Role of Silicon in Diatom Metabolism

The in vivo uptake of ³¹Si-silicic acid and ⁶⁸Ge-germanic acid by cell organelles of Nitzschia alba Lewin and Lewin and Cylindrotheca fusiformis Reimann and Lewin was demonstrated. The organelles were isolated by fractionation of pre-labeled cells by differential centrifugation in 0.4 M sucrose medium. Electron micrographs showed that the isolated organelles were intact, with the exception of the N. alba mitochondria which appeared swollen and the C. fusiformis chloroplasts which had ruptured outer membranes and lacked stroma material. The amount of ³¹Si or ⁶⁸Ge per mg protein of the subcellular fractions decreased in the following general order for both organisms: cell wall, mitochondria, chloroplasts, vesicles, and microsomes. A portion of the ³¹Si or ⁶⁸Ge in the organelle fractions could be extracted into a distilled water wash. The uptake of silicon by the organelles suggests that silicon may be involved in some processes of the compartmentalized systems of the cell.     

Cell organelles from crassulacean-acid-metabolism (CAM) plants

Cell organelles were isolated from the CAM plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen by isopycnic centrifugation in sucrose gradients. The inclusion of 2.5% Ficoll in the grinding medium proved to be essential for a satisfactory separation of cell organelles during the subsequent centrifugation. Peroxisomes, mitochondria, and whole and broken chloroplasts were at least partially resolved as judged by marker-enzyme-activity profiles. The isolated peroxisomes contained activities of glycollate oxidase, catalase, hydroxypyruvate reductase, glycine aminotransferase, serine-glyoxylate aminotransferase, and aspartate aminotransferase, comparable to activities found in spinach (Spinacia oleracea L.) leaf peroxisomes. In contrast to spinach, however, only little, if any, particulate malate dehydrogenase activity could be attributed to isolated peroxisomes of the three CAM plants.     

In-vitro interaction between chloroplasts and peroxisomes as controlled by inorganic phosphate

Peroxisomes, whole chloroplasts, mitochondria, and broken chloroplasts of spinach (Spinacia oleracea L.), each form 1 band at its typical density, when isolated in sucrose gradients by isopycnic centrifugation in glycylglycine buffer. In potassium-phosphate buffer peroxisomes form a 2nd band at the density of whole chloroplasts. The phosphate effect is half-saturated at a concentration of 10–20 mM. If whole chloroplasts are removed by differential centrifugation before isopycnic centrifugation no second band is formed. Arsenate can be substituted for potassium phosphate while KCl, NaCl, KNO₃ and glycolate cannot, showing inorganic phosphate to be the active ion. Evidence is presented showing that during isopycnic centrifugation more slowly sedimenting peroxisomes have to move through faster sedimented bands of whole and broken chloroplasts. In the presence of inorganic phosphate this leads to a specific interaction between whole chloroplasts and peroxisomes visible as a second peroxisomal band at the density of the whole chloroplasts. The relationship of the interaction in vitro to the known association of the two organelles in vivo is considered.     

Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to △pH-based translocation (△pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.     

Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to △pH-based translocafion (△pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.     

Regular arrangements of mitochondria and plastids during sporogenesis inEquisetum

Summary Plastids and mitochondria in premeiotic cells ofEquisetum were situated at random. By early prophase I all these organelles aggregated for a short period into one group at the nuclear envelope, but subsequently the organelles became again scattered. By late prophase I they aggregated into two groups at opposite sides of the nucleus, then moved towards the equator of the cell. By interphase plastids and mitochondria aggregated into a layer which divided each dyad into two parts. After telophase II the reorganized layer divided the tetrad into four parts. The organelle layer underwent differentiation into three strata. The cell plate was formed in the middle one which was constituted of mitochondria.     

Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts

The precursors of the F₁-ATPase -subunits fromNicotiana plumbaginifolia andNeurospora crassa were imported into isolated spinach (Spinacia oleracea L.) leaf mitochondria. Both F₁ precursors were imported and processed to mature size products. No import of the mitochondrial precursor proteins into isolated intact spinach chloroplasts was seen. Moreover, the precursor of the 33 kDa protein of photosynthetic water-splitting enzyme was not imported into the leaf mitochondria. This study provides the first experimental report ofin vitro import of precursor proteins into plant mitochondria isolated from photosynthetic tissue and enables studies of protein sorting between mitochondria and chloroplasts in a system which is homologous with respect to organelles. The results suggest a high organellar specificity in the plant cell for the cytoplasmically synthesized precursor proteins.     

Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C<Subscript>4</Subscript> photosynthesis in <Emphasis Type="Italic">Bienertia sinuspersici</Emphasis>

Bienertia sinuspersici Akhani has an unusual mechanism of C₄ photosynthesis which occurs within individual chlorenchyma cells. To perform C₄, the mature cells have two cytoplasmic compartments consisting of a central (CCC) and a peripheral (PCC) domain containing dimorphic chloroplasts which are interconnected by cytoplasmic channels. Based on leaf development studies, young chlorenchyma cells have not developed the two cytoplasmic compartments and dimorphic chloroplasts. Fluorescent dyes which are targeted to membranes or to specific organelles were used to follow changes in cell structure and organelle distribution during formation of C₄-type chlorenchyma. Chlorenchyma cell development was divided into four stages: 1—the nucleus and chloroplasts occupy much of the cytoplasmic space and only small vacuoles are formed; 2—development of larger vacuoles, formation of a pre-CCC with some scattered chloroplasts; 3—the vacuole expands, cells have directional growth; 4—mature stage, cells have become elongated, with a distinctive CCC and PCC joined by interconnecting cytoplasmic channels. By staining vacuoles with a fluorescent dye and constructing 3D images of chloroplasts, and by microinjecting a fluorescence dye into the vacuole of living cells, it was demonstrated that the mature cell has only one vacuole, which is traversed by cytoplasmic channels connecting the CCC with the PCC. Immunofluorescent studies on isolated chlorenchyma cells treated with cytoskeleton disrupting drugs suspended in different levels of osmoticum showed that both microtubules and actin filaments are important in maintaining the cytoplasmic domains. With prolonged exposure of plants to dim light, the cytoskeleton undergoes changes and there is a dramatic shift of the CCC from the center toward the distal end of the cell.     

Heterogeneity of chondrocyte mitochondria: A study of the Ca2+ concentration and density banding characteristics of normal and rachitic cartilage

Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca²⁺ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5–2.0 M) in the presence of inhibitors of Ca²⁺ transport (ruthenium red and 5,5′-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca²⁺ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca²⁺ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca²⁺ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca²⁺ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca²⁺ concentration necessary for initiation of the calcification mechanism was 4 μmol/mg protein.     

Fluorescence immunohistochemical localization of malate dehydrogenase isoenzymes in watermelon cotyledons : a developmental study of glyoxysomes and mitochondria

Monospecific antibodies to glyoxysomal, mitochondrial, and cytosolic I malate dehydrogenase were used for the fluorescence immunohistochemical localization of these isoenzymes in dark-grown watermelon (Citrullus vulgaris Schrad.) cotyledons. It was demonstrated that, with cell organelles isolated by sucrose density gradient centrifugation, antibodies to glyoxysomal malate dehydrogenase were specific markers for glyoxysomes, and similarly, antibodies to mitochondrial malate dehydrogenase were markers for mitochondria. The time course of the glyoxysomal malate dehydrogenase appearance and decline was not synchronous for the individual tissues and differed completely from that of the mitochondria. The cytosolic malate dehydrogenase I was confined to restricted regions of the lower epidermis. The activity which was definitively localized outside the cell organelles decreased during the first days of germination.     

CALCIFICATION IN THE GREEN ALGA HALIMEDA. I. AN ULTRASTRUCTURE STUDY OF THALLUS DEVELOPMENT1

The ultrastructure of 4 species of the calcareous, siphonaceous alga Halimeda (H. cylindracea Decaisne, H. discoidea Decaisne, H. macroloba Decaisne and H. tuna (Ellis & Solander) Lamour) has been studied, and the observed changes during growth and development are related to changes in the degree of calcification. A distinct gradient in the types and quantities of cell organelles exists in a growing apical filament. As these filaments grow, branch, and eventually develop into a mature segment, changes in the organization of organelles such as mitochondria and chloroplasts are observed. Calcification begins when the chloroplasts reach structural maturity and when the peripheral utricles adhere (fuse). This adhesion of the peripheral utricles isolates the intercellular space (ICS) in which calcification occurs from the external seawater. Calcification begins in the outermost (pilose) cell wall layer of the walls facing into the ICS. The cell walls at the thallus exterior undergo extensive changes after utricular fusion; the pilose layer is lost, the cuticles of adjacent utricles fuse forming a ridge at their junction, and multiple cuticles are formed. The aragonite (CaCO₃) crystals which are initially precipitated within the pilose wall layer, rapidly increase in size and number, eventually filling much of the ICS. Only the initial nucleation of aragonite is associated with the pilose wall layer, the later precipitation of aragonite is totally independent of the pilose layer. In older segments secondary deposition of CaCO₃ also occurs around existing aragonite needles.     

Intracellular Localization of Phosphoenolpyruvate Carboxykinase in Bundle Sheath Cells of C4 Plants

Bundle sheath protoplasts (BSP) were isolated and purified fromfour C₄ species of the phosphoenolpyruvate (PEP) carboxykinasetype (Panicum maximum, P. texanum, Chloris gayana and Eriochloaborumensis), and cell organellses were separated from the BSPextract by differential centrifugation or sucrose density gradientcentrifugation. Separation of the organelles was ascertainedby the distribution of marker enzymes for chloroplasts, mitochondria,peroxisomes and cytoplasm. Contrary to the previous report [Rathnamand Edwards (1975) Arch. Biochem. Biophys. 171: 214], the distributionof PEP carboxykinase in BSP of P. maximum was the same as thatof UDP-glucose pyrophosphorylase, a marker for cytoplasm, andPEP carboxykinase activity was not recovered in the intact chloroplasts.The same results were obtained with P. texanum, C. gayana andE. borumensis. Therefore, we conclude that PEP carboxykinase is exclusivelylocalized in the cytoplasm of bundle sheath cells of C₄ plants. (Received July 23, 1983; Accepted October 17, 1983)     

Heterogeneity of chondrocyte mitochondria: A study of the Ca concentration and density banding characteristics of normal and rachitic cartilage

Previous morphological studies of the mineralizing epiphysis suggested that some mitochondria were concerned with Ca²⁺ accumulation while others were associated with cellular energetics and metabolism. To determine if there was mitochondrial heterogeneity in chondrocytes of the epiphyseal growth plate, mitochondria were isolated from four different regions of the plate and subjected to continuous sucrose gradient centrifugation. Centrifugation of the organelles in a narrow density sucrose gradient (1.5–2.0 M) in the presence of inhibitors of Ca²⁺ transport (ruthenium red and 5,5′-dithiobis-(2-nitrobenzoic acid)) revealed that considerable heterogeneity existed. In the least calcified zone 20% of the mitochondria formed a low density band of low Ca²⁺ concentration (309 nmol/mg protein). Organelles isolated from more calcified tissue zones showed a concomitant increase in Ca²⁺ concentration (up to 5700 nmol/mg protein) as well as an increase in the total percentage of mitochondria sedimenting in 2.0 M sucrose. The banding patterns of mitochondria isolated from rachitic and hypertrophic cartilage were similar. In addition, similarities were also noted in the Ca²⁺ concentration and the cytochrome oxidase activities of mitochondria of these tissues. During recovery from the rachitic condition, there was a change in the density centrifugation characteristics of this tissue and a substantial increase was noted in the proportion of mitochondria sedimenting in 2.0 M sucrose. The Ca²⁺ concentration of mitochondria of this rapidly calcifying tissue suggested that the critical Ca²⁺ concentration necessary for initiation of the calcification mechanism was 4 μmol/mg protein.     

