expression patterns conferred by tyrosine ...opium poppy (papaver somniferum cv marianne) and...

Expression Patterns Conferred byTyrosine/Dihydroxyphenylalanine Decarboxylase Promotersfrom Opium Poppy Are Conserved in Transgenic Tobacco1

Peter J. Facchini*, Catherine Penzes-Yost, Nailish Samanani, and Brett Kowalchuk

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

Opium poppy (Papaver somniferum) contains a large family oftyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes in-volved in the biosynthesis of benzylisoquinoline alkaloids and cellwall-bound hydroxycinnamic acid amides. Eight members from twodistinct gene subfamilies have been isolated, tydc1, tydc4, tydc6,tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in theother. The tydc8 and tydc9 genes were located 3.2 kb apart on onegenomic clone, suggesting that the family is clustered. Transcriptsfor most tydc genes were detected only in roots. Only tydc2 andtydc7 revealed expression in both roots and shoots, and TYDC3mRNAs were the only specific transcripts detected in seedlings.TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, werenot detected in elicitor-treated opium poppy cultures. Expression oftydc4, which contains a premature termination codon, was notdetected under any conditions. Five tydc promoters were fused tothe b-glucuronidase (GUS) reporter gene in a binary vector. Allconstructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cellcultures. The organ- and tissue-specific expression pattern of tydcpromoter-GUS fusions in transgenic tobacco was generally parallelto that of corresponding tydc genes in opium poppy. GUS expres-sion was most abundant in the internal phloem of shoot organs andin the stele of roots. Select tydc promoter-GUS fusions were alsowound induced in transgenic tobacco, suggesting that the basicmechanisms of developmental and inducible tydc regulation areconserved across plant species.

Opium poppy (Papaver somniferum) remains an econom-ically important medicinal plant, because it is the onlycommercial source of several pharmaceutical alkaloids, in-cluding the analgesics morphine, codeine, and thebaine.The biosynthesis of these and other benzylisoquinolinealkaloids begins with the condensation of dopamine and4-HPAA to form the first committed alkaloid intermediate,(S)-norcoclaurine (Stadler et al., 1987). More than 2500benzylisoquinoline alkaloids have been isolated from fivemajor plant families, and all are derived from (S)-norcoclaurine (Stadler et al., 1987). Both dopamine and4-HPAA are simple derivatives of Tyr, but their synthesishas not been unequivocally characterized. The synthesis ofdopamine could result from either the decarboxylation of

dihydroxyphenylalanine or from the hydroxylation of tyra-mine, which is the product of Tyr decarboxylation (Ruefferand Zenk, 1987). The capacity of TYDC to decarboxylateboth Tyr and dihydroxyphenylalanine (Facchini andDe Luca, 1994, 1995a) suggests that dopamine might besynthesized by both routes. Similarly, the synthesis of4-HPAA could result from either the decarboxylation of4-hydroxyphenylpyruvate or the oxidation of tyramine(Rueffer and Zenk, 1987). Therefore, TYDC is probablyinvolved in the formation of both dopamine and 4-HPAAand could play a key role in the regulation of benzyliso-quinoline alkaloid biosynthesis (Fig. 1).

TYDC cDNAs have been reported from parsley (Kawal-leck et al., 1993), Arabidopsis (Trezzini et al., 1993), andopium poppy (Facchini and De Luca, 1994). In each case,TYDC mRNAs were shown to be rapidly and transientlyinduced in response to elicitor treatment (Kawalleck et al.,1993; Trezzini et al., 1993; Facchini et al., 1996) and patho-gen challenge (Schmelzer et al., 1989). Induction of TYDCmRNAs in plant species that do not accumulate Tyr-derived alkaloids, such as parsley and Arabidopsis, sug-gests that Tyr serves as the precursor to a ubiquitous classof plant defense-response metabolites. Recent studies sug-gest that the biosynthesis and deposition in the cell wall ofamides composed of hydroxycinnamic acid derivativesand tyramine are central to the defense response of manyplants (Negrel and Martin, 1984; Negrel and Jeandet, 1987;Negrel and Lherminier, 1987; Negrel et al., 1993, 1995;Negrel and Javelle, 1995). Amides, together with other cellwall-bound phenolics, are believed to reduce cell wall di-gestibility and/or directly inhibit pathogen growth. Hy-droxycinnamoyltyramines have been isolated from a vari-ety of plant species (Martin-Tanguy et al., 1978). Recently,the conversion of aromatic amines to both alkaloids andamides in elicitor-treated opium poppy cultures was dem-onstrated (Facchini, 1998). Tyramine hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase, which cata-lyzes the condensation of hydroxycinnamoyl-CoA andtyramine (Fig. 1), has been purified and characterized intobacco (Nicotiana tabacum; Negrel and Martin, 1984;Negrel and Javelle, 1997) and potato (Hohlfeld et al., 1995,1996) and isolated in opium poppy (Facchini, 1998).

1 This work was supported by grants from the Natural Sciencesand Engineering Research Council of Canada and the AlbertaAgricultural Research Institute to P.J.F.

* Corresponding author; e-mail [email protected]; fax1– 403–289 –9311.

Abbreviations: CaMV, cauliflower mosaic virus; 4-HPAA, 4-hydroxyphenylacetaldehyde; NOS, nopaline synthase; NPT II,neomycin phosphotransferase; ORF, open reading frame; TYDC,Tyr/dihydroxyphenylalanine decarboxylase.

Plant Physiol. (1998) 118: 69–81

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The tydc gene family in opium poppy contains approxi-mately 15 members that can be divided into two subfami-lies based on sequence identity and represented by tydc1and tydc2 (Facchini and De Luca, 1994). Each subfamily oftydc genes exhibits distinct and tissue-specific patterns ofdevelopmental and inducible expression in opium poppyplants and elicited cell cultures (Facchini and De Luca,1994, 1995b; Facchini et al., 1996). The dual role of tyramineas a precursor for benzylisoquinoline alkaloid and hy-droxycinnamic acid amide biosynthesis and the demon-stration that tydc genes are involved in the defense re-sponses of many plants suggest that different members ofthe large tydc gene family in opium poppy might displaycomplex patterns of regulation and diverse metabolic roles.

