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Divergent DNA-Binding Specificities of a Group ofETHYLENE RESPONSE FACTOR Transcription FactorsInvolved in Plant Defense1[C][W]

Tsubasa Shoji*, Masaki Mishima, and Takashi Hashimoto

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630–0101, Japan(T.S., T.H.); and Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Tokyo192–0397, Japan (M.M.)

Transcription factors (TFs) recognize target DNA sequences with distinct DNA-binding domains (DBDs). The DBD of Arabidopsis(Arabidopsis thaliana) ETHYLENE RESPONSE FACTOR1 (AtERF1) uses three consecutive b-strands to recognize a GCC-containingsequence, but tobacco (Nicotiana tabacum) ERF189 and periwinkle (Catharanthus roseus) Octadecanoid-derivative ResponsiveCatharanthus AP2-domain protein3 (ORCA3) of the same TF subgroup appear to target similar but divergent DNA sequences.Here, we examined how DNA-binding specificities of these TFs have diverged in each plant lineage to regulate distinct defensemetabolisms. Extensive mutational analyses of these DBDs suggest that two modes of protein-DNA interactions independentlycontribute to binding specificity and affinity. Substitution of a conserved arginine to lysine in the first b-strand of ERF189 relaxesits interaction with the second GC pair of the GCC DNA sequence. By contrast, an increased number of basic amino acids in thefirst two b-strands of ORCA3 allows this TF to recognize more than one GCC-related target, presumably via increasedelectrostatic interactions with the negatively charged phosphate backbone of DNA. Divergent DNA-binding specificities ofthe ERFs may have arisen through mutational changes of these amino acid residues.

Biological processes ranging from development tometabolism rely on reprogramming of the transcriptome,which is governed largely by transcription factors (TFs).TFs control gene expression at the level of transcriptionby recognizing specific DNA sequences, or cis-elements,in promoters of target genes through DNA-binding do-mains (DBDs) with various structural motifs (Yamasakiet al., 2012). Understanding specific binding of TFs to theDNA sequences and the underlying biophysical phe-nomena is necessary to interpret the genetic informa-tion in the genome directing gene regulation (Segal andWidom, 2009). Recently, systematic analyses of the DNAbinding of a large number of TFs using protein-bindingmicroarrays (Badis et al., 2009) and SELEX (Jolma et al.,2013) have shown promise in this direction.

TF genes account for over 5% of the total genes in theplant genome (Riechmann and Ratcliffe, 2000) and areusually present as large superfamilies, each member ofwhich serves a specific regulatory role. Plant-specific TFs

of the APETALA2/Ethylene Response Factor (AP2/ERF) superfamily are defined by the presence of aconserved AP2/ERF DBD of about 60 amino acid res-idues (Ohme-Takagi and Shinshi, 1995; Nakano et al.,2006). The AP2/ERF superfamily can be further dividedinto several subfamilies, including the most prevalentERF subfamily. Many members of the ERF subfamilyact as monomers that recognize cis-elements with aGCC box (59-AGCCGCC-39; Ohme-Takagi and Shinshi,1995; Hao et al., 1998; Fujimoto et al., 2000). An NMRstudy of the solution structure of the DBD of AtERF1 incomplex with a GCC box revealed a unique mode ofDNA-protein interaction (Allen et al., 1998). The DBDconsists of three-stranded antiparallel b-sheet and onea-helix parallel to the sheet (Fig. 1A; Supplemental Fig.S1). Amino acid residues, mainly Arg and Trp, thatdirectly contact base moieties of DNA are found in theb-sheet (Fig. 1B) and are important, along with otherresidues in the DBD, for DNA-binding properties (Allenet al., 1998; Sakuma et al., 2002; Hao et al., 2002; Liuet al., 2006; Wang et al., 2009).

The IXa group of the ERF subfamily (Nakano et al.,2006) includes a handful of TFs mainly involved in plantdefense, such as AtERF1 and AtERF13 from Arabidopsis(Arabidopsis thaliana; Fujimoto et al., 2000; Lee et al., 2010),Octadecanoid-derivative Responsive Catharanthus AP2-domain protein3 (ORCA3) from periwinkle (Catharanthusroseus; van der Fits and Memelink, 2000), ERF189,ERF115, ERF179, and ERF163 from tobacco (Nicotianatabacum; Shoji et al., 2010), and Sl1g90340 from tomato(Solanum lycopersicum; Fig. 1B). AtERF1 is a foundingmember of the AP2/ERF superfamily and recognizes a

1 This work was supported in part by the Japan Society for thePromotion of Science (Grant-in-Aid for Scientific Research [C] no.23570055 to T.S.).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Tsubasa Shoji ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.113.217455

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GCC box found in a broad range of defense genes,though its involvement in plant defense has yet to beconfirmed (Fujimoto et al., 2000; Oñate-Sánchez andSingh, 2002; Gutterson and Reuber, 2004; McGrath et al.,2005). ORCA3 is a regulator of jasmonate-inducible in-dole alkaloid biosynthesis in periwinkle (van der Fitsand Memelink, 2000), and multiple ERF genes in tobaccoare clustered at the nicotine-controlling NICOTINE2 lo-cus, as master regulators of nicotine alkaloid production,which is also inducible by jasmonate (Shoji et al., 2010).

Three GC-rich boxes in the promoters of target genesare known to be bound by group IXa ERFs. A P box(59-CCGCCCTCCA-39) in the promoter of the tobaccoputrescine N-methyltransferase2 (PMT2) gene is recognizedby ERF189, ERF115, ERF179, ERF163, ORCA3, andAtERF13, but not by AtERF1 (Shoji et al., 2010; Shoji andHashimoto, 2012a). A CS1 box (59-TAGACCGCCT-39) ispart of a jasmonate- and elicitor-responsive elementtargeted by ORCA3 in the promoter of the strictosidinesynthase (STR) gene (van der Fits and Memelink, 2001).Finally, AtERF1 recognizes a GCC box (59-AGCCGCC-39; Fujimoto et al., 2000; Gutterson and Reuber, 2004).

TFs with DBDs related in amino acid sequence oftenbind similar but distinct DNA sequences. Little is knownabout how such divergence of DNA-binding specificitiesarose and how it is translated into functional distinctionof the TFs. In this study, we examined the DNA-bindingspecificities of group IXa ERFs, demonstrating their

differences in binding to multiple GC-rich sequences.Mutational analysis of the ERFs revealed amino acidresidues important for the differential DNA binding,and these were found to be involved in interactionswith bases and phosphate backbones of DNA. The di-vergent DNA-binding specificities of group IXa ERFsappear to have arisen through mutational changes ofthese residues.

