INTRODUCTION

Protein phosphorylation is a key mechanism for regulating signal transduction pathways in both prokaryotes and eukaryotes. In eukaryotes, regulatory phosphorylation predominantly occurs at serine, threonine, and tyrosine residues (Hunter, 1995; Hunter and Plowman, 1997; Plowman et al., 1999). In contrast, many signal transduction pathways in bacteria employ a so-called “two-component system” that relies upon phosphorylation of histidine and aspartic-acid residues (Mizuno, 1997). Plants also make use of two-component systems and these perform important roles in growth and development (Schaller, 2000; Mizuno, 2005; To and Kieber, 2008).

Two-component systems confer upon bacteria the ability to sense and respond to environmental stimuli and are involved in such diverse processes as chemotaxis, osmotic sensing, oxygen sensing, and host recognition (Parkinson, 1993; Stock et al., 2000; Baker et al., 2006). The simplest form of a two-component system employs a receptor with histidine-kinase activity and a response regulator (Figure 1). The receptor is located in the plasma membrane of the bacterium. In response to an environmental stimulus, the histidine kinase autophosphorylates a conserved histidine residue. This phosphate is then transferred to a conserved aspartic acid residue within the receiver domain of the response regulator. Phosphorylation of the response regulator modulates its ability to mediate downstream signaling. In bacteria, many of the response regulators are transcription factors. Thus, two proteins create a signaling circuit, capable of converting an external stimulus into a change in transcription. There are permutations on the two-component system. Of particular relevance to the plant two-component systems are multi-step phosphorelays (Figure 1) (Swanson et al., 1994; Appleby et al., 1996). These make use of a “hybrid” kinase that contains both histidine kinase and receiver domains in one protein, a histidine-containing phosphotransfer (HPt) protein, and a separate response regulator (Appleby et al., 1996). In these multi-step phosphorelays, the phosphate is transferred from amino acid to amino acid in sequence His → Asp → His → Asp.

Figure 1.

Histidyl-Aspartyl Phosphorelay.

Histidine kinase domains are indicated by rectangles, receiver domains by ovals, HPt proteins by rounded rectangles, and transmembrane domains by black bars. Sites of phosphorylation upon histidine (H) and aspartic acid (D) residues are indicated. Terminology is based on that of Parkinson (1993).

(A) Simple two-component system that employs a histidine kinase and a response regulator.

(B) Multi-step phosphorelay that employs a hybrid histidine kinase with both histidine kinase and receiver domains, a histidine-containing phosphotransfer protein (HPt), and a response regulator.

Although originally identified in bacteria, two-component signaling elements have also been identified in fungi, slime molds, and plants (Swanson et al., 1994; Loomis et al., 1997; Schaller, 2000; Mizuno, 2005). Interestingly, the canonical histidyl-aspartyl phosphorelay is apparently not found in animals. Analysis using the SMART protein-domain search interface ( http://SMART.embl-heidelberg.de) (Schultz et al., 2000) indicates that bona-fide two-component signaling elements are lacking from the genome sequences of Homo sapiens, Drosophila melanogaster, and Caenorhabditis elegans.

In Arabidopsis, proteins with significant sequence similarities to all elements of the two-component system have been identified, including histidine kinases, response regulators, and HPt proteins (Figure 2) (Schaller, 2000; Mizuno, 2005). Phosphorylation activity has been confirmed for at least one example in each case (Gamble et al., 1998; Imamura et al., 1998; Miyata et al., 1998; Suzuki et al., 1998; Imamura et al., 1999). In recent years, multiple experimental approaches have demonstrated the action of a histidyl-aspartyl phosphorelay in mediating cytokinin signal transduction (Hwang and Sheen, 2001; Higuchi et al., 2004; Nishimura et al., 2004; Mason et al., 2005; Hutchison et al., 2006; Riefler et al., 2006; Yokoyama et al., 2007).

Figure 2.

Representative Domain Structures of Arabidopsis Two-Component Signaling Elements.

Histidine kinase domains are indicated by rectangles, receiver domains by ovals, HPt proteins by rounded rectangles, and transmembrane domains by black bars. Additional domains are as indicated and are described in the text.

(A) Bona-fide signaling elements likely to participate in phosphorelays. Sites of phosphorylation upon histidine (H) and aspartic acid (D) residues are indicated.

