The cytokinin regulatory system (CRS) is important for plant adaptation to adverse environmental conditions, including drought and heat stress. CRS represents a promising biotechnological target to improve crop tolerance to these stresses. Drought and heat are widespread natural phenomena leading to stress in plants and a significant yield reduction in crop plants. Tolerance of drought and heat determines the productivity of major crops. The global climate forecast predicts further temperature increase and worsening water shortage in the very near future. Plants can withstand heat stress and water scarcity using their adaptive hormonal system. Among the plant hormones, cytokinins (CKs), which are best known for their numerous functions in regulating growth and development, play a significant role in plants’ responses to heat and water stress. This review summarizes the effects of CK on drought and heat tolerance in common agricultural plants, and strategies to protect these plants are described. Particular attention is paid to biotechnological approaches targeting the cytokinin regulatory system (CRS) to preserve crop phenotype and yield under stress conditions. Interestingly, CKs have been reported to improve drought/heat resistance both by increased and decreased signaling. We propose a rational explanation for this apparent paradox of CK action. This review aims to be helpful to plant biotechnologists and genetic engineers in selecting suitable CRS targets that could serve as tools to generate new drought- and heat-tolerant crop varieties.

Recently, the existence of a new class of plant phosphotransfer proteins (HPts) with transmembrane (TM) domains was predicted by a large-scale bioinformatics method. These non-canonical proteins belong to the multistep phosphorelay (MSP) signal transduction system. The gene for one of these predicted TM-HPt was first cloned from tea (Camellia sinensis L.) plant cells. The membrane localization of the encoded protein (TM-CsHPt1) was confirmed using confocal microscopy and immunoblotting. These proteins were detected in the endoplasmic reticulum-enriched but not plasma membrane-enriched fractions. Using the BiFC method, the ability of TM-CsHPt1 to homodimerize was shown, similar to classical soluble HPt. However, heterodimerization between canonical and non-canonical CsHPts was not detected. Furthermore, TM-CsHPt1 was capable of specific interaction with the Arabidopsis cytokinin (CK) receptor AHK3, but not its paralogs AHK2 and AHK4. The obtained data are compatible with the involvement of TM-CsHPt1 in CK signaling (which utilizes the MSP system), possibly through a suggested non-canonical membrane branch. In addition, the key components of the CK signaling system in C. sinensis were uncovered and characterized by bioinformatics and phylogenetic analysis. The putative functions of the predicted MSP membrane branch in the tea plant are discussed.

Cytokinins (CKs) are important group of phytohormones that influence a vast number of processes throughout all stages of plant ontogenesis. Components of the CK signaling chain mainly act as negative regulators of plant adaptation to unfavorable environmental conditions. The fine tuning of the CK signaling, a targeted and careful decrease in CK activity in definite plant organs and tissues, can increase the survival and productivity of agricultural plants affected by stress factors. One of the promising options to achieve this goal is the use of anticytokinins (antiCKs) with pronounced receptor specificity. To date, many synthetic analogs of natural CKs are known; however, there is little progress in the development of CK antagonists. In this work, a large series of N6-substituted adenosine derivatives were synthesized and studied as potential antiCKs for individual CK receptors from Arabidopsis thaliana. To dissect the dependence of CK antagonist activity on the structure of the adenosine derivatives, we studied effects of modifications of the sugar moiety at the N9 position, as well as different substituents at the N6 position of the purine heterocycle. The cognate nucleobases for all tested nucleoside derivatives were also assessed for both CK agonist and antagonist activity. As a result, new receptor-specific antiCKs were found both among nucleoside derivatives and nucleobases. The activity of such compounds was shown to strictly depend on the structure of the N6-substituent. In several cases, the presence of the intact β-D-ribofuranose residue at N9 of purine heterocycle was critical for the manifestation of the antiCK properties, since cognate nucleobases exhibited not antagonist, but definite CK agonist activity. Most if not all, newly found antiCKs acted non-competitively at the receptor level by yet unknown molecular mechanism. Some of these regulators exhibited dual activities, usually acting as antiCKs with AHK2 and AHK4, but as CK agonists with AHK3 receptors. Such new class of cytokinin-related regulators with opposite activities depending on the receptor opens large prospects for their practical application in biotechnology and agriculture.

