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Plant Physiol. 2005 Jan; 137(1): 176–189.
PMCID: PMC548849

Annotations and Functional Analyses of the Rice WRKY Gene Superfamily Reveal Positive and Negative Regulators of Abscisic Acid Signaling in Aleurone Cells1,[w]


The WRKY proteins are a superfamily of regulators that control diverse developmental and physiological processes. This family was believed to be plant specific until the recent identification of WRKY genes in nonphotosynthetic eukaryotes. We have undertaken a comprehensive computational analysis of the rice (Oryza sativa) genomic sequences and predicted the structures of 81 OsWRKY genes, 48 of which are supported by full-length cDNA sequences. Eleven OsWRKY proteins contain two conserved WRKY domains, while the rest have only one. Phylogenetic analyses of the WRKY domain sequences provide support for the hypothesis that gene duplication of single- and two-domain WRKY genes, and loss of the WRKY domain, occurred in the evolutionary history of this gene family in rice. The phylogeny deduced from the WRKY domain peptide sequences is further supported by the position and phase of the intron in the regions encoding the WRKY domains. Analyses for chromosomal distributions reveal that 26% of the predicted OsWRKY genes are located on chromosome 1. Among the dozen genes tested, OsWRKY24, -51, -71, and -72 are induced by abscisic acid (ABA) in aleurone cells. Using a transient expression system, we have demonstrated that OsWRKY24 and -45 repress ABA induction of the HVA22 promoter-β-glucuronidase construct, while OsWRKY72 and -77 synergistically interact with ABA to activate this reporter construct. This study provides a solid base for functional genomics studies of this important superfamily of regulatory genes in monocotyledonous plants and reveals a novel function for WRKY genes, i.e. mediating plant responses to ABA.

The WRKY genes encode a large group of transcription factors. There are over 70 WRKY genes in Arabidopsis (Arabidopsis thaliana; Eulgem et al., 2000; Dong et al., 2003) and rice (Oryza sativa; Goff et al., 2002; Zhang et al., 2004). This family is defined by a domain of 60 amino acids, which contains the amino acid sequence WRKY at its amino-terminal end and a putative zinc finger motif at its carboxy-terminal end. Some of the WRKY proteins contain two WRKY domains, while others have only one. Most of the published WRKY proteins bind to the cognate cis-acting element (C/T)TGAC(T/C) in the promoter (for review, see Eulgem et al., 2000) or the 5′ untranslated regions of target genes (Yu et al., 2001). For the WRKY proteins containing two WRKY domains, such as PcWRKY1 (Eulgem et al., 1999), SPF1 (Ishiguro and Nakamura, 1994), and AtZAP1 (de Pater et al., 1996), the C-terminal domain has the major DNA-binding activities.

WRKY proteins function as transcriptional activators and repressors. For example, the PcWRKY1 protein functions as a transcription activator binding to the W-box in the PcWRKY1 promoter as shown in the assays with parsley (Petroselinum crispum) protoplasts and yeast (Saccharomyces cerevisiae) as LexA-WRKY1 fusion (Eulgem et al., 1999). Similarly, Arabidopsis ZAP1 binds to and activates a synthetic promoter containing the W-box in yeast and Catharanthus roseus suspension cells (de Pater et al., 1996). AtWRKY22 and AtWRKY29 transactivate the promoters of WRKY29 and FRK1 (FLG22-INDUCED RECEPTOR-LIKE KINASE 1), respectively, in the Arabidopsis protoplasts (Asai et al., 2002). Overexpression of AtWRKY18 in transgenic Arabidopsis plants results in enhanced expression of pathogenesis-related genes (Chen and Chen, 2002). Recently, we have demonstrated that rice WRKY71, a homolog of wild oat (Avena sativa subsp. fatua) ABF2 (Rushton et al., 1995), encodes a transcriptional repressor of GA signaling in aleurone cells (Zhang et al., 2004). Some WRKY genes can function as either a repressor or an activator. For instance, AtWRKY6 suppresses its own promoter as well as the promoter of a closely related WRKY family member, whereas it activates the promoters of a receptor-like protein kinase (SIRK) and the senescence- and pathogen defense-associated PR1 genes (Robatzek and Somssich, 2002).

WRKY genes play a variety of developmental and physiological roles in plants. The most reported studies for this superfamily of genes address their involvement in salicylic acid (SA) and disease responses (Chen and Chen, 2000; Dellagi et al., 2000; Du and Chen, 2000; Eulgem et al., 2000; Kim et al., 2000; Asai et al., 2002). In addition, WRKY genes are involved in plant responses to freezing (Huang and Duman, 2002), wounding (Hara et al., 2000), oxidative stress (Rizhsky et al., 2004), drought, salinity, cold, and heat (Pnueli et al., 2002; Rizhsky et al., 2002; Seki et al., 2002). Some WRKY genes regulate embryogenesis (Lagace and Matton, 2004), seed coat and trichome development (Johnson et al., 2002), and senescence (Hinderhofer and Zentgraf, 2001; Robatzek and Somssich, 2001). Biosynthesis of anthocyanin (Johnson et al., 2002), starch (Sun et al., 2003), and sesquiterpene (Xu et al., 2004) are also dependent on WRKY proteins. WRKY genes may control seed germination and postgermination growth as well, because two wild oat WRKY proteins (ABF1 and ABF2) bind to the box2/W-box of the GA-regulated α-Amy2 promoter (Rushton et al., 1995) and OsWRKY71 encodes a transcriptional repressor of GA signaling in aleurone cells (Zhang et al., 2004). The report of AtWRKY70 in mediating both SA and jasmonic acid responses indicates the importance of this gene in the cross-talk of hormone signaling (Li et al., 2004).

Previously, we reported the annotations of 77 OsWRKY genes (Zhang et al., 2004). Here we present more OsWRKY genes, along with the bioinformatic analysis data such as their chromosomal localizations, intron-exon structures, full-length cDNA (FL-cDNA) support, alternative splicing, phylogenetic relationships, and the related protein motifs for these 81 OsWRKY genes. Our studies also reveal that there are at least four OsWRKY genes involved in abscisic acid (ABA) signaling in aleurone cells, with two functioning as positive and two as negative regulators. To our knowledge, this is the first report of such activity by a WRKY protein.


