Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Turnover of cell wall components was examined in two growth phases of a batch suspension culture of Vinca rosea L. Three-day-cultured cells (cell division phase) and 5-day-cultured cells (cell expansion phase) were incubated with d -[U-¹⁴C]glucose. After various periods of incubation, extra-cellular polysaccharides (ECP) and cell walls were isolated, and then the cell walls were fractionated to pectic substance, hemicellulose, and cellulose fractions. The results of the measurement of radioactivities and amounts of total carbohydrate in the ECP and cell wall fractions indicated that synthesis of pectic substance was more active in the cell division phase than in the cell expansion phase. From the results of the pulse-chase experiments, in which cells prelabelled by incubation with d -[U-¹⁴C]glucose for 3 h were incubated in a medium containing unlabelled glucose for various periods, the gross degradation, net synthesis, and gross synthesis of cell wall components were estimated. Active degradation and synthesis were observed in the hemicellulose fraction, indicating that active turnover occurred in the hemicellulose fraction, while little degradation was found in the pectic substance and cellulose fractions.     

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up ∼90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of α-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.     

Cell wall polysaccharides from fern leaves: evidence for a mannan-rich Type III cell wall in Adiantum raddianum

Primary cell walls from plants are composites of cellulose tethered by cross-linking glycans and embedded in a matrix of pectins. Cell wall composition varies between plant species, reflecting in some instances the evolutionary distance between them. In this work the monosaccharide compositions of isolated primary cell walls of nine fern species and one lycophyte were characterized and compared with those from Equisetum and an angiosperm dicot. The relatively high abundance of mannose in these plants suggests that mannans may constitute the major cross-linking glycan in the primary walls of pteridophytes and lycophytes. Pectin-related polysaccharides contained mostly rhamnose and uronic acids, indicating the presence of rhamnogalacturonan I highly substituted with galactose and arabinose. Structural and fine-structural analyses of the hemicellulose fraction of leaves of Adiantum raddianum confirmed this hypothesis. Linkage analysis showed that the mannan contains mostly 4-Man with very little 4,6-Man, indicating a low percentage of branching with galactose. Treatment of the mannan-rich fractions with endo-β-mannanase produced characteristic mannan oligosaccharides. Minor amounts of xyloglucan and xylans were also detected. These data and those of others suggest that all vascular plants contain xyloglucans, arabinoxylans, and (gluco)mannans, but in different proportions that define cell wall types. Whereas xyloglucan and pectin-rich walls define Type I walls of dicots and many monocots, arabinoxylans and lower proportion of pectin define the Type II walls of commelinoid monocots. The mannan-rich primary walls with low pectins of many ferns and a lycopod indicate a fundamentally different wall type among land plants, the Type III wall.     

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans

 Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na₂CO₃, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides. Received: 29 May 1999 / Accepted: 19 October 1999     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Enzymic analysis of cell wall structure in apple fruit cortical tissue

Galactanase from Phytophthora infestans and an arabinosidase isoenzyme from Sclerotinia fructigena attacked the cortical cell walls of apple fruits liberating galactose and arabinose residues, respectively. Other arabinosidase isoenzymes from S. fructigena attacked cell walls very slowly. A S. fructigena polygalacturonase isoenzyme liberated half of the uronic acid residues with few associated neutral residues, while a second polygalacturonase isoenzyme released more uronic acid with a substantial proportion of arabinose and galactose and lesser amounts of xylose, rhamnose and glucose; reaction products of this enzyme could be further degraded by the first isoenzyme to give high MW fragments, rich in arabinose with most of the xylose, rhamnose and glucose, and low MW fragments rich in galactose and uronic acid. Endoglucanase from Trichoderma viride released a small proportion of the glucose residues from cell walls together with uronic acid, arabinose, xylose and galactose; more extensive degradation occurred if walls were pre-treated with the second polygalacturonase isoenzyme. Endoglucanase reaction products were separated into a high MW fraction, rich in arabinose, and lower MW fractions rich in galactose and glucose residues. The high MW polygalacturonase and endoglucanase products could be degraded with an arabinosidase isoenzyme to release about 75% of their arabinose. Cell walls from ripe fruit showed similar susceptibility to arabinosidase and galactanase to those from unripe apples. Cell walls from fruit, ripened detached from the tree were more susceptible to degradation by polygalacturonase than walls from unripe fruit or fruit ripened on the tree. Endoglucanase released less carbohydrate from ripe fruit cell walls than from unripe fruit cell walls.     

