Protein Chemistry & Function

NOTE: Link to Bioinformatics page.

Major Research Themes

• Determining the role of cell surface proteoglycans in plant growth and development.
• Functional genomics of cell surface proteoglycans
• Developing novel bioinformatics approaches for maximising information on plant genomes
• Understanding the post-translational modifications of AGP protein backbones
• Functional analysis of plant prolyl hydroxylases using a yeast expression system

Research Programmes

• Functional genomics of cell surface proteoglycans
  Our research program utilises the model plant, Arabidopsis thaliana, for determining the function(s) of arabinogalactan-proteins (AGPs) and other proteins involved in the assembly of the plant cell wall. AGPs are proteoglycans of the plant extracellular matrix and are implicated in a variety of roles in plant growth and development. The AGP gene family is large (more than 45 AGP genes have been identified). We are in the process of characterising these genes. We are attempting to stream line the analysis of publicly available genomics resources, e.g. DNA sequences (genomic and cDNA), microarrays and DNA insertion mutants to help us select specific subsets of genes for targeted experimental approaches. We are focusing our research efforts on candidate genes that are specifically implicated in plant development and in biotic and abiotic stress responses.

  Contact Researchers: Dr Carolyn Schultz, Bacic

  Funding: ARC large grant (2000-2002, Bacic and Schultz)

• Developing novel bioinformatics tools for maximising information on plant genomes
  As part of our work on the function of arabinogalactan-proteins (AGPs) in plant development we have developed two new bioinformatics tools. The protein backbones of AGPs are rich in Pro, Ala, Ser and Thr, but they generally have very low sequence similarity so they are difficult to identify.

  The first tool we developed enabled us to identify all of the AGP protein backbones from the Arabidopsis proteome. The proteome is all of the proteins predicted from the Arabidopsis genome. This program can be used to identify any proteins that have an unusual amino acid bias. This tool also enabled us to identify all of the AG-peptides, a subclass of AGPs that are very small.

  The second tool we developed allows us to reformat the Arabidopsis Functional Genomics Consortium (AFGC) microarray data. The microarray chip used by AFGC has approximately 8000 unique genes on it and contains 35 of the 47 AGP genes. Our customised software makes it possible to view the ratio data for all AFGC experiments and as many genes as desired in a single spreadsheet. The results for reciprocal experiments are grouped to simplify analysis and candidate AGPs involved in development or biotic and abiotic stress responses were readily identified.

  We are regularly monitoring publicly available genomic resources and developing more efficient ways of accessing and integrating emerging genomic information into our research program.

• Functional analysis of plant prolyl hydroxylases using a yeast expression system
  Molecules containing hydroxyproline are found in the cell wall of all plants. Many are differentially regulated during plant growth and in response to stress. There are nine candidate gene in Arabidopsis for the catalytic subunit of prolyl hydroxylase. A yeast expression system will be used to confirm the predicted function of these genes. Once established, this system will provide an efficient approach for determining the specificity of individual prolyl hydroxylases for candidate target proteins including extensins, arabinogalactan-proteins and chitinases. This knowledge will provide valuable information for choosing appropriate genes for over- and under-expression studies for the purpose of crop improvement.

• Understanding the post-translational modifications of AGP protein backbones
  AGPs undergo extensive post-translational modifications and it is not possible to accurately predict the modifications based on gene sequences. One of the key modifications of AGPs is the hydroxylation of most, but not all, of the proline (Pro) residues. There are some general "rules" (i.e. Pro-Val is always hydroxylated, but Lys-Pro is not), however they do not cover all of the motifs found in AGP protein backbones. Another modification of AGPs is the addition of a lipid anchor (called a glycosylphosphatidylinositol (GPI)-anchor). This modification involves cleavage of a C-terminal portion of the protein backbone. The precise cleavage site has only been determined for a few AGPs.

  The advent of tools such as matrix assisted laser disorption-time of flight (MALDI-TOF) mass spectrometry make it possible to analyse large numbers of protein samples quickly and accurately. Therefore, by purifying and characterising AGP protein backbones we will be able to determine the modifications made in vivo. This will allow us to more accurately predict two different forms of post-translational modifications in plants.