Dataset Information

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

ABSTRACT: 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 ?-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.

SUBMITTER: Klimes P

PROVIDER: S-EPMC5463364 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

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

Klimeš Pavel P Turek Dušan D Mazura Pavel P Gallová Lucia L Spíchal Lukáš L Brzobohatý Břetislav B

Frontiers in plant science 20170608

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 s ...[more]

PMID: 28642766

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Project description:Background and aimsExcess selenium (Se) is toxic to plants, but relatively little is known about the regulatory mechanism of plant Se tolerance. This study explored the role of the TPS22 gene in Se tolerance in Arabidopsis thaliana.MethodsArabidopsis wild type and XVE mutant seeds were grown on half-strength MS media containing Na2SeO3 for screening of the Se-tolerant mutant tps22. The XVE T-DNA-tagged genomic sequence in tps22 was identified by TAIL-PCR. The TPS22 gene was transformed into the mutant tps22 and wild type plants using the flower infiltration method. Wild type, tps22 mutant and transgenic seedlings were cultivated on vertical plates for phenotype analysis, physiological index measurement and gene expression analysis.Key resultsWe identified an Arabidopsis Se-tolerant mutant tps22 from the XVE pool lines, and cloned the gene which encodes the terpenoid synthase (TPS22). TPS22 was downregulated by Se stress, and loss-of-function of TPS22 resulted in decreased Se accumulation and enhanced Se tolerance; by contrast, overexpression of TPS22 showed similar traits to the wild type under Se stress. Further analysis revealed that TPS22 mediated Se tolerance through reduction of Se uptake and activation of metabolism detoxification, which decreased transcription of high-affinity transporters PHT1;1, PHT1;8 and PHT1;9 and significantly increased transcription of selenocysteine methyltransferase (SMT), respectively. Moreover, loss-of-function of TPS22 resulted in reduced cytokinin level and repression of cytokinin signalling components AHK3 and AHK4, and upregulation of ARR3, ARR15 and ARR16. Exogenous cytokinin increased transcription of PHT1;1, PHT2;1 and SMT and decreased Se tolerance of the tps22 mutant. In addition, enhanced Se resistance of the tps22 mutant was associated with glutathione (GSH).ConclusionsSe stress downregulated TPS22, which reduced endogenous cytokinin level, and then affected the key factors of Se uptake and metabolism detoxification. This cascade of events resulted in reduced Se accumulation and enhanced Se tolerance.

Dataset Information

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

Publications

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

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