Cell-wall synthesis and elongation growth in hypocotyls of Helianthus annuus L.

The relationship between growth and increase in cell-wall material (wall synthesis) was investigated in hypocotyls of sunflower seedlings (Helianthus annuus L.) that were either grown in the dark or irradiated with continuous white light (WL). The peripheral three to four cell layers comprised 30–50% of the entire wall material of the hypocotyl. The increase in wall material during growth in the dark and WL, respectively, was larger in the inner tissues than in the peripheral cell layers. The wall mass per length decreased continuously, indicating that wall thinning occurs during growth of the hypocotyl. When dark-grown seedlings were transfered to WL, a 70% inhibition of growth was observed, but the increase in wall mass was unaffected. Likewise, the composition of the cell walls (cellulose, hemicellulose, pectic substances) was not affected by WL irradiation. Upon transfer of dark-grown seedlings into WL a drastic increase in wall thickness and a concomitant decrease in cell-wall plasticity was measured. The results indicate that cell-wall synthesis and cell elongation are independent processes and that, as a result, WL irradiation of etiolated hypocotyls leads to a thickening and mechanical stiffening of the cell walls.     

Assessing the relative contributions of phytochelatins and the cell wall to cadmium resistance in white lupin

In response to Cd stress, higher plants utilise a number of defence systems, such as retention in cell walls, binding by organic molecules in the cytosol and sequestration in the vacuole. White lupin is a Cd-resistant legume that is of interest for phytoremediation of acidified and Cd-contaminated soils. The aim of this research was to evaluate the contributions of various mechanisms of Cd detoxification used by this species, focusing on cell-wall retention and binding by thiol-rich compounds. Retention of Cd by the cell wall of white lupin was well described by a Langmuir isotherm model. The percentage of total Cd adsorbed by the cell wall ranged from 29 to 47% in leaves, from 38 to 51% in stems and from 26 to 42% in roots depending on the Cd supply. Cadmium induced the synthesis of high levels of phytochelatins (PCs) in lupin plants, mainly in roots, with PC₃ being the major PC. The amount of Cd complexed by thiols accounted for approximately 20% of the total Cd in leaves, 40% in stems and 20% in roots. Therefore, cell-wall retention could account for more than twice the amount of Cd complexed by PCs in leaves and roots. In stems, both mechanisms contributed equally to Cd detoxification. These studies indicate that white lupin plants use cell-wall binding and, secondarily, the production of PCs, as effective mechanisms of Cd detoxification.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.     

Cell wall proteins of plant fibers

The cell wall of the same type - phloem fibers (Linum usitatissimum L.), active forming the thick secondary cell wall, - was obtained. Weakly bound cell wall proteins of phloem fibers were extracted and it subsequent separation and obtaining mass spectra was carried out. For identification and attachment of identified proteins to a specific cell compartments a variety ofbioinformatics methods was used. Were identified 93 proteins, many of which were defined as predicted, putative or hypothetical. At the same 21 proteins were identified as cell-wall protein. The absence of such marker proteins of the primary cell wall as xyloglucan-endotransglycosilase, expansins indirectly confirms that in the sample for extraction of proteins dominated the secondary cell wall.     

Fermentation characteristics of cell-wall sugars from soya bean meal,and from separated endosperm and hulls of soya beans

Two experiments were conducted to investigate the degradation of cell-wall sugars from soya bean meal (in situ), and soya bean endosperm and hulls (in vitro). Soya bean meal, soya bean endosperm, and soya bean hulls were extracted with different chemicals to obtain the cell-wall fraction. Soya bean meal cell walls were incubated in the rumen of a fistulated cow. The individual cell-wall sugars were degraded at different rates: galactose (13.6% h⁻¹), arabinose (7.8% h⁻¹), uronic acids (5.1% h⁻¹), xylose (3.5% h⁻¹) and glucose (3.2% h⁻¹). Microscopic evaluation of the cell walls and degraded material revealed the presence of two cell wall types, with distinctly different degradation characteristics: one originating from the hull (thick, slowly degraded) and one from the endosperm (thin, rapidly degraded). Furthermore, the cell-wall sugar composition of endosperm and hull cell walls was different, most markedly for galactose (281 vs. 12 g kg⁻¹) and glucose (132 vs. 508 g kg⁻¹). The degradation of endosperm and hull cell walls was measured in vitro by use of in vitro cumulative gas production. Degradation rates of the individual cell-wall sugars for hull cell walls were similar (ranging from 2.4% to 4.6% h⁻¹). For endosperm cell walls, the degradation rates of the individual sugars were different but with the same ranking as in the in situ experiment (ranging from 20.9% to 7.0% h⁻¹). It was concluded that for soya bean meal cell walls, the cell-wall sugar degradation pattern is influenced by the presence of two cell-wall types (hull and endosperm cell-wall), which differ in their rate of degradation and sugar composition. The difference in cell-wall sugar degradation pattern between hull and endosperm cell walls is likely to be caused by a combined effect of particle size and cell-wall thickness.     

