Showing papers by "Shigeyuki Kawano published in 1992"

Preferential mitochondrial and plastid DNA synthesis before multiple cell divisions in Nicotiana tabacum

Abstract: Organelle DNA synthesis in root meristem and cultured cell line BY-2, both derived from Nicotiana tabacum cv. Bright Yellow 2, was examined by immunofluorescence microscopy of Technovit sections with antibody against 5- bromodeoxyuridine (BrdU) and co-fluorescent staining with 4′,6-diamidino-2-phenylindole (DAPI) and quantitative Southern hybridization. In the root meristem, the mitochondrial DNAs (mtDNAs) were synthesized in a specific region near to the quiescent center, where a low frequency of DNA synthesis of cell nuclei was observed. The mitochondrial nuclei (nucleoids) changed morphologically from long ellipsoids with a high frequency of DNA synthesis, in the region just above the quiescent center, to granules with a low frequency of DNA synthesis, as cell distance from the quiescent center increased. Similar patterns were observed in the cultured tobacco cell line (BY-2), in which large amounts of preferential synthesis of DNA of both mitochondria and plastids occurred prior to cell nuclear DNA synthesis just after stationary phase cells were transferred to fresh medium. Granular mitochondria which vigorously synthesized mtDNA were observed in both lag phase and logarithmic growth phase cells. However, long, ellipsoidal mitochondria which showed a low frequency of mtDNA synthesis were observed in stationary phase cells. Morphological changes of plastids were more conspicuous than those of mitochondria. After the medium was renewed, spherical plastids became extremely elongated and string-like, for 24 h, but were divided into small pieces after the third day. Vigorous synthesis of plastid DNA (ptDNA) occurred during this period of plastids elongation.

Conversion of proplastids to amyloplasts in tobacco cultured cells is accompanied by changes in the transcriptional activities of plastid genes.

Abstract: When tobacco (Nicotiana tabacum L.) cultured cells (line BY-2) at the stationary phase were transferred to culture medium that contained cytokinin (benzyladenine) instead of auxin (2,4-dichlorophenoxyacetic acid), proplastids in the BY-2 cells were converted to amyloplasts within 48 h. The data obtained from in vitro transcription assays using isolated plastid-nucleoids (nuclei) strongly suggested that amyloplast formation in BY-2 cells was accompanied by changes in the transcriptional activities of plastid genes.

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen in Lilium longiflorum and Pelargonium zonale

Abstract: The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca2+-dependent nuclease and a 23 kDa Zn2+ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.

Fine structure of centrosome complex and its connection with cell nucleus in the slime mould, Physarum polycephalum

Abstract: Cell-nuclear migration during the amoebo-flagellate transformation of Physarum polycephalum is mediated by the centrosome. The centrosome is expected to be strongly connected to the cell nucleus. An electron micrograph of a thin section of intact amoeba cell suggested that one of the structures consisting centrosome complex, mtoc, was directly attached to the outer membrane the of cell nucleus without any intervening microtubules. To analyze the fine details of the centrosome perimeter, and especially the space between the cell nucleus and mtoc, we used 0.1% Triton X-100 and different concentrations of NaCl to remove obstructive cytoplasm around the centrosome and the cell nucleus. The centrosome complex was shown to be composed of 2 centrioles, mtoc, link, ppks, extension, crown and lattice. With 100mM NaCl, the connection between the centrosome complex and the cell nucleus remained intact and was visible as a cell-nuclear appendage even by light microscope. This linkage was maintained even when 2mM Ca2+ ion was added to the samples to depolymerize and eliminate the effects of microtubules. This shows that the structure which connects the centrosome complex to the cell nucleus is mtoc, not microtubules. Electron micrography using whole mounts and thin sections of the treated amoeba cells demonstrated that the cell nucleus retained traces of its outer and inner membranes, and that the mtoc of the centrosome complex was directly connected to the outer membrane of the cell nucleus.