The Arabidopsis Abscisic Acid Response Gene ABI5 Encodes a Basic Leucine Zipper Transcription Factor

Correspondence to: Ruth R. Finkelstein, finkelst{at}lifesci.ucsb.edu (E-mail), 805-893-4724 (fax)

	ABSTRACT

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The Arabidopsis abscisic acid (ABA)–insensitive abi5 mutants have pleiotropic defects in ABA response, including decreased sensitivity to ABA inhibition of germination and altered expression of some ABA-regulated genes. We isolated the ABI5 gene by using a positional cloning approach and found that it encodes a member of the basic leucine zipper transcription factor family. The previously characterized abi5-1 allele encodes a protein that lacks the DNA binding and dimerization domains required for ABI5 function. Analyses of ABI5 expression provide evidence for ABA regulation, cross-regulation by other ABI genes, and possibly autoregulation. Comparison of seed and ABA-inducible vegetative gene expression in wild-type and abi5-1 plants indicates that ABI5 regulates a subset of late embryogenesis–abundant genes during both developmental stages.

	INTRODUCTION

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Abscisic acid (ABA) regulates many agronomically important aspects of seed development, including synthesis of storage proteins and lipids (Finkelstein and Somerville 1988 ; Rock and Quatrano 1995 ) and acquisition of desiccation tolerance and dormancy (Black 1983 ; Karssen et al. 1983 ; Koornneef et al. 1989 ). In addition, vegetative responses to ABA include induction of stomatal closure and tolerance of drought, salt, and cold stresses (reviewed in Leung and Giraudat 1998 ). Molecular studies have identified many ABA-regulated genes and an array of corresponding transcriptional regulators (reviewed in Busk and Pages 1998 ). Genetic studies, especially those with Arabidopsis plants, have identified a large number of loci involved in responses to ABA. Mutants with defects at these loci are being characterized physiologically, and as the affected genes are cloned, their products are being characterized biochemically.

To date, six genes required for wild-type ABA response have been reported cloned. These genes represent four classes of protein: two orthologous transcriptional regulators (VIVIPAROUS1 [VP1] of maize and ABA INSENSITIVE3 [ABI3] of Arabidopsis) (McCarty et al. 1991 ; Giraudat et al. 1992 ), two highly homologous members of the protein phosphatase 2C family (ABI1 and ABI2 of Arabidopsis) (Leung et al. 1994 , Leung et al. 1997 ; Meyer et al. 1994 ), a member of the APETALA2 domain family (ABI4 of Arabidopsis) (Finkelstein et al. 1998 ), and a farnesyl transferase (ENHANCED RESPONSE TO ABA1 [ERA1] of Arabidopsis) (Cutler et al. 1996 ). Two additional genes demonstrated to interact with ABI3 in regulating seed maturation, FUSCA3 (FUS3) and LEAFY COTYLEDON1 (LEC1), have been found to encode presumed transcription factors (Lotan et al. 1998 ; Luerssen et al. 1998 ). To fully describe the molecular events during ABA signaling, we need to identify the biochemical functions of many more of the genes that are required for ABA response.

The Arabidopsis abi5 mutants, like many of the other ABA-insensitive mutants, were selected on the basis of ABA-resistant germination (Finkelstein 1994 ). Initial physiologic and genetic analyses suggested that ABI5 represented a new element of a signal transduction pathway involving two other ABA response loci: ABI3 and ABI4. Mutations affecting all three loci resulted in defects in seed ABA sensitivity and seed-specific gene expression but did not alter vegetative growth. In addition, in digenic mutant analyses, the abi3-1, abi4-1, and abi5-1 mutations all greatly enhanced the ABA resistance of abi1 mutants with respect to ABA inhibition of seed germination (Finkelstein and Somerville 1990 ; Finkelstein 1994 ). In contrast, the abi3-1 abi5-1 digenic mutant was only slightly more resistant to ABA than were its monogenic parents. Recently, we have found that ABI5 function is essential for the ABA hypersensitivity conferred by ectopically expressed ABI3 (R.R. Finkelstein, unpublished observations).

To address the molecular relationship between ABI5 and other components of the ABA signal transduction pathways, we used a positional cloning approach to identify the ABI5 gene. The predicted gene product showed structural similarities to the basic leucine zipper (bZIP) class of transcriptional regulators. Expression analyses showed that, like ABI4, ABI5 is expressed in vegetative as well as seed tissues, albeit at much lower levels, and is required for some ABA-regulated gene expression in vegetative tissue. In addition, ABI5 expression appears to be regulated by ABA, by most of the other known ABI genes, and possibly by itself.

