[in Japanese]

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Plant morphology and plant physiology are major subdisciplines in plant biology. Although the focus and approach differ each other, both morphological and physiological studies have discovered numerous important findings in the field of plant biology. In addition, microscopic techniques and analytical apparatuses are making remarkable advances and these advances, often involving molecular biological analyses, made researchers to understand plant cells more precisely than before. On the other hand, the gap between plant morphology and physiology is widening, because the techniques related to morphology and physiology are developed too high to be followed by a single researcher. In the 79th Annual Meeting of the Botanical Society of Japan, we had a joint symposium with the Japanese Society of Plant Morphology and IIRS focusing how we can integrate morphology and physiology.

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Plant seeds resume their physiological activities during imbibition, but little is known regarding the associated structural changes because in vivo observation is not easy. We employed X-ray micro-computed tomography (micro-CT) to investigate the inner structures of seeds. Tomograms of seeds of a leguminous model plant, Lotus miyakojimae, were obtained using the micro-CT at BL20B2, SPring-8. By using this method, we could examine the spatial and temporal distributions of calcium oxalate crystals during seed maturation and germination. Further, we could examine the development of intercellular spaces in the embryo during maturation and germination. Intercellular spaces in cotyledons were not detectable in dry seeds, but became detectable at 60 min after imbibition. We employed the X-ray micro-CT at BL20XU to examine embryo structures at the cellular level. In addition, we determined the three-dimensional cell and tissue shapes and geometries in the hypocotyl–root axis of a dry seed of Arabidopsis thaliana. On the basis of these observations, we discuss the effective utilization of X-ray micro-CT and its drawbacks.

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Super resolution microscopy enables us to acquire images possessing much higher resolution than optical diffraction unit. Photostability of fluorescent dye is a definitely important property in super resolution microscopy, although required properties to fluorescence dye are varied and specialized dependent on the different super resolution technologies. Especially for STED imaging, exceptional photostability surviving in intense STED laser is required for repeated super resolution imaging and development of suitable fluorescence dye is awaited. Here, we introduce a phosphole oxide based fluorescent dye possessing unprecedented photostability sufficient for continuous STED imaging.

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The use of fluorescent protein for live imaging is an effective method of determining the localization of various proteins and organelles in cells or in tissues. Fluorescence labeling of organelles is useful for the study and analysis of the ultrastructure of plant organelles. To obtain electron micrographs of fluorescently labeled organelles and cells, we developed a correlative light and electron microscopy and successfully visualized the green fluorescent protein (GFP)-labeled organelles in resin by using a combination of confocal laser scanning microscopy (LSM) and field-emission scanning electron microscopy (FE-SEM). Organs of an Arabidopsis thaliana plant expressing GFP-labeled organelles were fixed and then embedded in acrylic resin. The block was cut by the ultramicrotome, and 1 µm-thick sections were placed on a glass slide. Then the GFP fluorescence images were acquired by performing LSM. Subsequently, these sections were stained with heavy metals, and electron microscope images of stained sections were acquired by FE-SEM utilizing a high-sensitivity reflection electron detector. Finally, the ultrastructure of the GFP-labeled organelles was analyzed by merging the fluorescence image and the electron micrograph.

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Imaging mass spectrometry (IMS) is a new tool to map the spatial distribution of molecules on the biological materials. Recently, we applied IMS to intact plant tissues or thin slices by using a combination of manufactured apparatus and commercial mass spectrometers. The present work is concerned with the refinement of MALDI/laser desorption/ionization (LDI)-Fourier transform ion cyclotron resonance (FTICR)-MS incorporating certain specific features namely, ultra-high mass resolution (>100,000), ultra-high molecular mass accuracy (<1 ppm) and high spatial resolution (<10 μm) for imaging MS of plant tissues. The IMS analysis succeeded in visualizing of cellular localization of small molecules of intact Arabidopsis thaliana roots and Catharanthus roseus stem tissues glued to a small conductive glass with and without a matrix substance. The images of various small metabolites representing their two-dimensional distribution on the dried intact tissues were obtained.

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Plant organelles dynamically change their morphology responding to environmental signals. To investigate the correlation between the light-dependent morphological changes of peroxisomes and interaction force between peroxisomes and chloroplasts, we applied femtosecond laser to estimate the interaction force between peroxisomes and chloroplasts. Here, we introduce our studies based on recent researches about organelles interaction.

