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Circadian and Diurnal Calcium Oscillations Encode
Photoperiodic Information in Arabidopsis
John Love,1,2 Antony N. Dodd,1 and Alex A.R. Webb3
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom
We have tested the hypothesis that circadian oscillations in the concentration of cytosolic free calcium ([Ca21]cyt) can
encode information. We imaged oscillations of [Ca21]cyt in the cotyledons and leaves of Arabidopsis (Arabidopsis thaliana)
that have a 24-h period in light/dark cycles and also constant light. The amplitude, phase, and shape of the oscillations of
[Ca21]cyt and [Ca21]cyt at critical daily time points were controlled by the light/dark regimes in which the plants were grown.
These data provide evidence that 24-h oscillations in [Ca21]cyt encode information concerning daylength and light intensity,
which are two major regulators of plant growth and development.
INTRODUCTION
Calcium is a ubiquitous second messenger involved in the
transduction of many environmental and developmental stimuli
in plants and animals (Trewavas, 1999; Sanders et al., 2002;
Schuster et al., 2002). In response to diverse stimuli, cells generate transient increases in the concentration of cytosolic free
calcium ([Ca21]cyt) that vary in amplitude, frequency, duration,
cellular location, and timing (McAinsh et al., 1995; McAinsh and
Hetherington, 1998; Trewavas, 1999; Berridge et al., 2000; Evans
et al., 2001; Allen et al., 2001). Important information regarding
the nature of the stimulus may therefore be encoded in the
different spatiotemporal profiles of [Ca21]cyt increases (McAinsh
and Hetherington, 1998; Sanders et al., 2002; Schuster et al.,
2002). The majority of stimuli tested to date result in single, rapid
[Ca21]cyt spikes (also called bursts) or in complex [Ca21]cyt
oscillations recurring with a period of 1 to 20 min (Sanders et al.,
2002; Schuster et al., 2002). However, longer-term [Ca21]cyt
oscillations, characterized by a period of 24 h, occur in the
cytosol and chloroplast of Nicotiana plumbaginifolia, in the
cytosol of Arabidopsis (Arabidopsis thaliana; Johnson et al.,
1995), and in the cytosol of neurons of the mouse suprachiasmatic nucleus, which is the primary circadian pacemaker
in mammals (Ikeda et al., 2003). These circadian rhythms
of [Ca21]cyt are believed to be involved in signaling to or from
the endogenous circadian clock (Gómez and Simón, 1995;
Trewavas, 1999; Webb, 2003), although their precise role
remains unclear (Sai and Johnson, 1999). One possibility is that
1 These
authors contributed equally to this work.
address: School of Biological Sciences, The University of
Exeter, Washington Signer Laboratories, Perry Road, Exeter EX4
4QG, UK.
3 To whom correspondence should be addressed. E-mail alex.webb@
plantsci.cam.ac.uk; fax 44 (0)1223 333953.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Alex A.R. Webb
([email protected]).
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.020214.
2 Current
circadian oscillations of [Ca21]cyt encode information in a manner analogous to the short-period oscillations of [Ca21]cyt
induced by extracellular signals. Known regulators of circadian
behavior include light duration and intensity. Thus, circadian
oscillations of [Ca21]cyt might have the potential to encode
temporal information about the daily duration and intensity of
light, which are two major regulators of plant growth and
development.
Eukaryotic circadian clocks have similar basic structures;
input pathways integrate environmental cues such as the daily
(24-h) cycle of alternating light and dark intervals and temperature to entrain a core molecular oscillator that, in turn, regulates
multiple output pathways and transduces temporal information
(Harmer et al., 2001). In plants, the circadian clock controls
fundamental aspects of plant physiology and development,
including gene expression (Harmer et al., 2000) and the movements of stomata (Webb, 1998, 2003) and leaves (Gómez and
Simón, 1995). The circadian clock is also the internal chronometer by which photoperiodic or daylength sensitive responses,
such as seasonal growth, the induction and breaking of bud
dormancy, vegetative reproduction, and floral development, can
occur (Yanovsky and Kay, 2002; Hayama and Coupland, 2003).
Despite the wealth of knowledge concerning the input pathways
that entrain the circadian clock (Somers et al., 1998; Devlin,
2002; Fankhauser and Staiger, 2002), the molecular nature of the
core circadian oscillator (Alabadi et al., 2001; Somers 2001; Hall
et al., 2002; Hayama and Coupland, 2003) and the circadian and
photoperiodic responses that are regulated by the oscillator
(Lumsden, 1998; Hayama and Coupland, 2003; Webb 2003),
little is known about the signaling pathways by which the
circadian oscillator regulates cellular events (Morre et al., 2002;
Webb 2003). We hypothesized that circadian Ca21 oscillations
act as second messengers in circadian signaling, in a similar
manner to the more rapid [Ca21]cyt oscillations induced by extracellular stimuli (McAinsh et al., 1995; McAinsh and Hetherington,
1998; Trewavas, 1999; Berridge et al., 2000; Allen et al., 2001;
Evans et al., 2001). To test this hypothesis, we have examined
information encoding in 24-h oscillations of [Ca21]cyt to determine whether these could act as outputs in the circadian
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Figure 1. Imaging Circadian Oscillations of [Ca21]cyt in Leaves and Cotyledons of Arabidopsis.
