characterization of sanguinarine reductases from papaver

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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(Unpublished master's thesis).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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UNIVERSITY OF CALGARY

Characterization of sanguinarine reductases from Papaver somniferum

by

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

© Crystal Dawn Bross 2015

ii

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.

iii

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

v

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

vi

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

vii

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

viii

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

ix

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


roots................................................................................................................. 92


roots................................................................................................................. 96

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

opium poppy roots. ......................................................................................... 99


roots............................................................................................................... 101

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

roots............................................................................................................... 103


roots............................................................................................................... 105


roots............................................................................................................... 107

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

x

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

xi

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

xii

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

1

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.

2

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

3

Figure 1. Benzylisoquinoline alkaloid biosynthesis in Papaver somniferum.

4

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.

5

(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

6

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

7

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.

8

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

9

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

10

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

11

(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).

12

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

13

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.

14

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).

15

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).

16

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


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).

17

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).

18

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


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

19

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).

20

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.

21

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).

pTRV1

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

pTRV2

SanR VIGS Construct

LB RB 2x35S MCS CP NOSt Rz

22

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).

23

ECASANR

PSOSANR1

PSOSANR2

PSOSANR3

PSOSANR4

ECASANR

PSOSANR1

PSOSANR2

PSOSANR3

PSOSANR4

ECASANR

PSOSANR1

PSOSANR2

PSOSANR3

PSOSANR4

ECASANR

PSOSANR1

PSOSANR2

PSOSANR3

PSOSANR4

36

36

36

79

63

M - - - - - - - - - - - - - - - - - - - - - - - - - - - A D S S K K - - - - - - - - - - - - - - - - - L T V L L S G A S G L T G S L A F K K L K E R S D K F E V

M - - - - - - - - - - - - - - - - - - - - - - - - - - - A E S N Q K - - - - - - - - - - - - - - - - - IT V L V T G A S G L T G E IA F K K L K E R S D K F V V

M - - - - - - - - - - - - - - - - - - - - - - - - - - - A A LM Q K - - - - - - - - - - - - - - - - - IT V L V T G A S G L T G E IA F K K L K E R S D K F A A

M G L V T R V P L F S S P S S T F S P H K Y S S T T K L F S S S S S S S L S FQ R R T S V V V K A M A S T V IV T G A G G R T G Q IV Y K K L K E R A E - F V A

M R S V SQ IC L S L R N K S K M A C K R C S N K V A M A C S S P K - - - - - - - - - - - - - - - - - K T V L V T G A S G L T G Q F A F K K L K E R S D K L V V

114

113

113

140

139

R G L V R S E A S K Q K L G G G D E I F IG D I S D P K T L E P A M E G ID A L I I L T S A IP R M K P T E E F T A EM I S G G R S E D V ID A S F - - S G P M

R G L V R S E A S K Q R L G G G D E I F L G D V M D K K S L E T A M Q G ID A L I I L T S A V P K V V P G S Y P G A - - - D G K R A E D V F G E S F D F N G P M

R G L V R S E A S K Q K L G G G D E IY L G D IM D K K S L K H A M Q G ID G L V I L T S A V P K IV P G S Y P G A - - - D G K R A E D V F D D S F D Y S G P M

R G L V R T E E S K E K IG G A D D V F V A D IR D A E S IV P A IQ G V D A L V I L T S A V P K M K P G F D P T K - - - - G G R - - - - - - - - - - - - - - -

R G L V R S E G S K K K L G G G N E IY V G D V M K P E S L E P A M K G V D A L I I L T T A IP K M K P G S Y P A N I- - S G A R A E D L ID G S F - - Q G T I

194

193

193

220

219

P E F Y Y D E G Q Y P EQ V D W IG Q K N Q ID T A K K M G V K H IV L V G SM G G C D P D H F L N H M G N G N I L IW K R K A EQ Y L A D S G V P Y T I IR A

P E F Y Y E E G Q F P EQ ID W IG Q K N Q ID T A K S C G V K H IV L V G SM G G T D P N N F L N H M A N G N I L V W K R K A EQ Y L A D S G IP Y T I IR A

P E F F Y A E G Q Y P EQ ID W IG Q K N Q I E T A K A C G V K H IV L V G SM G G T D P N H F L N H M G N G N I L IW K R K A EQ Y L A D S G IP Y T I IR A

P E F F F E D G A N P EQ V D W IG Q K N Q ID A A K A A G V K Q IV L V G SM G G T N L N H P L N S IG N G N I L V W K R K A EQ Y L A D S G IP Y T I IR A

P E F Y F E G G Q Y P EQ V D W IG Q K N Q ID A A K A A G V K H I I L V S T M G S G D P N H P L N S L G N G N I L A W K R K A E E Y L A K S G V P Y T I L R A

273

271

271

299

298

G G L D N K A G G V R E L L V A K D D V L L P T E N G F IA R A D V A E A C V Q A L E I E E V K N K A F D L G S K P E G V G E A T K D F K A L F SQ V T T P F

G G L D N K V G G - R E L L V G K D D E L L S T E N H F IA R A D V A E A C V Q A LQ I E E S K F K A F D L G SM P E G V G E P T K D F K A L F SQ V T T P F

A A L D N K V G G - R E L L V G K D D E L L P T E N G Y IA R A D V A E A C V Q A LQ I E D C K F K A Y D L G S K P E G V G E P T K D F K A L F A L V T T R F

G G LQ D K D G G V R E L V V G K D D E L L E T D IR T IA R A D V A E V C IQ A L L L E E A K F K A L D L A S K P E G T G E P T K D F K T L F SQ I S T R F

G G L D N K Q G G K R Q L L IG K N D E L L P T E K G Y V A R E D V A E A C V Q A V Q L E E V K F K A F D L G SM P E G T G V P T K D F K A L F A P IT T C F

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.

24

Table 1. List of primers used for cloning procedures.

Namea

Sequenceb

RE sequence

Direction

Amplicon

size (bp)

Protein size

(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

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

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

25

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

Namea Sequenceb

RE sequence

Direction

Amplicon

size (bp)

Protein size

(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.

26

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).

27

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

28

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


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

29



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

30



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

31



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.

32

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.

V1: SR1-5UTR

218 bp

*V2:

SR1-3UTR

**V9: SR1-CDS

357 bp (100%)

816 bp 215 bp 218 bp

V3: SR2-UTR

167 bp

V5: SR2-3’UTR

249 bp

V4: SR2-CDS

433 bp

816 bp 299 bp 207 bp

*V7: SR3-3’UTR

352 bp

V6: SR3-CDS

133 bp

900 bp 352 bp 144 bp 147 bp

897 bp 174 bp

A

B

C

D

33

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.

34

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

35

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

36

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

37

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).

38

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

39

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).

40

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

41

(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

42

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

43

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

44

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).

45

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

46

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.

47

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.

48

M

1

U I

SanR

2

U I

3

U I U I

3B

27.0 kDa

34.6 kDa

66.4 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).

49

A

27

34

M 1 2 3 4 5 6 8 7 9 10 11 12 M S F 1 10 11 12

27

34

S F 1 10 11 12 M S F 1 10 11 12 S F 1 10 11 12

SanR1 SanR2 SanR3 SanR3B

23

30

46

B Imidazole concentration (mM)

27

34

Sa

nR

1

27

34

Sa

nR

2

27

34

Sa

nR

3

Sa

nR

3B

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.

50

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.

1 2 3 3B M

SanR

27.0 kDa

34.6 kDa

A

1 2 3 3B M

SanR

23 kDa

30 kDa

B

51

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).

52

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

Compound Structure [M]+ or

[M+H]+ Type

1,2-Dehydroreticuline

328 1-Benzylisoquinoline

Papaverine

340 1-Benzylisoquinoline

Noscapine

414 Phthalideisoquinoline

Berberine

336 Protoberberine

Cryptopine

370 Protopine

Thebaine

312 Morphinan

53

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

Compound Structure [M]+ or

[M+H]+ Type

Sanguinarine

332 Benzophenanthridine

Chelirubine


Macarpine


Chelerythrine


54

0

200

400

600

800

1000

0 5 10 15

LC

-MS

Cou

nts

(x 1

05)

Retention Time (min.)

0

1

2

3

4

0 5 10 15

LC

-MS

Cou

nts

(x 1

05)


0

5

10

15

0 5 10 15

LC

-MS

Cou

nts

(x

10

5)


0

5

10

15

20

0 5 10 15

LC

-MS

Cou

nts

(x

10

5)


A B

C D

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.

55

Origin

1

2

3

4

Origin

1 2

3

4

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.

56

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).

57

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.

0

10000

20000

30000

200 250 300 350 400

LC

-MS

Co

un

ts 332.2

317.2 304.1

274.1

Sanguinarine

0

10000

20000

30000

200 250 300 350 400

LC

-MS

Co

un

ts

Mass-to-Charge (m/z)

392.2

362.2

377.1

348.2

334.1

Macarpine

0

10000

20000

30000

200 250 300 350 400

LC

-MS

Co

un

ts

348.1 318.1

332.1

304.1

290.1

Chelerythrine

0

10000

20000

30000

200 250 300 350 400

LC

-MS

Co

un

ts

362.1

332.1

347.1

318.1

303.8

Chelirubine

58

y = 9E+06x + 1E+06

R² = 0.99999 0

20

40

60

80

100

0 50 100 150 LC

-MS

Co

un

ts (

x 1

07)

Sanguinarine

Concentration (µM)

y = 6E+06x + 1E+06

R² = 0.99874 0

2

4

6

8

0 5 10 15 LC

-MS

Co

un

ts (

x 1

07)

Dihydrosanguinarine

Concentration (µM)

y = 1E+07x - 258094

R² = 0.99998 0

2

4

6

8

10

12

0 5 10 15 LC

-MS

Cou

nts

(x

10

7)

Chelerythrine

Concentration (µM)

y = 2E+06x + 7E+06

R² = 0.99216 0

5

10

15

0 20 40 60 LC

-MS

Cou

nts

(x

10

7)

Chelirubine

Concentration (µM)

y = 2E+06x - 3E+06

R² = 0.99794 0

2

4

6

8

10

12

0 20 40 60 LC

-MS

Cou

nts

(x

10

7)

Macarpine

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.

