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The Regulation of Phosphoenolpyruvate Carboxylase in Stomatal Guard Cells of Commelina Communis

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The Regulation of Phosphoenolpyruvate Carboxylase in Stomatal Guard Cells of Commelina Communis The Regulation of Phosphoenolpyruvate Carboxylase in Stomatal Guard Cells of Commelina communis by John Paul S. Nelson Thesis submitted for the degree of doctor of philosophy Department of Biochemistry University of Glasgow 1994 ProQuest Number: 13833766 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13833766 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Contents of Thesis Page Contents ii List of figures vii List of tables ix Abbreviations x Acknowledgements xi Summary xii Chapter 1: Introduction 1.1 General features of stomata 1 1.1.1 Stomatal regulation of photosynthesis and 5 responses to environmental stimuli 1.1.2 The osmotic relations of guard cells 8 1.2 Guard cell ionic relations and metabolism. 9 1.2.1 K+ uptake and efflux 11 1.2.2 CL uptake and efflux 11 1.2.3 Malate accumulation and removal 12 1.2.4 Contribution of sugars to turgor changes in guard 17 cells 1.3 Phosphoenolpyruvate carboxylase - a key enzyme 19 in plant metabolism. 1.3.1 Phosphoenolpyruvate carboxylase activity in 19 plant tissues. 1.3.2. Structure 20 1.3.3 Regulation 21 1.4 Phosphorylation as a control mechanism in higher 24 plants 1.4.1 Protein kinases 24 1.4.2 Plant enzymes regulated by phosphorylation 26 1.4.3 Protein phosphatases 27 1.5 Stimulus-response coupling in guard cells 29 1.5.1 Signal transduction pathways 29 1.5.2 Evidence for Ca2+ as a signalling component in 30 plant cells, and its relevance to stomatal function 1.5.3 Evidence for the presence of the phosphoinositide 32 system in plants, and its relevance to stomatal function 1.6 Objectives 33 Chapter 2: Materials and Methods 2.1. Materials 35 2.2. Experimental tissues 36 2.2.1 Plant material 36 2.2.2 Preparation of epidermal strips and guard cell 36 protoplasts 2.3 General biochemical methods 38 2.3.1 pH calibrations 38 2.3.2 Glassware and plastics 38 2.3.3 Spectrophotometric assays 38 2.3.4 Centrifugation 38 2.3.4 Micropipetting 38 2.4. Methods for the extraction of enzyme activities 38 2.4.1 Epidermal strip extracts 38 2.4.2 Protoplast extracts 39 2.5 Assay procedures 39 2.5.1 Phosphoenolpyruvate carboxylase activity 39 2.5.2 Protein phosphatase activity 40 2.6 Polyacrylamide gel electrophoresis techniques 40 2.6.1 SDS/polyacrylamide gel 40 electrophoresis (discontinuous system) 2.6.2 Staining SDS/polyacrylamide gels 41 2.6.3 Drying and autoradiography of gels 41 2.6.4 Scanning of autoradiographs 41 2.6.5 Immunoblotting of polyacrylamide gels 42 2.7 Experimental manipulation of tissue 43 iii 2.7.1 Incubation of epidermal strips and the 43 measurement of stomatal apertures 2.7.2 Incubation of guard cell protoplasts 45 2.8 Miscellaneous methods 47 2.8.1 Immunoprecipitation of 47 phosphoenolpyruvate carboxylase 2.8.2 Vital staining of protoplasts 47 2.8.3. Labelling of phosphoenolpyruvate carboxylase 47 in extracts of guard cell protoplasts 2.8.4. Non radioactive incubations of guard cell 47 extracts with protein phosphatases and kinases Chapter 3: Results 3.1 Introduction 49 3.2 Responses ofCommelina communis stomata to various 50 stimuli 3.3 The phosphorylation of guard cell phosphoenolpyruvate 54 carboxylase 3.3.1 Introduction 54 3.3.2 Phosphorylation of phosphoenolpyruvate 60 carboxylase in guard cell extracts 3.3.3. Time course of labelling of the ATP pool in 60 guard cell protoplasts by incubation of the protoplasts in [32P]-orthophosphate 3.3.4. The effect of fusicoccin on the phosphorylation 65 state of phosphoenolpyruvate carboxylase 3.3.5 Time course of phosphorylation of guard 65 cell phosphoenolpyruvate carboxylase 3.3.6 The reversibility of phosphorylation of guard 70 cell phosphoenolpyruvate carboxylase 3.3.7. Stomatal opening was delayed and 72 phosphoenolpyruvate carboxylase phosphorylation was prevented by the protein synthesis inhibitor cycloheximide 3.3.8. The effects of okadaic acid and abscisic 75 acid on the phosphorylation of guard cell i v phosphoenolpyruvate carboxylase 3.4 Studies on the kinetic properties of guard cell 80 phosphoenolpyruvate carboxylase 3.4.1 The stability of phosphoenolpyruvate 81 carboxylase activity during preparation of guard cell protoplasts and in extracts 3.4.2 Some kinetic parameters of guard cell 83 phosphoenolpyruvate carboxylase 3.4.3 Attempts to alter the kinetic properties of 83 guard cell phosphoenolpyruvate carboxylase by incubating cells and extracts in conditions likely to cause a change in phosphorylation