A Tale of Three Cell Types: Alkaloid Biosynthesis Is Localized to Sieve Elements in Opium Poppy
David A. Bird,
a
Vincent R. Franceschi,
b
and Peter J. Facchini
a,1
a
Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4 Canada
b
School of Biological Sciences, Washington State University, Pullman, Washington 99164-4236
Opium poppy produces a diverse array of pharmaceutical alkaloids, including the narcotic analgesics morphine and codeine.The benzylisoquinoline alkaloids of opium poppy accumulate in the cytoplasm, or latex, of specialized laticifers that accom-pany vascular tissues throughout the plant. However, immunofluorescence labeling using affinity-purified antibodies showed
that three key enzymes, (
S
)-
N
-methylcoclaurine 3
�
-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinonereductase (COR), involved in the biosynthesis of morphine and the related antimicrobial alkaloid sanguinarine, are restrictedto the parietal region of sieve elements adjacent or proximal to laticifers. The localization of laticifers was demonstrated us-ing antibodies specific to the major latex protein (MLP), which is characteristic of the cell type. In situ hybridization showedthat CYP80B1, BBE, and COR gene transcripts were found in the companion cell paired with each sieve element, whereasMLP transcripts were restricted to laticifers. The biosynthesis and accumulation of alkaloids in opium poppy involves celltypes not implicated previously in plant secondary metabolism and dramatically extends the function of sieve elements be-yond the transport of solutes and information macromolecules in plants.
INTRODUCTION
The opium poppy is an ancient medicinal plant and the only com-mercial source for the narcotic analgesics morphine and codeine.Global production of morphine for licit pharmaceutical applica-
tions is
�
150 tons annually (United Nations, 2002a). However, theestimated illicit production of morphine for the synthesis of heroinis at least 10-fold higher and contributes to numerous social andpolitical problems throughout the world (United Nations, 2002b).Despite the widespread significance of opium poppy, many basicaspects of morphine and codeine metabolism are poorly under-stood, including the cellular localization of their biosynthesis inthe plant.
Morphine is a major component of the alkaloid-rich latex inopium poppy. Latex is the cytoplasm of specialized cells, or latici-fers, that form an internal secretory system associated with phloemtissues of the vascular system throughout the plant (Thureson-Klein, 1970). The manual lancing of unripe seed capsules is thetraditional method for the collection of opium poppy latex. Theair-dried latex, or opium, then is extracted with solvents to iso-late morphine. When damaged, laticifers release a copious vol-ume of latex because their cellular contents are under positiveturgor pressure similar to sieve elements of the phloem. More-over, despite the compound origin of opium poppy laticifers, perfo-rations develop between the lateral walls of adjacent cells, ensuringa contiguous network of latex vessels (Nessler and Mahlberg,1977). Opium poppy latex is characterized by an abundance ofmajor latex proteins (MLPs), which constitute a family of highly
conserved, low molecular weight polypeptides found exclusivelyin laticifers (Nessler et al., 1985).
Morphine and codeine are members of the large and diversegroup of benzylisoquinoline alkaloids, of which
�
2500 differentstructures have been identified in plants. Alkaloids are consid-ered secondary metabolites because they are not essential fornormal plant development but often play important ecophysio-logical roles. Morphine is most abundant in the latex of aerialorgans, whereas the antimicrobial agent sanguinarine is themajor alkaloid in opium poppy roots (Facchini and De Luca,1995). Morphine and sanguinarine share a common biosyn-thetic pathway (Figure 1), beginning with the condensation oftwo
L
-Tyr derivatives to produce the central precursor (
-methylcoclaurine (Pauli and Kutchan, 1998).The subsequent 4
�
-
O
-methylation of (
S
)-3
�
-hydroxy-
N
-methyl-coclaurine yields (
S
)-reticuline, the last common intermediate inthe biosynthesis of both sanguinarine and morphine. Berberinebridge enzyme (BBE) catalyzes the conversion of (
S
)-reticulineto (
S
)-scoulerine, the first committed step in the sanguinarinepathway (Facchini et al., 1996). Alternatively, (
S
)-reticuline canbe isomerized to its (
R
)-epimer as the first step in the formationof morphine. The NADPH-dependent enzyme codeinone reduc-tase (COR) converts (
�
)-codeinone to (
�
)-codeine as the penul-timate step in morphine biosynthesis (Unterlinner et al., 1999).
The accumulation of morphine and related secondary metab-olites in the large, membranous vesicles of opium poppy latexhas contributed to the long-standing assumption that alkaloidsare synthesized in laticifers (Fairbairn and Wassel, 1964; Fairbairnet al., 1968; Wilson and Coscia, 1975; Roberts et al., 1983). How-
1
To whom correspondence should be addressed. E-mail [email protected]; fax 403-289-9311.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.015396.
