| Cell walls are major determinants of plant growth, morphology, development, and interactions with the environment. Components of the plant cell wall include polysaccharides such as cellulose, pectins, and hemicelluloses as well as structural proteins. Although much is known about the architecture of the plant cell wall, far less is known about the biosynthesis of its structural components. The focus of this project is the biosynthesis of a hemicellulosic polysaccharide component of the plant primary cell wall, xyloglucan. Xyloglucan is comprised of a beta-(1,4)-glucan backbone decorated with side chains consisting of xylose, xylose and galactose, or xylose, galactose and fucose. An enzyme that adds the terminal fucose to galactose on xyloglucan in an alpha(1,2)-linkage, xyloglucan fucosyltransferase (XyG FUTase), was purified from rapidly elongating segments of etiolated pea epicotyls. Amino acid sequence data from this purified enzyme allowed the identification of an expressed sequence tagged cDNA (EST) encoding an Arabidopsis XyG FUTase. A full-length cDNA was obtained for this Arabidopsis gene and named Arabidopsis Fucosyltransferase 1 (AtFUT1). The biochemical function of AtFUT1 was confirmed by (1) using antibodies against the protein encoded by this cDNA to immunoprecipitate XyG FUTase activity from Arabidopsis microsomal proteins, and (2) expressing AtFUT1 in mammalian Cos-7 cells, thereby conferring XyG FUTase activity.; The regulation of XyG fucosylation was studied in Arabidopsis as a model cell wall biosynthetic process. The expression of AtFUT1, the distribution of XyG FUTase activity, and the fucosylation state of XyG was studied in seven Arabidopsis tissues. Gene expression and enzyme activity levels were highest in the uppermost regions of the expanding Arabidopsis inflorescence stem and lowest in fully expanded tissue such as mature rosette leaves. AtFUT1::GUS transgenic plants were used to determine the spatial regulation of AtFUT1 expression. Results generally paralleled gene expression data obtained by other techniques. A gradient of reporter gene expression was observed in the elongating inflorescence stem.; 35S::AtFUT1 transgenic plants were generated to study the effect of strong constitutive AtFUT1 expression on XyG structure. These plants show higher expression of AtFUT1 and XyG FUTase activity compared to wild-type controls. Altered XyG structure is also observed in the transgenic plants in comparison to wild-type controls; however, these alterations consist not of highly elevated levels of fucose content, but of approximately two-fold higher acetylation of galactose.; AtFUT1 is one of a small number of genes encoding a cell wall biosynthetic enzyme with confirmed biochemical function. The sequence information from this gene and enzyme, as well as gene expression data, should assist in the identification and evaluation of other plant cell wall biosynthetic genes. |
