Introduction

Plants undergo continuous exposure to various biotic and abiotic stresses in their natural environment. To respond optimally to environmental stresses including attack by microbial pathogens plants have evolved intricate mechanisms to perceive external signals and transduce them into the appropriate gene expression response (Pieterse et al. 2009). Perception of stress signals leads to the production of one or more of the secondary signaling molecules jasmonates (JAs), ethylene (ET), or salicylic acid (SA). JAs are a group of related lipid-derived signaling molecules including the namesake compound jasmonic acid (JA) (Wasternack 2007).

In general, it can be stated that SA-dependent gene expression responses are effective against pathogens with a biotrophic lifestyle, whereas defense genes activated by a combination of JA and ET are effective against pathogens with a necrotrophic lifestyle (Glazebrook 2005). The JA/ET-dependent defense response is characterized by the production of antimicrobial PLANT DEFENSIN (PDF) proteins (Penninckx et al. 1998). In plants grown under sterile conditions PDF1.2 mRNA accumulation is weakly induced by JA or ET, but is strongly induced by a combination of both hormones (Penninckx et al. 1998). In soil-grown plants, exogenous application of either JA or ethylene strongly induces PDF1.2 expression, presumably because the plants already produce low amounts of the signaling molecules. Mutant analysis shows that the response of soil-grown plants to either hormone is still dependent on functional JA and ET signaling pathways. Accumulation of PDF1.2 mRNA is commonly used as a marker for activation of the JA/ET signaling pathway. To understand what this marker actually reports it is crucial to understand the regulation of PDF1.2 mRNA accumulation.

A large number of transcription factors have been reported to affect PDF1.2 mRNA accumulation when overexpressed or when inactivated by mutation or by RNA interference (RNAi). Positive effects were observed upon overexpression of the APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factors ERF1 (Solano et al. 1998; Lorenzo et al. 2003), OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF-domain protein 59 (ORA59) (Pré et al. 2008), AtERF1 (Pré et al. 2008) or AtERF2 (Brown et al. 2003), or the basic leucine zipper (bZIP) transcription factor TGA5 (Zander et al. 2009). Knocking out the AtMYC2 gene encoding a basic helix-loop-helix (bHLH) transcription factor (Boter et al. 2004; Lorenzo et al. 2004) or the AtERF4 gene encoding an AP2/ERF-domain transcription factor with an ERF-associated repression (EAR) motif (McGrath et al. 2005) also stimulated the expression of the PDF1.2 gene. Negative effects were observed upon overexpression of AtERF4 (McGrath et al. 2005), the WRKY transcription factor WRKY70 (Li et al. 2004), upon triple knockout of the TGA2, 5 and 6 transcription factor genes (Zander et al. 2009) or upon downregulation of ORA59 expression via RNAi (Pré et al. 2008).

Direct interaction of these transcription factors with the PDF1.2 promoter has not been reported except for TGA2 (Spoel et al. 2003), which was shown to bind in vitro without a documented in vivo relevance. In fact the TGA factors are thought to act indirectly (Zander et al. 2009). Among the positive regulators, ORA59 and ERF1 were shown to activate the PDF1.2 promoter in transient assays in protoplasts (Pré et al. 2008), suggesting that they bind directly to the promoter. AtERF1 and AtERF2 were not active in this assay (Pré et al. 2008), indicating that they act indirectly. Analysis of transgenic plants in which ORA59 gene expression was silenced by RNAi, whereas the ERF1 gene was normally expressed, showed that PDF1.2 expression in response to JA and to JA/ET was abolished (Pré et al. 2008). This establishes ORA59 as the main positive regulator of PDF1.2 expression in response to JA/ET (Memelink 2009).

Studies of the PDF1.2 promoter in transgenic plants showed that it can confer JA-responsive gene expression to a β-glucuronidase (GUS) reporter gene (Brown et al. 2003; Manners et al. 1998). The promoter was reported to be unresponsive to ET (Manners et al. 1998). It was not studied whether the PDF1.2 promoter can confer synergistic gene expression to the combination of JA and ET. It is generally assumed that JA and ET affect PDF1.2 mRNA accumulation at the transcriptional level, but it cannot be excluded that synergism is caused by stimulation of PDF1.2 promoter activity by JA (Brown et al. 2003; Manners et al. 1998) and PDF1.2 mRNA stabilization by ET. In this context it should be noted that the ET signal transduction component ETHYLENE INSENSITIVE 5 (EIN5) is an exoribonuclease which affects stability of certain mRNAs (Olmedo et al. 2006). Brown et al. (2003) identified one GCC-box in the PDF1.2 promoter which is involved in the JA response. It was not studied whether this GCC box is also involved in the JA/ET response. GCC boxes are binding sites for AP2/ERF-domain transcription factors (Hao et al. 1998). However no binding studies were reported with the GCC box from the PDF1.2 promoter and AP2/ERF-domain proteins.

The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with the key positive regulator ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box.