	RESULTS

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Fine Mapping ABI5
Our initial mapping of ABI5 localized it to the lower arm of chromosome 2, near the phenotypic marker pyrimidine-requiring (py) (Finkelstein 1994 ). To generate fine-mapping populations with closely linked recombinations, we outcrossed abi5-1 and abi5-3 (in the Wassilewskija and Columbia [Col] backgrounds, respectively) to lines carrying the erecta (er) and py mutations (in the Landsberg erecta [Ler] background) and screened for recombinants with these phenotypically scored markers and the molecular marker nga168. Recombinant families were subsequently scored at a series of molecular markers to identify the region of chromosome 2 that was most tightly linked to ABI5. This enabled us to fine map ABI5 to a region of ~150 kb contained within two bacterial artificial chromosomes (BACs), TAMU 19H20 and TAMU 25C22 (Fig 1). The BAC fingerprint database (http://genome.wustl.edu/gsc/arab/arabidopsis.html) revealed a match with two of the BACs being sequenced, IGF 2H17 and IGF 1011, and the sequence data were made publicly available at the Institute for Genomic Research website (http://www.tigr.org/tdb/at/atgenome/atgenome.html). Likely genes were predicted by using GenScan analysis (Burge and Karlin 1997 ) via the Massachusetts Institute of Technology server (http://CCR-081.mit.edu/GENSCAN.html). Predicted amino acid sequences were then used for BLAST (Altschul et al. 1997 ) searches of all nonredundant protein sequences to identify possible functions of the predicted genes.

View larger version (20K): [in this window] [in a new window]   Figure 1. Fine Mapping of ABI5 on Chromosome 2. We screened five distinct mapping populations and isolated 106 recombinants across a 20-centimorgan interval surrounding abi5. Mapping results with a series of molecular markers across the interval are summarized schematically, indicating that ABI5 was in a region of ~150 kb. The yeast artificial chromosome (YAC) clones indicated are from the CIC library; BAC clones are from the TAMU library. Markers shown to have >n recombinants either were not polymorphic or were scored in all mapping populations. Dashed lines denote the positions of the molecular markers used for mapping. LE, left end of BAC; RE, right end of BAC.

Of the >20 predicted genes within the region between the two closest recombinations, only one appeared to be a strong candidate: a member of the bZIP transcription factor family, now designated gene F2H17.12 (GenBank accession number AC006921.5, PID g4510349). Consequently, we focused our attention on this gene. RNA gel blot analysis showed that transcript levels for this gene were severely decreased in the abi5-1 and abi5-2 mutants (Fig 2).

View larger version (53K): [in this window] [in a new window]   Figure 2. ABI5 Transcript Levels in Wild-Type and abi5 Siliques. Each lane contains 15 µg of total RNA isolated from pooled stages of developing siliques. The hybridization probe spanned 826 bp of the ABI5 coding region, starting at codon 63 and ending before the basic domain. Col, wild-type Columbia; Ws, wild-type Wassilewskija.

Identification of ABI5
After determining that the bZIP family member described above was underexpressed in some of the abi5 mutants, we subcloned a 6-kb HindIII fragment encompassing only the bZIP gene and found that this fragment was sufficient to complement the mutation (Table 1). To determine the nature of the lesions in the mutants, we sequenced the mutant alleles. We found a single base pair change in the coding sequence of abi5-1: a G-to-T substitution at nucleotide 34,017 of IGF 2H17 (Fig 3A). This substitution resulted in an early translation termination such that the mutant protein lacked 81 C-terminal amino acids, including the conserved basic and leucine zipper domains required for DNA binding and dimerization.

View larger version (51K): [in this window] [in a new window]   Figure 3. Sequence and Domain Structure of the ABI5 Gene. (A) The sequence displayed corresponds to the reverse complement of nucleotides 33,132 to 34,991 of BAC F2H17 (GenBank accession number AC006921.5). The coding sequence is shown in capital letters, with the predicted amino acid sequence below. Introns, identified by sequencing cDNAs, and untranslated regions are in lowercase letters. The basic domain and leucine repeats of the bZIP domain are double underlined, possible kinase recognition sites are single underlined, the position of the nested primer used for rapid amplification of cDNA ends is indicated by dashes, and the locations of poly(A)+ tracts in three independent cDNAs are indicated by carets. Nucleotides altered in the mutants are underlined. The asterisk denotes the position of the stop codon. (B) Domain structure of the ABI5 protein, including the bZIP (Basic L zipper) domain, a proline-rich (P rich) region, and the positions of the defects in two mutant alleles.

In addition to the base change within the coding sequence, abi5-1 has a small duplication that can be detected by DNA gel blot analysis. The duplicated region is contained within a 1-kb BglII-PstI fragment comprising 272 bp at the 5' end of the coding sequence and an additional 758 bp extending into the promoter region (data not shown); the precise endpoints of the duplication are not known. However, the abi5-1 genomic sequence is identical to that of its progenitor line for at least 1184 bp 5' to the initiating codon, extending into a region beyond the BglII site delimiting the duplicated region. This indicates that the duplication is not immediately adjacent to the ABI5 locus.

The abi5-1 and abi5-2 mutants have identical genomic rearrangements, indicating that they are probably siblings that were redistributed into independent T-DNA pools. Another allele, abi5-3, has a small rearrangement adjacent to the 5' splice site of the final exon, extending from nucleotides 33,454 to 33,439 of IGF 2H17 (Fig 3A). Although the abi5-3 mutation results in a failure to splice the final intron, it does not reduce the transcript accumulation (Fig 2). The combination of identified sequence mutations and functional evidence from complementation indicates that this gene is indeed ABI5.

ABI5 Shows Homology to bZIP Domain Proteins
The annotated database submission and the sequence of several independent cDNA clones obtained by the 3' rapid amplification of cDNA ends technique (Frohman 1995 ) indicate that the ABI5 gene is composed of four exons that encode a 442–amino acid protein (Fig 3). The first intron separates the regions that encode the basic and leucine zipper portions of the bZIP domain; the leucine zipper is assembled from sequences spread over the next two exons (Fig 3B). The 3' ends of the cDNA clones are heterogeneous, indicating that any of several possible polyadenylation signals can be used.