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Alkaloid Biosynthesis in Sieve Elements 2627
ever, several key enzymes involved in morphine biosynthesishave not been detected in latex (Gerardy and Zenk, 1993a,1993b), suggesting that although alkaloids accumulate in latici-fers, their synthesis occurs elsewhere. Using immunofluores-cence labeling and in situ hybridization to identify the cellular lo-calization of alkaloid biosynthesis in opium poppy, we show herethat key alkaloid biosynthetic enzymes and gene transcripts arefound in sieve elements and companion cells, respectively. Thesetwo phloem cell types have not been implicated previously inplant secondary metabolism. The implication of sieve elements inthe biosynthesis of complex alkaloids dramatically extends thefunction of the phloem beyond the transport of solutes and infor-mation macromolecules in plants.
RESULTS
Key Alkaloid Biosynthetic Enzymes Generally Are Located in All Plant Organs
Polyclonal antibodies were raised against recombinant CYP80B1,BBE, and COR in both mice and rabbits. Immunoblot analysis us-ing affinity-purified IgG fractions demonstrated the specificity ofthe antibodies. One band was detected in each lane of an immu-noblot containing crude protein extracts from different opium poppyorgans using affinity-purified mouse IgGs raised against CYP80B1,BBE, and COR (Figure 2). Identical results were obtained usingaffinity-purified rabbit IgGs (data not shown). The immunoreactiveproteins were consistent with the expected molecular masses ofCYP80B1 (54 kD), BBE (57 kD), and COR (36 kD). All three en-zymes were present in each organ except for BBE, which wasnot detected in the carpel (Figure 2). The highest level of eachprotein was found in roots. No signals were detected on immu-noblots probed with preimmune IgG fractions.
Immunolocalization Identifies a Distinct Cell Type Involved in Alkaloid Biosynthesis
Immunofluorescence labeling using resin-embedded cross-sections of various opium poppy organs showed the colocal-ization of CYP80B1, BBE, and COR to a specific cell type asso-ciated with vascular tissue throughout the plant (Figure 3). In roots,bundles composed of sieve element/companion cell pairs and la-ticifers were interspersed among parenchyma tissue throughoutthe secondary phloem, which surrounds a core of secondaryxylem (Figure 3A). The use of affinity-purified antibodies showedthat CYP80B1 (Figure 3B), BBE (Figure 3C), and COR (Figure3D) were localized to the same cells in the root vascular tissue.Colocalization using affinity-purified mouse anti-COR and rab-bit anti-MLP IgGs, which specifically labeled latex proteins,showed that laticifers were adjacent or proximal to cells con-taining CYP80B1, BBE, and COR (Figure 3D).
Identical results were obtained using sections of stem (Fig-ures 3E to 3H), leaf (Figures 3I to 3L), and carpel (Figures 3M to3P) from opium poppy. CYP80B1, BBE, and COR antibodieswere colocalized to the same cells adjacent or proximal to latic-ifers, which were identified clearly in stem (Figure 3H), leaf (Fig-ure 3J), and carpel (Figure 3P) using the MLP antibodies. Nosignals were detected in tissues probed with preimmune IgG
fractions. In stems, laticifers generally were larger than those inroots and located closer to the cortex than sieve elements andcompanion cells (Figure 3F). Similarly, the large laticifers inleaves were abaxial to other phloem tissues (Figure 3I). Across-section of a vascular bundle in the carpel shows the ex-tensive anastomosis that often occurs between adjacent latici-fers (Figure 3M). The size, angular shape, spatial distribution,and ubiquitous occurrence of cells labeled with the CYP80B1,BBE, and COR antibodies are consistent with their identifica-tion as sieve elements.
Figure 1. Sites of Action of (S)-N-Methylcoclaurine-3�-Hydroxylase(CYP80B1), Berberine Bridge Enzyme (BBE), and Codeinone Reductase(COR) in the Biosynthesis of Morphine and Sanguinarine.
Dashed arrows indicate multiple enzymatic steps.
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2628 The Plant Cell
Localization of Alkaloid Biosynthetic Gene Transcripts to Companion Cells
In situ hybridization using digoxigenin (DIG)-labeled antisenseRNA probes for CYP80B1 (Figure 4A), BBE (Figure 4B), and COR(Figure 4C) showed the accumulation of transcripts in compan-ion cells adjacent to large, angular sieve elements of the phloem.Transcripts for all three enzymes were detected in most organs,but only CYP80B1 and COR mRNAs were found in carpels. Weaklabeling also was detected in xylem parenchyma (data not shown)but was absent from all other tissue in all plant organs. Laticifers ineach section could be identified by a relatively thick cell wall, lessangular shape, and a diameter generally
�
30
�
m. A DIG-labeledantisense RNA probe for MLP hybridized specifically to laticifers(Figure 4D). MLP transcripts were not detected in other cell types,including companion cells.
