Snow algae of the Windmill Islands, continental Antarctica 3. Chloromonas polyptera (Volvocales, Chlorophyta)

A new name, Chloromonas hohamii, is proposed to accommodate a common North American snow alga previously incorrectly referred to as Chloromonas polyptera. Chloromonas hohamii differs in having the motile vegetative cells with a cup-shaped chloroplast opening in the anterior end of the cell, shorter, narrower, ellipsoidal to elongate to somewhat fusiform, sexual spores with non-spiralled wall flanges, shorter and narrower daughter cells derived from the spores, and it grows in snow of significantly lower pH and conductivity. Received: 29 August 1997 / Accepted: 24 April 1998     

Snow algae from northwest Svalbard: their identification, distribution, pigment and nutrient content

We mapped coloured snow during the summers of 1995 and 1996 at about 60 localities in the coastal region of northwest Spitsbergen. The colour was mainly induced by snow algae (Chlamydomonas spp. and Chloromonas spp.). In the late summer of 1996, snow algal fields of several hundred meters in size were observed along the west and north coasts. They had no preferred geographical orientation. We studied the abundance of primary pigments and secondary carotenoids from different developmental stages of the snow algae of Chlamydomonas spp. under natural conditions. Extensive accumulation of astaxanthin and its esters accompanied the transition from green biflagellated cells to orange spores, hypnozygotes and dark-red cysts. The photoprotective effect of the secondary carotenoids is enhanced by concentration in cytoplasmic lipid droplets around the nucleus and chloroplast. The nutrient content of melt-water and snow algae had no direct correlation with the content of secondary carotenoids. Relatively high Fe, Ca, P, K and Al contents of snow algae were found, suggesting a good supply of these mineral elements. Received: 20 May 1997 / Accepted: 18 March 1998     

Electrorotation of Isolated Generative and Vegetative Cells, and of Intact Pollen Grains of Lilium longiflorum

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m⁻¹. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques. Received: 9 June 1997/Revised: 4 August 1997     

Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form. Received: 19 March 1997 / Received revision: 19 May 1997 / Accepted: 1 June 1997     

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular “secretory” peroxidase activity (−73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed. Received: 14 October 1998 / Accepted: 31 May 1999     

Binding-Dissociation Properties of Potato Tuber Cell Wall-Associated {beta}-N-acetylglucosaminidase

The binding-dissociation properties of an endogenous cell wallprotein, ß-GlcNAcase, was compared to an artifactuallybound basic protein (cytochrome c) and acidic proteins (bovineserum albumin, -lactalbumin, ß-lactoglobulin and cytosolicß-GlcNAcase). Salt dissociation curves with monovalent,divalent and trivalent salts all indicated that the endogenouscell wall enzyme binds much tighter to the wall than does anyartificially bound protein. At high ionic strength (I=1.5),ammonium sulfate was as efficient as NaCl, KCl and LiCl in dissociatingthe cell wall enzyme. The pH of the dissociation medium onlyhas an effect on the dissociation of cell wall enzymes whenthe ionic strength of the buffer is low. The binding of proteinto purified cell walls is pH dependent in the physiologicalrange only if the protein has an acidic isoelectric pH. (Received May 27, 1992; Accepted August 3, 1992)     

Wall extensibility and cell hydraulic conductivity decrease in enlarging stem tissues at low water potentials

Measurements with a guillotine psychrometer (H Nonami, JS Boyer [1990] Plant Physiol 94: 1601-1609) indicate that the inhibition of stem growth at low water potentials (low ψ_w) is accompanied by decreases in cell wall extensibility and tissue hydraulic conductance to water that eventually limit growth rate in soybean (Glycine max L. Merr.). To check this conclusion, we measured cell wall properties and cell hydraulic conductivities with independent techniques in soybean seedlings grown and treated the same way, i.e. grown in the dark and exposed to low ψ_w by transplanting dark grown seedlings to vermiculite of low water content. Wall properties were measured with an extensiometer modified for intact plants, and conductances were measured with a cell pressure probe in intact plants. Theory was developed to relate the wall measurements to those with the psychrometer. In the elongation zone, the plastic deformability of the walls decreased when measured with the extensiometer while growth was inhibited at low ψ_w. It increased during a modest growth recovery. This behavior was the same as that for the wall extensibility observed previously with the psychrometer. Tissue that was killed before measurement with the extensiometer also showed a similar response, indicating that changes in wall extensibility represented changes in wall physical properties and not rates of wall biosynthesis. The elastic compliance (reciprocal of bulk elastic modulus) did not change in the elongating or mature tissue. The hydraulic conductivity of cortical cells decreased in the elongating tissue and increased slightly during growth recovery in a response similar to that observed with the psychrometer. We conclude that the plastic properties of the cell walls and the conductance of the cells to water were decreased at low ψ_w but that the elastic properties of the walls were of little consequence in this response.     

