Chloroplast-actin filaments decide the direction of chloroplast avoidance movement under strong light in Arabidopsis thaliana.

Chloroplast-actin (cp-actin) filaments are crucial for light-induced chloroplast movement, and appear in the front region of moving chloroplasts when visualized using GFP-mouse Talin. They are short and thick, exist between a chloroplast and the plasma membrane, and move actively and rapidly compared to cytoplasmic long actin filaments that run through a cell. The average period during which a cp-actin filament was observed at the same position was less than 0.5 s. The average lengths of the cp-actin filaments calculated from those at the front region of the moving chloroplast and those around the chloroplast periphery after stopping the movement were almost the same, approximately 0.8 µm. Each cp-actin filament is shown as a dotted line consisting of 4-5 dots. The vector sum of cp-actin filaments in a moving chloroplast is parallel to the moving direction of the chloroplast, suggesting that the direction of chloroplast movement is regulated by the vector sum of cp-actin filaments. However, once the chloroplasts stopped moving, the vector sum of the cp-actin filaments around the chloroplast periphery was close to zero, indicating that the direction of movement was undecided. To determine the precise structure of cp-actin filaments under electron microscopy, Arabidopsis leaves and fern Adiantum capillus-veneris gametophytes were frozen using a high-pressure freezer, and observed under electron microscopy. However, no bundled microfilaments were found, suggesting that the cp-actin filaments were unstable even under high-pressure freezing.

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.

Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.

Light-induced chloroplast movements in Oryza species.

Light-induced chloroplast movements control efficient light utilization in leaves, and thus, are essential for leaf photosynthesis and biomass production under fluctuating light conditions. Chloroplast movements have been intensively analyzed using wild-type and mutant plants of Arabidopsis thaliana. The molecular mechanism and the contribution to biomass production were elucidated. However, the knowledge of chloroplast movements is very scarce in other plant species, especially grass species including crop plants. Because chloroplast movements are efficient strategy to optimize light capture in leaves and thus promote leaf photosynthesis and biomass, analysis of chloroplast movements in crops is required for biomass production. Here, we analyzed chloroplast movements in a wide range of cultivated and wild species of genus Oryza. All examined Oryza species showed the blue-light-induced chloroplast movements. However, O. sativa and its ancestral species O. rufipogon, both of which are AA-genome species and usually grown in open condition where plants are exposed to full sunlight, showed the much weaker chloroplast movements than Oryza species that are usually grown under shade or semi-shade conditions, including O. officinalis, O. eichingeri, and O. granulata. Further detailed analyses of different O. officinalis accessions, including sun, semi-shade, and shade accessions, indicated that the difference in chloroplast movement strength between domesticated rice plants and wild species might result from the difference in habitat, and the shape of mesophyll chlorenchyma cells. The findings of this study provide useful information for optimizing Oryza growth conditions, and lay the groundwork for improving growth and yield in staple food crop Oryza sativa.

Phototropin2 Contributes to the Chloroplast Avoidance Response at the Chloroplast-Plasma Membrane Interface.

Blue-light-induced chloroplast movements play an important role in maximizing light utilization for photosynthesis in plants. Under a weak light condition, chloroplasts accumulate to the cell surface to capture light efficiently (chloroplast accumulation response). Conversely, chloroplasts escape from strong light and move to the side wall to reduce photodamage (chloroplast avoidance response). The blue light receptor phototropin (phot) regulates these chloroplast movements and optimizes leaf photosynthesis by controlling other responses in addition to chloroplast movements. Seed plants such as Arabidopsis (Arabidopsis thaliana) have phot1 and phot2. They redundantly mediate phototropism, stomatal opening, leaf flattening, and the chloroplast accumulation response. However, the chloroplast avoidance response is induced by strong blue light and regulated primarily by phot2. Phots are localized mainly on the plasma membrane. However, a substantial amount of phot2 resides on the chloroplast outer envelope. Therefore, differentially localized phot2 might have different functions. To determine the functions of plasma membrane- and chloroplast envelope-localized phot2, we tethered it to these structures with their respective targeting signals. Plasma membrane-localized phot2 regulated phototropism, leaf flattening, stomatal opening, and chloroplast movements. Chloroplast envelope-localized phot2 failed to mediate phototropism, leaf flattening, and the chloroplast accumulation response but partially regulated the chloroplast avoidance response and stomatal opening. Based on the present and previous findings, we propose that phot2 localized at the interface between the plasma membrane and the chloroplasts is required for the chloroplast avoidance response and possibly for stomatal opening as well.

