Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin

Oligogalacturonides [oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by 1H and 13C NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants.     

Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides

Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.     

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.     

Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.     

A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly.

Abbreviations: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II     

Polysaccharides from grape berry cell walls. Part I: tissue distribution and structural characterization of the pectic polysaccharides

Buffer-soluble arabinogalactan-proteins (AGPs) and pectins from grape berry skin and pulp tissues have been isolated and their structure has been partly determined. Pectic polysaccharides from the cell wall material were solubilized by treating pulp and skin cell walls with homogeneous glycosyl hydrolases. Homogalacturonans, rhamnogalacturonans I (RG-I), and rhamnogalacturonan II (RG-II) of each tissue have been fractionated by high resolution size exclusion chromatography and their relative distribution and major structural features have been determined. It has been shown that pulp tissue contains two-fold more buffer-soluble AGPs and pectins than skin tissue and we have determined that 75% of the grape berry walls originates from the skin tissue. There is three-fold more RG-I and RG-II in skin tissue than in pulp tissue and three-fold more RG-I than RG-II in the grape berry cell walls.

The results of this study have shown that the grape polysaccharide content of a wine is related to the type of tissue used for wine making and to the solubility of the grape polysaccharides and their resistance to fragmentation by grape and yeast glycanases.     

Oligogalacturonides inhibit the formation of roots on tobacco explants

α-1,4-Oligogalacturonides with degrees of polymerization (DPs) ranging from 6 to 18 or 2 to 8 were added to tobacco leaf explants and root formation was evaluated after 15 days of incubation. Auxin-induced formation of roots was inhibited by oligogalacturonides with DPs 6–18 but not by the oligogalacturonides with DPs 2–8. The inhibition of root formation by the larger oligogalacturonides was prevented by increasing the amount of auxin present in the medium. Oligogalacturonides (DPs 6–18) also inhibited root formation when added to tobacco thin cell-layer (TCL) explants in a medium that is known to induce the formation of roots. The addition of size-homogeneous oligogalacturonides, to either tobacco leaf explants or TCLs, established that oligogalacturonides with DPs between 10 and 14 were most active in inhibiting the formation of roots. These data suggest that oligogalacturonides of the same size as those known to elicit plant defense responses, and to affect floral development and membrane functions, also inhibit the induction of root morphogenesis in tobacco.     

Elicitation of H2O2 production in cucumber hypocotyl segments by oligo-1,4-α-D-galacturonides and an oligo-β-glucan preparation from cell walls of Phythophthora megasperma f. sp. glycinea

The production of H₂O₂ by cucumber hypocotyl segments ( Cucumis sativus L. cv. Wisconsin SMR 58) in response to α-1,4-linked oligomers of galacturonic acid and oligo-β-glucans from the cell walls of Phytophthora megasperma f. sp. glycinea was studied. Oligogalacturonides with degrees of polymerization of 9 to 13 elicited H₂O₂ production, the most effective being the deca-, undeca- and dodecamers. A similar relationship between size and effect was previously obtained when oligogalacturonides were tested for their ability to elicit lignification in cucumber hypocotyls. The oligogalacturonide-induced increase in H₂O₂ concentration was detected after 4 h, reaching a maximum after 10 h of incubation. The glucan elicitor induced lignification at a 100-fold lower concentration than the oligogalacturonides, but yielded only 10% of the maximum H₂O₂ accumulation seen with oligogalacturonides. The glucan elicitor-induced H₂O₂ production was detectable after 2 h, and reached a maximum after 4 to 6 h. Catalase abolished the elicitation of both phenol red oxidation and lignification in cucumber hypocotyls. At least part of the oligogalacturonide-induced H₂O₂ production appeared to be dependent upon de novo protein synthesis.     

Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells

The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified α-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.     

The nature of lignin-like compounds in cucumber hypocotyls induced by α-1,4-linked oligogalacturonides

The nature of the phloroglucinol-positive material, which is induced in cucumber ( Cucumis sativus L.) hypocotyls by α-1,4-linked oligogalacturonides, has been investigated by three different experimental approaches and they all suggest that the material may be classified as lignin. Firstly, formation of the material is inhibited by compounds which are known to inhibit the first (phenylalanine ammonia lyase) and the last (peroxidase) step in the biosynthesis of lignin. Secondly, the elicitor enhances incorporation of [¹⁴C]-cinnamic acid derivatives into the hypocotyl cell walls. Thirdly, the analyses of phenols released from cell walls by cupric oxide oxidation show that the walls of oligogalacturonide-treated hypocotyls contain more polyphenolic material than the walls from intact or wounded hypocotyls. The walls from the oligogalac-turonide-treated hypocotyls produce more p -hydroxybenzaldehyde, p -hydroxyace-tophenone, p -hydroxybenzoic acid and p -coumaric acid upon oxidation than the other two wall samples. These results suggest that the lignin formed in cucumber hypocotyls in response to oligogalacturonides is mainly derived from p -coumaryl alcohol.     

