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.     

Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls

Cell wall material (CWM) was prepared from nine fruit species at two ripening stages (unripe and ripe) and extracted sequentially with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na₂CO₃ and 4 M KOH. Each solubilised fraction and the CWM-residue remaining after 4 M KOH extraction was analysed for non-cellulosic sugar composition. A common pattern of distribution for polyuronide and pectin-associated neutral sugar was observed for all unripe fruit. Most polyuronide was extracted in the CDTA/Na₂CO₃ fractions while 70–93% of the neutral sugar was located on pectic polysaccharides in the 4 M KOH-soluble and CWM-residue fractions. During ripening, most of the galactose was lost from pectic polysaccharides in the CWM-residue. Partial solubilisation of these polysaccharides was achieved by treating the CWM-residue with endopolygalacturonase. The solubilised polysaccharides were separated into two fractions by ion-exchange chromatography. One of these contained polysaccharides with average molecular weights of 400 kDa or larger and consisted of between 70 and 90% arabinogalactan. The galactosyl residues were 80–90% β-1→4 linked, indicating largely unbranched side-chains. The arabinosyl residues were distributed among terminal, 3-, 5-, 2,5-, and 2,3,5-linked residues, indicating a highly ramified structure. The results are discussed with regard to the relationship between pectin solubilisation and galactose loss and their respective contribution to fruit softening. Received: 28 January 1997 / Accepted: 11 March 1997     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.     

Down-regulation of UDP-glucuronic acid biosynthesis leads to swollen plant cell walls and severe developmental defects associated with changes in pectic polysaccharides

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.     

Intracellular feruloylation of pectic polysaccharides

The pectic polysaccharides of spinach cell walls carry feruloyl groups on arabinose and galactose residues. The following experiments were designed to discover whether the arabinose residues are feruloylated intra-or extracellularly. Cultured spinach cells started to incorporate exogenous [³H]arabinose into polymers at a linear rate after a lag period of approx. 3–4 min, although radioactive polysaccharides and extensin did not start to appear outside the plasmalemma until after an approx. 25-min lag. In the same cells, polysaccharide-bound feruloyl-[³H]arabinose units starded to accumulate radioactivity at a linear rate after a lag period of approx. 4–5 min. Therefore, arabinose residues of polysaccharides began to be feruloylated while still intracellular. The rate of formation of polysaccharide-bound feruloyl-[³H]arabinose units did not appreciably increase after 25 min, showing that any additional extracellular feruloylation of the polysaccharide was relatively slow. This conclusion was supported by two different types of pulse-chase experiments, one of which was designed to detect feruloylation of polysaccharides up to 6 d after synthesis.Abbreviations Ara₂ 3-O–-L-arabinopyranosyl-L-arabinose - BAW butan-1-ol/acetic acid/water (12:3:5, by vol.) - BEW butan-1-ol/ethanol/water (20:5:11, by vol.) - EPW ethyl acetate/pyridine/water (8:2:1, by vol.) - Fer-Ara₂ 3-O–(3-O–feruloyl--L-arabinopyranosyl)-L-arabinose - Fer-Gal₂ 4-O–(6-O–feruloyl--D-galactopyranosyl)-D-galactose     

Effects of boron deficiency and calcium supply on the calcium metabolism in tomato plant

Summary Tomato plants were grown in water-culture with a different supply of Ca (10, 100 ppm) and B (0, 0.2 ppm), and the effects of B deficiency on the translocation and subcellular distribution of Ca in tomato plants were studied by using⁴⁵CaCl₂ as a carrier of Ca. Boron deficiency slight increased the total Ca uptake by the plant and inhibited the Ca translocation to the upper leaves. The incorporation of⁴⁵Ca into the cell wall in the upper leaves was increased by B deficiency at both Ca levels. As Ca supply decreased, the distribution of⁴⁵Ca in the 1N NaCl fraction of the cell wall increased only at 0.2 ppm B. As B supply decreased, the distribution of⁴⁵Ca in the 0.6N HCl fraction increased at both Ca levels. These results suggest that B deficiency inhibit the translocation of Ca, and induce the abnormal changes of the Ca metabolism in the cell wall.     

