Ripening-related perturbations in apple cell wall nuclear spin dynamics

Spin-lattice (¹HT₁, ²³Na⁺T₁) and rotating frame spin-lattice (¹HT_1p, ¹³CT_1p) relaxation times were measured on intact, critical point dried apple tissue at various degrees of ripeness using cross polarization and magic angle spinning (CPMAS) NMR techniques. Solid state carbonyl (δ172)¹³CT_1p and ²³Na⁺-carboxylate anion T₁ values, which are inversely proportional to carboxylate reorientation rates, decreased 15–19% during the time course study. Carbonyl resonance ¹HT₁s diminished by 63% as the tissue softened; a maximal decline of 42% was also observed in the ¹HT₁s of nonspecific carbohydrate ring carbon signals (δ74) indicating an increase in both acidic and neutral polymer motion. Treatment of the cell wall with polygalacturonase resulted in a significant decrease in both carbonyl and ring carbon ¹HT₁s (57 and 42%, respectively) demonstrating the important structural function of polyuronides not only in the middle lamellae but also in the primary cell wall.     

Auxin-indnced changes in barley coleoptile cell wall composition

Auxin induces extension growth of barley coleoptile segments,causing cell extension and cell wall loosening represented bya change in mechanical properties of the cell wall. This responsedecreased after the segments were starved for more than 12 hrin buffer solution. Auxin decreased the noncellulosic glucosecontent of the cell wall of the segments starved for 0 and 6hr, but very little that of segments starved for 12 and 18 hr.The contents of arabinose, xylose and galactose, among noncellulosicpolysaccharides, and -cellulose of the cell wall increased duringthe starvation, but auxin did not affect them. The auxin-induceddecrease in glucose content was inhibited by nojirimycin, apotent inhibitor of ß-glucanase, which inhibited auxin-inducedextension and changes in mechanical properties of the cell wall,suggesting that cell wall loosening, and thus cell extension,resulted from partial degradation of ß-glucan of thecell wall. (Received April 20, 1978; )     

Kinetin induced changes in cell wall composition of tobacco callus

Culture of tobacco callus on high or low kinetin in light or darkness leads to changed tissue texture and associated changes in cell wall composition. In particular, friable callus (low kinetin, darkness) cell walls have a greater extensin content and an altered composition of arabinose and xylose containing hemicelluloses compared with cell walls of compact callus (high kinetin, darkness). The possible importance of these differences in determining callus friability is discussed.     

Chemical composition and structure of cell wall teichoic acids of staphylococci

The cell wall teichoic acid structures of 22 staphylococci including 13 type strains were determined. Most of the strains contain a poly(polyolphosphate) teichoic acid with glycerol and/or ribitol as polyol component. The polyolphosphate backbone is partially substituted with various combinations of sugars and/or amino sugars. Most of the substituents occur in a monomeric form but some strains also contain dimers of N-acetylglucosamine as substituents. Staphylococcus hyicus subsp. hyicus NCTC 10350 and S. sciuri DSM 20352 revealed rather complex cell wall teichoic acids. They consist of repeating sequences of phosphate-glycerol-phosphate-N-acetylglucosamine. The amino sugar component is present in this case as a monomer or an oligomer (n less than or equal to 3). Moreover, the glycerol residues are partially substituted with N-acetylglucosamine. The cell wall teichoic acid of S. auricularis is a poly(N-acetylglucosaminyl-phosphate) polymer similar to that found in S. caseolyticus ATCC29750. The cell wall teichoic acid structures for type strains of S. auricularis, S. capitis, S. cohnii, S. haemolyticus, S. hominis, S. hyicus subsp. hyicus, S. sciuri, S. xylosus and S. warneri were determined for the first time in detail. The structures of some of the previously described teichoic acids had to be revised (S. epidermidis, S. simulans, S. aureus phage type 187).     

Ripening-related changes in the cell walls of Spanish pear (Pyrus communis)

Unripe Spanish pears ( Pyras commanis L. ev. Blanquilla ) were ripened at 18°C for 5 and 10 days. Softening of the cortical tissues was associated with swelling of parenchyma cell walls from 1 to more than 5 μm in 10 day ripe pears, by which time the pears were over ripe. However, there was little indication of cell separation and the middle lamella could be detected between most cell walls. Furthermore, cell separation was constrained by regions rich in plasmodesmata where wall swelling was prevented. Parenchyma cells in the 500 μm of tissue underlying the epidermis did not undergo ripening-related changes to the same extent as those of the cortex. These cells, in combination with a sub-epidermal layer of lignified sclereid clusters, constituted a relatively tough and protective skin. Ripening of the cortical tissues was associated with a depletion of alcohol-insoluble pectic polysaccharides, as indicated by the decrease in arabinose and uronic acid. Analysis of alcohol-insoluble cell wall preparations enriched in either parenchyma or sclereid cell walls indicated that this change was predominantly associated with the parenchyma walls. Such changes were less prominent in the peel. The decrease in pectic polysaccharides was accompanied by an increase in their solubility. During ripening, the sclereid clusters of the cortex continued in develop, as indicated by an increase in their size and yield of cell wall xylose and glucose. Cortical parenchyma cells radiating from the sclereids were firmly attached to the lignified cells. This was due to lignification extending from the sclereids into the primary walls of the parenchyma cells. We conclude that dissolution of pectic polysaccharides is one of several factors which determine softening during ripening of Spanish pears.     

Chitosan-mediated changes in cell wall composition, morphology and ultrastructure in two wood-inhabiting fungi

The effect of chitosan on cell wall deposition was investigated in the two wood-inhabiting fungal species Trichoderma harzianum (CBS 597.91) and Sphaeropsis sapinea (NZFS 2725). The study used three independent analytical techniques to quantify chitin in the fungal mycelium. A colorimetric method for the detection of d-glucosamine was compared with two gas chromatography–mass spectroscopy (GC-MS) methods employing alditol acetates analysis and pyrolysis. The latter used a stable-isotope-labelled internal standard, d₃-N-acetyl glucosamine. At least in the case of S. sapinea, the study provided evidence of an increase in the chitin content in the mycelium due to chitosan treatment, indicating that chitosan treatment affected cell wall deposition. Electron microscopy techniques showed alteration in surface morphology and cell wall texture due to chitosan treatment. The implications of these results are discussed with a view to analysing possible mechanisms for growth inhibitory effects of chitosan on fungal hyphae.     

Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata

Background and Aims

In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata.

Methods

Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples.

Key Results

Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly.

Conclusions

This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.     

Changes in the cell wall composition and structure of Streptococcus pyogenes during batch culture

Changes in S. pyogenes cells in the process of batch cultivation have been studied. The composition of S. pyogenes cell walls has been studied by amino acid analysis; besides, their resistance to enzymatic hydrolysis and the electric conductivity of cell-wall lysates have been determined at different phases of the growth of S. pyogenes. The molar amino acid composition, expressed in percent, is unrelated to the growth phase, while the content of amino acids in preparations changes in the process of growth and reaches its maximum in the middle and in the end of the logarithmic phase. At the same time the electric conductivity of cell-wall lysates reaches the minimum level at these growth stages. The authors suggest that additional electrically charged compositions are formed in the cell walls at the beginning of the logarithmic and stationary phases. A considerable increase in the initial rate of cell-wall lysis with muramidase has been found to occur at the end of the logarithmic phase. This difference in the initial rate in the initial rates of lysis of S. pyogenes cell walls at different growth phases decreases after previous treatment of the cell walls with streptolytin possessing proteolytic activity. Analysis of these data leads to a conclusion on the "loose" structure of the outer protein layer of the cell wall at the end of the logarithmic phase of the growth curve.     

Auxin-induced cell elongation and cell wall changes

It has been well known that auxin induces cell elongation through its effect on modifications of the cell wall. The present review will discuss cell wall modifications, physical and biochemical, as the background of the former, based on the experimental results from our laboratory and from others, with the historical background. Discussions will particularly put stress on the auxin effect on the cell wall in terms of the following studies, namely, (1) measurements of the mechanical property of the cell wall, and (2) biochemical studies on the polysaccharide molecules of the cell wall. This article is dedicated to Professor Anton N.J. Heyn for his 85th birthday.     

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with β-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.     

The relationship between changes in cell wall composition and the establishment of polarity in Fucus embryos

Changes in the appearance and location of fucoidin in the cell walls of Fucus embryos were related to embryo development. Fucoidin was not present in the cell wall until 10–14 hr after fertilization, when the embryos began to incorporate fucoidin preferentially into a localized area of the wall. At this time the site of rhizoid initiation was determined; that is, the embryos had undergone axis commitment. Germination of the single-celled embryo occurred between 12 and 16 hr, after fertilization, with all cell walls from germinated embryos showing fucoidin localization at the rhizoid end of the cell. The percentage of embryos with localized fucoidin at the time of axis fixation equaled the percentage of embryos that subsequently germinated. Culturing the embryos in sea water plus 0.8 M sucrose prevented the outgrowth of the rhizoid, but not the localization of fucoidin in the wall or axis commitment. Cycloheximide, nitroprusside, cytochalasin B, sulfate-free sea water, high levels of Ca²⁺, and a breakdown product of TIBA all prevented rhizoid growth and the specific localization of fucoidin. In addition, axis commitment could not be demonstrated in the presence of these inhibitors. DTNB, PCMBS, TIBA, HgCl₂, Mg²⁺ were ineffective as reversible inhibitors of rhizoid initiation. The authors propose that the fixation of axis commitment is accompanied by localized changes in the cell wall involving the incorporation of fucoidin as a structural component of the wall.     