Passive Electrical Properties of Microorganisms: II. Resistance of the Bacterial Membrane

Studies of the effective, homogeneous, dielectric constants of bacteria are used to show that the resistances of their cytoplasmic membranes are too great to explain the low-frequency conductivities which have been observed for these organisms. This reaffirms the conclusion that at low frequencies the conductivities of bacteria reflect properties of their cell walls. In the organisms studied, the conductivities of the cell wall region are as great as the conductivities of the cytoplasm. This is true even though the ion concentration in the environment is much less than that in the cells. The mobile ions of the wall are presumed to be counterions for fixed charges in this region.     

Teichoic acid content in different lineages of Staphylococcus aureus NCTC8325

A series of mec transformants of Staphylococcus aureus strain NCTC8325 were analysed for alterations in wall teichoic acid and lipoteichoic acid. Although the methicillin resistance determinant alters the autolytic behaviour of S. aureus, it had no effects on the cellular content, chain length, and alanine substitution of the lipoteichoic acid, or on the wall teichoic acid content and composition. However, independently of the presence or absence of the methicillin resistance determinant, level of methicillin resistance, or autolytic behaviour, a correlation was found between a 25% reduced cell wall phosphate content and either loss of prophages φ11 and 13 or a 30-kb deletion in the chomosmal SmaI-F fragment adjacent to the prophage φ11 attachment site. Received: 23 February 1998 / Accepted: 15 May 1998     

Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

 The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo. Received: 27 December 1999 / Accepted: 11 May 2000     

Novel type II cell wall architecture in dichlobenil-habituated maize calluses

Growth of maize (Zea mays L.) callus-culture cells was inhibited using dichlobenil (2,6 dichlorobenzonitrile, DCB) concentrations ≥1 μM; I ₅₀ value for the effect on inhibited fresh weight gain was 1.5 μM. By increasing the DCB concentration in the culture medium, DCB-habituated cells became 13 times more tolerant of the inhibitor (I ₅₀: 20 μM). In comparison with non-habituated calluses, DCB-habituated calluses grew slower, were less friable and were formed by irregularly shaped cells surrounded by a thicker cell wall. By using an extensive array of techniques, changes in type II cell wall composition and structure associated with DCB habituation were studied. Walls from DCB-habituated cells showed a reduction of up to 75% in cellulose content, which was compensated for by a net increase in arabinoxylan content. Arabinoxylans also showed a reduction in their extractability and a marked increase in their relative molecular mass. DCB habituation also involved a shift from ferulate to coumarate-rich cells walls, and enrichment in cell wall esterified hydroxycinnamates and dehydroferulates. The content of polymers such as mixed-glucan, xyloglucan, mannans, pectins or proteins did not vary or was reduced. These results prove that the architecture of type II cell walls is able to compensate for deficiencies in cellulose content with a more extensive and phenolic cross-linked network of arabinoxylans, without necessitating β-glucan or other polymer enhancement. As a consequence of this modified architecture, walls from DCB-habituated cells showed a reduction in their swelling capacity and an increase both in pore size and in resistance to polysaccharide hydrolytic enzymes.     

Structure peculiarities of cell walls of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>—an autotroph of cephalosporin C

Alterations of cell walls of Acremonium chrysogenum occurring at intensive synthesis of cephalosporin C has been studied. It is shown, using electron microscopy, that the cell wall of the cells of ATCC 11550 strain (“wild” type) became looser and thicker during growth. The cell wall of the cells of strain 26/8 (hyperautotroph of cephalosporin C) considerably degraded by the end of the stationary phase. Biochemical analysis has shown that these alterations entailed decrease of the proteins’ content covalently or noncovalently linked with the polysaccharides of cell walls of both strains. An increase of sensitivity of cell walls of the strain-hyperautotroph to an activity of lytic enzymes of chitinase, laminarinase, proteinase K, and lyticase preparation has been observed during the growth, but this increase has not been found in the case of “wild” type strain. The obtained results evidence to the structure failure of the cell wall of A. chrysogenum entailing the intensive creation of antibiotic.     

Differential Roles of the Two-Component Peptides of Lactocin 705 in Antimicrobial Activity

Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705α and Lac705β. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705α/Lac705β peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705β peptide alone. Furthermore, Lac705β increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705α protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Δψ and ΔpH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705α peptide is responsible for receptor recognition, and the Lac705β peptide is the active component on the cell membrane of CRL691 cells. Received: 12 April 2002 / Accepted: 24 May 2002     

T cell–tumor cell: a fatal interaction?

Fas (Apo-1/CD95) is a cell-surface protein that is responsible for initiating a cascade of proteases (caspases) culminating in apoptotic cell death in a variety of cell types. The function of the Fas/FasL system in the dampening of immune responses to infectious agents through the autocrine deletion of activated T cells has been well documented. More recently, it has been proposed that tumor cells express FasL, presumably to avoid immune detection. In this review, we focus on the role of the interaction of Fas and FasL in the modulation of antitumor responses. We critically examine the evidence that FasL is expressed by tumor cells and explore alternative explanations for the observed phenomena in vitro and in vivo. By reviewing data that we have generated in our laboratory as well as reports from the literature, we will argue that the Fas/FasL system is a generalized mechanism used in an autocrine fashion to regulate cell survival and expansion in response to environmental and cellular cues. We propose that FasL expression by tumor cells, when present, is indicative of a perturbed balance in the control of proliferation while “immune privilege” is established by “suicide” of activated antitumor T cells, a form of activation-induced cell death. Received: 5 May 1998 / Accepted: 20 May 1998     

Passive Electrical Properties of Microorganisms: I. Conductivity of Escherichia coli and Micrococcus lysodeikticus

Effective conductivities are reported for the bacteria Escherichia coli and Micrococcus lysodeikticus over a range of environmental conductivity. The apparent conductivities of the organisms can be explained in terms of the properties of the cell wall. At low conductivities of the environment, the conductivity of the cell appears to be dominated by the counterions of the fixed charge of the cell wall. At higher conductivities of the suspending medium, evidence suggests that ions from the environment invade the cell wall causing an increase in the effective conductivity of the cell so that it takes on values roughly proportional to that of the environment. The model points to the usefulness of dielectric techniques in studies of the properties of intact cell walls.     

Ecotypic Variation in the Osmotic Responses of Enteromorpha intestinalis (L.) Link.

Young, A. J., Collins, J. C. and Russell, G. 1987. Ecotypicvariation in the osmotic responses of Enteromorpha intestinalis(L.) Link.—J. exp. Bot. 38: 1309–1324. The physiological basis for salt tolerance has been studiedin the euryhaline marine alga Enteromorpha intestinalis. Adaptationto dilute and concentrated seawaters has been investigated inthree separate populations of this alga: marine, rock pool andestuarine. Internal K⁺, Na⁺ and Cl^– levels have been determined usingtracer efflux analyses. K⁺ has been shown to be the major osmoticsolute within this alga. Cellular levels of Cl^– and, inparticular, Na⁺ are low although levels in the cell wall arehigh. Levels of these ions varied considerably between the separateplants; K⁺ levels within marine plants of E. intestinalis aretwo to four times those found in the other populations. Thetertiary sulphonium compound ß-dimethylsulphonio-propionateis maintained at relatively high levels, although it remainsfairly insensitive to change in the external salinity. Changes in the tissue water content and cell volume are large,particularly within the estuarine plants. The thin cell wallsof these plants allow large changes in volume in the diluteconditions experienced in an estuary, while low turgor preventscell rupture. Thicker cell walls and small cells of the marineand rock pool plants assist in tolerating high and low externalosmotic potential—the estuarine plants respond poorlyto concentrated seawater. Key words: Enteromorpha, osmoregulation, ecotypes     

Effect of snow depth on under-ice irradiance and growth of <Emphasis Type="Italic">Aulacoseira baicalensis</Emphasis> in Lake Baikal

