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Clock Genes Keep Time to Daily RhythmBy Jason Bardi For a flowering plant, life is no bed of roses. Burning sunlight during the day, freezing cold at night, and too much shade from the competition offer various threats that plants must overcome to stay alive and reproduce. A single blade in a field of grass has no other choice but to work round the clock to stay alive. Plants have evolved the means to cope with the various challenges of their environment by reaching the point where they can alter or even predict their adaptive response to their niches day by day, as nature demands. Plants survive by expressing certain genes and proteins at optimal times of the day—as they are needed to protect against the sun’s damaging UV radiation or the night’s cold air, for instance. This so-called circadian rhythm follows the solar day, and a group of researchers at The Scripps Research Institute (TSRI) have been studying the rhythm in one small, leafy, weed-like relative of the mustard plant, Arabidopsis. “When you can’t go indoors when it’s cold, or find some shade when it’s sunny,” says Cell Biology Professor Steve Kay, “you organize your stress responses on a daily basis. Plants need to anticipate changes in their environment, not just respond to them.” Kay says what they do is make their own equivalent of sunscreen in the morning and warm clothes at night. Arabidopsis uses all the genetic tools at its disposal to do the daily work of adapting to the varying stresses of its environment in order to stay alive. Timing on the Nano-ScaleUnlike chronometers, which keep the same time continuously, plant clocks have to change continuously. Plants must sense changes in their environment, such as length of the day, temperature, and the foliage around them that may be blocking the sun and entrain their clocks to adjust accordingly. All such timing takes place on the level of individual cells, with molecules ebbing and flowing throughout the day and year as they are needed. Photoreceptors, for instance, need to be assembled in plant cells in the morning and afternoon, but not in the evening. Plant cells also need protection from freezing at night but not often during the day. Circadian rhythms can be identified, then, by observing this pendulum swing of molecular expression throughout the day. But that’s only the beginning of the story—call it the loud ticking and turning hands that keeps the time on the face of the clock. The real mechanism that drives the circadian rhythm is the intricate and elegant genetic machinery that expresses the molecules that control the ticking of the ticking genes. The clockworks. Clockworks comprise a number of complicated feedback switches involving the expression of sometimes large numbers of genes, mostly transcription factors, which regulate the ticking genes by binding to their promoter regions, their mRNA, and otherwise turning them on and off as needed. Kay’s laboratory is particularly interested in these clockworks and has been tinkering with and studying them in Arabidopsis for some time. Members of the laboratory vary the plants’ environments—the amount of light these test plants receive, for instance—then ask how the plants adjust their own clocks to keep abreast of these changes, looking for which genes are turned on and off when and what other molecules are persistently present. “We’ve begun to identify some of those transcription factors,” Kay says. His laboratory identified several genes that are under tight circadian control in a recent issue of the journal Science.
The Molecules are the MessagePlants use the blue light photoreceptor, cryptochrome, to measure the overall intensity of daylight and activated cryptochromes transmit this information throughout the organism. They have another set of receptors for red light, the phytochromes, that work in the same way. And Arabidopsis uses the ratio of activated red:blue photoreceptors to tell them how crowded they are in their microenvironment. The number of phytochromes and cryptochromes that are transported into cell nuclei influences how nascent transcription factors will respond to changing light conditions by altering genomic expression to accommodate the updated environment and provide the correct response. Arabidopsis plants also produce phenylpropanoids to protect them from harmful UV radiation, analogous to how the phenyl compounds in commercial sunscreens work. Production of these phenylpropanoids peaks just prior to dawn. Chlorophyll-binding proteins are also turned on in the early morning in anticipation of the coming sun. Lipid desaturation enzymes and other molecules that confer cold and frost resistance throughout the night are not needed throughout the day, and so by mid-day, the expression of various transcription factors that down-regulate these cold resistance genes peak. Arabidopsis also down-regulates the synthesis of enzymes that store, transport, and break down the sugars the plant produces throughout the day until they are needed. Nitrogen and sulfur assimilation and amino acid production is timed so that these energy-intensive reactions can be performed in the morning when the plants have the most energy available to them. Cell elongation genes are expressed in the late afternoon and early evening and then put to rest by nighttime so that other enzymes can begin to use the available energy to synthesize the polysaccharides needed to reinforce the newly elongated cell walls, working through the night to finish. All and all, Kay and his colleagues found that of the 8,200 genes they surveyed, some 453—about six percent—moved to a circadian beat. The expression of these 453 ebbed and flowed with a period of 24 hours. About one quarter of the circadian genes they identified had no known function. “It’s really rather cool,” says Kay. With so many uncharacterized circadian genes, we must assume that much of the biochemical detail of the clock story is not yet known in exhaustive detail. Biology in Broad StrokesKay and his colleagues look at which genes are turned on and off by using DNA chips made available through the Novartis Agricultural Discovery Institute (now a part of Syngenta), a collaboration