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 mor- phine in aerial organs and roots, whereas sanguinarine, which is derived from a distinct branch pathway, accumulates only in roots. Expression of the TYDC gene family in opium poppy was investigated in relation to the organ-specific bio- synthesis 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 in stems and are also present in roots, whereas TYDCl-like transcripts are abundant only in roots. In situ hybridization anal- ysis revealed that the expression of TYDC genes is developmentally regulated. TYDC transcripts are associated with vascular tissue in mature roots and stems but are also expressed in cortical tissues at earlier stages of development. Expression of TYDC genes is restricted to metaphloem and to protoxylem in the vascular bundles of mature aerial organs. Localization of TYDC transcripts in the phloem is 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 con- trolled 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. Phar- maceutical 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 pre- vent 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 ac- tive 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 Biological Sciences, University of Calgary, 2500 Univer- sity Drive N.W., Calgary, Alberta, T2N 1N4 Canada.

Morphine belongs to the largest and most diverse class of naturally occurring alkaloids, which includes compounds based on the tetrahydroisoquinoline nucleus. In total, some 40 iso- quinoline 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). Ma- ture laticifers possess numerous isoquinoline alkaloid-rich (Fairbairn and Djote, 1970; Fairbairn et al., 1974; Rush et al., 1985) and membrane-bound vesicles that are derived from di- lations 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 sanguina- rine (Phillipson, 1983; Preininger, 1985).

Classic radiotracer experiments and modern applications of plant cell culture technology have contributed to our knowledge

1812 The Plant Cell

of the chemistry and enzymology of specific tetrahydroiso- quinoline branch pathways. For example, the unique biosynthetic pathways from the common branch point intermediate (S)-reti- culine 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 cata- lyze 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 en- zymes 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-hydroxy- phenylacetaldehyde (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 de- carboxylase; 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 metabo- lism. 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 chem- istry and biosynthesis of morphine and related alkaloids, much remains to be learned about the basic biology of alkaloid bio- genesis 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 biosyn- thesis, 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 localiza- tion of TYDC genes, we used in situ RNA hybridization techniques with probes specific for two differentially expressed subgroups of WDC genes (represented by WDC7 and WDCP). We demonstrated that TYDC gene expression becomes de- velopmentally 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)-norcoclaurine and the branch pathways from (S)-reticuline to the benzophenanthridine alkaloid san- guinarine and the phenanthrene alkaloids codeine and morphine. The sites of action of tyrosine/dopa decarboxylase (TYDCIDODC) and ber- berine 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 accumu- lation of specific alkaloids in these organs suggest that different developmental programs may regulate the biosynthesis of spe- cific 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 sepa- rated by agarose gel electrophoresis, and two identical blots were probed independently with radiolabeled cTYDCl and

Localization of Alkaloid Biosynthetic Genes 1813

CTYDC2 as shown in Figure 2A. The stringent hybridizationconditions depicted in Figure 2A were identical to those usedfor 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 andcTYDCS share >90% nucleotide sequence identity in regionsthat overlap. In contrast, cTYDCI and cTYDC2, as represen-tatives of the two groups, share <73°/o nucleotide sequenceidentity. Thus, the cTYDCI and cTYDC2 probes revealed onlygenes with a minimum sequence identity of ~85% (Figure 2A).

The differential and organ-specific expression of TYDC genesin mature opium poppy plants is shown in Figure 2B. The pre-dominant site of expression for TYDC7-like genes is the root,although much lower levels of expression were also detectedin stems. In contrast, rYDC2-like genes are expressed pre-dominantly in stems, but significant expression was alsodetected in roots. Lower levels of TVDCZ-like transcripts wereobserved in total RNA from sepals 1 day before anthesis andfrom carpels 1 day after anthesis. No signals were detectedwith the cTYDCI or CTYDC2 probes in total RNA isolated fromthe leaf blade (minus midrib), anthers, or petals collected 1day after anthesis. The length of rVDC2-like transcripts in bothroots and sepals appeared to be slightly shorter than thosein stems and carpels (Figure 2B), suggesting further differen-tial and organ-specific expression of the TYDC gene family inopium poppy.

