GENE EXPRESSION PROFILING IN SINGLE CELL C4 AND RELATED PHOTOSYNTHETIC SPECIES IN SUAEDOIDEAE By RICHARD MATTHEW SHARPE A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY WASHINGTON STATE UNIVERSITY Program in Molecular Plant Sciences DECEMBER 2014 © Copyright by RICHARD MATTHEW SHARPE, 2014 All Rights Reserved 137 i i © Copyright by RICHARD MATTHEW SHARPE, 2014 All Rights Reserve To the Faculty of Washington State University: The members of the Committee appointed to examine the dissertation of RICHARD MATTHEW SHARPE find it satisfactory and recommend it be accepted. i i ______________________________ Gerald E. Edwards, PhD. Co-Chair ______________________________ Amit Dhingra, PhD. Co-Chair ______________________________ Thomas W. Okita, PhD. ii ACKNOWLEDGMENTS Gerald E. Edwards, Amit Dhingra, Thomas W. Okita, Sascha Offermann, Helmut Kirchhoff, Miroslava Herbstova, Robert Yarbrough, Tyson Koepke, Derick Jiwan, Christopher Hendrickson, Maxim Kapralov, Chuck Cody, Artemus Harper, John Grimes, Marco Galli, Mio Satoh-Cruz, Ananth Kalyanaraman, Katherine Evans, David Kramer, Scott Schaeffer, Nuria Koteyeva, Elena Voznesenskaya, National Science Foundation, Washington State University i i Program in Molecular Plant Sciences i iii GENE EXPRESSION PROFILING IN SINGLE CELL C4 AND RELATED PHOTOSYNTHETIC SPECIES IN SUAEDOIDEAE Abstract by Richard Matthew Sharpe, Ph.D. Washington State University December 2014 Co-Chair: Gerald E. Edwards Co-Chair: Amit Dhingra Slightly over a decade ago Suaeda aralocaspica, a higher land plant species that performs C4 photosynthesis in a single cell was discovered. Subsequent to this discovery three additional i species in the Bienertia genus, a sister clade to the Suaeda genus, were reported that perform the v C4 photosynthetic function in a single chlorenchyma cell. Since the discovery of these plants with a novel form of anatomy associated with photosynthesis, the genetic resources required for the advancement of knowledge of this phenomenon have been lacking. The goal of providing the genetic resources required to advance the knowledge of how these species attain the capability to perform C4 photosynthesis in a single cell has been the focus of this research. The advent and maturing of High Throughput Sequencing (HTS) technologies has allowed for the generation of the massive amount of genomic and transcriptomic sequence information required to provide the resources required to investigate the unique genetic landscape of these single cell C4 (SCC4) species. The state of current knowledge about the SCC4 species is provided as well as the use of HTS technologies to elucidate the transcriptomic landscape of the developing Bienertia sinuspersici leaf and a photosynthesis-centric transcriptome comparison iv between the different structural C4 and C3 type photosynthetic species in the Suaedoideae subfamily is detailed. The B. sinuspersici developmental profile indicates that the young leaf tissue devotes the majority of the transcriptional energy in cell division, transcription and regulation whereas the transcriptional energy in the mature tissue is focused towards maintenance of the photosynthetic processes. Differential translational and chloroplast import components between the tissues are quite evident as well. Species level photosynthetic comparisons indicated differential isoform recruitment into the various pathways. The identification and characterization of the induction and regulation of genes required to develop dimorphic chloroplasts in a single cell will enable efforts to instill C4 traits into C3 species. v v TABLE OF CONTENTS ACKNOWLEDGMENTS ...…………………………..............…………………………………iii ABSTRACT ...………………….……………...……………...……..…………………………..iv LIST OF TABLES …………………….…………………………………....…………….………x LIST OF FIGURES ………………………………….…………………………....…….……….xi SUPPLEMENTARY DATA….………………………………………………………..….…….xii CHAPTER ONE …………………………………………...…………………………..…………1 INTRODUCTION …………………………………………………………………….………….1 Conservation of Energy …………………………………………………………….…….1 Plant Research ……………………………………………………………………..……..2 Molecular Plant Research ………………………………………….……….…………….3 Photosynthesis ……………………………………………..……….…………………….4 v i Focus of Research ………………………………………………………………………..5 “One decade after the discovery of single-cell C4 species in terrestrial plants - What did we learn about the minimal requirements of C4 photosynthesis?” ……………………………………………………………………...…5 Summary………………………………………………………………………………..39 Chapter References ……………………………………………………………..………40 CHAPTER 2……………………………………………………………………………….……41 DEVELOPMENTAL TRANSCRIPTOMES OF THE SINGLE CELL C4 PHOTOSYNTHETIC TYPE PLANT BIENERTIA SINUSPERSICI Abstract …………………………………………………………………………………41 Introduction ……………………………………………………………………………..42 vi Results …………………………………………………………………………………..46 Read Quality, Trimming, Mapping and Overall Transcriptome Expression …..46 Read Trimming Parameters …………………………………………..