A Role for the GCC-Box in Jasmonate-Mediated
Activation of the PDF1.2 Gene of Arabidopsis1
Rebecca L. Brown, Kemal Kazan*, Ken C. McGrath, Don J. Maclean, and John M. Manners
Cooperative Research Centre for Tropical Plant Protection (R.L.B., K.K., K.C.M., D.J.M., J.M.M.), and
Department of Biochemistry and Molecular Biology (R.L.B., K.M., D.J.M.), The University of Queensland,
Brisbane, Queensland 4072, Australia; and Commonwealth Scientific and Industrial Research Organisation,
Plant Industry, Queensland Bioscience Precinct, St. Lucia, Brisbane, Queensland 4067, Australia (K.K.,
J.M.M.)
The PDF1.2 gene of Arabidopsis encoding a plant defensin is commonly used as a marker for characterization of the
jasmonate-dependent defense responses. Here, using PDF1.2 promoter-deletion lines linked to the ␤-glucoronidase-reporter
gene, we examined putative promoter elements associated with jasmonate-responsive expression of this gene. Using stably
transformed plants, we first characterized the extended promoter region that positively regulates basal expression from the
PDF1.2 promoter. Second, using promoter deletion constructs including one from which the GCC-box region was deleted,
we observed a substantially lower response to jasmonate than lines carrying this motif. In addition, point mutations
introduced into the core GCC-box sequence substantially reduced jasmonate responsiveness, whereas addition of a
20-nucleotide-long promoter element carrying the core GCC-box and flanking nucleotides provided jasmonate responsiveness to a 35S minimal promoter. Taken together, these results indicated that the GCC-box plays a key role in conferring
jasmonate responsiveness to the PDF1.2 promoter. However, deletion or specific mutations introduced into the core
GCC-box did not completely abolish the jasmonate responsiveness of the promoter, suggesting that the other promoter
elements lying downstream from the GCC-box region may also contribute to jasmonate responsiveness. In other experiments, we identified a jasmonate- and pathogen-responsive ethylene response factor transcription factor, AtERF2, which
when overexpressed in transgenic Arabidopsis plants activated transcription from the PDF1.2, Thi2.1, and PR4 (basic
chitinase) genes, all of which contain a GCC-box sequence in their promoters. Our results suggest that in addition to their
roles in regulating ethylene-mediated gene expression, ethylene response factors also appear to play important roles in
regulating jasmonate-responsive gene expression, possibly via interaction with the GCC-box.
Diseases caused by fungal, bacterial, and viral
pathogens result in enormous reductions in yield and
quality from agricultural crops around the world.
However, although plants are continuously challenged by pathogenic microorganisms, the development of disease is a relatively rare event. It is clear,
therefore, that plants possess efficient defense systems. Effective induction of the defense response requires pathogen recognition followed by a network
of signal transduction processes, resulting in the
rapid activation of defense gene expression. A number of secondary signal molecules such as salicylic
acid (SA), jasmonic acid (JA) and its methyl ester,
methyl jasmonate (MeJA), and ethylene act to amplify and regulate defense responses after initial activation (Dong, 1998; Reymond and Farmer, 1998;
Schenk et al., 2000). Interestingly, the SA- and
jasmonate/ethylene-dependent signaling pathways
appear to modulate plant defense responses against
different classes of pathogens (for review, see
1
This work was supported by the Grains Research and Development Corporation (postgraduate fellowship to R.L.B.).
* Corresponding author; e-mail [email protected]; fax 61–
7–3214 –2950.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.102.017814.
1020
Thomma et al., 2001). Treatment with MeJA provides
Arabidopsis plants with protection against subsequent inoculation with a range of necrotrophic fungi,
but not against the biotrophic pathogen Peronospora
parasitica (Thomma et al., 1998). Similarly, several
studies of Arabidopsis mutants including ein2, coi1,
jar1, and fad3/7/8, in which the jasmonate/ethyleneresponse is disrupted, have indicated that resistance
to a variety of necrotrophic pathogens requires a
functional jasmonate/ethylene-signaling pathway
(Staswick et al., 1998; Vijayan et al., 1998; Thomma et
al., 1999). In contrast, resistance to the biotroph P.
parasitica but not to the necrotroph Alternaria brassicicola is enhanced by pretreatment with isonicotinic
acid, a functional analog of SA (Thomma et al., 1998).
A number of classes of regulatory proteins and
transcription factors are known to play important
roles in relaying the pathogen-initiated signals to
downstream components for the activation of plant
defense responses. Among these, ethylene response
factor domain-containing transcription factors (ERFs)
are implicated as key regulators of plant defense
responses. For example, the tomato (Lycopersicon esculentum) gene Pti4 encodes an ERF protein that is
induced by defense-signaling molecules and by the
bacterial pathogen Pseudomonas syringae pv. tomato
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GCC-Box and Jasmonate-Mediated Activation of the PDF1.2 Gene
(Thara et al., 1999; Gu et al., 2000). Pti4 and other
tomato ERFs Pti5 and Pti6 are phosphorylated by Pto
kinase, and this provides a direct link between
R-gene mediated pathogen recognition and subsequent defense gene activation (Zhou et al., 1997; Gu
et al., 2000). Transgenic expression of ERFs such as
ERF1 of Arabidopsis, Pti4 of tomato, and Tsi1 of
tobacco (Nicotiana tabacum) activates defense gene
expression and provides enhanced resistance against
fungal and bacterial pathogens (Park et al., 2001;
Berrocal-Lobo et al., 2002; Gu et al., 2002). Expression
of ERFs may also provide resistance to abiotic
stresses (e.g. osmotic stress), indicating that multiple
signaling pathways may converge on ERFs to regulate gene expression in response to a number of biotic
and abiotic stresses (Fujimoto et al., 2000; Park et al.,
2001; Memelink et al., 2001).
Several members of the ERF family bind specifically to the sequence AGCCGCC through the conserved ERF domain (Hao et al., 1998). This cis-acting
sequence, known as the GCC-box, is found in the
promoters of many pathogen-responsive genes such
as PDF1.2, Thi2.1, and PR4 (basic chitinase; Zhou et
al., 1997; Manners et al., 1998). The GCC-box has been
shown to function as an ethylene-responsive element
that is necessary, and in some cases sufficient, for the
regulation of transcription by ethylene (OhmeTakagi and Shinshi, 1995; Shinshi et al., 1995). In
addition, the GCC-box is implicated in ozoneresponsive expression of tobacco basic-type
pathogenesis-related PR-1 protein gene via ethylenedependent signaling (Grimmig et al., 2003).
More recently, an ERF subfamily of transcription
factors called octadecanoid-responsive Catharanthus
AP2s (ORCAs) has been implicated in jasmonateregulated expression of secondary metabolic pathways in Madagascar periwinkle (Catharanthus roseus;
Menke et al., 1999a; van der Fits and Memelink,
2000). MeJA treatment and fungal elicitors rapidly
induce transcript levels of Orca2 and Orca3. The
ORCA2 and ORCA3 proteins regulate overlapping
but distinct sets of genes associated with secondary
metabolism via specific binding to a promoter element called the jasmonate- and elicitor-responsive
element, which contains a core GCC-box (Menke et
al., 1999a; van der Fits and Memelink, 2000).
The PDF1.2 gene of Arabidopsis encodes a plant
defensin, and its expression is induced by pathogen
challenge both locally at the site of inoculation by
incompatible fungal pathogen and systemically in
remote noninoculated regions of the plant (Penninckx et al., 1996). This activation occurs via a
jasmonate/ethylene-mediated signaling pathway,
rather than via the SA-dependent pathway of defense
gene activation (Penninckx et al., 1996, 1998). Although the promoter of the PDF1.2 gene contains
several putative cis-acting sequence elements, such
as a GCC-box (Manners et al., 1998), the roles of these
elements and their respective trans-acting factors in
Plant Physiol. Vol. 132, 2003
regulation of the PDF1.2 gene by jasmonates have not
been investigated directly. We previously reported
on the jasmonate and pathogen-mediated activation
of the PDF1.2 promoter as promoter-reporter transgene that mimicked the expression patterns of the
native PDF1.2 gene (Manners et al., 1998; Mitter et al.,
1998). Examination of the 1,183-bp region upstream
of the transcription start site of the PDF1.2 gene
revealed several putative motifs with homology to
known cis-elements involved in the transcriptional
regulation of other genes, including those with particular relevance for jasmonate-mediated expression.