Comparison of the predicted ABI5 amino acid sequence with those of other gene products in databases showed that ABI5 shares greatest sequence similarities with the bZIP class of proteins. The protein most similar to ABI5 (62% of predicted amino acids similar or identical) was a member of the Dc3–promoter binding factor family (DPBF-1) from sunflower embryos (Kim et al. 1997 ). The predicted amino acid sequence of TRAB1 (for transcription factor responsible for ABA regulation), a rice protein that interacts with VP1 (Hobo et al. 1999 ), was 55% similar to that of ABI5. Somewhat weaker similarity of predicted amino acid sequences was observed in comparisons with other members of the DPBF family (53 to 55% similar) (Kim and Thomas 1998 ), with another rice seed transcription factor (OSE2; 44% similar) (GenBank accession number U25283), with an Arabidopsis G-box binding factor thought to participate in light-regulated transcription (GBF4; 46% similar) (Menkens and Cashmore 1994 ), and with two predicted bZIP factors (50 to 59% similar) whose genes were discovered during sequencing of the Arabidopsis genome.

Whereas overall amino acid similarity with the closest homolog, DPBF-1, was only 62%, the highly conserved bZIP regions of ABI5 and DPBF-1 are 96% similar (Fig 4). This domain is thought to be involved in DNA binding and potential dimerization of bZIP transcription factors (reviewed in Hurst 1995 ). Additional conserved domains are present at both 5' and 3' of the bZIP domain, and three of six predicted serine/threonine phosphorylation sites (Woodget et al. 1986 ; Pinna 1990 ) are located in these conserved regions. Therefore, we hypothesize that the ABI5 protein is also a transcription factor and may be the Arabidopsis ortholog of DPBF-1. Consistent with this hypothesis, ABI5 is identical to AtDPBF-1, a clone that was isolated on the basis of hybridization to DPBF-1 (T. Thomas, personal communication). An ABI5 cDNA has not appeared in the Arabidopsis expressed sequence tag collection, but that is consistent with its presence as a low-abundance transcript.

View larger version (83K): [in this window] [in a new window]   Figure 4. Comparison of ABI5 and Its Two Closest Homologs. Alignment of the predicted amino acid sequences for ABI5 and its two closest homologs currently in the database, DPBF-1 and TRAB1. Alignment was done by the Pileup and PrettyBox programs. Identical residues are indicated by black boxes, and conservative substitutions are indicated in boldface. Potential phosphorylation sites are marked by asterisks; the canonical basic domain and the leucine repeats are outlined by boxes. Dashes denote positions of amino acids absent from the N termini of DPBF-1 and TRAB1.

To determine whether ABI5 is likely to have more closely related family members than those present in the sequence databases, we performed reduced stringency DNA gel blot hybridizations of genomic DNA. The hybridization probe was a region of the coding sequence that excluded the sequence encoding the highly conserved portion of the bZIP domain. This probe hybridized very weakly, regardless of stringency, to only four to seven fragments other than the one containing ABI5, indicating that few Arabidopsis genes are homologous to ABI5 beyond the region encoding the bZIP domain (data not shown). Both of the two predicted Arabidopsis genes identified by BLAST searches (GenBank accession numbers AC004261 and AL031032) contain four regions encoding conserved domains of 20 to 30 amino acids each (data not shown), which probably correspond to some of the weakly hybridizing fragments. When the Arabidopsis genome sequence is complete, it will be possible to definitively identify the closest homolog by searching the database. Whether any of the more weakly homologous family members participate in the same regulatory processes as ABI5 is not known. However, the DPBF subfamily of sunflower is also rather divergent outside the conserved basic domain, yet all members of this subfamily were identified by using a one-hybrid activation screen, indicating that they bind to the same promoter fragment and may be functionally redundant.

ABI5 Expression
As described earlier, our previous genetic and physiological studies suggested that ABI5 expression was likely to be most abundant in seeds of wild-type plants and possibly regulated by ABA, ABI3, or ABI4. To test whether accumulation of ABI5 transcripts fit these predictions, we compared ABI5 transcript levels in developing siliques and dry seeds of a variety of genotypes and in vegetative tissues of wild-type plants (Fig 5). Although the hybridization probe included some of the conserved regions of the gene, the stringency used for RNA gel blot analyses produced gene-specific hybridization during DNA gel blot analyses. We found that the ABI5 transcript is much more abundant in developing siliques than in vegetative tissue, with the greatest amounts being observed in desiccating and dry seeds (Fig 5A). A comparison of ABI5 transcript levels in dry seeds showed that accumulation is diminished, to various extents, in ABA-deficient (aba1-1) seeds and all of the abi mutant seeds tested (Fig 5B). Consistent with this is the finding that ectopic expression of ABI3 confers ABA-inducible vegetative expression of ABI5 to levels even higher than those found in wild-type siliques (Fig 5C). These results indicate cross-regulation of ABI5 expression by other known ABA response loci.