In situ hybridization was not detected in tissues exposed tosense RNA probes, as shown for CYP80B1 (Figure 4E) and MLP(Figure 4F). Up to fivefold higher concentration of sense RNAprobe was used relative to that of the corresponding antisenseRNA probe. The lack of signal in sections exposed to sense RNAprobes supports the specific hybridization between the anti-sense RNA probes and gene transcripts localized in either com-panion cells or laticifers.
Presence of Sieve Plates Confirms the Identity ofSieve Elements
The colocalization of callose was used to positively identity com-panion cells and sieve elements proximal or adjacent to laticifersas the cell types containing transcripts and enzymes, respectively,involved in alkaloid biosynthesis (Figure 5). Callose, a
�
-1,3-linkedglucan lining the walls of plasmodesmata and characteristicallyassociated with sieve plates, can be detected readily by fluo-
rescence microscopy using a monospecific antibody or afterstaining with aniline blue (Smith and McCully, 1978). Serial over-lays of stem longitudinal sections exposed to COR, MLP, andcallose antibodies showed the localization of COR along the pe-ripheral cytoplasm of two adjoining sieve elements separated bya well-defined sieve plate (Figure 5A). The COR-containing sieveelements are adjacent or proximal to laticifers, identified by thepresence of MLP (Figure 5A). The staining of callose using anilineblue in root longitudinal sections confirmed the presence of sieveelements adjacent to a typically elongated companion cell towhich CYP80B1 transcripts were localized (Figures 5B and 5C).A field of plasmodesmata frequently was found between adja-cent sieve elements (Figure 5C). BBE transcripts also were local-ized specifically to companion cells in root cross-sections (Fig-ure 5D) stained with aniline blue to show the location of a sieveplate and numerous plasmodesmata between DIG-labeled com-panion cells and adjacent sieve elements (Figure 5E).
Immunoreactive Proteins Are Associated with the Parietal Region of Sieve Elements
The localization of CYP80B1, BBE, and COR to the cytoplasm ofsieve elements was confirmed by counterstaining with calcofluorwhite, which binds specifically to cellulose and thus demarcatescell walls. All three enzymes were localized to the parietal regionof the sieve element cytoplasm, whereas MLP was found dis-persed throughout the cytoplasm of laticifers (Figure 6). Immuno-fluorescence labeling clearly was not associated with cell walls.
DISCUSSION
A Tale of Three Cell Types
We have shown that the biosynthesis and accumulation of al-kaloids in opium poppy involves three cell types of the phloem,two of which have not been implicated previously in plant sec-ondary metabolism. Three key alkaloid biosynthetic genes areexpressed in companion cells of the phloem, as shown by theaccumulation of the gene transcripts in this cell type through-out the plant (Figures 4 and 5). The relative abundance ofCYP80B1, BBE, and COR transcript accumulation (Facchini etal., 1996; Unterlinner et al., 1999; Huang and Kutchan, 2000) isconsistent with the level of each protein in various organs ofopium poppy (Figure 2). The localization of the CYP80B1, BBE,and COR enzymes to sieve elements (Figure 3) implies that thecorresponding gene transcripts are translated in companioncells and that the proteins are transported to adjacent sieveelements. Mature angiosperm sieve elements lack a variety ofcellular organelles, including a nucleus and ribosomes, and thusare incapable of basic transcriptional and translational processes.As a result, a sieve element depends on its paired companion cellfor survival. Companion cell–specific gene expression and trans-lation of mRNAs that encode proteins found in sieve elements,such as lectins, are well established (Bostwick et al., 1992). Themovement of fluorescently labeled phloem proteins from compan-ion cells to sieve elements has been demonstrated (Balachandranet al., 1997).
Figure 2. Immunospecificity of Affinity-Purified Antibodies.
Immunoblot showing the levels of CYP80B1, BBE, and COR in crudeprotein extracts of various opium poppy organs. Protein blots wereprobed with affinity-purified polyclonal antibodies. Data are representa-tive of three independent experiments. R, root; S, stem; L, leaf; C, carpel.
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Alkaloid Biosynthesis in Sieve Elements 2629
Symplastic connections among adjoining sieve elements cre-ate the contiguous sieve tubes that allow solutes to be trans-ported systemically throughout the plant. The bulk flow ofphloem sap in these conductive sieve tubes produces consider-able shear forces (Fisher, 1990); thus, alkaloid biosynthetic en-zymes and other proteins must be anchored to the parietal regionof sieve elements to prevent dislodging and translocation. Manysieve element proteins, such as P proteins, are not translocatedalong the solute stream (Knoblauch and van Bel, 1998), probablybecause they are anchored to the sieve element reticulum (SER)(Oparka and Turgeon, 1999). Ultrastructural observations suggestthat small protein anchors immobilize the parietal SER and othercellular organelles, forming a channel adjacent to the plasma
membrane (Ehlers et al., 2000). Sieve element proteins havebeen suggested to reside in this channel and along the parietalSER. Immunofluorescence labeling of CYP80B1, BBE, and CORalong the cellular periphery supports the localization of alkaloidbiosynthesis to the parietal layer of sieve elements (Figures 3, 5,and 6).
