Investigations of mannan and glucan in the cell wall ofCandida boidinii cultivated on methanol

The polysaccharide components (mannan and glucan) in the cell wall ofCandida boidinii M 363 grown on methanol and glucose as control were investigated using electron microscopy, cytochemical and biochemical methods. An ultrastructural rearrangement of the polymers in the cell wall of yeasts cultivated on methanol in comparison to those cultivated on glucose was established. The morphological changes correlate to the quantitative changes in the polysaccharide constituents of the cell wall. The forming and the role of thiosemihydrocarbazide (TSHC) — negative zones in theCandida boidinii cell wall cultivated on methanol media are discussed.     

ULTRASTRUCTURE OF SPONGIOCHLORIS TYPICA DURING SENESCENCE12

The ultrastructural changes which occurred during senescence in the stationary phase of growth of the unicellular green alga Spongiochloris typica were observed. The cell wall consists of a membrane like primary wall and an inner secondary wall which becomes progressively thickened with age of the culture. During senescence the lamellae become more compact within the chloroplast. The major feature of aging is the appearance of lipid bodies which eventually come to occupy a major portion of the cell lumen. The ultrastructural changes observed to occur during senescence are discussed in relation to physiological data.     

A MORPHOMETRIC ANALYSIS OF CYTOLOGICAL CHANGES DURING SPORE MATURATION IN THE MYXOMYCETE DIDYMIUM IRIDIS

Ultrastructure of spore maturation in the myxomycete Didymium iridis was investigated using morphometric analytical techniques. Changes in actual volume (μm³) and relative volume (Vv) of nuclei, autophagic vacuoles, mitochondria, microbodies, lipid droplets, and spore wall were described for spores in three stages of development. Stage I spores were newly formed, surrounded only by the cell membrane. Stage II spores were approximately 1 hr older than Stage I spores and possessed surface spines, but little if any additional wall material. Stage III spores were 24 hr old and possessed a fully formed, multilayered wall. The results of this study indicate that spore maturation in D. iridis is a multistep process involving a decrease in spore volume and coordinated changes in specific organelle compartments. From Stage I to Stage III, mean spore volume decreased by more than 50%. Percent volume data (Vv) showed that Stage I spores allocated volume equally to all measured organelles except microbodies and the spore wall, the latter of which had not yet begun to develop. By Stage II, only the nucleus and spore wall showed significant changes in Vv values, both increasing. In terms of actual volume, the nucleus, autophagic vacuole and spore wall increased by Stage II. Between Stages II and III the cell wall was the only component to increase in volume, all others decreased in volume. Our data indicate a close relationship between a decrease in organelle volume and an increase in cell wall volume in the Stage III spore. The autophagic vacuole and the cell wall dominated the volume of the Stage III spore while the remaining volume was allocated unequally to the other components.     

THE EFFECTS OF MECHANICAL STIMULATION AND ETIOLATION ON THE COLLENCHYMA OF DATURA STRAMONIUM

Walker , Waldo S. (Grinnell College, Grinnell, Iowa.) The effects of mechanical stimulation and etiolation on the collenchyma of Datura stramonium. Amer. Jour. Bot. 47(9) : 717–724. Illus. 1960.–In an effort to determine the effect of mechanical stimulation on collenchyma tissue, plants of Datura stramonium L. were placed on a mechanical agitator and subjected to intensive shaking for 9 hr. per day for 40 days. Measurements indicated that such stimulation greatly increased the amount of wall thickening per cell, as observed in transverse section. Measurements also indicated that such stimulation may inhibit collenchyma cell elongation. A second group of Datura stramonium plants was placed in total darkness to determine the effect of such treatment on the quantity of wall thickening in the collenchyma tissue. Measurements indicated that when plants were placed in the dark for extended periods a great reduction of wall thickening resulted. It is suggested that reduction of wall material was due to its utilization as substrate for respiratory processes which occur in the plant under such extreme conditions. The composition and structure of the collenchyma cell walls are discussed.     