In an attempt to better understand the regulation of tydcgenes in plants, in this paper we describe the organizationand gene-specific patterns of developmental and inducibleexpression of eight tydc genes from opium poppy. Theregulation of five opium poppy tydc gene promotors isfurther studied in transgenic tobacco plants that containtydc promoter-GUS fusions. Our results show that mem-bers of the tydc gene family exhibit different patterns ofdevelopmental and inducible expression and that thetissue-specific and wound-induced regulation of tydc pro-moters is conserved across plant species. The highly par-allel regulation of opium poppy tydc promoters in tobaccosuggests that TYDC plays significant and ubiquitous rolesin both the development and defense response of plants.

MATERIALS AND METHODS

Plants and Cell Cultures

Opium poppy (Papaver somniferum cv Marianne) andtobacco (Nicotiana tabacum cv Xanthi) plants were grownunder greenhouse conditions at a day/night temperature

of 20°C/18°C. Seedlings were grown at 23°C in sterile Petriplates containing moist filter paper. Seeds were surfacesterilized with 20% (v/v) bleach for 15 min, washed thor-oughly with sterile, distilled water, and allowed to imbibewater for 24 h (d 0). Seeds were kept in the dark for 3 dfollowing imbibition and were then transferred to a pho-toperiod of 16 h light/8 h dark.

Opium poppy and tobacco cell-suspension cultures weremaintained in diffuse light at 23°C on 1B5C medium (Gam-borg et al., 1968) consisting of B5 salts and vitamins plus100 mg L21 myo-inositol, 1 g L21 hydrolyzed casein, 20 gL21 Suc, and 1 mg L21 2,4-D. Cells were subcultured every6 d using a 1:4 dilution of inoculum to fresh medium.Cultured cells in rapid growth phase (2–3 d after subcul-ture) were used for all experiments.

Elicitor Treatment of Opium Poppy Cell Cultures

Fungal elicitor was prepared from Botrytis sp. accordingto the method of Eilert et al. (1985). A 1-cm2 section ofmycelia grown on potato dextrose agar was cultivated in 50mL of 1B5C medium, including supplements but excluding2,4-D, on a gyratory shaker (120 rpm) at 22°C in the darkfor 6 d. Mycelia and medium were homogenized with aPolytron (Brinkmann), autoclaved (121°C) for 20 min, andsubsequently centrifuged under sterile conditions with thesupernatant serving as elicitor. Elicitor treatments wereinitiated by the addition of 1 mL of fungal homogenate per50 mL of cell culture. Cells were subsequently collected byvacuum filtration, frozen in liquid N2, and stored at 280°C.

Genomic Library Construction and Screening

A lEMBL3 (Stratagene) library was constructed fromopium poppy genomic DNA partially digested with MboI(Sambrook et al., 1989). A primary library of 1.1 3 107

plaques was obtained (Facchini and De Luca, 1994), and2.5 3 109 plaques of the amplified library were indepen-dently screened at high stringency, as described below,with random-primer 32P-labeled probes synthesized fromthe full-length coding region of the TYDC1 and TYDC2cDNAs from opium poppy (Facchini and De Luca, 1994).Isolated lEMBL3 genomic clones for tydc3, tydc6, tydc7,tydc8, and tydc9 were subcloned into pBluescript andmapped for restriction endonuclease cleavage sites andgene location.

Isolation and Analysis of Nucleic Acids

Genomic DNA was isolated from young leaves of opiumpoppy plants (Murray and Thompson, 1980). Total RNAfor gel-blot analysis was isolated according to the methodof Logemann et al. (1987), and 15 mg was fractionated on1.0% formaldehyde agarose gels before transfer to nylonmembranes (Sambrook et al., 1989). RNA gel blots werehybridized with random-primer 32P-labeled (Feinberg andVogelstein, 1984) full-length probes for TYDC1 and TYDC2or gene-specific probes for tydc1/tydc8, tydc2/tydc7, tydc3,tydc4, tydc6, tydc7, and tydc9. A list of oligonucleotide prim-ers used to isolate gene-specific 39 flanking regions by PCR

Figure 1. Schematic representation of the early steps in the biosyn-thetic pathways leading to benzylisoquinoline alkaloids and hydroxy-cinnamic acid amides of tyramine showing the sites of action ofkey gene products. NS, (S)-Norcoclaurine synthase; THT, tyraminehydroxycinnamoylCoA:tyramine hydroxycinnamoyltransferase; Dopa,dihydroxyphenylalanine.

70 Facchini et al. Plant Physiol. Vol. 118, 1998

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is shown in Table I. Hybridizations were performed at 65°Cin 0.25 m sodium phosphate buffer, pH 8.0, 7% (w/v) SDS,1% (w/v) BSA, and 1 mm EDTA. Blots were washed at65°C, twice with 23 SSC and 0.1% (w/v) SDS and twicewith 0.23 SSC and 0.1% (w/v) SDS (Sambrook et al., 1989;13 SSC 5 0.15 m NaCl, 0.015 m sodium citrate, pH 7.0), andautoradiographed with an intensifying screen at 280°Cfor 48 h.

Double-stranded DNA was sequenced using thedideoxynucleotide chain-termination method (Sanger etal., 1977) and a recombinant T7 DNA polymerase (UnitedStates Biochemical). Sequences were aligned using theFASTA program package (Pearson and Lipman, 1988).

Transient Expression and Stable Transformation Vectors

A binary vector, designated pBI 102, was used to con-struct promoter-GUS fusions for transient expression as-says in microprojectile-bombarded cell cultures and for thestable transformation of tobacco. Restriction sites for ApaI,XhoI, and KpnI were included in pBI 102 by inserting anadapter fragment into the SmaI site of pBI 101 (Jefferson etal., 1987). Binary vectors were maintained in Escherichia colistrain DH10b and mobilized in Agrobacterium tumefaciensstrain LB4404 by direct DNA transfer (An, 1987).

