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Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine Decarboxylase Promoters from Opium Poppy Are Conserved in Transgenic Tobacco

Abstract

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the β-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Opium poppy (Papaver somniferum) remains an economically important medicinal plant, because it is the only commercial source of several pharmaceutical alkaloids, including the analgesics morphine, codeine, and thebaine. The biosynthesis of these and other benzylisoquinoline alkaloids begins with the condensation of dopamine and 4-HPAA to form the first committed alkaloid intermediate, (S)-norcoclaurine (Stadler et al., 1987). More than 2500 benzylisoquinoline alkaloids have been isolated from five major plant families, and all are derived from (S)-norcoclaurine (Stadler et al., 1987). Both dopamine and 4-HPAA are simple derivatives of Tyr, but their synthesis has not been unequivocally characterized. The synthesis of dopamine could result from either the decarboxylation of dihydroxyphenylalanine or from the hydroxylation of tyramine, which is the product of Tyr decarboxylation (Rueffer and Zenk, 1987). The capacity of TYDC to decarboxylate both Tyr and dihydroxyphenylalanine (Facchini and De Luca, 1994, 1995a) suggests that dopamine might be synthesized by both routes. Similarly, the synthesis of 4-HPAA could result from either the decarboxylation of 4-hydroxyphenylpyruvate or the oxidation of tyramine (Rueffer and Zenk, 1987). Therefore, TYDC is probably involved in the formation of both dopamine and 4-HPAA and could play a key role in the regulation of benzylisoquinoline alkaloid biosynthesis (Fig. 1).

Figure 1.

Figure 1

Schematic representation of the early steps in the biosynthetic pathways leading to benzylisoquinoline alkaloids and hydroxycinnamic acid amides of tyramine showing the sites of action of key gene products. NS, (S)-Norcoclaurine synthase; THT, tyramine hydroxycinnamoylCoA:tyramine hydroxycinnamoyltransferase; Dopa, dihydroxyphenylalanine.

TYDC cDNAs have been reported from parsley (Kawalleck et al., 1993), Arabidopsis (Trezzini et al., 1993), and opium poppy (Facchini and De Luca, 1994). In each case, TYDC mRNAs were shown to be rapidly and transiently induced in response to elicitor treatment (Kawalleck et al., 1993; Trezzini et al., 1993; Facchini et al., 1996) and pathogen challenge (Schmelzer et al., 1989). Induction of TYDC mRNAs in plant species that do not accumulate Tyr-derived alkaloids, such as parsley and Arabidopsis, suggests that Tyr serves as the precursor to a ubiquitous class of plant defense-response metabolites. Recent studies suggest that the biosynthesis and deposition in the cell wall of amides composed of hydroxycinnamic acid derivatives and tyramine are central to the defense response of many plants (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 cell wall-bound phenolics, are believed to reduce cell wall digestibility and/or directly inhibit pathogen growth. Hydroxycinnamoyltyramines have been isolated from a variety of plant species (Martin-Tanguy et al., 1978). Recently, the conversion of aromatic amines to both alkaloids and amides in elicitor-treated opium poppy cultures was demonstrated (Facchini, 1998). Tyramine hydroxycinnamoylCoA:tyramine hydroxycinnamoyltransferase, which catalyzes the condensation of hydroxycinnamoyl-CoA and tyramine (Fig. 1), has been purified and characterized in tobacco (Nicotiana tabacum; Negrel and Martin, 1984; Negrel and Javelle, 1997) and potato (Hohlfeld et al., 1995, 1996) and isolated in opium poppy (Facchini, 1998).

The tydc gene family in opium poppy contains approximately 15 members that can be divided into two subfamilies based on sequence identity and represented by tydc1 and tydc2 (Facchini and De Luca, 1994). Each subfamily of tydc genes exhibits distinct and tissue-specific patterns of developmental and inducible expression in opium poppy plants and elicited cell cultures (Facchini and De Luca, 1994, 1995b; Facchini et al., 1996). The dual role of tyramine as a precursor for benzylisoquinoline alkaloid and hydroxycinnamic acid amide biosynthesis and the demonstration that tydc genes are involved in the defense responses of many plants suggest that different members of the large tydc gene family in opium poppy might display complex patterns of regulation and diverse metabolic roles.

In an attempt to better understand the regulation of tydc genes in plants, in this paper we describe the organization and gene-specific patterns of developmental and inducible expression of eight tydc genes from opium poppy. The regulation of five opium poppy tydc gene promotors is further studied in transgenic tobacco plants that contain tydc promoter-GUS fusions. Our results show that members of the tydc gene family exhibit different patterns of developmental and inducible expression and that the tissue-specific and wound-induced regulation of tydc promoters is conserved across plant species. The highly parallel regulation of opium poppy tydc promoters in tobacco suggests that TYDC plays significant and ubiquitous roles in both the development and defense response of plants.

