Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism

We used loss-of-function mutants to study three Arabidopsis thaliana sensor histidine kinases, AHK2, AHK3, and CRE1/AHK4, known to be cytokinin receptors. Mutant seeds had more rapid germination, reduced requirement for light, and decreased far-red light sensitivity, unraveling cytokinin functions in seed germination control. Triple mutant seeds were more than twice as large as wild-type seeds. Genetic analysis indicated a cytokinin-dependent endospermal and/or maternal control of embryo size. Unchanged red light sensitivity of mutant hypocotyl elongation suggests that previously reported modulation of red light signaling by A-type response regulators may not depend on cytokinin. Combined loss of AHK2 and AHK3 led to the most prominent changes during vegetative development. Leaves of ahk2 ahk3 mutants formed fewer cells, had reduced chlorophyll content, and lacked the cytokinin-dependent inhibition of dark-induced chlorophyll loss, indicating a prominent role of AHK2 and, particularly, AHK3 in the control of leaf development. ahk2 ahk3 double mutants developed a strongly enhanced root system through faster growth of the primary root and, more importantly, increased branching. This result supports a negative regulatory role for cytokinin in root growth regulation. Increased cytokinin content of receptor mutants indicates a homeostatic control of steady state cytokinin levels through signaling. Together, the analyses reveal partially redundant functions of the cytokinin receptors and prominent roles for the AHK2/AHK3 receptor combination in quantitative control of organ growth in plants, with opposite regulatory functions in roots and shoots.     

Membrane transport and cytokinin action in root hairs of Medicago sativa

In an effort to develop a cellular model for studying cytokinin action in higher plants, we investigated the effect of cytokinins on growth and membrane transport in root hairs of alfalfa (Medicago sativa␣L.) seedlings. Alfalfa seedlings grown for 24 h in the presence of cytokinins showed increased root hair length and formation of root hairs close to the root cap. Increased growth of root hairs was observed within 10 min of cytokinin application and was accompanied by a hyperpolarization of the plasma membrane. The ion-transport inhibitor diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) blocked both cytokinin-induced root hair growth and hyperpolarization but did not, by itself, alter growth or membrane potential. Finally, hyperpolarization was induced by extracellular cytokinin but not by injection of cytokinin into the cytoplasm. These findings show that root hairs undergo several rapid responses to cytokinin, including changes in membrane transport and growth. We conclude that multiple cytokinin response in root hairs may be mediated by events involving perception and ion transport at the plasma membrane. Received: 18 July 1996 / Accepted: 1 October 1996     

The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling

Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.     

N-glucosyltransferase UGT76C2 is involved in cytokinin homeostasis and cytokinin response in Arabidopsis thaliana

Cytokinins are a class of phytohormones that play a crucial role in plant growth and development. The gene UGT76C2 encoding cytokinin N-glucosyltransferase of Arabidopsis thaliana has been previously identified. To determine the in planta role of UGT76C2 in cytokinin metabolism and response, we analyzed the phenotypes of its loss-of-function mutant (ugt76c2) and its overexpressors. The accumulation level of the cytokinin N-glucosides was significantly decreased in ugt76c2, but substantially increased in UGT76C2 overexpressors compared with the wild type. When treated with exogenously applied cytokinin, ugt76c2 showed more sensitivity and UGT76C2 overexpressors showed less sensitivity to cytokinin in primary root elongation, lateral root formation, Chl retention and anthocyanin accumulation. Under normal growth conditions ugt76c2 had smaller seeds than the wild type, with accompanying lowered levels of active and N-glucosylated cytokinin forms. The expression levels of cytokinin-related genes such as AHK2, AHK3, ARR1, IPT5 and CKX3 were changed in ugt76c2, suggesting homeostatic control of cytokinin activity. Studies of spatiotemporal expression patterns showed that UGT76C2 was expressed at a relatively higher level in the seedling and developing seed. In their entirety, our data, based mainly on this comparison and opposite phenotypes of knockout and overexpressors, strongly suggest that UGT76C2 is involved in cytokinin homeostasis and cytokinin response in planta through cytokinin N-glucosylation.     

