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MIAMI UNIVERSITY the Graduate School MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Isha Kalra Candidate for the Degree Doctor of Philosophy ______________________________________ Rachael Morgan-Kiss, Director ______________________________________ Xin Wang, Reader ______________________________________ Donald J. Ferguson, Reader ______________________________________ Annette Bollmann, Reader ______________________________________ Carole Dabney-Smith, Graduate School Representative ABSTRACT ROLE OF CYCLIC ELECTRON FLOW (CEF) AND PHOTOSYSTEM I (PSI) SUPERCOMPLEX FORMATION DURING ACCLIMATION TO LONG-TERM SALINITY STRESS IN GREEN ALGAE: A COMPARATIVE STUDY by Isha Kalra Photosynthesis is one of the most important processes on Earth by which organisms convert solar energy into usable forms of energy. Linear electron flow (LEF) and cyclic electron flow (CEF) constitute two major pathways in photosynthesis. While LEF leads to production of both ATP and NADPH, CEF only produces ATP that helps balance the ATP:NADPH ratio required for carbon fixation. CEF also plays a major role during acclimation to several environmental stressors. However, the regulation and mechanism by which CEF operates is still not clearly understood. Recent studies have shown that formation of a protein supercomplex with PSI appears to be essential for induction of CEF in several model organisms. However, both supercomplex formation and CEF induction have been mainly studied under short-term, transitory stress conditions. In addition, the role and mechanism by which organisms may rely on CEF to survive in their natural habitat and acclimate to stress over a long period of time has not been considered. In this study we compared how three photosynthetic organisms (one model alga, Chlamydomonas reinhardtii; two extremophiles, C sp. UWO241 and C. sp. ICE-MDV) utilize CEF to cope with their natural environment and adapt to steady-state environmental stress. To that end, the objectives of this thesis were i) to elucidate the role of CEF in long-term salinity acclimation ii) to understand the downstream changes associated with increased CEF, and iii) to identify whether PSI-supercomplexes are associated with increased CEF during salinity acclimation. We hypothesized that a stable PSI-supercomplex is required for high CEF, which in turn supports strong carbon fixation capacity for production of downstream metabolic products important for long-term acclimation to salinity stress. We showed for the first time, that increased CEF in UWO241 leads to excess ATP production and rewiring of downstream metabolism under high salinity. Next, we showed that a laboratory evolved salinity-tolerant strain of model C. reinhardtii uses constitutive upregulation of CEF to deal with salinity stress, which is in-turn associated with increased non-photochemical quenching and rewired carbon metabolism. Last, we show that CEF is involved in salinity acclimation in all three Chlamydomonas species, regardless of their salinity tolerance. We also show that PSI-supercomplexes are associated with increased CEF in these species. Characterization of high-salt supercomplex of C. reinhardtii revealed that it shares many similarities with the extensively described state 2 supercomplex, and that supercomplex composition might be species dependent rather than stress dependent. ROLE OF CYCLIC ELECTRON FLOW (CEF) AND PHOTOSYSTEM I (PSI) SUPERCOMPLEX FORMATION DURING ACCLIMATION TO LONG-TERM SALINITY STRESS IN GREEN ALGAE: A COMPARATIVE STUDY A DISSERTATION Presented to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Microbiology by Isha Kalra The Graduate School Miami University Oxford, Ohio 2021 Dissertation Director: Rachael Morgan-Kiss © Isha Kalra 2021 TABLE OF CONTENTS LIST OF TABLES VI LIST OF FIGURES VII DEDICATION VIII ACKNOWLEDGEMENTS IX CHAPTER I. INTRODUCTION 2 1.1 INTRODUCTION 2 1.2 PHOTOSTASIS AND ENVIRONMENTAL STRESS RESPONSE 3 1.3 CYCLIC ELECTRON FLOW 5 1.3.1 PSI-Supercomplex formation 6 1.4 EXTREMOPHILES 8 1.4.1 Psychrophilic phototrophs of McMurdo Dry Valleys, Antarctica 8 1.4.1.1 The model for psychrophilic photosynthesis: Chlamydomonas sp. UWO241 9 1.4.1.2 New phototroph from Lake Bonney: Chlamydomonas sp. ICE-MDV 10 1.5 OBJECTIVES OF THE THESIS 11 1.6 REFERENCES 16 CHAPTER II. CHLAMYDOMONAS SP. UWO241 EXHIBITS CONSTITUTIVELY HIGH CYCLIC ELECTRON FLOW AND REWIRED METABOLISM UNDER HIGH SALINITY. 34 2.1 INTRODUCTION 34 2.2 MATERIALS AND METHODS 36 2.2.1 Culture conditions, growth physiology. 36 2.2.2 Low temperature (77K) fluorescence spectra. 36 2.2.3 P700 oxidation-reduction and cyclic electron flow. 37 2.2.4 In vivo spectroscopy measurements. 37 2.2.6 Thylakoid isolation. 38 2.2.7 SDS-PAGE and Immunoblotting. 38 2.2.8 Supercomplex isolation. 38 2.2.9 Sample preparation for proteomics. 39 2.2.10 Proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) 40 2.2.11 Gas Chromatography - Mass Spectrometry. 41 2.3 RESULTS 42 2.3.1 UWO241 is adapted to low temperature and high salt. 42 iii 2.3.2 UWO241 possesses constitutively high rates of CEF. 42 2.3.3 Isolation of a PSI-supercomplex in UWO241. 