The localization of CYP80B1, BBE, and COR gene transcriptsand enzymes to companion cells and sieve elements, respec-tively, is consistent with the association of Tyr/dopa decarbox-ylase (TYDC) to vascular tissues in opium poppy (Facchini andDe Luca, 1995; El-Ahmady and Nessler, 2001). Although TYDCis involved in other biochemical processes in addition to cata-lyzing the first steps in alkaloid formation and thus is not neces-
Figure 3. Alkaloid Biosynthetic Enzymes Are Localized to a Specific Cell Type Adjacent or Proximal to Laticifers in Opium Poppy.
(A) to (D) Anatomical staining and immunofluorescence localization of CYP80B1 (red), BBE (green), COR (blue), and MLP (yellow) in the phloem of se-rial root cross-sections.(E) to (H) Phloem of serial stem cross-sections.(I) to (L) Phloem of serial leaf cross-sections.(M) to (P) Phloem of serial carpel cross-sections.Asterisks show the locations of several laticifers in sections (A), (E), (I), and (M) stained with toluidine blue O. Bars � 25 �m.
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2630 The Plant Cell
sarily a direct marker for morphine and sanguinarine biosynthe-sis, it is notable that TYDC gene expression was not detected inlaticifers (Facchini and De Luca, 1995; El-Ahmady and Nessler,2001). It also is notable that low levels of TYDC mRNAs werefound in xylem parenchyma (Facchini and De Luca, 1995), aswere CYP80B1, BBE, and COR transcripts. Although CYP80B1,BBE, and COR represent only three of many biosynthetic en-zymes, we suggest that alkaloid formation is restricted to sieveelements. This notion is supported by the positions in the alka-loid biosynthetic pathway of CYP80B1 at a common early step,BBE at the branch point in the sanguinarine pathway, and CORat the penultimate stage in morphine biosynthesis. The colocal-
ization of CYP80B1, BBE, and COR to the same sieve elementsshows that both morphine and sanguinarine biosynthesis oc-curs in the same cell type and implies that different alkaloidsaccumulate in the same laticifers. Because morphine and san-guinarine biosynthesis requires a common pathway intermedi-ate, (
S
)-reticuline, the localization of both branch pathways tothe same cell has regulatory implications with respect to therelative accumulation of each alkaloid in the plant.
The involvement of multiple, adjacent cell types in alkaloidbiosynthesis and accumulation in opium poppy raises intrigu-ing questions about the transport of products from sieve ele-ments to laticifers. Recently, a multidrug-resistance-type, ATPbinding cassette (ABC) protein from
Coptis japonica
(CjMDR1)was shown to transport the benzylisoquinoline alkaloid berberine(Shitan et al., 2003). In situ hybridization showed that CjMDR1transcripts were most abundant in rhizome xylem tissues; thus,the transporter could function in the translocation of berberinefrom sites of synthesis to the rhizome, a major site of alkaloidaccumulation in
C. japonica
. A membrane-bound ABC trans-porter also might reside at the interface between sieve elementsand laticifers in opium poppy and participate in the transport ofalkaloids between these cell types. It is noteworthy that an ABCtransport protein transcript was identified in rice phloem (Asanoet al., 2002). However, symplastic transport of alkaloids also mustbe considered, although plasmodesmata connecting the two celltypes have not been reported.
Laticifers Function in Alkaloid Accumulation,Not Biosynthesis
The absence of CYP80B1, BBE, and COR proteins (Figure 3) ortranscripts (Figure 4) in laticifers shows that the latex is incapa-ble of alkaloid biosynthesis and, instead, serves strictly as thesite of alkaloid accumulation. The alkaloid-rich latex of opiumpoppy was the focus of many early efforts to identify the tissue-specific sites of alkaloid biosynthesis (Roberts et al., 1983). Ini-tial studies suggested that the radiolabeled precursors
L
-Tyrand
L
-dopa were incorporated into alkaloids when incubatedwith isolated latex (Fairbairn and Wassel, 1964; Fairbairn et al.,1968). However, a later study reported that the incorporation ofradiolabeled precursors was better with, but not restricted to,isolated latex vesicles (Wilson and Coscia, 1975). Recently, al-most 100 isolated proteins were partially sequenced after two-dimensional gel electrophoresis of the cytosolic and vesicularfractions of opium poppy latex (Decker et al., 2000). Severalenzymes associated with primary metabolism and various pro-teins implicated in general cellular processes were identified. How-ever, only one enzyme involved in alkaloid biosynthesis, COR, wasdetected. The localization of CYP80B1, BBE, and COR to sieve el-ements suggests that previous studies involving isolated latexwere tainted by contamination from the phloem sap. Both thelatex and phloem sap exhibit similar positive pressure; thus, thecrude lancing of plant organs is certain to cause the exudationof unanchored proteins from both cell types. The absence ofmembrane-bound enzymes of alkaloid biosynthesis in isolated la-tex is consistent with this suggestion (Gerardy and Zenk, 1993a,1993b).
