WO2000067558A1 - Regulation of gene expression in tobacco for manipulation of plant growth and secondary metabolism - Google Patents

Abstract

This invention relates to enzymes involved in alkaloid, and specifically nicotine, formation in tobacco plants. The invention is based, at least in part, on the nucleotide sequences encoding four variants of putrescine N-methyltransferase (PMT1, PMT2, PMT3, and PMT4), two variants of arginine decarboxylase (ADC1 and ADC2), ornithine decarboxylase (ODC), S-adenosylmethionine synthetase (SAMS), a fragment of NADH dehydrogenase, and a fragment of phosphoribosylanthranilate isomerase. The invention also relates to proteins expressed by these nucleotides, promoter regions of these nucleotides, use of these promoter regions to culture transgenic plant cells and to produce transgenic plants, sense and antisense nucleotides complementary to all or portions of these nucleotide sequences, use of sense and antisense nucleotides to regulate gene expression, and assays using proteins involved in alkaloid formation in tobacco plants.

Description

REGULATION OF GENE EXPRESSION IN TOBACCO FOR MANIPULATION OF PLANT GROWTH AND SECONDARY METABOLISM

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a contmuation-m-part of US Patent Application Ser. No. 60/ 132,919, filed

May 6, 1999, now abandoned, which is hereby incorporated by reference in its entirety herein.

FIELD OF THE INVENTION This invention relates to enzymes involved in alkaloid, and specifically nicotine, formation in tobacco plants. The invention is based, at least in part, on the nucleotide sequences encoding four vaπants of putrescine N-methyltransferase (PMT1, PMT2, PMT3, and PMT4), two vaπants of arginine decarboxylase (ADC 1 and ADC2), omithine decarboxylase (ODC), S-adenosylmethionme synthetase (SAMS), a fragment of NADH dehydrogenase, and a fragment of phosphoπbosylanthranilate isomerase. The invention also relates to proteins expressed by these nucleotides, promoter regions of these nucleotides, use of these promoter regions to culture transgenic plant cells and to produce transgenic plants, sense and antisense nucleotides complementary to all or portions of these nucleotide sequences, use of sense and antisense nucleotides to regulate gene expression, and assays using proteins involved in alkaloid formation m tobacco plants.

BACKGROUND OF THE INVENTION I. Alkaloid Formation

Alkaloids are one of the most diverse groups of secondary compounds found in plants and they are the product of a complex biosynthesis pathway (Hashimoto and Yamada, 1994; Chou and Kutchan, 1998; Waterman, 1998). Why plants accumulate these compounds and in so many different forms is not known. Moreover, for many alkaloids, the exact site of synthesis and the factors that control their intercellular distribution and accumulation remain to be determined (Hashimoto and Yamada, 1994; Kutchan, 1995; Chou and Kutchan, 1998)

Nicotine is the most abundant alkaloid present m cultivated tobacco. Nicotine is formed pπmaπly in the roots of the tobacco plant and subsequently is transported to the leaves, where it is stored (Tso, Physiology and Biochemistry of Tobacco Plants, pp. 233-34, Dowden, Hutchmson & Ross, Stroudsburg, Pa. (1972))

The synthesis and accumulation of nicotine and other tobacco alkaloids are known to be controlled by vaπous developmental, environmental, and chemical cues. Changes in phytohormone (e.g., auxin, cytokinin) levels and/or ratios as a consequence of developmental age (Hashimoto and Yamada, 1994; Kutchan, 1995) or by direct manipulation of plant cell culture conditions have been shown to affect the synthesis and accumulation of nicotine and various tobacco alkaloids (Hashimoto and Yamada, 1994; Hibi et al, 1994; Eilbert, 1998). Various abiotic factors (wounding, drought stress, pH imbalance, etc.) [Hashimoto and Yamada, 1994; Kutchan, 1998; Waterman, 1998) 1, 2, 4], as well as biotic factors, such as herbivory, insect feeding, and attack by various microbial and fungal pathogens, are known elicit increased production of nicotine and other alkaloids in the leaves of wild and cultivated tobacco species (Baldwin, 1989; Saito and Murakoishi, 1998; Baldwin and Prestin, 1999). In addition, the commercial practice of topping (i.e., removal of flowering head and young leaves at the upper portions of the plant), results in increases in nicotine and the amount and complexity total alkaloids present in the leaves of Nicotiana tabacum (Hashimoto and Yamada, 1994; Hibi et al., 1994). The factors controlling the topping-induced increase in alkaloid biosynthesis are not known, but likely involve a complex physiological response in the plant as a result of altered phytohormones and wound induced signaling (Akehurst, 1981; Hibi et al, 1994; Kutchan, 1998). In this regard, considerable evidence now exists indicating that a jasmonic acid (JA)- mediated signal transduction pathway may play a role in regulation of gene expression contributing to this increase in alkaloid biosynthesis (Baldwin et al, 1994, 1996,1997; Ohnmeiss et al., 1997; Imanishi et al, 19 _a, 1998b).

The nicotine molecule is comprised of two heterocyclic rings, a pyridine moiety and a pyπolidine moiety, each of which is derived from a separate biochemical pathway. The pyridine moiety of nicotine is derived from nicotinic acid. The pyrrolidine moiety nl^' nicotine is provided through a pathway leading from putrescine to N-methylputrescine and then to N-methylpyrrolme. (Goodwin and Mercer, Introduction to Plant Biochemistry, pp. 488-91, Pergamon Press, New York, (1983)). Putrescine is formed in plants by one of two pathways (Chattopadhyay and Ghosh, 1998). It can be synthesized directly from omithine, in a reaction catalyzed by the enzyme omithine decarboxylase (ODC, EC 4.1.1.17), or formed indirectly from arginine in a reaction sequence initiated by arginine decarboxylase (ADC, EC 4.1.1.19). Putrescine formed by the ADC and/or ODC pathway serves as precursor in the synthesis of the higher polyamines, spermine and spermidine, catalyzed by the enzymes spermine synthase and spermidine synthase, respectively, or it is converted to N-methylputrescine by the action of putrescine N-methyltransferase (PMT), the first committed step in nicotine biosynthesis (Hashimoto and Yamada, 1994; Kutchan, 1995; Chattopadhyay and Ghosh, 1998). N-methyl putrescine is oxidized by a diamine oxidase and cyclized to form the 1- methyl-Δ'-pyrrolium cation, which is condensed with nicotinic acid or its derivative to form nicotine (Hashimoto and Yamada, 1994).

Putrescene is a precursor for N-methylputrescine, which then forms N-methylpyrroline. Conversion of putrescine to N-methylputrescine is catalyzed by the enzyme putrescine Ν-methyltransferase ("PMT"), with S-adenosylmethionine serving as the methyl group donor. PMT appears to be the rate-limiting enzyme in the pathway supplying Ν-methylpyrroline for nicotine synthesis in tobacco (Feth et al., "Regulation in Tobacco Callus of Enzyme Activities of the Nicotine Pathway", Planta, 168, pp. 402-07 (1986); Wagner et al., "The Regulation of Enzyme Activities of the Nicotine Pathway in Tobacco", Physiol. Plant., 68, pp. 667-72 (1986)).

II. TRANSGENIC PLANTS

The methods of nicotine formation in tobacco and the genes involved have been studied both to better understand differential gene expression during tobacco growth and development, and also to discover tools useful for creating transgenic plants. For example, the regulatory sequences that modify protein expression in tobacco may be useful in creating transgenic tobacco or other transgenic plants.

It has already been demonstrated that tissues of many plant species may be transformed by exogenous, typically chimeric, genes which are effective to stably transform cells of the tissues. For several species, tissues transformed in this fashion may be regenerated to give rise to whole transqenic or genetically engineered plants. The engineered traits introduced into the transgenic plants by these techniques have proven to be stable and have also proven to be transmissible through normal Mendellian inheritance to the progeny of the regenerated plants. One such desirable trait is the production in the plant cells of desired gene products in vivo in the cells of the transqenic plants. For a chimeric gene to be effective, the foreign DNA sequence containing a coding region should be flanked by appropriate promotion and control regions. Commonly used plant cell transcription promoters include the nopaline synthase promoter from the T-DNA of A. tumefaciens and the 35S promoter from the cauliflower mosaic vims.

In order for the newly inserted chimeric gene to express the protein for which it codes in the plant cell, the proper regulatory signals must be present and in the proper location with respect to the gene. These regulatory signals include a promoter region, a 5' non-translated leader sequence and a 3' polyadenylation sequence. A promoter is a DNA sequence that directs the cellular machinery of a plant to produce RNA from the contiguous structural coding sequence downstream (3') to the promoter. The promoter region influences the rate at which the RNA product of the gene and resultant protein product of the gene is made. The 3' polyadenylation signal is a non-translated region that functions in the plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA to enable the mRNA to be transported to the cytoplasm and to stabilize the mRNA for subsequent translation of the RNA to produce protein.

Other plant cell transformation techniques are directed toward the direct insertion of DNA into the cytoplasm of plant cells from which it is taken up, by an uncharacteπzed mechanism, into the genome of the plant. One such technique is electroporation, in which electric shock causes disruption of the cellular membranes of individual plant cells. Plant protoplasts m aqueous solution when subject to electroporation will uptake DNA from the surrounding medium. Another technique involves the physical acceleration of DNA, coated onto small inert particles, either into reqenerable plant tissues or into plant germlme cells.

