An Examination of Centrifugation as a Method of Extracting an Extracellular Solution from Peas, and Its Use for the Study of Indoleacetic Acid-induced Growth

A technique of centrifuging pea epicotyl sections which extracts water-soluble cell wall polysaccharides with less than 1.5% cytoplasmic contamination as revealed by malate dehydrogenase activity determinations was developed. Tests for protein, hexose, pentose, and malate dehydrogenase indicate that significant damage to the cells occurs above 3,000g. Below this force, there is little damage, as evidenced by the similar growth rates of centrifuged and noncentrifuged sections. Centrifugation at 1,000g extracts polysaccharides containing rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose. An increase in xylose and glucose, presumably xyloglucan, is induced by treating sections with indoleacetic acid. Much of the alcohol-insoluble, water-soluble polysaccharide within the wall is extractable by centrifugation, since nearly as much arabinose and xylose are extractable by centrifugation as by homogenization. The utility of this method for the study of cell wall metabolism is discussed.     

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.     

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.     

Galactosyl- and fucosyltransferases in etiolated pea epicotyls: product identification and sub-cellular localisation

Particulate membrane preparations from etiolated pea epicotyls were found to contain fucosyltransferases, which transferred fucose from GDP-fucose onto xyloglucan and N-linked glycoprotein, and galactosyltransferases, which transferred galactose from UDP-galactose onto galactan, xyloglucan, and N-linked glycoprotein. The products were characterised by specific enzyme degradation and by acid and alkaline hydrolysis. All the enzymes were found to be concentrated in the Golgi apparatus. The Golgi apparatus was further fractionated into membranes of low, medium and high-density. The glycoprotein fucosyltransferase activity was present in highest amounts in the medium-density Golgi membranes, while the majority of the xyloglucan fucosyltransferase was present in the low-density Golgi membranes. The majority of the galactan galactosyltransferase (galactan synthase) was found in the low-density membranes, while the glycoprotein galactosyltransferase was equally distributed in all three subfractions.     

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.     

Biologically Active Oligosaccharides from Pectins of Pisum sativum L. Seedlings Affecting Root Generation

Two physiologically active oligosaccharide fractions were isolated from pectin of Pisum sativum L. cell wall after its partial acid hydrolysis. These fractions displayed stimulating and inhibiting effects on root formation in thin-layer explants. The subsequent separation of these fractions by gel permeation and anion-exchange chromatography resulted in fractions with effective concentrations two orders of magnitude lower than the concentrations of the initial fractions. The resulting oligosaccharides displayed their effect on the earliest stage of the rhizogenesis associated with formation of root primordias. The rhizogenesis-inhibiting fraction suppressed cell division by 30-50%. The stimulating fraction mainly contained fragments of xyloglucan and galactan, and the inhibiting fraction contained fragments of xyloglucan, galactan, and arabinan. The polymerization degrees of the stimulating and of the inhibiting oligosaccharides were 10-11 and 5-6, respectively.     

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.     

Loss of tomato cell wall galactan may involve reduced rate of synthesis

Changes in the galactose content of the noncellulosic polysaccharides of tomato (Mill) fruit cell walls were analyzed under various conditions. On the plant, galactan decreased gradually during fruit growth. As normal fruits ripened, the loss of galactan increased sharply; this was not observed in attached rin fruits beyond the fully mature stage. The ability to produce new wall galactan in vitro was retained in mature fruit tissue but declined with ripening. Normal tomatoes ripening on the plant showed a transient increase in galactan content at the climacteric. It is suggested that the decline in wall galactan is partly due to reduced synthesis in senescing, normal fruits and in detached rin tomatoes.     

Promotion of Xyloglucan Metabolism by Acid pH

Like indoleacetic acid, buffers of acidic pH, which stimulate elongation of pea (Pisum sativum var. Alaska) stem tissue, induce the appearance within the tissue of a watersoluble xyloglucan polymer that probably arises from previously deposited wall material. Neutral pH buffers, which inhibit the elongation response to indoleacetic acid in this tissue, inhibit indoleacetic acid-induced increase in soluble xyloglucan. The findings provide further evidence that release of soluble xyloglucan from the cell walls of pea results from the biochemical action on the cell wall that is responsible for wall extension. The data also indicate that treatment of tissue with either auxin or acidic pH has a similar biochemical effect on the cell wall. This is consistent with the H⁺ secretion theory of auxin action.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

Changes in Cell Wall Polysaccharides During the Growth of Phaseolus vulgaris Leaves

