Abstract. A development-speci®c and elicitor-inducibleacyltransferase [hydroxycinnamoyl-CoA: tyramineN-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110)]that catalyzes the transfer of hydroxycinnamic acidsfrom hydroxycinnamoyl-CoA esters to hydroxyphen-ethylamines was puri®ed 988-fold to apparent homoge-neity from opium poppy (Papaver somniferum L.)cell-suspension cultures. The puri®cation procedure,which resulted in a 6.8% yield, involved hydrophobicinteraction and anion-exchange chromatography, fol-lowed by a�nity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) aseluent. Puri®ed THT had an isoelectric point of 5.2, anative molecular mass of approximately 50 kDa, andconsisted of two apparently identical 25-kDa subunitsas determined by two-dimensional polyacrylamidegel electrophoresis. The puri®ed enzyme was able tosynthesize a variety of amides due to a relativelylow speci®city for cinnamoyl-CoA derivatives andhydroxyphenethylamines. The best substrates wereferuloyl-CoA (V Kÿ1m 13.4 mkat g)1 M)1) and tyramine(V Kÿ1m 6.57 mkat g)1 M)1). The THT activity increasedduring development of opium poppy seedlings, occurredat high levels in roots and stems of mature plants, andwas induced in cell-suspension cultures after treatmentwith a pathogen-derived elicitor. Immunoblot analysisusing THT mouse polyclonal antibodies did not alwaysshow a correlation between THT polypeptide andenzyme activity levels. For example, despite low THTactivity in leaves, an abundant 25-kDa immunoreactivepolypeptide was detected. Immunohistochemical local-ization showed that THT polypeptides occur in corticaland xylem parenchyma, immature xylem vessel ele-ments, root periderm, anthers, ovules, and the inner

layer of the seed coat, but are most abundant in phloemsieve-tube members in roots, stems, leaves, and anther®laments.

Key words: Cell suspension culture (poppy) ± Elicitor ±Hydroxycinnamoyl-CoA: tyramine N-(hydroxycinna-moyl)transferase ± Opium poppy ±Papaver (acyltrans-ferase) ± Protein puri®cation

Introduction

The biosynthesis of hydroxycinnamic acid amides, andtheir subsequent peroxidative polymerization in the cellwall, have been suggested as integral and ubiquitouscomponents of plant defense responses to pathogenchallenge and wounding (Negrel and Martin 1984;Negrel and Jeandet 1987; Negrel and Lherminier 1987;Negrel et al. 1993; Schmidt et al. 1998). The depositionof amides, and other phenolics, in the cell wall isbelieved to create a barrier against pathogens byreducing cell wall digestibility and/or by directly inhib-iting the growth of fungal hyphae (Grandmaison et al.1993). Amides are widespread in the plant kingdom(Martin-Tanguy et al. 1978; Martin-Tanguy 1985), andare formed by the transfer of hydroxycinnamic acidsfrom hydroxycinnamoyl-CoA esters to hydroxyphen-ethylamines such as tyramine (Negrel and Martin 1984),or to polyamines such as putrescine and spermidine(Leubner-Metzger and Amrhein 1993). Hydroxy-cinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)trans-ferase (THT; EC 2.3.1.110) was ®rst described intobacco-mosaic-virus-inoculated leaves of tobacco (Ne-grel and Martin 1984), and speci®cally catalyzes thecondensation of various hydroxycinnamoyl-CoA esterswith tyramine and its derivatives (Fig. 1).

Pathogen-derived elicitors (Villegas and Brodelius1990; Holhfeld et al. 1995), wounding (Negrel et al.1993), and cell-wall-degrading enzymes (Negrel andJavelle 1995) can induce THT activity in cell-suspension

Abbreviations: 2-ME = 2-mercaptoethanol; dopamine = 3,4-di-hydroxyphenethylamine; THT = hydroxycinnamoyl-CoA: tyr-amine N-(hydroxycinnamoyl)transferase; TYDC = tyrosine/dihydroxyphenylalanine decarboxylase

Correspondence to: P.J. Facchini; E-mail: pfacchin@ucalgary.ca;Fax: 1 (403) 289 9311

Planta (1999) 209: 33±44

Puri®cation, characterization, and immunolocalization ofhydroxycinnamoyl-CoA: tyramine N-(hydroxycinnamoyl)transferasefrom opium poppy

Min Yu, Peter J. Facchini

Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada

Received: 19 January 1999 /Accepted: 3 March 1999

cultures of several plant species, including opium poppy(Facchini 1998). The induction of THT activity, coupledwith the hydrophobic nature of the protein, led to thedevelopment of rapid and e�cient puri®cation methodsfor THT from potato (Hohlfeld et al. 1995, 1996) andtobacco (Negrel and Javelle 1997) cell-suspension cul-tures. It was found that THT is a dimer consisting of twoidentical, or similar, subunits of 24 to 25 kDa. Puri®edTHT from both species was able to catalyze thesynthesis of a wide range of amides due to a relativelylow speci®city for cinnamoyl-CoA derivatives andhydroxyphenethylamines, but the best substrates wereferuloyl-CoA and tyramine (Fig. 1). The Km fortyramine was 15- to 20-fold lower than that for 3,4-dihydroxphyenethylamine (dopamine), but increased10-fold when 4-coumaroyl-CoA was used instead offeruloyl-CoA as the aryl donor. Unlike potato andtobacco, opium poppy accumulates copious amounts ofdopamine as a precursor to benzylisoquinoline alka-loids, such as morphine and sanguinarine (Roberts et al.1983). The e�cient use of dopamine as a substrate forhydroxycinnamic acid amide biosynthesis would beprecluded if THT in opium poppy also exhibits lowspeci®city for this aromatic amine.