(B) Divergent signaling elements from Arabidopsis that lack residues normally associated with participation in a two-component phosphorelay. An X in a histidine-kinase-like domain indicates that it lacks multiple residues implicated in histidine kinase activity. The divergent HPt (APHP1) contains an Asn (N) substitution for the His that normally gets phosphorylated. The divergent response regulators typically contain a Glu (E) substitution for the Asp that normally gets phosphorylated.

Arabidopsis also contains divergent two-component-like elements that are unlikely to function in histidyl-aspartyl phosphorelays (e.g. phytochromes and pseudo-response regulators) as they are missing one or more key amino-acid residues involved in a phosphorelay (Figure 2) (Makino et al., 2000; Schaller, 2000). Plants, like animals, contain pyruvate dehydrogenase kinase; this enzyme is related to histidine kinases but has an altered specificity such that it now phosphorylates serine residues (Popov et al., 1993). Thus, there is evidence from eukaryotes that two-component signaling elements have evolved to fill new functions that no longer rely upon phosphorylation of histidine and aspartic acid residues.

HISTIDINE KINASES

Analysis of the Arabidopsis genome supports the existence of eight histidine kinases that contain all the conserved residues required for enzymatic activity (Table 1, Figures 2 and 3), as well as additional diverged histidine-kinase like proteins that lack residues essential to histidine kinase activity. Some of the functional histidine-kinases have been identified as receptors for the plant hormones cytokinin (AHK2, AHK3, and AHK4) and ethylene (ETR1 and ERS1), but the ligands for the other three bona-fide histidine kinases (AHK1, CKI1, and CKI2) have yet to be determined.

Table 1.

Histidine Kinase-like Proteins of Arabidopsis

Figure 3.

Phylogenetic Relationship of Histidine Kinases.

Sequences containing conserved histidine-kinase domains (open circles) and diverged histidine kinase domains (filled squares) were identified in Arabidopsis. An unrooted bootstrapped tree is shown based on amino-acid alignment of the histidine-kinase domain sequences. The histidine kinase domain was defined as the sequence extending from the phospho-acceptor domain through the ATPase domain.

CKI1

CKI1 was initially identified in a mutant screen where it was found that its ectopic expression results in cytokinin-independent greening and shoot induction in callus cultures (Kakimoto, 1996). However CKI1 is not closely related to the cytokinin-receptor family and lacks the CHASE domain involved in cytokinin binding. Thus, the cytokinin-related phenotype resulting from CKI over-expression may arise due to non-physiological cross-talk with the cytokinin-signaling pathway. Analysis of loss-of-function mutations has clarified the role of CKI1 in plant growth and development, although a regulatory ligand for this putative receptor has not been identified. The homozygous cki1 mutation is lethal and examination of the mutant indicates that CKI1 is required for megagametogenesis, consistent with expression data that indicate CKI1 is expressed in developing ovules (Pischke et al., 2002; Hejatko et al., 2003).

CKI2

CKI2, like CKI1, can induce cytokinin responses when ectopically expressed (Kakimoto, 1996), but is also not thought to be directly involved in cytokinin signaling. CKI2 shows strong expression in the root and weaker expression in flowers (Iwami et al., 2007). Loss-of-function mutations in CKI2 exert a subtle phenotype in which root elongation is more sensitive to growth inhibition in response to ethylene, suggesting possible cross-talk with the ethylene receptors (Iwami et al., 2007).

AHK1

AHK1 was initially proposed to function as a plant osmosensor based on its ability to complement function of the yeast osmosensor SLN1, a histidine kinase that regulates the HOG1 MAP-kinase pathway for osmosensing in yeast (Urao et al., 1999). However it was subsequently found that other histidine kinases of Arabidopsis, including the cytokinin receptors, can complement yeast SLN1 mutations, indicating that this assay is not necessarily diagnostic of plant function (Reiser et al., 2003; Tran et al., 2007). Rather, genetic analysis indicates that AHK1 modulates plant growth and stress responses based on the finding that an ahk1,ahk2,ahk3 triple mutant (i.e. a combination of ahk1 with two cytokinin receptor mutations) is reduced in size compared to the ahk2,ahk3 double mutant (Tran et al., 2007). AHK1 is induced by dehydration and acts as a positive regulator of drought and salt stress responses, in contrast to the cytokinin receptors which are also induced by dehydration but act as negative regulators of these responses (Tran et al., 2007).

