From the Center for Agricultural Biotechnology and the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742
Received for publication, June 14, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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As-1-type
cis-elements augment transcription of both nuclear and
pathogen genes in response to stress and defense cues in plants.
Basic/leucine zipper proteins termed "TGA factors" that specifically bind as-1 elements are likely candidates for
mediating these transcription activities. Our earlier work has shown
that 2,4-dichlorophenoxyacetic acid-induced xenobiotic stress enhances trans-activation by a chimeric fusion protein of the yeast
Gal4 binding domain and TGA1a, a TGA factor of tobacco. Here we
demonstrate that xenobiotic stress also enhances the ability of native
TGA1a to bind as-1 and activate transcription of a known
target gene. In addition, the previously identified xenobiotic
stress-responsive domain of TGA1a was found to inhibit this factor's
trans-activation potential by a mechanism that appears to
involve stimulus-reversible interactions with a nuclear repressor
protein. Results from these and other studies can now be placed in the
context of a working model to explain basal and xenobiotic
stress-induced activities of TGA1a through its cognate
cis-acting element.
As-1-type elements contribute to the expression of both
pathogen and nuclear genes in plants (1-9). When inserted upstream of
a minimal promoter and A number of genes have been cloned that encode for
as-1-binding proteins, both in the same and different plant
species. These "TGA factors" share considerable homology and belong
to the basic/leucine zipper
(bZIP)1 family of
transcription factors. Efforts to understand the contributions of TGA
factors to as-1-dependent transcription will
require knowledge of their cellular distribution and molecular
properties. To this end, we initially chose to study TGA1a because
close homologues of this tobacco TGA factor exist in other plants,
suggesting a conserved and perhaps important biological role. In prior
studies, we found that a chimeric protein comprised of the yeast Gal4
binding domain and TGA1a could potentiate transcription through the
GAL4 cis-element in response to xenobiotic stress. These
data implicate TGA1a in the expression of plant genes involved in
chemical defense (12). Consistent with this notion,
TGA1a transcripts and protein are preferentially
coexpressed in root meristem cells with transcripts of
as-1-regulated genes (e.g. GNT35) (11)
that encode for type III glutathione S-transferase (GST)
isoenzymes. Although the biological function of these
as-1-regulated GSTs is unknown, this class of enzymes has
been specifically implicated in xenobiotic detoxification and
resistance in plants (13-15).
Gain of function assays with the yeast Gal4 binding domain led to the
identification of specific TGA1a domains involved in basal and
xenobiotic stress-activated transcription (12). By this assay, it was
shown that the amino-terminal (NT) domain (residues 1-86) of TGA1a
confers constitutive trans-activation, whereas the
carboxyl-terminal (CT) domain (residues 142-373) of this factor largely enhances transcription in response to xenobiotic stress. Here
we show that xenobiotic stress rapidly and transiently affects the
ability of native TGA1a to bind its cognate as-1 element and to activate transcription through a mechanism involving
stimulus-reversible repression and this factor's regulatory CT domain.
Additional evidence suggests that this regulatory mechanism is likely
to occur through stimulus-reversible interactions of the CT domain with
a putative corepressor protein.
Tobacco Suspension Cell Cultures
Cultures (100 ml) of tobacco BY-2 suspension cells were grown
and maintained in flasks at 28 °C in the dark as described
previously (12). For the preparation of labeled nuclear proteins, cells were cultured for 4 days and adjusted to a packed volume of 50%, and 4 ml of the cell culture were transferred to each of 5 wells in an 8-well
culture plate (Costar). After 24 h, 200 µCi of
[35S]methionine (6000 Ci/mmol; PerkinElmer Life
Sciences) were added to each sample, which was then incubated
for an additional 20 h. Where indicated, cells were exposed to
xenobiotic stress induced by treatment for 0-8 h with 100 µM 2,4-dichlorophenoxyacetic acid (2, 4-D) in 0.1%
ethanol carrier, collected by vacuum filtration, frozen in liquid
nitrogen, and stored at Effector and Reporter Gene Constructs
Reporter gene constructs and the effector construct of
FLAG epitope-tagged TGA1a were prepared as described previously
(12). TGA1a For each of the TGA1a mutants, the carboxyl-terminal primer used
to make the final construct contained a TAA stop codon in-frame after
the last TGA1a residue. All PCR reactions were run as described, and
the resultant products were treated with proteinase K, digested with
EcoRI and ClaI, agarose gel-purified, and ligated
into cognate restriction sites in KS-FLAG vector (12). The TGA1a
constructs were sequenced and tested for their ability to express the
expected FLAG epitope-tagged protein using a coupled in
vitro transcription/translation system (Promega). The FLAG-tagged
cDNA was excised with BamHI and ClaI,
blunt-ended with Klenow enzyme and deoxynucleotide triphosphates, and
subcloned into the plant expression vector pMON999 as described previously (12).
