XvGoIS, a galactinol synthase is transcriptionally upregulated under water deficit : the role of raffinosaccharides in abiotic stress tolerance in the resurrection plant Xerophyta viscosa (Baker)

11 Chapter One . 13 Introduction . 13 1.1 Leaf after death . 13 1.2 Compatible solutes: contributors to stress tolerance . 16 1.3 Photosynthesis: the origin of plant carbohydrates . 17 1.4 The raffinose family of oligosaccharides (RFOs) are important sucrosyl carbohydrates . 19 1.4.1 RFOs have been identified in the phloem translocation stream . 20 1.4.2 The "Polymer Trap" relies on RFO biosynthesis . 21 1.4.3 Gol is not a component of the translocation stream . 23 1.4.4 GolS is not the master regulator ofRFO biosynthesis . 23 1.4.5 A role for RFOs in stress tolerance . 24 1.4. 6 Are combinations of sugars necessary to facilitate stress tolerance? 25 1.5 Sugars emerge as important role players in signal transduction . 26 1.5. 1 The master stress hormone may link stress and sugar signaling . 29 1.6 Targeting carbohydrate and carbohydrate based metabolic pathways for improved stress tolerance . 30 1.6. 1 A broad variety of carbohydrate species increases during abiotic stress . 31 1.6. 2 "Sweet" success . 32 1.6. 3 Raffinosacharides as targets for metabolic engineering . 33 Chapter Two . 35 Materials and Methods . 35 2.1 Plant material . 36 2.1.1 Plant collection . 36 2.1.2 In vitro propagation . 36 2.2 General methods . 37 2.2.1 Endonuclease restrictions and ligations . 37 Un ive rsi ty of Ca pe To wn 2.2.2 Polymerase chain reaction (PCR) . 37 2.2.3 Transformations, plasmid isolations, PCR product purification and isolation of agarose bound DNA . 37 2.2.4 Visualisation of ethidium bromide stained gels . 37 2.2.5 Sequencing analysis . 3 8 2.2.6 Nucleic acid transfers . 38 2.2.7 Soluble carbohydrate extraction . 38 2.2.8 Enzyme extractions and GolS activityassays . 39 2.2.9 Desalting for HPLC analysis . 39 2.2.10 HPLC-PAD analysis and quantification of carbohydrates . 39 2.3 Cloning and in silico characterisation . 40 2.3.1 Isolation and cloning of Xv Go IS cDNA . 40 2.3.2 Bioinformatic analysis of X vGoIS . 40 2.4 DNA isolation and Southern blot hybridisation . 41 2.5 Northern blot analysis . 41 2.6 Western blot analysis . 42 2.6. 1 Protein purification and antiserum production . 42 2.6. 2 Protein extraction and western transfer. 42 2.6. 3 Immunoblotting . 43 2.7 Heterologous expression in E. coli . 43 2.7. 1 Subcloning . 43 2.7. 2 Expression of recombinant XvGolS . 44 2.7. 3 Enzymatic hydrolysis . 44 2.8 Biochemical characterisation of recombinant XvGolS . 45 2.8. 1 Determination of the time linearity . 45 2.8. 2 Substrate kinetics . 45 2.9 Plant stress treatments . 45 2.9. 1 ABA . 45 2.9. 2 Water deficit . 45 2.9. 3 Low temperature . 46 2.9. 3.1 Low temperature shock . 46 2.9. 3.2 Low temperature acclimation . 46 2.10 Electron microscopy . 46 2.10. 1 Tissue fixation . 46 Un ive rs ty of Ca pe To wn 2.10. 2 Staining and microscopy . 47 2.11 Evaluation of a phloem exudation technique for X viscosa . 4 7 Chapter Three . 48 Results . 48 3.1 In silico analysis of Xv Go IS . 49 3.2 Southern blot analysis . 53 3.3 Northern and western blot analysis . 54 3.4 Functional expression of Xv Go IS . 55 3.5 Changes in carbohydrate levels during stress treatments . 60 3.5. 1 Low temperature acclimation . 60 3.5. 2 Low temperature shock treatments . 61 3.5. 3 Carbohydrate profiles of plants at full turgor and during water deficit are variable . 64 3.5. 3.1 Full turgor . 64 3.5. 3.1 Water deficit. 64 3.6 Evaluation of a phloem exudation technique . 67 Chapter 4 . 70 Discussion . 70 4.1 XvGolS shows identity to known GolS enzymes . 71 4.2 Transcript and protein levels increase in response to stress . 72 4.3 Carbohydrate profiles vary between plants at full turgor . 73 4.4 Low temperature shock . 77 4.5 XvGolS is functionally identified as a bonafide GoIS .