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The Arabidopsis Circadian Clock Circadian clocks have evolved to provide an adaptive advantage to most organisms that live under alternating cycles of light and darkness (Ouyang et al., 1998; Woelfle et al., 2004; Dodd et al., 2005). The clock provides a mechanism whereby physiological and behavioral processes can be performed at the appropriate times of day or night. Furthermore, the evolution of a true oscillator, rather than just a light-responsive sand-timer, enables organisms to predict the alterations that occur in relative proportion to day and night throughout the year in most clines. In mammals, many aspects of behavior and physiology are regulated by endogenous circadian clocks and are subject to daily oscillations (Hastings et al., 2003). In higher plants, the circadian clock regulates many key physiological processes, ranging from flowering time (Yanovsky and Kay, 2003; Imaizumi and Kay, 2006) and growth (Dowson-Day and Millar, 1999) to stomatal opening and CO2 assimilation (Hennessey and Field, 1991). Moreover, in Arabidopsis thaliana the expression of at least 6% of the transcriptome is regulated by the circadian clock (Harmer et al., 2000; Schaffer et al., 2001; Michael and McClung, 2003), and recent findings show that the matching of internal and external cycles optimizes growth and survival (Dodd et al., 2005). Figure 1 Circadian systems can be thought of consisting of 3 parts (Figure 1). The imput pathways are involved in the entrainment or reprograming of the central oscillator which is the core of the circadian system. In turn this molecular self-sustained oscillator regulates the different physiological processes by regulating output pathways. The mechanisms regulating the circadian oscillator Although circadian clocks in different species utilize distinct proteins, they appear to maintain the same overall pattern of regulation based on the combination of transcriptional regulatory loops with post-translational regulation. Therefore, the elucidation of the molecular mechanisms regulating the clock in plants will help understand the general principles governing circadian oscillators. Circadian systems can be thought of consisting of 3 parts (Figure 1). The imput pathways are involved in the entrainment or reprograming of the central oscillator which is the core of the circadian system. In turn this molecular self-sustained oscillator regulates the different physiological processes by regulating output pathways. Transcriptional feedback loops The first regulatory feedback loop described in plants consisted of the repression by the morning expressed CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and LHY (LATE ELONGATED HYPOCOTYL) of the evening expressed TOC1 (TIMING OF CAB EXPRESSION). In turn, TOC1 has been shown to be necessary for CCA1 and LHY activation (Alabadi et al., 2001) (Figure 2). In addition to TOC1, there are several other evening expressed genes that are necessary for the morning expression of CCA1 and LHY. These include the putative MYB transcription factor LUX (LUX ARRYTHMO)(Hazen et al., 2005), as well as ELF3 and ELF4 (EARLY FLOWERING 3, 4) (Schaffer et al., 1998; Doyle et al., 2002; Kikis et al., 2005)(Figure 1).TOC1 belongs to the multigene family of circadian regulated PSEUDO-RESPONSE REGULATORS (PRR), which, in Arabidopsis consists of four other members: PRR3, PRR5, PRR7, PRR9 (Matsushika et al., 2000). We discovered that PRR7 and PRR9 form additional transcriptional feedback loops with CCA1 and LHY indicated that the plant circadian clock might have a multiloop structure similar to that of other eukaryotes (Farre et al., 2005). Using mathematical modeling we and others could show that this network structure was able to reproduce many of the system’s characteristics (Locke et al., 2006; Zeilinger et al., 2006). Figure 2 The importance of post-translational regulation In animals and fungi protein degradation, phosphorylation and subcellular localization form part of the molecular mechanisms governing circadian oscillations. In plants recent results have