the journal of bio~ical chemistry vol. 269, no. 43, of …pharmacological effects. most isoquinoline...

0 1994 by The American Society for T H E JOURNAL OF B I O ~ I C A L CHEMISTRY

Biochemistry and Molecular Biology, Inc. Vol. 269, No. 43, Issue of October 28, pp. 26684-26690, 1994

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Differential and Tissue-specific Expression of a Gene Family for TyrosineDopa Decarboxylase in Opium Poppy*

(Received for publication, April 13, 1994, and in revised form, July 19, 1994)

Peter J. FacchiniO and Vlncenzo De Lucag From the Znstitut de Recherche en Biologie Vegetale, Dbpartement de Sciences Biologiques, Universite de Montreal, Montreal, Quebec, HlX2B2 Canada

' b o early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) in- volvedecarboxylationofL-tyrosineandL-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, re- spective1y.ADNAfragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases. A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDCI DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTyDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic library was screened with clYDC1 and two intronless genomic clones (gTYDC1 and gTyDC4) were isolated. The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were divided into two subsets (cTyDC1 andgTYDC4; clYDC2 and clYDC3) of proteins with predicted M, = 56,983 and 59,323, respectively. Within each subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity. Expression of gTYDCl and cTYDC2 as /3-galactosidase fusion proteins in Escherichia coli resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies. Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates. Genomic DNA blot- hybridization analysis revealed 6 to 8 genes homologous to cTyDCl and 4 to 6 genes homologous to cTyDC2 in the tetraploid poppy genome. A premature translation stop codon was found in the glYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes specific to the gTYDC1- or cTyDC2- like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.

Research Council of Canada strategic and operating grants (to V. D. L.). *This work was funded by Natural Sciences and Engineering

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTMIEMBL Data Bank with accession numbercs) U08597, The nucleotide sequencers) reported in this paper has been submitted

U08598, U08599, and U08600. $ Supported by a Natural Sciences and Engineering Research Council

of Canada Postdoctoral Fellowship. 9 To whom correspondence should be addressed: Institut de Recher-

est, Montreal, Quebec, H1X 2B2 Canada. Tel.: 514-872-8492; Fax: 514- che en Biologie Vegetale, Universit4 de Montreal, 4101 rue Sherbrooke

872-9406.

Aromatic amino acids are important precursors to many secondary metabolites in higher plants, and enzymes responsible for diverting these essential primary metabolites into secondary metabolic pathways typically have key regulatory func- tions. For example, phenylalanine ammonia-lyase (PAL'; EC 4.3.1.5) catalyzes the conversion of L-phenylalanine to trans- cinnamic acid and its regulation, primarily at the transcriptional level, controls general phenylpropanoid metabolism (Hahlbrock and Scheel, 1989). The phenylpropanoid pathway supplies the substrate for functionally diverse products such as flavonoids, lignin, and antimicrobial coumarins. Similarly, tryptophan decarboxylase (TDC; EC 4.1.1.28) which converts L-tryptophan into tryptamine, is a highly regulated branch- point enzyme involved in the biosynthesis of various alkaloids (Waller and Dermer, 1981). TDC was the first plant aromatic amino acid decarboxylase to be cloned (De Luca et al., 1989) and subsequent work has revealed a complex regulation pattern at both the transcriptional (Pasquali et al., 1992; Goddijn et al., 1992) and post-translational levels (Fernandez and De Luca, 1994).

Tyramine and dopamine, the decarboxylation products of L- tyrosine and L-dihydroxyphenylalanine (L-dopa), serve as dis- tant precursors to isoquinoline alkaloids (Fig. 1) and as more immediate precursors to various amines and amides. Isoquino- line alkaloids comprise a structurally diverse class of natural products found mainly in the Papaveraceae, Berberidaceae, Ranunculaceae, and Menispermaceae and exhibit a variety of pharmacological effects. Most isoquinoline alkaloids are derived from (S)-norcoclaurine (Stadler et al., 1987, 1989), which results from the condensation of dopamine and 4-hydroxyphenylacetaldehyde (Fig. 1). Despite an extensive appreciation for the chemistry of plant isoquinoline alkaloid biosynthesis, the enzymology and regulation of the early steps in the isoquinoline pathway are not well understood. Although many enzymes of specific isoquinoline branch pathways have been measured in plants, few enzymes suspected of being rate-limiting have been identified. Furthermore, in contrast to the extensive molecular analysis of phenylpropanoid metabolism, little is known about the mechanisms regulating the activity of plant isoquinoline alkaloid biosynthetic enzymes. Important regulatory func- tions similar to those of PAL and TDC have been suggested for tyrosine (TYDC; 4.1.1.25) and dopa (DODC) decarboxylases (Marques and Brodelius, 1988b; Kawalleck et al., 1993).

Soluble tyramine has also been found to be incorporated, either directly or following conjugation with 4-coumaroyl or feruloyl residues, into the plant cell wall (Negrel and Jeandet,

DODC, dopa decarboxylase; TDC, tryptophan decarboxylase; TYDC, The abbreviations used are: PAL, phenylalanine ammonia-lyase;

tyrosine decarboxylase; PCR, polymerase chain reaction; kb, kilo- base(s); IPTG, isopropyl-P-D-thiogalactopyranoside; ORF, open reading frame; Bis-Tris, 2-[bis(2-hydroxyethyI~aminol-2-~hydrox~ethy~~-pro- pane-1,3-diol.