The changes in pectic and hemicellulosic polysaccharides, and-cellulose during the expansion growth of the primary leavesof Phaseolus vulgaris L. var. Pinta have been studied. -Celluloseincreased continuously with age, while pectic and water-solublehemicellulose extracted with 4% KOH fractions slightly decreased.The water-soluble hemicelluloses extracted with 24% KOH showedthe most conspicuous changes, increasing until the 8th day,when the absolute growth rate was maximal, and thereafter decreasing.Furthermore, the study of the molecular mass distribution ofpectin, and water-soluble polysaccharides extracted with 4%and 24% KOH, showed an increase in the degree of polymerizationof polyuronic acid and xylan, and an important depolymerizationof galactan and xyloglucan. Accordingly, the mechanism of cellwall loosening in the leaf cell walls is similar to that describedfor plant axes. Key words: Cell wall, growth, leaf     

Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Antisense Polygalacturonase Gene on Cell Wall Changes Accompanying Ripening in Transgenic Tomatoes)

Cell walls of tomato (Lycopersicon esculentum Mill.) fruit, prepared so as to minimize residual hydrolytic activity and autolysis, exhibit increasing solubilization of pectins as ripening proceeds, and this process is not evident in fruit from transgenic plants with the antisense gene for polygalacturonase (PG). A comparison of activities of a number of possible cell wall hydrolases indicated that antisense fruit differ from control fruit specifically in their low PG activity. The composition of cell wall fractions of mature green fruit from transgenic and control (wild-type) plants were indistinguishable except for trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid (CDTA)-soluble pectins of transgenic fruit, which had elevated levels of arabinose and galactose. Neutral polysaccharides and polyuronides increased in the water-soluble fraction of wild-type fruit during ripening, and this was matched by a decline in Na2CO3-soluble pectins, equal in magnitude and timing. This, together with compositional analysis showing increasing galactose, arabinose, and rhamnose in the water-soluble fraction, mirrored by a decline of these same residues in the Na2CO3-soluble pectins, suggests that the polyuronides and neutral polysaccharides solubilized by PG come from the Na2CO3-soluble fraction of the tomato cell wall. In addition to the loss of galactose from the cell wall as a result of PG activity, both antisense and control fruit exhibit an independent decline in galactose in both the CDTA-soluble and Na2CO3-soluble fractions, which may play a role in fruit softening.     

CHARACTERIZATION OF CELL WALL POLYSACCHARIDES OF THE COENCOCYTIC GREEN SEAWEED BRYOPSIS PLUMOSA (BRYOPSIDACEAE,CHLOROPHYTA) FROM THE ARGENTINE COAST1

Bryopsis sp. from a restricted area of the rocky shore of Mar del Plata (Argentina) on the Atlantic coast was identified as Bryopsis plumosa (Hudson) C. Agardh (Bryopsidales, Chlorophyta) based on morphological characters and rbcL and tufA DNA barcodes. To analyze the cell wall polysaccharides of this seaweed, the major room temperature (B1) and 90°C (X1) water extracts were studied. By linkage analysis and NMR spectroscopy, the structure of a sulfated galactan was determined, and putative sulfated rhamnan structures and furanosidic nonsulfated arabinan structures were also found. By anion exchange chromatography of X1, a fraction (F4), comprising a sulfated galactan as major structure was isolated. Structural analysis showed a linear backbone constituted of 3‐linked β‐d ‐galactose units, partially sulfated on C‐6 and partially substituted with pyruvic acid forming an acetal linked to O‐4 and O‐6. This galactan has common structural features with those of green seaweeds of the genus Codium (Bryopsidales, Chlorophyta), but some important differences were also found. This is the first report about the structure of the water‐soluble polysaccharides biosynthesized by seaweeds of the genus Bryopsis. These sulfated galactans and rhamnans were in situ localized mostly in two layers, one close to the plasma membrane and the other close to the apoplast, leaving a middle amorphous, unstained cell wall zone. In addition, fibrillar polysaccharides, comprising (1→3)‐β‐d ‐xylans and cellulose, were obtained by treatment of the residue from the water extractions with an LiCl/DMSO solution at high temperature. These polymers were also localized in a bilayer arrangement.     

