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Item Structural and functional analysis of plant ELO-like elongase for fatty acid elongation(Plant Molecular Biology, 2024-08) Meesapyodsuk, Dauenpen; Sun, Kaiwen; Qiu, XiaoELO-like elongase is a condensing enzyme elongating long chain fatty acids in eukaryotes. Eranthis hyemalis ELO-like elongase (EhELO1) is the first higher plant ELO-type elongase that is highly active in elongating a wide range of polyunsaturated fatty acids (PUFAs) and some monounsaturated fatty acids (MUFAs). This study attempted using domain swapping and site-directed mutagenesis of EhELO1 and EhELO2, a close homologue of EhELO1 but with no apparent elongase activity, to elucidate the structural determinants critical for catalytic activity and substrate specificity. Domain swapping analysis of the two showed that subdomain B in the C-terminal half of EhELO1 is essential for MUFA elongation while subdomain C in the C-terminal half of EhELO1 is essential for both PUFA and MUFA elongations, implying these regions are critical in defining the architecture of the substrate tunnel for substrate specificity. Site-directed mutagenesis showed that the glycine at position 220 in the subdomain C plays a key role in differentiating the function of the two elongases. In addition, valine at 161 and cysteine at 165 in subdomain A also play critical roles in defining the architecture of the deep substrate tunnel, thereby contributing significantly to the acceptance of, and interaction with primer substrates.Item Biosynthetic mechanisms of omega-3 polyunsaturated fatty acids in microalgae(Journal of Biological Chemistry, 2024-08) Sun, Kaiwen; Meesapyodsuk, Dauenpen; Qiu, XiaoMarine microalgae are the primary producers of u3 poly- unsaturated fatty acids (PUFAs), such as octadecapentaenoic acid (OPA, 18:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) for food chains. However, the biosynthetic mechanisms of these PUFAs in the algae remain elusive. To study how these fatty acids are synthesized in microalgae, a series of radio- labeled precursors were used to trace the biosynthetic process of PUFAs in Emiliania huxleyi. Feeding the alga with 14C- labeled acetic acid in a time course showed that OPA was solely found in glycoglycerolipids such as monogalactosyldiacylgly- cerol (MGDG) and digalactosyldiacylglycerol (DGDG) synthe- sized plastidically by sequential desaturations while DHA was exclusively found in phospholipids synthesized extraplastidi- cally. Feeding the alga with 14C-labeled a-linolenic acid (ALA), linoleic acid (LA), and oleic acid (OA) showed that DHA was synthesized extraplastidically from fed ALA and LA, but not from OA, implying that the aerobic pathway of DHA biosyn- thesis is incomplete with missing a D12 desaturation step. The in vitro enzymatic assays with 14C-labeled malonyl-CoA showed that DHA was synthesized from acetic acid by a PUFA synthase. These results provide the first and conclusive biochemistry evidence that OPA is synthesized by a plastidic aerobic pathway through sequential desaturations with the last step of D3 desaturation, while DHA is synthesized by an extraplastidic anaerobic pathway catalyzed by a PUFA synthase in the microalga.Item Molecular cloning and functional analysis of a plastidial ω3 desaturase from Emiliania huxleyi(Frontiers in Microbiology, 2024-07) Sun, Kaiwen Meesapyodsuk, Dauenpen Qiu, XiaoEmiliania huxleyi is a marine microalga playing a significant ecological and biogeochemical role in oceans. It can produce several polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, 22:6–4,7,10,13,16,19) and octadecapentaenoic acid (OPA, 18:5–3,6,9,12,15), providing a primary source for nutritionally important ω3 PUFAs in the marine food chain. However, the biosynthesis of these PUFAs in this organism is not well understood. In this study, a full length plastidial ω3 desaturase cDNA (EhN3) was cloned from this alga. Heterologous expression of EhN3 with and without the chloroplast targeting peptide (cTP) in cyanobacterium Synechococcus elongatus showed that it possessed high desaturation activity toward C18-ω6 PUFAs, linoleic acid (LA, 18:2–9,12), γ-linolenic acid (GLA, 18:3–6,9,12), and C20-ω6 PUFAs, dihomo-γ- linolenic acid (DGLA, 20:3–8,11,14) and arachidonic acid (ARA, 20:4–5,8,11,14) that were exogenously supplied. Desaturation