Metabolism of cell wall polysaccharides in cell suspension cultures of Populus alba in relation to cell growth

Changes in the composition of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Populus alba cells. Three growth phases, namely the cell division phase, cell elongation phase and stationary phase, were distinguished. The active deposition of polysaccharides in cell wall fractions (50 m M Na₂CO₃-, 1 M KOH-, 4 M KOH-soluble and 4 M KOH-insoluble) was observed during the elongation phase. A 50 m M Na₂CO₃-soluble pectic fraction mainly composed of 1,4-linked galactan and arabinan except acidic sugars. The 1,4-linked galactan decreased markedly during elongation. In 1 and 4 M KOH-soluble hemicellulosic fractions, non-cellulosic 1,4-glucan and xyloglucan were observed as major components, respectively. These polysaccharides also decreased during elongation. A large amount of polysaccharides was secreted into the medium as ECP. Neutral sugars were detected predominantly by sugar composition analysis. Acidic sugars, such as galacturonic acid, were less than 12% of total. In this study, active metabolism of pectic polysaccharides in addition to hemicellulosic polysaccharides, especially neutral side chains of pectin, during cell growth, was clarified.     

Production and composition of extracellular polysaccharide from cell suspension cultures of Mentha

A suspension culture of Mentha was established from callus which formed on the tips of young shoots of a Mentha hybrid (M. arvenis × M. spicata). Changes in growth parameters during a culture cycle were recorded. The general appearance of cells during division and growth, including the changes in cell form, was also represented.Suspension-cultured cells of Mentha hybrid released a large amount of extracellular polysaccharides (ECP) mainly at the logarithmic phase of the growth cycle. The ECP contained galacturonic acid as major components and arabinose, galactose, glucose, xylose, rhamnose and mannose as minor components. The ratio of the uronic acid content to total sugar content in the ECP was below 40% at day 7, but increased up to 90% at day 21. The relative contents of xylose and glucose in the ECP decreased during the culture period, while the arabinose content increased and those of rhamnose, mannose and galactose remained constant.The IR spectrum suggested that the ECP were low-methoxylated pectic polysaccharides. The presence of lignin and related compounds in the ECP was not detected. The protein content of the ECP was about 10% and the main amino acids were alanine, proline, hydroxyproline, valine, asparticacid and serine, in that order.     

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.     

Changes in Cell Wall Composition and Water-soluble Polysaccharides During Kiwifruit Development

Changes in both cell wall and water-soluble polysaccharide compositionduring the growth of kiwifruits [Actinidia deliciosa (A. chev.) C. F. Liang and A. R. Ferguson var. deliciosa ‘Hayward’]were investigated. Cellulose was the major wall polysaccharide,with galactose and uronics the main non-cellulosic sugars. Muchsolubilization of cell wall pectic polysaccharides was detected.While wall-galactose solubilization started 3 months after anthesis,polyuronide degradation did not start until the fifth month,1 month prior to the harvest date. Parallel to these processes,a linear increase in water-soluble polysaccharides was detected.These mainly comprised galactose-rich polymers in the first3 months and little-branched polyuronides after the fifth month.Two different mechanisms for galactose and uronic acid solubilizationfrom kiwifruit cell walls during fruit development are proposed. Actinidia deliciosa ; cell wall; fruit growth; kiwifruit; water-soluble polysaccharides     

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.     

Changes in the Molecular Weight Distribution of the Hemicellulosic Polysaccharides from Rice Coleoptiles Growing Under Different Conditions

The changes in the molecular weight distribution of water-solubleand water-insoluble hemicelluloses from the cell walls of ricecoleoptiles growing in air and under water were studied. Thegrowth of rice coleoptiles was remarkably enhanced by growingunder water. Water-insoluble hemicellulose, mainly constitutedby xyloglucan, suffered an important depolymerization duringcoleoptile growth. On the other hand, ß-glucan andarabinoxylan, the two main polysaccharides of the water-solublehemicelluloses showed different changes during coleoptile growth.ß-glucan showed an increase in its degree of polymerizationduring the coleoptile fast growth phase and it decreased beforecoleoptile growth ceased. Arabinoxylan did not show importantdifferences in its mass-average molecular weight. Thus, xyloglucanand ß-glucan are the two hemicellulosic polysaccharidesinvolved in the cell wall loosening mechanism during coleoptilegrowth under both culture conditions. Key words: Arabinoxylan, cell wall, ß-glucan, xyloglucan     

