Ion-exchange properties of the cell wall of reindeer lichen <Emphasis Type="Italic">Cladonia rangiferina</Emphasis>

Ion-exchange properties of cell walls were investigated in reindeer lichen Cladonia rangiferina (L.) F. H. Wigg. In order to isolate cell walls, we used living parts of podetia as well as young parts (four upper internodes of podetia) and old parts (from the 4th to the 8th internode). We studied functional dependences of cell wall ion-exchange capacity on pH in the range from 2 to 12 and constant ionic strength of solution equal to 10 mM. It was found that three-dimensional structure of C. rangiferina cell walls comprised three types of ionogenic groups, which determine ion-exchange properties of the cell walls. They are amino groups with pK_a of about 3, carboxyl groups with pK_a of about 7, and phenolic OH-groups with pK_a of about 10. The content of groups of each type and their ionization constants were determined, and it was shown that, in the cell walls of young parts, the content of amino groups and carboxylic groups was greater than in old parts of podetia (by 1.5 and 2.0 times, respectively). It was found that with age the content of nitrogen and the proportion of deacetylated amino groups in the cell walls changed from 34% (young parts of podetia) to 40% (old parts of podetia). It was shown that in C. rangiferina N-acetyl glucosamine and glucosamine are not the main monomers of cell wall polymers because both in thalli and in the cell walls isolated therefrom the content of total nitrogen was less than 1%.     

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.     

植物叶片氮分配及其影响因子研究进展

氮是植物生长的基本限制性因子,它的有效利用可以增加植物的适应性。叶片氮分配是指氮在植物叶片细胞各细胞结构以及游离化合物中所分配的比例。叶片氮的分配方式决定了叶片光合作用的强弱,影响叶片的坚韧程度以及化学防御强度,因此研究氮在植物叶片内的分配方式具有重要意义。阐述了叶片氮分配的方式,分析了影响叶片氮分配的生物和非生物因子(CO2,光,土壤养分),介绍了常用的叶片氮分配的研究方法,并对未来的研究进行了展望。     

Effect of nitrogen deficiency on the ion-exchange properties of cell wall polymers from wheat roots

The ion-exchange properties of cell wall polymers isolated from the roots of wheat (Triticum aestivum L.) plants grown on either nitrate-free (N-deficient) or nitrate-containing (+N) hydroponic nutrient medium have been investigated. Irrespective of the nitrogen nutrition regimen, the studied cell walls contained four types of ion-exchange groups: primary amino groups of structural proteins (pK_a < 3), carboxyl groups of polygalacturonic acid in pectin (pK _a ~4.7), carboxyl groups of hydroxycinnamic acids (pK _a ~7.3), and phenolic OH-groups of lignin (pK_a ~10.2). The quantitative ratio between these types of ion-exchange groups, the mass fraction of cell walls in the dry weight of roots, and the swelling coefficient of cell walls depended on the nitrate presence in the growing medium. Compared to the +N variant, the N-deficient variant was characterized by a 2.4 times higher content of phenolic OH-groups in cell walls and 1.24 times higher mass fraction of cell walls; at the same time, the swelling coefficient for this variant was lower by 10%. The obtained data indicate that nitrogen deficiency results in a formation of thicker root cell walls with a higher degree of polymer cross-linking that may be caused by the increased lignin content.     

Characterization of cell wall properties in needles from Scotch pine trees of various vigor

Ion-exchange properties of needle cell walls were studied on healthy and severely weakened Scotch pine (Pinus sylvestris L.) trees subjected to industrial pollutions with sulfur and heavy metals. Three types of cation-exchange groups were identified: carboxylic groups of polygalacturonic acid, carboxylic groups unrelated to polygalacturonic acid, and phenolic OH-groups. The needles of impaired trees (vigor state IV) differed from needles of healthy plants (vigor state I) by a higher coefficient of cell wall swelling, higher nitrogen content, and lower content of ion-exchanging groups in the cell wall structure. It is supposed that differences in ion-exchange capacity of cell walls could be among the causes for distribution of pine trees into several groups according to their vigor.     