MATERIALS AND METHODS

Plants and Cell Cultures

Opium poppy (Papaver somniferum cv Marianne) and tobacco (Nicotiana tabacum cv Xanthi) plants were grown under greenhouse conditions at a day/night temperature of 20°C/18°C. Seedlings were grown at 23°C in sterile Petri plates containing moist filter paper. Seeds were surface sterilized with 20% (v/v) bleach for 15 min, washed thoroughly with sterile, distilled water, and allowed to imbibe water for 24 h (d 0). Seeds were kept in the dark for 3 d following imbibition and were then transferred to a photoperiod of 16 h light/8 h dark.

Opium poppy and tobacco cell-suspension cultures were maintained in diffuse light at 23°C on 1B5C medium (Gamborg et al., 1968) consisting of B5 salts and vitamins plus 100 mg L−1 myo-inositol, 1 g L−1 hydrolyzed casein, 20 g L−1 Suc, and 1 mg L−1 2,4-D. Cells were subcultured every 6 d using a 1:4 dilution of inoculum to fresh medium. Cultured cells in rapid growth phase (2–3 d after subculture) were used for all experiments.

Elicitor Treatment of Opium Poppy Cell Cultures

Fungal elicitor was prepared from Botrytis sp. according to the method of Eilert et al. (1985). A 1-cm2 section of mycelia grown on potato dextrose agar was cultivated in 50 mL of 1B5C medium, including supplements but excluding 2,4-D, on a gyratory shaker (120 rpm) at 22°C in the dark for 6 d. Mycelia and medium were homogenized with a Polytron (Brinkmann), autoclaved (121°C) for 20 min, and subsequently centrifuged under sterile conditions with the supernatant serving as elicitor. Elicitor treatments were initiated by the addition of 1 mL of fungal homogenate per 50 mL of cell culture. Cells were subsequently collected by vacuum filtration, frozen in liquid N2, and stored at −80°C.

Genomic Library Construction and Screening

A λEMBL3 (Stratagene) library was constructed from opium poppy genomic DNA partially digested with MboI (Sambrook et al., 1989). A primary library of 1.1 × 107 plaques was obtained (Facchini and De Luca, 1994), and 2.5 × 109 plaques of the amplified library were independently screened at high stringency, as described below, with random-primer 32P-labeled probes synthesized from the full-length coding region of the TYDC1 and TYDC2 cDNAs from opium poppy (Facchini and De Luca, 1994). Isolated λEMBL3 genomic clones for tydc3, tydc6, tydc7, tydc8, and tydc9 were subcloned into pBluescript and mapped for restriction endonuclease cleavage sites and gene location.

Isolation and Analysis of Nucleic Acids

Genomic DNA was isolated from young leaves of opium poppy plants (Murray and Thompson, 1980). Total RNA for gel-blot analysis was isolated according to the method of Logemann et al. (1987), and 15 μg was fractionated on 1.0% formaldehyde agarose gels before transfer to nylon membranes (Sambrook et al., 1989). RNA gel blots were hybridized with random-primer 32P-labeled (Feinberg and Vogelstein, 1984) full-length probes for TYDC1 and TYDC2 or gene-specific probes for tydc1/tydc8, tydc2/tydc7, tydc3, tydc4, tydc6, tydc7, and tydc9. A list of oligonucleotide primers used to isolate gene-specific 3′ flanking regions by PCR is shown in Table I. Hybridizations were performed at 65°C in 0.25 m sodium phosphate buffer, pH 8.0, 7% (w/v) SDS, 1% (w/v) BSA, and 1 mm EDTA. Blots were washed at 65°C, twice with 2× SSC and 0.1% (w/v) SDS and twice with 0.2× SSC and 0.1% (w/v) SDS (Sambrook et al., 1989; 1× SSC = 0.15 m NaCl, 0.015 m sodium citrate, pH 7.0), and autoradiographed with an intensifying screen at −80°C for 48 h.

Table I.

Oligonucleotide primers used to amplify gene-specific 3′ flanking 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 113
TYDC1/8-2 GAA ATG AAT GGT AAT TTC
TYDC2/7-1 AGG AAC GCC ATG TTA TTC 400
TYDC2/7-2 CAT AAC ATC TAA CAT TAA
TYDC3-1 CGC GTA TCT TGT GGT TGA 320
TYDC3-2 CTG ATG AAC AAC TTT GGT
TYDC4-1 GGT ACA GCG GAG ATG AAG 177
TYDC4-2 TGT TTC CTT AGG CTC AAC
TYDC6-1 GGA GAG AGA AAG GAA ACG 280
TYDC6-2 TAC GAT GCA AAC GGA ACC
TYDC9-1 ACA GAT GCC ATA CTT GGT 191
TYDC9-2 ATA AGT AGT TAT GAG TAT

Double-stranded DNA was sequenced using the dideoxynucleotide chain-termination method (Sanger et al., 1977) and a recombinant T7 DNA polymerase (United States Biochemical). Sequences were aligned using the FASTA program package (Pearson and Lipman, 1988).

Transient Expression and Stable Transformation Vectors

A binary vector, designated pBI 102, was used to construct promoter-GUS fusions for transient expression assays in microprojectile-bombarded cell cultures and for the stable transformation of tobacco. Restriction sites for ApaI, XhoI, and KpnI were included in pBI 102 by inserting an adapter fragment into the SmaI site of pBI 101 (Jefferson et al., 1987). Binary vectors were maintained in Escherichia coli strain DH10β and mobilized in Agrobacterium tumefaciens strain LB4404 by direct DNA transfer (An, 1987).

