Brief irradiation with red or blue light induces orientational movement of chloroplasts in dark-adapted prothallial cells of the fernAdiantum

Orientational movement of chloroplasts was induced by a brief irradiation with red light (R) or blue light (B) in dark-adapted prothallial cells ofAdiantum, whose chloroplasts had gathered along the cell dividing wall (i.e., the anticlinal wall). When the whole dark-adapted prothallia were irradiated from a horizontal direction (i.e., from their lobes) with horizontally vibrating polarized R (H pol. R) for 10 or 3 min, the chloroplast left the anticlinal walls and spread over the prothallial surface (i.e., the periclinal walls) within 1–2 hr after the onset of irradiation, returning to the anticlinal wall (dark-position) within 10 hr. However, vertically vibrating polarized R (V pol. R) for 10 min did not induce the movement towards periclinal walls. The R effect was cancelled by non-polarized far-red light (FR) irradiation just after the R irradiation. Irradiation with H pol. B for 10 or 3 min but not with V pol. B could also induce a similar movement of chloroplasts, although the chloroplasts returned within 4 hr. The effect of H pol. B, however, was not cancelled by the subsequent FR irradiation. When a part of the dark-adapted cell at the prothallial surface was irradiated from above with a microbeam of R or B for 1 min, chloroplasts of the cell in the dark-position moved towards the irradiated locus in subsequent darkness. However, in the neighboring cells, orientational movement was not induced by either R or B microbeams. These results show that in dark-adapted prothallial cells, both brief irradiation with R and B can induce chloroplast photo-orientation and that the photoreceptors are phytochrome and blue light-absorbing pigment, respectively. It is also clear that effects of both R and B irradiation do not transfer to neighboring cells.     

Phytochrome- and blue-light-absorbing pigment-mediated directional movement of chloroplasts in dark-adapted prothallial cells of fernAdiantum as analyzed by microbeam irradiation

When prothalli ofAdiantum capillus-veneris L. were kept for 2 d in the dark, chloroplasts gathered along the anticlinal walls (Kagawa and Wada, 1994, J Plant Res 107: 389–398). In these dark-adapted prothallial cells, irradiation with a microbeam (10 gm in diameter) of red (R) or blue light (B) for 60 s moved the chloroplasts towards the irradiated locus during a subsequent dark period. Chloroplasts located less than 20 gm from the center of the R microbeam (18 J·m^–2) moved towards the irradiated locus. The higher the fluence of the light, the greater the distance from which chloroplasts could be attracted. The B microbeam was less effective than the R microbeam. Chloroplasts started to move anytime up to 20 min after the R stimulus, but with the B microbeam the effect of the stimulus was usually apparent within 10 min after irradiation. The velocity of chloroplast migration was independent of light-fluence in both R and B and was about - 0.3 m·min^–1 between 15 min and 30 min after irradiation. Whole-cell irradiation with far-red light immediately after R- and B-microbeam irradiations demonstrated that these responses were mediated by phytochrome and a blue-light-absorbing pigment, respectively. Sequential treatment with R and B microbeams, whose fluence rates were less than the threshold values when applied separately, resulted in an additive effect and induced chloroplast movement, strongly suggesting that signals from phytochrome and the blue-light-absorbing pigment could interact at some point before the induction of chloroplast movement.Abbreviations B blue light - FR far-red light - IR infrared light - R red light     

Phototropins and neochrome1 mediate nuclear movement in the fern Adiantum capillus-veneris

In gametophytic cells (prothalli) of the fern Adiantum capillus-veneris, nuclei as well as chloroplasts change their position according to light conditions. Nuclei reside on anticlinal walls in darkness and move to periclinal or anticlinal walls under weak or strong light conditions, respectively. Here we reveal that red light-induced nuclear movement is mediated by neochrome1 (neo1), blue light-induced movement is redundantly mediated by neo1, phototropin2 (phot2) and possibly phot1, and dark positioning of both nuclei and chloroplasts is mediated by phot2. Thus, both the nuclear and chloroplast photorelocation movements share common photoreceptor systems.     

Low temperature-induced chloroplast relocation mediated by a blue light receptor,phototropin 2, in fern gametophytes

Chloroplast movement in response to light has been known more than 100 years. Chloroplasts move towards weak light and move away from strong light. Dark-induced relocation, called dark positioning, has also been shown. However, the effects of other stimuli on chloroplast movement have not been well characterized. Here we studied low temperature-induced chloroplast relocation (termed cold positioning) in prothallial cells of the gametophytes of the fern Adiantum capillus-veneris. Under weak light chloroplasts in prothallial cells accumulated along the periclinal wall at 25 degrees C, but they moved towards anticlinal walls when the prothalli were subsequently transferred to 4 degrees C. A temperature shift from 25 degrees to 10 degrees C or lower was enough to induce cold positioning, and high-intensity light enhanced the response. Nuclei also relocated from the periclinal position (a position along periclinal walls) to the anticlinal position (a position along anticlinal walls) under cold temperature, whereas mitochondria did not. Cold positioning was not observed in mutant fern gametophytes defective of the blue light photoreceptor, phototropin 2.     

