Characterization of cDNAs differentially expressed in roots of
tobacco (
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 probes generated 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 alkaloid biosynthesis. The other cDNAs isolated were placed into one of several categories and encode metabolic enzymes, proteins involved in transcription and translation, components of signal transduction pathways, and homologs of genes whose expression has been shown to be regulated by phytohormones (i.e. auxin, ABA), wounding or other stress responses. RNA gel blot analysis showed that theADCandODCtranscripts were preferentially expressed in the roots and floral tissues of mature tobacco plants, whereasSAMStranscripts were detected in all tissues examined. The steady-state levels of theADCandODCmRNAs increased in the roots of wild-type tobacco plants during the 24 h period after topping, whereas little change was observed in the abundance of the SAMS transcripts in these tissues. The possible factors associated with the regulation of expression of these genes are discussed. © 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 of secondary compounds found in plants and they are the product of a complex biosynthesis pathway [1 – 3]. 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 inter-cellular distribution and accumulation remain to be determined [2 – 4].
Nicotine is the most abundant alkaloid present in cultivated tobacco and, like other alkaloids
found in Nicotiana and related plant species, its
biosynthetic origin begins with the plant
polyamine putrescine [2,4]. Putrescine is formed in plants by one of two pathways [5]. It can be synthesized directly from ornithine, in a reaction catalyzed by the enzyme ornithine 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).
Pu-trescine formed by the ADC and/or ODC
path-way serves as precursor in the synthesis of the higher polyamines, spermine and spermidine, cata-lyzed by the enzymes spermine synthase and sper-midine synthase, respectively, or it is converted to
N-methylputrescine by the action of putrescine
N-methyltransferase (PMT), the first committed
step in nicotine biosynthesis [2,4,5]. N-methyl
pu-trescine is oxidized by a diamine oxidase and
cyclized to form the 1-methyl-D1-pyrrolium cation,
* Corresponding author. Tel.: +1-804-9825817; fax: + 1-804-9825626.
E-mail address:[email protected] (M.P. Timko).
which is condensed with nicotinic acid or its derivative to form nicotine [2]. Other amino acids, such as tyrosine, tryptophan, phenylala-nine, and related compounds (e.g. anthranilic acid, nicotinic acid, and purines) can also serve as biosynthetic precursors to some classes of al-kaloids [5].
The synthesis and accumulation of nicotine and other tobacco alkaloids are known to be controlled by various developmental, environ-mental, and chemical cues [1,2,4]. Changes in
phytohormone (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 the synthesis and accumulation of nicotine and vari-ous tobacco alkaloids [2,6,7]. Varivari-ous abiotic fac-tors (wounding, drought stress, pH imbalance, etc.) [1,2,4], as well as biotic factors, such as herbivory, insect feeding, and attack by various microbial and fungal pathogens, are known to elicit increased production of nicotine and other alkaloids in the leaves of wild and cultivated to-bacco species [8,9]. 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 of total alkaloids present
in the leaves of Nicotiana tabacum [2,6]. The
fac-tors 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 [6,10] . In this regard, considerable evidence now exists indicating that a jasmonic acid (JA)-mediated signal transduc-tion pathway may play a role in regulatransduc-tion of gene expression contributing to this increase in alkaloid 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 literature
as Nic1 and Nic2 [6] . Nic1 and Nic2 are
semi-dominant and operate synergistically to control plant alkaloid content, with mutations within these genes resulting in plants with reduced lev-els of nicotine and total leaf alkaloids
(wild-type\nic1\nic2\nic1 nic2) [16,17]. Although
no information is available on the nature of their encoded products, it has been speculated
that Nic1 and Nic2 likely encode transcriptional
regulators capable of globally interacting with a
subset of genes encoding components of
polyamine 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 related
alkaloids have been isolated from a number of plant species [2,5,6,15,18,19]. Initially, most at-tempts at the cloning of cDNAs for enzymes involved 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 a
library with synthetic oligonucleotide probes
based upon the defined amino acid sequence [19]. A few attempts at alternative approaches, such as the use of differential hybridization screening [6,15,20] or the use of PCR based strategies [21], have also been reported. In the present study, we used a subtractive hybridiza-tion screening strategy to identify cDNAs whose encoded proteins are differentially expressed in the roots of Burley 21 tobacco plants 3 days after topping, a developmental time known to be active in nicotine and leaf alkaloid formation. We report here the results of our studies and describe the nature of the gene products and their expression in wild-type tobacco plants.
