[in Japanese]

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Rapid freezing method has been used as a powerful tool to analyze fine structure of cell organelles which are the base of various cell functions. In case of plants, however, this method can applied to limited materials because of the presence of cell wall which prevents quick freezing. By using high pressure freezing method, plant tissues can be analyzed at the ultrafine level. In the last annual meeting of the Botanical Society of Japan, we had a joint symposium with the Japanese Society of Plant Morphology supported by Bio-imaging Center, Japan Women’s University. In this symposium, real structure of various plant cell organelles will be presented and discussed.

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The goal of specimen preparation for electron microscopy of biological tissues and cells is to preserve fine structure as close to their native state as possible. Cryofixation is now generally accepted as the best initial fixation step providing superior preservation of not only fine structure but also antigenicity essential for immunoelectron microscopy. Currently, high-pressure freezing followed by freeze-substitution is the most reliable method to obtain a high yield of vitreous (ice-crystal damage-free) freezing. The application of high-pressure (in the range of 2,100 bars) lowers the freezing point and reduces the rate of ice nucleation and ice crystal growth. It has also been reported that vitrification depth could reach several hundred micrometer under high-pressure. Such a deep vitrification enables us to apply the frozen specimens to not only conventional electron microscopy of resin embedded specimen but also electron microscopic tomography, freeze-fracture method, and cryo-electron microscopy of vitrous sections (CEMOVIS). This review describes the practical basis and advantages of high-pressure freezing technique for fine structural electron microscopy.

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Plant cells contain several types of single-membrane-bound organelles. These organelles differ in function, size, and shape, as well as in their dynamics. High-pressure freezing and freeze-substitution (HPF-FS) is a method for preparing samples for transmission electron microscopic analysis that are now used widely in plant cell biology, but some technical hurdles remain. In this mini-review, I summarize the characteristics of plant organelles and the strengths and weaknesses of these sample preparation methods. HPF-FS is generally superior to chemical fixation in terms of preserving structural integrity, but HPF-FS is not ideal for large and watery organelles, such as the vacuole, as it distorts the membrane. Notably, organelles with complex shapes that are dynamic by nature, such as the Golgi apparatus, should be analyzed using HPF-FS, as there are clear differences in terms of the morphology of the Golgi apparatus when HPF-FS is used instead of conventional chemical fixation.

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High-pressure freezing techniques have been developed in recent decades, and have proven to be immensely powerful for ultrastructural analyses of plant tissues. An increasing number of researchers and students of plant biology are interested in these techniques, and several different types of high-pressure freezing machine are available. The aim of this mini review is to provide the minimum basic knowledge for researchers who intend to freeze plant tissues by using high-pressure freezing techniques.

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Four topics obtained by a cryofixation technique for electron microscopic research are introduced. 1. The cell ultrastructures of high pressure and/or liquid propane frozen twenty species including 2 higher plants, 14 algae, 2 fungi and 2 animals were examined and their definitions on electron micrographs were compared. 2. In Euglena gracilis, the number of chloroplast envelopes has been considered to be three according to the electron microscopic specimens by conventional chemical fixation. However, this idea turned out to be reexamined by high pressure frozen samples preserving membrane structure. By the latter, a lipid bilayer structure of plasma membrane and membrane of Golgi cisterna was clearly visualized. On the other hand, the unit-membrane of mitochondria and chloroplast envelopes was not seen as a bilayer. 3. Among oleaginous microalgae, Botryococcus braunii has attracted attention for obtaining a renewable biofuel, because it accumulates especially large amounts of hydrocarbons around cell surfaces. However, this surrounding oil interfered with electron microscopic preparation using a chemical fixation. In contrast, we optimized high pressure and/or liquid propane freeze-substitution methods for B. braunii that showed the best definitions among examined species (except 2 animals) listed above. As a result, we were able to progress the cytological researches on lipid synthesis and secretion in B. braunii. 4. The COPⅡcoat of coated vesicles and clathrin coated pids at plasma membrane were especially well-defined in the specimens prepared by cryofixation technique.

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High pressure freezing (HPF) can be fixed the fission yeast cells as vitrified bulk specimens constantly. HPF-freeze substitution method can preserve the fine structures of cell organelles such as cell membranes and cytoskeletons. We identified the presence of the autophagic pathway in fission yeast. We successfully applied the HPF ultra-low temperature low voltage scanning electron microscopy (SEM) and HPF-fracture SEM to observe the fine structures of fission yeast. As the results, the 3D structure of the yeast during cell cycle can be indicated. We also developed HPF-freeze substitution method for immuno-electron microscopy of fission yeast for molecular anatomic analysis. By using ultra-low concentration of fixative, fine structure of the cells was visualized and simultaneously antigen was detected on the same thin section. This method revealed that beta-1, 3-glucan synthase is in fact located on the cell membrane of the septum. And the electron dense area beneath the cell membrane was firstly appeared in the future site of invagination for the initiation of the septum. We showed various ultra-structural methods for fission yeast, Schizosaccharomyces pombe, using HPF.

