning 1 (chup1), in A. thaliana deficient in chloroplast photo-relocation movement. In palisade mesophyll cells of the chup1 mutant, chloroplasts constitutively form an aggregate at the bottom of the cell. The chup1 gene encodes a protein equipped with several domains exhibiting putative functions, namely, the N-terminal hydrophobic, coiled-coil, F-actin-binding, proline-rich, and C-terminal conserved domains. CHUP1 protein translated in vitro was shown to associate with actin and profilin (Schmidt von Braun and Schleiff, 2008). However, whether CHUP1 can modulate the polymerization–depolymerization state of actin was not examined. Oikawa et al. (2008) precisely analysed the functions of N-terminal hydrophobic and coiled-coil domains in A. thaliana. In palisade mesophyll cells, CHUP1 is localized on the outer chloroplast envelope. This localization is dependent on the presence of its N-terminal hydrophobic domain, and is impaired by over-expression of GFP fused to the N-terminal hydrophobic domain, concomitant with the expression of the chup1 phenotype. On the other hand, the coiled-coil domain is important in determining the distribution of chloroplasts along the anticlinal walls of the cells. Oikawa et al. (2008) proposed that CHUP1 is an indispensable factor for the association of chloroplasts with the cortical cytoplasm and the actin cytoskeleton. Further Chloroplast anchoring | 3305 characterization of CHUP1, especially the modes of regulation of its functions and localization, will provide critical information on the mechanisms of chloroplast movement and positioning (Wada and Suetsugu, 2004). Augustynowicz et al. (2001) examined chloroplast movement in isolated protoplasts from tobacco leaves. They found that the responsiveness to light was markedly enhanced when intact leaves were subjected to cold stress before the isolation of protoplasts. The protoplasts isolated from cold-stressed leaves exhibited an increased rigidity, which was greatly antagonized by treatment with an actindepolymerizing reagent. Although cold-induced factors involved in regulation of the rigidity of protoplasts have not yet been identified, these results may support the idea that the actin cytoskeleton is an important structural component of the cortical cytoplasm, and that the interaction with intact cortical cytoplasm is required for proper chloroplast movement and positioning. Dissection of chloroplast anchoring using spinach To test the hypothesis that chloroplasts under physiologically stationary conditions are anchored on the cortical cytoplasm in an actin-dependent manner, cell biological dissection of the subject may be necessary. For this purpose, spinach was chosen as the preferred experimental system for the following reasons; first, blue-light-dependent chloroplast movement was observed (Inoue and Shibata, 1973; Kumatani et al., 2006) concomitant with the reorganization of the actin cytoskeleton (Kumatani et al., 2006; Fig. 1); second, immunoblotting analyses of biochemically prepared crudemicrosome and PM fractions detected actin as well as phot1 (Fig. 2). In A. thaliana, both phot1 and phot2 are predominantly localized on the PM, and some populations of them are relocalized to endomembranes after photoperception (Sakamoto and Briggs, 2002; Harada et al., 2003; Kong et al., 2006; Wan et al., 2008). Third, procedures for the preparation of protoplasts and isolated chloroplasts are well established (Walker, 1987). In this section, examination of the hypothesis described above is summarized using spinach, comparing results obtained in other plant species, together with future prospects. As described, the interaction of actin filaments with chloroplasts in vivo has been suggested in various types of plant cells (Takagi, 2003), while attempts to detect the interaction in vitro were assessed for the first time in spinach. In the final chloroplast fraction isolated from spinach leaves (Spinacia oleracea L. cv. Torai), endogenous actin was not detected, although these chloroplasts cosedimented with exogenously added skeletal muscle filamentous actin in vitro (Kumatani et al., 2006). Binding was strictly dependent on the intactness of the outer envelope of isolated chloroplasts, and was at least partially light sensitive (Kumatani et al., 2006). The interaction of exogenously added actin, regardless of its polymerization state, with isolated chloroplasts was demonstrated in Fig. 1. Actin organization in spinach mesophyll cells under different light conditions. Actin filaments in mesophyll cells either kept under