View larger version (52K): [in this window] [in a new window]   Figure 5. Expression of ABI5. (A) Developmental time course of embryonic ABI5 expression. RNA was isolated from maturation phase (M), postabscission (P), late embryogenesis (L), or dry seeds (D) of wild-type Ler plants. Each lane contains 15 µg of total RNA. (B) Comparison of ABI5 expression in seeds of ABA-deficient (aba) or ABA-insensitive (abi) mutants and their respective wild-type backgrounds. RNA was isolated from dry seeds of the indicated genotypes. Each lane contains 5 µg of total RNA. Ws, Wassilewskija. (C) ABI5 expression in siliques compared with ABI5 expression in untransformed and EN35S::ABI3 (ABI3c, for constitutive ABI3) vegetative tissue. Results with both control and ABA-treated tissue are shown. Each lane contains 15 µg of total RNA. RNA was isolated from pooled stages of developing siliques of wild-type Wassilewskija plants and from 13-day-old or 3-week-old plants. The plants used to compare Col and ABI3c were incubated with (+) or without (-) 50 µM ABA for 48 hr before harvest. Comparable expression was observed in wild-type plants of the Col and C24 ecotypes (data not shown).

ABI5-Regulated Gene Expression
Our initial characterization of the abi5-1 mutant indicated that ABI5 regulated at least one gene expressed late in embryogenesis, AtEm6 (Finkelstein 1994 ). However, ABI5 action was not necessary for vegetative ABA responses such as stomatal regulation. Having found that ABI5 encoded a member of the bZIP family of transcriptional regulators and was expressed in both vegetative tissue and seeds, we compared gene expression in wild-type and abi5-1 mutants at three ages: late embryogenesis, dry seed, and 13-day-old plants (Fig 6). Of seven LATE EMBRYOGENESIS ABUNDANT (LEA) genes assayed, three were underexpressed in abi5-1 plants (AtEm1, AtEm6, and the LeaD34 homolog), three showed little or no change (the RAB18, vicilin, and oleosin2 homologs), and one had substantially increased expression (M17) (Fig 6A). Of the two ABA-inducible genes assayed in young plants, only AtEm1 appeared to depend on ABI5 function for wild-type induction (Fig 6B). These results indicate that ABI5 is important for regulation of some but not all LEA genes and that it may act as a positive or negative regulator, depending on the target gene. In addition, the low amount of vegetative AB15 expression appears to be physiologically relevant because ABA induction of vegetative AtEm1 expression is decreased in the abi5-1 mutant.

View larger version (56K): [in this window] [in a new window]   Figure 6. ABI5-Regulated Gene Expression. RNA was hybridized sequentially with probes corresponding to genes of the identity or homology indicated at left. (A) Comparison of LEA gene expression in late embryogenesis (L; 17 to 21 DPA) and mature dry seeds (D) of wild-type (Wassilewskija [Ws]) and abi5-1 mutant plants. (B) Comparison of ABA-inducible LEA gene expression in 13-day-old wild-type (Ws) and abi5-1 plants incubated with (+) or without (-) 50 µM ABA for 48 hr before harvest.

	DISCUSSION

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Regulation of seed development and ABA signaling has been analyzed by using biochemical and genetic approaches. Many transcription factors correlated with seed-specific and ABA-responsive gene expression have been identified biochemically (reviewed in Busk and Pages 1998 ). The strategies used have included identification of products of stress-induced transcripts and identification of factors that bind to cis-elements required for seed-specific or ABA-inducible gene expression. However, no available genetic evidence indicates whether most of these are required for the correlated response or any other ABA-regulated processes. In fact, although several of the bZIP family factors show similar DNA binding specificities in vitro, they are probably involved in transducing different signals. For example, ABA-responsive elements and sequences required for light regulation both contain an ACGT core, also known as the G-box. Consequently, at least one factor (GBF3) initially identified in a biochemical screen for regulators of light-induced expression was subsequently implicated in ABA-regulated expression (Lu et al. 1996 ).

Genetic studies have identified signaling elements required for seed maturation, ABA responses, or both. Several of these have now been shown to encode probable transcription factors. These transcription factors include three members of the B3 domain family (Vp1, ABI3, and FUS3) (McCarty et al. 1991 ; Giraudat et al. 1992 ; Luerssen et al. 1998 ), a homolog of the CAAT-box binding factors (LEAFY COTYLEDON1) (Lotan et al. 1998 ), and an APETALA2 domain family member (ABI4) (Finkelstein et al. 1998 ). In this study, we report the positional cloning of the ABI5 gene, which encodes a member of the bZIP domain transcription factor family.

A comparison with other members of the bZIP family shows four large blocks and several small blocks of homology between ABI5 and its closest homologs, DPBF-1 and TRAB1 (Fig 4). However, ABI5 differs from these proteins by the presence of a 64–amino acid N-terminal peptide that shows some homology to another predicted Arabidopsis bZIP factor (GenBank accession number AL031032). It is not clear whether this peptide is truly missing from DPBF-1 and TRAB1 or whether the available predicted amino acid sequences are based on less than full-length cDNA clones. In addition to the bZIP domain, the conserved regions include a proline-rich (33% of residues 221 to 241) domain that could function in transcriptional activation and three possible casein kinase II phosphorylation sites (Pinna 1990 ) that could modulate ABI5 activity by changes in phosphorylation status. Several casein kinases have been identified from plants, and a variety of plant bZIP proteins have been shown to be either activated (e.g., GBF1) or inactivated (e.g., Opaque2) by phosphorylation of specific residues (reviewed in Schwechheimer et al. 1998 ). The conservation of the serine or threonine residues (or both) at the putative casein kinase II target sites suggests that these residues may be functionally relevant.

