Uncovering cold disruption of the circadian clock in poplar

Dormancy is an adaptive mechanism that allows woody plants to survive at low temperatures during the winter. Disruption of circadian clock genes in winter or under low temperatures, both in long days as in short days, were described in our group few years ago (Ramos et al., 2005). Basic mechanisms of the circadian clock function are similar in herbaceous as well as in woody plants although there are differences in their response to low temperatures (Bieniawska et al., 2008). Woody plants growing in daylight conditions should have a specific transcriptional control above the circadian clock genes, which is responsible of their constitutive transcriptional activation observed under low temperatures conditions. In order to understand this regulatory process, we are analyzing the behavior of a circadian clock gene in poplar. To this aim, we have isolated its promoter region and fused to the luciferase reporter gene. This construct has been transformed into Populus tremula x P. alba 717-1B4 INRA clone. Here we present the characterization of these transgenic lines under different conditions of light and temperature.

A swarm intelligence approach based on coupled oscillators: an application in demand side management with photovoltaic distributed generation

Esta Tesis aborda los problemas de eficiencia de las redes eléctrica desde el punto de vista del consumo. En particular, dicha eficiencia es mejorada mediante el suavizado de la curva de consumo agregado. Este objetivo de suavizado de consumo implica dos grandes mejoras en el uso de las redes eléctricas: i) a corto plazo, un mejor uso de la infraestructura existente y ii) a largo plazo, la reducción de la infraestructura necesaria para suplir las mismas necesidades energéticas. Además, esta Tesis se enfrenta a un nuevo paradigma energético, donde la presencia de generación distribuida está muy extendida en las redes eléctricas, en particular, la generación fotovoltaica (FV). Este tipo de fuente energética afecta al funcionamiento de la red, incrementando su variabilidad. Esto implica que altas tasas de penetración de electricidad de origen fotovoltaico es perjudicial para la estabilidad de la red eléctrica. Esta Tesis trata de suavizar la curva de consumo agregado considerando esta fuente energética. Por lo tanto, no sólo se mejora la eficiencia de la red eléctrica, sino que también puede ser aumentada la penetración de electricidad de origen fotovoltaico en la red. Esta propuesta conlleva grandes beneficios en los campos económicos, social y ambiental. Las acciones que influyen en el modo en que los consumidores hacen uso de la electricidad con el objetivo producir un ahorro energético o un aumento de eficiencia son llamadas Gestión de la Demanda Eléctrica (GDE). Esta Tesis propone dos algoritmos de GDE diferentes para cumplir con el objetivo de suavizado de la curva de consumo agregado. La diferencia entre ambos algoritmos de GDE reside en el marco en el cual estos tienen lugar: el marco local y el marco de red. Dependiendo de este marco de GDE, el objetivo energético y la forma en la que se alcanza este objetivo son diferentes. En el marco local, el algoritmo de GDE sólo usa información local. Este no tiene en cuenta a otros consumidores o a la curva de consumo agregado de la red eléctrica. Aunque esta afirmación pueda diferir de la definición general de GDE, esta vuelve a tomar sentido en instalaciones locales equipadas con Recursos Energéticos Distribuidos (REDs). En este caso, la GDE está enfocada en la maximización del uso de la energía local, reduciéndose la dependencia con la red. El algoritmo de GDE propuesto mejora significativamente el auto-consumo del generador FV local. Experimentos simulados y reales muestran que el auto-consumo es una importante estrategia de gestión energética, reduciendo el transporte de electricidad y alentando al usuario a controlar su comportamiento energético. Sin embargo, a pesar de todas las ventajas del aumento de auto-consumo, éstas no contribuyen al suavizado del consumo agregado. Se han estudiado los efectos de las instalaciones locales en la red eléctrica cuando el algoritmo de GDE está enfocado en el aumento del auto-consumo. Este enfoque puede tener efectos no deseados, incrementando la variabilidad en el consumo agregado en vez de reducirlo. Este efecto se produce porque el algoritmo de GDE sólo considera variables locales en el marco local. Los resultados sugieren que se requiere una coordinación entre las instalaciones. A través de esta coordinación, el consumo debe ser modificado teniendo en cuenta otros elementos de la red y buscando el suavizado del consumo agregado. En el marco de la red, el algoritmo de GDE tiene en cuenta tanto información local como de la red eléctrica. En esta Tesis se ha desarrollado un algoritmo autoorganizado para controlar el consumo de la red eléctrica de manera distribuida. El objetivo de este algoritmo es el suavizado del consumo agregado, como en las implementaciones clásicas de GDE. El enfoque distribuido significa que la GDE se realiza desde el lado de los consumidores sin seguir órdenes directas emitidas por una entidad central. Por lo tanto, esta Tesis propone una estructura de gestión paralela en lugar de una jerárquica como en las redes eléctricas clásicas. Esto implica que se requiere un mecanismo de coordinación entre instalaciones. Esta Tesis pretende minimizar la cantidad de información necesaria para esta coordinación. Para lograr este objetivo, se han utilizado dos técnicas de coordinación colectiva: osciladores acoplados e inteligencia de enjambre. La combinación de estas técnicas para llevar a cabo la coordinación de un sistema con las características de la red eléctrica es en sí mismo un enfoque novedoso. Por lo tanto, este objetivo de coordinación no es sólo una contribución en el campo de la gestión energética, sino también en el campo de los sistemas colectivos. Los resultados muestran que el algoritmo de GDE propuesto reduce la diferencia entre máximos y mínimos de la red eléctrica en proporción a la cantidad de energía controlada por el algoritmo. Por lo tanto, conforme mayor es la cantidad de energía controlada por el algoritmo, mayor es la mejora de eficiencia en la red eléctrica. Además de las ventajas resultantes del suavizado del consumo agregado, otras ventajas surgen de la solución distribuida seguida en esta Tesis. Estas ventajas se resumen en las siguientes características del algoritmo de GDE propuesto: • Robustez: en un sistema centralizado, un fallo o rotura del nodo central provoca un mal funcionamiento de todo el sistema. La gestión de una red desde un punto de vista distribuido implica que no existe un nodo de control central. Un fallo en cualquier instalación no afecta el funcionamiento global de la red. • Privacidad de datos: el uso de una topología distribuida causa de que no hay un nodo central con información sensible de todos los consumidores. Esta Tesis va más allá y el algoritmo propuesto de GDE no utiliza