Alterations of iron distribution in Arabidopsis tissues infected by Dickeya dadantii

Dickeya dadantii is a plant‐pathogenic enterobacterium responsible for plant soft rot disease in a wide range of hosts, including the model plant Arabidopsis thaliana. Iron distribution in infected A. thaliana was investigated at the cellular scale using the Perls'–diaminobenzidine–H₂O₂ (PDH) method. Iron visualization during infection reveals a loss of iron from cellular compartments and plant cell walls. During symptom progression, two distinct zones are clearly visible: a macerated zone displaying weak iron content and a healthy zone displaying strong iron content. Immunolabelling of cell wall methylated pectin shows that pectin degradation is correlated with iron release from cell walls, indicating a strong relationship between cell wall integrity and iron in plant tissues. Using a D. dadantii lipopolysaccharide antibody, we show that bacteria are restricted to the infected tissue, and that they accumulate iron in planta. In conclusion, weak iron content is strictly correlated with bacterial cell localization in the infected tissues, indicating a crucial role of this element during the interaction. This is the first report of iron localization at the cellular level during a plant–microbe interaction and shows that PDH is a method of choice in this type of investigation.     

Chitin-glucan complex in cell walls of the Peltigera aphthosa lichen

Cell walls and chitin-glucan complexes isolated from uneven-aged components of the thallus of the Peltigera aphthosa lichen were studied. The mass fraction of the cell wall and chitin-glucan complexes increased with age, but the content of nitrogen in these structures decreased with age. The basal area of the thallus was characterized by the largest mass fraction of the chitin-glucan complex from the dry mass of the thallus; the apical area, by the largest mass fraction of chitin in the complex. It was demonstrated that in P. aphthosa, the degree of deacetylation of chitin in the complex (depending on the age) was 33 and 54% in the apical and basal areas, respectively. The suggested method of functional analysis of chitin-glucan complexes for the presence of free amino groups in them can be used for studying other lichenified fungi.     

Chitin-glucan complex in cell walls of the Peltigera aphthosa lichen

Cell walls and chitin-glucan complexes isolated from uneven-aged components of the thallus of the Peltigera aphthosa lichen were studied. The mass fraction of the cell wall and chitin-glucan complexes increased with age, but the content of nitrogen in these structures decreased with age. The basal area of the thallus was characterized by the largest mass fraction of the chitin-glucan complex from the dry mass of the thallus; the apical area, by the largest mass fraction of chitin in the complex. It was demonstrated that in P. aphthosa, the degree of deacetylation of chitin in the complex (depending on the age) was 33 and 54% in the apical and basal areas, respectively. The suggested method of functional analysis of chitin-glucan complexes for the presence of free amino groups in them can be used for studying other lichenified fungi.     

Chemical basis for selectivity of metal ions by the Bacillus subtilis cell wall.

The use of equilibrium dialysis techniques established that isolated cell walls of Bacillus subtilis possess selective affinities for several cations. The binding of these cations to the cell wall was influenced by the presence of various functional groups in the peptidoglycan matrix. Selective chemical modification of the free carboxyl and amino groups showed that when amino groups were replaced by neutral, bulky, or negatively charged groups, the sites available for cation complexing generally increased. Introduction of positive charges into the wall resulted in a marked decrease in the numbers of metal binding sites and usually a decrease in the apparent association constants. Both teichoic acid and peptidoglycan contribute to the sites available for interaction with metals. Hill plots of equilibrium dialysis data suggest that metal binding to cell walls involves negative cooperativity. Competition between various metals for binding sites suggested that the cations complex with identical sites on the cell walls. When the hydrogen ion concentration was increased, the affinity of the walls for metals decreased, but the numbers of metal binding sites remained constant, suggesting that cations and protons also compete for the same sites.     

