JXB Advance Access originally published online on October 30, 2006
Journal of Experimental Botany 2006 57(15):4051-4058; doi:10.1093/jxb/erl179
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay
1Free University of Berlin, Institute of Biology/Applied Genetics, Albrecht-Thaer-Weg 6, D-14195 Berlin, Germany
2Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia
* To whom correspondence should be addressed. E-mail: gar{at}ippras.ru or gromanov{at}yahoo.com
Received 14 June 2006; Accepted 30 August 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The cytokinin receptor AHK3 of Arabidopsis thaliana plays a predominant role in shoot development. A study of the hormone-binding characteristics of AHK3 compared with the mainly root-confined receptor CRE1/AHK4 has been accomplished using a live-cell binding assay on transgenic bacteria expressing individual receptor proteins. Both receptors bound trans-zeatin (tZ) with high affinity. Scatchard analysis showed a linear function corresponding to an apparent KD of 1–2 nM for the AHK3 receptor–hormone complex, which is close to the KD (2–4 nM) for the CRE1/AHK4 receptor–hormone complex. The specific binding of tZ to both receptors was pH dependent, AHK3 being more sensitive to pH changes than CRE1/AHK4. Hormone binding was reversible, at least for the bulk of 3H-zeatin, and influenced by monovalent cations, while divalent cations (Ca2+, Mg2+, Mn2+) at physiological concentrations had no significant effect. AHK3 differed significantly from CRE1/AHK4 in relative affinity to some cytokinins. AHK3 had an approximately 10-fold lower affinity to isopentenyladenine (iP) and its riboside, but a higher affinity to dihydrozeatin than CRE1/AHK4. For AHK3, cytokinin ribosides (tZR, iPR) and cis-zeatin had true binding activity, although lower than that of tZ. The phenylurea-derived cytokinin thidiazuron was a strong competitor and bound to the same site as did adenine-derived cytokinins. The inhibitor of cytokinin action butan-1-ol had little effect on cytokinin–receptor complex formation. The revealed properties of AHK3 suggest its specific function in root-to-shoot communication.
Key words: AHK3, Arabidopsis thaliana, binding assay, CRE1/AHK4, cytokinin, hormone–receptor complex, receptor, sensor histidine kinase, zeatin
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Arabidopsis thaliana possesses three cytokinin receptors (AHK2, AHK3, and CRE1/AHK4), which are all membrane-located sensor histidine kinases (Kakimoto, 2003; Heyl and Schmülling, 2003; Ferreira and Kieber, 2005; Hwang and Sakakibara, 2006; Heyl et al., 2006). The developmental and physiological roles of these receptors are under extensive study (Higuchi et al., 2004; Nishimura et al., 2004; Kim et al., 2006; Riefler et al., 2006). It was shown recently that AHK3 plays a predominant role in different aspects of shoot development, including the regulation of leaf and shoot growth, chloroplast development, shoot de-etiolation, far-red light resistance, leaf senescence, and chlorophyll retention. By contrast, the CRE1/AHK4 receptor is of primary importance in root development and tissue culture (Inoue et al., 2001; Riefler et al., 2006). However, biochemical data on the interaction of these receptors with their ligands, i.e. cytokinins, remain scarce. So far, only CRE1/AHK4 protein was studied in direct binding assays, and some essential parameters of cytokinin binding to this receptor were determined (Yamada et al., 2001; Romanov et al., 2005). Thus only the CRE1/AHK4 protein was proven to possess a true cytokinin-binding ability by direct biochemical methods. Another approach showed that CRE1/AHK4 or AHK3 expression in yeast or bacteria makes these transformed cells sensitive to cytokinins (Inoue et al., 2001; Suzuki et al., 2001; Ueguchi et al., 2001b; Spíchal et al., 2004). The latter paper (Spíchal et al., 2004) provided evidence that AHK3 and CRE1/AHK4 differ in ligand specificity. Difference in ligand preference of cytokinin receptors from another species, maize, was also revealed in a promoter activation test on transformed bacteria (Yonekura-Sakakibara et al., 2004). However, long-lasting functional assays relying on heterologous signal transduction generate mostly qualitative results which can not replace direct binding data.
