Research Outline :
Functional Characterization of Wavelet Events in the Arabidopsis Genome
The structure and evolution of plant genomes can be studied in detail following the published availability of high-quality and comprehensive genome sequences for the model species Arabidopsis thaliana and, more recently, the model crop species Oryza sativa (Rice). Strategies have been developed for the numerical representation of genome sequence data in, which allows for the analysis of sequence patterning using numerical signal processing algorithms such as Wavelet Analysis. We have applied these strategies to Arabidopsis to identify Wavelet events that define perturbations in sequence pattern in the genome as a matter of evolutionary interest. We are also pursuing functional studies of Wavelet events via the identification of lines of Arabidopsis that are variant for select wavelet events – with special emphasis on events that lie outside known or predicted genes or other transcript units and their associated cis-regulatory regions.
Molecular Function of ASK Proteins in the Regulation of Plant Development
The model plant Arabidopsis thaliana is a powerful system for the genetic investigation of plant patterning and development. Recently, a number of plant growth and developmental response pathways including auxin signalling, pathogen response, florigenesis and maintenance of circadian rhythm have been found to be regulated by genes encoding components of the SCF complexes in this organism. SCF complexes, as E3 ubiquitin ligases, direct the post-translational ubiquitination and degradation of target proteins.
The Arabidopsis genome contains an unusually large number of genes that encode known or predicted subunits of SCF. Among these are an estimated 21 independent genes (ASK genes) that encode Skp1-like subunits in this species. We and others have provided evidence that ASK proteins in Arabidopsis may be functionally specialized, since certain of the subunits exhibit selective protein:protein interactions with other components of SCF complexes, as revealed by yeast 2-hybrid approaches.
We are characterizing loss-of-function alleles of members of the ASK gene family, both alone and in combination, for their affect on Arabidopsis morphology and development. In the long term, we seek to understand the functional contribution of individual members of this protein family, and to understand in molecular terms their role the unique adaptive strategy inherent to plants.
Development of Whole-Genome Mutational Resources in Arabidopsis
The power of reverse genetics in plant model systems like Arabidopsis has been fully realized with the advent of large insertion-mutagenesis populations such as the Salk/NRC, Syngenta and GABI T-DNA mutant collections. Together, these resources tag about 80% of known or predicted genes in Arabidopsis. Notwithstanding the number of independent insertions involved in the unified T-DNA insertion collection (about 150,000 lines), there is gathering evidence that a significant subset of these are less than fully genetically penetrant as evidenced by the absence of discernible phenotypes. In some cases, evidence of genetic redundancy has provided a ready explanation for the lack of phenotype for select insertions, where in other cases the evidence points to partial inactivation of gene expression or gene product function. Overall, about 30% of known and predicted genes likely exhibit partial inactivation phenotypes across the consolidated Arabidopsis collection.
We are developing a complimentary resource based on plant lines subjected to fast-neutron (FN) irradiation, where chromosomal deletions are the dominant lesion We anticipate the FN resource to present a complimentary source of genetic variation that will likely expand the availability of true null mutations in select genes.
Currently the lab has two main research themes:
1- Functional Analysis of SKP1 orthologs in Arabidopsis:
Protein degradation is an important post-transcriptional gene regulatory process that allows cells to respond rapidly to changing environmental conditions by adjusting the steady state abundance of key proteins that regulate environmental responses, patterning and development. One major process for targeted proteolysis in eukaryotes involves the Ubiquitin (Ub)/26S proteasome pathway, in which proteins destined for degradation become modified by the covalent attachment of multiple Ub molecules under the action of E3 Ligase complexes, followed by degradation via the 26s proteosome.
SCF ligase a well charctrized subclass of E3 Ligases exhibits a quaternary structure that includes Cullin1/Cdc53, Rbx1/Roc1/Hrt1, Skp1, and F-box protein subunits. Structural analysis of a human SCF complex has shown that the Skp1p component acts as an adapter that associates Cullin to the F-box protein in the functional complex.
The Arabidopsis genome contains 21 known or predicted Skp1-like (ASK) genes compared to only a single gene in both Baker's Yeast (S. cerevisiae) and Humans. The large number of ASK genes may reflect the unique adaptive strategy of plants versus animals, where plants are well-adapted to rapidly adjust their metabolic and developmental profiles in response to changing environmental conditions. All known or predicted products of the ASK gene family are believed to be part of SCF complexes (E3 Ligases) but this has been directly demonstrated for only two of the genes in Arabidopsis - ASK1 and ASK2.
Functional studies of a subset of the predicted ASK family of genes are being carried out using initially a reverse genetic approach. Artificial miRNA (amiRNA) constructs will be used to simultaneously interfere with the expression of single or multiple genes via a Dicer mechanism targeting common transcript sequences, we are also in the process of developing novel approacehes to assess and characterize the regulatory pathways that are subject to hierarchical control via the 26S ubiquitinylation-mediated activity of select ASK genes.
2-Castor as a Next-Generation Crop for the Biorenewable Chemical and Energy Sectors:
A combination of market pressures have resulted in significant increases in consumption and associated cost of petroleum-based energy. These forces have co-emerged with improvements in fuel production technology, such that fuels derived from biorenewable plant-based chemical feedstocks are now a viable opportunity within the global energy sector.
The global biorenewable fuels industry, while expanding rapidly in both size and value, is nevertheless confronted with significant obstacles to future growth. Chief among these is the limited availability and cost of input agricultural feedstocks – particularly those based on food crops (e.g. corn starch, Canola oil and soybean oil) for ethanol or biodiesel production
Castor meets all these criteria, and is particularly attractive as a novel source of biorenewable oil feedstocks for the liquid fuel transportation sector. In this project we propose to develop and use advanced genomics tools to develop the Castor plant (Ricinus communis L.) as a non-food source of biorenewable energy. The specific project objectives include completion of the Castor genome sequence to a high quality, as a prerequisite to developing expression arrays, whole genome tiling arrays and reverse-genetic resources that will accelerate development of the crop into the Ontario regional and national sectors.