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.

Microsatellite Dataset for Cultivar Discrimination in Spring Orchid (Cymbidium goeringii).

Cymbidium goeringii Reichb. fil., locally known as the spring orchid in the Republic of Korea, is one of the most important and popular horticultural species in the family Orchidaceae. C. goeringii cultivars originated from plants with rare phenotypes in wild mountains where pine trees commonly grow. This study aimed to determine the cultivar-specific combined genotypes (CGs) of short sequence repeats (SSRs) by analyzing multiple samples per cultivar of C. goeringii. In this study, we collected more than 4000 samples from 67 cultivars and determined the genotypes of 12 SSRs. Based on the most frequent combined genotypes (CG1s), the average observed allele number and combined matching probability were 11.8 per marker and 3.118 × 10-11, respectively. Frequencies of the CG1 in 50 cultivars (n ≥ 10) ranged from 40.9% to 100.0%, with an average of 70.1%. Assuming that individuals with the CG1 are genuine in the corresponding cultivars, approximately 30% of C. goeringii on the farms and markets may be not genuine. The dendrogram of the phylogenetic tree and principal coordinate analysis largely divided the cultivars into three groups according to their countries of origin; however, the genetic distances were not great among the cultivars. In conclusion, this dataset of C. goeringii cultivar-specific SSR profiles could be used for ecogenetic studies and forensic authentication. This study suggests that genetic authentication should be introduced for the sale of expensive C. goeringii cultivars. We believe that this study will help establish a genetic method for the forensic authentication of C. goeringii cultivars.

Enhancement of the CRISPR/Cas9-Based Genome Editing System in Lettuce (Lactuca sativa L.) Using the Endogenous U6 Promoter.

The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter and proved that the PHOT2 gene is a versatile target gene. We isolated the LsU6-10 promoter from 10 U6 snRNA genes identified from the lettuce genome database for comparison with the AtU6-26 promoter that has been used to drive sgRNAs in lettuce. Two CRISPR/Cas9 vectors were constructed using the LsU6-10 and AtU6-26 promoters to drive sgRNA361 to target the PHOT2 gene. The chloroplast avoidance response was defective in lettuces with biallelic mutations in the targeted PHOT2 gene, as in the Arabidopsis phot2 mutant. The PHOT2 gene mutations were stably heritable from the R0 to R2 generations, and the high gene-editing efficiency enabled the selection of transgene-free lines in the R1 generation and the establishment of independent phot2 mutants in the R2 generation. Our results suggest that the LsU6-10 promoter is more effective than the AtU6-26 promoter in driving sgRNA for the CRISPR/Cas9 system in lettuce and that PHOT2 is a useful target gene to verify gene editing efficiency without any detrimental effects on plant growth, which is often a consideration in conventional target genes.

Guard-Cell-Specific Expression of Phototropin2 C-Terminal Fragment Enhances Leaf Transpiration.

Phototropins (phot1 and phot2) are plant-specific blue light receptors that mediate chloroplast movement, stomatal opening, and phototropism. Phototropin is composed of the N-terminus LOV1 and LOV2 domains and the C-terminus Ser/Thr kinase domain. In previous studies, 35-P2CG transgenic plants expressing the phot2 C-terminal fragment-GFP fusion protein (P2CG) under the control of 35S promoter showed constitutive phot2 responses, including chloroplast avoidance response, stomatal opening, and reduced hypocotyl phototropism regardless of blue light, and some detrimental growth phenotypes. In this study, to exclude the detrimental growth phenotypes caused by the ectopic expression of P2C and to improve leaf transpiration, we used the PHOT2 promoter for the endogenous expression of GFP-fused P2C (GP2C) (P2-GP2C) and the BLUS1 promoter for the guard-cell-specific expression of GP2C (B1-GP2C), respectively. In P2-GP2C plants, GP2C expression induced constitutive phototropin responses and a relatively dwarf phenotype as in 35-P2CG plants. In contrast, B1-GP2C plants showed the guard-cell-specific P2C expression that induced constitutive stomatal opening with normal phototropism, chloroplast movement, and growth phenotype. Interestingly, leaf transpiration was significantly improved in B1-GP2C plants compared to that in P2-GP2C plants and WT. Taken together, this transgenic approach could be applied to improve leaf transpiration in indoor plants.

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.

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.

Molecular basis of chloroplast photorelocation movement.

