Single Cell Wall Research Articles

Biological affinities of Neoproterozoic acritarchs from Australia: microscopic and chemical characterisation

A range of well-preserved Neoproterozoic acanthomorph and sphaeromorph acritarchs from the Australian Centralian Superbasin have been individually analysed by combined microscopic and chemical methods. The acanthomorph acritarchs generally exhibit two or more cell wall categories and have fine laminations with no obvious trilaminar sheath structure, whereas the sphaeromorph acritarchs are characterised by a single cell wall structure with no distinct laminations. The significant organic carbon content of the acritarchs is evident by the EDS analysis. Petrological indices (Thermal Alteration Index⩽2.8, and strong yellow fluorescence) suggest a low thermal maturity for both coexisting acritarch groups. The biomarker composition of the enclosing-rock is similarly indicative of low thermal maturity, although most of the rock extract has probably originated from other sources including the lipid fraction of the acritarchs' cell membrane and cytoplasm rather than the preserved cell walls. In contrast, however, very limited chemical data was obtained directly from the morphologically-preserved component of the acritarchs by micro-FTIR and pyrolysis GC–MS analyses, and these results are consistent with a polyaromatic biomacromolecule of high recalcitrance. A large signal attributable to significant carbon-ordering — a characteristic of polyaromatic structures — was detected by laser micro-Raman spectroscopy. A unique ultrastructural and molecular entity of the studied Neoproterozoic acritarchs is indicated although a distinct fibrillar multilayered wall and a condensed polyaromatic macromolecular structure suggest a possible link between acanthomorph taxa and dinoflagellates. The contrasting maturity values indicated by the respective petrographic and chemical data highlight the need for a more rigorous correlation of these different maturity parameters.

Imaging of the helical arrangement of cellulose fibrils in wood by synchrotron X-ray microdiffraction

A complete image of the helical arrangement of cellulose fibrils in the S2 layer of adjacent wood cells ofPicea abies(Norwegian spruce) was obtained by applying position-resolved synchrotron X-ray microdiffraction on cells in cross section. In contrast to conventional fiber diffraction studies, the incident beam was parallel to the longitudinal cell axis, resulting in a glancing angle μ far from 90° with respect to the cellulose fibrils. This special choice of diffraction geometry allowed us to take advantage of an asymmetry effect in the two-dimensional diffraction patterns arising from the curvature of the Ewald sphere to obtain information on the local orientation of the cellulose fibrils. The small size of the beam, smaller than the thickness of a single cell wall, allowed mesh scans over intact transverse sections of adjacent wood cells with a microscopic position resolution. The scan yielded a map of diffraction patterns that could readily serve as a microscopic image. Each of the diffraction patterns was then used to evaluate the local orientation of the cellulose fibrils at the actual beam position. The combination of these results gave an image of cellulose fibrils forming (Z) helices in several adjacent wood cells.

Selection and Evaluation of Smooth Bromegrass Clones with Divergent Lignin or Etherified Ferulic Acid Concentration

ABSTRACTLignin and etherified ferulic acid (EthFA) are cell wall constituents believed to have important negative impacts on digestibility of forage cell walls. Our objective was to identify genotypes of smooth bromegrass (Bromus inermis Leyss) with unconfounded divergence in lignin and EthFA, such that their independent effects could be determined on in vitro fiber digestibility (IVFD). Eight clones were selected from each of four populations (‘Alpha’, WB19e, ‘Lincoln’, and WB88S). Selection was successful for EthFA in all populations except WB88S, creating repeatable divergence of 11.2 to 12.5%. Selection was unsuccessful for lignin concentration per se, most likely because of large genotype × environment interactions. Nevertheless, the resulting clones showed significant variation for both EthFA and lignin concentrations and these two variables were nearly independent among the selected clones. Both lignin and EthFA had significant negative effects on 96‐h IVFD, regardless of the statistical estimation method used. Across estimation methods, there was not a clear difference in the magnitude of the lignin and EthFA effects on IVFD. Both the total amount of lignin in the cell wall and the amount of ferulic acid cross‐linking lignin to polysaccharides via ether bonds appear to be under genetic control, and both components play a role in regulating the genetic potential for rumen degradation of cell walls in smooth bromegrass. These clones are a potential tool for conducting animal feeding trials using forages with relatively unconfounded differences in a single cell wall characteristic.