which Kay values. “NADI has done a lot to enhance plant research in academic labs,” he says. “Those [DNA chips] allow us to do many things.” DNA “chips” are glass or silicon wafers onto which are deposited arrays of nucleic acid oligos, sometimes to concentrations of tens of thousand per square centimeter. One can quite easily put all or most of the oligos that correspond to known genes in an organism on one of these so-called affinity matrices and can then look for expression of every known gene in a genome for entire tissues at a time. There are limitations to the method. Post-translational modification of expressed proteins into multiple isozymes—phosphorylated, glycosylated, and enzyme modified variants, for instance—cannot be observed. Also genes that are not annotated, or only expressed in tiny amounts but nevertheless play a role in the clockworks, may be missed. But the chips are perfect for looking at total mRNA expression of a tissue at a time, which is exactly what Kay is doing. Kay also uses more traditional though no less enlightening methods of looking at the pattern of expression for single genes. He employs Agrobacterium tumefaciens as a vector to attach markers onto genes in Arabidopsis without disturbing the plant’s other functions. “We do this famous trick of making the plants glow in the dark,” he says. By attaching a luminescent enzyme from fireflies, called luciferase, to specific genes in various tissues in the Arabidopsis plants, Kay and his colleagues can monitor when those genes are turned on or off and how they respond to environmental stimuli. They vary the amount of light a plant receives, for instance, and observe how the gene expression and flowering response of the plant varies accordingly. A Model SystemThe report Kay and his colleagues published appeared at the same time as the report of the Arabidopsis genome was published in the journal Nature, another milestone in the history of genomics, since Arabidopsis is the first plant to have its full genome sequenced. There are many reasons why this work is important. Genetic variations between particular strains of Arabidopsis may shed light. It may enable us to understand why some strains of a particular plant are different than others and find ways to use those differences to, for instance, make the plants flower more often. Sequencing several plant genomes will allow us to access diversity in plants. The genetic codes of several different species can be compared to one another in the not too distant future, and the rice genome is right around the corner, due to be finished some time in the next couple of years. Another genome, the legume Lotus japonicus, is being sequenced along with its nitrogen fixing bacteria in the interest of uncovering the genetic basis for this cooperative nitrogen fixing. Such comparisons promise to be directly relevant to our lives in many ways, not the least of which is the possibility that we will use what we learn to boost food production. “The danger of running out of arable land is very real,” says Kay, “and we have to solve the problem of feeding an rapidly increasing population in the next 10 years.” Certain crop species may eventually be modified by incorporating diverse plant genes into crop plant genomes so that the conditions under which they can be grown are broader. Perhaps plants can be made to bear fruit faster and in larger and more nutritious yields. “Understanding plant diversity from genome sequences is going to have a huge effect on the whole of biology,” says Kay. “People should not put their heads in the sand.” Arabidopsis is a good model organism for several reasons. It is tiny and has a fast generation time, both of which fit well in the modern tight-on-space-and-time laboratory. It also produces an overabundance of seeds at the end of its reproductive cycle. Finally, as a weed, Arabidopsis is easily grown. Plant genomics stands to benefit more than just the vegetables of the world. As more and more genomes are solved, comparisons across genomes will become commonplace, with discoveries in one informing on mysteries in others. Studies involving the circadian control of Arabidopsis have direct relevance to studies in humans because, in theory, genes similar to those that exhibit daily fluctuations in another species could undergo the same sort of rhythms. The first human circadian disorder, a mutation in the gene hPer2, was identified this year in people with familial advanced sleep phase syndrome, a type of insomnia. There is a similar gene to hPer2 in Drosophila melanogaster, which makes the fruit fly an excellent system for subsequent studies that will not just seek to understand sleep and wake cycles but find ways to address disorders associated with those cycles. Likewise other human genes will no doubt find their long lost cousins in Arabidopsis. Genetically, the tiny Arabidopsis plants are more similar to humans than the genomes of yeast and the nematode. There are 11,000 different types of gene families in the Arabidopsis genome, and many of these genes have their counterparts in humans. With its 25,498 genes spread over some 115 million base pairs of DNA and organized into 5 chromosomes, Arabidopsis has a very dense genome, which Kay speculates is insurance against the deleterious effect of mutations or genetic diversity to combat pathogens and fungi. “Quite a large part of this genome seems to be dedicated to combating things that nibble on it,” he says. Moreover, says Kay, there are remarkable similarities between the innate immunity of plants and that of humans. A lot of the molecules that plants use to recognize pathogens are similar to molecules in the human body that perform tasks of innate immunity recognition performed by phagocytes. At the same time, there are some 8,000 other genes that have been annotated but have no known function and no corresponding gene in some other organisms. Only time will tell what these genes will tell us. But Kay sees a bright future. “The Arabidopsis genome will ultimately have equal effect on improving the health and welfare of the population as the human genome sequence,” he says. “I truly believe that.”
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