Cytopathologie von viroid-infiziertem Pflanzengewebe

Cytopathology of viroid-infected plant tissue II. Light- and electron microscopical investigations on the leaf tissue of the Chrysanthemum morifolium cultivar “Mistletoe” after infection with the chrysanthemum stunt viroid (CSV) The infection of the Chrysanthemum morifolium cultivar “Mistletoe” with the chrysanthemum stunt viroid (CSV) leads to the appearance of numerous yellowish leaf spots 2–5 mm in diameter. The cells of these chlorotic leaf areas were investigated by phase contrast- and electron microscopy and compared with the cells of the adjacent green tissue and the tissue of healthy plants. Phase contrast microscopy showed that the chlorotic tissue containes about 50 % more cells per area and that their size is reduced by 30–60 %. The parenchymatic cells of the xylem and phloem are irregular and their walls are malformed. In these cells the chloroplasts are reduced to about half in their size and number. In the electron microscope an accumulation of osmiophilic material between the thylakoid membranes of the chloroplasts of the chlorotic cells and a deterioration of the chloroplast stroma can be observed. Moreover, malformations of the cell wall and in the cell wall-associated plasmalemma-somes are found, which lead to an increase in contrast and to irregularities of their surface and internal structure. The most prominent CSV-specific cytopathic effect in cells of the vascular tissue is the extreme accumulation of microfilament bundles which were analysed in detail with the aid of a goniometer. The observed viroid-induced ultrastructural changes are compared with previously described changes caused by conventional plant viruses and the possible functional implications are discussed.     

CRYPTIC DIVERSITY IN PHYTOPLANKTON CULTURES IS REVEALED USING A SIMPLE PLATING TECHNIQUE1

A simple technique has been developed to probe the occurrence of cryptic genetic diversity in populations of laboratory‐maintained phytoplankton cultures. This agarose‐based method allows the investigator to plate a broad range of fragile algae, including representatives of the Raphidophyceae, Synurophyceae, Prymnesiophyceae, and Dinophyceae. Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara was selected as a model system for our genetic diversity study. Further optimization of the plating technique for this alga demonstrated that colony formation was independent of the physiological state of the parent culture and yet dependent on incident light intensity. The density at which cells were plated affected colony formation and the rate of growth, with intermediate densities (～10³ cells per plate) performing best. High‐metal stress was used as the selective screen for assessing genetic variability within a single H. akashiwo culture. To this end, “clonal” lines (quotation marks used to indicate that within‐clone diversity was expected) were generated from individual plated colonies, and their tolerance was measured by plating on selective medium. Results of these experiments suggest the following: (i) “clonal” lines generated from a single H. akashiwo culture displayed a significant variation in stress tolerance; (ii) “clonal” lines chosen for their ability to grow on selective plates retained this tolerance in the absence of stress, indicating that the observed variation is heritable; and (iii) genetic variation is continually generated in growing cultures. Our results are consistent with a conceptual model, presented here, in which stress tolerance among the individuals in a culture has a genetic basis that varies over a continuous spectrum.     

AN ANALYSIS OF THE EFFECT OF "CONDITIONED MEDIUM" UPON THE CELL CULTURE AT LOW DENSITY

A favorable effect of “conditioned medium” upon outgrowth of the cell culture with low density in vitro was analysed with the cells of chicken embryos. For preparing “conditioned medium”, cultures with a large number of cells were made with muscle, kidney, lung, liver and skin, while the biological activity of the medium was assayed by using the culture of a small number of the lung secondary cells. A use of “conditioned medium” was found to be necessary for encouraging the outgrowth of the cultured cells below a critical inoculum size. Of the various types of the media tested, the medium conditioned with muscle was most effective. “Conditioned medium” contained at least two different active factors, the first to enhance the plating efficiency of the inoculated cells to the surface of the culture dish, and the second to promote further outgrowth of the plated cells. “Conditioned medium” taken out of the mass culture at its exponentially growing phase had only the second factor, while that taken out of that at its stationary phase contained both factors. An activity of the first factor was not detected, when the mass culture was kept in such condition that the collagen synthesis was inhibited. The factor for enhancing the plating efficiency was eliminated from “conditioned medium” by preincubating the cells, before assaying the effect of the medium.     