Changes in molecular size of previously deposited and newly synthesized pea cell wall matrix polysaccharides : effects of auxin and turgor

Effects of indoleacetic acid (IAA) and of turgor changes on the apparent molecular mass (M_r) distributions of cell wall matrix polysaccharides from etiolated pea (Pisum sativum L.) epicotyl segments were determined by gel filtration chromatography. IAA causes a two- to threefold decline in the peak M_r of xyloglucan, relative to minus-auxin controls, to occur within 0.5 hour. IAA causes an even larger decrease in the peak M_r concurrently biosynthesized xyloglucan, as determined by [³H]fucose labeling, but this effect begins only after 1 hour. In contrast, IAA does not appreciably affect the M_r distributions of pectic polyuronides or hemicellulosic arabinose/galactose polysaccharides within 1.5 hours. However, after epicotyl segments are cut, their peak polyuronide M_r increases and later decreases, possibly as part of a wound response. Xyloglucan also undergoes IAA-independent changes in its M_r distribution after cutting segments. In addition, the peak M_r of newly deposited xyloglucan increases from about 9 kilodaltons shortly after deposition to about 30 kilodaltons within 0.5 hour. This may represent a process of integration into the cell wall. A step increase in turgor causes the peak M_r of previously deposited xyloglucan (but not of the other major polymers) to increase about 10-fold within 0.5 hour, returning to its initial value by 1.5 hours. This upshift may comprise a feedback mechanism that decreases wall extensibility when the rate of wall extension suddenly increases. IAA-induced reduction of xyloglucan M_r might cause wall loosening that leads to cell enlargement, as has been suggested previously, but the lack of a simple relation between xyloglucan M_r and elongation rate indicates that loosening must also involve other wall factors, one of which might be the deposition of new xyloglucan of much smaller size. Although the M_r shifts in polyuronides may represent changes in noncovalent association, and for xyloglucan this cannot be completely excluded, xyloglucan seems to participate in a dynamic process that can both decrease and increase its chain length, possible mechanisms for which are suggested.     

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.     

Increase in XET activity in bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) cells habituated to dichlobenil

Bean (Phaseolus vulgaris L.) cells have been habituated to grow in lethal concentrations of dichlobenil (DCB), a specific inhibitor of cellulose biosynthesis. Bean callus cells were successively cultured in increasing DCB concentrations up to 2 μM. The 2-μM DCB habituated cells were impoverished in cellulose and xyloglucan, had an increased xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity, together with an increased growth rate and a decreased molecular size of xyloglucan. However, the application of lethal concentrations of two different cellulose-biosynthesis inhibitors (DCB and isoxaben) for a short period of time produced little effect on XET activity and xyloglucan molecular size. We propose that the weakening of plant cell wall provoked by decrease in cellulose content might promote the xyloglucan tethers and increase the ability of xyloglucan to bind to cellulose in order to give rigidity to the wall.     

Hypocotyl growth of Pinus pinaster seedlings. Changes in the molecular weight distribution of hemicellulosic polysaccharides

Lorences, E. P., Suárez, L. and Zarra, I. 1987. Hypocotyl growth of Pinus pinaster seedlings. Changes in the molecular weight distribution of hemicellulosic polysaccharides.
The changes in the molecular weight distribution of water-soluble hemicelluloses and xyloglucan during hypocotyl growth of intact seedlings of Pinus pinaster Aiton were investigated. The mass-average molecular weight of total polysaccharides of the hemicellulose fraction soluble in 4% KOH dramatically increased during hypocotyl growth while xyloglucan slightly decreased. These phenomena were due to an increase in the degree of polymerization of an arabinogalactan and a slight depolymer-ization in the xyloglucan present in this fraction. In the hemicellulose fraction soluble in 24% KOH, xyloglucan increased its degree of polymerization from day 7 to 10 after which it decreased slightly. The xyloglucan of the hemicellulose fraction soluble in 4% KOH may thus be involved in cell wall loosening which makes cell wall expansion possible during hypocotyl growth.     

