Arabidopsis Response Regulator, ARR22, Ectopic Expression of Which Results in Phenotypes Similar to the wol Cytokinin-Receptor Mutant

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Abstract
Arabidopsis thaliana has a number of response regulators (ARRs) implicated in the histidine (His)→aspartate (Asp) phosphorelay signal transduction. According to the current consistent model, both the type-A and type-B ARR family members play crucial roles in the cytokinin signaling circuitry. However, this higher plant has a few extra ARRs, on which no attention has been paid so far. Characterization of these extra ARRs might provide us with new insight into the His→Asp phosphorelay signal transduction in plants. For this reason, in this study we extensively examined the natures of such a representative (named ARR22). Transcripts of ARR22 were expressed predominantly in reproductive organs, and a GFP::ARR22 fusion protein was localized in the cytoplasmic space in onion epidermal cells. The purified ARR22 protein had the ability to undergo phosphorylation in vitro, when incubated with phospho-AHP5, indicating that ARR22 has the fundamental ability to participate into a His-Asp phosphorelay pathway in its own right. In plants, transgenic lines overexpressing ARR22 were characterized (referred to as ARR22-ox), which showed the characteristic dwarf phenotypes with poorly developed root systems. The results of Northern blot hybridization with selected sets of hormone-responsive genes suggested that cytokinin responses are selectively attenuated in ARR22-ox, while other hormone responses (auxin, ABA and ethylene) occur normally. The results of microarray analyses with cytokinin-treated wild-type and ARR22-ox plants further supported the view that cytokinin responses are globally attenuated in ARR22-ox, at least, at the level of gene regulation. Finally, we demonstrated that the dwarf phenotypes of ARR22-ox are very similar to those of the wooden leg (wol) mutant, which has a severe lesion in the AHK4/CRE1 cytokinin-receptor of histidine protein kinase. These results suggested that ARR22 might also be implicated, directly or indirectly, in the cytokinin-responsive His→Asp phophorelay signal transduction.