The Use of the Luciferase Reporter System for in Planta Gene Expression Studies

Abstract

The properties of the firefly luciferase (LUC) make it a very good non-destructive reporter to quantify and image transgene promoter activity in plants. The short half-life of the LUC mRNA and protein, and the very limited regeneration of the LUC protein after reacting with luciferin, enables monitoring of changes in gene activity with a high time resolution. However, the ease at which luciferase activity is measured in planta, using a light sensitive camera system (2D-luminometer), contrasts sharply with the complications that arise from interpreting the results. A variegated pattern of luciferase activity, that is often observed in in planta measurements, might either be caused by differences in influx, availability of the substrates (luciferin, oxygen, ATP) or by local differences in reporter gene activity. Here we tested the possible contribution of differences in the availability of each substrate to the variegated in planta luciferase activity, and we show when in planta luciferase activity is measured under substrate equilibrium conditions and can be related to the promoter activity of the reporter gene. Furthermore, we demonstrate the effects of protein stability, apparent half-life of luciferase activity, regeneration of luciferase and pH on the in vivo and in vitro luciferase measurements. The combined results give the prerequisites for the correct utilisation of the luciferase reporter system, especially for in vivo gene expression studies in plant research.