METABOLITE ANALYSIS OF RESURRECTION PLANTS Master thesis in Plant Sciences- Laboratory of Plant Physiology (PPH) at Wageningen University and Department of Molecular and Cell Biology at University of Cape Town (UCT) Supervised by: Dr. Henk Hilhorst Prof. Jill Margaret Farrant Halford Dace Celeste Righi Ricco 23/07/2018 Table of contents ............................................................................................................................................................. 0 List of figures ........................................................................................................................................ 1 List of tables ......................................................................................................................................... 1 Acknowledgments ................................................................................................................................ 2 Abstract ................................................................................................................................................ 3 1. Introduction ................................................................................................................................. 4 1.1 Desiccation tolerance ............................................................................................................ 4 1.2 Metabolites analysis in resurrection plants .......................................................................... 7 1.3 Numerical analysis ............................................................................................................... 10 1.4 Plants under study and objectives of the research ............................................................. 11 1.5 Research questions ............................................................................................................. 13 2 Materials and methods ................................................................................................................ 1 2.1 Plant materials and maintenance ......................................................................................... 1 2.2 Sampling procedures ............................................................................................................. 8 2.3 Freeze drying ......................................................................................................................... 9 2.4 Metabolites extraction .......................................................................................................... 9 2.5 Drying extracts in vacuum ................................................................................................... 10 2.6 Derivatisation ...................................................................................................................... 10 2.7 GC-MS analysis .................................................................................................................... 10 2.8 Numerical analysis ............................................................................................................... 10 3 Results and discussion ............................................................................................................... 12 3.1 Drying process ..................................................................................................................... 12 3.2 Morphological changes upon dehydration ......................................................................... 12 3.3 Principal component analysis of GC-MS data ..................................................................... 15 3.4 Heat map ............................................................................................................................. 25 3.5 Biochemistry and adaptations ............................................................................................ 28 3.6 Suggestion for future research ............................................................................................ 30 4 Conclusions ................................................................................................................................ 31 5 References.................................................................................................................................. 32 List of figures Figure 1 :Selaginella spp. .................................................................................................................................................... 2 Figure 2: Xerophyta viscosa ................................................................................................................................................ 2 Figure 3: Xerophyta elegans. .............................................................................................................................................. 3 Figure 4: Xerophyta humili .................................................................................................................................................. 4 Figure 5: Eragrostis nindensis tiller. .................................................................................................................................... 5 Figure 6: Eragrostis nindensis ............................................................................................................................................. 5 Figure 7: Craterostigma pumilum ....................................................................................................................................... 6 Figure 8: Myrothamnus flabellifolia. ................................................................................................................................... 7 Figure 9:Eragrostis tef ......................................................................................................................................................... 7 Figure 10: Arabidopsis thaliana .......................................................................................................................................... 7 Figure 11: Cumulative and relative variance per Principal Component for GC-MS peaks of Xerophyta viscosa. ............. 15 Figure 12:PCA of Xerophyta viscosa ................................................................................................................................. 16 Figure 13: Boxplot of Xerophyta viscosa for the first principle component. ..................................................................... 16 Figure 14:Cumulative and relative variance per Principal Component for GC-MS peaks of Xerophyta humilis. .............. 17 Figure 15:PCA of Xerophyta humilis . ................................................................................................................................ 17 Figure 16: Boxplot of Xerophyta humilis for the first principle component. ..................................................................... 18 Figure 17: Cumulative and relative variance per Principal Component for GC-MS peaks of Myrothamnus flabellifolia.. 18 Figure 18:PCA of Myrothamnus flabellifolia .................................................................................................................... 19 Figure 19: Boxplot of Myrothamnus flabellifolia for the first principle component. ......................................................... 19 Figure 20: Cumulative and relative variance per Principal Component for GC-MS peaks of Craterostigma pumilum. .... 19 Figure 21:PCA of Craterostigma pumilum ........................................................................................................................ 20 Figure 22:PCA of Craterostigma pumilum ....................................................................................................................... 21 Figure 23: Boxplot of Craterostigma pumilum for the first principle component. ............................................................ 21 Figure 24: Cumulative and relative variance per Principal Component for GC-MS peaks of Arabidopsis thaliana. ......... 22 Figure 25: PCA of Arabidopsis thaliana . ........................................................................................................................... 22 Figure 26: Boxplot of Arabidopsis thaliana for the first principle component. ................................................................. 23 Figure 27: Cumulative and relative variance per Principal Component for GC-MS peaks of all species together. ........... 23 Figure 28:Total PCA .......................................................................................................................................................... 24 Figure 29:Total PCA. ......................................................................................................................................................... 24 Figure 30: Heat map. ........................................................................................................................................................ 25 List of tables Table 1:Pprimary metabolites classes with examples and function explanation ............................................................. 10 Table 2: Plants under study and their phylogenetic classification. ................................................................................... 12 Table 3: Harvesting specificities . ........................................................................................................................................ 8 Table 4:Harvesting