Enzymic degradation of apple pectins

Purified apple pectins extracted under mild conditions were degraded by purified pectin lyase (EC 4.2.2.10) and pectate lyase (EC 4.2.2.2). The degraded pectins were fractionated by gel permeation chromatography and the degree of esterification and the sugar composition determined for each fraction. More than 90% of the uronic acid residues can be isolated as homogalacturonan chains. The neutral sugar residues can be detected in the column eluates as high molecular weight fragments. Models of the apple pectin molecules are presented. In the models the neutral sugars are present as side chains, arranged in blocks (in so-called ‘hairy regions’). The galacturonate residues in the hairy regions are esterified with methanol.     

Extraction and purification of pectins from Alcohol Insoluble Solids from ripe and unripe apples

Pectins are extracted from Alcohol Insoluble Solids of ripe and unripe apples and fractionated by ion exchange chromatography and gelfiltration. In the extracts mainly pectins with neutral sugar contents of 0·15, 0·24 and 0·53 mol neutral sugar residues/mole galacturonate residues are present. The pectin molecules contain rhamnose, arabinose, xylose, galactose, glucose and galacturonic acid residues. No mannose could be detected. The neutral sugar composition of the glycans bound to the galacturonan was found to be constant, except for the relative amount of galactose. During ripening the neutral sugar composition of the extractable pectin does not change.     

Apple juice pectic substances

Apple juice pectic substances are characterised by a high content of galacturonic acids (75·6%), a high degree of esterification (83), a low content of neutral sugars (0·109 moles/mole of galacturonate residues) and a high molecular weight ($\text{M}{}_{\mathrm{v}}\text{= 95 000}$). Degradation by heat (β-elimination) and by endopolygalacturonase showed that the pectic macromolecules consist of long homogalacturonan regions (92% of total galacturonides) and of ‘hairy’ regions in which neutral sugars form long or short side-chains.     

Simultaneous in vivo truncation of pectic side chains

Despite the wide occurrence of pectin in nature only a few source materials have been used to produce commercial pectins. One of the reasons for this is that many plant species contain pectins with high levels of neutral sugar side chains or that are highly substituted with acetyl or other groups. These modifications often prevent gelation, which has been a major functional requirement of commercial pectins until recently. We have previously shown that modification of pectin is possible through heterologous expression of pectin degrading enzymes in planta. To test the effect of simultaneous modification of the two main neutral pectic side chains in pectic rhamnogalacturonan I (RGI), we constitutively expressed two different enzymes in Arabidopsis thaliana that would either modify the galactan or the arabinan side chains, or both side chains simultaneously. Our analysis showed that the simultaneous truncation of arabinan and galactan side chains is achievable and does not severely affect the growth of Arabidopsis thaliana.     

Composition, Properties and Localisation of Pectins in Young and Mature Cells of the Mung Bean Hypocotyl

Two pectic fractions were extracted along the growth gradientof the mung bean hypocotyl. The first one (PF₁) contained pectinssoluble in boiling water and characterized by low uronic acids/cationsratio, high esterification degree and high neutral sugars/acidicsugars ratio. The second one (PF₂) contained pectins insolublein boiling water characterized by high uronic acids/cationsratio, low esterification degree, very low neutral sugar contentand a high selectivity for calcium. Water soluble pectins werepresent in all the wall area whereas the other ones were detectedmainly in the middle lamella. The PF₁/PF₂ ratio was high infast growing tissues but low in mature, slowly growing tissues.The development of the cell wall exchange properties along thegrowth gradient was related to the changes observed in the PF₁/PF₂balance. (Received July 31, 1985; Accepted January 20, 1986)     

Effects of Hexametaphosphate on Soybean Pectic Polysaccharide Extraction

The effects of sodium hexametaphosphate concentration, incubation temperature, and pH on the extraction of polysaccharide from soybean okara (soybean curd waste) were examined. The results suggest that the soybean polysaccharides were almost completely extracted with 50 times their volume of a 2% hexametaphosphate solution at 100°C for 2h, avoiding protein contamination. The viscosity, molecular weight, and sugar composition of the polysaccharides were compared with those of the commercially available samples. No differences were observed in the molecular weight or neutral sugar composition. However, it was found that the galacturonate distribution in the molecules of the two polysaccharides was different. The viscosity of the polysaccharide solution was changed by adding small amount of salt.     

