WRKY transcription factors (pronounced ‘worky’) are proteins that bind DNA. They are transcription factors that regulate many processes in plants and algae (Viridiplantae), such as the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination and some developmental processesRushton PJ, Somssich IE, Ringler P, Shen QJ: WRKY transcription factors. Trends in plant science 2010, 15(5):247-258Bakshi and Oelmüller (2014) WRKY transcription factors: Jack of many trades in plants. Plant Signaling & Behavior. 9(1). e27700 but also contribute to secondary metabolism.Schluttenhofer and Yuan (2014) Regulation of Specialized Metabolism by WRKY Transcription Factors. Plant Physiology.
Like many transcription factors, WRKY transcription factors are defined by the presence of a DNA-binding domain; in this case, it is the WRKY domain. The WRKY domain was named in 1996 after the almost invariant WRKY amino acid sequence at the N-terminus and is about 60 residues in length. In addition to containing the ‘WRKY signature’, WRKY domains also possess an atypical zinc-finger structure at the C-terminus (either Cx4-5Cx22-23HxH or Cx7Cx23HxC). Most WRKY transcription factors bind to the W-box promoter element that has a consensus sequence of TTGACC/T.
Individual WRKY proteins do appear in the human protozoan parasite Giardia lamblia and slime mold Dictyostelium discoideum.Wu, Guo, Wang and Li (2005) The WRKY Family of Transcription Factors in Rice and Arabidopsis and Their Origins. DNA Research. 12(1). 9-26
Structural diversity WRKY transcription factors are denoted by a 60-70 amino acid WRKY protein domain composed of a conserved WRKYGQK motif and a zinc-finger region.Eulgem, Rushton, Robatzek and Somssich (2000) The WRKY superfamily of plant transcription factors. Trends in Plant Science. 5(5). 199-206 Based on the amino acid sequence WRKY transcription factors are classified into three major categories, group I, group II, and group III. Group I WRKY proteins are primarily denoted by the presence of two WRKY protein domains, whereas both groups II and III each possess only one domain. Group III WRKY proteins have a C2HC zinc finger instead of the Cys2His2 motif of group I and II factors. The structure of several plant WRKY domains has been elucidated using crystallographyDuan, Nan, Liang, Mao, Lu, et al. (2007) DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein. Nucleic Acids Research. 35(4). 1145-1154 and nuclear magnetic resonance spectroscopy.Yamasaki, Kigawa, Inoue, Tateno, Yamasaki, et al. (2005) Solution Structure of an Arabidopsis WRKY DNA Binding Domain. The Plant Cell. 17(3). 944-956, Yamasaki, Kigawa, Watanabe, Inoue, Yamasaki, et al. (2012) Structural Basis for Sequence-specific DNA Recognition by an Arabidopsis WRKY Transcription Factor. Journal of Biological Chemistry. 287(10). 7683-7691
As soon as the WRKY domain was characterized, it was suggested that it contained a novel zinc finger structure and the first evidence to support this came from studies with 2-phenanthroline that chelates zinc ions. Addition of 2-phenenthroline to gel retardation assays that contained E. coli expressed WRKY proteins resulted in a loss of binding to the W-box target sequence. The other suggestion was that the WRKY signature amino acid sequence at the N-terminus of the WRKY domain directly binds to the W-box sequence in the DNA of target promoters. These suggestions were shown to be correct by publication of the solution structure of the C-terminal WRKY domain of the Arabidopsis WRKY4 protein. The WRKY domain was found to form a four-stranded β-sheet.Yamasaki K, Kigawa T, Watanabe S, Inoue M, Yamasaki T, Seki M, Shinozaki K and Yokoyama S (2012) Structural basis for sequence-specific DNA recognition by an Arabidopsis WRKY transcription factor. J Biol Chem 287: 7683–7691 Soon afterwards, a crystal structure of the C-terminal WRKY domain of the Arabidopsis WRKY1 protein was reported. This showed a similar result to the solution structure except that it may contain an additional β-strand at the N-terminus of the domain.Duan MR, Nan J, Liang YH, Mao P, Lu L, Li L, Wei C, Lai L, Li Y and Su XD (2007) DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein. Nucleic Acids Res. 35:1145-1154 From these two studies it appears that the conserved WRKYGQK signature amino acid sequence enters the major groove of the DNA to bind to the W-Box. Recently, the first structural determination of the WRKY domain complexed with a W-Box was reported. The NMR solution structure of the WRKY DNA-binding domain of Arabidopsis WRKY4 in complex with W Box DNA revealed that part of a four-stranded β-sheet enters the major groove of DNA in an atypical mode that the authors named the β-wedge, where this sheet is almost perpendicular to the DNA helical axis. As initially predicted, amino acids in the conserved WRKYGQK signature motif contact the W Box DNA bases mainly through extensive apolar contacts with thymine methyl groups. These structural data explain the conservation of both the WRKY signature sequence at the N-terminus of the WRKY domain and the conserved cysteine and histidine residues. It also provides the molecular basis for the previously noted remarkable conservation of both the WRKY amino acid signature sequence and the W Box DNA sequence.Eulgem T, Rushton PJ, Robatzek S and Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199-206
