Cell Walls Of Higher Plants Research Articles

Digestive enzymes of the saltmarsh periwinkleLittorina irrorata (Mollusca: Gastropoda)

The saltmarsh periwinkleLittorina irrorata is well adapted for the digestion of a wide range of polysaccharides. Enzyme extracts attacked cellulose, pectin, xylan, bean gum and mannan (common in cell walls of higher plants), as well as starch and laminarin (representative of major polysaccharide classes in fungal, algal, and animal tissues). Activities were generally highes at a ph of 5 or 6. There was no evidence that chitin was digested, but 19 other enzymes, active toward various carbohydrates, lipids and peptides, were demonstrated. Enzymatic activity toward Azocoll, a general substrate for proteinase activity, was weak compared to that of other aquatic detritivores. The maximum was reached at a pH of 8. Enzymatic activities were generally measured with extracts of the entire visceral hump. Separate stomach or intestine extracts also gave strong activities. The stomach was the most acidic section of the digestive system with an average pH of 5.8; the intestine had an average pH of 7.3.

An extrinsic marker for higher plant cell walls

Cytochromec was ionically bound to cell walls as a useful extrinsic marker. The marker binds rapidly, can readily be removed by salt treatment, and can be quantitated by conventional spectrophotometric procedures. The use of the marker allows each step of a cell wall purification to be monitored qualitatively and, furthermore, it provides a rapid, simple quantitative method to determine the yield of purified cell walls once isolated from the cell.

Xylanase and β‐xylosidase production by Aspergillus ochraceus during growth on lignocelluloses

Xylan is a polysaccharide located in the cell wall of higher plants which is one of the major components of lignocellulosic materials. In some higher plants and agricultural wastes the xylan is 20-40% of the dry material.' It consists of p-1, 4-linked D-xylose chain. The breakdown of hemicellulose is accomplished by the synergistic action of xylanase (1,4+-~-xylan xylanohydrolase, EC 3.2.1.8) and P-xylosidase (P-D-xyloside xylohydrolase, EC 3.2.1.37). Such enzymes are useful in the degradation of biomaterials containing hemicellulose for the production of monosaccharides from which single-cell protein, single-cell oil, or ethanol could be produced. '-I1 The production and properties of enzymes capable of breaking down cellulose have been extensively st~died. '~-~' Although there are reports on the production of xylanase,'8-26 the behavior of xylanases and its involvement in the utilization of complex lignocellulosic material is still incomplete. Therefore, the present communication deals with the utilization of some lignocellulosic materials by A . ochraceus for xylanase and pxylosidase production. The use of purified xylan enhances the cost of enzyme production and is a major limitation to the economic feasibility of bioconversion and utilization of lignocellulosic materials. Therefore, in this work, easily available substrates like wheat straw, wheat bran, and sugarcane bagasse were used in the experiment to obtain optimum levels of xylanases by the fungal strain A . ochruceus.

Molecular shape and cytochemical characterization of hemicelluloses from higher plant cell walls

Molecular shape and cytochemical characterization of hemicelluloses from higher plant cell walls

Pea Xyloglucan and Cellulose

Since xyloglucan is believed to bind to cellulose microfibrils in the primary cell walls of higher plants and, when isolated from the walls, can also bind to cellulose in vitro, the binding mechanism of xyloglucan to cellulose was further investigated using radioiodinated pea xyloglucan. A time course for the binding showed that the radioiodinated xyloglucan continued to be bound for at least 4 hours at 40 degrees C. Binding was inhibited above pH 6. Binding capacity was shown to vary for celluloses of different origin and was directly related to the relative surface area of the microfibrils. The binding of xyloglucan to cellulose was very specific and was not affected by the presence of a 10-fold excess of (1-->2)-beta-glucan, (1-->3)-beta-glucan, (1-->6)-beta-glucan, (1-->3, 1-->4)-beta-glucan, arabinogalactan, or pectin. When xyloglucan (0.1%) was added to a cellulose-forming culture of Acetobacter xylinum, cellulose ribbon structure was partially disrupted indicating an association of xyloglucan with cellulose at the time of synthesis. Such a result suggests that the small size of primary wall microfibrils in higher plants may well be due to the binding of xyloglucan to cellulose during synthesis which prevents fasciation of small fibrils into larger bundles. Fluorescent xyloglucan was used to stain pea cell wall ghosts prepared to contain only the native xyloglucan:cellulose network or only cellulose. Ghosts containing only cellulose showed strong fluorescence when prepared before or after elongation; as predicted, the presence of native xyloglucan in the ghosts repressed binding of added fluorescent xyloglucan. Such ghosts, prepared after elongation when the ratio of native xyloglucan:cellulose is substantially reduced, still showed only faint fluorescence, indicating that microfibrils continue to be coated with xyloglucan throughout the growth period.

