Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.     

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.     

Lemon juice improves the extractability and quality characteristics of pectin from yellow passion fruit by-product as compared with commercial citric acid extractant

An environment-friendly procedure, allowing the extraction of safe pectin products with good functional properties from yellow passion fruit by-product, was developed using two natural acid extractants, namely, pure lemon juice and citric acid solvent. The results show that both of them solubilise, from cell wall material, pectins characterised by high galacturonic acid content (64–78% w/w), degree of esterification (52–73), viscosity-average molecular weight (70–95 kDa) and capable of forming gels in the presence of high soluble solids (sucrose) content and acid. However, compared to pure citric acid solvents, lemon natural juice and its concentrate isolate, under similar extraction conditions, pectins of superior quality characteristics, i.e., higher galacturonic acid content, degree of esterification, viscosity-average molecular weight and gelling power.     

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

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

Tomato fruit cell wall : I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins

Cell wall isolation procedures were evaluated to determine their effect on the total pectin content and the degree of methylesterification of tomato (Lycopersicon esculentum L.) fruit cell walls. Water homogenates liberate substantial amounts of buffer soluble uronic acid, 5.2 milligrams uronic acid/100 milligrams wall. Solubilization appears to be a consequence of autohydrolysis mediated by polygalacturonase II, isoenzymes A and B, since the uronic acid release from the wall residue can be suppressed by homogenization in the presence of 50% ethanol followed by heating. The extent of methylesterification in heat-inactivated cell walls, 94 mole%, was significantly greater than with water homogenates, 56 mole%. The results suggest that autohydrolysis, mediated by cell wall-associated enzymes, accounts for the solubilization of tomato fruit pectin in vitro. Endogenous enzymes also account for a decrease in the methylesterification during the cell wall preparation. The heat-inactivated cell wall preparation was superior to the other methods studied since it reduces β-elimination during heating and inactivates constitutive enzymes that may modify pectin structure. This heat-inactivated cell wall preparation was used in subsequent enzymatic analysis of the pectin structure. Purified tomato fruit polygalacturonase and partially purified pectinmethylesterase were used to assess changes in constitutive substrates during tomato fruit ripening. Polygalacturonase treatment of heat-inactivated cell walls from mature green and breaker stages released 14% of the uronic acid. The extent of the release of polyuronides by polygalacturonase was fruit development stage dependent. At the turning stage, 21% of the pectin fraction was released, a value which increased to a maximum of 28% of the uronides at the red ripe stage. Pretreatment of the walls with purified tomato pectinesterase rendered walls from all ripening stages equally susceptible to polygalacturonase. Quantitatively, the release of uronides by polygalacturonase from all pectinesterase treated cell walls was equivalent to polygalacturonase treatment of walls at the ripe stage. Uronide polymers released by polygalacturonase contain galacturonic acid, rhamnose, galactose, arabinose, xylose, and glucose. As a function of development, an increase in the release of galacturonic acid and rhamnose was observed (40 and 6% of these polymers at the mature green stage to 54 and 15% at the red ripe stage, respectively). The amount of galactose and arabinose released by exogenous polygalacturonase decreased during development (41 and 11% from walls of mature green fruit to 11 and 6% at the red ripe stage, respectively). Minor amounts of glucose and xylose released from the wall by exogenous polygalacturonase (4-7%) remained constant throughout fruit development.     

Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β-subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β-subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β-subunit protein. Cell walls prepared from mature-green fruit were employed for binding and assay of PG2. Walls were provided with rate-limiting quantities of purified PG2 and incubated at 100 mM KCl, pH 4.5, or 25 mM KCl, pH 6.0. Cell walls of both β-subunit antisense and wild-type fruit retained comparable quantities of added PG2. The release of pectin from PG2-loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β-Subunit-antisense cell walls released 2- to 3-fold higher levels of pectin in response to PG2 than did wild-type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2-mediated hydrolysis of β-subunit-antisense compared with wild-type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG-mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 mM CDTA (trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid), and were more extensive for the β-subunit-antisense cell walls compared with the wild-type walls. Pectic polymers derived from ethanol insoluble-powders showed molecular mass downshifts during ripening but differences between the β-subunit-antisense and wild-type fruits were not observed.     