ORIENTATION OF THE PLANE OF CELL DIVISION IN FERN GAMETOPHYTES: THE ROLES OF CELL SHAPE AND STRESS

Apical cells of Onoclea sensibilis L. protonemata were measured to determine areas of new walls which were formed during both transverse and longitudinal cell division. Actual wall areas were compared with calculated areas of hypothetical walls oriented in the opposite sense (i.e., an actual transverse wall compared with a hypothetical longitudinal wall, and the reverse). Among 87 out of 90 cells which were analyzed the actual walls had the least area. Thus, the minimal area hypothesis of cell partitioning accurately predicts wall orientation in this instance, although it appears, on other grounds, that the hypothesis does not furnish a plausible mechanism for wall orientation. The application of Lintilhac's concept of the orientation of cell walls in response to anisotropic stresses in the cell was explored. Photographs of apical cells during deplasmolysis indicated that unequal stresses might be generated in apical cells as a result of the osmotic distension of the elastic protoplast. It is concluded that the primary factor which determines the plane of cell division in the apical cell, and the transition from one- to two-dimensional growth, is the local pattern of stress which exists at the position of the nucleus at the time of onset of cell division and wall formation. Calculations of some geometrical properties of idealized model cells are interpreted to mean that the accuracy of the minimal area hypothesis results from a coincidence of its predictions with predictions of Lintilhac's hypothesis, and no causal significance is attributed to wall areas.     

The Regulator RamA Influences cmytA Transcription and Cell Morphology of Corynebacterium ammoniagenes

RamA plays a regulatory role for acetate utilization and S-layer biosynthesis in Corynebacterium glutamicum. Looking for any additional role, the function of RamA was analyzed in Corynebacterium ammoniagenes, which is closely related to C. glutamicum. In this study, we showed that the ΔramA mutant constructed by a markerless knockout strategy possessed increased cell surface hydrophobicity, leading to the formation of aggregated cell masses in liquid media. In addition, the mutant exhibited an elongated cell shape as observed by SEM, suggesting that cell wall-associated proteins might be influenced. Furthermore, cell surface proteome analysis revealed that the expression of cmytA gene encoding corynomycoloyl transferase required for cell wall biosynthesis was down-regulated in the mutant, supporting the regulatory role of RamA in cell wall assembly. These studies support a novel regulatory role of RamA in inducing the expression of proteins required for cell wall assembly.     

THE ULTRASTRUCTURE OF THE QUIESCENT CENTER IN THE APEX OF CULTURED ROOTS OF CONVOLVULUS ARVENSIS L.

Cultured roots of the common bindweed, Convolvulus arvensis L. growing at the rate of 15–30 mm/day in sterile nutrient medium were fixed for electron microscopic analysis. The ultrastructure of the quiescent center, the initials of the ground meristem, and the initials of the procambium were studied in order to determine whether sequential structural changes could be correlated with models for specifying the mechanisms by which cell differentiation and cell division might be controlled. The differentiation of cells in the root proper occurs very gradually in linear files from the site of the quiescent center proximally into the different tissue regions. Major structural changes, such as the orientation and subsequent elongation of cells along the longitudinal axis of the root and cell wall changes, indicate that the control of differentiation and perhaps cell division occurs in radial gradients outwardly from the quiescent center.     

THE INITIAL STRUCTURAL LESION OF PENICILLIN ACTION IN BACILLUS MEGATERIUM

The effect of penicillin on the structure of Bacillus megaterium cells was followed in media with and without osmotic stabilization. In peptone without osmotic support the cells showed a distortion of the normal membrane-wall relationship by 20 minutes. This appeared to be a combination of both membrane distortion and cytoplasmic leakage. Lytic changes quickly followed. With osmotic support a clean-cut lesion at the transverse-septal site developed by 10 minutes' growth in penicillin. The membrane lost its normal relationship to the cell wall and formed a pocket which was filled with a fibrous material which appeared to be unorganized wall mucopeptide. The pocket of fibers enlarged until the cell either lysed or formed a protoplast.     

