Characterization of Sanguinarine Reductases from Papaver Somniferum (Unpublished Master's Thesis)

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2015-05-20 Characterization of sanguinarine reductases from Papaver somniferum

Bross, Crystal

Bross, C. (2015). Characterization of sanguinarine reductases from Papaver somniferum (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25290 http://hdl.handle.net/11023/2260 master thesis

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Characterization of sanguinarine reductases from Papaver somniferum

Crystal Dawn Bross

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF BIOLOGICAL SCIENCES

CALGARY, ALBERTA

MAY, 2015

Abstract

Papaver somniferum (opium poppy) produces several pharmacologically relevant benzylisoquinoline alkaloids, such as the analgesics codeine and morphine, the muscle relaxant papaverine, the potential anti-cancer drug noscapine, and the antimicrobial agent sanguinarine. Sanguinarine is a highly cytotoxic benzophenanthridine alkaloid synthesized by the plant to defend against herbivory and pathogens. However, sanguinarine can bind DNA, induce apoptosis, and will inhibit the growth of plant cell cultures that do not synthesize benzophenanthridine alkaloids. Therefore, it is proposed that sanguinarine reductase (SanR) exists in plants that synthesize benzophenanthridine alkaloids to facilitate the detoxification of sanguinarine through its reduction to dihydrosanguinarine. Three transcripts encoding SanRs were identified in opium poppy transcriptome databases and were characterized biochemically and physiologically using enzyme assays, virus-induced gene silencing, and immunolocalization to gain insight into the role of SanR as an enzyme of detoxification.

Acknowledgements

I would like to thank those who supported me during the completion of my thesis.

Thank you to my lab members, especially Guillaume Beaudoin, Thu-Thuy Dang, Scott

Farrow, Donald Dinsmore, Xue Chen, Eun-Jeong Lee, and Jeremy Morris, for their guidance and assistance, and willingness to help in any way. And thank you to my friends, Ramya Singh and Bonnie McNeil, for keeping me grounded, and offering an outside perspective on my research.

I would also like to thank my committee, Dr. Doug Muench and Dr. Marcus Samuel, for their guidance. And thank you to Dr. Ed Yeung, Dr. Christoph Sensen, and Ye Zhang for their expertise in botany and phylogeny.

Lastly, a special thank you to my parents for their unconditional love, support, and encouragement. You were always there when I needed you, and I am forever grateful.

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Table of Contents

Abstract ...... ii Acknowledgements ...... iii Table of Contents ...... iv List of Tables ...... vii List of Figures and Illustrations ...... viii List of Symbols, Abbreviations, and Nomenclatures ...... x

1 INTRODUCTION...... 1 1.1 Alkaloids ...... 1 1.2 Benzylisoquinoline alkaloid biosynthesis ...... 2 1.3 Epimerization of reticuline and morphine biosynthesis ...... 5 1.4 Sanguinarine biosynthesis ...... 7 1.5 Sanguinarine is a cytotoxic compound ...... 9 1.6 Localization of alkaloids and biosynthetic enzymes in planta ...... 11 1.7 Objectives ...... 13

2 MATERIALS AND METHODS ...... 14 2.1 Media ...... 14 2.1.1 Lysogeny broth (LB) media ...... 14 2.1.2 Antibiotics ...... 14 2.1.3 Blue/white selection ...... 14 2.1.4 Agrobacterium tumefaciens induction medium ...... 15 2.1.5 Agrobacterium tumefaciens infiltration solution ...... 15 2.2 Buffers...... 15 2.2.1 Plasmid DNA isolation buffers ...... 15 2.2.2 2X CTAB RNA extraction buffer ...... 15 2.2.3 2X SDS-PAGE sample buffer ...... 16 2.2.4 10X SDS-PAGE electrode buffer ...... 16 2.2.5 10X Western blot transfer buffer ...... 16 2.2.6 1X Transfer buffer for Western blotting ...... 16 2.2.7 10X Tris-buffered saline (TBS) buffer ...... 16 2.2.8 1X TBS-Tween ...... 16 2.2.9 Plant protein extraction buffer ...... 16 2.2.10 Sodium phosphate buffer, pH 7.6 (100 mM) ...... 16 2.2.11 Coomassie stain ...... 17 2.2.12 Solvent A mass spectrometry running buffer ...... 17 2.3 Gel electrophoresis...... 17 2.3.1 50X TAE buffer ...... 17 2.3.2 30% Acrylamide solution ...... 17 2.3.3 Separating gel (12%) ...... 17 2.3.4 Resolving gel (4%) ...... 18

2.4 Organisms ...... 18 2.4.1 Bacteria ...... 18 2.4.2 Plants ...... 18 2.5 Plasmids ...... 19 2.5.1 Subcloning plasmid ...... 19 2.5.2 Recombinant protein expression plasmid ...... 20 2.5.3 Virus-induced gene silencing (VIGS) plasmids ...... 20 2.6 Cloning and Transformations ...... 22 2.6.1 Sequence identification and primer design ...... 22 2.6.2 PCR amplification of DNA and ligation ...... 33 2.6.3 Bacterial transformation ...... 34 2.6.4 Plant transformation ...... 35 2.7 Escherichia coli protein induction, purification, and detection ...... 36 2.8 Plant protein purification, and detection ...... 38 2.9 Alkaloids ...... 38 2.9.1 Isolation of benzophenanthridine alkaloids ...... 39 2.10 Enzyme assays ...... 40 2.11 Antibody production ...... 40 2.11.1 Dot blots ...... 41 2.12 Immunolocalization ...... 41 2.12.1 Tissue fixation and embedding ...... 41 2.12.2 Immunohistochemistry ...... 42 2.12.3 Microscopy ...... 42 2.13 Virus-induced gene silencing ...... 43 2.13.1 RNA extraction and cDNA synthesis ...... 43 2.13.2 Quantitative real-time PCR ...... 44 2.13.3 Root alkaloid extraction ...... 45 2.14 Liquid chromatography-mass spectrometry ...... 45 2.15 Statistical analysis ...... 46

3 RESULTS ...... 47 3.1 Sanguinarine reductase identification, expression, and purification ...... 47 3.2 Biochemical characterization of sanguinarine reductases in vitro ...... 51 3.2.1 Sanguinarine reductase does not reduce 1,2-dehydroreticuline ...... 51 3.2.2 Purification of benzophenanthridine alkaloids ...... 51 3.2.3 Sanguinarine reductases reduce benzophenanthridine alkaloids...... 56 3.2.4 Temperature curves ...... 56 3.2.5 Michaelis-Menten kinetic analysis ...... 66 3.3 Immunolocalization of sanguinarine reductases ...... 66 3.3.1 Antibody production & dot blots ...... 66 3.3.2 Sanguinarine reductase expression in planta ...... 70 3.3.3 Epifluorescence microscopy ...... 70 3.4 Virus-induced gene silencing of sanguinarine reductases ...... 75 3.4.1 Quantitative PCR primer and probe specificity towards SanRs ...... 75 3.4.2 Sanguinarine reductase expression in planta ...... 75 3.4.3 Knocking down expression in planta using VIGS ...... 79

4 DISCUSSION ...... 109 4.1 Sanguinarine reductase identification, expression, and purification ...... 109 4.2 Biochemical characterization of sanguinarine reductases in vitro ...... 111 4.3 Short-chain dehydrogenase/reductases ...... 115 4.4 Protein localization of sanguinarine reductases in planta ...... 117 4.5 Expression of sanguinarine reductases in planta ...... 120 4.6 Biological roles of sanguinarine reductases ...... 127

5 CONCLUSION ...... 134

Bibliography ...... 137 List of Appendix Tables and Figures ...... 148 Appendix A1: Cloning dehydroreticuline reductase candidates ...... 149 Appendix A2: Biochemical characterization of SanRs...... 167 Appendix A3: SanR expression in transcriptome libraries ...... 170 Appendix A4: Phylogenetic analysis ...... 174

List of Tables

Table 1. List of primers used for cloning procedures...... 23

Table 2. List of VIGS and qPCR primers...... 27

Table 3. Benzylisoquinoline alkaloids tested as potential substrates of SanRs...... 52

Table 4. Quantitative PCR primer and probe specificity towards SanR genes...... 76

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List of Figures and Illustrations

Figure 1. Benzylisoquinoline alkaloid biosynthesis in Papaver somniferum...... 2

Figure 2. TRV-based virus-induced gene silencing vectors ...... 21

Figure 3. Sequence alignment of sanguinarine reductases ...... 23

Figure 4. Constructs designed to silence sanguinarine reductases (SanRs) using virus-induced gene silencing (VIGS) ...... 32

Figure 5. Expression of recombinant sanguinarine reductases...... 48

Figure 6. Purification of sanguinarine reductases using TALON metal affinity resin .... 49

Figure 7. Purification of recombinant sanguinarine reductases ...... 50

Figure 8. Sanguinarine reductases do not reduce 1,2-dehydroreticuline ...... 54

Figure 9. TLC separation of benzophenanthridine alkaloids ...... 55

Figure 10. Collision-induced dissociation spectra for benzophenanthridines ...... 57

Figure 12. Non-enzymatic reduction of benzophenanthridines ...... 59

Figure 13. Sanguinarine reductases reduce benzophenanthridine alkaloids...... 60

Figure 14. SanR2 reduces benzophenanthridine alkaloids using NADPH or NADH ..... 64

Figure 15. Temperature curve for SanR2 ...... 65

Figure 16. Michaelis-Menten enzyme kinetics for SanR1 and SanR3B ...... 67

Figure 17. Generation of antibodies against sanguinarine reductases ...... 68

Figure 18. Specificity of antibodies generated against recombinant sanguinarine reductases ...... 69

Figure 19. Sanguinarine reductases are present in all opium poppy tissues...... 71

Figure 20. Sanguinarine reductases localized to the phloem ...... 74

Figure 21. Relative gene expression of opium poppy sanguinarine reductases in different tissues ...... 77

Figure 22. Presence of coat protein RNA in plants transformed with VIGS constructs .. 80

Figure 23. Root gene expression profiles of empty vector control opium poppy plants...... 82

viii

Figure 24. Retention times of benzylisoquinoline alkaloid authentic standards ...... 84

Figure 25. Example chromatographs for VIGS metabolite analysis ...... 86

Figure 26. Gene expression and metabolite profiles of SanR1-silenced opium poppy roots...... 89

Figure 27. Gene expression and metabolite profiles of SanR2-silenced opium poppy roots...... 92

Figure 28. Gene expression and metabolite profiles of SanR3-silenced opium poppy roots...... 96

Figure 29. Gene expression and metabolite profiles of SanR1- and SanR3-silenced opium poppy roots...... 99

Figure 30. Gene expression and metabolite profiles of SanR1-silenced opium poppy roots...... 101

Figure 31. Gene expression and metabolite profiles of SanR-silenced opium poppy roots...... 103

Figure 32. Gene expression and metabolite profiles of SanR-silenced opium poppy roots...... 105

Figure 33. Gene expression and metabolite profiles of SanR-silenced opium poppy roots...... 107

Figure 34. Predicted model for opium poppy SanRs...... 131

List of Symbols, Abbreviations, and Nomenclatures

*Standard SI units not listed

Symbol Definition [M]+ or [M+H]+ Parent ion 4-HPAA 4-hydroxyphenylacetaldehyde 4’OMT 4’-O-methyltranferase 6xHis tag composed of 6 consecutive histidine residues 6OMT 6-O-methyltransferase A260 absorbance at 260 nm APS ammonium persulfate AhR aryl hydrocarbon receptor BIA benzylisoquinoline alkaloid BBE berberine bridge enzyme bp base pairs CaMV cauliflower mosaic virus cDNA complementary DNA CID collision-induced dissociation CFS cheilanthifoline synthase CNMT coclaurine N-methyltransferase CODM codeine O-demethylase COR codeinone reductase CP coat protein CTAB cetrimonium bromide cv. cultivar CYP cytochrome P450 oxidase DBOX dihydrobenzophenanthridine oxidase (DBOX) DEPC diethylpyrocarbonate DMSO dimethyl sulfoxide DRR dehydroreticuline reductase DRS dehydroreticuline synthase ECL enhanced chemiluminescence EDTA ethylenediaminetetraacetic acid EIC extracted ion chromatograph EMS ethyl methanesulfonate ER endoplasmic reticulum FADOX FAD-dependent oxidoreductase FAM fluorescein GAPDH glyceraldehyde 3-phosphate dehydrogenase HO heme oxidase HRP horseradish peroxidase IPTG isopropyl-beta-D-thiogalactopyranoside LB lysogeny broth LC-MS liquid chromatography-mass spectrometry MCS multiple cloning site

MES 2-(N-morpholino)ethanesulfonic acid MGB minor groove binder M-MLV Moloney Murine Leukemia Virus MLP major latex protein MP movement protein MSH (S)-cis-N-methylstylopine 14-hydroxylase m/z mass-to-charge ratio NADH Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate NCS norcoclaurine synthase NFQ non-fluorescent quencher Ni-NTA nickel-nitrilotriacetic acid NMCH (S)-N-methylcoclaurine 3’-hydroxylase NOS noscapine synthase NQO NAD(P)H quinone oxidoreductase OD600 optical density at 600 nm P6H protopine 6-hydroxylase PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PDS phytoene desaturase PFA paraformaldehyde PHYLIP PHYLogeny Inference Package PIPES piperazine-N,N′-bis(2-ethanesulfonic acid) PMSF phenylmethylsulfonyl fluoride PVP polyvinylpyrrolidone PVPP polyvinylpolypyrrolidone qPCR quantitative real-time PCR RE restriction enzyme RPM revolutions per minute RT reverse transcriptase SalAT salutaridinol 7-O-acetyltransferase SanR sanguinarine reductase SalR salutaridine reductase SalSyn salutaridine synthase SD standard deviation SDR short-chain dehydrogenase/reductase SDS sodium dodecyl sulphate SEM standard error of the mean SPD spermidine SPS stylopine synthase T6OM thebaine 6-O-demethylase TAE Tris-acetate-EDTA TBS Tris-buffered saline TEMED tetramethylethylenediamine TIC total ion chromatograph TLC thin-layer chromatography

TNMT tetrahydroprotoberberine cis-N-methyltransferase TRV tobacco rattle virus UTR untranslated region VIGS virus-induced gene silencing X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

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1 INTRODUCTION

1.1 Alkaloids

Alkaloids are naturally occurring, low molecular weight compounds that are difficult to categorize (Ziegler and Facchini, 2008). Alkaloid are often derived from an amino acid with the nitrogen atom in a heterocyclic ring, but this definition does not hold true for all alkaloids. Alkaloids are grouped into different classes based upon their carbon skeletal structures, including, but not limited to, pyridine alkaloids (e.g. nicotine), purine alkaloids (e.g. caffeine), tropane alkaloids (e.g. cocaine), indole alkaloids

(e.g. vinblastine), and benzylisoquinoline alkaloids (e.g. morphine).

Alkaloids are a structurally and functionally diverse group of secondary metabolites found in approximately 20% of plant species (Facchini and De Luca, 2008).

Alkaloids synthesized by plants may serve as defence molecules against herbivores or pathogens, but alkaloids are often used as pharmaceuticals due to their potent pharmacological properties. For example, Nicotiana attenuate synthesizes nicotine, which acts as a deterrent for herbivory (Steppuhn et al., 2004). Nicotine was once used as an insecticide to control pests in agriculture (Soloway, 1976), but nicotine is also smoked recreationally for its mood altering effects as both a stimulant and relaxant. Conversely, the exact role of morphine in planta remains unknown. Morphine may play a defence role, whereby it is quickly metabolized to bismorphine upon mechanical damage

(Morimoto et al., 2001). Bismorphine accumulates in the cell wall and crosslinks with pectin to increase resistance to hydrolysis by pectinases. Nonetheless, morphine is an important analgesic, and to date Papaver somniferum (opium poppy) remains its sole commercial source.

Like morphine, many benzylisoquinoline alkaloids display potent pharmacological activities, including codeine as a cough suppressant, papaverine as a muscle relaxant, noscapine as an anti-cancer drug, and sanguinarine as an antimicrobial agent (Ziegler and Facchini, 2008). Morphine precursors are also used as precursors to several semi-synthetic drugs. For example, thebaine is used to produce semi-synthetic drugs, such as the analgesic oxycodone, and naltrexone and naloxone, which are used to treat opiate addiction (Millgate et al., 2004).

1.2 Benzylisoquinoline alkaloid biosynthesis

All benzylisoquinoline alkaloids (BIAs) are synthesized from two derivatives of the aromatic amino acid tyrosine, and are produced mainly by plants in the Papaveraceae,

Ranunculaceae, Berberidaceae, and Menispermaceae families (Facchini and De Luca,

2008). Although, BIAs are extensively studied in Papaver somniferum (opium poppy),

Eschscholzia californica (California poppy), Thalictrum species and Coptis japonica, and, to date, approximately 2,500 BIA structures have been elucidated. In opium poppy, norcoclaurine synthase (NCS) catalyzes the first committal step in BIA biosynthesis through the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) to form (S)-norcoclaurine (Fig. 1) (Lee and Facchini, 2010; Samanani et al., 2004).

(S)-Norcoclaurine undergoes three methylations and a hydroxylation to form

(S)-reticuline. First, (S)-norcoclaurine is methylated by norcoclaurine

6-O-methyltransferase (6OMT) to (S)-coclaurine, which is then N-methylated to

(S)-N-methylcoclaurine by coclaurine N-methyltransferase (CNMT) (Ounaroon et al.,

2003; Choi et al., 2002; Sato et al., 1994). (S)-N-Methylcoclaurine is hydroxylated by

Figure 1. Benzylisoquinoline alkaloid biosynthesis in Papaver somniferum.

Figure 1 (continued). Benzylisoquinoline alkaloid biosynthesis in Papaver somniferum.

Schematic of the biosynthetic pathways leading to sanguinarine and morphine in opium poppy. All enzymes that have been identified (bold) except for those responsible for the epimerization of reticuline. Sanguinarine reductase (SanR, blue) is characterized in this thesis. Not pictured are the biosynthetic pathways for papaverine, which is derived from

(S)-coclaurine, and noscapine, which is derived from (S)-reticuline. NCS: norcoclaurine synthase, 6OMT: norcoclaurine 6-O-methyltrans-ferase, CNMT: coclaurine

N-methyltransferase, NMCH: N-methylcoclaurine 3′-hydroxylase, 4′OMT:

(S)-3’-hydroxy N-methylcoclaurine 4’-O-methyltranferase, BBE: berberine bridge enzyme, CFS: cheilanthifoline synthase, SPS: stylopine synthase, MSH:

N-methylstylopine 14-hydroxylase, P6H: protopine 6-hydroxylase, DBOX: dihydrosanguinarine oxidase, DRS: dehydroreticuline synthase, DRR: dehydroreticuline reductase, SalSyn: salutaridine synthase, SalR: salutaridine reductase, SalAT: salutaridinol 7-O-acetyltransferase, T6ODM: thebaine 6-O-demethylase, CODM: codeine

O-demethylase, COR: codeinone reductase.

(S)-N-methylcoclaurine 3’-hydroxylase (NMCH) to form (S)-3’-hydroxy-

N-methylcoclaurine, which undergoes another O-methylation by (S)-3’-hydroxy-

N-methylcoclaurine 4’-O-methyltranferase (4’OMT) to form (S)-reticuline (Ziegler et al.,

2005; Morishige et al., 2000; Pauli and Kutchan, 1998). Many BIAs are derived from

(S)-reticuline, and is considered a major branch-point intermediate (Fig. 1) (Ziegler et al.,

2009). (S)-Reticuline is 7-O-methylated to laudanine, or oxidized by the berberine bridge enzyme (BBE) to form (S)-scoulerine (Ounaroon et al., 2003; Facchini et al., 1996).

Formation of (S)-scoulerine is the first committal step to several classes of BIAs, including protoberberines (e.g. berberine) phthalideisoquinolines (e.g. noscapine), and benzophenanthridines (e.g. sanguinarine) (Chen et al., 2015; Fossati et al., 2014;

Facchini et al., 1996). Alternatively, (S)-reticuline can be epimerized to (R)-reticuline, which is the first committal step to morphine biosynthesis (Hirata et al., 2004;

De-Eknamkul and Zenk, 1992; Battersby et al., 1965).

1.3 Epimerization of reticuline and morphine biosynthesis

The natural occurrence of morphine has only been confirmed in Papaver decaisnei, Papaver setigerum, and Papaver somniferum, which are all members of the family Papaveraceae (Theuns et al., 1986). Interestingly, the production of morphine from Papaver rhoeas callus culture has also been reported (Sarin, 2003). However, salutaridine and/or its derivatives have been confirmed in several members of

Papaveraceae (e.g. Papaver bracteatum), and some members of Euphorbiace (e.g. Croton balsamifera) and Apocynace (e.g. Rauvolfia serpentina) (Theuns et al., 1986).

In opium poppy, morphine biosynthesis begins with the epimerization of

(S)-reticuline to (R)-reticuline. Feeding studies with radiolabeled 1,2-dehydroreticuline

5 and (R)-reticuline showed both were incorporation into thebaine, codeine, and morphine

(Borkowski et al., 1978; Battersby et al., 1965). Therefore, the mechanism for epimerization was proposed to occur via an intermediate 1,2-dehydroreticulinium ion

(1,2-dehydroreticuline) (Hirata et al., 2004; De-Eknamkul and Zenk, 1992; Borkowski et al., 1978; Battersby et al., 1965). Both dehydroreticuline synthase (DRS) and dehydroreticuline reductase (DRR) have been partially purified and characterized, but the encoding genes have not been identified (Hirata et al., 2004; De-Eknamkul and Zenk,

1992). DRS was shown to accept reticuline in vitro to form dehydroreticuline in absence of a co-factor, and was predicted to be a FAD-dependent oxidoreductase (FADOX)

(Hirata et al., 2004). Several opium poppy FADOXs were identified, however, they do not accept (S)-reticuline in vitro, and silencing FADOXs had no apparent effect on reticuline or thebaine levels in planta (Hagel et al., 2012). Purified DRR was highly specific, and only accepted 1,2-dehydroreticuline as a substrate to form (R)-reticuline

(De-Eknamkul and Zenk, 1992). The purified DRR was approximately 30 kDa, appeared to be cytosolic, and was only present in crude enzyme extracts of differentiated

P. somniferum and P. bracteatum plants, which produce morphinan alkaloids

(De-Eknamkul and Zenk, 1992). Following the epimerization of reticuline, (R)-reticuline is converted to salutaridine by salutaridine synthase (SalSyn), a cytochrome P450

(CYP719B1) (Fig. 1) (Gesell et al., 2009). Salutaridine is reduced to salutaridinol by salutaridine reductase (SalR), and acetylated by salutaridinol 7-O-acetyltransferase

(SalAT) to salutaridinol 7-O-acetate, which spontaneously rearranges to thebaine (Ziegler et al., 2006; Grothe et al., 2001; Lenz and Zenk, 1994; Gerardy and Zenk, 1993).

Thebaine is then O-demethylated at position 6 by thebaine 6-O-demethylase (T6ODM) or

6 position 3 by codeine O-demethylase (CODM) to form neopinone or oripavine, respectively (Hagel and Facchini, 2010). Neopinone spontaneously rearranges to codeinone, which is then reduced to codeine by codeinone reductase (COR), and CODM demethylates codeine to form morphine (Hagel and Facchini, 2010). Alternatively, oripavine can be demethylated by T6ODM to produce morphinone, which is reduced by

COR to form morphine (Hagel and Facchini, 2010).

1.4 Sanguinarine biosynthesis

(S)-Scoulerine is the first committal step to several classes of BIAs, including the benzophenanthridine alkaloid sanguinarine (Fig. 1). (S)-Scoulerine is formed from

(S)-reticuline by BBE (Fossati et al., 2014; Facchini et al., 1996; Dittrich and Kutchan,

1991). In opium poppy, (S)-scoulerine undergoes two oxidization reactions by cheilanthifoline synthase (CFS) and stylopine synthase (SPS) to form cheilanthifoline and stylopine, respectively (Fossati et al., 2014; Hagel and Facchini, 2012). (S)-Stylopine is then N-methylated by tetrahydroprotoberberine cis-N-methyltransferase (TNMT) to form

(S)-cis-N-methylstylopine, which is converted by (S)-cis-N-methylstylopine

14-hydroxylase (MSH) to form protopine (Beaudoin and Facchini, 2013; Liscombe and

Facchini, 2007). Protopine 6-hydroxylase (P6H) hydroxylates protopine to

6-hydroxyprotopine, which spontaneous rearranges to dihydrosanguinarine (Beaudoin and Facchini, 2013). Dihydrosanguinarine is oxidized to sanguinarine by the FADOX dihydrobenzophenanthridine oxidase (DBOX) (Hagel et al., 2012). Additionally, dihydrosanguinarine can be oxidized and methylated one or two times to form dihydrochelirubine and dihydromacarpine, respectively. However, these benzophenanthridine alkaloids have not been detected in opium poppy.

Sanguinarine can be reduced to dihydrosanguinarine by a sanguinarine reductase

(SanR). It has been suggested that the role of SanR in planta is to detoxify the cytotoxic sanguinarine through its reduction to the seemingly non-toxic dihydrosanguinarine. Initial experiments conducted by Dr. Jill Hagel showed silencing SanRs resulted in an accumulation of reticuline in the latex (data not shown). Since 1,2-dehydroreticuline and sanguinarine are both quaternary ammonium compounds, it was proposed that SanR might also be responsible for catalyzing the reduction of 1,2-dehydroreticuline to form

(R)-reticuline in opium poppy. However, SanR(s) from Papaver somniferum have not been characterized.

To date, only SanR from Eschscholzia californica has been characterized (Vogel et al., 2010; Weiss et al., 2006). Sanguinarine reductase was first purified from

E. californica cell cultures treated with a yeast elicitor (Vogel et al., 2010; Weiss et al.,

2006). Upon treatment with the microbial elicitor, total alkaloid content in culture increases approximately four-fold within 24 hours, with at least a quarter being benzophenanthridine alkaloids. However, the majority of benzophenanthridine alkaloids are excreted into the medium, while the corresponding dihydrobenzophenanthridine alkaloids are retained within the cells. Therefore, only dihydrosanguinarine, not sanguinarine, is detected in elicited E. californica cells. Furthermore, addition of sanguinarine to cell suspensions results in the disappearance of sanguinarine from the medium, and an increase in dihydrosanguinarine within the cell. Therefore, elicited

E. californica cell cultures were used to purify, sanguinarine reductase (SanR), which is a

29.5 kDa short-chain dehydrogenase/reductase (SDR) that reduces sanguinarine to dihydrosanguinarine. The purified SanR was sequenced using Edman degradation, and its

8 encoding cDNA was identified in an E. californica cDNA library (Vogel et al., 2010).

Recombinant SanR from E. californica reduces the quaternary amine in sanguinarine to form dihydrosanguinarine using NADPH or NADH as a hydrogen donor (Vogel et al.,

2010; Weiss et al., 2006). However, the catalytic properties of SanR are dependent on the hydrogen donor and the concentration of substrate. The reaction velocity is about threefold higher with NADPH than with NADH when the substrate concentration is below 10 μM, and higher substrate concentrations show reduced reaction velocities with

NADPH, but not NADH. E. californica SanR is also able to reduce chelerythrine to dihydrochelerythrine. Interestingly, the maximum conversion rates of sanguinarine are observed using NADPH as the reducing agent, whereas the maximum conversion rates of chelerythrine are observed with NADH.

1.5 Sanguinarine is a cytotoxic compound

Sanguinarine is a highly cytotoxic compound that can bind DNA, inhibit DNA synthesis, and induce apoptosis (Basu and Suresh Kumar, 2015; Schmeller et al., 1997).

However, these same properties also make sanguinarine, and its derivatives, potential anticancer compounds (Cao et al., 2015). Similarly, sanguinarine was once used in

Viadent oral health products as a treatment for oral plaque and gingivitis (Vlachojannis et al., 2012). However, correlational studies linked the use of sanguinarine-based oral health products to leukoplakia (Damm et al., 1999). Consequently, sanguinarine was removed from Viadent products in the early 2000s.

The role of sanguinarine in planta is likely as a defense strategy against pathogens and/or herbivory. Eschscholtzia californica, Papaver somniferum, and Papaver bracteatum cell cultures will accumulate sanguinarine in response to treatment with a

9 microbial elicitor (Weiss et al., 2006; Cline and Coscia, 1988; Schumacher et al., 1987;

Eilert and Constabel, 1985). However, due to the cytotoxic nature of sanguinarine it is detoxified in plants through the action of SanR (Weiss et al., 2006). Addition of sanguinarine to Eschscholzia californica cell cultures had no effect on growth, and sanguinarine was converted to dihydrosanguinarine. However, addition of sanguinarine to Nicotiana tobacum or Arabidopsis thaliana cell cultures resulted in growth inhibition with no significant conversion of sanguinarine. Therefore, sanguinarine is cytotoxic to plants that do not synthesize benzophenanthridine alkaloids, and SanR has likely evolved to prevent self-intoxication of benzophenanthridine-producing species.

Studies have shown insects and mammals are also able to metabolize sanguinarine to dihydrosanguinarine (Schütz et al., 2014; Wu et al., 2013; Dvorák and

Simánek, 2007). Frankliniella occidentalis (thrips) will metabolize consumed sanguinarine to dihydrosanguinarine (Schütz et al., 2014). However, thrips will avoid feeding on leaf discs from plants that accumulate benzophenanthridine alkaloids, such as

Eschscholzia californica and Chelidonium majus, in favour for the non- benzophenanthridine accumulating Phaseolus vulgaris (common bean). Thrips will also avoid feeding from sugar solutions containing sanguinarine. However, the mechanism of sanguinarine detoxification in both insects and mammals remains unclear. In mammals, sanguinarine detoxification may be mediated by the aryl hydrocarbon receptor

(AhR)/CYP1A pathway (Nguyen et al., 2009; Dvorák and Simánek, 2007). Phase I liver detoxification is mediated by cytochrome P450 oxidases (CYPs). CYP-expressing cell lines were tested for sanguinarine reductase activity in vitro, and of 10 tested only

CYP1A1 and CYP1A2 were able to metabolize sanguinarine to dihydrosanguinarine

(Deroussent et al., 2010). In addition to dihydrosanguinarine, CYP1A formed several other sanguinarine metabolites, which likely mediate phase II detoxification reactions

(Deroussent et al., 2010). Phase II detoxification results in activation of Nrf2 and downstream antioxidant response elements, such as heme oxygenase-1 (HO-1) and

NAD(P)H quinone oxidoreductase 1 (NQO1). Studies have shown that sanguinarine induces the expression of proteins HO-1 and NQO1 (Park et al., 2014; Wu et al., 2013).

Furthermore, treatment of rat liver preparation with dicoumarol, an inhibitor of NQO1, resulted in significantly less dihydrosanguinarine production from sanguinarine as compared to the control (Wu et al., 2013). Together these data support the detoxification of sanguinarine in mammalian livers via the AhR/CYP1A pathway.

1.6 Localization of alkaloids and biosynthetic enzymes in planta

In addition to detoxification by SanR, sanguinarine is also compartmentalized in planta. In elicited opium poppy cell cultures, sanguinarine accumulates in the vacuole

(Alcantara et al., 2005). Conversely, sanguinarine accumulates along the cell wall in elicited E. californica cell cultures (Weiss et al., 2006). Fluorescence microscopy indicated the addition of exogenous sanguinarine to E. californica cell cultures results in its localization to the cell wall followed by its reduction to dihydrosanguinarine, which accumulates in cytosol then enters the vacuole (Weiss et al., 2006). Although cell culture may not accurately reflect alkaloid subcellular localization in intact plants, vacuole localization of alkaloids has been observed for terpenoid indole alkaloids in

Catharanthus roseus, berberine in Coptis japonica, and nicotine in Nicotiana tabacum

(Carqueijeiro et al., 2013; Morita et al., 2009; Otani et al., 2005).

Enzymes involved in sanguinarine biosynthesis have been localized in opium poppy cell culture (Hagel and Facchini, 2012; Alcantara et al., 2005). Both NCS and

BBE have been shown to localize to the endoplasmic reticulum (ER), and DBOX has a putative ER-targeting signal peptide, which implicates sanguinarine biosynthesis is associated with the ER (Hagel and Facchini, 2012; Hagel et al., 2012; Emanuelsson et al., 2007; Alcantara et al., 2005). Therefore, oxidation of dihydrosanguinarine in the ER could facilitate the vesicle-mediated transport of sanguinarine to the vacuole.

Conversely, morphinan alkaloids are only detected in differentiated opium poppy plants, not in cell culture, and many BIAs, especially morphinan alkaloids, accumulate in laticifers (Onoyovwe et al., 2013; Desgagné-Penix et al., 2012; Alcantara et al., 2005).

Laticifers are specialized cells that contain a unique cytoplasm, referred to as latex, and store specialized metabolites. Laticifers are classified by their origin, development, and anatomy (Hagel et al., 2008). In opium poppy, morphine is stored within large, irregularly shaped vesicles derived from the ER that are housed in a large central vacuole within the laticifer (Nessler and Mahlberg, 1977; Fairbairn et al., 1974; Thureson-Klein,

1970). Furthermore, extensive research has been conducted to localize the transcripts and proteins involved in morphinan biosynthesis. Transcripts encoding biosynthetic enzymes are localized to companion cells, biosynthetic enzymes are localized to sieve elements and laticifers, and alkaloids are stored within laticifers (Onoyovwe et al., 2013; Hagel and Facchini, 2010; Lee and Facchini, 2010; Samanani et al., 2006; Weid et al., 2004;

Bird et al., 2003; Facchini and De Luca, 1995). However, sanguinarine is not found in the latex, and dihydrosanguinarine and sanguinarine are only detected in the roots of opium poppy (Desgagné-Penix et al., 2012; Facchini et al., 1996). Therefore, neither

12 sanguinarine nor SanR have been localized in intact plants (Desgagné-Penix et al., 2012;

Facchini et al., 1996). However, shotgun proteomics revealed that at least one SanR is present in opium poppy latex (personal communication; Onoyovwe et al., 2013).

1.7 Objectives

All the cDNAs encoding enzymes in the morphine pathway from norcoclaurine to morphine have been cloned, except for those encoding DRS and DRR. The original goal of my thesis was to clone and characterize DRR. It was hypothesized that sanguinarine reductase (SanR) may catalyze the reduction of 1,2-dehydroreticuline to (R)-reticuline since both sanguinarine and dehydroreticuline are quaternary ammonium compounds.

However, in this thesis I have shown that SanRs do not accept dehydroreticuline as a substrate in vitro (Fig. 8). Additional attempts to clone DRR are outlined in Appendix A1.

Although SanR do not exhibit DRR activity it is important to understand the role of sanguinarine reductases in planta. Therefore, the objective of this work was to characterize sanguinarine reductases from opium poppy to discern differences between their activities both in vitro and in vivo. Specifically, (1) opium poppy SanRs were identified in transcriptome libraries based on homology to the previously characterized

Eschscholzia californica SanR; (2) three opium poppy SanRs were cloned and expressed as recombinant proteins for biochemical assays and enzyme kinetics, as well as for antibody production in mice; (3) SanR proteins were localized in various opium poppy tissues using Western blot analysis immunolocalization; and (4) SanR gene expression was analyzed in various tissues, but effects of silencing SanR(s) was only analyzed in root tissue, the site of sanguinarine accumulation. Together these data were used to gain insight into the role of SanR as an enzyme of detoxification in planta.