Hormone–receptor interaction is a first and crucial step of hormone signalling in the cell. Therefore the characteristics of such an interaction are very important for the subsequent hormone-triggered intracellular events. The purpose of this study was to fill the gap in our knowledge on the interaction of cytokinins with their receptors. Various biochemical parameters of cytokinin binding to the receptor AHK3 compared to CRE1/AHK4 have been investigated. To this end, a hormone binding assay was developed that uses living Escherichia coli cells expressing distinct receptor proteins from Arabidopsis. This assay has been shown to be specific and to yield biochemical data similar to those obtained with isolated receptor-containing membrane fractions (Romanov et al., 2005). The results provide ultimate proof that AHK3 specifically binds active cytokinins, although its ligand specificity and some essential parameters of hormone binding differ substantially from those of CRE1/AHK4. Differences in their functional properties suggest specific functions of these receptors in cytokinin-regulated processes.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Chemicals
Cytokinins and related compounds were obtained from OlChemim (Olomouc, Czech Republic) and from Sigma Aldrich Chemie GmbH (Munich, Germany). In addition, cis-zeatin was purified by HPLC on a C18 column and checked for the absence of contamination with possible traces of trans-zeatin (tZ) in the laboratory of Dr M Strnad at the Palack
University (Olomouc, Czech Republic). Highly labelled trans-[2-3H]zeatin (3H-tZ) (592 GBq mmol–1) was a kind gift of the Isotope laboratory of the Institute of Experimental Botany (Prague, Czech Republic). The radiochemical purity was >99%.
Bacterial clones
The Escherichia coli strain KMI001 carrying vector pSTV28 expressing AHK3 (Yamada et al., 2001; Spíchal et al., 2004) or vector pINIII
EH expressing CRE1/AHK4 was used in the experiments. E. coli strain KMI001 carrying the empty vector pINIII was used as a control. Bacterial strains were kindly provided by Dr T Mizuno (Nagoya, Japan). Bacteria were grown overnight in M9 medium (pH 7) with 0.1% casamino acids (Sambrook and Russell, 2001) and 25 µg ml–1 carbenicillin or chloramphenicol at 24±1 °C with extensive shaking. Culture density was controlled by measuring the absorption at 600 nm (OD600).
Hormone binding assay
The binding of hormones to receptors was tested directly on intact bacteria using a recently developed method (Romanov et al., 2005) without the isolation of a particular fraction or protein. For this purpose, a homogenous bacterial suspension (OD600 ~ 1) was aliquoted (0.75–1.2 ml) to Eppendorf tubes containing 2 pmol of 3H-tZ (c. 40 000 cpm) with or without unlabelled tZ. Routine incubations were done on ice for 30 min. To discriminate between specific saturable binding and non-specific non-saturable binding, each probe with 3H-tZ had its counterpart where 3H-tZ was mixed with a large (~ 3500-fold) excess of non-labelled tZ. Probes with only 3H-zeatin assessed total binding while probes with an excess of non-labelled zeatin measured non-specific binding. The difference between total and non-specific binding was taken as a measure of specific binding. After incubation, bacteria were pelleted at 13 000 rpm for 2 min in the cold, supernatants were carefully removed and 0.24 ml of 99% ethanol was added to each pellet. Absence of protein in the supernatant was controlled by the Bradford method (Bradford, 1976). The pellet was extracted for at least 1 h with shaking, briefly centrifuged, and the radioactivity of 0.2 ml ethanol extract was determined using ACS-II scintillation cocktail (Amersham Biosciences, UK).