Identification of 81 WRKY Genes in the Rice Genome

A systematic analysis was carried out to identify WRKY genes in the rice genome using the publicly available genomic sequences of rice L. subsp. japonica (Goff et al., 2002) and subsp. indica (Yu et al., 2002) that were downloaded from GenBank and from the Web site of the Beijing Genomics Institute (http://btn.genomics.org.cn/rice). Using GENSCAN, a gene prediction program (Burge and Karlin, 1997), we constructed databases of the predicted rice coding regions and peptides (Zhang et al., 2004). WRKY genes were identified by using the HMMsearch program and a Hidden Markov Model (PF03106; http://pfam.wustl.edu), with a cutoff E-value of 0.1. A total of 158 WRKY genes were identified in japonica and indica rice genome sequences. Manual inspections eliminated 32 candidate genes because the deduced proteins for these genes do not contain any of the conserved amino acid residues, W(R/K)(K/R)Y, in the N terminus of the WRKY domain. The corresponding coding region sequences (CRSs) of the remaining 126 sequences were analyzed for redundant sequences by performing pairwise comparisons. An arbitrary cutoff value of 95% was used to take into account sequencing errors and natural variations in the japonica and indica rice genomes. Thus, if the identity between 2 CRSs (including both exon and intron sequences between the start and stop codons) is equal to or higher than 95%, they are considered to be redundant and only 1 is kept. This analysis eliminated 54 sequences. The WRKY domain peptide sequences of the remaining 72 putative WRKY genes were aligned using ClustalW (Thompson et al., 1994). The alignment output was used to build a rice-specific WRKY HMM model using HMMbuild and HMMcalibrate in the HMMER package. Scanning with this new model against the rice peptide databases (Zhang et al., 2004), also with a cutoff E-value of 0.1, resulted in 178 candidates. Twenty-nine of these candidate sequences were eliminated because they did not contain the conserved WRKY motif. One gene identified in Contig96377 (accession no. AAAA01094809.1) was eliminated because it was very short, missing both the 5′ and 3′ regions of the gene. The CRSs of the remaining 148 candidates were retrieved and a redundancy analysis was carried out as described, eliminating 67 sequences (see Supplemental Table I). In total, 81 WRKY genes were identified, with 45 in the japonica genome sequences and 36 in the indica genome sequences. The detailed information on these 81 OsWRKY genes is listed in Supplemental Table II.

Manual Inspections Reveal Annotation Errors and Alternative Spliced Open Reading Frames

Approximately 36% of Arabidopsis genes identified through the automated predictions contained errors (Haas et al., 2002; Meyers et al., 2003). We took several approaches to refine the accuracy of the collected OsWRKY genes. First of all, three genes (OsWRKY41, -43, and -44) were reannotated using FGENESH (www.softberry.com), because the first introns of these genes were too small (Supplemental Table III). Second, two partial genes published previously (Zhang et al., 2004), OsWRKY46 (missing both the 5′ and 3′ ends) and OsWRKY63 (missing the 3′ end), were replaced with full-length genes we identified in newly available bacterial artificial chromosome (BAC) sequences. However, no new sequences for OsWRKY50 have been identified yet and hence it is still short at the 3′ end. Third, the predicted genes were analyzed by comparison to DNA and deduced amino acid sequences of the rice FL-cDNA sequences (Kikuchi et al., 2003) using BLAST analyses. An annotation is considered to have a significant FL-cDNA match if (1) the predicted coding sequence is ≥95% identical to the corresponding region of its FL-cDNA sequence at both the DNA and deduced amino acid sequence levels; and (2) the length of the identical region between the predicted and FL-cDNA sequences is ≥95% of the length of the entire sequence at both the DNA and deduced amino acid sequence levels. In contrast, a cDNA is considered as a partial match if the identity is ≥95%, but the length of the identical region is between 50% and 94% (Supplemental Table IV). Among the 81 predicted OsWRKY genes, 36 had significant matches. This analysis also revealed that 17 predicted sequences had annotation errors, including incorrect intron-exon splice boundaries, incorrect number of exons, and wrong start/stop codons (Supplemental Table III). After manual corrections, 48 OsWRKY genes had significant matches, two had partial matches, and 31 did not have any matches. The accession numbers of the matched FL-cDNA sequences are listed in Supplemental Table II.

Recently, it has been reported that about 27% of 18,933 transcription units in rice genomes contain 2 or more transcripts (Kikuchi et al., 2003) due to alternative splicing, which results in alternative open reading frames (ORFs) that differ in initiation and termination sites, as well as splice donor and acceptor sites. To date, three Arabidopsis WRKY genes (AtWRKY1, -4, and -43) have been identified as having two ORFs. Because 66 out of 81 OsWRKY genes in our collection have more than 2 exons, we tested whether they contain alternative ORFs. In this study, we found that five OsWRKY loci (OsWRKY1, -8, -35, -39, and -57) contain alternative structures (Fig. 1). In two of these OsWRKY genes, OsWRKY1 and OsWRKY57, one of the alternative peptides does not contain a WRKY domain. In another one (OsWRKY35), the alternative splice variant retains part (54 out of 88 nucleotides) of the fourth intron located in the C-terminal WRKY domain, resulting in the addition of 18 amino acids to the deduced peptide. The predicted gene structure of OsWRKY39 is supported by a FL-cDNA (accession AK066775). In addition, an alternative structure (variant 1) is revealed by another FL-cDNA (AK119593). This splice variant retains the first intron of the predicted structure, which introduced a stop codon right after the 127th codon, thereby eliminating the WRKY domain that lies downstream of codon 127. OsWRKY34 has a significant FL-cDNA match (AK072906), but it retains the third predicted intron. No FL-cDNA that completely matches the predicted structure has been found yet. These results suggest that alternative splicing might be involved in modulating the function of WRKY proteins.

