Dartmouth Researchers Find 
2 Circadian Clocks in the Same Plant Tissue 

SUSAN KNAPP / Dartmouth College Office of Public Affairs 7may03

[Abstract below]

Dartmouth researchers have found evidence of two circadian clocks working within the same tissue of the plant Arabidopsis thaliana, a flowering plant often used in genetic studies. Their results suggest that plants can integrate information from at least two environmental signals, light and temperature, which is important in order to respond to seasonal changes.

The study, published this week, appears in the online edition of the Proceedings of the National Academy of Sciences.

"Having two clocks with different sensitivities to light and to temperature is a better way to ensure that both signals of environmental input are fully understood by the plant," says C. Robertson McClung, professor of biological sciences and an author on the paper. "The plant can then process the data and make decisions about flowering, which is a very critical decision. Arabidopsis flowers in response to the lengthening days of spring, but if it were to flower too soon and there is a nasty frost, the blossoms will die. Early spring is cool, so it makes sense for a plant to clue in to more than one environmental signal."

Arabidopsis thaliana wildtype flower. 
It is approximately 5 mm in size.

The researchers, which included McClung, Todd Michael, a former graduate student who is now a postdoctoral fellow at the Salk Institute in San Diego, and , a graduate student, followed rhythms in two kinds of genes - one kind that encodes for photosynthesis and another not involved in photosynthesis. The genes in this study are both found in the mesophyll, the spongy inner layer of tissue in leaves.

To measure gene expression, McClung and his students manipulated the clock-controlled genes they were studying and put them in control of luciferase, the enzyme that makes fireflies glow, and then introduced that new gene into Arabidopsis. Each plant in the study had only one altered, light-making gene. When that gene was stimulated, light production was captured by a very sensitive camera. McClung and his team used this method to test how Arabidopsis responded to conflicting signals, such as a cycle of cool days and warm nights.

"We found if we gave them warm nights and cool days, the photosynthetic gene ignored the temperature signal and behaved as if it was only seeing the light signal, which makes sense because photosynthesis absolutely depends on daylight," says McClung. "But the other gene ignored the light signal and responded to the temperature signal. That kind of surprised us."

McClung and his students continued the study by examining how the circadian clocks were reset by different stimuli. For example, people respond to a pulse of light prior to dawn by readjusting their internal clocks a few hours ahead. The same pulse of light administered after dusk delays the clock. The researchers found that the non-photosynthetic gene, which favored temperature signals, showed an exaggerated response to pulses of cold air relative to the photosynthetic gene that responded more to light signals.

"This could only occur if the two genes were responding to two different clocks," says McClung. "Since both the genes are expressed in the mesophyll, it's clear that both clocks are operating in that tissue. This is exciting because this is the first good example of two clocks operating within a single tissue in any multicellular organism. We're not quite at the point where we can find out if there are two clocks operating in a single cell, but that's our goal."

This research is supported by the National Science Foundation.

ABSTRACT

May 7, 2003

Proc. Natl. Acad. Sci. USA, 10.1073/pnas.1131995100

Plant Biology

Two Arabidopsis circadian oscillators can be distinguished by differential temperature sensitivity

Todd P. Michael, Patrice A. Salomé, and C. Robertson McClung * Department of Biological Sciences, Dartmouth College, Hanover, NH 03755

Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved April 8, 2003 (received for review April 5, 2003)

Circadian rhythms are widespread in nature and reflect the activity of an endogenous biological clock. In metazoans, the circadian system includes a central circadian clock in the brain as well as distinct clocks in peripheral tissues such as the retina or liver. Similarly, plants have distinct clocks in different cell layers and tissues. Here, we show that two different circadian clocks, distinguishable by their sensitivity to environmental temperature signals, regulate the transcription of genes that are expressed in the Arabidopsis thaliana cotyledon. One oscillator, which regulates CAB2 expression, responds preferentially to light-dark versus temperature cycles and fails to respond to the temperature step associated with release from stratification. The second oscillator, which regulates CAT3 expression, responds preferentially to temperature versus light-dark cycles and entrains to the release from stratification. Finally, the phase response curves of these two oscillators to cold pulses are distinct. The phase response curve of the oscillator component TOC1 to cold pulses is similar to that of CAB2, indicating that CAB2 is regulated by a TOC1-containing clock. The existence of two clocks, distinguishable on the basis of their sensitivity to temperature, provides an additional means by which plants may integrate both photoperiodic and temperature signals to respond to the changing seasons.

*To whom correspondence should be addressed.

E-mail: mcclung@dartmouth.edu.

www.pnas.org/cgi/doi/10.1073/pnas.1131995100