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The conventional sample preparation for electron microscopy makes the artifacts in the process of dehydration and substitution, such as the changes of ultrastructure of cells and outflow of soluble components in the cells. Cryofixation has been proposed as the method to overcome the inconvenient and unexpected problems, by freezing the cells very rapidly. Because water freezes to be ice crystals with increase of the volume, and those formed in the cells destroy the ultrastructure. To preserve the ultrastructure of native cells at the resolution of electron microscopic level, it is required to make amorphous ice at the process of cryofixation. As the methods for it, rapid freezing to cool samples rapidly in normal pressure and high pressure freezing to freeze under 2100 bar have been proposed. In this review, the information of the latest high pressure freezing systems and their application has been introduced. The depth of good freezing in amorphous ice made by rapid freezing is 5 to 20 μm, on the other hand, that of high pressure freezing is about 200 μm. So the high pressure freezing can be applied for the electron microscopic analyses of cells and tissues at closed-to-native state. Workflows after cryofixation are, for example, freeze substitution and cryo-TEM observation of vitreous ice sections, or CEMOVIS. Furthermore, the surface of cryo-sectioned frozen samples has been observed by cryo-SEM.

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Imaging techniques have contributed to reveal the mechanism of biological dynamic phenomena. “Integrated imaging techniques” beyond the classical imaging techniques are emerged through the improvement of imaging hardware and combination of computer science, optics and nanotechnology. In this review, rising stars who actively work using integrated imaging techniques introduce their recent achievements.

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Differentiating xylem vessels develop secondary cell walls in distinct patterns following dramatic re-arrangement of cortical microtubules. Because xylem cells are formed in the center of plant organs, analysis of cellular events in xylem cells, in particular, in living xylem cells, has long been difficult. Recently, we established an in vitro Arabidopsis cell culture system, in which estrogen-induced expression of a xylem master transcription factor triggers xylem differentiation. This system enabled us to analyze the molecular dynamics during xylem differentiation. Using this system, we performed various lines of analysis such as microarray analysis, high-throughput live imaging, in vivo detection of protein-protein interaction, and functional analysis of novel genes. These analyses revealed that a ROP GTPase and MIDD1, which is a novel microtubule-associated protein, are essential components of secondary wall patterning. In this review, we summarize recent knowledge of molecular components governing secondary wall patterning, with emphasizing MIDD1 and a ROP GTPase.

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Chromophore-assisted light inactivation (CALI) enables us to inactivate proteins in specific cells or induce target cell death based on light-inducible and tissue-selective cell ablation. KillerRed can produce at least 1000 times as much oxygen as EGFP. CALI using proteins fused with KillerRed is a useful optogenetic method that has revealed the spatiotemporal function of proteins in vivo. Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. An RNA-binding motif protein encoded on the X chromosome (RBMX) was identified as a chromosomal protein responsible for chromosome cohesion. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation. In order to confirm the G2 phase-specific function of RBMX in nuclei, we performed spatiotemporal knockdown analyses by CALI using KillerRed fused with RBMX. We transfected a KillerRed-RBMX expression vector into HeLa cells expressing EGFP fused with histone-H1.2 and monitored chromosome dynamics by live cell imaging. At the consequence, we confirmed that RBMX is a novel cohesion regulator that maintains the proper cohesion of sister chromatids at G2 phase.

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Cellular functions are fundamentally regulated by intracellular temperature. However, the method to analyze intracellular temperature had been completely lacked. We have developed a novel method to visualize temperature distribution inside living cells using a novel fluorescent thermometer and fluorescence lifetime imaging microscopy. Here, we describe the detail of this intracellular temperature imaging method, particularly about the fluorescence lifetime imaging microscopy. We also discuss the results obtained from the intracellular temperature imaging and briefly introduce our current efforts in aim to have this method easily available to other researchers and to introduce a new point of view – temperature – into the biological research.

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Live imaging inside the living tissues and cells has become possible, in recent years, owing to the advancement of microscopic technologies. On the other hand, techniques to handle and manipulate samples during live imaging have many issues to be overcome. Microfabrication technologies represented by MEMS (Micro-Electro-Mechanical-Systems) allow us to produce custommade miniaturized experimental systems at the micrometer to sub-micrometer scale, which is as same scale as tissues or cells. This review introduces studies of observation and manipulation of enzymes, motor proteins, roots and pollen tubes using microfabricated systems to show the merits on applying those technologies to biological assays. Finally, the review discusses advantages of microsystem-based bioexperiments in general and future prospects of combination with single molecule nano-manipulation technique and stereolithographic technology at the millimeter scale.