2. Materials and methods
2.1. Plant growth, tissue preparation, and RNA isolation
N. tabacum cv. Burley 21 plants were grown hydroponically in a dilute (half-strength) Peters nutrient solution with continuous aeration of the roots under natural lighting conditions in the
greenhouse. The total and polyA+mRNA were
2.2. cDNA library construction and differential screening
cDNAs were generated using oligo-d(T) primers
and the cDNA synthesis kit (purchased from Stratagene). The cDNAs were cloned into lambda Zap II vector DNA linearized by digestion with
EcoRI and XhoI. The library of recombinant
clones was screened by differential hybridization
of duplicate nitrocellulose filters [23] using a-32
P-dCTP labeled cDNA as probe [24]. Probes used for screening consisted of radioactively labeled cDNAs prepared from tobacco root and leaf RNAs, and root RNA from tomato (as a het-erologous control probe). cDNAs showing strong hybridization with probes prepared from roots of topped plants were recovered and their nucleotide sequence determined.
2.3. DNA sequencing and analysis
Nucleotide sequencing was carried out manually using the Sequenase Version 2.0 protocols accord-ing to the manufacturer’s protocol (United States Biochemical) or with an ABI 310 Genetic Ana-lyzer (PE Applied Biosystems) using
double-stranded plasmid DNA templates prepared
utilizing 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 out using the PILEUP program (Genetics Computer Group Sequence Analysis Package, Version 9.0, Madison, WI) and the various gene sequences available in the NCBI (National Center for Bio-technology Information, Bethesda, MD) nucle-otide and protein sequence database. Manual adjustment of the sequence alignments was carried out 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 the various tissues were fractionated by electrophore-sis through a 1.2% agarose – formaldehyde gel and blotted onto Nytran nylon membranes (Schleicher
and Schuell) using 10×SSC [24]. The transferred
RNA was UV cross-linked to the membrane using a UV Stratalinker (Stratagene) and the mem-branes were prehybridized in 7% SDS, 0.25 M
Na2HPO4, pH 7.2 for 2 – 4 h at 65°C.
Hybridiza-tion was carried out in the same buffer in the
presence of a-32P-dCTP labeled probes for 16 h at
65°C. The membranes were washed under high stringency conditions and subject to
autoradiogra-phy at −80°C for 48 h.
Restriction fragments derived from cDNA clones of interest were separated by agarose gel electrophoresis, the DNA was purified, and
quantified spectrophotometrically. The a-32
P-dCTP labeled probes were prepared from 25 – 50 ng of insert DNA by random primed labeling
(Random Primed Labeling Kit, Boehringer
Mannheim, IN). As a control probe used to quan-tify and normalize RNA levels in each lane, blots were hybridized with a 400-bp portion of the
cDNA encoding the b-subunit of mitochondrial
ATPase [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 and Metzlaff [28]. Total genomic DNA (15 mg) was
digested to completion withEcoRI orHindIII, the
digestion products were fractionated by
elec-trophoresis through a 0.8% (w/v) agarose gel, and
transferred onto Nytran nylon membrane (Schle-icher and Schuell) in the presence of 0.4 N NaOH [24]. Following transfer, the membrane was rinsed
in 2×SSC, the DNA was UV cross-linked to the
membrane, and the membrane was prehybridized and 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 gene products in the roots of topped tobacco plants
Using a subtractive hybridization strategy, a
group of 60 cDNAs were isolated that showed
differential levels of expression in the roots of
tobacco (N. tabacum cv. Burley 21) plants before
head and upper leaves and stem of the plant). The nucleotide sequence of the individual cDNAs was determined and both the nucleotide and predicted amino acid sequences of the various cDNAs were analyzed using BLAST sequence analysis programs [25,26]. In most cases, the complete nucleotide sequence of the individual cDNA was determined. Where multiple clones were available, only full length clones were fully sequenced. Following our analysis, the majority of the cDNAs isolated could be placed into one of several categories based upon their similarity to known genes or their encoded products already present in the NCBI databases. The results of our study are presented in Table 1. Among the largest subset of related cDNAs recovered were clones encoding enzymes involved in polyamine biosynthesis and alkaloid formation (Group I). Among this group were both full length and partial cDNAs encoding ADC, ODC, and
S-adenosylmethionine synthetase (SAMS). The
fur-ther characterization of these gene products is described in greater detail below.