Promoter-GUS Constructs

Promoters of tydc3, tydc6, tydc7, tydc8, and tydc9 wereamplified by PCR using specific primers designed to add aHindIII and either a BamHI or XhoI restriction site at the 59and 39 ends, respectively, of each promoter fragment. A listof oligonucleotide primers used to isolate and subclone 59

flanking regions is shown in Table II. The isolated tydc3,tydc6, tydc7, tydc8, and tydc9 promoters extended approxi-mately 3.5, 3.0, 1.2, 1.2 and 2.1 kb, respectively, upstream ofthe putative translation start codon in each gene. The PCR-generated tydc3, tydc6, tydc8, and tydc9 promoter fragmentswere inserted into pBI 102 between the HindIII and BamHIsites to yield the TYDC3::GUS, TYDC6::GUS, TYDC8::GUS,and TYDC9::GUS constructs, whereas the tydc7 promoterfragment was inserted into pBI 102 between the HindIIIand XhoI sites to yield the TYDC7::GUS construct. Theassembly of all constructs was verified by sequencingthrough the promoter-GUS junction. The CaMV 35Spromoter-GUS fusion in pBI 121 (Jefferson et al., 1987) andthe promoterless pBI 102 vector were used as positive andnegative controls, respectively.

Transient luciferase activity was introduced into cul-tured cells using pCaLucNOS, which harbors the CaMV35S promoter fused to the luciferase-coding region, fol-lowed by the NOS polyadenylation signal in pUC 19. Allplasmids were purified before microprojectile bombard-ment by PEG precipitation, phenol/chloroform extraction,LiCl precipitation, and RNase digestion and then wereextracted again with phenol/chloroform and precipitatedwith ethanol.

Microprojectile Bombardment of Cultured Cells

Gold particles (60 mg, 1.6 mm in diameter, Bio-Rad) weresterilized by vortexing in 1 mL of 100% ethanol for 5 min,washed twice with sterile, distilled water, and resuspendedin 1 mL of sterile, distilled water. A 50-mL aliquot of thesuspension was removed and 15 mg of each plasmid DNA,50 mL of 2.5 m CaCl2, and 20 mL of 0.1 m spermidine wereadded successively. The gold particles were incubated onice for 5 min after each addition. The mixture was thenvortexed at room temperature for 4 min, washed twicewith ethanol, and resuspended in 45 mL of 100% ethanol.For each bombardment, 15 mL of the particle suspension (1mg of particles per shot) was pipetted onto microcarriers,sterilized with 100% ethanol, and used after all of theethanol had evaporated.

Cultured cells were collected on microfiber filters(GF/D, Whatman) by gentle vacuum filtration to form athin cell layer approximately 2 cm in diameter. Filterscontaining the plant cells were placed in sterile Petri platesand positioned below a microprojectile-stopping screen.Bombardments were performed using a particle-accel-eration device (PDS 1000/He, Bio-Rad) under a chamberpressure of 26 mm of Hg, at a distance of 1.5, 2.0, and 6.5cm from the rupture disc to the microcarriers to the stop-ping screen to the target, respectively, and at a He pressureof 1100 p.s.i. After bombardment, the cultured cell layerswere incubated at 23°C in the sterile Petri plates. Elicitortreatments 24 h after bombardment consisted of the addi-tion of 0.25 mL of either the Botrytis sp. elicitor or a solutionof 0.3 mg mL21 cellulase to opium poppy and tobacco cells,respectively.

Table I. Oligonucleotide primers used to amplify gene-specific 39flanking regions from tydc1/tydc8, tydc2/tydc7, tydc3, tydc4,tydc6, and tydc9a

Name Sequence Product Sizeb

bp

TYDC1/8-1c TCC TGG GAT TAA CAG AAA 113TYDC1/8-2 GAA ATG AAT GGT AAT TTCTYDC2/7-1 AGG AAC GCC ATG TTA TTC 400TYDC2/7-2 CAT AAC ATC TAA CAT TAATYDC3-1 CGC GTA TCT TGT GGT TGA 320TYDC3-2 CTG ATG AAC AAC TTT GGTTYDC4-1 GGT ACA GCG GAG ATG AAG 177TYDC4-2 TGT TTC CTT AGG CTC AACTYDC6-1 GGA GAG AGA AAG GAA ACG 280TYDC6-2 TAC GAT GCA AAC GGA ACCTYDC9-1 ACA GAT GCC ATA CTT GGT 191TYDC9-2 ATA AGT AGT TAT GAG TAT

a Nucleotide sequences of the 59 and 39 flanking regions of tydc1and tydc8 were identical; thus, it was not possible to isolate probesthat could distinguish between these genes. The same was true fortydc2 and tydc7. b The product size is that resulting from PCRusing the matching set of sense and antisense primers with thecorresponding template. c Designation of the oligonucleotide as“-1” indicates the sense primer, whereas “-2” indicates the antisenseprimer.

Regulation of Opium Poppy tydc Promoters in Tobacco 71



Tobacco Transformation

Transformation of tobacco with tydc promoter-GUS con-structs in pBI 102 was performed with the A. tumefaciensstrain LB4404 using the leaf disc method (Horsch et al.,1985). All binary vectors used in this study harbor theNOS::NPT II gene in the T-DNA region, which confersresistance to kanamycin in transgenic plants (Jefferson etal., 1987). Tobacco plants were regenerated from transgeniccalli according to standard protocols (Rogers et al., 1986),transferred to soil, self-pollinated, and allowed to set seed.Transformation of kanamycin-resistant plants was verifiedby PCR using gene-specific primers (Table I) and by directassay for NPT II enzyme activity (Radke et al., 1988).

Wounding of Transgenic Tobacco Plants

Young leaves from transgenic tobacco plants grown invitro were placed on two layers of moist filter paper insterile Petri plates. Wounding was performed by punctur-ing leaves with sterile pins approximately once per squaremillimeter. Wounded and control samples were collectedafter 48 h.

GUS and Luciferase Assays

Transgenic tobacco tissues and cultured cells collectedby vacuum filtration 48 h after microprojectile bombard-ment were ground with extraction buffer consisting of 50mm KPO4 buffer, pH 7.0, 1 mm EDTA, and 10 mmb-mercaptoethanol. The GUS fluorometric assay bufferconsisted of 50 mm NaPO4 buffer, pH 7.0, 10 mmb-mercaptoethanol, 10 mm EDTA, 0.1% (w/v) sodiumlauryl sarcosine, and 0.1% (w/v) Triton X-100.4-Methylumbelliferyl-b-d-glucuronide was added at a fi-nal concentration of 0.44 mg mL21. Assays were per-formed on 80 mL of bombarded cell culture extract for 3 hat 37°C and stopped with a 103 volume of 0.2 m Na2CO3.