Relationship Between Ultrastructural Topochemistry of Lignin and Wood Properties

The main subunits of lignin could be visualized by transmission electron microscopy (TEM) with antibodies raised against synthetic lignin model polymers. Thus, immunological probes against p-hydroxyphenyl propane, guaiacyl and mixed guaiacyl-syringyl units allowed to specifically localize the qualitative distribution of lignins in plant cell tissues . Depending on the mode of preparation of the synthetic lignin antigens , the corresponding antibodies showed specificity for condensed or noncondensed interunits linkages . This specificity is illustrated with the different labellings provided by the antibodies when applied to various wood and nonwoody materials . The results c1early show the heterogeneity of lignification between tissues but also demonstrate the microheterogeneity of lignin deposition within a a single wood cell wall. Our immunological markers were successfully applied to transgenic plants in which lignin synthesis pathways had been modified, to tissues from reaction wood, as well as to materials degraded by ligninolytic fungi.

Specific cell wall proteins confer resistance to nisin upon yeast cells.

The cell wall of a yeast cell forms a barrier for various proteinaceous and nonproteinaceous molecules. Nisin, a small polypeptide and a well-known preservative active against gram-positive bacteria, was tested with wild-type Saccharomyces cerevisiae. This peptide had no effect on intact cells. However, removal of the cell wall facilitated access of nisin to the membrane and led to cell rupture. The roles of individual components of the cell wall in protection against nisin were studied by using synchronized cultures. Variation in nisin sensitivity was observed during the cell cycle. In the S phase, which is the phase in the cell cycle in which the permeability of the yeast wall to fluorescein isothiocyanate dextrans is highest, the cells were most sensitive to nisin. In contrast, the cells were most resistant to nisin after a peak in expression of the mRNA of cell wall protein 2 (Cwp2p), which coincided with the G2 phase of the cell cycle. A mutant lacking Cwp2p has been shown to be more sensitive to cell wall-interfering compounds and Zymolyase (J. M. Van der Vaart, L. H. Caro, J. W. Chapman, F. M. Klis, and C. T. Verrips, J. Bacteriol. 177:3104-3110, 1995). Here we show that of the single cell wall protein knockouts, a Cwp2p-deficient mutant is most sensitive to nisin. A mutant with a double knockout of Cwp1p and Cwp2p is hypersensitive to the peptide. Finally, in yeast mutants with impaired cell wall structure, expression of both CWP1 and CWP2 was modified. We concluded that Cwp2p plays a prominent role in protection of cells against antimicrobial peptides, such as nisin, and that Cwp1p and Cwp2p play a key role in the formation of a normal cell wall.

Structural Changes of Zoospores During and After Net Formation in Hydrodictyon reticulatum

Changes in the cell structure during and after net formation of zoospores in Hydrodictyon reticulatum were investigated by electron and fluorescence microscopy. Many small vesicles were accumulated around zoospores, and these vesicles later restricted the movement of zoospores. Immediately after cessation of the movement of polyhedral zoospores, the plasma membrane evaginated to form thread-like projections around the closely approaching regions of adacent zoospores. Then the projections seemed to fuse with the adjacent zoospores. Once the connections between the adacent cells were formed by the proections, the contact between two zoospores became progressively tighter. Cell wall materials were deposited on the plasma membrane about 10 minutes after cessation of the cell movement. Cell wall materials appeared firstly as electron-dense discontinuous patches on the plasma membrane. The patches then expanded to connect with one another, finally forming a single electron-dense cell wall layer which covered the cell surface. Netcells were finally linked by their cell walls. Cell wall substances continued to be accumulated in the space between the electron-dense cell wall layer and the plasma membrane.