The availability of cloned nucleic acid sequences encoding an enzyme involved m alkaloid synthesis allows for the potential manipulation of alkaloid contents. Furthermore, the availability of promoters useful for expressing genes in plants allows for the creation of chimeπc molecules and transgenic plants, which in turn result in possible native plant production of desirable proteins Previously reported work discloses cloning nucleotides encoding proteins involved in the biosynthesis of nicotine, and isolating such proteins. Approximately twenty or more cDNAs and/or genomic DNA fragments encoding different enzymes involved with alkaloid formation have been isolated (Chattopadhyay and Ghosh, 1998) For example, successful cloning of partial or full-length cDNA encoding ODC activity from tobacco was disclosed by (Malik et al., J Plant Biochem &Bιotech 5 109-112 (1996)). Also, a relatively cmde preparation of PMT (30-fold puπfication) has been subjected to limited characteπzation (Mizusaki et al., "Phytochemical Studies on Tobacco Alkaloids XIV. The Occurrence and Properties of Putrescine N-Methyltransferase in Tobacco Plants", Plant Cell Physiol., 12, pp. 633-40 (1971)). A process for purifying PMT is disclosed m US Patent No 5,369,023, "Method of puπfymg putrescine n-methyltransferase from tobacco plant extract with an amon exchange medium", hereby incorporated by reference m its entirety herein

Several laboratoπes have reported the cloning of partial or full-length cDNAs encoding ADC (Bell and Malmberg ,1990; Rostogi et al., 1993; Perez-Amador et al., 1995; Nam et al., 1997, Watson and Malmberg, 1996). Compaπsons of the ammo acid sequences of ADC from vaπous plants revealed a high degree of conservation among the vaπous proteins, as well as homology to ODC (Malmberg et al., 1998).

It is an object of the present invention to characteπze the nucleotide and ammo acid sequences of enzymes involved in the biosynthesis of nicotine in tobacco. It is also an object of the present invention to provide plant promoter regions that are capable of conferπng high levels of transcπption in rapidly dividing cells of transformed plants when coupled with a heterologous coding sequence in a chimeπc gene. Further, the invention is directed to chimeπc genes incorporating such promoter regions, stable transfection of plants with these chimeπc genes, and the plants and cells that are transfected, as well as seeds of such transfected plants. It is a further object to charactenze sense and antisense nucleotides capable of regulating expression of genes encoding for enzymes involved in the biosynthesis of alkaloids.

SUMMARY OF THE INVENTION Proteins involved in the biosynthesis of nicotine in tobacco N. tabacum are the subject of this invention. More specifically, the invention concerns four variants of putrescine N-methyltransferase (PMT1 , PMT2, PMT3, and PMT4), two vaπants of arginine decarboxylase (ADC 1 and ADC2), omithine decarboxylase (ODC), S-adenosylmethionme synthetase (SAMS), NADH dehydrogenase, and phosphoπbosylanthramlate isomerase.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 Genomic DNA gel blot analysis of the PMT gene family in N tabacum cv. Xanthi.

Total genomic DNA (30 μg) was digested with Kpril, EcoRl, or EcoRI and Kpnl, separated by agarose gel electrophoresis, and transferred to nylon membranes. The membrane was hybπdized with a 32p-iabeled antisense strand probe coveπng the complete coding region of the NtPMTla cDNA. Identity of the hybndizmg bands as determined by compaπson to phage DNA digests is indicated. Molecular weights are given in kb. Note that Kpnl shifts only the NtPMTlb band m the gel blot because this restriction site is present ony in Exon 1 of NtPMTlb and not NtPMTla.

Figure 2 Ammo acid sequence alignment of N tabacum PMTs. Shown is a PILEUP alignment of the predicted ammo acid sequences of the vaπous tobacco PMTs. Amino acid residues that diffeπng among the PMTs are shaded. NtPMTla, NtPMT2, NtPMT3, and NtPMT4 refer to the deduced ammo acid sequences of the PMTs encoded by the NtPMTla, NtPMTl, NtPMT3, and NtPMT4 genes, respectively, isolated from N tabacum cv. Xanthi genomic DNA; cNtPMTla is the predicted amino acid sequence of the A411 cDNA (Accession No. D28506) isolated from N tabacum cv Burley 21 by Hibi et al. (1994). The location of the exon-intron boundaπes are indicated by the dark vertical line. The nucleotide sequences for NtPMTla, NtPMT2, NtPMT3, and NtPMT4 appear m GenBank under the accession numbers AF126810, AF126809, AF126811, and AF126812, respectively

Figure 3. Polyacrylamide gel electrophoresis analysis of PCR amplified genomic DNA fragments encoding Exon 1 of PMT from vaπous species of Nicotiana. PCR amplification was earned out as descπbed in the Mateπals and Methods using Exon 1 -specific pnmers 1 and 2 and total genomic DNA isolated from N. tabacum, N otophora, and N. tomentosiformis . The amplification products were separated by electrophoresis on 6.5% polyacrylamide gels, the gels fixed, and subject to autoradiography. The amplification products isolated from N. tabacum cv. Burley 21 and N tabacum cv. Xanthi were identical and only the amphcation products from the reactions with N. tabacum cv. Burley 21 DNA are shown. Standards were generated in identical reaction conditions pπmed with plasmid DNA encoding the vanous PMT genes isolated in this study.

Figure 4 Nucleotide sequence alignment of the 5'-flankιng regions of the N. tabacum PMT genes. Shown is a PILEUP alignment of the nucleotide sequences upstream of the initiating methionine (MET) codon of the four PMT genes isolated from N. tabacum cv. Xanthi. The proposed start site for transcπption of the NtPMTla gene is indicated by the +1 above the sequences. The TATA-box and CCAAT-box motifs are boxed. Potential transcπptional regulatory elements identified by MOTIF search programs are also boxed and indicated by the following abbreviations:. PAL: pahndromic sequences; G-Box: G-Box homologous sequences; MRE: metal-responsive element homolog. Nucleotides identical in three or more sequences are shaded. The polyguanme- nch region is underlined. Numbenng is indicated to the πght and is relative to the proposed start site of each gene.

Figure 5. RNA gel blot analysis of EΛ/T transcnpt levels m various tissue , Total RNA was isolated from vanous tissues of mature N tabacum cv. Burley 21 and analyzed by gel blot analysis using a ³²P-labeled NtPMTla cDNA coding region (Εxons 2 to 8) probe capable of detecting all PMT transcπpts. A. PMT transcnpt levels in vanous tobacco plant tissues and/or organs.

B. Induction of PMT expression m tobacco roots following topping. Abbreviations: HP, wild-type (NιclNic2) Burley 21; LP, low alkaloid (niclnid) mutant. The β-subunit of mitochondnal ATPase (β-ATPase) served as a control.

Figure 6. Semi-quantitative RT-PCR analysis of PMT gene expression in roots of tobacco plant before and after topping. A. Shown is relative abundance of the individual PMT gene transcnpts before and after topping. RT- PCR was carried out as described m the Material and methods using Εxon 1 specific pnmers. Messenger RNA was amplified from total RNA isolated from the roots of wild-type (HP, NiclNic2) Burley 21 and low alkaloid (LP, niclnic2) Burley 21 tobacco plants. Far right lane represents size standards for the genes isolated by PCR amplification from plasmid DNA. The β- subunit of mitochondrial ATPase (β-ATPase) served as a control.

. Bar graphs showing relative expression of the individual PMT genes following topping in both HP and LP tobacco roots. Abbreviations: HP, wild-type (NiclNic2) Burley 21; LP, low alkaloid (niclnicl) mutant.

Figure 7. The nucleotide and predicted amino acid sequences of the transcribed portions of the N. tabacum cv Xanthi NtADCl and NtADC2 genes. Shown are the complete nucleotide and predicted amino acid sequence of the N. tabacum cv Xanthi NtADCl gene and where it differs from the NtADC2 gene sequence. The dots indicate nucleotide sequence identity and the stars indicate amino acid sequence identity. The proposed polyadenylation signal is underlined. The sequences terminate at the point of polyadenylation found in the full length cDNA (Wang, 1999; AF127239).The complete nucleotide sequences for the N. tabacum cv Xanthi NtADCl (AF 127240) and NtADC2 (AF127241) including the 5' and 3' flanking sequences appear in Genbank.

Fig. 8. Comparison of the predicted amino acid sequences of arginine decarboxylases (ADCs) from various species. Shown is a PILEUP alignment of the predicted amino acid sequence of the N. tabacum cv Xanthi NtADCl gene (AF 127240) aligned to the predicted ADC protein sequences from N. sylvestris (AB12873), Arabidopsis thaliana (AF009647), Avena sativa (oat) (X56802), Lycopersicon esculentum (tomato) (LI 6582) and Escherichia coli (M31770). Amino acid residues conserved among the various ADC are shaded.

Fig. 9. Gel blot analysis of ADC transcript levels in the roots of wild-type and low alkaloid mutant Burley 21 tobacco before and after topping. Total RNA was isolated from the roots of mature wild- type and low alkaloid mutant N. tabacum cv. Burley 21 and analyzed by gel blot analysis using [α- ³²P]-dCTP labeled probes recognizing the coding region of ADC or the β-subunit of tobacco mitochondrial ATP synthase (Boutry and Chua, 1985). Quantitation was carried out by phosphorimaging using a Molecular Dynamics Phosphorlmager. Values were normalized relative to the intensities of the atp2 control band in each lane. The experiment was conducted twice with different total RNA samples. Fig. 10. Differential expression of NtADC-1 and NtADC-2 in various tissues of tobacco. Expression of the NtADC-1 and NtADC-2 genes was analyzed using semi-quantitative RT-PCR and gene specific primers capable of discriminating between transcripts arising from the two genes. Panel A shows control reactions demonstrating primer specificity in the PCR reactions using plasmids containing the NtADC-1 and NtADC-2 coding sequences. The numbers above the lane refer to the specific primer combinations as described in the Materia and methods. Panel B shows the results of RT-PCR reactions using first strand cDNA synthesized from total RNA extracted from either root, leaf, or flowers. As a internal control, primers specific for the atp2 gene transcript were include in the amplification reactions. All reactions were carried out within the linear range of template amplification as determined by varying template amount, amplification time, and temperature as described in Riechers and Timko (1999).

Fig. 11. Genomic DNA gel blot analysis of the ODC gene family in N. tabacum. Total genomic DNA (30 μg) was digested with EcøRI or Hindlll, fractionated by agarose gel electrophoresis, transferred to nylon membranes and hybridized with an α-³²P-dCTP labeled probe encoding full- length ODC cDNA as described in the Materials. The mobility of molecular weights standards are given to the right of the figure in kilobases (kb).

Fig 12. Comparison of the nucleotide and predicted amino acid sequences of the NtODC-1 and NtODC-2 genes. Shown are the nucleotide and predicted amino acid sequences of the NtODC-1

(AF233850) and NtODC-2 (AF233849) genes. In the figure, the complete amino acid sequence of the pODC2 is given and the pODC 1 sequence is given only where it differs. The start site of transcription is designated as +1 and the poly(A) addition site is indicated by the arrow. Within the relevant regions of homology, nucleotide differences between the NtODC-1 and NtODC-2 genes are in bold lettering. The proposed TATA-box, and polyadenylation signal are shaded.

