The origin and early evolution of cytokinin signaling.

Angiosperms, especially Arabidopsis and rice, have long been at the center of plant research. However, technological advances in sequencing have led to a dramatic increase in genome and transcriptome data availability across land plants and, more recently, among green algae. These data allowed for an in-depth study of the evolution of different protein families - including those involved in the metabolism and signaling of phytohormones. While most early studies on phytohormone evolution were phylogenetic, those studies have started to be complemented by genetic and biochemical studies in recent years. Examples of such functional analyses focused on ethylene, jasmonic acid, abscisic acid, and auxin. These data have been summarized recently. In this review, we will focus on the progress in our understanding of cytokinin biology. We will use these data to synthesize key points about the evolution of cytokinin metabolism and signaling, which might apply to the evolution of other phytohormones as well.

Cytokinin Response Factor 9 Represses Cytokinin Responses in Flower Development.

A multi-step phosphorelay system is the main conduit of cytokinin signal transduction. However, several groups of additional factors that also play a role in this signaling pathway have been found-among them the Cytokinin Response Factors (CRFs). In a genetic screen, CRF9 was identified as a regulator of the transcriptional cytokinin response. It is mainly expressed in flowers. Mutational analysis indicates that CRF9 plays a role in the transition from vegetative to reproductive growth and silique development. The CRF9 protein is localized in the nucleus and functions as a transcriptional repressor of Arabidopsis Response Regulator 6 (ARR6)-a primary response gene for cytokinin signaling. The experimental data suggest that CRF9 functions as a repressor of cytokinin during reproductive development.

Cytokinin Perception in Ancient Plants beyond Angiospermae.

Cytokinins (CKs) control many plant developmental processes and responses to environmental cues. Although the CK signaling is well understood, we are only beginning to decipher its evolution. Here, we investigated the CK perception apparatus in early-divergent plant species such as bryophyte Physcomitrium patens, lycophyte Selaginella moellendorffii, and gymnosperm Picea abies. Of the eight CHASE-domain containing histidine kinases (CHKs) examined, two CHKs, PpCHK3 and PpCHK4, did not bind CKs. All other CHK receptors showed high-affinity CK binding (KD of nM range), with a strong preference for isopentenyladenine over other CK nucleobases in the moss and for trans-zeatin over cis-zeatin in the gymnosperm. The pH dependences of CK binding for these six CHKs showed a wide range, which may indicate different subcellular localization of these receptors at either the plasma- or endoplasmic reticulum membrane. Thus, the properties of the whole CK perception apparatuses in early-divergent lineages were demonstrated. Data show that during land plant evolution there was a diversification of the ligand specificity of various CHKs, in particular, the rise in preference for trans-zeatin over cis-zeatin, which indicates a steadily increasing specialization of receptors to various CKs. Finally, this distinct preference of individual receptors to different CK versions culminated in vascular plants, especially angiosperms.

Characterization of CHARK, an unusual cytokinin receptor of rice.

The signal transduction of the plant hormone cytokinin is mediated by a His-to-Asp phosphorelay. The canonical cytokinin receptor consists of an extra cytoplasmic hormone binding domain named cyclase/histidine kinase associated sensory extracellular (CHASE) and cytoplasmic histidine kinase and receiver domains. In addition to classical cytokinin receptors, a different type receptor-named CHASE domain receptor serine/threonine kinase (CHARK)-is also present in rice. It contains the same ligand binding domain as other cytokinin receptors but has a predicted Ser/Thr-instead of a His-kinase domain. Bioinformatic analysis indicates that CHARK is a retrogene and a product of trans-splicing. Here, we analyzed whether CHARK can function as a bona fide cytokinin receptor. A biochemical assay demonstrated its ability to bind cytokinin. Transient expression of CHARK in protoplasts increased their response to cytokinin. Expression of CHARK in an Arabidopsis receptor double mutant complemented its growth defects and restored the ability to activate cytokinin response genes, clearly demonstrating that CHARK functions as a cytokinin receptor. We propose that the CHARK gene presents an evolutionary novelty in the cytokinin signaling system.