The leucine zipper domain of bZIP proteins is involved in dimerization, which precedes DNA binding and therefore affects the relative affinities for possible binding sites. The potential interactions of each bZIP protein are determined by the charge distribution of the ZIP region as well as the identities of the available partners in any given tissue (Hurst 1995 ). The residues present at the positions involved in stabilizing the ABI5 leucine zipper by interhelical salt bridges have a heterogeneous charge distribution, compatible with either homodimer or heterodimer formation. Consistent with function as a homodimer, the highly homologous DPBF-1 was identified by interaction with a target promoter fragment in a one-hybrid screen, where it presumably bound as a homodimer (Kim et al. 1997 ). In addition, DPBF-1 was shown to form heterodimers with DPBF-2. Identification of the ABI5 and AtDPBF gene products, in combination with the availability of the abi5-1 mutant, should allow us to determine whether ABI5 is truly the ortholog of DPBF-1 and whether the orthologs of the other DPBFs are functionally redundant in vivo, as implied by the similarity of their binding specificity.

RNA gel blot analyses (Fig 5) showed that ABI5 expression is strongest during the later stages of embryogenesis. This differs from ABI3, which is expressed at a relatively constant level throughout embryo development (Parcy et al. 1994 ) and at low levels in some vegetative tissues (Rhode et al. 1999 ). Low-level ABI5 expression was also observed in vegetative tissue. Thus, although the abi5 mutations were initially characterized as having seed-specific effects (Finkelstein 1994 ), ABI5 expression is not seed specific. Consistent with this result, we have found that ABI5 function is required for full induction of some LEA genes expressed at low levels in ABA-treated vegetative tissue. In addition, the ABA hypersensitivity conferred by ectopic ABI3 expression in vegetative tissues appears to be partially mediated by hyperinduction of ABI5 expression (Fig 5C; R.R. Finkelstein, unpublished observations). Comparison of ABI5 transcript amounts in various mutant backgrounds also supports the hypothesis that ABI5 expression is regulated by ABA and by most, if not all, of the known ABI genes. Products of three of these genes, ABI3, ABI4, and ABI5 itself, are presumed to be transcription factors (Giraudat et al. 1992 ; Finkelstein et al. 1998 ) and could regulate ABI5 expression directly. The other two genes, ABI1 and ABI2, encode members of the serine/threonine protein phosphatase 2C family (Leung et al. 1994 , Leung et al. 1997 ) and could regulate ABI5 expression by altering phosphorylation and, concomitantly, activity of these or other transcription factors. Although specific substrates for ABI1 and ABI2 have not been identified, the cloning of genes demonstrated to interact genetically with ABI1 and ABI2 should allow us to test for such interactions.

Analysis of the abi5-1 allele indicated that it encodes a truncated product lacking the bZIP domain required for dimerization and DNA binding. This suggests that the abi5-1 gene product is probably inactive unless it can interact with other classes of transcriptional regulators through any of the domains remaining in the truncated protein. However, probably very little ABI5-1 protein is present because the sibling alleles abi5-1 and abi5-2 have low abi5 transcript levels. This could reflect autoregulation, as has been documented for numerous other transcription factors (e.g., DEFICIENS; Schwarz-Sommer et al. 1992 ), or the mRNA could be destabilized as a result of poor translation (reviewed in Abler and Green 1996 ). The abi5-3 allele has a small rearrangement at the 5' splice site of the fourth exon. Sequence analysis of cDNA from the abi5-3 mutant shows that it fails to splice the third intron and thereby replaces most of the amino acids encoded by the last exon with the intron-encoded product. Although this splice defect disrupts the coding sequence, it would be unlikely to eliminate function, which is consistent with the weak phenotype of this mutant. Consequently, the strong expression of the abi5-3 allele does not rule out the possibility of autoregulation.

Unlike the severe alleles of ABI3 (e.g., abi3-4), which fail to complete seed maturation and consequently produce green, desiccation-intolerant seeds that are very insensitive to ABA (Ooms et al. 1993 ), the abi5-1 mutant has a relatively weak phenotype (Finkelstein 1994 ). abi5-1 mutant seeds are desiccation tolerant and weakly dormant, but they are only slightly resistant to ABA inhibition of germination, and their accumulation of embryonic or ABA-inducible transcripts is altered for only a subset of the transcripts. Although it is not clear why ABI5 acts as a positive regulator of some genes but negatively regulates other coordinately expressed genes, there is ample precedent for specific bZIP factors to act as both positive and negative regulators (reviewed in Hurst 1995 ). Further analysis of the promoter structures of various ABI5-regulated genes and identification of other transcriptional regulators controlling their expression could clarify the mechanism of this differential regulation.

