Plant Physiol. (1998) 117: 533-543
Tissue Culture-Specific Expression of a Naturally
Occurring Tobacco Feedback-Insensitive Anthranilate
Synthase1
Hee-Sook Song,
Jeffrey E. Brotherton,
Robert A. Gonzales, and
Jack M. Widholm*
Department of Crop Sciences, University of Illinois, Edward R. Madigan Laboratory, 1201 West Gregory, Urbana, Illinois 61801 (H.-S.S.,
J.E.B., J.M.W.); and The Samuel Roberts Noble Foundation, Inc., P.O.
Box 2180, 2510 Sam Noble Parkway, Ardmore, Oklahoma 73402 (R.A.G.)
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ABSTRACT |
A cDNA and corresponding promoter
region for a naturally occurring, feedback-insensitive anthranilate
synthase (AS) 
-subunit gene, ASA2, has been isolated
from an unselected, but 5-methyl-tryptophan-resistant (5MTr), tobacco (Nicotiana tabacum) cell
line (AB15-12-1). The ASA2 cDNA contains a putative
transit peptide sequence, and Southern hybridization shows that more
than one closely related sequence is present in the tobacco genome. The
ASA2 cDNA complemented a trpE nonsense
mutant Escherichia coli strain, allowing growth on 300 µM 5MT-containing minimal medium without tryptophan, and cell extracts contained feedback-insensitive AS activity. The 5MTr was lost when the E. coli strain was
transformed with an ASA2 site-directed mutant
(phenylalanine-107-arginine-108 
serine-107-glutamine-108). Identical nucleotide sequences encoding the
phenylalanine-107-arginine-108 region have been found in polymerase
chain reaction-amplified 326-bp ASA2 genomic fragments
of wild-type (5-methyl-tryptophan-sensitive [5MTs])
tobacco and a progenitor species. High-level ASA2
transcriptional expression was detected only in
5MTr-cultured cells, not in 5MTs cells or in
plants. Promoter studies indicate that tissue specificity of
ASA2 is controlled by the promoter region between 
2252
and 607. Since the ASA2 promoter sequences are not
substantially different in the 5MTr and 5MTs
lines, the increased levels of ASA2 mRNA in the
5MTr lines are most likely due to changes in a regulatory
gene affecting ASA2 expression.
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INTRODUCTION |
Many aromatic compounds are synthesized via the shikimate pathway
in higher plants (Haslam, 1993
; Herrmann, 1995). Chorismate is located
at a branch point of this pathway and is the last common precursor of
many of these compounds. AS catalyzes the first committed reaction in
the Trp-biosynthesis branch by converting chorismate to anthranilate
and is feedback inhibited by the end product, Trp (Haslam, 1993; Romero
et al., 1995). That AS is the control point in the Trp branch in plant
cells is indicated by (a) pathway-intermediate feeding studies
(Widholm, 1974), (b) enzyme activity levels, (c) feedback inhibition of
the respective enzyme activities (Singh and Widholm, 1974), and (d) 5MT
resistance selection that yielded lines with altered feedback-inhibited
AS and higher free Trp (Widholm, 1972a, 1972b).
Plant cell culture systems have been useful for studying the regulatory
mechanisms of amino acid biosynthesis (Widholm, 1972a, 1972b).
Selecting cells resistant to amino acid analogs such as 5MT can produce
lines with increased amounts of the end product, Trp, because of an
alteration of the allosteric regulatory enzyme, causing less inhibition
of the enzyme by the analog and Trp (Widholm, 1972a, 1972b).
Feedback-insensitive AS has been found in 5MT-selected carrot
(Daucus carota; Brotherton et al., 1986), Datura
innoxia (Ranch et al., 1983), potato (Solanum
tuberosum; Carlson and Widholm, 1978), and tobacco
(Nicotiana tabacum; Brotherton et al., 1986). The
5MTr tobacco cell line (TX2-4) contained
Trp-insensitive AS and higher levels of free Trp than wild-type cells.
Similar results were obtained with 5MT-selected cultured potato cells
(Carlson and Widholm, 1978). These characteristics were not found in
the regenerated tobacco plants but were recovered again in cultured
cells induced from leaves of the regenerants (Brotherton et al., 1986).
These results suggest that expression of this altered AS is regulated in a tissue-specific manner and that selection for
5MTr produces lines with increased amounts of the
feedback-insensitive AS form that is expressed in cultured cells but
not in plants.
Recently, AS was purified from cultured plant cells or plant tissues
(Poulsen et al., 1993; Bohlmann et al., 1995; Romero and Roberts, 1996)
and found to have a subunit composition similar to AS from some
microbes, i.e. nonidentical large (, component I) and small (
,
component II) subunits (Zalkin et al., 1984; Yanofsky and Crawford,
1987; Crawford, 1989). AS genes encoding an -subunit (Niyogi and
Fink, 1992; Bohlmann et al., 1995) and -subunit (Niyogi et al.,
1993) have been cloned from Arabidopsis and Ruta graveolens.
The two AS genes encoding the -subunit of the enzyme cloned from
Arabidopsis and R. graveolens have been designated
ASA1/ASA2 and
AS1/AS2, respectively (Niyogi and Fink, 1992; Bohlmann et al., 1995). These AS genes have
similar gene-expression patterns, since ASA1 and
AS1 expression is induced by wounding and/or elicitor
treatment, whereas the ASA2 and AS2 genes are expressed constitutively at low levels (Niyogi and Fink, 1992; Bohlmann
et al., 1995).
