ARABINOGALACTAN FROM ZEA SHOOTS

The stucture of the arbinogalactan obtained from the buffer-homogenate of Zca mays L. (hybrid B73 x Mol7) shoots has been studied. The purified polysaccharide was investigated by methylation analysis before and after controlled acid hydrolysis. Aabinogactan-l consists of nrabinose, plactose, xylose, uronic acid, and glucose in the molar rtio of 37.1:55.8. 3.0A. l:trace, and aabinolactan-2 consists of the same sugars in the ratio of 35A:53.9:1.69. 2:trace. A trace of protein was detected in aabinoglactan-1 and about 0.2% was present in 2. About 20% of the galactose residues in arbinogaactan-l constitute a (1 -_ 3)-linked galactan chain and approximately 60% constitute a (1 _.* 6)-Hinked gaactan sequence. About 15% of the glactose residues in aabinoglactan-l are substituted by galactose in the 3and 6-positions, thereby constituting branch points of the galactan framework. The remainder (5%) of the galactose residues in arabilctnl are located at nonreduciDng terminal positions. About 85% of the (1 -_ 6)-gctosyl sequence is substituted, mostly by single arabinose residues. Nonreducig terminal glucuronic acid is attached to C-6 of glactose residues. The basic structure of arabinoglactan-2 is similar to that of arabin gaactan-1. The monocotyledonous coleoptile has been widely used as an experimental material in investigation of cell elongation mediated by a plant hormone, IAA. A number ofstudies have focused on the monsaccharide composition of various kinds ofcoleoptile cell walls and on the changes in their composition during growth (2, 6, 14, 16, 18, 19, 22-24, 26, 30, 33). Although information is available regarding structures ofsome components of polysaccharides in the growing monocot cell walls (7, 10-13, 15, 20, 21, 28, 31, 32), the complete structure of cell wall polysaccharides of any growing monocot has not been elucidated. To obtain detailed structural features of cell walls in growing monocots, we have examined Zea shoots which were excised at the coleoptile node 96 h after imbibition. Structural analyses were conducted on soluble polysaccharides obtained in the course of preparation of cell walls and on certain components released from isolated Zea shoot cell walls upon treatment with enzymes. This paper is the first in a series dealing with the polysaccharides ofZea shoots and describes the structure of arabino-3,6-galactan. MATERIALS AND METHODS Plant Material. Imbibed corn caryopses (Zea mays L., hybrid B73 x Mol7; Brayton Seed Co., Ames, IA) were soaked in running water for 2 h, sown on moist vermiculite, and maintained at 26°C under darkness. After 96 h, entire seedling shoots above the coleoptile node were excised and stored at -20°C. Preparation of Zea Shoot Cell Walls and Water-Soluble Pol'Supported in part by National Science Foundation Research Grant PCM 7818588. ysaccharide Fractions. Approximately 850 g of seedling shoots were homogenized in 2 L of ice-cold 0.3% NaCl in 10 mm Naphosphate buffer (pH 6.5) for 5 min using a Waring Blendor. The homogenate was filtered through premoistened Miracloth. The insoluble material was washed twice with 2 L of ice-cold 0.3% NaCl in 10 mm Na-phosphate buffer (pH 6.5). The combined filtrate and washings was made 5% with respect to TCA and after 10 h at 0°C was centrifuged for 30 min. The supernatant was dialyzed against distilled H20, concentrated to an appropriate volume, and freeze-dried to give WSP-I.2 The cell wall material recovered from the homogenate was suspended in 200 ml of 3 M LiCl in 50 mM Mcllvaine buffer (pH 6.8) and maintained for 48 h at 4°C. Then the suspension was filtered through Miracloth and washed with a small volume of the same buffer. The combined filtrate and washing were heated in a boiling water bath for 10 min, dialyzed against distilled H20, and finally freeze-dried to give WSP-II. The LiCl-treated cell wall material suspended in water was heated in a boiling water bath for 15 min and filtered through Miracloth. Three volumes ofmethanol were added to the filtrate. Precipitated material was recovered by centrifugation, washed successively with methanol and acetone, and dried in vacuo to give WSP-III. The cell wall material obtained by this procedure was freezedried to give Zea shoot cell walls. Resolution ofWSP-I by Chromatography on DEAE4Sephadex A-25. WSP-I (400 mg) was dissolved in 10 ml of 20 mM Naphosphate buffer (pH 6.0) and centrifuged. The supernatant was applied to a column (2.1 x 30 cm) of DEAE -Sephadex A-25 (Pharmacia Fine Chemicals) pre-equilibrated with the same buffer, and then the column was washed with the buffer. The adsorbed materials were eluted with a salt gradient (0-0.8 M in Na-phosphate buffer (followed by elution with 0.5 N NaOH. Seventeen-ml fractions were collected. Each tube was assayed for carbohydrate and protein. Tubes 3 to 5, 18 to 20, 21 to 26, and 27 to 40 were separately combined, dialyzed against distilled H20, and freeze-dried. Tubes 3 to 5 and 21 to 26 were designated WSP-I-1 and WSP-I-2, respectively. Purification of WSP-I-1 and WSP-I-2 by Chromatography on Bio-Gel A-1.5m. WSP-I-1 (30 mg) was dissolved in 2 ml of 20 mm Na-phosphate buffer (pH 6.0) and centrifuged. The supernatant was applied to a column (2.2 x 52 cm) of Bio-Gel A1.5m (100-200 mesh, Bio-Rad) pre-equilibrated with 20 mm Naphosphate buffer (pH 6.0) followed by filtration with the same buffer, 0.9-ml fractions were collected. Each tube was assayed for carbohydrate and protein. Tubes 79 to 91 were combined, dialyzed against distilled H20, and freeze-dried to give purified WSP-I-1 (AG-1). WSP-I-2 (20 mg) was chromatographed under the same conditions. Tubes 82 to 95 were combined, dialyzed against distilled H20, and freeze-dried to give purified WSP-I-2 (AG-2). Partial Acid Hydrolysis of AG-1. AG-I (11 mg) was hydro2Abbreviations: WSP-I, -II, and -III, water-soluble polysaccharide fractions, I, II, and III; AG, arabinogalactan.