Fig. 13. Protein sequences alignment of omithine decarboxylases (ODCs) from various species. Shown is a PILEUP alignment of the predicted amino acid sequences of the N. tabacum cv. Xanthi pODC2 protein (AF233849) with the ODCs from N. tabacum cv. SC58 (Y10472) and cv. BY-2 (ABO31066), Lycopersicon esculentum (tomato) (AF030292), Datura stramonium (jimsonweed) (X87847), Saccharomyces cerevisiae (NP_012737), and humans (Homo sapiens; AAA59966). Amino acid residues conserved among the various ODCs are shaded. Fig. 14. Gel blot analysis of ODC transcnpt levels in vanous tissues of mature tobacco plants and in the roots before and after topping. Total RNA was isolated from vaπous tissues of mature N tabacum cv. Burley 21 and analyzed by gel blot analysis using an α-³²P-dCTP labeled coding region probes for ODC. (A) Transcnpt levels in vanous organs of wild-type tobacco: R, root: S, stem ; L, leaf ; SE, sepal; PE, petal; O, ovary; S, stamen; and AN, anther. (B) Transcript levels in roots of Burley 21 tobacco plants before and after topping. RNA gel blot analysis of the tissues-specific distribution and post-topping expression of transcripts encoding ODC in tobacco. As a control, the blots were also probed with radioactively labeled probes encoding the alkaloid biosynthesis enzyme putrescine N-methyltransferase (PMT) and a root specific β-glucosidase (TBG-1).

DETAILED DESCRIPTION OF THE INVENTION Nucleic acid sequences have been isolated from tobacco that encode important enzymes in nicotine and total alkaloid formation, including PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, and SAMS. Also identified are cDNA fragments encoding partial segments of NADH dehydrogenase and phosphonbosilanthromlate isomerase. Also identified are promoter regions for the nucleotides encoding PMTl, PMT2, PMT3, PMT4, and ADC2. All of these nucleic acids are isolated from Nicotiana tabacum L.

"Promoter" and "promoter region" are terms used interchangeably herein to refer to a DNA sequence that regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in cells. The term also encompasses the 5 'untranslated region that may be transcnbed into mRNA but is not translated.

"Protein", "polypeptide", and "peptide" are used interchangeably herein when referring to a gene product.

In one aspect, the invention features isolated nucleic acid molecules encoding for PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, and SAMS, a fragment of NADH dehydrogenase and a fragment of phosphonbosilanthromlate isomerase. The disclosed molecules can be non-coding (e.g. probe, antisense or πbozyme molecules) or can code for a functional enzyme. In one embodiment, the nucleic acid molecules can hybndize to the nucleic acid sequences encoding for PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, SAMS, a fragment of NADH dehydrogenase, or a fragment of phosphonbosilanthromlate isomerase or to the complements of these nucleic acid sequences. In a preferred embodiment, the hybndization is conducted under mildly stπngent or stπngent conditions In further embodiments, the nucleic acid molecule is at least 50%, 60%, 70%, 80% and more preferably at least 90% or 95% homologous in sequence to the nucleic acid sequences encoding for PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, SAMS, a fragment of NADH dehydrogenase, or a fragment of phosphoribosilanthronilate isomerase or to the complements of these nucleic acid sequences. In another embodiment, the nucleic acid encodes a polypeptide that is at least 50%, 60%, 70%, 80% and more preferably at least 90% or 95% similar in sequence to the amino acid sequence of PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, SAMS, the fragment disclosed herein of NADH dehydrogenase, or the fragment of phosphoribosilanthronilate isomerase disclosed herein. In another embodiment, the invention features isolated polypeptides, preferably substantially pure preparations, encoded for by the nucleic acid sequences of the invention. Particularly preferred are those polypeptides encoded for by the nucleic acid sequences identified by (SEQ. ID. NO. 2), (SEQ. ID. NO. 5), (SEQ. ID. NO. 8), (SEQ. ID. NO. 11), (SEQ. ID. NO. 13), (SEQ. ID. NO. 15), (SEQ. ID. NO. 18), (SEQ. ID. NO. 21), (SEQ. ID. NO. 23), (SEQ. ID. NO. 25) or (SEQ. ID. NO. 26) or comprising a nucleotide sequence encoding the amino acid sequence encoded by (SEQ ID NO. 3), (SEQ. ID. NO. 6), (SEQ ID. NO. 9), (SEQ. ID. NO. 12), (SEQ. ID. NO. 14), (SEQ. ID. NO. 16), (SEQ. ID. NO. 19), (SEQ. ID. NO. 22) or (SEQ. ID. NO. 24). In particularly prefened embodiments, the subject polypeptides can aid in regulating the production of alkaloids, particularly nicotine, in plants. In one embodiment, the polypeptide is identical to or similar to the protein represented by the amino acid sequences of (SEQ ID NO. 3), (SEQ. ID. NO. 6), (SEQ ID. NO. 9), (SEQ. ID. NO. 12), (SEQ. ID. NO. 14), (SEQ. ID. NO. 16), (SEQ. LD. NO. 19), (SEQ. ID. NO. 22) or (SEQ. ID. NO. 24). In a preferred embodiment, the polypeptide is encoded by a nucleic acid that hybridizes with a nucleic acid represented in. The polypeptides of the present invention can comprise full length proteins, such as represented by (SEQ ID NO. 3), (SEQ. ID. NO. 6), (SEQ ID. NO. 9), (SEQ. ID. NO. 12), (SEQ. ID. NO. 14), (SEQ. ID. NO. 16), (SEQ. ID. NO. 19), (SEQ. ID. NO. 22) and (SEQ. ID. NO. 24) , or can comprise one or more fragments conesponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150, or 200 amino acids in length. Another aspect of the invention features chimeric genes comprised of a promoter for the genes for PMT2, PMTl, PMT3, PMT4, or ADC2. Yet another aspect of the invention features chimeric genes or chimeric molecules comprised respectively of the functional gene encoding for or the protein PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, SAMS, NADH dehydrogenase and/or phosphoribosilanthronilate isomerase. The invention also concerns isolated and purified promoter regions for tobacco Beta- glucosidase and their use in chimeric genes or chimeric molecules.

Another aspect of the invention involves vectors capable of transporting another nucleic acid to which a vector has been linked. Preferably, the vectors comprise the nucleic acid sequences of the invention or their complements. The invention also features transgenic plants and their seeds that include (and preferably express) a heterologous form of PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, SAMS, NADH dehydrogenase and/or phosphoribosilanthronilate isomerase. The present invention also encompasses transgenic plants that contain in their genome a chimeric gene construction incorporating the nucleic acid encoding PMTl, PMT2, PMT3, PMT4, ADCl, ADC2, ODC, SAMS, NADH dehydrogenase and/or phosphoribosilanthronilate isomerase. Such transgenic plants and their seeds may be useful to natively produce enhanced quantities of desirable exogenous proteins, such as compounds useful for pharmaceutical purposes, or proteins that provide herbicide resistance.

Another feature of the invention is the use as probes of the DNA sequences disclosed herein or oligonucleotide fragments thereof. Probes may be useful to obtain additional gene family members or locate homologous genes in tobacco or other plant species. Copies of related genes can be obtained from existing genomic libraries or the genomic libraries can be constructed. In one embodiment, an isolated DNA sequence comprising about a fifteen to about a twenty-five base pair oligonucleotide sequence identical to any consecutive about fifteen to about twenty-five base pair sequence found in the sequences of the invention is used as a probe.

Another feature is use of the polypeptides of the invention in an assay, such as an assay to identify modulators of enzyme activity in plants.

Other features and advantages of the invention will be apparent to those of skill in the art. The nucleotide and amino acid sequences of the invention are disclosed herein in the Sequence Listing, text, and the figures. Specific sequences of the invention are provided in the attached Sequence Listing and can be understood to represent promoters, nucleic acids, and proteins respectively relating to the following proteins: PMT2 (SEQ. ID. NOS. 1, 2, and 3); PMTl (SEQ. ID. NOS. 4, 5, and 6); PMT3 (SEQ. ID. NOS. 7, 8, and 9); PMT4 (SEQ. ID. NOS. 10, 11, and 12); SAMS (SEQ. ID. NOS. 13 and 14 ); ODC (SEQ. ID. NOS. 15 and 16); ADCl (SEQ. ID. NOS. 17, 18, and 19); ADC2 (SEQ. ID. NOS. 20, 21, and 22); ADCl mRNA (SEQ. ID. NOS. 23 and 24); NADH dehydrongenase (SEQ. ID. NO. 25); and PAI (SEQ. ID. NO. 26). If only two sequence identifiers are provided for a protein, those sequences represent the nucleic acid and the protein respectively. If three identifiers are provided, the identifiers represent promoter, genomic or cDNA nucleic acid, and peptide sequences, respectively. If only one identifier is provided, it represents a DNA fragment coding for the protein or portions of it.

For other reference, the sequences may be found at the following records in GenBank at the following Accession Numbers, which records are hereby incorporated in their entirety herein: AF126810 (NtPMTl); AF126809 (NtPMT2); AF126811 (NtPMT3); AF126812 (NtPMT4), AF176908 (NtomPMT)(Nicotiana tomentosiformis); AF76909 (NotoPMT)(Nicotiana otophora); AF127239 (ADC); AF127240 (ADCl); AF127241 (ADC2); AF127242 (ODC); AF233849 (ODC2); AF233850 (ODC1); and AF127243 (SAMS).

The following experimental discussion is presented to better illustrate the invention. I. PMT The present invention features the characterization of four members of the nuclear gene family encoding PMT in tobacco N. tabacum. The nucleic acid sequences encoding PMT and the amino acid sequences for the proteins are reported herein and can also be found in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers for NtPMTla, NtPMT2, NtPMT3, and NtPMT4 at AF126810, AF126809, AF126811, and AF126812, respectively. The complete coding region and immediate 5'- and 3'- flanking regions are characterized.

