Light-dependent chloroplast relocation in wild strawberry (Fragaria vesca).

Chloroplast photorelocation is a vital organellar response that optimizes photosynthesis in plants amid fluctuating environmental conditions. Chloroplasts exhibit an accumulation response, in which they move toward weak light to enhance photoreception, and an avoidance response, in which they move away from strong light to avoid photodamage. Although chloroplast photorelocation has been extensively studied in model plants such as Arabidopsis thaliana, little is known about this process in the economically important crop strawberry. Here, we investigated chloroplast photorelocation in leaf mesophyll cells of wild strawberry (Fragaria vesca), a diploid relative of commercially cultivated octoploid strawberry (F. × ananassa). Microscopy observation revealed that the periclinal area of leaf mesophyll cells in F. vesca is considerably smaller than that of A. thaliana. Given this small cell size, we investigated chloroplast photorelocation in F. vesca by measuring light transmittance in leaves. Weak blue light induced the accumulation response, whereas strong blue light induced the avoidance response. Unexpectedly, strong red light also induced the accumulation response in F. vesca. These findings shed light on chloroplast photorelocation as an intracellular response, laying the foundation for enhancing photosynthesis and productivity in Fragaria.

Bilirubin Distribution in Plants at the Subcellular and Tissue Levels.

In heterotrophs, heme degradation produces bilirubin, a tetrapyrrole compound that has antioxidant activity. In plants, heme is degraded in plastids and is believed to be converted to phytochromobilin rather than bilirubin. Recently, we used the bilirubin-inducible fluorescent protein UnaG to reveal that plants produce bilirubin via a non-enzymatic reaction with NADPH. In the present study, we used an UnaG-based live imaging system to visualize bilirubin accumulation in Arabidopsis thaliana and Nicotiana benthamiana at the organelle and tissue levels. In chloroplasts, bilirubin preferentially accumulated in the stroma, and the stromal bilirubin level increased upon dark treatment. Investigation of intracellular bilirubin distribution in leaves and roots showed that it accumulated mostly in plastids, with low levels detected in the cytosol and other organelles, such as peroxisomes, mitochondria and the endoplasmic reticulum. A treatment that increased bilirubin production in chloroplasts decreased the bilirubin level in peroxisomes, implying that a bilirubin precursor is transported between the two organelles. At the cell and tissue levels, bilirubin showed substantial accumulation in the root elongation region but little or none in the root cap and guard cells. Intermediate bilirubin accumulation was observed in other shoot and root tissues, with lower levels in shoot tissues. Our data revealed the distribution of bilirubin in plants, which has implications for the transport and physiological function of tetrapyrroles.

A tool for live-cell confocal imaging of temperature-dependent organelle dynamics.

Intracellular organelles alter their morphology in response to ambient conditions such as temperature to optimize physiological activities in cells. Observing organelle dynamics at various temperatures deepens our understanding of cellular responses to the environment. Confocal laser microscopy is a powerful tool for live-cell imaging of fluorescently labeled organelles. However, the large contact area between the specimen and the ambient air on the microscope stage makes it difficult to maintain accurate cellular temperatures. Here, we present a method for precisely controlling cellular temperatures using a custom-made adaptor that can be installed on a commercially available temperature-controlled microscope stage. Using this adaptor, we observed temperature-dependent organelle dynamics in living plant cells; morphological changes in chloroplasts and peroxisomes were temperature dependent. This newly developed adaptor can easily be placed on a temperature-controlled stage to capture intracellular responses to temperature at unprecedentedly high resolution.

Fluorescein staining of chloroplast starch granules in living plants.

Chloroplast starch granules (cpSGs) store energy harvested through photosynthesis in plants, and cpSG dynamics have important roles in plant energy metabolism and stress responses. To date, cpSGs have been visualized using several methods, such as iodine staining; however, no method can be used to specifically visualize cpSGs in living cells from various plant species. Here, we report a simple method to visualize cpSGs in living plant cells in various species by staining with fluorescein, a commonly used fluorescent dye. We show that fluorescein is taken up into chloroplasts and interacts with cpSGs similarly to iodine. Fluorescein also interacts with refined starch in vitro. Using a fluorescein derivative for ultrabright cpSG imaging, we produced high-quality 3D reconstructions of cpSGs and evaluated their accumulation in multiple plant species. As fluorescein is well known and readily purchasable, our fluorescein-based staining method should contribute to all research regarding starch.

Functional comparison of phototropin from the liverworts Apopellia endiviifolia and Marchantia polymorpha.

