Human use of synthetic chemical compounds, such as pharmaceuticals and personal care products, is on the rise in developed countries. Many of these chemicals are discharged into freshwater ecosystems via, for example, municipal wastewater effluents. While the concentrations in surface waters are often unlikely to cause direct mortality, there is rising concern about how chronic exposure to sub-lethal amounts of these pollutants may directly or indirectly affect animal fitness. I will discuss a series of experiments I conducted addressing how pharmaceuticals and municipal wastewater effluents impact wild fish across scales of biological organization, from physiology to behaviour to changes in fish community composition. I will compare my results from controlled laboratory studies with findings from in situ field exposures and from studies using animal tracking technologies. Our current understanding of how chemical contaminants affect aquatic organisms is largely based on individual-level responses. Yet, understanding and mitigating the impacts of anthropogenic pollution requires knowledge of how pollutants affect animals in their natural social environment and habitats. I will therefore highlight how my research program is closing these knowledge gaps by focusing on complex inter- and intra-specific interactions (e.g., predator-prey, social dominance) in realistic environments.

Ecological and evolutionary success of animals depends on the expression of phenotypes that are compatible with their environment. In order to understand how specific physiological phenotypes arise, and the degree of plasticity or flexibility of these phenotypes, it is critical that we integrate the study of physiology across different levels of biological organisation. In my research, I have used ionoregulatory systems (salt and water balance) of freshwater fishes as a model to demonstrate how the genome and environment interact to influence phenotypes and how these interactions change over life history to shape physiological systems. In particular, I have studied how fishes sustain Na+ absorption, a process critical to maintaining internal ion and water balance, over development and in response to a range of environmental conditions (ionic strength, pH, contaminant exposure). In this presentation, I will discuss the molecular mechanisms of Na+ absorption by rainbow trout, how they change over development, and the implications that this has in understanding how fish at different stages of life history respond to changes in environmental conditions. I will also discuss the use of CRISPR/Cas9 gene editing as a tool to knock out genes that regulate Na+ absorption in zebrafish and explore how the resulting reduction in genetic complexity influences the expression and plasticity of phenotypes. This research highlights the importance of integrating molecular, organismal, and environmental physiology to understand how fishes occupy different environmental niches and how they respond to environmental change.

Ayo Adurogbangba
PhD. Candidate
University of Sherbrooke

RNA silencing is a major mechanism of constitutive antiviral defense in plants. Arabidopsis encodes four Dicer-like (DCL) proteins and ten different Argonaute (AGO) proteins that are specialized to function in different RNA silencing-related mechanisms. We have previously shown that AGO2 plays an important role in protecting plants against viruses, including Potato virus X (PVX) in Arabidopsis. However, despite having a functional AGO2 protein, Nicotiana benthiana is highly susceptible to PVX. Using a transient expression system in N. benthamiana leaves, we found that Arabidopsis AGO2 (AtAGO2), but not N. benthamiana (NbAGO2), possesses antiviral activity against PVX. Consistent with this, we find that activity of NbAGO2, but not AtAGO2, is supressed by the PVX suppressor of RNA silencing, P25. Therefore, we generated transgenic tomato and N. benthamiana plants containing AtAGO2 and NbAGO2 constructs. These transgenic lines were inoculated with PVX and viral accumulation levels were assessed. Recent results of these assays will be discussed. In addition, we have observed natural variation in AtAGO2 in naturally occurring Iberian Arabidopsis wild accessions and found that certain polymorphisms correlate with the degree of susceptibility to PVX and to Turnip mosaic virus (TuMV) but not with susceptibility to Cucumber mosaic virus (CMV). Our results indicate that natural variation in AGO2 can have important effects on infection for some, but not all viruses.

Christian Danve M. Castroverde
Department of Biology, Wilfrid Laurier University
Website: castroverdelab.ca
Twitter: @DanveC

Climate change combined with plant diseases pose serious threats to global agriculture and food security. Because the molecular mechanisms by which environmental factors affect plant immunity and disease are poorly understood, my research program aims to address this critical knowledge gap. We have recently reported that salicylic acid (SA) production is a temperature-sensitive pathway in the plant immune system (Nature Communications 2017 vol 8:1808). SA is an important plant hormone mediating immune responses against a broad range of pathogens.
 