HISTIDINE-CONTAINING PHOSPHOTRANSFER PROTEINS

Histidine-containing phosphotransfer (HPt) proteins function in multi-step phosphorelays, acting as signaling intermediates between hybrid histidine kinase and response regulators (Figure 1). The Arabidopsis genome encodes five HPt proteins (AHP1 through 5) that contain the conserved residues required for activity, as well as one pseudo-HPt (APHP1/AHP6) that lacks the histidine phosphorylation site (Table 2, Figures 2 and 4) (Miyata et al., 1998; Suzuki et al., 1998; Suzuki et al., 2000). The HPt proteins have been shown capable of participating in a phosphorelay with Arabidopsis response regulators (Suzuki et al., 1998). In addition, two-hybrid analysis has demonstrated their ability to interact with both hybrid histidine kinases and response regulators (Imamura et al., 1999; Urao et al., 2000; Tanaka et al., 2004; Dortay et al., 2007), consistent with an ability to function in a multi-step phosphorelay.

Table 2.

HPt Proteins of Arabidopsis

Figure 4.

Phylogenetic Relationship of HPt Proteins.

An unrooted bootstrapped tree is shown based on amino-acid alignment of HPt domain sequences.

Analysis of loss-of-function mutations has revealed that AHP1, AHP2, AHP3, and AHP5 function as redundant positive regulators of cytokinin signaling (Hutchison et al., 2006). AHP4 only contributes slightly to the cytokinin responses, and in some cases appears to act as a negative regulator (Hutchison et al., 2006). The AHPs have been shown to accumulate in the nucleus in response to exogenous cytokinin (Hwang and Sheen, 2001; Yamada et al., 2004). APHP1, which encodes a pseudo-HPt lacking the phosphorylation site, acts as a negative regulator of cytokinin responses (Mähönen et al., 2006b). APHP1 expression is induced by cytokinin and thus functions as part of a negative feedback loop to reduce the plants sensitivity to cytokinin, with APHP1 potentially interacting with the receiver domains of the cytokinin receptors to prevent phosphotransfer to bona-fide AHPs.

RESPONSE REGULATORS

There are 23 genes in the Arabidopsis genome encoding proteins predicted to be functional response regulators (Table 3, Figures 2 and 5). These authentic response regulators (ARRs) can be divided into three classes based on phylogenetic analysis and function: type-A type-B, and type-C response regulators (Imamura et al., 1999; Schaller et al., 2007). In addition, there are nine genes encoding response regulators that lack the conserved Asp for phosphorylation, and are called pseudo-response regulators (PRRs) (Makino et al., 2000).

Table 3.

Response Regulators of Arabidopsis

Figure 5.

Phylogenetic Relationship of Receiver Domains from Response Regulators and Hybrid Kinases.

An unrooted bootstrapped tree is shown based on amino-acid alignment of receiver and pseudoreceiver domain sequences. The different families of receiver domains are highlighted, with subfamilies 1, 2 and 3 being indicated for the type-B ARRs. Note that the tree has some low bootstrap values indicating that regions of this tree should be considered to be simply radiations.

SIGNALING THROUGH PHOSPHORELAYS

A multi-step phosphorelay, rather than a simple two-component system, appears to be the major His-Asp signaling circuit employed by Arabidopsis. This possibility was initially raised by analysis of the Arabidopsis genome, which revealed a preponderance of hybrid kinases in Arabidopsis along with the presence of HPt proteins (Schaller et al., 2002). Subsequent analyses of protein-protein interactions using the yeast two-hybrid system supports interactions among the hybrid kinases with HPt proteins, and of the HPt proteins with both type-A and type-B response regulators, consistent with what would be expected in a multi-step phosphorelay (Imamura et al., 1999; Urao et al., 2000; Dortay et al., 2007). Finally, as described below, analysis of the cytokinin signaling pathway supports use of the multi-step phosphorelay in planta.