low-fluence-rate white light (0.9 W m 2) (A) or exposed to high-fluence-rate blue light (460 nm, 15 W m 2) (C) were visualized by staining with fluorescent phalloidin. Under dim light, many fine bundles of actin filaments are associated with chloroplasts (B), whereas under strong blue light, much thicker, linear actin bundles run in the cytoplasm (D). Bar: 10 lm. a similar way in A. thaliana (Schmidt von Braun and Schleiff, 2008). Involvement of actin-binding proteins, such as CHUP1, in this interaction should be urgently examined. The interaction between chloroplasts and the cortical cytoplasm and/or PM may be crucial for proper chloroplast anchoring. The structure and function of plant cortical cytoplasm have been studied using PM ghosts. PM ghosts are prepared after rupture or lysis of protoplasts so as to expose the cortical cytoplasm underlying the PM. Experimental systems using PM ghosts have markedly contributed to our understanding of the cortical microtubule organization (Cyr, 1991; Sonobe and Takahashi, 1994), actin organization (Kobayashi, 1996), and the interaction between actin and microtubule cytoskeletons (Collings et al., 1998). During the development of tracheary elements in cultured mesophyll cells of Zinnia elegans, Kobayashi et al. (1987) observed reticulated arrays of actin filaments between chloroplasts and the PM. Since those structures disappeared at the stage of development when chloroplasts are detached from the PM, Kobayashi et al. (1987) concluded that the reticulated array of actin filaments functions to anchor chloroplasts to the cortical cytoplasm and/or PM. Kobayashi 3306 | Takagi et al. Fig. 2. Immunodetection of phototropins and actin in spinach leaf cells. The crude-microsome (CM) and plasma-membrane (PM) fractions prepared from spinach leaves were separated by SDSPAGE (10 lg in each lane). A polypeptide of 120 kDa was detected with the anti-phot1 antibody, but not with the anti-phot2 antibody. A polypeptide of 43 kDa was detected with the anti-actin antibody. (1996) confirmed the presence and disappearance of similar arrays of actin filaments on PM ghosts prepared from the mesophyll cells at various developmental stages. He further demonstrated that such arrays of actin filaments could be removed from the PM ghosts by washing with a high concentration of salt, suggesting an involvement of protein– protein interaction (Kobayashi, 1996). On PM ghosts prepared from spinach mesophyll protoplasts kept under dim white light, from which most of the chloroplasts were removed after gentle treatment with a detergent (Fig. 3A), fine bundles of actin filaments were seen in configurations suggesting that they had surrounded individual chloroplasts (Fig. 3B). By contrast, when PM ghosts were prepared from protoplasts exposed to highfluence-rate blue light (Fig. 3C), much thicker and straighter bundles of actin filaments were detected (Fig. 3D). These distinctive configurations of actin filaments are reminiscent of those observed in living spinach cells under similar light conditions (Fig. 1). The results suggest that light-induced reorganization of the actin cytoskeleton maintained some association with the cortical cytoplasm and/or PM, and especially, the reticulated array of actin filaments plays an important role to anchor chloroplasts on the cortical cytoplasm in spinach as well as Z. elegans. To date, there has been no indication that suggests an involvement of the activity of motor proteins in chloroplast anchoring. Without detergent treatment, more native PM ghosts, to which chloroplasts remain attached, can be prepared from spinach mesophyll cells (Fig. 4A). The actin cytoskeleton is also retained (Fig. 4B), and its organization seems to Fig. 3. Actin organization on PM ghosts prepared from spinach mesophyll protoplasts. After chemical fixation, PM ghosts were treated with a detergent to remove chloroplasts and then labeled with an anti-actin antibody. On the PM ghosts prepared from protoplasts kept under dim white light (A), the actin filaments have surrounded each chloroplast (B), as visualized in living cells (see Fig. 1B). By contrast, on the PM ghosts prepared from protoplasts exposed to strong blue light (C), the actin filaments form thicker, linear bundles (D), very similar to those observed in living cells (see Fig. 1D). Bar: 10 lm. surround each chloroplast on the PM ghost. When such PM ghosts were treated with an actin-depolymerizing reagent (Fig. 4C) or Ca2+ at physiologically relevant concentrations (Fig. 4D), detachment of chloroplasts from the PM ghosts was