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Morphine Biosynthesis in Opium Poppy Involves Two Cell
Types: Sieve Elements and Laticifers
W OPEN
Akpevwe Onoyovwe,a Jillian M. Hagel,a Xue Chen,a Morgan F. Khan,b David C. Schriemer,b and Peter J. Facchinia,1
a Department
b Department
of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T4N 1N2, Canada
ORCID ID: 0000-0002-7693-290X (P.J.F.).
Immunofluorescence labeling and shotgun proteomics were used to establish the cell type–specific localization of morphine
biosynthesis in opium poppy (Papaver somniferum). Polyclonal antibodies for each of six enzymes involved in converting (R)reticuline to morphine detected corresponding antigens in sieve elements of the phloem, as described previously for all
upstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stem
and latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to nine
morphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymes
were detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The final
three enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limited
occurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases in
the latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieve
elements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-rich
latex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellular
localization of morphine biosynthesis in opium poppy.
INTRODUCTION
Opium poppy (Papaver somniferum) produces several benzylisoquinoline alkaloids (BIAs) of pharmaceutical importance, including the narcotic analgesics morphine and codeine, the
anticancer drug noscapine, and the vasodilator papaverine. The
five chiral centers in the morphinan alkaloid backbone preclude
chemical synthesis as an alternative to crop cultivation for the
commercial production of pharmaceutical opiates. Alkaloids accumulate in the latex, which is the cytoplasm of highly specialized
cells known as laticifers that are associated with the phloem
throughout the plant. The traditional method for the isolation of
opiates from cultivated plants involves lancing the unripe seed
capsules and collecting the exuded latex, which oxidizes and
dries yielding raw opium. Whole seed capsules and stem segments from licit commercial opium poppy crops are also harvested as straw after the plants have desiccated in the field,
locking the alkaloid-rich latex in the dry biomass.
BIA biosynthesis begins with the condensation by norcoclaurine synthase (NCS) of two Tyr derivatives, dopamine and
4-hydroxyphenylacetaldehyde, yielding the primary intermediate
(S)-norcoclaurine (Samanani et al., 2004; Liscombe et al., 2005)
(Figure 1). (S)-Norcoclaurine is converted to (S)-reticuline through the
successive action of norcoclaurine 6-O-methyltransferase (6OMT),
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Peter J. Facchini
([email protected]).
W
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www.plantcell.org/cgi/doi/10.1105/tpc.113.115113
(S)-coclaurine N-methyltransferase (CNMT), N-methylcoclaurine
39-hyroxylase (NMCH), and 39-hydroxy N-methylcoclaurine 4’-Omethyltransferase (4’OMT) (Ounaroon et al., 2003; Facchini and
Park, 2003; Ziegler et al., 2005). (S)-Reticuline is a branch-point
intermediate in the biosynthesis of several BIA structural subgroups (Hagel and Facchini, 2013). Uniquely, morphine biosynthesis requires the epimerization of (S)-reticuline. Salutaridine,
the first tetracyclic promorphinan alkaloid, is formed via intramolecular carbon-carbon phenol coupling of (R)-reticuline catalyzed by the cytochrome P450 monooxygenase salutaridine
synthase (SalSyn; Gesell et al., 2009). The NADPH-dependent
salutaridine reductase (SalR) reduces the C7 keto group of salutaridine in a stereospecific manner, yielding salutaridinol (Ziegler
et al., 2006), which undergoes stoichiometric transfer of an acetyl
group to the C7 hydroxyl moiety by the acetyl-CoA–dependent
salutaridinol 7-O-acetyltransferase (SalAT) to form salutaridinol
7-O-acetate (Grothe et al., 2001). Spontaneous loss of the acetyl
group results in a rearrangement to thebaine, the first pentacyclic
morphinan alkaloid (Lenz and Zenk, 1995). Thebaine is O-demethylated
by thebaine 6-O-demethylase (T6ODM) to neopinone, which is
spontaneously converted to codeinone (Hagel and Facchini,
2010). The NADPH-dependent codeinone reductase (COR) reduces codeinone to codeine (Unterlinner et al., 1999), which is
O-demethylated by codeine-O-demethylase (CODM), yielding
morphine (Hagel and Facchini, 2010). As a minor alternative route,
thebaine undergoes 3-O-demethylation by CODM to oripavine
prior to 6-O-demethylation by T6ODM to morphinone, which is
ultimately reduced by COR to morphine (Brochmann-Hanssen,
1984). Presently, cDNAs encoding 10 enzymes involved in the
conversion of (S)-norcoclaurine to morphine have been isolated
(Figure 1). Only the epimerization of reticuline has not been
characterized at the molecular biochemical level (De-Eknamkul
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The Plant Cell
and Zenk, 1992). Additional cDNAs encoding several other BIA
biosynthetic enzymes have been characterized from opium
poppy (see Supplemental Figure 1 online), including reticuline 7-O-methyltransferase (7OMT) (Ounaroon et al., 2003),
norreticuline 7-O-methyltransferase (N7OMT) (Pienkny et al.,
2009), scoulerine O-methyltransferase (SOMT) (Dang and
Facchini, 2012; Winzer et al., 2012), berberine bridge enzyme
(Facchini et al., 1996), stylopine synthase (Díaz-Chávez et al., 2011),
tetrahydroprotoberberine N-methyltransferase (TNMT) (Liscombe
and Facchini, 2007), pavine N-methyltransferase (PavNMT) (Liscombe
et al., 2009), and sanguinarine reductase (Vogel et al., 2010).
We previously reported the localization of several BIA biosynthetic enzymes (NCS, 6OMT, CNMT, NMCH, 4’OMT, SalAT,
and COR) exclusively to sieve elements of the phloem by immunofluorescence labeling of resin-embedded tissue sections of
opium poppy using polyclonal antibodies (Bird et al., 2003;
Samanani et al., 2006; Lee and Facchini, 2010). Corresponding
gene transcripts for each enzyme were localized to adjacent
companion cells by in situ RNA hybridization. No enzymes or
corresponding mRNAs were detected in laticifers, leading to
a model suggesting that the transcription and translation of BIA
biosynthetic genes occur in companion cells followed by the
transport of functional enzymes to sieve elements, which serve as
the site of alkaloid biosynthesis. Subsequently, alkaloids are
transported to laticifers for storage in large vesicles of the latex.
However, a similar study based on the use of polyclonal antibodies to localize BIA biosynthetic enzymes (4’OMT, SalAT, COR,
and 7OMT) in resin-embedded tissue sections of opium poppy
suggested that laticifers and cells defined as phloem parenchyma
are the sites of alkaloid biosynthesis (Weid et al., 2004). An
alternative model purported that the early stages of BIA biosynthesis occur in phloem parenchyma cells and that downstream intermediates leading to morphine are transported to
laticifers for final biosynthetic transformations and product
accumulation. Considerable effort has been focused on the
identification of immunofluorescent cells as sieve elements
rather than phloem parenchyma (Samanani et al., 2006), although the localization of COR remained a discrepancy between the two studies, with one suggesting its occurrence in
laticifers (Weid et al., 2004) and the other its exclusive association with sieve elements (Bird et al., 2003). As such, the cell
type–specific localization of morphine biosynthesis in opium
poppy remains controversial.
