Plant Science 158 (2000) 19–32

Characterization of cDNAs differentially expressed in roots oftobacco (Nicotiana tabacum cv Burley 21) during the early stages

of alkaloid biosynthesis

Jianmin Wang, Moira Sheehan, Heather Brookman, Michael P. Timko*Department of Biology, Uni6ersity of Virginia, Charlottes6ille, VA 22903, USA

Received 13 March 2000; received in revised form 9 May 2000; accepted 9 May 2000

Abstract

A set of 60 cDNAs were isolated by subtractive hybridization screening of a phage library using radioactively-labeled probesgenerated from root mRNAs isolated from tobacco (Nicotiana tabacum cv Burley 21) plants before and 3 days after topping.Among the differentially expressed gene products were full-length and partial cDNAs encoding arginine decarboxylase (ADC),ornithine decarboxylase (ODC), and S-adenosylmethionine synthetase (SAMS), enzymes involved in polyamine and alkaloidbiosynthesis. The other cDNAs isolated were placed into one of several categories and encode metabolic enzymes, proteinsinvolved in transcription and translation, components of signal transduction pathways, and homologs of genes whose expressionhas been shown to be regulated by phytohormones (i.e. auxin, ABA), wounding or other stress responses. RNA gel blot analysisshowed that the ADC and ODC transcripts were preferentially expressed in the roots and floral tissues of mature tobacco plants,whereas SAMS transcripts were detected in all tissues examined. The steady-state levels of the ADC and ODC mRNAs increasedin the roots of wild-type tobacco plants during the 24 h period after topping, whereas little change was observed in the abundanceof the SAMS transcripts in these tissues. The possible factors associated with the regulation of expression of these genes arediscussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Nicotine; Alkaloid biosynthesis; Arginine decarboxylase; Ornithine decarboxylase; S-adenosyl methionine synthetase

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1. Introduction

Alkaloids are one of the most diverse groups ofsecondary compounds found in plants and theyare the product of a complex biosynthesis pathway[1–3]. Why plants accumulate these compoundsand in so many different forms is not known.Moreover, for many alkaloids, the exact site ofsynthesis and the factors that control their inter-cellular distribution and accumulation remain tobe determined [2–4].

Nicotine is the most abundant alkaloid presentin cultivated tobacco and, like other alkaloidsfound in Nicotiana and related plant species, its

biosynthetic origin begins with the plantpolyamine putrescine [2,4]. Putrescine is formed inplants by one of two pathways [5]. It can besynthesized directly from ornithine, in a reactioncatalyzed by the enzyme ornithine decarboxylase(ODC, EC 4.1.1.17), or formed indirectly fromarginine in a reaction sequence initiated byarginine decarboxylase (ADC, EC 4.1.1.19). Pu-trescine formed by the ADC and/or ODC path-way serves as precursor in the synthesis of thehigher polyamines, spermine and spermidine, cata-lyzed by the enzymes spermine synthase and sper-midine synthase, respectively, or it is converted toN-methylputrescine by the action of putrescineN-methyltransferase (PMT), the first committedstep in nicotine biosynthesis [2,4,5]. N-methyl pu-trescine is oxidized by a diamine oxidase andcyclized to form the 1-methyl-D1-pyrrolium cation,

* Corresponding author. Tel.: +1-804-9825817; fax: +1-804-9825626.

E-mail address: mpt9g@virginia.edu (M.P. Timko).

0168-9452/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0 1 6 8 -9452 (00 )00293 -4

J. Wang et al. / Plant Science 158 (2000) 19–3220

which is condensed with nicotinic acid or itsderivative to form nicotine [2]. Other aminoacids, such as tyrosine, tryptophan, phenylala-nine, and related compounds (e.g. anthranilicacid, nicotinic acid, and purines) can also serveas biosynthetic precursors to some classes of al-kaloids [5].

The synthesis and accumulation of nicotineand other tobacco alkaloids are known to becontrolled by various developmental, environ-mental, and chemical cues [1,2,4]. Changes inphytohormone (e.g. auxin, cytokinin) levels and/or ratios as a consequence of developmental age[2,4] or by direct manipulation of plant cell cul-ture conditions have been shown to affect thesynthesis and accumulation of nicotine and vari-ous tobacco alkaloids [2,6,7]. Various abiotic fac-tors (wounding, drought stress, pH imbalance,etc.) [1,2,4], as well as biotic factors, such asherbivory, insect feeding, and attack by variousmicrobial and fungal pathogens, are known toelicit increased production of nicotine and otheralkaloids in the leaves of wild and cultivated to-bacco species [8,9]. In addition, the commercialpractice of topping (i.e. removal of floweringhead and young leaves at the upper portions ofthe plant), results in increases in nicotine and theamount and complexity of total alkaloids presentin the leaves of Nicotiana tabacum [2,6]. The fac-tors controlling the topping-induced increase inalkaloid biosynthesis are not known, but likelyinvolve a complex physiological response in theplant as a result of altered phytohormones andwound induced signaling [6,10] . In this regard,considerable evidence now exists indicating thata jasmonic acid (JA)-mediated signal transduc-tion pathway may play a role in regulation ofgene expression contributing to this increase inalkaloid biosynthesis [11–15].