The fact that the abi5-1 mutant has a weak phenotype, even though the abi5-1 allele is grossly underexpressed and encodes a transcription factor lacking its presumed DNA binding and dimerization domains, suggests that loss of ABI5 function may be masked by the presence of proteins having at least partially redundant activity. There are a large number of candidates for the source of this redundancy. Many bZIP factors have been implicated in ABA-regulated seed gene expression, including EmBP-1, DPBF factors, and TRAB1 (Guiltinan et al. 1990 ; Kim and Thomas 1998 ; Hobo et al. 1999 ). Other bZIP factors such as GBF3 have been implicated in ABA-regulated but not seed-specific gene expression (Lu et al. 1996 ). Conversely, the bZIP factor Opaque2 is required for endosperm-specific but not ABA-regulated zein expression (Muller et al. 1997 ); presumably, comparable factors are required for seed-specific but hormone-independent gene expression in Arabidopsis. In addition, transcription factors belonging to other protein families (e.g., ABI3, ABI4, and FUS3) have been shown to regulate many of the same genes as ABI5 (Baeumlein et al. 1994 ; Keith et al. 1994 ; Parcy et al. 1994 , Parcy et al. 1997 ; R.R. Finkelstein, unpublished observations). VP1 has been shown to interact with EmBP-1, TRAB1, and various other transcription factors (Hattori et al. 1992 ; Hill et al. 1996 ; Hobo et al. 1999 ), indicating that this coordinate regulation may involve direct interactions among disparate classes of transcription factors, as has been described for a variety of yeast and mammalian transcriptional regulators (reviewed in Wolberger 1998 ). The modular nature of promoters (e.g., Shen et al. 1996 ) also provides opportunities for different factors to bind adjacent sites independently, according to their recognition specificity. Altogether, gene expression in seeds appears to depend on the combinatorial action of a large number of transcription factors. This high degree of genetic redundancy could permit subtle variations in gene expression during seed set that might be critical for plants growing outside of the controlled environment of the laboratory.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plant Material
The abscisic acid (ABA)–insensitive abi5-1 and abi5-2 mutants were isolated from T-DNA insertion lines of the Arabidopsis thaliana ecotype Wassilewskija, as described previously (Finkelstein 1994 ); the mutations did not cosegregate with the T-DNA insertions. The abi5-3 mutant was isolated from a mutagenized population derived from fast-neutron–irradiated seeds of the A. thaliana ecotype Columbia (Col; obtained from Lehle Seeds, Round Rock, TX). The EN35S::ABI3 transgenic line (isolate C7A19), which contains an ABI3 cDNA controlled by a double-enhanced cauliflower mosaic virus 35S promoter, was constructed as described by Parcy et al. 1994 . Marker lines used for mapping were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus).

For germination assays scoring ABA sensitivity, 20 to 100 seeds per treatment were surface sterilized in 5% hypochlorite and 0.02% Triton X-100. Seeds were then rinsed three or four times with sterile water before plating on minimal medium (Haughn and Somerville 1986 ) containing 0.7% agar and ABA (mixed isomers; Sigma) at 3 or 5 µM in 15 x 100-mm Petri dishes. For scoring kanamycin resistance, the medium included 0.5 x Murashige and Skoog salts (Murashige and Skoog 1962 ), 1% sucrose, 0.05% Mes, and 50 µg/mL kanamycin. The dishes were incubated for 1 to 3 days at 4°C to break any residual dormancy, and then they were transferred to 22°C in continuous light (50 to 70 µE m^-2 sec^-1).

For DNA isolation, plants were grown in pots of soil (a 1:1:1 mix of vermiculite, perlite, and peat moss) supplemented with nutrient salts at 22°C in continuous light or in 16-hr light/8-hr dark cycles; shoots and rosette leaves were harvested when the shoots began bolting. For RNA isolation from siliques or 3-week-old plants, plants were grown as described for DNA isolation. Siliques were harvested as a pooled mixture of developmental stages spanning the full period of embryogeny (i.e., all stages from flower buds to dry seeds) or as subpools corresponding to four developmental stages: maturation (8 to 11 days postanthesis [DPA]), postabscission (12 to 16 DPA), late embryogenesis (17 to 21 DPA), and dry seed (>21 DPA). Three-week-old plants were sprayed to runoff with 0.05% Triton X-100 supplemented with 0 or 50 µM ABA and then incubated another 2 days before harvest. Seedlings were grown aseptically on Murashige and Skoog medium (Murashige and Skoog 1962 ) containing 1% sucrose and 0.55% agar for 11 days at 22°C in continuous light (50 to 70 µE m^–2 sec^–1); then they were transferred to fresh Murashige and Skoog medium containing 1% sucrose, 0.7% agar, and 0 or 50 µM ABA for an additional 2 days before harvest. All tissues harvested for nucleic acid extraction were weighed, frozen in liquid nitrogen, and stored frozen at –70°C until extracted.

Isolation of Recombinant Plants
The abi5 mutants were outcrossed to a Landsberg erecta (Ler) marker line carrying the er and py mutations. Mapping populations were produced by selecting ABA-insensitive F₂ progeny and then screening the resulting F₃ families for recombinations with the markers er and py, which could be scored phenotypically. In addition, F₂ progeny of the abi5-3 outcross were screened for recombination with nga168, a simple sequence length polymorphism that could be scored using polymerase chain reaction (PCR) (Bell and Ecker 1994 ). To allow direct selection of recombinants between the ABI5 and PY loci, we backcrossed an abi5-1 py recombinant to wild-type Ler; ABA-insensitive F₂ progeny that were viable without a thiamine supplement were abi5 PY recombinants. Those with recombinations tightly linked to ABI5 were identified by scoring their genotypes with the cleaved amplified polymorphic sequence (CAPS) marker m323a.

