Screening for gene regulation mutants by bioluminescence imaging.
Because plants cannot move, they have evolved complex sensing and response systems to cope with the physical environment. Adverse environmental conditions, such as those causing abiotic stress, often cause significant losses in crop productivity and quality. Because of a paucity of well-defined visible phenotypes, conventional genetic screens have not been very successful in isolating abiotic stress signal transduction mutants of plants. Here, we describe a reporter gene-based strategy to screen for mutants affected in abiotic stress-regulated gene transcription. Our genetic screen uses the firefly luciferase reporter gene driven by the cold, drought, salt, and abscisic acid (ABA)-responsive RD29A promoter (RD29A::LUC). Arabidopsis plants transformed with the RD29A::LUC reporter emit bioluminescence in response to cold, drought, salt, or ABA treatment. After mutagenesis of these plants with ethyl methanesulfonate (EMS), mutants can be screened from the M2 population by monitoring the level of stress-inducible bioluminescence with a high-throughput, low-light imaging system. This protocol describes in detail the procedures for this luciferase reporter-based genetic screen for Arabidopsis mutants defective in abiotic stress signaling.
Chinnusamy
Viswanathan
V
Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA.
Stevenson
Becky
B
Lee
Byeong-ha
BH
Zhu
Jian-Kang
JK
D000040:Abscisic Acid / Q000494:pharmacology; D017360:Arabidopsis / Q000235:genetics; D019677:Artificial Gene Fusion; D003080:Cold Temperature / Q000009:adverse effects; D004190:Disasters; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000235:genetics*; D017343:Genes, Plant / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008163:Luminescent Measurements; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D012492:Salts / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods
D009153:Mutagens; D012492:Salts; D000040:Abscisic Acid; D005020:Ethyl Methanesulfonate; D008156:Luciferases