Figure 1.
Schematic diagrams of alternative spliced gene structures. Accession numbers of the significantly matched FL-cDNA sequences are shown in parentheses. Rectangles with numbers represent exons; thick lines represent introns; hatched regions represent WRKY ...

OsWRKY Proteins Are Classified into Four Subgroups Based on Their WRKY Domain Sequences

The most prominent structural feature of these proteins is the WRKY domain of 60 amino acid residues. Among 93 WRKY domains found in WRKY proteins, 87 domains contain the highly conserved WRKY motif, including 77 with WRKYGQK, 6 with WRKYGKK, and 4 with WRKYGEK. However, 6 domains in OsWRKY41, -60, -61, -63, and -81 have either 1 or 2 mismatched amino acids within the WRKY motif (referred to as an atypical WRKY motif). Thus, we suggest that W(R/K)(K/R)Y be considered as a new consensus WRKY motif. Similar to the Arabidopsis WRKY proteins (Eulgem et al., 2000), OsWRKY proteins were classified into 4 groups (Fig. 2): 11 members with 2 WRKY domains belong to group I; the WRKY domains of 46 group II members have a C2H2-type (C-X4–5-C-X22–24-H-X1–2-H) zinc finger motif; 19 WRKY proteins belong to group III with a C2HC-type (C-X6–7-C-X23–33-H-X1-C) zinc finger motif; and 5 group IV WRKY proteins contain no zinc finger motif. The zinc finger motif patterns in group II and III OsWRKY proteins are slightly different from those in group II (C-X4–5-C-X22–23-H-X1-H) and group III (C-X7-C-X23-H-X1-C) of Arabidopsis WRKY proteins, respectively (Eulgem et al., 2000). In addition, based on their zinc finger motifs, members in groups I and IV can be further divided into four subgroups (Ia, Ib, IVa, and IVb). Group Ia proteins contain a C2H2-type zinc finger motif with a common WRKY motif; group Ib proteins contain a C2HC-type zinc finger motif, usually with an atypical WRKY motif; group IVa proteins have only a partial zinc finger motif (CX4C); and group IVb proteins do not contain the conserved Cys or His residues at all. With the exception of OsWRKY56, which is supported by a FL-cDNA sequence, group IV genes might contain annotation errors caused by genomic sequencing or gene prediction software, or they might be pseudogenes with no biological functions.

Figure 2.Figure 2.
Multiple sequence alignment of WRKY domains. Eighty-one OsWRKY proteins were divided into four groups based on the features of their WRKY domains. Groups I and IV were further divided into subgroups (Ia, Ib, IVa, and IVb). The names in bold highlight ...

Phylogenetic Analysis of the OsWRKY Gene Family

To study the phylogenic relationship among the OsWRKY genes and generate an evolutionary framework of this gene family, a multiple peptide sequence alignment of all 93 WRKY domains, including both C-terminal and N-terminal domains of group I genes, was constructed using ClustalW (Thompson et al., 1994). For the WRKY genes that have alternatively spliced ORFs, only variant 2 ORFs of these genes (OsWRKY1v2, -8v2, -35v2, and -57v2) were included in the phylogenetic analysis, because the sequences and structures of variant 2 ORFs were more conserved than those of variant 1 ORFs. The sequence alignment output was used to reconstruct the unrooted majority rule consensus phylogenetic tree within the Bayesian framework (Fig. 3). Although the basal nodes of the tree are not well resolved, there are several patterns visible in the branches with 95% to 100% Bayesian posterior probabilities, as described in the next paragraph.

Figure 3.
Phylogenetic analyses of OsWRKY proteins. The majority rule consensus phylogenetic unrooted tree was reconstructed based on the WRKY domain peptide sequences by the MrBayes 3.0 program (Huelsenbeck et al., 2000). Statistical support values are shown above ...

Recently, it was reported that group I WRKY genes may represent the ancestral form of WRKY genes (Ulker and Somssich, 2004). The loss of one WRKY domain in the origin of several group II genes (Fig. 3, names in black) that evolved from group Ia genes (Fig. 3, names in red) is evident in the separate monophyletic clusters of all C- and N-terminal WRKY domains. The C-terminal WRKY domain branch includes genes that have lost the N-terminal domain, such as two group IVa genes (OsWRKY33 and -38) and a group II gene (OsWRKY80). In contrast, in the C-terminal WRKY domain cluster, a group II gene (OsWRKY57v2) appears to be evolved from group Ia genes by loss of the C-terminal domain. Of the remaining 44 group II genes, 40 cluster in 5 well-supported branches that share an unresolved basal node with the group Ia genes, suggesting that either group II genes evolved from the ancestral form of group Ia genes or two-domain group Ia genes evolved from gene duplication of group II genes. Both WRKY domains of the group Ib genes (Fig. 3, names in purple) are imbedded within a cluster of group III genes (Fig. 3, names in green). Although these relationships are not as well supported as the patterns described for group Ia, the N-terminal WRKY domains of group Ib genes form a monophyletic cluster and the C-terminal WRKY domains of group Ib genes connect basally to form a monophyletic cluster of all Ib genes and 4 group III genes with 82% Bayesian posterior probability, suggesting a WRKY domain duplication event of a group III gene gave rise to group Ib genes. The three group IVa WRKY genes (OsWRKY52, -56, and -58; Fig. 3, names in blue) are scattered throughout the tree and branch, with low resolution, from group II and III genes.