To reconcile these apparently disparate results, we have performed immunofluorescence labeling of serial cross sections
using polyclonal antibodies raised against all six biosynthetic
enzymes of the morphine branch pathway in opium poppy. As an
independent approach to enzyme identification, we also used
shotgun proteomics to confirm the relative abundance of the final
three enzymes in laticifers. Although all six enzymes were
detected in sieve elements by immunofluorescence labeling,
Figure 1. Biosynthesis of Morphine in Opium Poppy from the Tyr
Derivatives Dopamine and 4-Hydroxyphenylacetaldehyde.
Corresponding cDNAs have been isolated for all enzymes shown in blue.
The dotted arrow refers to a conversion catalyzed by unknown enzymes.
Chemical conversions catalyzed by each enzyme are shown in red.
Compounds in bold are major accumulating alkaloids in opium poppy
latex.
Morphine Pathway Localization in Poppy
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shotgun proteomics and immunoblot analyses supported the
abundance in laticifers of the final three enzymes involved in the
conversion of thebaine to morphine. The occurrence of active
enzymes was confirmed by the conversion of exogenous thebaine to downstream intermediates and morphine in cell-free latex
protein extracts. By contrast, the formation of salutaridine from (S)norcoclaurine occurs exclusively in sieve elements, indicating a requirement for the intercellular translocation of pathway intermediates.
RESULTS
Immunoblot Analysis of Morphine Pathway Enzymes
The relative abundance of the final six enzymes in morphine biosynthesis was determined by immunoblot analysis of total proteins extracted from various organs of the opium poppy
chemotypes 40 and T using polyclonal antibodies raised against
purified recombinant enzymes. Each antibody detected a single
band with a molecular mass expected for the various enzymes:
SalSyn (56 kD), SalR (34 kD), SalAT (52 kD), T6ODM (41 kD), COR
(39 kD), and CODM (41 kD) (Figure 2). SalR and COR were detected at relatively similar abundance in all organs of both chemotypes, with roots and carpels consistently showing the highest
levels (Figure 2A). SalSyn was most abundant in stems and carpels and was detected at lower levels in leaves and roots. By
contrast, SalAT was most abundant in stems and roots and was
present at lower levels in leaves and carpels. T6ODM levels were
similar in roots, stems, and carpels of the codeine/morphineproducing chemotype 40 but were lower in leaves. However,
T6ODM was not detected in any organs of the thebaine/oripavineaccumulating chemotype T. CODM was found in all aerial organs
but was not detected in roots. In isolated latex, all enzymes except
SalSyn were detected (Figure 2B). Simultaneous probing of protein blots with major latex protein (MLP) antibodies was used to
normalize the signal intensity of bands corresponding to the different biosynthetic enzymes. To ensure that antibodies raised
against T6ODM and CODM, which share 72% amino acid sequence identity (Hagel and Facchini, 2010), did not cross-react on
immunoblots, different amounts of the purified recombinant enzymes were probed with polyclonal antisera under identical
conditions used to analyze plant protein extracts. No crossreactivity was observed when 100 ng of antigen was loaded on
the blot (see Supplemental Figure 2 online).
Detection of Morphine Pathway Enzymes by
Immunofluorescence Labeling
Owing to the amino acid sequence similarity between T6ODM and
CODM, each polyclonal antiserum was scrubbed with the nonspecific protein to maximize antigen specificity by reducing the
levels of IgGs recognizing common epitopes. Immunofluorescence labeling using resin-embedded serial cross sections of
various organs from the opium poppy chemotype 40 (DesgagnéPenix et al., 2012) showed the colocalization of all enzymes catalyzing the conversion of (R)-reticuline to morphine to a cell type
associated with phloem tissue throughout the plant (Figure 3).
Previous immunolocalization experiments performed with the
SalAT antiserum used herein showed that the labeled cells were
Figure 2. Immunoblot Analysis of Morphine Pathway Enzymes in Opium
Poppy.
(A) Immunoblots showing the relative abundance of the final six morphine biosynthetic enzymes in the opium poppy chemotypes T and 40.
Equal amounts (50 µg) of total protein extracts from different organs were
separated by SDS-PAGE. Protein blots were probed with polyclonal
antibodies specific for each enzyme.
(B) Immunoblots showing the occurrence of the final six morphine biosynthetic enzymes in the latex of opium poppy chemotype 40. The blots
were probed simultaneously with MLP antibodies as a gel loading and
autoradiogram exposure control. Data are representative of three independent
experiments.
sieve elements of the phloem (Samanani et al., 2006). In agreement with the absence of a signal in root protein extracts by immunoblot analysis (Figure 2), no signal above background was
detected in root sections using CODM antibodies (Figure 3ZZ).
Interestingly, sieve elements were labeled in root cross sections
using SalSyn antibodies and in leaf cross sections using SalAT
antibodies despite the apparent lack of a signal in the corresponding protein extracts (Figure 2). MLP antibodies showed the
distinct labeling of laticifers rather than sieve elements in corresponding serial cross sections of each organ (Figures 3AA to
3DD). Fluorescent cells in sections immunolabeled with MLP
antibodies correlated with laticifers discernable in toluidine blue
O–stained serial sections based on their position, size, and relatively thick primary cell walls (Figures 3A to 3D). The fluorescent
signal in some laticifers was weak owing to the loss of the highturgor latex during tissue fixation.
Shotgun Proteomics Identifies Morphine
Biosynthetic Enzymes
Shotgun proteomics methods were used to perform deep
analyses aimed at determining the occurrence and relative
Figure 3. Immunolocalization of Morphine Biosynthetic Enzymes in Different Opium Poppy Organs.
Polyclonal antibodies were raised against: SalSyn ([E] to [H]), SalR ([I] to [L]), SalAT ([M] to [P]), T6ODM ([O] to [T]), COR ([U] to [X]), and CODM ([Y] to
[ZZ]). Serial cross sections of resin-embedded tissues from opium poppy chemotype 40 were 0.5 µm in thickness. One serial section for each organ
was stained with toluidine blue O to show the anatomical organization of the phloem, with several laticifers indicated by red asterisks ([A] to [D]).
Immunofluorescence labeling of laticifers was performed using MLP polyclonal antibodies ([AA] to [DD]). Bars = 25 µm.
Morphine Pathway Localization in Poppy
Figure 4. SDS-PAGE of Whole-Stem and Latex Protein Extracts Used
for Shotgun Proteomics Analysis.
