Sergey N. Lomin

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.

Key message 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. Abstract 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.

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.

A new class of plant phosphotransfer proteins belonging to multistep phosphorelay (MSP) system was discovered by using 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 membranes. To date, 95 predicted TM-containing phosphotransmitter (TM-HPt) homologs were found in 61 plant species belonging to different clades, taxa and groups: bryophytes, gymnosperms, mono- and dicotyledons. The conserved HPt motif with phosphorylatable histidine was preserved in the most of TM-HPts under study that allowed us to consider these proteins potentially active in MSP signaling. For the found 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 the 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. Possible roles of TM-HPts as modulators of 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.

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 (pal1), which shows decreased panicle size due to reduced inflorescence meristem 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 inflorescence meristem 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 two 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.

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.

It has long been known that 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 analyzes of deduced proteins were supplemented by their expression profiling and 3D molecular modeling. The canonical and ABP1-mediated pathways of auxin signaling appeared to be well conserved. Total number of potato genes/proteins presumably involved in canonical auxin signaling is 46 and 108 in monoploid DM and tetraploid Otava, respectively. Among 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 be low probable in potato. This was equally applied to all cultivars used irrespective of their ploidy. Thus, some important features of the (noncanonical) auxin signaling pathways may be variable and species-specific.

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.