As the discussion below shows, all four PMT genes present in the N. tabacum genome are expressed in the roots of wild-type plants and differentially regulated in tobacco lines expressing either high or low total alkaloid contents.

Materials and Methods

Plant materials

Seeds of N. sylvestris, N. otophora, and N. tomentosiformis were obtained from the USDA-ARS national tobacco germplasm collection (Oxford, NC). N. tabacum cv. Burley 21 and N. tabacum cv. Xanthi seeds were kindly provided by Glenn Collins, University of Kentucky. Tobacco plants used for DNA isolation were grown in a soi vermiculite mixture in the greenhouse under natural lighting conditions. Plants used for RNA extraction were grown in Moltan Plus (Moltan Co., Middleton, TO).

Gel blot analysis of genomic DNA

Young leaves were collected from greenhouse grown tobacco (N. tabacum cv. Xanthi) plants and total genomic DNA was isolated from freshly-harvested tissues using a modification of the CTAB extraction method (Dellaporta et al., 1983). Approximately 30 μg of total DNA was digested with EcoRI, Kpnl, or EcoRI and Kpnl and the digestion products separated by electrophoresis through a 0.75% agarose gel. Restricted and size-fractionated DNA was denatured and transferred to Nytran+ nylon membranes (Schleicher and Schuell, Keene, NH) by capillary blotting in 0.4N NaOH overnight. Membranes were prehybridized in 0.25M Na₂HP0₄ (pH 7.4), 7% SDS, 1 mM NajΕDTA for at least 2 hr, then hybndized overnight at 65 °C m the same buffer with 2-3 x 10⁶ cprn/mL of a ³²P-labeled single-stranded probe (antisense DNA strand). The probe was prepared by the method of Bednarczuk et al. (1991) using a pnmer (Table 1, pnmer 4) designed from the 3' end of the NtPMTla coding region (Exon 8) and the full-length coding region of the NtPMTla cDNA as template. The NtPMTla cDNA was generated by RT-PCR using synthetic oligonucleotide pnmers based on the N- and C-terminal sequences of the A411 cDNA reported by Hibi et al. (1994) and RNA template isolated from N tabacum cv. Burley 21 roots. Membranes were washed at a final stringency of 0.1 x SSC, 0.1% SDS at 65 °C. Hybndizmg bands were visualized by autoradiography and/or imaged using a Molecular Dynamics Phosphorlmager (Model 445 SI, Sunnyvale, CA).

Genomic library construction and phage isolation

A library of N tabacum cv. Xanthi genomic DNA fragments constructed in EMBL3 was purchased from Clontech (Palo Alto, CA) and a total of 1.1 x 10 recombinant phage were screened by plaque hybndization using random-pnmed 32p_ι_abeled NtPMTla cDNA as probe (Sambrook et al , 1989) Prehybπdization, hybndization, and washing conditions were as descnbed above Positive hybndizmg phage were plaque punfied by subsequent rounds of rescreening and DNA was prepared from 18 independently isolated phage. The phage DNA was charactenzed by restnction analysis and DNA gel blot analysis and fragments containing the sequences encoding PMT were subcloned into pBluescπpt KS vectors for further analysis.

Companson of the hybndizmg fragments present in the 18 recombinant phage to the hybndization pattern obtained by genomic DNA blot analysis indicated that only three of the PMT genes suspected to be present m the N tabacum genome were recovered from the library screen To obtain sequences encoding NtPMTla, a subgenomic library was constructed from N tabacum cv Xanthi DNA. The library consisted of gel-punfied 2.5-3 5 kb EcoRI fragments ligated into λ_ZAP II vector arms and packaged using Gigapack III Gold packaging extracts according to the manufacturer's instructions (Stratagene, La Jolla, CA) The pπmary library was amplified once in E coh XL 1 -Blue MRF' strain (Stratagene) and screened as descnbed above, except that a random- pnmed 32p_ι_ab_eι_ed NtPMTla cDNA Εxon 1 -specific probe was used (Table 1). Εxon 1 had previously been amplified by PCR using pnmers 1 and 2 (Table 1) and the NtPMTla cDNA as template. The recombinant phage that hybndized with the probe was isolated from the subhbrary by two more rounds of plaque punfication, and the pBluescπpt phagemid containing the approximate 3.1 kb EcoRI genomic fragment with the NtPMTla gene was excised from the λ_ZAP II phage vector using the in vivo excision protocol descnbed by Stratagene. DNA sequence analysis

Unless otherwise noted, DNA sequencing was performed with double-stranded plasmid DNA templates using fluorescent dye terminator technology (dRhodamine Terminator Cycle Sequencing Ready Reaction kit) on an ABI 310 DNA sequencer (Perkin-Elmer Applied Biosystems). For analysis of PCR products, following electrophoretic separation of amplification reaction products, the bands of interest were excised from the polyacrylamide gels, the DNA extracted using the Quiagen Gel Extraction Kit, and the recovered DNA used as sequencing template. Sequencing was performed using AmpliTaq DNA polymerase and fluorescent dye terminator technology (as described above) and primers 1 and 2 (Table 1) specific for Exon 1. Nucleotide and amino acid sequences were analyzed and aligned using either the Clustal method and Lasergene software (DNAStar Inc., Madison, Wl) or the PILEUP and ALSCRIPT (Genetics Computer Group, Madison, Wl) sequence analysis package (Version 9.0). Transcription factor binding site homologies were identified in promoter DNA sequences by searching the transcription factor database using the GCG program.

RNA gel blot analysis

For RNA analysis, roots and other tissues were harvested from mature wild-type (HP; NiclNic2) and low alkaloid mutant (LP; niclnic2) Burley 21 tobacco plants. For topping experiments, the stem was cut and the top one-third of the plant was removed just prior to flower opening. Roots were harvested just prior to topping (0 hr control) and at various times after decapitation. The tissue was immediately frozen in liquid nitrogen and stored at -80 °C until RNA extraction and isolation. Total RNA was isolated from vegetative organs and floral structures of HP and LP Burley 21 tobacco using the TRI-reagent (Molecular Research Center Inc., Cincinnati, OH) and quantified spectrophotometrically by measuring A269. Total RNA (5 μg) was electrophoresed through 1.2% agarose gels (containing 0.4 M formaldehyde) and transferred to Nytran+ nylon membranes. Following prehybridization the membranes were hybridized with a single-stranded NtPMTla cDNA antisense probe (corresponding to the antisense strand of Exons 2 to 8 of the NtPMTla cDNA coding region) as described above. As a control to quantify and normalize RNA levels in each lane, the blot was hybridized with a 400-bp probe derived from the β-ATPase cDNA using primers 6 and 7 (Table 1) as described below. Semi-quantitative RT-PCR analysis of individual PMT transcript levels

Total RNA (1 μg) extracted from the roots of HP and LP Burley 21 tobacco plants was reverse- transcribed into first-strand cDNA at 42 °C using Superscript II reverse transcriptase (Gibco BRL) according to the manufacturer's protocol. Two gene-specific primers were employed in the reactions: primer 5 capable of recognizing Exon 3 of the PMT genes and primer 8 specific for Exon 8 of the nuclear gene encoding the β-subunit of mitochondrial ATPase from N. plumbaginifolia (NpATP2.1) andN. sylvestris (NsATP2.1) (Boutry and Chua, 1985; Lalanne et al., 1998). The β-ATPase transcript served as an internal reference (constitutively-expressed control) to determine loading accuracy and to normalize expression levels (Kinoshita et al., 1992) Following first strand cDNA synthesis, two sets of nested primers (0.4 μM each primer) were used to amplify the PMT and β- ATPase transcripts: primers 1 and 2 (Table 1) recognized Exon 1 in all five PMT transcripts and gave products ranging in size from 220 bp to 420 bp and primers 6 and 7 amplified an approximately 400-bp region encompassing a portion of Exons 6 to 8 of the β-ATPase coding region. Amplification was carried out for 25 cycles using the following reaction conditions: denaturation at 95 °C for 1 min, primer annealing at 60°C for 35 sec, and extension at 72°C for 1.5 min; a final extension was conducted at 72 °C for 6 min. Amplification products were radioactively labeled by spiking the PCR reaction with 10 μCi 32P-dCTP. Aliquots of the PCR reaction were analyzed on a 6.5% non-denaturing polyacrylamide/lX TBE gel and electrophoresed at 600 volts. The reaction conditions were optimized to provide amplification of both PMT and $-ATPase transcripts in the linear range of the reaction by varying the levels of first strand cDNA template, annealing temperature, and number of cycles of amplification as described in Kinoshita et al. (1992). Molecular weight standards were prepared by PCR amplification using the same primers and protocol described above and plasmid DNA templates containing the PMT encoding genomic fragments, as well as genomic DNA from the various Nicotiana species indicated in the text.

Following electrophoresis, the polyacrylamide gels were fixed in 5% MeOH, 7.5% acetic acid for 30 min, dried overnight, and used to expose X-ray film. PMT band intensities were quantified using phosphorimager analysis (Molecular Dynamics) and normalized relative to the intensities of the β-ATPase control band in each lane. The experiment was conducted twice with different total RNA samples, and representative results are presented from one of the two experiments.

Results

PMT gene structure and organization in N. tabacum Gel blot analysis of total genomic DNA isolated from N. tabacum cv. Xanthi, hybridized with a radioactively-labeled cDNA (NtPMTla) encoding the complete coding region of putrescine N- methyltransferase (PMT) showed the presence of five major hybridizing bands in Kpnl or EcoRI digested DNA, consistent with the presence of a small multigene family in the N. tabacum genome (Figure 1).

