Higher plants are virtual factories for the synthesis of polysaccharides. For the major reserve polysaccharide, starch, considerable information exists concerning the genes involved as well as the mechanism and regulation of synthesis. However, for the major polysaccharides involved in the synthesis of plant cell walls, very little similar information exists. However, for the most abundant of all plant polysaccharides, cellulose, a breakthrough recently has occurred in identification of a class of genes called CelA that encodes the catalytic subunit of the cellulose synthase. Preliminary analyses indicate that the CelA genes comprise a large gene family in plants, and, furthermore, there are now indications that other glycosyltransferases that synthesize related beta-glycans in plants may share sequence homology and that an even larger "superfamily" of CelA-like genes exists in plants.
The overall objective is to carry out an extensive analysis of the large CelA superfamily of genes in plants with initial concentration on the specific class of CelAs, that encode the catalytic subunit of the cellulose synthase. Efforts will be concentrated on the important crop plants corn and cotton, but comparisons will also be made with genes from the model plant Arabidopsis. Specific questions to be answered include: How many distinct CelA genes can be identified? What is the chromosomal location of the genes, are there indications of gene clustering, and do these genes show similarities in genomic organization in different plants? What are the intron/exon relationships among the genes, and can this information provide clues as to how the genes function, how they evolved, and their relationship to other glycosyltransferases? What is the pattern of expression and function of each of the members of the gene family and what is the consequence to the plant of mutating individual members of the gene family? These latter studies will use three approaches: i) characterization of corn and Arabidopsis mutants which have a specific CelA gene disrupted by insertion of a foreign piece of DNA; ii) In situ hybridization to localize mRNA and immunocytochemistry to localize CelA gene products in wild-type and mutant plants as well chemical, physical and cytological analyses of mutant phenotypes; and iii) development of a system for domain swapping in which the portion of the sequence encoding the catalytic domain of each identified gene is used to replace the catalytic domain of a known glycosyltransferase-a hyaluronan synthase of mammals, and to test for appearance of a new enzyme activity in a transient expression system. This last method is proposed as a way to confirm the function of each of the encoded proteins under study.
By such studies, the goal is to understand why plants have so many CelA genes, their function, how they may have evolved, and what the consequences to the plant would be when each is disrupted. Such information is critical for future design of strategies for the engineering of plants that would have altered cellulose properties and/or altered timing or extent of deposition of cellulose or related polysaccharides. The development of the novel domain swapping technique should prove extremely valuable for the entire scientific community to use for assessment of function of individual domains within such enzymes and other important membrane proteins and also for identification of other genes encoding related glycosyltransferases in plants.
This is a multi-institutional proposal involving collaborations with scientists from Academia, industry, and a USDA laboratory. The project will also fulfill an important training role for an undergraduate and graduate student and a post-doctoral scholar who will also participate in the research.