Effect of fluchloralin on protein synthesis, free amino acids and hydroxyproline content in groundnut (Arachis hypogaea L.)

Cultivar TMV-2 of groundnut plant {Arachis hypogaea L.) was grown in a nutrient solution containing fluchloralin at the rate of either 2 mg litre^-1 or 4 mg litre“¹. Protein synthesis and hydroxyproline content in the cell walls of roots, stem and leaves were determined. Free amino acids content and total ammonia in leaves and roots were also analysed. Presence of fluchloralin did not adversely affect protein synthesis. No significant effect of herbicide was observed on hydroxyproline content of a purified cell wall fraction of groundnut roots, stem and leaves. The total amount of ammonia increased in roots and leaves of plants which received the higher concentration of fluchloralin. With the exception of aspartic acid, asparagine, glutamic acid and glutamine, free amino acids content decreased considerably with herbicide treatment. Alanine and glycine were strongly reduced. It is suggested that transamination reactions could be affected and the process of senescence may be enhanced.     

Elevated cell wall serine in pleiotropic staphylococcal mutants

Korman, Ruth Z. (Cornell University, Ithaca, N.Y.). Elevated cell wall serine in pleiotropic staphylococcal mutants. J. Bacteriol. 92:762-768. 1966.-Physically purified cell walls were prepared from two staphylococcal strains and from pleiotropic variants derived from them. The quantitative amino acid and amino sugar content of these walls is reported. The pleiotypes, which are identified culturally by their failure to elaborate coagulase, their resistance to bacteriophage, and their sensitivity to mannitol, have altered molar ratios of amino acids and amino sugars in their cell walls. In comparison with lysine content, the serine content of the mutant wall is elevated and the glycine content is reduced. The glucosamine content is reduced also. It is postulated that the pleiotropic mutants possess an altered cell wall biosynthetic pathway.     

Plant biotechnology for lignocellulosic biofuel production

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin–carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall‐degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat‐stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.     

Isolation and characterisation of the homogalacturonan from type II cell walls of the commelinoid monocot wheat using HF-solvolysis

In contrast to the typical type I cell wall of the dicot plants, the type II cell wall of the commelinoid monocot plants is known to be relatively poor in pectins. Assuming a critical role for the remaining pectins in terms of cell wall architecture and/or as a reservoir of signalling molecules, we have compared different protocols for the isolation of the main pectin polymer, homogalacturonan, from wheat leaf cell walls. Pectin was detected in these cell walls immunochemically using the monoclonal antibodies JIM5 and JIM7, and biochemically by monosaccharide analysis. The Ca(++)-chelators CDTA and imidazole extracted a pectin rich fraction from isolated cell walls which was however contaminated with significant amounts of hemicelluloses. Pretreatment of the cell walls with anhydrous hydrogen fluoride at controlled low temperatures followed by HF/ether- and water-extraction prior to imidazole-extraction of pectins yielded a purer homogalacturonan fraction. The near absence of rhamnosyl residues proved that the isolated homogalacturonan fraction was free of rhamnogalacturonans. If HF-solvolysis was performed at -23 degrees C, the resulting homogalacturonan had a degree of methyl esterification identical to that of the pectins in the initial wheat cell wall. The antibodies JIM5 and JIM7 as well as PAM1 and LM5 proved that the isolated homogalacturonan had a low methyl ester content, was polymeric and free of galactan side chains. We can thus isolate native homogalacturonan from the type II wheat cell walls with the original in muro pattern of methyl esterification still intact, to further investigate e.g., its degradability by plant or microbial pectic enzymes.     

Cell wall composition of novobiocin-resistant pleiotropic mutant staphylococci.