Stress physiology functions of the Arabidopsis histidine kinase cytokinin receptors

Cytokinin signaling has complex effects on abiotic stress responses that remain to be fully elucidated. The Arabidopsis histidine kinases (AHKs), AHK2, AHK3 and CRE1 (cytokinin response1/AHK4) are the principle cytokinin receptors of Arabidopsis. Using a set of ahk mutants, we found dramatic differences in response to low water potential and salt stress among the AHKs. ahk3‐3 mutants had increased root elongation after transfer to low water potential media. Conversely ahk2‐2 was hypersensitive to salt stress in terms of root growth and fresh weight and accumulated higher than wild‐type levels of proline specifically under salt stress. Strongly reduced proline accumulation in ahk double mutants after low water potential treatment indicated a more general role of cytokinin signaling in proline metabolism. Reduced P5CS1 (Δ¹‐pyrroline‐5‐carboxylate synthetase1) gene expression may have contributed to this reduced proline accumulation. Low water potential phenotypes of ahk mutants were not caused by altered abscisic acid (ABA) accumulation as all ahk mutants had wild‐type ABA levels, despite the observation that ahk double mutants had reduced NCED3 (9‐cis‐epoxycartenoid dioxygenase3) expression when exposed to low water potential. No difference in osmoregulatory solute accumulation was detected in any of the ahk mutants indicating that they do not affect drought responsive osmotic adjustment. Overall, our examination of ahk mutants found specific phenotypes associated with AHK2 and AHK3 as well as a general function of cytokinin signaling in proline accumulation and low water potential induction of P5CS1 and NCED3 expression. These results show the stress physiology function of AHKs at a new level of detail.     

Cytokinins regulate a bidirectional phosphorelay network in Arabidopsis

The cytokinin class of plant hormones plays key roles in regulating diverse developmental and physiological processes. Arabidopsis perceives cytokinins with three related and partially redundant receptor histidine kinases (HKs): CRE1 (the same protein as WOL and AHK4), AHK2, and AHK3 (CRE-family receptors). It is suggested that binding of cytokinins induces autophosphorylation of these HKs and subsequent transfer of the phosphoryl group to a histidine phosphotransfer protein (HPt) and then to a response regulator (RR), ultimately regulating downstream signaling events. Here we demonstrate that, in vitro and in a yeast system, CRE1 is not only a kinase that phosphorylates HPts in the presence of cytokinin but is also a phosphatase that dephosphorylates HPts in the absence of cytokinin. To explore the roles of these activities in planta, we replaced CRE1 with mutant versions of the gene or with AHK2. Replacing CRE1 with CRE1(T278I), which lacks cytokinin binding activity and is locked in the phosphatase form, decreased cytokinin sensitivity. Conversely, replacing CRE1 with AHK2, which favors kinase activity, increased cytokinin sensitivity. These results indicate that in the presence of cytokinins, cytokinin receptors feed phosphate to phosphorelay-integrating HPt proteins. In the absence of cytokinins, CRE1 removes phosphate from HPt proteins, decreasing the system phosphoload.     

Design and synthesis of photolabile caged cytokinin

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.     

Plant growth promotion by Bacillus megaterium involves cytokinin signaling

Accumulating evidence indicates that plant growth promoting rhizobacteria (PGPR) influence plant growth and development by the production of phytohormones such as auxins, gibberellins, and cytokinins. Little is known on the genetic basis and signal transduction components that mediate the beneficial effects of PGPRs in plants. We recently reported the identification of a Bacillus megaterium strain that promoted growth of A. thaliana and P. vulgaris seedlings. In this addendum, the role of cytokinin signaling in mediating the plant responses to bacterial inoculation was investigated using A. thaliana mutants lacking one, two or three of the putative cytokinin receptors CRE1, AHK2 and AHK3, and RPN12 a gene involved in cytokinin signaling. We show that plant growth promotion by B. megaterium is reduced in AHK2-2 single and double mutant combinations and in RPN12. Furthermore, the triple cytokinin-receptor CRE1-12/AHK2-2/AHK3-3 knockout was insensitive to inoculation in terms of growth promotion and root developmental responses. Our results indicate that cytokinin receptors play a complimentary role in plant growth promotion by B. megaterium.Key words: Arabidopsis, plant growth stimulation, root development, rhizobacteria     

The subcellular distribution of the Arabidopsis histidine phosphotransfer proteins is independent of cytokinin signaling

Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two‐component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine‐kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear‐localized response regulators (Type‐A and Type‐B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.     