Effect of media conditions on hormone binding
For pH-dependence studies, 30 ml of bacterial suspension in M9 medium was pelleted and resuspended in 15 ml of a non-buffered M9 medium analogue containing the same concentrations of basic salts as the original M9 medium (Ca2+, 0.1 mM; Mg2+, 2 mM; K+, 53.5 mM; Na+, 253.2 mM; 
46.4 mM; 
2 mM; Cl–, 238.2 mM). For the pH range 5–7, 0.1 M MES buffer was adjusted to a defined pH at 4 °C by 5 N NaOH. For the pH range 7–9, 0.1 M TRIS buffer was adjusted to a defined pH at 4 °C by 5 N HCl. Buffer aliquots of defined pH were mixed with the same volume of bacterial suspension in non-buffered M9 analogue and then distributed to cold Eppendorf tubes containing labelled tZ (with or without non-labelled tZ). The binding was then measured as described above.
To assess a divalent salt effect on zeatin-receptor interaction, 0.5 M solutions of CaCl2, MgSO4, and MnSO4 were prepared. Bacterial suspensions were centrifuged, the M9 medium discarded and the bacterial pellet washed thoroughly in 0.05 M TRIS, pH 7, 0.15 M NaCl. Then the washing solution was discarded and the bacterial pellet was resuspended in fresh TRIS-NaCl buffer. This suspension was used in a routine binding assay with the addition of microaliquots of divalent salt solutions. Control probes were supplemented with 1 mM EGTA (in the case of the Ca2+ assay) or 1 mM EDTA (in the case of the Mg2+ or Mn2+ assays).
Competition assays
Stock solutions of cytokinins (0.1 M) and related compounds in 40% DMSO were stored at –20 °C. Serial dilutions were made in 40% DMSO identically for all ligands. Microaliquots of diluted ligands were mixed with microaliquots of 3H-tZ, tubes were transferred on ice and then aliquots of cold bacterial suspension were added and briefly shaken. The final concentration of DMSO was less than 0.1%; nevertheless control samples with the same DMSO content were always included in the series. The binding assay was performed as described above.
Computation and statistical analysis
Calculations of binding parameters were accomplished according to Scatchard (1949) as interpreted by Varfolomeev and Gurevich (1999) and confirmed by the ligand binding option of the SigmaPlot8 program. Mean values and standard errors were calculated using Excel or TTEST programs.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Various biochemical parameters of cytokinin binding to AHK3 were investigated. The specific 3H-trans-zeatin (3H-tZ) binding of the receptor-expressing bacteria was influenced by temperature. In the interval of 0–37 °C it was maximal at 0 °C (Fig. 1), similarly to CRE1/AHK4 (Romanov et al., 2005). By contrast, non-specific binding was minimally affected by temperature. At 37 °C, the binding to AHK3 markedly decreased after 5–15 min of incubation. Figure 1 shows that total binding at 0 °C was rapid, reaching the apparent equilibrium between 5–15 min and stable over the period 15–60 min, therefore this temperature was used in all subsequent experiments.
|
Figure 2 shows the pH-dependence of 3H-tZ binding between pH 5 and pH 9. MES and TRIS were used to buffer the pH intervals 5–7 and 7–9, respectively. AHK3 showed an apparent maximum of zeatin specific binding at pH 8.5 with a strong decrease of binding with decreasing pH. At pH 5 the zeatin-specific binding of AHK3 was close to zero (Fig. 2). This pH-dependent decrease in binding was, to a large extent, reversible (not shown). For CRE1/AHK4, the zeatin specific binding was also maximal at alkaline pH (8.5–9). However, the changes in binding with decreasing pH were much less pronounced. A local maximum was also noticeable between pH 6 and pH 7. The specific binding at pH 5 was 70% of the binding at pH 7 (Fig. 2).
|
Mono- and divalent salts play an important role in cellular metabolism and regulation, therefore the effect of different salts on 3H-tZ binding to the receptors was tested. The dependence of the binding capacity on the concentration of monovalent salts (NaCl or KCl) differed for AHK3 and CRE1/AHK4 (Fig. 3). Zeatin binding to AHK3 was 20–50% enhanced by increasing salt concentrations, while binding to CRE1/AHK4 clones was decreased in an almost linear fashion. By contrast, divalent cations (Ca2+, Mg2+, and Mn2+) in a concentration range of 0.2–12 mM produced very minor effects on zeatin binding (not shown).
|
To test the reversibility of zeatin binding, a 15.5-fold excess of non-labelled trans-zeatin (tZ) was added to bacteria either simultaneously with radiolabelled tZ or 30 min later, when the binding equilibrium of 3H-tZ was already established. Non-labelled zeatin was able effectively to displace its radioactive analogue from the hormone–receptor complex. The displacement was more pronounced for CRE1/AHK4 achieving 90% compared to ~70% for AHK3. It was concluded that 3H-tZ binding to Arabidopsis receptors is easily reversible, especially in the case of the CRE1/AHK4 receptor.
