Structural basis for cytokinin recognition by Arabidopsis thaliana histidine kinase 4

Cytokinins are classic hormones that orchestrate plant growth and development and the integrity of stem cell populations. Cytokinin receptors are eukaryotic sensor histidine kinases that are activated by both naturally occurring adenine-type cytokinins and urea-based synthetic compounds. Crystal structures of the Arabidopsis thaliana histidine kinase 4 sensor domain in complex with different cytokinin ligands now rationalize the hormone-binding specificity of the receptor and may spur the design of new cytokinin ligands.     

Identification of amino acid substitutions that render the Arabidopsis cytokinin receptor histidine kinase AHK4 constitutively active

In Arabidopsis, three genes (AHK2, AHK3 and AHK4/CRE1) encode histidine kinases (His-kinases), which serve as cytokinin receptors. To understand how the external cytokinin signal activates the His-kinase across the cell membrane, we exploited the power of microbial genetics to isolate several AHK4 mutants that function independently of cytokinin in both prokaryotic and eukaryotic assay systems. In each mutant, a single amino acid substitution within the second membrane-spanning segment, or within the region around the phosphorylation His site, renders the His-kinase constitutively active. These mutant receptors appear to have a 'locked-on' conformation, even in the absence of stimulus. We discuss the implications of these data for the structure and function of the cytokinin receptor His-kinases in plants.     

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.     

Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis

Cytokinins are plant hormones that may play essential and crucial roles in various aspects of plant growth and development. Although the functional significance of exogenous cytokinins as to the proliferation and differentiation of cells has been well documented, the biological roles of endogenous cytokinins have remained largely unknown. The recent discovery of the Arabidopsis Histidine Kinase 4 (AHK4)/CRE1/WOL cytokinin receptor in Arabidopsis thaliana strongly suggested that the cellular response to cytokinins involves a two-component signal transduction system. However, the lack of an apparent phenotype in the mutant, presumably because of genetic redundancy, prevented us from determining the in planta roles of the cytokinin receptor. To gain insight into the molecular functions of the three AHK genes AHK2, AHK3, and AHK4 in this study, we identified mutational alleles of the AHK2 and AHK3 genes, both of which encode sensor histidine kinases closely related to AHK4, and constructed a set of multiple ahk mutants. Application of exogenous cytokinins to the resultant strains revealed that both AHK2 and AHK3 function as positive regulators for cytokinin signaling similar to AHK4. The ahk2 ahk4 and ahk3 ahk4 double mutants and the ahk single mutants grew normally, whereas the ahk2 ahk3 double mutants exhibited a semidwarf phenotype as to shoots, such as a reduced leaf size and a reduced influorescence stem length. The growth and development of the ahk2 ahk3 ahk4 triple mutant were markedly inhibited in various tissues and organs, including the roots and leaves in the vegetative growth phase and the influorescence meristem in the reproductive phase. We showed that the inhibition of growth is associated with reduced meristematic activity of cells. Expression analysis involving AHK:beta-glucuronidase fusion genes suggested that the AHK genes are expressed ubiquitously in various tissues during postembryonic growth and development. Our results thus strongly suggest that the primary functions of AHK genes, and those of endogenous cytokinins, are triggering of the cell division and maintenance of the meristematic competence of cells to prevent subsequent differentiation until a sufficient number of cells has accumulated during organogenesis.     

Adenosine kinase contributes to cytokinin interconversion in Arabidopsis

Purine salvage enzymes have been implicated, but not proven, to be involved in the interconversion of cytokinin (CK) bases, ribosides, and nucleotides. Here, we use Arabidopsis (Arabidopsis thaliana) lines silenced in adenosine kinase (ADK) expression to understand the contributions of this enzyme activity to in vivo CK metabolism. Both small interfering RNA- and artificial microRNA-mediated silencing of ADK led to impaired root growth, small, crinkled rosette leaves, and reduced apical dominance. Further examination of ADK-deficient roots and leaves revealed their irregular cell division. Root tips had uneven arrangements of root cap cells, reduced meristem sizes, and enlarged cells in the elongation zone; rosette leaves exhibited decreased cell size but increased cell abundance. Expression patterns of the cyclinB1;1::β-glucuronidase and Arabidopsis Response Regulator5::β-glucuronidase reporters in the ADK-deficient background were consistent with altered cell division and an increase in CK activity, respectively. In vivo feeding of ADK-deficient leaves with radiolabeled CK ribosides of isopentenyladenosine and zeatin showed a decreased flux into the corresponding CK nucleotides. Comprehensive high-performance liquid chromatography-tandem mass spectrometry analysis detected significantly higher levels of active CK ribosides in both sense ADK and artificial microADK. Taken together, these metabolic and phenotypic analyses of ADK-deficient lines indicate that ADK contributes to CK homeostasis in vivo.     