Modification of chemical properties of cell wall polysaccharides in the inner tissues by white light in relation to the decrease in tissue tension in Pisum sativum epicotyls

When white light irradiation inhibits shoot growth in derooted pea ( Pisum sativum L. cv. Alaska) cuttings, it decreases tissue tension, a driving force for shoot growth, by making the cell wall of the inner tissues mechanically rigid. To elucidate the mechanism by which light affects the mechanical properties of the cell wall in the inner tissues, its effect on the chemical properties of the cell walls was studied by analyzing qualitatively and quantitatively cell wall polysaccharides in the epdidermis and inner tissue of pea epicotyls grown in both dark and light. The amount of polysaccharides per subhook in the cell walls of both tissues increased during a 4-h dark incubation. Light suppressed the increase in hemicellulosic (HC)-II and cellulosic polysaccharides in the inner tissues, while it did not affect the increase in other wall fractions in either the epidermal or subepidermal tissues. No light effect was observed on the neutral sugar compositions of pectin, HC-I or HC-II fractions in either of the tissues. Light increased the mass-average molecular mass of xyloglucan and rhamnoarabinogalactan in HC-II fractions in the inner tissues, while such an effect was not observed in the epidermis. These facts suggest that the light-induced decrease in the tissue tension in pea epicotyls is caused by an increase in the molecular size of xyloglucan, rhamnoarabinogalactan in the HC-II fraction and/or the suppression of the synthesis of HC-II and cellulosic polysaccharides in the inner tissues.     

Constitutive overexpression of a ripening-related pepper endo-1,4-β-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening

The ripening-related pepper endo-1,4--D-glucanase (EGase) CaCel1 was over-expressed in transgenic tomato plants under the control of the constitutive 35S promoter to investigate the effects on plant growth and fruit softening of high levels of a potential cell wall-degrading activity. In transgenic fruit, recombinant CaCel1 protein was associated with a high-salt putative cell wall fraction, and extractable CMCase activity was increased by up to 20-fold relative to controls. However, the effects of high levels of EGase activity on fruit cell wall metabolism were relatively small. The largest consequence observed was a decrease of up to 20% in the amount of matrix glycans in a 24% KOH-soluble fraction consisting of polysaccharides tightly bound to cellulose. This decrease was confined to polysaccharides other than xyloglucan, did not affect the size distribution of remaining molecules, and was not correlated with a corresponding increase in glycans in a 4% KOH-soluble fraction loosely bound to cellulose, suggesting that the missing polymers had been degraded to fragments small enough to be lost from the extracts. The amount of matrix glycans in the 4% KOH-soluble fraction was not substantially changed, but the size distribution showed a small relative increase in the amount of polymers in a peak eluting close to a linear dextran marker of 71 kDa. This could be due either to an increase in the amount of polymers of this size, or to a loss from the extract of other polymers present in peaks of higher molecular weight. Transgenic fruit were not softer than controls but appeared the same or slightly firmer at both green and red developmental stages, and no differences in plant vegetative growth were observed. CaCel1 did not cause depolymerization of tomato fruit xyloglucan in vivo, but differences in the amount or molecular weight profile of other matrix glycans were observed. The data suggest that degradation of a proportion of matrix glycans other than xyloglucan does not result in fruit softening, and that fruit softening is not limited by the amount of EGase activity present during ripening.     

Changes in Cell Wall Composition during Ripening of Grape Berries

Cell walls were isolated from the mesocarp of grape (Vitis vinifera L.) berries at developmental stages from before veraison through to the final ripe berry. Fluorescence and light microscopy of intact berries revealed no measurable change in cell wall thickness as the mesocarp cells expanded in the ripening fruit. Isolated walls were analyzed for their protein contents and amino acid compositions, and for changes in the composition and solubility of constituent polysaccharides during development. Increases in protein content after veraison were accompanied by an approximate 3-fold increase in hydroxyproline content. The type I arabinogalactan content of the pectic polysaccharides decreased from approximately 20 mol % of total wall polysaccharides to about 4 mol % of wall polysaccharides during berry development. Galacturonan content increased from 26 to 41 mol % of wall polysaccharides, and the galacturonan appeared to become more soluble as ripening progressed. After an initial decrease in the degree of esterification of pectic polysaccharides, no further changes were observed nor were there large variations in cellulose (30–35 mol % of wall polysaccharides) or xyloglucan (approximately 10 mol % of wall polysaccharides) contents. Overall, the results indicate that no major changes in cell wall polysaccharide composition occurred during softening of ripening grape berries, but that significant modification of specific polysaccharide components were observed, together with large changes in protein composition.     