Two of these motifs, located between ⫺277 and ⫺495
of the PDF1.2 promoter, included the jasmonateresponsive element, TGACG (⫺392 to ⫺396) present
in the Lox1 gene of barley (Hordeum vulgare; Rouster
et al., 1997), and a 9-bp sequence, AAATGTTGT
(⫺410 to ⫺419), similar to a sequence within the
jasmonate-responsive region of the promoter of the
soybean (Glycine max) gene VspB (Mason et al., 1993).
In the present study, we set out to delineate the
regions of the PDF1.2 promoter that control jasmonate responsiveness. We report here on the identification of regions of the PDF1.2 promoter including
a GCC-box that actively modulate the expression of
this gene in response to jasmonates. In addition, we
identified a jasmonate-, ethylene-, and pathogenresponsive Arabidopsis ERF transcription factor
whose transgenic overexpression in Arabidopsis resulted in transcriptional activation of the PDF1.2
gene.
RESULTS
Production of Transgenic Lines Carrying PDF1.2
Promoter Deletion Constructs and Identification of a
Positive Regulatory Element in the PDF1.2 Promoter
We generated a series of 5⬘ deletions of the PDF1.2
promoter fused in frame with the ␤-glucoronidase
(GUS, UidA) reporter gene in a binary vector for use
in plant transformation (Fig. 1C). The locations of the
putative cis sequences that may be associated with
jasmonate responses were taken into account when
designing the 5⬘-deletion series. The deletion series
included, in addition to the complete P1 construct
(⫺1,183), three constructs P2 (⫺789), P3 (⫺495), and
P4 (⫺278), in which relatively large deletions of 394,
688, and 905 nucleotides from the P1 construct were
made. The constructs P5 (⫺262) and P6 (⫺255) removed only 16 and 23 nucleotides from the previous
smallest construct P4 and were designed to examine
the role of the GCC-box (GCCGCC between ⫺255 to
⫺261). We generated between seven and 12 independent transgenic lines stably transformed for each of
the PDF1.2-promoter deletion constructs. We randomly selected five independently transformed lines
for each PDF1.2 promoter deletion construct and analyzed GUS activity in homogenates from whole
sterile-grown seedlings as described in “Materials
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1021
Brown et al.
Figure 1. Basal and MeJA-responsive expression of GUS activity from the PDF1.2 promoter deletion lines. A, Average basal
GUS activity (fluorescence units per milligram of protein) in each of the PDF1.2 promoter deletion constructs. Each bar
represent data from five independent transgenic lines derived from six replicates in two independent experiments. Shaded
bars, Plants grown in sterile conditions; black bars, plants grown in soil. B, The PDF1.2 promoter reporter constructs that
were placed in the binary vector pBI101.3 used to generate transgenic plant lines. C, Average induction values from each
promoter deletion construct in MeJA-treated plants. Induction data were generated from five independent transgenic lines
for sterile-grown seedlings, whereas two representative lines were used for soil-grown plants. GUS activity assays were
performed on 20 T2 plants for each transgenic line. Bars indicate SE.
and Methods.” Average GUS expression data from
five transgenic lines carrying the same construct revealed that basal expression (i.e. absence of pathogen
challenge and MeJA treatment) of the reporter genes
in plants carrying the longer P1, P2, and P3 PDF1.2
promoter deletions was significantly higher (P ⬍
0.01) than that observed for plants carrying the
shorter P4, P5, and P6 constructs (Fig. 1A, shaded
bars). Comparison of average GUS activity in untreated plants carrying the P3 and P4 deletion constructs indicated that deletion of the ⫺495 to ⫺279
region of the PDF1.2 promoter resulted in a 97%
reduction in expression. This suggested that a putative positive regulatory element is apparently located
within the ⫺495 to ⫺278 region of the PDF1.2
promoter.
We also examined the basal expression of the reporter gene in rosette leaves of soil-grown 6-weekold plants (two lines for each PDF1.2 promoter deletion construct; Fig. 1A, black bars). Despite some
differences in the basal expression levels driven by
the PDF1.2 promoter deletions P1, P2, and P3 between the two growth conditions, the overall effect of
the putative enhancer element revealed in the seedling studies was still readily detectable in the soilgrown plants
To further delineate the presence of a putative
positive regulatory element within the ⫺495 to ⫺278
region of the PDF1.2 promoter, we performed transient expression assays. In these assays, we cobombarded the leaves of wild-type Arabidopsis plants
with three constructs, which in addition to each promoter deletion construct included the 35S-green fluorescent protein (GFP) and 35S-Luc plasmids. After
bombardment, leaves were examined by fluorescent
microscopy. Leaf sections displaying a large number
of GFP-expressing cells (⬎60) were selected for assay
of luciferase activity in leaf homogenates, which was
1022
then used to normalize GUS activity measurements
for the same sample. Results (Fig. 2) showed that
constructs P3 and Ea did not differ significantly in
expression, but displayed approximately 2-fold
higher expression than constructs Eb, Ec, and P5 (P ⬎
0.01). This suggested that the putative enhancer element of the PDF1.2 promoter is located downstream
of ⫺465. Because no significant difference was observed among Eb, Ec, and P5, these results indicate
that the 68-nucleotide region of the PDF1.2 promoter
lying between ⫺465 and ⫺398, or spanning the ⫺398
region, is critical for high-level expression.
Jasmonate Responsiveness Conferred by the PDF1.2
Promoter Deletion Constructs
We next examined transgenic plants carrying the
PDF1.2 promoter deletion constructs to identify regions of the promoter that may regulate the response
Figure 2. Delineation of enhancer element using quantitative transient expression analysis of the PDF1.2-promoter deletion constructs
in Arabidopsis leaves. Relative expression data for each construct
represent the mean of GUS/luciferase ratios for at least six separate
leaf samples. Bars indicate SE. The data shown here are compiled
from several independent bombardment experiments that produced
similar results.
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Plant Physiol. Vol. 132, 2003
GCC-Box and Jasmonate-Mediated Activation of the PDF1.2 Gene
to jasmonate. In both sterile- and soil-grown plants,
although GUS activity levels were higher after MeJA
treatment, differences between constructs appeared
to mirror the results from untreated plants in that
specific GUS activities were considerably higher in
lines carrying constructs P1, P2, and P3 than lines
carrying constructs P4, P5, and P6 (data not shown).
However, a different pattern emerged when -fold
induction ratios were compared (Fig. 1C). In sterilegrown plants, constructs P3 and P4 did not differ
significantly from one another in -fold induction ratios and showed significantly higher -fold induction
by MeJA than P1 and P2 (P ⬍ 0.01). Similarly, in
soil-grown plants, lines carrying the P3 construct
showed a higher level of induction than lines carrying P1 and P2 constructs.
Using sterile-grown transgenic plants, the deletion
of the 15-bp region between constructs P4 and P5
resulted in a significant decrease in induction by
MeJA (P ⬍ 0.01; Fig. 1C). A further decrease in induction was observed in P6 with the deletion of an
additional 7 bp comprising the core GCC motif of
AGCCGCC. In contrast to sterile-grown plants, the
stably transformed soil-grown plants carrying the P5
construct gave a similar -fold induction of the reporter gene to plants carrying the P3 or P4. However,
similar to sterile-grown plants, deletion of the core
GCC-box (P6 construct) significantly reduced the
level of MeJA-mediated induction of GUS activity.
Although these results indicate the importance of this
GCC-box motif for induction of the PDF1.2 gene by
jasmonate, deletion of this box did not completely
abolish jasmonate responsiveness, indicating that
other elements downstream of ⫺255 may also be
involved in the regulation of this response. The bio-
logical significance of the difference between sterileand soil-grown plants carrying construct P5 is not
known but if confirmed, could be related to additional positive signaling pathways initiated by biotic
or abiotic factors.
In an effort to confirm the role of the GCC-box in
mediating jasmonate responsiveness, we generated
two additional PDF1.2 promoter constructs, mGCC
and pIB (Fig. 3B). The mGCC consisted of the ⫺495
PDF1.2 promoter fragment with substitution of the
core GCC-box sequence (GCCGCC) with TCCTCA.
The pIB construct was aimed at determining whether
the GCC region alone was sufficient to confer
jasmonate-responsive gene expression. This construct was a fusion of the extended GCC-box region
(⫺250 to ⫺270) upstream of a truncated 35S (⫺46)
promoter derived from CaMV.
