Patterns of asexual reproduction in <Emphasis Type="Italic">Nannochloris bacillaris</Emphasis> and <Emphasis Type="Italic">Marvania geminata</Emphasis> (Chlorophyta,Trebouxiophyceae)

Non-flagellated vegetative green algae of the Trebouxiophyceae propagate mainly by autosporulation. In this manner, the mother cell wall is shed following division of the protoplast in each round of cell division. Binary fission type Nannochloris and budding type Marvania are also included in the Trebouxiophyceae. Phylogenetic trees based on the actin sequences of Trebouxiophyceae members revealed that the binary fission type Nannochloris bacillaris and the budding type Marvania geminata are closely related in a distal monophyletic group. Our results suggest that autosporulation is the ancestral mode of cell division in Trebouxiophyceae. To elucidate how non-autosporulative mechanisms such as binary fission and budding evolved, we focused on the cleavage of the mother cell wall. Cell wall development was analyzed using a cell wall-specific fluorescent dye, Fluostain I. Exfoliation of the mother cell wall was not observed in either N. bacillaris or M. geminata. We then compared the two algae by transmission electron microscopy with rapid freeze fixation and freeze substitution; in both algae, the mother cell wall was cleaved at the site of cell division, but remained adhered to the daughter cell wall. In N. bacillaris, the cleaved mother cell wall gradually degenerated and was not observed in the next cell cycle. In contrast, M. geminata daughter cells entered the growth phase of the next cell cycle bearing the mother and grandmother cell walls, causing the uncovered portion of the plane of division to bulge outward. Such a delay in the degeneration and shedding of the mother cell wall probably led to the development of binary fission and budding.     

Glycoprotein conformation in plant cell walls

The major structural glycoprotein of the cell wall of Chlamydomonas reinhardii has a protein core, at least 50% of which is in the unusual polyproline II conformation. This has been demonstrated by examining the circular dichroism of the cell wall, its constituent glycoproteins, and thermolysin released wall glycopeptides. One of these glycopeptides, T2, has a high hydroxyproline and sugar content, and possesses upward of 85% polyproline II structure. The main extracellular matrix glycoprotein therefore has a rigid, rod-like structure and the significance of this and its relation to higher plant cell wall glycoproteins is discussed. The unusual conformation appears to confer great stability on the glycoprotein as it is unchanged either by certain denaturing agents or during the transition from protomer to assembled cell wall.Abbreviations CD circular dichroism - HP 4-hydroxy-L-proline - PP poly-L-proline - SDS sodium dodecylsulphate This is the eight paper in a series entitled Structure, Composition and Morphogenesis of the Cell Wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1978)     

Variations of the Surface Sculpture and Cell Wall Ultrastructure of the Zygospores in Chlamydomonas geitleri (Chlorophyta)

The superficial cell wall ornamentation in the zygospores of the alga Chlamydomonas geitleri Ettl (Chlorophyta) is formed by thickenings of the cell wall which are shaped into a network of anastomosing ribs, sometimes with local wart-like protuberances. Clearly different sculpture patterns (given by presence, arrangement and/or morphological modification of sculpture elements) were accompanied by many transient forms. Sculpture variations occurred even in clonal cultures. In the zygospore cell wall of C. geitleri, the inner, outer and middle layer can be distinguished from the morphological point of view. The relatively thin outer (sporopollenin) layer covers the whole surface of the zygospore wall. The thicker inner layer adhering to the zygospore protoplast forms, either solely or together with the middle layer (possessing a fine meshwork substructure), variously shaped thickening of the zygospore cell wall. Discussed are the ultrastructural morphology of the cell wall in Chlamydomonas zygospores, the striking similarity of the cell wall ultrastructure of zygospores in C. geitleri to the ultrastructure of the cell wall of vegetative cells in some green algae (subfamily Scotiellocystoideae), as well as the extensive morphological variability of the zygospore wall sculpture in C geitleri and its species specificity.     

Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans

The multilayered cell wall of the cyanobacterium Anacystis nidulans was studied by the freezeetching technique. A characteristic fracture face in the outer cell wall was demonstrated which is densely packed with particles of a diameter of 60–75 Å. This particle layer is comparable with layers which have been described in many cell walls of Gram-negative prokaryotes.The outer membrane of the cell wall was solubilised by extraction with phenol/water or sodium dodecyl sulfate (SDS). In the SDS-extract 31 bands were separated by polyacrylamide gel electrophoresis, among them 3–5 major proteins with molecular weights of approximately 60, 40, and 10 kdaltons, respectively. Several polypeptides of the Anacystis cell wall were comparable in their mobility with polypeptides extracted from cell walls of different Gramnegative bacteria. The analysis of the SDS-unsoluble electron dense layer (sacculi) revealed the typical components of peptidoglycan diaminopimelic acid, muramic acid, glutamic acid, glucosamine and alamine in the molar ratio of 1.0:0.9:1.1:1.5:1.9. In addition, other amino acids (molar ratio from 0.05–0.36), mannosamine (molar ratio 0.54), and lipopolysaccharide components were detected in low concentration.Abbreviations SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetate     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

Studies on Chlorella protoplasts

Protoplasts of Chlorella saccharophila (Krüger) Nadson were obtained by cellulase digestion of the microfibrillar inner compount of the cell wall after the resistant outermost layer had been scratched with sea sand. The absence of the cell wall was demonstrated immunologically, electron microscopically and by staining, thus confirming the protoplastic nature of the treated cells. After transfer to an enzyme-free medium regeneration of a thin cell wall was observed. The regeneration of the cell wall obviously followed the same steps as does the cell wall development of the autospores. At least 50% of the protoplasts were able to form colonies when plated on a suitable agar medium.     

Alterations in the cell wall ofSaccharomyces cerevisiae induced by the alpha sex factor or a mutation in the cell cycle

We performed experiments in parallel to study the rate of synthesis of cell wall polysaccharides and the activity of glycosyl transferases inSaccharomyces cerevisiae after arrest of acdc 28 mutant in G1 phase by either addition of alpha-factor or transfer to the non-permissive temperature. Both effectors brought about similar time-dependent increases in the rate of synthesis and deposition of the cell wall polysaccharides chitin, glucan and mannan. These changes in cell wall composition were accompanied by an increase in the specific activities of glucan and chitin synthetases. This increase was inhibited by cycloheximide suggesting that it representedde novo enzyme biosynthesis and not enzyme activation. Our data are consistent with the notion that both alpha-factor and thecdc 28 mutation affect the same stage-specific function that controls the temporal expression of glycosyl transferases.Abbreviations GlcNAc N-acetyl glucosamine - UDPGIcNAc uridine-diphosphate-N-acetyl glucosamine - UDPGlc uridine-diphosphate glucose - TCA trichloroacetic acid - EDTA ethylene diamino tetraacetate - TAME tosyl-L-arginyl methyl ester - GTP guanosine triphosphate - WGA wheat germ agglutinin     

Regeneration of the cell wall in protoplasts of Candida albicans

To assess the dynamics of synthesis of the wall by regenerating Candida albicans protoplasts deposition of chitin and mannoproteins were investigated ultrastructurally using wheat germ agglutinin conjugated with either horseradish peroxidase or colloidal gold, and Concanavalin A coupled to ferritin respectively.Freshly prepared protoplasts lacked wheat germ agglutinin receptor sites but after 1–2 h of regeneration, they were detected. After 4–5 h of regeneration, the cell wall showed a discrete structure which was only labelled with wheat germ agglutinin in thin sections. At this stage of regeneration the outermost layer of the wall was labelled with clusters of Concanavalin A-ferritin particles.After 8 h regeneration, the cell wall appeared compact, and homogenously marked with wheat germ agglutinin whereas only the surface layers appeared consistently labelled with Concanavalin A-ferritin.From these observations we conclude that C. albicans protoplasts are able to regenerate in liquid medium a cell wall consisting of a network of chitin fibrils and mannoproteins at least (glucan polymers were not determined in the present cytological study). The former are the fundamental component of the inner layers at early stages of regeneration, whereas the latter molecules are predominant in the outer layers of the wall.Abbreviations WGA-HRP wheat germ agglutinin conjugated with horseradish peroxidase - WGA-Au wheat germ agglutinin conjugated with colloidal gold - Con A-ferritin Concanavalin A coupled to ferritin     

The Cell Wall of Fusarium oxysporum

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50–60% of the total mass of the wall. X-ray diffraction studies showed the presence of α-1,3-glucan in the alkali-soluble cell wall fraction and of β-1,3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.     

