The cytokinin receptor OHK4/OsHK4 regulates inflorescence architecture in rice via an IDEAL PLANT ARCHITECTURE1/WEALTHY FARMER'S PANICLE-mediated positive feedback circuit.

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.

Canonical and Alternative Auxin Signaling Systems in Mono-, Di-, and Tetraploid Potatoes.

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.

A Model of the Full-Length Cytokinin Receptor: New Insights and Prospects.

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.

In Planta, In Vitro and In Silico Studies of Chiral N6-Benzyladenine Derivatives: Discovery of Receptor-Specific S-Enantiomers with Cytokinin or Anticytokinin Activities.

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.

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.

Global View on the Cytokinin Regulatory System in Potato.

Cytokinins (CKs) were earlier shown to promote potato tuberization. Our study aimed to identify and characterize CK-related genes which constitute CK regulatory system in the core potato (Solanum tuberosum) genome. For that, CK-related genes were retrieved from the sequenced genome of the S. tuberosum doubled monoploid (DM) Phureja group, classified and compared with Arabidopsis orthologs. Analysis of selected gene expression was performed with a transcriptome database for the S. tuberosum heterozygous diploid line RH89-039-16. Genes responsible for CK signaling, biosynthesis, transport, and metabolism were categorized in an organ-specific fashion. According to this database, CK receptors StHK2/3 predominate in leaves and flowers, StHK4 in roots. Among phosphotransmitters, StHP1a expression largely predominates. Surprisingly, two pseudo-phosphotransmitters intended to suppress CK effects are hardly expressed in studied organs. Among B-type RR genes, StRR1b, StRR11, and StRR18a are actively expressed, with StRR1b expressing most uniformly in all organs and StRR11 exhibiting the highest expression in roots. By cluster analysis four types of prevailing CK-signaling chains were identified in (1) leaves and flowers, StHK2/3→S t H P1a→StRR1b/+; (2) shoot apical meristems, stolons, and mature tubers, StHK2/4→S t H P1a→StRR1b/+; (3) stems and young tubers, StHK2/4→S t H P1a→StRR1b/11/18a; and (4) roots and tuber sprouts, StHK4→S t H P1a→StRR11/18a. CK synthesis genes StIPT3/5 and StCYP735A are expressed mainly in roots followed by tuber sprouts, but rather weakly in stolons and tubers. By contrast, CK-activation genes StLOGs are active in stolons, and StLOG3b expression is even stolon-confined. Apparently, the main CK effects on tuber initiation are realized via activity of StLOG1/3a/3b/7c/8a genes in stolons. Current advances and future directions in potato research are discussed.

Modeling of Protein⁻Protein Interactions in Cytokinin Signal Transduction.

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 in potato: molecular aspects and emerging roles in tuber formation and stress resistance.

The study of the effects of auxins on potato tuberization corresponds to one of the oldest experimental systems in plant biology, which has remained relevant for over 70 years. However, only recently, in the postgenomic era, the role of auxin in tuber formation and other vital processes in potatoes has begun to emerge. This review describes the main results obtained over the entire period of auxin-potato research, including the effects of exogenous auxin; the content and dynamics of endogenous auxins; the effects of manipulating endogenous auxin content; the molecular mechanisms of auxin signaling, transport and inactivation; the role and position of auxin among other tuberigenic factors; the effects of auxin on tuber dormancy; the prospects for auxin use in potato biotechnology. Special attention is paid to recent insights into auxin function in potato tuberization and stress resistance. Taken together, the data discussed here leave no doubt on the important role of auxin in potato tuberization, particularly in the processes of tuber initiation, growth and sprouting. A new integrative model for the stage-dependent auxin action on tuberization is presented. In addition, auxin is shown to differentially affects the potato resistance to biotrophic and necrotrophic biopathogens. Thus, the modern auxin biology opens up new perspectives for further biotechnological improvement of potato crops.

Cytokinin perception in potato: new features of canonical players.

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 studied 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 required for CK signal transduction were identified in the 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 the main characteristics of encoded proteins were determined, in particular their consensus motifs, modelled structure, ligand-binding properties, and ability to transmit CK signals. 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.

Studies of cytokinin receptor-phosphotransmitter interaction provide evidences for the initiation of cytokinin signalling in the endoplasmic reticulum.

Cytokinin receptors were shown recently to be localised mainly to the endoplasmic reticulum (ER); however, the activity of ER-located receptors was not proven. We have therefore tested the functionality of ER-located Arabidopsis receptors. The first step of cytokinin signal transduction is the transfer of a phosphoryl group from the activated receptor to a phosphotransfer protein. To determine the subcellular localisation of receptor-phosphotransmitter interaction in planta, BiFC experiments were performed. Receptors ARABIDOPSIS HISTIDINE KINASE 2 (AHK2), AHK3 and AHK4 (CRE1) and phosphotransmitters ARABIDOPSIS HISTIDINE-CONTAINING PHOSPHOTRANSMITTER 1 (AHP1), AHP2 and AHP3 fused to split-eYFP were transiently expressed in Nicotiana benthamiana leaves. Receptor-phosphotransmitter pairs were shown to interact in every possible combination in a pattern reflecting the ER. Receptor dimers, an active form of the receptors, were also detected in the ER. According to BiFC and protease protection data, the catalytic part of AHK3 was located in the cytoplasm whereas the hormone binding module faced the ER lumen. This topology is consistent with receptor signalling from the ER membrane. Finally, the functionality of receptors in different membrane fractions was tested using an in vitro kinase assay visualising the phosphorylation of phosphotransfer proteins. The detected cytokinin-dependent phosphotransfer activity was confined mainly to the ER-enriched fraction. Collectively, our data demonstrate that ER-located cytokinin receptors are active in cytokinin signal transduction. Hence, intracellular cytokinins appear to play an essential role in cytokinin signalling. An updated model for the spatial organisation of cytokinin transport form activation, intracellular trafficking and signalling from the ER is proposed.

Plant membrane assays with cytokinin receptors underpin the unique role of free cytokinin bases as biologically active ligands.

Cytokinin receptors play a key role in cytokinin-dependent processes regulating plant growth, development, and adaptation; therefore, the functional properties of these receptors are of great importance. Previously the properties of cytokinin receptors were investigated in heterologous assay systems using unicellular microorganisms, mainly bacteria, expressing receptor proteins. However, within microorganisms receptors reside in an alien environment that might distort the receptor properties. Therefore, a new assay system has been developed allowing studies of individual receptors within plant membranes (i.e. closer to their natural environment). The main ligand-binding characteristics of receptors from Arabidopsis [AHK2, AHK3, and AHK4] and maize (ZmHK1) were refined in this new system, and the properties of full-length Arabidopsis receptor AHK2 were characterized for the first time. Ligand specificity profiles of receptors towards cytokinin bases were comparable with the profiles retrieved in bacterial assay systems. In contrast, cytokinin-9-ribosides displayed a strongly reduced affinity for receptors in the plant assay system, indicating that ribosides as the common transport form of cytokinins have no or very weak cytokinin activity. This underpins the central role of free bases as the sole biologically active cytokinin compounds. According to molecular modelling and docking studies, N (9)-ribosylation alters the bonding pattern in cytokinin-receptor interaction and prevents ß6-ß7 loop movement important for tight hormone binding. A common feature of all receptors was a greatly reduced ligand binding at low (5.0-5.5) pH. The particularly high sensitivity of ZmHK1 to pH changes leads to the suggestion that some cytokinin receptors may play an additional role as pH sensors in the lumen of the endoplasmic reticulum.