Lanes containing whole-stem and latex proteins were sectioned into 48
and 41 segments, respectively, which were individually subjected to ingel digestion with trypsin. Numbers on the left show the molecular
masses of protein markers in kilodaltons.
abundance of BIA biosynthetic enzymes in whole stems and
laticifers of opium poppy. SDS-PAGE of whole-stem and latex
protein extracts from the opium poppy chemotype Roxanne
(Desgagné-Penix et al., 2012) contrasted the complexity of each
subproteome and revealed the abundance of low molecular
mass MLPs in laticifers (Figure 4). Using a gel-based liquid
chromatography-tandem mass spectrometry approach (Schirle
et al., 2003; Vasilj et al., 2012), 1518 and 511 distinct protein
families were identified from the whole-stem and latex samples,
respectively. Identification was based on searches of all available
plant entries in the National Center for Biotechnology Information
nonredundant (NCBInr) database. When searched against
a transcriptome database built using 415,818 independent nucleotide sequences from opium poppy and partially annotated
by BLAST analysis of the National Center for Biotechnology Information Viridiplantae database (Desgagné-Penix et al., 2012),
2031 and 830 distinct protein families were identified from whole
stem and latex, respectively. The transcriptome database search
did not increase coverage of the biosynthetic pathway enzymes,
and since a large fraction of the transcriptome hits were established only from partial sequences, the NCBInr hit sets were
chosen for analysis of pathway enzymes. The exponentially
modified protein abundance index (emPAI) strategy (Ishihama
et al., 2005; Shinoda et al., 2009) was used for this purpose, and its
validity was corroborated using the top three quantification
method on a subset of enzymes. This comparatively quantitative
method is based on chromatographic peak intensities of the three
most abundant peptides for a given protein (Silva et al., 2006;
Grossmann et al., 2010). For example, COR was detected in the
latex at 7 times the level of the whole stem using the top-three
method and at 5 times the level using the emPAI approach.
Quantification data for the biosynthetic pathway enzymes
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discovered in the latex and whole-stem samples are displayed in
Figure 5.
Among the most abundant identified polypeptides in each
subproteome were several primary metabolic enzymes, defense
proteins, and cell structural components (see Supplemental
Table 1 online). As expected, ribulose-1,5-bisphosphate carboxylase was the most abundant protein in the photosynthetic whole
stem, whereas MLP was the most abundant protein in the latex.
Five known BIA biosynthetic enzymes (NCS, COR, SalR, 4’OMT2,
and CODM) were among the top 30 most abundant proteins in the
whole stem. In the latex, COR and CODM were joined by T6ODM
and 7OMT as four of the eight most abundant proteins. Annotated
sequences in each subproteome were assigned to one of 10
functional categories based on putative eukaryotic cellular
processes. The representation of proteins in each category was
determined as either the number of proteins annotated or the
sum of emPAI scores for all assigned proteins as an approximation of protein abundance (see Supplemental Figure 3 online).
More than half of the total number of proteins could be functionally annotated. When considered in terms of relative abundance based on emPAI scores, almost three-quarters of each
protein extract could be assigned a putative function. In both the
whole-stem and latex subproteomes, the percentage of proteins
of unknown function was lower when considered in terms of
Figure 5. Relative Abundance of Alkaloid Biosynthetic Enzymes in
Opium Poppy Based on emPAI Scores.
The emPAI scores were derived from shotgun proteomics performed on
whole-stem (A) and latex (B) total protein extracts.
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relative abundance as opposed to the absolute number of polypeptides, which is consistent with the generally low emPAI
scores for such proteins. By contrast, most other functional
categories included proteins of higher relative abundance than
the overall number of annotated polypeptides. Of note are enzymes involved in primary metabolism in both the whole stem
and latex as well as secondary metabolic enzymes, defense
proteins, and MLPs in the latex. Interestingly, MLPs represented
55 mol % of the latex subproteome, which correlates with the
relative abundance suggested by SDS-PAGE (Figure 4). By
contrast, MLPs represented 2.3 mol % of the whole stem subproteome corresponding to 4.9% contamination with the latex
subproteome. The absence of chloroplast-specific proteins
suggests that the latex subproteome was not contaminated with
the contents of photosynthetic cells in the stem. Although proteins associated with, but not exclusive to, sieve elements were
identified, including monodehydroascorbate reductase, glutathione
reductase, and allene oxide cyclase (Walz et al., 2004; Vilaine
et al., 2003), phloem-specific P-proteins (Xonconostle-Cazares
et al., 1999) were not detected, further validating the purity of
the latex sample.
Most known morphine biosynthetic enzymes were detected in
the whole stem subproteome with the exception of tyrosine/
DOPA decarboxylase, NMCH, and SalAT (Figure 5). The combined mol % of all known BIA biosynthetic enzymes was 9.4 and
4.7 in the latex and stem subproteomes, respectively. The relative
abundance of enzymes in the whole-stem proteome was variable
with NCS, 4’OMT, SalR, and COR detected at the highest levels
and SalSyn detected at a relatively low level. In the latex subproteome, no enzymes operating on pathway intermediates upstream of salutaridine were detected (Figure 5). However, four of
the final five enzymes in the morphine branch pathway were
identified, although the SalR levels were substantially lower than
those of T6ODM, COR, and CODM. COR displayed the highest
emPAI score, but T6ODM and CODM were also abundant. Several BIA biosynthetic enzymes operating in other branch
pathways were also detected in the whole stem subproteome,
although only 7OMT and PavNMT were detected in the latex
subproteome (Figure 5).
Transcripts and Enzyme Activities in Latex
Quantitative RT-PCR was performed using gene-specific primers
and total RNA isolated from either whole stem or latex. Primer
specificity was confirmed using first-strand cDNA generated from
whole-stem total RNA, which yielded single PCR amplicons with
similar signal intensities and predicted fragment sizes (Figure 6;
see Supplemental Table 2 online). By contrast, first-strand cDNA
generated from latex total RNA showed that T6ODM, COR, and
CODM transcripts were substantially more abundant compared
with mRNAs encoding SalSyn, SalR, and SalAT in the 40 chemotype. SalSyn and SalAT transcript levels were low to undetected.
A similar result was obtained for the T chemotype, except that
T6ODM transcripts were not detected, whereas SalAT and SalR
transcripts were found at similar levels (Figure 6).
In the presence of NADPH, Fe2+, and 2-oxoglutarate, native
cell-free latex protein extracts converted exogenous thebaine to
downstream intermediates and morphine, whereas no increase in
Figure 6. Relative Abundance of Gene Transcripts Encoding the Final
Six Enzymes of Morphine Biosynthesis in Opium Poppy.
Quantitative RT-PCR was performed using total RNA isolated from the
whole stem and latex of opium poppy chemotypes T (A) and 40 (B). The
experiment was performed in triplicate and produced similar results each
time.
endogenous alkaloid levels was detected using denatured latex
protein extracts (see Supplemental Figure 4 online). Compared
with denatured samples, native cell-free latex protein extracts
showed reduced thebaine and increased morphinone, codeinone, codeine, and morphine compared with denatured latex
extracts. Collision-induced dissociation spectra of all enzymatic
reaction products were compared with those of authentic standards to confirm compound identities (see Supplemental Table 3 and
Supplemental References 1 online).
O-Demethylase Isoforms in Latex
The gel-based liquid chromatography-tandem mass spectrometry data were processed at the individual band level to determine
the molecular mass distribution of all biosynthetic enzymes
detected in each subproteome (see Supplemental Figure 5 online). Several enzymes (6OMT, 4’OMT, SalR, T6ODM, COR,
CODM, and 7OMT) migrated over wide molecular mass ranges,
suggesting a combination of different isoforms, posttranslational
processing, and/or proteome-specific degradation. It should also
be noted that the dynamic range of the data is approximately three
orders of magnitude and was normalized to the most abundant
entry (COR). As such, the apparent background in some cases
resulted from (1) limitations associated with the visual presentation of the substantial dynamic range and (2) the potential for
the reduced accuracy of emPAI values at higher protein intensity
levels.
