Identification of Proteins Involved in Salinity Tolerance in Salicornia bigelovii by Octavio Rubén Salazar Moya In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy of Science King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia November, 2017 2 EXAMINATION COMMITTEE PAGE The dissertation of Octavio Rubén Salazar Moya is approved by the examination committee. Committee Chairperson: Mark Tester Committee Members: Enrico Martinoia, Heribert Hirt, and Timothy Ravasi 3 COPYRIGHT PAGE © November, 2017 Octavio Rubén Salazar Moya All Rights Reserved 4 ABSTRACT With a global growing demand in food production, agricultural output must increase accordingly. An increased use of saline soils and brackish water would contribute to the required increase in world food production. Abiotic stresses, such as salinity and drought, are also major limiters of crop growth globally - most crops are relatively salt sensitive and are significantly affected when exposed to salt in the range of 50 to 200 mM NaCl. Genomic resources from plants that naturally thrive in highly saline environments have the potential to be valuable in the generation of salt tolerant crops; however, these resources have been largely unexplored. Salicornia bigelovii is a plant native to Mexico and the United States that grows in salt marshes and coastal regions. It can thrive in environments with salt concentrations higher than seawater. In contrast to most crops, S. bigelovii is able to accumulate very high concentrations (in the order of 1.5 M) of Na+ and Cl- in its photosynthetically active succulent shoots. Part of this tolerance is likely to include the storage of Na+ in the vacuoles of the shoots, making S. bigelovii a good model for understanding mechanisms of Na+ compartmentalization in the vacuoles and a good resource for gene discovery. In this research project, phenotypic, genomic, transcriptomic, and proteomic approaches have been used for the identification of candidate genes involved in salinity tolerance in S. bigelovii. The genomes and transcriptomes of 5 three Salicornia species have been sequenced. This information has been used to support the characterization of the salt-induced transcriptome of S. bigelovii shoots and the salt-induced proteome of various organellar membrane enriched fractions from S. bigelovii shoots, which led to the creation of organellar membrane proteomes. Yeast spot assays at different salt concentrations revealed several proteins increasing or decreasing yeast salt tolerance. This work aims to create the basis for Salicornia research by providing a genome, transcriptomes, and organellar proteomes, contributing to salinity tolerance research overall. We identified a set of candidate genes for salinity tolerance with the aim of shedding some light on the mechanisms by which this plant thrives in highly saline environments. 6 ACKNOWLEDGEMENTS I would like to thank Prof. Mark Tester for accepting me in his lab when I was facing the unfortunate situation of my previous supervisor having to leave the university. Without his support, I would not have been able to continue my career in my field of interest. I am truly grateful to have received this lifeline. Of course, I also appreciate all the time we spent together and the discussions that we had. I would also like to thank Dr. Sandra Schmöckel for her assistance and support throughout my Ph.D., she basically acted as my co-supervisor. All the discussions that we had, helped us shape the project. I thank the support of all the “Salties”, particularly to all the students whose conversations always helped to get a smile back into my face. I would like to thank Prof. Nina Fedoroff, she was the reason why I joined KAUST and she started the project with Salicornia. Dr. Suhail Khan was also involved at the start of the Salicornia project and we spent time working together. Dr. Alain Lesimple taught me and worked with me in analytical chemistry, even while he was facing a difficult time. Dr. Muppala Reddy was in charge of Salicornia growth in the greenhouse and helped me understand more about Salicornia. My undergraduate student José Otero, for spending all those long nights in the lab working with me. All the lab support team, who make all the work in the laboratory possible. All the friends that I made in KAUST were very important for me. DooLi Kim, for all her unconditional support during the hard times. Of course, to my committee members for all their time and input while 7 assessing my research project. I would like to thank KAUST for giving me the opportunity to study and fund my research. I would like to thank my family members, who have been always with me. My mother, who has always done her best to support me in any way possible. I would not have been able to do this Ph.D. without all the sacrifices that she has done during her life for me. Thank you, mother, you deserve all the credit. To my aunt, who basically has acted as a second mother throughout my life. You have always supported me and I am very happy that you have looked out for me throughout my life. I also hope this will encourage my cousin, Raúl, whatever you decide to do in life, give it everything. To all my friends back in my country, for their constant support, despite being afar, every time we meet is as if not a single day has passed. There are many people who assisted me and is impossible to mention every single person, but I am grateful to all of you. 8 TABLE OF CONTENTS EXAMINATION COMMITTEE PAGE ....................................................................... 2 COPYRIGHT PAGE ....................................................................................................... 3 ABSTRACT ....................................................................................................................... 4 ACKNOWLEDGEMENTS ............................................................................................ 6 LIST OF ABBREVIATIONS ........................................................................................ 12 LIST OF GENES AND PROTEINS ............................................................................ 17 LIST OF ILLUSTRATIONS ......................................................................................... 20 LIST OF TABLES ........................................................................................................... 24 Chapter 1. Literature review ....................................................................................... 27 1.1 Introduction ..................................................................................................... 27 1.2 Salinity tolerance mechanisms of land plants ............................................. 32 1.2.1 Shoot ion independent toxicity .................................................................. 33 1.2.2 Shoot ion toxicity ......................................................................................... 37 1.3 The role and mechanisms of Na+ accumulation in the vacuoles .............. 41 1.3.1 Ion transport across the tonoplast ............................................................. 42 1.3.2 Proteins involved in Na+ accumulation in vacuoles............................... 45 1.4 Salicornia as a model plant for understanding salinity tolerance ............. 56 1.4.1 Salinity tolerance in Salicornia .................................................................... 58 1.4.2 Evidence of vacuolar Na+/H+ antiporter activity in Salicornia ............. 61 1.5 Aim of the project ............................................................................................ 64 Chapter 2. Genome assembly and annotation of Salicornia species....................... 66 9 2.1 Introduction ..................................................................................................... 66 2.2 Materials and methods ................................................................................... 69 2.2.1 Plant material, library preparation, and genome sequencing ............... 69 2.2.2 Read trimming and genome assembly ..................................................... 71 2.2.3 Genome selection, completeness, and annotation .................................. 72 2.3 Results and discussion .................................................................................... 73 2.3.1 Genome sequencing .................................................................................... 73 2.3.2 Genome assembly using Illumina reads .................................................. 76 2.3.3 Salicornia bigelovii genome assembly using SMRT sequencing and hybrid assemblies .................................................................................................... 88 2.4 Conclusion ........................................................................................................ 90 Chapter 3. Salicornia bigelovii phenotype under salinity ......................................... 92 3.1 Introduction ..................................................................................................... 92 3.2 Materials and methods ................................................................................... 94 3.2.1 Plant material and growth .......................................................................... 94 3.2.2 Plant ion content and phenotype .............................................................. 95 3.3 Results and discussion ...................................................................................