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The phytohormone abscisic acid (ABA) can regulate many agronomically important aspects of plant growth, including seed maturation, onset of seed dormancy, and adaptation to assorted environmental stresses such as drought and salinity (Leung and Giraudat, 1998). Genetic studies have identified many loci required for wild-type ABA response; the best characterized are the ABA insensitive (ABI) and enhanced response to ABA (ERA) genes of Arabidopsis and the VP1 genes of maize and other cereals (Holdsworth et al., 1999). Many additional presumed ABA-signaling components have been identified biochemically, based on binding to cis-acting regulatory elements such as ABREs (ABA-response elements; Busk and Pages, 1998); one- or two-hybrid screens in yeast (Choi et al., 2000; Hobo et al., 1999; Kim et al., 1997; Kurup et al., 2000; Uno et al., 2000); or correlations between kinase or phosphatase activity and ABA response (Leung and Giraudat, 1998). However, in the absence of genetic tests of function the roles of most of these components remain speculative.
Three of the ABA insensitive loci, ABI3, ABI4 and ABI5, encode transcription factors of the B3-, AP2- and bZIP-domain families, respectively, and regulate overlapping subsets of seed-specific and/or ABA-inducible genes (Finkelstein and Lynch, 2000; Finkelstein et al., 1998; Giraudat et al., 1992). Two of these, ABI4 and ABI5, contain presumed DNA-binding and dimerization domains: the AP2 and bZIP domains, respectively. ABI3 can activate transcription in vivo, but the intact purified protein does not specifically bind DNA in vitro, suggesting that it interacts with other proteins that mediate DNA binding (Suzuki et al., 1997). Mutational analyses of VP1/ABI3-responsive promoters have shown that G-box elements such as those present in the Em1a and Em1b elements, although required for ABA regulation and consequently designated ABREs, are sufficient but not necessary for VP1 transactivation (Vasil et al., 1995). These studies suggest that VP1 activates transcription through multiple cis-acting sequences, only some of which are ABREs. It was subsequently shown that both VP1 and EmBP1, an Em1a-binding bZIP protein, specifically interact with GF14, a 14-3-3 protein which may provide a structural link between these transcription factors (Schultz et al., 1998). Although ABI5 is similar to EmBP1 in that they are both bZIP proteins correlated with ABA response, ABI5 is a member of the DPBF subfamily (Finkelstein and Lynch, 2000). This subfamily has a broader consensus-binding site than the other bZIP proteins in that its members tolerate variability in the ACGT core element essential to the ABRE G-box (Kim et al., 1997). Furthermore, the homology between ABI5 and EmBP1 is limited to the bZIP domain. Consequently, any interactions between ABI5 and VP1/ABI3 need not be mechanistically similar to the previously described VP1–EmBP1 interactions.
Physiological studies have shown that the ABI3, ABI4 and ABI5 loci have similar qualitative effects on seed development and ABA sensitivity, consistent with action in a common pathway, but that null mutations in ABI3 are more severe than those in ABI4 or ABI5 (Finkelstein and Lynch, 2000; Finkelstein et al., 1998; Parcy et al., 1994). Action in a common pathway was also suggested by genetic studies showing that digenic mutants combining the leaky abi3-1 alleles with severe mutations in either ABI4 or ABI5 produced seeds that were only slightly more resistant to ABA than their monogenic parents (Finkelstein, 1994). Recent studies show extensive cross-regulation of expression among ABI3, ABI4 and ABI5 (Söderman et al., 2000). Furthermore, ectopic expression of either ABI3 or ABI4 results in ABA hypersensitivity of vegetative tissues which is partly dependent on increased ABI5 expression (Parcy et al., 1994; Söderman et al., 2000). Taken together, these results suggest that these three transcription factors participate in combinatorial control of gene expression, possibly by forming a regulatory complex mediating seed-specific or ABA-inducible expression. Consistent with this, rice homologs of ABI3 and ABI5 (OSVP1 and TRAB1, respectively) have been shown to interact in a yeast two-hybrid assay, as well as in transient assays in plant cells (Hobo et al., 1999).
The two remaining cloned ABI loci, ABI1 and ABI2, were initially identified by dominant negative mutations resulting in decreased sensitivity to ABA (Gosti et al., 1999; Koornneef et al., 1984). These loci encode highly homologous members of the PP2C family of ser/thr protein phosphatases, and it has been suggested that they might act on overlapping subsets of substrates (Leung et al., 1997). However, none of their substrates has been identified to date. Both the ABI4 and ABI5 gene products contain ser/thr-rich domains that could be sites of phosphorylation (Finkelstein and Lynch, 2000; Finkelstein et al., 1998), consistent with the possibility that either might be a substrate for the ABI PP2Cs. Recently, two ABI5-related transcription factors, AREB1 and AREB2, were shown to promote ABA-activation of target gene expression (Uno et al., 2000). Although this activation was repressed either by protein kinase inhibitor treatment of wild-type cells or by the dominant negative abi1-1 mutation, it is not known whether this reflects a direct or indirect effect on the phosphorylation status of these transcription factors.
Our study makes use of yeast two-hybrid assays to test for direct physical interactions among ABI gene products previously demonstrated to interact genetically. Only two of these proteins interacted directly: ABI3 and ABI5. In addition, ABI5 interacted with itself, presumably reflecting an ability to form homodimers. We mapped the interacting domains by assaying reporter activation by combination of various subdomains of ABI3 and ABI5. In addition, we tested for transactivation of the AtEm6 promoter by ABI3, ABI4 and ABI5 in a yeast one-hybrid system.
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