Turnover of cell wall polysaccharides of a Vinca rosea suspension culture

Three-day-cultured cells of Vinca rosea L. (in the cell division phase) and 5-day-cultured cells (in the cell expansion phase) prelabelled with d -[U-¹⁴C] glucose were incubated in a medium containing unlabelled glucose. After various periods of chase, extra-cellular polysaccharides (ECP) and cell walls were isolated, and cell walls were fractionated into pectic substances, hemicellulose, and cellulose fractions. After acid hydrolysis, the radioactive constituents in the pectic substances and hemicellulose fractions were analyzed. Active turnover was observed in arabinose and galactose in the hemicellulose fraction of cell walls, while the constituents of the pectic substances, and xylose and glucose in the hemicellulose fraction did not undergo active turnover. The proportion of radioactivities of arabinose and galactose in total radioactivity of ECP increased markedly after chasing. These results indicate that arabinogalactan was synthesized, deposited in the cell wall, degraded rapidly, and made soluble in the medium as a part of ECP.     

Water stress and cell wall polysaccharides in the apical root zone of wheat cultivars varying in drought tolerance

Glycosyl composition and linkage analysis of cell wall polysaccharides were examined in apical root zones excised from water-stressed and unstressed wheat seedlings (Triticum durum Desf.) cv. Capeiti ("drought-tolerant") and cv. Creso ("drought sensitive"). Wall polysaccharides were sequentially solubilized to obtain three fractions: CDTA+Na(2)CO(3) extract, KOH extract and the insoluble residue (alpha-cellulose). A comparison between the two genotypes showed only small variations in the percentages of matrix polysaccharides (CDTA+Na(2)CO(3) plus KOH extract) and of the insoluble residues (alpha-cellulose) in water-stressed and unstressed conditions. Xylosyl, glucosyl and arabinosyl residues represented more than 90mol% of the matrix polysaccharides. The linkage analysis of matrix polysaccharides showed high levels of xyloglucans (23-39mol%), and arabinoxylans (38-48mol%) and a low amount of pectins and (1-->3), (1-->4)-beta-d-glucans. The high level of xyloglucans was supported by the release of the diagnostic disaccharide isoprimeverose after Driselase digestion of KOH-extracted polysaccharides. In the "drought-tolerant" cv. Capeiti the mol% of side chains of rhamnogalacturonan I and II significantly increased in response to water stress, whereas in cv. Creso, this increase did not occur. The results support a role of the pectic side chains during water stress response in a drought-tolerant wheat cultivar.     

The functions of cell wall polysaccharides in composition and architecture revealed through mutations

The plant cell wall is a highly organized composite of many different polysaccharides, proteins and aromatic substances. These complex matrices define the shape of each individual cell, and ultimately, they are the determinants of plant morphology. The fine structures of the major angiosperm cell wall polysaccharides have been characterized, but it is not well understood how these polysaccharides are assembled into a metabolically active architecture. Cell wall biogenesis and remodeling may be partitioned into six major stages of development (precursor synthesis, polymerization, secretion, assembly, rearrangement and disassembly), and to date, a handful of mutations have been identified that affect the composition and structure in each of these stages. To greatly augment this collection, we have initiated a program to use Fourier transform infrared spectroscopy as a high through-put screen to identify a broad range of cell-wall mutants of Arabidopsis and maize. We anticipate that such mutants will be useful to probe the impact of the individual components and their metabolism on basic processes of plant growth and development. The structures of dicot and grass walls, the identification of representative cell wall mutants, and the use of a novel spectroscopic screen to identify many more cell wall mutants, are briefly reviewed.     

