Hisashi Matsushima

EFFECTS OF CO2 CONCENTRATION DURING GROWTH ON THE INTRACELLULAR STRUCTURE OF CHLORELLA AND SCENEDESMUS (CHLOROPHYTA)

Effects of CO2 concentration during growth on intracellular structure were studied with ftve species of Chlorella and Scenedesmus obliquus. Cells grown under ordinary air conditions (low‐CO2 cells) had a well developed pyrenoid surrounded by starch, while those grown under high CO2 conditions (high‐CO2 cells) had a less developed pyrenoid or no detectable pyrenoid. Two mitochondria, one at each side of the neck of the projection of the chloroplast close to the pyrenoid, were found in low CO2 cells of C. vulgaris 11h. Usually, lamellar stacks extended in parallel in the chloroplast of low‐CO2 cells of C. vulgaris 11h, while a grana‐like structure was found in high‐CO2 cells. However, in C. pyrenoidosa, grana like structures were found more commonly in low‐CO2 cells than in high‐CO2 cells. These results suggest that development of pyrenoid starch is generally correlated with growth under low CO2 conditions, whereas CO2‐effects on lamellar stacking are species dependent.

A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.

Scanning electron microscopic study on the in vitro organogenesis of saffron stigma- and style-like structures

Abstract In vitro organogenesis of saffron stigma- and style-like structures was studied by scanning electron microscopy. Several patterns of stigma- and style-like primordium formation were observed in sections of several kinds of floral tissue. In most cases, primordium formation was initiated from the cut edge of the ventral epidermis of the carpel or hypanthium and extended to the whole surface except for the dorsal epidermis of the ovary wall and the dorsal epidermis of the hypantium. Various types of primordia, developing into individual stigma- and style-like structures, were apparent. The stigmatic surfaces of these structures was morphologically highly mature and biologically functional as pollen receptors whereas the epidermal cells on the styles of these structures were rather immature.

Enhancement of Cell Division in Marchanda Protoplast Culture by Activated Charcoal

Summary Protoplasts isolated from callus culture of liverwort (Marchantia polymorpha L.) were cultured in the presence of activated charcoal. The rate of cell division of regenerated protoplasts was enhanced by an addition of activated charcoal to the culture medium. Partial but considerable enhancement of cell division was observed with frequent washing of the protoplasts and replacement of the medium during the initial stage of culture. These results suggest that a certain substance(s) inhibitory to cell division and absorbed by activated charcoal is released from protoplasts and/or regenerated protoplasts.

Cultured green cells of tobacco as a useful material for the study of chloroplast replication

Chloroplast replication in cultured cells of tobacco (Nicotiana tabacum cv. Samsun NN) was investigated by electron microscopy in comparison with that of green leaves. The structure of chloroplasts in cultured cells changed conspicuously during cell growth especially in photoautotrophic cells. The frequency of dumbbell-shaped chloroplasts (intermediate of chloroplast division) was the maximum (about 23% of total chloroplast) in photoautotrophic cells at 3 days after inoculation, before the cells had started to grow. By contrast, in photomixotrophically cultured cells, the highest frequency of dividing chloroplasts was observed at the early exponential phase (about 7 days after inoculation). The dividing chloroplast was hardly detected in green leaves even at a young stage. The advantages of cultured cells for the study of chloroplast replication and ultrastructural development are discussed.

Histological and morphological observation of sporophytic shoot of Equisetum arvense produced from gametophytic cells by exogenously supplied cytokinin.

Summary Histological and morphological analysis were carried out during the sequence of sporophytic shoot formation of Equisetum arvense which was induced by exogenously supplied cytokinin. Internal histological observation revealed that meristemoid from the division of small and cytoplasmic dense cells formed on the marginal or surface part of gametophyte tissue and subsequently developed to a sporophytic shoot bud with an apical cell and leaf primordia. These result suggest that sporophytic shoot is produced by cytokinin apogamously. Using a low-temperature SEM method, we could obtained three-dimensional information during the morphogenesis of an apogamous shoot bud. The single apical cell, which is characteristic of the genus Equisetum , was recognized with high resolution of cellular dynamics by the SEM.

Effect of the Embryonic Axis on the Development of Glyoxysomes, Plastids and Mitochondria in Cotyledons of Germinated Cucumber Seeds

Summary The effect of the presence of the embryonic axis was examined on the following parameters of activities of cellular organelles in dark-grown cucumber cotyledons: activities of malate synthase, isocitrate lyase and 3-hydroxy-acyl-CoA dehydrogenase with glyoxysomes, ribulose bis-phosphate carboxylase activity and rate of chlorophyll formation with plastids, and activities of some respiratory enzymes (succinate and malate dehydrogenase and cytochrome oxidase) and respiratory activities (O 2 uptake and its cyanide-sensitivity, and respiratory control) with mitochondria. The development of these parameters depends upon the presence of the axis, and benzyladenine can bring about a full replacement of the action of the axis on the development of all the parameters except the two, respiratory control and cyanide-sensitivity of mitochondrial respiration. As to these two parameters, benzyladenine showed little effect on their development. On the basis of these results, the role of the axis in the development of the organelles in cotyledons is discussed.

Observation by SEM of the attachment of Agrobacterium tumefaciens to the surface of vinca, Asparagus and rice cells

Transformation of vinca cells was performed by the co-cultivation of cell-wall regenerated vinca protoplasts withAgrobacterium tumefaciens. Using thisin vitro and single cell system, attachment of the bacteria to the surface of vinca cells was observed by scanning electron microscopy (SEM). Figures of the bacteria polarly binding to the plant cell wall were often observed. AsEscherichia coli does not attach to the plant cells at all, the observed attachment ofA. tumefaciens is suggested as a characteristic feature in crown gall induction. Even though no evidence of transformation was obtained by the co-cultivation methods, a similar attachment was observed in the cell-wall regenerated protoplasts of rice. The bacteria also attached to the surface of isolated mesophyll cells of asparagus and root hairs of rice. From these observation, we concluded that the attachment is not the limiting step of crown gall induction byA. tumefaciens in monocotyledonous plants. Extracellular fibrils like pili were observed with a few strains of A.tumefaciens for the first time. These fibrils were observed regardless of their ability of attachment and infectivity.

Changes in membrane glycoproteins during fruit-body formation inPhysarum polycephalum

Sporangia formation ofPhysarum polycephalum was induced by starvation and illumination, and the morphogenic process during the differentiation was studied by scanning electron microscopy. Plasma membranes were prepared from these differentiating plasmodia and the membrane proteins were analyzed by polyacrylamide gel electrophoresis. Many glycoproteins appeared during the fruit-body formation. Of these a protein of apparent molecular mass of 66 kD was prominent in sporangia forming stage which showed a high affinity to RCA lectin. Inhibition of the glycosylation and processing of these glycoproteins resulted in the prevention of fruit-body formation suggesting that the synthesis of these membrane components is a prerequisite process for the sporangia formation in the slime mold.