Comparative Analysis of the MAX Pathway Joanna Alex Hepworth, MA Cantab. Submitted in part fulfilment for the degree of Doctor of Philosophy The University of York Department of Biology March 2012 2 3 Abstract The pattern of branch outgrowth is a key determinant of the plant body plan. In most angiosperms branching is flexible, as branches are produced from axillary meristems which can either remain dormant or grow out. Strigolactones (SLs), a new class of plant hormones, repress branching in a range of angiosperms, including Arabidopsis, and there is increasing evidence that SLs are regulators of plant development in response to nutrient stress. This study has exploited genetic and physiological methods to investigate the evolution of SL biosynthesis and roles across the four major lineages of vascular plants. The cytochrome P450 family member MAX1 in Arabidopsis is required for the synthesis of SLs, and forms part of a signalling pathway containing at least four other genes in Arabidopsis and five in rice. Most other components of the strigolactone signalling pathway are conserved throughout the land plants, but MAX1 orthologues are absent from the moss Physcomitrella patens, which nevertheless produces SLs. Unlike other members of the pathway MAX1 orthologues have radiated in the angiosperms, particularly in the monocots. By use of complementation analysis this study presents evidence that MAX1 catalytic function is conserved in lycopodiophytes and gymnosperms, and that it may therefore have been incorporated into the SL pathway before the division of the vascular plant groups. In angiosperms the radiation of MAX1 gene copies has led to different evolutionary fates, of conservation of catalytic function in monocots, but divergence in dicots. Deletions of MAX1 orthologues have also contributed to natural variation in shoot architecture in domestic rice. In addition, this study presents evidence that the action of D27 in the biosynthetic pathway of SLs in rice is conserved in Arabidopsis. These genetic approaches are complemented with physiological investigation of the actions of strigolactones in non-angiosperm species, including spruce, fern and Selaginella species. 4 Table of Contents Abstract .......................................................................................................... 3 Table of Contents ........................................................................................... 4 Acknowledgements ...................................................................................... 10 Author’s Declaration .................................................................................... 12 Chapter 1. Introduction ................................................................................ 13 1.1 Shoot branching ................................................................................. 14 1.1.1 Shoot meristems .......................................................................... 16 1.1.2 Hormone pathways ..................................................................... 20 1.2 The MAX pathway and Strigolactones ............................................... 25 1.2.1 Discovery .................................................................................... 25 1.2.2 Phenotypes and functions ........................................................... 27 1.2.3 Regulation, signal transduction and transport ............................. 29 1.2.4 Biochemical structure and hormone pathway ............................. 31 1.3 Evolution of shoot branching ............................................................. 37 1.4 Evolution of strigolactones ................................................................ 41 1.5 Aims ................................................................................................... 43 Chapter 2. Methods and Materials ............................................................... 45 2.1 Definition of terms ............................................................................. 45 2.1.1 Nomenclature of duplicated genes .............................................. 45 2.1.2 Gene and protein naming conventions ........................................ 45 5 2.2 Molecular cloning techniques ............................................................ 46 2.2.1 dH20 ............................................................................................ 46 2.2.2 RNA extraction ........................................................................... 46 2.2.3 DNA extraction from plants ........................................................ 46 2.2.4 cDNA synthesis ........................................................................... 47 2.2.5 3’RACE ....................................................................................... 47 2.2.6 5’RACE ....................................................................................... 47 2.2.7 Sequencing .................................................................................. 47 2.2.8 PCR ............................................................................................. 48 2.2.9 Error-free PCR ............................................................................ 49 2.2.10 Gel electrophoresis .................................................................... 50 2.2.11 PCR Primers .............................................................................. 51 2.2.12 Q-PCR ....................................................................................... 51 2.2.13 Restriction digestion .................................................................. 51 2.2.14 Ligation ..................................................................................... 52 2.2.15 Cloning from PCR products ...................................................... 53 2.3 Bioinformatics .................................................................................... 53 2.3.1 Orthologue identification ............................................................ 53 2.3.2 Coding sequence prediction ........................................................ 53 2.3.3 Alignments .................................................................................. 54 2.4 Constructs ........................................................................................... 54 6 2.4.1 Overexpression constructs .......................................................... 54 2.4.2 Pre-transcriptional repression construct ...................................... 54 2.5 Production of Transgenic Organisms ................................................. 55 2.5.1 Bacterial selection and growth .................................................... 55 2.5.2 Escherichia coli transformation .................................................. 55 2.5.3 Agrobacterium tumefaciens transformation ................................ 56 2.5.4 Plant transformation .................................................................... 56 2.6 Plant growth and experimentation ..................................................... 58 2.6.1 Plant material .............................................................................. 58 2.6.2 Growing conditions ..................................................................... 60 2.6.3 Hormone treatments .................................................................... 60 2.6.4 Arabidopsis ................................................................................. 61 2.6.5 Medicago ..................................................................................... 63 2.6.6 White Spruce ............................................................................... 63 2.6.7 Selaginella kraussiana ................................................................ 67 2.6.8 Ceratopteris richardii ................................................................. 68 2.7 Statistical analysis and representation of data.................................... 69 2.7.1 Statistical analysis ....................................................................... 69 2.7.2 Graphs & Thesis ......................................................................... 69 Chapter 3. MAX1 Incorporation into the MAX pathway .............................. 70 3.1 Introduction to the evolution of MAX1 .............................................. 70 7 3.1.1 Phenotype .................................................................................... 71 3.2 Dose response curves ......................................................................... 72 3.3 The ‘Brassicaceae-specific’ hypothesis ............................................. 74 3.4 MAX1 complementation by non-angiosperm species ........................ 78 3.4.1 Branch phenotype ........................................................................ 89 3.4.2 Leaf phenotype ............................................................................ 92 3.5 Discussion .......................................................................................... 97 Chapter 4. Roles for Strigolactones in Non-Angiosperm Species ............. 102 4.1 Gymnosperms - Picea glauca .......................................................... 104 4.1.1 Initial decapitation studies and protocol development .............. 106 4.1.2 Long term effects of SL application .......................................... 111 4.1.3 SL effects on dormant apical bud formation ............................. 115 4.1.4 SL effects on outgrowth after decapitation ............................... 116 4.1.5 SL genes and phosphate response ............................................