Promoter-GUS Constructs

Promoters of tydc3, tydc6, tydc7, tydc8, and tydc9 were amplified by PCR using specific primers designed to add a HindIII and either a BamHI or XhoI restriction site at the 5′ and 3′ ends, respectively, of each promoter fragment. A list of oligonucleotide primers used to isolate and subclone 5′ flanking regions is shown in Table II. The isolated tydc3, tydc6, tydc7, tydc8, and tydc9 promoters extended approximately 3.5, 3.0, 1.2, 1.2 and 2.1 kb, respectively, upstream of the putative translation start codon in each gene. The PCR-generated tydc3, tydc6, tydc8, and tydc9 promoter fragments were inserted into pBI 102 between the HindIII and BamHI sites to yield the TYDC3::GUS, TYDC6::GUS, TYDC8::GUS, and TYDC9::GUS constructs, whereas the tydc7 promoter fragment was inserted into pBI 102 between the HindIII and XhoI sites to yield the TYDC7::GUS construct. The assembly of all constructs was verified by sequencing through the promoter-GUS junction. The CaMV 35S promoter-GUS fusion in pBI 121 (Jefferson et al., 1987) and the promoterless pBI 102 vector were used as positive and negative controls, respectively.

Table II.

Oligonucleotide primers used to amplify and subclone 5′ 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 3.5
TYDC3-4 GGG GGG AAG CTT CTG TGT GCC AAC CCG CGA TA
TYDC6-3 GGG GGG GGA TCC TTG CTG ATT AGT GAG GGA GA 3.0
TYDC6-4 GGG GGG AAG CTT ATA GAA GTT GTT GGG AGA TA
TYDC7-3 GGG GGG CTC GAG CAG GTG AAA GAA GGT TAT TG 1.2
TYDC7-4 GGG GGG AAG CTT TTA TCC ACA CCC AAC TCA TC
TYDC8-3 GGG GGG GGA TCC CGT TAC TAT CAG TTT TGA TG 1.2
TYDC8-4 T3 primerd
TYDC9-3 GGG GGG GGA TCC TGT TAC TGG TTT TGC TAA TG 2.1
TYDC9-4 GGG GGG GTC GAC CAA ATG AGG ACC CAA ATC TG

Transient luciferase activity was introduced into cultured cells using pCaLucNOS, which harbors the CaMV 35S promoter fused to the luciferase-coding region, followed by the NOS polyadenylation signal in pUC 19. All plasmids were purified before microprojectile bombardment by PEG precipitation, phenol/chloroform extraction, LiCl precipitation, and RNase digestion and then were extracted again with phenol/chloroform and precipitated with ethanol.

Microprojectile Bombardment of Cultured Cells

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

Cultured cells were collected on microfiber filters (GF/D, Whatman) by gentle vacuum filtration to form a thin cell layer approximately 2 cm in diameter. Filters containing the plant cells were placed in sterile Petri plates and positioned below a microprojectile-stopping screen. Bombardments were performed using a particle-acceleration device (PDS 1000/He, Bio-Rad) under a chamber pressure of 26 mm of Hg, at a distance of 1.5, 2.0, and 6.5 cm from the rupture disc to the microcarriers to the stopping screen to the target, respectively, and at a He pressure of 1100 p.s.i. After bombardment, the cultured cell layers were incubated at 23°C in the sterile Petri plates. Elicitor treatments 24 h after bombardment consisted of the addition of 0.25 mL of either the Botrytis sp. elicitor or a solution of 0.3 μg mL−1 cellulase to opium poppy and tobacco cells, respectively.

Tobacco Transformation

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

Wounding of Transgenic Tobacco Plants

Young leaves from transgenic tobacco plants grown in vitro were placed on two layers of moist filter paper in sterile Petri plates. Wounding was performed by puncturing leaves with sterile pins approximately once per square millimeter. Wounded and control samples were collected after 48 h.

GUS and Luciferase Assays

Transgenic tobacco tissues and cultured cells collected by vacuum filtration 48 h after microprojectile bombardment were ground with extraction buffer consisting of 50 mm KPO4 buffer, pH 7.0, 1 mm EDTA, and 10 mm β-mercaptoethanol. The GUS fluorometric assay buffer consisted of 50 mm NaPO4 buffer, pH 7.0, 10 mm β-mercaptoethanol, 10 mm EDTA, 0.1% (w/v) sodium lauryl sarcosine, and 0.1% (w/v) Triton X-100. 4-Methylumbelliferyl-β-d-glucuronide was added at a final concentration of 0.44 mg mL−1. Assays were performed on 80 μL of bombarded cell culture extract for 3 h at 37°C and stopped with a 10× volume of 0.2 m Na2CO3. A fluorescence spectrophotometer (model F-2000, Hitachi, Tokyo, Japan) was used to quantify the amount of 4-methylumbelliferone cleaved from 4-methylumbelliferyl-β-d-glucuronide.

