A Developmentally Regulated Hydroxyproline-Rich Glycoprotein in Maize Pericarp Cell Walls1

We have studied the accumulation of peptidyl hydroxyproline in the pericarp of developing maize (Zea mays L., Golden cross Bantam sweet corn) kernels. Although this hydroxyproline accumulates throughout development, it is most soluble and its content per milligram dry weight greatest at midmaturation stages of development. Salt-soluble proteins containing this hydroxyproline from isolated cell wails of developing kernels were fractionated on a CsCI density gradient and on a Chromatofocusing column, resulting in the purification of an hydroxyproline-rich glycoprotein, PC-1. PC-1 is a basic protein of approximately 65 to 70 kilodaltons in molecular weight with an isoelectric point of at least 10.2 and a density of 1.38 to 1.39 in CsCl. Amino acid composition data indicate that it is rich in hydroxyproline, threonine, proline, lysine, and glycine. Its relation to dicot extensin is discussed. Plant form is determined largely by the rigid walls surrounding the cells. In addition, cell walls are important in disease resistance and in protection against physical damage. In the deliberate design of plants with increased resistance to disease and physical damage, we need to know more about how the primary cell wall works. Primary walls constitute a large fraction of edible plants and are therefore the major determinant of the character of dietary fiber. A correct understanding of the nutritional role of fiber, again, requires a more complete understanding of the primary wall. Because of the evolutionary distance of monocots and dicots, differences in cell wall structure between them are likely. As might be expected, the glycoprotein abundance in primary walls differs between dicots and monocots (especially as represented by thegraminaceous cereals), but these differences are not well studied and understood (11). Analysis of cell wall proteins in dicots has suggested possible roles of structural proteins and wallassociated enzymes in growth, development, and defense (16, 20). Similar types of studies with monocots will be equally informative. A structural protein component, extensin, has been characterized in primary walls of several dicot species (16, 20), and molecular biological studies of its expression have begun (20, 23). Extensin is an important component of the cell wall matrix, comprising in some species as much as 18% of the wall's mass (15). A salt-extractable monomer extensin has been isolated from aerated carrot disks (21). This has allowed the characterization of the protein and carbohydrate moieties of the monomer (21, 26) as well as a determination of the time course for its insolubilization in the wall (8). In addition to structural roles, extensins also appear to be involved in the plant's defense against pathogen I Supported by United States Department of Energy grant No. DEFG02-84ER13255 to J. E. V. attack and wounding (6, 18, 19). But just how these rod-like extensin molecules fit into the polysaccharide matrix, become insolubilized, or are involved in defense remains to be elucidated (7). To understand better the role of these interesting cell wall glycoproteins, a study of their similarities and differences and of their developmental regulation in divergent plants is necessary. We used pericarp tissue for this study because it is an important protective tissue and because seed coat/pericarp tissue in many species had been reported by Van Etten et al. (25) to be relatively rich in hydroxyproline. In addition, Boundy et al. (1) extracted peptides from mature dent corn pericarp that were rich in hydroxyproline, although the proteins from which the peptides are derived were not identified. We have isolated and characterized an hydroxyproline-rich glycoprotein from maize, a monocot that is widely diverged taxonomically from those plants in which cell wall proteins have been characterized to date. This protein may be developmentally regulated and has an amino acid composition quite different from that of the dicot extensins. MATERIALS AND METHODS Plant Material. Golden cross Bantam sweet corn (Zea mays L.) was greenhouse grown and the ears harvested at several times after self pollination. For some experiments, sweet corn at a midmaturation stage of development was obtained from a local supermarket. Cell Wall Preparation. Pericarps (approximately 25 per batch) were peeled from frozen maize seed and the aleurone layer removed. The pericarps were ground in a glass homogenizer with buffer containing 0.1% K-acetate (pH 5.0) and 4 mM Na,S,O,. Walls were washed with 0.5% Nonidet P-40 (Sigma) containing 2 mm Na2S2O and were resuspended and centrifuged six times with 5 ml each of cold 2 mM Na2S2O5. The wall pellet was extracted with 2 ml 0.2 M CaCl2 overnight with stirring at 4°C. Hydroxyproline-Rich Glycoprotein Isolation. For some experiments, solid CsCl was added to the CaC12 extract to final density of 1.4 g cc1. Gradients were established by spinning at 250,000g for 72 h in a Beckman SW-65 Ti rotor. Gradients were fractionated, and hydroxyproline assays and density determinations were performed on the fractions. In other experiments, the CaCl, extract was desalted and concentrated with a Centricon-30 apparatus (Amicon Division, W.R. Grace and Co., Danvers, MA). Proteins were then subjected to fractionation on a Chromatofocusing column (Pharmacia; Uppsala, Sweden). The column was packed with polybuffer exchanger PBE 118 and equilibrated with 0.025 M triethylamine-HCl (pH 11) (TEA). The protein sample was lyophilized and brought to 1 ml in the TEA buffer before being applied to the column. The pH profile was developed with pH 8.0 Pharmalyte pH 8 to 10.5-HCl (Pharmacia). The pH gradient typically ran from 10.5 to 8.0 in 50 to 65 ml elution volume from a 12 x 0.5 cm column. Protein-containing fractions were identified by absorbance at 280 nm. 138 www.plantphysiol.org on October 15, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. MAIZE PERICARP CELL WALL HYDROXYPROLINE-RICH GLYCOPROTEIN Gel Electrophoresis. SDS-PAGE was by the method of Laemmli (14). Mini-gels of 0.75 mm thickness and 8 cm x 10 cm in size were used. Cationic neutral polyacrylamide gel electrophoresis (22) for separation of basic proteins was performed with gels of the same dimensions. Gels were silver stained using the method of Neilsen and Brown (17). Chemical Assays. Tissue or protein samples were hydrolyzed in 6 N HCI for 24 h at 1 10°C. Colorimetric hydroxyproline determinations were done by the method of Drozdz et al. (9) on these hydrolyzed samples. Total protein was determined by the microassay method developed by Bradford (2) with commercial reagent from Bio-Rad (Richmond, CA). Amino acid analyses were performed on samples hydrolyzed for 24 h at 110°C in constant boiling HCl (Pierce) in glass ampules flushed with N2 gas. Samples were prepared for HPLC analysis by passing each over a C,8 SEP-PAK cartridge (Waters Associates, Milford, MA).