To assess the affinity and specificity of hormone–receptor binding, a series of dose-dependent binding assays was performed. A typical dose-dependent 3H-tZ binding is shown in Fig. 4. The binding curves clearly show that binding is saturable, which means that the number of binding sites is limited. Scatchard analysis (Scatchard, 1949) of these data produced linear functions and indicated high affinity binding, with an apparent dissociation constant (KD) for AHK3 in the range of 1–2 nM, very close to the KD for CRE1/AHK4 (2–4 nM). These data agree well with the one-site saturation model, with no indication of any co-operativity of binding. Similar results were obtained from Scatchard analysis performed on the basis of displacement of a constant amount of 3H-tZ by various doses of unlabelled zeatin (not shown). AHK3 showed clear cytokinin specificity as, among a large number of different phytohormones and signalling substances, only cytokinins were able to compete for binding with radioactive zeatin (see supplementary material Fig. S1 at JXB online), similar to CRE1/AHK4 (Romanov et al., 2005).
|
The ligand specificity of AHK proteins was studied using a wide range of naturally occurring and some synthetic cytokinins. The ability of different cytokinins to displace 3H-tZ from the hormone–AHK3 complex varied by several orders of magnitude reflecting the very different affinities of these compounds for this cytokinin receptor (Fig. 5; see supplementary material Fig. S2 at JXB online). On the basis of data from competition assays (Fig. 5; see supplementary material Fig. S2 at JXB online), ligand concentrations resulting in 50% displacement of labelled zeatin from the complex with receptors were calculated (Table 1). These values are identical or close to the apparent binding constants (KD) for these ligands. Large variations of more than three orders of magnitude were found for the KD of different cytokinins. Similar to CRE1/AHK4, tZ was the most effective competitor. All modifications of tZ lowered its affinity for receptors to various degrees depending on the type of modification. The removal of the aliphatic side chain (producing adenine) almost completely abolished the ability to interact with the receptors. This was also the case when the side chain was O-glucosylated (tZOG). tZ acetylated on the side chain (tZOAc) showed very low affinity and was able to compete with 3H-tZ only at high concentrations. The cis-isomer of zeatin was much less active compared with its trans analogue although it was still able to replace 3H-tZ from the hormone–receptor complex. Surprisingly, 6-benzyladenine (BA), an aromatic cytokinin which was usually considered one of the most potent cytokinins, showed a rather low affinity, especially for AHK3. Ribosides (iPR, tZR) were substantially less effective than the corresponding free bases tZ and isopentenyladenine (iP) but nevertheless bound to both receptors more strongly than many other cytokinin compounds (Fig. 5; Table 1; see supplementary material Fig. S2 at JXB online).
|
|
A sequence of cytokinin preference was created for each receptor based on the relative position of competition curves. According to our data, this sequence for AHK3 is: tZ>TD>tZR>DZ>iP>cZ>BA>iPR~AcZ>Ade~tZOG, compared to an analogous sequence for CRE1/AHK4: tZ>iP>TD>tZR>iPR>BA>DZ>cZ>AcZ>Ade~tZOG. These sequences show that the relative affinities to many of the tested cytokinins are similar for AHK3 and CRE1/AHK4. However, some cytokinins differ in relative positions, notably iP, iPR and DZ. The most prominent difference was in binding iP and its riboside (iPR). iP had a relatively weak affinity for AHK3, rather intermediate among other tested cytokinins, and the competition of iPR was as weak as that of tZOAc. By contrast, iP was one of the most potent ligands for CRE1/AHK4, with an affinity close to that of tZ (Fig. 5; Table 1; see supplementary material Fig. S2 at JXB online). Similarly, the iPR competition curve in the case of CRE1/AHK4 was positioned next to that of ZR (see supplementary material Fig. S2 at JXB online). On the other hand, dihydrozeatin (DZ), the product of zeatin side chain hydrogenation, was shown to be a much stronger competitor with AHK3 than with CRE1/AHK4 (Fig. 5).
