Download Phloem-Specific Expression of Tyrosine/Dopa

The Plant Cell, Vol. 7, 1811-1821, November 1995 O 1995 American Society of Plant Physiologists
Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase
Genes and the Biosynthesis of lsoquinoline Alkaloids in
Opium Poppy
Peter J. Facchini’ and Vincenzo De Luca
lnstitut de Recherche en Biologie Végétale, 4101 rue Sherbrooke Est, Département de Sciences Biologiques, Universite de
Montréal, Montréal, Québec, H1X 262 Canada
Tyrosineklopa decarboxylase (TYDC) catalyzes the formation of tyramine and dopamine and represents the first steps
in the biosynthesis of the large and diverse group of tetrahydroisoquinoline alkaloids. Opium poppy accumulates morphine in aerial organs and roots, whereas sanguinarine, which is derived from a distinct branch pathway, accumulates
only i n roots. Expression of the TYDC gene family in opium poppy was investigated i n relation to the organ-specific biosynthesis of these different types of alkaloids. Members of the TYDC gene family are classified into two groups (represented
by TYDCl and TYDCP) and are differentially expressed. In the mature plant, TYDCP-like transcripts are predominant i n
stems and are also present in roots, whereas TYDCl-like transcripts are abundant only i n roots. I n situ hybridization analysis revealed that the expression of TYDC genes i s developmentally regulated. TYDC transcripts are associated with
vascular tissue i n mature roots and stems but are also expressed in cortical tissues at earlier stages of development.
Expression of TYDC genes i s restricted t o metaphloem and to protoxylem i n the vascular bundles of mature aerial organs.
Localization of TYDC transcripts i n the phloem i s consistent with the expected developmental origin of laticifers, which
are specialized internal secretory cells that accompany vascular tissues in all organs of select species and that contain
the alkaloid-rich latex in aerial organs. The differential expression of TYDC genes and the organ-dependent accumulation
of different alkaloids suggest a coordinated regulation of specific alkaloid biosynthetic genes that are ultimately controlled by specific developmental programs.
INTRODUCTION
Few plants possess the combined historic and contemporary
importance, notoriety,and mystique of the opium poppy. Among
the first civilizations to cultivate poppy were the Sumerians,
inhabitants, at the end of the third millennium B.C., of what is
now Iraq. The Sumerian name for opium poppy was hul gil
or “plant of joy”(Brownstein, 1993), suggesting that the opium
extracted from its seed capsules was used as a narcotic. Pharmaceutical applications of opium, or opium derivatives, have
occurred throughout human history. In 1500 B.C. the Ebers
Papyrus (Brownstein, 1993) described it as a remedy to prevent the excessive crying of children. In 1898, with the synthesis
and widespread availability of 0,O-diacetylmorphine (heroin),
it was used as a “cough suppressant.” Today, worldwide use
of opium is estimated to be in excess of one million kilograms
annually (Roberts, 1988). lsolation in 1806 of the principal active ingredient in opium, a weak base named morphine after
the Greek god of dreams, also marked the beginning of study
in the field of plant alkaloid chemistry and biosynthesis.
1 To whom correspondence should be addressed. Current address:
Department of BiologicalSciences, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, T2N 1N4 Canada.
Morphine belongs to the largest and most diverse class of
naturally occurring alkaloids,which includes compoundsbased
on the tetrahydroisoquinoline nucleus. In total, some 40 isoquinoline alkaloids, representing many different structural
types, have been isolated from opium (Preininger, 1985).Opium
consists of the dried latex exuded from unripe seed capsules
of opium poppy. The latex represents the multinucleate
cytoplasm of fused cells, known as laticifers, that comprise
a highly specialized internal secretory system (Fairbairn and
Kapoor, 1960; Nessler and Mahlberg, 1977, 1978, 1979). Mature laticifers possess numerous isoquinoline alkaloid-rich
(Fairbairn and Djote, 1970; Fairbairn et al., 1974; Rush et al.,
1985) and membrane-boundvesicles that are derived from dilations of the endoplasmic reticulum (Nessler and Mahlberg,
1977, 1978, 1979). In addition to the phenanthrene alkaloids,
which include the analgesics morphine and codeine, other
important classes of tetrahydroisoquinoline alkaloids found in
opium poppy include the benzylisoquinolines, such as the
vasodilator papaverine and the antispasmodic noscapine, and
the benzophenanthri- dines, such as the antibiotic sanguinarine (Phillipson, 1983; Preininger, 1985).
Classic radiotracer experiments and modern applications of
plant cell culture technology have contributed to our knowledge
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of the chemistry and enzymology of specific tetrahydroisoquinoline branch pathways. For example, the unique biosynthetic
pathways from the common branch point intermediate(S)-reticuline to either morphine (reviewed in Brochmann-Hanssen,
1985) or sanguinarine (reviewed in Kutchan and Zenk, 1993)
are well established (Figure 1). Many of the enzymes that catalyze these multistep pathways have also been characterized
(Zenk, 1985; Gerardy and Zenk, 1993; Kutchan and Zenk,
1993). However, despite extensive work on alkaloid chemistry
and the characterization of many biosynthetic enzymes, the
molecular mechanisms that regulate the activity of key enzymes and control the synthesis of specific alkaloids are not
well understood.
The precise chemistry and the nature of enzymes involved
in the early steps of isoquinoline alkaloid biosynthesis are less
well characterized. Most tetrahydroisoquinoline alkaloids are
derived from (S)-norcoclaurine (Stadler et al., 1987, 1989), which
is formed via the condensation of dopamine and 4-hydroxyphenylacetaldehyde(Figure 1). Both precursors originate from
L-tyrosine, or ~-3,4-dihydroxyphenylalanine(dopa) in the case
of dopamine, with the initial and potentially rate-limiting steps
catalyzed by a tyrosineldopa decarboxylase (TYDCldopa decarboxylase; EC 4.1.1.25; Figure 1). Key regulatory functions
are often associated with enzymes, such as phenylalanine
ammonia-lyase (EC 4.3.1.5; Hahlbrock and Scheel, 1989), that
operate at the interface of primary and secondary metabolism. A similar regulatory function has been suggested for
TYDC and dopa decarboxylase (Marques and Brodelius, 1988;
Kawalleck et al., 1993).