59

0

1

2

3

6.5 7.5 8.5 9.5

Cou

nts

(x

10

6)

Retention Time (minutes)

Retention Time (minutes)

0

2

4

6

6.5 7.5 8.5 9.5

Cou

nts

(x

10

6)

0

2

4

6

8

6.5 7.5 8.5 9.5

Cou

nts

(x

10

6)

DCHR DMAC

SAN CHE CHR MAC

DSAN

0

1

2

6.5 7.5 8.5 9.5

Co

un

ts (

x 1

06)

DCHE

0

2

4

6

6.5 7.5 8.5 9.5

Co

un

ts (

x 1

06)

0

1

2

3

4

6.5 7.5 8.5 9.5

Co

un

ts (

x 1

06)

0

2

4

6

8

6.5 7.5 8.5 9.5

Co

un

ts (

x 1

06)

0

5

10

15

6.5 7.5 8.5 9.5

Cou

nts

(x

10

6)

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).

60

0

5

10

15

20

25

30

0 5 10 15

Co

un

ts (

x 1

04)

Retention Time (min)

G

0

5

10

15

20

25

30

0 5 10 15

Co

un

ts (

x 1

04)


H

A

0

10

20

30

40

0 5 10 15

Co

un

ts (

x 1

04)


B

0

10

20

30

40

0 5 10 15

Co

un

ts (

x 1

04)


D

0

5

10

15

20

25

30

0 5 10 15

Co

un

ts (

x 1

04)


C

0

50

100

150

200

0 5 10 15

Co

un

ts (

x 1

04)


E

0

10

20

30

40

50

60

0 5 10 15

Co

un

ts (

x 1

04)


F

0

10

20

30

40

0 5 10 15

Co

un

ts (

x 1

04)


+SanR1

+SAN

+NADPH

+SanR2

+SAN

+NADPH

+SanR3B

+SAN

+NADPH

No enzyme

+SAN

+NADPH

+SanR1

+SAN

+NADH

+SanR2

+SAN

+NADH

+SanR3B

+SAN

+NADH

No enzyme

+SAN

+NADH

Figure 13. Sanguinarine reductases reduce benzophenanthridine alkaloids.

61

I J

K L

N M

O P

0

5

10

15

20

0 5 10 15

Co

un

ts (

x 1

04)


0

5

10

15

20

0 5 10 15

Co

un

ts (

x 1

04)


0

20

40

60

80

100

0 5 10 15

Co

un

ts (

x 1

04)


0

20

40

60

80

100

0 5 10 15

Co

un

ts (

x 1

04)


0

50

100

150

0 5 10 15

Co

un

ts (

x 1

04)


0

10

20

30

40

50

0 5 10 15 C

ou

nts

(x 1

04)


0

20

40

60

80

0 5 10 15

Co

un

ts (

x 1

04)


0

50

100

150

0 5 10 15

Co

un

ts (

x 1

04)


+SanR1

+CHE

+NADPH

+SanR2

+CHE

+NADPH

+SanR3B

+CHE

+NADPH

+SanR1

+CHE

+NADH

+SanR2

+CHE

+NADH

+SanR3B

+CHE

+NADH

+SanR1

+CHR

+NADPH

+SanR1

+CHR

+NADH

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

62

Q R

S T

V U

W X

0

50

100

150

200

0 5 10 15

Co

un

ts (

x 1

04)


0

50

100

150

200

0 5 10 15

Co

un

ts (

x 1

04)


0

100

200

300

400

0 5 10 15

Co

un

ts (

x 1

04)


0

50

100

150

200

0 5 10 15

Co

un

ts (

x 1

04)


0

10

20

30

40

0 5 10 15

Co

un

ts (

x 1

04)


0

10

20

30

40

50

0 5 10 15

Co

un

ts (

x 1

04)


0

200

400

600

800

1000

0 5 10 15

Co

un

ts (

x 1

04)


0

100

200

300

400

500

600

0 5 10 15

Co

un

ts (

x 1

04)


+SanR2

+CHR

+NADPH

+SanR3B

+CHR

+NADPH

+SanR1

+MAC

+NADPH

+SanR2

+CHR

+NADH

+SanR3B

+CHR

+NADH

+SanR1

+MAC

+NADH

+SanR2

+MAC

+NADPH

+SanR2

+MAC

+NADH


63

Y Z

0

200

400

600

800

1000

1200

0 5 10 15

Cou

nts

(x 1

04)


0

500

1000

1500

0 5 10 15

Cou

nts

(x 1

04)


+SanR3B

+MAC

+NADPH

+SanR3B

+MAC

+NADH


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).

64

0

2

4

6

8

10

12

14

16

18

20

Sanguinarine Chelerythrine Chelirubine Macarpine

Am

ou

nt

of

dih

yd

rob

enzo

ph

ena

nth

rid

ine

pro

du

ct f

orm

ed (

Co

un

ts,

x 1

06)

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.

65

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70

Dih

yd

rosa

ng

uin

ari

ne

Cou

nts

(x

10

6)

Temperature (°C)

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25 30

Am

ou

nt

of

pro

du

ct f

orm

ed

(µ

M d

ihy

dro

san

gu

ina

rin

e)

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.

66

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).

67

Michaelis-Menten

Sanguinarine Concentration (μM)

0 20 40 60 80 100 120

Rat

e

0.0

0.2

0.4

0.6

0.8

Vmax = 0.6955 Km = 7.3

Ra

te (

µm

ol

ho

ur-1

µg

-1)

Sanguinarine Concentration (µM)

Michaelis-Menten


0 20 40 60 80 100 120

Rat

e (M

S C

ou

nts

ho

ur-

1 μ

g-1

)

0

1e+6

2e+6

3e+6

4e+6

5e+6

6e+6

Vmax = 4.450e+6 Km = 1.6

Ra

te (

LC

-MS

co

un

ts h

ou

r-1 µ

g-1

)


Michaelis-Menten


0 20 40 60 80 100 120

Rat

e

0

1e+6

2e+6

3e+6

4e+6

5e+6

6e+6

Vmax = 5.831e+6 Km = 19.9

Rate

(L

C-M

S c

ou

nts

ho

ur-1

µg

-1)


A

B

C

Figure 16. Michaelis-Menten 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.

68

Antiserum

Tes

t

ble

ed #

1

Tes

t

ble

ed #

2

Tes

t

ble

ed #

3

SanR1 SanR2 SanR3B

Fin

al

ble

ed

+

SanR1 SanR2

SanR3B

2 3 1 2 3 1

2 3 1

A

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.

69

46

23

30

46

23

30

46

23

30

Before After

Sa

nR

1

An

tise

rum

Sa

nR

2

An

tise

rum

Sa

nR

3B

An

tise

rum

1 2 3B 1 2 3B

SanR SanR

A B

46

23

30

An

ti-H

is

1 2 3B

SanR C

oom

ass

ie

Sta

in

27

34

M M M

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).

70

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).

71

A

Ma

Cultivar

R M 40 T L P B V Eca

B

C

D

27

34

27

34 46

30

23

46

30

23

27

34 46

30

23

27

34

Ma

Cultivar

R M 40 T L P B V Eca

E

27

34

46

23

30

30

23

46

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

72

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).

74

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).

75

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.

76

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

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

Template

Concentration Template Average Ct Template Average Ct Template Average Ct

1 μg

SanR1 12.9 SanR1 No signal SanR1 23.3

SanR2 21.3 SanR2 14.1 SanR2 23.7

SanR3 22.9 SanR3 23.1 SanR3 13.5

1 ng

SanR1 17.5 SanR1 26.7 SanR1 26.6

SanR2 27.2 SanR2 18.8 SanR2 28.6

SanR3 26.3 SanR3 26.7 SanR3 16.3

0.1 ng

SanR1 21.1 SanR1 26.5 SanR1 30.1

SanR2 23.0 SanR2 22.4 SanR2 30.4

SanR3 23.0 SanR3 26.7 SanR3 20.2

0.01 ng

SanR1 22.9 SanR1 26.6 SanR1 30.4

SanR2 23.0 SanR2 26.4 SanR2 30.6

SanR3 22.8 SanR3 27.1 SanR3 23.6

77

0.000

0.005

0.010

0.015

0.020

0.025

Stem Root Leaf Bud

Rel

ati

ve

Ex

pre

ssio

n L

evel

Opium Poppy Tissue

SanR1

SanR2

SanR3

0.000

0.200

0.400

0.600

0.800

1.000

1.200

Stem Root Leaf Bud

Rel

ati

ve

Ex

pre

ssio

n L

evel

Opium Poppy Tissue

SanR1

SanR2

SanR3

A B

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.

79

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).