Analysis of the mean GUS expression in MeJAtreated sterile-grown whole seedlings stably transformed with the mGCC construct indicated that there
was no significant induction by MeJA compared with
mock-treated controls (Fig. 3C). This experiment included plants stably transformed with construct P3
as control, which showed the same highly significant
increase in MeJA-responsive induction (P ⬍ 0.01) as
the previous experiment described in Figure 1. This
result directly indicates that the GCC-box sequence
confers jasmonate responsiveness to the PDF1.2
promoter.
Next, we examined the jasmonate responsiveness
conferred by the pIB construct linking the GCC-box
to a minimal 35S promoter in stably transformed
sterile-grown plants. After MeJA treatment, these
plants showed some jasmonate response, but the
level of induction was significantly less than that
Figure 3. A through C, Further confirmation that the GCC-box confers jasmonate responsiveness to the PDF1.2 promoter
using quantitative analysis of stably transformed plants. C, Results are presented as average -fold induction values from two
to five transgenic lines harboring each PDF1.2 promoter deletion construct. A, The basal expression of GUS from each
construct is also given. The mGCC construct is the same as construct P3 but otherwise contains three substitutions in the first
(G–T), fourth (G–T), and sixth (C–A) nucleotides (shown in bold) to convert the core GCCGCC box sequence to TCCTCA.
B, Construct pIB contains a 20-nucleotide-long sequence including the GCC-box and the surrounding nucleotides from the
⫺270 to ⫺250 of the PDF1.2 promoter linked to a truncated 35S cauliflower mosaic virus (CaMV) promoter. SE bars are also
shown for each construct.
Plant Physiol. Vol. 132, 2003
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Brown et al.
measured for construct P3 (P ⬍ 0.05). Plants transformed with a 35S minimal promoter construct alone
did not show any significant induction of GUS activity after MeJA treatment (Fig. 3C).
Identification of MeJA-Responsive Arabidopsis ERFs
Jasmonate-responsive expression of GUS activity
in plants stably transformed with PDF1.2 promoter
constructs including the GCC-box suggested that this
conserved sequence element might act as binding site
for jasmonate-responsive ERF transcription factors.
To identify gene family members encoding putative
Arabidopsis ERFs, we used the consensus amino acid
sequence of the DNA-binding domain of all known
ERF members that bind to the GCC-box domain as a
query sequence for BLASTP searches of Arabidopsis
sequences available on GenBank. The searches returned 145 sequences representing unique proteins
(see also Sakuma et al., 2002), of which 131 contained
a single AP2/ERF-type DNA-binding domain. Because of the relatively large size of this transcription
factor family, we selected representatives from sequence clusters for expression profiling. With the
exception of APETALA2, all of the genes were selected from the subfamily that contains only a single
ERF domain. The selected sequences included ERF1
(Solano et al., 1998), AtERF2, AtERF3, AtERF4,
AtERF5 (Fujimoto et al., 2000), and 12 other ERFs (see
“Materials and Methods” for locus numbers) for
which no expression data were available in the literature. Of the latter, AP6 was selected because it had
the highest homology of any Arabidopsis ERF to
ORCA2. DNA sequences for each of these 18 ERFs
were generated by PCR amplification from divergent
regions outside the conserved DNA-binding domain,
to avoid cross-hybridization among the different
members of the gene family.
To identify jasmonate-responsive Arabidopsis
ERFs, we first examined the expression patterns of
the 18 selected Arabidopsis ERFs by reverse northern
hybridization analysis of a macroarray of the variable
sequence from each gene. Expression patterns of
ERFs showing induction after MeJA treatment were
then confirmed by northern-blot analysis. Only five
(ERF1, AtERF2, AtERF3, AtERF4, and RAP2.10) of the
18 AP2/ERF gene family members present on the
macroarray, hybridized with cDNA prepared from
MeJA-treated plants and their associated controls.
All of these five genes showed induction by MeJA at
0.5 h, with a peak in induction after 1 h of MeJA
exposure (data not shown). With the exception of
AtERF4, which remained induced until 4 h, the transcription levels of all genes had returned to those
observed in control plants by 2 h (data not shown).
Examination of expression patterns by northern blotting confirmed the results obtained from macroarray
analysis (Fig. 4A, left panel). Induction of PDF1.2 was
observed after 4 h of exposure to MeJA in these
experiments (Fig. 4A, left panel).
1024
Figure 4. A, Expression profiling of selected ERF genes by northern
blot in a time-course analysis. Left panel, Expression in response to
MeJA; right panel, expression in response to ethylene for each gene.
Top and bottom panels represent RNA from control (C) and treated
(T) plants, respectively. B, Induction ratios of the Arabidopsis ERF1,
AtERF2, and PDF1.2 genes obtained by real-time quantitative reverse
transcriptase (RT)-PCR after MeJA treatment.
We further quantified the induction ratios of ERF1,
AtERF2, and PDF1.2 after MeJA treatment by quantitative real-time RT-PCR, which is particularly suitable for specific detection of transcripts from the
members of multigene families (Charrier et al., 2002).
In these time-course experiments, we used RNA isolated from plants grown and treated at separate occasions. Results from these experiments which included early as well as late time-points (e.g. 24 and
48 h) were in agreement with the results from
northern-blot and macroarray analyses as both ERF1
and AtERF2 displayed distinct yet overlapping expression patterns (Fig. 4B). AtERF2 was substantially
more responsive to MeJA treatment than ERF1, especially at earlier time points (e.g. 3 and 6 h), whereas
a sustained level of expression was observed for both
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Plant Physiol. Vol. 132, 2003
GCC-Box and Jasmonate-Mediated Activation of the PDF1.2 Gene
AtERF2, AtERF3, AtERF4, and RAP2.10 was induced
in both local and systemic tissue 5 h after A. brassicicola inoculation (Fig. 5A). For AtERF2 and RAP2.10,
induction was substantially higher in local compared
with systemic tissue at this early time point. No
significant induction was observed from any of the
other ERFs tested or the known defense genes PDF1.2
and PR-1 (data not shown). Inoculation with A. brassicicola has been previously demonstrated to induce
expression of these defense genes (Penninckx et al.,
1996; Thomma et al., 1998), but at later time periods
after inoculation (48–72 h). We further quantified the
induction ratios of AtERF2 and PDF1.2 by real-time
quantitative RT-PCR after inoculation with A. brassicicola. Analysis of RNA samples obtained from an
independent (i.e. RNA isolated from plants grown
and inoculated in a different occasion) time-course
experiment showed induction of both ERF1 and
AtERF2 by this incompatible pathogen at all time
points examined although ERF1 induction was substantially higher than that observed for AtERF2 (Fig.
5B).
Overexpression of AtERF2 in Arabidopsis Activates
PDF1.2, Thi2.1, and PR4 Expression
Figure 5. Arabidopsis ERF genes respond to fungal inoculation. A,
Macroarray analysis showing -fold induction of four Arabidopsis
ERFs after challenge by incompatible fungal pathogen A. brassicicola
in inoculated (local) leaves and uninoculated (systemic) leaves of the
same plant. Samples were collected 5 h after inoculation. Bars show
-fold induction of each gene compared with the equivalent tissue
from inoculated plants. Expression was determined using ImageQuant software, and values were adjusted against ␤-tubulin, as a
loading control. B, Induction ratios (local) of the Arabidopsis AtERF2
and PDF1.2 genes obtained by real-time quantitative RT-PCR after
inoculation with A. brassicicola.
genes at late time points. Consistent with known
expression pattern of PDF1.2, we detected a strong
induction of the PDF1.2 transcripts starting at 6 to
12 h after treatment.
We also tested the ethylene response profiles of
those ERFs that were induced by MeJA by northernblot analysis. These analyses showed that AtERF2,
AtERF3, AtERF4, and RAP2.10 responded in a similar
manner to ethylene as observed for MeJA (Fig. 4A,
right panel). They displayed rapid induction on ethylene exposure, with expression returning to control
levels after one to 2 h. ERF1 expression after ethylene
treatment was more sustained and continued to increase over the time points tested.
We next used macroarray analysis to investigate
the effect of inoculation by fungal pathogen A. brassicicola on the expression profiles of selected ERF
genes in Arabidopsis plants 5 h after inoculation.
