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 [...

Types, frequencies and value of intra-varietal genotypic non-uniformity in common wheat cultivars: authentic biotypes and foreign seeds

Seed-by-seed electrophoretic analysis of the seed storage-protein gliadin was used to identify two major types of intra-varietal non-uniformity in grain samples of 756 registered Triticum aestivum cultivars: authentic biotypes (genotypes arising from the original cross performed by the breeder) and foreign seeds (admixed alien genotypes or products of accidental cross-pollination). Authentic biotypes of a cultivar, distinct in alleles at one or more of eight Gli loci (Gli-1, Gli-2, Gli-A3, Gli-B3), occurred in more than 30% of the cultivars studied. Four biotypes of the cultivar Saratovskaya-29 (distinct in alleles at the Gli-A1 and Gli-A2 loci) differed significantly in 1000-kernel mass and dough quality. The relative frequency of biotypes changed with time when the cultivar grew in the region where it was bred and, more strongly, in alien regions. There is thus potential value in characterizing multiple biotypes to facilitate the adaptation of a cultivar to given eco-climatic environments. Changes in the frequencies of biotypes alter the grain quality of a cultivar as a whole. Ignorance of possible wheat intra-varietal non-uniformity (biotypes and foreign genotypes) in a grain material may confuse the interpretation of experimental results in field trails and grain-quality assessments

Technical university students’ readiness for sustainable learning in terms of self-directed learning

The article analyzes the readiness of technical university students for sustainable learning in terms of self-oriented learning based on the conducted research data. The authors conducted a respondents’ survey, in which they studied the technical university students’ perception of the self-oriented learning basic indicators. The obtained data analysis showed how the technical university students understand and develop their readiness for self-oriented learning. The results of the research work confirm that the technical university students are ready for sustainable learning partially. Despite the fact that the technical university students have the necessary digital competencies for effective participation in the learning process, the data obtained indicate the tendency of the technical university students to procrastinate in the learning process. The authors of the research work noted the importance of transformative learning in the sustainable development of the technical university students, analysed possible lack of time management skills problems and tendency to procrastination, which may have been contributed by the high level of stress caused by the negative effects of the COVID-19 pandemic and the unplanned transition to online learning. In recent decades, higher education has increasingly focused on student self-orientation. Self-oriented learning is effective only if the technical university students are capable of self-oriented learning. Self-oriented students are able not only to manage their own learning, but also to take responsibility, understand and realize the significance of learning and cognitively control their learning. A facilitator is able to help learners become fully self-directed online learners