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Keywords:

  • ABA-insensitive (ABI) loci;
  • ABA response;
  • Arabidopsis;
  • two-hybrid assays;
  • bZIP factors;
  • B3 domain factors

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Genetic and physiological studies have shown that the Arabidopsis thaliana abscisic acid-insensitive (ABI) loci interact to regulate seed-specific and/or ABA-inducible gene expression. We have used the yeast two-hybrid assay to determine whether any of these genetic interactions reflect direct physical interactions. By this criterion, only ABI3 and ABI5 physically interact with each other, and ABI5 can form homodimers. The B1 domain of ABI3 is essential for this interaction; this is the first specific function ascribed to this domain of the ABI3/VP1 family. The ABI5 domains required for interaction with ABI3 include two conserved charged domains in the amino-terminal half of the protein. An additional conserved charged domain appears to have intrinsic transcription activation function in this assay. Yeast one-hybrid assays with a lacZ reporter gene under control of the late embryogenesis-abundant AtEm6 promoter show that only ABI5 binds directly to this promoter fragment.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

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.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Two-hybrid assays of interactions among the ABI gene products

The ABA insensitive (ABI) loci of Arabidopsis have been shown to interact genetically in regulating processes such as germination and ABA-inducible gene expression. To determine whether ABI4 or ABI5 can physically interact with themselves or any of the other ABIs, we tested pairwise combinations in yeast two-hybrid interaction assays. The assay system was comprised of fusions between the ABI proteins and either the DNA-binding domain or the transcription-activation domain of GAL4. Direct interaction of the ABI protein fusions would bring the GAL4 domains of the fusions into close enough proximity to transactivate a β-galactosidase gene controlled by a GAL4-responsive promoter (James et al., 1996).

The GAL4-binding domain (BD)-ABI4 construct encoded a slightly truncated form of ABI4 (amino acids 3–287) because a full-length ABI4 fusion provides very strong transcription-activation function in the absence of any activation domain (AD) fusion (Söderman et al., 2000). Although the truncation reduces target gene expression roughly threefold, the remaining basal level of expression is still approximately tenfold higher than that produced by the GAL4-BD alone (Figure 1a). The BD-ABI5 construct encoded all but the first eight amino acids of ABI5, thus including all conserved domains, but produced a much lower basal expression of the reporter genes (only fourfold that produced by the vector control) (Figure 1b). Combination of these GAL4-BD fusions with GAL4-AD fusions to full-length ABIs produced a broad range of results (Figure 1). ABI1 and ABI4 did not interact with either ABI4 or ABI5, but both ABI3 and ABI5 strongly interacted with ABI5.

image

Figure 1. Tests for pairwise interactions among ABI proteins.

β-galactosidase activity of yeast carrying plasmids encoding the GAL4-AD (pGAD) or GAL4-AD fusions to the ABIs indicated and GAL4-BD (pGBD); or (a) a GAL4-BD-ABI4 fusion (BD-ABI4); or (b) a GAL4-BD-ABI5 (BD-ABI5) fusion.

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Given the documented genetic interactions and the similarities in effects on marker gene expression, we were surprised by the apparent lack of physical interaction between ABI4 and any other ABIs. Therefore we tested whether such interactions might either be masked by the high level of basal expression, or require formation of a ternary complex. Although further truncations of ABI4 in the BD fusion resulted in lower basal reporter-gene expression, none interacted with the AD-ABIs (data not shown). While deletion of various carboxy-terminal regions might remove domains required for interaction with other proteins, all the truncations encoded the complete AP2 domain, including the potential dimerization domain. Although it is also possible that these truncations are misfolded and/or unstable, and therefore not available for interaction, one of the ABI4 truncations in a BD fusion has been used successfully as a ‘bait’ construct in a two-hybrid screen (S.N., unpublished results).

To determine whether ABI4 might participate in a ternary complex with ABI3 and ABI5, we introduced plasmids encoding full-length native ABI3, ABI4 or ABI5 into yeast with ABI fusions to the GAL4-BD and -AD, then assayed reporter activity (Figure 2). Addition of ABI5 does not promote a BD-ABI4/AD-ABI3 interaction. Similarly, addition of ABI4 does not enhance the already strong activation produced by the BD-ABI5/AD-ABI3 interaction. Although ABI3 interacts with BD-ABI5 and strongly activates reporter expression, this interaction is not enhanced by the presence of an AD-ABI4 fusion.

image

Figure 2. Tests for ternary interactions among ABI3, ABI4 and ABI5.

