The signaling of cytokinins (CKs), classical plant hormones, is based on the interaction of proteins that constitute the multistep phosphorelay system (MSP): catalytic receptors—sensor histidine kinases (HKs), phosphotransmitters (HPts), and transcription factors—response regulators (RRs). Any CK receptor was shown to interact in vivo with any of the studied HPts and vice versa. In addition, both of these proteins tend to form a homodimer or a heterodimeric complex with protein-paralog. Our study was aimed at explaining by molecular modeling the observed features of in planta protein–protein interactions, accompanying CK signaling. For this purpose, models of CK-signaling proteins’ structure from Arabidopsis and potato were built. The modeled interaction interfaces were formed by rather conserved areas of protein surfaces, complementary in hydrophobicity and electrostatic potential. Hot spots amino acids, determining specificity and strength of the interaction, were identified. Virtual phosphorylation of conserved Asp or His residues affected this complementation, increasing (Asp-P in HK) or decreasing (His-P in HPt) the affinity of interacting proteins. The HK–HPt and HPt–HPt interfaces overlapped, sharing some of the hot spots. MSP proteins from Arabidopsis and potato exhibited similar properties. The structural features of the modeled protein complexes were consistent with the experimental data.

Auxins and cytokinins create versatile regulatory network controlling virtually all aspects of plant growth and development. These hormonal systems act in close contact, synergistically or antagonistically, determining plant phenotype, resistance and productivity. However, the current knowledge about molecular interactions of these systems is still scarce. Our study with potato plants aimed at deciphering potential interactions between auxin and cytokinin signaling pathways at the level of respective gene expression. Potato plants grown on sterile medium with 1.5% (vegetation) or 5% (tuberization) sucrose were treated for 1 h with auxin or cytokinin. Effects of these two hormones on expression profiles of genes belonging to main signaling pathways of auxin and cytokinin were quantified by RT-qPCR. As a result, several signaling genes were found to respond to auxin and/or cytokinin by up- or down-regulation. The observed effects were largely organ-specific and depended on sucrose content. Auxin strongly reduced cytokinin perception apparatus while reciprocal cytokinin effect was ambiguous and sucrose-dependent. In many cases, functional clustering of genes of the same family was observed. Promoters in some clusters are enriched with canonic hormone-response cis-elements supporting their direct sensitivity to hormones. Collectively, our data shed new light on the crosstalk between auxin- and cytokinin signaling pathways.

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 (K_D 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.

Plants are autotrophic organisms that rely on light to produce sugars. The sugars serve as structural components and energy sources throughout plant life. Since the availability of carbohydrates is an important factor for survival, plants have evolved complex mechanisms for sugar sensing and signaling during evolution. The signals transmitted through HXK1, RGS1, T6P, SUC2/SUC4, SnRK1, and TOR provide flexible regulation of plant growth and metabolism and allow efficient redistribution of resources by adapting to changing levels of endogenous sugars and unfavorable environmental conditions. Besides sugars, phytohormones, such as cytokinins, play an important role in plant development. Cytokinins have a variety of physiological effects and, like sugars, are essential for plant growth and development. One of the main effects of cytokinins is the stimulation of cell division. Cytokinin signal transduction is analogous to bacterial two-component signaling system and is accomplished by multistep phosphorelay. The cytokinin signaling system consists of sensor histidine kinases, phosphotransmitters, and response regulators, the latter (B-type) are transcription factors. This paper presents the overview of the interaction between cytokinin and sugar signaling pathways in the regulation of seed germination, seedling formation, root and shoot growth, flowering, leaf senescence and tuberization.

Potatoes are one of the most important agricultural crops in the world. The formation of potato tubers is regulated by phytohormones, including cytokinins. Transduction of the cytokinin signal in plant cells is accomplished by interprotein multistep phosphorelay. The role of cytokinin receptors is performed by sensor histidine kinases. Three groups of cytokinin receptors have been identified in potato Solanum tuberosum: StHK2, StHK3, and StHK4. In this work, the expression features of a reporter gene GUS under the control of potato cytokinin receptor gene promoters StHK2a, StHK3a, and StHK4b in potato plants were investigated. GUS activity was detected in all potato organs. In transgenic plants proStHK2a::GUS, a weaker staining of all tissues was observed compared to proStHK3a::GUS and proStHK4b::GUS. Promoters proStHK2a and proStHK3a provided high activity of the reporter gene in vascular bundles of leaves, stems, and roots. Expression of promoter proStHK3a was found in the mesophyll cells of leaves. GUS gene activity under the control of promoter proStHK4b was found in the apical and lateral root meristems as well as in root tissues. In the stem, GUS staining of endoderm and pericycle cells was observed. According to the results of bioinformatics analysis, in the structure of proStHK2a, proStHK3a, and proStHK4b, 18 regulatory motifs were identified, some of which occurring in all promoters. In addition, unique cis-elements were found in the nucleotide sequence of individual promoters.

Potato is the most economically important non-cereal food crop. Tuber formation in potato is regulated by phytohormones, cytokinins (CKs) in particular. The present work was aimed to study CK signal perception in potato. The sequenced potato genome of doubled monoploid Phureja was used for bioinformatic analysis and as a tool for identification of putative CK receptors from autotetraploid potato cv. Désirée. All basic elements of multistep phosphorelay (MSP) required for CK signal transduction were identified in Phureja genome, including three genes orthologous to three CK receptor genes ( AHK 2-4 ) of Arabidopsis. As distinct from Phureja, autotetraploid potato contains at least two allelic isoforms of each receptor type. Putative receptor genes from Désirée plants were cloned, sequenced and expressed, and main characteristics of encoded proteins, firstly their consensus motifs, structure models, ligand-binding properties, and the ability to transmit CK signal, were determined. In all studied aspects the predicted sensor histidine kinases met the requirements for genuine CK receptors. Expression of potato CK receptors was found to be organ-specific and sensitive to growth conditions, particularly to sucrose content. Our results provide a solid basis for further in-depth study of CK signaling system and biotechnological improvement of potato.