cDNAs were also identified that encode ho-mologs of various cell wall-associated proteins or enzymes involved in wall formation (Group II). Among these are three distinct extensins and two different proline-rich proteins (PRPs). The recovery of homologs of extensin and PRPs is not surprising. Members of both gene families have been reported to be expressed during root growth and regenera-tion, and the steady state levels of both classes of transcripts have been shown to increase in response to wounding [29]. The third recognizable category of cDNAs (Group III) contains homologs of proteins previously shown to be involved in tran-scription or translation, or to be components of signal transduction pathways. Notable among these clones is the ethylene responsive element binding (EREB) protein. The role of ethylene in the control of gene expression both independently and in con-junction with other phytohormones (e.g. auxin) is well documented [30].
cDNAs encoding enzymes known to be involved in general cellular processes (i.e. cell structure, intra-and intercellular communication intra-and transport, protein turnover, etc.) constitute Group IV. In-cluded among these are transmembrane proteins with similarity to phosphate transporters and intrin-sic water channel proteins (e.g. PR12 and PR16) whose activities have been previously suggested to be modulated in response to various stress responses
or changes in phytohormone levels or ratios. Changes in some of these components, such as fructose-1,6-bisphosphate aldolase and ribose-5-phosphate isomerase, may simply reflect the gross alteration in whole plant physiology upon topping. A number of cDNAs encode proteins for which no function has yet to be defined in plants (Group V), or for which no match of any significance could be found within the databases (Group VI). There-fore, the function of these cDNAs and their impor-tance to the regulation of alkaloid formation and distribution remains unknown.
3.2. Characterization of ADC, ODC, and SAMS proteins from N. tabacum L. c6. Burley 21
Within the differentially expressed gene products cloned in our investigation were both partial and full-length cDNAs encoding ADC, ODC, and
SAMS. PR24 encodes the full-length N. tabacum
ADC cDNA. The cDNA contains an open reading frame of 2163 bp coding for a protein 720 amino
acids in length. The ADC transcript contains an
unusually long 5%-untranslated region (UTR) of 431
nucleotides and a short 3%-UTR of 84 nucleotides
with a near consensus polyadenylation signal
(AATAATA) 30 nucleotides upstream of the
poly(A)n tract.
The N. tabacum ADC is 82% identical to the
ADC of its evolutionary progenitor species N.
syl6estris [Genbank Accession No. AB012873] and
86% identical to the ADC from tomato (Lycopersi
-con esculentum) [31], another member of the Solanaceae family (Fig. 1). As might be expected, theN. tabacumADC shares considerably less sim-ilarity to ADCs isolated from species more distantly
related evolutionarily, such as Arabidopsis, 67%
identical [32,33]; soybean, 67% identical [34]; oat,
42% identical [35]; andEscherichia coli, 29%
identi-cal [36].
The predicted protein coding region for the N.
tabacum ADC is substantially longer than those
reported for the ADC proteins of N. syl6estrisand
L. esculentum[31], but is similar in length to those
reported in other higher plant species (e.g. Ara
-bidopsis, oat, soybean) [32 – 35] and for the E. coli enzyme [36]. The difference in overall length ap-pears to arise from an apparent nucleotide dele-tion
in the N. syl6estris and tomato cDNA sequences
Table 1
Summary 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 biosynthesis
PR-1 1400 (219) SAMS, partial cds. (6e-84) 1200 (168) SAMS, partial cds. (8e-29) PR-2
PR-3 1300 (701) SAMS, partial cds. (1e-138) AF127243
1636 (1636) SAMS, full length coding PR-6
PR-7 600 (600) SAMS, partial cds.
