Separation of vascular cell walls from cortical cell walls of plant roots

Summary The cortex was physically separated from the stele of corn roots. The isolated walls from cortical cells were less dense than the walls isolated from stelar cells. The cell walls from each tissue were centrifuged on a step gradient composed of 50 and 60% sucrose for 5 min at 900 g. After the short centrifugation time, the cortical cell walls banded at the 50/60% interface while the vascular tissue walls pelleted through 60% sucrose. An aliquot of vascular cell walls was then marked with cytochromec. The marked cell walls were mixed with cortical cell walls and centrifuged on a 50/60% sucrose gradient and after 5 min, the vascular tissue walls were cleanly separated from the cortical cell walls. The experiment was repeated without prior separation of tissue types with another corn variety, carrot roots grown in culture, and pea roots. A clean separation of cell wall types was achieved after homogenization of intact roots in liquid nitrogen, extrusion from a nitrogen bomb, and centrifugation in sucrose gradients.     

Taxonomic significance of cellulosic cell walls in the bangiales (Rhodophyta)

Cellulose has been characterized from isolated cell walls of the conchocelis phases of both Porphyra umbilicalis and P. leucostricta. Evidence for cellulose II (regenerated cellulose) in Schweitzer's reagent extracts was provided by X-ray powder analysis and paper chromatography of partial hydrolyzates. The presence of cellulose in the conchocelis phase of species of Porphyra provides evidence for the continuity of cell wall composition within the Rhodophyta. Adoption of a classification scheme incorporating consolidation of all red algal orders under the single class Rhodophyceae is proposed.     

Fractionation of hemicelluloses from maize cell walls with increasing concentrations of alkali

Hemicelluloses were solubilized from depectinated walls of maize coleoptiles and leaves with increasing concentrations of alkali to yield three major fractions of polymers. A highly-substituted glucuronoarabinoxylan released by dilute alkali from walls of coleoptiles was present only in very small amounts in the walls of the leaves. The stepwise extractions with increasing concentrations of alkali resolved a relatively unbranched xylan from a mixture of mixed-linked glucan, xyloglucan and additional xylan from walls of young leaves. Delignification in acidic sodium chlorite solubilized a small amount of substituted xylan from walls of both coleoptiles and leaves, and rendered about one-half of the unextracted hemicellulose soluble in only 0.02 M potassium hydroxide solution. Delignification prevented the detection of highly-substituted xylans released by dilute alkali.     

Aromatic aldehyde constituents of graminaceous cell walls

p-Hydroxybenzaldehyde, vanillin and syringaldehyde were released as their sodium salts from graminaceous cell walls by treatment with sodium hydroxide. Treatment of the walls with ‘cellulase’ having both cellulase and hemicellulase activity released the aldehydes in bound form apparently linked at their phenolic groups to the wall polysaccharides. These findings are discussed in relation to tests for lignin using phloroglucinol-HC1 and alkaline nitrobenzene reagents.     

Phenolic components and degradability of cell walls of grass and legume species

Cell walls separated from the aerial parts of Lolium multiflorum, Lolium perenne and Phleum pratense contained bound cis and trans ferulic and p-coumaric acids and diferulic acid which were released from the walls by treatment with sodium hydroxide. The total content of these acids in L. multiflorum ranged from 5 to 16.8 mg/g of wall, the trans-ferulic acid content varying between 2.8 and 8.9 mg/g of wall. In addition, small amounts of p-hydroxybenzoic acid were released from senescent leaf blade plus sheath parts. Cell walls from legume species gave much smaller amounts of the acids, the total content of aerial parts of Trifolium pratense being <0.8 mg/g of wall. The degra dability of the cell walls with a commercial cellulase preparation was determined and the water-soluble phenolic compounds released were estimated by UV absorption spectroscopy.     