información específica acerca de los comportamientos de los consumidores, siendo la coordinación entre las instalaciones completamente anónimos. • Escalabilidad: el algoritmo propuesto de GDE opera con cualquier número de instalaciones. Esto implica que se permite la incorporación de nuevas instalaciones sin afectar a su funcionamiento. • Bajo coste: el algoritmo de GDE propuesto se adapta a las redes actuales sin requisitos topológicos. Además, todas las instalaciones calculan su propia gestión con un bajo requerimiento computacional. Por lo tanto, no se requiere un nodo central con un alto poder de cómputo. • Rápido despliegue: las características de escalabilidad y bajo coste de los algoritmos de GDE propuestos permiten una implementación rápida. No se requiere una planificación compleja para el despliegue de este sistema. ABSTRACT This Thesis addresses the efficiency problems of the electrical grids from the consumption point of view. In particular, such efficiency is improved by means of the aggregated consumption smoothing. This objective of consumption smoothing entails two major improvements in the use of electrical grids: i) in the short term, a better use of the existing infrastructure and ii) in long term, the reduction of the required infrastructure to supply the same energy needs. In addition, this Thesis faces a new energy paradigm, where the presence of distributed generation is widespread over the electrical grids, in particular, the Photovoltaic (PV) generation. This kind of energy source affects to the operation of the grid by increasing its variability. This implies that a high penetration rate of photovoltaic electricity is pernicious for the electrical grid stability. This Thesis seeks to smooth the aggregated consumption considering this energy source. Therefore, not only the efficiency of the electrical grid is improved, but also the penetration of photovoltaic electricity into the grid can be increased. This proposal brings great benefits in the economic, social and environmental fields. The actions that influence the way that consumers use electricity in order to achieve energy savings or higher efficiency in energy use are called Demand-Side Management (DSM). This Thesis proposes two different DSM algorithms to meet the aggregated consumption smoothing objective. The difference between both DSM algorithms lie in the framework in which they take place: the local framework and the grid framework. Depending on the DSM framework, the energy goal and the procedure to reach this goal are different. In the local framework, the DSM algorithm only uses local information. It does not take into account other consumers or the aggregated consumption of the electrical grid. Although this statement may differ from the general definition of DSM, it makes sense in local facilities equipped with Distributed Energy Resources (DERs). In this case, the DSM is focused on the maximization of the local energy use, reducing the grid dependence. The proposed DSM algorithm significantly improves the self-consumption of the local PV generator. Simulated and real experiments show that self-consumption serves as an important energy management strategy, reducing the electricity transport and encouraging the user to control his energy behavior. However, despite all the advantages of the self-consumption increase, they do not contribute to the smooth of the aggregated consumption. The effects of the local facilities on the electrical grid are studied when the DSM algorithm is focused on self-consumption maximization. This approach may have undesirable effects, increasing the variability in the aggregated consumption instead of reducing it. This effect occurs because the algorithm only considers local variables in the local framework. The results suggest that coordination between these facilities is required. Through this coordination, the consumption should be modified by taking into account other elements of the grid and seeking for an aggregated consumption smoothing. In the grid framework, the DSM algorithm takes into account both local and grid information. This Thesis develops a self-organized algorithm to manage the consumption of an electrical grid in a distributed way. The goal of this algorithm is the aggregated consumption smoothing, as the classical DSM implementations. The distributed approach means that the DSM is performed from the consumers side without following direct commands issued by a central entity. Therefore, this Thesis proposes a parallel management structure rather than a hierarchical one as in the classical electrical grids. This implies that a coordination mechanism between facilities is required. This Thesis seeks for minimizing the amount of information necessary for this coordination. To achieve this objective, two collective coordination techniques have been used: coupled oscillators and swarm intelligence. The combination of these techniques to perform the coordination of a system with the characteristics of the electric grid is itself a novel approach. Therefore, this coordination objective is not only a contribution in the energy management field, but in the collective systems too. Results show that the proposed DSM algorithm reduces the difference between the maximums and minimums of the electrical grid proportionally to the amount of energy controlled by the system. Thus, the greater the amount of energy controlled by the algorithm, the greater the improvement of the efficiency of the electrical grid. In addition to the advantages resulting from the smoothing of the aggregated consumption, other advantages arise from the distributed approach followed in this Thesis. These advantages are summarized in the following features of the proposed DSM algorithm: • Robustness: in a centralized system, a failure or breakage of the central node causes a malfunction of the whole system. The management of a grid from a distributed point of view implies that there is not a central control node. A failure in any facility does not affect the overall operation of the grid. • Data privacy: the use of a distributed topology causes that there is not a central node with sensitive information of all consumers. This Thesis goes a step further and the proposed DSM algorithm does not use specific information about the consumer behaviors, being the coordination between facilities completely anonymous. • Scalability: the proposed DSM algorithm operates with any number of facilities. This implies that it allows the incorporation of new facilities without affecting its operation. • Low cost: the proposed DSM algorithm adapts to the current grids without any topological requirements. In addition, every facility calculates its own management with low computational requirements. Thus, a central computational node with a high computational power is not required. • Quick deployment: the scalability and low cost features of the proposed DSM algorithms allow a quick deployment. A complex schedule of the deployment of this system is not required.

