Just Scratching the Surface - The Evolution of the Plant Cuticle Author: Flor Spaans Supervisor: Dr. Samuel Brockington August 2012 Thesis submitted in partial fulfilment for the MSc in the Biodiversity and Taxonomy of Plants. i Abstract The cuticle consists of a thin waxy layer on the epidermal surfaces of plant tissues, and is present in all land plants including extant bryophytes. The cuticle performs a range of functions in flowering plants and is controlled by a complex system of regulation, but little is known about its function and regulation in bryophytes The regulatory genes AtMYB96 and SHN are involved in cuticle regulation in Arabidopsis thaliana and consequently are candidates for cuticle regulation in bryophytes. Phylogenetic trees were constructed to elucidate the evolutionary history of the AtMYB96 and SHN1/WIN1 gene lineages and to identify putative homologs in the model bryophyte Physcomitrella patens. The phylogeny of both gene lineages is complex with numerous inferred gene duplication events. AtMYB96 was shown to be a Brassicaceae-specific representative of a gene lineage that could only be traced to the origin of seed plants, however SHN homologs were found in Physcomitrella patens, indicating a deep evolutionary history for this gene lineage. Gene expression analysis revealed that the P. patens SHN homologs were expressed in both protonemal tissues and gametophyte tissue, were differentially expressed in correlation with the onset of cuticle formation, and that the expression of both homologs was affected by treatment with abscisic acid. Future work will further characterise these SHN genes in bryophytes and explore the relationship between abscisic acid regulation and SHN function. ii iii Acknowledgements I would like to thank my supervisor, Dr Samuel Brockington for the huge effort he has put into this project, Dr. Beverly Glover for allowing me to spend time at her lab, and members of the Glover lab for their support. Furthermore, I thank all my course-mates for their support during this project and Chadtip Rodtassana in particular. My gratitude as well to the MSc course director, Louis Ronse de Craene, and the staff at the Royal Botanical Gardens, Edinburgh. I would also like to mention my family who have given me their continued support. Finally, I should also mention that this work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number OCI-1053575. iv v Table of Contents Abstract...................................................................................................... i Acknowledgements....................................................................................iii Table of Contents........................................................................................v Lists of Tables and Figures........................................................................viii Aims and Objectives...................................................................................ix Chapter 1 -Introduction........................................................................1 Overview......................................................................................................................1 The plant cuticle..........................................................................................................1 Structure.................................................................................................1 Function.................................................................................................2 The early evolution of the plant cuticle ....................................................................4 The fossil record......................................................................................4 Bryophyte cuticles....................................................................................4 Abscisic acid as a regulator...................................................................6 Genetic control of the plant cuticle............................................................................7 Transcription factors...............................................................................8 MYB gene family transcription factors....................................................9 R2R3 subgroup 1....................................................................................9 AP2/ERF gene family transcription factors...........................................11 AP2 SHN genes.....................................................................................12 Rationale for this study .............................................................................................13 Finding gene homologs.........................................................................13 Physcomitrella patens (Hedw.) Bruch & Schimp. as a test organism......14 Chapter 2 -Materials & Methods.........................................................19 Phylogenetic analysis.................................................................................................19 Data retrieval.......................................................................................19 Sequence alignments............................................................................20 Phylogenetic analysis...........................................................................20 Gene expression analysis............................................................................................22 vi Plant growth........................................................................................22 Harvesting...........................................................................................22 RNA extractions...................................................................................23 RNA Purification.................................................................................23 cDNA synthesis....................................................................................24 qPCR...................................................................................................25 Statistics..............................................................................................26 Chapter 3 - Results...............................................................................29 Phylogenies.......................................................................................................29 Gene expression analysis...................................................................................31 Chapter 4 - Discussion..........................................................................37 MYB gene trees...........................................................................................................38 SHN gene trees............................................................................................................42 ABA-regulation of putative cuticle homologs in early land plants........................47 Conclusions...........................................................................................49 Bibliography..........................................................................................51 Appendices............................................................................................65 Appendix I- Sequence alignments Appendix II – Gene accession numbers Appendix III- Moss medium Appendix IV – qPR loading plan vii List of Tables and Figures Tables Table I – Table summarizing statistics of the four datasets and resulting phylogenetic trees. Table II – Gene expression analysis results from data gathered using Pp1s58 primers. Table III – Gene expression analysis results from data gathered using Pp1s21 primers. Table IV- ANOVA summary table for Pp1s58 expression analysis results. Table V-ANOVA summary table for Pp1s58 expression analysis results. Figures Figure 1 – Moss life-cycle. Figure 2 – Gel visualization of samples after DNase treatment. Figure 3 – Gel visualization of synthesized DNA. Figure 4 – Possible topologies for angiosperm SHN clades. Colour Plates Plate I- MYB Subgroup 1.1 ML tree. Plate II-MYB Subgroup 1.1 Bayesian tree. Plate III-MYB Subgroup 1.1 MP tree. Plate IV-MYB60 ML tree. Plate V-MYB60 Bayesian tree. Plate VI-MYB60 MP tree. Plate VII-Angiosperm SHN ML tree. Plate VIII-Angiosperm SHN Bayesian tree. Plate IX-Angiosperm SHN MP tree. Plate X-SHN linker tree Plate XI-Early diverging SHN ML tree. Plate XII-Early diverging SHN Bayesian tree. Plate XIII-Early diverging SHN MP tree. Plate XIV-Bar charts showing gene expression analysis results. viii ix Aims and Objectives Aims The project was part of a wider research programme into the genetic mechanisms that underlie the origin and evolution of the land plant cuticle. Therefore, the overall aim of the project was to attain a better understanding of the genetic mechanisms underlying the evolution of the plant cuticle. More specifically, this project sought to identify key genetic regulators that may have been significant in the early evolution of the plant cuticle. In order to achieve this, phylogenetic trees were constructed of genes known to be involved in regulation of the cuticle in Arabidopsis thaliana (L.) Heynh., in order to identify corresponding homologs in the bryophyte model Physcomitrella patens (Hedw.) Bruch & Schimp. Furthermore when gene functions are considered in the context of phylogeny, much can be revealed about the evolutionary history of the gene and its role in evolutionary innovation. Therefore, one