β-galactosidase activity of yeast carrying plasmids encoding the GAL4-AD fusion indicated (pGAD, AD-ABI3, AD-ABI4), the GAL4-BD fusion indicated (BD-ABI5 or BD-ABI4), and a third plasmid encoding a full-length ABI protein under control of the ADH promoter (p26 derivatives).

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Mapping the interacting domains of ABI3 and ABI5

Having established that ABI3 and ABI5 interact in the yeast two-hybrid assay, we tested a series of subdomain fusions for each to map the interacting domains. The ABI3/VP1 subfamily of the B3-domain protein family contains four conserved domains: an acidic domain presumed to function in transcription activation (A), and three basic domains (B1, B2 and B3) (Giraudat et al., 1992; McCarty et al., 1991; Suzuki et al., 1997). Fusions between the GAL4 AD and various subsets of these domains were tested for interaction with the BD-ABI5 fusion (Figure 3). Strong interactions were observed only when the B1 domain was present, although the B1 domain alone was not sufficient for strong interactions. It is likely that the enhanced reporter activation with the A/B1 construct reflects the ability of the A domain to serve as a strong activator in yeast. We do not know whether the strong activation with the B1B2 construct is due to synergy between these domains, or to a stabilizing effect of the larger construct.

image

Figure 3. Mapping interacting domains of ABI3.

(a) Domain structure of ABI3 showing conserved acidic (A) and basic (B1, B2 and B3) domains. Heavy underlines show extent of domains present in GAL4-AD-ABI3 fusions.

(b) β-galactosidase activity of yeast carrying plasmids encoding the GAL4-AD-ABI3 fusions depicted in (a) and either a GAL4-BD-ABI5 (BD-ABI5) fusion or GAL4-BD (pGBD).

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The ABI5/DPBF/TRAB1/ABF subfamily of the bZIP transcription factor family contains four conserved domains: three charged domains in the amino-half of the protein (corresponding to amino acids 36–79, 137–164, and 198–220 of ABI5) (Figure 4), each including several possible phosphorylation sites; and the bZIP domain presumed involved in DNA binding and dimerization (Finkelstein and Lynch, 2000; Lopez-Molina and Chua, 2000). Fusions between the GAL4-BD and various subsets of these domains were tested for basal activity in combination with GAL4-AD or activation by interaction with a GAL4-AD fusion to either full-length ABI3 or ABI5 (Figure 5). Although fusions containing the first amino-terminal conserved domain alone (residues 9–122 or 64–122) produced a high level of basal activity, possibly by revealing an activation domain, this domain did not appear to interact with ABI3 or ABI5. In contrast, fusions containing the second and third conserved charged domains (residues 9–361, 123–371 or 123–442) had low basal activity, but continued to interact strongly with ABI3. As anticipated, the bZIP domain was critical for dimerization of ABI5. However, we were surprised to find that presence of the leucine zipper domain was not sufficient for dimerization of ABI5; although deletion of residues 91–122 had no effect on interactions with ABI3, loss of this unconserved domain eliminated interactions with AD-ABI5. Given that a weak ABI5 dimerization interaction was observed with an even shorter BD-ABI5 construct (including residues 262–442), the lack of dimerization by the aa 123–442 construct might reflect inaccessibility of the bZIP domain.

image

Figure 4. Three conserved domains in amino-half of ABI5 and homologs.

Comparison of conserved regions of ABI5 and the closest homologs present in the database. The consensus sequence is shown below the underline, with invariant residues capitalized, conserved residues in lowercase, and positions that usually contain negatively charged residues indicated as minus signs. C1 consists of a highly conserved region (double underlined) and a region that is missing from the other members of this subfamily (single underline). Sites of potential phosphorylation are indicated by asterisks. GenBank accession numbers for the sequences encoding these proteins are provided in parentheses after the gene names: ABI5 (AC006921), DPBF1 (AF001453), ABF1 (AF093544), ABF4/AREB2 (AF093547), ABF3 (AF093546), ABF2/AREB1 (AF093545), TRAB1 (AB023288).

