Various optical microscope technologies for visualization of specific molecules such as fluorescence imaging have been developed recently, however there are various biological phenomena that can be taken only by the electron microscopy. In the 82nd Annual Meeting of the Botanical Society of Japan, we had a joint symposium with the Japanese Society of Plant Morphology and IIRS, we arranged a symposium with speakers who are analyzing ultrastructural level by using various electron microscopic techniques for various biological materials from unicellular algae to trees. After presentation by the speakers, we held a symposium to discuss current morphology issues and ways to solve them.

A variety of eukaryotic phytoflagellates belonging to green algae, chromalveolates, and excavates exhibit three-dimensional phototaxis, movements directed toward (positive) or away from (negative) the light source along a light vector. Most phototactic algae possess a characteristic structure called ‘eyespot’ (also called ‘stigma’) in the chloroplast close to the flagella or the plane of flagellar beat. The eyespot is a cluster of lipid globules containing carotenoids and is mostly located close to the actual photoreceptor for photomovements (phototaxis and photophobic responses). In most algae, the eyespot has been believed to function as a reflective device providing a periodic illumination contrast for photoreceptors in combination with helical swimming patterns. Eyespots considerably vary in shape, size, and position among algal groups, and seem to have been independently acquired by those algae. Euglenophyta, photosynthetic euglenoids belonging to supergroup excavates, also possesses the eyespot and exhibits negative and positive phototaxis. Unlike other algal eyespots, eyespot globules of Euglenophyta are arranged irregularly in the cytoplasm and seemed to lack reflective properties. The eyespot function and its significance in the phototaxis of Euglenophyta have not been fully proved and have still been controversial. Here we summarize studies on the structure of eyespots and its function in the phototaxis of phytoflagellates focusing on green algae and Euglenophyta, one of the best studied algae in the eyespot and photomovement researches. In addition, we introduce our ongoing researches aiming at the elucidation of the eyespot function in the phototaxis of Euglena gracilis, one of the best studied Euglenophyta.

Motile dinoflagellates are bound by a complex cell covering, termed amphiesma. The cell covering consists of the plasma membrane, amphiesmal vesicles, and microtubules arrays lying under the amphiesmal vesicles. Armored dinoflagellates have amphiesmal vesicles containing thecal plates. Thecal plate pattern defined by the number, shape and arrangement of thecal plates is one of the most important characters to classify these armored dinoflagellate species. The present study demonstrated how amphiesmal vesicles develop after ecdysis in the armored dinoflagellate Scrippsiella hexapraecingula. In this report, we show the three-dimensional aspects of cortical membrane systems including the developing amphiesmal vesicles, which were visualized by both freeze-fracture and thin sectioning methods. Furthermore, the possible role of cortical microtubules in dinoflagellates was studied using high-pressure treatments. During the nonmotile phase, one or two daughter cells are produced inside the pellicle, and they develop new amphiesmal vesicles. The amphiesmal vesicles are empty, but are arranged in the same pattern as the developing thecal plates. Thecal plates are produced in the amphiesmal vesicles of motile cells, and therefore thecal plate pattern is determined at the time of development of amphiesmal vesicles in nonmotile cells. After nonmotile cells were exposed to high-pressure treatments sufficient to disorganize the cortical microtubules, they produced new motile cells with thecal plate patterns that differed considerably from the pattern known for this species.

Carotenoids in plants and algae are known for their role as photosynthetic antenna pigments or antioxidant effect. A unicellular green alga Hematococcus produces a red carotenoid, astaxanthin, from β-carotene as a precursor and accumulates in the cell. Recent studies have revealed that astaxanthin is contained in lipid droplets and localized outside the chloroplast. It is suggested that astaxanthin has the other functions essentially different from β-carotene and lutein localized in the chloroplast. When time-lapse imaging analysis was performed, astaxanthin contained lipid droplets actively in the cell. In the Hematococcus cell, it was observed that the strong light was avoided by shielding the light directly. As a result of hyperspectral and freeze-fracture replica imaging, it became clear that Haematococcus developed a successful adaptive strategy for light intensity using astaxanthin-containing oil droplets to avoid strong light.