Circadian Calcium Signaling
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signaling pathway carrying temporal and other information. We
show that circadian (measurements performed in constant light
[LL]) and diurnal (measurements performed in light/dark [LD]
cycles) oscillations in [Ca21]cyt in the cotyledons and leaves of
Arabidopsis have the potential to encode essential information
about the LD cycle in which plants are grown.
in the plant and any cells not responding will result in the mean
peak whole-plant [Ca21]cyt being lower than the peak of [Ca21]cyt
attained in the cells.
RESULTS
We hypothesized that if circadian [Ca21]cyt oscillations act in the
circadian signaling network, then these oscillations should
encode information that regulates circadian behavior, such as
light intensity and daylength. First, we examined whether circadian oscillations in [Ca21]cyt were modulated by light intensity.
Seedlings were entrained for 11 d in a 12-h-light/12-h-dark (12L/
12D) regime with a photon flux density (PFD) of 60 mmol m2 s1.
Seedlings were transferred to LL with a PFD of 60 mmol m2 s1
or 110 mmol m2 s1, and aequorin luminescence imaged. The
amplitude of the circadian rhythm of aequorin luminescence for
seedlings exposed to a LL PFD of 60 mmol m2 s1 was consistently lower than for seedlings exposed to 110 mmol m2 s1
(Figure 2). The peak height of the circadian oscillations of
mean whole-plant [Ca21]cyt was 50 nmol L1 higher in
110 mmol m2 s1 than in 60 mmol m2 s1. However, other
characteristics of the circadian [Ca21]cyt rhythms were not
altered by PFD. The period of the circadian oscillations was not
significantly different in the two PFD. The periods were 21.93 6
1.85 h (n ¼ 16) in 60 mmol m2 s1 LL and 23.8 6 2.25 h (n ¼ 14) in
110 mmol m2 s1 LL. In addition, there was no difference in the
phase of the oscillations in the two light intensities. These
data suggest that the amplitude of circadian [Ca21]cyt oscillations
was sensitive to the light intensity of LL and may encode information about the PFD.
Circadian Oscillations of [Ca21]cyt in the Leaves of
Arabidopsis
Aequorin bioluminescence was measured in vivo, using photoncounting imaging (Figure 1). Seedlings in LL had circadian
oscillations in aequorin luminescence that increased during
the subjective day to a peak value and decreased during the
subjective night to a minimum. The circadian alteration in the
bioluminescent signal was localized to the cotyledons and
the emerging leaves (Figure 1). The changes in aequorin
bioluminescence reflected alterations in [Ca21]cyt because
oscillations of luminescence were not detected when we imaged
untransformed plants treated with coelenterazine or transformed
plants without coelenterazine. The oscillations were not a consequence of alterations in the abundance of cytosolic aequorin
because the total aequorin pool measured using antibodies
(Johnson et al., 1995) and photon counting of the luminescence
of the entire pool following discharge in excess Ca21 (data not
shown) did not change in a circadian manner. No differences in
growth rate, flowering time, and morphology were detected
between wild-type and transgenic plants, demonstrating that
transgene insertion and expression had no effect on circadian
behavior (data not shown). The period of the [Ca21]cyt oscillations
was 24 h and predicted both subjective dawn and subjective
dusk, which are characteristics of circadian rhythms (Millar and
Kay, 1996).
The mean whole-plant [Ca21]cyt typically oscillated between
100 nmol L1 at the trough and 300 nmol L1 at the peak.
However, this may be an underestimate of the peak of [Ca21]cyt
occurring in individual cells in response to circadian signals
because (1) the [Ca21]cyt of different populations of cells oscillate
with different circadian phases (Wood et al., 2001), and (2)
individual cells in a population can have different circadian
phases (see Webb, 2003). The multiple phases of individual cells
The Amplitude of Circadian Oscillations of [Ca21]cyt Is
Modulated by Photon Flux Density
The Phase of Circadian Oscillations of [Ca21]cyt Is
Modulated by the Photoperiod of Entrainment
We tested whether circadian [Ca21]cyt oscillations were modulated by changes in daylength (photoperiod). Seedlings were
entrained in 6L/18D, 8L/16D, 12L/12D, 16L/8D, and 20L/4D.
Seedlings entrained in 6L/18D, 8L/16D, 12L/12D, and 16L/8D
had free-running rhythms in aequorin luminescence (Figure 3)
with an approximate period of 24 h (Figure 4B). By contrast,
seedlings entrained in 20L/4D had no rhythmic component to the
bioluminescence signal (Figures 4A and 4B). The phase of the
Figure 1. (continued).
(A) Bright-field (BF) and pseudocolored photon-counting image of aequorin luminescence (AL; [C]) of Arabidopsis seedlings expressing aequorin.
(B) Aequorin luminescence of Arabidopsis seedlings in circadian free-run (LL) at a PFD of 110 mmol m2 s1. Seedlings were entrained to a 12L/12D
photoperiod at 60 mmol m2 s1 PFD for 11 d before LL. Closed black circles represent the mean luminescence of 12 seedling clusters with standard
error bars. Closed red circles represent the luminescence from a single seedling cluster. The numbers beside the points during the second circadian
cycle indicate the number of hours into LL that each photon-counting image was acquired and correspond to the images presented in (C). The
theoretical best-fit curve for FFT at 95% confidence probability is shown in blue. The bar above the abscissa indicates the subjective light regime during
LL. Open areas (in the bar) represent subjective day, and hatched areas represent subjective night. The closed black area represents the final dark
period before LL.
