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Investigating montbretin A biosynthesis and elucidating acyltransferase in Crocosmia x crocosmiiflora Jo, Seohyun 2018

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INVESTIGATING MONTBRETIN A BIOSYNTHESIS AND ELUCIDATING ACYLTRANSFERASE IN CROCOSMIA X CROCOSMIIFLORA   by  Seohyun Jo  B.Sc., University of Manitoba, 2016    A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  In  The Faculty of Graduate and Postdoctoral Studies (Botany)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)    June 2018 © Seohyun Jo, 2018  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Investigating Montbretin A Biosynthesis and Elucidating Acyltransferase in Crocosmia x crocosmiiflora   submitted by  Seohyun Jo in partial fulfillment of the requirements for the degree of Master of Science  in Botany   Examining Committee: Dr. Jörg Bohlmann  Supervisor  Dr. Simone Castellarin  Supervisory Committee Member  Dr. Reinhard Jetter Supervisory Committee Member Dr. Yvonne Lamers  University Examiner iii  Abstract Plant specialized metabolites have been historically used in traditional medicine, flavours and fragrances throughout centuries, and they still serve as a valuable source for new pharmaceutical and nutraceutical development. Montbretin A (MbA) is an acylated flavonol glycoside produced by the ornamental plant montbretia (Crocosmia x crocosmiiflora), and mainly accumulates in the underground storage organs, called corms. This unique metabolite is a highly specific inhibitor of the human pancreatic α-amylase (HPA), thus making it a promising candidate for drug development against type-2 diabetes. However, a production system for obtaining large quantities of MbA is currently unavailable. Metabolic engineering of MbA in alternative microbial or plant systems may lead to the large-scale production of MbA.  The main goal of this thesis was to obtain foundational insight on MbA biosynthesis. I first examined the growth and development of the montbretia plant, and performed detailed metabolite analysis focusing on the corms. The results of the metabolite profiling revealed the spatiotemporal patterns of MbA biosynthesis. This provided a foundational knowledge for the development of the montbretia transcriptome by Irmisch et al. (submitted). Furthermore, based on the activity of corm protein extracts, I identified that a member of the BAHD-AT family is involved in the acylation step of MbA biosynthesis. The candidate BAHD-ATs were identified using the established montbretia transcriptome, and were cloned and heterologously expressed in Escherichia coli for functional characterization. Of the seven candidate ATs tested, two candidates, CcAT1 and CcAT2 catalyzed the formation of mini-MbA, the product of the third step of MbA biosynthesis. Additionally, CcAT1 and CcAT2 were transiently expressed in the leaves of Nicotiana benthamiana, which led to the formation of a surrogate mini-MbA compound. This provided preliminary insight towards the metabolic engineering of MbA in N. benthamiana. Furthermore, qRT-PCR analyses were performed to investigate the transcript abundance patterns of CcAT1 and CcAT2 during montbretia corm development. The transcript profiling of CcAT1 and CcAT2 further supported their in vivo roles of MbA biosynthesis. Overall, the results presented in this thesis provide new knowledge on specialized plant metabolism in a non-model plant species.  iv  Lay Summary Throughout centuries, plant specialized metabolites have been a source of valuable chemicals that are potentially beneficial to human health. Montbretin A (MbA) found in the ornamental plant montbretia (Crocosmia x crocosmiiflora) has been discovered to be a promising new drug candidate against type-2 diabetes. However, a production system for obtaining large quantities of MbA is currently not available. This thesis investigated the biosynthesis of MbA in montbretia plants. I first observed the growth and development of the montbretia plant, and looked at the metabolite levels in the corms. This revealed that MbA biosynthesis is regulated both spatially and temporally. Furthermore, using available montbretia transcriptomic resources, I discovered two BAHD-ATs that are involved in the third step of MbA biosynthesis, leading to the formation of mini-MbA. Overall, this thesis offers new knowledge on the biosynthesis of MbA in montbretia that may lead to the increased production of MbA.  v  Preface My MSc thesis comprises original and unpublished work. The overall project of which this thesis is a part was conceived by my supervisor Dr. Jörg Bohlmann and Dr. Stephen G. Withers (UBC). I designed and performed experiments, data analysis, and prepared the presentation of the results under the supervision of Dr. Jörg Bohlman and Dr. Sandra Irmisch (UBC). Work performed by others included the MALDI-FTICR analysis by Dr. Jun Han (University of Victoria and Genome BC Proteomics Centre) with samples that I generated. I performed the transcriptome analysis of the montbretia BAHD-ATs together with Mr. Macaire MS Yuen (UBC) and Dr. Sandra Irmisch.  vi  Table of Contents Abstract .............................................................................................................................iii Lay Summary .....................................................................................................................iv Preface ............................................................................................................................... v Table of Contents ..............................................................................................................vi List of Tables ................................................................................................................... viii List of Figures ...................................................................................................................ix List of Symbols and Abbreviations .................................................................................. x Acknowledgements ...........................................................................................................xi Dedication .........................................................................................................................xii Chapter 1: Introduction ..................................................................................................... 1 1.1 Plant Specialized Metabolites .......................................................................... 1 1.2 Type-2 Diabetes and Montbretin A ................................................................... 1 1.3 MbA Biosynthetic Pathway ............................................................................... 5 1.4 BAHD-Acyltransferases ................................................................................... 8 1.5 Research Objectives ........................................................................................ 9 Chapter 2: Materials and Methods ..................................................................................10 2.1 Montbretia Plant Material and Collection .........................................................10 2.2 Metabolite Extraction .......................................................................................10 2.3 LC-MS Analysis ..............................................................................................11 2.4 MALDI Analysis ...............................................................................................11 2.5 Total Protein Extraction and AT Enzyme Activity Assays ................................12 2.6 Identification of BAHD-AT Transcripts .............................................................13 2.7 Differential Expression and Haystack Analysis ................................................13 2.8 BAHD-AT Sequence Analysis and Phylogeny Construction ............................14 2.9 cDNA Synthesis and BAHD-AT Cloning ..........................................................14 2.10 Heterologous BAHD-AT Expression in E. coli .................................................15 2.11 Recombinant BAHD-AT Enzyme Assays ........................................................17 2.12 Transient BADH-AT Expression in N. benthamiana ........................................18 vii  2.13 Real-time RT-PCR ..........................................................................................20 2.14 Statistical Analysis ..........................................................................................21 Chapter 3: Results - Investigation of development of montbretia corms and MbA accumulation pattern .......................................................................................................22 3.1 Vegetative Propagation of Montbretia Plants and Corm Development ............22 3.2 Spatiotemporal Accumulation Pattern of MbA .................................................24 3.3 Putative Intermediates in MbA Biosynthesis ....................................................28 3.4 Distribution Pattern of Metabolites within Montbretia Corms ............................33 Chapter 4: Results - Discovery and Characterization of ATs Involved in MbA Biosynthesis .....................................................................................................................34 4.1 Acylation Activity of Total Corm Protein ...........................................................34 4.2 Differential Expression of BAHD-ATs in Montbretia Transcriptome .................35 4.3 Cloning and Heterologous Expression of Candidate BAHD-ATs in E. coli .......37 4.4 Transient Expression of Candidate BAHD-ATs in N. benthamiana ..................42 4.5 Transcript Abundance Pattern of BAHD-ATs in Montbretia Corms ..................45 Chapter 5: Discussion .....................................................................................................46 5.1 Spatiotemporal Regulation of MbA Biosynthesis and Plant Defense ...............46 5.2 Substrate Specificity of CcAT1 and CcAT2 .....................................................48 5.3 Alternative MbA Production Systems ..............................................................51 Chapter 6: Conclusion and Future Directions ................................................................53 Bibliography .....................................................................................................................55 Appendix ...........................................................................................................................60 Supplementary Figure ................................................................................................60 Supplementary Tables ................................................................................................61    viii  List of Tables Table 1. MbA concentration in dried montbretia corm samples ..........................................27 Table 2. Total amount of MbA in individual old and young corms of montbretia plants .......27 Table 3. MbA levels in different organs of montbretia .........................................................28 Table S4. Targeted m/z for MALDI analysis of old and young corms of montbretia plants ..61 Table S5. BAHD acyltransferases used in phylogenetic analyses ......................................62 Table S6. Differential expression of AT transcripts in montbretia corms .............................63 Table S7. Primer oligonucleotide sequences ......................................................................65 Table S8. Metabolite levels in old and young corms of montbretia plants during Feb. 2016 – Oct. 2017 ...........................................................................................................................67 Table S9. Gene expression correlation analysis of BAHD-ATs ...........................................70 Table S10. Statistical analysis of MbA levels in montbretia corms ......................................73 Table S11. Statistical analysis of CcAT1 and CcAT2 relative transcript abundance in montbretia corms ...............................................................................................................75 Table S12. Statistical analysis of MbA levels in dried montbretia corms .............................77 Table S13. Statistical analysis of total MbA levels per corm ...............................................78   ix  List of Figures Figure 1. Anti-diabetic compound montbretin A (MbA) found in the corms of montbretia. .... 3 Figure 2. Schematic of potential intermediates in MbA biosynthesis. ................................... 6 Figure 3. Biosynthetic pathway of MbA. ............................................................................... 7 Figure 4. The development of montbretia corms and MbA accumulation in corms .............23 Figure 5. Corm extracts analyzed by LC-MS/UV in negative ionization mode.....................25 Figure 6. Potential intermediates in MbA biosynthesis present in montbretia corm extracts29 Figure 7. Accumulation pattern of potential MbA biosynthesis intermediates in montbretia corms .................................................................................................................................32 Figure 8. MALDI-FTICR analysis of MbA and potential intermediates in a 1-year-old corm and a young corm of montbretia .........................................................................................33 Figure 9. BAHD-AT activity in total protein extracts of montbretia corms ............................35 Figure 10. Phylogenetic analysis of montbretia BAHD-ATs ................................................36 Figure 11. Western blot analysis of AT protein expression .................................................37 Figure 12. Activity of CcATs heterologously expressed in E. coli ........................................38 Figure 13. Acyl donor specificity of CcAT1 and CcAT2 .......................................................40 Figure 14. Acyl acceptor specificity of CcAT1 and CcAT2 ..................................................42 Figure 15. Activity of CcAT1 and CcAT2 transiently expressed in N. benthamiana ............44 Figure 16. Relative transcript abundance of CcAT1 and CcAT2 in old corm (oC) and young corm (yC) of montbretia plant .............................................................................................45 Figure 17. Amino acid sequence alignment of montbretia BAHD-ATs and literature BAHD-ATs from other plants .........................................................................................................49 Figure S18. Schematic drawing of montbretia plant development over a vegetative cycle ..60   x  List of Symbols and Abbreviations   α  Alpha  ANOVA Analysis of variance AT Acyltransferase β Beta  BAHD Benzyl alcohol O-acetyltransferase-anthocyanin O-hydroxycinnamoyltransferase-N-hydroxycinnamoyl/benzoyltransferase-deacetylvindoline 4-O-acetyltransferase  BLASTP Basic local alignment search tool for proteins CcAT Crocosmia x crocosmiiflora acyltransferase  cDNA Complementary deoxyribonucleic acid  CoA Coenzyme A DW Dry weight FTICR Fourier-transform ion cyclotron resonance  HPA Human pancreatic α-amylase  HPLC High-performance liquid chromatography  Ki Inhibitory constant   LC-MS Liquid chromatography-mass spectrometry  M Myricetin  MALDI Matrix-assisted laser desorption ionization MbA Montbretin A MbA-C Myricetin 3-O-glucosyl glucosyl rhamnoside 4’-O-rhamnosyl xyloside MbA-XR2 Myricetin 3-O-(6-O-caffeoyl)-glucosyl glucosyl rhamnoside MbB Montbretin B MbC Montbretin C MG Myricetin 3-O-glucoside  Mini-MbA Myricetin 3-O-(6-O-caffeoyl)-glucosyl rhamnoside  MR Myricetin 3-O-rhamnoside  MRG Myricetin 3-O-glucosyl rhamnoside MS Mass spectrometry  MS/MS Tandem mass spectrometry  oC Old corm π Pi  PCR Polymerase chain reaction QGG Quercetin 3-O-glucosyl glucoside  qRT-PCR  Real-time reverse transcription polymerase chain reaction RNA Ribonucleic acid  RNA-Seq Transcriptome sequencing  SCPL Serine carboxypeptidase-like  SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis  T2D Type-2 diabetes  UDP Uridine 5’-diphosphate  UGT Uridine 5’-diphosphate glycosyltransferase  USER Uracil-specific excision reagent  UV Ultraviolet  yC Young corm  xi  Acknowledgements A thousand ‘thank you’s would not be enough to express my gratitude to my supervisor, Dr. Jörg Bohlmann, for his support and guidance throughout my Master’s degree.  His kindness and patience have always amazed me, and I feel truly honoured to have been a part of his research group. I would also like to thank my committee members, Dr. Reinhard Jetter and Dr. Simone Castellarin, for their expert advice and invaluable feedback on various stages of my project.  I would also like to express my gratitude to everyone in the Bohlmann Lab, for their wonderful friendship and support. A heartfelt thank you to Dr. Sandra Irmisch, who has been an incredible mentor and a friend. Without your invaluable expertise and continuous encouragement, I would not have been able to achieve what I have. I would also like to thank Dr. Carol Ritland for her project management but also for her warm compassion. Thank you also to Mack Yuen for his assistance on the transcriptomic analysis, and to Lina Madilao, for her expertise with LC-MS. I am also grateful to Angela Chiang for her amazing lab management and to Sharon Jancsik for all her technical support. Thank you also to Kristina Kshatriya for being a fantastic friend, I will miss our lunch dates.    Lastly, I am forever grateful to my parents, who have dedicated their lives to provide me with countless opportunities. Thank you for your continuous love and support.     xii  Dedication          To all the montbretia plants that were sacrificed. 1  Chapter 1: Introduction  1.1 Plant Specialized Metabolites Plants produce thousands of different metabolites collectively termed plant specialized metabolites. Many of the specialized metabolites may not always be essential for the growth and development of the plant, but they may be critical for the plant’s survival under specific environmental conditions (Hartmann, 2007). For example, specialized metabolites may protect plants against abiotic and biotic stresses such as UV radiation, disease, herbivory, and may function in a diversity of plant interactions with other organisms (Bourgaud et al., 2001). Due to diverse biological activities of these compounds, many of them have been employed by humans as medicines, flavours, or fragrances. For example, derivatives of artemisinin, which was first isolated from Chinese wormwood, are used as anti-malaria treatments (White 2008). Another example is Taxol, a diterpene discovered in the bark of yew trees, used in cancer chemotherapy (McGuire et al., 1989). Plant metabolites continue to be a valuable source for new pharmaceutical development, and the discovery of these high-value compounds continues to address global health concerns.    1.2 Type-2 Diabetes and Montbretin A  Type-2 diabetes (T2D) is one of the growing worldwide epidemics, affecting over 320 million people globally (http://www.who.int). Patients suffering from T2D are unable to control blood glucose levels due to the body’s ineffective use of insulin, leading to hyperglycaemia and associated long-term complications. Thus one approach for T2D treatment is to control blood glucose levels (Crandall et al., 2008). The body mainly obtains glucose from digested carbohydrates in foods, such as starch. The pancreatic α-amylase (HPA) and gut α-glycosidases are responsible for digestion of starch into oligosaccharides and subsequently to monosaccharaides, respectively. Currently available drugs such as Acarbose, Miglitol and Voglibose inhibit α-glucosidases to reduce sugar uptake. However, these drugs lead to the formation of oligosaccharide fragments that become fermented by the microbiome in the colon. This results in unpleasant gastrointestinal side effects such as abdominal pain, diarrhea, flatulence, and ultimately leading to patient noncompliance (Scheen, 1997; Garcia-Perez et al., 2013). To address these problems and minimize potential side effects, development of specific drugs inhibiting HPA, the enzyme that initializes starch degradation, would be advantageous.  