The cytokinin signaling pathway gives us our clearest picture of how two-component signaling elements have been adapted to signaling in plants (Hwang and Sheen, 2001; Haberer and Kieber, 2002; Heyl and Schmülling, 2003; Kakimoto, 2003; To and Kieber, 2008). Genetic analyses using loss-of-function mutations indicate that the primary cytokinin signaling pathway is a positive regulatory circuit that requires hybrid histidine kinases (AHK2, AHK3, and AHK4) (Inoue et al., 2001; Suzuki et al., 2001; Ueguchi et al., 2001; Yamada et al., 2001; Kakimoto, 2003; Kim et al., 2006), HPt proteins (AHP1, AHP2, AHP3, AHP5) (Hutchison et al., 2006), and type-B response regulators (ARR1, ARR2, ARR10, ARR11, and ARR12) (Sakai et al., 2001; Mason et al., 2005; Yokoyama et al., 2007; Ishida et al., 2008) (Figure 6). The function of type-B ARRs as transcription factors indicates that signals may move from histidine kinase to the nucleus solely through elements of a two-component system, the same type of signaling circuit employed by many prokaryotes. The mobile element of this signaling circuit are the AHPs, which relocate from cytosol to nucleus in response to cytokinin (Hwang and Sheen, 2001; Yamada et al., 2004). It should be noted that additional two-component signaling elements (such as other type-B ARRs) may also contribute to this cytokinin signaling circuit but this has not yet been confirmed genetically.

Figure 6.

Cytokinin Signal Transduction Occurs Through a Multi-Step Phosphorelay.

Cytokinin receptors, AHPs, and type-B ARRs function as a positive regulatory circuit to relay the cytokinin signal from plasma membrane to nucleus. Type-B ARRs act as transcription factors, one target being genes encoding type-A ARRs. The type-A ARRs feed back to inhibit their own transcription and may also mediate other cytokinin responses. The roles of the listed cytokinin receptors, AHPs, type-B response regulators, and type-A response regulators in cytokinin signal transduction have each been confirmed by the analysis of T-DNA insertion mutations.

Several negative regulatory circuits are also employed in cytokinin signaling. First, AHK4, like some bacterial histidine kinases, has both kinase and phosphatase activities (Mähönen et al., 2006a). In the absence of cytokinin, AHK4 acts as a phosphatase to dephosphorylate AHPs, thereby decreasing signaling through the phosphorelay. Upon cytokinin binding, AHK4 switches to act as a histidine kinase to initiate the multi-step phosphorelay, resulting in phosphorylation of AHPs and downstream response regulators. Second, one of the initial transcriptional responses mediated by the type-B ARRs in response to cytokinin is to induce the expression of the type-A ARRs (Brandstatter and Kieber, 1998; Imamura et al., 1998; D'Agostino et al., 2000; Mason et al., 2005; Rashotte et al., 2006; Taniguchi et al., 2007; Yokoyama et al., 2007). The type-A ARRs then negatively regulate cytokinin signaling, potentially by competing with the type-B ARRs for phosphorylation by the AHPs (Kiba et al., 2003; To et al., 2004; Leibfried et al., 2005; Lee et al., 2007; To et al., 2007). Furthermore, multiple type-A proteins are stabilized by cytokinin, further increasing this negative feedback loop (To et al., 2007). Third, the pseudo-HPt, APHP1, is induced by cytokinin and acts as a negative regulator of cytokinin responses (Mähönen et al., 2006b).

The presence of additional hybrid histidine-kinases in Arabidopsis, such as ETR1, AHK1, CKI1, and CKI2, suggests that multi-step phosphorelays function in relaying signals other than cytokinin. This raises the question as to which downstream components are involved in relaying the additional signals, given that a majority of the two-component signaling elements have already been implicated in cytokinin signaling. While there exists a formal possibility for unique combinations of signaling elements, it is far more likely that downstream signaling elements are shared among the receptors. In some cases, different input signals may be channeled into the same downstream signaling pathway to regulate a common response. Alternatively, a response tailored to a specific signal input could still be obtained even with shared signaling elements. One way to accomplish this would be to have varying affinities between the receptors and the AHPs, and between the AHPs and the response regulators, such that each signal would differentially activate the downstream components. Yeast two-hybrid analysis does suggest some specificity to the interactions between receptors and the AHPs (Urao et al., 2000), but the physiological relevance of these differences has not been determined. An alternative possibility would be for specificity of the downstream signaling elements to be modified by covalent modification or protein-protein interaction in ways unique to the signal input. It is also possible that protein subsets are sequestered in unique signaling complexes by scaffold proteins. Genetic, molecular, and proteomic approaches should clarify how this network of interactions functions in plants.