199 trends in plant science Reviews May 2000, Vol. 5, No. 5 1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(00)01600-9 O ne of the apparent fundamental principles of biological evolution is that the progression from ancient to advanced life forms is inseparably connected to an increase in regu- latory capacity. Genome-sequencing efforts have provided evi- dence for a positive correlation between the proportion of genes involved in information processing and the complexity of organ- isms. More than 20% of the genes within the sequence available for the Arabidopsis thaliana genome appear to encode proteins that play a role in signal transduction or transcription 1 , whereas only 12% of the genome of the single-celled yeast Saccharomyces cerevisiae contains genes of this type 2 . This increase in biological complexity coincides with the appearance or expansion of specific groups of regulator genes. One example is the nuclear-receptor-gene family, which is com- pletely absent in yeast but highly represented in metazoan organ- isms 3 . The evolution of nuclear receptors is believed to be a key event in the development of intercellular communication, a pre- requisite for the multicellularity of metazoans 4 . Similarly, the establishment of a complex animal body plan was driven by the amplification and divergence of ancestral homeobox genes, thereby generating a sophisticated regulatory system of function- ally interconnected transcriptional regulators 5 . To meet their disparate biological requirements, plants and ani- mals have evolved unique regulatory mechanisms. This was partly achieved by combining functional domains from pre-existing fac- tors to build new regulators, as exemplified by the MADS-box fac- tors, which play a central role in determining floral and organ identity in plants 6 . In addition, completely new factors have arisen and we focus here on the potential biological roles of WRKY (pro- nounced ‘worky’) proteins, a large family of transcriptional regu- lators that has to date only been found in plants. The abundance of information provided by the Arabidopsis sequencing projects is an ideal basis for comparative analysis of this superfamily within one plant species. Although their precise regulatory functions are largely unknown, the fact that these factors appear to be specific to plants, with probably up to 100 members in Arabidopsis, suggests that they play an important role during plant evolution. Biochemical properties of WRKY proteins The first WRKY cDNAs were cloned from sweet potato (Ipomoea batatas; SPF1), wild oat (Avena fatua; ABF1,2), parsley (Petro- selinum crispum; PcWRKY1,2,3) and Arabidopsis (ZAP1), based on the ability of the encoded proteins to bind specifically to the DNA sequence motif (T)(T)TGAC(C/T), which is known as the W box 7–10 . It has been suggested that the cognate binding site for SPF1 is different from other WRKY proteins. However, the oligonucleotide used to isolate SPF1 does have a W box in the flanking sequence 7 . The name of the WRKY family is derived from the most promi- nent feature of these proteins, the WRKY domain, a 60 amino acid region that is highly conserved amongst family members. The emerging picture is that these proteins are regulatory transcription factors with a binding preference for the W box, but with the potential to differentially regulate the expression of a variety of target genes. Consistent with a role as transcription factors, PcWRKY1 and WIZZ (from tobacco) have been shown to be tar- geted to the nucleus 11,12 . The WRKY domain and the W box The WRKY domain is defined by the conserved amino acid sequence WRKYGQK at its N-terminal end, together with a novel zinc-finger-like motif 8 (Fig. 1). Because of the clear binding pref- erence of all characterized WRKY proteins for the same DNA motif, it has been assumed that the WRKY domain, as their only conserved structural feature, constitutes a DNA-binding domain. Indeed, it has recently been shown that an isolated WRKY domain has sequence-specific DNA-binding activity 12 . The divalent metal chelators 1,10-o-phenanthroline and EDTA abolish in vitro DNA binding, which is taken as strong support for a zinc-finger struc- ture within the WRKY domain 8,10,11 . However, it has not yet been proven that zinc is actually complexed in the WRKY domain. In addition, nothing is known about the function of the WRKYGQK heptapeptide stretch, the hallmark of this superfamily. All known WRKY proteins contain either one or two WRKY domains. They can be classified on the basis of both the number of WRKY domains and the features of their zinc-finger-like motif. WRKY proteins with two WRKY domains belong to group I, whereas most proteins with one WRKY domain belong to group II (Fig. 2). Generally, the WRKY domains of group I and group II members have the same type of finger motif, whose pattern of potential zinc ligands (C –X 4–5 –C –X 22–23 –H –X 1 –H ; Fig. 1) is unique among all described zinc-finger-like motifs 13 . The single finger motif of a small subset of WRKY proteins is distinct from that of group I and II members. Instead of a C 2 –H 2 pattern, their WRKY domains contain a C 2 –HC motif (C –X 7 –C –X 23 –H –X 1 –C ; Fig. 1). Owing to this distinction, they were recently assigned to the newly defined group III. Nevertheless, experimental evidence has shown that members of all three groups bind sequence specifically to vari- ous W box elements (R.S. Cormack et al., unpublished). The two WRKY domains of group I members appear to be functionally distinct. As has been shown for SPF1, ZAP1 and PcWRKY1, sequence-specific binding to their target DNA sequences is mediated mainly by the C-terminal WRKY domain 7,10,12 . The function of the N-terminal WRKY domain remains unclear. Because protein regions outside of the C-termi- nal WRKY domain contribute to the overall strength of DNA The WRKY superfamily of plant transcription factors Thomas Eulgem, Paul J. Rushton, Silke Robatzek and Imre E. Somssich The WRKY proteins are a superfamily of transcription factors with up to 100 representatives in Arabidopsis. Family members appear to be involved in the regulation of various physio- logical programs that are unique to plants, including pathogen defense, senescence and trichome development. In spite of the strong conservation of their DNA-binding domain, the overall structures of WRKY proteins are highly divergent and can be categorized into distinct groups, which might reflect their different functions.