The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis

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.

AtGRP7, a nuclear RNA-binding protein as a component of a circadian-regulated negative feedback loop in Arabidopsis thaliana

The endogenous clock that drives circadian rhythms is thought to communicate temporal information within the cell via cycling downstream transcripts. A transcript encoding a glycine-rich RNA-binding protein, Atgrp7, in Arabidopsis thaliana undergoes circadian oscillations with peak levels in the evening. The AtGRP7 protein also cycles with a time delay so that Atgrp7 transcript levels decline when the AtGRP7 protein accumulates to high levels. After AtGRP7 protein concentration has fallen to trough levels, Atgrp7 transcript starts to reaccumulate. Overexpression of AtGRP7 in transgenic Arabidopsis plants severely depresses cycling of the endogenous Atgrp7 transcript. These data establish both transcript and protein as components of a negative feedback circuit capable of generating a stable oscillation. AtGRP7 overexpression also depresses the oscillation of the circadian-regulated transcript encoding the related RNA-binding protein AtGRP8 but does not affect the oscillation of transcripts such as cab or catalase mRNAs. We propose that the AtGRP7 autoregulatory loop represents a “slave” oscillator in Arabidopsis that receives temporal information from a central “master” oscillator, conserves the rhythmicity by negative feedback, and transduces it to the output pathway by regulating a subset of clock-controlled transcripts.

Pituitary adenylyl cyclase-activating peptide: A pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock

The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep–wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6–38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6–38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light.

Circadian clock-controlled genes isolated from Neurospora crassa are late night- to early morning-specific

An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs preferentially accumulate during the late night to early morning, although they vary with respect to steady-state mRNA levels and amplitude of the rhythm. Sequencing of the ends of the new ccg cDNAs revealed that ccg-12 is identical to N. crassa cmt encoding copper metallothionein, providing the suggestion that not all clock-regulated genes in N. crassa are specifically involved in the development of conidia. This was supported by finding that half of the new ccgs, including cmt(ccg-12), are not transcriptionally induced by developmental or light signals. These data suggest a major role for the clock in the regulation of biological processes distinct from development.