The occurrence of multiple O-demethylase isoforms was investigated further. Despite the occurrence of only single transcripts encoding T6ODM and CODM (see Supplemental Figures
6A and 6C online), a previous opium poppy proteomics study
based on two-dimensional (2D) SDS-PAGE and Edman degradation sequencing revealed a large collection of proteins with
similar molecular masses but variable isoelectric points, annotated as senescence-related gene (SRG) products (Decker et al.,
2000). The peptide sequences obtained from these SRG proteins matched the amino acid sequences of T6ODM and CODM.
To support the occurrence of multiple charge isoforms of these
Morphine Pathway Localization in Poppy
O-demethylases in opium poppy, protein blots of latex proteins
separated by 2D SDS-PAGE were probed with scrubbed
T6ODM and CODM polyclonal antibodies. Two sets of ;43-kD
proteins were detected between pH 4.5 and 5.3 (Figure 7). CODM
isoforms showed marginally lower molecular mass than T6ODM
isoforms.
DISCUSSION
Opium poppy is the only plant known to produce the narcotic
analgesics codeine and morphine, which accumulate at copious
levels in specialized laticifers that accompany sieve elements of
the phloem in all organs. The ability to synthesize a specialized
metabolite, such as morphine, depends on the evolution of
several biosynthetic enzymes via the recruitment of genes
arising through duplication events in the genome (Pichersky and
Lewinsohn, 2011). Morphine biosynthesis in opium poppy was
made possible by the emergence of enzymes capable of catalyzing key reactions leading to (1) the formation of the tetracyclic
promorphinan ring system of salutaridine; (2) the reduction of the
carbonyl group in salutaridine and subsequent O-acetylation of
the resulting hydroxyl moiety causing molecular rearrangement
and, ultimately, formation of the pentacyclic morphinan backbone; and (3) the double regiospecific O-demethylation and
associated reduction of the carbonyl moiety to convert the nonnarcotic intermediate thebaine to morphine (Figure 1). Based on
the occurrence of specific metabolites and some molecular
biochemical characterization (Ziegler et al., 2006; Gesell et al.,
2009), the first three enzymes (SalSyn, SalR, and SalAT) appear
restricted to a small number of related members in the genus
Papaver. By contrast, at least one of the final three enzymes
(T6ODM, COR, and CODM) are likely unique to opium poppy.
However, abundant product accumulation is also expected to
require the appropriate cellular and subcellular localization of
enzymes to divert (R)-reticuline, the ubiquitous stereoisomer of
Figure 7. Immunoblot Analysis of Opium Poppy Latex Proteins Separated by 2D SDS-PAGE Showing Numerous Charge Isoforms of T6ODM
and CODM.
Latex proteins (50 µg) were subjected to isoelectric focusing followed by
SDS-PAGE and transferred to nitrocellulose membranes. Duplicate
protein blots were probed with polyclonal antibodies raised against
T6ODM or CODM.
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the central pathway intermediate (S)-reticuline, to morphine.
Competition for (R,S)-reticuline could be regulated by the transport
of branch pathway intermediates to separate compartments, including different cell types. Laticifers in Papaveraceae undoubtedly
evolved many features to function as an effective site of alkaloid
accumulation. However, data presented here show that laticifers
also play a major role in the final transformations leading to morphine and potentially other alkaloids. Such information provides
a crucial basis for metabolic engineering or molecular breeding
efforts in opium poppy.
Efforts to determine the cellular localization of BIA biosynthesis
in opium poppy have focused on the immunofluorescence labeling of tissue sections using antibodies raised against recombinant enzymes (Bird et al., 2003; Weid et al., 2004; Samanani
et al., 2006; Lee and Facchini, 2010). Although the approach is
widely used, and despite many similarities in the reported results,
various incongruities and contradictory interpretations have resulted in controversial localization models based on two major
issues. The first involves identification of the phloem cell type
linked to all previously studied biosynthetic enzymes as either
sieve elements or parenchyma. The detection of unique cellular
features, including sieve plates, a characteristic cytoplasmic
architecture, and the localization of a sieve element–specific
H+-ATPase isoform, is the strongest evidence in support of the
labeled cells being sieve elements (Bird et al., 2003; Samanani
et al., 2006). In a separate study, the assignment of the cell type as
parenchyma was based on the reported occurrence of sieve
plates in tissues that were not labeled (Weid et al., 2004). The
second point of contention was the role of laticifers in BIA biosynthesis. Although most investigated enzymes were not associated with laticifers by immunofluorescence labeling (Bird et al.,
2003; Weid et al., 2004; Samanani et al., 2006; Lee and Facchini,
2010), COR was colocalized to both laticifers and, less abundantly, to parenchyma or sieve elements (Weid et al., 2004). The
occurrence in laticifers of the penultimate step in the morphine
pathway would suggest the colocalization of other biosynthetic
enzymes. We used a combination of (1) immunofluorescence
labeling and (2) shotgun proteomics to determine the cellular
localization and relative abundance of the final six enzymes involved in morphine biosynthesis.
Immunofluorescence labeling results were essentially identical to those reported previously (Bird et al., 2003), anchored by
the use of SalAT as a positive control (Samanani et al., 2006) to
confirm the localization of all six biosynthetic enzymes to sieve
elements in roots, stems, leaves, and carpels (Figure 3). However, stem cross sections exposed to CODM antibodies consistently showed the simultaneous, yet relatively weak, labeling
of laticifers based on the colocalization of MLP (Figures 3YY and
3BB; see Supplemental Figure 7 online). Despite the previous
report of a similar result for COR (Weid et al., 2004), the labeling
of laticifers with our COR polyclonal antibodies (Bird et al.,
2003), which were prepared independently for this study, was
not reliably observed (Figure 3V). T6ODM antibodies also failed
to label laticifers in support of the antiserum scrubbing efforts
used to enhance immunospecificity with respect to CODM
(see Supplemental Figure 2 online). Immunoblot (Figure 2) and
immunofluorescence labeling (Figure 3) results were generally
consistent.
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Advanced shotgun proteomics methods have the potential to
penetrate deeply into the proteome of plant organs and, in some
cases, specific cell types. Previously, we used shotgun proteomics to analyze opium poppy cell cultures producing the antimicrobial alkaloid sanguinarine (Zulak et al., 2009; Desgagné-Penix
et al., 2010) but have only now extended the approach to analyze
intact plant tissues. The positive turgor of laticifers facilitates the
specific isolation of latex with minimal contamination from surrounding cells (Hagel et al., 2008). Latex subproteomes of various
depths have been reported for rubber tree (Hevea brasiliensis)
(D’Amato et al., 2010), the rubber-producing plant Taraxacum
brevicorniculatum (Wahler et al., 2012), lettuce (Lactuca sativa)
(Cho et al., 2010), papaya (Carica papaya) (Dhouib et al., 2011),
greater celandine (Chelidonium majus) (Nawrot et al., 2007), and
opium poppy (Decker et al., 2000). Laticifers in various plant
families appear to have evolved independently (Hagel et al., 2008),
allowing interesting functional, if not phylogenetic, comparisons
based on proteomics. An opium poppy latex subproteome generated using Edman degradation sequencing of cytosolic and
vesicle-associated proteins separated by 2D SDS-PAGE included 98 annotated polypeptides (Decker et al., 2000). Our
shotgun proteomics approach resulted in the annotation of up to
eightfold more latex proteins and many additional whole stem
proteins, including most known BIA biosynthetic enzymes.