1600 (198) SAMS, partial cds. (2e-58) PR-8
1300 (135)
PR-9 SAMS, partial cds. (4e-27)
1400 (174) SAMS, partial cds. (2e-25) PR-10
1600 (299)
PR-11 SAMS, partial cds. (1e-102)
PR-17 959 (959) U59812 ODC (partial clone) 1000 (201) SAMS, partial cds. (2e-08) PR-21
PR-23 228 (228) SAMS, partial cds. (1e-77) AF127239
2694 (2694) ADC, full length coding PR-24
PR-37 1600 (140) SAMS, partial cds
AF127242 ODC, full length coding 1596 (1596)
PR-46
Group II. Cell wall related proteins
AF156371
3500 (559) Extensin 1, partial cds. (0.96) PR-29
659 (659)
PR-38 AF154651 Extensin 2, partial cds. (3e-89) AF154653
696 (696) Extensin 3, partial cds. (11e-3) PR-41
AF154654
PR-42 734 (734) Extensin precursor, partial cds. (1e-17) AF154655
661 (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) AF154669
1500 (901) Proline cell wall-associated protein, partial cds. (7e-26) PR-64
Group III. Transcription, translation, signal transduction 673 (673)
PR-5 AF154636 40S ribosomal S4 protein, partial cds. (1e-56) AF154644
705 (705) Glycine-rich RNA-binding protein, ABA-inducible, partial cds (3e-31) PR-20
AF156367
PR-22 128 (128) Glycine-rich RNA binding protein, partial cds. (4e-19)
500 (172) 26S ribosomal RNA
PR-31
1078 (1078)
PR-47 AF154656 Putative ethylene responsive element binding (EREB) protein (3e-22) AF154657
1122 (1122) Putative serine/threonine protein kinase, partial cds. (2e-08) PR-48
708 (708)
PR-50 AF154659 40S ribosomal S12 protein, partial cds. (8e-42) AF154660
PR-51 948 (948) Putative elongation factor EF-1a; (vitronectin-like adhesion protein) (5e-65)
AF154663
PR-55 888 (888) 60S ribosomal L15 protein, partial cds. (2e-99)
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 genes
AF154637 Putative inorganic phosphate transporter, partial cds. (9e-08) PR-12 450 (430)
AF154640
1451 (1451) Actin, partial cds (1e-180) PR-15
AF154641 Intrinsic plasmamembrane protein (water channel), partial cds. (1e-106) PR-16 1100 (1100)
AF154647
637 (637) Poly-ubiquitin (2e-76) PR-32
AF154648
PR-33 1313 (1313) Fructose-1,6-bisphosphate aldolase, partial cds. (1e-160) AF154650
638 (638) Ubiquitin conjugating enzyme E2, partial cds. (7e-69) PR-35
PR-36 737 (737) X00945 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 plants AF154635
685 (685) Putative N7 protein homolog, partial cds. (2e-19) PR-4
PR-13 722 (722) AF154638 dnaJ homolog, partial cds. (2e-37) AF154642
057 (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)
Table 1 (Continued)
Insert size (bp) Homology/Identity (blast score)
Plasmid Accession no.
(amt. seq.) designation
1083 (1083) AF154666
PR-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 protein AF156363
PR-25 900 (171) 1141 (1141)
PR-26 AF154645 Hypothetical topping-induced protein Hypothetical topping-induced protein AF156370
PR-28 1200 (106) 1217 (1217)
PR-M AF154646 Hypothetical topping-induced protein 750 (536) AF154659
PR-34 Hypothetical topping-induced protein
Hypothetical topping-induced protein AF154661
PR-52 871 (871)
429 (429) AF154662
PR-53 Hypothetical topping-induced protein
429 (429) (same as PR53)
PR-54 Hypothetical topping-induced protein
900 (705)
PR-56 AF154664 Hypothetical topping-induced protein PR-63 1500 (986) AF154668 Hypothetical topping-induced protein
Fig. 1. Comparison of the predicted amino acid sequences of arginine decarboxylases (ADCs) from various species. Shown is a PILEUP 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),
for both the N. syl6estris and tomato
cDNAs, 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 frame and introduces a premature termination to the predicted coding region. Using the sequence infor-mation 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 ADC and those of
other plant ADCs as indicated in Fig. 1. We have also included in the alignment shown in Fig. 1, the correction at the N-terminus of the predicted tomato ADC protein sequence noted by Pe´rez-Amado et al. [37], allowing better alignment of all of the higher plant sequences.