The luciferase assay buffer consisted of 25 mm Tricine, pH 7.8, 15 mm MgCl2, 5 mm ATP, 0.5 mg mL−1 BSA, and 7 mm β-mercaptoethanol. Bombarded cell extract (20 μL) was mixed with 200 μL of assay buffer and incubated at room temperature for 15 min (de Wet et al., 1987). Luciferin (100 μL of 0.5 mm diluted with 1 mm Tricine, pH 7.8, from 10 mm stock, Boehringer Mannheim) was injected into the reaction mixture, and the light emitted within the first 10 s was quantified using a luminometer (Monolight 2010, Analytical Luminescence Laboratories, San Diego, CA). The protein concentration was determined by the method of Bradford (1976) using BSA as a standard.

GUS Histochemical Staining

GUS activity was localized histochemically by standard protocols (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, pH 7.5, and 300 mm mannitol for 1 h at 20°C, rinsed three times in 50 mm sodium phosphate, pH 7.5, and subsequently incubated in 50 mm sodium phosphate, pH 7.5, 2 mm 5-bromo-4-chloro-3-indolyl-β-d-glucuronide cyclohexylammonium salt, and 20% (v/v) methanol for 6 to 12 h at 37°C. Stained tissues were rinsed extensively in 70% ethanol to remove chlorophyll.

RESULTS

Structure and Organization of tydc Genes in Opium Poppy

Screening an opium poppy λEMBL3 genomic library with TYDC1 and TYDC2 cDNAs (Facchini and De Luca, 1994) resulted in the isolation of genomic clones that contained five homologous full-length genes designated tydc3, tydc6, tydc7, tydc8, and tydc9 (Fig. 2). Nucleotide sequence analysis showed that tydc3 was identical to a previously reported partial TYDC3 cDNA (Facchini and De Luca, 1994). The coding region of tydc3 was highly homologous to tydc7 (93% identity), and both genes shared extensive nucleotide identity with the ORF of the TYDC2 cDNA (93% for tydc3 and 97% for tydc7). The tydc6, tydc8, and tydc9 genes displayed strong nucleotide identity with the ORF of the TYDC1 cDNA (96% for tydc6, 93% for tydc8, and 93% for tydc9). In contrast, ORF alignments between any member of the tydc1, tydc6, tydc8, and tydc9 subfamily with any member of the tydc2, tydc3, or tydc7 subfamily revealed only 70% to 73% identity. None of the ORFs of isolated tydc genes was interrupted by intervening sequences, suggesting that all members of the gene family lack introns.

Figure 2.

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 approximate location and direction of transcription initiation. The horizontal brackets show the regions amplified by PCR and used as gene-specific probes. B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Sp, SpeI; Xb, XbaI; Xh, XhoI.

The complete genes for tydc8 and tydc9 were localized on one genomic clone with inversely oriented transcription units (Fig. 2). The ORFs were separated by a 3.2-kb DNA segment that contained both of the putative tydc8 and tydc9 promoters. Transcription would be expected to initiate in opposite directions. Despite the extensive homology between the ORFs of tydc8 and tydc9, the 5′ and 3′ flanking sequences were highly divergent. In contrast, the 5′ and 3′ flanking regions of tydc8 were identical to those of the TYDC1 cDNA. Similarly, the 5′ and 3′ flanking regions of tydc7 were identical to those of the TYDC2 cDNA. Alignment of predicted amino acid sequences for tydc3, tydc6, tydc7, tydc8, and tydc9 with those for tydc4 (Facchini and De Luca, 1994), tydc5 (Maldonado-Mendoza et al., 1996), and the TYDC1 and TYDC2 cDNAs showed that all reported members of the tydc gene family from opium poppy share extensive homology with one of two subfamilies that can be represented by tydc1 and tydc2 (Fig. 3). These data demonstrate that the large tydc gene family in opium poppy is at least partially clustered and probably evolved as the result of extensive duplication of two relatively divergent ancestral genes.

Figure 3.

Figure 3

Alignment of predicted amino acid sequences from isolated members of the tydc gene family in opium poppy. Amino acid sequences for TYDC1, TYDC2, and TYDC4 were reported by Facchini and De Luca (1994). The asterisk at position 56 in the TYDC4 sequence represents a premature termination codon. The TYDC5 amino acid sequence was also reported previously (Maldonado-Mendoza et al., 1996).

