Plant Cell Physiol. 44(3): 360–365 (2003)
JSPP © 2003
Short Communication
Cell Autonomous Circadian Waves of the APRR1/TOC1 Quintet in an
Established Cell Line of Arabidopsis thaliana
Norihito Nakamichi, Akinori Matsushika, Takafumi Yamashino and Takeshi Mizuno 1
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan
;
Keywords: Arabidopsis thaliana — Circadian rhythms —
Cultured cells.
Abbreviations: APRR, ARABIDOPSIS PSEUDO RESPONSE
REGULATOR; TOC1, TIMING OF CAB EXPRESSION 1; CCA1, CIRCADIAN CLOCK ASSOCIATED 1; CCR2, COLD CIRCADIAN
RHYTHM RNA BINDING 2; DD, continuous darkness; LD, 12 h light/
12 h dark; LL, continuous white light; LHY, LATE ELONGATED
HYPOCOTYL.
In general, circadian rhythms are driven by an endogenous biological clock(s) that regulates many biochemical,
physiological and behavioral processes in a wide variety of
1
Corresponding author: E-mail, [email protected]; Fax, +81-52-789-4091.
360
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organisms (Dunlap 1999). In higher plants too, there are a wide
range of biological processes that are controlled through such a
circadian clock. They include movement of organs such as
leaves and petals, opening of stomata, daily fluctuations of
metabolic activities such as respiration, photosynthesis, and
gene expression (Harmer et al. 2000, Schaffer et al. 2001).
Thus, clarification of the biological clock (central oscillator) is
a paradigm of current plant molecular biology (Barak et al.
2000, Murtas and Millar 2000, McClung 2000, Samach and
Coupland 2000, Putterill 2001, Somers 2001, Carre 2001,
Mouradov et al. 2002, and references therein). With regard to
the Arabidopsis circadian clock, several genes were proposed
to encode potential components. Both the CIRCADIAN
CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED
HYPOCOTYL (LHY) genes are implicated in a part of a feedback loop that is closely associated with the circadian clock
(Wang and Tobin 1998, Schaffer et al. 1998, Green and Tobin
1999). These homologous transcription factors with a single
Myb-domain are the best candidates for such clock components. Another gene, TIMING OF CAB EXPRESSION 1
(TOC1), is also believed to encode a component of the central
oscillator (Somers et al. 1998). Furthermore, reciprocal and
intimate interactions were observed between the functions of
CCA1/LHY and TOC1 (Alabadi et al. 2001, Alabadi et al.
2002, Makino et al. 2002, Matsushika et al. 2002a, Mizoguchi
et al. 2002).
We have been extensively characterizing a novel family
of proteins, termed Arabidopsis pseudo response regulators
(APRRs), with special reference to circadian rhythms
(Makino et al. 2000, Makino et al. 2001, Makino et al. 2002,
Matsushika et al. 2000, Matsushika et al. 2002a, Matsushika et
al. 2002b, Murakami-Kojima et al. 2002, Sato et al. 2002),
based on the fact that one of them (APRR1) is identical to
TOC1 (Matsushika et al. 2000, Strayer et al. 2000). More
interestingly, each transcript of APRRs starts accumulating after
dawn rhythmically and one after another at intervals in the
order of APRR9 ® APRR7 ® APRR5 ® APRR3 ® APRR1/
TOC1. This event was referred to as “circadian waves of the
APRR1/TOC1 quintet”. It was thus assumed that not only
APRR1/TOC1 itself, but also other APRR1/TOC1 quintet
members might be implicated as part of an Arabidopsis circadian clock. Indeed, several lines of solid evidence have already
A small family of genes, named ARABIDOPSIS
PSEUDO RESPONSE REGULATOR (APRR), are intriguing with special reference to circadian rhythms in plants,
based on the fact that one of the members (APRR1) is identical to TOC1 (TIMING OF CAB EXPRESSION 1) that is
believed to encode a clock component. In Arabidopsis plants,
each transcript of the APRR1/TOC1 quintet genes starts
accumulating after dawn rhythmically and one after another
at intervals in the order of APRR9 ® APRR7 ® APRR5 ®
APRR3 ® APRR1/TOC1. To characterize such intriguing
circadian-associated events, we employed an established
Arabidopsis cell line (named T87). When T87 cells were
grown in an appropriate light and dark cycle, cell autonomous diurnal oscillations of the APRR1/TOC1 quintet genes
were observed at the level of transcription, as seen in intact
plants. After transfer to the conditions without any environmental time cues, particularly in constant dark, we further showed that free-running circadian rhythms persisted
in the cultured cells, not only for the APRR1/TOC1 quintet
genes, but also other typical circadian-controlled genes
including CCA1 (CIRCADIAN CLOCK ASSOCIATED 1),
LHY (LATE ELONGATED HYPOCOTYL) and CCR2
(COLD CIRCADIAN RHYTHM RNA BINDING 2). To our
knowledge, this is the first indication of cell autonomous
circadian rhythms in cultured cells in Arabidopsis thaliana,
which will provide us with an alternative and advantageous means to characterize the plant biological clock.
