On the biological activity of cytokinin free bases and their ribosides

Main conclusion The free bases of cytokinins are the biologically active forms of the hormone while cytokinin ribosides become active only upon removal of the ribose residue. Abstract Cytokinins (CKs) belong to the classical plant hormones. They were discovered more than 65 years ago, but which molecular forms possess genuine CK activity is still matter of debate. Numerous studies support the view that only the free bases are the biologically active molecules. This standpoint has been challenged in a recent review (Nguyen et al. in Planta 254: 45, 2021) proposing that also CK ribosides may have genuine own CK activity. Here we critically discuss the pros and cons of this viewpoint considering the results of biological assays, CK binding studies, 3D structural data of CK-receptor interaction and mutant analyses. It is concluded that all types of study provide clear and convincing evidence only for biological activity of free bases and not ribosides; the latter are rather a transport form of the hormone without their own biological activity

Evolutionary proteomics identifies amino acids essential for ligand-binding of the cytokinin receptor CHASE domain

BACKGROUND: In plants the hormone cytokinin is perceived by members of a small cytokinin receptor family, which are hybrid sensor histidine kinases. While the immediate downstream signaling pathway is well characterized, the domain of the receptor responsible for ligand binding and which residues are involved in this process has not been determined experimentally. RESULTS: Using a live cell hormone-binding assay, we show that cytokinin is bound by a receptor domain predicted to be extracellular, the so called CHASE (cyclases, histidine kinase associated sensory extracellular) domain. The CHASE domain occurs not only in plant cytokinin receptors but also in numerous orphan receptors in lower eukaryotes and bacteria. Taking advantage of this fact, we used an evolutionary proteomics approach to identify amino acids important for cytokinin binding by looking for residues conserved in cytokinin receptors, but not in other receptors. By comparing differences in evolutionary rates, we predicted five amino acids within the plant CHASE domains to be crucial for cytokinin binding. Mutagenesis of the predicted sites and subsequent binding assays confirmed the relevance of four of the selected amino acids, showing the biological significance of site-specific evolutionary rate differences. CONCLUSION: This work demonstrates the use of a bioinformatic analysis to mine the huge set of genomic data from different taxa in order to generate a testable hypothesis. We verified the hypothesis experimentally and identified four amino acids which are to a different degree required for ligand-binding of a plant hormone receptor

A rapid cytokinin response assay in Arabidopsis indicates a role for phospholipase D in cytokinin signalling

AbstractSeedlings of Arabidopsis thaliana harboring a fusion of the cytokinin-responsive ARR5 gene promoter and the GUS reporter gene were used for a pharmacological approach to study cytokinin signal transduction. The assay was shown to be rapid, sensitive, dose-dependent and highly specific for cytokinins, both adenine and phenylurea derivatives. Numerous inhibitors of known signalling pathways were tested and some were shown to suppress reporter gene induction. Particularly, primary alcohols that specifically inhibit phospholipase D (PLD) partially prevented cytokinin-induced GUS activity and reduced the accumulation of ARR5 gene transcripts. This indicates a role for PLD early during cytokinin signalling

Ligand-binding properties and subcellular localization of maize cytokinin receptors

The ligand-binding properties of the maize (Zea mays L.) cytokinin receptors ZmHK1, ZmHK2, and ZmHK3a have been characterized using cytokinin binding assays with living cells or membrane fractions. According to affinity measurements, ZmHK1 preferred N6-(Δ2-isopentenyl)adenine (iP) and had nearly equal affinities to trans-zeatin (tZ) and cis-zeatin (cZ). ZmHK2 preferred tZ and iP to cZ, while ZmHK3a preferred iP. Only ZmHK2 had a high affinity to dihydrozeatin (DZ). Analysis of subcellular fractions from leaves and roots of maize seedlings revealed specific binding of tZ in the microsome fraction but not in chloroplasts or mitochondria. In competitive binding assays with microsomes, tZ and iP were potent competitors of [3H]tZ while cZ demonstrated significantly lower affinity; adenine was almost ineffective. The binding specificities of microsomes from leaf and root cells for cytokinins were consistent with the expression pattern of the ZmHKs and our results on individual receptor properties. Aqueous two-phase partitioning and sucrose density-gradient centrifugation followed by immunological detection with monoclonal antibody showed that ZmHK1 was associated with the endoplasmic reticulum (ER). This was corroborated by observations of the subcellular localization of ZmHK1 fusions with green fluorescent protein in maize protoplasts. All these data strongly suggest that at least a part of cytokinin perception occurs in the ER

Perception, Transduction and Crosstalk of Auxin and Cytokinin Signals

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

Empirically-specified Environmental Assisted Cracking Model

AbstractBased on various mechanisms of hydrogen diffusion in metals, different crack propagation models have been developed. Here, empirically specified model of crack propagation in structure components, which are simultaneously subjected to aggressive hydrogen environment influence and cycling, is presented. The model uses two different approaches to problem solving, i.e. the influence of aggressive hydrogen environment on material and the fatigue. A developed environmental assisted cracking model assumes that either the influence of aggressive hydrogen environment or the fatigue initiates a local fracture of structure component. This model enables estimating a structure component life

The project approach specifics in relation to the student self-organisation development

The study is based on the concept of shaping students' self-organisation in the digital learning environment of a technical university. The recommendations for creating an effective learning system can be used in the creation of effective education systems. They will be created through the use of digital technologies in the educational process of the university to create a culture of self-organization of students. According to these provisions, the author of the work should identify the characteristics of educational activities and ensure their effective implementation during the formation of the culture of students of a technical university. Scientific research on students' self-organization culture formation will be carried out considering the specifics of students' interaction and information environment of the university. The direction of the project approach in pedagogy is "pedagogical planning". The authors have identified the stages of pedagogical planning necessary for successful implementation of the project methodology and the problem under the study. They are necessary in the formation process of university students' self-organization culture. To conduct the research, it is necessary to determine the way in which the ability to create a culture of self-organization in the digital educational environment of a technical university can be developed. The authors have set a goal to provide students with the opportunity to realize their creative potential, to form self-organization. To enable students to design projects independently, the authors suggest that they show their autonomy in designing. They plan means to organize an effective learning process and ensure its effectiveness, which in modern conditions is possible through the introduction of digital technologies in the educational process of the university to create self-organization of students. The article describes technological methods of students' self-organization culture formation in the conditions of digital educational environment of a technical university, namely, project technologies, which are based on the relationship of a teacher and a group