2  In 2008, Tarling et al. screened a library of approximately 30,000 terrestrial and marine natural product extracts in search for new potential HPA inhibitors. In this process, montbretin A (MbA) from the ornamental plant montbretia (Crocosmia x crocosmiiflora) was found to be a potent and specific HPA inhibitor with a Ki of 8.1 nM (Tarling et al., 2008). MbA is a complex acylated flavonol glycoside composed of the building blocks myricetin, caffeic acid, UDP-rhamnose, UDP-glucose and UDP-xylose (Fig. 1A). The 3-hydroxyl of the myricetin core is α-linked to the 1-hydroxyl of the linear trisaccharide D-glucosyl-(β1→2)-D-glucosyl-(β1→2)-L-rhamnoside, with a caffeic acid moiety attached to the 6-hydroxyl of the central glucosyl sugar. The 4’-hydroxyl of myricetin is β-linked to the linear disaccharide L-rhamnosyl-(α1→4)-D-xyloside. 3   Figure 1. Anti-diabetic compound montbretin A (MbA) found in the corms of montbretia. (A) Chemical structure of MbA. MbA is composed of the building blocks myricetin, caffeic acid, rhamnose (Rha), glucose (Glc) and xylose (Xyl). Position of the hydroxyl groups of myricetin and A,B,C designation of the aromatic rings are shown. Dashed rectangle highlights the structure of mini-MbA (myricetin 3-O-[6-O-caffeoyl]-β-D-glucosyl (12)-α-L-rhamnoside). (B) Representative montbretia plant. (i) Whole image of flowering montbretia plant. (ii) Different organs of the montbretia plant. Plant organs are labeled: leaves (L); stem (s); seed buds (sb); flower (f); old corm (oC); young corm (yC); stolon (st); roots (r). Scale bar = 1 cm. B (i)MyricetinAcyl GroupXylRhaRhaGlcGlcBCA3573’4’5’ARAcyl Group RMontbretin A Caffeoyl OHMontbretin B Coumaroyl HMontbretin C Feruloyl OMel srstyCoCfsb1cm(ii)4  To understand the importance of individual building blocks of the complex MbA molecule, Williams et al. (2015) investigated various MbA derivatives. Montbretin B (MbB) and montbretin C (MbC) are natural analogs of MbA found in the montbretia plant. Instead of the caffeoyl moiety, MbB and MbC have coumaroyl and feruloyl moiety, respectively. The HPA inhibitory activity of MbB and MbC was more than 400 times weaker than MbA (Williams et al., 2015).  Furthermore, Williams et al. (2015) synthesized and investigated the MbA precursor molecule mini-MbA (myricetin 3-O-[6-O-caffeoyl]-β-D-glucosyl (12)-α-L-rhamnoside) (Fig. 1A). Compared to MbA, mini-MbA is a less complex molecule lacking the 4’ disaccharide chain and the terminal glucose, but was a potent and specific HPA inhibitor with Ki of 93.3 nM (Williams et al., 2015). These findings suggested the importance of the caffeoyl moiety in HPA inhibition. Further in depth X-ray structural analysis on the MbA-HPA complex revealed that the π-stacking interaction between the two aromatic moieties, myricetin and caffeoyl moiety is crucial for the HPA inhibition of MbA (Williams et al., 2015). The highly effective and specific inhibition of HPA makes MbA an attractive new drug candidate for the treatment of T2D. To explore the potential advantages of MbA as a glucose lowering agent, trials with “zucker diabetic fatty” rats have been conducted by Yuen et al. (2016). Compared to Acarbose, a commercially available α-glucosidase inhibitor, lower doses of MbA was required to be an effective T2D treatment (Yuen et al., 2016). However, in order to develop MbA as an anti-diabetic drug, large quantities need to be readily accessible. Due to the complex chemical structure of MbA, chemical synthesis of this compound is currently not feasible. An alternative method of obtaining large quantities of MbA is directly extracting MbA from the natural source.  MbA was predominantly found in the corms of montbretia (Andersen et al., 2009; Roach, 2017).  The corms are stem-derived underground storage organs that function in the vegetative propagation of the plant. MbA was found to accumulate in rather low amounts (1.85 – 4.31 mg x g-1 FW) in montbretia corms (Roach, 2017). In addition to the low abundance of MbA in planta, harvesting the corms would effectively destroy the plant. For these reasons, extraction of MbA from montbretia plants is likely not currently a realistic method for large volume MbA production. Natural harvesting of MbA may be a feasible method for obtaining large quantities of MbA if high MbA producing montbretia cultivars can be developed. Alternatively, metabolic engineering of MbA in microbial or other plant systems may also lead to the large-scale production of MbA. The Bohlmann laboratory has been investigating the later method of MbA production. In order for metabolic engineering of 5  MbA in alternative systems, fundamental knowledge on the MbA biosynthetic pathway is required.   1.3 MbA Biosynthetic Pathway The predicted biosynthetic pathway of MbA can conceptually be divided into two stages: (i) the biosynthesis of the building blocks of MbA and (ii) the assembly of the building blocks into the complex MbA molecule (Roach, 2017). Three different metabolic pathways are likely to be involved in the production of MbA building blocks. This includes the employment of the flavonoid pathway to synthesize myricetin, the phenylpropanoid pathway to produce caffeoyl-CoA, and the nucleotide sugar pathway to obtain UDP-glucose, UDP-rhamnose and UDP-xylose. Theoretically, the building blocks can be assembled into MbA by 29 different ways, resulting in many potential intermediate compounds (Fig. 2A). The stepwise assembly of the MbA molecule was predicted to involve five UDP-glycosyltransferases (UGTs) and one acyltransferase (AT) (Roach, 2017). Building on this hypothesis, the first two steps of MbA assembly was revealed by Irmisch et al. (submitted) (Fig. 3). Myricetin is first rhamnosylated by UGT1 at the 3-hydroxyl position, resulting in the formation of myricetin 3-O-rhamnoside (MR). The second step of MbA assembly is the addition of glucose to myricetin 3-O-rhamnoside by UGT2 to form myricetin 3-O-glucosyl rhamnoside (MRG) (Irmisch et al., submitted). The acylation of MRG involving the addition of the caffeoyl moiety to the 6-hydroxyl of the central glucosyl sugar was predicted as the third step of MbA assembly (Irmisch et al., submitted). However, the enzyme responsible for this reaction remained unknown. The caffeoyl moiety was revealed to play a critical role in the inhibition of HPA (Williams et al., 2015). Thus elucidating the genes and enzymes responsible for the addition of the caffeoyl moiety to MRG is essential for future metabolic engineering of MbA or the simplified derivative, mini-MbA.  6   Figure 2. Schematic of potential intermediates in MbA biosynthesis. (A) Schematic structures of the potential intermediates in steps I-VII of MbA assembly are shown. M, myricetin (pink); R, rhamnose (yellow); G, glucose (green); X, xylose (orange); C, caffeic acid (blue). (B) Corresponding mass of the potential intermediates. Intermediates identified in the plant extracts are shown in bold. (M-H)-, molecular mass in negative ion mode.   Compound (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (n) (o) (p) (q) (r) (M-H) 317 463 449 625 595 595 787 787 757 741 949 919 919 903 1081 1065 1065 1227IIIIII IV VVIVIIMRRXGGCMRRXGCMRRXGGMRXGGCMRXGCMRRXGMRXGGMRGGCMRGCMRGGMRXGMRRXMRXMRXMRGMXMRM(a)(b)(c)(d)(e)(f)(g)(h)(i)(j)(k)(l)(m)(n)(o)(p)(q)(r)AB-7   Figure 3. Biosynthetic pathway of MbA. Myricetin is first rhamnosylated by UGT1 to produce myricetin 3-O-rhamnoside (MRG), which is subsequently glucosylated by UGT2, resulting in the formation of myricetin 3-O-glucosyl rhamnoside (MRG). CcAT1 and CcAT2 catalyze the formation of myricetin 3-O-(6-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) from myricetin 3-O-glucosyl rhamnoside (MRG) and caffeoyl-CoA. Solid arrows indicate characterized steps; the dashed arrow indicates so far unknown downstream steps of MbA biosynthesis. MR, myricetin 3-O-rhamnoside.  (Irmisch et al., submitted)(Irmisch et al., submitted)UGT1MRMRGMyricetinMbAMini-MbAUGT2CcAT1 / CcAT28  1.4 BAHD-Acyltransferases Acylation is the transfer of an acyl group from an activated energy-rich donor to an acceptor. It is a common modification of plant specialized metabolites, influencing functions and properties of the compound, such as solubility and stability (Bontpart et al., 2015). There are two families of ATs that catalyze the acylation of specialized plant metabolites: BEAT-AHCT-HCBT-DAT (BAHD) and Serine CarboxyPeptidase-Like (SCPL) acyltransferases. There seem to be no species or compound specificity between the two AT families. Both AT families are able to acylate a variety of compounds belonging to the same families in the same plant species (Bontpart et al., 2015). The main differences between the two families are the localization and the type of energy-rich donors they use. The SCPL-ATs are vacuolar and use 1-O-β-glucose ester donors while BAHD-ATs are cytosolic and use acyl-CoA thioester donors (Bontpart et al., 2015). Currently, there are only a handful of characterized SCPL-ATs while there are over 60 BAHD-ATs that are characterized (D’Auria, 2006; Bontpart et al., 2015). The members of the BAHD-AT family share a low protein sequence identity except for the two conserved motifs, the HXXXD motif and the DFGWG motif (St-Pierre and De Luca, 2000). The BAHD-ATs are grouped into five major phylogenetic clades (D’Auria, 2006). Clade I contains all of the currently characterized BAHD-ATs involved in the acylation of flavonoids, except for the malonyltransferase from Salvia splendens, Ss5MAT2, in clade III (Bontpart et al., 2015; Suzuki et al., 2004). Clade II is a small clade composed of only two members currently involved in the elongation of cuticular waxes (Xia et al., 1996; Tacke et al., 1995). Clade III is composed mostly of members that use aliphatic acyl donors, but also accept a diversity of acyl acceptors (D’Auria, 2006). Clade IV is the smallest BAHD-AT clade, composed of only one known member, the argmatine coumaroyltransferase, ACT, from Hordeum vulgare (Burhenne et al., 2003). Clade V members are known for their versatile range of substrates including terpenoids, medium-chain alcohols, and quinic acids (Tuominen et al., 2011). Generally, members within each clade show more specificity towards acyl acceptors in comparison to acyl donors (D’Auria, 2006; Bontpart et al., 2015). For example, the hydroxylcinnamoyl-transferase from Arabidopsis thaliana, At3AT1, and the malonyltransferase from Dahlia variabilis, Dv3MAT, both utilize flavonoids as the acyl acceptor, but use either aromatic or aliphatic acyl donors, respectively (Luo et al., 2007; Suzuki et al., 2002). A phylogenetic analysis of BAHD-ATs across different plant taxa 9  revealed that clade V and clade I are generally the most represented clades (Tuominen et al., 2011).  1.5 Research Objectives The overall goal of my thesis was to gain fundamental insight on MbA biosynthesis. The first objective of my project was to determine where and when MbA was biosynthesized in the montbretia plant. As MbA was predominantly found in the corms of montbretia plant, I hypothesized that the corm is a likely site of MbA biosynthesis. To determine this, I first observed the growth and development of the montbretia plant with emphasis on the corms, followed by a thorough metabolite analysis. As my objective was to investigate the site and the time point of MbA biosynthesis, I monitored the accumulation pattern of both MbA and all of its potential intermediates. The results of my first objective provided a foundational knowledge on the MbA biosynthesis in montbretia corms in order to study the biosynthesis of MbA at a molecular level.  The second objective of my project was to discover and characterize the AT involved in the third step of MbA assembly. As the majority of known ATs involved in the modification of flavonoid glucosides belong to the BAHD-AT family, I hypothesized that the acylation step in MbA biosynthesis is likely catalyzed by a BAHD-AT rather than a SCPL-AT. I confirmed the involvement of a BAHD-AT in MbA biosynthesis through looking at the activity of the corm protein extracts. Using an established montbretia transcriptomic database (Irmisch et al., submitted), I identified candidate montbretia BAHD-ATs. Subsequently, the candidate ATs were cloned and heterologously and transiently expressed in Escherichia coli and Nicotiana benthamiana, respectively.  The results of my project will offer groundwork required to facilitate a large-scale production of MbA and/or mini-MbA through metabolic engineering in alternative systems in the future. Alongside with the practical application to human health, this study will provide new fundamental knowledge on specialized metabolite biosynthesis in a non-model plant species, montbretia.   10  Chapter 2: Materials and Methods  2.1 Montbretia Plant Material and Collection  The corms of the “Emily McKenzie” variety of montbretia (Crocosmia x crocosmiiflora) were obtained from Dr. Gary Brayer’s private collection in Richmond, British Columbia, Canada in July 2010. The montbretia plants were separated by the individual corms and were maintained in 4 L pots with perennial potting soil. The plants were grown in a partially shaded outdoor patio of the University of British Columbia Horticulture Greenhouse. As part of annual plant maintenance, decaying above ground tissues such as leaves and stems were removed in November. The pots were covered for over-wintering. In November or February, plants were re-potted by separating the clusters of corms and individually placing each corm in a separate pot. Corms were collected throughout February 2016 – October 2018. Four to six biological replicates were harvested at least once a month. The original corm was designated as the old corm (oC), and the newly developed corms during the growing season were designated as the young corms (yC). For each biological replicate, the yC were pooled and were collected separately from the oC. Other parts of the plant were collected during the summer months (June 27th, July 22nd, August 16th of 2016), including stems, leaves, flower buds, flowers, seed pods, stolons and roots. All plant samples were sectioned with a razor blade, flash frozen in liquid nitrogen and stored in -80 ºC until further use.    2.2 Metabolite Extraction  Frozen plant samples were ground into fine powder using a mortar and pestle with liquid nitrogen. The metabolite extraction protocol was established by Irmisch et al. (submitted). For extraction, 60 mg of the ground samples were extracted using 1 mL of 50% (v/v) MeOH/H2O for 2 h shaking at 21 ºC. Following centrifugation at 15,871 x g for 30 min at 21 ºC, supernatant was collected and the remaining material was re-extracted. Supernatants from the first and second extraction were combined. A volume of 200 uL of the combined sample was diluted 1:10 in 50% (v/v) MeOH/H2O. Undiluted and 1:10 diluted samples from four to six biological replicates from each time point and sample type were used for metabolite analyses. MbA levels of dried oC and yC were analyzed using samples harvested from June 27th, July 22nd and September 12th of 2016. To obtain dry samples, 60 11  mg of the ground corm samples were dried at 50 ºC for three days. The dry weight (DW) was measured and the dried samples were extracted as described above. All extracts were analyzed with LC-MS as described below.   2.3 LC-MS Analysis Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the plant extracts and enzyme assays. LC was performed using an Agilent 1100 High Performance Liquid Chromatography (HPLC) system (Agilent Technologies GmbH, Waldbronn, Germany) with Agilent ZORBAX SB-C18 column (50 x 4.6 mm, 1.8 μm particle size) (Merck, Darmstadt, 370 Germany). Aqueous formic acid (0.2% v/v) was used as mobile phase A, and acetonitrile with formic acid (0.2% v/v) was used as mobile phase B. The elution profile was as follows: 0 – 0.5 min 95% A; 0.5 – 5 min, 5 – 20% B in A; 5 – 7 min 90% B in A; 7.1 – 10 min 95% A. The flow rate was 0.8 mL x min-1 at a column temperature of 50 ºC. LC was coupled to an Agilent mass spectrometry detector (MSD) Trap (XCT-Plus) equipped with an electro-spray operated in negative ionization mode (capillary voltage, 4000 eV; temp, 350 ºC; nebulizing gas, 60 psi; dry gas 12 L/min). Diode array detector (DAD; J&M Analytik AG, Aalen, Germany) was used to monitor wavelengths from 200 – 700 nm. MS/MS was used to monitor the precursor ion formation. The LC MSD Trap Data Analysis software was used for data acquisition and processing. Plant metabolites and enzyme products were quantified using an external MbA standard curve, as MbA was the only authentic standard available in sufficient amounts to create a standard curve. The MbA standard curve was composed of ten different concentrations of MbA standards (1, 2.5, 5, 7.5, 10, 15, 25, 50, 75 100 mg/uL). MbA intermediates such as myricetin 3-O-glucosyl glucosyl rhamnoside 4’-O-rhamnosyl xyloside (MbA-C), myricetin 3-O-glucosyl rhamnoside (MRG), myricetin 3-O-rhamnoside (MR), and enzyme formation of mini-MbA were identified with authentic standards and MS/MS fragmentation pattern. MbA, MbA-C and mini-MbA were provided by Dr. Stephen Withers (UBC). MRG was provided by Dr. Sandra Irmisch in the Bohlmann laboratory. MR was obtained from Sigma-Aldrich (http://www.sigmaaldrich.com).  2.4 MALDI Analysis  Matrix Assisted Laser Desorption/Ionization (MALDI) imaging was performed at the University of Victoria and Genome BC Proteomics Centre. The oC and yC samples collected 12  on September 6th, 2017 were used for analyses. The fresh corm samples were gradually frozen to -20 ºC for at least 1 h. The frozen corms were sectioned at -20 ºC within a Microm HM500 cryostat into 20 μm and 40 μm slices for yC and oC, respectively. The matrix solution was prepared using 10 mg x mL-1 of 2-mercaptobenzothiazole (2-MBT) in 80% (v/v) MeOH containing 2% (v/v) formic acid. The corm sections were spray coated with the matrix solution using a Bruker Daltonic ImagePrep electronic matrix sprayer with the MCAEF technique in negative mode (Wang et al., 2015). The MALDI-mass spectrometry (MS) data was obtained in negative ion mode using the Bruker Apex-Qe 12-Tesla hybrid quadrupole-fourier transform-ion cyclotron resonance (FTICR) as the mass spectrometer. The instrument was equipped with a 355 nm UV laser, and the mass detection ranged from m/z 250 – 2300. The target compounds for detection are listed in Table S4. For tissue imaging, a laser raster step size of 200 μm and 300 μm for yC and oC was used, respectively, with the laser beam size of about 200 μm. The mass spectral datasets were processed with Bruker DataAnalysis. The MS ion images were constructed with Bruker FlexImaging.   