Many types of microscopic technics have been developed and enable to study various properties of biological phenomena. Currently, these techniques are extensively used to study various biological events at tissue- or cellular-level, as well as ultrastructural observations, for various biological materials from a single cell to multicellular species. In the 83rd Annual Meeting of the Botanical Society of Japan, we organized a joint symposium with the Japanese Society of Plant Morphology and IIRS, aiming to introduce the recent progress in various microscopic techniques, such as latest technique for electron microscope, multidimensional image data analysis, fluorescence imaging, imaging mass spectrometry, and laser microdissection. After presentation by the speakers, we held a symposium to discuss current morphology issues and ways to solve them.

The scanning electron microscopy (SEM) produces images by scanning the sample surface with an electron beam to detect backscattered electrons and/or secondary electrons. SEM has been widely used for the imaging tiny structures that are difficult to observe with optical microscopes. In recent years, SEM has been improved on various kinds of parts such as detectors and electron guns and can be used for the analysis of biological samples like tissues and cells by improving sample preparation methods such as fixation, heavy metal staining, resin embedding and coating. In this article, we describe new techniques for small organisms, plant tissues and cells using SEM and introduce new findings obtained from this observation.

The scanning electron microscopy (SEM) produces images by scanning the sample surface with an electron beam to detect backscattered electrons and/or secondary electrons. SEM has been widely used for the imaging tiny structures that are difficult to observe with optical microscopes. In recent years, SEM has been improved on various kinds of parts such as detectors and electron guns and can be used for the analysis of biological samples like tissues and cells by improving sample preparation methods such as fixation, heavy metal staining, resin embedding and coating. In this article, we describe new techniques for small organisms, plant tissues and cells using SEM and introduce new findings obtained from this observation.

To observe internal structures, specimens need to be sliced into thin sections. Sectional images only provide two-dimensional (2D) data. However, the stacking of micrographs of serial sections provides three-dimensional (3D) volume data. In this review, we introduce the 3D reconstruction method using a focused ion beam scanning electron microscope (FIB-SEM) that provides multiple serial sections automatically and accurately. We examined rice leaves with FIB-SEM and reconstructed 3D models for whole mesophyll cells, revealing their intricate cell shape and intracellular distribution of chloroplasts. The 3D models enable us to quantitatively analyse volume and surface area, which are difficult to estimate based on 2D sections. This review emphasises the importance of the 3D anatomy that comprehensively captures the objects, compared to 2D observations of sections that are only parts of the object.

Chlorophyll within chloroplast emits high-intensity autofluorescence, which interferes with the observation of exogenous fluorescent substance such as fluorescent protein in the plant cell. We previously reported that clear fluorescence observation can be done by using the time-gated method that eliminates the autofluorescence. Recently, we succeeded in tracking the subcellular localization process of the blue-light photoreceptor phototropin (Phot) to the chloroplast periphery in the liverwort Marchantia polymorpha by a combination of the confocal laser microscopy for the time-gated method and a photoconvertible fluorescent protein. Here, as an example of the time-gated fluorescence imaging, we introduce a study about intracellular localization and movement of Phot in M. polymorpha.

Imaging mass spectrometry (IMS) is an analytical method for visualizing the localization of metabolites. IMS analysis involves performing spatial mass spectrometry in coordinates specified on cross-section or longitudinal section of organisms, including plants. The localization of detected metabolites can be visualized using the m/z value and signal intensity acquired in IMS analysis. The development of genome or transcriptome sequencing technologies enables us to perform extremely local part analyses at the level of the cells, tissues, or organs. Using the sequencing technologies, phytochemical genomics researches have shown that specialized metabolic pathways have associations between gene expression and metabolite accumulation in a tissue- or organ-specific manner. These findings suggest that the identification of specialized metabolites in the local parts through IMS analysis makes narrowing down biosynthetic genes more simplified. The IMS analysis is potentially capable of increasing the efficiency and accuracy in phytochemical genomics. Herein, I provide recent updates on IMS analysis in plants.

