An isoelectric focusing study of the effect of methyl-esterified pectic substances on the production of extracellular pectin isoenzymes by soft rot Erwinia spp.

The production of extracellular pectic isoenzymes by seven strains of soft rot bacteria, Erwinia carotovora subsp. carotovora, E.c. atroseptica and E. chrysanthemi , when grown in media containing four different pectic substances with different degrees of methylation or with potato tuber cell-wall extract was examined by isoelectric focusing activity staining. In addition to the isoenzymes of pectate lyase, polygalacturonase and pectin methyl esterase produced constitutively or following induction by polygalacturonic acid (PGA) and coded by known genes, between two and seven novel isoenzymes of the three enzymes with a wider pI range were apparently induced by the pectins and cell-wall extract. Pectin lyase, which is induced in vitro by DNA-damaging agents, was not produced in the absence of mitomycin C in a medium containing PGA but up to two isoenzymes were found with pectin or cell-wall extract. In contrast, cellulase isoenzyme production was not affected by pectin or cell-wall extract. A greater number of novel isoenzymes of all pectic enzymes except pectin lyase tended to be produced in media containing Link pectin, which is PGA methylated to 98%, than the other pectic substances and cell-wall extract. Pectate lyase and polygalacturonase were induced by pectin lyase-degraded products of highly methylated pectin but not by PGA in an E. chrysanthemi strain with all its known pei and peh genes mutated. The results suggest that the production of novel pectic isoenzymes could be related to the presence of CH⁺₃ groups and that their induction differs from that for isomers induced by PGA-degraded products and DNA-damaging agents or produced constitutively.     

Polysaccharide compositions of primary cell walls of the palms Phoenix canariensis and Rhopalostylis sapida

The polysaccharide compositions of unlignified primary cell walls from two species of palms were examined. Cell-wall preparations were isolated from the stem apex, including the pre-emergent leaflets and rachides, of Phoenix canariensis (Canary Island date palm), and from leaflets and rachides dissected from pre-emergent leaves in the stem apex of Rhopalostylis sapida (Nikau palm). The non-cellulosic polysaccharides in the cell-wall preparations from both species had similar monosaccharide compositions, with arabinose and galactose being the predominant neutral monosaccharides, together with large amounts of galacturonic acid. These monosaccharide compositions indicated the presence of large proportions of pectic polysaccharides, including homogalacturonans. This was confirmed by linkage analyses of the cell-wall preparations which showed the presence of large proportions of pectic arabinans, together with pectic galactans and/or Type I arabinogalactans. Evidence for rhamnogalacturonan I and small amounts of rhamnogalacturonan II was also obtained. In addition to pectic polysaccharides, the cell-wall preparations contained smaller amounts of xyloglucans and even smaller amounts of heteroxylans, probably glucuronoarabinoxylans, and glucomannans and/or galactoglucomannans; (1→3,1→4)-β-D-glucans were not present. Although palms (Arecaceae) are commelinoid monocotyledons, the polysaccharide compositions of their primary cell walls resemble those of non-commelinoid monocotyledons and dicotyledons. These compositions contrast with those of primary cell walls of other commelinoid families which have glucuronoarabinoxylans rather than pectic polysaccharides as the major non-cellulosic polysaccharides. The results are discussed in relation to the possible evolution of the composition of primary cell walls of monocotyledons.     

Structural features of cell-wall polymers of the apple

Cell-wall material was isolated from ripe-apple cortical tissues by sequential extraction with aqueous 1.5% sodium dodecyl sulphate and aqueous 90% methyl sulphoxide. The wall material, which contained ～1% of protein, with proline and hydroxyproline as the preponderant amino acids, was sequentially extracted with water at 80°, oxalate at 80°, m KOH at 1°, and m and 4m KOH at 20°, to leave a residue of α-cellulose, which was associated with an appreciable amount of arabinose-rich pectic material. The depectinated material was also extracted with 6m guanidinium thiocyanate at 20° to solubilise preferentially polysaccharides rich in mannose. The hot-water-soluble pectic substances were richer in arabinose compared with the oxalate-soluble ones and were resolved into five fractions by anion-exchange chromatography. The bulk of the hemicelluloses, which were xyloglucans, were solubilised by 4m KOH. The alkali-soluble hemicellulose polymers were resolved by anion-exchange chromatography into polysaccharides, mainly xyloglucans, arabinoxylan-pectic-xyloglucan, and arabinoxylan-pectic complexes. Small amounts of polysaccharide-protein-polyphenol complexes (where the polysaccharide moieties were arabinoxylans), pectic substances, and xyloglucans were also present. The glycosidic linkages of the above polymers were determined by methylation analysis. The general structural features of the cell-wall polymers are discussed.     

Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea

The biochemistry of cell-wall regeneration in protoplasts obtained from Vinca rosea L. (Catharanthus roseus (L.) G. Don) cells grown in suspension culture by isolating the regenerated wall and the extracellular polysaccharides of protoplasts cultured for various periods, and investigating their composition. Gas-liquid chromatography and tracer studies with D-[U-¹⁴C]glucose showed that the sugar composition of the extracellular polysaccharides was similar to that of the original cell culture, consisting mainly of polyuronide and 3,6-linked arabinogalactan. the regenerated cell wall was composed of non-cellulosic glucans having 1,3- and 1,4-linkages, while its content in pectic and hemicellulosic components was very low.     

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.     

Heterogenous Degradation of Myofibrillar Proteins

Pectic substances were extracted from the vegetables with oxalate buffer of pH 4.25 and, after saponification, fractionated into two components, weakly acidic pectic polysaccharide (WAP) and pectic acid, by DEAE-cellulose and Sephadex G-100 chromatographies. The galacturonic acid content (17.3~25.8%) of WAPs was much lower than that of pectic acids, though the neutral sugar compositions of both pectic substances were almost the same. The arabinose-galactose side chains were found to be very long or highly branched in WAPs compared with those in pectic acids.

All the WAPs were appreciably hydrolyzed by exo- and endopolygalacturonases. The limited-degradation products (the residual polysaccharides; i.e., the rhamnogalacturonan segments) obtained by endopolygalacturonase from both WAPs and pectic acids showed a similar behavior on Sephadex G-100 and Sepharose CL-4B gel filtrations; each of the rhamnogalacturonan segments was eluted in the void volume of the Sephadex G-100 column. From these results, we concluded that WAPs are probably an inherent pectic component of the cell walls of the vegetables.     

Effect of Abscisic Acid on Cell-Wall Metabolism in Guard Cells of Vicia faba L.

Cell-wall synthesis in guard cells of Vicia faba L. was examinedusing sonicated epidermal strips incubated with [¹⁴C]glucose.The cell walls of the guard cells incorporated [¹⁴C]glucoseat a lower level in the dark than in the light. Stomatal aperturein the epidermal strips was reduced by application of 1 µmabscisic acid (ABA) in the light but not in the dark. The ABAtreatment reduced the incorporation of [¹⁴C]glucose into thecell walls especially in the light. Fractionation of the labeledcell-wall components revealed that ABA inhibited the synthesisof pectic substances and cellulose, but did not affect hemicellulosesynthesis. Microautoradiographs of the cell-wall fraction ofthe epidermal strips showed that a large amount of radioactivitywas distributed at both ends of the guard cells in the absenceof ABA and that removal of pectic substances from the cell-wallfraction resulted in uniform distribution of the radioactivityin the cell walls of the guard cells. These results indicatedthat the synthesis of pectic substances was active at both endsof the guard cells and was inhibited by ABA. Measurement ofspecific activities of neutral sugars in the guard-cell wallsshowed that polymers composed of galactose underwent activeturnover and that synthesis of glucans was inhibited by ABA.These results revealed a strong correlation between the stomatalmovement and the synthesis of pectic substances and cellulosein the guard cells, suggesting that the cell-wall metabolismin the guard cells may play a role in the regulation of stomatalmovement. (Received October 9, 1987; Accepted March 9, 1988)     

Modification of cell-wall polymers of onion waste: III. Effect of extrusion-cooking on cell-wall material of outer fleshy tissues

