1. Cytokinin homeostasis
Cytokinins are N6-prenylated adenine derivatives. In higher plants, the predominant cytokinins, known as isoprenoid cytokinins, are isopentenyladenine (iP) trans-zeatin (tZ), cis-zeatin, and dihydrozeatin, iP and tZ being the major forms in Arabidopsis thaliana (Sakakibara, 2006). The maintenance of an optimum cellular concentration of active cytokinin is controlled by the balance among biosynthesis, activation and catabolism. The spatial and temporal regulation of these processes is of key importance for correct growth (Werner et al., 2003; Miyawaki et al., 2004; Sakakibara, 2006; Kuroha et al., 2009), although the precise role of individual members of each family is still to be understood, mainly because of gene redundancy.
The initial, rate-limiting step in cytokinin biosynthesis is catalysed by ATP/ADP-ISOPENTENYLTRANSFERASE (IPT) (Fig. 1). In A. thaliana, there are nine IPT genes (AtIPT1–9). IPTs catalyse the transfer of an isopentenyl group from dimethylallyl diphosphate to an adenine nucleotide (ATP, ADP, or AMP) (Kakimoto, 2001; Takei et al., 2001). From their overall expression patterns it can be deduced that cytokinins are synthesized throughout the plant, including the root, shoot, and immature seeds. However, the expression domain of each IPT gene is tissue- and organ-specific (Miyawaki et al., 2004), suggesting gene-specific functions. Expression of AtIPT3, AtIPT5, and AtIPT7 is relatively high in the vegetative organs. AtIPT4 and AtIPT8 are expressed in immature seeds and AtIPT1 is expressed in ovules and vegetative organs. Nevertheless, no mutant of a single IPT gene exhibits a visible phenotype, suggesting functional redundancy within this gene family (Miyawaki et al., 2006). Combinations of multiple ipt mutants, however, display a drastic decrease in cytokinin concentrations, severely inhibiting shoot growth, while enhancing the production of root tissue (Miyawaki et al., 2006).
Figure 1. Schematic representation of the cytokinin molecular pathway and homeostasis network. Cytokinin (CK) action is based on a two-component signalling pathway. Cytokinin binding (1) to its receptor ARABIDOPSIS HIS KINASE (AHK) starts a phosphorelay cascade (2), which, through the intermediate factors ARABIDOPSIS HIS PHOSPHOTRANSFER 1–5 (AHP1-5), activates two classes of ARABIDOPSIS RESPONSE REGULATOR (ARR). Type-B ARRs are transcription factors that positively regulate the transcri-ption of cytokinin-responsive genes (3). Type-A ARRs are negative regulators of cytokinin signalling (4), via a still unclear mechanism. AHP6 is an AHP that lacks the conserved histidine residue required for phosphotransfer and therefore acts as a negative regulator of cytokinin signalling (5). The maintenance of an optimum cellular concentration of active cytokinin is controlled by the balance among biosynthesis, activation and catabolism. The initial, rate-limiting step in cytokinin biosynthesis (6) is catalysed by ATP/ADP-ISOPENTENYLTRANSFERASE (IPT). Members of the LONELY GUY (LOG) protein family then catalyse an additional modification in the production of bioactive cytokinin (7). Cytokinin breakdown (8) is controlled by the activity of CYTOKININ OXIDASE/DEHYDROGENASEs (CKXs). Type-A ARRs, IPTs and CKXs are components of the negative feedback loops of the cytokinin response, being direct targets of type-B ARRs (9).
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An additional step in the production of bioactive cytokinin is the removal of a ribose 5′-monophosphate group. Initially identified in rice (Oryza sativa), the LONELY GUY (LOG) genes encode enzymes with phosphoribohydrolase activity that releases cytokinin from the inactive precursor cytokinin riboside 5′-monophosphate (Fig. 1). In rice, LOG is specifically expressed in the shoot meristem tips where it regulates stem cell activity through the regulation of cytokinin concentrations (Kurakawa et al., 2007). In A. thaliana, nine homologues of the rice LOG gene have been isolated (AtLOG1–9), the products of seven of which retain enzymatic activity equivalent to that of the rice LOG (Kuroha et al., 2009). Analysis of spatial distribution patterns of AtLOGs revealed that, like the IPTs, these genes are expressed throughout the plant during development, although with different and overlapping expression domains (Kuroha et al., 2009). In some tissues, such as in the lateral root primordia and root vasculature, the expression of AtLOGs is concomitant with the expression of IPTs (Miyawaki et al., 2004; Kuroha et al., 2009). This suggests that cytokinins may be synthesized and subsequently activated by the AtLOGs in the same tissue, and then act as autocrine or paracrine signals. However, some AtLOG genes are expressed in tissues, such as root hairs and the root elongation zone epidermis, in which no expression of IPT genes can be observed (Miyawaki et al., 2004; Kuroha et al., 2009). This could indicate that, in these cases, cytokinin precursors translocate from cell to cell and/or over a long range, before being activated in these tissues. Multiple AtLOG mutants show inhibition of shoot meristem size (Kuroha et al., 2009; Tokunaga et al., 2012), resembling the phenotype of multiple loss-of-function ipt mutants (Miyawaki et al., 2006), strongly suggesting that cytokinin activation is essential to complete different developmental processes. While multiple ipt mutants show an increased production of root tissue, multiple AtLOG mutants display shorter roots compared with wild-type plants as a result of the lack of phloematic tissue, which is still present in the multiple ipt mutants (Matsumoto-Kitano et al., 2008; Tokunaga et al., 2012).
