General Research Interests

My research addresses the physiology, biochemistry, and molecular biology of metal resistance in plants. The major focus of current work is on aluminum, the primary growth-limiting factor in acid soils, although work on cadmium is becoming increasingly important. We are seeking to understand the physiological and biochemical mechanisms by which certain plant species, cultivars, and ecotypes can grow in the face of severe metal stress, as well as the mechanisms of metal transport across the plasma membrane. We are also investigating potential interactions between phytotoxic metals under conditions of low ionic strength. Research is interdisciplinary in nature, utilizing a wide variety of physiological, biochemical, molecular, and genetic techniques on whole plant and cell culture systems.

Understanding the physiological, cellular, and molecular basis of resistance to Al. Physiological, cellular, and molecular aspects of resistance to Al in higher plants

Funded by the Natural Sciences and Engineering Research Council of Canada (NSERC), Research Grants Program.

Aluminum-resistant cultivars of wheat yield 10-fold higher than Al-sensitive cultivars under conditions of Al stress. This resistance appears to be achieved in part by exclusion of Al from the cytoplasm. I am using new technologies (single cell micro-extraction and ²⁶Al-Accelerator Mass Spectroscopy) to provide conclusive evidence that plants can exclude Al from the cytoplasm. I am also seeking to identify mechanisms which account for exclusion and mediate the resistance phenotype. A growing body of evidence suggests that enhanced synthesis and export of malate to the root surface plays a role in exclusion. I am using two approaches to test this hypothesis. In the first I am overexpressing malate synthase in Al-sensitive canola and testing the effect of the transgene on malate export, Al influx, and resistance. In the second, I am disabling malate export in Al resistant wheat and transgenic canola using transport inhibitors. If enhanced export of malate plays a role in resistance, these inhibitors should increase Al influx and render plants more sensitive to Al.

I am also conducting experiments that bring together my work on transport physiology and protein production. Net Al transport reflects uptake into both the plant cytoplasm and its vacuole. My work with giant algal cells suggests that transport to the vacuole becomes rate limiting soon after exposure. This provides impetus for cloning the gene for an Al-induced, tonoplast-bound protein which co-segregates with the Al resistance phenotype in wheat. By expressing this gene in Al-sensitive canola, I can test the role that the protein plays in mediating the resistance phenotype.

In the final part of this project, I am attempting to reconcile apparently conflicting data regarding the effect of Al on callose synthase. Callose accumulation in roots of Al-sensitive genotypes increases 3-fold after 2 h of exposure to Al. However, the behaviour of the enzyme depends on conditions of exposure. In isolated plasma membranes, Al exerts a powerful inhibitory effect. I have hypothesized that the different effects of Al on in vivo and in vitro systems reflect an Al-induced increase in cytosolic Ca (which only occurs in vivo) and the extent to which Al has (or does not have) access to the cytosolic face of the enzyme. I am testing this hypothesis by examining the sensitivity of callose synthase in right-side-out and inside-out plasma membrane vesicles, using detergents and ionophores to facilitate Al and/or Ca entry to the vesicle interior.

 

Using proteomics to establish an Extracytosolic Plant Proteins database (EPPdb) and identify root-specific promoters for improved stress tolerance in plants.

Funded in part by the Alberta Agricultural Research Institute.   Application pending with the  Natural Sciences and Engineering Research Council of Canada (NSERC), Strategic Grants Program.

The ability to secrete a wide array of compounds into the apoplasm/rhizosphere is one of the most fascinating metabolic features of plant roots. Rhizosecretion has been shown to affect a number of plant processes, including protection from pathogens, nutrient acquisition, communication with other soil organisms and resistance to disease and toxic metals. Identification and characterisation of these exudates will provide an important means of increasing our understanding of the mechanisms used by plants to respond to biotic and abiotic stresses.

We have demonstrated that roots of wheat and canola exude a large suite of proteins into the apoplast rhizosphere. We are using proteomic tools (2-D gel electrophoresis, MALDI/MS, ESI/MS and bioinformatics) to analyze extracytosolic proteins and establish an Extracytosolic Plant Proteins database (EPPdb) for canola, an important crop species in Canada. This EPPdb will be made available to the scientific community through the world wide web, where it will provide a source of integrated, value-added information for plant biologists, and a platform to easily retrieve and add more information. The availability of the database to the plant biology community and the knowledge of genes involved in production of extracytosolic proteins will offer new opportunities in metabolic engineering and pharmacological engineering. Identification of these proteins will also provide a vehicle for identifying a suite of stress-induced genes and their regulatory sequences. One of the key requirements for genetic improvement of plants using recombinant DNA technology is a suite of promoters that will allow efficient, tissue-specific expression of a particular gene of interest. Promoters that regulate expression of extracytosolic proteins will be characterized using promoter/GUS (or GFP) fusions to analyze their tissue-specific and stress-specific expression patterns. Benefits to industry and society will ultimately include development of crop plants with improved stress tolerance and reduced yield losses resulting from root-specific diseases. This will benefit society by increasing producer yields and reducing the levels of chemical inputs into agricultural systems.