Phototropin (phot) is a blue light (BL) receptor and thermosensor that mediates chloroplast movements in plants. Liverworts, as early-diverging plant species, have a single copy of PHOT gene, and the phot protein in each liverwort activates the signaling pathway adapted to its specific growing environment. In this study, we functionally compared phot from two different liverworts species: Apopellia endiviifolia (Aephot) and Marchantia polymorpha (Mpphot). The BL-dependent photochemical activity of Aephot was similar to that of Mpphot, whereas the thermochemical activity of Aephot was lower than that of Mpphot. Therefore, the phot-mediated signaling pathways of the two plant species may differ more in response to temperature than to BL. Furthermore, we analyzed the functional compatibility of Aephot and Mpphot in chloroplast movements by transiently expressing AePHOT or MpPHOT. The transient expression of AePHOT did not mediate chloroplast movement in M. polymorpha, showing the incompatibility of Aephot with the signaling pathway of M. polymorpha. By contrast, the transient expression of MpPHOT mediated chloroplast movement in A. endiviifolia, indicating the compatibility of Mpphot with the signaling pathway of A. endiviifolia. Our findings reveal both functional similarities and differences between Aephot and Mpphot proteins from the closely related liverworts.

Statistical analysis of organelle movement using state-space models.

BACKGROUND: Organelle motility is essential for the correct cellular function of various eukaryotic cells. In plant cells, chloroplasts move towards the intracellular area irradiated by a weak light to maximise photosynthesis. To initiate this process, an unknown signal is transferred from the irradiated area to distant chloroplasts. Quantification of this chloroplast movement has been performed using visual estimations that are analyst-dependent and labour-intensive. Therefore, an objective and faster method is required. RESULTS: In this study, we developed the cellssm package of R ( https://github.com/hnishio/cellssm.git ), which is a user-friendly tool for state-space modelling to statistically analyse the directional movement of cells or organelles. Our method showed a high accuracy in estimating the start time of chloroplast movement in the liverwort Marchantia polymorpha over a short period. The tool indicated that chloroplast movement accelerates during transport to the irradiated area and that signal transfer speed is uneven within a cell. We also developed a method to estimate the common dynamics among multiple chloroplasts in each cell, which clarified different characteristics among cells. CONCLUSIONS: We demonstrated that state-space modelling is a powerful method to understand organelle movement in eukaryotic cells. The cellssm package can be applied to various directional movements (both accumulation and avoidance) at cellular and subcellular levels to estimate the true transition of states behind the time-series data.

C-Terminal Domain Controls Protein Quality and Secretion of Spider Silk in Tobacco Cells.

The remarkable mechanical strength and extensibility of spider dragline silk spidroins are attributed to the major ampullate silk proteins (MaSp). Although fragmented MaSp molecules have been extensively produced in various heterologous expression platforms for biotechnological applications, complete MaSp molecules are required to achieve instinctive spinning of spidroin fibers from aqueous solutions. Here, a plant cell-based expression platform for extracellular production of the entire MaSp2 protein is developed, which exhibits remarkable self-assembly properties to form spider silk nanofibrils. The engineered transgenic Bright-yellow 2 (BY-2) cell lines overexpressing recombinant secretory MaSp2 proteins yield 0.6-1.3  µg L-1 at 22 days post-inoculation, which is four times higher than those of cytosolic expressions. However, only 10-15% of these secretory MaSp2 proteins are discharged into the culture media. Surprisingly, expression of functional domain-truncated MaSp2 proteins lacking the C-terminal domain in transgenic BY-2 cells increases recombinant protein secretion incredibly, from 0.9 to 2.8 mg L-1 per day within 7 days. These findings demonstrate significant improvement in the extracellular production of recombinant biopolymers such as spider silk spidroins using plant cells. In addition, the results reveal the regulatory roles of the C-terminal domain of MaSp2 proteins in controlling their protein quality and secretion.

Peroxisomes undergo morphological changes in a light-dependent manner with proximity to the nucleus.

The size and shape of organelles can influence the rate of biochemical reactions in cells. Previous studies have suggested that organelle morphology changes due to intra- and extracellular environmental responses, affecting the metabolic efficiency of and signal transduction emanating from neighboring organelles. In this study, we tested the possibility that intracellularly distributed organelles exhibit a heterogeneous response to intra- and extracellular environments. We detected a high correlation between peroxisome morphology and distance to the nucleus in light-exposed cells. Moreover, the proximity area between chloroplasts and peroxisomes varied with distance to the nucleus. These results indicate that peroxisome morphology varies with proximity to the nucleus, suggesting the presence of a nucleus-peroxisome signal transduction cascade mediated by chloroplasts.