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image

Figure 5. Mapping interacting domains of ABI5.

(a) Domain structure of ABI5 showing conserved domains (C1, C2, C3 and bZIP). Heavy underlines show extent of domains present in GAL4-BD fusions. Numbers over lines indicate amino acid residues present in fusion.

(b) β-galactosidase activity of yeast carrying plasmids encoding the GAL4-BD-ABI5 fusion depicted in (a) and GAL4-AD (pGAD), GAL4-AD-ABI5 (AD-ABI5), or GAL4-AD-ABI3 (AD-ABI3) fusions.

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Interactions between ABI transcription factors and an endogenous plant promoter

Previous studies of ABI-dependent gene expression have shown that the late embryogenesis abundant gene AtEm6 is regulated by ABI3, ABI4 and ABI5 (Finkelstein, 1993; Finkelstein, 1994; Finkelstein and Lynch, 2000; Finkelstein et al., 1998; Parcy et al., 1994). To determine whether this reflects a direct interaction between the AtEm6 promoter and any of these ABI transcription factors, we established a one-hybrid assay in yeast with a 1.3 kb fragment of the AtEm6 promoter and 5′-UTR fused to a lacZ reporter gene. Previous studies with transgenic plants carrying an AtEm6::GUS fusion have shown that this fragment is sufficient to induce strong GUS expression throughout the embryo (R.R.F., unpublished results). Each ABI gene product was introduced individually as part of a GAL4-AD fusion, and β-galactosidase expression was compared with that induced by a GAL4-AD vector control. Neither the ABI3 nor the ABI4 fusion had any effect on expression, but addition of the ABI5 fusion produced a threefold increase in β-galactosidase activity, reflecting binding of ABI5 to this promoter fragment (Figure 6).

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Figure 6. Transactivation of the AtEm6 promoter in yeast.

β-galactosidase activity of yeast carrying an AtEm6::lacZ reporter and plasmids encoding GAL4-AD (pGAD) or GAL4-AD fusions to the indicated ABIs.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Genetic and biochemical approaches have been used to identify and clone regulators of seed development and ABA signaling. The ABI loci of Arabidopsis cloned to date can be divided into genes encoding two major classes of biochemical function: protein phosphatases (ABI1 and ABI2), and transcription factors (ABI3, ABI4 and ABI5). These loci have been well characterized in terms of their roles in marker gene expression and ABA sensitivity of growth, by analysis of mutants and transgenic ectopic expression lines, both singly and in various combinations (Finkelstein and Lynch, 2000; Finkelstein et al., 1998; Parcy and Giraudat, 1997; Parcy et al., 1994; Söderman et al., 2000). These studies have shown that ABI3, ABI4 and ABI5 have similar physiological roles and show some cross-regulation of expression, leading to the suggestion that they act combinatorially. In contrast, the effects of the dominant negative abi1-1 mutation have relatively limited overlap with those of mutations in ABI3, ABI4 or ABI5. Although double mutants combining abi1-1 with any of the transcription factor mutations show significantly enhanced resistance to ABA (Finkelstein, 1994; Finkelstein and Somerville, 1990), suggesting action in separate pathways, the abi1-1 mutation can block the ABA hypersensitivity produced by overexpression of ABI3, consistent with action in the same pathway (Parcy and Giraudat, 1997).