A fluorescence spectrophotometer (model F-2000, Hitachi,Tokyo, Japan) was used to quantify the amount of4-methylumbelliferone cleaved from 4-methylumbelliferyl-b-d-glucuronide.

The luciferase assay buffer consisted of 25 mm Tricine,pH 7.8, 15 mm MgCl2, 5 mm ATP, 0.5 mg mL21 BSA, and7 mm b-mercaptoethanol. Bombarded cell extract (20 mL)was mixed with 200 mL of assay buffer and incubated atroom temperature for 15 min (de Wet et al., 1987). Luciferin(100 mL of 0.5 mm diluted with 1 mm Tricine, pH 7.8, from10 mm stock, Boehringer Mannheim) was injected into thereaction mixture, and the light emitted within the first 10 swas quantified using a luminometer (Monolight 2010, An-alytical Luminescence Laboratories, San Diego, CA). Theprotein concentration was determined by the method ofBradford (1976) using BSA as a standard.

GUS Histochemical Staining

GUS activity was localized histochemically by standardprotocols (Jefferson, 1987; Martin et al., 1992). Hand-sectioned tissues or whole plant parts were fixed in a 0.35%(v/v) formaldehyde solution containing 10 mm Mes, pH7.5, and 300 mm mannitol for 1 h at 20°C, rinsed threetimes in 50 mm sodium phosphate, pH 7.5, and subse-quently incubated in 50 mm sodium phosphate, pH7.5, 2 mm 5-bromo-4-chloro-3-indolyl-b-d-glucuronide cy-clohexylammonium salt, and 20% (v/v) methanol for 6 to12 h at 37°C. Stained tissues were rinsed extensively in 70%ethanol to remove chlorophyll.

RESULTS

Structure and Organization of tydc Genes inOpium Poppy

Screening an opium poppy lEMBL3 genomic librarywith TYDC1 and TYDC2 cDNAs (Facchini and De Luca,

Table II. Oligonucleotide primers used to amplify and subclone 59 flanking regions from tydc3,tydc6, tydc7, tydc8, and tydc9

Name Sequencea Product Sizeb

kb

TYDC3-3c GGG GGG GGA TCC GAA GAA GAA AGA GAG GTG GT 3.5TYDC3-4 GGG GGG AAG CTT CTG TGT GCC AAC CCG CGA TATYDC6-3 GGG GGG GGA TCC TTG CTG ATT AGT GAG GGA GA 3.0TYDC6-4 GGG GGG AAG CTT ATA GAA GTT GTT GGG AGA TATYDC7-3 GGG GGG CTC GAG CAG GTG AAA GAA GGT TAT TG 1.2TYDC7-4 GGG GGG AAG CTT TTA TCC ACA CCC AAC TCA TCTYDC8-3 GGG GGG GGA TCC CGT TAC TAT CAG TTT TGA TG 1.2TYDC8-4 T3 primerd

TYDC9-3 GGG GGG GGA TCC TGT TAC TGG TTT TGC TAA TG 2.1TYDC9-4 GGG GGG GTC GAC CAA ATG AGG ACC CAA ATC TGa Underlined sequences represent restriction endonuclease sites introduced into each primer to

allow efficient cloning of the PCR product into the appropriate vector. b The product size is thatresulting from PCR using the matching set of sense and antisense primers with the correspondingtemplate. c Designation of the oligonucleotide as “-3” indicates the sense primer, whereas “-4”indicates the antisense primer. d The antisense primer used to amplify the 59 flanking region oftydc8, subcloned into the XhoI site, was the standard pBluescript T3 primer. The HindIII site from thepBluescript polylinker was used for ligation of the PCR product.




1994) resulted in the isolation of genomic clones that con-tained five homologous full-length genes designated tydc3,tydc6, tydc7, tydc8, and tydc9 (Fig. 2). Nucleotide sequenceanalysis showed that tydc3 was identical to a previouslyreported partial TYDC3 cDNA (Facchini and De Luca,1994). The coding region of tydc3 was highly homologousto tydc7 (93% identity), and both genes shared extensivenucleotide identity with the ORF of the TYDC2 cDNA (93%for tydc3 and 97% for tydc7). The tydc6, tydc8, and tydc9genes displayed strong nucleotide identity with the ORF ofthe TYDC1 cDNA (96% for tydc6, 93% for tydc8, and 93%for tydc9). In contrast, ORF alignments between any mem-ber of the tydc1, tydc6, tydc8, and tydc9 subfamily with anymember of the tydc2, tydc3, or tydc7 subfamily revealedonly 70% to 73% identity. None of the ORFs of isolated tydcgenes was interrupted by intervening sequences, suggest-ing that all members of the gene family lack introns.

The complete genes for tydc8 and tydc9 were localized onone genomic clone with inversely oriented transcriptionunits (Fig. 2). The ORFs were separated by a 3.2-kb DNAsegment that contained both of the putative tydc8 and tydc9promoters. Transcription would be expected to initiate inopposite directions. Despite the extensive homology be-tween the ORFs of tydc8 and tydc9, the 59 and 39 flankingsequences were highly divergent. In contrast, the 59 and 39flanking regions of tydc8 were identical to those of theTYDC1 cDNA. Similarly, the 59 and 39 flanking regions oftydc7 were identical to those of the TYDC2 cDNA. Align-ment of predicted amino acid sequences for tydc3, tydc6,tydc7, tydc8, and tydc9 with those for tydc4 (Facchini and DeLuca, 1994), tydc5 (Maldonado-Mendoza et al., 1996), andthe TYDC1 and TYDC2 cDNAs showed that all reportedmembers of the tydc gene family from opium poppy shareextensive homology with one of two subfamilies that canbe represented by tydc1 and tydc2 (Fig. 3). These datademonstrate that the large tydc gene family in opiumpoppy is at least partially clustered and probably evolvedas the result of extensive duplication of two relativelydivergent ancestral genes.

