ABR1, an APETALA2-domain transcription factor that functions as a repressor of ABA response in Arabidopsis

Abstract

The plant hormone abscisic acid (ABA) plays an important role in plant development and stress responses. An important step of ABA action is activation or inactivation of gene expression. Although several transcription factors are identified to function as positive regulators of ABA-induced gene expression, little is known about the negative regulators of ABA-regulated gene expression. Here, we have identified an APETALA2 (AP2) domain transcription factor that serves as a repressor of ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis (Arabidopsis thaliana). The expression of the AP2-like ABA repressor 1 (ABR1) gene itself was responsive to ABA and stress conditions including cold, high salt, and drought. Disruption of ABR1 led to hypersensitive response to ABA in seed germination and root growth assays. The mutant plants were also hypersensitive to osmotic stress conditions, such as high salt and high concentrations of mannitol. Further analyses indicated that increased stress sensitivity may result from hypersensitivity to ABA as ABA biosynthesis inhibitor rescued the stress hypersensitivity phenotype. The abr1 mutant plants accumulated significantly higher levels of ABA- and stress-inducible gene transcripts as compared to the wild-type plants, supporting the hypothesis that this AP2 domain protein serves as a repressor of ABA-regulated gene expression.

Figure 1.

Expression pattern of the ABR1 gene. A, RT-PCR analysis of ABR1 transcripts during seed germination and in different organs of Arabidopsis plants. Total RNA was isolated from various tissues and stages (root, rosette leaf, cauline leaf, flower, silique, stem, and dry seeds) of wild-type plants grown under long-day conditions or from germinating seeds and young seedlings (2, 5, and 21 d after sowing). Thirty-five cycles of RT-PCR were performed with ABR1-specific primers (top gel) or Actin2-specific primers (bottom gel). B, Four-day-old seedlings grown on MS medium were treated with ABA (100 μm), NaCl (300 mm), cold (4°C), mannitol (400 mm), and drought (dehydration), and 25 cycles of RT-PCR analyses performed with ABR1 gene-specific primers. Actin2-specific primers were used for control RT-PCR analyses.

Figure 2.

Isolation of the abr1 T-DNA insertional mutants. A, Intron-exon organization of the Arabidopsis ABR1 gene (coding region) and T-DNA locations. Gray shaded boxes indicate untranslated regions, and solid black boxes and line indicate coding regions and intron, respectively. The position of the T-DNA insertion in two alleles, abr-1 and abr1-2, are indicated by triangles (not to scale). B, RT-PCR analysis of ABR1 transcripts in wild-type (Col-0) and mutant (abr1-1 and abr1-2) alleles. Expression of Actin2 was analyzed as a loading control.

Figure 3.

Germination of abr1 mutant seeds is hypersensitive to ABA, Glc, and osmotic stress conditions. Wild-type (Col-0), abr1-1, and abr1-2 mutant seeds on MS agar medium or MS agar medium containing 0.7 μm ABA, 125 mm NaCl, 375 mm mannitol, or 4% Glc were incubated at 4°C for 6 d before transfer to 23°C for germination. The photograph was taken on day 12 after transfer to 23°C.

Figure 4.

Germination time courses (days after incubation at 23°C) on medium containing 0.7 μm ABA, 150 mm NaCl, 350 mm mannitol, or 4% Glc. Wild-type (Col-0) and abr1-1 and abr1-2 mutant alleles are represented as black squares, black circles, and white circles, respectively. Results in A to D are presented as average values with standard errors from three experiments.

Figure 5.

Effect of the ABA biosynthesis inhibitor NF on germination. Germination of wild-type and abr1 mutant seeds on medium containing 150 mm NaCl or 400 mm mannitol with or without 100 μm NF. Seed germination rate was scored 3 d after transfer to a 23°C growth chamber.

Figure 6.

Postgermination root growth in abr1 mutants was hypersensitive to ABA. Three-day-old seedlings of Col-0 and abr1 mutant alleles were transferred from MS medium to MS medium supplemented with various concentrations of ABA. The root length was measured 2 weeks after the transfer. Results presented as average values with standard errors from three experiments.

Figure 7.

Expression of stress-responsive genes in wild-type (Col-0) and abr1 mutants (abr1-1 and abr1-2) after ABA treatment. Four-day-old seedlings grown on MS medium were treated with ABA (100 μm), and 25 cycles of semiquantitative RT-PCR analyses were performed with respective stress marker gene-specific primers. Actin2-specific primers were used in the RT-PCR analysis as an internal quantitative control.

Figure 8.

Expression of stress-responsive genes in wild-type (Col-0) and abr1 (abr1-1 and abr1-2) mutants after NaCl treatment. Semiquantitative RT-PCR analyses (25 cycles) with RNA isolated from NaCl-treated plants. Actin2-specific primers were used in the RT-PCR analysis as internal quantitative control. Experiments were repeated three times and results from one representative experiment are shown.