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

• Published in: Bio-protocol Vol. 15; no. 1372; p. e5319
• Main Authors: Kitawaki, Kohei, Mihara, Ryota, Ito-Inaba, Yasuko, Inaba, Takehito
• Format: Journal Article
• Language: English
• Published: United States Bio-Protocol 20.05.2025; Bio-protocol LLC
• Subjects: Biology; Methods; Arabidopsis thaliana; Chemical screening; Cold-regulated gene expression; Submerged culture; Luciferase; Microplate reader; Chemiluminescence imaging device
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.5319

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 . Specifically, we generated plants harboring a COLD-REGULATED 15A promoter::luciferase ( ) construct and grew them in a submerged liquid culture. Using a single true leaf excised from plants and 96-well culture plates, we performed high-throughput screening of chemical compounds that inhibit cold-induction of . 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 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.