Dimeric calcium complexes of arabinan-rich pectic polysaccharides from Olea europaea L. cell walls

The study carried out in this work concerns the pectic polysaccharides of olive cell walls as present in olive pulp and that remained entrapped in the cellulosic residue after sequential extraction of the cell wall material (CWM) with imidazole, carbonate and KOH aqueous solutions. These polymers, obtained after neutralisation and dialysis of an aqueous suspension of the residue (sn-CR fraction), extracted with 4 M KOH, were arabinan-rich pectic polysaccharides. They accounted for 11–19% of the total pectic polysaccharides found in the olive pulp cell walls of fruits collected in two years and in three stages of ripening (green, cherry and black). The analysis by powder X-ray diffraction highlighted the existence, in all sn-CR fractions, of crystalline phases related with the presence of calcium-pectic polysaccharide complexes (CPPC) occurring in an amorphous carbohydrate network. The relative crystallinity of the CPPC varied linearly with the Ca²⁺/GalA molar ratio until a maximum of 0.57. Size-exclusion chromatography showed that sn-CR fractions possessed a bimodal molecular weight distribution. The lower molecular weight fraction of sn-CR (M_w = 70–135 kDa) was independent on the ripening stage of olive fruit, whereas the higher molecular weight fraction showed values of 1.1, 0.6–0.9 and 0.5–0.7 MDa, respectively, for green, cherry and black olives. Treatment of the sn-CR pectic polysaccharides with a 2 M imidazole solution disrupted the CPPC crystalline network showing the loss of low molecular weight galacturonan-rich material during dialysis (12–14 kDa cut off) and the decrease of molecular weight of the polymers to roughly half. These results allowed to infer the presence of oligogalacturonides held within cell walls by calcium ions and that the pectic polysaccharides of sn-CR fraction occurred in olive pulp cell walls as calcium bridged macrodimers.     

植物硼钙效应及其在细胞壁中互作机制的研究

植物营养元素之间存在着相互作用,其作用机理一直是相关学者研究的重点。硼是植物必需的营养元素,近年来,有关硼与其它元素之间的相关性研究已取得了一系列成果。本文综述了国内外关于植物在不同硼、钙条件下的形态发育、代谢组学、细胞壁果胶网络中的交联机制等方面的研究进展,并对如何充分利用代谢组学手段探究硼钙之间相互作用的机制以及硼钙互作对植物生长发育的调控作用,尤其是两者在细胞壁的互作机制方面的研究进行了展望。     

Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. var. botrytis) stems

Pectic substances are a major component of cell walls in vegetable plants and have an important influence on plant food texture. Cauliflower (Brassica oleracea L. var. botrytis) stem sections at different regions of the mature plant stem have been monitored for tissue-related changes in the native pectic polysaccharides. Chemical analysis detected appreciable differences in the degree of methyl-esterification (ME) of pectic polysaccharides. About 65% of galacturonic acid (GalpA) residues were methyl-esterified in floret tissues. Relative ME showed a basipetal decrease, from 94% in the upper stem to 51% in the lower-stem vascular tissues. The decrease was not related to a basipetal increase in glucuronic acid (GlcpA) residues. The monoclonal antibodies, JIM 5 and JIM 7, produced distinct labelling patterns for the relatively low-methyl-esterified and high-methyl-esterified pectin epitopes, respectively. Labelling was related to cell type and tissue location in the stem. Floret cell walls contained epitopes for both JIM 5 and JIM 7 throughout the wall. Stem vascular tissues labelled more strongly with JIM 5. Whereas pith parenchyma in the upper stem labelled more strongly with JIM 7, in the lower-stem pith parenchyma, JIM 5 labelling predominated. Localization of pectic polysaccharide epitopes in cell walls provides an insight into how structural modifications might relate to the textural and nutritional properties of cell walls. Received: 16 August 1997 / Accepted: 20 December 1997     

