Fluorescence-based High-throughput Method Research Articles

A fluorescence-based high-throughput screening method for cytokinin translocation mutants

BackgroundCytokinins are one kind of phytohormones essential for plant growth, development and stress responses. In the past half century, significant progresses have been made in the studies of cytokinin signal transduction and metobolic pathways, but the mechanism of cytokinin translocation is poorly understood. Arabidopsis (Arabidopsis thaliana) response regulator 5 (ARR5) is a type-A response factor in cytokinin signaling which is induced by cytokinins and has been used as a reporter gene for the endogenous cytokinins in Arabidopsis. Here, we report a fluorescence-based high-throughput method to screen cytokinin translocation mutants using an ethyl methyl sulfone (EMS) mutagenesis library generated with ARR5::eGFP transgenic plants.ResultsThe seedlings with enhanced green fluorescent protein (GFP) signal in roots were screened in a luminescence imaging system (LIS) in large scale to obtain mutants with over-accumulated cytokinins in roots. The selected mutants were confirmed under a fluorescence microscopy and then performed phenotypic analysis. In this way, we obtained twelve mutants with elevated GFP signal in the roots and further found three of them displayed reduced GFP signal in the aerial tissues. Two of the mutants were characterized and proved to be the atabcg14 allelic mutants which are defective in the long-distance translocation of root-synthesized cytokinins.ConclusionsWe provide a strategy for screening mutants defective in cytokinin translocation, distribution or signaling. The strategy can be adapted to establish a system for screening mutants defective in other hormone transporters or signaling components using a fluorescence reporter.

Chemical Mutagenesis and Fluorescence-Based High-Throughput Screening for Enhanced Accumulation of Carotenoids in a Model Marine Diatom Phaeodactylum tricornutum.

Diatoms are a major group of unicellular algae that are rich in lipids and carotenoids. However, sustained research efforts are needed to improve the strain performance for high product yields towards commercialization. In this study, we generated a number of mutants of the model diatom Phaeodactylum tricornutum, a cosmopolitan species that has also been found in Nordic region, using the chemical mutagens ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG). We found that both chlorophyll a and neutral lipids had a significant correlation with carotenoid content and these correlations were better during exponential growth than in the stationary growth phase. Then, we studied P. tricornutum common metabolic pathways and analyzed correlated enzymatic reactions between fucoxanthin synthesis and pigmentation or lipid metabolism through a genome-scale metabolic model. The integration of the computational results with liquid chromatography-mass spectrometry data revealed key compounds underlying the correlative metabolic pathways. Approximately 1000 strains were screened using fluorescence-based high-throughput method and five mutants selected had 33% or higher total carotenoids than the wild type, in which four strains remained stable in the long term and the top mutant exhibited an increase of 69.3% in fucoxanthin content compared to the wild type. The platform described in this study may be applied to the screening of other high performing diatom strains for industrial applications.

Evaluation of the effects of 18 non-synonymous single-nucleotide polymorphisms of CYP450 2C19 on in vitro drug inhibition potential by a fluorescence-based high-throughput assay

To comprehensively understand the effects of CYP2C19 genetic polymorphisms on inhibition-based drug–drug interactions (DDIs), 18 human CYP2C19 non-synonymous single-nucleotide polymorphic variants and the wild-type isoform (CYP2C19.1A) were expressed in yeast cells. Using a fluorescence-based high-throughput method, the kinetic constants of these variants, as well as the inhibition constants for 10 drugs, were determined.CYP2C19.5B and CYP2C19.6 showed no activity towards CEC (3-cyano-7-ethoxycoumarin) O-deethylation. CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.16, CYP2C19.19, E122A and A161P* (an allele containing both A161P and I331V) exhibited significantly reduced catalytic activities compared with CYP2C19.1A. The inhibition assay showed that the CYP2C19 genotype significantly affected the in vitro drug inhibition potential. Although the effect on drug inhibition potential is genotype- and inhibitor-dependent, there was an obvious trend: drugs tended to exhibit higher IC50 values (i.e. decreased inhibition potential) towards variants with reduced activity compared with variants with normal activity. This indicated that patients with reduced-function alleles may be less susceptible to CYP2C19-related DDIs.In this study, we provided the first in vitro evidence of CYP2C19 genotype-dependent effects on drug inhibition potential. This work greatly extends our understanding of the functional consequences of CYP2C19 genetic polymorphisms, and thus should prove valuable for CYP2C19 genotype-based therapy.