26684

Qrosine I Dopa Decarboxylase Gene Family in Opium Poppy 26685

FIG. 1. Proposed biosynthetic pathway from prephenate and L-tyrosine to (S)-norcoclaurine, the accepted precursor to most isoquinoline alkaloids in higher plants. Independent metabolic channels are illustrated to represent the possibility that dopamine and 4-hydroxyphenylpyruvate have different biosynthetic origins. The enzymes involved are: I , phenoloxidase; 2, pheno- lase; 3, L-tyrosine decarboxylase; 4, L-dopa decarboxylase; 5, 4-hydroxyphenylpyruvate decarboxylase; 6 , L-tyrosine transaminase; 7, tyramine transaminase; 8, (S)-norcoclaurine synthase.

Prephenale 4-Hydroxyphenylpyruvate 4-Hydroxyphenylacetaldehyde (S)-Normclaurine

HO

1987). Following oxidative polymerization, tyramine or its de- rivatives may function to reinforce cell walls rendering them less susceptible to penetration by pathogens. Recently, a group of TYDC cDNAs have been isolated from parsley (Petroselinum crispum) (Schmelzer et al., 1989; Kawalleck et al., 1993) and Arabidopsis thaliana ("rezzini et al., 1993) and were shown to be transcriptionally activated upon fungal infection or elicitor treatment.

Undifferentiated cell suspension cultures of opium poppy (Papaver somniferum) also respond to elicitor treatment by an induction of TYDC activity and the de nouo synthesis of anti- biotic isoquinolines, such as the phytoalexin sanguinarine (Eilert et al., 1985). In addition, mature seed capsules of opium poppy plants contain numerous isoquinoline alkaloids including the pharmacologically active phenanthrene alkaloids, morphine and codeine. The isoquinolines in poppy accumulate only in the latex, which is contained in structurally and physiologi- cally specialized cells known as laticifers (Roberts et al., 1983; Rush et al., 1985). Thus, the expression of TYDC genes involved in isoquinoline alkaloid biosynthesis and in the defense response may be controlled by specific environmental and developmental cues. Further investigation of the regulation of the early steps of isoquinoline alkaloid biosynthesis will require detailed molecular analysis of the TYDC gene(s), especially those sequences involved in transcriptional regulation. In this paper the isolation and characterization of cDNA and genomic clones for L-tyrosine and L-dopa decarboxylase from opium poppy is described, and the differential and tissue-specific regulation of members of the gene family is demonstrated.

MATERIALS AND METHODS Plant Material-€? somniferum L. cv. Marianne plants were grown

under standard greenhouse conditions. Tissue samples were collected from mature plants with fully expanded flowers and used immediately. Seedlings were germinated on moist sterile filter paper in the dark for 3 days, then transferred to a 16-h photoperiod for 4 days prior to RNA extraction.

Isolation and Analysis of Nucleic Acid-Total RNA for cDNA library construction was prepared from 7-day-old seedlings by phenol/ chloroform extraction of powdered frozen tissue followed by lithium chloride precipitation (Jones et al., 1985), and poly(A)' RNA was isolated by oligo(dT) cellulose chromatography (Gubler and Hoffman, 1983). Total RNA for gel blot analysis was isolated according to Loge- mann et al. (19871, and 15 pg were fractionated on 1.0% formaldehyde agarose gels before transfer to nitrocellulose membranes (Sambrook et al., 1989). Poppy leaf genomic DNA was isolated (Murray and Thomp

L-Tyrosine COZ Tyramine

son, 19801, digested with various restriction endonucleases, electro- phoresed on 1.0% agarose gels and transferred to nylon membranes. RNA and DNA blots were hybridized with random primer 32P-labeled (Feinberg and Vogelstein, 1984) cTYDCI or cTYDC2 inserts at 65 "C in 0.25 M sodium phosphate buffer, pH 8.0, 7% SDS, 1% bovine serum albumin, 1 mM EDTA. Blots were washed at 55 "C, twice with 2 x SSC, 0.1% SDS and twice with 0.2 x SSC, 0.1% SDS (Sambrook et al., 1989) (1 x SSC = 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0). RNA and DNA blots were autoradiographed with an intensifying screen on Fuji RxlOO x-ray film for 4 days at -80 "C.

Library Constructions and Screening-A unidirectional oligo(dT)- primed cDNA library was constructed according to the manufacturers instructions in a AUni-ZAPII XR vector (Stratagene) from poly(A)+ RNA prepared from 7-day-old light-grown poppy seedlings. The primary library contained 1 x lo6 plaques and was amplified prior to screening. A 32P-labeled cDNA for TDC from Catharanthus roseus (De Luca et al., 1989; GenBankTM accession no. J04521) was used as a probe to screen 2.5 x lo5 plaques of the amplified cDNA library at moderate stringency (45 "C in the hybridization buffer used for nucleic acid blot analyses as described above). An additional 2.5 x lo5 plaques were screened using a 32P-labeled 490-bp PCR product amplified from poppy genomic DNA using aromatic amino acid decarboxylase-specific degenerate primers. Plasmids were rescued from phage that gave a positive signal with R408 helper phage (Short et al., 1988).

AAEMBLB (Stratagene) genomic library was constructed from poppy leaf DNA partially digested with MboI (Sambrook et al., 1989). A primary library of 1.1 x lo' plaques was obtained and 2.5 x lo5 plaques of the amplified library were screened with 3T"labeled cTYDCI insert at moderate stringency as described above. Genomic DNAfragments were subsequently subcloned into pBluescript (SK-) (Stratagene) for further characterization (Sambrook et al., 1989).

Double-stranded DNAs were sequenced using the dideoxynucleotide chain-termination method (Sanger et al., 1977) using a recombinant T7 DNA polymerase (Amersham). Both strands of insert DNAs were sequenced from their ends using M13 universal and reverse primers. In addition, 20-mer oligonucleotides synthesized according to obtained sequence information were used directly as primers for further sequenc- ing. Comparative sequences were obtained from published reports or from the GenBankTM (release no. 79) and Swiss-Prot (release no. 26) sequence data bases. Sequence compilation and analysis was performed using the FASTA program package (Pearson and Lipman, 1988).