2 MATERIALS AND METHODS

2.1 Media

2.1.1 Lysogeny broth (LB) media

Media was prepared in either liquid or solid form. Per 1 L: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 200 μl 5 N NaOH (Sambrook and Russell, 2001). For solid media, 15 g of agar was added. Media was supplemented with antibiotics, as required.

2.1.2 Antibiotics

Stock solutions of ampicillin (100 mg/mL), kanamycin (50 mg/mL), and gentamicin (20 mg/mL) were prepared by dissolving each antibiotic in distilled water.

Stock solutions of rifampicin (50 mg/mL) were prepared by dissolving the antibiotic in dimethyl sulfoxide (DMSO). Aliquots were stored at -20°C. Working concentrations for ampicillin, kanamycin, gentamicin and rifampicin are 100 µg/mL, 20 µg/mL, 50 µg/mL, and 50 µg/mL, respectively.

2.1.3 Blue/white selection

Blue/white selection was performed on solid LB agar media containing 5-bromo-

4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropyl-beta-D- thiogalactopyranoside (IPTG). Stock solutions of X-gal (20 mg/mL) were prepared by dissolving the compound in DMSO. Stock solutions of IPTG (100 mM) were prepared by dissolving the compound in water. Aliquots were stored at -20°C. X-gal was stored in the dark. X-gal and IPTG (100 μL each) were spread on LB media then allowed to dry before plating bacteria (Sambrook and Russell, 2001).

2.1.4 Agrobacterium tumefaciens induction medium

Per 100 mL of LB media: 100 µL 50 mg/mL kanamycin, 1 mL 1 M

2-(N-morpholino)ethanesulfonic acid (MES), 20 µL 100 mM acetosyringone (Hileman et al., 2005).

2.1.5 Agrobacterium tumefaciens infiltration solution

Per 500 mL of infiltration solution: 5 mL 1 M MES, 5 mL 1 M MgCl2, 1 mL

100 mM acetosyringone (Hileman et al., 2005).

2.2 Buffers

2.2.1 Plasmid DNA isolation buffers

Plasmid DNA was extracted through alkaline lysis using resuspension, lysis and neutralization buffers (modified from Birnboim and Doly, 1979). Per 100 mL of resuspension buffer: 5 mL 1 M Tris-HCl (pH 8.0), 2 mL 0.5 M EDTA (pH 8.0), 1 mL 10 mg/mL RNase A. Per 100 mL of lysis buffer: 2 mL 10 M NaOH, 10 mL 10% (w/v) sodium dodecyl sulphate (SDS). Per 100 mL of neutralization buffer: Dissolve 40.8 g sodium acetate trihydrate in ~70 ml of distilled water. Adjust pH to 5.2 with glacial acetic acid. Bring up volume to 100 mL with water.

2.2.2 2X CTAB RNA extraction buffer

Per 100 mL: 2 g cetrimonium bromide (CTAB), 10 mL 1 M Tris (pH 8.0), 4 mL

0.5 M EDTA (pH 8.0), 8.18 g NaCl, 1 g polyvinylpyrrolidone (PVP). Adjust volume to

100 mL with diethylpyrocarbonate (DEPC)-treated distilled water, and autoclave. Add

50 µL 100X SPD (0.05g/mL spermidine trihydrochloride), and 10 µL -mercaptoethanol before use (Meisel et al., 2005).

2.2.3 2X SDS-PAGE sample buffer

Per 10 mL: 3.55 mL water, 1.25 mL 0.5 M Tris-HCl (pH 6.8), 2.5 mL glycerol,

2.0 mL 10% (w/v) SDS, 0.2 mL 0.5% (w/v) bromophenol blue, 50 µL -mercaptoethanol

(BioRad Mini-PROTEAN® Tetra Cell Instruction Manual #10007296).

2.2.4 10X SDS-PAGE electrode buffer

Per 1 L: 30.3 g Tris base, 144 g glycine, 10 g SDS. Adjust to pH 8.3 with HCl

(BioRad Mini-PROTEAN® Tetra Cell Instruction Manual #10007296).

2.2.5 10X Western blot transfer buffer

Per 1 L: 30.4 g Tris-HCl, 144 g glycine.

2.2.6 1X Transfer buffer for Western blotting

Per 1 L: 100 mL 10X Western blot transfer buffer, 200 mL methanol. Bring up to

1 L with distilled water (Sambrook and Russell, 2001; Tovey and Baldo, 1987)

2.2.7 10X Tris-buffered saline (TBS) buffer

Per 1 L: 24.2 g Tris, 80.06 g NaCl (Sambrook and Russell, 2001).

2.2.8 1X TBS-Tween

Per 1 L: 100 mL 10X TBS, 1 mL Tween-20 (Sambrook and Russell, 2001).

2.2.9 Plant protein extraction buffer

Per 1 L: 50 mL 1 M Tris-HCl (pH 7.5), 4 mL 0.5 M EDTA (pH 8.0), 10 g polyvinylpolypyrrolidone (PVPP).

2.2.10 Sodium phosphate buffer, pH 7.6 (100 mM)

Per 1 L: 84.5 mL 1 M Na2HPO4 (141.96 g/L), 15.5 mL 1 M NaH2PO4

(119.98 g/L). Bring volume to 1 L with distilled water (Sambrook and Russell, 2001).

2.2.11 Coomassie stain

Per 1 L: 2 g Coomassie Brilliant Blue R250, 500 mL methanol, 100 mL glacial acetic acid. Bring volume to 1 L with distilled water (Sambrook and Russell, 2001).

2.2.12 Solvent A mass spectrometry running buffer

Per 1 L: Add 0.7708 g ammonium acetate ~800 mL LC-MS grade water. Adjust pH to 5.5 with glacial acetic acid. Add 50 mL acetonitrile then bring volume to 1 L with

LC-MS grade water (Farrow et al., 2012).

2.3 Gel electrophoresis

Agarose (1-2%) gels made with Tris-acetate-EDTA (TAE) buffer were used to size separate DNA or RNA (Sambrook and Russell, 2001). SDS-polyacrylamide gel electrophoresis system (PAGE) was employed to size separate proteins (Laemmli, 1970).

2.3.1 50X TAE buffer

Per 1 L: 242 g Tris base, 57.1 mL glacial acetic acid, 100 mL 0.5 M EDTA

(pH 8.0). The working solution of TAE is 1X (Sambrook and Russell, 2001).

2.3.2 30% Acrylamide solution

Per 100 mL: 29.2 g acrylamide and 0.8 g N’N’-bis-methylene-acrylamide. Store at 4°C in the dark (BioRad Mini-PROTEAN® Tetra Cell Instruction Manual

#10007296).

2.3.3 Separating gel (12%)

Per 10 mL: 3.4 mL water, 4.0 mL 30% acrylamide solution, 2.5 mL 1.5 M

Tris-HCl (pH 8.8), 0.1 mL 10% (w/v) SDS, 0.1 mL 10% (w/v) ammonium persulfate

(APS), 5 µL tetramethylethylenediamine (TEMED) (BioRad Mini-PROTEAN® Tetra

Cell Instruction Manual #10007296).

2.3.4 Resolving gel (4%)

Per 10 mL: 6.1 mL water, 1.3 mL 30% acrylamide solution, 2.5 mL 0.5 M

Tris-HCl (pH 6.8), 0.1 mL 10% (w/v) SDS, 0.1 mL 10% (w/v) APS, 10 µL TEMED

(BioRad Mini-PROTEAN® Tetra Cell Instruction Manual #10007296).

2.4 Organisms

2.4.1 Bacteria

Escherichia coli (E. coli) strain XL1-Blue (Agilent, Cat. No. 200249) was used for the maintenance, and propagation of plasmid DNA. E. coli strain SG13009 (Qiagen,

Cat. No. 34210) was used for recombinant protein expression. E. coli strain SG13009 harbours the plasmid pREP4, which confers kanamycin resistance. Agrobacterium tumefaciens (A. tumefaciens) strain GV3101 was used to transform Papaver somniferum.

A. tumefaciens strain GV3101 is resistant to gentamycin and rifampicin, and carries a disarmed Ti plasmid to facilitate T-DNA transfer. E. coli and A. tumefaciens strains were maintained at 37°C and 28°C, respectively, in liquid or on solid LB media supplemented with the appropriate antibiotics. Bacterial cultures grown in liquid media were shaken at

200 RPM.

2.4.2 Plants

Papaver somniferum cv. Bea’s Choice (The Basement Shaman, Woodstock, IL, http://www.basementshaman.com) was used for VIGS, and expression analysis (qPCR) experiments; P. somniferum cultivars 40, Veronica, and Marianne were used for localization experiments; tissues from Bea’s Choice, as well as cultivars Veronica,

Roxanne, Marianne, 40, T, L, and Przemko provided by Dr. Peter Facchini, were harvested for purification of total soluble proteins (Desgagné-Penix et al., 2012). Papaver

18 somniferum seeds were germinated in growth chambers (Conviron, Winnipeg, MB) under a 16-hour photoperiod using a combination of fluorescent and incandescent lights. Day and night temperatures were set to 20°C and 18°C, respectively.

Elicited Eschscholzia californica (E. californica) cell cultures were used to purify benzophenanthridine alkaloids. E. californica cell cultures (Deutsche Sammlung von

Mikroorganismen und Zellkulturen, Cat. No. PC-1096) were grown by Guillaume

Beaudoin in liquid Gamborg’s B5 media (Phytotechnology Laboratories, Cat. No. G398) supplemented with 20 g/L sucrose, 1 g/L casein hydrolysate, and 1 mg/L

2,4-dichlorophenoxyacetic acid. Cultures were grown at room temperature on a gyratory shaker (125 RPM), and elicited with yeast extract. Cell filtrate was collected 96-hours post elicitation.

2.5 Plasmids

2.5.1 Subcloning plasmid

The TA-cloning vector pGEM-T (Promega, Cat. No. A3600) was used for the non-directional subcloning of polymerase chain reaction (PCR) products. The pGEM-T vector is a high-copy number vector that confers ampicillin resistance to E. coli.

Advantages to pGEM-T subcloning include easy PCR cloning via T-overhangs, and blue/white selection when transformed into E. coli containing a mutant lacZ gene (β- galactosidase), such as XL1-Blue. The multiple cloning site (MCS) is within the α- peptide coding region of β-galactosidase. Successful ligation of amplicons into pGEM-T disrupts β-galactosidase, and can be identified as white colonies on solid LB media containing X-gal and IPTG. Amplicons in pGEM-T were analyzed prior to downstream applications (e.g. recombinant protein expression, and VIGS).

2.5.2 Recombinant protein expression plasmid

E. coli expression vector pQE30 (Qiagen, Cat. No. 32915) was used express recombinant SanR proteins in E. coli strains harbouring the pREP4 plasmid, such as

SG13009. The pREP4 plasmid encodes the lacI gene (repressor) to regulate expression from pQE vectors (Farabaugh, 1978). The repressor binds the two lacO (lac operator) sequences immediately following the T5 promoter (Gilbert and Müller-Hill, 1967).

Consequently, expression from pQE30 is IPTG-inducible. Genes expressed from pQE30 result in the production of N-terminally polyhistidine (6xHis)-tagged recombinant proteins that can be purified using TALON cobalt resin (Clontech, Cat. No. 635501).

2.5.3 Virus-induced gene silencing (VIGS) plasmids

The tobacco rattle virus (TRV)-based VIGS vector system was previously developed to mediate gene silencing in opium poppy plants (Hileman et al., 2005;

Dinesh-Kumar et al., 2003; Liu et al., 2002). TRV is a bipartite RNA virus composed to two single-stranded RNAs (MacFarlane, 1999). RNA1 encodes two replicase proteins and a movement protein (MP) for the multiplication and movement of the virus. RNA2 encodes the coat protein (CP) for the generation of virus particles. RNA1 and RNA2

TRV sequences were modified and introduced into Agrobacterium T-DNA vectors generating pTRV1 and pTRV2, respectively (Fig. 2) (Liu et al., 2002). The nonessential structural genes encoded by RNA2 were removed from pTRV2 and replaced with a MCS for insertion of the VIGS target gene. Phytoene desaturase (PDS) was previously cloned into pTRV2 (pTRV2-PDS) as a visual control for the efficiency of gene silencing

(Hileman et al., 2005). Both pTRV1 and pTRV2 were individually transformed into

A. tumefaciens strain GV3101 for co-infiltration of poppy seedlings.

pTRV1

LB 2x35S 134K 194K MP 16K Rz NOSt RB

pTRV2

LB 2x35S CP MCS Rz NOSt RB

SanR VIGS Construct

Figure 2. TRV-based virus-induced gene silencing vectors. The T-DNA vectors position

TRV cDNA clones between the left and right borders (LB and RB, respectively), and under the control of the duplicated cauliflower mosaic virus 35S promoter (2x35S) and nopaline synthase terminator (NOSt). The pTRV1 and pTRV2 encodes two replicase proteins (134K and 194K), movement protein (MP), 16-kDa cysteine-rich protein (16K), and coat protein (CP) necessary to propagate viral particles in planta. MCS: multiple cloning site, Rz: self-cleaving ribozyme. Modified from Hileman et al. (2005).

2.6 Cloning and Transformations

2.6.1 Sequence identification and primer design

Primers were designed to amplify sequences for recombinant protein expression, gene expression analysis, VIGS, genetic screening, and sequencing. All primers, except those used for quantitative real-time PCR (qPCR), were tested for self-complementarity, primer pair complementarity, balanced GC content and similar melting temperature for primer pairs using DNAMAN (Lynnon BioSoft, Version 8). Primers used for qPCR were designed in Primer Express (Life Technologies, Version 3).

Papaver somniferum cv. Bea’s Choice transcriptome databases were searched for sequences with a high degree of amino acid sequence similarity to previously characterized E. californica SanR (GenBank Accession No. GU338458) (Xiao et al.,

2013; Vogel et al., 2010; Weiss et al., 2006). Four opium poppy SanRs were identified with greater than 60% amino acid sequence similarity to E. californica SanR (Fig. 3).

Primers were designed to amplify full-length SanRs, as well as a N-terminally truncated form of SanR3 (SanR3B), from opium poppy cDNA. Primers designed to amplify SanR1-

SanR3 for insertion into the pQE30 expression vector introduced 5’-BamHI and 3’-KpnI restriction enzyme (RE) cut sites to allow for directional cloning (Table 1). These RE cut sites were used, if necessary, to also facilitate the directional cloning of SanRs into the pRSET A expression vector (Invitrogen, Cat. No. V351-20). However, primers used to amplify SanR4 introduced 5’-SphI and 3’-SalI. Primers complementary to regions flanking the pQE30 MCS (pQE30-F and pQE30-R) were designed to facilitate sequencing and/or colony PCR (Table 1). T7 and SP6 primer sequences were used to sequence pGEM-T inserts and/or for use in colony PCR (Table 1).

22 ECASANR M------A DSS K K ------LT VLL S G ASGLT GSL AFK KLKERSD KFE V 36 PSOSANR1 M------AES N QK------IT VLV T G ASGLT GEIAFK KLKERSD KFV V 36 PSOSANR2 M------A ALM QK------IT VLV T G ASGLT GEIAFK KLKERSD KFA A 36 PSOSANR3 MGLV T R V PLF S S PSS TFS P H K YSS T T KLF S S S S S S S L S FQRR TSV V V K A MAST V IV T G A G G R T G Q IV Y K KLKER AE-FV A 79 PSOSANR4 MRSVSQ ICLS L R N KSK MAC K R CSN K V A MACSS P K ------K T VLV T G ASGLT G Q F AFK KLKERSD KLV V 63

ECASANR RGL VRSEA S K Q K L GGGDE I F IGD I S DPKTLEPAM E G IDAL IIL T S A IPRM KPTEEFTAEM I S GGRSEDV IDASF--S GPM 114 PSOSANR1 RGL VRSEA S K Q R L GGGDE I FLGDVM DKKSLETAMQG IDAL IIL T S AVPKVVPGS YPGA---DGKRA E DVF G ESFD F NGPM 113 PSOSANR2 RGL VRSEA S K Q K L GGGDE IY L GD IM DKKSLKHAMQG IDGL V I L T S AVPK IVPGS YPGA---DGKRA E DVF DDSFDYS GPM 113 PSOSANR3 RGL VRTEESK E K IGGADDVF VAD IRDAESIVPA IQ GVDAL V I L T S AVPKM KPGF DPTK----GGR------140 PSOSANR4 RGL VRSEG S KKKL GGGNE IYVGDVM KPESLEPAM KGVDAL IIL TTA IPKM KPGS YPAN I--S GARAE D L IDGSF--Q GT I 139

ECASANR PEF Y Y DEG QYPEQVD W IG QKN Q ID T A K K MGV K H IVLV GSMGG C D P D HFL N H MGN G N I LIWKR K AEQYLA DSG V P Y T I IR A 194 PSOSANR1 PEF Y YEE G Q F PEQ ID W IG QKN Q ID T A KSC G V K H IVLV GSMGG T D P N NFL N H MAN G N ILVW KRKAEQ Y L ADS G IPYT IIRA 193 PSOSANR2 PEF F Y AEG QYPEQ ID W IG QKN Q I E T A K A C G V K H IVLV GSMGG T D P N HFL N H MGN G N I LIWKR K AEQYLA DSG IP Y T I IR A 193 PSOSANR3 PEF F F E D G A N PEQVD W IG QKN Q ID A A K A A G V K Q IVLV GSMGG T NLN H PLNSIG N G N ILVW KRKAEQ Y L ADS G IPYT IIRA 220 PSOSANR4 PEF YFE G G QYPEQVD W IG QKN Q ID A A K A A G V K H I I L VST MGSG D P N H PLNSL G N G N ILAW KRKAEEY L AKS GVPYT I L RA 219

ECASANR GGL DNKAGGVRELLVAKDDVLLPTE NGF IARADVAE ACVQ A LEI EEVKNKAF D L G S KPE GVGE ATKDF KALFSQ VTTPF 273 PSOSANR1 GGL DNKVGG-RELLVGKDDELLST E NHF IARADVAE ACVQ A LQ I EESK F KAF D L G SM P E GVGE PTKDF KALFSQ VTTPF 271 PSOSANR2 AAL DNKVGG-RELLVGKDDELLPTE NGY IARADVAE ACVQ A LQ I E DCKF KAYDL G S KPE GVGE PTKDF KALFA L VTTRF 271 PSOSANR3 GGLQ DKDGGVRELVVGKDDELLETD IRT IARADVAE VC IQ A LLLEEAKF KAL D L A S KPE GTGE PTKDF KTLFSQ I S TRF 299 PSOSANR4 GGL DNKQ GGKRQ LLIGKNDELLPTE KGYVARE DVAE ACVQ AVQ LEEVKF KAF D L G SM P E GTGVPTKD F KALFAP ITTCF 298

Figure 3. Sequence alignment of sanguinarine reductases. Eschscholzia californica SanR (ECASANR), and Papaver somniferum

SanR (PSOSANR1-PSOSANR4) amino acid sequences were aligned using the M-coffee server (www.tcoffee.org) then colour-coded

in Jalview to visualize percent similarity (Waterhouse et al., 2009; Notredame et al., 2000). Unshaded amino acids share less than

40% sequence similarity. Amino acids shaded with light, medium, and dark gray share 40-59%, 60-79%, and 80-100% sequence

similarity, respectively. Box outlines N-terminal extension of SanR3, compared to SanR1 and SanR2, which is absent in the SanR3B

construct.

Table 1. List of primers used for cloning procedures.

Amplicon Protein size Namea Sequenceb RE sequence Direction size (bp) (kDa)

SanR1-F 5’-GGATCCATGGCAGAATCAAATCAAAAAATC-3’ BamHI Forward 816 29.4 SanR1-R 5’-GGTACCTCAGAAACGAGTGGTGACTAGAGC-3’ KpnI Reverse

SanR2-F 5’-GGATCCATGGCAGCATTAATGCAAAAG-3’ BamHI Forward 816 29.4 SanR2-R 5’-GGTACCTCAGAAAGGAGTAGTGACTTGCG-3’ KpnI Reverse

SanR3-F 5’-GGATCCATGGGTTTAGTGACACGTGTTCC-3’ BamHI Forward

SanR3B-F 5’-GGATCCATGGCGAGTACTGTGATTGTTACTG-3’ BamHI Forward 900 or 753 32.1 or 26.8

SanR3-R 5’-GGTACCTCAGAATCGTGTAGAGATTTGAGAAAAG-3’ KpnI Reverse

SanR4-F 5’-GCATGCATGAGGTCTGTCTCTCAAATTTG-3' SphI Forward 897 32.0 SanR4-R 5’-GTCGACTTAGAAACAAGTAGTGATTGGGG-3’ SalI Reverse

SanR4-F2 5'-GCATGCATGGCATGTTCAAGTCC-3' SphI Forward 897 32.0 SanR4-R2 5'-GTCGACTTAGAAACAAGTAGTGATTGG-3' SalI Reverse

Table 1 (continued). List of primers used for cloning procedures.

Amplicon Protein size Namea Sequenceb RE sequence Direction size (bp) (kDa)

T7 5'-AATACGACTCACTATAGG-3' N/A N/A 160 N/A Sp6 5'-ATTTAGGTGACACTATAG-3' N/A N/A

pQE30-F 5'-GATTCAATTGTGAGCGGATAA-3' N/A Forward 198 N/A pQE30-R 5'-CCAGATGGAGTTCTGAGG-3' N/A Reverse

aSanR: amplifies SanR coding sequences for expression from pQE30; T7 and Sp6: sequence constructs in pGEM-T vector, or for use in colony PCR (empty

vector amplicon is 160 bp); pQE30: sequence constructs in pQE30 vector, or for use in colony PCR (empty vector amplicon is 198 bp); F: forward primer

complementary to 5’-end of sequence; R: reverse primer complementary to 3’-end of sequence; bItalics: restriction enzyme (RE) recognition sequence;

unformatted: template sequence.

Small regions (~150 to 400 bp) of the SanR coding or untranslated region (UTR) sequences were amplified for insertion into pTRV2 (Table 2; Fig. 4). Primers designed to amplify these small regions introduced 5’-EcoRI and 3’-XhoI RE cut sites. In order to silence multiple SanRs, primers were designed to introduce 5’-EcoRI and 3’-KpnI cut sites in one construct, and 5’-KpnI and 3’-XhoI RE cut sites in another so that they could be ligated together. Primers were also designed to screen opium poppy plants the presence of TRV1 (MP, GenBank Accession No. AF166084), and TRV2 (CP, GenBank

Accession No. AF034621) (Table 2). Primers flanking the TRV2 MCS (PYL156F,

PYL156R) were also used to screen poppy plants post-infiltration, as well as to sequence constructs in the TRV2 vector (Table 2) (Hileman et al., 2005). Primers were also designed to amplify glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Table 2).

Primers were designed for conventional and MGB TaqMan qPCR methods

(Table 2). Primers were also designed to analyze the expression of select cytochrome

P450s and reductases, and endogenous reference genes from Papaver somniferum and

Papaver rhoeas (Appendix A1, Table A1.1).

Several Papaver somniferum DRR candidates were also amplified (Appendix A1,

Table A1.1 and Table A1.2). Primers introduced 5’-BamHI and 3’-KpnI RE cut sites to allow for directional cloning into the pQE30 expression vector. Opium poppy transcriptome databases were also searched for sequences with a high degree of amino acid sequence similarity to previously characterized reductases involved in BIA biosynthesis (Appendix A1, Table A1.1).

26 Table 2. List of VIGS and qPCR primers.

Namea Sequenceb RE Direction Amplicon size (bp)

5UTR-SanR1-F 5'-GAATTCCTAGGCTATATTTTTTCTTATAATATTC-3' EcoRI N/A 210 5UTR-SanR1-R 5'-CTCGAGTTATTTTGTAAGTCTGTAAAAAC-3' XhoI N/A

3UTR-SanR1-F 5'-GAATTCATGTTGAGATCCAAGAACAACTTATCATC-3' EcoRI N/A 215 3UTR-SanR1-R 5'-GAATTCATGTTGAGATCCAAGAACAACTTATCATC-3' XhoI N/A

5UTR-SanR2-F 5'-GAATTCCCTAGAAGAAAGTTTGAATTTTCG-3' EcoRI N/A 167 5UTR-SanR2-R 5'-CTCGAGCTGTGGAAACAAGATGTAATTG-3' XhoI N/A

CDS-SanR2-F 5'-GAATTCTTGGATTGGACAAAAGAAC-3' EcoRI N/A 433 CDS-SanR2-R 5'-CTCGAGTCAGAAAGGAGTAGTGACTTG-3' XhoI N/A

3UTR-SanR2-F 5'-GAATTCAATCTGAGATCCAAGAGCAATTTAG-3' EcoRI N/A 249 3UTR-SanR2-R 5'-CTCGAGCAAAGACCTGACCTCCAAGG-3' XhoI N/A

3UTR-SanR3-F 5'-GAATTCGATTCCATATGCGGTATGTTCTGATTG-3' EcoRI N/A 352 3UTR-SanR3-R 5'-CTCGAGAGTGTTCACAAGCACGATGAAC-3' XhoI N/A

Table 2 (continued). List of VIGS and qPCR primers.

Namea Sequenceb RE Direction Amplicon size (bp)

CDS-SanR3-F 5'-GAATTCATGGGTTTAGTGACACGTGTTC-3' EcoRI N/A 133 CDS-SanR3-R 5'-CTCGAGCTGAAGTTCTCCTTTGAAATG-3' XhoI N/A

UTR-SanR1-F 5'-GAATTCATGTTGAGATCCAAGAACAACTTATCATC-3' EcoRI N/A 215 UTR-SanR1-R 5'-GGTACCACTTTAATGCAACTGCAACTATAGG-3' KpnI N/A

UTR-SanR3-F 5'-GGTACCGATTCCATATGCGGTATGTTCTGATTG-3' KpnI N/A 352 UTR-SanR3-R 5'-CTCGAGAGTGTTCACAAGCACGATGAAC-3' XhoI N/A

PYL156F 5'-GGTCAAGGTACGTAGTAGAG-3' N/A Forward 390 PYL156R 5'-CGAGAATGTCAATCTCGTAGG-3' N/A Reverse

OYL195 5'-CTTGAAGAAGAAGACTTTCGAAGTCTC-3' N/A Forward 936 OYL198 5'-GTAAAATCATTGATAACAACACAGACAAAC-3' N/A Reverse

TRV2-CP-F 5'-CTGACTTGATGGACGATTC-3' N/A Forward 305 TRV2-CP-R 5'-TGTGTTTGGATTCGCAG-3' N/A Reverse

Table 2 (continued). List of VIGS and qPCR primers.

Namea Sequenceb RE Direction Amplicon size (bp)

TRV1-MP-F 5'-ATGGAAGACAAGTCATTGGTC-3' N/A Forward 759 TRV1-MP-R 5'-TTAAGACGAGTTTTTCTTATTAGACG-3' N/A Reverse

GADPH-F 5'-CTCATTTGAAGGGTGGAGC-3' N/A Forward 216 GADPH-R 5'-GTCATTGCGTGGACAGTGG-3' N/A Reverse

Taqman-SanR1-F 5'-AGATAACAAGGTAGGTGGCAG-3' N/A Forward 106 Taqman-SanR1-R 5'-AACGCAAGCTTCAGCAAC-3' N/A Reverse

Taqman-SanR1-P 5'-GAGAGAAGCTCATCATCCTTCCCGACCA-3' N/A N/A N/A

Taqman-SanR2-F 5'-AAGAGAAAAGCTGAGCAGTATC-3' N/A Forward 109 Taqman-SanR2-R 5'-CCTTTCCAACCAACAACTCC-3' N/A Reverse

Taqman-SanR2-P 5'-CCCACCTTGTTATCTAGAGCAGCAGCTCTTAT-3' N/A N/A N/A

Table 2 (continued). List of VIGS and qPCR primers.

Namea Sequenceb RE Direction Amplicon size (bp)

Taqman-SanR3-F 5'-TGAACAGCATTGGAAACGG-3' N/A Forward 140 Taqman-SanR3-R 5'-AACAACAAGCTCTCTCACAC-3' N/A Reverse

Taqman-SanR3-P 5'-ATATTGCTCCGCCTTCCTCTTCCACAC-3' N/A N/A N/A

Taqman-UBI-F 5'-CTCTCGCTGATTACAACATCC-3' N/A Forward 93 Taqman-UBI-R 5'-TGAAACACCATCAACAGACAC-3' N/A Reverse

Taqman-UBI-P 5'-AGACGAAGGACAAGGTGAAGGGTGGA-3' N/A N/A N/A

MGB-SanR1-F 5'-TGGTCGGGAAGGATGATGAG-3' N/A Forward 78 MGB-SanR1-R 5'-GAACGCAAGCTTCAGCAACA-3' N/A Reverse

MGB-SanR1-P 5'-CTCTACTGAAAACCATTT-3' N/A N/A N/A

MGB-SanR2-F 5'-CCTGGTGCTGATGGCAAAA-3' N/A Forward 71 MGB-SanR2-R 5-TCAGGCATTGGACCACTGTAAT-3' N/A Reverse

Table 2 (continued). List of VIGS and qPCR primers.

Namea Sequenceb RE Direction Amplicon size (bp)

MGB-SanR2-P 5'-AGATGTGTTTGATGATT-3' N/A N/A N/A

MGB-SanR3-F 5'-TTGGGCAGAAGAATCAAATAGATG-3' N/A Forward 72 MGB-SanR3-R 5'-CCATAGACCCAACCAAAACAATC-3' N/A Reverse

MGB-SanR3-P 5'-CAAAAGCAGCGGGAG-3' N/A N/A N/A

MGB-UBI-F 5'-GTACTCTCGCTGATTACAACATCCA-3' N/A Forward 69 MGB-UBI-R 5'-TACCACCACGAAGACGAAGGA-3' N/A Reverse

MGB-UBI-P 5'-TCCACCCTTCACCT-3' N/A N/A N/A

a5UTR, 3UTR, and CDS: amplifies small regions of SanR 5’ UTR, 3’ UTR, and coding sequence for ligation into pTRV2; UTR-SanR1 and UTR-SanR3

amplicons were combined in pTRV2 to make a single construct; PYL156: amplifies pTRV2 multiple cloning site (empty vector amplicons is 390 bp);

OYL195/OYL198: amplifies pTRV1; CP: amplifies pTRV2 coat protein; MP: amplifies pTRV1 movement protein; GADPH: glyceraldehyde 3-phosphate

dehydrogenase; UBI: ubiquitin; MGB: TaqMan qPCR primers (F or R) and probe (P); F: forward primer complementary to 5’-end of sequence; R: reverse primer

complementary to 3’-end of sequence; P: qPCR probe with FAM reporter and BHQ1 (conventional) or TAMRA (MGB) quencher. bItalics: restriction enzyme

(RE) recognition sequence; unformatted: template sequence.

A 218 bp 816 bp 215 bp

V1: SR1-5UTR **V9: SR1-CDS *V2: 218 bp 357 bp (100%) SR1-3UTR

B 207 bp 816 bp 299 bp

V3: SR2-UTR V4: SR2-CDS V5: SR2-3’UTR 167 bp 433 bp 249 bp

C 144 bp 147 bp 900 bp 352 bp

V6: SR3-CDS *V7: SR3-3’UTR 133 bp 352 bp

D 897 bp 174 bp

Figure 4. Constructs designed to silence sanguinarine reductases (SanRs) using virus-induced gene silencing (VIGS). Double-ended

arrows indicate the small regions (~100-300 bp) of (A) SanR1, (B) SanR2, (C) SanR3, or (D) SanR4 that were cloned into pTRV2.

VIGS constructs are labeled as V1-V8. *Construct V8 was created by combining V2 and V7. **Additional constructs V9-V12

designed by Guillaume Beaudoin. V9 is 100% identical to a region in SanR1 coding sequence (CDS). V10, V11 and V12 share 75 and

34%, 21 and 86%, and 20% and 16% sequence similarity to SanR1 and SanR2, respectively. SanR schematic is to scale. Black and

white boxes indicate UTR and CDS, respectively. Gray box represents predicted transit peptide of SanR3.

Primers were designed to amplify sequences with greater than 50%, 35%, and 50% amino acid sequence similarity to P. somniferum COR (GenBank Accession No.

AF108432), NOS (GenBank Accession No. JQ659007), and SalR (GenBank Accession

No. DQ316261), respectively (Winzer et al., 2012; Ziegler et al., 2006; Unterlinner et al.,

1999). Primers were designed to introduce 5’-BamHI and 3’-KpnI, or 5’-SphI and 3’-SalI

RE cut sites to allow for directional cloning of reductases into the pQE30 expression vector.

2.6.2 PCR amplification of DNA and ligation

High-fidelity polymerases were used for amplification from cDNA template when sequence integrity was necessary (KAPA HiFi HotStart DNA Polymerase, Kapa

Biosystems, Cat. No. KK2501; Platinum Pfx DNA Polymerase, Invitrogen, Cat. No.

11708-013; TaKaRa Ex Taq, Clontech, Cat. No. RR001A; or Phusion high-fidelity DNA polymerase, NEB, Cat. No. M0530). Green Taq DNA polymerase (GenScript, Cat. No.

E00043) was used for routine PCR reactions, such as screening bacterial colonies for construct insertion into pGEM-T or pQE30 vectors, or presence of TRV vectors. Green

Taq DNA polymerase was also used to A-tail PCR products amplified by high-fidelity polymerases with 3’ to 5’ exonuclease activity. PCR reactions and thermocycler conditions were set according to the manufacturer’s instructions.

A-tailed PCR products were directly ligated into pGEM-T according to manufacturer’s instructions (Promega, Cat. No. A3600). Constructs to be ligated into pQE30 or pTRV2 were excised from pGEM-T using the appropriate REs, and digests were incubated at 37°C for 1 hour. PCR products and RE digests were size separated using 1% (w/v) agarose gels, containing 0.5 μg/mL ethidium bromide, in TAE buffer.

Bands corresponding to DNA of the appropriate size were excised and purified using the

AxyPrep DNA Gel Extraction Kit (Axygen, Cat. No. AP-GX-250) then ligated into pQE30 or pTRV2 using T4 DNA ligase (NEB, Cat. No. M0202) according to manufacturer’s instructions, except that reactions were incubated at 4°C overnight.

2.6.3 Bacterial transformation

Chemically competent E. coli were prepared according to the CaCl2 method, and were transformed with plasmid DNA using the heat shock transformation method

(Sambrook and Russell, 2001). For each transformation reaction, 100 μL competent cells were thawed on ice then mixed with ~1 μg plasmid DNA or an entire ligation reaction

(10 μL). The cells were incubated on ice 10 minutes then heat shocked for 30 seconds at

42°C, and immediately placed back on ice. Pre-warmed LB media (1 mL) was added to the transformation reaction, and incubated for one hour at 37°C with shaking. Only

~100 μL of transformation reactions with plasmid DNA was plated on solid LB media supplemented with the appropriate antibiotics, and X-gal and IPTG, if necessary.

However, entire transformation reactions were plated when E. coli were transformed with ligation products. Plates were incubated at 37°C overnight or until the formation of single bacterial colonies. Positive clones were identified using blue-white screening (pGEM-T only), antibiotic resistance, colony PCR and/or restriction enzyme digestion, and sequencing methods. For colony PCR, individual clones were spotted onto a solid plate of LB media for reference, and then added to water in the PCR reaction. The initial denaturation step (94°C for 10 min) was extended to ensure complete E. coli lysis.