80

300 bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

EV

L

16 17 1 2 3 4 5 6 7 8 9 10 11 12 13

V1

L

EV

300 bp

14 15 16 17 18 19 20 21 22

V1

L

300 bp

300 bp

5 6 7 8 9 10 11 12 13 14 15 16 17 19 20

V2

L

300 bp

V3

1 2 3 4 16 6 7 8 9 10 11 12 13 14 15 L

1 2 3 5 6 7 8 9 10 11 12 13 14 15 16

V7

300 bp

L

6 7 8 9 10 11 12 13 14 15 16 1 2 3

V6

4

V2

300 bp

L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

V8

300 bp

L

17 18 19 20 21 22 23 24 25 26 1 2 3 4 5

V7 V6

300 bp

L

16 17 1 2 3 4 5 6 8 9 10 1 2 3 4

V5 V4 V8

300 bp

L

5 6 7 8

V4

1 2 3 4 6 7 8 9 10 11 12

V10

300 bp

L

13 14 1 2 3 4 5 6 7 8 9 10 11 1 2

V11 V10 V12

300 bp L

3 4 5 6 7 8 9 10 11 12

V12

1 2 3 4 5

V9

300 bp L

V3

17 18 19 20

300 bp

L 6 7 8 9 10 11 12

V9

300 bp

L

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

81

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.

82

A B

C D

0

0.005

0.01

0.015

0.02

SanR1 SanR2 SanR3

Rel

ati

ve

Gen

e E

xp

ress

ion

Gene

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

EV

1

EV

2

EV

3

EV

4

EV

5

EV

6

EV

7

EV

8

EV

9

EV

10

EV

11

EV

12

EV

13

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

0.002

0.004

0.006

0.008

0.01

0.012

0.014

EV

1

EV

2

EV

3

EV

4

EV

5

EV

6

EV

7

EV

8

EV

9

EV

10

EV

11

EV

12

EV

13

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

0

0.005

0.01

0.015

0.02

EV

1

EV

2

EV

3

EV

4

EV

5

EV

6

EV

7

EV

8

EV

9

EV

10

EV

11

EV

12

EV

13

Rel

ati

ve SanR3 E

xp

ress

ion

VIGS Construct

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

83

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).

84

0

50

100

150

0 1 2 3 4 5 6 7 8 9 10

LC

-MS

Co

un

ts x

10

6


0

20

40

60

0 1 2 3 4 5 6 7 8 9 10

LC

-MS

Co

un

ts x

10

6


0

50

100

1 2 3 4 5 6 7 8 9 10

LC

-MS

Co

un

ts x

10

6


0

5

10

15

20

0 1 2 3 4 5 6 7 8 9 10

LC

-MS

Co

un

ts x

10

6


0

20

40

60

0 1 2 3 4 5 6 7 8 9 10

LC

-MS

Cou

nts

x1

06


A B

E

C D

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.

85

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).

86

0

10

20

30

40

50

60

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

LC

-MS

Cou

nts

x1

06


0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

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

LC

-MS

Co

un

ts x

10

6


EIC312

EIC330

EIC332

EIC334

EIC340

EIC370

EIC414

A

B

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).

87

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

88

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).

89

0

0.005

0.01

0.015

0.02

0.025

0.03

V1

-1

V1

-2

V1

-3

V1

-4

V1

-5

V1

-6

V1

-7

V1

-8

V1

-9

V1

-10

V1

-11

V1

-12

V1

-13

V1

-14

V1

-15

Rel

ati

ve

Gen

e E

xp

ress

ion

VIGS Construct

0

0.005

0.01

0.015

0.02

EV V1

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

10

20

30

40

50

N P R T M C S DHS Am

ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea,

Arb

itary

Un

its,

Mil

lio

ns)

Metabolite

EV

V1

A B

C D

0

5

10

15

20

25

30

35

40

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V1

*

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

90

E F

G H

*

0

0.005

0.01

0.015

0.02

0.025

V2

-1

V2

-2

V2

-3

V2

-4

V2

-5

V2

-6

V2

-7

V2

-8

V2

-9

V2

-10

V2

-11

V2

-12

V2

-13

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

0.005

0.01

0.015

0.02

EV V2

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

20

40

60

80

100

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V2

0

10

20

30

40

50

60


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea,

Arb

itary

Un

its,

Mil

lio

ns)

Metabolite

EV

V2

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

91

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.

92

*

0

10

20

30

40

50

60

70

80


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea,

Arb

itary

Un

its,

Mil

lio

ns)

Metabolite

EV

V3

A B

C D

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

V3

-1

V3

-2

V3

-3

V3

-4

V3

-5

V3

-6

V3

-7

V3

-8

V3

-9

V3

-10

V3

-11

V3

-12

V3

-13

V3

-14

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

0

5

10

15

20

25

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V3

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

EV V3

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct


93

E F

G H

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

EV V4

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

*

0

10

20

30

40

50


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea,

Arb

itary

Un

its,

Mil

lio

ns)

Metabolite

EV

V4

0

0.001

0.002

0.003

0.004

0.005

0.006

V4-1 V4-2 V4-3 V4-4 V4-5

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

0

10

20

30

40

50

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V4


94

I J

K L

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

0.0016

V5-1 V5-2 V5-3 V5-4 V5-5 V5-6 V5-7 V5-8

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

EV V5

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

*

0

10

20

30

40

50


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea,

Arb

itary

Un

its,

Mil

lio

ns)

Metabolite

EV

V5

0

5

10

15

20

25

30

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V5

*


95


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.

96

A B

C D

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

0.004

V6

-1

V6

-2

V6

-3

V6

-4

V6

-5

V6

-6

V6

-7

V6

-8

V6

-9

V6

-10

V6

-11

V6

-12

V6

-13

Rel

ati

ve SanR3

Exp

ress

ion

VIGS Construct

0

0.002

0.004

0.006

0.008

0.01

EV V6

Rel

ati

ve SanR3

Exp

ress

ion

VIGS Construct

*

0

10

20

30

40

50

60


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea,

Arb

itary

Un

its,

Mil

lio

ns)

Metabolite

EV

V6

0

5

10

15

20

25

30

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V6

*

* *

*


97

E F

G H

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

V7

-1

V7

-2

V7

-3

V7

-4

V7

-5

V7

-6

V7

-7

V7

-8

V7

-9

V7

-10

V7

-11

V7

-12

V7

-13

V7

-14

Rel

ati

ve SanR3

Exp

ress

ion

VIGS Construct

0

0.002

0.004

0.006

0.008

0.01

EV V7

Rel

ati

ve SanR3

Exp

ress

ion

VIGS Construct

*

0

5

10

15

20

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V7

0

10

20

30

40

50

60

70

80


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea,

Arb

itary

Un

its,

Mil

lio

ns)

Metabolite

EV

V7

*

* *


98


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



99

A B

C D

0

0.01

0.02

0.03

0.04

0.05

V8-

1

V8-

2

V8-

3

V8-

4

V8-

5

V8-

6

V8-

7

V8-

8

V8-

9

V8-

10

V8-

11

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

0.001

0.002

0.003

0.004

0.005

0.006

V8-

1

V8-

2

V8-

3

V8-

4

V8-

5

V8-

6

V8-

7

V8-

8

V8-

9

V8-

10

V8-

11

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

0

0.002

0.004

0.006

0.008

0.01

0.012

V8-

1

V8-

2

V8-

3

V8-

4

V8-

5

V8-

6

V8-

7

V8-

8

V8-

9

V8-

10

V8-

11

Rel

ati

ve SanR3 E

xp

ress

ion

VIGS Construct

* * 0

0.005

0.01

0.015

0.02

0.025

EV V8

Rel

ati

ve

Gen

e E

xp

ress

ion

VIGS Construct

SanR1

SanR2

SanR3

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

100

E F

0

5

10

15

20

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V8

0

10

20

30

40

50


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea

,

Arb

ita

ry U

nit

s, M

illi

on

s)

Metabolite

EV

V8 *

*

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.

101

A B

C D

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

V9-1 V9-2 V9-3 V9-4 V9-5 V9-6 V9-7 V9-8 V9-9

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

0.0014

V9-1 V9-2 V9-3 V9-4 V9-5 V9-6 V9-7 V9-8 V9-9

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

0

0.005

0.01

0.015

0.02

0.025

V9-1 V9-2 V9-3 V9-4 V9-5 V9-6 V9-7 V9-8 V9-9

Rel

ati

ve SanR3 E

xp

ress

ion

VIGS Construct

*

* 0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

EV V9

Rel

ati

ve

Gen

e E

xp

ress

ion

VIGS Construct

SanR1

SanR2

SanR3


102

E F

0

10

20

30

40

50

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V9

0

20

40

60

80

100


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea

,

Arb

ita

ry U

nit

s, M

illi

on

s)

Metabolite

EV

V9

*

* *

*

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



103

A B

C D

0

0.002

0.004

0.006

0.008

0.01

0.012

V10

-1

V10

-2

V10

-3

V10

-4

V10

-5

V10

-6

V10

-7

V10

-8

V10

-9

V10

-10

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

0.0035

V10

-1

V10

-2

V10

-3

V10

-4

V10

-5

V10

-6

V10

-7

V10

-8

V10

-9

V10

-10

Rel

ati

ve SanR2

Exp

ress

ion

VIGS Construct

0

0.0002

0.0004

0.0006

0.0008

0.001

0.0012

V10

-1

V10

-2

V10

-3

V10

-4

V10

-5

V10

-6

V10

-7

V10

-8

V10

-9

V10

-10

Rel

ati

ve SanR3 E

xp

ress

ion

VIGS Construct

0

0.005

0.01

0.015

0.02

EV V10

Rel

ati

ve

Gen

e E

xp

ress

ion

VIGS Construct

SanR1

SanR2

SanR3 *

* *

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

104

E F

0

5

10

15

20

25

S DHS

Co

nce

ntr

ati

on

(µ

M)

Metabolite

EV

V10

0

10

20

30

40

50


ou

nt

of

Alk

alo

id (

LC

-MS

Inte

gra

ted

Pea

k A

rea

,

Arb

ita

ry U

nit

s, M

illi

on

s)

Metabolite

EV

V10

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.