Shown in Fig. 2 is the predicted amino acid
sequence for theN. tabacum ODC encoded by the
full-length cDNA PR46 in a PILEUP alignment with ODC proteins isolated from other plant and animal species. The predicted amino acid sequence
for theN.tabacumODC protein encoded in PR 46
is identical to the partial N. tabacum ODC cDNA
sequence (PR17) reported earlier [38], but differs at eight amino acids (98% identity) from the protein sequence of an ODC isolated from the high alkaloid
cultivar,N.tabacumcv. SC58 [Genbank Accession
No. Y10472.1]. The two tobacco proteins are 88 – 89% identical to the ODC from tomato and
jimson-weed (Datura stramonium) [39,40], but substantially
less similar to other ODCs from non-photosynthetic
eukaryotes and prokaryotes (e.g. C. elegans, 32%
similarity; Xenopus lae6is, 31%).
All eukaryotic ODCs share several structural features in common, including a conserved lysine involved in binding of pyridoxyl phosphate cofactor
(LYS-96 in the N. tabacum ODC sequence) and a
conserved cysteine (CYS-378), which is the attach-ment site for DMFO, a potent inhibitor of enzyme function [41]. The ODCs characterized from eu-karyotic animal cells also contain a C-terminal extension relative to the enzymes present in prokaryotes that is thought to be involved in the
rapid degradation/turnover of the protein [41]. As
evidenced from the alignment shown in Fig. 3, the tobacco ODC lacks this extension, as do the ODCs from other plant species [39].
It has been previously noted that plant ADCs and ODCs share domains in common and, therefore, it is likely that they share a common evolutionary origin [5,41]. Among the more highly conserved
Fig. 2. Comparison of the predicted amino acid sequences of ornithine decarboxylases (ODCs) from various species. Shown is a PILEUP alignment of the predicted amino acid sequences of theN.tabacumcv. Burley 21 ODC encoded on plasmid PR46 (AF127242) with the ODCs fromN.tabacum
cv. SC58 (Y10472), Lycopersicon esculentum (tomato) (AF029349),Datura stramonium (jimsonweed) (X87847), and
Fig. 3. Comparison of the predicted amino acid sequences of
S-adenosylmethionine synthetases (SAMS) from various spe-cies. Shown is a PILEUP alignment of the predicted amino acid sequences of the N. tabacum cv Burley 21 SAMS en-coded on plasmid PR6 (AF127243), with the SAMS from
Lycopersicon esculentum (tomato) (Z24743), Catharanthus roseus (Z71272), Arabidopsis thaliana (M55077), Pisum sa
-ti6um(pea) (L36681),Oryza sati6a(rice) ( Z26867), andHomo
sapiens(humans) ( X68836 ). Amino acid residues conserved among the various SAMS are shaded.
regions of the ADC and ODC proteins of N.
tabacum is the proposed active site involved in decarboxylation of arginine and orinithine, respec-tively. This sequence, DTGGGL in ADC and DVGGGF in ODC is similar to the consensus motif
DI/VGGGL/F observed in ADCs, ODCs, and the
related protein diaminopimelic acid decarboxylases, in other species of plants, animals, and microbes [5,41,42].