Protoplast Transient Transfection Assay
Protoplasts were prepared from BY-2 suspension culture cells and
transfected with the indicated plasmid reporter and effector DNAs as
described previously (12). Transfected protoplasts were collected by
brief centrifugation and then treated with lysis buffer according to
the manufacturer's instructions (Promega). Lysates were assayed for
reporter gene activity by luciferase or chloramphenicol acetyl
transferase (CAT) enzyme assays (12, 16). To correct for differences in
transfection efficiency, reporter gene activity of -90-CAT was
normalized to that of CHS-LUC, a luciferase reporter gene that is
transcribed from a bean chalcone synthase promoter (17). Data shown are
the mean and standard error from three or more independent experiments.
Nuclear Run-on Assay
Six days after transfer into fresh medium, BY-2 cells were
incubated with 100 µM 2,4-D in 0.1% ethanol carrier for
0-8 h, collected by vacuum filtration, frozen in liquid nitrogen, and stored at Preparation of Nuclear Extracts
BY-2 cells were ground with a mortar and pestle to a fine powder
under liquid nitrogen. Nuclear proteins were extracted from this
material as described previously (7), followed by dialysis against two
changes of 500 ml of dialysis buffer (20 mM HEPES, pH 7.9, 25 mM NaCl, 1 mM EDTA, 20% (v/v) glycerol, and
1 mM dithiothreitol). Dialyzed extracts were clarified at
5,000 × g for 5 min, divided into aliquots, frozen in
liquid nitrogen, and stored at Preparation of Recombinant TGA1a
Recombinant TGA1a was synthesized using a coupled TnT/T7
transcription/translation system (Promega) with pBluescript KS vector containing full-length cDNA of TGA1a as template (12).
DNA-affinity Chromatography
Preparation of Labeled Nuclear Extracts--
BY-2 cells cultured
for 6 days were incubated with 200 µCi/ml
[35S]methionine (6000 Ci/mmol; PerkinElmer Life Sciences)
for 24 h and then treated for 1 h with either 100 µM 2,4-D in 0.1% ethanol carrier (xenobiotic stress) or
0.1% ethanol carrier alone (mock). Labeled nuclear protein extracts
were prepared as described above.
Preparation of DNA-affinity Resins--
Oligonucleotide
concatamers of wild-type or mutant as-1 sequences were
coupled to cyanogen bromide-activated Sepharose according to the
manufacturer's instructions (Amersham Pharmacia Biotech). The
coupling efficiency of these oligonucleotides to Sepharose was ~4.5
µg DNA/ml resin. For each binding reaction, 125 ng of immobilized DNA
were used. Immobilized DNA was equilibrated in binding buffer (20 mM HEPES, pH 7.9, 100 mM NaCl, 0.2 mM EDTA, 25 µg/ml poly(dI·dC), 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol, and 20% (v/v) glycerol) for 15 min on ice.
Assay Conditions--
Wild-type or mutant immobilized DNAs were
then added to nuclear extracts (1.0 × 106 cpm
incorporated [35S]methionine) from BY-2 cells treated for
1 h with either 100 µM 2,4-D in 0.1% ethanol
carrier (xenobiotic stress) or 0.1% ethanol carrier alone (mock).
After incubation on ice for 1 h, immobilized DNA-protein complexes
were washed repeatedly with binding buffer. Bound proteins were then
eluted with RIPA (21) and analyzed for the presence of TGA1a by immunoprecipitation.
Immunoprecipitation
Protein extracts were clarified by brief centrifugation at
14,000 × g and incubated on ice for 1 h with 5 µl of either preimmune sera or immune sera prepared against NT
residues 57-72 of TGA1a (11). Immune complexes were recovered with 10 µl of Gammabind Plus Sepharose (Amersham Pharmacia Biotech) in RIPA
buffer solution and 2% bovine serum albumin (Sigma) during gentle
mixing for 30 min on ice. Bound protein was resuspended in 25 µl of
Laemmli loading buffer, denatured by boiling, fractionated by SDS-PAGE on a 10% running gel, and detected by fluorography with
En3hance according to the manufacturer's instructions
(PerkinElmer Life Sciences).
GST Binding Assay
GST Fusion Proteins--
PCR amplification of TGA1a and its
derivatives was done using the following sets of primers and KS-TGA1a
as template (12): (a) for TGA1a-CT,
5'-ACTTGGGAATTCTCTTCAACGTACACCCAATTT-3' and 5'-TTTATCGATGTCAGGTAGGCTCACGT AGACG-3'; and (b) for
TGA1a-NT, 5'-TTTGAATTCTCTCGTCGTGCATCTGTTAATTCTTCAACGTAC-3' and
5'-TTTATCGATGTCATTCATATCTGTTAGAAGT-3'. The PCR products were
gel-purified, digested with EcoRI and ClaI, and
subcloned into pBluescript KS+. After amplification in the
XL-1 blue strain of Escherichia coli, plasmid DNA was
isolated and digested with EcoRI and XhoI.
Inserts were sequenced to confirm their identity and subcloned into
pGEX-4T1 (Amersham Pharmacia Biotech). GST fusion proteins were
expressed and isolated from an E. coli BL-21 strain
according to the manufacturer's instructions (Amersham
Pharmacia Biotech).