Differences in cell wall polysaccharides of several species of Eupenicillium

Abstract A β-(1–5)-galactofuran was isolated and characterized from fraction F1S (alkali- and water-soluble) of the cell wall of most of the species of Eupenicillium . In E. cryptum, E. euglaucum and E. nepalense the galactan contained galactofuranose with different linkages in addition to β-(1–5). Fraction F1I (alkali-soluble, water-insoluble) was an α-glucan in certain species while in other it was a =gb-glucan. Xylose was detected in some species in F1I or in F3 (alkali-soluble at 70°C). The most abundant fraction (F4), resistant to the alkali treatment, was a β-glucan-chitin complex. Excepting this component, the β-(1–5)-galactofuran was the polysaccharide which appeared more frequently in the cell wall of species of Eupencillium and it may have chemotaxonomic relevance.     

Subcellular location and composition of the wall and secreted extracellular sulphated polysaccharides/proteoglycans of the diatomStauroneis amphioxys Gregory

Summary Extracellular polysaccharide/proteoglycan (EPS) mucilages play a crucial role in maintaining the structure of the extensive algal sheets that appear along the undersurface of nearshore Antarctic sea ice during the austral spring. In this study we have determined the composition and ultrastructural location of a family of novel sulphated polysaccharides/proteoglycans from the pennate ice diatomStauroneis amphioxys Gregory. They occur as soluble EPS in the culture supernatant, as an intercellular mucilage sheet, and as components of a distinct organic layer (diatotepum) underlying the silicious cell wall. The ultrastructural location and quantitative extraction of the mucilage EPS and the major diatotepum polysaccharides with hot water and alkali, respectively, was monitored by light and electron microscopy. The EPS and wall components were purified by Ultrafiltration, anion exchange and gel filtration chromatographies, and their monosaccharide composition was determined by gas-chro-matography mass spectrometry. The soluble and mucilage EPS, and major diatotepum polysaccharides/proteoglycans had an apparent molecular mass greater than 2 × 10⁶ Da on gel. They contained a similar complex monosaccharide composition that includes glucuronic acid and galactose as the major sugars and significant levels of rhamnose, fucose, arabinose, xylose, mannose, glucose and the mono-O-methylated monosaccharides 3-O-methylrhamnose, 3-O-methylfucose, 3-O- and 4-O-methylxylose. The ratios of Gal to GlcA, which together account for 45% of the monosaccharides, varied from 0.8 (in the soluble EPS) to 2.3 (in diatotepum polysaccharides). The level of sulphation also varied from 5–15% (w/w), with the mucilage EPS being the most highly sulphated. The soluble EPS also contains a small amount of protein (ca. 5%, v/w) which cochromatographs with the polysaccharide during gel filtration and anion exchange chromatographies suggesting that it may be a sulphated proteoglycan. They are clearly distinct from a sulphated glucuronomannan that remained in the alkali-insoluble fraction and may be tightly associated with the silica wall components. The amount of mucilage EPS increased during logarithmic growth but decreased during stationary phase, when most of the EPS was found in the soluble pool. These changes correlate with the breakdown of the mucilage sheet and dispersal of diatom colonies during stationary growth. Interestingly, the soluble EPS from stationary-growth cultures was indistinguishable from the mucilage EPS of logarithmic- or stationary-phase cells, suggesting that the dissolution of the intercellular mucilage was not due to a change in EPS composition. The possibility that cell motility may be required for mucilage formation and the significance of these polysaccharides in the under-ice community is discussed.     

Identification of lucerne stem cell wall traits related to in vitro neutral detergent fibre digestibility