AUTOGAMOUS AUXOSPORULATION IN PINNULARIA NODOSA (BACILLARIOPHYCEAE)1

Auxosporulation of the freshwater epipelic diatom Pinnularia nodosa (Ehrenb.) W. Sm. was studied in a clonal culture. Interphase cells possessed two chloroplasts with invaginated pyrenoids. The nucleus contained a single small body of heterochromatin at one end, also visible during most of meiotic prophase. During auxosporulation, induced by transfer of stationary‐phase cells to fresh medium and suppressed by high nitrogen (N), an unpaired mother cell produced a single auxospore. Although meiosis II and nuclear fusion were not observed, indirect evidence indicated that auxosporulation was autogamous (rarely reported in pennate diatoms), rather than apomictic; paedogamy was excluded. The protoplast produced after meiosis either (1) matured into a “pseudozygote,” via an asymmetrical contraction after meiosis I to form a single spherical cell at one end of the mother cell (pathway 1); or (2) constricted into two spherical cells (pathway 2). In pathway 2, the “pseudogametes” never fused and only one or none developed into a pseudozygote and then into an auxospore. Pathway 2 could be suppressed by continuous light. During metamorphosis of the spherical pseudozygote into an elongate young auxospore, a complete covering of thin siliceous incunabular strips was formed, separate from the organic wall formed around the pseudozygote when first formed and from the perizonium. Mature auxospores produced via pathway 2 had 60% of the volume as those produced via pathway 1 and had smaller chloroplasts (through loss of fragments during protoplast cleavage), but they achieved exactly the same lengths, suggesting that absolute length is monitored during expansion.     

LIGHT AND ELECTRON MICROSCOPIC CHARACTERIZATION OF TWO TYPES OF PYRENOIDS IN GONIUM (GONIACEAE,CHLOROPHYTA)1

The single, basal pyrenoids of Gonium quadratum Pringsheim ex Nozaki and G. pectorale Müller (Goniaceae, Chlorophyta) differed in appearance when vegetative colonies were cultured photoheterotrophically in medium containing sodium acetate. Chloroplasts of G. quadratum had distinct pyrenoids when grown in medium without major carbon compounds. However, the pyrenoids degenerated and were markedly reduced in size when such cells were inoculated into a medium containing 400 mg·L^?1 of sodium acetate. No pyrenoids were visible under the light microscope; however, with electron microscopy small pyrenoids and electron-dense bodies were visible within the degenerating chloroplasts, which had only single layers of thylakoid lamellae at the periphery. The chloroplasts subsequently developed distinct pyrenoids and several layers of thylakoid lamellae as the culture aged. In contrast, vegetative cells of G. pectorale always showed distinct pyrenoids when cells were inoculated into medium containing sodium acetate, sodium pyruvic acid, sodium lactate, and/or yeast extract. Therefore, we propose two terms, “unstable pyrenoids” and “stable pyrenoids,” for pyrenoids of G. quadratum and G. pectorale, respectively. Chloroplasts of the colonial green flagellates should thus be examined under various culture conditions in order to determine whether their pyrenoids are unstable or stable when pyrenoids are used as taxonomic indicators. Immunogold electron microscopy showed that the ratios of gold particle density of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) between pyrenoid matrix and chloroplast stroma in G. quadratum grown in medium with or without sodium acetate were lower than those of G. pectorale. Heavy labeling by anti-RuBisCO was observed in both the electron-dense bodies and pyrenoid matrix of G. quadratum. This is the first electron microscopic demonstration of degeneration and development of both pyrenoids and thylakoid lamellae in the chloroplast as a function of culture condition in green algae.     

The effect of abscisic acid on cold tolerance and chloroplasts ultrastructure in wheat under optimal and cold stress conditions

The effect of abscisic acid (0.1 mM) on cold tolerance of leaf cells and ultrastructure of chloroplasts in wheat (Triticum aestivum L.) under optimal (22 °C) and cold stress conditions (4 °C) was studied. Results indicated that exogenous abscisic acid induces a rise in the cold tolerance of wheat along with a number of significant ultrastructural changes in chloroplasts both at 22 and at 4 °C. Some of them (increase in density of chloroplasts stroma, formation of “distorted” and “dilated” thylakoids, appearance of invaginations, changes in the shape of chloroplasts and increase of their dimension owing to the stroma area) were common to the two types of treatments. At the same time, the character of changes in the membrane system of plastids was temperature specific, i.e. if at 22 °C the hormone caused a considerable increase in the length of photosynthetic membranes in chloroplast owing the length of both appressed and non-appressed membranes of thylakoids, then in cold stress conditions observed an increase in the number of grana and the length of appressed membranes of thylakoids. These results suggested that the rise in the cold tolerance of abscisic acid-treated plants is associated with the ultrastructural reorganization of chloroplasts aimed to defense plant cells against chilling injury and to maintain the activity of the photosynthetic system.     

