The type-A response regulator,ARR15, acts as a negative regulator in the cytokinin-mediated signal transduction in Arabidopsis thaliana

The Arabidopsis thaliana AHK4 histidine kinase (also known as CRE1 or WOL) acts as a cytokinin signal transducer, presumably, in concert with downstream components, such as histidine-containing phosphotransfer factors (AHPs) and response regulators (ARRs), through the histidine-to-aspartate (His-->Asp) phosphorelay. Among 10 members of the type-A ARR family, the cytokinin-induced expression of ARR15 in roots is selectively impaired in the cre1-1 mutant, which carries a mutation in the AHK4 gene, suggesting a link between this type-A response regulator and the AHK4-mediated cytokinin signal transduction in roots. To address this issue further, we characterized a T-DNA insertion mutant of ARR15, and also constructed transgenic lines (referred to as ARR15-ox) that overexpress the ARR15 gene in a manner independent of cytokinin. While the T-DNA insertion mutant (arr15-1) showed no apparent phenotype, the cytokinin-independent overexpression of ARR15 in ARR15-ox plants resulted in a reduced sensitivity toward exogenously applied cytokinin, not only in elongation of roots in plants, but also in green callus formation (or shoot formation) in explants. Cytokinin-induced expressions of certain type-A ARRs were also down-regulated in ARR15-ox plants. These results support the view that ARR15 acts as a repressor that mediates a negative feedback loop in the cytokinin and AHK4-mediated His-->Asp phosphorelay.     

Two types of putative nuclear factors that physically interact with histidine-containing phosphotransfer (Hpt) domains, signaling mediators in His-to-Asp phosphorelay, in Arabidopsis thaliana

The higher plant, Arabidopsis thaliana, has a large number of genes, each of which encodes a component of His-to-Asp phosphorelay signal transduction systems. One type of such signal transducers are the histidine-containing phosphotransmitters (termed AHPs), which presumably mediate His-to-Asp phosphorelay. Here we attempted to isolate a factor or factors that interact with AHP1, AHP2 and AHP3 by means of a yeast two-hybrid system. This allowed us to identify two types of nuclear-localizing proteins. They are the members of the type-B family of response regulators (specifically, ARR1, APP2 and ARR10), and a novel protein named TCP10. The binding of ARR1 to AHP2 was also confirmed by in vitro binding assays. Moreover, dephosphorylation of AHP2 was observed in a manner dependent on ARR in vitro. A subset of AHPs appeared to also interact with a protein that contains a TCP domain, a recently proposed basic helix-loop-helix motif. Because several factors carrying the TCP domain have been implicated in the regulation of growth and development in lateral organs, the binding of TCP10 to this subset of AHPs suggests a possible linkage between the His-to-Asp phosphorelay systems and plant growth regulation.     

Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction.

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.     

l-Serine Production Improved by Analogue-resistant Mutants of a Methanol-utilizing Bacterium

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

Cellular localization of the signaling components of Arabidopsis His-to-Asp phosphorelay.

In the higher plant, Arabidopsis thaliana, histidine-to-aspartate (His-to-Asp) phosphorelay signal transduction systems play crucial roles in propagation of environmental stimuli, including plant hormones. This plant has 11 sensor His-kinases, 5 histidine-containing phosphotransfer (HPt) factors (AHPs), and 20 response regulators (ARRs). To gain new insight into the functions of these phosphorelay components, their intracellular localization was examined with use of GFP-fusion proteins, constructed for certain representatives of HPt factors (AHP2) and type-A and type-B ARRs (ARR6/ARR7 and ARR10, respectively). The results showed that AHP2 is mainly located in the cytoplasmic space, while both the types of ARRs have an ability to enter preferentially into the nuclei, if not exclusively. Together with the results from an in vitro phosphorelay assay with AHP2 and ARRs, these results are discussed, in terms of a geneal framework of the Arabidopsis His-to-Asp phosphorelay network.     

The Arabidopsis sensor His-kinase, AHk4, can respond to cytokinins

His-to-Asp (His-->Asp) phosphorelay mechanisms are presumably involved in propagation of certain environmental stimuli, including phytohormones, in Arabidopsis thaliana. In addition to the previously characterized His-kinases, namely, the ETR1 family of ethylene receptors, CKI1 cytokinin-sensor, and ATHK1 osomo-sensor, this higher plant has three more His-kinases (named AHK2, AHK3, and AHK4). By employing the well-known His-->Asp phosphorelay systems in both the fission yeast and Escherichia coli, evidence is presented showing that the AHK4 His-kinase has an ability to serve as a cytokinin-responsive environmental sensor. Taking advantage of this AHK4-dependent His-->Asp phosphorelay system in E. coli, a phosphorelay interaction between the Arabidopsis His-kinase and histidine-containing phosphotransmitters (AHPs) was also demonstrated for the first time.     