Despite almost 200 years of intensive research on the chemistry and biosynthesisof morphine and related alkaloids, much
remains to be learned about the basic biology of alkaloid biogenesis in poppy and other plants. For example, it has long
been known that isolated latex can convert radioactive amino
acid and amine precursors into phenanthrene alkaloids
(Fairbairn and Wassel, 1964a, 1964b; Fairbairn et al., 1968;
Fairbairn and Djote, 1970). In addition, enzymes of both general
metabolism (Antoun and Roberts, 1975) and alkaloid biosynthesis, including TYDC (Roberts, 1971, 1974; Roberts and
Antoun, 1978; Roberts et al., 1983), have been detected in
poppy latex. However, little is known about the relationships
between plant development, cellular differentiation, alkaloid
biosynthetic gene expression, and accumulation Of specific
alkaloids in opium poppy.
In this study, we examined the differential and tissue-specific
expression of the TYDC gene family (Facchini and De Luca,
1994) as a putative marker for the localization and developmental
regulation of tetrahydroisoquinoline alkaloid biosynthesis in
opium poppy. To determine the precise cell-specific localization of TYDC genes, we used in situ RNA hybridization
techniques with probes specific for two differentiallyexpressed
subgroups of WDC genes (representedby WDC7 and WDCP).
We demonstrated that TYDC gene expression becomes developmentally restricted to the vascular tissues in stems and
roots. WDC7-like transcripts are most abundant in root phloem,
whereas TYDCP-like transcripts are predominant in the
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Figure 1. Biosynthesis of Morphine, Codeine, and Sanguinarine in
Opium Poppy.
The proposed biosynthetic pathway from L-tyrosine to the common
tetrahydroisoquinoline intermediate (S)-norcoclaurineand the branch
pathways from (S)-reticulineto the benzophenanthridine alkaloid sanguinarine and the phenanthrenealkaloids codeine and morphine. The
sites of action of tyrosine/dopa decarboxylase (TYDCIDODC)and berberine bridge enzyme (BBE) are indicated.
metaphloem of the mature stem. The low leve1 of TYDC gene
expression in carpels contrasts sharply with the abundance
of alkaloid-rich latex that can be extracted from seed capsules.
These data suggest that carpelslcapsules are sites of alkaloid
accumulation and that stems and roots are more likely the sites
of alkaloid biosynthesis. The major alkaloids found in aerial
organs were morphine and noscapine, whereas sanguinarine
was found at high levels only in roots. Thus, the.differentia1
expression of TYDC genes in stems and roots and the accumulation of specific alkaloids in these organs suggest that different
developmental programs may regulate the biosynthesisof specific secondary products.
RESULTS
The TYDC Gene Family Exhibits Differential and
Organ-Specific Expression
Four members of the TYDC gene family in opium poppy have
been cloned and characterized previously (Facchini and
De Luca, 1994). Full- and partial-length cDNA and genomic
clones for cTY DC1, cTY DC2, cTY DC3, and TYDC4 were separated by agarose gel electrophoresis, and two identical blots
were probed independently with radiolabeled cTYDCl and
Localization of Alkaloid Biosynthetic Genes
CTYDC2 as shown in Figure 2A. The stringent hybridization
conditions depicted in Figure 2A were identical to those used
for all other nucleic acid hybridizations reported in this study.
In previous work, we showed that cTYDCI and TYDC4 share
>84% nucleotide sequence identity. Similarly, cTYDC2 and
cTYDCS share >90% nucleotide sequence identity in regions
that overlap. In contrast, cTYDCI and cTYDC2, as representatives of the two groups, share <73°/o nucleotide sequence
identity. Thus, the cTYDCI and cTYDC2 probes revealed only
genes with a minimum sequence identity of ~85% (Figure 2A).
The differential and organ-specific expression of TYDC genes
in mature opium poppy plants is shown in Figure 2B. The predominant site of expression for TYDC7-like genes is the root,
although much lower levels of expression were also detected
in stems. In contrast, rYDC2-like genes are expressed predominantly in stems, but significant expression was also
detected in roots. Lower levels of TVDCZ-like transcripts were
observed in total RNA from sepals 1 day before anthesis and
from carpels 1 day after anthesis. No signals were detected
with the cTYDCI or CTYDC2 probes in total RNA isolated from
the leaf blade (minus midrib), anthers, or petals collected 1
day after anthesis. The length of rVDC2-like transcripts in both
roots and sepals appeared to be slightly shorter than those
in stems and carpels (Figure 2B), suggesting further differential and organ-specific expression of the TYDC gene family in
opium poppy.
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TYDC1 TYDC2 TYDC3 TYDC4
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TYDC Genes Are Expressed in All Internodes
of the Stem
The localization of abundant TYDC transcripts in stems and
roots suggested that these organs are potential primary sites
fortetrahydroisoquinoline alkaloid biosynthesis in opium poppy.
Poppy has a predominantly fibrous root system, with relatively
few roots >1 to 2 mm in diameter emanating from the central
axis. The observed pattern of expression in small fibrous roots
(<1 mm in diameter) and larger roots (5 to 10 mm in diameter)
was identical (data not shown). 7YDC transcript levels along
the length of the stem at successive internodes are shown in
Figure 3. Expression of 7~YDC7-like genes was observed from
the first to sixth internode and rapidly declined in subsequent
internodes to the base of the stem. At least two 7YDC7-like
transcripts that differ in size by several hundred nucleotides
were detected. The larger transcript(s) appeared to be similar
in size to the major 7VDC7-like transcript(s) occurring in the
root. Levels of rVOC2-like transcripts decreased gradually in
successive internodes of the stem. Low levels of rVDC2-like
transcripts were detectable even in the final internodes just
above the initiation of root tissue. The relative abundance of
TVDC transcripts in roots and carpels is shown for comparison. Furthermore, 7YDC7-like transcript levels in the root, as
determined by scanning laser densitometry, were at least 250fold higher than those in the stem (Figure 3), whereas TYDC2like transcript levels were at least twofold higher in some parts
of the stem than in roots (Figure 3, compare lanes 2 and 10).