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

Full-length TYDC1 and TYDC2 cDNAs are sufficiently different in nucleotide sequence to prevent cross-hybridization when used as probes for RNA gel-blot hybridization analyses (Facchini and De Luca, 1994). Such probes were used previously to show that tydc gene subfamilies in opium poppy display development-specific expression in the plant and temporal-specific expression in elicitor-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 and stems. The levels of individual TYDC transcripts in various organs from opium poppy plants are shown in Figure 4. Gene-specific probes were isolated from 3′ flanking regions of tydc3, tydc4, tydc6, and tydc9. However, the untranslated 3′ and 5′ flanking regions of the TYDC1 cDNA and tydc8 gene were identical, as were the 3′ and 5′ flanking regions of the TYDC2 cDNA and tydc7 gene. Therefore, the 3′ flanking region used as a tydc1/8-specific probe could not discriminate between TYDC1 and TYDC8 transcripts, and the 3′ flanking region used as a tydc2/7-specific probe could not discriminate between TYDC2 and TYDC7 transcripts. Using these probes, we found that TYDC1/8 and TYDC3 mRNAs occurred abundantly and specifically in opium poppy roots (Fig. 4). Lower levels of TYDC6 and TYDC9 transcripts were also detected only in roots, whereas TYDC2/7 mRNAs occurred at detectable levels in both stems and roots. TYDC4 transcripts were not detected in any plant organ or tissue.

Figure 4.

Figure 4

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

TYDC2-like but not TYDC1-like mRNAs were detected at low levels in developing opium poppy seedlings (Fig. 5). The maximum relative abundance of TYDC2-like mRNAs occurred 3 d postimbibition and then decreased steadily. Using gene-specific probes, we detected only TYDC3 mRNAs by northern-blot hybridization analysis in developing opium poppy seedlings. TYDC2/7 transcripts were not detected at any stage of seedling development (Fig. 5). TYDC1/8, TYDC4, TYDC6, and TYDC9 transcripts were also not detected (data not shown).

Figure 5.

Figure 5

RNA gel-blot hybridization analysis for various members of the tydc gene family during opium poppy seedling development. Fifteen micrograms of total RNA was fractionated on 1.0% formaldehyde agarose gels, transferred to nylon membranes, and hybridized at high stringency with 32P-labeled full-length probes for tydc1 and tydc2 or gene-specific probes for tydc2/7 and tydc3. To ensure equal loading, gels were stained with ethidium bromide before blotting.

TYDC1- and TYDC2-like genes also exhibit differential and temporal-specific expression in elicitor-treated opium poppy cell cultures (Facchini et al., 1996). TYDC1-like genes were activated rapidly after the addition of elicitor, reaching maximum levels between 1 and 2 h and then rapidly declining (Fig. 6). In contrast, TYDC2-like mRNAs accumulated more slowly, reaching maximum levels 10 h after elicitor treatment, but were maintained at a high level for an extended period. Using gene-specific probes, we found that tydc3 was abundantly expressed and tydc2/7 and tydc6 were expressed at lower levels in elicitor-treated opium poppy cultures (Fig. 6). TYDC1/8, TYDC4, and TYDC9 transcripts were not detected in response to elicitor treatment (Fig. 6).

Figure 6.

Figure 6

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

Transient Expression of Opium Poppy tydc Promoter-GUS Fusions in Cultured Cells

The relative activity of isolated promoter regions from five opium poppy tydc genes was tested directly by measuring the transient expression of tydc promoter-GUS fusions in microprojectile-bombarded opium poppy cell cultures. CaMV 35S promoter-GUS and promoterless-GUS constructs were used as positive and negative controls, respectively. As shown in Figure 7A, all of the tydc promoter-GUS fusions produced higher levels of GUS activity in bombarded cell cultures than the CaMV 35S-GUS construct. The tydc3 promoter directed the highest level of GUS expression, which was 10-fold greater than the CaMV 35S promoter (Fig. 7A). The tydc9, tydc7, tydc6, and tydc8 promoters produced levels of GUS activity that were approximately 5-, 3-, 2-, and 1.5-fold higher, respectively, than the CaMV 35S promoter. GUS activity was not detected in opium poppy cells bombarded with the promoterless-GUS construct (Fig. 7A).

Figure 7.

Figure 7

Activity of various tydc gene promoters determined by transient expression of promoter-GUS fusions in opium poppy (A) and tobacco (B) cell cultures. Bars represent normalized GUS activity in cultured cells 48 h after microprojectile bombardment with the following constructs: pBI 102 (promoterless), 35S::GUS (CaMV 35S promoter), TYDC3::GUS (tydc3 promoter), TYDC6::GUS (tydc6 promoter), TYDC7::GUS (tydc7 promoter), TYDC8::GUS (tydc8 promoter), and TYDC9::GUS (tydc9 promoter). Values represent the means ± se of three independent experiments whereby cultured cells were co-bombarded with promoter-GUS and CaMV 35S-luciferase constructs and GUS activity was normalized against luciferase activity. MU, 4-Methylumbelliferone.

Transient expression of tydc promoter-GUS fusions was also tested in cultured tobacco cells (Fig. 7B). The relative pattern of GUS activity produced by tydc promoters in bombarded tobacco cultures was qualitatively similar but quantitatively lower than in opium poppy cultures. The tydc3 promoter directed the highest level of GUS expression in tobacco cells, which was 10-fold greater than the CaMV 35S promoter but 2-fold lower than the level produced by the tydc3 promoter-GUS fusion in opium poppy cells (Fig. 7B). Relative GUS activities produced by the tydc7 and tydc9 promoters in tobacco cultures were 2-fold higher and 4-fold lower, respectively, than in opium poppy cultures. The CaMV 35S-GUS construct also produced approximately 2-fold lower GUS activity in tobacco cells.