Consequently, ABI1, ABI3, ABI4 and ABI5 were all legitimate candidates for proteins that might be involved in direct physical interactions. We have tested for such interactions among these ABI gene products by yeast two-hybrid assays; as this is a completely heterologous system, any apparent interactions should be dependent on the plant genes included in the assay. Despite the apparent similarities of the genetic interactions involving ABI3, ABI4 and ABI5, only two of the ABI proteins interact in this assay: ABI3 and ABI5. Recent studies have shown that ABI5 interacts synergistically with ABA and co-expressed VP1 to transactivate an Em-GUS reporter in transfected rice protoplasts, providing further support for the hypothesis that these proteins interact in plant cells (Gampala et al., 2001). The failure of ABI1 to interact with any of the transcription factors might reflect either a simple lack of physical interaction between these proteins, or a requirement for a phosphorylated substrate which is not provided by heterologous expression in yeast. The lack of interaction between ABI4 and any of the other ABIs, despite the observed similarities in genetic interactions and physiological effects, does not exclude the possibility of participating in the same regulatory complex, but does show that these factors are probably not in direct contact. We have mapped the ABI3–ABI5 interaction to the B1 domain of ABI3 and a region of ABI5 containing two conserved charged domains, and several possible sites of ser/thr phosphorylation. In addition, ABI5 can form homodimers; this interaction appears to be dependent on a weakly conserved hydrophilic domain as well as the bZIP domain. ABI5/AtDPBF1 homodimer formation has also been demonstrated by in vitro binding to the Dc3 promoter (T. Thomas, Texas A & M University, personal communication).

The functional domains of ABI3 and its homologs, for example, the VP1 proteins of cereals, have been analyzed in terms of their roles in DNA binding and transactivation of target promoters. These studies have shown that the B2 domain is involved in regulation of some late embryogenesis-abundant (LEA) and storage protein genes (Bies-Etheve et al., 1999), and enhances DNA binding of a variety of bZIP factors, but binds DNA only weakly and non-specifically by itself. In contrast, the B3 domain can bind DNA directly (Suzuki et al., 1997), but is not required for ABA-dependent gene regulation (Carson et al., 1997). Both the B2 and B3 domains were recently shown to be necessary for expression of a Brassica napus gene encoding the storage protein napin, apparently by interaction with distinct cis-elements, leading the authors to suggest that B2 tethers ABI3 to a seed-specific ABRE via protein–protein interactions (Ezcurra et al., 2000). Two-hybrid screens for proteins interacting with oat VP1 or Arabidopsis ABI3 using bait constructs containing only the B2 and B3 domains identified several transcription factors, but did not result in isolation of an ABI5 fusion (Jones et al., 2000; Kurup et al., 2000). In contrast, a two-hybrid screen using the B1 and B2 domains of the rice OSVP1 in the bait construct identified TRAB-1 (Hobo et al., 1999), a rice bZIP factor with strong homology to ABI5 (55% similar). Although the interacting domains of TRAB-1 and OSVP1 were not specifically mapped, these results are consistent with the importance of the B1 domain to the interaction.

The functional domains of ABI5 are less well characterized. ABI5 belongs to a subfamily of the bZIP transcription factor family whose members have been identified by mutation (Finkelstein and Lynch, 2000; Lopez-Molina and Chua, 2000), one-hybrid screens with repeats of the ABRE motif (Choi et al., 2000; Uno et al., 2000) or a fragment of the Dc3 promoter (Kim et al., 1997); or a two-hybrid screen with OSVP1 as ‘bait’ (Hobo et al., 1999). Within this subfamily, expression patterns vary in terms of relative abundance in seed versus vegetative tissues or induction by salt, drought or ABA, but all appear correlated with ABA response. To date, the only member of the family for which mutants have been identified is ABI5. The most severe available mutations result in truncation at aa 361 (abi5-1) (Finkelstein and Lynch, 2000) or after only 110 amino acids of the wild-type coding sequence (abi5-4) (Lopez-Molina and Chua, 2000); the former includes all but the bZIP domain, while the latter is lacking all but the first conserved charged domain. However, both mutant lines show similar, limited ABA resistance and comparable effects on seed gene expression, suggesting that both are effectively null mutations. Given that both lack the bZIP domain required for dimerization and DNA binding, this result is neither surprising nor informative regarding other potentially important domains. In contrast, deletion analyses of the sunflower DPBF-1 protein have shown that in vitro DNA binding is retained in constructs lacking one or all three of the charged domains in the amino half of the protein (Kim et al., 1997). This implies that the region of DPBF-1 corresponding to amino acids 1–248 of ABI5 is not required for homodimer formation. Although sunflower DPBF-1 shows the strongest overall homology to ABI5 of all sequences currently in the database, the Arabidopsis ABF/AREB proteins share with ABI5 a larger conserved region at the amino-terminal end. In our studies, this conserved region appears to function as a transcription-activation domain. Although a truncation including only the most conserved part of this domain (aa 64–122) still functions as an activator, it is attenuated approximately twofold relative to the construct including amino acids 9–122. Recently, all three of the conserved domains in the amino-terminal halves of AREB1 and AREB2 were shown to undergo ABA-dependent phosphorylation in an in-gel kinase assay (Uno et al., 2000). Furthermore, transactivation of reporter genes in protoplasts required the presence of both an AREB and ABA, but could be blocked by protein kinase inhibitors, implying that ABA-dependent phosphorylation is essential for activity of the AREBs. However, in vivo phosphorylation of the AREBs has not been assayed directly.