Remarkably, thidiazuron (TD), a phenylurea-derived artificial cytokinin, which is structurally unrelated to the purine-type cytokinins, was, for both receptors, one of the most effective competitors of tZ (see supplementary material Fig. S2 at JXB online). To find out whether these two compounds bind to the same or different sites of the receptors, competition assays were performed in the presence or absence of a constant concentration of thidiazuron. The results of these experiments were plotted on a graph in which the position of the interception point of competition lines is indicative of the identity or non-identity of binding sites for the tested compounds (Varfolomeev and Gurevich, 1999). Figure 6 shows that the interception points were located close to the abscissa indicating that thidiazuron binds to the same receptor site as tZ.
|
Earlier it was shown that butan-1-ol (at a concentration of 0.2–1.0%) suppressed the cytokinin-induced transcript accumulation of primary response genes in Arabidopsis, whereas butan-2-ol was almost inactive (Romanov et al., 2002). Butan-1-ol could interfere with either cytokinin signal perception or signal transduction. To test the first possibility, binding assays were performed in the presence of different concentrations (0.2–1.0%) of butan-1-ol or butan-2-ol. Results show that the effect of butanols on cytokinin–receptor complex formation was rather weak, especially in the case of AHK3, and that there was no difference in action between butan-1-ol or butan-2-ol (see supplementary material Fig. S3 at JXB online).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
For binding assays, intact E. coli expressing individual receptors were used. These bacteria were able to detect cytokinin activity because the receptor couples to an E. coli phosphotransmitter which in turn activates a cps::lacZ reporter gene (Suzuki et al., 2001; Yamada et al., 2001). This system was previously used to test a wide range of cytokinin-related compounds in a promoter activation test (Spíchal et al., 2004) and proved that receptors retained physiological activity. An important point is that our binding assays were performed under conditions (low temperature, short time) which practically exclude metabolic degradation of ligands or receptors. Therefore the results reflect genuine cytokinin–receptor interaction, not distorted by any kind of cellular metabolism.
Affinity constants are in accordance with natural cytokinin concentrations
Our work provides missing quantitative data on the affinity of numerous cytokinin compounds for AHK3 and to some extent for CRE1/AHK4 as well (Table 1). tZ is distinguished by showing the highest affinity (KD 1–4 nM) for AHK3 and CRE1/AHK4. These KD values are in reasonable accordance with estimations of the endogenous cytokinin content in plants. For wild-type Arabidopsis, recent determinations (Takei et al., 2004; Riefler et al., 2006) yielded a cytokinin content of 0.6–1.3 and 1.0–2.0 pmol g–1 fresh weight for the most active cytokinins tZ and iP, respectively. If one assumes a water content in most plant organs of about 80–90% (Flindt, 1988), the average concentrations of these cytokinins should vary between 0.7–2.5 nM, which is close to the experimentally measured KD. Similar cytokinin concentrations were found in maize xylem sap, with a maximum for tZ and tZR at 0.7 and 2 nM, respectively (Takei et al., 2001). Together, the accordance of the apparent KD with natural cytokinin concentrations and the reasonable correlation between binding and functional activities of different cytokinins support the biological relevance of the obtained data.