TYDC2
Figure 2. Specificity of TYDC Probes and Differential Expression of
TYDC Genes in Opium Poppy.
(A) Each lane contains 100 ng of DNA representing cTYDCI, CTYDC2,
cTYDCS, and a fragment of TYDC4. The gel was stained with ethidium
bromide before blotting. Blots were hybridized with the 32P-labeled
probes indicated at left.
(B) RNA gel blot analysis of TYDC transcript levels in various organs
from mature plants at anthesis. Each lane contains 15 ng of total RNA
fractionated on formaldehyde-agarose gels, transferred to nitrocellulose membranes, and hybridized with 32P-labeled cTYDCI or cTYDC2
probes at high stringency Gels were stained with ethidium bromide
before blotting to ensure equal loading of samples. The conditions
for hybridization and washing were identical for blots shown in (A) and
(B), and blots were developed after a 12-hr film exposure.
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The autoradiograms shown in Figure 3 were exposed fourfold
longer than those shown in Figure 2. Although this increased
the background of the blot, it improved detection of message
levels in several different tissues. With the longer exposure
time, low levels of TVDC7- and TYDC2-like transcripts in carpel tissue were clearly detectable, and all parts of the stem
appeared to express TYDC genes.
TYDC2-Like Genes Are Expressed at Low Levels in
Floral Buds before Anthesis
The traditional method used to harvest the alkaloid-rich latex
of opium poppy involves making incisions in the unripe seed
capsule and collecting the white exudate. Results presented
in Figures 2B and 3 demonstrate that TYDC transcripts are
not abundant in carpels at anthesis. To determine the possible
relationship of TYDC gene expression to alkaloid biosynthesis and accumulation, we studied the accumulation of TYDC
transcripts in carpels dissected from floral structures at different stages of development, as shown in Figure 4A. The carpels
of opium poppy consist of a large stigma and a short style,
and each contains many ovules. After pollination, the carpels
expand over a period of a few weeks (Figure 4A, stages 3 to
6) to form the large seed capsule containing hundreds of seeds.
Incisions made on the surface of the mature, but still unripe
seed capsule (stage 6) result in the exudation of considerable
amounts of latex.
Results shown in Figure 4B demonstrate that the levels of
expression of both TYDC1- and TVDC2-like genes continued
to decrease as the carpel developed and as the seed capsule
matured. The level of 7YDC2-like transcripts is higher in carpels than the level of 7VDC7-like transcripts, as shown in
Figures 2B and 3. In previous work (Facchini and De Luca,
1994), we demonstrated that the specific activity of TYDC in
carpels of plants at anthesis is much lower than that in stems
and roots. A decline in the level of TYDC activity correlates
with the decrease in the levels of TYDC transcripts during carpel and seed capsule development (data not shown). Thus,
although the laticifers of unripe seed capsules accumulate and
store isoquinoline alkaloids, the low level of IVDC expression
suggests that this tissue may not be the primary site of alkaloid biosynthesis.
TYDC mRNAs Are Localized in the Vasculature of Stem
and Root Tissues
We examined the spatial distribution of TYDC transcripts in
various tissues of opium poppy by in situ hybridization using
cTYDCl and cTYDC2 sense and antisense probes, as shown
in Figure 5. Transcripts of both TYDC1- and 7YDC2-like genes
are widely expressed throughout the phloem and selectively
expressed in many fewer cells in the primary xylem (Figure
5B) but are not expressed in the secondary xylem or in the
250-
TYDC1
TYDC2
Figure 3. Expression of TYDC Genes in Different Internodal Stem
Segments.
Each lane contains 15 ng of total RNA fractionated on formaldehydeagarose gels, transferred to nitrocellulose membranes, and hybridized with 32P-labeled cTYDCl or CTYDC2 probes at high stringency.
Gels were stained with ethidium bromide before blotting to ensure equal
loading of samples. Lanes 1 contain total RNA isolated from the carpel dissected from a flower at anthesis. Lanes 2 to 9 contain total RNA
isolated from eight internodal stem segments of a flowering opium poppy
plant at anthesis. Lanes 10 contain total RNA isolated from secondary
roots ~2 to 3 mm in diameter. Blots were exposed fourfold longer than
in Figure 2 to improve detection of TYDC transcripts in stem samples.
Localization of Alkaloid Biosynthetic Genes
periderm of well-differentiated mature poppy roots (Figures 5A
to 5D). No hybridization was detected when the CTYDC1 (Figure 5D) and cTYDC2 (data not shown) sense probes were used.
In situ hybridization of cross-sections of stems from the internode below the first leaf near the shoot apex revealed that
rVDCMike transcripts are localized to both the abaxial and
adaxial sides of the vascular cambium in all vascular bundles,
whereas 7VDC2-like transcripts are more abundant in the
phloem than in any other tissue (Figures 5E to 5H). Although
the spatial patterns of expression using cTYDCI or CTYDC2
antisense RNA probes were similar, the development time
Carpel Developmental Stage
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required to visualize the insoluble blue-purple reaction products
of the alkaline phosphatase-conjugated anti-digoxigenin antibody was considerably longer when the cTYDCI antisense
RNA probe was used (Figure 5F). No hybridization of either
antisense RNA probe was apparent in parenchymatous cortical cells adaxial to the vascular bundles or in the sclerenchyma
peripheral to the vasculature. Closer examination of vascular
bundles revealed that both TYDC1- and r/DC2-like transcripts
(Figures 5F and 5G) are associated predominantly with the
metaphloem and to a lesser extent with the protoxylem; however, the transcripts were not detected in cells of the cambial
layer and not clearly detected in cells associated with the protophloem and metaxylem. No hybridization was apparent when
the cTYDCI (data not shown) and CTYDC2 (Figure 5H) sense
probes were used.
