Imbibition, but Not Release from Stratification, Sets the Circadian Clock in Arabidopsis Seedlings

Correspondence to: C. Robertson McClung, mcclung{at}dartmouth.edu (E-mail), 603-646-1347 (fax).

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Circadian rhythms in the abundance of the CAT2 catalase mRNA were not seen in etiolated seedlings but developed upon illumination. These circadian oscillations were preceded by a rapid and transient induction of CAT2 mRNA abundance that varied strikingly according to the timing (circadian phase) of the onset of illumination. This variation oscillated with a circadian periodicity of ~28 hr, indicating that the circadian oscillator is running in etiolated seedlings and regulates (gates) the induction of CAT2 by light. Moreover, because we assayed populations of seedlings, we infer that the individual clocks among populations of etiolated seedlings were synchronized before the onset of illumination. What developmental or environmental signals synchronized the clocks among seedlings? Varying the phase of the onset of illumination relative to release from stratification failed to affect the acute induction of CAT2, indicating that the temperature step from 4 to 22°C associated with release from stratification did not reset the circadian clock. However, the acute induction of CAT2 mRNA varied with time after imbibition, demonstrating that imbibition provides a signal capable of resetting the circadian clock and of synchronizing the clocks among populations of seedlings.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

The biological circadian clock generates circadian rhythms with periods of ~24 hr. Plants are richly rhythmic, and the clock provides endogenous timing information that is used to coordinate numerous aspects of plant development and physiology (Sweeney 1987 ; Kreps and Kay 1997 ; Lumsden and Millar 1998 ). The circadian clock regulates the expression of many plant genes that serve as molecular markers of the status of the clock. These circadian rhythms are not unique to plants but are widespread, if not ubiquitous, among eukaryotes and prokaryotes (Edmunds 1988 ; Dunlap 1996 ; Golden et al. 1997 ).

The catalase (CAT) gene family of Arabidopsis provides a particularly useful set of molecular markers for the circadian clock because the individual members of this small family of three genes each respond differently to temporal information supplied by the circadian clock (McClung 1997 ). Although CAT1 mRNA abundance does not exhibit circadian oscillation (Frugoli et al. 1996 ), there are circadian oscillations in the mRNA abundance of both CAT2 and CAT3. It is intriguing that the peaks in mRNA abundance of CAT2 and CAT3 are gated to distinct phases (times of day) by the circadian clock, with CAT2 mRNA being most abundant at dawn and CAT3 mRNA being most abundant at dusk (Zhong et al. 1994 ; Zhong and McClung 1996 ). The physiological significance of the circadian regulation of CAT mRNA abundance is not yet apparent. Catalase (H₂O₂:H₂O₂ oxidoreductase; EC 1.11.1.6) belongs to a group of enzymes involved in regulating the cellular levels of active oxygen species. Catalase is present in all aerobic organisms and protects cells from the damaging effects of H₂O₂ by converting H₂O₂ to O₂ and H₂O. However, it remains to be determined whether the circadian oscillations in CAT2 and CAT3 mRNA abundance confer temporal variation in the resistance of the plant to oxidative stresses; it is not yet known whether the abundance or activity of catalase proteins varies over the circadian cycle.

One hallmark of circadian rhythms is persistence under constant conditions (e.g., continuous light or continuous dark) in which the organism is deprived of environmental time information; this feature constitutes part of the logic by which it is concluded that circadian rhythms are innately generated by an endogenous biological clock (Pittendrigh 1981b ; Edmunds 1988 ; Hastings et al. 1991 ). Although circadian oscillations persist in constant conditions, environmental input can have pronounced effects on the state of the oscillator. Two prominent environmental zeitgebers (environmental time cues or, literally, "time givers"; Aschoff 1960 ) are light and temperature. For example, light–dark cycles or temperature cycles can entrain the oscillator to the environmental cycle. Pulses of light or temperature are effective at shifting the phase of the circadian oscillator (Pittendrigh 1981a ; Edmunds 1988 ; Johnson 1992 ). In addition, light intensity and spectral quality can have pronounced effects on the amplitude and period of the oscillation (Aschoff 1960 ; Millar et al. 1995 ; Somers et al. 1998 ).

Although the clock continues to oscillate in constant conditions, under some conditions the amplitude of the overt rhythm can damp below the threshold of detection. For example, the circadian oscillations in mRNA abundance of light-inducible genes, such as those encoding the chlorophyll a/b binding protein (CAB) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA), and CAT2 (Nagy et al. 1988 ; Pilgrim and McClung 1993 ; Zhong et al. 1997 ), damp to low level expression after several cycles in extended dark. This has been attributed to the loss of Pfr phytochrome, which serves as a positive regulator of gene expression and whose activity is gated by the circadian clock (Kay and Millar 1993 ). Curiously, the circadian oscillations in CAT3 mRNA abundance also damp in extended dark, but they damp to high steady state mRNA abundance in a process that requires signaling through both phytochrome A and cryptochrome1 (Zhong et al. 1997 ).

In this work, we investigated the signals required for the initiation of rhythmicity in CAT mRNA abundance in Arabidopsis seedlings. We show that circadian oscillations of both CAT2 and CAT3 mRNA abundance can be detected in developing seedlings grown in continuous light but are not seen in etiolated (grown in continuous dark) seedlings. However, circadian oscillations become evident after illuminating etiolated seedlings with continuous white light. This circadian accumulation is preceded by an acute, transient induction of CAT2 mRNA abundance. This acute response to illumination prompted us to ask whether the circadian clock is running in etiolated seedlings before the onset of illumination.

