High Throughput Screening Method for Identifying Potential Agonists and Antagonists of Arabidopsis thaliana Cytokinin Receptor CRE1/AHK4

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

Simulation of random clone picking in randomized codon based saturation mutagenesis and calculation of the average number of clones needed to detect a new variant in libraries generated by NNK, NNN and NNM randomization.

<p>Simulation of random clone picking in randomized codon based saturation mutagenesis and calculation of the average number of clones needed to detect a new variant in libraries generated by NNK, NNN and NNM randomization.</p

Hydrolysis rates for substrates <i>p</i>NPG (A) and <i>t</i>ZOG (B) by β-glucosidase Zm-p60.1 variants produced in the course of saturation mutagenesis of the W373position.

<p>Hydrolysis rates for substrates <i>p</i>NPG (A) and <i>t</i>ZOG (B) by β-glucosidase Zm-p60.1 variants produced in the course of saturation mutagenesis of the W373position.</p

Comparison of projected costs of randomized codon-based saturation mutagenesis library creation and one-by-one site-directed mutagenesis (SDM).

<p>Empirically determined results for the combined β-glucosidase Zm-p60.1 mutagenesis approach are also shown (experimental).</p

Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants

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