Circadian influence in plants more widespread than previously thought

While picking apart the genetic makeup of the plant Arabidopsis, two Dartmouth researchers made a startling discovery. They found that approximately 36 percent of its genome is potentially regulated by the circadian clock, which is three and a half times more than had previously been estimated.

The study, which appears in the June issue of Plant Physiology, was conducted by C. Robertson McClung, Dartmouth professor of biological sciences, and Todd Michael, a former Dartmouth graduate student who is now a postdoctoral fellow at the Salk Institute in La Jolla, Calif. Their research on circadian-controlled genes contributes to efforts to help improve plant productivity and can possibly lead to growing crops that are more resistant to stressful soil or climate conditions.

McClung and Michael used a technique called “gene trapping” or “enhancer trapping” to measure how much mRNA is produced or synthesized by large sections in the genome. According to McClung, a great deal of gene regulation occurs in the gene’s ability to synthesize mRNA, which then is translated into proteins that perform the critical metabolic activities of a cell.

In this study, the researchers randomly inserted a gene that encodes an easily measurable element, in this case luciferase (the enzyme that makes fireflies glow), into the genome to see what genes would be involved in mRNA synthesis. Luciferase is only expressed when it is inserted adjacent to an active plant gene, and it takes on that native gene’s expression. With this method the researchers found new regions of the genome under circadian control.

“In terms of clock control of mRNA synthesis, it does appear that it’s more widespread than we had estimated,” says McClung. “It runs contrary to accepted dogma. It’s a new look at this from a slightly different perspective and it gives a slightly different answer. I think our study points out some of the limitations of microarray analysis.”

Previous comprehensive genetic studies utilized microarray analysis and had only measured about 10% of the mRNAs in the organism showing circadian oscillations. Microarray studies look at the total abundance of mRNA, including both synthesis and degradation. McClung and Michael’s measurements focus specifically on the rate of mRNA synthesis.

Questions still remain about the discrepancy between the number of genes whose mRNA synthesis is clock regulated and the number whose mRNA abundance exhibits circadian oscillations.

“The answer might lie with the stability of mRNA; if it’s too stable, then the rhythms disappear because the vast majority of the mRNA persists, leading to a pattern of apparent continuous accumulation,” says McClung. “It’s also possible that we picked up orphan circadian elements that aren’t actually regulating anything.”