Amplification of TYDC by PCR-Degenerate PCR primers (sense primer = 5'-CCIGCIGCNACNGA(G/A)(T/C)TNGA-3'; antisense primer = 5'-CCIGC(G/A)TANGCNGC(G/A)TC-3'; I = inosine, N = all bases), designed from two highly conserved domains found in other aromatic amino acid decarboxylases, were used to amplify a 490-bp fragment of poppy genomic DNA at a primer annealing temperature of 40 "C and a chain reaction of 35 cycles. The PCR product was purified by electrophoresis on agarose gel, random primer-labeled, and used to screen the cDNA library.

26686 TyrosinelDopa Decarboxylase Gene Family in Opium Poppy

maps for TYDC cDNA and genomic FIG. 2. Restriction and structural

clones. Coding regions are represented by open boxes. The vertical arrowhead indicates the position of an internal translation termination codon in gTYDC4. Re- striction sites are as follows: A, AccI; B, BamHI; C, ClaI; D, DraI; E, EcoRI; H, HindIII; P, PstI; R, EcoRV; V, PuuII.

H E R R

cTYDC1

gTYDC1

E V DV PBAC A H A R

cTYDC2

V D PB H A D D

200bp AAAAA cTYDC3

H E Y R V P R A H A D I ' gTYDC4

A

Expression of TYDCs in Escherichia coli-The original clones of gTYDCl and cTYDC2 in pBluescript (SK-) were not in the correct reading frame to allow expression of TYDC polypeptides as P-galactosidase fusion proteins in E. coli. Therefore, the reading frames were shifted by oligo-directed mutagenesis to allow re-insertion of the open reading frames (ORFs) into pBluescript (SK') such that the ORF of the first 28 amino acids of the P-galactosidase polypeptide continued unin- terrupted into the TYDC proteins. Oligonucleotides specific to the ter- mini of the ORFs of gTYDCl (A, 5'-GCCGCTCTAGAAATGGGAA- GTCTTCCAGCT-3' and B, 5'-TGCAGGTCGACTCAACAAACATCCT- CACCTAG-3') and cTYDC2 (C, 5"GCCGCTCTAGAAATGGGTAGTCT- TAACACTGA-3' and (D, 5'-TGCAGGTCGACTTATGGATGACCCG- GAATGAT-3') were designed to incorporate 5'XbaI (A and C) and 3'SaZI (B and D) restriction sites into the amplified PCR products with gryDCl and cTYDC2 as templates, respectively. PCR products were inserted into the XbaUSulI sites of pBluescript (SK'). Expected ligation junctions were verified by DNA sequence analysis. E. coli XL-1 Blue cells harboring the pBluescript-TYDCl and pBluescript-TYDC2 constructs were grown a t 30 "C in Luna-Bertani medium to A,,, = 0.5, and expression of the fusion proteins was induced by addition of IFTG to a final concentration of 1 m. Cells were collected 1 h after addition of IPTG, centrifuged to remove the medium, and the pellets were washed with 50 mM Bis-Tris, pH 7.2, then frozen a t -80 "C until used for analysis. As controls, E. coli XL-1 Blue cells harboringpTDC5, a pBluescript vector containing a C. roseus TDC cDNA capable of directing the expression of a catalytically active TDC, and non-recombinant pBluescript (SK') were grown, induced, and processed under identical conditions. Bacterial proteins (approximately 100 pg) were solubilized in SDS sample buffer (0.1 M Tris-HC1, pH 6.8,1.6% (v/v) glycerol, 0.008% brom- phenol blue, 4 mM EDTA, 10 mM p-mercaptoethanol, 3% (w/v) SDS), separated by SDSPAGE, transferred to nitrocellulose, and immunode- tected with TDC antiserum (1:lOOO dilution) as described by Leary et al. (1983).

Enzyme Assays-Transformed E. coli extracts were assayed for decarboxylase activity by measuring the release of [l4C10, from L-carboxyl- 14C-labeled tyrosine, dihydroxyphenylalanine (dopa), and phenylalanine as described previously (Palavan and Galston, 1982; Cohen et al., 1982). Bacterial cells were lysed by sonication in 200 m~ Bis-"is, pH 7.2, the debris removed by centrifugation and the supernatant was desalted by passage through a PD-IO column (Pharmacia Biotech Inc.). The standard assay mixture for decarboxylase activity contained 50 mM Bis-Tris, pH 7.2, 1 mM EDTA, 25 p~ pyridoxal 1-phosphate, 0.1 pCi (specific activity, 55 mCi/mmol; 1 Ci = 37 GBq) I4C-labeled aromatic amino acid substrate, and 250 pl of protein extract in a total volume of 1 ml. Tryptophan decarboxylase activity was determined by monitoring the conversion of ~-methylene-['~Cltryptophan to [l4CItryptamine. As- says were terminated by the addition of 100 pl of 0.1 N NaOH, and radioactive tryptamine was extracted with ethyl acetate and quanti- tated by liquid scintillation counting.

Plant tissues used for decarboxylase activity measurements were ground to a fine powder under liquid N, and extracted in the presence of polyvinylpolypyrrolidone in 200 mM Bis-Tris, pH 7.2,l m~ EDTA, and 28 mM p-mercaptoethanol. Debris was removed by centrifugation and the supernatant was desalted on a PD-10 column. The assay mixture contained 50 mM Bis-Tris, pH 7.2, 1 mM EDTA, 25 p~ pyridoxal l-phosphate, 28 mM P-mercaptoethanol, 0.1 pCi of ~-carboxyl-['~Cltyrosine, and 250 pl of protein extract in a total volume of 1 ml. Enzyme activity was determined by measuring the release of [14C10, as described above.

