His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.

In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-to-Asp phosphorelay mechanisms are involved presumably in propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we identified and characterized a set of novel Arabidopsis genes whose products considerably resemble the authentic response regulators (ARR-series) of Arabidopsis in the sense that they have a phospho-accepting receiver-like domain. However, they should be discriminated from the classical ones in the strict sense that they lack the invariant phospho-accepting aspartate site. They were thus named APRRs (Arabidopsis pseudo-response regulators). Two such representatives, APRR1 and APRR2, were characterized extensively through cloning of the corresponding cDNAs, in terms of their structural designs, biochemical properties, subcellular localization in plant cells, and expression profiles at the transcriptional level. The result of in vitro phosphorylation experiment with the Arabidopsis AHP phosphotransmitter suggested that the pseudo-receivers have no ability to undergo phosphorylation. The result of transient expression assay with onion epidermal cells showed that the GFP-APRR1 fusion protein has an ability to enter into the nuclei. The C-terminal domain of APRR1, termed CONSTANS-motif, appears to be responsible for the nuclear-localization. The most intriguing result was that the accumulation of APRR1 transcript is subjected to a circadian rhythm. The APRR1 protein is identical to the one that was recently suggested to interact with the ABI3 (ABISCISIC ACID INSENSITIVE3) protein. These are discussed with special reference to the His-to-Asp phosphorelay signal transduction and circadian rhythm in Arabidopsis thaliana.

The His to Asp phosphotransfer signal transduction mechanism involves three common signaling domains: the transmitter (or His-kinase), the receiver, and the histidine-containing phototransfer (HPt) domain. Typically, a sensor kinase has a His-kinase domain and a response regulator has a receiver domain containing a phosphoaccepting aspartate, whereas a histidine-containing phototransfer domain serves as a mediator of the histidine-to-aspartate phosphotransfer. This signal transduction mechanism was thought to be restricted to prokaryotes. However, many examples have been discovered in diverse eukaryotic species including higher plants. In Arabidopsis, three sensor kinases have been characterized, namely, ETR1, ERS, and CKI1, which were suggested to be involved in ethylene-and cytokinin-dependent signal transduction pathways, respectively. To date, no response regulator has been discovered in higher plants. We identify five distinct Arabidopsis response regulator genes, each encoding a protein containing a receiver-like domain. In vivo and in vitro evidence that ARRs can function as phosphoaccepting response regulators was obtained by employing the Escherichia coli His-Asp phosphotransfer signaling system.

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.z 1998 Federation of European Biochemical Societies.