AHK3 and CRE1/AHK4 show a different preference for tZ- and iP-type cytokinins
Even minor changes in the structure of tZ reduce its affinity for the receptors, the degree of reduction depends on the type of change and is partly different for both receptors. The most prominent differences in ligand binding specificities of the receptors concerned iP, iPR, and DZ. iP and iPR bind approximately 10-fold more tightly to CRE1/AHK4 than to AHK3, whereas DZ has a much higher affinity for AHK3 than to CRE1/AHK4 (Fig. 5; Table 1; see supplementary material Fig. S2 at JXB online). Recent data on the ligand specificity of maize cytokinin receptors (sensor histidine kinases ZmHK1-ZmHK3, Yonekura-Sakakibara et al., 2004) allowed a preliminary comparison of the cytokinin perception systems in evolutionary distant plant species. According to sequence homology analysis, ZmHK1, ZmHK2, and ZmHK3 are closely related to CRE1/AHK4, AHK3, and AHK2, respectively. Qualitative studies on the basis of the bacterial promoter activation test have shown that ZmHK1-expressing bacteria strongly responded to iP, even more strongly than to tZ (Yonekura-Sakakibara et al., 2004). By contrast, in the case of ZmHK2, tZ was more active than iP. Thus, in terms of the relative effectiveness of tZ and iP, maize cytokinin receptors seem to resemble their Arabidopsis counterparts.
Ribosides, cis-zeatin and phenylurea derivatives have genuine affinities for AHK3
Direct binding assays provided ultimate proof that cytokinin ribosides (tZR, iPR), cis-zeatin and the non-adenine compound thidiazuron are recognized by AHK3. It is of particular relevance that our test-system prevents the enzymatic riboside cleavage (to produce free bases) and cis-trans isomerization of zeatins. cZ was purified from possible tZ contamination and showed measurable activity though much less (200–300-fold) compared with tZ (Table 1).
Thidiazurone, a phenylurea derivative, was shown to possess high affinity for both cytokinin receptors and bind to the same site as tZ (Fig. 6). Taking into account data that thidiazurone exerted a clear cytokinin effect in a promoter activation test (Inoue et al., 2001; Yamada et al., 2001; Spíchal et al., 2004, Yonekura-Sakakibara, 2004) as well as in planta (Spichal et al., 2004), the genuine cytokinin activity of this artificial growth regulator is now beyond question.
Cytokinin receptors differ in biochemical properties
The two cytokinin receptors differ not only in ligand specificity but in some biochemical properties as well. Cytokinin–receptor interaction is pH-, salt- and temperature-dependent. tZ-binding of AHK3 was much more pH-dependent than that of CRE1/AHK4, its binding to AHK3 sharply decreased at a lower pH (Fig. 2). The affinity of AHK3 for tZ was enhanced by Na+ and K+, while the affinity of CRE1/AHK4 was decreased (Fig. 3). By contrast, common divalent cations including Ca2+, Mg2+, and Mn2+ had, in physiological concentrations, no marked effect on binding. Also, AHK3 appeared to be less stable at elevated temperatures (Fig. 1) than CRE1/AHK4.
The pH dependence of the receptor–cytokinin association may have a role in crosstalk between different signals, in particular between cytokinin and auxin. Auxin is known to influence the pH in plant tissues through induction of a proton efflux from cells and acidification of the surrounding medium (Cleland, 2004). Such acidification might strongly reduce the affinity of receptors for cytokinins, in particular of AHK3, and may thus contribute to the biological cytokinin–antagonistic activity of auxin. In addition, the enhanced cytokinin binding to both receptors at neutral and alkaline pH could indicate some receptor function inside the plant cell, although data showing the location of AHK3 mainly on the plasma membrane were recently presented (Kim et al., 2006).
Cytokinin binding to receptors is resistant to primary alcohol
Evidence was found that butan-1-ol, which was shown to be an effective inhibitor of early cytokinin effects in different plants including Arabidopsis (Romanov et al., 2002), does not interfere directly with cytokinin perception. Different effectiveness of butan-1-ol and butan-2-ol led to the suggestion that phospholipase D, which is exclusively sensitive to primary and not to secondary alcohols, is involved in cytokinin signal transduction (Romanov et al., 2002). Our results show that concentrations of butan-1-ol, which effectively block the cytokinin effect in planta, are unable to suppress the binding of cytokinin to its receptors. These data indicate that not hormone perception but rather an unknown early stage of signal transduction is probably a target for primary alcohols.
