Swedish University of Agricultural Sciences Faculty of Forest Sciences Department of Forest Genetics and Plant Physiology Characterization of the metacaspase gene family in Arabidopsis thaliana Paige Cox Master’s thesis • 30 hec • Advanced level Programme/education • Masters programme in Plant and Forest Biotechnology Place of publication • Umeå Characterization of the metacaspase gene family in Arabidopsis thaliana Paige Cox Supervisor: Hannele Tuominen, Umeå University, Department of Plant Physiology Assistant Supervisor: Benjamin Bollhöner, Umeå University, Department of Plant Physiology Examiner: Karin Ljung, SLU, Department of Forest Genetics and Plant Physiology Credits: 30 hec Level: Advanced level Course title: Master thesis in Biology at the dept of Forest Genetics and Plant Physiology Course code: EX0634 Programme/education: Masters programme in Plant and Forest Biotechnology Place of publication: Umeå Year of publication: 2011 Online publication: http://stud.epsilon.slu.se Key Words: Programmed cell death, caspase, metacaspase, xylem, Arabidopsis thaliana , Genevestigator, beta-galactosidase, phenotype Swedish University of Agricultural Sciences Faculty of Forest Sciences Department of Forest Genetics and Plant Physiology Table of Contents List of Figures ..................................................................................................................... 5 List of Tables ...................................................................................................................... 6 Acknowledgments ............................................................................................................... 7 Abstract ............................................................................................................................... 8 1 Introduction .................................................................................................................. 9 1.1 Introduction to the components of programmed cell death ............................................. 9 1.1.1 Introduction to programmed cell death ................................................................... 9 1.1.2 Enzymes involved in PCD ....................................................................................... 9 1.1.3 Caspases and proteins with caspase-like activity .................................................. 10 1.1.4 Introduction to metacaspases ................................................................................. 11 1.1.5 Similarities and differences between caspases, and MCs ...................................... 12 1.2 Analysis of MC functions .............................................................................................. 13 1.2.1 Enzyme activity and substrate/inhibitor analyses .................................................. 13 1.2.2 Developmental functional characterisation ........................................................... 16 1.3 My Thesis: The value in determining the function of MC genes and their connection to programmed cell death in Arabidopsis thaliana ................................................................... 17 1.3.1 Value of the project ............................................................................................... 17 1.3.2 Why study MCs? ................................................................................................... 18 1.3.3 Objectives and goals of the thesis .......................................................................... 18 2 Materials and methods ............................................................................................... 20 2.1 Plant material ................................................................................................................. 20 2.2 Growth conditions ......................................................................................................... 20 2.3 DNA extraction ............................................................................................................. 20 2.4 Gene expression studies ................................................................................................ 20 2.5 GUS histochemical staining .......................................................................................... 20 2.6 Genotyping .................................................................................................................... 21 2.7 Phenotyping ................................................................................................................... 22 2.7.1 Mutants and experimental design .......................................................................... 22 2.7.2 Phenotyping data collection ................................................................................... 23 2.7.3 Data analysis .......................................................................................................... 23 2.8 Production of promoter::gene::GUS constructs............................................................. 24 2.8.1 Primer design and PCR of AtMC1-9 ..................................................................... 24 2.8.2 Production of constructs using Gateway® cloning ............................................... 25 2.8.3 Transformation of E. coli and Agrobacterium ....................................................... 26 2.9 Sequencing .................................................................................................................... 26 2.10 Sequence analysis .......................................................................................................... 26 2.11 T-DNA insertion sites .................................................................................................... 27 2.12 Supplemental material and methods .............................................................................. 29 3 Results........................................................................................................................ 30 3.1 Isolation and validation of AtMC mutants ..................................................................... 30 3.2 In silico expression analysis of the AtMC gene family .................................................. 30 3.3 Reporter gene analysis of the AtMC gene family .......................................................... 33 3.4 Functional characterization of the AtMC gene family by reverse genetic approaches .. 34 4 Discussion .................................................................................................................. 37 4.1 Future work ................................................................................................................... 40 4.2 Conclusion ..................................................................................................................... 43 5 References .................................................................................................................. 45 List of Figures Figure 1.1: AtMC phylogenetic tree. ............................................................................... 13 Figure 2.1: Schematic representation of the DNA insertion sites for all AtMCs . ............ 28 Figure 3.1 Expression Analysis of AtMC1-9.. ................................................................. 32 Figure 3.2: Expression of promoter::gene::GUS constructs in Arabidopsis . ................... 33 Figure 3.3 : Morphological changes in AtMC single and double mutants. ....................... 36 5 List of Tables Table 2.1 Primers used for genotyping. ........................................................................... 21 Table 2.2 AtMC single and double mutants analyzed ...................................................... 22 Table 2.3 Forward, reverse and inset primer sequences................................................... 24 Table 3.1 RT-PCR expression analysis. .......................................................................... 31 Table 3.2 Highest expression summary of AtMC1-9 ....................................................... 31 Table 3.3 Summary of mutant lines with statistically significant differences ................. 34 6 Acknowledgments First and foremost I would like to give a special thank you to my family and friends. These are the people who I can always count on to make me laugh when I need a pick- me-up, who have inspired me to become a better person, and who I have come to treasure over the years for each of their unique personalities. Without the support of these people, I would not be who I am, or have made it to where I am, today. Secondly, I would like to thank my adviser at the Umeå Plant Science Centre in Umeå Sweden, Hannele Tuominen, for accepting me as her Master’s student. She was a joy to work with and has been a wonderful inspiration and role model both in science and in life. Her guidance and encouragement have been invaluable throughout my whole Master’s degree process, and I am extremely proud to have worked with her and learned from her. Another role model I am honoured to have met during my academic career is my Michigan Tech co-adviser, Victor Busov. I would first like to thank him for providing me with the initial opportunity to work in his lab, an opportunity which helped me to find my direction in life, and for helping to further encourage my future scientific aspirations and career. Victor supported me during the hardest moments of this thesis and I will never forget his kindness. Thank you to all the other MS, PhD, post doctorial, and professors