Structural study of bergenan,a pectin from <Emphasis Type="Italic">Bergenia crassifolia</Emphasis>

A pectin polysaccharide named bergenan was isolated from the freshly collected leaves of the leather bergenia Bergenia crassifolia by extraction with an aqueous solution of ammonium oxalate. The main component of its carbohydrate chain was shown to be the residues of D-galacturonic acid (about 80%). In addition, the polysaccharide contains the residues of galactose, arabinose, and rhamnose; their total content is less than 15%. It was shown that the bergenan samples from bergenia leaves collected at different vegetation periods (from July to September) do not substantially differ either in monosaccharide composition or in the viscosity of their aqueous solutions. The results of enzymatic hydrolysis by α-1,4-galacturonase (pectinase), partial acidic hydrolysis, NMR spectroscopy, and methylation with subsequent analysis of the results by GC-MS indicate that the bergenan macromolecule contains the regions of a linear α-1,4-D-galactopyranosyluronan and rhamnogalacturonan-I (RG-I). Galacturonan responds for a greater part of the macromolecule. A considerable amount of its constitutent galacturonic acid residues are present as methyl esters. The side chains in RG-I are attached to the rhamnopyranose residues of the backbone by a 1,4-linkages and are composed of the residues of terminal arabinofuranose and galactopyranose, 1,5-linked α-arabinofuranose, and 1,4- and 1,6-linked β-galactopyranose. The branching points of the side chains of the RG-I molecule are 3,4- and 3,6-di-O-substituted galactose residues.     

植物细胞壁同聚半乳糖醛酸的代谢与功能

果胶是细胞壁多糖的重要组成成分,对植物正常的生长发育十分重要。作为初生细胞壁中果胶的一种主要组成成分,同聚半乳糖醛酸（homogalacturonan,HG）是由α-D-半乳糖醛酸单体经α-（1,4）-糖苷键连接起来的一种长链大分子物质。HG的合成和降解参与了细胞壁中的多糖代谢,影响了细胞壁的结构和功能。同时,HG精确的去甲酯化以及HG所参与的细胞壁关联激酶（WAKs）和促分裂原活化蛋白激酶（MAPKs）相关的信号转导途径,在植物生长发育中也发挥着重要作用。该文主要从HG的合成、降解和循环利用以及HG的作用等方面对植物细胞壁中HG的研究进展进行了阐述。     

Oligogalacturonides: novel signaling molecules in Rhizobium-legume communications

Oligogalacturonides are pectic fragments of the plant cell wall, whose signaling role has been described thus far during plant development and plant-pathogen interactions. In the present work, we evaluated the potential involvement of oligogalacturonides in the molecular communications between legumes and rhizobia during the establishment of nitrogen-fixing symbiosis. Oligogalacturonides with a degree of polymerization of 10 to 15 were found to trigger a rapid intracellular production of reactive oxygen species in Rhizobium leguminosarum bv. viciae 3841. Accumulation of H(2)O(2), detected by both 2',7'-dichlorodihydrofluorescein diacetate-based fluorescence and electron-dense deposits of cerium perhydroxides, was transient and did not affect bacterial cell viability, due to the prompt activation of the katG gene encoding a catalase. Calcium measurements carried out in R. leguminosarum transformed with the bioluminescent Ca(2+) reporter aequorin demonstrated the induction of a rapid and remarkable intracellular Ca(2+) increase in response to oligogalacturonides. When applied jointly with naringenin, oligogalacturonides effectively inhibited flavonoid-induced nod gene expression, indicating an antagonistic interplay between oligogalacturonides and inducing flavonoids in the early stages of plant root colonization. The above data suggest a novel role for oligogalacturonides as signaling molecules released in the rhizosphere in the initial rhizobium-legume interaction.     

Structural characterization of cell wall polysaccharides from two plant species endemic to central Africa,Fleurya aestuans and Phragmenthera capitata

Fleurya aestuans (Linnaeus) Miquel and Phragmenthera capitata (Spreng) are two plants endemic to central Africa that are used in traditional medicine. However, information on their molecular constituents is lacking. In the present study and as part of our research on the structure/bioactivity relationship of plant cell wall molecules, we investigated the structure of polysaccharides isolated from leaf cell walls of both plant species. To this end, we used sequential extraction of polysaccharides, gas chromatography, matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and immuno-dot assays. Our data indicate the presence of both pectin and hemicellulosic polysaccharides in the cell walls of both plants. In particular, cell wall of F. aestuans leaves appears to contain much more pectin than those of P. capitata. Structural analysis of hemicellulosic polysaccharides revealed differences in the structure of xyloglucan isolated from both species. While only the XXXG-type was found in P. capitata, both XXXG and XXGG types were detected in F. aestuans. No arabinosylated subunits were found in any of the xyloglucan isolated from both plant species. In addition, xylan structure with non methylated-α-d-glucuronic acid on side chains was only detected in F. aestuans leaf cell walls. Finally, structural analysis of rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) shows that unlike RG-II, RG-I is qualitatively different between F. aestuans and P. capitata leaves.     

