Chemical Composition of Hypodermal and Endodermal Cell Walls and Xylem Vessels Isolated from Clivia miniata (Identification of the Biopolymers Lignin and Suberin)

The occurrence of the biopolymers lignin and suberin was investigated with hypodermal (HCW) and endodermal cell walls (ECW) and xylem vessels (XV) isolated from Clivia miniata Reg. roots. Both biopolymers were detected in HCW and ECW, whereas in XV, typical aliphatic suberin monomers were missing and only representative lignin monomers such as guaiacyl (G) and syringyl (S) units could be detected. The absolute amounts of lignin were about one order of magnitude higher compared with suberin in both HCW and ECW. The ratios of the two aromatic lignin units (G/S) decreased from 39 in XV and 10 in HCW to about 1 in ECW, indicating significant differences in lignin structure and function between the three investigated samples. Additionally, compared with the detectable lignin-derived aromatic units G and S, significantly higher amounts of esterified p-coumaric acid-derived aromatic monomers were obtained with HCW, but not with ECW. This is interpreted as a functional adaption of HCW toward pathogen defense at the root/soil interface. The final aim of this study was to provide a thorough chemical characterization of the composition of HCW, ECW, and XV, which in turn will form the basis for a better understanding of the relevant barriers toward the passive, radial, and apoplastic diffusion of solutes from the soil across the root cortex into the root cylinder.     

Modifications of Cortical Cell Walls in Roots of Seedless Vascular Plants

Forty-three species of seedless vascular plants were assessed for modifications to root cortical cell walls. All species except Lycopodium had an endodermis with distinct Casparian bands. Experiments with the apoplastic tracer berberine hemisulfate showed that walls of all root cortical cells in the two Lycopodium species tested were permeable to this tracer. Although most species examined lacked a hypodermis several Equisetum species had a hypodermis with modified walls. Three Selaginella species had distinct Casparian bands in this cortical cell layer. This layer, therefore, is an exodermis in Selaginella and its presence limited the inward diffusion of the apoplastic tracer berberine hemisulfate.     

Chemical Composition of the Cell Walls of Bacillus stearothermophilus

Cell walls were isolated by mechanical disruption of mid-log phase cells of Bacillus stearothermophilus NCA 1503-4R grown in Trypticase-yeast extract-fructose medium at 55 C. The cell walls were purified by treatment with sodium dodecyl sulfate (SDS) and incubation with deoxyribonuclease and trypsin. The cell wall peptidoglycan contained glucosamine, muramic acid, alpha, epsilon-diaminopimelic acid, and glutamic acid. Low amounts of glycine, galactosamine, serine, aspartic acid, lysine, and valine were also present. The relative mole ratios of glutamic acid-alpha, epsilon-diaminopimelic acid-glycine-alanine were 1.00:1.26:0.08:1.55. The cell walls were free from ribonucleic acid and deoxyribonucleic acid and contained less than 0.2% chloroform-methanol extractable lipid and 0.09 mumole of phosphorus per mg of cell wall. Teichoic acid was not detected in the cell walls of this organism. Cell walls isolated without treatment with SDS contained 7.5% chloroform-methanol extractable lipid, 0.24 mumole of phosphorus per mg of cell wall, and relatively high concentrations of all amino acids. These results suggest that the extracted lipid is not a cell wall component per se, but a contaminant from the lipoprotein cell membrane.     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Determination of Subunit Composition of Clostridium cellulovorans Cellulosomes That Degrade Plant Cell Walls

Clostridium cellulovorans produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of C. cellulovorans and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.     

Preparation and Chemical Composition of the Cell Walls of Mature Infectious Dense Forms of Meningopneumonitis Organisms

Relatively large-scale production and purification of meningopneumonitis organisms was developed for chemical and immunological studies on cell walls of the infectious dense forms. By disruption of purified organisms with glass beads in a Mickle shaker, highly purified preparations of cell walls were obtained by sucrose density gradient centrifugation, enzyme digestion, and sodium dodecyl sulfate treatment. The dry-weight recovery of purified cell walls from intact organisms was about 13%. When (32)P-labeled preparations of cell walls were fractionated into acid-soluble, lipid, ribonucleic acid (RNA), deoxyribonucleic (DNA), and residual fractions, about 80% of the (32)P in cell wall preparations was recovered in the phospholipid fraction, which corresponded to about 3% of the total phospholipid in the intact organisms. About 7% of the (32)P in purified cell walls was recovered in the RNA and DNA fractions respectively, but this corresponds to only about 0.4% of the (32)P found in those fractions in intact organisms. From dry-weight determinations, it was calculated that the purified cell wall preparations contained only 0.6% total nucleic acids, and these are probably not true cell wall constituents. These cell walls contained 70 to 75% protein, corresponding to about 14% of the protein in intact organisms. Amino acid analysis of these protein showed the existence of all common amino acids, glucosamine, and galactosamine. However, no muramic acid was detected by the methods employed.     

