The Physiology and Transcriptomics Underlying Dahlia Flower Senescence A thesis submitted to Royal Holloway, University of London for the degree of Doctor of Philosophy By Matthew Casey 2018 1 Declaration of Authorship I, Matthew Casey, hereby declare that this thesis and the work presented in it is entirely my own. Where I have consulted the work of others, this is always clearly stated. Signed: Date: 2 Abstract Dahlias are a popular commercial flower due to their variety in colour and morphology. Unfortunately, they are currently not commercially viable as cut flowers due to an unreliable vase-life. Analysis of the cut flower trade suggests that minimum longevity needs to be 10-14 days from harvest to provide time for transport to a pack house and on to the supermarket, three days in-store shelf life, and still allow a 5-day consumer guarantee. Dahlia inflorescences present unique challenges due to their complex composite floral structure: florets develop sequentially within the capitulum hence in each flower head flowers of different ages are represented. Another problem has been the limited data on dahlia and the lack of conclusive data regarding ethylene sensitivity. Therefore it was necessary to address some key questions on dahlia during this project. Firstly, dahlia flower senescence was characterised and cut flower senescence compared to senescence on the plant. The response of cut dahlia to traditional and novel postharvest treatments, including ethylene inhibitors, exogenous phytohormones and sugars, and application methods, including sprays, pulses, and holding solutions was also analysed. It was found that treatment with ethylene action inhibitors and exogenous cytokinins may significantly extend dahlia vase life. Finally, RNA-sequencing has been carried out on material from three different developmental stages of dahlia flowers to examine transcriptomic changes during the process and analyse the process at a molecular level. The differential expression analysis of this data found upregulation of putative genes including ethylene biosynthetic genes and downregulation of cytokinin biosynthetic genes, along with changes in expression in the signalling pathways of both cytokinins and ethylene. This work will provide a database for further research on dahlia flower senescence. 3 Contents Declaration of Authorship 2 Abstract 3 Contents 4 List of Figures 9 List of Tables 10 List of Abbreviations 11 Acknowledgements & Dedication 12 1. Introduction 13 1.1. The cut flower industry 13 1.2. Dahlia physiology and genetics 15 1.3. Floral senescence 20 1.3.1. Autophagy 21 1.3.2. Phytohormones 21 1.3.3. Macromolecule breakdown 23 1.3.4. Remobilisation 25 1.3.5. Reactive oxygen species (ROS) 25 1.4. Composite flower senescence 26 1.5. Transcriptomics and flower senescence 27 1.6. Methods to improve floral vase life: pre- harvest and harvesting 28 1.7. Methods to improve floral vase life: post- harvest 30 1.7.1. Transportation and storage 30 1.7.2. Ethylene inhibition 31 1.7.3. Exogenous cytokinins 32 1.7.4. Carbohydrates 34 1.8. Summary 34 1.9. Objectives 35 2. Materials and Methods 36 2.1. Plant material growth and harvest 36 2.2. Postharvest treatments 40 2.3. Physiological Measurements 42 2.3.1. Vase life 42 4 2.3.2. Floret mass 42 2.3.3. Conductivity 43 2.3.4. Protein content: extraction and assay 43 2.4. RNA extraction from dahlia florets 44 2.4.1. Extracting RNA using RNEasy Plant MiniKit (QIAGEN) 44 2.4.2. Extracting RNA using NucleoSpin® RNA Plant (Macherey-Nagel) 45 2.4.3. RNA yield and quality 46 2.5. Polymerase chain-reaction 46 2.5.1. Primer design 46 2.5.2. Genomic DNA removal and cDNA synthesis (reverse transcription) 46 2.5.3. Reverse transcription polymerase chain-reaction (RT- PCR) and gel electrophoresis 47 2.5.4. Sequencing of PCR products 47 2.5.5. Quantitative reverse transcription polymerase chain-reaction (qRT-PCR) and analysis 47 3. RNA-sequencing and de novo assembly of the Dahlia floret transcriptome 49 3.1. Introduction 49 3.2. Aims 53 3.3. Materials and Methods 53 3.3.1. Harvesting material 53 3.3.2. RNA extraction 54 3.3.3. Genomic DNA removal 54 3.3.4. RNA-sequencing 54 3.3.5. De novo assembly and analysis of transcriptome 55 3..3.6. Expression analysis 58 3.4. Results 61 5 3.4.1. Sequencing and de novo transcriptome assembly 61 3.4.2. Differential expression analysis 63 3.4.2.1. Differential expression analysis: abscisic acid and senescence associated enzymes 66 3.4.2.2. Differential expression analysis: ethylene 72 3.4.2.3. Differential expression analysis: cytokinins 78 3.5. Discussion 81 4. Physiology of Dahlia flower senescence on the plant 92 4.1. Introduction 92 4.2. Aims 96 4.3. Materials