Intron Phases and Positions Support the Phylogeny of OsWRKY Genes

The position and phase of the intron in each region encoding the WRKY domain provides additional support to the phylogeny in Figure 3. An intron position refers to a codon into which an intron is inserted, whereas an intron phase refers to where in a codon an intron is inserted: immediately upstream (phase 0), after the first nucleotide (phase 1), or after the second nucleotide (phase 2) of a codon. About 71% (66 out of 93) of WRKY domains found in OsWRKY genes contain an intron in a conserved position, including both WRKY domains of group Ib genes (Fig. 2, red boxes and Fig. 3, right). This phase 2 intron is localized at the 11th codon downstream of the WRKY motif, interrupting the codon encoding Arg (91% of 66 genes), Ser (5%), Lys (3%), or Asn (1%; Fig. 2). The presence of the intron in both WRKY domains of group Ib genes and in all other gene groups suggests that its origin precedes much of the gene duplication in this family. Of the 20 WRKY domains that do not have this conserved intron, 8 are in the monophyletic cluster of N-terminal WRKY domains of group Ia genes, 3 domains are in a monophyletic cluster of group II genes (OsWRKY1v2 to OsWRKY 62; Fig. 3), and 5 domains share a branch tip among group II genes (OsWRKY2, -10, -25, -31, and -44; Fig. 3). The remaining domains that lack the conserved intron belong to group III genes (OsWRKY21, -48, -54, and -69; Fig. 3). If this intron is ancestral to most gene duplication events in the WRKY gene family, only seven independent losses are required to account for the intron distribution. The absence of this phase 2 intron from the N-terminal WRKY domains of all group Ia proteins is another feature that distinguishes group Ia proteins from group Ib proteins (Fig. 2). Another type of intron within the WRKY domain occurs only in a group II gene cluster. This phase 0 intron is localized between the 28th and 29th codons downstream of the WRKY motif, and is found only in eight group II WRKY genes, including OsWRKY1v2, -5, -9, -32, -43, -62, -71, and -73. The 28th and 29th codons were conserved, encoding Gln or Lys and Val or Leu, respectively (Fig. 2).

Chromosomal Localization of OsWRKY Genes

Forty-five WRKY genes revealed in the BAC sequences of the japonica genome were localized on chromosomes based on the BAC information obtained from National Center for Biotechnology Information (NCBI). To investigate the chromosomal localizations of OsWRKY genes identified in the indica genome, in which draft sequence was produced by whole-genome shotgun sequencing (Yu et al., 2002), WRKY genes containing BAC sequences were used for BLAST analyses against the japonica genome sequence. Twenty OsWRKY genes found in the indica genome were also identified in the japonica genome and hence localized on chromosomes. The detailed chromosomal information of OsWRKY genes is listed in Supplemental Table V. OsWRKY genes in this collection were distributed across all 12 chromosomes (Fig. 4A). Interestingly, 21 of the 81 OsWRKY genes were found on chromosome 1, including 2 tandem repeats of OsWRKY15. In other words, approximately 26% of the identified OsWRKY genes are on chromosome 1, yet the length of chromosome 1 only accounts for 11% of the rice genome. These results suggest that there might be WRKY gene hot spots in the rice genome, with the highest density on chromosome 1. Based on the sequence of chromosome 1, the physical positions of 21 OsWRKY genes were determined by BLAST analyses and are shown in Figure 4B. Eighteen of the 21 OsWRKY genes were clustered into 6 regions (A–F) on chromosome 1. Most of the OsWRKY genes within the same region are highly conserved. Furthermore, three possible tandem gene duplication events were revealed in regions D, E, and F because those genes were clustered on the deepest branches of the tree, suggesting a close phylogenetic relationship (Fig. 3). It will be interesting to analyze whether these regions with a higher density of OsWRKY genes are in genome tandem arrays or intrachromosomally and interchromosomally duplicated areas.

Figure 4.
Chromosomal distribution of OsWRKY Genes. A, The distribution of OsWRKY genes in the rice genome. The chromosomal information for OsWRKY genes identified in the japonica genome was determined based on BAC information obtained from the NCBI (http://www.ncbi.nlm.nih.gov ...

Motif Analyses of OsWRKY Proteins

Identification of the conserved protein motifs might help elucidate protein functions. It has been suggested that several Arabidopsis group IIb WRKY proteins contain a conserved Leu zipper motif (Eulgem et al., 2000), which is characterized by a region containing several repeats of seven hydrophobic residues with a strong preference for Leu at every seventh position, although other small hydrophobic amino acids are also acceptable (Pu and Struhl, 1991; Acharya et al., 2002). Among 81 OsWRKY proteins, 4 group II proteins (OsWRKY1, -9, -43, and -71) contain Leu zipper motifs (Supplemental Fig. 1). Experimental evidence is necessary to determine whether these proteins can indeed dimerize. Another notable motif, the HARF motif, is found in group IId AtWRKY proteins, although its function is still unknown (Eulgem et al., 2000). This HARF motif was only found in a few group II OsWRKY proteins (OsWRKY25, -44, -51, and -68; Fig. 3). OsWRKY27 and -80 contain a hydroxylation motif, which is present in proteins such as HIF-1α (hypoxia-inducible factor 1α) and is involved in sensing oxygen levels in cells (Huang et al., 2002). Recently, sumoylation is shown to be involved in both positive and negative regulations of transcription by modulating the ability of transcription factors to interact with their partners, alter their patterns of subcellular localization, and control their stability, leading to positive or negative regulation of transcription (for review see Seeler and Dejean, 2003; Verger et al., 2003). The consensus sequence of the sumoylation motif is ΨKXD/E, where Ψ is often an aliphatic residue but sometimes can be basic or aromatic amino acids such as Lys and Phe, respectively (Zhou et al., 2004). We found that 35 OsWRKY peptide sequences contained one or more sumoylation motif(s). Furthermore, four WRKY proteins (OsWRKY1, -8, -35, and -57), which have alternative ORFs, contain one or more sumoylation motifs. It will be interesting to determine whether these proteins can be sumoylated and whether the function of these proteins can be altered through this modification.