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.     

Properties, functions and evolution of cytokinin receptors

The discovery of cytokinin receptors of Arabidopsis thaliana ten years ago was a milestone in plant hormone research. Since then, research has yielded insights into the biochemical properties and functions of these sensor histidine kinases. Their affinities to both trans-zeatin and isopentenyladenine are in the low nM range. Cytokinin ribosides, cis-zeatin and thidiazuron were established as compounds with genuine cytokinin activity and the first cytokinin antagonists were identified. Numerous functions of cytokinin receptors in plant development, as well as in the plant's responses to the environment, have been elucidated and are summarized. Finally, we address the question how the receptors have evolved during plant evolution.     

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.     

Different cytokinin histidine kinase receptors regulate nodule initiation as well as later nodule developmental stages in Medicago truncatula

Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen‐fixing soil bacteria to form a new specific organ: the nitrogen‐fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other putative CHASE‐domain containing histidine kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCHK1/MtCRE1 has the strongest expression at all stages. Mutant and knock‐down analyses revealed that other CHKs than MtCHK1/CRE1 are positively involved in nodule initiation, which explains the delayed nodulation phenotype of the chk1/cre1 mutant. In addition, cre1 nodules exhibit an increased growth, whereas other chk mutants have no detectable phenotype, and the maintained nitrogen fixation capacity in cre1 requires other CHK genes. Interestingly, an AHK4/CRE1 genomic locus from the aposymbiotic Arabidopsis plant rescues nodule initiation but not the nitrogen fixation capacity. This indicates that different CHK cytokinin signalling pathways regulate not only nodule initiation but also later developmental stages, and that legume‐specific determinants encoded by the MtCRE1 gene are required for later nodulation stages than initiation.     

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.     

Advances in upstream players of cytokinin phosphorelay: receptors and histidine phosphotransfer proteins

Cytokinins are a class of plant hormones that have been linked to numerous growth and developmental aspects in plants. The cytokinin signal is perceived by sensor histidine kinase receptors and transmitted via histidine phosphotransfer proteins (HPts) to downstream response regulators. Since their discovery, cytokinin receptors have been a focus of interest for many researchers. Ongoing research on these transmembrane receptors has greatly broadened our knowledge in terms of cytokinin–receptor interaction, receptor specificity, receptor cellular localization, and receptor functions in cytokinin related growth and developmental processes. This review focuses on the recent advances on the cytokinin receptors and HPt proteins in Arabidopsis.     

Novel family of sensor histidine kinase genes in Arabidopsis thaliana

We identified three novel, highly homologous, sensor histidine kinases that possibly function in the plasma membrane of Arabidopsis thaliana, i.e. AHK2, 3 and 4. While AHK2 and 3 are expressed in several organs, AHK4 is mainly expressed in roots. AHK3 suppresses a sensor histidine kinase mutant of yeast.     

Azospirillum brasilense Sp245 triggers cytokinin signaling in root tips and improves biomass accumulation in Arabidopsis through canonical cytokinin receptors