GST Binding Conditions--
Reactions (40 µl) contained
Binding buffer (20 mM HEPES, pH 7.9, 25 mM
NaCl, 1 mM EDTA, 20% (v/v) glycerol, 0.1 mM
phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol),
2 × 108 cpm of labeled nuclear protein extract, and
10 µg of GST fusion protein on
glutathione-Sepharose resin (Pharmacia). After gentle
mixing for 1 h, the Sepharose resins were recovered by brief
centrifugation at 2000 g and repeatedly washed with Binding Buffer. Bound proteins were eluted with Laemmli loading buffer and
fractionated by SDS-PAGE. Staining with Coomassie blue was used to
detect GST proteins. Labeled proteins in gels were detected by
fluorography using En3hance according to the
manufacturer's instructions (NEN), with an intensifying screen
at-80 °C and 3-7 days exposure to x-ray film (Kodak).
Gel-shift Binding Assay
To compare as-1 binding activities of ASF-1 from
mock- and 2,4-D-treated cells, a double-stranded as-1
oligonucleotide (5'-ATCTCCACTGACGTAAGGGATGACGCACAATCCCACAAA-3') was
end-labeled with [ As-1-dependent Activation by TGA1a Is Repressed through
Its CT Domain--
Our previous work using chimeric Gal4-binding
proteins led to the suggestion that the CT domain of TGA1a might
function as a stimulus-reversible repressor of transcription. To
further test this notion, we examined here whether removal of the
regulatory CT domain from TGA1a enhanced its
as-1-dependent transcriptional activity.
Effector constructs of wild-type and mutant forms of TGA1a were
"epitope-tagged" with the octapeptide FLAG epitope at their amino
termini (Fig. 1) to facilitate
immunological monitoring of steady-state amounts of these proteins.
Gel-shift binding assays revealed that a mutant TGA1a factor, termed
TGA1a
Having ascertained that the modified TGA1a factors were capable of
binding as-1, we next examined their effect on
as-1-dependent transcription. We found that
TGA1a-CREB
The results above, however, might have arisen from differences in the
steady-state amounts of these TGA1a proteins in transfected protoplasts. To test this possibility, we incubated transfected protoplasts with [35S]methionine and prepared nuclear
extracts for immunoprecipitation analysis. Using this approach, we
found that steady-state amounts of these TGA1a factors were generally
similar among transfected protoplasts (Fig.
4). Thus, differences in the activities
of these factors were not likely due to their relative abundance.
We were also interested in determining whether accessory proteins with
potential regulatory activity interact with TGA1a because we previously
observed that a 120-kDa protein was bound to transiently expressed
TGA1a.2 Immunoprecipitation
assays here confirmed this observation (Fig. 4, lane 2). We
also noted that TGA1a-CREB The CT Domain of TGA1a Is Necessary and Sufficient for
Stimulus-responsive Recruitment of a 120-kDa Nuclear
Protein--
Results here indicate that the CT domain of TGA1a
inhibits trans-activation under basal conditions. This is
consistent with our earlier work showing that this domain confers
transcription in response to xenobiotic stress by a mechanism involving
stimulus-induced de-repression (12). We thus examined here whether this
regulatory domain is necessary and sufficient for recruiting the
TGA1a-binding protein. In this experiment, radiolabeled nuclear
proteins were incubated with GST fusion proteins of NT or CT domain
polypeptides of TGA1a, washed to remove unbound material, fractionated
by SDS-PAGE, and detected with fluorography (Fig.
5a). Due to its high degree of
insolubility, recombinant GST-TGA1a was not tested with this assay.
Comparable amounts of each GST fusion protein were programmed in the
binding reactions, as evidenced by staining of the SDS-PAGE gel with
Coomassie Blue (Fig. 5b). Results show that GST-CT, but not
GST or GST-NT, bound a 120-kDa protein from nuclear extracts of
mock-treated cells. This interaction was not detected with extracts
from cells that had been treated for 0.5 h with 2,4-D, suggesting
that xenobiotic stress affects recruitment by TGA1a of a 120-kDa
nuclear protein.
Binding by TGA1a to a 120-kDa Protein Is Inversely Correlated with
Stimulus-induced Changes in the Rate of as-1-dependent
Transcription--
The results above showed that recruitment of a
120-kDa nuclear protein in vitro by recombinant CT domain
protein was responsive to xenobiotic stress. Here we examined whether
changes in TGA1a activity, and in its association with the 120-kDa
protein, are correlated. Because time-course studies are difficult to
perform with transient transfection systems, we used an alternative
approach to address this question. Nuclear extracts were prepared from [35S]methionine-labeled suspension cells that had been
treated for 0-8 h with 100 µM 2,4-D. TGA1a in these
extracts was recovered by immunoprecipitation with anti-TGA1a peptide
(residues 57-72) antibodies that are monospecific for TGA1a (11). We
note that although the calculated mass of TGA1a is 41 kDa, plant
transiently expressed and recombinant forms of this factor appear to
migrate as 46- and 47-kDa proteins, respectively, on SDS-PAGE gels
(12). This small difference in mobility is thought to arise from
differences in the posttranslational state of these factors. As
expected, control reactions with preimmune sera did not bind either
recombinant TGA1a (data not shown) or a nuclear protein with an
apparent mass of 46 kDa, which is similar to that of TGA1a (Fig.
