THERE’S a strange glow about Steve Kay’s lab these days – a green glow. Packed into dishes several hundred at a time, tiny seedlings of a nondescript weed begin to glimmer a few hours before dawn, growing steadily brighter through the morning and steadily weaker as the day wears on.
The glow comes from a spray whose chemical and optical properties fall and rise in time with the plant’s own daily biological rhythms. Kay and his team at the University of Virginia in Charlottesville have used the spray to identify plants carrying genetic mutations that disrupt those rhythms – mutant seedlings that can’t keep proper time. Normal plants raise and lower their leaves once every 24 hours, and they switch on certain genes to a similar drumbeat. But not Kay’s mutants. Some rush, with “days” as short as 21 hours; others slouch with 28-hour days.
Until now, this poor sense of rhythm has been a private affair. No longer. Published for all to see in this week’s Science, Kay’s approach is being hailed as a milestone in the science of “rhythm genetics”, the push by geneticists to understand the mysterious daily, or “circadian”, clocks that tick inside most living things. “All of a sudden we’ve got access to a plant that’s as genetically handleable as a fruit fly,” says Mike Menaker, a veteran of circadian clock research at the University of Virginia, in Charlottesville.
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Key to the clock
The excitement is understandable. Circadian clocks choreograph a host of biological events: when to wake, when to sleep, when to emerge from a burrow; when to put out a spore, open a flower or raise a leaf. Researchers like Kay want to find the genes that control the biochemical processes behind these rhythmic events. Such genes, they reason, should hold the key to discovering how circadian clocks work. They may also help researchers to tackle the fundamental question of whether all circadian clocks are built of the same chemical cogs and springs, or whether they vary from species to species. In other words, whether today’s biological clocks are all descendants of a primitive ancestral timekeeper, or whether they have evolved independently.
One day it may also be possible to manipulate biological clocks to tick slower or faster. And that, in turn, could have a host of applications for farmers and horticulturalists – from decorative plants that flower in winter to strawberry plants that fruit three times a year. The reason is that plants use their clocks to sense the seasons, via the length of day, ensuring that flowers emerge at the right time of year. Some plants, such as wheat, flower when days get longer. Others, such as barley, flower when days shorten.
At the moment, bulb-growers in the Netherlands who want to produce year-round tulips have to drive around on tractors in the middle of the night with big booms of red light that illuminate the plants and induce flowering. The same kind of unseasonal flowering could be produced by genetically altering the plant, says Kay. His research on the rhythms of Arabidopsis thaliana (thale cress) marks a step in this direction. By analysing DNA from their 21 mutant plants, the researchers should be able to track down the mutant genes responsible for the overly short or long daily rhythms. That will lead them to the proteins whose biochemical actions somehow produce these rhythms inside plant cells. The researchers’ choice of the Arabidopsis weed is no accident. Its relatively small genome has made it a favourite subject for research.
The first known experiment on circadian rhythms was done by the 18th-century astronomer Jean Jacques d’Ortous de Mairan. He kept mimosa plants away from the regular rising and setting of the Sun, and found that the plant’s regular leaf movements persisted nonetheless. That showed that plants don’t simply say to themselves, “Oh, the Sun just came up, I’ll lift up my leaves.” Plants consult their own, custom-made clocks instead – and for obvious reasons. By “knowing” when the Sun will rise, a plant can be ready to greet it, leaves unfurled, photosynthetic enzymes cranked up.
Kay believes he has stumbled on at least one of the clock genes that make this happen. One of the 21 mutations to thale cress DNA disrupts the rhythms of the plant’s leaf movements as well as those of certain gene activities. When the gene affected by this mutation is firmly identified, the most pressing task will be to compare it to the clock genes of other organisms. Already, researchers have found such a gene in the fruit fly – known as period, or per for short – and one in pink bread mould (Neurospora crassa) – known as frequency, or frq.
