Structure of Plant Cell Walls: VIII. A New Pectic Polysaccharide

This paper describes the isolation and characterization of rhamnogalacturonan II, a hitherto unobserved component of the primary cell walls of dicotyledonous plants. Rhamnogalacturonan II constitutes 3 to 4% of the primary cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells. Rhamnogalacturonan II is a very complex polysaccharide yielding, upon hydrolysis, 10 different monosaccharides including the rarely observed sugars apiose, 2-O-methylxylose, and 2-O-methylfucose. In addition, rhamnogalacturonan II is characterized by the rarely observed glycosyl interconnections of 2-linked glucuronosyl, 3,4-linked fucosyl, and 3-linked rhamnosyl residues. These glycosyl linkages have never previously been detected in primary sycamore cell walls. Evidence is presented which suggests that polysaccharides similar to rhamnogalacturonan II are present in the primary cell walls of the three other dicotyledonous plants examined.     

Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides

Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-α-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-α-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na₂CO₃ at 1 and 22°C. These previously uncharacterized polysaccharides accounted for ~4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO₃-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na₂CO₃ at 1°C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.     

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-α-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-α-1,4-polygalacturonase-treated walls by treatment with an endo-β-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-β-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.     

The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells

The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed “amyloid” xyloglucans.     

The Structure of Plant Cell Walls: III. A Model of the Walls of Suspension-cultured Sycamore Cells Based on the Interconnections of the Macromolecular Components

Degradative enzymes have been used to obtain defined fragments of the isolated cell walls of suspension-cultured sycamore cells. These fragments have been purified and structurally characterized. Fragments released from endopolygalacturonase-pretreated cell walls by a purified endoglucanase and the fragments extracted from these walls by urea and alkali provide evidence for a covalent connection between the xyloglucan and pectic polysaccharides. Fragments released by a protease from endopolygalacturonase-endoglucanase-pretreated cell walls provide evidence for a covalent connection between the pectic polysaccharides and the structural protein of the cell wall. Based on these interconnections and the strong binding which occurs between the xyloglucan and cellulose, a tentative structure of the cell wall is proposed.     

Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS

The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.     

The Structure of Plant Cell Walls: IV. A Structural Comparison of the Wall Hemicellulose of Cell Suspension Cultures of Sycamore (Acer PseudoPlatAnus) and of Red Kidney Bean (Phaseolus Vulgaris)

The molecular structure and chemical properties of the hemicellulose present in the isolated cell walls of suspension cultures of sycamore (Acer pseudoplatanus) cells has recently been described by Bauer et al. (Plant Physiol. 51: 174-187). The hemicellulose of the sycamore primary cell wall is a xyloglucan. This polymer functions as an important cross-link in the structure of the cell wall; the xyloglucan is hydrogen-bonded to cellulose and covalently attached to the pectic polymers.     

Structure of Plant Cell Walls: X. RHAMNOGALACTURONAN I, A STRUCTURALLY COMPLEX PECTIC POLYSACCHARIDE IN THE WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS

The purification and characterization of a pectic polymer, rhamnogalacturonan I, present in the primary cell walls of dicots is described. Rhamnogalacturonan I accounts for approximately 7% of the mass of the walls isolated from suspension-cultured sycamore cells. As purified, rhamnogalacturonan I has a molecular weight of approximately 200,000 and is composed primarily of l-rhamnosyl, d-galacturonosyl, l-arabinosyl, and d-galactosyl residues. The backbone of rhamnogalacturonan I is thought to be composed predominantly of d-galacturonosyl and l-rhamnosyl residues in a ratio of approximately 2:1. About half of the l-rhamnosyl residues are 2-linked and are glycosidically attached to C(4) of a d-galacturonosyl residue. The other half of the l-rhamnosyl residues are 2,4-linked and have a d-galacturonosyl residue glycosidically attached at C(2). Sidechains averaging 6 residues in length are attached to C(4) of the l-rhamnosyl residues. There are many different sidechains, containing variously linked l-arabinosyl, and/or d-galactosyl residues.     