Constructs in pGEM-T and pQE30 were amplified using T7 and SP6, or pQE30-F and pQE30-R primer pairs, respectively (Table 1). For RE digest, E. coli colonies were grown

34 overnight at 37°C in LB media supplemented with appropriate antibiotics, then cells were pelleted, and plasmid DNA was isolated using a modified alkaline lysis method

(Birnboim and Doly, 1979). For E. coli clones containing the correct construct of the correct size, plasmid DNA was extracted with a kit (e.g. AccuPrep Plasmid Mini

Extraction Kit, Molecular Biology Products Inc., Cat. No. K-3030) then sent for sequencing by Eurofins MWG Operon (Eurofins Genomics, Huntsville, AL.). All constructs were checked for correct reading frame, and nucleotide sequence using

DNAMAN (Lynnon Biosoft, Version 8).

Electrocompetent A. tumefaciens were prepared (Sambrook and Russell, 2001), then transformed using the Gene Pulser II System (Bio-Rad) set to 2.0 kV, 25 µF capacitance, and 400 Ω resistance. Plasmid DNA was ethanol precipitated and resuspended in double-distilled water to remove excess salt. Immediately following electroporation, A. tumefaciens recovered in LB for an hour at 28°C with shaking. Cells were plated on LB media containing gentamycin, rifampicin, and kanamycin. Plates were incubated 28°C for 2-3 days or until the formation of single colonies. Positive transformants were identified as colonies that grew on selective media. Bacterial stocks were frozen in 25% glycerol and stored at -80°C.

2.6.4 Plant transformation

Papaver somniferum were transformed via Agrobacterium infiltration (modified from Hileman et al., 2005). Seedlings were transplanted to allow for one plant per pot then allowed to recover a couple days prior to transformation. A. tumefaciens harbouring pTRV1, pTRV2-SanR constructs, pTRV2-PDS, or empty pTRV2 were grown at 28°C overnight with shaking in LB media supplemented with gentamycin, rifampicin, and

35 kanamycin. Overnight cultures were used to inoculate induction medium (LB media containing kanamycin, MES, and acetosyringone), and cultures were grown overnight at

28°C with shaking. A. tumefaciens were pelleted at 3,000 g, and resuspended in infiltration solution to an absorbance at 260 nm (A260) of 1.5. Agrobacterium were incubated at room temperature, with shaking, for four hours before poppy infiltration.

P. somniferum seedlings at the 2 to 4 leaf stage were infiltrated at the apical meristem with a 1:1 mixture of A. tumefaciens harbouring pTRV1 and pTRV2-SanR constructs. As controls, seedlings were also infiltrated with 1:1 mixtures of A. tumefaciens harbouring pTRV1 and pTRV2-PDS, or A. tumefaciens harbouring pTRV1 and empty pTRV2.

Plants were grown for 6 weeks post-infiltration then roots were harvested for VIGS analysis.

2.7 Escherichia coli protein induction, purification, and detection

Recombinant 6xHis-tagged proteins were expressed, and purified using the

QIAexpressionist™ handbook as a guide (5th Ed., Qiagen). E. coli strain SG13009 harbouring pQE30-SanR constructs were grown in 3 mL LB media supplemented with ampicillin and kanamycin were grown overnight at 37°C with shaking. Overnight cultures were used to inoculate 1 L LB containing ampicillin and kanamycin. Cultures were grown at 37°C until optical density at 600 nm (OD600) reached 0.4 to 0.6. Cultures were induced with 1 mM IPTG then grown at 30°C with shaking for 4 hours. E. coli were pelleted at 6,000 g, and stored -80°C until purification.

For purification, the E. coli pellet was thawed on ice, and resuspended in 100 mM sodium phosphate, pH 7.5 containing 1 mM phenylmethylsulfonyl fluoride (PMSF).

Cells were sonicated on ice using the Microson XL2000 Ultrasonic Homogenizer (Fisher

Scientific, Cat. No. 15-338-274). Supernatant was collected by centrifugation at

10,000 x g for 15 minutes then added to TALON® metal-affinity resin (Clontech, Cat.

No. 635501), which was pre-equilibrated with 100 mM sodium phosphate, pH 7.5. Resin and supernatant were shaken on ice for 1 hour. The protein-charged resin was washed three times with 100 mM sodium phosphate, pH 7.5, and then proteins were eluted stepwise with 100 mM sodium phosphate supplemented with 25, 50, 75 and 200 mM imidazole. The 50 mM imidazole fractions were desalted with 100 mM sodium phosphate using PD-10 desalting columns (GE Healthcare, Cat. No. 17-0851-01).

Protein concentration of imidazole fractions was determined using Bradford assays (Bradford, 1976), and protein was size-separated on SDS-PAGE then stained with

Coomassie to visualize total protein, or transferred to nitrocellulose membrane (VWR,

Cat. No. CA27376-991) using the conventional wet (tank) transfer method for Western blot analysis (Tovey and Baldo, 1987). Following protein transfer, nitrocellulose membrane was blocked with 5% skim milk power in TBS-tween. Recombinant 6xHis- tagged proteins were detected using 0.2 µg/ml mouse anti-His antibodies (GenScript, Cat.

No. A00186-100), and SanR proteins were detected using a 1:10,000 dilution of the polyclonal mouse SanR antisera (see section 2.11). Both recombinant and native proteins were secondarily probed with goat anti-mouse horseradish peroxidase (HRP)-conjugated antibodies (1:10,000 dilution; BioRad, Cat. No. 170-5047) for visualization using

SuperSignal™ West Pico Chemiluminescent Substrate, an enhanced chemiluminescence

(ECL) detection system (Thermo Scientific, Cat. No. 34077). Western blots were imaged using X-ray film (VWR Cat. No. IB1651454).

2.8 Plant protein purification, and detection

Stem, root, leaf, and capsule/flower bud tissue were flash frozen in liquid nitrogen, then ground to a fine powder using a TissueLyser II (Qiagen, Cat. No. 85300) fitted with 35-mL stainless steel grinding jars with 20 mm grinding balls (Retsch, Cat.

Nos. 01.462.0214 and 05.368.0062, respectively) cooled in liquid nitrogen. Plant protein extraction buffer was added to equal volumes of powdered tissue then centrifuged at

5,000 x g to remove debris. Proteins were precipitated in 90% (NH4)2SO4 overnight at

4°C. Proteins were pelleted by centrifugation at 5,000 x g, supernatant was removed, and pellet was resuspended in 50 mM Tris, pH 7.5, and 2 mM EDTA. Protein concentration was determined using Quick Start™ Bradford 1x Dye Reagent according to manufacturer’s instructions (BioRad, Cat. No. 500-0205) (Bradford, 1976). Plant proteins were size-separated on SDS-PAGE then stained with Coomassie or transferred to nitrocellulose membrane for Western blot analysis. SanR proteins were detected with a

1:100 dilution of mouse SanR3B antisera (see section 2.11), and secondarily probed with goat anti-mouse HRP-conjugated antibody (1:5,000) for visualization using an ECL detection system. Western blots were imaged using X-ray film.

2.9 Alkaloids

Various alkaloids were used as substrates for SanR enzyme assays, and as standards for analyzing VIGS results. Sanguinarine, chelerythrine, papaverine, berberine, noscapine (Sigma-Aldrich, Cat. Nos. S5890, C2932, P3510, B3251, and 363960, respectively), canadine (ChromaDex, Cat. No. ASB-00020155), cryptopine (MP

Biomedicals, Cat. No. 0520114201), and 1,2-dehydroreticuline (Toronto Research

Chemicals, Cat. No. D230065) were all purchased. Morphine and codeine, and reticuline

38 were gifts from Sanofi-Aventis (Paris, France; http://en.sanofi-aventis.com), and

Tasmanian Alkaloids (Westbury, Australia; http://www.tasalk.com.au), respectively.

Thebaine was prepared by Dr. Jill Hagel from P. somniferum latex (Hagel and Facchini,

2010). Chelirubine and macarpine were purified from elicited E. californica cell culture filtrate by thin-layer chromatography (TLC) (see section 2.9.1). Dihydrosanguinarine, dihydrochelerythrine, dihydrochelirubine, and dihydromacarpine were produced by sodium borohydride reduction of sanguinarine, chelerythrine, chelirubine, and macarpine, respectively.

2.9.1 Isolation of benzophenanthridine alkaloids

Eschscholzia californica cell cultures treated with yeast extract were harvested

96-hours post-elicitation. Cell filtrate (media) was collected using vacuum filtration, freeze-dried then resuspended in methanol (300 µL/10mg) to extract alkaloids. Alkaloid extraction was spotted onto silica gel 60 F254 TLC plates (EMD Millipore, Cat. No.

105735), and allowed to dry before being developed using toluene:methanol (9:1)

(protocol modified from Baerheim-Svendsen and Verpoorte, 1983). Plates were visualized using long-wave UV light (365 nm) to identify benzophenanthridine alkaloids by color: chelirubine is a purple-red, sanguinarine is orange, macarpine is red, and chelerythrine is a yellow-green (Shamma, 1972). Individual spots corresponding to the benzophenanthridines were scraped off the TLC plate, and incubated in methanol at 65°C for 30 minutes to extract alkaloids from silica. Extract was filtered using Millex-GV

Syringe Filters, 0.22 µm (EMD Millipore) then speed vacuumed to concentrate sample.

Alkaloid identities were confirmed by LC-MS (see section 2.13), and concentrations were calculated using extinction coefficients (Krane et al., 1984).

2.10 Enzyme assays

In vitro enzyme assays were performed as 50-μL reactions in 100 mM sodium phosphate, pH 7.5, using 1 μg recombinant SanR (desalted 50 mM-imidazole fraction),

1 mM NADPH or NADH (BioShop Cat. Nos. NAD004 and NAD002), and 0.1 to 10 mM alkaloid substrate. To determine temperature and pH optima, and substrate range, alkaloid concentration was 5 μM. For temperature optima, 100 mM sodium phosphate buffer, pH 7.5, was incubated at 4, 16, 21, 30, 37, 42, 55 and 65°C, prior to the addition of protein and NAD(P)H. For pH optima, various 100 mM buffers were used to obtain a pH range of 5-10: citrate (pH 5), sodium phosphate (pH 6, 7, and 8), Tris (pH 8), and glycine (pH 9 and 10). Assays were incubated at room temperature for pH curves, and substrate range. Assays were incubated for one hour then reactions were stopped by adding 950 μL methanol.

To determine the Michaelis constant (Km) of SanRs, 50-μL reactions were performed in 100 mM sodium phosphate, pH 7.5, with 1 mM NADPH, 1 μg recombinant

SanR, and varying amounts of sanguinarine (0.1 to 10 mM). Reactions were incubated at room temperature for one hour then quenched with methanol. Michaelis-Menten kinetic constants (Km, and Vmax) were determined using the Enzyme Kinetics Wizard package from SigmaPlot 12.0 (Systat Software; San Jose, CA).

2.11 Antibody production

Recombinant SanRs were purified as described in section 2.7. The 50 mM imidazole fraction resuspended in 0.85% saline solution using dialysis tubing. Proteins were diluted to 100-300 μg/mL, and stored as 500 μL aliquots at -80°C. Five-week-old female Swiss Webster mice (Charles River) were injected with 100 μL SanR antigen

(100-300 μg/mL in 0.85% saline) (LESARC, Calgary, AB.). Following the initial injection, mice were injected 3 more times every 3 weeks. Test bleeds were taken at the first (pre-immune serum), second, third, and forth injection. Mice were exsanguinated two weeks after the forth injection. Blood was centrifuged at 300 x g for 5 minutes at

4°C, and supernatant (serum) was collected and stored at -20°C.

2.11.1 Dot blots

Dot blots were used to determine specificity of SanR antisera. Recombinant SanR protein in 0.85% saline solution (100-300 μg/mL) was diluted 1/10, 1/100, and 1/1000.

The four dilutions, for SanR1, SanR2, and SanR3B, were spotted (1 μL) on nitrocellulose membrane, as well as 0.5 μL of mouse antisera as a positive control, and allowed to dry.

Membranes were blocked with 5% skim milk in TBS-Tween then incubated with SanR antisera (1:10,000 dilution), and secondarily probed with goat anti-mouse HRP- conjugated antibodies (1:10,000 dilution). Blots were visualized using ECL to expose

X-ray film.

2.12 Immunolocalization

2.12.1 Tissue fixation and embedding

Large sections of opium poppy stem, root, leaf, and capsule from cultivars

Marianne, Veronica, and 40 were immediately placed into 50 mM piperazine-N,N′- bis(2-ethanesulfonic acid) (PIPES), pH 7.0, containing 4% paraformaldehyde (PFA) under vacuum for a couple hours. Tissues were cut into approximately 5 mm sections then placed into glass vials containing with fresh 4% PFA, and fixed overnight. Tissues were placed under vacuum, and then rinsed in 50 mM PIPES, pH 7.0 before incubation in

50%, 70%, 90%, and 100% (v/v) ethanol for 2 hours in each solution to facilitate

41 dehydration. Tissue was placed in 100% ethanol overnight, and then placed under vacuum the next morning before incubation with embedding medium. Tissues were infiltrated with increasing concentrations of LR White (Electron Microscopy Sciences,

Cat. No. 14381) or Technovit 8100 (supplied by Dr. Ed Yeung). Resin concentration was changed from 50% to 75% to 87.5% to 100% over two days. Tissue sections were trimmed, and infiltrated with 100% embedding medium for another day. Tissue fixation and embedding was performed at 4°C. To polymerize resin, tissues were immersed in

100% resin contained in 1-mL gelatin capsules, and incubated at 58°C for 24 hours.

Sections were cut to 1.0 to 2.0 μm thickness using a Sorvall MT-I Ultramicrotome, and mounted on SuperFrost Plus glass slides (Electron Microscopy Sciences, Cat. No.

71869).

2.12.2 Immunohistochemistry

Tissue sections mounted on glass slides were blocked using 5% skim milk powder in TBS containing 1% (w/v) Tween 20. Sections were incubated with a 1:50 dilution of

SanR3B antisera (see section 2.11) for one hour in a humid chamber, and then rinsed three times with TBS-Tween. Sections were incubated with a 1:100 dilution of Alexa

488-conjugated goat anti-mouse secondary antibody (Life Technologies, Cat. No.

A-11001) for one hour in a humid chamber, and then rinsed three times with TBS-Tween.

2.12.3 Microscopy

Immunofluorescence labeling was viewed using a Leica DM RXA2 microscope

(Leica Microsystems, Wetzlar, Germany), and images were acquired with a Retiga EX digital camera (QImaging, Burnaby, British Columbia, Canada). Alexa 488 labels were detected using Leica L5 filter. The xylem was visualized using UV light. False-coloured

42 images were generated in Photoshop (Adobe, CS5, Version 12.0). Light microscopy images were captured using the Leica microscope and the Retiga camera mounted with a

RGB color liquid crystal filter (QImaging).

2.13 Virus-induced gene silencing

Papaver somniferum seedlings, grown to the 2-4 leaf stage, were pressure- infiltrated with Agrobacterium (see section 2.6.4) at the apical meristem. A. tumefaciens harbouring pTRV1 were mixed, in equal volumes, with A. tumefaciens harbouring pTRV2-SanR constructs designed to silence one or multiple SanRs (Table 2; Fig. 4).

Previous pTRV2-SanR constructs cloned by Guillaume Beaudoin were also infiltrated into opium poppy. Six-weeks post-infiltration, root tissue from individual plants was harvested. Roots were washed to remove excess soil then flash frozen in liquid nitrogen, and stored at -80°C until RNA extraction.

2.13.1 RNA extraction and cDNA synthesis

RNA was extracted from opium poppy tissues (stem, root, leaf, and capsule/flower bud) using CTAB (modified from Meisel et al., 2005). Either TissueLyser

Adapter Sets (2 x 24; Qiagen, Cat. No. 69982), or 35-mL stainless steel grinding jars with

20 mm grinding balls (Retsch, Cat. Nos. 01.462.0214 and 05.368.0062, respectively) were pre-cooled in liquid nitrogen for use with a TissueLyser II (Qiagen, Cat. No. 85300) to grind plant tissue to a fine powder. Oscillation frequency was set to 30 Hz for 1-2 minutes. 2X CTAB RNA extraction buffer pre-heated to 65°C then 500 mL was added to

100-200 μL ground tissue, and incubated at 65°C for 10 minutes. Nucleic acids were extracted with chloroform:isoamyl alcohol (24:1) until the aqueous phase was free of particulate matter, then the aqueous phase incubated with 0.25X 10 M LiCl overnight at

4°C to precipitate RNA (Sambrook and Russell, 2001; Barlow et al., 1963). The supernatant was discarded, or incubated with 100% ethanol to precipitate DNA, and the pellet was washed with 70% ethanol then resuspended in DEPC-treated water. RNA was treated with DNase I according to the manufacturer’s instructions (NEB, Cat. No.

M0303S) then RNA quality and quantity was determined using a NanoDrop ND-1000

UV-Vis Spectrophotometer (Thermo Scientific) (Bustin et al., 2009). Complementary

DNA (cDNA) was synthesized from 1 μg total RNA using the Moloney Murine

Leukemia Virus reverse transcriptase (M-MLV RT; Invitrogen, Cat. No. 28025-013) according to the manufacturer’s instructions, and was diluted with equal volumes of

DEPC-treated water for use in qPCR.

2.13.2 Quantitative real-time PCR

Primer and TaqMan minor groove binder (MGB) probe pairs were designed to amplify target gene of interest (e.g. SanR) or ubiquitin as an endogenous reference gene

(Table 2). TaqMan MGB probes were ordered with a 5’ 6-FAM reporter dye, and a 3’ non-fluorescent quencher (NFQ) dye (Life Technologies). Quantitative real-time PCR

(qPCR) reactions were performed in an Applied Biosystems 7300 real-time PCR system (Life Technologies) using 1 μL diluted cDNA (see section 2.11.1), 250 nM forward primer, 250 nM reserve primer, 250 nM TaqMan MGB probe, and 0.5X

PerfeCTa qPCR FastMix II with Rox (Quanta Biosciences, Cat. No. 95118). Initial denaturation occurred at 95°C for 3 minutes, followed by 40 cycles of 10 seconds at 95°C then 60 seconds at 60°C. Data was collected each cycle at the end of the 60°C incubation.

Relative gene expression was calculated using the comparative CT method (Livak and

Schmittgen, 2001).

2.13.3 Root alkaloid extraction

Opium poppy roots were flash-frozen in liquid nitrogen, and ground to a fine powder with a TissueLyser II (Qiagen), fitted with 2 x 24 TissueLyser Adapter Sets pre-cooled at -80°C, at 30 Hz for 2 minutes. Alkaloids were extracted in methanol

(20 mL/g dry weight) (modified from Farrow and Facchini, 2013). Extracts were sonicated then incubated overnight at -20°C. Extracts were centrifuged at 14,000 g for 10 minutes at 4 °C to remove debris. A 1:20 dilution was prepared LC-MS analysis.

2.14 Liquid chromatography-mass spectrometry

Enzyme assays, VIGS samples, and TLC-purified alkaloids were analyzed by liquid chromatography-mass spectrometry (LC-MS) using a 1200 Liquid Chromatograph and a 6410 Triple Quadruple Mass Spectrometer (Agilent Technologies, Santa Clara,

CA) (protocol modified from (Dang and Facchini, 2014; Farrow et al., 2012). Samples (1 to 10 μL) were injected onto a Poroshell 120 SB C18 column (2.1 mm × 50 mm, 2.7 μm particle size, Agilent Technologies), and eluted at a flow rate of 0.7 mL/min over a gradient of solvent A (95:5 10mM ammonium acetate, pH 5.5:acetonitrile) and solvent B

(100% acetonitrile) as follows: 0-30% solvent B from 0 to 6 minutes, 30-60% solvent B from 6 to 7 minutes, 60-99% solvent B from 7 to 10 minutes, 99% solvent B from 10 to

14 minutes, 99-0% solvent B from 14 to 14.1 minutes, and 0% solvent B from 14.1 to

19.1 minutes. Eluent from the HPLC column was introduced to the electrospray ionization source (ESI) operating in positive ion mode, and full-scan mass spectrometry data was acquired in the range of m/z 200-700. For collision-induced dissociation (CID) analysis, the precursor m/z was selected, and collision energy of 25 eV was applied.

Retention times and fragmentation spectra were compared to those of authentic BIA

45 standards and published reference spectra for the identification (Farrow et al., 2012). The concentration of an alkaloid, except for sanguinarine and dihydrosanguinarine, was estimated by integrating the extracted ion chromatogram (EIC) for the alkaloid of interest based on their m/z and retention time. The concentration of sanguinarine and dihydrosanguinarine was determined using a standard curve.

2.15 Statistical analysis

Statistical analyses were performed using unpaired, two-tailed Student’s t-test in

GraphPad Prism 5 (GraphPad Software, San Diego, California, USA) to determine if two sets of data are significantly different from each other. If variance between the two groups were unequal, then Welch’s correction was applied.

3 RESULTS

3.1 Sanguinarine reductase identification, expression, and purification

Four SanRs (SanR1, SanR2, SanR3, and SanR4) were identified in the

Papaver somniferum cv. Bea’s Choice transcriptomes when queried with E. californica

SanR (GenBank Accession No. GU338458) (Fig. 3). SanR1, SanR2, SanR3, and SanR4 share 79.2, 77.3, 62.7, and 71.2% amino acid sequence similarity to E. californica SanR

(DNAMAN). SanR3 was predicted to localize to chloroplasts, therefore a truncated form of SanR3, SanR3B, was PCR amplified to remove the sequence corresponding to the 48 amino acids predicted to encode the putative transit peptide (Table 1) (Horton et al.,

2007; Emanuelsson et al., 2007). SanR1, SanR2, SanR3, SanR3B, and SanR4 were all successfully cloned into the pQE30 expression vector, and transformed into E. coli strains

M15 and SG13009. Initial attempts to amplify SanR4 were unsuccessful; consequently,

SanR4 was never heterologously expressed as a recombinant protein. Expression of

N-terminally 6xHis-tagged SanRs was IPTG-inducible in E. coli SG13009 cultures harbouring pQE30-SanR constructs (Fig. 5), but no recombinant protein expression was observed for induced E. coli M15 harbouring pQE30-SanR constructs (data not shown).

N-terminally 6xHis-tagged SanRs were purified using TALON metal affinity resin, and predominantly eluted with buffer containing 50 mM imidazole (Fig. 6). Therefore, SanRs were eluted with 0.1 M sodium phosphate buffer containing 50 mM imidazole (Fig. 7) then desalted for use in enzyme assays and to produce antibodies in mice.

47 SanR

1 2 3 3B

M U I U I U I U I

66.4 kDa

34.6 kDa

27.0 kDa

Figure 5. Expression of recombinant sanguinarine reductases. Three full-length sanguinarine reductases (SanR1, SanR2, and SanR3) and a N-terminally truncated form of SanR3 (SanR3B) were cloned into the pQE30 vector then transformed into E. coli strain SG13009. Soluble proteins from uninduced (U) and IPTG-induced (I) E. coli were size-separated using 12% SDS-PAGE, and proteins were visualized by Coomassie staining. Only E. coli induced with 1 mM IPTG expressed recombinant SanRs. The predicted sizes of SanR1, SanR2, SanR3, and SanR3B are 29.4, 29.4, 32.1, and 26.8 kDa, respectively. M: protein marker, broad range (NEB, Cat. No. P7702).

48 A Imidazole concentration (mM) B SanR1 SanR2 SanR3 SanR3B M 1 2 3 4 5 6 7 8 9 10 11 12 M S F 1 10 11 12 S F 1 10 11 12 M S F 1 10 11 12 S F 1 10 11 12

34 n

1 27 34

a 27

34 n

n 46 34 R

3 27 30

34 n

3 23 27 B

Figure 6. Purification of sanguinarine reductases using TALON metal affinity resin. IPTG-induced E. coli expressing P. somniferum

sanguinarine reductases (SanRs) were lysed, and supernatant (S) was applied to a gravity column containing TALON resin. Flow

through (F) was collected then proteins were eluted with sodium phosphate buffer containing various concentrations of imidazole.

Proteins were size-separated on 12% SDS-PAGE, and total proteins were stained with Coomassie (A, B: top row). His-tagged proteins

were detected using a mouse anti-His antibody, and secondarily probed with a horseradish-peroxidase conjugated goat anti-mouse

antibody. Western blots were visualized using an enhanced chemiluminescent system (B: bottom row). M: protein marker (NEB, Cat.

Nos. P7702 and P7709) with approximate molecular weights (kDa) indicated on left-hand side of gel or blot; 1-10: 5 mM stepwise

increase in imidazole concentration from 5 to 50 mM; 11 and 12: 75 and 100 mM imidazole, respectively.

A B SanR SanR

M 1 2 3 3B M 1 2 3 3B

34.6 kDa 30 kDa

27.0 kDa

23 kDa

Figure 7. Purification of recombinant sanguinarine reductases. Sanguinarine reductases (SanRs) were purified using TALON metal

affinity resin eluted with sodium phosphate buffer containing 50 mM imidazole, desalted then quantified using Bradford reagent.

Purified proteins (5 µg) were size-separated on 12% SDS-PAGE then stained with Coomassie (A), or transferred to nitrocellulose

membrane for Western blot analysis (B). Recombinant proteins were detected with an anti-His antibody then secondarily probed with

a horseradish-peroxidase conjugated goat anti-mouse antibody, and visualized using an enhanced chemiluminescent system.

3.2 Biochemical characterization of sanguinarine reductases in vitro

Several benzylisoquinoline alkaloids were tested as potential substrates for opium poppy SanRs, including 1-benzylisoquinoline, phthalideisoquinoline, protoberberine, protopine, morphinan, and benzophenanthridine alkaloids (Table 3).

3.2.1 Sanguinarine reductase does not reduce 1,2-dehydroreticuline

SanR1, SanR2, or SanR3B did not reduce 1,2-dehydroreticuline to reticuline

(Table 3; Fig. 8). Neither assays with cell lysate (data not shown), nor assays with purified proteins resulted in the enzymatic reduction of 1,2-dehydroreticuline (Fig. 8).

3.2.2 Purification of benzophenanthridine alkaloids

E. californica root and cell culture filtrate alkaloid extract was separated by TLC then alkaloid composition of individual bands, as visualized by long-wave UV light, was analyzed by mass spectrometry (Fig. 9 and 10). Several TLC solvent systems were used in an attempt to separate benzophenanthridine alkaloids within the root alkaloid extract, such as toluene:acetone:ethyl acetate (7:2:1), toluene:methanol (9:1), and chloroform:ethyl acetate:methanol (2:2:1) (Schumacher et al., 1987; Baerheim-Svendsen and Verpoorte, 1983). Mass spectrometry analysis indicated that benzophenanthridine alkaloids were present, in high abundance relative to other compounds, only in a single band when separated using chloroform:ethyl acetate:methanol (2:2:1). Therefore, alkaloids within this band were further separated by TLC using toluene:methanol (9:1), which was able to resolve sanguinarine, chelerythrine, chelirubine, and macarpine as individual bands on the TLC plate (Fig. 9). On the other hand, the cell filtrate alkaloid extract only separated by TLC using toluene:methanol (9:1) was able to resolve sanguinarine, chelerythrine, chelirubine, and macarpine (Fig. 9).

51 Table 3. Benzylisoquinoline alkaloids tested as potential substrates of SanRs.

[M]+ or Compound Structure Type [M+H]+

1,2-Dehydroreticuline 328 1-Benzylisoquinoline

Papaverine 340 1-Benzylisoquinoline

Noscapine 414 Phthalideisoquinoline

Berberine 336 Protoberberine

Cryptopine 370 Protopine

Thebaine 312 Morphinan

Table 3 (continued). Benzylisoquinoline alkaloids tested as potential substrates of SanRs.

[M]+ or Compound Structure Type [M+H]+

Sanguinarine 332 Benzophenanthridine

Chelirubine 362 Benzophenanthridine

Macarpine 392 Benzophenanthridine

Chelerythrine 348 Benzophenanthridine

53 A B

) 1000 ) 4

5 5

0 0

1 800 1 3

x x

( (

600

s s

t t 2

n 400 n

u u o 200 o 1

C C

S 0 S 0

M M - 0 5 10 15 - 0 5 10 15

C C

L Retention Time (min.) L Retention Time (min.) C D

) 15 ) 20

5 5

0 0

1 1

x 10 x

( (

s s

t t 10

n n

u 5 u

o o 5

C C

S 0 S 0

M M - 0 5 10 15 - 0 5 10 15

C C

L Retention Time (min.) L Retention Time (min.)

Figure 8. Sanguinarine reductases do not reduce 1,2-dehydroreticuline. (A) Extracted ion chromatographs (EIC) for authentic

standards 1,2-dehydroreticuline ([M]+ 328; retention time ~3.6 min.; black), and reticuline ([M+H]+ 330; retention time ~4.0 min.;

gray). (B-D) Enzyme assays for SanR1, SanR2, and SanR3B, respectively, with 5 μM 1,2-dehydroreticuline as a substrate.

1 1 2 2

3 3

4 4

Origin Origin Origin

Figure 9. TLC separation of benzophenanthridine alkaloids. E. californica root alkaloid extract was spotted onto silica gel 60 F254 plates then separated using the solvent system chloroform:ethyl acetate:methanol (2:2:1). Benzophenanthridine alkaloids were present in the single, orange band indicated by the arrow (left), and were further separated using toluene:methanol (9:1) (middle). E. californica cell filtrate alkaloid extract was separated only by the toluene:methanol (9:1) solvent system to separate benzophenanthridine alkaloids (right). TLC plates were visualized with long-wave UV light (365 nm).

1: Chelirubine, 2: sanguinarine, 3: macarpine, 4: chelerythrine.

55 CID analysis confirmed the identities of sanguinarine (m/z 332.2), chelerythrine

(m/z 348.2), chelirubine (m/z 362.2), and macarpine (m/z 392.2) (Fig. 10). Standard curves were generated to correlate LC-MS counts to concentration of benzophenanthridines as determined using extinction coefficients (Krane et al., 1984)

(Fig. 11). Benzophenanthridine alkaloids were treated with sodium borohydride to reduce sanguinarine, chelerythrine, chelirubine, and macarpine to dihydrosanguinarine, dihydrochelerythrine, dihydrochelirubine, and dihydromacarpine, respectively (Fig. 12).

3.2.3 Sanguinarine reductases reduce benzophenanthridine alkaloids

SanR1, SanR2, and SanR3B reduce the benzophenanthridine alkaloids sanguinarine (m/z 332.2), chelerythrine (m/z 348.2), chelirubine (m/z 362.2), and macarpine (m/z 392.2) to dihydrosanguinarine (m/z 333.4), dihydrochelerythrine

(m/z 349.4), dihydrochelirubine (m/z 363.4), and dihydromacarpine (m/z 393.4), respectively, using either NADPH or NADH as a co-factor (Table 3; Fig. 13). However,

SanRs produce more dihydrobenzophenanthridine when NADPH is used a co-factor

(Fig. 14). No activity was observed for SanR1, SanR2, or SanR3B with other benzylisoquinoline alkaloids papaverine (m/z 339.4), noscapine (m/z 413), berberine (m/z

336.4), cryptopine (m/z 369.4), or thebaine (m/z 312.1) (Table 3; data not shown).

3.2.4 Temperature curves

SanRs were assayed with sanguinarine over a range of temperatures and pHs to determine optimal conditions. A standard curve (Fig. 11) was used to convert dihydrosanguinarine LC-MS counts to concentration. Only SanR2 assays with 5 μM sanguinarine at various temperatures were analyzed by LC-MS. The temperature optimum of SanR2 is approximately 18°C (Fig. 15).

30000 s 332.2

n Sanguinarine

o 20000

S 10000 304.1

- 274.1 317.2

L 0 200 250 300 350 400

30000

n Chelerythrine

u 332.1

o 20000

S 304.1 10000 348.1

M 318.1 - 290.1

L 0 200 250 300 350 400

30000

n Chelirubine

u 347.1

o 20000 362.1

S 10000 318.1

M - 332.1

C 303.8

L 0 200 250 300 350 400

30000 s 392.2

t 377.1

n Macarpine

o 20000

S 10000 348.2

- 362.2

C 334.1

L 0 200 250 300 350 400 Mass-to-Charge (m/z)

Figure 10. Collision-induced dissociation spectra for benzophenanthridines. Alkaloids extracted from E californica cell filtrate were separated using TLC. Identities of sanguinarine (m/z 332), chelerythrine (m/z 348), chelirubine (m/z 362), and macarpine

(m/z 392) were confirmed by collision-induced dissociation. Ions were scanned from m/z

0-400, but only m/z 200-400 are shown for clarity. Diamond indicates parent ion.

) 100 ) 8

7 7

0 0

1 1

x x 6

( (

s 60 s

t t

n n 4

u u 40

o o

C C y = 9E+06x + 1E+06 2 y = 6E+06x + 1E+06 S 20 S R² = 0.99999 R² = 0.99874

M M - 0 - 0

C C

L 0 50 100 150 L 0 5 10 15 Sanguinarine Dihydrosanguinarine Concentration (µM) Concentration (µM)

) 15 ) 12

7 7

0 0

1 1 10

x x

( (

10 8

s s

t t

n n 6

u u o 5 o 4

C C y = 2E+06x + 7E+06 y = 2E+06x - 3E+06 S S 2 R² = 0.99216 R² = 0.99794

M M - 0 - 0

C C

L 0 20 40 60 L 0 20 40 60 Chelirubine Macarpine Concentration (µM) Concentration (µM)

) 12

1 10

(

n 6

u o 4

C y = 1E+07x - 258094 S 2 R² = 0.99998

M - 0

L 0 5 10 15 Chelerythrine Concentration (µM)

Figure 11. Standards curves for benzophenanthridine alkaloids. Various concentrations

(0.001-100 μM) of sanguinarine, dihydrosanguinarine, chelerythrine, chelirubine, macarpine were run on LC-MS to determine linear range for each alkaloid.

58 SAN CHE CHR MAC 3 15 6 8

) ) ) )

6 6 6 6

0 0 0 0

1 1 1 1 6

x 2 x 10 x 4 x

( ( ( (

s s s s 4

t t t t

n 1 n 5 n 2 n

u u u u

o o o o 2

C C C C 0 0 0 0 6.5 7.5 8.5 9.5 6.5 7.5 8.5 9.5 6.5 7.5 8.5 9.5 6.5 7.5 8.5 9.5 Retention Time (minutes)

DSAN DCHE DCHR DMAC 2 6 4 8

) ) ) )

6 6 6 6

0 0 0 0

1 1 1 3 1 6

x x 4 x x

( ( ( (

s 1 s s 2 s 4

t t t t

n n 2 n n

u u u u

o o o 1 o 2

C C C C 0 0 0 0 6.5 7.5 8.5 9.5 6.5 7.5 8.5 9.5 6.5 7.5 8.5 9.5 6.5 7.5 8.5 9.5 Retention Time (minutes)

Figure 12. Non-enzymatic reduction of benzophenanthridines. Benzophenanthridine alkaloids purified from E. californica cell filtrate

(top row) were reduced to dihydrobenzophenathridine alkaloids (bottom row) using sodium borohydride. Samples were analyzed by

LC-MS, and all compounds eluted between 7 and 9 minutes. Extracted-ion chromatographs are shown for sanguinarine (SAN, [M]+

332), dihydrosanguinarine (DSAN, [M+H]+ 334), chelerythrine (CHE, [M]+ 348), dihydrochelerythrine (DCHE, [M+H]+ 350),

chelirubine (CHR, [M]+ 362), dihydrochelirubine (DCHR, [M+H]+ 364), macarpine (MAC, [M]+ 392), and dihydromacarpine

(DMAC, [M+H]+ 394).