How elevated temperature intercepts the SA pathway is currently unclear, and strategies to counter temperature-mediated suppression of SA production are also lacking. My continued research into this central biological question has shown that elevated temperature has a profound effect on gene expression of a calmodulin-binding master transcription factor (TF) controlling various immune regulators in plants. Remarkably, constitutive gene expression of this master TF was sufficient to recover SA production and immunity at elevated temperature. Global transcriptome analyses revealed that a significant suite of defense-related target genes were downregulated at elevated temperature. My research has identified a crucial temperature-regulated component of the plant immune system.
 
The upstream mechanisms involved in temperature regulation of this master TF are unknown. Whether temperature-sensitive immunity observed in the model plant Arabidopsis thaliana occurs in other plant species is also unclear. These are important next-level questions that I am currently investigating. By integrating molecular, genetic and biochemical approaches, my research program aims to understand plant-pathogen interactions in a dynamically changing environment, in order to shed light on plant resilience mechanisms.

The use of plants to remediate contaminated lands is of growing interest to the scientific community because of its ease of implementation, cost effectiveness and ability to stabilize contaminated soils. A novel Populus tremula x Populus alba mutant named fuzzy with an increased trichome density, an elevated growth rate, enhanced pest resistance and a predicted root phenotype was investigated for its ability to tolerate and accumulate arsenic and cadmium in soil. The chlorophyll, hydrogen peroxide and proline contents of fuzzy tissues were assayed, its tissue metal content was measured using ICP-MS and its wet biomass was quantified. Transgenic Arabidopsis thaliana overexpressing homologous gene of that overexpressed in the fuzzy poplar were grown on gel media and their roots were measured to corroborate the root phenotype of fuzzy poplars. The fuzzy poplar was found to have lower levels of hydrogen peroxide in its tissues after arsenic and cadmium exposure than control poplars, suggesting it has a reduced oxidative stress response when exposed to high levels of arsenic and cadmium. This low hydrogen peroxide content was not due to reduced arsenic and cadmium accumulation in fuzzy trees, as they did not accumulate different amounts of arsenic or cadmium than control plants. Proline and chlorophyll levels were not significantly different between metal treatments, or between fuzzy and control poplars. fuzzy poplars exhibited significantly higher root biomass than control poplars, and transgenic Arabidopsis thaliana overexpressing a homologous gene to the one that causes the fuzzy phenotype had significantly longer roots than control plants. All poplars exposed to arsenic and cadmium in this trial contained the majority of these compounds in their tissues in roots. The root biomass phenotype, low hydrogen peroxide content and root metal accumulation in the fuzzy poplar make it an ideal candidate for bioremediation and soil stabilization of contaminated soils.
 

 Mehran Dastmalchi
Department of Biological Sciences,
Brock University, St. Catharines, ON

Opium poppy has been in use as a medicinal plant since before the dawn of civilization. Today, it remains the only source for natural opiate analgesics, codeine and morphine, and precursors used to synthesize derivatives such as the overdose antidote, naloxone. Total chemical synthesis of opiates is not commercially feasible due to complex stereochemistry. Engineering microbes with alkaloid biosynthetic machinery from opium poppy has the potential of providing a sustainable global supply of opiate pharmaceuticals. However, microbial bioproduction often suffers from carbon loss to alternative pathways. In the latter stages of opiate biosynthesis, thebaine is converted, via codeine, to morphine. When this pathway is simulated in vitro or in microbial systems, two undesirable isomers accumulate: neopine and neomorphine. We addressed this aberrant pathway in two studies: 1) In the penultimate step of morphine biosynthesis COR reduces codeinone to codeine. However, codeinone exists in an apparent equilibrium with its isomeric form, neopinone. We showed that COR irreversibly reduces neopinone to neopine. Using natural and synthetic protein variation we also identified four residues that can confer COR with higher protein stability and performance, improving metabolic flux. 2) We sought an explanation for why the plant does not accumulate the isomeric byproducts. Using a proteomics approach, we found a novel enzyme, neopinone isomerase (NISO) that catalyzes the assumed spontaneous conversion of neopinone to codeinone. NISO provides the substrate for COR to produce codeine and precludes the formation of neopine. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products and avoids carbon loss.

