Kinetics of the assembly of gas vacuoles in the blue-green alga Microcystis aeruginosa Kuetz. emend. Elekin.

Summary In exponentially growing cultures of the blue-green alga Microcystis aeruginosa the number of gas vacuoles per cell decreases and reaches a value of 4.5×10³ at 1.2×10⁷ cells per ml. The assembly of gas vacuoles with respect to number and length was followed after the organelles were caused to collapse by a pulse of ultrasound. The change in the number N of gas vacuoles per cell is N=224.8×t ^0.757, 0<t<24 h. After 24 h 50% of the value of non-sonicated cultures is reached. The changes in the length L of the organelles is expressed by L=87.06 ×t ^0.4084, 0<t<24 h. After 24 h 85% of the control value is reached.Abbreviations used EDTA ethylene diamine tetraacetate - BSA bovine serum albumine - P probability - N number of gas vacuoles - L length of gas vacuoles in nm - t time in hours     

Physiological effects of the presence and absence of gas vacuoles in the blue-green alga,Microcystis aeruginosa Kuetz. emend. Elenkin

Physiological evidence was obtained for a light shielding role for gas vacuoles inMicrocystis aeruginosa Kuetz. emend. Elenkin, by comparing photosynthetic oxygen evolution, growth behaviour and pigment composition of cells with intact or collapsed gas vacuoles. The oxygen evolution rates were strongly dependent on cell concentration, a maximum rate for cells with intact gas vacuoles occurring at about 1.4×10⁹ cells/ml and for cells with collapsed gas vacuoles at about 2.5×10⁹ cells/ml. By using light saturation curves for oxygen evolution, it was estimated that at low light intensities up to 30% of the photosynthetically useable light was shielded at a cell concentration of 6×10⁸ cells/ml. Collapsing the gas vacuoles twice daily did not alter the initial growth rate of the cultures, but enabled them to reach a higher final cell density. Collapsing of gas vacuoles during growth for about four generations resulted in a lower level of all acetone soluble pigments with a greater relative reduction in carotenoids than in chlorophyll a. Collapse of the gas vacuoles does not alter the cell volume. Various optical interactions which could account for light shielding are discussed.     

Isolation and chemical characterization of gas-vacuole membranes fromMicrocystis aeruginosa Kuetz. emend. Elenkin

Summary A method involving penicillin treatment was developed to osmotically lyse the cells of the blue-green alga,Microcystis aeruginosa Kuetz. emend. Elenkin, and release the pressure-sensitive gas vacuoles intact. The gas vacuoles were purified by liquid-polymer partitioning or by macromolecular sieving and centrifugation. The degree of purification of the gas vacuoles was followed by observation in the electron microscope and by the use of C¹⁴-labeled vacuolated and nonvacuolated strains ofM. aeruginosa. The gas-vacuole membrane is composed of only protein consisting of 10% basic, 18% acidic and 52% non-polar amino acids.Supported by U.S. Atomic Energy Commission, Contract No. AT(11-1)-1338.     

An investigation into the possible light-shielding role of gas vacuoles in a planktonic blue-green alga

When the gas vacuoles of Anabaena flos-aquae Bréb. ex Born. et Flah. are collapsed, the optical properties of the alga change. While this may suggest a light-shielding role, photosynthetic measurements indicate that intact gas vacuoles reduce the light falling on the thylakoids by only 4%, or less. Intact gas vacuoles offer no protection against the lethal effects of ultraviolet light. When the alga is grown at high light intensity the gas vacuoles are fewer in number but are oriented peripherally in the cells. However, this does not markedly affect their light shielding efficiency. Spectrophotometric measurements carried out by others indicate a light shielding role by gas vacuoles in a non-planktonic blue-green alga, Nostoc muscorum Kütz., but do not give a quantitative estimate of this effect. In Anabaena no definite evidence of light-shielding is obtained by such a method. All of the experiments described were conducted with dilute algal suspensions to investigate shielding effects in individual cells. Possible self-shading effects in dense suspensions and surface water blooms require further investigation.     