Cultured cells bombarded with select tydc promoter-GUS constructs showed an increase in GUS activity after elicitor treatment relative to untreated controls (data not shown). GUS activity in both opium poppy and tobacco cells bombarded with the TYDC3::GUS, TYDC6::GUS, or TYDC7::GUS constructs increased between 1.5- and 2-fold after addition of elicitor. GUS activity was not significantly affected by elicitor treatment in cell cultures bombarded with the TYDC8::GUS or TYDC9::GUS constructs or with the 35S::GUS control. It should also be noted that the Botrytis sp. and cellulase elicitors were effective with only opium poppy and tobacco cultures, respectively.

Cell cultures were co-bombarded with pCaLucNOS so that GUS activity could be normalized against luciferase activity to account for differences in expression efficiency between bombardments. The specific luciferase activity was similar for each bombardment.

Expression of Opium Poppy tydc Promoter-GUS Fusions in Transgenic Tobacco

The binary vectors harboring the tydc promoter-GUS fusions used to test promoter activity by transient expression analysis in opium poppy and tobacco cell cultures were mobilized in A. tumefaciens and used for tobacco transformation. Transgenic tobacco plants resistant to kanamycin were tested for the presence of transgenes by PCR and by direct NPT II enzyme assay. GUS activity was measured in 10-d transgenic seedlings and in plants grown in vitro or under greenhouse conditions. Results presented in Figure 8 represent the mean and se of triplicate measurements on each of three independent transgenic lines for each construct. The CaMV 35S promoter-GUS fusion, used as a positive control, exhibited strong GUS activity in all transgenic tobacco organs and in seedlings. The tydc3 promoter-GUS fusion resulted in weak GUS activity in roots and only very low levels of activity in shoot organs and seedlings (Fig. 8). The tydc6, tydc8, and tydc9 promoter-GUS fusions produced moderate to high levels of GUS activity in young transgenic tobacco roots, but only low levels of GUS activity were detected in shoot organs or seedlings. Among these, the level of GUS activity was highest in young roots of plants transformed with TYDC8::GUS and lowest in plants transformed with TYDC9::GUS (Fig. 8). The tydc7 promoter-GUS fusion produced strong GUS activity in all transgenic tobacco organs and in seedlings (Fig. 8).

Figure 8.

Figure 8

GUS activity in mature plant organs and in 10-d-old seedlings of transgenic tobacco expressing tydc promoter-GUS constructs. GUS activity levels in transgenic tobacco expressing a CaMV 35S promoter-GUS fusion are shown for comparison. MU, 4-Methylumbelliferone.

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, and TYDC7::GUS tobacco relative to unwounded controls (Fig. 8). In contrast, no significant change in GUS activity occurred in response to wounding in TYDC8::GUS, TYDC9::GUS, or 35S::GUS plants.

Histochemical staining for GUS activity showed that the developmental expression of all tydc promoter-GUS fusions was concentrated in the vascular tissues of all transgenic tobacco organs (Fig. 9). Young stems and petioles from TYDC7::GUS tobacco showed GUS activity restricted to the internal phloem (Fig. 9, A and B). GUS activity was absent from mature stems and petioles that exhibited substantial secondary growth. Petioles of TYDC7::GUS tobacco also showed GUS activity in an adaxial layer of cortex (Fig. 9B). Leaves from TYDC7::GUS plants displayed a similar pattern of expression, with the strongest GUS activity localized in veins and lower levels of activity detected in mesophyll tissue between veins (data not shown). TYDC7::GUS roots showed the strongest staining for GUS activity in dividing meristematic tissues (Fig. 9C). As root development proceeded through the zones of elongation and maturation, GUS activity become progressively restricted to the stele (Fig. 9C). GUS activity was significantly reduced but still restricted to vascular tissues in older roots (data not shown). In contrast, strong GUS activity was clearly detected in TYDC7::GUS tobacco during the early stages of lateral root development (Fig. 9D). TYDC7::GUS plants were the only transformants that showed significant GUS activity in shoot organs and in meristematic regions of roots. Strong GUS activity was also detected in the cotyledons, shoot apical meristem, root meristem, and developing root vascular tissues of TYDC7::GUS seedlings (Fig. 9E).

Figure 9.

Figure 9

(Figure appears on facing page.)

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

TYDC6::GUS, TYDC8::GUS, and TYDC9::GUS tobacco showed similar patterns of localization, with GUS activity strictly localized in root vascular tissues. In contrast to TYDC7::GUS plants, strong GUS activity was detected in the stele of the main root axis in TYDC6::GUS plants (Fig. 9F). Examination of the root-shoot transition zone of TYDC6::GUS plants showed that GUS activity in the stele terminated as vascular bundles emerged in the stem (Fig. 9F). Also, unlike TYDC7::GUS plants, GUS activity was absent in dividing meristematic tissues of TYDC6::GUS roots (Fig. 9G).

