Cell Wall Regions Research Articles

Evidence that actin reinforces the extensible hyphal apex of the oomyceteSaprolegnia ferax

Filamentous actin in the apices of growing hyphae of the oomyceteSaprolegnia ferax is distributed such that it could compensate for weakness in the expanding apical cell wall and thus play a role in morphogenesis of the tip. The tapered extensible portion of the hyphal tip where the cell wall is plastic contains a cap of actin which differs in organization from the actin in subapical, inextensible regions of the hypha. Rapidly growing hyphae which are expected to have a longer plastic cell wall region contain longer actin caps. Furthermore, the weakest point in the hyphal apex, demonstrated by osmotic shock-induced bursting, was within the taper where the wall is plastic but never in the extreme apex where actin was most densely packed and presumably the strongest. Treatment of hyphae with cytochalasin E/dimethyl sulphoxide induced rapid changes in actin caps. Cap disruption was accompanied by transient growth rate increases, subsequent rounding and swelling of apices and a shift of osmotically induced burst points closer to the apex. These correlated changes are consistent with a role for the actin cap in tip morphogenesis. The association between regions of plasticity in the apical cell wall, the extent of the actin cap, the location of the weakest point in the apex and the effects of damage to the actin cap suggest that the cap functions to support the apex in regions where the cell wall is weak.

Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on <italic>K</italic><sub><italic>1/2</italic></sub> (CO<sup>2</sup>) for Photosynthesis in <italic>Chlorella ellipsoidea</italic> C-27

The ratio of the extracellular to the intracellular activity of carbonic anhydrase (CA) in cells of Chlorella ellipsoidea C-27, adapted to low levels of CO2 for 24 h (low-CO2 cells), was about one to one. Treatment of intact cells with Pronase P inactivated about one-half of the extracellular CA activity without affecting photosynthetic activity. The CA activity in cell homogenates and in cell-wall ghosts liberated during cell division was completely inactivated by the same treatment. Pretreatment with Glycosidase mix, Chitosanase and Macerozyme enhanced the inactivation of the CA activity in intact cells. These results suggest that extracellular CA is evenly distributed throughout the whole cell-wall region. The apparent K1/2 for dissolved inorganic carbon (DIC) in low-CO2 cells doubled when extracellular CA was inactivated by treatment with Pronase P, but the K1/2 obtained was still one-half of that in high-CO2 cells. Photosynthetic 14CO2-fixation in low-CO2 cells was enhanced by acetazolamide, whereas H14CO3−-fixation was suppressed. The results suggest that CO2 is a dominant substrate utilized by cells and that HCO3− is utilized after conversion to CO2. The present results show that both intracellular and extracellular CA contribute to the increase in affinity for DIC during photosynthesis in low-CO2 cells of Chlorella ellipsoidea C-27.

Cuticular Properties and Postharvest Calcium Applications Influence Cracking of Sweet Cherries

Abstract The influence of stomata, the stylar scar, cuticular fractures, and Ca2+ on susceptibility of ‘Bing’ sweet cherry fruit (Prunus avium L.) to water injury was studied. Water injury was first detected as an increase in cell turgor. Water penetration caused separation of the cuticle from the epidermal cell wall. Swelling in the epidermal cell wall region resulted in cuticular fracturing that generally preceded fruit cracking. Uncracked fruit that had cuticular fractures softened rapidly. Stomata were sparsely distributed on the fruit surface and were often fixed in an open or partially open position. Water injury was not visible at stomata even when injury occurred adjacent to the stomatal region. Initial signs of injury were commonly visible near the stylar scar. Histochemical studies revealed that the surface of the stylar scar was devoid of a cuticle covering and was rich in insoluble carbohydrates. Greater penetration of solute containing 45Ca2+ occurred at the stylar scar. Fine fractures in the cuticle surface were observed in fruit at harvest time in 1985 and 1986. Cherry fruit with cuticular fractures had a higher water absorption rate than unfractured fruit. In immersion tests, Ca2+ reduced cherry cracking. EGTA increased fruit cracking; this increase was negated by adding Ca2+. Neither Ca2+ nor EGTA affected the water absorption rate of the fruit. EGTA decreased the cracking threshold of the fruit, while Ca2+ increased it. Soluble pectin content of the immersion solution rose with increasing incubation times. EGTA increased while Ca2+ markedly decreased soluble pectin concentration in the immersion solution. Histochemical studies indicated a breakdown of the cell wall structure in the epidermal region of water-injured fruit. Autoradiographs of fruit immersed in a solution containing 45Ca2+ showed the epidermal region to be the site of Ca2+ action in altering fruit cracking. Chemical name used: Ethyleneglycol-bis-(β-aminoethyl ether) N,N,N,N-tetraacetic acid (EGTA)