TYDC Genes Are Expressed in All Internodesof the Stem

The localization of abundant TYDC transcripts in stems androots suggested that these organs are potential primary sitesfortetrahydroisoquinoline alkaloid biosynthesis in opium poppy.Poppy has a predominantly fibrous root system, with relativelyfew roots >1 to 2 mm in diameter emanating from the centralaxis. 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 alongthe length of the stem at successive internodes are shown inFigure 3. Expression of 7~YDC7-like genes was observed fromthe first to sixth internode and rapidly declined in subsequentinternodes to the base of the stem. At least two 7YDC7-liketranscripts that differ in size by several hundred nucleotideswere detected. The larger transcript(s) appeared to be similarin size to the major 7VDC7-like transcript(s) occurring in theroot. Levels of rVOC2-like transcripts decreased gradually insuccessive internodes of the stem. Low levels of rVDC2-liketranscripts were detectable even in the final internodes justabove the initiation of root tissue. The relative abundance ofTVDC transcripts in roots and carpels is shown for compari-son. Furthermore, 7YDC7-like transcript levels in the root, asdetermined by scanning laser densitometry, were at least 250-fold higher than those in the stem (Figure 3), whereas TYDC2-like transcript levels were at least twofold higher in some partsof the stem than in roots (Figure 3, compare lanes 2 and 10).

TYDC1 TYDC2 TYDC3 TYDC4

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TYDC2

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Figure 2. Specificity of TYDC Probes and Differential Expression ofTYDC 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 ethidiumbromide before blotting. Blots were hybridized with the 32P-labeledprobes indicated at left.(B) RNA gel blot analysis of TYDC transcript levels in various organsfrom mature plants at anthesis. Each lane contains 15 ng of total RNAfractionated on formaldehyde-agarose gels, transferred to nitrocellu-lose membranes, and hybridized with 32P-labeled cTYDCI or cTYDC2probes at high stringency Gels were stained with ethidium bromidebefore blotting to ensure equal loading of samples. The conditionsfor hybridization and washing were identical for blots shown in (A) and(B), and blots were developed after a 12-hr film exposure.

1814 The Plant Cell

The autoradiograms shown in Figure 3 were exposed fourfoldlonger than those shown in Figure 2. Although this increasedthe background of the blot, it improved detection of messagelevels in several different tissues. With the longer exposuretime, low levels of TVDC7- and TYDC2-like transcripts in car-pel tissue were clearly detectable, and all parts of the stemappeared to express TYDC genes.

TYDC2-Like Genes Are Expressed at Low Levels inFloral Buds before Anthesis

The traditional method used to harvest the alkaloid-rich latexof opium poppy involves making incisions in the unripe seedcapsule and collecting the white exudate. Results presentedin Figures 2B and 3 demonstrate that TYDC transcripts arenot abundant in carpels at anthesis. To determine the possiblerelationship of TYDC gene expression to alkaloid biosynthe-sis and accumulation, we studied the accumulation of TYDCtranscripts in carpels dissected from floral structures at differ-ent stages of development, as shown in Figure 4A. The carpelsof opium poppy consist of a large stigma and a short style,and each contains many ovules. After pollination, the carpelsexpand over a period of a few weeks (Figure 4A, stages 3 to6) to form the large seed capsule containing hundreds of seeds.Incisions made on the surface of the mature, but still unripeseed capsule (stage 6) result in the exudation of considerableamounts of latex.

Results shown in Figure 4B demonstrate that the levels ofexpression of both TYDC1- and TVDC2-like genes continuedto decrease as the carpel developed and as the seed capsulematured. The level of 7YDC2-like transcripts is higher in car-pels than the level of 7VDC7-like transcripts, as shown inFigures 2B and 3. In previous work (Facchini and De Luca,1994), we demonstrated that the specific activity of TYDC incarpels of plants at anthesis is much lower than that in stemsand roots. A decline in the level of TYDC activity correlateswith the decrease in the levels of TYDC transcripts during car-pel and seed capsule development (data not shown). Thus,although the laticifers of unripe seed capsules accumulate andstore isoquinoline alkaloids, the low level of IVDC expressionsuggests that this tissue may not be the primary site of alka-loid biosynthesis.

TYDC mRNAs Are Localized in the Vasculature of Stemand Root Tissues

We examined the spatial distribution of TYDC transcripts invarious tissues of opium poppy by in situ hybridization usingcTYDCl and cTYDC2 sense and antisense probes, as shownin Figure 5. Transcripts of both TYDC1- and 7YDC2-like genesare widely expressed throughout the phloem and selectivelyexpressed in many fewer cells in the primary xylem (Figure5B) but are not expressed in the secondary xylem or in the

250-

TYDC1

TYDC2

Figure 3. Expression of TYDC Genes in Different Internodal StemSegments.

