Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana
The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2015-01-01
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| Series: | Frontiers in Plant Science |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00013/full |
| _version_ | 1818149090100248576 |
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| author | Vivek eHalder Erich eKombrink |
| author_facet | Vivek eHalder Erich eKombrink |
| author_sort | Vivek eHalder |
| collection | DOAJ |
| description | The use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as ‘chemical genetics’, has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical’s bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line. |
| first_indexed | 2024-12-11T13:01:30Z |
| format | Article |
| id | doaj.art-25786af8471548da9165c00b1923844d |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-11T13:01:30Z |
| publishDate | 2015-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-25786af8471548da9165c00b1923844d2022-12-22T01:06:27ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2015-01-01610.3389/fpls.2015.00013125786Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thalianaVivek eHalder0Erich eKombrink1Max Planck Institute for Plant Breeding ResearchMax Planck Institute for Plant Breeding ResearchThe use of biologically active small molecules to perturb biological functions holds enormous potential for investigating complex signaling networks. However, in contrast to animal systems, the search for and application of chemical tools for basic discovery in the plant sciences, generally referred to as ‘chemical genetics’, has only recently gained momentum. In addition to cultured cells, the well-characterized, small-sized model plant Arabidopsis thaliana is suitable for cultivation in microplates, which allows employing diverse cell- or phenotype-based chemical screens. In such screens, a chemical’s bioactivity is typically assessed either through scoring its impact on morphological traits or quantifying molecular attributes such as enzyme or reporter activities. Here, we describe a facile forward chemical screening methodology for intact Arabidopsis seedlings harboring the β-glucuronidase (GUS) reporter by directly quantifying GUS activity in situ with 4-methylumbelliferyl-β-D-glucuronide (4-MUG) as substrate. The quantitative nature of this screening assay has an obvious advantage over the also convenient histochemical GUS staining method, as it allows application of statistical procedures and unbiased hit selection based on threshold values as well as distinction between compounds with strong or weak bioactivity. At the same time, the in situ bioassay is very convenient requiring less effort and time for sample handling in comparison to the conventional quantitative in vitro GUS assay using 4-MUG, as validated with several Arabidopsis lines harboring different GUS reporter constructs. To demonstrate that the developed assays is particularly suitable for large-scale screening projects, we performed a pilot screen for chemical activators or inhibitors of salicylic acid-mediated defense signaling using the Arabidopsis PR1p::GUS line. Importantly, the screening methodology provided here can be adopted for any inducible GUS reporter line.http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00013/fullChemical GeneticsHigh-Throughput Screeningchemical screeningreporter gene expressionβ-glucuronidase activitybioactive small molecules |
| spellingShingle | Vivek eHalder Erich eKombrink Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana Frontiers in Plant Science Chemical Genetics High-Throughput Screening chemical screening reporter gene expression β-glucuronidase activity bioactive small molecules |
| title | Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana |
| title_full | Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana |
| title_fullStr | Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana |
| title_full_unstemmed | Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana |
| title_short | Facile high-throughput forward chemical genetic screening by in situ monitoring of glucuronidase-based reporter gene expression in Arabidopsis thaliana |
| title_sort | facile high throughput forward chemical genetic screening by in situ monitoring of glucuronidase based reporter gene expression in arabidopsis thaliana |
| topic | Chemical Genetics High-Throughput Screening chemical screening reporter gene expression β-glucuronidase activity bioactive small molecules |
| url | http://journal.frontiersin.org/Journal/10.3389/fpls.2015.00013/full |
| work_keys_str_mv | AT vivekehalder facilehighthroughputforwardchemicalgeneticscreeningbyinsitumonitoringofglucuronidasebasedreportergeneexpressioninarabidopsisthaliana AT erichekombrink facilehighthroughputforwardchemicalgeneticscreeningbyinsitumonitoringofglucuronidasebasedreportergeneexpressioninarabidopsisthaliana |