COR has previously been associated with the latex proteome
along with T6ODM and CODM, but the O-demethylases were
hitherto unknown and the corresponding proteins were annotated
as SRGs (Decker et al., 2000). The occurrence and relative
abundance of T6ODM, COR, and CODM in laticifers, compared
with SalSyn, SalR, and SalAT, is supported by the detection of
corresponding gene transcripts in latex (Figure 6). Unlike the adjacent sieve elements, the articulated, anastomosing laticifers in
opium poppy contain nuclei and ribosomes and do not appear to
rely on other cells for gene expression and protein synthesis
(Hagel et al., 2008). The relative abundance in latex of transcripts
corresponding to SalR, T6ODM, COR, and CODM is consistent
with the comparative levels of these enzymes determined using
shotgun proteomics (Figure 5). The low to undetected levels of
SalSyn and SalAT transcripts in the latex is also consistent with
the lack of detection of the corresponding enzymes in the latex
subproteome. However, the relative abundance of all tested
transcripts was similar in whole stems, despite the detection of all
proteins except SalAT in the corresponding subproteome. Minor
differences are apparent in addition to the expected absence of
T6ODM protein (Figure 2) and transcript (Figure 6) in the T chemotype (Hagel and Facchini, 2010). Interestingly, SalR and SalAT
appeared relatively abundant in latex by immunoblot analysis
(Figure 2B), suggesting that similar short-chain dehydrogenase/
reductase and acyltransferase proteins distinguishable using
shotgun proteomics, but cross-reactive with polyclonal antisera,
occur in laticifers.
The multiple proteins of similar molecular mass, but with different isoelectric points annotated as SRGs (Decker et al., 2000),
were confirmed as T6ODM and CODM isoforms by 2D immunoblot analysis (Figure 7). Contigs represented in our 454 pyrosequencing transcriptome databases predicted single T6ODM
and CODM isoforms (see Supplemental Figure 6 online), suggesting that the numerous charge isoforms were the result of
posttranslational modification. The enzymatic conversion of thebaine to downstream intermediates and morphine in latex protein
extracts confirms that the T6ODM, COR, and CODM polypeptides detected by shotgun proteomics are active catalysts
(see Supplemental Figure 4 online). Morphine biosynthesis from
14C-Tyr in isolated opium poppy latex was reported in several
landmark investigations (Stermitz and Rapoport, 1961; Fairbairn
and Wassel, 1964; Kirby, 1967). However, in these studies, latex
was collected from the base of decapitated capsules, which likely
resulted in substantial contamination with sieve element sap and
the inclusion of enzymes upstream of T6ODM. By contrast, the
carpel lancing method used herein resulted in the collection of
latex free of phloem proteins.
A cDNA encoding 7OMT from opium poppy was originally
isolated based on peptide amino acid sequence data obtained
via latex proteomics analysis (Ounaroon et al., 2003). 7OMT was
Figure 8. Model Summarizing the Localization and Relative Abundance
of Morphine Biosynthetic Enzymes in Sieve Elements and Laticifers of
Opium Poppy.
The cellular localization of biosynthetic enzymes was based on immunolocalization and shotgun proteomics data. The font size used for each
enzyme shown in blue was adjusted to reflect the estimated relative
abundance in sieve elements and laticifers. The thickness of vertical
arrows suggests the proposed relative flux through various conversions
in each cell type. The dashed vertical arrow represents an unknown
enzyme. Horizontal arrows indicate alkaloids that are putatively transported between sieve elements and laticifers with thebaine suggested as
the major translocated intermediate.
Morphine Pathway Localization in Poppy
also identified using our shotgun proteomics method (Figure 5).
However, immunofluorescence labeling using 7OMT polyclonal
antibodies previously failed to detect the enzyme in laticifers
(Weid et al., 2004), similar to the incongruity in the immunolocalization results for COR resulting from two independent studies
(Bird et al., 2003; Weid et al., 2004). Our proteomics analysis
showed that both COR and 7OMT are abundant in laticifers
(Figure 5), indicating that immunofluorescence labeling is not
a reliable method for protein localization in opium poppy laticifers.
Immunolocalization has proven useful for the detection of BIA
biosynthetic enzymes in sieve elements. The ineffectiveness of
the technique with respect to laticifers is likely related to the
unique nature of the vesicle- and MLP-rich (Figure 4) latex, which
could mask proteins from immunological detection in fixed and
resin-embedded tissues, at least using paraformaldehyde-based
methods (Figure 3) (Bird et al., 2003; Weid et al., 2004; Samanani
et al., 2006). The dual use of immunofluorescence labeling and
shotgun proteomics confirmed or showed that (1) the central
pathway from (S)-norcoclaurine to (S)-reticuline operates exclusively in sieve elements, (2) the early morphinan branch pathway
enzymes converting salutaridine to thebaine are primarily in sieve
elements, but can also occur in laticifers, and (3) the final three
enzymes involved in the conversion of thebaine to morphine are
abundant in laticifers but also likely occur in sieve elements.
A model summarizing the cellular localization of morphine
biosynthesis in opium poppy is presented in Figure 8. In this
model, thebaine represents the major alkaloid transported from
sieve elements to laticifers, although the detection of many BIA
pathway intermediates in latex (Desgagné-Penix et al., 2012)
suggests that the translocation process is not specific. Support
for the formation of salutaridine in sieve elements is based on the
following: (1) SalSyn was not detected in the latex subproteome
and displayed a low emPAI score in the whole stem subproteome
(Figure 5), (2) the enzyme was localized by immunofluorescence
labeling to sieve elements (Figure 3) and immunoblot analysis did
not detect the enzyme in latex protein extracts (Figure 2), and (3)
SalSyn transcripts were detected at only trace levels in latex
(Figure 6). Similarly, although immunoblot analysis suggested the
occurrence of SalR and SalAT in latex (Figure 2), SalAT was not
detected in the latex subproteome, and the emPAI score for SalR
was low compared with the scores for T6ODM, COR, and CODM
(Figure 5). Moreover, the low to undetectable levels of SalR and
SalAT transcripts in latex (Figure 6), the demonstrated protein
interaction between SalR and SalAT (Kempe et al., 2009), and the
relative abundance of SalR in the whole stem subproteome (see
Supplemental Table 1 online) suggest that thebaine is formed
primarily in sieve elements. Intermediates upstream of salutaridine or involved in different branch pathways are likely metabolized to a variety of substituted alkaloids via other enzymes
localized to laticifers, including 7OMT and PavNMT (Figure 5).
Interestingly, these late substitutions appear to primarily involve
the addition of O- and N-linked methyl groups. The colocalization
of T6ODM and CODM, which catalyze unique O-demethylation
reactions, further suggests that a major role for laticifers involves
altering the methylation status of various alkaloids. However,
O-methylation of 1-benzylisoquinolines at C6 and C4’ via 6OMT
and 4’OMT, respectively [leading to (S)-reticuline], 9-O-methylation
of (S)-scoulerine by SOMT (leading to noscapine) (Dang and
9 of 13
Facchini, 2012), and 7-O-methylation of (S)-norreticuline by
N7OMT (leading to papaverine) (Pienkny et al., 2009; DesgagnéPenix and Facchini, 2012) were not associated with laticifers
(Figure 5), indicating that other major alkaloids in opium poppy are
produced largely or exclusively in sieve elements.