Glycoprotein conformation in plant cell walls

The major structural glycoprotein of the cell wall of Chlamydomonas reinhardii has a protein core, at least 50% of which is in the unusual polyproline II conformation. This has been demonstrated by examining the circular dichroism of the cell wall, its constituent glycoproteins, and thermolysin released wall glycopeptides. One of these glycopeptides, T2, has a high hydroxyproline and sugar content, and possesses upward of 85% polyproline II structure. The main extracellular matrix glycoprotein therefore has a rigid, rod-like structure and the significance of this and its relation to higher plant cell wall glycoproteins is discussed. The unusual conformation appears to confer great stability on the glycoprotein as it is unchanged either by certain denaturing agents or during the transition from protomer to assembled cell wall.Abbreviations CD circular dichroism - HP 4-hydroxy-L-proline - PP poly-L-proline - SDS sodium dodecylsulphate This is the eight paper in a series entitled Structure, Composition and Morphogenesis of the Cell Wall of Chlamydomonas reinhardii. The last paper in this series was Catt et al. (1978)     

The pectic polysaccharide rhamnogalacturonan II is present as a dimer in pectic populations of bilberries and black currants in muro and in juice

Rhamnogalacturonan II (RG II) can play an important role during processing of berries due to its enzyme resistance and its possible role as a pectic cross-linker. This article describes the presence of RG II in cell walls, in juice and in press cake of bilberries and black currants. RG II was identified and quantified via its diagnostic sugar residues. RG II, which was released from homogalacturonan, was probably present in its dimeric form in muro. Juice contained the free RG II dimer, while from press cake dimeric RG II was released by enzymatic degradation of homogalacturonan. A higher amount of RG II was present in juice than in press cake. During juice processing a cross-linker RG II might improve gel formation, which hinders the processability of berries. In addition, enzymes used during juice processing release dimeric RG II from pectin molecules and accumulate RG II in the juice.     

ESR evidence for sequential divalent cation binding in higher plant cell walls

Electron spin resonance linewidth measurements have been made on intact cell walls exchanged with various combinations of Mn²⁺ and Ca²⁺. These experiments were performed to find the Mn²⁺ nearest-neighbor distance and thereby determine whether carboxylate-Mn²⁺ complexes potentiate ion association at adjacent sites on cell wall polyuronides. Our results show that as the fraction of available binding sites occupied by Mn²⁺ increased from 2% to 27%, the nearest-neighbor distance parameter decreased only from 14 to 11 Å. These distances are close to polyuronide interanionic spacings. The small change in the distance parameter with concentration is evidence for sequential rather than random binding. Competitive ion-exchange with Ca²⁺ was found to reduce the Mn²⁺ spin-spin line broadening at similar total bound Mn²⁺ concentrations. This is expected only if Ca²⁺ competes at adjacent sites. The data presented offer strong support for the hypothesis that carboxylate groups near already occupied sites have a greater affinity for divalent cations than other sites along the polyuronide main chain.     

Alteration of pectic polysaccharides in cell walls, extracellular polysaccharides, and glycan-hydrolytic enzymes of growth-restricted carrot cells under calcium deficiency

Suspension-cultured carrot ( Daucus carota L. cv. Kintoki) cells were grown in calcium (Ca²⁺)-deficient and normal liquid media. Cell growth was limited by the Ca²⁺ deficiency. Similar amounts of pectic fractions were extracted from the walls of control and Ca²⁺-deprived cells, but the fractions from the walls of Ca²⁺-deprived cells showed a substantial decrease in galacturonic acid content. However, after 15 days of culture, Ca²⁺-deprived cells released galacturonic acid-rich extracellular polysaccharides at twice the rate of control cells. The polysaccharides consisted of a mixture of several polymers containing predominantly arabinose, galactose and galacturonic acid. Ca²⁺-deprived cells also secreted three times more extracellular proteins, containing many glycan-hydrolytic enzymes, into the medium than did normal cells. SDS-PAGE analysis revealed several distinct changes in the polypeptide pattern in the medium of control and Ca²⁺-deprived cells. Activities of α -galactosidase, β -glucosidase and exo- polygalacturonase increased considerably during Ca²⁺ deficiency, whereas α - l -arabinofuranosidase and β -galactosidase activities were much reduced.     