Development of a filter assay for measuring homogalacturonan: alpha-(1,4)-Galacturonosyltransferase activity

Alpha-(1,4)-galacturonosyltransferases (GalATs) catalyze the addition of (1,4)-linked alpha-D-galacturonosyl residues onto the nonreducing end of homogalacturonan chains. The nucleotide-sugar donor for the enzymatic reaction is uridine diphospho-D-galactopyranosyluronic acid (UDP-D-GalpA). Many GalAT activity assays are based on the incorporation of D-[(14)C]GalpA from UDP-D-[(14)C]GalpA onto exogenously added homogalacturonan acceptors. Reactions based on this method can be time-consuming because multiple labor-intensive centrifugations and washes with organic solvents are required to remove the unincorporated UDP-D-[(14)C]GalpA from the (14)C-labeled products. Here we report the development of an alternative GalAT filter assay based on the ability of homogalacturonan to bind to cetylpyridinium chloride (CPC). GalAT assay reaction products made using radish (Raphanus sativus) microsomal membranes or solubilized proteins from tobacco (Nicotiana tabacum L. cv. Samsun) and Arabidopsis thaliana (cv. Columbia) were spotted onto Whatman 3MM paper treated with 2.5% (w/v) CPC. Unincorporated UDP-D-[(14)C]GalpA was selectively removed from the filters by washing with 150-250 mM NaCl. The versatility of this assay is demonstrated by using it to identify GalAT activity in fractions obtained during the partial purification of tobacco GalAT by SP Sepharose cation exchange chromatography and by detecting the GalAT-catalyzed incorporation of D-[(14)C]GalpA onto endogenous acceptors from Arabidopsis membranes.     

Solubilization and characterization of a galacturonosyltransferase that synthesizes the pectic polysaccharide homogalacturonan

Polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT), the glycosyltransferase that synthesizes the plant cell wall pectic polysaccharide homogalacturonan, has previously been identified and partially characterized in tobacco membranes. Membrane bound PGA-GalAT catalyzes the transfer of galacturonic acid from UDP-galacturonic acid (UDP-GalA) onto an endogenous acceptor to produce polymeric homogalacturonan ( Doong et al. (1995) Plant Physiol. 109, 141 –152). It is shown here that a galacturonosyltransferase is solubilized from tobacco membranes with a HEPES buffer, pH 6.8, containing 40 mM CHAPS and 2 mM EDTA. The solubilized galacturonosyltransferase was identified as putative PGA-GalAT because it transfered [¹⁴C]GalA from UDP-[¹⁴C]GalA onto exogenous homogalacturonan acceptors with degrees of polymerization (DP) of ≥ 10. Maximal solubilized PGA-GalAT activity in the presence of 0.9 μM UDP-[¹⁴C]GalA required approximately 125 μM exogenous homogalacturonan acceptor [e.g. oligogalacturonide (OGA) of DP 15]. Solubilized PGA-GalAT was active over a broad pH range of 6.3–7.8, and had an apparent K_m for UDP-GalA of 37 μM and a V_max of 290 pmol min^–1 mg^–1 protein. Approximately 44% of the PGA-GalAT activity in detergent-dispersed membranes, corresponding to 21% of the PGA-GalAT activity in intact membranes, was solubilized. PGA-GalAT solubilized with 40 mM CHAPS was shown, by exopolygalacturonase treatment in combination with size exclusion and high performance anion exchange chromatographies, to add a single α-1,4-linked galacturonic acid residue onto an OGA exogenous acceptor of DP 15 to yield an OGA product of DP 16. The significance of the apparent lack of processivity of the solubilized PGA-GalAT is discussed.     

Boron nutrition of cultured tobacco BY-2 cells. III. Characterization of the boron-rhamnogalacturonan II complex in cells acclimated to low levels of boron

Cultured cells of tobacco (Nicotiana tabacum L.) BY-2 which could propagate at the same rate as the parent cells (1 mg B liter(-1)) under a lower level of boron (0.01 mg B liter(-1)) were obtained. The selected cells had swollen cell walls. In the parent cells, all the RG-II occurred as a B-RG-II complex, however, two-thirds of the RG-II occurred in a monomeric form in the selected cells.     

Ginseng root water-extracted pectic polysaccharides originate from secretory cavities

A range of molecular probes for cell wall polysaccharides has been used to explore the structure and location of water-extracted pectic polysaccharides occurring in fractions isolated from ginseng roots. The LM19 homogalacturonan (HG) epitope was abundant in an HG fraction and analysis of LM19 binding to a rhamnogalacturonan-I (RG-I) rich-fraction indicated that the LM19 epitope is sensitive to acetylation. A specific RG-I epitope (LM16), four arabinogalactan-protein (AGP) epitopes (LM2, LM14, JIM16, MAC207) and an extensin epitope (JIM20) were found to be abundant and co-located in several isolated polysaccharide fractions including an arabinogalactan fraction and two RG-I fractions. Detection of the RG-I, AGP and extensin epitopes identified in isolated polysaccharide fractions in sections of ginseng roots indicated that they were most abundant in secretory cavities found in the cortical regions of ginseng roots. In addition, the immunocytochemical study indicated that polysaccharide epitope masking is a widespread phenomenon in the primary cell walls of ginseng roots.