Circadian rhythms in Arabidopsis
T87 cells were grown in liquid JPL medium (100 ml) with
500 ml flask, under continuous light at 22°C (Fig. 1A, right
panel, note that cells are chemomixotrophic, i.e. they derive
their carbon source partially from photosynthetic fixation of
CO2 and partially from sucrose in the medium). Aliquots were
spread on Gamborg’s B5 agar-plates, and then they were incubated (or entrained) under the 12 h light/12 h dark (LD) cycle
for 18 d (Fig. 1A, left panel). The well-grown cells were harvested at intervals (3 h), and RNA fractions were prepared. We
first examined whether or not each expression of the APRR1/
TOC1 quintet genes shows a diurnal oscillation in the cells cultured under the LD cycle (i.e. in entraining LD). Northern blot
hybridization analyses were carried out with probes specific for
each of APRR9, APRR7, APRR5, APRR3, APRR1/TOC1 (Fig.
1B). The hybridized bands were also quantified (Fig. 1C),
based on the UBQ10-value as a loading and internal reference
(Fig. 1E). In the cultured cells, the APRR9 transcript was rapidly induced immediately after dawn, whereas that of APRR1
started accumulating upon the onset of evening. It was clearly
seen that these APRR1/TOC1 quintet genes were expressed in a
sequential and rhythmic manner in the characteristic order of
APRR9 ® APRR7 ® APRR5 ® APRR3 ® APRR1/TOC1.
Fig. 1 Northern blot hybridization analyses of the transcripts of the
APRR1/TOC1 quintet genes and other circadian-regulated genes with
the cells cultured in entraining light and dark cycle. T87 cells were
first grown with liquid JPL medium (100 ml) containing 1.5% sucrose
and 1-naphthalenacetic acid (1 mM NAA) in 500 ml flask, by shaking
on a rotary shaker (120 rpm), under continuous white light (25 mmol
m–2 s–1) at 22°C. The contents of JPL medium are exactly the same as
those described by Axelos et al. (1992). (A, right) A representative of
such suspension culture is shown. Then, aliquots were spread on Gamborg’s B5 agar-plates (containing 3% sucrose and 1 mM NAA), and
then they were incubated (or entrained) under the 12 h light/12 h dark
cycle for 18 d at 22°C. Gamborg’s B5 medium salt mixture was purchased from Wako Co. (Osaka, Japan). (A, left) Resulting well-grown
cells on the plate are shown. Cells were harvested at intervals (3 h),
and RNA fractions were prepared from each single plate, as described
previously (Taniguchi et al. 1998, Imamura et al. 1999). Note that the
procedures for RNA preparation were essentially the same as those
used for plants. (B) Northern blot hybridization analyses were carried
out with probes each specific for the APRR1/TOC1 quintet genes, as
described previously (Matsushika et al. 2002b, Sato et al. 2002). The
hybridized bands were detected with a phosphoimage analyzer (BAS2500) (FujiXerox, Tokyo, Japan). In these experiments, the transcript
of UBQ10 was also detected as an internal reference (see panel E). (C)
The detected bands were also quantified. The measured intensities of
each band were normalized (by dividing with the UBO10-value).
Based on these values, the relative amounts of mRNA (or transcript)
were calculated and expressed as arbitrarily units, in which the maximum level of each transcript was taken as 10 arbitrarily, in order to
clarify the profiles. The color-coordinated lines were each denoted by
the corresponding number (e.g. the red line denoted by 9 is the profile
of APRR9). (D) Similarly, Northern blot hybridization analyses were
carried out with probes each specific for CCA1, LHY, CAB2 and
CCR2, respectively. Details for the probes used in this study were
described previously (Matsushika et al. 2002b, Sato et al. 2002). (E)
The results of UBQ10, analyzed as internal references, are collectively
presented as a single panel at bottom, for clarify of this figure.