Extrusion of Protoplasm and Protein Bodies through Pores in Cell Walls of Pea, Bean, and Faha Bean Cotyledons during Imbibition

Several processes contribute to leakage of intracellular substances from legume seeds during imbibition. This study was conducted to determine whether protoplasm and protein bodies also leak from cotyledonary cells by a process analogous to pressure‐driven extrusion from multicellular blisters. The effects of soaking, and air‐drying or fixing excised cotyledons and intact seeds of field‐grown bean (Phaseolus vulgaris L.), pea (Pisum sativum L.), and faba bean (Vicia faba L.) were investigated using scanning electron microscopy. When dissolution and dispersion of intracellular substances were minimized by removing cotyledons from water, cells of excised ‘OSU 605’ pea cotyledons provided direct evidence for cellular pressure‐driven extrusion. Streams of intracellular substances from OSU 605 ranged from 0.2 to 1 μm in diameter. Similar extrusion streams were not found in situ on cotyledons of other genera, cultivars, or treatments; however, projections from protoplast surfaces, pores through cell walls, and orifices and protein bodies in intercellular spaces were consistent with cellular extrusion. The path for cellular extrusion probably was pores through contact cell walls (regions of cell wall in contact with adjacent cells), and then channels along the middle lamella to orifices at intercellular spaces or cotyledonary surfaces.

Structural Polysaccharides in Forages and Their Degradability

AbstractPlant cell walls are organized into complex matrices composed primarily of carbohydrates that represent a potential energy source for ruminants. An overview of the basic structural features of the major groups of polysaccharides that make up forage cell walls, metabolic changes during cell wall development, cell wall component interactions, and some aspects of cell wall degradation are presented. Polysaccharide components that compose the cell wall matrix can be disassociated and fractionated according to similar physio‐chemical properties. Plant structural polysaccharides vary in monosaccharide composition, substitution patterns, and glycosidic linkages between monomers. Chemical fractionation schemes typically separate structural polysaccharides into three major groups. These include pectic polysaccharides rich in galacturonic acid residues, hemicellulosic polysaccharides typically rich in xylose, and the cellulose fraction. Mature walls can be envisioned as a layered matrix with cellulose microfibrils embedded in an amorphous material composed of pectic and hemicellulosic polysaccharides. The amorphous regions of cell walls show the greatest variation during development. Interactions among individual components of the cell wall are key to the formation and structural integrity of the matrix. These include bonding interactions that range from weak hydrogen bonds to covalent cross‐links. During growth of plant cells there is a rearrangement of structural elements within the wall. The matrix undergoes reorganization by disruption of selected intra‐ and intermolecular bonds and de novo synthesis and insertion of new wall polymers. Individual polysaccharides may vary in rates and possibly extent of degradation by rumen microorganisms. These differences are due to the degree of hydrogen bonding of the individual polysaccharides, branching patterns, and associations with other wall constituents.

Ultrastructural immunolocalization of developmental proteins in phloem cell wall regions of Pisum Sativum l. shoot tissue

The spatial and temporal distribution of 2 antigenic proteins have been examined in shoot apical and internode tissues of pea seedlings in the context of protein expression during cell differentiation. Monoclonal antibodies were raised against the proteins of interest extracted from pea internode tissue and were employed as cytochemical probes. Goat anti-mouse second antibody conjugated with colloidal gold was used for labelling the proteins. A 32-kDa protein appeared as concentrated aggregations in cell wall areas between phloem parenchyma and sieve elements, and in the cell wall junctions between phloem parenchyma cells. A 47-kDa protein was also observed in the cell walls of the phloem tissue only, but was more dispersed. Both the 32-kDa and the 47-kDa proteins were absent from apical tissue. The observation of such specific localizations and the time of their appearance suggests possible developmental roles for the antigenic proteins.