(C) Pseudocolored photon-counting images of the luminescence emitted by a single cluster of Arabidopsis seedlings expressing aequorin. Numbers
indicate the time in LL when each image was recorded. Cold colors (blue and green) represent regions of low luminescence counts, corresponding to
low [Ca21]cyt. Warm colors (yellow and orange) represent regions of more intense luminescence, indicating higher [Ca21]cyt. The closed red circles in (B)
represent the integrated luminescence emitted by this seedling cluster at the indicated times.
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Figure 2. Light Intensity Modulates the Amplitude of Circadian
Oscillations of [Ca21]cyt.
Points represent the mean luminescence from seedling clusters, with
standard error bars shown. Seedlings were entrained to a 12L/12D
photoperiod at 60 mmol m2 s1 for 11 d before transfer to LL at a PFD of
110 mmol m2 s1 (closed circles; n ¼ 10) or 60 mmol m2 s1 (closed
triangles; n ¼ 8). Open areas indicate subjective day, and shaded areas
indicate subjective night. The closed area represents the final dark period
before LL.
circadian [Ca21]cyt oscillations was dependent on the entrainment photoperiod, with peak [Ca21]cyt occurring later per 24-h
cycle in plants entrained in long photoperiods than in plants
entrained in short photoperiods (Figure 5A). The luminescence of
seedlings entrained in 6L/18D peaked at a mean of 4.09 6 0.19 h
(n ¼ 24) after subjective dawn for circadian cycle 2 (24 to 48 h
after transfer to LL). Seedlings entrained in 8L/16D had peak
luminescence at 5.39 6 0.22 h (n ¼ 36) after subjective dawn,
seedlings entrained in 12L/12D had peak luminescence at 6.59 6
0.37 h (n ¼ 44) after subjective dawn, and seedlings entrained in
16L/8D were maximally luminescent 8.16 6 0.90 h (n ¼ 24) after
subjective dawn, for circadian cycle 2. Thus, the longer the
photoperiod during entrainment, the later the peak luminescence
after subjective dawn in LL. This trend was repeated in the
subsequent cycles of circadian free-run (e.g., cycle 3, from 48 to
72 h, and cycle 4, from 72 to 96 h after transfer to LL). The timing
of minimum luminescence in LL was unaffected by the entrainment daylength and occurred 19 to 20 h after subjective dawn
in seedlings from all entrainment regimes (Figure 5C). Consequently, the circadian [Ca21]cyt oscillations were asymmetric,
and the asymmetry depended on the entrainment photoperiod
because the longer the photoperiod, the shorter the time difference between maximum and minimum [Ca21]cyt. Therefore,
the daylength during entrainment controlled both the phase and
the shape (symmetry) of the [Ca21]cyt oscillations during circadian free-run in LL.
The photoperiod-dependent phase shift of the circadian
[Ca21]cyt rhythms had significant consequences for [Ca21]cyt at
subjective dusk. At subjective dusk, relative luminescence (as
a percentage of the maximum luminescence for each circadian
cycle) was dependent on the duration of the entrainment
photoperiod. The relative luminescence at subjective dusk of
seedlings entrained to 6L/18D was 72.59 6 10.92% (n ¼ 24), that
of seedlings entrained in 8L/16D was 59.82 6 11.95% (n ¼ 36),
that of seedlings entrained in 12L/12D was 55.06 6 11.10% (n ¼
44), and for seedlings entrained to 16L/8D, relative luminescence
was 24.17 6 8.91% (n ¼ 24). Thus, at subjective dusk, [Ca21]cyt
was relatively high in plants entrained in short photoperiods and
low in plants entrained in long photoperiods. These differences in
[Ca21]cyt at subjective dusk are a consequence of photoperiodic
entrainment. Conversely, at subjective dawn, there was no
significant difference in relative luminescence for seedlings
entrained in the different LD regimes.
We next tested whether the phase and shape of circadian
[Ca21]cyt oscillations were set by the length of the dark period by
transferring the seedlings to LL after a dark period equal to that
they were entrained in (12 h) or following a final dark period that
had been extended by 5 h (17 h total; Figure 6A). Extending the
length of the final dark period did not alter the circadian period.
The period estimate of the circadian rhythm of seedlings
transferred to LL after a 5-h delay was 22.6 6 1.47 h (n ¼ 4),
which was not significantly different to that of seedlings transferred to LL without delay; 21.9 6 1.85 h (n ¼ 10). Extending the
length of the final dark period resulted in a shift in phase of the
oscillations relative to each other, but the phase of the rhythms
relative to the final subjective dawn was unaffected (Figure 6B).
The timing of maximum luminescence relative to subjective
dawn in the first three circadian cycles was not significantly
different between the treatments (Figure 6B). The characteristics
of the circadian luminescence oscillations were therefore determined by the entrainment regime and not by the length of the
final dark period.
Leaf [Ca21]cyt Oscillations in LD Cycles Are Modulated by
Photoperiod and Light Intensity
Although important information regarding biological clocks is
obtained from studying organisms under constant conditions,
such conditions are largely artificial. We therefore examined
[Ca21]cyt dynamics in more natural LD cycles to determine, firstly,
whether [Ca21]cyt oscillations with a 24-h period exist under
these conditions and, secondly, whether the [Ca21]cyt oscillations encode environmental information. Arabidopsis expressing
aequorin was grown in 8L/16D or 16L/8D and luminescence
measured in these same LD regimes rather than in LL
(Figure 7). Plants grown in 16L/8D were exposed to a PFD of
60 mmol m2 s1. Plants grown in the 8L/16D regime were exposed to 60 mmol m2 s1 or to 110 mmol m2 s1, thus the latter
received similar daily integrated photons as plants in 16L/8D.