The circadian clock controls the expression pattern of the circadian input photoreceptor, phytochrome B

Developmental and physiological responses are regulated by light throughout the entire life cycle of higher plants. To sense changes in the light environment, plants have developed various photoreceptors, including the red/far-red light-absorbing phytochromes and blue light-absorbing cryptochromes. A wide variety of physiological responses, including most light responses, also are modulated by circadian rhythms that are generated by an endogenous oscillator, the circadian clock. To provide information on local time, circadian clocks are synchronized and entrained by environmental time cues, of which light is among the most important. Light-driven entrainment of the Arabidopsis circadian clock has been shown to be mediated by phytochrome A (phyA), phytochrome B (phyB), and cryptochromes 1 and 2, thus affirming the roles of these photoreceptors as input regulators to the plant circadian clock. Here we show that the expression of PHYB∷LUC reporter genes containing the promoter and 5′ untranslated region of the tobacco NtPHYB1 or Arabidopsis AtPHYB genes fused to the luciferase (LUC) gene exhibit robust circadian oscillations in transgenic plants. We demonstrate that the abundance of PHYB RNA retains this circadian regulation and use a PHYB∷Luc fusion protein to show that the rate of PHYB synthesis is also rhythmic. The abundance of bulk PHYB protein, however, exhibits only weak circadian rhythmicity, if any. These data suggest that photoreceptor gene expression patterns may be significant in the daily regulation of plant physiology and indicate an unexpectedly intimate relationship between the components of the input pathway and the putative circadian clock mechanism in higher plants.

Rapid down-regulation of mammalian Period genes during behavioral resetting of the circadian clock

The pervasive role of circadian clocks in regulating physiology and behavior is widely recognized. Their adaptive value is their ability to be entrained by environmental cues such that the internal circadian phase is a reliable predictor of solar time. In mammals, both light and nonphotic behavioral cues can entrain the principal oscillator of the hypothalamic suprachiasmatic nuclei (SCN). However, although light can advance or delay the clock during circadian night, behavioral events trigger phase advances during the subjective day, when the clock is insensitive to light. The recent identification of Period (Per) genes in mammals, homologues of dperiod, which encodes a core element of the circadian clockwork in Drosophila, now provides the opportunity to explain circadian timing and entrainment at a molecular level. In mice, expression of mPer1 and mPer2 in the SCN is rhythmic and acutely up-regulated by light. Moreover, the temporal relations between mRNA and protein cycles are consistent with a clock based on a transcriptional/translational feedback loop. Here we describe circadian oscillations of Per1 and Per2 in the SCN of the Syrian hamster, showing that PER1 protein and mRNA cycles again behave in a manner consistent with a negative-feedback oscillator. Furthermore, we demonstrate that nonphotic resetting has the opposite effect to light: acutely down-regulating these genes. Their sensitivity to nonphotic resetting cues supports their proposed role as core elements of the circadian oscillator. Moreover, this study provides an explanation at the molecular level for the contrasting but convergent effects of photic and nonphotic cues on the clock.

Conserved circadian elements in phylogenetically diverse algae

Circadian expression of the luciferin-binding protein (LBP) from the dinoflagellate Gonyaulax polyedra is regulated at the translational level. A small interval in the lbp 3′-untranslated region, which contains seven UG-repeats, serves as a cis-acting element to which a trans-acting factor (CCTR) binds in a circadian manner. Its binding activity correlates negatively with the circadian expression of LBP. Here I report the identification of a protein in the green alga Chlamydomonas reinhardtii that represents a CCTR analog. It binds both specifically and under control of the circadian clock to the UG-repeat region. The data show for the first time that circadian cis-elements implicated in translational regulation have been conserved during evolution.

Identification of a novel vertebrate circadian clock-regulated gene encoding the protein nocturnin

Photoreceptors of the Xenopus laevis retina are the site of a circadian clock. As part of a differential display screen for rhythmic gene products in this system, we have identified a photoreceptor-specific mRNA expressed in peak abundance at night. cDNA cloning revealed an open reading frame encoding a putative 388 amino acid protein that we have named “nocturnin” (for night-factor). This protein has strong sequence similarity to the C-terminal domain of the yeast transcription factor, CCR4, as well as a leucine zipper-like dimerization motif. Nocturnin mRNA levels exhibit a high amplitude circadian rhythm and nuclear run-on analysis indicates that it is controlled by the retinal circadian clock at the level of transcription. Our observations suggest that nocturnin may function through protein–protein interaction either as a component of the circadian clock or as a downstream effector of clock function.