Physically purified cell walls were prepared from selected pleiotropic novobiocin-resistant staphylococcal strains. The quantitative amino acid, amino sugar, and phosphorus contents of these walls are reported. This pleiotype was culturally diagnosed by its inability to support the growth of typing phages, inability to release latent bacteriophage, failure to elaborate coagulase, altered sugar catabolic pattern, and resistance to novobiocin. The strains were divided into two groups on the basis of wall composition. The walls of both groups of strains appeared to possess at least two phosphorus-containing polymers. On group of strains contained elevated levels of phosphorus in the cell walls. The second group contained the novel amino sugar galactosamine in the cell walls. This amino sugar is probably associated with one of the phosphorus-containing wall polymers of this group. On the basis of the data presented, it is suggested that the pleiotropy of these strains is the result of genetic change in the control of the biosynthesis of teichoic acids.     

Passive Electrical Properties of Microorganisms: III. Conductivity of Isolated Bacterial Cell Walls

The dielectric properties of isolated Micrococcus lysodeikticus cell walls have been studied to establish more firmly the view that wall-associated ions play a major role in the conduction of low frequency electric current by intact bacterial cells. The conductivity of isolated walls was found to be about 0.40 mho/m. If counterions associated with fixed, ionized groups in the wall have average mobilities equal to that of sodium ions in free solution, the fixed charge concentration required to account for the measured conductivity is between 75 and 95 meq/liter of wet wall volume. Estimates of the numbers of titratable amino and carboxyl groups in wall polymers indicate that conductivity is more closely related to net wall charge than to total wall charge. The measured wall conductivity was used to predict a value of 0.15 ± 0.03 mho/m for whole cell conductivity. This prediction is close to the measured value of 0.25 ± 0.05 mho/m and it is thought that much of the disparity in values is related to changes in wall structure and composition during the isolation procedures.     

Ion-exchange properties of cell walls in chickpea cultivars with different sensitivities to salinity

Ion-exchange properties of polymeric matrices were compared for cell wall preparations isolated from roots and shoots of two cultivars of Cicer arietinum L. (cvs. Bivanij and ILC 482) with different sensitivities to salinity. Irrespective of growth conditions, the cell walls contained four types of ionogenic groups: amino groups, carboxyl groups of uronic and hydroxycinnamic acids, and phenolic hydroxyl groups. Regardless of the salt concentration in the medium, the cells walls of different chickpea cultivars and from different organs of the same plant were similar in qualitative composition of ionogenic groups, although quantities of ionogenic groups per unit dry wt of cell walls varied depending on external and internal factors. Irrespective of the external medium salinity, the cation-exchange capacity of cell walls, expressed per unit dry wt, decreased in a sequence: stem > root ∼ bottom leaves > upper leaves. The volume of chickpea cell walls was found to vary depending on ionic composition and pH of the incubation medium. The results were analyzed in the context of cell wall involvement in responses of C. arietinum to elevated salinity.     

Cell wall composition in the ascomycete sphaerostilbe repens at different developmental stages

Changes in the biochemical composition of isolated cell walls were analysed during the differentiation of coremia and rhizomorphs in Sphaerostilbe repens.Differentiation was accompanied by exclusively quantitative variations of the wall components: the content in carbohydrates, chitin and free amino sugars increased; on the contrary, amino acids, uronic acids, lipids and mineral substances decreased.Carbohydrates were composed of glucose, galactose and mannose; glucosamine was the main component of amino sugars. The predominant amino acid in the walls was cysteine the amount of which increased during hyphal aggregation, while quantities of the sixteen other determined amino acids decreased.Mineral matter was present in large quantities in the walls of the fungus, especially in vegetative mycelium. Iron, phosphorus and calcium were the most abundant elements.Possible relations between the variations in chemical composition of the wall and the capability of hyphae to aggregate are discussed.     

Evolution and diversity of green plant cell walls

Plant cells are surrounded by a dynamic cell wall that performs many essential biological roles, including regulation of cell expansion, the control of tissue cohesion, ion-exchange and defence against microbes. Recent evidence shows that the suite of polysaccharides and wall proteins from which the plant cell wall is composed shows variation between monophyletic plant taxa. This is likely to have been generated during the evolution of plant groups in response to environmental stress. Understanding the natural variation and diversity that exists between cell walls from different taxa is key to facilitating their future exploitation and manipulation, for example by increasing lignocellulosic content or reducing its recalcitrance for use in biofuel generation.     

