A Protein Phosphatase 2A Catalytic Subunit Modulates Blue Light-Induced Chloroplast Avoidance Movements through Regulating Actin Cytoskeleton in Arabidopsis

Chloroplast avoidance movements mediated by phototropin 2 (phot2) are one of most important physiological events in the response to high-fluence blue light (BL), which reduces damage to the photosynthetic machinery under excess light. Protein phosphatase 2A-2 (PP2A-2) is an isoform of the catalytic subunit of PP2A, which regulates a number of developmental processes. To investigate whether PP2A-2 was involved in high-fluence BL-induced chloroplast avoidance movements, we first analyzed chloroplast migration in the leaves of the pp2a-2 mutant in response to BL. The data showed that PP2A-2 might act as a positive regulator in phot2-mediated chloroplast avoidance movements, but not in phot1-mediated chloroplast accumulation movements. Then, the effect of okadaic acid (OA) and cantharidin (selective PP2A inhibitors) on high-fluence BL response was further investigated in Arabidopsis thaliana mesophyll cells. Within a certain concentration range, exogenously applied OA or cantharidin inhibited the high-fluence BL-induced chloroplast movements in a concentration-dependent manner. Actin depolymerizing factor (ADF)/cofilin phosphorylation assays demonstrated that PP2A-2 can activate/dephosphorylate ADF/cofilin, an actin-binding protein, in Arabidopsis mesophyll cells. Consistent with this observation, the experiments showed that OA could inhibit ADF1 binding to the actin and suppress the reorganization of the actin cytoskeleton after high-fluence BL irradiation. The adf1 and adf3 mutants also exhibited reduced high-fluence BL-induced chloroplast avoidance movements. In conclusion, we identified that PP2A-2 regulated the activation of ADF/cofilin, which, in turn, regulated actin cytoskeleton remodeling and was involved in phot2-mediated chloroplast avoidance movements.     

Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement

The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.     

Phototropin Mediated Relocation of Myosins in Arabidopsis thaliana

Background

The mechanism of the light-dependent movements of chloroplasts is based on actin and myosin but its details are largely unknown. The movements are activated by blue light in terrestrial angiosperms. The aim of the present study was to determine the role of myosin associated with the chloroplast surface in the light-induced chloroplast responses in Arabidopsis thaliana. The localization of myosins was investigated under blue light intensities generating avoidance and accumulation responses of chloroplasts. The localization was compared in wild type plants and in phot2 mutant lacking the avoidance response.

Results

Wild type and phot2 mutant plants were irradiated with strong (36 µEm⁻²s⁻¹) and/or weak (0.8 µEm⁻²s⁻¹) blue light. The leaf tissue was immunolabeled with antimyosin antibodies. Different arrangements of myosins were observed in the mesophyll depending on the fluence rate in wild type plants. In tissue irradiated with weak blue light myosins were associated with chloroplast envelopes. In contrast, in tissue irradiated with strong blue light chloroplasts were almost myosin-free. The effect did not occur in red light and in the phot2 mutant.

Conclusions

Myosin displacement is blue light specific, i.e., it is associated with the activation of a specific blue-light photoreceptor. We suggest that the reorganization of myosins is essential for chloroplast movement. Myosins appear to be the final step of the signal transduction pathway starting with phototropin2 and leading to chloroplast movements.Key Words: Arabidopsis, blue light, chloroplast movements, myosins, phototropins     

Blue light-induced chloroplast relocation

Chloroplast relocation movement is induced by blue light in most plants tested. Under weak light, chloroplasts move toward a brighter area in a cell (called low-fluence-rate response or accumulation movement), but they avoid strong light and move away from the light (called high-fluence-rate response or avoidance movement). Recently, mutants deficient in the chloroplast avoidance movement were isolated from Arabidopsis thaliana. The results of mutant analyses revealed that the phototropin photoreceptors phot1 and phot2 both control chloroplast accumulation while phot2 alone controls the avoidance movements.     

