Research Projects

What comprises the central oscillator in plants? How does it function?

Since there is no apparent homology between the clock-associated genes found in plants and those in other types of organisms, we cannot use homology-based approaches to identify plant clock genes. Instead, we are using forward genetics to find mutant plants with altered circadian rhythms and then performing positional cloning to identify the altered genes. Although we could monitor leaf movement rhythms or other clock outputs in these experiments, we most often make use of a luciferase reporter assay. We generate transgenic Arabidopsis plants that express firefly luciferase under the control of a clock-regulated promoter. These plants are then monitored with a sensitive CCD camera in constant environmental conditions; the rhythms in luciferase activity reflect the activity of the endogenous clock.

In our screen, we mutagenized plants carrying a luciferase reporter gene with EMS and assayed rhythmicity of the M2 generation in constant darkness. Here is an example of a mutant with a short-period, low-amplitude phenotype. We isolated 10 mutants whose clocks run either faster or slower than wild type plants and are currently characterizing these plants and mapping the underlying mutations.

How does the circadian oscillator influence plant physiology?

We are interested in how the clock influences plant physiology at both the mechanistic and descriptive levels. What processes are influenced by the clock? How does the clock regulate its many outputs so that each occurs at the most appropriate time of day? We have taken a genomic approach to address both kinds of questions.
Using DNA microarrays, we have found that in young seedlings grown in constant light and temperature, about 10% of expressed genes show circadian variation in steady-state mRNA levels. Peak expression of these genes occurs at a wide range of times, just as clock regulated physiological pathways show peak activity at diverse times of day.

To better understand how the circadian clock controls gene expression, we examined the promoters of clock-regulated genes to see if there were any conserved promoter motifs. We found one motif to be highly over-represented in the promoters of evening-phased genes. Subsequently, we showed that this motif is necessary and sufficient to confer evening-phased reporter gene expression in transgenic plants and thus termed it the evening element, or EE. The EE is bound by both repressors and activators of transcription; we are currently using biochemical, genetic, and genomic approaches to identify these trans-acting factors.

 

How does the circadian clock regulate this myriad of target genes? Does a single transcription factor regulate all the genes that act in a specific pathway? Are most or all of the genes with a given phase regulated by the same transcription factor? We are investigating the function of specific clock-regulated transcription factors to address these questions.

Finally, we have used our microarray data to investigate the role of the clock in pathways not previously recognized as circadian regulated. We found that many genes implicated in plant responses to the hormone auxin are also clock regulated. This spurred us to investigate whether the circadian clock might modulate auxin signal transduction. We indeed found that plants are more responsive to treatment with exogenous auxin when it is applied just before subjective dawn than when it is applied at other times.

We have also found that either endogenous levels or responsiveness to auxin is clock regulated. We are currently studying the mechanism of circadian regulation of auxin signaling and its implications for plant growth and development.