Determination of Phototropism and Polarotropism in Fern Protonemal Cells.

Fern protonemal cells grow at their apices as long, undivided filamentous cells toward red (or weak white) light and change their growth direction if the light direction is changed (i.e., phototropism). When protonemata growing between an agar surface and cover glass are irradiated with polarized red light through the glass on the protonemal side, they start growing at the point where the direction of the vibration plane of polarized light and the transition moment of the photoreceptor, which is parallel to the plasma membrane of the cell's apical part, are equal (i.e., polarotropism). Herein, the methods on how to induce and observe this protonemal phototropism and polarotropism are described.

Cytoskeleton Observation in Phototropism and Polarotropism of Adiantum Protonema.

The distribution patterns of the cytoskeleton, i.e., microtubules and actin filaments, in the apical part of protonemal cells are unique and differ from those of other apical growing cells, such as moss and liverwort protonemata, fungal hyphae, and angiosperm pollen tubes. A ring structure composed of microtubules and actin filaments exists at the basal part of the apical dome of protonemal cells. The structure may control the protonemal diameter and growth direction. Herein, the methods of staining of both microtubules and actin filaments are described.

Light-dependent spatiotemporal control of plant cell development and organelle movement in fern gametophytes.

The haploid gametophyte generation of ferns is an excellent experimental material for cell biology studies because of its simple structure and high sensitivity to light. Each step of the developmental process, such as cell growth, cell cycle and the direction of cell division, is controlled, step by step, by light, unlike what happens in complex seed plant tissues. To perform analyses at the cell or organelle level, we have developed special tools, instruments and techniques, such as a cuvette suitable for repeated centrifugation in particular directions, microbeam irradiators for partial cell irradiation and single-cell ligation technique to create enucleated cells. Some of our main discoveries are as follows: (1) changes in the intracellular position of the nucleus in long protonemal cells by centrifugation revealed that the nuclear position or a factor(s) that is/are co-centrifuged with the nucleus is important for the decision regarding the place of the formation of preprophase bands and the timing of their disappearance, which determines the position where the new cell wall attaches to the mother cell wall; (2) even within a single cell, various phenomena could be induced by blue or red light, with the localization of the blue or red light receptors being different depending on the phenomenon; (3) de novo mRNA synthesis is not involved in the signal transduction pathways underlying light-induced chloroplast movements. In this review article, various microscopic techniques, in addition to the results of physiology studies in fern gametophytes, are described.

Chloroplast Accumulation Response Enhances Leaf Photosynthesis and Plant Biomass Production.

Under high light intensity, chloroplasts avoid absorbing excess light by moving to anticlinal cell walls (avoidance response), but under low light intensity, chloroplasts accumulate along periclinal cell walls (accumulation response). In most plant species, these responses are induced by blue light and are mediated by the blue light photoreceptor, phototropin, which also regulates phototropism, leaf flattening, and stomatal opening. These phototropin-mediated responses could enhance photosynthesis and biomass production. Here, using various Arabidopsis (Arabidopsis thaliana) mutants deficient in chloroplast movement, we demonstrated that the accumulation response enhances leaf photosynthesis and plant biomass production. Conspicuously, phototropin2 mutant plants specifically defective in the avoidance response but not in other phototropin-mediated responses displayed a constitutive accumulation response irrespective of light intensities, enhanced leaf photosynthesis, and increased plant biomass production. Therefore, our findings provide clear experimental evidence of the importance of the chloroplast accumulation response in leaf photosynthesis and biomass production.

Palisade cell shape affects the light-induced chloroplast movements and leaf photosynthesis.