and Methods 97 4.3.1. Plant growth and harvesting 97 4.3.2. Floret mass 97 4.3.3. Conductivity 97 4.3.4. Protein content: extraction and assay 97 4.4. Results 98 4.4.1. Qualitative comparison of on- plant and cut flowers 98 4.4.2. Conductivity 100 4.4.3. Floret mass 102 4.4.4. Protein content 104 4.5. Discussion 105 5. Role of ethylene in Dahlia flower senescence 112 5.1. Introduction 112 5.2. Aims 116 5.3. Materials and Methods 117 5.3.1. Plant growth and harvesting 117 5.3.2. Postharvest treatments 117 5.3.3. Floret mass 117 5.3.4. Conductivity 117 6 5.3.5. Protein content: extraction and assay 117 5.3.6. RNA extraction 118 5.3.8. Polymerase chain-reaction 118 5.4. Results 119 5.4.1. Vase life 119 5.4.2. Conductivity 129 5.4.3. Floret mass 132 5.4.4. Protein content 135 5.4.5. Quantitative reverse transcription polymerase chain-reaction (qRT-PCR) 137 5.5. Discussion 139 6. Role of cytokinins in Dahlia flower senescence 145 6.1. Introduction 148 6.2. Aims 149 6.3. Materials and Methods 149 6.3.1. Plant growth and harvesting 149 6.3.2. Postharvest treatments 149 6.3.3. Floret mass 149 6.3.4. Conductivity 149 6.3.5. Protein content: extraction and assay 150 6.3.6. RNA extraction 150 6.3.8. Polymerase chain-reaction 150 6.4. Results 152 6.4.1. Vase life 152 6.4.2. Conductivity 165 6.4.3. Floret mass 167 6.4.4. Protein content 169 6.4.5. Quantitative reverse transcription polymerase chain-reaction (qRT-PCR) 171 6.5. Discussion 174 7. General discussion and conclusion 181 7 7.1. Comparison of senescence in cut Dahlia flowers vs. on-plant Dahlia flowers 181 7.2. Interaction of phytohormones during Dahlia floral senescence 186 7.3. Inter-cultivar comparison 187 7.4. A summary of Dahlia flower senescence: from closed bud to senesced florets 191 7.5. Recommendations for improving Dahlia vase life 193 7.6. Future work 193 7.7. Conclusion 197 Bibliography 198 Appendix I. Weather Data 228 Appendix II. Primers 231 Appendix III. ANOVA tables 241 8 List of Figures 1 Structure of composite inflorescence 2 Summary of phytohormone crosstalk 3 Asteraceae phylogenetic tree 4 Developmental stages of dahlia inflorescences 5 Floret stages of cv. ‘Sylvia’ harvested for RNA-sequencing 6 Pfaffl equation 7 Trinity pipeline flowchart 8 Flowchart of transcriptome assembly and read mapping 9 Flowchart of differential expression analysis 10 Mean sequence quality of reads from sample 2a 11 Differentially expressed genes when compared to A. thaliana or H. annuus 12 Heatmap of genes significantly up or downregulated in all sample comparisons 13 Expression changes in ABA (abscisic acid) signal transduction pathway 14 Expression changes in ABA biosynthesis pathway 15 Expression of Arabidopsis thaliana senescence associated enzymes 16 Expression changes in ethylene signal transduction pathway 17 Expression changes in ethylene biosynthesis pathway 18 Expression of A. thaliana lipases homologous to dahlia genes 19 Expression changes in cytokinin signal transduction pathway 20 Expression changes in cytokinin biosynthesis and breakdown pathway 21 Summary of ethylene biosynthesis and signalling 22 Summary of cytokinin biosynthesis and signalling 23 A summarised scheme of types of senescence, and the common processes that lead to symptoms of senescence 24 Photographic comparison of control and on-plant dahlias 25 Conductivity of control and on-plant dahlias 26 Floret mass of control and on-plant dahlias 27 Protein content of on-plant and control dahlias 28 Scheme showing the reaction of relevant plant species to either exogenous ethylene, inhibition of an ethylene biosynthetic enzyme or a transformed and non-functional ethylene biosynthetic enzyme 29 Vase life of STS or STS & BA treated dahlias 30 Vase life of CEPA treated dahlias 31 Vase life of STS and sucrose treated dahlias 32 Vase life of sucrose treated dahlias 33 Photographic comparison of cv. ‘Gerrie Hoek’ dahlias treated with STS, CEPA, or STS & BA 34 Photographic comparison of cv. ‘Karma Prospero’ dahlias treated with STS, CEPA, or STS & BA 35 Photographic comparison of cv. ‘Onesta’ dahlias treated with STS, CEPA, or STS & BA 36 Photographic comparison of cv. ‘Sylvia’ dahlias treated with STS, CEPA, or STS & BA 37 Conductivity of dahlias treated as controls or with STS or CEPA 38 Mass of dahlia florets treated as controls or with STS or CEPA 39 Protein content of control or STS treated dahlias. 40 Relative expression of DvACO4 and DvACS6 in cv. ‘Karma Prospero’ 41 Relative expression of DvACO4 and DvACS6 in cv. ‘Onesta’ 42 Vase life of cv. ‘Gerrie Hoek’ treated with BA solutions, sprays or pulses 43 Vase life of cv. ‘Karma Prospero’ treated with BA solutions, sprays or pulses 44 Vase life of cv.