History In 1994 and 1995, the first two reports of WRKY transcription factors appeared. They described newly discovered but as yet ill-defined DNA binding proteins that played potential roles in the regulation of gene expression by sucrose (SPF1) Ishiguro, S. and Nakamura, K. (1994) Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 50 upstream regions of genes coding for sporamin and beta-amylase from sweet potato. Mol. Gen. Genet. 244, 563–571 or during germination (ABF1 and ABF2).Rushton, P.J. et al. (1995) Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of alpha- Amy2 genes. Plant Mol. Biol. 29, 691–702 A third report appeared in 1996 that identified WRKY1, WRKY2 and WRKY3 from parsley. The authors named the new transcription factor family the WRKY family (pronounced ‘worky’) after a conserved amino acid sequence at the N-terminus of the DNA-binding domain.Rushton, P.J., Torres, J.T., Parniske, M., Wernert, P., Hahlbrock, K. and Somssich, I.E. (1996) Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 15, 5690–5700 The parsley WRKY proteins also provided the first evidence that WRKY transcription factors play roles in regulating plant responses to pathogens. Numerous papers have now shown this to be a major function of WRKY transcription factors. Since these initial publications, it has become clear that the WRKY family is among the ten largest families of transcription factors in higher plants and that these transcription factors play key roles in regulating a number of plant processes including the responses to biotic and abiotic stresses, germination, senescence, and some developmental processes.Eulgem, T. and Somssich, I.E. (2007) Networks of WRKY transcription factors in defense signaling. Curr. Opin. Plant Biol. 10, 366–371, Ulker, B. and Somssich, I.E. (2004) WRKY transcription factors: from DNA binding towards biological function. Curr. Opin. Plant Biol. 7, 491–498
Evolution WRKY transcription factor genes are found throughout the plant lineage and also outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa.Charles I. Rinerson, Roel C. Rabara, Prateek Tripathi Qingxi J Shen, and Paul J. Rushton (2015) Structure and evolution of WRKY transcription factors. In Plant Transcription Factors: Evolutionary, Structural and Functional Aspects Edited by: Daniel H. Gonzalez. Elsevier 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, where there are seven well-defined groups, Groups I + IIc, Groups IIa + IIb, Groups IId + IIe, and Group III. Flowering plants also 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.
Several early reports proposed that a group I WRKY transcription factor was the progenitor of the family.Wu, Guo, Wang and Li (2005) The WRKY Family of Transcription Factors in Rice and Arabidopsis and Their Origins. DNA Research. 12(1). 9-26, Zhang and Wang (2005) The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evolutionary Biology. 5(1). 1 It was thought that a single group I WRKY domain occurred first and then duplicated to form the original ancestral WRKY transcription factor. However, more recent evidence suggests that WRKY transcription factors evolved from a single group IIc-like gene, which then diversified into group I, group IIc, and group IIa+b domains.Brand, Fischer, Harter, Kohlbacher and Wanke (2013) Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays. Nucleic Acids Research. 41(21). 9764-9778 The original WRKY protein domain has been proposed to have arisen from the GCM1 and FLYWCH zinc finger factors.Babu, Iyer, Balaji and Aravind (2006) The natural history of the WRKY–GCM1 zinc fingers and the relationship between transcription factors and transposons. Nucleic Acids Research. 34(22). 6505-6520 GCM1 and FLYWCH are proposed ancestral proteins base on their crystal structural similarity to the WRKY domain.Babu, Iyer, Balaji and Aravind (2006) The natural history of the WRKY–GCM1 zinc fingers and the relationship between transcription factors and transposons. Nucleic Acids Research. 34(22). 6505-6520 Both GCM1 and FLYWCH belong to families of DNA-binding factors found in metazoan. The plant specific NAC transcription factor family also shares a common structural shape and origin with WRKY transcription factors.Welner, Lindemose, Grossmann, Møllegaard, Olsen, et al. (2012) DNA binding by the plant-specific NAC transcription factors in crystal and solution: a firm link to WRKY and GCM transcription factors. Biochemical Journal. 444(3). 395-404