The steady-state concentrations of active cytokinins are negatively regulated by cytokinin catabolic genes. In A. thaliana, cytokinin breakdown is controlled by the activity of seven CYTOKININ OXIDASE/DEHYDROGENASE (CKX) genes (CKX1 to CKX7) (Fig. 1). The N6 side chain of cytokinin, which confers high cytokinin activity (Whitty & Hall, 1974), is the target of CKXs, which catalyse its cleavage (Mok & Mok, 2001; Schmulling et al., 2003). Like IPT and LOG genes, each CKX gene displays a specific expression pattern during plant development, but in general CKX expression can be found in regions of active growth, in the shoot and root meristems, and in emerging leaves (Werner et al., 2003). Overexpression of CKX genes phenocopies the effect of combined ipt and multiple log mutations in the shoot, and ipt mutations in the root, confirming the importance of the fine-tuning of cytokinin concentration for plant development (Werner et al., 2003; Miyawaki et al., 2006).
In order to modulate cytokinin signalling, many phytohormones, including cytokinin itself, auxin and abscisic acid, regulate the expression of IPT and CKX genes. In A. thaliana, cytokinins inhibit the transcription of AtIPT1, AtIPT3, AtIPT5, and AtIPT7 (Fig. 1), whereas auxin promotes the accumulation of the transcripts of AtIPT5 and AtIPT7 in roots (Miyawaki et al., 2004). In maize, CKX genes are up-regulated by cytokinins and abscisic acid (Brugiere et al., 2003) (Fig. 1).
Cytokinin homeostasis varies with time. Promoter-GUS fusion analyses have revealed changes in the expression of members of each family (Kuroha et al., 2009). In the embryo, AtIPT1 is only expressed in mature embryos, while AtIPT4 and AtIPT8 exhibit expression at an early stage of seed development, but while AtIPT4 promoter activity disappears at the early heart stage, AtIPT8 promoter activity decreases when the embryo is at the late heart stage. In the primary root, AtIPT5 is expressed at the transition zone (TZ) and in the columella root cap of the primary root, where its promoter activity decreases with time and is undetectable at 7 d post germination (dpg) (Kuroha et al., 2009). AtCKX4 is also expressed in the root cap, but its expression increases with development of the primary root. LOG8 expression is detectable only in the quiescent centre (QC) of young roots (Kuroha et al., 2009). AtCKX1 and AtCKX6 are expressed at lateral root emergence sites, and the activity of their promoter has been found to gradually increase with increasing elongation of the lateral root (Kuroha et al., 2009). This dynamicity reflects the importance of tight temporal regulation of the concentrations of active cytokinin for plant development.
2. Biochemistry of cytokinin signalling
Cytokinin signalling involves a multistep phosphotransfer cascade similar to the bacterial two-component system (Kakimoto, 2003; Argueso et al., 2010). In its simplest form, a two-component system comprises two elements, a histidine protein kinase and a response regulator protein, that are phosphorylated at conserved His and Asp residues, respectively. Phosphotransfer from the histidine kinase to the response regulator results in activation of the latter. More complex versions of the two-component system, like cytokinin signalling, simply contain multiple phosphotransfer steps between different proteins. The activation of the first element in such a signalling pathway generates a phosphorelay cascade between all the downstream components.