Two groups have described a mutant Arabidopsis AS gene encoding a
feedback-insensitive -subunit of the enzyme (Kreps et al., 1996; Li
and Last, 1996). Sequencing of these mutant AS genes showed that a
single amino acid, Asp-341, was replaced by Asn in both cases. These
mutant AS genes were expressed in both cultured cells and leaves, with
slightly higher expression in cultured cells (Kreps et al., 1996). In
contrast to these Arabidopsis mutants, the AS1 gene from
R. graveolens that encodes a feedback-insensitive -subunit was isolated from wild-type young shoots (Bohlmann et al.,
1996). Based on sequence comparison, it was postulated that a single
amino acid substitution in the AS1 gene, Arg-138 for Gln-138, may cause feedback insensitivity. The residues potentially affecting feedback inhibition in higher plants do not align with those
of bacteria (Matsui et al., 1987; Caliguri and Bauerle, 1991) and yeast
(Graf et al., 1993), but the altered amino acids are located within or
very close to the conserved motifs Leu-Leu-Glu-Ser-X10-Ser and Asn-Pro-Ser-Pro-Tyr-Met, which are important for feedback inhibition (Matsui et al., 1987; Caliguri and Bauerle, 1991; Graf et
al., 1993).
The tobacco plants regenerated from the 5MTr
suspension cultures described by Brotherton et al. (1986) were not
fertile; therefore, the selection was repeated with new cultures to
obtain fertile plants (S. Schechter and J.M. Widholm, unpublished
data). One of nine plants regenerated from an unselected control
culture produced a suspension culture from a leaf that was
5MTr. This resistance was inherited by progeny,
and the cell line studied here, AB15-12-1, was initiated from a
fifth-generation plant. This cell line has the typical characteristics
of 5MTr cells, feedback-insensitive AS and
increased free Trp. We report the cloning and characterization of the
wild-type tobacco ASA2 gene encoding a feedback-insensitive
-subunit of the enzyme and analysis of the role of promoter regions
in tissue-specific expression of this gene. We show that
ASA2 mRNA is specifically elevated in
5MTr lines and propose that this increased level
of expression is due to enhanced transcription of the ASA2
gene in the 5MTr lines.
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MATERIALS AND METHODS |
Suspension cultures were maintained by weekly transfers
into 50 mL of liquid MX medium (MS basal medium [Murashige and Skoog, 1962] with 1.8 µM 2,4-D). The 5MTr
AB15-12-1 cell line originated from a tobacco (Nicotiana
tabacum cv Xanthi) plant regenerated from unselected
suspension-cultured cells. Originally, nine plants were regenerated by
placing the unselected cells on MS agar-solidified medium with 5.7 µM IAA and 2 µM kinetin. Callus and
subsequent suspension cultures were initiated from the leaves of these
plants. One plant produced a suspension culture that showed resistance
to 5MT when 0.5 g fresh weight of cells was incubated for 9 to
12 d in MX liquid medium containing 46 and 137 µM
5MT; other wild-type cells did not grow in the same medium. Because
this plant was male-sterile, it was pollinated with wild-type pollen
and the resulting progeny were self-pollinated four times to obtain
AB15-12-1. Additional tobacco cell-suspension cultures were
subsequently initiated from AB15-12-1 seedlings and maintained on MX
or 5MT-containing medium as indicated.
Nucleic Acid Analysis
Genomic DNA was isolated from 1-week-old suspension-cultured cells
using the procedures of Dellaporta et al. (1983) and purified by
CsCl-gradient centrifugation (Ausubel et al., 1989; Sambrook et al.,
1989).
Total RNA was prepared using a phenol-extraction method (Wang et al.,
1994) from 1-week-old suspension-cultured cells, mature seeds, and
leaves, roots, and stems harvested from 3-week-old shoot cultures grown
on MS basal medium solidified with 0.8% agar.
DNA and RNA gels were blotted onto a nylon membrane (Hybond
N+, Amersham) following a general
capillary-transfer method (Sambrook et al., 1989). The
Arabidopsis thaliana ASA1 (pKN41) cDNA clone (obtained from K. Niyogi [Niyogi and Fink, 1992]) or other tobacco AS
cDNA clones obtained in this work were used as a probe following labeling with a Megaprime DNA-labeling system (Amersham) with [-32P]dCTP (3000 Ci/mmol). Southern and
northern hybridizations were done at 42°C with hybridization solution
(50% formamide, 5× SSPE, 5× Denhardt's solution [Sambrook et al.,
1989], 0.1% SDS, and 100 µg/mL salmon-sperm DNA). The membranes
were washed at high stringency twice at room temperature with 2× SSC
and 0.5% SDS for 20 min each time and at 65°C with 0.1× SSC and
0.1% SDS until the background signal disappeared and were then exposed
to radiographic film overnight with an intensifying screen.
Cloning of AS cDNA
Tobacco AS cDNAs were isolated using 5
and 3 RACE (GIBCO-BRL).
Primers 1 and 2 for cloning the 5 end of the ASA2 cDNA were designed based on the sequence of the Arabidopsis ASA1
genomic clone (GenBank accession no. M92353). The sequences of primers used for cloning are listed in Table I.
For 5 RACE, first-strand cDNA was synthesized with antisense primer 1 at nucleotide position 5403 to 5382 of the Arabidopsis ASA1
genomic clone. The 5 end of the first-strand cDNA was oligo-dA tailed
(200 µM dATP) using terminal deoxynucleotidyl transferase
(0.4 unit/µL).