In many systems, the circadian clock influences the responsiveness of the organism to environmental stimuli, such as light (Pittendrigh 1981a ; Johnson 1992 ; Millar and Kay 1996 ). We reasoned that if the circadian clock regulated the acute responses of CAT expression to illumination, the kinetics or the amplitude of the acute response would vary according to the circadian phase of the onset of illumination. When etiolated seedlings were illuminated at intervals over three circadian cycles, the acute induction of CAT2 expression varied strikingly according to the timing of the onset of illumination. The magnitude of the acute response in CAT2 mRNA abundance exhibited a circadian oscillation with a period of ~28 hr, which is consistent with the periodicity seen in CAB transcription in extended dark (Nagy et al. 1988 ; Millar et al. 1992a , Millar et al. 1995 ; Millar and Kay 1996 ). Because the circadian clock gates the acute response of CAT2 expression to illumination, we conclude that the circadian oscillator is running in etiolated seedlings. We illuminated and assayed mRNA abundance in populations of seedlings, and therefore, we infer that the clocks of individual etiolated seedlings are synchronized. We further show that imbibition, but not the temperature shift associated with release from stratification, provides a signal that synchronizes the clocks among the populations of etiolated seedlings.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

A Circadian Rhythm Is Detected in CAT2 and in CAT3 Expression in Light-Grown Seedlings but Not in Etiolated Seedlings
We have established that CAT2 and CAT3 mRNA abundance is regulated by the circadian clock in plants released from light–dark cycles into continuous light (Zhong et al. 1994 ; Zhong and McClung 1996 ). We investigated whether circadian oscillations could also be observed in seedlings that have never been exposed to 24-hr light–dark cycles. Seedlings were imbibed, surface-sterilized, plated, and stratified for 3 days at 4°C in the dark and then released into 22°C. RNA gel and slot blot analyses indicate that CAT3 mRNA was abundant and increased over time in seedlings grown in continuous dark (Figure 1A and Figure 1B). When statistical analysis (FFT-NLLS analysis; see Methods) was applied to these data, no circadian oscillations were detected (at a 95% significance level) in CAT3 mRNA abundance in these etiolated seedlings. Similarly, mRNA abundance for two other catalase genes, Arabidopsis CAT2 and maize Cat3, also fails to oscillate in etiolated seedlings (Acevedo et al. 1991 ; Zhong et al. 1994 ; Boldt and Scandalios 1995 ), yet both genes exhibit circadian oscillations in mRNA abundance in seedlings transferred from light–dark cycles into continuous light (Redinbaugh et al. 1990 ; Zhong et al. 1994 ).

View larger version (35K): [in this window] [in a new window]   Figure 1. Circadian Oscillations in CAT3 mRNA Abundance Are Not Detected in Etiolated Seedlings. (A) Analysis of steady state abundance of CAT3 and ARP mRNA in etiolated Arabidopsis seedlings grown in continuous dark at 22°C after release from stratification (72 hr at 4°C) and harvested at 4-hr intervals from 72 to 144 hr. RNA gel blots containing 5 µg of total RNA per lane were hybridized with CAT3, stripped, and rehybridized with ARP, a ribosomal protein gene (Kim et al. 1990 ) that does not exhibit circadian oscillations in transcript abundance and hence serves as a loading control. (B) Quantification of slot blot analysis of steady state abundance of CAT3 and ARP mRNA in etiolated Arabidopsis seedlings grown and harvested as described in (A). Blots were hybridized with CAT3, stripped, and rehybridized with ARP. Data presented represent mean values derived from two independent experiments (similar results were obtained in each experiment). The most abundant signal is defined as 1.0 (arbitrary units), and other abundances are expressed relative to that value.

In contrast, circadian oscillations in mRNA abundance were detected for both CAT2 and CAT3 when Arabidopsis seedlings were germinated and grown in continuous light for 7 days after the release from stratification. In the first 2 days after release from stratification, CAT2 mRNA accumulated to high levels (Figure 2A) and exhibited no circadian periodicity. However, by the third day, oscillations with period lengths in the circadian range were detected in CAT2 mRNA abundance. Circadian oscillations in CAT3 mRNA abundance were detectable by the second day after release from stratification (Figure 2B). Therefore, we conclude that neither light–dark cycles nor transitions from light into dark or from dark into light are necessary to induce the circadian rhythms in CAT2 and CAT3 mRNA abundance in Arabidopsis. The period lengths in seedlings grown in continuous light were more variable than we have observed in plants that have been entrained to light–dark cycles before release into continuous light (e.g., Zhong et al. 1994 ; Zhong and McClung 1996 ). This suggests a role for light–dark cycles or transitions from light to dark in the establishment of a precise 24-hr-period length rather than as prerequisites for the establishment of rhythmicity.