TYDC transcripts were not detected in total RNA extracted
from young leaf blade tissue, as shown in Figure 2B. However,
in situ hybridization analysis of cross-sections through the
midrib of a young leaf and through young axillary buds demonstrated that both cTYDCI and CTYDC2 antisense RNA probes
hybridized to mRNAs present in cells in the vascular bundles
as well as in cortical and epidermal cells of younger tissues
(data not shown).
These results suggest that the tissue specificity of TYDC
gene expression is associated with the developmental stage
of the plant. In emerging axillary buds and in young leaves,
TYDC transcripts appeared to be most abundant in the develop-
ing vascular bundles but were also found at lower levels in
other cell types, whereas in mature roots (Figures 5B and 5C)
and stems (Figures 5F and 5G), TYDC gene expression is
clearly localized to the vasculature.
B
1
2
3
4
5
6
TYDC1
TYDC2
Figure 4. Expression of TYDC Genes during Carpel Development.
(A) Opium poppy reproductive organ development from floral bud (stage
1) through anthesis (stage 3) to maturation of the seed capsule just
before desiccation and seed ripening (stage 6).
(B) TYDC mRNA levels in carpel tissue at six stages (lanes 1 to 6)
of development. Ovules were removed from the carpels before RNA
extraction. Each lane contains 15 ng of total RNA fractionated on
formaldehyde-agarose gels, transferred to nitrocellulose membranes,
and hybridized with 32P-labeled cTYDCI or cTYDC2 probes at high
stringency. Gels were stained with ethidium bromide before blotting
to ensure equal loading of samples.
Roots and Aerial Organs Accumulate Different Major
Isoquinoline Alkaloids
Opium poppy accumulates three major structural types of wellcharacterized tetrahydroisoquinoline alkaloids. In aerial organs,
the alkaloids are contained within the vesicle-rich cytoplasm
of mature laticifers (Fairbairn and Djote, 1970; Fairbairn et al.,
1974; Roberts et al., 1983; Nessler et al., 1985; Rush et al.,
1985). Latex collected from unripe seed capsules (Figure 4,
stage 6), stems, or young leaves exhibited a profile of extractable alkaloids that was both quantitatively and qualitatively
similar, as shown in Figure 6. In contrast, the alkaloid profile
of the root consisting mainly of sanguinarine and morphine
was different from that of the latex of carpels, stems, and leaves
in which no sanguinarine was detected.
The identities of morphine and sanguinarine were compared
with authentic standards and verified by mass spectrometry
(see Methods). No sanguinarine was detected in latex samples by HPLC (data not shown). The levels of morphine and
sanguinarine in capsule, stem, and leaf latex as well as in roots
were approximated by comparison with authentic standards
using laser densitometry of thin-layer chromatograms. The levels
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The Plant Cell
Figure 5. Localization of TYDC Transcripts in the Root and Stem of Opium Poppy by in Situ RNA Hybridization.
Localization of Alkaloid Biosynthetic Genes
of morphine in capsules, stems, and leaves varied between
43 and 51 ug/mL, whereas roots contained 9.7 and 9.3 mg/g
dry weight, respectively, of morphine and sanguinarine. The
specific localization of sanguinarine accumulation in roots and
the more complex alkaloid pattern found in the latex of aerial
organs are representative of the dichotomy of alkaloid accumulation in opium poppy.
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DISCUSSION
In this study, we examined the differential and tissue-specific
expression of TYDC genes in opium poppy and compared the
localization of TYDC transcripts with the accumulation of different alkaloids produced by the plant. The alkaloids synthesized
by opium poppy and related species are of the tetrahydroisoquinoline type. In the Papaveraceae, the major alkaloids of
aerial organs are sequestered to vesicles found in the
cytoplasm of articulated and anastomized internal secretory
cells known as laticifers (Fairbairn et al., 1974; Messier and
Mahlberg, 1977,1978,1979). Although laticifers are associated
with vascular tissue in all organs, the principal alkaloids that
accumulate in roots of various species are often different from
those of aerial organs (Phillipson, 1983; Preininger, 1985). In
this study, we found that among several major latex alkaloids
found in all aerial organs of opium poppy, only morphine also
occurred as a major root alkaloid, whereas significant levels
of sanguinarine occurred only in poppy roots. All of these
compounds represent end products of specific tetrahydroisoquinoline alkaloid branch pathways that originate from common
intermediates. TYDC represents the first of these common biosynthetic steps that channel the aromatic amino acids
L-tyrosine and L-dopa into the alkaloid biosynthetic pathways
(Rueffer and Zenk, 1986; Stadler et al., 1987, 1989).
TYDC in opium poppy is encoded by a family of 10 to 14
genes, of which we have cloned four members (Facchini and
De Luca, 1994). Within the TYDC gene family, two subgroups
based on sequence identity are evident. Each subgroup consists of approximately six members, all of which share ^90%
identity at the nucleotide and amino acid levels. In contrast,
comparison of subgroup members (represented by TYDC1 and
TYDC2) reveals sequence identities of <75°/o. Although the
catalytic properties of the different TYDC isoforms are similar
Origin
Latex
Figure 6. Thin-Layer Chromatographic Analysis of Alkaloids Found
in Latex and in Root Extracts of Mature Opium Poppy Plants.
Alkaloids were extracted in methanol from an equal volume of latex
collected from mature seed capsules, leaves, and stems. Roots were
extracted in methanol from 100 mg of frozen powdered tissue. Samples were applied to silica gel 60 F254 thin-layer chromatography
plates for separation and identification of alkaloids. Authentic standards
of sanguinarine (S), noscapine, papaverine, codeine, morphine (M),
and opium were also run. Alkaloids were detected by their absorbance
or fluorescence after illumination at 365 nm.