Leaf photosynthesis is regulated by multiple factors that help the plant to adapt to fluctuating light conditions. Leaves of sun-light-grown plants are thicker and contain more columnar palisade cells than those of shade-grown plants. Light-induced chloroplast movements are also essential for efficient leaf photosynthesis and facilitate efficient light utilization in leaf cells. Previous studies have demonstrated that leaves of most of the sun-grown plants exhibited no or very weak chloroplast movements and could accomplish efficient photosynthesis under strong light. To examine the relationship between palisade cell shape, chloroplast movement and distribution, and leaf photosynthesis, we used an Arabidopsis thaliana mutant, angustifolia (an), which has thick leaves that contain columnar palisade cells similar to those in the sun-grown plants. In the highly columnar cells of an mutant leaves, chloroplast movements were restricted. Nevertheless, under white light condition (at 120 µmol m-2 s-1), the an mutant plants showed higher chlorophyll content per unit leaf area and, thus, higher light absorption by the leaves than the wild type, which resulted in enhanced photosynthesis per unit leaf area. Our findings indicate that coordinated regulation of leaf cell shape and chloroplast movement according to the light conditions is pivotal for efficient leaf photosynthesis.

Actin-mediated movement of chloroplasts.

Plants are sessile and require diverse strategies to adapt to fluctuations in the surrounding light conditions. Consequently, the photorelocation movement of chloroplasts is essential to prevent damages that are induced by intense light (avoidance response) and to ensure efficient photosynthetic activities under weak light conditions (accumulation response). The mechanisms that underlie chloroplast movements have been revealed through analysis of the behavior of individual chloroplasts and it has been found that these organelles can move in any direction without turning. This implies that any part of the chloroplast periphery can function as the leading or trailing edge during movement. This ability is mediated by a special structure, which consists of short actin filaments that are polymerized at the leading edge of moving chloroplasts and are specifically localized in the space between the chloroplast and the plasma membrane, and is called chloroplast-actin. In addition, several of the genes that encode proteins that are involved in chloroplast-actin polymerization or maintenance have been identified. In this Review, we discuss the mechanisms that regulate chloroplast movements through polymerization of the chloroplast-actin and propose a model for actin-driven chloroplast photorelocation movement.

Nuclear movement and positioning in plant cells.

Plant cells connected to adjacent cells with rigid cell wall cannot change their position, so that appropriate nuclear positioning according to nuclear movement is indispensable for cellular development involving unequal cell division. Sessile plants are severely affected by fluctuating environmental conditions, so that movement of organelles including nucleus is fundamental to accomplish physiological functions. The mechanisms of nuclear movement and their purposes studied recently with Arabidopsis thaliana, the model plants for genetics and molecular biology, and the nuclear behavior in fern gametophytes, an apical growing protonemal cell and a two-dimensional prothallus of Adiantum capillus-veneris, the model plants for cell biology and photobiology are described in this review.

Two Coiled-Coil Proteins, WEB1 and PMI2, Suppress the Signaling Pathway of Chloroplast Accumulation Response that Is Mediated by Two Phototropin-Interacting Proteins, RPT2 and NCH1, in Seed Plants.

Chloroplast movement is induced by blue light in a broad range of plant species. Weak light induces the chloroplast accumulation response and strong light induces the chloroplast avoidance response. Both responses are essential for efficient photosynthesis and are mediated by phototropin blue-light receptors. J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST ACCUMULATION RESPONSE 1 (JAC1) and two coiled-coil domain proteins WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT 1 (WEB1) and PLASTID MOVEMENT IMPAIRED 2 (PMI2) are required for phototropin-mediated chloroplast movement. Genetic analysis suggests that JAC1 is essential for the accumulation response and WEB1/PMI2 inhibit the accumulation response through the suppression of JAC1 activity under the strong light. We recently identified two phototropin-interacting proteins, ROOT PHOTOTROPISM 2 (RPT2) and NPH3/RPT2-like (NRL) PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1) as the signaling components involved in chloroplast accumulation response. However, the relationship between RPT2/NCH1, JAC1 and WEB1/PMI2 remained to be determined. Here, we performed genetic analysis between RPT2/NCH1, JAC1, and WEB1/PMI2 to elucidate the signal transduction pathway.