A new class of plant phosphotransfer proteins belonging to the multistep phosphorelay (MSP) system implicated in phytohormone cytokinin signaling was discovered based on large-scale bioinformatics methods. Unlike the canonical soluble nucleo-cytosolic forms, these proteins were predicted to have transmembrane (TM) domains and, apparently, should be localized on some kind of cell membrane. To date, 94 predicted TM-containing phosphotransmitter (TM-HPt) homologs were found in 62 plant species belonging to different clades, taxa, and groups of embryophytes: bryophytes, gymnosperms, and mono- and dicotyledons. The conserved HPt motif with phosphorylatable histidine was preserved in most of the TM-HPts under study, which allowed us to consider these proteins potentially active in MSP signaling. For the identified TM-HPts, a Bayesian analysis at the DNA level was performed, and a relevant phylogenetic tree was constructed. According to evolutionary relationships, plant TM-HPts were divided into two main groups corresponding to Arabidopsis AHP1-3,5,6, and AHP4 orthologs. Transcriptomic analysis confirmed the expression of most of the investigated TM-HPt-encoding genes. Their moderate-to-low overall transcription rate may be a consequence of inducible and/or tissue-specific expression. Using molecular modeling methods, a variety of potential spatial organizations of several such proteins are demonstrated. The ability of the uncovered TM domains to tether HPts to membranes was supported by molecular dynamic simulation. Possible roles of TM-HPts as modulators of the MSP signaling pathway and corresponding putative mechanisms of their action are suggested.

The multistep phosphorelay (MSP) is a conserved signaling system that allows plants to sense and respond to a variety of cues under rapidly changing environmental conditions. The MSP system comprises three main protein types: sensor histidine kinases, phosphotransmitters, and response regulators. There are numerous signaling pathways that use, in whole or in part, this set of proteins to transduce diverse signals. Among them, the cytokinin signal transduction system is the best-studied pathway, which utilizes the entire MSP cascade. Focusing on this system, we review here protein–protein interaction of MSP components that are not directly related to cytokinin signaling. These interactions are likely to play an essential role in hormonal crosstalk and may be promising targets for fine-tuning plant development. In addition, in light of recent advances in the study of cytokinin signaling, we discuss new insights into the putative molecular mechanisms that mediate the pleiotropic action of cytokinins and provide specificity for distinct MSP signals. A detailed network of known non-canonical protein–protein interactions related to cytokinin signaling was demonstrated.

Cytokinins (CK) are one of the most important classes of phytohormones that regulate a wide range of processes in plants. A CK receptor, a sensor hybrid histidine kinase, was discovered more than 20 years ago, but the structural basis for its signaling is still a challenge for plant biologists. To date, only two fragments of the CK receptor structure, the sensory module and the receiver domain, were experimentally resolved. Some other regions were built up by molecular modeling based on structures of proteins homologous to CK receptors. However, in the long term, these data have proven insufficient for solving the structure of the full-sized CK receptor. The functional unit of CK receptor is the receptor dimer. In this article, a molecular structure of the dimeric form of the full-length CK receptor based on AlphaFold Multimer and ColabFold modeling is presented for the first time. Structural changes of the receptor upon interacting with phosphotransfer protein are visualized. According to mathematical simulation and available data, both types of dimeric receptor complexes with hormones, either half- or fully liganded, appear to be active in triggering signals. In addition, the prospects of using this and similar models to address remaining fundamental problems of CK signaling were outlined.