Expression patterns were assessed in local, inoculated tissue and systemic noninoculated tissue of inoculated plants and were compared with equivalent
tissues of control, uninoculated plants. Expression of
Plant Physiol. Vol. 132, 2003
As described above, transcripts of some AtERF2
genes substantially increased at earlier time points
after MeJA treatment than the first detection of elevated levels of transcripts of the PDF1.2 gene. To
examine whether there was causal relationship between elevated levels of ERF transcripts and subsequent induction of the PDF1.2 gene, we stably transformed Arabidopsis plants with the AtERF2 gene
driven by the constitutive 35S promoter. Examination
of the transcript levels T2 plants in four stably transformed lines by real-time quantitative RT-PCR revealed that two of the lines were overexpressing
AtERF2 3- to 5-fold over untransformed control
plants (Fig. 6). Expression levels of the AtERF2 gene
in two other stably transformed lines were similar to
Figure 6. Overexpression of AtERF2 activates defense gene expression in Arabidopsis. Shown here are -fold expression values of the
AtERF2, PDF1.2, Thi2.1, and PR4 genes in two independent stably
transformed transgenic T2 lines containing the 35S-AtERF2 transgene
over those observed in untransformed control plants. Error bars are
also shown for each line.
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1025
Brown et al.
that observed in untransformed control plants (data
not shown). We also detected a 16- and 51-fold increase in PDF1.2, an 80- and 50-fold increase in
Thi2.1, and 2- and 3-fold increase in PR4 (basic chitinase) transcript levels, respectively, in these two
AtERF2 overexpressing lines (Fig. 6), whereas no
such increase in the expression of these genes was
evident in untransformed control plants and in two
other transgenic lines with wild-type levels of
AtERF2 transcripts. Overall, these results suggest
that the AtERF2 protein is capable of activating the
PDF1.2, Thi2.1, and PR4 genes when overexpressed
in transgenic plants.
AtERF2 Transcript Level Remains Unchanged in
Arabidopsis Mutants Constitutively Expressing the
PDF1.2 Gene
To determine whether enhanced levels of the
AtERF2 transcript can be found in Arabidopsis
defense-signaling mutants that constitutively express
the PDF1.2 and Thi2.1 genes, we examined the transcript levels of AtERF2 by real-time quantitative RTPCR analysis in Arabidopsis defense-signaling mutants cev-1 and cpr5 (Bowling et al., 1997; Ellis and
Turner, 2001). These analyses showed no significant
change in the transcript level of AtERF2 in these
mutants (data not shown). However, consistent with
previous reports, we detected enhanced transcript
levels of PDF1.2 in both cev-1 and cpr5 mutants (data
not shown).
DISCUSSION
The GCC-Box Is a Major Element Conferring the
Jasmonate Responsiveness of the PDF1.2 Promoter
In this report, we have identified the ⫺255 to ⫺277
promoter region as being critical for regulation of the
PDF1.2 gene by jasmonate. Plant lines carrying constructs with deletions in this region (P5 and particularly P6) showed significantly lower induction by
MeJA in comparison with all other deletion constructs. Penninckx et al. (1998) and Manners et al.
(1998) have reported that the level induction of
PDF1.2 by both MeJA and ethylene in tissue culturegrown plants was increased by growth of the plants
under non-sterile conditions. Therefore, we used
both types of growth conditions in assessing the expression from the promoter-deletion constructs. In
general, results reported in this manuscript supported this observation for the induction of PDF1.2
promoter-driven reporter gene expression by MeJA
in soil-grown plants. Augmented induction in soilgrown plants may be a consequence of physiological
differences between plants grown in soil and in vitro
or as a result of potentiation by soil-associated biological factors.
The ⫺255 to ⫺277 region contains the GCC-box,
which is a common motif in the promoters of various
1026
genes encoding defense-related proteins, and has
been previously identified as the specific binding site
for members of the ERF subfamily of AP2/ERF transcription factors (Ohme-Takagi and Shinshi, 1995;
Buttner and Singh, 1997; Zhou et al., 1997). Analysis
of Arabidopsis lines stably transformed with a
PDF1.2 promoter-GUS reporter transgene revealed a
stepwise reduction in induction by jasmonate as the
sequences immediately preceding the GCC-box (P4
and P5) were deleted, with a further reduction of
induction after deletion of the core GCC motif itself
(P6). In other studies, a 21-nucleotide sequence containing the GCC-box in the promoter of the PDF1.2
promoter provided jasmonate responsiveness when
linked to a minimal 35S CaMV promoter, and mutations introduced into the GCC-box significantly reduced the jasmonate responsiveness of the plants
carrying P3 construct. Taken together, these results
indicate that the GCC-box in the promoter of the
PDF1.2 gene modulates the jasmonate responsiveness of the PDF1.2 gene. Similar to our findings
reported here, Menke et al. (1999a) have recently
shown that jasmonate- and elicitor-responsive elements found in the promoter of the secondary metabolite biosynthetic gene, Str, of Madagascar periwinkle, contains a GCC-like box that is both
necessary and sufficient for jasmonate- and elicitorresponsive expression.
It has been well established that members of ERF
transcription factor family interact with the GCC-box
sequence. Detailed analysis of the in vitro binding
requirements of three ERF transcription factors from
tobacco indicated that whereas the contact of specific
amino acid residues of the ERFs are confined to the
6-bp core GCCGCC region of the GCC-box, surrounding nucleotides are necessary for high-affinity
binding (Hao et al., 1998). These sequences may also
modulate differential binding by different ERFs. This
may explain the observation that plants carrying the
P5 construct, with a deletion of the 5⬘-flanking sequence to the core GCC motif, showed decreased
responsiveness to jasmonate but significantly higher
basal expression levels compared with the P4 plants
carrying an extended GCC motif. The removal of the
nucleotides specifying recruitment of the specific
ERFs that up-regulate PDF1.2 transcription in response to jasmonate could allow competitive binding
of other ERF subfamily members, which although
unresponsive to jasmonate, may increase basal gene
expression.
Although the results presented here identify the
⫺255 to ⫺277 region, containing the core GCC-box,
and flanking sequences as critical for gene regulation,
they do not exclude the additional significance of
cis-elements located further downstream in the
PDF1.2 promoter. In fact, other cis-elements also appear to contribute to jasmonate responsiveness, as
indicated by residual induction displayed by lines
carrying the P6 construct. Interestingly, in P6, a par-
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Plant Physiol. Vol. 132, 2003
GCC-Box and Jasmonate-Mediated Activation of the PDF1.2 Gene
tial GCC-box sequence (AGCCGC, ⫺219 to ⫺214)
occurs in the PDF1.2 promoter downstream from the
consensus GCC-box at ⫺255 to ⫺277. It is possible
that some transcription factors can bind to this GCCbox like region and activate gene expression through
interactions with ERFs. Coregulation of gene expression by the GCC-box and other promoter elements
has also been observed previously. For example, the
GCC-box is required but not sufficient for high-level
induction of the tobacco osmotin gene by ethylene
(Raghothama et al., 1997). Also, a G-box is associated
with jasmonate responsiveness in defense-associated
genes in various plant species (Kim et al., 1992;
Mason et al., 1993). In the PDF1.2 promoter, the
GCC-box also directly follows a putative G-box motif (⫺255 to ⫺250) with the sequence CATGTG
(Manners et al., 1998). Close proximity of these
G- and GCC-box elements is a common feature of
tobacco PR genes and is postulated to allow crosslinking of ERF and bZIP transcription factors for
regulation of gene expression during plant defense
responses (Sessa et al., 1995; Buttner and Singh,
1997). Although the G-box has been shown to be
important in regulating the expression of certain
jasmonate-responsive genes (Kim et al., 1992; Mason
et al., 1993), the putative G-box in the PDF1.2 promoter lacks the exact ACGT core sequence characteristic of bZIP transcription factor binding sites
(Izawa et al., 1993) and thus may not represent a
functional G box. Further work would be needed to
determine the role of these additional elements in
the regulation of the PDF1.2 gene.
AtERF2, a Jasmonate/Ethylene- and Pathogen-Inducible
ERF, Constitutively Activates Transcription from the
GCC-Box Containing Defense Genes
The deletion analysis of the PDF1.2 promoter suggested that the GCC-box is critical for jasmonatemediated regulation of the PDF1.2 gene expression. It
thus seemed a reasonable hypothesis that one or
more members of the Arabidopsis ERF transcription
factor family homologous to the periwinkle ORCA2
and ORCA3 transcription factors (Menke et al.,
1999a; van der Fits and Memelink, 2000) might act to
regulate the expression of PDF1.2 in response to jasmonates. Therefore, we examined the mRNA levels
of selected ERFs in Arabidopsis plants after treatment with inducers of PDF1.2 expression and identified ERFs that show distinct induction profiles.
ERF1, AtERF2, AtERF3, AtERF4, and RAP2.10 all displayed immediate early induction, with mRNA accumulating after only 0.5 h of exposure to MeJA. MeJAinduced expression of each of these genes occurred
earlier than that observed for PDF1.2, suggesting that
one or more of these ERFs may regulate PDF1.2
expression. Rapid and transient induction of AtERF2
by MeJA mimicked the expression patterns of the
Orca2 and Orca3 transcripts, which also showed a
Plant Physiol. Vol. 132, 2003
strong induction 0.5 h after exposure to MeJA
(Menke et al., 1999a, 1999b; van der Fits and
Memelink, 2000, 2001). In addition, the deduced
amino acid sequence of AtERF2 is highly similar to
that of the ORCA3, particularly around the Ser-rich
region, a putative phosphorylation site located at the
C terminus of ORCA3 protein (van der Fits and
Memelink, 2000). These similarities between AtERF2
and ORCA2/3 suggested that AtERF2 might play a
role in MeJA-mediated induction of the PDF1.2 gene.
We specifically quantified expression from the
PDF1.2 gene by quantitative real-time RT-PCR to
distinguish the expression of PDF1.2 from three other
PDF1.2-like expressed sequences that exist in Arabidopsis. The predicted mature peptides encoded by
genes PDF1.2a (same as PDF1.2), PDF1.2b, and
PDF1.2c are identical (R.L. Brown and K. Karzan,
unpublished data; Thomma et al., 2002). Moreover,
the upstream regulatory sequences of the latter two
genes contain regions with substantial homology to
the GCC-box and neighboring sequences found in
the promoter of the PDF1.2 gene (R.L. Brown and K.
Karzan, unpublished data). The identification of multiple family members homologous to PDF1.2 means
that studies of the expression of this gene based
solely on hybridization signals may be misleading.
This was especially critical to rule out the possibility
that in AtERF2-overexpressing plants, expression of
PDF1.2 but not any other PDF1.2-like genes is detected because it is possible that other PDF1.2-like
genes might also be activated due to having GCC-box
like sequences in their promoters. Our analysis
showed that overexpression of AtERF2 activated
transcription of defense genes such as PDF1.2, Thi2.1,
and PR4. All of these genes contain GCC-boxes in
their promoters (Epple et al., 1995; Zhou et al., 1997;
Manners et al., 1998), and their activation by AtERF2
probably occurs via the GCC-box. Fujimoto et al.
(2000) have demonstrated the ability of AtERF2 to
interact with the GCC-box for the activation of reporter gene expression in transient expression assays.
The AtERF2 protein also shares high amino acid
sequence identity with the tomato ERF, Pti4, whose
overexpression in Arabidopsis also results in elevated expression of PDF1.2, Thi2.1, and PR4 and
enhanced resistance against the fungal pathogen Erysiphe orontii and the bacterial pathogen P. syringae pv.
tomato (Gu et al., 2002). Defense gene activation and
an enhanced disease resistance phenotype were also
reported for Arabidopsis plants overexpressing ERF1
(Berrocal-Lobo et al., 2002; Lorenzo et al., 2003). This
may indicate that ERF1 and AtERF2 may have overlapping or redundant functions. Although we have
not tested the level of disease resistance in plants
overexpressing AtERF2, we predict that these plants
may also show elevated levels of disease resistance
due to constitutive overexpression of a number of
defense genes which may be further induced during
infection due to availability of additional positive
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1027
Brown et al.
regulators of defense genes, which may require induction by a pathogen.
Convergence of Multiple Signaling Pathways on ERFs
Recent studies (Fujimoto et al., 2000; Ohta et al.,
2000; Park et al., 2001; Cheong et al., 2002; Gu et al.,
2002; Mazarei et al., 2002; Nishiuchi et al., 2002;
Oñate-Sánchez and Singh, 2002; Wu et al., 2002) suggest that ERFs are regulated differentially at both the
transcriptional and posttranscriptional levels and
that different ERF proteins may regulate various subsets of GCC-box-containing genes. For instance, the
ERF genes studied here including AtERF2 were responsive to the known inducers of the PDF1.2 gene,
namely MeJA and ethylene (Fujimoto et al., 2000;
Lorenzo et al., 2003; this study), and inoculation with
fungal pathogen (this study). Identification of
jasmonate-, ethylene-, pathogen-, wounding-,
nematode-, and stress-inducible ERF transcription
factors suggest that ERFs can spatially and temporally regulate gene expression in response to biotic
and abiotic cues. As recently suggested by Fujimoto
et al. (2000), both ethylene and stress-related signaling pathways seem to converge on a subset of different ERFs, which subsequently regulate a subset of
GCC-box containing genes. Results reported here,
together with studies on ORCA2/3 proteins, and
ERF1 (Lorenzo et al., 2003) suggest that components
of jasmonate-signaling pathways converge with
those of other pathways (e.g. abiotic stress, wounding, ethylene, and pathogen/elicitor) on ERF transcription factors in regulating gene expression.
Interestingly, so far a number of ERF proteins have
been shown to be able to trans-activate the PDF1.2
expression when expressed stably or transiently in
Arabidopsis (Fujimoto et al., 2000; Ohta et al., 2001;
Berrocal-Lobo et al., 2002; Gu et al., 2002; Wu et al.,
2002; this study). However, it is not clear which ERFs
might be responsible for activating the PDF1.2 expression in planta in response to jasmonate or pathogen signals. More recently, Lorenzo et al. (2003) have
suggested that ERF1 and AtERF2 may have redundant functions. It is also possible that the activation
of the PDF1.2 gene in transgenic plants constitutively
expressing a GCC-box binding protein occurs somewhat nonspecifically. In this study, AtERF2, a
jasmonate-, pathogen-, and ethylene-inducible ERF,
was able to activate the expression from the PDF1.2
gene when overexpressed in transgenic plants. However, examination of the constitutive PDF1.2expressing Arabidopsis mutants, cev1 and cpr5, did
not reveal any increase in the level of AtERF2 transcript. These results are consistent with those of
Oñate-Sánchez and Singh (2002), who have recently
reported that the expression of a number of
jasmonate-, pathogen-, and SA-inducible ERFs, including AtERF2 have not changed in cep1 and cpr5-2
mutants. Taken together, these data suggest that
1028
PDF1.2 overexpression observed in these mutants
may be mediated by other ERFs. Alternatively, as
suggested by Gu et al. (2002), relatively low levels of
AtERF2 may be activated posttranslationally by a
constitutively expressed putative kinase without any
detectable increase at the transcript level. Such a
phosphorylation event could influence the localization of AtERF2 to nucleus and modulate its DNAbinding ability or its interaction with other transcription factors. Phosphorylation by Pto kinase enhances
binding of the tomato ERF Pti4 to the GCC-box (Gu et
al., 2000). Several other AP2/ERFs, such as CBF1,
CBF2, CBF3 ORCA2, ORCA3, and AtERF5, contain
potential recognition sites for protein kinases, which
may play important roles in modulating the function
of these proteins (Medina et al., 1999; Yamamoto et
al., 1999; Fujimoto et al., 2000).
Alternatively to ERF activation, inactivation of a
putative repressor protein, perhaps also an ERF,
might be responsible for constitutive expression of
the PDF1.2 gene in the cev1 and cpr5 mutants noted
above. Recent studies have indicated that some ERFs
may act as repressors rather than activators of transcription (Fujimoto et al., 2000; Ohta et al., 2001). A
conserved sequence motif called ethylene-associated
amphiphilic repression within the C-terminal region
of these transcription factors seems to be essential for
repression by such repressor ERFs (Ohta et al., 2001).
For example, the AtERF3, AtERF4, and RAP2.10 proteins all contain the ethylene-associated amphiphilic
repression motif, and AtERF3 and AtERF4 have been
shown to function as repressors in in vitro assays
(Fujimoto et al., 2000). Thus, even though the MeJA-,
ethylene-, and A. brassicicola-induced expression of
AtERF3, AtERF4, and RAP2.10 precedes induction of
PDF1.2 in the research reported herein, it cannot be
concluded that these genes are involved in the specific activation of PDF1.2.
Enhancer Region in the PDF1.2 Promoter
In this paper, we have also reported on promoter
elements other than the GCC region, that appear to
play roles in regulating the overall levels of expression of the PDF1.2 gene of Arabidopsis. Analysis of
reporter gene expression in multiple transgenic lines
for each promoter-reporter deletion line showed that
a strong enhancer element is located within the ⫺277
to ⫺495 region of the PDF1.2 promoter. Further deletion analysis of the PDF1.2 promoter using the
transient expression system allowed delineation of
this element to a 68-nucleotide region located between ⫺398 and ⫺465. This region of the PDF1.2
promoter contains several motifs with similarity to
regulatory elements of other genes, and/or known
recognition sites for transcription factors. These included the sequences TTCGAC (⫺398 to ⫺404), involved in the SA responsiveness to the PR-2d gene of
tobacco (Shah and Klessig, 1996), and CGACG (⫺399
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Plant Physiol. Vol. 132, 2003
GCC-Box and Jasmonate-Mediated Activation of the PDF1.2 Gene
to ⫺403), necessary for high-level expression of the
rice (Oryza sativa) Amy3D gene under conditions of
sugar starvation (Hwang et al., 1998). In addition,
this 68-nucleotide region contains the sequence
CAACA (⫺454 to ⫺458), which was revealed by in
vitro binding experiments as the DNA recognition
sequence for the RAV1 transcription factor of Arabidopsis (Kagaya et al., 1999). The RAV1 protein binds
to this motif through a protein domain that strongly
resembles the AP2 domain found in AP2/ERF proteins (Kagaya et al., 1999). Also located within the
68-nucleotide putative enhancer region of the PDF1.2
promoter are sequences with homology to the GTand GATA (I box) elements, which mediate lightresponsive expression of many genes (Zhou, 1999).
GT-elements also exist in many light-unresponsive
genes, indicating a diverse range of functions and,
depending on promoter structure, may have a positive or negative effect on transcription (Zhou, 1999).
Further experimentation is necessary to determine
whether one or more of the elements described above
modulate high-level expression on the PDF1.2 promoter, or whether the putative enhancer contained
within this region of the PDF1.2 promoter represents
a novel element.
In conclusion, our results have demonstrated a role
for the GCC-box in conferring jasmonate responsiveness to the PDF1.2 promoter. This motif and its surrounding bases most probably interact with several
members of the ERF family of transcription factors,
some of which may be also induced by jasmonates.
Analysis of the PDF1.2 promoter has also indicated
the existence of other, probably interactive, cis elements necessary for high-level jasmonate-mediated
regulation of PDF1.2 expression during plant defense
responses.
MATERIALS AND METHODS
Production of the PDF1.2 Promoter Constructs
The cloned promoter of the PDF1.2 gene described in Manners et al.
(1998) was used as template for the production of all PDF1.2-promoter
deletions. Promoter fragments of varying lengths were amplified by PCR
using the following forward primers for constructs P2, P3, P4, P5, and P6,
respectively (Fig. 1B): 5⬘-ATCAAAGCT TTGAGCCTCTCACTTGCGGTC-3⬘,
5⬘-ATCAAA GCTTTGCTGCTCTTGAGATCAACC-3⬘, 5⬘-GCCGAAGCTTCCATTCAGATTAACCAGCC-3⬘, 5⬘-ATCAAAGCTTAGCCGCCCATGTGAACGATG-3⬘, 5⬘-ATCAAAGCTTCATGTGAACGATGTAGCATTAGC-3⬘,
and a common reverse primer 5⬘-GAGAGAGGATCCTGATGGAAGCAAACTTAGCCATG-3⬘ for all constructs. The PDF1.2-promoter deletions were
cloned into the HindIII-BamHI site of the binary vector pBI101.3 (CLONTECH, Hampshire, UK), in frame with the UidA gene, using standard
molecular biology techniques (Sambrook et al., 1989). The pA35S construct
was made by amplifying the ⫺46 to ⫹10 region of the CaMV 35S promoter
using the primer pair 5⬘-CACTATGTCGACCAAGACCTTTCCTCTATATAAG-3⬘ and 5⬘-CCCGGGGATCCGTCCTCTCAAAATGAATGGAAC-3⬘.
Amplification product was digested with SalI and BamHI and inserted in
front of the UidA gene in pBI101.3 also digested with SalI and BamHI. This
construct is termed pB35S. The transgene cassette in pB35S was then removed by digesting HindIII and EcoRI and inserted into HindIII/EcoRI
digested binary vector pAOV, which carries a gene for Basta resistance
(Mylne and Botella, 1998). The pIB construct was made by amplifying the
⫺270 to ⫺250 region of the PDF1.2 promoter using primers 5⬘-
Plant Physiol. Vol. 132, 2003
TTAATCAAGCTTGATTAACCAGCCGCC-3⬘ and 5⬘-TGCTACGTCGACCACATGGGCGGCTGG-3⬘. Amplification product was then digested with
SalI and HindIII and ligated onto SalI/HindIII-digested pB35S, which was
called pB6. The transgene cassette in pB6 was then removed by digesting
with HindIII/EcoRI and cloned into HindIII/EcoRI-digested pAOV. The
mGCC construct was generated via a two-step PCR process using primer
pairs: (a) 5⬘-ATCAAAGCTTTGCTGCTCTTGAGATCAACC-3⬘ and 5⬘CGTTCACATGTGAGGATGGTTAATC-3⬘ and (b) 5⬘-GATTAACCATCCTCACATGTGAACG-3⬘ and 5⬘-GAGAGAGGATCCTGATGGAAGCAAACTTAGCCATG-3⬘. The amplification product was digested with BamHI and
HindIII and was cloned into BamHI/HindIII-digested binary vector
PBI101.3. The transgene cassette was then removed from this plasmid by
HindIII/EcoRI digest and cloned into HindIII/EcoRI digested pAOV. Plasmids were introduced into Escherichia coli DH5-␣ cells via heat shock transformation (Sambrook et al., 1989). The fusion constructs were verified by
sequencing using the antisense primer (5⬘-CAGTTTTCGCGATCCAGACTG-3⬘) for the UidA gene. Binary vectors were transferred to Agrobacterium tumefaciens via triparental mating. The procedure outlined by Valvekens et al. (1988) was used for the production of transgenic Arabidopsis
plants of C24 ecotype, and transformants were selected based on kanamycin
resistance. The mGCC, pIB, pA35, and an additional P3 construct were
introduced into Col-0 plants by using the flower-dip transformation procedure (Clough and Bent, 1998).
Plant Treatments for Promoter Activity Assays
For sterile-grown plants, Arabidopsis seeds were surface sterilized and
evenly positioned across petri dishes containing germination medium
(Valvekens et al., 1988) at a density of 70 seeds per plate. Seeds were
stratified at 4°C for 3 d, and the petri dishes were transferred to growth
cabinets with a 12-h photoperiod, at 24°C. Induction experiments were
carried out on 16-d-old seedlings. For soil-grown plants, seeds were germinated on germination medium in petri dishes, and 10-d-old seedlings were
transferred to soil, with five individuals planted in each 4- ⫻ 4-cm pot for
induction experiments using exogenous chemicals. For ease of inoculation,
only four individuals were transplanted per pot for experiments when
plants were challenged by the fungal pathogen Alternaria brassicicola. Plants
were grown under short days (8-h photoperiod) to delay bolting. Daytime
temperature was 21°C with 70% humidity, and nighttime temperature was
24°C, with 60% humidity. Induction experiments were performed on 5- to
6-week-old plants.
For plant treatments, the plants pots or tissue culture plates were placed
into 1-L phytocon vessels (Sigma-Aldrich, St. Louis), which were sealed
around the rim with plasticine. Ethylene was applied at a concentration of
200 ␮L L⫺1 by injection of gaseous ethylene through a rubber septum. MeJA
was applied at a concentration of 0.1 ␮mol L⫺1 air, by inoculation of 5 ␮L of
a 21.8 mm solution of MeJA (in ethanol) to a ball of cotton wool taped to the
inside of the container. Control plants were placed in identical chambers,
but without the addition of inducing agents. Samples were collected after
24 h of incubation in continuous light. For the seedling-based assay, 20
individual whole transgenic plants (T2 generation) were bulked for each
replicate sample. For each treatment, three replicate sets of samples were
taken from each of two independent experiments. Five plants were bulked
for each replicate in the mature plant assays. MeJA treatment of plants used
in additional real-time quantitative RT-PCR time-course experiments was
done according to Schenk et al. (2000).
Fungal Inoculation
Leaves of soil-grown plants were inoculated with 5-␮L drops of a freshly
harvested conidial spore suspension (5 ⫻ 105 spores mL⫺1 in water) of A.
brassicicola (isolate UQ4273). The plants were placed in a propagator flat
under a clear polystyrene lid to maintain high humidity. The treated leaves
were collected separately from the untreated leaves of the same plant, 5 h
after inoculation.
Quantitative GUS Assays for Promoter Activity
Transgenic plants at T2 generation (20 individuals) stably transformed
with each promoter reporter construct were used. Promoter activity was
assessed by quantitative GUS assays of the UidA gene, using 4-methyl-
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1029
Brown et al.
umbelliferyl-␤-d-glucuronide (Sigma-Aldrich) as a substrate. The assay was
carried out according to the procedure of Jefferson (1987), adapted for use
with microtitre plates and the Fluoroskan Ascent fluorometer (Labsystems,
Helsinki). The total protein content of samples was assessed using the
dye-binding protein assay adapted for microtitre plates (Bio-Rad Laboratories, Hercules, CA).
Transient Expression Assays
The pBINm-GFP5-ER plasmid (Haseloff et al., 1997) containing a synthetic GFP gene under the control of the 35S promoter (CaMV) was used as
a transformation control in all experiments. The pSP35Slucnos plasmid
containing the firefly luciferase gene under the control of the 35S promoter
(CaMV) was a gift from Dr. Robert Birch (Department of Botany, The
University of Queensland, Australia). PCR amplified PDF1.2-promoter
deletion fragments using the forward primers 5⬘-AACAAGCTTTTTAGTCCACAACG-3⬘, 5⬘-TGTAAGCTTATGACGAAGGTC-3⬘, and 5⬘-AAGCTTGACTATGAACTGC-3⬘ for constructs Ea, Eb, and Ec, respectively, and
a common reverse primer 5⬘-GAGAGAGGATCCTGATGGAAGCAAATTAGCCATG-3⬘ for all constructs was cloned, in frame with the UidA gene,
into the 4.87-kb vector pGEM4.GUS3 using standard molecular biology
techniques (Sambrook et al., 1989). Bombardments were carried out using a
custom-made helium pressure-driven particle inflow gun as previously
described (Schenk et al., 1998). GFP-expressing cells in the bombarded
leaves were viewed using an MZ6 stereomicroscope with fluorescence GFP
Plus filter module (Leica Microscopy and Scientific Instruments, Bannockburn, IL), containing a 480/40-nm excitation filter, a 505-nm long pass
dichromatic beam-splitting mirror, and a 510-nm long pass barrier filter.
Luciferase activity was detected using Luciferase Assay Reagent (Promega,
Madison, WI) in a Fluoroskan Ascent FL luminometer (Labsystems).
GACTCATTAG-3⬘; for putative protein (At4g31060), 5⬘-AGGCAACCAAAAAGTGGCG-3⬘ and 5⬘-CAACCATTCCGTGTTCTCCATC-3⬘; and for
APETALA2 (At4g36920), 5⬘-AACGACGCACCACACCAAAC-3⬘ and 5⬘AAAGCCAGAAGCAACACCACC-3⬘. For normalization purposes, the
macroarray filters also contained DNA sequences from the following genes
amplified using the following primer pairs: PR-1, 5⬘-CGGCGACAAGACTACCTTGATG-3⬘ and 5⬘-TGGCACATCCAACCCACTCTG-3⬘; PDF1.2,
5⬘-CGCACCGGCAATGGTGGAAG-3⬘ and 5⬘-CACACGATTTAGCACCAAAG-3⬘; BGL-2, 5⬘-GTAAAACGACGGCCAGT-3⬘ and 5⬘-GGAAACAGCTATGACCATG-3⬘; UidA, 5⬘-ACCCTTACGCTGAAGAGATGC-3⬘,
and 5⬘-AACGTATCCACGCCGTATTCG-3⬘; Actin, 5⬘-GGATGCTTATGTTGGCGATG-3⬘ and 5⬘-GCTGGTTTTGGCTGTCTCG-3⬘; and Tubulin ␤-8 chain,
5⬘-GTAAAACGACGGCCAGT-3⬘ and 5⬘-GGAAACAGCTATGACCATG-3⬘.
Macroarray filters were prepared as follows. Probe DNA (40 ng spot⫺1) was
denatured and transferred in duplicate to a Hybond N⫹ membrane (Amersham Biosciences, Piscataway, NJ) using the Bio-Dot microfiltration apparatus (Bio-Rad Laboratories), according to the manufacturer’s instructions. Total cDNA synthesis was performed using 750 ng of purified
poly(A⫹) RNA using Superscript II RT (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. The total cDNA prepared was labeled
either using the Megaprime DNA labeling system (Amersham Biosciences)
or the Strip-EZ system (Ambion, Austin TX) as instructed by the manufacturer. Blots were hybridized as previously described (Mitter et al., 1998) and
were visualized by exposure to a PhosporImaging system, and data were
processed with the ImageQuant analysis software (Molecular Dynamics,
Sunnyvale, CA). For each hybridization blot, a reference figure was calculated by averaging the hybridization signal from the duplicate ␤-tubulin
spots minus the background. Data for probes from all other genes were
determined similarly and then divided by the ␤-tubulin reference for that
blot to allow comparison of relative expression levels of the different genes.
Northern-Blot Analysis
Macroarray Analysis
The consensus amino acid sequence for the DNA-binding domain of the
AP2/ERF transcription factor family (YRGV RRPW GKWA AEIR DPKR
VWLG TFTA EEAA RAYD AARG KALN FP; Riechmann and Meyerowitz,
1998) was used as a query sequence in a BLASTP search of the nonredundant
GenBank protein database, limited to Arabidopsis. Subfamily allocation was
determined by searching for the conserved linker sequence (E-V—LRR-S—FRG; Klucher et al., 1996) occurring between the two conserved AP2 domains
of AP2 subfamily members. ClustalW was used to analyze sequence similarity within the conserved domain and to produce a guide tree of relatedness.
For macroarray construction, genomic DNA was extracted from Arabidopsis
ecotype Col-0 plants using the CTAB method. The following forward and
reverse primers sequences were used for amplification of divergent regions of
AP2/ERF sequences from the Arabidopsis genome: for ERF1 (At3g23240),
5⬘-CGGCGGAGAGAGTTCAAGAGTC-3⬘ and 5⬘-TCCCACTATTTTCAGAAGACCCC-3⬘; for AtERF2 (At5g47220), 5⬘-TTGGGGAGGTTTGCCATTG-3⬘
and 5⬘-TCCATCGCCGTAAAGTTCTCAG-3⬘; for AtERF3 (At1g50640),
5⬘-CCGACCATCAACAACAGTTCCC-3⬘ and 5⬘-CGACGAATCGCAATCGCTG-3⬘; for EREBP4/RAP2.5/AtERF4 (At3g15210), 5⬘-TCTACTTTTTGGACCTGATGGGG-3⬘ and 5⬘-TCGCTTTGGGCACCACAAG3⬘; for AtERF5 (At5g47230), 5⬘-ACGAGGCTTCTCCTGTGGCTAC-3⬘ and
5⬘-GCGAAATCTTCAATGGCGG-3⬘; for putative EREBP (At2g44970), 5⬘CGATAACTGGAGCGACTTGCC-3⬘ and 5⬘-GGAGAGGTAACGGGAGGAACG-3⬘; for TINY isolog (At1g22810), 5⬘-TCCTTCACTCGTTTCCAGAACTTC-3⬘ and 5⬘-GCCGCCCAGATAATCATACACTG-3⬘; for gene
similar to tobacco EREBP3 (At1g12980), 5⬘-AGAAATCTTCGCCGTCTGCTC-3⬘ and 5⬘-TCTTGATACCCCCACTCGTTTG-3⬘; for AP9 (At1g80580),
5⬘-AACCCGAAAACACGCATC-3⬘ and 5⬘-GCTTCACACCACCTTCTTTGG-3⬘; for RAP2.10 (At4g36900), 5⬘-AGGAGGAGTGAACGGTGGTG-3⬘
and 5⬘-GATGATGATGATGACGATTCCC-3⬘; for putative AP2 (At1g63030),
5⬘-ATTCTGCTTGGAGGTTGCCG-3⬘ and 5⬘-CTCCAAAGTGACAAATCTTC-3⬘; for putative AP2 (At2g35700), 5⬘-CATCTCCCACAGTTACGGAAAC-3⬘ and 5⬘-AAAAGAGTCCCAGAAGCCATC-3⬘; for putative AP2
(At1g63040), 5⬘-TCTTACCCCATTCCCCTTTCC-3⬘ and 5⬘-AGCCACCATCATCCCTTTGG-3⬘; for putative AP2 (At2g46310), 5⬘-CGAGGGAATCGCAAATCAGTC-3⬘ and 5⬘-CGAGAAACAAAGGGTCAGGGG-3⬘; for AP2
transcription factor (At2g40340), 5⬘-CAAGTTCAGGTTTTGGTCAGGTG-3⬘
and 5⬘-GCAATCTCCATAGGGTTGAGGC-3⬘; for putative AP2 (At2g22200),
5⬘-TGAATCCTCTCCCTTCCTCTGTTG-3⬘ and 5⬘-CATCGCTTCTCGGT-
1030
Arabidopsis ecotype Col-0 plants were grown under short-day conditions
(8 of h light, 16 h of dark) for 3 weeks and treated with MeJA, ethylene, or
nothing (controls), as described above for GUS activity assays. Total cellular
RNA was extracted from the aerial portion of the plants using the guanidine
thiocyanate method and a 20-␮g sample of total RNA was run on the gel and
blotted onto a Hybond N⫹ membrane (Amersham Biosciences). Probes were
amplified by PCR and purified using the Qiaquick PCR purification kit
(Qiagen USA, Valencia, CA). The ERF probes were amplified using the primer
sequences given above. The PDF1.2 probe was amplified from Clone 10a
(Manners et al., 1998) using the primers 5⬘-CGCACCGGCAATGGTGGAAG-3⬘ and 5⬘-CACACGATTTAGCACCAAAG-3⬘. The tubulin ␤-8 chain
probe was amplified from EST clone number 108K21T7 (GenBank accession
no. T41808; Arabidopsis Biological Resource Center, Columbus, OH) using
the M13 forward and M13 reverse primers. The hybridizations and analysis of
hybridization signals were done as described for macroarray hybridizations.
Real-Time Quantitative RT-PCR Analysis
Total cellular RNA was extracted from the aerial portion of the plants
using Plant RNA extraction kit (Bio-Rad Laboratories). First-strand cDNA
was synthesized using 2 ␮g of total RNA, Superscript II RT (Invitrogen), and
random hexamer primers to a total volume of 10 ␮L. The reaction mix was
incubated at 42°C for 50 min, before heat inactivation of the enzyme at 70°C
for 15 min. The cDNA template was then diluted by adding 240 ␮L of milli-Q
water, to give a final concentration of 8 ng ␮L⫺1 for use in real-time PCR
assays. A second 2-␮g aliquot of each RNA sample was subjected to the same
conditions, without the addition of RT (the minus RT controls). Real-time PCR
amplification and detection was carried out in an ABI model 7700 Sequence
Detection System (Applied Biosystems, Foster City, CA). Reactions were in
final volumes of 25 ␮L and contained 40 ng of cDNA template and 5 ␮L of
primer mix containing a 1 ␮m solution of each of the AtERF2 primers,
5⬘-TGAGGTTAATTCCGGTGAACC-3⬘ and 5⬘-TCAACTTCCCGTTTTCAGACGA-3⬘; ERF1 primers, 5⬘-CGAGAAGCTCGGGTGGTAGT-3⬘ and
5⬘-GCCGTGCATCCTTTTCC-3⬘; PDF1.2 (At5g44420) primers, 5⬘-TTGCTGCTTTCGACGCA-3⬘ and 5⬘-TGTCCCACTTGGCTTCTCG-3⬘; Thi2.1
(L41244.1) primers (Epple et al., 1995), 5⬘-CTCAGCTGATGCTACCAATGAGC-3⬘ and 5⬘-GCTCCATTCACAATTTCACTTGC-3⬘; or PR4
(At3g12500) primers, 5⬘-ATCAGCGCTGCAAAGTCCTTC-3⬘ and 5⬘-
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Copyright © 2003 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 132, 2003
GCC-Box and Jasmonate-Mediated Activation of the PDF1.2 Gene
GTGCTGTAGCCCATCCACCTG-3⬘; and 12.5 ␮L of SYBR Green PCR master mix (Applied Biosystems). The thermal cycling conditions were: 10 min
at 95°C, 45 cycles of 15 s at 95°C and 1 min at 60°C. Duplicate SYBR Green
assays for each gene target were performed on both plus and minus RT
samples. No-template controls for each primer pair were included in each
run. Expression detected from three ␤-actin genes of Arabidopsis, ␤-actin-2
(At3g18780), ␤-actin-7 (At5g09810), and ␤-actin-8 (At1g49240) used the
following mixture of primers (R. Simpson, personal communication; a
universal actin forward primer 5⬘-AGTGGTCGTACAACCGGTATTGT-3⬘
and specific reverse primers 5⬘-GATGGCATGGAGGAAGAGAGAAAC3⬘, 5⬘-GAGGAAGAGCATTCCCCTCGTA-3⬘, and 5⬘-GAGGATAGCATGTGGAACTGAGAA-3⬘, respectively) were used as combined internal
standards to normalize small differences in template amounts. Real-time
PCR results were captured and analyzed using the Sequence Detector
Software (SDS v1.7, Applied Biosystems) and data analysis procedures
described in the User Bulletin 2 for the ABI model 7700 Sequence Detection
System.
Construction of the 35S-AtERF2 Binary Vector and
Plant Transformation
The AtERF2 (AT5g47220) gene of Arabidopsis was amplified from the
Col-0 ecotype using the primers 5⬘-CTGAAAATGTACGGACAGTGC-3⬘
and 5⬘-AACTTATGAACCAATAACTC-3⬘. The amplification product was
treated with 1 unit of polynucleotide kinase (Roche Diagnostics, Mannheim,
Germany), ligated into the binary vector pKEN cut by HindIII, filled in by
Klenow DNA polymerase (Roche Diagnostics), and dephosphorylated.
pKEN was constructed by cloning a SacI/XhoI fragment containing the
double 35S promoter/terminator cassette from the plasmid pJIT163 (Hellens
et al., 2000) into SacI/XhoI-digested pGreen229 (Hellens et al., 2000). The
recombinant plasmid was electroporated into competent A. tumefaciens
(strain AGL-1) cells previously transformed with pSoup plasmid (Hellens et
al., 2000). Arabidopsis Col-0 plants were then transformed using the floral
dip transformation procedure (Clough and Bent, 1998), and the transformants were selected based on their resistance to Basta.
Statistical Analysis of Data
The JMP IN Statistics package v3.2.6 (SAS Institute Inc., Cary, NC) was
used for all statistical analyses. The significance of differences between
transgenic plants carrying the various promoter deletion constructs and
independent transgenic lines within these constructs was determined by
nested ANOVA. The non-parametrical Wilcoxen-Mann-Whitney test was
used to determine statistical significance of differences between constructs
in bombardment experiments. Biological significance was assigned at P ⬍
0.05.
ACKNOWLEDGMENTS
We thank Dr. John Turner and the Arabidopsis Stock Center at Ohio State
University for cev1 and cpr5 seeds, respectively; Jonathan Anderson for cpr5
cDNA samples; Dr. Peer Schenk for advice in plasmid construction and
some of the cDNA samples; Anca Rusu for GUS activity analyses of some of
the promoter-reporter lines; Dr. Philip Mullineaux for the pGreen0229,
pSoup, and pJIT163 plasmids; Dr. Robert Birch for the pSP35Slucnos plasmid; Dr. Jimmy Botella for the pAOV plasmid; Dr. Jim Haseloff for the
pBINm-GFP5-ER plasmid; and Dr. Willem Broekaert for his advice on plant
treatments and collaboration.
Received November 14, 2002; returned for revision January 4, 2003; accepted
March 5, 2003.
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