(Facchini and De Luca, 1994,1995), the TYDC gene family exhibits differential and organ-specific expression (Facchini and
De Luca, 1994). In this study, we determined that the tissuespecific expression of TYDC genes is developmentally regulated, because the expression of TYDC genes is strictly
associated with vascular tissues in mature roots and stems
Figure 5. (continued).
Tissues were fixed, dehydrated, embedded in paraffin, cut into 10-nm cross-sections, and hybridized at high stringency with digoxigenin-labeled
cTYDd or cTYDC2 RNA probes. Hybridization was detected with anti-digoxigenin antibodies conjugated to alkaline phosphatase and visualized
after conversion of enzyme substrates to the insoluble blue-purple product.
(A) Root cross-section stained with aniline safranme and astra blue.
(B) to (D) Root cross-sections hybridized with cTYDCt (B) and CTYDC2 (C) antisense probes and the cTYDC! (D) sense probe.
(E) Stem cross-section stained with aniline safranme and astra blue.
(F) to (H) Stem cross-sections hybridized with cTYDCt (F) and CTYDC2 (G) antisense probes and the cTYDC2 (H) sense probe. Bar in (H) =
0.5 mm. pd. penderm; ph. phloem: vc, vascular cambrium; xy, xylem.
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The Plant Cell
but is also expressed in cortical and epidermal cells of tissues
from young leaves and axillary buds at earlier stages of development. In roots, TYDC!-like genes are preferentially
expressed, whereas in stems and other aerial organs, TYDCPlike genes exhibit higher expression levels. In mature aerial
organs, TYDC gene expression appears to predominate in
metaphloem and to a lesser extent in protoxylem of vascular bundles.
Laticifers of opium poppy are a highly specialized cell type
that has been shown to possess alkaloid biosynthetic enzymes,
including TYDC (Fairbairn and Wassel, 1964b; Fairbairn et al.,
1968; Roberts et al., 1983). Thus, localizationof abundant TYDC
transcripts in the phloem is consistent with fhe expected developmental origins of laticifers. Although not distinguishable
in the tissue sections shown in Figure 5, the location of laticifers
in mature roots, stems, and leaves is always associated with
the phloem, and they are often positioned adjacent to sieve
tube members (Nessler and Mahlberg, 1977, 1978, 1979).
Our data support the role of vascular tissue as the primary
site of isoquinoline alkaloid biosynthesis. In situ detection of
TYDC transcripts in developing phloem, together with previous reports on the presence of TYDC and other alkaloid
biosynthetic enzymes in the cytoplasm of phloem-derived
laticifers (Roberts, 1971, 1974; Roberts and Antoun, 1978;
Roberts et al., 1983), suggests that isoquinoline alkaloid biosynthesis occurs in these tissues in stems and roots. We have
found that the developing carpel lacks abundant TYDC transcripts and has relatively low enzyme activity compared with
other tissues (Facchini and De Luca, 1994). Salutaridine synthase (SS) activity, which catalyzes the sixth to last step in the
biosynthesis of morphine, is also low in the developing carpel
(Gerardy and Zenk, 1993), suggesting that alkaloids accumulate, but are not synthesized, in the latex of the seed capsule.
The articulated laticifers of opium poppy contain perforated
transverse walls between adjoining cells that would allow for
bulk flow of latex (Nessler and Mahlberg, 1977,1978,1979) and
tissue-specific alkaloid accumulation. To account for the high
levels of alkaloids found in poppy capsules, it has been suggested that stem latex, where the alkaloids are synthesized,
is then translocated into the growing capsule during its rapid
expansion after peta1 fall (Fairbairn et al., 1974).
These results establish provocativecorrelations betweenthe
differential expression of TYDC genes, the tissue-specific localization of TYDC transcripts in mature organs, and the
accumulation of specific alkaloids in opium poppy. For example, TYDC7-like genes are expressed predominantly in tissue
corresponding to the phloem in roots, where sanguinarine and
morphine are the principal alkaloids. The lack of detectable
quantities of sanguinarine in latex and the low levels of TYDC7like transcripts suggest that the expression of TYDCI-like genes
may be coordinately regulated with that of other genes specifically involved in benzophenanthridine alkaloid biosynthesis.
The abundance of morphine in latex and its presence in root
extracts, together with the high levels of expression and
localization of TYDCP-like transcripts in the phloem of mature
stems and roots, suggest a possible association between the
regulation of TYDCP-like genes and others specific to the biosynthesis of phenanthrene alkaloids. However, the reguiation
of other alkaloid biosynthetic enzymes in opium poppy and
related species is poorly understood. Only the spatial pattern
of SS activity has been determined (Gerardy and Zenk, 1993),
and it resembles that of TYDC (Facchini and De Luca, 1994).
A key step in sanguinarine biosynthesis, the berberine bridge
enzyme (BBE), has been cloned and characterized from
Eschscholzia californica (Dittrich and Kutchan, 1991). ExpresSion of BBE has been shown to be induced by various elicitors
in E. californica cell cultures; however, the tissue-specific expression of BBE has not been investigated. The availability
of genes, such as SS and BBE, from specific branch pathways
is essential for further study of the coordinated regulation of
specific genes and the developmental, temporal, and spatial
patterns of isoquinoline alkaloid biosynthesis.
Because the functions in plants of most alkaloids are not
known, interpretations of the occurrence of specific alkaloids
in specific organs are highly speculative. Most isoquinoline
alkaloids, including morphine and noscapine, induce potent
pharmacological effects, and their accumulation in aerial organs could serve as a deterrent to herbivores. Sanguinarine
has been shown to be an active fungitoxin at concentrations
as low as 10 mM, although its role as a phytoalexin in plants
has not been verified unequivocally (Cline and Coscia, 1988).
The occurrence of sanguinarine in the root of opium poppy
and other Papaveraceae may confer protection against soilborne pathogens. It is also likely that the protective potency
and adaptability of the chemical arsenal of plants increase as
a function of the structural diversity of the products that they
accumulate. The complex development-, organ-, and tissuespecific regulation oí alkaloid biosynthesis illustrated in this
and other reports suggests that the roles of plant secondary
metabolites may be more significant than previously suspected.