WRKY proteins can function as activators and repressors of many different biological processes (de Pater et al., 1996; Eulgem et al., 1999; Asai et al., 2002; Chen and Chen, 2002). Some can function as activators in one pathway but as repressors in another (Robatzek and Somssich, 2002). To predict putative activating and repressing activities of OsWRKY proteins, the deduced peptide sequences were searched for the consensus coactivator motif, LXXLL, where L is Leu and X is any amino acid (Savkur and Burris, 2004) and the active repressor motif, LXLXLX (Tiwari et al., 2004). Sixteen OsWRKY proteins (OsWRKY2, -11, -17, -20, -21, -36, -40, -41, -45, -48, -50, -54, -57, -65, -73, and -75) contain the coactivator motif, and nine proteins (OsWRKY27, -28, -29, -32, -40, -51, -64, -71, and 73) contain the active repressor motifs. Moreover, OsWRKY40 and -73 contain both coactivator and active repressor motifs. The motif structures of OsWRKY24, -45, -51, -71, -72, and -77, whose functions were experimentally analyzed in aleurone cells as detailed below, are shown in Figure 5.

Figure 5.
Schematic diagrams of conserved motifs found in the OsWRKY peptide sequences. Conserved motifs of OsWRKY24, -45, -51, -71, -72, and -77 were listed above. Other OsWRKY proteins were shown in Supplemental Figure 1. WRKY, WRKY motif; zinc finger, zinc finger ...

Expression Patterns of OsWRKY Genes in Rice Aleurone Cells

To investigate WRKY genes expressed in rice aleurone cells, northern-blot analyses were carried out using total RNA isolated from aleurone cells derived from rice embryoless half-seeds treated with or without the hormones, GA3, ABA, or both. Steady-state mRNA levels for several OsWRKY genes were determined and compared to those of RAmy1A (a GA-inducible α-amylase gene; Karrer et al., 1991), RAB21 (an ABA-inducible gene; Mundy and Chua, 1988), and OsActin1 (a constitutively expressed rice gene; McElroy et al., 1990). As shown in Figure 6, the expression of RAmy1A and RAB21 was strongly induced after GA3 and ABA treatments at 24 and 48 h, respectively. The steady-state mRNA levels of OsActin1 were similar in all samples, suggesting that an equal amount of RNA was used on the blots. The steady-state mRNA signals of 5 WRKY genes, OsWRKY24, -45, -51, -71, and -72, were detectable in rice aleurone cells. OsWRKY24, -51, and -71 transcripts were relatively abundant compared to those of OsWRKY45 and -72. The low level of OsWRKY45 expression was not affected by either ABA or GA3, but OsWRKY72 demonstrates a clear induction upon treatment with ABA (Fig. 6). The abundant transcripts of OsWRKY24, -51, and -71 were enhanced upon ABA treatment. Furthermore, the mRNA levels of OsWRKY51 and -71 were down-regulated in response to exogenous GA3 treatments at 24- and 48-h time points. OsWRKY24 was slightly down-regulated by GA at 4- and 24-h time points.

Figure 6.
Expression patterns of OsWRKY genes in rice aleurone cells. After being imbibed for 2 d, about one-tenth of rice embryoless half-seeds were harvested as a control (0 h), and the rest were further treated without hormone (control) or with 1 μm ...

We also examined OsWRKY77 by northern-blot analysis but could not detect a signal. The signal of OsWRKY77 transcription, however, can be detected by reverse transcription-PCR (Zhang et al., 2004).

Functional Analyses of OsWRKY Genes in Aleurone Cells

Because at least four out of six OsWRKY genes (OsWRKY24, -45, -51, -71, -72, and -77) expressed in aleurone cells are induced by ABA, we investigated whether they are involved in ABA signaling by transient expression of these OsWRKY genes in aleurone cells. The genomic sequences of these six OsWRKY genes were cloned into an expression vector driven by the maize (Zea mays) ubiquitin promoter (Fig. 7A). We have previously demonstrated that the genomic coding sequences (including all exons and introns between the start and stop codons) of several effector genes, including PKABA1 and GAMYB, function as well as their corresponding cDNA clones in the transient expression system (Zhang et al., 2004). The ABA-inducible reporter construct, HVA22-GUS (Shen and Ho, 1995), was used to study the function of OsWRKY proteins in ABA signaling. The OsWRKY effector constructs, along with the HVA22-GUS reporter and UBI-LUC internal control constructs, were introduced into barley (Hordeum vulgare) aleurone cells by particle bombardment. The HVA22-GUS reporter is induced 31-fold in the presence of ABA (Fig. 7B). Transient expression of UBI-OsWRKY71 had little effect on ABA induction of HVA22-GUS (Zhang et al., 2004). However, OsWRKY24 and -45 repressed ABA induction of HVA22-GUS. OsWRKY24 almost completely blocked ABA induction of HVA22-GUS, while transient expression of UBI-OsWRKY45 partially inhibited ABA-dependent induction of HVA22-GUS. In contrast, coexpression of UBI-OsWRKY72 and UBI-OsWRKY77 induced the expression of HVA22 to 4- and 11-fold, respectively, in the absence of ABA. With the ABA treatment, the induction levels were increased to 58- and 53-fold, respectively. These data suggest that OsWRKY72 and -77 synergistically interact with ABA in activating the HVA22 promoter in aleurone cells. Finally, transient expression of UBI-OsWRKY51 had little effect on ABA induction of HVA22-GUS.

Figure 7.
Functional analyses of OsWRKY proteins in ABA signaling. A, Schematic diagrams of the reporter and effector constructs used in the cobombardment experiments. The gene structures were drawn to scale. Shaded rectangles represent exons; lines between exons ...


This article reports the identification of 81 OsWRKY genes (Supplemental Table II) by reiterative bioinformatic analyses with the GENSCAN program followed by (1) removing redundant OsWRKY genes identified in the japonica and indica genome sequences (Supplemental Table I); (2) verification with FGENESH and manual inspections (Supplemental Table III); and (3) searching for supporting FL-cDNA sequences (Supplemental Tables II and IV). Most of the predicted OsWRKY genes are supported by FL-cDNA sequences, as demonstrated by the vigorous analyses that also led to the identification of OsWRKY mRNA variants resulting from alternative splicing (Fig. 1). Sequence alignment studies allowed the classification of OsWRKY proteins into four groups, which differ in the number of WRKY domains in the proteins and in the sequences of the WRKY and zinc finger motifs (Fig. 2). Phylogenetic analyses revealed both loss and gain of the WRKY domain in the evolutionary history of this gene family in rice (Fig. 3). Although OsWRKY genes have been found on each of the 12 rice chromosomes, they are not evenly distributed. Instead, chromosome 1 contains more than 25% of OsWRKY genes identified so far (Fig. 4). We demonstrated that OsWRKY51 and -71 were enhanced by ABA and suppressed by GA (Fig. 6). Finally, our studies identified two OsWRKY genes as negative regulators and two other OsWRKY genes as positive regulators of ABA signaling (Fig. 7).