A 40 B 40 +SanR1 +SanR1

) ) 4 +SAN 4 +SAN 0 30 0 30

1 1 +NADPH +NADH

x x

( (

s 20 s 20

t t

n n

u u

o 10 o 10

C C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) C 200 +SanR2 D 30 +SanR2

) ) 4 +SAN 4 25 +SAN 0 150 0

1 1 +NADPH 20 +NADH

x x

( (

s 100 s 15

t t

n n

u u 10

o 50 o

C C 5 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) E 60 +SanR3B F 40 +SanR3B

) ) 4 50 +SAN 4 +SAN 0 0 30

1 1 40 +NADPH +NADH

x x

( (

s 30 s 20

t t

n n

u 20 u

o o 10

C 10 C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) G 30 H 30 No enzyme No enzyme

) ) 4 25 +SAN 4 25 +SAN

0 0

1 1 20 +NADPH 20 +NADH

x x

( (

s 15 s 15

t t

n n

u 10 u 10

o o

C 5 C 5 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min)

Figure 13. Sanguinarine reductases reduce benzophenanthridine alkaloids.

60 I 20 J 20 +SanR1 +SanR1

) ) 4 +CHE 4 +CHE 0 15 0 15

1 1 +NADPH +NADH

x x

( (

s 10 s 10

t t

n n

u u

o 5 o 5

C C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) K 150 +SanR2 L 50 +SanR2

)

4 4 +CHE +CHE

0 0 40

100 +NADPH +NADH

( ( 30

t t

n n 20

u 50 u o o 10

C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) M 80 +SanR3B N 150 +SanR3B

) ) 4 +CHE 4 +CHE 0 60 0

1 1 +NADPH 100 +NADH

x x

( (

s 40 s

t t

n n

u u 50

o 20 o

C C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) O 100 P 100 +SanR1 +SanR1

) ) 4 +CHR 4 +CHR 0 80 0 80

1 1 +NADPH +NADH

x x

( 60 ( 60

s s

t t

n 40 n 40

u u o 20 o 20

C C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min)

Figure 13 (continued). Sanguinarine reductases reduce benzophenanthridine alkaloids.

61 Q 400 +SanR2 R 200 +SanR2

) ) 4 +CHR 4 +CHR 0 300 0 150

1 1 +NADPH +NADH

x x

( (

s 200 s 100

t t

n n

u u

o 100 o 50

C C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) S 40 +SanR3B T 50 +SanR3B

) ) 4 +CHR 4 +CHR 0 30 0 40

1 1 +NADPH +NADH

x x

( ( 30

s 20 s

t t

n n 20

u u o 10 o 10

C C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) U 200 V 200 +SanR1 +SanR1

) ) 4 +MAC 4 +MAC 0 150 0 150

1 1 +NADPH +NADH

x x

( (

s 100 s 100

t t

n n

u u

o 50 o 50

C C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min) W 1000 +SanR2 X 600 +SanR2

) ) 4 +MAC 4 500 +MAC 0 800 0

1 1 +NADPH 400 +NADH

x x

( 600 (

s s 300

t t

n 400 n

u u 200 o 200 o C C 100 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min)

Figure 13 (continued). Sanguinarine reductases reduce benzophenanthridine alkaloids.

62 Y 1200 +SanR3B Z 1500 +SanR3B

) ) 4 1000 +MAC 4 +MAC

0 0

1 1 800 +NADPH 1000 +NADH

x x

( (

s 600 s

t t

n n

u 400 u 500

o o

C 200 C 0 0 0 5 10 15 0 5 10 15 Retention Time (min) Retention Time (min)

Figure 13 (continued). Sanguinarine reductases reduce benzophenanthridine alkaloids.

Purified SanRs (1 μg) were incubated with benzophenanthridines (5 μM) and 1 mM

NAD(P)H co-factor for one hour at room temperature, quenched with methanol then analyzed by LC-MS. Extracted-ion chromatograms for benzophenanthridine substrates

(coloured) and dihydrobenzophenathridine products (black) are shown. SanRs reduce benzophenanthridines using NADPH (left column) or NADH (right column). SanR1

(A-B), SanR2 (C-D), and SanR3B (E-F) reduce sanguinarine to dihydrosanguinarine

(arrow). Without enzyme, sanguinarine (5 μM) incubated with NADPH (G) or NADH

(H) is not converted to dihydrosanguinarine. SanR1 (I-J), SanR2 (K-L), and SanR3B

(M-N) reduce chelerythrine to dihydrochelerythrine (arrow). SanR1 (O-P), SanR2 (Q-R), and SanR3B (S-T) reduce chelirubine to dihydrochelirubine (arrow). SanR1 (U-V),

SanR2 (W-X), and SanR3B (Y-Z) reduce macarpine to dihydromacarpine (arrow).

Approximate retention times are 7.0 min. for sanguinarine (SAN, [M]+ 332), 8.9 min. for dihydrosanguinarine ([M+H]+ 334), 7.6 min. for chelerythrine (CHE, [M]+ 348), 8.9 min. for dihydrochelerythrine ([M+H]+ 350), 8.0 min. for chelirubine (CHR, [M]+ 362), 8.9 min. for dihydrochelirubine ([M+H]+ 364), 8.3 min. for macarpine (MAC, [M]+ 392), and

9.0 min for dihydromacarpine ([M+H]+ 394).

63 20

d 18

)

1 16

e 14

o o 12

(

b d 10

y 8

c 6

n r 4

m 2

A 0 Sanguinarine Chelerythrine Chelirubine Macarpine Substrate

Figure 14. SanR2 reduces benzophenanthridine alkaloids using NADPH or NADH as a co-factor. SanR2 reduces sanguinarine, chelerythrine, chelirubine, and macarpine to their corresponding dihydrobenzophenanthridine products dihydrosanguinarine, dihydrochelerythrine, dihydrochelirubine, and dihydromacarpine, respectively, using

NADPH (black) or NADH (gray) as a co-factor. More product is formed when NADPH, as compared to NADH, is used a co-factor. Errors bars indicate SD for three technical replicates.

64 7

e 6

)

6 a 5

( 4

s n 3

y C 2

D 1 0 0 10 20 30 40 50 60 70 Temperature (°C)

1.0

)

a 0.8

n 0.6

f d 0.4

o 0.2

(

0.0 0 5 10 15 20 25 30 Temperature (°C)

Figure 15. Temperature curve for SanR2. The temperature optimum of SanR2 is approximately 18°C. Purified SanR2 (1 μg) was incubated with 5 μM sanguinarine and 1 mM NADPH for one hour at 7, 16, 21, 28.5, 37, 42, 55, and 65°C. Assays were analyzed by LC-MS for amount of dihydrosanguinarine produced. Counts of dihydrosanguinarine

(top) were converted to concentration (bottom) using s standard curve (y=6E+06x +

1E+06). Dihydrosanguinarine counts for assays incubated at 37, 42, 55, and 65°C were below the linear range, and were omitted from the bottom graph. Error bars indicate SD for three technical replicates.

65 3.2.5 Michaelis-Menten kinetic analysis

Michaelis–Menten model was used to analyze enzyme kinetics of SanR1 and

SanR3B with sanguinarine as the substrate (Fig. 16). Using LC-MS count values for dihydrosanguinarine, the Michaelis constant, Km, for SanR1 or SanR3B was calculated as

1.6 and 19.9 μM sanguinarine, respectively (Fig. 16A,B). The concentration of dihydrosanguinarine was determined using a standard curve (Fig. 11). However, the majority of count values for dihydrosanguinarine produced by SanR3B were below linear range. Omitting data points outside linear range for SanR1, resulted in a Michaelis constant, Km, of 7.3 μM sanguinarine (Fig. 16C). Similarly, the Vmax for SanR1 was calculated as 4.450 x 10-6 counts hour-1 μg-1 or 0.6955 μmol hour-1 μg-1 using dihydrosanguinarine counts or concentration, respectively (Fig. 16A,C). SanR3B Vmax,

5.831 x 10-6 counts hour-1 μg-1, could only be calculated based using counts (Fig. 16B).

3.3 Immunolocalization of sanguinarine reductases

3.3.1 Antibody production & dot blots

Antisera from test bleeds collected from mice at the time of booster injection with purified recombinant SanRs showed little to no antigen specificity (Fig. 17). Therefore, antisera were subjected to antibody scrubbing (Fig. 18). For example, a 1:10,000 dilution of SanR3B antiserum was non-specific and could detect all SanRs. SanR3B antiserum was incubated with high concentrations of recombinant SanR1 and SanR2 on nitrocellulose membrane to remove non-specific antibodies. As a result, SanR3B antiserum specificity was increased towards recombinant SanR3B. Overall, antibody scrubbing with excess recombinant enzyme was success in increasing antigen specificity of SanR1, SanR2, and SanR3B antisera (Fig. 18).

66 Michaelis-Menten

)

- 6e+6

)

A -

o 5e+6

C 4e+6

(

u a 3e+6

S 2e+6

( 1e+6

R 0 020 40 60 80 100 120 Michaelis-Menten SanguinarSaingeui nCarionne Ccoenncetrntratiatioonn (μ M(µM) )

6e+6

) Vmax = 4.450e+6

1 B - Km = 1.6

µ 5e+6

h 4e+6

a 3e+6

S 2e+6

(

1e+6

R 0 020 40Michaelis-6Me0 nten 80 100 120 SanguinarSaninguein aCrinone Ccoencnentrtraatitionon (μM ()µM) 0.8 Vmax = 5.831e+6 C Km = 19.9

)

- 0.6

a 0.4

( 0.2

0.0 020 40 60 80 100 120 SanguinarSaingeu inCaronine cCeonctrentatiratioonn (μ M(µM) ) Vmax = 0.6955 Figure 16. Michaelis Km =- Menten7.3 enzyme kinetics for SanR1 and SanR3B. LC-MS counts of dihydrosanguinarine produced from sanguinarine by (A) SanR1 and (B) SanR3B were used to calculate Km and Vmax values. (C) Dihydrosanguinarine counts for SanR1 were converted to concentration using a standard curve. Graphs were made in SigmaPlot 12.0.

67 A SanR1 1 2 3 1 2 3 SanR2 + 1 2 3 SanR3B

B Antiserum SanR1 SanR2 SanR3B

T e

F b

Figure 17. Generation of antibodies against sanguinarine reductases. (A) Legend outlining pattern used to spot 1 μL recombinant SanRs on nitrocellulose membrane

(1: 100 ng, 2: 10 ng, 3: 1 ng), and positive (+) control (mouse antiserum). (B) Test bleeds were obtained 3 weeks after an injection, and the final bleed was obtained by exsanguination. SanRs were detected using antisera (1:10,000) secondarily probed with a

HRP-conjugated goat anti-mouse antibody. Blots were visualized using an ECL system and X-ray film. Only representative dot blots are shown to demonstrate immunogenicity of mouse antisera.

A B Before After SanR SanR SanR M 1 2 3B M 1 2 3B M 1 2 3B

34 46

a i

n s 30

m t 27

C A 23

n 30 s 30

n 23 A 23

n 30

S A 23

Figure 18. Specificity of antibodies generated against recombinant sanguinarine reductases. (A) Coomassie stain showing equal loading of 200 ng of recombinant SanR proteins (top). All recombinant 6xHis-tagged SanRs are recognized by the anti-His antibody (bottom). (B) Antibodies generated against recombinant SanRs show cross- reactivity towards other SanRs (left), and specificity is increased after scrubbing (right).

Anti-His antibody was diluted to 0.2 μg/mL, and SanR antisera were diluted 1:10,000 to detect recombinant SanR proteins. Blots were secondarily probed with a horseradish peroxidases-conjugated goat anti-mouse antibody (1:10,000), then visualized using an enhanced chemiluminescence system and X-ray film. . M: marker indicating approximate molecular weight (kDa).

3.3.2 Sanguinarine reductase expression in planta

Antibodies generated against recombinant SanR3B (see section 3.3.1) were used to detect SanRs in plant protein extracts. SanRs are present in stem, root, leaf, capsule, and latex of Papaver somniferum cultivars (Fig. 19). SanRs present in root tissue of opium poppy appear to be larger in size than those found in other tissues (Fig. 19). The molecular weight of SanRs in root tissue is approximately 30 kDa (Fig. 19), whereas the molecular weight of SanRs present in stem, leaf, and capsule/bud is a less than 30 kDa

(Fig. 19). The size of SanR in opium poppy latex is smaller than those found in other tissues, and is between 23 and 30 kDa, and were only detected in the latex of cultivars

Veronica, Marianne, and Bea’s Choice (Fig. 19E). Soluble proteins were only extracted from aerial organs (stem and leaf combined), and root tissue of Eschscholzia californica.

Opium poppy SanR antibodies only detected protein in the aerial organs, not the root tissue, and the molecular weight of SanRs in E. californica aerial organs is greater than

30 kDa (Fig. 19A,B).

3.3.3 Epifluorescence microscopy

Antibodies raised against recombinant SanR3B were used to detect sanguinarine reductases in planta, and localization was visualized using epifluorescence microscopy.

Opium poppy SanRs appear to be localized to phloem in stem, root, and capsule (Fig.

20). No reliable signal was obtained for leaf tissue (data not shown). SanRs were also detected in seeds within in the capsule (Fig. 20D).

Cultivar Cultivar

Ma V R M 40 T L P B Eca Ma V R M 40 T L P B Eca A

34 46 27 30

B 46 30 34 27 23

C 46 34 30 27 23

D 46 34 27 30 23

E 46 34 30 27 23

Figure 19. Sanguinarine reductases are present in all opium poppy tissues.

Figure 19 (continued). Sanguinarine reductases are present in all opium poppy tissues.

Soluble proteins were extracted from different tissues of various cultivars of Papaver somniferum (Veronica (V), Roxanne (R), Marianne (M), 40, T, L, Przemko (P), and

Bea’s Choice) and Eschscholzia californica (A: root, B: stem, C: leaf, D: capsule,

E: latex). Approximately 5 μg of protein was size-separated on 12% SDS-PAGE then stained with Coomassie to visualize total proteins (left) or transferred to nitrocellulose membrane for Western blot analysis (right). SanRs were detected using SanR3B antisera diluted 1:100. Blots were probed with a HRP-conjugated goat anti-mouse secondary antibody (1:5,000), then visualized using a ECL system and X-ray film. Ma: marker indicating approximate molecular weight (kDa).

73 A B C D

E F G H

Figure 20. Sanguinarine reductases localized to the phloem. SanRs are present in the stem (A), root (B), and capsule (C), and seeds

(D). (A-D) Xylem was visualized with UV light, and is false-coloured blue. SanRs were detected with a polyclonal antibody generated

against recombinant SanRs, and an Alexa Fluor 488-labeled secondary antibody. Signal was visualized using the L5 filter on a Leica

DM RXA2 microscope, and are false-coloured yellow. Corresponding light microscope images are shown below (E-H).

3.4 Virus-induced gene silencing of sanguinarine reductases

3.4.1 Quantitative PCR primer and probe specificity towards SanRs

To study sanguinarine reductase (SanR) expression in planta, primers and

TaqMan MGB probes were designed to specifically amplify an individual gene (see section 2.6.1; Table 2). Primer and probe set specificity was tested using purified plasmid

(pQE30) containing SanR1, SanR2, or SanR3 as a template for qPCR (Table 4). Plasmid template was diluted approximately to 1 μg, 1 ng, 0.1 ng, and 0.01 ng. As template concentration decreased, the CT value increased for a given primer and probe set. With sufficient template DNA, primer and probe sets specifically amplified the SanR gene they were designed against (Table 4). For example, amplification using the SanR3 primer and probe set resulted in average CT values of 26.6, 28.6, and 16.3 when using 1 ng of pQE30-SanR1, pQE30-SanR2, and pQE30-SanR3 as a template, respectively.

3.4.2 Sanguinarine reductase expression in planta

To determine relative expression of SanRs, expression level was compared to ubiquitin as an endogenous control. A primer and probe set was designed against ubiquitin (Table 2), and stem and root cDNA was used as a template. The CT values obtained using stem and root were 15.4 and 15.7, 16.4 and 17.0, and 17.4 and 18.1 for templates diluted to 1, 0.5, and 0.25 μg, respectively. Therefore, the average CT values obtained from qPCR reactions were consistent between stem and root tissues.

To determine SanR expression within four different tissues, stem, root, leaf, and flower bud/capsule cDNA was diluted to 1, 0.5, and 0.1 μg. Relative gene expression was determined using the comparative ΔΔCT method (Fig. 21). SanR1 and SanR2 are more highly expressed in root tissue, as compared to stem, leaf, or flower bud/capsule.

75 Table 4. Quantitative PCR primer and probe specificity towards SanR genes.

SanR1 primer & probe set SanR2 primer & probe set SanR3 primer & probe set

Template Template Average Ct Template Average Ct Template Average Ct Concentration

SanR1 12.9 SanR1 No signal SanR1 23.3

1 μg SanR2 21.3 SanR2 14.1 SanR2 23.7

SanR3 22.9 SanR3 23.1 SanR3 13.5

SanR1 17.5 SanR1 26.7 SanR1 26.6

1 ng SanR2 27.2 SanR2 18.8 SanR2 28.6

SanR3 26.3 SanR3 26.7 SanR3 16.3

SanR1 21.1 SanR1 26.5 SanR1 30.1

0.1 ng SanR2 23.0 SanR2 22.4 SanR2 30.4

SanR3 23.0 SanR3 26.7 SanR3 20.2

SanR1 22.9 SanR1 26.6 SanR1 30.4

0.01 ng SanR2 23.0 SanR2 26.4 SanR2 30.6

SanR3 22.8 SanR3 27.1 SanR3 23.6

A 0.025 B 1.200

l l

e e

v v

e e 1.000

L 0.020 L

n n

o o

i i 0.800

s s

s 0.015 s

e e r SanR1 r 0.600 SanR1

p p

x x

E 0.010 E SanR2 SanR2 e e 0.400

v v

i i

t t

a 0.005 SanR3 a SanR3 l l 0.200

e e

R R 0.000 0.000 Stem Root Leaf Bud Stem Root Leaf Bud Opium Poppy Tissue Opium Poppy Tissue

Figure 21. Relative gene expression of opium poppy sanguinarine reductases in different tissues. Total RNA was extracted from

stem, root, leaf, and flower bud/capsule, and was used as a template for cDNA synthesis. Gene expression was analyzed by TaqMan

qPCR using MGB probes. Relative gene expression was calculated using ubiquitin as an endogenous control (A: ΔCT plotted) and the

comparative ΔΔCT method (B: gene expression to the highest expressed gene). Error bars indicate SEM for three technical replicates.

Conversely, SanR3 is more highly expressed in leaf, as compared to stem tissue, and is expressed at low levels or not at all in root and flower bud/capsule. Alternatively, it appears that only SanR1 and SanR3 are expressed in the stem; all SanRs are expressed in root tissue, but SanR1 is expressed approximately 4 times more than SanR2 or SanR3; only SanR1 and SanR3 are expressed in leaf tissue; and only SanR1 is expressed in the flower bud/capsule (Fig. 21).

3.4.3 Knocking down expression in planta using VIGS

VIGS constructs were designed complementary to the 5’- or 3’-untranslated regions (UTR) or coding sequences of SanRs in order to silence one or multiple genes in planta (Fig. 4). Approximately 200 poppies were infiltrated with VIGS constructs. RNA was isolated from transformed plants, reverse-transcribed into cDNA, and analyzed for presence of coat protein (CP) by PCR. Root gene expression and metabolite composition were further analyzed for approximately 130 plants CP positive plants (Fig. 22). As a control, poppies were infiltrated with empty pTRV2 vector (EV) to establish baseline

SanR expression and root metabolite profiles. All SanRs are expressed in root tissue with

SanR1 expression levels approximately four times greater than that of SanR2 or SanR3

(Fig. 23). Metabolite identities of root alkaloids were determined by extracting the appropriate ion mass and comparing retention times to authentic standards (Fig. 24).

Alkaloids extracted from root samples (20 mL methanol per gram dry weight) were diluted in methanol (1/20) for LC-MS analysis. Levels of sanguinarine and dihydrosanguinarine from root samples were in linear range according to the standards curves (Fig. 11).

79 EV V6 V2 L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 L 6 7 8 9 10 11 12 13 14 15 16 1 2 3 4 300 bp 300 bp EV V1 V8 L 16 17 1 2 3 4 5 6 7 8 9 10 11 12 13 L 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 300 bp 300 bp V1 V8 V5 V4 L 14 15 16 17 18 19 20 21 22 L 16 17 1 2 3 4 5 6 8 9 10 1 2 3 4 300 bp 300 bp V2 V4 V10 L 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 L 5 6 7 8 1 2 3 4 6 7 8 9 10 11 12 300 bp 300 bp V3 V10 V11 V12 L 1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 L 13 14 1 2 3 4 5 6 7 8 9 10 11 1 2 300 bp 300 bp V7 V12 V9 L 1 2 3 5 6 7 8 9 10 11 12 13 14 15 16 L 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 300 bp 300 bp V7 V6 V9 V3 L 17 18 19 20 21 22 23 24 25 26 1 2 3 4 5 L 6 7 8 9 10 11 12 L 17 18 19 20 300 bp 300 bp 300 bp

Figure 22. Presence of coat protein RNA in plants transformed with VIGS constructs.

Figure 22 (continued). Presence of coat protein RNA in plants transformed with VIGS constructs. RNA was extracted from root

tissue of plants transformed with pTRV2-VIGS constructs. RNA was reverse transcribed into cDNA and was amplified with primers

designed to amplify coat protein (CP), encoded by the pTRV2 vector. PCR amplicons were size-separated on a 2% agarose gel. DNA

was stained with ethidium bromide and visualized with UV light. Expected CP amplicon size is 305 bp. L: 100 bp ladder; EV: empty

vector; V1 through V12: pTRV2-SanR VIGS constructs #1-12 designed to silence one or multiple SanR genes.

A 0.035 B 0.014

i i 0.03 0.012

r r 0.025 0.01

x x 0.02 0.008

2 1 0.015 0.006

a a 0.01 0.004

e e 0.005 0.002

0 0

3 2 1 0 9 8 7 6 5 3 2 1

1 1 1

V V V V V V V V

V V V

E E E E E E E E

E E E

E E E VIGS Construct VIGS Construct

C 0.02 D 0.02

e e 0.015

r r 0.015

0.01

e 3 0.01

a 0.005

e 0.005

3 2 1 0 9 8 7 6 5 3 2 1

1 1 1 0

V V V V V V V V

V V V

E E E E E E E E

E E E SanR1 SanR2 SanR3 VIGS Construct Gene

Figure 23. Root gene expression profiles of empty vector control opium poppy plants.

Figure 23 (continued). Root gene expression profiles of empty vector control opium poppy plants. Expression of SanR1, SanR2, and

SanR3 in the roots of poppies transformed with an empty pTRV2 vector (EV). Expression levels of (A) SanR1, (B) SanR2, and (C)

SanR3 in the roots of 13 different EV control plants. Error bars represent the SEM of three technical replicates. (D) Average root

expression levels SanR1, SanR2, and SanR3 in control (EV) plants. Error bars represent SEM (n=13).

A B

150 60

6 6

0 0

1 1

x x 100 40

s s

t t

n n

u u

o 50 o 20

C C

S 0 S 0

M M - 0 1 2 3 4 5 6 7 8 9 10 - 0 1 2 3 4 5 6 7 8 9 10

C C L Retention Time (min) L Retention Time (min)

C D

100 20

6 6

0 0

1 1

x x 15

s s

t t

n 50 n 10

u u

o o

C C 5

S 0 S 0

M M - 1 2 3 4 5 6 7 8 9 10 - 0 1 2 3 4 5 6 7 8 9 10

C C L Retention Time (min) L Retention Time (min)

x 40

o 20

S 0

M - 0 1 2 3 4 5 6 7 8 9 10

C L Retention Time (min)

Figure 24. Retention times of benzylisoquinoline alkaloid authentic standards. Extracted ion chromatographs showing retention times (RT) of (A) noscapine ([M+H]+ 414,

RT=8.0 min), (B) papaverine ([M+H]+ 340, RT=6.9 min), (C) reticuline ([M+H]+ 330,

RT=3.9 min), (D) thebaine ([M+H]+ 312, RT=4.9 min), and (E) cryptopine ([M+H]+

370, RT=5.2). Method time is 19.1 minutes, but only results for time 0-10 minutes are shown for clarity.

Levels of the alkaloids noscapine, papaverine, reticuline, thebaine, morphine, and cryptopine were determined to be in linear range using the standard curves previously generated by Guillaume Beaudoin (data not shown). Examples of a total ion chromatograph (TIC) and extracted ion chromatographs (EICs) used to analyze root metabolite data are shown for one plant transformed with an empty pTRV2 vector (EV1;

Fig. 25).

Two constructs that were designed to specifically silence SanR1 were complementary to the 5’ UTR (V1) and 3’ UTR (V2) (Fig. 26). Both constructs resulted in a significant reduction in SanR1 expression compared to the EV control according to

Student’s t-test (p<0.05; V1, p=0.01; V2, p=0.0004) (Fig. 26B,F). However, no significant changes in metabolite composition were observed (Fig. 26C,G).

Three constructs that were designed to silence SanR2, and they were complementary to the 5’ UTR (V3), coding sequence (V4) and 3’ UTR (V5) (Fig. 27).

All constructs resulted in a significant reduction in SanR2 expression compared to the EV control according to Student’s t-test (p<0.05; V3, p=0.0000001; V4; p=0.004; V5, p=0.00003) (Fig. 27B,F,J). The V4 construct resulted in an increase in sanguinarine, but using Student’s t-test with Welch’s correction resulted in p>0.05. Therefore, the increase in sanguinarine is not significant. However, the V5 construct did result in a significant increase in morphine levels (p=0.005).

Two constructs that were designed to specifically silence SanR3 were complementary to the 5’ UTR (V6) and 3’ UTR (V7) (Fig. 28). Both constructs resulted in a significant reduction in SanR3 expression compared to the EV control according to

Student’s t-test (p<0.05; V6 and V7, p=0.00005) (Fig. 28B,F).

A 60

6 50

t 40

u o 30

M 20

L 10

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Retention Time (min)

B 5 4.5

0 4

1 EIC312

3.5

s t EIC330

n 3

u o 2.5 EIC332

S 2 EIC334

M - 1.5 C EIC340 L 1 0.5 EIC370 0 EIC414 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Retention Time (min)

Figure 25. Example chromatographs for VIGS metabolite analysis. Root alkaloids from a plant transformed with an empty pTRV2 vector (EV1) were extracted in methanol (20 mL methanol per gram dry weight), then diluted 1 in 20 for LC-MS analysis. (A) Total ion chromatograph. (B) Extracted ion chromatographs for thebaine ([M+H]+ 312), reticuline ([M+H]+ 330), sanguinarine ([M]+ 332), dihydrosanguinarine ([M+H]+ 334), papaverine ([M+H]+ 340), cryptopine ([M+H]+ 370), and noscapine ([M+H]+ 414).

86 Both constructs resulted in a significant increase in sanguinarine (V6 and V7, p=0.02).

V6 plants also exhibited a significant increase in cryptopine (p=0.04), and a decrease in noscapine (p=0.04). In addition, V7 plants showed a significant increase reticuline

(p=0.009). In addition, V7 plants showed a significant increase reticuline (p=0.009).

One construct was designed to silence SanR1 and SanR3 (V8) (Fig. 29). The construct was designed by combining V2 and V5, which are complementary to the

3’ UTRs of SanR1 and SanR3, respectively. Interestingly, only SanR2 and SanR3 expression levels are significantly decreased relative to the EV control according to

Student’s t-test (p-values are 0.0005 and 0.004, respectively; Fig. 29D). SanR1 expression levels do not differ from the EV control (p=0.8). A significant decrease in noscapine (p=0.01) and papaverine (p=0.04) levels was observed (Fig. 29E).

Guillaume Beaudoin designed constructs V9 through V12 (Fig. 30-33). V9 was

100% complementary to a small region of Bea’s choice SanR1 coding sequence, but constructs V10-V12 were not entirely identical to regions of Bea’s choice SanRs. V10 shares 75 and 34% sequence similarity to SanR1 and SanR2, respectively; V11 shares 21 and 86%, sequence similarity to SanR1 and SanR2, respectively; and V12 shares 20% and 16% sequence similarity to SanR1 and SanR2, respectively. Although, construct V9 was designed to silence SanR1, SanR1 expression levels were significantly increased compared to the EV control (p=0.0001 (Fig. 30). As well, SanR2 expression levels were significantly decreased (p=0.000007) (Fig. 30D). Increasing expression levels of SanR1, and decreasing expression levels of SanR2 resulted in a significant increase in the levels of reticuline (p=0.00009), thebaine (p=0.01), and sanguinarine (p=0.008) compared to the

EV control (Fig. 20E). Both V10 and V11 constructs resulted in a significant reduction in

SanR1, SanR2, and SanR3 expression levels (Fig. 31 and 32). However, no significant changes in metabolite levels were observed in plants transformed with V10 or V11 constructs (Fig. 31 and 32). Lastly, V12 resulted in a significant reduction in SanR2

(p=0.00004) and SanR3 (p=0.009) expression levels compared to the control (Fig. 33).

However, Student’s t-test with Welch’s correction indicated no significant changes in metabolite levels between plants transformed with V12 constructs or an EV (Fig. 33).

A 0.03 B 0.02

s 0.025 s

r r 0.015

p 0.02 p *

0.015

e 1 0.01

R e 0.01

e 0.005

5 4 3 2 1 0 9 8 7 6 5 4 3 2 1

------

1 1 1 1 1

- - - - - 1 1 1 1 1 1 1 1 1 0

1 1 1 1 1

V V V V V V V V V

V V V V V EV V1 VIGS Construct VIGS Construct

C 50 D 40

)

, 35

a 40 M

( 30

(

d o 25

M i 30 i

, o

a s 20

k EV EV

n d 20 15

y V1 n V1 o 10

t 10

t 5

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 26. Gene expression and metabolite profiles of SanR1-silenced opium poppy roots.

E 0.025 F 0.02

s s 0.02

r r 0.015

x x 0.015

1 1 0.01 0.01

S S 0.005

e e 0.005

3 2 1 0 9 8 7 6 5 4 3 2 1

------

1 1 1

- - - 2 2 2 2 2 2 2 2 2 0

2 2 2

V V V V V V V V V

V V V EV V2 VIGS Construct VIGS Construct

G 60 H 100

)

a 50 M 80

(

M i i 60

, o

a s 30

k EV EV

d 40

U A 20

y V2 n V2

t 20

t 10

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 26 (continued). Gene expression and metabolite profiles of SanR1-silenced opium poppy roots.

Figure 26 (continued). Gene expression and metabolite profiles of SanR1-silenced opium poppy roots. Relative SanR1 gene

expression in the roots of poppy plants transformed with a VIGS construct designed against the (A) 5’ UTR (V1, n=15) or (E) 3’ UTR

(V2, n=10) of SanR1. Error bars represent the SEM of three technical replicates. Average relative SanR1 expression in EV control

(n=13) versus (B) V1 (p=0.01) or (F) V2 (p=0.0004). Error bars represent SEM. Root alkaloid metabolite profile of EV control versus

(C) V1 or (G) V2. (D, H) LC-MS counts of sanguinarine and dihydrosanguinarine were converted to concentration (μM) using a

standard curve. Significance was calculated using Student’s two-tailed t-test, and asterisk (*) indicates p<0.05. N: noscapine,

P: papaverine, R: reticuline, T: thebaine, M: morphine, C: cryptopine, S: sanguinarine, DHS: dihydrosanguinarine.

A 0.0014 B 0.007

n n

o o i 0.0012 i s s 0.006

s s

e e r 0.001 r 0.005

p p

x 0.0008 x

E E

0.004

2 0.0006 2 R R 0.003 n 0.0004 n

a a

S S

0.002

e 0.0002 e

v v

i i

t t 0.001 *

a a

1 2 3 4 5 6 7 8 9 0 1 2 3 4

l l

------

e e

1 1 1 1

- 3 3 3 3 3 3 3 3 3 - - - - 0

R R

3 3 3 3

V V V V V V V V V

V V V V V EV V3 VIGS Construct VIGS Construct

C 80 D 25

)

, 70

a M 20

60 (

(

d 50 o

i M i 15

o ,

a s 40

k EV EV

n d 30 10

y V3 n V3 o 20

t 5

t 10

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 27. Gene expression and metabolite profiles of SanR2-silenced opium poppy roots.

E 0.006 F 0.007

s s 0.006

s s 0.005

r r 0.005

p p 0.004

0.004

2 2 0.003

R R 0.003

a a 0.002 *

0.002

i i 0.001

t t 0.001

e e 0 0

R R V4-1 V4-2 V4-3 V4-4 V4-5 EV V4 VIGS Construct VIGS Construct

G 50 H 50

)

a 40 M 40

(

M i 30 i 30

, o

k EV EV

d 20 20

y V4 n V4

t 10 10

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 27 (continued). Gene expression and metabolite profiles of SanR2-silenced opium poppy roots.

I 0.0016 J 0.007

n n

o o i 0.0014 i s s 0.006

s s

e e r 0.0012 r 0.005

p p

x 0.001 x

E E

0.004

2 0.0008 2 R R 0.003 n 0.0006 n

a a

S S

0.002

e 0.0004 e

v v

i i t 0.0002 t 0.001 *

a a

l l e 0 e 0 R V5-1 V5-2 V5-3 V5-4 V5-5 V5-6 V5-7 V5-8 R EV V5 VIGS Construct VIGS Construct

K 50 L 30

)

a 25 40 M

(

i M 30 i

o ,

a s 15

k EV EV

d 20

U A 10

y V5 n V5

t 10

t 5

b *

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 27 (continued). Gene expression and metabolite profiles of SanR2-silenced opium poppy roots.

Figure 27 (continued). Gene expression and metabolite profiles of SanR2-silenced opium poppy roots. Relative SanR2 gene

expression in the roots of poppy plants transformed with a VIGS construct designed against the (A) 5’ UTR (V3, n=14), coding

sequence (E) (V4, n=5), or (I) 3’ UTR (V5, n=8) of SanR2. Error bars represent the SEM of three technical replicates. Average

relative SanR2 expression in EV control (n=13) versus (B) V3 (p=0.0000001), (F) V4 (p=0.004), or (J) V5 (p=0.00003). Error bars

represent SEM. Root alkaloid metabolite profile of EV control versus (C) V3, (G) V4, or (K) V5. Silencing SanR2 in V4 plants

resulted in an increase in sanguinarine (p=0.0005). However, due to unequal variance between EV and V4, Welch’s correction was

applied resulting in a p-value of 0.052. Therefore, the increase in sanguinarine is insignificant. Silencing SanR2 in V5 resulted in an

increase in morphine (p=0.005). (D, H, L) LC-MS counts of sanguinarine and dihydrosanguinarine were converted to concentration

(μM) using a standard curve. Significance was calculated using Student’s two-tailed t-test, and asterisk (*) indicates p<0.05.