Blue light-induced chloroplast relocation in Arabidopsis thaliana as analyzed by microbeam irradiation

Chloroplast relocation in mesophyll cells of Arabidopsis thaliana was observed microscopically and analyzed by microbeam irradiation. Chloroplasts located along the anticlinal walls in dark-adapted cells. When part of a cell was irradiated with a microbeam of high fluence rate blue light (B) simultaneously with background red light (R) on the whole cell, the chloroplasts moved towards the B-irradiated area, but did not enter the beam. The background R illumination activated cytoplasmic motility as well as chloroplast movement. Without R illumination, there was little chloroplast relocation. In light-adapted cells in which the chloroplasts were spread over the cell surface perpendicular to the incident light, R-illumination had the same effect. Under background R, the chloroplasts moved out of the area irradiated with a B microbeam of 8 or 30 W m(-2) (avoidance response), but chloroplasts outside the beam moved towards the area irradiated with the B microbeam (accumulation response). These results suggest that the signals for accumulation and avoidance responses were generated in a single cell by high fluence rate B. cry1cry2, npq1 and nph1 mutants showed B-induced chloroplast relocation. Both the accumulation and avoidance responses were observed in all the mutants, although in the nph1 mutant, the sensitivity of accumulation movement was slightly lower than that of the wild type. We discuss the possible photoreceptor for B-induced chloroplast relocation.     

Actin-based mechanisms for light-dependent intracellular positioning of nuclei and chloroplasts in Arabidopsis

The plant organelles, chloroplast and nucleus, change their position in response to light. In Arabidopsis thaliana leaf cells, chloroplasts and nuclei are distributed along the inner periclinal wall in darkness. In strong blue light, they become positioned along the anticlinal wall, while in weak blue light, only chloroplasts are accumulated along the inner and outer periclinal walls. Blue-light dependent positioning of both organelles is mediated by the blue-light receptor phototropin and controlled by the actin cytoskeleton. Interestingly, however, it seems that chloroplast movement requires short, fine actin filaments organized at the chloroplast edge, whereas nuclear movement does cytoplasmic, thick actin bundles intimately associated with the nucleus. Although there are many similarities between photo-relocation movements of chloroplasts and nuclei, plant cells appear to have evolved distinct mechanisms to regulate actin organization required for driving the movements of these organelles.Key words: actin, Arabidopsis, blue light, chloroplast positioning, phototropin, nuclear positioning     

Photosynthesis-dependent but neochrome1-independent light positioning of chloroplasts and nuclei in the fern Adiantum capillus-veneris

Chloroplasts change their positions in the cell depending on the light conditions. In the dark, chloroplasts in fern prothallia locate along the anticlinal wall (dark position). However, chloroplasts become relocated to the periclinal wall (light position) when the light shines perpendicularly to the prothallia. Red light is effective in inducing this relocation in Adiantum capillus-veneris, and neochrome1 (neo1) has been identified as the red light receptor regulating this movement. Nevertheless, we found here that chloroplasts in neo1 mutants still become relocated from the dark position to the light position under red light. We tested four neo1 mutant alleles (neo1-1, neo1-2, neo1-3, and neo1-4), and all of them showed the red-light-induced chloroplast relocation. Furthermore, chloroplast light positioning under red light occurred also in Pteris vittata, another fern species naturally lacking the neo1-dependent phenomenon. The light positioning of chloroplasts occurred independently of the direction of red light, a response different to that of the neo1-dependent movement. Photosynthesis inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-isopropyl-6-methyl-p-benzoquinone blocked this movement. Addition of sucrose (Suc) or glucose to the culture medium induced migration of the chloroplasts to the periclinal wall in darkness. Furthermore, Suc could override the effects of 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Interestingly, the same light positioning was evident for nuclei under red light in the neo1 mutant. The nuclear light positioning was also induced in darkness with the addition of Suc or glucose. These results indicate that photosynthesis-dependent nondirectional movement contributes to the light positioning of these organelles in addition to the neo1-dependent directional movement toward light.     