Arabidopsis thaliana leaves with altered chloroplast numbers and chloroplast movement exhibit impaired adjustments to both low and high light

The effects of chloroplast number and size on the capacity for blue light-dependent chloroplast movement, the ability to increase light absorption under low light, and the susceptibility to photoinhibition were investigated in Arabidopsis thaliana. Leaves of wild-type and chloroplast number mutants with mean chloroplast numbers ranging from 120 to two per mesophyll cell were analysed. Chloroplast movement was monitored as changes in light transmission through the leaves. Light transmission was used as an indicator of the ability of leaves to optimize light absorption. The ability of leaves to deal with 3 h of high light stress at 10 degrees C and their capacity to recover in low light was determined by measuring photochemical efficiencies of PSII using chlorophyll a fluorescence. Chloroplast movement was comparable in leaves ranging in chloroplast numbers from 120 to 30 per mesophyll cell: the final light transmission levels after exposure to 0.1 (accumulation response) and 100 micromol photons m(-2) s(-1) (avoidance response) were indistinguishable, the chloroplasts responded quickly to small increases in light intensity and the kinetics of movement were similar. However, when chloroplast numbers per mesophyll cell decreased to 18 or below, the accumulation response was significantly reduced. The avoidance response was only impaired in mutants with nine or fewer chloroplasts, both in terms of final transmission levels and the speed of movement. Only mutants lacking both blue light receptors (phot1/phot2) or those with drastically reduced chloroplast numbers and severely impacted avoidance responses showed a reduced ability to recover from high light stress.     

Rapid Severing and Motility of Chloroplast-Actin Filaments Are Required for the Chloroplast Avoidance Response in Arabidopsis

Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light–induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light’s intensity and position.     

In vivo reorganization of the actin cytoskeleton in leaves of Nicotiana tabacum L. transformed with plastin-GFP. Correlation with light-activated chloroplast responses

Background  

The actin cytoskeleton is involved in the responses of plants to environmental signals. Actin bundles play the role of tracks in chloroplast movements activated by light. Chloroplasts redistribute in response to blue light in the mesophyll cells of Nicotiana tabacum. The aim of this work was to study the relationship between chloroplast responses and the organization of actin cytoskeleton in living tobacco cells. Chloroplast movements were measured photometrically as changes in light transmission through the leaves. The actin cytoskeleton, labeled with plastin-GFP, was visualised by confocal microscopy.     

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     

Chloroplast photorelocation movement mediated by phototropin family proteins in green plants

Chloroplasts gather in areas irradiated with weak light to maximize photosynthesis (the accumulation response). They move away from areas irradiated with strong light to minimize damage of the photosynthetic apparatus (the avoidance response). The processes underlying these chloroplast movements can be divided into three parts: photoperception, signal transduction, and chloroplast movement. Photoreceptors for chloroplast movement have been identified recently in various plant species. A blue light receptor phototropin (phot) mediates chloroplast photorelocation movement in the seed plant Arabidopsis thaliana, the fern Adiantum capillus-veneris, the moss Physcomitrella patens and possibly the green alga Mougeotia scalaris. A chimeric photoreceptor between phytochrome and phototropin, neochrome (neo), was found in some advanced ferns and in the green alga M. scalaris. While the mechanism of chloroplast movement is not well understood, it is known that actin filaments play an important role in this process. To understand the molecular mechanisms associated with chloroplast movement, several mutants were isolated in A. thaliana (jac1 and chup1) and the corresponding genes were cloned. In this review, recent progress in photoreceptor research into chloroplast movement in various plant species and the possible factors functioning in signal transduction or the regulation of actin filaments identified in A. thaliana is discussed.     