Genetic improvement of forage digestibility, especially utilizing marker assisted selection and recombinant DNA techniques, requires identification of specific biochemical traits and associated genes that impact digestibility. We undertook a study to identify cell wall (CW) traits of lucerne (Medicago sativa L.) stems that were consistently and strongly correlated with in vitro neutral detergent fibre (NDF) digestibility, a measurement that has been shown to correlate with animal performance. Spring and summer harvested lucerne stem material, for 2 years, from 24 individual plants in each of two germplasm sources were analyzed for 16 and 96 h in vitro NDF digestibility, and cell wall concentration and composition (monosaccharide constituents of cellulose, hemicellulose, and pectin; and Klason lignin (KL)) by the Uppsala dietary fibre method using near-infrared reflectance spectroscopy (NIRS). Pearson correlation coefficients were calculated for the relationships among these cell wall traits and with in vitro NDF digestibility. Concentrations of the pectin monosaccharide components were all negatively correlated (r=−0.73 to −0.94) with total cell wall concentration. In contrast, the three most abundant cell wall components glucose (Glc), xylose (Xyl) and Klason lignin were not correlated, or only weakly positively correlated (r<0.35), with cell wall concentration. Cell wall concentration was consistently negatively correlated (r=−0.60 to −0.94) with both 16 and 96 h in vitro NDF digestibility. In contrast, Klason lignin concentration was only marginally correlated (r<0.30) with 16 h in vitro NDF digestibility, but strongly negatively correlated (r=−0.71 to −0.74) with 96 h in vitro NDF digestibility. This is consistent with previous reports which show that lignin affects potential extent of digestion, but not rate. Cell wall glucose and xylose concentrations were inconsistently correlated with fibre digestibility. The monosaccharide components of pectin were consistently positively correlated (r=0.54–0.90) with in vitro NDF digestibility, except for 96 h in vitro NDF digestibility of spring harvested stems. Growth environment (year) and germplasm source had only minor impacts on the preceding correlation patterns, whereas spring versus summer harvests accounted for the inconsistencies observed among correlations for cell wall traits. The results of this study indicate that genetic improvement of fibre digestibility of lucerne stems should target genes that reduce total cell wall concentration, perhaps by reducing the rate of xylem tissue deposition during maturation, and reduce Klason lignin and increase pectin concentrations in the cell wall to improve potential extent and rate of fibre digestibility, respectively.     

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.     

The combined effects of black oxidising table olive process and ripening on the cell wall polysaccharides of olive pulp

Olive fruits harvested at cherry and black stages of ripening were processed according the table olive black oxidising processing and sampled after the three main steps: storage in brine, lye treatment and thermal treatment (final product). The results show that the storage in brine contributed positively to the stabilisation of cell wall polysaccharides of olive pulp as the amounts of main polysaccharides practically were maintained in both stages of ripening. The lye treatment introduced degradation of cell walls due to the generalised loss of pectic and hemicellulosic polysaccharides and cellulose, caused by the breakage of ester and hydrogen bonds. On the other hand, the lye treatment introduced shifts in the solubilisation of polysaccharides rendering them more difficult to extract by alkali solutions and enabling their retention in the cellulosic residue, which should contribute positively to cell wall firmness. The thermal treatment introduced degradation of cellulose and increased the solubilisation of polysaccharides with a higher extent in the black olives. This work showed that the differences on the cell wall polysaccharides between stages of ripening are magnified after processing and allowed to conclude that the stage of ripening of olive fruits is determinant for obtaining a final product with adequate texture properties for table olive consumption.     

Changes in the rate of synthesis of wall polysaccharides during the cell cycle of yeast

Reevaluation and comparison of seemingly contradictory literature data on the mode of synthesis of wall polysaccharides during the cell cycle ofSaccharomyces cerevisiae explained the source of discrepancies and demonstrated their general consonance in the following points: 1. The rate of synthesis of glucan and mannan is not constant and does not increase continuously throughout the entire cell cycle. 2. The rate of synthesis of both polysaccharides is considerably reduced at the time of cell division and in the prebudding phase.     

Mode of increase in cell wall polysaccharides in synchronously growing Saccharomyces cerevisiae

The mode of increase in cell wall polysaccharides of yeast (glucan and mannan) during cell cycle was analyzed using cell wall samples obtained from a synchronous culture. The increase in mannan and total glucan proceeded almost linearly throughout the cell cycle except for a short period of their leveling off at the time of cell division. However, the constituents of glucan behaved characteristically: Alkalisoluble glucan and insoluble glucan increased mainly in the former and the latter half of the cell cycle, respectively.     