Structural characterization of oligosaccharides isolated from the pectic polysaccharide rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic ( d-galactosyl-uronic acid-rich) polysaccharide that is present in the primary (growing) cell-walls of higher plants. RG-II is composed of ∼60 glycosyl residues. The isolation and structural characterization of 23 oligosaccharide fragments of the residue of RG-II that remained after removal of hepta- and di-saccharides by partial hydrolysis with acid are reported. In order to obtain the oligosaccharide fragments characterized herein, the carboxyl groups of RG-II were dideuterio-reduced, and the carboxyl-reduced polysaccharide was per- O-methylated. The per- O-methylated polysaccharide was fragmented by partial hydrolysis with acid, producing partially O-methylated oligosaccharides. These derivatized oligosaccharides were reduced, to afford a mixture of partially O-methylated oligoglycosyl-alditols, which was then per- O-methylated. The structures of the resulting per- O-methylated oligoglycosylalditols were determined by chemical-ionization mass spectrometry, electron-impact mass spectrometry, fast-atom-bombardment mass spectrometry, 1H-n.m.r. spectroscopy, and analysis of corresponding, partially O-acetylated, partially O-methylated alditols. Seventeen of the oligosaccharides isolated from RG-II were parts of a single heptasaccharide, namely.

高等植物におけるセルロースの生合成 現状と問題点

Cellulose is a polymer of 1, 4-β-glucan which is the most abundant organic substance on earth. It is deposited in crystalline microfibrils in the cell walls of higher plants during growth. Plant membranes may contain at least two UDP-glucose 4-β-glucosyltransferases, one with UDP-xylose that is involved in xyloglucan synthesis and the other may form cellulose. No one has not succeeded in the synthesis of cellulose at cell free system, although cellulose shouhd occur in plasma membranes. The question is “Where is the cellulose synthase in higher plants? and where do we go?”

3-deoxy- d- manno-2-octulosonic acid (KDO) is a component of rhamnogalacturonan II, a pectic polysaccharide in the primary cell walls of plants

3-Deoxy- d- manno-2-octulosonic acid (KDO), a sugar previously presumed to occur only as a glycosyl residue in polysaccharides produced by Gram-negative bacteria, was found to be a component of the cell walls of higher plants. In the form of the disaccharide α- l-Rha p-(1→5)- d-KDO, KDO was released by mild hydrolysis with acid from the purified cell wall polysaccharide rhamnogalacturonan II. KDO was shown to be present in purified cell walls of several plants, including dicots, a monocot, and a gymnosperm. Improved methods for detecting and quantitating KDO residues in polysaccharides were developed during this investigation.

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.

Cell wall glycoproteins: structure and function.

Hydroxyproline-rich glycoproteins form an important, but little understood, structural component of most cell walls. Their occurrence, chemistry, synthesis, secretion, cross-linking and functions in higher plant cell walls will be briefly reviewed. Similar molecules also occur in other groups of plants; in particular, in the algae. In many of these they form highly ordered cell surface arrays, and we have studied these by high-resolution electron microscopy and computer image reconstruction. Some resulting three-dimensional models of these are presented. One particular glycoprotein, the major structural component of the cell wall of Chlamydomonas reinhardii, has been investigated in some detail. The chemistry and structure of this glycoprotein, which we have called volvin, has been studied and a family of monoclonal antibodies has been raised against it. Some of these antibodies appear to be specific to oligosaccharide side-chains and allow the localization of these substituents and their sites of synthesis. Immunofluorescence studies have shown that the expression of some of these antigenic determinants is developmentally regulated or cell-cycle-dependent. Immunogold labelling of thin sections has enabled the sites of synthesis and the method of secretion to be determined. These results will be discussed in the context of other cell wall glycoproteins, their relation to other glycoproteins, such as the mating agglutinin, and to their possible functions.

ESR evidence for sequential divalent cation binding in higher plant cell walls

Electron spin resonance linewidth measurements have been made on intact cell walls exchanged with various combinations of Mn 2+ and Ca 2+. These experiments were performed to find the Mn 2+ nearest-neighbor distance and thereby determine whether carboxylate-Mn 2+ complexes potentiate ion association at adjacent sites on cell wall polyuronides. Our results show that as the fraction of available binding sites occupied by Mn 2+ increased from 2% to 27%, the nearest-neighbor distance parameter decreased only from 14 to 11 Å. These distances are close to polyuronide interanionic spacings. The small change in the distance parameter with concentration is evidence for sequential rather than random binding. Competitive ion-exchange with Ca 2+ was found to reduce the Mn 2+ spin-spin line broadening at similar total bound Mn 2+ concentrations. This is expected only if Ca 2+ competes at adjacent sites. The data presented offer strong support for the hypothesis that carboxylate groups near already occupied sites have a greater affinity for divalent cations than other sites along the polyuronide main chain.