As part of our initial characterization of the gene family encoding PMT in N. tabacum, an ΕMBL3 genomic library, prepared from N. tabacum cv. Xanthi DNA, was screened using the NtPMTla A411 homologous) cDNA as probe. From a total of 18 recombinant phage isolated, three phage were recovered that contained genomic fragments encoding the NtPMT2, NtPMT3 and NtPMT4 genes. The three PMT genes were completely encoded within a unique sized EcoRI fragment within the phage DNA insert which allowed for the coπelation of each with a hybridizing restriction fragment on the gel blot of N tabacum genomic DNA (Figure 1). The complete coding region and immediate 5' and 3' non-coding sequences of the three genes were determined and found to encode full-length PMT proteins (Figure 2). Each PMT gene consisted of 8 exons and 7 introns, consistent with the gene structure reported previously for the PMT genes from N. sylvestris

(Hashimoto et al., 1998a). Comparison of the deduced amino acid sequences (Figure 2) revealed that the encoded PMT proteins were extremely similar over their entire length, with the only significant variability in primary sequence localized to the extreme N-terminal region of the protein. This region, completely encoded within Exon 1 , contains a variable number of an 11 amino acid repeat with a consensus sequence of NGHQNGTSEHQ. The function of the repeated sequence is unknown, but is apparently inconsequential to enzyme function, since the number of repeats does not influence activity and PMTs characterized from other species do not contain the repeated element (Hashimoto et al., 1998a; Suzuki et al., 1999a).

Multiple rounds of screening of the EMBL3 genomic library failed to yield additional hybridizing phage containing sequences encoding the other two PMT genes thought to be present in the N. tabacum genome and, therefore, a directed cloning approach was pursued using a subgenomic library constructed from EcoRI fragments isolated from N. tabacum cv. Xanthi. From this hybridization screening, a phage containing the approximately 3.1 kb ΕcoRI fragment encoding NtPMTla was recovered. The coding region of the NtPMTla gene was found to be identical to the A411 cDNA (Hibi et al., 1994), with the exception of a single base change in Εxon 6 that results in a conservative amino acid substitution. This difference could be the result of minor differences among cultivars used in the two studies (i.e., Xanthi vs. Burley 21). Translation of the open reading frame contained in NtPMTla showed that it encoded a protein containing four N-terminal 11 amino acid repeats, similar to Εxon 1 of the PMT gene present in N. tomentosiformis (Hashimoto et al., 1998a). Given the observation that NtPMTla encoded a homolog of the PMT gene present in N. tomentosiformis, the nature and possible evolutionary origin of the remaining PMT gene present in the N. tabacum genome was brought into question. From our expression studies (described in detail below), we had determined that five distinct PMT encoding transcripts were present in the roots of N. tabacum, four of which could be accounted for based upon the length of the Exon I coding region in the four PMT genes isolated and characterized in our studies described above. The fifth transcript was similar in size to that encoded by NtPMTla and, therefore, was designated NtPMTlb. Since the variability tii EAETgene stmcture is primarily localized within Εxon 1, we used a PCR-based strategy to analyze the PMT gene stmcture and family size in N. otophora, the other possible progenitor of N. tabacum. As shown in Figure 3, five distinct PCR products were detected in the electrophoretic pattern of amplification products generated from N. tabacum genomic DNA using Εxon 1 specific primers (Table 1). Consistent with our studies described above and the previous work of Hashimoto et al. (1998a), three PCR products were detected in the electrophoretic pattern of amplification products generated from N. sylvestris genomic DNA, and a single band was recovered from N. tomentosiformis genomic DNA. Amplification of genomic DNA from N. otophora using Εxon 1 specific primers also yielded only a single band, whose electrophoretic mobility was most similar to that of the NtPMTlb derived product.

Analysis of PMT gene intron and flanking sequences

The location of the seven introns within the protein coding region of the five PMT genes in N. tabacum is identical and appears to be conserved among PMT genes from different Nicotiana species. There is also little variation in the nucleotide sequences that comprise the Εxon-Intron splice junctions in the various PMT genes in N. tabacum (Table 2). The high degree of nucleotide sequence similarity recognized among PMT genes within their coding regions is also present within their introns and immediate 5' and 3' flanking sequences (Table 2 and Figure 4). In general, a greater level of sequence identity is found in the introns of the NtPMT2, NtPMT3, and NtPMT4 genes, than in pair- wise comparisons among the introns of the other members of the N. tabacum PMT gene family. The observed conservation in the intron sequences of the NtPMT2, NtPMT3, and NtPMT4 genes is consistent with their origin from the same progenitor species (N. sylvestris). One interesting exception occurs within Intron 6, where the length of the intron and the sequence similarity is more conserved between NtPMTla and NtPMT4, than between NtPMT4 and NtPMT2 or NtPMT3.

Approximately 1 kb of nucleotide sequence was determined 5' to the coding regions of the NtPMTla, NtPMT2, NtPMT3, and NtPMT4 genes (Figure 4). By comparison to the 5'-untranslated region (UTR) present in the A411 cDNA, we set the start site for transcription initiation at approximately 57 nucleotides upstream of the MET start codon in NtPMTla and NtPMT3, and either 69 or 60 nucleotides upstream in NtPMT2 and NtPMT4. The major distinguishing feature between the 5'-UTRs in the various genes is the presence or absence of a 17 bp sequence in the gene. An appropriately placed TATA-box can be easily recognized 45 bp 5' to the initiation site in all four genes. Within the first 200-250 bp upstream of the TATA box, a high level of sequence conservation is found to exist among the promoter regions in the four genes. After this point, a clear difference can be observed between the NtPMTla promoter and the remaining three genes, and by 400 bp upstream, little similarity can be found among any of the gene family members. Analyzing the proximal regions of the various PMT promoters with various motif scanning software identified several G-box-like sequences (Foster et al., 1994; Kim et al., 1992; Menkens et al., 1995; Staiger et al., 1989; Williams et al., 1992) at various positions among the PMT promoters, and a potential metal response element (MRE) (positions -75 to -66; numbering relative to the NtPMTla promoter sequence) in three of the four PMTs (Cizewski-Culotta and Hamer, 1989; Thiele, 1992). An unusual 17 nucleotide stretch of guanine occurs at positions -259 to -243 in the NtPMTla gene promoter followed upstream by a purine-rich region (positions -332 to -263). In the NtPMT3 promoter a 14 bp palindromic sequence (positions -497 to -484) was detected. PMT gene expression has been reported to increase in root tissues following treatment with methyl jasmonate (Imanishi et al., 1998). However, none of the sequence motifs reported to confer methyl jasmonate- responsiveness in other plant genes (Mason et al., 1993; Rouster et al., 1997) were detected in the PMT promoters.

Comparison of the available nucleotide sequence information from the 3 '-flanking regions of the various PMT genes in N tabacum revealed that the 3'-UTRs in the NtPMT2, NtPMT3, and NtPMT4 genes of N tabacum share approximately 81-94% identity with each other and are essentially identical to those reported for N. sylvestris PMTs by Hashimoto et al. (1998a). The major distinguishing feature among the various genes is the presence of two short (20 bp and 4 bp) deletions in the NtPMT2 gene, which lowers the percent identity. The 3'-UTR of NtPMTla is identical to that reported for the A411 cDNA (Hibi et al, 1994) and 81-94% identical to the other PMT genes in the N. tabacum genome. Unfortunately, no sequence information is currently available for the 3'-UTR of the N. otophora or N. tomentosiformis PMT genes.

Regulation of PMT gene expression

To determine whether the members of the PMT gene family in N. tabacum were differentially expressed, a series of experiments were carried out to define the temporal and spatial distribution of transcripts arising from the five genes. Shown in Figure 5A are the results of gel blot analysis of total RNA extracted from various tissues of mature Burley 21 tobacco plants hybridized with radioactively-labeled probe capable of detecting all five PMT transcripts. Consistent with previous studies (Hashimoto et al, 1998b; Hibi et al., 1994), PMT expression is localized exclusively to roots. When maturing wild-type (HP) Burley 21 plants are topped (i.e., the floral meristem and upper 1/3 of the stem are removed), a dramatic increase in EA T transcript abundance is observed within 2 hr, reaching a maximal level of accumulation by 12-24 br. Two size transcripts are detected on the gel blots, reflecting the small difference in message size that occurs as a result of the difference in size of Εxon 1 among the genes.

In addition to examining PMT gene expression in wild-type plants, we also examined expression in a low nicotine-producing (LP) mutant of Burley 21 (Legg and Collins, 1971). The low nicotine Burley 21 line harbors mutations at two independent loci (nicl and nic2) thought to be global regulators of gene expression involved in alkaloid formation. As shown in Figure 6B, topping of the low nicotine mutant (niclnic2) Burley 21 did not cause an increase in PMT transcript abundance as observed in wild type plants. Thus, it appears that Nicl and Nic2 are likely involved in regulation of PMT expression in the very least, and may also be involved in the regulation of other genes in the alkaloid biosynthetic pathway. Whether this is a direct effect (e.g., transcriptional activation) or indirect remains to be determined. In order to determine the extent to which the individual members of the gene family contributed to the general pattern of expression described above, a semi-quantitative RT-PCR strategy (Kinoshita et al., 1992) was used to detect and quantify the levels of the individual PMT transcripts in the roots of both wild-type (HP) and low alkaloid (LP) Burley 21 tobacco. This approach takes advantage of the fact that Εxon 1 is variable in length among the various PMT genes (Figure 2), allowing for their individual detection and quantitation following polyacrylamide gel electrophoresis and autoradiography.

Five RT-PCR products (representing Εxon 1 from each of the five genes present in N. tabacum) were detected in the electrophoretic profiles of amplification products derived from reactions using either HP or LP Burley 21 root RNA (Figure 6A). All five PMT genes present in the N. tabacum genome were expressed in the roots of wild-type plants, and topping resulted in a differential accumulation of transcripts derived from each gene. Among the five genes, transcripts derived from the NtPMT2 and NtPMTlb showed the greatest increase in abundance rising approximately 3-fold during the first 24 hr post-topping, whereas levels of the NtPMTla and NtPMT4 transcripts changed little in response to topping (Figure 6B). In the LP mutant, little or no effect was observed on the levels of the various PMT transcripts following topping, although in some cases (e.g., NtPMTla) a small but likely insignificant depression in transcript abundance was detected. Thus, it appears that all five genes contribute to PMT activity levels within the root.

II. ADC

The present invention features the characterization of two members of the nuclear gene family encoding ADC in tobacco N. tabacum L. As the following discussion shows, ADC2 is preferentially expressed in roots and accounts for the major portion of ADC transcripts present. Furthermore, analysis of ADC transcript levels in roots of low and high nicotine producing lines showed that ADC expression is under the control of the Nicl Nic2 regulatory loci.