Restriction Fragment Length Polymorphism Analysis
Restriction fragment length polymorphism mapping was conducted with F₃ and F₄ recombinant families. Plant DNA was extracted (Jhingan 1992 ), and ~2 µg was digested with an appropriate enzyme to distinguish between the parental DNAs. The digested DNA was fractionated according to size on an 0.8% agarose gel, denatured, and transferred to Zeta Probe (Bio-Rad) membranes as described previously (Finkelstein 1993 ). Mapping probes included cosmid clones obtained from the Arabidopsis Biological Resource Center and bacterial artificial chromosome (BAC) ends derived from plasmid rescue or inverse PCR for right and left ends, respectively (Woo et al. 1994 ). Cosmid and plasmid DNA was isolated as described by Sambrook et al. 1989 . DNA templates were labeled by random priming (Hodgson and Fisk 1987 ). Filters were hybridized in 5 x SSPE (1 x SSPE is 0.15 M NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4), 5 x Denhardt's solution (1 x Denhardt's solution is 0.02% each of Ficoll, PVP, and BSA), 0.5% SDS, and 200 µg/mL herring testes DNA (Sambrook et al. 1989 ) with a probe added at 10⁶ to 4 x 10⁶ cpm/mL.

CAPS Analysis
Two CAPS markers were used to fine map ABI5: m323a and veo17a. Map positions of these markers, as well as the primers and enzymes used to score them, were as described by E. Drenkard and F. Ausubel (http://www.arabidopsis.org/aboutcaps.html). Reaction and cycling conditions were as described by Konieczny and Ausubel 1993 .

Simple Sequence Length Polymorphism Analysis
The microsatellite sequence nga168 (Bell and Ecker 1994 ) is polymorphic between the Col and Ler ecotypes, which are the genetic backgrounds for the abi5-3 mutant and the marker line, respectively. DNA from F₂ individuals (10 to 50 mg of leaf tissue per plant) and F₃ or F₄ families was used as a template in PCR amplification of the satellite sequences. PCR mixtures contained ~10 ng of DNA, 2.5 pmol of each primer (MapPairs; Research Genetics, Huntsville, AL), 10 mM Tris, pH 9.0, 50 mM KCl, 2 mM MgCl₂, 0.01% gelatin, 0.1% Triton X-100, 200 µM deoxynucleotide triphosphates, and 0.25 units of Taq polymerase in a 10-µL reaction volume. Reaction products were size-fractionated by electrophoresis through a 6% polyacrylamide-Tris-borate-EDTA gel (Sambrook et al. 1989 ).

Construction of Clones for Complementation Studies
The 6-kb HindIII fragment from a complete HindIII digest of TAMU 19H20 was isolated from a gel and then purified with a QIAQuick Gel extraction kit (Qiagen, Chatsworth, CA). The pBIN19 (Bevan 1984 ) binary plasmid vector DNA (~2 µg) was digested with HindIII and dephosphorylated. The digested BAC DNA was ligated to the dephosphorylated vector DNA and transformed into Escherichia coli DH5 cells. Transformants were selected on kanamycin/X-gal/Luria-Bertani agar plates; white colonies were screened for the presence of appropriate inserts by restriction mapping of plasmid DNA.

Construction of Transgenic Plants
abi5-1 plants were grown at a density of five to 10 plants per 5-inch pot under photoperiods of 14 hr of light and 10 hr of darkness to produce large, leafy plants. Plants were vacuum-infiltrated with an Agrobacterium tumefaciens culture carrying an appropriate plasmid essentially as described by Bent et al. 1994 . Seeds were harvested from individual pots and plated on selection medium (0.5 x Murashige and Skoog salts, 1% sucrose, and 50 µg/mL kanamycin) to identify transgenic progeny. ABA sensitivity and antibiotic resistance were scored in the next generation.

RNA Isolation and Gel Blot Analysis
RNA was isolated from dry seeds, 13-day-old plants, and 3-week-old plants by hot phenol extraction as described previously (Finkelstein et al. 1985 ). Silique RNA was isolated by grinding the siliques to a fine powder in liquid nitrogen, followed by incubation for 1 hr at 37°C in 3 to 5 mL of extraction buffer (0.2 M Tris, pH 9.0, 0.4 M NaCl, 25 mM EDTA, 1% SDS, 5 mg/mL polyvinylpolypyrrolidone, and 0.5 mg/mL proteinase K) per gram of tissue. Proteins and polysaccharides were precipitated by incubation on ice with 18.3 mg/mL BaCl₂ and 150 mM KCl. After the mixture was cleared by a 10-min centrifugation at 9000g, RNA was isolated from the supernatant by LiCl precipitation. The pellets were washed in 2 M LiCl and then resuspended and reprecipitated with ethanol and sodium acetate before a final resuspension in Tris-EDTA. The RNA concentration was estimated from the absorbance of the suspension at 260 and 280 nm.