105

A B

C D

0

0.005

0.01

0.015

0.02

0.025

V11

-1

V11

-2

V11

-3

V11

-4

V11

-5

V11

-6

V11

-7

V11

-8

V11

-9

V11

-10

V11

-11

Rel

ati

ve SanR1

Exp

ress

ion

VIGS Construct

0

0.0005

0.001

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E F

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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.

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A B

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E F

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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,


109

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,

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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.

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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

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(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,

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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

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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

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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

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(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-

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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

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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

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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

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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

123

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

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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

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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).

127

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

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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

129

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.

131

A B

SanR1

SanR2

SanR3

SanR1

SanR3

SanR1

SanR3

SanR1

Sanguinarine DHS

Phloem cells

Laticifer Sieve

element

Companion

cell

Vacuole

ER SanR1

SanR2 SanR3

Figure 34. Predicted model for opium poppy SanRs.

132

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.

133

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.

134

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;

135

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

136

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.

137

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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

149

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

150

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).

151

Table A1.1. List of primers designed to amplify Papaver somniferum reductases.

Namea Sequenceb RE Direction Amplicon

size (bp)

Protein size

(kDa)

PsoCOR2-qPCR-F1 5'-ATCCCTCCAGCTGTGAATCAA-3' N/A Forward 74

N/A

N/A PsoCOR2-qPCR-R1 5'-GCGTTGCAATACTCTCTCAGCTT -3' N/A Reverse

PsoCOR2-qPCR-F2 5'-TGCAATCTCTGTACTGGGATCAA-3' N/A Forward

66

N/A

N/A PsoCOR2-qPCR-R2 5'-ACCTCAGAACCCAAAACTGCAT-3' N/A Reverse

PsoCOR2-qPCR-F3 5'-CCATGCTCTGGTGCACTGAT-3' N/A Forward

69

N/A

N/A PsoCOR2-qPCR-R3 5'-GATTCCTCAGCGAATTCTGAAGA-3' N/A Reverse

PsoCOR2-qPCR-F4 5'-TGCTCTGGTGCACTGATGCT-3' N/A Forward

74

N/A

N/A PsoCOR2-qPCR-R4 5'-CAATTTAAGATTCCTCAGCGAATTC-3' N/A Reverse

PsoCYP82-qPCR-F1 5'-GACCACCATCTGGACCCTTTC-3' N/A Forward

72

N/A

N/A PsoCYP82-qPCR-R1 5'-TCCACTTCTTGTTTTGCCTTGTC-3' N/A Reverse

PsoCYP82-qPCR-F2 5'-GACACCACAAAACTGACCACCAT-3' N/A Forward

61

N/A

N/A PsoCYP82-qPCR-R2 5'-TTTTGCCTTGTCCAACACATG-3 N/A Reverse

152

Table A1.1 (continued). List of primers designed to amplify Papaver somniferum reductases.


size (bp)

Protein size

(kDa)

PsoCYP82-qPCR-F3 5'-CCAGGCAATCATCAAAGAATCA-3' N/A Forward

77

N/A

N/A PsoCYP82-qPCR-R3 5'-CACAATCTTCGCCGCTCAGT-3' N/A Reverse

PsoCYP82-qPCR-F4 5'-TCGCCGAATTATCGTTCAATG-3' N/A Forward

80

N/A

N/A PsoCYP82-qPCR-R4 5'-CCTGCTTGGAGCACCTGTCT-3' N/A Reverse

Pso-qPCR-ELF-F1 5'-TTTGAGGCCGGTATCTCTAAGG-3' N/A Forward

74

N/A

N/A Pso-qPCR-ELF-R1 5'-TGCTTGACACCAAGGGTGAA-3' N/A Reverse

Pso-qPCR-ELF-F2 5'-CCTCCCAGGTCATCATCATGA-3' N/A Forward

84

N/A

N/A Pso-qPCR-ELF-R2 5'-CAATGTGAGATGTGTGACAGTCAAG-3' N/A Reverse

Pso-qPCR-ELF-F3 5'-TGAGCCTAAGAGACCCACAGACA-3' N/A Forward

87

N/A

N/A Pso-qPCR-ELF-R3 5'-CCCACTGGCACAGTTCCAA-3' N/A Reverse

Pso-qPCR-UBC-F1 5'-CAGCTTCTGGATGAGCCATCA-3' N/A Forward

69

N/A

N/A Pso-qPCR-UBC-R1 5'-GGAGCCCTGCTTGGACTCT-3' N/A Reverse

153



size (bp)

Protein size

(kDa)

Pso-qPCR-UBC-F2 5'-TCGTCGCACACAAGTTGGA-3' N/A Forward 77

N/A

N/A Pso-qPCR-UBC-R2 5'-GCTAGGCTCTTCAAGGAATACAAAGA-3' N/A Reverse

Pso-qPCR-ACT-F1 5'-GTCTGGATTGGTGGGTCCAT-3' N/A Forward

68

N/A

N/A Pso-qPCR-ACT-R1 5'-TCAGCCTTGGAGATCCACATC-3' N/A Reverse

Pso-qPCR-ACT-F2 5'-GTGCCAATCTATGAGGGTTATGC-3' N/A Forward

70

N/A

N/A Pso-qPCR-ACT-R2 5'-TCAGATCACGGCCAGCAA-3' N/A Reverse

Pso-qPCR-ACT-F3 5'-GGGATCGCAGACCGTATGA-3' N/A Forward

74

N/A

N/A Pso-qPCR-ACT-R3 5'-GGTGCAACCACTTTGATTTTCA-3' N/A Reverse

Pso-qPCR-UBQ10-F1 5'-TGGATGTTGTAATCAGCGAGAGTAC-3' N/A Forward

74

N/A

N/A Pso-qPCR-UBQ10-R1 5'-CCAGACCAGCAACGTTTGATTT-3' N/A Reverse

Pso-qPCR-UBQ10-F2 5'-GGTGGACTCCTTCTGGATGTTG-3' N/A Forward

75

N/A

N/A Pso-qPCR-UBQ10-R2 5'-CGTTTGATTTTCGCAGGAAAG-3' N/A Reverse

154



size (bp)

Protein size

(kDa)

Pso-qPCR-UBQ10-F3 5'-GGGAACACAAACGACACCAAA-3' N/A Forward 80

N/A

N/A Pso-qPCR-UBQ10-R3 5'-TCGTCTTCGTGGTGGTAACTAGAG-3' N/A Reverse

Pso-qPCR-GAPC-F 5'-CCCAGCACTTAATGGAAAATTGAC-3' N/A Forward

80

N/A

N/A Pso-qPCR-GAPC-R 5'-TCACAGTAAGATCAACCACTGAAACA-3’ N/A Reverse

PrhCYP82-qPCR-F1 5'-GGGTCTCGTTGAACCTTCCAT-3’ N/A Forward

76

N/A

N/A PrhCYP82-qPCR-R1 5'-ATTGTGTGGTTGGTGGGTTTC-3' N/A Reverse

PrhCYP82-qPCR-F2 5'-CGATGCTTCATCAATTGCTACTG-3' N/A Forward

77

N/A

N/A PrhCYP82-qPCR-R2 5'-GCGGATTCCGATCAGACACT-3' N/A Reverse

PrhCYP82-qPCR-F3 5'-CCTGGATGTAGACAAGGCTACGA-3' N/A Forward

77

N/A

N/A PrhCYP82-qPCR-R3 5'-TGGAAAGAAAAGGAGTTCAACCA-3' N/A Reverse

PrhCOR-qPCR-F1 5'-TGATTCACAGCTGGTGGGATAT-3' N/A Forward

72

N/A

N/A PrhCOR-qPCR-R1 5'-TGTCAGCAATTTCTCCTGCAA-3 N/A Reverse

155



size (bp)

Protein size

(kDa)

PrhCOR-qPCR-F2 5'-CCTGAAGCTTTTTGCAGGAGAA-3 N/A Forward 79

N/A

N/A PrhCOR-qPCR-R2 5'-GCAGCCATGGAAGAGTGTCA-3' N/A Reverse

Prh-qPCR-ELF-F 5'-GCAGCCTCCTTCTCGAACCT-3' N/A Forward

75

N/A

N/A Prh-qPCR-ELF-R 5'-CACCACTGGTCACTTGATCTACAAG-3' N/A Reverse

Prh-qPCR-TUB-F1 5'-CTCATTCCCTTCCCTCGTCTT-3' N/A Forward

69

N/A

N/A Prh-qPCR-TUB-R1 5'-CTGGGAACCCCGAGATGTG-3' N/A Reverse

Prh-qPCR-TUB-F2 5'-GCAGATGTGGGACACCAAGAA-3' N/A Forward

74

N/A

N/A Prh-qPCR-TUB-R2 5'- TGGCTGAGGCAGTGAGGTATC-3' N/A Reverse

Prh-qPCR-UBC-F1 5'-TGTCATCGCACACAAGTTGGA-3' N/A Forward

80

N/A

N/A Prh-qPCR-UBC-R1 5'-GGCTAGGCTCTTCAAGGAATACAA-3' N/A Reverse

Prh-qPCR-UBC-F2 5'-GTTAGTGTCATCGCACACAAGTTG-3' N/A Forward

84

N/A

N/A Prh-qPCR-UBC-R2 5'-GCTAGGCTCTTCAAGGAATACAAAGA-3' N/A Reverse

156



size (bp)

