Localization of monoterpenoid indole alkaloid (MIA) biosynthesis by in situ hybridization (ISH) By Elizabeth Edmunds, Hons. B.Sc. A Thesis Submitted to the Department of Biotechnology In partial fulfillment of the requirements For the degree of Masters of Science August, 2012 Brock University St. Catha rines, Ontario ©Elizabeth Edmunds, 2012 ii Acknowledgments First and foremost I would like to thank Dr. Vincenzo Deluca for the opportunity to work in his laboratory under his mentorship. I have appreciated the helpful insight that has guided me through the course of this project. I have gained a valuable experience being able to learn from such an established and knowledgeable researcher. Secondly, I would like to thank my committee members Dr. Jeffrey Atkinson and Dr. Heather Gordon for their support and advice and their time to serve on my advisory committee. Thirdly, I would like to thank my colleagues and co-workers for their patience and helpful advice throughout my project. Particular mention must be given to Dr. Carlone's lab for their assistance and insight into in situ hybridization techniques. Finally, I would like to express my sincerest gratitude and appreciation towards my family and friends for their support. I would not be where I am today without the support and love from my mother and father, as well as Craig Easton. iii Abstract Monoterpenoid indole alkaloids (MIA) are among the largest and most complex group of nitrogen containing secondary metabolites that are characteristic of the Apocynaceae plant family including the most notable Catharanthus roseus. These compounds have demonstrated activity as successful drugs for treating various cancers, neurological disorders and cardiovascular conditions. Due to the low yields of these compounds and high pharmacological value, their biosynthesis is a major topic of study. Previous work highlighting the leaf epidermis and leaf surface as a highly active area in MIA biosynthesis and MIA accumulation has made the epidermis a major focus of this thesis. This thesis provides an in-depth analysis of the valuable technique of RNA in situ hybridization (ISH) and demonstrates the application of the technique to analyze the location of the biosynthetic steps involved in the production of MIAs. The work presented in this thesis demonstrates that most of the MIAs of Eurasian Vinca minor, African Tabernaemontana e/egans and five Amsonia species, including North American Amsonia hubrichitii and Mediterranean A. orienta/is, accumulate in leaf wax exudates, while the rest of the leaf is almost devoid of alkaloids. Biochemical studies on Vinca minor displayed high tryptophan decarboxylase (TOe) enzyme activity and protein expression in the leaf epidermis compared to whole leaves. ISH studies aimed at localizing TOe and strictosidine synthase suggest the upper and lower epidermis of V. minor and T. e/egans as probable significant production sites for MIAs that will accumulate on the leaf surface, however the results don't eliminate the possibility of the involvement of other cell types. The monoterpenoid precursor to all MIAs, secologanin, is produced through the MEP pathway occurring in two cell types, the IPAP cells (Gl0H) and epidermal cells (LAMT and SLS). iv The work presented in this thesis, localizes a novel enzymatic step, UDPG-7-deoxyloganetic acid glucosyltransferase (UGT8) to the IPAP cells of Catharanthus longifolius. These results enable the suggestion that all steps from Gl0H up to and including UGT8 occur in the IPAP cells of the leaf, making the IPAP cells the main site for the majority of secologanin biosynthesis. It also makes the IPAP cells a likely cell type to begin searching for the gene of the uncharacterized steps between Gl0H and UGT8. It also narrows the compound to be transported from the IPAP cells to either 7-deoxyloganic acid or loganic acid, which aids in the identification of the transportation mechanism. v Table of Contents Page Acknowledgements ii Abstract iii Table of Contents v List of Figures ix Chapter 1. Literature Review 1.1 In situ hybridization 1.1.1 The power of localization 2 1.1.2 Localization with the technique of in situ hybridization 4 1.1.3 Applications of in situ hybridization 5 1.1.4 Isotopic and non-isotopic systems for performing ISH 6 1.1.5 Detection of the DIG label 8 1.1.6 Probe choice and synthesis 9 1.1.7 Tissue and slide preparation 13 1.2 Alkaloid producing Apocynaceae family 1.2.1 Secondary metabolism 17 1.2.2 Catharanthus raseus and Catharanthus longifolius taxonomy 18 and origin 1.2.3 Vinca minor taxonomy and origin 19 1.2.4 Tabernaemontana elegans taxonomy and origin 21 1.2.5 Pharmacological value of monoterpenoid indole alkaloids 22 1.3 Previous work on Catharanthus raseus 1.3.1 DNA in situ hybridization on Catharanthus roseus 25 vi Table of Contents Page 1.3.2 Monoterpenoid indole alkaloid biosynthetic pathway 27 1.3.3 Shikimate pathway 31 1.3.4 2-C-methyl-D-erythritoI4-phosphate (MEP) pathway 36 1.4 Concluding remarks 41 Chapter 2. Epidermome enriched biosynthesis of monoterpenoid indole alkaloids and secretion to the plant surface may be common in the Aocynaceae family of plants. 