state of the enzyme 3.4.4 Significant changes in the kinetic properties of 87 guard cell phosphoenolpyruvate carboxylase in response to environmental stimuli were observed using altered assay conditions 3.5 Conclusions 95 Chapter 4: General Discussion 4.1 The control of guard cell phosphoenolpyruvate 96 carboxylase by reversible phosphorylation 4.2 The effects of cycloheximide on stomatal 98 aperture and phosphorylation of phosphoenolpyruvate carboxylase 4.3 Similarities and differences of the phosphoenolpyruvate 99 carboxylase phosphorylation systems of guard cells and the C4 and CAM systems 4.4 The variability of data from experiments involving 100 stomata and guard cells v 4.5 Possible signalling mechanisms involved 101 in the phosphorylation/dephosphorylation of guard cell phosphoenolpyruvate carboxylase Chapter 5: References 105 vi List of figures Chapter 1 Page 1.1a Viciafaba stomata 2 1.1b Commelina communis stomata 2 1.2 Transverse section through an elliptical stoma 3 1.3 Distribution of cell wall micellae in an elliptical stoma 4 1.4a Graminaceous stomata ofZea mays 6 1.4b The distribution of micellae in cell walls of a graminaceous 6 stoma 1.5 Malate metabolism in guard cells 14 1.6 Ion fluxes in guard cells 18 Chapter 2 2.1 Epidermis incubation apparatus 44 2.2 Apparatus for illumination of guard cell protoplasts 46 Chapter 3 3.1 The responses of mean stomatal aperture to opening 51 and closing stimuli 3.2 Frequency distribution of stomatal apertures in stomal 52 populations of different mean apertures 3.3 Effect of KC1 concentration on opening and closure of 53 stomata in response to light/low C02 and darkness/normal co2 3.4 The effect of fusicoccin on stomatal aperture 55 3.5 Immunoprecipitation of guard cell phosphoenolpyruvate 58 carboxylase protein 3.6 The phosphorylation of phosphoenolpyruvate carboxylase 61 in guard cell extracts 3.7 Time course of labelling of total protein in guard cell 64 protoplasts 3.8 The effect of fusicoccin on phosphorylation of guard cell 66 phosphoenolpyruvate carboxylase 3.9 Timecourse of phosphorylation of phosphoenolpyruvate 67 carboxylase vii 3.10 Timecourse of phosphorylation of phosphoenolpyruvate 68 carboxylase in guard cell protoplasts incubated in the light and dark 3.11 The reversibility of light-stimulated phosphorylation of 70 phosphoenolpyruvate carboxylase 3.12 The effect of cycloheximide on fusicoccin stimulated 74 phosphorylation of phosphoenolpyruvate carboxylase 3.13 The effect of cycloheximide on light-stimulated stomatal 75 opening 3.14 The effect of cycloheximide on fusicoccin-stimulated 76 stomatal opening 3.15 The viability of guard cells during production of guard cell 77 protoplasts and their subsequent incubation with cycloheximide 3.16 The effects of okadaic acid and ABA on phosphorylation 79 guard cell phosphoenolpyruvate carboxylase in the dark 3.17 Variation of Ki for malate of epidermal and guard cell 81 phosphoenolpyruvate carboxylase during preparation of guard cell protoplasts and incubation of epidermal extract 3.18 Activity of phosphoenolpyruvate carboxylase and 83 sensitivity to inhibition by malate at different pH values 3.19 Variation of Ki of phosphoenolpyruvate carboxylase at 84 different concentrations of PEP 3.20 Variation of Ki (malate) of guard cell phosphoenolpyruvate 88 carboxylase during incubation of guard cell protoplast extract with PP2A 3.21 The effect of incubation of extracts with PP2A and PP1 on 89 the malate sensitivity of guard cell phosphoenolpyruvate carboxylase 3.22 The effect of protein kinases on the Ki (malate) of guard 90 cell phosphoenolpyruvate carboxylase 3.23 The effect of okadaic acid and protein kinase A catalytic 91 subunits on Ki (malate) of guard cell phosphoenolpyruvate carboxylase Chapter 4 4.1 Model of possible signalling pathways in guard cells 103 viii List of tables Chapter 1 Page 1.1 K+ concentrations in guard cells of various species 10 1.2 Kinetic properties of guard cell phosphoenolpyruvate 16 carboxylase Chapter 3 3.1 The distribution of phosphoenolpyruvate carboxylase between different cells in the epidermis of Commelina 55 communis 3.2 Immunoprecipitation of phosphoenolpyruvate 59 carboxylase activity from guard cell extracts 3.3 Ca2+ has no effect on phosphorylation of 62 phosphoenolpyruvate carboxylase in guard cell extracts 3.4 Phosphocasein phosphatase activity in guard cell extract 72 3.5 Variation of Km (PEP) and Vmax of guard cell 85 phosphoenolpyruvate carboxylase at different malate concentrations 3.6 Ki (malate) values measured using assay buffer A of 87 phosphoenolpyruvate carboxylase from guard cells incubated under different
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