Molecular and Cellular Basis of Oriented Cellulose Microfibril Deposition in Boergesenia

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The giant algal Cell, Boergesenia forbesii, will be used to investigate the molecular and cellular basis of oriented cellulose microfibril deposition. Advantages of this model system are: (a) large scale cultivation of axenic cultures; (b) the cell responds to wounding to synchronously form numerous protoplasts; (c) protein synthesis is initiated, allowing segregation of developmental stages for biochemical analysis; (d) highly crystalline cellulose microfibril assembly is initiated by linear aggregation of ordered plasma membrane particles known as terminal complexes (TCs); and (e) TC size is sufficiently large for microscopic detection. Plasma membranes of young protoplasts and thin walled aplanospores will be purified by advanced biochemical techniques (including free flow electrophoresis). Differences in protein patterns will be correlated with onset of cellulose biosynthesis. Monoclonal antibodies will be produced against purified plasma membrane proteins. Due to large TC size, binding antibodies can be screened using fluorescence microscopy. Protein will be identified by western blotting and purified by immunoaffinity columns. Antibodies will be used to study TC function in vitro and in vivo. The antibody approach leading to identification of proteins in the TC will allow comparative analysis of TC proteins from other plants, prokaryotes, and possibly some animals which synthesize cellulose. The question of how cellulosic wall is laid down is one of the most fundamental, and least well understood, problems in plant biology. The question of wall growth is key to problems of plant cell growth and division, as well as whole-plant development. The work to be carried out under this award may also result in advances in our understanding of cellulose synthesis which may be of commercial significance.