The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors

Arabidopsis thaliana has three membrane‐located cytokinin receptors (AHK2, AHK3 and CRE1/AHK4), which are sensor histidine kinases containing a ligand‐binding CHASE domain. Despite their structural similarity the role of these receptors differs in planta. Here we have explored which parameters contribute to signal specification. In a bacterial assay, the CHASE domain of AHK2 has a similar ligand binding spectrum as CRE1/AHK4. It shows the highest affinity for isopentenyladenine (iP) and trans‐zeatin (tZ) with an apparent K_D of 1.4 and 4.0 nm , respectively. Real‐time PCR analysis of cytokinin primary response genes in double mutants retaining only single receptors revealed that all receptors are activated in planta by cytokinin concentrations in the low nanomolar range. However, there are differences in sensitivity towards the principal cytokinins iP and tZ. The activation of the cytokinin‐sensitive P_ARR5:GUS reporter gene in three different double mutants shows specific, but also overlapping, spatial domains of activity, which were for all receptors predominantly in the shoot apical meristems and root cap columella. AHK2 and AHK3 signal specifically in leaf parenchyma cells, AHK3 in stomata cells, and CRE1/AHK4 in the root vasculature. Promoter‐swap experiments demonstrate that CRE1/AHK4 can functionally replace AHK2 but not AHK3. However, the cytoplasmic AHK3 histidine kinase (Hk) domain can be replaced by the CRE1/AHK4 Hk domain, which suggests that functionality is mediated in this case by the extracytosolic domain. Together, the data show that both differential gene expression and ligand preference contribute to specify the receptor activity.     

Two cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, differ in their ligand specificity in a bacterial assay

Strains of Escherichia coli that express two different cytokinin receptors of Arabidopsis thaliana, CRE1/AHK4 and AHK3, were used to study the relative sensitivity of these receptors to various cytokinins. Both receptors were most sensitive to the bases of the isoprenoid-type cytokinins trans-zeatin and isopentenyladenine but differed significantly in the recognition of other cytokinin compounds. In particular, CRE1/AHK4 recognized at 1 microm concentration only trans-zeatin while AHK3 recognized cis-zeatin and dihydrozeatin as well, although with a lower sensitivity. Similarly, CRE1/AHK4 was not activated by cytokinin ribosides and ribotides, but AHK3 was. Comparisons using the ARR5::GUS fusion gene as a cytokinin reporter in Arabidopsis showed similar relative degrees of responses in planta, except that cytokinins with aromatic side chains showed much higher activities than in the bacterial assay. These results indicate that the diverse cytokinin compounds might have specific functions in the numerous cytokinin-regulated processes, which may depend in turn on different receptors and their associated signalling pathways. The importance of precise control of local concentrations of defined cytokinin metabolites to regulate the respective downstream event is corroborated.     

A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation

Cytokinin is an adenine derivative plant hormone that generally regulates plant cell division and differentiation in conjunction with auxin. We report that a major cue for the negative regulation of sulfur acquisition is executed by cytokinin response 1 (CRE1)/wooden leg (WOL)/Arabidopsis histidine kinase 4 (AHK4) cytokinin receptor in Arabidopsis root. We constructed a green fluorescent protein (GFP) reporter system that generally displays the expression of the high-affinity sulfate transporter SULTR1;2 in Arabidopsis roots. GFP under the control of SULTR1;2 promoter showed typical sulfur responses that correlate with the changes in SULTR1;2 mRNA levels; accumulation of GFP was induced by sulfur limitation (-S), but was repressed in the presence of reduced sulfur compounds. Among the plant hormones tested, cytokinin significantly downregulated the expression of SULTR1;2. SULTR1;1 conducting sulfate uptake in sultr1;2 mutant was similarly downregulated by cytokinin. Downregulation of SULTR1;1 and SULTR1;2 by cytokinin correlated with the decrease in sulfate uptake activities in roots. The effect of cytokinin on sulfate uptake was moderated in the cre1-1 mutant, providing genetic evidence for involvement of CRE1/WOL/AHK4 in the negative regulation of high-affinity sulfate transporters. These data demonstrated the physiological importance of the cytokinin-dependent regulatory pathway in acquisition of sulfate in roots. Our results suggested that two different modes of regulation, represented as the -S induction and the cytokinin-dependent repression of sulfate transporters, independently control the uptake of sulfate in Arabidopsis roots.     

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.