RESULTS

Zsolation and Characterization of Clones-Degenerate oligo- nucleotide primers were synthesized for direct PCR amplification of a L-tyrosine or L-dopa decarboxylase gene fragment from opium poppy genomic DNA. PCR primers were designed from two highly conserved domains found in TDC from the plant C. roseus (De Luca et al., 1989) and in DODC from fruit fly (Drosophila melanogaster) (Eveleth et al., 1986; Morgan et al., 19861, human (Homo sapiens) (Ichinose et al., 19891, and rat (Rattus noruegicus) (Tanaka et al., 1989). The sense primer (degeneracy = 256) corresponded to the motif PA(A/C)TELE (residues 130 to 136 in TDC), and the antisense primer (degeneracy = 64) to the motif DAAYAG, (residues 287 to 292 in TDC). A 490 bp fragment was amplified from both poppy or C. roseus genomic DNA, as well as the authentic TDC clone (pTDC5) (De Luca et al., 1989) using the degenerate primers. The TDC insert frompTDC5 and the 490-bp poppy PCR product were used to screen a AUni-ZAP11 XR poppy seedling cDNA library at moderate stringency. Two different cDNAs (cTYDC2 and cTYDC3) were isolated using TDC cDNA as probe, while a third independent cDNA (cTYDC1) was recovered with the PCR-generated fragment as probe (Fig. 2). Comparison of amino acid sequences deduced from the longest open reading frame (ORF) on each clone to proteins in the Swiss-Prot data base, revealed extensive homology between all three cDNA clones and previously reported aromatic amino acid decarboxylases. Only cTYDC2 possessed the full-length ORF, while cTYDC 1 and cTYDC 3 were partial-length clones (Fig. 2).

The 1.9-kb full-length cTYDC2 clone consisted of an ORF of 1593 bp flanked by a 140-bp 5' leader sequence and a 143-bp 3"untranslated region followed by a polyadenylate tract. The predicted translation product initiated at the first in frame ATG had a molecular mass of 59.3 kDa. The partial-length ORFs of cTYDCl and cTYDC3 were 1554 and 927 bp, respectively, followed by 127- and 357-bp untranslated regions, respectively. Amino acid sequence alignments of the three cDNAs showed that cTYDC2 and cTYDC3 were 90% identical in regions of overlap, but that cTYDCl and cTYDC2 were less than 75% identical over the residues present in the partial cTYDCl clone (Fig. 3). Since the proteins encoded by cTYDCl and either cTYDC2 or cTYDC3 were significantly divergent in amino acid sequence, a full-length clone of TYDCl was sought for a comparative functional analysis of the translation products. Genomic clones were believed to be a good source of the full- length ORF since the TDC gene from C. roseus was recently isolated and shown to lack introns (Goddijn et al., 1994).

A poppy genomic library in AEMBL3 was screened with cTYDCl as probe at moderate stringency, and 50 positive plaques were recovered. Two plaques, one which exhibited a

Consensus TYDCl TYDC4 TYDCZ

Consensus TYDCl TYDC4 TYDC2

Consensus TYDCl TYDC4 TYDCZ

Consensus

TYDC4 TYDCl

TYDCZ TYDC3

Consensus TYDC1 TYDC4 TYDC2 TYDC3

Consensus TYDCl TYDC4 TYDC2 TYDC3

consensus TYDCl

TYDC2 TYDC4

TYDC3

Consensus TYDCl TYDCQ TYDCZ TYDC3



Consensus TYDCl

TYDCZ TYDC4

TYDC3

HGSL .....- E-S ...... NPLDP.EFRRQGHMIIDFLADYY..VE.YPV .... PANNF .... MSLCSQ ..... D.. ............... KN..K... .... PTGNL .... MSISSQ ..... D.. ............... KN..S... .... NTEDVL.N.SAFGVT ..... E ................. RD..K. . .

R.QV.PG.L.KRLPE.AP.N.ESIETIL.DVT..IIPGLTHnQ.PNY.AY .T .. D..Y.K.....S..Y.P.......E...ND.........S...F.. .S .. E..X.R.....S..N.S.......Q...ND.........N...F.. .S . . E..Y.R.....T..Y.P.......Q...TE.........S...Y..

FPSSGS..GFLGEMLSTGFNVVGFNWMSSPAATELES.VM.W.G.ML.LP ...... IA ............................. I..N.L.Q..T.. ...... IT. ............................ I..N.L.Q..M.. ...... VA ............................. V..D.F.K..N..

K . . . . SSDG.SG ........ T. ............ KM ....... N.NK... .SFLF....S..GGGVLQGT.CEAILCTLTAARD..LNKIGRE.I..LVV

K .... SSDG.SG ........ T. ........... YKM.T. .... N.NK. .. E ....----. GS. ....... S.............RK.......H.GR... ...... RK ....... H.GR. .. Y.SDQTHCALQKAAQIAGINeKNFRAI.T.KA .. FGLS...L ... IL.DI .A .... LS ................ L..A.S..TN .... PNS.QST..A.. .A ......................... A.S..TN .... PNS.HST..A.. .G ............. V.. ......... K.F.ENS .... AAT.REV..E.. .G ........................ VK.F .. NS ... AAST.REV..E..

E.GL.PLF.C.TVGTTSSTAVDPIGP.CEVAK.Y..WVH.DAAYAGSACI .S .. V...L.A...............L.A...LHGI...I.......... .S .. V...L.A...............L.....M.GI...V.......... . A .. I...V.P.... ......... S.I.. ... E.EM ... V. ......... .A . . I...V.P.... ........... I.. ... E.EM...I .......... CPEFRHFIDGVEEADSFSLNAHKWFFTTLDCCCLWVKD..SLVKALSTNP ............ D. ........................ SD.... .... S . ...................................... SD .......... ...................................... PSA ......... ...................................... PS ..........

EYL.NKATES.QV.DYKDWQIALSRRFRSMKLW,VLRSYG..NLR.FLRS ... K....D.K .. I ................... L......IA...T.... ... K.. .... K..I.. L IA... T ... R ...... R..V. .............. L...M......VT...N.... ... R ...... R..V.........I.........M......VT...N.... ................. ...... ....

HVKHAK.F.GLIGMD.RFE1.VPRTFAMVCFRL.P .... K.. ..-.... N ...... H.Q ...... N .... V............K.AAIFRK---. KIVED ...... H.Q ...... N .. GNVF ........... K.TA1F.Q---.KIV E. ...... T.E ... C .. G....T............L.PKTI.VYDN.GVH Q. . . R . . . T.E .. V.A.R . . . . T............L.PTTV.VCGENGVH Q.

........... ...... ... DH----IEAQT..-"VNA S S.KI...............

NE KL. LE.VNA.G..YMTHAVVGGVYMIRF

EY ---- IEAQT ..--- TNA. S ... S.RI............... GNGVIAVLRNE..ELVLAN .. NQVY.RQ.K.T.SV ...............

... GNGWP-LRDE..NLVLAN .. NQVY .. T ... T.SV...............

AVG.TLTEERHV .. AWKV.QEH.D.IL .... E..... ... A ........ TG .... V...T.A..GALG.DVC-- ... A ..... H..TG....V . . . T.T.. SALDATTAPEIVG ... S ........ IY . . . IL . . . A.L..GKF S.ADFSS ... S... ..... IH .. E.L ... A.L..SKFD.ANFSS

50 48 48 50

100 90

100 98

150 148 148 150

200 198 198 196 22

250 248 240 246 72

300 298 290 296 122

350 340

346 348

172

400 398 398

222 396

444 450

444 445 272

500 403 483 4 94 322

537 518 523 531 359

poppy TYDC genes. Dots represent amino acid residues identical to FIG. 3. Comparison of deduced amino acid sequences for four

the consensus sequence. A translation termination codon at position 55 of the gTYDC4 sequence is marked with an X .

strong hybridization signal and one which showed a weak signal, were purified and subcloned into pBluescript (SK') for further characterization. The nucleotide sequence of the clone which exhibited the stronger signal was identical to cTYDCl in the region of overlap and was thus designatedgTYDC1 (Fig. 2). The weaker hybridizing clone shared 90% nucleotide identity with gTYDC1, and was designated gTYDC4 as it represented the fourth TYDC-like gene cloned from poppy (Fig. 2). The predicted translation product ofgTYDCl had a molecular mass of 57.0 kDa which is 2.5 kDa smaller than the predicted cTYDC2 protein. However, the anticipated ORF of gTYDC4 contained an internal TAA translation termination codon 165 bp (55 amino acids) downstream from the analogous ATG used to initiate the gTYDCl ORF (Fig. 2). Subsequent to this premature stop codon, the ORF continues to a termination codon in a position analogous to that found in gTYDCl (Fig. 3). Since gTYDC4 was isolated as a genomic clone and not a cDNA, and appears to contain a point mutation resulting in premature termination of TYDC translation, this clone may represent a pseudogene.

Sequence alignments of the proteins encoded by gTYDCl and cTYDC2 with previously reported aromatic amino acid

Ps-TYDCI P3-TXDC2 Pc-TYDC2 Cr-TDC Dm-DODC

PS-TYDCI P,-TYDC2

CZ-TDC PC-TYDC2

Dm-DODC

PI-TYDC1 PI-TYDC2 PC-TYDCZ Cr-TDC Dm-DODC

PS-TYDCI Ps-TYDC2

Cr-TDC PC-TYDC2

Dm-DODC

PD-TYDCI PS-TYDC2 PC-TYDCZ Cr-TDC Dm-DODC

PI-TYDCI Ps-TYDC2

Cr-TDC PC-TYDCZ

Dm-DODC

PS-TYDCI Ps-TYDCZ

Cr-TDC PC-TYDC2

Dm-DODC

Ps-TYDC2

Cr-TDC PC-TYDC2

Dm-DODC

Pa-TYDC1

Ps-TYDC1 P1-TYDCZ PC-TYDCZ Cr-TDC Dm-DODC

Pa-TYDCI P,-TYDCZ

Cr-TDC PC-TYDCZ

Dm-DODC

PS-TYDCI Ps-TYDC2

Cr-TDC Dm-DODC

PC-TYDC2

PD-TYDCI ps-1YDc2 PC-TYDC2 Cr-TDC Dm-DODC

LPANNF--ES MSLCSQNPLD P LNTCDVLENS SWGWNPLD P IDNLTC-KLA SQIPM-NTLE P IDSTNVMSN SPVGEFKPLE

AIATSKATNF G L S P N ~ w v v

AIKTFKENSF GLSM AIETTKSSNF QLCPK LIPTTVETDF GISPQ ""- QSENH M G KA IE

PLCAVAKLHG PICEVAKEYE ALTEVAKKYD SLSEIANEFG ECGPVGNKHN

-GALGEDVC

DDTFTSNKLV EVLS -GKFSLADFS S

""