The discovery of other clock genes could be just around the corner. A mutant mouse with circadian rhythms showed its face last April. And in November, a team of researchers from Japan, Texas and Tennessee reported the discovery of 17 mutants of blue-green algae – the only bacteria known to have circadian clocks – with daily cycles ranging from 16 to 60 hours. Given the speed with which bacterial genes can be isolated and manipulated, this discovery may open the gates to a flood of new findings. It may also produce telling clues about the evolutionary origins of biological clocks.
“In the past, there’s been a lot of progress in many areas of circadian rhythms – how clocks behave, where they’re located in animals’ brains, why clocks are evolutionarily useful,” says Carl Johnson of Vanderbilt University in Nashville, Tennessee, a member of the team that found clock mutants in blue-green algae. “But understanding the cellular basis of circadian rhythms – that’s been a very long row to hoe.”
Certainly, spotting plants with mutant clocks was a tough job. With fruit flies, researchers can look at activity levels. Depending on how the per gene is damaged, the flies move and rest with abnormally long or short rhythms, or simply at random. And bread moulds with mutant frq genes will, obligingly, make spores with similar, wrong-headed rhythms. But monitoring leaf movements for days on end would be hellish. So Kay and his colleagues had to be sly.
They simply took the gene for a firefly enzyme, luciferase, and put it under the control of a gene that is known to switch on and off with a daily rhythm. Luciferase acts on a chemical luciferin, emitting light in the process. So when one of the genetically-engineered plants is sprayed with luciferin, the plant glows green by day, starting just before dawn, then darkens each night. All Kay’s team had to do was ply their thale cress seeds with noxious, DNA-damaging chemicals, and then look for the seedlings that were glowing with the wrong rhythm. It was still very tedious, but it worked.
Geneticists want to do more than just fill up their coffers with new clock mutants. Both the per and frq genes have been cloned. And in the last few years, a rough picture has emerged about how both set up daily rhythms. It’s a strikingly similar story, for two strikingly dissimilar genes and two very different life forms.
The model goes something like this. To start with, the frq (or per) gene is turned on, and copies of it, in the form of molecules of RNA, are shuttled off to guide the construction of a FRQ protein. As more and more RNA is made, the FRQ protein slowly accumulates in the cell. That protein has work to do, and one of its jobs is to turn off its own gene. When levels of the FRQ protein climb high enough in the cell, the frq gene turns off. As a result, levels of the FRQ protein begin to plummet. Eventually, there isn’t enough FRQ protein in the cell to keep the frq gene turned off. Once more, levels of frq RNA and FRQ protein begin to rise. And so the cycle continues, in a 24-hour rhythm.
At the same time, the FRQ and PER proteins probably switch other genes off and on. These would be the genes that ultimately affect all the things that are rhythmic in a fly or fungus: when Neurospora makes its spores; when Drosophila walks and rests or emerge from a pupal case.
The model is attractive, but also preliminary, and only one small part of the story. Other genes and proteins must be involved. Not only that but the clock must keep steady time even as temperatures rise and fall – a tricky feat, since the rate of biochemical reactions vary greatly with temperature.
Enter Michael Rosbash and his colleagues at Brandeis University in Waltham, Massachusetts. In this week’s Science, they suggest how the fruit fly clock might maintain a steady rhythm in the face of fluctuating temperatures. The PER protein does two things, they propose. It binds to other proteins, and does its work, or it folds up on itself, and refrains from work. As the temperature climbs, both events speed up equally, so PER’s work rate stays fixed and the fly’s clock keeps good time.
Yet even if this model turns out to be right, there will still be many questions to answer, not least how organisms use light to reset their clocks each day.
Circadian clocks would soon be out of step with the cycles of light and dark if they didn’t make daily adjustments. But for the time being, rhythm researchers are celebrating. In the words of Carl Johnson: “We’re finally on the verge of getting handles on the concrete, molecular components of the clock.”