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First published online September 17, 2002; 10.1105/tpc.003400 American Society of Plant Biologists Maize ABI4 Binds Coupling Element1 in Abscisic Acid and Sugar Response GenesAgronomic Traits, Trait and Technology Development, Pioneer Hi-Bred International, Johnston, Iowa 50131-0552 1 To whom correspondence should be addressed. E-mail nic.bate{at}pioneer.com; fax 515-334-4788
Significant progress has been made in elucidating the mechanism of abscisic acid (ABA)–regulated gene expression, including the characterization of an ABA-responsive element (ABRE), which is regulated by basic domain/Leu zipper transcription factors. In addition to the ABRE, a coupling element (CE1) has been demonstrated to be involved in ABA-induced expression. However, a trans factor that interacts with CE1 has yet to be characterized. We report the isolation of a seed-specific maize ABI4 homolog and demonstrate, using a PCR-based in vitro selection procedure, that the maize ABI4 protein binds to the CE-1 like sequence CACCG. Using electrophoretic mobility shift assays, we demonstrate that recombinant ZmABI4 protein binds to the CE1 element in a number of ABA-related genes. ZmABI4 also binds to the promoter of the sugar-responsive ADH1 gene, demonstrating the ability of this protein to regulate both ABA- and sugar-regulated pathways. ZmABI4 complements Arabidopsis ABI4 function, because abi4 mutant plants transformed with the ZmABI4 gene have an ABA- and sugar-sensitive phenotype. Identification of the maize ABI4 ortholog and the demonstration of its binding to a known ABA response element provide a link between ABA-mediated kernel development and the regulation of ABA response genes.
Abscisic acid (ABA) plays an important role in a number of agronomically important traits, including the plant's response to abiotic stress and the deposition of storage protein and starch during seed development (Bewley and Black, 1994  ; Davies, 1995; Wobus and Weber, 1999). A greater understanding of the mechanism of ABA-regulated gene expression has been achieved in genetic and molecular studies (Grill and Himmelbach, 1998; McCourt, 1999). The dissection of the signal transduction pathway from ABA to gene activation has led to the identification of cis-acting elements important to ABA-induced gene expression. Most of these studies have identified ABA-responsive element (ABRE) or G-box–like elements as the important cis element for ABA-induced gene expression (Shen and Ho, 1995; Shen et al., 1996; Busk et al., 1997; Hobo et al., 1999a).
A substantial body of biochemical work has identified basic domain/Leu zipper (bZIP)–type transcription factors in the regulation of ABA-responsive genes because they have high binding affinity for ABRE/G-box (Guiltinan et al., 1990
In addition to ABI5/bZIP transcription factors that interact with ABREs, two other transcription factors (ABI3/VP1 and ABI4) have been characterized that play a role in regulating the ABA response during seed development (Finkelstein and Somerville, 1990
Cloning of ABI4 from Arabidopsis revealed an AP2 domain characteristic of a class of transcription factors known to regulate plant development (Finkelstein and Goodman, 1998 In this article, we describe a seed-specific gene from maize that has homology with the Arabidopsis ABI4 gene. Using an in vitro selection technique, we demonstrate that this protein binds to the sequence CACCG, which corresponds to the previously characterized CE1 element found in ABA-responsive genes. Transformation of the maize protein into the Arabidopsis abi4 mutant rescues the ABA- and sugar-sensitive phenotype, demonstrating that maize ABI4 can complement the Arabidopsis gene.
Isolation and Sequence Analysis of the ZmABI4 Gene Three ESTs with a deduced protein sequence highly homologous with that of the AP2 domain of the Arabidopsis ABI4 protein were identified from the Pioneer/Dupont database. All three ESTs are from 20-days after pollination (DAP) maize embryo libraries and represent transcripts of the same gene. After sequencing of the full insert from the longest clone, an open reading frame of 747 bp was detected that, when translated, included the AP2 domain in frame. This sequence was used subsequently to isolate an upstream fragment of 2521 bp 5' of the ATG start codon by genome walking. The sequence upstream of the open reading frame contains several potential TATA boxes, including one located 71 bp upstream of the longest cDNA (Figure 1) . Genomic sequence analysis by PCR also indicated that the maize ABI4 homolog has no intron (data not shown). Based on sequence homology, we designated the maize homolog ZmABI4.