Figure 4. Alkaloid Biosynthetic Gene Transcripts Are Localized to theCompanion Cells Paired with Sieve Elements in Opium Poppy.
(A) to (D) In situ hybridization using DIG-labeled antisense probes forCYP80B1 (A), BBE (B), COR (C), and MLP (D) performed on stem ([A]and [B]) and carpel ([C] and [D]) sections.(E) and (F) In situ hybridization using DIG-labeled sense probes forCYP80B1 (E) and MLP (F) performed on stem (E) and carpel (F) sec-tions.Asterisks and arrowheads show the locations of several laticifers and la-beled companion cells, respectively. Bars � 25 �m.
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Alkaloid Biosynthesis in Sieve Elements 2631
Alkaloid Biosynthesis in Plants Involves Diverse Cell Types
Plant secondary metabolism and accumulation are associatedwith a diverse array of cell types, which are well represented inthe biosynthesis of several distinct alkaloids. Particularly surpris-ing are the alkaloid biosynthetic pathways in which individualenzymes reside in different cell types. Early enzymes of monoter-penoid indole alkaloid formation are localized to leaf epidermis in
Catharanthus roseus
, whereas late enzymes are found severalcell layers away in laticifers and idioblasts, in which the products
Figure 5. Colocalization of MLP, Biosynthetic Enzymes or Gene Tran-scripts, and Callose Confirms the Role of Sieve Elements and Compan-ion Cells in Alkaloid Biosynthesis.
(A) Immunofluorescence localization of COR (blue), MLP (yellow), andcallose (red) in a serial overlay of LR White–embedded, longitudinal rootsections (0.3 �m thick) of opium poppy. Callose was localized using a�-1,3-linked glucan monoclonal antibody.(B) and (C) In situ hybridization using a DIG-labeled antisense probe forCYP80B1 (B) and localization of callose using aniline blue (C) in a rootlongitudinal section. Closed arrowheads point to two sieve plates, andthe open arrowhead shows a DIG-labeled companion cell.(D) and (E) In situ hybridization using a DIG-labeled antisense probe forBBE (D) and localization of callose using aniline blue (E) in a root cross-section. Arrowheads show the locations of a sieve plate and pit fields.Bars � 15 �m.
Figure 6. Immunofluorescence Localization of Alkaloid BiosyntheticEnzymes to the Parietal Layer of Sieve Elements.
Root cross-sections were counterstained with calcofluor white.(A) CYP80B1 (red) and MLP (yellow).(B) BBE (green) and MLP (yellow).(C) COR (blue) and MLP (yellow). Bar � 25 �m.
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2632 The Plant Cell
accumulate (St-Pierre et al., 1999). The first committed and lastenzymes in tropane alkaloid biosynthesis in
Atropa belladonna
and
Hyoscyamus niger
occur in the pericycle, whereas an inter-mediate enzyme is found in the adjacent endodermis (Hashimotoet al., 1991; Nakajima and Hashimoto, 1999; Suzuki et al., 1999).Homospermidine synthase, the first step of the pyrrolizidinepathway in
Senecio vernalis
, is restricted to distinct groups ofendodermis and neighboring cortical cells located opposite thephloem (Moll et al., 2002). Although their biosynthetic enzymesare restricted to roots, tropane and pyrrolizidine alkaloids aretranslocated systemically and accumulate in the cells of other or-gans (Hartmann et al., 1989; Hashimoto et al., 1991). Despite theevolutionary independence of these alkaloid biosynthetic path-ways, the emerging paradigm clearly implicates multiple celltypes and the intercellular translocation of pathway intermedi-ates or products.