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Table I.
Nucleotide sequences of the primers used for cloning
Restriction enzyme site for cloning at 5 end of the primers is
underlined. Boldfaced nucleotides in the primers 7 and 8 represent mismatch nucleotides for site-directed mutagenesis.
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A nested PCR was performed with the adapter primer
5-GGCCACGCGTCGACTAGTAC(T)17-3 (GIBCO-BRL) and
antisense primer 2 at nucleotides 4430 to 4409 of the Arabidopsis
ASA1 genomic clone. The first-strand cDNA was used as a
template for the nested PCR. A fragment (approximately 1.1 kb) was
amplified and cloned into the pGEM-T vector and sequenced. The
procedures used to isolate the 3 end of the ASA2 cDNA were the same as for 5 RACE except for the primers and dATP tailing at the
5 end of the cDNA. Primer 3 was designed based on the sequence of the
5 ASA2 cDNA clone at nucleotides +148 to +168 (adenine of
ATG was counted as +1, since the transcription start site was not
determined in this work). An approximately 1.9-kb fragment was
amplified and strongly hybridized with the 5 ASA2 cDNA
clone as a probe. This fragment was cloned into the pGEM-T vector and
sequenced.
Analysis of the sequence alignment of the two fragments using Clustal
(Higgins and Sharp, 1989) shows that the 828-bp overlapping region is
identical. There is only one XbaI site in the 828-bp overlapping region, only one NsiI site in the pGEM-T vector,
and no NsiI site in either the 5 or the 3 fragments. These
two restriction enzyme sites were used to construct the full-length
tobacco ASA2 cDNA. Sequencing of the cDNA clones was
performed by the Genetic Engineering Laboratory of the University of
Illinois (Urbana-Champaign) using the dideoxynucleotide-sequencing
method.
Complementation Tests and Site-Directed Mutagenesis
The ASA2 cDNA without the presumed transit-peptide
sequence (amino acid sequence 1-60) was amplified using Pfu
DNA polymerase (Stratagene) with primers 4 and 5 (Table I) containing
BamHI and KpnI overhangs, respectively, and
ligated into an expression vector (pQE30 containing a 6-His tag-coding
sequence [6xHis, Qiagen, Santa Clarita, CA]) followed by
restriction-enzyme digestion.
Site-directed mutagenesis was performed by PCR using a primer
containing mismatched nucleotide sequences by changing four nucleotides
(CC
GGTTC
ACC
GGTTC
A)
at amino acid residues 105 to 108. The first two mismatched
nucleotides, Pro-105 and Gly-106, do not change the amino acid codon
but create an SmaI site. The last two mismatched nucleotides
change Phe-107 and Arg-108 to Ser-107 and Gln-108. Two PCR products
were obtained using primers 6 and 7 and primers 8 and 5 (Table I).
These two PCR fragments were ligated, digested by SmaI, and
ligated in-frame into the pQE30 vector.
The chimeric constructs were transformed into trpE mutant
Escherichia coli (trpE5972, nonsense mutant)
using CaCl2 transformation (Sambrook et al.,
1989). Complemented strains were plated on M9 minimal medium containing
100 µg/mL ampicillin and 0.1 mM IPTG but no Trp. For the
inhibition test, 300 µM 5MT was added to the minimal
medium described previously.
Expression of 6xHis-Tagged ASA2 Protein in E. coli
The E. coli trpE mutant (trpE5972)
transformed with the 6xHis-tagged ASA2 chimeric construct
was grown in 1 L of Luria-Bertani medium supplemented with 100 µg
mL
1 ampicillin and 0.1 mM Trp. IPTG
(0.1 mM) and PMSF (0.1 mM) were added to a
mid-logarithm culture. After 3 h of further incubation at 30°C
and 150 rpm, the cells were harvested by centrifugation, resuspended in
20 mL of extraction buffer (Bernasconi et al., 1994) with 0.1 mM PMSF added, disrupted using a French press (two passages
at 20,000 p.s.i.), and centrifuged to remove cell debris. The
supernatant was held overnight on ice and then combined with 2 volumes of saturated, room-temperature
(NH4)2SO4.
The resulting protein precipitate was collected by centrifugation at
4°C and resuspended in 10 mL of 50 mM
NaH2PO4, 300 mM NaCl, and
2 mM DTT, pH 8.0. The 6xHis-tagged ASA2 protein was
partially purified after binding to Ni-nitrilotriacetic acid resin
(Qiagen) and elution with 100 mM imidazole in 50 mM NaH2PO4, 300 mM NaCl, 2 mM DTT, and 10% glycerol, pH 6.0.
Arabidopsis ASA1 protein fused with glutathione
S-transferase was expressed using the pSCI1674 construct
cloned into E. coli MC1061 by Bernasconi et al. (1994). The
procedure was the same as described above except that the resuspended
(NH4)2SO4
pellet was used without further purification.
AS Enzyme Assay
Cell extracts were prepared as described by Brotherton et al.
(1986) from mid-logarithm-phase cell cultures and were used the same
day. The resuspended
(NH4)2SO4
fraction was desalted using Sephadex G-25.