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been provided to support the notion that APRR9 and APRR5
(together with APRR1/TOC1) must also be taken into consideration for a better understanding of the molecular links
between circadian rhythms, control of flowering time through
the photoperiodic long-day pathway, and also light signalingcontrolled plant development (Matsushika et al. 2002b, Sato et
al. 2002).
In our previous studies, we mainly adopted Northern blot
hybridization analyses in order to examine the expression profiles of certain circadian-regulated genes, including the APRR1/
TOC1 quintet genes, for which mRNA was prepared exclusively from intact plants. Alternatively, in this study we
employed an Arabidopsis cultured cell line in the hope of gaining further insight into the functions of the APRR1/TOC1 quintet. Axelos et al. (1992) have previously established a cell line
(named T87) from the ecotype Columbia plant. With this cell
line, we showed that cell autonomous circadian rhythms were
observed, not only for the APRR1/TOC1 quintet genes, but also
for CCA1 and LHY, under the conditions without any environmental time cues (particularly, in constant dark).
361
362
Circadian rhythms in Arabidopsis
These rhythmic profiles with an approximately 24 h period
were essentially the same as those previously observed in intact
plants (Matsushika et al. 2000). In other words, this is indeed
the event that has been referred to as “circadian waves of the
APRR1/TOC1 quintet”. It should be also noted that the same
circadian events were seen, even when the suspension-cultured
cells in the liquid medium (instead of the solid-cultured cells
on the agar medium) were directly analyzed in entraining LD
(data not shown).
As mentioned above, several genes have been proposed to
encode proteins that function within, or close to, the central
oscillator. Among them, CCA1 and LHY are particularly
intriguing, because these homologous gene products are the
best candidates for central oscillator components, as mentioned above. It is thus of interest to examine whether or not
the CCA1 and LHY transcripts also show oscillated expression
profiles in T87 cells in entraining LD. The results showed that
both CCA1 and LHY exhibited a diurnal and robust oscillation
with a peak at dawn, respectively (Fig. 1D, upper two panels).
We then examined more examples of circadian-regulated
genes. In plants, a well-known hallmark of circadian-regulated
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Fig. 2 Northern blot hybridization analyses of the transcripts of the
APRR1/TOC1 quintet genes with the cells grown under constant white
light. T87 cells were first grown with liquid JPL medium. Aliquots
were spread on Gamborg’s B5 agar-plates, and then they were incubated (or entrained) under the 12 h light/12 h dark cycle for 18 d at
22°C. Details were the same as those given in the legend to Fig. 1.
Plates were transferred to constant white light (LL), as schematically
shown (see the horizontal bars). Cells were harvested at intervals (3 h),
and RNA fractions were prepared. (A) Northern blot hybridization
analyses were carried out with probes each specific for the APRR1/
TOC1 quintet genes. (B) The detected bands were also quantified.
Other detains were essentially the same as those given in the legend to
Fig. 1.
events is a rhythmic expression of CAB2 (encoding lightharvesting chlorophyll-a/b-binding protein) (Millar et al. 1995).
The CAB2 gene with its peak at morning is the most downstream target of a circadian output pathway. Another hallmark
of clock-regulated transcriptional events is a rhythmic expression of the COLD CIRCADIAN RHYTHM RNA BINDING 2
(CCR2) gene (also known as AtGRP7), the peak of which is
seen at evening in plants (Carpenter et al. 1994, Heintzen et al.
1997). These well-known circadian-controlled genes were also
examined in T87 cells (Fig. 1D, lower two panels). They each
showed a diurnal oscillation with a peak at the anticipated
timing, respectively, as can be seen in intact plants. Taken
together, as far as the entraining LD conditions are concerned,
T87 cells have the ability to generate diurnal rhythms to oscillate the transcription of certain genes, such as APRR1/TOC1,
CCA1, LHY, CAB2 and CCR2, all of which are known to be
circadian-controlled ones in intact plants.
The most critical view as to the circadian rhythm in plants
is that when plants are deprived of environmental time cues
(e.g. light/dark cycle and temperature cycle) and placed in constant (“free-running”) environmental conditions, circadian
rhythms must persist with a period of around 24 h, often for
many days. We next needed to address this critical issue. T87
cells were grown under the entraining LD conditions as
described above, and then they were transferred to continuous
white light (LL conditions). For the APRR1/TOC1 quintet
genes, no free-running rhythm was observed in T87 cells
(Fig. 2). In other words, after transfer to LL, their rhythms were
dampened very rapidly. The APRR9 transcript no longer
appeared, while others were expressed more or less constitutively. These events in T87 cells were quite different form
those seen in intact plants.