2.5 Total Protein Extraction and AT Enzyme Activity Assays   An amount of 300 mg of powdered corm samples were extracted using 1.5 mL of extraction buffer [100 mM sodium phosphate buffer (NaPi), pH 7.4, 5 mM ascorbic acid, 5 mM sodium bisulfite, 5 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone (PVP), 4% (w/v) polyvinylpolypyrrolidon (PVPP), 4% (w/v) Amberlite XAD-4, 0.1% (v/v) Tween)] for 1 h at 4 ºC. After centrifugation at 4000 x g for 30 min at 4 ºC, the supernatant was collected and desalted three times into assay buffer (10 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol (DTT), 10% (v/v) glycerol) using NAP-5 columns (GE-Healthcare). Enzyme activity assays were composed of 75 μL of the desalted protein extract, 50 μM of MRG as the acyl-acceptor and 120 μM of caffeoyl-CoA (TransMIT, http://www.plantmetachem.com/) as the acyl-donor in a total volume of 100 μL of assay buffer. MRG and caffeoyl-CoA were prepared in 50% (v/v) dimethyl sulfoxide (DMSO)/H2O. Control assays were set up in the same way as the enzyme activity assays, but using coumaroyl-CoA instead of caffeoyl-CoA as the acyl donor. The purpose of the control assay was to monitor the presence of mini-MbA from the corm protein extracts. The assays were incubated with gentle shaking at 21 ºC for 6 h in a Teflon-sealed, screw-capped 1-mL GC glass vial. Assays were stopped by the addition of 100 μL of MeOH and samples were 13  immediately placed on ice. Following centrifugation at 4300 x g for 20 min at 4 ºC, the supernatant was transferred into a glass insert and assays were analyzed using LC-MS.   2.6 Identification of BAHD-AT Transcripts  The montbretia transcriptome database developed by Irmisch et al. (submitted) was used to identify putative montbretia BAHD-AT transcripts. This transcriptome resource was established from two biological replicates of oC and yC collected on June 10th, 2016. BLASTP search was performed against the translated montbretia protein database using published BAHD-ATs as queries representing different BAHD-ATs from all reported BAHD-AT clades (I-V). The literature BAHD-ATs used as queries can be found in Table S5. A phylogenetic tree was constructed using the obtained putative montbretia BAHD-ATs and literature BAHD-ATs from other plant species (Table S5) to identify representative montbretia BAHD-ATs from each clade. Reciprocal BLASTP search was performed with the representative montbretia BAHD-ATs to obtain all putative montbretia BAHD-AT candidates. To filter the results, sequences with 98% similarity were removed using CD-HIT (v 4.6.1, http://cd-hit.org), and short sequences with a length less than 250 amino acids (aa) were removed. The list of putative BAHD-ATs was manually assessed to remove likely chimeric or misassembled genes, resulting in a total of 59 montbretia BAHD-AT sequences.   2.7 Differential Expression and Haystack Analysis   The differential expression analysis of montbretia BAHD-ATs was performed as described in Irmisch et al. (submitted). Of the total 59 putative BAHD-ATs in montbretia, 50 putative BAHD-ATs were differentially expressed between oC and yC (Table S6). This list was filtered by identifying BAHD-ATs showing at least 2-fold higher expression in yC compared to oC, resulting in a refined list of 27 putative BAHD-ATs. Additionally, BAHD-AT expression in other organs of montbretia (leaf, stem, stolon) was assessed using RNA-Seq data developed by Roach (2017). The Haystack (http://haystack.mocklerlab.org/) software was used to study the expression pattern of putative BAHD-ATs. To identify putative ATs that show correlating expression pattern with enzymes involved in MbA biosynthesis, UGT1 and UGT2 (Irmisch et al., submitted) were used as baits for Haystack analysis. The Haystack parameters were set as follows: correlation cut off of 0.5, fold cut-off of 1, P-value cut-off of 0.05, background cut-off of 1.   14  2.8 BAHD-AT Sequence Analysis and Phylogeny Construction  All sequence analysis and phylogenetic tree construction was performed using MEGA6 (http://www.megasoftware.net). An amino acid alignment of the 27 putative montbretia BAHD-ATs and published BAHD-ATs from other plant species (Table S4) was created using the MUSCLE algorithm (gap open, -2.9; gap extend, 0; hydrophobicity multiplier, 1.5; clustering method, upgmb) (Tamura et al., 2011). This alignment was used to create a phylogenetic tree using the neighbour-joining algorithm (Poisson model) with a bootstrap value of 1000.  To visualize the conserved HXXXD and DFGWG motifs in CcAT1 and CcAT2, an amino acid sequence alignment of CcAT1, CcAT2 and literature BAHD-ATs were created using BioEdit (http://www.mbio.ncsu.edu/bioedit/bioedit.html) and the ClustelW algorithm available within the program. Accession numbers and details of the literature BAHD-ATs (At3AT1, NtMAT1, TAT, CHAT, TpHCT1A) are listed in Table S5.  2.9 cDNA Synthesis and BAHD-AT Cloning Total RNA of montbretia corm samples were provided by Dr. Sandra Irmisch and are described in Irmisch et al. (submitted). cDNA was synthesized from 650 ng of total RNA using Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific, https://www.thermofisher.com) according to manufacturer’s instructions. The synthesized cDNA was diluted 1:5 in water for further use. For cloning of candidate BAHD-ATs, cDNA pool was generated from oC and yC harvested from Jun 2016 – October 2016. Target montbretia BAHD-ATs were PCR amplified from the cDNA pool using Phusion High-Fidelity DNA Polymerase (ThermoFisher Scientific) following manufacturer’s instructions. Gene specific primers listed in Table S7 were used to amplify the open reading frames of candidate BAHD-ATs. The PCR products were separated by agarose gel electrophoresis. The product bands corresponding to the expected molecular weight of the PCR products were identified, and the bands were gel extracted using QIAquick Gel Extraction Kit following manufacturer’s instructions (Qiagen, http://www.qiagen.com). The extracted PCR fragments were cloned into pJET1.2/blunt vector using CloneJET PCR Cloning Kit (ThermoFisher Scientific) according to manufacturer’s instructions. The pJET vectors carrying the target BAHD-ATs were subsequently heat-shock transformed into chemically competent E. coli TOP10 cells (Invitrogen, http://www.thermofisher.com). Transformed E. coli cells were grown on Lysogeny Broth (LB) agar plates containing ampicillin (100 mg x L-1) 15  at 37 ºC O/N. Individual colonies were selected to inoculate 3 mL of LB media with ampicillin (100 mg x L-1), and the cultures were grown shaking at 37 ºC O/N. Plasmid preparation from the overnight cultures were performed using QIAprep Spin Miniprep Kit (Qiagen) according to manufacturer’s instructions, and were sequenced for sequence verification  2.10 Heterologous BAHD-AT Expression in E. coli  The open reading frames of the candidate BAHD-ATs were cloned into pASK-IBA37+ expression vector (IBA-GmbH, Gottingen, Germany) with N-terminal His6-tag. Type IIS restriction endonucleases were used for directional cloning of the candidate BAHD-ATs into the expression vector. The sequence verified pJET vector constructs containing the target BAHD-ATs were diluted 1:100 in water, and were used as templates for pASK-IBA37+ cloning. Candidate BAHD-ATs were PCR amplified from the diluted pJET constructs using Phusion High-Fidelity DNA Polymerase, following manufacturer’s instructions. The pASK-IBA37+ vector was cut with BsaI (NEB, http://www.neb.com) to create non-palindromic overhangs. Primers used to PCR amplify the target BAHD-ATs were designed using Primer D’Signer (http://en.bio-soft.net/pcr/DSigner.html). The gene specific primers contained overhang adaptors corresponding to the pASK-IBA37+ vector, and restriction sites of BsaI, BspMI or BsmBI (Table S7). Following PCR amplification, the amplified gene products were verified and separated by agarose gel electrophoresis. The product bands corresponding to the estimated molecular weight of the amplified genes were identified and gel extracted as described in Section 2.9. The concentrations of the extracted gel products were measured using the absorbance at 260 nm with NanoDrop 1000 (ThermoFisher Scientific). Total of 200 ng of the amplified DNA was digested with respective restriction enzyme (BsaI, BspMI or BsmBI; NEB). Digested fragments were ligated with pre-cut pASK-IBA37+ vector using T4 ligase (ThermoFisher Scientific) following manufacturer’s protocol, and were subsequently heat-shock transformed into E. coli TOP10 cells. Transformed E. coli cells were grown on LB agar plates containing 100 mg x L-1 ampicillin at 37 ºC O/N. Individual colonies were selected to inoculate 3 mL of LB media with ampicillin (100 mg x L-1), and were grown shaking O/N at 37 ºC. Overnight cultures were used for plasmid preparations using QIAprep Spin Miniprep Kit according to manufacturer’s instructions. Resulting plasmids were sequenced to verify the in-frame position of the gene with the N-terminal His6-tag.    16  After sequence verification, individual colonies were grown in 3 mL of Terrific Broth (TB) media (12 g tryptone, 24 g yeast extract, 8 mL glycerol, 0.17 M KH2PO4 and 0.72 M KHPO4 per L media, pH 7.0) containing ampicillin (100 mg x L-1) at 37 ºC, shaking O/N. For protein expression, 3 mL of the O/N cultures were used to inoculate 47 mL of TB media containing ampicillin (100 mg x L-1). Cultures were grown at 22 ºC, 220 rpm until OD600 of 0.5 was reached. Cultures were induced with 200 μg x L-1 anhydrotetracycline (Sigma-Aldrich) and were grown for another 20 h at 18 ºC, 180 rpm. The cells were harvested by centrifugation at 4000 x g for 20 min at 4 ºC, and the supernatant was removed. The cell pellets were re-suspended in 3 mL of ice-cold extraction buffer (50 mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 10 mM MgCl2, 5 mM dithiothreitol, 1/40 x mL-1 PierceTM Protease Inhibitor Tablet EDTA-free (ThermoFisher Scientific), 0.1 μL x mL-1 Benzonase Nuclease (MilliporeSigma, http://www.emdmillipore.com), 0.2 mg x mL-1 lysozyme). The re-suspended cells were ruptured through five freeze and thaw cycles using liquid nitrogen, followed by centrifugation at 15,871 x g for 20 min at 4 ºC to remove cell debris. The obtained supernatants were desalted into assay buffer (10 mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 1 mM dithiothreitol) using Econopac 10DG columns (BioRad, https://www.bio-rad.com/), resulting in crude bacterial protein extracts.  For protein purification, protein expression and harvesting was performed as described above using a modified extraction buffer [50 mM Tris-HCl, pH 7.5, 2% (v/v) glycerol, 10 mM MgCl2, 5 mM dithiothreitol, 150 mM NaCl, 20 mM imidazole, 1/40 tablet x mL-1 PierceTM Protease Inhibitor Tablet EDTA-free (ThermoFisher Scientific), 0.1 μL x mL-1 Benzonase Nuclease (MilliporeSigma), 0.2 mg x mL-1 lysozyme]. Protein purifications were performed using Ni-NTA agarose columns (Qiagen). Supernatants from the cell lysates were loaded onto Ni-NTA agarose columns equilibrated in re-suspension buffer [50 mM Tris, 2% (v/v) glycerol, 10 mM MgCl2, 5 mM DTT, 150 mM NaCl, 20 mM imidazole]. After sample loading, the columns were flushed once using re-suspension buffer. Soluble protein was eluted from the column using elution buffer [50 mM Tris, 2% (v/v) glycerol, 10 mM MgCl2, 5 mM DTT, 400 mM imidazole] for a total of three times. The first elution fraction was discarded, and the second and third elution fractions were collected separately. The elution fractions were individually desalted into assay buffer using NAP-5 columns (GE-Healthcare). Concentrations of the purified proteins were calculated by dividing the concentration obtained using UV absorption at 280 nm by the extinction coefficient of the protein. The protein extinction coefficients were determined using ExPASY ProtParam tool 17  (https://web.expasy.org/protparam/). Based on the concentrations, the second elution fraction contained majority of the soluble protein sample and was used for further analyses.  Successful protein expressions were confirmed by SDS-PAGE and Western blot. The Western blots were performed with His•Tag® Antibody HRP Conjugate Kit (MilliporeSigma), and detected with Clarity Western ECL Blotting Substrate (Bio-Rad) following manufacturer’s instructions.  2.11 Recombinant BAHD-AT Enzyme Assays  Crude bacterial protein extracts were used to test for enzyme activity. Unless otherwise stated, the 100 μL standard activity assays were composed of 98 μL of crude protein extract, 1 μL of 6 mM caffeoyl-CoA (final concentration of 60 μM) and 1 μL of 12 mM MRG (final concentration of 120 μM) in a Teflon-sealed, screw-capped 1 mL GC glass vial.  Assays were incubated O/N gently shaking at 21 ºC, and stopped by the addition of 100 μL MeOH. After centrifugation at 15,871 x g for 20 min at 4 ºC, the soluble fraction was transferred into an insert for LC-MS analysis. Control for all enzyme assays were performed using protein extracts from E. coli expressing the empty vector.   For further enzyme characterization, Ni-NTA purified CcAT1 and CcAT2 proteins were used. Acyl acceptor specificity was tested against myricetin (M; TCI, http://www.tcichemicals.com/en/ap/), myricetin 3-O-glucoside (MG; Extrasynthese, https://www.extrasynthese.com/), myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), quercetin 3-O-sophoroside (QGG; Sigma-Alderich), rutin (Sigma-Aldrich), arbutin (Sigma-Aldrich) and salicin (Sigma-Aldrich). M, MG, MR, QGG and rutin were prepared in DMSO. MRG was prepared in 50% (v/v) DMSO. Arbutin and salicin were prepared in water. Acyl-acceptor specificity assays were performed in 100 μL volume containing 1 μg of purified CcAT1 or CcAT2, 100 μM acyl acceptor listed above and 150 μM caffeoyl-CoA, shaking for 1 h at 21 ºC. Assays were stopped by the addition of 100 μL MeOH, followed by centrifugation at 15,871 x g for 20 min at 4 ºC. The soluble fractions were transferred into an insert and analyzed by LC-MS. Acyl donor specificity was tested for caffeoyl-CoA, coumaroyl-CoA (TransMIT), feruloyl-CoA (TransMIT), acetyl-CoA (Sigma-Aldrich) and malonyl-CoA (Sigma-Aldrich). All acyl donors except acetyl-CoA and malonyl-CoA were prepared in 50% (v/v) DMSO/H2O. Acetyl-CoA and malonyl-CoA were prepared in water. Acyl donor specificity was tested in 100 μL assays containing 80 ng of purified CcAT1 or CcAT2, 25 μM MRG and 37.5 μM acyl 18  donor listed above, shaking for 10 min at 21 ºC. Assays were stopped by the addition of 100 μL MeOH. The soluble fractions were obtained by centrifugation at 15,871 x g for 20 min at 4 ºC, and were used for LC-MS analysis. To determine relative turnover rates for the different acyl donors, external MbA standard curve was used for product quantification. Enzyme concentration and incubation times were selected at a range where the reaction velocity was linear.    2.12 Transient BADH-AT Expression in N. benthamiana  N. benthamiana plants were grown in potting soil at 26 ºC during the day and at 22 ºC during the night under 16 h light/8 h dark cycle. Leaves of 4-week old N. benthamiana plants were used for transient expression of target ATs. For transient expression in N. benthamiana, the coding regions of CcAT1 and CcAT2 were cloned into pCAMBIA2300U vector as described in Nour-Eldin et al. (2006). The target regions of CcAT1 and CcAT2 were amplified using PfuTurbo Cx Hotstart DNA Polymerase (Agilent) following manufacturer’s instructions. The target regions were amplified using gene specific primers containing uracil residue and vector compatible site (Table S6). PCR products were digested with DpnI (NEB) to remove any residual bacterial DNA. The pCAMBIA vector was prepared for USER cloning by digestion with PacI (NEB) followed by subsequent nicking using Nt.BbvCI (NEB) as described in Nour-Eldin et al. (2006), producing compatible overhangs. To facilitate insertion of the PCR fragment into the pCAMBIA vector, 20 ng of the PacI/Nt.BbvCI digested vector and 1U of the USERTM enzyme (MilliporeSigma) were added to the DpnI digested PCR mixtures. The reaction mixtures were incubated at 37 ºC for 20 min, followed by 25 ºC for 20 min. After incubation, the reaction mixtures were used to transform chemically competent E. coli TOP10 cells. Transformed E. coli cells were grown on LB agar plates containing kanamycin (50 mg x L-1) at 37 ºC O/N. Selection of individual colonies were plasmid purified as described in Section 2.9. The orientation of the insert DNA was verified by sequencing.  After sequence verification, individual pCAMBIA constructs containing CcAT1, CcAT2, UGT1, UGT2, enhanced green fluorescence protein (eGFP) and pBIN::p19 (Voinnet et al., 2003)  were separately transformed into Agrobacterium tumefaciens strain LBA4404. The pCAMBIA vectors containing UGT1, UGT2, eGFP and pBIN::p19 were provided by Dr. Sandra Irmisch in the Bohlmann laboratory. For transformation, 1 µg of the pCAMBIA constructs were added to 100 µL of frozen A. tumefaciens cells. The mixtures were allowed 19  to thaw in 37 ºC water bath, followed by freezing in liquid nitrogen for 5 min, and were subsequently thawed in 37 ºC water bath. After incubating on ice for 20 min, 1 mL of LB media was added and incubated at 28 ºC for 2 h at 220 rpm. The transformed A. tumefaciens cells were collected by centrifugation at 15,871 x g for 1 min. The cells were re-suspended in 100 µL of LB media, and were transferred and grown on LB agar plates containing rifampicin (20 mg x L-1), kanamycin (50 mg x L-1) and gentamycin (20 mg x L-1) for 2 days at 28 ºC. Individual colonies were selected and grown O/N at 28 ºC, 220 rpm in 10 mL of LB media fortified with rifampicin (20 mg x L-1), kanamycin (50 mg x L-1) and gentamycin (20 mg x L-1). Using 1 mL of the overnight cultures, new overnight cultures were inoculated in 9 mL of LB media containing rifampicin (20 mg x L-1), kanamycin (50 mg x L-1) and gentamycin (20 mg x L-1), and were grown at 28 ºC, 220 rpm. Cells from the overnight cultures were collected by centrifugation at 4000 x g for 5 min, and were re-suspended in 10 mL of infiltration buffer (10 mM MES, 10 mM MgCl2, 100 µM acetosyringone pH 5.6) to a final OD600 of 0.5. The suspensions were incubated at 21 ºC for 3 h at 220 rpm. For N. benthamiana leaf infiltration, the following mixtures of transformed A. tumefaciens were prepared:  (i) A. tumefaciens 35S::eGFP + A. tumefaciens pBIN::p19;  (ii) A. tumefaciens 35S::UGT1 + A. tumefaciens 35S::UGT2 + A. tumefaciens pBIN::p19; (iii) A. tumefaciens 35S::UGT1 + A. tumefaciens 35S::UGT2 + A. tumefaciens 35S::CcAT1 + A. tumefaciens pBIN::p19;  (iv) A. tumefaciens 35S::UGT1 + A. tumefaciens 35S::UGT2 + A. tumefaciens 35S::CcAT2 + A. tumefaciens pBIN::p19;  (v) A. tumefaciens 35S::CcAT1 + A. tumefaciens pBIN::p19;  (vi) A. tumefaciens 35S::CcAT2 + A. tumefaciens pBIN::p19.   Equal volumes of each transformed A. tumefaciens suspensions were used to prepare the mixtures. The abaxial surface of the N. benthamiana leaves were infiltrated with appropriate A. tumefaciens mixtures using a needle-free 1 mL syringe. Infiltrated leaves were marked with tape and harvested 4 days after infiltration. Harvested leaves were flash frozen in liquid nitrogen and stored in -80 ºC until further use.   Frozen N. benthamiana leaf samples were ground into fine powder using motor and pestle with liquid nitrogen. For metabolite extraction, 150 mg of the ground samples were 20  extracted with 400 µL of 50% (v/v) MeOH/H2O, shaking for 2 h at 21 ºC. The extraction mixtures were vortexed every 30 min to re-suspend any sediments. Following centrifugation at 4000 x g at 21 ºC for 30 min, the supernatants were used for LC-MS analysis.         