Screening for gene regulation mutants by bioluminescence
imaging.
Chinnusamy V, Stevenson B, Lee BH, Zhu JK.
Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA.
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.