Ion-Exchange Properties of Cell Walls Isolated from Lupine Roots

Acid–base properties of cell walls isolated from various root tissues of 7-day-old lupine seedlings and 14-day-old lupine plants grown in various media were studied. The ion-exchange capacity of root cell walls was estimated at various pH values (from 2 to 12) and constant ionic strength (10 mM). The parameters determining the qualitative and quantitative composition of cell wall ionogenic groups along the root length and in its radial direction were estimated using Gregor's model. This model fits the experimental data reasonably well. Four types of ionogenic groups were found in the cell walls: an amino group (pK _a 3), two types of carboxylic groups (pK _a 5 and 7.3, the first being the carboxylic group of galacturonic acid), and a phenolic group (pK _a 10). The number of functional groups of each type was estimated, and the corresponding ionization constant values were calculated. It is shown that the chemical composition of the ionogenic groups was constant along the root length as well as in its radial direction and did not depend on either physiological state or root nutrition, while the number of different groups varied. The content of carboxylic groups of -D-polygalacturonic acid in the root cell walls of 14-day-old plants was shown to depend on the distance from the root tip, being maximal in the zone of lateral roots. The number of these groups was 10- and 2-fold less in the central cylinder compared to that of cortex for 14-day-old plants and 7-day-old seedlings, respectively.     

Development of the Hypodermal Casparian Band in Corn and Onion Roots

A hypodermal Casparian band develops 40–50 mm from theroot tip in corn and 30–40 mm from the root tip in onion.In both plants, the endodermal Casparian band matures about20 mm closer to the root tip than the hypodermal Casparian band.Using the apoplastic fluorescent dye, Calcofluor white M2R (CFW),a permeability barrier could be distinguished in the radialwalls of the hypodermis 40–50 mm from the root tip incorn and onion. In progressively younger regions of the roots,CFW was first excluded from the outer tangential hypodermalwalls and the inner tangential epidermal walls, then the radialepidermal walls so that in very young regions only the outertangential epidermal walls were permeated. In contrast to CFW,the symplastic fluorescent dye, uranin, was translocated fromthe epidermis into the stele at all distances tested (5.0–50mm from the root tips). CFW and uranin at a concentration of0.01% proved nontoxic to corn and onion roots on the basis ofroot growth tests. Key words: Zea mays, Casparian band, Hypodermis, Allium cepa     

Cation Exchange Capacity, Chemical Composition and the Balance of Carboxylic Acids in the Floral Parts of Various Plant Species

Floral and other parts of antirrhinum, fuchsia, daffodil, andmaize have been analysed for cation-exchange capacity (CEC),major and trace-element constituents, and carboxylic acids.The two dicot species, but not the monocot species, have a gradientof CEC, Ca, and Ca/K increasing along the style and into theovary. Trace-element levels in anthers and ovaries are oftenhigher than in the adjacent filaments and styles. The floralparts of daffodil, with the exception of ovary, ovary wall,and anthers, have closely related K/Ca and citric/malic acidratios which are comparable with those for the rest of the plant.     

Dissociation of Cortical Cell Walls and Enhancement of Cellulase Activity during the Emergence of Callus from Rice Roots in the Presence of 2,4-D

The separation of cortical cells from root explants of rice(Oryza saliva L. cv. Sasanishiki) was stimulated by 2,4-D andboth the plastic and the elastic properties of cell walls increasedduring the formation of callus. These events, in particular,the separation of cortical cells, may be important for the generationof clumps of callus cells that are initiated at the interiorof root tissues, specifically around the vessels. Buffer-solublecellulase activity was significantly enhanced by treatment with2,4-D (6- to 10-fold) at the early stage of formation of rootcallus (1–2 days after the start of treatment with 2,4-D).The optimum concentration of 2,4-D and time course studies indicatedthat this enhacement was correlated with and preceded both theseparation of cortical cells and loosening of the cell wall.The enhancement of cellulase activity by 2,4-D in rice is thesuch first finding in a monocot. (Received September 24, 1992; Accepted February 18, 1993)     

Effect of Ethylenediaminetetraacetic Acid, Triton X-100, and Lysozyme on the Morphology and Chemical Composition of Isolated Cell Walls of Escherichia coli

