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Identification of a Cis-Regulatory Module for Bundle Sheath-Specific Gene Expression and Transcriptome Analysis of the Chilling Response of the C4 Grass Zea Mays

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Identification of a Cis-Regulatory Module for Bundle Sheath-Specific Gene Expression and Transcriptome Analysis of the Chilling Response of the C4 Grass Zea Mays Identification of a cis-regulatory module for bundle sheath-specific gene expression and transcriptome analysis of the chilling response of the C4 grass Zea mays Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Sandra Kirschner aus Neuss Düsseldorf, August 2017 aus dem Institut für Entwicklungs- und Molekularbiologie der Pflanzen der Heinrich-Heine-Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. Peter Westhoff Korreferentin: Prof. Dr. Maria von Korff Schmising Tag der mündlichen Prüfung: 09.11.2017 Erklärung Ich versichere an Eides Statt, dass die Dissertation von mir selbstständig und ohne unzulässige fremde Hilfe unter Beachtung der “Grundsätze zur Sicherung guter wissenschaftlicher Praxis an der Heinrich-Heine-Universität Düsseldorf“ erstellt worden ist. Die Dissertation habe ich in der vorgelegten oder in ähnlicher Form noch bei keiner anderen Institution eingereicht. Ich habe bisher keine erfolglose Promotionsversuche unternommen. Neuss, 04.08.2017 Sandra Kirschner Contents I Contents I. Introduction ................................................................................................... 1 1. C4 photosynthesis .............................................................................................................. 1 1.1. The dark side of Rubisco.......................................................................................................... 1 1.2. C4 plants have a distinct anatomy and concentrate CO2 around Rubisco to supress photorespiration .................................................................................................................................. 2 1.3. The benefits of the CO2 concentrating mechanism .................................................................. 5 1.4. The convergent evolution of C4 photosynthesis ....................................................................... 9 1.5. A model for the step-by-step evolution of C4 photosynthesis ................................................ 10 2. Cis-regulatory modules for BS- and M-specific expression ........................................ 14 2.1. Implementation of the C4 cycle in C3 plants .......................................................................... 14 2.2. Modules conferring BS- and M-specific gene expression on diverse regulatory levels ........ 15 2.3. More modules for C4 expression patterns are required .......................................................... 19 3. C4 photosynthesis and temperature............................................................................... 20 3.1. C4 crop plants are often susceptible to low temperatures ....................................................... 20 3.2. Chilling susceptibility in relation to photosynthetic enzymes ............................................... 21 3.3. Low temperature tolerance of C4 grasses ............................................................................... 22 3.4. The need for chilling tolerant crops ....................................................................................... 24 II. Scientific Aims ............................................................................................. 25 III.A Summary ................................................................................................... 26 III.B Zusammenfassung .................................................................................... 28 IV. Literature ..................................................................................................... 30 V. Manuscripts ................................................................................................. 39 1. Sandra Kirschner, Helen Woodfield, Katharina Prusko, Maria Koczor, Udo Gowik, Julian M. Hibberd, Peter Westhoff (2017). The SULTR2;2 promoter from Arabidopsis thaliana contains a positive regulator for gene expression in the bundle sheath. In progress. ............................................................................................................................ 40 2. Transcriptome analysis of maize subjected to chilling stress ................................. 73 VI. Addendum .................................................................................................. 123 1. Bundle sheath cells are more susceptible to chilling than mesophyll cells .......... 124 2. ke-1 contains a functional mitochondrial target sequence .................................... 