Segregation structures of melt-spun CuSiB alloys and their high temperature deformation behaviour

Rapidly-solidified microcrystalline alloys typically possess a fine-grained matrix with solute distributed in a cellular arrangement, either in the form of enriched cell-wall regions or as second-phase particles at the original cell walls. It is unclear whether optimal strength would be achieved when a second phase is distributed as discrete particles or as a continuous film. The microstructures and mechanical properties of a series of CuSiB alloys were examined both at room temperature and at high temperature. Depending on the alloy considered, fairly stable amorphous films, or discrete particles, arranged in a cell structure, are formed by rapid solidification. The continuous second-phase film is shown to lead to higher average internal stresses and material deformation process, during hot testing, appears to be the same as that controlling deformation in the particle-strengthened materials.

Degradation of delignified Eucalyptus maculata by Cellvibrio mixtus

Electron microscopic evidence is presented to show that the cellulolytic bacterium Cellvibrio mixtus degrades delignified Eucalyptus maculata, initiating the decay in the S region of the fibre cell wall.

Haustorial mother cell development by Uromyces vignae on collodion membranes

The development of infection structures by the rust fungus Uromyces vignae was observed on oil-containing collodion membranes. About 40% of infection hyphae formed a haustorial mother cell, but this structure commonly senesced and died more rapidly than the infection hypha to which it was attached. These data suggest that the continued development of the haustorial mother cell requires some component normally provided by the host plant. Before they died, many haustorial mother cells apparently formed the thickened region of the wall which normally is traversed by the penetration peg during haustorium formation. Such a peg was observed in the centre of up to 40% of these thickened regions. However, no pegs protruded beyond the haustorial mother cell far enough to be called a haustorial neck. The thickened region of the haustorial mother cell wall could be differentiated from the rest of the wall by its lack of fluorescence under ultraviolet irradiation when mounted in Calcofluor or SITS (4-acetomido-4′-iso-thiocyanatostilbene-2,2′-disulphonic acid). Treatment with alkali, acid, chloroform–methanol, protease, and laminarinase did not affect this differential fluorescence, and the haustorial mother cell wall stained uniformly for proteins, carbohydrates, and chitin. Since Calcofluor normally binds to chitin, these data suggest that the thickened region of the haustorial mother cell wall may physically exclude the dye or may contain potential binding sites that are masked by other wall components.

Delignification studies on spruce wood tissue fractions isolated from outer and inner cell wall regions

Article Delignification studies on spruce wood tissue fractions isolated from outer and inner cell wall regions was published on August 1, 1987 in the journal Nordic Pulp & Paper Research Journal (volume 2, issue 3).

Ultrastructural localization of cell surface sugar residues in ericoid mycorrhizal fungi by gold-labeled lectins

Surface sugar residues were ultrastructurally localized in two strains ofHymenoscyphus ericae, one having a strong tendency to form ericoid mycorrhiza, the other, very little. The strains were studied both in the presence and absence of the host plant. Wheat germ agglutinin (WGA) and Concanavalin A (Con A)-colloidal gold complexes were used as cytochemical markers. N-acetylglucosamine residues were localized exclusively on septa and on the inner electron-transparent layer of longitudinal walls, confirming the presence of chitin in well defined regions of the fungal cell wall, both in the infective and in the noninfective strain. Con A-binding sites were detected on extracellular material commonly radiating from the wall of the infective strain. They were particularly abundant when the infective strain was in contact with the host, but were uncommon on the surface of the noninfective strain, whether this was in contact with the host or not. The extracellular material presumed to contain glucose and mannose residues appears to be important in establishing contact between fungus and host.

A comparison of relative lignin concentration as determined by interference microscopy and bromination/EDXA

The lignin concentrations of the compound and cell corner middle lamella regions of the tracheid cell wall in radiata pine were determined relative to the S2 region using (i) interference microscopy and (ii) bromination in conjunction with energy dispersive X-ray microanalysis. Relative lignin concentration values for the cell corner middle lamella as determined by interference microscopy were 3.39 and 4.23 for the two specimens examined, while the corresponding values determined by bromination/EDXA using Br K X-rays were 1.95 and 1.21. Values for the compound middle lamella were 1.54 and 0.86 as determined by bromination/EDXA. Possible reasons for the poor agreement between the two techniques are discussed.