The immunolocalization of all tested BIA biosynthetic enzymes
showed fluorescent labeling only in peripheral cytoplasmic regions of sieve elements (Figure 3) (Bird et al., 2003; Samanani
et al., 2006) and/or laticifers (see Supplemental Figure 7 online)
(Weid et al., 2004). SalAT and several cytosolic enzymes involved
in the formation of (S)-reticuline were localized proximal to the
sieve element reticulum (Samanani et al., 2006), similar to the
association of sanguinarine biosynthesis with the endoplasmic
reticulum in cultured opium poppy cells (Alcantara et al., 2005).
Alkaloid translocation from sieve elements to laticifers could occur via symplastic transport through plasmodesmata (Facchini
and De Luca, 2008) or apoplastic movement mediated by one or
more transporters. A plasma membrane ATP binding cassette
transporter (Shitan et al., 2003) and a vacuolar H+-antiporter
(Otani et al., 2005) have been implicated in BIA metabolism in
Japanese goldthread (Coptis japonica). The involvement of
transporters in opium poppy would not only require export from
sieve elements and import into laticifers, but also endomembrane
translocation owing to the accumulation of BIAs in latex vesicles.
The peripheral localization of enzymes converting salutaridine to
morphine in laticifers could indicate an association with plasma
membrane transporters to minimize the migration of pathway intermediates directly to latex vesicles, which would preclude further metabolism. An analysis of cytosolic and vesicular latex
proteins by 2D SDS-PAGE showed the occurrence of T6ODM and
CODM along with COR in the cytosol (Decker et al., 2000). The
mechanism of transport between sieve elements and latex remains an unresolved aspect of morphine biosynthesis.
METHODS
Plant Material
Opium poppy (Papaver somniferum) chemotypes 40, T, and Roxanne
(Desgagné-Penix et al., 2012) were cultivated in a growth chamber under
a photoperiod of 16 h light/8 h dark at 20/18°C. Plant tissues were
harvested 2 to 3 d after anthesis and flash frozen in liquid N2. Latex was
isolated by lancing unripe seed capsules with a razor blade ;7 d after
anthesis, and corresponding whole stem segments were collected by
immersing undetached organs directly in liquid N2 to preserve the latex.
Polyclonal Antibodies
Recombinant T6ODM and CODM were prepared as described previously
(Hagel and Facchini, 2010). SalR (Ziegler et al., 2006), COR (Unterlinner
et al., 1999), and SalSyn (Gesell et al., 2009) cDNAs were inserted into
pQE30 (see Supplemental Table 2 online) and expressed using Escherichia coli strain SG13005. To improve solubility, the SalSyn cDNA was
truncated to remove 56 hydrophobic residues from the N terminus of the
corresponding polypeptide. Luria-Bertani broth (170 mM NaCl, 10 g L21
tryptone, and 5 g L21 yeast extract) containing 50 mg L21 kanamycin and
100 mg L21 ampicillin was inoculated with overnight bacterial cultures and
incubated at 4°C (COR) or 25°C (SalSyn and SalR) to optimize protein
solubility. At a density of OD600 = 0.4, the cultures were induced for 4 h
with 300 µM isopropyl-b-D-thiogalactopyranoside. Bacterial cells were
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The Plant Cell
lysed using a French press, and soluble SalR and COR proteins were
purified as described for T6ODM and CODM (Hagel and Facchini, 2010).
Bacterial extracts containing truncated SalSyn as inclusion bodies were
resuspended in denaturing buffer (8.0 M urea, 300 mM NaCl, and 300 mM
sodium phosphate) and sonicated. Cell debris was removed by centrifugation, and the pellet was reextracted in denaturing buffer. Recombinant proteins were purified using a Talon Co2+-affinity column (Clontech).
His-tagged SalSyn was eluted with denaturing buffer containing 150 mM
imidazole and desalted using a PD10 column (GE Healthcare Life Sciences). MLP and SalAT antibodies were described previously (Griffing and
Nessler, 1988; Bird et al., 2003; Samanani et al., 2006). Other antibodies
were raised against purified recombinant proteins. Antigens were subcutaneously injected into mice at a concentration of 100 µg mL21 after
dialysis with 146 mM NaCl. Preimmune sera were collected from mice
prior to the initial injection of antigen. Four booster injections were performed every 3 weeks. Final antisera were centrifuged at 1000g to isolate
plasma.
For immunofluorescence labeling, the T6ODM and CODM antibodies
used were each scrubbed against the other antigen to reduce cross
reactivity. One hundred micrograms of purified CODM and T6ODM
proteins were separated by SDS-PAGE, transferred to separate blots, and
blocked in TBST (10 mM Tris-HCl, pH 7.2, 500 mM NaCl, and 0.3% [v/v]
Tween 20) containing 1% (w/v) BSA. The CODM blot was incubated in
blocking solution containing 0.5% (v/v) anti-T6ODM, whereas the T6ODM
blot was incubated in blocking solution containing 0.5% (v/v) anti-CODM,
both overnight at 4°C. The scrubbed blocking solutions containing antiT6ODM or anti-CODM were used for immunolocalization. Blots were
washed three times in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM sodium
phosphate, 1.76 mM potassium phosphate, and 0.1% [v/v] Tween 20) for
15 min, incubated with secondary antibody, washed in PBST, incubated
with chemiluminescence substrate, and developed on Kodak XAR film to
verify the absence of nonspecific binding.
Immunoblot Analysis
Tissues from opium poppy chemotype 40 were extracted with 50 mM TrisHCl, pH 7.5, 100 mM NaCl, 0.05% (v/v) Tween 20, 1 mM EDTA, and 250 µM
phenylmethylsulfonyl fluoride. Protein concentration was determined using
the Bradford assay. Fifty micrograms of soluble protein from various plant
organs was added to sample buffer (Laemmli, 1970) containing 5% (w/v)
SDS and incubated in boiling water for 5 min. Proteins were separated by
SDS-PAGE on a 12% (w/v) gel and transferred to a nitrocellulose membrane. Gel transfer was performed at 100 V for 1 h in transfer buffer (25 mM
Tris-HCl, pH 8.3, and 192 mM Gly). After transfer, blots were blocked in
PBST containing 5% (w/v) skim milk powder for 1 h, with gyratory shaking.
Primary antibodies (0.01% [v/v] antisera) were added, and blots were incubated overnight at 4°C and subsequently washed three times in PBST for
15 min with shaking. Blots were then incubated for 1 h with horseradish
peroxidase–conjugated goat anti-mousesecondary antibody (0.001% [v/v]
in blocking buffer) with shaking. Blots were triplicate washed in PBST,
incubated with SuperSignal West Pico chemiluminescence substrate
(Thermo Scientific) for 5 min, and exposed on Kodak XAR film.
