Expression Assays of Soybean Promoters of WRKY Transcription Factors

We have cloned and tested 12 soybean WRKY promoters driven GFP expression intensities
via lima bean transient assays and soybean hairy root expression assay.

Introduction on WRKY Transcription Factor

WRKY transcription factors are one of the largest multigene families of transcriptional regulators in plants and are almost green lineage specific, being absent from prokaryotes, animals and fungi. The first reports of members of the family date from the mid 90s when Ishiguro and Nakamura (1994) [1], Rushton et al. (1995) [2] and Rushton et al. (1996) [3] reported WRKY genes from sweet potato, wild oat and parsley. The transcription factors were named WRKYs in 1996 by Rushton et al. after the conserved WRKY amino acid sequence in their DNA binding domains. The DNA binding domain was called the WRKY domain and the binding sites were named W boxes.

WRKY Domain Structure WRKY Domain Introduction W-box Binding Site Description on W-box Binding site
Each WRKY protein has at least one WRKY domain of approximately 60 amino acids containing the conserved amino acid sequence WRKYGQK at its N-terminus (from which the factors take their name) and a novel zinc finger motif at its C-terminus [2-4]. The WRKY domain consists of four beta-strands. The first beta-strand contains the WRKYGQK amino acid sequence and this appears to directly contact the W box binding site on the promoters of target genes. The other three beta-strands form a novel zinc finger structure [5].
For most WRKY transcription factors, the binding site is the W box (C/T)TGAC(T/C), an element found in the promoters of many stress-related plant genes. W boxes are often over-represented and clustered in the promoters of stress inducible genes as shown by many transcriptome studies, starting with Maleck et al. (2000) [6]. Although WRKYs bind W boxes, some specificity is dependent on sequences flanking the W box sequence (Ciolkowski et al., 2008) [7].

The WRKY Family across the Plant Kingdom

The ancestral-type WRKY transcription factor is postulated to be a Group I gene that contains two WRKY domains (N-terminal and C-terminal). All other genes contain one WRKY domain and were originally classified into groups IIa, IIb, IIc, IId, IIe and III based on their primary amino acid sequence and structure of their zinc finger motifs. It is now clear that Group II is not monophyletic and that a better classification is Groups I, IIa + b, IIc, IId + e and III.

The Role of WRKYs in Plant Defence Responses

Taken from Asai et al. (2002) [8]: "Rushton et al. (1996) presented the first evidence that WRKY transcription factors play roles in plant defence when they presented data showing that parsley WRKY1 and WRKY3 are rapidly and transiently induced by fungal elicitor treatment. There is now considerable evidence that WRKYs play key regulatory roles in responses to biotic stresses. WRKYs play key roles in signaling due to PAMPs such as flagellin and some WRKYs also interact with resistance proteins (R proteins). In Arabidopsis, most of the 74 WRKY genes are transcriptionally upregulated by defence-related stimuli. This illustrates that a major role of WRKY genes in flowering plants is to mediate defence responses".

The Roles of WRKYs in Regulating Abiotic Stress Responses

There is now considerable evidence that WRKY transcription factors play roles in responses not only to biotic stresses but also abiotic stresses such as wounding, drought and cold adaptation. Less effort has gone into the roles of WRKYs in these processes but evidence is now accumulating that illustrates a key role of WRKY transcription factors in the responses to abiotic stresses. The extent of similarities and cross talk between the signaling networks involved in the responses to biotic and abiotic stress is not yet clear. WRKYs also play major roles in the plant responses to mechanical wounding and during senescence.

The Role of WRKYs in Regulating Germination:

The WRKY transcription factors ABF1 and ABF2 were implicated in regulating seed germination at the start of research into WRKYs (Rushton et al., 1995) [2]. It took almost a decade before more evidence accumulated and Jeff Shen's group at the University of Nevada at Las Vegas has now produced convincing evidence showing a role for WRKYs in regulating germination in rice. Other more recent work suggests that rice WRKY51 and WRKY71 [10] and Arabidoposis WRKY2 [11] are negative regulators of seed germination.

WRKYs and Development

Unlike some other large families of transcription factors, WRKYs rarely seem to be regulators of developmental processes. One exception is the Transparent Tests Glabra 2 (TTG2) gene that codes for AtWRKY44. TTG2 affects both seed pigmentation and trichome development [12, 13]. Another example is the Arabidopsis gene MINISEED3 (MINI3), that is a regulator of seed size [14].


It has recently been suggested that WRKY DNA-binding domains are related to the widespread GCM1 superfamily as both contain a four-stranded fold [15]. However, this is controvertial as any relatedness is very distant. No WRKY sequence is present in GCM1 proteins and as this amino acid sequence is absolutely conserved in WRKY transcription factors, then it seems that they are unrelated to GCM1 proteins.

Updated by Dr. Jeff Chen on September 14, 2009.