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 be easily placed on a temperature-controlled stage to capture intracellular responses to temperature at unprecedentedly high resolution.

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.

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.

Three-dimensional nanoscale analysis of light-dependent organelle changes in Arabidopsis mesophyll cells.

Different organelles function coordinately in numerous intracellular processes. Photorespiration incidental to photosynthetic carbon fixation is organized across three subcellular compartments: chloroplasts, peroxisomes, and mitochondria. Under light conditions, these three organelles often form a ternary organellar complex in close proximity, suggesting a connection with metabolism during photorespiration. However, due to the heterogeneity of intercellular organelle localization and morphology, organelles' responses to changes in the external environment remain poorly understood. Here, we used array tomography by field emission scanning electron microscopy to image organelles inside the whole plant cell at nanometer resolution, generating a three-dimensional (3D) spatial map of the light-dependent positioning of chloroplasts, peroxisomes, nuclei, and vacuoles. Our results show, in light-treated cells, the volume of peroxisomes increased, and mitochondria were simplified. In addition, the population of free organelles decreased, and the ternary complex centered on chloroplasts increased. Moreover, our results emphasized the expansion of the proximity area rather than the increase in the number of proximity sites interorganelles. All of these phenomena were quantified for the first time on the basis of nanoscale spatial maps. In summary, we provide the first 3D reconstruction of Arabidopsis mesophyll cells, together with nanoscale quantified organelle morphology and their positioning via proximity areas, and then evidence of their light-dependent changes.

Cellular internalization mechanism of novel Raman probes designed for plant cells.

Diphenylacetylene derivatives containing different polymeric components, poly(l-lysine) (pLys) or tetra(ethylene glycol) (TEG) were designed as novel Raman imaging probes with high Raman sensitivity and low cytotoxicity in living plant cells. The pLys-conjugated probe is internalized via an endocytosis-dependent pathway, whereas TEG-conjugated probe most likely induces direct penetration into the plant cells.

Vacuum/Compression Infiltration-mediated Permeation Pathway of a Peptide-pDNA Complex as a Non-Viral Carrier for Gene Delivery in Planta.

Non-viral gene carriers have been extensively investigated as alternatives to viral vectors for gene delivery systems into animal and plant cells. A non-viral gene carrier containing a cell-penetrating peptide and a cationic sequence was previously developed for use in intact plants and plant cells; however, the permeation pathway of the gene carrier into plant cells is yet to be elucidated, which would facilitate the improvement of the gene delivery efficiency. Here, we identified the vacuum/compression infiltration-mediated permeation pathway of a non-viral gene carrier into plant tissues and cells using a complex of plasmid DNA and a peptide-based gene carrier. This complex was taken up via the hydathodes in Arabidopsis thaliana, and from root hairs in Nicotiana benthamiana. Remarkably, these structurally weak tissues are also routes of bacterial invasion in nature, suggesting that peptide-pDNA complexes invade intact plants through similar pathways as bacterial pathogens.

Oryza sativa Brittle Culm 1-like 6 modulates ß-glucan levels in the endosperm cell wall.

The endosperm cell wall affects post-harvest grain quality by affecting the mechanical fragility and water absorption of the grain. Therefore, understanding the mechanism underlying endosperm cell wall synthesis is important for determining the growth and quality of cereals. However, the molecular machinery mediating endosperm cell wall biosynthesis is not well understood. In this study, we investigated the role of Oryza sativa Brittle Culm 1-like 6 (OsBC1L6), a member of the COBRA-like protein family, in cellulose synthesis in rice. OsBC1L6 mRNA was expressed in ripening seeds during endosperm enlargement. When OsBC1L6-RFP was expressed in Arabidopsis cell cultures, this fusion protein was transported to the plasma membrane. To investigate the target molecules of OsBC1L6, we analyzed the binding interactions of OsBC1L6 with cellohexaose and the analogs using surface plasmon resonance, determining that cellohexaose bound to OsBC1L6. The ß-glucan contents were significantly reduced in OsBC1L6-RNAi calli and OsBC1L6-deficient seeds from a Tos insertion mutant, compared to their wild-type counterparts. These findings suggest that OsBC1L6 modulates ß-glucan synthesis during endosperm cell wall formation by interacting with cellulose moieties on the plasma membrane during seed ripening.

Additional nitrogen fertilization at heading time of rice down-regulates cellulose synthesis in seed endosperm.

The balance between carbon and nitrogen is a key determinant of seed storage components, and thus, is of great importance to rice and other seed-based food crops. To clarify the influence of the rhizosphere carbon/nitrogen balance during the maturation stage of several seed components, transcriptome analysis was performed on the seeds from rice plants that were provided additional nitrogen fertilization at heading time. As a result, it was assessed that genes associated with molecular processes such as photosynthesis, trehalose metabolism, carbon fixation, amino acid metabolism, and cell wall metabolism were differentially expressed. Moreover, cellulose and sucrose synthases, which are involved in cellulose synthesis, were down-regulated. Therefore, we compared cellulose content of mature seeds that were treated with additional nitrogen fertilization with those from control plants using calcofluor staining. In these experiments, cellulose content in endosperm from plants receiving additional nitrogen fertilization was less than that in control endosperm. Other starch synthesis-related genes such as starch synthase 1, starch phosphorylase 2, and branching enzyme 3 were also down-regulated, whereas some α-amylase and ß-amylase genes were up-regulated. On the other hand, mRNA expression of amino acid biosynthesis-related molecules was up-regulated. Moreover, additional nitrogen fertilization caused accumulation of storage proteins and up-regulated Cys-poor prolamin mRNA expression. These data suggest that additional nitrogen fertilization at heading time changes the expression of some storage substance-related genes and reduces cellulose levels in endosperm.