6). In contrast, immune sera bound
recombinant TGA1a (data not shown; Ref. 11) and a 46-kDa nuclear
protein that migrated slightly faster than recombinant TGA1a by
SDS-PAGE electrophoresis. Immunoprecipitation of the 46-kDa protein was
blocked (data not shown) by addition of the TGA1a peptide epitope.
Immunospecificity of anti-TGA1a antibodies was also confirmed using a
similar NT peptide from PG13, a tobacco TGA factor that shares a strong
(>85%) overall identity with TGA1a. Although the PG13 peptide
has 11 of 15 residues identical to those of the TGA1a peptide
epitope, the PG13 peptide had no effect on immunoprecipitation of
TGA1a. These results indicate that the 46-kDa protein in nuclear
extracts is identical in both apparent mass and antigenic properties to
TGA1a.
The relative amount of TGA1a that was recovered by immunoprecipitation
from extracts of cells treated for 0-8 h with 2,4-D varied little
between samples, consistent with the results of earlier studies of
TGA1a in planta (11). Also, a 120-kDa nuclear protein was
recovered with TGA1a in nuclear extracts of mock-treated (i.e. 0 h) cells. This association was not observed in
extracts obtained from cells treated for 0.5-2 h with 2,4-D. Longer
treatments with 2,4-D of 4 and 8 h, respectively, led to a partial
or complete restoration of the association between TGA1a and the
120-kDa protein. Other proteins with molecular masses that were
slightly smaller or larger than that of the 120-kDa protein might
reflect either its posttranslational modification or proteolysis or
might simply reflect the presence of unrelated proteins.
We next examined whether this transient association between TGA1a and
the 120-kDa protein correlates with changes in the rate of
as-1-dependent transcription (Fig.
7). To this end, we used a nuclear run-on
assay to monitor the rate of accumulation of de novo
transcripts from GNT35, a tobacco GST gene whose expression is both enhanced by 2,4-D through as-1 and preferentially
localized in planta with that of TGA1a (5, 8, 11). Tobacco
cells treated with 2,4-D showed a strong increase in GNT35
transcription by 0.5 h, with maximal activity occurring by 2 h. Longer treatments with 2,4-D resulted in a rapid decline in the rate
of GNT35 transcription. Because expression of
TGA1a has been found to be unaffected by xenobiotic stress
(11), de novo transcription from this gene served as the
internal control. We observed that the rate of transcription of
TGA1a was initially similar to that of GNT35 and
unaffected by 2,4-D. Collectively, these findings indicate that changes
in the rate of transcription of GNT35 were inversely
correlated with the degree of association between TGA1a and a 120-kDa
nuclear protein.
Stimulus-induced Changes in the as-1 Binding Activity of
TGA1a--
One means by which xenobiotic stress might affect the
activity of TGA1a is by inhibiting its ability to bind as-1.
However, gel-shift experiments with nuclear extracts from mock- or
2,4-D-treated cells showed comparable amounts of a nuclear
as-1 binding activity, termed "ASF-1" (data not shown).
ASF-1 from these suspension cells is derived from the contributions of
a number of different TGA factors (e.g. TGA2.1 and TGA2.2)
that are more abundant than TGA1a (23). To therefore determine the
specific contribution of the comparatively small amount of TGA1a to
this activity, we used a sequential enrichment procedure involving
DNA-affinity chromatography and anti-TGA1a immunoprecipitation (Fig.
8a). Results indicate that
TGA1a from mock-treated cells failed to bind immobilized as-1 DNA (Fig. 8b, lane 4), unlike the activity
of TGA1a from 2,4-D-treated cells (Fig. 8b, lane
10). Based on results with a mutant as-1 sequence,
TGA1a binding was specific for as-1 (Fig. 8b,
lanes 6 and 12). As expected, control reactions
with preimmune sera did not recover detectable amounts of TGA1a from
either of these extracts.
Interestingly, crude input fractions from mock-treated cells revealed
the presence of a 120-kDa nuclear protein bound to TGA1a (Fig.
8b, lane 2), whereas TGA1a from the input nuclear
fraction from 2,4-D-treated cells was not associated with this protein (Fig. 8b, lane 8). Based on the relative amounts
of TGA1a in lanes 8 and 10 of Fig. 8b,
we estimate that nearly all of the TGA1a present in the input fraction
was recovered using as-1 DNA-affinity chromatography. The
above-mentioned findings contrast sharply with the apparent inability
of the TGA1a-120-kDa protein complex to bind as-1 (Fig.
8b, lane 4).
Plant transcription involving as-1-type
cis-elements and their cognate TGA factors is activated by
diverse cues including defense hormones, wounding, and xenobiotic
stress. Of the known TGA factors, tobacco TGA1a is among the best
characterized with regard to its contribution to transcription. TGA1a
promotes as-1-dependent transcription in
vitro (24) and as a transiently transfected chimeric factor in
response to xenobiotic stress (12). In tobacco seedlings, TGA1a is
preferentially coexpressed in root tip meristem cells along with
several as-1-regulated genes, including GNT35, whose activities are further enhanced by xenobiotic stress (11). Gain
of function studies with a heterologous system involving the yeast
Gal4-binding protein indicate that the CT domain of TGA1a confers
transcription through the GAL4 cis-element in response to
xenobiotic stress through a mechanism involving a change in trans-activation potential (12). Evidence here significantly extends our understanding of how TGA1a is regulated by showing that the
CT domain and xenobiotic stress play additional regulatory roles in
TGA1a activity.
Transient transfection assays with TGA1a-CREB During these studies, we observed that a 120-kDa protein bound TGA1a
and that this interaction is responsive to xenobiotic stress. Evidence
from several lines of investigation suggests that this TGA1a-binding
protein may be a putative corepressor. First, its association with
TGA1a was transiently affected by xenobiotic stress to a degree that
was inversely correlated with the rate of expression of a TGA1a target
gene, suggesting an inhibitory function. Second, binding by this
120-kDa protein to TGA1a specifically involved the regulatory CT domain
of this factor, which mediates transcription in a heterologous system
in response to xenobiotic stress. Deleting this domain from TGA1a
abolished both its association with the 120-kDa protein and its ability
to repress basal transcription activity, thus providing support for the
notion that this TGA1a-binding protein has potential inhibitory activity.
In addition to the TGA1a-binding protein identified here, several other
plant proteins have been shown to bind TGA factors. A ~26-kDa
Arabidopsis protein termed OBP1 associates with a subset of
Arabidopsis TGA factors to facilitate their as-1
binding activity. OBP1 itself is a DNA-binding factor and binds to AAGG
motifs in as-1-regulated promoters (25, 26). A second
Arabidopsis protein of ~66 kDa termed NPR1 (or NIM1) is a
positive regulator of the as-1-regulated PR1 gene
in response to pathogens and plant defense cues, such as salicylic
acid. Like OBP1, the NPR1 protein binds to a subset of TGA factors
(27-29). Mutations in NPR1 that disrupt this binding activity also
impair salicylic acid-induced expression of PR1, thus
linking NPR1 activity to as-1. Intriguingly, Zhang et
al. (27) have suggested a model whereby NPR1 potentiates transcription by reversing the binding of a repressor to one or more
TGA factors. The apparent mass of the 120-kDa protein and its proposed
mechanism of action appear to distinguish it from either OBP1 or NPR1.
Results from this study indicate that TGA1a belongs to a subset of bZIP
transcription factors whose activities are regulated through a
mechanism involving stimulus-reversible repression. In the bZIP factor
ATF-2, intramolecular binding under basal conditions occurs between the
activation and bZIP domains and inhibits this factor's DNA binding and
trans-activation potential (30). In vivo, these
inhibitory interactions in ATF-2 are alleviated by the coactivator
CBP (31). A different type of regulatory mechanism occurs with
CCAAT/enhancer-binding protein, a bZIP factor that negatively regulates
its trans-activation potential via intramolecular interactions between activation and repressor domains through a
cellular repressor protein (32). Transcription factors of the estrogen
hormone receptor class represent yet another type of repression (33).
When bound in the nucleus by the 90-kDa heat shock protein (hsp90), the
estrogen receptor is unable to bind its cognate cis-element.
Estrogens reverse this effect by inducing a conformational change in
the receptor, thus promoting its release of hsp90 and subsequent
binding to DNA.
Thus, how might xenobiotic stress affect the activity of TGA1a? One
likely mechanism suggested here involves stimulus-induced changes in
as-1 binding by TGA1a, perhaps through its
stimulus-reversible association with a putative corepressor protein.
Despite the fact that the 120-kDa protein bound to TGA1a in nuclear
extracts from mock-treated cells, we were unable to detect this complex
by gel-shift binding assay, suggesting that this complex may not bind
DNA. This view is directly supported by evidence from an alternative TGA1a detection assay involving sequential DNA-affinity chromatography and immunoprecipitation. By this approach, xenobiotic stress was found
to potentiate the as-1-binding activity of TGA1a, presumably by inducing a change in this factor's association with a 120-kDa protein. However, knowledge of the proportion of cellular TGA1a bound
by this protein will be necessary before definitive conclusions regarding the regulatory contribution of this interaction to
as-1 binding by TGA1a can be made.
Based on present and previous findings, we propose the following
working model to explain how TGA1a affects
as-1-dependent transcription. In the absence of
xenobiotic stress, as-1 binding and
trans-activation functions of TGA1a are inhibited. TGA1a's loss of basal transcription activity occurs through this factor's inhibitory CT domain, by a mechanism that may involve its
stimulus-reversible interaction with a putative corepressor protein. In
response to xenobiotic stress, TGA1a trans-activation and
as-1 binding activities are enhanced. These changes occur in
parallel with the release of the putative corepressor protein. Longer
exposure to xenobiotic stress promotes reassociation of TGA1a with this
protein and a concomitant decline in
as-1-dependent transcription.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucuronidase reporter gene, these elements drive
-glucuronidase expression predominantly in primary and lateral root tips of tobacco and Arabidopsis seedlings,
and in the root and shoot vascular system of older plants (2, 10, 11).
This transcription activity is further augmented in response to plant
defense hormones (e.g. salicylic and jasmonic acids), wounding, and xenobiotic stress, thus indicating that diverse stimuli
affect the activity of one or more cognate transcription factors
through these elements.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
CT, which lacks CT residues 142-373 of TGA1a, was
synthesized through PCR using TGA1a as template and the following set
of primers: 5'-ACTTGGGAATTCTCTTCAACGTACACCCAATTT-3' and
5'-AAAATCGATTCAGCGTTCTAGTTCTTG-3', corresponding to residues 1-142.
TGA1a-CREB
CT was made by separately PCR-amplifying residues 1-142
of TGA1a, as described above, and two leucine zipper repeats (residues
311-315) of CREB using the primers
5'-AAGGCCTATGTTCAGCAGTTAGAGAACAGAGTG-3' and
5'AAAATCGATTCAGTCCTTAAGTGCTTT-3' with Rous sarcoma virus CREB as
template. Full-length PCR products were gel-purified, combined, and
amplified with primers corresponding to the 5' and 3' termini of TGA1a
and CREB leucine zipper sequences, respectively, to yield a single
intact translational fusion product. TGA1a-CREB also required a more
complex subcloning strategy involving separate and sequential PCR
amplifications. TGA1a-CREB
CT was used as template with the following
primers to generate fragment 1:
5'-ACTTGGGAATTCTCTTCAACGTACACCCAATTT-3' and
5'-GTCCTTAAGTGCTTTTAGCTCCTCAATCAATG-3'. TGA1a was used as template with
the following primers to generate fragment 2:
5'-ACATTGATTGAGGAGCTAAAAGCACTTAAGGACGTAGATGC TAGCCAGCTA-3' and
5'-TTTATCGATGTCAGGTAGGCTCACGTAGACG-3'. Both DNA fragments were agarose
gel-purified and combined in equal amounts to serve as template for PCR
reactions with the following primers:
5'-ACTTGGGAATTCTCTTCAACGTACACCCAATTT-3' and
5'-TTTATCGATGTCAGGTAGGCTCACGTAGACG-3'.
80 °C. Nuclei were isolated essentially as described by
Dröge-Laser et al. (18); nascent transcripts from
these nuclei were radiolabeled as described by Lawton and Lamb (19) and
isolated according to the work of McKnight and Palmiter (20). Labeled
transcripts (~4 × 109 cpm) were hybridized as
described by Lawton and Lamb (19) against Nytran-immobilized
full-length cDNAs (200 ng) of the tobacco GNT35 and
TGA1a genes. Radioactivity associated with hybridized
transcripts was quantified with a PhosphorImager (Molecular Dynamics).
80 °C.
-32P]ATP (6000 Ci/mmol; Amersham
Pharmacia Biotech) using polynucleotide kinase. The specific activity
of the labeled oligonucleotide was ~1 × 108
cpm/µg. This oligonucleotide was subsequently annealed with its unlabeled complement (5'-TTTGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGAT-3'). Binding reactions (25 µl) containing 20 mM HEPES, pH 7, 40 mM KCl, 1 mM EDTA, 2 mM
MgCl2, 1 µg of bovine serum albumin, 20% (v/v) glycerol,
1 µg of poly(dI·dC), and 500 ng of nuclear protein were
incubated on ice for 15 min, and then 1 ng of the radiolabeled double-stranded probe was added, and incubation was continued for 15 min. To identify as-1-bound complexes, samples were
fractionated on 4% native polyacrylamide gels in 0.5× Tris-borate
EDTA and analyzed by autoradiography as described previously (22). DNA binding competition assays were done with 200 ng of unlabeled as-1 wild-type (see above) or mutant as-1
double-stranded oligonucleotides (5'-ATCTCCACTGCTGTAAGGGATGCTGCACAATCCCACAAA-3', the sense strand is shown, and mutated bases that differ from those of as-1 are underlined).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CT, which lacks the entire CT domain (residues 142-373), was
unable to bind as-1 (Fig. 2).
This loss of function was due to the fact that CT residues appear to be
essential for promoting formation of the DNA-binding dimer of TGA1a, as
suggested by Katagiri et al. (22). Because the leucine
zipper of TGA1a alone was unable to efficiently confer dimer formation,
we pursued an alternative strategy to distinguish between dimer
stabilization and repressor functions of the CT domain. This strategy
involved exchanging the three heptad repeats that comprise the leucine
zipper region of TGA1a
CT for three heptad repeats from the leucine
zipper of the mammalian bZIP factor CREB. The resultant chimeric
protein, TGA1a-CREB
CT, bound as-1 as well as wild-type
TGA1a (Fig. 2, lane 3). As a control, we also tested whether
the same leucine zipper exchange made in full-length TGA1a affected
as-1-dependent binding by this factor. Results of gel-shift binding assays indicate that this protein, TGA1a-CREB, bound as-1 to a degree that was equal to or greater than
that of TGA1a (Fig. 2, lane 4). These data imply that the
inability of the leucine zipper of TGA1a to efficiently promote
as-1 binding is due to its weaker dimerization potential
compared with that of a similar length region of the CREB zipper.
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Fig. 1.
TGA1a effector and reporter gene
constructs. Structural features of TGA1a: NT domain (residues
1-86), bZIP domain (residues 87-141), and CT domain (residues
142-373). The position of the three heptad repeats that comprise the
leucine zipper is indicated by the solid letter L. Effector constructs: cDNAs encoding for wild-type and
modified TGA1a proteins fused downstream of the octapeptide FLAG
epitope (Kodak) were subcloned into the pBluescript KS+
vector for sequencing and for in vitro protein synthesis. In
both TGA1a-CREB CT and TGA1a-CREB, the three heptad repeats that
comprise the leucine zipper of TGA1a were exchanged for three heptad
repeats from the leucine zipper of CREB (indicated by the open
letter L). For plant protoplast expression studies, the cDNAs
were excised from the pBluescript vector and inserted as shown between
the CaMV 35S promoter and nos 3' polyadenylation/termination
sequences in the plant expression vector pMON999. Reporter
constructs: a single copy of as-1 (indicated by
two tandem TGAC motifs) was placed just upstream of the TATA
box and the start site of the CaMV 35S minimal promoter. This promoter
fragment was then ligated upstream of the CAT reporter gene. A DNA
fragment of sequences
326 to +180 bp of the bean chalcone synthase
promoter was ligated upstream of the LUC reporter gene
(CHS-LUC) and served as an internal control for transfection
efficiency.
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Fig. 2.
As-1-binding by wild-type and modified TGA1a
factors. a, fluorographic image of a SDS-PAGE gel
showing in vitro synthesized,
[35S]methionine-labeled TGA1a factors. Arrows
indicate the expected position of the full-length proteins.
b, gel-shift mobility assays were performed with
32P-labeled as-1 oligonucleotide as probe and 2 µl each of unlabeled reactions corresponding to those indicated
immediately above each lane in a. P,
probe only. DNA-protein complexes were detected by
autoradiography.
CT, which lacks the regulatory CT domain, significantly
enhanced as-1-dependent transcription in the
absence of xenobiotic stress, i.e. under basal conditions (Fig. 3). In contrast, neither TGA1a nor
TGA1a-CREB had significant effects on basal transcription, even when
increasing amounts of their respective effector genes were tested (data
not shown). TGA1a
CT, which lacks the ability to bind
as-1, as expected, had little or no effect on transcription
through this element.
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Fig. 3.
Effect of wild-type and modified TGA1a
factors on as-1-dependent
transcription. Tobacco protoplasts were transfected as described
with pMON999 vector alone (mock) or with pMON999 effector DNAs encoding
the indicated TGA1a factors. In addition to the
as-1-regulated 90-CAT reporter gene, the CHS-LUC reporter
gene was included in all transfection experiments as an internal
control. After 20 h of incubation, homogenates of the protoplasts
were assayed for CAT and luciferase activities. CAT activity was
expressed as the percentage of conversion of nonacetylated to
acetylated forms of [14C]chloramphenicol and then
normalized to luciferase values. Values shown are the mean and S.E.,
expressed as the normalized percentage of conversion of chloramphenicol
substrate.
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Fig. 4.
Association of wild-type and modified TGA1a
factors with a 120-kDa protein. Tobacco protoplasts were
transfected with pMON999 vector alone (mock) or with pMON999 effector
DNAs encoding the indicated TGA1a factors and incubated overnight with
[35S]methionine to label de novo proteins.
Nuclei were isolated, and protein was extracted with RIPA buffer. The
FLAG-tagged TGA1a factors were recovered from these extracts by
immunoprecipitation with anti-FLAG monoclonal antibody, fractionated by
SDS-PAGE, and detected by fluorography. The arrowhead
indicates the position of the 120-kDa protein. Apparent molecular
masses of prestained protein markers are shown in kDa.
CT, which lacks the regulatory CT domain
of TGA1a, was not associated with the 120-kDa protein. This loss of
activity was not due to the presence of the CREB leucine zipper in
TGA1a-CREB
CT because the TGA1a-CREB control factor, which contains
the same zipper substitution, bound the 120-kDa protein as well as
TGA1a. These observations prompted us to test whether the CT domain
alone can recruit this protein.
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Fig. 5.
Stimulus-reversible binding by a 120-kDa
protein to the regulatory CT domain of TGA1a. a,
nuclear protein extracts from [35S]methionine-labeled
tobacco suspension cells were incubated on ice with recombinant GST
fusion proteins of NT and CT domains of TGA1a or with GST alone as a
control. GST proteins were recovered on glutathione-Sepharose,
washed to remove unbound material, fractionated by SDS-PAGE, and
analyzed for the presence of labeled nuclear proteins by fluorography.
