Isolation of β-galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

β-Galactosidase (EC 3.2.1.23) was purified from the cell wall of the fruit of Japanese pear ( Pyrus serotina Rehder var. culta Rehder cv. Hosui) and characterized. Five peaks of β-galactosidase activity, designated as Gal I to V, were separated by hydrophobic chromatography on butyl toyopearl and ion exchange chromatography on Mono S. These isolated β-galactosidases were investigated with regard to their abilities to release monomeric galactose from the fractionated polymers of native cell wall (cyclo-hexane-trans-1,2-diamine tetraacetic acid-, Na₂CO₃-, guanidine thiocyanate- and KOH-soluble fractions) and arabinogalactan (from larch wood). All the β-galactosidase fractions were active against native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall and arabinogalactan. Gal II released much galactose only from KOH-soluble polymers and arabinogalactan. Gal III released the most galactose. from cyclohexane- trans -1,2-diamine tetraacetic acid-, Na₂CO₃- and guanidine thiocyanate-soluble cell wall polymers which probably contained galactosyl side chains of pectic polymers, although it did not react much to arabinogalactan. In addition, the activity of Gal Ill dramatically increased as ripening proceeded. Furthermore, Gal III was purified to homogeneity by gel filtration on TSKgel 3000SW and the size of a polypeptide was 80 kDa on SDS-PAGE.     

Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Mango ripening--chemical and structural characterization of pectic and hemicellulosic polysaccharides

Ripening of mango is characterized by a gradual, but natural softening of the fruit, which is due to progressive depolymerization of pectic and hemicellulosic polysaccharides with significant loss of galactose, arabinose and mannose residues at the ripe stage. Structural characterization employing permethylation followed by GC-MS analysis, IR and ¹³C NMR measurements revealed the major CWS fractions of both unripe and ripe mangoes to be of variable molecular weights and having a 1,4-linked galactan/galacturonan backbone, which is occasionally involved in side chain branches consisting of single residues of galactose and arabinose or oligomeric 1,5-linked arabinofuranose residues linked through 1,3-linkages; whereas the major hemicellulosic fractions of unripe mango to be of xyloglucan-type having 1,4-linked glucan backbone with branching by non-reducing terminal arabinose and xylose residues.     

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.     

Characterisation of the extractable pectins and hemicelluloses of the cell wall of carrot

Pectic and hemicellulosic polysaccharides were successively extracted from an alcohol-insoluble residue (AIR) from carrot root by the actions of Pronase, hot dilute acid, cold dilute alkali, and concentrated alkali in yields corresponding to 12.6, 13.5, 21.7, and 6.7% of AIR, respectively. The first two products were fractionated further by ion-exchange chromatography. Carrot pectins contained 61.3–66.0% of galacturonic acid and 16.0–19.9% of neutral sugars, mainly galactose, arabinose, and rhamnose. Except for the alkali-soluble pectins, the degrees of methylation were high (62.9–67.1) and there was a significant degree of acetylation (7.2–13.5). Pectin fractions were homogeneous in gel-filtration chromatography with viscosity-average molecular weights varying between 36,200 and 56,500. Methylation analysis indicated the presence of arabinogalactans in the pectins extracted during the proteolysis, and fairly long chains of (1→4)-linked galactan with a branched arabinan in the two other pectic fractions. The hemicellulose fraction was mainly composed of (1→4)-linked glucan, (1→4)-linked mannan, (1→4)-linked xylan, and small but significant amounts of pectic polysaccharides. The possible association of cell-wall polymers is discussed.     

Quantitative and qualitative changes in cell wall polysaccharides in relation to growth and cell wall loosening in Lactuca sativa hypocotyls