2.13 Real-time RT-PCR  The cDNA synthesized as described in Section 2.9 was used for real-time RT-PCR (qRT-PCR) analysis. Each primer pair for CcAT1 and CcAT2 was designed to amplify approximately 170 bp length fragments, Tm around 60 ºC, GC content of 45 – 60%, and primer length of 20 – 25 nt (Table S6). qRT-PCR primers were used to amplify the target gene fragments using Phusion high-Fidelity DNA Polymerase according to manufacturer’s instructions. PCR amplifications were verified by agarose gel electrophoresis to observe the presence of a single band corresponding to the expected molecular weights. The PCR products were cloned into pJET1.2/blunt vector and were transformed into E. coli TOP10 cells for sequence verification, using procedures described in Section 2.9. Primer specificity was verified by sequencing of qRT-PCR products and standard curve analysis. The standard curve was composed of six 1:3 serial dilutions of the template cDNA pool. qRT-PCR analysis was performed on the standard curve using SsoFastTM EvaGreen® Supermix (BioRad) following manufacture instructions. The reactions were performed on Bio-Rad CF96TM (BioRad), loaded in duplicates in 96-well optical reaction plates (Bio-Rad). The standard curve analysis was repeated three times.  After verifying primer specificity, qRT-PCR analyses were performed on oC and yC from June 10th, June 27th, July 22nd, August 16th, September 12th, and October 06th of 2016 with three biological replicates for each sampling date. qRT-PCR reactions were performed on Bio-Rad CFX96TM (BioRad), loaded in duplicates in 96-well optical reaction plates (Bio-Rad). The reactions were prepared with SsoFastTM EvaGreen® Supermix (BioRad) according to manufacturer’s instructions. The thermo-cycling conditions for qRT-PCR was as follows: initial denaturation at 95 ºC for 30 s; 40 cycles of denaturation at 95 ºC for 5 s, and annealing at 60 ºC for 10 s. Fluorescence was measured during the annealing and the extension step of each cycle, and data for the melting curves were recorded at the end of cycling from 55 ºC to 95 ºC. Primers for Serin-incooperator (MEP) and zinc-finger protein (ZF) were provided by Dr. Sandra Irmisch (Irmish et al., submitted) to be used as reference house-keeping genes.   21  2.14 Statistical Analysis  All statistical analyses were performed using SigmaPlot 11.0 for Windows (Systat Software Inc. 2008). Two-way analysis of variance (ANOVA) was performed followed by a Tukey Test to test for significant differences in the MbA accumulation pattern, AT relative transcript levels, and total amount of MbA in oC and yC of different time points. Significant differences in MbA levels in dried and fresh corms were also tested by a two-way ANOVA followed by a Tukey test. Statistical analyses of MbA accumulation levels in corms harvested Feb. 16th 2016 – Feb. 03rd 2017 were performed previously (Irmisch et al., submitted). The data for the AT relative transcript abundance, and the total amount of MbA per corm was transformed (^0.25) to normalize the data.     22  Chapter 3: Results - Investigation of development of montbretia corms and MbA accumulation pattern  3.1 Vegetative Propagation of Montbretia Plants and Corm Development   The corms of the montbretia plant are the only known natural source of MbA. As a part to investigate when and where MbA biosynthesis was occurring, I first observed the growth cycle of the montbretia plant, specifically to observe the development of the corms.  Montbretia plants vegetativley propagate via the use of corms (Fig. 4A, Fig. S18). In early spring, the overwintering corm began to produce multiple stolons which continued to grow and elongate until they reached the surface of the soil (Fig. 4A). From the tip of the stolon, a new montbretia plant including leaves, stems and flowers developed over the growing season (Fig. S18). Around late May, the basal stem area of the newly grown montbretia plant expanded due to a developing new corm. The newly formed corms were termed as young corms (yC), and the original corm was termed as old corms (oC). The yC continued to expand in diameter until the size and the appearance of yC eventually resembled oC around late August (Fig. 4B). In the above ground portion of the plant, flowers started to wither while seedpods developed throughout August (Fig. S18). As seeds continued to ripen, rest of the above ground organs of the montbretia plant, stem and leaves, withered during September to October. Also during this time, the underground stolon connecting oC and yC desiccated while yC started to form stolons of their own (Fig. 4B). These stolons gave rise to new montbretia plants with corresponding yC in the following growth cycle. In average, about five yC developed from a single oC.  23   Figure 4. The development of montbretia corms and MbA accumulation in corms. (A) Schematic drawing summarizing the vegetative growth and development of montbretia 0 10 20 30 40 50 60 Dates (May 2017 – Oct. 2017) MbA mg x corm-1 [FW] F * a a a,b a,b a,b a,b b c c,e e,f,g d,g d d,f d d * a,b oC yC yC oC Mar 08       Apr 10      May 03       May 31        Jun 16        Jul 10        Aug 08         Aug 25      Sept 22       Oct 31  1cm A B C D st st yC oC r Dates (Feb. 2016 – Feb. 2017) 0 1 2 3 4 5 6 7 MbA mg x g-1 [FW] oC 2016 yC 2016 a,b a a,c a b,c b b,c b,c * MbA mg x g-1 [FW] 0 1 2 3 4 5 6 7 oC 2017 yC 2017 Dates (Mar. 2017 – Oct. 2017) a,b,c a,b,c a,b,c a,b a,b,c a,c a,b,c a,c a,c a,c d d,i d,i e,g e,h e,f e,f g,h,i f,g,h d,h,i * * * E Dates (Jun. 2016 – Sept. 2016) 0 2 4 6 8 10 12 14 16 18 oC yC MbA mg x g-1 [DW] a a b * * * 24  corms.  Different plant organs are labeled: young corm (yC); old corm (oC); stolon (st); roots (r). (B) Representative images of oC and yC collected through Mar. 2017 – Oct. 2017. Scale bar = 1 cm. MbA accumulation in corms of montbretia collected during (C) Feb. 2016 – Feb. 2017 (D) Mar. 2017 – Oct. 2017. (E) MbA levels in dried montbretia corms. Ground corm samples harvested from Jun. 27th, Jul. 22nd and Sept. 12th 2016 were dried at 50 °C for three days. (F) Total concentration of MbA per corm.  All samples were analyzed using LC-MS in negative ionization mode. MbA levels were quantified from corm extracts using an external MbA standard curve. Each data point represents the average of four to six biological replicates. Error bars represent standard error. Different letters indicate significant differences between time points. Asterisks (*) indicate significant differences between yC and oC of the corresponding time point. FW, fresh weight. DW, dry weight.   3.2 Spatiotemporal Accumulation Pattern of MbA   After observing the different developmental stages of the corms, I monitored the metabolite accumulation in oC and yC for spatial and temporal patterns. The metabolite analysis was performed on corms harvested during February 16th, 2016 – October 31st, 2017. LC-MS was used to analyze metabolites in the corm extracts. MbA in the corm extracts was identified using an authentic MbA standard (Fig. 5). Peak 1 (m/z 1227) was detected in the corm extracts, and the retention time of this peak matched with the retention time of peak 1 (m/z 1227) of the MbA standard. Furthermore, the MS/MS fragmentation pattern of peak 1 (m/z 1227) in both the corm extract and the MbA standard was identical. Based on the retention time and the MS/MS fragmentation pattern in comparison to the authentic MbA standard, peak 1 (m/z 1227) was identified as MbA.  25   Figure 5. Corm extracts analyzed by LC-MS/UV in negative ionization mode. (A) LC-UV (350-370 nm) chromatogram of a representative corm extract. (B) Extracted ion chromatograms (EIC) m/z 1227. (C) Mass spectra (MS) m/z 1227, peak 1. (D) MS/MS fragmentation pattern of MbA (m/z 1227, peak 1). Extracted ion chromatogram (EIC) and MS/MS fragmentation pattern of an authentic standard and a representative corm extract are shown. Star (★) indicate precursor ion for the MS/MS. Peak 1, MbA.  The first year of collection included twelve time points from February 16th, 2016 – February 3rd, 2017 (Fig. 4C). The oC were of unknown age and yC were first collected on June 10th, 2016. The MbA accumulation level in oC remained relatively stable throughout the year, with an average of 2.8 ± 0.1 mg MbA x g-1 FW (Table S8). In contrast, yC showed significantly higher levels of MbA compared to oC during the summer months from June to August (Fig. 4C). The yC collected early June contained 4.9 ± 0.2 mg MbA x g-1 FW. The highest level of MbA in the yC was reached in late June, with MbA content of 6.1 ± 0.3 mg x 0.51.50.51.52.54.5 5.5 6.5 7.5 8.5Relative intensity(EIC x 100 000)Retention time (min)11EIC 1227MbA standardCorm extractB2 4 6 80102030Intensity (mAUx 100)Retention time (min)A UV Chromatogram, 350-370 nm1Corm extractStandardExtract47461312270.51.52.5400 800 1200 1600 m/zPeak 1, MS47461312270.20.61.0200 1000 1800 m/zRelative intensity (x 1 000)CRelative intensity (x 1 000)787949106526200 600 1000 m/z★787949106526200 600 1000 m/z★Peak 1, MS/MS 1227StandardExtractD26  g-1 FW, and remained in a similar range until August. In September, MbA levels in yC decreased significantly to 3.5 ± 0.3 mg x g-1 FW, resembling the MbA content of oC of the same time point. The MbA concentration in yC remained similar to oC throughout the rest of the first sampling year. In November, the yC were separated from the mother plant and were individually re-potted. These re-potted yC served as the 1-year-old oC of the following sampling year.  A similar MbA accumulation pattern was observed in oC and yC harvested during the second year. Ten time points were collected throughout March 8th, 2017 – October 31st, 2017 (Fig. 4D). The oC were the yC developed during the first year, and new yC in the second year were first collected in early March. Similar to the first year, MbA concentration in oC showed no significant changes throughout the sampling dates (Fig. 4D). The yC from the second year showed a similar MbA accumulation pattern as yC harvested in the first year. The yC harvested on March 8th, 2017 contained the lowest MbA concentration of 0.6 ± 0.2 mg x g-1 FW, which remained similar throughout April and early May (Table S8). The yC harvested during March to early May contained significantly lower amounts of MbA in comparison to oC of the same time points (Fig. 4D). However from early to late May, the yC MbA content increased significantly from 1.4 ± 0.1 mg x g-1 FW to 4.3 ± 0.5 mg x g-1 FW, which resembled the corresponding oC MbA concentration of 4.1 ± 0.5 mg x g-1 FW. Similar to the first year of collection, yC collected in the second year also showed significantly higher levels of MbA during the summer (June, July, August) compared to the respective oC of the same time point. Highest concentration of MbA in yC was seen on July 10th, with MbA level of 5.8 ± 0.4 mg x g-1 FW (Table S8). Between early and late August, the MbA concentration in yC decreased significantly from 4.8 ± 0.6 mg x g-1 FW to 2.9 ± 0.3 mg x g-1 FW, which resembled the corresponding oC with MbA content of 2.3 ± 0.6 mg x g-1 FW (Table S8). No significant changes in MbA content was observed from yC collected during late August to October.  To validate that the observed MbA accumulation pattern was not due to the differences of water content in the corms, a subset of corm samples from June 27th, July 22nd and September 12th, 2016 were dried and analyzed for MbA content. The same MbA accumulation pattern was observed in fresh and dried corm samples (Fig. 4E, Table 1). During late June and July, yC accumulated significantly higher amounts of MbA compared to oC, followed by the subsequent decrease of MbA concentration in yC from July to September (Fig. 4E, Table 1). 27  Table 1. MbA concentration in dried montbretia corm samples. The average and standard error of 3 biological replicates are shown. DW, dry weight.   MbA [mg x g-1 (DW)] Water Loss (%) Sampling Date (2016) Old Corm Young Corm Old Corm Young Corm Jun. 27 2.90 ± 0.46 15.12 ± 0.60 70.10 ± 3.26 71.20 ± 0.33 Jul. 22 3.76 ± 0.61 13.54 ± 0.69 65.88 ± 3.26 71.99 ± 0.47 Sept. 12 2.19 ± 1.12 3.84 ± 0.61 53.94 ± 0.82 61.77 ± 0.84 In both sampling years, MbA content in yC decreased significantly from early August to September. To determine if this decrease in MbA level is from the degradation of MbA or a dilution effect due to corm growth, total amount of MbA per corm was monitored throughout March 8th, 2017 – October 31st, 2017. The total MbA content in the oC showed no obvious pattern (Fig. 4F, Table 2). In comparison, total amount of MbA in yC increased significantly from 0.43 ± 0.3 mg x g-1 FW to 16.3 ± 5.1 mg x g-1 FW from early May to August (Fig. 4F, Table 2). Following the significant increase, the total amount of MbA in yC remained consistent throughout August to October (Fig. 4F, Table 2).  Table 2. Total amount of MbA in individual old and young corms of montbretia plants.  The average and standard error of four to six biological replicates are shown.  MbA [mg x (Fresh Corm)-1] Sampling Date (2017) Old Corm Young Corm May 03 41.92 ± 6.66 0.43 ± 0.29 May 31 41.78 ± 9.56 0.91 ± 0.13 Jun. 16 18.91 ± 2.37 3.53 ± 0.40 Jul. 10 18.14 ± 3.21 9.14 ± 1.77 Aug. 08 31.92 ± 8.50 16.33 ± 5.06 Aug. 25 14.06 ± 6.06 13.79 ± 5.33 Sept. 22 20.89 ± 4.51 17.23 ± 1.67 Oct. 31 19.96 ± 4.57 16.00 ± 2.54 In addition to the corms, other montbretia organs were harvested during late June 2016 – August 2016 for MbA analysis. These organs included roots, stolons, leaves, stem, flowers and seed buds (Fig. 1B). MbA was not detectable in roots and leaves, and low levels of MbA were found in stolons, stem, flowers and seed buds (Table 3). Of the additional 28  montbretia organs analyzed for MbA content, flowers harvested in late June showed the highest MbA concentration. The MbA content in flowers harvested in late June was 0.20 ± 0.03 mg x g-1, which was less than 4% of the MbA content in yC collected at the same time point (Table 3, Table S8).  Table 3. MbA levels in different organs of montbretia. The average and standard error of four to six biological replicates are shown. FW, fresh weight; n.d. not detectable; (-) organ not analyzed or present.   MbA [mg x g-1 (FW)] Sampling Date (2016) Flower Seed Buds Leaf Root Stem Stolon Jun. 27 0.20 ± 0.03 - n.d. n.d. 0.09 ± 0.01 0.05 ± 0.02 Jul. 22 0.07 ± 0.02 0.13 ± 0.01 n.d. n.d. 0.08 ± 0.01 0.04 ± 0.01 Aug. 16 0.08 ± 0.01 0.13 ± 0.02 n.d. n.d. 0.10 ± 0.01 0.08 ± 0.02  3.3 Putative Intermediates in MbA Biosynthesis  MbA is an acylated flavonol glycoside consisting of seven building blocks that in theory can be assembled through many different routes resulting in numerous potential intermediate compounds (Fig. 2A). The accumulation pattern of these intermediate compounds may enlighten the site and the time point of MbA biosynthesis. As such, I searched for all potential intermediates of MbA biosynthesis listed in Fig. 2A in the extracts of montbretia corms harvested from February 16th, 2016 – October 31st, 2017. LC-MS was used to screen for masses of theoretical MbA intermediates, and metabolites were identified using authentic standards and MS/MS fragmentation patterns (Fig. 6).  29   Figure 6. Potential intermediates in MbA biosynthesis present in montbretia corm extracts. (A) Metabolite m/z 463 peak 1, myricetin 3-O-rhamnoside (MR). (B) Metabolite m/z 625 peak 3, myricetin 3-O-glucosyl rhamnoside (MRG). (C) Metabolite m/z 1065 peak 4, myricetin 3-O-glucosyl glucosyl rhamnoside 4’-O-rhamnosyl xyloside (MbA-C). (i) The (147)(316)(147)(162)(316)H(147)(162)(162)(132)(146)(316)Relative intensity (EIC x 1 000)316★1030100 300 500 m/z316★0.51.5100 300 500 m/zStandardPeak 3, MS/MS 625316★0.20.6100 300 500 m/zExtractPeak 2, MS/MS 62526100.10.34.5 5.5 6.5 7.5 8.5Retention time (min)Corm extractMRG standard 332Relative intensity (EIC x 100 000)bEIC 625ExtractPeak 3, MS/MS 625Relative intensity (EIC x 100 000)26100.10.30.54.5 5.5 6.5 7.5 8.5Retention time (min)Corm extractMR standard 11Relative intensity (EIC x 10 000)3161050200 400 m/z★StandardPeak 1, MS/MS 463aEIC 463316★0.52.5200 400 m/zExtractPeak 1, MS/MS 4632462464.5 5.5 6.5 7.5 8.5Relative intensity (EIC x 100 000)Retention time (min)Corm extractMbA-C standard 447870.250.751.25200 600 1000 m/z★316StandardPeak 4, MS/MS 10657870.250.751.25200 600 1000 m/z316 ★ExtractPeak 4, MS/MS 1065Relative intensity (EIC x 100  000)EIC 1065ABC (ii)(iii)(i)(ii)(iii)(i)(ii)(iii)(i)30  chemical structure of each metabolite. The dotted line indicates the predicted breakage cite when MS/MS is performed. The mass of myricetin and sugar moieties are in brackets. (ii) The extracted ion chromatograms (EIC) and (iii) MS/MS fragmentation pattern of authentic standards and representative corm extracts are also shown. Samples were analyzed using LC-MS in negative ionization mode. Peak 2, tentatively identified as myricetin 3-O-rhamnosyl glucoside. Peak a, b, unidentified. Star (★) indicates precursor ion for the MS/MS.  In the corm extracts, three potential intermediates in MbA biosynthesis were detected: MR, MRG, and myricetin 3-O-glucosyl glucosyl 4’-O-rhamnosyl xyloside (MbA-C). Peak 1 (m/z 463) detected in the corm extracts corresponded with the retention time and the MS/MS fragmentation pattern of the peak detected in the authentic MR standard (Fig. 6Aii, 6Aiii). The MS/MS fragmentation resulted in the formation of a product ion with m/z 316 from the precursor ion (m/z 463), suggesting the loss of rhamnose (Fig. 6Ai). Based on the comparisons with the authentic standard, peak 1 (m/z 463) in the corm extract was identified as MR. Two metabolite peaks (peak 2 and peak 3) with m/z 625 were observed during the LC-MS analysis of the corm extracts (Fig. 6B). Peak 2 was previously identified as myricetin 3-O-rhamnosyl glucoside (Irmisch et al., submitted). The retention time and MS/MS fragmentation pattern of peak 3 (m/z 625) in the corm extracts corresponded to peak detected in the authentic MRG standard (Fig. 6Bii, Fig. 6Biii). The MS/MS fragmentation showed that the precursor ion (m/z 625) of peak 3 fragmented to m/z 316, corresponding to the mass of myricetin. The loss difference from the precursor to product ion corresponded to the mass of a disaccharide chain composed of rhamnose and glucose (Fig. 6Bi). Based on the comparison with the authentic standard, peak 3 in the corm extract was identified as MRG. Along with MR and MRG, a third metabolite with m/z 1065 was detected in the corm extracts. The retention time of this metabolite matched of with the peak detected in the myricetin 3-O-glucosyl glucosyl 4’-O-rhamnosyl xyloside (MbA-C) standard, which is MbA without the caffeoyl-moiety (Fig. 6Cii). Peak 3 (m/z 1065) in the corm extracts also showed the same MS/MS fragmentation pattern as the authentic MbA-C standard (Fig. 6Ciii). The precursor ion (m/z 1065) fragmented into m/z 787, and the loss difference corresponded to the mass of a caffeoyl-moiety. The product ion (m/z 787) further fragmented into m/z 316, corresponding to the mass of myricetin (Fig. 6Ci). Based on the comparison with the authentic standard, peak 4 (m/z 1065) was identified as MbA-C.    The accumulation profile of MR in the corms harvested during year 1 (February 16th, 2016 – February 3rd, 2017) showed a similar pattern as MbA accumulation (Fig. 7A). Higher amount of MR accumulated in yC compared to oC of the same time point during the summer (June – August). From August to September, MR concentration in yC decreased, and 31  remained similar to the amount of MR in oC (Fig. 7A). The yC harvested in year 2 (March 8th, 2017 – October 31st, 2017) displayed a different pattern. Although the MR concentration in yC increased by 10 times from March to June, no higher accumulation of MR was observed in the yC compared to oC (Fig. 7B, Table S8). Accumulation of MRG in oC and yC also resembled the accumulation pattern of MbA in both year 1 and year 2 (Fig. 7). During the summer (June – August), yC contained higher amounts of MRG compared to oC of the same time point. MRG concentration in yC subsequently decreased from August to September (Fig. 7). Unlike MR and MRG, the accumulation pattern of MbA-C in the corms harvested throughout February 16th, 2016 – October 31st, 2017 did not resemble the MbA accumulation pattern (Fig. 7). Aside from MR, MRG and MbA-C, no other potential intermediates of MbA biosynthesis were detectable in the corm extracts.   