""QEQ KEA

31 33 31

41 33

81

81 83

97 83

131 133 131 133 I11

101 179 177 119 1 9 1

223 221 219 221 214

271 213

211 269

289

323 3 2 1 319 321 339

373 371 369 311 387

423 421 419 421 131

471 460

447 450

451

510 521 500 497 501

518 531 514 500 510

TYDCl and Ps-TyDC2, two tyrosineldopa decarboxylases from FIG. 4. Alignment of deduced amino acid sequences for Ps-

opium poppy; PC-TYDC2, a tyrosine decarboxylase from parsley

from C. roseus (De Luca et al., 1989); and Dm-DODC, a dopa (Kawalleck et al., 1993); Cr-TDC, a tryptophan decarboxylase

decarboxylase from D. melanogaster (Eveleth et al., 1986; Morgan et al., 1986). Boxes indicate residues identical in all five proteins. Gaps introduced into sequences to maximize alignments are represented by dashes. Conserved amino acid positions found in all pyri-

triangles. The closed triangle indicates the putative pyridoxal phos- doxal phosphate-dependent decarboxylases are marked by open

phate binding site.

decarboxylases (Fig. 4 ) showed 64% and 62% identity, respectively, to an L-tyrosine decarboxylase from parsley (Kawalleck et al., 1993); 52 and 51% identity, respectively, to L-tryptophan decarboxylase from C. roseus (De Luca et al., 1989); and 38 and 37% identity, respectively, to a L-dopa decarboxylase from D. melanogaster (Eveleth et al., 1986; Morgan et al., 1986). The

Tyrosine I Dopa Decarboxylase Gene Family in Opium Poppy 26688

kb-

8.0-

5.7-

4.4-

3.7-

2.4-

1.9-

1.2-

0.7-

n - m

WDCl WDC2

FIG. 5. DNAgel blot analysis of TYDC genes in poppy.A, genomic DNA was digested with XbaI, HindIII, EcoRI, and KpnI; separated by electrophoresis; transferred to a nylon membrane; and duplicate blots probed with either 32P-labeled cTYDCl or cTYDC2 a t high stringency. B, specificity of TYDCl and TYDC2 probes demonstrated by their lack

both unlabeled cDNAs. Hybridization conditions were identical in A of cross-hybridization at high stringency. Each lane contained 2.5 pg of

and B.

poppy TYDC proteins also contain amino acid positions conserved in all other previously reported pyridoxal phosphate- dependent decarboxylases including L-histidine decarboxylase (Zahnow et al., 1991) and L-glutamate decarboxylase (Baum et al., 1993). However, the region of homology for L-glutamate decarboxylase is restricted to 50 residues which comprise the probable active domain and the putative pyridoxal phosphate binding site. The L-glutamate decarboxylases are the only non- aromatic amino acid decarboxylases with clear homology to the poppy TYDC proteins.

Organization of the TYDC Gene Family in Opium Poppy- Poppy genomic DNA digested with eitherXba1, EcoRI, HindIII, or KpnI and probed with cTYDCl at high stringency revealed between 6 to 8 bands for each digestion (Fig. 5A ). An identical genomic DNA blot probed with cTYDC2 at high stringency revealed an entirely different pattern of 4 to 6 bands for each digestion (Fig. 5A). These results suggest that there are 6-8 and 4-6 copies, respectively, of TYDCl- and TYDC2-like genes. The specificity of each probe for their homologous sequences was verified on separate blots which contained 2.5 pg of both cTYDCl (1.4 kb) and cTYDC2 (1.9 kb) using the same conditions employed for the genomic DNA blot hybridizations (Fig. 5B). The amount of DNA on the blots in Fig. 5B was approximately equivalent to that expected for single copy genes on the genomic DNA blots (Fig. 5A). Cross hybridization under these conditions was negligible.

DNA blot hybridization analysis, with cTYDCl as probe, of the two 12-kb AEMBLS genomic clones indicated that the structural genes were located on HindIII fragments of 3.5 kb (gTYDC1) and 1.9 and 1.1 kb (gTYDC4). The genomic fragments corresponding to gTYDC4 are not observed as expected on the genomic DNA blot probed with cTYDCl a t high stringency (Fig. 5A 1. An identical blot probed with cTYDCl a t moderate stringency (45 "C hybridization) revealed additional bands in HindIII-digested DNA a t 1.1 and 1.9 kb (data not shown). Since gTYDC4 was 90% identical to cTYDCl but did not cross hybridize a t high stringency, all bands revealed in Fig. 5A must represent genes with very strong (>go%) homology to the respective probes. Thus, the family of TYDC-like genes in

kDa

106 80

50

33

28

19

7 4

M I 2 3 4 5 6 7 8

FIG. 6. Immunoblot detection of TYDC1, TYDC2, and TDC proteins, each fused to a p-galactosidase N-terminal polypeptide, in cell homogenates of IF'TG-induced E. coli transformants harboring the pBluescript-TYDC1, pBluescript-TYDC2, and pTDC5 (De Luca et al., 1989) constructs, respectively. Lanes: M , molecular weight standards; 1 and 2, uninduced (lane 1) and IF'TG-induced (lane 2) XL-1 Blue cells transformed with pBluescript-TYDCl; 3 and 4, uninduced (lane 3 ) and IPTG-induced (lane 4 ) X L - 1 Blue cells transformed with pBluescript-TYDC2; 5 and 6 , uninduced (lane 5 ) and IF'TG- induced (lane 6 ) XL-1 Blue cells transformed with pTDC5; 7 and 8, uninduced (lane 7) and IPTG-induced (lane 8) XL-1 Blue cells transformed with nonrecombinant pBluescript (SK').

opium poppy consists of two subsets. Within each subset (represented by gTYDC1 and cTYDC2) the clones exhibit >90% nucleotide identity, but clones between subsets are less than 75% identical. The possibility that additional TYDC-like genes may be present cannot be ruled out by these experiments.

