Gene expression under the control of molecular clocks occurs earlier in plant development than previously thought, finds Dartmouth professor C. Robertson McClung, the associate dean of faculty for the sciences. In a recent article in Plant Physiology, he reports that circadian function can be detected in some proteins just two days after hydration of the dried seed.
According to the article, as much as 80 to 90 percent of the Arabidopsis thaliana genes could be under circadian control. Genes that exhibit these circadian dependencies are said to be under clock control. A small subset of genes, called clock genes, provides the circadian oscillations that control the output of all the genes under circadian control.
Clock genes and circadian dependence are especially important in experimentation, because the time of the day when an experiment is performed can mask or amplify the results. In an interview with the DUJS, McClung explained, “if a clock is gating the responsiveness to a stimulus, then the time the stimulus is administered matters.”
In a simple example, administering a pulse of light to a person at noon would have very different effects than administering the same pulse of light before dawn. In the first case, the extra light has no noticeable effect, while in the second case the subject believes that dawn is occurring earlier, and advances his internal clock. This simple example illustrates the profound effect the timing of a stimulus can have on an organism’s response to it.
In developmental biology experiments using Arabidopsis thaliana, researchers often let etiolated (grown in the dark) seedlings grow for two days and then start experiments, ignoring possible clock effects at such early stages in the plant’s development. Quantifying exactly when the circadian rhythm kicks in by observing new mRNA production can be challenging for a variety of reasons. In particular, population effects and the small amplitude of the oscillations in mRNA levels can obfuscate the results.
In order to discover when circadian rhythms were established in the absence of normal entraining cues, such as light and temperature cycles, seedlings were grown under conditions that eliminated these cues, making the amplitude of the oscillations hard to detect.
The other, less straightforward difficulty is addressing population effects. McClung demonstrates that seed hydration is enough to start synchronized clock function in the seedlings. Although seedlings begin synchronized, small variations in circadian period can have an amplified effect after a few days, making seedlings out of sync with one another. This effect occurs not only on the organismal level, but also between cells within the same organism, obscuring the oscillations of the mRNA products under clock control. In normal growing conditions, continued synchronization is achieved through entraining cues like light.
Using a luciferase fusion protein, McClung was able to observe new mRNA transcription in real time. Luciferase is the light-generating enzyme originally found in fireflies.
Using these methods, McClung discovered that clock function is present only two days after the initial hydration of the seedling. McClung said that he felt these findings were likely to make researchers take a second look at their results, whether they were positive or negative, and to always control for time.