N: noscapine, P: papaverine, R: reticuline, T: thebaine, M: morphine, C: cryptopine, S: sanguinarine, DHS: dihydrosanguinarine.

A 0.004 B 0.01

n n

o o

i 0.0035 i

s s

s s 0.008 e 0.003 e

r r

p 0.0025 p x x 0.006

E E

0.002

3 3

R 0.0015 R

n n 0.004

a 0.001 a

S S

e 0.0005 e

v v 0.002

i i

t t *

a a

1 2 3 4 5 6 7 8 9 0 1 2 3

l l

------

e e

1 1 1

- 6 6 6 6 6 6 6 6 6 - - - 0

R R

6 6 6

V V V V V V V V V

V V V V EV V6 VIGS Construct VIGS Construct

C 60 D 30

)

a 50 25

(

40 20

i M

a o , *

a s 30 15

k EV EV

n d *

U A 20 10

y V6 n V6

t 10 * 5

b n *

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 28. Gene expression and metabolite profiles of SanR3-silenced opium poppy roots.

E 0.0035 F 0.01

i i 0.003

s s 0.008

r r 0.0025

x x 0.002 0.006

3 3 0.0015

n n 0.001 0.004

e e 0.0005

v v 0.002

i i *

4 3 2 1 0 9 8 7 6 5 4 3 2 1

------

1 1 1 1

- - - - 7 7 7 7 7 7 7 7 7 0

7 7 7 7

V V V V V V V V V

V V V V EV V7 VIGS Construct VIGS Construct

G 80 H 20

)

, 70

60 ( 15

(

i *

d 50 o

M i

, o

a s 40 10

k EV EV

n d 30

y V7 n V7 o 20 5

* o

t 10

u *

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 28 (continued). Gene expression and metabolite profiles of SanR3-silenced opium poppy roots.

Figure 28 (continued). Gene expression and metabolite profiles of SanR3-silenced opium poppy roots. Relative SanR3 gene

expression in the roots of poppy plants transformed with a VIGS construct designed against the (A) 5’ UTR (V6, n=13), or

(E) 3’ UTR (V7, n=14) of SanR3. Error bars represent the SEM of three technical replicates. Average relative SanR3 expression in EV

control (n=13) versus (B) V6 (p=0.00005), or (F) V7 (p=0.00005). Error bars represent SEM. Root alkaloid metabolite profile of EV

control versus (C) V6, or (G) V7. Silencing SanR3 in V6 plants resulted in an increase in sanguinarine (p=0.02) and cryptopine

(p=0.04), and a decrease in noscapine (p=0.04). Silencing SanR3 in V7 plants resulted in an increase in sanguinarine (p=0.02) and

reticuline (p=0.009). (D, H) LC-MS counts of sanguinarine and dihydrosanguinarine were converted to concentration (μM) using a

standard curve. Significance was calculated using Student’s two-tailed t-test, and asterisk (*) indicates p<0.05. N: noscapine,

P: papaverine, R: reticuline, T: thebaine, M: morphine, C: cryptopine, S: sanguinarine, DHS: dihydrosanguinarine.

A 0.05 B 0.006

i i 0.005

s s 0.04

r r 0.004

x x 0.03 0.003

2 1 0.02 0.002

n n 0.001

S S 0.01

e e 0

i i

t t 0 -1 -2 -3 -4 -5 -6 -7 -8 -9 10 11

a a 8 8 8 8 8 8 8 8 8 - -

l l 8 8

e e 1 2 3 4 5 6 7 8 9 0 1 V V V V V V V V V V ------1 -1 V

R R 8 8 8 8 8 8 8 8 8 - V V V V V V V V 8 8 V V V VIGS Construct VIGS Construct

C 0.012 D 0.025

0.01 s

s s 0.02

r r 0.008

x x 0.006

E 0.015

e 3 0.004 SanR1

n 0.01

0.002 G

SanR2

e 0

t v 0.005

i SanR3

a t 1 2 3 4 5 6 7 8 9 0 1 ------1 1 l * *

a 8 8 8 8 8 8 8 8 8 - - e l 8 8

e V V V V V V V V V V V R 0

R VIGS Construct EV V8 VIGS Construct

Figure 29. Gene expression and metabolite profiles of SanR1- and SanR3-silenced opium poppy roots.

E 50 F 20

)

a 40 M

( 15

(

i M 30 i

o ,

a s 10

* t

k EV EV

d 20

U *

y V8 n V8 o 5

t 10

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 29 (continued). Gene expression and metabolite profiles of SanR1- and SanR3-silenced opium poppy roots. Relative

expression of (A) SanR1, (B), SanR2, and (C) SanR3 in the roots of poppy plants transformed with a VIGS construct designed against

the 3’ UTRs of SanR1 and SanR3 (V8, n=11). Error bars represent the SEM of three technical replicates. (D) Average relative SanR

expression in EV control (n=13) versus V8. SanR2 (p=0.0005) and SanR3 (0.004), but not SanR1 (p=0.8), were silenced as compared

to the control. Error bars represent SEM. (E) Root alkaloid metabolite profile of EV control versus V8. Both noscapine (p=0.01) and

papaverine (p=0.04) levels were decreased compared to the control. (F) LC-MS counts of sanguinarine and dihydrosanguinarine were

converted to concentration (μM) using a standard curve. Significance was calculated using Student’s two-tailed t-test, and asterisk (*)

indicates p<0.05. N: noscapine, P: papaverine, R: reticuline, T: thebaine, M: morphine, C: cryptopine, S: sanguinarine,

DHS: dihydrosanguinarine.

100

A 0.14 B 0.0014

s s 0.12 0.0012

r r 0.1 0.001

0.08 0.0008

R R 0.06 0.0006

0.04 0.0004

t t 0.02 0.0002

e e 0 0

R R V9-1 V9-2 V9-3 V9-4 V9-5 V9-6 V9-7 V9-8 V9-9 V9-1 V9-2 V9-3 V9-4 V9-5 V9-6 V9-7 V9-8 V9-9 VIGS Construct VIGS Construct

C 0.025 D 0.08

n n *

s 0.07

s s 0.02

r r 0.06

x x 0.05

0.015 E

e 3 0.04 SanR1

n 0.01

G 0.03

SanR2

e 0.02

t v 0.005

i SanR3

l 0.01

e 0 R 0 * R V9-1 V9-2 V9-3 V9-4 V9-5 V9-6 V9-7 V9-8 V9-9 EV V9 VIGS Construct VIGS Construct

Figure 30. Gene expression and metabolite profiles of SanR1-silenced opium poppy roots.

101

E 100 F 50

)

a 80 M 40

(

l *

(

k *

i M 60 i 30

o ,

k EV EV

d 40 20

y V9 n V9

* o

t 20 10

g a *

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 30 (continued). Gene expression and metabolite profiles of SanR1-silenced opium poppy roots. Relative expression of

(A) SanR1, (B), SanR2, and (C) SanR3 in the roots of poppy plants transformed with a VIGS construct designed against the coding

sequence of SanR1 (V9, n=9). Error bars represent the SEM of three technical replicates. (D) Average relative SanR expression in EV

control (n=13) versus V9. SanR1 expression was increased (p=0.00001), and SanR2 expression was decreased (p=0.000007)

compared to the control. No change in expression was observed for SanR3 (p=0.7). Error bars represent SEM. (E) Root alkaloid

metabolite profile of EV control versus V9. Reticuline (p=0.00009), thebaine (p=0.01), and sanguinarine (p=0.008) levels were all

increased compared to EV. (F) LC-MS counts of sanguinarine and dihydrosanguinarine were converted to concentration (μM) using a

standard curve. Significance was calculated using Student’s two-tailed t-test, and asterisk (*) indicates p<0.05. N: noscapine,

P: papaverine, R: reticuline, T: thebaine, M: morphine, C: cryptopine, S: sanguinarine, DHS: dihydrosanguinarine.

102

A 0.012 B 0.0035

n n

o o

i i 0.003

s 0.01 s

s s

e e 0.0025 r 0.008 r

p p

x x 0.002

E 0.006 E

1 2 0.0015

R 0.004 R

n n 0.001

a a

S 0.002 S

0.0005

e e

v v

i 0 i 0

t t

a a l 1 2 3 4 5 6 7 8 9 0 l 1 2 3 4 5 6 7 8 9 0

e ------e ------0 0 0 0- 0 0 0 0 0 -1 0 0 0 0- 0 0 0 0 0 -1

R 1 1 1 1 1 1 1 1 1 0 R 1 1 1 1 1 1 1 1 1 0 V V V V V V V V 1 V V V V V V V V 1 V V V V VIGS Construct VIGS Construct

C 0.0012 D 0.02

s s 0.001

r r 0.0008 0.015

E E 0.0006

e 3 0.01 SanR1

0.0004 e

SanR2

0.0002 e

e 0.005

i *

i 0 SanR3

e * l 1 2 3 4 5 6 7 8 9 0

e ------* 0 0 0 0 0 0 0 0 0 -1 R 0

R 1 1 1 1 1 1 1 1 1 0 V V V V V V V V 1 V V EV V10 VIGS Construct VIGS Construct

Figure 31. Gene expression and metabolite profiles of SanR-silenced opium poppy roots.

103

E 50 F 25

)

a 40 M 20

(

i M 30 i 15

o ,

k EV EV

d 20 10

y V10 n V10

t 10 5

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 31 (continued). Gene expression and metabolite profiles of SanR-silenced opium poppy roots. Relative expression of (A)

SanR1, (B), SanR2, and (C) SanR3 in the roots of poppy plants transformed with a VIGS construct designed against SanR1 (V10,

n=10). The Error bars represent the SEM of three technical replicates. (D) Average relative SanR expression in EV control (n=13)

versus V10. SanR1 (p=0.0000003), SanR2 (p=0.00004), and SanR3 (p=0.0001) expression levels were decreased compared to the

control. Error bars represent SEM. (E) Root alkaloid metabolite profile of EV control versus V10. (F) LC-MS counts of sanguinarine

and dihydrosanguinarine were converted to concentration (μM) using a standard curve. Significance was calculated using Student’s

two-tailed t-test, and asterisk (*) indicates p<0.05. N: noscapine, P: papaverine, R: reticuline, T: thebaine, M: morphine,

C: cryptopine, S: sanguinarine, DHS: dihydrosanguinarine.

104

A 0.025 B 0.003

n n

o o

i i

s s 0.0025

s 0.02 s

e e

r r 0.002 p 0.015 p

x x

E E 0.0015

1 0.01 2

R R 0.001

n n

a 0.005 a

S S 0.0005

e e

v v

i 0 i 0

t t

a a l 1 2 3 4 5 6 7 8 9 0 1 l 1 2 3 4 5 6 7 8 9 0 1

e ------e ------1 1 1 1 1 1 1 1 1 -1 -1 1 1 1 1 1 1 1 1 1 -1 -1

R 1 1 1 1 1 1 1 1 1 1 R 1 1 1 1 1 1 1 1 1 1 11 1 11 1 V V V V V V V V V V V V V V V V V V V V V V VIGS Construct VIGS Construct

C 0.003 D 0.02

s s 0.0025

r r 0.002 0.015

x x *

E E 0.0015

e 3 0.01 SanR1

0.001 e

SanR2

0.0005 e

e 0.005

i 0 SanR3

e * l 1 2 3 4 5 6 7 8 9 0 1 *

e ------1 1 1 1 1 1 1 1 1 -1 -1 R 0

R 1 1 1 1 1 1 1 1 1 1 11 1 V V V V V V V V V V V EV V11 VIGS Construct VIGS Construct

Figure 32. Gene expression and metabolite profiles of SanR-silenced opium poppy roots.

105

E 50 F 30

)

a 25 40 M

(

i M 30 i

o ,

a s 15

k EV EV

d 20

U A 10

y V11 n V11

t 10

t 5

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite

Figure 32 (continued). Gene expression and metabolite profiles of SanR2-silenced opium poppy roots. Relative expression of

(A) SanR1, (B), SanR2, and (C) SanR3 in the roots of poppy plants transformed with a VIGS construct designed against the coding

sequence of SanR2 (V11, n=11). Error bars represent the SEM of three technical replicates. (D) Average relative SanR expression in

EV control (n=13) versus V11. SanR1 (p=0.002), SanR2 (p=0.00002), and SanR3 (p=0.00007) expressed decreased relative to the

control. Error bars represent SEM. (E) Root alkaloid metabolite profile of EV control versus V11. (F) LC-MS counts of sanguinarine

and dihydrosanguinarine were converted to concentration (μM) using a standard curve. Significance was calculated using Student’s

two-tailed t-test, and asterisk (*) indicates p<0.05. N: noscapine, P: papaverine, R: reticuline, T: thebaine, M: morphine,

C: cryptopine, S: sanguinarine, DHS: dihydrosanguinarine.

106

A 0.06 B 0.0025

n n

o o

i i

s s

s 0.05 s 0.002

e e

r r

p 0.04 p

x x 0.0015

E E

1 0.03 2 0.001

R R

n n

a 0.02 a

S S 0.0005

e e

v v

i 0.01 i t t 0

a a

l l

e e 1 2 3 4 5 6 7 8 0 2- 2- 2- - 2- 2- 2- 2- R R 2 1 1 1 1 1 1 1 1 V12-1 V12-2 V12-3 V12-4 V12-5 V12-6 V12-7 V12-8 V V V V V V V V VIGS Construct VIGS Construct

C 0.008 D 0.03

0.007 s

s 0.025

r r 0.006

p p 0.02

x x 0.005

e 3 0.004 0.015 SanR1

0.003 G

a 0.01 SanR2

e 0.002

i 0.005 SanR3

0.001 l

a *

e l *

e 0 R 0 R V12-1V 12-2V 12-3V 12-4V 12-5V 12-6V 12-7V 12-8 EV V12 VIGS Construct VIGS Construct

Figure 33. Gene expression and metabolite profiles of SanR-silenced opium poppy roots.

107

E 70 F 80

)

, 70

- 60

( 60

l 50

(

d o 50

i M

t a 40

o ,

a s 40

k 30 EV EV

n d 30

a 20

y V12 n V12 o 20

n 10

t 10

r I 0 0

A N P R T M C S DHS S DHS Metabolite Metabolite Figure 33 (continued). Gene expression and metabolite profiles of SanR-silenced opium poppy roots. Relative expression of

(A) SanR1, (B) SanR2, and (C) SanR3 in the roots of poppy plants transformed with a VIGS construct designed by Guillaume

Beaudoin (V12, n=8). Error bars represent the SEM of three technical replicates. (D) Average relative expression in EV control (n=13)

versus V12. SanR2 (p=0.00004) and SanR3 (p=0.009) expression levels were decreased compared to EV. Error bars represent SEM.

(E) Root alkaloid metabolite profile of EV control versus V12. Welch’s correction was applied indicating no significant increase in

sanguinarine (p=0.07) and cryptopine (p=0.08). (F) LC-MS counts of sanguinarine and DHS were converted to concentration (μM)

using a standard curve. Significance was calculated using Student’s two-tailed t-test, and asterisk (*) indicates p<0.05. N: noscapine,

P: papaverine, R: reticuline, T: thebaine, M: morphine, C: cryptopine, S: sanguinarine, DHS: dihydrosanguinarine.

108

4 DISCUSSION

4.1 Sanguinarine reductase identification, expression, and purification

The Illumina stem and root transcriptomes for Papaver somniferum cultivar Bea’s

Choice encodes four sequences that share more than 60% amino acid sequence identity to the previously characterized E. californica SanR (Fig. 3) (Vogel et al., 2010; Weiss et al.,

2006), and have been named SanR1 through SanR4. Sequences encoding SanR1

(comp9568) were only found in the stem transcriptome, SanR2 (comp73923) was only found in the root transcriptome, and both SanR3 (comp1703 and comp80098) and SanR4

(comp26502 and comp72201) were found in the stem and root transcriptomes. However, the contig encoding SanR4 was incomplete in the root transcriptome. Primers were designed to amplify individual SanRs (Table 1). However, amplification of SanR4 was not successful. This suggested that the SanR4 contig might not be a real transcript, but the result of transcriptome misassembly. However, a second set of primers (SanR4-F2/R2;

Table 1) successfully amplified a product of expected size (data not shown), and sequencing confirmed its identity as SanR4. Therefore, SanR4 is expressed in planta, but was not further analyzed.

SanR coding sequences were successfully cloned into pQE30 expression vectors, but no recombinant protein expression was observed when constructs were transformed into E. coli strain M15 (data not shown). Recombinant SanR expression was only observed in cultures of induced E. coli strain SG13009 harbouring pQE30-SanR constructs (Fig. 5). However, E. coli strains M15 and SG13009 are very similar. Both strains are derived from E. coli strain K12, have the phenotype NalS, StrS, RifS, Thi-, Lac-,

Ara+, Gal+, Mtl-, F-, RecA+, Uvr+, Lon+, and harbor the pREP4 plasmid (Qiagen Manual,

109

Genotype analysis of E. coli strains SG13009 and M15). Interestingly, previous reports indicate proteins poorly expressed in M15 are produced to higher levels in SG13009, and proteins expressed in M15 are overproduced in SG13009 to a level that inhibits cell growth (Stüber, D., Matile, H., and Garotta, 1990).

To confirm the identity of overexpressed proteins in IPTG-induced cultures of

E. coli strain SG13009 harbouring pQE30-SanR constructs, soluble and purified proteins were extracted and size-separated on polyacrylamide gels (Fig. 5 and 7). Gels were stained with Coomassie, or transferred to nitrocellulose membrane for Western blot analysis. Coomassie stains proteins through its affinity for basic amino acids, such as arginine and lysine (Congdon et al., 1993). Recombinant 6xHis-tagged proteins were purified using the TALON cobalt resin instead of nickel-nitrilotriacetic acid (Ni-NTA) resin because cobalt ions are more selective for histidine tags than nickel ions to improve purity, and proteins can be eluted with lower imidazole concentrations (Chaga et al.,

1999). Interestingly, SanR1, SanR2, and SanR3 size-separate at approximately the same molecular weight, despite having predicted molecular weights of 29.4, 29.4, and 32.1 kDa respectively (Fig. 7). Overall, the size of the recombinant proteins stained with

Coomassie and detected with an anti-His antibody are consistent with the predicted size of SanRs: 29.4, 29.4, 32.1, and 26.8 kDa for SanR1, SanR2, SanR3, and SanR3B, respectively (Fig. 7). Together this data suggests that SanRs were successfully expressed in E. coli strain SG13009 from the pQE30, and were specifically purified using TALON metal affinity resin.

110

4.2 Biochemical characterization of sanguinarine reductases in vitro

Although both SanR and DRR accept quaternary alkaloids as substrates, opium poppy SanRs do not accept 1,2-dehydroreticuline as a substrate (Fig. 8; De-Eknamkul and Zenk, 1992; Weiss et al., 2006). However, consistent with previous reports of

E. californica SanR, all opium poppy SanRs reduce sanguinarine and chelerythrine to dihydrosanguinarine and dihydrochelerythrine, respectively (Fig. 13). To test if SanRs will reduce other benzophenanthridine alkaloids, chelirubine and macarpine were isolated from elicited E. californica cell culture filtrate extracts (Fig. 9 and 10) because they do not accumulate in opium poppy nor are they commercially available (Schumacher et al.,

1987). Initial attempts to isolate benzophenanthridines were performed using

E. californica root extract. However, fewer purification steps and increased purity was obtained using elicited E. californica cell culture, presumably due to fewer contaminates, such as chlorophyll. Benzophenanthridine alkaloids separated into four distinct bands when cell filtrate alkaloid extract was separated by TLC using 2:2:1 chloroform:ethyl acetate:methanol (Fig. 9). CID dissociation spectra for the TLC-isolated benzophenanthridines are consistent with previously reported CID spectra (Fig. 10) (Son et al., 2014; Liscombe et al., 2009). Consequently, chelirubine and macarpine could be used as substrates for SanR assays. As a control for the enzymatic assays, all benzophenanthridines (sanguinarine, chelerythrine, chelirubine, and macarpine) were converted to the corresponding dihydrobenzophenanthridines through treatment with sodium borohydride (Fig. 12). Treatment with excess sodium borohydride resulted in almost 100% conversion of sanguinarine, chelerythrine, and chelirubine to dihydrosanguinarine, dihydrochelerythrine, and dihydrochelirubine, respectively

111

(Fig. 12). However, not all macarpine (retention time approximately 8.4 minutes) was converted to dihydromacarpine (retention time approximately 9.0 minutes; Fig. 12).

There is a peak present in chromatographs (EIC m/z 394) before and after sodium borohydride treatment with a retention time of approximate 8.0 minutes (Fig. 12), but

CID analysis indicated that this peak is neither macarpine (m/z 392) nor dihydromacarpine (m/z 394) (data not shown).

In addition to sanguinarine and chelerythrine, SanRs from opium poppy also reduce chelirubine and macarpine to dihydrochelirubine and dihydromacarpine, respectively (Fig. 13). However, chelerythrine, chelirubine and macarpine are not known to accumulate in Papaver somniferum. Only dihydrochelirubine and dihydromacarpine are detected in E. californica cell cultures (Weiss et al., 2006). However, upon elicitation

E. californica cultures produce both chelirubine and macarpine. Furthermore, exogenous sanguinarine added to E. californica cells is immediately reduced to dihydrosanguinarine, then derivatized to dihydrochelirubine and dihydromacarpine (Weiss et al., 2006). Weiss et al. (2006) hypothesized elicited sanguinarine and chelerythrine only accumulates transiently before being converted to macarpine and chelilutine, respectively, as sanguinarine was reported to have a greater effect on growth inhibition of Candida albicans and Staphylococcus aureus as compared to chelerythrine or chelirubine.

However, recent studies have shown that both chelirubine and macarpine also inhibit growth and induce apoptosis in vitro (Slaninová et al., 2007). Macarpine inhibited human tumor cell line growth 5- to 10-times more than the other benzophenanthridine alkaloids, with chelerythrine and sanguinarine being the least effective. Chelirubine had the greatest proapoptotic effect and induced up to 90% apoptosis at concentrations as low as 1 μg/mL,

112 while sanguinarine was essentially non-toxic until concentrations were greater than

10 μg/mL. Interestingly, sanguinarine was detected in the roots of Macleaya cordata,

Macleaya microcarpa, Chelidonium majus, Sanguinaria canadensis, and Dicranostigma lactucoides at higher concentrations than chelirubine or macarpine using HPLC

(Suchomelová et al., 2007). Macarpine was only detected in the roots of Macleaya microcarpa, and Stylophorum lasiocarpum. Therefore, opium poppy SanRs may have evolved from an ancestral enzyme found in a plant that accumulates the more cytotoxic macarpine resulting in a wider benzophenanthridine substrate range (Fig. 13). However, macarpine was detected in 15 cell cultures that produce benzophenanthridines, including

Chelidonium majus and Sanguinaria canadensis, using LC-MS using a triple-quadrupole mass analyzer (Farrow et al., 2012). Thus, more sensitive detection methods may reveal a wider occurrence of macarpine in benzophenanthridine producing species. Enzymes involved in secondary metabolism are often promiscuous and less catalytically efficient, but have broader specificities to facilitate chemical diversity (Weng et al., 2012).

Consequently, SanRs may reduce chelerythrine, chelirubine and macarpine, though not detected in opium poppy, due to an accommodating conformation of the active site.

Additional experiments are required to determine the exact benzophenanthridine substrate preference of opium poppy SanRs.

Preliminary experiments using an unknown, but consistent concentration of substrate demonstrated that all opium poppy SanRs reduce benzophenanthridine alkaloids to the corresponding dihydrobenzophenanthridines using either NADPH or NADH as a co-factor (Appendix A2, Fig. A2.1). To gain further insight into co-factor preference,

SanR2 was incubated with 5 μM sanguinarine, chelerythrine, chelirubine or macarpine

113 and 1mM NADPH or NADH (Fig. 14). SanR2 produces more dihydrobenzophenanthridine compounds using NADPH, as compared to NADH, as a co-factor. These data mimic the trends observed for SanR1 and SanR3B in preliminary experiments (Fig. A2.1). Furthermore, these data are consistent with the results for

E. californica SanR, which catalyzes the reduction of sanguinarine and chelerythrine using either NADPH or NADH as a hydrogen donor (Weiss et al., 2006). Furthermore, no activity was observed for papaverine, noscapine, berberine, cryptopine, or thebaine

(data not shown). Similarly, E. californica SanR also does not reduce berberine (Weiss et al., 2006). Like benzophenanthridines, berberine is derived from (S)-scoulerine

(Beaudoin and Facchini, 2014). The inability of SanRs to reduce the quaternary ammonium ion in berberine, a protoberberine alkaloid, further supports its preference for the benzophenanthridine backbone. It would be interesting to test if SanRs are able to reduce (S)-cis-N-methylstylopine, an intermediate in sanguinarine biosynthesis, and structurally similar to berberine except for the presence of two methylenedioxy bridges

(Fig. 1; Table 4).

The temperature optima of SanRs were determined by incubating the enzyme with sanguinarine and NADPH for one hour at various temperatures. Preliminary experiments indicated that the temperature optimum for SanR1 and SanR3B as 21°C

(Appendix A2, Fig. A2.2). Similarly, the temperature optimum for SanR2 is 18°C

(Fig. 15). Temperature optimum of E. californica SanR was never explicitly stated, however, assays were incubated at 22°C (Weiss et al., 2006). Preliminary experiments to determine pH optima were inconclusive (data not shown). However, the pH optimum of

E. californica SanRs is 6.5 to 7.5 (Weiss et al., 2006). Therefore, sanguinarine reductase

114 assays were performed at pH 7.5, and typically in sodium phosphate buffer, instead of

Tris-HCl, as to not have temperature affect pH (Sambrook and Russell, 2001). The Km of

SanR1 and SanR3B were calculated as 1.6 and 19.9 μM sanguinarine, respectively

(Fig. 16). However, the LC-MS counts for amount of dihydrosanguinarine produced was below the linear range for most of the enzymatic reactions. Therefore, omitting these values, the Km for SanR1 was re-calculated as 7.3 μM (Fig. 16). The Km of E. californica

SanR was 9.5 μM sanguinarine (Weiss et al., 2006). However, the Km of E. californica

SanR was determined using 40 µM NADH, and may not have been saturating. The Km for E. californica SanR was calculated as 19 μM, when using NADPH and glutathione, and corrected for spontaneous product formation in the absence of enzyme (Vogel et al.,

2010). I have also observed the reduction of sanguinarine to dihydrosanguinarine in the presence of high concentrations of NAD(P)H, in absence of SanR (data not shown).

However, no conversion of sanguinarine to dihydrosanguinarine was observed in the presence of 1 mM NAD(P)H (Fig. 13G-H). Vogel et al. (2010) observed E. californica

SanR is irreversibly inhibited by its product, dihydrosanguinarine. Therefore, conversion of sanguinarine to dihydrosanguinarine is never saturated. Adding glutathione to the reaction allows a constant supply of sanguinarine in complex to prevent dihydrosanguinarine inhibition, and achieve saturation kinetics. Consequently, the affinity of opium poppy SanRs for sanguinarine may be higher than calculated (Fig. 16).

4.3 Short-chain dehydrogenase/reductases

To date, four reductases have been identified in opium poppy benzylisoquinoline biosynthesis. An aldo-keto reductase, codeinone reductase (COR), and three short-chain dehydrogenase/reductases (SDRs), noscapine synthase (NOS), salutaridine reductase

115

(SalR), and sanguinarine reductase (Marchler-Bauer et al., 2015; Chen and Facchini,

2014; Ziegler et al., 2006; Unterlinner et al., 1999). SDRs are a superfamily of

NAD(P)(H)-dependent oxidoreductases typically share three conserved features, a

Rossmann-fold motif, an N-terminal dinucleotide co-factor binding motif, and an active site with a catalytical YXXXK motif (Moummou et al., 2012). Furthermore, plant SDRs are classified into three major subgroups, classical, extended, and divergent. However, it is estimated that approximately 10% of predicted SDRs do not fall into one of these general categories, and are referred to as “atypical” SDRs, which typically include enzymes involved in secondary metabolism or developmental processes. Opium poppy

SanRs are defined as atypical SDRs, whereas NOS and SalR belong to the extended and classical subgroups, respectively (Marchler-Bauer et al., 2015; Chen and Facchini, 2014;

Ziegler et al., 2006). Both NOS and SalR exhibit high substrate specificity towards narcotinehemiacetal and salutaridine, respectively (Chen and Facchini, 2014; Ziegler et al., 2006). However, SanRs will reduce benzophenanthridines sanguinarine, chelerythrine, chelirubine, and macarpine (Fig. 13). Therefore, SanRs may have undergone more functional diversification compared to NOS or SalR, and could explain the ability of SanRs to reduce chelerythrine, chelirubine and macarpine, which are not known to accumulate in Papaver somniferum.

The N-terminal dinucleotide co-factor binding motif of SanRs (TGASGLTG or

TGAGGRTG) is very similar to the NADP-binding motif, [TS]GXXGXXG, of extended

SDRs (Marchler-Bauer et al., 2015; Kavanagh et al., 2008). However, SanRs will reduce benzophenanthridines using either NADPH or NADH as a co-factor (Fig. 13), and end-

116 point assays indicate SanRs may prefer NADPH to NADH (Fig. 14). Additional assays are required, including Km determination, to accurately conclude co-factor preference.

4.4 Protein localization of sanguinarine reductases in planta

Antibodies (antisera) were generated against recombinant 6xHis-tagged SanRs in mice. After four booster injections the mice antisera were not specific for a given SanR

(Fig. 17 and 18). This is expected due to the high degree of sequence similarity between opium poppy SanRs. To increase antisera specificity, antibodies could have been raised against a small region specific to a given SanR. However, pre-exposing antiserum to a high concentration of the non-target antigens (“antibody scrubbing”) was successful in increasing specificity towards a given SanR (Fig. 18). To increase specificity of the antisera towards a given SanRs in planta, a much higher concentration (1:50 or 1:100) of

“scrubbed” antiserum is needed compared to the concentration required to detect SanRs on a Western blot (1:10,000).

Soluble proteins were isolated from stem, root, leaf, and capsule/bud tissues, as well as latex, from 8 different Papaver somniferum cultivars. SanRs were detected in all tissue types using SanR3B antiserum, which is able to detect all three SanRs (Fig. 17 and

18). Although SanR3B antiserum is non-specific to a given SanR, the SanR found within a tissue type can be inferred based on molecular weight. The molecular weight of

SanR(s) detected in root tissue is consistent with that of SanR3 (32.1 kDa) than SanR1 and SanR2 (29.4 kDa) (Fig. 19), however SanR1, SanR2, and SanR3 separate at similar molecular weights in SDS-PAGE (Fig. 7). The molecular weight of SanR(s) present in stem, leaf, and capsule/bud are consistent with the size of SanR3B (26.8 kDa) (Fig. 7 and

19). The SanR(s) detected in latex is smaller than those found in all tissues, and its size is

117 not consistent with SanR1, Sanr2, or SanR3(B) (Fig. 7 and 19). Also, SanRs were only detected in the latex of poppy Veronica, Marianne, and Bea’s Choice (Fig. 19). However, latex signal was inconsistent, and difficult to reproduce. The smaller band observed in the latex Western blots with SanR antiserum may correspond to splice variants or cleaved

SanR1 or SanR2 proteins, and the larger band, greater than the 46 kDa molecular marker, in the latex Western blots, which may correspond to a post-translationally modified

SanRs. However, if these signals were representative of SanRs in the latex, it is expected that similar sized proteins would be detected in the Western blot for each of the tissues, as they all contained laticifers. Interestingly, shotgun proteomics implicated SanR1 and/or SanR2 in the latex (personal communication) (Onoyovwe et al., 2013).

Consistent with Western blot analysis, immunolocalization indicated SanRs are present in stem, root, and capsule tissues (Fig. 20). No reliable signal was obtained for leaf tissue, but absence of signal does not indicate absence of protein. Furthermore, multiple cultivars (40, Veronica, and Marianne) were used for immunolocalization, but all show similar results when probed with SanR3 antiserum (data not shown). Therefore, representative images using poppy cultivar 40 are shown (Fig. 20). Specifically, SanRs appear to localize to the phloem, which is consistent with the localization of other enzymes involved in BIA biosynthesis (Onoyovwe et al., 2013). Enzymes involved in morphinan biosynthesis (downstream of (R)-reticuline) predominantly localize to laticifers, whereas other BIA enzymes (upstream of (S)-reticuline) predominantly localize to sieve elements (Fig. 1) (Onoyovwe et al., 2013). In cell culture, DBOX (Accession

JX390714), the enzyme responsible for synthesizing sanguinarine from dihydrosanguinarine, is predicted to localize to secretory pathway, because it contains a

118 signal peptide targeting it to the ER (Hagel and Facchini, 2012; Emanuelsson et al.,

2007). Therefore, SanR, the enzyme responsible for the reverse reaction, reducing sanguinarine to dihydrosanguinarine, might also be targeted to the ER. During laticifer development, the ER differentiate in to alkaloid containing vesicles, which could indicate a potential route for SanR laticifer localization (Nessler and Mahlberg, 1977). However,

SanRs are not predicted to localize to the ER. Both SanR1 and SanR2 are predicted to localized to the cytosol, while SanR3 and SanR4 are predicted to localize to the chloroplast (Horton et al., 2007; Emanuelsson et al., 2007). Therefore, to definitively determine the phloem cell type SanRs are localized to sections need to be stained with aniline blue to detect sieve tubes (callose), and/or probed with a latex-specific protein, such as major latex protein (MLP) (Nessler et al., 1985; Currier, 1957). Furthermore, the

SanR3B antiserum was used to detect SanRs for immunolocalization experiments. Due to the cross-reactivity of this antiserum the identity of SanR(s) detected in the different tissues in unknown. A lower antiserum dilution (1:100) would need to be scrubbed to increase antisera specificity toward a given SanR.

Interestingly, the opium poppy SanR3B antiserum only detected proteins in the aerial tissues of E. californica, not root tissues, and has a molecule weight greater than 30 kDa (Fig. 19). This is surprising as sanguinarine is only predominantly found in the roots of California poppy. Therefore, the E. californica SanR(s) detected in aerial organs may have similar roles in planta to opium poppy SanR(s) found within the latex. Perhaps, aerial E. californica, and latex P. somniferum SanRs are responsible for reducing sanguinarine to dihydrosanguinarine for its transport throughout the plant. The characterized E. californica SanR shares 77%, 78%, and 59% sequence similarity to

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SanR1, SanR2, and SanR3, respectively. The molecular weight of the characterized

E. californica SanR isolated from cell culture is 29.5 kDa (Weiss et al., 2006). Analyzing our E. californica root transcriptome database identified 5 transcripts that encode proteins with more than 60% sequence similarity to the characterized SanR, and all encoded proteins ranging from 29.0 to 30.6 kDa. But we do not have any data on the expression of

SanRs in aerial tissue of E. californica. Therefore, the SanR(s) detected in the aerial organs is likely tissue-specific, and may be more similar to opium poppy SanRs than the characterized root-specific SanR, which could explain why SanR(s) were not detected in the root tissue of E. californica.

4.5 Expression of sanguinarine reductases in planta

Primers were originally designed to amplifying SanRs using the SYBR Green method of qPCR. However, primers sets were not specific for a given SanR, and would amplify all SanR templates (purified pQE30-SanR plasmids) (data not shown).

