Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

• Keywords
• Arabidopsis, N-glucosides, Zea mays, [2-3H]tZ9G, cis-zeatin, cytokinin, cytokinin oxidase/dehydrogenase, oat, senescence, trans-zeatin, β-D-glucosidase Zm-p60.1,
• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Cytokinins genetics   MeSH
• Glucosides genetics   MeSH
• Glucosyltransferases genetics   MeSH
• Zea mays genetics   metabolism   MeSH
• Evolution, Molecular * MeSH
• Oxidoreductases genetics   MeSH
• Gene Expression Regulation, Plant genetics   MeSH
• Plant Growth Regulators genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• Glucosides MeSH
• Glucosyltransferases MeSH
• Oxidoreductases MeSH
• Plant Growth Regulators MeSH

Cytokinin (CK) N-glucosides are the most abundant group of CK metabolites in many species; however, their physiological role in planta was for a long time perceived as irreversible storage CK forms only. Recently, a comprehensive screen showed that only vascular plants form CK N-glucosides in contrast to mosses, algae, and fungi. The formation of CK N-glucosides as biologically inactive CK conjugates thus represents an evolutionarily young mechanism for deactivation of CK bases. Even though CK N-glucosides are not biologically active themselves due to their inability to activate the CK perception system, new data on CK N-glucoside metabolism show that trans-zeatin (tZ) N7- and N9-glucosides are metabolized in vivo, efficiently releasing free CK bases that are most probably responsible for the biological activities observed in a number of bioassays. Moreover, CK N-glucosides' subcellular localization as well as their abundance in xylem both point to their possible plasma membrane transport and indicate a role also as CK transport forms. Identification of the enzyme(s) responsible for the hydrolysis of tZ N7- and N9-glucosides, as well as the discovery of putative CK N-glucoside plasma membrane transporter, would unveil important parts of the overall picture of CK metabolic interconversions and their physiological importance.

• Keywords
• UGT, cytokinin N-glucoside, cytokinin metabolism, cytokinin transport, isopentenyladenine N7-glucoside, isopentenyladenine N9-glucoside, zeatin N7-glucoside, zeatin N9-glucoside,
• Publication type
• Journal Article MeSH
• Review MeSH

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

• Keywords
• 5′ss, SMN1, U1 snRNA, cryptic splice sites, splicing-affecting mutation,
• MeSH
• Alternative Splicing * MeSH
• Cell Line MeSH
• Exons * MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• RNA Splice Sites MeSH
• Mutation * MeSH
• Mutagenesis MeSH
• Survival of Motor Neuron 1 Protein chemistry   genetics   metabolism   MeSH
• RNA, Small Nuclear chemistry   genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Computational Biology methods   MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA Splice Sites MeSH
• Survival of Motor Neuron 1 Protein MeSH
• RNA, Small Nuclear MeSH
• SMN1 protein, human MeSH Browser
• U1 small nuclear RNA MeSH Browser

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of β-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

• Keywords
• enzyme kinetics, fluorescence, glucose, glycoside hydrolases, lab automation, β-glucosidase,
• MeSH
• Automation MeSH
• beta-Glucosidase chemistry   MeSH
• Catalysis MeSH
• Kinetics MeSH
• Zea mays enzymology   MeSH
• Plant Proteins chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Glucosidase MeSH
• Plant Proteins MeSH

Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins' structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations' effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

• MeSH
• Enzyme Activation MeSH
• beta-Glucosidase genetics   metabolism   MeSH
• Databases, Protein MeSH
• Gene Library MeSH
• Hydrolysis MeSH
• Codon MeSH
• Zea mays genetics   metabolism   MeSH
• Mutagenesis MeSH
• Protein Engineering * methods   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Substrate Specificity MeSH
• Computational Biology MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta-Glucosidase MeSH
• Codon MeSH
• Plant Proteins MeSH

BACKGROUND: When applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N(6)-benzylaminopurine were further tested in planta. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants. CONCLUSIONS/SIGNIFICANCE: Tetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.

• MeSH
• Enzyme Activation MeSH
• Arabidopsis drug effects   genetics   growth & development   metabolism   MeSH
• Biomass MeSH
• Cytokinins chemistry   pharmacology   MeSH
• Plant Roots chemistry   drug effects   growth & development   MeSH
• Zea mays drug effects   genetics   growth & development   metabolism   MeSH
• Plant Leaves chemistry   drug effects   growth & development   MeSH
• Oxidoreductases metabolism   MeSH
• Cell Proliferation drug effects   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Growth Regulators chemistry   pharmacology   MeSH
• Seedlings drug effects   MeSH
• Gene Expression Profiling MeSH
• Xylem chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytokinins MeSH
• Oxidoreductases MeSH
• Plant Growth Regulators MeSH