Response of the Photosynthetic Apparatus of the Diatom Thallassiosira weisflogii to High Irradiance Light

The response of the photosynthetic apparatus to high irradiance illumination (440–2200 W/m²) was studied in the diatom Thallassiosira weisflogii by fluorescence methods. Changes in the photosynthetic apparatus were monitored by measuring characteristics of chlorophyll fluorescence F ₀, F _m, F _v/F _m, and qN for several hours after illumination of the alga with high-intensity light. Incubation of the alga with 2 mM DTT, an inhibitor of de-epoxidase of carotenoids in the diadinoxanthin cycle, led to a decrease in the nonphotochemical quenching of chlorophyll fluorescence and a drop in the F _v/F _m ratio, a characteristic that reflects the quantum efficiency of the functioning of the photosynthetic apparatus. Light-induced absorption changes associated with transformations of carotenoids of diadinoxanthin cycle were recorded in vivo in algal suspensions in the absence and in the presence of DTT. Using the microfluorometric method, we measured cell distribution over the efficiency of the primary processes of photosynthesis (F _v/F _m) after illumination. We found cells with a high tolerance of their photosynthetic apparatus to photooxidative damage. The relatively high tolerance of a portion of the cell population to high-light illumination can be related to light-induced transformation of carotenoids and to the functioning of other protective systems of the photosynthetic apparatus in diatoms.     

Distribution of the Golgi apparatus in the mitosis of cultured tobacco cells as revealed by DiOC6 fluorescence microscopy

Summary We developed a new method for distinguishing the Golgi apparatus from the other membranous organelles which contain DNA, such as mitochondria and chloroplasts, under a fluorescence microscope. Thin sections of cells embedded in Technovit 8100 resin were stained with both 3,3-dihexyloxacarbocyanine iodide (DiOC₆) and 4,6-diamidino-3-phenylindole (DAPI), and those three membranous organelles were observed under an epifluorescence microscope. The Golgi apparatus, which do not contain DNA, were easily recognized when the two images stained with DiOC₆ and DAPI were superimposed using an image processor. Using this method, we investigated the dynamics of cellular membranes and organelles during the mitotic cycle of synchronized cultured tobacco cells BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). The Golgi apparatus did not accumulate in the rim of the formating early cell plate at anaphase, while it accumulated near the maturing cell plate at telophase, and this accumulation seemed to be related to the maturation of cell plates. To confirm this hypothesis, synchronized BY-2 cells were treated with caffeine, which is known to inhibit the cell plate formation. Most of the cells treated with caffeine remained in a phase in which Golgi vesicles were accumulated at the equatorial plate, but the cell plate was only partially maturing. The Golgi apparatus accumulated only near the partially maturing cell plate, but not by the equatorial plate where the Golgi vesicles had accumulated.Abbreviations DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DAPI 4,6-diamidino-3-phenylindole - LSD a modified Linsmaier and Skoog's medium containing 2,4-D     

Synchronous intra-Golgi transport induces the release of Ca from the Golgi apparatus

The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca²⁺) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca²⁺ concentrations in the cell cytosol ([Ca²⁺]_cyt) and inside the lumen of the Golgi apparatus ([Ca²⁺]_GA), we have revealed transient increases in [Ca²⁺]_cyt during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca²⁺]_GA restoration ability. Thus, this redistribution of Ca²⁺ from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca²⁺-dependent phase of SNARE-regulated fusion of Golgi compartments.     

Localization of ATPase in the endoplasmic reticulum and golgi apparatus of barley aleurone

Summary The cytochemical localization of adenosine triphosphatase (ATPase) was studied in the aleurone layer of barley (Hordeum vulgare L. cv. Himalaya). Isolated barley aleurone layers secrete numerous enzymes having acid phosphatase activity, including ATPase. The secretion of these enzymes was stimulated by incubation of the aleurone layer in gibberellic acid (GA₃). ATPase was localized using the metal-salt method in tissue incubated in CaCl₂ with and without GA₃. In sections of tissue incubated without GA₃, cytochemical staining was confined to a narrow band of cytoplasm adjacent to the starchy endosperm and to the cell wall of the innermost tier of aleurone cells. Cytochemical staining was absent from the organelles of tissues not treated with GA₃. In tissue incubated in the presence of GA₃, cytochemical staining was evident throughout the cytoplasm and cell walls of the tissue. In the cell wall, electron-dense deposits were found only in digested channels. The cell-wall matrix of GA₃-treated aleurone did not stain, indicating that it does not permit diffusion of enzyme. In the cytoplasm of GA₃-treated aleurone, all organelles except microbodies, plastids, and spherosomes stained for ATPase activity; endoplasmic reticulum (ER), Golgi apparatus, and mitochondria showed intense deposits of stain. The ER of the aleurone is a complex system made up of flattened sheets of membrane, which may be associated with both the Golgi apparatus and the plasma membrane. The dictyosome did not stain uniformly for ATPase activity; rather there was a gradation in staining of the cisternae from thecis (lightly stained) to thetrans (heavily stained) face. Vesicles associated with dictyosome cisternae also stained intensely as did the protein bodies of GA₃-treated aleurone cells.     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Complete mechanical impedance increases the turgor of cells in the apex of pea roots