AS activity was measured using the ethyl-acetate-extraction method used
previously for tobacco cell extracts (Widholm, 1971) and incorporating
the modifications described by Li and Last (1996), except that the
assay buffer was 50 mM Hepes, pH 7.5, 10 mM
Gln, 2.0 mM MgCl2, 0.05 mM Na2EDTA, 2.0 mM DTT,
and 5% glycerol. The reaction was started by the addition of 50 µL
of cell extract. Chorismate and Trp concentrations were as indicated in
individual experiments. The production of anthranilate was linear with
respect to time for twice the standard incubation time and enzyme
concentration (data not shown). The conversion of chorismate to
anthranilate never exceeded 1% in any experiment. Chorismate was
produced using the fermentation method of Gibson (1970).
ASA2 Genomic Fragments Containing the
Phe-107-Arg-108 Region from Wild-Type Nicotiana sp.
ASA2 genomic fragments containing the Phe-108-Arg-108
region were PCR amplified and isolated from three wild-type
(5MTs) cultured cell lines, N. tabacum
(TXD), Nicotiana tomentosiformis (Nto), and Nicotiana
sylvestris (Ns), from leaves harvested from AB15-12-1 shoot
cultures, and from two 5MTs (H15-6, NRMX) and
one 5MTr (NR5MT) tobacco cell lines. The region
from 110 to +392 was first amplified using two sets of primers based
on the ASA2 cDNA and promoter sequences 9 and 10 (Table I).
Primers 4 and 10 were then used for a nested PCR amplification using
the first PCR product as the template to produce the 326-bp genomic
fragments (+181 to +392) that were then cloned into pBluescriptSK,
digested with EcoRV, and sequenced.
Cloning of AS Promoter and Construction of Chimeric AS Promoter-GUS
Constructs
The ASA2 promoter was isolated using inverse PCR
(Ochman et al., 1989). AB15-12-1 genomic DNA was digested with
HindIII, circularized with T4 DNA ligase after dilution to
100 ng/mL with distilled, deionized water and used as a template for
inverse PCR with primer 3 and antisense primer 11 based on the sequence
of the ASA2 cDNA at nucleotide +10 to 11. PCR
amplification was performed for 30 cycles (95°C, 1 min; 50°C,
40 s; 72°C, 2 min). A PCR fragment (approximately 2.3 kb) was
detected by Southern hybridization with the ASA2 cDNA as a
probe and cloned into pGEM-T vector. Sequencing of the ASA2
promoter (approximately 2.3 kb) was performed at the Molecular Analysis
and Synthesis Section of the Samuel Roberts Noble Foundation (Ardmore,
OK) using the dideoxynucleotide-sequencing method.
Based on database search results, deletions were made by using PCR
amplification with seven sets of primers as follows: antisense primer
12 with sense primers 13, 14, 15, or 16 (Table I) that amplified 2252-, 1356-, 606-, or 370-bp ASA2 promoter fragments (including the 5 UTR),
respectively. These PCR products were fused to the GUS reporter gene
with NOS terminator. The chimeric constructs were designated 2252, 1356, 606, and 370. Each primer contains a restriction enzyme site
overhang for cloning. Primer 12 contains an SmaI site and
primers 13, 14, 15, and 16 contain a PstI site. Each
restriction enzyme site was underlined in the primer sequences (Table
I). These five fragments were cloned into pBI221, replacing the CaMV
35S promoter.
GUS Assay
The constructed plasmid DNAs were isolated using a Plasmid Maxi
Kit (Qiagen) and biolistically transferred into tobacco
suspension-cultured cells (AB15-12-1) and leaves using a
particle-inflow gun (5 µg of DNA and 0.5 mg of 1.0-µm diameter gold
particles [Bio-Rad] shot at 80 p.s.i.; Vain et al., 1993).
Leaves (180-350 mg fresh weight) harvested from 3-week-old shoot
cultures were placed on 5-cm-diameter sterile Whatman no. 1 filter
paper discs. Five milliliters of 2-d-old AB15-12-1 suspension-cultured
cells (approximately 200 mg fresh weight) was collected on filter paper
using a vacuum before bombardment. The samples were transferred with
the filter paper onto MX solid medium after bombardment and incubated
at 24°C under fluorescent light (60 µE m2
s1) for 3 d. The promoter activity was
determined with the fluorometric MUG assay (Jefferson, 1987; Jefferson
et al., 1987). GUS-specific activity was determined as picomoles of
4-methylumbelliferone per hour per milligram of protein. The protein
concentration was determined using a protein dye-binding assay kit
(Bio-Rad).
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RESULTS |
ASA2 cDNA Cloning and Sequence Analysis
We have cloned a full-length ASA2 (2.16 kb including 5
and 3 UTR) cDNA using 5 and 3 RACE. The nucleotide and deduced amino
acid sequences are shown in Figure 1.
This AS gene is denoted ASA2, since its amino acid sequence
was 72% identical to the sequence of AS2 of R. graveolens. An apparent 5 UTR was found 89 nucleotides upstream
of a translation start codon (ATG) in the 5 end of the cDNA fragment.
The 3 RACE results indicate that polyadenylation begins 205 nucleotides downstream of the translation stop codon. No perfect match
to the consensus eukaryotic poly(A+) signal
sequence AAUAAA was found in the 3 UTR.
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| Figure 1.
Nucleotide and the predicted amino acid sequence
of the ASA2 cDNA. The coding region corresponds to amino
acid sequence 1 (ATG) to 616 (TAG). The putative translation start and
stop codons are indicated in boldface. The 89 and 205 bp of the
upstream and downstream coding region represent 5 and 3 UTRs,
respectively.