DISCUSSION

TYDC clones and/or enzyme activity have been isolated in several plant species, including opium poppy (Facchini and De Luca, 1994), Eschscholtzia californica, Thalictrum rugosum (Marques and Brodelius, 1988), Sanguinaria canadensis (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 even ubiquitous, in plants, its metabolic roles have not been fully characterized and its physiological importance is not well understood. TYDC is involved in the biosynthesis of cell wall-bound amides and, in select species including opium poppy, E. californica, T. rugosum, and S. canadensis, in the biogenesis of benzylisoquinoline alkaloids. The recent isolation of a jasmonic acid conjugate of tyramine suggests additional roles for TYDC (Miersch et al., 1998).

Unlike parsley (Kawalleck et al., 1993) and Arabidopsis (Trezzini et al., 1993), which possess relatively few tydc genes, TYDC in opium poppy is encoded by a large gene family (Facchini and De Luca, 1994). The large number of tydc genes in opium poppy might reflect the diverse roles for TYDC in the biosynthesis of both cell wall-bound amides and numerous benzylisoquinoline alkaloids (Facchini, 1998). TYDC isoforms in opium poppy do not exhibit any major differences in catalytic properties (Facchini and De Luca, 1995a). However, the role of Tyr as precursor to both amides and alkaloids suggests that tydc genes might be differentially regulated to ensure the optimum availability of aromatic amines for both pathways under a variety of developmental and environmental conditions.

The tydc gene family in opium poppy appears to have evolved from the duplication of two ancestral genes. All isolated tydc genes from opium poppy are highly homologous to one of two subfamilies (Fig. 3). Genes encoding tydc1, tydc4, tydc5, tydc6, tydc8, and tydc9 likely all resulted from duplication of one ancestral gene, whereas genes encoding tydc2, tydc3, and tydc7 all likely resulted from the duplication of a second ancestral tydc gene. The proximity of the tydc8 and tydc9 genes indicates that the duplication process resulted in at least partial clustering of the tydc gene family in the opium poppy genome.

Previous work has shown that the two tydc subfamilies in opium poppy are differentially regulated (Facchini and De Luca, 1994; Facchini et al., 1996). Using gene-specific probes, we found that individual members of each tydc subfamily exhibited different patterns of developmental and inducible expression (Figs. 4–6). Only TYDC3 transcripts were detected in the mature plant, developing seedlings, and elicitor-treated cell cultures. However, unlike the collective pattern of tydc2-like gene expression, TYDC3 mRNAs were not detected in stems. TYDC2 and TYDC7 transcripts were the only specific transcripts detected at low levels in stems (Fig. 4). The abundance of mRNAs detected with the full-length TYDC2 probe suggests that another tydc2-like gene that exhibits strong expression in stems remains to be isolated. The expression pattern of TYDC2/7 mRNAs was qualitatively similar but weaker than the collective pattern of the tydc2-like subfamily.

Despite the qualitative consistency in root-specific expression of tydc1 and/or tydc8, tydc6, and tydc9 (Fig. 4), low levels of only TYDC6 mRNAs were detected in elicitor-treated opium poppy cultures (Fig. 6). Additional members of the tydc1-like subfamily that display strong inducible expression must also exist in opium poppy. The absence of TYDC4 transcripts is consistent with the previous suggestion that tydc4 is a pseudogene (Facchini and De Luca, 1994). The premature stop codon (Fig. 3) is likely a mutation that occurred because of the lack of selection pressure for a functional gene. Our data clearly show a distinction among individual members of the opium poppy tydc gene family in terms of their expression patterns in response to developmental and/or environmental cues. A complex pattern of developmental and inducible tydc expression could ensure that the extensive requirements for aromatic amines in opium poppy are satisfied.

All tydc promoter-GUS fusions showed higher activity than the CaMV 35S::GUS construct in transient assays in both opium poppy and tobacco cell cultures (Fig. 7). Each construct assembled in the pBI 102 binary vector was also used for tobacco transformation. Data presented in Figure 7 demonstrate that each translational fusion was at least potentially functional in transgenic tobacco. The pattern of relative GUS activity was similar in opium poppy and tobacco cells, with the only exception being the lower activity of the tydc9 promoter in tobacco. TYDC mRNAs were present only at low levels in control cultures (Fig. 6), so the transient expression of tydc promoter-GUS fusions in opium poppy and tobacco cultures was likely not constitutive. Members of the opium poppy tydc gene family are induced by wounding, in addition to elicitor treatment (P. Facchini, unpublished data); therefore, it is likely that the tydc promoter-GUS fusions were activated by a wound signal caused by penetration of DNA-coated gold particles into cells.

The putative wound-induced activation of some tydc promoter-GUS fusions in opium poppy and tobacco cells after particle bombardment is supported by the relative increase in GUS activity after treatment of bombarded cultures with elicitor. Moreover, the same tydc promoter-GUS fusions that responded to elicitor treatment in bombarded opium poppy and tobacco cultures were also induced by wounding in transgenic tobacco. However, because of the limitations of transient expression systems, we cannot rule out the possible constitutive expression of tydc promoter-GUS fusions in bombarded cells. An unwounded control was not possible because particle bombardment inherently wounds cells. However, our data support the conclusion that at least some opium poppy tydc promoters are inducible by environmental signals such as elicitors and wounding in both opium poppy and tobacco.