The correlation between hypocotyl elongation, cell wall loosening and changes in cell wall polysaccharides was studied using intact lettuce seedlings grown in the dark or in light together with gibberellic acid (GA) and/or 5-fluorodeoxyuridine (FUDR). The following results were obtained:
1) The production of pectic, hemicellulosic and cellulosic polysaccharides look place in parallel with hypocotyl elongation, which was substantially affected by different growth conditions.
2) The mole percentage sugar composition of pectic and hemicellulosic polysaccharides changed in response to dark, light, GA, or FUDR treatments.
3) The amounts of xylose and glucose in hemicellulosic polysaccharides and those of galactosc, rhumnose and uronic acid in pectic polysaccharides increased in parallel with hypocotyl elongation.
4) Statistical analysis of the quantitative relationship between sugars composing polysaccharides revealed that the uronic acid content changed in parallel with those of rhamnose and galactose in pectic polysaccharides, and the content of xylose varied in parallel with those of fucose and glucose.
5) The content of hemicellulosic polysaccharides was correlated with cell wall loosening represented by a decrease in the minimum stress-relaxation time. Changes in the stress-relaxation time value were correlated with those in the content of araltinose and galactose in hemicellulosic polysaccharides.
Based on these results, the relationship between hypocotyl elongation, changes in cell wall polysaccharides, and cell wall loosening is discussed with respect to the effect of GA and FUDR on hypocotyl elongation.     

Cell wall changes in immature Asparagus stem tissue after excision.

Cell wall material (CWM) was prepared from sections of fresh and aerobically-stored asparagus (Asparagus officinalis, L. cv. Connovor Collossus) stems. Polymers were solubilized from the CWM by successive extraction with cyclohexane-trans-1,2-diamine-N N N' N'-tetraacetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue which contained a significant amount of cross-linked pectic polysaccharides. The polymers were fractionated by anion-exchange chromatography and selected fractions were subjected to methylation analysis. The storage-related decrease in (1-4)-linked Galp was detected in all the fractions rich in pectic polysaccharides, particularly in the CDTA, Na2CO3, 0.5 M KOH fractions and alpha-cellulose residue. A smaller decrease in Araf was also observed. This was mainly due to a decrease in (1-5)-linked Araf in the Na2CO3-1-soluble polymers, and terminal Araf in the alpha-cellulose residue. There was evidence for the occurrence of significant amounts of complexes containing pectic polysaccharides and xylans having a relatively low degree of polymerization in the dilute alkali-soluble polymers, and some of these contained phenolic compounds; the storage-induced increase in (1-4)-linked Xylp was confined to these polymers. Interestingly, no free acidic xylans could be detected in the 1 M and 4 M KOH-soluble polymers; instead, the bulk of the hemicellulosic polysaccharides appeared to be mixtures of xyloglucans and xylans in which the ratio of xyloglucan to xylan increased with increasing strength of alkali used for extraction of the polymers. The non-degradative extraction and fractionation procedures revealed heterogeneity in pectic polysaccharides, pectic polysaccharide-xylan complexes and xyloglucans in close association with xylans. The possible relationship between pectic polysaccharide-xylan-phenolic complexes and the onset of lignification in maturing tissues is discussed.     

Hypocotyl growth of Pinus pinaster seedlings. Changes in α-cellulose, and in pectic and hemicellulosic polysaccharides

Lorences, E. P., Suárez, L. and Zarra, I. 1987. Hypocotyl growth of Pinus pinaster seedlings. Changes in a-cellulose, and in pectic and hemicellulosic polysaccharides.
The changes in pectic and hemicellulosic polysaccharides of hypocotyl cell walls during the growth of intact seedlings of Pinus pinaster Aiton were investigated, α -Cellulose and the water-soluble hemicellulose presented the most conspicuous changes during hypocotyl growth. The relative amount of the water-soluble hemicellulose decreased from day 7 to day 13 when hypocotyls were in the rapid growth phase, and stabilized when hypocotyl growth ceased. In this fraction, the relative amount of non-cellulosic glucose decreased dramatically during hypocotyl growth, while the relative amount of xylose increased. We suggest that these changes may be due to partial degradation of xyloglucan present in the water_:soluble hemicellulose fraction, accompanied by the synthesis of a xylan.     

Auxin-induced elongation growth and expressions of cell wall-bound exo- and endo-beta-glucanases in barley coleoptiles

