The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis thaliana circadian clock to sucrose

Abstract

Circadian clocks are 24-h timing devices that phase cellular responses; coordinate growth, physiology, and metabolism; and anticipate the day–night cycle. Here we report sensitivity of the Arabidopsis thaliana circadian oscillator to sucrose, providing evidence that plant metabolism can regulate circadian function. We found that the Arabidopsis circadian system is particularly sensitive to sucrose in the dark. These data suggest that there is a feedback between the molecular components that comprise the circadian oscillator and plant metabolism, with the circadian clock both regulating and being regulated by metabolism. We used also simulations within a three-loop mathematical model of the Arabidopsis circadian oscillator to identify components of the circadian clock sensitive to sucrose. The mathematical studies identified GIGANTEA (GI) as being associated with sucrose sensing. Experimental validation of this prediction demonstrated that GI is required for the full response of the circadian clock to sucrose. We demonstrate that GI acts as part of the sucrose-signaling network and propose this role permits metabolic input into circadian timing in Arabidopsis.

The Arabidopsis thaliana circadian clock confers growth and competitive advantage (1). The phase of circadian rhythms in plants is adjusted by light signals to entrain the clock to dawn and dusk (2). Additionally, it has been proposed that the Arabidopsis circadian clock is sensitive to nitrogen acting as a nutritional cue (3) and phytohormones, possibly as an input from stress signaling and growth pathways (4). In Arabidopsis, circadian oscillations are generated and maintained by interlocking transcriptional–translational feedback loops and posttranslational regulation (1, 5–8). In the morning, light activates the expression of two Myb-like transcription factors, CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), leading to expression of PSEUDO RESPONSE REGULATORS (PRR) 7 and 9, which in turn repress CCA1 and LHY expression. This “morning” loop of the oscillator is connected to “evening” expressed genes through direct repression of the expression of TIMING OF CAB EXPRESSION 1 (TOC1/PRR1) by binding of LHY/CCA1 to the TOC1 promoter. TOC1 is expressed in the evening and feeds back to activate LHY/CCA1 through an unknown pathway. TOC1 physically interacts with and antagonizes CCA1 HIKING EXPEDITION (CHE), a transcriptional repressor of CCA1 (9). CCA1 and LHY are also coupled with TOC1 through GIGANTEA (GI). LHY/CCA1 repress GI and GI forms a loop with TOC1, GI activating TOC1 and TOC1 repressing GI. GI encodes a protein of unknown biochemical function but in blue light, GI physically interacts with and stabilizes ZEITLUPE (10), an F-box protein that targets TOC1 for degradation (11). To entrain the circadian oscillator, signals are incorporated from light, second messengers, and metabolites to alter circadian clock gene expression and modulate circadian function (7, 12–15).

Carbohydrates are a major energy store in most forms of life and in plants are synthesized as a result of photosynthesis. Sugars, such as sucrose, are also important signaling molecules regulating growth, development, gene expression, and metabolism. Supplying high exogenous concentrations of sucrose (e.g., 300 mM, 10% wt/vol) severely inhibits early development of Arabidopsis seedlings, resulting in small white or purple cotyledons (16). Lower concentrations of sucrose (30–90 mM) do not inhibit shoot development although can interfere with a range of signal transduction pathways. Exogenous sucrose abolishes circadian oscillations in the concentration of cytosolic-free Ca²⁺ ([Ca²⁺]_cyt) (17) and differentially perturbs daily oscillations of gene expression in roots compared with aerial tissue (18). Exogenous sucrose also increases expression of core central oscillator genes CCA1, GI, and TOC1 and reduces period in constant light (LL), although these behaviors are absent in sensitive to freezing 6 (sfr6) mutants (19). A large number of mutants have been identified that are insensitive to sucrose, although it is not clear how the circadian oscillator perceives sugar signals.

