Role of Plastid Terminal Oxidase (PTOX) as a safety valve for electrons in Hordeum vulgare (Barley) plants. A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in Plant Sciences in the Faculty of Science and Engineering and 2018 Mariela P. Aguilera Miranda School of Earth and Environmental Sciences Table of Contents List of figure .................................................................................................... 7 List of tables ................................................................................................. 10 Abbreviations ................................................................................................ 11 Abstract ........................................................................................................ 14 Declaration ................................................................................................... 15 Copyright statement ...................................................................................... 16 Acknowledgements ....................................................................................... 18 Chapter 1 ......................................................................................................... 19 General Introduction ......................................................................................... 19 Introduction ................................................................................................... 20 1. Photosynthesis ...................................................................................... 21 1.1 Light reactions ..................................................................................... 21 1.2 The Calvin Benson Bassham cycle ..................................................... 23 2. Reactive Oxygen Species production .................................................... 26 3. Mitigation of ROS through the antioxidant system ................................. 27 3.1 ROS scavenging enzymatic systems ................................................... 28 3.2 ROS scavenging non enzymatic system ............................................. 32 4. Prevention of ROS production ............................................................... 33 4.1 Non-photochemical quenching (NPQ) ................................................. 33 4.2 Cyclic electron transport (CET) ............................................................ 34 4.3 Mehler reaction ................................................................................... 35 2 5. The Plastid Terminal Oxidase (PTOX) ................................................... 35 5.1 Discovery of PTOX .............................................................................. 36 5.2 PTOX role in the carotenoid biosynthesis ............................................ 38 5.3 Chloroplast biogenesis ........................................................................ 41 5.4 Chlororespiration ................................................................................. 41 5.5 PTOX as a safety valve for electron transport ..................................... 42 5.6 PTOX localization and characterization ............................................... 43 5.7 PTOX activity regulation ...................................................................... 45 6. Aims and Objectives .............................................................................. 46 Chapter 2 ......................................................................................................... 48 Role of Plastid Terminal Oxidase (PTOX) as alternative electron sink in Hordeum vulgare .............................................................................................. 48 Introduction ................................................................................................... 49 Materials and Methods.................................................................................. 53 Plant growth .............................................................................................. 53 Measuring gas exchange .......................................................................... 53 Measuring chlorophyll fluorescence and electron transport to oxygen. ...... 53 Chlorophyll content ................................................................................... 56 Immunoblot analysis.................................................................................. 56 Reverse transcription polymerase chain reaction (rt-PCR) ........................ 58 Specific leaf area (SLA) ............................................................................. 59 Statistical analysis ..................................................................................... 59 Results ......................................................................................................... 60 3 Temperature and oxygen effects on P700 and chlorophyll fluorescence parameters ................................................................................................ 60 Effect of light intensities on chlorophyll fluorescence and P700 oxidation parameters across different temperatures and oxygen concentrations. ..... 62 PSII electron transport is sensitive to Plastid Terminal Oxidase inhibitor n- Propyl Gallate............................................................................................ 66 Characterization of developmental acclimation of H. vulgare at two different grown temperature, 10°C and 20°C. .......................................................... 70 Temperature and oxygen effects on chlorophyll fluorescence and P700 parameters of plants grown at 10 ºC ......................................................... 71 Effect of light intensity on chlorophyll fluorescence and P700 parameters across different temperatures and two O2 concentrations in plants grown at 10°C .......................................................................................................... 75 Barley developmental acclimation to cold does not induce Plastid Terminal Oxidase electron sink activity .................................................................... 77 Discussion .................................................................................................... 79 Conclusion .................................................................................................... 85 Chapter 3 ......................................................................................................... 86 Role of Plastid Terminal Oxidase (PTOX) as alternative electron sink in water restricted and salt-treated Hordeum vulgare plants .......................................... 86 Introduction ................................................................................................... 87 Materials and Methods.................................................................................. 91 Plant growth .............................................................................................. 91 Measuring gas exchange .......................................................................... 91 Measuring chlorophyll fluorescence and electron transport to oxygen. ...... 91 4 Chlorophyll content ................................................................................... 94 Immunoblot analysis.................................................................................. 94 Quantitative polymerase chain reaction (Q-PCR) ...................................... 96 Specific leaf area (SLA) ............................................................................. 97 Relative Water Content (RCW) ................................................................. 97 Statistical analysis ..................................................................................... 98 Results ......................................................................................................... 99 Characterization of acclimation of H. vulgare to water limitation or salinity 99 Effect of light intensity on chlorophyll fluorescence and P700 oxidation parameters at two O2 concentration in water restricted plants. ................ 103 PSII electron transport is sensitive to n-propyl gallate (n-PG), a Plastid Terminal Oxidase inhibitor, in water restricted plants ............................... 105 Effect of light intensities on chlorophyll fluorescence and P700 oxidation parameters at two O2 concentration in salt treated plants. ....................... 108 PSII electron transport is sensitive to n-propyl gallate (n-PG), a Plastid Terminal Oxidase inhibitor, in plants watered with 150 mM NaCl ........... 111 Discussion .................................................................................................. 114 Conclusion .................................................................................................. 119 Chapter 4 ....................................................................................................... 120 Dynamic responses of Plastid Terminal Oxidase (PTOX) to changing environmental conditions in Hordeum vulgare plants ...................................... 120 Introduction ................................................................................................. 121 Materials and Methods................................................................................ 124 Plant growth ............................................................................................ 124 5 Measuring chlorophyll fluorescence and electron transport