Genetic Variation in Photosynthesis as a Tool for Finding Principal Routes to Enhancing Photosynthetic Efficiency A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Nicholas J. Tomeo August 2017 © 2017 Nicholas J. Tomeo. All Rights Reserved. 2 This dissertation titled Genetic Variation in Photosynthesis as a Tool for Finding Principal Routes to Enhancing Photosynthetic Efficiency by NICHOLAS J. TOMEO has been approved for the Department of Environmental and Plant Biology and the College of Arts and Sciences by David M. Rosenthal Assistant Professor of Environmental and Plant Biology Robert Frank Dean, College of Arts and Sciences 3 Abstract TOMEO, NICHOLAS J., Ph.D., August 2017, Environmental and Plant Biology Genetic Variation in Photosynthesis as a Tool for Finding Principal Routes to Enhancing Photosynthetic Efficiency Director of Dissertation: David M. Rosenthal Throughout this dissertation I approach the long-term aim of improving photosynthesis through the lens of natural genetic variation for photosynthesis. To date few studies have directly asked how photosynthetic variation might inform or provide the genetic material required to enhance photosynthesis, despite the clear utility of this strategy for other types of agronomic improvement. Of the many traits underling variation in photosynthesis, mesophyll conductance – the diffusional flux of CO2 through the leaf interior – has potential to improve both photosynthesis and water use efficiency. I assess genetic variation for photosynthesis among ecotypes of the model plant Arabidopsis thaliana and cultivars of soybean (Glycine max). In both species, and across both controlled and field environments in soybean, I find heritable genetic variation for mesophyll conductance that is positively correlated to variation in photosynthetic rate, indicating that selection to enhance mesophyll conductance will increase photosynthesis. Genetic variation in mesophyll conductance though was largely unrelated to variance in water use efficiency as a result of phenotypic correlation between stomatal and mesophyll conductance. If trait variation is to prove useful for crop breeding, that trait must not have already been improved in the varieties currently used by farmers. In soybean, photosynthesis has improved slightly with breeding for yield across a historical set of cultivars. Mesophyll conductance is not responsible for this increase in photosynthesis; it remains unchanged after 75 years of selection for yield. 4 Stomatal conductance is greater in modern varieties and I show that this increase scales from the leaf to the canopy. Greater canopy conductance in modern soybeans resulted in lower canopy temperatures and reduced leaf heat-stress. Few leaf-level photosynthetic traits were improved across this historical set of soybean cultivars. Given that I observed heritable genetic variation for mesophyll conductance among a small sampling of available soybean germplasm, there is substantial opportunity to harness this variation for the improvement of photosynthetic efficiency. 5 Dedication For her unwavering support and motivation, this work is dedicated to my wife Tiffany. 6 Table of Contents Page Abstract ............................................................................................................................. 3 Dedication ......................................................................................................................... 5 List of Tables ..................................................................................................................... 8 List of Figures .................................................................................................................... 9 Chapter 1. Background Material and Introduction to Proceeding Chapters .................... 12 Background ................................................................................................................. 12 Introduction to chapters ............................................................................................... 18 References .................................................................................................................. 19 Chapter 2. Natural Genetic Variation in Photosynthesis and Photorespiration Among Arabidopsis thaliana Ecotypes ........................................................................................ 29 Abstract ....................................................................................................................... 29 Introduction .................................................................................................................. 30 Hypotheses and experimental overview .................................................................. 35 Methods ....................................................................................................................... 36 Plant material ........................................................................................................... 36 Gas exchange and associated measurements ....................................................... 38 Analysis of gas exchange data ................................................................................ 40 Statistical analysis ................................................................................................... 44 Results ......................................................................................................................... 44 Discussion ................................................................................................................... 48 Genetic variation in photosynthetic physiology ........................................................ 48 Coordination of photosynthetic drivers .................................................................... 49 Correlation of photosynthesis and photorespiration ................................................ 50 Drivers of variation in water use efficiency .............................................................. 51 Conclusions ................................................................................................................. 54 References .................................................................................................................. 55 Chapter 3. Mesophyll Conductance Among Soybean Cultivars Sets a Tradeoff Between Photosynthesis and Water-Use ....................................................................................... 78 Abstract ....................................................................................................................... 78 Introduction .................................................................................................................. 79 Results ......................................................................................................................... 83 Physiological and structural trait variation among cultivars ..................................... 85 Trait correlations with gm .......................................................................................... 86 Trait correlations were consistent between experiments ......................................... 89 Trait coordination ..................................................................................................... 89 Discussion ................................................................................................................... 91 Conclusions ................................................................................................................. 97 Methods ....................................................................................................................... 98 Controlled environment experiment ......................................................................... 98 7 Field experiment ...................................................................................................... 98 Gas exchange measurements ................................................................................. 99 AN-Ci curves ........................................................................................................... 101 Variable-J gm calculation ........................................................................................ 102 Leaf morphology .................................................................................................... 104 Statistical analysis ................................................................................................. 105 References ................................................................................................................ 107 Chapter 4. Physiological Changes Across a Selection of Historical Soybean Cultivars 132 Abstract ..................................................................................................................... 132 Introduction ................................................................................................................ 133 Methods ..................................................................................................................... 136 Results ....................................................................................................................... 145 Discussion ................................................................................................................. 147 Conclusions