Oligosaccharide side chains of wall molecules are essential for cell-wall lysis in Chlamydomonas reinhardtii

The glycoproteins of the cell walls of Chlamydomonas are lysed during the reproductive cycle by proteases (autolysins) which are specific for their substrates. The autolysin which digests the wall of sporangia to liberate the zoospore daughter cells in the vegetative life cycle is a collagenase-like enzyme which attacks only selected domains in its wall substrates containing (hydroxy)-proline clusters. Cell-wall fractions obtained by salt-extraction (NaClO₄) and oxidizing agents (NaClO₂) and the insoluble residue were tested as substrates. The most-crosslinked insoluble inner part of the wall is the best substrate for the sporangia autolysin. Oligosaccharides obtained from the insoluble cell-wall fraction of sporangia by hydrolysis with Ba(OH)₂ inhibit autolysin action. We conclude that the oligosaccharide side chains of wall substrates are essential for forming the reactive enzyme-substrate complex.Abbreviations CSW chlorite-soluble cell-wall fraction - ICW insoluble cell-wall fraction - PSW salt-soluble fraction - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Formation and regeneration of protoplasts of wild-typeNeurospora crassa

Trichoderma reesei was grown using purified cell walls ofNeurospora crassa as a primary source of carbon. The resulting culture medium contained an undefined mixture ofN. crassa cell-wall digesting enzymes. Protoplasts (cell lacking wall) were formed when youngN. crassa hyphae were treated withTrichoderma mixture. The vast majority of protoplasts resynthesized cell-wall material when washed free of cell-wall digesting enzyme; of these, about 40% regenerated a mycelium.     

In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus

Many surface proteins of Gram-positive bacteria are covalently anchored to the cell wall by a ubiquitous mechanism, involving a specific, C-terminal sorting signal. To achieve cell-wall immobilization of a normally secreted enzyme in vivo, we constructed a hybrid protein consisting of Staphylococcus hyicus lipase and the C-terminal region of Staphylococcus aureus fibronectin binding protein B (FnBPB). This region comprised the authentic cell-wall-spanning region and cell-wall sorting signal of FnBPB. Expression of the hybrid protein in Staphylococcus carnosus resulted in efficient cell-wall anchoring of enzymatically active lipase. The cell-wall-immobilized lipase (approximately 10000 molecules per cell) retained more than 80% of the specific activity, compared to the C-terminally unmodified S. hyicus lipase secreted by S. carnosus cells. After releasing the hybrid protein from the cell wall by lysostaphin treatment, its specific activity was indistinguishable from that of the unmodified lipase. Thus, the C-terminal region of FnBPB per se was fully compatible with folding of the lipase to an active conformation. To study the influence of the distance between the cell-wall sorting signal and the C-terminus of the lipase on the activity of the immobilized lipase, the length of this spacer region was varied. Reduction of the spacer length gradually reduced the activity of the surface-immobilized lipase. On the other hand, elongation of this spacer did not stimulate the activity of the immobilized lipase, indicating that the spacer must exceed a critical length of approx. 90 amino acids to allow efficient folding of the enzyme, which probably can only be achieved outside the pep-tidoglycan web of the cell wall. When the lipase was replaced by another enzyme, the Escherichia coliβ-lactamase, the resulting hybrid was also efficiently anchored in an active conformation to the cell wall of S, carnosus. These results demonstrate that it is possible to immobilize normally soluble enzymes on the cell wall of S. carnosus - without radically altering their catalytic activity - by fusing them to a cell-wall-immobilization unit, consisting of a suitable cellwall-spanning region and a standard cell-wall sorting signal.     

