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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 promoterb-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.

SummaryWRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination. For over 15 years, limited evidence has been available suggesting that WRKY TFs may play roles in regulating plant responses to the phytohormone abscisic acid (ABA), notably some WRKY TFs are ABA-inducible repressors of seed germination. However, the roles of WRKY TFs in other aspects of ABA signalling, and the mechanisms involved, have remained unclear. Recent significant progress in ABA research has now placed specific WRKY TFs firmly in ABA-responsive signalling pathways, where they act at multiple levels. In Arabidopsis, WRKY TFs appear to act downstream of at least two ABA receptors: the cytoplasmic PYR ⁄ PYL ⁄ RCARprotein phosphatase 2C-ABA complex and the chloroplast envelope-located ABAR-ABA complex. In vivo and in vitro promoter-binding studies show that the target genes for WRKY TFs that are involved in ABA signalling include well-known ABA-responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a and RAB18. Additional well-characterized stressinducible genes such as RD29A and COR47 are also found in signalling pathways downstream of WRKY TFs. These new insights also reveal that some WRKY TFs are positive regulators of ABA-mediated stomatal closure and hence drought responses. Conversely, many WRKY TFs are negative regulators of seed germination, and controlling seed germination appears a common function of a subset of WRKY TFs in flowering plants. Taken together, these new data demonstrate that WRKY TFs are key nodes in ABA-responsive signalling networks.

The molecular mechanism by which GA regulates plant growth and development has been a subject of active research. Analyses of the rice (Oryza sativa) genomic sequences identified 77 WRKY genes, among which OsWRKY71 is highly expressed in aleurone cells. Transient expression of OsWRKY71 by particle bombardment specifically represses GA-induced Amy32b  α-amylase promoter but not abscisic acid-induced HVA22 or HVA1 promoter activity in aleurone cells. Moreover, OsWRKY71 blocks the activation of the Amy32b promoter by the GA-inducible transcriptional activator OsGAMYB. Consistent with its role as a transcriptional repressor, OsWRKY71 is localized to nuclei of aleurone cells and binds specifically to functionally defined TGAC-containing W boxes of the Amy32b promoter in vitro. Mutation of the two W boxes prevents the binding of OsWRKY71 to the mutated promoter, and releases the suppression of the OsGAMYB-activated Amy32b expression by OsWRKY71, suggesting that OsWRKY71 blocks GA signaling by functionally interfering with OsGAMYB. Exogenous GA treatment decreases the steady-state mRNA level of OsWRKY71 and destabilizes the GFP:OsWRKY71 fusion protein. These findings suggest that OsWRKY71 encodes a transcriptional repressor of GA signaling in aleurone cells.

WRKY genes encode transcription factors that are involved in the regulation of various biological processes. These zinc-finger proteins, especially those members mediating stress responses, are uniquely expanded in plants. To facilitate the study of the evolutionary history and functions of this supergene family, we performed an exhaustive search for WRKY genes using HMMER and a Hidden Markov Model that was specifically trained for rice. This work resulted in a comprehensive list of WRKY gene models in Oryza sativa L. ssp. indica and L. ssp. japonica. Mapping of these genes to individual chromosomes facilitated elimination of the redundant, leading to the identification of 98 WRKY genes in japonica and 102 in indica rice. These genes were further categorized according to the number and structure of their zinc-finger domains. Based on a phylogenetic tree of the conserved WRKY domains and the graphic display of WRKY loci on corresponding indica and japonica chromosomes, we identified possible WRKY gene duplications within, and losses between the two closely related rice subspecies. Also reviewed are the roles of WRKY genes in disease resistance and responses to salicylic acid and jasmonic acid, seed development and germination mediated by gibberellins, other developmental processes including senescence, and responses to abiotic stresses and abscisic acid in rice and other plants. The signaling pathways mediating WRKY gene expression are also discussed. Available online at www.blackwell-synergy.com/links/toc/jipb, www.jipb.net Plants, because of their inability to escape predation or environmental changes, have evolved strategies to tolerate and adapt to biotic and abiotic stresses. Biotic stresses, such as fungal, bacterial, and viral challenges, and abiotic stresses, such as drought, cold, wounding, and salinity, are sensed through a complex signal transduction network and result in physiological, biochemical, and gene expression changes (Kunkel and Brooks 2002;Singh et al. 2002; Mahalingam et al. 2003; Katagiri 2004). Families of transcription factors, central to this process, have uniquely expanded and evolved in plants to coordinate gene expression. The WRKY class of zinc-finger proteins, similar to the ethylene-responsive-element binding factors (ERF) (Gutterson and Reuber 2004), DNA binding one finger (Dof;Yanagisawa 2002Yanagisawa , 2004 and basic domain leucine zipper (bZIP;Siberil et al. 2001; Jakoby et al. 2002) families, is a family of proteins that contains many members that mediate plant stress responses (previously reviewed in Eulgem et al. 2000;Ulker and Somssich 2004). In the present review, we present the signature and domain of the WRKY genes, their distribution and evolution history, comprehensive analyses of rice WRKY family, and the regulation and functions of WRKY genes in rice and other plants. The WRKY Signature and DomainWRKY domains were initially defined by a nearly 60 amino acid long motif possessing the well-conserved amino acid signature WRKYGQK at their N-terminus and conta...

