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

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.