Assessing Protein Stability Under Different Light and Circadian Conditions.

Plants use light as an indicator of time and space as well as the major energy source for photosynthesis. Due to the development of specific photoreceptors, plants can perceive a wide range of wavelengths and adjust their development accordingly to their surroundings. In addition to light, the circadian clock allows the anticipation of diurnal and seasonal changes thus providing organisms with the adequate physiological responses to ever changing surroundings, which are reflected in increased fitness and survival rate. Although initially described as a set of interconnected transcriptional loops, it is now accepted that posttranslational modifications are also important for proper clock function. In fact, not only the clock but also light signaling rely on posttranslational modifications, such as phosphorylation and ubiquitination, for proper signal transduction. We have designed a simple and yet reproducible method to determine protein stability and half-life under different light and circadian conditions. Our method only requires standard laboratory equipment, a relatively small amount of starting material and can be applied to young seedlings and mature plants. Besides our application to study light and circadian clock proteins, this protocol can be adapted to any other conditions that regulate protein stability.