Differences in cell wall polysaccharide composition between embryogenic and non-embryogenic calli of <Emphasis Type="Italic">Medicago arborea</Emphasis> L.

Analysis of cell wall polysaccharide composition of embryogenic and non-embryogenic calli obtained from hypocotyl and petiole explants from Medicago arborea L. revealed significant differences. For calli induced from both hypocotyls and petioles, levels of total sugars, pectins, and hemicelluloses were higher in embryogenic than in non-embryogenic calli. Whereas in the residual cellulose fraction, the highest levels of sugar were detected in non-embryogenic calli. When comparing the two donor sources of callus explants, the highest total sugar levels were detected in embryogenic calli induced from petioles, mainly in the pectin fraction and to a lesser extent in the hemicellulose fraction. Moreover, analysis of uronic acids revealed higher levels in embryogenic calli, primarily in the pectin fraction. Analysis of those sugars associated with cell walls of calli suggested that these polysaccharides consisted of pectic polysaccharides and glucans, and that their levels were higher in embryogenic than non-embryogenic calli.     

Purification and characterization of a soluble β-1,4-glucan from bean (Phaseolus vulgaris L.)-cultured cells dehabituated to dichlobenil

Bean cells habituated to grow in the presence of dichlobenil exhibited reduced cellulose and hemicellulose content and an increase in pectic polysaccharides. Furthermore, following the extraction of pectins and hemicelluloses, a large amount of neutral sugars was released. These sugars were found to be part of a soluble β-1,4-glucan in a preliminary characterization, as reported by Encina et al. (Physiol Plant 114:182–191, 2002). When habituated cells were subcultured in the absence of the herbicide (dehabituated cells), the release of neutral sugars after the extraction of pectins and hemicelluloses was maintained. In this study, we have isolated a soluble β-1,4-glucan from dehabituated cells by sonication of the wall residue (cellulose fraction) remaining after fractionation. Gel filtration chromatography revealed that its average molecular size was 14 kDa. Digestion of the sample with endocellulase revealed the presence of cellobiose, cellotriose, and cellotetraose. Methylation analysis showed that 4-linked glucose was the most abundant sugar residue, but 4,6-linked glucose, terminal arabinose and 4-linked galactose for xyloglucan, and arabinogalactan were also identified. NMR analysis showed that this 1,4-glucan may be composed of various kinds of substitutions along the glucan backbone together with acetyl groups linked to the OH group of sugar residues. Thus, despite its relatively high molecular mass, the β-glucan remains soluble because of its unique configuration. This is the first time that a glucan with such characteristics has been isolated and described. The discovery of new molecules, as this β-glucan with unique features, may help understand the composition and arrangement of the polymers within plant cell walls, contributing to a better understanding of this complex structure.     

Studies on the Pectic Substances of Plant Cell Walls: III. DEGRADATION OF CARROT ROOT CELL WALLS BY ENDOPECTATE LYASE PURIFIED FROM ERWINIA AROIDEAE

Pectate lyase was isolated from the cell extract of Erwinia aroideae. The enzyme was further purified to a high degree by a procedure involving ammonium sulfate fractionation and chromatography on CM-Sephadex C-50 and on Sephadex G-200. The enzyme attacked its substrate in an endo fashion and was more active on the sodium salt of acid-insoluble polygalacturonate or pectic acid than it was on the methoxylated pectin. The enzyme had an optimum pH at 9.3, was stimulated by calcium ions, and was completely inhibited by ethylenediaminetetraacetic acid. In addition, the reaction products showed an absorption maximum between 230 and 235 nm and reacted with thiobarbituric acid. These results indicate that the purified enzyme is an endopectate lyase. The endopectate lyase also had the ability to solubilize effectively the pectic fraction from the cell walls of carrot (Daucus carota) root tissue. The enzyme released 30.5% of the wall as soluble products and also liberated all of the galacturonic acid present in the walls. The total neutral sugars released by the enzyme were 10.6% of the walls, which corresponded to 71.5% of noncellulosic neutral sugars. The soluble products were separated into five fractions by DEAE-Sephadex A-50 column chromatography. Based on the analysis of sugar composition of each fraction, the pectic fraction of carrot cell wall is presented.     