The polymers of onion cell walls are known to be modified by heating, but there is little information on the effects of extrusion-cooking. This work investigates the effects of extrusion-cooking on the physico-chemical characteristics and microstructure of cell walls of onion waste in relation to cell-wall chemistry. Cell-wall material from white fleshy outer scale leaves of waste onions was extruded at a range of moisture contents, barrel temperatures and screw speeds through a co-rotating twin-screw extruder. Extrusion-cooking had little effect on the carbohydrate composition of cell-wall material. However, it resulted in an increase in the solubility of pectic polymers and hemicelluloses, and this was accompanied by an increase in swelling of the cell-wall material. The degree of solubility of the pectic polysaccharides was largely dependent on the barrel temperature, and involved depolymerisation.     

Isolation and Identification of Native Auxins in Marine Algae

Endosperm cell walls were isolated from rice grains and their chemical composition was analyzed. The cell walls were composed of cellulose microfibrils and matrix phase which consisted of hemicellulose and pectic substances. Hemicellulose mainly comprised arabinoxylan, accompanied by a small amount of glucose-containing polysaccharide. Pectic substances contained polygalacturonides, some of which had side chains containing neutral sugars such as galactose and arabinose. Amino acid analysis of these fractions suggested that hydroxyproline-containing glycoproteins were contained in these cell walls and firmly bound to cellulose microfibrils.     

Pectic polysaccharides of cabbage (Brassica oleracea)

Pectic substances extracted from cabbage cell walls with water, at 80°, and (NH₄)₂C₂O₄, at 80°, accounted for 45%(w/w) of the purified cell wall material. Only a small amount of neutral arabinan was isolated. Partial acid hydrolysis and methylation analysis revealed that the major pectic polysaccharide had a rhamnogalacturonan backbone to which a highly branched arabinan was linked, at C-4 of the rhamnose units, mainly through short chains of (1→4)-linked galactopyranose residues. The bulk of the soluble pectic substances had only small amounts of proteins associated with them. After further extraction of the depectinated material with 1M and 4M KOH, to remove the hemicelluloses, the cellulose residue was found to contain a pectic polysaccharide which was solubilized by treatment with cellulase. The general structural features of the pectic polymers are discussed in the light of these results.     

Effects of gibberellic Acid, calcium, kinetic, and ethylene on growth and cell wall composition of pea epicotyls

The influence of gibberellic acid (GA), calcium, kinetin, and ethylene on growth and cell-wall composition of decapitated pea epicotyls (Pisum sativum L. var. Alaska) was investigated. Calcium, kinetin, and ethylene each caused an inhibition of GA-induced elongation of pea stems. Gibberellic acid did not reverse the induction of swelling by Ca²⁺, kinetin, or ethylene. Both Ca²⁺ and ethylene significantly inhibited the stimulatory effects of GA on the formation of residual wall material. Although GA promoted the development of walls relatively low in pectic substances and pectic uronic acid, Ca²⁺, kinetin, and ethylene favored the formation of walls rich in these constituents. Calcium, kinetin, and GA, alone or in combination, had no effect on the production of ethylene by pea epicotyls.     

A comparative study of the extracellular and cell-wall polysaccharides of someCandida species

Candida bogoriensis, C.buffonii, C.diffluens, C.foliarum andC.javanica, produce an extracellular polysaccharide which contains galactose, glucose, mannose, fucose and rhamnose. These sugars were also found in cell-wall preparations of the same yeast species. The cell-wall preparations ofC.diffluens andC.foliarum included capsular material. The similarity in composition of the extracellular polysaccharides and components of the cell wall suggests that both are synthetized by the same enzymatic system. The fiveCandida species may be closely related.     

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.     

Light-scattering studies of pectic substances in aqueous solution

The behaviour of pectic substances in aqueous solution has been studied by the light-scattering technique. Pectic substances were found to be present as high molecular aggregates having strong, intermolecular interactions, and their behaviour depends upon the nature of the carbohydrate chain. Additives can cause dissociation of the polysaccharide aggregates, resulting, in some cases, in particles having similar weight-average ( _w) and number-average ( _n) molecular weights.     