2.1 Abstract 44 2.2 Introduction 45 2.3 Materials and Methods 2.3.1 Plant material and alkaloid extraction 49 2.3.2 Alkaloid analysis 50 2.3.3 Isolation and Identification of vincadifformine from 51 Amsonia hubrichtii 2.3.4 Crude protein preparation and enzyme assays 52 2.3.5 Construction and sequence analysis of cDNA libraries 52 2.3.6 Real-time quantitative PCR 53 2.3.7 Molecular cloning and functional characterization 54 2.3.8 Tissue fixation and embedding 55 2.3.9 In situ hybridization 55 2.4 Results 2.4.1 Five Amsonia species accumulate their MIAs exclusively 56 in their leaf surface. vii Table of Contents Page 2.4.2 Vinca minor accumulates plumeran- and eburnan- type 58 MIAs on the leaf and stem surfaces. 2.4.3 MIA biosynthetic enzymes are preferentially expressed 60 in young developing leaves in V. minor. 2.4.4 Tabernaemontana elegans accumulates the majority of 62 MIAs on the leaf surfaces. 2.4.S MIA biosynthesis is regulated differently in 63 Tabernaemontana elegans compared to Vinca minor or Catharanthus rase us. 2.4.6 Biochemical and in situ hybridization localizes TOC and 65 STR gene expression close to the upper and lower leaf epidermis of T. elegans and V. minor 2.S Discussion 68 2.6 Acknowledgements 72 Chapter 3. UDPG-7-deoxyloganetic acid glycosyltransferase 3.1 Abstract 74 3.2 Introduction 75 3.3 Materials and Methods 3.3.1 In situ hybridization 80 3.3.2 Whole leaf and Epidermal Protein Extraction 81 3.3.3 Tryptophan decarboxylase activity assay 82 3.3.4 Western Immunoblots 83 viii Table of Contents Page 3.4 Results 3.4.1 Preferential expression of CrUGT8 in plant organs and 87 leaf cells 3.4.2 ISH to detect CrUGT8 in young leaves of Catharanthus 89 roseus 3.4.3 The cell-specific organization of MIA biosynthesis in 91 Catharanthus roseus and in Catharonthus longifolius. 3.4.4 ISH to detect CrUGT8 in young leaves of Catharonthus 92 longifolius 3.5 Discussion 92 3.6 Acknowl edgement 97 References 98 Appendix I : Chapter 2 Supplemental Figures 106 ix List of Figures Figures Title Page Chapter 1 1.1 Model of NBT/BCIP immunological detection of 10 digoxigenin (DIG) labeled ssRNA probes. 1.2 Monoterpenoid indole alkaloid producing plants. 20 1.3 Pharmacologically valuable indole-indoline dimeric alkaloids. 24 1.4 Application of DNA ISH for the cytogenetic analyses of C. roseus 26 on mitotic spreads prepared from root tip cells. 1.5 Biosynthesis of monoterpenoid indole alkaloids. 28 1.6 Examples ofthe typical biosynthetic MIAs of biosynthesis of 30 V. minor, A. hubrichtii, C. roseus and T. elegans from the central strictosidine pathway. 1.7 Previous RNA ISH work on longitudinal sections to localize the 32 MIA biosynthetic steps of tdc, strl, d4h and dat mRNA in developing leaves. 1.8 Spatial separation of the distribution of catharanthine and 35 vindoline on the surface of and within the third leaf of Catharonthus species. 1.9 Summary of previous work on the localization of gene 38 expression of the biosynthetic pathway for monoterpenoid indole alkaloids in C.roseus aerial organs. 1.10 The biosynthetic steps in the production of the iridoid 39 secologanin from geraniol. Chapter 2 2.1 The biosynthetic of typical V. minor, A. hubrichtii, 46 C. roseus and T. elegans MIAs from the central strictosidine pathway. x List of Figures Figures Title Page 2.2 Amsonia hubrichtii secretes vincadifformine into 57 leaf surface. 2.3 Vinca minor secretes most MIAs into leaf surface. 59 2.4 Tabernaemontana elegans secretes MIAs into leaf 61 surface except for some dimers. 2.5 Functional verification of Vinca minor and Tabernaemontana 64 elegans recombinant TDCs and STRs expressed in E. coli. 2.6 Biochemical and in situ localization of tdc and str mRNA 66 in young developing leaves of Tabernanamonta elegans and Vinca minor. 2.7 Model for the biosynthesis and secretion of MIAs in leaves 69 of the Apocynaceae. Chapter 3 3.1 Preferential expression of CrUGT8 in Catharanthus roseus 84 leaves with highest activity in first leaf pair. 3.2 CrUGT8 mRNA not detectable in young developing leaves of 86 Catharanthus roseus 3.3 Catharanthus longifolius demonstrates similar cellular 88 organization of the MIA biosynthetic steps, TDC and D4H, as Catharanthus roseus. 3.4 Localization of ugt8 mRNA in young developing leaves of 90 Catharanthus longifolius to the IPAP cells. 3.5 Spatial model of iridoid biosynthesis and translocation in 93 Catharanthus leaves.