Circadian rhythms have been demonstrated in organisms across the taxonomic spectrum. In view of their widespread occurrence, the adaptive significance of these rhythms is of interest. We have previously shown that constitutive expression of the CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) gene in Arabidopsis plants (CCA1-ox) results in loss of circadian rhythmicity. Here, we demonstrate that these CCA1-ox plants retain the ability to respond to diurnal changes in light. Thus, transcript levels of several circadian-regulated genes, as well as CCA1 itself and the closely related LHY, oscillate robustly if CCA1-ox plants are grown under diurnal conditions. However, in contrast with wild-type plants in which transcript levels change in anticipation of the dark/light transitions, the CCA1-ox plants have lost the ability to anticipate this daily change in their environment. We have used CCA1-ox lines to examine the effects of loss of circadian regulation on the fitness of an organism. CCA1-ox plants flowered later, especially under long-day conditions, and were less viable under very short-day conditions than their wild-type counterparts. In addition, we demonstrate that two other circadian rhythm mutants, LHY-ox and elf3, have low-viability phenotypes. Our findings demonstrate the adaptive advantage of circadian rhythms in Arabidopsis.First described in plants more than 270 years ago, circadian rhythms have been found in the vast majority of eukaryotes examined and in many prokaryotes. The basic mechanisms that are responsible for circadian rhythms are being elucidated in several model species. At the core of a circadian system is a molecular oscillator that generates a period of rhythmicity of about 24 h. The oscillator is self-sustaining, but it is influenced by environmental cues such as changes in light and temperature conditions (Dunlap, 1999). The oscillator controls a plethora of biological processes including nitrogen fixation in cyanobacteria (Johnson et al., 1996), scent emission in plants ( Kolosova et al., 2001), conidiation in Neurospora crassa. (Pittendrigh et al., 1959), olfactory responses of Drosophila melanogaster (Krishnan et al., 1999), luteinizing hormone levels in birds (Follett et al., 1974), and wheel running activity in hamsters (Mesocricetus auratus; Ralph and Menaker, 1988). The fact that circadian regulation is ubiquitous across the taxonomic spectrum and that its importance was even recognized in the Hebrew Bible (Ancoli-Israel, 2001) suggests that it is important for optimizing an organism's response to its environment and enhancing its fitness.The circadian clock plays a number of different roles. One is in the regulation of photoperiodism, which is the detection and response to changes in the duration of days and nights that enables organisms to adapt to seasonal changes in their environment (Thomas, 1998). Photoperiodism controls reproduction in many organisms, including flowering time in many plants ( Thomas and Vince-Prue, 1997). This photoperiodic response ensures that plants flower duri...

Circadian (24 h) clock regulated biological rhythms have been identified in a wide range of organisms from prokaryotic unicellular cyanobacteria to higher mammals. These rhythms regulate an enormous variety of processes including gene expression, metabolic processes, activity and reproduction. Given the widespread occurrence of circadian systems it is not surprising that extensive efforts have been directed at understanding the adaptive significance of circadian rhythms. In this review we discuss the approaches and findings that have resulted. In studies on organisms in their natural environments, some species show adaptations in their circadian systems that correlate with living at different latitudes, such as clines in circadian clock properties. Additionally, some species show plasticity in their circadian systems suggested to match the demands of their physical and social environment. A number of experiments, both in the field and in the laboratory, have examined the effects of having a circadian system that does not resonate with the organismÕs environment. We conclude that the results of these studies suggest that having a circadian system that matches the oscillating environment is adaptive.

The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (␤) subunit of the protein kinase CK2 and have designated it as CKB3. CKB3 is the only reported example of a third ␤-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay. Other subunits of CK2 also show an interaction with CCA1 in vitro. CK2 ␤-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo.

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis. C ircadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological, and behavioral processes in a wide variety of organisms. According to current models, circadian clocks regulating these rhythms consist of input pathways, a central oscillator, and output pathways (1-3). Oscillators are thought to generate rhythms by a transcription-translation negative feedback loop (4-8). Studies in cyanobacteria, Neurospora, Drosophila, and mouse have found that both positive and negative elements that activate and inhibit the transcription of clock genes are required to maintain the loop. In addition, posttranscriptional and posttranslational regulation play important roles in circadian clocks in Drosophila and Neurospora (7-10). The oscillator can be entrained by input pathways from environmental cues such as light and temperature, and, in turn, regulates specific cellular events such as expression of clock-controlled genes (1-3).Until recently, little was known about the molecular mechanisms of circadian clocks in plants (11-13). In Arabidopsis thaliana, the toc1 mutation affects the period of many circadian rhythms (14,15). Although the corresponding gene has not yet been cloned, it is thought that TOC1 encodes a component of the oscillator. The ELF3 gene is proposed to act in the input pathway (16). Two Myb-related genes, circadian clock-associated 1 (CCA1) and late elongated hypocotyl (LHY), have also been identified as potential clock genes (17, 18), and CCA1 was found to act as a transcription factor for Lhcb gene expression (19). Expression of CCA1 and LHY oscillates with a circadian rhythm. Constitutive expression of CCA1 was shown to abolish several distinct circadian rhythms, suppress its own expression as well as that of LHY, and delay flowering substa...