Hetero‐trans‐β‐glucanase,an enzyme unique to Equisetum plants,functionalizes cellulose

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that ‘cut and paste’ certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell‐wall polysaccharides (xyloglucan, mannans, mixed‐linkage β‐glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero‐trans‐β‐glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early‐diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero‐transglycosylation: its preferred donor substrates (cellulose or mixed‐linkage β‐glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose–xyloglucan and mixed‐linkage β‐glucan–xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter‐linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better‐known xyloglucan‐acting homo‐transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable ‘green’ technology for industrially manipulating biomass.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening

Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato β-galactosidase.     

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN‐LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)1

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan‐proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the β‐d ‐glycosyl‐Yariv (β‐GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). Epitopes of alkali‐soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti‐xyloglucan (anti‐XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC‐M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti‐XG antibody at the trans‐sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)‐β‐glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)‐β‐glucan (callose) could not be detected. The analytical results revealed that alkali‐soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)‐β‐d ‐glucan.     

Growth capacity and molecular mass distribution of cell wall polysaccharides along the hypocotyl of Pinus pinaster

The changes in the endogenous growth as well as in the cell wall composition were studied along the hypocotyl of Pinus pinaster Aiton. Cell elongation decreased as the distance from the cotyledonary node increased. Pectic polysaccharides underwent an important depolymerization accompanied by a decrease in their uronic acid content from the apical to basal region of the hypocotyl. Additionally, the molecular mass of pectic polysaccharides strongly decreased from the apical to the basal regions. Watersoluble hemicellulosic polysaccharides extracted with 4% KOH decreased notably from the cotyledonary node towards the base, while water-soluble polysaccharides extracted with 24% KOH showed few differences along the hypocotyl. The molecular mass of xyloglucan present in both hemicellulosic fractions was lower in the upper hypocotyl region as compared with the basal region. These findings are in agreement with an active xyloglucan depolymerization in the upper region as would be expected in a region exhibiting very active growth.     

Arabidopsis XXT5 gene encodes a putative alpha-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis

The function of a putative xyloglucan xylosyltransferase from Arabidopsis thaliana (At1g74380; XXT5) was studied. The XXT5 gene is expressed in all plant tissues, with higher levels of expression in roots, stems and cauline leaves. A T-DNA insertion in the XXT5 gene generates a readily visible root hair phenotype (root hairs are shorter and form bubble-like extrusions at the tip), and also causes the alteration of the main root cellular morphology. Biochemical characterization of cell wall polysaccharides isolated from xxt5 mutant seedlings demonstrated decreased xyloglucan quantity and reduced glucan backbone substitution with xylosyl residues. Immunohistochemical analyses of xxt5 plants revealed a selective decrease in some xyloglucan epitopes, whereas the distribution patterns of epitopes characteristic for other cell wall polysaccharides remained undisturbed. Transformation of xxt5 plants with a 35S::HA-XXT5 construct resulted in complementation of the morphological, biochemical and immunological phenotypes, restoring xyloglucan content and composition to wild-type levels. These data provide evidence that XXT5 is a xyloglucan alpha-1,6-xylosyltransferase, and functions in the biosynthesis of xyloglucan.     

Biosynthesis and cell-wall deposition of a pectin–xyloglucan complex in pea

Golgi-enriched enzyme preparations prepared from etiolated pea epicotyls incorporated [U–¹⁴C]galactose from UDP-[U–¹⁴C]galactose into the 1,4--galactan sidechains of a pectin–xyloglucan complex. This complex could bind to paper and was degraded both by pectin-degrading enzymes and by a xyloglucan-specific endoglucanase. Gel permeation chromatography was used to assess the molecular size of the complex and of enzymically-degraded, galactan-containing fragments of it. Etiolated pea stems were labelled with [U–¹⁴C]sucrose for 1 h, and the newly-synthesised cell wall polysaccharides were extracted with EDTA or NaOH and fractionated by ion-exchange chromatography. The NaOH-extracted, acidic radioactive polysaccharides obtained in this way were also degraded both by pectin-degrading enzymes and by xyloglucan-specific endoglucanase. Analysis of the radioactive sugar composition indicated that neutral sugars characteristic of both pectin and xyloglucan were present. Analysis of the total non-radioactive, neutral sugar composition of the NaOH-extracted, acidic cell-wall polysaccharides indicated that pectin–xyloglucan complexes were a general feature of the cell wall in this tissue