Characterization of two cell-envelope fractions from chemotrophically grownRhodospirillum rubrum

Two cell-envelope fractions were isolated from chemotrophically grown cells ofRhodospirillum rubrum. On the basis of electron-microscopic investigations, chemical analysis, distribution of components involved in respiration, and polyacrylamide gel electrophoresis, the heavy fraction (ρ²⁰=1.246 g per cm³) was identified as cell-wall, and the light fraction (ρ²⁰=1.145 g per cm³) as cytoplasmic-membrane fragments. Electron micrographs showed cell-wall fragments as open structures while cytoplasmic-membrane preparations were composed of closed membrane vesicles. With respect to the main classes of chemical compounds, cell wall could be distinguished from cytoplasmic membranes by a rather low ratio of phospholipids per protein and a high ratio of carbohydrates per protein. The relative proportion of individual neutral sugars as well as phospholipids (except for lysophosphatidyl ethanolamine) revealed no significant differences between both envelope fractions. Fatty acid analysis demonstrated a higher proportion of saturated fatty acids in cell-wall than in cytoplasmic-membrane fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions showed distinct protein compositions. While in cell-wall preparations polypeptides of 43000 and 14000 daltons predominated, 56000- and 52000-dalton polypeptides were the main protein subunits of cytoplasmic membranes. Cross contaminations of both cell-envelope fractions were defined.     

Effect of tomicide and the cell wall biopolymers of Streptococcus sp. Thom-1606 on mast cell degranulation

The influence of tomicide and biopolymers obtained from the cell wall of Streptococcus sp. TOM-1606 on the degranulation of mast cells was studied. Among the biopolymers of the streptococcal cell-wall polysaccharide was shown to induce the highest destruction of mast cells (14.84 +/- 6.8%). The alteration of mast cells under the effect of peptidoglycan and teichoic acid was mildly positive (11.85 +/- 5.8% and 12.1 +/- 6.2%). At the same time the destruction induced by the complex of noninfectious allergen and the patient's serum was 33.2 +/- 3.8% respectively. Other preparations induced destruction on the level of spontaneous degranulation. The study of the action of the allergen-antibody complex in combination with tomicide and biopolymers obtained from the cell wall of Streptococcus sp. TOM-1606 revealed a decrease in the rate of mast cell degranulation almost to the background level (24.7 +/- 0.55% for the allergen-antibody complex and 8.4 +/- 4.2% to 11.8 +/- 5.3% for streptococcal biopolymers).     

Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses

Development, abiotic and biotic stress each affect the physical architecture and chemical composition of the plant cell wall, making maintenance of cell-wall integrity an important component of many plant processes. Cellulose biosynthesis inhibition (CBI) was employed to impair the functional integrity of the cell wall, and the plant's response to this specific stress was characterized in an Arabidopsis seedling model system. CBI caused changes in the expression of genes involved in mechanoperception, the response to microbial challenge, and lignin and cell-wall polysaccharide biosynthesis. Following CBI, activation of a UDP- d -xylose 4-epimerase gene correlated with increases in arabinose and uronic acid content in seedling cell walls. Activation of pathogen response genes, lignin deposition and lesion formation were dependent on externally supplied sugars and were suppressed by osmotic support. Lignin deposition in the root elongation zone caused by CBI was reduced in atrbohd (NADPH oxidase) mutant seedlings but increased in jasmonic acid resistant1 ( jar1-1 ) mutant seedlings. Phytohormone measurements showed that CBI-induced increases in jasmonic (JA) and salicylic acids were dependent on sugar availability and prevented by osmotic support. We show that CBI activates responses commonly attributed to both abiotic and microbial challenges. Glucose/sucrose and turgor pressure are critical components in maintenance of cell-wall integrity and the regulation of induced responses, including JA biosynthesis. Lignin deposition induced by CBI is regulated by JAR1-1 and NADPH oxidase-dependent signalling processes. Our results identify components of the mechanism that mediates the response to impairment of cell-wall integrity in Arabidopsis thaliana .     