Plants, like many other organisms, have endogenous biological clocks that enable them to organize their physiological, metabolic and developmental processes so that they occur at optimal times. The best studied of these biological clocks are the circadian systems that regulate daily (∼ 24 h) rhythms. At the core of the circadian system in every organism are oscillators responsible for generating circadian rhythms. These oscillators can be entrained (set) by cues from the environment, such as daily changes in light and temperature. Completing the circadian clock model are the output pathways that provide a link between the oscillator and the various biological processes whose rhythms it controls. Over the past few years there has been a tremendous increase in our understanding of the mechanisms of the oscillator and entrainment pathways in plants and many useful reviews on the subject. In this review we focus on the output pathways by which the oscillator regulates rhythmic plant processes. In the first part of the review we describe the role of the circadian system in regulation at all stages of a plant's development, from germination and growth to reproductive development as well as in multiple cellular processes. Indeed, the importance of a circadian clock for plants can be gauged by the fact that so many facets of plant development are under its control. In the second part of the review we describe what is known about the mechanisms by which the circadian system regulates these output processes.

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As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian (approximately 24 h) clocks. In plants, the circadian clock regulates a diverse range of cellular and physiological events from gene expression and protein phosphorylation to cellular calcium oscillations, hypocotyl growth, leaf movements, and photoperiod-dependent flowering. In Arabidopsis (Arabidopsis thaliana), as in other model organisms, such as Drosophila (Drosophila melanogaster) and mice, circadian rhythms are generated by molecular oscillators that consist of interlocking feedback loops involving a number of elements. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYLS (LHY) are closely related single myb transcription factors that have been identified as key elements in the Arabidopsis oscillator. Research in other model organisms has shown that posttranslational regulation of oscillator components plays a critical role in the generation of the approximately 24-h cycles. To examine the role of posttranslational regulation of CCA1 and LHY in the Arabidopsis oscillator, we generated transgenic plants with tagged CCA1 and LHY under the control of their own promoters. We have shown that these tagged proteins are functional and can restore normal circadian rhythms to CCA1-and LHY-null plants. Using the tagged proteins, we demonstrate that CCA1 can form both homodimers and heterodimers with LHY. Furthermore, we also show that CCA1 is localized to the nucleus in vivo and that there is no significant delay between the translation of CCA1 and its translocation to the nucleus. We discuss our findings in the context of the functioning of the Arabidopsis oscillator.The circadian, approximately 24 h, clock has an enormous influence on the biology of plants and controls a plethora of processes, including hypocotyl growth, shade avoidance, leaf movements, scent production, and stomatal opening (Yakir et al., 2007). Consistent with the role of the circadian clock in the regulation of a wide range of activities, transcription from approximately one-third of the genome (including noncoding genes) of the model plant Arabidopsis (Arabidopsis thaliana) is under circadian control (Michael and McClung, 2003; Covington et al., 2008;Michael et al., 2008;Hazen et al., 2009). In addition, the circadian clock serves as a timekeeper to regulate daylengthdependent processes, such as flowering time and tuberization. The circadian systems responsible for generating circadian rhythms are ubiquitous in eukaryotes and have also been found in some prokaryotes (Dunlap, 1999). Conceptually, a circadian system can be divided into three parts: the oscillator mechanism, input pathways, and output pathways. Interestingly, although their components differ, the basic oscillator mechanism appears to be well conserved in all the eukaryotic model organisms that have been studied (Dunlap, 2006). Oscillators are comprised of interlocking positive/negative feedback loops made from clock proteins that control t...

SUMMARYThe circadian system of plants regulates a wide range of rhythmic physiological and cellular output processes with a period of about 24 h. The rhythms are generated by an oscillator mechanism that, in Arabidopsis, consists of interlocking feedback loops of several components including CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION 1 (TOC1) and CCA1 HIKING EXPEDITION (CHE). Over recent years, researchers have gained a detailed picture of the clock mechanism at the resolution of the whole plant and several tissue types, but little information is known about the specificities of the clock mechanism at the level of individual cells. In this paper we have addressed the question of cell-typespecific differences in circadian systems. Using transgenic Arabidopsis plants with fluorescence-tagged CCA1 to measure rhythmicity in individual leaf cells in intact living plants, we showed that stomatal guard cells have a different period from surrounding epidermal and mesophyll leaf cells. By comparing transcript levels in guard cells with whole plants, we identified differences in the expression of some oscillator genes that may underlie cell-specific differences in clock properties. In addition, we demonstrated that the oscillators of individual cells in the leaf are robust, but become partially desynchronized in constant conditions. Taken together our results suggest that, at the level of individual cells, there are differences in the canonical oscillator mechanism that has been described for plants.