43 2.3.4 Protein composition of the supercomplex. 44 2.3.5 Whole cell proteome analysis. 45 2.3.6. Primary metabolome analysis. 46 2.4 DISCUSSION 47 2.5 REFERENCES 64 2.6 APPENDIX 74 CHAPTER III. COORDINATED RESPONSE OF CYCLIC ELECTRON FLOW, PHOTORESPIRATION AND TRANSIENT STARCH SYNTHESIS IN A HIGH SALT-EVOLVED STRAIN OF CHLAMYDOMONAS REINHARDTII 102 3.1 INTRODUCTION 102 3.2 METHODS 106 3.2.1 Growth conditions and evolution of high salt evolved strain of C. reinhardtii 106 3.2.2 Experimental set-up 106 3.2.3 PSII measurements and oxygen evolution rates 107 3.2.4 P700 photo-oxidation and cyclic electron flow 107 3.2.5 Protein extraction and sample preparation for proteomics 108 3.2.6 Proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) 109 3.3 RESULTS 109 3.3.1 The high salinity evolved strain has faster growth rate 109 3.3.2 The evolved strain maintains high photosynthetic capacity and constitutive upregulated NPQ under low and high salinity 110 3.3.3 The evolved strain has higher oxygen evolution and respiration rates compared to the parent strain 111 3.3.4 The evolved strain displays constitutive high rates of PSI-CEF 111 3.3.5 Proteomic comparison of the evolved and the wild type strains under high salinity 112 3.3.5.1 Sub-cellular localization 112 3.3.5.2 Gene ontology 113 3.3.5.2 Kegg Orthology Biological Pathway 113 3.4 DISCUSSION 118 3.5 REFERENCES 153 iv 3.6 APPENDIX 167 CHAPTER IV. IMPACT OF SALINITY-TOLERANCE VERSUS-ACCLIMATION ON THE STRUCTURE AND FUNCTION OF THE PHOTOCHEMICAL APPARATUS: A COMPARATIVE STUDY 170 4.1 INTRODUCTION 170 4.2 METHODS: 174 4.2.1 Culture conditions, growth physiology. 174 4.2.2 State transition induction 174 4.2.3 Low temperature (77K) fluorescence spectra. 175 4.2.4 PSII fluorescence state transition measurement 175 4.2.5 SDS-PAGE and Immunoblotting. 175 4.2.6 P700 oxidation-reduction kinetics 176 4.2.7 Supercomplex isolation. 176 4.2.8 Sample preparation for proteomics. 177 4.2.9 Proteomic analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) 177 4.3 RESULTS 178 4.3.1 Salinity tolerance the three Chlamydomonas species 178 4.3.2 Photosystem I activity 179 4.3.3 Effect of long-term stress high salinity acclimation on short-term state transition response 180 4.3.4 Thylakoid protein phosphorylation 182 4.3.5 Assembly of protein supercomplexes under high salinity 183 4.3.6 Proteome analysis of high salinity-associated supercomplexes 185 4.4 DISCUSSION 186 4.5 REFERENCES 199 4.6 APPENDIX 213 CHAPTER V. CONCLUSION 216 v LIST OF TABLES 2.1 Components of UWO241 supercomplex under high salinity 53 3.1 Growth characteristics of Wildtype and Evolved strain under low and high salinity 136 3.2 Significantly regulated proteins of the Wildtype strain under high salinity 137 3.3 Significantly regulated proteins of the Evolved strain under high salinity 144 4.1 Major proteins involved in high salt and state 2 supercomplexes of C. reinhardtii 198 vi LIST OF FIGURES 1.1 Two modes of photosynthetic electron transport 14 1.2 Differences between stress, acclimation and adaptation 15 1.3 Model of East Lake Bonney 16 2.1 P700 re-reduction kinetics of UWO241 54 2.2 Photosynthetic properties of UWO241 using Electrochomic shift 55 2.3 Supercomplex formation in UWO241 vs C. reinhardtii 56 2.4 77K fluorescence spectra of protein complex of UWO241 and C. reinhardtii 57 2.5 Heatmap of proteins of UWO241 under low and high salinity 59 2.6 Changes in photosynthesis and metabolism of UWO241 under high salinity 61 2.7 Model of photosynthesis and metabolism of UWO241 under high salinity 62 3.1 Comparison of growth of different C. reinhardtii strains 125 3.2 PSII parameters of Wildtype and evolved strain under low and high salinity 126 3.3 Oxygen evolution and dark respiration rates of wildtype and evolved strain 127 3.4 P700 kinetics of wildtype and evolved strain of C. reinhardtii 128 3.5 Heatmap of proteins of Wildtype and evolved strain under high salinity 129 3.6 Analysis of significantly regulated proteins of Wildtype and Evolved strain 130 3.7 Categorization of differentially regulated proteins into gene ontology 131 3.8 Categorization of differentially regulated proteins into Kegg Orthology 132 3.9 Analysis of common proteins of Wt and Ev strain 133 3.10 Analysis of unique proteins of Wt and Ev strain 135 4.1 Growth curve under salinity gradient for the three Chlamydomonas species 192 4.2 P700 oxidation/reduction kinetics of the three Chlamydomonas species 193 4.3 State transition tests after acclimation to low and high salinity 194 4.4 Thylakoid phosphorylation pattern of the three Chlamydomonas species 195 4.5 Isolation of supercomplexes from conditions promoting CEF in Chlamydomonas species 196 4.6 Proteome comparison of supercomplex fractions from state 2 and high salt cultures 197 5.1 Changes in photosynthesis and metabolism after acclimation and adaptation 220 vii DEDICATION This thesis is dedicated to my younger brother and only sibling, Akshay, who was the light and the joy of my life.
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