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Plant Physiol. (1998) 118: 69-81
Expression Patterns Conferred by Tyrosine/Dihydroxyphenylalanine
Decarboxylase Promoters from 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
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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
 -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.
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INTRODUCTION |
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).
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| 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.
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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.
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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
L1 hydrolyzed casein, 20 g
L1 Suc, and 1 mg L1
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  EMBL3 (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 EMBL3 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.
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Table I.
Oligonucleotide primers used to amplify
gene-specific 3 flanking regions from tydc1/tydc8, tydc2/tydc7, tydc3,
tydc4, tydc6, and tydc9a
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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 DH10 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.
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Table II.
Oligonucleotide primers used to amplify and
subclone 5 flanking regions from tydc3, tydc6, tydc7, tydc8, and tydc9
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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 mL1 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 -mercaptoethanol. The GUS fluorometric assay buffer consisted of 50 mM NaPO4
buffer, pH 7.0, 10 mM -mercaptoethanol, 10 mM EDTA, 0.1% (w/v) sodium lauryl sarcosine, and 0.1%
(w/v) Triton X-100. 4-Methylumbelliferyl--D-glucuronide
was added at a final concentration of 0.44 mg
mL1. 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--D-glucuronide.
The luciferase assay buffer consisted of 25 mM Tricine, pH
7.8, 15 mM MgCl2, 5 mM
ATP, 0.5 mg mL1 BSA, and 7 mM
-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--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.
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RESULTS |
Structure and Organization of tydc Genes
in Opium Poppy
Screening an opium poppy EMBL3 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.
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| 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.
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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.
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| 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).
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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.
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| 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.
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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).
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| 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.
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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).
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| 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.
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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).
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| 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).
View larger version (31K):
[in this window]
[in a new window]
| 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).
View larger version (94K):
[in this window]
[in a new window]
| 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.
| |
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.
*
Corresponding author; e-mail pfacchin{at}acs.ucalgary.ca; fax
1-403-289-9311.
Received February 18, 1998;
accepted June 8, 1998.
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).
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
4-HPAA, 4-hydroxyphenylacetaldehyde.
NOS, nopaline synthase.
NPT II, neomycin phosphotransferase.
ORF, open reading frame.
TYDC, Tyr/dihydroxyphenylalanine decarboxylase.
| |
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.
| |
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DNA sequencing with chain-terminating inhibitors.
Proc Natl Acad Sci USA
74:
5463-5467
[Abstract/Free Full Text]
Schmelzer E,
Krüger-Lebus S,
Hahlbrock K
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Temporal and spatial patterns of gene expression around sites of attempted fungal infection in parsley leaves.
Plant Cell
1:
993-1001
[Abstract/Free Full Text]
Stadler R,
Kut |
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Abstract
Introduction