METHODS
Plant Material
Plants (Papaversomniferum cv Marianne)were grown under standard
greenhouse conditions at a daylnight temperature regime of 20/18"C.
Tissue samples were harvested from mature flowering plants 1 day
after anthesis or at specific stages of carpel development as indicated.
Latex was isolated from capsules, stems, and leaves by cutting the
organ from the plant and collecting the white exudate with a glass
capillary.
Tyrosine/Dopa Decarboxylase Probes
Previously,we describedthe isolation of full- and partial-lengthcDNA
and genomic clones for tyrosine/dopadecarboxylasefrom ?f somniferum. They are designated cTYDC1, TYDC1, cTYDC2, cTYDC3, and
TYDC4 (Facchini and De Luca, 1994).AI1 inserts were cloned using
Localization of Alkaloid Biosynthetic Genes
StratagenepBluescript SK+ (genomicclones)or SK- (cDNAs). Probes
for DNA and RNA gel blot hybridizationswere synthesizedfrom cDNA
inserts isolated by digestingplasmidswith EcoRl and Xhol. The partiallength cTYDCl and cTYDC3 inserts were 1554 and 927 bp, respectively, whereas the full-length cTYDC2 insert was 1876 bp. A 1.9-kb
genomic fragment containing the 3’ translated region of TYDC4 was
subcloned and subsequently isolated using Hindlll. Probesfor in situ
RNA hybridizationanalysiswere synthesizedfrom plasmids that were
linearizedwith either Sacl (antisense) or Kpnl (sense) and treated with
2 mg mL-’ proteinase K for 30 min at 37%.
Nucleic Acid lsolation and Analysis
lsolated DNA inserts were separated on 1.0% agarose gels and immobilized on nylon membranes. Total RNA for gel blot analysis was
isolated according to Logemann et al. (1987), and 15 pg was fractionated on 1.O% formaldehyde-agarosegels before being transferred to
nitrocellulose membranes(Sambrooket al., 1989). DNA and RNAgel
blots were hybridized with random primed 3ZP-labeled-DNAinserts
(Feinbergand Vogelstein, 1984) at 65OC in 0.25 M sodium phosphate
buffer, pH 8.0, 7% SDS, 1% BSA, 1 mM EDTA. Blots were washed at
55OC twice with 2 x SSC (1 x SSC is 0.15 M NaCI, 0.015 M sodium
citrate, pH 7.0), 0.1% SDS, and twice with 0.2 x SSC, 0.1% SDS
(Sambrook et al., 1989). Blots were autoradiographed with an intensifying screen on RXlOO x-ray film (Fuji, Montreal, Canada) for 24 hr
or longer.
In Situ Hybridization
In situ RNA hybridizations were performed essentially as described
by Cox and Goldberg (1988), with modifications used by Wang et al.
(1992). Tissue samples were fixed in FAA (50% ethanol, 5% acetic
acid, 10% formalin) at room temperature for 1 to 2 days, depending
on the size of the material. After dehydration with tert-butanol and
embedding in paraffin, samples were cut into 10-pm sections and
mountedon replicateslides coated with poly-L-lysine. Tissue sections
were deparaffinized, hydrated, and subjected to the following series
of treatments: 0.2 N HCI for 20 min at room temperature, 2 pg mL-l
proteinase K for 30 min at 37% and 0.25% acetic anhydride for 10
min at room temperature. Sense and antisensetranscriptsfor cTYDCl
and cTYDC2 were synthesized and labeled in vitro with digoxigenin11-UTP(Boehringer Mannheim)usingT7 or T3 RNA polymerase. Slides
were prehybridized for 1 hr at 5OoC in 50% formamide, 30 mM NaCI,
10 mM Tris-HCI, pH 6.8, 10 mM sodium phosphate, pH 6.8,5 mM EDTA,
10% dextran sulfate, 10 mM DTT, 150 mg mL-l yeast tRNA, and 500
mg mL-’ polyadenylic acid. Tissue sections were hybridized in the
same solution containing 0.2 mg mL-’ digoxigenin-labeled probe for
15 hr at 50OC. After hybridization, slides were treated with 50 pg mL-’
RNaseA for 30 min at 37OC to digest nonhybridized probe and subsequently washed in 2 x SSC for 2 hr at room temperature, 1 x SSC
for 2 hr at room temperature, and 0.1 x SSC for 1 hr at 65%. Hybridized probe was detectedwith anti-digoxigenin antibodies conjugated
to alkaline phosphatase(Boehringer Mannheim)and visualized by color
developmentusing5-bromo-4-chlorc-3-indolylphosphateand nitro blue
tetrazolium as substrates. Sections were photographed with a Leitz
Ortholux II microscope(Leica Canada Ltd., Toronto, Canada)on Kodak
RoyalGold 100 film.
1819
Alkaloid Extraction and Thin-Layer Chromatography
Alkaloids were extracted from fresh latex (50 pL) collected from mature poppy seed capsules, stems, or leaves with 0.2 mL of methanol
for 10 min at 100OC. Roots (100 mg) were ground to a fine powder under liquid nitrogen and extracted with 0.5 mL of methanol for 10 min
at 100OC.Debrisfrom all sampleswas remwed by centrifugation.Samples (10 pL) of plant extracts were applied to silica gel 60 FZs4
thin-layer chromatography plates (EM Separations, Darmstadt,
Germany) and developed in a solvent system consistingof acetone/toluene/ethanol/concentratedammonia (45:45:7:3 [v/v]) (Wagner et al.,
1984). Standards for sanguinarine, noscapine, and papaverine were
purchasedfrom Sigma. Morphine, codeine, and opium standards were
a kind gift from U. Eilert (Technische Universitat, Braunschweig, Germany).Plateswere dried at room temperatureand photographedunder
ultraviolet illumination at 365 nm on Kodak RoyalGold 100 film. Extracts were separated by HPLC (600E HPLC and 991 photodiodearray
detector; Waters, Montreal, Canada) on a Waters Nova Pak C18 reverse phase (3.9 x 300 mm) column using an isocratic gradient of
methanol/water (6:4) containing 0.1% triethanolamine (Kraml and
DiCosmo, 1993). The retention time for sanguinarine in root extracts
was 16.1min and was identical with that obtained for the corresponding standard. Furthermore, an absorption spectrum identical to that
of sanguinarine standard was obtainedfrom the root-derivedalkaloids
eluting at this retention time.
