Structure of Plant Cell Wails

This paper describes the isolation and characterization of rhamnogalacturonan II, a hitherto unobserved component of the primary ceo wails of dicotyledonous plants. Rhamnogalacturonan II constitutes 3 to 4% of the primary ceo wais of suspension-cultured sycamore (Acerpseudoplatanus) cels. Rhamnogalacturonan II is a very complex polysaccharide yielding, upon hydrolysis, 10 different monosaccharides including the rarely observed sugars apiose, 2-0-methylxylose, and 20-methylfucose. In adtion, rhamnogalacturonan II is characterized by the rarely observed glycosyl interconnections of 2-linked glucuronosyl, 3,4-inked fucosyl, and 3-lunked rhamnosyl residues. These glycosyl lunkages have never previously been detected in primary sycamore ceol walls. Evidence is presented which suggests that polysaccharldes similar to rhamnogalacturonan II are present in the primary celH wails of the three other dicotyledonous plants examined. A structural model of the primary cell wall of suspensioncultured sycamore (Acer pseudoplatanus) cells has been proposed previously (7, 20, 27). A continuing study of the pectic polymers of the primary walls of sycamore cells has now revealed the presence in these walls of a complex polysaccharide which had not been detected previously. This paper describes the extraction and purification of this polysaccharide and the unusually linked glycosyl residues of which it is comprised. MATERIALS AND METHODS Isolation of Cell Walls. The walls were prepared from suspension-cultured sycamore (Acer pseudoplatanus) cells as previously described (27). Enzyme Purification. Endo-a-1,4-polygalacturonase from Colletotrichum lindemuthianum was purified as previously described ( 14). The purified enzyme was shown to be free off?1,4-galactanase, 81,4-glucanase, and arabanase by the inability of a large excess of the endopolygalacturonase to degrade a f3-l,4-galactan (22), CMC-cellulose (Hercules Inc.), and beet araban (18). Enzymic Extraction of Pectic Polymers. A large proportion of the sycamore cell wall pectic polysaccharides were extracted by subjecting the walls three successive times to the action of the C. lindemuthianum endopolygalacturonase. This procedure was carried out by first washing 3.6 g of the walls three times with 200 ml of 10 mm Na-acetate (pH 5.2). The washed walls were suspended in 200 ml of this buffer and 8,400 units of the purified endopolygalacturonase was added. The wall-enzyme suspension was incubated at 30 C for 3 hr, after which the walls were removed from the solubilized material by centrifugation at l0,OOOg for 15 min. ' Research was supported by Department of Energy (formerly ERDA) Grant EY-76-S-02-1426. 2 To whom correspondence should be addressed. This extraction procedure was repeated with another 8,400 units of the endopolygalacturonase and, finally, with 4,200 units of the enzyme. The solubilized material from the three enzymic extracts was combined (total volume 600 ml) and filtered through GF/C glass filter paper. This solution was dialyzed exhaustively against distilled H20 and then lyophilized to yield 12% of the mass of the original cell walls. Small oligosaccharides, consisting mainly of mono-, di-, and trigalacturonic acid (27), were lost during dialysis. This was the only significant amount of carbohydrate lost during the fractionation and purification procedures. Gel Filtration. Gel filtration was accomplished on an agarose 5m column (3 x 50 cm) and then on a Bio-Gel P-10 column (2 x 23 cm). Both columns were equilibrated and eluted with 50 mm Na-acetate (pH 5.2). The void and included volumes of the columns were determined with blue dextran (Sigma) and NaCl, respectively. Colorimetric Techniques. Neutral sugar concentrations were determined by the anthrone method of Dische (12), and uronic acid concentrations were determined by the m-hydroxydiphenyl method of Blumenkrantz and Asboe-Hansen (10). Analysis of Glycosyl and Glycosyl Linkage Compositions. Glycosyl compositions were determined by the alditol acetate method of Albersheim et al. (1). Glycosyl linkage compositions were determined by combined gas chromatographic-mass spectrometric analysis of the partially methylated alditol acetate derivatives (9, 24). Polysaccharide methylations were performed using a modification (27) of the procedure reported by Hakomori (15). Kdimethyl sulphinyl anion was used instead of Na-dimethyl sulphinyl anion because, in our hands, the K anion works more rapidly and results in a more complete methylation. The K anion is made as reported for the Na anion (15) except for the use of K hydride instead of Na hydride and the omission of heating during anion preparation. The hydrolysis of the methylated polysaccharide was accomplished by heating at 100 C for 5 hr in 88% formic acid followed by heating at 121 C for I hr with 2 M trifluoroacetic acid. Partially methylated aldoses were reduced with Na-borodeuteride rather than Na-borohydride to aid in mass spectral analysis. The uronosyl derivatives of RG-II:3 were analyzed by reduction to the corresponding 6,6-di-deutero hexoses; the reduction was accomplished by the method of Taylor and Conrad (28) using Naborodeuteride and deuterium oxide instead of Na-borohydride and water. The samples were then analyzed for glycosyl residues and glycosyl linkages as described above. The quantities of unlabeled hexose and of dideutero-labeled hexose, resulting from the reduction of the corresponding uronic acid, were determined by quantitation of the appropriate fragment ions obtained by mass spectrometric analysis of the alditol acetate derivative. Methylation with trideuteromethyliodide was used to determine the glycosyl linkages of the endogenously methylated glycosyl residues of RG-II. | Abbreviations: RG-I1: rhamnogalacturonan 11; GC-MS: gas chromatography-mass spectrometry. 418 www.plantphysiol.org on December 30, 2017 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. STRUCTURE OF PLANT CELL WALLS. VIII GD-MS. GC analysis of the alditol acetates was performed on column A, which contained 0.2% ethylene glycol succinate, 0.2% ethylene glycol adipate, and 0.4% XF1150 on Gas-chrom P (1). GC analysis of the partially methylated alditol acetates was performed on column A, on column B, which contained 0.3% OV275 and 0.4% XF1150 on Gas-chrom Q (I 1), and on column C, a 25-m open tubular glass capillary column containing SE 30 (LKB, Broma, Sweden). All GC-MS analyses were carried out on a Hewlett-Packard GC-MS system (model 5980A) coupled to a Hewlett-Packard (model 5933A) data system. The GC flame-ionization detector peaks were quantitated by a computer-assisted electronic technique (H. Albert and P. Albersheim, unpublished results). All of the GC flame-ionization responses to partially methylated alditol acetates were corrected to mol responses as described by Sweet et al. (26).