Immunofluorescence Labeling
Organs from opium poppy chemotype 40 were fixed in 2% (v/v) paraformaldehyde in 100 mM phosphate buffer, pH 7.4, overnight at 4°C. After
fixation, the tissues were rinsed twice in 100 mM phosphate buffer for 10 min
each time and dehydrated as follows: 30% (v/v) ethanol in water for 1 h, 50%
for 1 h, 60% for 90 min, 70% for 2 h, 80% overnight at 4°C, 90% for 2 h, and
100% for 4 h. Tissues were infiltrated with LR white resin (London Resin
Company) as follows: 33% (v/v) resin in ethanol for 2 h, 50% for 2 h, fresh
50% resin overnight, and 100% resin overnight. Tissues were cast in 1-mL
gelatin capsules and polymerizedat 60°C for 20 h. Serial cross sections (0.3-
to 0.5-µm thickness) were prepared using a Reichert-Jung Ultracut E
microtome (Leica Microsystems) and mounted on gelatin-coated slides.
Tissue sections were blocked for 1 h with Tris-buffered saline and Tween
20 (TBST) containing 1% (w/v) BSA in a humid chamber. The blocking
solution was replaced with primary antisera (10% [v/v]) in fresh TBST
containing 1% (w/v) BSA and incubated for 3 h at room temperature.
Blocking solutions containing scrubbed anti-T6ODM and anti-CODM
antibodies were used directly. Slides were washed with TBST containing
1% (w/v) BSA, four times for 15 min, and sections were incubated for 1 h with
Alexa 488–conjugated goat anti-mouse IgGs or, in the case of MLP primary
antiserum, Alexa 594–conjugated goat anti-rabbit IgGs (Molecular Probes)
diluted with blocking solution. Slides were washed three times with TBST
containing 1% (w/v) BSA for 15 min, three times with distilled water for
5 min, and finally sealed with Fluoro-Gel mounting medium (Electron Microscopy Sciences). Some serial sections were stained in 10 mM sodium
benzoate, pH 4.4, containing 0.1% (w/v) toluidine blue O. Alexa 488 and
594 fluorescent probes were visualized using Leica L5 and TX2 filters, respectively, on a DM RXA2 microscope (Leica Microsystems). Images were
captured with a Retiga EX digital camera (QImaging), and false-color imaging
was performed using Improvision Open Lab version 2.09 (Perkin-Elmer).
Shotgun Proteomics
Latex from opium poppy chemotype Roxanne was extracted in 50 mM
potassium phosphate, pH 7.0, and 500 mM mannitol. Corresponding
whole-stem proteins were isolated from 1 g of ground tissue in 50 mM
Tris-HCl, 100 mM NaCl, 0.05% (v/v) Tween 20, 1 mM EDTA, and 250 µM
phenylmethylsulfonyl fluoride. Extracted proteins were mixed with sample
buffer (Laemmli, 1970) containing 5% (w/v) SDS and incubated in boiling
water for 5 min, and 50 mg of the mixture was separated by SDS-PAGE on
a 12% (w/v) gel. The gels were subsequently stained with Coomassie
Brilliant Blue R 250. Gels were fully sectioned into multiple segments: 48
for the whole-stem sample and 41 for the latex sample. Each gel segment
was rinsed once with 200 mL of HPLC-grade water and twice with 200 mL
of 50 mM ammonium bicarbonate in 50% (v/v) acetonitrile. Each segment
was then treated with 50 mL of 10 mM DTT in 100 mM ammonium bicarbonate at 56°C for 1 h. The supernatant was removed and replaced
with 50 mL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate
and incubated at room temperature and in the dark for 30 min to alkylate
free disulfides. Supernatants were removed, and the gel segments were
washed twice with 200 mL of 100 mM ammonium bicarbonate for 15 min.
Gel segments were then reduced to dryness, followed by rehydration in
12.5 ng µL21 trypsin in 25 mM ammonium bicarbonate, pH 8.0. Subsequently, 25 mM ammonium bicarbonate (;10 to 20 µL) was added to
cover the gel segments, and the segments were then incubated overnight
at 37°C. Extraction of peptides was performed twice using 50 mL of 1%
(v/v) formic acid in 1:1 acetonitrile:water. Supernatants were pooled, reduced to dryness, and reconstituted in mobile phase A for HPLC injection.
Mass Spectrometry, Identification, and Quantification of Proteins
Tryptic protein digests were analyzed using an Orbitrap Velos (Thermo
Scientific). Injected samples were desalted on an Acclaim PepMap trapping
column (3-µm silica particle size, 2-cm length 3 75-µm inner diameter;
Dionex) for 4 min with 3% (v/v) acetonitrile/0.2% (v/v) formic acid delivered
at 4 mL min21. Peptides were reverse eluted from the trapping column and
separated on an Acclaim Pepmap analytical column (2-µm silica particle
size, 15-cm length 3 75-µm inside diameter) at a rate of 0.3 mL min21. Datadependent acquisition of collision-induced dissociation tandem mass
spectrometry (MS/MS) spectra was used, with parent ion scans over
a mass-to-charge ratio range of 300 to 1750. Raw files were converted to.
mgf files using the MM conversion tool (www.massmatrix.net/mm-cgi/
home.py). Converted files for all proteins from one gel lane were combined
into one .mgf file, which was searched with Mascot version 2.3 (Matrix
Morphine Pathway Localization in Poppy
11 of 13
Science) using the following parameters: 10 ppm mass spectrometry error,
0.8 DMS/MS error, one potential missed cleavage, fixed modification of Cys
carbamidomethylation (C), and variable modification of Met oxidation.
Protein identification was performed by searching all available plant entries
in the NCBInr database and, separately, using a transcriptome database
constructed from 415,818 independent nucleotide sequences from opium
poppy partially annotated by BLAST analysis of the National Center for
Biotechnology Information Viridiplantae database (Desgagné-Penix et al.,
2012). For the NCBInr database search of the concatenated .mgf files,
individual peptide scores were readjusted with the Percolator algorithm for
improved sensitivity, and peptide spectrum matches were reported as
significant based on posterior error probabilities of <0.05. Globally, this
approach generated false discovery rates of 0.87% (latex) and 0.73%
(whole stem). Protein families were established conservatively, using
dendrogram cutoffs of 200 (whole stem) and 250 (latex) in Mascot and
representing a given family by the highest scoring protein. All MS/MS data
sets were submitted to ProteomeXchange. The abundance of each protein
was estimated by calculating the emPAI from the Mascot database search
results (Ishihama et al., 2005; Shinoda et al., 2009). emPAI values were
derived from the two (whole stem and latex) concatenated data sets (Figure 5)
and from the individual gel bands of each sample (see Supplemental Figure 5
online). The whole-stem (www.peptideatlas.org/PASS/PASS00312) and
latex (www.peptideatlas/PASS/PASS00309) proteomics data are available
publicly at PeptideAtlas.
20 s, 56°C for 30 s, and 72°C for 45 s. The final cycle ended at 72°C for 5 min.
PCR amplification products were visualized using ethidium bromide.