Results

To investigate the effect of sucrose on the Arabidopsis circadian clock network, we compared rhythmic behaviors in the presence or absence of 90 mM (3% wt/vol) exogenous sucrose. Most studies of circadian behavior in Arabidopsis have been performed using seedlings grown in agar media containing 90 mM exogenous sucrose, and the mathematical models describing the Arabidopsis circadian clock are based on experimental data accordingly (20–24). Experiments from our laboratory and others investigating circadian rhythms of [Ca²⁺]_cyt demonstrate that the Arabidopsis circadian clock also functions in the absence of exogenous sucrose (7, 17). We first compared rhythms of CCA1::luc, TOC1::luc, and GI::luc in LL with or without 90 mM sucrose added to the growth media (Fig. 1 A and C). In LL, sucrose caused a small but statistically significantly period increase in TOC1::luc rhythms (period estimates: 90 mM sucrose, 23.83 h; 90 mM mannitol, 23.36 h, P = 0.0023; H₂O, 23.23 h, P = 0.0043) and period decrease in GI::luc rhythms (period estimates: 90 mM sucrose, 24.75 h; 90 mM mannitol, 25.78 h, P = 0.0099; dH₂O, 25.63 h, P = 0.0041; Fig. 1C). In constant dark (DD), CCA1::luc, TOC1::luc, and GI::luc were rhythmic in the presence of 90 mM sucrose (period estimates: CCA1::luc, 23.76 h; TOC1::luc, 26.50 h; GI::luc, 27.39 h) but the rhythms were either very low amplitude or undetectable in the absence of exogenous sucrose (Fig. 1 B and D). Without exogenous sucrose, CCA1::luc luminescence oscillated in 12 h light, 12 h dark (LD) cycles, increased 12 h after transfer to DD in anticipation of subjective dawn, and then decreased to a constant low level (Fig. 1B). TOC1::luc and GI::luc luminescence also oscillated in LD before oscillating with decreasing amplitude in DD in the absence of exogenous sucrose (Fig. 1B). The luc reporter was active in DD in the absence of exogenous sucrose because we were able to detect low temperature induction of CCA1::luc after 60 h in DD, and the presence or absence of 90 mM sucrose did not increase the degree of induction compared with H₂O controls (Fig. S1).

Fig. 1.

Exogenous supply of sucrose influences period and amplitude of circadian oscillations in clock gene expression. (A and B) CCA1::luc (circles), TOC1::luc (triangles) and GI::luc (squares) luminescence measured for seedlings entrained to LD cycles and transferred to (A) LL at t = 0 h or (B) DD at t = 0 h. Data were normalized by dividing each point by the mean over the whole trace. Bars on the abscissa are open for light, solid for darkness, and hatched for (A) subjective night or (B) subjective day. (C and D) Circadian period estimates of CCA1::luc, TOC1::luc, and GI::luc in (C) LL and (D) DD. Relative amplitude error quantifies the rhythmic robustness (0 is sine wave, 1 indicates no oscillation). Agar media supplements: solid is H₂O control, open is 90 mM mannitol control, and shaded is 90 mM sucrose.

Our data demonstrate that the amplitude of circadian oscillations in DD was increased by the continual supply of exogenous sucrose in the growth media. To understand sucrose sensitivity in a more physiological setting, we were interested to test whether the circadian clock also responds to step changes in sucrose concentration, such as might occur following a dark to light transition as plants start photosynthesizing and consequently produce sucrose. A total of 90 mM sucrose was applied to entrained seedlings that had previously been exposed to prolonged darkness (60–72 h). Before sucrose treatment, oscillations of CCA1::luc, TOC1::luc and CHLOROPHYLL A/B-BINDING PROTEIN 2 (CAB2)::luc were barely detectable and oscillations of GI::luc were low amplitude (Fig. 2). Sucrose treatment led to a large induction of all circadian markers, resulting in renewed circadian oscillations (Fig. 2). Therefore, sucrose has both a long-term effect on circadian rhythms, potentially incorporating metabolic status, and a short term effect that may be related to intercellular signaling, as has been speculated to exist between roots and aerial tissue (18).

Fig. 2.

Sucrose entrains circadian oscillations of CCA1, TOC1, GI, and CAB2 promoter activity in DD. Seedlings were grown in LD12/12 cycles before transfer to constant dark (DD). After (A and B) 60 h (CT0) or (C) 72 h (CT12) in DD, 90 mM sucrose (open circles) or 90 mM mannitol (solid circles) was applied to aerial tissue. Luminescence of (A) CCA1::luc, TOC1::luc, and GI::luc or (B and C) CAB2::luc was measured for 4–12 clusters of two to three seedlings. Bars on the abscissa are solid for the subjective night and hatched for the subjective day.

To determine whether sucrose was amplifying low amplitude oscillations, such as observed in GI::luc, or was resetting the phase of the oscillator, sucrose treatment was administered at different times through the circadian cycle. When sucrose treatment coincided with subjective dawn (CT0), CAB2::luc transiently increased before continuing with a large amplitude circadian rhythm that peaked 26.75 h after sucrose addition (Fig. 2B). When sucrose treatment coincided instead with subjective dusk (CT12), an initial transient oscillation of CAB2::luc luminescence was not observed (Fig. 2C). However, similar to the CT0 treatment, transient dynamics were followed by a circadian rhythm that peaked 25.25 h after sucrose addition (Fig. 2C), qualitatively similar to the gated response of CAB2 induction to transient pulses of red light (25). Therefore, although the timing of the sucrose treatment led to a differential transient response of CAB2::luc expression, the resulting circadian oscillation peaked ~24 h after sucrose addition irrespective of the subjective time of application, suggesting that sucrose, like light, has the potential to act as a zeitgeber (“time giver”), a signal that is capable of reestablishing rhythms and setting the phase of the circadian oscillator.

To investigate these experimental findings in a quantitative framework, we considered mathematical models of the Arabidopsis circadian clock. Currently published models simulate the behavior of the circadian oscillator in seedlings grown with exogenous sucrose in the growth media (20–24). A three-loop model of the central oscillator (22), from now on referred to as the nominal model, yields stable oscillations with a period of 23.81 h in LL and a period of 23.75 h in DD, reflecting the wealth of LL and DD data available for seedlings grown with exogenous sucrose (note the most recent Arabidopsis clock model, presented in ref. 24, was not published at the time of this research). To identify potential targets for the long-term sucrose effect on the circadian system we exploited the differences in system behavior observed in DD. In the absence of sucrose the system tended toward a steady state, whereas in the presence of sucrose there were stable oscillations (Fig. 1 B and D), which are also predicted by the nominal model (22).

We investigated whether changes in the kinetic parameters of the nominal model could account for the dynamical differences of central oscillator gene expression in the absence of exogenous sucrose. The kinetic parameters describe rates of transcription, translation, light activation, degradation, and nuclear transport (22) (Table S1). The model parameters were first varied singly, implicitly assuming that sucrose targets only a single mechanism. Over a wide range of values, the oscillation period and peak/trough fold ratio were computed for simulated [LHY/CCA1 mRNA] (Fig. S2A). Twenty-six of the 71 model parameters had regions of values that led to a loss of rhythms (peak/trough fold ratio <1.1) in DD, while retaining oscillations in LL with a period between 20 and 30 h (Fig. S2B), reminiscent of the behavior of the circadian oscillator in the absence of exogenous sucrose. Simulations using the 26 parameter alterations were examined in more detail to identify those parameter alterations that could also describe the dynamics seen in DD in the absence of sucrose, including anticipation of subjective dawn in the first cycle after transfer to DD and the fast tendency to a low steady state of CCA1 expression (Fig. 3A). Only a reduction in n₅ (maximum light-independent rate of Y transcription; reduction to 25% of the nominal value) could account for both the anticipation of subjective dawn after 12 h of darkness and the fast tendency to a low steady state of CCA1 expression (Fig. 3A, S2C). In this reduced-n₅ model (from now on referred to as the N5 model), simulated [TOC1 mRNA] was also constantly low after transfer to DD, agreeing with measured TOC1::luc in the absence of exogenous sucrose (Fig. 3A). There were stable oscillations of both [LHY/CCA1 mRNA] and [TOC1 mRNA] in LL, with an early phase compared with the nominal model (Fig. S3A). The relative phase advance in the N5 model resulted in a poorer fit of simulated [LHY/CCA1 mRNA] and [TOC1 mRNA] to the corresponding experimental data (Fig. S3A). Although significant quantitatively, this difference is very minor qualitatively compared with the requirement that parameter alterations should abolish rhythms in DD. Also, if sucrose targets more than one process, there may be compensatory effects that maintain the relative timing of gene expression.

Fig. 3.

Mathematical modeling suggests the hypothetical component Y mediates sucrose modulation of the clock. (A and C) Normalized CCA1::luc (solid circles, Upper) and TOC1::luc (solid triangles, Lower) luminescence in LD and DD compared against equivalent simulated [LHY/CCA1 mRNA] and [TOC1 mRNA] (solid lines) from variants of the three-loop model (20). (A) N5 is the three-loop model with the maximum rate of light-independent Y transcription (n₅) reduced to 0.25 of its nominal value. (B) Average alterations to each parameter of the three-loop model after parameter reoptimization to account for wild-type luminescence data from seedlings without exogenous sucrose (SI Materials and Methods). A simulated annealing algorithm was run 400 times, producing 400 parameter sets (annealed solutions). On the graph, the bars indicate the logarithm of the average of the top 100 annealed parameter sets. Largest increase (n₄) and largest decrease (n₅) are indicated on the graph. (C) PostGI is the three-loop model with increased maximum degradation of cytoplasmic and nuclear Y (possibly GI) protein in the absence of light to five times the nominal rate. Bars on the abscissa are open for light, solid for darkness, and hatched for dark in a subjective day.

In a contrasting analysis, all model parameters were allowed to vary. Enabling multiple-parameter changes had the advantage of considering potential interactions in model parameters that are sucrose dependent. Modified parameter sets were generated by optimizing a cost function that penalizes deviations in simulated [mRNA] for LHY/CCA1 and TOC1 to CCA1::luc and TOC1::luc luminescence data, respectively, in both LL and DD after transfer from LD cycles. Approximate solutions to the optimization problem were obtained using a modified Metropolis–Hastings simulated annealing algorithm, which was run independently 400 times for 1,000 iterations each (SI Materials and Methods). For each trial run, the lowest cost parameter set (annealed solution) was retained for further analysis (for example models, see Fig. S4 and Table S2). To establish which parameters should be modified to account for sucrose absence, the mean of each parameter was computed over the best 100 annealed solutions and analyzed for their deviation from the nominal parameter value (Fig. 3B, Fig. S5, Table S2, and Table S3). The strategy of averaging over the trial optimal parameter sets was chosen for two reasons. First, many parameter sets returned cost function values that were close to the best observed set; the distribution was bimodal, so we retained only the better half of the low cost mode (Fig. S5A). Second, some parameter values were not constrained by the experimental data, as their distributions across low cost parameter sets were broad (Fig. S5B). Therefore, taking the average of this distribution distinguishes between parameters that are required to be different from their nominal value and parameters that are poorly constrained by the data and on average will not deviate from the nominal value.

The nominal model considers Y transcription to be constitutive with maximum rate n₄ + n₅ nM/h during light and rate n₅ nM/h during darkness (n₄ is the light-dependent rate of Y transcription, and n₅ is the light-independent rate) and inhibited by TOC1 and LHY/CCA1 (22). The parameter that was decreased the most on average to account for the removal of sucrose was n₅ (down 39%), and the parameter that on average was increased the most was n₄ (up 93%). As n₅ > n₄, this equated to an increase of n₄ + n₅ (total rate of Y transcription in light) of only 6% to account for the absence of sucrose. Therefore, the mathematical analysis suggested that Y transcription is less light dependent when sucrose is supplied exogenously and more light dependent in the absence of exogenous sucrose. The concentration of intracellular sucrose ([Suc]_i) is high during the day and low at night in the absence of exogenous sucrose (26). Also, supplying sucrose exogenously increases [Suc]_i (27). Therefore, our mathematical modeling predicts the supply of exogenous sucrose will increase the rate of Y transcription in the dark (when endogenous sucrose is low), but will have little effect on Y transcription in the light (when endogenous sucrose is high). Consequently, the rate of Y transcription varies coincident to the endogenous intracellular concentration of sucrose.

Considering parameter alterations to the nominal model of the Arabidopsis circadian oscillator led to the hypothesis that sucrose up-regulates Y transcription. At least some of the biological function described by the hypothetical component Y is fulfilled by GIGANTEA (GI) (22). Therefore, we wanted to consider in more detail how perturbations to the expression of GI could be reconciled by corresponding simulations of perturbations to Y. In particular, we wanted to investigate whether light/dark-dependent posttranslational modification of Y could instead be a target mechanism of sucrose, as the protein turnover of GI is light dependent (28) and our hypothesis that sucrose up-regulates Y depends upon the light/dark dependency of Y expression. This is important because the nominal model does not have specific terms to describe the regulation of Y protein degradation. To address this alternative hypothesis, the nominal model equations governing Y protein dynamics were modified to include a dark-dependent degradation term (from now on, the PostGI model; SI Materials and Methods). As the rate of dark-dependent degradation was increased (without changing the existing parameters), circadian oscillations of simulated [LHY/CCA1 mRNA] and [TOC1 mRNA] were suppressed in DD (Fig. 3C), similar to the luc luminescence data without exogenous sucrose. A single oscillation in simulated [LHY/CCA1 mRNA] peaking 24 h after transfer from LD cycles was retained, as observed for CCA1::luc luminescence (Figs. 1B and 3C), and stable oscillations were retained in LL (Fig. S3B). This suggested a hypothesis whereby GI degradation is high when [Suc]_i is low and invariant to light availability when [Suc]_i is buffered with exogenous sucrose.

Our mathematical modeling suggested two competing hypotheses for the long-term sucrose regulation of Arabidopsis circadian rhythms. On the basis of a parameter perturbation to the nominal model it was hypothesized that sucrose increases Y transcription (N5 model). Alternatively, incorporation of light/dark-dependent posttranslational modification terms into the nominal model suggested that Y protein degradation is invariant when [Suc]_i is buffered by exogenous sucrose supply (PostGI model). To distinguish between the behaviors of the N5 and PostGI model hypotheses, we simulated topical sucrose treatment after a period of prolonged darkness. Both models were simulated in entrained LD cycles before transfer to DD for 60 or 72 h. Sucrose treatment was then simulated by switching to the nominal (sucrose supplied) model (22). In the N5 model simulated sucrose treatment caused a large induction of both [TOC1 mRNA] and [Y mRNA] that preceded reestablished oscillations in all model components (Fig. S6A). In contrast, simulated sucrose treatment in the PostGI model led to an initial decrease in [Y mRNA], followed by a reestablishment of circadian oscillations (Fig. S6B). Sucrose addition in DD caused an immediate increase in GI::luc (Fig. 2) as predicted for Y/GI by the N5 model and the initial decrease in Y/GI predicted by the PostGI model was not observed. These data experimentally validate the N5 model on the basis of the assumption that the behavior of the hypothetical component Y describes the behavior of GI and that GI fulfills at least some of the functions of Y (22). However, the assumption that GI fulfills the function of Y has been questioned (29). To address this question and determine the functional significance for the circadian system of our findings, we further examined the model hypotheses experimentally by testing the effects of sucrose on GI promoter activity and the response of gi mutants to sucrose.

We experimentally tested the hypothesis that GI acts as part of a long term sucrose-sensing pathway for the circadian clock by comparing circadian behavior in gi-11 null mutants (30) grown in the presence and absence of exogenous sucrose. Luminescence of CAB2::luc was measured in DD after transfer from LD cycles. As expected from earlier experiments measuring CCA1::luc (Fig. 1B), wild-type plants had circadian oscillations of CAB2::luc expression in DD when 90 mM sucrose was supplied in the media and rhythms were undetectable in media supplied with water or mannitol as a control (Fig. 4A). However, in gi-11 mutants addition of sucrose to the growth media did not permit oscillations of CAB2::luc in DD (Fig. 4A). Similarly, CCR2 expression was arrhythmic in the gi-3 point mutant in the presence of sucrose in DD (31). These observations demonstrate that functional GI is required for the circadian system to integrate long-term sucrose signals and implicate GI in sucrose sensing in DD. As we had observed a GI–sucrose interaction in DD, we were interested to see whether this interaction was also observable in LL. We compared two seed lines of gi-11 expressing CAB2::luc with corresponding wild-type WS and found statistically significant long circadian periods in both mutant lines (−Suc: WS, 23.50 h; gi-11/1, 26.37 h, P = 0.006; gi-11/2, 25.35 h, P = 0.002; +Suc: WS, 23.91 h; gi-11/1, 26.21 h, P = 4.5 × 10⁻⁵; gi-11/2, 24.88 h, P = 0.03l; Fig. 4 B and C). However, sucrose had no effect on the period of CAB2::luc rhythms in the wild-type and both gi-11 mutant lines (Fig. 4C). Therefore, we conclude that the putative long term GI–sucrose interaction is not active in LL.

Fig. 4.

GI is required for long-term but not short-term modulation by sucrose. (A) Normalized CAB2::luc luminescence in LD and DD for wild-type WS (Upper) and gi-11 (Lower) seedlings, grown on agar media containing 90 mM sucrose (open circles) or 90 mM mannitol (solid circles). (B) CAB2::luc luminescence in LD and LL comparing wild-type WS (solid symbols) and two progreny of a single gi-11 transformant (gi-11/1 and gi-11/2; open symbols), grown on agar media with (Upper) or without (Lower) 90 mM sucrose. (C) Circadian period estimates of CAB2::luc in LL for wild-type (filled symbols) and gi-11 (open symbols) seedlings, grown on agar media containing 90 mM sucrose (triangles) or 90 mM mannitol (circles). Relative amplitude error quantifies the rhythmic robustness (0 is sine wave, 1 indicates no oscillation). (D) Wild-type (Upper) or gi-11 mutant seedlings were grown in entrained LD12/12 cycles before transfer to constant dark (DD). After 60 h (CT0), 90 mM sucrose (open circles) or 90 mM mannitol (solid circles) was applied to aerial tissue. Bars on the abscissa are open for light, solid for darkness, and hatched for dark in the subjective day.

Finally, to ascertain whether GI is involved in the short-term response of the circadian system to sucrose, we investigated the response of wild-type and gi-11 plants expressing CAB2::luc to a step change in exogenous sucrose in DD (Fig. 4D). In both wild type and gi-11 the response to topical addition of sucrose were almost identical, indicating GI is not involved in short-term sucrose signaling and that there is a functional difference between the experimentally observed short- and long-term responses of the circadian network to sucrose.

Discussion

We have demonstrated that the primary metabolite sucrose modulates the functioning of the Arabidopsis circadian oscillator, the effects of which are most pronounced when endogenous sucrose is low, in DD. Continual supply of exogenous sucrose contributes to sustained oscillations in DD (Fig. 1), indicating the oscillator integrates long-term metabolic signals. The advantage of incorporating long-term metabolic status could be related to why circadian clocks enhance growth (1, 32). Topical sucrose supply reestablished rhythms in plants grown without exogenous sucrose (Fig. 2), indicating the oscillator also integrates short-term metabolic signals.

The suggested regulation of plant clock function by metabolic status is reminiscent of the situation in mammals where it is proposed that circadian oscillations of NAD levels feed back to regulate circadian function and provide input concerning the metabolic status of the cell (33). NAD has profound effects on the circadian systems of plants and animals. Treatment with nicotinamide, which is a product and inhibitor of NAD consumption, increases the period of both the plant and the mammalian circadian clock (7, 34). The effects of NAD on circadian systems have been proposed to be mediated by deacetylases, poly-ADP ribose, and cyclic ADP ribose and it is likely that all contribute to modulate circadian function in both kingdoms (7, 33, 35, 36).

Short-term sucrose signals might confer intercellular communication; for example, the coupling of oscillators in the shoots and roots of plants is perturbed by addition of sucrose (18). Another possibility is that sucrose feedback into the oscillator provides a further example of circadian loop formation, because the circadian clock regulates photosynthetic capacity (1) and we demonstrate that sucrose affects clock function (Fig. 1). It has been proposed that forming circadian clocks of multiple feedback loops increases robustness (37). Our data show that sucrose treatment in LL has a relatively small effect on the circadian oscillator (Fig. 1A), which is consistent with a previous study (19). In LL, the small effects of sucrose included a statistically significant, but slight decrease in the period of GI:luc rhythms and also a small, but significant increase in the period of TOC1::luc (Fig. 1). This result could be interpreted as evidence for uncoupled cell-specific oscillators (18) responding differently to the sucrose signal. Alternatively, these apparently opposite responses to sucrose in LL might be a consequence of measuring small responses close to the limit of resolution of the data capture and the statistical analysis packages and so might not be of physiological consequence. We found the effect of sucrose was much greater in DD. It is possible that this effect represents an example of circadian gating of input sensitivity, similar to that seen with the circadian gating of light input (38). However, gating is more often associated with short-term signals, and we have identified a mechanism that perceives long-term signals. Therefore, we favor the interpretation that the circadian system is insensitive to sucrose in the light because the effects of the exogenous sucrose are buffered by the high intracellular sucrose produced as a result of photosynthesis. The buffering of the effects of exogenous sucrose by intracellular sucrose in the light might explain why corresponding observations have been elusive until now and why relatively high concentrations of sugars have been required to investigate sugar signaling in plants.

Significant mechanistic understanding of the interactions between central oscillator components has been realized in the past two decades. Whereas cross-talk with second messengers such as cyclic ADP ribose (7) and metabolites such as sucrose (19) has been demonstrated, mechanistic understandings of the links between clocks and signaling remain few. We exploited the qualitative differences between plants grown in the presence and absence of exogenous sucrose to explore with mathematical modeling how the long-term sucrose signals are integrated into the clock. Mathematical modeling allowed us to identify the hypothetical component Y as being sensitive to sucrose perturbations. Our simulations and modeling predictions suggested that sucrose increases the rate of Y transcription and consequently we hypothesized that Y is required for the response of the oscillator to sucrose. In the nominal model, it is thought that at least 70% of the function of Y can be ascribed to GI (22). In a more recent model of the Arabidopsis central oscillator (24), the functions of GI have been separated from the component Y to reflect additional knowledge, although both Y and GI are modeled as being transcribed in response to the same transcription factors (TOC1 and LHY/CCA1), consistent with the previous model (22). In contrast, however, GI is transcribed only during light (24). Therefore, a reduction in the rate of Y but not GI transcription in the new model (24) might cause suppressed rhythms, as observed in the absence of exogenous sucrose (Fig. 1B).

On the basis of our analysis using the three-loop model, we hypothesized that if GI does fulfill the role of Y, then sucrose sensing by the circadian oscillator should be compromised in gi nulls. This proved to be the case for long-term responses to sucrose (Fig. 4), demonstrating GI is required for metabolic sensing by the plant circadian clock as suggested by overaccumulation of starch in gi nulls (39). However, transcriptional responses of the oscillator to sucrose were unaffected by gi-11 mutations (Fig. 4), which was not consistent with our predictions based on the N5 model. The difference in response of the gi-11 nulls to long-term and transient changes in sucrose is consistent with there being a functional difference between the biochemical networks that perceive the fast and slow changes in intracellular sucrose imposed in this study. The difference in the response of the gi-11 nulls to transient changes in sucrose compared with that predicted by the N5 model might also reflect that GI does not fulfill all of the functions of the hypothetical model component Y (24, 29). By recognizing GI as a necessary regulator and demonstrating assays that admit significant sucrose sensitivity, it should now be possible to characterize more completely the link(s) between metabolism and circadian rhythms in plants.

Materials and Methods

Imaging of Luciferase Activity for Sucrose-Supplemented Growth Media Assay.

Circadian rhythms of luciferase bioluminescence were measured using a Photek IFS32 photon counting camera as described previously (40). Circadian period estimates and relative amplitude errors (RAE) were obtained using fast Fourier transform (nonlinear least-squares method) conducted with the Biological Rhythms Analysis Software System (BRASS) (41).

Imaging of Luciferase Activity for Topical Sucrose Treatment.

Circadian rhythms of luciferase luminescence were measured before and after treatment of 90 mM sucrose (Fisher Scientific) or 90 mM mannitol (BDH Laboratory Supplies). Arabidopsis WS seeds were surface sterilized, stratified at 4 °C for 2 d in darkness, and germinated on half-strength Murashige and Skoog medium (Duchefa Biochemie), 0.8% (wt/vol) agar, and no sucrose. Sixteen clusters of 3–5 seedlings were grown in 12 h light/12 h dark (LD12/12) for 10 d at constant 19 °C and then treated with 100 μL of 1 mM luciferin and placed in darkness for 60 h or 72 h. Photon counting was conducted as above for 48 h before sucrose/mannitol treatment. A total of 100 μL of 90 mM sucrose/mannitol was then applied to seedling clusters in low intensity green light to prevent light entrainment (12 clusters of sucrose treatment, 4 clusters with mannitol treatment). Photon counting was then resumed immediately and up to 48 h following.