Western University Scholarship@Western Electronic Thesis and Dissertation Repository 9-5-2018 12:30 PM Phylogeny, Heat-Stress and Enzymatic Heat-Sensitivity in the Antarctic Psychrophile, Chlamydomonas sp. UWO 241 Marc Possmayer The University of Western Ontario Supervisor Hüner, Norman P.A. The University of Western Ontario Co-Supervisor Maxwell, Denis P. The University of Western Ontario Graduate Program in Biology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Marc Possmayer 2018 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Plant Sciences Commons Recommended Citation Possmayer, Marc, "Phylogeny, Heat-Stress and Enzymatic Heat-Sensitivity in the Antarctic Psychrophile, Chlamydomonas sp. UWO 241" (2018). Electronic Thesis and Dissertation Repository. 5737. https://ir.lib.uwo.ca/etd/5737 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract Psychrophilic species, micro-organisms which are unable to grow at temperatures of or above 20°C, are abundant in perennially cold ecosystems across the globe. Intensifying scientific investigation of these organisms from ecological to molecular scales has underscored the ability of life on Earth to adapt to environments which seem inhospitable due to high or low temperatures, high salinity, pressure and light, and ultra-low nutrient availability. Psychrophilic organisms that are also photosynthetic represent a much more limited group than psychrophiles generally, as their habitats must be well buffered against the warming influence of the infra-red energy accompanying sunlight. Photosynthetic psychrophiles face the added challenge of balancing the biophysical capture and biochemical transformation of light energy. The psychrophilic green alga Chlamydomonas sp. UWO 241 (hereafter UWO 241) has been the subject of numerous publications, however these are mainly focused on adaptations which facilitate life at low temperature as opposed to restricting it from moderate ones. In this thesis I critically examine previously published phylogenies placing UWO 241 as a strain of Chlamydomonas raudensis, investigate the changes in mRNA abundance in UWO 241 in response to high steady-state growth temperatures and heat shock using targeted and transcriptome-wide approaches, and investigate the regulatory enzymes of the carbon-fixing Calvin-Benson-Bassham (CBB) cycle for signs of heat sensitivity using the techniques of Homology Modelling and Molecular Dynamics. These investigations revealed that, contrary to previously published reports, UWO 241 is not closely related to C. raudensis SAG 49.72. Here, I also show that mRNA abundance in UWO 241 does not respond to different steady-state growth temperatures or a heat-shock temperature shift regime in ways similar to the related mesophile, Chlamydomonas reinhardtii. Additionally, analysis of the regulatory CBB cycle enzymes of UWO 241 for indications of heat-lability indicated that glyceraldehyde-3- phosphate dehydrogenase may be the heat-limited enzyme in this pathway. These findings deepen our understanding of the reasons that this Antarctic alga is unable to grow at moderate temperatures. i Keywords Chlamydomonas sp. UWO 241, psychrophile, phylogenetics, heat stress, heat shock, Calvin cycle, molecular dynamics ii Co-Authorship Statement Chapter 2 was previously published in the Journal of Phycology with Rajesh K. Gupta, Dr. Beth Szyszka-Mroz, Dr. Denis P. Maxwell, Dr. Marc‐André Lachance, Dr. Norman P.A. Hüner and Dr. David R. Smith as co-authors. Rajesh Gupta provided scientific rationale, Dr. Szyszka-Mroz assisted with the collection of light-micrographs, Dr. Lachance provided scientific rationale and assisted with the phylogenetic analyses, Dr. Maxwell, provided funding and editorial comments, Dr. Hüner provided funding and assisted with the revisions of the manuscript, and Dr. Smith provided funding and helped write the manuscript. Chapter 3 was conceived of, performed by and written by Marc Possmayer with the following exception. Dr. Marina Cvetkovska and Nina Malczewski isolated protein, performed SDS-PAGE, Western Blotting and densitometry analysis for various Heat Shock Proteins in Chlamydomonas reinhardtii and C. sp. UWO 241 grown at steady-state temperatures. Dr. Marina Cvetkovska wrote methods text detailing these procedures. iii Acknowledgments To my supervisors, Drs. Denis Maxwell and Norman Hüner, I owe an immense debt of gratitude for their support, mentorship, scientific insights and contagious enthusiasm during my studies. Their consistent, patient confidence in me was essential in the performance of the work described in this thesis as was the inspiration I derived from their leadership. I believe they considerably exceeded the expectations of their role as supervisors. Thank-you Norm and Denis, you have my heartfelt thanks. I thank my advisory committee members, Drs. Robert Cumming, David R. Smith and Charlie G. Trick, for their encouragement and suggestions. In particular, David Smith was inspiring as senior author of Chapter 2 of this thesis, and Charlie Trick not only provided equipment used in experiments described here, but also ensured that the results were interpreted with a high level of scientific rigour. Fellow authors of the publications I have written and co-authored are due thanks for their contributions. Specifically, the spirit of inquiry and analytic expertise of Dr. Marc‐André Lachance was essential in motivating and producing Chapter 2 of this thesis. Fellow members, past and present, of the Maxwell and Hüner labs were invariably a pleasure to work with and helped create not only an atmosphere conducive to scientific inquiry but also one of excitement. Specifically, I thank Drs. Marina Cvetkovska, Alex Ivanov, Shanmugasundaram Pakkiriswami and Beth Szyszka-Mroz for their insights and comradery. I also thank current and former students of the Environmental Stress Biology Group of the Biology Department for sharing their expertise, enthusiasm and company. iv Table of Contents Abstract ................................................................................................................................ i Co-Authorship Statement................................................................................................... iii Acknowledgments.............................................................................................................. iv Table of Contents ................................................................................................................ v List of Tables ..................................................................................................................... ix List of Figures .................................................................................................................... xi List of Abbreviations ....................................................................................................... xvi Chapter 1 ............................................................................................................................. 1 1 General Introduction and Literature Review.................................................................. 1 1.1 Psychrophiles: Environment and Diversity ............................................................ 1 1.2 Characteristics of Psychrophiles ............................................................................. 2 1.2.1 Cellular Membranes .................................................................................... 2 1.2.2 Antifreeze Proteins and Compatible Solutes .............................................. 3 1.2.3 Cold-active Enzymes .................................................................................. 3 1.3 Photosynthetic Psychrophiles ................................................................................. 5 1.4 Psychrophilic Chlamydomonad Habitats and Diversity ....................................... 11 1.5 Characteristics of Chlamydomonad Psychrophiles .............................................. 11 1.6 Characteristics of Chlamydomonas sp. UWO 241 ............................................... 14 1.7 Phylogeny of UWO 241........................................................................................ 17 1.8 Evolution of Cold Adaptation and Psychrophily in Microorganisms ................... 18 1.9 Heat Stress ............................................................................................................ 19 1.9.1 Characteristics of the Heat Shock Response in C. reinhardtii .................. 19 1.9.2 Heat Shock and the Photosynthetic Apparatus ......................................... 23 1.9.3 Heat Stress and Heat Shock in Psychrophiles........................................... 24 v 1.10 Molecular Dynamics .......................................................................................... 25 1.11 Thesis objectives ................................................................................................ 26 1.12 References .......................................................................................................... 28 Chapter 2 ........................................................................................................................... 38 2 Resolving