View larger version (35K): [in this window] [in a new window]   Figure 2. Circadian Oscillations in CAT2 and CAT3 mRNA Abundance Are Detected in Arabidopsis Seedlings Germinated and Grown in Continuous Light. (A) Seedlings were stratified for 72 hr in the dark at 4°C and then transferred to continuous light at 22°C. Seedlings were harvested at 3-hr intervals from the onset of illumination. Total RNA was hybridized with CAT2, stripped, and rehybridized with rDNA. CAT2 mRNA abundance was normalized to rRNA, which did not fluctuate with circadian period, and served as a loading control. The most abundant signal is defined as 1.0 (arbitrary units), and other abundances are expressed relative to that value. Data are presented as mean values derived from two independent experiments (similar results were obtained in each experiment). (B) The blots described in (A) were stripped, rehybridized with CAT3, and quantified as described in (A). Thus, comparison of absolute abundances between CAT2 and CAT3 is not valid.

When etiolated seedlings were illuminated, robust circadian oscillations, with peaks 24, 44, and 68 hr after the onset of illumination, became apparent in CAT2 mRNA abundance (Figure 3A; Zhong et al. 1994 ). Similar results were obtained with CAT3 mRNA abundance; robust circadian oscillations with peaks 32 and 56 hr after the onset of illumination were detected after illumination of etiolated seedlings (Figure 3B). Several conclusions may be drawn from these results. First, the circadian clock regulates CAT2 and CAT3 mRNA abundance in light-grown seedlings. Second, the periodicity of the circadian oscillation is innate and does not require exposure to an environmental cycle with a 24-hr period or indeed to even a single transition from light to dark or dark to light to confer a 24-hr periodicity upon the oscillations in CAT2 and CAT3 mRNA abundance. Third, light itself, or some signal associated with the dark-to-light transition, is necessary for the detection of circadian oscillations in both CAT2 and CAT3 mRNA abundance, which were not seen in etiolated seedlings.

View larger version (35K): [in this window] [in a new window]   Figure 3. Illumination of Etiolated Seedlings Allows Detection of Circadian Oscillations in CAT2 and CAT3 mRNA Regardless of the Timing (Circadian Phase) of the Onset of Illumination. (A) and (B) Seedlings were imbibed, plated, and stratified for 72 hr at 4°C in the dark, transferred to continuous darkness, and grown for exactly 8 days (192 hr) at 22°C. Seedlings were then transferred to continuous white light. After illumination, seedlings were harvested at 0, 1, and 4 hr and at successive 4-hr intervals. CAT2 (cyan lines in [A]) and CAT3 (magenta lines in [B]) mRNA abundances were determined as described in Figure 2, except that the least abundant signal was defined as 1.0 (arbitrary units), and other abundances are expressed relative to that minimum. (C) to (L) At 4-hr intervals after the illumination of the seedlings in (A) and (B), scanning through one circadian cycle, sets of etiolated seedlings were transferred to continuous white light. CAT2 ([C], [E], [G], [I], and [K]) and CAT3 ([D], [F], [H], [J], and [L]) mRNA abundances were determined as described in (A) and (B). To facilitate the comparison of the effects of illumination at successive 4-hr intervals, the data plotted in (A) and (B) for each gene (CAT2, [C], [E], [G], [I], and [K]; CAT3, [D], [F], [H], [J], and [L]) are replotted as the black line in each of the lower graphs. The light regime is indicated by the bars at the base of the graphs, where a black bar indicates dark and a white bar indicates light. Total RNA was prepared from seedlings harvested at the indicated times and hybridized with CAT2, stripped, and rehybridized first with CAT3 and then stripped and rehybridized with rDNA. CAT2 (left) and CAT3 (right) mRNA abundances were normalized to rRNA abundance. Each experiment was repeated four times with similar results; data are presented as mean values derived from four independent replicates.

The Circadian Clock Is Running in Etiolated Seedlings and Gates the Acute Response of CAT2 Expression to Illumination
In addition to the relatively long-term circadian effects of illumination, expression of both CAT2 and CAT3 exhibited acute (detectable within 1 to 4 hr) responses to illumination (Figure 3). In response to illumination, CAT3 mRNA initially declined in abundance and subsequently returned to a level of abundance similar to that seen before illumination (Figure 3B). In contrast, CAT2 mRNA rapidly accumulated to a transient peak 4 hr after light onset (Figure 3A).

It is well established that the sensitivity of the clock to phase shifting stimuli, such as light, varies over the circadian cycle (Pittendrigh 1981a ; Johnson 1992 ). Therefore, if the clock were running in etiolated seedlings, it is possible that the magnitude or the kinetics of the acute response in CAT2 or CAT3 expression to illumination might vary according to the circadian phase at which illumination begins. To test this possibility, after 8 days (192 hr) of etiolated growth, we transferred sets of seedlings into continuous light at 4-hr intervals, spanning a circadian cycle (Figure 3). In each case, circadian rhythmicity was detected after illumination. The circadian oscillations in CAT2 and CAT3 mRNA were out of phase with one another: CAT2 mRNA was maximal in the early subjective morning (~24, 44 to 48, and 68 to 72 hr after the onset of illumination), and CAT3 mRNA was maximal in the subjective evening (~12 to 16, 32 to 36, and 52 to 56 hr after the onset of illumination). This is consistent with our observations in mature plants (Zhong et al. 1994 ; Zhong and McClung 1996 ). Regardless of the timing of the onset of illumination, the phases of the ensuing circadian peaks in CAT2 and CAT3 mRNA abundance were consistent with respect to the onset of illumination. This suggests that transfer into bright continuous white light serves as a strong zeitgeber, in each case resetting the clock to dawn (type 0 or strong resetting; Winfree 1980 ). The first peak of CAT3 mRNA after illumination was somewhat delayed relative to the second and third peaks and to the timing of the peak in plants growing in a light–dark cycle. This probably represents a transient, because it often takes several cycles for the rhythm to adopt a new stable phase after a phase-shifting stimulus (Pittendrigh 1981a ).