Seedlings in LD had [Ca21]cyt oscillations that were strikingly
similar to those observed in circadian free-run (LL). In LD,
[Ca21]cyt increased during the day to a peak value, peaked
before dusk, and then decreased to a minimum during the dark
period. The period of the [Ca21]cyt oscillations in LD also was
24 h. Importantly, the timing of peak luminescence (phase) in
LD was consistent with that in LL and dependent on the
photoperiod (Figure 7). At dusk, the relative luminescence of
seedlings in 8L/16D was 59.57 6 15.02% (n ¼ 22) compared with
24.89 6 4.34% (n ¼ 22) in 16L/8D. The relative intensity at
subjective dusk was very similar whether measured in LD or LL.
Seedlings entrained to 8L/16D had dusk relative luminescence
values of 59% in both LD and in LL. Moreover, the relative
luminescence at subjective or true dusk of seedlings entrained to
Circadian Calcium Signaling
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Figure 3. Circadian Oscillations of [Ca21]cyt in Arabidopsis Seedlings Entrained to Different Photoperiods.
Aequorin luminescence emitted by seedlings in 110 mmol m2 s1 LL. Seedlings were entrained in 6L/18D (A), 8L/16D (B), 12L/12D (C), and 16L/8D (D)
for 11 d before LL. During the entrainment light period, the PFD was 60 mmol m2 s1. Points represent the mean bioluminescence of 12 seedling
clusters 6SE. Open areas indicate the subjective day, and shaded areas indicate the subjective night. The closed area represents the final dark period of
the entrainment.
16L/8D was 24% in both LL and LD. Additionally, the amplitude
of the [Ca21]cyt oscillations in LD was consistently larger in the
higher PFD (Figure 7A). These data demonstrate that leaf
[Ca21]cyt oscillates with a period of 24 h in light and dark cycles
that are characteristic of natural environments and that the
pattern of these oscillations was dependent on the photoperiod
and the intensity of light.
DISCUSSION
We have examined both the location and potential for information encoding in circadian oscillations of [Ca21]cyt. The
spatial resolution provided by photon-counting imaging enabled
localization of circadian [Ca21]cyt oscillations to the leaves and
cotyledons of Arabidopsis in both LL and LD. It is likely that both
the epidermal and mesophyll cells contribute to the circadian
oscillations of [Ca21]cyt in the leaves (Wood et al., 2001).
However, the imaging system we used did not allow us to
investigate the localization of the [Ca21]cyt oscillations at a cellular
level. These data demonstrate that the circadian oscillations of
[Ca21]cyt reported previously for whole plants (Johnson et al.,
1995) are present in the photosynthetic organs, where light
stimuli are perceived and integrated.
The localization of 24-h oscillations of [Ca21]cyt to the
cotyledons and leaves suggests that they might have a role in
transducing circadian and photoperiodic information (Lumsden,
1998). Our estimate of the amplitude of the circadian [Ca21]cyt
oscillation (300 nmol L1) and that of Johnson et al. (1995;
600 nmol L1) are comparable to the [Ca21]cyt increases after
excitation by extracellular stimuli in plant cells and are sufficient
to activate many Ca21-dependent signaling mechanisms (Webb
et al., 2001; Hetherington and Woodward, 2003). More accurate
measurement of the circadian control of [Ca21]cyt in single
cells requires experimental and technical advances. [Ca21]cyt
oscillations might serve in the transduction of light input to the
circadian oscillator through the phytochrome and cryptochrome
photoreceptors (Shacklock et al., 1992; Bowler, et al., 1994;
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Figure 4. Circadian Oscillations of [Ca21]cyt Were Arrhythmic in Very
Long Photoperiods.
(A) Aequorin luminescence emitted by Arabidopsis seedlings entrained
in 60 mmol m2 s1 16L/8D (open circles) or 20L/4D (closed squares) for
11 d before transfer to 110 mmol m2 s1 LL. Points represent the mean
of 12 seedling clusters 6SE. The period of the mean oscillation in
aequorin bioluminescence was 26 h (relative amplitude error ¼ 0.31) for
seedlings entrained in 16L/8D. However, no rhythmic component to the
mean bioluminescence of seedlings entrained in 20L/4D was determined
by FFT-NLLS.
(B) Table showing the mean 6 SE of the period of bioluminescence for
seedling clusters expressing aequorin in LL. The period of bioluminescence was calculated for each cluster using FFT-NLLS. Seedlings
were entrained in the photoperiods indicated at 60 mmol m2 s1 for
11 d before transfer to 110 mmol m2 s1 LL.
Somers et al., 1998; Baum et al., 1999; Devlin and Kay, 2000;
Guo et al., 2001; Fankhauser and Staiger, 2002) or as a regulatory
output from the clock (Johnson et al., 1995; Sai and Johnson,
1999; Webb, 2003). Candidates for regulation by 24-h oscillations of [Ca21]cyt include physiological and developmental
processes in the leaf that are known to be regulated by both the
circadian clock and alterations in [Ca21]cyt (Bakrim et al., 2001;
Jung et al., 2002; Webb, 2003). However, the role of [Ca21]cyt
oscillations has limitations: Circadian [Ca21]cyt rhythms are not
involved in the circadian regulation of the LIGHT-HARVESTING
COMPLEX B (LHCB) promoter. The rhythms of LHCB promoter
activity and [Ca21]cyt are asynchronous in LL and uncoupled
such that in callus, rhythms of LHCB promoter activity can be
detected in the absence of [Ca21]cyt rhythms (Sai and Johnson,
1999).