The chloroplast avoidance response decreases internal conductance to CO2 diffusion in Arabidopsis thaliana leaves

The relationship between chloroplast arrangement and diffusion of CO(2) from substomatal cavities to the chloroplast stroma was investigated in Arabidopsis thaliana. Chloroplast position was manipulated by varying the amount of blue light and by cytochalasin D (CytD) treatment. We also investigated two chloroplast positioning mutants. Chloroplast arrangement was assessed by the surface area of chloroplasts adjacent to intercellular airspaces (S(c)). Although it has been previously shown that long-term acclimation to high light is linked with a large S(c), we found that the short-term chloroplast avoidance response reduces S(c). This effect was not apparent in the blue-light-insensitive phot2 mutant, which did not show the avoidance response. As expected, the smaller S(c) induced by the avoidance response was coupled to a similar decrease in internal conductance. This reduction in internal conductance resulted in an increased limitation of the rate of photosynthesis. The limiting effect of S(c) on internal conductance and photosynthesis was also shown in chup1, a mutant with a constant small S(c) as the result of an unusual chloroplast arrangement. We conclude that chloroplast movements in A. thaliana can rapidly alter leaf morphological parameters, and this has significant consequences for the diffusion of CO(2) through the mesophyll.     

Blue light-induced orientation movements of chloroplasts in higher plants: Recent progress in the study of their mechanisms

The discovery of phototropins, photoreceptors for chloroplast responses in Arabidopsis thaliana, brought about renewed interest in these blue light-controlled movements. Recent progress in research on their mechanisms in higher plants is briefly summarized. Phototropins mediate phototropism, chloroplast relocations and stomatal movements. Their functions are partially overlapping, with phot1 active predominantly in weak light and phot2 active in strong light. The accumulation response of chloroplasts appears to be mediated by phot1 and phot2 whereas the avoidance response is controlled by phot2. The role of Ca²⁺ as a potential intracellular messenger has been discussed in view of the recently demonstrated blue light-induced transient increases in the cytosolic Ca²⁺ mediated differently by phot1 and phot2. Differential inhibition of accumulation and avoidance responses by wortmannin, the inhibitor of phosphoinositide-3 kinases, in Lemna trisulca points to an important role of these enzymes in the signal transduction. A new, multi-domain component controlling chloroplast positioning and movement, CHUP1, encodes an actin-binding protein in Arabidopsis.     

Light-induced chloroplast movements in Lemna trisulca. Identification of the motile system

In mesophyll cells of the water plant Lemna trisulca L. chloroplasts redistribute in response to blue light. In the present study it is shown that an actin depolymerizing agent cytochalasin D, a crosslinker of actin subunits in F-actin m-maleimidobenzoic acid N-hydroxysuccinimide ester (MBS) as well as N-ethylmaleimide (NEM)—a sulfhydryl group reagent, are potent inhibitors of these blue light-induced chloroplast movements in Lemna. Extraction with cold, buffered glycerol solution preserves light-induced chloroplast arrangements within cells producing permeabilized cell models. ‘Reactivation’ of these cell models by Mg-ATP results in remarkable movements which can be inhibited by treatment with NEM and cytochalasin D. Immunofluorescence microscopy demonstrates that a component which is associated with isolated Lemna chloroplasts cross-reacts with antibodies directed against bovine myosin. These results indicate that a contractile actomyosin system is involved in blue light-induced chloroplast movements in Lemna and a putative motor protein, similar to myosin, is associated with the surface of Lemna chloroplasts.     

A plant-specific protein essential for blue-light-induced chloroplast movements

In Arabidopsis (Arabidopsis thaliana), light-dependent chloroplast movements are induced by blue light. When exposed to low fluence rates of light, chloroplasts accumulate in periclinal layers perpendicular to the direction of light, presumably to optimize light absorption by exposing more chloroplast area to the light. Under high light conditions, chloroplasts become positioned parallel to the incoming light in a response that can reduce exposure to light intensities that may damage the photosynthetic machinery. To identify components of the pathway downstream of the photoreceptors that mediate chloroplast movements (i.e. phototropins), we conducted a mutant screen that has led to the isolation of several Arabidopsis mutants displaying altered chloroplast movements. The plastid movement impaired1 (pmi1) mutant exhibits severely attenuated chloroplast movements under all tested fluence rates of light, suggesting that it is a necessary component for both the low- and high-light-dependant chloroplast movement responses. Analysis of pmi1 leaf cross sections revealed that regardless of the light condition, chloroplasts are more evenly distributed in leaf mesophyll cells than in the wild type. The pmi1-1 mutant was found to contain a single nonsense mutation within the open reading frame of At1g42550. This gene encodes a plant-specific protein of unknown function that appears to be conserved among angiosperms. Sequence analysis of the protein suggests that it may be involved in calcium-mediated signal transduction, possibly through protein-protein interactions.     