The control of cellulose microfibril deposition in the cell wall of higher plants

In maize (Zea mays L.) and pine (Pinus taeda L.) seedlings, cellulose microfibril impressions are present on freeze-fractured plasma membranes. It has been proposed that impressions of newly synthesized microfibrils are a record of the movement of terminal synthesizing complexes through the plasma membrane (Mueller and Brown, 1980, J. Cell Biol. 84, 315-326). The association of terminal complexes with the ends of microfibril impressions or with the ends of microfibrils torn through the membrane indicates the orientation of microfibril tips. Unidirectionally-oriented microfibril tips (all pointing in the same direction) are associated with the organized deposition of parallel arrays of microfibrils. Multidirectionally-oriented microfibril tips were observed in a cell in which microfibril deposition was unusually disorganized. Microfibril patterns around pit fields are asymmetric and resemble flow patterns. Unidirectionally-oriented tears are associated with these microfibrils. Although microfibril orientations are deflected around pit fields, the main axis of microfibril orientation is maintained across the surface of the cell. The hypothesis is proposed that the interaction of a flowing plasma membrane with microfibril synthesizing complexes in the plane of the membrane may result in unidirectional deposition and asymmetric microfibril impressions around pit fields.

The control of cellulose microfibril deposition in the cell wall of higher plants

Cells of maize (Zea mays L.) seedling that are not fixed or cryoprotected contain the impressions of cellulose microfibrils on freeze-fractured plasma membranes. Impressions of the most recently deposited microfibrils have terminal complexes associated with them (see preceding paper). The orientations of microtubules in cytoplasmic fractures are parallel to the newest microfibrils observed on adjacent plasma membrane fractures. Small groups of microfibrils, distinguished from the next older layer by their new orientation, are sometimes observed directly adjacent and parallel to individual microtubules. Whereas microtubules are parallel to microfibril orientations which vary from transverse to occasionally longitudinal, microfilaments are parallel to the longitudinal cell axis. After colchicine treatment, cytoplasmic microtubules are absent, as are the bands of microfibrils that are observed on the membrane of control cells. Parallel orientations of microfibrils and normal pitfield outlines are often still observed after colchicine treatment. However, on some membranes, multidirectionally-oriented microfibril tips occur, associated with perturbations of microfibril orientation and rounded pit-field outlines. In ethylene-treated cells, some membranes have microfibril tips oriented in only one direction in new layers of longitudinal microfibrils. On other membranes, longitudinal bands of microfibril tips are oriented in opposing directions. We propose that after colchicine treatment, the patterns of microfibrils reflect an orientation mechanism which has been uncoupled from the influence of microtubules but which is still under some other form of cellular control. We propose that membrane flow could orient the lateral movement of synthesizing complexes in the membrane and that microtubules modulate this movement, apparently organizing the microfibrils into parallel bands in newly-forming wall layers.

Ultrastructural and biochemical effects of endopectate lyase on cell walls from cell suspension cultures of bean and rice

Cell walls prepared from suspension cell cultures of bean and rice in log-phase growth were used to examine the effects of endopectate lyase (PL) on the solubilization of cell wall carbohydrates and concomitant ultrastructural alterations. Cell wall preparations from both plant sources were heterogeneous and contained a range of wall types from primary walls to xylem elements with spiral, secondary wall thickenings. Marked differences in wall thickness and number of wall laminations typified both preparations.Bean cell walls were more susceptible to degradation by PL than were those of rice. Upon treatment of the former with 2.3 × 10−3 units/mL of PL (1 unit released 1 μmol of unsaturated uronide/min at 30 °C from polygalacturonic acid at pH 8.5), 27% of the noncellulosic wall carbohydrate was solubilized in 1 h. This represented 50% of the PL susceptible carbohydrate in the preparation. Only 3% of the noncellulosic carbohydrate was released from rice cell walls in 1 h when treated with 115 × 10−3 units of PL/mL. This accounted for 60% of the PL susceptible wall fractions. Only uronic acid, rhamnose, galactose, and arabinose were solubilized from both preparations by PL.Cell walls in the bean and rice preparations were affected differentially by the PL. Those walls with secondary thickenings did not appear to be degraded, while the distinct fibrillar appearance of both bean and rice walls tended to fade or disappear. The middle lamella tended to dissolve to varying degrees in the presence of PL. Bean walls were more severely degraded than were the rice walls and many exhibited swelling, separation of wall layers, markedly reduced staining intensity, and (or) a granular ultrastructure.This study has demonstrated that as PL acts on susceptible cell walls there are major changes evoked in cell wall structure which suggest that the rhamnogalacturonan fraction of the higher plant cell wall contributes significantly to cell wall integrity. This study also emphasizes the need for cell wall material of proven uniformity for investigations of both cell wall composition and effects of specific polysaccharide degrading enzymes on cell wall integrity. Preliminary studies indicate that tobacco pith may provide more uniform cell walls than do cell suspension cultures.