Materials and methods

Plant growth and tissue preparation

Seeds of N. tabacum cv. Xanthi, wild-type and low alkaloid nicl nic2 mutant N. tabacum cv. Burley 21 were obtained from Dr. G. Collins (University of Kentucky, Lexington). Tobacco plants used for DNA isolation were grown in soihvermiculite mixture in the greenhouse under natural lighting conditions. Plants used for RNA extraction were grown either in Moltan Plus (Moltan Co., Middleton, TN) or hydoponically in a dilute (half-strength) Peters nutrient solution with continuous aeration of the roots under natural lighting conditions in the greenhouse. Topping experiments were conducted by removing the floral meristem, leaves and stem (approximately the upper 1/3 of the plant) from tobacco plants just prior to blooming. Plant tissues were collected from fully matured individuals, frozen in liquid nitrogen, and stored at -80 °C until used for RNA preparation (see below).

Screening of genomic libraries and phage characterization

A genomic library constructed in λ EMBL3 from N. tabacum cv. Xanthi leaf DNA (Clonetech, Inc., Palo Alto, CA) was screened by plaque hybridization (Sambrook et al, 1989) using an [ - ³²P]- dCTP-labeled, 2.7 kb EcoRI-.A7.oI fragment from plasmid PR24 as probe. PR24 encodes a full length ADC cDNA isolated from the roots of wild-type N tabacum cv. Burley 21 (Wang, 1999). Hybridization was performed at 65 ^CC for 16 h in a solution containing 0.25 M Na₂HP0₄ (pH 7.2) and 7% (w/v) SDS. Following hybridization, the membranes were washed twice in 2 x SSC, 0.1% SDS for 15 mm at room temperature, once m 0.2 x SSC, 0.1% SDS for 30 mm at 65°C. Hybndizmg phage were picked and plaque punfied through three subsequent rounds of hybndization screening Phage DNA was isolated from plaque punfied phage using a Qiagen Phage Midi Preparation Kit (Qiagen, Germany) and insert DNA charactenzed by restriction mapping and DNA gel blot analysis The relevant hybndizmg bands in each phage were cloned into pBluescnpt SK+ vectors for further analysis.

Nucleic acid sequencing and analysis

Nucleotide sequencing was earned out manually using the Sequenase Version 2.0 protocols according to the manufacturer's protocol (United States Biochemical, Cleveland, OH) or with an ABI 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA) using double-stranded plasmid DNA templates prepared utilizing the Qiaprep Spin Plasmid Kit (Qiagen USA, Valencia, CA). The nucleotide and predicted ammo acid sequences of the vanous cDNAs were analyzed using BLAST sequence analysis programs (Altschul et al , 1990; Gish and States, 1993) and protein sequence alignments were earned out using the PILEUP program (Genetics Computer Group Sequence Analysis package, Version 9.0 (GCG, University of Wisconsin, Madison, Wl) and the vanous gene sequences available in the NCBI (National Center for Biotechnology Information, Bethesda, MD) nucleotide and protein sequence database Manual adjustment of the sequence alignments were earned out as necessary.

RNA isolation and gel blot analysis

Total RNA was extracted from tobacco roots, leaves, and floral parts using Tn-Reagent (Molecular Research Center, USA, Cincinnati, OH) according to the manufacturer's protocol. For RNA gel blot analysis, ahquots (10 μg) of total RNA extracted from the vanous tissues were fractionated by electrophoresis through a 1.2% agarose-formaldehyde gel and blotted onto Nytran nylon membranes (Schleicher & Schuell, Keene, NH) using 10 X SSC The transferred RNA was UV cross-linked to the membrane using a UV Stratahnker (Stratagene, La Jolla, CA) and the membranes were prehybndized in 7% SDS, 0.25 M Na_jHPO,,, pH 7.2 for 2-4 hours at 65 °C

Hybndization was earned out in the same buffer in the presence of ³²P-labeled probes for 16 hr at 65 °C. The membranes were washed under high stnngency conditions and subject to autoradiography at -80°C for approximately 48 h.

For gel blot analysis, [a- ³²P]-dCTP -labeled probes were prepared by random primed labeling (Random Pπmed Labeling Kit, Boehnnger Mannheim, Indianapolis, IN) using 25-50 ng of a 2.7 kb EcoRI-ΛTzoI fragment denved from PR24 and a 460 bp fragment amplified from the β- subunit of the tobacco mitochondrial ATP synthase gene (atp2) (Boutry and Chua, 1985).

Semi-quantitative RT-PCR analysis of NtADCl and NtADC2 transcript levels

Total RNA (2 μg) from roots, leaves, or floral parts was reverse transcnbe at 40 °C for 1 h in a reaction cocktail containing 200 units of Superscπptll reverse transcnptase (RNase H-, Gibco BRL, USA), 10 units RNase inhibitor (Perkm Elmer), 200 μm dNTPs and 40 pmol of pnmer, m total volume of 20μl. For first strand cDNA synthesis, a single pnmer [5'-

AGAAAAACATCACCAACT-3'] capable of hybndizmg to both the ADCl andADC2 transcnpts was used in the reaction As a control, a pnmer ( 5'-GCAACTGTCATCTTATCATCTTC-3') specific for the β-subunit of the tobacco mitochondnal ATP synthase gene apt2 (Boutry and Chua, 1985) was used in the reverse transcnptase reaction Following reverse transcnption, the single stranded cDNA products were senally diluted over a concentration range between 1 to 50 ng RNA, and PCR amplification was earned out for 25 cycles of 45 s at 94 °C, 1 mm at 64 °C and 1 mm at 72° C in a Genemate thermocycler (ISC Bioexpress, UT). The reaction mixture contained cDNA template, 1 x PCR buffer (Boehnnger Mannheim), 100 μM dNTPs, 25 pmol of each forward and reverse primer and 1 unit Taq DNA polymerase. The PCR reactions specific for ADCl transcnpts contained the following pnmers: ADCl-forward, 5'- CGTAGACGCTACTGTTTC-3' and ADC 1 -reverse, 5'-TGGACAAC TGTGGAGGCG-3'. Reactions specific for ADC2 transcnpts contained pnmers ADC2-forward, 5'- TGTAGATGCTGCTGTTGTTT-3', and ADC2-reverse, 5'-TGAACAAC TGCGGAGGCA-3 ' Control reactions for normalization of amplification products contained 25 pmol of pnmers specific for the tobacco apt2 transcnpts: atp2 forward, 5 '-GTATATGGTCAAATGAATGAGCC-3', and atp2 reverse.mt, 5'-GCAGTATTGTAGTGATCCTCTCC-3'. For quantitation purposes, amplification reactions were supplemented with lμCi ³²P-dCTP. PCR products were separated by electrophoresis through 1.2% agarose gels, the fractionated reaction products transferred onto a Hybond N+ membranes, dned and subject to autoradiography at -70° C. Quantitation was earned out by phosphonmagmg using a Molecular Dynamics Phosphorlmager. Values were normalized relative to the intensities of the atp2 control band m each lane. The expenment was conducted twice with different total RNA samples, and representative results are presented from one of the two expeπments. Results

These studies show the stmcture and expression of individual members of the ADC gene family in tobacco. An α- ³²P-dCTP-labeled 2.7 kb EcoRI-XhoI fragment from PR24 encoding the ADC coding region was used to screen an λ EMBL3 phage genomic library. From a screen of approximately 3 X10⁵ phage, seventeen hybridizing phage were recovered, of which five were fully characterized by restriction mapping and DNA gel blot analysis. These phage fell into two groups based on their restriction profile. The relevant hybridizing fragments from the various phage were cloned into pBluescript and their nucleotide sequence determined. Presented in Figure 7 are the nucleotide and predicted amino acid sequences of NtADC-1 and

NtADC-2 genes. Both genes contain a single open reading frame, uninterrupted by introns. The nucleotide and amino acid sequence encoded in NtADC-1 is identical to that of PR24, the full length cDNA isolated from N. tabacum cv Burley 21. There are 84 nucleotide differences within the NtADC-1 and NtADC-2 coding regions, resulting in 23 amino acid differences between the ADCl and ADC2 proteins, respectively. The ADC 1 protein is one amino acid shorter in length, missing Val-13.

By comparison to the full-length cDNA, the 5 '-untranslated region (UTR) present in NtADC-1 and NtADC-2 are 431 bp and 432 bp long, respectively. The size of the 5'-UTR in the ADC transcripts is considerably larger than the average size of the plant leader sequence (Joshi, 1987). In contrast, the 3 ' -UTRs present in NtADC- 1 and NtADC-2 are relatively short, approximately 84 nucleotides in length. In both gene sequences, a conserved polyadenylation signal (AATAATA) can be recognized 23 nucleotides from the site of polyadenylation site found in the PR24 cDNA.

Pairwise comparison of the N. tabacum ADCl and ADC2 proteins with the ADCs of other plant species showed that the N tabacum proteins are approximately 82% identical to the ADC of its evolutionary progenitor species N. sylvestris [Genbank Accession No. ABO 12873] and 86% identical to the ADC from tomato (Lycopersicon esculentum) [31], another member of the Solanaceae family (Figure 2). As might be expected, the N. tabacum ADC shares considerably less similarity to ADCs isolated from species more distantly related evolutionarily, such as Arabidopsis - 67% identical [32, 33], soybean- 67% identical [34], and oat - 42% identical [35] and is only 29% identical to the enzyme from Escherichia coli - [36].

The predicted protein coding regions for the N tabacum ADCs are substantially longer than those reported for the ADC proteins of N. sylvestris and L. esculentum [31], but are similar in length to those reported in Arabidopsis, oat, soybean [32-35] and for the E. coli enzyme [36]. The difference in overall length appears to arise from an apparent nucleotide deletion in the N. sylvestris and tomato cDNA sequences relative to the ADCl and ADC2 predicted sequence and those in other plants. In the nucleotide sequences reported for both the N. sylvestris and tomato cDNAs, a guanine residue (position 2295 in the N. sylvestris sequence and 1531 in the tomato sequence) is missing [Genbank Accession No. AB012873]. This deletion changes the reading frame and introduces a premature termination to the predicted coding region. Using the sequence information available in the NCBI database, correcting for this error allowed us to extend the predicted C-terminus of the both ADC proteins, yielding the alignment to the N. tabacum ADCs and those of other plant ADCs as indicated in Figure 8. We have also included in the alignment shown in Figure 8, the correction at the N-terminus of the predicted tomato ADC protein sequence noted by Perez- Amado et al. [37], allowing better alignment of all of the higher plant sequences.