Isolation and characterization of an extracellular hydroxyproline-rich glycoprotein and a mannose-rich polysaccharide from Eudorina californica (Shaw)

Mucilage and colony walls of E. californica were separated from the cells by homogenization, filtration, and differential centrifugation. The chief components of the mucilage were a high-molecular-weight (MW) hydroxyproline-rich glycoprotein and a very high-MW polysaccharide in the proportions 47% and 34%, respectively. The glycoprotein consisted of galactose, arabinose, xylose and an unidentified neutral sugar; and the amino acids cysteine, aspartic acid, glutamic acid, arginine, lysine, glycine, serine, methionine, histidine, alanine, proline, hydroxyproline, tyrosine, threonine, valine, phenylalanine, isoleucine and leucine. The principal sugar of the polysaccharide was mannose. The chemical composition of the colony walls was essentially the same as that of the glycoprotein in the mucilage except that there was almost twice as much hydroxyproline. Also the protein content of the colony walls was 34% while that of the glycoprotein in the mucilage was 22%. No glucose, sugar acids or nucleic acids were found in the extracellular matrix.     

THE COMPOSITION OF THE DEVELOPING PRIMARY WALL IN ONION ROOT TIP CELLS. II. CYTOCHEMICAL LOCALIZATION

Jensen , William A. (U. California, Berkeley.) The composition of the developing primary wall in onion root tip cells. II. Cytochemical localization. Amer. Jour. Bot. 47(4) : 287—295. Illus. 1960.–The composition of the developing cell wall in the first 2 mm. of the onion root tip was studied using a cytochemical technique that permitted the detection of hemicellulose and the noncellulosic polysaccharides as well as the pectic substances and cellulose. The technique is based on the combination of a differential extraction procedure with the periodic acid-Schiff reaction for carbohydrates. The data obtained indicate that the cells of the apical initials are low in all wall substances but that all of the wall materials are present to some extent. Early in cell development, differences appear in the composition of the walls of the various tissues. The cortical cells are relatively high in the noncellulosic polysaccharides and cellulose while relatively low in the pectic substances and hemicellulose. Very early in development the protoderm is similar to the cortex, but differences develop during the radial enlargement of the cells. During this stage the walls of the protodermal cells are low in the noncellulosic polysaccharides and cellulose and high in pectic substances and hemicellulose. As elongation progresses, these differences are lost and the 2 tissues become very similar. The vascular cell walls are low in the noncellulosic polysaccharides and cellulose and are high in pectic substances and hemicellulose early in development. Later, hemicellulose becomes relatively more important. When the cell wall materials are sequentially extracted, no change in the general morphology of the cell occurs until only the noncellulosic polysaccharides and the cellulose remained. When the noncellulosic polysaccharides are then removed, the cells remain intact but are 30% less in diameter. This suggests that while cellulose is of critical importance, the noncellulosic polysaccharides may play a major role in determining the physical characteristics of the wall.     

FT-IR study of the Chara corallina cell wall under deformation

Fourier-transform infrared (FT-IR) microspectroscopy was used to investigate both the chemical composition of, and the effects of an applied strain on, the structure of the Chara corallina cell wall. The inner layers of the cell wall are known to have a transverse cellulose orientation with a gradient through the thickness to longitudinal orientation in the older layers. In both the native state and following the removal of various biopolymers by a sequential extraction infrared dichroism was used to examine the orientation of different biopolymers in cell-wall samples subjected to longitudinal strain. In the Chara system, cellulose microfibrils were found to be aligned predominantly transverse to the long axis of the cell and became orientated increasingly transversely as longitudinal strain increased. Simultaneously, the pectic polysaccharide matrix underwent molecular orientation parallel to the direction of strain. Following extraction in CDTA, microfibrils were orientated transversely to the strain direction, and again the degree of transverse orientation increased with increasing strain. However, the pectic polysaccharides of the matrix were not detected in the dichroic difference spectra. After a full sequential extraction, the cellulose microfibrils, now with greatly reduced crystallinity, were detected in a longitudinal direction and they became orientated increasingly parallel to the direction of strain as it increased.     