ldentification of Morphine and Sanguinarine by
Mass Spectrometry
Morphine and sanguinarine standards as well as morphins from latex, morphine from roots, and sanguinarine from roots were purified
by thin-layer chromatography.Spots corresponding to morphine and
sanguinarinestandards were removed, and alkaloids were redissolved
in methanol. lnsoluble silica was removed by centrifugation, and the
supernatants were subjected to mass spectrometry. Low-resolution
mass spectra (direct probe mass spectrometry with a VG 7070F gas
chromatography/massspectrometer)(V.G. Analytical Ltd., Manchester,
UK) obtained for morphine (mlz: 285 [IOO], 268 [l8]. 215 127,174 [19],
162 [27], and 124 [26]) in latex were similar to those obtained for morphine in roots, and these were identical to those obtained for the
morphine standard(mlz: 285 [lOO], 268 [13], 215 [%I, 174 [IA, 162 [34],
and 124 [lg]). The low-resolutionmassspectraobtained for sanguinarine ( m h : 332 [IOO], 317 [34], 194 [26], and 149 [IOO]) in roots were
identical to those obtainedfor sanguinarine standards ( m h : 332 [lOO],
317 [30], 194 [38], and 149 [32]).
When a sample is placed in a mass spectrometer, it is bombarded
with a beam of energetic electrons that causes the molecules to ionize and break down into many fragments. Reported above are the
masses of the major ions obtained from the morphine standard, with
285 being the mass of the largest ion produced and 100 being the
relativevalue for the largest peak. Subsequent masses(268,215, 174,
162, and 124) representother major ions produced that are representative of morphine. The numbers in bracketsrepresentthe intensities
of each ion relative to the largest peak, which is 100.
ACKNOWLEDGMENTS
We are grateful to Drs. Udo Eilert for the generous gifts of alkaloid
standards, Joachim Vieth (Institut de Rechercheen Biologie VkgBtale)
1820
The Plant Cell
for his invaluable assistancewith the interpretation of anatomicaldata,
James Berry (State University of New York at Buffalo) for providing
us with the in situ hybridization protocol used in his laboratory, Edward
YeUng (University of Calgary) for assistance with photomicrography,
and Gijs van Rooijen for assistancewith the laser densitization of thinlayer chromatography results. We also thank Sylvain Lebeurier for maintaining plants in the greenhouse, Julie Poupartfor technical assistance
with alkaloid extractions, Christiane Morisset for advice on the preparation of tissue sections, Jean-Luc Verville for photographic work, and
Drs. Normand Brisson and Benoit St.-Pierre for critical reading of the
manuscript and helpful discussions. This work was funded by Natural
Sciences and Engineering ResearchCouncil of Canadaoperating and
strategic grants to V.D.L. P.J.F. was supported in part by a Natural
Sciences and Engineering Research Council of Canada Postdoctoral
FellowshiD.
Fairbairn,J.W., and Wassel, G. (1964b). The alkaloids of fapaversomniferum L. (I. Biosynthesis in isolated latex. Phytochemistry 3,
583-585.
Fairbairn, J.W., Djote, M., and Paterson, A. (1968).The alkaloids of
fapaver somniferum L. VIL Biosynthetic activity of the isolated latex. Phytochemistry 7, 2111-2116.
Fairbairn, J.W., Hakim, F., and El Kheir, Y. (1974).Alkaloidal storage,
metabolism, and translocation in the vesicles of fapaver somniferum latex. Phytochemistry 13, 1133-1139.
Feinberg, A.P., and Vogelstein, B. (1984).A techniquefor radiolabeling
DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem. 137, 266-269.
Gerardy, R., and Zenk, M.H. (1993). Formation of salutaridine from
(R)-reticulineby a membrane-boundcytochrome P-450 enzyme from
fapaver somniferum. Phytochemistry 32, 79-86.
Hahlbrock, K., and Scheel, D. (1989).Physiology and molecular bi-
Received August 11, 1995; accepted August 30, 1995.
REFERENCES
ology of phenylpropanoid metabolism. Annu. Rev. Plant Physiol.
Plant MOI.Biol. 40, 347-369.
Kawalleck, P., Keller, H., Hahlbrock, K., Scheel, D., and Somssich,
I.E. (1993). A pathogen-responsive gene of parsley encodes tyrosine decarboxylase. J. Biol. Chem. 268, 2189-2194.
Kraml, M.M., and DiCosmo, F. (1993).A rapid high performance liq-
Antoun, M.D., and Roberts, M.F. (1975).Some enzymes of general
metabolism in the latex of fapaversomniferum. Phytochemistry 14,
909-914.
Brochmann-Hanssen,E. (1985). Biosynthesisof morphinan alkaloids.
In The Chemistry and Biology of lsoquinoline Alkaloids, J.D.
Phillipson, M.F. Roberts, and M.H. Zenk, eds (Berlin: SpringerVerlag), pp. 229-239.
Brownstein, M.J. (1993). A brief history of opiates, opioid peptides,
and opioid receptors. Proc. Natl. Acad. Sci. USA 90, 5391-5393.
Cline, S.D., and Coscia, C.J. (1988). Stimulation of sanguinarine
production by combined funga1 elicitation and hormonal deprivation in cell suspensioncultures of fapaverbracteatum. Plant Physiol.
86, 161-165.
Cox, K.H., and Goldberg, R.B. (1988). Analysis of plant gene expression. In Plant Molecular Biology: A Practical Approach, C.H. Shaw,
ed (Oxford: IRL Press), pp. 1-35.
