Arabidopsis ABI5 subfamily members have distinct DNA-binding and transcriptional activities

Abstract

A small family of novel basic leucine zipper proteins that includes abscisic acid (ABA)-INSENSITIVE 5 (ABI5) binds to the promoter region of the lea class gene Dc3. The factors, referred to as AtDPBFs (Arabidopsis Dc3 promoter-binding factors), were isolated from an immature seed cDNA library. AtDPBFs bind to the embryo specification and ABA-responsive elements in the Dc3 promoter and are unique in that they can interact with cis-elements that do not contain the ACGT core sequence required for the binding of most other plant basic leucine zipper proteins. Analysis of full-length cDNAs showed that at least five different Dc3 promoter-binding factors are present in Arabidopsis seeds; one of these, AtDPBF-1, is identical to ABI5. As expected, AtDPBF-1/ABI5 mRNA is inducible by exogenous ABA in seedlings. Despite the near identity in their basic domains, AtDPBFs are distinct in their DNA-binding, dimerization, and transcriptional activity.

Figure 1

Alignment and sequence relationships of the ABI5 subfamily members. A, Deduced amino acid sequences of ABI5 subfamily members were aligned with ClustalW (http://clustalw.genome.ad.jp/); the aligned regions including the bZIP domain are shown. The basic domains are in bold, and the Leu residues defining the Leu zipper are in red. B, Sequence relationships among the ABI5 bZIP subfamily. Results of ClustalW were used to generate the dendrogram. ABI5/AtDPBF-1, AC006921/AF334206; AtDPBF-2, AF334207; AtDPBF-3/AREB3, AF334208/AB017162; AtDPBF-4, AF334209; AtDPBF-5/ABF3, AF334210/AF093546; ABF1, AF093544; ABF2/AREB1, AF093545/AB017160; ABF3, AF093546; ABF4/AREB2, AF093547/017161.

Figure 2

Expression pattern of AtDPBFs. A, RNA gel blot analysis of AtDPBF transcripts. Ten micrograms of total RNA from various tissues was run on 1.2% (w/v) agarose/formaldehyde gels, transferred to nylon membranes, and hybridized with gene-specific probes. St, Stems; Si, silique coats; Se, 3- to 5-DAF seeds; S1, 1-DAF seeds; R, root; L, leaf; F, flower. B, ABA inducibility of AtDPBF-1. Poly(A⁺) RNA from ABA-treated Arabidopsis seedlings (1.5 μg) was run on an agarose/formaldehyde gel, transferred to a nylon membrane, and hybridized to an AtDPBF-1 probe. The numbers indicate the duration of ABA treatment in hours. Probes, Upper, AtDPBF-1; lower, EF1α.

Figure 3

Binding of AtDPBFs to the Dc3 PPR in vitro. Binding of AtDPBFs was examined by a mobility shift assay employing in vitro translation products of AtDPBFs and Dc3 PPR as a probe. P, Probe only; FP, free probe; pCITE, in vitro translation product of the pCITE expression vector.

Figure 4

Heterodimerization of AtDPBFs. Dimerization between AtDPBFs was examined by EMSAs, using cotranslation products of AtDPBFs and the Dc3 PPR. A, Dimerization between AtDPBF-1, -3, and -4. B, Dimerization between AtDPBF-2 and other AtDPBFs. Numbers above each lane indicate corresponding AtDPBFs. d, Deletion constructs of AtDPBFs (see text and “Materials and Methods”). Shifted bands with intermediate mobility are highlighted by arrows. FP, Free probe.

Figure 5

Transcriptional activation function of AtDPBFs. Individual AtDPBFs were fused to a GAL4 DNA-binding domain in a yeast expression vector pGBT9 and the resulting constructs were introduced into a yeast strain harboring a lacZ reporter gene fused to GAL4-binding sites. The reporter activity was monitored by a filter lift assay.