Effects of two species of macroalgae—Ulva pertusa and Gracilaria lemaneiformis—on growth of Heterosigma akashiwo (Raphidophyceae)

The effects of fresh thalli, culture filtrate, water-soluble extract and dry powder of two species of macroalgae, Ulva pertusa (Chlorophyta) and Gracilaria lemaneiformis (Rhodophyta), on the growth of a bloom-forming microalga, Heterosigma akashiwo, were studied in co-culture under controlled laboratory conditions. Both fresh thalli and culture filtrate of U. pertusa and G. lemaneiformis, particularly in the form of fresh thalli, significantly inhibited microalgal growth; indeed, the microalga was completely killed during the course of the experiment. A clear concentration-dependent relationship was observed between the initial concentration of fresh thalli (either U. pertusa or G. lemaneiformis) and its inhibitory effect on H. akashiwo. Simultaneous nutrient assays showed that nitrate and phosphate were almost exhausted in G. lemaneiformis fresh thalli co-culture but remained well above nutrient limitation for microalgal growth in U. pertusa co-culture, in which the microalgal cells were completely killed. However, daily f/2 medium repletion would obviously alleviate the growth inhibition in G. lemaneiformis co-culture. Since the present study was carried out under controlled conditions, fluctuations in environmental factors (i.e., light, temperature, carbon limitation, bacterial presence and pH) were limited during the experiment. We thus concluded that allelopathy was the most likely explanation for microalgal growth inhibition in U. pertusa co-culture, while the combined roles of allelopathy and nutrient limitation were responsible for growth inhibition in G. lemaneiformis co-culture. Similarly, macroalgal water-soluble extracts and dry powders affected the co-cultured H. akashiwo greatly, with more obvious effects observed in water-soluble extract co-cultures. A dose-dependent relationship was also observed over the course of the experiment. It can be concluded that macroalgal thalli contain some bioactive compounds. The results of the present study suggest that U. pertusa and G. lemaneiformis, especially in the form of fresh thalli, effectively inhibit the growth of H. akashiwo and could thus be potential candidates for use in the control and mitigation of H. akashiwo blooms.     

DINOPHYSIS CAUDATA (DINOPHYCEAE) SEQUESTERS AND RETAINS PLASTIDS FROM THE MIXOTROPHIC CILIATE PREY MESODINIUM RUBRUM1

“Phototrophic”Dinophysis Ehrenberg species are well known to have chloroplasts of a cryptophyte origin, more specifically of the cryptophyte genus complex Teleaulax/Geminigera. Nonetheless, whether chloroplasts of “phototrophic”Dinophysis are permanent plastids or periodically derived kleptoplastids (stolen chloroplasts) has not been confirmed. Indeed, molecular sequence data and ultrastructural data lead to contradictory interpretations about the status of Dinophysis plastids. Here, we used established cultures of D. caudata strain DC‐LOHABE01 and M. rubrum strain MR‐MAL01 to address the status of Dinophysis plastids. Our approach was to experimentally generate D. caudata with “green” plastids and then follow the ingestion and fate of “reddish‐brown” prey plastids using light microscopy, time‐lapse videography, and single‐cell TEM. Our results for D. caudata resolve the apparent discrepancy between morphological and molecular data by showing that plastids acquired when feeding on M. rubrum are structurally modified and retained as stellate compound chloroplasts characteristic of Dinophysis species.     

GRASS LEAF ULTRASTRUCTURAL VARIATIONS

The results of an ultrastructural examination of leaf anatomy and cytology of plastids of both non-Kranz and Kranz grasses are presented. Variations of Kranz anatomy examined include agranal and two granal conditions of kranz-cell chloroplasts, details of the two typical parenchyma sheaths of Aristida, and the “distinctive cells” of species of some small panicoid tribes. These conditions are discussed in relation to C₄ photosynthesis, evolution, and classification.     