Materials and methods

Growth conditions and treatments

Arabidopsis thaliana ecotype Columbia (Col-0) was the genetic background for all wild type and transgenic plants. Following stratification for 3 days at 4°C, surface-sterilized seeds were germinated for 10 days at 21°C in a growth chamber (16 h light/8 h dark, 2,500 lux at 70% humidity) on plates containing MA medium (Masson and Paszkowski 1992) with 0.6% agar supplemented with 20 mg/l hygromycin for selection of transgenic plants. Batches of 15–20 seedlings were transferred to 50 ml polypropylene tubes (Sarstedt) containing 10 ml liquid MA medium without antibiotic and the tubes were incubated on a shaker at 120 rpm for 4 additional days before treatments. Transgenic plants carrying an XVE expression module containing the ORA59 gene fused to the TAP tag were treated for 16 h with 4 μM estradiol. As control, seedlings were treated with dimethyl sulfoxide (DMSO; 0.1% v/v final concentration). Transgenic seedlings carrying the PDF1.2 promoter derivatives LF, SF, m1, m2 or dm or the wild-type and mutant GCC box tetramers fused to GUS were treated for 24 h with 50 μM JA (Sigma–Aldrich, St. Louis, MO) dissolved in DMSO (0.1% v/v final concentration), 1 mM of the ethylene-releasing compound ethephon (Sigma–Aldrich) dissolved in 50 mM sodium phosphate pH 7 (0.5 mM final concentration) or a combination of JA and ethephon. As control, seedlings were treated with 0.1% DMSO and 0.5 mM sodium phosphate pH 7.

PDF1.2 promoter and constitutive overexpression constructs

Arabidopsis genomic DNA was used as template for the amplification of PDF1.2 (At5g44420) promoter fragments LF and SF with forward primers 5′-CGG GAT CCA TGC AGC ATG CAT CGC CGC ATC-3′ or 5′ CGG GAT CCC CAT TCA GAT TAA CCA GCC GCC C-3′, respectively, and the reverse primer 5′-GCG TCG ACG ATG ATT ATT ACT ATT TTG TTT TCA ATG-3′. Amplified products were digested with BamHI and SalI and cloned in plasmid GusXX (Pasquali et al. 1994). Mutations m1, m2 and dm were generated according to the QuickChange Site-Directed Mutagenesis protocol (Stratagene) and primers 5′-CCA TTC AGA TTA ACC ATC CTC ACC TGT GAA CGA TG-3′ or 5′-CAT TAG CTA AAA GCC GAA TCA TCC TCT TAG GTT ACT TTA GAT ATC G-3′, and their respective reverse complementary primers. For the construction of the GCC box tetramers, wild-type and mutant GCC box monomers of the PDF1.2 promoter were cloned by annealing the oligonucleotides 5′-GATC CTT AAC CAG CCG CCC ATG TGA-3′ and 5′-GAT CTC ACA TGG GCG GCT GGT TAA G-3′, and 5′-GATC CTT AAC CAT CCT CAC ATG TGA-3′ and 5′-GAT CTC ACA TGT GAG GAT GGT TAA G-3′, respectively, and ligating them into the plasmid pIC-20H (Marsh et al. 1984) digested with BamHI and BglII. Monomers were then tetramerized in a head-to-tail configuration using the BamHI and BglII sites. The tetramers were cloned as BamHI/BglII fragments in the plasmid GusSH-47 (Pasquali et al. 1994) digested with BamHI such that the orientation of the GCC-boxes relative to the downstream ORF was the same as in the PDF1.2 promoter. The ORA59 (At1g06160) open reading frame (ORF) was PCR-amplified from Arabidopsis genomic DNA using the primer set 5′-CGG GAT CCA TAT GGA ATA TCA AAC TAA CTT C-3′ and 5′-CGG GAT CCT CAA GAA CAT GAT CTC ATA AG-3′, digested with BamHI and cloned in pRT101 (Töpfer et al. 1987). The ERF1 (At3g23240) ORF was PCR-amplified using the primer set 5′-GAA GAT CTT CAT CAC CAA GTC CCA CTA TTT TC-3′ and 5′-GAA GAT CTC ATA TGG ACC CAT TTT TAA TTC AGT CC-3′, digested with BglII and cloned in BamHI-digested pRT101. The ORA47 (At1g74930) ORF was PCR-amplified from Arabidopsis genomic DNA using the primer set 5′-GAA GAT CTC ATA TGG TGA AGC AAG CGA TGA AG-3′ and 5′-GAA GAT CTT CAA AAA TCC CAA AGA ATC AAA G-3′ and following digestion with BglII cloned in pIC-20R (Marsh et al. 1984). The ORA47 insert was excised with BglII and inserted into BamHI-digested pMOG183 (Mogen International, Leiden, The Netherlands), a pUC18 derivative carrying a double-enhanced CaMV 35 S promoter and the nos terminator separated by a BamHI site.