Previously, circadian oscillations in [Ca21]cyt have been
detected in N. plumbaginifolia and in Arabidopsis (Johnson
et al., 1995; Wood et al., 2001). In those studies, plants were
entrained in 16L/8D, and [Ca21]cyt peaked just before subjective
dusk. The data presented here indicated an earlier peak in
[Ca21]cyt for plants entrained in 16L/8D that occurred between
8 h and 10 h after subjective dawn. Our study differs in the light
intensity supplied to the plants (22 mmol m2 s1 in the previous
studies [Johnson et al., 1995; Wood et al., 2001] compared with
60 or 110 mmol m2 s1 for our measurements), suggesting one
possible explanation for the different phasing of the circadian
[Ca21]cyt oscillations in the two studies. However, when we
tested the effect of varying PFD, we found that PFD affected the
amplitude but not the period or the phase of the circadian bioluminescence oscillations (Figure 2). The discrepancy between
this investigation and the earlier studies may instead be as
a result of the different methods of bioluminescence detection
used. Aequorin luminescence was previously quantified by
luminometry, as an integrated signal from whole seedlings
(Johnson et al., 1995; Wood et al., 2001). However, different
tissues have differently phased circadian [Ca21]cyt oscillations
(Wood et al., 2001) that may summate to produce the integrated
signal detected using the luminometer. Conversely, in this study,
aequorin bioluminescence was imaged from only the cotyledons
and emerging leaves of the seedlings. Thus, the enhanced resolution of photon-counting imaging may explain the differences
between these and earlier data.
Our data provide new evidence that circadian and photoperiodic information can be encoded in the pattern of [Ca21]cyt
oscillations in both circadian free-run in LL and in LD cycles. This
has important implications for understanding signaling in plants.
We have demonstrated that the phase (or timing of the peak of
[Ca21]cyt) and shape of free-running circadian oscillations of
[Ca21]cyt in LL were dependent on the length of the light and dark
cycles during entrainment. The peak of [Ca21]cyt after subjective
dawn in LL occurred later in those plants that had been entrained
to long light periods than the peak in plants entrained to short
light periods. These data demonstrate that the phase of the
circadian oscillations of [Ca21]cyt was sensitive to the length of
the light and/or dark cycles during entrainment. However, the
timing of the trough of [Ca21]cyt relative to subjective dawn was
not altered by the photoperiodic entrainment.
Another effect of the phase shift of the [Ca21]cyt rhythms was
that the timing of the peak was altered in relation to subjective
dusk. The peak of [Ca21]cyt in LL in plants entrained to 6L/18D
occurred at the end of the subjective light period, 0 to 2 h before
subjective dusk. By comparison, the peak in plants entrained to
16L/8D occurred in the middle of the subjective light period, 6 to
8 h before subjective dusk. Plants entrained in 6L/18D therefore
emitted 80% of the peak luminescence level at subjective
dusk, whereas plants entrained in 16L/8D emitted only 25% of
the peak luminescence level at subjective dusk.
These data demonstrate that the entrainment photoperiod
regulates the phase and shape of the circadian [Ca21]cyt oscillations and importantly, the [Ca21]cyt at subjective dusk. It is
therefore possible that these characteristics of circadian
[Ca21]cyt oscillations encode information that affects the integration and outcomes of Ca21-based signaling networks
(Berridge et al., 2000; Evans et al., 2001; Sanders et al., 2002;
Schuster et al., 2002; Hetherington and Woodward, 2003).
Two aspects of circadian [Ca21]cyt oscillations in Arabidopsis
seedlings, the phase and the amplitude of the oscillations, are
Circadian Calcium Signaling
Figure 5. The Photoperiod of Entrainment Shifts the Phase of Circadian
Oscillations of [Ca21]cyt in Constant Light.
(A) Aequorin luminescence of Arabidopsis seedlings in 110 mmol m2 s1
LL. Seedlings were entrained in 6L/18D (dashed line) or in 16L/8D (solid
line) at a PFD of 60 mmol m2 s1 for 11 d before LL. For clarity, the points
representing the mean values do not appear. The thin, vertical lines that
intersect the abscissa at 0, 24, 48, and 72 h indicate subjective dawn for
all seedlings.
(B) Timing of maximum aequorin luminescence, relative to subjective
dawn, of Arabidopsis seedlings in LL, entrained to 6L/18D (open bars),
8L/16D (light-shaded bars), 12L/12D (dark-shaded bars), and 16L/8D
(closed bars). Bars represent the mean luminescence of 24 to 36
seedling clusters, with standard errors shown. Circadian cycles are
indicated below each set of bars: Cycle 2 corresponds to 24 to 48 h in LL,
and cycle 3 corresponds to 48 to 72 h in LL.