In this paper we describe an experimental approach which allows turgor (p) in an impeded root to be measured without the need to remove the root from the impeding environment. The maximum axial growth pressure (σ_max) generated by completely impeded pea (Pisum sativum L.) roots was measured using a novel apparatus incorporating a force transducer. The apparatus was designed so that it was possible to gain access to the impeded root with the microcapillary of a pressure probe and so obtain in situ measurements of P. Turgor in cells in the apical region of impeded roots was 0.78 MPa, compared with 0.55 MPa in unimpeded roots. In impeded roots, σ_max was 0.52 MPa, showing that the pressure component resulting from cell wall tension (W, where W=P–σ) decreased from 0.55 to 0.26 MPa as the roots became impeded. When impeded roots were removed from the apparatus, there was no decrease in P over the following 90 min. Impedance did not cause P to change in the non-elongating part of the roots further from the apex.     

DISTINCTIVE LECTIN LABELING OF THE FLAGELLAR APPARATUS OF TETRASELMIS (PRASINOPHYCEAE) AND DUNALIELLA (CHLOROPHYCEAE)1

Fluorescent labeling of the flagellar apparatus of Tetraselmis (Prasinophyceae) and Dunaliella (Polyblepharidaceae, Chlorophyceae) were successfully performed using fluorescein isothiocyanate–labeled lectins from Arachis hypogaea and Glycine maxima. These lectins specifically bound to the flagella and kinetosome of the cell but did not bind to the cell surface. Lectin binding on the flagellar apparatus remained constant under different culture media, temperatures, irradiances, cell division cycle, and culture aging. All the Tetraselmis and Dunaliella analyzed (five species, 20 clones) showed intense labeling of the flagellar apparatus. In contrast, no other species analyzed (46 clones of 25 species from four classes) showed binding to their flagellar apparatus. After the lectin treatment, many cells remained alive, and they were able to swim with the flagellar apparatus intensely labeled. The lectin binding indicates that the flagella and kinetosome of Tetraselmis are rich in Gal and GalNH₂ moieties and that the flagella of Dunaliella are rich in Gal and GalNAc moieties. Apparently, this feature seems to be specific to these species.     

Working design for a 5-liter controlled continuous culture apparatus

A continuous culture apparatus of 5-liter capacity is described which is capable of control of dissolved oxygen. Combined turbidostat and constant feed operation permit control of cell population density and one or more nutrients. A system for the measurement of oxygen uptake and CO₂ output is described. Sufficient detail is supplied to enable construction and operation of the apparatus.     

Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus

The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1–2 h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A₂ inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A₂ was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus.     

The role of microtubules during the cytokinetic events of cell wall ontogenesis and papilla development inCarteria crucifera

Summary An ultrastructural study of cytokinesis, cell wall ontogenesis, and papilla development/form inCarteria crucifera Korsh. andChloromonas rosae Ettl was undertaken. After typical phycoplast-mediated cytokinesis, wall ontogenesis begins at the level of Golgi apparatus activation and secretion to the outside of the daughter cells of fibrillar wall precursors which self assemble into the typical chlamydomonad wall (sensuRoberts 1974). As wall ontogenesis approaches the flagellar region of the cell, several precisely timed events occur: flagellar apparatus formation, flagellar emergence, protoplasmic extension in the future papilla area underlined by series of parallel aligned microtubules, wall formation (at least the W₂–W₆ layers), retraction of the protoplasmic extension and loss of underlying microtubules, and final wall modification (gap filling by W₁ material) to yield the characteristic wall papilla. The transient cytoplasmic extensions mimic the shape of the future wall papilla and are maintained, at least inCarteria, by underlying microtubules. Structural and developmental properties of the papilla are characterized and phylogenetic implications are discussed.This research was supported by National Science Foundation Grant DEB 78-0554.     

