AHK5 histidine kinase regulates root elongation through an ETR1-dependent abscisic acid and ethylene signaling pathway in Arabidopsis thaliana

The Arabidopsis thaliana genome encodes a small family of histidine (His) protein kinases, some of which have redundant functions as ethylene receptors, whereas others serve as cytokinin receptors. The most poorly characterized of these is authentic histidine kinase 5 (AHK5; also known as cytokinin-independent 2, CKI2). Here we characterize three independent ahk5 mutants, and show that they have a common phenotype. Our results suggest that AHK5 His-kinase acts as a negative regulator in the signaling pathway in which ethylene and ABA inhibit the root elongation through ETR1 (an ethylene receptor).     

The Arabidopsis sensor His-kinase, AHk4, can respond to cytokinins

His-to-Asp (His-->Asp) phosphorelay mechanisms are presumably involved in propagation of certain environmental stimuli, including phytohormones, in Arabidopsis thaliana. In addition to the previously characterized His-kinases, namely, the ETR1 family of ethylene receptors, CKI1 cytokinin-sensor, and ATHK1 osomo-sensor, this higher plant has three more His-kinases (named AHK2, AHK3, and AHK4). By employing the well-known His-->Asp phosphorelay systems in both the fission yeast and Escherichia coli, evidence is presented showing that the AHK4 His-kinase has an ability to serve as a cytokinin-responsive environmental sensor. Taking advantage of this AHK4-dependent His-->Asp phosphorelay system in E. coli, a phosphorelay interaction between the Arabidopsis His-kinase and histidine-containing phosphotransmitters (AHPs) was also demonstrated for the first time.     

Cytokinin receptor antagonists derived from 6-benzylaminopurine

Recently we reported 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) as the first molecule to antagonize cytokinin activity at the receptor level. Here we report the synthesis and in vitro biological testing of eleven BAP derivatives substituted in the benzyl ring and in the C2, N7 and N9 positions of the purine moiety. The ability of the compounds to interact with Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 was tested in bacterial receptor and in live-cell binding assays, and in an Arabidopsis ARR5:GUS (Arabidopsis response regulator 5) reporter gene assay. Cytokinin activity of the compounds was determined in classical cytokinin biotests (tobacco callus, wheat leaf senescence and Amaranthus bioassays). 6-(2,5-Dihydroxybenzylamino)purine (LGR-991) was identified as a cytokinin receptor antagonist. At the molecular level LGR-991 blocks the cytokinin receptor CRE1/AHK4 with the same potency as PI-55. Moreover, LGR-991 acts as a competitive inhibitor of AHK3, and importantly shows reduced agonistic effects in comparison to PI-55 in the ARR5:GUS reporter gene assay and in cytokinin bioassays. LGR-991 causes more rapid germination of Arabidopsis seeds and increases hypocotyl length of dark-grown seedlings, which are characteristics of plants with a reduced cytokinin status. LGR-991 exhibits a structural motive that might lead to preparation of cytokinin antagonists with a broader specificity and reduced agonistic properties.     

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.     

Cytokinin receptors are required for normal development of auxin-transporting vascular tissues in the hypocotyl but not in adventitious roots

Plants alter the architecture of their root systems to adapt to the environment by modulating post-embryonic (lateral and adventitious) root formation and growth. To understand better the genetic basis of this regulation, we screened ethylmethane sulfonate-mutagenized lines of Arabidopsis thaliana for adventitious rooting mutants. One mutant showed retardation of the primary root growth, no production of lateral roots and enhanced formation of adventitious roots. Mapping and genetic complementation revealed that this mutant named wooden leg-3 (wol-3) was an allele of ARABIDOPSIS HISTIDINE KINASE 4 (AHK4), a locus known to encode a cytokinin receptor. Although the vascular system of the primary root and hypocotyl in the wol-3 mutant was aborted, that of the adventitious roots was normally developed. In the hypocotyl of the wol-3 mutant, auxin signals accumulated around the aborted vascular system. The application of auxin to primary roots induced lateral root formation in the wol-3 mutant. Transport of radiolabeled auxin from the top of the hypocotyl to the primary root was inhibited in wol-3. Although only a single amino acid alteration had occurred in AHK4, the root morphology in the wol-3 mutant was quite similar to that in the ahk2 ahk3 ahk4 triple mutant, which is a loss-of-function mutant of the three cytokinin receptors. This implies that the functional disturbance of AHK4 affects the function of the other receptors. Our results suggest that cytokinin receptors are necessary for the formation of auxin-transporting vascular tissues in the hypocotyl, but not in adventitious roots.     