Meghan Rains, PhD student
Algoma University / Queen's University

Pathogens, climate change, and pollution represent important stressors that plants face continuously. To better prepare for a changing terrestrial landscape, we must understand how plants adapt and cope with various environmental stressors. The plant cuticle and suberized cells (cork) are among the critical adaptations that plants developed when they moved from an aqueous environment to land. These cell wall-specific extracellular lipid barriers provide the first line of defense against pathogens and control water exchange. The cuticle covers most aerial plant surfaces and is composed of a polymer of fatty acids and waxes. The periderms of roots, tubers, and tree bark, contain waxes and suberin –an esterified network of glycerol and fatty acid derivatives that is bound to a lignin-like polymer. Although structural models have been inferred from chemical depolymerizations, the insoluble nature of these polymers makes analyses challenging, and consequently, the native structure of suberin remains unclear. Much of the current research on suberin biosynthesis has focused on the model plant Arabidopsis thaliana. However, the complete pathways have not yet been fully characterized, and it is unclear how knowledge derived from Arabidopsis translates to woody tree periderms. This dissertation work used a combination of chemical and molecular approaches to identify candidate genes for suberin biosynthesis and to investigate the structure of the polyester using the model tree, Populus trichocarpa (Poplar). The results from this research further our understanding of suberin structure in tree bark, establish improved methodologies, and generate hypotheses for future targeted studies.

Dr. Kenji Nakahara, Lecturer
Pathogen-Plant Interactions Group, Research Faculty of Agriculture,
Hokkaido University, Sapporo, Hokkaido 060-8589, Japan

RNA silencing is one of major antiviral systems in plants and most plant viruses encode RNA silencing suppressors (RSS) to facilitate their infection of plants by inhibiting the plant’s endogenous antiviral RNA-silencing machinery. Previously, a tobacco calmodulin-like protein (CML), termed rgs-CaM, has been reported to interact with HC-Pro and 2b, which are RSSs encoded by members of the genus Potyvirus and Cucumovirus, respectively. We have shown that the tobacco CML counteractively functions as an antiviral defense factor to direct degradation of its interacting RSS proteins via autophagy. Further studies suggest that the rgs-CaM-mediated counterdefense against RSSs involves salicylic acid signaling. Plants encode dozens of CMLs (50 and 32 CMLs in Arabidopsis and rice, respectively). Several CMLs of tobacco and other plants are similar to rgs-CaM in their amino acid sequences, suggesting possible binding to viral RSSs and involvement with antiviral defense. We have been investigating whether Arabidopsis CML orthologs of rgs-CaM may be involved in plant/virus interaction and I will present data from research in Japan and recent collaborative work in the Snedden lab here at Queen’s.

Gustavo MacIntosh // Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology; Iowa State University, Ames, IA, USA
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Ribosomes are essential cellular components, and a large proportion of cellular resources are dedicated to their synthesis. Yet, the pathways involved in the turnover of normal ribosomes remain poorly studied. We have shown that the Arabidopsis ribonuclease RNS2 functions in the vacuolar degradation of rRNA. Mutants lacking this RNase activity have rRNA with a longer half-life, which accumulates in the vacuole. rns2 mutants also have constitutive activation of the autophagy pathway, possibly as an attempt to compensate for the loss of rRNA degradation. A functional autophagy pathway is also necessary to maintain normal RNA levels in Arabidopsis, suggesting that plants use an autophagy-dependent mechanism to transport ribosomes to the vacuole for recycling. However, differential rRNA accumulation in vacuoles of specific atg mutants suggest that rRNA or ribosome transport to the organelle may normally occur through a selective mechanism that utilizes some, but not all, the autophagy core components. In addition to dissecting the rRNA decay pathway and the mechanisms of rRNA transport to the vacuole, we are interested in understanding why rRNA is recycled. Metabolome and transcriptome analyses indicated that carbon flux through the pentose phosphate pathways is altered in mutants that cannot recycle rRNA properly. Our results suggest that rRNA turnover is necessary to maintain cellular homeostasis, likely as part of the nucleoside salvage pathway. When this salvage pathway is blocked, the PPP is rerouted to produce ribose-5-P for de novo nucleoside synthesis. This change in carbon flux, in turn, causes growth phenotypes and the production of reactive oxygen species that are responsible for activation of the general autophagy pathway in rns2 mutants.