The involvement of multiple cell types and differential sites ofproduct formation and accumulation also are features of ben-zylisoquinoline alkaloid biosynthesis in opium poppy. However,the localization of alkaloid biosynthetic enzymes to sieve ele-ments and their corresponding gene transcripts to companioncells is unique among plant secondary metabolic pathways. Noother biosynthetic pathway has been localized to sieve elements,which have been shown to possess only a limited number of en-zymes. Sieve element cytoplasm typically includes only severalhundred polypeptides, few of which have been identified (Kehr etal., 1999). Metabolites and enzymes associated with sieve ele-ments include ascorbate and monodehydroascorbate reduc-tases, which help to maintain an antioxidative environment (Walzet al., 2002), and glutathione, glutaredoxin, and glutathione re-ductase, which suggest the capacity for glutathione-dependentthiol reduction (Alosi et al., 1988; Szederkenyi et al., 1997). Theability of sieve elements to harbor a complex metabolic pathwayhas not seriously been considered. Nevertheless, the isolationof glutathione reductase (Alosi et al., 1988), mannitol dehydro-genase (Zamski et al., 1996), and other NAD(P)H-dependent en-zymes (Walz et al., 2002) supports the catalytic functionality ofCOR in sieve elements.
The localization of benzylisoquinoline alkaloid biosynthesis tosieve elements in opium poppy demonstrates the unexpectedmetabolic competence of this unusual cell type. Our resultsextend the fundamental physiological role of sieve elements be-yond the transport of solutes and information macromolecules.It is interesting to speculate on whether or not benzylisoquino-line alkaloid biosynthesis, in general, is localized to sieve ele-ments or if other secondary metabolic pathways display similarcell type–specific localization. A more general role for these celltypes could emerge as additional pathways are localized at thecellular level.
METHODS
Plant Material
Opium poppy (
Papaver somniferum
cv Marianne) plants were main-tained in a growth chamber at 23
�
C with a photoperiod of 14 h. Plant or-
gans were harvested 2 to 3 d after anthesis except for carpels, whichwere collected 3 to 5 d after anthesis.
Heterologous Expression and Purification of Proteins
CYP80B1
(Huang and Kutchan, 2000) and
BBE1
(Facchini et al., 1996)open reading frames were inserted in frame into pET29 (Novagen, Mad-ison, WI), and the constructs were introduced into
Escherichia coli
strainBL21(DE3). The
COR
(Unterlinner et al., 1999) open reading frame wasinserted in frame into pRSET, and the constructs were introduced into E.coli strain ER2566 (New England Biolabs, Boston, MA). Heterologous ex-pression was performed according to the pET29 manual. Briefly, 1 L of NZYbroth (86 mM NaCl, 20 mM MgSO4, 5 mg/L yeast extract, and 10 mg/Lcasein hydrolysate) containing 50 mg/L kanamycin (pET29-BBE) or 25 mg/Lampicillin (pRSET-CYP80B1 and pRSET-COR) was inoculated with 5 mL ofovernight bacterial culture and incubated at 37�C. At a density of OD600 �0.5, the cultures were induced for 4 h with 400 �M isopropyl-�-D-thioga-lactopyranoside. Cells were pelleted, resuspended in homogenizationbuffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 10 �m phenylmethylsul-fonyl fluoride [PMSF], and 5 mM 2-mercaptoethanol), and ruptured usinga French press (Spectronic Instruments, Rochester, NY). Cell debris andprotein inclusion bodies were recovered by centrifugation. The rinsedpellet was solubilized in homogenization buffer containing 6 M urea, andthe solution was passed through a 0.20-�m filter. Recombinant proteinswere affinity purified using a Ni2-charged HiTrap column according tothe manufacturer’s instructions (Pharmacia Biotech).
Preparation of Antibodies
Antibodies were prepared from purified antigens using repeated subcu-taneous injections as described by Harlow and Lane (1988). Antigen pro-teins were dialyzed against 146 mM NaCl, resuspended at a concentra-tion of 400 �g/mL, emulsified 1:1 with Freund’s complete adjuvant, andinjected into mice (100 �L) or rabbits (500 �L). Preimmune sera were col-lected from each animal, and IgG fractions were purified using an Affi-Gel Protein A MAPSII Kit (Bio-Rad). Booster injections were performedevery 3 weeks until a sufficient titer was achieved. Antibodies againstBBE, CYP80B1, and COR were affinity-purified using purified protein im-mobilized on nitrocellulose membranes (Smith and Fisher, 1984). Serawere incubated with the immobilized antigen for 3 h, rinsed in TBST (20mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% [v/v] Tween 20), andeluted with 50 mM Gly buffer, pH 2.3. Purified IgGs were neutralized in1 M Tris-HCl, pH 8.8, dialyzed against TBS (20 mM Tris-HCl, pH 7.5, and150 mM NaCl) containing 0.2% (w/v) sodium azide, and concentratedusing Centricon YM10 spin columns (Millipore, Bedford, MA).