The dual role of hydroxyphenethylamines as precur-sors for hydroxycinnamic acid amide and benzyl-isoquinoline alkaloid biosynthesis suggests a complexand interactive pattern of regulation in plant species thatproduce both types of secondary product. Cell-suspen-sion cultures of opium poppy respond to treatment withpathogen-derived elicitors by accumulating the alkaloidphytoalexin sanguinarine. The biosynthesis of sang-uinarine, and other benzylisoquinoline alkaloids, beginswith the condensation of dopamine and 4-hydroxyphe-nylacetaldehyde (4-HPAA) to form the ®rst committedalkaloid intermediate, (S)-norcoclaurine (Stadler et al.1987). The synthesis of dopamine could result fromeither decarboxylation of 3,4-dihydroxyphenylalanine

(Dopa) or hydroxylation of tyramine, which is producedby tyrosine decarboxylation. The capacity of tyrosine/dihydroxyphenylalanine decarboxylase (TYDC) to de-carboxylate both tyrosine and Dopa (Facchini and DeLuca 1994) suggests that dopamine might be synthesizedby both routes. Similarly, the biosynthesis of 4-HPAAby TYDC could result from either decarboxylation of4-hydroxyphenylpyruvate, or oxidation of tyramine;thus, TYDC is involved in the formation of tyramine,dopamine, and 4-HPAA, and could play a key role inthe regulation of both benzylisoquinoline alkaloidand hydroxycinnamic acid amide biosynthesis. Pulse-labeling experiments showed that substantially moreexogenous [14C]tyramine or [14C]dopamine was insolu-bilized in cell walls, at least partially as hydroxycinnamicacid amides, than was incorporated into sanguinarine inelicitor-treated opium poppy cell cultures (Facchini1998); thus, control of THT activity might not onlyregulate the ¯ux of tyrosine into hydroxycinnamic acidamides, but could also in¯uence the availability ofhydroxyphenethylamines for benzylisoquinoline alka-loid biosynthesis. An improved understanding of themechanisms that control THT activity in opium poppywill advance our studies on the overall regulation oftyrosine-derived secondary metabolism in plants.

A combination of hydrophobic interaction, anionexchange, and dye-ligand a�nity chromatography, wasused to purify THT to apparent homogeneity fromelicitor-treated opium poppy cell-suspension cultures. Inthis paper, we describe the opium poppy THT puri®ca-tion protocol, and characterize the substrate speci®city ofthe pur®ed enzyme. Our results support the suggestionthat THT is a homodimer (Negrel and Javelle 1997), asdetermined by size-exclusion chromatography and two-dimensional gel electrophoresis. Puri®ed THT was usedto generate mouse polyclonal antibodies that were usedto determine if developmental and inducible di�erencesin THT activity correlate with absolute changes in the

Fig. 1. Reaction scheme for THT activityin opium poppy cell-suspension cultures.The potential in-vivo substrates investi-gated in this study are shown

34 M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy

amount of THT polypeptide. Antibodies to THT werealso used to identify the tissue-speci®c localization ofTHT polypeptides in opium poppy plants.

Materials and methods

Plants and cell cultures. Opium poppy (Papaver somniferum L. cv.Marianne) plants were grown under greenhouse conditions at aday/night temperature of 20/18 °C. Seedlings were grown in sterilePetri plates on Phytagar (Gibco) containing B5 salts and vitamins(Gamborg et al. 1968) under a photoperiod of 16 h light/8 h darkat 23 °C. Before germination on agar, the seeds were surface-sterilized with 20% (v/v) sodium hypochlorite for 15 min andthoroughly rinsed with sterile water.

Opium poppy cell-suspension cultures were maintained indi�use light at 23 °C on 1B5C medium consisting of B5 salts andvitamins (Gamborg et al. 1968) plus 100 mg L)1 myo-inositol,1 g L)1 hydrolyzed casein, 20 g L)1 sucrose, and 1 mg L)1 2,4dichlorophenoxyacetic acid (2,4-D). Cells were subcultured every6 d using a 1:4 dilution of inoculum to fresh medium.

Elicitor treatment of cell cultures. Fungal elicitor was prepared fromBotrytis sp. according to Eilert et al. (1985). A section (1 cm2) ofmycelia grown on potato dextrose agar was cultivated in 50 mL1B5C medium, including supplements described above but exclud-ing 2,4-D, on a gyratory shaker (120 rpm) at 22 °C in the dark for6 d. Mycelia and medium were homogenized with a Polytron(Brinkmann), autoclaved (121 °C) for 20 min, and subsequentlycentrifuged under sterile conditions with the supernatant serving aselicitor. Elicitor treatments were initiated by the addition of 0.5 mLfungal homogenate per 50 mL cultured cells in rapid growth phase(i.e., 2±3 d after subculture). Cells were subsequently collected byvacuum ®ltration, frozen in liquid N2, and stored at )80 °C.

Chemicals and substrates. [7-14C]Tyramine (278 GBq mol)1) and[8-14C]dopamine (577 GBq mol)1) were purchased from Sigma.Cinnamoyl-CoA derivatives were enzymatically synthesized usingtotal soluble protein extracts from Escherichia coli harboring theplasmid pQE19, which directs the expression of recombinanttobacco 4-coumarate:coenzyme A ligase (Lee and Douglas 1996).Synthesis reactions consisted of 0.2 mM of various cinnamic acidderivatives, 0.1 mM coenzyme A, 2.5 mM ATP, 1 mM dit-hiothreitol, and approximately 250±300 mg total bacterial proteinextract (Facchini 1998). After 1 h incubation, the synthesizedcinnamoyl-CoA derivatives were separated from the remainingreaction components using a Sep-Pak C18 column (Waters),concentrated under vacuum, and subjected to HPLC analysis todetermine purity and UV spectra (StoÈ ckigt and Zenk 1975).

Phenyl-Sepharose, Q-Sepharose, and Superose-12 were ob-tained from Pharmacia. t-Butyl HIC was purchased from Bio-Rad,and Reactive-Yellow-3-Agarose was obtained from Sigma.N-Hydroxycinnamoyltyramine standards were a gift from Vi-ncenzo De Luca (Universite de Montre al). Protein standards forthe determination of native and denatured molecular mass, andisoelectric point, were puchased from Sigma.