The Arabidopsis AHK4 histidine kinase is a cytokinin-binding receptor that transduces cytokinin signals across the membrane

Common histidine-to-aspartate (His-->Asp) phosphorelay is a paradigm of signal transduction in both prokaryotes and eukaryotes for the propagation of certain environmental stimuli, in which histidine (His)-kinases play central roles as sensors for environmental signals. For the higher plant, Arabidopsis thaliana, it was recently suggested that the His-kinase (AHK4 / CRE1 / WOL) is a sensor for cytokinins, which are a class of plant hormones important for the regulation of cell division and differentiation. Interestingly, AHK4 is capable of functioning as a cytokinin sensor in the eubacterium, Escherichia coli (Suzuki et al. 2001, Plant Cell Physiol. 42: 107). Here we further show that AHK4 is a primary receptor that directly binds a variety of natural and synthetic cytokinins (e.g. not only N(6)-substituted aminopurines such as isopentenyl-adenine, trans-zeatin, benzyl-adenine, but also diphenylurea derivatives such as thidiazuron), in a highly specific manner (K(d) = 4.55+/-0.48x10(-9) M). AHK4 has a presumed extracellular domain, within which a single amino acid substitution (Thr-301 to Ile) was shown to result in loss of its ability to bind cytokinins. This particular mutation corresponds to the previously reported wol allele (wooden leg) that causes a striking phenotype defective in vascular morphogenesis. Collectively, evidence is presented that AHK4 and its homologues (AHK3 and possibly AHK2) are receptor kinases that can transduce cytokinin signals across the plasma membrane of A. thaliana.     

Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay

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 K(D) of 1-2 nM for the AHK3 receptor-hormone complex, which is close to the K(D) (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 (3)H-zeatin, and influenced by monovalent cations, while divalent cations (Ca(2+), Mg(2+), Mn(2+)) 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.     

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.     

Interaction between phosphate-starvation, sugar, and cytokinin signaling in Arabidopsis and the roles of cytokinin receptors CRE1/AHK4 and AHK3

Cytokinins control key processes during plant growth and development, and cytokinin receptors CYTOKININ RESPONSE 1/WOODEN LEG/ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/WOL/AHK4), AHK2, and AHK3 have been shown to play a crucial role in this control. The involvement of cytokinins in signaling the status of several nutrients, such as sugar, nitrogen, sulfur, and phosphate (Pi), has also been highlighted, although the full physiological relevance of this role remains unclear. To gain further insights into this aspect of cytokinin action, we characterized a mutant with reduced sensitivity to cytokinin repression of a Pi starvation-responsive reporter gene and show it corresponds to AHK3. As expected, ahk3 displayed reduced responsiveness to cytokinin in callus proliferation and plant growth assays. In addition, ahk3 showed reduced cytokinin repression of several Pi starvation-responsive genes and increased sucrose sensitivity. These effects of the ahk3 mutation were especially evident in combination with the cre1 mutation, indicating partial functional redundancy between these receptors. We examined the effect of these mutations on Pi-starvation responses and found that the double mutant is not significantly affected in long-distance systemic repression of these responses. Remarkably, we found that expression of many Pi-responsive genes is stimulated by sucrose in shoots and to a lesser extent in roots, and the sugar effect in shoots of Pi-starved plants was particularly enhanced in the cre1 ahk3 double mutant. Altogether, these results indicate the existence of multidirectional cross regulation between cytokinin, sugar, and Pi-starvation signaling, thus underlining the role of cytokinin signaling in nutrient sensing and the relative importance of Pi-starvation signaling in the control of plant metabolism and development.     