Each lane contains 15 ng of total RNA fractionated on formaldehyde-agarose gels, transferred to nitrocellulose membranes, and hybrid-ized with 32P-labeled cTYDCl or CTYDC2 probes at high stringency.Gels were stained with ethidium bromide before blotting to ensure equalloading of samples. Lanes 1 contain total RNA isolated from the car-pel dissected from a flower at anthesis. Lanes 2 to 9 contain total RNAisolated from eight internodal stem segments of a flowering opium poppyplant at anthesis. Lanes 10 contain total RNA isolated from secondaryroots ~2 to 3 mm in diameter. Blots were exposed fourfold longer thanin Figure 2 to improve detection of TYDC transcripts in stem samples.

Localization of Alkaloid Biosynthetic Genes 1815

periderm of well-differentiated mature poppy roots (Figures 5Ato 5D). No hybridization was detected when the CTYDC1 (Fig-ure 5D) and cTYDC2 (data not shown) sense probes were used.

In situ hybridization of cross-sections of stems from the in-ternode below the first leaf near the shoot apex revealed thatrVDCMike transcripts are localized to both the abaxial andadaxial sides of the vascular cambium in all vascular bundles,whereas 7VDC2-like transcripts are more abundant in thephloem than in any other tissue (Figures 5E to 5H). Althoughthe spatial patterns of expression using cTYDCI or CTYDC2antisense RNA probes were similar, the development time

Carpel Developmental Stage

B

TYDC1

1 2 3 4 5 6

TYDC2

Figure 4. Expression of TYDC Genes during Carpel Development.

(A) Opium poppy reproductive organ development from floral bud (stage1) through anthesis (stage 3) to maturation of the seed capsule justbefore 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 RNAextraction. Each lane contains 15 ng of total RNA fractionated onformaldehyde-agarose gels, transferred to nitrocellulose membranes,and hybridized with 32P-labeled cTYDCI or cTYDC2 probes at highstringency. Gels were stained with ethidium bromide before blottingto ensure equal loading of samples.

required to visualize the insoluble blue-purple reaction productsof the alkaline phosphatase-conjugated anti-digoxigenin an-tibody was considerably longer when the cTYDCI antisenseRNA probe was used (Figure 5F). No hybridization of eitherantisense RNA probe was apparent in parenchymatous corti-cal cells adaxial to the vascular bundles or in the sclerenchymaperipheral to the vasculature. Closer examination of vascularbundles revealed that both TYDC1- and r/DC2-like transcripts(Figures 5F and 5G) are associated predominantly with themetaphloem and to a lesser extent with the protoxylem; how-ever, the transcripts were not detected in cells of the cambiallayer and not clearly detected in cells associated with the proto-phloem and metaxylem. No hybridization was apparent whenthe cTYDCI (data not shown) and CTYDC2 (Figure 5H) senseprobes were used.

TYDC transcripts were not detected in total RNA extractedfrom young leaf blade tissue, as shown in Figure 2B. However,in situ hybridization analysis of cross-sections through themidrib of a young leaf and through young axillary buds demon-strated that both cTYDCI and CTYDC2 antisense RNA probeshybridized to mRNAs present in cells in the vascular bundlesas well as in cortical and epidermal cells of younger tissues(data not shown).

These results suggest that the tissue specificity of TYDCgene expression is associated with the developmental stageof 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 inother cell types, whereas in mature roots (Figures 5B and 5C)and stems (Figures 5F and 5G), TYDC gene expression isclearly localized to the vasculature.

Roots and Aerial Organs Accumulate Different MajorIsoquinoline Alkaloids

Opium poppy accumulates three major structural types of well-characterized tetrahydroisoquinoline alkaloids. In aerial organs,the alkaloids are contained within the vesicle-rich cytoplasmof 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 extract-able alkaloids that was both quantitatively and qualitativelysimilar, as shown in Figure 6. In contrast, the alkaloid profileof the root consisting mainly of sanguinarine and morphinewas different from that of the latex of carpels, stems, and leavesin which no sanguinarine was detected.