Bu�ers. The following bu�ers were used for enzyme puri®cationand activity measurements: (A) 0.2 M Tris-HCl (pH 7.5), contain-ing 14 mM 2-mercaptoethanol (2-ME); (B) 0.05 M Tris-HCl (pH7.5), containing 35% (w/v) (NH4)2SO4 and 14 mM 2-ME; (C)0.05 M Tris-HCl (pH 7.5), containing 10% (w/v) (NH4)2SO4 and14 mM 2-ME; (D) 0.05 M Tris-HCl (pH 7.5), containing 14 mM2-ME; (E) 0.05 M Tris-HCl (pH 7.5); (F) 0.05 M Tris-HCl (pH7.5), containing 0.1 M feruloyl-CoA. Bu�ers (200 mM) used todetermine optimum pH were potassium phosphate (pH 5±7), Tris-HCl (pH 7±9) and glycine-NaOH (pH 9±11).

Enzyme assays. The activity of THT was measured as describedpreviously (Facchini 1998). Routinely, 50 lL of each protein

fraction was incubated for 1 h with 6.7 nmol (1.85 kBq) [7-14C]tyr-amine and 40 lmol feruloyl-CoA, unless stated otherwise. Reac-tions were stopped by the addition of 1.0 M HCl, and 20 lL wasapplied to a silica gel 60 F254 TLC plate that was subsequentlydeveloped in a solvent system consisting of chloroform:methanol(3:1 w/v). The TLC plate was then autoradiographed for 12 h.Radiolabeled spots with the same Rf values as authenticN-hydroxycinnamoyltyramine standards were scraped o� the plate,and the radioactivity was quanti®ed by liquid scintillationcounting.

Protein determination. Protein concentration was determinedaccording to the method of Bradford (1976) using bovine serumalbumin as the standard.

Puri®cation of THT. All procedures were performed at 4 °C, withthe exception of Reactive-Yellow-3-Agarose chromatographywhich was conducted at room temperature.

Preparation of crude extract. Frozen opium poppy cells (100 g)were ground to a ®ne powder with a mortar and pestle andextracted in bu�er A, the slurry was centrifuged at 20,000 g for20 min, and solid (NH4)2SO4 was dissolved in the supernatant to35% (w/v) saturation.

Phenyl-Sepharose chromatography. The crude protein extract in35% (w/v) (NH4)2SO4 was loaded onto a Phenyl-Sepharosecolumn (150 mm long, 16 mm i.d.) equilibrated in bu�er B. Thecolumn was washed with bu�er C before elution of the boundprotein fraction with bu�er D.

t-Butyl HIC chromatography. The THT fraction from the Phenyl-Sepharose column was adjusted to 35% (w/v) (NH4)2SO4 andloaded onto a t-Butyl HIC column (100 mm long, 12 mm i.d.)equilibrated in bu�er B. The column was washed with bu�er Bbefore applying a linear (NH4)2SO4 gradient (35% to 0%, w/v)at a ¯ow rate of 1 mL min)1 using a BioSys 510 protein chroma-tography system (Beckman). Fractions (2 mL each) were collectedand assayed for THT activity.

Q-Sepharose chromatography. The most active fractions fromt-Butyl HIC chromatography were pooled, dialyzed against bu�erD, and added to a Q-Sepharose column (150 mm long, 16 mm i.d.),equilibrated in bu�er D, before applying a linear NaCl gradient (0±1.0 M) at a ¯ow rate of 1 mL min)1 using the BioSys 510 proteinchromatography system. Fractions (2 mL each) were collected andassayed for THT activity.

Reactive-Yellow-3-Agarose chromatography. Active protein fromQ-Sepharose chromatography was dialyzed against bu�er D andtransferred to a Reactive-Yellow-3-Agarose column (50 mm long,15 mm i.d.) equilibrated with bu�er E. The column was washedwith bu�er E before elution of bound protein with 1 mL of bu�er Ffollowed by bu�er E at a ¯ow rate of 1 mL min)1. Fractions (1 mLeach) were collected and assayed for THT activity. Active fractionswere pooled and concentrated on Centricon-10 ultra®ltrationmembranes (Amicon) and stored at )80 °C.

Gel electrophoresis. Protein preparations at di�erent stages ofpuri®cation were visualized by gel electrophoresis under denaturingconditions according to the method of Laemmli (1970) using 12%polyacrylamide gels. The molecular mass of puri®ed THT wasdetermined by SDS-PAGE from a graph of log molecular massversus migration distance of protein standards. Proteins weresilver-stained using the method of Blum et al. (1987). Highresolution two-dimentional electrophoresis was performed accord-ing to the method of O'Farrell (1975).

Estimation of molecular mass. The molecular mass of native THTwas estimated by size-exclusion chromatography a on Superose-12column (300 mm long, 5 mm i.d.) using the BioSys 510 protein

M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy 35

chromatography system. The column was calibrated using bovineserum albumin (Mr 67,000), ovalbumin (Mr 45,000), chymotryp-sinogen A (Mr 25,000), and ribonuclease A (Mr 13,500) as referenceproteins. The void volume of the column was measured by theelution of blue-dextran 2000.

Antibody preparation. Three mice were immunized with a primaryintraperitoneal injection of 20 lg puri®ed THT in completeFreund's adjuvant. Two secondary injections, each containing20 lg puri®ed THT in incomplete Freund's adjuvant, wereadministered at 2- to 3-week intervals before the mice were bledand the high-titer THT antiserum was collected.

