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.     

The evolutionarily conserved OsPRR quintet: rice pseudo-response regulators implicated in circadian rhythm

In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION 1) that is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms, control of flowering time through photoperiodic pathways, and also photosensory signal transduction in this dicotyledonous plant. In this respect, both the dicotyledonous (e.g. A. thaliana) and monocotyledonous (e.g. Oryza sativa) plants might share the evolutionarily conserved molecular mechanism underlying the circadian rhythm. Based on such an assumption, and as the main objective of this study, we asked the question of whether rice also has a set of pseudo-response regulators, and if so, whether or not they are associated with the circadian rhythm. Here we showed that rice has five members of the OsPRR family (Oryza sativa Pseudo-Response Regulator), and also that the expressions of these OsPRR genes are under the control of circadian rhythm. They are expressed in a diurnal and sequential manner in the order of OsPRR73 (OsPRR37)-->OsPRR95 (OsPRR59)-->OsPRR1, which is reminiscent of the circadian waves of the APRR1/TOC1 quintet in A. thaliana. These and other results of this study suggested that the OsPRR quintet, including the ortholog of APRR1/TOC1, might play important roles within, or close to, the circadian clock of rice.     

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.     

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 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.     

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.     

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.     

Pseudo-Response Regulators (PRRs) or True Oscillator Components (TOCs)

In Arabidopsis thaliana, AUTHENTIC RESPONSE REGULATORS (ARRs) act as downstream components of the His-to-Asp phosphorelay (two-component) signaling pathway that is propagated primarily by the cytokinin receptor kinases, AUTHENTIC HIS-KINASES (AHK2, AHK3 and AHK4/CRE1). Thus, this bacterial type of signaling system is essential for responses to a class of hormones in plants. Interestingly, this higher plant has also evolved its own atypical (or unique) variants of two-component signal transducers, PSEUDO-RESPONSE REGULATORS (PRRs). Several lines of recent results suggest that the functions of PRRs are closely relevant to the plant clock (oscillator) that is central to circadian rhythms, the underlying mechanisms of which have long been the subject of debate. Through an overview of recent results, the main issue addressed here is whether or not the pseudo-response regulators (PRRs) are true oscillator components (TOCs).     

Histidine-containing phosphotransfer (HPt) signal transducers implicated in His-to-Asp phosphorelay in Arabidopsis

His to Asp phosphorelay signal transduction mechanisms involve three types of widespread signaling components: a sensor His-kinase, a response regulator, and a histidine-containing phosphotransfer (HPt) domain. In Arabidopsis, several sensor His-kinases have recently been discovered (e.g., ETR1 and CKI1) through extensive genetic studies. Furthermore, a recent search for response regulators in this higher plant revealed that it possesses a group of response regulators (ARR-series), each of which exhibits the phospho-accepting receiver function. However, no signal transducer containing the HPt domain has been reported. Here we identify three distinct Arabidopsis genes (AHP1 to AHP3), each encoding a signal transducer containing a HPt domain. Both in vivo and in vitro evidence that each AHP can function as a phospho-transmitting HPt domain with an active histidine site was obtained by employing both the Escherichia coli and yeast His-Asp phosphorelay systems. It was demonstrated that AHP1 exhibits in vivo ability to complement a mutational lesion of the yeast YPD1 gene, encoding a typical HPt domain involved in an osmosensing signal transduction. It was also demonstrated that AHPs can interact in vitro with ARRs through the His-Asp phosphotransfer reaction. It was thus suggested that the uncovered sensors-AHPs-ARRs lineups may play important roles in propagating environmental stimuli through the multistep His-Asp phosphorelay in Arabidopsis.     

Comparative genetic studies on the APRR5 and APRR7 genes belonging to the APRR1/TOC1 quintet implicated in circadian rhythm, control of flowering time, and early photomorphogenesis

In Arabidopsis thaliana, a number of circadian-associated factors have been identified. Among those, TOC1 (TIMING OF CAB EXPRESSION 1) is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as Arabidopsis PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). Nonetheless, it is not very clear whether or not the APRR family members other than APRR1/TOC1 are also implicated in the mechanisms underlying the circadian rhythm. To address this issue further, here we characterized a set of T-DNA insertion mutants, each of which is assumed to have a severe lesion in each one of the quintet genes (i.e. APRR5 and APRR7). For each of these mutants (aprr5-11 and aprr7-11) we demonstrate that a given mutation singly, if not directly, affects the circadian-associated biological events simultaneously: (i) flowering time in the long-day photoperiod conditions, (ii) red light sensitivity of seedlings during the early photomorphogenesis, and (iii) the period of free-running rhythms of certain clock-controlled genes including CCA1 and APRR1/TOC1 in constant white light. These results suggest that, although the quintet members other than APRR1/TOC1 may not be directly integrated into the framework of the central oscillator, they are crucial for a better understanding of the molecular mechanisms underlying the Arabidopsis circadian clock.     

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.