TYDCl and TYDC2 Encode L-Tyrosinel L-Dopa Decarboxyl- ases-The ORFs of gTYDCl and cTYDC2 were amplified by PCR using specific primers designed to allow reinsertion of the fragments into pBluescript (SK') such that the TYDC polypeptides could be expressed as p-galactosidase fusions in E. coli. The N terminus of each TYDC protein was fused to a 28-amino acid N-terminal P-galactosidase polypeptide. A similar con- struct (pTDC5) capable of directing the expression of catalytically active C. roseus TDC, fused to an additional 64 amino acids comprised of N-terminal P-galactosidase and TDC cDNA 5' leader sequence-encoded residues (De Luca et al., 19891, was used as a positive control. Non-recombinant pBluescript (SK') provided the negative control. Polyclonal antiserum raised against purified TDC (Fernandez et al., 1989) was used to immunodetect IPTG-inducible proteins in the 55-60-kDa range in extracts from E. coli cells harboring the pBluescript-TYDCl, pBluescript-TYDC2, and pTDC5 constructs (Fig. 6). Thus, a t least some TDC-specific immunoglobulins recognize similar epitopes found on TYDC polypeptides. Immunoblots on soluble fractions demonstrated that the majority of the immunodetectable fusion proteins were associated with the bacterial pellet as inclusion bodies (data not shown). However, sufficient catalytically active soluble enzyme was available to measure decarboxylase activity in total protein extracts.

The highest specific activity in total protein extracts from E. coli expressing poppy TYDCl or TYDC2 was observed using L-dopa as substrate (Table I). Relative to L-dopa, the rate of L-tyrosine decarboxylation was 90 and 65% for TYDCl and TYDC2, respectively. L-Phenylalanine was a poor substrate and L-tryptophan was not accepted in both cases. In contrast, decarboxylase activity in total protein extracts from E. coli expressing C. roseus TDC was observed only with L-tryptophan as substrate (Table I). No L-tyrosine, L-dopa, L-phenylalanine, or L-tryptophan decarboxylase activity was detected in total protein extracts from IPTG-induced E. coli transformed with non- recombinant pBluescript (SK').

Tissue-specific Expression of TYDC Genes in Opium Poppy- RNAgel blot analysis revealed that members of the TYDC gene

Tyrosine JDopa Decarboxylase Gene Family in Opium Poppy TABLE I

Relative decarboxylase actiuity in total protein extracts from E. coli transformed with pBluescript-TYDCI, pBluescript-

TYDC2, pTDC5, or non-recombinant pBluescript (SF) for various aromatic amino acid substrates

Specific activities for the preferred substrate are as follows: TYDCI = 250, TYDC2 = 25, and TDC = 16 femtokataldmg, respectively.

Substrate Relative specific activity

TYDCl TYDC2 TDC Bluescript

%

L-Tyrosine 90 65 0 0 L-Dopa 100 100 0 0 L-Phenylalanine <1 <1 0 0 1,-Tryptophan 0 0 100 0

family are differentially expressed in various mature poppy tissues (Fig. 7A). Hybridization conditions were identical to those employed for genomic DNA gel blot analysis (Fig. 5) to allow for independent detection of TYDCl-like and TYDC2-like transcripts. The same RNA blot was first probed with cTYDC2 and after stripping it was re-probed with cTYDCl (Fig. 7A). TYDCl-like transcripts were detected predominantly in roots while TYDC2-like transcripts were detected mostly in roots and stems, and to a much lesser extent in the sepals and carpels of fully expanded flowers, and in cultured callus tissue. Furthermore, the size of the predominant TYDC2-like mRNAs in roots and stems were different. The major hybridizing mRNA in stems was larger than the detectable mRNA band in roots. Since the two TYDC2-like cDNAs (cTYDC2 and cTYDC3) isolated from the poppy seedling library had 3' untranslated regions differing by 214 bp, the predominant TYDC2-like mRNAs detected in roots and stems may represent the tissue- specific expression of different members of the TYDC2-like subset of genes. Direct measurement of TYDC enzyme activity in mature poppy tissues confirmed the RNAblots with the highest levels being found in roots (Fig. 7B 1. Lower levels were detected in stems, carpels and petals from fully expanded flowers, and in cultured callus tissue.

DISCUSSION

A PCR product amplified with aromatic amino acid-specific degenerate primers, and a plant TDC cDNA (De Luca et al., 1989), were used as probes to isolate three cDNA and two genomic clones encoding L-tyrosine and L-dopa decarboxylase from opium poppy. The predicted molecular masses of 57.0- 59.3 kDa for the various isoforms agrees with estimates of 56.3 kDa based on SDS-polyacrylamide gel electrophoresis with a purified TYDC from Thalictrum rugosum (Marques and Brode- lius, 1988a). The gene family consists of a t least 10-14 members, which can be categorized into two subsets of similar clones (>go% amino acid identity), containing 6-8 and 4-6 members, respectively. In contrast, TYDC in parsley (Kawal- leck et al., 1993) is encoded by four similar genes whereas TYDC from Arabidopsis thaliana (Trezzini et al., 1993) and TDC from C. roseus (Goddijn et al., 1994) are encoded by single copy genes. The clones were overexpressed in E. coli, immuno- detected with TDC-specific polyclonal antibodies, and were subsequently characterized for their substrate specificity. Al- though representative members of each subset (TYDC1 and TYDCS) shared only 73% amino acid identity, they were functionally similar. Both enzymes exhibited marginally higher activity with L-dopa than with L-tyrosine as substrate whereas neither L-tryptophan nor L-phenylalanine were accepted as substrates. Despite the apparently similar in vitro catalytic function of the heterologously expressed proteins, RNA blot hybridization analysis demonstrated that members of the gene family are differentially expressed in various mature plant tissues and may thus play different roles.

