9805960 Kieliszewski Hydroxyproline-rich glycoproteins (HRGPs) are ubiquitous architectural components of growing plant cell walls, often accounting for 10-20% of their dry weight; polysaccharides account for the bulk. HRGPs are implicated in all aspects of plant growth and development including responses to stress. The HRGP superfamily contains three major groups representing a continuum of glycosylation and periodicity. They range from the repetitive proline-rich proteins that are lightly glycosylated and highly periodic, through the crosslinked extensins that are highly arabinosylated and periodic, to the arabinogalactan-proteins that are the most highly glycosylated and least periodic. The repetitive units are small, often only 4-6 residue glycosylated motifs viewed hypothetically as functional modules. The function of these unique plant glycoproteins remains largely unexplored. We propose to define their roles through functional analysis of single glycopeptide modules when expresssed as repetitive glycopolypeptides in tomato cultures. We shall insert synthetic genes encoding HRGP glycopeptide modules into two plant transformation vectors both derived from pBI121; one with a reporter gene, the other, for test for module function, lacking the reporter. We shall use these plasmids to transform cell cultures of tomato, which contain the plant prolylhydroxylases and glycosyltransferases required for module posttranslational modifications. Expression and isolation of these modules will enable us to test general and specific hypotheses for glycosylation and molecular 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 polygalactosylation of repetitive noncontiguous Hyp residues that typify AGPs. 2. Carbohydrate-mediated alignment of extensin monomers is necessary for their orderly assembly and crosslinking roles in cell wall self-assembly.