Chloroplast photorelocation movement is an essential physiological response for sessile plant survival and the optimization of photosynthetic ability. Simple but effective experiments on the physiological, cell biological and molecular genetic aspects have been widely used to investigate the signaling components of chloroplast photorelocation movement in Arabidopsis for the past few decades. Although recent knowledge on chloroplast photorelocation movement has led us to a deeper understanding of its physiological and molecular basis, the biochemical roles of the downstream factors remain largely unknown. In this review, we briefly summarize recent advances regarding chloroplast photorelocation movement and propose that a new high-resolution approach is necessary to investigate the molecular mechanism underlying actin-based chloroplast photorelocation movement.

Distinct Biochemical Properties of Arabidopsis thaliana Actin Isoforms.

Plants and animals express multiple actin isoforms in a manner that is dependent on tissues, organs and the stage of development. Previous genetic analyses suggested that individual actin isoforms have specific roles in cells, but there is little biochemical evidence to support this hypothesis. In this study, we purified four recombinant Arabidopsis actin isoforms, two major vegetative actin isoforms, ACT2 and ACT7, and two major reproductive isoforms, ACT1 and ACT11, and characterized them biochemically. Phalloidin bound normally to the filaments of the two reproductive actins as well as to the filaments of skeletal muscle actin. However, phalloidin bound only weakly to ACT7 filaments and hardly at all to ACT2 filaments, despite the conserved sequence of the phalloidin-binding site. Polymerization and phosphate release rates among these four actin isoforms were also significantly different. Moreover, interactions with profilin (PRF) were also different among the four Arabidopsis actin isoforms. PRF1 and PRF2 inhibited the polymerization of ACT1, ACT11 and ACT7, while ACT2 was only weakly affected. Plant actin isoforms have different biochemical properties. This result supports the idea that actin isoforms play specific roles to achieve multiple cell functions.

PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei.

Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.

Photoinduced oligomerization of Arabidopsis thaliana phototropin 2 LOV1.

Phototropins are blue-light-sensitive photoreceptor proteins in plants. Phototropins consist of two LOV (light, oxygen, and voltage sensor) domains (LOV1 and LOV2) that undergo photochemical reactions. Although the photochemical reaction of the LOV2 domain has been investigated extensively, the reaction of the LOV1 domain remains unresolved. In this study, the reactions of the Arabidopsis phototropin 2 LOV1 (phot2LOV1) domain were revealed by the transient grating (TG) method. The TG signal showed a significant diffusion coefficient (D) change upon photoexcitation. This change was sensitive to the protein concentration and the observation time range. These observations were explained by assuming that there are reactive and nonreactive forms, and the fraction of these species is concentration dependent. From the concentration dependence of the dynamics, the monomer was found to form a dimer; however, the dimer does not exhibit an observable reaction. In the dark state, both species were in equilibrium and were not distinguishable spectroscopically. For the LOV1 domain with the hinge domain, the reaction scheme was the same as the LOV1 domain sample, but the D change was affected by the presence of the hinge region. This observation suggested that the hinge region undergoes a conformational change during the photoreaction.

Actin-dependent plastid movement is required for motive force generation in directional nuclear movement in plants.

Nuclear movement and positioning are indispensable for most cellular functions. In plants, strong light-induced chloroplast movement to the side walls of the cell is essential for minimizing damage from strong visible light. Strong light-induced nuclear movement to the side walls also has been suggested to play an important role in minimizing damage from strong UV light. Although both movements are regulated by the same photoreceptor, phototropin, the precise cytoskeleton-based force generation mechanism for nuclear movement is unknown, in contrast to the short actin-based mechanism of chloroplast movement. Here we show that actin-dependent movement of plastids attached to the nucleus is essential for light-induced nuclear movement in the Arabidopsis leaf epidermal cell. We found that nuclei are always associated with some plastids, and that light-induced nuclear movement is correlated with the dynamics of short actin filaments associated with plastids. Indeed, nuclei without plastid attachments do not exhibit blue light-induced directional movement. Our results demonstrate that nuclei are incapable of autonomously moving in response to light, whereas attached plastids carry nuclei via the short actin filament-based movement. Thus, the close association between nuclei and plastids is essential for their cooperative movements and functions.

Recent advances in understanding the molecular mechanism of chloroplast photorelocation movement.

Plants are photosynthetic organisms that have evolved unique systems to adapt fluctuating environmental light conditions. In addition to well-known movement responses such as phototropism, stomatal opening, and nastic leaf movements, chloroplast photorelocation movement is one of the essential cellular responses to optimize photosynthetic ability and avoid photodamage. For these adaptations, chloroplasts accumulate at the areas of cells illuminated with low light (called accumulation response), while they scatter from the area illuminated with strong light (called avoidance response). Plant-specific photoreceptors (phototropin, phytochrome, and/or neochrome) mediate these dynamic directional movements in response to incident light position and intensity. Several factors involved in the mechanisms underlying the processes from light perception to actin-based movements have also been identified through molecular genetic approach. This review aims to discuss recent findings in the field relating to how chloroplasts move at molecular levels. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.

