Research Area

RESEARCH PROGRAM

1. Summary: Secondary metabolism; Plant defense against insects and insect-associated pathogens; Forestry and grapevine genomics

Research in my group is developing a genomic and functional biochemical characterization of conifer defense systems that are active against insect pests and insect-associated fungal pathogens. Some of this research is based on our studies of the molecular biochemistry of plant terpenoid secondary metabolism (see below). Our research has laid the foundation for Canada’s first large-scale forestry genomics program (Treenomix, www.treenomix.ca; funded by Genome Canada, Genome British Columbia and the Province of British Columbia) of which I have been a project leader since 2001. With the Treenomix project I have extended my program to include research on poplar as a model system for tree biology. My lab has been leading genomics research on defense against insects in long-lived, woody plants. In 2006, our forestry genomics program has been refunded with a new project “Conifer Forest Health” genomics. While my research program is primarily designed as fundamental and curiosity-driven research, the results of our research program can potentially be translated into enormous economic, social, and environmental benefit through the long-term sustainability of Canada’s forests. These forests are already under pressure from insect pests, of which the current mountain pine beetle epidemic in British Columbia is just one example. Under predicted scenarios of global climate change forest insect pests will continue to increase as a major threat to Canada’s forests. Our research program on forest health involves collaboration with entomologists targeting the molecular biochemistry of bark beetle pheromone systems (initially supported with an HFSP young investigators award), and collaboration with microbiologists targeting the interacting genomes of bark beetle-associated fungal pathogens and pine host trees (supported with an NSERC strategic grant). The original foundation of my research in plant secondary metabolism, specifically my research on the molecular biochemistry of terpenoid natural products, allowed me to develop several other successful lines of related research. These research activities include research on grapevine (Vitis vinifera) flavor and aroma biochemistry (supported with a Genome Canada and Genome Spain international program grant), collaborative research on floral scent biochemistry, and research on the role and regulation of terpenoids in Arabidopsis thaliana.

2. Research on the molecular genetic, biochemical, and chemical characterization of terpenoid defenses in conifers and poplars against insect pests and pathogens; Terpenoid synthases and cytochrome P450 enzymes

Insect pests and pathogens cause annual losses of billions of dollars to conifer-based forest economies in North America and Europe. To address this issue, my group has established a program on the molecular genetic and biochemical characterization of chemical defenses in conifers. As some of the oldest and longest living plants, conifers have evolved complex, terpenoid-based defense mechanisms against insect herbivores and pathogens. These chemical defenses occur both as preformed defenses and as inducible defenses. In my postdoctoral research I characterized a family of terpene synthase (TPS) genes and enzymes from the conifer grand fir, Abies grandis. The TPS are responsible for the formation of oleoresin terpenes in this system. I isolated cDNAs for a multiple member TPS gene family, expressed the cDNAs in E. coli for in vitro characterization of recombinant enzymes, analyzed TPS gene expression in wound-induced trees and reconstructed a phylogeny of the plant TPS gene family. This work provided an understanding of highly complex defense mechanisms in conifers, relevant to forest biotechnology, and can lead to new strategies for pest control. With my group at at UBC, I have build on our knowledge from the grand fir system to explore more comprehensively terpenoid defenses in the economically important conifer species of spruce (Picea spp.), pines (Pinus spp.), and Douglas fir (Pseudotsuga menziesii). Like other conifers, species of spruce accumulate terpenoids constitutively in preformed cortical resin ducts. In addition, upon insect feeding or pathogen challenge, these trees also respond with an unusual induced de novo differentiation of traumatic resin ducts (TDs) in the developing xylem. Research in my group showed that treatment of trees with the defense signal compound methyl jasmonate (MeJA) induces increased resin terpenoid biosynthesis and traumatic resin accumulation, as well as de novo development of TDs. A suite of chemically defined elicitors has been evaluated for their effect on activation of spruce defenses in pretreatment studies. To further analyze in detail the molecular regulation of resin terpenoid defenses in species of spruce, we isolated cDNAs for a large family of TPS genes from Norway spruce (P. abies) and Sitka spruce (P. sitchensis). We functionally characterized many of these genes and analyzed gene expression in response to insect herbivory, wounding, or treatment of trees with MeJA. We then found that Norway spruce and Sitka spruce trees also respond to treatment with MeJA and to insect attack with the emission of volatile organic compounds. The emission of volatiles was in addition to activation of direct, resin-based terpenoid defenses. Such volatiles can serve as info-chemicals in indirect defense systems of plants. Based on a detailed understanding of the signals and mechanisms of induced direct and indirect terpenoid defenses in spruce we are now developing new strategies for protection of forest trees against insect pests. To complement our research on conifer-insect interactions with a comparative analysis of a conifer-pathogen system, we have developed in parallel a successful characterization of chemical defenses of Douglas fir (Pseudotsuga menziesii) trees that are hosts to certain root rot fungi. Recently we have expanded our program on terpenoid biochemistry in conifers with the discovery of a new group of cytochrome P450 genes in spruce and in loblolly pine (Pinus taeda). Using an integrated functional genomics, FL-cDNA, and metabolite profiling approach, we successfully identified and characterized a new gymnosperm cytochrome P450 gene. We characterized the corresponding recombinant P450 enzyme as an unusual multifunctional and multi-substrate diterpene oxidase with a metabolic function in diterpene resin acid biosynthesis. Other research with conifers that is ongoing in my group has been studying molecular and biochemical mechanisms of induced defenses in lodgepole pine (Pinus contorta) against its fungal pathogen (Ophiostoma clavigerum), a blue stain fungus that is associated with the mountain pine beetle (Dendroctonus ponderosa). The mountain pine beetle epidemic is the largest recorded bark beetle outbreak in Canada causing major economic losses to the forest industry. In a comparative analysis of terpenoid defenses in gymnosperm and angiosperm forest trees, we have investigated indirect tritrophic defense systems in the poplar (Populus) / forest tent caterpillar (FTC) system. In poplars, we identified spatial and temporal patterns of FTC-induced systemic volatile emissions and the TPS gene and enzyme responsible for FTC-induced, diurnal emission of ()-germacrene D, a sesquiterpene.