Immunoblot Analysis
Plant tissues were frozen in liquid nitrogen and ground to a fine powderin the presence of 100 mg/g (fresh weight) polyvinyl polypyrrolidone. Tis-sues were suspended in extraction buffer (50 mM Tris-HCl, pH 7.5, 5mM EDTA, 5 �M PMSF, and 5 mM 2-mercaptoethanol) and incubatedon ice, and the supernatant was collected by centrifugation. Soluble pro-teins (25 �g) were fractionated by SDS-PAGE (Laemmli, 1970) and trans-ferred to nitrocellulose membranes. Protein blots were incubated with 10�g/mL CYP80B1, 5 �g/mL BBE, or 25 �g/mL COR antiserum for 3 h,washed in TBST, and incubated for 2 h with either alkaline phosphatase(AP)–conjugated anti-rabbit or anti-mouse secondary antibodies (Bio-Rad). The membranes were washed in TBST and developed in AP buffer(100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2) containing
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Alkaloid Biosynthesis in Sieve Elements 2633
20 �M nitroblue tetrazolium and 20 �M 5-bromo-4-chloro-3-indolylphosphate as substrates (Sambrook et al., 1989).
Tissue Fixation and Embedding for Immunocytochemical Localization
Tissue fixation and immunocytochemical localization were performed asdescribed previously (Voznesenskaya et al., 1999). Briefly, tissues wereimmersed in fixation buffer (50 mM Pipes, pH 7.0, 1.25% [v/v] glutaral-dehyde, 2% [v/v] paraformaldehyde, and 5 �M PMSF), cut with a razorblade into 1.5- to 2-mm sections, fixed for 2 h, and rinsed in 50 mMPipes, pH 7.0, containing 5 �M PMSF. The tissues were dehydrated us-ing a 30 to 100% (v/v) ethanol series with a 2-h incubation in each solu-tion. After dehydration, LR White resin (London Resin Company, London,UK) was introduced into the ethanol series at an initial ratio of 1:4 (v/v)and gradually increased to 1:3, 1:2, 1:1, 2:1, and 3:1 (v/v). Finally, tissueswere immersed in pure resin, cast into 1-mL gelatin capsules, and incu-bated at 60�C for 16 h. Sections were cut 1.0 �m thick using a Reichert-Jung Ultracut E microtome (Leica Microsystems, Wetzlar, Germany).
Tissue Fixation and Embedding for In Situ Hybridization
Organs were immersed in FAA (50% [v/v] ethanol, 5% [v/v] acetic acid,and 3.7% [v/v] formaldehyde), cut with a razor blade into 2- to 5-mm seg-ments, and fixed overnight at 4�C. Tissues were dehydrated using an eth-anol/tertiary butanol (t-butanol) series (4:1:5, 5:2:3, 5:3.5:1.5, 4.5:5.5:0,2.5:7.5:0, and 0:1:0 ethanol:t-butanol:water) with a 2-h incubation in eachsolution except for the final step, which was overnight. Paraplast Plus(Oxford Labware, St. Louis, MO) was added to a paraffin infiltration se-ries (1:1, 6.7:3.3, and 1:0 wax:t-butanol) with overnight incubations foreach step. Embedded tissues were cut into 10-�m sections using anAmerican Optical 620 microtome (Buffalo, NY). Sections were placedonto aminopropyltriethoxysilane-coated slides and incubated overnightat 37�C to promote the firm adhesion of sections to the slides.
Immunocytochemical Localization
Affinity-purified anti-BBE, anti-CYP80B1, and anti-COR IgGs were usedat concentrations of 20 �g/mL, 45 �g/mL, and 35 �g/mL, respectively.The anti-MLP (Griffing and Nessler, 1989) IgG fraction was purified usingthe Affi-Gel Protein A MAPSII Kit (Bio-Rad) and used at a concentrationof 20 �g/mL. A mouse monoclonal antibody specific to callose (Biosup-plies, Parkville, Australia) was used at a concentration of 15 �g/mL. Tissuesections were incubated with primary antibodies for 2 h and rinsed threetimes in TBS containing 1% (w/v) BSA (BSA Fraction V; Roche Diagnos-tics, Mannheim, Germany) and twice in TBS for 10 min. Sections were in-cubated for 1 h with either Alexa 488–conjugated goat anti-mouse IgG orAlexa 594–conjugated goat anti-rabbit IgG (Molecular Probes, Eugene,OR) and then rinsed in TBS and water. Slides were sealed using Aqua-perm (ThermoShandon, Pittsburgh, PA).
In Situ Hybridization
In situ hybridization was performed as described by St-Pierre et al.(1999). Briefly, CYP80B1, BBE, COR, and MLP (Nessler and VonderHaar, 1990) open reading frames in pBluescript SK� (Stratagene) servedas templates for the synthesis of sense and antisense digoxigenin (DIG)-labeled RNA probes using T3 and T7 RNA polymerases. DIG-labeledprobes were hydrolyzed at 60�C in 40 mM sodium carbonate buffer, pH10, to produce fragments 200 to 400 nucleotides in length. The pH wasneutralized using 10% (v/v) acetic acid, and the RNA was resuspendedin 50 �L of deionized water.