Separation of macromolecular components of plant cell walls: electrophoretic methods

Qualitative quantitative and preparative electrophoretic methods of separating polymeric substances derived from plant cell walls are described. Analyt     

Promotion of tick cell growth by proline and fractions from tick eggs

Tick cell lines representing three genera (Rhipicephalus appendiculatus, Dermacentor variabilis and Boophilus microplus) were grown in basal medium containing different supplements. Their effect on cell growth was measured by determining the rate at which cultures were transferred. The optimal concentration of the foetal bovine serum was 5% for the Boophilus line and 10% for the other two. Addition.of tick egg extract (TEE) increased the splitting rate 1.5–2.4 times during a period of 30 days, and changed the morphology of some cells. The crude TEE was delipidized and the protein and lipid fractions were analyzed for biological activity. The delipidized protein fraction of the TEE improved the splitting rate to about the same degree as the crude TEE. Adding l-proline to the basal medium increased the splitting rate 1.2–1.9-fold, or nearly to the same degree as did the crude TEE. The addition of the lipid and aqueous polar fractions of TEE did not improve cell growth.     

Chemical and enzymatic extraction of heavy metal binding polymers from isolated cell walls of Saccharomyces cerevisiae

Isolated cell walls of the yeast Saccharomyces cerevisiae were treated by either chemical (alkali and acid) or enzymatic (protease, mannanase or beta-glucuronidase) processes to yield partially purified products. These products were partially characterized by infrared analysis. They were subsequently reacted with heavy metal cation solutions and the quantity of metal accumulated by the cell wall material determined. The Cu(2+) ion (0.24, 0.36, 1.12, and 0.60 mumol/mg) was accumulated to a greater extent than either Co(2+) (0.13, 0.32, 0.43, and 0.32 mumol/mg) or Cd(2+) (0.17, 0.34, 0.39, and 0.32 mumol/mg) by yeast cell walls, glucan, mannan, and chitin, respectively The isolated components each accumulated greater quantities of the cations than the intact cell wall. Removal of the protein component of the yeast cell walls by Pronase caused a 29.5% decrease in metal accumulation by yeast cell walls per mass, indicating the protein is a heavy metal accumulating component. The data indicate that the outer mannan-protein layer of the yeast cell wall is more important than the inner glucan-chitin layer in heavy metal action accumulation. (c) 1994 John Wiley & Sons, Inc.     

Self-assembly of plant cell walls

The object of this paper is to define criteria for distinguishing between self-assembly and template-based assembly in plant cell walls. The example of cellulose shows that cell wall polymers biosynthesized at a membrane may retain parallel chain packing arrangements that are thermodynamically unstable and cannot be reproduced in vitro, making the experimental testing of the self-assembly hypothesis difficult. Also, natural cellulose is ordered on a number of scales of pattern, each of which may be constructed by either self- or template-based assembly independently of the rest. These conceptual problems apply equally to the self-assembly of complete cell walls and other cell wall polymers. It is suggested that the self-assembly concept should be applied only to one stage or level in the synthesis of a cell wall, and that an additional concept of parallel assembly may be useful for understanding the synthesis of some polysaccharides.     

Diferulic acid as a component of cell walls of Lolium multiflorum

Trans,trans-, cis,trans- and cis,cis-diferulic acids were released from cell walls of Lolium multiflorum by treatment with sodium hydroxide. The isomers were apparently bound via ester links to the structural carbohydrates of the cell walls. Sodium hydroxide treatment gave, per g of wall, 0.18 mg trans,trans-diferulic, 0.02 mg cis,trans-diferulic and a trace of cis,cis-diferulic acids compared with 5.3 mg trans-ferulic, 1.2 mg cis-ferulic, 0.78 mg trans-p-coumaric and 0.12 mg cis-p-coumaric acids. The significance of these acids in lignin biosynthesis is discussed. The effect of UV light on the trans,trans isomer and its fully silylated trimethylsilyl either derivative was also investigated.     

Safranine fluorescent staining of wood cell walls

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.     

Chemical composition of the cell walls of Lolium multiflorum endosperm

Cell walls isolated from Lolium multiflorum endosperm grown in liquid suspension culture contain 90% carbohydrate (as anhydro-glucose), 0·3 nitrogen, 1·9% lipid and 4·3% ash. The relative proportions of neutral sugars present in hydrolysates of the wall polysaccharides are glucose, 50%; arabinose, 19%; xylose, 26% and galactose, 5%. Extraction of the wall with 7 M urea solubilizes a polysaccharide representing 19% of the wall and composed of glucose and minor amounts of pentoses. This fraction has been examined by acid and enzymic hydrolysis and by periodate oxidation, and was shown to be a β-1,3; 1,4-glucan with approx. 79% 1,4-linkages. A specific β-glucan hydrolase has been used to determine the content of this mixed-linked glucan in isolated endosperm cell walls.     