Developmental and Inducible Expression of Individualtydc Genes in Opium Poppy Plants and Cell Cultures

Full-length TYDC1 and TYDC2 cDNAs are sufficientlydifferent in nucleotide sequence to prevent cross-hybridization when used as probes for RNA gel-blot hy-bridization analyses (Facchini and De Luca, 1994). Suchprobes were used previously to show that tydc gene sub-families in opium poppy display development-specific ex-pression in the plant and temporal-specific expression inelicitor-treated cell cultures (Facchini and De Luca, 1994,1995b; Facchini et al., 1996). In mature opium poppy plants,TYDC1-like genes are predominantly expressed in roots,whereas TYDC2-like genes are expressed in both roots andstems. The levels of individual TYDC transcripts in variousorgans from opium poppy plants are shown in Figure 4.Gene-specific probes were isolated from 39 flanking regionsof tydc3, tydc4, tydc6, and tydc9. However, the untranslated39 and 59 flanking regions of the TYDC1 cDNA and tydc8gene were identical, as were the 39 and 59 flanking regionsof the TYDC2 cDNA and tydc7 gene. Therefore, the 39flanking region used as a tydc1/8-specific probe could notdiscriminate between TYDC1 and TYDC8 transcripts, andthe 39 flanking region used as a tydc2/7-specific probe couldnot discriminate between TYDC2 and TYDC7 transcripts.Using these probes, we found that TYDC1/8 and TYDC3mRNAs occurred abundantly and specifically in opiumpoppy roots (Fig. 4). Lower levels of TYDC6 and TYDC9transcripts were also detected only in roots, whereasTYDC2/7 mRNAs occurred at detectable levels in bothstems and roots. TYDC4 transcripts were not detected inany plant organ or tissue.

TYDC2-like but not TYDC1-like mRNAs were detectedat low levels in developing opium poppy seedlings (Fig. 5).The maximum relative abundance of TYDC2-like mRNAsoccurred 3 d postimbibition and then decreased steadily.Using gene-specific probes, we detected only TYDC3mRNAs by northern-blot hybridization analysis in devel-oping opium poppy seedlings. TYDC2/7 transcripts werenot detected at any stage of seedling development (Fig. 5).

Figure 2. Structural and restriction endonuclease maps for regions of genomic clones containing the tydc3, tydc6, tydc7,tydc8, and tydc9 genes from opium poppy. The open boxes represent ORFs and the bent arrows show the approximatelocation and direction of transcription initiation. The horizontal brackets show the regions amplified by PCR and used asgene-specific probes. B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Sp, SpeI; Xb, XbaI; Xh, XhoI.




TYDC1/8, TYDC4, TYDC6, and TYDC9 transcripts werealso not detected (data not shown).

TYDC1- and TYDC2-like genes also exhibit differentialand temporal-specific expression in elicitor-treated opiumpoppy cell cultures (Facchini et al., 1996). TYDC1-likegenes were activated rapidly after the addition of elicitor,reaching maximum levels between 1 and 2 h and thenrapidly declining (Fig. 6). In contrast, TYDC2-like mRNAsaccumulated more slowly, reaching maximum levels 10 hafter elicitor treatment, but were maintained at a high levelfor an extended period. Using gene-specific probes, wefound that tydc3 was abundantly expressed and tydc2/7 andtydc6 were expressed at lower levels in elicitor-treatedopium poppy cultures (Fig. 6). TYDC1/8, TYDC4, andTYDC9 transcripts were not detected in response to elicitortreatment (Fig. 6).

Transient Expression of Opium Poppy tydc Promoter-GUSFusions in Cultured Cells

The relative activity of isolated promoter regions fromfive opium poppy tydc genes was tested directly by mea-suring the transient expression of tydc promoter-GUS fu-sions in microprojectile-bombarded opium poppy cell cul-tures. CaMV 35S promoter-GUS and promoterless-GUSconstructs were used as positive and negative controls,respectively. As shown in Figure 7A, all of the tydcpromoter-GUS fusions produced higher levels of GUS ac-tivity in bombarded cell cultures than the CaMV 35S-GUSconstruct. The tydc3 promoter directed the highest level ofGUS expression, which was 10-fold greater than the CaMV35S promoter (Fig. 7A). The tydc9, tydc7, tydc6, and tydc8promoters produced levels of GUS activity that were ap-proximately 5-, 3-, 2-, and 1.5-fold higher, respectively,than the CaMV 35S promoter. GUS activity was not de-tected in opium poppy cells bombarded with thepromoterless-GUS construct (Fig. 7A).

Transient expression of tydc promoter-GUS fusions wasalso tested in cultured tobacco cells (Fig. 7B). The relativepattern of GUS activity produced by tydc promoters inbombarded tobacco cultures was qualitatively similar butquantitatively lower than in opium poppy cultures. Thetydc3 promoter directed the highest level of GUS expres-sion in tobacco cells, which was 10-fold greater than theCaMV 35S promoter but 2-fold lower than the level pro-duced by the tydc3 promoter-GUS fusion in opium poppycells (Fig. 7B). Relative GUS activities produced by thetydc7 and tydc9 promoters in tobacco cultures were 2-foldhigher and 4-fold lower, respectively, than in opium poppycultures. The CaMV 35S-GUS construct also produced ap-proximately 2-fold lower GUS activity in tobacco cells.

Figure 3. Alignment of predicted amino acid sequences from iso-lated members of the tydc gene family in opium poppy. Amino acidsequences for TYDC1, TYDC2, and TYDC4 were reported by Fac-chini and De Luca (1994). The asterisk at position 56 in the TYDC4sequence represents a premature termination codon. The TYDC5amino acid sequence was also reported previously (Maldonado-Mendoza et al., 1996).




Cultured cells bombarded with select tydc promoter-GUS constructs showed an increase in GUS activity afterelicitor treatment relative to untreated controls (data notshown). GUS activity in both opium poppy and tobaccocells bombarded with the TYDC3::GUS, TYDC6::GUS, orTYDC7::GUS constructs increased between 1.5- and 2-foldafter addition of elicitor. GUS activity was not significantlyaffected by elicitor treatment in cell cultures bombarded

with the TYDC8::GUS or TYDC9::GUS constructs or withthe 35S::GUS control. It should also be noted that theBotrytis sp. and cellulase elicitors were effective with onlyopium poppy and tobacco cultures, respectively.

Cell cultures were co-bombarded with pCaLucNOS sothat GUS activity could be normalized against luciferaseactivity to account for differences in expression efficiencybetween bombardments. The specific luciferase activitywas similar for each bombardment.