Sections were deparaffinized and rehydrated using an ethanol series(1:0, 1:0, 9.5:0.5, 7:3, and 1:1 ethanol:water) with a 5-min incubation ineach solution. Sections were incubated in prehybridization buffer (100mM Tris-HCl, pH 8.0, and 50 mM EDTA) containing 5 �g/mL proteinaseK (Roche Diagnostics) for 30 min and then blocked in TBS (10 mM Tris-HCl, pH 7.5, and 150 mM NaCl) containing 2 mg/mL Gly. Sections werepostfixed in 3.7% (v/v) formaldehyde in PBS (100 mM sodium phosphatebuffer, pH 7.2, and 140 mM NaCl), incubated in 100 mM triethanolaminebuffer, pH 8.0, containing 0.25% (v/v) acetic anhydride, and finally rinsedin TBS. The slides were inverted onto 100 �L of hybridization buffer (10mM Tris-HCl, pH 6.8, 10 mM sodium phosphate buffer, pH 6.8, 40% [v/v]deionized formamide, 10% [w/v] dextran sulfate, 300 mM NaCl, 5 mMEDTA, 1 mg/mL yeast tRNA, 500 ng/mL DIG-RNA, and 0.8 unit/mL RNaseinhibitor [Invitrogen, Carlsbad, CA]) spread over a cover slip. Slides weresealed in a Petri dish lined with filter paper soaked in 50% (v/v) formamideand incubated overnight at 50�C.
Slides were immersed in 2 SSC (1 SSC � 300 mM NaCl and 30 mMsodium citrate, pH 7.0) at 37�C until the cover slips fell off. Sections wereincubated in 50 mg/mL RNase A (Roche Diagnostics) in 500 mM NaCl, 10mM Tris-HCl, pH 7.5, and 1 mM EDTA for 30 min at 37�C. Slides werewashed in 2 L of the following solutions for 1 h: 2 SSC and 1 SSC atroom temperature and 0.1 SSC at 60�C. Slides were rinsed in TBST andblocked for 1 h in TBST containing 2% (w/v) BSA. Slides were invertedonto cover slips carrying 100 �L of goat anti-DIG-AP conjugate (Roche Di-agnostics) diluted 1:200 in TBST containing 1% (w/v) BSA and incubatedfor 2 h in sealed Petri dishes lined with filter paper soaked in TBST. After in-cubation, the slides were rinsed in TBST and AP buffer. Colorimetric devel-opment was performed in AP buffer containing 400 �M 5-bromo-4-chloro-3-indolyl phosphate and 428 �M nitroblue tetrazolium for 2 to 24 h.
Aniline Blue, Toluidine Blue O, and Calcofluor White Staining
Deparaffinized and rehydrated tissue sections were stained in 67 mMphosphate buffer, pH 8.5, containing 0.05% (w/v) aniline blue to detectcallose. Sections were stained in 0.5% (w/v) calcofluor white to localizecell walls. For general anatomy, LR White sections were stained in ben-zoate buffer (10 mM sodium benzoate, pH 4.4) containing 0.1% (w/v)toluidine blue O.
Fluorescence and Light Microscopy
Immunofluorescence labeling was viewed using a Leica DM RXA2 mi-croscope (Leica Microsystems, Wetzlar, Germany), and images were ac-quired with a Retiga EX digital camera (QImaging, Burnaby, British Co-lumbia, Canada). Alexa 488 and Alexa 594 fluorescent labels weredetected using Leica L5 and TX2 filters, respectively. Aniline blue andcalcofluor white were detected using a Leica A1 filter. Deconvolution andfalse-color imaging was performed using Open Lab version 2.09 (Impro-vision, Coventry, UK). Light microscopy images were captured using theLeica microscope and the Retiga camera mounted with a RGB color liq-uid crystal filter (QImaging).
Upon request, materials integral to the findings of this publication willbe made available in a timely manner to all investigators on similar termsfor noncommercial research purposes. To obtain materials, please con-tact P.J. Facchini, [email protected].
ACKNOWLEDGMENTS
We thank Craig Nessler for the gift of the MLP cDNA and antibodies.P.J.F. is the Canada Research Chair in Plant Metabolic Processes Bio-technology. This work was funded by a grant from the Natural Sciencesand Engineering Research Council of Canada to P.J.F.
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2634 The Plant Cell
Received July 9, 2003; accepted August 18, 2003.
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DOI 10.1105/tpc.015396; originally published online September 24, 2003; 2003;15;2626-2635Plant Cell
David A. Bird, Vincent R. Franceschi and Peter J. FacchiniA Tale of Three Cell Types: Alkaloid Biosynthesis Is Localized to Sieve Elements in Opium Poppy
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