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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, Peter J. Facchini

Published October 2013. DOI: https://doi.org/10.1105/tpc.113.115113

Abstract

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), (S)-coclaurine N-methyltransferase (CNMT), N-methylcoclaurine 3′-hyroxylase (NMCH), and 3′-hydroxy N-methylcoclaurine 4’-O-methyltransferase (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 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).

Figure 1.
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.

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, 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/morphine-producing chemotype 40 but were lower in leaves. However, T6ODM was not detected in any organs of the thebaine/oripavine-accumulating 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 cross-reactivity was observed when 100 ng of antigen was loaded on the blot (see Supplemental Figure 2 online).

Figure 2.
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.

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 (Desgagné-Penix 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 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 high-turgor latex during tissue fixation.

Figure 3.
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.

Shotgun Proteomics Identifies Morphine Biosynthetic Enzymes

Shotgun proteomics methods were used to perform deep analyses aimed at determining the occurrence and relative 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 discovered in the latex and whole-stem samples are displayed in Figure 5.

Figure 4.
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 in-gel digestion with trypsin. Numbers on the left show the molecular masses of protein markers in kilodaltons.

Figure 5.
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.

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 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).

Figure 6.
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.

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 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 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.

Figure 7.
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.

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 non-narcotic 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 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.

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 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 Facchini, 2012), and 7-O-methylation of (S)-norreticuline by N7OMT (leading to papaverine) (Pienkny et al., 2009; Desgagné-Penix 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.

Figure 8.
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.

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 L−1 tryptone, and 5 g L−1 yeast extract) containing 50 mg L−1 kanamycin and 100 mg L−1 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-β-d-thiogalactopyranoside. Bacterial cells were 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 mL−1 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 anti-T6ODM 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 Tris-HCl, 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-mouse secondary 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 polymerized at 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 μg 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 μL of HPLC-grade water and twice with 200 μL of 50 mM ammonium bicarbonate in 50% (v/v) acetonitrile. Each segment was then treated with 50 μL of 10 mM DTT in 100 mM ammonium bicarbonate at 56°C for 1 h. The supernatant was removed and replaced with 50 μL 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 μL of 100 mM ammonium bicarbonate for 15 min. Gel segments were then reduced to dryness, followed by rehydration in 12.5 ng µL−1 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 μL 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 × 75-µm inner diameter; Dionex) for 4 min with 3% (v/v) acetonitrile/0.2% (v/v) formic acid delivered at 4 μL min−1. Peptides were reverse eluted from the trapping column and separated on an Acclaim Pepmap analytical column (2-µm silica particle size, 15-cm length × 75-µm inside diameter) at a rate of 0.3 μL min−1. Data-dependent 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 Science) using the following parameters: 10 ppm mass spectrometry error, 0.8 D MS/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.

Cell-Free Latex Assays

Latex was mixed with chilled assay buffer (50 mM Tris-HCl, pH 6.8, 10% [v/v] glycerol, and 2 mM polyvinylpyrrolidone), 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 μL of a 3.2-mg mL−1 solution) was incubated with 100 μM thebaine, 200 μM 2-oxoglutarate, 5 mM sodium ascorbate, 0.5 mM FeSO4, and 200 μM 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 min−1 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.

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 μg of total RNA per 20-μL reaction. For quantitative PCR, individual cDNAs were amplified using gene-specific primers (see Supplemental Table 2 online), 2 μL 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 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.

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 h−1. 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.

Accession Numbers

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).

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.

  • 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.

Footnotes

  • 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 (pfacchin{at}ucalgary.ca).

  • www.plantcell.org/cgi/doi/10.1105/tpc.113.115113

  • ↵[OPEN] Articles can be viewed online without a subscription.

  • ↵[W] Online version contains Web-only data.

Glossary

BIA
benzylisoquinoline alkaloid
NCS
norcoclaurine synthase
CNMT
(S)-coclaurine N-methyltransferase
6OMT
6-O-methyltransferase
NMCH
N-methylcoclaurine 3′-hyroxylase
4’OMT
4’-O-methyltransferase
SalR
salutaridine reductase
SalAT
salutaridinol 7-O-acetlytransferase
T6ODM
thebaine 6-O-demethylase
COR
codeinone reductase
CODM
codeine-O-demethylase
7OMT
7-O-methyltransferase
N7OMT
norreticuline 7-O-methyltransferase
SOMT
scoulerine O-methyltransferase
TNMT
tetrahydroprotoberberine N-methyltransferase
PavNMT
pavine N-methyltransferase
MLP
major latex protein
NCBInr
National Center for Biotechnology Information nonredundant
emPAI
exponentially modified protein abundance index
2D
two-dimensional
SRG
senescence-related gene
MS/MS
tandem mass spectrometry
SalSyn
salutaridine synthase
  • Received June 19, 2013.
  • Revised August 30, 2013.
  • Accepted September 21, 2013.
  • Published October 8, 2013.

References

  1. ↵
    1. Alcantara J.,
    2. Bird D.A.,
    3. Francheschi V.R.,
    4. Facchini P.J.
    (2005). Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment. Plant Physiol. 138: 173–183.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Bird D.A.,
    2. Franceschi V.R.,
    3. Facchini P.J.
    (2003). A tale of three cell types: Alkaloid biosynthesis is localized to sieve elements in opium poppy. Plant Cell 15: 2626–2635.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Brochmann-Hanssen E.
    (1984). A second pathway for the terminal steps in the biosynthesis of morphine. Planta Med. 50: 343–345.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Cho W.K.,
    2. Chen X.Y.,
    3. Rim Y.,
    4. Chu H.,
    5. Jo Y.,
    6. Kim S.,
    7. Park Z.-Y.,
    8. Kim J.Y.
    (2010). Extended latex proteome analysis deciphers additional roles of the lettuce laticifer. Plant Biotechnol. Rep. 4: 311–319.
    OpenUrlCrossRef
  5. ↵
    1. D’Amato A.,
    2. Bachi A.,
    3. Fasoli E.,
    4. Boschetti E.,
    5. Peltre G.,
    6. Sénéchal H.,
    7. Sutra J.P.,
    8. Citterio A.,
    9. Righetti P.G.
    (2010). In-depth exploration of Hevea brasiliensis latex proteome and “hidden allergens” via combinatorial peptide ligand libraries. J. Proteomics 73: 1368–1380.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Dang T.T.T.,
    2. Facchini P.J.
    (2012). Characterization of three O-methyltransferases involved in noscapine biosynthesis in opium poppy. Plant Physiol. 159: 618–631.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Decker G.,
    2. Wanner G.,
    3. Zenk M.H.,
    4. Lottspeich F.
    (2000). Characterization of proteins in latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and microsequencing. Electrophoresis 21: 3500–3516.
    OpenUrlCrossRefPubMed
  8. ↵
    1. DeEknamkul W.,
    2. Zenk M.H.
    (1992). Purification and properties of 1,2-dehydroreticuline reductase from Papaver somniferum seedlings. Phytochemistry 31: 813–821.
    OpenUrlCrossRef
  9. ↵
    1. Desgagné-Penix I.,
    2. Facchini P.J.
    (2012). Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy. Plant J. 72: 331–344.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Desgagné-Penix I.,
    2. Farrow S.C.,
    3. Cram D.,
    4. Nowak J.,
    5. Facchini P.J.
    (2012). Integration of deep transcript and targeted metabolite profiles for eight cultivars of opium poppy. Plant Mol. Biol. 79: 295–313.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Desgagné-Penix I.,
    2. Khan M.F.,
    3. Schriemer D.C.,
    4. Cram D.,
    5. Nowak J.,
    6. Facchini P.J.
    (2010). Integration of deep transcriptome and proteome analysis reveals the components of alkaloid metabolism in opium poppy cell cultures. BMC Plant Biol. 10: 252.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Dhouib R.,
    2. Laroche-Traineau J.,
    3. Shaha R.,
    4. Lapaillerie D.,
    5. Solier E.,
    6. Rualés J.,
    7. Pina M.,
    8. Villeneuve P.,
    9. Carrière F.,
    10. Bonneu M.,
    11. Arondel V.
    (2011). Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics. FEBS J. 278: 97–110.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Díaz Chávez M.L.,
    2. Rolf M.,
    3. Gesell A.,
    4. Kutchan T.M.
    (2011). Characterization of two methylenedioxy bridge-forming cytochrome P450-dependent enzymes of alkaloid formation in the Mexican prickly poppy Argemone mexicana. Arch. Biochem. Biophys. 507: 186–193.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Fairbairn J.W.,
    2. Wassel G.
    (1964). The alkaloids of Papaver somniferum L.–III. Biosynthesis in the isolated latex. Phytochemistry 3: 583–585.
    OpenUrlCrossRef
  15. ↵
    1. Facchini P.J.,
    2. De Luca V.
    (2008). Opium poppy and Madagascar periwinkle: Model non-model systems to investigate alkaloid biosynthesis in plants. Plant J. 54: 763–784.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Facchini P.J.,
    2. Park S.U.
    (2003). Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy. Phytochemistry 64: 177–186.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Facchini P.J.,
    2. Penzes C.,
    3. Johnson A.G.,
    4. Bull D.
    (1996). Molecular characterization of berberine bridge enzyme genes from opium poppy. Plant Physiol. 112: 1669–1677.
    OpenUrlAbstract
  18. ↵
    1. Farrow S.C.,
    2. Hagel J.M.,
    3. Facchini P.J.
    (2012). Transcript and metabolite profiling in cell cultures of 18 plant species that produce benzylisoquinoline alkaloids. Phytochemistry 77: 79–88.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Gesell A.,
    2. Rolf M.,
    3. Ziegler J.,
    4. Díaz Chávez M.L.,
    5. Huang F.C.,
    6. Kutchan T.M.
    (2009). CYP719B1 is salutaridine synthase, the C–C phenol-coupling enzyme of morphine biosynthesis in opium poppy. J. Biol. Chem. 284: 24432–24442.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Griffing L.R.,
    2. Nessler C.L.
    (1988). Immunolocalization of the major latex proteins in developing laticifers of opium poppy (Papaver somniferum). J. Plant Physiol. 134: 357–363.
    OpenUrl
  21. ↵
    1. Grossmann J.,
    2. Roschitzki B.,
    3. Panse C.,
    4. Fortes C.,
    5. Rutishauser D.,
    6. Barkow-Oesterreicher S.,
    7. Schlapbach R.
    (2010). Implementation and evaluation of relative and absolute quantification in shotgun proteomics with label-free methods. J. Proteomics 73: 1740–1746.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Grothe T.,
    2. Lenz R.,
    3. Kutchan T.M.
    (2001). Molecular characterization of the salutaridinol 7-O-acetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum. J. Biol. Chem. 276: 30717–30723.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Hagel J.M.,
    2. Facchini P.J.
    (2010). Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy. Nat. Chem. Biol. 6: 273–275.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Hagel J.M.,
    2. Facchini P.J.
    (2013). Benzylisoquinoline alkaloid metabolism: A century of discovery and a brave new world. Plant Cell Physiol. 54: 647–672.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Hagel J.M.,
    2. Yeung E.C.,
    3. Facchini P.J.
    (2008). Got milk? The secret life of laticifers. Trends Plant Sci. 13: 631–639.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Ishihama Y.,
    2. Oda Y.,
    3. Tabata T.,
    4. Sato T.,
    5. Nagasu T.,
    6. Rappsilber J.,
    7. Mann M.
    (2005). Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell. Proteomics 4: 13256–13272.
    OpenUrl
  27. ↵
    1. Kempe K.,
    2. Higashi Y.,
    3. Frick S.,
    4. Sabarna K.,
    5. Kutchan T.M.
    (2009). RNAi suppression of the morphine biosynthetic gene salAT and evidence of association of pathway enzymes. Phytochemistry 70: 579–589.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Kirby G.W.
    (1967). Biosynthesis of morphine alkaloids. Science 155: 170–173.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Laemmli U.K.
    (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Lee E.-J.,
    2. Facchini P.J.
    (2010). Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family. Plant Cell 22: 3489–3503.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Lenz R.,
    2. Zenk M.H.
    (1995). Acetyl coenzyme A:salutaridinol-7-O-acetyltransferase from Papaver somniferum plant cell cultures. The enzyme catalyzing the formation of thebaine in morphine biosynthesis. J. Biol. Chem. 270: 31091–31096.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Liscombe D.K.,
    2. Facchini P.J.
    (2007). Molecular cloning and characterization of tetrahydroprotoberberine cis-N-methyltransferase, an enzyme involved in alkaloid biosynthesis in opium poppy. J. Biol. Chem. 282: 14741–14751.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Liscombe D.K.,
    2. MacLeod B.P.,
    3. Loukanina N.,
    4. Nandi O.I.,
    5. Facchini P.J.
    (2005). Evidence for the monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms. Phytochemistry 66: 2501–2520.
    OpenUrlPubMed
  34. ↵
    1. Liscombe D.K.,
    2. Ziegler J.,
    3. Schmidt J.,
    4. Ammer C.,
    5. Facchini P.J.
    (2009). Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species. Plant J. 60: 729–743.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Nawrot R.,
    2. Kalinowski A.,
    3. Gozdzicka-Jozefiak A.
    (2007). Proteomic analysis of Chelidonium majus sap using two-dimensional gel electrophoresis and tandem mass spectrometry. Phytochemistry 68: 1612–1622.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Otani M.,
    2. Shitan N.,
    3. Sakai K.,
    4. Martinoia E.,
    5. Sato F.,
    6. Yazaki K.
    (2005). Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica. Plant Physiol. 138: 1939–1946.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Ounaroon A.,
    2. Decker G.,
    3. Schmidt J.,
    4. Lottspeich F.,
    5. Kutchan T.M.
    (2003). (R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy. Plant J. 36: 808–819.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Pichersky E.,
    2. Lewinsohn E.
    (2011). Convergent evolution in plant specialized metabolism. Annu. Rev. Plant Biol. 62: 549–566.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Pienkny S.,
    2. Brandt W.,
    3. Schmidt J.,
    4. Kramell R.,
    5. Ziegler J.
    (2009). Functional characterization of a novel benzylisoquinoline-O-methyltransferase suggests its involvement in papaverine biosynthesis in opium poppy (Papaver somniferum L). Plant J. 60: 56–67.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Raith K.,
    2. Neubert R.,
    3. Poeaknapo C.,
    4. Böttcher C.,
    5. Schmidt J.,
    6. Zenk M.H.
    (2003). Electrospray tandem spectrometric investigations of morphinans. Am. Soc. Mass Spectrom. 14: 1262–1269.
    OpenUrlCrossRef
  41. ↵
    1. Samanani N.,
    2. Alcantara J.,
    3. Bourgault R.,
    4. Zulak K.G.,
    5. Facchini P.J.
    (2006). Role of sieve elements and laticifers in the biosynthesis of accumulation of alkaloids in opium poppy. Plant J. 47: 547–564.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Samanani N.,
    2. Liscombe D.K.,
    3. Facchini P.J.
    (2004). Molecular cloning and characterization of norcoclaurine synthase, an enzyme catalyzing the first committed step in benzylisoquinoline alkaloid biosynthesis. Plant J. 40: 302–313.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Schirle M.,
    2. Heurtier M.-A.,
    3. Kuster B.
    (2003). Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry. Mol. Cell. Proteomics 2: 1297–1305.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Shinoda K.,
    2. Tomita M.,
    3. Ishihama Y.
    (2009). emPAI-Calc--For the estimation of protein abundance from large-scale identification data by liquid chromatography-tandem mass spectrometry. Bioinformatics 26: 576–577.
    OpenUrlPubMed
  45. ↵
    1. Shitan N.,
    2. Bazin I.,
    3. Dan K.,
    4. Obata K.,
    5. Kigawa K.,
    6. Ueda K.,
    7. Sato F.,
    8. Forestier C.,
    9. Yazaki K.
    (2003). Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica. Proc. Natl. Acad. Sci. USA 100: 751–756.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Silva J.C.,
    2. Gorenstein M.V.,
    3. Li G.Z.,
    4. Vissers J.P.,
    5. Geromanos S.J.
    (2006). Absolute quantification of proteins by LCMSE: A virtue of parallel MS acquisition. Mol. Cell. Proteomics 5: 144–156.
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Stermitz F.R.,
    2. Rapoport H.
    (1961). O-demethylation as a biosynthetic pattern in the formation of opium alkaloids. Nature 189: 310–311.
    OpenUrlCrossRef
  48. ↵
    1. Unterlinner B.,
    2. Lenz R.,
    3. Kutchan T.M.
    (1999). Molecular cloning and functional expression of codeinone reductase: The penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum. Plant J. 18: 465–475.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Vasilj A.,
    2. Gentzel M.,
    3. Ueberham E.,
    4. Gebhardt R.,
    5. Shevchenko A.
    (2012). Tissue proteomics by one-dimensional gel electrophoresis combined with label-free protein quantification. J. Proteome Res. 11: 3680–3689.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Vilaine F.,
    2. Palauqui J.C.,
    3. Amselem J.,
    4. Kusiak C.,
    5. Lemoine R.,
    6. Dinant S.
    (2003). Towards deciphering phloem: A transcriptome analysis of the phloem of Apium graveolens. Plant J. 36: 67–81.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Vogel M.,
    2. Lawson M.,
    3. Sippl W.,
    4. Conrad U.,
    5. Roos W.
    (2010). Structure and mechanism of sanguinarine reductase, an enzyme of alkaloid detoxification. J. Biol. Chem. 285: 18397–18406.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Wahler D.,
    2. Colby T.,
    3. Kowalski N.A.,
    4. Harzen A.,
    5. Wotzka S.Y.,
    6. Hillebrand A.,
    7. Fischer R.,
    8. Helsper J.,
    9. Schmidt J.,
    10. Schulze-Gronover C.,
    11. Prüfer D.
    (2012). Proteomic analysis of latex from the rubber-producing plant Taraxacum brevicorniculatum. Proteomics 12: 901–905.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Walz C.,
    2. Giavalisco P.,
    3. Schad M.,
    4. Juenger M.,
    5. Klose J.,
    6. Kehr J.
    (2004). Proteomics of curcurbit phloem exudate reveals a network of defence proteins. Phytochemistry 65: 1795–1804.
    OpenUrlCrossRefPubMed
  54. ↵
    1. Weid M.,
    2. Ziegler J.,
    3. Kutchan T.M.
    (2004). The roles of latex and the vascular bundle in morphine biosynthesis in the opium poppy, Papaver somniferum. Proc. Natl. Acad. Sci. USA 101: 13957–13962.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Winzer T.,
    2. et al
    . (2012). A Papaver somniferum 10-gene cluster for synthesis of the anticancer alkaloid noscapine. Science 336: 1704–1708.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Xonconostle-Cazares B.,
    2. Xiang B.,
    3. Ruiz-Medrano R.,
    4. Wang H.L.,
    5. Monzer J.,
    6. Yoo B.C.,
    7. McFarland K.C.,
    8. Francheschi V.R.,
    9. Lucas W.J.
    (1999). Plant paralog to viral movement protein that potentiates transport of mRNA into the phloem. Science 283: 94–98.
    OpenUrlAbstract/FREE Full Text
  57. ↵
    1. Ziegler J.,
    2. Diaz Chávez M.L.,
    3. Kramell R.,
    4. Ammer C.,
    5. Kutchan T.M.
    (2005). Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis. Planta 222: 458–471.
    OpenUrlCrossRefPubMed
  58. ↵
    1. Ziegler J.,
    2. Voigtländer S.,
    3. Schmidt J.,
    4. Kramell R.,
    5. Miersch O.,
    6. Ammer C.,
    7. Gesell A.,
    8. Kutchan T.M.
    (2006). Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis. Plant J. 48: 177–192.
    OpenUrlCrossRefPubMed
  59. ↵
    1. Zulak K.G.,
    2. Khan M.F.,
    3. Alcantara J.,
    4. Schriemer D.C.,
    5. Facchini P.J.
    (2009). Plant defense responses in opium poppy cell cultures revealed by liquid chromatography-tandem mass spectrometry proteomics. Mol. Cell. Proteomics 8: 86–98.
    OpenUrlAbstract/FREE Full Text
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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, Peter J. Facchini
The Plant Cell Oct 2013, 25 (10) 4110-4122; DOI: 10.1105/tpc.113.115113
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