Protein size

(kDa)

Prh-qPCR-ACT-F1 5'-GACACGGAGCTCATTGTAGAAGGT-3' N/A Forward 78

N/A

N/A Prh-qPCR-ACT-R1 5'-ATTGAGCATGGTATTGTCAGCAA-3' N/A Reverse

Prh-qPCR-ACT-F2 5'-GCGACACGGAGCTCATTGT-3' N/A Forward

80

N/A

N/A Prh-qPCR-ACT-R2 5'-ATTGAGCATGGTATTGTCAGCAA-3' N/A Reverse

Prh-qPCR-ACT-F3 5'-CACAGTCCCCATCTATGAAGGTT-3' N/A Forward

75

N/A

N/A Prh-qPCR-ACT-R3 5'-GTCAGATCCCGTCCAGCAA-3' N/A Reverse

Prh-qPCR-UBQ10-F 5'-TGATTTGCAGGAAGTGCTATGC-3' N/A Forward

79

N/A

N/A Prh-qPCR-UBQ10-R 5'-TGGTTGCTGTGACCACACTTCT-3' N/A Reverse

Prh-qPCR-GAPC-F1 5'-CAACGTGGACGATCAAGTCAATAA-3' N/A Forward

79

N/A

N/A Prh-qPCR-GAPC-R1 5'-AACTTCGTCAAGCTTGTGTCATG-3' N/A Reverse

Prh-qPCR-GAPC-F2 5'-CATCATCTCTCTGTAGGGCAACTC-3' N/A Forward

79

N/A

N/A Prh-qPCR-GAPC-R2 5'-GCAAAGATCAAGATCGGAATCAA-3' N/A Reverse

157



size (bp)

Protein

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

158



size (bp)

Protein

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

159



size (bp)

Protein size

(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

160



size (bp)

Protein size

(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

161



size (bp)

Protein size

(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

162

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.

163

Table A1.2. Predicted open reading frame of P. somniferum dehydroreticuline reductase candidates.

Candidate Sequence (5’ to 3’)

DRR1

ATGAATTATGCACAAAGTAGTAGTGATCCAAGTCTTAGATGGTCTCTTAAGGGAAAGACTGCACTTGTCACTGGCGGAACCAAAGGGATTGGACACGCTATC

GTCGAGGAGTTGGCTGGATTTGGTGCAGCTGTTCATATCACTTCTCGACATGAAAATGAAATCGAGGAGTGCTTGCGAAATTGGAGAGAAAAAGGCTATACA

GTGACTGGGTCGGTCAGTGATGCTTCAGTTCCGGTTGATAGAGAGAAATTAATCAAGACTATTTCATCTGTTTTTGGGGGCAAACTAAACATCCTTGTTAACA

ACGTTGGGGGAGCTGTGTTCAAAGACACCGTGGATTACACAGATGAGGACCATGCGAAAGTTATGTCTACTAACTTCGACTCTAGCTACTATTTATCAAAACT

AGCACACCCTCTCTTGAAAGCCTCTGGCTCAGGAAGCATTGTGTTCATTACATCTGTTGCGGGCATTGTTGCTGCACCGAAGGTGTCCAGTTATGCGGCATGT

AATGGAGCTATTAATCAAGTCACAAAGAACTTTGCTTGTGAATGGGCGAAAGATAAGATACGGGTCAATAGCGTAGCACCATGGTATATTAGAACCAGGCTT

GCAGAACATGATCTGCTATTATTTGAAGGGCTTGAAGACTCTATCGTAGCTAGAACTCCTATGAGGCGTCTTGGGAACCCAAATGAAGTTTCATCTCTTGTGG

CATTCTTGTCCCTACCTTGTGCTTCGTACATCACTGGCCAAGTCATTTGTGTCGATGGTGGATTTGCAGTAAATGGTTTCTTCCCTACACAAGATTGA

DRR2*

ATGGAGGTTGAGAAAGTAGAGAGTAGTAGCAGTAAGAAGATTACAGTTGCTGGAGGAAATGGAAGATGGTCTCTCAATGGAATGACTGCTCTAGTCACTGGT

GGTACTAGAGGAATCGGATATGCTGTTGTGGAGGAATTGGCTGGATTTGGGGCAAAAGTACATACTTGTTCAAGAAATGAAATTGAACTTAATCGATGTTTA

CAAGAATGGAAGCAAAAGGGTTTTCAAGTTACTGGCTCTGTTTGTGATGTTTCGTCTTCAGAGGGTCGTGTCAAGCTCATGGATTCTGTGTCTGGTCTTTATAA

CACCAAGCTCAATATCCTTGTTAATAATGTTGGCACAAATATAAGAAAACCGTCGGTGGAGTACACTGCTGAAGAATACTCGAAACTGATGTCTACCAACTTG

GAATCCTGTTACCACCTATGCCAACTTGCACACCCGCTTCTGAAATCTTCAGGGATGGGAAGTATTGTGTTTATCTCTTCTGTCGCTGGTGTGGCGGCATTGGC

TACTGGGAGTATTTATGGAGCAACTAAAGGAGCAATGAATCAACTCGCAAAAAGTTTGGCATGTGAATGGGCGAAAGACCACATTAGGTGTAACTCTGTTGC

ACCTTGGTACATCAAAACCTCGCTTGTCGAACATTTGCTTGAAGACAAAGAATTTGTAGATAGAGTAATCGCCCGTACTCCTCTTCGACGAGTTGGAGAACCG

CAGGAGGTTGCATCACTGGTTGCCTATCTTTGCTTACCTGCTTCCTCTTACATCACGGGGCAGACTATCTCTGTTGATGGAGGGATGACTGTCAATGGGTTTTT

TCCAACTCATGACTAG

DRR3

ATGGAGTCTCCATTCAAGGCTGATATAGTGAAAGGGAAAGTAGCTTTGATTACTGGAGGAGGATCAGGAATTGGGTTTGAGATTACTAAAGAATTTGGTAGA

CATGGAGCTTCTGTTGCTATCATGGGCAGACGCAAATCTGTTCTTGATTCTGCTGTCTCCTCTCTTCGCTCTCTCGGTATCCAGGCAATTGGATTAGAGGGAGA

TGTGCGGAAGAAAGAAGACGCGGCTAGAGTTGTCGATTCAACATTTGAGCATTTTGGGAGGATAGACATTCTTGTTAATGCTGCTGCTGGAAATTTTCTTGTG

ACTGCTGAGGATTTGTCCCCAAATGGATTTAAAACAGTTATGGATATTGATTCCGTTGGCACATTTACAATGTGCCACACAGCGTTGAAGTATATAAAGAAAG

GGGGTCTTGGGAGGGGTATGTCTGGTGGTGGAACCATAATGAACATAAGCGCTACGCTACATTATACAGCAGCTTGGTATCAAATCCATGTATCTGCCGCTAA

GGCAGCTGTTGATGCCATTACAAGGTCGTTGGCGTTAGAGTGGGGTACGGATTATGATATAAGAGTCAACGGGATTGCACCAGGACCAATTGGTGACACTCC

TGGCTTGAGTAAGCTTGCTCCCGATGAAATGAAAATCAACCATTCTGAAGACGCCAGGCCTCTGTATAAAGCAGGAGAGAAATGGGATATTGCTATGGCTGC

TCTCTACCTAGCTTCAGATGCAGCCAAGTACATCAACGGTATGACACTTGTGGTCGATGGAGGAAACTGGTTGAGCCGGCCCCGCCACATTTCAAAAGAGGC

AGTGAAGGAATTGTCTCGGGTTGTGGAAAAGAGATCCAGATCAGGTGCACCAGCACCTGCCAGGGGAGTTCCAAGCCGCAGTAAAATGTAG

DRR4

ATGGCTACAATCCAATGCATCAAGGCTCGTCAGATCTTTGATAGTCGTGGTAACCCAACCGTTGAGGTTGATATTAAACTATCCAATGGAACTTTCGCCAGAG

CCGCTGTTCCAAGTGGTGCATCTACTGGTGTTTACGAAGCTCTTGAACTACGTGATGGAGGTTCAGAATACCTAGGAAAGGGAGTTTCCAAGGCTGTTGACAA

TGTTAACTCCATCATTGGGCCTGCATTGATCGGAAAGGACCCAACACAACAAACCGAAATTGATAACTTCATGGTGCGAGAACTTGACGGAACTACCAACGA

GTGGGGTTGGTGCAAGCAAAAGCTTGGAGCCAATGCTATCCTAGCAGTGTCTCTTGCCGTTTGCAAAGCTGGAGCCAGTGTTTTGGACATTCCCCTTTACAAG

CATATTGCCAACCTTGCTGGTAACAAGAACTTGGTACTTCCAGTTCCTGCTTTCAATGTTATTAATGGAGGATCGCACGCAGGAAACAAGCTTGCAATGCAAG

AGTTCATGATCCTTCCCGTTGGAGCCAAATCCTTCAAGGAGGCAATGAAAATGGGAGTTGAAGTATACCACAATTTGAAGTCCGTCATCAAGAAGAAGTACG

GTCAAGATGCAACCAATGTTGGTGATGAAGGTGGCTTTGCTCCCAACATCCAAGAGAACAAGGAAGGACTTGAGTTGCTTAAGACTGCTATTGCTAAAGCTG

GCTACACTAAAGAAGTTGTCATCGGAATGGATGTTGCTGCCTCAGAGTTTTACGGATCAGACAAAACCTATGACTTGAACTTCAAGAAGAGAACAACAACGG

AGCAGCAAAGATCTCCGGTGAGAAATTGA

164

Table A1.2 (continued). Predicted open reading frame of P. somniferum dehydroreticuline reductase candidates.