32   Figure 7. Accumulation pattern of potential MbA biosynthesis intermediates in montbretia corms. Concentration of potential intermediates in MbA biosynthesis were measured in old corms (oC) and young corms (yC) collected during (A) Feb. 2016 – Feb. 2017 and (B) Mar. 2017 – Oct. 2017. Samples were analyzed using LC-MS in negative ionization mode. All metabolites were identified using authentic standards. An external MbA standard curve was used to quantify each compound in the corm extracts. Each data point shows the average of four to six biological replicates. Error bars represent standard error. MR, myricetin 3-O-rhamnoside; MRG, myricetin 3-O-glucosyl rhamnoside; MbA-C, myricetin 3-O-glucosyl glucosyl rhamnoside 4’-O-rhamnosyl xyloside. FW, fresh weight.  Dates0.10.30.5mgx g-1[FW]Relative amounts based on MbAstandard curveoCyCAMRMRGMbA-C(Feb. 2016- Feb. 2017)0.010.030.050.070.050.150.250.0050.0150.0250.0350.0450.050.150.250.35MRGMRMbA-C(Mar. 2017 – Oct. 2017)BoCyC0.020.060.1033  3.4 Distribution Pattern of Metabolites within Montbretia Corms   The distribution of MbA and potential intermediates in MbA biosynthesis within the oC and yC was investigated. Fresh whole corm samples were sent to the University of Victoria Genome BC Proteomic Centre, where corms were transversely sectioned and analyzed with MALDI-FTICR. Although earlier time point samples were sent, due to problems at the facility, data was only gathered from the 1-year-old oC and yC harvested from September 6th, 2017. Fig. S18 lists the MbA and MbA related metabolites targeted for MALDI analysis. Of the targeted compounds, only MbA (m/z 1227), MbA-C (m/z 1065) and myricetin 3-O-(6-O-caffeoyl)-glucosyl glucosyl rhamnoside (MbA-XR2; m/z 949) were detectable. The distribution of MbA and MbA-XR2 showed similar patterns. Equal distribution of MbA and MbA-XR2 were observed throughout the yC (Fig. 8). However, within the 1-year-old oC, MbA and MbA-XR2 accumulated in higher amounts near the peripheral area compared to the central vascular bundle area (Fig. 8). In contrast to MbA and MbA-XR2, MbA-C showed equal distribution pattern in both oC and yC (Fig. 8).   Figure 8. MALDI-FTICR analysis of MbA and potential intermediates in a 1-year-old corm and a young corm of montbretia. Corm samples harvested on Sept. 6th 2017 were transversely sectioned and analyzed using MALDI-FTICR in negative ion mode. MALDI images were reconstructed from extracted ion chromatograms (EIC) of m/z 1227.32 (MbA), 1065.29 (MbA-C; myricetin 3-O-glucosyl glucosyl rhamnoside 4’-O-rhamnosyl xyloside) and 949.22 (MbA-XR; myricetin 3-O-(6-O-caffeoyl)-glucosyl glucosyl rhamnoside). Scale bar = 5 mm. Young Corm1 YearOld Corm1227 m/z                1065 m/z                 949 m/zMbA                      MbA-C                  MbA-XR5 mm234  Chapter 4: Results - Discovery and Characterization of ATs Involved in MbA Biosynthesis    4.1 Acylation Activity of Total Corm Protein  The third step of MbA assembly was predicted to be the addition of the caffeoyl-moiety to MRG to form mini-MbA (Irmisch et al., submitted). To confirm this reaction and to test my hypothesis that a BAHD-AT is involved in this acylation step, I investigated the activity of the corm protein extracts. As I also hypothesized that the corms are the site of MbA biosynthesis, enzymes involved in MbA biosynthesis may be present in the protein extract of the corms. Furthermore, if the corm protein extracts show acylation activity with acyl-CoA thioester donors, this would indicate the activity of a BAHD-AT, as SCPL-ATs use 1-O-β-glucose ester donors. The total protein was extracted from oC and yC samples collected on July 10th, 2017. Activity of the corm protein extracts were tested using MRG as the acyl acceptor and caffeoyl-CoA as the acyl donor. The assays were analyzed using LC-MS. The formation of peak with m/z 787 was observed in the corm protein assays (Fig. 9). The retention time and MS/MS fragmentation pattern of peak 1 (m/z 787) produced by the corm protein extract assays corresponded to the authentic mini-MbA standard (Fig. 9). The MS/MS fragmentation showed that the precursor ion (m/z 787) fragmented into m/z 625, corresponding to the mass of MRG, and was further fragmented into m/z 316, corresponding to the mass of myricetin (Fig. 9B). Based on the comparison with the authentic standard, peak 1 (m/z 787) in the corm protein assays was identified as mini-MbA. The formation of mini-MbA was observed in both yC and oC protein assays, but product formation was about 27 times greater in protein extracts from yC compared to oC. Mini-MbA (peak 1, m/z 787) was not detected in the control corm protein assays performed with the corm protein extracts of yC and oC incubated with coumaroyl-CoA and MRG. The role of the control assays was to test for potential residual mini-MbA from the corm protein extracts.  35   Figure 9. BAHD-AT activity in total protein extracts of montbretia corms. (A) Corm protein extract catalyzes the formation of mini-MbA (peak 1, m/z 787) from myricetin 3-O-glucosyl rhamnoside (MRG) and caffeoyl-CoA (Caff-CoA). Formation of mini-MbA was 27 times greater in protein extracts from yC (black trace) compared to oC (grey trace). Samples were analyzed using LC-MS in negative ionization mode. Extracted ion chromatogram (EIC) at m/z 787 is shown. Control assays showed no residual mini-MbA from protein extracts of yC (black trace) and oC (grey trace). (B) MS/MS of peak 1 (m/z 787). Star (★) indicate precursor ion for MS/MS. Peak 1, mini-MbA.  4.2 Differential Expression of BAHD-ATs in Montbretia Transcriptome   A montbretia transcriptome database was previously developed in the Bohlmann laboratory using oC and yC harvested on June 10th, 2016 (Irmisch et al., submitted). In order to identify putative montbretia BAHD-AT sequences, a BLASTP search was performed against the translated montbretia protein database. Published BAHD-ATs from other plants representing each BAHD-AT clade (I-V) were used as search queries (Table S5). Subsequently, a reciprocal BLASTP search was performed to obtain all putative BAHD-ATs in the montbretia transcriptome. A total of 59 putative BAHD-ATs were identified in the montbretia transcriptome (Table S9). Considering that higher accumulation of MbA was seen in yC compared to oC, and that the yC protein extract displayed higher activity compared to the oC protein extract, candidate BAHD-ATs were filtered by their relative expression in oC compared to yC. Of the 59 putative BAHD-ATs, 27 candidates showing greater transcript abundance in yC compared to oC by at least 2-folds were identified. A phylogenetic tree was constructed using the refined list of 27 BAHD-ATs and BAHD-ATs EIC 787Relative Intensity (x 10 000) 625135100 300 500 700 m/z316 ★Standard625135100 300 500 700 m/z316 ★Protein assayPeak 1, MS/MS 787B4.5 5.5 6.5 7.5 8.50.20.60.51.50.51.51.0Retention time (min)Relative intensity (EIC x 100 000) Mini-MbA standardCorm protein assayCorm extract control11AMRG + Caff-CoA 36  from other plant species (Fig. 10, Table S5). Based on the phylogenetic tree, 20, 6, 2, and 1 putative montbretia BAHD-ATs clustered into clade V, I, IV, and II of respectively (Fig. 10).    Figure 10. Phylogenetic analysis of montbretia BAHD-ATs. Montbretia BAHD-ATs that show higher abundance in young corms (yC) compared to old corms (oC) by at least two-folds are shown. The neighbour-joining tree was produced using an amino acid alignment of 27 putative montbretia BAHD-ATs and published BAHD-ATs from other plant species using MEGA6 (bootstrap value = 1,000). The amino acid alignment was created using MUSCLE algorithm. BAHD-AT clades (I-V) are labeled outside the circle. Montbretia BAHD-ATs investigated in this work are labeled in bold. Clade II was designated as the outgroup. The corresponding contig number of the labeled montbretia BAHD-ATs can be found in Table S5. Accession numbers and details of literature BAHD-ATs used in the phylogenetic analysis can be found in Table S4.  IIIIIIIVVCcAT237  To further refine and prioritize candidate BAHD-ATs for cloning, co-expression analysis was performed using Haystack. RNA-seq data from different montbretia organs (oC, flower, leaf, stem and stolon) available in the Bohlmann laboratory (Irmisch et al., submitted) was used to correlate the expression pattern of putative BAHD-ATs with the expression pattern of UGT1 and UGT2. Total of 7 candidate BAHD-ATs showing a strong correlation (>0.85) with UGT1 and UGT2 expression, as well as 10-folds higher transcript abundance in yC compared to oC were selected (Table S9). Within the 7 BAHD-ATs, 3 candidates (DN66658_c0_g1_i2, DN66658_c0_g1_i1, DN29265_c0_g1_i1) clustered into clade V, another 3 candidates (DN69556_c0_g2_i1, DN63150_c0_g1_i1, DN61381_c0_g1_i1) clustered into clade I, and 1 candidate (DN37908_c0_g1_i1) clustered into clade IV (Fig. 10, Table S6).   4.3 Cloning and Heterologous Expression of Candidate BAHD-ATs in E. coli Candidate montbretia BAHD-AT cDNAs selected through the correlation analysis were amplified and cloned into E. coli. The amplified BAHD-ATs were numbered CcAT1-7. The deducted amino acid sequence of CcAT1, CcAT3, CcAT4, CcAT5, CcAT6 and CcAT7 corresponded 100% to the translated amino acids of the transcriptome contigs DN66658_c0_g1_i2, DN69556_c0_g2_i1, DN63150_c0_g1_i1, DN29265_c0_g1_i1, DN37908_c0_g1_i1 and DN61381_c0_g1_i1, respectively. CcAT2 shared 99.22% amino acid identity with DN66658_c0_g1_i1, and 96.31% amino acid identity with CcAT1. The 7 BAHD-ATs were heterologously expressed in E. coli., and the protein expression was confirmed using Western blot analysis (Fig. 11).   Figure 11. Western blot analysis of AT protein expression. ATs were heterologously expressed in E. coli and analyzed using a His6-tag specific antibody. Lane 1, CcAT1; Lane 2, CcAT2; Lane 3, CcAT3; Lane 4, CcAT4; Lane 5, CcAT5; Lane 6, CcAT6, Lane 7, CcAT7. L, protein ladder.  kDa75503725L          1          2 L          6        7kDa75503725kDaL           3        4    5  7550372538  To screen for enzyme activity, crude protein extracts from E. coli heterologously expressing CcATs was used in activity assays. Substrates of the activity assays included MRG as the acyl acceptor and caffeoyl-CoA as the acyl donor. The enzyme assays were analyzed using LC-MS. The activity assays for CcAT1 and CcAT2 resulted in the formation of a single product peak with m/z 787 (Fig. 12). The retention time and the MS/MS fragmentation pattern of peak 1 (m/z 787) produced by CcAT1 and CcAT2 corresponded to the authentic mini-MbA standard (Fig. 12). The MS/MS fragmentation showed that the precursor ion (m/z 787) fragmented into m/z 625, corresponding to the mass of MRG, and was further fragmented into m/z 316, corresponding to the mass of myricetin (Fig. 12B). No activity was observed for CcAT3-7 or the empty vector control.   Figure 12. Activity of CcATs heterologously expressed in E. coli. Crude protein extracts from E. coli heterologously expressing CcATs were tested for activity using myricetin 3-O-Relative intensity (EIC x 100 000)Mini-MbA standardCcAT1MRG + Caff-CoA 1114.5 5.5 6.5 7.5Empty vectorMRG + Caff-CoA260.20.60.20.60.20.60.20.60.20.60.20.60.20.60.20.6CcAT2MRG + Caff-CoA CcAT3MRG + Caff-CoA CcAT4MRG + Caff-CoA CcAT5MRG + Caff-CoA CcAT6MRG + Caff-CoA CcAT7MRG + Caff-CoA EIC 787Retention time (min)ABRelative Intensity (x 10 000) 6251030100 300 500 700 m/z316 ★StandardPeak 1, MS/MS 78762513100 300 500 700 m/z316 ★CcAT1Peak 1, MS/MS 78762513100 300 500 700 m/z316 ★CcAT2Peak 1, MS/MS 78739  glucosyl rhamnoside (MRG) and caffeoyl-CoA (Caff-CoA) as substrates. E. coli extract expressing the empty vector were used as control. Enzyme assays were analyzed using LC-MS in negative ionization mode. (A) Extracted ion chromatogram (EIC) of m/z 787 is shown. Peak 1 was identified as mini-MbA using an authentic standard. (B) MS/MS fragmentation pattern of peak 1 (m/z 787). The precursor ion for MS/MS is marked with a star (★). Peak 1, mini-MbA. The activities of CcAT1 and CcAT2 were further investigated using the purified proteins, which showed the same activity as the crude E. coli protein with MRG and caffeoyl-CoA (Fig. 13). The acyl donor specificity of CcAT1 and CcAT2 was tested. The purified CcAT1 or CcAT2 was incubated with MRG as the acyl acceptor, and either caffeoyl-CoA, coumaroyl-CoA, feruloyl-CoA, acetyl-CoA or malonyl-CoA as the acyl donor. LC-MS was used to analyze the enzyme assays. CcAT1 and CcAT2 showed similar activity with the different acyl donors (Fig. 13). Peaks 1-4 were produced by CcAT1 and CcAT2 when caffeoyl-CoA, coumaroyl-CoA, feruloyl-CoA and acetyl-CoA were used as the acyl donor, respectively (Fig. 13Ai, Fig. 13Bi). The precursor ions of peaks 1-4 (m/z 787, m/z 771, m/z 801, m/z 667) all fragmented into m/z 625, corresponding to the mass of MRG. The resulting loss difference of 162, 146, 176, 42, corresponded to the mass of caffeoyl, coumaroyl, feruloyl or acetyl moiety, respectively (Fig. 13Aiii, Fig. 13Biii). Peak 1 was identified above as mini-MbA using the authentic mini-MbA standard. Peaks 2-4 were tentatively identified based on the MS/MS fragmentation pattern as myricetin 3-O-(6-O-coumaroyl)-glucosyl rhamnoside, myricetin 3-O-(6-O-feruloyl)-glucosyl rhamnoside and myricetin 3-O-(6-O-acetyl)-glucosyl rhamnoside, respectively. To compare the activity of CcAT1 and CcAT2 with different acyl donors, relative turnover rates were determined. For CcAT1, the lowest turnover rate was seen with acetyl-CoA of 47.6 ± 8.6 ng x min-1 x μg-1 (Fig. 13Aii). The turnover rate for feruloyl-CoA was about twice as the turnover rate for acetyl-CoA, and caffeoyl-CoA and coumaroyl-CoA resulted in a turnover rate about 10 times the turnover rate of acetyl-CoA (Fig. 13Aii). The turnover rates for CcAT2 were similar to those of CcAT1, except the lowest turnover rate was seen with feruloyl-CoA of 93.5 ± 1.9 ng x min-1 x μg-1 (Fig. 13Bii). The turnover rate for acetyl-CoA was twice as the turnover rate for feruloyl-CoA. Caffeoyl-CoA and coumaroyl-CoA resulted in a turnover rate about 5 and 10 times the turnover rate of feruloyl-CoA, respectively (Fig. 13Bii). No activity was observed when malonyl-CoA was used as the acyl acceptor, as well as the empty vector control.  40   Figure 13. Acyl donor specificity of CcAT1 and CcAT2. ATs were heterologously expressed in E. coli, Ni-purified and tested for their acyl donor specificity. Enzyme assays were composed of purified (A) CcAT1 or (B) CcAT2, MRG as the acyl acceptor and different acyl donors (Caff-CoA, Cou-CoA, Fe-CoA, Ac-CoA). Product formation was monitored using LC-MS in negative ionization mode. (i) The extracted ion chromatograms (EIC), (ii) relative turn-over rates of production formation and (iii) MS/MS fragmentation patterns are shown for (A) and (B). Enzyme assays using E. coli expressing the empty vector were used as controls (grey trace), and were overlaid with AT enzyme assays (black trace). Relative turn-over Product ng x min-1 x μg-1(CcAT1)MRG+Caff 456.81  25.69MRG+Cou 438.32  58.97MRG+Fe 103.72  11.11MRG+Ac 47.58  8.55MRG+Ma No product formationProduct ng x min-1 x μg-1(CcAT2)MRG+Caff 510.90  17.47MRG+Cou 882.38  22.72MRG+Fe 93.53  1.87MRG+Ac 181.15  17.85MRG+Ma No product formation2626240.51.54.5 5.5 6.5 7.5 8.5Relative intensity (EIC x 10 000) EIC 787EIC 771EIC 801EIC 667MRG + Caff-CoA MRG + Cou-CoA MRG + Fe-CoA MRG + Ac-CoA (i)1234A (ii)Retention time (min)Relative Intensity (x 1 000)BMS/MS 771316625★13100 300 500 700 m/z5Peak 2316625639135100 300 500 700 900m/z★Peak 3MS/MS 8012713166076250.51.5100 300 500 700 m/z★Peak 4MS/MS 66762513100 300 500 700 m/z316 ★MS/MS 778Peak 1(iii)(i) (ii)2651013134.5 5.5 6.5 7.5 8.5EIC 787EIC 771EIC 801EIC 667MRG + Caff-CoA MRG + Cou-CoA MRG + Fe-CoA MRG + Ac-CoA Relative intensity (EIC x 10 000)Retention time (min)1234Relative Intensity (x 1 000)315625639 ★26100 300 500 700 900 m/z271316607625★0.51.5100 300 500 700 m/z316625★2610100 300 500 700 m/zMS/MS 771 MS/MS 801 MS/MS 667Peak 2 Peak 3 Peak 462513100 300 500 700 m/z316 ★MS/MS 778Peak 1(iii)41  rates of product formation were calculated using an external MbA standard curve. Star (★) indicate precursor ion for MS/MS. Peak 1 m/z 787, myricetin 3-O-(6-O-caffeoyl)-glucosyl rhamnoside (mini-MbA); Peak 2-4 were tentatively identified, Peak 2 m/z 771, myricetin 3-O-(6-O-coumaroyl)-glucosyl rhamnoside; Peak 3 m/z 801, myricetin 3-O-(6-O-feruloyl)-glucosyl rhamnoside; Peak 4 m/z 667, myricetin 3-O-(6-O-acetyl)-glucosyl rhamnoside. MRG, myricetin 3-O-glucosyl rhamnoside; Caff, caffeoyl; Cou, coumaroyl; Fe, feruloyl; Ac, acetyl; Ma, malonyl. In addition to acyl donors, the acyl acceptor specificity of CcAT1 and CcAT2 was tested. Ni-purified CcAT1 or CcAT2 was incubated with caffeoyl-CoA as the acyl donor, and various acyl acceptors: Myricetin (M), myricetin 3-O-glucoside (MG), myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), quercetin 3-O-glucosyl glucoside (QGG), rutin, arbutin and salicin. LC-MS was used to analyze the enzyme assays. CcAT1 and CcAT2 showed the same acyl acceptor specificities. Formation of a peak 1 and peak 2 was observed when MRG and QGG were used as the acyl acceptors, respectively (Fig. 14). Peak 1 was identified above as mini-MbA using the authentic standard. Peak 2 was identified as quercetin 3-O-(6-O-caffeoyl)-glucosyl glucoside based on the MS/MS fragmentation pattern. The precursor ion of peak 2 (m/z 787) fragmented into m/z 625, corresponding to the mass of MRG (Fig. 14). The loss difference of 162 matched the mass of a caffeoyl-moiety. The product ion (m/z 652) further fragmented into m/z 301, corresponding to the mass of quercetin. The loss difference of 324 corresponded to the mass of two glucose moieties. Except for MRG and QGG, no activity was observed from the rest of the substrates tested as the acyl acceptor, or the empty vector control.  42   Figure 14. Acyl acceptor specificity of CcAT1 and CcAT2. ATs were heterologously expressed in E. coli, Ni-purified and tested for activity with different acyl acceptors. Enzyme assays were composed of purified CcAT1 or CcAT2, caffeoyl-CoA (Caff-CoA) as the acyl donor and different acyl acceptors. (A) Summary of CcAT1 and CcAT2 enzyme activity with various acyl acceptors. (+) indicates positive enzyme activity; (-) indicates negative enzyme activity. The extracted ion chromatograms (EIC) and MS/MS fragmentation pattern of m/z 787 (peak 1 and peak 2) for positive (B) CcAT1 and (C) CcAT2 activity are shown. Product formation was monitored using LC-MS in negative ionization mode. Enzyme assays with empty vector protein extracts were used as controls (grey trace), and were overlaid with AT enzyme assays (black trace). Star (★) indicate precursor ion for MS/MS. Peak 1, myricetin 3-O-(6-O-caffeoyl)-glucosyl rhamnoside (mini-MbA); Peak 2, tentatively identified as quercetin 3-O-(6-O-caffeoyl)-glucosyl glucoside. M, myricetin; MG, myricetin 3-O-glucoside; MR, myricetin 3-O-glucosyl rhamnoside; QGG, quercetin 3-O-sophoroside.   4.4 Transient Expression of Candidate BAHD-ATs in N. benthamiana   In addition to characterizing CcAT1 and CcAT2 heterologously expressed in E. coli, the activity of transiently expressed CcAT1 and CcAT2 in N. benthamiana was investigated. The full ORF of CcAT1, CcAT2, UGT1, UGT2 and the gene for enhanced green fluorescence (eGFP) were individually fused with the 35S promoter and transformed into Acyl acceptor CcAT1 CcAT2M - -MG - -MR - -MRG + +QGG + +Rutin - -Arbutin - -Salicin - -Relative intensity (EIC x 100 000)B26264.5 5.5 6.5 7.5 8.5EIC 787MRG + Caff-CoA QGG + Caff-CoA CcAT112Retention time (min)Relative Intensity(x 100 000)625★0.51.5100 300 500 700 m/z300Peak 2MS/MS 77862513100 300 500 700 m/z316 ★Peak 1MS/MS 778Relative intensity (EIC x 100 000)C13526104.5 5.5 6.5 7.5 8.5EIC 787MRG + Caff-CoA QGG + Caff-CoA CcAT212Retention time (min)Relative Intensity(x 100 000)625★13100 300 500 700 m/z300Peak 2MS/MS 77862513100 300 500 700 m/z316 ★Peak 1MS/MS 778A43  Agrobacterium tumefaciens. The leaves of N. benthamiana were infiltrated with mixtures of differently transformed A. tumefaciens, resulting in leaves expressing: (i) 35S::CcAT1; (ii) 35S::CcAT2; (iii) 35S::UGT1 + 35S::UGT2; (iv) 35S::UGT1 + 35S::UGT2 + 35S::CcAT1; (v) 35S::UGT1 + 35S::UGT2 + 35S::CcAT2 and (vi) 35S:: eGFP. Leaves were harvested 4 days after infiltration, and metabolites were extracted and analyzed using LC-MS.  