Degradation of forage chicory by ruminal fibrolytic bacteria

Aims: Determine the susceptibility of forage chicory (Cichorium intybus L.) to degradation by ruminal fibrolytic bacteria and measure the effects on cell-wall pectic polysaccharides. Methods and Results: Large segments of fresh forage chicory were degraded in vitro by Lachnospira multiparus and Fibrobacter succinogenes, but not by Ruminococcus flavefaciens or Butyrivibrio hungatei. Cell-wall pectins were degraded extensively (95%) and rapidly by L. multiparus with a simultaneous release of uronic acids and the pectin-derived neutral monosaccharides arabinose, galactose and rhamnose. Fibrobacter succinogenes also degraded cell-wall pectins extensively, but at a slower rate than L. multiparus. Immunofluorescence microscopy using monoclonal antibodies revealed that, after incubation, homogalacturonans with both low and high degrees of methyl esterification were almost completely lost from walls of all cell types and from the middle lamella between cells. Conclusions: Only two of the four ruminal bacteria with pectinolytic activity degraded fresh chicory leaves, and each showed a different pattern of pectin breakdown. Degradation was greatest for F. succinogenes which also had cellulolytic activity. Significance and Impact of the Study: The finding of extensive removal of pectic polysaccharides from the middle lamella and the consequent decrease in particle size may explain the decreased rumination and the increased intake observed in ruminants grazing forage chicory.     

Comparative studies of the neutral sugar composition of cell wall polysaccharides between cultured cells and mother plant tissues of soybean and carrot

The cell walls obtained from cultured cells grown under variousconditions and their mother plant tissues or organs were comparedwith regard to the neutral sugar composition of polysaccharides.In the pectic fraction of cell walls from soybean hypocotyl,greater amounts of galactose than other neutral sugars weredetected, and the relative sugar ratio did not change in theelongating and non-elongating portions of the tissue. On theother hand, more arabinose than other sugars was detected inthe pectic fraction of cell walls from cultured cells from soybeanhypocotyl tissue, and no significant change in the sugar ratioof polysaccharides was found in this fraction during the cultureperiod. A higher arabinose content in cell walls from culturedcells from root tissues as compared to the mother tissues ororgans was also detected in carrot. The neutral sugar ratioof cell walls from cultured cells was similar in cells grownon agar or liquid medium, with indoleacetic acid, or with orwithout 2,4-dichlorophenoxyacetic acid (2,4-D). These factssuggest that the high arabinose content of cell walls is notcommon to the cell walls of young, growing, undifferentiatedcells, and that 2,4-D is not required for the maintenance ofthe higher content of arabinose in cell walls of cultured cells. ¹A preliminary report of this work was presented for the annualmeeting of the American Society of Plant Physiologist at OhioState University, August, 1979. [Plant Physiol., 63(5) 118 (1979)]. ⁴Department of Agricultural Chemistry, Washington State University,Pullman, Washington 99164, U.S.A. (Received October 6, 1979; )     

Differences in pectic polysaccharides between carrot embryogenic and non-embryogenic calli

Summary Cell walls and media were obtained from three kinds of carrot cell culture, namely, embryogenic callus (EC), non-embryogenic callus (NC) and somatic embryos (SE), and analyzed for their sugar content and sugar composition by electrophoresis and gas chromatography. EC formed large cell clusters while NC formed small clusters. Observations under the light microscope revealed that the intercellular contacts in NC were much more limited than those in EC. The analysis of pectic polysaccharides revealed that the level of neutral sugars was higher than that of acidic sugars in EC, while the opposite was true in NC. Gaschromatographic analysis of neutral sugars in pectic fractions revealed that EC and SE were rich in arabinose, while NC was rich in galactose. On the basis of these results, we discuss the possible involvement of neutral sugars, and of arabinose and galactose in particular, in pectic polysaccharides in intercellular contacts.Abbreviations EC embryogenic callus - NC non-embryogenic callus - SE somatic embryo - MS Murashige and Skoog - PAS periodic acid-Schiff s reagent     