The charophycean green algae provide insights into the early origins of plant cell walls

Numerous evolutionary innovations were required to enable freshwater green algae to colonize terrestrial habitats and thereby initiate the evolution of land plants (embryophytes). These adaptations probably included changes in cell-wall composition and architecture that were to become essential for embryophyte development and radiation. However, it is not known to what extent the polymers that are characteristic of embryophyte cell walls, including pectins, hemicelluloses, glycoproteins and lignin, evolved in response to the demands of the terrestrial environment or whether they pre-existed in their algal ancestors. Here we show that members of the advanced charophycean green algae (CGA), including the Charales, Coleochaetales and Zygnematales, but not basal CGA (Klebsormidiales and Chlorokybales), have cell walls that are comparable in several respects to the primary walls of embryophytes. Moreover, we provide both chemical and immunocytochemical evidence that selected Coleochaete species have cell walls that contain small amounts of lignin or lignin-like polymers derived from radical coupling of hydroxycinnamyl alcohols. Thus, the ability to synthesize many of the components that characterize extant embryophyte walls evolved during divergence within CGA. Our study provides new insight into the evolutionary window during which the structurally complex walls of embryophytes originated, and the significance of the advanced CGA during these events.     

Neutral and Amino sugars from the cell walls of Oomycetes

A survey of the wall sugars of 41 species of Oomycetes has been carried out to indicate whether they could provide information of taxonomic significance. These species contain one or more glucans with β1→3, β1→6 or β1→4 linkages. No evidence for a β1→2 linkages was obtained. Apart from the possible exceptions of mannose and galactose, neutral sugars appear to be similar at family, genus and species levels within the Oomycetes. Amino sugar levels were more distinctive. Amounts of hexosamine were greater in Achlya, Apodachlya and Leptomitus than in Pythium, Phytophthora or Sapromyces; amounts in Saprolegnia were intermediate. The data support the view based on other criteria that the two families of Leptomitales are only distantly related.     

Incorporation of proline and aromatic amino acids into cell walls of maize coleoptiles

Sections excised from maize coleoptiles incorporated radioactivity from proline, tyrosine, and phenylalanine into structural components of the cell wall. Only about 2% of radioactivity from proline taken up by sections was incorporated into cell wall; about 24% of that incorporated was in hydroxyproline and the rest remained in proline. In contrast, as much as 40% of the radioactivity from phenylalanine and 30% from tyrosine was incorporated into cell wall material. Most of this radioactivity was in saponifiable ferulic acid. Small amounts of p-coumaric and diferulic acid were found, but only a small fraction of the hemicellulose can possibly be immobilized directly through cross-linking of diferulic esters. Substantial amounts of radioactivity from aromatic amino acids remained insoluble after strong alkali extractions of wall material, and a large fraction of polysaccharide was solubilized by dilute alkali following oxidation of phenolics by acidic NaClO₂. Hence, hemicellulosic material in the cell walls of maize coleoptiles may be organized and cross-linked primarily through alkali-resistant etherified aromatics.     

Freezing stresses and hydration of isolated cell walls

The hydration of the cell walls of the giant alga Chara australis was measured as a function of temperature using quantitative deuterium nuclear magnetic resonance (NMR) of samples hydrated with D2O. At temperatures 23-5K below freezing, the hydration ratio (the ratio of mass of unfrozen water in microscopic phases in the cell wall to the dry mass) increases slowly with increasing temperature from about 0.2 to 0.4. It then rises rapidly with temperature in the few Kelvin below the freezing temperature. The linewidth of the NMR signal varies approximately linearly with the reciprocal of the hydration ratio, and with the freezing point depression or water potential. These empirical relations may be useful in estimating cell wall water contents in heterogeneous samples.     

