The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor

Abstract

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.

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.

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.

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.

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.

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 (ABI3^c, 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 ABI3^c 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).

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.