The formation of nicotine and total leaf alka-loids in tobacco is known to be under the con-trol of at least two independent genetic loci[16,17], referred to most recently in the literatureas Nic1 and Nic2 [6] . Nic1 and Nic2 are semi-dominant and operate synergistically to controlplant alkaloid content, with mutations withinthese genes resulting in plants with reduced lev-els of nicotine and total leaf alkaloids (wild-type\nic1\nic2\nic1 nic2) [16,17]. Althoughno information is available on the nature oftheir encoded products, it has been speculated

that Nic1 and Nic2 likely encode transcriptionalregulators capable of globally interacting with asubset of genes encoding components ofpolyamine and alkaloid biosynthesis [6].

cDNAs and/or genomic DNA fragments en-coding enzymes involved in polyamine biosynthe-sis and/or the formation of nicotine and relatedalkaloids have been isolated from a number ofplant species [2,5,6,15,18,19]. Initially, most at-tempts at the cloning of cDNAs for enzymesinvolved in alkaloid formation involved immuno-screening of phage expression libraries with anti-bodies prepared against purified enzyme protein,or by sequencing the protein and screening alibrary with synthetic oligonucleotide probesbased upon the defined amino acid sequence[19]. A few attempts at alternative approaches,such as the use of differential hybridizationscreening [6,15,20] or the use of PCR basedstrategies [21], have also been reported. In thepresent study, we used a subtractive hybridiza-tion screening strategy to identify cDNAs whoseencoded proteins are differentially expressed inthe roots of Burley 21 tobacco plants 3 daysafter topping, a developmental time known to beactive in nicotine and leaf alkaloid formation.We report here the results of our studies anddescribe the nature of the gene products andtheir expression in wild-type tobacco plants.

2. Materials and methods

2.1. Plant growth, tissue preparation, and RNAisolation

N. tabacum cv. Burley 21 plants were grownhydroponically in a dilute (half-strength) Petersnutrient solution with continuous aeration of theroots under natural lighting conditions in thegreenhouse. The total and polyA+mRNA wereisolated from root tissues according to the meth-ods of Chomczynski and Sacchi [22]. For prepa-ration of RNA, root tissues were harvested frommature Burley 21 plants, immature plants withunopened floral meristems, and immature plantsfrom which the floral meristems and upper thirdof the stem tissues were removed (referred to inthe text as topped). RNA was also preparedfrom leaf tissues and floral parts of matureplants.

J. Wang et al. / Plant Science 158 (2000) 19–32 21

2.2. cDNA library construction and differentialscreening

cDNAs were generated using oligo-d(T) primersand the cDNA synthesis kit (purchased fromStratagene). The cDNAs were cloned into lambdaZap II vector DNA linearized by digestion withEcoRI and XhoI. The library of recombinantclones was screened by differential hybridizationof duplicate nitrocellulose filters [23] using a-32P-dCTP labeled cDNA as probe [24]. Probes usedfor screening consisted of radioactively labeledcDNAs prepared from tobacco root and leafRNAs, and root RNA from tomato (as a het-erologous control probe). cDNAs showing stronghybridization with probes prepared from roots oftopped plants were recovered and their nucleotidesequence determined.

2.3. DNA sequencing and analysis

Nucleotide sequencing was carried out manuallyusing the Sequenase Version 2.0 protocols accord-ing to the manufacturer’s protocol (United StatesBiochemical) or with an ABI 310 Genetic Ana-lyzer (PE Applied Biosystems) using double-stranded plasmid DNA templates preparedutilizing the Qiaprep Spin Plasmid Kit (Qiagen).The nucleotide and predicted amino acid se-quences of the various cDNAs were analyzed us-ing BLAST sequence analysis programs [25,26]and protein sequence alignments were carried outusing the PILEUP program (Genetics ComputerGroup Sequence Analysis Package, Version 9.0,Madison, WI) and the various gene sequencesavailable in the NCBI (National Center for Bio-technology Information, Bethesda, MD) nucle-otide and protein sequence database. Manualadjustment of the sequence alignments was carriedout as necessary.

2.4. RNA gel blot analysis

Total RNA was extracted from tobacco roots,leaves, and floral parts using Tri-Reagent (Molec-ular Research Center) according to the manufac-turer’s protocol. For RNA gel blot analysis,aliquots (10 mg) of total RNA extracted from thevarious tissues were fractionated by electrophore-sis through a 1.2% agarose–formaldehyde gel andblotted onto Nytran nylon membranes (Schleicher

and Schuell) using 10×SSC [24]. The transferredRNA was UV cross-linked to the membrane usinga UV Stratalinker (Stratagene) and the mem-branes were prehybridized in 7% SDS, 0.25 MNa2HPO4, pH 7.2 for 2–4 h at 65°C. Hybridiza-tion was carried out in the same buffer in thepresence of a-32P-dCTP labeled probes for 16 h at65°C. The membranes were washed under highstringency conditions and subject to autoradiogra-phy at −80°C for �48 h.