The plant growth promoting rhizobacterium Azospirillum brasilense Sp245 enhances biomass production in cereals and horticultural species and is an interesting model to study the physiology of the phytostimulation program. Although auxin production by Azospirillum appears to be critical for root architectural readjustments, the role of cytokinins in the growth promoting effects of Azospirillum remains unclear. Here, Arabidopsis thaliana seedlings were co-cultivated in vitro with A. brasilense Sp245 to assess whether direct contact of roots with bacterial colonies or exposure to the bacterial volatiles using divided Petri plates would affect biomass production and root organogenesis. Both interaction types increased root and shoot fresh weight but had contrasting effects on primary root length, lateral root formation and root hair development. Cell proliferation in root meristems analyzed with the CYCB1;1::GUS reporter decreased over time with direct contact, but was augmented by plant exposure to volatiles. Noteworthy, the expression of the cytokinin-inducible reporters TCS::GFP and ARR5::GUS increased in root tips in response to bacterial contact, without being affected by the volatiles. In A. thaliana having single (cre1-12, ahk2-2, ahk3-3), double (cre1-12/ahk2-2, cre1-12/ahk3-3, ahk2-2/ahk3-3) or triple (cre1-12/ahk2-2/ahk3-3) mutations in canonical cytokinin receptors, only the triple mutant had a marked effect on plant growth in response to A. brasilense. These results show that different mechanisms are elicited by A. brasilense, which influence the cytokinin-signaling pathway.     

Characterization of the ARR15 and ARR16 response regulators with special reference to the cytokinin signaling pathway mediated by the AHK4 histidine kinase in roots of Arabidopsis thaliana

The cytokinin receptor AHK4 histidine kinase, identified in Arabidopsis thaliana, presumably acts in concert with downstream components, such as histidine-containing phosphotransfer (HPt) factors (AHPs) and response regulators (ARRs). In this respect, we characterized a loss-of-function mutant of the AHK4 gene, named cre1-1, which showed a reduced cell number within the vascular tissues in roots. Among the 10 type-A ARR members, the expression of ARR15 and ARR16 in roots was specifically and markedly reduced in cre1-1, suggesting a link between these response regulators and the AHK4-mediated signal transduction in roots. The results for transgenic plants expressing promoter::GUS or promoter::LUC fusion genes showed that both the ARR15 and the ARR16 gene products are accumulated upon cytokinin treatment in roots. The results of GFP-fusion experiments with onion epidermal cells further showed that ARR15 was found in the nucleus, and ARR16 mainly in the cytoplasm. Together, it was suggested that ARR15 and ARR16 are distinctly implicated in the presumed AHK4-mediated signaling pathway in roots.     

AtSKIP functions as a mediator between cytokinin and light signaling pathway in Arabidopsis thaliana

Key message

AtSKIP participated in cytokinin-regulated leaf initiation. Putative phosphorylated AtSKIP (AtSKIP ^DD ) displayed the opposite function in the leaf development from AtSKIP transgenic seedlings.

Abstract

AtSKIP, as a multiple protein, is involved in many physiological processes, such as flowering, cell cycle regulator, photomorphogenesis and stress tolerance. However, the mechanism of AtSKIP in these processes is unclear. Here, we identify one gene, AtSKIP, which is associated with cytokinin-regulated leaf growth process in Arabidopsis. The expression of AtSKIP was regulated by cytokinin. Leaf development in AtSKIP overproduced seedlings was independent of light, but promoted by cytokinin, and phosphorylation of AtSKIP (AtSKIP^DD) partially interfered with AtSKIP function as a positive regulator in cytokinin signaling, indicative of true leaf formation, and the defects of AtSKIP^DD in the true leaf formation could be recovered to some extent by the addition of cytokinin. Moreover, different cytokinin-responsive gene Authentic Response Regulator 7 (ARR7) promoter-GUS activity further proved that expression of AtSKIP or AtSKIP^DD altered endogenous cytokinin signaling in plants. Together, these data indicate that AtSKIP participates in cytokinin-regulated promotion of leaf growth in photomorphogenesis, and that phosphorylation interferes with AtSKIP normal function.     

A two-component histidine kinase gene that functions in Dictyostelium development.

A mutant which failed to complete development was isolated from a population of cells that had been subjected to insertional mutagenesis using restriction enzyme-mediated integration. The disrupted gene, dhkA, encodes the conserved motifs of a histidine kinase as well as the response regulator domain. It is likely that the histidine in DhkA is autophosphorylated and the phosphate passed to one or more response regulators. Such two-component systems function in a variety of bacterial signal transduction pathways and have been characterized recently in yeast and Arabidopsis. In Dictyostelium, we found that DhkA functions both in the regulation of prestalk gene expression and in the control of the terminal differentiation of prespore cells.