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The ASA2 cDNA clone hybridized under high-stringency
conditions to several fragments of genomic DNA digested with nine
restriction enzymes (Fig. 2A). Three
major bands at approximately 3.3, 4.0, and 24 kb were found in
KpnI-digested genomic DNA of the amphidiploid N. tabacum leaves and the two cultured cell lines, whereas the diploid tobacco progenitors, N. sylvestris and N. tomentosiformis, showed different patterns (Fig. 2B). The
hybridization pattern of N. tabacum was more like N. tomentosiformis than N. sylvestris, since both had
bands at approximately 3.3 and 4.0 kb. Two faint bands hybridized to
the ASA2 cDNA clone in N. sylvestris. The 24-kb
fragment in N. tabacum shows very weak hybridization and is
not found in either N. sylvestris or N. tomentosiformis.
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| Figure 2.
Genomic DNA-blot analysis. A, Twenty micrograms of
AB15-12-1 genomic DNA was digested with nine restriction enzymes.
Lanes from left to right correspond to BamHI,
EcoRI, EcoRV, HincII, HindIII, KpnI, PstI,
ScaI, and XbaI. B, Ten micrograms of each genomic DNA obtained from leaves of N. sylvestris,
N. tomentosiformis, N. tabacum, and
suspension-cultured cells of the 5MTs and 5MTr
tobacco cell line (AB15-12-1) was digested with KpnI.
The full-length ASA2 cDNA clone was used as a probe in
both A and B. Fragment sizes were determined by a 1-kb ladder
(GIBCO-BRL).
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Comparison of Amino Acid Sequences of AS Genes
Based on BLAST analysis (Altschul et al., 1990), the five best
matches to the predicted amino acid sequence of the tobacco ASA2 gene are aligned in Figure
3. The 1851 nucleotide
ASA2-coding region would encode a 616-amino acid polypeptide
with a calculated molecular mass of 69,035 D. The tobacco
ASA2 gene encodes 60 amino acids beyond the amino terminus
of the aligned microbial homologs. The sequence
Ile-58-Glu-59-Ala-60-Ser-61 is similar to the consensus cleavage-site
motif ([Val/ Ile]X[Ala/Cys]
Ala) for plant chloroplast transit
peptides (von Heijne et al., 1989; Gavel and von Heijne, 1990). The
transit peptide sequences of the five plant AS genes show little
homology to each other. The tobacco ASA2 gene shows 72 and
70, 67 and 66, and 32% amino acid identity to R. graveolens AS2 and AS1, A. thaliana ASA1 and
ASA2, and Clostridium thermocellum trpE genes,
respectively (Sato et al., 1989; Niyogi and Fink, 1992; Niyogi et al.,
1993; Bohlmann et al., 1995).
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| Figure 3.
Amino acid sequence alignment of AS genes from
plants and a prokaryote. TASA2, RASA1, RASA2, AASA1, AASA2, and CTRPE
correspond to N. tabacum ASA2, R. graveolens
AS1 and AS2, A. thaliana
ASA1 and ASA2, and C. thermocellum
trpE cDNA clones, respectively. Dashes within sequences
indicate gaps. Asterisks under the sequence represent identical amino
acids among these six different AS sequences. Dots under the sequence
indicate similar amino acids. Two consensus motifs,
Leu-Leu-Glu-Phe-X10-Ser and Asn-Pro-Ser-Pro-Tyr-Met,
affecting feedback inhibition based on microorganisms and yeast, are
indicated in boldface. The amino acid(s) substitutions Phe-Arg and Arg, which caused feedback insensitivity in N. tabacum and
R. graveolens, are indicated in boldface with a diamond
mark at positions 107 and 108, respectively. A single amino acid change
in an Arabidopsis mutant (Asp to Asn) is indicated in boldface with a
dagger at position 326. A consensus sequence motif for plant
chloroplast transit peptides in the tobacco ASA2 is underlined at
positions 58 to 61. The amino acid numbers indicated are based on the
tobacco ASA2 amino acid sequence.
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In bacteria, fungi, and yeast two conserved amino acid motifs,
Leu-Leu-Glu-Ser-X10-Ser and Asn-Pro-Ser-Pro-Tyr-Met, are
associated with Trp feedback inhibition (Matsui et al., 1987; Caliguri
and Bauerle, 1991; Graf et al., 1993). The six AS genes in Figure 3
showed no amino acid changes in these conserved regions except in the
Leu-Leu-Glu-Ser-X10-Ser motif, where the Ser and Gln
residues found in feedback-sensitive AS of higher plants were changed
to Phe-107 and Arg-108 (based on the tobacco ASA2 amino acid
sequence) in the tobacco ASA2 and to Arg-108 in the
AS1 gene of R. graveolens. These changes
appear to correlate with the feedback insensitivity found in the latter
two enzymes.
AS Gene Expression
The tobacco ASA2 cDNA probe detected high levels of a
2.2-kb transcript on northern blots only in 5MTr
cultured tobacco cell lines, including 5MTr N. sylvestris but not in 5MTs-cultured cell
lines, leaves, roots, stems, and seeds after overnight exposure (Fig.
4).
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| Figure 4.
