Daylily (Hemerocallis) as a model system for the study of ethylene-insensitive flower senescence: tissue culture and aspects of the development of proteolytic enzyme activities, with special emphasis on leucine aminopeptidase. A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy in Biotechnology in the University of Canterbury by Mahagamage Gesha Patalee Mahagamasekera University of Canterbury 2000 October · I " I I ) To my father who to me was a beacon of great achievement And To my mother for laying my course this far, I gratefully dedicate this. I CONTENTS Page Contents ............................. , ................ ............................. ... I List of figures ........................................................................ VI List of tables ........................................................................................ VIII List of plates ........................................................................................ X Abbreviations ....................................................................................... XI Abstract ............................................................................... XIII Acknowledgments ................................................................................ XIV CHAPTERl ........................................................................ 1 INTRODUCTION 1.1 Senescence of Plants and Plant Parts 1.1.1 Senescence symptoms ...... ... .. 2 1.1.1.1 Changes in fresh and dry weights ........................... 3 1.1.1.2 Changes in pigmentation 1.1.1.3 Changes in membranes and fine structures 1.1.1.4 Changes in macromolecul ar components ................. 4 1.1.1.4.1 Hydrolysis and translocati on of cell components 1.1.1.4.2 Proteins ................................................. 5 1.1.1.4.3 Nucleic acids 1.1.1.4.4 Carbohydrates .......................................... 6 1.1.1.5 Changes in respiration ........ .. .. .... .. .... .. .. .... ........ 7 1.1.1.6 Changes in photosynthesi s .................................. 8 1.1.1.7 Changes in plant hormone s 1.1.1.8 Changes in reactive oxyge n species and free radicals 11 1.1.2 Factors affecting senescence ...................................... 12 1.1.3 Flower senescence .................................................. 15 1.1.4 Senescence mechanisms ........................................... 17 1.2 Protein Degradation in Plants ............................................ 20 1.2.1 Proteolytic enzymes ................................................ 22 1.2.1.1 Endopeptidases ............................................... 23 1.2.1.2 Carboxypeptidases 1.2.1.3 Aminopeptidases ........................................... 26 1.2.1.3.1 Leucine aminopeptidase (LAP) ..................... 27 1.2.2 Developmental regulation of proteolytic enzymes ............. 28 1.2.3 Inhibitors of proteolytic enzymes .............................. 32 1.2.3.1 Synthetic inhibitors 1.2.3.2 Endogenous inhibitors of proteolytic enzymes from plant sources ........................................ '. .. 35 1.3 Micropropagation of Plants .......................................... ..... 36 1.4 Daylilies .................................................................. 37 1.4.1 Origin, history and spread of daylilies 1.4.2 Physical characteristics .......................................... 38 1.4.3 Classification ...................................................... 39 1.4.4 Modern daylilies 1.4.5 Uses of day lilies ............ ......................................... 40 II 1.4.6 Propagation of daylily .............................................. 41 1.4.7 Tissue culture of daylily for micropropagation ................. 42 1.5 Aims and Objectives ...................................................... 46 CHAPTER2 ........................................................................ 50 MATERIALS AND METHODS 2.1 Plant Material 2.1.1 Maintenance of daylily pI ants in a glasshouse or growth room 2.2 Micropropagation of Dayl ily 2.2.1 Types of explants 2.2.2 Culture initiation .................................................... 51 2.2.3 Media ................................................................. 52 2.2.3.1 Media preparation 2.2.3.2 Media tested for shoot ini tiation ........................... 55 2.2.3.3 Media for root initiation and maintenance of shoots and/or plantlets 2.2.4 Establishment of tissue cultured plantlets in soil 2.2.5 Effect of sucrose concentr ation on rooting ...................... 56 2.3 Proteins Associated with Development and Senescence of Day lily Petals ........................................................................ 58 2.3.1 Harvesting of flowers, lea ves and roots 2.3.2 Extraction of proteins from plant material 2.3.3 Determination of total protein concentration .................... 