Light microscopic detection of PAS-positive substances with thiosemicarbazide in freeze-substituted ovaries of Paspalum longifolium before pollination

Summary Localization of polysaccharides in the freeze-substituted, Eponembedded ovaries of Paspalum longifolium prior to pollination was carried out by periodic acid-Schiff's (PAS), periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-AgPr) and periodic acid-thiosemicarbazide-osmium (PA-TSC-OsO₄) reactions. The specificities of these three reactions were also studied. These three reactions are all effective for light microscopic demonstration of polysaccharides in the filiform apparatus, starch grains in the cells and PAS substance in the micropylar region. Nonspecific staining of the nucleoli of the egg and polar nuclei was observed in the PAS reaction. The PA-TSC-AgPr reaction is very specific for polysaccharides but its overall reaction takes a much longer period of time than the PAS reaction. The PA-TSC-OsO₄ reaction colors the cytoplasm and nuclei of most cells and therefore stains of the cell walls, especially those of the egg cell and synergids, do not stand out clearly. The synergid cytoplasm contains some amorphous polysaccharides and thus it colors even in PAS and PA-TSC-AgPr preparations. In the mature embryo sac, the egg and central cell as well as antipodals are vacuolated but the two synergids have no visible vacuoles under light microscope. Each synergid has a prominent filiform apparatus at the micropylar end, which stains intensely in all three preparations. The walls of the central cell and antipodals adjacent to the nucellar cells have many inward papillae which are also intensely stained in all three preparations. Starch grains are abundant in the ovary wall and usually absent in the nucellus and integuments. They are present in the egg, central cell and antipodals, but not in the two synergids.     

Cell morphology, ultrastructure, and calcification pattern of Oocardium stratum, a peculiar lotic desmid

Cell morphology and ultrastructure of the desmid Oocardium stratum and its habitat conditions in two limestone-precipitating spring habitats in the Alps were studied. In spite of specific cell geometry, we found ultrastructural features (nucleus with nucleolus, Golgi apparatus, chloroplast structure, lipid bodies, cell wall texture) closely related to other desmids. The type of the mucilage pore apparatus perforating in high densities extended areas of the cell wall of Oocardium is of the Cosmarium type. Oocardium contrasts to Cosmarium by a peculiar bilateral cell geometry (lateral sphenoid shape) which is combined with a dislocated nucleus. Although the cell features of Oocardium did not differ between the two habitats, different calcification types (rhombohedral calcite versus fascicular-fibrous calcite) and calcification intensities were recorded. The spatial positioning and extension of the Oocardium niches differed considerably between the two springs in spite of high CO₂ oversaturation at both sites.     

Anaerobic Growth of Purple Nonsulfur Bacteria Under Dark Conditions

Purple nonsulfur photosynthetic bacteria were cultured anaerobically in the absence of light by a modification of the Hungate technique. Growth was slow and resembled that of fastidious anaerobes; on yeast extract-peptone-agar medium, each cell produced about 16 descendants in 15 to 20 days. Growth was stimulated by addition of ethyl alcohol, acetate and H₂, or pyruvate and H₂. Cells grown in the presence of pyruvate and H₂ produced acetate and CO₂; each cell produced approximately 10 descendants in 24 hr under anaerobic, dark conditions. Spectrophotometric evidence obtained from cells which were the product of five generations suggests no difference between the bacteriochlorophyll and carotenoids synthesized by cells grown anaerobically under dark or light conditions. Likewise, the ultrastructure of the photosynthetic apparatus in cells grown anaerobically in the dark and in the light appears similar.     

CYTOCHEMICAL LOCALIZATION OF ADENOSINE TRIPHOSPHATASE IN THE MITOTIC APPARATUS OF HELA AND SARCOMA 180 TISSUE CULTURE CELLS

ATPase was localized in distinct regions of the mitotic apparatus of HeLa and Sarcoma 180 tissue culture cells. ATPase was demonstrated in the metaphase spindle of HeLa and Sarcoma 180 cells fixed in cold buffered 2 per cent formalin (pH 6.5 to 6.8) containing 2 x 10^-3 M CaCl₂. A high concentration of ATPase was frequently observed at the poles of the spindle. ATPase was also demonstrated in the interzonal region of both cell types during anaphase. The narrowing of the band of ATPase activity localized in the interzonal region during telophase indicates that ATPase activity is associated with the central spindle. In polar views of Sarcoma 180 cells fixed in cold, unbuffered, 2 per cent formalin, ATPase was frequently localized in granules in the region of the inner circumference of the ring of chromosomes formed at metaphase. ATPase in the mitotic apparatus of HeLa and Sarcoma 180 cells was shown not to be due to non-specific alkaline phosphatase. Mitotic apparatus ATPase in Sarcoma 180 cells was suppressed by an —SH inhibitor.     