Mannan structural complexity is decreased when Candida albicans is cultivated in blood or serum at physiological temperature

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D ¹H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man-(1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.     

Effects of Hydroxyproline and other Amino Acid Analogues on the Growth of Pea Root Segments

Changes in the lengths and growth rates of isolated 2–4 mm pea root segments, cultured in sucrose media under aseptic conditions, were paralleled by changes in invertase development and in chloride and leucine uptakes. The amino acid analogues o-, m- and p-fluorophenylalanine, azetidine-2-carboxylic acid and ethionine inhibited growth with corresponding changes in invertase activity and in chloride and leucine uptakes. In contrast hydroxyproline, which under the conditions used may be regarded as an analogue of proline, enhanced both the growth rate and duration of growth but had little effect on the several parameters of protein synthesis which were measured. No amino acid tested affected changes in growth, invertase activity or the uptake of chloride and leucine, but they prevented the effects of the corresponding analogues. The results show that although extension growth is dependent on continuous protein synthesis, only specific proteins, probably in the cell wall, play a key role in this process.     

The inactivation of pectin depolymerase associated with isolated tomato fruit cell wall: implications for the analysis of pectin solubility and molecular weight

An approach commonly employed to assess the potential role of the enzyme polygalacturonase (PG, EC 3.2.1.15) in tomato fruit cell-wall pectin metabolism includes correlating levels of extractable PG with changes in specific characteristics of cell wall pectins, most notably solubility and molecular weight. Since information on these features of pectins is generally derived from analyses of subfractions of isolated cell wall, assurance of inactivation of the various isoforms of wall-associated PG is imperative. In the present study, cell wall prepared from ripe tomato (Lycopersicon esculentum Mill. cv. Rutgers) fruit was examined for the presence of active PG and for the ability of phenolic solvents to inactivate the enzyme. Using pectin solubility and M_r (relative molecular mass) changes as criteria for the presence of wall-associated PG activity, pectins from phenol-treated and nonphenol-treated (enzymically active) cell wall from ripe fruit incubated in 50 mM Na-acetate, 50 mM cyclohexanetrans-1,2-diamine tetraacetic acid (CDTA), pH 6.5 (outside the catalytic range of PG), were of similar M_r and exhibited no change in size with incubation time. Wall prepared without exposure to the phenolic protein-denaturants exhibited extensive pectin solubilization and depolymerization when incubated in 50 mM Na-acetate, 50 mM CDTA at pH 4.5, indicating the presence of active PG. Based on the changes in the M_r of pectins solubilized in 50 mM Na-acetate, 50 mM CDTA, pH 4.5, active PG was also detected in wall exposed during isolation to phenolacetic acid-water (PAW, 2:1:1, w/v/v), a solvent commonly employed as an enzyme denaturant. Although the depolymerization of pectins in PAW-treated wall was extensive, oligouronides constituted minor reaction products. Interestingly, PAW-treated wall did not exhibit PG-mediated pectin release when incubated under conditions (30 mM Na-acetate, 150 mM NaCl, pH 4.5) in which nonphenol-treated cell wall exhibited high autolytic activity. In an alternative protocol designed to inactivate PG, cell wall was exposed to Tris-buffered phenol (BP). In contrast to pectins released from PAW-treated wall, pectins solubilized from BP-treated wall at pH 4.5 were indistinguishable in M_r from those recovered from BP-treated wall at pH 6.5 Even when incubated at pH 4.5 at 34°C, conditions under which pectins from PAW-treated wall underwent more rapid and extensive depolymerization, pectins from BP-treated wall exhibited no change in M_r, providing evidence that active PG was not present in these wall preparations. The implications of this study in interpreting the solubility and M_r of pectin in cell wall from ripening fruit are discussed.     