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     

Temporal Sequence of Cell Wall Disassembly in Rapidly Ripening Melon Fruit

The Charentais variety of melon (Cucumis melo cv Reticulatus F1 Alpha) was observed to undergo very rapid ripening, with the transition from the preripe to overripe stage occurring within 24 to 48 h. During this time, the flesh first softened and then exhibited substantial disintegration, suggesting that Charentais may represent a useful model system to examine the temporal sequence of changes in cell wall composition that typically take place in softening fruit. The total amount of pectin in the cell wall showed little reduction during ripening but its solubility changed substantially. Initial changes in pectin solubility coincided with a loss of galactose from tightly bound pectins, but preceded the expression of polygalacturonase (PG) mRNAs, suggesting early, PG-independent modification of pectin structure. Depolymerization of polyuronides occurred predominantly in the later ripening stages, and after the appearance of PG mRNAs, suggesting the existence of PG-dependent pectin degradation in later stages. Depolymerization of hemicelluloses was observed throughout ripening, and degradation of a tightly bound xyloglucan fraction was detected at the early onset of softening. Thus, metabolism of xyloglucan that may be closely associated with cellulose microfibrils may contribute to the initial stages of fruit softening. A model is presented of the temporal sequence of cell wall changes during cell wall disassembly in ripening Charentais melon.     

Polar constituents of the ascidia Botryllus schlosseri

Eighteen compounds were identified by GC-MS of their trimethylsilyl derivatives in n-butanolic extract from the biomass of Botryllus schlosseri. Three of them, 5-oxoproline, 5-hydroxyhydantoin, and kinurenic acid, were found in marine invertebrates for the first time. In addition to cellulose, the biomass was also shown to contain complex water-soluble sulfated polysaccharides. These were extracted and fractionated, and sulfate content and monosaccharide composition were determined in the fractions; fucose, xylose, galactose, mannose, glucose, glucosamine, galactosamine, and uronic acids were found. Unlike several other tunicate species, Botryllus schlosseri does not seem to contain any simple galactan sulfate.     

Enzymatic degradation of cell wall polysaccharides from soybean meal

Soybean meal, soybean water unextractable solids (WUS) and extracts thereof, which contain particular cell wall polysaccharides, were incubated with a number of cell wall degrading enzymes. The intact cell wall polysaccharides in the meal and WUS were hardly degradable, while the extracts from WUS were well degraded. The arabinogalactan side chains in the pectin-rich ChSS fraction (Chelating agent Soluble Solids) could to a large extent be removed from the pectins by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase and arabinofuranosidase B. The remaining polymer was isolated and represented 30% of the polysaccharides in the ChSS fraction. Determination of the sugar composition showed these polymers to be very highly substituted pectic structures. It still contained 5 mol% of arabinose and 12 mol% of galactose, representing 7% and 12%, respectively, of the arabinose and galactose present in the ChSS fraction before degradation. Further, the presence of uronic acid (50 mol%) and of xylose (18 mol%) indicated the presence of a xylogalacturonan.     

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.     

Structural and functional analysis of tomato β‐galactosidase 4: insight into the substrate specificity of the fruit softening‐related enzyme

Plant β‐galactosidases hydrolyze cell wall β‐(1,4)‐galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β‐galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β‐(1,4)‐galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1–7 that belongs to the β‐galactosidase/exo‐β‐(1,4)‐galactanase subfamily. The enzyme can hydrolyze a wide range of plant‐derived (1,4)‐ or 4‐linked polysaccharides, and shows a strong ability to attack β‐(1,4)‐galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β‐d ‐galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β‐sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β‐galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β‐(1,6)‐galactobiose, and ~0.6‐fold activity towards β‐(1,4)‐galactobiose, compared with wild‐type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β‐galactosidases towards β‐1,4 and β‐1,6 linkages.     

The fermentation of cell wall substances in grass silage and in potato pulp

Summary The amount of acid formed in grass silage was greater than could have been formed from the soluble sugars present, even when only a lactic fermentation took place. This seemed to point to fermentation of cell wall substances by lactic acid bacteria. Lactic acid fermentation in potato pulp always takes place with cell wall substances as substrates, as sugars are absent. It was found that galactose, probably occurring as galactan, and also some pectic acid were fermented in potato pulp. Some lactobacilli were isolated from potato pulp; streptobacteria which could ferment galactan but no pectic or galacturonic acid, and betabacteria which could ferment galacturonic acid but no galactan or pectic acid. A number of homofermentative lactobacilli were all found to belong to the speciesStreptobacterium casei. It was shown that a strain of this species could ferment galactan in potato pulp sterilised previously with ethylene oxide. Part of this work was carried out at the Netherlands Institute for Dairy Research, Ede, Netherlands.