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. 2025 May 20;15(10):e5319.
doi: 10.21769/BioProtoc.5319.

High-Throughput Screening Identification of Chemical Compounds That Affect Cold-Regulated Gene Expression in Arabidopsis thaliana Using an Excised Single Leaf

Kohei Kitawaki  1 , Ryota Mihara  1 , Yausko Ito-Inaba  1   2 , Takehito Inaba  1
Affiliations

High-Throughput Screening Identification of Chemical Compounds That Affect Cold-Regulated Gene Expression in Arabidopsis thaliana Using an Excised Single Leaf

Kohei Kitawaki et al. Bio Protoc. .

Abstract

The identification of chemical compounds that affect intracellular processes has greatly contributed to the understanding of developmental regulation in plants. In this protocol, we describe a method for identifying chemical compounds that affect cold-regulated gene expression in Arabidopsis thaliana. Specifically, we generated Arabidopsis plants harboring a COLD-REGULATED 15A (COR15A) promoter::luciferase (COR15Apro::LUC) construct and grew them in a submerged liquid culture. Using a single true leaf excised from COR15Apro::LUC plants and 96-well culture plates, we performed high-throughput screening of chemical compounds that inhibit cold-induction of COR15Apro::LUC. Luciferase activity was detected using a microplate reader and a chemiluminescence imaging device. This protocol can be easily used for the identification of chemical compounds that regulate other processes, being versatile with respect to equipment. Key features • High-throughput screening of chemical compounds that affect cold-regulated gene expression is possible using a single leaf excised from Arabidopsis grown in a submerged culture. • Screening is based on luciferase activity derived from an excised single leaf. • Direct measurement of luciferase activity is possible using a microplate reader and a chemiluminescence imaging device. • This protocol can be easily used for the identification of chemical compounds that regulate other processes.

Keywords: Arabidopsis thaliana; Chemical screening; Chemiluminescence imaging device; Cold-regulated gene expression; Luciferase; Microplate reader; Submerged culture.

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Conflict of interest statement

Competing interestsAuthors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.. Submerged Arabidopsis thaliana culture in a 100 mL flask
Figure 2.
Figure 2.. Overview of the preparation of a 96-well culture plate containing a single leaf excised from Arabidopsis.
This procedure can be performed under sterile conditions if necessary.
Figure 3.
Figure 3.. Overview of the incubation of each single leaf with the compound
Figure 4.
Figure 4.. Summary of data analysis.
Raw data should be converted to a percentile of negative control (DMSO) to identify compounds that inhibit cold-induction of COR15Apro::LUC.
Figure 5.
Figure 5.. Chemiluminescence image of the 96-well culture plate containing a single leaf of COR15Apro::LUC Arabidopsis after cold treatment for 24 h.
Dark wells contain inhibitors of cold-induction of COR15Apro::LUC identified in the previous study [7].
See this image and copyright information in PMC

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