DRR5

ATGGTATTCCTCCAAACTCATTCTCTCACAAGAAACATCAAATCCCCCTTTACTTCATCTTCATCAGCATCACCAATTGTTAACCACTCCAAACCTTTCTCTTCT

GCTAACACATCTATTAGAGCTACAACAACAAAGATGGAAAACACAGGAATTGAGGGTTCAAATGTAAATGACAATAAAAGGAAAATATTTGTGGCTGGGGC

TACTGGAAGTACTGGTAAAAGAATCGTTGAACAACTTTTAGCCAAGGGTTTTGCAGTTAAGGCTGGTGTTCGTGATGTTGACAAAGCTAAGACTACTTTTTCA

GATAACCCATTTCTCCAAATCGTGAAGGCTGATGTAACTGAGGGTTCAGCTAAGTTAGCTGAAGCTATTGGTAATGATGCTGAGGCTGTTATTTGTGCTACCG

GATTTCGTCCTTCATTGGATTTGTTTACTCCTTGGAAGGTCGATAATTTTGGCACAGTAAACCTTGTAGATGCATGCAAGAAAAGTAATGTGAATAGATTTATT

CTCATCAGTTCCATTCTAGTCAATGGTGCTGCAATGGGACAAATCTTTAATCCAGCTTACCTATTCCTAAACGTATTTGGACTAACGTTGATAGCCAAACTACA

AGCGGAGAATTATATCAGGAAATCGGGTATAAACTACACAATTATAAGGCCTGGTGGATTGAAAAATGACCCCCCAAGTGGAAATTTAGTCATGGAACCTGA

GGATACTCTGTCTACAGGTTCTGTATCTAGAGATCTGGTTGCAGAAGTAGCTGTTGAGGCATTAGGCCACTCTGAATCTTCTTACAAGGTAGTGGAGATAGTT

TCCCGTCCTGAAGCTCCAAAACGCTCATTTGAAGATCTCTTTGGCTCTATCAAACAACGCTAA

DRR6

ATGGCAGAAGCACTCCTCAATTCTGAAGTAAAGAGGTGTGCAGTAGTTACAGGTGCCAACAAGGGGATTGGTTTGGAGATTTGTAGGCAATTAGTTTTTAAT

GGAATCTTTGTTGTATTGACATCTAGAGATAGAAACAAGGGTCTTGAAGCTGTTGAAAATCTCAAAAAATCTGGACTCCCAAATGTGATTTTTCATCAATTAG

ATGTTATGAATCTAACTTCTGTCTCATCCTTGGCTGAATTCATCAAAACCCACTTTGGAAAACTTGATATCTTGGTGAATAATGCAGGCATTGGTGGGGTGAG

AATAGTAGACAAGGATCAATTAAAGGTTCTGATGCTTGAGGATGAAAAGTATTTGGAGAACCCAAAGTTGAAACAGATATGGACAGAACCGTATGACGACG

CAGAAAAATGTCTTAAAACAAATTACTACGGCGTCAAGGCGGTGACTGAAGCCTTTATTCCTTTACTTGAACTATCTGATTCAAGAATAATTGTCAATGTTTCT

TCTGCCATGGGGATGCTGAAGAATGTTGGCAATGAAAAGGCTTTCGAAGTGCTCAGCGATGCTGATTGTTTAACGGAAGAAAGAATAGATGTGGTGGTGAAT

ACATTTCTAAACGATCTTAAAGAGGGTTGCTTAGAAGCAAAAGGTTGGCCAACATTACTTTCTGCTTATACAATCTCAAAAGCATCAGTTAATGCGTACACTA

GGATTTTGGCAAAAAAATTCCCTACTTTTCGCATAAACTGTGTTTGTCCTGGTTTTGTCAAAACCGATATAAACTTTAACAGTGGGGTCTTGACTGTTGAACAA

GGTGCTAAAAGTCCTGTAAGATTAGCTCTTTTACCAGATAACATAACTTCTTCTGGGCTCTTCTTTGTTCGTGAAGAGGTATCGTCATTTTGA

DRR7*

ATGGAAGAAACCATCTCTACAACAGCAGAAAAGAGGTGTGCGGTTGTTACAGGAGCTAATAAAGGAATAGGACTAGAGATATGTCGTCAGCTCGCTATTAAT

GGTATCACCGTTATACTAACCGCAAGAGATGAGACCAGGGGAACTCGATCTGTTGAAGCTCTTAAAGGGTCAGGACTTGCTGGTGTGATTTTTCATCAGCTTG

ATGTAAACGATGCAACTAGTGTTACTTCATTGGCTGGTTTCATCAGAACTCGATACGGGAAACTTGACATCTTGGTAAACAATGCAGGGGACAACGGAGTAA

GACTACAAATTACTGAGGAGGCTATGAAAGACATGAACTTTCGATATGGTGATGAGAATGATGAGATTGCCAAGTTGTTGGAGGAATCCATTGAGGAGACAT

ACGAGAGGGCAAAACAATCCATAGAAACAAATTACTATGGAACCATAAGAATAACTGAAGCACTGCTTCCACTTCTTCAACTCTCCAATTCAGCAAGAATTG

TGAATGTTTCCTCCGTATATGGTCAACTAAAGTTTATCTCGAGTGAGACGATTAAAGAGGAGCTAAGAAATGTTGATTGCTTAACGGTAGAGAAACTGGACG

AGCTTATGCAGAAGTTCTTAAAGGATTTTAAGGATGGGATGTTGGAGACTAATGGATGGCCTGTATTAGTTTCTGCGTATAAAGTCTCGAAAGCTGCTATCAA

TGCCTACACTCGAATTCTTGCGAGGAAGTTCCCAACTTTTCGTGTTAATGTTGTTCATCCTGGTTTGGTTAAAACAGATATTGCATTCCAACAAGGTAACTTAA

CACCGGATGAAGGAGCTAAAGCACCGGTTATGGTGGCATTGTTGCCTTCTGATGGCCCTTCTGGTTTCTACTTTGACCAGATGCAGATTTCAACCTTTTGA

DRR8*

ATGGCATTAAAAGAAGTTAACGAGCCCTCTGCTTCCTTAACTAGGTGGTGGTCGGGAAACACCGTAGCGGTTGTGACCGGAGGAAATAGAGGGATCGGATTC

TCTCTAGTTAAGAAACTCGCCGAGCTTGGATTAACTGTAGTCCTAACTTGTAGAGATGATTCTAAAGGTCAAGAAGCAATTGAATCACTCAAATCTCAAGGAC

TCAATAATGTTCGATTCTTCCGATTAGATGTTATGGACACTGCTTGCATCAATGAGCTGGTTTCATGGTTGAAGGAAGCATTTGGAGGTCTTGATATTCTTGTG

AATAATGCTGCTGTGTCGTTCAACGAGATCAACGAGAACTCGATGCAACACGCCGAAACCACCATCAAGACAAACTACTATGGACCGAAGTTGTTAACCGAA

GCTCTGCTACCACTGTTTCGGCGTTCGGAATCCGTAAGCAGGATTTTGAATGTTAGCTCGCGTCTTGGCTTGTTGAACAAGGTGAGTAATCCTGTTGTAAGGG

AGTTATTAGAAGACGAAGACAGATTATGCGAAGAACGTATAGATTTTGTTGTAAATCGATTTCTTGAAGATGTTACTACTGGTACTTGGGAAAGAGAAGGAT

GGCCAAAGGTATGGACAGATTACGCAGTCTCGAAAGTAGGATTGAATGCGTACTCTCGAGTTTTAGCTAAGCGTTATGATGGGTTGGGATTATCTGTCAATTG

TTTATGCCCCGGGTTTACACAGACAGACATGACCGCCGGAAAAGGAAATCACTCGGCGGATTCAGCTGCGGAAATGGCTGCACAAATTGTCTTACTACCACC

TGAGAAACTTCCCACTGGCAAGTTTTATATAAAAAACAAACCATTCTTGTACTCGAAGATATAA

165

Table A1.2 (continued). Predicted open reading frame of P. somniferum dehydroreticuline reductase candidates.