Extraction of a partially purified preparation of cell walls from Escherichia coli with the nonionic detergent Triton X-100 removed all cytoplasmic membrane contamination but did not affect the normal morphology of the cell wall. This Triton-treated preparation, termed the “Triton-insoluble cell wall,” contained all of the protein of the cell wall but only about half of the lipopolysaccharide and one-third of the phospholipid of the cell wall. This Triton-insoluble cell wall preparation was used as a starting material in an investigation of several further treatments. Reextraction of the Triton-insoluble cell wall with either Triton X-100 or ethylenediaminetetraacetic acid (EDTA) caused no further solubilization of protein. However, when the Triton-insoluble cell wall was extracted with a combination of Triton X-100 and EDTA, about half of the protein and all of the remaining lipopolysaccharide and phospholipid were solubilized. The material which remained insoluble after this combined Triton and EDTA extraction still retained some of the morphological features of the intact cell wall. Treatment of the Triton-insoluble cell wall with lysozyme resulted in a destruction of the peptidoglycan layer as seen in the electron microscope and in a release of diaminopimelic acid from the cell wall but did not solubilize any cell wall protein. Extraction of this lysozyme-treated preparation with a combination of Triton X-100 and EDTA again solubilized about half of the cell wall protein but resulted in a drastic change in the morphology of the Triton-EDTA-insoluble material. After this treatment, the insoluble material formed lamellar structures. These results are interpreted in terms of the types of noncovalent bonds involved in maintaining the organized structure of the cell wall and suggest that the main forces involved are hydrophobic protein-protein interactions between the cell wall proteins and to a lesser degree a stabilization of protein-protein and protein-lipopolysaccharide interactions by divalent cations. A model for the structure of the E. coli cell wall is presented.     

Differential Staining for Cellulosic and Modified Plant Cell Walls

A simple method to enhance the staining of cell wall components for fluorescence microscopy is described. In stems of Nicotiana tabacum and needles of Pinus eldarica lignin, the cuticle and unsaturated lipids are indicated by a purple-red fluorescence while pectocellulosic components fluorescc pale blue.     

Cell Walls of Seed Hairs fromLygeum spartum: Ultrastructure, Composition and Mechanical Properties

The silky hairs covering the whole surface ofLygeum spartumdispersalunits are unicellular, unlignified epidermal structures whoselength varies from 5 to 17 mm. The cell walls present a stratifiedtexture after extraction of matrix material. Their compositiondepends on the geographical origin of the grasses. Polysaccharidesextracted with boiling water represent approx. 18% of the cellwall dry matter in samples harvested in Algerian arid high-plateausand only 3% in samples harvested in semi-arid coastal zones.In the latter case, hemicellulose and cellulose contents amountedto approx. 50 and 40%, respectively. Slightly lower values werefound in high-plateau samples. Cinnamic acids were detectedonly in coastal region specimens which exhibited a higher mechanicalresistance and a lower extensibility. These results supportthe existence of two distinct populations colonizing arid orsemi-arid areas respectively.Copyright 1998 Annals of BotanyCompany Cell wall; extensibility; polysaccharides;Lygeum spartum; seed hairs.     

Composition of Regenerated Cell Walls of Reverting Aureobasidium pullulans Protoplasts

Protoplasts of Aureobasidium pullulans, formed by treating normal blastospores with polysaccharide-hydrolyzing enzymes, synthesized glycans when incubated in shallow culture. The polysaccharides formed a loose, thick coating on the cells. The glycans that were formed are similar to the glycans in normal cell walls; however, they lack the branching that is normally found. This result is consistent with the findings of others who have studied the cell wall composition of reverting yeast protoplasts.     

Isolation and Chemical Structure of the Peptidoglycan of Spirillum serpens Cell Walls

The peptidoglycan layer of Spirillum serpens cell walls was isolated from intact cells after treatment with sodium dodecylsulfate and digestion with Pronase. The isolated peptidoglycan contained glucosamine, muramic acid, alanine, glutamic acid, and meso-diaminopimelic acid in the approximate molar ratio of 1:1:2:1:1. Aspartic acid and glycine were the only other amino acids found in significant quantities. N-terminal amino acid analyses of the tetrapeptide amino acids in the peptidoglycan revealed that 54% of the diaminopimelic acid molecules are involved in cross-linkage between tetrapeptides. This amount of cross-linkage is greater than that found in the peptidoglycan of previously studied cell walls of gram-negative bacteria. The polysaccharide backbone was isolated, after myxobacter AL-1 enzyme digestion of the peptidoglycan, by fractionation with ECTEOLA-cellulose and Sephadex G-100. An average length of 99 hexosamines for the polysaccharide chains was found (ratio of total hexosamines to reducing end groups).