134 VII. Acknowledgements ................................................................................................ 144 Abbreviations II Abbreviations °C Degree Celsius 3-PGA 3-Phosphoglycerate 3’/5’UTR 3’/5’ Untranslated region 5’RACE Rapid amplification of 5' complementary DNA ends A Rate of CO2 uptake A. thaliana Arabidopsis thaliana ABA Abscisic Acid ACR8 ACT domain repeat 8 ARF Auxin response factor Asat Light-saturated rate of CO2 uptake ATP Adenosine triphosphate AuxRE Auxin response element bp Base pairs BS Bundle sheath bZIP Basic leucine zipper C2, C3, C4 Two-, three-, four-carbon molecule CA Carbonic anhydrase CAM Crassulaceaen acid metabolism CaMV35S 35S promoter of the cauliflower mosaic virus CBP Calmodulin-binding protein cDNA Complementary DNA CDS Coding sequence cm Centimetre cM Centimorgan CO2 Carbon dioxide Col-0 Columbia-0 Cyt f Cytochrome f DAT Days after transplanting dH2O Distilled water DNA Desoxyribonucleic acid DNase Desoxyribonuclease DR Distal region of Mem1 Abbreviations III DST element Downstream element DW Dry weight ETIF3SU4 Eukaryotic translation initiation factor 3 subunit 4 F. bidentis/Fb Flaveria bidentis F. pringlei/Fp Flaveria pringlei F. trinervia/Ft Flaveria trinervia FDR False discovery rate G. gynandra/Gg Gynandropsis gynandra GDC Glycine decarboxylase complex GFP Green fluorescent protein GLDPA Glycine decarboxylase PA gene of Flaveria trinervia GO Gene ontology GSH Glutathione GST Glutathione S-transferase GUS β-glucuronidase h Hour(s) H2B Histone 2B H2O2 Hydrogen peroxide – HCO3 Bicarbonate HSF Heat shock factor Hz Hertz kb Kilo base pairs kcat Catalytic turnover rate kDa Kilodalton ke-1 Kühlempfindlich-1 Km Michaelis constant l Litre LSU Large subunit of Rubisco LT50 50 % rhizome mortality M Mesophyll M x g Miscanthus x giganteus MAP Mitogen activated protein Mb Mega base pairs MDH Malate dehydrogenase Abbreviations IV ME Malic enzyme Mem1/2 Mesophyll expression module 1/2 MeSA Methyl salicylate mg Milligram min Minute(s) MJ Megajoule ml Millilitre mRNA Messenger ribonucleic acid Mt Megatonne MU 4-methylumbelliferone MUG 4-methylumbelliferone β-D-glucuronide my Million years mya Million years ago N. benthamiana Nicotiana benthamiana NAD Nicotinamide adenine dinucleotide NAD-ME Nicotinamide adenine dinucleotide-malic enzyme NADP Nicotinamide adenine dinucleotide phosphate NADP-MDH Nicotinamide adenine dinucleotide phosphate-malate dehydrogenase NADP-ME Nicotinamide adenine dinucleotide phosphate-malic enzyme NADPH Nicotinamide adenine dinucleotide phosphate, reduced form NH3 Ammonia NIL Near isogenic line nm Nanometre nM Nanomolar NUE Nitrogen use efficiency O2 Molecular oxygen OAA Oxalacetate PCA Principal component analysis pCO2 Carbon dioxide partial pressure PCR Polymerase chain reaction PEP Phosphoenolpyruvate PEP-CK Phosphoenolpyruvate carboxykinase PEPC Phosphoenolpyruvate carboxylase PFD Photon flux density Abbreviations V PG Phosphoglycolate pH Potential of hydrogen PIP Plasma membrane intrinsic protein PNUE Photosynthetic nitrogen use efficiency ppcA Phosphoenolpyruvate carboxylase gene A of Flaveria PPDK Pyruvate, orthophosphate dikinase ppm Parts per million PPFD Photosynthetic photon flux density PR Proximal region of Mem1 PSII Photosystem II qRT-PCR Quantitative real-time polymerase chain reaction QTL Quantitative trait locus R2 Coefficient of determination RNA Ribonucleic acid RNase Ribonuclease ROS Reactive oxygen species RPKM Reads per million mapped reads and kb transcript length Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase RuBP Ribulose-1,5-bisphosphate RUE Radiation use efficiency s Second(s) S. pectinata Spartina pectinata SAUR SMALL AUXIN-UP RNA SDS-PAGE Sodium dodecyl sulfate polyacrylamid gel electrophoresis SULTR2;2 Sulfate transporter gene 2;2 TCA Tricarboxylic acid TEL50 50 % electrolyte leakage THI Thiamine thiazole synthase TSS Transcription start site Vmax Substrate saturated enzyme activity WUE Water use efficiency YFP Yellow fluorescent protein Z. mays/Zm Zea mays θ Light response curve Abbreviations VI µg Microgram µl Microliter Φ Maximum quantum efficiency ΦCO2 Efficiency of CO2 assimilation Nucleobases of the DNA A Adenine C Cytosine G Guanine T Thymine Amino acids A Alanine M Methionine C Cysteine N Asparagine D Aspartic acid P Proline E Glutamic acid Q Glutamine F Phenylalanine R Arginine G Glycine S Serine H Histidine T Threonine I Isoleucine V Valine K Lysine W Tryptophan L Leucine Y Tyrosine Introduction 1 I. Introduction 1. C4 photosynthesis 1.1. The dark side of Rubisco Photosynthesis is an elementary process performed by plants, algae and some bacteria. Atmospheric and inorganic CO2 as well as water are transformed in dependence of light energy into organic carbohydrates and O2. Three photosynthetic mechanisms are known: C3, C4 and crassulacean acid metabolism (CAM) that can be differentiated by the location and time of CO2 fixation. The majority
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