Mechanical properties and polysaccharide nature of the cell walls of maize root

Changes in mechanical properties and chemical nature of the cell walls of the different zones along elongating maize ( Zea mays L. cv. LG 11) roots were analyzed and the following results were obtained. (1) The apical region 2 to 5 mm from the tip of 15 mm long roots showed rapid elongation whereas the region 8–10 mm from the tip showed very little growth. (2) The minimum stress-relaxation time (To) and the mean stress-relaxation rate (R) of the cell wall were small whereas the maximum stress-relaxation time (Tm) was large in the region where cell elongation was optimum. The To and R increased and the Tm decreased gradually towards the base of the root. (3) The amounts of non-cellulosic polysaccharides of the cell wall were highest in the region 1.5–2.5 mm from the tip, decreasing until 5 mm from the tip, and then increasing towards the base. However, the proportion of this fraction in the total cell wall polysaccharides was highest in the extreme tip (cap and meristem, 0–1 mm) and decreased towards the base. (4) Major neutral sugars constituting the non-cellulosic polysaccharides of the cell wall were xylose, arabinose, galactose and glucose, with minor amounts of rhamnosc. mannose and fucose. The 1–15 mm region was on the whole rich in glucose and xylose and contained arabinose to a lesser extent. However, the chemical nature in the apical region, (0–2 mm, was rather special, being rich in galactose and fucose. (5) The cell wall of maize roots contained, as a whole, only little pectic substances but was high in hemicellulose 1 (rich in xylose, arabinose and glucose) and hemicellulose 2 (rich in glucose and xylose). (6) It appeared that in the elongating region (apical 2 to 5 mm) the cell elongation rate (CET) showed a rather good correlation with the parameters of mechanical properties (To, Tm and R) and with neutral sugar compositions in the non-cellulosic polysaccharides.     

Electron-energy-loss spectroscopic imaging of calcium and nitrogen in the cell walls of apple fruits

Changes in texture are an integral part of ripening in most fleshy fruits and these changes are thought to be determined, primarily, by alterations in cell wall structure. Electron energy loss spectroscopy (EELS) imaging was used to obtain quantitative information on the levels of calcium and nitrogen in the cell walls of apple (Malus domestica Borkh. cv. Cox's Orange Pippin) fruits. Samples of fruit cortex were prepared for EELS by high-pressure freezing and molecular distillation drying to minimize loss and redistribution of soluble cell wall components such as calcium. The EELS imaging successfully resolved calcium and nitrogen levels in the middle lamella and primary cell wall. When the elemental compositions of the cell walls of Cox's apples from two sites in the UK were compared at harvest or after 6 months storage, the orchard which always produced consistently firmer fruit had significantly lower levels of cell wall calcium and higher levels of cell wall nitrogen. This result was unexpected since firm texture in apples and other fruits has been commonly associated with elevated levels of fruit calcium. The nitrogen-rich material in the sections used for EELS was insoluble in acidified methanol, indicating that it represented a high-molecular-weight component in the cell wall. Furthermore, total tissue hydroxyproline levels were greatest in material with elevated cell wall nitrogen, suggesting enhanced levels of wall structural proteins in the tissue. These data indicate a correlation between increased amounts of cell wall nitrogen and firm fruit texture. The possible role of cell wall proteins in determining the textural properties of fruit tissue is discussed. Received: 19 November 1998 / Accepted: 28 January 1999     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

Biosynthesis of ferulic acid esters of plant cell wall polysaccharides in endomembranes from parsley cells

A microsomal preparation from suspension-cultured parsley cells is able to transfer ferulic acid from the respective CoA thioester to endogenous acceptors. The reaction is not enhanced by digitonin but stimulated by Mg2+, Ca2+ and Co2+. Spermine can partly replace divalent ions. Solubility properties and degradation by polysaccharide hydrolases suggest that the products are polymeric cell wall carbohydrates. Sucrose density gradient centrifugation revealed that the most active vesicle fraction is distinct from plasma membranes but does also not peak with IDPase. It is suggested that a subfraction of the Golgi-apparatus is the source of enzyme and acceptors.     