Actin-organelle interaction: association with chloroplast in arabidopsis leaf mesophyll cells.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.     

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.     

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.     

Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement

Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.     

Actin Reorganization Underlies Phototropin-Dependent Positioning of Nuclei in Arabidopsis Leaf Cells

In epidermal and mesophyll cells of Arabidopsis (Arabidopsis thaliana) leaves, nuclei become relocated in response to strong blue light. We previously reported that nuclear positions both in darkness and in strong blue light are regulated by the blue light receptor phototropin2 in mesophyll cells. Here, we investigate the involvement of phototropin and the actin cytoskeleton in nuclear positioning in epidermal cells. Analysis of geometrical parameters revealed that, in darkness, nuclei were distributed near the center of the cell, adjacent to the inner periclinal wall, independent of cell shape. Dividing the anticlinal wall into concave, convex, and intermediate regions indicated that, in strong blue light, nuclei became relocated preferably to a concave region of the anticlinal wall, nearest the center of the cell. Mutant analyses verified that light-dependent nuclear positioning was regulated by phototropin2, while dark positioning of nuclei was independent of phototropin. Nuclear movement was inhibited by an actin-depolymerizing reagent, latrunculin B, but not by a microtubule-disrupting reagent, propyzamide. Imaging actin organization by immunofluorescence microscopy revealed that thick actin bundles, periclinally arranged parallel to the longest axis of the epidermal cell, were associated with the nucleus in darkness, whereas under strong blue light, the actin bundles, especially in the vicinity of the nucleus, became arranged close to the anticlinal walls. Light-dependent changes in the actin organization were clear in phot1 mutant but not in phot2 and phot1phot2 mutants. We propose that, in Arabidopsis, blue-light-dependent nuclear positioning is regulated by phototropin2-dependent reorganization of the actin cytoskeleton.Positioning organelles is essential for cellular activities. The nucleus changes its position in a programmatic way during development and the cell cycle (Britz, 1979; Nagai, 1993; Chytilova et al., 2000). For example, before asymmetrical divisions that give rise to the formation of root hair cells or guard mother cells, the nucleus migrates to the future division plane (Britz, 1979). In elongating root hair cells of Arabidopsis (Arabidopsis thaliana), the nucleus is maintained at a fixed distance from the apex (Ketelaar et al., 2002).While the nuclear migrations before mitosis and in root hairs are developmental, nuclear positioning is also regulated environmentally. In the fern, Adiantum capillus-veneris, nuclei in prothallial cells change their intracellular positions in response to light (Kagawa and Wada, 1993, 1995). The nuclei are located along the anticlinal walls in darkness and move toward the outer periclinal walls in weak light and to the anticlinal walls in strong light (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). This response is called light-dependent nuclear positioning. Since the response is induced in cells that exhibit neither cell division nor expansion, it is believed to have a physiological role, distinct from the nuclear positioning associated with development.Recently, light-dependent nuclear positioning was reported in the spermatophyte Arabidopsis (Iwabuchi et al., 2007). In epidermal and mesophyll cells of dark-treated leaves, nuclei are distributed along the inner periclinal wall. Under strong light, they become located along the anticlinal walls. In mesophyll cells, nuclear movement from inner periclinal to anticlinal walls is induced repeatedly and specifically by blue light of high-fluence rate (more than 50 μ mol m⁻² s⁻¹) and is regulated by the blue light receptor phototropin2. Interestingly, mesophyll cells of the phot2 mutant have aberrantly positioned nuclei even in darkness. By contrast, the involvement of phototropins in nuclear positioning has not yet been examined for epidermal cells.Phototropin