An Arabidopsis root cultured for 9 days was stained with a new DNA saining dye based on N-aryl pyrido cyanine backbone (PC1), designated Kakshine PC1, at 1 µM. The stained root was observed by two-photon excitation microscoy (LSM780-DUO-NLO; Zeiss) excited at 1000 nm using a LD C-Apochromat 40x/1.10 water immersion objective lens. It should be noted that Kakshine PC1 penetrate deep root region and stained mitochondrial DNA as well as nuclear DNA.

Technologies using "light" such as super-resolution microscopic imaging, single-molecule measurement, and protein crystal structure analysis have developed remarkably in the last ten years, and unravel the real functions of intracellular structures in plant cells. These analytical techniques have brought major changes in plant science. In the 85th Annual Meeting of the Botanical Society of Japan, we organized the symposium entitled “Real functions of intracellular structures in plant cells pioneered by light technology” under the auspices of the Japanese Society of Plant Morphology, aiming to introduce these technologies using "light". Researchers who utilize the latest nano-level structure visualization techniques have gathered to introduce the basic system of these new technologies, and discuss future perspective in plant science.

In the secretory pathway of eukaryotic cells, cargo proteins and lipids are transported first to the Golgi apparatus after leaving the ER. The morphology and spatial-temporal dynamics of the compartments that are involved in this transport step at the ER-Golgi interface looks greatly variable depending on species or cell types because of the difference of the Golgi structure. However, comparison of the data derived from plants with those from the other organisms highlights a compartment that has the shared nature and roles among eukaryotes in spite of the difference in its morphological appearance. In this review, I will introduce the knowledge about the membranous compartments that function at the ER-Golgi interface in plant cells and discuss about the difference and common points compared to the other species.

Correlative light and electron microscopy (CLEM) is an analytical technique used to evaluate the correlation between fluorescence images of identical samples and regions of fluorescent-labeled organelles captured using light microscopy and electron microscopy. By utilizing various methods during sample preparation, the fluorescence images of fluorescent-labeled organelles are juxtaposed with ultra-structures of the same region obtained using scanning electron microscopy (SEM) or transmission electron microscopy. Unlike animal tissues and adherent cells, visualizing the organelles detected in plant samples via CLEM analysis is difficult because of the non-adherence of plant tissue and cultured cells to glass plates and the size of the cells. Therefore, we combine the serial section SEM method (Array tomography) based on ultra-thin sections and CLEM to identify fluorescent-labeled organelles in plant cells and reconstruct them in three dimensions (3D). In particular, as osmium (Os) epoxy-tolerant fluorescent proteins have recently been reported to be resistant to Os fixation and epoxy resin embedding in animal adherent cells, we applied these fluorescent tags to plant samples. In this paper, we introduce the sample preparation method for the CLEM analysis of organelles labeled with Os epoxy-tolerant fluorescent proteins in plant tissues, alongside the method for obtaining CLEM images and the combined 3D-CLEM analysis method comprising CLEM and array tomography.

Live cell imaging is a powerful tool to understand the molecular mechanisms of a wide range of cellular processes. However, it is difficult to detect very small objects such as transport vesicles (< 100 nm in diameter) by light microscopy because of the diffraction limit of light. To overcome this problem, several superresolution technologies such as STED, SIM, and STORM/PALM has been developed so far, but the time resolution of these methods is not sufficient to track the small vesicles undergoing fast movement inside the cell. In the present study, we developed a highspeed, superresolution, and multicolor optical microscope, which we call SCLIM2 (the second generation superresolution confocal live cell imaging microscopy). SCLIM2 consists of an inverted microscope equipped with piezo actuator for objective lens, a spinningdisk confocal scanner, an emission splitter unit, cooled image intensifiers, and ultrafast CMOS cameras (1000 frames/s). Using this system, we acquired fluorescence images of specimens at a single photon precision. We also developed a mathematical method for 4D (xyz and time) image reconstruction and novel deconvolution algorism based on the probability calculation. The resulting lateral resolution was less than 71 nm. We then applied SCLIM2 to observe a variety of membrane traffic events in live cells. We succeeded in visualizing a large number of small vesicles (< 100 nm in diameter) were moving around the Golgi apparatus.

Plants have generated divergent peptide ligands and corresponding membrane receptors to control their growth and development, as well as various aspects of physiology. Secreted-peptide ligands are mainly recognized by membrane receptor kinases that mediate cell-cell communication. In Arabidopsis, more than 1,000 genes are putative secreted peptides and ~600 genes are annotated as receptor-like kinases. However, several pairs of the peptide ligands and the cognate receptors are identified and mechanistically characterized, many of them remain orphan ligands and receptors. In the past 5 years, structural and biochemical studies have revealed how the short liner peptides with post-translational modification are perceived by the corresponding receptors with leucine-rich repeats (LRR) ectodomain at a molecular level. The short linear peptides are recognized by the LRR-type receptors and co-receptors in a conserved manner. Moreover, the recent structural study has implied a new mode of peptide recognition by LRR receptors. This short review summarizes recognition mode of the secreted peptides by the LRR-type receptors and receptor-activation mechanisms that have been structurally characterized in recent studies.

The simple cellular structure of the unicellular alga Cyanidioschyzon merolae consists of one nucleus, one mitochondrion, one chloroplast, and one peroxisome per cell and offers unique advantages to investigate mechanisms of organellar proliferation and the cell cycle. Here, we describe an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, CZON-cutter, for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease to target editing at a locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants by fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows validation of phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner.

Most animal cells harbor spherical and oval nuclei, whereas the nuclei in terrestrial plants do not only show spherical and oval shapes but also show various morphologies such as spindle-shaped and rod-shaped. The morphology of animal nuclei is regulated by lamin proteins, which form the nuclear lamina. In contrast, lamin orthologs are not conserved in plants; therefore, nuclear lamina proteins that regulate nuclear morphology in plants have not been identified. In my research, I detected CROWDED NUCLEI (CRWN) proteins in crude nuclear lamina fraction prepared from Arabidopsis thaliana. Phenotype analysis of crwn disruptants and the subcellular localization analysis of CRWNs demonstrated that CRWNs are plant-specific nuclear lamina proteins and play essential roles in regulating nuclear morphology. In addition, I found that CRWNs govern the transcriptional control of many environmental stress-responsive genes. Particularly, I revealed that CRWNs alter the position of the CA gene cluster in the nucleus affecting CA gene expression, contributing to tolerance to excess copper.

Stomata have long been playing an important role since plant ancestor invaded the land. The stomatal guard cells are produced by a simple developmental process, in which the meristemoids, stomatal-lineage stem cells, undergo asymmetric divisions prior to differentiation. Since the 2000s, stomatal development has been lively studied in the model plant Arabidopsis, which led to the elucidation of its molecular basis, particularly the pivotal role of three stomatal key transcription factors, namely SPEECHLESS, MUTE, and FAMA. On the other hand, when looking at non-model plants, people will notice that the developmental process of stomata is considerably diverged among species. While such diversity in stomatal development has been well documented by plant morphologists from early times, its molecular basis and ecological significance remained poorly known. In recent years, however, accumulating knowledge in model plants and the development of research technologies applicable to non-model plants have made stomata accessible to studies of ecological evolutionary developmental biology (eco-evo-devo). This review provides an overview of the diversity in the form and development of stomata in land plants, introduces the eco-evo-devo research applied on stomata, such as (1) the research on the unique stomatal complex in grass species, and (2) our recent work on the intrageneric diversity of stomatal development in Callitriche (Plantaginaceae), and discusses the prospects of this emerging field.

DNA detection by synthetic fluorescent dye is an essential technology in all aspects of life science research. In the field of fluorescence live imaging, DNA stain is also an indispensable technique, and many researchers use DNA fluorescent dyes such as for cell cycle analysis, analysis of cell nucleus and organelle DNA dynamics, and the functional analysis of nuclear regulatory factors. In this paper, we describe the development of DNA staining dyes that further improve on the advantages of conventional excellent dyes and overcome their disadvantages. In addition, we also demonstrate their usefulness for plant cell research.

The pollen tube is a male reproductive tissue in angiosperms that transports sperm cells to the ovule where double fertilization takes place. It was believed that the nuclei, always present at the apical region of the pollen tube, were responsible for the pollen-tube growth and directional control during its long journey to the ovule. However, we recently showed that pollen tubes could reach the ovule even when de novo transcripts are not supplied to a tip during pollen-tube elongation. This review focuses on recent topics about the directional control of pollen tubes including our research and discusses the importance of these studies for plant reproduction.