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 Structure of Plant Cell Walls: VI. A Survey of the Walls of Suspension-cultured Monocots

The primary cell walls of six suspension-cultured monocots and of a single suspension-cultured gymnosperm have been investigated with the following results: (a) the compositions of all six monocot cell walls are remarkably similar, despite the fact that the cell cultures were derived from diverse tissues; (b) the cell walls of suspension-cultured monocots differ substantially from those of suspension-cultured dicots and from the suspension-cultured gymnosperm; (c) an arabinoxylan is a major component (40% or more by weight) of monocot primary cell walls; (d) mixed β-1,3; β-1,4-glucans were found only in the cell wall preparations of rye grass endosperm cells, and not in the cell walls of any of the other five monocot cell cultures nor in the walls of suspension-cultured Douglas fir cells; (e) the monocot primary cell walls studied contain from 9 to 14% cellulose, 7 to 18% uronic acids, and 7 to 17% protein; (f) hydroxyproline accounts for less than 0.2% of the cell walls of monocots. Similar data on the soluble extracellular polysaccharides secreted by these cells are included.     

Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins

A bioassay to measure the incorporation of [¹⁴C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [¹⁴C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [¹⁴C]leucine uptake. The effectiveness of the wall fragments in inhibiting [¹⁴C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.     

Strategies To Discover the Structural Components of Cyst and Oocyst Walls

Cysts of Giardia lamblia and Entamoeba histolytica and oocysts of Toxoplasma gondii and Cryptosporidium parvum are the infectious and sometimes diagnostic forms of these parasites. To discover the structural components of cyst and oocyst walls, we have developed strategies based upon a few simple assumptions. Briefly, the most abundant wall proteins are identified by monoclonal antibodies or mass spectrometry. Structural components include a sugar polysaccharide (chitin for Entamoeba, β-1,3-linked glucose for Toxoplasma, and β-1,3-linked GalNAc for Giardia) and/or acid-fast lipids (Toxoplasma and Cryptosporidium). Because Entamoeba cysts and Toxoplasma oocysts are difficult to obtain, studies of walls of nonhuman pathogens (E. invadens and Eimeria, respectively) accelerate discovery. Biochemical methods to dissect fungal walls work well for cyst and oocyst walls, although the results are often unexpected. For example, echinocandins, which inhibit glucan synthases and kill fungi, arrest the development of oocyst walls and block their release into the intestinal lumen. Candida walls are coated with mannans, while Entamoeba cysts are coated in a dextran-like glucose polymer. Models for cyst and oocyst walls derive from their structural components and organization within the wall. Cyst walls are composed of chitin fibrils and lectins that bind chitin (Entamoeba) or fibrils of the β-1,3-GalNAc polymer and lectins that bind the polymer (Giardia). Oocyst walls of Toxoplasma have two distinct layers that resemble those of fungi (β-1,3-glucan in the inner layer) or mycobacteria (acid-fast lipids in the outer layer). Oocyst walls of Cryptosporidium have a rigid bilayer of acid-fast lipids and inner layer of oocyst wall proteins.     

Host-Pathogen Interactions : XXII. A Galacturonic Acid Oligosaccharide from Plant Cell Walls Elicits Phytoalexins

Elicitors of phytoalexin accumulation in soybean (Glycine max L. Merr., cv Wayne) cotyledons were released from soybean cell walls and from citrus pectin by partial acid hydrolysis. These two hydrolysates yielded nearly identical distributions of elicitor activity when fractionated on anion-exchange columns. Chromatography of the pectin elicitor on gel filtration and high-pressure anion-exchange columns did not further purify the elicitor. Elicitor activity of the preparation was lost by treatment with either endo-α-1,4-polygalacturonase or pectate lyase. Glycosyl residue compositions of the purified elicitors from cell walls and pectin were both found to be approximately 98% galacturonosyl residues. Linkage analysis of the pectin elicitor showed that most, if not all, of the galacturonosyl residues were α-1,4-linked. The high-mass molecular ions detected by fast atom bombardment-mass spectrometry of the most active elicitor fractions from cell walls and pectin both corresponded precisely to a molecule composed of 12 galacturonosyl residues. These results suggest that dodeca-α-1,4-d-galacturonide is the active elicitor, but the possibility remains that the active component could be a slightly modified oligogalacturonide present, but not detected, in the purified fractions.     