3. Forestry Genomics: Treenomix, Canada’s first large-scale forestry genomics project (2001 – 2005); Conifer Forest Health Genomics (since 2006) funded by Genome Canada, Genome British Columbia, and the Province of British Columbia

Our studies on chemical defense against insects and pathogens in conifers and poplars provided a biological and functional foundation for Canada’s first large-scale Forestry Genome project “Treenomix”(www.treenomix.ca) and a new genomics project, “Conifer Forest Health”. I am directing the “Conifer Forest Health” project together with Dr. Kermit Ritland (UBC, Dept. Forest Sciences). Collaborators at UBC are Drs. Sally. Aitken, Gary Bull, Thomas Maness, Shawn Mansfield, Paul Wood. Our international partners are at the University of California, Davis (Dr. David Neale), at the Max Planck Institute for Chemical Ecology, Germany (Drs. Jonathan Gershenzon and Michael Phillips), at the Umeå Plant Sciences Centre, Sweden (Drs. Rhishi Bhalerao, Thomas Moritz, Ove Nilsson, Göran Sandberg, Björn Sundberg, and others), and Forest Research Institute in the UK (Dr. Steven Lee). In our forestry genomics program we are using a combination of genomic, proteomic, and metabolite profiling tools to decipher constitutive and induced defense mechanisms and genetic resistance in forest trees, with a primary emphasis on species of spruce, Sitka spruce (Picea sitchensis), White spruce (P. glauca), and Norway spruce (P. abies). In addition, we are using Arabidopsis thaliana and poplar as reference and model systems. Our program has developed essential genomic tools to increase the scientific community’s understanding about trees’ built-in defense mechanisms against insect pests, insect-assocaited pathogens, and environmental stress. The program uses a vertically integrated approach that transfers information between the diverse species of spruce, species of pines, poplar, and Arabidopsis. Specifically, cDNA ESTs and full-length cDNAs from spruce and poplar have been sequenced, obtaining a database and cDNA clone collection of more than 350,000 ESTs from each tree species. The project builds further upon the whole-genome sequence of Arabidopsis and poplar, and on the ability of these plants to display insect-induced defense responses. EST sequences are used to study gene expression underlying important traits in tree defense using microarrays, identifying similarities and differences across resistant and susceptible genotypes. To this end we have developed a 15.5k poplar cDNA microarray and a 22k spruce cDNA microarray. We have identified differentially expressed proteins related to defense against insects in spruce, poplar and Arabidopsis by 2-D gel analysis coupled with Q-TOF and MALDI-TOF MS, and by ICAT and iTRAQ technologies. For spruce and poplar libraries of more than ten thousand genes have been cloned as full-length (FL) cDNAs and in each species more than 4,000 non-redundant FL-cDNAs have been completely sequenced for future functional characterization. Bioinformatic comparisons are being made of sequence changes across this group of species, and correlated with trait evolution. The project is also identifying genetic markers to help in seed orchard and breeding programs, and to locate and identity major genes underlying economically important traits in forestry. As a contribution to the whole-genome sequencing of poplar, a physical map of this tree has been generated from the fingerprinting of 48,000 BAC clones and 96,000 BAC end-sequences. With this work and the FL-cDNAs from poplar, our project has contributed to an international effort with the US Department of Energy (DOE) Joint Genomics Institute (JGI), the Umeå Plant Science Center in Sweden, and several other international partners to sequence, assemble and annotate the poplar genome. A first draft poplar genome sequence was released in September of 2004. This work in tree genomics has been the first of its kind in Canada and is internationally recognized to be at the forefront of forestry genomics. Most of our research on functional genomics is now targeted at identifying and characterizing forest tree defense systems against insects and insect-associated fungi. Specifically we are identifying defense and possible resistance mechanisms of Sitka spruce and white spruce against the white pine weevil and spruce budworm; defense mechanisms of lodgepole pine against the bark beetle associated blue stain fungus Ophiostoma clavigerum, and defense mechanisms of poplars against forest tent caterpillar and willow borer.

