RESOURCE USE EFFICIENCY OF C4 GRASSES WITH DIFFERENT EVOLUTIONARY ORIGINS Harshini Sugandika Uswattha Liyanange Pinto A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Degree Hawkesbury Institute for the Environment University of Western Sydney Australia SEPTEMBER 2015 This thesis is dedicated to my parents and beloved husband for their endless support and encouragement. ACKNOWLEDGEMENTS It is with great pleasure that I wish to express my utmost gratitude to my principal supervisor, Dr. Oula Ghannoum for her continuous encouragement, advice, and guidance. She has been a source of generosity, insight and inspiration; guiding me in all my efforts throughout my candidature. I owe my research achievements to her enthusiastic supervision. I acknowledge with great gratitude my co-supervisors Prof. David Tissue, Prof. Jann Conroy and Dr. Robert Sharwood who provided me with the unflinching encouragement, support and feedback during the candidature. Successful completion of this thesis would not have been possible without your invaluable insights and comments on my work. I am also thankful to Dr. Jeff Powell, Dr. Barbara Drigo (HIE-UWS), Dr. Pascal- Antoine Christin and Dr. Rebecca Atkinson (Sheffield University, UK) for their generous support in statistical analysis. I would like to thank Ms. Liz Kabanoff and Dr. Anya Salih (UWS) for their kind help and support on microscopy. I am also thankful to Dr. Kristine Crous and Dr. Craig Barton (UWS) for their assistance with the tuneable diode laser and carbon isotope discrimination measurements. I gratefully acknowledge the University of Western Sydney and the Hawkesbury Institute for the Environment for granting me the Australian Postgraduate Award. My gratitude also goes to staff at Hawkesbury Institute for the Environments, Dr. David Harland, Ms. Gillian Wilkins, Ms. Patricia Hellier, Mr. Gavin Mckenzie and Dr. Kaushal Tewari for their generous support on administrative and laboratory work. I wish to thank my colleagues Mr. Balasaheb Sonawane, Ms. Renee Smith, Ms. Josephine Ontedhu, Dr. Honglang Duan and Mr. Guomin Huang for their friendship and support throughout my candidature. As always, my heartfelt gratitude goes to my parents for their love and constant support throughout my life and for motivating me to pursue an academic career. Finally, my most sincere thanks go to my loving husband, Uthpala Pinto, who has been a shadow behind all my success throughout my life. STATEMENT OF AUTHENTICATION The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution Author’s Signature TABLE OF CONTENTS ACKNOWLEDGEMENTS .......................................................................................................................... I STATEMENT OF AUTHENTICATION ......................................................................................................... I TABLE OF CONTENTS .............................................................................................................................. I LIST OF FIGURES .................................................................................................................................... V LIST OF TABLES .................................................................................................................................... VII ABBREVIATIONS ................................................................................................................................... IX ABSTRACT ............................................................................................................................................. 1 CHAPTER 1 GENERAL INTRODUCTION ................................................................................................... 5 1.1 C3 PHOTOSYNTHETIC PATHWAY AND ITS INEFFICIENCIES ........................................................................... 6 1.2 EVOLUTION OF C4 PLANTS ................................................................................................................. 9 1.3 ANATOMY AND PHYSIOLOGY OF C4 BIOCHEMICAL SUBTYPES ................................................................... 12 1.3.1 NADP-ME subtype ................................................................................................................ 13 1.3.2 NAD-ME subtype .................................................................................................................. 14 1.3.3 PCK subtype .......................................................................................................................... 15 1.4 PHYSIOLOGY OF THE C3 AND C4 PATHWAYS ......................................................................................... 17 1.4.1 Water use efficiency and carbon isotope discrimination ...................................................... 19 1.4.2 Nitrogen use efficiency and Rubisco ..................................................................................... 22 1.5 PHYSIOLOGY OF THE C4 SUBTYPES ..................................................................................................... 24 1.5.1 Water use efficiency of the C4 subtypes ............................................................................... 24 1.5.2 Nitrogen use efficiency of the C4 subtypes ........................................................................... 25 1.6 GAPS IN THE LITERATURE ................................................................................................................. 27 1.7 AIMS AND OBJECTIVES OF THIS RESEARCH ........................................................................................... 29 1.8 FORMAT OF THE THESIS ................................................................................................................... 30 CHAPTER 2 PHOTOSYNTHETIC PHYSIOLOGY OF C4 GRASSES REFLECTS AT INTER-GLACIAL CO2 THE BIOCHEMICAL SUBTYPE AND EVOLUTIONARY ORIGIN .........................................................................32 ABSTRACT ................................................................................................................................................. 33 2.1 INTRODUCTION ............................................................................................................................. 34 2.2 MATERIALS AND METHODS ............................................................................................................. 38 2.2.1 Plant culture and water use measurements ......................................................................... 38 2.2.2 Gas exchange measurements ............................................................................................... 40 2.2.3 Rubisco, PEPC activity and soluble protein ........................................................................... 40 i 2.2.4 Growth and nitrogen analyses ............................................................................................. 41 2.2.5 WUE and NUE calculations ................................................................................................... 41 2.2.6 Statistical analysis ................................................................................................................ 42 2.3 RESULTS....................................................................................................................................... 44 2.3.1 Summary of the linear mixed effect (lme) statistical analysis .............................................. 44 2.3.2 Leaf water use efficiency ...................................................................................................... 44 2.3.3 Leaf N use efficiency ............................................................................................................. 45 2.3.4 Plant water and nitrogen use efficiency ............................................................................... 45 2.3.5 Activity and content of photosynthetic enzymes .................................................................. 46 2.3.6 The RDA for inter-glacial CO2 and ambient CO2 ................................................................... 47 2.3.7 The phylogenetic effect on species under inter-glacial CO2 and ambient CO2 ..................... 47 2.4 DISCUSSION .................................................................................................................................. 60 2.4.1 Variations in photosynthetic nitrogen use efficiency among C4 grasses are associated with the biochemical subtype .................................................................................................................... 60 2.4.2 Variations in photosynthetic water use efficiency among C4 grasses are associated with the evolutionary origin ............................................................................................................................ 61 2.4.3 Response of photosynthesis to inter-glacial [CO2] ................................................................ 62 2.4.4 Response of stomatal conductance to inter-glacial [CO2] .................................................... 63 2.4.5 Response of plant DM and WUE to inter-glacial [CO2] ......................................................... 64 2.5 CONCLUSIONS ..............................................................................................................................