Extracts were prepared from cells that had been treated for 30 min with
either 0.1% ethanol carrier solvent ( ) or 100 µM 2,4-D
(+). The arrowhead indicates the expected position of the
120-kDa protein. b, the same gel stained with Coomassie Blue
to detect GST proteins.
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Fig. 6.
Xenobiotic stress induces transient changes
in the association of a 120-kDa protein with TGA1a. Preimmune
(lanes 1-5) and anti-TGA1a immune (lanes 7-11)
sera were incubated with nuclear extracts from
[35S]methionine-labeled tobacco suspension cells, and
protein-antibody complexes were subsequently recovered with Gammabind
(protein G) Plus Sepharose resin, fractionated by SDS-PAGE, and
visualized with fluorography. Nuclear extracts used were from cells
treated with 100 µM 2,4-D for 0-8 h, as indicated.
Lane T is [35S]methionine-labeled recombinant
TGA1a synthesized in vitro. The open arrowhead
indicates the 120-kDa protein, and the solid arrowhead
indicates endogenous TGA1a. The apparent molecular masses of prestained
protein markers are shown in kDa.
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Fig. 7.
Xenobiotic stress induces transient changes
in the rate of transcription of an as-1-regulated
target gene of TGA1a. Results of nuclear run-on assays of de
novo transcription from GNT35 and TGA1a
genes are shown. Tobacco suspension cells were treated with 100 µM 2,4-D for 0, 0.5, 2, 4, and 8 h to induce
xenobiotic stress, and nuclei were then isolated and used to generate
labeled nascent transcripts as hybridization probes. The amount of
labeled RNA (in cpm) that hybridized to Nytran-bound GNT35
and TGA1a cDNAs is shown. Due to its constitutive and
xenobiotic stress-insensitive expression, transcription of
TGA1a served as an internal control for differences in RNA
recovery and labeling between samples.
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Fig. 8.
Changes in as-1 binding
activity of nuclear TGA1a and its association with a 120-kDa
protein. a, flow diagram of the experiment.
b, labeled nuclear proteins from tobacco suspension cells
treated with 0.1% ethanol carrier solvent (Mock) or 100 µM 2,4-D (Xenobiotic stress) were analyzed
directly as described below (input) or were incubated with
wild-type (as-1) or mutant (mtas-1) immobilized
oligonucleotides, washed, and then extracted from the DNA with
radioimmune precipitation buffer (eluate). Input and eluate protein
fractions were incubated with preimmune (P) or anti-TGA1a
immune (I) sera, collected on Gammabind (protein G) Plus
Sepharose resin, fractionated by SDS-PAGE, and detected by
fluorography. The solid arrowhead indicates endogenous
TGA1a, and the open arrowhead indicates the expected
position of the 120-kDa protein. The apparent molecular masses of
prestained protein markers are shown in kDa.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
CT have demonstrated
here an inhibitory role for the CT domain in TGA1a
trans-activity. The presence of the leucine zipper of CREB
in TGA1a-CREB
CT promoted efficient dimerization and DNA binding in
the absence of a "dimer stabilization" function conferred by the
missing CT domain (22). This modification allowed us to identify
separate contributions made by dimer stabilization and repression
activities of the CT domain to as-1-dependent
transcription. Enhanced trans-activity by TGA1a-CREB
CT
was shown to be largely due to the absence of the inhibitory CT domain
and not due to the presence of the leucine zipper of CREB, as evidenced
by the relatively poor trans-activity of the TGA1a-CREB
control. In addition, immunoprecipitation assays indicated that the
enhanced activity of TGA1a-CREB
CT was not due to significant
comparative differences in its steady-state concentration, a finding
that is further supported by our observations that further increasing
expression of TGA1a or TGA1a-CREB had little additional stimulatory
effect on transcription. We conclude from these data that the CT domain
represses the basal activity of TGA1a.
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ACKNOWLEDGEMENTS |
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We thank Jennifer Miller for preparing the as-1 oligonucleotide resins and both Marisela Morales and Doug Julin for helpful comments regarding the manuscript.
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FOOTNOTES |
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* This work was supported by the University of Maryland Biotechnology Institute and by Grant MCB-9527364 from the National Science Foundation (to J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Maryland
Biotechnology Institute, 5115 Plant Sciences Bldg., College Park, MD 20742. Tel.: (301) 405-5353; Fax: (301) 314-9075; E-mail: arias@umbi.umd.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005143200
2 J. Arias, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: bZIP, basic/leucine zipper; 2, 4-D, 2,4-dichlorophenoxyacetic acid; GST, glutathione S-transferase; NT, amino-terminal; CT, carboxyl-terminal; PCR, polymerase chain reaction; CREB, cAMP-response element-binding protein; CAT, chloramphenicol acetyl transferase; PAGE, polyacrylamide gel electrophoresis.
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