shown that post-translational regulation of protein levels plays a key role in the control of the plant circadian clock. In Arabidopsis the ZEITLUPE (ZTL) family of F-box proteins is involved the turnover of clock and flowering time proteins. ZTL regulates the degradation of both TOC1 and PRR5 but not PRR9, PRR7, and PRR3 (Mas et al., 2003; Ito et al., 2007; Kiba et al., 2007; Fujiwara et al., 2008) (Figure 2). LKP2 (LOV KELCH PROTEIN 2) is a homologue of ZTL whose arrhythmic overexpression phenotype indicates a key role in regulating circadian rhythms (Schultz et al., 2001) but LKP targets remain unknown. This ZTL family of F-box proteins that also includes FKF1 (FLAVIN BINDING, KELCH REPEAT, F-BOX 1), a protein involved in the regulation of flowering time (Nelson et al., 2000; Imaizumi et al., 2003; Imaizumi et al., 2005), contains a LOV/PAS domain that mediates blue light dependent protein-protein interactions (Kim et al., 2007; Sawa et al., 2007) (Figure 2). Several clock proteins in Arabidopsis have been shown to be phosphorylated. The phosphorylation of the transcription factor CCA1 by CKBII is necessary for its activity (Daniel et al., 2004). It has been shown recently that all PRR are phosphorylated in vivo (Farre and Kay, 2007; Fujiwara et al., 2008). Phosphorylation is necessary for the interaction between TOC1 and PRR3 proteins (Fujiwara et al., 2008), however the role of the phosphorylation in the other PRRs remains to be elucidated. The circadian regulated pseudo-response regulators Arabidopsis has five circadian regulated PRRs. Overexpression and mutant analyses indicate that all of them play a role in the regulation of the circadian clock in Arabidopsis (Mizuno, 2004, 2005). Furthermore, experiments performed in rice, wheat and barley show that the PRRs also play a role in the regulation of the circadian rhythms and flowering time in these species (Murakami et al., 2005; Tago et al., 2005; Turner et al., 2005; Beales et al., 2007; Murakami et al., 2007b; Murakami et al., 2007a). The expression of all PRRs is clock regulated with each peaking at a different time of the day or night (Matsushika et al., 2000). As indicated above, recent results also suggest that post-translational regulation plays a key role in the function of these proteins (Mas et al., 2003; Farre and Kay, 2007; Ito et al., 2007; Kiba et al., 2007). Genetic analyses have shown that PRR7, PRR9 and PRR5 play partially overlapping functions (Farre et al., 2005; Nakamichi et al., 2005b; Nakamichi et al., 2005a; Salome and McClung, 2005). PRRs share two conserved protein domains. The pseudo-receiver domain (PR) shares homology with the receiver domains found in response regulators involved in the two-component signaling pathways found in bacteria and plants (Hwang and Sheen, 2002; Oka et al., 2002). However, it lacks the specific aspartate residue that becomes phosphorylated in canonical receiver domains and in vitro experiments suggest that unlike other plant response regulators it cannot be phosphorylated by sensor histidine kinases (Makino et al., 2000; Strayer et al., 2000). The CCT domain (CONSTANS, CONSTANTS-LIKE, TOC1) contains a putative nuclear localization signal and shares some homology with the DNA binding domain of yeast HEME activator protein 2 (HAP2), which is a subunit of the HAP2/HAP3/HAP5 trimeric complex that binds to CCAAT boxes in eukaryotic promoters (Wenkel et al., 2006). However, the DNA binding capacity of CCT domains has not been reported. Recent results have found TOC1 associated with the CCA1 promoter and indicate that it might block the inhibitory activity of the TCP transcription factor CHROMATIN HIKING EXPEDITION 1 (CHE1) on CCA1 expression (Pruneda-Paz et al., 2009; Figure 2). PRR3 is involved in the regulation of TOC1 stability(Para et al., 2007), which represents the first report of the biochemical function of a PRRs (Figure 2). The biochemical function of the other PRRs remains to be elucidated. Bibliography Alabadi, D., Oyama, T., Yanovsky, M.J., Harmon, F.G., Mas, P., and Kay, S.A. (2001). 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