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face     

Evaluation of effectiveness of the methods for isolation of cell wall polysaccharides during cell elongation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> seedlings

Phaseolus vulgaris seedlings were grown in light with or without chromium. Changes in cell wall components i.e. pectic polysaccharides and xyloglucan contents were looked into during cell elongation, by two different methods in order to find the most suitable method for isolation of cell wall polysaccharides. The first method was short and easy. It made use of organic solvents for preparation of cell wall components and ammonium oxalate and oxalic acid buffer and high temperature for extracting pectic polysaccharides; 0.7 M and 4.3 M KOH was used for extracting low and high molecular weight xyloglucans respectively. On the other hand, in the second method, cell wall components were fractionated by sequential treatments with different inorganic solvents, chelating agents, sodium lauryl sulphate, etc. KOH (1 M and 4 M) was used for extracting xyloglucans. The advantage of using the second method for extracting cell wall polysaccharides especially pectic polysaccharides is discussed.     

产朊假丝酵母细胞壁33 ku蛋白的功能研究

通过胰蛋白酶和枯草杆菌蛋白酶对产朊假丝酵母Candida utilis细胞壁的酶解,发现一种分子质量为33 ku的酵母细胞壁主要结构蛋白. 研究显示,在细胞壁上这种蛋白质与细胞壁绝大多数蛋白质成分不同, 它不被胰蛋白酶水解,但对枯草杆菌蛋白酶的作用敏感.33 ku蛋白存在于酵母菌整个对数生长期的细胞壁中,特别是在对数早期细胞壁中,它是唯一的对胰蛋白酶作用不敏感的蛋白质成分.实验证明,该蛋白质对维系酵母细胞壁骨架成分葡聚糖的相互连接和细胞壁的完整结构,具有重要作用,是一种重要的酵母细胞壁嵌合蛋白.     

Regeneration and maturation of daughter cell walls in the autospore-forming green alga<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis> (Chlorophyta,Trebouxiophyceae)

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17–21 nm) emerged as a small, but complete, autospore.     

The wall ofPinus sylvestris L. pollen tubes

Summary The wall ofPinus sylvestris pollen and pollen tubes was studied by electron microscopy after both rapid-freeze fixation and freeze-substitution (RF-FS) and chemical fixation. Fluorescent probes and antibodies (JIM7 and JIM5) were used to study the distribution of esterified pectin, acidic pectin and callose. The wall texture was studied on shadow-casted whole mounts of pollen tubes after extraction of the wall matrix. The results were compared to current data of angiosperms. TheP. sylvestris pollen wall consists of a sculptured and a nonsculptured exine. The intine consists of a striated outer layer, that stretches partly over the pollen tube wall at the germination side, and a striated inner layer, which is continuous with the pollen tube wall and is likely to be partly deposited after germination. Variable amounts of callose are present in the entire intine. No esterified pectin is detected in the intine and acidic pectin is present in the outer intine layer only. The wall of the antheridial cell contains callose, but no pectin is detectable. The wall between antheridial and tube cell contains numerous plasmodesmata and is bordered by coated pits, indicating intensive communication with the tube cell. Callose and esterified pectin are present in the tip and the younger parts of the pollen tubes, but both ultimately disappear from the tube. Sometimes traces in the form of bands remain present. No acidic pectin is detected in either tip or tube. The wall of the pollen tube tip has a homogenous appearance, but gradually attains a fibrillar character at aging, perhaps because of the disappearance of callose and pectin. No secondary wall formation or callose lining can be seen wilh the electron microscope. The densily of the cellulose microfibrils (CMF) is much lower in the tip than in the tube. Both show CMF in all but axial and nontransverse orientations. In conclusion,P. sylvestris and angiosperm pollen tubes share the presence of esterified pectin in the tip, the oblique orientations of the CMF, and the gradual differentiation of the pollen tube wall, indicating a possible relation to tip growth. The presence of acidic pectin and the deposition of a secondary-wall or callose layer in angiosperms but not inP. sylvestris indicales that these characteristics are not related to tip growth, but probably represent adaptations to the fast and intrastylar growth of angiosperms.Abbreviations CMF cellulose microfibrils - II inner intine - NE nonsculptured exine - OI outer intine - RF-FS rapid-freeze fixation freeze-substitution - SE sculptured exine - SER smooth endoplasmic reliculum - SV secretory vesicles     