Restriction fragments derived from cDNAclones of interest were separated by agarose gelelectrophoresis, the DNA was purified, andquantified spectrophotometrically. The a-32P-dCTP labeled probes were prepared from 25–50ng of insert DNA by random primed labeling(Random Primed Labeling Kit, BoehringerMannheim, IN). As a control probe used to quan-tify and normalize RNA levels in each lane, blotswere hybridized with a 400-bp portion of thecDNA encoding the b-subunit of mitochondrialATPase [27]

2.5. Genomic DNA isolation and gel blot analysis

Tobacco genomic DNA was isolated from to-bacco leaf tissue by the method of Junghans andMetzlaff [28]. Total genomic DNA (15 mg) wasdigested to completion with EcoRI or HindIII, thedigestion products were fractionated by elec-trophoresis through a 0.8% (w/v) agarose gel, andtransferred onto Nytran nylon membrane (Schle-icher and Schuell) in the presence of 0.4 N NaOH[24]. Following transfer, the membrane was rinsedin 2×SSC, the DNA was UV cross-linked to themembrane, and the membrane was prehybridizedand hybridized as described above. Following hy-bridization and washing, the membranes were sub-jected to autoradiography at −80°C.

3. Results

3.1. Analysis of differentially expressed geneproducts in the roots of topped tobacco plants

Using a subtractive hybridization strategy, agroup of �60 cDNAs were isolated that showeddifferential levels of expression in the roots oftobacco (N. tabacum cv. Burley 21) plants beforeand 3 days after topping (i.e. removal of the flower

J. Wang et al. / Plant Science 158 (2000) 19–3222

head and upper leaves and stem of the plant). Thenucleotide sequence of the individual cDNAs wasdetermined and both the nucleotide and predictedamino acid sequences of the various cDNAs wereanalyzed using BLAST sequence analysis programs[25,26]. In most cases, the complete nucleotidesequence of the individual cDNA was determined.Where multiple clones were available, only fulllength clones were fully sequenced. Following ouranalysis, the majority of the cDNAs isolated couldbe placed into one of several categories based upontheir similarity to known genes or their encodedproducts already present in the NCBI databases.The results of our study are presented in Table 1.

Among the largest subset of related cDNAsrecovered were clones encoding enzymes involved inpolyamine biosynthesis and alkaloid formation(Group I). Among this group were both full lengthand partial cDNAs encoding ADC, ODC, andS-adenosylmethionine synthetase (SAMS). The fur-ther characterization of these gene products isdescribed in greater detail below.

cDNAs were also identified that encode ho-mologs of various cell wall-associated proteins orenzymes involved in wall formation (Group II).Among these are three distinct extensins and twodifferent proline-rich proteins (PRPs). The recoveryof homologs of extensin and PRPs is not surprising.Members of both gene families have been reportedto be expressed during root growth and regenera-tion, and the steady state levels of both classes oftranscripts have been shown to increase in responseto wounding [29]. The third recognizable categoryof cDNAs (Group III) contains homologs ofproteins previously shown to be involved in tran-scription or translation, or to be components ofsignal transduction pathways. Notable among theseclones is the ethylene responsive element binding(EREB) protein. The role of ethylene in the controlof gene expression both independently and in con-junction with other phytohormones (e.g. auxin) iswell documented [30].

cDNAs encoding enzymes known to be involvedin general cellular processes (i.e. cell structure, intra-and intercellular communication and transport,protein turnover, etc.) constitute Group IV. In-cluded among these are transmembrane proteinswith similarity to phosphate transporters and intrin-sic water channel proteins (e.g. PR12 and PR16)whose activities have been previously suggested tobe modulated in response to various stress responses

or changes in phytohormone levels or ratios.Changes in some of these components, such asfructose-1,6-bisphosphate aldolase and ribose-5-phosphate isomerase, may simply reflect the grossalteration in whole plant physiology upon topping.

A number of cDNAs encode proteins for whichno function has yet to be defined in plants (GroupV), or for which no match of any significance couldbe found within the databases (Group VI). There-fore, the function of these cDNAs and their impor-tance to the regulation of alkaloid formation anddistribution remains unknown.

3.2. Characterization of ADC, ODC, and SAMSproteins from N. tabacum L. c6. Burley 21

Within the differentially expressed gene productscloned in our investigation were both partial andfull-length cDNAs encoding ADC, ODC, andSAMS. PR24 encodes the full-length N. tabacumADC cDNA. The cDNA contains an open readingframe of 2163 bp coding for a protein 720 aminoacids in length. The ADC transcript contains anunusually long 5%-untranslated region (UTR) of 431nucleotides and a short 3%-UTR of 84 nucleotideswith a near consensus polyadenylation signal(AATAATA) �30 nucleotides upstream of thepoly(A)n tract.

The N. tabacum ADC is �82% identical to theADC of its evolutionary progenitor species N.syl6estris [Genbank Accession No. AB012873] and86% identical to the ADC from tomato (Lycopersi-con esculentum) [31], another member of theSolanaceae family (Fig. 1). As might be expected,the N. tabacum ADC shares considerably less sim-ilarity to ADCs isolated from species more distantlyrelated evolutionarily, such as Arabidopsis, 67%identical [32,33]; soybean, 67% identical [34]; oat,42% identical [35]; and Escherichia coli, 29% identi-cal [36].