The largest number of cDNAs recovered by our differential hybridization screening were partial and full-length clones encoding SAMS. Based upon a comparison of available nucleotide sequences from
the coding and 3%-UTRs of the various clones, it
appears that there are at least five different
ex-pressed gene products inN.tabacum roots. Shown
in Fig. 3 is the predicted amino acid sequence for
the full-length N. tabacumSAMS encoded in PR6
compared to the predicted protein sequences of
SAMS from other species. The N. tabacum SAMS
is most similar to the SAMS of tomato (90% identical)[43] and has between 85 and 88% identity to the SAMS from plant species, including pea [44], Arabidopsis[45] andCatharanthus[46]. Significantly less sequence similarity is found between the SAMS ofN. tabacumand those present in
non-photosyn-thetic eukaryotes (e.g. 60% identity to SAMS
from yeast [47] and humans [48]).
It has been reported previously that the members of the SAMS gene families present within various plant species can be divided into evolutionary groupings based upon the conserved na-ture of specific amino acid residues within the SAMS primary protein sequence [46]. Comparison of the predicted amino acid sequence for PR6 encoded SAMS from tobacco with SAMS from other plant species indicates that the PR6-encoded protein falls into the Type II cluster, as defined by Schru˚der et al. [46]. Unfortunately, the partial cDNAs encoding SAMS isolated in our studies do not provide sufficient sequence information and therefore we can not predict their phylogenetic placement with any accuracy.
3.3. Genomic complexity of the ADC, ODC, and SAMS gene families
Both ADC and ODC are encoded by small gene
families in theN.tabacumgenome. Gel blots of total
with eitherEcoRI orHindIII. Five to seven major bands and several minor bands of sufficient size to encode full-length genes were detected when the same blots were hybridized with radioactively-la-beled cDNA probes encoding ODC. It has been reported previously that ADC is encoded by a single gene or low-copy number nuclear gene in other plants species [31 – 33,42]. For example, tomato and soybean are reported to contain a
single ADC gene [31,42], two copies of ADC are
present in many Brassicaceae [49], including Ara
-bidopsis [33]. ODC has also been reported to be encoded by a small gene family in other plant
species, such as Datura, where three to five family
members are reported to be present [39]. The presence of multiple genes encoding ADC and
ODC in N. tabacum is consistent with its
evolu-tionary origin from the hybridization of three
different progenitor species (i.e. N. syl6estris, N.
tomentosiformis, and N. otophora), each of which
could contribute a locus to theN.tabacumgenome
[50,51].
The recovery of multiple expressed SAMS cD-NAs is consistent with our genomic DNA gel blot analysis (Fig. 4) showing eight to ten hybridizing
bands in both EcoRI- or HindIII- digested total
genomic DNA samples probed with a radioac-tively-labeled SAMS coding region probe. The
gene family encoding SAMS activity in N.
tabacum appears to be of greater complexity than
that observed for either the ADC or ODC gene
family in this species. The existence of multiple
genes encoding SAMS in N. tabacum is consistent
with previous reports of multiple expressed SAMS
genes in other plant species, including pea [44], Arabidopsis [45], and tomato [43], although it
should be noted that the SAMS gene family in
tobacco appears to be substantially larger than those present in the other plant species.
3.4. RNA gel blot analysis of ADC, ODC, and SAMS expression in tobacco
The distribution and abundance ofADC, ODC,
and SAMS transcripts in mature tobacco plants
was analyzed by gel blots using total RNA pre-pared from the roots, stems, leaves, and various floral parts of mature Burley 21 tobacco plants. As shown in Fig. 5, transcripts encoding ODC were highly expressed in the roots and present to a lesser extent in floral portions of the plant. Little or no expression of these transcripts was detected
in other tissues. Only very low levels of ADC
transcripts were present in roots and among floral tissues significant levels were only observed in sepals. The tissue specific distribution of ODC and ADC transcripts was similar but not identical to
that found for transcripts encoding putrescine N
-methyl transferase (PMT), an enzyme controlling the flow of precursors between polyamine and nicotine biosynthesis [6,50]. In contrast, transcripts encoding SAMS were easily detected in all tissues with slightly higher levels observed in photosyn-thetic versus non-photosynphotosyn-thetic tissues.