Total RNA (5 to 15 µg per lane) was size-fractionated on 1% agarose Mops–formaldehyde gels (Sambrook et al. 1989 ) and then transferred to Nytran (Schleicher and Schuell) membranes with 20 x SSPE used as blotting buffer. RNA was bound to the filters by UV cross-linking (120 mJ cm^-2 at 254 nm). Uniformity of loading and transfer was assayed qualitatively by methylene blue staining of the filters (Herrin and Schmidt 1988 ). The ABI5 mRNA was detected by hybridization with a PCR product corresponding to ~60% of the 5' part of the coding sequence (nucleotides 33,878 to 34,703 of IGF 2H17; GenBank accession number AC006921.5) and labeled by random priming to a specific activity of 10⁸ cpm/µg (Hodgson and Fisk 1987 ). Hybridization conditions included incubation in 7% SDS, 0.5 M sodium phosphate, pH 7.2, 1 mM EDTA, and 1% BSA for 16 to 24 hr at 65°C (Church and Gilbert 1984 ) in a rotisserie oven (Hyb-Aid, Teddington, UK), with probe being added at 2 x 10⁶ to 4 x 10⁶ cpm/mL. Washing was performed with 40 mM sodium phosphate, pH 7.2, 5% SDS, and 1 mM EDTA, and then with 40 mM sodium phosphate, pH 7.2, 1% SDS, and 1 mM EDTA, with a final wash in 0.2 x SSC (1 x SSC is 0.015 M NaCl and 0.015 M sodium citrate) and 0.1% SDS. Each wash step was performed for 10 to 60 min at 65°C. Exposure times were 2 to 8 days. The LEA gene transcripts were detected by hybridization with cDNA clones designated PAP085 (vicilin homolog), PAP140 (LeaD34 homolog), PAP147 (oleosin2 homolog), PAP023 (RAB18 homolog), and AtEm1 (generous gifts of M. Delseny), and a genomic clone comprising AtEm6 (Finkelstein 1993 ). Hybridization conditions for the LEA transcript analyses were either as given above or were 50% formamide, 5 x SSPE, 5 x Denhardt's solution, 0.1% SDS, and 200 µg/mL DNA at 43°C in a Hyb-Aid rotisserie oven. Filters were washed twice at 60°C in 2 x SSC and 0.1% SDS and once at 60°C in 0.2 x SSC and 0.1% SDS for 10 to 60 min each.

DNA Sequence Analysis
Plasmid DNA containing cDNA clones was isolated by using QIAprep Spin Miniprep kits (Qiagen) and then dissolved in double-distilled water to be used as DNA templates for sequencing. PCR primers were designed to amplify 5', internal, and 3' regions of the ABI5 gene from genomic DNA of the mutant alleles and their progenitor lines. DNA was sequenced on a model 310 DNA sequencer (ABI Prism; ABI, Foster City, CA) using BigDye Terminator mix (ABI).

Comparison of Predicted Amino Acid Sequences for ABI5 and Closely Related Basic Leucine Zipper Proteins
Predicted amino acid sequences for the indicated proteins were compared using the Pileup and Gap programs of the Genetics Computer Group (Madison, WI) sequence analysis software package. Overall percentage similarity to ABI5 was calculated using the Gap program. GenBank accession numbers for the genes compared are given in parentheses: ABI5 (AC006921), DPBF-1 (AF001453), TRAB1 (AB023288), DPBF-2 (AF001454), DPBF-3 (AF061870), OSE2 (U25283), and GBF4 (P42777).

3' Rapid Amplification of cDNA Ends
Total RNA (5 µg) was used as the template for reverse transcription with a kit for 3' rapid amplification of cDNA ends (Gibco BRL). After first-strand synthesis and RNase H treatment, the cDNAs were amplified by using the universal adapter primer and a gene-specific primer annealing 51 nucleotides 5' to the start of the ABI5 open reading frame. Amplified products were size-fractionated on a 1% agarose gel, blotted, and hybridized with an internal fragment of the ABI5 gene. A nested primer, annealing 23 nucleotides 5' to the presumed initiating codon, was used to amplify cDNAs from a plug of sized DNA corresponding to the region of hybridization. The amplification reactions contained 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl₂, 200 nM each primer, 200 µM each deoxynucleotide triphosphate, and 0.05 units/mL Taq polymerase. The polymerase was added after a 3-min incubation at 94°C for "hot start" amplification. Cycling conditions for the first round of amplification were 30 cycles of 45 sec at 94°C, 1 min at 46°C, and 2 min at 72°C. Cycling conditions for the nested amplifications used a 57°C annealing step but were otherwise identical to those in the first round.

The cDNA ends were blunted by fill-in reactions with the Klenow fragment of DNA polymerase I, treated with T4 polynucleotide kinase as described by Sambrook et al. 1989 , and then gel-purified with QIAQuick Gel extraction kit (Qiagen) according to the manufacturer's instructions. The cDNAs were ligated into pBluescript KS+ (Stratagene, La Jolla, CA) after digestion with EcoRV and dephosphorylation and then transformed into E. coli DH5. Transformants were selected on ampicillin/X-gal/Luria-Bertani agar plates; white colonies were screened for the presence of appropriate inserts by restriction mapping of plasmid DNA.

	ACKNOWLEDGMENTS

We thank Dr. Douglas Bush for critical review of the manuscript, the Institute for Genomic Research for providing rapid public access to genomic sequence data, Dr. Jerome Giraudat for providing the EN35S::ABI3 transgenic line, Dr. Michel Delseny for providing cDNA clones used in gene expression studies, and the Arabidopsis Biological Resource Center and Drs. Howard Goodman and Ming-Li Wang for BAC clones used for mapping and subcloning. This work was supported by grants to R.R.F. from the National Science Foundation (No. DCB-9105241) and the U.S. Department of Agriculture (No. 95-37304-2217).

Received February 3, 2000; accepted February 28, 2000.

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