Disintegration of dried yeast cells and its effect on protein extractability,sedimentation property,and viscosity of the cell suspension

The morphology of dried Candida lipolytica yeast suspended in aqueous solutions (H₂O, 0.4% NaOH, 2N HCl, and 6N HCl) and organic solvents (95% alcohol and acetone) was studied using a scanning electron microscope (SEM) and an optical microscope. The effect of high-pressure homogenization on cell-wall structure and cell clumps was also determined. The protein extractability, sedimentation property, and viscosity of cells subjected to different mechanical and chemical treatments were also investigaged. The dried yeast cells were in a spherical agglomeration consisting of 100s of closely bound cells. The clump was resistant to water, aqueous 2N HCl solution at 25°C, 95% alcohol and acetone, but vulnerable to 6N HCl, aqueous 0.4% NaOH solution, and homogenization. The homogenization of the cell suspension not only broke the clump but also cracked the cell-wall structure. The aqueous alkaline solution could have weakened the cell wall and increased the solubility of the protein released through the cracks in the cell wall. The destruction of the agglomeration and the cell-wall structure increased the hydration of the cell and thereby increased the stability of the suspension. The sedimentation and the viscosity of the cell suspension corresponded to the morphological changes and the extractability of protein in the cell suspensions with different treatments.     

Silica nanocasts of wood fibers: a study of cell-wall accessibility and structure

The porosity and the available surface area of a lignocellulosic fiber can influence the accessibility and reactivity in derivatization and modification reactions because the porous cell-wall network determines the upper size limit for molecules that can penetrate and react with the interior of the wall. To obtain information concerning the accessibility of the porous cell wall of wood fibers, surfactant-templated sol-gel mineralization has been examined. Wood and kraft pulp samples of Norway spruce were impregnated with a silica sol-gel and subsequently heated (calcined) and transformed into structured mesoporous silica. Microscopy studies (environmental scanning electron microscopy, transmission electron microsopy, TEM) on the silica casts showed that the three-dimensional architecture of the wood and pulp fiber cell wall was revealed down to the nanometer level. Image analysis of TEM micrographs of silica fragments from the never-dried pulp revealed complete infiltration of the cell-wall voids and microcavities (mean pore width 4.7 +/- 2 nm) by the sol-gel and the presence of cellulose fibrils with a width of 3.6 +/- 1 nm. Cellulose fibrils of the same width as that shown by image analysis were also identified by nitrogen adsorption measurements of the pore size distribution in the replicas.     

Synthesis and Turnover of Cell-Wall Polysaccharides and Starch in Photosynthetic Soybean Suspension Cultures

Soybean (Glycine max [L.] Merr.) suspension cultures grown under photoautotrophic and photomixotrophic (1% sucrose) culture conditions were used in 14CO2 pulse-chase experiments to follow cell-wall polysaccharide and starch biosynthesis and turnover. Following a 30-min pulse with 14CO2, about one-fourth of the 14C of the photoautotrophic cells was incorporated into the cell wall; this increased to about 80% during a 96-h chase in unlabeled CO2. Cells early in the cell culture cycle (3 d) incorporated more 14C per sample and also exhibited greater turnover of the pectin and hemicellulose fractions as shown by loss of 14C during the 96-h chase than did 10- and 16-d cells. When the chase occurred in the dark, less 14C was incorporated into the cell wall because of the cessation of growth and higher respiratory loss. The dark effect was much less pronounced with the photomixotrophic cells. Even though the cell starch levels were much lower than in leaves, high 14C incorporation was found during the pulse, especially in older cells. The label was largely lost during the chase, indicating that starch is involved in the short-term storage of photosynthate. Thus, these easily labeled and manipulated photosynthetic cells demonstrated extensive turnover of the cell-wall pectin and hemicellulose fractions and starch during the normal growth process.     

Extraction by lithium chloride of hydroxyproline-rich glycoproteins from intact cells of Chlamydomonas reinhardii