We then adopted constant dark (DD) conditions, under
which the light signal was completely deprived. Provided that
T87 cells were transferred to DD, free-running rhythms were
seen, to some extent, with regard to the APRR1/TOC1 quintet
genes (Fig. 3). In contrast to LL, the sustained APRR9 peak
appeared at the anticipated phase (i.e. subjected dawn) on the
first day in DD, and it persisted even on the second day (Fig.
3A, B). A similar event was also seen for the APRR7 transcript. Including APRR5, APRR3 and APRR1 together, the freerunning circadian waves were seen in DD, at least for the first
day (Fig. 3A, B). To evaluate these events, the circadian waves
of the APRR1/TOC1 quintet in intact plants were examined in
DD, as critical references. Wild-type plants (Columbia) were
grown for 22 d under the 12 h light/12 h dark cycle, and then
they were transferred to constant dark. RNA samples were prepared from leaves at appropriate intervals (3 h), and then
Northern blot hybridization analyses were carried out with
regard to the APRR1/TOC1 quintet genes (Fig. 3C). The profiles observed for the APRR1/TOC1 quintet genes in plants in
DD were considerably similar to those observed for T87 cells
in DD (compare panels B and C). For instance, the quantified
profiles of the free-running APRR7 and APRR3 rhythms in T87
Circadian rhythms in Arabidopsis
plants, were very similar to each other. The first robust peak of
CCA1 appeared at the anticipated timing (subjected morning),
which was followed by the less robust second peak with an
approximate 24 h period. Indeed, the quantified profile of the
CCA1 rhythm in T87 cells in DD was well superimposed to
that in plants (Fig. 4D, red lines for cells, and blue lines for
plants). Essentially the same event was observed for another
circadian-associated gene, LHY. The same was true for another
hallmarked gene, CCR2, although the free-running rhythm of
CCR2 showed a different phase (i.e. with peak at subjective
evening), as compared with CCA1 and LHY (with peak at subjective morning). Note also that these circadian events,
observed here for plants, were well consistent with those
reported previously for the CCA1, LHY and CCR2 rhythms in
DD (Wang and Tobin 1998, Park et al. 1999).
Within the last 5 years, intensive molecular studies have
been conducted to understand the mechanisms by which the
model Arabidopsis plant generates free-running circadian
rhythms (Barak et al. 2000, Murtas and Millar 2000, McClung
2000, Samach and Coupland 2000, Putterill 2001, Somers
2001, Carre 2001, Mouradov et al. 2002, and references
therein). These studies were conducted exclusively with use of
intact plants or detached organs. In this study, we employed an
established Arabidopsis cultured cell line (T87) (Axelos et al.
1992), with which Yuasa et al. (2001) recently succeeded in
demonstrating that oxidative stress activates an Arabidopsis
homologue of mitogen-activated protein kinase in the cultured
cells. To our knowledge, we succeeded for the first time in
demonstrating that this established cell line T87 also retains the
ability to generate circadian rhythms, at least in part. T87 cells
entrained in LD generated robust and diurnal oscillations with
regard to certain circadian-regulated genes, such as the APRR1/
TOC1 quintet genes, the CCA1 and LHY clock-component
genes, and the typical CAB2 and CCR2 output genes (Fig. 1),
Fig. 3 Northern blot hybridization analyses of the transcripts of the
APRR1/TOC1 quintet genes with the cells grown under constant dark.
T87 cells were first grown with liquid JPL medium. Aliquots were
spread on Gamborg’s B5 agar-plates, and then they were incubated (or
entrained) under the 12 h light/12 h dark cycle for 18 d at 22°C.
Details were the same as those given in the legend to Fig. 1. Plates
were transferred to constant dark (DD), as schematically shown (see
the horizontal bars). Cells were harvested at intervals (3 h), and RNA
fractions were prepared. (A and B) Northern blot hybridization analyses were carried out with probes each specific for the APRR1/TOC1
quintet genes (for references, see the UBQ10 profiles in panel E). The
detected bands were also quantified. (C) As references, wild-type
plants (Columbia) were grown for 22 d under the 12 h light/12 h dark
cycle, and then they were transferred to constant dark (DD). RNA
samples were prepared from leaves at appropriate intervals (3 h), and
then Northern blot hybridization analyses were carried out with regard
to the APRR1/TOC1 quintet genes, as described previously (Matsushika et al. 2000). The detected bands were also quantified. (D) The
resulting quantified profiles were compared between those of cells (red
lines) and plants (blue lines), with regard to APRR7 and APRR3. Other
details were essentially the same as those given in the legend to Fig. 1.