The predicted protein coding region for the N.tabacum ADC is substantially longer than thosereported for the ADC proteins of N. syl6estris andL. esculentum [31], but is similar in length to thosereported in other higher plant species (e.g. Ara-bidopsis, oat, soybean) [32–35] and for the E. colienzyme [36]. The difference in overall length ap-pears to arise from an apparent nucleotide dele-tionin the N. syl6estris and tomato cDNA sequencesrelative to the PR24 sequence and those in otherplants. In the nucleotide sequences reported

J. Wang et al. / Plant Science 158 (2000) 19–32 23

Table 1Summary of differentially expressed cDNAs isolated from roots of wild-type Burley 21 tobacco after topping

Insert size (bp) Homology/Identity (blast score)Plasmid Accession no.designation (amt. seq.)

Group I. Alkaloid biosynthesisPR-1 SAMS, partial cds. (6e-84)1400 (219)

1200 (168) SAMS, partial cds. (8e-29)PR-2PR-3 1300 (701) SAMS, partial cds. (1e-138)

AF1272431636 (1636) SAMS, full length codingPR-6PR-7 SAMS, partial cds.600 (600)

1600 (198) SAMS, partial cds. (2e-58)PR-81300 (135)PR-9 SAMS, partial cds. (4e-27)1400 (174) SAMS, partial cds. (2e-25)PR-101600 (299)PR-11 SAMS, partial cds. (1e-102)

PR-17 U59812959 (959) ODC (partial clone)1000 (201) SAMS, partial cds. (2e-08)PR-21

PR-23 SAMS, partial cds. (1e-77)228 (228)AF1272392694 (2694) ADC, full length codingPR-24

PR-37 1600 (140) SAMS, partial cdsAF127242 ODC, full length coding1596 (1596)PR-46

Group II. Cell wall related proteinsAF1563713500 (559) Extensin 1, partial cds. (0.96)PR-29

659 (659)PR-38 AF154651 Extensin 2, partial cds. (3e-89)AF154653696 (696) Extensin 3, partial cds. (11e-3)PR-41AF154654PR-42 Extensin precursor, partial cds. (1e-17)734 (734)AF154655661 (661) Lignin-forming anionic peroxidase, partial cds. (3e-72)PR-45

PR-59 832 (832) AF154667 Proline-rich cell wall-associated protein, partial cds. (2e-94)AF1546691500 (901) Proline cell wall-associated protein, partial cds. (7e-26)PR-64

Group III. Transcription, translation, signal transduction673 (673)PR-5 AF154636 40S ribosomal S4 protein, partial cds. (1e-56)

AF154644705 (705) Glycine-rich RNA-binding protein, ABA-inducible, partial cds (3e-31)PR-20AF156367PR-22 Glycine-rich RNA binding protein, partial cds. (4e-19)128 (128)

500 (172) 26S ribosomal RNAPR-311078 (1078)PR-47 AF154656 Putative ethylene responsive element binding (EREB) protein (3e-22)

AF1546571122 (1122) Putative serine/threonine protein kinase, partial cds. (2e-08)PR-48708 (708)PR-50 AF154659 40S ribosomal S12 protein, partial cds. (8e-42)

AF154660PR-51 Putative elongation factor EF-1a; (vitronectin-like adhesion protein)948 (948)(5e-65)

AF154663PR-55 60S ribosomal L15 protein, partial cds. (2e-99)888 (888)PR-57 687 (687) AF154665 Glycine-rich RNA-binding protein (wound repressed), partial cds. (1e-35)

AF156372 60S cytoplasmic ribosomal protein L2, partial cds. (2e-71)PR-60 600 (440)

Group IV. General metabolic function housekeeping and structural genesAF154637 Putative inorganic phosphate transporter, partial cds. (9e-08)PR-12 450 (430)AF1546401451 (1451) Actin, partial cds (1e-180)PR-15AF154641 Intrinsic plasmamembrane protein (water channel), partial cds. (1e-106)PR-16 1100 (1100)AF154647637 (637) Poly-ubiquitin (2e-76)PR-32AF154648PR-33 Fructose-1,6-bisphosphate aldolase, partial cds. (1e-160)1313 (1313)AF154650638 (638) Ubiquitin conjugating enzyme E2, partial cds. (7e-69)PR-35

PR-36 X00945737 (737) a-1 Protease inhibitor (antitrypsin), partial cds. (8e-49)AF154658 Ribose-5-phosphate isomerase, partial cds. (8e-50)1084 (1084)PR-49

Group V. Unknown function in plantsAF154635685 (685) Putative N7 protein homolog, partial cds. (2e-19)PR-4

PR-13 722 (722) AF154638 dnaJ homolog, partial cds. (2e-37)AF154642057 (1057) CF2 protein homolog; partial cds. (4e-20)PR-18