Infrared microspectroscopy: Sampling heterogeneity in plant cell wall composition and architecture

The use of probes such as monoclonal and polyclonal antibodies to specific cell wall components, at both the light and electron microscope levels, has demonstrated the diversity in cell wall composition between species, between tissues, between different regions of the cell surface, and even within a single wall. Traditional methods of cell wall analysis have provided valuable information on wall composition and architecture, but, by having to rely on the use of bulk samples, have averaged out this intrinsic heterogeneity. Fourier Transform Infrared (FTIR) microspectroscopy addresses this problem by providing chemical information from an area as small as 10×10 μm of a single cell wall fragment or area of a tissue section that has been imaged with a microscope accessory.We have used FTIR microspectroscopy as a powerful and extremely rapid assay for wall components and putative cross‐links in muro. The spectra are sensitive to polymer conformation, and the use of polarisers in the microscope accessory allows the orientation of particular functional groups to be determined, with respect to the long axis of elongating cells. The spectra constitute species and tissue‐specific ‘fingerprints’, and the use of classical discriminant analysis may provide the opportunity for correlating spectral features with chemical, architectural or rheological wall properties. Spectral mapping of an area of a specimen allows the morphological features resulting from cell growth and differentiation to be characterised chemically at the single cell level.

Role of the ABC transporter Ste6 in cell fusion during yeast conjugation.

Though early stages of yeast conjugation are well-mimicked by treatment with pheromones, the final degradation of the cell wall and membrane fusion of mating that leads to cytoplasmic mixing may require separate signals. Mutations that blocked cell fusion during mating in Saccharomyces cerevisiae were identified in a multipartite screen. The three tightest mutations proved to be partial-function alleles of the ABC-transporter gene STE6 required for transport of a-factor. The ste6(cefl-1) allele was recovered and sequenced. The ste6(cefl-1) allele contained a stop codon predicted to truncate Ste6 at amino acid residue 862 (of 1290). The ste6(cef) mutations reduced, but did not eliminate, expression of a-factor. Light and electron microscopy revealed that unlike ste6 null mutations which block mating before the formation of mating pairs, the ste6(cef) (cell fusion) alleles permitted early steps in mating to proceed normally but blocked at a late stage in conjugation where mating partners were encased by a single cell wall and separated by only a thin layer of cell wall material we term the fusion wall. Morphologically the prezygotes appeared symmetrical with successful cell wall fusion at the periphery of the region of cell contact. Responses to a-factor were efficiently induced in partner cells under mating conditions as expected given the symmetric appearance of the prezygotes. A strain expressing a ste6(K1093A) mutation that conferred export of a twofold to fourfold higher level of a-factor than ste6(cef) did not accumulate prezygotes during mating which could indicate a tight threshold of a-factor signaling required for mating. However, mating to an sst2 partner which has a greatly increased sensitivity to a-factor did not suppress the fusion defect of a ste6(cef) strain. Overexpression of the structural gene for a-factor also did not suppress the fusion defect. It is possible that a-factor or STE6 play more complex roles in cell fusion.

Old and new ways to probe plant cell-wall architecture

Wall structure has been analysed by a process of careful demolition, in which chemical extradants are used to remove specific polymers for sugar and linkage analysis, gel-permeation or ion-exchange chromatography, and nuclear magnetic resonance spectroscopy. Sequence-dependent endoglycanases cleave certain polysaccharides into oligomers that can be sequenced completely and give a clear picture of the repetitive units used to make fundamental polymers. We have also developed and adapted new chemical procedures and pulse-labelling techniques to give more information on the ways that wall polymers are subtly modified during growth. In this report, we review these conventional means of carbohydrate analyses together with newer methods of selective enzymic hydrolysis, separation of large oligosaccharides by high pH anion-exchange chromatography, and detection of molecular mass of several thousand daltons by electrospray mass spectrometry. These new technologies have already given much valuable information about the polymeric building blocks, but little information on how these polymers are arranged in space. For this, we adapted new cryopreservation techniques for electron microscopy that can image the wall in as close to the in vivo state as possible. In addition to defining anomeric linkages and linkage structures in preparations of native polymers, nuclear magnetic resonance spectroscopy can also determine the relative mobility of particular polymers within the structure of hydrated cell walls. The generation of antibodies to particular cell wall epitopes has enabled us to define architectural differences among species, among tissue types, and even among domains within a single wall. Our awareness of the diversity and complexity of primary cell wall architecture has driven a search for methodologies such as Fourier transform infrared and Fourier transform Raman microspectroscopies, which are suitable for analysis at the single cell wall level. Key words: cell walls, polysaccharides, gas – liquid chromatography – mass spectrometry, Fourier transform infrared microspectroscopy, nuclear magnetic resonance spectroscopy, immunocytochemistry.