DIFFERENTIATION OF ULVA MUTABILIS (CHLOROPHYTA) GAMETANGIA AND GAMETE RELEASE ARE CONTROLLED BY EXTRACELLULAR INHIBITORS1

Blade cells of Ulva mutabilis Føyn (Chlorophyta) excrete regulatory factors into their cell walls and into the environment. These factors are essential for the maintenance of the vegetative state. “Sporulation inhibitor-1a” (SI-1a) is a glycoprotein that was isolated from the culture medium of axenic Ulva growing as an undifferentiated callus. This protein was unusually stable to denaturing treatments and showed an extremely high apparent molecular mass (M_r) of 1–4 × 10⁷ daltons estimated by size exclusion chromatography. The glycosylation was not essential for activity. SI-1a suppressed gametogenesis completely at concentrations lower than 10^?14 M. When Ulva developed normally in the presence of their symbiotic bacteria, smaller forms of SI-1 accumulated in the medium (10⁴–10⁶ daltons). Sporulation inhibitors of the same size spectrum and with similar properties were also extracted from crude cell walls of nonaxenic Ulva. A class of different nonprotein sporulation inhibitors (SI-2) of very low M_r and yet unknown structure was isolated from the inner space between the two blade cell layers. Excretion of all SI-1 forms decreased with maturation of the thallus, whereas the overall concentration of SI-2 in the thallus stayed constant throughout the life cycle. The SI-2 affected different Ulva species whereas SI-1 was species-specific. Gametogenesis was induced upon removal of both Sporulation inhibitors from small single-layered fragments of mature blades. After a “determination phase” of 23–46 h, dependent on the time of induction within the cell cycle, the cells became irreversibly committed to differentiation and were no longer susceptible to SI-1 or SI-2. Subsequently, during a 28-h “differentiation phase,” 16 progametes were formed by synchronous genome doublings and cell divisions and differentiated into mature gametes. These became motile and were released from the gametangia when the concentration of a “swarming inhibitor” of low M_r, excreted mainly during the “determination phase,” declined below a threshold concentration. The biochemical properties of these regulatory factors and their effects on gametogenesis and gamete release are described.     

ULTRASTRUCTURAL CYTOLOGY OF CALLUS CULTURES OF STREPTANTHUS TORTUOSUS AS AFFECTED BY TEMPERATURE

Tissue culture cells of Streptanthus tortuosus var. orbiculatus (Cruciferae) which have acquired a spherical viruslike particle located in their nucleoli, designated cell line STV, developed supergranal chloroplasts and lost the ability to differentiate vascular tissues. The effect of temperature on the ultrastructural cytology of one line of the STV tissue, STV-I, was compared with the effect of temperature on the ultrastructural cytology of tissue culture cells lacking the viruslike particles (control cell lines). At 4 C, the cellular and ultrastructural appearance of control tissue culture cells differed from that of tissue grown at 22 C by producing increased amounts of endoplasmic reticulum and dictyosomes and by reduction of chloroplast thylakoids. STV-I cells were generally moribund as a result of 4 C treatment. Chloroplast thylakoids were also reduced in control tissue following growth at 10 C and the apparent quantities of endoplasmic reticulum and dictyosomes were similar to those observed in control cells grown at the control temperature (22 C), but less than those observed in tissue subjected to 4 C. STV-I tissue grown at 10 C demonstrated increased endoplasmic reticulum and dictyosomes and reduction of polysomal configurations. The mitochondrial morphology was variable and the cells contained supergranal chloroplasts and proplastids. At the control temperature (22 C), the fine structural appearance of control tissue culture cells was typical of parenchyma cells, but STV-I cells contained mitochondria of variable morphology and two types of chloroplasts— normal and supergranal. Control tissue grown at 30 C also contained proplastids, but these proplastids contained starch in contrast to the proplastids in control tissue grown at low temperatures. The ultrastructural cytology of STV-I cells grown at elevated temperature (30 C) was characterized by enlarged mitochondria containing massive lipid bodies and the presence of protoplastids with starch and supergranal chloroplasts.     

Cortical actin filaments fragment and aggregate to form chloroplast-associated and free F-actin rings in mechanically isolatedZinnia mesophyll cells

