Implications of leaf anatomy and stomatal responses in the Clusia genus for the evolution of Crassulacean Acid Metabolism 1 To those who believe in science as a tool for a better future 2 Declaration I hereby certify that this thesis is the result of my own investigations and that no part of it has been submitted for any degree other than the Doctor of Philosophy at the University of Newcastle upon Tyne. All references to the work of others are duly acknowledged. Victoria Andrea Barrera Zambrano 3 Table of Contents Acknowledgments ...................................................................................................... 11 Abbreviations ............................................................................................................. 12 Abstract ...................................................................................................................... 15 Chapter 1: Introduction .............................................................................................. 16 1.1 The Clusia genus .................................................................................................. 17 1.2 CAM evolution ..................................................................................................... 22 1.2.1 Evolution of CAM in Clusia .......................................................... 23 1.3 Crassulacean Acid Metabolism ............................................................................ 25 1.3.1 Carbohydrate metabolism and enzyme control in the CAM pathway 29 1.3.2 Circadian control and plasticity of the CAM pathway .................... 31 1.3.3 Water Use Efficiency .................................................................... 35 1.3.4 Leaf anatomy ................................................................................ 36 1.3.4.1 Stomatal size and density and functions ..................................................... 38 1.4 Photoreceptors ...................................................................................................... 40 1.4.1 Cryptochromes (cry) ..................................................................... 40 1.4.2 Phototropins (phot) ....................................................................... 42 1.4.3 Phytochromes (phy) ...................................................................... 44 1.4.4 Photoreceptors and the circadian clock .......................................... 46 1.5 Aims of thesis and hypotheses to be tested .......................................................... 47 Chapter 2: Functional leaf anatomy of Clusia ........................................................... 48 2.2 Materials and Methods ......................................................................................... 51 2.2.1 Plant Material ............................................................................... 51 2.2.2 Gas exchange profiles ....................................................................................... 55 2.2.3 Leaf anatomy and stomatal characteristics ....................................................... 55 2.2.4 Protein extraction and western blotting ............................................................. 58 4 2.2.4.1 Protein extraction ........................................................................................ 58 2.2.4.2 Discontinuous SDS – PAGE ....................................................................... 58 2.2.4.3 Western blotting .......................................................................................... 59 2.2.5 Immuno-labelling for PEPC and Rubisco in leaf sections ............... 60 2.2.6 Statistical Analysis ............................................................................................ 61 2.3.1 Gas exchange profiles ....................................................................................... 62 2.3.1.1 Degree of CAM and relationship to specific leaf area (SLA) and instantaneous water use efficiency (WUE) ............................................................. 62 2.3.2 Leaf Anatomy .................................................................................................... 69 2.3.2.1 Leaf tissue characteristics and implications for the degree of CAM .......... 69 2.3.2.2 Stomatal size and density and photosynthetic mode ................................... 78 2.3.3.1 Abundance of PEPC and Rubisco proteins ....................................... 85 2.4 Discussion ....................................................................................... 87 2.4.1 Leaf anatomy and implications for the degree of CAM in Clusia ................. 87 2.4.2 Anatomical traits and implications for internal CO2 conductance ................. 89 2.4.3 Localization of carboxylases .......................................................................... 90 2.4.4 Stomatal size and density and photosynthetic physiology in Clusia .............. 91 Chapter 3: Stomatal Responses to Light in C3 and CAM species of Clusia ............. 93 3.1 Introduction .......................................................................................................... 93 3.2 Materials and methods ......................................................................................... 96 3.2.1 Gas exchange under different light regimes ...................................................... 97 3.2.2 Speed of response of stomata to changes in light intensity ............................... 97 3.3 Results .................................................................................................................. 98 3.3.1 Gas exchange profiles under different light regimes ........................................ 98 3.3.2 Speed of response of stomata to light ............................................................. 107 3.3.3 Stomatal guard cell chloroplasts ..................................................................... 110 3.4 Discussion .......................................................................................................... 111 5 3.4.1. Circadian control of stomatal conductance is more robust in the CAM Clusia compared to the C3 Clusia ....................................................................................... 111 3.4.2. Circadian control of stomatal conductance is mediated via photosynthesis and by photoreceptors in Clusia ...................................................................................... 112 3.4.3. Water use efficiency is altered under constant light regimes in CAM Clusia (C. rosea) ........................................................................................................................ 115 3.4.4 Stomata of the CAM Clusia responded faster to changes in PFD imposed during the photoperiod compared to the C3 Clusia ............................................................. 116 Chapter 4: Circadian regulation of the expression of photoreceptors in a C3 and CAM species of Clusia ....................................................................................................... 118 4.1 Introduction ........................................................................................................ 118 4.2 Materials and methods ....................................................................................... 122 4.2.1 Plant material .................................................................................................. 122 4.2.2 Experimental set up: Light Treatments ........................................................... 122 4.2.3 Genes selection and primers design ................................................................ 122 4.2.4 RNA extraction ............................................................................................... 124 4.2.5 Sequencing of photoreceptors ......................................................................... 126 4.2.6 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) ...................... 127 4.2.7 Real time PCR ................................................................................................. 129 4.2.8 Titratable acidity ............................................................................................. 131 4.3 Results ................................................................................................................ 132 4.3.1 DNA sequences of photoreceptors .................................................................. 132 4.3.2 Gene expression .............................................................................................. 133 4.3.2.1 Phot1.......................................................................................................... 133 4.3.2.2 Phot2.......................................................................................................... 133 4.3.2.3 Cry 2 .......................................................................................................... 133 4.3.2.4 PhyA .......................................................................................................... 134 4.3.4 Real time PCR ................................................................................................. 139 4.3.5 Titratable acidity ............................................................................................. 141 6 4.4 Discussion .......................................................................................................... 144 4.4.1 Transcript abundance of phot1 and phot2 ....................................................... 144 4.4.2