RESULTS

Group IXa ERF Genes in Various Flowering Plants

To clarify the distribution of group IXa ERF genesamong flowering plants, all genes of the group in thegenomes of rice (Oryza sativa), maize (Zea mays), Arab-idopsis, Brassica rapa, poplar (Populus trichocarpa), andtomato were retrieved from public databases, and therelationship of these and other members from tobacco,periwinkle, and Artemisia annua was examined byaligning the sequences of the DBD (Supplemental Fig.S1) and generating a phylogenetic tree (Fig. 2). Thisanalysis divided group IXa into two clades, with clade1 represented by AtERF1. Clade 2 was further dividedinto four subclades, clade 2-1, including ERF189 andERF115, clade 2-2, including ERF179, clade 2-2b, in-cluding Sl1g90340, and clade 2-3, including ERF163,ORCA3, and AtERF13. The alignment for the mentioned

Figure 1. The DBD of group IXa ERF proteins. A, A three-dimensional structure of the DBD of AtERF1 bound with GCC box(1GCC.pdb). Each segment of secondary structure is a different color. B, Multiple sequence alignment of the DBDs. The sequenceswere aligned with ClustalW (Thompson et al., 1994). Residues identical or similar at least in four sequences are shaded in black orgray, respectively, and dashes indicate gaps introduced to maximize the alignment. Asterisks below the alignment denote residuesthat directly contact base moieties of DNA based on a structural study (Allen et al., 1998), and secondary structures are indicated asfollows: arrows denote b-strands and the bar shows a-helix. The residues (numbered from the N-terminal end of the domain) thatwere examined in following mutational analyses are marked with arrowheads. AtERF1 (At4g17500) and AtERF13 (At2g44840) arefrom Arabidopsis (At), ORCA3 (EU072424) from periwinkle (Cr), Sl1g90340 (Solyc01g090340) from tomato (Sl), and the others arefrom tobacco (Nt). Only for the Arabidopsis and tomato genes, the abbreviations for organisms are included in gene names asprefixes. Sequences of tobacco ERFs can be found in the Database of Tobacco Transcription Factors (Rushton et al., 2008) underthe names used here. [See online article for color version of this figure.]

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members is shown in Figure 1B. Note that clade 2-3 isdefined more loosely than other subclades. Althoughthree rice ERF genes had been classified into group IXapreviously (Nakano et al., 2006), reexamination hererevealed that only two of them,Os2g43790 andOs4g46220,are in group IXa, while the other one, Os1g54890, actuallybelongs to group IXc.

The positions of group IXa ERF genes on chromo-somes in rice, maize, Arabidopsis, B. rapa, poplar, andtomato are illustrated in Supplemental Figure S2. Allclade 1 ERFs, except for Br022115 in B. rapa, are locatedin close proximity of group IXb members on thechromosomes, whereas clade 2 ERFs are far fromthem, indicating a clear distinction between the two

Figure 2. Phylogenetic tree of group IXa ERFs. Amino acid sequences of the DBD were aligned with ClustalW (Thompson et al.,1994; Supplemental Fig. S1), and based on the alignment, a tree was generated using MEGA4 software (Tamura et al., 2007)with the neighbor-joining algorithm. Bootstrap values are indicated at branch nodes, and the scale bar indicates the number ofamino acid substitution per site. According to species, gene names are denoted with prefixes. Aa, A. annua; At, Arabidopsis, Br,B. rapa; Cr, periwinkle; Nt, tobacco; Os, rice; Pt, poplar; Zm, maize. Group VIII AtERF4, group IXb AtERF5, and group IXcAtORA59 are included as outgroup members. [See online article for color version of this figure.]

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DNA-Binding Specificities of ERF Transcription Factors





clades in terms of gene evolution. We also found tan-dem clustering of multiple clade 2 ERF genes in poplarand tomato, which had been speculated for tobaccoERFs at the NICOTINE2 locus (Shoji et al., 2010). Al-though the majority of clade 2 ERFs are in the clusters,a few genes of clade 2, e.g. AtERF13 in Arabidopsis,Br037630 and Br004873 in B. rapa, and Sl5g50790 intomato, are present as singletons.

Differential Binding of Group IXa ERFs to ThreeGC-Rich Boxes

To compare the in vitro binding of group IXa ERFsto the P, CS1, and GCC boxes, electrophoresis mobilityshift assays (EMSAs) were carried out using selectgroup IXa ERFs (ERF189, ERF115, ERF179, Sl1g90340,ERF163, ORCA3, AtERF13, and AtERF1) and threeprobes, P, CS1, and GCC, each containing one of theboxes. The purities of the recombinant ERFs are shownin Supplemental Figure S3. As the GCC probe, a 10-bpsequence with a GCC box (59-AGAGCCGCCA-39, thebox is underlined) from the tobacco b-1,3-glucanase(GLN2) promoter was used (Table I). The GCC probewas bound by all ERFs except clade 2-1 membersERF189 and ERF115, whereas the CS1 probe wasbound only by clade 2-3 members ERF163, ORCA3,and AtERF13 (Fig. 3A). Consistent with previous re-ports, the P probe was recognized by all but clade1 AtERF1 (Fig. 3A). These results, summarized in Figure3B, suggest similar but distinct DNA-binding specific-ities of the group IXa ERFs.

There are other non-GCC boxes that are recognizedby AP2/ERF TFs: Dehydration-Responsive Element

(DRE; 59-[A/G)]CCGAC-39; Liu et al., 1998), C-Repeat/DRE/HVCBF2 (CBF2; 59-GTCGAC-39; Xue, 2003; Yuet al., 2012), RAV1/AAT (RAA; 59-CAACA-39; Kagayaet al., 1999; Yu et al., 2012), and Coupled Element1(CE1; 59-CCACC-39; Shen and Ho, 1995; Lee et al.,2010). No clear binding to probes containing DRE,CBF2, RAA, or CE1 boxes was detected for ERFs ex-amined by EMSA (Supplemental Fig. S4).

In Vitro Binding of Mutant Versions of ERF189, ORCA3,and AtERF1

Each of the three probes was recognized by a uniquesubset of group IXa ERFs (Fig. 3). This led us to askwhat structural differences among the ERF subsetsaccount for the probe discrimination. To address this,we focused on the DNA-contacting N-terminal half ofthe DBD (Fig. 1B), where the three b-strands form aninterface to bind the DNA (Allen et al., 1998). Only sixresidues in that region differ among the subsets ofgroup IXa ERFs (Fig. 1B).

To clarify the amino acid residues that are critical forDNA binding, EMSA was performed with mutant ver-sions of ORCA3 (mORCA3), in which each of the sixresidues was substituted with that of other clade ERFs(Fig. 4). ORCA3 bound all the three probes as shown(Fig. 3A), as did mORCA3/Arg-1-to-His (R1H) andmORCA3/Lys-3-to-Arg (K3R; Supplemental Fig. S5A),indicating no detectable influence of the R1H and K3Rsubstitutions. By contrast, the Lys-3-to-Ile (K3I) substi-tution caused nearly complete loss of binding to CS1(Fig. 3A). The Arg-6-to-Lys (R6K) and Arg-7-to-Glu(R7Q) substitutions disrupted binding of all probes

Table I. Sequences bound by group IXa ERFs in vitro

Prefixes to gene names indicate organisms (tobacco [Nt] and periwinkle [Cr]). Positions of 59 ends of the indicated sequences relative to the firstATG and orientations (forward [F] and reverse [R]) of boxes in the context of promoters are indicated to the left of the sequences. TESS scores areindicated as follows: a, based on data from ERF189 at the P box; b, ORCA3 at the P box; c, ORCA3 at the CS1; d, ORCA3 at the GCC box, and e,AtERF1 at the GCC box. Sequences of 7 bp that give rise to the scores in d and e are in the rightmost column. Nd, Not detected.

Name Gene Sequence (59 to 39)TESS Scores

Sequence for Scores d and ea b c d e

P typeP NtPMT2 2133 F CCGCCCTCCA 7.45 11.56 nd 4.39 3.42 (CGCCCTC)Q1 NtQPT2 2131 R TAGCACTCCA 5.58 10.96 nd nd ndQ2 NtQPT2 2160 F AAGCACTCCA 7.18 10.69 nd nd ndQ3 NtQPT2 2238 R TAGCACTCCA 5.58 10.96 nd nd ndO3 NtODC1 2969 R TAGCCAGCCT 7.45 8.92 nd 3.02 nd (GCCAGCC)O5 NtODC2 2919 R TAGCCAGCCT 7.45 8.92 nd 3.02 nd (GCCAGCC)M1 NtMATE1 2206 R TAGCACTCCA 7.75 7.44 nd 2.99 2.89 (AGCACCC)M2 NtMATE1 2704 R GAGCACACCT 9.04 9.27 nd nd ndCC3 CrCPR 2346 R ACGCCTACCA 3.58 8.79 nd nd nd

Consensus A A AN/GC/NNCC/C C C

CS1 typeCS1 CrSTR 2146 F TAGACCGCCT 6.20 nd 9.11 5.85 7.29 (GACCGCC)CC2 CrCPR 2342 R AAGAACGCCT 7.36 nd 12.36 2.54 4.73 (GAACGCC)CC1 CrCPR 2224 F ACGCCGGCGA nd 7.28 nd 6.16 4.97 (CGCCGGC)

GCCGCC NtGLN2 21179 F AGAGCCGCCA nd nd nd 10.29 10.50 (AGCCGCC)


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except P and GCC, respectively (Fig. 4A). Both Lys-12-to-Thr (K12T) and Ala-14-to-Ser (A14S) substitutions ham-pered binding to all three probes (Fig. 4A). Even doublesubstitution of these close-together residues (K12T/A14S), which mimicked the sequences of clade 2-1 and2-2b ERFs in that region (Fig. 1B), did not lead tobinding (Supplemental Fig. S5A).Because three substitutions K3I, R6K, and R7Q in

ORCA3, wherein the original residues were changedto the corresponding residues of ERF179, ERF189, andAtERF1, respectively, altered binding of ORCA3 to theprobes, we examined how the reverse substitutions,Ile-3-to-Arg (I3K) in ERF179, Lys-6-to-Arg (K6R) inERF189, and Glu-7-to-Arg (Q7R) in AtERF1, affectedthe binding patterns of the original ERFs (Fig. 4A;Supplemental Fig. S5A). Two substitutions, I3K in ERF179and Q7R in AtERF1, did not affect the binding patterns(compare Fig. 3A with Supplemental Fig. S5A). However,in contrast to the wild-type ERF189 binding of only the Pbox, the R6K substitution in ERF189 enabled strongbinding to all three probes (Fig. 4A).Overall, these results suggest that the amino acid

residues at positions 3, 6, 7, 12, and 14 are important forERF binding to the probes. In particular, the changes ofbinding patterns that were caused by substitutions K3I,R6K, and R7Q in ORCA3 and K6R in ERF189 weremostly consistent with the distinct DNA-binding spec-ificities of the corresponding ERFs. On the other hand,

two reverse substitutions (I3K in ERF179 and Q7R inAtERF1) had no effects on the binding patterns.

In Vivo Binding of ERF189, ORCA3, and AtERF1 and TheirMutant Derivatives

We then asked whether and how well the in vitrobinding results (Figs. 3 and 4) reflected in vivo DNA-protein interaction. First, we performed transient trans-activation assays using tobacco Bright Yellow-2 (BY-2)cultured cells, into which combinations of reporter genesunder the control of a P, CS1, or GCC box and ERF ef-fector genes were delivered by particle bombardment.To examine P box-dependent expression, the PMT2promoter, including the box, was tested for activationby ERF189, ORCA3, AtERF1, and their mutant deriva-tives (Fig. 5). As previously reported (Shoji et al., 2010;Shoji and Hashimoto, 2012a), ERF189 and ORCA3 ac-tivated the reporter about 8- and 5-fold, respectively,whereas AtERF1 did not. The R7Q substitution inORCA3 markedly diminished the induction to 1.2-fold, whereas other substitutions, K3I and R6K inORCA3 and K6R in ERF189, did not significantly alterthe induction, in accordance with the in vitro bindingresults (Fig. 4A; Supplemental Fig. S5A). No inductionwas observed of a mutant reporter in which the P boxwas disrupted (Fig. 5), indicating the requirement of

Figure 3. In vitro binding of group IXa ERFs to P, CS1, and GCC boxes. A, EMSA was performed to examine the binding be-tween ERFs and oligonucleotide probes containing a P box (59-CCGCCCTCCA-39) from the tobacco PMT2 promoter, a CS1 box(59-TAGACCGCCT-39) from the periwinkle STR promoter, or a GCC box (59-AGCCGCC-39) from the tobacco GLN2 promoter.Sequences and other information about the boxes are in Table I. Clade numbers are indicated in parentheses. The relative levelsof binding of the P probe by different ERFs, except AtERF1, which did not bind the probe, compared in the same blots are shownin Supplemental Figure S9C. B, Summary of the results shown in A. Nucleotides in P and CS1 boxes that are different from thosein the corresponding positions of the GCC box are underlined.










the box for the responses. To monitor the expressiondependent on CS1 and GCC boxes, promoters con-taining four copies of each box were used to constructreporter vectors CS1x4-35Smini-GUS and GCCx4-35Smini-GUS. Unexpectedly, even ORCA3 and AtERF1 failed toactivate CS1x4-35Smini-GUS and GCCx4-35Smini-GUS,respectively (Supplemental Fig. S6). Activation of asimilarly designed reporter construct with a GCC boxtetramer by AtERF1 was reported (Fujimoto et al.,2000), and we could not find any particular reasons forthe inconsistency other than different experimentalconditions.

Next, we carried out transactivation assays in yeast(Saccharomyces cerevisiae). A modified firefly luciferase(mLUC; see “Materials and Methods”) was placedunder the control of promoters containing four copiesof the P box in Px4-mini-mLUC, the CS1 box in CS1x4-mini-mLUC, or the GCC box in GCCx4-mini-mLUC.Yeast harboring a pair of reporter and ERF effectorplasmids was grown, and LUC activity of the culturewas determined by measuring luminescence emittedafter addition of luciferin (Fig. 6). CS1x4-mini-mLUCwas significantly activated by ORCA3 and mERF189/K6R but not by the others, and the activity induced byORCA3 was about 5 times that induced by mERF189/

K6R. GCCx4-mini-mLUC was activated by AtERF1,ORCA3, mERF189/K6R, and mORCA3/R7Q to vary-ing extents. The lack of significant activation of reportersmutated in the CS1 or GCC box demonstrated the de-pendence of the activation on the boxes. High LUC ac-tivity was detected for Px4-mini-mLUC, even with theempty effector vector (Fig. 6B), and thus we did notperform the assay with this reporter.

In Vitro Binding Specificities of ERF189, ORCA3,and AtERF1 Determined with a Series of SingleNucleotide-Substituted Probes

To determine in vitro binding specificities of ERF189,ORCA3, and AtERF1, EMSAwas performed with seriesof wild-type and mutant probes that contained everypossible nucleotide substitution (A, T, G, or C) in theboxes (Supplemental Figs. S7 and S8), as in Shoji andHashimoto (2011b), except that the sequences flankingthe box were randomized in this study (see “Materialsand Methods”). Binding specificities of TFs are oftenexpressed as position weight matrices (PWMs), whichare based on the assumption that interactions betweeneach base of DNA and a TF are independent (Benos

Figure 4. In vitro binding of mutant versions of ORCA3 and ERF189 to P, CS1, and GCC boxes. A, EMSA was performed toexamine binding of ORCA3, ERF189, and their mutant versions to P, CS1, and GCC probes. To indicate the positions ofsubstitutions, amino acid residues are numbered from the N terminus of the AP2/ERF DBD (Fig. 1B). The relative abundance ofthe complexes on each blot was determined by comparing the intensities of the retarded bands and is indicated above theimages. The values for P probe (or GCC probe for mORCA3/R7Q) were set to 100. B, Relative levels of binding of different ERFsto P probe for ORCA3, ERF189, and their mutant versions, compared in the same blots.


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et al., 2002). Based on PWMs reflecting the bindingprofiles obtained by EMSA, the binding specificities ofERF189, ORCA3, and AtERF1 were represented as se-quence logos (Fig. 7).The obtained binding specificity of ORCA3 to the P

box, 59-(C/A)GC(C/A)NNCC-39, was quite similar tothat of ERF189 to the same box (59-[A/C]GC[C/A][C/A]NCC-39; Shoji and Hashimoto, 2011b; Fig. 7). Thebinding specificity of ORCA3 to the CS1 box was 59-GNACGCC-39, which was clearly distinct from those ofORCA3 to the P and GCC boxes (Fig. 7). ERF189,ORCA3, and AtERF1 were also examined by EMSAwitha set of probes based on the GCC box (Supplemental Fig.S8). ERF189 did not bind the GCC box, and only weakbinding of ERF189 to a small number of mutant probeswas observed. ORCA3 and AtERF1 robustly bound theprobes, allowing us to determine their specificities. Theyshowed nearly identical specificities (59-GCCGCC-39),which were less flexible than those at other sites andclearly different from those of ORCA3 for P and CS1boxes (Fig. 7).

In Vitro Binding Sequences of Group IXa ERFs inPromoters of Alkaloid Biosynthesis Genes

The in vitro binding specificities of ERF189, ORCA3,and AtERF1 (Shoji and Hashimoto, 2011b; Fig. 7) allowedus to predict potential binding sites in possible targetpromoters. The promoter sequences (up to 1 kb from thefirst ATG) of alkaloid biosynthesis and transport genesfrom tobacco and periwinkle, which are presumed to beregulated by relevant group IXa ERFs, were searchedcomputationally using Transcriptional Element SearchSoftware (TESS) software, which gave scores to candi-date sequences based on the EMSA-derived PWMs. Allsequences with scores higher than 7.0 in any searches arelisted in Table I. For the GCC box, a core 7-bp sequencewas used for the searches. Tobacco A622, encoding aPIP-family reductase (Kajikawa et al., 2009), periwinkleTrp decarboxylase (TDC), and periwinkle desacetoxyvindo-line 4-hydroxylase (D4H) are also regulated by group IXaERFs, but no binding sites were predicted in their pro-moters (up to approximately 1.4 kb of A622, 1.0 kb ofTDC, and 0.6 kb of D4H were examined).

In tobacco, all eight sequences (P, Q1, Q2, Q3, O3, O5,M1, and M2) that have been reported as ERF189-bindingsequences (Shoji and Hashimoto, 2011b, 2012a), but noneapart from those, were identified in promoters of PMT2,quinolinate phosphoribosyl transferase2 (QPT2), Orn decarbox-ylase (ODC), and multidrug and toxic compound extrusion-type transporter1 (MATE1; Shoji et al., 2009; Table I). Inperiwinkle, four sequences, CS1 in the STR promoterand CC1 to CC3 in the cytochrome P450 reductase (CPR)promoter, were predicted (Table I).

To validate the predictions, binding between probescontaining each predicted sequence and group IXaERFs was examined by EMSA (Fig. 8; SupplementalFig. S9). Clade 2-1 ERF189 and ERF115 were excludedfrom this assay, because binding between ERF189 andthe tested sequences has previously been examined(Shoji and Hashimoto, 2012a). The eight tobacco probeswere bound by all but AtERF1, though the bindingstrengths varied. AtERF13 displayed relatively weakbinding to most of the probes (Supplemental Fig. S9, Aand C). Three of the probes from periwinkle, CS1, CC1,and CC2, were bound by clade 2-3 members ERF163,ORCA3, and AtERF13, while the fourth, CC3, wasbound by all but AtERF1, similar to the results for thetobacco probes (Fig. 8; Supplemental Fig. S9B).

Based on the similarity of the binding patterns to Pand CS1, the binding sequences found in the alkaloidgene promoters could be classified into two types, P typeand CS1 type (Fig. 8; Table I). The P type included theeight tobacco sequences and CC3, which were scoredhighly by TESS with specificities obtained at the P boxand had 59-(A/C)GC(A/C)NNCCA-39 as a consensus(Table I). The consensus matched especially well withthe specificity of ORCA3 obtained for the P box (Fig. 7).The CS1 type included three sequences, CS1, CC2, andCC3, none of which matched the P-type consensus (TableI). Although CS1 and CC2 are similar and have 59-(A/T)AGA(A/C)CGCCT-39 in common, the CC1 sequence

Figure 5. Transient transactivation of the PMT2 promoter with ERFeffectors and their mutant derivatives in tobacco BY-2 cells. Culturedtobacco BY-2 cells were bombarded with a combination of a GUS-expressing reporter plasmid, a LUC-expressing reference plasmid, andeither an ERF-expressing effector or an empty plasmid (EV). The re-porter GUS gene was driven either by the wild-type PMT2 promoter(PMT2pro236-GUS) or by a PMT2 promoter in which the ERF-bindingP box was mutated (PMT2pro236m4-GUS). GUS activity in the cellextracts is shown relative to the LUC activity. The values for the emptyplasmid are set to 1. Error bars indicate the SD for three independentbiological replicates. SDs among the effectors were determined at P ,0.05 by one-way ANOVA, followed by the Tukey-Kramer test, and areindicated by different letters.












is quite divergent from them, and a consensus of thethree could not be defined. The CS1-type sequenceswere identified by the prediction program as putativebinding sites, not only based on the specificity for theCS1 box, but also with the specificities for P and GCCboxes (Table I), reflecting intermediate nature of theCS1 type between the P type and the GCC box. On thecontrary, for the GCC box, which is not present in thesearched alkaloid gene promoter regions, high bindingscores were predicted only with the less flexible spec-ificities obtained from the bona fide GCC box (Fig. 7;Table I).

DISCUSSION

Divergent DNA-Binding Specificities of Group IXa ERFs

We demonstrated that group IXa ERFs differentiallybind to multiple GC-rich sequences, which can be grou-ped into three types, namely P type, CS1 type, and theGCC box, indicating divergent DNA-binding speci-ficities of these TFs (Figs. 3, 5, 6, and 8; Table I). In vitro

DNA-binding specificities of ERF189, ORCA3, andAtERF1were determined using series of single nucleotide-substituted probes at the defined binding boxes (Fig.7). Based on the resulting specificities, sequences of Pand CS1 types could be identified in promoters of al-kaloid biosynthesis genes and added to the repertoireof known GC-rich sequences bound by these TFs (Fig.8; Table I).

Every binding sequence that was predicted compu-tationally (TESS scores . 7.0) was demonstrated to bebound by more than one of the examined ERFs,demonstrating the reliability of the predictions. Fur-thermore, TESS software gave scores of less than 5.0 tosequences of probes including non-GCC boxes (DRE,CBF2, RAV, and CE1), which were shown not to bebound by the ERFs (Supplemental Table S1). Thereliability of the scoring was improved over previousattempts (Shoji and Hashimoto, 2011b, 2012a), in partbecause of the addition of four specificities (Fig. 7) andthe adoption of a stricter cutoff value. In vivo rele-vance of the binding sequences as cis-elements hasbeen demonstrated for the P box in PMT, the Q1, Q2,and Q3 boxes in QPT2, and the CS1 box in STR (van

Figure 6. Transactivation assay in yeast. Yeast Y187 strain was transformed with a combination of a mLUC-expressing reporterand either an ERF-expressing effector or an empty plasmid (EV). A, The reporter mLUC gene was driven by either four copies ofthe CS1 or the GCC box placed upstream of a minimal promoter of the HIS3 locus (CS1x4-mini-mLUC, GCCx4-mini-mLUC) ortheir mutant derivatives (mCS1x4-mini-mLUC,mGCCx4-mini-mLUC). B, The reportermLUC gene was driven by four copies ofthe P box placed upstream of a minimal promoter of the HIS3 locus (Px4-mini-mLUC). LUC activities of yeast cultures fromthree independent colonies were measured, and error bars indicate the SD. Only the values above those of negative controls,which were measured using buffer without luciferin, are considered significant. The value for CS1x4-mini-LUC activated byORCA3 was set to 100. SDs among the effectors were determined at P , 0.05 by one-way ANOVA, followed by the Tukey-Kramer test, and are indicated by different letters. ND, Not detected.


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der Fits and Memelink, 2001; Shoji et al., 2010; Shojiand Hashimoto, 2011b), but experimental proof re-mains necessary for the other sequences.The importance of probe sequence may explain the

disparities between our results and those obtained inprevious studies, which pointed to specific nucleotides(the second G, fifth G, and seventh C [Hao et al., 1998]or a CG step in the center [Yang et al., 2009] of theGCC box [59-AGCCGCC-39]) as being critical or in-dispensable for recognition. Although such tendenciescould be observed in our results, there were small butsignificant discrepancies among these findings andours. In addition, no binding sequences were predictedin A622, TDC, and D4H promoter regions. Althoughthere is no experimental evidence of direct binding ofERFs to A622 and D4H promoter regions, in vitrobinding of ORCA3 to the proximal TDC promoterhas been clearly demonstrated (van der Fits andMemelink, 2000). It is possible that the promoter regionsthat we searched were not long enough to include thebinding sequences or that sequences with low scoreswere omitted by our cutoff. As exemplified by the factthat the ORCA3 specificities were variable among thethree boxes (Fig. 7), the results also imply the existenceof multiple distinct sequence motifs recognized by asingle TF (Badis et al., 2009). As such, we cannot ruleout the existence of still more sequences that these TFs

can bind. In addition, our methodology using probessubstituted at one nucleotide position and PWMs ig-nores position interdependence (Badis et al., 2009; Jolmaet al., 2013) and therefore could fail to detect somesubsets of possible binding sequences.

Structural Basis of Divergent DNA-Binding Specificities

We found that the similar but different DNA-bindingspecificities of group IXa ERFs are closely related to theamino acid sequences of the DBD (Figs. 1, 3, 5, 6, and 8).Through mutational analyses, a few amino acid resi-dues critical for such differences were defined: at leastsix substitutions at five positions (K3I, R6K, R7Q, K12T,and A14S in ORCA3 and K6R in ERF189) influencedDNA-binding specificities (Figs. 4–6). The only struc-ture available for this superfamily is that of the DBD ofAtERF1, a member of group IXa, bound to a GCC box(Allen et al., 1998). This prompted us to model thestructures of representative group IXa ERFs ORCA3and ERF189 with DNA and compare their structures toelucidate the structural effects of the critical residueson DNA binding. Using the structure of the AtERF1complex (1GCC.pdb) as a template, homology modelsof ORCA3 and ERF189 complexes were generated.

In the AtERF1 and ORCA3 complexes, the guanidylgroup of Arg-6 (R6) contacts the O6 atom of the guaninebase of a CG pair at the third position in the GCC boxthrough a hydrogen bond. The side chain of R6 alsointeracts with the cytosine base moiety of a GC pair atthe fourth position in the box through hydrophobicinteraction (Fig. 9, A and B; Allen et al., 1998). This Argresidue in the first b-strand is conserved in all AP2/ERF TFs except clade 2-1 ERFs (Fig. 1A; SupplementalFig. S1), suggesting the requirement of R6 for recog-nition by most ERFs, including AtERF1. However, thefindings that clade 2-3 ERFs also can bind to P-typesequences without the GC pair (Figs. 3 and 8; Table I)and that the specificity of ORCA3 for the P box is notstringent at the fourth position (Fig. 7), indicate thatthe GC pair is dispensable for recognition by ORCA3and related ERFs. As discussed below, stronger affin-ity of this ERF subset may allow such relaxed recog-nition. In contrast to R6, Lys-6 (K6) in clade 2-1 ERF189may favor the adenine of a TA pair at the fourth po-sition, which appears in the P box and four otherP-type sequences (Table I), because the «-amino groupof K6 can interact with the N7 atom of adeninethrough hydrogen bonding (Fig. 9B). Such pairingsbetween Lys and adenine are prevalent in reportedstructures (Mao et al., 2003). ERF189 also recognizesP-type sequences without the TA pair (Table I), and itsspecificity for the P box did not show a firm preferencefor a TA pair at the position (Fig. 7), indicating thatthe pairing is favored but not exclusive. Apart from R6and K6, there are no apparent changes in residuesdirectly contacting the bases of DNA among the ERFstructures, not even in the region responsible for rec-ognition of nearly one-half of the binding sequences,

Figure 7. Sequence logos representing in vitro binding specificities ofERF189, ORCA3, and AtERF1. EMSA was used to examine bindingbetween ERF and a set of wild-type and single nucleotide-substitutedmutant probes (Supplemental Figs. S7 and S8). The sequences of thewild-type probes are indicated below the logos, and sequences of theboxes are underlined and shown in black letters. The binding profilesobtained by EMSA were expressed as PWMs. Multiple alignments of100 hypothetical sequences reflecting the EMSA-derived PWMs wereused to generate sequence logos by WebLogo (Crooks et al., 2004).Lowercase letters (a–e) in parentheses indicate TESS scores listed inTable I, which were calculated based on the PWMs. The bindingspecificity of ERF189 to the P box-containing sequence was obtainedin a previous study (Shoji and Hashimoto, 2011b). [See online articlefor color version of this figure.]








where P, CS1, and GCC boxes also show distinctionsbetween each other to some extent (Figs. 3B and 7).

In addition to sequence-specific recognition depen-dent on interaction with base moieties of DNA, which isexemplified by cases mentioned above, TFs also interactwith the backbone of DNA in a non-sequence-specificmanner (von Hippel and Berg, 1989). Basic amino acidsat positions 3, 7, and 12 (K3, R7, and K12 in ORCA3, R3and K12 in AtERF1, and R7 in ERF189) in the first twob-strands may enhance the ERFs’ general affinities forDNA by electrostatically interacting with its negativelycharged phosphate backbone (Fig. 9A). Three basicresidues, K3, R7, and K12, may enable ORCA3 to bindstrongly to P, CS1, and GCC boxes, and therefore singlesubstitutions to nonbasic residues at any of these posi-tions (K3I, R7Q, and K12T) could cause loss of affinity.The K12T substitution caused nearly complete loss ofbinding to all of the probes, while K3I and R7Q sub-stitutions disrupted binding to certain but not all probes(CS1 for K3I and CS1 and P for R7Q). The differentialloss of binding may be explained by different affinitiesof ORCA3 for the individual probes, which remains tobe examined with quantitative analyses.

Evolution of Group IXa ERFs

Mutations of TFs and TF-targeted cis-elements alter ex-pression profiles of targeted genes, resulting in rearrange-ment of regulatory networks (Dowell, 2010). Because such

regulatory mutations are difficult to define due to ourlimited understanding of DNA-TF interactions, theirsignificance tends to be underestimated. After arisingfrom an original copy through gene duplication, novelTF genes usually undergo functional diversification bychanging expression patterns or functional propertiesbased on protein structure, such as DNA-bindingspecificity and transactivation activity. In general,DNA-binding specificities of TFs evolve very slowly(Amoutzias et al., 2007). Dimer orientation and spac-ing preferences are sometime divergent among relatedTFs that bind to DNA as dimers (Jolma et al., 2013).Because ERFs bind to DNA as monomers (Hao et al.,1998), the divergence that we found here relates to theprimary binding specificities of the TFs as monomersand seems rare in that sense (Maerkl and Quake, 2009;Baker et al., 2011).

Group IXa ERF genes from various flowering plantsare grouped into five clades. Signature residues forthe clades critical to DNA-binding specificities alongwith other properties are summarized in Figure 10.The residues are conserved in nearly all examined se-quences, except for some members mostly from clade1 (ERF168, Pt14s04630, Br024954, Br040159, Sl8g78180,ERF34, ERF66, ERF123, ERF125, and ERF10/ERF108/ERF146; DBD sequences of the latter three are identi-cal). Based on distribution of the genes in the species,we infer dicot-specific existence of clade 2 ERFs andthat clade 2-3 ERFs exist widely in various lineages of

Figure 8. In vitro binding of group IXa ERFs to predicted binding sequences in the promoters of alkaloid biosynthesis genes.EMSA was performed to examine binding between ERFs and oligonucleotide probes containing each binding sequence pre-dicted in promoters of alkaloid biosynthesis genes in tobacco (P, Q1/Q3, Q2, O3/O5, M1, and M2) and periwinkle (CS1, CC1,CC2, and CC3; Supplemental Fig. S9). Sequences and other information about the predicted sequences are in Table I. Relativeabundance of the complexes on each blot was determined by comparing the intensities of the retarded bands (SupplementalFig. S9). As a reference, probe P (or the GCC probe for AtERF1) was included in the blots. The values for the P probe (or GCCprobe for AtERF1) were set to 100. Asterisks are above the bars for AtERF1. Binding of ERF115 to the P box-type probes was notexamined. Relative levels of binding to the GCC box are not shown. Clade numbers are indicated in parentheses. Nd, Notdetermined.


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dicot plants, including woody poplar, whereas clade 2members outside of clade 2-3 are found only in thefamily Solanaceae. In particular, clade 2-1 ERFs bear-ing a unique K6 are specific to the genus Nicotianarepresented by tobacco and not found in other Sola-naceae species, such as tomato. This Nicotiana spp.-specific distribution was also confirmed by extensivedatabase searching at the SOL Genomics Network(http://solgenomics.net/).Divergent DNA-binding specificities of group IXa

ERFs may mirror their functional divergence in biolog-ical contexts. Group IXa ERFs play regulatory roles invarious forms of jasmonate-dependent chemical defense(De Geyter et al., 2012; Shoji and Hashimoto, 2012b).Tobacco ERF189 and periwinkle ORCA3 are under thecontrol of the basic Helix-Loop-Helix family TF MYC2in jasmonate signaling (Shoji and Hashimoto, 2011a;Zhang et al., 2011), regulating distinct alkaloid pathwaysthat have evolved independently in each plant lineage(i.e. tobacco and periwinkle belong to the families So-lanaceae and Apocynaceae, respectively). Despite theirconserved action in the same signaling cascade, ERF189and ORCA3 have recruited different sets of downstreamtarget genes constituting individual metabolic path-ways. Mutational changes are more likely to occur incis-elements than in TFs, in part because of the shortand degenerate nature of cis-elements and the limitedeffects of the mutations, which influence only thegenes controlled by those cis-elements (Wray, 2007). Gainand loss of cis-elements allow dynamic rearrangement ofthe connections among TFs and their target genes andthus enable recruitment of the genes into regulons underthe control of certain TFs (Shoji and Hashimoto, 2011b). Adominant role of clade 2-1 ERFs, including ERF189, asnicotine regulators (Shoji et al., 2010) may explain the

presence of only P-type sequences, exclusive targets ofthis clade of ERFs, in the promoters of tobacco alkaloidgenes. On the other hand, elimination of the GCC boxfrom the promoters may keep alkaloid regulation freefrom influence by a large number of GCC box-recognizingERFs in tobacco and periwinkle.

Figure 9. Structure of AtERF1 and modelstructures of ORCA3 and ERF189 in complexwith the GCC box (GCC) or its substitutedderivative (TCC). The model structures werebuilt by the SWISS-MODEL server, using thestructure of AtERF1 bound with GCC box(1GCC.pdb) as a template. In the ERF189complex, a GC pair in the GCC box-containingDNA molecule (59-TAGCCGCCAGC-39; thepair is underlined) was replaced with a TA pairusing the 3D-DART server. Base moieties of thepairs are labeled. Amino acid residues of in-terest are shown in sticks and labeled. Nitrogenand oxygen atoms are indicated in blue andred, respectively. To show possible access tothe nearby phosphate backbone, side chains ofR7 and Q7 were moved arbitrarily and coloredin yellow in A. Hydrogen bonding between thebasic side chain of K6 and the adenine basemoiety is shown as a red dotted line, and hy-drophobic interaction between the side chainof R6 and the base moiety of cytosine is shownas a blue dotted curve in B.

Figure 10. Properties of group IXa ERFs. Characteristics of each cladeare summarized. The amino acid residues at four positions (3, 6, 7, and12; numbered from the N terminus of the DBD) that are important forDNA-binding specificity, and thus clade distinction, are shown. Basicresidues at three positions that interact with the phosphate backboneof DNA are shown with asterisks, while the K6 unique to clade 2-1 isshown in gray. In clade 2-3 ERFs, position 3 is occupied by Arg or Lys,but here only Arg is shown. Confirmed presence of ERF genes of theindicated types in five plant species is indicated by bars. [See onlinearticle for color version of this figure.]





Functions of group IXa ERFs are not restricted to al-kaloid regulation, of course. The GCC box-dependentregulation by clade 1 and some clade 2 ERFs, some ofwhich are inducible by jasmonate as well, is responsiblefor the expression of a wide range of defense genes,such as genes for Pathogenesis-Related proteins (Guttersonand Reuber, 2004). In addition, clade 1 AaERF1 fromA. annua regulates jasmonate-inducible production ofartemisinin, a sesquiterpene lactone used for malariatreatment (Yu et al., 2012). Apart from jasmonate re-sponses, clade 2-3 AtERF13 is assumed to be involved inabiotic stress tolerance, probably controlling multipledownstream genes, because its overexpression conferredhypersensitivity to abscisic acid (Lee et al., 2010).

The different but partially overlapping binding pat-terns of group IXa ERFs to three types of sequences,P type, CS1 type, and the GCC box (Fig. 10), suggest agradual transition of binding specificity during evolu-tion, where TFs with intermediate or broader speci-ficities appeared in between TFs with distinct narrowerspecificities. Such a partial overlap of binding specific-ities is observed for other TFs, and gradual evolution oftheir specificities is also postulated (Slattery et al., 2011).According to the presumed distribution of group IXaERF genes among flowering plants (Fig. 10), we caninfer gene appearance during evolution in the followingorder: clade 1, clade 2-3, clade 2-2, and clade 2-2b orclade 2-1. In this scenario, clade 2-3 ERFs that can bindto all three types of sequence first arose from clade1 ERFs that bind only to the GCC box by acquiringbroader DNA-binding specificities around the time ofdifferentiation between monocots and dicots. Next,partial loss of such broad binding activity led to thegeneration of clades 2-2, 2-2b, and 2-1 in certain dicotlineages (e.g. Solanaceae and Nicotiana spp.).

Clustering of multiple clade 2 ERFs in a certain chro-mosomal region was speculated to occur in tobacco (Shojiet al., 2010) and was confirmed in poplar and tomatogenomes (Supplemental Fig. S2). There are relativelylarge numbers of clade 2 genes in some species (e.g.five in poplar, six in tomato, and at least 13 in tobacco),in which most of the ERFs are clustered in tandem, im-plying relatively recent generation of the genes throughrepeated gene duplications. In tomato and tobacco, be-cause the ERF clusters include members from differentclades (e.g. clades 2-3, 2-2, 2-2b, and 2-1), DNA-bindingspecificities are divergent even among the clusteredgenes, and that fact implies their rapid diversification,which could occur by subtle mutational changes in arelatively small number of the signature residues (Fig.10). Why did clade 2 ERF genes extensively duplicate incertain lineages? An increase in gene number, or genedosage, leads to higher accumulation of the gene prod-ucts, or proteins (Kondrashov et al., 2002). In line withthis notion, the increased number of tobacco ERF genesmay match the requirement for substantial nicotineproduction in this species. Gene duplications also allowthe functional diversification of the genes (Innan andKondrashov, 2010). The clustered ERF genes of to-bacco may have overlapping but distinct roles in

nicotine regulation, possibly reflecting divergent DNA-binding specificities, because patterns of jasmonate-dependent induction and effects of overexpression onnicotine production are different among the genes (Shojiet al., 2010). Little is known regarding the biologicalsignificance of the lineage-specific expansion and di-versification of clade 2, which is especially apparent inthe family Solanaceae (i.e. tomato and tobacco). Eluci-dation of clade 2 ERFs’ functions other than in nicotineregulation, which occurs only in the genus Nicotiana, isawaited.

MATERIALS AND METHODS

EMSA

The pET32-based expression vectors for ERF189, ERF115, ERF179, ERF163,ORCA3, AtERF13, and AtERF1 to express recombinant proteins fused to athioredoxin, an S-tag, and a His-tag at their N-terminal ends have been de-scribed (Shoji et al., 2010; Shoji and Hashimoto, 2012a). Because we failed toexpress the fusion of full-length Sl1g90340 as a soluble protein using the samevector arrangement, an expression vector was generated by cloning a portion ofSl1g90340 (corresponding to 40–219 amino acid residues) into the BamHI andEcoRI of pET32b. To generate the mutant versions, PCR-based site-directed mu-tagenesis (Hemsley et al., 1989) using a high-fidelity Prime Star Max DNA poly-merase (Takara) was performed, with the relevant expression vectors as templates.Sequences of the primers used for the mutagenesis are listed in SupplementalTable S2. Recombinant protein was expressed in Escherichia coli BL21 Star (DE3;Novagen), affinity purified, quantified, and stained with Coomassie Brilliant BlueR250 after separation on a 12% (w/v) SDS-PAGE gel (Shoji et al., 2010). Thepurities of the recombinant proteins are shown in Supplemental Figure S3.

Sense oligonucleotides containing the 10 base sequences shown in Table I andtheir mutant versions, as described in the text, were flanked by 59-NNNNNNNN-39 and 59-NNNNCCTCGG-39, where N represents any nucleotide. Sequencesincluding non-GCC boxes shown in Supplemental Table S1 were likewise placedin the center of the oligonucleotides. An antisense oligonucleotide (59-ACACC-GAGG-39) was biotin-labeled at the 59 end and annealed to the sense oligonu-cleotides to generate double-stranded probes (Shoji et al., 2010). The DNA-proteinbinding assay, gel separation, and detection of DNA-protein complexes havebeen described (Shoji et al., 2010). The biotin-labeled DNA probes (20 femtomoles)and purified recombinant proteins (2 mg) were used for each binding reaction.

Computational Prediction of ERF Binding Sequences

TESS (http://www.cbil.uppenn.edu./tess) was used to search for and scoreputative ERF binding sequences in the query promoters by weight matrix scoring,adapting EMSA-derived PWMs (Shoji and Hashimoto, 2011b; Supplemental Figs.S7 and S8). Minimum log-likelihood ratio and maximum log-likelihood deficit wereset to 2.0 and 8.0, respectively, and the expert parameters were used in the defaultsetting. The promoter sequences from this article can be found in the GenBank/EMBL data libraries under accession numbers AB004323 (PMT2), AJ748263 (QPT2),AB031066 (ODC1), AF233849 (ODC2), AB071165 (A622), AB286963 (MATE1),X53600 (GLN2), Y09417 (CPR), Y10182 (STR), X67662 (TDC), AF008597 (D4H), andL19119 (Hordeum vulgare HVA22)

Transient Transactivation Assay in Tobacco BY-2 cells

The reporter plasmids for PMT2pro236-GUS with a PMT2 promoter frag-ment (–236 to –1; numbered from the first ATG) and its mutant derivativePMT2pro236m4-GUS have been described (Shoji et al., 2010). To generateCS1x4-35Smini-GUS and GCCx4-35Smini-GUS, sense and antisense oligonu-cleotides that contained four copies of the CS1 or GCC box (SupplementalTable S3) were annealed to generate double-stranded oligonucleotides withcohesive tails for HindIII and SpeI at each end, and the resultant oligonucle-otides were inserted into corresponding restriction sites upstream of a minimalCauliflower mosaic virus (CaMV) 35S promoter (–46 to –1) generated by PCRmutagenesis in pBI221. The effector plasmids for 35S-ERF189, 35S-ORCA3,and 35S-AtERF1, all of which contain the CaMV 35S promoter, have been


Shoji et al.







http://www.cbil.uppenn.edu./tess






described (Shoji et al., 2010; Shoji and Hashimoto, 2012a). To introduce themutations, PCR-based mutagenesis was performed with the appropriateprimers (Supplemental Table S2) and the relevant effector vectors as tem-plates. The pBI221-LUC vector harboring LUC under the control of the CaMV35S promoter was cotransformed as an internal standard. Particle bombard-ment and subsequent measurement of GUS and LUC activities in extracts ofthe bombarded tobacco (Nicotiana tabacum) BY-2 cells have been described(Shoji et al., 2010).

Transactivation Assay in Yeast

The vectors pHIS2.1 and pGAD-Rec2-53 included in the Matchmaker One-Hybrid Screening Kit (Clontech) were manipulated. To replace HIS3 inpHIS2.1, the coding sequence of LUC lacking a 9-bp sequence correspondingto the C-terminal three amino acid residues, or mLUC, was amplified frompBI221-LUC by PCR with primers including the appropriate restriction sites.The amplification product was inserted into ApaI and KpnI sites generatedby PCR-based mutagenesis between the minimal promoter and the 39-untranslated region of HIS3 locus in pHIS2.1, generating mini-mLUC. The removalof the C-terminal residues of LUC is intended to allow mLUC protein to be cy-toplasmic to increase its access to the luciferin substrate applied exogenously(Leskinen et al., 2003). Sense and antisense oligonucleotides containing four copiesof the P, CS1, or GCC box or their mutant derivatives (Supplemental Table S3)were annealed to generate double-stranded oligonucleotides with cohesive tails forEcoRI and SpeI at each end. The resultant oligonucleotides were placed at thecorresponding sites upstream of mini-mLUC to generate the Px4-mini-mLUC,CS1x4-mini-mLUC, and GCCx4-mini-mLUC reporters. To generate the effectorplasmids, the p53 coding sequence in pGAD-Rec2-53 was removed andreplaced with BamHI and SpeI sites by PCR mutagenesis, into which full-length sequences of ERF189, ORCA3, AtERF1, and their mutant versionsamplified from the relevant bacterial expression vectors by PCR with primersattaching a BglII site compatible with BamHI and a SpeI site were inserted.Sequence information for primers used for vector construction other thanthose already listed in Supplemental Tables S2 and S3 are available uponrequest.

After the reporter and effector plasmids were cotransformed into yeast(Saccharomyces cerevisiae) Y187 strain (Clontech) following a lithium acetateprotocol, the transformed colonies were grown on agar plates containing yeastsynthetic dropout medium lacking Trp and Leu. Yeast cells were grown in thesame liquid medium for 48 h. After the optical density at 600 nm of the culturewas adjusted to 0.5 in a total volume of 1.5 mL, the cells were further grownuntil the optical density reached 1.0. Of this culture, 100-mL aliquots wereplaced into a 96-well plate and 100 mL 1 mM D-luciferin in 0.1 M sodium citratebuffer (pH 3.0) was added. After briefly shaking the plate, luminescence fromthe solutions was measured for 10 s using a LAS-4000 (Fujifilm).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Multiple sequence alignment of the DBD of groupIXa ERF proteins.

Supplemental Figure S2. Positions of group IXa ERF genes on chromosomesin rice, maize, Arabidopsis, B. rapa, popular, and tomato.

Supplemental Figure S3. Purity of recombinant ERF proteins.

Supplemental Figure S4. In vitro binding of group IXa ERFs to DRE, CBF2,RAV, and CE1 boxes.

Supplemental Figure S5. In vitro binding of mutant versions of ORCA3,ERF179, and AtERF1 to P, CS1, and GCC boxes.

Supplemental Figure S6. Transient transactivation of CS1x4-35Smini-GUSby ORCA3 and GCCx4-35Smini-GUS by AtERF1 in tobacco BY-2 cells.

Supplemental Figure S7. In vitro binding profiles of ORCA3 at P and CS1boxes.

Supplemental Figure S8. In vitro binding profiles of ORCA3 and AtERF1at GCC box.

Supplemental Figure S9. In vitro binding of group IXa ERFs to predictedbinding sequences in promoters of alkaloid biosynthesis genes.

Supplemental Table S1. Sequences of non-GCC boxes.

Supplemental Table S2. Oligonucleotides used for PCR-based site-directedmutagenesis.

Supplemental Table S3. Oligonucleotides including four copies of P, CS1,and GCC boxes.

ACKNOWLEDGMENTS

We thank Kazuyuki Hiratsuka (Yokohama National University) for pro-viding the pBI221-LUC plasmid and advising on the LUC assay in yeast andMasaru Ohme-Takagi (Saitama University) for consulting on vectors contain-ing four copies of the GCC box.

Received March 18, 2013; accepted April 25, 2013; published April 29, 2013.

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divergent dna-binding speciﬁcities of a group of ethylene ... · divergent dna-binding...

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