Abnormal formation of Candida albicans walls produced by Calcofluor white: An ultrastructural and stereologic study

The effect of Calcofluor white on the formation of the cell wall was studied in yeast and mycelial cells of Candida albicans using uv microscopy, conventional electron microscopy (including qualitative and stereologic analyses), and freeze-fracture. The uv microscopy of semithin sections showed that abnormal deposition of chitin occurs in both yeast and mycelial cells. Ultrathin sections revealed that abnormal chitin deposition in yeast cells results in a wall thickening which protrudes into the cytoplasm. The thickening appeared in the region of the yeast cell wall where formation of the bud would take place. In mycelial cells, one or more bulges were observed, mainly located at the position of the septum. In freeze-fracture replicas, abnormally deposited wall material from the bulge displayed a rougher appearance than that normally deposited. Moreover, no significant differences with respect to intramembranous particles were observed between plasma membranes of control and Calcofluor-treated cells. In contrast, stereologic analysis on ultrathin sections of control and Calcofluor-treated cells showed significant differences in the volume of the cell wall between these cell populations. These results support the previously reported hypothesis that Calcofluor white interferes with deposition of chitin but not with its synthesis.

Degradation of carbohydrates and lignins in buried woods

Spruce, alder, and oak woods deposited in coastal sediments were characterized versus their modern counterparts by quantification of individual neutral sugars and lignin-derived phenols as well as by scanning electron microscopy, 13C NMR, and elemental analysis. The buried spruce wood from a 2500 yr old deposit was unaltered whereas an alder wood from the same horizon and an oak wood from an open ocean sediment were profoundly degraded. Individual sugar and lignin phenol analyses indicate that at least 90 and 98 wt% of the initial total polysaccharides in the buried alder and oak woods, respectively, have been degraded along with 15–25 wt% of the lignin. At least 75% of the degraded biopolymer has been physically lost from these samples. This evidence is supported by the SEM, 13C NMR and elemental analyses, all of which indicate selective loss of the carbohydrate moiety. The following order of stability was observed for the major biochemical constituents of both buried hardwoods: vanillyl and p- hydroxyl lignin structural units > syringyl lignin structural units > pectin > α-cellulose > hemicellulose. This sequence can be explained by selective preservation of the compound middle lamella regions of the wood cell walls. The magnitude and selectivity of the indicated diagenetic reactions are sufficient to cause major changes in the chemical compositions of wood-rich sedimentary organic mixtures and to provide a potentially large in situ nutrient source.

Cytochemical staining of a yeast polyphosphate fraction, localized outside the plasma membrane

Primary aldehyde fixation in the presence of Ca2+ and Mg2+ followed by alkaline Pb2+ staining leads to electron microscopical visualization of lead precipitates in the yeastKluyveromyces marxianus. These lead precipitates are found in vacuoles, cytoplasm, and on the outside of the plasma membrane in the periplasmic and inner cell wall regions.

Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis.

C-reactive protein (CRP) is a serum protein that shows rapid increases of as much as 1000-fold in concentration in response to infection, traumatic injury, or inflammation. CRP reacts with the phosphocholine moiety of pneumococcal cell wall C-polysaccharide, and this reaction can lead to complement activation in vitro and protection against pneumococcal infection in vivo. We have previously studied the chemiluminescence response of human neutrophils to Streptococcus pneumoniae as a measure of in vitro opsonophagocytosis by CRP and complement. CRP in the presence of complement was an effective opsonin for S. pneumoniae serotype 27 (Pn27), but not for serotypes 3 or 6. Because Pn27 differs from most serotypes of S. pneumoniae in containing phosphocholine in its capsular polysaccharide, we have determined the sites of CRP and C3 fixation to Pn27 and S. pneumoniae serotype 4 (Pn4), and related these to the ability of CRP and complement to opsonize these serotypes in vitro. By using a chemiluminescence (CL) assay to measure opsonophagocytosis, CRP was shown to enhance the response of human neutrophils and monocytes to Pn27 in the presence of normal human serum. The CL response of neutrophils and monocytes to Pn4 was not affected by the addition of CRP to serum. The addition of anti-capsular antibody to Pn4 and Pn27 enhanced the CL responses of both neutrophils and monocytes to both bacteria. The localization of bound CRP and C3 on Pn4 and Pn27 was determined by immunoelectron microscopy. CRP bound to Pn4 only in the cell wall region and C3 was located in this area whether or not CRP was present. Anti-capsular antibody deposited C3 in the capsule of Pn4. In contrast, Pn27 bound CRP throughout the capsule and cell wall areas. C3 was deposited in the cell wall region of Pn27 by serum alone and in the cell wall region and capsule when CRP or anti-capsular antibody was present. Because C3 fixation to the capsule was consistently associated with enhanced responses by phagocytic cells, it appears that the site of CRP binding and subsequent complement activation may be critical in the opsonophagocytosis of S. pneumoniae. These findings extend the correlation between capsular C3 and opsonization to a nonimmune system. By using CRP and different pneumococcal serotypes we have shown that the same molecules that are effective in the stimulation of phagocytic cells when bound to the capsule are not effective when bound to the cell wall.

