Aya Imamura

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

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.

An Arabidopsis protein that interacts with the cytokinin‐inducible response regulator, ARR4, implicated in the His‐Asp phosphorylay signal transduction

Previously, Arabidopsis thaliana was shown to possess a set of response regulators (ARR‐series), which are implicated in the prokaryotic type of signal transduction mechanism, generally referred to as the His‐Asp phosphorylay. Among them, ARR4 is a typical phospho‐accepting response regulator, whose expression was recently demonstrated to be rapidly induced by a cytokinin‐treatment of the plant. To gain insight into the presumed His‐Asp phosphotransfer signaling mechanism as well as the role of ARR4 in this higher plant, in this study we adopt the widely used yeast two‐hybrid system, and report the identification of an Arabidopsis protein that has an ability to interact physically with the cytokinin‐inducible ARR4 response regulator.