Physiological properties and cytological features of protoplasts prepared fromChlorella saccharophila

Summary Protoplasts were prepared from cells ofChlorella saccharophila by treatment with a mixture of pectinase and cellulase. The yield of protoplasts is dependent upon the culture conditions prior to cell wall digestion. In thin section chemically-fixed protoplasts were without wall remnants at the surface of the plasma membrane. Of particular interest is the relationship between the Golgi apparatus and a nuclear envelope-endoplasmic reticulum continuum. Protoplasts have a photosynthetic capacity lying between 70 and 80% of that of normal cells, but show the same response towards CO₂ concentration and DCMU inhibition. Protoplasts also respond to changes in the osmolarity of the surrounding medium in accordance with the Boylevan't Hoff equation as if they are an osmometer. The nonosmotic volume (NOV) was calculated.Abbreviations GA Golgi apparatus - ER endoplasmic reticulum - NE nuclear envelope - PM plasma membrane - N nucleus - S starch - M mitochondria - V vacuole     

Ultrastructural localization of adenosine triphosphatase activity in HeLa cells at various stages of the cell cycle

Summary HeLa cells in a monolayer culture were synchronized to S, G₂ and mitotic phases by use of excess (2.5 mM) deoxythymidine double-block technique. The localizations of Ca⁺⁺-activated adenosine triphosphatase (ATPase) at different phases of the cell cycle were studied using light and electron-microscopic histochemical techniques, and microphotometric comparisons of the densities of reaction products. Enzyme reaction product was always localized in the endoplasmic reticulum, nuclear membrane, mitochondria and Golgi apparatus, but there were qualitative and quantitative differences related to the phases of the cell cycle. In S phase the activity was mainly concentrated in a perinuclear area of the cytoplasm whereas in G₂ and mitosis the activity was scattered throughout the cell. The total activity per cell was maximal in G₂, was less in S phase and least in mitosis. Activity in the mitochondria and endoplasmic reticulum was distinctly less in mitosis than in other phases of the cell cycle. The mitochondrial ATPase differed from the ATPase at other sites in ion dependence and sensitivity to oligomycin. The results suggest that there may be several distinct ATPases in proliferating cells.     

Changes in the activity of the Golgi apparatus during the cell cycle in antheridial filaments ofChara vulgaris L.

Summary The number of dictyosomes found in one central cell section in antheridial filaments ofChara vulgaris increases proportionally to the cell length during interphase. The activity of Golgi apparatus was expressed by a number of Golgi vesicles surrounding a single dictyosome. These vesicles are most numerous during mitosis and cytokinesis,i.e., prior to and during cell plate formation. In the middle and late S phase the number of Golgi vesicles decreases by about 25%; subsequently, during the early and middle G₂, it increases again. At the end of the G₂ phase, Golgi vesicles are the scarcest.The increase in the number of Golgi vesicles during the G₂ phase coincides with the period of intense cellular elongation, and, thus, it is probably related to the enhanced synthesis of cell wall components.Coated vesicles are most numerous in prophase, metaphase, and early telophase, and during interphase in both late S and G₂ phase. It was found that the number of coated vesicles is proportional to the degree of condensation of nuclear chromatin.This work was supported by the Polish Academy of Sciences within the project 09.7.3.1.4.     

Human alveolar macrophages spontaneous reduction of BSPT salt

Summary Alveolar macrophages of non smoking and smoking human adults reduce BSPT salt spontaneously. The staining obtained is located on three cell membrane systems: the endoplasmic reticulum, the Golgi apparatus and the nuclear envelope. Methylene blue MB inhibits BSPT reduction. The smokers alveolar macrophages have less positivity than those of the non smokers. The endogenous cell substrate revealed in this work is the initial common pathway of two different oxidative chains bounded to the microsome. One acts with cytochrome P 450 for chemical detoxification by hydroxylation, the other one acts with cytochrome B₅ for lipid oxidation or peroxidation and both may be connected with the cell bactericidal system.     

Changes in the photosynthetic apparatus of maize in response to simulated natural temperature fluctuations

The response of the photosynthetic apparatus to low temperature periods differed among three hybrids of maize (Zea mays L.) grown in a phytotron. Light-saturated photosynthetic rates, leaf chlorophyll content, and mesophyll cell photosynthetic unit density all declined with increasing duration of low temperature. No single metabolic or physiological parameter appeared to control the response of the three hybrids to low temperature stress. Among all temperature treatments, net photosynthetic rate on a leaf area basis was more closely correlated with leaf chlorophyll content than with any other measured parameter. Final shoot dry weight was most highly correlated with stomatal conductance to CO₂.