Absence of gas vesicle protein in a mutant of Anabaena flos-aquae

Filaments without gas vacuoles arose spontaneously in the gas-vacuolate alga Anabaena flos-aquae. The non-vacuolate mutant was enriched by repeated sedimentation and subsequently cloned by microsyringe transfer. No revertants have been observed. In the gas-vacuolate wild-type alga the gas vesicle protein was clearly distinguished by gel electrophoresis as one of the ten most abundant protein species present in whole cell extracts. Electrophoresis indicated that the mutant had lost the ability to synthesize the gas vesicle protein. A second mutant partially defective in production of gas vacuoles and gas vesicle protein has been isolated.Abbreviations gv gas vesicle protein - pb phycobilin - TCA trichloracetic acid     

Intracellular Bubble Formation: Differences in Gas Supersaturation Tolerances Between Tetrahymena and Euglena1

Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.     

Disappearance of gas vacuoles in the blue-green alga Microcystis aeruginosa

Abstract In a culture of Microcystis aeruginosa, which had been transferred from a mineral medium into distilled water, the number of gas vacuoles per cell decreased and reached a value of 20% of the control 24 h after transfer. In senescent cells grown on a mineral agar for several weeks, the gas vacuoles also disappeared. The disappearance of the gas vacuoles may be a response to a nutrient deficiency in both cultures.     

Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens

The aim of this study was to show the potential of Thlaspi caerulescens in the cleaning‐up of a moderately Zn ‐contaminated soil and to elucidate tolerance mechanisms at the cellular and subcellular level for the detoxification of the accumulated metal within the leaf. Measured Zn concentrations in shoots were high and reached a maximum value of 83 mmol kg ^{? 1} dry mass, whereas total concentrations of Zn in the roots were lower (up to 13 mmol kg ^{? 1}). In order to visualize and quantify Zn at the subcellular level in roots and leaves, ultrathin cryosections were analysed using energy‐dispersive X‐ray micro‐analysis. Elemental maps of ultrathin cryosections showed that T. caerulescens mainly accumulated Zn in the vacuoles of epidermal leaf cells and Zn was almost absent from the vacuoles of the cells from the stomatal complex, thereby protecting the guard and subsidiary cells from high Zn concentrations. Observed patterns of Zn distribution between the functionally different epidermal cells were the same in both the upper and lower epidermis, and were independent of the total Zn content of the plant. Zinc stored in vacuoles was evenly distributed and no Zn‐containing crystals or deposits were observed. From the elemental maps there was no indication that P, S or Cl was associated with the high Zn concentrations in the vacuoles. In addition, Zn also accumulated in high concentrations in both the cell walls of epidermal cells and in the mesophyll cells, indicating that apoplastic compartmentation is another important mechanism involved in zinc tolerance in the leaves of T. caerulescens.     

Specific inhibition by NH4Cl of autophagy-associated proteolysis in cultured fibroblasts

Rat embryo fibroblasts, prelabeled with [¹⁴C]leucine, showed an enhanced degradation of cell protein as well as increased peptide release when placed in a serum-deficient medium. NH₄Cl inhibited only the induced proteolysis, but had no effect on basal protein turnover. Electron microscopy studies showed that enhanced proteolysis was associated with an increase in autophagic vacuoles containing amorphous and membranous debris, and that NH₄Cl markedly increased the number of these intracellular vacuoles. Upon release from NH₄Cl inhibition, these cells showed a compensatory enhanced release of ¹⁴C into the medium and a decrease in the number of intracellular degradative vacuoles. We conclude that enhanced proteolysis reflects an activation of the autophagic-lysosomal system in these cells and that NH₄Cl inhibits the final hydrolysis and release steps in this mechanism.     

Vacuolar citrate/H+ symporter of citrus juice cells

We have isolated a cDNA, designated Citrus sinensis citrate transporter 1 CsCit1 encoding a novel vacuolar citrate/symporter. Immunoblots using antibodies raised against CsCit1 showed that the protein is localized to the juice sac cell vacuoles. The highest expression of CsCit1 and the amount of protein in the juice sac cell vacuoles coincided with the developmental stage at which the vacuolar citrate content began declining with the concomitant increase in vacuolar pH. Vacuoles from Sacharomyces cereviseae expressing CsCit1 displayed a citrate-dependent H⁺ efflux, and our results clearly demonstrate that CsCit1 is able to mediate the electroneutral co-transport of H⁺ and citrate ions, since the citrate-dependent H⁺ fluxes are not affected by changing the electrical potential difference across the tonoplast. The roles of CsCit1 in mediating citrate efflux from the vacuole and on citric acid homoestasis in Citrus juice sac cells are discussed. T. Shimada and R. Nakano contributed equally to this work.     

Identification and mitotic partitioning strategies of vacuoles in the unicellular red alga <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis>

Cyanidioschyzon merolae is considered as a suitable model system for studies of organelle differentiation, proliferation and partitioning. Here, we have identified and characterized vacuoles in this organism and examined the partitioning of vacuoles using fluorescence and electron microscopy. Vacuoles were stained with the fluorescent aminopeptidase substrate 7-amino-4-chloromethylcoumarin l-arginine amide, acidotrophic dyes quinacrine and LysoTracker, and 4′,6-diamidino-2-phenyl indole, which, at a high concentration, stains polyphosphate. Vacuoles have been shown to be approximately 500 nm in diameter with a mean of around five per interphase cell. The vacuolar H⁺-ATPase inhibitor concanamycin A blocked the accumulation of quinacrine in the vacuoles, suggesting the presence of the enzyme on these membranes. Electron microscopy revealed that the vacuoles were single membrane-bound organelles with an electron-dense substance, often containing a thick layer surrounding the membrane. Immunoelectron microscopy using an anti-vacuolar-H⁺-pyrophosphatase antibody revealed the presence of the enzyme on these membranes. In interphase cells, vacuoles were distributed in the cytoplasm, while in mitotic cells they were localized adjacent to the mitochondria. Filamentous structures were observed between vacuoles and mitochondria. Vacuoles were distributed almost evenly to daughter cells and redistributed in the cytoplasm after cytokinesis. The change in localization of vacuoles also happened in microtubule-disrupted cells. Since no actin protein or filaments have been detected in C. merolae, this result suggests an intrinsic mechanism for the movement of vacuoles that differs from commonly known mechanisms mediated by microtubules and actin filaments.     

Uptake and accumulation of tritiated uteroglobin by day-6 rabbit blastocysts

Summary Uptake of uteroglobin (UGL) by day-6 rabbit blastocysts and the intracellular fate of this protein were studied by light- and electron-microscopic autoradiography, immunocytochemistry and acid-phosphatase cytochemistry. UGL, labelled with N-succinimidyl-(2-3-³H)-propionate, was administered to embryos in vitro for 15 min to 4 h. The kinetics, determined from light-microscopic autoradiographs, showed a continuous uptake of the labeled protein over a 4-h period of incubation. At the ultrastructural level, increasing numbers of silver grains and an intense UGL immunoreaction in protein vacuoles and crystalloid bodies of trophoblast cells indicated that ³H-UGL had accumulated in these organelles. The presence of crystalloid inclusions in protein vacuoles suggests their origin by a condensation of the protein content, including UGL. Lysosomes containing radioactivity were rarely found, suggesting a very low degradation rate of the ³H-UGL. Protein vacuoles and crystalloid bodies exhibited no acid phosphatase reaction. The enzyme was mainly found outside the basal and lateral cell membranes of trophoblast cells, and on the rough endoplasmic reticulum of endoderm cells.     

Sucrose uptake into vacuoles of sugarcane suspension cells

Uptake of sucrose into vacuoles of suspension cells of Saccharum sp. (sugarcane) was investigated using a vacuole-isolation method based on osmotic- and pH-dependent lysis of protoplasts. Vacuoles took up sucrose at high rates without the influence of tonoplast energization on sucrose transport. Neither addition of ATP or pyrophosphate nor dissipation of the membrane potential or the pH gradient by ionophores changed uptake rates appreciably. Generation of an ATP-dependent pH gradient across the tonoplast was measured in vacuoles and tonoplast vesicles by fluorescence quenching of quinacrine. No H⁺ efflux could be measured by addition of sucrose to energized vacuoles or vesicles so that there was no evidence for a sucrose/H⁺ antiport system. Uptake rates of glucose and other sugars were similar to those of sucrose indicating a relatively non-specific sugar uptake into the vacuoles. Sucrose uptake was concentration-dependent, but no clear saturation kinetics were found. Strict dependence on medium pH and inhibition of sucrose transport by p-chloromercuriphenylsulfonic acid (PCMBS) indicate that sucrose uptake into sugarcane vacuoles is a passive, carrier-mediated process.Abbreviations FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - Mes 2-(N-morpholino)ethanesulfonic acid - Mops 3-(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuriphenylsulfonic acid - PPi pyrophosphate This research was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of H. Schroer is gratefully acknowledged.     

Effect of inhibitors and uncouplers on light-induced potential changes triggering photophobic responses

Light-energy absorption by Microcystis aeruginosa with and without gas vacuoles was observed, respectively by using an integrating sphere photometer. As far as the concentration of cell suspension of the order of 10⁶⁷ cells/ml in this work was concerned, the performance of gas vacuoles to shield incident light was most unlikely. Referring to a correlation secured by the integrating sphere photometer between light absorption and cell concentration of the suspension, a turbidostat culture of the blue-green alga demonstrated that the growth efficiency, Y _kJ defined as g cells harvested per kJ of light energy absorbed by the cells was nearly 0.004. This value of Y _kJ was almost the same as that of Spirulina platensis.Abbreviation vvm volume of air per volume of medium per min     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Isolation of gas vacuole-less mutants of the blue-green alga Anabaenopsis raciborskii

From a bloom forming blue-green alga, Anabaenopsis raciborskii, spontaneous mutants, which had lost the ability to form gas vacuoles have been isolated; the mutant frequency was 4.8×10^-3. The filaments of gas vacuole-less mutants settled at the bottom of flasks in liquid culture media unlike the parent alga. The growth and nitrogen fixation were comparatively poor in the mutants.     

Macrophages Rapidly Transfer Pathogens from Lipid Raft Vacuoles to Autophagosomes

Macrophages activate autophagy as an immediate response to Legionella pneumophila infection, but what marks the pathogen phagosome as a target for the autophagy machinery is not known. Because a variety of bacteria, parasites, viruses, and toxins that associate with the endoplasmic reticulum enter host cells by a cholesterol-dependent route, we tested the hypothesis that autophagy is triggered when microbes engage components of lipid raft domains. As the intracellular respiratory pathogen L. pneumophila or the extracellular uropathogen FimH⁺ Escherichia coli entered macrophages by a cholesterol-sensitive mechanism, they immediately resided in vacuoles rich in glycosylphosphatidylinositol moieties and the autophagy enzyme Atg7. As expected for autophagosomes, the vacuoles sequentially acquired the endoplasmic reticulum protein BiP, the autophagy markers Atg8 and monodansyl-cadaverine, and the lysosomal protein LAMP-1. A robust macrophage response to the pathogens was cholesterol-dependent, since fewer Atg7-rich vacuoles were observed when macrophages were pre-treated with methyl-beta-cyclodextrin or filipin. A model in which macrophages exploit autophagy to capture pathogens within the lipid raft pathway for antigen presentation prior to disposal in lysosomes is discussed.     

Na+/H+-transporter, H+-pumps and an aquaporin in light and heavy tonoplast membranes from organic acid and NaCl accumulating vacuoles of the annual facultative CAM plant and halophyte Mesembryanthemum crystallinum L.

