Hydroxyproline-rich glycoproteins (HRGPS), ubiquitous structural components (up to 10-20% dw) of growing cell walls, are implicated in all aspects of plant growth and development including responses to stress. Three major groups of HRGPs represent a continuum of glycosylation and polypeptide periodicity ranging from the lightly glycosylated repetitive proline-rich proteins (RPRPs), through the highly arabinosylated crosslinked extensins, to the arabinogalactan-proteins (AGPs) that are the most highly glycosylated and least periodic. Repetitive units are generally small 4-6 residue glycosylated motifs viewed hypothetically as functional modules. However, HRGP function remains largely unexplored at the molecular level. This research will define their roles through functional analysis of single glycopeptide modules expressed as repetitive glycopolypeptides in tobacco and tomato cultures. These include the (Ser-Hyp4)n, (Ser-Hyp3)n, (Ser-Hyp2)n, (Ser-Hyp)n, (Ala-Hyp)n, and related motifs of extensins and AGPs, as well as the 19-residue consensus sequence of another glycoprotein (GAGP). This involves construction of synthetic gene cassettes based on ligating sets of overlapping oligonucleotide complementary internal repeat pairs and complementary external 3'- and 5'-linker pairs to form long duplex DNA encoding a given number of module repeats. Insertion of these synthetic genes into a modified pBIl2l plant transformation vector containing an extensin signal sequence and the enhanced green fluorescent protein (EGFP) reporter gene will be used to transform cell cultures of tobacco and tomato that contain the plant prolyl hydroxylases and glycosyltransferases required for module posttranslational modifications. Expression and isolation of these modules will permit tests of the following general and specific hypotheses for glycosylation and glycomodule function: 1. Glycosylation codes predicted by the Hyp-contiguity Hypothesis specify precise saccharide addition to specific HRGP Hyp residues, namely: tetraarabinose to the most highly contiguous residues, arabinosylation decreasing with decreasing Hyp-contiguity; and arabinogalactan polysaccharide addition to repetitive noncontiguous Hyp residues that typify AGPs including GAGP. 2. Carbohydrate-mediated alignment of extensin monomers is necessary for their orderly assembly and crosslinking roles in cell wall self-assembly.
As analogs of highly periodic glycoproteins, "designer extensins" and "designer gums" will help relate site-specific glycosylation to essential molecular functions like crosslinking, self-association and cell wall self-assembly. These analogs also provide a unique approach to the design of novel genetically engineered gums and glycoprotein polymers of controlled structure and properties for enhancing plant disease resistance, and potential use in the agricultural, food and nanotechnology industries. Finally, this research will give students access to a wide range of tools and concepts in molecular biology, ranging from gene manipulation, microbiology and plant biology, to protein, glycoprotein and carbohydrate chemistry, and an excellent opportunity to learn how to integrate them to make significant scientific contributions.