Speed of signal transfer in the chloroplast accumulation response

Chloroplast photorelocation movement is important for plants to perform efficient photosynthesis. Phototropins were identified as blue-light receptors for chloroplast movement in Arabidopsis thaliana and in the fern Adiantum capillus-veneris, whereas neochrome functions as a dual red/blue light receptor in the latter. However, the signal transduction pathways involved in chloroplast movement remain to be clarified. To investigate the kinetic properties of signalling from these photoreceptors to the chloroplasts, we deduced the speed of signal transfer using Adiantum capillus-veneris gametophytes. When a region of dark-adapted gametophyte cells was subjected to microbeam irradiation, chloroplasts moved towards the irradiated area even in subsequent darkness. We therefore recorded the movement and calculated the speeds of signal transfer by time-lapse imaging. Movement speeds under red or blue light were similar, e.g., about 1.0 μm min⁻¹ in prothallial cells. However, speeds varied according to cell polarity in protonemal cells. The speed of signal transfer from the protonemal apex to the base was approximately 0.7 μm min⁻¹, but roughly 2.3 μm min⁻¹ in the opposite direction. The speed of signal transfer in Arabidopsis thaliana mesophyll cells was approximately 0.8 μm min⁻¹ by comparison. Surprisingly, chloroplasts located farthest away from the microbeam were found to move faster than those in close proximity to the site of irradiation both in Adiantum capillus-veneris and A. thaliana.     

Light,genotype, and abscisic acid affect chloroplast positioning in guard cells of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves in distinct ways

The goal of this study was to investigate the effects of light intensity, genotype, and various chemical treatments on chloroplast movement in guard cells of Arabidopsis thaliana leaves. After treatment at various light intensities (dark, low, and high light), leaf discs were fixed with glutaraldehyde, and imaged using confocal laser microscopy. Each chloroplast was assigned a horizontal (close to pore, center, or epidermal side) and vertical (outer, middle, inner) position. White light had a distinct effect on chloroplast positioning, most notably under high light (HL) when chloroplasts on the upper leaf surface of wild-type (WT) moved from epidermal and center positions toward the pore. This was not the case for phot1-5/phot2-1 or phot2-1 plants, thus phototropins are essential for chloroplast positioning in guard cells. In npq1-2 mutants, fewer chloroplasts moved to the pore position under HL than in WT plants, indicating that white light can affect chloroplast positioning also in a zeaxanthin-dependent way. Cytochalasin B inhibited the movement of chloroplasts to the pore under HL, while oryzalin did not, supporting the idea that actin plays a role in the movement. The movement along actin cables is dependent on CHUP1 since chloroplast positioning in chup1 was significantly altered. Abscisic acid (ABA) caused most chloroplasts in WT and phot1-5/phot2-1 to be localized in the center, middle part of the guard cells irrespective of light treatment. This indicates that not only light but also water stress influences chloroplast positioning.     

Chloroplasts can move in any direction to avoid strong light

Chloroplasts migrate in response to different light intensities. Under weak light, chloroplasts gather at an illuminated area to maximize light absorption and photosynthesis rates (the accumulation response). In contrast, chloroplasts escape from strong light to avoid photodamage (the avoidance response). Photoreceptors involved in these phenomena have been identified in Arabidopsis thaliana and Adiantum capillus-veneris. Chloroplast behavior has been studied in detail during the accumulation response, but not for the avoidance response. Hence, we analyzed the chloroplast avoidance response in detail using dark-adapted Adiantum capillus-veneris gametophyte cells and partial cell irradiation with a microbeam of blue light. Chloroplasts escaped from an irradiated spot. Both duration of this response and the distance of the migrated chloroplasts were proportional to the total fluence irradiated. The speed of movement during the avoidance response was dependent on the fluence rate, but the speed of the accumulation response towards the microbeam from cell periphery was constant irrespective of fluence rate. When a chloroplast was only partially irradiated with a strong microbeam, it moved away towards the non-irradiated region within a few minutes. During this avoidance response two additional microbeam irradiations were applied to different locus of the same chloroplast. Under these conditions the chloroplast changed the moving direction after a lag time of a few minutes without rolling. Taken together, these findings indicate that chloroplasts can move in any direction and never have an intrinsic polarity. Similar phenomenon was observed in chloroplasts of Arabidopsis thaliana palisade cells.     