Laser microdissection (LMD), also called as Laser capture microdissection (LCM), is a method for isolating different tissue cells or specific single cells from wide variety of tissue samples under direct microscopic observation. LMD method enables to harvest the cells of interest region or specific cells for several analysis such as DNA/RNA analysis, proteomics, metabolomics and other molecular analysis. Currently, this approach can be used to study various biological events at tissue- or cellular-level, LMD has been used in the wide field of research. In this report, we describe techniques for isolation different tissues/specific cells from cryosections of Arabidopsis incised flowering stem by LMD for spatio-temporal gene expression analyses.

Among plants and animals, cell size is restricted at the tissue level. Especially in a tissue where cells are actively dividing, they maintain a uniform size by the coordination of cell division and cell growth. In the Arabidopsis inflorescence meristem, which can be observed for several days under confocal microscopy, cells often divide unevenly and cell size variation of daughter cells is greater than that of mother cells. This variation is, however, reduced in subsequent cell cycles by changing cell cycle length or relative growth rate (RGR), depending on cell size. Several studies combined observation with mathematical models and indicated that cell size-dependent cell cycle regulation could maintain cell size regardless of variation in daughter cell size or RGR. In addition, cells need to properly regulate their size in response to environmental stress. TOR (target of rapamycin) pathway or responses to phytohormones would be involved in this regulation because those are activated by environmental signals and affect cell cycle or cell growth. It is also suggested that altered meristem cell size may influence gene expression pattern or tissue-level molecule gradient. Therefore, cell size regulation in the meristem should have an important role in organogenesis.

Epigenetic priming is one of the potential systems that genes are poised for activation by external signal inputs. Although the priming does not alter the gene expression, it is considered to induce the open structure of chromatin and the poised state for the future transcription. This priming is involved in stem cell differentiation, cancer development and drug action but remains unclear in plant regeneration. Through integrated analysis of genome-wide histone modifications and gene expression profiles, we successfully identified an epigenetic priming by a histone demethylase LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3) that specifically eliminates H3K4me2 during formation of callus derived from roots of Arabidopsis thaliana. While LDL3-mediated H3K4me2 removal does not immediately affect gene expression, it does facilitate the later activation of genes that act to form shoot progenitors after shoot induction. This finding gives insights into plant regenerative competency with epigenetic priming.

GTPase dynamin-related protein (Dnm1)-mediated membrane fission is an important membrane remodeling event supporting the proliferation and housekeeping function of semiautonomous organelles such as the mitochondrion and peroxisome. Dnm1 is at the heart of the membrane fission machinery, which constricts the neck of the dividing organelles. Similar to classical dynamin protein, Dnm1 hydrolyzes GTP, an energy source, thereby generating a constriction force to sever the neck. To complete this process, replenishment of GTP to Dnm1 needs to be done in a regulated and timely manner. However, the molecular mechanisms that provide GTP to Dnm1 are not known. In this review, we present the evidence for emerging consensus on Dnm1 function and our recent work demonstrating that: (1) The ATP-GTP converting, nucleoside diphosphate kinase-like protein DYNAMO1 is present in the mitochondrial and peroxisomal membrane fission machinery. (2) DYNAMO1 facilitates enzyme kinetics of Dnm1 and locally provides GTP to Dnm1 on the membrane fission machinery. (3) The membrane fission machinery spends more GTP on constriction than on recruitment, as seen by the in vivo experiments and in vitro reconstitution of the Dnm1 structure. Summarizing these data, this review would help to understand the mechanism by which Dnm1 promotes membrane fission using GTP as an energy source. We also discuss how future research might solve the remaining open questions regarding the energy issues presently under discussion.