Interaction between avoidance of photon absorption, excess energy dissipation and zeaxanthin synthesis against photooxidative stress in Arabidopsis.

Plants evolved photoprotective mechanisms in order to counteract the damaging effects of excess light in oxygenic environments. Among them, chloroplast avoidance and non-photochemical quenching concur in reducing the concentration of chlorophyll excited states in the photosynthetic apparatus to avoid photooxidation. We evaluated their relative importance in regulating excitation pressure on photosystem II. To this aim, genotypes were constructed carrying mutations impairing the chloroplast avoidance response (phot2) as well as mutations affecting the biosynthesis of the photoprotective xanthophyll zeaxanthin (npq1) or the activation of non-photochemical quenching (npq4), followed by evaluation of their photosensitivity in vivo. Suppression of avoidance response resulted in oxidative stress under excess light at low temperature, while removing either zeaxanthin or PsbS had a milder effect. The double mutants phot2 npq1 and phot2 npq4 showed the highest sensitivity to photooxidative stress, indicating that xanthophyll cycle and qE have additive effects over the avoidance response. The interactions between non-photochemical quenching and avoidance responses were studied by analyzing the kinetics of fluorescence decay and recovery at different light intensities. phot2 fluorescence decay lacked a component, here named as qM. This kinetic component linearly correlated with the leaf transmittance changes due to chloroplast relocation induced by white light and was absent when red light was used as actinic source. On these basis we conclude that a decrease in leaf optical density affects the apparent non-photochemical quenching (NPQ) rise kinetic. Thus, excess light-induced fluorescence decrease is in part due to avoidance of photon absorption rather than to a genuine quenching process.

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.

A C-terminal membrane association domain of phototropin 2 is necessary for chloroplast movement.

Phototropins (phot1 and phot2), plant-specific blue light receptor kinases, mediate a range of physiological responses in Arabidopsis, including phototropism, chloroplast photorelocation movement, stomatal opening and leaf flattening. Phototropins consist of two photoreceptive domains at their N-terminus, LOV1 (light, oxygen or voltage 1) and LOV2, and a serine/threonine kinase domain at their C-terminus. Here, we determined the molecular moiety for the membrane association of phototropins using the yeast CytoTrap and Arabidopsis protoplast systems. We then examined the physiological significance of the membrane association of phototropins. This detailed study with serial deletions narrowed down the association domain to a relatively small part of the C-terminal domain of phototropin. The functional analysis of phot2 deletion mutants in the phot2-deficient Adiantum and Arabidopsis mutants revealed that the ability to mediate the chloroplast avoidance response correlated well with phot2's membrane association, especially with the Golgi apparatus. Taken together, our data suggest that a small part of the C-terminal domain of phototropins is necessary not only for membrane association but also for the physiological activities that elicit phototropin-specific responses.

Both phototropin 1 and 2 localize on the chloroplast outer membrane with distinct localization activity.

Chloroplasts change their position to adapt cellular activities to fluctuating environmental light conditions. Phototropins (phot1 and phot2 in Arabidopsis) are plant-specific blue light photoreceptors that perceive changes in light intensity and direction, and mediate actin-based chloroplast photorelocation movements. Both phot1 and phot2 regulate the chloroplast accumulation response, while phot2 is mostly responsible for the regulation of the avoidance response. Although it has been widely accepted that distinct intracellular localizations of phototropins are implicated in the specificity, the mechanism underlying the phot2-specific avoidance response has remained elusive. In this study, we examined the relationship of the phot2 localization pattern to the chloroplast photorelocation movement. First, the fusion of a nuclear localization signal with phot2, which effectively reduced the amount of phot2 in the cytoplasm, retained the activity for both the accumulation and avoidance responses, indicating that membrane-localized phot2 but not cytoplasmic phot2 is functional to mediate the responses. Importantly, some fractions of phot2, and of phot1 to a lesser extent, were localized on the chloroplast outer membrane. Moreover, the deletion of the C-terminal region of phot2, which was previously shown to be defective in blue light-induced Golgi localization and avoidance response, affected the localization pattern on the chloroplast outer membrane. Taken together, these results suggest that dynamic phot2 trafficking from the plasma membrane to the Golgi apparatus and the chloroplast outer membrane might be involved in the avoidance response.