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

Shuttling of Entire Libraries from an Entry Vector to a Destination Vector of the Gateway System.

Large-scale experiments are the basis of functional genomics, the investigating of several hundred or even thousand genes or proteins in parallel. A prerequisite for such experiments is the ability to clone several thousand genes simultaneously into a vector of choice to investigate different aspects of protein function, e.g., protein interactions, or subcellular localization. In the recent past several such cloning systems have been developed and successfully used. Of the commercially available systems, the Gateway™ system is the most widely used.This protocol describes how to shuttle a library from an Entry vector to a destination vector of the Gateway™ system. Emphasis is placed on the efficiency of the shuttling process to avoid loss of complexity and on reproducibility of the method.

The effects of repeated whole genome duplication events on the evolution of cytokinin signaling pathway.

BACKGROUND: It is thought that after whole-genome duplications (WGDs), a large fraction of the duplicated gene copies is lost over time while few duplicates are retained. Which factors promote survival or death of a duplicate remains unclear and the underlying mechanisms are poorly understood. According to the model of gene dosage balance, genes encoding interacting proteins are predicted to be preferentially co-retained after WGDs. Among these are genes encoding proteins involved in complexes or in signal transduction. RESULTS: We have investigated the way that repeated WGDs during land plant evolution have affected cytokinin signaling to study patterns of gene duplicability and co-retention in this important signal transduction pathway. Through the integration of phylogenetic analyses with comparisons of genome collinearity, we have found that signal input mediated by cytokinin receptors proved to be highly conserved over long evolutionary time-scales, with receptors showing predominantly gene loss after repeated WGDs. However, the downstream elements, e,g. response regulators, were mainly retained after WGDs and thereby formed gene families in most plant lineages. CONCLUSIONS: Gene dosage balance between the interacting components indicated by co-retention after WGDs seems to play a minor role in the evolution of cytokinin signaling pathway. Overall, core genes of cytokinin signaling show a highly heterogeneous pattern of gene retention after WGD, reflecting complex relationships between the various factors that shape the long-term fate of a duplicated gene.

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium.

Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

Novel in vivo screening design for the rapid and cost-effective identification of transcriptional regulators.

Genetic screens are a common tool to identify new modulators in a defined context, e.g. hormonal response or environmental stress. However, most screens are either in vitro or laborious and time-and-space inefficient. Here we present a novel in planta screening approach that shortens the time from the actual screening process to the identification of a new modulator and simultaneously reduces space requirements and costs. The basic features of this screening approach are the creation of luciferase reporter plants which enable a non-invasive readout in a streamlined multiplate reader process, the transformation of those plants with an inducible, Gateway™-compatible expression vector, and a screening setup, in which whole plants at the seedling stage are screened in 96-multiwell plates in the first transformed generation without the use of an expensive charge-coupled device (CCD) camera system. The screening itself and the verification of candidates can be done in as little as 2-3 weeks. The screen enables the analysis of reporter gene activity upon different treatments. Primary positive plants can immediately be selected and grown further. In this study a fast, simple, cost- and space-efficient in planta screening system to detect novel mediators of a given transcriptional response was developed and successfully tested using the cytokinin signal transduction as a test case.

CHASE domain-containing receptors play an essential role in the cytokinin response of the moss Physcomitrella patens.