induced. More intriguingly, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7) almost completely blocked the effect of Ca2+ (Fig. 4E), while its derivative N-(6-aminohexyl)-1naphthalenesulphonamide (W-5) did not (Fig. 4F), suggesting that the mechanism of chloroplast anchoring in spinach mesophyll cells is under the control of the Ca2+-calmodulin system. Consequently, the PM ghosts prepared from spinach mesophyll cells preserve the intactness in terms of regulation of chloroplast anchoring on the cortical cytoplasm. Although the involvement of calmodulin in bluelight-dependent chloroplast movement was suggested in Lemna trisulca (Tla1ka and Fricker, 1999), its specific target process was not clear. The mode of regulation of actin organization by the Ca2+-calmodulin system and a possible involvement of actin-binding proteins should be investigated. Finally, elementary processes are proposed that are associated with the redistribution of chloroplasts in plant cells induced by environmental stimuli (Fig. 5). Under physiologically stable conditions, chloroplasts are anchored to the cortical cytoplasm and/or PM through their interaction with actin filaments. Although numerous Chloroplast anchoring | 3307 Fig. 5. A schematic model for the elementary processes associated with the redistribution of chloroplasts in plant cells. Environmental stimuli, such as blue light and mechanical touch, induce the redistribution of chloroplasts through the modulation of actin organization in the cortical cytoplasm. For further details, see the text. ABPs, actin-binding proteins; [Ca2+]cyt, cytoplasmic Ca2+ concentration. Fig. 4. Bright-field images of PM ghosts prepared from spinach mesophyll cells. When PM ghosts were prepared in the absence of detergent, the ghosts were occupied by numerous chloroplasts (A). Those chloroplasts seem to be tightly associated with actin filaments (red: chlorophyll autofluorescence, green: actin) (B). When the prepared PM ghosts were treated with the actindepolymerizing reagent latrunculin B (C, +LatB) or Ca2+ at 10 6 M (D, +Ca), many of the chloroplasts were detached from the PM ghosts. The effect of Ca2+ was antagonized by the calmodulin antagonist W-7 (E) but not by its derivative W-5 (F). Bar: 10 lm. functional actin-binding proteins have been identified and characterized in plant cells (Staiger, 2000; Higaki et al., 2007), at present, the only known factor involved in chloroplast anchoring is CHUP1. Essentially, there is no information about protein factors mediating the interaction of the actin cytoskeleton with the cortical cytoplasm and/or PM. Various kinds of environmental stimuli, including blue light (Harada and Shimazaki, 2007) and mechanical touch (Knight, 2000), which is also known to induce chloroplast movement (Makita and Shihira-Ishikawa, 1997; Sato et al., 1999, 2003), trigger a transient increase in the cytoplasmic Ca2+ concentration. Ca2+-sensitive actin-binding proteins may modulate the actin organization in the cortical cytoplasm, inducing a release of chloroplasts from the anchored state (de-anchoring). According to signals produced from the receptors, for example, phototropins and mechanosensory systems (Nakagawa et al., 2007), chloroplasts migrate to their intracellular destinations depending on the interaction of cytoskeletal components with specific motor proteins such as actomyosin systems (Shimmen and Yokota, 2004). Little is known about the identity of the various signals regulating chloroplast movements, namely, the direction and velocity of movement, to date. In their proper positions, chloroplasts are re-anchored through the reorganization of the actin cytoskeleton. Although an involvement of photosynthetic activity in the regulation has been suggested in several plant species, the factors involved in chloroplast re-anchoring are also elusive. Further multidisciplinary approaches to these elementary processes should shed light on the mechanism of actin-dependent chloroplast anchoring and positioning in plant cells and its significance for plant life. Acknowledgements We are grateful to the anonymous reviewers for numerous invaluable comments to improve this article. Thanks also go to Louis John Irving of Tohoku University for his critical reading of the manuscript. References Augustynowicz J, Lekka M, Burda K, Gabryś H. 2001. Correlation between chloroplast motility and elastic properties of tobacco mesophyll protoplasts. Acta Physiologiae Plantarum 23, 291–302. Chuong SDX, Franceschi VR, Edwards GE. 2006. 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