Integration of circadian and phototransduction pathways in the network controlling CAB gene transcription in Arabidopsis

The transcription of CAB genes, encoding the chlorophyll a/b-binding proteins, is rapidly induced in dark-grown Arabidopsis seedlings following a light pulse. The transient induction is followed by several cycles of a circadian rhythm. Seedlings transferred to continuous light are known to exhibit a robust circadian rhythm of CAB expression. The precise waveform of CAB expression in light–dark cycles, however, reflects a regulatory network that integrates information from photoreceptors, from the circadian clock and possibly from a developmental program. We have used the luciferase reporter system to investigate CAB expression with high time resolution. We demonstrate that CAB expression in light-grown plants exhibits a transient induction following light onset, similar to the response in dark-grown seedlings. The circadian rhythm modulates the magnitude and the kinetics of the response to light, such that the CAB promoter is not light responsive during the subjective night. A signaling pathway from the circadian oscillator must therefore antagonize the phototransduction pathways controlling the CAB promoter. We have further demonstrated that the phase of maximal CAB expression is delayed in light–dark cycles with long photoperiods, due to the entrainment of the circadian oscillator. Under short photoperiods, this pattern of entrainment ensures that dawn coincides with a phase of high light responsiveness, whereas under long photoperiods, the light response at dawn is reduced.

Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock

Under free running conditions, FREQUENCY (FRQ) protein, a central component of the Neurospora circadian clock, is progressively phosphorylated, becoming highly phosphorylated before its degradation late in the circadian day. To understand the biological function of FRQ phosphorylation, kinase inhibitors were used to block FRQ phosphorylation in vivo and the effects on FRQ and the clock observed. 6-dimethylaminopurine (a general kinase inhibitor) is able to block FRQ phosphorylation in vivo, reducing the rate of phosphorylation and the degradation of FRQ and lengthening the period of the clock in a dose-dependent manner. To confirm the role of FRQ phosphorylation in this clock effect, phosphorylation sites in FRQ were identified by systematic mutagenesis of the FRQ ORF. The mutation of one phosphorylation site at Ser-513 leads to a dramatic reduction of the rate of FRQ degradation and a very long period (>30 hr) of the clock. Taken together, these data strongly suggest that FRQ phosphorylation triggers its degradation, and the degradation rate of FRQ is a major determining factor for the period length of the Neurospora circadian clock.

Circadian rhythms in Neurospora crassa: Lipid deficiencies restore robust rhythmicity to null frequency and white-collar mutants

The conidiation rhythm in the fungus Neurospora crassa is a model system for investigating the genetics of circadian clocks. Null mutants at the frq (frequency) locus (frq9 and frq10) make no functional frq gene products and are arrhythmic under standard conditions. The white-collar strains (wc-1 and wc-2) are insensitive to most effects of light, and are also arrhythmic. All three genes are proposed to be central components of the circadian oscillator. We have been investigating two mutants, cel (chain-elongation) and chol-1 (choline-requirer), which are defective in lipid synthesis and affect the period and temperature compensation of the rhythm. We have constructed the double mutant strains chol-1 frq9, chol-1 frq10, chol-1 wc-1, chol-1 wc-2, cel frq9, cel frq10, and cel wc-2. We find that these double mutant strains are robustly rhythmic when assayed under lipid-deficient conditions, indicating that free-running rhythmicity does not require the frq, wc-1, or wc-2 gene products. The rhythms in the double mutant strains are similar to the cel and chol-1 parents, except that they are less sensitive to light. This suggests that the frq, wc-1, and wc-2 gene products may be components of a pathway that normally supplies input to a core oscillator to transduce light signals and sustain rhythmicity. This pathway can be bypassed when lipid metabolism is altered.

Nucleotide binding and autophosphorylation of the clock protein KaiC as a circadian timing process of cyanobacteria

A negative feedback control of kaiC expression by KaiC protein has been proposed to generate a basic oscillation of the circadian clock in the cyanobacterium Synechococcus sp. PCC 7942. KaiC has two P loops or Walker's motif As, that are potential ATP-/GTP-binding motifs and DXXG motifs conserved in various GTP-binding proteins. Herein, we demonstrate that in vitro KaiC binds ATP and, with lower affinity, GTP. Point mutation by site-directed mutagenesis of P loop 1 completely nullified the circadian rhythm of kaiBC expression and markedly reduced ATP-binding activity. Moreover, KaiC can be autophosphorylated in vitro. These results suggest that the nucleotide-binding activity of KaiC plays important roles in the generation of circadian oscillation in cyanobacteria.