Binding of Tau-derived peptide-fused GFP to plant microtubules in Arabidopsis thaliana.

Studies on how exogenous molecules modulate properties of plant microtubules, such as their stability, structure, and dynamics, are important for understanding and modulating microtubule functions in plants. We have developed a Tau-derived peptide (TP) that binds to microtubules and modulates their properties by binding of TP-conjugated molecules in vitro. However, there was no investigation of TPs on microtubules in planta. Here, we generated transgenic Arabidopsis thaliana plants stably expressing TP-fused superfolder GFP (sfGFP-TP) and explored the binding properties and effects of sfGFP-TP on plant microtubules. Our results indicate that the expressed sfGFP-TP binds to the plant microtubules without inhibiting plant growth. A transgenic line strongly expressing sfGFP-TP produced thick fibrous structures that were stable under conditions where microtubules normally depolymerize. This study generates a new tool for analyzing and modulating plant microtubules.

Bilirubin is produced nonenzymatically in plants to maintain chloroplast redox status.

Bilirubin, a potent antioxidant, is a product of heme catabolism in heterotrophs. Heterotrophs mitigate oxidative stress resulting from free heme by catabolism into bilirubin via biliverdin. Although plants also convert heme to biliverdin, they are generally thought to be incapable of producing bilirubin because they lack biliverdin reductase, the enzyme responsible for bilirubin biosynthesis in heterotrophs. Here, we demonstrate that bilirubin is produced in plant chloroplasts. Live-cell imaging using the bilirubin-dependent fluorescent protein UnaG revealed that bilirubin accumulated in chloroplasts. In vitro, bilirubin was produced nonenzymatically through a reaction between biliverdin and reduced form of nicotinamide adenine dinucleotide phosphate at concentrations comparable to those in chloroplasts. In addition, increased bilirubin production led to lower reactive oxygen species levels in chloroplasts. Our data refute the generally accepted pathway of heme degradation in plants and suggest that bilirubin contributes to the maintenance of redox status in chloroplasts.

Deuterium- and Alkyne-Based Bioorthogonal Raman Probes for In Situ Quantitative Metabolic Imaging of Lipids within Plants.

Plants can rapidly respond to different stresses by activating multiple signaling and defense pathways. The ability to directly visualize and quantify these pathways in real time using bioorthogonal probes would have practical applications, including characterizing plant responses to both abiotic and biotic stress. Fluorescence-based labels are widely used for tagging of small biomolecules but are relatively bulky and with potential effects on their endogenous localization and metabolism. This work describes the use of deuterium- and alkyne-derived fatty acid Raman probes to visualize and track the real-time response of plants to abiotic stress within the roots. Relative quantification of the respective signals could be used to track their localization and overall real-time responses in their fatty acid pools due to drought and heat stress without labor-intensive isolation procedures. Their overall usability and low toxicity suggest that Raman probes have great untapped potential in the field of plant bioengineering.

Dark-induced chloroplast relocation depends on actin filaments in the liverwort Apopellia endiviifolia along with the light- and cold-induced relocations.

Chloroplasts move to the periclinal walls of cells under weak light to harness light energy for photosynthesis and to anticlinal walls to avoid strong light. These responses involve the cytoskeleton components microtubules and/or actin filaments. In the dark, chloroplasts move to the anticlinal cell walls bordering neighbouring cells (dark-positioning response), but this response in various plants normally requires a prolonged dark incubation period, which has hampered analysis. However, we recently demonstrated the dark-positioning response that can be induced after a short period of dark incubation in the liverwort Apopellia endiviifolia. Here, we investigated whether the cytoskeleton components function in the dark-positioning response of A. endiviifolia cells. Microtubules and actin filaments were fluorescently visualised in A. endiviifolia cells and were disrupted following treatment with the microtubule and actin filament polymerisation inhibitors. The dark-positioning response was unaffected in the cells with disrupted microtubules. By contrast, the dark-positioning response was inhibited by the disruption of actin filaments. The disruption of actin filaments also restricted chloroplast mobility during light- and cold-dependent chloroplast movements in A. endiviifolia. Therefore, the dark-positioning response of A. endiviifolia depends solely on an actin filament-associated motility mechanism, as do the light- and cold-dependent chloroplast responses.

Using the organelle glue technique to engineer the plant cell metabolome.

KEY MESSAGE: By using the organelle glue technique, we artificially manipulated organelle interactions and controlled the plant metabolome at the pathway level. Plant cell metabolic activity changes with fluctuating environmental conditions, in part via adjustments in the arrangement and interaction of organelles. This hints at the potential for designing plants with desirable metabolic activities for food and pharmaceutical industries by artificially controlling the interaction of organelles through genetic modification. We previously developed a method called the organelle glue technique, in which chloroplast-chloroplast adhesion is induced in plant cells using the multimerization properties of split fluorescent proteins. Here, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants in which chloroplasts adhere to each other and performed metabolome analysis to examine the metabolic changes in these lines. In plant cells expressing a construct encoding the red fluorescent protein mCherry targeted to the chloroplast outer envelope by fusion with a signal sequence (cTP-mCherry), chloroplasts adhered to each other and formed chloroplast aggregations. Mitochondria and peroxisomes were embedded in the aggregates, suggesting that normal interactions between chloroplasts and these organelles were also affected. Metabolome analysis of the cTP-mCherry-expressing Arabidopsis shoots revealed significantly higher levels of glycine, serine, and glycerate compared to control plants. Notably, these are photorespiratory metabolites that are normally transported between chloroplasts, mitochondria, and peroxisomes. Together, our data indicate that chloroplast-chloroplast adhesion alters organellar interactions with mitochondria and peroxisomes and disrupts photorespiratory metabolite transport. These results highlight the possibility of controlling plant metabolism at the pathway level by manipulating organelle interactions.

A high-throughput quantitative method to evaluate peroxisome-chloroplast interactions in Arabidopsis thaliana.

Organelles contribute to plant growth via their movements and interactions, which ensure efficient metabolic flow and help plants adapt to environmental stress. Live-cell imaging of the interactions of organelles has been performed in yeast, plant, and animal cells. However, high-throughput quantitative methods are needed to simultaneously analyze the interactions of many organelles in living plant cells. Here, we developed a semi-automatic high-throughput method to quantitatively evaluate the interactions between peroxisomes and chloroplasts using a distance transformation algorithm and high-resolution 3D fluorescent images taken by confocal laser scanning microscopy. Using this method, we measured the 3D distance between the center of peroxisome and chloroplast surface in Arabidopsis thaliana. We then compared the distances between these organelles in leaf mesophyll cells under light and dark conditions. This distance was shorter in the light than in the dark, which is in agreement with the findings of previous studies. We used our method to evaluate peroxisome-chloroplast (plastid) interactions in different cell types in the light and dark, including guard, stem, and root cells. Like in mesophyll cells, the distance between the peroxisome and chloroplast was shorter in the light in guard and stem cells, but not in root cells, suggesting that photosynthetic plastids (chloroplasts) play important roles in these interactions. When leaf mesophyll cells were incubated under high-intensity light, the frequency of shorter distances between peroxisomes and chloroplasts significantly increased. Our high-throughput, semi-automatic method represents a powerful tool for evaluating peroxisome-chloroplast interactions in different types of plant cells under various environmental conditions.

Live-cell imaging of the chloroplast outer envelope membrane using fluorescent dyes.

Chloroplasts are organelles composed of sub-organellar compartments-stroma, thylakoids, and starch granules-and are surrounded by outer and inner envelope membranes (OEM and IEM, respectively). The chloroplast OEM and IEM play key roles not only as a barrier separating the chloroplast components from the cytosol but also in the interchange of numerous metabolites and proteins between the chloroplast interior and the cytosol. Fluorescent protein markers for the chloroplast OEM have been widely used to visualize the outermost border of chloroplasts. However, the use of marker proteins requires an established cellular genetic transformation method, which limits the plant species in which marker proteins can be used. Moreover, the high accumulation of OEM marker proteins often elicits abnormal morphological phenotypes of the OEM. Because the OEM can currently only be visualized using exogenous marker proteins, the behaviors of the chloroplast and/or its OEM remain unknown in wild-type cells of various plant species. Here, we visualized the OEM using live-cell staining with the fluorescent dyes rhodamine B and Nile red in several plant species, including crops. We propose rhodamine B and Nile red as new tools for visualizing the chloroplast OEM in living plant cells that do not require genetic transformation. Significance Statement: We established a live-cell imaging method to visualize the chloroplast outer envelope membrane by staining living cells with fluorescent dyes. This method does not require genetic transformation and allows the observation of the chloroplast outer envelope membrane in various plant species.