Lake Baikal freezes for 4–5 months each year; yet the planktonic diatoms that grow under the ice include some of the largest species found in freshwater. An important factor influencing their growth is the depth of snow. In this study, a population of Aulacoseira baicalensis developed where there was little or no snow on the ice but declined where there was 10 cm of snow, because 99% of the available light was attenuated. Culture studies of light response showed that A. baicalensis was adapted to relatively low light intensities (<40 μmol m⁻² s⁻¹) that were close to the average that a cell experiences in L. Baikal when mixed vertically by convection to depths that can exceed 100 m. On sunny days, cell division could be inhibited down to >10 m depth but narrow (<15 μm) diameter cells trapped in high light intensities in sub-ice layers switched to auxosporulation and size regeneration.     

Effects of the medium ammonium-nitrate ratio on competence for asparagus cell division induced by phytosulfokine-α

The effects of medium ammonium-nitrate ratio on cell proliferation were investigated using a low cell-density culture of Asparagus officinalis L., which was triggered by a peptidal plant growth factor, phytosulfokine-α (PSK-α). The asparagus cells proliferated the most in a medium with an ammonium-nitrate ratio of 0:30 mM and could be maintained without significant loss of responsiveness to PSK-α for at least 96 h from the beginning of culture. In this medium, single cells gave rise to microcalli at initial densities as low as 3.2×10² cells/ml as long as PSK-α was present in the medium. Increasing the ammonium-nitrate ratio resulted in severe inhibition of cell proliferation at a low cell density, even if PSK-α was added to the medium. Received: 6 May 1997 / Revision received: 5 August 1997 / Accepted: 2 September 1997     

In vivo antioxidant role of astaxanthin under oxidative stress in the green alga Haematococcus pluvialis

Intracellular production of active oxygen in the green alga Haematococcus pluvialis was studied by measuring the capacity for in vivo conversion of 2′,7′-dichlorohydrofluorescein diacetate to the fluorescent dye dichlorofluorescein in different algal cell types (i.e., vegetative, immature cyst and mature cyst cells). The increase in formation of dichlorofluorescein by methyl viologen (superoxide anion radical generator) was linear for 2 h in immature cyst cells (low astaxanthin) in a methyl viologen-concentration-dependent manner, while no production was detected in mature (high astaxanthin) cysts. Compared to cyst cells, formation of dichlorofluorescein in vegetative cells (no astaxanthin) was markedly increased by methyl viologen. The formation of dichlorofluorescein in cyst cells was decreased with higher astaxanthin content under excessive oxidative stress. All of the active oxygen species tested (singlet oxygen, superoxide anion radical, hydrogen peroxide and peroxy radical) at 10⁻³ M increased the intracellular dichlorofluorescein formation in immature cysts, but did not increase the dichlorofluorescein content of mature cysts. Therefore, astaxanthin in cyst cells appeared to function as an antioxidant agent against oxidative stress. Received: 26 January 2000 / Received revision: 5 April 2000 / Accepted: 1 May 2000     

Electric Conductivity and Internal Osmolality of Intact Bacterial Cells

Intact cells of Streptococcus faecalis and Micrococcus lysodeikticus were found to have high-frequency electric conductivities of 0.90 and 0.68 mho/m, respectively. These measured values, which reflect movements of ions both within the cytoplasm and within the cell wall space, were only about one-third of those calculated on the basis of determinations of the amounts and types of small ions within the cells. Concentrated suspensions of bacteria with damaged membranes showed similarly large disparities between measured and predicted conductivities, whereas the conductivities of diluted suspensions were about equal to predicted values. Thus, the low mobilities of intracellular ions appeared to be interpretable in terms of the physicochemical behavior of electrolytes in concentrated mixtures of small ions and cell polymers. In contrast to the low measured values for conductivity of intact bacteria, values for intracellular osmolality measured by means of a quantitative plasmolysis technique were higher than expected. For example, the plasmolysis threshold for S. faecalis cells indicated an internal osmolality of about 1.0 osmol/kg, compared with a value of only 0.81 osmol/liter of cell water calculated from a knowledge of the cell content and the distribution of small solutes. In all, our results indicate that most of the small ions within vegetative bacterial cells are free to move in an electric field and that they contribute to cytoplasmic osmolality.