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Root apical growth is altered by genetic and/or environmental influences, which is attributable to changes in cell proliferation and volume growth. For investigating spatial profiles of these two parameters, “cell flux”-based kinematic analysis offers a powerful tool. Kinematic analysis of the root growth answers the question of where and at what rates cells proliferate and increase their volumes. The first step of kinematic analysis is to determine the spatial profile of root elongation rates from time-lapse images of a steadily growing root that are captured by a videomicroscope at a certain interval. Next, cell length is measured along the root axis on a differential interference contrast microscopic image, and the collected cell length data are used together with the spatial profile of elongation rates to determine the spatial profile of cell proliferation rates. We further analyze these profiles by an original mathematical model, which assumes several relationships between cell number, cell proliferation rate, and volume growth rate, to estimate costs of various aspects of the root growth. Here we describe the methodology of kinematic analysis of the root growth and application of our mathematical model.

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Advances in imaging techniques have yield massive images into the biology. Along with the increase of dimension and data size of bioimages in the research field, a need for computer-aided image analysis becomes clear. However, software environments are not utilized enough for image analysis. This is because the versatility of purposes and the diversity of bioimages. With this situation,we have developed an adaptive classification system for bioimages, named “clustering-aided rapid training agent (CARTA)”. The CARTA is applicable to various bioimage classification that facilitates annotation and selection of features. The CARTA interactively collects information from experts and generates the customized classifier for the specified bioimages. In this review, approaches for development of bioimage classifiers are outlined as follows: clustering, rule-based classifier, supervised learning and active learning. The explanation of CARTA is followed as an example of active learning approach.

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In animal fertilization, numerous numbers of sperms are required for successful fertilization. In plants, however, it had long been thought that an ovule accepts only one pollen tube to complete fertilization even though huge amount of pollen is pollinated to stigmata. Recently, we reported a ‘fertilization recovery system’ in flowering plants that actively rescues failed fertilization of a defective mutant pollen tube by attracting a second, functional pollen tube. In typical flowering plants, two synergid cells beside the egg cell attract pollen tubes, one of which degenerates upon pollen tube discharge. We observed that fertilization was rescued when the second synergid cell accepted a wild-type pollen tube. Our results suggest that flowering plants precisely control the number of pollen tubes that arrive at each ovule and use a fertilization recovery mechanism to maximize the likelihood of successful seed set.

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Plants in the genus Asparagus have determinate leaf-like organs called cladodes in axils of degenerated foliage leaves. Because of their leaf-like morphology and axillary position, it has been unclear how cladodes have evolved and diversified. To reveal the evolutionary processes of cladodes, we have studied cladodes of A. asparagoides, which is sister to all other species in Asparagus and has leaf-like cladodes. The results indicated that cladodes are modified axillary shoots and the co-option of pre-existing genetic regulatory networks (GRN) involved in leaf development transferred the leaf-like form to axillary shoots. Moreover, to reveal the processes of the diversification, we also studied cladodes of A. officinalis, which has cylindrical cladodes. The results imply that altered expression pattern of leaf polarity genes led to the cylindrical morphology of cladodes in the A. officinalis clade. Thus, these results indicated that the co-option and alteration of pre-existing GRN play an important role in acquisition and subsequent morphological diversification of cladodes, respectively. We take the cladodes in the genus Asparagus as an example and discuss the acquisition and diversification of leaf-like organ.

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Flowers play roles in transferring the genetic information to the next generation. Androecium and gynoecium are involved in the reproduction, while calyx protects the inner floral organs from outer environment, and corolla attracts pollinators such as insects and birds. For this role the corolla has evolved to be the most variable plant organ with their shape, color, and fragrance. Genetic mechanism of floral organ development has been clarified with the use of Arabidopsis thaliana, a model plant for the molecular research. Here I introduce the molecular system of the petal development, with including the recent findings.

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Although grasses basically have a distichous phyllotaxy, the vegetative shoots of Cynodon dactylon seem to show two types of phyllotaxy, including opposite phyllotaxy. These phyllotaxies, however, may be formed by internodal growth after initiation of leaf primordia. To determine the initial phyllotaxy of C. dactylon, we observed the development and anatomy of the shoot apices of vertical and creeping vegetative shoots. The former seem to have an opposite phyllotaxy and the latter have a triserial phyllotaxy. In both vertical and creeping shoots, the initiation pattern of the leaf primordia was clearly distichous and the leaf primordia on shoot apices were more concentrated than those of other grasses. Therefore, apparent phyllotaxies, including opposite phyllotaxy, can be derived in C. dactylon from the regulation of internodal growth after concentrated initiation of distichous leaf primordia.

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