Inhibitors of cytokinin oxidase/dehydrogenase (CKX) reduce the degradation of cytokinins in plants. This effect can be applied in agriculture as well as in plant tissue culture-based industries. In this work, we present the structure-activity relationship study of two series of CKX inhibitors based on diphenylurea. The compounds of Series I are derived from recently published CKX inhibitors - 3TFM-2HM and 3TFM-2HE. Here, we have identified key substituents with increased selectivity for maize ZmCKX1 and ZmCKX4a over AtCKX2 from Arabidopsis. Series II contains compounds that further excel in CKX inhibitory activity as well as in the ease of their synthesis. The best inhibitors exhibit IC50 in low nanomolar ranges with ZmCKX1 and especially with ZmCKX4a, which is generally more resistant to inhibition. The activity of the key compounds was verified in a tobacco and lobelia leaf disk assay, where N  6-isopentenyladenine was protected from degradation and promoted shoot regeneration. All prepared compounds were further tested for toxicity against Caenorhabditis elegans. The assay revealed clear differences in toxicity between compounds with and without a hydroxyalkyl group. In a broader perspective, this work increases our understanding of CKX inhibition and provides a more extensive portfolio of compounds suitable for agricultural and biotechnological research.

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.

The CRE1/AHK4 cytokinin receptor is an important component of plants’ hormone signaling systems, and compounds that can alter its activity have potential utility for studying the receptor’s functions and/or developing new plant growth regulators. A high throughput method was developed for screening compounds with agonist or antagonist properties toward the CRE1/AHK4 cytokinin receptor in a single experiment using the Nanodrop II liquid handling system and 384-well plates. Potential ligands are screened directly, using a reporter system in which receptor signaling activity triggers expression of β-galactosidase in Escherichia coli. This enzyme generates a fluorescent product from a non-fluorescent substrate, allowing the agonistic/antagonistic behavior of tested compounds to be assayed in relation to that of an internal standard (here the natural ligand, trans-zeatin). The method includes a robust control procedure to determine false positive or false negative effects of the tested compounds arising from their fluorescent or fluorescent-quenching properties. The presented method enables robust, automated screening of large libraries of compounds for ability to activate or inhibit the Arabidopsis thaliana cytokinin receptor CRE1/AHK4.