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cells in DD were well superimposed with those seen in plants
(Fig. 3D, red lines for cells, and blue lines for plants). These
results suggested that the circadian-regulated free-running
waves of the APRR1/TOC1 quintet genes persist in DD, at least
partly, in T87 cultured cells, in a similar manner as those
observed in intact plants.
To further verify the above notion, the transcripts of
CCA1, LHY and CCR2 were also examined in T87 cells, grown
in either LL or DD (Fig. 4). As in the case of the APRR1/TOC1
quintet genes, the free-running rhythms of CCA1, LHY and
CCR2 did not persist in LL (see each upper panel in A, B and
C). In contrast, their rhythms were clearly sustained at least for
2 d in DD (see each middle panel in A, B and C). These circadian profiles in T87 cells were compared with those in intact
plants in DD (see each pair of lower panels in A, B and C). The
free-running rhythms of CCA1, observed for both cells and
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Circadian rhythms in Arabidopsis
as do the intact plants. More importantly, such cell autonomous rhythms persist, even without environmental time cues
(i.e. in DD), as can be seen in the intact plants (Fig. 3, 4).
Unfortunately, such free-running rhythms were not observed in
LL. The reason for this is not clear. In any event, as far as both
the LD and DD conditions were concerned, T87 cells will
provide us with alternative and advantageous means to characterize the Arabidopsis biological clock at the molecular level.
For instance, light-pulse phase response curves in circadian
rhythms could be easily examined, and pharmacological
approaches to circadian physiology could also easily be taken.
As has been extensively debated, central circadian pacemakers that control animal behaviors are located in the brains
of insects and rodents. Nevertheless, the location of such a central pacemaker has not been determined in plants. Rather, isolated plant organs and tissues support circadian rhythms, indicating that these explants each contain a circadian clock (Sai
and Johnson 1999, Thain et al. 2000, Thain et al. 2002). It has
been also proposed that the circadian systems of organs and
localized areas of tissues in plants appear to be functionally
independent on each other (Thain et al. 2002). In this context,
our findings of cell autonomous circadian rhythms in cultured
cells are consistent with the above view, and also will shed
more light on such longstanding debates.
Acknowledgments
This study was supported by Grants-in-Aid for scientific research
on a priority area (12142201 to TM) and COE from the Ministry of
Education, Science, Sports and Culture of Japan. We thank K. Shinozaki and R. Yoshida (RIKEN, Japan) for providing T87 cells, and their
helpful advices. Thanks are also due to T. Oyama (Nagoya University) for valuable discussion.
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Fig. 4 Northern blot hybridization analyses of the CCA1, LHY
and CCR2 transcripts with the cells grown under either constant light or dark. T87 cells were first grown with liquid JPL
medium. Aliquots were spread on Gamborg’s B5 agar-plates,
and then they were incubated (or entrained) under the 12 h
light/12 h dark cycle for 18 d at 22°C. Plates were transferred to
either constant light (LL) or dark (DD), as schematically shown
(see horizontal bars). Cells were harvested at intervals (3 h),
and RNA fractions were prepared. Northern blot hybridization
analyses were carried out with probes each specific for (A)
CCA1, (B) LHY, (C) CCR2. The detected bands were also
quantified (for references, see the UBQ10 profiles in panel E).
As references, wild-type plants (Columbia) were grown for
22 d under the 12 h light/12 h dark cycle, and then they were
transferred to constant dark. RNA samples were prepared from
leaves at appropriate intervals (3 h), and then Northern blot
hybridization analyses were carried out with regard to the
CCA1, LHY and CCR2 genes (see each panel at bottom in A, B
and C). The detected bands were also quantified. (D) The
resulting quantified profiles were compared between those of
cells (red lines) and plants (blue lines), with regard to CCA1,
LHY and CCR2, as indicated. Other details were essentially the
same as those given in the legend to Fig. 1.
Circadian rhythms in Arabidopsis
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(Received November 6, 2002; Accepted January 8, 2003)
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