1034 (1034)PR-19 AF154643 Formamidopyrimidine-DNA glycosylase, partial cds. (2e-79)AF156369 a-2-HS-glycoprotein homolog, partial cds. (3e-58)PR-27 159 (159)AF154652 Auxin regulated mRNA (glycine max), partial cds. (2e-90)824 (824)PR-39

J. Wang et al. / Plant Science 158 (2000) 19–3224

Table 1 (Continued)

Insert size (bp) Homology/Identity (blast score)Plasmid Accession no.(amt. seq.)designation

1083 (1083) AF154666PR-58 Putative auxin-regulated mRNA, partial cds. (4e-15)

Group VI. Unique (no matches in database)1085 (1085)PR-14 AF154639 Hypothetical topping-induced protein

Hypothetical topping-induced proteinAF156363PR-25 900 (171)1141 (1141)PR-26 AF154645 Hypothetical topping-induced protein

Hypothetical topping-induced proteinAF156370PR-28 1200 (106)1217 (1217)PR-M AF154646 Hypothetical topping-induced protein

750 (536) AF154659PR-34 Hypothetical topping-induced proteinHypothetical topping-induced proteinAF154661PR-52 871 (871)

429 (429) AF154662PR-53 Hypothetical topping-induced protein429 (429) (same as PR53)PR-54 Hypothetical topping-induced protein900 (705)PR-56 AF154664 Hypothetical topping-induced protein

PR-63 AF154668 Hypothetical topping-induced protein1500 (986)

Fig. 1. Comparison of the predicted amino acid sequences of arginine decarboxylases (ADCs) from various species. Shown is aPILEUP alignment of the predicted amino acid sequences of the N. tabacum cv Burley 21 ADC encoded on plasmid PR24(AF127239) with the ADCs from N. syl6estris (AB12873), Arabidopsis thaliana (AF009647), A6ena sati6a (oat) (X56802),Lycopersicon esculentum (tomato) (L16582) and E. coli (M31770). Amino acid residues conserved among the various ADC areshaded.

J. Wang et al. / Plant Science 158 (2000) 19–32 25

for both the N. syl6estris and tomatocDNAs, a guanine residue (position 2295 in the N.syl6estris sequence and 1531 in the tomato se-quence) is missing [Genbank Accession No.AB012873]. This deletion changes the reading frameand introduces a premature termination to thepredicted coding region. Using the sequence infor-mation available in the NCBI database, correctingfor this error allowed us to extend the predictedC-terminus of the both ADC proteins, yielding thealignment to the N. tabacum ADC and those ofother plant ADCs as indicated in Fig. 1. We havealso included in the alignment shown in Fig. 1, thecorrection at the N-terminus of the predictedtomato ADC protein sequence noted by Perez-Amado et al. [37], allowing better alignment of allof the higher plant sequences.

Shown in Fig. 2 is the predicted amino acidsequence for the N. tabacum ODC encoded by thefull-length cDNA PR46 in a PILEUP alignmentwith ODC proteins isolated from other plant andanimal species. The predicted amino acid sequencefor the N. tabacum ODC protein encoded in PR 46is identical to the partial N. tabacum ODC cDNAsequence (PR17) reported earlier [38], but differs ateight amino acids (98% identity) from the proteinsequence of an ODC isolated from the high alkaloidcultivar, N. tabacum cv. SC58 [Genbank AccessionNo. Y10472.1]. The two tobacco proteins are 88–89% identical to the ODC from tomato and jimson-weed (Datura stramonium) [39,40], but substantiallyless similar to other ODCs from non-photosyntheticeukaryotes and prokaryotes (e.g. C. elegans, 32%similarity; Xenopus lae6is, 31%).

All eukaryotic ODCs share several structuralfeatures in common, including a conserved lysineinvolved in binding of pyridoxyl phosphate cofactor(LYS-96 in the N. tabacum ODC sequence) and aconserved cysteine (CYS-378), which is the attach-ment site for DMFO, a potent inhibitor of enzymefunction [41]. The ODCs characterized from eu-karyotic animal cells also contain a C-terminalextension relative to the enzymes present inprokaryotes that is thought to be involved in therapid degradation/turnover of the protein [41]. Asevidenced from the alignment shown in Fig. 3, thetobacco ODC lacks this extension, as do the ODCsfrom other plant species [39].

It has been previously noted that plant ADCs andODCs share domains in common and, therefore, itis likely that they share a common evolutionaryorigin [5,41]. Among the more highly conserved

Fig. 2. Comparison of the predicted amino acid sequences ofornithine decarboxylases (ODCs) from various species.Shown is a PILEUP alignment of the predicted amino acidsequences of the N. tabacum cv. Burley 21 ODC encoded onplasmid PR46 (AF127242) with the ODCs from N. tabacumcv. SC58 (Y10472), Lycopersicon esculentum (tomato)(AF029349), Datura stramonium (jimsonweed) (X87847), andC. elegans (U03059). Amino acid residues conserved amongthe various ODCs are shaded.