The report demonstrates that the manual reannotation of genes is an essential component of genome analysis because of the limitations inherited in automated annotations and the complex nature of gene structures. This has also been demonstrated in the analysis of Arabidopsis R genes (Meyers et al., 2003). Our study shows that about 36% of predicted OsWRKY genes that have cDNA support contain errors of one type or another, assuming that all alternative forms of the cDNAs have been identified (Supplemental Table III). This assumption, of course, is likely to be an underestimation. The most frequent error we found was the wrong annotation of start and stop codons, which accounted for approximately 65% of the misannotations. The next frequent categories were the incorrect number of exons (approximately 18%) and incorrect placement of intron-exon splice boundaries (approximately 18%). FL-cDNA sequences are essential for the reannotation process; 64% of the predicted OsWRKY genes reported herein are supported by FL-cDNA sequences.

The OsWRKY genes encode proteins containing one or two WRKY domains. These two classes of WRKY genes may have arisen by gene duplication of the one-domain proteins, deletion of one WRKY domain in a two-domain protein, or a combination of both, i.e. deletion followed by duplication. Recently, WRKY expressed sequence tags have been identified from lower plants such as ferns (Ceratopteris richardii), mosses (Physcomitrella patens), and green algae (Chlamydomonas reinhardtii), and from the slime mold (Dictyostelium discoideum) and the unicellular protist (Giardia lamblia; Ulker and Somssich, 2004). Group I-like WRKY genes are present in the lower organisms, implying that the group I-like WRKY genes may represent the ancestral form of the WRKY family that originated approximately 1.5 to 2 billion years ago in eukaryotes, before the divergence of the plant phyla (Ulker and Somssich, 2004). However, rice group I WRKY genes can be further classified into two subgroups (group Ia and Ib). Two obvious features can distinguish group Ia from group Ib WRKY genes in rice. First, the zinc finger motif in both the N- and the C-terminal WRKY domain in group Ia genes is C2H2 type, while the zinc finger motif in both the N- and the C-terminal domain in group Ib genes is C2HC type. Second, the C- but not the N-terminal domain of group Ia genes contains the conserved intron in the region encoding the WRKY domain. In contrast, both the N- and the C-terminal WRKY domains of the group Ib genes contain the conserved intron (Fig. 2). By comparing their domain sequences, Arabidopsis group I and the slime mold WRKY genes are more similar to rice group Ia genes than to rice group Ib genes (data not shown). Hence, only group Ia genes, but not group Ib genes, appear to be directly derived from ancestral forms of the OsWRKY genes in plants. By referring to the domain feature (Fig. 2), the phylogenic tree (Fig. 3, left), and the intron pattern (Fig. 3, right), we deduced three major steps of OsWRKY gene evolution that involve both loss and gain of the WRKY domain (Fig. 8). Among OsWRKY genes, we find evidence of loss of the N terminus in the evolution of single-domain group II and IV genes from group Ia genes and independent loss of the C terminus in the evolution of a single-domain group II gene. Other group II genes may have evolved by this process. The domain loss scenario is also supported by studies of WRKY genes in Arabidopsis and tomato. AtWRKY10 has only one WRKY domain, but it is clustered with the two-domain group I AtWRKY genes (Eulgem et al., 2000). However, its ortholog from tomato has two WRKY domains, suggesting that a recent likely loss of the N-terminal WRKY domain occurred during evolution (Rossberg et al., 2001). The domain duplication scenario is deduced based on the observation that the two zinc fingers in each group I gene are either C2H2-C2H2 or C2HC-C2HC type; no hybrid double zinc finger structures (C2HC-C2H2 or C2H2-C2HC) have been identified. Therefore, group Ib OsWRKY genes are likely to have evolved by intramolecular duplication of a group III WRKY domain that had already evolved the C2HC-type zinc finger (Fig. 8). Further phylogenetic analyses based on WRKY domain DNA sequences are needed to confirm the deduced evolutionary history of this gene family.

Figure 8.
Deduced evolutionary framework of the OsWRKY gene family. An evolutionary framework of the OsWRKY gene family was deduced based on the phylogenetic analysis and the features of the WRKY domain. Rectangles designate the N- or C-terminal WRKY domains with ...

SA and H2O2 strongly induce the expression of WRKY genes in several plant species including rice (Kim et al., 2000; Wen et al., 2003). In Arabidopsis, 49 out of 72 tested WRKY genes respond to SA treatment or bacterial infection (Dong et al., 2003). The involvement of WRKY genes in abiotic stress response (Hara et al., 2000; Huang and Duman, 2002; Pnueli et al., 2002; Rizhsky et al., 2002, 2004; Seki et al., 2002) suggests that some of them might be responsive to ABA. Indeed, 4 Arabidopsis WRKY genes, AtWRKY25 (At2g30250), -28 (At4g18170), -40 (At1g80840), and -75 (At5g13080), are ABA inducible in vegetative tissues (Seki et al., 2002). In this study, 4 OsWRKY genes, OsWRKY24, -51, -71, and -72, were shown to be ABA responsive (Fig. 6). Multiple sequence alignment data (Z. Xie and Q.J. Shen, unpublished data) indicate these four OsWRKY genes are unlikely to be the orthologs of those ABA-inducible Arabidopsis WRKY genes. Hence, it is likely that other WRKY genes, which have not yet been analyzed experimentally, are also responsive to ABA. By having different temporal and spatial expression patterns, these WRKY genes might modulate ABA signaling in different tissues or different developmental stages.

