Summary: We developed a method for isolating chloroplast-nuclei(nucleoids)from a unicellular green alga, Chlamydomonas reinhardtii, using cell-wall-deficient mutant(cw15)cells. Because the cells drastically change the structure of their chloroplast-nuclei during the cell division cycle, we synchronized the cell division under a 12: 12-h light: dark regimen, and collected cells in the 8th h during the dark period for use as starting material, when chloroplast-nuclei were granular in shape and scattered randomly within individual chloroplasts. The cells were converted to protoplasts and disrupted by repeated passages through a narrow-bore needle, and the intact chloroplasts were purified by Percoll density-gradient centrifugation. The chloroplast-nuclei were isolated from the purified chloroplasts following lysis with Nonidet P-40. The results of Southern and northern hybridization analyses suggested that not only structure, but also DNA synthesis and transcriptional activities of the chloroplast-nuclei might fluctuate during the cell division cycle of C. reinhardtii. Thus, the chloroplast-nuclei of C. reinhardtii may provide an ideal system for analyzing the structure-function relationships of DNA-protein complexes.

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Summay. High-pressure freezing-freeze substitution methods(HPF-FS)were applied to germinating pea leaves. Good ultrastructural preservation without visible freezing damage was obtained up to 200μm in thickness. Compared to conventional chemical fixation(CF), cellular membranes were smoother without undulation, and organelles appeared more turgid. The matrices of cytoplasm and organelles were denser and more homogeneous. These features imply that HPF-FS samples retain more substances and ultrastructure closer to the living state. There were differences in membrane stainability among organelles in HPF-FS specimens, which were not seen after CF. Bundles of microfilaments were observed frequently after HPF-FS. Dynamic ultrastructural change during the first 24h of imbibition of dry seeds was revealed by this method.

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Summary: A high pressure freeze-substitution method was developed for immunoelectron microscopy(immuno-EM)of S. pombe. The method used a low concentration of OsO4 or uranyl acetate(UA)provided a high contrast in the specimen, while retaining the fine structure of the cells. In the image fixed with 0.01% OsO4-acetone, the cell wall and cytoskeleton were visible, while 0.1% UA-acetone was superior in the membranous structure and cytoplasm. The new method makes it possible to visualize the ultrastructure and localization of the material on one thin section for the immuno-EM.

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Summary: After cytokinesis, the septum protorude a daughter half cell which is at first bulge formed and later develops into 3, and 5 lobes. Daughter half cells incresed in volume due to water penetration resulted from the decrease in the wall pressure of young half cells. Young half cells stopped growth in isotopic or hypertonic solutions, and small half cells were obtained. The irregular shaped small half cells produced normal shaped and normal sized descendant cells. Double cells produced anucleate cells or multinucleate cells, by which the role of the nuclei and of the cytoplasm in the morphogenesis was analyzed. Plasmolysis was not seen at the concave regions of the lobes. It is speculated that this regions stopped the out growth by inhibiting of Golgi vesicles to pass through the plasma menbrane.The localization of microtubules implies that they porevent the spheriacal expansion of lobes. The two factors, the connecting substancs at the concave regions and the microtubules, seem to regulate the morphogensis in Micrasterias.

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Summary: An in vitro system to analyze light signal transduction inducing the morphogenesis of perithecia was developed. A crude membrane fraction prepared from wild type(band)mycelia was mixed with [γ-32P]ATP and irradiated with blue light for 1 sec at 0°C. Five sec after illumination, the reaction was stopped and the proteins were separated by SDS-polyacrylamide gel electrophoresis. The increase in the phosphorylation of 15 kDa protein was detected and the increase lasted till 15 sec after illumination. The 15 kDa protein was purified, identified to be nucleoside diphosphate kinase and designated to be NDK-1. The cDNA and genomic DNA for NDK-1 were isolated. A mutant with no phosphorylation activity of NDK-1 was detected among wc-1 strains as a double mutant. The mutation designated psp(phosphorylation of small protein)was separated from the wc-1 and mapped on LGVR. Wild type and psp formed perithecial beak at random places of perithecia in darkness. Under directional light parallel to the solid medium the wild type perithecia formed the beak facing upward. However, under the same light condition psp formed the perithecial beak at random places on the perithecia. The cDNA for NDK-1 isolated from psp was sequenced. We found that ndk-1 gene in psp included Pro 72 His replacement, and finally psp was determined to be ndk-1Pro72His mutation.

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Summary: In gymnosperms chromosomes were analyzed by fluorescence banding using DAPI and chromomycin As and in situ hybridization with some probes in addition to conventional cytogenetic technique to be revealed as follows; Several valuable information on chromosomes in Chinese conifers such as comparative FISH analysis in Picea koreiensis and P. crassifolia. In Larix species examined were divided into six groups by fluorescence banding pattern and the DNA sequence localizing at the proximal DAPI-band was analyzed to be a tandem repeated AT-rich sequence and the sequence was presented in all Larix species examined. Sex chromosomes were discovered in Podocarpus macrophyllus and Cycas revoluta. In Pinus densiflora and P. thunbergii the DNA sequences localized at the fluorescent bands were revealed. The multi color FISH using four DNA probes allows to be identified each chromosome in each species and homoeologous chromosomes between these species.

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Summary: Senescence of the higher plant is genetically programmed as the last stage of the development. As the first step to clarify the whole mechanism of senescence, we characterized the pattern of senescence at both the tissue and cellular levels. The coleoptile of rice, which has a short life span and a simple structure, has been used mainly as the material for this study. A series of morphological analyses revealed two different types of programmed cell death in the coleoptile; natural senescence and aerenchyma formation. Although the progression at the tissue level differed largely between two types of death, initial and last events at cellular level were highly conserved in both processes. These facts suggest that the program of cell death would be identical in all cells of the coleoptile and the progression of the program would be affected largely by the surroundings.

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Summary: The structure of the Casparian strip was first described by the German botanist Robert Caspary in the 19th century. The function of the Casparian strip as an apoplastic barrier for solute transport is now fairly well understood. However, up to now the formation and the development of the Casparian strip is still an open question not only for plant physiologists but also for plant morphologists. Therefore, in the following I will try to rise some new questions and I will try to point out some new experimental approaches, which will be necessary in future in order to find answers to these questions concerning Casparian strip formation and development.

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