Hydraulic conductivity ofNitella cells using the intracellular perfusion technique

The hydraulic conductivity of the intracellularly-perfused internodal cell ofNitella flexilis was measured by establishing and maintaining osmotic and hydrostatic pressure gradients between the inside and the outside of the cell. The osmotic filtration coefficient (LPD) determined at zero hydrostatic pressure difference varied between 1.55 and 2.32×10−5 cm/sec/atm. Under internal perfusion conditions no polarity between endosmotic and exosmotic flow was observed. The overall hydrostatic filtration coefficient (LP) was determined with a step change in hydrostatic pressure up to 0.2 atm, while the osmotic pressure difference was maintained at zero.LP was considerably greater than theLPD, i.e., 14.1 to 19.2×10−5 cm/sec/atm. Theoverall LP of such internodes, which showed protoplasmic streaming and action potentials was the same as that of the isolated cell walls, the latter being 13.2 to 19.9×10−5 cm/sec/atm. Some of these results are consistent with previous results onNitella using different techniques. The situation inNitella where at abnormallylow internal pressure the barrier to hydrostatic pressure-driven water flow does not reside in the plasmalemma but in an in-series structure is comparable to that in the squid axon where the normal internal pressure is close to zero. An interpretation is offered for the finding in the alga that athigh internal pressures the plasmalemma becomes the rate-limiting structure for hydrostatic pressure-driven water flow. It is suggested that the internal pressure pushes a large fraction of the plasmalemma against skeletal nonporous regions of the cell wall. This suggestion entailing a pressure-dependent cell wall-plasmalemma juxtaposition was also deployed in interpreting previous observations in plant cells on water flow polarity (i.e., observations showing that exosmotic rates are less than endosmotic).

Mannan‐Lokalisation durch Concanavalin A im Zusammenhang mit elektronenmikroskopischen und chemisch‐analytischen Untersuchungen an unterschiedlich präparierten Zellwänden der Futtereiweißhefe Candida spec. H

AbstractRegions of different electron densities after concanavalin A‐peroxidase‐reaction have been observed in cell walls of intact cells as well as in isolated cell walls: In the peripheral region of intact cell walls two mannan layers appear. Treating isolated cell walls with pronase, NaOH, ethylendiamine or citrate buffer, respectively, we find con A binding spots in the inner cell wall regions which appear electrontransparent in the untreated cell walls. Apparently the mannan component of the inner cell wall regions is masked by proteins. In correspondence with chemical analysis our results cannot confirm the existence of inner cell wall regions which are free of mannan.

Mannan localization with concanavalin A in conjunction with electron microscopy and chemical analysis of differently prepared cell walls of the food protein yeast Candida spec. H

Regions of different electron densities after concanavalin A-peroxidase-reaction have been observed in cell walls of intact cells as well as in isolated cell walls: In the peripheral region of intact cell walls two mannan layers appear. Treating isolated cell wall with pronase, NaOH, ethylendiamine or citrate buffer, respectively, we find on A binding spots in the inner cell wall regions which appear electron transparent in the untreated cell walls. Apparently the mannan component of the inner cell wall regions is masked by proteins. In correspondence with chemical analysis our results cannot confirm the existence of inner cell wall regions which are free of mannan.

The Fine Structrue of Chlamydomonas bullosaButcher

Summary The ultrastructure of Chlamydomonas bullosa Butcher is described with special reference to the papilla and cell wall. Comparisons are made with other species of the genus. C. bullosa has an inner granular layer in the cell wall region, which is an extension of the cytoplasm. It has a selliform papilla.