While the molecular basis for cytokinin action is quite well understood in flowering plants, little is known about the cytokinin signal transduction in early diverging land plants. The genome of the bryophyte Physcomitrella patens (Hedw.) B.S. encodes three classical cytokinin receptors, the CHASE domain-containing histidine kinases, CHK1, CHK2, and CHK3. In a complementation assay with protoplasts of receptor-deficient Arabidopsis thaliana as well as in cytokinin binding assays, we found evidence that CHK1 and CHK2 receptors can function in cytokinin perception. Using gene targeting, we generated a collection of CHK knockout mutants comprising single (Δchk1, Δchk2, Δchk3), double (Δchk1,2, Δchk1,3, Δchk2,3), and triple (Δchk1,2,3) mutants. Mutants were characterized for their cytokinin response and differentiation capacities. While the wild type did not grow on high doses of cytokinin (1 µM benzyladenine), the Δchk1,2,3 mutant exhibited normal protonema growth. Bud induction assays showed that all three cytokinin receptors contribute to the triggering of budding, albeit to different extents. Furthermore, while the triple mutant showed no response in this bioassay, the remaining mutants displayed budding responses in a diverse manner to different types and concentrations of cytokinins. Determination of cytokinin levels in mutants showed no drastic changes for any of the cytokinins; thus, in contrast to Arabidopsis, revealing only small impacts of cytokinin signaling on homeostasis. In summary, our study provides a first insight into the molecular action of cytokinin in an early diverging land plant and demonstrates that CHK receptors play an essential role in bud induction and gametophore development.

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

Members of a recently discovered subfamily of cytokinin receptors display differences and similarities to their classical counterparts.

Cytokinins represent a group of plant hormones that have been shown to be essential for plant growth and development. A recent large-scale phylogenetic analysis of components of the cytokinin signal transduction pathway revealed, among other findings, the existence of a second, previously unknown subfamily of cytokinin receptors. Here we report that the cytokinin binding domains of the members of the 2 subfamilies contain residues that are highly conserved in either or in both subfamilies. Experiments using fluorescence microscopy hint at an ER and a plasma membrane localization for 2 members of the newly identified subfamily. These data provide new insights in the conservation of sequence and localization properties among the 2 subfamilies.

A subfamily of putative cytokinin receptors is revealed by an analysis of the evolution of the two-component signaling system of plants.

The two-component signaling system--the major signaling pathway of bacteria--is found among higher eukaryotes only in plants, where it regulates diverse processes, such as the signaling of the phytohormone cytokinin. Cytokinin is perceived by a hybrid histidine (His) kinase receptor, and the signal is transduced by a multistep phosphorelay system of His phosphotransfer proteins and different classes of response regulators (RRs). To shed light on the origin and evolution of the two-component signaling system members in plants, we conducted a comprehensive domain-based phylogenetic study across the relevant kingdoms, including Charophyceae algae, the group of green algae giving rise to land plants. Surprisingly, we identified a subfamily of cytokinin receptors with members only from the early diverging land plants Marchantia polymorpha and Physcomitrella patens and then experimentally characterized two members of this subfamily. His phosphotransfer proteins of Charophyceae seemed to be more closely related to land plants than to other groups of green algae. Farther down the signaling pathway, the type-B RRs were found across all plant clades, but many members lack either the canonical Asp residue or the DNA binding domain. In contrast, the type-A RRs seemed to be limited to land plants. Finally, the analysis provided hints that one additional group of RRs, the type-C RRs, might be degenerated receptors and thus, of a different evolutionary origin than bona fide RRs.

Updates on the model and the evolution of cytokinin signaling.

Cytokinins represent a class of phytohormones, which are key players not only in many processes important for plant growth and development, but also in the response to changes in their environment. The model for the cytokinin signaling pathway was established at the turn of the last century and many experiments confirmed its validity. In recent years several changes and extensions to the model were necessary to accommodate new findings concerning its components, such as subcellular localization, selective protein degradation and new modes of cross talk. In addition phylogenetic analyses of components of the cytokinin circuitry started to reveal the origin and evolution of the cytokinin regulatory system.

In planta analysis of a cis-regulatory cytokinin response motif in Arabidopsis and identification of a novel enhancer sequence.