Expression of Opium Poppy tydc Promoter-GUS Fusions inTransgenic Tobacco

The binary vectors harboring the tydc promoter-GUSfusions used to test promoter activity by transient expres-sion analysis in opium poppy and tobacco cell cultureswere mobilized in A. tumefaciens and used for tobaccotransformation. Transgenic tobacco plants resistant tokanamycin were tested for the presence of transgenes byPCR and by direct NPT II enzyme assay. GUS activity wasmeasured in 10-d transgenic seedlings and in plants grownin vitro or under greenhouse conditions. Results presentedin Figure 8 represent the mean and se of triplicate mea-surements on each of three independent transgenic linesfor each construct. The CaMV 35S promoter-GUS fusion,used as a positive control, exhibited strong GUS activity inall transgenic tobacco organs and in seedlings. The tydc3promoter-GUS fusion resulted in weak GUS activity inroots and only very low levels of activity in shoot organs

Figure 4. RNA gel-blot hybridization analysis for various members ofthe tydc gene family in mature opium poppy organs. Fifteen micro-grams of total RNA was fractionated on 1.0% formaldehyde agarosegels, transferred to nylon membranes, and hybridized at high strin-gency with 32P-labeled full-length probes for tydc1 and tydc2 orgene-specific probes for tydc1/8, tydc2/7, tydc3, tydc4, tydc6, andtydc9. To ensure equal loading, gels were stained with ethidiumbromide before blotting.

Figure 5. RNA gel-blot hybridization analysis for various members ofthe tydc gene family during opium poppy seedling development.Fifteen micrograms of total RNA was fractionated on 1.0% formal-dehyde agarose gels, transferred to nylon membranes, and hybrid-ized at high stringency with 32P-labeled full-length probes for tydc1and tydc2 or gene-specific probes for tydc2/7 and tydc3. To ensureequal loading, gels were stained with ethidium bromide beforeblotting.




and seedlings (Fig. 8). The tydc6, tydc8, and tydc9 promoter-GUS fusions produced moderate to high levels of GUSactivity in young transgenic tobacco roots, but only lowlevels of GUS activity were detected in shoot organs orseedlings. Among these, the level of GUS activity washighest in young roots of plants transformed withTYDC8::GUS and lowest in plants transformed withTYDC9::GUS (Fig. 8). The tydc7 promoter-GUS fusion pro-duced strong GUS activity in all transgenic tobacco organsand in seedlings (Fig. 8).

Select tydc promoter-GUS fusions exhibited wound-induced expression in young transgenic tobacco leaves(data not shown). GUS activity increased between 3- and

5-fold in wounded TYDC3::GUS, TYDC6::GUS, andTYDC7::GUS tobacco relative to unwounded controls (Fig.8). In contrast, no significant change in GUS activity oc-curred in response to wounding in TYDC8::GUS,TYDC9::GUS, or 35S::GUS plants.

Histochemical staining for GUS activity showed that thedevelopmental expression of all tydc promoter-GUS fu-sions was concentrated in the vascular tissues of all trans-genic tobacco organs (Fig. 9). Young stems and petiolesfrom TYDC7::GUS tobacco showed GUS activity restrictedto the internal phloem (Fig. 9, A and B). GUS activity wasabsent from mature stems and petioles that exhibited sub-stantial secondary growth. Petioles of TYDC7::GUS tobaccoalso showed GUS activity in an adaxial layer of cortex (Fig.9B). Leaves from TYDC7::GUS plants displayed a similar

Figure 6. RNA gel-blot hybridization analysis for various members ofthe tydc gene family in elicitor-treated opium poppy cell-suspensioncultures. Fifteen micrograms of total RNA was fractionated on 1.0%formaldehyde agarose gels, transferred to nylon membranes, andhybridized at high stringency with 32P-labeled full-length probes fortydc1 and tydc2 or gene-specific probes for tydc1/8, tydc2/7, tydc3,tydc4, tydc6, and tydc9. To ensure equal loading, gels were stainedwith ethidium bromide before blotting.

Figure 7. Activity of various tydc gene promoters determined bytransient expression of promoter-GUS fusions in opium poppy (A)and tobacco (B) cell cultures. Bars represent normalized GUS activityin cultured cells 48 h after microprojectile bombardment with thefollowing constructs: pBI 102 (promoterless), 35S::GUS (CaMV 35Spromoter), TYDC3::GUS (tydc3 promoter), TYDC6::GUS (tydc6 pro-moter), TYDC7::GUS (tydc7 promoter), TYDC8::GUS (tydc8 promot-er), and TYDC9::GUS (tydc9 promoter). Values represent themeans 6 SE of three independent experiments whereby cultured cellswere co-bombarded with promoter-GUS and CaMV 35S-luciferaseconstructs and GUS activity was normalized against luciferase activ-ity. MU, 4-Methylumbelliferone.




pattern of expression, with the strongest GUS activity lo-calized in veins and lower levels of activity detected inmesophyll tissue between veins (data not shown).TYDC7::GUS roots showed the strongest staining for GUSactivity in dividing meristematic tissues (Fig. 9C). As rootdevelopment proceeded through the zones of elongationand maturation, GUS activity become progressively re-stricted to the stele (Fig. 9C). GUS activity was significantlyreduced but still restricted to vascular tissues in older roots(data not shown). In contrast, strong GUS activity wasclearly detected in TYDC7::GUS tobacco during the earlystages of lateral root development (Fig. 9D). TYDC7::GUSplants were the only transformants that showed significantGUS activity in shoot organs and in meristematic regions ofroots. Strong GUS activity was also detected in the cotyle-dons, shoot apical meristem, root meristem, and develop-ing root vascular tissues of TYDC7::GUS seedlings (Fig. 9E).

TYDC6::GUS, TYDC8::GUS, and TYDC9::GUS tobaccoshowed similar patterns of localization, with GUS activitystrictly localized in root vascular tissues. In contrast toTYDC7::GUS plants, strong GUS activity was detected inthe stele of the main root axis in TYDC6::GUS plants (Fig.9F). Examination of the root-shoot transition zone ofTYDC6::GUS plants showed that GUS activity in the steleterminated as vascular bundles emerged in the stem (Fig.9F). Also, unlike TYDC7::GUS plants, GUS activity wasabsent in dividing meristematic tissues of TYDC6::GUSroots (Fig. 9G).