Role of calcium during biosynthesis,secretion and organization of cell-wall polysaccharides

Abstract

The role of calcium during the synthesis, secretion and molecular organization of the primary cell-wall polysaccharides is the topic of this review. With the exception of callose synthase, the in vitro activity of all polysaccharide synthases is not controlled by Ca²⁺ ions. However, changes in the intracellular Ca²⁺ level could control the rate of exocytotic fusion of the secretory vesicles containing cell-wall matrix polysaccharides. In particular, the ability of Ca²⁺ to regulate the fusion of secretory vesicles with the plasma membrane is due to a class of Ca²⁺-dependent phospholipid-binding proteins known as annexins. The ionic interactions between calcium and the negatively charged homogalacturonan domains of the pectins are important not only for the mechanical properties of the wall but also for the gel-properties of these complex biopolymers.     

水稻和小麦根尖细胞壁多糖的铝积累能力比较

采用水培法比较4种禾本科植物水稻（Oryza sativa L.）、玉米（Zea mays L.）、高粱（Sorghum bicolor（L.）Moench）和小麦（Triticum aestivum L.）8个基因型的抗铝（Al）能力,并对他们在Al积累后细胞壁的多糖组分进行分析。结果显示,在5~200 μmol/L Al处理下,水稻抗Al能力较强,而小麦抗Al能力较弱。在50 μmol/L Al处理下,小麦根尖的果胶和半纤维素1含量的增幅明显高于水稻。水稻基因型‘日本晴’与‘浙辐802’的细胞壁Al含量分别占根尖总Al含量的78.7%和91.6%;小麦基因型‘扬麦18’与‘扬麦16’Al含量分别占根尖总Al含量的64.9%和72.1%。Al吸附-解吸实验结果显示,小麦根尖细胞壁上Al的吸附量高于水稻。研究结果表明,细胞壁是Al积累的主要部位,对Al敏感的水稻和小麦基因型细胞壁中的Al主要分布在果胶中;而对Al耐性较强的水稻和小麦基因型细胞壁中的Al主要分布在半纤维素1中。     

The role of wall calcium in the extension of cell walls of soybean hypocotyls

Calcium crosslinks are load-bearing bonds in soybean (Glycine max (L.) Merr.) hypocotyl cell walls, but they are not the same load-bearing bonds that are broken during acid-mediated cell elongation. This conclusion is reached by studying the relationship between wall calcium, pH and the facilitated creep of frozenthawed soybean hypocotyl sections. Supporting data include the following observations: 1) 2-[(2-bis-[carboxy-methyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[carboxymethyl]aminoquinoline (Quin 2) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) caused only limited facilitated creep as compared with acid, despite removal of comparable or larger amounts of wall calcium; 2) the pH-response curves for calcium removal and acid-facilitated creep were different; 3) reversible acid-extension occurred even after removal of almost all wall calcium with Quin 2; and 4) growth of abraded sections did not involve a proportional loss of wall calcium. Removal of wall calcium, however, increased the capacity of the walls to undergo acid-facilitated creep. These data indicate that breakage of calcium crosslinks is not a major mechanism of cell-wall loosening in soybean hypocotyl tissues. This research was supported by Department of Energy grant DE-FG06-88ER13830 and NASA grant NAGW 1394. The authors are grateful to Dr. David Rayle (San Diego State University, Cal.) for stimulating discussions and comments during the course of this work.