Furthermore, no silencing of SanRs in VIGS-treated plants was observed using these primers (data not shown). Presumably, the primers were able to amplify the non-silenced

SanRs to mask the reduced expression of SanRs targeted by VIGS constructs. Therefore, primers and MGB probes were designed for TaqMan qPCR to increase specificity

(Table 4). In addition, ubiquitin (GenBank Accession: JN402989) was used an endogenous reference gene because its expression level (CT value) was consistent between tissues. The relative expression level of each SanR in four tissue types was determined using the comparative ΔΔCT method, whereby the average CT value for ubiquitin is subtracted from the average CT value for SanR1, SanR2, or SanR3 (ΔCT).

ΔCT values can be directly converted to relative expression level using the equation

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2^(-ΔCT), or compared to control samples to obtain a ΔΔCT value (Fig. 21). Overall,

SanR1 is expressed in all tissue, SanR2 is expressed predominantly in root tissue, and

SanR3 is expressed in all tissues except capsule/flower bud (Fig. 21). The expression profiles of SanR2 and SanR3 are consistent with the Illumina transcriptomes: SanR2 was only present in the root transcriptome, and SanR3 was present in both the stem and root transcriptomes. On the other hand, SanR1 is expressed in both stem and root tissue, but was only present in the stem transcriptome. However, since there is evidence that one contig representing SanR2 in the root transcriptome is poorly assembled (personal communication), and SanR1 and SanR2 share 88% sequence similarity at the nucleotide level that the absence of SanR1 in the root transcriptome is the result of assembly error.

For simplicity, I have chosen to represent gene expression data relative to the endogenous gene only. Papaver somniferum cultivar Bea’s Choice does not have an isogenic background, and exhibits natural variation in gene expression. For example,

SanR3 expression levels for control, non-silenced plants are highly variable, and expression is three-times higher in some plants than the average (Fig. 23). Therefore, it is difficult to choose a single reference to compare all VIGS plants. As well, the natural variation in SanR expression in Bea’s Choice likely contributes to the variation in the silenced phenotypes observed. VIGS is designed to knock down, not knock out, gene expression, therefore, silencing effects may be masked in plant that naturally display higher baseline expression levels.

VIGS constructs were designed complementary to the 5’- or 3’-untranslated region (UTR) or coding sequence of SanRs (Fig. 4). However, there were discrepancies in the UTRs of SanR1 and SanR2 in the Illumina transcriptome (Appendix A3). The two

121 contigs representing SanR1 or SanR2 had different 3’ UTR sequences. Therefore, the

Roche-454 stem transcriptome was consulted to clarify assembly error. Constructs designed to silence SanRs were cloned into the pTRV2 and transformed into

Agrobacterium. Cultures of Agrobacterium harbouring pTRV2-SanR VIGS constructs mixed with equal volumes of Agrobacterium harbouring pTRV1 were infiltrated into opium poppy seedlings. To confirm poppies were successfully infiltrated and transformed with pTRV2, RNA was isolated from poppy root tissue, synthesized into cDNA, and

PCR amplified with primers designed to amplify viral coat protein (CP) (Table 2;

Fig. 22). Approximately 200 poppies were infiltrated with VIGS constructs, and 130 plants tested positive CP, which is an approximately 65% success rate. Poppy cDNA should have also been checked for the presence of transcripts encoded by pTRV1, and

PCR amplified using primers designed against the movement protein (Fig. 2; Table 2).

However, plant RNA isolated from previous VIGS experiments always displayed co- expression of genes encoded by pTRV1 and pTRV2 together (data not shown).

To analyze root metabolites in VIGS-treated plants, retention times from extracted-ion chromatographs of alkaloid authentic standards were compared to authentic standards to identify unknown alkaloids in the roots of SanR-silenced plants (Fig. 24 and

25). Electrospray ionisation mass spectrometry readily detects the alkaloids noscapine, papaverine, reticuline, thebaine, sanguinarine, and dihydrosanguinarine (Hagel et al.,

2012). However, a more targeted search for less abundant alkaloids, such as stylopine, an intermediate in sanguinarine biosynthesis, and oripavine, an intermediate in morphine biosynthesis, is required for detection (Hagel et al., 2012; Hagel and Facchini, 2010;

Ikezawa et al., 2007). Therefore, root alkaloids extracted from VIGS plants either need to

122 be re-analyzed at a lower dilution, or re-analyzed using selected ion monitoring, in order to assess changes in the accumulation of these low-abundance alkaloids. Consequently, a caveat of my analysis is that I may be seeing little to no effect in metabolite levels as a result of VIGS treatment, perhaps because I did not investigated changes in lower abundance alkaloids. As well, dihydrosanguinarine spontaneously converts to sanguinarine in the presence of heat. Therefore, changes in dihydrosanguinarine and sanguinarine may have been lost during analysis, because the LC-MS does not have a sample-cooling chamber. Furthermore, Student’s t-test was used to determine if two data sets were significantly different from each other, but this test can only be used when the two distributions have the same variance. In some cases, the variation within the empty vector control samples and SanR-silenced treatment groups was not equal, and Welch’s correction was applied in attempt to correct for unequal variance (GraphPad Prism).

Consequently, unequal variance between groups abolished the significance implicated with Student’s t-test.

Silencing SanR1 resulted in no significant changes in metabolite profile (Fig. 26).

Similarly, silencing SanR2 using VIGS constructs complementary to the 5’ UTR (V3) and coding sequence (V4) did not affect root metabolite profile (Fig. 27). However, silencing SanR2 using VIGS constructs complementary the 3’ UTR (V5) resulted a significant increase in morphine, even though the knocked down expression level is comparable to that of V3-treated plants (Fig. 27). Interestingly, silencing T6ODM and/or

CODM, which encode enzymes involved in morphine biosynthesis, in opium poppy roots resulted in reduced levels of sanguinarine (Farrow and Facchini, 2013). Together, these

123 data may indicate an indirect relationship between morphine and sanguinarine biosynthesis.

Silencing SanR3 resulted in a significant increase in sanguinarine, but in some cases cryptopine or reticuline levels were increased, while noscapine levels were decreased (Fig. 28). This supports the role of SanR3 as sanguinarine reductase in planta.

It is expected that knocking down the expression of SanR3 would result in less translated protein, and an accumulation of sanguinarine, because it cannot be effectively reduced to dihydrosanguinarine. Furthermore, sanguinarine biosynthesis begins with the formation of scoulerine from reticuline by BBE, and cryptopine is a protopine derivative also originating from scoulerine (Beaudoin and Facchini, 2014). Therefore, silencing SanR3 resulted in a build up of upstream metabolites in the sanguinarine biosynthetic pathway.

Surprisingly, the VIGS construct designed to silence SanR1 and SanR3 (V8) resulted in reduced expression levels of SanR2 and SanR3, but no change in the expression level of SanR1 (Fig. 29). The V8 construct was designed to target the 3’ UTRs of SanR1 and SanR3, and it has been noted that as little as seven perfect matches between siRNA and 3' UTR is sufficient to facilitate off-targeting silencing effects (Birmingham et al., 2006). I assumed that 3’ UTR sequence similarity of SanR1 contig comp9568_c0_seq1 and SanR2 contig comp73923_c0_seq1, and SanR1 contig comp9568_c0_seq2 and SanR2 contig comp73923_c0_seq2 was an assembly error in the

Illumina transcriptome through comparison with the transcripts in the Roche-454 transcriptome (Appendix A3). Therefore, if the Illumina library is accurate then the construct designed to silence SanR1 is 100% complementary to 3’ UTR of both SanR1 and SanR2, and reduced expression levels of SanR2 is not unexpected. Furthermore, the

124 expression level of SanR2 was not determined in V1 or V2 plants, and may have also been silenced in addition to SanR1 (Fig. 26). Therefore, another caveat of my VIGS analysis is that only expression levels of targeted SanR were checked, and it is clear that expression of non-targeted SanRs may be affected. Nevertheless, silencing SanR2 and

SanR3 resulted in decreased noscapine, and papaverine levels (Fig. 29), and the decreased noscapine levels are consistent with SanR3-silencing in V6-treated plants

(Fig. 28). Reduced noscapine and papaverine levels were also observed in T6ODM and/or

CODM silenced plants (Farrow and Facchini, 2013).

Guillaume Beaudoin (V9-V12) designed four VIGS constructs, but only V9 showed 100% sequence similarity to Bea’s Choice SanR1. Therefore, expression levels of all SanRs were analyzed. Interestingly, the V9 construct, which shared 100% sequence similarity to SanR1 coding sequence, resulted in an increase in SanR1 expression, and a decrease in SanR2 expression (Fig. 30). Although silencing SanR1 did not alter metabolite levels in the roots, increasing the expression of SanR1 increased reticuline, thebaine, and sanguinarine levels (Fig. 26 and 30). However, no change in morphine levels was observed as seen in other SanR2-silenced plants (Fig. 27). Both V12 and V8 plants showed decreased levels in both SanR2 and SanR3 expression (Fig. 33). However, silencing in V8 plants was correlated to a decrease in noscapine and papaverine levels, while no significant change in metabolite levels was observed in V12 plants (Fig. 33).

The only difference between the V8 and V12 construct is that they target the 3’ UTR and coding sequence, respectively. Lastly, both V10 and V11 plants resulted in a significant reduction of all SanR expression levels (Fig. 31 and 32). Yet no significant changes in metabolite levels were observed (Fig. 31 and 32).

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Interestingly, in few cases did silencing one or more SanRs result in increased sanguinarine levels compared to the empty vector control. Due to the highly cytotoxic nature of sanguinarine, pests, such as Frankliniella occidentalis (Western Flower thrips), will preferentially feed on Phaseolus vulgaris (common bean) instead of the benzophenanthridine-producing Chelidonium majus, and will also avoid sugar solutions containing sanguinarine (Schütz et al., 2014). However, when forced to feed on leaves from Chelidonium majus or Eschscholzia californica, two members of the Papaveraceae family, thrips induced enhanced alkaloid production in the plants. After feeding for three days, the leaves of C. majus and E. californica showed a marked increase in benzophenanthridine alkaloid content, specifically sanguinarine (Schütz et al., 2014).

Induction and/or increase in sanguinarine production as a result of herbivory is consistent with the responses evoked by microbial elicitors in cell culture (Schumacher et al., 1987).

Opium poppy plants used for VIGS analysis were often severely infested with aphids. As thrips and aphids are both small, soft-bodied insects that feed on plant phloem, it likely that aphid infection would also induce an enhanced production of sanguinarine.

Therefore, pest herbivory may have induced alternative regulatory mechanisms to mask the decrease in sanguinarine levels expected from silencing SanRs in planta.

In addition to observing metabolite changes in root tissues of VIGS-treated plants, changes in metabolite levels of other tissues, or at least latex, should have been investigated. SanRs are also expressed in stem, leaf, and capsule/flower bud (Fig. 21); therefore, silencing SanRs in planta would likely affect the metabolite profile in other tissues as well. My initial VIGS experiments only analyzed latex, not root, metabolites when checking if SanRs may be responsible for DRR activity in planta (data not shown).

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However, initial VIGS analyses were performed with non-specific qPCR primers to analyze gene expression. The SYBR Green method of qPCR was not specific enough to identify individual SanRs, and, presumably is the cause for lack of gene silencing observed (data not shown). Therefore, samples could have been re-analyzed using primer and probe sets for TaqMan qPCR. As well, it is clear that little to no changes in sanguinarine or dihydrosanguinarine levels are observed in roots. This could be attributed to the constant aphid infestation problems seen in both the growth chambers and greenhouse, or it may be an accurate reflection of SanR silencing. Nevertheless, root extracts from VIGS plants should have been re-analyzed for changes in other alkaloids, using both full ion scanning and multiple reaction monitoring methods on the LC-MS.

Combined with the 130 plants analyzed here, a more detailed, and controlled analysis of previous VIGS experiments would help to gain further insight into the role of sanguinarine in planta.

4.6 Biological roles of sanguinarine reductases

To gain insight into the role of SanRs, both substrate and enzyme need to be localized in planta. SanR cellular localization has been described in this thesis, but sanguinarine has yet to be localized. Localization of sanguinarine in C. majus and

E. californica can be inferred in unstained root sections as cells containing sanguinarine will appear yellow in colour. However, sanguinarine in P. somniferum does not accumulate to such levels, and opium poppy roots do not exhibit any visible yellow coloration. Therefore, sanguinarine in opium poppy can only be detected using more sensitive methods, such as TLC or LC-MS. Laser microdissection could be used to

127 isolate individual cell types for LC-MS analysis in order to gain further insight into sanguinarine cellular localization (Abbott et al., 2010).

As sanguinarine is a highly cytotoxic compound, compartmentalization is also important in planta, in addition to possessing an enzyme capable of reducing this metabolite. Both Chelidonium majus and Eschscholzia californica accumulate benzophenanthridine alkaloids. C. majus sequesters benzophenanthridine alkaloids into laticifers, specifically latex vesicles, whereas E. californica accumulates the alkaloids in idioblasts of the root cortex and/or laticifers (personal communication; Hauser and Wink,

1990; Schütz et al., 2014). In opium poppy plants, sanguinarine and dihydrosanguinarine are the major alkaloids detected in the roots, and account for 20% and 80% of the benzophenanthridine alkaloid content in the roots of opium poppy cultivar Marianne, respectively (Frick et al., 2005; Facchini et al., 1996). Neither sanguinarine, nor dihydrosanguinarine, are detected in the latex of opium poppy, but trace amount of both alkaloids have been detected in the stems and leaves of a narcotic cultivar, C048-6-14-64, in which morphine, codeine, and thebaine account for 91.2% of the latex alkaloids (Frick et al., 2005). Only trace amounts of dihydrosanguinarine were detected in the leaves of

Marianne, a low morphine cultivar. However, it is still unclear as to the presence of sanguinarine in the capsule.

In Papaver somniferum cell cultures, sanguinarine localizes to the central vacuole and ER-derived vesicles (Alcantara et al., 2005). However, sanguinarine only accumulates in elicited microbial cells, whereas sanguinarine is abundant in the roots of opium poppy plants (Alcantara et al., 2005). Moreover, sanguinarine is mostly excreted into the media of elicited E. californica cell cultures, while small quantities are retained

128 around the cell wall (Weiss et al., 2006). Also, exogenous sanguinarine taken up

E. californica cell cultures localizes to the cell wall, which is converted to dihydrosanguinarine and targeted to the vacuole (Weiss et al., 2006). Similarly, elicited

P. bracteatum cell cultures secrete sanguinarine into the medium, and cells exhibit a yellow fluorescence within the peripheral regions of the cell (Cline and Coscia, 1989;

Cline and Coscia, 1988). Unlike control cells, elicited cells display an extensive network of elongated and dilated ER, and electron-dense aggregates associated with the tonoplast.

The aggregates appeared to migrate to and enter the tonoplast forming vesicles within the vacuole (Cline and Coscia, 1989). Therefore, it is difficult to use cell cultures to predict the cellular localization of sanguinarine in opium poppy plants, specifically root tissue, as sanguinarine is not detected in the latex of opium poppy. However, the alkaloid- containing vesicles in opium poppy laticifers are formed from ER dilations (Nessler and

Mahlberg, 1977). Perhaps in absence of different cell types in culture, sanguinarine is targeted to the vacuole for storage via ER-derived vesicles, while sanguinarine is targeted to laticifers for storage in planta.

As both BBE, which catalyzes the first committal step in sanguinarine biosynthesis, and SanRs, which catalyzes the reduction sanguinarine, are detected in all tissues (Fig. 19, 20, and 21) (Bird et al., 2003), sanguinarine biosynthesis may occur throughout the plant, but, due to its cytotoxicity, sanguinarine is reduced to dihydrosanguinarine in the aerial organs for transport, via the phloem, to the roots for storage. Based on previous cell culture experiments, sanguinarine and dihydrosanguinarine transport is likely mediated by the ER, and is stored within vacuoles

(Weiss et al., 2006; Alcantara et al., 2005).

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Together, gene expression and Western blot analyses indicates SanR1 and/or

SanR3 is mediating the reduction of sanguinarine in aerial organs (stem and leaf).

Western blot analysis suggests the predominantly expressed SanR is less than 30 kDa, which correlates to the predicted size of SanR3B (Fig. 19), but previous shotgun proteomics data indicated SanR1 and/or SanR2 are present in the latex (personal communication) (Onoyovwe et al., 2013). The gene expression profile of SanR2 is consistent with other sanguinarine reductase biosynthetic enzymes, where DBOX is only expressed in roots, but not stem, leaf, or capsule (Fig. 21) (Hagel et al., 2012). Therefore,

I hypothesize that SanR1 is restricted to laticifers of all tissues, SanR3 is present in another phloem cell type, such as companion cells, in all tissues except the capsule/flower bud, and SanR2 is restricted to the root (Fig. 34). Co-localization with cell-type specific antibodies, such as major latex protein (MLP), and using SanR-specific antibodies is necessary to determine the precise tissue and cellular localization of SanR.

As sanguinarine and dihydrosanguinarine are both found to some extent in stem, leaf, and root tissue, but not the capsule, I predict that SanR2 plays a direct role in sanguinarine detoxification in the roots, while SanR1 and SanR3 may have additional, uncharacterized roles.

130 A B Phloem cells Companion Sieve Laticifer cell element

Vacuole

SanR1 SanR1 ER SanR3 SanR2 SanR1 SanR3

SanR1 SanR3

SanR1 SanR2 SanR3 Sanguinarine DHS Figure 34. Predicted model for opium poppy SanRs.

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Figure 34 (continued). Predicted model for opium poppy SanRs. Schematic outlining the putative roles of sanguinarine reductase

(SanR) through gene expression and protein localization studies in planta. (A) SanRs are expressed in all tissues, and (B) the encoded proteins are localized to the phloem. SanR3 is expressed in all tissues, except the capsule and/or flower bud, which are not known to contain sanguinarine or dihydrosanguinarine (orange). SanR3 also contains a putative transit peptide, which may signal it to the endoplasmic reticulum (ER), the site of sanguinarine biosynthesis. Therefore, sanguinarine biosynthesis may occur in stem, leaf, and root tissues, and SanR3 reduces sanguinarine to dihydrosanguinarine for transport shoot-to-root via the phloem (sieve element), or storage in the vacuole. SanR1 and SanR2 are found in the laticifers, which accumulate morphinan alkaloids (blue), and may have an additional role outside sanguinarine detoxification.

132 There is a clear segregation between benzophenanthridine and morphine biosynthetic pathways in planta. Sanguinarine accumulates in the roots, but is not detected in the latex, whereas morphinan alkaloids accumulate to high levels in laticifers, but are only detected at low levels in the roots (Frick et al., 2005; Facchini and De Luca,

1995). As well, dedifferentiated opium poppy cell cultures do not constitutively accumulate alkaloids, such as morphine, but will produce sanguinarine in response to treatment with a fungal elicitor (Alcantara et al., 2005). Interestingly, silencing the genes encode T6ODM and/or CODM, which convert thebaine to codeinone and oripavine, respectively (Fig. 1), results in increased thebaine levels in latex, and reduced levels of sanguinarine in the roots, despite increased protopine levels (Farrow and Facchini, 2013).

It would be interesting to test the expression levels of all three SanRs to determine if silencing T6ODM and/or CODM had an effect on expression. In addition, an ethyl methanesulfonate (EMS)-induced mutant displaying an unusual accumulation of sanguinarine in the latex also exhibited lower thebaine levels compared to the untreated control (Desgagné-Penix et al., 2009). Together these data suggest a currently unstudied relationship between benzophenanthridine and morphine biosynthesis. Perhaps the presence of sanguinarine in latex excludes the biosynthesis of morphinan alkaloids, such as thebaine, and SanRs, particularly SanR1, are required to mediate morphinan biosynthesis in planta, as well as cellular detoxification of sanguinarine.

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5 CONCLUSION

Sanguinarine is able to intercalate DNA, inhibit DNA synthesis, and affects membrane permeability (Schmeller et al., 1997), which also makes sanguinarine, and its derivatives, a potential anticancer compound (Cao et al., 2015). Therefore, sanguinarine reductase (SanR) is considered an enzyme of detoxification due to its substrate’s highly cytotoxic nature. Thrips will avoid feeding on leaf discs from plants that accumulate benzophenanthridine alkaloids, and avoid feeding from sugar solutions containing sanguinarine (Schütz et al., 2014). However, if consumed, thrips will metabolize sanguinarine to dihydrosanguinarine (Schütz et al., 2014). Furthermore, addition of exogenous sanguinarine will inhibit growth of plant cell cultures that do not produce benzophenanthridines (Weiss et al., 2006). Microbial elicited E. californica cells will accumulate sanguinarine along the cell wall, secrete it into the medium, then re-absorb it for reduction to dihydrosanguinarine (Weiss et al., 2006). Therefore, dihydrosanguinarine appears to be less toxic than sanguinarine, and SanR may have evolved to prevent self- intoxication by benzophenanthridine producing species.

In cell culture, the sanguinarine biosynthetic pathway is associated with the ER

(Hagel and Facchini, 2012). BBE, the enzyme that converts reticuline to scoulerine, is associated with the ER, and is transported to the central vacuole (Bird and Facchini,

2001), and membrane-bound P450s, CFS and SPS, are associated with the endomembrane (Hagel and Facchini, 2012). However, MSH, P6H, DBOX, and SanR have not been localized at the cellular level (Beaudoin and Facchini, 2013; Hagel et al.,

2012). It is likely that MSH and P6H, which are the P450s, and DBOX, which has a putative ER signal peptide, will also be targeted to the ER (Beaudoin and Facchini, 2013;

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Emanuelsson et al., 2007). Only two of the four opium poppy SanRs are predicted to localize to the cytosol (Emanuelsson et al., 2007; Horton et al., 2007). Therefore, sanguinarine biosynthesis likely occurs in the ER, and ER-derived vesicles transport sanguinarine to the vacuole for storage, and/or become the precursors to alkaloid- containing vesicles in laticifers, and/or are targeted to the cell wall for exocytosis as a defense mechanism. SanRs localized in the cytosol may act against self-cytotoxicity upon reabsorption of secreted sanguinarine. For example, upon herbivory sanguinarine contained within the vacuoles and/or laticifers is released and transported across the membrane of intact, undamaged cells. Perhaps SanRs have evolved to reduce the “free” sanguinarine in undamaged cell to prevent self-cytotoxicity.

The roles of all four opium poppy SanRs in planta are unclear. Many questions remain, such as do the four SanRs have unique roles, or are they functionally redundant?

Characterization of opium poppy SanRs have revealed both similarities and differences between them. I have also shown SanRs are expressed in, and the encoding proteins are localized to the phloem of all tissues (Fig. 20 and 21). However, co-localization with cellular markers, and antibody scrubbing is required to discern the specific details regarding SanR localization (Fig. 20). Since sanguinarine only accumulates in opium poppy roots, seedlings, and fungal elicitor-treated cell cultures, it is expected that SanRs responsible for reducing sanguinarine in planta would be expressed in the roots (Facchini et al., 1996). Genes encoding other enzymes involved in the biosynthesis of sanguinarine, such as BBE, MSH, CFS, SPS and DBOX, expressed predominantly in roots, with little to no expression in stem, leaf, or flower bud (Fossati et al., 2014; Beaudoin and Facchini,

2013; Hagel et al., 2012). The only opium poppy SanR to mimic this expression profile is

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SanR2 (Fig. 21). Therefore, SanR2 likely plays a direct role in sanguinarine biosynthesis in the roots of opium poppy.

Sanguinarine, chelirubine, and macarpine are most abundant in members of the

Papaveraceae, but also present at low levels in most of the BIA-producing cell cultures

(Farrow et al., 2012). However, the reduced forms (dihydrobenzophenanthridines) of these alkaloids are detected almost exclusively in members of the Papaveraceae (Farrow et al., 2012). Phylogenetic analysis revealed the characterized SanR from E. californica clustered with SanR homologs from other Papaveraceae family members that accumulate benzophenanthridine alkaloids, such as Sanguinaria canadensis, Chelidonium majus,

Corydalis chelianthifolia, but did not cluster with SanR homologs from Ranunculaceae,

Berberidaceae, or Menispermaceae family members (Appendix A4). Interestingly,

P. somniferum SanR1, SanR2, and SanR4 cluster with E. californica SanR, whereas

P. somniferum SanR3 clusters with homologs from Ranunculaceae, Berberidaceae, and

Menispermaceae families (data not shown). I have shown that P. somniferum SanRs are capable of reducing sanguinarine, as well as chelerythrine, chelirubine, and macarpine, to the corresponding dihydrobenzophenanthridines alkaloids in vitro (Fig. 13), which is consistent with the ability of E. californica SanR to reduce both sanguinarine and chelerythrine (Weiss et al., 2006).

Although silencing SanR in planta rarely resulted in an increase in sanguinarine, this is likely due to aphid infestation. Together, biochemical characterization of SanRs, gene expression analysis, and immunolocalization data support SanRs as a detoxifying enzyme in planta. However, additional experiments are required to differentiate, and characterize the individual roles of four SanRs from Papaver somniferum.

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List of Appendix Tables and Figures

Table A1.1. List of primers designed to amplify Papaver somniferum reductases ...... 151

Table A1.2. Predicted open reading frame of P. somniferum dehydroreticuline reductase candidates ...... 163

Figure A1.1. Expression of dehydroreticuline reductase (DRR) candidates in vivo ..... 166

Figure A2.1. Preliminary SanR assays using NADPH or NADH as a co-factor ...... 168

Figure A2.2. Preliminary temperature curves for San1 and SanR3B ...... 169

Figure A4.1. Multiple sequence alignment of sanguinarine reductase candidates from 20 BIA-producing species...... 176

Figure A4.2. Phylogenetic trees for enzymes involved in BIA biosynthesis...... 178

148 Appendix A1: Cloning dehydroreticuline reductase candidates

Primers were designed to analyze the expression of select cytochrome P450s and reductases from P. somniferum using the SYBR Green method of qPCR (Table A1.1).

Primers were also designed to amplify putative endogenous reference genes from

P. somniferum and P. rhoeas using the SYBR Green method of qPCR (Table A1.1).

Several primer pairs were tested, but melting curves were analyzed to determine best set for downstream applications.

Primers were designed to amplify several P. somniferum DRR candidates

(Table A1.1 and A1.2). Using microarray data, DRR candidates were chosen as transcripts predicted to encode reductases or epimerases, and were upregulated in morphinan-producing cultivars, T, 40 or Marianne (M), versus a low alkaloid cultivar,

Przemko (P) (Desgagné-Penix et al., 2012). Cultivar T contains high levels of thebaine and oripavine, but low levels of codeine and morphine; cultivar 40 contains high codeine and morphine levels; and cultivar Marianne contains codeine and morphine along with high noscapine levels (Desgagné-Penix et al., 2012). As the partially purified dehydroreticuline reductase had an apparent molecular weight of 30 kDa, twelve candidates (DRR1-DRR12) unregulated in M, 40 or T with predicted molecular weights ranging from 28 to 34 kDa were cloned into the pGEM-T subcloning vector (De-

Eknamkul and Zenk, 1992). DDR2, DRR3, DRR5, DRR7-DDR12 amplicons were 100% identical to sequences in the transcriptome, but mutations in the restriction enzyme sequence of DDR9 prevented downstream cloning into pQE30. DRR1 and DDR4 were not identical to sequences in the transcriptome, and DDR6 could not be amplified.

Therefore, nine of the twelve DDR candidates were cloned into the pQE30 expression

149 vector. However, only six candidates were expressed when cultures were induced with

IPTG (Fig. A1.1). Crude extracts were used to assay dehydroreticuline activity. None of the six DDR candidates were able to reduce dehydroreticuline to reticuline (data not shown).

Alternatively, genes encoding proteins that share a high degree of sequence similarity to previously characterized reductases, noscapine synthase (NOS), SalR, COR, and SanR, were to be cloned from Papaver somniferum and tested for DRR activity

(Table A1.1). Sixteen sequences were identified in the stem and/or root transcriptomes with more than 50, 50, and 35% amino acid sequence similarity to COR (GenBank

Accession No. AF108432), SalR (GenBank Accession No. DQ316261), and NOS

(GenBank Accession No. Q659007), respectively. Many of the sequences were cloned into pGEM-T or pQE30 with the ultimate goal to express and characterize each of these enzymes, and compare activity to the previously characterized enzymes (Winzer et al.,

2012; Ziegler et al., 2006; Unterlinner et al., 1999).

150 Table A1.1. List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

PsoCOR2-qPCR-F1 5'-ATCCCTCCAGCTGTGAATCAA-3' N/A Forward N/A 74 PsoCOR2-qPCR-R1 5'-GCGTTGCAATACTCTCTCAGCTT -3' N/A Reverse N/A

PsoCOR2-qPCR-F2 5'-TGCAATCTCTGTACTGGGATCAA-3' N/A Forward N/A 66 PsoCOR2-qPCR-R2 5'-ACCTCAGAACCCAAAACTGCAT-3' N/A Reverse N/A

PsoCOR2-qPCR-F3 5'-CCATGCTCTGGTGCACTGAT-3' N/A Forward N/A 69 PsoCOR2-qPCR-R3 5'-GATTCCTCAGCGAATTCTGAAGA-3' N/A Reverse N/A

PsoCOR2-qPCR-F4 5'-TGCTCTGGTGCACTGATGCT-3' N/A Forward N/A 74 PsoCOR2-qPCR-R4 5'-CAATTTAAGATTCCTCAGCGAATTC-3' N/A Reverse N/A

PsoCYP82-qPCR-F1 5'-GACCACCATCTGGACCCTTTC-3' N/A Forward N/A 72 PsoCYP82-qPCR-R1 5'-TCCACTTCTTGTTTTGCCTTGTC-3' N/A Reverse N/A

PsoCYP82-qPCR-F2 5'-GACACCACAAAACTGACCACCAT-3' N/A Forward N/A 61 PsoCYP82-qPCR-R2 5'-TTTTGCCTTGTCCAACACATG-3 N/A Reverse N/A

151

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Namea Sequenceb RE Direction Amplicon Protein size size (bp) (kDa)

PsoCYP82-qPCR-F3 5'-CCAGGCAATCATCAAAGAATCA-3' N/A Forward N/A

PsoCYP82-qPCR-R3 5'-CACAATCTTCGCCGCTCAGT-3' N/A Reverse 77 N/A

PsoCYP82-qPCR-F4 5'-TCGCCGAATTATCGTTCAATG-3' N/A Forward N/A 80 PsoCYP82-qPCR-R4 5'-CCTGCTTGGAGCACCTGTCT-3' N/A Reverse N/A

Pso-qPCR-ELF-F1 5'-TTTGAGGCCGGTATCTCTAAGG-3' N/A Forward N/A 74 Pso-qPCR-ELF-R1 5'-TGCTTGACACCAAGGGTGAA-3' N/A Reverse N/A

Pso-qPCR-ELF-F2 5'-CCTCCCAGGTCATCATCATGA-3' N/A Forward N/A 84 Pso-qPCR-ELF-R2 5'-CAATGTGAGATGTGTGACAGTCAAG-3' N/A Reverse N/A

Pso-qPCR-ELF-F3 5'-TGAGCCTAAGAGACCCACAGACA-3' N/A Forward N/A 87 Pso-qPCR-ELF-R3 5'-CCCACTGGCACAGTTCCAA-3' N/A Reverse N/A

Pso-qPCR-UBC-F1 5'-CAGCTTCTGGATGAGCCATCA-3' N/A Forward N/A 69 Pso-qPCR-UBC-R1 5'-GGAGCCCTGCTTGGACTCT-3' N/A Reverse N/A

152

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

Pso-qPCR-UBC-F2 5'-TCGTCGCACACAAGTTGGA-3' N/A Forward N/A 77 Pso-qPCR-UBC-R2 5'-GCTAGGCTCTTCAAGGAATACAAAGA-3' N/A Reverse N/A

Pso-qPCR-ACT-F1 5'-GTCTGGATTGGTGGGTCCAT-3' N/A Forward N/A 68 Pso-qPCR-ACT-R1 5'-TCAGCCTTGGAGATCCACATC-3' N/A Reverse N/A

Pso-qPCR-ACT-F2 5'-GTGCCAATCTATGAGGGTTATGC-3' N/A Forward N/A 70 Pso-qPCR-ACT-R2 5'-TCAGATCACGGCCAGCAA-3' N/A Reverse N/A

Pso-qPCR-ACT-F3 5'-GGGATCGCAGACCGTATGA-3' N/A Forward N/A 74 Pso-qPCR-ACT-R3 5'-GGTGCAACCACTTTGATTTTCA-3' N/A Reverse N/A

Pso-qPCR-UBQ10-F1 5'-TGGATGTTGTAATCAGCGAGAGTAC-3' N/A Forward N/A 74 Pso-qPCR-UBQ10-R1 5'-CCAGACCAGCAACGTTTGATTT-3' N/A Reverse N/A

Pso-qPCR-UBQ10-F2 5'-GGTGGACTCCTTCTGGATGTTG-3' N/A Forward N/A 75 Pso-qPCR-UBQ10-R2 5'-CGTTTGATTTTCGCAGGAAAG-3' N/A Reverse N/A

153

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

Pso-qPCR-UBQ10-F3 5'-GGGAACACAAACGACACCAAA-3' N/A Forward N/A 80 Pso-qPCR-UBQ10-R3 5'-TCGTCTTCGTGGTGGTAACTAGAG-3' N/A Reverse N/A

Pso-qPCR-GAPC-F 5'-CCCAGCACTTAATGGAAAATTGAC-3' N/A Forward N/A 80 Pso-qPCR-GAPC-R 5'-TCACAGTAAGATCAACCACTGAAACA-3’ N/A Reverse N/A

PrhCYP82-qPCR-F1 5'-GGGTCTCGTTGAACCTTCCAT-3’ N/A Forward N/A 76 PrhCYP82-qPCR-R1 5'-ATTGTGTGGTTGGTGGGTTTC-3' N/A Reverse N/A

PrhCYP82-qPCR-F2 5'-CGATGCTTCATCAATTGCTACTG-3' N/A Forward N/A 77 PrhCYP82-qPCR-R2 5'-GCGGATTCCGATCAGACACT-3' N/A Reverse N/A

PrhCYP82-qPCR-F3 5'-CCTGGATGTAGACAAGGCTACGA-3' N/A Forward N/A 77 PrhCYP82-qPCR-R3 5'-TGGAAAGAAAAGGAGTTCAACCA-3' N/A Reverse N/A

PrhCOR-qPCR-F1 5'-TGATTCACAGCTGGTGGGATAT-3' N/A Forward N/A 72 PrhCOR-qPCR-R1 5'-TGTCAGCAATTTCTCCTGCAA-3 N/A Reverse N/A

154

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

PrhCOR-qPCR-F2 5'-CCTGAAGCTTTTTGCAGGAGAA-3 N/A Forward N/A 79 PrhCOR-qPCR-R2 5'-GCAGCCATGGAAGAGTGTCA-3' N/A Reverse N/A

Prh-qPCR-ELF-F 5'-GCAGCCTCCTTCTCGAACCT-3' N/A Forward N/A 75 Prh-qPCR-ELF-R 5'-CACCACTGGTCACTTGATCTACAAG-3' N/A Reverse N/A

Prh-qPCR-TUB-F1 5'-CTCATTCCCTTCCCTCGTCTT-3' N/A Forward N/A 69 Prh-qPCR-TUB-R1 5'-CTGGGAACCCCGAGATGTG-3' N/A Reverse N/A