The identities of morphine and sanguinarine were comparedwith authentic standards and verified by mass spectrometry(see Methods). No sanguinarine was detected in latex sam-ples by HPLC (data not shown). The levels of morphine andsanguinarine in capsule, stem, and leaf latex as well as in rootswere approximated by comparison with authentic standardsusing laser densitometry of thin-layer chromatograms. The levels

1816 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 1817

of morphine in capsules, stems, and leaves varied between43 and 51 ug/mL, whereas roots contained 9.7 and 9.3 mg/gdry weight, respectively, of morphine and sanguinarine. Thespecific localization of sanguinarine accumulation in roots andthe more complex alkaloid pattern found in the latex of aerialorgans are representative of the dichotomy of alkaloid accumu-lation in opium poppy.

DISCUSSION

In this study, we examined the differential and tissue-specificexpression of TYDC genes in opium poppy and compared thelocalization of TYDC transcripts with the accumulation of differ-ent alkaloids produced by the plant. The alkaloids synthesizedby opium poppy and related species are of the tetrahydroiso-quinoline type. In the Papaveraceae, the major alkaloids ofaerial organs are sequestered to vesicles found in thecytoplasm of articulated and anastomized internal secretorycells known as laticifers (Fairbairn et al., 1974; Messier andMahlberg, 1977,1978,1979). Although laticifers are associatedwith vascular tissue in all organs, the principal alkaloids thataccumulate in roots of various species are often different fromthose of aerial organs (Phillipson, 1983; Preininger, 1985). Inthis study, we found that among several major latex alkaloidsfound in all aerial organs of opium poppy, only morphine alsooccurred as a major root alkaloid, whereas significant levelsof sanguinarine occurred only in poppy roots. All of thesecompounds represent end products of specific tetrahydroiso-quinoline alkaloid branch pathways that originate from commonintermediates. TYDC represents the first of these common bio-synthetic steps that channel the aromatic amino acidsL-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 14genes, of which we have cloned four members (Facchini andDe Luca, 1994). Within the TYDC gene family, two subgroupsbased on sequence identity are evident. Each subgroup con-sists 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 andTYDC2) reveals sequence identities of <75°/o. Although thecatalytic properties of the different TYDC isoforms are similar

Front

Origin

Latex

Figure 6. Thin-Layer Chromatographic Analysis of Alkaloids Foundin Latex and in Root Extracts of Mature Opium Poppy Plants.

Alkaloids were extracted in methanol from an equal volume of latexcollected from mature seed capsules, leaves, and stems. Roots wereextracted in methanol from 100 mg of frozen powdered tissue. Sam-ples were applied to silica gel 60 F254 thin-layer chromatographyplates for separation and identification of alkaloids. Authentic standardsof sanguinarine (S), noscapine, papaverine, codeine, morphine (M),and opium were also run. Alkaloids were detected by their absorbanceor fluorescence after illumination at 365 nm.

(Facchini and De Luca, 1994,1995), the TYDC gene family ex-hibits differential and organ-specific expression (Facchini andDe Luca, 1994). In this study, we determined that the tissue-specific expression of TYDC genes is developmentally regu-lated, because the expression of TYDC genes is strictlyassociated 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-labeledcTYDd or cTYDC2 RNA probes. Hybridization was detected with anti-digoxigenin antibodies conjugated to alkaline phosphatase and visualizedafter 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.

1818 The Plant Cell

but is also expressed in cortical and epidermal cells of tissues from young leaves and axillary buds at earlier stages of de- velopment. In roots, TYDC!-like genes are preferentially expressed, whereas in stems and other aerial organs, TYDCP- like 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 vascu- lar 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, localization of abundant TYDC transcripts in the phloem is consistent with fhe expected de- velopmental 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 previ- ous 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 bio- synthesis occurs in these tissues in stems and roots. We have found that the developing carpel lacks abundant TYDC tran- scripts and has relatively low enzyme activity compared with other tissues (Facchini and De Luca, 1994). Salutaridine syn- thase (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 accumu- late, 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 sug- gested 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 provocative correlations between the differential expression of TYDC genes, the tissue-specific lo- calization of TYDC transcripts in mature organs, and the accumulation of specific alkaloids in opium poppy. For exam- ple, 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 TYDC7- like transcripts suggest that the expression of TYDCI-like genes may be coordinately regulated with that of other genes specif- ically 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 bio- synthesis 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). Expres- Sion of BBE has been shown to be induced by various elicitors in E. californica cell cultures; however, the tissue-specific ex- pression 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 or- gans 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 soil- borne 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 tissue- specific 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 described the isolation of full- and partial-length cDNA and genomic clones for tyrosine/dopa decarboxylase from f? somnife- rum. They are designated cTYDC1, TYDC1, cTYDC2, cTYDC3, and TYDC4 (Facchini and De Luca, 1994). AI1 inserts were cloned using