Different receptor properties suggest specific functions in root-to-shoot communication
Different affinities of AHK3 and CRE1/AHK4 for different ligands may reflect their specific roles in root-to-shoot communication. An important feature of the AHK3 receptor is a strong preference of tZ-type cytokinins as compared to iP-type cytokinins. tZ-type cytokinins are predominant in the xylem sap (Takei et al., 2001, and references therein) moving from root to shoot, where AHK3 is predominantly expressed (Ueguchi et al., 2001a; Higuchi et al., 2004). On the other hand, in the phloem sap, iP-type cytokinins prevail (Corbesier et al., 2003). Thus it appears that AHK3 is tuned to respond to a long-distance signal coming from the roots. In this context it is interesting to note that AHK3 has an important role in regulating cytokinin-controlled processes in the shoot, such as leaf growth and leaf senescence (Riefler et al., 2006; Kim et al., 2006), which thus may be at least partly controlled by the root. Also, Z-type cytokinins induce a primary response gene (ZmRR1) in maize leaves much stronger that iP-type cytokinins (Takei et al., 2001). It is noteworthy that CRE1/AHK4, which is predominantly expressed in the root and fulfils important functions in vasculature development there (Mähönen et al., 2000; Inoue et al., 2001; Ueguchi et al., 2001a), recognizes tZ and iP very well. Therefore, this receptor may also react to phloem-derived cytokinin.
Finally, during recent years a body of evidence has accumulated showing that cytokinin regulation is very flexible and the signalling level might be finely adjusted for each tissue or even each individual cell (Riefler et al., 2006; Hwang and Sakakibara, 2006). Such an adjustment can be achieved through different molecular mechanisms. The results presented highlight the potential of the regulation at the initial stage of hormone signalling, i.e. hormone–receptor interaction.
|
Acknowledgements |
|---|
We acknowledge support of the DFG within the frame of the Collaborative Research Initiative ‘Structure and function of membrane receptors’ (Sfb 449) and the Russian Foundation for Basic Research project number 04-04-49120.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Bradford MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72 248–254.[CrossRef][ISI][Medline]
Cleland RE. (2004) Auxin and cell elongation. In Davies PJ (Ed.). Plant hormones: biosynthesis, signal transduction, actionDordrecht/Boston/London Kluwer Academic Publishers pp. 204–220.
Corbesier L, Prinsen E, Jacqmard A, Lejeune P, Van Onckelen H, Perilleux C, Bernier G. (2003) Cytokinin levels in leaves, leaf exudate and shoot apical meristem of Arabidopsis thaliana during floral transition. Journal of Experimental Botany 54 2511–2517.
Ferreira FJ and Kieber JJ. (2005) Cytokinin signaling. Current Opinion in Plant Biology 8 518–525.[CrossRef][ISI][Medline]
Flindt R. (1988) Biologie in ZahlenStuttgart, NY Gustav Fisher Verlag.
Heyl A and Schmülling T. (2003) Cytokinin signal perception and transduction. Current Opinion in Plant Biology 6 480–488.[CrossRef][ISI][Medline]
Heyl A, Werner T, Schmülling T. (2006) Cytokinin metabolism and signal transduction. In Hedden P and Thomas S (Eds.). Plant hormone signalling. Annual plant reviewsOxford Blackwell Publishing Ltd pp. 93–123.
Higuchi M, Pischke MS, Mähönen AP, et al. (2004) In planta functions of the Arabidopsis cytokinin receptor family. Proceedings of the National Academy of Sciences, USA 101 8821–8826.
Hwang I and Sakakibara H. (2006) Cytokinin biosynthesis and perception. Physiologia Plantarum 126 528–538.
Inoue T, Higuchi M, Hashimoto Y, Seki M, Kobayashi M, Kato T, Tabata S, Shinozaki K, Kakimoto T. (2001) Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409 1060–1063.[CrossRef][Medline]
Kakimoto T. (2003) Perception and signal transduction of cytokinins. Annual Review of Plant Biology 54 605–627.[CrossRef][Medline]
Kim HJ, Ryu H, Hong SH, Woo HR, Lim PO, Lee IC, Sheen J, Nam HG, Hwang I. (2006) Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis. Proceedings of the National Academy of Sciences, USA 103 814–819.