DRR9

ATGCCTGCACCAGTAATGACTCATGAGAATGTAGCAGCATCCATTCAAGGATCAGGAATGAACCATGTCATGAACTCTCCTGCACCAAGAAGGTTGGAAGGA

AAAGTTGCGATCATCACCGGTGGTGCGAGGGGGATTGGGGAAGCAACTGTAAGACTCTTTGTAAGACAAGGTGCAAAAGTAGTCATTGCTGATGTTGAAGAT

GCTACTGGAACTTCACTTGCAAATTCATTAGCTCCTTCAGCTACATTTGTACATTGTGATGTCACCAGAGAAGAAGATATCGAGAACCTAATCGATTCAACAA

TAGCACATTACGGGCGACTGGATATACTTTTCAACAACGCTGGTATTCTCGGAAACCAATCAAAACGGAAAAGCATTTTGAACTTTGATGCTGATGAGTTCGA

CTCAGTTATGCGTGTTAATGTGCGAGGGACTGCATTAGGTATGAAACACGCTGCACGAGTTATGATGCCAAGAGGTACCGGATGTATCATCTCAACAGCCAG

TGTGGCCGGAGTCATGGGAGGATTTGGACCTCACGGGTACACGGCTTCGAAGCATGCCATCGTCGGACTTACAAAGAATACGGCTTGTGAATTAGGAAGGTA

TGGGATTAGAGTTAACTGCATTTCCCCATTTGGTGTTGCAACTTCAATGCTTGTTAATGCATGGAGGAAAGTTGAAGATGAAGATGAAGAAGATAGTATGGAT

TTTGGAGGACCTTCTGAAAAAGAAGTTGAGAAAACGGAGGAGTTTGTGCGTGGTTTAGCAGATCTTAAAGGAACAACCCTTAAACCAAGAGATATTGCTGAG

GCTGCTTTGTTTCTTGCTAGTGATGAATCAAGATATGTAAGTGGTCATAATTTAGTTGTGGATGGAGGAGTTACTACTTCAAGAAACTGTGTAGGATTGTAG

DRR10*

ATGTCTAAACTAAGATTAGAAGGTAAAGTAGCTATAATTACTGGAGCAGCAAGTGGTATTGGCGAAGCAACAGCGAGGCTATTCGTCGAACATGGTGCGTTC

GTTGTAGTTGCAGACATTCAAGACGAATTAGGGGATCAAGTTGTATCTTCAATTGGTAAAGAGAAAGCTAGTTACAAACATTGCGATGTAAGTGTCGAAAAA

CAAGTTGAAGAAACAGTAGCATTTGCTTTAGAGAAATATGGATCTCTAGATATTGTGTATAGCAATGCAGGTATGGGCGGATCTTTTTCGAGTATCCTTGATT

TCAGCTTGGAAGATTTTAACAAGATTATTGCTACAAACGTATCCGGTGCAGCATTAATGATCAAACACGCTGCTCGTGCGATGTTAGACAGAAAAATCCGTGG

CTCGATTATATGTACTGCGAGTGTAGCTGCAGTTCAAGCTGGATTTGCACCACACTGTTACACAGCGTCTAAACACGCTGTGCTAGGATTGGTTCAATCAGCT

TGTAGCGAACTTGGTGCTTACGGAATAAGGGTGAATTGTATTTCTCCATCTGGAGTTGGAACACCATTAGCCTGTGATATAGGCAAGATTAGTGCAAGGCATG

TTGAAGAATATACAGCAAAAATGAGTCTTCTGAAAGGGATTATTTTGAAAGCTAAACACATTGCTGATGCTGCATTGTTTCTTGCATCCGATGATTCGGTTTAT

CTGAATGGACATAATCTTGTTGTTGATGGCGGATTTACAGTTGCGGCTAGTAGCTTTCCCATTAATGTTAGTTCTGCTACTTCATGA

DRR11*

ATGGCGCTGGATAATGCGAACGCAACCCCTTCTTCTTCCTCCCTCCTACTGGAAAACCGGGTGGCGATAGTCACAGGTGCATCAGGTGGAATCGGTGGTGCAA

TCGCCCGTCACCTTGCCTCTCTCGGTGCAAAATTAGTCCTCAGTTACTCCAGTAACTCAACCCAAACTGATCTCCTTGCCACTGAACTCAACAACTCTTCATCA

TCATCCTCACAGCCAAAAGCCATATCAATCAAAGCCAATGTTTCAGATCCAGACCAAGTCAAATCGTTATTCGATCACGCCGAGAAGGTTTTCAACTCGCAAC

CACATATCTTAGTTAACTCTGCCGGAGTATTAGATCCGAAATACCCTACAATCTCCAACACCAAGATCGAAGATTTTGATCACATATTCAACATAAACGCAAA

AGGAGCGTTCTTATGCGCCAGAGAAGCTGCTAACCGGTTGGTACGCGGTGGTGGTGGACGGATTATATTGATTTCATCGTCTATGGTTGGCGGATTGAAACCT

GGGTTCGGTGCTTATGCTGCGTCGAAGGCGGCGGTGGAGACCATGATGAGAATTCTTGCGAAAGAATTGAAAGGGACTGGAATTACAGCTAATTGTGTTGCG

CCTGGACCTATTGCAACTGACATGTTTTATGCAGGGAAAGGAGAAGAGCAAATTAAGAATGTGATTGCAGAATGTCCGTTGAGTCGACTCGGTGAAACTAAA

GATGTTGCTCCTGTTGTTGGGTTTTTGGCTGGGGATGCTAGTGAGTGGGTTAATGGACAGGTTATCCGTGTTAATGGCGGATATGTTTAG

DRR12*

ATGGCAGAAGCCATAGTTGTTAAAAATGAGAAGAAGAAGCTGGAAGGAAAAGTGGTTATCATCACCGGAGGAGCCAGCGGTATTGGAGAGGCAACCGCAA

GGCTATTCGCCAATCATAACCCAAGTATGATTGTCATTGCAGACATCCAAGACCAAAAAGGCCACGCGGTAGCAACGTCCATTGGTTCACAAATTTGTTCCTA

CATCCACTGCGATGTCTCCGATGAACTACAAGTCAAATCGATGGTGGATTCCACAGTGAAGAGCTACGGCGGACTCGATATCATGTTTAGCAACGCTGGTATT

GCTAACGGATGTCACCAAACAATCCTTGATATAGATTTGGCTGATTATGATCGTCTCATGGATATCAACACCCGTGGGATGCTTGCTTGCGTGAAACATGCTG

CCAAGGCCATGGTTGACGGCGGGGTGAAAGGTAGTATAGTTTGTACGGCAAGCACTGCAGCAACCTCGGCGCTTGATGGATACTTGGATTACACTATTTCTA

AGCATGCAGTTTTGGGGTTGATGAGATCAGCTAGTCAACAACTCGGCAAATACGGTATTAGAGTGAATTCTGTATCTCCATCAGCTGTAGGAACTGCGATGCC

ATGCAAGACCTATGGTACTGATGCAGAGGGCATTGAGAAGATGTTCATGAGTTCCACTGTCCTAGGAGGTGCCGGATTAGTTTTAAAAGTGAATCATGTGGCT

GAAGCTGTGTTGTTCTTGGCTTCTGACGATTCTGCCTTCATTACTGGCCATAATTTGATGGTTGATGGTGGCTCTCTTCATGGATAA

*Dehydroreticuline reductase (DRR) candidates were assayed for activity. None were able to reduce dehydroreticuline to reticuline.

166

DRR11 DRR2 DRR8 DRR12 DRR7 DRR10

M

27.0

34.6

42.7

U I S U I S U I S U I S U I S U I S

A

B

11 2 8 12 7 10 M

DRR Candidate

27.0

34.6

23

30

46

11 2 8 12 7 10 M

DRR Candidate

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.

167

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).

168

0

10

20

30

40

50

60

70

80

90

100

No

Enzyme

SanR1 SanR2 SanR3B

Am

ou

nt

of

Su

bst

rate

Co

nv

ersi

on

(%

) Chelirubine+NADPH

Chelirubine+NADH

Macarpine+NADPH

Macarpine+NADH

Chelerythrine+NADPH

Chelerythrine+NADH

0

5

10

15

20

25

30

35

40

SanR1 SanR2 SanR3B

Co

nv

ersi

on

of

Sa

ngu

ina

rin

e

to D

HS

(%

)

NADPH

NADH

A

B

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.

169

0

1000000

2000000

3000000

4000000

5000000

6000000

0 10 20 30 40 50 60 70 80

Am

ou

nt

of

DH

S F

orm

ed

(LC

-MS

Co

un

ts)

Temperature (°C)

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 10 20 30 40 50 60 70 80

Am

ou

nt

of

DH

S

(LC

-MS

Cou

nts

)

Temperature (°C)

A

B

SanR1

SanR3B

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.

170

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:


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


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).