Changes in cell wall architecture during cell elongation

Abstract A model of the plant cell wall of dicotyledons and non-graminaceous monocotyledons contains two principal structural polysaccharide networks: a stretch-resistant load-bearing cellulose/xyloglucan network, and a compression-resistant pectic polysaccharide network. Structural modifications to either polysaccharide network are likely to have profound consequences for cell wall properties, including tensile strength and extensibility. We have described dynamic changes in the pattern of esterification of pectic polysaccharides that correlate with cell growth in both carrot and tobacco suspension cells. During elongation, cellulose microfibrils become oriented transversely with respect to the long axis of the cell. Other matrix polymers such as pectin and protein also acquire a net orientation, but in a tissue-specific manner. Preliminary results from Zinnia mesophyll cells suggest that some wall modifications such as the removal of fucose from xyloglucan may be developmentally regulated, but are not required for elongation per se. Our observations lead us to propose a tentative model for the molecular rearrangements that occur within these walls during cell elongation. Our use of methodologies which are suitable for analysis at the single cell wall level has demonstrated that the apoplastic continuum is a mosaic of different architectures, with major architectural differences between species, between tissues, and between domains within a single wall, and we discuss the implications of this heterogeneity for our model.

Changes in pectin structure and localization during the growth of unadapted and NaCl‐adapted tobacco cells

SummaryTobacco cells adapted to grow in high concentrations of NaCl exhibit a drastically altered growth physiology that results in cells whose fully expanded volume is only one‐fifth to one‐eighth those of unadapted cells. Comparison between NaCl‐adapted and unadapted tobacco cells provides an opportunity to evaluate current concepts of the structural and mechanical determinants of cell wall expansion. Both biochemical studies of pectic polymers and the ultrastructural localization of pectic epitopes at three specific phases of cell culture, maximal cell division, maximal elongation, and stationary phase are reported here.One‐half of the galactosyluronic acid units in wall polymers of NaCl‐adapted cells are esterified throughout the culture period, while wall polymers of unadapted cells show a rise in esterified polygalacturonic acid from 50 to 80% during elongation and then a decrease to 70% at stationary phase. Methyl esters account for only a proportion of the total esterified polygalacturonic acid at any stage in both unadapted and NaCl‐adapted cell walls. Using monoclonal antibodies, we show differences in the localization of relatively methyl‐esterified and unesterified pectic epitopes at different stages of growth and corroborate the chemical determinations. Fourier transform infrared (FTIR) microspectroscopy of representative walls of both NaCl‐adapted and unadapted cells confirms, at the single cell wall level, that results obtained from chemical analysis of bulk samples are applicable to the entire cell population.FTIR microspectroscopy also reveals an increase in wall protein in the walls of adapted cells. Images obtained by the fast‐freeze, deep‐etch, rotary‐shadowed replica technique show clearly different cell wall architectures in NaCl‐adapted compared with unadapted cells; walls of elongating unadapted cells contain long, thin fibres that show a net orientation with respect to the long axis of the cell, whereas walls of adapted cells have thicker, flatter bundles of fibres with no clear net orientation. Polarized FTIR microspectroscopy indicates that, in unadapted tobacco cells during elongation, pectin molecules may be oriented within the wall in a similar manner to cellulose. Possible ways in which pectin structure and conformation may affect the behaviour of the cellulose‐xyloglucan network are discussed.

Silicification of wood-cell walls

Cellular organic structures such as wood can be used as scaffolds for the synthesis of complex structures of organic/ceramic nanocomposites. The wood cell is a fiber-reinforced resin composite of cellulose fibers in a lignin matrix. A single cell wall, containing several layers of different fiber orientations and lignin content, is separated from its neighboring wall by the middle lamella, a lignin-rich region. In order to achieve total mineralization, deposition on and in the cell wall must be achieved. Geological fossilization of wood occurs as permineralization (filling the void spaces with mineral) and petrifaction (mineralizing the cell wall as the organic component decays) through infiltration of wood with inorganics after growth. Conversely, living plants can incorporate inorganics into their cells and in some cases into the cell walls during growth. In a recent study, we mimicked geological fossilization by infiltrating inorganic precursors into wood cells in order to enhance the properties of wood. In the current work, we use electron microscopy to examine the structure of silica formed in the cell walls after infiltration of tetraethoxysilane (TEOS).