Dittrich, H., and Kutchan, T.M. (1991).Molecular cloning, expression,
and induction of berberine bridge enzyme, an enzyme essential to
the formation of benzophenanthridine alkaloids in the response
of plants to pathogenic attack. Proc. Natl. Acad. Sci. USA 88,
9969-9973.
Facchini, P.J., and De Luca, V. (1994).Differential and tissue-specific
expressionof a gene family for tyrosine/dopa decarboxylase in opium
poppy. J. Biol. Chem. 269, 26684-26690.
Facchini, P.J., and De Luca, V. (1995).Expression in Escherichia coli
and partia1 characterization of two tyrosine/dopa decarboxylases
from opium poppy. Phytochemistry 38, 1119-1126.
Fairbairn, J.W., and Djote, M. (1970). Alkaloid biosynthesis and
metabolism in an organelle fraction of fapaver somniferum.
Phytochemistry 9, 739-742.
Fairbairn, J.W., and Kapoor, L. (1960). The laticiferous vessels of
fapaver somniferum L. Planta Med. 13, 1133-1139.
Fairbairn, J.W., and Wassel, G. (1964a).The alkaloidsof fapaversomniferum L. I. Evidence for a rapid turnover of the major alkaloids.
Phytochemistry 3, 253-258.
uid chromatography method for the separation of the alkaloid
precursor L-tyrosine and six tetrahydroisoquinoline alkaloids of
fapaver somniferum. Phytochem. Anal. 4, 103-104.
Kutchan, T.M., and Zenk, M.H. (1993). Enzymology and molecular
biology of benzophenanthridine alkaloid biosynthesis.J. Plant Res.
3, 165-173.
Logemann, J., Schell, J., and Willmitzer, L. (1987). lmprovedmethod
for the isolationof RNAfrom plant tissues. Anal. Biochem.163, 16-20.
Marques, I.A., and Brodelius, P. (1988). Elicitor-induced L-tyrosine
decarboxylase from plant cell suspension cultures. 11. Partia1characterization. Plant Physiol. 88, 52-55.
Nessler, C.L., and Mahlberg, P.G. (1977). Cell wall perforations in
laticifers of fapaver somniferum L. Bot. Gaz. 138, 402-408.
Nessler, C.L., and Mahlberg, P.G. (1978).Ontogeny and cytochemistry of alkaloid vesicles in laticifers of fapaver somniferum L. Am.
J. Bot. 64, 541-551.
Nessler, C.L., and Mahlberg, P.G. (1979).Ultrastructure of laticifers
in redifferentiated organs on callus from fapaver somniferum
(Papaveraceae).Can. J. Bot. 57, 675-685.
Nessler, C.L., Allen, R.D., and Galewsky, S. (1985). ldentification
and characterization of latex-specific proteins in opium poppy. Plant
Physiol. 79, 499-504.
Phillipson, J.D. (1983).lntraspecific variation and alkaloids of fapaver
species. Planta Med. 48, 187-192.
Preininger, V. (1985).Chemotaxonomyof the Papaveraceaealkaloids.
In The Chemistry and Biology of lsoquinoline Alkaloids, J.D.
Phillipson, M.F. Roberts, and M.H. Zenk, eds (Berlin: SpringerVerlag), pp. 23-37.
Roberts, M.F. (1971). Polyphenolasesin the 1OOOgfractions of fapaver
somniferumlatex. Phytochemistry 10, 3021-3027.
Roberts, M.F. (1974). Oxidation of tyrosine by fapaver somniferum latex. Phytochemistry 13, 119-123.
Roberts, M.F. (1988). lsoquinolines (fapaver alkaloids). In Cell Cul-
ture and Somatic Cell Genetics of Plants, Vol. 5, F. Constabel and
I.K. Vasil, eds (New York: Academic Press), pp. 315-334.
Localization of Alkaloid Biosynthetic Genes
Roberts, M.F., and Antoun, M.D. (1978). The relationship between
L-dopadecarboxylase in the latex of Papaversomniferum and alkaloid formation. Phytochemistry 17, 1083-1087.
Roberts, M.F., McCarthy, D., Kutchan, T.M., and Coscia, C.J. (1983).
Localization of enzymes and alkaloidal metabolites in Papaver latex. Arch. Biochem. Biophys. 222, 599-609.
Rueffer, M., and Zenk, M.H. (1986). Distant precursors of benzylisoquinoline alkaloids and their enzymatic formation. 2. Naturforsch.
Sect. C Biosci. 42, 319-332.
Rush, M.D., Kutchan, T.M., and Coscia, C.J. (1985). Correlation of
the appearance of morphinan alkaloids and laticifer cells in germinating Papaver bracteatum seedlings. Plant Cell Rep. 4, 237-240.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory).
1821
Stadler, R., Kutchan, T.M., Loeffler, S.,Nagakura, N., Cassels, B.,
and Zenk, M.H. (1987). Revision of the early steps of reticuline biosynthesis. Tetrahedron Lett. 28, 1251-1254.
Stadler, R., Kutchan, T.M., and Zenk, M.H. (1989).(S)-Norcoclaurine
is the central intermediate in benzylisoquinolinealkaloidbiosynthesis.
Phytochemistry 28, 1083-1086.
Wagner, H., Bladt, S.,and Zgainski, E.M. (1984). Plant Drug Analysis. (Berlin: Springer-Verlag), pp. 78-79.
Wang, J.-L., Klessig, D.F., and Berry, J.O. (1992). Regulation of C4
gene expression tn developing Amaranth leaves. Plant Cell 4,
173-184.
Zenk, M.H. (1985).Enzymology of benzylisoquinoline alkaloid formation. In The Chemistry and Biology of lsoquinoline Alkaloids, J.D.
Phillipson, M.F. Roberts, and M.H. Zenk, eds (Berlin: SpringerVerlag), pp. 240-256.
Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase Genes and the Biosynthesis of
Isoquinoline Alkaloids in Opium Poppy.
P. J. Facchini and V. De Luca
Plant Cell 1995;7;1811-1821
DOI 10.1105/tpc.7.11.1811
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