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.

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.

Distinct Peculiarities of In Planta Synthesis of Isoprenoid and Aromatic Cytokinins.

The biosynthesis of aromatic cytokinins in planta, unlike isoprenoid cytokinins, is still unknown. To compare the final steps of biosynthesis pathways of aromatic and isoprenoid cytokinins, we synthesized a series of nucleoside derivatives of natural cytokinins starting from acyl-protected ribofuranosyl-, 2'-deoxyribofuranosyl- and 5'-deoxyribofuranosyladenine derivatives using stereoselective alkylation with further deblocking. Their cytokinin activity was determined in two bioassays based on model plants Arabidopsis thaliana and Amaranthus caudatus. Unlike cytokinins, cytokinin nucleosides lack the hormonal activity until the ribose moiety is removed. According to our experiments, ribo-, 2'-deoxyribo- and 5'-deoxyribo-derivatives of isoprenoid cytokinin N6-isopentenyladenine turned in planta into active cytokinins with clear hormonal activity. As for aromatic cytokinins, both 2'-deoxyribo- and 5'-deoxyribo-derivatives did not exhibit analogous activity in Arabidopsis. The 5'-deoxyribo-derivatives cannot be phosphorylated enzymatically in vivo; therefore, they cannot be "activated" by the direct LOG-mediated cleavage, largely occurring with cytokinin ribonucleotides in plant cells. The contrasting effects exerted by deoxyribonucleosides of isoprenoid (true hormonal activity) and aromatic (almost no activity) cytokinins indicates a significant difference in the biosynthesis of these compounds.

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.

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.

Cytokinin activity of N6-benzyladenine derivatives assayed by interaction with the receptors in planta, in vitro, and in silico.

Biological effects of hormones in both plants and animals are based on high-affinity interaction with cognate receptors resulting in their activation. The signal of cytokinins, classical plant hormones, is perceived in Arabidopsis by three homologous membrane receptors: AHK2, AHK3, and CRE1/AHK4. To study the cytokinin-receptor interaction, we used 25 derivatives of potent cytokinin N6-benzyladenine (BA) with substituents in the purine heterocycle and/or in the side chain. The study was focused primarily on individual cytokinin receptors from Arabidopsis. The main in planta assay system was based on Arabidopsis double mutants retaining only one isoform of cytokinin receptors and harboring cytokinin-sensitive reporter gene. Classical cytokinin biotest with Amaranthus seedlings was used as an additional biotest. In parallel, the binding of ligands to individual cytokinin receptors was assessed in the in vitro test system. Quantitative comparison of results of different assays confirmed the partial similarity of ligand-binding properties of receptor isoforms. Substituents at positions 8 and 9 of adenine moiety, elongated linker up to 4 methylene units, and replacement of N6 by sulfur or oxygen have resulted in the suppression of cytokinin activity of the derivative toward all receptors. Introduction of a halogen into position 2 of adenine moiety, on the contrary, often increased the ligand activity, especially toward AHK3. Features both common and distinctive of cytokinin receptors in Arabidopsis and Amaranthus were revealed, highlighting species specificity of the cytokinin perception apparatus. Correlations between the extent to which a compound binds to a receptor in vitro and its ability to activate the same receptor in planta were evaluated for each AHK protein. Interaction patterns between individual receptors and ligands were rationalized by structure analysis and molecular docking in sensory modules of AHK receptors. The best correlation between docking scores and specific binding was observed for AHK3. In addition, receptor-specific ligands have been discovered with unique properties to predominantly activate or block distinct cytokinin receptors. These ligands are promising for practical application and as molecular tools in the study of the cytokinin perception by plant cells.