Study of pectin esterase and changes in pectin methylation during normal and abnormal peach ripening

Peach fruit ( Prunus persica cv. Hermosa) were allowed to ripen immediately after harvest or after 30 days of 0°C storage. The fruits lost 75–80% of their firmness after 5 days at 20°C. During ripening after harvest there was a loss of both uronic acid and methyl groups from the cell wall. Cell wall labelling with JIM 7, a monoclonal antibody which recognized pectins with a high degree of methylation, was lower in ripe fruits than in freshly harvested fruits. However, ripe fruit cell walls did not cross-react with JIM 5, which recognizes pectins with low methylation. During storage, de-methylation occurred and in fruit ripened after storage there was little further change in pectin methylation or pectin content in the cell walls. The labelling of stored or stored plus ripened cell walls with JIM 7 was similar, but the cell walls of fruit ripened after storage showed some low cross-reactivity with JIM 5. The in vitro activity and mRNA abundance of pectin esterase (EC 3.1.1.11) was not correlated with the amount of de-esterification as measured chemically or by immuno-labelling in the cell walls. Eighty percent of the fruits which ripened after storage developed a woolly texture. It is suggested that woolliness is due to de-esterification of pectins, not accompanied by depolymerization, which leads to the formation of a gel-like structure in the cell wall.     

桃果实采后贮藏过程中细胞壁多糖的变化

以‘雨花三号’水蜜桃果实为试材,分别在5℃（低温）和20℃（常温）贮藏一段时间后,研究桃果实采后细胞壁多糖降解、硬度以及乙烯释放速率的变化特征。结果表明,乙烯释放高峰明显滞后于果实采后硬度的快速下降期。不同温度下贮藏过程中果实细胞壁多糖变化的对比表明,低温抑制了细胞壁果胶和细胞壁其余组分的变化,从而抑制了果实的软化。富含半乳糖醛酸的果胶主链断裂。果胶和细胞壁其余组分也发生了半乳糖和阿拉伯糖等中性糖的损失,说明果胶和细胞壁其余组分的增溶及其侧链中性糖的降解也是桃果实采后软化的重要因素,这可能是由多种相关多糖降解酶的作用所导致的。但半纤维素多糖中中性糖的降解对桃果实采后软化的进程并没有影响。     

Autolysis-Iike Release of Pectic Polysaccharides from Regions of Cell Walls Other Than the Middle Lamella

Cell walls of a storage organ (potato tubers) showed autolysis-likeactivity. After 20 h of incubation in water at 35°C, thepurified cell walls released approximately 10% of the cell walldry weight as pectic polysaccharides containing about 40% ofthe total galacturonic acid present in the cell walls. Virtuallyno neutral polysaccharides were found in the soluble fraction.The pectic polysaccharides were heterogeneous in galacturonicacid content and had a very large molecular size. The releaseof pectic polymers was caused neither by enzymatic reactionsnor by ß-elimination, but by a chelation of Ca²⁺ and/orother metal ions during the cell wall isolation. Ultrastructuralobservations clearly showed that these pectic polysaccharideswere released not from the middle lamella, but from the primarycell wall adjacent to the plasma membrane. These results indicatethat nearly half of cell wall pectic polysaccharides are heldin the primary wall only by Ca²⁺- and/or other metal-bridgesand that these pectic polymers are not associated with the middlelamella. (Received March 20, 1989; Accepted October 3, 1989)     

HISTOLOGICAL AND HISTOCHEMICAL CHANGES IN DEVELOPING AND RIPENING PEACHES. II. THE CELL WALLS AND PECTINS

Reeve , R. M. (U.S.D.A., Albany, California.) Histological and histochemical changes in developing and ripening peaches. II. The cell walls and pectins. Amer. Jour. Bot. 46(4): 241–247. Illus. 1959.—Histological and histochemical observations on developing and ripening clingstone and freestone peaches have revealed that, after cell divisions have ceased in the mesocarp, cell wall thickening and cell enlargement in the mesocarp parenchyma increase until the fruit is nearly full cell size. The cell walls then decrease in thickness as the fruit ripens and softens. Degree of methyl esterification of the pectic substances, as estimated histochemically, remains at about 75–80% in immature fruits during their cell-enlargement phase of growth. Percent of methyl esterification apparently is much lower, or amounts of esterified pectates are very low during the meristematic phases of fruit growth. Just prior to ripening, degree of esterification increases and approaches 100% in hard, ripe fruit at about the same time that the parenchyma cell walls exhibit their greatest thickness or degree of hydration. The degree of esterification of the pectic substances then rapidly decreases and the cell walls become appreciably thinner as the ripening fruit softens. Further relation of these changes in wall thickness, in degree of esterification of the pectins, and in other cell wall carbohydrates to the textural qualities of ripening fruits is discussed. Interpretations concerning cell wall plasticity, cell growth and relation between auxin and changes in pectins also are presented.     

