Differential Extraction and Protein Sequencing Reveals Major Differences in Patterns of Primary Cell Wall Proteins from Plants

Abstract

The proteins of the primary cell walls of suspension cultured cells of five plant species, Arabidopsis, carrot, French bean, tomato, and tobacco, have been compared. The approach that has been adopted is differential extraction followed by SDS-polyacrylamide gel electrophoresis (PAGE), rather than two-dimensional gel analysis, to facilitate protein sequencing. Whole cells were washed sequentially with the following aqueous solutions, CaCl 2 , CDTA (cyclohexane diaminotetraacetic acid, DTT (dithiothreitol), NaCl, and borate. SDS-PAGE analysis showed consistent differences between species. From the 233 proteins that were selected for sequencing, 63% gave N-terminal data. This analysis shows that (i) patterns of proteins revealed by SDS-PAGE are strikingly different for all five species, (ii) a large number of these proteins cannot be identified by data base searches indicating that a significant proportion of wall proteins have not been previously described, (iii) the major proteins that can be identified belong to very different classes of proteins, (iv) the majority of proteins found in the extracellular growth media are absent from their respective cell wall extracts, and (v) the results of the extraction process are indicative of higher order structure. It appears that aspects of speciation reside in the complement of extracellular wall proteins. The data represent a protein resource for cell wall studies complementary to EST (expressed sequence tag) and DNA sequencing strategies.The plant cell wall is a dynamic system generally considered to be composed of more than 90% carbohydrate polymers. Proteins, phenolics and possibly lipids make up the remainder of the wall (1-3). To date, most research interest has been in the carbohydrate components because of considerations of their structural role and commercial interest. This has led to a number of models for the integration, interpolymeric association, and assembly of the wall (3, 4). By comparison, our knowledge of the complexity of protein in the plant cell wall is in a less advanced state. Much of the understanding of the range of structural wall proteins has come from cDNA and genomic cloning exercises and has led to the identification of glycine-, cysteine-, proline-, and hydroxyproline-rich subsets of wall proteins. In addition, many extracellular enzymes have been identified that are required for the restructuring and modification of this dynamic extracellular matrix which underpin its role in defense, detoxification, signaling, cell-cell recognition, cell expansion, cell adhesion, cell separation, translocation, differentiation, and morphogenesis (2, 5, 6). However, there is a lack of direct studies on the proteins themselves and the true range of extracellular proteins and their species differences remains to be elucidated. The present work describes the systematic extraction and sequencing of the major primary wall proteins from five species representing four families of plants.Since whole plant tissue is complicated by the presence of different tissue...

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