 

Regulation of cadmium accumulation in the grain of durum wheat

Application pending with the  Natural Sciences and Engineering Research Council of Canada (NSERC), Strategic Grants Program.

High levels of cadmium (Cd) have been found in crops such as wheat, flax, sunflower and potato. Because of the importance of these crops in the human diet, the Codex Alimentarius Commission (international food standards organisation) is currently considering a 0.1 mg Cd per kg grain guideline for cereals, pulses and legumes. Several countries have already adopted this limit in their food standards regulations. Recent widescale analysis of Canadian durum wheat by the Canadian Grain Commission indicates that Prairie durum wheats consistently exceed the proposed limit. Consequently, Canadian durum wheat exports, which are worth an estimated one billion a year, would be threatened if the standard were adopted. The physiological basis for high accumulation of Cd in durum grain is poorly understood. Our recent research suggests Cd accumulation in durum wheat grain may be regulated at several stages, including translocation from root to shoot, and remobilization from leaves to maturing grain. However, it remains unknown to what extent these processes contribute to overall Cd accumulation in grain. In collaboration with Agrium Inc., we are planning to undertake a research program designed to better understand the regulation of Cd accumulation in durum wheat grain. The project will concentrate on determining the relative contribution of leaf, stem and root pools of Cd to Cd accumulation by grain. Using chelator-buffered hydroponic culture to regulate Cd availability to agriculturally relevant concentrations, we intend to measure whole-plant Cd accumulation over several stages of vegetative and reproductive growth. By combining this approach with isotopic (109-Cd) labelling techniques, we will be able to determine the relative contribution of different Cd pools to grain Cd, identify the routes of Cd remobilization, and determine the relative mobility of Cd from the various Cd pools. The time-course of Cd remobilization will also be studied under field conditions. Quantifying the pathways and timing of remobilization of plant Cd-pools to grain will provide information that is directly applicable to the development of management strategies that may minimise Cd accumulation. In the long run, Canada will benefit through greater competitiveness in international durum wheat markets.

 

Degradation of petroleum hydrocarbons at the soil/root interface in contaminated soils

Funded by C.O.U.R.S.E.

Petroleum hydrocarbons can be a major soil pollution problem resulting from oil spills, pipeline leaks and improper storage of fuels. While clean up methods such as thermal desorption (incineration) and slurry treatment exist, they are costly, pose environmental risk to water and soil, and require transportation of contaminated soils to a remote site. These shortcomings have prompted research into the use of in situ (on-site) biological methods of decontamination, including bioremediation and phytoremediation. Bioremediation describes the use of microorganisms to degrade contaminants, while phytoremediation techniques use plants for the same purpose. Recently it has been suggested that the two methods should be considered together. Plant roots can improve soil structure, increase surface area for colonization, and provide a source of carbon for soil microorganisms, which are often efficient at degrading contaminants. Few studies have focused on the relationship between plant roots and microbes for remediation purposes, even though it is recognized that microbial density is higher in vegetated soil than in unvegetated soil

It has been hypothesized that elevated microbial populations in vegetated soils can be attributed to sugars, amino acids and proteins released into soil by plant roots. Microorganisms can use these compounds as sources of carbon, allowing for increased microbial activity in soil surrounding the root (rhizosphere). This proposed research will measure and compare rates of degradation of petroleum hydrocarbons in contaminated bulk and rhizosphere soils that have been sterilized and inoculated, or contain indigenous microbes from a selected site. Analysis of degradation rates will be conducted using radiolabeled compounds to analyze the fate of a mixture of model contaminants (e.g. benzene, toluene, naphthalene, phenanthrene) in a model plant/microorganism system. It is expected that degradation rates will be higher in the rhizosphere soil than in bulk soil. This research will be the first step in understanding the soil-root interface and its potential role in soil clean up technologies.