Immunoblot analysis. Total protein was extracted from plant tissuesin 100 mM Tris-HCl (pH 7.5), fractionated by SDS-PAGE, andtransferred to nitrocellulose membranes. Protein blots wereblocked for 1 h with 5% non-fat dry milk in TBS [20 mM Tris-HCl (pH 7.5), 150 mM NaCl] and incubated for 3 h with THTantiserum (diluted 1:1000 in TBS). After rinsing in TBS containing5% non-fat dry milk, the blots were incubated for 1 h in TBScontaining goat anti-mouse IgGs conjugated to alkaline phospha-tase (diluted 1:3000 in TBS), then rinsed again with TBS. Colordevelopment was performed in AP bu�er [100 mM Tris-HCl (pH9.5), 100 mM NaCl, 5 mM MgCl2] using 5-bromo-4-chloro-3-indolyl phosphate (BCIP; 0.005%, w/v) and nitroblue tetrazo-lium (NBT; 0.01%, w/v) as substrates.

Immunohistochemical localization of THT. Plant tissues were ®xedin FAA (50% ethanol, 5% acetic acid, 10% formalin), dehydratedwith tert-butanol and embedded in para�n. Tissues were cut into10-lm sections and mounted on replicate microscope slides.Sections were depara�nized in xylenes, rehydrated, blocked withPBS [10 mM NaHPO4 (pH 7.5), 500 mM NaCl] containing 3%nonfat dry milk for 1 h, and incubated with THT antiserum orpreimmune serum (diluted 1:100 in PBS) for 1 h. Tissue sectionswere then incubated with goat anti-mouse IgG conjugated toalkaline phosphatase (diluted 1:300 in TBS) for 1 h. Colordevelopment was performed in AP bu�er using BCIP (0.005%,w/v) and NBT (0.01%, w/v) as substrates. The developed sectionswere dehydrated, mounted, and photographed on Kodak Gold 200®lm using an Aristoplan microscope (Leitz).

Results

Puri®cation of opium poppy THT. Incubation of [14C]tyr-amine and feruloyl-CoA with a crude protein extractfrom elicited opium poppy cell-suspension culturesresulted in the formation of a single radiolabeledproduct with an Rf of 0.82 on silica gel TLC platesdeveloped in chloroform:methanol (3:1 w/v) (Fig. 2).The radiolabeled product co-migrated with an authenticN-feruloyltyramine standard using three di�erent TLCsolvent systems (Fig. 2). The enzyme-catalyzed reactionin the THT assay was linear with time for at least60 min. Boiled controls, and controls without enzyme,did not produce products. These data con®rm that theradiometric assay used in this study is a simple andsensitive procedure that is speci®c for the formation ofhydroxycinnamic acid amides from 14C-labeled hy-droxyphenethylamines and cinnamoyl-CoA ester deriv-atives.

A summary of the protocol used for the puri®cationof THT from opium poppy cell- suspension cultures isshown in Table 1. Typically, the homogenate from 100 gof cells was centrifuged at 13,000 g to remove insolublecellular debris. The THT activity was never found

associated with the 13,000 g pellet. The 13,000-g super-natant was dissolved in 35% (w/v) (NH4)2SO4 andloaded onto a Phenyl-Sepharose column equilibratedwith bu�er containing 35% (w/v) (NH4)2SO4. Afterwashing the column thoroughly with bu�er containing10% (w/v) (NH4)2SO4, the remaining protein, whichincluded all the THT activity, was eluted with salt-freebu�er. This initial column chromatography step resultedin only a 10% loss of total activity, but enriched THTspeci®c activity over 3-fold and appreciably reduced thelevel of phenolic contaminants in the protein extract.Protein recovered from the Phenyl-Sepharose columnwas subjected to hydrophobic interaction and anion-exchange chromatography as shown by the elutionpro®les in Fig. 3. Opium poppy THT proved to behydrophobic and eluted from the t-Butyl HIC column asa single peak of activity between approximately 25 and15% (NH4)2SO4 (Fig. 3A). The THT activity also elutedas a single peak from the Q-Sepharose column betweenapproximately 400 and 700 mM NaCl (Fig. 3B). Thespeci®c activity of THT increased 116-fold, compared tothe crude extract, after both hydrophobic interactionand anion-exchange chromatography (Table 1).

Fig. 2A±C. Speci®city of the radiometric assay used to detect THTactivity in protein extracts from opium poppy cell-suspension cultures.A The radiolabeled spot with Rf = 0.82 is visible only when proteinextract from elicited opium poppy cells was added, and was not seenwhen the enzyme preparation was boiled before addition to the assaymixture. The position of radiolabeled spots corresponded to themigration distance of authentic feruloyltyramine (arrowheads). Thin-layer chromatography plates were developed in: A chloroform:meth-anol (3:1); B chloroform:methanol (24:1); C ethyl acetate:methanol(1:1). In each case, the radiolabeled spot with the lowest Rf value is[14C]tyramine. The aryl donor for all assays was feruloyl-CoA.O, Sample origin; F, solvent front

36 M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy

The ®nal and most e�ective puri®cation step wasa�nity chromatography on Reactive-Yellow-3-Agaroseusing feruloyl-CoA as eluent. Using tyramine as eluent,THT could not be eluted from the a�nity column, whichis consistent with the reported behavior of potato THTon Reactive-Yellow-3-Agarose (Hohlfeld et al. 1996).The a�nity step resulted in an increase in THT speci®cactivity of almost 10-fold, but only about 10% of the

activity loaded onto the column was recovered (Ta-ble 1). As the puri®cation proceeded, a 24-kDa proteinband became prominent on silver-stained SDS-poly-acrylamide gels and was the only detectable proteinrecovered from the Reactive-Yellow-3-Agarose a�nitychromatography step (Fig. 4).

General properties of THT. The Mr of native THT wasdetermined by chromatography on a Superose-12 col-umn calibrated with molecular-mass standards. TheTHT eluted with an apparent Mr of 50 kDa, suggestingthat the native protein was a dimer. Puri®ed THT wassubjected to two-dimensional PAGE using an isoelec-tric-focusing tube gel with a pH gradient of from 3 to 10.Only one protein charge isoform with an isoelectricpoint of 5.2 was visualized after SDS-PAGE on a silver-stained gel (Fig. 5). These data con®rm that native THTconsists of two very similar, if not identical, subunits.