The co-expression of UGT1 and UGT2 in N. benthamiana led to the formation of two peaks with m/z 593 (Fig. 15A). Previous work in the Bohlmann laboratory identified peak 1 as kaempferol 3-O-rhamnosyl glucoside (KGR), and peak 2 as kaempferol 3-O-glucosyl rhamnoside (KRG) (Irmisch et al., submitted). Peak 1 was present in all N. benthamiana samples, including N. benthamiana leaves expressing the control eGFP, while peak 2 was specific to leaves co-expressing UGT1 and UGT2 (Fig. 15A). When either CcAT1 or CcAT2 was co-expressed with UGT1 and UGT2, depletion of peak 2 and the formation of peak 3 (m/z 793) was observed (Fig. 15). The MS/MS fragmentation of peak 3 (m/z 739) resulted in the formation of product ions m/z 593 and m/z 285, which corresponded to the mass of KRG and kaempferol, respectively (Fig. 15B). The mass difference from m/z 739 to m/z 593, corresponded to the mass of a coumaroyl-moiety. Additionally, the loss difference from m/z 593 to m/z 285, corresponded to the mass of a disaccharide chain composed of rhamnose and glucose. Based on this fragmentation pattern, peak 3 (m/z 739) was tentatively identified as kaempferol 3-O-(6-O-coumaroyl) glucosyl rhamnoside. Formation of peak 3 (m/z 739) was not observed when UGTs were co-expressed without ATs or when ATs were independently expressed without UGTs, as well as in the control N. benthamiana samples expressing eGFP. 44   Figure 15. Activity of CcAT1 and CcAT2 transiently expressed in N. benthamiana. Tobacco leaves were infiltrated with A. tumefaciens transformed with plasmids containing 35S::UGT1, 35S::UGT2, 35S::CcAT1, 35S::CcAT2 or the gene for enhanced green fluorescence (eGFP). Metabolites were extracted from tobacco leaves 4 days after infiltration and were analyzed using LC-MS in negative ionization mode. The extracted ion chromatograms (EIC) and MS/MS fragmentation pattern of (A) m/z 593 and (B) m/z 793 are shown. Compounds are tentatively identified based on MS/MS fragmentation patterns. Star (★)  indicate precursor ion for MS/MS. Peak 1, kaempferol 3-O-rhamnosyl glucoside; Peak 2, kaempferol 3-O-glucosyl rhamnoside; Peak 3, quercetin 3-O(6-O-coumaroyl)-glucosyl rhamnoside; Peak a, unidentified.   2272552854134310.51.5100 300 500 m/zPeak 2, MS/MS 593★229267285135100 300 500 m/zPeak 1, MS/MS 593★Relative intensity (EIC x 100 000)Relative intensity (EIC x 100 000)A2624122612244.5 5.5 6.5 7.5 8.5UGT1 + UGT2UGT1 + UGT2 + CcAT1eGFPCcAT 1CcAT 2UGT1 + UGT2 + CcAT21111112EIC 593Retention time (min)aaaaaa5930.51.5100 300 500 700 m/z285 575 ★Relative intensity (EIC x 10 000)413Peak 3, MS/MS 739Relative intensity (EIC x 100 000)BEIC 793Retention time (min)134.5 5.5 6.5 7.5 8.5131313CcAT2UGT1 + UGT2eGFPUGT1 + UGT2 + CcAT21313CcAT13UGT1 + UGT2 + CcAT1345  4.5 Transcript Abundance Pattern of BAHD-ATs in Montbretia Corms   The relative transcript abundance of CcAT1 and CcAT2 in oC and yC during corm development was determined using quantitative RT-PCR (qRT-PCR). cDNA isolated from corm samples harvested during June 10th, 2016 – October 6th, 2016 were used for qRT-PCR analysis. The transcript abundance profiles of CcAT1 and CcAT2 (Fig. 16) showed similar pattern with mbA accumulation in montbretia corms (Fig. 4C). In oC, the transcript abundance of CcAT1 and CcAT2 were barely detectable and remained unchanged throughout June – October (Fig. 16). In yC throughout June – October, the transcript levels of CcAT1 and CcAT2 were significantly higher compared to oC of the same time point (Fig. 16). Highest transcript levels were observed in yC of June for both ATs. From June to July, the transcript levels of CcAT1 and CcAT2 in yC decreased significantly, and remained unchanged throughout August – October (Fig. 16).   Figure 16. Relative transcript abundance of CcAT1 and CcAT2 in old corm (oC) and young corm (yC) of montbretia plant. Relative transcript abundance of (A) CcAT1 and (B) CcAT2 was determined by qRT-PCR. Corms harvested at six different time points in 2016 were used as material for qRT-PCR analysis. Averages of three biological replicates are shown. Error bars indicate standard error. Asterisk (*) indicates significant differences between oC and yC of the corresponding time point. Different letters indicate significant differences between time points. Serin-incorporator (MEP) and zinc-finger protein (ZF) were used as reference house-keeping genes.    020406080100120140160180oCyCDates(Jun. 2016 – Oct. 2016)Relative transcript abundanceCcAT1aa,cbbb,cbA******050100150200oCyCDates(Jun. 2016 – Oct. 2016)Relative transcript abundanceCcAT2Ba,cab,cbb,cb******020406080100120140160180oCyCDates(Jun. 2016 – Oct. 2016)Relative transcript abundanceCcAT1aa,cbbb,cbA******050100150200oCyCDates(Jun. 2016 – Oct. 2016)Relative transcript abundanceCcAT2Ba,cab,cbb,cb******46  Chapter 5: Discussion   5.1 Spatiotemporal Regulation of MbA Biosynthesis and Plant Defense As a foundation for my research on MbA biosynthesis in montbretia, I first examined the growth and development of montbretia plants, with emphasis on the corms where MbA was predominantly found. In parallel, I performed metabolite analysis on the montbretia corms over two separate years to monitor the levels of MbA and possible intermediates of MbA biosynthesis.  The metabolite analysis of the montbretia plant showed that MbA mainly accumulates in the corms compared to other organs, as previously reported by Andersen et al. (2009) and Roach (2017). MbA accumulation in the yC during June to August was significantly higher than in the oC of the same harvesting time points (Fig. 4). This difference in MbA accumulation pattern between yC and oC was seen in both fresh and dry corm samples, indicating that the observed difference in MbA level was not due to the difference in the water content of the corms (Fig. 4E). Potential intermediates of MbA biosynthesis, MR and MRG, showed similar patterns of accumulation as MbA. In addition to the metabolite analysis, whole protein extracted from yC showed higher activity in the in vitro formation of mini-MbA compared to oC of the same time points. Furthermore, the transcript abundance pattern of CcAT1 and CcAT2 corresponded with the MbA accumulation pattern. The transcript abundance of CcAT1 and CcAT2 was significantly higher in yC compared to oC, suggesting higher MbA biosynthetic activity in the yC (Fig. 16). Based on the combination of these results, it is likely that MbA biosynthesis occurs predominantly during the development of yC in early summer (June – August).  As the yC grew into the size of the oC in late summer/early fall, MbA biosynthesis seemed to cease as indicated by the significant decrease of the MbA levels in the yC between August and September (Fig. 4). Despite the decrease of MbA concentration in the yC, the total amount of MbA per yC remained unchanged from August to September (Fig. 4F). These observations suggest that as MbA biosynthesis slows down, the remaining MbA may be diluted as the yC continue to grow in volume. This was supported by the results of the MALDI analysis which showed the differences in the pattern of MbA distribution in oC and yC. MbA was distributed evenly throughout the yC. In comparison, MbA in the oC mainly accumulated in the peripheral regions compared to the central vascular bundle area (Fig. 8). The distribution of MbA-XR2, a potential intermediate in MbA biosynthesis, matched 47  the distribution pattern of MbA. These observations may suggest that MbA biosynthesis likely occurs throughout the yC, and as MbA biosynthesis stops and corm growth continues, the cells already containing MbA may be moved out to the peripheral area of the corm. Investigating the growth and development of the corms on a cellular level in the future may further enlighten the spatial biosynthesis of the MbA in the corms.  In addition to the MALDI imaging, the transcript abundance of CcAT1 and CcAT2 in yC also significantly decreased from June to August, suggesting lower activity of MbA biosynthetic genes in the fall (Fig. 16). The proposed spatiotemporal patterns of MbA biosynthesis served to guide the development of the montbretia transcriptome, which was used to search for putative genes involved in MbA biosynthesis.  The biosynthesis of plant specialized metabolites often shows organ and/or time specific patterns over the development of the plant (Hartmann, 1996). For example, the biosynthesis of flavonoids responsible for flower pigmentation in petunia (Petunia hybrida) is coordinated with flower development, resulting in differential accumulation of flavonoids in different floral parts (Tunen et al., 1988). In another example, the biosynthesis of monoterpenes in spearmint (Mentha spicata) is restricted to the glandular trichomes that are formed early in leaf development, resulting in higher concentration of monoterpenes in young leaves compared to mature leaves (Gershenzon 1989). The early biosynthesis of monoterpenes is thought to provide protection for the vulnerable young leaves against herbivores (Gershenzon 2000). The spatiotemporal biosynthesis of MbA in montbretia corms may also indicate the protective role of MbA. The biosynthesis of MbA likely occurs during the development of the yC, and although MbA biosynthesis gradually stops, MbA is continuously stored in the oC. Corms are starch accumulating overwintering organs and contribute to the vegetative propagation of the montbretia plant.  It is therefore reasonable to suggest that protection of the corms is relevant for the plant’s survival and asexual reproduction. When mammalian herbivores ingest the corms, the α-amylase inhibitory activity of MbA may interfere with the degradation of starch from the corms. The absence of nutrient uptake may discourage the future feeding by the herbivores. Other starch-rich underground organs such as cassava (Manihot esculenta) roots and potato (Solanum tuberosum) tubers accumulate toxic cynogenic glycosides and glycoalkaloids, respectively that provide defense against herbivory (Flanders et al., 1992; McMahon et al., 1995). Future herbivory experiments on montbretia plants may enlighten the ecological role of MbA in the corms.   48  5.2 Substrate Specificity of CcAT1 and CcAT2  The third step of MbA assembly was identified as the acylation of MRG involving the addition of the caffeic acid (Irmisch et al., submitted). The activity of corm protein extracts led to the in vitro formation of mini-MbA from MRG and caffeoyl-CoA. This suggests that the enzyme involved in the acylation step of MbA assembly is present in the total corm protein extracts, but also suggests the enzyme responsible for the acylation activity is a BAHD-AT based on the use of the acyl-CoA thioesters as acyl donors (Fig. 9).  The montbretia corm transcriptome revealed a total of 59 putative BAHD-ATs. Similar numbers of BAHD-ATs were found in the genomes of Arabidopsis thaliana (55 BAHD-ATs), Medicago truncatula (50 BAHD-ATs), and Vitis vinifera (52 BAHD-ATs) (Tuominen et al., 2011). Using information on the spatiotemporal pattern of MbA biosynthesis and BAHD enzyme activities, seven BAHD-AT candidates were identified in the transcriptome as target for functional characterization. Of these, only CcAT1 and CcAT2 catalyzed the formation of mini-MbA when the candidate BAHD-ATs were heterologously expressed in E. coli. CcAT1 and CcAT2 contained the two conserved motifs of BAHD-ATs, and clustered with the members of the clade V BAHD-ATs (Fig. 17, Fig. 10). Clade V is a large clade that is involved in the biosynthesis of diverse range of products (D’Auria, 2006). For example, the hexenol acetyltransferase, CHAT, from A. thaliana is involved in the production of volatile esters, while the taxadienol acetyltransferase, TAT, from Taxus cuspidate is involved in the production of paclitaxel (D’Auria, 2002; Walker et al., 1999). Furthermore, a subgroup of clade V consists of enzymes involved in the production of p-coumaroyl-shikimate quinate esters such as the hydroxylcinnamoyl transferases from A. thaliana, AtHCT, and Trifolium pratense, TpHCT1A (Hoffmann et al., 2005; Sullivan, 2009). Interestingly, all of the currently characterized BAHD-ATs involved in the acylation of flavonoids belong to clade I, except for the malonyltransferase from Salvia splendens, Ss5MAT2 in clade III (Bontpart et al., 2015; Suzuki et al., 2004). Based on the literature search, CcAT1 and CcAT2 may be the first members of clade V that use flavonoids as the acyl acceptor.  49   Figure 17. Amino acid sequence alignment of montbretia BAHD-ATs and literature BAHD-ATs from other plants. Sequence alignment was produced in BioEdit using the ClustelW algorithm. Highly conserved amino acids are shaded in black, similar amino acid residues are shaded in grey. The two BAHD-AT conserved motifs, HXXXD (motif 1) and DFGWG (motif 2) are outlined in red. Accession numbers and details of the literature BAHD-ATs are listed in Table S4.  Characterization of CcAT1 and CcAT2 revealed a lack of absolute substrate specificities for the acyl donor (Fig. 13). The ability of CcAT1 and CcAT2 to utilize caffeoyl-CoA, coumaroyl-CoA and feruloyl-CoA as acyl donors suggests that CcAT1 and CcAT2 may also be involved in the production of MbB and MbC as well as MbA. It is not uncommon for enzymes involved in the biosynthesis of plant specialized metabolites to lack strict substrate -----------MVAHLQPPKIIETCHISPPKGTVPSTTLPLTFFD-APWLSLPLADSLFFFSYQNSTESFLQDFVPNLKH 68 -----------------MASVIEQCQVVPSPGSATELTLPLTYFD-HVWLAFHRMRRILFYKLPISRPDFVQTIIPTLKD 62 -----------MEKTDLHVNLIEKVMVGPSPP-LPKTTLQLSSIDNLPGVRGSIFNALLIYNASPSPTMISADPAKPIRE 68 MDHQVSLPQSTTTGLSFKVHRQQRELVTPAKP-TPRELKPLSDIDDQQGLRFQIP-VIFFYRPNLSS---DLDPVQVIKK 75 ----------------MIINVRDSTMVRPSEE-VTRRTLWNSNVD--LVVPNFHTPSVYFYRSNNGTSN--FFDAKIMKE 59 --------------MSFTVTKTAPALITPSEPTPSGHILPLSFFDRLPFLRVFLVDMIMVYGRGDQP-------AKVIKE 59 --------------MSFTVTKTAPAVITPSEPTPSGHILPLSFFDRLPFLRVFLVDMIMVYGHGDQP-------AKVIKE 59  SLSITLQHFFPYAGKLIIPPRPD-PPYLHYNDGQDSLVFTVAESTETDFDQLKSDSPKDISVLHGVLPKLPPPHVSPEGI 147 SLSLTLKYYLPLAGNVACPQDWSGYPELRYVTG--NSVSVIFSESDMDFNYLIGYHPRNTKDFYHFVPQLAEPKDAP-GV 139 ALAKILVYYPPFAGRLR-ETENGDLEVECTGEG----AMFLEAMADNELSVLG-----DFDDSNPSFQQLLFSLPLDTNF 138 ALADALVYYYPFAGRLR-ELSNRKLAVDCTGEG----VLFIEAEADVALAELEEAD--ALLPPFPFLEELLFDVEGSSDV 148 ALSKVLVPFYPMAGRLR-RDEDGRVEIDCDGQG----VLFVEADTGAVIDDFG------DFAPTLELRQLIPAVDYSRGI 128 AVAKALVHYYPLAGRLTTDTDDGELSVACTGEG----VWFVEATADCRMEDVN-----YLQVEPLMIPKEQMLPSHPEGV 130 AVAKALVHYYPLAGRLTTDTDDGELSVACTGEG----VWFVEATADCRMEDVN-----YLQVEPLMIPKEQILPSHPEGV 130  QMRPI-MAMQVTIFPGAGICIGNSATHVVADGVTFSHFMKYWMSLTKSSGKDPATVLLPSLPIHSCRNMIKDPGEVGAGH 226 QLAPV-LAIQVTLFPNHGISIGFTNHHVAGDGATIVKFVRAWALLNKFGGDE--QFLANEFIPFYDRSVIKDPNGVGMSI 216 KDLSL-LVVQVTRFTCGGFVVGVSFHHGVCDGRGAAQFLKGLAEMARG-------EVKLSLEPIWNRELVKLD------- 203 LNTPL-LLVQVTRLKCCGFIFALRFNHTMTDGAGLSLFLKSLCELACG-------LHAPSVPPVWNRHLLTVS--ASEAR 218 ESYPL-LVLQVTYFKCGGVSLGVGMQHHVADGASGLHFINTWSDVARG--------LDVSIPPFIDRTLLRARDPPRPVF 199 DPYTLPLMIQVTQFRCGGFAFATRANHAVFDGIGAGQIKVAIGEMARG-------LKHPTVKPVWCRDVIRKPIPSQISA 203 DPYTLPFMIQVTQFRCGGFTFATRANHAVFDGIGAGQIKVAIGEMARG-------LKHPTVKPVWCRDVIRKPIPSQISA 203  LERFWS-QNSAKHSSHVT---PENMVRATFTLSRKQIDNLKSWVTEQSENQSPVSTFVVTLAFIWVSLIKTLVQDSETKA 302 WNEMKKYKHMMKMSDVVT---PPDKVRGTFIITRHDIGKLKNLVLTRRPKLTHVTSFTVTCAYVWTCIIKSEAATG---- 289 -DPKYLQFFHFEFLRAPSI--VEKIVQTYFIIDFETINYIKQSVMEECK--EFCSSFEVASAMTWIARTRAFQIPE---- 274 VTHTHREYDDQVGIDVVAT--GHPLVSRSFFFRAEEISAIRKLLPPDLHN----TSFEALSSFLWRCRTIALNPDP---- 288 DHIEYKPPPSMKTTQQSTKPGSDGAAVSIFKLTREQLSTLKAKSKEAGNT-IHYSSYEMLAGHVWRSVCKARSLPD---- 274 TEPHDTDLSPVSDIKFTNN--QTNIECCSFDLSLDHINHLKDRFAKEVG--KICSVFDVITAKLWQSRTRAIGLQP---- 275 TEPHDTDLSSIPDLKFTNN--QTNIECCSFDLSLDHINHLKDHFAKEVG--KICSVFDVIAAKLWQSRTRAIGLQP---- 275  NEEDKDEVFHLMINVDCRNRLKYTQPIPQTYFGNCMAPGIVSVKKHDLLGEKCVLAASDAITARIKDMLSSDLLKTAPRW 382 EEIDENGMEFFGCAADCR--AQFNPPLPPSYFGNALVGYVARTRQVDLAGKEGFTIAVELIGEAIRKRMKDEEWILSGSW 367 -------SEYVKILFGMDMRNSFNPPLPSGYYGNSIGTACAVDNVQDLLSGS-LLRAIMIIKKSKVSLNDNFKSRAVVKP 346 ------NTEMRLTCIINSRSKLRNPPLEPGYYGNVFVIPAAIATARDLIEKP-LEFALRLIQETKSSVTEDYVRSVTALM 361 -------DQETKLYIATDGRARLQPPPPPGYFGNVIFTTTPIAIAGDLMSKP-TWYAASRIHNALSRMDNEYLRSALDFL 346 ----QTEVSLTFLLNIRQVVLHNELPPDGGYYGNCLVPLVNKAPSGQIANAP-LFEIVRLIKEAKDDLLRKDSASLIGGM 350 ----QTEVSLTFLLNIRQVVLHNELPPDGGYYGNCLVPLINKAPSGQIANAP-LFEIVRLIKEAKDDLLSKDSASLIGEM 350  GQG-------VRKWVMSHYPTSIAGAPKLGLYDMDFG-LGKPCKMEIVHIE-TGG--SIAFSESRDGSNG--VEIGIALE 449 FK--------EYDKVDAKRSLSVAGSPKLDLYAADFG-WGRPEKLEFVSID-NDDGISMSLSKSKDSDGD--LEIGLSLS 435 SE--------LDVNMNHENVVAFADWSRLGFDEVDFG-WGNAVSVSPVQQQSALAMQNYFLFLKPSKNKPDGIKILMFLP 417 ATR-------GRPMFVASGNYIISDLRHFDLGKIDFGPWGKPVYGGTAKAGIALFPGVSFYVPFKNKKGETGTVVAISLP 434 ELQPDLKALVRGAHTFKCPNLGITSWARLPIHDADFG-WGRPIFMGPGGIA-----YEGLSFIIPSSTNDGSLSVAIALQ 420 PP----------YKKPSYADLSIVDWRRLGLYEADFG-WGGPMFLVPLNEH-TVTSCSTYLFKSPVASKKDVCLVTYCIV 418 PP----------YKKPSYADLSIVDWRRLGLYEADFG-WGGPMFLVPLNEH-TVTSCSTYLFKSPVASKKDVRLVTYCIV 418  KKKMDVFDSILQQGIKKFAT-- 469---------------------------------------------------------- KTRMNAFAAMFTHGISFL---- 453---------------------------------------------------------- LSKMKSFKIEMEAMMKKYVAKV 439---------------------------------------------------------- VRAMETFVAELNGVLNVSKG-- 454---------------------------------------------------------- HEHMKVFKEFLYDI-------- 434---------------------------------------------------------- KEHLEAFRAEMNDFT------- 433---------------------------------------------------------- KEHLEAFRDEMNDFT------- 433----------------------------------------------------------At3AT1 NtMAT1 TAT CHAT TpHCT1A CcAT1 CcAT2 At3AT1 NtMAT1 TAT CHAT TpHCT1A CcAT1 CcAT2 At3AT1 NtMAT1 TAT CHAT TpHCT1A CcAT1 CcAT2 At3AT1 NtMAT1 TAT CHAT TpHCT1A CcAT1 CcAT2 At3AT1 NtMAT1 TAT CHAT TpHCT1A CcAT1 CcAT2 At3AT1 NtMAT1 TAT CHAT TpHCT1A CcAT1 CcAT2 At3AT1 NtMAT1 TAT CHAT TpHCT1A CcAT1 CcAT2 Motif 1Motif 250  specificity, and the availability of different substrates may determine the type and the amount of product formed (Pichersky, 2006). For example, cocaine synthase from Erythroxylum coca is able to form cocaine and cinnamoylcocaine based on the availability of benzoyl-CoA and cinnamoyl-CoA, respectively (Schmidt et al., 2015). The spatial regulation of substrates was further illustrated by the differential accumulation pattern of cocaine and cinnamoyl-cocaine in the leaves of E. coca. Cocaine was found accumulating in the adaxial side of the E. coca leaves, while cinnamoylcocaine was found in the abaxial side of the leaf (Schmidt et al., 2015). Similarly, as the three different montbretins MbA, MbB and MbC only differ in the type of the acyl-group (Fig. 1), the availability of caffeoyl-CoA, coumaroyl-CoA and feruloyl-CoA may be a key factor in determining the formation of MbA, MbB and MbC, respectively. CcAT1 and CcAT2 were also able to utilize acetyl-CoA as the acyl donor (Fig. 13). However, there are no known montbretin analog with an acetyl moiety. The activity of CcAT1 and CcAT2 with acetyl-CoA may be an artifact of the in vitro assay conditions. Furthermore, CcAT1 and CcAT2 may be specific towards the flavonol 3-O-disaccharide structure of the acyl acceptor rather than the flavonol core. Along with myricetin 3-O-glucosyl rhamnoside (MRG), CcAT1 and CcAT2 were able to use quercetin 3-O-glucosyl glucoside (QGG) as the acyl acceptor but not myricetin 3-O-glucoside (MG) (Fig. 14). A similar characteristic was seen in an anthocyanin 5-aromatic acyltransferase from Gentiana triflora (Gt5AT) (Fujiwara et al., 1998). Gt5AT was able to acylate various 3,5-O-diglucoside of anthocyanidins including delphinidin, cyanidin and pelargonidin, but did not utilize anthocyanidin 3-O-monoglucosides or anthocyanidin 3,7,3’-O-triglucosides (Fujiwara et al., 1998). However, no acylation activity of CcAT1 and CcAT2 was detected when rutin (quercetin 3-O-rhamnosyl glucoside) was used as the acyl acceptor. This may indicate the specificity of CcAT1 and CcAT2 towards the 6-hydroxyl of the second glucose moiety, which is absent in rhamnose. Another explanation of the ability of CcAT1 and CcAT2 to utilize QGG but not rutin as the acyl acceptor may be due to the difference in the glycosidic linkage of the disaccharide chain. The sugars in the disaccharide chain of MRG and QGG are 12 linked, while they are 16 linked in rutin. Based on the substrate specificities of CcAT1 and CcAT2, the biosynthesis of MbA is likely to be regulated by both substrate availability and enzyme specificity.   