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.     

Evidence for the localization of the Arabidopsis cytokinin receptors AHK3 and AHK4 in the endoplasmic reticulum

Cytokinins are hormones that are involved in various processes of plant growth and development. The model of cytokinin signalling starts with hormone perception through membrane-localized histidine kinase receptors. Although the biochemical properties and functions of these receptors have been extensively studied, there is no solid proof of their subcellular localization. Here, cell biological and biochemical evidence for the localization of functional fluorophor-tagged fusions of Arabidopsis histidine kinase 3 (AHK3) and 4 (AHK4), members of the cytokinin receptor family, in the endoplasmic reticulum (ER) is provided. Furthermore, membrane-bound AHK3 interacts with AHK4 in vivo. The ER localization and putative function of cytokinin receptors from the ER have major impacts on the concept of cytokinin perception and signalling, and hormonal cross-talk in plants.     

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.     

Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: A structural study

Cytokinin oxidase/dehydrogenase (CKO) is a flavoenzyme, which irreversibly degrades the plant hormones cytokinins and thereby participates in their homeostasis. Several synthetic cytokinins including urea derivatives are known CKO inhibitors but structural data explaining enzyme–inhibitor interactions are lacking. Thus, an inhibitory study with numerous urea derivatives was undertaken using the maize enzyme (ZmCKO1) and the crystal structure of ZmCKO1 in a complex with N-(2-chloro-pyridin-4-yl)-N′-phenylurea (CPPU) was solved. CPPU binds in a planar conformation and competes for the same binding site with natural substrates like N⁶-(2-isopentenyl)adenine (iP) and zeatin (Z). Nitrogens at the urea backbone are hydrogen bonded to the putative active site base Asp169. Subsequently, site-directed mutagenesis of L492 and E381 residues involved in the inhibitor binding was performed. The crystal structures of L492A mutant in a complex with CPPU and N-(2-chloro-pyridin-4-yl)-N′-benzylurea (CPBU) were solved and confirm the importance of a stacking interaction between the 2-chloro-4-pyridinyl ring of the inhibitor and the isoalloxazine ring of the FAD cofactor. Amino derivatives like N-(2-amino-pyridin-4-yl)-N′-phenylurea (APPU) inhibited ZmCKO1 more efficiently than CPPU, as opposed to the inhibition of E381A/S mutants, emphasizing the importance of this residue for inhibitor binding. As highly specific CKO inhibitors without undesired side effects are of major interest for physiological studies, all studied compounds were further analyzed for cytokinin activity in the Amaranthus bioassay and for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4. By contrast to CPPU itself, APPU and several benzylureas bind only negligibly to the receptors and exhibit weak cytokinin activity.     

The cytokinin receptors of Arabidopsis are located mainly to the endoplasmic reticulum

The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Arabidopsis (Arabidopsis thaliana) possesses three cytokinin receptors: ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1/AHK4. The current model predicts perception of the cytokinin signal at the plasma membrane. However, cytokinin-binding studies with membrane fractions separated by two-phase partitioning showed that in the wild type, as well as in mutants retaining only single cytokinin receptors, the major part of specific cytokinin binding was associated with endomembranes. Leaf epidermal cells of tobacco (Nicotiana benthamiana) expressing receptor-green fluorescent protein fusion proteins and bimolecular fluorescence complementation analysis showed strong fluorescence of the endoplasmic reticulum (ER) network for all three receptors. Furthermore, separation of the microsomal fraction of Arabidopsis plants expressing Myc-tagged AHK2 and AHK3 receptors by sucrose gradient centrifugation followed by immunoblotting displayed the Mg2?-dependent density shift typical of ER membrane proteins. Cytokinin-binding assays, fluorescent fusion proteins, and biochemical fractionation all showed that the large majority of cytokinin receptors are localized to the ER, suggesting a central role of this compartment in cytokinin signaling. A modified model for cytokinin signaling is proposed.     

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.     

Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.