…46 EST Gene Ontology ……………………………………………………..48 EST Gen Ontology Classifications ….…………………………………..50 Representation of ccGO ….….…………………………………………..52 Representation of mfGO ….….……………………………………….....54 Representation of bpGO ….….…………...……………………………..55 Discussion ……………………………………………………………………………….57 Materials and Methods …………………………………………………………………..62 Plant Material ……………………………………………………………………62 RNA Extraction …………………………………………………………………62 v Illumina Sequencing …………………………………………………………….63 i i 454 Sequencing ………………………………………………………………….64 Bioinformatics …………………………………………………………………..64 Initial Assembler Comparison …………………………………………..64 Data assembly …………………………………………………………...65 Annotation ……………………………………………..………………..65 References ……………………………………………………………………………….67 CHAPTER 3 …………………………………………………………………………………….81 COMPARATIVE TRANSCRIPTOMICS OF SINGLE CELL C4, KRANZ C4 AND C3 PHOTOSYNTHETIC TYPES IN SUAEDOIDEAE Abstract ………………………………………………………………………………....81 vii Introduction ……………………………………………………………………………..82 Results ……………………………………………………………….………………….85 Post-translational Components ………………………………….………………85 14-3-3 chaperones ……………………………………….………………85 HSP 70 …………………………………………………………………..86 Chloroplast Import ………………………………………………………………87 Photosynthetic Import TOC components ……………………………….87 Housekeeping Import TOC components ………………………………..88 C4 pathway components ………………………………………………………..89 Alternate C4 Biochemical Enzymes ……………………………………………91 Discussion ……………………………………………………………….………………91 Material and Methods ………………………………………………….….…………….96 v Plant Material …………………………………………………………………...96 i i Genome size estimates ………………………………………………………….96 i RNA Extraction ………………………………………………………...……….96 Illumina Sequencing …………………………………………………………….97 454 Sequencing ………………………………………………………………….98 Bioinformatics …………………………………………………………………………..98 Data Assembly …………………………………………………………..……...98 Annotation ………………………………………………………………………99 Proteomics and Transcriptomics Comparison …………………………………100 References …………………..………………………………………………………….101 CHAPTER 4…………………………………………………………………………...……….112 viii Conclusions and Future Perspectives…………………………………………………..112 References……………………………………………………………………...………114 i x ix LIST OF TABLES CHAPTER 2 Table 1. B. Sinuspersici Developmental Stage ESTs & Percentage of ESTs Per Fold Change ...72 Table 2. Gene Ontology Enrichment in young and mature Bienertia sinuspersici……………...74 Table 3. GO term enrichment and number of ESTs representing enriched GO term……………76 x x LIST OF FIGURES CHAPTER 1 Figure 1. Comparison of C3 and the two structural types of single-cell C4 photosynthesis ……27 Figure 2. Biochemistry of SCC4 species ………………………………………………..………28 CHAPTER 2 Figure 1. de novo Transcriptome Build Workflow ……………...………………………………71 Figure 2. B. Sinuspersici Developmental Stage ESTs & Percentage of ESTs Per Fold Change...73 Figure 3. Enriched GO term relationships……………………………………………………….75 Figure 4. Over-represented cellular component gene ontology distribution…………………….79 CHAPTER 3 Figure 1. 14-3-3 Identification and Expression Values………………………………….……..104 Figure 2. Heat Shock Protein Identity and RPKM Values………………….………….………105 x Figure 3. Photosynthetic Pre-protein Import TOC Component Expression…………..……….106 i Figure 4. Initial analysis of carbonic anhydrase isoforms…………………………….…….....108 Figure 5. Phosphoenolpyruvate carboxylase (PEPC) expression values………………………109 Figure 6. Transaminating and decarboxylation enzymes………………………………………110 Figure 7. Alternate C4 Biochemical Decarboxylases………………………………………….111 xi SUPPLEMENTARY DATA SUPPLEMENTARY DATA 1 Flow Cytometric Estimation of Nuclear DNA Content of B. sinuspersici Leaf Samples……...116 Supplementary Data 1 Table 1. Flow cytometry results of 16 B. sinuspersici replicates……...117 SUPPLEMENTARY DATA 2 Supplementary Data 2 Figure 1………………………………………………………………...121 Supplementary Data 2 Table 1. Bienertia sinuspersici top species blastx to blastn comparisons with e-values of 0.00……….…………………………………………………………………..122 Supplementary Data 2 Figure 2. Bienertia sinuspersici Gene Ontology Characterization…...123 References……………………………………………………………………………………..124 Supplementary Data 2 Table 2…………………………………………………………………125 Supplementary Data 2 Table 3…………………………………………………………………128 x ASSEMBLY OF DIFFERENT GENE STRUCTURES i i Pyruvate, orthophosphate dikinase……………………………………………………………..137 Supplementary Data 3 Figure 1. Suaedoideae species PPDK characterization……………......138 Supplementary Data 3 Figure 2. Serine-Glyoxylate Transaminase……………………………139 References………………………………………………………………………………………140 xii Chapter 1 Introduction Why would anyone think plant research is cool?