The deduced amino acid sequence revealed a significant degree of identity between the ZmABI4 and AtABI4 proteins in the AP2 domain (50 of 55 residues identical). However, the ZmABI4 protein is smaller, and outside of the AP2 domain, the sequence is divergent (Figure 2A) . The overall structural features of AtABI4 are largely present in ZmABI4. For example, a Ser/Thr-rich sequence is present (9 of 24 residues, compared with 19 of 28 for Arabidopsis), as is a small Gln-rich domain (3 of 14 residues, compared with 13 of 21). The C-terminal half of the ZmABI4 protein is Pro rich, but a defined Pro domain is missing from the maize sequence. Lastly, the C-terminal 40 residues are high in acidic residues, similar to the Arabidopsis sequence. The identity between ZmABI4 and AtABI4 in the AP2 domain is significantly higher than that between ZmABI4 and other AP2 domain proteins in maize or Arabidopsis, and the overall size and structure are more similar between the ABI4 proteins than between the ABI4 proteins other AP2 proteins (Figure 2B).
Recently, a draft sequence of the rice genome was reported (Yu et al., 2002 ), and within the rice database, a single sequence shares significant homology with the AtABI4 AP2 domain, indicating that a single ABI4 homolog exists in rice. The rice ABI4 (OsABI4) also is highly homologous with ZmABI4, sharing 100% identity in the AP2 domain and the same structural features described above. Therefore, within available databases, the deduced maize protein sequence presented here represents the closest structural homolog of the Arabidopsis ABI4 protein.
Expression of the ZmABI4 Gene
Determination of the ZmABI4 Binding Sites To determine the sequences to which the ABI4 protein may bind, and to gain a greater understanding of the function of the ABI4 protein, we used a PCR-assisted method to determine the binding site for this putative DNA binding protein. Recombinant ZmABI4 protein was incubated with a library of random double-stranded oligonucleotides in a binding reaction. After gel electrophoresis, the shifted protein/DNA complex was excised from the gel, purified, and subjected to PCR amplification. This procedure was repeated five additional times to enrich the sequences bound by the ZmABI4 protein. The enriched sequences from the sixth round of selection were cloned, and 52 independent clones were sequenced. The selected sequences are shown in Figure 4 .
Of 52 sequences, the majority (42) have the 5-bp consensus sequence CACCG, suggesting that the ZmABI4 protein has affinity for this pentamer sequence (Figure 4). We found that the CE1 site, a cis element shown previously to be functionally important to ABA-regulated gene expression (Shen and Ho, 1995 ), resembles the ZmABI4 binding sites. The CE1 element in the HVA22 promoter and the DRE2 element in the rab17 promoter both were identified in the selected sequences (Figure 4), and the ABI4 selected binding site also is present in the promoters of a number of other ABA-regulated genes (Figure 5A)
. Interestingly, a putative binding site was present in the maize ABI4 upstream region (Figures 4 and 5A), and a highly conserved ABI4 binding site also was present in the upstream sequence of the Arabidopsis and rice ABI4, immediately downstream from a putative TATA box, as observed in the maize upstream region (Figure 5B).
ZmABI4 Binds to CE1-Like Elements in the Promoters of ABA- and Sugar-Responsive Genes and to Its Own Upstream Region To confirm the binding of ZmABI4 to the selected site, double-stranded DNA probes were created to correspond to the CE1-like sequences found in ABA-related genes from a range of species. Figure 5C shows that recombinant ZmABI4 protein binds to a range of promoter elements, including those from the ZmABI4, maize rab28, maize rab17, barley HVA22, barley HVA1, and rice rab16b genes. Among these promoter elements, HVA22 CE1 and rab17 DRE2 elements have been shown to be important for ABA induction (Shen and Ho, 1995 ; Busk et al., 1997).