J. Wang et al. / Plant Science 158 (2000) 19–3226

Fig. 3. Comparison of the predicted amino acid sequences ofS-adenosylmethionine synthetases (SAMS) from various spe-cies. Shown is a PILEUP alignment of the predicted aminoacid sequences of the N. tabacum cv Burley 21 SAMS en-coded on plasmid PR6 (AF127243), with the SAMS fromLycopersicon esculentum (tomato) (Z24743), Catharanthusroseus (Z71272), Arabidopsis thaliana (M55077), Pisum sa-ti6um (pea) (L36681), Oryza sati6a (rice) ( Z26867), and Homosapiens (humans) ( X68836 ). Amino acid residues conservedamong the various SAMS are shaded.

regions of the ADC and ODC proteins of N.tabacum is the proposed active site involved indecarboxylation of arginine and orinithine, respec-tively. This sequence, DTGGGL in ADC andDVGGGF in ODC is similar to the consensus motifDI/VGGGL/F observed in ADCs, ODCs, and therelated protein diaminopimelic acid decarboxylases,in other species of plants, animals, and microbes[5,41,42].

The largest number of cDNAs recovered by ourdifferential hybridization screening were partial andfull-length clones encoding SAMS. Based upon acomparison of available nucleotide sequences fromthe coding and 3%-UTRs of the various clones, itappears that there are at least five different ex-pressed gene products in N. tabacum roots. Shownin Fig. 3 is the predicted amino acid sequence forthe full-length N. tabacum SAMS encoded in PR6compared to the predicted protein sequences ofSAMS from other species. The N. tabacum SAMSis most similar to the SAMS of tomato (90%identical)[43] and has between 85 and 88% identityto the SAMS from plant species, including pea [44],Arabidopsis [45] and Catharanthus [46]. Significantlyless sequence similarity is found between the SAMSof N. tabacum and those present in non-photosyn-thetic eukaryotes (e.g. �60% identity to SAMSfrom yeast [47] and humans [48]).

It has been reported previously that the membersof the SAMS gene families present within variousplant species can be divided into evolutionarygroupings based upon the conserved na-ture ofspecific amino acid residues within the SAMSprimary protein sequence [46]. Comparison of thepredicted amino acid sequence for PR6 encodedSAMS from tobacco with SAMS from other plantspecies indicates that the PR6-encoded protein fallsinto the Type II cluster, as defined by Schruder etal. [46]. Unfortunately, the partial cDNAs encodingSAMS isolated in our studies do not providesufficient sequence information and therefore wecan not predict their phylogenetic placement withany accuracy.

3.3. Genomic complexity of the ADC, ODC, andSAMS gene families

Both ADC and ODC are encoded by small genefamilies in the N. tabacum genome. Gel blots of totalgenomic DNA hybridized with radioactively-la-beled ADC cDNA probes detected two major bandsand several minor bands in DNA samples digested

J. Wang et al. / Plant Science 158 (2000) 19–32 27

with either EcoRI or HindIII. Five to seven majorbands and several minor bands of sufficient size toencode full-length genes were detected when thesame blots were hybridized with radioactively-la-beled cDNA probes encoding ODC. It has beenreported previously that ADC is encoded by asingle gene or low-copy number nuclear gene inother plants species [31–33,42]. For example,tomato and soybean are reported to contain asingle ADC gene [31,42], two copies of ADC arepresent in many Brassicaceae [49], including Ara-bidopsis [33]. ODC has also been reported to beencoded by a small gene family in other plantspecies, such as Datura, where three to five familymembers are reported to be present [39]. Thepresence of multiple genes encoding ADC andODC in N. tabacum is consistent with its evolu-tionary origin from the hybridization of threedifferent progenitor species (i.e. N. syl6estris, N.tomentosiformis, and N. otophora), each of whichcould contribute a locus to the N. tabacum genome[50,51].

The recovery of multiple expressed SAMS cD-NAs is consistent with our genomic DNA gel blotanalysis (Fig. 4) showing eight to ten hybridizingbands in both EcoRI- or HindIII- digested totalgenomic DNA samples probed with a radioac-tively-labeled SAMS coding region probe. Thegene family encoding SAMS activity in N.

tabacum appears to be of greater complexity thanthat observed for either the ADC or ODC genefamily in this species. The existence of multiplegenes encoding SAMS in N. tabacum is consistentwith previous reports of multiple expressed SAMSgenes in other plant species, including pea [44],Arabidopsis [45], and tomato [43], although itshould be noted that the SAMS gene family intobacco appears to be substantially larger thanthose present in the other plant species.

3.4. RNA gel blot analysis of ADC, ODC, andSAMS expression in tobacco

The distribution and abundance of ADC, ODC,and SAMS transcripts in mature tobacco plantswas analyzed by gel blots using total RNA pre-pared from the roots, stems, leaves, and variousfloral parts of mature Burley 21 tobacco plants. Asshown in Fig. 5, transcripts encoding ODC werehighly expressed in the roots and present to alesser extent in floral portions of the plant. Littleor no expression of these transcripts was detectedin other tissues. Only very low levels of ADCtranscripts were present in roots and among floraltissues significant levels were only observed insepals. The tissue specific distribution of ODC andADC transcripts was similar but not identical tothat found for transcripts encoding putrescine N-methyl transferase (PMT), an enzyme controllingthe flow of precursors between polyamine andnicotine biosynthesis [6,50]. In contrast, transcriptsencoding SAMS were easily detected in all tissueswith slightly higher levels observed in photosyn-thetic versus non-photosynthetic tissues.