Binary constructs and plant transformation

The TAP insert was excised from pBS1479 (Puig et al. 2001) with BamHI and cloned into pC1300intB-35SnosBK (accession number AY560326) digested with BglII. pC1300intB-35SnosBK is a derivative of the binary vector pCAMBIA1300 carrying a CaMV 35S expression cassette. The ORA59 ORF lacking the stop codon (ORA59-Δstop) was amplified by PCR with the primer set 5′-ACG CGT CGA CAA AAT GGA ATA TCA AAC TAA CTT C-3′ and 5′ CCG CTC GAG CCT TGA GAA CAT GAT CTC ATA AG-3′ and cloned in pGEM-T Easy (Promega). The ORA59 ORF was excised from pGEM-T Easy with SalI/XhoI and cloned into pC1300intB-35SnosBK-TAP. The ORA59-TAP fusion was excised with SalI/SpeI from pC1300intB-35SnosBK-ORA59-TAP and introduced into the binary vector pER8 (Zuo et al. 2000) digested with XhoI/SpeI. The PDF1.2 promoter derivatives SF, m1, m2 and dm fused to GUS and the tetrameric constructs 4xGCC:GUS and 4xmGCC:GUS were cloned into binary vector pMOG22λCAT (Pasquali et al. 1994; Menke et al. 1999) with XbaI/XhoI and XbaI/HindIII, respectively. The pMOG22λCAT binary vectors were introduced into Agrobacterium tumefaciens strain EHA105 (Hood et al. 1993). Arabidopsis plants were transformed using the floral dip method (Clough and Bent 1998). Transgenic plants were selected on MA medium containing 100 mg/l timentin and 20 mg/l hygromycin.

Protein production and immunoblot analysis

ORA59, ERF1 and ORA47 proteins were produced with N- and C-terminal Strep and His tags, respectively. ORA59 was amplified with the primer set 5′-CGG AAT TCA ATG GAA TAT CAA ACT AAC TTC-3′ and 5′-CGG TCG ACC CTT GAG AAC ATG ATC TCA TAA G-3′, digested with EcoRI and SalI and cloned in pASK-IBA45plus (IBA Biotagnology, Göttingen, Germany). ERF1 was amplified with the primer set 5′-CGG AAT TCA ATG GAC CCA TTT TTA ATT CAG-3′ and 5′-CGG TCG ACC CTT GCC AAG TCC CAC TAT TTT C-3′, digested with EcoRI and XhoI and cloned in pASK-IBA45 digested with EcoRI and SalI. ORA47 was amplified with the primer set 5′-CGG AAT TCA ATG GTG AAG CAA GCG ATG AAG-3′ and 5′-CGG TCG ACC CTT GAA AAT CCC AAA GAA TC-3′, digested with EcoRI and SalI and cloned in pASK-IBA45plus. The proteins were expressed in Escherichia coli strain BL21(DE3)pLysS (Novagen). Since the large majority of ORA59 and ERF1 proteins was insoluble and the remaining soluble part was mostly degraded, proteins were purified from inclusion bodies harvested by centrifugation of cells lysed by freeze-thawing by denaturation in Ni–NTA binding buffer (5 mM imidazole, 0.5 M NaCl, 40 mM Tris–HCl pH 8.0) with 6 M urea and re-folded by a quick tenfold dilution in binding buffer without urea followed by 16 h dialysis against binding buffer. All proteins were purified by sequential Ni–NTA agarose (Qiagen) and Strep-Tactin Sepharose (IBA) chromatography according to the Novagen His tag and the IBA Strep tag purification protocols. Proteins were separated by 10% (w/v) SDS–PAGE and transferred to Protan nitrocellulose (Schleicher & Schuell) by semi-dry electroblotting. Recombinant proteins isolated from E. coli were detected with Penta-His HRP antibody conjugate (Qiagen 1:20000), following blocking with Penta-His HRP blocking agent. TAP-tagged proteins expressed in plants were detected with peroxidase anti-peroxidase (PAP; Sigma–Aldrich 1:10000) antibodies and 5% nonfat dry milk as blocking agent. Plant proteins were extracted by grinding frozen tissue samples (0.2 g) in liquid nitrogen and thawing the powder in 0.25 ml protein extraction buffer (PBS buffer; 137 mM NaCl, 27 mM KCl, 100 mM NaHPO4, 2 mM K2HPO4, pH 7.4, 1× Complete protease inhibitor Cocktail (Roche) and 0.5% v/v Triton ×100). After centrifugation at 15,000×g for 10 min at 4°C, supernatants were transferred to clean tubes, frozen in liquid nitrogen, and stored at −80°C. Protein concentrations were determined using the Bio-Rad protein assay reagent with bovine serum albumin as the standard. Detection was carried out by incubating the blots in 10 ml luminol solution (250 μM sodium luminol (Sigma–Aldrich), 0.1 M Tris–HCl pH 8.6, 0.01% H2O2) mixed with 60 μl enhancer solution [67 μM p-hydroxy coumaric acid (Sigma–Aldrich)] in DMSO and exposure to X-ray films (Fuji, Tokyo, Japan).