Northern analysis. Twenty micrograms of total RNA
per lane was used for northern hybridization. A full-length
ASA2 cDNA clone (ASA2) and rRNA (rRNA) were used as
probes for hybridization. A, Total RNAs were prepared from 1-week-old
N. tabacum suspension-cultured cells (SC) of four
5MTs (s) and four 5MTr (r) cell lines,
5MTs N. tabacum leaves (L) harvested from
3-week-old AB15-12-1 shoot cultures, and 1-week-old
5MTr N. sylvestris suspension-cultured
cells. B, Different plant organs were used to determine tissue
specificity. AB15-12-1 leaves, roots, and stems harvested from
3-week-old shoot cultures and dried mature seeds and
suspension-cultured 5MTs cells (TXD) and
5MTr cells (AB15-12-1) were used to prepare total RNA.
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Complementation and Inhibition Tests
The E. coli trpE5972 nonsense mutant transformed with
the tobacco ASA2 cDNA and with the site-directed mutant ASA2
form (Phe-107-Arg-108 changed to Ser-107-Gln-108) both grew on minimal
medium containing ampicillin and IPTG but no Trp (Fig.
5A). The complemented strain transformed
with the site-directed mutant, however, did not grow on 300 µM 5MT containing minimal medium without Trp (Fig. 5B), whereas the growth of the strain transformed with the ASA2
cDNA was not inhibited by 300 µM 5MT.
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| Figure 5.
Complementation of E. coli trpE5972
by the tobacco ASA2 and a site-directed mutant. The
pQE30/ASA2 and pQE30/ASA2 mutants were
transformed into the trpE nonsense mutant E. coli strain (trpE5972) and plated on M9 minimal
medium containing 100 µg/mL ampicillin (A) and 0.1 mM
IPTG without and with 300 µM 5MT (B). The picture was
taken 2 d after streaking.
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AS Kinetic Constants
Trp inhibition of the partially purified product of the E. coli-expressed tobacco ASA2 and Arabidopsis
ASA1 genes is shown in Figure
6A. The ASA2 gene product is
still 50% active at 100 µM Trp, similar to AS in
5MT-selected tobacco cells and to purified R. graveolens
AS1 protein, which was 80% active at this Trp concentration (Bohlmann et al., 1996).
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| Figure 6.
Kinetics of AS from wild-type ( ) and
5MT-selected ( ) tobacco cells and from E. coli
transformed with the Arabidopsis ASA1 gene ( ) or the
tobacco ASA2 gene ( ). A, Relative AS activity in the
presence of Trp was measured as described in ``Materials and Methods'' (with 100 µM chorismate) and is expressed as a
percentage of the activity with no Trp added. The specific activity
with no Trp for AS from each tobacco line was 73 and 169 nmol
anthranilate min1 mg1 protein,
respectively. B. Lineweaver-Burk plot of AS activity. Velocity is
expressed as nanomoles per minute per milligram of protein.
|
|
When AS activity was measured in extracts from wild-type and
5MTr tobacco cell-suspension cultures the
resistant cultures had a portion of the activity that is more feedback
insensitive than that of the wild-type cells (Fig. 6A). The apparent
Ki values for Trp are 2 and 300 µM, respectively, for AS from wild-type and 5MT-selected
tobacco cells when estimated from the Trp concentration, resulting in
50% inhibition, as shown in Figure 6A. Similar values were reported by
Bohlmann et al. (1996) for R. graveolens Trp-sensitive and
-insensitive AS (2.8 and >100 µM, respectively). AS from
wild-type Arabidopsis had an apparent Ki of
3 µM, whereas AS from a 6-methyl-Trp-selected mutant had
a Ki of 8 µM (Li and Last,
1996).
The apparent chorismate Km value was lower
for AS from 5MT-selected cells (6 µM) than for the
wild-type cells (22 µM) when measured using 10 mM Gln as the second substrate (Fig. 6B). These values are
comparable to those obtained for AS from Arabidopsis (10 and 21 µM using Gln [Li and Last, 1996]) and R. graveolens (17 and 29 µM using
NH4+ [Bohlmann et al., 1996]).
In all three species the chorismate Km
value (first value listed) is lower for the feedback-insensitive AS.
Comparison of ASA2 and Wild-Type Gene Sequences
The ASA2 fragment between +181 and +392, which contains
the Phe-107-Arg-108 residues, was PCR amplified and cloned from
5MTs and 5MTr genomic DNAs.
When the nucleotide sequence of the fragment from AB15-12-1 was
aligned with those from the three wild-type Nicotiana sp.
cell lines (Fig. 7) and compared with the
ASA2 cDNA, the genomic fragments were found to contain a
115-bp intron; therefore, the cloned fragments were 326 bp in length.
The nucleotide sequences of this fragment were 100% identical in all
cases except for eight nucleotides in N. sylvestris. This
eight-nucleotide difference did not change the encoded amino acids.
Amino acid sequences in this region showed 66% identity to four other
plant AS genes from Arabidopsis and R. graveolens.
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| Figure 7.
Nucleotide sequence alignment of
ASA2 genomic DNA fragments. The 326-bp
ASA2 genomic DNA fragments, corresponding to the
ASA2 cDNA sequence from +181 to +392, were amplified
from the genomic DNAs of tobacco leaves (ASA2) harvested from
AB15-12-1 shoot cultures and three wild-type (5MTs)
Nicotiana sp. cultured cell lines, N. tabacum (TXD), N. tomentosiformis (Nto), and
N. sylvestris (Ns) using primers 4 (sense) and 10 (antisense), which are shown in bold. These genomic DNA fragments
contain a 115-bp intron, which is underlined. Eight nucleotides,
indicated by (+) above the sequence, are different in the N. sylvestris genomic fragment compared with those in N. tabacum and N. tomentosiformis. The nucleotide
sequences corresponding to Phe-107-Arg-108 are indicated in boldface
and underlined with the one-letter amino acid codon abbreviations F and
R above the sequence.
|
|
Promoter-Sequencing Analysis
The 99 nucleotides at the 3 end of the promoter region and 5 end
of the ASA2 cDNA, including the 5 UTR, were identical (Fig.