Changes in cell wall polysaccharides in the internodes of submerged floating rice

In excised stem segments of floating rice (Oryza sativa L.), as well as in intact plants, submergence greatly stimulates the elongation of internodes. The differences in the composition of cell wall polysaccharides along the highest internodes of submerged and air-grown stem segments were examined. The newly elongated parts of internodes that had been submerged for two days contained considerably less cellulosic and noncellulosic polysaccharides than air-grown internodes, an indication that the cell walls of the newly elongated parts of submerged internodes are extremely thin. In the young parts of both air-grown and submerged internodes, the relative amounts of noncellulosic polysaccharides were equal to those of -cellulose, whereas the relative amounts of -cellulose were higher than those of noncellulosic polysaccharides in the upper, old parts. In the cell-elongation zones of both air-grown and submerged internodes, glucose was predominant among the noncellulosic neutral sugars of cell wall. The relative amount of glucose in noncellulosic neutral sugars decreased toward the upper, old parts of internodes, whereas that of xylose increased.     

Changes in composition of the outer epidermal cell wall of pea stems during auxin-induced growth

The gross composition of the outer epidermal cell wall from third internodes of Pisum sativum L. cv. Alaska grown in dim red light, and the effect of auxin on that composition, was investigated using interference microscopy. Pea outer epidermal walls contain as much cellulose as typical secondary walls, but the proportion of pectin to hemicellulose resembles that found in primary walls. The pectin and hemicellulose fractions from epidermal peels, which are enriched for outer epidermal wall but contain internal tissue as well, are composed of a much higher percentage of glucose and glucose-related sugars than has been found previously for pea primary walls, similar to non-cellulosic carbohydrate fractions of secondary walls. The epidermal outer wall thus has a composition rather like that of secondary walls, while still being capable of elongation. Auxin induces a massive breakdown of hemicellulose in the outer epidermal wall; nearly half the hemicellulose present is lost during 4 h of growth in the absence of exogenous sugar. The percentage breakdown is much greater than has been seen previously for whole pea stems. It has been proposed that a breakdown of xyloglucan could be the basis for the mechanical loosening of the outer wall. This study provides the first evidence that such a breakdown could be occurring in the outer wall.M.S. Bret-Harte would like to thank Dr. Peter M. Ray, of Stanford University, for helpful discussions and for technical and editorial assistance, Dr. Winslow R. Briggs, of the Camegie Institude of Washington, for the use of experimental facilities and for helpful discussions, Dr. Wendy K. Silk, of the University of California, Davis, for helpful discussions and financial support, Dr. Paul B. Green for financial support, and Drs. John M. Labavitch and L.C. Greve, of the University of California, Davis, for performing the -cellulose analysis on short notice, in response to a request by an anonymous reviewer. This work was supported by a National Science Foundation Graduate Fellowship to M.S. B.-H., National Science Foundation Grant DCB8801493 to Paul B. Green, and the generosity of Wendy K. Silk (Department of Land, Air, and Water Resources, University of California, Davis) during the final writing.     

Distribution of the anionic sites in the cell wall of apple fruit after calcium treatment

Summary The ripening and softening of fleshy fruits involves biochemical changes in the cell wall. These changes reduce cell wall strength and lead to cell separation and the formation of intercellular spaces. Calcium, a constituent of the cell wall, plays an important role in interacting with pectic acid polymers to form cross-bridges that influence cell wall strength. In the present study, cationic colloidal gold was used for light and electron microscopic examinations to determine whether the frequency and distribution of anionic binding sites in the walls of parenchyma cells in the apple were influenced by calcium, which was pressure infiltrated into mature fruits. Controls were designed to determine the specificity of this method for in muro labelling of the anionic sites on the pectin polymers. The results indicate that two areas of the cell wall were transformed by the calcium treatment: the primary cell walls on either side of the middle lamella and the middle lamella intersects that delineate the intercellular spaces. The data suggest that calcium ions reduce fruit softening by strengthening the cell walls, thereby preventing cell separation that results in formation of intercellular spaces.Abbreviations EDTA ethylenediaminotetraacetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate