Two groups of MYB transcription factors share a motif which enhances trans-activation activity

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Biochemical and Biophysical Research Communications 341 (2006) 1155–1163

BBRC

Two groups of MYB transcription factors share a motifwhich enhances trans-activation activity

Jigang Li a,1, Xiaoyuan Yang a,1, Yan Wang a, Xiaojuan Li a, Zhaofeng Gao a,Meng Pei a, Zhangliang Chen a,b, Li-Jia Qu a,b, Hongya Gu a,b,*

a Peking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering and Plant

Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, PR Chinab National Plant Gene Research Center (Beijing), Beijing 100084, PR China

Received 9 January 2006Available online 26 January 2006

Abstract

MYB transcription factors play important roles in many plant developmental processes and various defense responses. Two groups ofMYB transcription factors were found to share a W/Y-MDDIW motif. This motif alone shows no trans-activation activity in yeast,however, it enhances the trans-activation activity of the neighbor regions greatly. We further show that all the genes in the group 1,including the previously reported genes AtMYB21, PsMYB26, AmMYB305, and AmMYB340, are predominantly expressed in flowers.Furthermore, we found that these two groups ofMYB transcription factor genes might be regulated by light, and probably preferentiallyexpressed in the darkness, suggesting that they may play roles in the light signaling pathway.� 2006 Elsevier Inc. All rights reserved.

Keywords: MYB transcription factors; Motif; Trans-activation activity; Light regulation

Transcription factors play a central role in the regula-tion of developmental and metabolic programs. A typicalplant transcription factor contains, with few exceptions, aDNA-binding region, an oligomerization site, a transcrip-tion-regulation domain, and a nuclear localization signal[1]. On the basis of similarities in the DNA-bindingdomain, transcription factors have been classified into mul-tiple families [2]. In plants, MYB proteins form one of thelargest transcription factor super-families, which are char-acterized by a MYB domain (DNA-binding domain) com-posed of one to three imperfect repeats, each about 52amino acid residues [3–6]. According to the number ofthe repeats in the MYB domain, plant MYB transcriptionfactors can be divided into three families: R2R3-MYB withtwo adjacent repeats, R1R2R3-MYB proteins with three

0006-291X/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.bbrc.2006.01.077

* Corresponding author. Fax: +86 10 6275 1841.E-mail address: [email protected] (H. Gu).

1 These authors contributed equally to this work.

adjacent repeats, and MYB-related proteins with onerepeat or two separated repeats [6–8].

In addition to the conserved MYB domain, more than20 conserved motifs were found in the C-termini of theR2R3-MYB proteins [3,6,9]. On the basis of sequencesimilarity of these motifs, R2R3-MYB proteins were cat-egorized into different groups [3,6,9]. Although therehave been extensive studies on the expression, function,and evolution of the R2R3-MYB transcription factors,few studies were conducted on the function of the vari-ous conserved motifs. For example, evidence for theimportance of some motifs came from the studies onthe GLABROUS1 encoded by GL1 and the non-func-tional protein encoded by the allele gl1-2 which lackeda motif of 19-aa at the C-terminal region [3,10]. Thefunctional analysis on the conserved motifs may facilitatethe identification of functionally important sequencesoutside the MYB DNA-binding domains in MYBproteins.

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1156 J. Li et al. / Biochemical and Biophysical Research Communications 341 (2006) 1155–1163

The correlation between the sequence similarity andfunction has been found in some groups of MYB genes[3,6,8,9]. GL1, WER, and AtMYB23, members of a groupin the R2R3-MYB family, share the same C-terminalmotifs, and are all involved in the epidermal cell patterning[10–13]. Another example for this correlation comes fromthe studies on three R2R3-MYB homologues, AtMYB91/AS1 from Arabidopsis thaliana, AmMYBPHAN fromsnapdragon (Antirrhinum majus), and ZmMYBRS2 frommaize (Zea mays), all of which have been shown to nega-tively regulate KNOX (KNOTTED1-like homeobox) geneexpression in organ primordia [14–17].

In a previous study, we analyzed the phylogenetic rela-tionships among all the members in the MYB proteinsuper-family in Arabidopsis and rice, and found nearly100 putative motifs among them [8]. Here, we report thatone of the motifs (W/Y-MDDIW), shared by two phyloge-netic groups of R2R3-MYB proteins, involves in theenhancement of the trans-activation activity of the corre-sponding transcription factors. We also found the correla-tion between sequence similarities and gene expressionpattern both based on the results of the previously reportedstudies and that of our own experiments.

Materials and methods

Plant materials and growth conditions. Arabidopsis thaliana ecotypeColumbia was used for all the experiments. Mutant lines hy5-215 andcop1-4 (Columbia background) were provided by Professor X.W. Deng(Yale University, New Haven, CT). Arabidopsis seeds for germinationwere surface sterilized and sown on growth medium (half strength MSwith 1% sucrose) at 4 �C for 2–4 days in the dark for vernalization. Thenthe seeds were put in a growth chamber at 22 �C in complete darkness orcontinuous white light, or in a normal growth condition (16 h of light and8 h of darkness).

RNA extraction and cDNA synthesis. Total RNAs were isolated fromroots, rosette leaves, cauline leaves, inflorescences, and siliques of4-week-old Arabidopsis plants in normal growth condition or from the5-day-old seedlings grown in continuous white light or complete darknessusing RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manu-facturer’s instructions. First-strand cDNAs were synthesized using theSuperscript II RNase H-Reverse Transcriptase Kit (Invitrogen, Carlsbad,CA) according to the manufacturer’s instructions, and then used as thetemplates for RT-PCR amplification.

RT-PCR amplification. Specific primers were designed to amplify thefragments of target genes in Arabidopsis by RT-PCR with Ex Taq DNApolymerase (Takara). The following primers were used:

AtMYB57-F: 5 0-GTG GTC GAA GAT TGC GAA GCA T-3 0

AtMYB57-R: 5 0-GAG AAG CTC TCA GCC GTG TCT A-3 0

AtMYB21-F: 5 0-ACA ACA TCG TCC GTT GGA TCT C-3 0

AtMYB21-R: 5 0-TCA TCA ACG GTC ATC GGT ACT G-3 0

AtMYB24-F: 5 0-TCA TCA AGA GCG GAG AAA CGA C-3 0

AtMYB24-R: 5 0-TCA TGA TCG AAC CGG ATT CAG G-3 0

AtMYB116-F: 5 0-GAT CAC GAG GGG GTA GTA GTC A-3 0

AtMYB116-R: 5 0-TAC TTG GAA TCA GCC ACG TGA C-3 0

AtMYB62-F: 5 0-GGC TCG ATG TGT TTC CAT GAA G-3 0

AtMYB62-R: 5 0-CAT CCT CGT ACG CTG AAT AAC C-3 0

AtMYB48-F: 5 0-TTC TGT TTC CAA CAC AGT AGA TC-30

AtMYB48-R: 5 0-AGA AAG TGG GAT CAC AAG ACT TT-3 0

AtMYB59-F: 5 0-CTA AGT TTT GAA CAT GGT AGT GG-3 0

AtMYB59-R: 5 0-CAC AGA AAC TTC AAA AGT CTA TT-3 0

As multiple bands were obtained due to the alternative splicing ofAtMYB59 and AtMYB48 in an attempt to amplify the full-length cDNAsof these two genes [18], we designed the specific primers to amplify the3 0-UTRs of these two genes, so as to obtain a single band for each gene.PCR was performed for 35 cycles, each with 94 �C for 50 s, 60 �C for 50 s,and 72 �C for 2 min.

Trans-activation activity assays. Full-length coding region and variousdeletion mutants of AtMYB59 and AtMYB21 were amplified by PCRusing Pfu DNA polymerase (Takara) and appropriate primers as follows:

AtMYB59 (1)-F: 5 0-TTA GTC GAC AAA TGA AAC TTG TGCAAG AAG-30

AtMYB59 (112)-F: 5 0-TTA GTC GAC AGG CTC AAG AGA AGAAGC GA-30

AtMYB59 (151)-F: 5 0-TTA GTC GAC AAG AAT GCG AAG ACGGGT ACT-30

AtMYB59 (181)-F: 5 0-TTA GTC GAC ACT ACT ACT CAG AGCAAA GCT-3 0

AtMYB59 (150)-R: 5 0-TTA GAG CTC CTA TTG ATT CAT TTTCCC GTT-30

AtMYB59 (180)-R: 5 0-TTA GAG CTC CTA GTT GTC TTT TACCGG TTT-3 0

AtMYB59 (235)-R: 5 0-TTA GAG CTC CTA AAG GCG ACC ACTACC ATG-3 0

AtMYB21 (1)-F: 5 0-AGA GTC GAC AAA TGG AGA AAA GAGGAG GAG-3 0

AtMYB21 (124)-F: 5 0-AGA GTC GAC AAA TCA AGC AAT CGGATG TA-3 0

AtMYB21 (195)-F: 5 0-AGA GTC GAC AAG ATT ATC CAG TACCGA T-30

AtMYB21 (194)-R: 5 0-TTA GTC GAC CTA CAC GGT TGC CGTCAC TG-3 0

AtMYB21 (226)-R: 5 0-TTA GTC GAC CTA ATT ACC ATT CAATAA AT-3 0

The numbers in the brackets indicate the corresponding amino acidsites of respective primers, and the underlined nucleotides correspond toSalI and SacI restriction sites, respectively. Inserts were fused in-frame tothe sequences encoding the GAL4 DNA-binding domain by cloning theminto pYF503 [19]. All the constructs were confirmed by sequencing. Yeaststrain EGY48, and the reporter vector pG221, which harbors the CYC1

core promoter and b-galactosidase (LacZ) reporter gene, were used forthis assay [19]. Yeast LiAc-mediated transformations and b-galactosidasefilter assays using X-gal were performed as described in the ClontechYeast Protocols Handbook (Palo Alto, CA). Yeast transformants werescreened on the synthetic dropout SD/-Ura and SD/-Ura/-Trp medium.

Phylogenetic analysis.Alignments were conducted using the CLUSTALprogram in the CLUSTAL W package [20] and the phylogenetic tree wasconstructed using the parsimony method in PHYLIP package [21], with abootstrap of 100 samples.MEME [22] was used to identify the sharedmotifsin the MYB protein sequences.

Promoter–reporter gene fusion and GUS activity analysis. The promoterregions of AtMYB59 (2078 bp), AtMYB48 (1880 bp), AtMYB21

(1996 bp), and AtMYB24 (2189 bp) were amplified from Arabidopsis

genomic DNA by PCR using the following primers:

AtMYB59-GUS-F: 5 0-TAA GGA TCC AGA CGT TTA GGG GATAAG-30

AtMYB59-GUS-R: 5 0-CCA CTG CAG TTT ACG GTA TTC TTCTTG-30

AtMYB48-GUS-F: 5 0-GTC GGA TCC CGC ACC ACG GGC T-30

AtMYB48-GUS-R: 5 0-TTT CTG CAG TCC CTC CTC TTG CATCAT-30

AtMYB21-GUS-F: 5 0-TTG GCA GAA TTC TAT GGT CAC-3 0

AtMYB21-GUS-R: 5 0-CCT CCA CTG CAG CCT CCT CCT CTT-3 0

AtMYB24-GUS-F: 5 0-TGA TCA GAA TTC GAG AGT CTA C-3 0

AtMYB24-GUS-R: 5 0-CCA CCA CTG CAG TCT CTT TTC TCC-3 0

search Communications 341 (2006) 1155–1163 1157

The underlined nucleotides correspond to BamHI, PstI, and
EcoRI restriction sites, respectively. The amplified fragments werecloned into pCAMBIA 1381 (CAMBIA, Canberra, Australia)respectively, and transformed into Arabidopsis. The light shift treat-ments were conducted by following the methods described previously[23,24], i.e., one group of transgenic seedlings were grown for 5 daysin continuous darkness or light, and the other group of transgenicseedlings were grown in light or darkness for 4 days and then shiftedto the opposite conditions, respectively, for 1 day. The two groups ofseedlings were then examined for the GUS activity as describedpreviously [25,26].
Northern hybridization. The coding sequences corresponding to theC-terminal region of AtMYB57 (204 bp) and AtMYB21 (221 bp) wasamplified by the gene-specific primers:

AtMYB57-F: 5 0-GTG GTC GAA GAT TGC GAA GCA T-3 0

AtMYB57-R: 5 0-GAG AAG CTC TCA GCC GTG TCT A-3 0

AtMYB21-F: 5 0-ACA ACA TCG TCC GTT GGA TCT C-3 0

AtMYB21-R: 5 0-TCA TCA ACG GTC ATC GGT ACT G-3 0

The resulting fragments were cloned into pBlueScript vectors and usedas probes for Northern hybridization. The total RNA samples (15 lg perlane) were loaded on 1.2% (w/v) agarose gels, and Northern blot andhybridization were carried out as described previously [27].

J. Li et al. / Biochemical and Biophysical Re

Fig. 1. (A) Phylogenetic tree of two groups of MYB transcription factors whichthe light signaling pathway, AtMYB61 and LAF1 [34,36], were selected as ouamino acid positions (from the translation start codon). The arrow indicates thecodon. (C) Positions of the W/Y-MDDIW motif (shown as boxes) in the fuindicates the incompleteness of its cDNA.

Results

Seventeen MYB transcription factors share a conserved

W/Y-MDDIW motif

Our previous study identified about 100 motifs in theMYB protein sequences and one of them, designated motif52, was shared by some members of the R2R3-MYB genefamily in Arabidopsis and rice [8]. We performed sequencealignment of the closely related proteins with the motif 52based on the phylogenetic tree [8]. Finally, 12 members inArabidopsis and rice were confirmed to share the motif 52(Figs. 1A and B). Extensive BLAST search against Gen-Bank was also conducted, and 5 more members in otherplant species were found to share the motif 52:AY104893 from maize, BT009536 from wheat, PsMYB26from pea, and AmMYB305 and AmMYB340 from snap-dragon. Taken together, 17 R2R3-MYB transcription fac-tors from monocots and dicots were finally found to harborthe motif 52 (Figs. 1A and B).

share a motif W/Y-MDDIW. Two MYB transcription factors involved intgroups. (B) Alignment of the motif sequences. Numbers at right indicateboundary of the two groups. The star mark represents a translational stopll-length proteins. Question mark at the N-terminal region of BT009536


On the basis of the phylogenetic analysis, these 17proteins are divided into two groups (Fig. 1A). Sequencealignment of these proteins showed the consensus sequenceW/Y-MDDIW (Fig. 1B). We found two characteristics ofthe consensus sequence. First, the two groups are charac-terized by the first amino acid residue, respectively: W ingroup 1 and Y in group 2 (Fig. 1B). And second, the lastamino acid residue W is conserved in all members of thetwo groups. The location of the W/Y-MDDIW motif isalso different in two groups: in group 1, the motifs are alllocalized to the end of the C-terminal region, but in group2, the motifs are all localized in the middle of the C-termi-nal region (Fig. 1C).

The motif enhances trans-activation activity

Because the motif W/Y-MDDIW is conserved in someMYB transcription factors, it might be functionally impor-tant for the transcriptional activity. To determine whetherthismotif involves in trans-activation activity, we performedyeast one hybrid assay for two representative proteins,AtMYB21 in the group 1 and AtMYB59 in the group 2.

Fig. 2. (A) Sequence alignment of AtMYB21, AtMYB24, AmMYB305, and Ain gray. The two MYB repeats (R2 and R3) are indicated with sets of arrows, aright indicate amino acid positions (from the translation start codon) and the blmentioned in (B). The motif region is underlined, with the conserved amino amarks. (B) b-galactosidase filter assay of yeast carrying a pG221 reporter vectoframe with the GAL4 DNA-binding domain by using X-gal as a substrate. Thras the negative control, and pYF504, which harbors the full-length GAL4 geneto color in this figure legend, the reader is referred to the web version of this

Based on the sequence alignment, we found that the W/Y-MDDIW motif is the only conserved motif in additionto the MYB domain among the members in the group 1(data not shown; only the alignment of AtMYB21,AtMYB24, AmMYB305, and AmMYB340 was shown inFig. 2A). To study the function of this motif in the trans-activation activity, the constructs harboring different frag-ments of AtMYB21 were generated (Fig. 2B). The resultsof yeast one hybrid assay showed that, the full-lengthsequence of AtMYB21, and the C-terminal regions whichlacks the R2R3-MYB DNA-binding domain, have thetrans-activation activity (Fig. 2B). However, the deletionof a fragment of 32 amino acid residues in the C-terminus,which contains the conserved W/Y-MDDIW motif, result-ed in the complete loss of the trans-activation activity(Fig. 2B). It indicated that this motif was very importantfor the transcriptional function of AtMYB21. Interesting-ly, the fragment of 32 amino acid residues alone couldnot activate the transcription of the reporter gene (Fig. 2B).

As described above, the motif 52 locates at the middle ofthe C-terminal region of the proteins in the group 2. Bycomparing their protein sequences, we found three

mMYB340. Identical amino acids are shaded in black, conserved changesnd the critical tryptophan residues are indicated by star marks. Numbers atue arrows with numbers indicate the positions of the respective amino acidscid marked by dots. The critical tryptophan residues are indicated by starr and a pYF503 vector with different AtMYB21 cDNA fragments fused in-ee independent transformants were shown. Empty pYF503 vector was used, was used as the positive control [19]. (For interpretation of the referencespaper.)

J. Li et al. / Biochemical and Biophysical Research Communications 341 (2006) 1155–1163 1159

conserved motifs, including the motif 52, among the pro-teins of monocots and dicots in the group 2 (data notshown; only the alignment of proteins from Arabidopsis

and rice was shown in Fig. 3A). Therefore, the constructsfor the yeast one hybrid assay were made based on theseconserved motifs, each containing one or a combinationof them (Fig. 3). The results of the assays showed thatthe constructs containing the sequences 112–235 (with allthree motifs) and 151–235 (with the last two motifs, includ-ing motif 52) showed the highest levels of trans-activationactivity, but interestingly, the full-length protein ofAtMYB59 has lower transcriptional activity (Fig. 3B).The construct containing the sequence 181–235 (with thelast motif) alone has a very low level of trans-activationactivity (Fig. 3B). However, the expression of the reportergene was not detected in the constructs containing thesequences 112–180 (with the first two motifs, includingmotif 52), 112–150 (with the first motif), or 151–180 (with

Fig. 3. (A) Sequence alignment of AtMYB59, AtMYB48, OsMYBAS1, andrespective amino acids mentioned in (B). The motif regions are underlined,localization signals [18] are indicated with lines. The critical tryptophan residcarrying a pG221 reporter vector and a pYF503 vector with different AtMYB59using X-gal as a substrate. Three independent transformants were shown. Emptthe positive control [19]. (For interpretation of the references to color in this

the second motif, motif 52) (Fig. 3B). These results indicat-ed that the motif 52 alone has no trans-activation activity,but it greatly enhances trans-activation activity togetherwith its C-terminal sequence, residues 181–235, in this case,which is consistent with the results of the assay forAtMYB21.

Genes in the group 1 are predominantly expressed in flowers

Previous studies showed that some members in thegroup 1 were specifically expressed in the flowers [28–30].To investigate whether the other genes in the group 1 haveorgan-specific expression, we performed RT-PCR analysisto study the expression pattern of all the Arabidopsis genesin the group 1: AtMYB57, AtMYB21, AtMYB24,AtMYB116, and AtMYB62. The results showed that, allthese five genes were predominantly expressed in the flow-ers (Fig. 4A). We performed RNA gel blot analysis to

OsMYBAS2. The blue arrows with numbers indicate the positions of thewith the conserved amino acid marked by dots. The bipartite nuclearues are indicated by star marks. (B) b-galactosidase filter assay of yeastcDNA fragments fused in-frame with the GAL4 DNA-binding domain byy pYF503 vector was used as the negative control and pYF504 was used asfigure legend, the reader is referred to the web version of this paper.)

Fig. 5. Expression pattern analysis of all the Arabidopsis genes in thegroup 1 and 2 in 5-day-old wild type and mutant (cop1 and hy5) seedlingsgrown under complete darkness (D) or continuous white light (L) by RT-PCR. Arabidopsis ubiquitin10 gene was used as a positive internal control.

Fig. 4. Genes in the group 1 are predominantly expressed in flowers. (A)Expression pattern analysis of AtMYB57, AtMYB21, AtMYB24,AtMYB116, and AtMYB62 in different organs by RT-PCR. Arabidopsisubiquitin10 gene was used as a positive internal control. (B) RNA gel blotanalysis of AtMYB57 and AtMYB21 in different organs. Fifteen micro-gram RNA samples were loaded per lane.


confirm the expressions of AtMYB57 and AtMYB21, andthe result was consistent with that of RT-PCR (Fig. 4B).Our results are also supported by the microarray data inthe GENEVESTIGATOR database [31].

The members in both group 1 and 2 are regulated by light

AtMYB21, the member in the group 1, was also shownto be regulated by COP1 [30]. To determine whether theother genes in the group 1 and/or the genes in the group2 are regulated by light, and/or by the key regulators,COP1 and HY5 [32,33], in the light signaling pathway,we examined their expressions in the wild-type and mutant(cop1 and hy5) seedlings grown under continuous whitelight or complete darkness. Our results showed that theexpressions of all the Arabidopsis members in the group1, AtMYB57, AtMYB21, AtMYB24, and AtMYB116

(except AtMYB62), were undetectable either in the wildtype or in the mutants. The expression of AtMYB62 wasonly detectable in the seedlings grown under continuouswhite light (Fig. 5). Our data are consistent with the previ-ous report that the expression of AtMYB21 could not bedetected by RT-PCR in the wild-type seedlings under lightor dark conditions [30].

To further examine whether the genes in the group 1 areinvolved in light signaling pathway, we performed lightshift assay on the transgenic plants with promoter-GUSconstructs from AtMYB21 and AtMYB24. The resultsshowed that, although expressed at low levels under contin-uous white light, the expressions of AtMYB21 andAtMYB24 increased rapidly in cotyledons and hypocotylsonly 1 day after shifting from continuous white light tocomplete darkness (Fig. 6).

We also examined whether the expressions of the genes inthe group 2, e.g., AtMYB59 and AtMYB48, were regulatedby light. Contrary to most genes in the group 1, the expres-sions of AtMYB59 and AtMYB48 could be detected in both

wild-type and mutant seedlings grown under different lightconditions, but expressed preferentially in complete dark-ness (Fig. 5).We also conducted light shift assays to confirmthe RT-PCR data. The results showed that like AtMYB21and AtMYB24, AtMYB59 and AtMYB48 are alsoexpressed preferentially in darkness (Fig. 6).

Discussion

In this study, we found that two groups of MYB tran-scription factors share a motif W/Y-MDDIW. One repre-sentative from each group, AtMYB21 and AtMYB59,respectively, was studied for the trans-activation activityin yeast, and the results showed that although theW/Y-MDDIW motif alone could not activate the expres-sion of the reporter gene, it was indispensable for thetrans-activation activity of the C-terminal sequences.Moreover, this motif is the only conserved sequence inthe C-termini among the proteins in the group 1, suggestingits functional importance.

In the trans-activation activity assay, we found thatboth full-length proteins of AtMYB21 and AtMYB59had less trans-activation activity than their C-termini with-out the MYB domain (Figs. 2 and 3). This phenomenonwas also found in other MYB proteins. For example,LAF1, an R2R3-MYB transcription factor in Arabidopsis

involved in the phytochrome A signaling, was reported toact as a transcriptional activator, and interestingly, thefull-length LAF1 protein showed 100-fold reduction inb-galactosidase activity than its C-terminus alone [34]. Itseems that the MYB domain more or less interferes withthe C-terminal trans-activation activity of the MYB tran-scription factors. More studies are needed to draw a gener-al conclusion on the correlation between MYB domain andtrans-activation activity, or to understand the molecularmechanism of the interference.

Fig. 6. Expressions of AtMYB21, AtMYB24, AtMYB48, and AtMYB59 are repressed by light. Germinated seedlings grown for 5 days under continuouswhite light (Light) and complete dark (Dark) conditions, and grown in white light or darkness for 4 days and then shifted to the opposite conditions for 1day (Lightfi Dark, and Darkfi Light, respectively), were stained simultaneously for GUS activities. Bars indicate 1 mm.


AtMYB59 and AtMYB48 were found to undergohighly conserved alternative splicing and produce fourdistinctively spliced transcripts [18]. Interestingly, theencoded proteins of AtMYB59-1–3 mainly differ in theirMYB domains. Our data show that the correspondingproteins encoded by AtMYB59-1–3 transcripts are tran-scriptional activators, suggesting that these transcriptionfactors might activate different groups of target genes,by changing their DNA binding domains via alternativesplicing.

It has been previously reported that AtMYB21,PsMYB26, AmMYB305, and AmMYB340, the membersin the group 1, were specifically expressed in flowers,and directly activated the expression of genes involvedin the phenylpropanoid metabolism [28–30,35]. In thisstudy, we found that other Arabidopsis MYB genes inthe group 1, i.e., AtMYB24, AtMYB57, AtMYB62, andAtMYB116, were also predominantly expressed in flow-

ers (Fig. 4), suggesting that these homologous genes inthe group 1 may be involved in the regulation of floraldevelopment.

The expressions of AtMYB21 and AtMYB24 were notdetectable by RT-PCR in the wild-type or mutant seedlingsgrown under various light conditions. However, the expres-sions of these two genes were much enhanced in the lightshift experiment in the promoter-GUS transgenic lines.This could be caused by a very low expression level of thesetwo genes in vivo, while the exogenous GUS protein,whose gene was driven by the promoter of AtMYB21

and AtMYB24, might be degraded slowly in the transgenicArabidopsis plants. Therefore, slight induction in genes oflow expression level could be detected in the promoter-GUS assay. Taken together, our data showed that thesetwo groups of MYB transcription factor genes, at least inArabidopsis, are most likely to play roles in the light signal-ing pathway.


MYB transcription factor genes play important roles inmany plant developmental processes and various defenseresponses. Our study showed that two groups of MYBtranscription factors share an important motif for theirtrans-activation activity. Our data also suggested a possiblecorrelation of the W/Y-MDDIW motif-containing tran-scription factors and their involvement in the floral devel-opment and light signaling pathway.

Acknowledgments

This study was supported by a Natural Science Founda-tion Grant to Gu (30370093). We thank Professor MeihuaLiu for her advise in yeast one hybrid assay, and Miss LiZhang for technical assistance.

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Two groups of MYB transcription factors share a motif which enhances trans-activation activity

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