Leaf anatomy and photosynthesis: Unravelling the CO diffusion pathway in C leaves 2 3 Herman Nicolaas Cornelis Berghuijs Thesis committee Promotors Prof. Dr P.C. Struik Professor of Crop Physiology Wageningen University Prof. Dr B.M. Nicolaï Professor of Biosystems Engineering KU Leuven, Belgium Co-promotor Dr X. Yin Senior scientist, Centre for Crop Systems Analysis Wageningen University Other members Prof. Dr J. Molenaar, Wageningen UR Prof. Dr B.M. Mulder, Wageningen UR Prof. Dr M. de Proft, KU Leuven, Belgium Prof. Dr W. Saeys, KU Leuven, Belgium This research was conducted under the joined auspices of the C.T. de Wit Graduate School for Production Ecology and Resource Conservation and the Arenberg Doctoral School. Leaf anatomy and photosynthesis: Unravelling the CO diffusion pathway in C leaves 2 3 Herman Nicolaas Cornelis Berghuijs Thesis submitted in fulfilment of the requirements for the joint degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.P.J. Mol and doctor in de bio-ingenieurswetenschappen van de KU Leuven by the authority of the Director of the Arenberg Doctoral School Prof. Dr G. Govers in the presence of the thesis committee appointed by the Academic Board of Wageningen University and the Director of the Arenberg Doctoral School of the KU Leuven to be defended in public on Wednesday May 25, 2016 at 1:30 p.m. in the Aula, Wageningen University Berghuijs, H.N.C. Leaf anatomy and photosynthesis; unravelling the CO diffusion pathway in C leaves 2 3 Joint PhD thesis, Wageningen University, Wageningen, NL; KU Leuven, Leuven, BE (2016), 286 pages With references, with summaries in English and Dutch DOI: 10.18174/379249 ISBN: 978-94-6257-794-7 Abstract Herman Nicolaas Cornelis Berghuijs (2016). Leaf anatomy and photosynthesis; unravelling the CO diffusion pathway in C leaves. PhD thesis. Wageningen 2 3 University, Wageningen, The Netherlands, with summaries in English and Dutch. 286 pages Optimizing photosynthesis can contribute to improving crop yield, which is necessary to meet the increasing global demand for food, fibre, and bioenergy. One way to optimize photosynthesis in C plants is to enhance the efficiency of CO transport 3 2 from the intercellular air space to Rubisco. The drawdown of CO between these 2 locations is commonly modelled by Fick's first law of diffusion. This law states that the flux from the air spaces to Rubisco is proportional to the difference in partial pressure between these locations. The proportionality constant is the mesophyll conductance. Its inverse is mesophyll resistance. Mesophyll resistance is a complex trait, which lumps various structural barriers for CO transport and processes that add 2 or remove CO along the diffusion pathway. In order to better understand how and to 2 what extent these factors affect photosynthesis, it is necessary to find a more mechanistic description of CO transport in the mesophyll. The aim of this dissertation 2 is to investigate how leaf anatomical properties and CO sources and sinks along the 2 CO diffusion pathway in C leaves affect the photosynthetic capacity of these leaves. 2 3 In this study, Solanum lycopersicum was used as a model organism. In a first approach, we developed a model in which we partitioned mesophyll resistance into two sub-resistances. The model assumed that CO produced by respiration and 2 photorespiration was released between the two sub-resistance components. By quantifying these resistances using measured thicknesses, exposed mesophyll and chloroplast surfaces, and assumed diffusive properties, we were able to simulate the effect of various anatomical properties on photosynthesis. A disadvantage of this two- resistance approach is that it assumes either that (photo)respiratory CO release takes 2 place in the outer cytosol or that there is no CO gradient in the cytosol. Therefore, in 2 a second approach we modelled CO transport, production and consumption by use of 2 a reaction-diffusion model. This model is more flexible in terms of determining the v Abstract location of CO sources and sinks. We developed methods to estimate physiological 2 parameters of this model using combined gas exchange and chlorophyll fluorescence measurements on leaves. The results suggest that the rate of respiration depends on the oxygen partial pressure, which is often not considered in previous photosynthesis models. We also presented a method to calculate the fraction of (photo)respiratory CO that is re-assimilated. We found that this fraction strongly depends on both 2 environmental factors (CO , irradiance), the location of mitochondria relative to the 2 chloroplast, stomatal conductance and various physiological parameters. The reaction- diffusion model and associated methods presented in this study provide a more mechanistic framework to describe the CO diffusion pathway in C leaves. This 2 3 model could, therefore, contribute to identifying targets to increase mesophyll conductance in future research. Keywords: CO diffusion, C photosynthesis, mesophyll conductance, mesophyll 2 3 resistance, re-assimilation, photorespiration, respiration, tomato vi Table of contents Chapter 1 General introduction 1 Chapter 2 Reaction-diffusion models extend our understanding of C leaf 13 3 photosynthesis: opportunities and challenges. Chapter 3 Modelling the relationship between CO assimilation and leaf 45 2 anatomical properties in tomato leaves Chapter 4 Localization of (photo)respiration and CO re-assimilation in 95 2 tomato leaves investigated with a reaction-diffusion model Chapter 5 Quantitative analysis of the effects of environmental and 145 physiological factors on the re-assimilation of (photo)respired CO , 2 using a reaction-diffusion model Chapter 6 General discussion 221 References 245 Summary 259 Samenvatting 267 Acknowledgements 275 Curriculum vitae 281 List of publications 283 PE&RC Training and Education Statement 285 Funding 286 CHAPTER 1 General introduction Chapter 1 1.1 Introduction Photosynthesis can be defined as the process in which light energy is converted into chemical energy (Reece et al., 2011). This process is of vital importance for life on Earth; photosynthesis allows phototrophic organisms to convert sun light and inorganic carbon into biomass. More specifically, in green plants, photosynthesis refers to the conversion of CO from the atmosphere into sugars and other organic 2 compounds. This assimilation of CO consumes energy. Green plants obtain this 2 energy by the absorption of photosynthetically active radiation (PAR). Understanding the mechanisms of photosynthesis in green plants is of interest from an agronomical perspective. In 2009, the Food and Agricultural Organization (FAO) expected the global population to increase by 34% to 9 billion people in 2050 (FAO, 2009a). In order to fulfil this global demand for more food and production due to the growing world population, the FAO estimated that the global food production had to increase by 70% from 2009 to 2050 to meet the global demand for food, feed, and fibres (FAO, 2009b). This can be achieved in two ways; using larger areas of land for crop production or increasing the efficiency of the production process (Ort et al., 2015). Increasing of the efficiency of the process can be done by increasing the efficiency of light absorption by crops, by increasing the conversion of absorbed light energy into biomass and by increasing the harvest index (Long et al., 2006). During the second half of the 20th Century, there have already been major improvements in increasing the efficiency of the production process. Between 1960 and 2005, the global food production has been increased by 160%, while the total area of cropland has only increased by 27% (Burney et al., 2010). This increase of the global food production can mainly be explained by the increase in harvest index. Although there is some potential to further increase the efficiency of light absorption and the harvest index, the scope of possibilities to further improve these is very limited (Long et al., 2006). Therefore, further increase of crop yield can mainly be achieved by increasing the conversion efficiency of absorbed light into biomass; i.e. by optimizing photosynthesis. Zhu et al. (2010) identified several possibilities to further increase the efficiency of photosynthesis. These possibilities include alterations at the canopy level, 2