Both LOV1 and LOV2 domains of phototropin2 function as the photosensory domain for hypocotyl phototropic responses in Arabidopsis thaliana (Brassicaceae).

PREMISE OF STUDY: Phototropins (phot) are blue light receptor proteins that mediate phototropism and control photomovement responses, such as chloroplast photorelocation movement and stomatal opening. Arabidopsis thaliana has two phototropins, phot1 and phot2. Although both phot1 and phot2 redundantly mediate photomovement responses, phot2 uniquely regulates phototropism and the chloroplast avoidance response under high-intensity blue light. However, compared to that of phot1, the mechanistic basis of phot2 function is poorly understood, and in particular, the importance of the LOV2 domain in phot2 function has not been clearly demonstrated. Indeed, photocycle-deficient LOV2 transgenic lines expressing phot2 in a phot1phot2 mutant background retained phototropism, although with less sensitivity than wild-type plants. METHODS: We isolated 11 alleles of phot2 mutants and determined the molecular lesion in each allele. We analyzed hypocotyl phototropism, chloroplast photorelocation movement, and leaf flattening in the phot2 mutant and the respective phot1phot2 double mutant plants. KEY RESULTS: We demonstrated that unlike the phot2 null mutant, the phot2-10 mutant, which has the defective phot2 LOV2 domain, retained the phototropic response and had unusual chloroplast movement. Mutants phot2-2 and phot2-6, which have a missense mutation in the kinase activation loop of phot2, had the phot2-null mutant phenotype. Furthermore, we convincingly demonstrated that the commonly used phot2-1 mutant allele is a phot2-null mutant. CONCLUSIONS: The analyses of the multiple phot2 mutant alleles provided strong evidence for the importance of both LOV domains and the kinase activation loop of phot2 in phototropism and other phot-dependent responses and also demonstrated that phot2-1 allele is a null mutant.

The RS domain of Arabidopsis splicing factor RRC1 is required for phytochrome B signal transduction.

Plants monitor the light environment through informational photoreceptors that include phytochromes. In seedling de-etiolation, phytochrome B (phyB), which is the most important member of the phytochrome family, interacts with transcription factors to regulate gene expression and transduce light signals. In this study, we identified rrc1 (reduced red-light responses in cry1cry2 background 1), an Arabidopsis mutant that is impaired in phyB-mediated light responses. A genetic analysis demonstrated that RRC1 affected light signaling in a phyB-dependent manner. RRC1 encodes an ortholog of the human potential splicing factor SR140. The RRC1 polypeptide contains a C-terminal arginine/serine-rich (RS) domain that is important for the regulation of alternative splicing. Although the complete loss of RRC1 caused pleiotropic developmental abnormalities, the deletion of the RS domain specifically reduced phyB signaling and caused aberrant alternative splicing of several SR protein genes. Moreover, semi-quantitative RT-PCR analysis revealed that the alternative splicing patterns of some of the SR protein genes were altered in a red-light-dependent manner, and that these responses were reduced in both phyB and rrc1 mutants. These findings suggest that the regulation of alternative splicing by the RS domain of RRC1 plays an important role in phyB signal transduction.

Tissue-autonomous promotion of palisade cell development by phototropin 2 in Arabidopsis.

Light is an important environmental information source that plants use to modify their growth and development. Palisade parenchyma cells in leaves develop cylindrical shapes in response to blue light; however, the photosensory mechanism for this response has not been elucidated. In this study, we analyzed the palisade cell response in phototropin-deficient mutants. First, we found that two different light-sensing mechanisms contributed to the response in different proportions depending on the light intensity. One response observed under lower intensities of blue light was mediated exclusively by a blue light photoreceptor, phototropin 2 (PHOT2). Another response was elicited under higher intensities of light in a phototropin-independent manner. To determine the tissue in which PHOT2 perceives the light stimulus to regulate the response, green fluorescent protein (GFP)-tagged PHOT2 (P2G) was expressed under the control of tissue-specific promoters in the phot1 phot2 mutant background. The results revealed that the expression of P2G in the mesophyll, but not in the epidermis, promoted palisade cell development. Furthermore, a constitutively active C-terminal kinase fragment of PHOT2 fused to GFP (P2CG) promoted the development of cylindrical palisade cells in the proper direction without the directional cue provided by light. Hence, in response to blue light, PHOT2 promotes the development of cylindrical palisade cells along a predetermined axis in a tissue-autonomous manner.