8). The first possible TATA box (TATAAA)
is located 121 bp upstream from the translation start site (ATG). BLAST
analysis shows that the region from 1187 to 769 exhibits 81 to 83%
nucleotide sequence identity to the promoter region of the N. tabacum plant-defense-related Str246C gene (Froissard
et al., 1994).
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| Figure 8.
The nucleotide sequence of the ASA2 promoter
region. The first possible TATA box (TATAAA), translation start codon
(ATG), is indicated in boldface. The 98 bp at the 3 end of the ASA2 promoter region, which is identical to the nucleotide sequence at 5
end of the ASA2 cDNA, is underlined.
|
|
Chimeric GUS Gene Expression
When different deleted fragments of the ASA2 promoter region were
tested, the transient GUS expression controlled by the promoter region
denoted 606 was stronger in both suspension-cultured cells and in
leaves than that driven by the CaMV 35S promoter (Fig. 9). The 1356 and 2252 regions still
showed high GUS activity in suspension-cultured cells but very low
activity in leaves. The 1356 region showed approximately 5 times higher
GUS expression than that driven by the CaMV 35S promoter in
5MTr suspension-cultured cells.
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| Figure 9.
Chimeric ASA2 promoter-GUS
constructs and transient GUS assays with tobacco leaves and
suspension-cultured cells. Four deleted promoter fragments were fused
to the GUS reporter gene with NOS terminator in pUC19 and designated
2252, 1356, 606, and 370. These chimeric constructs and the CaMV
35S-GUS construct as a control were bombarded into 2-d-old AB15-12-1
cultured cells (black bars) and leaves (white bars) harvested from
3-week-old shoot cultures. GUS transient expression was measured using
the MUG assay 3 d after bombardment. The average values after
subtracting the background from four separate experiments with two
replicates are presented. GUS-specific activity was determined as
picomoles of 4-methylumbelliferone per hour per milligram of protein.
|
|
To determine whether the tissue culture-specific expression controlled
by the ASA2 promoter was caused by transcription factor(s) in the
5MTr cell lines, we performed separate
experiments using suspension-cultured cells from
5MTs N. tabacum and from
5MTs and 5MTr N. sylvestris. The transient GUS activities in the N. tabacum 5MTr suspension-cultured cells
(AB15-12-1) controlled by 35S, 606, 1356, and 2252 promoters were
approximately 1.8-, 7.4-, 3.7-, and 3.6-fold that of the
5MTs tobacco suspension-cultured cells,
respectively. In the case of N. sylvestris, 3.4-, 10.6-, 6.4-, and 9.1-fold GUS activities were detected in the
5MTr suspension-cultured cells in comparison with
the 5MTs suspension-cultured cells, respectively.
That the ASA2 promoter shows tissue-specific expression was also shown
by stable transformation experiments with tobacco using Agrobacterium tumefaciens (data not shown).
| |
DISCUSSION |
We have cloned the tobacco ASA2 cDNA encoding the
-subunit of a feedback-insensitive AS and the promoter region.
Southern hybridization analysis shows that more than one closely
related sequence is present in the tobacco genome (Fig. 2). Like all
plant AS genes described to date, the tobacco ASA2 has a
putative transit peptide sequence; therefore, the enzyme is apparently
localized in the plastids.
Our results support the conclusion that ASA2 is a wild-type
gene encoding the -subunit of a feedback-insensitive AS in tobacco. These results include the feedback-insensitive ASA2 gene
expression in the trpE mutant E. coli strain
(Fig. 5), enzyme kinetic analysis (Fig. 6), the nucleotide sequence of
wild-type ASA2 genomic fragments (Fig. 7), and high-level
expression in 5MTr suspension-cultured cells
(Fig. 4).
Several regions of the AS amino acid sequence have been shown to affect
feedback inhibition (Bohlmann et al., 1996; Kreps et al., 1996; Li and
Last, 1996). In tobacco ASA2 from both wild-type and
5MTr cell lines, the two amino acids Phe-107 and
Arg-108, found in the same region as in AS1 (Bohlmann et
al., 1996), were different from those found in feedback-sensitive AS,
Ser-107 and Gln-108. When we changed the ASA2 Phe-107 and
Arg-108 residues to Ser-107 and Gln-108 by site-directed mutagenesis,
the gene product was still active in E. coli, but this
complemented E. coli strain could not grow on 300 µM 5MT containing minimal medium without Trp, whereas the
original ASA2 complemented strain could. In addition, the E. coli-expressed ASA2 gene product was feedback
insensitive like the AS found in 5MTr tobacco
cell extracts. These results indicate that the Phe-107 and Arg-108
residues are important in the control of feedback inhibition. Further
mutagenesis and kinetic and binding studies will be necessary to
determine the effect of other amino acid changes in this region.