The phytohormone cytokinin plays a key role in regulating plant growth and development, and is involved in numerous physiological responses to environmental changes. The type-B response regulators, which regulate the transcription of cytokinin response genes, are a part of the cytokinin signaling system. Arabidopsis thaliana encodes 11 type-B response regulators (type-B ARRs), and some of them were shown to bind in vitro to the core cytokinin response motif (CRM) 5'-(A/G)GAT(T/C)-3' or, in the case of ARR1, to an extended motif (ECRM), 5'-AAGAT(T/C)TT-3'. Here we obtained in planta proof for the functionality of the latter motif. Promoter deletion analysis of the primary cytokinin response gene ARR6 showed that a combination of two extended motifs within the promoter is required to mediate the full transcriptional activation by ARR1 and other type-B ARRs. CRMs were found to be over-represented in the vicinity of ECRMs in the promoters of cytokinin-regulated genes, suggesting their functional relevance. Moreover, an evolutionarily conserved 27 bp long T-rich region between -220 and -193 bp was identified and shown to be required for the full activation by type-B ARRs and the response to cytokinin. This novel enhancer is not bound by the DNA-binding domain of ARR1, indicating that additional proteins might be involved in mediating the transcriptional cytokinin response. Furthermore, genome-wide expression profiling identified genes, among them ARR16, whose induction by cytokinin depends on both ARR1 and other specific type-B ARRs. This together with the ECRM/CRM sequence clustering indicates cooperative action of different type-B ARRs for the activation of particular target genes.

Gene regulation by cytokinin in Arabidopsis.

The plant hormone cytokinin realizes at least part of its signaling output through the regulation of gene expression. A great part of the early transcriptional regulation is mediated by type-B response regulators, which are transcription factors of the MYB family. Other transcription factors, such as the cytokinin response factors of the AP2/ERF family, have also been shown to be involved in this process. Additional transcription factors mediate distinct parts of the cytokinin response through tissue- and cell-specific downstream transcriptional cascades. In Arabidopsis, only a single cytokinin response element, to which type-B response regulators bind, has been clearly proven so far, which has 5'-GAT(T/C)-3' as a core sequence. This motif has served to construct a synthetic cytokinin-sensitive two-component system response element, which is useful for monitoring the cellular cytokinin status. Insight into the extent of transcriptional regulation has been gained by genome-wide gene expression analyses following cytokinin treatment and from plants having an altered cytokinin content or signaling. This review presents a meta analysis of such microarray data resulting in a core list of cytokinin response genes. Genes encoding type-A response regulators displayed the most stable response to cytokinin, but a number of cytokinin metabolism genes (CKX4, CKX5, CYP735A2, UGT76C2) also belong to them, indicating homeostatic mechanisms operating at the transcriptional level. The cytokinin core response genes are also the target of other hormones as well as biotic and abiotic stresses, documenting crosstalk of the cytokinin system with other hormonal and environmental signaling pathways. The multiple links of cytokinin to diverse functions, ranging from control of meristem activity, hormonal crosstalk, nutrient acquisition, and various stress responses, are also corroborated by a compilation of genes that have been repeatedly found by independent gene expression profiling studies. Such functions are, at least in part, supported by genetic studies.

Calcium and zinc DTPA administration for internal contamination with plutonium-238 and americium-241.

The accidental or intentional release of plutonium or americium can cause acute and long term adverse health effects if they enter the human body by ingestion, inhalation, or injection. These effects can be prevented by rapid removal of these radionuclides by chelators such as calcium or zinc diethylenetriaminepentaacetate (calcium or zinc DTPA). These compounds have been shown to be efficacious in enhancing the elimination of members of the actinide family particularly plutonium and americium when administered intravenously or by nebulizer. The efficacy and adverse effects profile depend on several factors that include the route of internalization of the actinide, the type, and route time of administration of the chelator, and whether the calcium or zinc salt of DTPA is used. Current and future research efforts should be directed at overcoming limitations associated with the use of these complex drugs by using innovative methods that can enhance their structural and therapeutic properties.

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 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 Mg²âº-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.