Involvement of AtWRKY6 in senescence and plant defence
Senescence is the terminal stage of leaf development, and is considered as a type of programmed cell death controlled by both external and internal signals (Quirino et al., 1999a). This switch from anabolism to catabolism is characterized by severe morphological, physiological, biochemical and molecular changes that are highly co-ordinated, and include not only down-regulation, but also up-regulation of gene expression, as shown for SAG12 and SAG13, and de novo protein synthesis (Buchanan-Wollaston, 1997; Quirino et al., 1999b). Although several hormones are implicated, very little else is known about the signalling and regulatory components required for proper progression of senescence. Transcription factors mediating senescence-associated gene expression remain to be identified. According to our present studies, AtWRKY6 may be one such mediator, as its expression is not only strongly induced during leaf senescence, but is also associated with early to intermediate senescence stages (Buchanan-Wollaston, 1997; Quirino et al., 1999b). Thus it is likely that AtWRKY6 is involved in regulating the expression of a number of leaf senescence-associated genes during this time period.
AtWRKY6 expression was observed in anthers, in the stigma, and in floral organ abscission zones. Maturation of reproductive floral organs and abscission of floral organs are considered to be senescence processes (Bleecker and Patterson, 1997), and certain leaf senescence-associated genes are also expressed in flowers (Quirino et al., 1999b). Thus AtWRKY6 may be part of the regulatory network common to various plant senescence programs.
WRKY factors appear to play a key role in regulating the plant-pathogen defence program (Eulgem et al., 2000; Maleck et al., 2000). Induced expression of AtWRKY6, upon challenge with avirulent or virulent bacteria, also implicates AtWRKY6 in regulating some aspects of the plant defence response. A number of plant defence-related genes, such as ELI3 and PR1, are also induced during senescence (Morris et al., 2000; Quirino et al., 1999b). Similarly, the expression of senescence-associated genes is affected in mutants such as PAD4 and NPR1, which are impaired in certain plant defence responses (Morris et al., 2000). As suggested by Quirino et al. (1999a), such genes may have been recruited during evolution from the pathogen defence to the senescence program. Possibly these genes are responding to signalling pathways common to both programs, with AtWRKY6 being one of its regulatory constituents. Indeed, salicylic acid, another inducer of AtWRKY6 expression, is a crucial signalling compound in plant defence, and also appears to play a role in regulating some genes during leaf senescence (Morris et al., 2000; Quirino et al., 1999b).
Several hormones, such as cytokinin, auxin, gibberellic acid, ethylene, jasmonic acid and abscisic acid, alter the expression of senescence-associated genes (Nam, 1997; Park et al., 1998; Quirino et al., 1999a). Of these, ethylene and jasmonic acid are also signalling components of plant defence (Pieterse and van Loon, 1999) and both of these hormones positively influenced AtWRKY6 expression, as did wounding. In nature, herbivore attack or fungal invasion often causes a wound response, which is closely associated with plant defence and involves jasmonic acid as a key signalling component (Titarenko et al., 1997).
Whether AtWRKY6 expression in roots is actually constitutive, or whether its expression is induced under some as-yet undefined, stress-related conditions, remains to be demonstrated. Our studies using transgenic AtWRKY6 promoter–GUS lines grown under sterile conditions argues against induction by soilborne micro-organisms. Still, as was previously demonstrated for parsley PcWRKY1, application of mechanical pressure to plant cells alone can induce WRKY gene expression (Eulgem et al., 2000).
The AtWRKY6 promoter
The 1188 bp of the AtWRKY6 promoter directed GUS activities in a spatial and tissue-specific manner coincident with the expression patterns exhibited by its mRNA. Thus histochemical analyses of transgenic plants containing AtWRKY6 promoter deletion constructs enabled us to define distinct regions containing relevant cis-acting elements. These studies revealed that the complex expression pattern of AtWRKY6 can be partly dissected, and is mediated by different promoter regions. Relevant elements required for anther/pollen and stigmatic tissue-specific expression reside between −1188 and −726 bp. One potential candidate within this region is the sequence motif, TTTTGGTTTTATA (−932 to −919 bp), which shares strong homology to a previously described anther/pollen-specific element, TTTTGGTTTTCCA, identified in the Arabidopsis TYKY gene promoter (Zabaleta et al., 1998). Apart from this, no other such functionally defined elements are known at present.
Promoter deletions down to −726 and −345 bp resulted in an enhancement of the wound response, as seen in the control treatments of 6p3::GUS plants in Figure 4(c), indicating that a negative element may be present upstream. One candidate element may be one of the W boxes, TTGACT (−830 and −1109 bp), as such a motif has been shown to negatively influence expression of the AtPR-1 gene (Lebel et al., 1998).
Elements necessary for pathogen inducibility are located between −345 and −217 bp. This AtWRKY6 promoter region contains one reverse as1-like element CGTCA (−270 to −265 bp), together with a directly adjacent, inverted MYB recognition element CTGTTA (−262 to −257 bp; Rushton and Somssich, 1998). As1-like elements are bound by TGA factors (Zhang et al., 1999), and have been functionally shown to mediate gene expression on treatment with salicylic acid, jasmonic acid or auxin (Chen and Singh, 1999; Lebel et al., 1998). Each of these signalling molecules also affects AtWRKY6 expression, implying the involvement of TGA factors in this response. Whether the single as1-like element is sufficient to mediate expression, or acts in conjunction with the MYB recognition element, remains to be elucidated. Alternatively, interaction among factors binding to these elements and those bound by the as1-like tandem TGACG-n7-TGACG (−150 to −134 bp) may be required.
In addition to pathogen inducibility, the promoter region between −345 and −217 bp mediated abscission zone-specific expression. Whether any of the elements discussed above plays a role in this process is an open question, as very few promoters have been studied in this respect (Hong et al., 2000) and no defined abscission zone-specific promoter elements have been reported.
Apart from the as1-like elements, the AtWRKY6 promoter region that still allows root-specific and leaf senescence-associated expression contains no sequence motifs known to mediate these types of responses. Comparison of this promoter region to the only well studied senescence-specific SAG12 promoter (Noh and Amasino, 1999) revealed no reliable homologies.
Our results clearly show that the complex expression patterns observed for AtWRKY6 involve several cis-acting elements that are probably bound by different types of trans-acting factors. This study, however, also illustrates how limited our knowledge is concerning the DNA elements and cognate DNA-binding factors required for transcriptional regulation.
Functional nuclear localization signal
Nuclear localization has been demonstrated for WRKY factors from parsley and tobacco (Eulgem et al., 1999; Hara et al., 2000), but the identification of the NLS motifs responsible was not addressed. NLS sequences are typically rich in basic amino acids, but can differ in their actual sequence (Nakielny and Dreyfuss, 1999). The most commonly studied NLS motifs are those of the monopartite and bipartite type (Hicks et al., 1995). Based on computer predictions, AtWRKY6 contains both types. However, our functional data clearly show that none of these predicted motifs was mediating nuclear translocation. Instead, one region bearing no obvious similarity with defined NLS motifs was shown to be required. Our experiments show that the region identified must represent a novel type of NLS motif involving at least one basic amino acid between positions 258 and 278. Protein alignments with the closely related AtWRKY factors 31 and 42 (Eulgem et al., 2000) underline the conservation of the five basic amino acids KR-n2−3-R-n9−16-K-n2-K located within this peptide stretch, indicating that these amino acids represent a novel type of NLS motif.