Light perception and the role of the xanthophyll cycle in blue-light-dependent chloroplast movements in lemna trisulca L

In most higher plants, chloroplasts move towards the periclinal cell walls in weak blue light (WBL) to increase light harvesting for photosynthesis, and towards the anticlinal walls as an escape reaction, thus avoiding photo-damage in strong blue light (SBL). The photo- receptor(s) triggering these responses have not yet been identified. In this study, the role of zeaxanthin as a blue-light photoreceptor in chloroplast movements was investigated. Time-lapse 3D confocal imaging in Lemna trisulca showed that individual chloroplasts responded to local illumination when one half of the cell was treated with light of different intensity or spectral quality to that received by the other half, or was maintained in darkness. Thus the complete signal perception, transduction and effector system has a high degree of spatial resolution and is consistent with localization of part of the transduction chain in the chloroplasts. Turnover of xanthophylls was determined using HPLC, and a parallel increase was observed between zeaxanthin and chloroplast movements in SBL. Ascorbate stimulated both a transient increase in zeaxanthin levels and chloroplast movement to profile in physiological darkness. Conversely, dithiothreitol blocked zeaxanthin production and responses to SBL and, to a lesser extent, WBL. Norflurazon preferentially inhibited SBL-dependent chloroplast movements. Increases in zeaxanthin were also observed in strong red light (SRL) when no directional chloroplast movements occurred. Thus it appears that a combination of zeaxanthin and blue light is required to trigger responses. Blue light can cause cis-trans isomerization of xanthophylls, thus photo-isomerization may be a critical link in the signal transduction pathway.     

Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells

The plant nucleus changes its intracellular position not only upon cell division and cell growth but also in response to environmental stimuli such as light. We found that the nucleus takes different intracellular positions depending on blue light in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in mesophyll cells were positioned at the center of the bottom of cells (dark position). Under blue light at 100 mumol m(-2) s(-1), in contrast, nuclei were located along the anticlinal walls (light position). The nuclear positioning from the dark position to the light position was fully induced within a few hours of blue light illumination, and it was a reversible response. The response was also observed in epidermal cells, which have no chloroplasts, suggesting that the nucleus has the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced specifically by blue light at >50 mumol m(-2) s(-1). Furthermore, the response to blue light was induced in phot1 but not in phot2 and phot1phot2 mutants. Unexpectedly, we also found that nuclei as well as chloroplasts in phot2 and phot1phot2 mutants took unusual intracellular positions under both dark and light conditions. The lack of the response and the unusual positioning of nuclei and chloroplasts in the phot2 mutant were recovered by externally introducing the PHOT2 gene into the mutant. These results indicate that phot2 mediates the blue light-dependent nuclear positioning and the proper positioning of nuclei and chloroplasts. This is the first characterization of light-dependent nuclear positioning in spermatophytes.     

Chloroplasts do not have a polarity for light-induced accumulation movement

Chloroplast photorelocation movement in green plants is generally mediated by blue light. However, in cryptogam plants, including ferns, mosses, and algae, both red light and blue light are effective. Although the photoreceptors required for this phenomenon have been identified, the mechanisms underlying this movement response are not yet known. In order to analyze this response in more detail, chloroplast movement was induced in dark-adapted Adiantum capillus-veneris gametophyte cells by partial cell irradiation with a microbeam of red and/or blue light. In each case, chloroplasts were found to move toward the microbeam-irradiated area. A second microbeam was also applied to the cell at a separate location before the chloroplasts had reached the destination of the first microbeam. Under these conditions, chloroplasts were found to change their direction of movement without turning and move toward the second microbeam-irradiated area after a lag time of a few minutes. These findings indicate that chloroplasts can move in any direction and do not exhibit a polarity for chloroplast accumulation movement. This phenomenon was analyzed in detail in Adiantum and subsequently confirmed in Arabidopsis thaliana palisade cells. Interestingly, the lag time for direction change toward the second microbeam in Adiantum was longer in the red light than in the blue light. However, the reason for this discrepancy is not yet understood. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Chloroplast avoidance movement is not functional in plants grown under strong sunlight

Chloroplast movement in nine climbing plant species was investigated. It is thought that chloroplasts generally escape from strong light to avoid photodamage but accumulate towards weak light to perform photosynthesis effectively. Unexpectedly, however, the leaves of climbing plants grown under strong sunlight showed very low or no chloroplast photorelocation responses to either weak or strong blue light when detected by red light transmittance through leaves. Direct observations of Cayratia japonica leaves, for example, revealed that the average number of chloroplasts in upper periclinal walls of palisade tissue cells was only 1.2 after weak blue‐light irradiation and almost all of the chloroplasts remained at the anticlinal wall, the state of chloroplast avoidance response. The leaves grown under strong light have thin and columnar palisade tissue cells comparing with the leaves grown under low light. Depending on our analyses and our schematic model, the thinner cells in a unit leaf area have a wider total plasma membrane area, such that more chloroplasts can exist on the plasma membrane in the thinner cells than in the thicker cells in a unit leaf‐area basis. The same strategy might be used in other plant leaves grown under direct sunlight.     

