Plant Biochemistry, Proteomics & Molecular Biology.
Long-term research goals are to elucidate the molecular, regulatory, and functional properties of key enzymes of plant carbohydrate and phosphate metabolism. Genome sequencing provides a crucial blueprint for systematic metabolic studies, but also reveals that plant metabolism and its control is incredibly complex and poorly understood. Many genes encode unknown ENZs &/or multiple isozymes having unidentified individual properties/roles. Metabolic deciphering requires integrated studies of gene expression, metabolite levels and in vivo fluxes, transgenic plants with altered ENZ expression, and individual ENZ molecular/regulatory properties and cellular/subcellular location. Post-translational protein modification by protein-kinase mediated phosphorylation is crucial since this can control an ENZ’s activity, subcellular location, protein interactions, &/or turnover in response to various extra- or intracellular signals.
Research Approach & Recent Advances Our key discovery tool has been the use of fast protein liquid chromatography (FPLC) for the high-yield purification of native enzymes, followed by detailed studies of their molecular, immunological, and kinetic/regulatory properties. However, over the past 10 years numerous innovative strategies have been incorporated into our research, particularly: i) key proteomic tools such as use of MALDI- & Q-TOF-mass spectrometry (for protein peptide mass fingerprinting & sequencing, and mapping of enzyme phosphorylation- and ubiquitination-sites), 2-D and phosphate-affinity polyacrylamide gel electrophoresis, and co-immunopurification, ii) anti-phosphorylation-site specific-antibody production for ENZ phosphorylation-dephosphorylation and protein kinase studies, & iii) isolation and use of relevant cDNAs as probes with respective antibodies to assess isozyme expression changes, and for heterologous (recombinant) ENZ expression in E. coli, and iv) use of Arabidopsis T-DNA insertional 'loss of function' mutants to test the physiological/metabolic function(s) of several enzymes that we have thoroughly characterized at the biochemical level. Thanks to collaborators, the dedicated efforts of my students and post-docs, and generous NSERC support these innovations have recently led to several remarkable discoveries that have set the stage for our current research.
Supervision of BIOL537 students Supervision of two well-qualified BIOL537 students would be ideal. Starting date is flexible. However, a SWEP position (http://careers.queensu.ca) is available that could be used to employ an incoming BIOL537 student as a full-time research assistant during the upcoming spring/summer, prior to their enrollment in BIOL537 in Sept. Our BIOL537students receive training in the theoretical and practical aspects of various lab techniques needed for their thesis research. Student supervision is provided by myself, as well as a research associate, post-doc, and senior grad students.
Student Qualifications Previous lecture courses &/or practical lab experience in biochemistry (BIOL334 is recommended), chemistry, or plant physiology will be an asset. The main prerequisites are that the student) is a highly motivated individual with excellent 'work ethic', communication, & interpersonal skills, (ii) has lots of initiative and is well organized, & (iii) enjoys lab work. Preference is given to students who: (i) have taken (or will be taking, prior to next Sept.) any of Biology’s 400-level practical lab courses (BIOL401*, 402*, 403*, & 404*) &/or have previous summer or volunteer experience working in a ‘cell & molecular’ oriented research lab, and (ii) might be interested in pursuing post-graduate (MSc/PhD) research in the plant sciences following completion of their BSc. Those who wish to work with us during the summer of 2013 should submit an application to the SWEP program, for the Research Assistant in Plant Biochemistry & Molecular Biology position (application deadline = Feb. 15, 2013).
Potential Projects There are a number of potential projects available, with the final choice depending upon the student's background and specific interests, and starting date. Systems we currently study include developing and germinating castor oilseeds, and suspension cell cultures and seedlings of the model plant Arabidopsis thaliana. Projects range from ENZ purification and characterization, to metabolite extraction and quantification, to the use of 2-dimensional gel electrophoresis, ENZ kinetic studies, immunological techniques (western blotting &/or co-immunoprecipitation using monospecific antibodies) or molecular techniques (transcript profiling via RT-PCR, screening & characterization of transgenic plants, etc) to assess the influence of seed development or environmental stressors such as phosphate starvation on ENZ regulation or expression. Possible projects also include screening and analyzing transgenic Arabidopsis plants in which several of the ENZs that we are studying have been 'knocked out' (loss-of-function) or over-expressed.
Significance of Our Research Understanding the organization and control of plant carbohydrate and phosphate metabolism is crucial to ongoing biotech efforts to improve Canada’s crops. Our discovery of novel Class-1 & Class-2 phosphoenolpyruvate carboxylase isoforms in castor oilseeds and their control by allosteric effectors, protein:protein interactions, reversible phosphorylation, and monoubiquitination may lead to strategies for modifying levels of storage proteins vs. vegetable oil content in transgenic seeds. Similarly, our studies of the biochemical adaptations of phosphate-starved plants has generated knowledge and molecular tools that will facilitate the development of phosphate-efficient transgenic crops, urgently needed to reduce mankind’s over-reliance on unsustainable, polluting, and non-renewable phosphate-containing fertilizers in agriculture.
STARTING DATE: Flexible