The Involvement of Glycosidases in the Cell Wall Metabolism of Suspension-cultured Acer pseudoplatanus Cells

Several glycosidases have been isolated from suspensioncultured sycamore (Acer pseudoplatanus) cells. These include an α-galactosidase, an α-mannosidase, a β-N-acetyl-glucosaminidase, a β-glucosidase, and two β-galactosidases. The pH optimum of each of these enzymes was determined. The pH optima, together with inhibition studies, suggest that each observed glycosidase activity represents a separate enzyme. Three of these enzymes, β-glucosidase, α-galactosidase, and one of the β-galactosidases, have been shown to be associated with the cell surface. The enzyme activities associated with the cell surface were shown to possess the ability to degrade to a limited extent isolated sycamore cell walls. It was found that the activities of β-glucosidase and of one of the β-galactosidases increase as the cells go through a period of growth and decrease as cell growth ceases.     

Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells

Plant cell walls serve several functions: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.     

Reversal of the Vancomycin Inhibition of Peptidoglycan Synthesis by Cell Walls

Addition of cell walls to the peptidoglycan synthetase-acceptor system containing vancomycin (50 μg/ml) prevented the inhibition by the antibiotic. In addition, the inhibition of incorporation of [¹⁴C]muramyl-pentapeptide into peptidoglycan in the presence of vancomycin was reversed by the addition of cell walls to the assay mixture at 60 min. Cell walls previously saturated with vancomycin lost their ability to reverse the inhibition by the antibiotic. The inhibition of peptidoglycan synthesis by ristocetin was partially reversed by the addition of cell walls. The initial stage in peptidoglycan synthesis is catalyzed by phospho-N-acetyl(NAc)muramyl-pentapeptide translocase (uridine 5′-phosphate) according to the reaction: UDP-NAc-muramyl-pentapeptide + acceptor acceptor-phospho-NAc-muramyl-pentapeptide + UMP where acceptor is C₅₅-isoprenoid alcohol phosphate. Vancomycin stimulates the transfer of phospho-NAc-muramyl-pentapeptide to the acceptor, and the addition of cell walls to this assay mixture prevented the stimulation of transfer. In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate (UMP) with UDP-NAc-muramyl-pentapeptide. The exchange reaction is effectively inhibited by vancomycin. For example, 60 μg of vancomycin per ml inhibited the rate of exchange by 50%. Addition of cell walls restored the exchange of UMP with the UMP moiety of UDP-NAc-muramyl-pentapeptide. Thus, cell walls appeared to have a higher affinity for vancomycin than did either the peptidoglycan synthetase-acceptor system or phospho-NAc-muramyl-pentapeptide translocase. These results provide support for the proposal made by Best and Durham that the effective binding of vancomycin to the cell wall could result in the inhibition of transfer of membrane-associated peptidoglycan chains to the growing wall.     

Treatment of rhamnogalacturonan I with lithium in ethylenediamine

Rhamnogalacturonan I is a pectic polysaccharide that is solubilized from the walls of suspension-cultured sycamore cells (Acer pseudoplatanus) by the action of a highly purified endo-1,4-α-polygalacturonanase. Rhamnogalacturonan I has a linear backbone consisting of the diglycosyl repeating unit, →4)-α-d-GalpA-(1→2)-α-l-Rhap-(1→. Approximately half of the α-l-rhamnosyl residues of the backbone are branched at O-4. Selective cleavage at the galactosyluronic acid residues of the backbone by treatment of rhamnogalacturonan I wit lithium in ethylenediamine resulted in the release of the neutral glycosyl-residue sidechains that had been attached to the backbone. Various analytical techniques, including combined liquid chromatography-mass spectrometry, combined gas-liquid chromatography-mass spectrometry, and ¹H-nuclear magnetic resonance spectroscopy, were used to determine the structure of the side chains. The majority of the sidechains were isolated as oligoglycosylalditols, with rhamnitol at the “reducing” end. Terminal 2-, 4-, or 6-linked galactosyl residues were found attached to O-4 of the rhamnitol residues The 2-, 4-, and 6-linked galactosyl residues had terminal or 2-linked arabinosyl, or additional galactosyl, residues attached to them. Based on the results of fast-atom-bombardment mass spectrometry, the side chains were found to range in size from one to fourteen glycosyl residues. The side-chain structures suggest that there are four or more distinct families of side chains attached to the backbone of rhamnogalacturonan I.     