When auxin stimulates rapid cell elongation growth of cereal coleoptiles, it causes a degradation of 1,3:1,4-beta-glucan in hemicellulosic polysaccharides. We examined gene expressions of endo-1,3:1,4-beta-glucanase (EI) and exo-beta-glucanase (ExoII), of which optimum pH are about 5, and molecular distribution of hemicellulosic polysaccharides in barley (Hordeum vulgare L.) coleoptile segments treated with or without IAA. IAA (10(-5) M) stimulated the gene expression of EI, while it did not affect that of ExoII. IAA induced gene expression of EI after 4 h and increased wall-bound glucanase activity after 8 h. The molecular weight distribution of hemicellulosic polysaccharides from coleoptile cell walls was shifted to lower molecular weight region by 2 h of IAA treatment. Fusicoccin (10(-6) M) mimicked IAA-induced elongation growth and the decrease in molecular weight of hemicellulosic 1,3:1,4-beta-glucan of coleoptiles in the first 4 h, but it did not promote elongation growth thereafter. These facts suggest that acidification of barley cell walls by IAA action enhances pre-existing cell wall-bound glucanase activity in the early first phase of IAA-induced growth and the late second phase involves the gene expression of EI by IAA.     

Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Effect of osmotic stress on the growth of epicotyls of Cicer arietinum in relation to changes in cell wall composition

The inhibition of growth by polyethlene glycol (PEG)-induced osmotic stress led to modifications in the changes taking place in cell wall composition during normal growth of epicotyls of Cicer arietinum L. cv. Castellana. Epicotyls growing under normal conditions showed a decrease in the amount of pectic fractions and an increase in the hemicellulosic fractions and α-cellulose that led to an increase in the rigidity and a loss in growth capacity. Among the hemicellulosic fractions, the KI-2 fraction (insoluble fraction of 10% KOH-extracted hemicelluloses) seemed to be the only one related to the elongation process and subsequent rigidity. During normal growth a decrease was observed in the total amount of galactose, mainly from the pectic fractions. The inhibition of elongation led to an increase in the amount of the cell walls, due to inhibition of cellular elongation. PEG prevented the increase in the hemicelluloses and the α-cellulose, indicating that these changes were related to elongation. Thus, during the inhibition of elongation there is probably an inhibition of new synthesis that prevents cell wall rigidity and maintains cell wall growth capacity. Changes in the pectic fractions during growth were not affected by the inhibition of elongation, showing that these fractions are related to cell wall loosening rather than to elongation. Study of the cell wall composition confirms the idea that the autolytic process is regulated by changes in the cell wall structure during epicotyl growth     

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.     

Polysaccharides from the callus tissue of the rowan tree Sorbus aucuparia L. stem

Polysaccharide fractions SAcI and SAcII were isolated from callus tissues of rowan tree stems. The SAcI fraction was shown to contain compounds belonging to the arabinogalactan II group. The SAcII fraction, called sorban, comprised pectic polysaccharides composing the protopectin complex of the cell wall callus. The SAcII fraction was found to contain a large amount of galacturonic acid residues and a set of neutral sugars characteristic of rhamnogalacturonan I. The composition and properties allowed a suggestion that the sorban backbone is mainly formed by 1,4-α-D-galactopyranosyluronic acid residues, while the neutral sugars are represented by 1,4-linked glucopyranose and xylopyranose residues, 1,5-linked arabinofuranose, 1,6-linked galactopyranose and mannofuranose residues as well as terminal glucopyranose and xylopyranose residues. The callus growth was shown to be associated with nearly a constant content of galacturonic acid and neutral sugar residues in sorban (fraction SAcII).     

Alkaline extractability of pectic arabinan and galactan and their mobility in sugar beet and potato cell walls

The organisation of sugar beet and potato cell walls was studied using alkaline extractions following a response surface methodology, simultaneously with solid-state ¹³C NMR spectroscopy. The influence of two extraction parameters: NaOH concentration (0.05, 0.275, 0.5 M) and temperature (40, 65, 90 °C) on the composition (neutral and acidic sugars) of the residues recovered was established. Treatments of increasing harshness progressively washed off non-cellulosic polysaccharides from the cell walls. Alkaline treatments applied to sugar beet cell wall material (SB-CWM) revealed the presence of diverse pectin populations. The existence of distinct pectin populations in potato cell wall material (P-CWM) was less outstanding. Solid-state ¹³C NMR applied to SB-CWM and P-CWM and residues after treatment by 0.275 M NaOH at 65 °C revealed two fractions of pectic arabinan and galactan side chains. One fraction was highly mobile, whereas the other one displayed restricted mobility.     

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.     