Localization of Alkaloid Biosynthetic Genes 1819

Stratagene pBluescript SK+ (genomic clones) or SK- (cDNAs). Probes for DNA and RNA gel blot hybridizations were synthesized from cDNA inserts isolated by digesting plasmids with EcoRl and Xhol. The partial- length cTYDCl and cTYDC3 inserts were 1554 and 927 bp, respec- tively, 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. Probes for in situ RNA hybridization analysis were synthesized from plasmids that were linearized with 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 im- mobilized on nylon membranes. Total RNA for gel blot analysis was isolated according to Logemann et al. (1987), and 15 pg was fraction- ated on 1 .O% formaldehyde-agarose gels before being transferred to nitrocellulose membranes (Sambrook et al., 1989). DNA and RNAgel blots were hybridized with random primed 3ZP-labeled-DNA inserts (Feinberg and 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 inten- sifying 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 mounted on replicate slides 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 antisense transcripts for cTYDCl and cTYDC2 were synthesized and labeled in vitro with digoxigenin- 11-UTP (Boehringer Mannheim) using T7 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-’ RNase A for 30 min at 37OC to digest nonhybridized probe and subse- quently 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%. Hybrid- ized probe was detected with anti-digoxigenin antibodies conjugated to alkaline phosphatase (Boehringer Mannheim) and visualized by color development using 5-bromo-4-chlorc-3-indolyl phosphate and nitro blue tetrazolium as substrates. Sections were photographed with a Leitz Ortholux II microscope (Leica Canada Ltd., Toronto, Canada) on Kodak RoyalGold 100 film.

Alkaloid Extraction and Thin-Layer Chromatography

Alkaloids were extracted from fresh latex (50 pL) collected from ma- ture 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 un- der liquid nitrogen and extracted with 0.5 mL of methanol for 10 min at 100OC. Debris from all samples was remwed by centrifugation. Sam- ples (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 consisting of acetone/tol- uene/ethanol/concentrated ammonia (45:45:7:3 [v/v]) (Wagner et al., 1984). Standards for sanguinarine, noscapine, and papaverine were purchased from Sigma. Morphine, codeine, and opium standards were a kind gift from U. Eilert (Technische Universitat, Braunschweig, Ger- many). Plates were dried at room temperature and photographed under ultraviolet illumination at 365 nm on Kodak RoyalGold 100 film. Ex- tracts were separated by HPLC (600E HPLC and 991 photodiode array detector; Waters, Montreal, Canada) on a Waters Nova Pak C18 re- verse 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 16.1 min and was identical with that obtained for the correspond- ing standard. Furthermore, an absorption spectrum identical to that of sanguinarine standard was obtained from the root-derived alkaloids eluting at this retention time.

ldentification of Morphine and Sanguinarine by Mass Spectrometry

Morphine and sanguinarine standards as well as morphins from la- tex, morphine from roots, and sanguinarine from roots were purified by thin-layer chromatography. Spots corresponding to morphine and sanguinarine standards 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/mass spectrometer) (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 mor- phine 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-resolution mass spectra obtained for sanguina- rine (mh: 332 [IOO], 317 [34], 194 [26], and 149 [IOO]) in roots were identical to those obtained for sanguinarine standards (mh: 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 ion- ize 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 relative value for the largest peak. Subsequent masses (268,215, 174, 162, and 124) represent other major ions produced that are represen- tative of morphine. The numbers in brackets represent the 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 Recherche en Biologie VkgBtale)

1820 The Plant Cell

for his invaluable assistance with the interpretation of anatomical data, 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 assistance with the laser densitization of thin- layer chromatography results. We also thank Sylvain Lebeurier for main- taining plants in the greenhouse, Julie Poupart for technical assistance with alkaloid extractions, Christiane Morisset for advice on the prepa- ration 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 Research Council of Canada operating 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.

Received August 11, 1995; accepted August 30, 1995.

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DOI 10.1105/tpc.7.11.1811 1995;7;1811-1821Plant Cell

P. J. Facchini and V. De LucaIsoquinoline Alkaloids in Opium Poppy.

Phloem-Specific Expression of Tyrosine/Dopa Decarboxylase Genes and the Biosynthesis of

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