Crassulacean acid metabolism (CAM) was induced in Mesembryanthemum crystallinum L. by either NaCl- or high light (HL)- stress. This generated in mesophyll cells predominantly of NaCl-stressed plants two different types of vacuoles: the generic acidic vacuoles for malic acid accumulation and additionally less acidic (“neutral”) vacuoles for NaCl sequestration. To examine differences in the tonoplast properties of the two types of vacuoles, we separated microsomal membranes of HL- and NaCl-stressed M. crystallinum plants by centrifugation in sucrose density gradients. Positive immunoreactions of a set of antibodies directed against tonoplast specific proteins and tonoplast specific ATP- and PP_i-hydrolytic activity were used as markers for vacuolar membranes. With these criteria tonoplast membranes were detected in both HL- and NaCl-stressed plants in association with the characteristic low sucrose density but also at an unusual high sucrose density. In HL-stressed plants most of the ATP- and PP_i-hydrolytic activity and cross reactivity with antibodies including that directed against the Na⁺/H⁺-antiporter from Arabidopsis thaliana was detected with light sucrose density. This relationship was inverted in NaCl-stressed plants; they exhibited most pump activity and immunoreactivity in the heavy fraction. The relative abundance of the heavy membrane fraction reflects the relative occurrence of “neutral” vacuoles in either HL- or NaCl-stressed plants. This suggests that tonoplasts of the “neutral” vacuoles sediment at high sucrose densities. This is consistent with the view that this type of vacuoles serves for Na⁺ sequestration and is accordingly equipped with a high capacity of proton pumping and Na⁺ uptake via the Na⁺/H⁺-antiporter.     

Nitrate interference with potassium-selective microelectrodes

Initial attempts to measure K⁺ activity (ak) in vacuoles of barley leaf epidermal cells using triple-barrelled K⁺-selective microelectrodes gave values that were only about one-third of those expected. This was due to high (c. 200 mM) NO3^- concentrations in the vacuoles interfering with the K⁺-sensor. The effect of NO3^- was on 1,2,-dimethyl-3-nitrobenzene (DNB) used as a plasticizer in the K⁺-sensor. Replacing DNB with dibutyl sebacate, but not with 2-nitrophenyl octyl ether, overcame this problem and the modified sensor gave acceptable calibration curves with no interference acceptable calibration curves with no interference from physiological concentrations of other ions. These electrodes were used successfully to measure a mean ak of 235 mM in vacuoles of epidermal cells of K⁺-replete barley leaves.Keywords: Leaf epidermis, potassium activity, potassium-selective microelectrodes, vacuoles, nitrate interference.      

Heterologous expression of vacuolar H+-PPase enhances the electrochemical gradient across the vacuolar membrane and improves tobacco cell salt tolerance

Summary. The vacuolar H⁺-translocating inorganic pyrophosphatase (H⁺-PPase) uses pyrophosphate as substrate to generate the proton electrochemical gradient across the vacuolar membrane to acidify vacuoles in plant cells. The heterologous expression of H⁺-PPase genes (TsVP from Thellungiella halophila and AVP1 from Arabidopsis thaliana) improved the salt tolerance of tobacco plants. Under salt stress, the transgenic seedlings showed much better growth and greater fresh weight than wild-type plants, and their protoplasts had a normal appearance and greater vigor. The cytoplasmic and vacuolar pH in transgenic and wild-type cells were measured with a pH-sensitive fluorescence indicator. The results showed that heterologous expression of H⁺-PPase produced an enhanced proton electrochemical gradient across the vacuolar membrane, which accelerated the sequestration of sodium ions into the vacuole. More Na⁺ accumulated in the vacuoles of transgenic cells under salt (NaCl) stress, revealed by staining with the fluorescent indicator Sodium Green. It was concluded that the tonoplast-resident H⁺-PPase plays important roles in the maintenance of the proton gradient across the vacuolar membrane and the compartmentation of Na⁺ within vacuoles, and heterologous expression of this protein enhanced the electrochemical gradient across the vacuolar membrane, thereby improving the salt tolerance of tobacco cells. Correspondence: J.-R. Zhang, School of Life Science, Shandong University, 27 Shanda South Road, Jinan, People’s Republic of China 250100.