Developmental regulation of arginine decarboxylase expression

It has been shown that nicotine formation can be activated in the roots of maturing tobacco plants by topping, that is, removal of the flower head and several young leaves (Akehurst, 1981; Hibi, et al., 1994). Coincident with the activation of nicotine formation, there is an increase in the levels of transcripts encoding ODC, PMT and spermidine synthase (SPS) over the subsequent 24 hr period in wild-type plants (Hibi et al., 1994; Riechers and Timko, 1999). To determine the effects of topping on ADC expression in roots, Burley 21 plants were grown in the greenhouse to the bud stage at which point the upper 1/3 of the plant was removed and samples of root > tissues were collected before and at various times post-topping. As shown in Figure 9, ADC message abundance increased in the roots of topped Burley 21 plants during the 24 hr period after topping. Low alkaloid (LA) mutants of Burley 21 show a much lower level of ADC expression in their roots, and no induction of ADC transcript accumulation after topping. The lack of ADC induction in the low-alkaloid mutant is consistent with previous studies (Hibi et al, 1994; Riechers and Timko, 1999; Wang, 1999) showing a general inability to activate gene expression leading to increased polyamine formation and alkaloid biosynthesis as a result of the mutation of the Nicl and Nic2 regulatory genes.

NtADC-2 is predominately expressed in roots of wild-type plants.

Due to the high degree of identity between the NtADC-1 and NtADC-2 transcripts (e.g., 95.8% coding regions, 94.4% and 96.4% in 5'- and 3'-UTRs, respectively), it is impossible to distinguish between the two transcripts by RNA gel bot analysis. Therefore, we employed a RT-PCR based strategy and gene specific oligonucleotide primers. Total RNA was extracted from tobacco roots, leaves and flowers, and single-stranded cDNA synthesized using an oligonucleotide primer capable of hybridizing to both ADCl and ADC2 transcripts. As an internal control for amplification, a gene specific primer recognizing the atp2 transcript encoding the β-subunit of the tobacco mitochondrial ATPase was include in the reactions. Under experimental conditions providing amplification in the linear range of the PCR reaction, gene specific forward and reverse primers were used to specifically amplify either ADCl or ADC2 cDNAs. Test reactions (Figure 10A) using plasmid DNA encoding NtADCl or NtADC2 as template demonstrated the specificity of the primers. As shown in Figure 10B, the main transcripts detectable in all tissues tested are derived from NtADC-2. Flowers express the highest level of ADC, and leaves lowest. In the flowers, although ADCl is detectable, far less than ADC2 Roots also express a significant level of ADC.

ADC transcript levels are highest in the roots and floral organs, and low in other plant tissues. The two ADC genes investigated appear to have different modes of regulation, with ADC2 being predominately expressed in the roots and other organs. At the present time, only limited information is available on the nature of regulatory regions in the promoters of genes encoding enzymes of alkaloid biosynthesis. The availability of cloned genomic fragments encoding ADC allows one to begin mapping regulatory sequences within members of these genes responsible for tissue specific, developmental, and inducible expression.

III. ODC

The present invention features the genes of two members of the nuclear gene family encoding ODC in tobacco N. tabacum. As the following experimental discussion shows, the ODC-2 gene is preferentially expressed in roots and floral tissues. Furthermore, the abundance of ODC transcripts in root tissues is affected by topping. Furthermore, analysis of ODC transcript levels in roots of low and high nicotine producing lines shows that ODC expression is under the control of the Nicl Nic2 regulatory loci. Materials and methods

Plant growth and tissue preparation

Seeds of N. tabacum cv. Xanthi, wild-type and low alkaloid nicl nic2 mutant N. tabacum cv. Burley 21 were obtained from Dr. G. Collins (University of Kentucky, Lexington). Tobacco plants used for DNA isolation were grown in soihvermiculite mixture in the greenhouse under natural lighting conditions. Plants used for RNA extraction were grown either in Moltan Plus (Moltan Co., Middleton, TN) or hydroponically in a dilute (half-strength) Peters nutrient solution with continuous aeration of the roots under natural lighting conditions in the greenhouse. Topping experiments were conducted by removing the floral meristem, leaves and stem (approximately the upper 1/3 of the plant) from tobacco plants just prior to blooming. Floral parts and other plant tissues were collected from fully matured individuals, frozen in liquid nitrogen, and stored at -80 °C until used for RNA preparation (see below).

Screening of genomic libraries and phage characterization A genomic library constructed in EMBL3 from N tabacum cv. Xanthi leaf DNA (Clonetech, Inc., Palo Alto, CA) was screened by plaque hybridization (Sambrook et al, 1989) using a³²P- radiolabeled, 1.6 kb EcoRI-ATzoI insert from plasmid PR46 as probe. PR46 encodes a full length ODC cDNA previously isolated by differential screening of plasmid libraries prepared from mRNA isolated from the roots of wild-type Burley 21 plants before and 3-days post-topping (Wang, J., Sheehan, M., Bookman, H. and Timko, M.P., unpublished data). Hybridization was performed at 65 °C for 16 h in a solution containing 0.25 M Na_jHPO,, (pH 7.2) and 7% (w/v) SDS. Following hybridization, the membranes were washed twice in 2 x SSC, 0.1% SDS for 15 min at room temperature, once in 0.2 x SSC, 0.1% SDS for 30 min at 65 °C. Hybridizing phage were picked and plaque purified through three subsequent rounds of hybridization screening. Phage DNA was isolated from plaque purified phage using a Qiagen Phage Midi Preparation Kit (Qiagen USA, Valencia, CA) and insert DNA characterized by restriction mapping and DNA gel blot analysis. The relevant hybridizing bands in each phage were cloned into pBluescript SK+ vectors for further analysis.

Nucleic acid sequencing and analysis Nucleotide sequencing was carried out manually using the Sequenase Version 2.0 protocols according to the manufacturer's protocol (United States Biochemical, Cleveland, OH) or with an ABI 310 Genetic Analyzer (PΕ Applied Biosystems, Foster City, CA) using double-stranded plasmid DNA templates prepared utilizing the Qiaprep Spin Plasmid Kit (Qiagen USA, Valencia, CA). The nucleotide and predicted amino acid sequences of the various cDNAs were analyzed using BLAST sequence analysis programs (Altschul et al, 1990; Gish and States, 1993) and protein sequence alignments were carried out using the PILEUP program (Genetics Computer Group Sequence Analysis package, Version 9.0 (GCG, University of Wisconsin, Madison, Wl) and the various gene sequences available in the NCBI (National Center for Biotechnology Information, Bethesda, MD) nucleotide and protein sequence database. Manual adjustment of the sequence alignments were carried out as necessary.

RNA isolation and gel blot analysis

Total RNA was extracted from tobacco roots, leaves, and floral parts using Tri-Reagent (Molecular Research Center, USA, Cincinnati, OH) according to the manufacturer's protocol. For RNA gel blot analysis, aliquots (10 μg) of total RNA extracted from the various tissues were fractionated by electrophoresis through a 1.2% agarose-formaldehyde gel and blotted onto Nytran nylon membranes (Schleicher & Schuell, Keene, NH) using 10 X SSC. The transferred RNA was UV cross-linked to the membrane using a UV Stratalinker (Stratagene, La Jolla, CA) and the membranes were prehybridized in 7% SDS, 0.25 M Na₂HP0₄, pH 7.2 for 2-4 hours at 65 °C.

Hybridization was carried out in the same buffer in the presence of ³²P -labeled probes for 16 hr at 65°C. The membranes were washed under high stringency conditions and subject to autoradiography at - 80° C for approximately 48 h.

Restriction fragments derived from cDNA clones of interest were separated by agarose gel electrophoresis, the DNA was purified, and quantified by spectrophotometry. [³²P]-dCTP -labeled probes were prepared from 25-50 ng of insert DNA by random primed labeling (Random Primed Labeling Kit, Boehringer Mannheim, Indianapolis, IN). As a control, the blots were also probed with radioactively labeled probes encoding the alkaloid biosynthesis enzyme putrescine N- methyltransferase (PMT) (Riechers and Timko, 1999), a root specific, topping inducible β- glucosidase encoding cDNA (TBG-1) (Riechers, D.E. and Timko, M.P., unpublished data), 26S rRNA (PR31) or 28 S rRNA fragments.

Genomic DNA isolation and gel blot analysis

Tobacco genomic DNA was prepared from tobacco leaf tissue by the method of Junghans and Metzlaff ( 1990). Total genomic DNA ( 15 μg) was digested to completion with EcoRI or Hindlll, the digestion products were fractionated by electrophoresis through a 0.8% (w/v) agarose gel, and transfeπed onto Nytran nylon membrane (Schleicher & Schuell, Keene, NH) in the presence of 0.4 N NaOH (Sambrook et al, 1989). Following transfer, the membrane was rinsed in 2 X SSC , the DNA was UV cross-linked to the membrane, and the membrane was prehybridized and hybridized as described above. Following hybridization and washing, the membranes were subjected to autoradiography at -80 °C.

Results and discussion Gel blot analysis of tobacco genomic DNA cut with various restriction enzymes and hybridized with an [ - ³²P]-dCTP-labeled 1.6 kb EcoRI-ΛTioI cDNA fragment (PR46) encoding the full-length ODC protein from N. tabacum cv Burley 21 (Wang, J., Sheehan, M., Bookman, H. and Timko, M.P., unpublished data) indicated ODC is encoded by small gene family in the N. tabacum genome (Fig. 11). Four to five major bands and several minor bands of sufficient size to encode full- length genes are detected in either EcoRI or Hindlll digested tobacco DNA.