Experimental inhibition of cell wall formation and of reversion inNadsonia elongata andSchizosaccharomyces pombe protoplasts

Summary The growth, cell wall regeneration, and the reversion of the protoplasts ofNadsonia elongata andSchizosaccbaromyces pombe cultivated in nutrient media containing snail enzyme was studied by light and electron microscopy. The protoplasts grew in the presence of snail enzyme and an incomplete cell wall composed of fibrils was formed on their surface. Thus, the presence of snail enzyme inhibited the completion of cell wall structure and, consequently, the reversion of the protoplasts to normal cells. The transfer of these protoplasts to medium free from snail enzyme led first to the completion of the cell wall and then to the reversion of the protoplasts to normal cells. The reported experiments confirmed that the regeneration of the complete cell wall preceded the protoplast reversion.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

A close-up view of wood structure and properties across a growth ring of Norway spruce (Picea abies [L] Karst.)

A growth ring of an adult Norway spruce (Picea abies [L] Karst.) was analyzed to a high resolution at the single cell level with respect to structural and mechanical changes during the growth period. For this purpose structural characterization was performed by means of light microscopy, scanning electron microscopy and wide angle X-ray diffraction for investigating the geometry of cells, their cell wall fractions and cellulose microfibril angles (MFA). The mechanical properties were determined in microtensile tests on individual tracheids which had been taken from sequentially cut tangential slices. The results revealed pronounced differences in tensile stiffness between earlywood and latewood cells but only minor differences in tensile stiffness between the cell walls of both tissue types. These comparatively small changes in cell wall stiffness across the growth ring were caused by slight changes in MFA. The findings suggest that trees mainly vary cell size to optimize water transport and mechanical stability during the growth period and that modification of the cell wall organisation plays a minor role.     

COMPARATIVE STUDIES ON THE CELL WALLS OF SEXUAL AND ASEXUAL BANGIA ATROPURPUREA (RHODOPHYTA). I. HISTOCHEMISTRY OF POLYSACCHARIDES1

A comparative histochemical study of the nature and distribution of acidic and neutral cell wall polysaccharides was conducted on marine sexual and asexual filaments of the red alga Bangia atropurpurea (Roth) C. Ag. Outer and inner walls of this species were clearly partitioned according to staining and transmission electron microscopic characteristics. Neutral polysaccharides were detected in the outer coating (cuticle) but were absent from outer and inner walls of all filaments. Acidic polysaccharides were noted in the outer wall material but not in the inner wall layers of any filaments at any developmental stages. The major staining component of vegetative regions of sexual material and all regions of asexual filaments was a highly sulfated polymer. During sexual reproduction only there was a generalized change in the nature of the acidic component, characterized by a decrease in intensity of staining for sulfates in both male and female filaments and the appearance, in female filaments only, of polysaccharides which presumably were carboxylated. Spermatia attached to both male and female filaments in regions where sexual differentiation was initiated and where changes in the outer wall components commenced.     

Microscopical observations onAvena coleoptile cell walls after stretching on a tensile-tester

In order to see if there are any structural damages or modifications of the cell wall ofAvena coleoptiles due to the stretching on an Instron tensile-tester when mechanical properties of the cell wall are measured, we made microscopic observations using cell wall specimens which had been stretched. As far as seen by light microscopic and scanning electron microscopic (SEM) observations there are no appreciable changes in the cell wall, such as breakage, tearing or formation of crack on the surface, due to the stretching which produced 10–30 g stress. It is concluded that stretching of this range causes no artifactitious changes in the wall and hence mechanical properties of the cell wall can be measured by stretching the wall specimen on a tensile-tester.     

The effects of tracheid dimensions on variations in maximum density of Picea glehnii and relationships to climatic factors

An investigation was made of the effects of tracheid dimensions on variations in the maximum density of Picea glehnii Mast., which were associated with climatic changes. Radial cell diameter and the thickness of the tangential cell walls of the last-formed cells in 90 annual rings of nine trees with different annual ring widths were analyzed by image analysis. Correlations between maximum density and tracheid dimensions indicated that changes in maximum density were due mainly to changes in cell wall thickness of the last-formed cells in annual rings and were not due to changes in radial cell diameter. The effects of climatic factors on tracheid dimensions were examined by application of dendroclimatological techniques. A chronology of cell wall thickness that represented common signals among trees was established. Simple correlation and response function analyses of the chronology revealed that cell wall thickness was influenced positively by summer temperature and negatively by precipitation in August, and these responses were similar to those of maximum density. The study demonstrated that variations in maximum density were due to variations in the cell wall thickness of the last-formed cells, which varied depending on the weather in summer. Received: 8 February 1999 / Accepted: 7 October 1999     