51  5.3 Alternative MbA Production Systems  Type-2 diabetes has become a major health problem in the 20th and 21st century, affecting over 300 million people worldwide. Although MbA has potential as an anti-diabetes compound, a sustainable production system for large quantities of MbA for further drug research and development is currently lacking. Specifically, chemical synthesis of MbA is not feasible due to the complex structure of MbA. And the only known natural source of MbA, which is the montbretia corms (Asada et al., 1988; Tarling et al., 2008; Roach 2017), is also limiting for MbA production. Investigation on the growth and development of the montbretia plant revealed that a single plant produces less than ten yC originating for a single oC. The average total MbA content in yC is 25.95 ± 3.91 mg x g-1 FW (Table 2), resulting in total of less than 300 mg of MbA per plant.Animal study with “zucker diabetic fatty” rats showed that a slightly lower dosage of MbA is required to lower blood glucose levels compared to acarbose, a commercially available α-glucosidase inhibitor (Yuen et al., 2016). Compared to 10 mg of Acarbose, 7.5 mg of MbA per kg body weight of rat per day was required to show therapeutic results (Yuen et al., 2016). T2D patients treated with Acarbose are prescribed 75 mg – 300 mg of Acarbose per day, gradually increasing the dosage as required (https://dailymed.nlm.nih.gov). Although no clinical information is available for the dosage of MbA, it may be roughly estimated using the lower dosage range of Acarbose, 75 mg per day. Based on this assumption, one montbretia plant can treat a single patient for four days. This number can be calculated with the number of days per year, given the annual growth cycle of montbretia, and the number of patients to get an estimate of the number of plants that would have to be cultivated and harvested.  If only one million patients were to receive a daily dose of MbA over the period of a year, 108 plants would be roughly needed. Even this low estimate is not currently supported with montbretia cultivation, which is focused on horticultural production. Yields of corms or MbA levels may be increased with improved growing conditions or through plant breeding as with other medicinal plants (Canter et al., 2006). For example, although artemisinin content of Artemisia annua is low (0.01 – 1% dry weight), breeding of A. annua led to hybrids with artemisinin content of up to 2% dry weight (Ferreira et al., 2005; Simonnet et al., 2006). Furthermore, the accumulation of plant specialized metabolites can be enhanced by environmental stresses and plant growth hormones (Akula and Ravishankar, 2011). For instance, increased accumulation of total phenolic compounds in pea (Pisum sativum), red pepper (Capsicum annuum) and sweet basil (Ocimum basillicum) were observed after 52  drought, salinity and methyl jasmonate treatments, respectively (Alexieva et al., 2001; Navarro et al., 2006; Kim et al., 2006). The production of high MbA yielding montbretia cultivars in combination with stress and elicitor treatments may improve the accumulation of MbA levels in montbretia corms, thus increasing the feasibility of natural harvesting of MbA.  Metabolic engineering in alternative microbial or plant systems is another possibility for improved MbA production. The reconstitution of plant metabolic pathways in heterologous hosts has been explored to produce various plant natural products (Owen et al., 2017). Recent achievements include the biosynthesis of opioids in yeast (Saccharomyces cerevisiae), and the production of taxadiene in tobaccao (Nicotiana benthamiana) (Galanie et al., 2016; Hasan et al., 2014). Furthermore, the production of salidroside, a therapeutic agent found in the overharvested Rhodiola rosea plants, was achieved in both yeast and tobacco through the transgenic expression of native salidroside biosynthetic genes (Torrens-Spence et al., 2018). However, metabolic engineering of plant specialized metabolites requires an extensive knowledge on the biosynthesis pathway (Hussain et al., 2011). With the discovery of the MbA biosynthetic genes and enzymes UGT1 and UGT2 (Irmisch et al., 2018) as well as CcAT1 and CcAT2 discussed in this thesis, the first three steps involved in the MbA specific biosynthesis are now known (Fig. 3). These enzymes are also the complete set of enzymes required for the biosynthesis of mini-MbA, which is the simpler precursor of MbA that still show potent and specific HPA inhibitory activity (Williams et al., 2015). The co-expression of UGT1, UGT2 and CcAT1/CcAT2 in tobacco leaves led to the formation of a compound similar to mini-MbA (Fig. 15). This mini-MbA surrogate is likely composed of kaempferol instead of myricetin, and coumaric acid instead of caffeic acid. Kaempferol based phenolic glycosides have been naturally found in tobacco leaves, and coumaroyl-CoA is generally abundantly found in plants due to its central role in the phenylpropanoid pathway (Chen et al., 2012; Vogt, 2010). As mentioned above, MbA biosynthesis is likely to be regulated by the combination of enzyme specificity and substrate availability. The result of the tobacco co-expression experiment suggests that if myricetin and caffeoyl-CoA are adequately present, co-expression of UGT1, UGT2, and CcAT1/CcAT2 in tobacco leaves will lead to the formation of mini-MbA. As such, new work in the Bohlmann laboratory aims to engineer the production of myricetin and caffeoyl-CoA in tobacco leaves through the overexpression of genes involved in the flavonoid and phenylpropanoid pathway, respectively.   53  Chapter 6: Conclusion and Future Directions    The main goal of this thesis was to gain insight into MbA biosynthesis in montbretia through the use of metabolite profiling, transcriptome analysis and enzyme characterization. The first objective of this project was to gain knowledge on the time profile of MbA biosynthesis by performing metabolite analyses using LC-MS and MALDI-FTICR. One of the major limitations in this study is the sensitivity of the LC-MS and MALDI-FTICR instruments. Metabolites in small abundance may not been detected due to the detection limit of the instruments. Furthermore, as the MbA standard curve was used to quantify the MbA intermediate compounds, the quantification of the MbA intermediates present in this study may not represent the accurate measurements. Nonetheless, the overall accumulation pattern of MbA and MbA intermediates will likely be unchanged. The combination of the metabolite analyses and gene expression profile of CcAT1 and CcAT2 provide strong evidence that MbA is likely biosynthesized during the development of the yC. It may be interesting to investigate the biosynthesis of MbA on a cellular level in the future. Furthermore, the spatiotemporal biosynthesis of MbA may serve as a method of protection against herbivores. Future studies on the effect of MbA on potential pests of the montbretia plant may help further enlighten the ecological role of MbA in the corms.  The second objective of my thesis was to elucidate and characterize the AT involved in the third step of MbA assembly, resulting in the formation of mini-MbA. Through the differential expression analysis on the montbretia transcriptome, I discovered two BAHD-ATs, CcAT1 and CcAT2, involved in the acylation step of MbA assembly. The transcript abundance pattern of CcAT2 and CcAT2 corresponded with the MbA accumulation pattern in the corms, suggesting the involvement of CcAT1 and CcAT2 in MbA biosynthesis. The substrate specificities of CcAT1 and CcAT2 indicated that MbA biosynthesis in the montbretia plant may be regulated by both substrate availability and enzyme specificity. Furthermore, the co-expression of previously characterized UGT1 and UGT2 (Irmisch et al., submitted) with CcAT1/CcAT2 in tobacco leaves led to the formation of a surrogate mini-MbA compound. This suggests that if myricetin and caffeoyl-CoA are adequately present in tobacco leaves, the co-expression of UGT1, UGT2 and CcAT1/CcAT2 may lead to the formation of mini-MbA. The Bohlmann laboratory has already started to explore the production of myricetin and caffeoyl-CoA in tobacco leaves through the overexpression of genes involved in the flavonoid and phenylpropanoid pathway, respectively.  54  The transient expression of currently identified MbA biosynthetic genes in tobacco leaves provided promising preliminary data on the metabolic engineering of MbA in an alternative system. However, natural harvesting of MbA should not be completely eliminated as a method of MbA production. Future studies should investigate the effect of stress and elicitor treatments on the production of MbA, in efforts to increase the accumulation of MbA in the montbretia plant. Furthermore, breeding of high MbA yielding montbretia cultivars may also increase the feasibility of natural harvesting of MbA.  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The corm of the montbretia plant is the source of vegetative plant propagation. Stolons grow from a corm, and each stolon tip grows and develops into a new individual plant developing a young corm at the base.     61  Supplementary Tables  Table S4. Targeted m/z for MALDI analysis of old and young corms of montbretia plants. (M-H)-, monoisotopic mass in negative ion mode. MbA, montbretin A; MbB, montbretin B; MbC, montbretin C; MbA-C, myricetin 3-O-glucosyl glucosyl rhamnoside 4’-O-rhamnosyl xyloside; MbA-XR2, myricetin 3-O-(6-O-caffeoyl)-glucosyl glucosyl rhamnoside; MRGC, myricetin 3-O-(6-O-caffeoyl)-glucosyl rhamnoside; MRGG, myricetin 3-O-glucosyl glucosyl rhamnoside; MRG, myricetin 3-O-glucosyl rhamnoside; MR, myricetin 3-O-rhamnoside.  Compound Molecular Formula Monoisotopic Mass (M-H)- MbA C53H64O33 1228.332984 1227.324611 MbB C53H64O32 1212.338070 1211.329696 MbC C54H66O33 1242.348635 1241.340261 MbA-C C44H58O30 1066.301290 1065.292917 MbA-XR2 C42H46O25 950.232817 949.224443 MRGC C36H36O20 788.180000 787.171627 MRGG C33H40O22 788.201123 787.192749 MRG C27H30O17 626.148299 625.139926 MR C21H20O12 464.095476 463.087102     62  Table S5. BAHD acyltransferases used in phylogenetic analyses. NCBI accession identification, major acyl CoA donor, major product formed, BAHD clade identification and species identified from are shown. ATs used as search queries to identify BAHD-ATs in the montbretia transcriptome database are marked with asterisks (*). AT Name NCBI Accession  ID Major Acyl-CoA Donor Major Formed Product Species Reference Clade At3AT1 NP_171890 Hydroxy-cinnamoyl Anthocyanins, flavonols Arabidopsis thaliana Luo et al. (2007) I Gt5AT* BAA74428 Hydroxy-cinnamoyl Anthocyanins Gentiana triflora Fujiwara et al. (1998) I Dv3MAT* AAO12206 Malonyl Anthocyanins Dahlia variabilis Suzuki et al. (2002) I Sc3MaT* AAO38058 Malonyl Anthocyanins Pericallis cruenta Suzuki et al. (2003) I NtMAT1 BAD93691 Malonyl Flavonoid and napthol glucosides Nicotiana tabacum Taguchi et al. (2005) I Glossy2* CAA61258 Unknown C32 epicuticular waxes Zea mays Tacke et al. (1995) II CER2* CAA61258 Unknown C30 epicuticular waxes Arabidopsis thaliana Xia et al. (1996) II Ss5MaT2 AAR26385 Malonyl Anthocyanins Salvia splendens Suzuki et al. (2004) III VAAT* CAC09062 Acetyl Short to medium chain aliphatic Fragaria vesca Beekwilder et al. (2004) III SalAT* AAK73661 Acetyl Thebaine Papaver somniferum Grothe et al. (2001) III Pun1* AAV66311 Unknown Capsaicin pathway Capsicum annum Stewart et al. (2005) III ACT* AAO73071 Hydroxy-cinnamoyl Hydroxy-cinnamoyl agamatine derviatives Hordeum vulgare Burhenne et al. (2003) IV AtHCT* NP_199704 Hydroxy-cinnamoyl Chlorogenic acid and derivatives Arabidopsis thaliana Hoffmann et al. (2005) V CHAT* AAN09797 Acetyl (Z)-3-hexen-1-yl acetate Arabidopsis thaliana D'Auria et al. (2002) V TAT* AAF34254 Acetyl Taxa-4(20),11(12)-dien-5a-yl acetate Taxus cuspidata Walker et al. (2002) V TpHCT1A ACI16630 Caffeoyl or p-coumaroyl Caffeoyl or p-coumaroyl shikimate Trifolium pratense Sullivan (2009) V 63  Table S6. Differential expression of AT transcripts in montbretia corms. ATs cloned and heterologously expressed in this work are labeled and marked with asterisks (*). Negative logFC (log of fold change) indicate higher transcript abundance in young corms (yC) compared to old corms (oC). Contig logFC (oC vs yC) Average Expression t P Value Adjusted P Value B DN66658_c0_g1_i2* (CcAT1) -8.596 3.357 -5.750 0.001 0.018 -0.397 DN37908_c0_g1_i1* (CcAT6) -8.462 -2.906 -6.015 0.001 0.016 -1.388 DN10363_c0_g1_i1 -7.752 -3.274 -6.390 0.001 0.014 -1.283 DN62405_c4_g2_i5 -7.410 2.158 -6.015 0.001 0.016 -0.351 DN66658_c0_g1_i1* (CcAT2) -7.047 1.912 -6.260 0.001 0.015 -0.223 DN71469_c0_g1_i1 -6.578 1.819 -3.872 0.008 0.046 -2.120 DN61381_c0_g1_i1* (CcAT7) -5.825 0.203 -5.949 0.001 0.017 -0.904 DN57075_c0_g4_i2 -5.512 4.014 -10.428 0.000 0.006 2.778 DN29265_c0_g1_i1* (CcAT5) -4.965 0.175 -5.931 0.001 0.017 -0.722 DN61013_c0_g1_i1 -4.682 -0.481 -2.575 0.042 0.116 -3.480 DN63150_c0_g1_i1* (CcAT4) -4.617 4.273 -17.771 0.000 0.003 5.583 DN69576_c1_g5_i2 -4.189 0.516 -3.366 0.015 0.064 -2.656 DN69556_c0_g2_i1* (CcAT3) -3.753 1.505 -6.563 0.001 0.014 0.283 DN57284_c0_g1_i1 -3.475 7.189 -5.371 0.002 0.021 -1.272 DN52484_c0_g1_i2 -3.229 -0.590 -3.294 0.017 0.067 -2.729 DN65558_c1_g2_i1 -3.028 1.927 -5.272 0.002 0.022 -0.861 DN68748_c0_g2_i1 -2.996 -0.053 -3.375 0.015 0.063 -2.644 DN65558_c1_g2_i3 -2.766 1.508 -4.979 0.003 0.025 -1.108 DN67599_c0_g1_i2 -2.721 3.007 -8.105 0.000 0.009 1.368 DN62872_c1_g2_i1 -2.717 4.749 -6.853 0.000 0.013 0.223 DN69576_c1_g5_i1 -2.617 0.561 -3.715 0.010 0.050 -2.330 DN67599_c0_g1_i3 -2.060 1.403 -4.300 0.005 0.036 -1.851 DN67599_c0_g1_i1 -1.775 3.132 -5.338 0.002 0.022 -1.041 DN51611_c0_g1_i1 -1.668 1.966 -5.832 0.001 0.018 -0.394 DN47966_c0_g1_i1 -1.642 1.940 -3.471 0.014 0.059 -2.991 DN58645_c0_g1_i1 -1.503 7.715 -3.651 0.011 0.053 -3.305 DN65718_c0_g1_i1 -1.262 0.837 -2.391 0.054 0.136 -4.186 DN61381_c0_g1_i2 -0.523 1.269 -1.398 0.212 0.342 -5.648 DN62872_c1_g1_i1 -0.200 2.615 -0.354 0.736 0.820 -6.877 DN56685_c0_g1_i1 -0.185 1.925 -0.483 0.647 0.752 -6.654 DN66624_c0_g3_i1 -0.179 2.696 -0.747 0.483 0.614 -6.658 DN67400_c1_g1_i2 -0.079 5.085 -0.093 0.929 0.956 -7.322 DN62269_c0_g1_i1 -0.042 3.566 -0.154 0.882 0.924 -7.132 DN67486_c0_g5_i5 -0.025 1.705 -0.081 0.938 0.961 -6.730 DN63748_c0_g1_i1 0.069 5.643 0.266 0.799 0.866 -7.315 64  DN60396_c0_g1_i1 0.239 5.183 0.809 0.450 0.583 -6.972 DN47966_c0_g1_i2 0.255 1.920 0.510 0.628 0.737 -6.640 DN59104_c0_g1_i1 0.493 5.752 1.828 0.118 0.228 -5.819 DN69541_c1_g1_i6 0.945 0.501 1.141 0.298 0.436 -5.697 DN67722_c0_g1_i1 1.285 2.136 4.790 0.003 0.027 -1.486 DN57845_c0_g1_i1 1.308 1.095 2.883 0.028 0.091 -3.602 DN68748_c0_g1_i1 1.611 0.180 2.317 0.060 0.145 -4.097 DN69541_c1_g1_i3 1.694 2.339 3.880 0.008 0.046 -2.561 DN69541_c1_g1_i7 1.699 1.138 3.287 0.017 0.067 -3.058 DN67722_c0_g1_i4 2.620 2.315 6.798 0.001 0.013 0.428 DN60210_c0_g1_i2 2.631 2.383 6.925 0.000 0.012 0.525 DN57845_c0_g2_i1 4.116 3.507 7.252 0.000 0.011 0.735 DN63931_c0_g1_i1 4.519 1.348 7.004 0.000 0.012 0.505 DN63931_c0_g1_i2 5.356 0.404 4.325 0.005 0.035 -1.747 DN60210_c0_g1_i3 6.405 -0.786 3.170 0.020 0.073 -2.978     65  Table S7. Primer oligonucleotide sequences.   Primer Name  Sequence   Gene specific     AT1-fwd ATGAGCTTCACAGTGACCAAG AT1-rev CTAGGTAAAATCATTCATCTCGG AT2-fwd ATGAGCTTCACAGTGACCAAG AT2-rev CTAGGTAAAATCATTCATCTCGG AT3-fwd ATGGTGAAGGGAGAAGTGACA AT3-rev TCAATTAGCAACTGCTTCCATGAA AT4-fwd ATGGCAGTAGAGGTAGAGGC AT4-rev TCAAGCACCCACATTGAGAAAC AT5-fwd ATGACACCTTCCCTATCCTTC AT5-rev TCAAAGTGTAGAAAGAGTAAATGGG AT6-fwd ATGAGCTTCACAGTGACCAAG AT6-rev TTAAGCTACAGCCTCGTAGAAC AT7-fwd ATGGGTAAGGGAGAAGTCATAATT AT7-rev TTAACCAACTGCCTCCATGAAC   pIBA     AT1-fwd-BsaI ATGGTAGGTCTCAGCGCATGAGCTTCACAGTGACCAAGAC AT1-rev-BsaI ATGGTAGGTCTCATATCAGGTAAAATCATTCATCTCGGCGC AT2-fwd-BsaI ATGGTAGGTCTCAGCGCATGAGCTTCACAGTGACCAAGAC AT2-rev-BsaI ATGGTAGGTCTCATATCAGGTGAAATCATTCATCTCGTCGC AT3-fwd-BsaI ATGGTAGGTCTCAGCGCATGGTGAAGGGAGAAGTGACAATC AT3-rev-BsaI ATGGTAGGTCTCATATCAATTAGCAACTGCTTCCATGAACTC AT4-fwd-BsaI ATGGTAGGTCTCAGCGCATGGCAGTAGAGGTAGAGGCAAA AT4-rev-BsaI ATGGTAGGTCTCATATCAAGCACCCACATTGAGAAACTGCT AT5-fwd-BsaI ATGGTAGGTCTCAGCGCATGACACCTTCCCTATCCTTCCC AT5-rev-BsaI ATGGTAGGTCTCATATCAAAGTGTAGAAAGAGTAAATGGGACA AT6-fwd-BspMI ATGGTAACCTGCATTAGCGCATGGAAGCCTCAAATGGAAACCC AT6-rev-BspMI ATGGTAACCTGCATTATATCAAGCTACAGCCTCGTAGAACAGTT AT7-fwd-BspMI ATGGTAACCTGCATTAGCGCATGGGTAAGGGAGAAGTCATAATTC AT7-rev-BspMI ATGGTAACCTGCATTATATCAACCAACTGCCTCCATGAACTCTC   pCAMBIA    AT1-fwd-USER GGCTTAAUATGAGCTTCACAGTGACCAAG AT1-rev-USER GGTTTAAUCTAGGTAAAATCATTCATCTCGG AT2-fwd-USER GGCTTAAUATGAGCTTCACAGTGACCAAG AT2-rev-USER GGTTTAAUCTAGGTGAAATCATTCATCTCGT   qRT-PCR     qrt-AT1-fwd CGGATCTGTCACCAGTGTCTGATAT 66  qrt-AT1-rev GGATTGCCATAGCTTCGCTGT qrt-AT2-fwd CGGATCTGTCATCAATTCCTGATT qrt-AT2-rev AGATTGCCATAGCTTGGCTGC MEP1-fwd GTCGTCGGCTTCTTCGAGATG MEP1-rev CACATAGCAAACTAGGGACACAAC ZF1-fwd GTCCGTCTCGAATCTGGTGAG ZF1-rev GAGCAACACAATTCTTAGGCGTG    67  Table S8. Metabolite levels in old and young corms of montbretia plants during Feb. 2016 – Oct. 2017. The averages and standard error of four to six biological replicates are shown. MbA standard curve was used for metabolite quantification. FW, fresh weight; (-), organ not analyzed or present. MbA, montbretin A; MR, myricetin 3-O-rhamnoside; MGR, myricetin 3-O-rhamnosyl glucoside, MRG, myricetin 3-O-glucosyl rhamnoside; MbA-C, myricetin 3-O-glucosyl glucosyl rhamnoside 4’-O-rhamnosyl xyloside. Sampling Date Old Corm (2016) Young Corm (2016) Young Corm (2017)   m/z 1227, MbA [mg x g-1 (FW)]      Feb. 16 (2016) 2.3513 ± 0.4626 - - Mar. 11 2.0833 ± 0.1725 - - Apr. 12 3.2332 ± 0.5526 - - May 04 3.5527 ± 0.7065 - - Jun. 10 2.3462 ± 0.5437 4.8494 ± 0.2224 - Jun. 27 2.4158 ± 0.0953 6.1072 ± 0.3191 - Jul. 22 2.3642 ± 0.6000 5.5217 ± 0.2769 - Aug. 16 2.8017 ± 0.4031 5.8323 ± 0.4693 - Sept. 12 3.1311 ± 0.7469 3.5328 ± 0.3063 - Oct. 06 3.1670 ± 0.4500 3.5159 ± 0.3526 - Nov. 08 3.1025 ± 0.3665 3.6091 ± 0.2804 - Dec. 06 2.4552 ± 0.4657 3.1029 ± 0.3362 - Jan. 04 (2017) 2.1963 ± 0.4127 3.1957 ± 0.7512 - Feb. 03 3.0038 ± 0.3351 3.1099 ± 0.3417 - Mar. 08 - 3.5477 ± 0.4122 0.5793 ± 0.2035 Apr. 10 - 3.1400 ± 0.4393 1.0019 ± 0.1431 May 03 - 3.7902 ± 0.8038 1.3594 ± 0.0968 May 31 - 4.0880 ± 0.4703 4.3177 ± 0.5028 Jun. 16 - 2.8734 ± 0.2416 4.2714 ± 0.4154 Jul. 10 - 2.2218 ± 0.3222 5.8021 ± 0.4071 Aug. 08 - 2.9496 ± 0.4970 4.7917 ± 0.6465 Aug. 25 - 2.2872 ± 0.6031 2.8855 ± 0.2737 Sept. 22 - 2.1127 ± 0.3195 3.3394 ± 0.2762 Oct. 31 - 2.2602 ± 0.1685 