Penicillin-induced changes in the cell wall composition of Staphylococcus aureus before the onset of bacteriolysis

To analyze if chemical cell wall alterations contribute to penicillin-induced bacteriolysis, changes in the amount, stability, and chemical composition of staphylococcal cell walls were investigated. All analyses were performed before onset of bacteriolysis i.e. during the first 60 min following addition of different penicillin G doses. Only a slight reduction of the amount of cell wall material incorporated after penicillin addition at the optimal lytic concentration was observed as compared to control cells. However, the presence of higher penicillin G concentrations reduced the incorporation of wall material progressively without bacteriolysis. Losses of wall material during isolation of dodecylsulfate insoluble cell walls were monitored to assess the stability of the wall material following penicillin addition. Wall material grown at the lytic penicillin concentration was least stable but about 30% of the newly incorporated wall material withstood even the harsh conditions of mechanical breakage and dodecylsulfate treatment. Dodecylsulfate insoluble cell walls were used for chemical analyses. While peptidoglycan chain length was unaffected in the presence of penicillin, other wall parameters were considerably altered: peptide cross-linking was reduced in the wall material synthesized after addition of penicillin; reductions from approx. 85% in controls to about 60% were similar for lytic and also for very high penicillin concentrations leading to nonlytic death. O-acetylation was also reduced after treatment with penicillin; this effect paralleled the occurence of subsequent bacteriolysis at different drug concentrations. The results are not consistent with hypotheses explaining penicillin-induced lysis as a result of an overall weakened cell wall structure or an overall activation of autolytic wall enzymes but not conflicting with the model that ascribes penicillin-induced bacteriolysis as the result of a very restricted, local perforation of the peripheral cell wall (murosome-induced bacteriolysis).Abbreviations CL Cross-linking - DNFB 2,4-dinitro-1-fluorobenzole - MIC Minimal inhibitory concentration - OD Optical density at 578 nm - PEN Penicillin G     

Morphology, wood structure and cell wall composition of rolC transgenic and non-transformed aspen trees

Morphology, wood structure and cell wall composition of 35S-rolC transgenic hybrid aspen (P. tremula2tremuloides) were compared with non-transformed control trees. The transgenics are characterised by stunted growth, altered physiological parameters and light green leaves of reduced size. Histometric measurements revealed thinner fibre walls as compared to the controls. UV microspectrophotometry of individual wall layers did not reveal distinctive differences in the lignification of xylem cells, but in the extremely thin-walled fibres of the transgenics the secondary walls were less lignified as revealed by KMnO₄ staining in transmission electron microscopy. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to only a few rows. Immunocytochemical analyses revealed the deposition of lignins in less differentiated xylem cells as compared to the controls. The first labelling of condensed lignin appeared in cell corners and of non-condensed lignin in secondary walls near cell corners during the deposition of S1 polysaccharides. Because of alterations in the formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful for studies on molecular factors controlling the differentiation continuum.     

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure

A family 15 carbohydrate esterase (CE15) from the white‐rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col‐0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl‐4‐O‐methyl‐d ‐glucopyranuronate and could target ester linkages that contribute to lignin–carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf‐yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT‐IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross‐linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross‐linking within plant cell walls.     

Growth and cell wall changes in rice coleoptiles

A fractionation of non-cellulosic sugars of Oryza sativa L. coleoptile cell walls was carried out and the composition of each fraction was studied during coleoptile growth.Percentages of fractions extracted with boiling water and with oxalate (pectic substances) were almost constant throughout development. An increase in the K II hemicellulosic fraction (extracted with 24% KOH) content, and a decrease in the K I hemicellulosic fraction (extracted with 10% KOH) were detected, when coleoptile growth finished.The percentage of glucose content in the K I hemicellulosic fraction was highest in young coleoptiles and lowest in old ones. Furthermore, a highly significant linear relationship between amounts of glucose and growth rate was obtained, while a inverse relationship between the amount of xylose and arabinose and growth rate was attained.Abreviations GLC gas liquid chromatography - IAA indole-3-acetic-acid - TFA trifluoroacetic acid - T_o minimum stress-relaxation time