It has been previously shown that removal of the flower head and several young leaves (i.e. topping) leads to activation of nicotine formation in the roots of decapitated plants [6,10]. It has also been reported that coincident with nicotine accu-mulation over the subsequent 24 h period, there is an increase in the levels of transcripts encoding PMT and spermidine synthase (SPS) in wild-type plants [6,50]. To determine the effects of topping
on ADC, ODC, and SAMS expression in roots,
Burley 21 plants were grown in the greenhouse to the bud stage at which point the upper third of the plant was removed and samples of roots tissues were collected before and at various times post-topping. As shown in Fig. 5(B), low levels of the
ODC and ADC transcripts were found in roots
Fig. 4. Gel blot analysis of genomic DNA fromN.tabacum
cv Burley 21 probed with radioactively-labeled cDNA encod-ing ADC, ODC, and SAMS. Total genomic DNA (30 mg) was digested withEcoRI orHindIII, fractionated by agarose gel electrophoresis, transferred to nylon membranes and hy-bridized with a-32P-dCTP labeled probes encoding tobacco
Fig. 5. Gel blot analysis of the ADC, ODC, and SAMS
transcript levels in various tissues of mature tobacco plants and in the roots before and after topping. Total RNA was isolated from various tissues of matureN.tabacumcv. Burley 21 and analyzed by gel blot analysis usinga-32P-dCTP labeled
coding region probes for ADC (PR24), ODC (PR46), and SAMS (PR10). As a control, the blots were also probed with radioactively labeled probes encoding the alkaloid biosynthe-sis enzyme putrescine PMT [50], the b-subunit of coupling factor CF1-ATPase (b-ATPase) [27] and 26S rRNA (PR31).
Panel A. Transcript levels in various organs of wild-type tobacco. R, Root; St, Stem; ML, Mature Leaf; YL, Young Leaf; Se, Sepal; C, Carpel; MS, Mature Stamen; P, Petal; Et Br, ethidium bromide stained. Panel B. Transcript levels in roots of Burley 21 tobacco plants before and after topping.
under stress will show higher levels of ODC prior to topping. Low but detectable amounts of mR-NAs encoding SAMS are present in the roots of
untopped plants, and the level of SAMS
tran-scripts changed little over the 24 h period after topping.
No significant expression of theODC and ADC
transcripts was observed in leaf tissues before or after topping. In contrast, transcripts encoding SAMS were moderately abundant in young leaves and present at low levels in the mature leaves and other photosynthetic tissues. This observation is consistent with increased requirement for methyl groups during light-induced leaf development and the general requirement of SAM for a wide range of cellular processes.
4. Discussion
A large number of endogenous and exogenous factors are known to affect gene expression lead-ing to alkaloid formation in plants includlead-ing de-velopmental age, phytohormones, and various biotic and abiotic stresses [1,2,4,8,41]. In the present study, we describe the cloning and charac-terization of a group of cDNAs, differentially expressed in the roots of tobacco after topping, a process involving the removal of the flowering head and young leaves and stem from the upper portion of a maturing tobacco plant. Topping is a common practice during commercial tobacco pro-duction and serves several purposes [10]. It re-leases the plant from apical dominance, causing increased root growth, stimulation of alkaloid biosynthesis in the roots and accumulation of these compounds in the leaves, and acceleration of senescence of the mature leaves near the base of the plant. Consistent with these changes, among the main categories of differentially expressed gene products 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 and the transduction pathways operating to bring about differential plant growth and activation of alkaloid metabolism are not yet known, among the most obvious factors are alterations in
phyto-hormone levels and/or ratios within the plant as a
consequence of topping. Meristems and young leaves are known to be a major source of auxin prior to topping and message abundance increased
and their removal would be expected to lead to significant changes in auxin availability, as well as to an alteration of auxin to cytokinin ratios within the shoots and roots. Such changes in phytohor-mone levels and ratios are known to result in the activation of expression of some genes and repres-sion of others. For example, previous studies have shown that auxin has a negative effect on nicotine and total alkaloid biosynthesis in plants and cul-tured cells, with the effects seen both at the level of enzyme activation and gene expression [6,15,52 – 55]. Expression of putrescine PMT, a key enzyme in nicotine and tropane alkaloid formation, was reported to be repressed in cultured tobacco roots by the presence of indolebutyric acid (IBA) in the growth media [6]. Removal of the IBA from the growth medium led to an increase of both PMT activity and steady state levels of mRNA encoding the enzyme. Similarly, addition of auxin to