efficiency could reach almost 100% in a time course. On the other hand, when expressed in Saccharomyces cerevisiae, EhN3 with and without cTP did not exhibit any activity. Lipid analysis of Synechococcus transformants expressing EhN3 showed that it utilized galactolipids as substrates. Transcriptional expression analysis revealed that the expression of the gene increased while the growth temperature decreased, which was correlated with the increased production of ω3-PUFAs, particularly OPA. This is the first report of a plastidial ω3 desaturase from microalgae that can effectively introduce an ω3 double bond into both C18-ω6 and C20-ω6 PUFAs. EhN3 might also be one of the key enzymes involved in the biosynthesis of OPA in E. huxleyi through the plastidial aerobic pathway.Item Kuhn-Tucker and Multiple Discrete-Continuous Extreme Value Model Estimation and Simulation in R: The rmdcev Package(The R Foundation, 2020) Lloyd-Smith, PatrickThis paper introduces the package rmdcev in R for estimation and simulation of Kuhn-Tucker demand models with individual heterogeneity. The models supported by rmdcev are the multiple-discrete continuous extreme value (MDCEV) model and Kuhn-Tucker specification common in the environmental economics literature on recreation demand. Latent class and random parameters specifications can be implemented and the models are fit using maximum likelihood estimation or Bayesian estimation. The rmdcev package also implements demand forecasting and welfare calculation for policy simulation. The purpose of this paper is to describe the model estimation and simulation framework and to demonstrate the functionalities of rmdcev using real datasets.Item Keeping phosphorus on the land: Workshopping a bridge between agricultural production and water quality(University of Saskatchewan, 2022) Liu, Jian; Elliott, Jane; Baulch, HelenManagement of the phosphorus (P) in prairie soils presents a challenging dilemma. Phosphorus is critical to continued agronomic productivity. Yet, management of P in prairie landscapes is also crucial to the protection of prairie lakes and reservoirs, which are highly vulnerable to issues of nutrient pollution and harmful algal blooms. Here we present detailed insights from a 2-day workshop “Keeping Phosphorus on the Land” where we worked to bridge the disciplines of water quality, and agronomy, and better understand issues, and opportunities within and across these areas of work as they relate to managing soil P. This report includes detailed insights and recommendations that reflect outcomes of presentations, panels, and discussions engaging researchers and practitioners in government, industry and universities from each of Canada’s three prairie provinces. It includes recommendations on ‘actionable’ areas, and areas where further research and dialogue is required. Readers are also directed to our short synthesis report, available here: Liu, J., H.M. Baulch, and J.A. Elliott. 2021. Keeping Phosphorus on the Land: Main Takeaways for Managing Soil Phosphorus in the Prairies. University of Saskatchewan, Saskatoon, Canada. DOI:10.23688/1gvs-5333.Item Keeping phosphorus on the land: Main takeaways for managing soil phosphorus in the Prairies(University of Saskatchewan, 2022) Liu, Jian; Baulch, Helen; Elliott, JaneManagement of the phosphorus (P) in prairie soils faces a challenging dilemma. Phosphorus is critical to continued agronomic productivity. Yet, management of P in prairie landscapes is also crucial to the protection of prairie lakes and reservoirs, which are highly vulnerable to issues of nutrient pollution and harmful algal blooms. In our workshop, “Keeping Phosphorus on the Land” (February 22 & March 2, 2021), we worked to bridge the disciplines of water quality, and agronomy, and better understand issues, and opportunities within and across these areas of work as they relate to managing soil P. After two half-days of meetings that engaged researchers and practitioners in government, industry and universities, we gained a long list of ideas to act on, and a number of crucial new insights. This two-page synthesis highlights several key points that came from the workshop. Readers are directed to our full report to learn about these key points and other areas in more detail, and for a full list of recommendations resulting from the meeting.