Optimizing the Use of a Liquid Handling Robot to Conduct a High Throughput Forward Chemical Genetics Screen of <em>Arabidopsis thaliana</em>

Abstract

Chemical genetics is increasingly being employed to decode traits in plants that may be recalcitrant to traditional genetics due to gene redundancy or lethality. However, the probability of a synthetic small molecule being bioactive is low; therefore, thousands of molecules must be tested in order to find those of interest. Liquid handling robotics systems are designed to handle large numbers of samples, increasing the speed with which a chemical library can be screened in addition to minimizing/standardizing error. To achieve a high-throughput forward chemical genetics screen of a library of 50,000 small molecules on Arabidopsis thaliana (Arabidopsis), protocols using a bench-top multichannel liquid handling robot were developed that require minimal technician involvement. With these protocols, 3,271 small molecules were discovered that caused visible phenotypic alterations. 1,563 compounds induced short roots, 1,148 compounds altered coloration, 383 compounds caused root hair and other, non-categorized, alterations, and 177 compounds inhibited germination.

Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis

“…A ChemBridge DIVERSet library was screened at 100 μM and 3271 of the 50,000 compounds caused aberrant phenotypes. Exactly 1,563 of these phenotypes were categorized as either short roots or very short roots (Figure B,C) . All seedlings that exhibited such phenotypes were investigated for the presence of ectopic lignification using the colorimetric phloroglucinol assay (Figure D).…”

“…The screen was performed as stated in a previous report. 32 Confocal Microscopy. Florescent plant lines were grown on 0.5× MS agar.…”

“…Each of the small molecules was solubilized in DMSO. The screen was performed as stated in a previous report …”

“…Exactly 1,563 of these phenotypes were categorized as either short roots or very short roots (Figure 1B,C). 32 All seedlings that exhibited such phenotypes were investigated for the presence of ectopic lignification using the colorimetric phloroglucinol assay (Figure 1D). From this, we identified 33 small molecules induced ectopic lignification at and below a concentration of 100 μM (Table S1).…”

ScreenSeed as a novel high throughput seed germination phenotyping method

“…However, several pipetting robots are presently available (e.g. the OT-2 Robot ), and therefore there would not be any difficulty in equipping the present ScreenSeed automate with such a pipetting robot, notably for applications aiming at screening large chemical libraries to find novel molecules affecting the seed germination process (rate, uniformity, damping off....) that can be used for the development of new priming and/or treatment procedures 49 . We anticipate that the present technology can be used to investigate the germination behavior of a broad panel of seeds from crop species.…”