Taken together with our two-hybrid interaction results, these studies suggest that the transcription-activation domain of ABI5 is located in the most amino-terminal conserved domain, but is normally inactive until phosphorylation induces a conformational change that exposes the domain to potential interaction partners. In our truncation studies, removal of surrounding domains by deletion of their coding regions might produce the same effect as that suggested for phosphorylation: exposure and resulting transcription-activation function in yeast. In contrast, the second and third conserved domains appear more significant in interacting with ABI3, but have no intrinsic activator function of their own. We do not know whether this interaction depends on phosphorylation of the BD-ABI5 truncations in yeast, but they are certainly not exhibiting ABA-dependent phosphorylation in this system. Analysis of the predicted protein structure for ABI5 shows that the three conserved regions in the amino half have a relatively low probability of being exposed on the surface compared with adjacent domains in the primary structure. This is consistent with a requirement for a phosphorylation-induced conformational change to expose the transcription activation and ABI3-interacting domains.

The studies described above all use the GAL4 DNA-binding domain to target transcription activation due to any interacting proteins to a GAL4-responsive promoter. To determine whether any of the ABI-transcription factors could interact directly with a known ABI-responsive plant promoter in a completely heterologous system, we tested their ability to activate an AtEm6::lacZ fusion in yeast. The promoter fragment used contains two consensus binding sites (ACACNNG) for the sunflower homologs of ABI5 (Kim et al., 1997) within ≈230 bp of the transcription start site, and it is likely that the observed activation by ABI5 reflects binding to these sites. In contrast, intact ABI3 (or VP1) has never been shown to bind directly to DNA, so the failure of ABI3 to transactivate the reporter gene is not surprising. Similarly, although ABI4 belongs to the EREBP/DREB/CBF subfamily of the APETALA2 domain family of transcription factors, most of which were identified by binding to similar DNA sequences, a binding site for ABI4 has not yet been identified. The failure of ABI4 to transactivate the AtEm6::lacZ reporter suggests that an ABI4 binding site either is not contained within this 1.3 kb fragment, or is too far from the transcription start site to efficiently recruit RNA polymerase to initiate transcription of this gene.

In summary, our studies demonstrate a direct physical interaction between two transcription factors previously shown to interact genetically, cross-regulate each other's expression, and regulate an overlapping set of physiological events (Finkelstein, 1994; Finkelstein and Lynch, 2000; Parcy et al., 1994; Söderman et al., 2000). These are likely to be present in regulatory complexes of varying composition, as appropriate for each target promoter they regulate at any given stage in development. Other possible participants in these complexes have already been identified by co-purification or two-hybrid screens (Kurup et al., 2000; Schultz et al., 1998), but the specific composition of any complex and its corresponding promoter have yet to be defined. Microarray analyses of gene expression in lines with loss or gain of function for specific transcription factors should help elucidate these interactions.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Construction of gene fusions for two-hybrid assays

Translational fusions between varying portions of the ABI genes and the GAL4 activation and DNA-binding domains were constructed in the pGAD-C(x) and pGBD-C(x) vectors, respectively (James et al., 1996). The activation domain was fused to ‘full-length’ cDNAs such that the ABI1 fusion encodes the entire ABI1 protein plus 24 amino acids derived from the 5′-UTR sequence; the ABI3 fusion encodes the entire ABI3 protein plus three amino acids derived from a multiple cloning site; the ABI4 fusion lacks the first two and last one amino acid of ABI4; and the ABI5 fusion lacks the first eight amino acids of ABI5. The same fragment of ABI5 was used in the ‘full-length’ fusion to the DNA-binding domain. The ABI4-GAL4 DNA binding-domain fusion terminated at a BamHI site, thereby truncating the C-terminal 41 amino acids. The smallest ‘B1 domain’ fusion for ABI3 contained a PCR-generated fragment encoding amino acids 217–302. Restriction sites used for the additional truncation products and the extent of each clone are described in Table 1.