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To determine the exact mode of inheritance of mitochondrial DNA (mtDNA) in Chlamydomonas reinhardtii, suitable markers to determine the origin of parental mitochondrial genes are necessary. For this goal, the difference in mtDNA between a C. reinhardtii strain and a C. smithii strain was used. These two species were serially backcrossed to ensure matching of the background of their nuclear genomes. The results showed strict paternal inheritance of mtDNA. Next, the fate of mt⁺ (female) and mt^- (male) mtDNA was followed using three methods: quantitative real-time PCR, Southern blotting hybridization, and fluorescence in situ hybridization (FISH). The disappearance of mt⁺ mtDNA was observed at the beginning of meiosis. In the mutant bp31, in which the maternal inheritance of chloroplast DNA (cpDNA) is disrupted, the paternal inheritance of mtDNA was also found to be disrupted, indicating commonality between cpDNA and mtDNA in part of the process leading to uniparental inheritance. Mitochondrial nucleoids and mitochondria were observed in living vegetative cells. Organellar nucleoids were stained with SYBR Green I, while mitochondria were stained with DiOC_6. During the cell cycle, mitochondrial nucleoids in living cells were granular, and mitochondria formed tangled threads. When mature zygotes were exposed to the light, meiosis began. Living zygotes with thick walls was also stained by SYBR Green I and DiOC₆. At the diakinesis stage of meiosis, the total number of chromosomes was 18. When mature zygotes were exposed to the light, granular mitochondria began to form short thread-like structures and then assembled around the cell nucleus. Once assembled, the mitochondria began to scatter in the opposite hemisphere, forming long and tangled thread-like structures. Mitochondrial nucleoids were usually granular, but some of them developed bead-like structures, probably by dividing. The results from the molecular biochemical experiments suggested that mt⁺ mitochondrial nucleoids disappear at the stage of assembly and the scattering of mitochondria. Then, the first and second nuclear divisions proceed, producing tetrads.

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In angiosperm, single pollen tube carrying sperm cells precisely targets to an ovule during fertilization processes. To ensure this one-on-one pairing, fertilized ovule inactivates the synergid cell to terminate the pollen tube attraction. We recently found a novel mechanism of inactivating the synergid cell by cell-fusion between the synergid cell and the endosperm. In this review, we describe the cell-fusion and other synergid inactivation mechanisms in Arabidopsis thaliana.

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Glaucophytes are one of the three major lineages of primary photosynthetic eukaryotes (Archaeplastida), together with red algae and Chloroplastida (green algae and embryophytes). Within Archaeplastida, glaucophytes are thought to retain ancestral features of the first photosynthetic eukaryote. The biflagellate glaucophyte genus Cyanophora has been widely studied as a model organism of primitive phototrophs, with released nuclear genome sequence, but no morphological study has clearly shown ultrastructural diversity to delimit glaucophyte species. Recently, we applied advanced electron microscopic (EM) methodologies for comparative morphology in a purpose of delineating the glaucophyte species. On the basis of ultra-high resolution field emission scanning EM (FE-SEM), we demonstrated that the cell surface of Cyanophora was ornamented with angular fenestrations framed by ridges. As supported by observation using several transmission EM methods, these ridges were formed by the edges of overlapping or attaching outermost plate vesicles that distributed throughout the cell periphery just underneath the cell membrane. Based on differences in the pattern of surface ornamentations detected by FE-SEM, Cyanophora biloba and C. sudae were distinguished from each other. FE-SEM, however, cannot be applied for the native protoplast surfaces that are enclosed by a thick cell wall or extracellular matrix as in the immotile glaucophyte genus Glaucocystis. Alternatively, we very recently used the advanced ultra-high voltage EM tomography to unveil the in situ peripheral ultrastructure of protoplasts in Glaucocystis and revealed the ultrastructural diversity in this genus. Furthermore, Glaucocystis cells have numerous, leaflet-like flattened vesicles distributed throughout the protoplast periphery just underneath the cell membrane, as in Cyanophora as well as some secondary photosynthetic eukaryotes. Thus, a similar 3D peripheral ultrastructure might have appeared in the common ancestor of glaucophytes and/or the first photosynthetic eukaryote.

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Insertion of a sheet of cover glass in the lid of a glass-based dish is a useful technique for observation by differential interference contrast microscopy. However, microscopic observations of algal cultures using this glass-based dish are often interfered with by dew condensation on the glass surface of the lid when the culture is incubated for a long time. To prevent dew development on the lid surface during incubation, we employed a glass-based dish whose inner surface of the glass-lid is coated with 2-methacryloyloxyethyl phosphorylcholine polymer. Using this modified dish, we could observe thalli development of Coleochaete scutata successively for four weeks without dew condensation on the lid.

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