Research Programs and Interests


Biochemistry, Molecular Biology, and Ecology of Herbivore Defense and Phenolic Metabolism in Trees

Forest health is a major concern in Canada and worldwide. In the natural environment, trees are faced with a diversity of pests and pathogens, yet survive because they have evolved sophisticated defenses to thwart them. The aim of our research is to understand the biochemical and molecular basis of tree defense, and in particular the role of phenolic secondary metabolites and flavonoids in this process. We are primarily studying these responses and pathways in the genus Populus (poplars and aspens), since the leaves of these trees respond to insect damage with strong inducible defense reactions and also contain many diverse phenolic phytochemicals.

Of particular interest are the proanthocyanidins, or condensed tannins, polymeric flavonoids which are induced by stress and have many interesting ecological activities. We have shown how a single gene (a MYB transcription factor) controls the entire proanthocyanidin pathway, and we are now studying other regulatory genes and proteins hypothesized to act in concert with this MYB. We are also investigating biosynthetic genes such as flavonoid glycosyl transferases and acyl transferases, which are hypothesized to contribute to poplar defense and phenolic synthesis. Understanding the synthesis of the proanthocyanidin has broad implications, since these chemicals are found in food plants (such as apples and blueberries) and are important in the maintenance of human health.

A key feature of the plant response to herbivory is a rapid shift in gene expression, leading to the upregulation of hundreds of genes and the rapid accumulation of defense proteins. Much of our previous work has been to characterize these  herbivore-induced genes, in order to obtain clues as what poplars do to actively defend themselves. We have used diverse molecular techniques including differential hybridization, EST sequencing, cDNA arrays, and genomics. We now have a catalog of dozens of putative herbivore-defense genes, and because the genome of Populus has been sequenced, we can study entire gene families.  Prominent among these are protease inhibitors, which interfere with insect digestive processes, and polyphenol oxidases. The defense response also upregulates lipases, phosphatases, chitinases and other hydrolases.

A major challenge is to determine how poplar defense proteins and regulators actually work. We are studying putative defense genes by biochemically testing recombinant proteins produced in E. coli and by overexpressing them in transgenic poplar, tobacco, and Arabidopsis. Transcriptional regulation of proanthocyanidin pathway is being studied using in vivo gene activation assays as well as in transgenic plants. In our efforts to characterize these genes and their effects, we collaborate with insect physiologists, chemists, and ecologists.

Several of my research projects on phenolic metabolism and carbon allocation, in particular its genetic control, will be part of the new NSERC CREATE Program in Forests and Climate Change (ForCC), which provides outstanding training and research opportunities for graduate students, undergraduates, and post-docs. Click here for more information.

Current Research Projects


Herbivore- and stress-induced proanthocyanidin biosynthesis in poplar

Some poplars accumulate substantial concentrations of polymeric flavonoids called proanthocyanidins, and their synthesis can be further induced by herbivores and other stresses, for example UV light. We are studying the regulatory genes that are involved in proanthocyanidin induction in response to stress using expression profiling, bioinformatics, and expression in transgenic poplars. The regulation of flavonoid metabolism is complex, as there are many flavonoid branch pathways, yet they are controlled independently. We are currently using in vivo transcriptional activation assays to test flavonoid promoter- transcription factor interactions (Kazuko Yoshida, post-doctoral fellow; Amy Franklin,MSc candidate; David Ma, MSc candidate).

Effects of poplar phenolics on aphids

As part of our work on gene regulation, we have created transgenic plants with very high proanthocyanidin levels. These plants are great tools to directly test the effects of these phytochemicals, long hypothesized  on diverse insect pests. We are now undertaking a study to determine the effects of proanthocyanidins on poplar aphids and their symbionts (Alpha Wong, MSc candidate).

Functions of poplar BAHD acyl transferases

The diversity of poplar phenolic glycosides suggests the involvement of BAHD acyl transferases. We have identified several acyl transferases, which may contribute to poplar defense or phenolic synthesis. These genes are being cloned into in E. coli for functional analysis, and we plan to test their biological activities in transgenic poplar plants (Russ Chedgy, PhD candidate).
   

Recent Research Projects


Biosynthesis of proanthocyanidins in apple and blueberry fruit

Flavonoids and proanthocyanidins in the human diet are important for preventing cardiovascular disease. Based on our knowledge of proanthocyanidin synthesis and its regulation in poplar, we have beenidentifying the corresponding genes in apple and blueberry. These fruits are major sources of health-promoting flavonoids in the North American diet, and important crops in British Columbia. We have profiled the expression of the genes responsible for proanthocyanidin synthesis in blueberry. We are also interested in elucidating unknown steps in the proanthocyanidin pathway (Mike Zifkin, MSc 2011; Andreas Gesell, post-doctoral fellow).

Functions of poplar flavonoid glycosyl transferases

During our studies of defense responses in poplar, we have identified flavonoid glycosyl transferases, which may contribute to poplar defense or proanthocyanidin synthesis. These genes are being expressed in bacteria in order to test the enzymological properties of the encoded proteins, and are also being modulated in transgenic poplar plants (Vasko Veljanovski, PhD 2012).

Analysis and expression of polyphenol oxidases in poplar

Polyphenol oxidases (PPOs) are common plant enzymes with a diversity of function, including defense against pests and pathogens. We used genome databases to study the entire PPO gene family in poplar and other plants. We have discovered a novel type of PPO, and have shown that it is unique in being localized to the vacuole (Lan Tran, MSc 2011).

Gene expression profiling in herbivore defense

Using macroarrays and other tools for gene expression profiling, we identified more than 100 genes strongly upregulated by herbivory. We have studied global patterns of regulation of these genes in response to wounding and to insect elicitors, substances found in insect regurgitant. We work to understand the role that many of these induced genes play in plant defense and how the corresponding proteins work as defensive agents (Ian Major, PhD 2007).

Proteomics of poplar phloem

Phloem sap contains at least 200 distinct proteins, but few have been characterized. We have used a proteomics approach to identify proteins in poplar phloem. In collaboration with the UVic Proteomics Centre, we have identified almost 100 such proteins using a combination of MALDI-TOF and tandem mass spectrometry. Our aim is to better understand the role of phloem proteins in plants, many of which are annotatd as plant defense proteins (Nicole Dafoe, PhD 2009).

Manipulation of oxidative enzymes for plant defense

Plant phenolic compounds are known to affect some insects more than others, and this is likely due to chemical environment in the insect midgut. As collaborative project with Dr Ray Barbehenn at the University of Michigan, we have generated poplar plants which overexpress plant oxidative enzymes. These are used for physiological experiments with herbivorous insects.