Composition of cell walls from cotyledons of Pisum sativum,Vicia faba and Glycine max

Cell walls from cotyledons of smooth field pea, broad bean and soya bean contain ca 55% pectic polysaccharides associated with 9% cellulose. Arabinose is the major pectic sugar of pea and broad bean walls whereas soya bean pectic polymers are constituted of galactose and arabinose in the ratio (2:1). Galacturonic acid represents ca 20% of the walls. In addition, pea and broad bean cell walls contain, respectively, 12% and 6% of non-starchy and non-cellulosic glucans bearing 4,6-linked and 3-linked glycosyl units. EDTA-soluble acidic pectic substances are distinct rhamnogalacturonans bearing decreasing proportions of interrupting rhamnose from highly interrupted moieties to nearly homogenous homogalacturonans. Pea and broad bean rhamnogalacturonans are associated with arabinose-containing polymers of average DP ca 30–35 whereas soya bean ones have side chains of arabinose and galactose of DP ca 40.     

Changes in the Endosperm Cell Walls of Two Datura Species before Radicle Protrusion

The possibility of an association between changes in cell walls of the micropylar portion of the endosperm and the induction of germination was explored in seeds of Datura ferox and Datura stramonium. The structure of the inner surface of the endosperm was studied by scanning electron microscopy and the composition of cell wall polysaccharides analyzed by gas chromatography and gas chromatography-mass spectrometry. Both scanning electron microscope images and chemical analysis showed changes in the micropylar portion of the endosperm in induced seeds before radicle protrusion. The inner surface of the endosperm appeared eroded, and in some areas, wall material seemed to be missing. The content of the main component of the cell wall polysaccharides, containing predominantly 4-linked mannose, decreased well before the emergence of the radicle through the endosperm. We propose that the degradation of a mannan type polysaccharide is an important factor in the reduction in mechanical strength of the endosperm, thus facilitating germination.     

Structure of Plant Cell Walls: IX. Purification and Partial Characterization of a Wall-degrading Endo-Arabanase and an Arabinosidase from Bacillus subtilis

Wild type Bacillus subtilis, when grown on beet araban, secretes into its culture medium an endo-arabanase and two arabinosidases. An alternate procedure to one previously described (Kaji A, T Saheki 1975 Biochim Biophys Acta 410: 354-360) has been developed for the purification of the endo-arabanase. The purified endo-arabanase is shown to be homogeneous by sodium dodecyl sulfate-urea disc gel electrophoresis (molecular weight approximately 32,000) and by isoelectric focusing (pI = 9.3). The endo-arabanase, acting on a branched araban substrate, has maximal activity at pH 6.0 and preferentially cleaves 5-linked arabinosyl residues. One of the arabinosidases (molecular weight approximately 65,000, pI = 5.3) has been purified to the point that it contains only one quantitatively minor contaminant, as shown by sodium dodecyl sulfate-urea disc gel electrophoresis and isoelectric focusing. The purified arabinosidase, acting on p-nitrophenyl-alpha-l-arabinofuranoside, has maximal activity at pH 6.5, and, when acting on a branched araban substrate, preferentially attacks nonreducing terminal arabinosyl residues linked to the 2 or 3 position of other arabinosyl residues. Neither of the two purified enzymes is capable of hydrolyzing a variety of carbohydrate substrates which lack arabinosidic linkages. The purified endo-arabinase is shown to be capable of releasing arabinosyl oligomers from the walls of suspension-cultured sycamore cells, thereby suggesting its usefulness as a probe in studying the structure of the araban component of primary cell walls.