Cytokinins are essential plant hormones that control cell division, shoot meristem initia-tion, leaf and root differentiation, chloroplast biogenesis, stress tolerance, and senescence. Together with auxin, another plant hormone, cytokinins can reprogram terminally differen-tiated leaf cells into stem cells and support shoot regeneration indefinitely in plant tissue culture (1, 2). Thus, cytokinins are master regulators of plant growth and development, which are highly plastic and adaptive, as well as remarkably resilient and perpetual. Research interest in the signaling pathways activated by cytokinins has increased recently because of new information arising from studies of Arabidopsis and the completion of its genome sequence. However, the importance of this pathway is given additional weight because it represents two-component signaling, a canonical mechanism that mediates diverse biological responses in many taxa. The specific Cytokinin Signaling Pathway (3) details the pathway as it has been elucidated in Arabidopsis; the canonical Cytokinin Signaling Pathway presents the general view (4).

In the Arabidopsis cytokinin signal transduction pathway, hybrid histidine protein kinases (AHKs) serve as cytokinin receptors and histidine phosphotransfer proteins (AHPs) transmit the signal from AHKs to nuclear response regulators (ARRs), which can activate or repress transcription (5–10). Similar components are also found in maize, suggesting a conservation of the cytokinin signaling mechanism in plants (11). There are four major steps to cytokinin signaling: AHK sensing and signaling, AHP nuclear translocation, ARR transcription activation, and a negative feedback loop through cytokinin-inducible ARR gene products (Fig. 1). Analyses of mutants and transgenic tissues and plants support the importance of this central signaling pathway in diverse cytokinin responses (5–10). The multistep two-component phosphorelay mechanism found in Arabidopsis is reminiscent of the bacterial two-component signaling system (12), but it is linked by AHPs, which shuttle from the cytoplasm to the nucleus in a cytokinin-dependent manner (6). Although conserved motifs for two-component phosphorelay systems have been identified in plant hormone ethylene receptors (13), phytochrome photoreceptors (14), and a putative osmosensor (15), until recently the importance of histidine protein kinase activity and phosphorelay had not been demonstrated in plant cells. Functional analyses of AHKs, AHPs, and ARRs in Escherichia coli, yeasts, plants, and a leaf protoplast system, and protein-protein interactions in yeast two-hybrid assays, have provided compelling evidence for the importance of multistep two-component phosphorelay in cytokinin signaling (5–10, 16–18).

In Arabidopsis, at least three genes encode cytokinin receptors: AHK4 [also known as CYTOKININ RESPONSE 1 (CRE1) and WOODEN LEG (WOL)], AHK2, and AHK3 (7, 19, 20). Other Arabidopsis histidine protein kinases, cytokinin independent 1 (CKI1) and CKI2 (also known as AHK5), can also activate cytokinin responses in the absence of exogenously added cytokinin (5, 6). Quantitative transcription analyses based on cytokinin-inducible ARR6-LUC reporter gene activity suggest that CKI1 and AHKs act through different cytokinin perception mechanisms. CKI1 is constitutively active, but AHK4, AHK2, and AHK3 require extracellular cytokinin for their activation (6). The function of AHK4 has been thoroughly demonstrated by direct cytokinin binding (21) and by the isolation of cre1 and wol mutants that exhibit defects in cytokinin-mediated shoot induction from callus and root vascular morphogenesis, respectively (7, 19). The lack of shoot phenotypes in cre1 and wol suggests that the functions of AHK2 and AHK3 may overlap with that of AHK4 (20). Further analyses of cellular expression patterns, cytokinin binding, and chimeric AHKs with swapped domains should clarify the underlying mechanism of each AHK action in cytokinin signaling.

The analysis of fusions between green fluorescent protein (GFP) and AHP (AHP-GFP) has provided the first visual, in vivo evidence that AHP1 and AHP2 are translocated into the nucleus in a cytokinin-dependent manner (6). In Arabidopsis, there are more AHKs, ARRs, and related proteins than there are AHPs (18, 22),suggesting that multiple two-component signaling pathways may share AHPs (6, 10). The cytokinin pathway does not follow the established eukaryotic histidine protein kinase and mitogen-activated protein kinase (MAPK) cascade paradigm (23), but rather integrates multiple AHK activities to common AHPs, which then modulate distinct ARRs in the nucleus (6).

