All living organisms, from bacteria to humans, have an internal clock that prompts them to wake, become active, feed, and sleep on a fixed 24-hour schedule called a circadian rhythm. Daily and seasonal changes in the light–dark cycle adjust the clock so that circadian rhythms in metabolic, physiological, and developmental processes are appropriately in phase with fluctuations in environmental conditions. However, our understanding of the molecular mechanisms through which light interacts with the circadian clock remains incomplete. Given that plants depend on sunlight for growth and development and that much is known about their photoreceptor and phototransduction mechanisms, they are an ideal system in which to analyze the interaction between light signaling and circadian networks.
Light Perception and the Ability to Keep Track of Time
To maintain an anticipatory function throughout the year, circadian clocks must be adjusted daily. Such entrainment is effected, in part, through signaling pathways that transmit information from light–dark transitions to the clock. In Neurospora and mammals, light resets the clock by regulating the expression of clock genes; in flies, light regulates the stability of clock proteins. Plants possess at least four groups of photoreceptors: the phytochromes (operating predominantly in the red and far-red spectral range), the blue/UVA photoreceptors cryptochromes and phototropins, and unknown UVB photoreceptors. We and others have provided photobiological and genetic evidence that phytochromes and cryptochromes control the circadian oscillator in plants, but the signaling mechanisms that connect the photoreceptors with the central oscillator remain elusive. We are using whole-genome Arabidopsis microarrays to identify genes whose expression is regulated by light at times of the day when light's effects on the pace of the clock are most pronounced. We then use reverse genetics to evaluate the role of candidate genes in light regulation of clock entrainment. We are also selecting mutants affected in light-dependent responses and testing whether the mutants show alterations in clock function. One such mutant is csa1, which shows defects in red light signaling similar to those observed in phytochrome B (phyB) mutants. Indeed, csa1-like phyB mutants show normal periodicity but the phase of their circadian rhythms is advanced relative to light–dark cycles. The mutant phenotype, which also involves alterations in numerous light-regulated developmental processes, is caused by a dominant negative mutation in a TIR-NBS-LRR gene. This gene is related to the Toll gene of Drosophila and to mammalian Toll-like receptors. Until now, members of this gene family were thought to mediate only innate immunity processes in plants. Our finding indicates that, like their Drosophila counterparts, TIR domain–containing proteins have dual roles regulating innate immunity and developmental processes. We also isolated the mutant lic1 (light input to the clock 1), which is defective in developmental responses to red, blue, and far-red light. Circadian rhythms in this mutant display a long-period phenotype. This gene is likely to mediate clock entrainment by light, acting downstream of all known photoreceptors. Identification of the LIC1 gene through positional cloning will allow us to deepen our understanding of the molecular mechanisms underlying clock entrainment by light–dark cycles in plants.
It has been proposed that, in addition to its role in clock entrainment, the light–dark cycle selects for circadian oscillators. According to this hypothesis, circadian clocks evolved to allow organisms to avoid the damaging effects of light, particularly its UV component. In support of this idea, circadian rhythms sensitive to UV light have been reported in some organisms. Furthermore, DNA photolyases, molecules that repair DNA damage in a light-dependent manner, share extensive homology with the blue light photoreceptors known as cryptochromes, which lack photolyase activity but play critical roles in the circadian system of plants and animals. However, we do not know whether photoreceptors are essential components of the central oscillatory mechanism or merely mediate entrainment to light–dark cycles. In mammals, cryptochromes are required to sustain circadian rhythms even in complete darkness. We have shown that light regulation of developmental processes is severely impaired in plants lacking the two cryptochromes, as well as phytochromes A and B, which are the most abundant red and far-red light photoreceptors, even though such mutants display robust circadian rhythms, which are still entrained by light–dark cycles. To evaluate the strength of the connection between photoreceptor molecules and the central oscillatory mechanism in plants, we are generating higher-order mutants that lack all known plant photoreceptors.
How Plants Tell Time
In Neurospora, Arabidopsis, Drosophila, and mice, circadian oscillations are thought to be generated by transcriptional–translational feedback loops in which cycling gene products negatively control their own expression by opposing the action of positive factors. Arabidopsis does not contain true homologs of any of the clock genes found in these organisms, but forward genetic approaches have identified TOC1, CCA1, and LHY as key components of transcriptional feedback loops closely associated with the oscillator. Almost all clock components identified so far have been shown to play roles in light signaling in addition to clock regulation. This is true even for the putative core oscillator components TOC1, CCA1, and LHY. Thus, it is not clear whether these components are involved in generating circadian oscillations or in linking the central oscillatory mechanism to the light environment, or whether the clock oscillatory mechanism cannot be separated from light perception and signaling. One possibility is that components involved in clock regulation that are independent of light conditions have not yet been identified because loss-of-function mutations in those genes are either lethal or have no phenotype because of genetic redundancy. We are using overexpression screening to overcome such limitations.
Anticipating Seasonal Changes
Seasonal changes in light, temperature, and rainfall have strongly influenced the evolution of life on earth. Most organisms adjust critical developmental processes to occur at times of the year that maximize their chances of survival and reproductive success. Changes in day length, or photoperiod, trigger the onset of flowering in many species. We and others showed that the acceleration of flowering time triggered by long days in Arabidopsis results in part from direct effects of light, via cryptochrome 2 and phytochrome A, on the expression of FLOWERING LOCUS T (FT), a gene that triggers the transition from vegetative to reproductive development when expressed above a certain threshold. This effect requires CONSTANS (CO), a transcriptional regulator whose expression is regulated by the clock. Thus, the overlap between high levels of CO mRNA and the illuminated part of the day is minimal on short days and maximal on long days. CONSTANS protein is unstable in the dark, and light in the late afternoon of a long day promotes FT expression by stabilizing CONSTANS.
Changes in photoperiod trigger several other responses that help plants cope with seasonal changes in the environment. These responses include bud dormancy, cold hardiness, and the formation of tubers and bulbs. While significant progress has been made in recent years toward understanding the molecular mechanism by which model plants such as Arabidopsis measure day length and regulate flowering time accordingly, little is known about the molecular changes underlying more general biochemical, physiological, and developmental adaptations triggered by changes in photoperiod. We are using comparative functional genomic approaches to identify genes that mediate the photoperiodic regulation of different processes in different plant species such as Arabidopsis, potato, and tobacco. We are identifying not only novel transcription factors that regulate flowering time but also regulatory mechanisms that mediate the acclimatization of plants to the high light conditions of summer, the limited energy resources of winter, and physiological processes that have a strong impact on crop productivity such as the rate of leaf initiation.
Last updated August 2007