It has been previously shown that removal ofthe flower head and several young leaves (i.e.topping) leads to activation of nicotine formationin the roots of decapitated plants [6,10]. It has alsobeen reported that coincident with nicotine accu-mulation over the subsequent 24 h period, there isan increase in the levels of transcripts encodingPMT and spermidine synthase (SPS) in wild-typeplants [6,50]. To determine the effects of toppingon ADC, ODC, and SAMS expression in roots,Burley 21 plants were grown in the greenhouse tothe bud stage at which point the upper third of theplant was removed and samples of roots tissueswere collected before and at various times post-topping. As shown in Fig. 5(B), low levels of theODC and ADC transcripts were found in roots

Fig. 4. Gel blot analysis of genomic DNA from N. tabacumcv Burley 21 probed with radioactively-labeled cDNA encod-ing ADC, ODC, and SAMS. Total genomic DNA (30 mg)was digested with EcoRI or HindIII, fractionated by agarosegel electrophoresis, transferred to nylon membranes and hy-bridized with a-32P-dCTP labeled probes encoding tobaccoADC, ODC and SAMS as described in the Materials andmethods. The mobility of molecular weights standards aregiven to the right of the figure in kilobases (kb).

J. Wang et al. / Plant Science 158 (2000) 19–3228

Fig. 5. Gel blot analysis of the ADC, ODC, and SAMStranscript levels in various tissues of mature tobacco plantsand in the roots before and after topping. Total RNA wasisolated from various tissues of mature N. tabacum cv. Burley21 and analyzed by gel blot analysis using a-32P-dCTP labeledcoding region probes for ADC (PR24), ODC (PR46), andSAMS (PR10). As a control, the blots were also probed withradioactively labeled probes encoding the alkaloid biosynthe-sis enzyme putrescine PMT [50], the b-subunit of couplingfactor CF1-ATPase (b-ATPase) [27] and 26S rRNA (PR31).Panel A. Transcript levels in various organs of wild-typetobacco. R, Root; St, Stem; ML, Mature Leaf; YL, YoungLeaf; Se, Sepal; C, Carpel; MS, Mature Stamen; P, Petal; EtBr, ethidium bromide stained. Panel B. Transcript levels inroots of Burley 21 tobacco plants before and after topping.

under stress will show higher levels of ODC priorto topping. Low but detectable amounts of mR-NAs encoding SAMS are present in the roots ofuntopped plants, and the level of SAMS tran-scripts changed little over the 24 h period aftertopping.

No significant expression of the ODC and ADCtranscripts was observed in leaf tissues before orafter topping. In contrast, transcripts encodingSAMS were moderately abundant in young leavesand present at low levels in the mature leaves andother photosynthetic tissues. This observation isconsistent with increased requirement for methylgroups during light-induced leaf development andthe general requirement of SAM for a wide rangeof cellular processes.

4. Discussion

A large number of endogenous and exogenousfactors are known to affect gene expression lead-ing to alkaloid formation in plants including de-velopmental age, phytohormones, and variousbiotic and abiotic stresses [1,2,4,8,41]. In thepresent study, we describe the cloning and charac-terization of a group of cDNAs, differentiallyexpressed in the roots of tobacco after topping, aprocess involving the removal of the floweringhead and young leaves and stem from the upperportion of a maturing tobacco plant. Topping is acommon practice during commercial tobacco pro-duction and serves several purposes [10]. It re-leases the plant from apical dominance, causingincreased root growth, stimulation of alkaloidbiosynthesis in the roots and accumulation ofthese compounds in the leaves, and acceleration ofsenescence of the mature leaves near the base ofthe plant. Consistent with these changes, amongthe main categories of differentially expressed geneproducts recovered were cDNAs encoding en-zymes of polyamine biosynthesis and alkaloid for-mation (e.g. ADC, ODC, and SAMS).

Although the nature of the cellular signals andthe transduction pathways operating to bringabout differential plant growth and activation ofalkaloid metabolism are not yet known, amongthe most obvious factors are alterations in phyto-hormone levels and/or ratios within the plant as aconsequence of topping. Meristems and youngleaves are known to be a major source of auxin

prior to topping and message abundance increasedin the roots of topped Burley 21 plants signifi-cantly during the 24 h period after topping. Thelevel of ODC transcripts found in roots prior totopping is dependent on the developmental age ofthe plant and is influenced by other a number offactors (e.g. water and nitrogen availability, tem-perature stress). In general, older plants or plants