(C) Timing of minimum aequorin luminescence, relative to subjective
dawn, emitted by Arabidopsis seedlings in LL, entrained to 6L/18D (open
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modulated by light quantity and entrainment photoperiod. The
modulation of the phase and amplitude of circadian oscillations
of [Ca21]cyt by environmental stimuli is consistent with the
hypothesis that information is encoded in the dynamics of
circadian [Ca21]cyt oscillations (Jaffe, 1991; McAinsh et al., 1995;
Ehrhardt et al., 1996; McAinsh and Hetherington, 1998; Allen
et al., 2001; Evans et al., 2001; Shaw and Long, 2003). If
information is encoded, there must also be mechanisms for
decoding circadian [Ca21]cyt oscillations. In plants these mechanisms remain obscure, but models exist based on counteracting and differentially sensitive regulatory enzymes and
posttranslational and biochemical modification of Ca21-sensitive proteins in phase with [Ca21]cyt oscillations (McAinsh and
Hetherington, 1998; Schuster et al., 2002; Cullen, 2003).
We also imaged the daily [Ca21]cyt dynamics of the leaves and
cotyledons of seedlings in light and dark cycles more representative of the natural environment. The [Ca21]cyt oscillations in LD
were strikingly similar to those observed in LL: [Ca21]cyt rose
during the day, peaked several hours after dawn, and then
decreased, attaining a minimal value during the night. Additionally, the photoperiod-dependent phase shift of [Ca21]cyt oscillations and amplitude modulation observed in LL occurred also in
LD. In 8L/16D, the peak [Ca21]cyt occurred at 6 h after dawn but in
16L/8D, the peak [Ca21]cyt occurred at 8 h after dawn. Higher
PFD in 8L/16D increased the amplitude of the oscillations in
[Ca21]cyt, in agreement with our observations of circadian
[Ca21]cyt oscillations in LL, but PFD did not affect the phase of
the [Ca21]cyt oscillations in LD. Thus, the effects of daylength on
[Ca21]cyt in LD were because of the duration of the light and dark
cycles and not the amount of light to which the seedlings were
exposed. These data demonstrate that [Ca21]cyt in cotyledon
and leaf cells oscillated with a 24-h period in LD cycles and that
the phase of these oscillations was sensitive to the entrainment
regime and the amplitude was sensitive to PFD. Importantly, the
level of [Ca21]cyt at dusk was relatively high in plants in short days
and low in plants in long days. This observation strongly
suggests that [Ca21]cyt oscillations in LD are not directly
regulated by light but are instead associated with the circadian
clock and may play a role in circadian signaling in response to LD
cycles.
There was one major difference between the oscillations of
[Ca21]cyt in LL and those in LD. The luminescence of seedlings in
LD did not increase significantly before dawn but only rose after
illumination. This difference between the Ca21 dynamics of
plants in LD or in LL can be explained by the observation that
[Ca21]cyt is arrhythmic in darkness (Johnson et al., 1995).
Therefore, it appears that [Ca21]cyt can rise in anticipation of
dawn only in the light, which is why the circadian anticipation of
dawn was only detected in LL but not LD cycles.
The slow rates of increase and decrease of [Ca21]cyt in LD
suggest that the daily [Ca21]cyt oscillations were not generated
bars), 8L/16D (light-shaded bars), 12L/12D (dark-shaded bars), and 16L/
8D (closed bars). As in (B), bars represent the mean luminescence of 24
to 36 seedling clusters, with standard errors and the circadian cycles
indicated below each set of bars.
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The Plant Cell
PHOT2 phototropin-mediated signaling (Baum et al., 1999;
Stoelzle et al., 2003). Organellar [Ca21] also has an acute
response to illumination. In the chloroplast, the dark-light
transition generates a rapid increase in stromal [Ca21] that
reaches a peak value between 25 to 30 min after dawn (Sai and
Figure 6. The Effect of the Dark Period Length on Circadian Oscillations
of [Ca21]cyt.
(A) Aequorin luminescence of Arabidopsis seedlings in 110 mmol m2 s1
LL. Seedlings expressing apoaequorin were entrained in 60 mmol m2
s1 12L/12D for 11 d. Closed circles represent the mean luminescence
with standard errors emitted by 10 seedling clusters that experienced the
normal 12L/12D on day 11, before transfer to LL. Open circles represent
the mean luminescence with standard errors emitted by four seedling
clusters that received an additional 5 h of darkness before transfer to LL,
hence a 12-h-light period followed by a 17-h-dark period on day 11. The
bars on the abscissa indicate the subjective light regime during LL and
are color-coded to match the graph. Open areas represent subjective
day, hatched areas subjective night, and closed areas the final dark
period before LL. For all seedlings, the time of transfer to LL is defined as
the start (t ¼ 0) of subjective dawn.
(B) Timing from subjective dawn of maximum aequorin luminescence
emitted by Arabidopsis seedlings entrained in 60 mmol m2 s1 12L/12D.
Bars represent the mean luminescence emitted by seedling clusters
6SE. Closed bars (n ¼ 10) indicate that seedlings received a 12-h-dark
period before transfer to LL, and shaded bars (n ¼ 4) denote seedlings for
which the transfer from darkness to LL was delayed by 5 h. As for (A), the
time of transfer to LL is defined as the t ¼ 0 of subjective dawn.
as an immediate, acute response to illumination but instead by
a complex signaling network cued to LD cycles and the circadian
clock (Millar and Kay, 1996; Hall et al., 2002). Nevertheless,
short-term or acute responses of [Ca21]cyt have been reported.
In Triticum aestivum (wheat) protoplasts, dark-light transitions
generate brief elevations in [Ca21]cyt lasting <5 min in response to
red light (Shacklock et al., 1992). In Arabidopsis and Nicotiana
tabacum (tobacco), blue light elevates [Ca21]cyt via PHOT1 and
Figure 7. Light Intensity and Duration Modulates [Ca21]cyt Oscillations in
Light and Dark Cycles.
