Dual DNA binding property of ABA insensitive 3 like factors targeted to promoters responsive to ABA and auxin Ronita Nag, Manas Kanti Maity and Maitrayee DasGupta* Department of Biochemistry, Calcutta University, 35, Ballygunge Circular Road, 700019, Calcutta (*author for correspondence; e-mail maitrayee_d@hotmail.com or ronitanag@rediffmail.com) Received 14 June 2005; accepted in revised form 25 July 2005 Key words: ABA signaling, abscisic acid insensitive 3, Arachis hypogea, auxin signaling, PvAlf Abstract The ABA responsive ABI3 and the auxin responsive ARF family of transcription factors bind the CATGCATG (Sph) and TGTCTC core motifs in ABA and auxin response elements (ABRE and AuxRE), respectively. Several evidences indicate ABI3s to act downstream to auxin too. Because DNA binding domain of ABI3s shows significant overlap with ARFs we enquired whether auxin responsiveness through ABI3s could be mediated by their binding to canonical AuxREs. Investigations were undertaken through in vitro gel mobility shift assays (GMSA) using the DNA binding domain B3 of PvAlf (Phaseolus vulgaris ABI3 like factor) and upstream regions of auxin responsive gene GH3 ()267 to )141) and ABA responsive gene Em ()316 to )146) harboring AuxRE and ABRE, respectively. We demonstrate that B3 domain of PvAlf could bind AuxRE only when B3 was associated with its flanking domain B2 (B2B3). Such strict requirement of B2 domain was not observed with ABRE, where B3 could bind with or without being associated with B2. This dual specificity in DNA binding of ABI3s was also demonstrated with nuclear extracts of cultured cells of Arachis hypogea. Supershift analysis of ABRE and AuxRE bound nuclear proteins with antibodies raised against B2B3 domains of PvAlf revealed that ABI3 associated complexes were detectable in association with both cis elements. Competition GMSA confirmed the same complexes to bind ABRE and AuxRE. This dual specificity of ABI3 like factors in DNA binding targeted to natural promoters responsive to ABA and auxin suggests them to have a potential role in conferring crosstalk between these two phytohormones. Abbreviations: ABA, abscisic acid; ABI3, ABA insensitive 3; BAP, benzylamino purine; DBD, DNA binding domain; FUS3, FUSCA3; NAA, 1-naphthaline acetic acid; PvAlf, Phaseolus vulgaris ABI3 like factor; RAV1, related to ABI3/VP1; VP1, viviparous 1 Introduction Phytohormone responses are emerging to resemble complex interaction webs rather than simple linear pathways that connect inputs and outputs (Moller and Chua, 1999; Gazzarrini and McCourt, 2003). The stress hormone ABA and the growth hormone auxin are involved in a wide variety of develop- mental processes throughout the lifecycle of higher plants (Kende and Zeevart, 1997) and several overlaps between the signaling pathways working downstream to these two phytohormones provide strong evidence in favor of existence of such networks. Recent evidences have suggested a role for ABA insensitive 3 (ABI3) family of transcription factors in auxin–ABA interactions (Suzuki et al., 2001; Brady et al., 2003). These factors are mainly implicated for imposition of dormancy in seeds but are also detectable in various quiescent tissues in the whole plant (McCarty et al., 1991; Parcy et al., Plant Molecular Biology (2005) 59:821–838 Ó Springer 2005 DOI 10.1007/s11103-005-1387-z