Sample fixation of electron microscopy is roughly classified into a conventional chemical fixation and freeze fixation method. Freeze fixation method has several advantages as compared to chemical fixation, for example, preventing outflow of cell substances and deformation of cell structures, high temporal resolution, holding antigens by immunoelectron microscopy. The high-pressure freezing method is suitable for plant samples which have large cells. To date, we have examined electron microscopic analysis of Arabidopsis thaliana and tobacco tissues and cultured cells as well as unicellular algae by high-pressure freezing/freeze substitution method. In this mini review, we will introduce basic application and technical findings of highpressure freezing/freeze substitution method, and discuss technical problems.

Rice contains the storage proteins consisting of 7~8 % of rice seed. Rice storage proteins are classified three types, such as glutelin, prolamine and globulin, and they are deposited in two kinds of protein bodies (PBI and PBII) in the starchy endosperm. Glutelin and globulin are accumulated within the vacuolar PBII. On the other hand, prolamine is accumulated within PBI within ER lumen. The content and property of rice storage proteins affect highly the qualities of sake and rice-bred, therefore the elucidation of genes participating to biosynthesis, intracellular transport and deposition of rice storage proteins contributes to improvement of rice grain qualities. We have identified several genes concerning to biosynthesis, transport and deposition of glutelin and analyzed them. We introduce the knowledge about intracellular transport mechanism of glutelin based on the results of histological analysis.

Organisms exhibit life cycles in which they change their morphological features extensively by means of reproduction. The life cycle is a fundamental and impressive attribute of organisms. Freshwater green algae are very suitable for the life cycle research because methods for cultivation and induction of sexual reproduction have been published in these algae. Here, our previous morphological studies of freshwater algae carried out in these 40 years are reviewed, especially in the life cycle and micromorphology. Results of my light microscopic observations of the colonial volvocalean life cycles in the last century triggered and based our subsequent studies of the phylogeny, sex-specific genes and multicellularity, using the molecular genetic, genome and microscopic data of the colonial Volvocales. In addition, our electron microscopic observations of freshwater green alga Carteria and glaucophytes Cyanophora and Glaucocystis demonstrated their more natural three-dimensional ultrastructural features that contributed much to the species taxonomy of these genera. Since missing link species of the colonial Volvocales recently found from the natural habitats exhibited very interesting morphological features of the life cycle, further field works are very fruitful for future studies of morphology and diversity of the freshwater algae.

The independent origin of roots in lycophytes and euphyllophytes has been proposed based on evidence from fossilized plants. I previously reported how roots evolved within these lineages by analyzing the organization of the root apical meristem (RAM) in extant lycophytes, which is key to understanding the early evolution of roots. The RAM in lycophytes (Lycopodiaceae, Isoetaceae, and Selaginellaceae) has been classified into four types based on cell division activities and anatomy. Each of these four types is comparable to the RAM organization in euphyllophytes. The type I RAM found in Lycopodium is characterized by the presence of the common initial zone, a region with a very low frequency of cell division, reminiscent of the quiescent center (QC) in angiosperm roots. Although types II and III and the apical cell type in lycophytes are structurally similar to the RAM types in euphyllophytes, they have no QC-like area. The organization of the RAM in extant lycophytes is more diverse than expected. These results support the paleobotanical hypothesis that roots evolved several times in lycophytes and euphyllophytes.

Boron (B) is an essential micronutrient for plants that contributes to cell elongation through mediating cross-linking of the pectic polysaccharide rhamnogalacturonan II (RG-II). However, as with many of other nutrients, excess level of B becomes toxic to plants, resulting in the impaired growth and consequently the reduction in yield of seeds. So far, numerous studies have described physiological defects caused by excess B, while the molecular mechanisms underlying B toxicity remains poorly understood. Hence I characterized the mutants of Arabidopsis thaliana exhibiting extremely reduced root growth and abnormal root morphology under the excess B condition and identified a chromosomal protein complex condensin II and an active proteolytic device 26S proteasome as novel factors required for the tolerance to B toxicity. Through the functional analyses of these factors, I found that excess B induces DNA damage. In addition, I revealed the generation and suppressing mechanism of excess B-dependent DNA damage. Here I report the new aspects of B toxicity at molecular level.