The e�ect of pH on the catalytic e�ciency of THTwas tested from pH 5 to 11. Optimum activity of puri®edTHT occurred between pH 7.5 and 8.0, with half-

Fig. 3A,B. Elution pro®les of protein and THT activity A from an(NH4)2SO4 gradient [35% (w/v) saturation to 0] on t-Butyl HIC (A)and from an NaCl gradient (0 to 1.0 M) on Q-Sepharose (B)

Table 1. Puri®cation of THT from elicitor-treated opium poppy cell-suspension cultures

Puri®cation step Total protein(mg)

Total activity(nkat)

Speci®c activity(nkat mg)1 protein)

Yield(%)

Puri®cation(-fold)

Crude extracta 250 32.5 0.131 100 1Phenyl-Sepharose 70 29.4 0.846 90 3.3t-Butyl HIC 3.4 28.6 8.40 88 64.3Q-Sepharose 1.5 22.6 15.1 69 115.8Reactive Yellow-3-Agarose 0.017 2.2 129.4 6.8 988

aCrude extract was prepared from 100 g of frozen cells

Fig. 4. Analysis by SDS-PAGE of protein fractions from elicitedopium poppy cell-suspension cultures exhibiting THT activity atdi�erent stages of puri®cation. Lanes: 1, crude extract (»10 lg); 2,Phenyl-Sepharose (»10 lg); 3, t-Butyl HIC (»5 lg); 4, Q-Sepharose(»2 lg); 5, Reactive Yellow-3-Agarose (»1 lg). The position and sizeof molecular-mass markers are shown on the left in kDa. Gelsconsisted of 12% polyacrylamide and were silver-stained afterelectrophoresis

M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy 37

maximal activity at pH 6.5 and 9.5. The speci®c activityof puri®ed THT was not a�ected by the presence of upto 10 mM CaCl2 or MgCl2, or by up to 5 mM MnSO4.These data are consistent with those reported fortobacco THT (Negrel and Javelle 1997), but disagreewith the reported increase in potato THT activity inresponse to the presence of these cations (Hohlfeld et al.1995). The addition of 1 mM CuSO4, FeSO4, or ZnCl2to the reaction mixture inhibited THT activity by morethan 95% in agreement with the results reported forpotato THT (Hohlfeld et al. 1995). The addition of upto 10% ethanol had no e�ect on opium poppy THTactivity, in contrast to the 2-fold ethanol-inducedactivation of tobacco THT activity (Negrel and Javelle1997). Puri®ed THT could be stored, in the presence of2-ME, at 4 °C for 14 d, at )20 °C for two months, or at)80 °C for at least four months without any appreciableloss of activity.

Substrate speci®city of THT. The substrate speci®city ofpuri®ed THT was tested with various cinnamoyl-CoAesters as acyl donors and hydroxyphenethylamines asacyl acceptors and is summarized in Table 2. Tofacilitate comparison, the Km values for tyramine anddopamine were determined with feruloyl-CoA at satu-ration. Opium poppy THT showed similar a�nity forboth tyramine (Km 76 lM) and dopamine (Km 78 lM),but the latter substrate exhibited a relatively lowreaction velocity (V). As previously reported for THTfrom potato (Hohlfeld et al. 1995) and tobacco (Negreland Javelle 1997), the a�nity of opium poppy THT forhydroxyphenethylamines was dependent on the sub-strate used as the acyl donor; thus, the Km value fortyramine increased to 454 lM in the presence of4-coumaroyl-CoA. In contrast, the Km values forcinnamoyl-CoA derivatives showed little di�erence

when either tyramine or dopamine was used as the acylacceptor. While THT had the highest a�nity forcinnamoyl-CoA (Km 2 lM), followed by 4-coumaroyl-CoA (Km 17 lM), sinapoyl-CoA (Km 21 lM), andferuloyl-CoA (Km 62 lM), the enzyme showed thehighest speci®city (V Kÿ1m ) for feruloyl-CoA, followedby cinnamoyl-CoA (41%), sinapoyl-CoA (32%), and4-coumaroyl-CoA (22%). Ca�eoyl-CoA was a poorsubstrate for the puri®ed opium poppy THT in agree-ment with the behavior of tobacco THT (Negrel andJavelle 1997), but in contrast to that of potato THT(Hohlfeld et al. 1995).

Correlation between THT activity and polypeptide levels.The speci®c activity of THT was measured, usingferuloyl-CoA and [14C]tyramine as substrates, in totalsoluble protein extracts from various organs of matureopium poppy plants, seedlings, and elicitor-treated cell-suspension cultures (Fig. 6). In the plant, the highestlevels of THT activity were found in mature roots andstems, followed by young stems and root tips. Only lowlevels of THT activity were detected in ¯ower buds andleaves (Fig. 6A). The speci®c activity of THT increasedin developing seedlings for 13 d after imbibition, butbegan to decrease thereafter (Fig. 6B). The level of THTactivity also increased rapidly and transiently in cell-suspension cultures treated with a pathogen-derivedelicitor, reaching maximum levels within 10 h (Fig. 6C).The maximum levels of THT speci®c activity in matureroots (165 pkat mg)1 protein) and 2-week-old seedlings(122 pkat mg)1 protein) were similar to the maximumelicitor-induced level of activity in cell-suspension cul-tures (135 pkat mg)1 protein).