Summary Changes in F-actin organization following mechanical isolation ofZinnia mesophyll cells were documented by rhodamine-phalloidin staining. Immediately after isolation, most cells contained irregular cortical actin fragments of varying lengths, and less than 5% of cells contained intact cortical filaments. During the first 8 h of culture, filament fragments were replaced by actin rings, stellate actin aggregates, and bundled filament fragments. Some of these aggregates had no association with organelles (free actin aggregates). Other aggregates were associated with chloroplasts, which changed in shape and location at the same time actin aggregates appeared. F-actin was concentrated within or around the nucleus in a small percentage of cells. After 12 h in culture, the percentage of cells with free actin rings and chloroplast-associated actin aggregates began to decline and the percentage of cells having intact cortical actin filaments increased greatly. Intermediate images were recorded that strongly indicate that free actin rings, chloroplast-associated actin rings, and other actin aggregates self-assemble by successive bundling of actin filament fragments. The fragmentation and bundling of F-actin observed in mechanically isolatedZinnia cells resembles changes in F-actin distribution reported after diverse forms of cell disturbance and appears to be an example of a generalized response of the actin cytoskeleton to cell stress.Abbreviations FITC fluorescein isothiocyanate - MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - RhPh tetramethylrhodamine isothiocyanate-phalloidin     

Influence of storage time and nutrient medium on recovery of fibroblast-like cells from refrigerated collared peccary (<Emphasis Type="Italic">Pecari tajacu</Emphasis> Linnaeus, 1758) skin

Animal cloning is a promising technology for biodiversity conservation, and its success depends on the recovery of nucleus donor cells. Specifically for collared peccaries, found sometimes in regions that are difficult to access, the storage at 4–6°C of skin tissues would be an alternative for the conservation of genetic material. Therefore, we aimed to evaluate different storage periods and the presence of a nutrient medium at 4–6°C on the recovery of somatic cells from the skin of collared peccaries. To analyze cell recovery rates, ear explants were distributed in non-refrigerated samples and samples refrigerated for 10, 30, and 50 d in the absence or presence of nutrient medium. All explants were analyzed by histologically and cultured. Only the fragments stored for 50 d without medium showed an increase in the total thickness of skin. Moreover, increased storage period, regardless of the presence of medium, increased the halo number and reduced the metabolic activity. After culture, only the fragments stored without medium for 50 d did not yield any somatic cells. Cells recovered from explants stored for 10 d showed similar characteristics to these recovered from non-refrigerated explants, regardless of the presence of medium, including the day at which explants achieved attachment and the total time to reach subconfluence. In conclusion, viable cells can be recovered from somatic tissues of collared peccaries stored for up to 50 d in the presence of medium, and tissues refrigerated for up to 10 d in the presence of medium yielded more viable cells.     

DESCRIPTION OF VIRIDILOBUS MARINUS (GEN. ET SP. NOV.), A NEW RAPHIDOPHYTE FROM DELAWARE'S INLAND BAYS

Delaware's Inland Bays (DIB), USA, are subject to blooms of potentially harmful raphidophytes, including Heterosigma akashiwo. In 2004, a dense bloom was observed in a low salinity tributary of the DIB. Light microscopy initially suggested that the species was H. akashiwo; however, the cells were smaller than anticipated. 18S rDNA sequences of isolated cultures differed substantially from all raphidophyte sequences in GenBank. Phylogenetic analysis placed it approximately equidistant from Chattonella and Heterosigma with only ~96% sequence homology with either group. Here, we describe this marine raphidophyte as a novel genus and species, Viridilobus marinus (gen. et sp. nov.). We also compared this species with H. akashiwo, because both species are superficially similar with respect to morphology and their ecological niches overlap. V. marinus cells are ovoid to spherical (11.4 × 9.4 μm), and the average number of chloroplasts (4 per cell) is lower than in H. akashiwo (15 per cell). Pigment analysis of V. marinus revealed the presence of fucoxanthin, violaxanthin, and zeaxanthin, which are characteristic of marine raphidophytes within the family Chattonellaceae of the Raphidophyceae. TEM and confocal microscopy, however, revealed diagnostic microscopic and ultrastructural characteristics that distinguish it from other raphidophytes. Chloroplasts were in close association with the nucleus and thylakoids were arranged either parallel or perpendicular to the cell surface. Putative mucocysts were identified, but trichocysts were not observed. These features, along with DNA sequence data, distinguish this species from all other raphidophyte genera within the family Chattonellaceae of the Raphidophyceae.     

AN ELECTRON MICROSCOPIC STUDY OF THE SPOROPHYTIC AND GAMETOPHYTIC PLANTS OF PADINA VICKERSIAE HOYT1

An electron microscopic comparison was made of the “isomorphic” sporophytic and gametophytic plants of Padina vickersiae. Light microscopic studies of the 2 generations showed few differences. This observation was also supported by studies at the ultrastructural level. However, minor differences in the gametophytic plant have been noted: (1) a well-defined nucleolus in the nucleus of the non-meristematic cell; (2) a general lack of osmiophilic globules in the chloroplasts; and (3) a clumping of chloroplasts in the maturing oogonium. The first and second differences are considered in terms of growth activity.     