Orientation of macromolecules in the walls of elongating carrot cells

When round cells from a carrot cell suspension culture are diluted into fresh medium without auxin, the cells elongate to almost 50 times their original diameter within three days. This process of elongation is accompanied by changes in both the composition and the orientation of cell wall polymers. We have obtained information on the orientation of wall polymers in elongating cells by two complementary techniques, one using microscopy and one spectroscopy. Images obtained by the fast-freeze, deep-etch, rotary-shadowed replica technique show that walls of round carrot cells have no net orientation of cellulose microfibrils, and that many thin fibres can be seen cross-linking microfibrils. Walls of elongated carrot cells, in contrast, show a marked net orientation of microfibrils at right angles to the axis of elongation. Fourier Transform Infrared (FTIR) spectra obtained from defined areas of single cell walls show that walls of round carrot cells contain more protein, esters and phenolics in a given area (10 microns x 10 microns) than walls of elongated carrot cells, that contain proportionally more carbohydrate. The orientation of particular functional groups, with respect to the direction of elongation of the cell, can be determined by inserting a polariser into the path of the infrared beam, before it passes through a cell wall sample mounted on the stage of the microscope accessory. In the walls of elongated cells, ester bands, amide bands characteristic of proteins, and stretching frequencies in the carbohydrate region of the spectrum all show a net orientation transverse to the long axis of the cells. In the walls of round carrot cells, however, there is no such net orientation of polymers. Spectra obtained from 25 microns-thick fresh sections of the etiolated stem of a carrot seedling show that different wall components are polarised in different tissue types. These techniques have therefore enabled us to define differences in both the composition and the architecture of walls of elongating cells at the level of a single cell, and to suggest that polymers not previously thought to be ordered, such as pectin and protein, are strictly oriented in some wall types.

Transport of carbon and phosphorus compounds about Sphagnum

The bog mosses, Sphagnum , lack any obvious anatomical specialization inside the stem but have a well-developed system of water conduction in capillary spaces among pendent branches around the stem. It has hitherto been assumed that this was the main route for solute transfer too. We describe experiments showing that there is rapid and quantitatively important transport within the stems. The tracers 32 P and 14 C were supplied to S. recurvum . When applied below the top of the plant they moved to the apex whether external mass flow was upward or downward. Autoradiographs showed high concentration of tracers in the stem. Steaming the stem above and below the site of application prevented tracer movement. An experiment lasting four weeks on S. papillosum showed that after 14 C labelling almost all that part of the alcohol-soluble fraction that moved did so from older parts to the apex, with very little transfer to the insoluble fraction, to neighbours, or into the gas phase. For the soluble fraction in the capitulum, the conductance for 14 C from below was about the same as that of its loss in respiration. The conductance to the insoluble fraction was about twice as great, and to neighbours about half as much. A longer experiment showed that predominately acropetal transport continued for at least 22 weeks in five species. By that time about 25% of the remaining 14 C label was incorporated in the new tissues. The stem of S. recurvum contains a central mass of parenchyma 20-50 cells across. The end walls of the parenchyma cells have perforations approximately 100 nm across at a density of 7-13 μm -2 . A single cell wall has about 1500 perforations. They probably house plasmodesmata. These results indicate that Sphagnum has an effective mechanism for retaining and relocating solutes within it. This property, coupled with the ability to grow in a very low supply of solutes, and to make the environment acid by cation exchange, may be seen as causes of the widespread success of Sphagnum .

Modeling and simulation of viscoelastic behavior (tensile strain) of wood under moisture change

A hypothesis was proposed on the mechanism of the characteristic viscoelastic behavior of wood under moisture change. The hypothesis was based on the inference in which the characteristic behavior might be attributed to the looseness of the interface between the S1 and S2 layers in a cell wall. A mechanical model representing the behavior of a single cell wall on basis of the hypothesis and the results of simulation by the use of the mechanical model with the computer were shown in this report. The characteristics of the viscoelastic strain obtained from the simulation agreed well with those of published experimental results.