Prh-qPCR-TUB-F2 5'-GCAGATGTGGGACACCAAGAA-3' N/A Forward N/A 74 Prh-qPCR-TUB-R2 5'- TGGCTGAGGCAGTGAGGTATC-3' N/A Reverse N/A

Prh-qPCR-UBC-F1 5'-TGTCATCGCACACAAGTTGGA-3' N/A Forward N/A 80 Prh-qPCR-UBC-R1 5'-GGCTAGGCTCTTCAAGGAATACAA-3' N/A Reverse N/A

Prh-qPCR-UBC-F2 5'-GTTAGTGTCATCGCACACAAGTTG-3' N/A Forward N/A 84 Prh-qPCR-UBC-R2 5'-GCTAGGCTCTTCAAGGAATACAAAGA-3' N/A Reverse N/A

155

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

Prh-qPCR-ACT-F1 5'-GACACGGAGCTCATTGTAGAAGGT-3' N/A Forward N/A 78 Prh-qPCR-ACT-R1 5'-ATTGAGCATGGTATTGTCAGCAA-3' N/A Reverse N/A

Prh-qPCR-ACT-F2 5'-GCGACACGGAGCTCATTGT-3' N/A Forward N/A 80 Prh-qPCR-ACT-R2 5'-ATTGAGCATGGTATTGTCAGCAA-3' N/A Reverse N/A

Prh-qPCR-ACT-F3 5'-CACAGTCCCCATCTATGAAGGTT-3' N/A Forward N/A 75 Prh-qPCR-ACT-R3 5'-GTCAGATCCCGTCCAGCAA-3' N/A Reverse N/A

Prh-qPCR-UBQ10-F 5'-TGATTTGCAGGAAGTGCTATGC-3' N/A Forward N/A 79 Prh-qPCR-UBQ10-R 5'-TGGTTGCTGTGACCACACTTCT-3' N/A Reverse N/A

Prh-qPCR-GAPC-F1 5'-CAACGTGGACGATCAAGTCAATAA-3' N/A Forward N/A 79 Prh-qPCR-GAPC-R1 5'-AACTTCGTCAAGCTTGTGTCATG-3' N/A Reverse N/A

Prh-qPCR-GAPC-F2 5'-CATCATCTCTCTGTAGGGCAACTC-3' N/A Forward N/A 79 Prh-qPCR-GAPC-R2 5'-GCAAAGATCAAGATCGGAATCAA-3' N/A Reverse N/A

156

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein Namea Sequenceb RE Direction size (bp) size (kDa)

DRR1-F 5'-GGATCCATGAATTATGCACAAAGTAGTAGTG-3' BamHI Forward 816 29.2 DRR2-R 5'-GGTACCTCAATCTTGTGTAGGGAAGAAAG-3' KpnI Reverse

DRR2-F 5'-GGATCCATGGAGGTTGAGAAAGTAGAGAG-3' BamHI Forward 840 30.0 DRR2-R 5'-GGTACCCTAGTCATGAGTTGGAAAAAACC-3' KpnI Reverse

DRR3-F 5'-GGATCCATGGAGTCTCCATTCAAGG-3' BamHI Forward 912 32.2 DRR3-R 5'-GGTACCCTACATTTTACTGCGGCTTG-3' KpnI Reverse

DRR4-F 5'-GGATCCATGGCTACAATCCAATGC-3' BamHI Forward 849 30.3 DRR4-R 5'-GGTACCTCAATTTCTCACCGGAGATC-3' KpnI Reverse

DRR5-F 5'-GGATCCATGGTATTCCTCCAAACTCATTC-3' BamHI Forward 888 31.7 DRR5-R 5'-GGTACCTTAGCGTTGTTTGATAGAGCC-3' KpnI Reverse

DRR6-F 5'-GGATCCATGGCAGAAGCACTCCTC-3' BamHI Forward 915 33.5 DRR6-R 5'-GGTACCTCAAAATGACGATACCTCTTC-3' KpnI Reverse

157

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein Namea Sequenceb RE Direction size (bp) size (kDa)

DRR7-F 5'-GGATCCATGGAAGAAACCATCTCTACAAC-3' BamHI Forward 921 33.8 DRR7-R 5'-GGTACCTCAAAAGGTTGAAATCTGCATC-3' KpnI Reverse

DRR8-F 5'-GGATCCATGGCATTAAAAGAAGTTAACG-3' BamHI Forward 885 32.6 DRR8-R 5'-GGTACCTTATATCTTCGAGTACAAGAATGG-3' KpnI Reverse

DRR9-F 5'-GGATCCATGCCTGCACCAGTAATG-3' BamHI Forward 921 32.5 DRR9-R 5'-GGTACCCTACAATCCTACACAGTTTCTTG-3' KpnI Reverse

DRR10-F 5'-GGATCCATGTCTAAACTAAGATTAGAAGGTAAAGTAGC-3' BamHI Forward 807 27.9 DRR10-R 5'-GGTACCTCATGAAGTAGCAGAACTAACATTAATG-3' KpnI Reverse

DRR11-F 5'-GGATCCATGGCGCTGGATAATGC-3' BamHI Forward 810 27.9 DRR11-R 5'-GGTACCCTAAACATATCCGCCATTAACAC-3' KpnI Reverse

DRR12-F 5'-GGATCCATGGCAGAAGCCATAGTTG-3' BamHI Forward 804 27.7 DRR12-R 5'-GGTACCTTATCCATGAAGAGAGCCAC-3' KpnI Reverse

158

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

COR-F* 5'-GGATCCATGGAGAGTAATGGTGTTCCTATG-3' BamHI Forward 966 35.8 COR-R* 5'-GGTACCTCAATCCTTCTCATCCCAGAAC-3' KpnI Reverse

COR2-F 5'-GGATCCATGGAGAGTAGTGGTGTACC-3' BamHI Forward 966 35.7 COR2-R 5'-GGTACCTCAAGCTTCATCATCCCAC-3' KpnI Reverse

COR3-F 5'-GGATCCATGGAGAGTAATGGTGTTCC-3' BamHI Forward 969 36.2 COR3-R 5'-GGTACCTCAAACTTCTCCGTCCCAG-3' KpnI Reverse

COR4-F 5'-GGATCCATGGTGAACACTGGTGTACC-3' BamHI Forward 972 35.9 COR4-R 5'-GGTACCTCAAGCAGCTTTTTCATCC-3' KpnI Reverse

COR5-F 5'-GGATCCATGGAGAATGTAATACCTGCAG-3' BamHI Forward 966 36.0 COR5-R 5'-GGTACCTCAAACTTCTCCGTCCCAG-3' KpnI Reverse

COR6-R 5'-GGATCCATGCCTATTTTAGGTATGGGAAC-3' BamHI Forward 961 35.6 COR6-R 5'-GGTACCTCAGACTTCGTCATCCCAG-3' KpnI Reverse

159

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

COR7-F 5'-GGATCCATGGAGGCAATTACCATGG-3' BamHI Forward 969 35.6 COR7-R 5'-GGTACCTCAGAGCTCAGCTTCCATC-3' KpnI Reverse

SALR2-F 5'-GCATGCATGCCTGAAACATGTCC-3' SphI Forward 936 34.2 SALR2-R 5'-GTCGACTCAAAATGCAGATAGTTCTGAAC-3' SalI Reverse

SALR3-F 5'-GGATCCATGGGATTTCAGTTGCG-3' BamHI Forward 820 33.2 SALR3-R 5'-GGTACCTCAAAATGGTGTTATTTCTCC-3' KpnI Reverse

SALR4-F 5'-GGATCCATGGATTCAAAAACTGAAAC-3' BamHI Forward 915 33.0 SALR4-R 5'-GGTACCTCAAAATGCAGATAGTTCTG-3' KpnI Reverse

SALR5-F 5'-GGATCCATGAATATGACAGAAACACTCC-3' BamHI Forward 921 33.8 SALR5-R 5'-GGTACCTCAAAATGACGATACCTCTTC-3' KpnI Reverse

SALR6-F 5'-GGATCCATGGAAGAAACCATCTCTACAAC-3' BamHI Reverse 921 33.8 SALR6-R 5'-GGTACCTCAAAAGGTTGAAATCTCCATC-3' KpnI Reverse

160

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.

Amplicon Protein size Namea Sequenceb RE Direction size (bp) (kDa)

NOS2-F 5'-GGATCCATGGAAGGTGGCGGAAATG-3' BamHI Forward 993 36.0 NOS2-R 5'-GGTACCCTACAAGAAACCCTTTTCTTTACAGC-3' KpnI Reverse

NOS3-F 5'-GCATGCATGGCCGATTCAAAGAAG-3' SphI Forward 1020 36.9 NOS3-R 5'-GTCGACTCATTTCTGCAGAAGTCCTAC-3' SalI Reverse

NOS4-F 5'-GGATCCATGGGTTCTATTGGCATTATTG-3' BamHI Forward 1119 41.7 NOS4-R 5'-GGTACCTTATTCCAAAACGGCAGG-3' KpnI Reverse

NOS5-F 5'-GGATCCATGCCTGAATACTGTGTAACG-3' BamHI Forward 963 35.2 NOS5-R 5'-GGTACCTCAGAGAAACCCCTTGTCTTG-3' KpnI Reverse

NOS6-F 5'-GGATCCATGGAGAAACAAAGGGTTTG-3' BamHI Forward 951 34.8 NOS6-R 5'-GGTACCTCAAAGCAAGATACCCTTCTC-3 KpnI Reverse

161

aPrimers were designed to amplify P. somniferum (Pso) or P. rhoeas (Phr) codeinone reductases (CORs) or cytochrome P450s (CYP82) for expression analysis using the SYBR Green method of qPCR. Several endogenous reference genes were tested: elongation factor (ELF), ubiquitin C (UBC), actin (ACT), polyubiqutin10 (UBQ10), glyceraldehyde-3-phosphate (GAPC), or tubulin (TUB). Coding sequences of dehydroreticuline reductase (DRR) candidates, codeinone reductases (COR), salutaridine reductases (SALR), and noscapine synthase (NOS) from P. somniferum were amplified for downstream cloning into pQE30 or pRSET expression vectors. F: forward primer complementary to 5’-end of sequence; R: reverse primer complementary to 3’-end of sequence; forward and reverse primer pairs are labeled with the same number; *primers designed to amplified previously characterized COR. bItalics: restriction enzyme (RE) recognition sequence; unformatted: template sequence.

162

Table A1.2. Predicted open reading frame of P. somniferum dehydroreticuline reductase candidates. Candidate Sequence (5’ to 3’)

ATGAATTATGCACAAAGTAGTAGTGATCCAAGTCTTAGATGGTCTCTTAAGGGAAAGACTGCACTTGTCACTGGCGGAACCAAAGGGATTGGACACGCTATC GTCGAGGAGTTGGCTGGATTTGGTGCAGCTGTTCATATCACTTCTCGACATGAAAATGAAATCGAGGAGTGCTTGCGAAATTGGAGAGAAAAAGGCTATACA GTGACTGGGTCGGTCAGTGATGCTTCAGTTCCGGTTGATAGAGAGAAATTAATCAAGACTATTTCATCTGTTTTTGGGGGCAAACTAAACATCCTTGTTAACA ACGTTGGGGGAGCTGTGTTCAAAGACACCGTGGATTACACAGATGAGGACCATGCGAAAGTTATGTCTACTAACTTCGACTCTAGCTACTATTTATCAAAACT DRR1 AGCACACCCTCTCTTGAAAGCCTCTGGCTCAGGAAGCATTGTGTTCATTACATCTGTTGCGGGCATTGTTGCTGCACCGAAGGTGTCCAGTTATGCGGCATGT AATGGAGCTATTAATCAAGTCACAAAGAACTTTGCTTGTGAATGGGCGAAAGATAAGATACGGGTCAATAGCGTAGCACCATGGTATATTAGAACCAGGCTT GCAGAACATGATCTGCTATTATTTGAAGGGCTTGAAGACTCTATCGTAGCTAGAACTCCTATGAGGCGTCTTGGGAACCCAAATGAAGTTTCATCTCTTGTGG CATTCTTGTCCCTACCTTGTGCTTCGTACATCACTGGCCAAGTCATTTGTGTCGATGGTGGATTTGCAGTAAATGGTTTCTTCCCTACACAAGATTGA

ATGGAGGTTGAGAAAGTAGAGAGTAGTAGCAGTAAGAAGATTACAGTTGCTGGAGGAAATGGAAGATGGTCTCTCAATGGAATGACTGCTCTAGTCACTGGT GGTACTAGAGGAATCGGATATGCTGTTGTGGAGGAATTGGCTGGATTTGGGGCAAAAGTACATACTTGTTCAAGAAATGAAATTGAACTTAATCGATGTTTA CAAGAATGGAAGCAAAAGGGTTTTCAAGTTACTGGCTCTGTTTGTGATGTTTCGTCTTCAGAGGGTCGTGTCAAGCTCATGGATTCTGTGTCTGGTCTTTATAA CACCAAGCTCAATATCCTTGTTAATAATGTTGGCACAAATATAAGAAAACCGTCGGTGGAGTACACTGCTGAAGAATACTCGAAACTGATGTCTACCAACTTG DRR2* GAATCCTGTTACCACCTATGCCAACTTGCACACCCGCTTCTGAAATCTTCAGGGATGGGAAGTATTGTGTTTATCTCTTCTGTCGCTGGTGTGGCGGCATTGGC TACTGGGAGTATTTATGGAGCAACTAAAGGAGCAATGAATCAACTCGCAAAAAGTTTGGCATGTGAATGGGCGAAAGACCACATTAGGTGTAACTCTGTTGC ACCTTGGTACATCAAAACCTCGCTTGTCGAACATTTGCTTGAAGACAAAGAATTTGTAGATAGAGTAATCGCCCGTACTCCTCTTCGACGAGTTGGAGAACCG CAGGAGGTTGCATCACTGGTTGCCTATCTTTGCTTACCTGCTTCCTCTTACATCACGGGGCAGACTATCTCTGTTGATGGAGGGATGACTGTCAATGGGTTTTT TCCAACTCATGACTAG

ATGGAGTCTCCATTCAAGGCTGATATAGTGAAAGGGAAAGTAGCTTTGATTACTGGAGGAGGATCAGGAATTGGGTTTGAGATTACTAAAGAATTTGGTAGA CATGGAGCTTCTGTTGCTATCATGGGCAGACGCAAATCTGTTCTTGATTCTGCTGTCTCCTCTCTTCGCTCTCTCGGTATCCAGGCAATTGGATTAGAGGGAGA TGTGCGGAAGAAAGAAGACGCGGCTAGAGTTGTCGATTCAACATTTGAGCATTTTGGGAGGATAGACATTCTTGTTAATGCTGCTGCTGGAAATTTTCTTGTG ACTGCTGAGGATTTGTCCCCAAATGGATTTAAAACAGTTATGGATATTGATTCCGTTGGCACATTTACAATGTGCCACACAGCGTTGAAGTATATAAAGAAAG DRR3 GGGGTCTTGGGAGGGGTATGTCTGGTGGTGGAACCATAATGAACATAAGCGCTACGCTACATTATACAGCAGCTTGGTATCAAATCCATGTATCTGCCGCTAA GGCAGCTGTTGATGCCATTACAAGGTCGTTGGCGTTAGAGTGGGGTACGGATTATGATATAAGAGTCAACGGGATTGCACCAGGACCAATTGGTGACACTCC TGGCTTGAGTAAGCTTGCTCCCGATGAAATGAAAATCAACCATTCTGAAGACGCCAGGCCTCTGTATAAAGCAGGAGAGAAATGGGATATTGCTATGGCTGC TCTCTACCTAGCTTCAGATGCAGCCAAGTACATCAACGGTATGACACTTGTGGTCGATGGAGGAAACTGGTTGAGCCGGCCCCGCCACATTTCAAAAGAGGC AGTGAAGGAATTGTCTCGGGTTGTGGAAAAGAGATCCAGATCAGGTGCACCAGCACCTGCCAGGGGAGTTCCAAGCCGCAGTAAAATGTAG

ATGGCTACAATCCAATGCATCAAGGCTCGTCAGATCTTTGATAGTCGTGGTAACCCAACCGTTGAGGTTGATATTAAACTATCCAATGGAACTTTCGCCAGAG CCGCTGTTCCAAGTGGTGCATCTACTGGTGTTTACGAAGCTCTTGAACTACGTGATGGAGGTTCAGAATACCTAGGAAAGGGAGTTTCCAAGGCTGTTGACAA TGTTAACTCCATCATTGGGCCTGCATTGATCGGAAAGGACCCAACACAACAAACCGAAATTGATAACTTCATGGTGCGAGAACTTGACGGAACTACCAACGA GTGGGGTTGGTGCAAGCAAAAGCTTGGAGCCAATGCTATCCTAGCAGTGTCTCTTGCCGTTTGCAAAGCTGGAGCCAGTGTTTTGGACATTCCCCTTTACAAG DRR4 CATATTGCCAACCTTGCTGGTAACAAGAACTTGGTACTTCCAGTTCCTGCTTTCAATGTTATTAATGGAGGATCGCACGCAGGAAACAAGCTTGCAATGCAAG AGTTCATGATCCTTCCCGTTGGAGCCAAATCCTTCAAGGAGGCAATGAAAATGGGAGTTGAAGTATACCACAATTTGAAGTCCGTCATCAAGAAGAAGTACG GTCAAGATGCAACCAATGTTGGTGATGAAGGTGGCTTTGCTCCCAACATCCAAGAGAACAAGGAAGGACTTGAGTTGCTTAAGACTGCTATTGCTAAAGCTG GCTACACTAAAGAAGTTGTCATCGGAATGGATGTTGCTGCCTCAGAGTTTTACGGATCAGACAAAACCTATGACTTGAACTTCAAGAAGAGAACAACAACGG AGCAGCAAAGATCTCCGGTGAGAAATTGA

163

Table A1.2 (continued). Predicted open reading frame of P. somniferum dehydroreticuline reductase candidates.

Candidate Sequence (5’ to 3’)

ATGGTATTCCTCCAAACTCATTCTCTCACAAGAAACATCAAATCCCCCTTTACTTCATCTTCATCAGCATCACCAATTGTTAACCACTCCAAACCTTTCTCTTCT GCTAACACATCTATTAGAGCTACAACAACAAAGATGGAAAACACAGGAATTGAGGGTTCAAATGTAAATGACAATAAAAGGAAAATATTTGTGGCTGGGGC TACTGGAAGTACTGGTAAAAGAATCGTTGAACAACTTTTAGCCAAGGGTTTTGCAGTTAAGGCTGGTGTTCGTGATGTTGACAAAGCTAAGACTACTTTTTCA GATAACCCATTTCTCCAAATCGTGAAGGCTGATGTAACTGAGGGTTCAGCTAAGTTAGCTGAAGCTATTGGTAATGATGCTGAGGCTGTTATTTGTGCTACCG DRR5 GATTTCGTCCTTCATTGGATTTGTTTACTCCTTGGAAGGTCGATAATTTTGGCACAGTAAACCTTGTAGATGCATGCAAGAAAAGTAATGTGAATAGATTTATT CTCATCAGTTCCATTCTAGTCAATGGTGCTGCAATGGGACAAATCTTTAATCCAGCTTACCTATTCCTAAACGTATTTGGACTAACGTTGATAGCCAAACTACA AGCGGAGAATTATATCAGGAAATCGGGTATAAACTACACAATTATAAGGCCTGGTGGATTGAAAAATGACCCCCCAAGTGGAAATTTAGTCATGGAACCTGA GGATACTCTGTCTACAGGTTCTGTATCTAGAGATCTGGTTGCAGAAGTAGCTGTTGAGGCATTAGGCCACTCTGAATCTTCTTACAAGGTAGTGGAGATAGTT TCCCGTCCTGAAGCTCCAAAACGCTCATTTGAAGATCTCTTTGGCTCTATCAAACAACGCTAA

ATGGCAGAAGCACTCCTCAATTCTGAAGTAAAGAGGTGTGCAGTAGTTACAGGTGCCAACAAGGGGATTGGTTTGGAGATTTGTAGGCAATTAGTTTTTAAT GGAATCTTTGTTGTATTGACATCTAGAGATAGAAACAAGGGTCTTGAAGCTGTTGAAAATCTCAAAAAATCTGGACTCCCAAATGTGATTTTTCATCAATTAG ATGTTATGAATCTAACTTCTGTCTCATCCTTGGCTGAATTCATCAAAACCCACTTTGGAAAACTTGATATCTTGGTGAATAATGCAGGCATTGGTGGGGTGAG AATAGTAGACAAGGATCAATTAAAGGTTCTGATGCTTGAGGATGAAAAGTATTTGGAGAACCCAAAGTTGAAACAGATATGGACAGAACCGTATGACGACG DRR6 CAGAAAAATGTCTTAAAACAAATTACTACGGCGTCAAGGCGGTGACTGAAGCCTTTATTCCTTTACTTGAACTATCTGATTCAAGAATAATTGTCAATGTTTCT TCTGCCATGGGGATGCTGAAGAATGTTGGCAATGAAAAGGCTTTCGAAGTGCTCAGCGATGCTGATTGTTTAACGGAAGAAAGAATAGATGTGGTGGTGAAT ACATTTCTAAACGATCTTAAAGAGGGTTGCTTAGAAGCAAAAGGTTGGCCAACATTACTTTCTGCTTATACAATCTCAAAAGCATCAGTTAATGCGTACACTA GGATTTTGGCAAAAAAATTCCCTACTTTTCGCATAAACTGTGTTTGTCCTGGTTTTGTCAAAACCGATATAAACTTTAACAGTGGGGTCTTGACTGTTGAACAA GGTGCTAAAAGTCCTGTAAGATTAGCTCTTTTACCAGATAACATAACTTCTTCTGGGCTCTTCTTTGTTCGTGAAGAGGTATCGTCATTTTGA

ATGGAAGAAACCATCTCTACAACAGCAGAAAAGAGGTGTGCGGTTGTTACAGGAGCTAATAAAGGAATAGGACTAGAGATATGTCGTCAGCTCGCTATTAAT GGTATCACCGTTATACTAACCGCAAGAGATGAGACCAGGGGAACTCGATCTGTTGAAGCTCTTAAAGGGTCAGGACTTGCTGGTGTGATTTTTCATCAGCTTG ATGTAAACGATGCAACTAGTGTTACTTCATTGGCTGGTTTCATCAGAACTCGATACGGGAAACTTGACATCTTGGTAAACAATGCAGGGGACAACGGAGTAA GACTACAAATTACTGAGGAGGCTATGAAAGACATGAACTTTCGATATGGTGATGAGAATGATGAGATTGCCAAGTTGTTGGAGGAATCCATTGAGGAGACAT DRR7* ACGAGAGGGCAAAACAATCCATAGAAACAAATTACTATGGAACCATAAGAATAACTGAAGCACTGCTTCCACTTCTTCAACTCTCCAATTCAGCAAGAATTG TGAATGTTTCCTCCGTATATGGTCAACTAAAGTTTATCTCGAGTGAGACGATTAAAGAGGAGCTAAGAAATGTTGATTGCTTAACGGTAGAGAAACTGGACG AGCTTATGCAGAAGTTCTTAAAGGATTTTAAGGATGGGATGTTGGAGACTAATGGATGGCCTGTATTAGTTTCTGCGTATAAAGTCTCGAAAGCTGCTATCAA TGCCTACACTCGAATTCTTGCGAGGAAGTTCCCAACTTTTCGTGTTAATGTTGTTCATCCTGGTTTGGTTAAAACAGATATTGCATTCCAACAAGGTAACTTAA CACCGGATGAAGGAGCTAAAGCACCGGTTATGGTGGCATTGTTGCCTTCTGATGGCCCTTCTGGTTTCTACTTTGACCAGATGCAGATTTCAACCTTTTGA

ATGGCATTAAAAGAAGTTAACGAGCCCTCTGCTTCCTTAACTAGGTGGTGGTCGGGAAACACCGTAGCGGTTGTGACCGGAGGAAATAGAGGGATCGGATTC TCTCTAGTTAAGAAACTCGCCGAGCTTGGATTAACTGTAGTCCTAACTTGTAGAGATGATTCTAAAGGTCAAGAAGCAATTGAATCACTCAAATCTCAAGGAC TCAATAATGTTCGATTCTTCCGATTAGATGTTATGGACACTGCTTGCATCAATGAGCTGGTTTCATGGTTGAAGGAAGCATTTGGAGGTCTTGATATTCTTGTG AATAATGCTGCTGTGTCGTTCAACGAGATCAACGAGAACTCGATGCAACACGCCGAAACCACCATCAAGACAAACTACTATGGACCGAAGTTGTTAACCGAA DRR8* GCTCTGCTACCACTGTTTCGGCGTTCGGAATCCGTAAGCAGGATTTTGAATGTTAGCTCGCGTCTTGGCTTGTTGAACAAGGTGAGTAATCCTGTTGTAAGGG AGTTATTAGAAGACGAAGACAGATTATGCGAAGAACGTATAGATTTTGTTGTAAATCGATTTCTTGAAGATGTTACTACTGGTACTTGGGAAAGAGAAGGAT GGCCAAAGGTATGGACAGATTACGCAGTCTCGAAAGTAGGATTGAATGCGTACTCTCGAGTTTTAGCTAAGCGTTATGATGGGTTGGGATTATCTGTCAATTG TTTATGCCCCGGGTTTACACAGACAGACATGACCGCCGGAAAAGGAAATCACTCGGCGGATTCAGCTGCGGAAATGGCTGCACAAATTGTCTTACTACCACC TGAGAAACTTCCCACTGGCAAGTTTTATATAAAAAACAAACCATTCTTGTACTCGAAGATATAA

164

Table A1.2 (continued). Predicted open reading frame of P. somniferum dehydroreticuline reductase candidates.

Candidate Sequence (5’ to 3’)

ATGCCTGCACCAGTAATGACTCATGAGAATGTAGCAGCATCCATTCAAGGATCAGGAATGAACCATGTCATGAACTCTCCTGCACCAAGAAGGTTGGAAGGA AAAGTTGCGATCATCACCGGTGGTGCGAGGGGGATTGGGGAAGCAACTGTAAGACTCTTTGTAAGACAAGGTGCAAAAGTAGTCATTGCTGATGTTGAAGAT GCTACTGGAACTTCACTTGCAAATTCATTAGCTCCTTCAGCTACATTTGTACATTGTGATGTCACCAGAGAAGAAGATATCGAGAACCTAATCGATTCAACAA TAGCACATTACGGGCGACTGGATATACTTTTCAACAACGCTGGTATTCTCGGAAACCAATCAAAACGGAAAAGCATTTTGAACTTTGATGCTGATGAGTTCGA DRR9 CTCAGTTATGCGTGTTAATGTGCGAGGGACTGCATTAGGTATGAAACACGCTGCACGAGTTATGATGCCAAGAGGTACCGGATGTATCATCTCAACAGCCAG TGTGGCCGGAGTCATGGGAGGATTTGGACCTCACGGGTACACGGCTTCGAAGCATGCCATCGTCGGACTTACAAAGAATACGGCTTGTGAATTAGGAAGGTA TGGGATTAGAGTTAACTGCATTTCCCCATTTGGTGTTGCAACTTCAATGCTTGTTAATGCATGGAGGAAAGTTGAAGATGAAGATGAAGAAGATAGTATGGAT TTTGGAGGACCTTCTGAAAAAGAAGTTGAGAAAACGGAGGAGTTTGTGCGTGGTTTAGCAGATCTTAAAGGAACAACCCTTAAACCAAGAGATATTGCTGAG GCTGCTTTGTTTCTTGCTAGTGATGAATCAAGATATGTAAGTGGTCATAATTTAGTTGTGGATGGAGGAGTTACTACTTCAAGAAACTGTGTAGGATTGTAG

ATGTCTAAACTAAGATTAGAAGGTAAAGTAGCTATAATTACTGGAGCAGCAAGTGGTATTGGCGAAGCAACAGCGAGGCTATTCGTCGAACATGGTGCGTTC GTTGTAGTTGCAGACATTCAAGACGAATTAGGGGATCAAGTTGTATCTTCAATTGGTAAAGAGAAAGCTAGTTACAAACATTGCGATGTAAGTGTCGAAAAA CAAGTTGAAGAAACAGTAGCATTTGCTTTAGAGAAATATGGATCTCTAGATATTGTGTATAGCAATGCAGGTATGGGCGGATCTTTTTCGAGTATCCTTGATT TCAGCTTGGAAGATTTTAACAAGATTATTGCTACAAACGTATCCGGTGCAGCATTAATGATCAAACACGCTGCTCGTGCGATGTTAGACAGAAAAATCCGTGG DRR10* CTCGATTATATGTACTGCGAGTGTAGCTGCAGTTCAAGCTGGATTTGCACCACACTGTTACACAGCGTCTAAACACGCTGTGCTAGGATTGGTTCAATCAGCT TGTAGCGAACTTGGTGCTTACGGAATAAGGGTGAATTGTATTTCTCCATCTGGAGTTGGAACACCATTAGCCTGTGATATAGGCAAGATTAGTGCAAGGCATG TTGAAGAATATACAGCAAAAATGAGTCTTCTGAAAGGGATTATTTTGAAAGCTAAACACATTGCTGATGCTGCATTGTTTCTTGCATCCGATGATTCGGTTTAT CTGAATGGACATAATCTTGTTGTTGATGGCGGATTTACAGTTGCGGCTAGTAGCTTTCCCATTAATGTTAGTTCTGCTACTTCATGA

ATGGCGCTGGATAATGCGAACGCAACCCCTTCTTCTTCCTCCCTCCTACTGGAAAACCGGGTGGCGATAGTCACAGGTGCATCAGGTGGAATCGGTGGTGCAA TCGCCCGTCACCTTGCCTCTCTCGGTGCAAAATTAGTCCTCAGTTACTCCAGTAACTCAACCCAAACTGATCTCCTTGCCACTGAACTCAACAACTCTTCATCA TCATCCTCACAGCCAAAAGCCATATCAATCAAAGCCAATGTTTCAGATCCAGACCAAGTCAAATCGTTATTCGATCACGCCGAGAAGGTTTTCAACTCGCAAC CACATATCTTAGTTAACTCTGCCGGAGTATTAGATCCGAAATACCCTACAATCTCCAACACCAAGATCGAAGATTTTGATCACATATTCAACATAAACGCAAA DRR11* AGGAGCGTTCTTATGCGCCAGAGAAGCTGCTAACCGGTTGGTACGCGGTGGTGGTGGACGGATTATATTGATTTCATCGTCTATGGTTGGCGGATTGAAACCT GGGTTCGGTGCTTATGCTGCGTCGAAGGCGGCGGTGGAGACCATGATGAGAATTCTTGCGAAAGAATTGAAAGGGACTGGAATTACAGCTAATTGTGTTGCG CCTGGACCTATTGCAACTGACATGTTTTATGCAGGGAAAGGAGAAGAGCAAATTAAGAATGTGATTGCAGAATGTCCGTTGAGTCGACTCGGTGAAACTAAA GATGTTGCTCCTGTTGTTGGGTTTTTGGCTGGGGATGCTAGTGAGTGGGTTAATGGACAGGTTATCCGTGTTAATGGCGGATATGTTTAG

ATGGCAGAAGCCATAGTTGTTAAAAATGAGAAGAAGAAGCTGGAAGGAAAAGTGGTTATCATCACCGGAGGAGCCAGCGGTATTGGAGAGGCAACCGCAA GGCTATTCGCCAATCATAACCCAAGTATGATTGTCATTGCAGACATCCAAGACCAAAAAGGCCACGCGGTAGCAACGTCCATTGGTTCACAAATTTGTTCCTA CATCCACTGCGATGTCTCCGATGAACTACAAGTCAAATCGATGGTGGATTCCACAGTGAAGAGCTACGGCGGACTCGATATCATGTTTAGCAACGCTGGTATT GCTAACGGATGTCACCAAACAATCCTTGATATAGATTTGGCTGATTATGATCGTCTCATGGATATCAACACCCGTGGGATGCTTGCTTGCGTGAAACATGCTG DRR12* CCAAGGCCATGGTTGACGGCGGGGTGAAAGGTAGTATAGTTTGTACGGCAAGCACTGCAGCAACCTCGGCGCTTGATGGATACTTGGATTACACTATTTCTA AGCATGCAGTTTTGGGGTTGATGAGATCAGCTAGTCAACAACTCGGCAAATACGGTATTAGAGTGAATTCTGTATCTCCATCAGCTGTAGGAACTGCGATGCC ATGCAAGACCTATGGTACTGATGCAGAGGGCATTGAGAAGATGTTCATGAGTTCCACTGTCCTAGGAGGTGCCGGATTAGTTTTAAAAGTGAATCATGTGGCT GAAGCTGTGTTGTTCTTGGCTTCTGACGATTCTGCCTTCATTACTGGCCATAATTTGATGGTTGATGGTGGCTCTCTTCATGGATAA *Dehydroreticuline reductase (DRR) candidates were assayed for activity. None were able to reduce dehydroreticuline to reticuline.

165

A DRR11 DRR2 DRR8 DRR12 DRR7 DRR10

M U I S U I S U I S U I S U I S U I S

42.7 34.6

27.0

B DRR Candidate DRR Candidate

M 11 2 8 12 7 10 M 11 2 8 12 7 10

46 34.6 30 27.0 23

Figure A1.1. Expression of dehydroreticuline reductase (DRR) candidates in vivo.

(A) No expression was observed in uninduced (U) cell cultures. DRR candidates were expressed from the pQE30 vector only upon induction (I) with IPTG, and were present in the soluble fraction (S). (B) Recombinant DRR candidates are expressed upon induced with IPTG (left), and are detected on a Western blot using an anti-His antibody (right).

Expected sizes of DDR11, DRR2, DRR8, DRR12, DRR7, and DDR10 are 27.9, 30.0,

32.6, 27.7, 33.8, and 27.9 kDa, respectively.

166

Appendix A2: Biochemical characterization of SanRs

Preliminary data outlining the ability of SanRs to reduce benzophenanthridine alkaloids using NADPH or NADH as a hydrogen donor (Fig. A2.1). At the time, alkaloid concentration had not been determined spectrophotometrically. Only a constant volume was used in each assay for a given alkaloid. Assays were to be repeated using the same concentration of alkaloid for all assays in order to gain insight into substrate preference, and the perform enzyme kinetics (Km) for all substrates.

First time performing temperature curves, the reaction buffer was not pre-cooled or pre-heated to the appropriate temperature. Therefore, the majority of the reaction likely proceeded at room temperature, as assays were set up on the bench top. This would account for the little variation in dihydrosanguinarine production by SanR1 at 6, 16, 21,

31, and 38°C (Fig. A2.2).

167

A 100

) 80

% s 70 Chelirubine+NADPH

(

n u 60

i Chelirubine+NADH

f s 50

v 40 Macarpine+NADPH

o o 30 Macarpine+NADH

C m 20 A 10 Chelerythrine+NADPH 0 Chelerythrine+NADH SanR1 SanR2 SanR3B No Enzyme B 40

n i 35

i 30

)

n % 25

(

f 20 NADPH

o NADH o 15

e 10

o 5

C 0 SanR1 SanR2 SanR3B

Figure A2.1. Preliminary SanR assays using NADPH or NADH as a co-factor.