To further analyze the stmcture and regulation of members of the ODC gene family in tobacco, a λ ΕMBL3 phage genomic library constructed with DNA from N. tabacum cv Xanthi was screened using a [α- ³²P]-labeled probes prepared from PR46 (as described above). From a screen of approximately 3 X10⁵ phage, five hybridizing phage were recovered, of which three were fully characterized by restriction mapping and DNA gel blot analysis. Two phage proved to contain identical insert DNA and the third had a unique restriction digestion profile. Following DNA gel blot analysis, the hybridizing fragments were cloned into pBluescript and their nucleotide sequence determined. The complete NtODC-2 gene spans two Sail fragments of 2.7 kb and 6.5 kb. The coding region of the gene contains a singlel302 bp open reading frame unintempted by introns (Fig. 12). The nucleotide sequences of NtoDC-2 is identical within the coding and 5' and 3'- untranslated regions to the PR46 encoded cDNA, with the exception of four nucleotide changes (residues +2, +4, +6 and +8) in the 5'-untranslated region. These nucleotide differences likely reflect changes introduced during the cDNA synthesis reaction.

The predicted amino acid sequence for the NtODC-2 encoded protein (designated pODC2) (Fig. 13) is identical to the ODC characterized from Burley 21 tobacco encoded by PR46 (Wang, J., Sheehan, M., Bookman, H. and Timko, M.P., unpublished data) and to the partial N. tabacum ODC cDNA sequence (PR17) reported by Malik et al, (1996). Comparison of the predicted amino acid sequence for pODC2 with the ODC proteins characterized from two different tobacco cultivars showed that the pODC2 differs by 7 amino acid (98% identity) from the ODC protein characterized from the high alkaloid cultivar, N tabacum cv. SC58 [Genbank Accession No. Y 10472.1] and by 7 amino acid (98% identity) from ODC protein from BY-2 cells. The tobacco pODC2 is 89% and 90% identical to the ODCs from tomato (Lycopersicon esculentum) and jimsonweed (Datura stramonium), respectively, but substantially less similar to ODCs from yeast (35% identity) and humans (32% identity).

The NtODC-1 gene, contained on an 4.0 kb Xbal fragment, encodes a single open reading frame of 141 amino acids encompassing the amino terminal one-half of ODC (Fig. 12). Six amino acid residue changes distinguish the NtODC-2 and NtODC-1 encoded proteins over the homologous region of the proteins. Beginning at amino acid residue 130, the NtODC-1 encoded protein (pODCl) diverges from pODC2, with a stop codon present after residue 141. Scanning the available nucleotide sequence (> 1 kb) in the 3'-flanking region of the NtODC-1 gene failed to reveal any evidence for ODC homologous protein sequences in any of the three translational reading frames. Interestingly, a comparison of the 5'-flanking sequence of the NtODC-1 and NtODC-2 genes revealed that while the NtODC-2 gene has a clearly recognizable TATA-box properly located at approximately -35 bp from the transcriptional start site, no such regulatory motif is found in the NtODC-1 gene sequence. Consistent with this observation, RNA gel blot analysis performed using a hybridization probe prepared from NtOCD-1 immediately downstream of the frame shift, failed to detect any message in various tissues of mature tobacco plants (data not shown). Thus, it appears that NtODC-2 represents an unexpressed pseudogene in the N tabacum genome.

To determine the spatial pattern of expression of the NtODC-2 gene, gel blot analysis was carried out using total RΝA prepared from roots, stems, young and mature leaves, and various floral parts of Burley 21 tobacco plants. As shown in Fig 14, transcripts encoding ODC were easily detected in the roots, with little or no expression in other tissues except sepals, carpels, and mature stamens.

The formation of nicotine and total leaf alkaloids in tobacco is known to be under the control of at least two independent genetic loci (Legg et al, 1969; Legg and Collins, 1971), designated Nicl and Nic2 (Hibi et al, 1994). Nicl and Nic2 are semi dominant and operate synergistically to control plant alkaloid content, with mutations within these genes resulting in plants with reduced levels of nicotine and total leaf alkaloids (wild-type > nicl > nic2 > nicl nic2) (Legg et al, 1969; Legg and Collins, 1971). Although no information is available on the nature of their encoded products, it has been speculated that Nicl and Nic2 likely encode transcriptional regulators capable of globally interacting with a subset of genes encoding components of polyamine and alkaloid biosynthesis (Hibi et al. , 1994). Removal of the flower head and several young leaves (i.e., topping) leads to activation of nicotine formation in the roots of decapitated plants (Akehurst, 1981; Hibi et al, 1994). To determine the effects of topping on NtODC-1 expression in roots, Burley 21 plants were grown in the greenhouse to the bud stage at which point the upper 1/3 of the plant was removed and samples of roots tissues were collected before and at various times post-topping. As shown in Fig 14B, low levels of the ODC transcripts were found in roots prior to topping and message abundance increased approximately 2-fold in the roots of topped Burley 21 plants 4 hr after topping. By 24 hr after topping, ODC transcript levels return to their initial levels. Low alkaloid mutants of Burley 21 subjected to the same treatment show a much lower level of stimulation of ODC transcript accumulation after topping, and the enhanced transcript abundance does not persist beyond 4 hr. By comparison, transcripts encoding PMT and and a tobacco root-specific β-glucosidase (TBG-1) show patterns of accumulation similar to that observed for ODC transcripts in wild-type plants, but no induction in the low-alkaloid mutant, consistent with previous studies (Hibi et al, 1994; Riechers and Timko, 1999; Wang, 1999).

IV. SAMS

A single recombinant phage is identified as encoding for SAMS. This λ phage contains an approximately 15kB Sail insert. Restriction mapping and PCR analysis indicates that the insert DNA contains primarily the coding and 3'non-coding portions of the SAMS gene. The nucleotide sequences for the gene encoding SAMS can be found at GenBank Accession Nos. AF27243 (full length SAMS cDNA).

V. NADH dehydrogenase

A fragment of the cDNA encoding for NADH dehydrogenase in N. tabacuum shows high expression in the roots of mature wild-type HP plants compared to low alkaloid mutant LP plants.

VI. Phosphoribosylanthranilite isomerase (PAI)

The gene encoding for a fragment of phosphoribosylanthranilite isomerase in N tabacuum is a homolog of the Arabidopsis thaliana gene encoding PAI, an enzyme involved in tryptophan biosynthesis. This enzyme is involved in the overall formation of aromatic compounds in plants.

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Claims

What is claimed is:

I. An isolated DNA molecule comprising the nucleotide sequence of (SEQ. ID. NO. 2), (SEQ. ID. NO. 5), (SEQ. ID. NO. 8), (SEQ. ID. NO. 11), (SEQ. ID. NO. 13), (SEQ. ID. NO. 15), (SEQ. ID. NO. 18), (SEQ. ID. NO. 21), (SEQ. ID. NO. 23), (SEQ. ID. NO. 25) or (SEQ. ID. NO. 26) or comprising a nucleotide sequence encoding the amino acid sequence encoded by (SEQ ID NO. 3), (SEQ. ID. NO. 6), (SEQ ID. NO. 9), (SEQ. ID. NO. 12), (SEQ. ID. NO. 14), (SEQ. ID. NO. 16), (SEQ. ID. NO. 19), (SEQ. ID. NO. 22) OR (SEQ. ID. NO. 24).

2. A vector comprising the isolated DNA molecule of claim 1 operably linked to sequences capable of directing the transcription of a mRNA encoded by said isolated DNA molecule.

3. An isolated DNA molecule comprising a DNA sequence complementary to the nucleotide sequence of claim 1.

4. A vector comprising the isolated DNA molecule of claim 3 operably linked to sequences capable of directing the transcription of a mRNA encoded by said isolated DNA molecule.

5. A cultured transgenic tobacco cell stably transformed with the vector of claim 2.

6. A cultured transgenic tobacco cell stably transformed with the vector of claim 4.

7. A transgenic tobacco plant stably transformed with the vector of claim 2.

8. A transgenic tobacco plant stably transformed with the vector of claim 4.

9. The isolated DNA molecule of claim 1, wherein the isolated DNA molecule comprises the nucleotide sequence of (SEQ ID NO:).

10. A vector comprising the isolated DNA molecule of claim 9 operably linked to sequences capable of directing the transcription of a mRNA encoded by said isolated DNA molecule.

I I. An isolated DNA molecule comprising a DNA sequence complementary to the nucleotide sequence of the isolated DNA molecule of claim 9.

12. An isolated DNA sequence comprising about a fifteen to about a twenty-five base pair oligonucleotide sequence identical to any consecutive about fifteen to about twenty-five base pair sequence found in (SEQ. ID. NO. 2), (SEQ. ID. NO. 5), (SEQ. ID. NO. 8), (SEQ. ID. NO. 11), (SEQ. ID. NO. 13), (SEQ. ID. NO. 15), (SEQ. ID. NO. 18), (SEQ. ID. NO. 21), (SEQ. ID. NO. 23), (SEQ. ID. NO. 25) or (SEQ. ID. NO. 26).

13. A cultured transgenic tobacco cell stably transformed with the vector of claim 10.

14. A transgenic tobacco plant stably transformed with the vector of claim 10.

15. A vector comprising a DNA sequence which encodes an antisense mRNA which is complementary to a fragment of a mRNA encoded by the isolated DNA molecule of claim 1, wherein said sequence is operably linked to sequences capable of directing the transcription of said antisense mRNA in tobacco cells and wherein the expression of said antisense mRNA in tobacco cells is sufficient to provide for reduced nicotine content in tobacco cells which are stably transformed with said vector as compared to untransformed tobacco cells.

16. A cultured transgenic tobacco cell stably transformed with the vector of claim 15.

17. An isolated and purified protein comprising the amino acid sequence identified in (SEQ ID NO. 3), (SEQ. ID. NO. 6), (SEQ ID. NO. 9), (SEQ. ID. NO. 12), (SEQ. ID. NO. 14), (SEQ. ID. NO. 16), (SEQ. ID. NO. 19), (SEQ. ED. NO. 22) or (SEQ. ID. NO. 24).

18. A method for regulating gene expression in a plant comprising functionally linking an alkaloid gene promoter to a nucleic acid encoding a protein, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of the sequences identified in (SEQ ID NO. 1), (SEQ. ID. NO. 4), (SEQ ID. NO. 7), (SEQ. ID. NO. 10), (SEQ. ID. NO. 17),and (SEQ. ID. NO. 20).

19. The method of claim 18, wherein the nucleic acid encoding a protein encodes a protein involved in the biosynthesis of alkaloids in plants.

20. A plant transformed by the method of claim 18.