It has long been known that the phytohormone auxin plays a promoting role in tuber formation and stress tolerance in potatoes. Our study aimed to identify and characterize the complete sets of auxin-related genes that presumably constitute the entire auxin signaling system in potato (Solanum tuberosum L.). The corresponding genes were retrieved from sequenced genomes of the doubled monoploid S. tuberosum DM1-3-516-R44 (DM) of the Phureja group, the heterozygous diploid line RH89-039-16 (RH), and the autotetraploid cultivar Otava. Both canonical and noncanonical auxin signaling pathways were considered. Phylogenetic and domain analyses of deduced proteins were supplemented by expression profiling and 3D molecular modeling. The canonical and ABP1-mediated pathways of auxin signaling appeared to be well conserved. The total number of potato genes/proteins presumably involved in canonical auxin signaling is 46 and 108 in monoploid DM and tetraploid Otava, respectively. Among the studied potatoes, spectra of expressed genes obviously associated with auxin signaling were partly cultivar-specific and quite different from analogous spectrum in Arabidopsis. Most of the noncanonical pathways found in Arabidopsis appeared to have low probability in potato. This was equally true for all cultivars used irrespective of their ploidy. Thus, some important features of the (noncanonical) auxin signaling pathways may be variable and species-specific.

Inflorescence architecture is important for rice (Oryza sativa) grain yield. The phytohormone cytokinin (CK) has been shown to regulate rice inflorescence development; however, the underlying mechanism mediated by CK perception is still unclear. Employing a forward genetic approach, we isolated an inactive variant of the CK receptor OHK4/OsHK4 gene named panicle length1, which shows decreased panicle size due to reduced inflorescence meristem (IM) activity. A 2-amino acid deletion in the long α-helix stalk of the sensory module of OHK4 impairs the homodimerization and ligand-binding capacity of the receptor, even though the residues do not touch the ligand-binding domain or the dimerization interface. This deletion impairs CK signaling that occurs through the type-B response regulator OsRR21, which acts downstream of OHK4 in controlling inflorescence size. Meanwhile, we found that IDEAL PLANT ARCHITECTURE1(IPA1)/WEALTHY FARMER'S PANICLE (WFP), encoding a positive regulator of IM development, acts downstream of CK signaling and is directly activated by OsRR21. Additionally, we revealed that IPA1/WFP directly binds to the OHK4 promoter and upregulates its expression through interactions with 2 TCP transcription factors, forming a positive feedback circuit. Altogether, we identified the OHK4-OsRR21-IPA1 regulatory module, providing important insights into the role of CK signaling in regulating rice inflorescence architecture.

The main reserve polysaccharide of plants—starch—is undoubtedly important for humans. One of the main sources of starch is the potato tuber, which is able to preserve starch for a long time during the so-called dormancy period. However, accumulated data show that this dormancy is only relative, which raises the question of the possibility of some kind of starch restructuring during dormancy periods. Here, the effect of long-term periods of tuber rest (at 2–4 °C) on main parameters of starches of potato tubers grown in vivo or in vitro were studied. Along with non-transgenic potatoes, Arabidopsis phytochrome B (AtPHYB) transformants were investigated. Distinct changes in starch micro and macro structures—an increase in proportion of amorphous lamellae and of large-sized and irregular-shaped granules, as well as shifts in thickness of the crystalline lamellae—were detected. The degree of such alterations, more pronounced in AtPHYB-transgenic tubers, increased with the longevity of tuber dormancy. By contrast, the polymorphic crystalline structure (B-type) of starch remained unchanged regardless of dormancy duration. Collectively, our data support the hypothesis that potato starch remains metabolically and structurally labile during the entire tuber life including the dormancy period. The revealed starch remodeling may be considered a process of tuber preadaptation to the upcoming sprouting stage.