4. Bark beetle pheromone de novo biosynthesis

Bark beetles use a complex pheromone communication system in the successful attack of conifer host trees. Myrcene is an acyclic monoterpene that has long been associated with pheromone synthesis in pine bark beetles (e.g. the pine engraver, Ips pini). The logical source of myrcene for bark beetle pheromone biosynthesis has been the oleoresin of the host pines. Monoterpene synthases, which convert geranyl diphosphate to monoterpenoid natural products, have been frequently characterized from conifers and other plants. However, they have not previously been found in animals. We discovered a monoterpene synthase activity in two populations of an insect, I. pini. Cell-free assays of I. pini revealed that 3H-geranyl diphosphate is converted to 3H-myrcene in whole-body extracts from male, but not female, I. pini. Furthermore, the enzyme activity in males can be induced by prior treatment with juvenile hormone III (JH III) or by feeding on phloem from the host trees, Jeffrey pine (Pinus jeffreyi) or red pine (Pinus resinosa). The sex-specificity and endocrine induction of this activity argue for its involvement in the biosynthesis of monoterpenoid pheromones mediated by enzymes from I. pini rather than from microbial symbionts. This work has been in collaboration with Dr. Steven Seybold of the USDA Forests Service, Davis. We have received funding from the international HFSP young investigators program to support this interdisciplinary collaboration between our terpenoid biochemistry group and Dr. Seybold’s entomology group.

5. Molecular biochemistry and genomics of floral scents and fruit flavor and aroma

In collaboration with Dr. Natalia Dudareva (Purdue University, USA), my lab has contributed to the discovery of floral scent genes in snapdragon (Antirrhinum majus) and identified biochemical functions of the encoded enzymes and biochemical and molecular mechanisms of terpenoid floral scent emission in this system. As part of my faculty affiliation with the UBC Wine Research Centre, I started in 2002 a project on the biochemical characterization of monoterpenoid and sesquiterpenoid aroma genes in grape vine (Vitis vinifera). Relevant candidate genes have been identified in EST databases. Selected genes have been functionally identified [()--terpineol synthase; (+) valencene synthase; ()-germacrene D synthase]. Our research with grape has recently received major support with a 3.1 million dollar international grant from Genome Canada / Genome Spain. This new 3-year project started in the spring of 2004, under the direction of Dr. Steven Lund (UBC), and in collaboration with Dr. Patricia Bowen (PARC, Penticton) with international partners in Spain.

6. Functional genomics of terpenoid in Arabidopsis thaliana

While research on conifers and forest health is the major activity of my program, my group is also studying a few selected aspects of low molecular weight terpenoids in Arabidopsis thaliana. Based on a genomics approach, we started identification and functional characterization of TPS genes involved in secondary metabolism in Arabidopsis. In a systematic analysis of the Arabidopsis genome sequence we discovered a surprisingly large family of terpene synthase (AtTPS) genes in this model plant system. The genome-wide analysis of the AtTPS gene family provided novel insights into genome organization and evolution of the plant TPS gene family. The AtTPS genes resembled TPS genes previously described for plants that produce an array of monoterpene, sesquiterpene, and diterpene secondary metabolites in a context of chemical interaction with other organisms. While terpenoid secondary metabolites of this type had not been described for Arabidopsis prior to our work, our discovery of a large and highly conserved AtTPS gene family suggested functional roles of TPS genes and terpenoid secondary metabolites in Arabidopsis. This surprising finding may be implicative of common roles for low molecular weight terpenoid compounds in plants. Recent gene-specific microarray analysis of the complete AtTPS gene family revealed differential and often organ-specific expression of AtTPS genes in Arabidopsis and is guiding ongoing functional characterization of AtTPS genes and terpenoid natural products in this system.

7. Collaborations, Funding, Facilities

Our research projects involve national and international collaborations, several of which are mentioned in the summary of research (see above).

Funding for our research is from the Natural Sciences and Engineering Research Council of Canada (NSERC) through its Discovery and Strategic programs and student and postdoctoral fellowship programs, the Canadian Foundation for Innovation (CFI), the British Columbia Knowledge and Development Funds (BCKDF), Genome Canada, Genome BC, the Province of British Columbia, and other sources.

My group uses laboratory space in the new Michael Smith Laboratories, in the adjacent National Centre of Excellence (NCE), and in the Forest Sciences Centre. We have modern equipment and facilities for genomics, microarray gene expression profiling, proteomics sample preparation, plant molecular biology, enzyme biochemistry, and analytics. Together with Dr. Hennie van Vuuren, UBC Wine Research Centre, I have developed a dedicated mass spectrometry laboratory for analysis of small molecules (e.g. plant secondary metabolites, signaling compounds) equipped with new GC/MS, GC/FID, radio-GC, and LC/MS instruments.