The amplitude of the acute response in CAT2 mRNA abundance showed a dramatic dependence on the time of onset of illumination (Figure 3). To facilitate comparison among the treatments, we replotted the data of Figure 3A in black in each of the other CAT2 graphs (Figure 3C, Figure 3E, Figure 3G, Figure 3I, and Figure 3K) and the data of Figure 3B in black in each of the other CAT3 graphs (Figure 3D, Figure 3F, Figure 3H, Figure 3J, and Figure 3L). The acute induction of CAT2 mRNA was strongest when illumination occurred at or near subjective dawn (Figure 3A, Figure 3C, Figure 3E, and Figure 3K) and was not seen when illumination began in subjective evening (Figure 3G and Figure 3I). One interpretation of these data is that the circadian clock regulates the responsiveness of CAT2 expression to illumination to be maximal in the late subjective night/early subjective morning. However, it is also possible that we were observing a developmentally regulated loss of the ability to generate an acute response in the older seedlings. To distinguish between these possibilities, we repeated the experiment to assess the acute induction of CAT2 mRNA abundance on the day preceding and the day after the collection of data shown in Figure 3. Figure 4 clearly shows that the acute response of CAT2 mRNA abundance varies periodically over 3 days, with the maximal acute responses occurring at subjective dawn. To facilitate comparison among the treatments, we replotted the data of Figure 4F in black in Figure 4A to E and Figure 4G to P.

View larger version (45K): [in this window] [in a new window]   Figure 4. The Acute Induction of CAT2 Expression by Light Varies with Time from Release from Stratification. (A) Seedlings were imbibed, plated, and stratified for 72 hr at 4°C in the dark. All seedlings were then transferred to continuous darkness and grown at 22°C. Seedlings were then transferred to continuous white light in timed series at 4-hr intervals, starting after 172 hr of etiolated growth (i.e., 244 hr after imbibition). After illumination, seedlings were harvested at 0, 1, and 4 hr and at successive 4-hr intervals. CAT2 mRNA abundances were determined as described in Figure 2 and are presented as the cyan line. To facilitate comparison among treatments, the data from (F) are replotted as the black line. (B) to (P) At successive 4-hr intervals after the illumination of the seedlings in (A), scanning through three circadian cycles, sets of etiolated seedlings were transferred to continuous white light. CAT2 mRNA abundance (cyan lines) was determined as described in (A). Data are presented as mean values (±SE) derived from three independent replicates (four independent replicates for [F], [G], [H], [I], and [K]). To facilitate comparison among treatments, the data from (F) are replotted as the black line in each of the panels. The light regime is indicated by the bars at the base of the graphs, where a black bar indicates dark and a white bar indicates light.

To assess the periodicity of the variation in the acute response of CAT2 mRNA abundance to illumination, we expressed the magnitude of the acute response as the ratio of the mRNA abundance 4 hr after illumination to the mRNA abundance 12 hr after illumination. The magnitude of the acute response varied approximately three- to fourfold and exhibited a periodicity of ~28 hr (Figure 5). This is somewhat longer than the 24-hr period seen in the oscillations in mRNA abundance for CAT2 (Figure 3 and Figure 4; Zhong et al. 1994 , Zhong et al. 1997 ; Zhong and McClung 1996 ). However, it is well established that the period of CAB transcription lengthens in extended dark to ~30 hr (Nagy et al. 1988 ; Millar et al. 1992a , Millar et al. 1995 ; Millar and Kay 1996 ), which is consistent with our determination of an ~28-hr period.

View larger version (19K): [in this window] [in a new window]   Figure 5. The Circadian Clock Gates the Acute Induction of CAT2 Expression by Light. The magnitude of the acute response of CAT2 mRNA abundance to illumination, defined as the ratio of CAT2 mRNA abundance 4 hr after the onset of illumination to CAT2 mRNA abundance 12 hr after the onset of illumination, varies with the time after imbibition and with the time after the release from stratification, which follows imbibition by 72 hr. This measure of the acute response oscillates with a circadian period of ~28 hr. Vertical lines indicate 24-hr intervals after the release from stratification. Data are derived from the values presented in Figure 4.

The data presented in Figure 4 and Figure 5 provide strong evidence that the circadian clock is running in etiolated seedlings and that the circadian clock gates the acute response of CAT2 mRNA abundance to the illumination of etiolated seedlings. Furthermore, our analysis entailed the isolation of mRNA from populations of seedlings. That these populations showed a coherent acute response to illumination indicates that the clocks of the individuals within the populations of etiolated seedlings are synchronized with one another. Moreover, if there are distinct clocks running in the different organs or within the individual cells of a seedling, these clocks must also be synchronized. The acute response of CAT3 mRNA abundance did not vary dramatically with time of onset of illumination; possibly the stability of CAT3 mRNA makes the acute effects of the clock on CAT3 mRNA abundance less obvious, or perhaps the acute effects of light on CAT3 mRNA abundance are not clock regulated.

Imbibition, but Not Release from Stratification, Synchronizes the Circadian Clocks among Seedlings
What signal or signals serve to initiate or to synchronize the clocks among the populations of etiolated seedlings? Our experimental protocol provides two obvious candidates: imbibition and the temperature shift from 4 to 22°C associated with release from stratification.

