The Role of Small RNAs in C4 Photosynthesis E.L.C. Gage Magdalene College University of Cambridge A thesis submitted for the degree of Doctor of Philosophy June 2012 Contents Declaration ........................................................................................................................... i Acknowledgements ............................................................................................................ iii Abstract ............................................................................................................................... v List of Figures .................................................................................................................... vii List of Tables .................................................................................................................... viii Abbreviations ..................................................................................................................... ix 1. Introduction ...................................................................................................................... 1 1.1: A Requirement for Improved Crop Productivity ....................................................... 1 1.2: The Effects of Photorespiration ................................................................................. 1 1.3: The C4 Cycle .............................................................................................................. 3 1.4: miRNA Regulation of the C4 Cycle ......................................................................... 14 1.5: miRNA Regulation .................................................................................................... 15 1.6: Experimental Approach ........................................................................................... 17 1.7: Project Aims .............................................................................................................. 21 2. Materials and Methods ................................................................................................. 23 2.1: General Plant Cultivation........................................................................................ 23 2.2: Molecular Techniques ............................................................................................ 24 2.3: Quantification of Protein ....................................................................................... 30 2.4: Plasmid Design and Construction ......................................................................... 32 2.5: Microbial Techniques ..............................................................................................35 2.6: Transformation of Cleome gynandra ..................................................................... 37 2.7: Transformation of Arabidopsis thaliana ................................................................ 39 2.8: Analysis of Transgenics .......................................................................................... 40 2.9: Anatomical Analysis ................................................................................................ 41 2.10: Photosynthetic Analysis ........................................................................................ 42 2.11: Hybridisations of C. hassleriana and C. gynandra ................................................ 43 2.11.2: In vitro Ovule Culture ......................................................................................... 44 3. Do miRNAs regulate the C4 Cycle? Analysis of sRNAs and their Inhibition ............. 45 3.1: Introduction ............................................................................................................. 45 3.1.4: Experimental Design ............................................................................................ 48 3.3: Discussion ............................................................................................................... 76 4. Changes in Gene Regulation during Cotyledon Development ................................... 87 4.1: Introduction ............................................................................................................ 87 4.2: Results ..................................................................................................................... 90 4.3: Discussion ............................................................................................................... 96 5. The Generation of Hybrids within Cleome ................................................................. 103 5.1: Introduction ............................................................................................................ 103 5.2: Results .................................................................................................................... 106 5.3: Discussion ............................................................................................................... 110 6. Final Discussion ............................................................................................................ 115 6.1: The potential for miRNAs to regulate the C4 Cycle .............................................. 115 6.2: miRNAs actively regulate Development in C. gynandra ...................................... 115 6.3: sRNAs may act as Regulators of the C4 Cycle in C. gynandra .............................. 116 6.4: Light Influences Development in C. gynandra .................................................... 120 6.5: Final Conclusions ................................................................................................... 121 7. References ..................................................................................................................... 123 Appendix 1: Primer List ..................................................................................................... 141 Appendix 2: sRNA Target List ......................................................................................... 144 Appendix 3: Plasmid Maps .............................................................................................. 147 Declaration This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. It does not exceed the word limit set out by the Degree Committee for Biology and is not substantially the same as any work that has been, or is being submitted to any other university for any degree, diploma or any other qualification. E.L.C. Gage i ii Acknowledgements I wish to thank my supervisor Julian Hibberd for his invaluable instruction and support throughout this project. Secondly, John Grey, John Carr and Howard Griffiths have all provided excellent advice and motivation throughout the project. Thanks are due to Kaiser Kajala, Naomi Brown and Holly Astley for advice on molecular techniques, Christine Newell for instruction on in vitro techniques, Padubidri Shiaprasad for advice and assistance on small RNA sequencing, Risha Narayan and Kim Rutherford for assistance with the bioinformatic analysis, Maria Herse and Jessica Royles for advice regarding photosynthetic analysis and to Susan Stanley and Britta Kuempers for advice on histological techniques. Thanks are due to all the members of the Molecular Physiology group for making this project a uniquely enjoyable experience and for putting up with my eccentricities as a scientist. Helen Woodfield and Jane Knerova have made working in the lab a constant pleasure, while Ben Williams and Marek Szecowka never fail to brighten my day. Considerable thanks are due to my parents, Harriet Dickinson, Alice Chang, Peter Brierley, Miranda de Graaf and Cheryl Snowdon for their unfailing support, advice and all-round-greatness. Finally, I would like to thank the Biotechnology and Biological Sciences Research Council, the Department of Plant Sciences, Magdalene College and the Cambridge Philosophical Society for the funding and support that made this work possible. Si scientia modo per ruinam confecta est, completa scientia modo per ruinam hominis ipsius confecta est. Immolatio operi factus sum, sed scientia mentis, corporis et plantae perfecta est. iii iv Abstract The C4 cycle represents a series of biochemical and anatomical modifications that are targeted to overcome the effects of photorespiration caused by the oxygenase capability of Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO). The cycle has evolved independently in over 60 lineages, which suggests that recruitment of genes into the C4 cycle is a relatively easy process. However, the mechanisms by which the anatomy and cell-specificity of the components of the C4 cycle is achieved is poorly understood. Preliminary work in maize indicated several components of the C4 cycle may be targeted by microRNAs (miRNAs). To explore this, a library of sRNA sequences from mature leaf tissue of the model C4 species Cleome gynandra L. was generated and then searched against a list of expressed sequence tag sequences for candidate genes of the C4 cycle. To complement this, transgenic C. gynandra containing the viral p19 protein, which is capable of suppressing miRNA activity, were produced. A limited subset of the candidate C4 genes showed a high level of sRNA read alignment. In C. gynandra plants expressing p19 photosynthesis was compromised and transcripts of several genes (most notably RbcS and RCA) were upregulated. These data were complemented by examining the effect