Molecular Structure of the GARP Family of Plant Myb-Related DNA Binding Motifs of the Arabidopsis Response Regulators

Abstract

The B motif is a signature of type-B response regulators (ARRs) involved in His-to-Asp phosphorelay signal transduction systems in Arabidopsis. Homologous motifs occur widely in the GARP family of plant transcription factors. To gain general insight into the structure and function of B motifs (or GARP motifs), we characterized the B motif derived from a representative ARR, ARR10, which led to a number of intriguing findings. First, the B motif of ARR10 (named ARR10-B and extending from Thr-179 to Ser-242) possesses a nuclear localization signal, as indicated by the intracellular localization of a green fluorescent protein-ARR10-B fusion protein in onion epidermal cells. Second, the purified ARR10-B molecule binds specifically in vitro to DNA with the core sequence AGATT. This was demonstrated by several in vitro approaches, including PCR-assisted DNA binding site selection, gel retardation assays, and surface plasmon resonance analysis. Finally, the three-dimensional structure of ARR10-B in solution was determined by NMR spectroscopy, showing that it contains a helix-turn-helix structure. Furthermore, the mode of interaction between ARR10-B and the target DNA was assessed extensively by NMR spectroscopy. Together, these results lead us to propose that the mechanism of DNA recognition by ARR10-B is essentially the same as that of homeodomains. We conclude that the B motif is a multifunctional domain responsible for both nuclear localization and DNA binding and suggest that these insights could be applicable generally to the large GARP family of plant transcription factors.

Multiple Type-B Response Regulators Mediate Cytokinin Signal Transduction in Arabidopsis

“…For example, several members of subfamily I (ARR1, ARR2, ARR10, and ARR11) can bind specific DNA sequences and enhance transcription (Sakai et al, 2000(Sakai et al, , 2001Hosoda et al, 2002;Imamura et al, 2003). In addition, studies of subcellular localization indicate that members of subfamily I (ARR1, ARR2, ARR10, and ARR12) are localized to the nucleus (Sakai et al, 2000;Hwang and Sheen, 2001;Imamura et al, 2001;Lohrmann et al, 2001;Hosoda et al, 2002;Mason et al, 2004). Among the transcriptional targets for the type-B ARRs are members of the type-A ARR family.…”

“…Type-A ARRs contain a central receiver domain along with short N-and C-terminal extensions and are transcriptionally induced by cytokinin. Type-B ARRs contain a receiver domain followed by a DNA binding domain and, based on several lines of evidence, function as transcription factors (Sakai et al, 2000;Lohrmann et al, 2001;Hosoda et al, 2002).…”

Characterization of a nitrate-inducible transcriptional repressor NIGT1 provides new insights into DNA recognition by the GARP family proteins

“…Structural analysis of an Arabidopsis ARR-B protein, ARR10, by NMR spectroscopy has shown that its GARP domain contains a helix-turnhelix structure and binds to an AGATT sequence. 10 Through comparisons of the DNA sequences of the target promoters of GLK1 and GLK2, the binding sequence for these two GARP proteins was suggested to be CCAATC. 8 The ARR10-and GLK1/2-binding sequences are similar as they both contain an AATC sequence (in the case of ARR10, this is present in the complementary strand).…”

Of Blades and Branches: Understanding and Expanding the Arabidopsis Ad/Abaxial Regulatory Network through Target Gene Identification

“…KANADI proteins belong to the GARP subfamily of MYB domain proteins (Hosoda et al 2002). These proteins have a single DNA binding MYB repeat.…”

“…A small handful of genes to date are known to control the development of ad/abaxial polarity in the leaf (reviewed by Husbands et al 2009). Genes required for adaxial leaf development are three of the five HD-ZIPIII genes (REVOLUTA, PHABULOSA, and PHAVOLUTA) and the ASYMMETRIC2 gene.…”