Temperature steps are known to provide effective phase-shifting stimuli in many systems (e.g., Kloppstech et al. 1991 ; Beator et al. 1992 ; Heintzen et al. 1994a , Heintzen et al. 1994b ). Therefore, we first tested whether release from stratification served as the synchronizing signal. Seedlings were imbibed and plated as described in the previous experiments. Half of the seedlings were stratified at 4°C for 72 hr, released into the dark at 22°C for 196 hr, and then illuminated with white light (Figure 6A and Figure 6B). The rest of the seedlings were released from stratification 12 hr after the first set and thus were stratified for 84 hr. This second set of seedlings was grown for 184 hr and then illuminated with white light (Figure 6A and Figure 6C). Thus, both sets of seedlings were illuminated at the same time, at 268 hr after imbibition, but the two sets of seedlings were illuminated at different phases relative to release from stratification.

View larger version (26K): [in this window] [in a new window]   Figure 6. Release from Stratification Does Not Synchronize the Clocks among Seedlings. (A) Diagram illustrating the experimental protocol for (B) at top and (C) in the middle. Hatched boxes indicate stratification in the dark at 4°C. Filled boxes indicate etiolated growth in the dark at 22°C. Open boxes indicate illuminated growth at 22°C. The open box at bottom indicates the harvesting regime after the onset of illumination. Seedlings were imbibed and plated as described in the previous experiments. (B) and (C) Half of the seedlings, shown in (B), were stratified at 4°C for 72 hr, released into the dark at 22°C for 196 hr, and then illuminated with white light (~125 µmol m-2 sec-1). The rest of the seedlings, shown in (C), were released from stratification 12 hr after the set in (B) and thus were stratified for 84 hr. This second set of seedlings was grown in the dark at 22°C for 184 hr and then illuminated with white light at the same time as the seedlings in (B). Thus, both sets of seedlings were illuminated 268 hr after imbibition, but the seedlings in (B) were illuminated 196 hr after release from stratification, and the seedlings in (C) were illuminated 184 hr after release from stratification. CAT2 mRNA was quantified and normalized to rRNA, as described in the legend to Figure 2, except that the least abundant signal was defined as 1 (arbitrary units), and other values are expressed relative to that value. The light regime is indicated by the bars at the base of the graphs, where a black bar indicates dark and a white bar indicates light.

If release from stratification synchronized the clocks among seedlings, one would predict that the amplitudes of the acute responses of the two sets of seedlings would differ. If, however, release from stratification did not synchronize the clocks among seedlings, one would predict that the amplitude of the acute induction of CAT2 mRNA would be the same in the two sets of seedlings, because each was illuminated at the same time after imbibition (268 hr). The amplitude of the acute induction of CAT2 mRNA was the same in the two sets of seedlings (Figure 6), indicating that release from stratification does not synchronize the circadian clocks among individuals. We have repeated this type of experiment three times, and in no case have we seen any indication that release from stratification affects the phase of the circadian oscillator. The results shown in Figure 6 as well as those of other experiments, however, are consistent with the hypothesis that imbibition synchronizes the circadian clocks among individual seedlings.

To more precisely test the hypothesis that imbibition synchronizes the clocks among seedlings, we sought to vary the time at which seedlings were imbibed but to release from stratification and to illuminate sets of seedlings at the same time, such that only the time after imbibition varied among the populations of seedlings. Therefore, sets of seeds were imbibed at successive 4-hr intervals (Figure 7A). After imbibition, all seedlings were stratified at 4°C in the dark. All seedlings were released from stratification at the same time, which was 72 hr after the final set of seeds was imbibed. Thus, the first set of seeds, which had been imbibed 20 hr before the last set of seeds, was stratified 20 hr longer (92 hr). All seedlings were illuminated at the same time, which was 196 hr after release from stratification. If imbibition synchronized the clocks among seedlings, one would predict that the amplitude of the acute induction of CAT2 mRNA would vary among the sets of seedlings, because the sets of seedlings had been imbibed at successive 4-hr intervals, at 268 to 288 hr before the onset of illumination.

View larger version (18K): [in this window] [in a new window]   Figure 7. Imbibition Synchronizes the Clocks among Seedlings. (A) Diagram illustrating the experimental protocol for (B) to (G). Hatched boxes indicate stratification in the dark at 4°C. Filled boxes indicate etiolated growth in the dark at 22°C. Open boxes indicate illuminated growth at 22°C. The open box at bottom indicates the harvesting regime after the onset of illumination. (B) to (G) Seedlings were imbibed and plated as described in the previous experiments. To more precisely test the hypothesis that imbibition synchronizes the clocks among seedlings, we sought to vary the time at which seedlings were imbibed and to illuminate the sets of seedlings at the same time, such that only the time after imbibition varied among the populations of seedlings. Sets of seeds were imbibed at successive 4-hr intervals. After imbibition, all seedlings were stratified at 4°C in the dark. All seedlings were released from stratification at the same time, which was 72 hr after the final set of seeds was imbibed. Thus, the first set of seeds, which was imbibed 20 hr before the last set of seeds, was stratified 20 hr longer (92 hr). All seedlings were illuminated at the same time at 196 hr (i.e., 8 days and 4 hr) after release from stratification. Because the sets of seedlings had been imbibed at 4-hr intervals, the sets of seedlings therefore were illuminated at 4-hr intervals 268 to 288 hr after imbibition. CAT2 mRNA was quantified and normalized to rRNA, as described in the legend to Figure 6. The light regime is indicated by the bars at the base of the graphs in (B) to (G), where a black bar indicates dark and a white bar indicates light.

The amplitude of the acute induction of CAT2 mRNA varied according to the time after imbibition (Figure 7B to G) and was strongest when illumination began at or near subjective dawn (Figure 7B and Figure 7C) and weakest when illumination began at or near subjective dusk (Figure 7F). The acute response of CAT2 mRNA abundance, defined by the ratio of abundance 4 hr after illumination/abundance 12 hr after illumination (see Figure 5), was calculated from the data presented in Figure 7B to G and is presented in Figure 8. Collectively, Figure 7 and Figure 8 show that the amplitude of the acute induction of CAT2 mRNA varied according to the time after imbibition, indicating that imbibition synchronizes the circadian clocks among individuals. This result also confirms the conclusions illustrated in Figure 6: the release from stratification does not synchronize (reset) the clocks. If release from stratification synchronized the clocks among seedlings, one would predict that each set of seedlings would exhibit the same acute response, because each set of seedlings was released from stratification at the same time and was illuminated at the same time, 196 hr after release from stratification. This is clearly inconsistent with the data presented in Figure 7 and Figure 8.

View larger version (17K): [in this window] [in a new window]   Figure 8. Imbibition Synchronizes the Clock. The acute response of CAT2 mRNA abundance, defined by the ratio of abundance 4 hr after illumination/abundance 12 hr after illumination (see the legend to Figure 5), was calculated from the data presented in Figure 7. The amplitude of the acute induction of CAT2 mRNA varied according to the time after imbibition, indicating that imbibition synchronizes the circadian clocks among individuals.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

We have asked how soon after imbibition can circadian clock activity be demonstrated in Arabidopsis and what developmental or environmental signals provide temporal information to the clock in young seedlings. Circadian rhythms in CAT2 and CAT3 mRNA abundance were not observed in etiolated seedlings, although both mRNAs were readily detected, yet rhythms became apparent upon illumination. Moreover, circadian oscillations in CAT2 and CAT3 mRNA abundance were also detected in seedlings germinated and grown in continuous light, which is consistent with CAB mRNA abundance in wheat grown in continuous light (Nagy et al. 1988 ). However, growth in continuous light is not always sufficient for detection of circadian rhythmicity. When mustard (Sinapis alba) plants are germinated and grown in continuous light, no circadian oscillations have been observed in mRNA abundance for a gene (Saglp) encoding a germin-like protein (Heintzen et al. 1994a ) or for genes (Sagrp1 and Saglp2) encoding glycine-rich putative RNA binding proteins (Heintzen et al. 1994b ), yet these genes show circadian regulation when transferred into continuous conditions from a diurnal light–dark cycle. Similar observations have been made for a light- and clock-regulated rice transcript of unknown function (Reimann and Dudler 1993 ). The clock-regulated maize Cat3 transcript does not exhibit circadian oscillations in seedlings grown either in continuous dark or continuous light (Acevedo et al. 1991 ; Boldt and Scandalios 1995 ).

Several hypotheses can be proposed to explain the detection of circadian oscillations in CAT2 and CAT3 mRNA abundance after illumination of etiolated seedlings. The transition from dark to light, or perhaps the presence of light itself, might be required to initiate clock function. Early studies suggested that plants maintained throughout their lifetimes in constant conditions fail to exhibit circadian oscillations, although a single stimulus (for example, a light or a dark pulse or a light-to-dark or dark-to-light transition) is sufficient to induce oscillations (Bunning 1931 ). It could be that the clock is functioning in etiolated seedlings but that an environmental signal is required to synchronize the clocks of individual plants in a population of seedlings. For example, in Drosophila, dark-reared flies are individually rhythmic in locomotor activity (Sehgal et al. 1992 ), although the proportion of rhythmic flies is reduced in comparison with light- or light–dark-reared flies (Dowse and Ringo 1989 ; Power et al. 1995a , Power et al. 1995b ). However, the progeny of dark-reared Drosophila fail to exhibit a circadian rhythm in eclosion because the flies, although individually rhythmic, are not synchronized to one another. A single light pulse synchronizes the clocks of the individual flies and allows detection of a rhythm in eclosion in a population of flies (Sehgal et al. 1992 ).

One also might speculate that an environmental signal could also serve to synchronize individual oscillators in separate cells, tissues, or organs that are running out of phase with one another. However, multiple oscillators have yet to be demonstrated experimentally in Arabidopsis (for further discussion, see Millar 1998 ). Alternatively, it could be that the clock is running in etiolated seedlings but is uncoupled from CAT2 and CAT3 expression. Light might be required for the functioning of an output pathway to provide temporal information to CAT2 and CAT3. It is also possible that the clock is running and coupled to CAT2 and CAT3 expression, but the effects of the clock are somehow obscured in extended dark.

This is an attractive hypothesis given our recent demonstration that oscillations in CAT3 mRNA abundance are obscured in extended dark by a mechanism that requires synergistic signaling through both phytochrome A and cryptochrome1 (Zhong et al. 1997 ). Moreover, this hypothesis is consistent with the suggestion that the circadian clock imposes periodic negative regulation on otherwise constitutive expression of the maize Cat3 gene (Acevedo et al. 1991 ).

If the circadian clock were functioning in etiolated seedlings, we reasoned that the responsiveness of the clock to light might vary according to the circadian phase at the onset of illumination. For example, the clock gates the acute induction by light of Arabidopsis CAB2, and the rhythm in inducibility is in phase with the circadian rhythm in mRNA abundance (Millar and Kay 1996 ). Similarly, the rate of greening in barley in response to illumination oscillates in phase with the rhythm in Cab mRNA abundance (Beator and Kloppstech 1993 ). Hence, if the clock were regulating the response of CAT expression to light, the amplitude or the kinetics of the acute induction or repression would vary according to the timing of the onset of illumination of etiolated seedlings. Indeed, we observed that the acute response of CAT2 expression varied strikingly according to the timing of the onset of illumination. These data demonstrate that a circadian clock is running in etiolated seedlings and that the circadian clock regulates the light responsiveness of CAT2 expression. Because our assay entailed the isolation of RNA from populations of seedlings, we can further infer that the clocks of the individual seedlings comprising these populations were synchronized before illumination.

In our experiments, we induced acute responses in CAT2 and CAT3 mRNA abundance with shifts from darkness into continuous light. However, as little as a single light pulse is sufficient to induce an acute response followed by circadian oscillations of cab mRNA abundance in etiolated bean, tobacco, and wheat (Paulsen and Bogorad 1988 ; Tavladoraki et al. 1989 ; Wehmeyer et al. 1990 ). Moreover, neither light nor dark-to-light transitions are essential for detection of circadian oscillations in CAB mRNA. Circadian oscillations have been detected in CAB mRNA abundance in etiolated wheat (Nagy et al. 1993 ) and tobacco (Kolar et al. 1995 ), and in the transcription of the Arabidopsis CAB2 gene, monitored either in etiolated transgenic tobacco seedlings (Millar et al. 1992b ; Anderson et al. 1994 ) or in Arabidopsis (Millar and Kay 1996 ).

Why, therefore, were we unable to detect oscillations in CAT mRNA abundance in etiolated seedlings? Either CAT expression is uncoupled from the clock output pathway or perhaps CAT mRNA oscillations are obscured in etiolated seedlings. We do not favor the hypothesis that the output pathways from the clock are not connected to the CAT genes, because we have established that the circadian clock gates the light responsiveness of CAT2 in etiolated seedlings. We prefer the hypothesis that clock-independent signal transduction pathways induce high levels of CAT mRNA, which obscure underlying CAT mRNA oscillations, similar to the damping in CAT3 mRNA oscillations seen in extended dark (Zhong et al. 1997 ). Consistent with this interpretation, CAB2 expression in etiolated plants is low, allowing a sensitive assay, such as light production in transgenic seedlings carrying CAB2::luciferase transcriptional fusions, to detect low amplitude oscillations in basal transcription (Millar et al. 1992a ; Anderson et al. 1994 ). Unlike CAB2 mRNA, CAT2 and CAT3 transcripts were readily detectable in etiolated seedlings, and it may be that this abundance prevented the detection of circadian oscillations in transcription. Circadian oscillations were not detected in CAT2 mRNA abundance during the first 2 days of growth in continuous light when a transient accumulation of CAT2 transcript is observed, whereas circadian oscillations were detectable in CAT3 mRNA abundance at this time. We think that this accumulation of CAT2 mRNA represents a short-term developmental response associated with germination that is consistent with a role of CAT2 protein in detoxifying H₂O₂ generated during ß-oxidation of the fatty acids stored in the seed. The regulatory signals associated with this response may block circadian regulation or may simply obscure the effects of circadian control.

It is clear that the clocks among individual etiolated Arabidopsis seedlings are synchronized because we see, in assays of populations of etiolated seedlings, circadian gating of light inducibility of CAT2 mRNA. What serves to synchronize the clocks among individual seedlings? Our experimental protocol provides two potential synchronizing signals. Our seeds were imbibed and then stratified at 4°C before growth in the dark at 22°C. We reasoned that either imbibition itself or the temperature shift from 4 to 22°C might provide the synchronizing signal. We systematically varied the time from either imbibition or release from stratification until the onset of illumination and found that the acute response was unaffected by the interval between release from stratification and illumination but varied according to the interval from imbibition to illumination. This variation in the magnitude of the acute response as a function of time from imbibition was consistent among independent experiments (e.g., cf. Figure 5 with Figure 8). Accordingly, we conclude that imbibition, but not the temperature shift associated with release from stratification, serves as the signal to synchronize the clocks among populations of etiolated seedlings. Because the phase of the ensuing rhythm in acute induction of CAT2 is established by imbibition, it follows that the clock must run at 4°C during stratification. Martino-Catt and Ort 1992 argued that low temperature stopped the circadian clock oscillation in tomato, a chilling-sensitive species. Arabidopsis is chilling tolerant (Thomashow 1994 ); apparently this chilling tolerance extends to the Arabidopsis circadian clock.

Temperature (cold) pulses are effective zeitgebers that shift the phase of the Arabidopsis clock (Kreps and Simon 1997 ), so why is the temperature shift from 4 to 22°C ineffective in resetting the clock in our experiments? We suspect that the Arabidopsis clock may initially be refractory to the phase-shifting effects of a temperature step and acquires competence to respond to the temperature step only at a later developmental stage. Similarly, it has been shown that extremely young tobacco seedlings are refractory to light pulses that shift the clock in older seedlings (Kolar et al. 1998 ).

We speculate that some physiological consequence of imbibition provides a cue to initiate or to synchronize the circadian oscillators in the population of seedlings germinated and grown in continuous illumination and hence not exposed to a dark-to-light transition. Experimental support for the time of sowing setting the phase of a circadian rhythm in CAB mRNA abundance has been obtained in tobacco (Kolar et al. 1995 , Kolar et al. 1998 ). It is also possible that the embryonic clock is set during seed development. Maternal entrainment is common in mammals (Moore-Ede et al. 1982 ). It is worth noting that a circadian rhythm has been detected in gas exchange in dry onion seeds in continuous dark (Bryant 1972 ), although we have been unable to reproduce this observation in Arabidopsis (E.V. Kearns and C.R. McClung, unpublished observations). Perhaps a very sensitive measure of gene expression, such as light production in transgenic plants expressing luciferase fusions, would allow one to detect circadian rhythms in gene expression during seed development. All three CAT mRNAs can be detected in siliques, although none is abundant (Frugoli et al. 1996 ). We are currently generating transgenic lines expressing CAT:: luciferase fusions, which we intend to examine for evidence of rhythmic CAT transcription during seed development.

The circadian clock regulates CAT2 and CAT3 mRNA abundance to distinct circadian phases both in Arabidopsis seedlings and in mature plants; CAT2 is morning specific, and CAT3 is evening specific. Furthermore, the mRNA abundance of both CAT2 and CAT3 exhibit opposite patterns of acute response to illumination; CAT2 mRNA abundance responds positively to illumination, but CAT3 mRNA abundance responds negatively to illumination. Thus, at least some components of the output pathways from the clock and from photoreceptors that control expression of CAT2 and CAT3 must differ, making further comparison of the clock- and light-regulatory properties of these two catalase genes worthwhile.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plant Materials and Growth Conditions
Seeds of Arabidopsis thaliana ecotype Columbia were soaked in water for 30 min. They were then surface-sterilized by soaking in 95% ethanol for 10 min and then in 10% bleach and 0.1% Triton X-100 for 10 min. After five washes with sterile distilled water, the seeds were spread onto plates containing Murashige and Skoog mineral salts (Murashige and Skoog 1962 ), 10 g/L agar, and 2% sucrose. After stratification at 4°C in the dark for 3 days, seeds were incubated at 22°C. White light (~125 µmol m^-2 sec^-1) was provided by a combination of white fluorescent and incandescent lights.

RNA Isolation and RNA Gel and Slot Blot Analyses
Tissue was homogenized with a handheld homogenizer (OMNI International, Waterbury, CT), and total RNA was isolated by phenol–SDS extraction and LiCl precipitation, as described previously (Zhong et al. 1994 ). RNA gel and slot blot analyses were as previously described (Zhong et al. 1994 ). Catalase CAT2 and CAT3 hybridization probes were as described previously (Zhong et al. 1994 ; Zhong and McClung 1996 ). CAT2 and CAT3 do not cross-hybridize under the high-stringency conditions employed (Frugoli et al. 1996 ). rRNA (Richards et al. 1991 ) and an Arabidopsis ribosomal protein (ARP) gene (Kim et al. 1990 ) hybridization probes served as loading controls. RNA abundance was quantified using a PhosphorImager with ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Statistical Analysis
Time-series data were quantitatively evaluated by an iterative, coupled fast Fourier transform–nonlinear least squares (FFT-NLLS) multicomponent cosine estimation algorithm (Johnson and Frasier 1985 ; Straume et al. 1991 ; Plautz et al. 1997 ). FFT-NLLS provides estimates of periods, phases, and amplitudes of rhythmic components, along with associated estimates of joint parameter confidence limits, as well as an index of determination regarding statistical significance of rhythmic amplitudes. The maximum number of statistically significant (i.e., with statistically significant [non-zero] amplitudes) cosine components supportable by the data is identified. FFT-NLLS accommodates noisy, short/sparse time series with occasional missing points and temporal nonstationarities (drifting measurements and nonconstant oscillatory amplitudes) while reliably detecting underlying rhythms. See Zhong et al. 1997 for a more detailed description.

	FOOTNOTES

¹ Current address: Howard Hughes Medical Institute Research Laboratories, Yale University School of Medicine, Section of Immuno-biology, 310 Cedar Street, New Haven, CT 06510.

	ACKNOWLEDGMENTS

We thank Mary Lou Guerinot and all members of our laboratories for criticism of the manuscript and for many helpful discussions. We also thank Paul Alloway, Dorota Balaban, Judy Meadows, and Jessicah Phillips for technical assistance. This work was supported by a Dartmouth College Cramer Fellowship awarded to H.H.Z., by grants from the National Science Foundation (No. NSF MCB-9316662) and from the United States Department of Agriculture (No. 9602632) to C.R.M., and by an institutional grant (No. CA23108) from the American Cancer Society to the Norris Cotton Cancer Center at Dartmouth. M.S. acknowledges support from the National Science Foundation Center for Biological Timing (No. NSF DIR-8920162) and the National Institutes of Health (No. R01 DK-51562).

Received August 4, 1998; accepted October 9, 1998.

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