Impairment of the Viability of Transformed Chinese Hamster Cells in a Nonsubcultured Culture under the Influence of Exogenous Oxidized Guanoside is Manifested Only in the Stationary Phase of Growth

Despite the fact that oxidation products of nucleotides and nucleosides are markers of oxidative stress, reports of the paradoxical ability of these compounds to protect cells from the harmful effects of reactive oxygen species began to appear more often. Among all nitrogenous bases, guanine is most susceptible to the influence of oxidative stress; therefore, guanosine is oxidized more often than other bases. In the present work, the effect of exogenous 8-oxo-2′-deoxyguanosine on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during cell proliferation slowing down within a single passage and subsequent “aging” in the stationary growth phase) of nonsubcultured transformed Chinese hamster cells was studied. We showed that the nucleoside is rapidly absorbed by the cells from the medium, but it does not affect the growth of the culture, and impairs the viability of the cells in the late stationary growth phase. Thus, no mitogenic or geroprotective effect of 8-oxo-2′-deoxyguanosine was found.     

The influence of ammonium and nitrate of nutrient medium on ultrastructural organization and photosynthesis of callus cells in Glycine max L

We studied the influence of exogenic ammonium on the functional activity and ultrastructural organization of cells of the mixotrophic soybean callus (Glycine max L.). Ammonium available in the nutrient medium increased the chlorophyll content, accelerating the rate of photosynthetic O2 evolution per unit of biomass. The presence of ammonium in the medium promoted formation of the protein-synthesizing system, which manifested itself as increased numbers of ribosomes, and thylakoids of chloroplasts, and higher electron density of the stroma in mitochondria and cytoplasm of mixotrophic cells. It has been concluded that the use of ammonium may lead to activation of protein synthesis, thus rising photosynthetic activity and favouring formation and development of membrane structures in chloroplasts.     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

Growth and senescence of antibody-forming cells

Studies were performed on the capacity of mice for hemagglutinating antibody production throughout their life-span. An in vivo culture method was used for assessment of primary and secondary antibody-forming potentials of spleen cells of mice ranging in age from 1 to 130 weeks. There was a marked growth of potential for antibody formation during neonatal and juvenile life followed by a gradual decline in potential with advancing age. It was possible to show that the changes in potential were principally due to changes in the number of competent progenitor cells and not to changes in their performance. Death of very old animals was correlated with decline in number of immunologically competent progenitor cells. The decay in number of progenitor cells during aging of mice was random. Loss of progenitor cells was not entirely attributable to either generative failure of the pool of progenitor cells or the capacity of the milieu of the animal to support such cells. Thus, spleen cells from aged animals displayed increasing capacity for primary antibody formation during a 3-week period of culture in young, irradiated mice; identical cultures in old, irradiated recipients failed in respect to growth of primary antibody-forming potential. Progressive imparirment of the milieu of aging animals was suggested by the fact that spleen cells from very old animals were “toxic” when infused into lightly irradiated recipients which were themselves of advanced age but far short of the senescent phase of their life-span. These results lead to the argument that senescence may be, to a major degree, the result of progressive loss of progenitor, or “stem,” cells which are normally utilized to replace terminally differentiated, dying cells.     

Variations in Zinc Content of Subcellular Fractions from Young and Old Roots, Stems and Leaves of Broad Beans (Vicia faba)

⁶⁵Zn labelled seeds of broad beans (Vicia faba L.) germinated and grown in a complete nutrient solution containing the radioisotope were used to investigate the change in the subcellular distribution of this trace element during subsequent development of the labelled seeds. Homogenates of leaves, stems and roots were fractionated into cell-wall debris, nuclei, chloroplasts, mitochondria, ribosomes and “soluble” fraction. The concentration of the radioisotope in these fractions, expressed as cpm/mg dry weight, was highest in the soluble fraction and in the ribosomes isolated from young tissues. However, as roots and leaves aged, a general decrease in the overall zinc concentration was followed by a similar drop in the concentration of the metal associated with the “soluble” fraction and ribosomes, causing a more or less uniform zinc distribution pattern. On the other hand, there was a general increase in the zinc concentration of the “soluble” and in the particulate fractions of relatively high sedimentation coefficient from older stem samples. Taking also into consideration the dry weight change per unit volume of the tissues under investigation, the relationship between these variations and the metabolism of the plant is discussed.