Currently, the mechanism by which OsWRKY72 and -77 synergistically interact with ABA to enhance the expression of HVA22-GUS (Fig. 7) remains unknown. Most of the published WRKY proteins bind to the cognate cis-acting element (C/T)TGAC(T/C) (Ishiguro and Nakamura, 1994; Rushton et al., 1995; Eulgem et al., 2000; Zhang et al., 2004). However, other binding sequences have been reported. For example, the barley SUSIBA2 protein binds to 3 other sequences, named SURE-a, SURE-b, and SURE-c, in addition to the W-box of the isoamylase1 promoter (Sun et al., 2003). The putative W-box has been found in the promoter regions of HVA22 (Shen et al., 1993) and ABF (Choi et al., 2000). However, the 49-bp promoter fragment in the reporter construct of this study does not include this W-box. Similarly, this promoter does not contain the SphI element that is bound by the C-terminal B3 domain of VP1 (Suzuki et al., 1997). Instead, only two elements are present in this promoter fragment: the ABA response element (ABRE) that is bound by ABI5 or its related bZIP proteins (Kim and Thomas, 1998; Hobo et al., 1999; Finkelstein and Lynch, 2000; Casaretto and Ho, 2003) and CE1 that is bound by an APETALA2 domain containing transcription factor ABI4 (Finkelstein et al., 1998; Niu et al., 2002). It is suggested (Shen et al., 1996) that the interaction of ABRE and CE-binding proteins might be mediated by VP1, which also interacts with 14-3-3 protein, ring (C3HC3-type) finger proteins, and RNA polymerase II subunit RPB5 (Schultz et al., 1998; Hobo et al., 1999; Jones et al., 2000; Kurup et al., 2000). Hence, OsWRKY72 and -77 might, as a non-DNA-binding component, form a complex with VP1, 14-3-3 protein, ring (C3HC3-type) finger proteins, ABI4, and ABI5 to modulate ABA signaling in aleurone cells.

In contrast, OsWRKY24 and -45 repressed ABA induction of HVA22-GUS (Fig. 7). If the model proposed for the OsWRKY positive regulators described above is correct, one possible repression mechanism would be the competition with the WRKY positive regulators in ABA signaling. In this regard, it is interesting to note that two rice bZIP proteins (OsZIP-2a and OsZIP-2b), which do not bind to the ABRE element by themselves, heterodimerize via the Leu zipper with EmBP-1 (another bZIP protein) and prevent it from binding ABRE. As a result, the EmBP-1 positive regulator is sequestered by these two negative regulators (Nantel and Quatrano, 1996). A similar mechanism has been found with helix-loop-helix (HLH) proteins, for which there are about 150 members in Arabidopsis (Toledo-Ortiz et al., 2003). Eighty-nine of them are predicted (Toledo-Ortiz et al., 2003) to bind to the G-box (CACGTG), which is similar to ABRE. Indeed, one of the Arabidopsis HLH proteins, AtMYC2, has been shown to be a transcriptional activator of ABA signaling (Abe et al., 2003). HLH proteins can function as negative regulators of basic HLH proteins by forming non-DNA-binding heterodimers with otherwise DNA-binding basic HLH proteins (Littewood and Evan, 1998). If this mode of action also holds true for the OsWRKY regulators, a different domain must be involved in dimerization because neither the positive nor the negative regulators contain a Leu zipper domain (Fig. 5; Supplemental Fig. 1) or a HLH domain. Of course, there are other possible mechanisms of repression for these repressors, as we suggested for OsWRKY71 in regulating GA signaling (Zhang et al., 2004).

In summary, the annotation of the OsWRKY genes has facilitated the identification of their gene structures and phylogenic relationships. In addition to SA (Chen and Chen, 2000; Dellagi et al., 2000; Du and Chen, 2000; Eulgem et al., 2000; Kim et al., 2000; Asai et al., 2002), jasmonic acid (Li et al., 2004), and GA (Rushton et al., 1992; Zhang et al., 2004), WRKY genes also modulate the responses of plant cells to ABA (this work). Recently, studies of parsley WRKY proteins using the chromatin immunoprecipitation technique indicate that the PcPR-1 promoter is constitutively repressed by some WRKY proteins. Elicitor treatments induce the expression of other WRKY family members and the displacement of the WRKY repressors by WRKY activators (Turck et al., 2004). The WRKY repressors and activators presented in the work will help test the model and contribute to the dissection of the transcriptional complex involved in ABA signaling.


Analyses of the Rice Genomic Sequences

About 287 Mb of rice (Oryza sativa) L. subsp. japonica BAC or P1 artificial chromosome sequences were downloaded from the International Rice Genome Sequencing Project (IRGSP; http://rgp.dna.affrc.go.jp/IRGSP/), which covers about 70% of the rice genome. About 361 Mb of rice indica contig sequences were downloaded from Beijing Genomics Institute (BGI; http://btn.genomics.org.cn/rice), which covers about 90% of the rice genome. A stand-alone version of GENSCAN (Burge and Karlin, 1997) was used for genome annotations. We used a model file for maize (Zea mays), which is also a monocot species that is close to rice on the taxonomy lineage because a model file trained on rice genes is not publicly available for GENSCAN. Rice peptide and coding region databases were constructed based on GENSCAN output files. Rice FL-cDNA sequences were obtained from the NCBI (http://www.ncbi.nlm.nih.gov). The sequence of rice chromosome 1 was downloaded from the Rice Genome Research Group (RGP; http://rgp.dna.affrc.go.jp/) to determine the locations of OsWRKY genes on chromosome 1.