Sequential solvent extraction and structural characterization of polysaccharides from the endosperm cell walls of barley grown in different environments

The objective of this study was to examine the composition and molecular structure of the endosperm cell walls (CW) derived from barley grain grown in three environments in Canada, and differing in grain hardness, protein, and total β-glucan contents. The endosperm CW were isolated from barley, cv. Metcalfe, grown in Davidson, SK (Sample A), Hythe, AB (sample B), and Hamiota, MB (sample C). The CW were sequentially extracted with water at 65 ^oC, saturated Ba(OH)₂, again with water at 25 ^oC, and 1 M NaOH, resulting in fractions designated WE65, BaE, Ba/WE, and NaE, respectively. The monosaccharide analysis indicated the presence of β-glucans, arabinoxylans, and small amounts of arabinogalactans, glucomannans, and xyloglucans. Cellulose was detected in the CW remnants. The CW of sample A, exhibiting a lower grain hardness than sample B, contained the lowest amount of β-glucans, but the highest amount of arabinoxylans and the mannose-containing polysaccharides. The CW of sample C, characterized by very high protein content in the grain, contained the highest amount of β-glucans and the lowest amount of other polysaccharides. Polysaccharides in the CW of sample B, exhibiting the highest grain hardness, were characterized by the highest weight average molecular weights (M_w). β-Glucans in the CW of Sample B showed the highest ratio of DP3/DP4 and the longest cellulosic fragments in the polymeric chains. Of the three barley samples, arabinoxylans in the endosperm CW of sample A exhibited the lowest degree of branching, the highest amount of unsubstituted Xyl residues, and the highest ratio of singly to doubly substituted Xylp. The highest water solubility of the CW of sample C was associated with the highest concentration of β-glucans, the lowest DP3/DP4 ratio, and the lowest M_w of the polymeric constituents. Arabinoxylans with the lowest amount of doubly substituted but the highest amount of unsubstituted xylose residues and long sequences of unsubstituted xylan regions were found in the NaE fractions. The NaE fractions showed a high ratio of →4)-Glcp-(1→ to →3)-Glcp-(1→ linkages and some →4)-Manp-(1→ linkages, indicating a high level of long cellulosic regions in β-glucan chains and the presence of glucomannans.     

Polysaccharide from cell walls of Chlamydomonas reinhardtii

The cell wall fraction of the green alga Chlamydomonas reinhardtii contained about 25% carbohydrate after prolonged treatment with salivary amylase     

Tunicamycin-induced growth and inhibition of glucosamine incorporation into cell walls of rice coleoptiles

Tunicamycin, 0.25 to 2.5 μ M , promotes elongation of rice coleoptile sections after a 2 h lag. Tunicamycin decreased the minimum stress-relaxation time of the cell wall, T₀; and the wall loosening is recognized as the cause of this growth promotion. Bacitracin did not have significant effects on growth or T₀ except for inhibition of elongation at high concentration. Coleoptile sections were incubated with [¹⁴C]-glucosamine, and the synthetic pathway of the hexosamine-containing cell wall component was examined by a pulse-chase experiment. This component seems to be synthesized in the particulate fraction and secreted mainly into the hemicellulose I fraction. Tunicamycin strongly inhibited glucosamine incorporation into the particulate fraction and stopped the labeling of the cell wall. At 2.5 μ M , tunicamycin had no effect on incorporation of mannose, leucine or proline. These results suggest that the hexosamine-containing wall component is a kind of asparagine-linked glycoprotein, and that this component plays a principal role in formation of the cell wall network and in growth regulation.     

Attachment of phenoloxidase to fungal cell walls in arthropod immunity

In crayfish, phenoloxidase was located in the hemocytes. The plasma had infinitesimal enzyme activity. A phenoloxidase preparation from hemocytes precipitated spontaneously after approximately 1.5 hr at 22°C, which became attached spontaneously to glass, Plexiglas, and polystyrene plastic. The enzyme preparation could also become attached to Saccharomyces cerevisiae cell walls. Attachment was mediated by a proteinaceous substance, since trypsin significantly decreased the degree of attachment. Calcium ions were also necessary for attachment. A β-1,3-glucan, laminaran, partially prevented attachment to the fungal cell walls. Heparin caused precipitation of the phenoloxidase preparation from hemocytes. In crayfish cuticle, proteins with associated phenoloxidase activity were attached to cell walls of Aphanomyces astaci as well as to those of S. cerevisiae.