(A) Oscillations of aequorin luminescence from Arabidopsis seedlings in
LD. Seedlings expressing apoaequorin were grown in 60 mmol m2 s1
8L/16D (open circles, top graph), in 110 mmol m2 s1 8L/16D (open
triangles, middle graph), or in 60 mmol m2 s1 16L/8D (closed circles,
bottom graph) for 14 d. Points represent the mean of 12 seedling clusters
6SE. Open areas represent the light periods, and shaded areas represent
the dark periods experienced by the seedlings during bioluminescence
imaging.
(B) Timing from dawn of maximum aequorin luminescence emitted by
Arabidopsis in 60 mmol m2 s1 8L/16D (closed bars), in 110 mmol m2
s1 8L/16D (shaded bars), or in 60 mmol m2 s1 16L/8D (open bars).
Bars represent the mean luminescence of 12 seedling clusters 6SE.
Circadian Calcium Signaling
Johnson, 1999). Our assay was not designed to measure shortterm changes in [Ca21]cyt, and our measurements began 30 min
after the dark-to-light transition to avoid the possibility of reentraining the plants to a new rhythm. It is therefore possible that
short, transient increases in [Ca21]cyt immediately after dawn
were superimposed on the 24-h oscillation of [Ca21]cyt that
occurred in LD and were not detected by our method.
Perhaps one of the most important plant responses regulated
by daylength is the photoperiodic transition from vegetative
growth to the development of flowers (Yanovsky and Kay, 2002;
Hayama and Coupland, 2003). In LD, [Ca21]cyt oscillates with
amplitude and phase that are sensitive to light quantity and
entrainment photoperiod. Most importantly, these diurnal
[Ca21]cyt oscillations result in substantially different [Ca21]cyt at
critical periods of the day, depending on the length of the light
period. Consequently, it is compelling to consider a role for
[Ca21]cyt in the signal transduction pathways of photoperiodic
responses. Pharmacological studies (Friedman et al., 1989) and
direct measurement of [Ca21]cyt (Walczysko et al., 2000) support
a role for alterations in [Ca21]cyt in the transduction pathways
regulating floral induction in response to changes in photoperiod.
It has also been proposed that Ca21-dependent protein kinase
activity acts as a Ca21 sensor in photoperiodism (Jaworski et al.,
2003). Additionally, it has recently been suggested that extracellular Ca21 may have a role in regulating the switch to flowering
through the action of a cell-surface Ca21 sensor (Han et al.,
2003). Strikingly, this cell-surface Ca21 sensor also has a role in
generating oscillations of [Ca21]cyt in the guard cell in response to
elevations of external [Ca21]cyt (Han et al., 2003). Although our
data demonstrate that [Ca21]cyt oscillations encode circadian
and photoperiodic information, further work is required to determine whether this information is decoded and contributes to
physiological processes, such as the control of floral induction.
METHODS
Seedling Growth and Photoperiodic Entrainment
Transgenic Arabidopsis seeds (ecotype Wassilewskija) that constitutively
express the apoaequorin cDNA under the control of the 35S promoter of
Cauliflower mosaic virus were surface-sterilized and germinated on solid
medium (0.53 Murashige and Skoog medium and 0.8% (w/v) agar, pH ¼
6.8) in clusters of 15 to 20 plants. Surface sterilization was performed by
rinsing seeds for 1 min in 100% ethanol, then by incubating seeds for
10 min in 50% (v/v) sodium hypochlorite, followed by two washes in
sterile water. Seed germination was synchronized by vernalization at
48C for 2 d in the dark. Seedlings were grown in white light at a PFD of
60 mmol m2 s1 at 198C for 11 d. During the growth period, seedlings
were entrained to several photoperiods: 6L/18D, 8L/16D, 12L/12D,
16L/8D, or 20L/4D. Plants were then transferred to 60 mmol m2 s1 or
110 mmol m2 s1 of LL for imaging circadian [Ca21]cyt oscillations under
circadian free-run conditions. In a separate protocol, plants remained
under the appropriate entrainment regime for measurement of [Ca21]cyt
dynamics under LD conditions. During the dark period, plants were
handled in a dark room under green safelight.
Imaging Aequorin Bioluminescence
[Ca21]cyt was measured in vivo using the bioluminescent Ca21-reporter
aequorin. For plants in LL conditions, 100 mL of 5 mmol L1
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coelenterazine-free base (Prolume, Pinetop, AZ) was applied to each
seedling cluster 24 h and 12 h before imaging, to ensure high levels of
reconstituted aequorin. Photon counting was performed in a light-tight
box using a Photek (Hastings, UK) ICCD225 photon-counting camera
system mounted above the seedlings. The camera was controlled by
Photek IFS32 software. Immediately before photon counting, bright-field
images of the seedlings in position under the camera were captured. The
bright-field illumination was from low-voltage, biologically safe, green
light–emitting diodes, which provided a PFD <1 mmol m2 s1. The
bioluminescence emitted by each seedling cluster was recorded for
1500 s, every 2 h, for 4 d. The first 200 s of each photon-counting integration contained luminescence generated by the autofluorescence
decay of chlorophyll and was discarded. Eight-bit images of the photon
density were generated from the remaining 1300 s of each integration and
pseudocolored. Unless otherwise indicated, 10 mL of 5 mmol L1
coelenterazine was applied to each seedling cluster after imaging, to
maintain high levels of aequorin in the plants. The baseline of aequorin
luminescence increased during the experiments as a result of a combination of regular coelenterazine application and seedling growth. Coelenterazine treatment had no adverse effects on the seedlings, which
increased in size during the experiments, showed no visible signs of
damage, and could be successfully grown to seed. For plants imaged
under LD rather than LL conditions, seedlings were dosed twice daily
with 100 mL of 10 mmol L1 coelenterazine for 3 d before imaging. These
seedlings received no further coelenterazine during imaging.