Effects of fructose on human fibroblast metabolism: the application of DNA measurements as a basis for interpretation

Summary A fluorometric procedure for measuring DNA was used to study growth and metabolic responses of eight cell strains of human foreskin fibroblasts. In preliminary studies this procedure gave more precise specific activity changes inN-acetyl-β-d-glucosaminidase (NAG) than did a protein activity basis, when changes in this enzyme's specific activity were investigated as a function of experimental cell manipulation. When fibroblast growth in eight cell strains was compared in 134 mM d-fructose vs. 13.4 mM glucose-supplemented minimum essential media, a significant increase in cellular DNA (50%) and protein (45%) occurred over an 11-d period. No significant differences in media pH change, lactate production, or carbohydrate uptake occurred on a DNA basis when cell metabolism was compared over the last 24 h of culture in the two media. Cells grown in fructose-containing media tended to show a reduction in NAG specific activity when compared with those grown in glucose-containing media.     

Genetic diversity associated with variation in silage corn digestibility for three<Emphasis Type="Italic"> O</Emphasis>-methyltransferase genes involved in lignin biosynthesis

Polymorphisms within three candidate genes for lignin biosynthesis were investigated to identify alleles useful for the improvement of maize digestibility. The allelic diversity of two caffeoyl-CoA 3-O-methyltransferase genes, CCoAOMT2 and CCoAOMT1, as well as that of the aldehyde O-methyltransferase gene, AldOMT, was evaluated for 34 maize lines chosen for their varying degrees of cell wall digestibility. Frequency of nucleotide changes averaged one SNP every 35 bp. Ninety-one indels were identified in non-coding regions and only four in coding regions. Numerous distinct and highly diverse haplotypes were identified at each locus. Numerous sites were in linkage disequilibrium that declined rapidly within a few hundred bases. For F4, an early flint French line with high cell wall digestibility, the CCoAOMT2 first exon presented many non-synonymous polymorphisms. Notably we found an 18-bp indel, which resembled a microsatellite and was associated with cell wall digestibility variation. Additionally, the CCoAOMT2 gene co-localized with a QTL for cell wall digestibility and lignin content. Together, these results suggest that genetic diversity investigated on a broader genetic basis could contribute to the identification of favourable alleles to be used in the molecular breeding of elite maize germplasm.     

CHEMICAL COMPOSITION AND STRUCTURE OF THE CELL WALLS OF THE CONCHOCELIS AND THALLUS PHASES OF PORPHYRA TENERA (RHODOPHYCEAE)1,2

Purified cell walls were prepared from both the conchocelis and thallus phases of Porphyra tenera (Kjellm.). The nitrogen content of cell walls from the conchocelis was significantly greater than that for the thallus cell walls, being 3.35 ± 0.26% and 2.39± 0.03%, respectively. Amino acid analysis revealed important differences. The conchocelis cell wall hydrolyzates were richer in aspartic acid, glutamic acid, methionine, and basic amino acids. The thallus cell wall hydrolyzates, however, contained much more glycine and alanine than did those of the conchocelis. Hydroxyproline was not detected in cell walls of either phase. The neutral sugar content of cell wall hydrolyzates from the thallus was more than double that from the conchocelis being 83.6% and 34.5%, respectively. The former contained predominantly mannose which accounted for 72.2% of the neutral sugars while the latter was principally galactose (49.9%) and glucose (36.4%). Methylation analysis confirmed the presence of cellulose microfibrils in the conchocelis in contrast to xylan microfibrils in the thallus. The results establish that the conchocelis and thallus phases of P. tenera differ markedly in the structure and composition of the cell walls.