Properties of lead deposits in cell walls of radish (Raphanus sativus) roots

Various mechanisms are involved in detoxification of heavy metals such as lead (Pb) in plant cells. Most of the Pb taken up by plants accumulates in their roots. However, the detailed properties of Pb complexes in roots remain unclear. We have investigated the properties of Pb deposits in root cell walls of radish (Raphanus sativus L.) seedlings grown on glass beads bed containing Pb pellets, which are the source of Pb-contamination in shooting range soils. Pb deposits were tightly bound to cell walls. Cell wall fragments containing about 50,000 ppm Pb were prepared from the roots. After extracting Pb from the cell wall fragments using HCl, Pb ions were recombined with the Pb-extracted cell wall fragments in a solution containing Pb acetate. When the cell wall fragments were treated with pectinase (E.C. 3.2.1.15) and were chemically modified with 1-ethyl-3-dimethylamino-propylcarboimide, the Pb-rebinding ability of the treated cell wall fragments decreased. When acid-treated cell wall fragments were incubated in a solution containing Pb²⁺ and excess amounts of a chelating agent, Pb recombined with the cell wall fragments were measured to estimate the affinity between Pb²⁺ and the cell wall fragments. Our data show that Pb²⁺ binds to carboxyl groups of cell walls. The source of the carboxyl groups is suggested to be pectic compounds. A stability constant of the Pb-cell wall complex was estimated to be about 10⁸. The role of root cell walls in the mechanism underlying heavy metal tolerance was discussed.     

Comparative in vivo nitrogen-15 nuclear magnetic resonance study of the cell wall components of five Gram-positive bacteria.

The proton-decoupled 9.12 MHz 15N NMR spectra of 15N-labeled Bacillus subtilis, Bacillus licheniformis, Staphylococcus auresu, Streptococcus faecalis, and Micrococcus lysodeikticus intact cells, isolated cells walls, and cell wall digests have been examined. The general characteristics of Gram-positive bacteria 15N NMR spectra and described and spectral assignments are provided, which allow in vivo 15N NMR to be applied to a wide range of problems in bacterial cell wall research. The qualitative similarity of the intact cell and cell wall spectra found in each bacteria allowed the 15 N resonances observed in the proton broad-band noise-decoupled 15N NMR spectra of intact cells to be assigned to cell wall components. Each of the five Gram-positive bacteria displayed a unique set of cell wall 15N resonances, which reflected variations in the primary structure of peptidoglycans and the amounts of teichoic acid and teichuronic acid in the cell wall, as well as the dynamic properties of the cell wall polymers. Spectral assignments of cell wall 15 N resonances assigned to teichoic D-Ala residues, teichuronic acid and acetamido groups, and peptidoglycan acetamido, amide, peptide, and free amino groups have been made on the basis of specific isotopic labeling and dilution experiments, comparison of chemical shifts to literature values, determination of pH titration shifts, cell wall fractionation experiments, and comparative analysis of the cell wall lysozyme digest spectra in terms of the known primary sequences of peptide chains. All the peptidoglycan 15N peptide resonances observed in the intact cells and isolated cell walls could be accounted for by residues in the bridge or crossbar regions of the peptide chains, which indicated that only the cross-linking groups had a high degree of motional freedom. Thermal- and pH-induced conformational changes around the cross-linking D-Ala residues were detected in the B. licheniformis cell wall lysozyme digest products. Comparison of the proton broad-band noise-decoupled and gated decoupled intact cell and cell wall 15N spectra indicated that broad-band proton decoupling resulted in nulling of cytoplasmic resonances and enhancement of the cell wall resonances by the 15N [1H5 nuclear Overhauser effect.