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

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

Galactose metabolism in cell walls of opening and senescing petunia petals

Galactose was the major non-cellulosic neutral sugar present in the cell walls of ‘Mitchell’ petunia (Petunia axillaris × P. axillaris × P. hybrida) flower petals. Over the 24 h period associated with flower opening, there was a doubling of the galactose content of polymers strongly associated with cellulose and insoluble in strong alkali (‘residual’ fraction). By two days after flower opening, the galactose content of both the residual fraction and a Na₂CO₃-soluble pectin-rich cell wall fraction had sharply decreased, and continued to decline as flowers began to wilt. In contrast, amounts of other neutral sugars showed little change over this time, and depolymerisation of pectins and hemicelluloses was barely detectable throughout petal development. Size exclusion chromatography of Na₂CO₃-soluble pectins showed that there was a loss of neutral sugar relative to uronic acid content, consistent with a substantial loss of galactose from rhamnogalacturonan-I-type pectin. β-Galactosidase activity (EC 3.2.1.23) increased at bud opening, and remained high through to petal senescence. Two cDNAs encoding β-galactosidase were isolated from a mixed stage petal library. Both deduced proteins are β-galactosidases of Glycosyl Hydrolase Family 35, possessing lectin-like sugar-binding domains at their carboxyl terminus. PhBGAL1 was expressed at relatively high levels only during flower opening, while PhBGAL2 mRNA accumulation occurred at lower levels in mature and senescent petals. The data suggest that metabolism of cell wall-associated polymeric galactose is the major feature of both the opening and senescence of ‘Mitchell’ petunia flower petals.     

A copolymer analysis approach to estimate the neutral sugar distribution of sugar beet pectin using size exclusion chromatography

Partially degraded sugar beet (Beta vulgaris) pectins were characterised in terms of galacturonic acid, neutral sugar and ferulic acids contents. It was shown that the total neutral sugar content is correlated with the ferulic acid content. One pectin (C) was further characterised by size exclusion chromatography coupled to refractive index and UV detectors (SEC-RI-UV). This gave the opportunity to estimate how the ferulic acid and neutral sugar contents changed with hydrodynamic radius. Pectin C was found to be heterogeneous in composition with neutral sugar-rich fractions of both high and low hydrodynamic radii. A neutral sugar-poor fraction was found at intermediate hydrodynamic radii.     

Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site.     

Comparison of the structural features of apple and citrus pectic substances

Pectic substances were extracted from Alcohol Insoluble Solids from lemon peel (albedo) and fractionated by ion exchange chromatography and gelfiltration. The pectin molecules contained rhamnose, arabinose, galactose, glucose and galacturonic acid residues; xylose residues were almost absent. Degradation with purified pectolytic enzymes and subsequent gelfiltration of the resulting pectin fragments showed that the neutral sugar side chains were present in ‘hairy regions’ (blocks of neutral sugar side chains). The distribution of the methoxyl groups was studied by HPLC analysis of enzyme-degraded pectins. Some influence of native pectinesterase on the distribution of the methoxyl groups was found. The results are compared with those of similarly extracted and purified apple pectic substances.     

Cell wall polysaccharides in black currants and bilberries—characterisation in berries, juice, and press cake

Cell wall polysaccharides from black currants and bilberries were characterised in three approaches. First, compositions of skin, pulp, and seeds show the distribution of polysaccharides over these tissues. A sequential extraction of cell wall material with different aqueous extractants informs about the extractability of the different polysaccharides, viz. pectins, hemicellulose, and cellulose. Finally, by isolation of cell wall polysaccharides from juice and press cakes obtained by the conventional juice manufacturing. The polysaccharide distribution was followed during juice processing. The main difference between bilberries and black currants is the dominant sugar residue in seeds: mannose for black currants and xylose for bilberries. Most of the hemicellulolytic sugars and cellulose can be found back in the press cake. The sugar composition of the press cake is similar to the composition of the residue after sequential extraction. Black currants contain more pectic sugars than bilberries. Consequently, a commercial enzyme used during processing releases more pectic material into the juice.     

Structure of a pectic polysaccharide fraction from zea shoots

A pectic fraction, accounting for about 0.3% of the total cell wall polysaccharide, was derived from the hot water extract of an insoluble fraction of the buffer-homogenate of Zea shoots. The pectic polysaccharide fraction was characterized by fragmentation analysis after hydrolysis with acid and Erwinia carotovora pectate lyase. The results suggest that the fraction consists of mostly a linear homopolygalacturonan with neutral sugar components or a homogalacturonan and a rhamnogalacturonan with neutral sugar components.