Mouse polyclonal antibodies raised against puri®edTHT enzyme were used to probe immunoblots of totalsoluble protein extracted from various organs of matureopium poppy plants, seedlings, and elicitor-treatedcell-suspension cultures (Fig. 7). The THT antibodies

Fig. 5. Two-dimensional electrophoresis of puri®ed THT fromelicited opium poppy cell-suspension cultures. Isoelectric focusing,using ampholites in the range of pH 3 to 10, of puri®ed THT (»1 lg)was performed in the ®rst dimension and SDS-PAGE, on a 12%polyacrylamide gel, was conducted in the second dimension. Theposition and size of molecular-mass markers are shown on the right inkDa and the apparent isoelectric points are indicated on the top

Table 2. Substrate speci®city of THT from opium poppy

Substrate Va

(%)Kam

(lM)V Kÿ1m(mkat g)1 M)1)

Donorsb

Feruloyl-CoA 100c 62 13.4Coumaroyl-CoA 6 17 2.94Cinnamoyl-CoA 1 2 5.52Sinapoyl-CoA 11 21 4.31Ca�eoyl-CoA <1 n.d. ±

Acceptorsd

Tyraminee 100f 76 6.57Dopamine 10 78 0.64

aDetermined from Lineweaver-Burk plots used to determine var-iations in 1/V at constant and saturating concentration of onesubstratebWith tyramine as acyl acceptorc0.83 nkat mg)1 proteindWith feruloyl-CoA as acyl donoreKm value was 5-fold higher (454 lM) with 4-coumaroyl-CoA asacyl donorf0.05 nkat mg)1 protein

38 M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy

recognized only a single 25-kDa polypeptide in eachprotein extract, demonstrating the speci®city of theantiserum. Immunoreactive 25-kDa polypeptides weremost abundant in mature roots and root tips, followedby mature leaves, young leaves and young stems.However, the level of THT activity (Fig. 6A) could notalways be correlated with the relative level of THTpolypeptides in some plant organs (Fig. 7A). Forexample, despite the low level of THT activity in leaves,abundant 25-kDa immunoreactive polypeptides weredetected. The THT activity in leaf protein extracts wasreassessed using various cinnamoyl-CoA derivatives andhydroxyphenethylamines as substrates, but the speci®cactivity was always low. These data suggest that eitherTHT isozymes with unique substrate speci®city arepresent in leaves, or the immunoreactive polypeptides inleaves represent an inactive form of the enzyme. Indeveloping seedlings (Fig. 7B), the level of THT poly-peptides directly correlated with the level of enzymeactivity. Maximum levels of immunoreactive polypep-tides were detected in seedlings 13 d after imbibition. Inelicitor-treated cell-suspension cultures (Fig. 7C), THTpolypeptide and enzyme activity levels were correlatedduring the induction phase. But, THT activity began todecline 10 h after elicitor treatment, whereas THTpolypeptide levels continued to increase.

Antiserum to THT did not completely inhibit THTactivity in the in-vitro assay. Preimmune serum had noe�ect on THT activity, whereas 5 lL of THT antiseruminhibited the activity of approx. 0.5 lg of the puri®edenzyme by 10%, and 10 lL reduced THT activity by lessthan 20%. Since THT antibodies were raised against thepuri®ed native enzyme, these data suggest that peptidesequences which comprise the active site of the THTprotein were not su�ciently recognized as epitopes. TheTHT antiserum was also used to probe a `garden blot' oftotal soluble stem protein from a taxonomically diverseselection of dicots (beet, California poppy, canola, ¯ax,parsley, pea, periwinkle, and tobacco), a monocot (rice),and a gymnosperm (yew). Although THT activity wasdetected in each species, using feruloyl-CoA and[14C]tyramine as substrates, opium poppy THT anti-bodies did not reveal immunoreactive polypeptides inthe protein extracts (data not shown). This result wasespecially surprising for California poppy (Eschscholziacalifornica Cham.) which, like opium poppy, is amember of the Papaveraceae. These data are consistentwith the suggestion that the polyclonal THT antibodieswere raised against epitopes on the native protein thatdo not include highly conserved active-site domains.

Immunohistochemical localization of THT. The spatialdistribution of THT polypeptides in various tissues ofopium poppy was examined by immunohistochemicallocalization using the THT polyclonal antiserum(Fig. 8). The THT polypeptides were most abundant inconductive elements of the phloem in roots, stems,leaves, and anther ®laments (Fig. 8A±D). Lower levelsof THT polypeptides were detected in root periderm andxylem parenchyma (Fig. 8A), the cortical and xylemparenchyma of stems and leaves (Fig. 8B,C), stem

Fig. 6A±C. Speci®c THT activity in various organs of maturegreenhouse-grown opium poppy plants (A); in opium poppy seedlingsgrown under sterile conditions (B); and in elicitor-treated opiumpoppy cell-suspension cultures (C)

M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy 39

epidermis (Fig. 8B), the ovule nucellus (Fig. 8E), an-thers (Fig. 8F), and the inner layer of the seed coat(Fig. 8G). Abundant THT polypeptides were not de-tected in mature xylem vessel elements (Fig. 8A±C),secondary phloem parenchyma in roots (Fig. 8A),laticifers (Fig. 8A±C), and leaf epidermis (Fig. 8C).Developing vessel elements were sometimes found inwhich strong histochemical staining was detected(Fig. 8H), suggesting that THT polypeptides are presentduring secondary wall deposition in the xylem. Thislocalization pattern was consistent in all organs from avariety of developmental stages. No histochemicalstaining occurred in any tissue exposed to preimmuneserum (data not shown).

Discussion

Hydroxycinnamoyl-CoA: tyramine N-(hydroxycinna-moyl)transferase from elicitor-treated opium poppycell-suspension cultures was puri®ed to apparent homo-geneity using a four-step puri®cation procedure. Theprotocol described here resulted in an overall puri®ca-tion of 988-fold with a recovery of 6.8%. The pureenzyme had a speci®c activity of 129.4 nkat mg)1

protein. Based on the initial THT activity of the crudeextract, the enzyme constituted 0.1% of the solubleprotein. Due to the moderately low abundance of THTin opium poppy tissues, the use of an a�nity-chroma-tography step speci®c for the enzyme was e�cacious inthe puri®cation of THT. Reactive-Yellow-3-Agarosea�nity chromatography using the acyl donor feruloyl-CoA as eluent has previously been used for thepuri®cation of THT from elicited potato cell-suspensioncultures (Hohlfeld et al. 1996). However, THT was alsopuri®ed from elicited tobacco cell-suspension cultureswithout an a�nity chromatography step by takingadvantage of the fact that tobacco THT binds tightlyto hydroxylapatite (Negrel and Javelle 1997). Allreported THT puri®cation procedures have also utilizedhydrophobic interaction chromatography as an e�ectivepuri®cation step due to the hydrophobic nature of theenzyme.