Table 1.  Truncation fusions for mapping ABI3 and ABI5 interaction domains
ConstructABI amino acids presentDomains present
  1. Specific amino acids and conserved domains included in the AD- and BD-fusions represented in Figures 3 and 5. The B2 and/or B3 domains of ABI3 were recloned from ‘cassette’ subclones (a generous gift of F. Parcy) in which the coding sequences were flanked by BamHI sites. The end points of the ABI5 subclones were determined by the restriction sites indicated.

pGAD-ABI3-B1B2B3(Eco)216–670B1, B2, B3
pGAD-ABI3-B2B3390–670B2, B3
pGAD-ABI3-B1B2(Eco/Kpn)216–505B1, B2
pGAD-ABI3-B3506–670B3
pGAD-ABI3-AB11–388A, B1
pGAD-ABI3-B1S217–302B1
pGAD-ABI3-B1L216–388B1
pGAD-ABI3-A1–215A
pGBD-ABI5-Pst/Hinc91–442C2, C3, bZIP
pGBD-ABI5-Xba/Hinc123–442C2, C3, bZIP
pGBD-ABI5-Sma/Hinc262–442bZIP
pGBD-ABI5-Dde/Hind341–442bZIP
pGBD-ABI5-Hinc/PCR9–361C1, C2, C3
pGBD-ABI5-Xba/Bgl123–371C2, C3
pGBD-ABI5-Hinc/Xba9–122C1
pGBD-ABI5-NlaIV/Xba64–122C1

Transcriptional fusions to full-length cDNAs for ABI3, ABI4 and ABI5 were constructed in p26A (a gift from R. Ballester), a derivative of pRS426 (Christianson et al., 1992) containing an ADH1 promoter with an adjacent cloning site.

A translational fusion of AtEm6 and lacZ was constructed in pLGΔ178, a derivative of pLGΔ312 (Guarente and Mason, 1983). The AtEm6 fragment extended from an EcoRI site 1.2 kb upstream of the transcription start site to a PvuII site spanning codons 9–10. This fragment, plus a region of the pBS polylinker encoding eight additional amino acids, was fused in frame at codon 3 of the lacZ gene.

Restriction fragments of interest were generated by standard molecular biological techniques (Sambrook et al., 1989), gel-purified with a QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA, USA), and ligated to vectors linearized by restriction digestion at specific cloning sites and dephosphorylated by treatment with calf intestinal phosphatase. The resulting plasmids were transformed into Escherichia coli DH10 cells by electroporation. Transformants were selected by growth on Luria broth with ampicillin, and screened for colonies containing plasmids with appropriate restriction patterns. Junctions of translational fusions were sequenced to confirm that all fusions were in the correct reading frame. All the fusion constructs are available on request.

Assays of transcription activation function

All gene fusions were transformed into the yeast cell line PJ69-4A using the EZ yeast transformation kit, according to the manufacturer's instructions (Bio101, Vista, CA, USA). The pGAD-, pGBD- and p26A fusions were maintained in yeast by requiring growth in the absence of leucine, tryptophan and uracil, respectively. Quantitative assays of GAL4-BD-driven β-galactosidase gene expression were performed as described at http://www.fhcrc.org/~gottschling/Bgal.html. All data presented are the averages ± SD of assays on at least three independent transformants.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Dr R. Ballester for gifts of yeast-cloning vectors and technical advice regarding yeast two-hybrid experiments. We thank Drs J. Giraudat, J. Leung, and F. Parcy for gifts of ABI1 and ABI3 cDNA clones and valuable discussions of results. This work was supported by the National Science Foundation (grant nos. IBN-9728297 and IBN-9982779 to R.R.F.). S.N. was supported by the Partially Guaranteed researcher exchange program of the Science and Technology Agency in Japan.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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