Changes in metabolism of cell wall polysaccharides in oat leaves during senescence: relevance to the senescence-promoting effect of methyl jasmonate

Changes in cell wall polysaccharides in oat (Avena sativa L.) leaf segments during senescence promoted by methyl jasmonate (JA-Me) were studied. During the incubation with water at 25 °C in the dark, the loss of chlorophyll of the segments excised from the primary leaves of 8-day-old green seedlings was found dramatically just after leaf excision, and leaf color completely turned to yellow after the 3- to 4-day incubation in the dark. Application of 10 µM JA-Me substantially promoted the loss of chlorophyll corresponding with the chloroplast degradation. Cell wall polysaccharides in oat leaf segments mainly consisted of hemicellulosic and cellulosic ones. During the process of leaf senescence, the amount of hemicellulosic I and II, and cellulosic polysaccharides decreased, but little in pectic polysaccharides. JA-Me significantly enhanced the decrease in cellulosic polysaccharides, but little in hemicellulosic ones. Arabinose, xylose and glucose were identified as main constituents of neutral sugars of hemicellulosic polysaccharides. The neutral sugar compositions of hemicellulosic polysaccharides changed little during leaf senescence both in the presence or absence of JA-Me. These facts suggest that JA-Me affects sugar metabolism relating to cellulosic polysaccharides during leaf senescence.     

Changes in the Composition of Cell Wall Polysaccharides of Suspension-cultured Vinca rosea Cells during Culture

In a suspension culture of Vinca rosea L. three distinct growth phases were distinguished: a cell division phase, a cell expansion phase, and a stationary phase. At various stages of the growth cycle, extra-cellular polysaccharides (ECP) and cell wall were isolated and their compositions were investigated. ECP are mainly composed of xyloglucan, 3,6-linked arabinogalactan, and polyuronide. From the results of gas chromatographic analyses of sugars and partially methylated sugars in each fraction of cell wall polysaccharides, the following changes were observed in the composition of cell wall polysaccharides during culture: (1) a decrease in polyuronide content, (2) a decrease in non-cellulosic glucan content, (3) an increase in xyloglucan content, and (4) an increase in a-cellulose content. The significance of the changes is discussed in relation to the growth of cells.     

Changes in non-cellulosic cell-wall polysaccharides during the growth of carrot cells in suspension cultures

The cell-wall composition of carrot (Daucus carota L.) cells has been studied during their growth in suspension culture. Pectic and hemicellulosic polymers were fractionated according to molecular size by a Sepharose 4B column. Polyuronides in the pectic fraction were resolved into high- and low-molecular-weight components. The low-molecular-weight polyuronides were relatively free of neutral sugars and showed a marked increase during the growth of the cell wall. Hemicellulosic polysaccharides were of disperse molecular size. As cell expansion proceeded, the contents of glucose and xylose in the high-molecular-weight region increased while those in the low-molecular-weight fraction decreased. Removal of auxin from the medium apparently caused degradation of high-molecular-weight polymers in both the pectic and hemicellulosic fractions.     

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     

Implication of persimmon fruit hemicellulose metabolism in the softening process. Importance of xyloglucan endotransglycosylase

Hemicellulosic polysaccharides from persimmon fruit ( Diospyros kaki L.) pericarp were extracted from depectinated cell walls with 0.5, 1 and 4 M KOH at different stages of development: (I) maximal growth corresponding to the first sigmoidal growth phase; (II) cessation of growth corresponding to the lag between the first and the second sigmoidal phases; (III) maximal growth corresponding to the second sigmoidal phase; and (IV) cessation of growth when the fruit had reached its maximum size and the change in colour (green to red) had taken place. During fruit development the amount of total hemicelluloses per unit dry mass cell wall decreased twofold. Xyloglucan was present in the three hemicellulosic fractions, and also decreased with fruit age, although its amount relative to other hemicelluloses increased. The amount of xyloglucan was especially high in the hemicelluloses extracted with 4 M KOH, representing more than 50% at stages III and IV. The average molecular mass of xyloglucan increased from stage I through stage II (0.5 M hemicellulosic fraction) or through stage III (I and 4 M hemicellulosic fractions) and decreased after that. The xyloglucan endotransglycosylase (XET: EC 2.4.1.-) activity was measured as the incorporation of [³H]XXXGol (reduced xyloglucan heptasaccharide labelled at position 1 of the glucitol moiety) into partially purified persimmon fruit xyloglucan. XET specific activity increased greatly between stages I and II. The importance of this enzyme during fruit ripening is discussed.