The biochemical properties of the puri®ed opiumpoppy enzyme are generally similar to those reported forTHT from potato (Hohlfeld et al. 1995; 1996) andtobacco (Negrel and Javelle 1997), although there arenotable di�erences. Estimates of molecular mass andisoelectric point for native opium poppy THT are in

Fig. 7A±C. Immunological detection of THT polypeptides in variousorgans of mature greenhouse-grown opium poppy plants (A); inopium poppy seedlings grown under sterile conditions (B); and inelicitor-treated opium poppy cell-suspension cultures (C)

Fig. 8A±H. Immunohistochemical localization of THT polypeptidesin opium poppy plants. Excised opium poppy organs were ®xed inFAA, embedded in para�n, and sectioned at a thickness of 10 lmusing a microtome. The depara�nized and rehydrated sections werethen incubated with THT polyclonal antiserum. A Root cross section;B stem cross-section; C leaf cross-section; D cross-section of anther®laments; E cross-section of ovules; F cross-section of anthers; Gcross-section of developing seed; H cross-section of leaf vascularbundle. Abbreviations: cp, cortical parenchyma; ep, epidermis; la,laticifers; nu, nucellus, pd, periderm; ph, phloem conductive elements;po, pollen; sc, inner layer of seed coat; xp, xylem parenchyma; xy,xylem vessel elements. Bars = 0.5 mm

c

40 M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy

M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy 41

agreement with those reported for the potato andtobacco enzymes. The indication from our data thatTHT is a homodimer comprised of two identicalsubunits is consistent with the reported composition oftobacco THT (Negrel and Javelle 1997), whereas potatoTHT was suggested to consist of two di�erent subunitsalmost identical in size (Hohlfeld et al. 1996). Anotherdi�erence among these enzymes is that the potato THTwas reported to be stimulated by Ca2+ (Hohlfeld et al.1995), whereas the opium poppy and tobacco enzymeswere not a�ected by this cation. However, tobacco THTwas reported to be stimulated by ethanol (Negrel andJavelle 1997), but we found no such e�ect with theopium poppy THT. These di�erences could be associ-ated with the growth conditions of the cell-suspensioncultures used as an enzyme source for the puri®cation ofthe respective enzymes. But, it is possible that a calcium-binding subunit could bind a catalytic domain undercertain growth conditions (Roberts and Harmon 1992).Similarly, ethanol could change the conformation of theprotein by interacting with speci®c hydrophobic residues(Pakusch et al. 1991) and could facilitate dissociation ofthe relatively hydrophobic amide reaction product fromthe enzyme (Negrel and Javelle 1997).

The speci®city of opium poppy THT for cinnamoyl-CoA derivatives is similar in some respects to the potatoand tobacco enzymes, but di�erent in others. All threeenzymes showed a marked preference for feruloyl-CoAas the acyl donor, and a moderate acceptance ofsinapoyl-CoA and 4-coumaroyl-CoA. However, ca-�eoyl-CoA was a very poor substrate for THT fromopium poppy and tobacco (Negrel and Javelle 1997), butwas accepted by potato THT (Hohlfeld et al. 1995). Amajor di�erence in substrate speci®city among theseenzymes is that potato and tobacco THT exhibited amuch higher Km for dopamine than tyramine. Incontrast, tyramine (Km 76 lM) and dopamine (Km

78 lM) showed nearly equal a�nity as substrates foropium poppy THT, but the speci®city (V Kÿ1m ) fordopamine was relatively low. Opium poppy, unlikepotato and tobacco, produces copious amounts ofdopamine as a precursor for the biosynthesis of benzyl-isoquinoline alkaloids, such as morphine and sang-uinarine (Stadler et al. 1987). In P. bracteatum, forexample, a large dopamine pool (1±3 mg mL)1) waslocalized to speci®c alkaloid-rich vesicles in the latex ofthe plant and in cultured cells (Roberts et al. 1983).However, the low speci®city of THT for dopamine inopium poppy suggests that the large dopamine pool inthe latex would not be used extensively for hydroxycin-namic acid amide biosynthesis, even if the enzyme hadaccess to the substrate.

The mode of action of THT from potato (Hohlfeldet al. 1995) and tobacco (Negrel and Javelle 1997) wasdetermined to be an ordered bi bi mechanism; thus, thecinnamoyl-CoA derivative binds to the enzyme ®rst, andno products are released before the formation of theternary complex between the enzyme and the twosubstrates. Opium poppy THT followed Michalis-Men-ton kinetics in the presence of low concentrations ofcinnamoyl-CoA derivatives, but negative cooperativity

was detected when the acyl donor concentration washigher than 2±3 lM, which resulted in a signi®cantdecrease in the a�nity for tyramine (data not shown).Negrel and Javelle (1997) have suggested that thisnegative cooperativity, which was also reported fortobacco THT (Hohlfeld et al. 1995), represents a signi-®cant physiological mechanism involved in the regula-tion of hydroxycinnamic acid amide biosynthesis.Cellular concentrations of cinnamoyl-CoA derivativesare probably much lower than those of tyramine anddopamine (Hahlbrock and Scheel 1989). Cooperativitybetween acyl donors and acceptors implies that anincrease in the cellular pool of amines should lead to anincrease in amide formation, even at constant levels ofcinnamoyl-CoA derivatives. An increase in the cellularpool of cinnamoyl-CoA esters would result in increasedformation of amides only if phenethylamine levels alsoincrease; thus, the concentration of tyramine could playa major role in the regulation of amide biosynthesis. Inexperiments with Brassica napus (canola) transformedwith chimeric genes encoding TYDC from opium poppyand driven by the constitutive cauli¯ower mosaic virus35 S promoter, we have observed that an alteration inthe cellular pool of tyramine results in an inversevariation in the digestibility of cell walls possibly due,at least in part, to the formation of amides (data notshown). These data are consistent with proposed regu-latory roles of both THT and TYDC in the biosynthesisof hydroxycinnamic acid amides.

