Endogenous biological clocks drive daily rhythms enabling organisms to anticipate environmental changes as well as to coordinate and adapt their physiology in a synchronized manner. The circadian clock enables plants to anticipate as well as to respond to environmental variations and thus, improves their fitness. Both, environmental and metabolic signals feed into the clock, which is comprised of a network of genes, and keep it synchronized with day/night cycles. In return, the clock controls various pathways and ensures they get activated at the appropriate time of the day. Light is one of the so-called Zeitgebers, which can reset the clock.
Research on circadian rhythms benefits from real-time monitoring of reporter lines in which the promoter of a gene of interest drives the expression of luciferase pGENE::LUC+ in combination with sensitive imaging systems. Monitoring firefly luciferase expression using an in vivo imaging system allows non-invasive, real-time reporting of regulated gene expression. This technique is broadly applicable to the analysis of transcriptional, post-transcriptional, or translational regulation. Furthermore, the instability of luciferase activity allows the bioluminescent marker to recapitulate both increases and decreases in expression over a time course of several hours. In this manner, it is possible to investigate the tissue focus of the circadian clock and its characteristics, or screen for mutants defective in the spatial regulation of the expression of genes of interest.
The NightShade In Vivo Imaging System uses a low noise, high sensitivity CCD camera and is suitable for both luminescence and fluorescence. The NightShade has been designed specifically for plant imaging, and has numerous advantages compared to general-purpose cameras or imaging systems designed for animal research:
- Daylight simulation with flexible light quality and intensity.
- Intuitive software with powerful scheduler, which allows to program the desired intensity and quality of light during several days and capture images at the desired time points.
- Light-tight ports to insert tubing for irrigation (critical for imaging during several days) and other goals.
- Flexible sample size: from seedlings growing in petri dishes to plants in pots.
- Side camera mounting position to image whole plants, from leaf to roots.
- Anti-condensation tray with temperature control for high quality images when working with petri dishes.
In multicellular organisms, circadian clocks are naturally variable at individual, tissue as well as cellular level, culminating in noisy or inaccurate data. Therefore, robustness is required to accurately address key questions in circadian biology. This Application Note presents a simple protocol for circadian rhythms experiments with Arabidopsis thaliana reporter lines using the NightShade, which improves data quality, reduces luminescence variation between replicates and highly correlates with modelling predictions.