Dissecting the Molecular Mechanism of Pectin Synthesis in Arabidopsis

University Of Georgia Research Foundation Inc, Athens GA

Investigators

Abstract

Pectin is a structurally complex family of plant cell wall polysaccharides that have multiple functions in plant growth, development and plant defense against pathogens. Modifications of pectin structure due to mutations in genes encoding pectin biosynthetic or pectinolytic enzymes result in altered cell-cell adhesion, dwarfism, brittle leaves and reduced numbers of shoots and flowers, indicating multiple roles for pectin in plant growth and development. Pectin biosynthesis is predicted to require ~50 biosynthetic enzymes of the glycosyltransferase type, including four or more galacturonosyltransferases (GalATs). Galacturonic acid comprises ~70% of pectin and is present in the three pectic polysaccharides: homogalacturonan (HG), rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II). In previous studies, the researchers have identified a superfamily of 25 Arabidopsis thaliana genes encoding demonstrated and putative GalATs: 15 GAUT (GAlactUronosylTransferase) and 10 GATL (GAUT-Like) genes. Sequence alignment of the 25 GAUT1-related superfamily genes and phylogenetic analyses identified four clades, GAUT A, B, C and GATL. Cell wall analyses of homozygous Arabidopsis T-DNA insertion mutants of 13 GAUT genes show that most of the GAUT mutants have modified levels of galacturonic acid in their walls and glycosyl residue changes consistent with defects in HG, RG-I and/or RG-II synthesis. The specific function of the mutated GAUTs, however, is not known. The goal of the research is to determine the molecular function of three GAUT1-related family genes for which mutant plants show novel pectin-related phenotypes. The SPECIFC AIMS are to: (1) Biochemically characterize GAUT6, GAUT14 and GATL8 proteins. Polyclonal antibodies will be raised against the heterologously expressed proteins. The possibility that specific GAUT/GATLs exist in complexes will be tested using SDS-PAGE and immunoprecipitation in combination with Western blotting and LC-MS/MS to identify protein subunits. The enzymatic activities of complexes and individual subunits will be tested. (2) Analyze homozygous mutants to elucidate GAUT/GATL biological function. Mutation-associated specific changes in plant growth and development will be analyzed and wall structure will be determined by a combination of glycosyl residue and linkage analyses and by immunolabeling tissue sections using antibodies against specific wall carbohydrate epitopes. (3) Determine cell-type specific expression of each GAUT/GATL. EST, microarray, and PCR transcript analyses, together with GUS promoter fusion studies will be done to determine tissue and cellular location of the encoded proteins. If time allows, immunogold labeling using antibodies generated against recombinant or purified GAUTs/GATLs, or GFP-fusion protein expression will be carried out to define the subcellular location of the proteins within the cell and the Golgi complex. Broader Impacts. The outcome of this project is likely to facilitate the engineering of plants to produce pectins with modified structures and properties, and with improved agricultural, industrial and biomedical value. The project will involve training of future scientists by recruiting undergraduate and graduate students through the University of Georgia Summer Undergraduate Research Program (SURP) and SURP-Bridge program and by participating in undergraduate science national conferences to identify meritorious historically underrepresented graduate student candidates. The research experience of students participating in this project will provide them with a broad training in diverse genetic, biochemical, and cell biological techniques.