J. Wang et al. / Plant Science 158 (2000) 19–32 29

and their removal would be expected to lead tosignificant changes in auxin availability, as well asto an alteration of auxin to cytokinin ratios withinthe shoots and roots. Such changes in phytohor-mone levels and ratios are known to result in theactivation of expression of some genes and repres-sion of others. For example, previous studies haveshown that auxin has a negative effect on nicotineand total alkaloid biosynthesis in plants and cul-tured cells, with the effects seen both at the level ofenzyme activation and gene expression [6,15,52–55]. Expression of putrescine PMT, a key enzymein nicotine and tropane alkaloid formation, wasreported to be repressed in cultured tobacco rootsby the presence of indolebutyric acid (IBA) in thegrowth media [6]. Removal of the IBA from thegrowth medium led to an increase of both PMTactivity and steady state levels of mRNA encodingthe enzyme. Similarly, addition of auxin to hor-mone free media used for the culture of Catha-ranthus roseus cells led to a decrease in the levels ofstrictosidine synthase and tyrosine decarboxylasetranscription and a corresponding decrease in alka-loid accumulation [53]. It is perhaps interestingthat in addition to recovering enzymes directlyinvolved in alkaloid formation (i.e. ODC, ADC),we also recovered cDNAs encoding homologs ofproteins whose expression are known to be regu-lated directly by auxin (e.g. PR39, PR58) as well asproteins associated with cell enlargement and dif-ferentiation whose expression are linked to phy-tohormone driven growth and development (e.g.PR29, PR38, PR59, PR 64).

In pea, ADC expression is high in young devel-oping shoots, leaves and floral organs and levels ofboth ODC and ADC activity parallel growth ratesduring the early stages of fruit development [37,56].In contrast, mature tissues show very low levels ofactivity of these enzymes and little or no detectabletranscript levels. Increased ADC activity could bedetected following gibberellin treatment of unpolli-nated ovaries to induce parthenocarpic fruit devel-opment in [56]. However, not all of the observedchanges in the activity of these enzymes duringfruit development can be attributed to changes ingene expression [31,37]. In germinating soybeanseedlings, ADC activity and transcript levels werehighest in elongation zone, between the roots andhypocotyl hook, and lowest in the leaves [42].Cumulatively, these data fit well with our observa-tions that levels of ADC and ODC transcripts are

highest in the roots and floral organs, and low inother plant tissues.

Wounding of the leaves of N. syl6estris andother wild species of tobacco has been shown toinduce de novo formation of nicotine in the rootsand its rapid accumulation throughout the plant[9,11–13]. Several lines of evidence suggest that theexpression of genes involved in polyamine forma-tion and subsequent nicotine biosynthesis may beunder the control of a JA signal transductionpathway [8,9,12–15]. For example, direct applica-tion of JA or its methyl derivative (MeJA) to theleaves of intact tobacco plants results in increasednicotine formation [11–14]. Treatment of tobaccoBY-2 suspension cultures with MeJA resulted inincreased alkaloid formation and induced smalltransient increases in PMT, ODC and SAMSmRNA levels [15]. In contrast, steady state levelsof ADC and SAMDC were not affected, suggest-ing that not all genes encoding enzymes ofpolyamine and alkaloid formation are under thesame type of regulation. The observed increase inODC, SAMS and PMT transcript levels was lesswhen BY-2 cells were cultured in the presence of2,4-D than BA indicating that multiple regulatorycircuits must exist within the plant cell [15]. Fur-ther support for the presence of cross-talk amongregulatory pathways comes from work with Catha-ranthus roseus, where addition of JA, MeJA, andtraumatic acid to suspension cultures of Catha-ranthus cells led to increased alkaloid formationonly when cells were grown in auxin depletedmedium [55].

At the present time, only limited information isavailable on the nature of regulatory regions in thepromoters of genes encoding enzymes of alkaloidbiosynthesis. Experiments on transgenic plants us-ing various promoter-b-glucuronidase (GUS) re-porter gene fusions have identified regions withinthe promoter of the hyoscyamine 6b-hydroxylase[57], PMT [58], and tyrosine/dihydroxyphenylala-nine decarboxylase [59] gene promoters necessaryfor the cell-type specific and temporal specific reg-ulation. In addition, using a combination of pro-moter deletion analysis and gel retardation assays,evidence has been found which suggests that thepromoters of several genes whose products areinvolved in terpenoid indole alkaloid formation(e.g. tryptophan decarboxylase (tdc), strictosidinesynthase (Str1)) contain sites capable of interactingwith a common transcriptional regulatory factor

J. Wang et al. / Plant Science 158 (2000) 19–3230

[60,61]. The biosynthesis of nicotine has beenshown to be under the control of at least twounlinked genetic loci, termed Nic1 and Nic2[16,17]. Although no information is available onthe nature of their encoded products, it has beenspeculated that Nic1 and Nic2 likely encode tran-scriptional regulators capable of globally interact-ing with a subset of genes encoding components ofpolyamine and alkaloid biosynthesis [6]. Theavailability of cDNAs and cloned genomic frag-ments encoding enzymes in nicotine and alkaloidformation are the first step towards unravelingthese processes.

Acknowledgements

The authors wish to thank Maria Shulleeta andher colleagues at Philip Morris for providing uswith the cDNA library and cDNA cloning infor-mation used in these studies, Jacques Retief for hishelp with the nucleotide and amino acid sequenceanalysis and alignment programs, and Ved PalMalik for his helpful suggestions. This work wassupported by a grant from Philip Morris (Rich-mond, VA).

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