is a blue light receptor containing two light oxygen voltage domains at the N terminus, which bind an FMN chromophore, and a Ser/Thr kinase domain at the C terminus, which undergoes blue-light-dependent autophosphorylation (Briggs et al., 2001a; Christie, 2007). Arabidopsis possesses phototropins1 and 2 (Huala et al., 1997; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Phototropins are shown microscopically and biochemically to localize to the plasma membrane region (Briggs et al., 2001b; Sakamoto and Briggs, 2002; Kong et al., 2006) and mediate several responses, including phototropism (Liscum and Briggs, 1995; Sakai et al., 2001), stomatal opening (Kinoshita et al., 2001), and chloroplast movements (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). In general, phototropin1 is more sensitive to light than its paralog and mediates low-fluence-rate light responses, whereas phototropin2 functions predominantly under higher fluence rates (Sakai et al., 2001).While the photoreceptor eliciting these nuclear movements has been revealed, the motile system responsible for moving the nuclei is still unknown. In general, organelle movements depend on the cytoskeleton, with the specific roles for actin and microtubules dependent on the organelle and species (Wada and Suetsugu, 2004). In land plants, the actin cytoskeleton plays a pivotal role in positioning organelles, including nuclei, chloroplasts, mitochondria, and peroxisomes (Wada and Suetsugu, 2004; Takagi et al., 2009).The role of the cytoskeleton in developmental nuclear movements has been investigated. In growing root hairs of Arabidopsis, the nuclear movements are driven along actin filaments (Ketelaar et al., 2002), whereas, in tobacco (Nicotiana tabacum) BY-2 cells, the cell-cycle-based nuclear migration before mitosis is found to depend on microtubules (Katsuta et al., 1990). In interphase Spirogyra crassa cells, centering of nuclei is regulated by both actin filaments and microtubules, but in distinct ways (Grolig, 1998). To the best of our knowledge, the cytoskeletal basis of environmentally induced nuclear movements in land plants has not been elucidated.The best-characterized organelle movements are the light-induced orientation movements of chloroplasts, and although exceptions have been reported, this movement depends on actin (Britz, 1979; Takagi, 2003; Wada et al., 2003). Under weak light, chloroplasts gather at the periclinal walls, perpendicular to the direction of light (accumulation response), whereas under strong light, they become positioned along the anticlinal walls, parallel to the direction of light (avoidance response). Recently, for Arabidopsis, Kadota et al. (2009) characterized the nature of the actin filaments probably involved in these movements. With the onset of either accumulation or avoidance response, short actin filaments appear at the leading edge of each chloroplast.In Arabidopsis, light-dependent nuclear positioning shows similarities to the chloroplast avoidance response, with regard to the direction of movement, relevant photoreceptor (phototropin2), and effective fluence rate (Iwabuchi and Takagi, 2008). On the other hand, nuclei are larger than chloroplasts and might require thicker, more rigid actin bundles for effective motility. Here, we investigate the involvement of the actin cytoskeleton as well as phototropin in regulatory system for nuclear positioning in epidermal cells of Arabidopsis leaves.     

The phototropin family as photoreceptors for blue light-induced chloroplast relocation

Blue light-induced chloroplast accumulation and avoidance relocation movements are controlled by the blue light photoreceptor phototropin. The Arabidopsis thaliana genome has two phototropin genes encoding phot1 and phot2. Each of these photoreceptors contains two LOV (light oxygen and voltage) domains and a kinase domain. The LOV domains absorb blue light though an associated flavin mononucleotide chromophore, while the kinase domain is thought to be associated with signal transduction. The phototropins control not only chloroplast relocation movement, but also blue light-induced phototropic responses, leaf expansion and stomatal opening. Here I review the role of phototropin as a photoreceptor for chloroplast photorelocation movement. Electronic Publication