171

>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

TTGCTCTAGTCACCACTCGTTTCTGAATGTTGAGATCCAAGAACAACTTATCATCTGCTATGGATTCTTGAGCTTGTTTGTACTTTATGCTTCTTAAATTTACAGAATTACACAGAAATAATGTATTGCCTGTTTAAAATGACACTTTGCCACTATTCCTCATCCAAAACAATTCCAGGTGCTGTGCTAGACATGTAAACAGATCGTGACCAGTTACCTATAGTTGCAGTTGCATTAAAGT

>comp73923_c0_seq1

CTTTCTTTCCTTATTTCTTATATTCTTCTCTTCTAGAAGAAAGTTTGAATTTTCGGTGAAGAAAACTTCCGA

TCAGCGGTTTCTTTATAAATATTACTCATATCATCACTTTCTTCTGCAATTCATTTCACTATCATATCTAAA

TCTTTAAAGGAAAAAAAAAAGTTTCGTCAATTTCAATTACATCTTGTTTCCACAGATTTTAAAATGGCAGCA

TTAATGCAAAAGATTACAGTTCTTGTTACCGGGGCTTCAGGTTTAACTGGTGAGATTGCATTCAAGAAACTG

AAAGAAAGATCAGACAAATTTGCAGCAAGGGGTTTAGTAAGATCGGAAGCAAGTAAGCAAAAACTTGGGGGA

GGTGATGAAATTTATCTTGGTGATATAATGGATAAGAAAAGTCTAAAACATGCTATGCAAGGAATTGATGGC

TTAGTTATACTGACAAGCGCTGTACCGAAGATAGTACCTGGATCATATCCTGGTGCTGATGGCAAAAGAGCT

GAAGATGTGTTTGATGATTCATTTGATTACAGTGGTCCAATGCCTGAATTCTTTTATGCGGAAGGACAATAC

CCAGAACAGATTGATTGGATTGGACAAAAGAACCAGATCGAAACTGCTAAAGCTTGTGGCGTCAAACATATT

GTTTTGGTTGGATCAATGGGTGGAACAGACCCTAATCATTTCTTGAATCATATGGGCAATGGAAATATACTT

ATTTGGAAGAGAAAAGCTGAGCAGTATCTGGCTGATTCTGGAATCCCGTACACAATTATAAGAGCTGCTGCT

CTAGATAACAAGGTGGGTGGCAGGGAGTTGTTGGTTGGAAAGGATGATGAGCTTCTCCCTACTGAAAATGGA

TACATTGCTAGGGCAGATGTTGCTGAAGCTTGCGTTCAGGCTCTGCAAATCGAGGATTGCAAATTCAAAGCG

TATGATTTGGGATCAAAGCCAGAGGGAGTTGGTGAGCCAACAAAGGATTTCAAGGCTCTTTTTTCGCAAGTC

ACTACTCCTTTCTGAAATCTGAGATCCAAGAGCAATTTAGTACACGTTATGGTTGCTTGTGCTTGTTTGCATTTTTCGCTTCTTAAATATACATAACAGGATTGCACCGAAATAATGTATTACCTATTTAATGGTTGCTTGAATTAATGAAATCGCATTCTAATATATGGGGCAGACAGATTTGCGATGCCGGCTTATAGGTTTAGATGATCTCTTCTTGTAATTTTTCTTCTTTCCTTTCTTTCCTTGGAGGTCAGGTCTTTGCCCCGCTCCCTTTTTGTTTTCTTTTCTTTGATTAATAAAGCGTCCTTGTGAGTTTTTTTTTCTTTTTTTAAATCTTAGTTATGGCTCTTATATGTTTTAGATACTTTTGGTTGGATGCAACATATCGGATATGAAACAGTCCAGTCTTTTACAGAGTCCTAGAACTGT

TTTTTGTCTGACTCTTTCATTTACCGAGATGAATCTCTTGCTAAAATTGTTTTTAACAATACTTTCCTTTCC

ATTCTTGCAGAAGGGATTTGTTTCTTTAGCCGGAATGCGCTTCTGATAAGTATTCGTTTCTCAAGCACGAGT

ATGATCGAAGGTGGAGTAGAACACTTGAGTGCAGTTCCAATCCAGGGTTGAACTGAGATCAAAGTGTGGAAG

CTCAATTTTTCTAACGACTGACATACCAGTTTAGTGCTAAAGGAAATCTTATCTGAAGAATAATGTCATTTT

172

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

TGAGATTCCATATGCGGTATGTTCTGATTGAATTTTTGGTTGATGCCTAGCGATTTGTAATGCCACTGGCTATTAGCAAGAGGGAAACTAGTATTCTTTTTCCTCATTAGAAAACCAATGAGAGGCCATGAATAACGATGATAGTGTATTTTACATTTTGTGTTCCGTCTAACGTTGTTTGAGTTATGAGTGATTGCTTATCATGCCTAGTTAGGCTGAAGCATAGTCGCGTGATGTCTTCATTCAAATGCTGCGAAGAATATGACGGGGCTGGCAATACCCTTATCTCATTCCCCTCTGCGAGATTCGAGTTTCTAGTCCTACTGGTTGCGAGTTCATCGTGCTTGTGAACACTCAGTTCGTCGAAAAGGCTTATCTAATTCTAATCTAACATATGAACCATCAACCCCATTCAGGGATCAAATT >comp1703_c0_seq3

TAGATGAAACGGTTTTTGCAGAAAGGGAAGAGGATAAGAATAAGATCATCATCATCTTCAATACTTCCCGTACTGTTGGAATATCTTTTTCTCTCTGAATTACAGTCTCAGTCACAGAGTCAAGAGACCTTACCGACCTAAGTTTAGTTAGCAAATCCATCCATTAAAATCAGGGCAATGGGTTTAGTGACACGTGTTCCGTTATTCTCTTCACCTTCTTCAACTTTCTCTCCTCATAAATACTCTTCCACCACCAAACTATTCTCTTCTTCATCTTCATCTTCCCTT

TCATTTCAAAGGAGAACTTCAGTTGTAGTGAAAGCAATGGCGAGTACTGTGATTGTTACTGGTGCCGGTGGT

AGAACTGGGCAAATTGTTTACAAGAAACTGAAAGAGAGAGCTGAGTTTGTAGCAAGGGGGTTAGTAAGAACG

GAAGAAAGCAAAGAGAAAATTGGAGGAGCTGACGATGTTTTCGTTGCTGATATTAGGGATGCTGAGAGTATT

GTACCTGCAATCCAAGGAGTTGATGCTCTTGTCATTCTTACCAGTGCTGTCCCCAAAATGAAACCGGGGTTT

GATCCTACTAAAGGTGGAAGACCTGAGTTCTTTTTCGAAGATGGAGCTAATCCTGAACAGGTTGATTGGATT

GGGCAGAAGAATCAAATAGATGCTGCAAAAGC

>comp1703_combined

TAGATGAAACGGTTTTTGCAGAAAGGGAAGAGGATAAGAATAAGATCATCATCATCTTCAATACTTCCCGTA

CTGTTGGAATATCTTTTTCTCTCTGAATTACAGTCTCAGTCACAGAGTCAAGAGACCTTACCGACCTAAGTTTAGTTAGCAAATCCATCCATTAAAATCAGGGCAATGGGTTTAGTGACACGTGTTCCGTTATTCTCTTCACCTTCTTCAACTTTCTCTCCTCATAAATACTCTTCCACCACCAAACTATTCTCTTCTTCATCTTCATCTTCCCTT

TCATTTCAAAGGAGAACTTCAGTTGTAGTGAAAGCAATGGCGAGTACTGTGATTGTTACTGGTGCCGGTGGT

AGAACTGGGCAAATTGTTTACAAGAAACTGAAAGAGAGAGCTGAGTTTGTAGCAAGGGGGTTAGTAAGAACG

GAAGAAAGCAAAGAGAAAATTGGAGGAGCTGACGATGTTTTCGTTGCTGATATTAGGGATGCTGAGAGTATT

GTACCTGCAATCCAAGGAGTTGATGCTCTTGTCATTCTTACCAGTGCTGTCCCCAAAATGAAACCGGGGTTT

GATCCTACTAAAGGTGGAAGACCTGAGTTCTTTTTCGAAGATGGAGCTAATCCTGAACAGGTTGATTGGATT

GGGCAGAAGAATCAAATAGATGCTGCAAAAGCAGCGGGAGTGAAGCAGATTGTTTTGGTTGGGTCTATGGGT

GGAACGAACCTCAATCATCCCTTGAACAGCATTGGAAACGGAAACATATTGGTGTGGAAGAGGAAGGCGGAG

CAATATCTGGCCGACTCTGGTATACCATACACAATTATTAGAGCTGGAGGCTTACAAGACAAAGATGGGGGT

173

GTGAGAGAGCTTGTTGTTGGCAAAGATGACGAGCTTCTCGAGACTGACATAAGGACTATTGCTAGAGCCGAT

GTTGCAGAAGTCTGCATTCAGGCATTGCTGTTGGAAGAGGCTAAGTTTAAAGCATTGGATCTCGCTTCAAAA

CCAGAAGGAACTGGCGAGCCAACAAAAGATTTCAAGACTCTCTTTTCTCAAATCTCTACACGATTCTGAGATTCCATATGCGGTATGTTCTGATTGAATTTTTGGTTGATGCCTAGCGATTTGTAATGCCACTGGCTATTAGCAAGAGGGAAACTAGTATTCTTTTTCCTCATTAGAAAACCAATGAGAGGCCATGAATAACGATGATAGTGTATTTTACATTTTGTGTTCCGTCTAACGTTGTTTGAGTTATGAGTGATTGCTTATCATGCCTAGTTAGGCTGAAGCATAGTCGCGTGATGTCTTCATTCAAATGCTGCGAAGAATATGACGGGGCTGGCAATACCCTTATCTCATTCCCCTCTGCGAGATTCGAGTTTCTAGTCCTACTGGTTGCGAGTTCATCGTGCTTGTGAACACTCAGTTCGTCGAAAAGGCTTATCTAATTCTAATCTAACATATGAACCATCAACCCCATTCAGGGATCAAATT

>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

174

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

175

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).

176

Figure A4.1. Multiple sequence alignment of sanguinarine reductase candidates from 20 BIA-producing species.

177

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).

178

BBE

OAT

FADX

SALR

Figure A4.2. Phylogenetic trees for enzymes involved in BIA biosynthesis.

179

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA biosynthesis.

COR SANR

180


NOS CYP80

181


CYP82 CYP719

182


CXE NCS

183

Figure A4.2 (continued). Phylogenetic trees for enzymes involved in BIA

DIOX NMT

184

biosynthesis.

OMT


185

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

186

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.

characterization of sanguinarine reductases from papaver

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