For the first time, N 6 -(5-phenylpentan-1-yl)adenine, a synthetic adenine derivative with a receptor-specific anticytokinin effect, was obtained. This compound exhibits a pronounced anticytokinin effect, reducing cytokinin-induced expression of the GUS reporter gene when interacting with the cytokinin receptor CRE1/AHK4 of the model plant Arabidopsis thaliana . This effect manifests itself much weaker with the related AHK2 receptor and is not observed at all with the AHK3 receptor. We showed that N 6 -(5-phenylpentan-1-yl)adenine does not bind to the ligand-binding sites of the Arabidopsis cytokinin receptors, which does not allow it to be classified as a true cytokinin antagonist. Despite the currently unknown mechanism of action, this compound may find its use as a component of plant growth regulators. Like true anticytokinins, it enhances root growth of Arabidopsis seedlings, apparently suppressing the action of endogenous cytokinins on the “root” receptor CRE1/AHK4.

It has long been known that auxins play a promoting role in tuber formation and stress tolerance in potatoes. Our study aimed to identify and characterize the complete sets of auxin-related genes that presumably constitute the entire auxin signaling system in potato (Solanum tuberosum L.). The corresponding genes were retrieved from sequenced genomes of the doubled monoploid S. tuberosum DM1-3-516-R44 (DM) of the Phureja group, the heterozygous diploid line RH89-039-16 (RH), and the autotetraploid cultivar Otava. Both canonical and noncanonical auxin signaling pathways were considered. Phylogenetic and domain analyzes of deduced proteins were supplemented by their expression profiling and 3D molecular modeling. Total number of potato genes/proteins involved in canonical auxin signaling is 43and 100 for monoploid DM and tetraploid Otava, respectively. Among studied potatoes, spectra of expressed genes associated with auxin signaling were partly cultivar-specific and quite different from analogous spectrum in Arabidopsis. Orthologs of noncanonical auxin signaling genes have also been identified and characterized in detail. Results show that some known noncanonical pathways are low probable in potato. Thus, according to cumulative data, potatoes use a variety of pathways for auxin signaling, where some important features of these pathways may be variable and even species-specific.

Auxins and cytokinins are considered the most important plant hormones, responsible for fundamental traits of the plant organism [...].

The results of an experimental study of the dependence of the energy of a surface barrier discharge on the value of the supply sinusoidal voltage with a frequency of 4 and 20 kHz in the electrode system of parallel stripes with a distance between them of 5 and 30 mm are presented. At a distance of 5 mm between the strips, there is a self-limited surface discharge in the electrode system. Self-limiting of the discharge leads to the fact that with an increase in the applied voltage, the energy invested in the electrode system increases more slowly than in the case when the discharge exists without restrictions. The energy of a self-limited discharge is 15-40% lower than the energy of a discharge that exists without restriction. Keywords: surface discharge, energy, volt-coulomb characteristic, self-limited discharge.

Cytokinin receptors are key proteins that trigger signaling of cognate classical phytohormones possessing a broad-spectrum biological action. Previously, we developed two test systems to analyze the ligand-binding properties of cytokinin receptors: a heterologous binding assay based on transformed bacteria E. coli, and, later, a more appropriate homologous assay based on microsomes from transiently transformed tobacco (Nicotiana benthamiana Domin.) leaves. In both cases, a genetic construct encoding one or another cytokinin receptor under study, was ectopically expressed. In the present work, we provide a versatile experimental validation for the homologous method and propose a modified (simplified) protocol for obtaining plant microsomes carrying transmembrane proteins to be studied, namely cytokinin receptors. In contrast to the original method, the simplified procedure makes it possible to avoid ultracentrifugation and to save reagents and time when obtaining a microsomal fraction. The microsomes isolated by the modified method were comparable in quantity and quality to a similar fraction obtained in a standard way, in both cases cytokinin specific binding by endogenous receptors was close to zero. Whichever way the microsomes were originally obtained (using an ultra- or preparative centrifuge), they precipitated equally well when subsequently centrifuged at 16 000 g in a benchtop microcentrifuge. All the experimental series allowed us to recommend a wide use of this plant-based binding method, in particular its simplified version, in studies of membrane-bound phytohormone receptors.