Cell-Free Latex Assays
Accession Numbers
Latex was mixed with chilled assay buffer (50 mM Tris-HCl, pH 6.8, 10% [v/v]
glycerol, and 2 mMpolyvinylpyrrolidone), and insoluble debris was removed
by centrifugation at 10,000g for 10 min. The supernatant was desalted, and
protein was concentrated in assay buffer using an Amicon Ultra centrifugal
filtration unit (Millipore). Protein extract (100 mL of a 3.2-mg mL21 solution)
was incubated with 100 mM thebaine, 200 mM 2-oxoglutarate, 5 mM sodium
ascorbate, 0.5 mM FeSO4, and 200 mM NADPH at 30°C for 16 h. Protein
extract boiled for 15 min was used as a negative control. Reactions were
quenched with the addition of 1 mL of methanol:acetic acid (99:1). Since
desalting did not remove all endogenous compounds, protein extract was
added directly to quenching solution to determine alkaloid content prior to
incubation. Analysis was performed using an Agilent 6410 Triple Quadrupole liquid chromatograph–mass spectrometer. Three microliters of
quenched assay was separated on a Poroshell 120 SB-C18 HPLC column
(Agilent) at a flow rate of 0.7 mL min21 using a gradient of solvent A (10 mM
ammonium acetate, pH 5.5, and 5% [v/v] acetonitrile) and solvent B (100%
acetonitrile): 0 to 80% (v/v) solvent B from 0 to 6 min, 80 to 99% solvent B
from 6 to 7 min, isocratic 99% B from 7 to 8 min, 99 to 0% solvent B from 8 to
8.1 min, followed by 100% solvent A from 8.1 to 11.1 min. Electrospray
ionization, full-scan mass analyses (mass-to-charge ratio range 200 to 700),
and collisional MS/MS experiments were performed as described previously (Farrow et al., 2012). Collision-induced dissociation spectra of
morphinan alkaloids were acquired at 25 eV, and fragmentation patterns
were matched with those of authentic standards and/or published spectra
(Raith et al., 2003) to confirm compound identities.
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: TYDC (U08598), NCS (AY860500), 6OMT (AY217335), CNMT
(AY217336), NMCH (AF191772), 4’OMT (AY217334), SalSyn (EF451150),
SalR(DQ316261),SalAT(AF339913), T6ODM(GQ500139),COR (AF108432),
CODM (GQ500141), 7OMT (AY268893), N7OMT (FJ156103), SOMT1
(JQ658999), berberine bridge enzyme (AF025430), stylopine synthase
(GU325750), and TNMT (DQ028579).
Quantitative RT-PCR
Total RNA was extracted from latex using Ambion TRIzol reagent (Life
Technologies), followed by treatment with Ambion Turbo DNase to eliminate genomic DNA contamination. First-strand cDNA was synthesized
using Invitrogen M-MLV reverse transcriptase (Life Technologies) and 1.65
mg of total RNA per 20-mL reaction. For quantitative PCR, individual cDNAs
were amplified using gene-specific primers (see Supplemental Table 2
online), 2 mL of the first-strand synthesis reaction, and a thermal profile of
94°C for 2 min followed by 20 (latex) or 25 (whole stem) cycles of 94°C for
2D Immunoblot Analysis
Latex samples were extracted in 500 mM Tris-HCl, pH 7.5, 50 mM EDTA,
1% (w/v) SDS, and 2% (v/v) 2-mercaptoethanol and subsequently partitioned with phenol. Proteins were precipitated with methanol containing
100 mM ammonium acetate and 0.0068% (v/v) 2-mercaptoethanol,
rehydrated in 7.0 M urea, 2.0 M thiourea, 56 mM DTT, and 2.5% (w/v)
CHAPS, and subsequently treated with 150 mM iodoacetamide to alkylate sulfhydryl groups. Finally, solubilization buffer (8.0 M urea, 4% [w/v]
3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate, 0.2%
[v/v] carrier ampholites, pH 3 to 10, and 50 mM DTT) and a trace amount of
bromophenol blue were added. The mixture was incubated overnight with
a 17-cm immobilized pH gradient strip (pH 4 to 7; Bio-Rad) for passive
rehydration at room temperature. Isoelectric focusing was performed at
20°C using linear voltage ramping at 250 V for 15 min, 4000 V for 2 h, and
4000 V for 20,000 V h21. The strips were equilibrated in 6.0 M urea, 2%
(w/v) SDS, 0.05 M Tris-HCl, pH 8.8, 20% (v/v) glycerol, and 2% (w/v) DTT
for 1 h. An additional 1-h incubation was performed in the same solution
containing 2.5% (v/v) iodoacetamide instead of DTT. Strips were rinsed in
SDS-PAGE running buffer for 30 s, followed by second-dimension
separation for 12 h at 50 V using a 12% (w/v) gel. Proteins were transferred
to nitrocellulose membranes for immunoblot analysis.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Biosynthesis of the Major Benzylisoquinoline
Alkaloids Produced by Opium Poppy from Dopamine and
4-Hydroxyphenylacetaldehyde.
Supplemental Figure 2. Specificity of T6ODM and CODM Polyclonal
Antibodies.
Supplemental Figure 3. Functional Classification of Proteins Identified
by Shotgun Proteomics in Whole Stem and Latex of Opium Poppy Based
on the Total Number of Annotated Proteins or the Sum of Exponentially
Modified Protein Abundance Index Values for All Annotated Proteins
within Each Category.
Supplemental Figure 4. Cell-Free Conversion of Thebaine to Downstream Intermediates and Morphine in Opium Poppy Latex Protein
Extracts.
Supplemental Figure 5. Reconstructed SDS-PAGE of Whole-Stem
and Latex Proteins Based on emPAI Values for Each of the Biosynthetic
Enzymes Listed in Figure 5 and Using a Mascot Output Derived from
Database Searches for Each Contiguous Band.
Supplemental Figure 6. Amino Acid Sequence Alignment of Contigs
and Singletons from an Opium Poppy Transcriptome Database Encoding Three BIA Biosynthetic Enzymes Occurring in Latex.
Supplemental Figure 7. Immunolocalization of CODM in Laticifers
and Sieve Elements of Opium Poppy.
12 of 13
The Plant Cell
Supplemental Table 1. Top 30 Most Abundant Proteins Based on
emPAI Score in the Whole-Stem and Latex Subproteomes of Opium
Poppy.
Supplemental Table 2. Sequences of PCR Primers Used to Assemble
Expression Constructs and to Perform Semiquantitative RT-PCR.
Supplemental Table 3. Collision-Induced Dissociation Spectra for
Thebaine and Downstream Alkaloids Produced in Native Cell-Free
Latex Protein Extracts.
Supplemental References 1. Additional Reference for the Supplemental
Data.
ACKNOWLEDGMENTS
We thank Ye Zhang and Christoph Sensen at the Visual Genomics
Centre, University of Calgary, for formatting the transcriptome database
to perform Mascot searches. P.J.F. is a Canada Research Chair in Plant
Metabolic Processes Biotechnology. D.C.S. is a Canada Research Chair
in Chemical Biology. Funds to perform this work were provided through
an Natural Sciences and Engineering Research Council of Canada
Discovery Grant to P.J.F.
AUTHOR CONTRIBUTIONS
A.O., J.M.H., X.C., and M.F.K. performed the research and analyzed the
data. D.C.S. directed the proteomics analysis. P.J.F. designed the
research and wrote the article.
Received June 19, 2013; revised August 30, 2013; accepted September
21, 2013; published October 8, 2013.
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Morphine Biosynthesis in Opium Poppy Involves Two Cell Types: Sieve Elements and Laticifers
Akpevwe Onoyovwe, Jillian M. Hagel, Xue Chen, Morgan F. Khan, David C. Schriemer and Peter J.
Facchini
Plant Cell; originally published online October 8, 2013;
DOI 10.1105/tpc.113.115113
This information is current as of June 16, 2017
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