In Vivo Bioluminescence Analyses of Circadian Rhythms in Arabidopsis thaliana Using a Microplate Luminometer

Our understanding of the circadian clock function in plants has been markedly assisted by studies with the model species Arabidopsis thaliana. Molecular and genetics approaches have delivered a comprehensive view of the transcriptional regulatory networks underlying the Arabidopsis circadian system. The use of the luciferase as a reporter allowed the precise in vivo determination of circadian periods, phases, and amplitudes of clock promoter activities with unprecedented temporal resolution. An increasing repertoire of fine-tuned luciferases together with additional applications such as translational fusions or bioluminescence molecular complementation assays have considerably expanded our view of circadian protein expression and activity, far beyond transcriptional regulation. Further applications have focused on the in vivo simultaneous examination of rhythms in different parts of the plant. The use of intact versus excised plant organs has also provided a glimpse on both the organ-specific and autonomy of the clocks and the importance of long distance communication for circadian function. This chapter provides a basic protocol for in vivo high-throughput monitoring of circadian rhythms in Arabidopsis seedlings using bioluminescent reporters and a microplate luminometer.