Wall Analyses of Lophocolea Seta Cells (Bryophyta) Before and After Elongation

Lophocolea heterophylla (Schrad.) Dum. (a leafy liverwort) produces sporophytes with seta cells that elongate 50-fold in 3 to 4 days. Wall components of these cells have been characterized by microscopic histochemistry, colorimetry, and gas chromatography of neutral sugars. Seta cell walls are qualitatively similar to primary cell walls of higher plants. The pectic fraction, however, responds differently to standard histochemical staining and extraction. Quantitatively, mannose, fucose, and rhamnose are in higher percentage, and arabinose and xylose are lower than typically found in vascular plants. Hexuronic acids increase on a percentage basis during elongation; pentoses decrease slightly, while hexose levels remain about the same. Increase in total wall carbohydrate after 2,400% elongation of setae was 1.8-fold.

The use of an optical brightener in the study of plant structure.

An optical brightener Calcoflour White M2R New has been used to stain cell walls of higher plants. It can be used either as a vital stain for intact plants or for hand sections and plastic-embedded thin sections. Walls are brilliantly fluorescent while most cytoplasmic components are normally unstained. The brightener binds strongly to cellulose, carboxylated polysaccharides, and callose. Staining for 20 sec to 2 min in a 0.01% solution of the brightener is preferred for most purposes.

Release of enzymes from cell walls by an endopectate-trans-eliminase.

THE cell walls of higher plants were long regarded as essentially inert structures which functioned mainly as mechanical barriers to the expansion of protoplasts under turgor pressure and as skeletal supports in tissues. It is now known, however, that they contain a number of hydrolytic enzymes at least one of which is a hydroxyproline-rich protein1. These enzymes are often of types associated with the dynamic recycling functions of intracellular lysosomes2–4. They have usually been detected by the activity of cell wall preparations after comminution of tissues and removal of contaminating cytoplasm by washing1,2,5 or by the activity of intact or excised roots3,6. We now report on the release of wall bound enzymes from living cells by a pectic enzyme.

Synthesis and Secretion of Hypoxyproline-containing Macromolecules in Carrots

Using pulse-chase experiments with radioactive proline, it is possible to study the rapid transfer from the cytoplasm to the cell wall of the hydroxyproline-rich protein found in the cell walls of higher plants. The secretion of this protein is not obligatorily coupled to protein synthesis. Secretion is completely inhibited by uncouplers of oxidative phosphorylation and strongly inhibited by the inhibitors of electron transport, cyanide and azide. It is concluded that the transfer of proteins from the cytoplasm to the cell wall is an energy-requiring step.

Fine structure of natural polysaccharide systems

AbstractThe ultrastructure of cellulose in higher plant cell walls is discussed. The primary and secondary wall is built up of a framework of microfibrils composed of smaller strands which are termed elementary fibrils. In recent years a number of models for the chain‐arrangement of the cellulose molecules have been proposed. According to one view the cellulose chains are in a folded conformation. Based on a new method which allows the determination of the chain‐arrangement in the elementary fibrils it was found that the structural carbohydrate must be aggregated into continuous, extended‐chain filaments of nearly perfect crystallinity. Freezeeten profiles suggest that the mechanism of fibrillar formation is characterized by enzyme particles attached to the plasma membrane and involved in cellulose synthesis.

The presence of ribonucleic acid in the cell walls of higher plants.

A method for isolating extensively purified cell walls from higher plants is described; the preparations contain no detectable chloroplast or nuclear material and the protein content (2-5% of the dry wt. of walls) indicates that there is little contamination with cytoplasm. Incubation of purified cell walls with 0.3n-potassium hydroxide for 17hr. at 37 degrees liberates ribonucleotides, which can be purified by adsorption on charcoal and by ion-exchange chromatography. Ribonucleotides are also liberated by incubating the walls with ribonuclease, but not with deoxyribonuclease. The RNA content varies from 0.5 to 6mg./g. dry wt. of walls, depending on the nature and age of the tissue, and at 3mg./g. dry wt. of walls accounts for about 7% of the total RNA of the tissue. Less than 0.2% of the RNA of the walls is due to the presence of bacteria in the preparation. The base composition of the cell-wall RNA is identical with that of ribosomal RNA.