DISCUSSION

TYDC clones and/or enzyme activity have been isolatedin several plant species, including opium poppy (Facchiniand De Luca, 1994), Eschscholtzia californica, Thalictrumrugosum (Marques and Brodelius, 1988), Sanguinaria cana-densis (Chapple et al., 1986), parsley (Kawalleck et al.,1993), Arabidopsis (Trezzini et al., 1993), Cytisus scoparius(Tocher and Tocher, 1972), and barley (Hosoi et al., 1970;

Hosoi, 1974). Although TYDC is common, perhaps evenubiquitous, in plants, its metabolic roles have not beenfully characterized and its physiological importance is notwell understood. TYDC is involved in the biosynthesis ofcell wall-bound amides and, in select species includingopium poppy, E. californica, T. rugosum, and S. canadensis, inthe biogenesis of benzylisoquinoline alkaloids. The recentisolation of a jasmonic acid conjugate of tyramine suggestsadditional roles for TYDC (Miersch et al., 1998).

Unlike parsley (Kawalleck et al., 1993) and Arabidopsis(Trezzini et al., 1993), which possess relatively few tydcgenes, TYDC in opium poppy is encoded by a large genefamily (Facchini and De Luca, 1994). The large number oftydc genes in opium poppy might reflect the diverse rolesfor TYDC in the biosynthesis of both cell wall-boundamides and numerous benzylisoquinoline alkaloids (Fac-chini, 1998). TYDC isoforms in opium poppy do not exhibitany major differences in catalytic properties (Facchini andDe Luca, 1995a). However, the role of Tyr as precursor toboth amides and alkaloids suggests that tydc genes mightbe differentially regulated to ensure the optimum availabil-ity of aromatic amines for both pathways under a variety ofdevelopmental and environmental conditions.

The tydc gene family in opium poppy appears to haveevolved from the duplication of two ancestral genes. Allisolated tydc genes from opium poppy are highly homolo-gous to one of two subfamilies (Fig. 3). Genes encodingtydc1, tydc4, tydc5, tydc6, tydc8, and tydc9 likely all resultedfrom duplication of one ancestral gene, whereas genesencoding tydc2, tydc3, and tydc7 all likely resulted from theduplication of a second ancestral tydc gene. The proximityof the tydc8 and tydc9 genes indicates that the duplicationprocess resulted in at least partial clustering of the tydcgene family in the opium poppy genome.

Previous work has shown that the two tydc subfamiliesin opium poppy are differentially regulated (Facchini andDe Luca, 1994; Facchini et al., 1996). Using gene-specificprobes, we found that individual members of each tydc

Figure 8. GUS activity in mature plant organsand in 10-d-old seedlings of transgenic tobaccoexpressing tydc promoter-GUS constructs. GUSactivity levels in transgenic tobacco expressinga CaMV 35S promoter-GUS fusion are shownfor comparison. MU, 4-Methylumbelliferone.




Figure 9. (Legend appears on facing page.)




subfamily exhibited different patterns of developmentaland inducible expression (Figs. 4–6). Only TYDC3 tran-scripts were detected in the mature plant, developing seed-lings, and elicitor-treated cell cultures. However, unlike thecollective pattern of tydc2-like gene expression, TYDC3mRNAs were not detected in stems. TYDC2 and TYDC7transcripts were the only specific transcripts detected atlow levels in stems (Fig. 4). The abundance of mRNAsdetected with the full-length TYDC2 probe suggests thatanother tydc2-like gene that exhibits strong expression instems remains to be isolated. The expression pattern ofTYDC2/7 mRNAs was qualitatively similar but weakerthan the collective pattern of the tydc2-like subfamily.

Despite the qualitative consistency in root-specific ex-pression of tydc1 and/or tydc8, tydc6, and tydc9 (Fig. 4), lowlevels of only TYDC6 mRNAs were detected in elicitor-treated opium poppy cultures (Fig. 6). Additional membersof the tydc1-like subfamily that display strong inducibleexpression must also exist in opium poppy. The absence ofTYDC4 transcripts is consistent with the previous sugges-tion that tydc4 is a pseudogene (Facchini and De Luca,1994). The premature stop codon (Fig. 3) is likely a muta-tion that occurred because of the lack of selection pressurefor a functional gene. Our data clearly show a distinctionamong individual members of the opium poppy tydc genefamily in terms of their expression patterns in response todevelopmental and/or environmental cues. A complexpattern of developmental and inducible tydc expressioncould ensure that the extensive requirements for aromaticamines in opium poppy are satisfied.

All tydc promoter-GUS fusions showed higher activitythan the CaMV 35S::GUS construct in transient assays inboth opium poppy and tobacco cell cultures (Fig. 7). Eachconstruct assembled in the pBI 102 binary vector was alsoused for tobacco transformation. Data presented in Figure7 demonstrate that each translational fusion was at leastpotentially functional in transgenic tobacco. The pattern ofrelative GUS activity was similar in opium poppy andtobacco cells, with the only exception being the loweractivity of the tydc9 promoter in tobacco. TYDC mRNAswere present only at low levels in control cultures (Fig. 6),so the transient expression of tydc promoter-GUS fusions inopium poppy and tobacco cultures was likely not consti-tutive. Members of the opium poppy tydc gene family areinduced by wounding, in addition to elicitor treatment (P.Facchini, unpublished data); therefore, it is likely that thetydc promoter-GUS fusions were activated by a woundsignal caused by penetration of DNA-coated gold particlesinto cells.

The putative wound-induced activation of some tydcpromoter-GUS fusions in opium poppy and tobacco cellsafter particle bombardment is supported by the relativeincrease in GUS activity after treatment of bombardedcultures with elicitor. Moreover, the same tydc promoter-GUS fusions that responded to elicitor treatment in bom-barded opium poppy and tobacco cultures were also in-duced by wounding in transgenic tobacco. However,because of the limitations of transient expression systems,we cannot rule out the possible constitutive expression oftydc promoter-GUS fusions in bombarded cells. An un-wounded control was not possible because particle bom-bardment inherently wounds cells. However, our data sup-port the conclusion that at least some opium poppy tydcpromoters are inducible by environmental signals such aselicitors and wounding in both opium poppy and tobacco.