A procedure has been developed to isolate and analyse the cell-wall glycoproteins of Chlamydomonas reinhardii. Under appropriate conditions, cell-wall glycoproteins can be quantitatively extracted from intact cells by aqueous LiCl. Although proteins and glycoproteins, which are presumably not related to the cell wall, are coextracted with the cell-wall subunits, these components can be readily identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as demonstrated by comparative analysis of LiCl-extracts from wild-type cells and the cell-wall-deficient mutant CW-15. Apart from the high-molecular-weight cell-wall components, two glycoproteins with apparent molecular weights (M_rs) of 36000 and 66000 were found to be present in LiCl-extracts of wild-type cells but absent in LiCl-extracts from the cell-wall-less mutant. Pulse-labeling experiments with [³H]proline and [³⁵S]methionine revealed that the LiCl-extracts contained — in addition to the well-known cell-wall subunits — proteins of lower molecular weight, which are also preferentially labeled with [³H]proline. Protein components with M_rs of 68000, 44000, 36000, 26000 and 22000 were found to be more strongly labeled with [³H]proline than with [³⁵S]methionine, whereas protein components with M_rs of 57000 and 52000 were more prominent after labeling with [³⁵S]methionine. The portion of cell-wall subunits within the total amount of proteins extracted by LiCl was calculated to be at least 10% on the basis of the amount of hydroxyproline. Self-assembly of cell walls could be demonstrated after dialysis against water of a mixture of crude LiCl-extract and purified, insoluble, inner wall layers. Cell-wall glycoproteins could be enriched by gel exclusion chromatography of crude LiCl-extracts on Sepharose CL-4B columns equilibrated with 1 mol l^-1 LiCl.Abbreviations EDTA ethylenediaminetetraacetic-acid - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff's reagent - SDS sodium dodecyl sulfate - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

A barrier covering lignified cell walls of barley straw that restricts access by rumen microorganisms

In barley straw, all stem tissues except the chlorenchyma are lignified. Electronmicroscopic investigations employing staining for cell-wall constituents and replica techniques revealed the presence of a tertiary wall covering the secondary wall and a warty layer deposited on the tertiary wall. The tertiary wall was composed of cellulose fibrils orientated in all directions, which were embedded in matrix material. The warty layer was comprised of granules and globules that were in many instances associated with a thin, flat, continuous layer. Though the tertiary wall could be degraded, the warty layer was resistant to degradation by rumen microorganisms. Only where the warty layer was mechanically disrupted could underlying cell-wall material be degraded. Bacteria could also burrow beneath these layers from exposed cell walls at the cut edges of the plant material. The warty layer forms a barrier to cell-wall-degrading bacteria and limits their colonization of straw stem.     

Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves.

The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.     

Cell growth in expanding primary leaves of Phaseolus

Plants were grown at 25 and 20° C in 6, 12, and 18 h daylengths.Final area of the primary leaf pair ranged from 105 to 209 cm²,and for a given temperature was greatest in the 12 h and leastin 6 h daylength. Cell numbers per leaf were similar for alltreatments. In the 6 h daylength leaves were thinner, containedless chlorophyll and ethanol-insoluble dry matter, and had considerablysmaller cells than leaves on plants in the longer daylengths;final levels of protein and cell-wall material per cell werealso low, although levels of nucleic acid per cell were as highas, or higher than, those for leaves in 12 and 18 h days. Itis concluded that the low levels of protein and cell-wall materialare associated with a low level of photosynthesis, and thatthe small area of these leaves is a result of the reductionin cell size. In the 12- and 18-h daylenghts, protein and cell wall per cellincreased linearly with time, and when expansion of the laminawas completed, values for these parameters were found to besimilar. Cell size, as measured by fresh weight, was also similarat this stage, although small differences in lamina thicknesswere found. Thus the smaller area for leaves in 18-h days wasnot due to a reduction in mean cell size, although differencesin epidermal cell dimensions must be involved. From consideration of simple models it is concluded that increasein cell wall material during lamina expansion is associatedwith increase in wall area, but that the continued formationof wall material after lamina expansion has ceased is accountedfor by deposition on already existing walls. This continuedincrease in wall material occurs at a time when protein andnucleic acid levels per cell are declining.     

Further studies on the role of cell-wall-degrading enzymes in cell-wall loosening in oat coleoptiles

A fungal endo-ß-l,3-glucanase was compared with afungal exo-ß-1,3-glucanase with respect to their effectson elongation and cell-wall extensibility in oat coleoptilesegments. The exo-enzyme enhanced elongation and extensibilityof the cell wall. Its effect was not additive to the effectof indole-3-acetic acid when given together with the latter,at least during 3 hr of incubation. Endo-glucanase showed nosignificant effect on elongation and no interaction with theexo-enzyme. Auxin and exo-glucanase increased extensibilityof the cell wall. The exo-glucanase was separated by isoelectricfocusing. The two fractions which were separated and showedglucanase activity induced elongation and cell wall loosening. (Received March 16, 1970; )