Four of the five tydc promoters introduced as GUS fusions into transgenic tobacco produced developmental expression patterns that were both qualitatively and quantitatively similar to the expression of each gene in opium poppy plants (Fig. 8). The exception was the tydc3 promoter, which did not produce significant GUS activity in any of the numerous transgenic tobacco lines tested. Expression of the tydc3 promoter-GUS fusion might have been inhibited by a trans-silencing mechanism caused by the presence of homologous tobacco tydc genes (Matzke and Matzke, 1995). The ORFs of tydc genes from opium poppy (Facchini and De Luca, 1994) and parsley (Kawalleck et al., 1993) share >60% nucleotide identity that could be expected to extend into promoter regions. Similar homology between tydc genes from opium poppy and tobacco is likely. In general, however, the correct developmental expression of a transgene in a heterologous species has been reported for promoters of genes whose products are common to both source and recipient of the transgene (Benfey and Chua, 1989). It could be expected that the expression patterns of at least some opium poppy tydc promoters would be conserved in a heterologous species such as tobacco, since both plants produce hydroxycinnamic acid amides of tyramine (Martin-Tanguy et al., 1978; Negrel and Martin, 1984). Other promoters that display both developmental and inducible regulation, such as the isoflavone reductase promoter from alfalfa, often confer different patterns of developmental expression in homologous and heterologous transgenic plants (Oommen et al., 1994).

Developmental expression patterns of endogenous genes in opium poppy (Figs. 4 and 5) and tydc promoter-GUS transgenes in tobacco (Figs. 8 and 9) are remarkably similar. Promoters from tydc1-like genes (i.e. tydc6, tydc8, and tydc9) were active only in the roots in both opium poppy and transgenic tobacco. In contrast, the tydc7 promoter, which represents a tydc2-like gene, was active in the roots and shoots of both species. The expression of tydc promoter-GUS fusions in the vascular tissues of transgenic tobacco organs (Fig. 9) is consistent with the localization of TYDC mRNAs in the secondary phloem of opium poppy roots and stems (Facchini and De Luca, 1995b).

Promoters from root-specific tydc1-like genes were active only in the stele of transgenic tobacco roots. Hand sections showed that GUS staining was concentrated in peripheral regions corresponding to the primary phloem (data not shown). GUS expression in TYDC7::GUS tobacco roots also occurred in the stele but extended into the apical meristem and zone of cell division (Fig. 9). Root-specific promoter activity for tydc5, an opium poppy tydc1-like gene, was reported previously in transgenic tobacco (Maldonado-Mendoza et al., 1996). The detection of abundant TYDC5 mRNAs in opium poppy roots was consistent with the heterologous expression of the tydc5 promoter-GUS fusion in tobacco (Maldonado-Mendoza et al., 1996). However, the tydc5 promoter also showed strong activity in roots of transgenic tobacco seedlings. In contrast, the tydc6, tydc8, and tydc9 promoters exhibited no significant activity in tobacco seedlings.

The tydc7 promoter was not active in the phloem external to the vascular cambium in transgenic tobacco but, rather, was restricted to the unusual internal phloem found adaxially to the xylem in select species, such as tobacco. TYDC mRNAs in opium poppy stems were restricted to the metaphloem, which contains many alkaloid-rich laticifer derivatives (Facchini and De Luca, 1995b). Expression of tydc genes might be suppressed in normal phloem tissues, occurring only in specialized cambial derivatives such as laticifers and internal phloem. It is not known whether the activity of the tydc7 promoter in tissues near the root apex, in the leaf and petiole cortex, and in cotyledons represents ectopic expression in transgenic tobacco, since tydc expression patterns have not been examined in corresponding opium poppy tissues.

Conservation of the correct differential expression patterns for most opium poppy tydc promoters in transgenic tobacco suggests that the developmental signals involved in the activation of tydc genes in opium poppy are also present in unrelated species such as tobacco. Conversely, cis-elements in opium poppy tydc promoters involved in developmental and, in some cases, inducible expression appear to be recognized by transcription factors in tobacco and might be homologous in sequence. Our data suggest that a common mechanism for the developmental and inducible regulation of tydc genes exists across plant species. The apparently common, if not ubiquitous, presence of tydc genes in plants coupled with their conserved mechanisms of regulation in unrelated species suggests that they play fundamental roles in plant development and defense responses.

Opium poppy tydc promoters conferred strong expression on a reporter gene in transgenic tobacco. This property, coupled with the highly conserved patterns of tissue- and organ-specific expression in a heterologous transgenic species, suggests that tydc promoters might be a useful tool in plant genetic and metabolic engineering strategies.

ACKNOWLEDGMENTS

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

Abbreviations:

CaMV

cauliflower mosaic virus

4-HPAA

4-hydroxyphenylacetaldehyde

NOS

nopaline synthase

NPT II

neomycin phosphotransferase

ORF

open reading frame

TYDC

Tyr/dihydroxyphenylalanine decarboxylase

Footnotes

1

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Alberta Agricultural Research Institute to P.J.F.

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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