HMMER software package version 2.1.1 (Sonnhammer et al., 1998) was downloaded from http://hmmer.wustl.edu/ and applied to identify WRKY genes in the rice peptide database. A BLAST software package (Altschul et al., 1997) was applied to compare genomic and cDNA sequences and to determine the chromosomal locations of the OsWRKY genes. To identify redundant sequences of the predicted WRKY genes, ClustalW (Thompson et al., 1994) was used to build a maximum local alignment of the predicted WRKY gene sequences. Then the program fRedun, which was developed in our lab using the PERL language, was applied to calculate the percentage of identity between a pair of sequences. Those with 95% or greater identity are considered redundant sequences.

Phylogenetic Analyses and Display of Gene Structures

The maximum likelihood tree was reconstructed with the WRKY domain peptide sequences using version 3.0 of the MrBayes program (Huelsenbeck et al., 2000) under the JTT model of amino acid substitutions with autocorrelated rates across sites. The Bayesian analysis was run for 200,000 generations, using 4 Markov chains with default heating values. A tree was sampled every 50 generations. The Markov chains converged rapidly, and the first 1,000 sampled trees were discarded as burn in. The majority rule consensus unrooted tree was computed based on the rest of the 3,000 sampled trees in the program PAUP*4.0 (Swofford, 2000). The statistical support values were shown above every node.

Intron phases were divided into three groups based on the position of introns within the reading frame of a gene as described in previous studies (Sharp, 1981): phase 0 introns interrupt between two codons; phase 1 introns lie between the first and second bases in a codon, and phase 2 introns interrupt a codon between the second and third bases. Gene structures were drawn to scale according to the size of exons and introns. First, exon coordinates were computed based on the revised version of OsWRKY genes presented in this study, and then intron phases were calculated based on exon coordinates. HMMsearch was performed by using the revised OsWRKY peptide sequences, and domain coordinates were retrieved from the HMMsearch output file. The exon and domain coordinates and intron phase information were used to graphically display the structure of OsWRKY genes.

Plant Materials and Incubation Conditions

Rice seeds were kindly provided by Dr. Kent McKenzie at the California Rice Experiment Station (cv M202) and Mr. Jack de Wit from DeWit Firm (cv M104; Davis, CA). Barley (Hordeum vulgare) cv Himalaya seeds (1998 harvest) were purchased from Washington State University (Pullman, WA). The preparation and imbibition of the embryoless half-seeds were done as described previously (Shen et al., 1993). After being imbibed for 2 d, about one-tenth of rice embryoless half-seeds were harvested as a control, and the rest of them were further incubated in petri dishes containing 20 mm CaCl2, 20 mm sodium succinate without hormone (control) or with 1 μm GA3, 20 μm ABA, or 1 μm GA3 plus 20 μm ABA at 26°C for 4, 24, and 48 h. After harvest, rice embryoless half-seeds were immediately frozen in liquid nitrogen and stored at −80°C until further analysis.

RNA Gel-Blot Analyses

Total RNA was isolated from rice embryoless half-seeds using the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA). For northern analyses, 10 μg of total RNA were loaded into each lane, electrophoresed, and transferred onto nylon membranes. Gene-specific probes for OsWRKY24, -45, -51, -71, -72, -77, and OsActin1 were labeled in the presence of α-32P-dCTP by using the Prime-a-Gene Labeling System (Promega, Madison, WI). Gene-specific probes for RAmy1A and RAB21 were labeled with α-32P-dCTP by PCR. The sequences of primers used for northern analyses were listed in Supplemental Table VI. The blots were hybridized and washed according to the method of Church and Gilbert (1984). The storage phosphor screens were scanned with a Typhoon 9410 phosphoimager (Amersham Biosciences, Piscataway, NJ).

Construct Preparations

Genomic DNA, isolated from 10-d-old rice seedlings as described previously (Zhang et al., 2004), was used for preparation of effector constructs. The genomic CRSs of the tested OsWRKY genes were amplified by PCR and cloned into the expression vector (Zhang et al., 2004) containing the constitutive maize ubiquitin promoter. The detailed information about primers and restriction enzyme sites used to clone the effector constructs is listed in Supplemental Table VII.

Particle Bombardment and Transient Expression Assays

Three types of DNA constructs were used in the transient expression experiments: reporter, effector, and internal control. Plasmid HVA22-GUS (Shen et al., 1996) was used as the reporter construct to study the function of effectors in the ABA signal transduction pathway. Plasmid UBI-LUC, which contains the luciferase reporter gene driven by the maize ubiquitin promoter (Bruce et al., 1989), was used as an internal control construct to normalize GUS activities of the reporter construct. The procedure of transient expression experiments with the barley aleurone system by particle bombardment has been described previously (Lanahan et al., 1992). All experiments consisted of four replicates and the entire experiment was repeated at least twice with similar results.

Sequence data from this article (OsWRKY78–OsWRKY81) have been deposited with the EMBL/GenBank data libraries under accession numbers BK005212 to BK005215.

Supplementary Material

Supplemental Data:


We thank Mr. Everett Cook and Ms. Tracy Hall for their excellent technical assistance. The manuscript was critically reviewed by Drs. Andrew Andres, Frank van Breukelen, and Deborah Hoshizaki in our department, and by Dr. Imre E. Somssich at Max-Plank-Institute for Plant Breeding Research, Köln, Germany. We appreciate their help. The authors are also grateful to Dr. Tuan-Hua David Ho at Washington University in St. Louis for stimulating discussions and for his generosity in providing the reporter and internal control constructs used in this study. We also thank Dr. Shoshi Kikuchi at the National Institute of Agrobiological Sciences in Japan for sharing the full-length OsWRKY cDNA clones prior to publication.


1This work was supported by the U.S. Department of Agriculture (grant no. 02–35301–12066), by the National Institutes of Health (Biomedical Research Infrastructure Network seed grant no. P20 RR16464), and by the University of Las Vegas, Nevada (start-up funds to Q.J.S.). X.Z. was supported by a National Science Foundation Experimental Program to Stimulate Competitive Research (EPSCoR), Integrative Approaches to Abiotic Stress (EPS–0132556), graduate assistantship and Z.-L.Z. by a National Science Foundation EPSCoR Advanced Computing in Environmental Sciences postdoctoral fellowship.

[w]The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.054312.


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