Grass lignin acylation: <Emphasis Type="Italic">p</Emphasis>-coumaroyl transferase activity and cell wall characteristics of C3 and C4 grasses

Grasses are a predominant source of nutritional energy for livestock systems around the world. Grasses with high lignin content have lower energy conversion efficiencies for production of bioenergy either in the form of ethanol or to milk and meat through ruminants. Grass lignins are uniquely acylated with p-coumarates (pCA), resulting from the incorporation of monolignol p-coumarate conjugates into the growing lignin polymer within the cell wall matrix. The required acyl-transferase is a soluble enzyme (p-coumaroyl transferase, pCAT) that utilizes p-coumaroyl-CoenzymeA (pCA-CoA) as the activated donor molecule and sinapyl alcohol as the preferred acceptor molecule. Grasses (C3and C4) were evaluated for cell wall characteristics; pCA, lignin, pCAT activity, and neutral sugar composition. All C3 and C4 grasses had measurable pCAT activity, however the pCAT activities did not follow the same pattern as the pCA incorporation into lignin as expected.     

Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Cellulose and xylans in the interface capsule in symbiotic cells of actinorhizae

Summary Enzyme-gold affinity labeling was used to show that in mature infected cells of actinorhizal symbioses the capsule on the plant host side of the symbiotic interface contained cellulose and xylans. Host species examined for cellulose wereAlnus rubra, Casuarina equisetifolia, C. glauca, Ceanothus cuneata, C. velutinus, Elaeagnus pungens, andMyrica cerifera.. Cellulose was in the capsule throughout the infected cell, implying that during development cellulose synthase was present in the host cell membrane component of the symbiotic interface. Any possible degradation of capsule cellulose by the microsymbiont was either incomplete or transient, because the polymer was present in mature infected cells. Cellulose labeling inCeanothus andElaeagnus was less consistent than in the other species. Dual labeled capsules inCasuarina glauca andAlnus rubra showed a similar distribution of xylans and cellulose. Cytochemical studies indicate that the capsule contains three major classes of cell wall polysaccharides: cellulose, hemicellulose (xylans), and pectins (shown previously). This suggests that the capsule is essentially a thin, internal, tubular plant cell wall.Abbreviations Au₅ Au₁₅ colloidal gold particles with mean diameter of 5 and 15 nm, respectively - CBHI cellobiohydrolase I - CBHII cellobiohydrolase II - PBS phosphate-buffered saline     

The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts of Mougeotia

Protoplasts of the filamentous green alga Mougeotia sp. are spherical when isolated and revert to their normal cylindrical cell shape during regeneration of a cell wall. Sections of protoplasts show that cortical microtubules are present at all times but examination of osmotically ruptured protoplasts by negative staining shows that the microtubules are initially free and become progressively cross-bridged to the plasma membrane during the first 3 h of protoplast culture. Cell-wall microfibrils areoobserved within 60 min when protoplasts are returned to growth medium; deposition of microfibrils that is predominantly transverse to the future axis of elongation is detectable after about 6 h of culture. When regenerating protoplasts are treated with either colchicine or isopropyl-N-phenyl carbamate, drugs which interfere with microtubule polymerization, they remain spherical and develop cell walls in which the microfibrils are randomly oriented.     

Cytochemical evidence of a fungal cell wall alteration during infection of Eucalyptus roots by the ectomycorrhizal fungus Cenococcum geophilum

When the ectomycorrhizal fungus Cenococcum geophilum changes from a saprophytic to a symbiotic stage, its cell wall structure becomes simplified. The external hyphal wall layer which, in the saprophytic stage, is highly reactive to the Gomori-Swift test becomes poorly reactive and can no longer be distinguished from the internal wall layer in the Hartig net hyphae. The intensely stained external wall layer was also absent from pure cultures of Cenococcum geophilum grown on a medium with a low sugar content. This cell wall alteration could be due to a decrease in the amount of melanin or of melanin plus cystine-containing proteins. This change may be necessary for increased nutrient exchange between symbionts through hyphal walls.