Cytokinins, classical phytohormones, affect all stages of plant ontogenesis, but their application in agriculture is limited because of the lack of appropriate ligands, including those specific for individual cytokinin receptors. In this work, a series of chiral N6-benzyladenine derivatives were studied as potential cytokinins or anticytokinins. All compounds contained a methyl group at the α-carbon atom of the benzyl moiety, making them R- or S-enantiomers. Four pairs of chiral nucleobases and corresponding ribonucleosides containing various substituents at the C2 position of adenine heterocycle were synthesized. A nucleophilic substitution reaction by secondary optically active amines was used. A strong influence of the chirality of studied compounds on their interaction with individual cytokinin receptors of Arabidopsis thaliana was uncovered in in vivo and in vitro assays. The AHK2 and CRE1/AHK4 receptors were shown to have low affinity for the studied S-nucleobases while the AHK3 receptor exhibited significant affinity for most of them. Thereby, three synthetic AHK3-specific cytokinins were discovered: N6-((S)-α-methylbenzyl)adenine (S-MBA), 2-fluoro,N6-((S)-α-methylbenzyl)adenine (S-FMBA) and 2-chloro,N6-((S)-α-methylbenzyl)adenine (S-CMBA). Interaction patterns between individual receptors and specific enantiomers were rationalized by structure analysis and molecular docking. Two other S-enantiomers (N6-((S)-α-methylbenzyl)adenosine, 2-amino,N6-((S)-α-methylbenzyl)adenosine) were found to exhibit receptor-specific and chirality-dependent anticytokinin properties.

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.

The treatment of soft winter wheat seeds of long-term storage with products of low-temperature plasma of the surface discharge was carried out according to an indirect scheme, i.e. without a contact between the layer of stationary seeds and the surface discharge existing in a strip electrode system with a distance between the strips of 10 mm. The treatment was carried out at an exposure time of 60 seconds in two modes of feeding the electrodes: with a sinusoidal voltage of 16 kHz/1.5 kV and 4.4 kHz/2.4 kV. The biometric parameters of seedlings on the third and seventh days, the tolerance of cold hardened seedlings to low temperatures, and the seed contamination were evaluated. It was found that the performed treatment does not provide an increase in quality characteristics of germination and tolerance to low temperatures, and also does not reduce seed contamination in conditions of high mycobiota level.

The seeds of soft winter wheat for long-term storage are treated with low-temperature plasma of a surface discharge according to an indirect method, i.e., without contact between a layer of immobile seeds and a surface discharge in a strip electrode system with a distance of 10 mm between strips. The seeds are treated with a surface discharge for 60 s in two modes of feeding the electrodes with a sinusoidal voltage (16 kHz/1.5 kV and 4.4 kHz/2.4 kV). The biometric parameters of seedlings on the third and seventh days, the resistance of cold hardened seedlings to low temperatures, and the degree of seed infection are measured. It is found that seed treatment does not provide an increase in the quality characteristics of germination and freezing tolerance and also does not reduce the seed infection under the condition of a high mycobiotic load.

In this work, the experimental results of surface barrier discharge plasma products affection to winter rye seed germination at the initial stage of growth and seedling tolerance to low temperatures are shown. The treatment was carried out for 10, 60, and 180 s in the plane-parallel electrode system with sinusoidal voltage of 2.7 kV with frequency 4.4 kHz applied to the strip electrodes at a distance of 5 mm from each other. It is shown that the treatment has no effect on seed germinating ability. In 180 s exposure treatment mode the stimulation of 3-day seedling shoot and root system length occurs. The freeze tolerance response of two-stage cold hardened seedlings grown from treated seeds shows that both the exposure of seed and the freezing temperature affect on the formation of positive response.