Temperature-dependent signal transmission in chloroplast accumulation response.

Chloroplast photorelocation movement, well-characterized light-induced response found in various plant species from alga to higher plants, is an important phenomenon for plants to increase photosynthesis efficiency and avoid photodamage. The signal for chloroplast accumulation movement connecting the blue light receptor, phototropin, and chloroplasts remains to be identified, although the photoreceptors and the mechanism of movement via chloroplast actin filaments have now been revealed in land plants. The characteristics of the signal have been found; the speed of signal transfer is about 1 µm min-1 and that the signal for the accumulation response has a longer life and is transferred a longer distance than that of the avoidance response. Here, to collect the clues of the unknown signal substances, we studied the effect of temperature on the speed of signal transmission using the fern Adiantum capillus-veneris and found the possibility that the mechanism of signal transfer was not dependent on the simple diffusion of a substance; thus, some chemical reaction must also be involved. We also found new insights of signaling substances, such that microtubules are not involved in the signal transmission, and that the signal could even be transmitted through the narrow space between chloroplasts and the plasma membrane.

Ferns, mosses and liverworts as model systems for light-mediated chloroplast movements.

Light-induced chloroplast movement is found in most plant species, including algae and land plants. In land plants with multiple small chloroplasts, under weak light conditions, the chloroplasts move towards the light and accumulate on the periclinal cell walls to efficiently perceive light for photosynthesis (the accumulation response). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response). In most plant species, blue light induces chloroplast movement, and phototropin receptor kinases are the blue light receptors. Molecular mechanisms for photoreceptors, signal transduction and chloroplast motility systems are being studied using the model plant Arabidopsis thaliana. However, to further understand the molecular mechanisms and evolutionary history of chloroplast movement in green plants, analyses using other plant systems are required. Here, we review recent works on chloroplast movement in green algae, liverwort, mosses and ferns that provide new insights on chloroplast movement.

Correction: Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana.

[This corrects the article DOI: 10.1371/journal.pone.0157429.].

Chloroplast and nuclear photorelocation movements.

Chloroplasts move toward weak light to increase photosynthetic efficiency, and migrate away from strong light to protect chloroplasts from photodamage and eventual cell death. These chloroplast behaviors were first observed more than 100 years ago, but the underlying mechanism has only recently been identified. Ideal plant materials, such as fern gametophytes for photobiological and cell biological approaches, and Arabidopsis thaliana for genetic analyses, have been used along with sophisticated methods, such as partial cell irradiation and time-lapse video recording under infrared light to study chloroplast movement. These studies have revealed precise chloroplast behavior, and identified photoreceptors, other relevant protein components, and novel actin filament structures required for chloroplast movement. In this review, our findings regarding chloroplast and nuclear movements are described.

RPT2/NCH1 subfamily of NPH3-like proteins is essential for the chloroplast accumulation response in land plants.

In green plants, the blue light receptor kinase phototropin mediates various photomovements and developmental responses, such as phototropism, chloroplast photorelocation movements (accumulation and avoidance), stomatal opening, and leaf flattening, which facilitate photosynthesis. In Arabidopsis, two phototropins (phot1 and phot2) redundantly mediate these responses. Two phototropin-interacting proteins, NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and ROOT PHOTOTROPISM 2 (RPT2), which belong to the NPH3/RPT2-like (NRL) family of BTB (broad complex, tramtrack, and bric à brac) domain proteins, mediate phototropism and leaf flattening. However, the roles of NRL proteins in chloroplast photorelocation movement remain to be determined. Here, we show that another phototropin-interacting NRL protein, NRL PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1), and RPT2 redundantly mediate the chloroplast accumulation response but not the avoidance response. NPH3, RPT2, and NCH1 are not involved in the chloroplast avoidance response or stomatal opening. In the liverwort Marchantia polymorpha, the NCH1 ortholog, MpNCH1, is essential for the chloroplast accumulation response but not the avoidance response, indicating that the regulation of the phototropin-mediated chloroplast accumulation response by RPT2/NCH1 is conserved in land plants. Thus, the NRL protein combination could determine the specificity of diverse phototropin-mediated responses.

Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana.

Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements.