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The Plant Cell, Vol. 9, 1235-1244, July 1997 O 1997 American Society of Plant Physiologists

Coordination of Plant Metabolism and Development by the Circadian Clock

Joel A. Kreps and Steve A. Kay’ Department of Cell Biology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037

BIOLOGICAL CLOCKS

Many plant cellular activities occur with a daily rhythmicity. In some cases, the rhythmicity of these cellular activities is maintained in plants growing under constant environmental conditions, such as continuous light (LL) or darkness (DD) and constant temperature. Because rhythms can persist in the absence of externa1 time cues (known as free-running conditions), they must be driven by an internal oscillator. This oscillator, which is known as the circadian clock, gen- erates circadian rhythms. For the circadian clock to regulate rhythms such that they occur at the correct time of day throughout the year, it must be able to perceive the seasonal changes in day length. This adjustment of the clock is known as entrainment, and the environmental cues that are perceived are called Zeitgebers, from the German word meaning “time giver.” Thus, the circadian clock can be con- sidered to be an internal processor of temporal inputs from the environment (such as light and temperature). Output from the processor regulates the timing of metabolic and developmental events within the plant.

Although the molecular basis of the circadian clock in plants is not known, work from other systems has estab- lished a transcriptional negative feedback loop as a para- digm. In Drosophila, the clock components encoded by the period (per) and timeless (fim) genes function to regulate negatively their own transcription (Hardin et al., 1990; Sehgal et al., 1995). In this system, the accumulation of PER and TIM proteins eventually leads to the arrest of transcription from the corresponding genes, but how this is achieved is not known (Hunter-Ensor et al., 1996). The TIM protein is un- stable in the light, and this photoinstability may be part of the mechanism by which the Drosophila circadian clock en- trains to the photoperiod (Myers et al., 1996).

A clock gene, Frequency (Frq), has also been isolated from the fungus Neurospora crassa (McClung et al., 1989). As is the case for the clock genes in Drosophila, the Frq gene product also negatively regulates its own transcription (Aronson et al., 1994b), and FRQ protein levels cycle (Dunlap, 1996). Transcription of the Frq gene is upregulated in re-

To whom correspondence should be addressed. E-mail stevekQ scripps.edu; fax 61 9-784-9840.

sponse to light, suggesting that Frq is also involved in the entrainment pathway (Crosthwaite et al., 1995). Recently, two new clock genes have been cloned: white collar-2 (wc-2) from N. crassa (Linden and Macino, 1996; Crosthwaite et al., 1997) and Clock from the mouse (King et al., 1997). In- terestingly, both of these proteins contain sequences related to the PAS domain (for PER, ARNT, SIM; see Hall, 1995) that is present in many transcription factors, including PER. Thus, the PAS motif is the first conserved sequence shared among clock proteins; its presence may indicate a common evolu- tionary origin for elements of clock function (Kay, 1997).

Severa1 circadian rhythm mutants have been isolated from Arabidopsis (Millar et al., 1995a). The timing ofcab-7 (tocl) mutation defines a product that is required for normal cycling of severa1 rhythms, such as the gene-expression rhythms of the chlorophyll alb binding protein 2 gene (Lhcb7*7; here termed CABZ) (Millar et al., 1995a) and the cold, circadian rhythm RNA binding protein 1 and 2 genes (CCR7 and CCR2; Kreps and Simon, 1997) as well as a leaf movement rhythm (Millar et al., 1995a). The effects of toc l on multiple rhythms suggest that the TOCl protein may be a clock component. Thus, the cloning and analysis of the TOC7 gene are likely to provide nove1 insights into the molecular basis of the circadian clock in plants.

A single organism may have more than one clock or oscillator. For example, different oscillators have been found within the same cell in Gonyaulax polyedra (Roenneberg and Morse, 1993). Moreover, Hennessey and Field (1992) showed that bean has circadian rhythms in stomatal opening and leaf movement which have different free-running periods. This is formally possible only if there are different oscillators (Pittendrigh, 1993). However, another possibility is that these two rhythms in bean have different free-running periods resulting from cell-specific modification of the same clock components. Indeed, in Arabidopsis, different rhythms or outputs may share clock components (see above).

A model for circadian rhythms in plants that is based on these observations has input pathways, such as light and temperature, that interact with one or more central oscillators (Figure 1). The central oscillator(s) is then involved in generating output rhythms via a range of cellular or systemic signaling pathways.

1236 The Plant Cell

Central Oscillator(s)

Outputs

Figure 1. A General Model for Organization of the Circadian Clock System in Plants.

Multiple input pathways, such as light and temperature, connect the clock or central oscillator to the externa1 environment. The central oscillator is presented as a series of components (arrows) that are interdependent and comprise a feedback loop (Aronson et al., 1994; Kyriacou, 1994). Output from the central oscillator produces rhythms that can differ in phase from one another (Zhong and McClung, 1996; Kreps and Simon, 1997), as indicated on the right.

PHOTOTRANSDUCTION AS AN INPUT AND AN OUTPUT PATHWAY

A universal property of circadian rhythms in all organisms is the dramatic effect of light on rhythmicity, and one of the most intriguing problems in this field is to understand how light, via the activation of specific photoreceptors, interacts with the circadian clock. Multiple photoreceptors, including the phytochromes, blue light receptors, and at least one UV-B receptor, allow plants to sense the light environment (Chory, 1997, in this issue). These photoreceptors interact with circadian systems at severa1 levels in higher plants, although the details of these interactions are unknown. For example, red and/or blue light pulses control the phase of the clock, thereby mediating entrainment to the dayhight cycle (Simon et al., 1976). Moreover, in etiolated wheat seedlings, the cyclic regulation of CAB RNA levels is initi- ated by a very-low-fluence response of phytochrome (Nagy et al., 1993), as is the regulation of catalase3 (CAT3) RNA cycling in etiolated maize seedlings (Boldt and Scandalios, 1995).

In Arabidopsis, experiments based on the expression of a circadian clock-regulated form of luciferase (LUC; Millar et al., 1995b) in light-signaling mutants have begun to indicate how the phototransduction pathways may be connected to the plant clock. Arabidopsis seedlings carrying the CAB2 promoter-LUC fusion gene (CAB2::LUC) exhibit a circadian rhythm in LUC enzyme activity with a period of 24.7 _t 0.4 hr in LL. However, the period dramatically lengthens to 30 to 36 hr when seedlings are transferred to DD, and the mean CAB2::LUC transcription level decreases rapidly.

The lengthening of the circadian period upon changes in the light regime was used as an assay for light input path-

ways into the circadian clock. For example, analysis of the circadian period length of bioluminescence in constant red light in the phytochrome mutant long bypocotyll (byl; a chromophore mutant that has reduced levels of all phytochromes; Parks and Quail, 1991) showed lengthening to 26.5 hr, whereas a 24.5-hr period in LUC activity was observed in b y l under constant blue or white light (Millar et al., 1995b). These results demonstrate that phytochromes are indeed responsible for red light input into the clock and that blue light must be acting through a phytochrome-independent pathway. Possibilities include transduction pathways compromised in the mutant by4 (which has a defect in putative blue light receptor; Ahmad and Cashmore, 1993) or the nonpbototropic hypocotyll (npbl) mutant (which is defective in blue light perception; Liscum and Briggs, 1995).

The effects of mutations that act downstream of photoreceptors, such as deetiolatedl (detl), det2, and constitutively pbotomorphogenicl (copl), on CAB2::LUC expression were also analyzed in these experiments (Millar et al., 199513). These mutations, which result in the activation of light transduction pathways in darkness (see Chory, 1997, in this issue), led to a shortened period of LUC activity in DD and also to higher CAB2::LUC expression levels in DD. Thus, these mutations affect both the input pathway of the circadian clock as well as the mean expression level of CAB. These data point toward a model in which circadian period length is regulated by independent blue and red light input pathways to the circadian clock that act through transduction pathways influenced by DET7 and COPl (Figure 2; Millar et al., 1995b).

The circadian clock has been reported to modulate photoreceptor function, indicating that light perception and transduction are also influenced by the output pathways of the clock in plants. Millar and Kay (1 996) observed an attenuation or gating of the acute response (i,e., a rapid induction of CAB gene expression by light) of CAB2::LUC transcription in Arabidopsis seedlings in DD (Figure 2). This gating activity cycled in parallel to the rhythm in CAB expression level in these seedlings and was rhythmic in DD, indicating that the clock can regulate the attenuation of light-induced CAB transcription. However, it is not known which photoreceptor systems are being affected by the clock in these experiments. Gating of the photic induction of gene expression by the circadian clock also occurs in animal systems (reviewed in Takahashi, 1993).

In tobacco, the ability of light to reset the clock is developmentally regulated. A CAB expression rhythm can be detected in etiolated seedlings before light treatment (Millar et al., 1992; Kolar et al., 1995), and the phase of this rhythm is not changed by red light pulses during the first 44 hr after sowing (Kolar et al., 1995). Interestingly, a second rhythm in CAB expression was detected in the same seedlings given a red light pulse at 36 or 44 hr after sowing. This second rhythm was in phase with the light pulses &e., peaks oc- curred in 24-hr cycles from the time of the light pulse [Kolar et al., 19951). Thus, in etiolated tobacco seedlings, it is pos-

Circadian Control and Coordination 1237

Blue-light PhotoreceptorsB

Morphology

Phytochromes Gene ExpressionFigure 2. A Model for Light Signal Transduction Pathways Leading to the Plant Circadian Clock and Downstream Pathways Regulated by the Clock.

Red (R) and blue (B) light, perceived by the phytochromes and blue light photoreceptors, respectively, initiate signaling pathways leading to thecentral oscillator via the DET1 and COP1 transduction components (Millar et al., 1995b). Both forms of light are also shown as directly affectingphenomena downstream from the clock, such as morphology, which can be regulated by blue light (Liscum and Briggs, 1995), and the inductionof CAB gene expression, which is triggered by red light (Chory, 1 997, in this issue). The clock gating of light induction of CAB (Millar and Kay,1996) is shown as a negative output (T-bar) from the clock. Other outputs from the clock (arrows) lead to gene expression and to changes inmorphology (see text for details).

sible to detect two separate rhythms of CAB expression,only one of which is regulated by a phototransduction path-way that becomes activated after some critical stage inseedling development.

The effects of interactions between light and the Circadianclock on CAB gene expression provide an example in whichthe Circadian clock coordinates the temporal regulation of acellular event with the external light environment (Figure 2).In dark-adapted seedlings, CAB expression is low and therhythm in expression is not detected. However, after a shortlight pulse, CAB transcript levels rise rapidly, and this initial,light-triggered increase is followed by a second, clock-mediated peak in expression (Millar et al., 1992). Additionalstudies with the CAB2::LUC construct in Arabidopsisshowed that gating of this acute response combined withdirect clock regulation of CAB work together to produce apattern of expression in which peak levels of CAB expres-sion always occur at the middle of the light period, regard-less of its length (Millar and Kay, 1996).

OUTPUT PATHWAYS REGULATED BY THE CLOCK

Stomatal and Leaf Movements

Stomatal opening and closing and leaf movements arecontrolled by the Circadian clock in several plant species(Sweeney, 1987; Kim et al., 1993). Both phenomena are

based on changes in turgor pressure due to ion fluxes thatare mediated in part by transport proteins located in theplasma membrane (Assmann and Haubrick, 1996). Themechanisms underlying the clock regulation of leaf move-ments and Stomatal opening and closing are not known.However, some pathways that regulate the function of theseion channels have been investigated. For example, whiteand blue light, but not red light, hyperpolarized the mem-branes of protoplasts from Samanea saman extensor andflexor cells (these cells effect leaf movements [Kim et al.,1992]). The hyperpolarized extensor but not flexor proto-plast membranes could be depolarized with exogenous po-tassium, suggesting that the potassium channels from theextensor but not the flexor cells are open in the light. This isconsistent with the physiological roles of these cells in theleaf (Kim et al., 1992). Moreover, an inward potassium chan-nel activity in We/a faba guard cells was negatively affectedby treatments with nonhydrolyzable GTP analogs, suggest-ing that the transporter is regulated by a G-protein (Wu andAssmann, 1994). G-protein regulation of the inward potas-sium transporter was supported further by patch-clamp exper-iments showing that cholera and pertussis toxins inhibitedthe transporter (Wu and Assmann, 1994).

Calcium-dependent protein kinase (CDPK) activity hasbeen proposed to regulate H' -ATPase activity, which in turnis required for potassium pumping into guard cells (Schallerand Sussman, 1988; Kinoshita et al., 1995; Assmann andHaubrick, 1996). CDPK activity is known to regulate a vacu-olar chloride channel that is required for Stomatal opening in

1238 The Plant Cell

V. faba (Pei et al., 1996), and an inhibitor of calcium/calmodulin myosin light chain kinase can block blue light-induced stomatal opening in V. faba (Shimazaki et al., 1992). Thus, changes in intracellular calcium levels are involved in stomatal opening. These changes may provide a mechanism whereby the clock regulates stomatal opening (see below).

Future studies of leaf movement and stomatal opening in circadian rhythm mutants of Arabidopsis (Millar et al., 1995a) may provide insight into the organization of clocks in plants. For instance, do different cell types have separate clocks, or are the same clock components used throughout the plant? Genes for several H+-ATPases and potassium transporters have been cloned (reviewed in Assmann and Haubrick, 1996) as well as a gene for a CDPK (Harper et al., 1991). Al- though regulation of these activities seems to be at the protein level, it would be interesting to determine whether the clock is involved in regulating the expression of the corresponding genes or the activity of their protein products.

Circadian Clock-Regulated Gene Expression

The observed circadian control of the expression level of many plant genes is a further indication of the clock’s wide- spread temporal control of metabolism. Circadian rhythms have been observed in gene transcription (reviewed in McClung and Kay, 1994), mRNA translation (Mittag et al., 1994), and post-translational modification of proteins (Carter et al., 1991). In some cases, such as the components involved in photosynthesis, carbon fixation, and nitrogen metabolism, the need for proper temporal regulation is obvious. How- ever, there are other examples (such as the high mobility group 1 DNA binding protein gene from Pharbitis nil [Zheng et al., 19931) for which the rationale for circadian cycling of expression is not immediately obvious or where the gene products encode pioneer proteins without a known function (Reimmann and Dudler, 1993).

Genes lnvolved in Light Harvesting

The ability of an organism to anticipate a daily requirement for particular physiological functions, which is afforded by the circadian clock, may confer a selective advantage. This function of anticipation is clearly seen in the predawn increase in the mRNA levels of genes encoding components of the photosynthetic apparatus, as exemplified by the induction of CAB gene expression before lights-on (Millar and Kay, 1996). In fact, the first observations of clock-regulated gene expression came from studies of the genes encoding CAB, the small subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase (rubisco), and the photosynthetic reac- tion center component, PSAD (for photosystem I gene D; Kloppstech, 1985; Giuliano et al., 1988; Nagy et al., 1988). Since then, other genes encoding photosynthetic components have been shown to be clock regulated at one level or

another (reviewed in McClung and Kay, 1994). Interestingly, none of the components encoded by the chloroplast ge- nome that were assayed by RNA gel blot analysis showed any evidence of cycling (Adamska et al., 1991). However, the rate of transcriptional activity of several chloroplast genes in Chlamydomonas rheinhardtii was recently shown to exhibit circadian rhythmicity (Hwang et al., 1996). It will be interesting to determine whether clock regulation of organel- lar transcriptional activity also occurs in multicellular photosynthetic organisms.

Severa1 reports have indicated that the normal accumulation of pigment binding proteins requires the presence of chlorophyll (Apel and Kloppstech, 1978; Eichacker et al., 1990; Kim et al., 1994). Moreover, the level of the chlorophyll precursor 6-aminolevulinic acid (Beator and Kloppstech, 1993) and the expression of some of the genes encoding enzymes involved in chlorophyll biosynthesis were shown to be clock regulated in barley. Interestingly, the phase at which the expression of these genes cycle is slightly earlier than that at which CAB cycles (Bougri and Grimm, 1996; Jensen et al., 1996). This temporal coordination of chlorophyll biosynthesis with CAB expression exemplifies the in- volvement of the circadian clock in the orchestration of cellular biology.

Genes lnvolved in Carbon Fixation

Carbon fixation can also be clock regulated. In plants that fix carbon via the crassulacean acid metabolism pathway, carbon fixation is timed to occur at night to minimize water loss through open stomata (reviewed in Ting, 1987). Indeed, the activity of the key enzyme in the crassulacean acid metabolism pathway, phosphoenolpyruvate carboxylase (PEPCase), peaks at night (Ting, 1987). although its mRNA and protein levels do not change. PEPCase activity is regulated by phosphorylation, which renders the enzyme less sensitive to feedback inhibition by malate (Nimmo et al., 1995). Thus, in this example, the circadian clock controls an essential metabolic pathway through the activity of a kinase (Carter et al., 1991).

In a study using the inhibition of PEPCase by malate as an assay for kinase activity, the protein synthesis inhibitors cyclohexamide and puromycin blocked the activation of PEPCase, indicating that PEPCase kinase activity requires ongoing gene expression to operate (Carter et al., 1991). Additional experiments have shown that the circadian rhythm in the kinase activity is probably the result of a rhythm in its steady state mRNA level (Hartwell et al., 1996). Similarly, the expression of Cahl, a gene encoding carbonic anhydrase in C. reinhardtii, is clock regulated (Fujiwara et al., 1996). In C. reinhardtii, carbonic anhydrase is involved in C02 fixation during photosynthesis, and the phasing of the circadian rhythm of Cah7 mRNA results in levels of Cah7 RNA that are high during the photoperiod and low during the dark period.


Nitrogen Assimilation

Another metabolic pathway that operates nocturnally in some plants is nitrogen assimilation. In this pathway, nitrate (NO,-) is transported into roots cells, where it is reduced by nitrate reductase (NR) to nitrite (NO,-), which is then reduced to NH4+ by nitrite reductase (NiR; Sander et al., 1995). NH4+ can then be transported throughout the plant mainly in the form of asparagine (Lea and Miflin, 1980), which is produced by a glutamine-dependent asparagine synthetase (AS; Lam et al., 1994). Interestingly, NR and NiR have maximal expression and activity during the light phase, and their gene expression is circadian clock regulated in bean (NiR; Sander et al., 1995), tobacco (NR; Deng et al., 1990), and Arabidopsis (NR; Pilgrim et al., 1993). By contrast, AS mRNA levels peak during the night phase in pea (Tsai and Coruzzi, 1990) and Arabidopsis (Lam et al., 1994), although the accumulation of this enzyme does not appear to be clock regulated (Tsai and Coruzzi, 1990).

The differences in lighydark (LD) phase expression of NR, NiR, and AS may be a function of the energy requirements for the corresponding enzyme reactions (Lam et al., 1994; Sander et al., 1995). For example, NR and NiR enzymes re- quire large amounts of reducing power, which is readily available when the plants are photosynthesizing (Pilgrim et al., 1993; Sander et al., 1995); hence, the clock coordinates the maximal expression of these genes during the light phase. By contrast, the dark-phase regulation of AS may re- flect the altered nitrogen-to-carbon metabolic ratio in dark- grown versus light-grown tissue (Lam et al., 1994). Asparagine is the favored vehicle for nitrogen transport when the nitrogen-to-carbon ratio is high, as is the case in dark-grown tissue (Lea and Miflin, 1980; Lam et al., 1994), and so it would be advantageous for AS expression to be timed to peak during the dark phase.

Temperature Stresses

Circadian clock regulation of chilling tolerance has been studied extensively in cotton (McMillan and Rikin, 1990; Rikin, 1991, 1992), which is a chilling-sensitive plant. Inter- estingly, cotton was found to be more tolerant of chilling stresses given during the dark phase than it was of those given during the light phase (Rikin et al., 1993). Further analysis indicated a correlation between the chilling-tolerant phase and changes in fatty acid composition (Rikin et al., 1993). Thus, the circadian clock appears to be regulating lipid con- tent, which is likely to affect membrane structure. Plant membranes are a major site for chilling injury (Thomashow, 1994), and the clock-regulated alteration in membranes may result in plants being more resistant to reduced tempera- tures during the night, a time when the plant is more likely to experience chilling stress.

In tomato, another chilling-sensitive species, Martino-Catt and Ort (1992) observed that cycling of CA6 RNA and pro-

tein levels ceased when the plants were transferred to 4°C and incubated in constant darkness. However, once the plants were returned to light and normal growth temperature (25"C), cycling resumed at the same leve1 as when the plants were moved into the cold, suggesting that the circadian clock in tomato cannot function at 4°C. The authors postu- lated that the cold-mediated interruption of CA6 cycling could be involved in the negative effects of chilling on photosynthesis in tomato (Martino-Catt and Ort, 1992).

In Arabidopsis, a chilling-tolerant plant, low temperature (4°C) has a different effect on clock-regulated gene expression. CA6 and CCR transcript levels cycled in Arabidopsis seedlings grown for several days under LD conditions at 4°C (Kreps and Simon, 1997). However, during the first 24 hr at 4"C, CA6 mRNA levels were severely reduced, and CCR expression was much higher relative to the levels for both in control plants. Despite the different effects on transcript levels, the amplitude of the cyclical changes in steady state transcripts was reduced for both CCR and CA6. This result indicates that under LD conditions, when externa1 time cues are driving the cycling, the machinery used to regulate the RNA levels for these genes can function at least partially at 4°C.

When Arabidopsis seedlings growing under LL conditions were given short cold pulses (12 or 20 hr at 4"C), the clock regulating CAB and CCR expression did not stop for the du- ration of the cold pulse (Kreps and Simon, 1997), unlike the clock in tomato. Interestingly, the 12-hr cold pulse had little if any effect on the circadian clock, but the 20-hr cold pulse did have an effect. Specifically, the rhythms for both CAB and CCR expression were phase shifted by 12 hr in the 20- hr cold-pulsed seedlings relative to the rhythms in control seedlings. There are different possible interpretations for these results. For example, there may a phase of the clock that is affected by a 20-hr but not a 12-hr cold pulse during continuous illumination. Alternatively, the 20-hr pulse may act as a Zeitgeber to reset the clock. Additional experimentation is required to discriminate between these two possibilities.

Finally, although clock-regulated resistance to elevated temperature has been reported for some microorganisms, no firm results have been obtained with plants (Rensing and Monnerjahn, 1996).

Other Clock-Controlled Genes

There are several genes whose expression is regulated by the circadian clock but for which an obvious need for clock regulation is not apparent. In some cases, these genes encode proteins with known functions, such as high mobility group DNA binding protein 1 from P. nil (Zheng et al., 1993), cysteine protease 8 from tobacco (Linthorst et al., 1993), CCRl and CCR2 from Arabidopsis (Carpenter et al., 1994), ger- minlike protein from mustard (Heintzen et al., 1994a), glycine-rich RNA binding proteins l a and 2a from mustard (Heintzen et al., 1994b), ADP-ribosylation factor 1 from C. reinhardtii (Memon et al., 1995), and S-adenosylmethionine

1240 The Plant Cell

decarboxylase from Tritordeum (Dresselhaus et al., 1996). Expression from one of the three CAT genes is clock regulated in maize (Redinbaugh et al., 1990), and expression of two CAT genes is clock regulated in Arabidopsis (Zhong et al., 1994; Zhong and McClung, 1996), although no rhythms in CAT enzyme activity have been reported. One gene that is also clock regulated, LIR (for jight-jnduced !ice), encodes a putative pioneer protein that has no matches in the data- bases (Reimmann and Dudler, 1993).

Mechanism of Clock Control of Transcription

Of all the clock-controlled genes (CCGs) described above and reviewed in McClung and Kay (1994) and in Beator and Kloppstech (1996), only the promoters of the Arabidopsis CABP (Millar et al., 1992) and rubisco activase (RCA) genes (Liu et al., 1996) and the CAB-7 promoter from wheat (Fejes et al., 1990) have been shown to confer clock regulation upon a reporter gene. In the case of the CAB2 promoter, the circadian clock-responsive element (CCRE) has been nar- rowed down to a 36-bp domain within the promoter (Carré and Kay, 1995). Although three different factors bind within the CABP CCRE (Carré and Kay, 1995), the binding activities do not appear to cycle, suggesting that the circadian clock- regulated expression of CABP is not mediated simply by the presence or absence of one of these factors. Interestingly, a myb-like factor, CCA1, which is thought to be involved in the phytochrome regulation of the Arabidopsis Lhcb73 gene, was recently cloned (Wang et al., 1997). This factor binds to sequences that are also present in the CCRE of CAB2; therefore, it will be of interest to investigate the role of this factor in circadian clock regulation of CAB transcription.

CCREs in the promoters of N. crassa CCGs have been identified, but no similarities between the funga1 and plant CCREs have been reported (Bell-Pedersen et al., 1996). Comparisons between the promoters of clock-regulated genes in plants may be more fruitful. Recently, a 330-bp fragment from the Arabidopsis RCA promoter was shown to confer clock regulation on the reporter gene chlorampheni- col acetyl transferase (Liu et al., 1996). Comparisons between the 36-bp CABP CCRE sequence and the 330-bp RCA CCRE highlighted some regions of similarity. However, the functional significance for these sequences in the RCA promoter is not known (Liu et al., 1996).

Calcium and Circadian Rhythms

Changes in intracellular calcium levels are involved in regulating several phenomena in plants (see Trewavas and Malhó, 1997, in this issue) and have been implicated in the control of circadian rhythms. The role of calcium in circadian rhythms is unusual in that it may function in both the input and output pathways (Figure 1).

Severa1 lines of research have indicated that calcium is

part of a phytochrome signal transduction pathway in plants (see Chory, 1997, in this issue). For example, in etiolated wheat protoplasts, cytoplasmic calcium levels rose in response to a red light pulse that could initiate protoplast swelling (Shacklock et al., 1992). Moreover, Neuhaus et al. (1993) and Bowler et al. (1994a, 199413) used both CAB:: p-glucuronidase and chalcone synthase promoter::p-glucuronidase reporter genes to determine that activated phytochrome directly regulates CAB gene expression via calcium but regulates chalcone synthase via cGMP. The pathways mediated by calcium and cGMP also appear to regulate each other negatively in a manner described as reciproca1 control (Bowler et al., 1994a). As discussed above, the phytochrome pathway is one of the ways by which light feeds into the circadian clock. Given the data of Neuhaus et al. (1993) and Bowler et al. (1994a, 1994b), an obvious question is, which signal transduction pathways connect phytochrome to the clock? Studies in several organisms, such as fuglena (Tamponnet and Edmunds, 1990), N. crassa (Nakashima, 1984), C. reinhardtii (Bruce et al., 1991), and Trifolium repens (Bollig et al., 1978), have shown that changes in intracellular calcium can shift the phase of the clock. These results indicate that calcium is part of the light signal transduction chain leading to the clock as well as a mediator of direct induction of gene expression by light (Figure 2).

Interestingly, experiments using the calcium-dependent photoprotein aequorin as an indicator of calcium flux identified a circadian rhythm in intracellular calcium levels in tobacco and Arabidopsis (Johnson et al., 1995). But, is calcium also a clock component, or is it also an output from the clock? Pharmacological studies using a soybean cell sus- pension culture and assays for overt rhythms such as reporter gene cycling (S. Welsh and S.A. Kay, unpublished results) are likely to provide further insight into the role of calcium in circadian rhythms. For instance, whether cGMP is used by the clock to gate phytochrome induction of CAB gene expression could be investigated by evaluating the effects of cGMP agonists and antagonists in the gating assay.

CONCLUSIONS AND FUTURE DlRECTlONS

Given the broad action of circadian clocks in regulating multiple aspects of plant growth and metabolism, a better understanding of how clocks are built and integrated into regulatory networks in plant cells is necessary. A bona fide clock is required not only to ensure that many biological activities are correctly phased throughout the LD cycle but also to cope with the regulated timing of biological events in an environment of seasonally changing daylengths. This latter aspect of clock function contributes to photoperiodism, the ability of both plants and animals to alter their reproductive development under changing daylengths. Photoperiodism is relevant to understanding vegetative functions because


photoperiodic time measurement occurs in leaves. There- fore, it is highly likely that the role of the clock in the control of flowering time will be an area of intense interest in the next few years. Indeed, genetic studies have begun to iden- tify loci that control common genetic components of circadian regulation, phototransduction, and flowering. These include early-flowering3 (em), which is affected in flowering time, photoperception, and circadian rhythms (Hicks et al., 1996), and the phyfochrome signaling early flowering (pef) genes (Ahmad and Cashmore, 1996).

The cellular nature of the circadian clock in plants also needs to be explored further. Which cell types support circadian rhythmicity? Can clocks act cell autonomously in plants, and if so, how are the clocks coupled and synchro- nized at the tissue level? A combination of single-cell imaging techniques and available genetic backgrounds will be the key tools to answering these questions and will lead ulti- mately to a better understanding of the regulatory networks underlying vegetative development in plants.

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DOI 10.1105/tpc.9.7.1235 1997;9;1235-1244Plant Cell

J. A. Kreps and S. A. KayCoordination of Plant Metabolism and Development by the Circadian Clock.

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