Host-plant resistance of sorghum: Differential hydrolysis of sorghum pectic substances by polysaccharases of greenbug biotypes (Schizaphis graminum,homoptera: Aphididae)

Pectic substances extracted from different varieties of sorghum are hydrolyzed at differing rates by unfractionated polysaccharases isolated from two biotypes (C, GBC; and E, GBE) of the sorghum pest, Schizaphis graminum (the greenbug). A higher degree of susceptibility of a sorghum variety is associated with a greater rate of hydrolysis of sorghum pectic substances by a greenbug biotype. Increases in the specific activity of polysaccharases on the pectic substances from a resistant sorghum variety are dependent on the duration that a biotype is maintained as a colony on that variety. Polysaccharase activity of GBE on arabinogalactan was significantly greater than GBC. However, there were no differences between the biotypes on the depolymerization of a variety of other plant matrix polysaccharides and a synthetic polysaccharide. The sequence of substrates of increasing refractoriness to hydrolysis are: arabinogalactan < microcrystalline cellulose < xylan < pectin < 2,3-diacetyl pectin < α-1,4-galacturonan. Pectic substances from sorghum varieties resistant to GBC but susceptible to GBE are relatively lower in arabinogalactan with elevated levels of uronic acid (UA) compared to varieties susceptible to both biotypes. A sorghum variety resistant to both GBC and GBE was lowest in levels of arabinogalactan, highest in UA, and highest in fructan content, which in the other varieties occurred only in trace amounts. Pectic composition of rhamnose, xylose, and glucose showed no relationship to resistance. Bound phenolics (potential inhibitors of enzyme activity) were not detected in any of the sorghum pectic substances. The relationship of plant matrix polysaccharides to host-plant aphid biotype compatibility is discussed.     

A simple procedure for the synthesis of alpha-32P-nucleoside triphosphates.

Chemical analysis of grapefruit (Citrus paradisi) pectic polysaccharides demonstrated that galacturonic acid constitutes 78% by weight of the total carbohydrates found. The remaining 22% was accounted for by a number of sugars which include galactose, glucose, arabinose, xylose, and mannose and, by weight, galactose accounted for almost 50% of the total neutral sugar components found in these pectic polysaccharides. Treatment of pectic polysaccharides with galactose oxidase followed by reduction of oxidized galactose residues with tritiated potassium borohydride resulted in the labeling of pectic polysaccharides. Analysis of the labeled polysaccharides demonstrated that of the total radioactivity incorporated more than 90% was recovered in the galactose residues. These results clearly demonstrate the successful utilization of the galactose oxidase/tritiated potassium borohydride method in labeling plant pectic polysaccharide.     

Fractionation and characterization of polysaccharides from abaca fibre

Abaca fibre polysaccharides were fractionated into water soluble, pectic, 1% NaOH soluble, hemicellulosic and cellulose fractions by extraction with hot water, dilute hydrochloric acid (pH 1.6), aqueous 1% NaOH and 17.5% NaOH, respectively. Cellulose (60.4–63.6%) and hemicelluloses (20.8%) were the major polysaccharides in abaca fibres. The hot water soluble polysaccharides contained noticeable amounts of pectic substances and a large proportion of neutral polysaccharides. The pectic polysaccharide preparation was enriched in both galacturonic acid and neutral sugars, including xylose, glucose, galactose, arabinose, and rhamnose. Extraction of the fibre with aqueous 1% NaOH produced the hemicellulose–lignin complex, which was enriched in xylose and, to a lesser extent, glucose-, arabinose- and galactose-containing polysaccharides, together with 7.6% associated lignin. Further extraction of the delignified fibre residue with aqueous 17.5%. NaOH removed the hemicellulose fractions, which were strongly enriched in xylose-containing polysaccharides. Besides ferulic and p-coumaric acids, six other phenolic monomers were also detected in the mixtures of alkaline nitrobenzene oxidation of associated lignin in all the polysaccharide fractions. The content of bound lignin in water soluble, pectic, and 1% NaOH soluble polysaccharides (Fractions 1, 2, and 3), isolated directly from the lignified fibres, was 12 times that of the hemicellulosic preparations (Fractions 4 and 5) isolated from the delignified fibre residues.