The integrated 8-bit photon-counting images were processed using the
Photek IFS32 software. Regions of interest, based on the bright-field
image, were demarcated around each seedling cluster, and total photon
counts (luminescence) were quantified. Rhythms of aequorin luminescence, corresponding to rhythms in [Ca21]cyt, were analyzed using two
methods. First, the fast Fourier transform–nonlinear least-squares
method (FFT-NLLS) described by Plautz et al. (1997) was used to
determine the period of each circadian rhythm in luminescence of the
mean signal from 12 seedling clusters. The degree of confidence in the
FFT-NLLS is described by the relative amplitude error (rel-amp), which
ranges from 0 to 1, with 0 indicating a rhythm known to infinite precision
and 1 indicating a rhythm that was not statistically significant (Plautz et al.,
1997). Second, the timing of minimum and maximum bioluminescence
during each 24-h period was determined for each seedling cluster and
averaged. Cycle 1, from 0 to 24 h, immediately followed the last dark
period of the entrainment regime and was therefore not considered a true
circadian cycle. Cycles 2 and 3, from 24 to 48 h and from 48 to 72 h,
respectively, represented genuine circadian cycles. Cycle 4, from 72 to
96 h, was also a circadian cycle but was not used because not all data
sets continued until 96 h. n in the text refers to the number of seedling
clusters analyzed.
Estimation of the Total Aequorin Pool and Magnitude of the
Oscillations in [Ca21]cyt
The size of the aequorin pool was quantified in 30 seedlings at opposite
phases of the circadian cycle (t ¼ 7.5 and 19.5) by discharging the entire
pool with 1 M L1 CaCl2 in 10% (v/v) ethanol and quantifying the luminescent signal using a photon-counting luminometer (photon multiplier
tube 9899A cooled to 14 C with a FACT50 housing; Electron Tubes,
Middlesex, UK).
The long time scale of the measurements compromise estimation of
[Ca21]cyt based on the rate of aequorin consumption as a proportion of
the total pool (Johnson et al., 1995). Additionally, we could not measure
the total aequorin pool using the photon-counting camera because the
light emission after discharge of the entire pool (Fricker et al., 1999)
saturated the detector. Instead, we used the fold increase in light to
estimate the circadian changes in [Ca21]cyt (Cobbold and Rink, 1987).
Fold increases in light were converted to [Ca21]cyt by comparison with the
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The Plant Cell
fold increase in light during imaging of transient excursions of [Ca21]cyt in
response to NaCl stimulation of seedlings.
To generate a calibration curve, we used data obtained from photoncounting imaging of 7-d-old aequorin-expressing seedlings irrigated with
100, 150, 200, or 300 mol m3 NaCl in Murashige and Skoog medium
(n ¼ 20; F. Tracy and M. Tester, personal communication). Functional
aequorin had been generated by overnight incubation with 10 mmol L1
coelenterazine in the dark. The seedlings were imaged continuously
before, during, and after perfusion with NaCl containing solution. A timeresolved integration graph of the integrated photon count s1 was
plotted, and the peak fold increase in light was calculated. Previously, the
mean peak [Ca21]cyt increase in response to 100, 150, 200, or 300 mol L1
NaCl in Murashige and Skoog medium had been measured using photoncounting luminometry calibrated exactly as described in Fricker et al.
(1999; n ¼ 120; F. Tracy and M. Tester, personal communication). By
comparing the mean peak height of the [Ca21]cyt transients in response to
NaCl stimulation measured using luminometry with the mean peak fold
change in light detected by the imaging camera, we made a calibration
curve of estimated [Ca21]cyt increase against fold change in light detected
by the camera. We estimated the changes in [Ca21]cyt in response to
circadian and light signals by comparing of the fold increases in light
during the circadian experiments with the calibration curve generated
from the NaCl experiments.
ACKNOWLEDGMENTS
We thank Marc Knight of the University of Oxford (Oxford, UK)
Department of Plant Sciences for his kind gift of Arabidopsis expressing
apoaequorin, and Carl H. Johnson of Vanderbilt University (Nashville,
TN) and Enid MacRobbie and Julia Davies (University of Cambridge,
Cambridge, UK) for their helpful comments on the manuscript. We thank
Francis Tracey and Mark Tester (University of Cambridge) for providing
access to their unpublished data. We also acknowledge the financial
support of the Broodbank Fellowship Trust of the University of
Cambridge (to J.L.), the Royal Society of London, and the Biotechnology
and Biological Sciences Research Council of the United Kingdom (to
A.A.R.W).
Received December 18, 2003; accepted January 30, 2004.
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Circadian and Diurnal Calcium Oscillations Encode Photoperiodic Information in Arabidopsis
John Love, Antony N. Dodd and Alex A.R. Webb
Plant Cell; originally published online March 18, 2004;
DOI 10.1105/tpc.020214
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