Electrophoretic mobility shift assays

PDF1.2 promoter derivatives SF, m1, m2 and dm were isolated from the GusXX plasmid with Xba and BglII. Wild-type and mutated versions of a GCC-like box from the AOC2 promoter with the sequences 5′-GGA TCC TTT AGG GAC CGG CCA AAA GTA AGA TCT-3′ and 5′-GGA TCC TTT AGG GAT CGT CCA AAA GTA AGA TCT-3′ were cloned into pIC-20H digested with BamHI/BglII and fragments were excised with SalI and HindIII. Promoter fragments were labeled by filling in the overhangs with the Klenow fragment of DNA polymerase I and α-32P-dCTP. DNA binding reactions containing 0.1 ng of end-labeled DNA probe, 500 ng of poly(dAdT)-poly(dAdT), binding buffer (25 mM HEPES–KOH pH 7.2, 100 mM KCl, 0.1 mM EDTA, 10% glycerol), and protein extract in a 10 μl volume, were incubated for 30 min at room temperature before loading on 5% w/v acrylamide/bisacrylamide (37:1)-0.5× Tris–Borate-EDTA gels under tension. Binding buffer conditions were optimized for ORA59 and ORA47 protein by addition of 25 ng of sonicated herring sperm DNA and 1 mM or 0.25 mM DTT to the binding buffer, respectively. After electrophoresis at 125 V for 1 h, gels were dried on Whatman DE81 paper and exposed to Fuji X-ray films.

Transient expression assays

Protoplasts prepared from Arabidopsis cell suspension ecotype Col-0 were co-transformed with plasmids carrying one of the PDF1.2-promoter-GUS versions, effector plasmids carrying ORA59, ERF1 or ORA47 fused to the CaMV 35S promoter and the p2rL7 plasmid (De Sutter et al. 2005) carrying the Renilla reniformis luciferase (LUC) gene under the control of the CaMV 35S promoter. As controls, co-transformations of PDF1.2-promoter-GUS with the empty pRT101 expression vector and the p2rL7 plasmid were carried out. Protoplasts were transformed using polyethylene glycol as described previously (Schirawski et al. 2000) with the three constructs in a ratio of 2:2:6 (μg GUS:LUC:effector plasmid). To study a possible synergistic effect of ORA59 and ERF1 a ratio of 2:2:1 (μg GUS:LUC:effector plasmid) was chosen. The protoplasts were harvested 18 h after transformation and were frozen in liquid nitrogen. GUS and LUC activity assays were performed as described (van der Fits and Memelink 1997; Dyer et al. 2000). GUS activities were related to LUC activities in the same samples to correct for differences in transformation and protein extraction efficiencies. Alternatively, differences in protein extraction efficiencies were corrected for protein concentration.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments were performed according to Bowler et al. (2004) with some modifications. Two grams of 2 weeks-old seedlings constitutively overexpressing TAP (line #7) or seedlings from XVE-ORA59-TAP transgenic line #4 treated with 0.1% DMSO or 4 μM estradiol for 16 h in liquid MA medium were harvested. A small part of the samples was used for mRNA and protein detection. The rest was infiltrated with 1% formaldehyde to crosslink protein and DNA and chromatin sonicated to an average size of 400 bp was prepared. IgG Sepharose 6 fast flow (GE Healthcare) preabsorbed with salmon sperm DNA (0.1 mg/ml) and BSA (1 mg/ml) in ChIP dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) was added to chromatin preparations and the mixtures were rotated at 4°C for 6 h to bind TAP or TAP-fusion protein. After 5 times washing the beads (Bowler et al. 2004), DNA recovered from the beads and sonicated chromatin input were reverse cross linked and analyzed by semi-quantitative PCR. The PDF1.2 promoter was amplified for 34 cycles using the primer set 5′-TAT ACT TGT GTA ACT ATG GCT TGG-3′ and 5′-TGT TGA TGG CTG GTT TCT CC-3′ located up and down stream of the two GCC boxes. For amplification of the AOC2 promoter the primer set 5′-CAT GTA TTT TCA TTC CAA GAG CAG C-3′ and 5′-GAT GCT TTG GGA GGA ATT TGG-3′ was used at 34 or 36 cycles.

RNA extraction and Northern blot analysis

Total RNA was isolated from tissue ground in liquid nitrogen by extraction with two volumes of phenol buffer (1:1 mixture of phenol containing 0.1% w/v 8-hydroxyquinoline and buffer containing 100 mM LiCl, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 100 mM Tris) and one volume of chloroform. After centrifugation, the aqueous phase was re-extracted with one volume of chloroform. RNA was precipitated overnight with LiCl at a final concentration of 2 M, washed twice with 70% ethanol, and resuspended in water. Northern blot analyses were performed as described (Memelink et al. 1994). Briefly, 10 μg RNA samples were subjected to electrophoresis on 1.5% w/v agarose/1% v/v formaldehyde gels, and blotted to GeneScreen nylon membranes (Perkin-Elmer Life Sciences, Boston, MA). All probes were 32P-labeled by random priming. Pre-hybridization of blots, hybridization of probes and subsequent washing were performed as described (Memelink et al. 1994) with minor modifications. Blots were exposed to Fuji X-ray films. The PDF1.2 probe was PCR amplified from Arabidopsis genomic DNA using the primer set 5′-AAT GAG CTC TCA TGG CTA AGT TTG CTT CC-3′ and 5′-AAT CCA TGG AAT ACA CAC GAT TTA GCA CC-3′. The TAP probe was excised from pBS1479 (Puig et al. 2001) with BamHI.

Results

ORA59 and ERF1 trans-activate the PDF1.2 promoter in a dose-dependent manner

PDF1.2 promoter fragments containing 1,187 bp (LF) or 278 bp (SF) upstream of the probable transcription start site (Manners et al. 1998) were fused to the β-glucuronidase (GUS) reporter gene (Fig. 1).

Fig. 1
figure 1

PDF1.2 promoter constructs. Reporter constructs consisted of the GUS gene driven by wild-type or mutated LF (long fragment −1,187 to +48) or SF (short fragment −278 to +48) PDF1.2 promoter derivatives. Bold and lowercase nucleotides indicate point mutations in GCC boxes. Numbers indicate positions relative to the start site of transcription

Full size image

To study the dose–response relationship for trans-activation of the PDF1.2 promoter by ORA59 (At1g06160) and ERF1 (At3g23240), Arabidopsis protoplasts were co-transformed with the SF promoter derivative fused to GUS, and variable amounts of effector plasmids carrying the ORA59, ERF1 or ORA47 (At1g74930) genes fused to the CaMV 35S promoter (Fig. S1). ORA59 and ERF1 activated the SF-GUS reporter gene 40- or tenfold respectively, whereas the unrelated AP2/ERF-domain transcription factor ORA47 had no effect. ORA47 trans-activated the promoter of the ALLENE OXIDE CYCLASE 2 (AOC2) gene under these conditions in the protoplast assay (results not shown), demonstrating that ORA47 is expressed and active. Previously we have shown that AtERF1 (At4g17500) and AtERF2 (At5g47220) did not significantly trans-activate the SF promoter derivative in a similar experimental setup (Pré et al. 2008). Together these observations indicate that ORA59 and ERF1 have a specific activating effect on the PDF1.2 promoter. The trans-activation of the SF promoter was dose-dependent and increased up to 6 μg of effector plasmid, where after the response saturated. Based on these observations, in the following trans-activation experiments 6 μg of effector plasmids were used for maximum responses (Figs. 2a, 6a) and 0.5–1 μg for studies on synergism between ORA59 and ERF1 (Fig. 2b).

Fig. 2
figure 2

ORA59 and ERF1 trans-activate the PDF1.2 promoter independently via two GCC boxes. a Arabidopsis cell suspension protoplasts were co-transformed with plasmids carrying different versions of the PDF1.2 promoter shown in Fig. 1 fused to GUS and overexpression vectors without or with the ORA59 or ERF1 genes driven by the CaMV 35S promoter. Values represent means ± SE of triplicate experiments. b The SF-GUS reporter plasmid was co-transformed with 1 μg of overexpression vectors carrying ORA59 or ERF1, or with a combination of 0.5 μg of each overexpression plasmid. Values represent means ± SE of triplicate experiments and are expressed relative to the vector control. Letters above the bars indicate different groups as determined by one-way ANOVA (P < 0.05)

Full size image

ORA59 and ERF1 trans-activate the PDF1.2 -278 promoter independently via two GCC boxes

Transient expression assays revealed that the short SF derivative conferred GUS expression to a similar level as the long LF derivative both with ORA59 as well as ERF1 (Fig. 2a), indicating that all cis-acting elements interacting with these two transcription factors are contained within the SF derivative. In the SF derivative a GCC box (GCCGCC) at positions −262 to −257 was pointed out as being important for the JA-responsive activity of this promoter derivative (Brown et al. 2003). To study whether ORA59 and ERF1 act via this GCC box, we mutated it generating the SFm1 promoter derivative (Fig. 1). This mutation reduced GUS activity conferred by ORA59 and ERF1 1.5–twofold, indicating that it is important but that there are other sequences interacting with these transcription factors. Indeed, there is another GCC-like box (GCAGCCGCT) at positions −222 to −214 (Fig. 1). Therefore we generated SFm2 and SFdm promoter derivatives carrying mutations in the second GCC box and in both GCC boxes, respectively. The m2 mutation reduced PDF1.2 promoter activity about twofold, similar to the m1 mutation. The double mutant version was activated five to sixfold less efficiently by ORA59 and ERF1 than the wild-type derivative. These results indicate that the two GCC boxes are functionally equivalent and are the main sites interacting with ORA59 and ERF1 (Fig. 2a).

To find out whether is there is a synergistic effect of ORA59 and ERF1 on activation of the PDF1.2 promoter, we co-transformed identical amounts of effector plasmids carrying ORA59 or ERF1 alone or as a mixture with the SF-GUS reporter construct. The results show that the mixture conferred an activity which was intermediate between the activity of each separate effector, indicating that ORA59 and ERF1 act additively instead of synergistically (Fig. 2b).

ORA59 and ERF1 bind to the two GCC-boxes in the PDF1.2 promoter in vitro

To establish whether ORA59 and ERF1 indeed bind the GCC boxes in the PDF1.2 promoter as suggested by the trans-activation experiments, we produced recombinant proteins expressed in Escherichia coli with N- and C-terminal Strep and His tags, respectively. Levels of intact ORA59 protein in the soluble fraction of E. coli extracts were almost undetectable. However high amounts of ORA59 and ERF1 proteins were present in the insoluble fraction, presumably in inclusion bodies. Therefore proteins in the insoluble fraction were dissolved in buffer with 6 M urea, proteins were renatured by dilution and dialysis, and full-length ORA59 and ERF1 were isolated by sequential Ni–NTA and Strep-Tactin affinity chromatography. Analysis of the proteins by staining of an SDS-PAA gel with coomassie brilliant blue (not shown) or immunoblot analysis with anti-His antibodies conjugated to horseradish peroxidase (Fig. 3a) showed a single main reactive band in each protein preparation. Although the tagged ORA59 and ERF1 proteins have similar predicted sizes of 31 and 29 kDa, respectively, ORA59 migrated in the denaturing gel system at a position corresponding to 42 kDa, which might be due to a specific structure of the protein. ORA59 expressed in Arabidopsis protoplasts also migrated at the same position (results not shown), which makes it unlikely that the aberrant migration is an artifact due to the E. coli expression system.

Fig. 3
figure 3

ORA59 and ERF1 bind to two GCC boxes in the PDF1.2 promoter in vitro. a After SDS–PAGE and Western blotting recombinant proteins were detected with anti-His HRP-conjugated antibodies. Positions of protein size markers are indicated in k Dalton. b EMSAs were performed with recombinant ORA59 and ERF1 proteins and radio-labeled SF, m1, m2 or dm fragments. ORA47 protein and wild-type or mutated AOC2 promoter fragments were used as control. The arrowheads mark the positions of protein-DNA complexes (C) and free probes (F)

Full size image

Next, the binding of ORA59 and ERF1 proteins to radiolabeled wild type and mutated SF fragments was studied in electrophoretic mobility shift assays. The unrelated AP2/ERF-domain transcription factor ORA47 was used as a control at an amount that gave clear complex formation with a binding site from the AOC2 promoter (Fig. 3b). As shown in Fig. 3b, ORA59 and ERF1 were able to bind to the SF fragment, in contrast to ORA47. Binding of ORA59 and ERF1 was partially decreased when the GCC box at positions −262 to −257 was mutated, and completely abolished when both GCC boxes were mutated. Although the GCC box at positions −222 to −214 clearly contributed to binding to the SF fragment, mutation of this GCC box alone had relatively little effect on binding. These EMSA experiments confirm that these two GCC boxes are the main binding sites for ORA59 and ERF1 in the SF derivative of the PDF1.2 promoter.

ORA59 binds to the PDF1.2 promoter in vivo

The trans-activation experiments as well as the in vitro binding studies suggest that ORA59 binds directly to the PDF1.2 promoter in vivo to regulate gene expression. We wanted to confirm this directly using chromatin immunoprecipitation analysis (ChIP). Therefore we constructed plants expressing ORA59 with the tandem affinity purification (TAP; Puig et al. 2001) tag attached to its C-terminal end under control of the estradiol-inducible XVE system (Zuo et al. 2000). Following screening of the transformants for the ORA59-TAP mRNA level, line #4 was selected for further analysis.

We first verified that the ORA59-TAP fusion protein was expressed and functional. In addition we wanted to determine the optimal induction conditions prior to harvesting plant samples for ChIP analysis. Following addition of 4 μM estradiol or the solvent DMSO the kinetics of mRNA and protein accumulation were followed (Fig. S2). Maximum levels of ORA59-TAP mRNA and protein were observed after 16–24 h. Estradiol treatment also induced the ORA59 target gene PDF1.2, but with slower kinetics, showing that the ORA59-TAP fusion protein is functionally active. DMSO-treated transgenic plants did not express ORA59-TAP or PDF1.2. Estradiol treatment had no effect on PDF1.2 expression in control plants (Fig. 4b).

Fig. 4
figure 4

ORA59 binds to the PDF1.2 promoter in vivo. Arabidopsis seedlings from XVE-ORA59-TAP line #4 and 35S-TAP line #7 were treated with 4 μM estradiol (E) or 0.1% DMSO (D). RNA and protein was extracted for Northern and Western blot analysis of transgene expression. Sonicated chromatin prepared from the remainder of the tissue samples was used in ChIP with IgG Sepharose which has affinity for the TAP tag. a ChIP analysis. Input chromatin or recovered chromatin preparations were used as templates in PCR with cycle number and gene-specific primers as indicated. b Northern blot analysis with probes as indicated. c Western blot analysis with Peroxidase anti-Peroxidase (PAP) antibodies

Full size image

Based on the results from the expression analysis, seedlings treated with 4 μM estradiol or 0.1% DMSO for 16 h were used for ChIP analysis. Transgenic seedlings expressing only the TAP tag under control of the CaMV 35S promoter were similarly treated as controls. Protein and mRNA analysis of the harvested samples prior to formaldehyde cross linking showed that the ORA59-TAP fusion protein was induced by estradiol treatment and was functional as judged by the induction of PDF1.2 expression (Fig. 4b, c). The 35S-TAP seedlings expressed the TAP mRNA and protein, but as expected did not express the PDF1.2 gene. PCR analysis using PDF1.2 primers of the chromatin prepared following formaldehyde cross linking of the samples showed that equivalent amounts of DNA were present (Fig. 4a, input). ChIP was performed using IgG Sepharose beads, which have strong affinity for the protein A part of the TAP tag. PCR analysis of the recovered DNA with primers flanking the GCC boxes in the PDF1.2 promoter revealed that this genomic region was overrepresented in the preparation from XVE-ORA59-TAP seedlings treated with estradiol. Primers specific for the promoter of the unrelated AOC2 gene did not show amplification of a fragment after the same number of PCR cycles. After 36 PCR cycles an AOC2 fragment was amplified to similar levels in all samples, which indicates that based on this background contamination equivalent amounts of immuno-precipitated DNA were used for the PCR reactions (Fig. 4a). The results therefore show that the ORA59-TAP fusion protein binds directly to the PDF1.2 promoter in vivo.

Effects of GCC box mutations on JA- and ethephon-responsive expression of PDF1.2 promoter derivative SF in stably transformed Arabidopsis plants

The expression of the PDF1.2 gene is synergistically induced by a combination of JA and ET (Penninckx et al. 1998). To study the contribution of the two GCC boxes to JA- and ET-responsive activity of PDF1.2 promoter derivative SF, we generated stably transformed plants containing the GUS fusion constructs shown in Fig. 1 via Agrobacterium-mediated transformation. T2 seedlings from eight independent transgenic lines for each construct were treated with JA, the ET-releasing agent ethephon or both for 24 h. Consistent with the accumulation of endogenous PDF1.2 mRNA (Penninckx et al. 1998), PDF1.2 promoter activity was relatively weakly induced by JA or ethephon alone, but strongly induced by the combination (Fig. 5). Mutation of either GCC box dramatically decreased PDF1.2 promoter activity in response to JA or JA/ethephon. Mutation of the GCC box at positions −262 to −257 reduced activity to the level observed with the wild-type promoter after control treatment, whereas mutation of the GCC box at positions −222 to −214 left a very low residual response to JA/ethephon. Mutation of both GCC boxes strongly reduced PDF1.2 promoter activity to levels below the level of the wild-type promoter after control treatment. Therefore in stably transformed plants the two GCC boxes were functionally equivalent and were both necessary for JA- and JA/ethephon-responsive activity of PDF1.2 promoter derivative SF.

Fig. 5
figure 5

Both GCC boxes are essential for JA- and ethephon-responsive expression of PDF1.2 promoter derivative SF in stably transformed Arabidopsis plants. Each bar represents average GUS activity values determined in pools of 10 T2 seedlings from 8 independent transformed lines for each construct corrected for protein concentration ± SE. Seedlings were control-treated (C) or treated with 50 μM JA, 1 mM of the ET-releasing agent ethephon (E) or both (EJA) for 24 h. The asterisk marks the only value that was different from any of the others in a one-way ANOVA (P < 0.05)

Full size image

A GCC box tetramer is sufficient to confer JA- and ethephon-responsive expression in stably transformed Arabidopsis plants

The synergistic effect of JA and ET on PDF1.2 promoter activity could be due either to convergence of the signaling pathways on each of the two GCC boxes, or it could be due to the separate action of each of the signaling pathways on a distinct single GCC box. To distinguish between these possibilities, tetramers of the wild-type and mutant (m1) GCC box between positions −262 to −257 were generated and fused to the TATA box of the CaMV 35S promoter and the GUS reporter gene. We tested whether the GCC box tetramer could support transcriptional activation by ORA59 or ERF1 in a transient assay. ORA59 and ERF1 strongly trans-activated the artificial promoter construct with ORA59 as the strongest activator as with the native PDF1.2 promoter (Fig. 6a). The mutant GCC box tetramer was not activated, demonstrating that the transcription factors activated the artificial promoter via binding to the GCC boxes. Analysis of transgenic seedlings containing the tetramer constructs revealed that none of the lines transformed with the mutant GCC box tetramer showed GUS activity after hormone treatment (not shown). The wild-type GCC box tetramer conferred JA- and JA/ethephon-responsive gene expression which was qualitatively and quantitatively similar to the native PDF1.2 promoter with a strong synergistic effect of the JA/ethephon combination (Fig. 6b). The results show that the JA and ET signaling pathways converge to a single type of GCC box sequence.

Fig. 6
figure 6

A GCC box tetramer is sufficient to confer JA- and ethephon-responsive expression in stably transformed Arabidopsis plants. a Arabidopsis protoplasts were co-transformed with 2 μg of wild-type or mutated 4GCC-GUS and 6 μg of overexpression vectors containing ORA59 or ERF1 driven by the CaMV 35S promoter. The Renilla luciferase (LUC) gene fused to the CaMV 35S promoter served as a reference gene to correct for differences in transformation and protein extraction efficiencies. Values represent means ± SE of triplicate experiments and are expressed relative to the corresponding vector control set at 100%. b Each bar represents average GUS activity values determined in pools of 10 T2 seedlings from 4 independent transformed 4GCC lines corrected for protein concentration ± SE. Seedlings were control-treated (C) or treated with 50 μM JA, 1 mM of the ET-releasing agent ethephon (E) or both (EJA) for 24 h. Letters above the bars indicate different groups as determined by one-way ANOVA (P < 0.05)

Full size image

Discussion

In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PDF1.2 (Penninckx et al. 1998). The AP2/ERF-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression (Pré et al. 2008). Here we aimed at characterizing the PDF1.2 promoter sequences responsible for synergistic expression in response to JA/ET and for interaction with ORA59.

We show that two GCC boxes in the PDF1.2 promoter were essential for in vitro binding to ORA59 and for transactivation by ORA59 in Arabidopsis protoplasts. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. In transgenic plants both GCC boxes were also essential and mutation of either GCC box resulted in a dramatic reduction of the expression of the PDF1.2 promoter in response to JA/ethephon or to JA alone. This latter finding is consistent with the report of Brown et al. (2003) on the effect of the single mutation of the GCC box at positions −262 to −257 on JA-responsive gene expression. Combined with the data reported by Pré et al. (2008), our findings strengthen the notion that ORA59 is the key regulator of JA/ET-responsive PDF1.2 expression. Previously we have shown that ORA59 can activate the PDF1.2 promoter in a transient assay in protoplasts and upon constitutive or inducible overexpression in stably transformed plants (Pré et al. 2008). In addition downregulation of ORA59 expression via RNAi abolished JA/ET-responsive PDF1.2 expression (Pré et al. 2008).

Both in transient assays as well as in stably transformed plants the GCC boxes were functionally equivalent. However, a difference was that in the transient assay mutation of a single GCC box had a moderate effect, whereas in stably transformed plants mutation of a single GCC box abolished PDF1.2 promoter activity. One possible explanation is that the PDF1.2 promoter in the context of a stably established chromatin structure in transgenic plants requires two GCC boxes to be activated in response to JA/ET, whereas a single GCC box is not sufficient for opening up the chromatin structure for transcription. In transiently transformed protoplasts the introduced plasmids probably have not assembled into a proper chromatin structure. Another possibility is that in protoplasts the JA/ET signaling pathways are partially active due to wounding associated with cell wall removal, resulting in opening of the chromatin structure of the PDF1.2 promoter. In support of either possibility is the observation that the PDF1.2 promoter has a clear basal expression level in protoplasts in the absence of effectors, whereas in stably transformed plants there is no detectable basal expression.

The function of ERF1 is somewhat mysterious at the current level of understanding. We show here that ERF1 also bound to the GCC boxes in vitro and activated the PDF1.2 promoter through interaction with the GCC boxes in the protoplast assay. ERF1 can activate the expression of the PDF1.2 gene when constitutively (Solano et al. 1998; Lorenzo et al. 2003) or inducibly (Pré et al. 2008) overexpressed in stably transformed plants, but when ORA59 expression is knocked out by RNAi ERF1 is not able to support expression of PDF1.2 in response to JA, JA/ethephon or infection with Botrytis cinerea (Pré et al. 2008). One option could be that ERF1 acts synergistically with ORA59 on PDF1.2 expression, for example by differential binding of these two proteins to the two GCC boxes. We tested this idea by comparing PDF1.2 promoter activity levels in response to ORA59 and ERF1 separately or combined, but we did not find evidence for synergism. In addition the EMSA experiments did not indicate differential binding to the two GCC boxes, since the effects of GCC box mutations were similar for ORA59 and ERF1.

The PDF1.2 promoter conferred a synergistic gene expression response, demonstrating that JA and ET act synergistically at the transcriptional level. The synergistic effect of JA and ethylene on PDF1.2 promoter activity could be due either to convergence of the signaling pathways on each of the GCC boxes, or it could be due to the separate action of each of the signaling pathways on a single distinct GCC box. The wild-type GCC box tetramer conferred JA- and JA/ethephon-responsive gene expression, showing that the JA and ethylene signaling pathways converge to a single GCC box sequence. If the two GCC boxes would have been functionally different, a tetramer of a single GCC box would not have been able to confer a similar synergistic response to JA/ethephon as the native PDF1.2 promoter. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ethylene signaling pathways.

It is likely that the JA and ET signaling pathways converge on the transcription factor ORA59. The question remains then how the JA and ET signaling pathways act on ORA59. One mechanism is by synergistically stimulating the expression of the ORA59 gene (Pré et al. 2008), which implies that unidentified upstream transcription factors are activated. One of those transcription factors could be EIN3. There is evidence suggesting that EIN3 regulates the expression of the ERF1 gene (Solano et al. 1998), and the ERF1 and ORA59 genes have very similar patterns of expression (Lorenzo et al. 2003; Pré et al. 2008). In addition the JA and ET signaling pathways could also activate ORA59 at the protein level. The JA signaling pathway is thought to activate the transcription factor AtMYC2 by promoting the degradation of the Jasmonate ZIM domain (JAZ) repressors (Chini et al. 2007; Thines et al. 2007). It is possible that members of the JAZ protein family repress ORA59 activity, although there is no evidence reported for JAZ-ORA59 interaction. Alternatively distinct repressor proteins could regulate ORA59 activity in response to JA/ET. The GCC box tetramer construct may have applications as an artificial minimal JA/ET-responsive promoter to dissect mechanisms of the synergistic effect of JA and ET or of the antagonistic effect of JA and SA, since it is likely to show less complex regulation than the native PDF1.2 promoter, which contains also binding sites for other transcription factors such as the TGA proteins (Spoel et al. 2003).