hor-mone free media used for the culture of Catha
-ranthus roseuscells led to a decrease in the levels of strictosidine synthase and tyrosine decarboxylase transcription and a corresponding decrease in alka-loid accumulation [53]. It is perhaps interesting that in addition to recovering enzymes directly involved in alkaloid formation (i.e. ODC, ADC), we also recovered cDNAs encoding homologs of proteins whose expression are known to be regu-lated directly by auxin (e.g. PR39, PR58) as well as proteins 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 of both ODC and ADC activity parallel growth rates during the early stages of fruit development [37,56]. In contrast, mature tissues show very low levels of activity of these enzymes and little or no detectable transcript levels. Increased ADC activity could be detected following gibberellin treatment of unpolli-nated ovaries to induce parthenocarpic fruit devel-opment in [56]. However, not all of the observed changes in the activity of these enzymes during fruit development can be attributed to changes in gene expression [31,37]. In germinating soybean seedlings, ADC activity and transcript levels were highest in elongation zone, between the roots and hypocotyl 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 in other plant tissues.
Wounding of the leaves of N. syl6estris and
other wild species of tobacco has been shown to induce de novo formation of nicotine in the roots and its rapid accumulation throughout the plant [9,11 – 13]. Several lines of evidence suggest that the expression of genes involved in polyamine forma-tion and subsequent nicotine biosynthesis may be under the control of a JA signal transduction pathway [8,9,12 – 15]. For example, direct applica-tion of JA or its methyl derivative (MeJA) to the leaves of intact tobacco plants results in increased nicotine formation [11 – 14]. Treatment of tobacco BY-2 suspension cultures with MeJA resulted in increased alkaloid formation and induced small transient increases in PMT, ODC and SAMS mRNA levels [15]. In contrast, steady state levels of ADC and SAMDC were not affected, suggest-ing that not all genes encodsuggest-ing enzymes of polyamine and alkaloid formation are under the same type of regulation. The observed increase in
ODC, SAMS and PMT transcript levels was less
when BY-2 cells were cultured in the presence of 2,4-D than BA indicating that multiple regulatory circuits must exist within the plant cell [15]. Fur-ther support for the presence of cross-talk among
regulatory pathways comes from work withCatha
-ranthus roseus, where addition of JA, MeJA, and
traumatic acid to suspension cultures of Catha
-ranthus cells led to increased alkaloid formation only when cells were grown in auxin depleted medium [55].
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. Experiments on transgenic plants
us-ing various promoter-b-glucuronidase (GUS)
re-porter gene fusions have identified regions within
the promoter of the hyoscyamine 6b-hydroxylase
[57], PMT [58], and tyrosine/
dihydroxyphenylala-nine decarboxylase [59] gene promoters necessary for 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 the promoters of several genes whose products are involved in terpenoid indole alkaloid formation
(e.g. tryptophan decarboxylase (tdc), strictosidine
synthase (Str1)) contain sites capable of interacting
[60,61]. The biosynthesis of nicotine has been shown to be under the control of at least two
unlinked genetic loci, termed Nic1 and Nic2
[16,17]. Although no information is available on the nature of their encoded products, it has been
speculated that Nic1 and Nic2 likely encode
tran-scriptional regulators capable of globally interact-ing with a subset of genes encodinteract-ing components of polyamine and alkaloid biosynthesis [6]. The availability of cDNAs and cloned genomic frag-ments encoding enzymes in nicotine and alkaloid formation are the first step towards unraveling these processes.
Acknowledgements
The authors wish to thank Maria Shulleeta and her colleagues at Philip Morris for providing us with the cDNA library and cDNA cloning infor-mation used in these studies, Jacques Retief for his help with the nucleotide and amino acid sequence analysis and alignment programs, and Ved Pal Malik for his helpful suggestions. This work was supported by a grant from Philip Morris (Rich-mond, VA).
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