Compilation and characterization of histidine-containing phosphotransmitters implicated in His-to-Asp phosphorelay in plants: AHP signal transducers of Arabidopsis thaliana

Histidine (His)-to-Aspartate (Asp) phosphorelay signal transduction systems are generally made up of a "sensor histidine (His)-kinase", a "response regulator", and a "histidine-containing phosphotransmitter (HPt)". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that the His-to-Asp phosphorelay mechanism is at least partly responsible for propagation of environmental stimuli, such as phytohormones (e.g. ethylene and cytokinin). Here we compiled the members of the HPt family of phosphotransmitters in Arabidopsis thaliana (AHP-series, Arabidopsis HPt phosphotransmitters), based on both database and experimental analyses, in order to provide a comprehensive basis at the molecular level for understanding the function of the AHP phosphotransmitters that are implicated in the His-to-Asp phosphorelay of higher plants.     

Comparative studies of the AHP histidine-containing phosphotransmitters implicated in His-to-Asp phosphorelay in Arabidopsis thaliana

The evolutionarily-conserved histidine to aspartate (His-to-Asp) phosphorelay signal transduction is common in both prokaryotes and eukaryotes. Such a phosphorelay system is generally made up of 'a histidine (His)-kinase', 'a histidine-containing phosphotransmitter (HPt)', and 'a phospho-accepting response regulator (RR)'. In general, an HPt factor acts as an intermediate in a given multistep His-to-Asp phosphorelay. In Arabidopsis thaliana, this model higher plant has five genes (named AHP1 to AHP5), each of which seems to encode an HPt factor. Recent studies suggested that the His-to-Asp phosphorelay involving the AHP factors is at least partly implicated in signal transduction in response to cytokinin (a plant hormone). Nevertheless, the properties of AHPs have not yet been fully clarified. Here we did comparative studies of all the AHP factors, in terms of (i) expression profiles in plants, (ii) intracellular localization, (iii) ability to acquire a phosphoryl group in vitro, and (iv) ability to interact with the downstream components, ARRs (Arabidopsis response regulators). The results of this study provided us with a comprehensive view at the molecular level for understanding the functions of the AHP phosphotransmitters in the His-to-Asp phosphorelay.     

An Arabidopsis histidine-containing phosphotransfer (HPt) factor implicated in phosphorelay signal transduction: overexpression of AHP2 in plants results in hypersensitiveness to cytokinin

Histidine-containing phosphotransfer (HPt) factors from Arabidopsis thaliana, designated as AHPs, function most likely in concert with histidine (His)-kinases (HKs) and response regulators (RRs) in certain multistep histidine (His)-->aspartate (Asp) phosphorelays that are involved in the signal transduction mechanisms, by which plant cells appear to respond to certain hormonal stimuli, including cytokinin. Although some previous in vitro results from studies on Arabidopsis AHPs (AHP1 to AHP5) supported this hypothesis, it has not yet been proven. To this end, here we constructed transgenic plants that contained the AHP2 protein in a considerably higher amount than in wild-type plants. Such AHP2-overexpressing young seedlings were examined in comparison with wild-type plants, with special reference to hormone responses; particularly, their inhibitory effects on root elongation of plants grown on agar-plates, and also hypocotyl elongation of etiolated seedlings grown in the dark. The results of this study suggested that AHP2-overexpressing plants showed a characteristic phenotype of cytokinin-hypersensitive. These in vivo observations were best interpreted by assuming that the AHP factor(s) is somehow implicated, if not directly, in a cytokinin-mediated His-->Asp phosphorelay signaling in Arabidopsis.     

A link between cytokinin and ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) that belongs to the AS2/LOB (LATERAL ORGAN BOUNDARIES) family genes in Arabidopsis thaliana

In Arabidopsis thaliana, each member of a large family of AS2/LOB (ASYMMETRIC LEAVES 2/LATERAL ORGAN BOUNDARIES) genes encodes a plant specific protein. They are highly homologous to one other. A mutational lesion in the representative AS2 gene results in the development of anomalous asymmetric leaves, implying that these family members commonly play some roles in plant development. In this study, we found that ectopic overexpression of ASL9 (ASYMMETRIC LEAVES 2 LIKE 9) in transgenic plants displayed a markedly anomalous architecture during the development of adult plants. Then we found that among AS2/LOB family members, ASL9 is distinct from the others in that it is exclusively regulated by the plant hormone cytokinin in a manner dependent on His-Asp phosphorelay signal transduction. We further found that when supplied externally in a medium, cytokinin specifically affected the growth properties of ASL9-ox seedlings. Taken together, the results of this study suggest that the cytokinin-induced ASL9 gene is implicated in regulation of the development of Arabidopsis thaliana.