How and why do plant nuclei move in response to light?

We recently found that nuclei take different intracellular positions depending upon dark and light conditions in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in both epidermal and mesophyll cells are distributed baso-centrally within the cell (dark position). Under light conditions, in contrast, nuclei are distributed along the anticlinal walls (light position). Nuclear repositioning from the dark to light positions is induced specifically by blue light at >50 µmol m⁻² s⁻¹ in a reversible manner. Using analysis of mutant plants, it was demonstrated that the response is mediated by the blue-light photoreceptor phototropin2. Intriguingly, phototropin2 also seems to play an important role in the proper positioning of nuclei and chloroplasts under dark conditions. Light-dependent nuclear positioning is one of the organelle movements regulated by phototropin2. However, the mechanisms of organelle motility, physiological significance, and generality of the phenomenon are poorly understood. In this addendum, we discussed how and why nuclei move depending on light, together with future perspectives.Key words: actin, Arabidopsis, blue light, cytoskeleton, nuclear positioning, nucleus, phototropin     

Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.     

The role of calcium in blue-light-dependent chloroplast movement in lemna trisulca L

Chloroplast movements are a normal physiological response to changes in light intensity and provide a good model system to analyse the signal transduction pathways following light perception. Blue-light-dependent chloroplast movements were observed in Lemna trisulca using confocal optical sectioning and 3-D reconstruction or photometric measurements of leaf transmission. Chloroplasts moved away from strong blue light (SBL) towards the anticlinal walls (profile position), and towards the periclinal walls (face position) under weak blue light (WBL) over about 20-40 min. Cytoplasmic calcium ([Ca2 + ]cyt) forms part of the signalling system in response to SBL as movements were associated with small increases in [Ca2 + ]cyt and were blocked by antagonists of calcium homeostasis, including EGTA, nifedipine, verapamil, caffeine, thapsigargin, TFP (trifluoperazine), W7 and compound 48/80. Treatments predicted to affect internal Ca2 + stores gave the most rapid and pronounced effects. In addition, artificially increasing [Ca2 + ]cyt in darkness using the Ca2 + ionophore A23187 and high external Ca2 + (or Sr2 + ), triggered partial movement of chloroplasts to profile position analogous to a SBL response. These data are all consistent with [Ca2 + ]cyt acting as a signal in SBL responses; however, the situation is more complex given that both WBL and SBL responses were inhibited to a similar extent by all the Ca2 + -signalling antagonists used. As the direction of chloroplast movement in WBL is exactly opposite to that in SBL, we conclude that, whilst proper regulation of [Ca2 + ]cyt homeostasis is critical for both SBL and WBL responses, additional factors may be required to specify the direction of chloroplast movement.     

Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis

Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 micromol m(-2) s(-1) of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 micromol m(-2) s(-1) caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 micromol m(-2) s(-1), chloroplasts in pmi2 leaves move to the periclinal walls; 100 micromol m(-2) s(-1) of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.     

Photoinduction of formation of circular structures by microfilaments on chloroplasts during intracellular orientation in protonemal cells of the fernAdiantum capillus-veneris

Summary Changes in the organization of cortical actin microfilaments during phytochrome-mediated and blue light-induced photoorientation of chloroplasts were investigated by rhodamine-phalloidin staining in protonemal cells of the fernAdiantum capillusveneris. Low- and high-fluence rate responses were induced by partial irradiation of individual cells with a microbeam of 20 m in width. In the low-fluence rate responses to red and blue light, a circular structure composed of microfilaments was induced on the chloroplast concentrated in the irradiated region, on the side facing the plasma membrane, as already reported in the case of the low-fluence rate response induced by polarized red or blue light. Such a structure was not observed on the chloroplasts located far from the microbeam. Time-course studies revealed that the structure was induced after the chloroplasts gathered in the illuminated region and that the structure disappeared before chloroplasts moved out of this region when the microbeam was turned off. In the high-fluence rate response to blue light, chloroplasts avoided the irradiated site but accumulated in the shaded area adjacent the edges of microbeam. The circular structure made of microfilaments was also observed on the chloroplasts gathered in the area and it showed the same behavior with respect to its appearance and disappearance during a light/dark regime as in the case of the low-fluence rate response. However, no such circular structure was observed in the high-fluence rate response to red light, in which case the chloroplasts also avoided the illuminated region but no accumulation in the adjacent areas was induced. These results indicate that the circular structure composed of microfilaments may play a role in the anchorage of the chloroplast during intracellular photo-orientation.