Mähönen AP, Bonke M, Kaupinnen L, Riikonen M, Benfey PN, Helariutta Y. (2000) A novel two-component hybrid molecule regulates vascular morphogenesis of the Arabidopsis root. Genes and Development 14 2938–2943.
Nishimura C, Ohashi Y, Sato S, Kato T, Tabata S, Ueguchi C. (2004) Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. The Plant Cell 16 1365–1377.
Riefler M, Novak O, Strnad M, Schmülling T. (2006) Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development and cytokinin metabolism. The Plant Cell 18 40–54.
Romanov GA, Kieber JJ, Schmülling T. (2002) A rapid cytokinin response assay in Arabidopsis indicates a role for phospholipase D in cytokinin signalling. FEBS Letters 515 39–43.[CrossRef][ISI][Medline]
Romanov GA, Spíchal L, Lomin SN, Strnad M, Schmülling T. (2005) A live cell hormone-binding assay on transgenic bacteria expressing a eukaryotic receptor protein. Analytical Biochemistry 347 129–134.[CrossRef][ISI][Medline]
Sambrook J and Russell DW. (2001) Molecular cloning: a laboratory manual 3rd edn Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press.
Scatchard G. (1949) The attraction of proteins for small molecules and ions. Annals of the New York Academy of Sciences 51 660–672.[CrossRef][ISI]
Spíchal L, Rakova NY, Riefler M, Mizuno T, Romanov GA, Strnad M, Schmülling T. (2004) Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay. Plant and Cell Physiology 45 1299–1305.
Suzuki T, Miwa K, Ishikawa K, Yamada H, Aiba H, Mizuno T. (2001) The Arabidopsis sensor His-kinase, AHK4, can respond to cytokinins. Plant and Cell Physiology 42 107–113.
Takei K, Sakakibara H, Taniguchi M, Sugiyama T. (2001) Nitrogen-dependent accumulation of cytokinins in root and the translocation to leaf: implication of cytokinin species that induces gene expression of maize response regulator. Plant and Cell Physiology 42 85–93.
Takei K, Ueda N, Aoki K, Kuromori T, Hirayama T, Shinozaki K, Yamaya T, Sakakibara H. (2004) AtIPT3 is a key determinant of nitrate-dependent cytokinin biosynthesis in Arabidopsis. Plant and Cell Physiology 45 1053–1062.
Ueguchi C, Koizumi H, Suzuki T, Mizuno T. (2001a) Novel family of sensor histidine kinase genes in Arabidopsis thaliana. Plant and Cell Physiology 42 231–235.
Ueguchi C, Sato S, Kato T, Tabata S. (2001b) The AHK4 gene involved in the cytokinin-signaling pathway as a direct receptor molecule in Arabidopsis thaliana. Plant and Cell Physiology 42 751–755.
Varfolomeev SD and Gurevich KG. (1999) Biokinetics. Practical course. (In Russian). Moscow FAIR Press pp. 335–496.
Yamada H, Suzuki T, Terada K, Takei K, Ishikawa K, Miwa K, Yamashino T, Mizuno T. (2001) The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane. Plant and Cell Physiology 42 1017–1023.
Yonekura-Sakakibara K, Kojima M, Yamaya T, Sakakibara H. (2004) Molecular characterization of cytokinin-responsive histidine kinases in maize. Differential ligand preferences and response to cis-zeatin. Plant Physiology 134 1654–1661.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  N. Hirose, K. Takei, T. Kuroha, T. Kamada-Nobusada, H. Hayashi, and H. Sakakibara Regulation of cytokinin biosynthesis, compartmentalization and translocation J. Exp. Bot., January 1, 2008; 59(1): 75 - 83. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Muller and J. Sheen Arabidopsis Cytokinin Signaling Pathway Sci. Signal., October 9, 2007; 2007(407): cm5 - cm5. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Muller and J. Sheen Advances in Cytokinin Signaling Science, October 5, 2007; 318(5847): 68 - 69. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||