To determine the role of ZmABI4 in regulating sugar-sensitive genes, we studied the sugar-regulated maize ADH1 gene (Koch et al., 2000
Rescue of the Arabidopsis abi4 Mutant with ZmABI4 To demonstrate functional equivalence between AtABI4 and ZmABI4, we transferred the ZmABI4 gene, under the control of a constitutive promoter (the maize ubiquitin promoter, with glufosinate resistance as the selectable marker), into the abi4-1 mutant of Arabidopsis (obtained from the ABRC Stock Center, Columbus, OH). Glufosinate resistance was used to select a number of primary transformants, and seeds from these plants were used to establish copy number. Two independent single-insert lines of abi4/Ubi:ZmABI4 were selected for further characterization. To establish the ability of the ZmABI4 gene to rescue the Arabidopsis abi4 mutation, we used an abi4-1 line homozygous for the mutation and segregating for the presence of the Ubi::ZmABI4 transgene. Seeds from these lines were grown in the presence of repressive concentrations of ABA (5 µM) or Glc (7%). When germinated in the presence of 5 µM ABA, wild-type plants produced a radicle but did not develop further (Figure 7A , right). Similarly, wild-type plants germinated in the presence of 7% Glc did germinate but had a reduced frequency of cotyledon expansion and did not produce green true leaves (Figure 7A, left). Therefore, early development of seedlings under these repressive conditions provides an effective screen to assess the ability of the ZmABI4 gene to rescue the phenotype of the Arabidopsis abi4 mutation. Surface-sterilized mutant seeds segregating for the presence of the transgene were placed onto minimal medium plates containing 5 µM ABA or 7% Glc. Single-insert lines segregating for the transgene have a 3:1 ratio of wild-type to mutant phenotype if ZmABI4 rescues the Arabidopsis abi4 mutation. If the transgene does not rescue the Arabidopsis mutant, the seedlings all have the mutant phenotype.
Figure 7B indicates that in two independent lines of abi4 mutant Arabidopsis, ectopic expression of the ZmABI4 gene compensated for the genetic lesion in Arabidopsis abi4. Approximately 75% of the seedlings had the wild-type phenotype when grown on 5 µM ABA, in keeping with a 3:1 segregation of the transgene. Screening of the same segregating population for the ability to germinate and form true green leaves in the presence of inhibitory concentrations of Glc gave similar results (Figure 7C). Mutant seed lines containing a segregating transgene were treated as described above, except that 7% Glc was the inhibitory reagent. Plants were allowed to imbibe for 5 days to ensure that germination was initiated before plating. Both transgenic lines rescued the mutant phenotype in a ratio close to the expected 3:1 (Figure 7C). To further demonstrate cosegregation of the wild-type phenotype and the transgene, seedlings with the wild-type and mutant phenotypes were removed from the 7% Glc plates and placed onto nonselective minimal medium plates containing 2% Glc. After 1 week of recovery, the plants were assayed for the presence of the transgene by protein gel blot analysis with a ZmABI4-specific antibody. None of the plants (eight of eight surveyed) with a mutant phenotype contained the transgene, whereas seven of eight seedlings with the wild-type phenotype possessed a functional transgene when grown on 7% Glc (Figure 7D).
In this study, we present the characterization of the maize homolog of Arabidopsis ABI4 and demonstrate that it binds to a cis-regulatory site shown previously to be essential for the induction of genes by ABA. Although the protein sequence for the maize and Arabidopsis genes are divergent outside of the AP2 domain, the overall similarity is in line with orthologous proteins in maize and Arabidopsis, such as VP1 and ABI3. No EST is present in the Pioneer/Dupont or public maize databases that has greater homology with the Arabidopsis ABI4 gene, and there is significant structural divergence between the ABI4 proteins and other AP2 domain proteins from either species. Furthermore, screening of the Arabidopsis, maize, and rice databases with ABI4 sequences did not identify any other AP2 proteins with greater identity than AtABI4, ZmABI4, and OsABI4. All three ABI4 sequences share a stretch of significant homology, including an ABI4 binding site, immediately downstream of the TATA box (Figure 5B). Together, these results suggest that the AP2 protein presented here is the maize ortholog of the Arabidopsis ABI4 gene.