A PSYCHOACOUSTICAL THEORY OF CRISPNESS

ABSTRACTThe theory is proposed that crispness is primarily an acoustical sensation. The frequency spectra and the amplitude‐time characteristics of sounds produced by biting crisp foods were studied. The frequencies present in crisp sounds cover a broad range. Amplitude‐time plots show irregular variations in loudness with time. A model system involving a generalized cellular structure is proposed to explain the generation of a crisp sound. As a crisp cellular material is crushed, a series of sounds is produced. Each sound is the result of the rupture of a single cell or cell wall.

Structure, composition, and morphogenesis of the cell wall of Chiamydomonas reinhardi: II. Electron microscopy and optical diffraction analysis

As a prelude to in vitro experiments on the problem of assembly processes, the detailed three-dimensional architecture of the cell wall of Chlamydomonas reinhardi is being investigated. Optical diffraction techniques have been applied to electron micrographs of purified cell walls, and the Fourier transform derived from a single cell wall has been identified. Similar micrographs, subjected to photographic linear integration, or optical reconstitution after image filtering, have clarified the structures comprising the complex lattice within the wall. Optical diffraction techniques, applied to micrographs of rotary-shadowed cell walls, have enabled a correlation between the structure of the outer surface of the wall and the negatively stained wall to be established. Our results demonstrate the presence within the cell wall of a highly ordered complex glycoprotein lattice for which we have proposed a two-dimensional structural model to a resolution of about 2.5 nm. The repeating morphological unit of the model can be described as a parallelogram, composed of several discrete components, with sides of 28.5 nm and 23.6 nm and an angle of 80°. Such a highly crystalline cell wall component is at present unique among plant cells, but may be of more general occurrence as its extreme lability has made detection difficult by conventional methods.

ULTRASTRUCTURE OF SPERMATIUM LIBERATION IN THE MARINE RED ALGA PTILOTA DENSA

The differentiation of male gametes of the marine red alga Ptilota densa was studied by electron microscopy. Mature primary spermatangia are enveloped by a single cell wall and possess a clearly polar subcellular organization. The nucleus is situated apical to large, striated, fibrous vacuoles which are apparently formed by the repeated fusion of dictyosome vesicles. The transformation and liberation of spermatia from spermatangia involve both the secretion of the fibrous vacuoles at the base of the cell and the subsequent rupturing of the spermatangial cell wall. Liberated spermatia are coated with a thin mucilage layer and contain numerous small vesicles and several mitochondria and dictyosomes. The nucleus is cup-shaped and generally lacks a limiting envelope. These findings are discussed in relation to other light and electron microscopic studies of differentiating spermatangia in red algae.

ULTRASTRUCTURE OF SPERMATIUM LIBERATION IN THE MARINE RED ALGA PTILOTA DENSA1

SUMMARYThe differentiation of male gametes of the marine red alga Ptilota densa was studied by electron microscopy. Mature primary spermatangia are enveloped by a single cell wall and possess a clearly polar subcellular organization. The nucleus is situated apical to large, striated, fibrous vacuoles which are apparently formed by the repeated fusion of dictyosome vesicles. The transformation and liberation of spermatia from spermatangia involve both the secretion of the fibrous vacuoles at the base of the cell and the subsequent rupturing of the spermatangial cell wall. Liberated spermatia are coated with a thin mucilage layer and contain numerous small vesicles and several mitochondria and dictyosomes. The nucleus is cup‐shaped and generally lacks a limiting envelope. These findings are discussed in relation to other light and electron microscopic studies of differentiating spermatangia in red algae.

Demonstration by freeze-etching of a single cleavage plane in the cell wall of a gram-negative bacterium.

In the examination of protoplasts of a gram-negative bacterium classified as a Pseudomonas sp. by freeze-etching, we found a smooth external surface which is not seen if the preparations are not "etched." This external structure is seen as a sleeve surrounding and connecting the cells in unetched preparations, and we present evidence that it is a eutectic formed during the freezing of the specimen. In the system used in this study, the four layers of the cell wall of a gram-negative bacterium can be removed from the cell. The single cell wall cleavage plane is not affected by the removal of the loosely bound outer layer or of the peptidoglycan layer, but it is lost when the outer double track layer and the underlying soluble layer are simultaneously removed. Thus, we conclude that it is one of these two layers which is responsible for the cleavage plane which exposes variable areas of a smooth surface in the cell wall. This cell wall cleavage plane is more likely to deflect the actual cleavage of the frozen cell when cells are relatively old or when they are suspended in sucrose.