Compositional changes in cell wall polysaccharides from chilled and non-chilled cucumber fruit

Cell wall carbohydrate composition and 1-aminocyclopropane-1-carboxylic acid (ACC) content have been determined in chilled (2.5°) and non-chilled (12.5°) cucumber fruit. The major compositional change that accompanied the increased capability for ACC synthesis during chilling was a diminished loss of galactose residues, relative to the loss which occurred at 12.5°. However, the loss of galactose residues increased markedly when fruit were transferred from 2.5° to 20°, and wall galactose levels eventually declined to similar levels in both chilled and non-chilled fruit. Rhamnose, arabinose, xylose, mannose and cellulose content of walls was similar in chilled and non-chilled fruit and did not change substantially upon transfer of fruit to 20°. Upon transfer of chilled fruit from 2.5° to 20°, an increase in the relative amount of galacturonic acid in cell walls occurred; this change did not occur in non-chilled fruit. Thus, chilling stress results in a rapid change in the neutral sugar and galacturonic acid composition of cell wall pectic polysaccharides upon warming.     

QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis

Pectins are a highly complex family of cell wall polysaccharides. As a result of a lack of specific mutants, it has been difficult to study the biosynthesis of pectins and their role in vivo. We have isolated two allelic mutants, named quasimodo1 (qua1-1 and qua1-2), that are dwarfed and show reduced cell adhesion. Mutant cell walls showed a 25% reduction in galacturonic acid levels compared with the wild type, indicating reduced pectin content, whereas neutral sugars remained unchanged. Immersion immunofluorescence with the JIM5 and JIM7 monoclonal antibodies that recognize homogalacturonan epitopes revealed less labeling of mutant roots compared with the wild type. Both mutants carry a T-DNA insertion in a gene (QUA1) that encodes a putative membrane-bound glycosyltransferase of family 8. We present evidence for the possible involvement of a glycosyltransferase of this family in the synthesis of pectic polysaccharides, suggesting that other members of this large multigene family in Arabidopsis also may be important for pectin biosynthesis. The mutant phenotype is consistent with a central role for pectins in cell adhesion.     

Polypotency of the immunomodulatory effect of pectins

Pectins are the major component of plant cell walls, and they display diverse biological activities including immunomodulation. The pectin macromolecule contains fragments of linear and branched regions of polysaccharides such as homogalacturonan, rhamnogalacturonan-I, xylogalacturonan, and apiogalacturonan. These structural features determine the effect of pectins on the immune system. The backbones of pectic macromolecules have immunosuppressive activity. Pectins containing greater than 80% galacturonic acid residues were found to decrease macrophage activity and inhibit the delayed-type hypersensitivity reaction. Branched galacturonan fragments result in a biphasic immunomodulatory action. The branched region of pectins mediates both increased phagocytosis and antibody production. The fine structure of the galactan, arabinan, and apiogalacturonan side chains determines the stimulating interaction between pectin and immune cells. This review summarizes data regarding the relationship between the structure and immunomodulatory activity of pectins isolated from the plants of the European north of Russia and elucidates the concept of polypotency of pectins in native plant cell walls to both stimulate and suppress the immune response. The possible mechanisms of the immunostimulatory and anti-inflammatory effects of pectins are also discussed.     

Carrot arabinogalactan proteins are interlinked with pectins

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI-TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.     

The localisation of pectin in Sphagnum moss leaves and its role in preservation

The localisation of pectin in Sphagnum moss leaves and its role in preservation has been investigated. Light microscopy using ruthenium red to detect pectin in whole and sectioned Sphagnum papillosum leaves revealed it is abundant in hyaline cell walls, fibrils, papillae, chlorophyllous cell walls and thickenings around hyaline cell pores. Transmission electron microscopy of ultrathin cell walls labelled with poly-l-lysine colloidal gold revealed pectin was distributed throughout the cell wall. The preservative/microbiocidal properties of these pectins are explained by the acid-dissociation properties of galacturonic acid carboxyls and their incorporation in the unique cell arrangement of the Sphagnum leaf. Liquid from a salmon fillet absorbed into S. papillosum leaves and incubated at room temperature for 22 h had a pH around 4.85, was dominated by Lactobacillus sp. and smelled fresh compared to experimental controls. Chlorite-treated Sphagnum leaves could have a potential as a food tray pad that absorbs liquid and prevents the growth of spoilage bacteria inside it.     