Isolation and characterisation of developing chloroplasts from light-grown barley leaves

Developing chloroplasts were isolated from the basal region of green barley ( Hordeum vulgare L. cv. Menuet) leaves and their ultrastructure and biochemical composition were compared to those of mature chloroplasts from the tip of the same leaves, using two methods of purification on sucrose and Percoll gradients.
When examined and compared to mature chloroplasts, the developing chloroplasts showed well-developed grana stacks, but these last organelles were 2-fold smaller and contained lower amounts of chlorohylls and polar lipids. Only traces of trans -3-hexadecenoic acid could be detected in phosphatidylglycerol of developing plastids. The protein content of these plastids was higher than in mature plastids and showed an increased proportion of polypeptides linked to P-700 chlorophyll α-protein. The photosynthetic activity of these plastids was about 2-fold lower and their photosystem 1/photosystem II ratio higher than in mature chloroplasts.     

Separation of mitochondria from microbodies of Pisum sativum (L. cv. Alaska) cotyledons

A method is described for separating mitochondria from microbodies in cotyledon preparations of Pisum sativum L. cv. Alaska. Pure and intact mitochondria were obtained on a continuous: discontinuous sucrose density gradient as shown by marke-enzyme assay and electron microscopy. Manipulation of sucrose-gradient construction to widen the distance between organelles provided a quick method for the separation of the mitochondria from the microbodies. The shorter time of exposure of mitochondria to centrifugation and osmotic stress produces mitochondria free of contamination.     

sn-Glycerol-3-phosphate transacylase activity in Euglena gracilis organelles

sn-Glycerol-3-phosphate transacylase activity was demonstrated in Euglena mitochondria, chloroplasts, and microsomes. There was no activity in the 100,000g 1-h supernatant. Exposure of each of the isolated organelles to 1 × 10^?4% Triton X-100 resulted in release of substantial quantities of transacylase activity into the 100,000g supernatant. Products formed by catalysis by the membrane-bound transacylases were heterogenous, while those resulting from catalysis by the extracted enzymes were practically all lysophosphatidate.     

Isolation and Properties of Intact Chromoplasts from Tomato Fruits

Intact chromoplasts were isolated from tomato fruits at differentripening stages by Percoll density gradient centrifugation.The isolated chromoplast fractions were contaminated very littleby other organelles, although the fraction from fully ripenedfruits contained some mitochondria and microbodies. As the transformationof chloroplasts to chromoplasts proceeded, the density of theplastids decreased from 1.096 to 1.075 g-cm^-3 and the decreasewas related to a decrease in chlorophyll and an increase inlycopene in the plastids. (Received December 21, 1983; Accepted April 20, 1984)     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA     

Intracellular Localization of Lunularic Acid and Prelunularic Acid in Suspension Cultured Cells of Marchantia polymorpha

Intracellular localization of lunularic acid and prelunularic acid in suspension cultured cells of Marchantia polymorpha L. was studied. The sum of both compounds was determined as lunularic acid group (LNAs) because of the instability of prelunularic acid to convert into lunularic acid.

Mechanical disruption of the cells followed by differential centrifugation showed that LNAs was associated with the supernatant of 100,000g centrifugation. Protoplasts isolated from the cells were osmotically ruptured and the distribution of LNAs among the organelles was examined by discontinuous density gradient centrifugation of the protoplast contents. Successful isolation of intact chloroplasts, mitochondria and peroxisomes free from cytoplasm indicated that LNAs was not accumulated in these organelles. Flotation techniques resulted in an efficient isolation of pure vacuoles and revealed that LNAs was distributed almost equally in the vacuoles and cytoplasm.