The first step of cytokinin perception requires the binding of cytokinin to a transmembrane histidine kinase receptor (Inoue et al., 2001) (Fig. 1). This binding induces, within the receptor, the autophosphorylation of a conserved His residue in its kinase domain (Perraud et al., 1999). In A. thaliana, there are three histidine kinase receptors: ARABIDOPSIS HIS KINASE 2 (AHK2), AHK3, and AHK4/WOODENLEG (WOL)/CYTOKININ RESPONSE 1 (CRE1 ) (Hwang & Sheen, 2001; Inoue et al., 2001). The three AHKs show largely overlapping expression patterns and partially redundant functions in cytokinin perception (Higuchi et al., 2004; Nishimura et al., 2004; Riefler et al., 2006). However, the lack of embryo lethality in the triple mutant, while calling into question the importance of the role of cytokinin signalling in plant development, leaves open the possibility of the presence of still unidentified cytokinin receptors (Higuchi et al., 2004; Nishimura et al., 2004). Indeed, CYTOKININ INSENSITIVE 1 (CKI1) has been shown to contribute to cytokinin-regulated vascular development and possibly acts as a cytokinin receptor (Kakimoto, 1996; Hejatko et al., 2009).
Following autophosphorylation, the phosphate group is then transferred to a conserved Asp residue within the receiver domain of the AHK protein, and subsequently transferred to a member of the ARABIDOPSIS HIS PHOSPHOTRANSFER PROTEIN (AHP) family (Hwang & Sheen, 2001; Hutchison et al., 2006) (Fig. 1). While it was previously hypothesized that AHP proteins translocate from the cytosol into the nucleus in response to cytokinin (Hwang & Sheen, 2001), it has been recently observed that in vivo AHPs are continuously transported in and out of the nucleus independently of their phosphorylation status and of the cytokinin response (Punwani et al., 2010). In the nucleus, phosphorylated AHPs transfer the phosphate group to the nuclear-localized ARABIDOPSIS RESPONSE REGULATOR (ARR) proteins (Hutchison et al., 2006) (Fig. 1). AHP6 is an AHP that lacks the conserved histidine residue required for phosphotransfer and therefore acts as a negative regulator of cytokinin signalling (Mahonen et al., 2006) (Fig. 1).
The ARRs are encoded by a gene family. All ARRs share a similar receiver domain with conserved residues targeted for phosphorylation (To & Kieber, 2008). However, ARRs are classified into type A, type B and type C, depending on their protein domains and cytokinin responsiveness (To et al., 2007). Type-A ARRs (ARR3–ARR9 and ARR15–ARR17) possess short C-termini, while type-B ARRs (ARR1, ARR2, ARR10–ARR14 and ARR18–ARR21) have longer C-termini, which contain a conserved GARP (GOLDEN2/ARR/Psr1) DNA-binding domain. Type-A ARRs act as negative regulators of cytokinin responses, via an as yet unknown mechanism (To et al., 2004, 2007; Kim, 2008; Muller, 2011) (Fig. 1). Type-B ARRs, which are transcription factors, play a positive role in mediating cytokinin-regulated gene expression (Fig. 1). Moreover, sequence alignment has revealed stretches of nonconserved sequences, which could be responsible for gene-specific functions (Muller, 2011). The type-C ARRs are more distantly related to type-A and type-B ARRs. They are not transcriptionally regulated by cytokinin like type-A ARRs and do not contain the output domain of type-B ARRs; however, their overexpression results in reduced sensitivity to cytokinin, suggesting a negative role similar to that of type-A ARRs (Kiba et al., 2004).
AHPs also mediate the accumulation in the nucleus of other plant-specific cytokinin-responsive genes, the CYTOKININ RESPONSE FACTORS (CRFs), members of the A. thaliana AP2 (APETALA 2) family of transcription factors (Rashotte et al., 2006). In response to cytokinin, activated type-B ARRs and CRFs act together to regulate gene expression of common and unique targets, including type-A ARRs (Hwang & Sheen, 2001; Rashotte et al., 2006). Indeed, the transcriptional activation of the type-A ARRs represents a negative feedback loop required to dampen the response upon high or prolonged signal input (To et al., 2004). In addition to their transcriptional up-regulation, activated type-A ARR proteins become more stable in response to cytokinin (To et al., 2007).
Although the individual role of each family involved in the two-component cytokinin signalling pathway is well understood, little is known regarding the way in which each member mediates a specific cytokinin-dependent developmental output (Muller, 2011). The fact that every step of the signalling cascade is supported by a small gene family, all members of which present a specific and complex expression pattern and may differ in affinity to the downstream elements, provides a first level of response diversity. In addition, the pre-patterned context of the signal-receiving cell could affect the response triggered by cytokinin. Moreover, temporal variations in the topology of the cytokinin pathway components within a cell or a tissue have been shown to be crucial for the completion of developmental events (Moubayidin et al., 2010).