The THT enzyme activity (Fig. 6) and polypeptide(Fig. 7) levels could often be directly correlated, sug-gesting that the developmental and inducible regulationof THT involves de-novo protein synthesis. However,the incongruity between the levels of THT activity(Fig. 6C) and immunoreactive polypeptides (Fig. 7C) inelicitor-treated cell-suspension cultures, after the initialinduction phase, suggests that the THT enzyme isinactivated by a mechanism that does not result inimmediate polypeptide degradation. A similar inactiva-tion mechanism might operate in opium poppy leaves, assuggested by the abundance of THT polypeptides(Fig. 7A), but low levels of enzyme activity (Fig. 6A).Although we were unable to determine a biochemicalfunction for the immunoreactive polypeptides in leaves,we cannot rule out the possible existence of THTisozymes in opium poppy with unique substrate speci-®city. The mouse polyclonal antibodies recognized onlya single polypeptide in all opium poppy total solubleprotein extracts, demonstrating the speci®city of theantiserum; thus, our THT antibodies were an e�ectiveprobe to determine the tissue-speci®c localization ofTHT proteins.

Much of the work on the regulation of THT, andamide biosynthesis, has involved its induction in responseto elicitors in cell-suspension cultures (Villegas andBrodelius 1990; Negrel and Javelle 1995, 1997; Kelleret al. 1996; MuÈ hlenbeck et al. 1996; Facchini 1998;Schmidt et al. 1998). Results shown in Figs. 6±8 demon-strate that THT also plays an important developmentalrole in plants. The biosynthesis and oxidative cross-linking of hydroxycinnamic acid amides in the cell wall

42 M. Yu and P.J. Facchini: Tyramine hydroxycinnamoyltransferase from opium poppy

appear to occur as a normal component of cellulardevelopment (Fry 1986; Iiyama et al. 1994). In opiumpoppy, THT polypeptides were detected in many celltypes such as periderm, cortical parenchyma, and vascu-lar tissues, consistent with the role of hydroxycinnamicacid amides in cell wall maturation and reinforcement.Negrel and Lherminier (1987) reported that radioactivitywas incorporated around necrotic lesions and in xylemcell walls when [14C]tyramine was fed by petiolar uptaketo tobacco leaves infected with tobacco mosaic virus. Wealso detected the presence of THT polypeptides in thexylem parenchyma of both roots and shoots, and in somedeveloping xylem vessel elements in leaves and stems, butnot in mature vessel elements. The high constitutive levelof THT polypeptides in conductive elements of thephloem, in all organs of the plant, shows that hydroxy-cinnamic acid amides also play an important role inphloem development. The lack of THT polypeptides inthe alkaloid- and dopamine-rich laticifers of opiumpoppy is consistent with the low speci®city of the enzymefor dopamine, and con®rms that THT does not haveaccess to the dopamine pool in the latex. As determinedby in-situ hybridization, TYDC mRNAs were found tobe most abundant near laticifers (Facchini and De Luca1995). The high level of TYDC near laticifers would benecessary to maintain the large dopamine pool, and tosupply the precursors for benzylisoquinoline alkaloidbiosynthesis. Lower levels of TYDC would also beexpected in other cell types to provide tyramine for thebiosynthesis of hydroxycinnamic acid amides. The spatialsegregation of TYDC and THT in di�erent cell types ofthe phloem suggests that the major sites for the biosyn-thesis of benzylisoquinoline alkaloids and hydroxycin-namic acid amides in opium poppy are di�erent.

The THT polypeptides were also abundant in theovule nucellus (Fig. 8E), anthers (Fig. 8F), and the innerlayer of the seed coat (Fig. 8G). High levels of THTactivity were also detected in these ¯oral organs and inseeds (data not shown). Hydroxycinnamic acid amideshave been reported to occur in many of the tissues whereTHT polypeptides and enzyme activity were detected(Martin-Tanguy 1985). For example, 4-coumaroyltyr-amine has been identi®ed in anthers of tobacco(Cabanne et al. 1977). Tobacco seeds containferuloyltyramine (Cabanne et al. 1977), and both4-coumaroyl- and feruloyl-tyramine, together with4-coumaroyl- and feruloyl-octapamine were detected inroots of eggplant (Yoshihara et al. 1978). A variety ofamides have also been detected in ¯owers (Martin-Tanguy et al. 1978; Ponchet et al. 1982). It is probablethat the biosynthesis of hydroxycinnamic acid amidesplays the same role in the reinforcement of cell walls inreproductive tissues as it does in other cells of the plant.However, the possibility that amides serve additionalfunctions in the development of anthers, ovules, seeds,and other reproductive structures cannot be ruled out(Martin-Tanguy 1985).

The availability of puri®ed THT protein and THT-speci®c antibodies will facilitate the cloning of THTcDNA and genomic clones from opium poppy that willbe valuable in our continued investigation of the

complex regulation of tyrosine-derived hydroxycinnamicacid amides in opium poppy, and other plants.

We are grateful to Carl Douglas (University of British Columbia)for the pQE19 plasmid and Vincenzo De Luca (Universite deMontre al) for the N-hydroxycinnamoyltyramine standards. Wealso thank Edward Yeung for advice on the interpretation ofphotomicrographs, and Doug Muench for assistance with the two-dimensional gel electrophoresis. Research was supported by grantsfrom the Natural Sciences and Engineering Research Council(NSERC) of Canada and the Alberta Agricultural ResearchInstitute (AARI) to P.J.F.

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