59 2.3.4 Effect of storage at low temperature on the stability of proteolytic enzymes 2.3.4.1 LAP 2.3.4.2 Endopeptidase and carboxypeptidase ..................... 60 2.3.5 Determination of presence of endogenous proteolytic enzyme inhibitors and promoters in petal extracts 2.3.6 Presence oflow pH extractable proteolytic enzymes in petals, leaves and roots ...................................................... 61 2.3.7 LAP ........................ ... ... ... ... ............................... 62 2.3.7.1 Determination of LAP activity 2.3.7.2 LAP activity in daylily petals during development of flowers 2.3.7.3 Effect of pH on LAP activity in petals and leaves 63 2.3.7.4 Effect of temperature on LAP activity 2.3.7.5 Effect of different classes of proteolytic enzyme inhibitors and promoters on LAP activity ................. 65 2.3.7.6 Effect of damaging floral parts on LAP activity in petals 2.3.7.7 Effect of different chemic als on bud opening, longevity and LAP activity in buds, flowers, petals and leaves of cv. Stella D'oro ................................................ 66 2.3.8 Endopeptidase ....................................................... 67 2.3.8.1 Determination of endopeptidase activity 2.3.8.2 Endopeptidase activity in daylily petals during development of flowers ...................................... 68 2.3.8.3 Effect of pH on endopeptidase activity in petals III 2.3.9 Carboxypeptidase ................................................... 68 2.3.9.1 Determination of carboxy peptidase activity 2.3.9.2 Carboxypeptidase activity in daylily petals during development of flowers ...................................... 70 2.3.9.3 Effect of pH on carboxypeptidase activity in petals 2.3.1 0 Gel electrophoresis 2.3.10.1 SDS PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) for proteins 2.3.10.2 Non denaturing PAGE for endopeptidase activity with gelatin as the substrate ....................................... 72 2.3.10.3 Native isoelectric focusing (IEF) for isozymes of LAP 2.3.10.4 Coomassie blue staining procedure ...................... 73 2.3.10.5 Silver staining procedure 2.3.10.6 Staining for endopeptidase activity on gelatin ... ....... 74 2.3.10.7 Staining for LAP isozymes 2.3.10.8 Determination of pI value ................................. 75 2.3.11 Partial purification ofproteins 2.3.11.1 Ammonium sulfate ((NH4)2S 0 4) precipitation 2.3.11.2 Purification by DEAE cellulose column 2.4 Nucleic Acids Associated with Development and Senescence of Daylily Flowers ............................................................ 176 2.4.1 Bacterial strain and lap cD N A 2.4.2 Microbiological media and culture conditions 2.4.3 Extraction of plasmid cDN A from bacteria by alkaline-lysis method and recovery of lap DNA from low-melting- temperature agarose ................................................. 77 2.4.4 Isolation of plant nucleic ac ids .................................... 78 2.4.4.1 Isolation of RNA from plant tissues 2.4.4.2 Estimation of plant nuclei c acid concentration and testing for purity .............................................. 79 2.4.5 Hybridization of RNA and DNA 2.4.5.1 DNA probe preparation 2.4.5.2 Estimation of labelled lap probe concentration .......... 80 2.4.5.3 Template preparation for dot blots 2.4.5.4 DNA and RNA dot blot hybridization 2.4.5.5 Post-hybridization washes ........................ .......... 81 2.4.5.6 Detection procedure ....................................... 82 2.5 Data Handling CHAPTER3 ............................ ,........................................... 83 RESULTS 3.1 Micropropagation 3.1.1 Initiation of shoot primordia-like structures from floral explants 3.1.2 Effect of position of the petal explant on initiation of shoot primordia-like structures .......................................... 89 3.1.3 Regeneration of plantlets from shoot primordia-like structures ............................................................ 91 IV 3.1.4 Effect of sucrose on root formation in shoot cultures of cv. Stella D'oro ......................................................... 92 3.1.5 Establishment of aseptically grown plants in soil and production of flowers of cv. Stella D'oro ...................... 97 3.1.6 Use oftissue cultured plant material in experiments on flower senescence 3.2 Development of Daylily Flowers 3.2.1 Morphological changes 3.2.2 Change in fresh weight during flower development .......... 98 3.2.3 Changes in proteins during flower development .............. 99 3.2.3.1 Soluble proteins 3.3 Effect of Different Chemi c als on the Senescence of cv. Stella D'oro .............................. .......................................... 105 3.3.1 Effect of chemicals on the senescence