2.5012 ± 0.2631     m/z 463, MR  [mg x g-1 (FW)]      Feb. 16 (2016) 0.0569 ± 0.0154 - - Mar. 11 0.0819 ± 0.0207 - - Apr. 12 0.2044 ± 0.0427 - - May 04 0.1812 ± 0.0575 - - Jun. 10 0.0985 ± 0.0385 0.2056 ± 0.0078 - Jun. 27 0.1218 ± 0.0121 0.2593 ± 0.0269 - Jul. 22 0.0863 ± 0.0279 0.2278 ± 0.0156 - Aug. 16 0.0984 ± 0.0133 0.2309 ± 0.0103 - Sept. 12 0.0848 ± 0.0262 0.1368 ± 0.0162 - Oct. 06 0.0834 ± 0.0123 0.1606 ± 0.0322 - Nov. 08 0.1013 ± 0.0113 0.0884 ± 0.0164 - Dec. 06 0.0830 ± 0.0049 0.0876 ± 0.0096 - Jan. 04 (2017) 0.0646 ± 0.0196 0.1437 ± 0.0398 - Feb. 03 0.0771 ± 0.0060 0.0948 ± 0.0134 - Mar. 08 - 0.0533 ± 0.0092 0.0083 ± 0.0020 Apr. 10 - 0.0504 ± 0.0074 0.0099 ± 0.0020 68  May 03 - 0.0836 ± 0.0167 0.0167 ± 0.0030 May 31 - 0.0737 ± 0.0099 0.0202 ± 0.0035 Jun. 16 - 0.0500 ± 0.0078 0.0738 ± 0.0117 Jul. 10 - 0.0403 ± 0.0076 0.0534 ± 0.0070 Aug. 08 - 0.0459 ± 0.0129 0.0712 ± 0.0142 Aug. 25 - 0.0351 ± 0.0115 0.0390 ± 0.0100 Sept. 22 - 0.0388 ± 0.0078 0.0778 ± 0.0071 Oct. 31 - 0.0459 ± 0.0097 0.0388 ± 0.0060     peak 1 m/z 625, tentatively MGR  [mg x g-1 (FW)]      Feb. 16 (2016) 0.0029 ± 0.0005 - - Mar. 11 0.0021 ± 0.0004 - - Apr. 12 0.0110 ± 0.0029 - - May 04 0.0071 ± 0.0042 - - Jun. 10 0.0037 ± 0.0018 0.0059 ± 0.0007 - Jun. 27 0.0045 ± 0.0006  0.0086 ± 0.0012 - Jul. 22 0.0042 ± 0.0020 0.0081 ± 0.0008 - Aug. 16 0.0026 ± 0.0007 0.0101 ± 0.0005 - Sept. 12 0.0028 ± 0.0009 0.0041 ± 0.0008 - Oct. 06 0.0036 ± 0.0018 0.0079 ± 0.0016 - Nov. 08 0.0048 ± 0.0009 0.0047 ± 0.0011 - Dec. 06 0.0110 ± 0.0017 0.0097 ± 0.0016 - Jan. 04 (2017) 0.0171 ± 0.0068 0.0158 ± 0.0042 - Feb. 03 0.0101 ± 0.0013 0.0108 ± 0.0028 - Mar. 08 - 0.0167 ± 0.0041 0.0076 ± 0.0026 Apr. 10 - 0.0132 ± 0.0016 0.0128 ± 0.0013 May 03 - 0.0086 ± 0.0014 0.0049 ± 0.0009 May 31 - 0.0077 ± 0.0014 0.0031 ± 0.0002 Jun. 16 - 0.0048 ± 0.0005 0.0042 ± 0.0007 Jul. 10 - 0.0049 ± 0.0011 0.0058 ± 0.0012 Aug. 08 - 0.0065 ± 0.0017 0.0057 ± 0.0009 Aug. 25 - 0.0033 ± 0.0011 0.0035 ± 0.0008 Sept. 22 - 0.0026 ± 0.0008 0.0048 ± 0.0006 Oct. 31 - 0.0055 ± 0.0009 0.0032 ± 0.0005     peak 2 m/z 625, MRG  [mg x g-1 (FW)]      Feb. 16 (2016) 0.0086 ± 0.0023 - - Mar. 11 0.0076 ± 0.0013 - - Apr. 12 0.0211 ± 0.0034 - - May 04 0.0417 ± 0.0174 - - Jun. 10 0.0134 ± 0.0063 0.0399 ± 0.0074 - Jun. 27 0.0146 ± 0.0024 0.0412 ± 0.0047 - Jul. 22 0.0133 ± 0.0035 0.0266 ± 0.0020 - Aug. 16 0.0163 ± 0.0037 0.0267 ± 0.0011 - Sept. 12 0.0194 ± 0.0043 0.0144 ± 0.0025 - Oct. 06 0.0211 ± 0.0064 0.0199 ± 0.0033 - Nov. 08 0.0211 ± 0.0023 0.0123 ± 0.0014 - Dec. 06 0.0216 ± 0.0033 0.0168 ± 0.0025 - Jan. 04 (2017) 0.0160 ± 0.0044 0.0362 ± 0.0111 - Feb. 03 0.0278 ± 0.0043 0.0175 ± 0.0041 - Mar. 08 - 0.0093 ± 0.0007 0.0136 ± 0.0034 Apr. 10 - 0.0094 ± 0.0010 0.0139 ± 0.0016 69  May 03 - 0.0140 ± 0.0021 0.0118 ± 0.0024 May 31 - 0.0116 ± 0.0011 0.0125 ± 0.0014 Jun. 16 - 0.0097 ± 0.0014 0.0361 ± 0.0043 Jul. 10 - 0.0106 ± 0.0004 0.0300 ± 0.0037 Aug. 08 - 0.0119 ± 0.0025 0.0217 ± 0.0016 Aug. 25 - 0.0107 ± 0.0027 0.0135 ± 0.0020 Sept. 22 - 0.0105 ± 0.0010 0.0193 ± 0.0030 Oct. 31 - 0.0113 ± 0.0017 0.0111 ± 0.0020     m/z 1065, MbA-C [mg x g-1 (FW)]      Feb. 16 (2016) 0.2826 ± 0.0577 - - Mar. 11 0.1756 ± 0.0217 - - Apr. 12 0.2011 ± 0.0247 - - May 04 0.2833 ± 0.0517 - - Jun. 10 0.2128 ± 0.0320 0.1583 ± 0.0050 - Jun. 27 0.1945 ± 0.0126 0.1858 ± 0.0079 - Jul. 22 0.2150 ± 0.0522 0.2020 ± 0.0218 - Aug. 16 0.2603 ± 0.0370 0.2439 ± 0.0121 - Sept. 12 0.3470 ± 0.0911 0.2168 ± 0.0303 - Oct. 06 0.3459 ± 0.0566 0.2302 ± 0.0250 - Nov. 08 0.3096 ± 0.0357 0.2139 ± 0.0113 - Dec. 06 0.3324 ± 0.0129 0.3136 ± 0.0216 - Jan. 04 (2017) 0.2727 ± 0.0235 0.2679 ± 0.0287 - Feb. 03 0.3398 ± 0.0262 0.2297 ± 0.0257 - Mar. 08 - 0.1537 ± 0.0087 0.0240 ± 0.0050 Apr. 10 - 0.1443 ± 0.0302 0.0365 ± 0.0057 May 03 - 0.2127 ± 0.0536 0.0528 ± 0.0066 May 31 - 0.2183 ± 0.0136 0.0993 ± 0.0095 Jun. 16 - 0.2199 ± 0.0218 0.1193 ± 0.0051 Jul. 10 - 0.2195 ± 0.0415 0.2276 ± 0.0200 Aug. 08 - 0.2455 ± 0.0449 0.2672 ± 0.0187 Aug. 25 - 0.2345 ± 0.0511 0.2524 ± 0.0126 Sept. 22 - 0.2096 ± 0.0133 0.2869 ± 0.0155 Oct. 31 - 0.2387 ± 0.0207 0.1910 ± 0.0195     70  Table S9. Gene expression correlation analysis of BAHD-ATs. Haystack analysis was used to correlate the gene expression of predicted BAHD-ATs to UGT1 and UGT2. (Part A) Expression of UGT1 and UGT2 used as baits for Haystack analysis. (Part B) List of putative BAHD-AT contigs correlating with the gene expression pattern of UGT1 or UGT2. LogFC (log of fold change) of genes in old corms (oC) compared to young corms (yC) are additionally shown. Negative logFC indicate higher expression in yC compared to oC. ATs with correlation cut off of 0.85 and logFC cut off of around -3.4 are listed. (Part C) Raw read counts of montbretia BAHD-ATs in different organs of montbretia plant. ATs cloned and heterologously expressed in this work are labeled and marked with asterisks (*).  Part A  Contig Corm Flower Leaf Stem Stolon UGT1 12.25 377.33 11.67 75.00 94.67 UGT2 6.75 600.67 53.67 7.33 101.00 Part B  Contig  Correlation t-statistic p-value logFC (oC vs yC)      UGT 1          DN63150_c0_g1_i1* (CcAT4)  0.985 9.895 1.10E-03 -4.615 DN29265_c0_g1_i1* (CcAT5) 0.983 9.169 1.37E-03 -4.964 DN37908_c0_g1_i1* (CcAT6) 0.973 7.355 2.60E-03 -8.461 DN66658_c0_g1_i2* (CcAT1) 0.972 7.155 2.81E-03 -8.596 DN61381_c0_g1_i1* (CcAT7) 0.968 6.644 3.47E-03 -5.824 DN69556_c0_g2_i1* (CcAT3) 0.963 6.176 4.27E-03 -3.753 DN66658_c0_g1_i1* (CcAT2) 0.886 3.314 2.26E-02 -7.047      UGT 2           DN29265_c0_g1_i1* (CcAT5) 0.996 19.983 1.37E-04 -4.964 DN63150_c0_g1_i1* (CcAT4) 0.996 19.466 1.48E-04 -4.615 DN66658_c0_g1_i2* (CcAT1) 0.992 14.032 3.92E-04 -8.596 DN37908_c0_g1_i1* (CcAT6) 0.986 10.426 9.42E-04 -8.461 DN69556_c0_g2_i1* (CcAT3) 0.971 6.989 3.01E-03 -3.753 DN61381_c0_g1_i1* (CcAT7) 0.970 6.923 3.09E-03 -5.824 DN66658_c0_g1_i1* (CcAT2) 0.909 3.781 1.62E-02 -7.047 Part C  Contig Corm Flower Leaf Stem Stolon DN10363_c0_g1_i1 0.00 0.00 0.00 0.00 0.00 DN10363_c0_g2_i1 0.00 0.33 0.00 0.00 0.00 DN29265_c0_g1_i1* (CcAT5) 0.00 778.35 2.41 5.15 75.75 71  DN37908_c0_g1_i1* (CcAT6) 0.75 337.67 2.00 11.33 3.67 DN38181_c0_g1_i1 15.25 0.67 3.33 0.00 164.67 DN47966_c0_g1_i1 10.40 210.90 77.86 36.51 1050.91 DN47966_c0_g1_i2 12.35 118.76 146.81 42.82 48.09 DN51611_c0_g1_i1 3.25 1042.12 41.00 47.00 45.29 DN52484_c0_g1_i2 0.25 0.00 0.00 0.33 34.67 DN55446_c0_g3_i1 7.57 172.33 10.03 0.33 689.08 DN56685_c0_g1_i1 14.00 32.00 11.00 101.00 174.00 DN57075_c0_g4_i2 44.25 4.30 0.00 0.00 437.43 DN57284_c0_g1_i1 354.88 348.14 0.33 0.67 567.81 DN57845_c0_g1_i1 18.68 1.33 0.00 1.33 64.13 DN57845_c0_g2_i1 121.07 0.00 0.00 0.00 87.53 DN58645_c0_g1_i1 418.50 149.00 95.33 344.00 2024.33 DN59104_c0_g1_i1 177.50 692.33 21.67 1329.00 1665.00 DN60210_c0_g1_i2 29.78 3.67 0.00 11.88 807.32 DN60210_c0_g1_i3 15.73 5.66 1.33 3.39 261.82 DN60396_c0_g1_i1 259.50 731.67 71.67 107.00 470.33 DN61013_c0_g1_i1 1.47 1.53 0.00 0.67 106.69 DN61381_c0_g1_i1* (CcAT7) 0.44 62.92 4.09 3.10 24.82 DN61381_c0_g1_i2 3.06 3.41 2.58 7.56 3.51 DN62269_c0_g1_i1 40.50 348.00 121.67 193.00 218.67 DN62405_c4_g2_i5 8.75 428.67 1.00 1.00 1347.25 DN62872_c1_g1_i1 63.80 26.32 18.67 40.65 1846.96 DN62872_c1_g2_i1 52.71 608.35 0.67 0.68 840.42 DN63150_c0_g1_i1* (CcAT4) 7.26 1183.15 0.33 0.33 210.95 DN63748_c0_g1_i1 131.00 91.00 11.33 94.33 172.33 DN63931_c0_g1_i1 23.14 8.60 0.83 545.18 102.04 DN63931_c0_g1_i2 21.86 9.87 2.50 285.26 193.01 DN65558_c1_g2_i1 4.50 21.38 4.15 0.00 222.99 DN65558_c1_g2_i3 3.00 347.52 10.94 2.33 101.03 DN65718_c0_g1_i1 11.65 9.67 19.22 11.53 494.04 DN65718_c0_g1_i2 5.10 3.67 6.44 1.13 45.96 DN66624_c0_g3_i1 9.50 7.33 3.33 9.00 10.67 DN66658_c0_g1_i1* (CcAT1) 1.58 17.25 5.71 2.63 10.74 DN66658_c0_g1_i2* (CcAT2) 17.67 448.75 13.30 5.71 29.26 DN67400_c1_g1_i2 124.25 9.00 11.00 343.33 112.00 DN67486_c0_g5_i5 12.50 66.93 55.98 69.21 23.51 DN67599_c0_g1_i1 28.80 23.18 4.00 12.98 1170.33 DN67599_c0_g1_i2 18.32 22.31 4.50 22.05 1203.00 DN67599_c0_g1_i3 14.37 6.51 2.83 6.31 510.68 DN67722_c0_g1_i1 22.91 25.55 7.18 2.55 66.49 DN67722_c0_g1_i4 31.27 59.07 46.33 50.41 115.48 DN67722_c0_g1_i5 0.57 10.72 6.16 3.65 5.36 DN68748_c0_g1_i1 4.14 0.44 0.00 23.58 10.95 72  DN68748_c0_g1_i2 1.40 1.38 0.33 0.00 58.25 DN68748_c0_g1_i4 2.98 3.51 0.00 3.37 22.68 DN68748_c0_g1_i5 0.32 9.00 3.00 11.05 6.90 DN68748_c0_g2_i1 1.25 296.94 265.07 160.67 46.21 DN68748_c0_g2_i2 1.50 5.40 4.59 0.00 2.46 DN69541_c1_g1_i3 10.15 8.82 3.24 5.01 34.63 DN69541_c1_g1_i6 0.84 5.92 0.67 1.05 48.45 DN69541_c1_g1_i7 8.19 2.11 14.99 10.16 57.12 DN69556_c0_g2_i1* (CcAT3) 0.75 24.00 0.67 0.00 9.00 DN69576_c1_g5_i1 8.50 25.85 0.00 7.00 94.97 DN69576_c1_g5_i2 0.75 8.82 0.00 0.67 76.70 DN71469_c0_g1_i1 2.75 206.00 343.00 90.67 39.33      73  Table S10. Statistical analysis of MbA levels in montbretia corms. Corms were harvested from Mar. 2017 – Oct. 2017. To test for significant differences of MbA levels in old corms (oC) and young corms (yC), a two-way analysis of variance (ANOVA) and Tukey Test was performed. Time refers to the sampling dates; age refers to the age of the corm (old or young). P-values indicating significant differences are in bold. (Part A) Results of the two-way ANOVA analysis. (Part B) Tukey Test analysis using age within time as the comparison factor. (Part C) Tukey test analysis using time within age as the comparison factor.  Part A   Time Age Time x Age     MbA oC vs. yC        F 7.65 0.651 12.721 P <0.001 0.422 <0.001  Part B  q P-value  q P-value       MbA oC vs. yC            Mar. 08 6.855 <0.001 Jul. 10 8.868 <0.001 Apr. 10 5.419 <0.001 Aug. 08 4.562 0.002 May 03 5.677 <0.001 Aug. 25 1.482 0.298 May 31 0.623 0.661 Sept. 22 3.039 0.035 Jun. 16 3.463 0.017 Oct. 31 0.597 0.674  Part C   q P-value  q P-value       MbA oC   MbA yC         May 31 vs. Sept. 22 5.110 0.018 Jul. 10 vs. Mar. 08 12.398 <0.001 May 31 vs. Jul. 10 4.828 0.032 Jul. 10 vs. Apr. 10 10.998 <0.001 May 31 vs. Oct. 31 4.729 0.039 Jul. 10 vs. May 03 11.004 <0.001 May 31 vs. Aug. 25 4.659 0.045 Jul. 10 vs. Oct. 31 8.176 <0.001 May 31 vs. Jun. 16 3.142 0.451 Jul. 10 vs. Aug. 25 7.224 <0.001 May 31 vs. Aug. 08 2.945 0.545 Jul. 10 vs. Sept. 22 6.100 0.002 May 31 vs. Apr. 10 1.334 0.994 Jul. 10 vs. Jun. 16 3.791 0.200 May 31 vs. Mar. 08 1.356 0.994 Jul. 10 vs. May 31 3.840 0.186 May 31 vs. May 03 0.723 1.000 Jul. 10 vs. Aug. 08 2.502 0.752 May 03 vs. Sept. 22 3.918 0.165 Aug. 08 vs. Mar. 08 9.896 <0.001 May 03 vs. Jul. 10 3.663 0.240 Aug. 08 vs. Apr. 10 8.639 <0.001 74  May 03 vs. Oct. 31 3.573 0.271 Aug. 08 vs. May 03 8.501 <0.001 May 03 vs. Aug. 25 3.510 0.294 Aug. 08 vs. Oct. 31 5.673 0.005 May 03 vs .Jun. 16 2.141 0.883 Aug. 08 vs. Aug. 25 4.721 0.040 May 03 vs. Aug. 08 1.963 0.927 Aug. 08 vs. Sept. 22 3.597 0.262 May 03 vs. Apr. 10 0.558 1.000 Aug. 08 vs. Jun. 16 1.289 0.996 May 03 vs. Mar. 08 0.529 1.000 Aug. 08 vs. May 31 1.226 0.997 Mar. 08 vs. Sept. 22 3.594 0.263 May 31 vs. Mar. 08 9.110 <0.001 Mar. 08 vs. Jul. 10 3.324 0.370 May 31 vs. Apr. 10 7.828 <0.001 Mar. 08 vs. Oct. 31 3.229 0.411 May 31 vs. May 03 7.653 <0.001 Mar. 08 vs. Aug. 25 3.162 0.442 May 31 vs. Oct. 31 4.699 0.042 Mar. 08 vs. Jun. 16 1.710 0.969 May 31 vs. Aug. 25 3.705 0.226 Mar. 08 vs. Aug. 08 1.521 0.986 May 31 vs. Sept. 22 2.531 0.740 Mar. 08 vs. Apr. 10 0.059 1.000 May 31 vs. Jun. 16 0.120 1.00 Apr. 10 vs. Sept. 22 3.329 0.367 Jun. 16 vs. Mar. 08 8.607 <0.001 Apr. 10 vs. Jul. 10 3.074 0.483 Jun. 16 vs. Apr. 10 7.424 <0.001 Apr. 10 vs. Oct. 31 2.985 0.526 Jun. 16 vs. May 03 7.213 <0.001 Apr. 10 vs. Aug. 25 2.922 0.556 Jun. 16 vs. Oct. 31 4.384 0.075 Apr. 10 vs. Jun. 16 1.553 0.984 Jun. 16 vs. Aug. 25 3.433 0.324 Apr. 10 vs. Aug. 08 1.375 0.993 Jun. 16 vs. Sept. 22 2.308 0.828 Aug. 08 vs. Sept. 22 2.073 0.901 Sept. 22 vs. Mar. 08 6.299 0.001 Aug. 08 vs. Jul. 10 1.803 0.956 Sept. 22 vs. Apr. 10 5.248 0.013 Aug. 08 vs. Oct. 31 1.708 0.969 Sept. 22 vs. May 03 4.904 0.028 Aug. 08 vs. Aug. 25 1.641 0.976 Sept. 22 vs. Oct. 31 2.076 0.901 Aug. 08 vs. Jun. 16 0.189 1.000 Sept. 22 vs. Aug. 25 1.124 0.999 Jun. 16 vs. Sept. 22 1.884 0.943 Aug. 25 vs. Mar. 08 5.174 0.016 Jun. 16 vs. Jul. 10 1.614 0.979 Aug. 25 vs. Apr. 10 4.188 0.107 Jun. 16 vs. Oct. 31 1.519 0.986 Aug. 25 vs. May 03 3.780 0.203 Jun. 16 vs. Aug. 25 1.452 0.990 Aug. 25 vs. Oct. 31 0.952 1.000 Aug. 25 vs. Sept. 22 0.432 1.000 Oct. 31 vs. Mar. 08 4.223 0.100 Aug. 25 vs. Jul. 10 0.162 1.000 Oct. 31 vs. Apr. 10 3.290 0.384 Aug. 25 vs. Oct. 31 0.067 1.000 Oct. 31 vs. May 03 2.828 0.602 Oct. 31 vs. Sept. 22 0.365 1.000 May 03 vs. Mar. 08 1.395 0.992 Oct. 31 vs. Jul. 10 0.095 1.000 May 03 vs. Apr. 10 0.624 1.000 Jul. 10 vs. Sept. 22 0.270 1.000 Apr. 10 vs. Mar. 08 0.691 1.000      75  Table S11. Statistical analysis of CcAT1 and CcAT2 relative transcript abundance in montbretia corms. Relative transcript abundance was determined by qRT-PCR analysis using old corms (oC) and young corms (yC) collected Jun. 2016 – Oct. 2016. A two-way analysis of variance (ANOVA) and a Tukey test was used to test for significant differences in CcAT1 and CcAT2 transcript levels. Time refers to the sampling dates; age refers to the age of the corm (old or young). P-values indicating significant differences are in bold. (Part A) Results of the two-way ANOVA analysis. (Part B) Tukey test analysis using age within time as the comparison factor. Tukey test analysis of (Part C) CcAT1 and (Part D) CcAT2 transcripts using time within age as the comparison factor.  Part A   Time Age Time x Age     CcAT1        F 193.527 6.24 5.619 P <0.001 0.001 0.002     CcAT2        F 304.188 3.676 5.828 P <0.001 0.015 0.002 Part B  CcAT1 CcAT2  q P q P      oC vs. yC          Jun. 10  12.563 <0.001 13.613 <0.001 Jun. 27 13.049 <0.001 16.134 <0.001 Jul. 22 6.047 <0.001 8.88 <0.001 Aug. 16 5.097 0.002 6.711 <0.001 Sept. 12 7.338 <0.001 9.492 <0.001 Oct. 06  4.966 0.002 6.563 <0.001 Part C  q P  q P       CcAT1 oC    CcAT1 yC          Jul. 22 vs. Sept. 12 2.592 0.467 Jun. 10 vs. Oct. 06 7.786 <0.001 Jul. 22 vs. Jun. 27 2.619 0.456 Jun. 10 vs. Aug. 16 7.768 <0.001 Jul. 22 vs. Aug. 16 2.252 0.612 Jun. 10 vs. Sept. 12 5.643 0.008 Jul. 22 vs. Oct. 06 2.137 0.661 Jun. 10 vs. Jul. 22 4.902 0.024 Jul. 22 vs. Jun. 10 1.614 0.859 Jun. 10 vs. Jun. 27 0.519 0.999 Jun. 10 vs. Sept. 12 1.149 0.962 Jun. 27 vs. Oct. 06 7.267 <0.001 76  Jun. 10 vs. Jun. 27 1.005 0.979 Jun. 27 vs. Aug. 16 7.249 <0.001 Jun. 10 vs. Aug. 16 0.808 0.992 Jun. 27 vs. Sept. 12 5.124 0.017 Jun. 10 vs. Oct. 06 0.694 0.996 Jun. 27 vs. Jul. 22 4.383 0.053 Oct. 06 vs. Sept. 12 0.415 1.000 Jul. 22 vs. Oct. 06 2.885 0.355 Oct. 06 vs. Jun. 27 0.205 1.000 Jul. 22 vs. Aug. 16 2.866 0.361 Oct. 06 vs. Aug. 16 0.104 1.000 Jul. 22 vs. Sept. 12 0.741 0.995 Aug. 16 vs. Sept. 12 0.311 1.000 Sept. 12 vs. Oct. 06 2.144 0.659 Aug. 16 vs. Jun. 27 0.091 1.000 Sept. 12 vs. Aug. 16 2.125 0.667 Jun. 27 vs. Sept. 12 0.250 1.000 Aug. 16 vs. Oct. 06 0.019 1.000  Part D   q P  q P       CcAT2 oC    CcAT2 yC          Jul. 22 vs. Jun. 27 2.484 0.513 Jun. 27 vs. Aug. 16 7.377 <0.001 Jul. 22 vs. Sept. 12 1.532 0.883 Jun. 27 vs. Oct. 06 6.965 0.001 Jul. 22 vs. Aug. 16 1.100 0.968 Jun. 27 vs. Jul. 22 4.771 0.030 Jul. 22 vs. Jun. 10 0.695 0.996 Jun. 27 vs. Sept. 12 4.751 0.031 Jul. 22 vs. Oct. 06 0.584 0.998 Jun. 27 vs. Jun. 10 0.732 0.995 Oct. 06 vs. Jun. 27 1.637 0.851 Jun. 10 vs. Aug. 16 6.645 0.002 Oct. 06 vs. Sept. 12 0.866 0.989 Jun. 10 vs. Oct. 06 6.233 0.003 Oct. 06 vs. Aug. 16 0.471 0.999 Jun. 10 vs. Jul. 22 4.039 0.087 Oct. 06 vs. Jun. 10 0.037 1.000 Jun. 10 vs. Sept. 12 4.019 0.089 Jun. 10 vs. Jun. 27 1.789 0.800 Sept. 12 vs. Aug. 16 2.626 0.454 Jun. 10 vs. Sept. 12 0.911 0.986 Sept. 12 vs. Oct. 06 2.214 0.628 Jun. 10 vs. Aug. 16 0.479 0.999 Sept. 12 vs. Jul. 22 0.020 1.000 Aug. 16 vs. Jun. 27 1.121 0.966 Jul. 22 vs. Aug. 16 2.607 0.462 Aug. 16 vs. Sept. 12 0.394 1.000 Jul. 22 vs. Oct. 06 2.195 0.637 Sept. 12 vs. Jun. 27 0.689 0.996 Oct. 06 vs. Aug. 16 0.412 1.000     77  Table S12. Statistical analysis of MbA levels in dried montbretia corms. Ground corm samples harvested from Jun. 27th, Jul. 22nd and Sept. 12th 2016 were dried. To test for significant differences of MbA levels in dried old corms (oC) and young corms (yC) samples, a two-way analysis of variance (ANOVA) and Tukey Test was performed. Time refers to the sampling dates; age refers to the age of the corm (old or young). P-values indicating significant differences are in bold. (Part A) Results of the two-way ANOVA analysis. (Part B) Tukey Test analysis using age within time as the comparison factor. (Part C) Tukey test analysis using time within age as the comparison factor.  Part A   Time Age Time x Age     MbA oC vs. yC        F 39.263 150.298 26.385 P <0.001 <0.001 <0.001 Part B  q P-value    MbA oC vs. yC      Jun. 27 14.993 <0.001 Jul. 22 11.988 <0.001 Sept. 12 2.270 0.140 Part C   q P-value  q P-value       MbA oC   MbA yC         Jul. 22 vs. Sept. 12 1.936 0.392 Jun. 27  vs. Sept. 12 15.470 <0.001 Jul. 22 vs. Jun. 27 0.969 0.777 Jun. 27  vs. Jul. 22 2.172 0.316 Jun. 27  vs. Sept. 12 0.875 0.814 Jul. 22 vs. Sept. 12 13.298 <0.001      78  Table S13. Statistical analysis of total MbA levels per corm. Masses of corms harvested from May 2017 – Oct. 2017 were weighed to determine total MbA content per corm. To test for significant differences of MbA levels in old corms (oC) and young corms (yC), a two-way analysis of variance (ANOVA) and Tukey Test was performed. Time refers to the sampling dates; age refers to the age of the corm (old or young). P-values indicating significant differences are in bold. (Part A) Results of the two-way ANOVA analysis. (Part B) Tukey Test analysis using age within time as the comparison factor. (Part C) Tukey test analysis using time within age as the comparison factor.  Part A   Time Age Time x Age     MbA oC vs. yC        F 4.173 93.529 15.437 P <0.001 <0.001 <0.001 Part B  q P-value    MbA oC vs. yC      May 03 13.876 <0.001 May 31 12.307 <0.001 Jun. 16 5.742 <0.001 Jul. 10 2.639 0.067 Aug. 08 2.932 0.042 Aug. 25 0.212 0.881 Sept. 22 0.422 0.766 Oct. 31 0.437 0.758 Part C   q P-value   q P-value        MbA oC   MbA yC         May 03 vs. Aug 25 5.489 0.006 Sept. 22 vs. May 03 10.662 <0.001 May 03 vs. Oct. 31 3.783 0.150 Sept. 22 vs. May 31 8.603 <0.001 May 03 vs. Jul. 10  3.751 0.157 Sept. 22 vs. Jun. 16 5.395 0.007 May 03 vs. Jun. 16 3.497 0.226 Sept. 22 vs. Jul. 10  2.561 0.615 May 03 vs. Sept. 22 3.426 0.249 Sept. 22 vs. Aug 25 1.554 0.955 May 03 vs. Aug 08 1.657 0.937 Sept. 22 vs. Aug 08 0.633 1.000 May 03 vs. May 31 0.332 1.000 Sept. 22 vs. Oct. 31 0.394 1.000 May 31 vs. Aug 25 5.470 0.006 Oct. 31 vs. May 03 10.268 <0.001 May 31 vs. Oct. 31 3.660 0.180 Oct. 31 vs. May 31 8.209 <0.001 May 31 vs. Jul. 10  3.626 0.189 Oct. 31 vs. Jun. 16 5.001 0.017 79  May 31 vs. Jun. 16 3.357 0.272 Oct. 31 vs. Jul. 10  2.167 0.787 May 31 vs. Sept. 22 3.282 0.299 Oct. 31 vs. Aug 25 1.160 0.991 May 31 vs. Aug 08 1.405 0.974 Oct. 31 vs. Aug 08 0.239 1.000 Aug 08 vs. Aug 25 4.065 0.096 Aug 08 vs. May 03 10.029 <0.001 Aug 08 vs. Oct. 31 2.255 0.752 Aug 08 vs. May 31 7.970 <0.001 Aug 08 vs. Jul. 10  2.221 0.766 Aug 08 vs. Jun. 16 4.763 0.027 Aug 08 vs. Jun. 16 1.952 0.863 Aug 08 vs. Jul. 10  1.928 0.870 Aug 08 vs. Sept. 22 1.877 0.885 Aug 08 vs. Aug 25 0.921 0.998 Sept. 22 vs. Aug 25 2.188 0.779 Aug 25 vs. May 03 9.108 <0.001 Sept. 22 vs. Oct. 31 0.379 1.000 Aug 25 vs. May 31 7.049 <0.001 Sept. 22 vs. Jul. 10  0.344 1.000 Aug 25 vs. Jun. 16 3.842 0.137 Sept. 22 vs. Jun. 16 0.075 1.000 Aug 25 vs. Jul. 10  1.008 0.996 Jun. 16 vs. Aug 25 2.113 0.808 Jul. 10  vs. May 03 8.101 <0.001 Jun. 16 vs. Oct. 31 0.304 1.000 Jul. 10  vs. May 31 6.042 0.002 Jun. 16 vs. Jul. 10  0.269 1.000 Jul. 10  vs. Jun. 16 2.834 0.488 Jul. 10  vs. Aug 25 1.844 0.894 Jun. 16 vs. May 03 5.266 0.010 Jul. 10  vs. Oct. 31 0.034 1.000 Jun. 16 vs. May 31 3.208 0.327 Oct. 31 vs. Aug 25 1.810 0.903 May 31 vs. May 03 2.059 0.827  

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