26689

kb

m 3.4

TYDC1 I 1 L J

FIG. 7. Tissue-specific expression of the TYDCI- and TYDC2- like genes in opium poppy. Total RNA was isolated from poppy roots, stems, leaves, sepals, carpels, stamens, petals, and callus tissues. A, equal amounts of RNA (15 pgilane) were fractionated on formaldehyde agarose gels, transferred to nitrocellulose membranes, and hybridized with "P-labeled cTYDCl and cTYDC2 probes a t high stringency. The same membrane was used for hybridization to both probes. Stripping of the first probe was verified by autoradiography prior to the second hybridization. B, corresponding TYDC enzyme activity in various tissues.

Aromatic amino acid decarboxylases have been cloned and characterized from plants (De Luca et al., 1989; Kawalleck et al., 19931, insects (Eveleth et al., 1986; Morgan et al., 19861, and mammals (Albert et al., 1987; Zahnow et al., 1991). TYDC/ DODCs in opium poppy are characteristic of other plant aromatic amino acid decarboxylases, which exhibit a high substrate specificity (No6 et al., 1984; Marques and Brodelius, 1988b; Kawalleck et al., 19931, whereas the animal enzymes typically accept a broader range of substrates, which include L-dopa, L-tyrosine, L-tryptophan, L-phenylalanine, and L-histidine (Christenson et al., 1972). DODC in Cytisus scoparius (Tocher and Tocher, 1972) and TDC in C. roseus (No6 et al., 1984) exhibit specificity toward dopa and tryptophan, respectively, and both are virtually inactive toward tyrosine and phenylalanine. TYDC in parsley (Kawalleck et al., 1993) and TYDC in T rugosum and Eschscholtzia californica (Marques and Brodelius, 1988b) accept tyrosine, and to a lesser extent dopa, but not tryptophan or phenylalanine. The opium poppy aromatic amino acid decarboxylases are the first clones to be isolated from plants which exhibit their highest specific activity toward L-dopa. The remarkable degree of amino acid sequence identity, and conserved distribution of a-helices and p-sheets, among all characterized aromatic amino acid decarboxylases suggests an evolutionary relationship (Maneckjee and Baylin, 1983; De Luca et al., 1989). Indeed, sequence comparisons of pyridoxal phosphate-dependent decarboxylases in general suggest a common evolutionary origin (Jackson, 1990). The accumulating sequence data for plant aromatic amino acid decarboxylases may provide some insight into the variable protein domains responsible for conferring substrate specificity (Fig. 5).

The similar substrate specificity of the two subsets of opium poppy TYDC/DODCs measured in vitro is in contrast to the substrate specific activity of other homologous plant secondary metabolic enzymes that function at, or near, branch points. For example, two cloned flavonol sulfotransferases from Flaueria

26690 Tyrosine I Dopa Decarboxylase

chloraefolia share an overall identity of 69%, but catalyze different substrate and position-specific sequential reactions (Varin et al., 1992). Two tropinone reductases cloned from Datura stramonium share 64% identity, but catalyze different stereospecific reactions (Nakajima et al., 1993). However, in bean (Phaseolus vulgaris) PAL is encoded by a gene family of 6-8 members, comprised of three major classes based on amino acid sequence homology (Cramer et al., 1989). Two classes show only 72% identity in amino acid sequence, but differ only in their apparent K, for phenylalanine. The differential tissue- specific expression of the two classes of TYDC genes in poppy appears to mimic the complex regulatory patterns and functional variants of the PAL gene products.

Isoquinoline alkaloids in opium poppy, in particular the phenanthrenes (morphine and codeine) and the benzyliso- quinolines (papaverine and noscapine) accumulate in the latex. Poppy latex consists of whole cytoplasm exuded from a se- ries of cells, known as laticifers, whose adjacent walls degenerate to form continuous vessels (Nessler and Mahlberg, 1981; Nessler and Vonder Haar, 1990). Laticifers accompany the vascular tissue in all organs (Nessler and Mahlberg, 1976) and, like primary phloem and xylem development, do not ap- pear to be tissue dependent. The occurrence of DODC activity in the latex of opium poppy was suggested as evidence for its involvement in alkaloid biosynthesis in the latex (Roberts and Antoun, 1978). Results presented here demonstrate that the highest steady state levels of TYDC transcripts in mature plants are found in the roots and stems (Fig. 7). Since these organs have an abundance of vascular tissue and laticifers, the results support the suggested association of decarboxylase activity with the latex. However, only small amounts of cTYDC2-like mRNA and TYDC activity were detected in the developing carpel (Fig. 7). The carpel develops into the mature seed capsule which is traditionally the organ from which latex is collected for its abundance of morphinan alkaloids. Al- though the data presented here do not address the developmental expression of TYDC genes during capsule formation, they do suggest that the capsule may not be the only site of alkaloid synthesis (Kutchan et al., 1985; Roberts et al., 1983). Instead, the intermediates and/or products may be synthesized in stem laticifers and subsequently transported to other organs (Waller and Nowacki, 1978).

Although we do not as yet understand the significance of the TYDC gene family in poppy, valuable clues may be provided by identifying the temporal, spatial, developmental, and environmental cues that control expression of the various family members. The proposed multifunctional importance of the many products derived from phenylalanine and tyrosine through PAL and TYDC, respectively, may account for the complexity of the gene families. Phenylalanine-derived products include cell wall structural components, UV protectants, and phytoalexins. In addition to being the first committed step in isoquinoline alkaloid biosynthesis, TYDC genes show a rapid transcriptional induction by pathogens and elicitors which has also been implicated in the defense response (Kawalleck et al., 1993; Schmelzer et al., 1989). The genetic polymorphism of the TYDC gene family, like that of PAL, may position different enzymatic isoforms in specific regulatory networks that provide the optimum response to physiological or environmental cues.

Acknowledgments-We thank Drs. Benoit St-Pierre, David Morse, and Normand Brisson for critical reading of the manuscript and Sylvain Lebeurier for maintenance of plants in the greenhouse.

Gene Family in Opium Poppy

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