(A) Alkaloids were incubated without or with SanRs (500 ng) in 10 mM Tris buffer, pH

8.0, using 50 mM NADPH or NADH. Reactions were 100 μL each with no technical replicates. (B) SanRs were incubated with sanguinarine (2 μM) in 100 mM sodium phosphate with 100 μM NADPH or NADH for one hour. 50 μL reactions were quenched with 950 μL methanol, and 10 μL was injected into MS for analysis. 500 ng of SanR1 was used, versus 250 ng of SanR2 and SanR3B. Errors bars represent SD for three technical replicates.

168

A 6000000 SanR1

5000000

)

4000000

S 3000000

C n 2000000

(

m 1000000

A 0 0 10 20 30 40 50 60 70 80 Temperature (°C)

B 3500000 SanR3B 3000000

)

H 2500000

o 2000000

u 1500000

L 1000000

( 500000 0 0 10 20 30 40 50 60 70 80 Temperature (°C)

Figure A2.2. Preliminary temperature curves for San1 and SanR3B. (A) SanR1 or

(B) SanR3B was incubated with sanguinarine at 6, 16, 21, 31, 38, 42, 54, and 67°C. Error bars represent SD for two technical replicates. DHS: dihydrosanguinarine. Extrapolated temperature optimum for SanR1 and SanR3B is 21°C.

169

Appendix A3: SanR expression in transcriptome libraries

SanR1 is represented by two contigs in the stem Illumina library: comp9568_c0_seq1 (expression level 2.98) and comp9568_c0_seq2 (expression level

13.99). Coding sequences (no formatting) and 5’ UTR (double underline) are identical but differ in 3’ UTR region (single underline). Illumina data was compared to the Roche-

454 library (bold) to suggest that the 3’ UTR of SanR1 is more likely represented by comp9568_c0_seq2.

SanR2 is represented by two contigs in the root Illumina library: comp73923_c0_seq1 (expression level 17.19) and comp73923_c0_seq2 (expression level

80.05). Coding sequences (no formatting) and 5’ UTR (underline) are identical but differ in 3’ UTR region (double underline). Illumina data was compared to the Roche-454 library (bold) to suggest that the 3’ UTR of SanR2 is more likely represented by comp73923_c0_seq1.

SanR3 is represents by four contigs: comp1703_c0_seq1 (expression level 95.05) and comp1703_c0_seq3 (expression level 68.74) in the stem Illumina library, and comp80098_c0_seq1 (expression level 2.81) and comp80098_c2_seq1 (expression level

3.52) in the root Illumina library. The root contigs are identical in coding (no formatting) and 3’ UTR (single underline) sequences, but contig comp80098_c2_seq1 has a slightly extended 5’ UTR (double underline; extension is italicized). There is no 454 data for genes expressed in root tissue. Contigs comp1703_c0_seq1 and comp1703_c0_seq3 are partial, and are combined to form the complete SanR3 sequence. The 5’ UTR of SanR3 is consistent with the contig found in the 454 library (bold), except with a small extension at the 5’ end. Also, the 3’ UTR of SanR3 is consistent with the 454 database (bold).

170

>comp9568_c0_seq1 CTAGGCTATATTTTTTCTTATAATATTCTCTCTTCTAGAAGAAATTCGAATTTGGAGAAAAAACCCTTTGAT CAAAGGGTTTCTTTATAAAATCATACTCATCTCTTCACTTTCTTCTGCAAATTCATTTCACCACTAAATATA ATCAAAAAAGAAAAAAGGGTTCCTATATTTTCAATTAGATCTTGTTTTTACAGACTTACAAAATAAAAATAA AAATGGCAGAATCAAATCAAAAAATCACAGTCCTTGTCACTGGAGCTTCAGGCTTAACTGGTGAAATTGCAT TCAAGAAGCTGAAAGAAAGATCAGACAAATTTGTGGTTCGGGGTTTAGTAAGATCAGAAGCAAGTAAACAAA GACTTGGTGGAGGTGATGAAATTTTTCTAGGTGATGTCATGGATAAGAAAAGCCTTGAAACTGCTATGCAAG GAATTGATGCGTTGATTATACTAACAAGTGCTGTGCCAAAGGTAGTACCTGGTTCATATCCTGGTGCTGATG GCAAAAGAGCTGAGGATGTATTCGGTGAATCATTTGATTTCAATGGTCCAATGCCTGAATTCTATTACGAGG AAGGGCAATTCCCTGAACAGATTGATTGGATTGGACAAAAGAATCAGATCGATACTGCGAAATCTTGTGGTG TGAAACATATTGTTTTGGTTGGATCAATGGGTGGAACTGACCCTAATAATTTCTTGAATCACATGGCTAATG GAAACATACTTGTTTGGAAGAGAAAGGCTGAGCAATATTTGGCTGATTCTGGAATCCCATACACAATTATAA GGGCTGGTGGTTTAGATAACAAGGTAGGTGGCAGGGAATTGTTGGTCGGGAAGGATGATGAGCTTCTCTCTA CTGAAAACCATTTCATTGCTAGGGCTGATGTTGCTGAAGCTTGCGTTCAGGCTCTGCAGATTGAGGAAAGTA AATTCAAAGCGTTTGATTTGGGATCAATGCCAGAGGGAGTTGGTGAGCCAACAAAGGATTTCAAGGCTCTTT TTTCGCAAGTCACTACTCCTTTCTGAAATCTGAGATCCAAGAGCAATTTAGTACACGTTATGGTTGCTTGTG CTTGTTTGCATTTTTCGCTTCTTAAATATACATAACAGGATTGCACCGAAATAATGTATTACCTATTTAATG GTTGCTTGAATTAATGAAATCGCATTCTAATATATGGGGCAGACAGATTTGCGATGCCGGCTTATAGGTTTA GATGATCTCTTCTTGTAATTTTTCTTCTTTCCTTTCTTTCCTTGGAGGTCAGGTCTTTGCCCCGCTCCCTTT TTGTTTTCTTTTCTTTGATTAATA >comp9568_c0_seq2 CTAGGCTATATTTTTTCTTATAATATTCTCTCTTCTAGAAGAAATTCGAATTTGGAGAAAAAACCCTTTGAT CAAAGGGTTTCTTTATAAAATCATACTCATCTCTTCACTTTCTTCTGCAAATTCATTTCACCACTAAATATA ATCAAAAAAGAAAAAAGGGTTCCTATATTTTCAATTAGATCTTGTTTTTACAGACTTACAAAATAAAAATAA AAATGGCAGAATCAAATCAAAAAATCACAGTCCTTGTCACTGGAGCTTCAGGCTTAACTGGTGAAATTGCAT TCAAGAAGCTGAAAGAAAGATCAGACAAATTTGTGGTTCGGGGTTTAGTAAGATCAGAAGCAAGTAAACAAA GACTTGGTGGAGGTGATGAAATTTTTCTAGGTGATGTCATGGATAAGAAAAGCCTTGAAACTGCTATGCAAG GAATTGATGCGTTGATTATACTAACAAGTGCTGTGCCAAAGGTAGTACCTGGTTCATATCCTGGTGCTGATG GCAAAAGAGCTGAGGATGTATTCGGTGAATCATTTGATTTCAATGGTCCAATGCCTGAATTCTATTACGAGG AAGGGCAATTCCCTGAACAGATTGATTGGATTGGACAAAAGAATCAGATCGATACTGCGAAATCTTGTGGTG TGAAACATATTGTTTTGGTTGGATCAATGGGTGGAACTGACCCTAATAATTTCTTGAATCACATGGCTAATG GAAACATACTTGTTTGGAAGAGAAAGGCTGAGCAATATTTGGCTGATTCTGGAATCCCATACACAATTATAA GGGCTGGTGGTTTAGATAACAAGGTAGGTGGCAGGGAATTGTTGGTCGGGAAGGATGATGAGCTTCTCTCTA CTGAAAACCATTTCATTGCTAGGGCTGATGTTGCTGAAGCTTGCGTTCAGGCTCTGCAGATTGAGGAAAGTA AATTCAAAGCGTTTGATTTGGGATCAATGCCAGAGGGAGTTGGTGAGCCAACAAAGGATTTCAAGGCTCTTT TTGCTCTAGTCACCACTCGTTTCTGAATGTTGAGATCCAAGAACAACTTATCATCTGCTATGGATTCTTGAG CTTGTTTGTACTTTATGCTTCTTAAATTTACAGAATTACACAGAAATAATGTATTGCCTGTTTAAAATGACA CTTTGCCACTATTCCTCATCCAAAACAATTCCAGGTGCTGTGCTAGACATGTAAACAGATCGTGACCAGTTA CCTATAGTTGCAGTTGCATTAAAGT >comp73923_c0_seq1 CTTTCTTTCCTTATTTCTTATATTCTTCTCTTCTAGAAGAAAGTTTGAATTTTCGGTGAAGAAAACTTCCGA TCAGCGGTTTCTTTATAAATATTACTCATATCATCACTTTCTTCTGCAATTCATTTCACTATCATATCTAAA TCTTTAAAGGAAAAAAAAAAGTTTCGTCAATTTCAATTACATCTTGTTTCCACAGATTTTAAAATGGCAGCA TTAATGCAAAAGATTACAGTTCTTGTTACCGGGGCTTCAGGTTTAACTGGTGAGATTGCATTCAAGAAACTG AAAGAAAGATCAGACAAATTTGCAGCAAGGGGTTTAGTAAGATCGGAAGCAAGTAAGCAAAAACTTGGGGGA GGTGATGAAATTTATCTTGGTGATATAATGGATAAGAAAAGTCTAAAACATGCTATGCAAGGAATTGATGGC TTAGTTATACTGACAAGCGCTGTACCGAAGATAGTACCTGGATCATATCCTGGTGCTGATGGCAAAAGAGCT GAAGATGTGTTTGATGATTCATTTGATTACAGTGGTCCAATGCCTGAATTCTTTTATGCGGAAGGACAATAC CCAGAACAGATTGATTGGATTGGACAAAAGAACCAGATCGAAACTGCTAAAGCTTGTGGCGTCAAACATATT GTTTTGGTTGGATCAATGGGTGGAACAGACCCTAATCATTTCTTGAATCATATGGGCAATGGAAATATACTT ATTTGGAAGAGAAAAGCTGAGCAGTATCTGGCTGATTCTGGAATCCCGTACACAATTATAAGAGCTGCTGCT CTAGATAACAAGGTGGGTGGCAGGGAGTTGTTGGTTGGAAAGGATGATGAGCTTCTCCCTACTGAAAATGGA TACATTGCTAGGGCAGATGTTGCTGAAGCTTGCGTTCAGGCTCTGCAAATCGAGGATTGCAAATTCAAAGCG TATGATTTGGGATCAAAGCCAGAGGGAGTTGGTGAGCCAACAAAGGATTTCAAGGCTCTTTTTTCGCAAGTC ACTACTCCTTTCTGAAATCTGAGATCCAAGAGCAATTTAGTACACGTTATGGTTGCTTGTGCTTGTTTGCAT TTTTCGCTTCTTAAATATACATAACAGGATTGCACCGAAATAATGTATTACCTATTTAATGGTTGCTTGAAT TAATGAAATCGCATTCTAATATATGGGGCAGACAGATTTGCGATGCCGGCTTATAGGTTTAGATGATCTCTT CTTGTAATTTTTCTTCTTTCCTTTCTTTCCTTGGAGGTCAGGTCTTTGCCCCGCTCCCTTTTTGTTTTCTTT TCTTTGATTAATAAAGCGTCCTTGTGAGTTTTTTTTTCTTTTTTTAAATCTTAGTTATGGCTCTTATATGTT TTAGATACTTTTGGTTGGATGCAACATATCGGATATGAAACAGTCCAGTCTTTTACAGAGTCCTAGAACTGT TTTTTGTCTGACTCTTTCATTTACCGAGATGAATCTCTTGCTAAAATTGTTTTTAACAATACTTTCCTTTCC ATTCTTGCAGAAGGGATTTGTTTCTTTAGCCGGAATGCGCTTCTGATAAGTATTCGTTTCTCAAGCACGAGT ATGATCGAAGGTGGAGTAGAACACTTGAGTGCAGTTCCAATCCAGGGTTGAACTGAGATCAAAGTGTGGAAG CTCAATTTTTCTAACGACTGACATACCAGTTTAGTGCTAAAGGAAATCTTATCTGAAGAATAATGTCATTTT

171

CATTGCTTTTGATTGAGAAGAGAGATTAGGCATGGCTACTGTTTGGACATGGAAGTCACCAGGACAGATGGA CTGAGCGAGAATCAAATAAAGTAGCAACTAAACAATAACAGACAACACTAGTAGGACACCTTTTTATTTGCT GTAGGAACATAAAACATGAAATGTATAGATAATATCCAACTTCAGACAAAGTGTGCCTCTGTCCACCGGATA CAGAATAATCTTGCATTGGCTTGCTTGGATTGCATAGTTGAATAGTTGAGCTTCCAGAAGCTCAGATTCTGA ACCACTTGCCAAACTTTCTAGAAATATGAAATGACGTGTTTTATAGGTCTATATTTTGGTATCAGGTTCTTT ATCAAAAGCAGTGACT >comp73923_c0_seq2 CTTTCTTTCCTTATTTCTTATATTCTTCTCTTCTAGAAGAAAGTTTGAATTTTCGGTGAAGAAAACTTCCGA TCAGCGGTTTCTTTATAAATATTACTCATATCATCACTTTCTTCTGCAATTCATTTCACTATCATATCTAAA TCTTTAAAGGAAAAAAAAAAGTTTCGTCAATTTCAATTACATCTTGTTTCCACAGATTTTAAAATGGCAGCA TTAATGCAAAAGATTACAGTTCTTGTTACCGGGGCTTCAGGTTTAACTGGTGAGATTGCATTCAAGAAACTG AAAGAAAGATCAGACAAATTTGCAGCAAGGGGTTTAGTAAGATCGGAAGCAAGTAAGCAAAAACTTGGGGGA GGTGATGAAATTTATCTTGGTGATATAATGGATAAGAAAAGTCTAAAACATGCTATGCAAGGAATTGATGGC TTAGTTATACTGACAAGCGCTGTACCGAAGATAGTACCTGGATCATATCCTGGTGCTGATGGCAAAAGAGCT GAAGATGTGTTTGATGATTCATTTGATTACAGTGGTCCAATGCCTGAATTCTTTTATGCGGAAGGACAATAC CCAGAACAGATTGATTGGATTGGACAAAAGAACCAGATCGAAACTGCTAAAGCTTGTGGCGTCAAACATATT GTTTTGGTTGGATCAATGGGTGGAACAGACCCTAATCATTTCTTGAATCATATGGGCAATGGAAATATACTT ATTTGGAAGAGAAAAGCTGAGCAGTATCTGGCTGATTCTGGAATCCCGTACACAATTATAAGAGCTGCTGCT CTAGATAACAAGGTGGGTGGCAGGGAGTTGTTGGTTGGAAAGGATGATGAGCTTCTCCCTACTGAAAATGGA TACATTGCTAGGGCAGATGTTGCTGAAGCTTGCGTTCAGGCTCTGCAAATCGAGGATTGCAAATTCAAAGCG TATGATTTGGGATCAAAGCCAGAGGGAGTTGGTGAGCCAACAAAGGATTTCAAGGCTCTTTTTGCTCTAGTC ACCACTCGTTTCTGAATGTTGAGATCCAAGAACAACTTATCATCTGCTATGGATTCTTGAGCTTGTTTGTAC TTTATGCTTCTTAAATTTACAGAATTACACAGAAATAATGTATTGCCTGTTTAAAATGACACTTTGCCACTA TTCCTCATCCAAAACAATTCCAGGTGCTGTGCTAGACATGTAAACAGATCGTGACCAGTTACCTATAGTTGC AGTTGCATTAAAGTCATGGTACTATTGCTCATTGATGTTGTAGATTTTGGCGCATTTCTTAGATAAGATCCA AAACTATCCGACATAGA >comp1703_c0_seq1 GGGTTTGATCCTACTAAAGGTGGAAGACCTGAGTTCTTTTTCGAAGATGGAGCTAATCCTGAACAGGTTGAT TGGATTGGGCAGAAGAATCAAATAGATGCTGCAAAAGCAGCGGGAGTGAAGCAGATTGTTTTGGTTGGGTCT ATGGGTGGAACGAACCTCAATCATCCCTTGAACAGCATTGGAAACGGAAACATATTGGTGTGGAAGAGGAAG GCGGAGCAATATCTGGCCGACTCTGGTATACCATACACAATTATTAGAGCTGGAGGCTTACAAGACAAAGAT GGGGGTGTGAGAGAGCTTGTTGTTGGCAAAGATGACGAGCTTCTCGAGACTGACATAAGGACTATTGCTAGA GCCGATGTTGCAGAAGTCTGCATTCAGGCATTGCTGTTGGAAGAGGCTAAGTTTAAAGCATTGGATCTCGCT TCAAAACCAGAAGGAACTGGCGAGCCAACAAAAGATTTCAAGACTCTCTTTTCTCAAATCTCTACACGATTC TGAGATTCCATATGCGGTATGTTCTGATTGAATTTTTGGTTGATGCCTAGCGATTTGTAATGCCACTGGCTA TTAGCAAGAGGGAAACTAGTATTCTTTTTCCTCATTAGAAAACCAATGAGAGGCCATGAATAACGATGATAG TGTATTTTACATTTTGTGTTCCGTCTAACGTTGTTTGAGTTATGAGTGATTGCTTATCATGCCTAGTTAGGC TGAAGCATAGTCGCGTGATGTCTTCATTCAAATGCTGCGAAGAATATGACGGGGCTGGCAATACCCTTATCT CATTCCCCTCTGCGAGATTCGAGTTTCTAGTCCTACTGGTTGCGAGTTCATCGTGCTTGTGAACACTCAGTT CGTCGAAAAGGCTTATCTAATTCTAATCTAACATATGAACCATCAACCCCATTCAGGGATCAAATT >comp1703_c0_seq3 TAGATGAAACGGTTTTTGCAGAAAGGGAAGAGGATAAGAATAAGATCATCATCATCTTCAATACTTCCCGTA CTGTTGGAATATCTTTTTCTCTCTGAATTACAGTCTCAGTCACAGAGTCAAGAGACCTTACCGACCTAAGTT TAGTTAGCAAATCCATCCATTAAAATCAGGGCAATGGGTTTAGTGACACGTGTTCCGTTATTCTCTTCACCT TCTTCAACTTTCTCTCCTCATAAATACTCTTCCACCACCAAACTATTCTCTTCTTCATCTTCATCTTCCCTT TCATTTCAAAGGAGAACTTCAGTTGTAGTGAAAGCAATGGCGAGTACTGTGATTGTTACTGGTGCCGGTGGT AGAACTGGGCAAATTGTTTACAAGAAACTGAAAGAGAGAGCTGAGTTTGTAGCAAGGGGGTTAGTAAGAACG GAAGAAAGCAAAGAGAAAATTGGAGGAGCTGACGATGTTTTCGTTGCTGATATTAGGGATGCTGAGAGTATT GTACCTGCAATCCAAGGAGTTGATGCTCTTGTCATTCTTACCAGTGCTGTCCCCAAAATGAAACCGGGGTTT GATCCTACTAAAGGTGGAAGACCTGAGTTCTTTTTCGAAGATGGAGCTAATCCTGAACAGGTTGATTGGATT GGGCAGAAGAATCAAATAGATGCTGCAAAAGC >comp1703_combined TAGATGAAACGGTTTTTGCAGAAAGGGAAGAGGATAAGAATAAGATCATCATCATCTTCAATACTTCCCGTA CTGTTGGAATATCTTTTTCTCTCTGAATTACAGTCTCAGTCACAGAGTCAAGAGACCTTACCGACCTAAGTT TAGTTAGCAAATCCATCCATTAAAATCAGGGCAATGGGTTTAGTGACACGTGTTCCGTTATTCTCTTCACCT TCTTCAACTTTCTCTCCTCATAAATACTCTTCCACCACCAAACTATTCTCTTCTTCATCTTCATCTTCCCTT TCATTTCAAAGGAGAACTTCAGTTGTAGTGAAAGCAATGGCGAGTACTGTGATTGTTACTGGTGCCGGTGGT AGAACTGGGCAAATTGTTTACAAGAAACTGAAAGAGAGAGCTGAGTTTGTAGCAAGGGGGTTAGTAAGAACG GAAGAAAGCAAAGAGAAAATTGGAGGAGCTGACGATGTTTTCGTTGCTGATATTAGGGATGCTGAGAGTATT GTACCTGCAATCCAAGGAGTTGATGCTCTTGTCATTCTTACCAGTGCTGTCCCCAAAATGAAACCGGGGTTT GATCCTACTAAAGGTGGAAGACCTGAGTTCTTTTTCGAAGATGGAGCTAATCCTGAACAGGTTGATTGGATT GGGCAGAAGAATCAAATAGATGCTGCAAAAGCAGCGGGAGTGAAGCAGATTGTTTTGGTTGGGTCTATGGGT GGAACGAACCTCAATCATCCCTTGAACAGCATTGGAAACGGAAACATATTGGTGTGGAAGAGGAAGGCGGAG CAATATCTGGCCGACTCTGGTATACCATACACAATTATTAGAGCTGGAGGCTTACAAGACAAAGATGGGGGT

172

GTGAGAGAGCTTGTTGTTGGCAAAGATGACGAGCTTCTCGAGACTGACATAAGGACTATTGCTAGAGCCGAT GTTGCAGAAGTCTGCATTCAGGCATTGCTGTTGGAAGAGGCTAAGTTTAAAGCATTGGATCTCGCTTCAAAA CCAGAAGGAACTGGCGAGCCAACAAAAGATTTCAAGACTCTCTTTTCTCAAATCTCTACACGATTCTGAGAT TCCATATGCGGTATGTTCTGATTGAATTTTTGGTTGATGCCTAGCGATTTGTAATGCCACTGGCTATTAGCA AGAGGGAAACTAGTATTCTTTTTCCTCATTAGAAAACCAATGAGAGGCCATGAATAACGATGATAGTGTATT TTACATTTTGTGTTCCGTCTAACGTTGTTTGAGTTATGAGTGATTGCTTATCATGCCTAGTTAGGCTGAAGC ATAGTCGCGTGATGTCTTCATTCAAATGCTGCGAAGAATATGACGGGGCTGGCAATACCCTTATCTCATTCC CCTCTGCGAGATTCGAGTTTCTAGTCCTACTGGTTGCGAGTTCATCGTGCTTGTGAACACTCAGTTCGTCGA AAAGGCTTATCTAATTCTAATCTAACATATGAACCATCAACCCCATTCAGGGATCAAATT >comp80098_c0_seq1 TTCAATACTTCCCGTACTGTTGGAATATCTCTTTCTCTCTGAATTACAGTCTCAGTCACAGAGTCAAGAGAC CTTACCGACCTAAGTTTAGTTAGCAAATCCATCCATTAAAATCAGGGCAATGGGTTTAGTGACACGTGTTCC GTTATTCTCTTCACCTTCTTCAACTTTCTCTCCTCATAAATACTCTTCCACCACCAAACTATTCTCTTCTTC ATCTTCATCTTCCCTTTCATTTCAAAGGAGAACTTCAGTTGTAGTGAAAGCAATGGCGAGTACTGTGATTGT TACTGGTGCCGGTGGTAGAACTGGGCAAATTGTTTACAAGAAACTGAAAGAGAGAGATGAGTTTGTAGCAAG GGGGTTAGTAAGAACGGAAGAAAGCAAAGAGAAAATTGGAGGAGCTGACGATGTTTTCGTTGCTGATATTAG GGATGCTGAGAGTATTGTACCTGCAATCCAAGGAGTTGATGCTCTTGTTATTCTTACTAGTGCTGTCCCCAA AATGAAACCCGGGTTTGATCCTACTAAAGGTGGAAGACCTGAGTTCTTTTTCGAAGATGGAGCTAATCCTGA ACAGGTTGATTGGATTGGGCAGAAGAATCAAATAGATGCTGCAAAAGCAGCGGGAGTGAAGCAGATTGTTTT GGTTGGGTCTATGGGTGGAACGAACCTCAATCATCCCTTGAACAGCATTGGAAACGGAAACATATTGGTGTG GAAGAGGAAGGCGGAGCAATATCTGGCCGACTCTGGTATACCATACACAATTATTAGAGCTGGAGGCTTACA AGACAAAGATGGGGGTGTGAGAGAGCTTGTTGTTGGCAAAGATGACGAGCTTCTCGAGACTGACATAAGGAC TATTGCTAGAACCGATGTTGCAGAAGTCTGCATTCAGGCATTGCTGTTGGAAGAGGCTAAGTTCAAAGCATT GGATCTCGCTTCAAAACCAGAAGGAACTGGCGAGCCAACAAAAGATTTCAAGACTCTCTTTTCTCAAATCTC TACACGATTCTGAGATTCCATATGCGGTATGTTCTGATTGAATTTTTGGTTGATGCCTAGCGATTTGTAATG CCACTGGCTATTAGCAAGAGGGAAACTAGTATTCTTTTTCCTCATTAGAAAACCAATGAGAGGCCATGAATA ACGATGATAGTGTATTTTACATTTTGTGTTCCGTCTAACGTTGTTTGAGTTATGAGTGATTGCTTATCATGC CTAGTTAGGCTGAAGCATAGTCGCGTGATGTCTTCATTCAAATGCTGCGAAGAATATGACGGGGCTGGCAAT ACCCTTATCTCATTCCCCTCTGCGAGATTCGAGTTTCTAGTCCTACTGGTTGCGAGTTCATCGTGCTTGTGA ACACT >comp80098_c2_seq1 ATAAGATCATCATCATCATCATCTTCAATACTTCCCGTACTGTTGGAATATCTCTTTCTCTCTGAATTACAG TCTCAGTCACAGAGTCAAGAGACCTTACCGACCTAAGTTTAGTTAGCAAATCCATCCATTAAAATCAGGGCA ATGGGTTTAGTGACACGTGTTCCGTTATTCTCTTCACCTTCTTCAACTTTCTCTCCTCATAAATACTCTTCC ACCACCAAACTATTCTCTTCTTCATCTTCATCTTCCCTTTCATTTCAAAGGAGAACTTCAGTTGTAGTGAAA GCAATGGCGAGTACTGTGATTGTTACTGGTGCCGGTGGTAGAACTGGGCAAATTGTTTACAAGAAACTGAAA GAGAGAGATGAGTTTGTAGCAAGGGGGTTAGTAAGAACGGAAGAAAGCAAAGAGAAAATTGGAGGAGCTGAC GATGTTTTCGTTGCTGATATTAGGGATGCTGAGAGTATTGTACCTGCAATCCAAGGAGTTGATGCTCTTGTT ATTCTTACTAGTGCTGTCCCCAAAATGAAACCCGGGTTTGATCCTACTAAAGGTGGAAGACCTGAGTTCTTT TTCGAAGATGGAGCTAATCCTGAACAGGTTGATTGGATTGGGCAGAAGAATCAAATAGATGCTGCAAAAGCA GCGGGAGTGAAGCAGATTGTTTTGGTTGGGTCTATGGGTGGAACGAACCTCAATCATCCCTTGAACAGCATT GGAAACGGAAACATATTGGTGTGGAAGAGGAAGGCGGAGCAATATCTGGCCGACTCTGGTATACCATACACA ATTATTAGAGCTGGAGGCTTACAAGACAAAGATGGGGGTGTGAGAGAGCTTGTTGTTGGCAAAGATGACGAG CTTCTCGAGACTGACATAAGGACTATTGCTAGAACCGATGTTGCAGAAGTCTGCATTCAGGCATTGCTGTTG GAAGAGGCTAAGTTCAAAGCATTGGATCTCGCTTCAAAACCAGAAGGAACTGGCGAGCCAACAAAAGATTTC AAGACTCTCTTTTCTCAAATCTCTACACGATTCTGAGATTCCATATGCGGTATGTTCTGATTGAATTTTTGG TTGATGCCTAGCGATTTGTAATGCCACTGGCTATTAGCAAGAGGGAAACTAGTATTCTTTTTCCTCATTAGA AAACCAATGAGAGGCCATGAATAACGATGATAGTGTATTTTACATTTTGTGTTCCGTCTAACGTTGTTTGAG TTATGAGTGATTGCTTATCATGCCTAGTTAGGCTGAAGCATAGTCGCGTGATGTCTTCATTCAAATGCTGCG AAGAATATGACGGGGCTGGCAATACCCTTATCTCATTCCCCTCTGCGAGATTCGAGTTTCTAGTCCTACTGG TTGCGAGTTCATCGTGCTTGTGAACACT

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Appendix A4: Phylogenetic analysis

Phylogenetic analysis was performed for all biosynthetic enzyme families involved in benzylisoquinoline alkaloid biosynthesis for 20 different BIA-producing species. Both the 454 and Illumina transcriptomes of 20 BIA-producing species

(Argemone mexicana, Chelidonium majus, Corydalis chelianthifolia, Eschscholzia californica, Glaucium flavum, Papaver bracteatum, Sanguinaria canadensis,

Stylophorum diphyllum, Hydrastis canadensis, Nigella sativa, Thalictrum flavum,

Xanthorhiza simplicissima, Berberis thunbergii, Jeffersonia diphylla, Mahonia aquifolium, Nandina domestica, Cissampelos muscronata, Cocculus trilobus,

Menispermum canadense, and Tinospora cordifolia) were mined for potential candidates involved in BIA biosynthesis (Xiao et al., 2013). Transcriptomes were searched using the amino acid sequence of previously characterized enzymes, COR (GenBank Accession

No. AF108432), NOS (GenBank Accession No. JQ659007), SalR (GenBank Accession

No. DQ316261), and SanR (GenBank Accession No. GU338458) (Winzer et al., 2012;

Weiss et al., 2006; Ziegler et al., 2006; Unterlinner et al., 1999), and candidates were selected based on percent sequence similarity to the query sequence. Candidates with greater than 50, 35%, 50, and 60% amino acid sequence similarity to COR, NOS, SalR, and SanR, respectively, were used for phylogenetic analysis. As well, only putative full-length sequences were selected, and duplicated entries between the two transcriptomes were removed. Other members of the Facchini laboratory selected the candidates for BBE, FADOX, CXE, CYP80, CYP82, CYP719, DIOX, NCS, NMT,

OAT, and OMT enzymes families from the 20 BIA-producing species, but I selected the appropriate outgroup for each enzyme type, and generated the alignment used to build a

174 phylogenetic tree with the assistance of Ye Zhang (Department of Biochemistry and

Molecular Biology, University of Calgary, AB.). Sequences were aligned using the

M-Coffee server (www.tcoffee.org) then manually edited in Jalview (see Fig. A4.1 for an example alignment; (Waterhouse et al., 2009; Notredame et al., 2000). M-coffee was used to align amino acid sequences because it combines multiple alignment methods, including ClustalW, into a single result (Notredame et al., 2000). Evolutionary relationships were analyzed using the Neighbor-Joining method in the Phylogeny

Inference Package (PHYLIP, Version 3.69; distributed by Joe Felsenstein, University of

Washington, WA), which was hosted on the coe03 bioinformatics server at the University of Calgary (Saitou and Nei, 1987), except for BBE, whose phylogeny was analyzed using

MEGA5 (Tamura et al., 2011). Bootstrapped consensus distance trees were generated with 1000 replicates, and evolutionary distance was computed using the Jones-Taylor-

Thornton (JTT) matrix-based method ((Jones et al., 1992; Felsenstein, 1985).

Phylogenetic trees were visualized using TreeGraph (Version 2.0.47 Beta) (Fig. A4.2).

175

Figure A4.1. Multiple sequence alignment of sanguinarine reductase candidates from 20 BIA-producing species.

176

Figure A4.1 (continued). Multiple sequence alignment of sanguinarine reductase candidates from 20 BIA-producing species.

Sequences were aligned using the M-Coffee server, and manually edited in Jalview (Version 14.0). Amino acids are coloured according to the Clustalx scheme in Jalview. Outgroup is a Zea mays NAD-Dependent Epimerase/Dehydratase (ZMASDR, Accession

ACG33645).

177

BBE FADX

OAT SALR

Figure A4.2. Phylogenetic trees for enzymes involved in BIA biosynthesis.

178

COR SANR

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA biosynthesis.

179

NOS CYP80

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA biosynthesis.

180

CYP719 CYP82

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA biosynthesis.

181

NCS CXE

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA biosynthesis.

182

DIOX NMT

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA

183

OMT

biosynthesis. Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA biosynthesis.

184

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in benzylisoquinoline alkaloid (BIA) biosynthesis. Candidates for biosynthetic enzymes were collected from 20

BIA-producing species (AME: Argemone mexicana, CMA: Chelidonium majus, CCH:

Corydalis chelianthifolia, ECA: Eschscholzia californica, GFL: Glaucium flavum, PBR:

Papaver bracteatum, SCA: Sanguinaria canadensis, SDI: Stylophorum diphyllum, HCA:

Hydrastis canadensis, NSA: Nigella sativa, TFL: Thalictrum flavum, XSI: Xanthorhiza simplicissima, BTH: Berberis thunbergii, JDI: Jeffersonia diphylla, MAQ: Mahonia aquifolium, NDA: Nandina domestica, CMU: Cissampelos muscronata, CTR: Cocculus trilobus, MCA: Menispermum canadense, TCO: Tinospora cordifolia). Bootstrapped consensus distance trees were generated with 1000 replicates, and evolutionary distance was computed using the Jones-Taylor-Thornton (JTT) matrix-based method. Outgroups:

Cannabis sativa tetrahydrocannabinolic acid synthase (CSATHCAS, Accession

Q8GTB6) for berberine bridge enzyme (BBE); Zea mays deoxymugineic acid synthase 1

(ZMADMAS1, Accession NP_001105931) for codeinone reductase (COR); Actinidia eriantha carboxylesterase 1 (AERCXE, Accession Q0ZPV7) for carboxylesterase (CXE);

Homo sapiens Cytochrome P450 1B1 (HSACYP1B1, Accession NP_000095), Homo sapiens Cytochrome P450 1A2 (HSACYP1A2, Accession P05177), and Homo sapiens

Cytochrome P450 17A1 (HSACYP17A1, Accession AAA59984) for three cytochrome

P450 families CYP80, CYP82, and CYP719, respectively; Arabidopsis thaliana

LEUCOANTHOCYANIDIN DIOXYGENASE (ATHDIOX, Accession 2BRT_A) for dioxygenase (DIOX);Cannabis sativa tetrahydrocannabinolic acid synthase

(CSATHCAS, Accession Q8GTB6) for FAD-dependent oxidoreductase (FADX); Betula pendula major pollen allergen Bet V1 (BPEBETV1, Accession P43185) for

185 norcoclaurine synthase (NCS); Mycobacterium tuberculosis mycolic acid synthase

(MTUMMA2, Accession AAC44617) for N-methyltransferase (NMT); Arabidopsis thaliana BRI1-5 ENHANCED 1 (ATHBEN1, Accession NP_182064) for noscapine synthase (NOS); Rauvolfia serpentine vinorine synthase (RSEVS, Accession Q70PR7) for salutaridinol 7-O-acetyltransferase (OAT); Medicago sativa Isoflavone

O-methyltransferase (MSAOMT, Accession O24529) for O-methyltransferase (OMT);

Sus scrofa porcine testicular carbonyl reductase (SSCPTCR, Accession 1N5D_A) for salutaridine reductase (SALR); and Zea mays NAD-dependent epimerase/dehydratase

(ZMASDR, Accession ACG33645) for sanguinarine reductase (SANR). The phylogenetic tree for OMT was split in half for clarity.

186