Four of the five tydc promoters introduced as GUS fu-sions into transgenic tobacco produced developmental ex-pression patterns that were both qualitatively and quanti-tatively similar to the expression of each gene in opiumpoppy plants (Fig. 8). The exception was the tydc3 pro-moter, which did not produce significant GUS activity inany of the numerous transgenic tobacco lines tested. Ex-pression of the tydc3 promoter-GUS fusion might havebeen inhibited by a trans-silencing mechanism caused bythe presence of homologous tobacco tydc genes (Matzkeand Matzke, 1995). The ORFs of tydc genes from opiumpoppy (Facchini and De Luca, 1994) and parsley (Kawal-leck et al., 1993) share .60% nucleotide identity that couldbe expected to extend into promoter regions. Similar ho-mology between tydc genes from opium poppy and tobaccois likely. In general, however, the correct developmentalexpression of a transgene in a heterologous species hasbeen reported for promoters of genes whose products arecommon to both source and recipient of the transgene(Benfey and Chua, 1989). It could be expected that theexpression patterns of at least some opium poppy tydcpromoters would be conserved in a heterologous speciessuch as tobacco, since both plants produce hydroxycin-namic acid amides of tyramine (Martin-Tanguy et al., 1978;Negrel and Martin, 1984). Other promoters that displayboth developmental and inducible regulation, such as theisoflavone reductase promoter from alfalfa, often conferdifferent patterns of developmental expression in homolo-gous and heterologous transgenic plants (Oommen et al.,1994).

Developmental expression patterns of endogenous genesin opium poppy (Figs. 4 and 5) and tydc promoter-GUStransgenes in tobacco (Figs. 8 and 9) are remarkably simi-

Figure 9. (Figure appears on facing page.)Histochemical localization of GUS activity in transgenic tobacco expressing tydc promoter-GUS constructs. A, Cross-sectionof a young TYDC7::GUS stem showing GUS activity restricted to internal phloem. B, Cross-section of a young TYDC7::GUSpetiole with GUS activity localized in internal phloem and an adaxial layer of cortex. C, TYDC7::GUS roots displaying GUSactivity in dividing meristematic tissues and in the stele of elongation and maturation zones. D, TYDC7::GUS root showingGUS activity during the early stages of lateral root bud development. E, Ten-day-old TYDC7::GUS seedling with GUS activityin the cotyledons, shoot apical meristem, root meristem, and vascular tissue of the young root. F, Root-shoot transition zoneof a TYDC6::GUS plant showing that GUS activity in the stele ends as vascular bundles emerge in the stem. G, TYDC6::GUSroots showing the restriction of GUS activity to the stele. co, Cotyledons, ep, external phloem; ip, internal phloem; lr, lateralroot; rm, root meristem; rc, root cap; sm, shoot apical meristem; st, stele; xy, xylem. All bars represent 1 mm.




lar. Promoters from tydc1-like genes (i.e. tydc6, tydc8, andtydc9) were active only in the roots in both opium poppyand transgenic tobacco. In contrast, the tydc7 promoter,which represents a tydc2-like gene, was active in the rootsand shoots of both species. The expression of tydcpromoter-GUS fusions in the vascular tissues of transgenictobacco organs (Fig. 9) is consistent with the localization ofTYDC mRNAs in the secondary phloem of opium poppyroots and stems (Facchini and De Luca, 1995b).

Promoters from root-specific tydc1-like genes were activeonly in the stele of transgenic tobacco roots. Hand sectionsshowed that GUS staining was concentrated in peripheralregions corresponding to the primary phloem (data notshown). GUS expression in TYDC7::GUS tobacco roots alsooccurred in the stele but extended into the apical meristemand zone of cell division (Fig. 9). Root-specific promoteractivity for tydc5, an opium poppy tydc1-like gene, wasreported previously in transgenic tobacco (Maldonado-Mendoza et al., 1996). The detection of abundant TYDC5mRNAs in opium poppy roots was consistent with theheterologous expression of the tydc5 promoter-GUS fusionin tobacco (Maldonado-Mendoza et al., 1996). However,the tydc5 promoter also showed strong activity in roots oftransgenic tobacco seedlings. In contrast, the tydc6, tydc8,and tydc9 promoters exhibited no significant activity intobacco seedlings.

The tydc7 promoter was not active in the phloem externalto the vascular cambium in transgenic tobacco but, rather,was restricted to the unusual internal phloem found adaxi-ally to the xylem in select species, such as tobacco. TYDCmRNAs in opium poppy stems were restricted to themetaphloem, which contains many alkaloid-rich laticiferderivatives (Facchini and De Luca, 1995b). Expression oftydc genes might be suppressed in normal phloem tissues,occurring only in specialized cambial derivatives such aslaticifers and internal phloem. It is not known whether theactivity of the tydc7 promoter in tissues near the root apex,in the leaf and petiole cortex, and in cotyledons representsectopic expression in transgenic tobacco, since tydc expres-sion patterns have not been examined in correspondingopium poppy tissues.

Conservation of the correct differential expression pat-terns for most opium poppy tydc promoters in transgenictobacco suggests that the developmental signals involvedin the activation of tydc genes in opium poppy are alsopresent in unrelated species such as tobacco. Conversely,cis-elements in opium poppy tydc promoters involved indevelopmental and, in some cases, inducible expressionappear to be recognized by transcription factors in tobaccoand might be homologous in sequence. Our data suggestthat a common mechanism for the developmental and in-ducible regulation of tydc genes exists across plant species.The apparently common, if not ubiquitous, presence oftydc genes in plants coupled with their conserved mecha-nisms of regulation in unrelated species suggests that theyplay fundamental roles in plant development and defenseresponses.

Opium poppy tydc promoters conferred strong expres-sion on a reporter gene in transgenic tobacco. This prop-erty, coupled with the highly conserved patterns of tissue-

and organ-specific expression in a heterologous transgenicspecies, suggests that tydc promoters might be a useful toolin plant genetic and metabolic engineering strategies.

ACKNOWLEDGMENTS

We thank Min Yu, David Bird, and Sang-Un Park for technicalassistance, Ken Girard for maintenance of plants in the green-house, and Dr. Edward Yeung for helpful comments.

Received February 18, 1998; accepted June 8, 1998.Copyright Clearance Center: 0032–0889/98/118/0069/13.Nucleotide sequences reported in this paper have been submitted

to the GenBank and EMBL databases with the accession nos.AF025431 (tydc3), AF025435 (tydc6), AF025434 (tydc7), AF025432(tydc8), and AF025433 (tydc9).

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expression patterns conferred by tyrosine ...opium poppy (papaver somniferum cv marianne) and...

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