Organellar Glue: A Molecular Tool to Artificially Control Chloroplast-Chloroplast Interactions.

Organelles can physically interact to facilitate various cellular processes such as metabolite exchange. Artificially regulating these interactions represents a promising approach for synthetic biology. Here, we artificially controlled chloroplast-chloroplast interactions in living plant cells with our organelle glue (ORGL) technique, which is based on reconstitution of a split fluorescent protein. We simultaneously targeted N-terminal and C-terminal fragments of a fluorescent protein to the chloroplast outer envelope membrane or cytosol, respectively, which induced chloroplast-chloroplast interactions. The cytosolic C-terminal fragment likely functions as a bridge between two N-terminal fragments, thereby bringing the chloroplasts in close proximity to interact. We modulated the frequency of chloroplast-chloroplast interactions by altering the ratio of N- and C-terminal fragments. We conclude that the ORGL technique can successfully control chloroplast-chloroplast interactions in plants, providing a proof of concept for the artificial regulation of organelle interactions in living cells.

Polymer-coated carbon nanotube hybrids with functional peptides for gene delivery into plant mitochondria.

The delivery of genetic material into plants has been historically challenging due to the cell wall barrier, which blocks the passage of many biomolecules. Carbon nanotube-based delivery has emerged as a promising solution to this problem and has been shown to effectively deliver DNA and RNA into intact plants. Mitochondria are important targets due to their influence on agronomic traits, but delivery into this organelle has been limited to low efficiencies, restricting their potential in genetic engineering. This work describes the use of a carbon nanotube-polymer hybrid modified with functional peptides to deliver DNA into intact plant mitochondria with almost 30 times higher efficiency than existing methods. Genetic integration of a folate pathway gene in the mitochondria displays enhanced plant growth rates, suggesting its applications in metabolic engineering and the establishment of stable transformation in mitochondrial genomes. Furthermore, the flexibility of the polymer layer will also allow for the conjugation of other peptides and cargo targeting other organelles for broad applications in plant bioengineering.

The endoplasmic reticulum membrane-bending protein RETICULON facilitates chloroplast relocation movement in Marchantia polymorpha.

Plant cells alter the intracellular positions of chloroplasts to ensure efficient photosynthesis, a process controlled by the blue light receptor phototropin. Chloroplasts migrate toward weak light (accumulation response) and move away from excess light (avoidance response). Chloroplasts are encircled by the endoplasmic reticulum (ER), which forms a complex network throughout the cytoplasm. To ensure rapid chloroplast relocation, the ER must alter its structure in conjunction with chloroplast relocation movement, but little is known about the underlying mechanism. Here, we searched for interactors of phototropin in the liverwort Marchantia polymorpha and identified a RETICULON (RTN) family protein; RTN proteins play central roles in ER tubule formation and ER network maintenance by stabilizing the curvature of ER membranes in eukaryotic cells. Marchantia polymorpha RTN1 (MpRTN1) is localized to ER tubules and the rims of ER sheets, which is consistent with the localization of RTNs in other plants and heterotrophs. The Mprtn1 mutant showed an increased ER tubule diameter, pointing to a role for MpRTN1 in ER membrane constriction. Furthermore, Mprtn1 showed a delayed chloroplast avoidance response but a normal chloroplast accumulation response. The live cell imaging of ER dynamics revealed that ER restructuring was impaired in Mprtn1 during the chloroplast avoidance response. These results suggest that during the chloroplast avoidance response, MpRTN1 restructures the ER network and facilitates chloroplast movement via an interaction with phototropin. Our findings provide evidence that plant cells respond to fluctuating environmental conditions by controlling the movements of multiple organelles in a synchronized manner.

Temperature Sensing in Plants: On the Dawn of Molecular Thermosensor Research.

Although many studies on plant growth and development focus on the effects of light, a growing number of studies dissect plant responses to temperature and the underlying signaling pathways. The identity of plant thermosensing molecules (thermosensors) acting upstream of the signaling cascades in temperature responses was elusive until recently. During the past six years, a set of plant thermosensors has been discovered, representing a major turning point in the research on plant temperature responses and signaling. Here, we review these newly discovered plant thermosensors, which can be classified as sensors of warmth or cold. We compare between plant thermosensors and those from other organisms and attempt to define the subcellular thermosensing compartments in plants. In addition, we discuss the notion that photoreceptive thermosensors represent a novel class of thermosensors, the roles of which have yet to be described in non-plant systems.

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts.

Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems.