Morphine Biosynthesis in Opium Poppy Involves Two CellTypes: Sieve Elements and LaticifersW OPEN

AkpevweOnoyovwe,a JillianM. Hagel,a Xue Chen,a Morgan F. Khan,b David C. Schriemer,b and Peter J. Facchinia,1

a Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, CanadabDepartment 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 morphinebiosynthesis 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 allupstream enzymes transforming (S)-norcoclaurine to (S)-reticuline. Validated shotgun proteomics performed on whole-stemand latex total protein extracts generated 2031 and 830 distinct protein families, respectively. Proteins corresponding to ninemorphine biosynthetic enzymes were represented in the whole stem, whereas only four of the final five pathway enzymeswere detected in the latex. Salutaridine synthase was detected in the whole stem, but not in the latex subproteome. The finalthree enzymes converting thebaine to morphine were among the most abundant active latex proteins despite a limitedoccurrence in laticifers suggested by immunofluorescence labeling. Multiple charge isoforms of two key O-demethylases inthe latex were revealed by two-dimensional immunoblot analysis. Salutaridine biosynthesis appears to occur only in sieveelements, whereas conversion of thebaine to morphine is predominant in adjacent laticifers, which contain morphine-richlatex. Complementary use of immunofluorescence labeling and shotgun proteomics has substantially resolved the cellularlocalization of morphine biosynthesis in opium poppy.

INTRODUCTION

Opium poppy (Papaver somniferum) produces several benzyli-soquinoline alkaloids (BIAs) of pharmaceutical importance, in-cluding the narcotic analgesics morphine and codeine, theanticancer drug noscapine, and the vasodilator papaverine. Thefive chiral centers in the morphinan alkaloid backbone precludechemical synthesis as an alternative to crop cultivation for thecommercial production of pharmaceutical opiates. Alkaloids ac-cumulate in the latex, which is the cytoplasm of highly specializedcells known as laticifers that are associated with the phloemthroughout the plant. The traditional method for the isolation ofopiates from cultivated plants involves lancing the unripe seedcapsules and collecting the exuded latex, which oxidizes anddries yielding raw opium. Whole seed capsules and stem seg-ments from licit commercial opium poppy crops are also har-vested 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 norco-claurine synthase (NCS) of two Tyr derivatives, dopamine and4-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 thesuccessive action of norcoclaurine 6-O-methyltransferase (6OMT),

(S)-coclaurine N-methyltransferase (CNMT), N-methylcoclaurine39-hyroxylase (NMCH), and 39-hydroxyN-methylcoclaurine 4’-O-methyltransferase (4’OMT) (Ounaroon et al., 2003; Facchini andPark, 2003; Ziegler et al., 2005). (S)-Reticuline is a branch-pointintermediate in the biosynthesis of several BIA structural sub-groups (Hagel and Facchini, 2013). Uniquely, morphine bio-synthesis requires the epimerization of (S)-reticuline. Salutaridine,the first tetracyclic promorphinan alkaloid, is formed via intra-molecular carbon-carbon phenol coupling of (R)-reticuline cata-lyzed by the cytochrome P450 monooxygenase salutaridinesynthase (SalSyn; Gesell et al., 2009). The NADPH-dependentsalutaridine reductase (SalR) reduces the C7 keto group of salu-taridine in a stereospecific manner, yielding salutaridinol (Ziegleret al., 2006), which undergoes stoichiometric transfer of an acetylgroup to the C7 hydroxyl moiety by the acetyl-CoA–dependentsalutaridinol 7-O-acetyltransferase (SalAT) to form salutaridinol7-O-acetate (Grothe et al., 2001). Spontaneous loss of the acetylgroup results in a rearrangement to thebaine, the first pentacyclicmorphinanalkaloid (LenzandZenk,1995).Thebaine isO-demethylatedby thebaine 6-O-demethylase (T6ODM) to neopinone, which isspontaneously converted to codeinone (Hagel and Facchini,2010). The NADPH-dependent codeinone reductase (COR) re-duces codeinone to codeine (Unterlinner et al., 1999), which isO-demethylated by codeine-O-demethylase (CODM), yieldingmorphine (Hagel and Facchini, 2010). As aminor alternative route,thebaine undergoes 3-O-demethylation by CODM to oripavineprior to 6-O-demethylation by T6ODM to morphinone, which isultimately reduced by COR to morphine (Brochmann-Hanssen,1984). Presently, cDNAs encoding 10 enzymes involved in theconversion of (S)-norcoclaurine to morphine have been isolated(Figure 1). Only the epimerization of reticuline has not beencharacterized at the molecular biochemical level (De-Eknamkul

1 Address correspondence to pfacchin@ucalgary.ca.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Peter J. Facchini(pfacchin@ucalgary.ca).W Online version contains Web-only data.OPENArticles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.113.115113

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and Zenk, 1992). Additional cDNAs encoding several other BIAbiosynthetic enzymes have been characterized from opiumpoppy (see Supplemental Figure 1 online), including reticu-line 7-O-methyltransferase (7OMT) (Ounaroon et al., 2003),norreticuline 7-O-methyltransferase (N7OMT) (Pienkny et al.,2009), scoulerine O-methyltransferase (SOMT) (Dang andFacchini, 2012; Winzer et al., 2012), berberine bridge enzyme(Facchini et al., 1996), stylopine synthase (Díaz-Chávez et al., 2011),tetrahydroprotoberberine N-methyltransferase (TNMT) (LiscombeandFacchini, 2007), pavineN-methyltransferase (PavNMT) (Liscombeet al., 2009), and sanguinarine reductase (Vogel et al., 2010).We previously reported the localization of several BIA bio-

synthetic enzymes (NCS, 6OMT, CNMT, NMCH, 4’OMT, SalAT,and COR) exclusively to sieve elements of the phloem by immu-nofluorescence labeling of resin-embedded tissue sections ofopium poppy using polyclonal antibodies (Bird et al., 2003;Samanani et al., 2006; Lee and Facchini, 2010). Correspondinggene transcripts for each enzyme were localized to adjacentcompanion cells by in situ RNA hybridization. No enzymes orcorresponding mRNAs were detected in laticifers, leading toa model suggesting that the transcription and translation of BIAbiosynthetic genes occur in companion cells followed by thetransport of functional enzymes to sieve elements, which serve asthe site of alkaloid biosynthesis. Subsequently, alkaloids aretransported to laticifers for storage in large vesicles of the latex.However, a similar study based on the use of polyclonal anti-bodies to localize BIAbiosynthetic enzymes (4’OMT,SalAT, COR,and 7OMT) in resin-embedded tissue sections of opium poppysuggested that laticifers and cells defined as phloemparenchymaare the sites of alkaloid biosynthesis (Weid et al., 2004). Analternative model purported that the early stages of BIA bio-synthesis occur in phloem parenchyma cells and that down-stream intermediates leading to morphine are transported tolaticifers for final biosynthetic transformations and productaccumulation. Considerable effort has been focused on theidentification of immunofluorescent cells as sieve elementsrather than phloem parenchyma (Samanani et al., 2006), al-though the localization of COR remained a discrepancy be-tween the two studies, with one suggesting its occurrence inlaticifers (Weid et al., 2004) and the other its exclusive asso-ciation with sieve elements (Bird et al., 2003). As such, the celltype–specific localization of morphine biosynthesis in opiumpoppy remains controversial.To reconcile these apparently disparate results, we have per-

formed immunofluorescence labeling of serial cross sectionsusing polyclonal antibodies raised against all six biosyntheticenzymes of the morphine branch pathway in opium poppy. As anindependent approach to enzyme identification, we also usedshotgun proteomics to confirm the relative abundance of the finalthree enzymes in laticifers. Although all six enzymes weredetected in sieve elements by immunofluorescence labeling,

Figure 1. Biosynthesis of Morphine in Opium Poppy from the TyrDerivatives 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 poppylatex.

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shotgun proteomics and immunoblot analyses supported theabundance in laticifers of the final three enzymes involved in theconversion of thebaine to morphine. The occurrence of activeenzymes was confirmed by the conversion of exogenous the-baine to downstream intermediates and morphine in cell-free latexprotein extracts. By contrast, the formation of salutaridine from (S)-norcoclaurine occurs exclusively in sieve elements, indicating a re-quirement for the intercellular translocation of pathway intermediates.

RESULTS

Immunoblot Analysis of Morphine Pathway Enzymes

The relative abundance of the final six enzymes in morphine bio-synthesis was determined by immunoblot analysis of total pro-teins extracted from various organs of the opium poppychemotypes 40 and T using polyclonal antibodies raised againstpurified recombinant enzymes. Each antibody detected a singleband 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 de-tected at relatively similar abundance in all organs of both che-motypes, with roots and carpels consistently showing the highestlevels (Figure 2A). SalSyn was most abundant in stems and car-pels and was detected at lower levels in leaves and roots. Bycontrast, SalAT was most abundant in stems and roots and waspresent at lower levels in leaves and carpels. T6ODM levels weresimilar in roots, stems, and carpels of the codeine/morphine-producing chemotype 40 but were lower in leaves. However,T6ODMwasnot detected in any organs of the thebaine/oripavine-accumulating chemotype T. CODMwas found in all aerial organsbutwasnot detected in roots. In isolated latex, all enzymes exceptSalSyn were detected (Figure 2B). Simultaneous probing of pro-tein blots with major latex protein (MLP) antibodies was used tonormalize the signal intensity of bands corresponding to the dif-ferent biosynthetic enzymes. To ensure that antibodies raisedagainst T6ODM and CODM, which share 72% amino acid se-quence identity (Hagel and Facchini, 2010), did not cross-react onimmunoblots, different amounts of the purified recombinant en-zymes were probed with polyclonal antisera under identicalconditions used to analyze plant protein extracts. No cross-reactivity was observed when 100 ng of antigen was loaded onthe blot (see Supplemental Figure 2 online).

Detection of Morphine Pathway Enzymes byImmunofluorescence Labeling

Owing to the amino acid sequence similarity betweenT6ODMandCODM, each polyclonal antiserum was scrubbed with the non-specific protein to maximize antigen specificity by reducing thelevels of IgGs recognizing common epitopes. Immunofluores-cence labeling using resin-embedded serial cross sections ofvarious organs from the opium poppy chemotype 40 (Desgagné-Penix et al., 2012) showed the colocalization of all enzymes cat-alyzing the conversion of (R)-reticuline to morphine to a cell typeassociated with phloem tissue throughout the plant (Figure 3).Previous immunolocalization experiments performed with theSalAT antiserum used herein showed that the labeled cells were

sieve elements of the phloem (Samanani et al., 2006). In agree-ment with the absence of a signal in root protein extracts by im-munoblot analysis (Figure 2), no signal above background wasdetected in root sections using CODM antibodies (Figure 3ZZ).Interestingly, sieve elements were labeled in root cross sectionsusing SalSyn antibodies and in leaf cross sections using SalATantibodies despite the apparent lack of a signal in the corre-sponding protein extracts (Figure 2). MLP antibodies showed thedistinct labeling of laticifers rather than sieve elements in corre-sponding serial cross sections of each organ (Figures 3AA to3DD). Fluorescent cells in sections immunolabeled with MLPantibodies correlated with laticifers discernable in toluidine blueO–stained serial sections based on their position, size, and rela-tively thick primary cell walls (Figures 3A to 3D). The fluorescentsignal in some laticifers was weak owing to the loss of the high-turgor latex during tissue fixation.

Shotgun Proteomics Identifies MorphineBiosynthetic Enzymes

Shotgun proteomics methods were used to perform deepanalyses aimed at determining the occurrence and relative

Figure 2. Immunoblot Analysis of Morphine Pathway Enzymes in OpiumPoppy.

(A) Immunoblots showing the relative abundance of the final six mor-phine biosynthetic enzymes in the opium poppy chemotypes T and 40.Equal amounts (50 µg) of total protein extracts from different organs wereseparated by SDS-PAGE. Protein blots were probed with polyclonalantibodies specific for each enzyme.(B) Immunoblots showing the occurrence of the final six morphine bio-synthetic enzymes in the latex of opium poppy chemotype 40. The blotswere probed simultaneously with MLP antibodies as a gel loading andautoradiogram exposure control. Data are representative of three independentexperiments.

Morphine Pathway Localization in Poppy 3 of 13

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 organwas 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.

abundance of BIA biosynthetic enzymes in whole stems andlaticifers of opium poppy. SDS-PAGE of whole-stem and latexprotein extracts from the opium poppy chemotype Roxanne(Desgagné-Penix et al., 2012) contrasted the complexity of eachsubproteome and revealed the abundance of low molecularmass MLPs in laticifers (Figure 4). Using a gel-based liquidchromatography-tandem mass spectrometry approach (Schirleet al., 2003; Vasilj et al., 2012), 1518 and 511 distinct proteinfamilies were identified from the whole-stem and latex samples,respectively. Identification was based on searches of all availableplant entries in the National Center for Biotechnology Informationnonredundant (NCBInr) database. When searched againsta transcriptome database built using 415,818 independent nu-cleotide sequences from opium poppy and partially annotatedby BLAST analysis of the National Center for Biotechnology In-formation Viridiplantae database (Desgagné-Penix et al., 2012),2031 and 830 distinct protein families were identified from wholestem and latex, respectively. The transcriptome database searchdid not increase coverage of the biosynthetic pathway enzymes,and since a large fraction of the transcriptome hits were estab-lished only from partial sequences, the NCBInr hit sets werechosen for analysis of pathway enzymes. The exponentiallymodified protein abundance index (emPAI) strategy (Ishihamaet al., 2005; Shinodaet al., 2009)wasused for this purpose, and itsvalidity was corroborated using the top three quantificationmethod on a subset of enzymes. This comparatively quantitativemethod is based on chromatographic peak intensities of the threemost abundant peptides for a given protein (Silva et al., 2006;Grossmann et al., 2010). For example, COR was detected in thelatex at 7 times the level of the whole stem using the top-threemethod 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 inFigure 5.Among the most abundant identified polypeptides in each

subproteome were several primary metabolic enzymes, defenseproteins, and cell structural components (see SupplementalTable 1 online). As expected, ribulose-1,5-bisphosphate carbox-ylase was the most abundant protein in the photosynthetic wholestem, 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 thewhole stem. In the latex, COR and CODM were joined by T6ODMand 7OMT as four of the eight most abundant proteins. Annotatedsequences in each subproteome were assigned to one of 10functional categories based on putative eukaryotic cellularprocesses. The representation of proteins in each category wasdetermined as either the number of proteins annotated or thesum of emPAI scores for all assigned proteins as an approxi-mation of protein abundance (see Supplemental Figure 3 online).More than half of the total number of proteins could be func-tionally annotated. When considered in terms of relative abun-dance based on emPAI scores, almost three-quarters of eachprotein extract could be assigned a putative function. In both thewhole-stem and latex subproteomes, the percentage of proteinsof unknown function was lower when considered in terms of

Figure 4. SDS-PAGE of Whole-Stem and Latex Protein Extracts Usedfor Shotgun Proteomics Analysis.

Lanes containing whole-stem and latex proteins were sectioned into 48and 41 segments, respectively, which were individually subjected to in-gel digestion with trypsin. Numbers on the left show the molecularmasses of protein markers in kilodaltons.

Figure 5. Relative Abundance of Alkaloid Biosynthetic Enzymes inOpium Poppy Based on emPAI Scores.

The emPAI scores were derived from shotgun proteomics performed onwhole-stem (A) and latex (B) total protein extracts.

Morphine Pathway Localization in Poppy 5 of 13

relative abundance as opposed to the absolute number of poly-peptides, which is consistent with the generally low emPAIscores for such proteins. By contrast, most other functionalcategories included proteins of higher relative abundance thanthe overall number of annotated polypeptides. Of note are en-zymes involved in primary metabolism in both the whole stemand latex as well as secondary metabolic enzymes, defenseproteins, and MLPs in the latex. Interestingly, MLPs represented55 mol % of the latex subproteome, which correlates with therelative abundance suggested by SDS-PAGE (Figure 4). Bycontrast, MLPs represented 2.3 mol % of the whole stem sub-proteome corresponding to 4.9% contamination with the latexsubproteome. The absence of chloroplast-specific proteinssuggests that the latex subproteome was not contaminated withthe contents of photosynthetic cells in the stem. Although pro-teins associated with, but not exclusive to, sieve elements wereidentified, including monodehydroascorbate reductase, glutathionereductase, and allene oxide cyclase (Walz et al., 2004; Vilaineet al., 2003), phloem-specific P-proteins (Xonconostle-Cazareset al., 1999) were not detected, further validating the purity ofthe latex sample.

Most known morphine biosynthetic enzymes were detected inthe whole stem subproteome with the exception of tyrosine/DOPA decarboxylase, NMCH, and SalAT (Figure 5). The com-bined mol % of all known BIA biosynthetic enzymes was 9.4 and4.7 in the latex and stem subproteomes, respectively. The relativeabundance of enzymes in the whole-stem proteome was variablewith NCS, 4’OMT, SalR, and COR detected at the highest levelsand SalSyn detected at a relatively low level. In the latex sub-proteome, no enzymes operating on pathway intermediates up-stream of salutaridine were detected (Figure 5). However, four ofthe final five enzymes in the morphine branch pathway wereidentified, although the SalR levels were substantially lower thanthose of T6ODM, COR, and CODM. COR displayed the highestemPAI score, but T6ODM and CODM were also abundant. Sev-eral BIA biosynthetic enzymes operating in other branchpathways were also detected in the whole stem subproteome,although only 7OMT and PavNMT were detected in the latexsubproteome (Figure 5).

Transcripts and Enzyme Activities in Latex

Quantitative RT-PCRwas performed using gene-specific primersand total RNA isolated from either whole stem or latex. Primerspecificity was confirmed using first-strand cDNAgenerated fromwhole-stem total RNA, which yielded single PCR amplicons withsimilar signal intensities and predicted fragment sizes (Figure 6;see Supplemental Table 2 online). By contrast, first-strand cDNAgenerated from latex total RNA showed that T6ODM, COR, andCODM transcripts were substantially more abundant comparedwith mRNAs encoding SalSyn, SalR, and SalAT in the 40 chemo-type. SalSyn and SalAT transcript levels were low to undetected.A similar result was obtained for the T chemotype, except thatT6ODM transcripts were not detected, whereas SalAT and SalRtranscripts were found at similar levels (Figure 6).

In the presence of NADPH, Fe2+, and 2-oxoglutarate, nativecell-free latex protein extracts converted exogenous thebaine todownstream intermediates andmorphine, whereas no increase in

endogenous alkaloid levels was detected using denatured latexprotein extracts (see Supplemental Figure 4 online). Comparedwith denatured samples, native cell-free latex protein extractsshowed reduced thebaine and increased morphinone, codei-none, codeine, and morphine compared with denatured latexextracts. Collision-induced dissociation spectra of all enzymaticreaction products were compared with those of authentic stand-ards toconfirmcompound identities (seeSupplemental Table3andSupplemental References 1 online).

O-Demethylase Isoforms in Latex

The gel-based liquid chromatography-tandem mass spectrom-etry datawere processed at the individual band level to determinethe molecular mass distribution of all biosynthetic enzymesdetected in each subproteome (see Supplemental Figure 5 on-line). Several enzymes (6OMT, 4’OMT, SalR, T6ODM, COR,CODM, and 7OMT) migrated over wide molecular mass ranges,suggesting a combination of different isoforms, posttranslationalprocessing, and/or proteome-specific degradation. It should alsobenoted that thedynamic rangeof thedata is approximately threeorders of magnitude and was normalized to the most abundantentry (COR). As such, the apparent background in some casesresulted from (1) limitations associated with the visual pre-sentation of the substantial dynamic range and (2) the potential forthe reduced accuracy of emPAI values at higher protein intensitylevels.The occurrence of multiple O-demethylase isoforms was in-

vestigated further. Despite the occurrence of only single tran-scripts encoding T6ODM and CODM (see Supplemental Figures6A and 6C online), a previous opium poppy proteomics studybased on two-dimensional (2D) SDS-PAGE and Edman degra-dation sequencing revealed a large collection of proteins withsimilar molecular masses but variable isoelectric points, anno-tated as senescence-related gene (SRG) products (Decker et al.,2000). The peptide sequences obtained from these SRG pro-teins matched the amino acid sequences of T6ODM and CODM.To support the occurrence of multiple charge isoforms of these

Figure 6. Relative Abundance of Gene Transcripts Encoding the FinalSix Enzymes of Morphine Biosynthesis in Opium Poppy.

Quantitative RT-PCR was performed using total RNA isolated from thewhole stem and latex of opium poppy chemotypes T (A) and 40 (B). Theexperiment was performed in triplicate and produced similar results eachtime.

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O-demethylases in opium poppy, protein blots of latex proteinsseparated by 2D SDS-PAGE were probed with scrubbedT6ODM and CODM polyclonal antibodies. Two sets of ;43-kDproteins were detected between pH 4.5 and 5.3 (Figure 7). CODMisoforms showed marginally lower molecular mass than T6ODMisoforms.

DISCUSSION

Opium poppy is the only plant known to produce the narcoticanalgesics codeine and morphine, which accumulate at copiouslevels in specialized laticifers that accompany sieve elements ofthe phloem in all organs. The ability to synthesize a specializedmetabolite, such as morphine, depends on the evolution ofseveral biosynthetic enzymes via the recruitment of genesarising through duplication events in the genome (Pichersky andLewinsohn, 2011). Morphine biosynthesis in opium poppy wasmade possible by the emergence of enzymes capable of cata-lyzing key reactions leading to (1) the formation of the tetracyclicpromorphinan ring system of salutaridine; (2) the reduction of thecarbonyl group in salutaridine and subsequent O-acetylation ofthe resulting hydroxyl moiety causing molecular rearrangementand, ultimately, formation of the pentacyclic morphinan back-bone; and (3) the double regiospecific O-demethylation andassociated reduction of the carbonyl moiety to convert the non-narcotic intermediate thebaine to morphine (Figure 1). Based onthe occurrence of specific metabolites and some molecularbiochemical characterization (Ziegler et al., 2006; Gesell et al.,2009), the first three enzymes (SalSyn, SalR, and SalAT) appearrestricted to a small number of related members in the genusPapaver. 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 torequire the appropriate cellular and subcellular localization ofenzymes 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 transportof branch pathway intermediates to separate compartments, in-cluding different cell types. Laticifers in Papaveraceae undoubtedlyevolved many features to function as an effective site of alkaloidaccumulation. However, data presented here show that laticifersalso play a major role in the final transformations leading to mor-phine and potentially other alkaloids. Such information providesa crucial basis for metabolic engineering or molecular breedingefforts in opium poppy.Efforts to determine the cellular localization of BIA biosynthesis

in opium poppy have focused on the immunofluorescence la-beling of tissue sections using antibodies raised against re-combinant enzymes (Bird et al., 2003;Weid et al., 2004; Samananiet al., 2006; Lee and Facchini, 2010). Although the approach iswidely used, and despite many similarities in the reported results,various incongruities and contradictory interpretations have re-sulted in controversial localization models based on two majorissues. The first involves identification of the phloem cell typelinked to all previously studied biosynthetic enzymes as eithersieve elements or parenchyma. The detection of unique cellularfeatures, including sieve plates, a characteristic cytoplasmicarchitecture, and the localization of a sieve element–specificH+-ATPase isoform, is the strongest evidence in support of thelabeled cells being sieve elements (Bird et al., 2003; Samananiet al., 2006). In a separate study, the assignment of the cell type asparenchyma was based on the reported occurrence of sieveplates in tissues that were not labeled (Weid et al., 2004). Thesecond point of contention was the role of laticifers in BIA bio-synthesis. Although most investigated enzymes were not asso-ciated 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 abun-dantly, to parenchyma or sieve elements (Weid et al., 2004). Theoccurrence in laticifers of the penultimate step in the morphinepathway would suggest the colocalization of other biosyntheticenzymes. We used a combination of (1) immunofluorescencelabeling and (2) shotgun proteomics to determine the cellularlocalization and relative abundance of the final six enzymes in-volved in morphine biosynthesis.Immunofluorescence labeling results were essentially identi-

cal to those reported previously (Bird et al., 2003), anchored bythe use of SalAT as a positive control (Samanani et al., 2006) toconfirm the localization of all six biosynthetic enzymes to sieveelements in roots, stems, leaves, and carpels (Figure 3). How-ever, stem cross sections exposed to CODM antibodies con-sistently showed the simultaneous, yet relatively weak, labelingof laticifers based on the colocalization of MLP (Figures 3YY and3BB; see Supplemental Figure 7 online). Despite the previousreport of a similar result for COR (Weid et al., 2004), the labelingof laticifers with our COR polyclonal antibodies (Bird et al.,2003), which were prepared independently for this study, wasnot reliably observed (Figure 3V). T6ODM antibodies also failedto label laticifers in support of the antiserum scrubbing effortsused to enhance immunospecificity with respect to CODM(see Supplemental Figure 2 online). Immunoblot (Figure 2) andimmunofluorescence labeling (Figure 3) results were generallyconsistent.

Figure 7. Immunoblot Analysis of Opium Poppy Latex Proteins Sepa-rated by 2D SDS-PAGE Showing Numerous Charge Isoforms of T6ODMand CODM.

Latex proteins (50 µg) were subjected to isoelectric focusing followed bySDS-PAGE and transferred to nitrocellulose membranes. Duplicateprotein blots were probed with polyclonal antibodies raised againstT6ODM or CODM.

Morphine Pathway Localization in Poppy 7 of 13

Advanced shotgun proteomics methods have the potential topenetrate deeply into the proteome of plant organs and, in somecases, specific cell types. Previously, we used shotgun proteo-mics to analyze opium poppy cell cultures producing the antimi-crobial alkaloid sanguinarine (Zulak et al., 2009; Desgagné-Penixet al., 2010) but have only now extended the approach to analyzeintact plant tissues. The positive turgor of laticifers facilitates thespecific isolation of latex with minimal contamination from sur-rounding cells (Hagel et al., 2008). Latex subproteomes of variousdepths have been reported for rubber tree (Hevea brasiliensis)(D’Amato et al., 2010), the rubber-producing plant Taraxacumbrevicorniculatum (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), andopium poppy (Decker et al., 2000). Laticifers in various plantfamilies appear to haveevolved independently (Hagel et al., 2008),allowing interesting functional, if not phylogenetic, comparisonsbased on proteomics. An opium poppy latex subproteome gen-erated using Edman degradation sequencing of cytosolic andvesicle-associated proteins separated by 2D SDS-PAGE in-cluded 98 annotated polypeptides (Decker et al., 2000). Ourshotgun proteomics approach resulted in the annotation of up toeightfold more latex proteins and many additional whole stemproteins, including most known BIA biosynthetic enzymes.

COR has previously been associated with the latex proteomealong with T6ODM and CODM, but the O-demethylases werehitherto unknownand the corresponding proteinswere annotatedas SRGs (Decker et al., 2000). The occurrence and relativeabundance of T6ODM, COR, and CODM in laticifers, comparedwith SalSyn, SalR, and SalAT, is supported by the detection ofcorresponding gene transcripts in latex (Figure 6). Unlike the ad-jacent sieve elements, the articulated, anastomosing laticifers inopium poppy contain nuclei and ribosomes and do not appear torely on other cells for gene expression and protein synthesis(Hagel et al., 2008). The relative abundance in latex of transcriptscorresponding to SalR, T6ODM, COR, and CODM is consistentwith the comparative levels of these enzymes determined usingshotgun proteomics (Figure 5). The low to undetected levels ofSalSyn and SalAT transcripts in the latex is also consistent withthe lack of detection of the corresponding enzymes in the latexsubproteome. However, the relative abundance of all testedtranscripts was similar in whole stems, despite the detection of allproteins except SalAT in the corresponding subproteome. Minordifferences are apparent in addition to the expected absence ofT6ODM protein (Figure 2) and transcript (Figure 6) in the T che-motype (Hagel and Facchini, 2010). Interestingly, SalR and SalATappeared relatively abundant in latex by immunoblot analysis(Figure 2B), suggesting that similar short-chain dehydrogenase/reductase and acyltransferase proteins distinguishable usingshotgun proteomics, but cross-reactive with polyclonal antisera,occur in laticifers.

The multiple proteins of similar molecular mass, but with dif-ferent isoelectric points annotated as SRGs (Decker et al., 2000),were confirmed as T6ODM and CODM isoforms by 2D immu-noblot analysis (Figure 7). Contigs represented in our 454 py-rosequencing transcriptome databases predicted single T6ODMand CODM isoforms (see Supplemental Figure 6 online), sug-gesting that the numerous charge isoforms were the result of

posttranslational modification. The enzymatic conversion of the-baine to downstream intermediates andmorphine in latex proteinextracts confirms that the T6ODM, COR, and CODM poly-peptides detected by shotgun proteomics are active catalysts(see Supplemental Figure 4 online). Morphine biosynthesis from14C-Tyr in isolated opium poppy latex was reported in severallandmark investigations (Stermitz and Rapoport, 1961; Fairbairnand Wassel, 1964; Kirby, 1967). However, in these studies, latexwas collected from the base of decapitated capsules, which likelyresulted in substantial contamination with sieve element sap andthe inclusion of enzymes upstream of T6ODM. By contrast, thecarpel lancing method used herein resulted in the collection oflatex free of phloem proteins.A cDNA encoding 7OMT from opium poppy was originally

isolated based on peptide amino acid sequence data obtainedvia latex proteomics analysis (Ounaroon et al., 2003). 7OMT was

Figure 8. Model Summarizing the Localization and Relative Abundanceof Morphine Biosynthetic Enzymes in Sieve Elements and Laticifers ofOpium Poppy.

The cellular localization of biosynthetic enzymes was based on immu-nolocalization and shotgun proteomics data. The font size used for eachenzyme shown in blue was adjusted to reflect the estimated relativeabundance in sieve elements and laticifers. The thickness of verticalarrows suggests the proposed relative flux through various conversionsin each cell type. The dashed vertical arrow represents an unknownenzyme. Horizontal arrows indicate alkaloids that are putatively trans-ported between sieve elements and laticifers with thebaine suggested asthe major translocated intermediate.

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also identified using our shotgun proteomics method (Figure 5).However, immunofluorescence labeling using 7OMT polyclonalantibodies previously failed to detect the enzyme in laticifers(Weid et al., 2004), similar to the incongruity in the immunolocal-ization results for COR resulting from two independent studies(Bird et al., 2003; Weid et al., 2004). Our proteomics analysisshowed that both COR and 7OMT are abundant in laticifers(Figure 5), indicating that immunofluorescence labeling is nota reliablemethod for protein localization in opiumpoppy laticifers.Immunolocalization has proven useful for the detection of BIAbiosynthetic enzymes in sieve elements. The ineffectiveness ofthe technique with respect to laticifers is likely related to theunique nature of the vesicle- and MLP-rich (Figure 4) latex, whichcould mask proteins from immunological detection in fixed andresin-embedded tissues, at least using paraformaldehyde-basedmethods (Figure 3) (Bird et al., 2003; Weid et al., 2004; Samananiet al., 2006). The dual use of immunofluorescence labeling andshotgun proteomics confirmed or showed that (1) the centralpathway from (S)-norcoclaurine to (S)-reticuline operates exclu-sively in sieve elements, (2) the early morphinan branch pathwayenzymes converting salutaridine to thebaine are primarily in sieveelements, but can also occur in laticifers, and (3) the final threeenzymes involved in the conversion of thebaine to morphine areabundant in laticifers but also likely occur in sieve elements.

A model summarizing the cellular localization of morphinebiosynthesis in opium poppy is presented in Figure 8. In thismodel, thebaine represents the major alkaloid transported fromsieve elements to laticifers, although the detection of many BIApathway intermediates in latex (Desgagné-Penix et al., 2012)suggests that the translocation process is not specific. Supportfor the formation of salutaridine in sieve elements is based on thefollowing: (1) SalSyn was not detected in the latex subproteomeand displayed a low emPAI score in the whole stem subproteome(Figure 5), (2) the enzyme was localized by immunofluorescencelabeling to sieve elements (Figure 3) and immunoblot analysis didnot 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 theoccurrence of SalR and SalAT in latex (Figure 2), SalAT was notdetected in the latex subproteome, and the emPAI score for SalRwas low compared with the scores for T6ODM, COR, and CODM(Figure 5). Moreover, the low to undetectable levels of SalR andSalAT transcripts in latex (Figure 6), the demonstrated proteininteraction between SalR and SalAT (Kempe et al., 2009), and therelative abundance of SalR in the whole stem subproteome (seeSupplemental Table 1 online) suggest that thebaine is formedprimarily in sieve elements. Intermediates upstream of salutar-idine or involved in different branch pathways are likely metabo-lized to a variety of substituted alkaloids via other enzymeslocalized to laticifers, including 7OMT and PavNMT (Figure 5).Interestingly, these late substitutions appear to primarily involvethe addition ofO- andN-linked methyl groups. The colocalizationof T6ODM and CODM, which catalyze unique O-demethylationreactions, further suggests that a major role for laticifers involvesaltering the methylation status of various alkaloids. However,O-methylation of 1-benzylisoquinolines at C6 and C4’ via 6OMTand 4’OMT, respectively [leading to (S)-reticuline], 9-O-methylationof (S)-scoulerine by SOMT (leading to noscapine) (Dang and

Facchini, 2012), and 7-O-methylation of (S)-norreticuline byN7OMT (leading to papaverine) (Pienkny et al., 2009; Desgagné-Penix and Facchini, 2012) were not associated with laticifers(Figure 5), indicating that othermajor alkaloids in opiumpoppy areproduced largely or exclusively in sieve elements.The immunolocalization of all tested BIA biosynthetic enzymes

showed fluorescent labeling only in peripheral cytoplasmic re-gions of sieve elements (Figure 3) (Bird et al., 2003; Samananiet al., 2006) and/or laticifers (see Supplemental Figure 7 online)(Weid et al., 2004). SalAT and several cytosolic enzymes involvedin the formation of (S)-reticuline were localized proximal to thesieve element reticulum (Samanani et al., 2006), similar to theassociation of sanguinarine biosynthesis with the endoplasmicreticulum in cultured opium poppy cells (Alcantara et al., 2005).Alkaloid translocation from sieve elements to laticifers could oc-cur via symplastic transport through plasmodesmata (Facchiniand De Luca, 2008) or apoplastic movement mediated by one ormore transporters. A plasma membrane ATP binding cassettetransporter (Shitan et al., 2003) and a vacuolar H+-antiporter(Otani et al., 2005) have been implicated in BIA metabolism inJapanese goldthread (Coptis japonica). The involvement oftransporters in opium poppy would not only require export fromsieve elements and import into laticifers, but also endomembranetranslocation owing to the accumulation of BIAs in latex vesicles.The peripheral localization of enzymes converting salutaridine tomorphine in laticifers could indicate an association with plasmamembrane transporters to minimize the migration of pathway in-termediates directly to latex vesicles, which would preclude fur-ther metabolism. An analysis of cytosolic and vesicular latexproteins by 2DSDS-PAGEshowed the occurrence of T6ODMandCODM along with COR in the cytosol (Decker et al., 2000). Themechanism of transport between sieve elements and latex re-mains 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 undera photoperiod of 16 h light/8 h dark at 20/18°C. Plant tissues wereharvested 2 to 3 d after anthesis and flash frozen in liquid N2. Latex wasisolated by lancing unripe seed capsules with a razor blade ;7 d afteranthesis, and corresponding whole stem segments were collected byimmersing 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 (Unterlinneret al., 1999), and SalSyn (Gesell et al., 2009) cDNAs were inserted intopQE30 (see Supplemental Table 2 online) and expressed using Escher-ichia coli strain SG13005. To improve solubility, the SalSyn cDNA wastruncated to remove 56 hydrophobic residues from the N terminus of thecorresponding polypeptide. Luria-Bertani broth (170 mM NaCl, 10 g L21

tryptone, and 5 g L21 yeast extract) containing 50 mg L21 kanamycin and100mg L21 ampicillin was inoculatedwith overnight bacterial cultures andincubated at 4°C (COR) or 25°C (SalSyn and SalR) to optimize proteinsolubility. At a density of OD600 = 0.4, the cultures were induced for 4 hwith 300 µM isopropyl-b-D-thiogalactopyranoside. Bacterial cells were

Morphine Pathway Localization in Poppy 9 of 13

lysed using a French press, and soluble SalR and COR proteins werepurified as described for T6ODM and CODM (Hagel and Facchini, 2010).Bacterial extracts containing truncated SalSyn as inclusion bodies wereresuspended in denaturing buffer (8.0 M urea, 300 mMNaCl, and 300 mMsodium phosphate) and sonicated. Cell debris was removed by centri-fugation, and the pellet was reextracted in denaturing buffer. Recombi-nant proteins were purified using a Talon Co2+-affinity column (Clontech).His-tagged SalSyn was eluted with denaturing buffer containing 150 mMimidazole and desalted using a PD10 column (GE Healthcare Life Sci-ences). MLP and SalAT antibodies were described previously (Griffing andNessler, 1988; Bird et al., 2003; Samanani et al., 2006). Other antibodieswere raised against purified recombinant proteins. Antigens were sub-cutaneously injected into mice at a concentration of 100 µg mL21 afterdialysis with 146 mM NaCl. Preimmune sera were collected from miceprior to the initial injection of antigen. Four booster injections were per-formed every 3 weeks. Final antisera were centrifuged at 1000g to isolateplasma.

For immunofluorescence labeling, the T6ODM and CODM antibodiesused were each scrubbed against the other antigen to reduce crossreactivity. One hundred micrograms of purified CODM and T6ODMproteins were separated by SDS-PAGE, transferred to separate blots, andblocked 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 inblocking solution containing 0.5% (v/v) anti-T6ODM, whereas the T6ODMblot 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 werewashed three times in PBST (137 mM NaCl, 2.7 mM KCl, 10 mM sodiumphosphate, 1.76 mM potassium phosphate, and 0.1% [v/v] Tween 20) for15 min, incubated with secondary antibody, washed in PBST, incubatedwith chemiluminescence substrate, and developed on Kodak XAR film toverify the absence of nonspecific binding.

Immunoblot Analysis

Tissues fromopiumpoppy chemotype 40were extracted with 50mMTris-HCl, pH7.5, 100mMNaCl, 0.05% (v/v) Tween 20, 1mMEDTA, and250µMphenylmethylsulfonylfluoride.Proteinconcentrationwasdeterminedusingthe Bradford assay. Fifty micrograms of soluble protein from various plantorgans was added to sample buffer (Laemmli, 1970) containing 5% (w/v)SDS and incubated in boiling water for 5 min. Proteins were separated bySDS-PAGE on a 12% (w/v) gel and transferred to a nitrocellulose mem-brane. Gel transfer was performed at 100 V for 1 h in transfer buffer (25mMTris-HCl, pH 8.3, and 192 mM Gly). After transfer, blots were blocked inPBST containing 5% (w/v) skimmilk powder for 1 h, with gyratory shaking.Primary antibodies (0.01% [v/v] antisera) were added, and blots were in-cubatedovernightat 4°Candsubsequentlywashed three times inPBST for15 min with shaking. Blots were then incubated for 1 h with horseradishperoxidase–conjugatedgoatanti-mousesecondaryantibody(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) para-formaldehyde in 100mM phosphate buffer, pH 7.4, overnight at 4°C. Afterfixation, thetissueswererinsedtwice in100mMphosphatebuffer for10mineachtimeanddehydratedas follows:30%(v/v)ethanol inwater for1h,50%for 1 h, 60% for 90min, 70% for 2 h, 80%overnight at 4°C, 90% for 2 h, and100% for 4 h. Tissues were infiltrated with LR white resin (London ResinCompany) as follows: 33% (v/v) resin in ethanol for 2 h, 50% for 2 h, fresh50% resin overnight, and 100% resin overnight. Tissueswere cast in 1-mLgelatincapsulesandpolymerizedat60°Cfor20h.Serialcrosssections(0.3-

to 0.5-µm thickness) were prepared using a Reichert-Jung Ultracut Emicrotome (Leica Microsystems) and mounted on gelatin-coated slides.Tissue sections were blocked for 1 h with Tris-buffered saline and Tween20 (TBST) containing 1% (w/v) BSA in a humid chamber. The blockingsolution was replaced with primary antisera (10% [v/v]) in fresh TBSTcontaining 1% (w/v) BSA and incubated for 3 h at room temperature.Blocking solutions containing scrubbed anti-T6ODM and anti-CODMantibodies were used directly. Slides were washed with TBST containing1%(w/v)BSA, four timesfor15min,andsectionswere incubated for1hwithAlexa 488–conjugated goat anti-mouse IgGsor, in the caseofMLPprimaryantiserum, Alexa 594–conjugated goat anti-rabbit IgGs (Molecular Probes)diluted with blocking solution. Slides were washed three times with TBSTcontaining 1% (w/v) BSA for 15 min, three times with distilled water for5 min, and finally sealed with Fluoro-Gel mounting medium (Electron Mi-croscopy Sciences). Some serial sections were stained in 10 mM sodiumbenzoate, pH 4.4, containing 0.1% (w/v) toluidine blue O. Alexa 488 and594 fluorescent probes were visualized using Leica L5 and TX2 filters, re-spectively, on a DM RXA2 microscope (Leica Microsystems). Images werecapturedwithaRetigaEXdigital camera (QImaging), and false-color imagingwas performed using Improvision Open Lab version 2.09 (Perkin-Elmer).

Shotgun Proteomics

Latex from opium poppy chemotype Roxanne was extracted in 50 mMpotassium phosphate, pH 7.0, and 500 mM mannitol. Correspondingwhole-stem proteins were isolated from 1 g of ground tissue in 50 mMTris-HCl, 100 mM NaCl, 0.05% (v/v) Tween 20, 1 mM EDTA, and 250 µMphenylmethylsulfonyl fluoride. Extracted proteins were mixed with samplebuffer (Laemmli, 1970) containing 5% (w/v) SDS and incubated in boilingwater for 5 min, and 50 mg of the mixture was separated by SDS-PAGE ona 12% (w/v) gel. The gels were subsequently stained with CoomassieBrilliant Blue R 250. Gels were fully sectioned into multiple segments: 48for the whole-stem sample and 41 for the latex sample. Each gel segmentwas rinsed once with 200 mL of HPLC-grade water and twice with 200 mLof 50 mM ammonium bicarbonate in 50% (v/v) acetonitrile. Each segmentwas then treated with 50 mL of 10 mM DTT in 100 mM ammonium bi-carbonate at 56°C for 1 h. The supernatant was removed and replacedwith 50 mL of 50 mM iodoacetamide in 100 mM ammonium bicarbonateand incubated at room temperature and in the dark for 30 min to alkylatefree disulfides. Supernatants were removed, and the gel segments werewashed twice with 200 mL of 100 mM ammonium bicarbonate for 15 min.Gel segments were then reduced to dryness, followed by rehydration in12.5 ng µL21 trypsin in 25 mM ammonium bicarbonate, pH 8.0. Sub-sequently, 25 mM ammonium bicarbonate (;10 to 20 µL) was added tocover the gel segments, and the segments were then incubated overnightat 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, re-duced 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 (ThermoScientific). InjectedsamplesweredesaltedonanAcclaimPepMaptrappingcolumn (3-µm silica particle size, 2-cm length 3 75-µm inner diameter;Dionex) for 4min with 3% (v/v) acetonitrile/0.2% (v/v) formic acid deliveredat 4mLmin21. Peptides were reverse eluted from the trapping column andseparated on an Acclaim Pepmap analytical column (2-µm silica particlesize, 15-cm length375-µminsidediameter) at a rateof 0.3mLmin21.Data-dependent acquisition of collision-induced dissociation tandem massspectrometry (MS/MS) spectra was used, with parent ion scans overa 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 lanewere combinedinto one .mgf file, which was searched with Mascot version 2.3 (Matrix

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Science) using the following parameters: 10ppmmass spectrometry error,0.8DMS/MSerror,onepotentialmissedcleavage,fixedmodificationofCyscarbamidomethylation (C), and variable modification of Met oxidation.Protein identificationwasperformedbysearchingall available plant entriesin the NCBInr database and, separately, using a transcriptome databaseconstructed from 415,818 independent nucleotide sequences from opiumpoppy partially annotated by BLAST analysis of the National Center forBiotechnology Information Viridiplantae database (Desgagné-Penix et al.,2012). For the NCBInr database search of the concatenated .mgf files,individual peptide scoreswere readjustedwith the Percolator algorithm forimproved sensitivity, and peptide spectrum matches were reported assignificant based on posterior error probabilities of <0.05. Globally, thisapproach generated false discovery rates of 0.87% (latex) and 0.73%(whole stem). Protein families were established conservatively, usingdendrogram cutoffs of 200 (whole stem) and 250 (latex) in Mascot andrepresenting a given family by the highest scoring protein. All MS/MS datasetswere submitted toProteomeXchange. The abundance of each proteinwas estimated by calculating the emPAI from theMascot database searchresults (Ishihama et al., 2005; Shinoda et al., 2009). emPAI values werederived fromthetwo(wholestemandlatex)concatenateddatasets (Figure5)and from the individual gel bandsofeachsample (seeSupplemental Figure5online). The whole-stem (www.peptideatlas.org/PASS/PASS00312) andlatex (www.peptideatlas/PASS/PASS00309) proteomics data are availablepublicly at PeptideAtlas.

Cell-Free Latex Assays

Latexwasmixedwithchilledassaybuffer (50mMTris-HCl,pH6.8,10%[v/v]glycerol,and2mMpolyvinylpyrrolidone),andinsolubledebriswasremovedby centrifugation at 10,000g for 10min. The supernatantwasdesalted, andproteinwas concentrated in assay buffer using anAmiconUltra centrifugalfiltration unit (Millipore). Protein extract (100mL of a 3.2-mgmL21 solution)was incubatedwith100mMthebaine,200mM2-oxoglutarate,5mMsodiumascorbate, 0.5 mM FeSO4, and 200 mM NADPH at 30°C for 16 h. Proteinextract boiled for 15 min was used as a negative control. Reactions werequenched with the addition of 1 mL of methanol:acetic acid (99:1). Sincedesalting did not remove all endogenous compounds, protein extract wasadded directly to quenching solution to determine alkaloid content prior toincubation. Analysis was performed using an Agilent 6410 Triple Quad-rupole liquid chromatograph–mass spectrometer. Three microliters ofquenched assay was separated on a Poroshell 120 SB-C18 HPLC column(Agilent) at a flow rate of 0.7mLmin21 using a gradient of solvent A (10mMammonium 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 Bfrom6 to7min, isocratic99%Bfrom7 to8min, 99 to0%solventB from8 to8.1 min, followed by 100% solvent A from 8.1 to 11.1 min. Electrosprayionization, full-scanmassanalyses (mass-to-chargeratiorange200to700),and collisional MS/MS experiments were performed as described pre-viously (Farrow et al., 2012). Collision-induced dissociation spectra ofmorphinan alkaloids were acquired at 25 eV, and fragmentation patternswere 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 (LifeTechnologies), followed by treatment with Ambion Turbo DNase to elimi-nate genomic DNA contamination. First-strand cDNA was synthesizedusing InvitrogenM-MLV reverse transcriptase (Life Technologies) and 1.65mgof total RNAper 20-mL reaction. For quantitativePCR, individual cDNAswere amplified using gene-specific primers (see Supplemental Table 2online), 2 mL of the first-strand synthesis reaction, and a thermal profile of94°C for 2 min followed by 20 (latex) or 25 (whole stem) cycles of 94°C for

20s,56°C for30s, and72°C for45s.Thefinalcycleendedat72°C for5min.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 par-titioned with phenol. Proteins were precipitated with methanol containing100 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 al-kylate 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 50mMDTT) and a trace amount ofbromophenol blue were added. The mixture was incubated overnight witha 17-cm immobilized pH gradient strip (pH 4 to 7; Bio-Rad) for passiverehydration at room temperature. Isoelectric focusing was performed at20°C using linear voltage ramping at 250 V for 15 min, 4000 V for 2 h, and4000 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) DTTfor 1 h. An additional 1-h incubation was performed in the same solutioncontaining 2.5% (v/v) iodoacetamide instead of DTT. Strips were rinsed inSDS-PAGE running buffer for 30 s, followed by second-dimensionseparation for 12 h at 50 V using a 12% (w/v) gel. Proteins were transferredto nitrocellulose membranes for immunoblot analysis.

Accession Numbers

Sequence data from this article can be found in the Arabidopsis GenomeInitiative or GenBank/EMBL databases under the following accessionnumbers: 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 BenzylisoquinolineAlkaloids Produced by Opium Poppy from Dopamine and4-Hydroxyphenylacetaldehyde.

Supplemental Figure 2. Specificity of T6ODM and CODM PolyclonalAntibodies.

Supplemental Figure 3. Functional Classification of Proteins Identifiedby Shotgun Proteomics inWhole Stem and Latex of Opium Poppy Basedon the Total Number of Annotated Proteins or the Sum of ExponentiallyModified Protein Abundance Index Values for All Annotated Proteinswithin Each Category.

Supplemental Figure 4. Cell-Free Conversion of Thebaine to Down-stream Intermediates and Morphine in Opium Poppy Latex ProteinExtracts.

Supplemental Figure 5. Reconstructed SDS-PAGE of Whole-Stemand Latex Proteins Based on emPAI Values for Each of the BiosyntheticEnzymes Listed in Figure 5 and Using a Mascot Output Derived fromDatabase Searches for Each Contiguous Band.

Supplemental Figure 6. Amino Acid Sequence Alignment of Contigsand Singletons from an Opium Poppy Transcriptome Database Encod-ing Three BIA Biosynthetic Enzymes Occurring in Latex.

Supplemental Figure 7. Immunolocalization of CODM in Laticifersand Sieve Elements of Opium Poppy.

Morphine Pathway Localization in Poppy 11 of 13

Supplemental Table 1. Top 30 Most Abundant Proteins Based onemPAI Score in the Whole-Stem and Latex Subproteomes of OpiumPoppy.

Supplemental Table 2. Sequences of PCR Primers Used to AssembleExpression Constructs and to Perform Semiquantitative RT-PCR.

Supplemental Table 3. Collision-Induced Dissociation Spectra forThebaine and Downstream Alkaloids Produced in Native Cell-FreeLatex Protein Extracts.

Supplemental References 1. Additional Reference for the SupplementalData.

ACKNOWLEDGMENTS

We thank Ye Zhang and Christoph Sensen at the Visual GenomicsCentre, University of Calgary, for formatting the transcriptome databaseto perform Mascot searches. P.J.F. is a Canada Research Chair in PlantMetabolic Processes Biotechnology. D.C.S. is a Canada Research Chairin Chemical Biology. Funds to perform this work were provided throughan Natural Sciences and Engineering Research Council of CanadaDiscovery Grant to P.J.F.

AUTHOR CONTRIBUTIONS

A.O., J.M.H., X.C., and M.F.K. performed the research and analyzed thedata. D.C.S. directed the proteomics analysis. P.J.F. designed theresearch and wrote the article.

Received June 19, 2013; revised August 30, 2013; accepted September21, 2013; published October 8, 2013.

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