The B-type ARR transcription activators (ARR1, ARR2, and ARR10) carry MYB-like domains for DNA binding and a glutamine (Q)-rich domain for transcriptional activation (24, 25), and they activate cytokinin-responsive ARR6 transcription (6, 8). These activators appear to be the evolutionary products of domain shuffling, with ancestral modules originating from both prokaryotic and eukaryotic heritage. Mutation in the conserved aspartate residue of ARR2 does not abolish its function as a transcription activator for a cytokinin early-response gene ARR6 promoter, suggesting that phospho-rylation may not intrinsically activate the transcription factor (Fig. 1) (6). Consistently, deletion of the receiver domain of ARR1 results in higher transcription activity in plant cells and constitutive cytokinin phenotypes in transgenic plants (8, 24). Thus, phosphorylation of ARR1 and ARR2 likely eliminates negative regulation (Fig. 1). Ectopic expression in transgenic Arabidopsis of ARR2, one of the rate-limiting transcription factors in the response to cytokinin, is sufficient to mimic cytokinin in promoting shoot meristem proliferation and leaf differentiation, and in delaying leaf senescence (6). The lack of striking phenotypes in the arr1 mutant indicates that multiple B-type ARRs may serve similar functions (6, 8). Determining the target genes of these transcription factors using microarrays will add new insight into the molecular basis of cytokinin actions.

The products of the cytokinin-inducible A-type ARR4, ARR5, ARR6, and ARR7 genes inhibit transcription, which could mediate a negative feedback loop that controls the tran-sient induction of cytokinin primary response genes and allows resetting and/or fine-tuning of the physiological state of the cells (Fig. 1) (6, 16). Although the B-type ARRs with transcriptional activation activities are likely the major regulators of a broad spectrum of cytokinin target genes (26), the A-type ARRs could also contribute to the outputs of cytokinin signaling through protein-protein interactions (16, 17). Two-component elements could potentially be regulated by signals other than cytokinin and provide a cross-talk mechanism in plant signaling networks. For instance, expression of some ARRs is regulated by stress (27) and sugar signals (28). ARR4 also interacts with phytochrome B and modulates light signaling (29). Thus, two-component elements could serve as the molecular links in a complex plant signal transduction network that sensitively integrates central growth signals such as plant hormones, sugars, light, and other environmental cues.

The expression analysis of CYCLIN D (30) and an ARR5::GUS transgene (31)inArabidopsis has shown that root and shoot meristems are major sites of cytokinin actions. However, cytokinin responses can also occur in other cell types (6, 31). This broad cellular competence to cytokinin responses may explain the plasticity of plant development. The emerging short cytokinin signaling circuit could represent a conserved core signaling pathway in different cell types in response to cytokinin. However, additional cell type–specific components are likely to play important roles for cytokinin responses in different cell types and tissues, for example, in dividing and nondividing cells. Elucidation of the expression patterns and subcellular localization of AHKs, AHPs, and ARRs will contribute to a better understanding of their unique or overlapping roles in cytokinin responses and in other two-component signaling pathways in plants. The major challenge is to determine how a conserved cytokinin signal transduction pathway influences cell cycle, leaf senescence, shoot initiation, and leaf patterning in different cell types at various developmental stages.

The completion of the Arabidopsis genome sequence has revealed 54 genes encoding puta-tive AHKs, AHPs, ARRs, and related proteins, suggesting a substantial involvement of this signaling mechanism in many facets of plant cell regulation (17, 18, 32). The development of the Arabidopsis protoplast system has enabled a high-throughput functional genomic analysis of the two-component regulators (6). Because pronounced redundancy in the Arabidopsis genome is evident (18, 32), cellular analyses of the two-component elements would complement the characterization of a large number of insertion mutants that may not display overt phenotypes. Genetic, genomic, and biochemical experiments will elucidate the details in cytokinin perception, protein-protein interactions, and target gene expression essential in cytokinin signaling.

Legend: Colors indicate the localization of a component. (This is, of course, a rough guide for orientation on the map, as many components change locations during signaling.)

To see more detailed information about each component, click here.

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