The expression pattern of ZmABI4 is consistent with a transcription factor that plays a role in the initiation or maintenance of the storage phase of maize seed development. The prestorage-to-storage phase transition occurs at
Mapping results provide further evidence to link ZmABI4 with seed development and yield. ZmABI4 was mapped on the maize genome and found to be located on chromosome 6 (bin 5) (data not shown), which is a similar map location as that of rab17 (Frova et al., 1998
A model for the role of ABI4 in the regulation of ABA-responsive genes is presented in Figure 8
. Two hybrid screens using VP1 as bait isolated TRAB1, a bZIP protein (Hobo et al., 1999b
CE1-like elements also are present in the promoters of Arabidopsis sugar-responsive genes, including Em6, ADH1, PC, and ApL3, consistent with the fact that these genes have altered expression in the abi4 mutant. Significantly, PC and ApL3 promoter mutant screens identified ABI4 as a crucial regulatory factor in Arabidopsis (Huijser et al., 2000 ; Rook et al., 2001). The ability of ZmABI4 to rescue sugar sensitivity when transformed into mutant abi4-1 plants provides further evidence that ABI4-mediated transcriptional regulation of sugar-responsive genes through the CE1-like element is an important control point. In Arabidopsis, ABI4 is thought to be an important regulatory component in the progression of early seedling development, responding to sugar and ABA signals that act together or separately (Gibson, 2000). Although it is clear that ZmABI4 can rescue sugar response functions of the Arabidopsis abi4 mutant, the role of ZmABI4 in maize appears to be strictly developmental (i.e., responding to a kernel development program to regulate storage-related genes). Whether or not ZmABI4 function is mediated by ABA and sugars remains to be determined.
The presence of a strong binding site for ABI4 in its own promoter in maize, rice, and Arabidopsis is interesting in light of their conserved locations downstream of the putative TATA box. Binding of regulatory proteins to sites downstream of the TATA box has been reported to be important to the transcriptional regulation of Shrunken (Bellmann and Werr, 1992 Collectively, the data presented here provide strong evidence for the conservation of regulatory mechanisms that control seed development in plants. By establishing a link between mutant phenotype, gene expression, and DNA binding, the characterization of ZmABI4 provides further insight into the role of ABI4 in ABA and sugar signal transduction pathways and into the regulatory framework that controls seed development.
cDNA and Gene Isolation A sequence with a deduced AP2 domain homologous with the Arabidopsis thaliana ABI4 gene was identified from the Pioneer/Dupont EST databases. Genomic DNA upstream of the coding sequence was isolated using the Universal GenomeWalker Kit from Clontech (Palo Alto, CA). Additional sequence information was obtained by PCR analysis of genomic DNA and cDNA. Mapping of the maize (Zea mays) ABI4 gene (ZmABI4) was performed as described previously (Tarchini et al., 2000 ).
Expression Analysis by Reverse Transcriptase–Mediated PCR
Gel-Shift Assays and DNA Binding Site Selection
DNA binding site selection for maize ABI4 was essentially as described (Niu et al., 1999
Transgenic Construct and Plant Transformation
The floral dip method was used for Arabidopsis transformation essentially as described (Clough and Bent, 1998
Arabidopsis Growth Conditions and Rescue Experiments Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Numbers
The authors thank Michael Muszynski, Norbert Brugière, and Steven Rothstein for critical reading of the manuscript and members of the Yield Stability Group for useful discussion. We also thank the Arabidopsis Biological Resource Center for providing abi4-1 seeds. The Pioneer Hi-Bred facilities also are gratefully acknowledged for mapping and protein production.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003400. Received March 27, 2002; accepted July 10, 2002.
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INTRODUCTION
12 DAP), and peaked at 