The apparent Trp Ki values for
feedback-sensitive AS from tobacco, R. graveolens, and
Arabidopsis are similar, as one might expect considering the high
degree of identity in the regions known to affect feedback inhibition
of bacterial AS, which are also assumed to be important in plant AS
(Caliguri and Bauerle, 1991; Bohlmann et al., 1995). The apparent Trp
Ki values for the tobacco and R. graveolens feedback-insensitive AS enzymes are similar to each
other and are much higher than the Ki value
of the mutant Arabidopsis feedback-insensitive AS. This pattern of inhibition constants correlates with the sequence homologies observed and supports the hypothesis that the substitution of Arg-163 in R. graveolens (corresponding to Arg-108 of tobacco) and
Phe-107-Arg-108 in tobacco AS are related to higher feedback
insensitivity than that found in the Arabidopsis mutant AS when Asp-341
is changed to Asn.
That the apparent Km values for chorismate
for tobacco AS are similar to the values obtained for Arabidopsis and
R. graveolens AS is also not surprising considering the high
degree of similarity, especially in the region known to be involved in
catalysis for bacterial AS (Caliguri and Bauerle, 1991; Bohlmann et
al., 1995). In all three species the apparent
Km for chorismate of the
feedback-insensitive AS is lower than the
Km for feedback-sensitive AS. This suggests a similar mechanism of inhibition and feedback insensitivity. This
would be expected for tobacco and R. graveolens, since a similar sequence difference appears to be involved, but the Arabidopsis mutant contains a different structural change that produces an enzyme
that is still appreciably inhibited by Trp.
The results presented here also indicate that the tobacco ASA2 promoter
regulates tissue-specific gene expression. This conclusion is supported
by measurements of gene expression at the mRNA level (Fig. 4) and by
the deletion-analysis studies of the ASA2 promoter region (Fig. 9). The
high level of ASA2 gene expression was detected at the mRNA
level only in 5MTr suspension-cultured cells,
which is similar to the previous results found at the enzyme activity
level (Brotherton et al., 1986). In addition, the transient GUS
expression was higher in the 5MTr than in the
5MTs suspension-cultured cells and very low-level
expression was found in leaves. This tissue-specific expression was
controlled by the promoter region between 2252 and 607. These
results suggest that there could be changes in the amount or type of
regulatory protein(s) in the 5MTr
suspension-cultured cells that up-regulate the ASA2 gene
expression. Changes in a cis-acting element(s) in the
promoter region can probably be excluded, since we have found only a
few nucleotide differences in the ASA2 promoter region isolated from
three wild-type tobacco cell lines in comparison with ASA2. We also
found similar transient expression patterns with these promoters
compared with that found with the ASA2 promoter from AB15-12-1 (data
not shown).
In Arabidopsis and R. graveolens the ASA1 and
AS1 gene expression is inducible, whereas the
ASA2 and AS2 genes are expressed constitutively. Even though we have denoted our clone ASA2,
since the highest amino acid identity was with R. graveolens
AS2 (72%) and since we have isolated a truncated
ASA1 cDNA similar to the Arabidopsis ASA1 (data
not shown), the tobacco ASA2 is also very similar to
R. graveolens AS1 (70% identity). So far, we have not
been able to induce leaf ASA2 mRNA synthesis by treatment with auxin or salicylic acid (within 8 h) or by wounding (within 48 h). To help understand in vivo ASA2 gene expression in plants and the effect of environmental conditions on gene expression, we have
recently produced tobacco plants transformed with the GUS reporter gene
driven by the 2252-bp ASA2 promoter. The feedback-insensitive R. graveolens AS1 appears to be involved in the pathway
synthesizing anthranilate-derivative alkaloids (Bohlmann et al., 1996).
However, we are not aware of any tobacco anthranilate or indole-derived phytoalexins or alkaloids that have been identified; therefore, the
function of the ASA2 gene in tobacco has yet to be
determined.
In this paper we have reported the cloning of a wildtype tobacco
ASA2 gene encoding the -subunit of a
feedback-insensitive AS. Feedback insensitivity in the
5MTr suspension-cultured cells was correlated
with the high level of the ASA2 gene expression and with the
tissue-specific expression of this gene controlled by defined promoter
regions. The role of the feedback-insensitive AS in tobacco is still
unknown and further studies are needed to understand the regulation and
function of this enzyme in plants.
| |
FOOTNOTES |
1
This research was supported by funds from the
Illinois Council on Food and Agricultural Research and the Illinois
Agricultural Experiment Station.
*
Corresponding author; e-mail widholm{at}uiuc.edu; fax
1-217-333-4777.
Received October 22, 1997;
accepted March 3, 1998.
| |
ABBREVIATIONS |
Abbreviations:
AS, anthranilate synthase.
CaMV, cauliflower
mosaic virus.
IPTG, isopropylthiogalactoside.
MS, Murashige-Skoog.
5MT, 5-methyl-Trp.
5MTr, 5-methyl-Trp-resistant.
5MTs, 5-methyl-Trp-sensitive.
MUG, 4-methylumbelliferone
glucuronide.
RACE, rapid amplification of cDNA ends.
UTR, untranslated
region.
| |
ACKNOWLEDGMENTS |
We thank Krishna Niyogi for cDNA clones, Mani Subramanian for
the Arabidopsis ASA1-expressing E. coli line, Charles
Yanofsky and the Escherichia coli Genetic Stock Center for
the E. coli mutant strains, Rob Alleman for technical
assistance with AS kinetic analysis, and Liugen Zhu for technical
assistance with the MUG assays.
| |
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Abstract
Introduction