Almost all life on earth depend on photosynthesis performed by chloroplasts of algae and land plants. Chloroplasts possess their own genomic DNA (cpDNA), which are considered as a relic of the endosymbiotic cyanobacterium. A copy of cpDNA encodes ~200 proteins essential for photosynthesis and biogenesis, and are packaged in DNA-protein complex called chloroplast nucleoids (CPNs), a counterpart of nuclear chromosome. However, little is known about the evolution of CPN structure and the molecular mechanism that regulates CPN morphology. To deduce the evolution of CPN structure, we analyzed CPNs of algae and liverwort, and proposed a model that the recurrent modification of CPN organization by eukaryotic factors originally related to chromatin organization likely have been the driving force for the diversification of CPNs since the early stage of plant evolution. We also investigated sulfite reductase (SiR), a major component of CPNs in land plants, to reveal the molecular basis to bind and bend cpDNA. The combination of in silico and in vivo analysis revealed that C-terminally region of SiR (~50 aa) plays a key role to interact and compact cpDNA. Furthermore, we identified the novel gene MOC1, which regulates the CPN morphology in algae and land plants. MOC1 encodes the chloroplast-targeted Holliday junction resolvase, suggesting that MOC1 untangles cpDNA tangled via Holliday junctions to segregate chloroplast genome faithfully.

Genome stability is always challenged by exogenous and endogenous stresses. As a sessile organism, plants are surrounded by various environmental stresses that induce DNA damage and inhibit plant growth. Thus, the maintenance of genome stability has an important role in the the growth and development of plants. In eukaryotes, DNA is wrapped with histones to form chromatin that is high-ordered structure in nuclei. The chromatin structure dramatically changes in accordance with DNA damage. However, little is known about the mechanism controlling the chromatin structure in response to DNA damage in plants. In this study, I investigated the regulation mechanism of chromatin structure during DNA damage response of Arabidopsis, mainly using live cell imaging techniques. My imaging analysis showed that chromatin remodeling factor RAD54 is required for the change of chromatin structure with DNA damages. I also found that RAD54 forms subnuclear foci (named RAD54 foci) in response to DNA damage. Additionally, I identified histone demethylase LDL1 as novel interacting factor of RAD54, using interactome analysis based on co-immunoprecipitation and LC-MS/MS analysis. My biochemical and imaging analysis showed that LDL1 regulates the dynamics of RAD54 at damaged sites through the demethylation of H3K4me2.

In order to promote the species taxonomy in microalgae, both detailed comparative morphological analysis and molecular phylogeny using cultured material are essential. I have focused on the Chloromonadinia clade (Volvocales, Chlorophyceae), which consists of morphologically and physiologically diverse species, and our research group has conducted taxonomic studies of this clade at the species level, based on comparative morphological analyses at the ultrastructural level and molecular data. Our previous reports of Chloromonas reticulata and its relatives showed that the species of which vegetative cells are morphologically similar to each other under light microscope, could be distinguished to the species level, based on ultrastructural differences in the pyrenoid or the eyespot within the chloroplast. In snow-inhabiting species belonging to the snow algae group within the Chloromonadinia clade, zygote morphologies are treated as the important taxonomic characteristics at the species level. However, induction of zygote formation (i.e. sexual reproduction) is difficult in experimental condition. Our comparative morphological analyses and molecular data using 19 strains of the snow algae group demonstrated that the strains could be subdivided into 12 species, based on differences in vegetative cell shape and chloroplast morphology, as well as in the number of zoospores within the parental cell wall and the presence or absence of cell aggregates in long-term cultures. In addition, we established the method that can obtain sequences of multiple DNA regions from field-collected zygotes of the snow algae group. The detailed molecular identification based on the sequence data unveiled that the part of Japanese field-collected materials morphologically assigned to the zygotes of C. brevispina and C. nivalis actually belong to the other species of the snow algae group. Besides, field-emission scanning electron microscopy for the zygotes of which the species were accurately identified by molecular data indicated that the ultrastructure on their zygote wall might represent a useful taxonomic trait at the species level.