Temperature Entrainment of the Arabidopsis Circadian Clock

Most eukaryotic organisms as well as some prokaryotes (cyanobacteria) have an endogenous circadian clock that keeps track of the earth's 24-h day/night cycle and regulates the activity of numerous metabolic pathways. The existence of the circadian clock as an endogenous feature of organisms and not merely a response to the presence or absence of sunlight was recognized in plants as early as the 18th and 19th centuries. It had been known since ancient times that certain plants, notably the sensitive plant Mimosa pudica, fold their leaves shut at night and open them during the day. The French astronomer Jean de Mairan in 1729, and botanists Henri-Louis Duhamel and Augustin de Candolle in 1758 and 1832, respectively, were motivated to ask if this was an innate property of the plant as opposed to a passive response and conducted the simple experiments of observing plants maintained in constant darkness or constant light. Their observations that plants in constant conditions continued to open and shut their leaves at the appropriate times suggested the existence of an endogenous circadian clock. De Candolle showed that the period of leaf opening and folding in Mimosa plants was 22 h under constant light, demonstrating the free-running nature of circadian clocks (Coleman, 1986). Investigations of daily rhythms in the activities of insects and mice and the remarkable navigational abilities of birds revealed the existence of an endogenous circadian clock in animals in the early to mid 20th century (Pittendrigh, 1993).

There are three hallmarks of circadian clock–controlled rhythms: (1) they are free running and will continue to oscillate with an approximate 24-h period in the absence of external stimuli, (2) they are entrainable by external stimuli, such as light and temperature, and (3) in contrast with most metabolic processes that are strongly affected by changes in temperature, overall periodicity is unaffected by temperature (McClung et al., 2002). In addition, circadian clocks are remarkable in their persistence (>2 years in some rodents) and precision (the standard deviation of a mouse rhythm is ∼1 min, or 1/1000) (Pittendrigh, 1993). Numerous metabolic processes follow daily cycles of activity that are regulated by the circadian clock. The most well known are pathways that affect sleep and other behaviors in animals and photosynthetic carbon assimilation and carbon and nitrogen metabolism in plants. In recent years, increasing interest in circadian rhythms and improved methods of analysis have led to the identification of additional clock-controlled pathways as well as components of the clock mechanism and input pathways.

HOW CLOCKS WORK

In all organisms studied, including plants (Arabidopsis), fungi (Neurospora), mammals (mainly rodents, and to some extent humans), and insects (Drosophila), the central oscillator consists of a complex feedback loop, or multiple interwoven loops, involving both positive and negative regulation, essential features of which include posttranslational protein modifications that precisely control degradation and shuttling of principal factors between the nucleus and cytoplasm.

The mammalian circadian clock is a complex system composed of numerous tissue-specific clocks, all regulated by a master pacemaker that resides in the suprachiasmatic nuclei in the brain (reviewed in Albrecht and Eichele, 2003). The central pacemaker consists of multiple feedback loops involving the CLOCK/BMAL1 family of helix-loop-helix transcriptional activators and their target genes Period (Per), Cryptochrome (Cry), and Rev-erbα. These gene products, in turn, inhibit CLOCK/BMAL1 and thereby negatively regulate their own transcription (see figure). CLOCK/BMAL1 heterodimers may be some of the principal factors regulating clock output via interactions with various other proteins (Albrecht and Eichele, 2003). Key points of regulation have been proposed to lie with the formation of the PER-CRY heterodimer, which remains in the nucleus where it binds and inhibits CLOCK/BMAL1, and phosphorylation of PER, which targets it for cytoplasmic degradation via the proteasome pathway (Yagita et al., 2002). REV-ERBα also represses transcription of Bmal1 and influences period length and entrainment of the clock (Preitner et al., 2002).

Figure 1.

Simplified Models of Core Circadian Oscillator Components.

The models depicted show the basic activities of some of the principal components of the circadian clock central oscillator in mammals (A) and Arabidopsis (B). The most current models in mammals and Arabidopsis propose multiple interlocking positive and negative feedback loops. See Albrecht and Eichele (2003), Farré et al. (2005), and McClung et al. (2002) for more details.

The clockwork mechanism in higher plants also consists of positive and negative feedback loops, which in Arabidopsis involve the single-Myb domain transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE AND ELONGATED HYPOCOTYL (LHY) and the pseudoresponse regulator protein TIMING OF CAB EXPRESSION 1/PSEUDO-RESPONSE REGULATOR 1 (TOC1/PRR1) (Alabadí et al., 2001). CCA1 and LHY inhibit TOC1 transcription, whereas TOC1 directly or indirectly promotes transcription of CCA1 and LHY (see figure). The loops are regulated by turnover of CCA1 and LHY via an unidentified mechanism and of TOC1 by proteasomal degradation mediated by the F-box protein ZEITLUPE (ZTL; Más et al., 2003a; Han et al., 2004).

In addition to TOC1/PRR1, the transcription of four other Arabidopsis PRR genes (PRR3, 5, 7, and 9) oscillates with a circadian period. Matsushika et al. (2000) showed that transcription of all five of these genes (including TOC1/PRR1) peaks at different times during the day, leading them to propose that circadian waves of these PRR family members form the basis of the higher plant circadian clock, with each one regulating accumulation of the next (reviewed in Mizuno, 2004).

CLOCK ENTRAINMENT

Circadian clocks are continually entrained by environmental factors, notably light and temperature, which act continually to reset the clock mechanism and thus synchronize output rhythms with changes in daylight hours during the year. Light is the most powerful and best-characterized entrainment signal in plants. The phytochrome (PHYA-PHYE) and cryptochrome (CRY1 and CRY2) photoreceptors in Arabidopsis have all been shown to function in light entrainment of the clock (reviewed in Devlin and Kay, 2001). ZTL is an interesting multidomain protein that appears to play critical roles both in the regulation of core clock proteins and in light entrainment of the clock. In addition to the F-box domain, which is presumed to function in targeting of interacting partners (such as TOC1) for proteasomal degradation, ZTL contains a LOV domain similar to that of PHOT1 and other phototropins, suggesting that it may function as a photoreceptor, and KELCH repeats, which are known to be involved in protein–protein interactions in numerous other proteins. ZTL has been shown to interact with the PHYB and CRY1 photoreceptors in a yeast two-hybrid system (Jarillo et al., 2001), so it is suggested that ZTL may function in light entrainment of the clock.

Although circadian clocks are temperature compensated, which means that overall clock period is constant over a range of temperatures, temperature changes can act to entrain or reset the clock output patterns. However, very little is known about the mechanism of temperature entrainment. In this issue of The Plant Cell, Salomé and McClung (pages 791–803) analyze prr7 and prr9 single and double mutants and show that PRR7 and PRR9 have overlapping functions that play a key role in temperature entrainment of the circadian clock in Arabidopsis.

PSEUDO-RESPONSE REGULATORS AND THE ROLE OF PRR7 AND PRR9

Pseudo-response regulators are so named because they lack an Asp residue that is conserved in the receiver domain of typical two-component response regulators and is phosphorylated by the kinase partner of the two-component system (Hwang et al., 2002). Makino et al. (2000) showed that the receiver-like domains of TOC1 and PRR2 do not appear to have phosphate-accepting activity in vitro, so the mechanism of action of the PRR proteins remains unclear.

Salomé and McClung studied output rhythms of cotyledon movements and also the expression in transgenic plants of the reporter gene LUCIFERASE driven (individually) by the promoters of the clock-regulated genes CCA1, TOC1, LHY, PRR7, and PRR9. They show that these output rhythms can be reset or entrained by thermocycles (i.e., alternating warm and cool conditions). They next analyzed the response of prr3, prr5, prr7, and prr9 single mutants to a range of entraining thermocycles and found that prr7 and prr9 mutants were strongly affected, whereas prr3 and prr5 mutants were minimally affected in their response to thermocycles. In addition, prr7 prr9 double mutant plants lost the ability to reset the clock in response to temperature entrainment and failed to maintain rhythmicity in the dark. Interestingly, they also found that, in the absence of thermocycles, loss-of-function alleles of PRR3 and PRR5 shortened clock period (similar to what Más et al. [ 2003b] described for TOC1 loss-of-function mutants), whereas those of PRR7 and PRR9 lengthened clock period, further suggesting that PRR7 and PRR9 play a role in regulating the clock that is distinct from that of the other PRR genes. Analysis of the prr7 prr9 double mutant in contrast with each single mutant suggested that the two genes are partially redundant.

The results of Salomé and McClung as well as those recently reported by Farré et al. (2005) suggest that PRR7 and PRR9 proteins function very close to, if not within, the core clock mechanism. Farré et al. emphasize a role for PRR7 and PRR9 in light input to the clock as well as in the regulation of the oscillator. Salomé and McClung's results indicate that PRR7 and PRR9 also serve either as factors regulating temperature entrainment input to the central oscillator or as components of the core oscillator that accept temperature entrainment input signals from other factors. This work thus represents a major contribution to the study of temperature entrainment of the circadian clock and sheds light on the inner works of the clock mechanism.