Response of the leaf cell wall to desiccation in the resurrection plant Myrothamnus flabellifolius

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.     

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.     

Agrobacterium adherence involves the pectic portion of the host cell wall and is sensitive to the degree of pectin methylation

Cell walls isolated from dicotyledon tissues compete with natural plant host sites for Agrobacterium tumefaciens (strain B6) when co-inoculated with infectious bacteria, thereby reducing tumor initiation. Removal of the pectic fraction from the cell walls results in loss of inhibition and the soluble pectic fraction is inhibitory. On treatment with pectin methyl transferase plus S-adenosyl-L-methionine these cell walls become less inhibitory and this change is reversible by pectinesterase. Cell walls isolated from monocotyledons, crown gall tumors or embryonic dicotyledons do not compete for Agrobacterium in the infection assay. These cell walls become inhibitory on treatment with pectinesterase and this is partially reversed by pectin methyl transferase. These data indicate that the pectic portion of the host cell wall is involved in the Agrobacterium -host adherence which is essential for tumor initiation and that the degree of methylation of polygalacturonic acid is critical to this adherence.     

The polysaccharide structure of potato cell walls: Chemical fractionation

Cell walls of potato tubers were fractionated by successive extraction with various reagents. A slightly degraded pectic fraction with 77% galacturonic acid was extracted in hot, oxalate-citrate buffer at pH 4. A further, major pectic fraction with 38% galacturonic acid was extracted in cold 0.1 M Na₂CO₃ with little apparent degradation. These two pectic fractions together made up 52% of the cell wall. Most of the oxalate-citrate fraction could alternatively be extracted with cold acetate-N,N,N-tetracetic acid (CDTA) buffer, a non-degradative extractant which nevertheless removed essentially all the calcium ions. This fraction was therefore probably held only by calcium binding, and the remainder of the pectins by covalent bonds. Electrophoresis showed that both pectic fractions contained a range of molecular types differing in composition, with a high arabinose: galactose ratio as well as much galacturonic acid in the most extractable fractions. From methylation data, the main side-chains were 1,4-linked galactans and 1,5-linked arabinans, with smaller quantities of covalently attached xyloglucan. Extraction with NaOH-borate removed a small hemicellulose fraction and some cellulose. The main hemicelluloses were apparently a galactoxyloglucan, a mannan or glucomannan and an arabinogalactan.Abbreviations GLC gas-liquid chromatography - MS mass spectrometry - V₀ void volume - MW weight-average molecular weight - DMSO dimethylsulphoxide - EDTA ethylenediamine tetraacetic acid - TFA trifluoroacetic acid - CDTA N,N,N-tetraacetic acid     

Effect of hydroxyl radical on the scission of cellular wall polysaccharides in vitro of banana fruit at various ripening stages

Fruit ripening is generally attributed to disassembly of cellular wall, particularly due to solubilisation and depolymerisation of pectin and hemicellulose. Experiments were conducted to test effects of hydroxyl radicals (·OH) on the scission of cellular wall polysaccharides from pulp tissues of banana fruit at different ripening stage. Cellular wall materials were isolated from pulp tissues of banana fruit at different ripening stages. Two pectic fractions, water soluble pectin (WSP) and acid soluble pectin (ASP), and two hemicellulosic fractions, 1 M KOH soluble hemicellulose (HC1) and 4 M KOH soluble hemicellulos (HC2), were obtained from the cellular wall materials from pulp tissues, respectively. Effects of ·OH induced by the Fenton reaction on the scission of pectin and hemicellulose in vitro were investigated. As fruit ripening progressed, the sugar components of the WSP, HC1 and HC2 attacked by ·OH showed obvious molecular-mass downshifts. Thus, ·OH caused the disassembly of polysaccharides (WSP, ASP, HC1 and HC2) from cellular walls of pulp tissues of banana fruit, demonstrated by the reduced molecular mass distribution. Moreover, ·OH production in pulp tissues increased significantly as banana fruit ripened, which further help account for the role of ·OH in accelerated fruit ripening.