The modular nature of the abscisic acid response complex (ABRC), the promoter unit necessary and sufficient for abscisic acid (ABA) induction of gene expression in barley, is defined in this study. We investigated ABA induction of a barley late embrogenesis abundant (Lea) gene, HVA1, and found that the ABRC of this gene consists of a 10-bp box with an ACGT core (ACGT-box) and the 11 bp directly upstream, named coupling element 3 (CE3). Only one copy of this ABRC is sufficient to confer ABA induction when linked to a minimal promoter. Because we previously reported another ABRC in the barley HVA22 gene, which consists of an ACGT-box with a distal coupling element (CE1), exchange experiments were conducted to study the interaction among modular elements in these ABRCs. We show that ACGT-boxes in these ABRCs are interchangeable, indicating that an ACGT-box can interact with either a distal or a proximal coupling element to confer ABA response. However, the two coupling elements are not fully exchangeable. Although CE3 can function either proximal or distal to the ACGT-box, CE1 is only functional at the distal position. The presence of both the distal and the proximal coupling elements has a synergistic effect on the absolute level of expression as well as on ABA induction. These ABRCs function in both seed and vegetative tissues. In seeds, ABA induction of the ABRC containing the proximal CE3, but not the ABRC with the distal CE1, is enhanced in the presence of the transcription regulator Viviparous1, indicating that these two ABRCs are mediated by different ABA signal transduction pathways.

BackgroundThe availability of increasing numbers of sequenced genomes has necessitated a re-evaluation of the evolution of the WRKY transcription factor family. Modern day plants descended from a charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae that typically have 1-3 WRKY genes and mosses that contain 30-40. WRKY genes have been previously found in non-plant species but their occurrence has been difficult to explain.ResultsOnly two WRKY genes are present in the Klebsormidium flaccidum genome and the presence of a Group IIb gene was unexpected because it had previously been thought that Group IIb WRKY genes first appeared in mosses. We found WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants. Flowering plants contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Once formed, R protein WRKY genes may combine different components of signalling pathways that may either create new diversity in signalling or accelerate signalling by short circuiting signalling pathways.ConclusionsWe propose that the evolution of WRKY transcription factors includes early lateral gene transfers to non-plant organisms and the occurrence of algal WRKY genes that have no counterparts in flowering plants. We propose two alternative hypotheses of WRKY gene evolution: The “Group I Hypothesis” sees all WRKY genes evolving from Group I C-terminal WRKY domains. The alternative “IIa + b Separate Hypothesis” sees Groups IIa and IIb evolving directly from a single domain algal gene separate from the Group I-derived lineage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0456-y) contains supplementary material, which is available to authorized users.

SummaryGibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and aleurone cells. Over-expression of these two genes in aleurone cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of aleurone cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/ OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in aleurone cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.