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Plant Cell Physiol. 46(8): 1423–1427 (2005)
doi:10.1093/pcp/pci127, available online at www.pcp.oupjournals.org
JSPP © 2005
Short Communication
Circadian Rhythm of Circumnutation in Inflorescence Stems of Arabidopsis
Kanae Niinuma *, Nobutaka Someya 1, Makoto Kimura 2, Isamu Yamaguchi and Hiroshi Hamamoto
Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa, 230-0045 Japan
;
We investigate the modulation of circumnutation in
inflorescence stems of Arabidopsis to determine the circadian regulation of circumnutation. Under constant light
conditions (LL), circumnutation speed in wild-type plants
fluctuates, with the phase of the highest speed at subjective
dawn; the period length is close to 24 h. toc1 appears to
shorten the period and elf3 causes an arrhythmic phenotype in circumnutation speed in LL, suggesting that a common circadian clock may control both circumnutation
speed and other circadian outputs. These results highlight
for the first time a role for a circadian clock in the regulation of circumnutation based on genetic analysis of
Arabidopsis.
Keywords: Arabidopsis — Circadian rhythm — Circumnutation — early flowering 3 (elf3) — timing of cab expression 1
(toc1).
Abbreviations: CCR2, COLD CIRCADIAN RHYTHM RNA
BINDING 2; elf3, early flowering 3; FFT-NLLS, fast Fourier transform-non-linear least squares; FRP, free-running period; LD, 12 h
light, 12 h dark cycles; LL, constant white light condition; RAE, relative amplitude error; RT–PCR, reverse transcription–PCR; toc1, timing
of cab expression1; UBQ10, UBIQUITIN10;
Circumnutation is a revolving movement (Darwin and
Darwin 1880) of elongating plant organs, such as stems, hypocotyls and tendrils, that occurs in many plant families. The path
described by the circumnutating organ tip is pendulum-like,
elliptical or circular (Shuster and Engelmann 1997). The bending in circumnutation has usually been considered a consequence of unequal growth on opposite sides of the organ.
However, Caré et al. (1998) reported that the revolving movement is related to partly reversible length variations in the cells
of the bending zone. The variations are caused by differences
in water content between the convex and concave sides of the
bending zone, associated with turgor and ion concentration
differences (Lubkin 1994), as occur in stomatal aperture
(MacRobbie 1981) and in petal opening (Claus 1926). The
circumnutating organ has areas with rhythmically changing
1
2
*
cell volumes that have been shown to move around the organ
(Millet et al. 1988).
There are some common characteristics in plant organ
movements. One of them is circadian rhythm. Circadian
rhythms are oscillations in the biochemical, physiological and
behavioral functions of organisms that occur with a periodicity
of approximately 24 h (McClung and Kay 1994). They are
generated by a circadian clock that is synchronized by environmental cues. In higher plants, the circadian clock controls
a wide range of biological processes, including the expression of various genes (Schaffer et al. 2001), leaf movement
(Engelmann and Johnsson 1998), hypocotyl elongation
(Dowson-Day and Millar 1999) and inflorescence stem elongation (Jouve et al. 1998). Potential components of the circadian
clock or those closely associated with circadian clock functions have been identified in Arabidopsis (Alabadí et al. 2002,
Mizoguchi et al. 2002, Eriksson and Millar 2003, Schultz and
Kay 2003, Salomé and McClung 2004). TIMING OF CAB
EXPRESSION 1 (TOC1; also called Arabidopsis PSEUDORESPONSE REGULATOR 1, APRR1; Millar et al. 1995,
Makino et al. 2000) encodes one of the best candidates for a
plant circadian oscillator component. The EARLY FLOWERING 3 (ELF3) gene product was proposed to function in a light
input pathway to the circadian oscillator.
The two topics—circumnutation and circadian rhythm—
have been well studied, and there are some reports of the
relationship between circumnutation and biological rhythms.
Shuster and Engelmann (1997) reported that Arabidopsis seedlings showed a very wide range of circumnutation rhythm. In
Helianthus annus, circumnutation speed and trajectory length
exhibit daily modulation under 16 h light/ 8 h dark (Buda et al.
2003). However, there have been no studies genetically examining the involvement of the circadian clock in the modulation
of circumnutation using Arabidopsis mutants.
To reveal the presence of a circadian rhythm in circumnutation, we examined circumnutation as described below. First,
we adopted ‘the duration of one nutation’ as an index. There
have been many reports about circumnutation using various
indexes, including ‘trajectory length’ (Buda et al. 2003), ‘the
organ’s tip loci’ (Hatakeda et al. 2003) or ‘the duration of one
nutation’ (Buda et al. 2003). However, no standard index to
evaluate circumnutating plant organs has been established.
Present address: Institute of Biological Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572 Japan
Present address: National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604 Japan
Corresponding author: E-mail, [email protected].
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Circadian rhythm of circumnutation in Arabidopsis
Fig. 1 Acquisition of circumnutation data. (A) Illustration of one
complete nutation. The images were obtained at 5 min intervals, and
data at 10 min intervals are shown. The coordinates of the inflorescence stem tip (closed circle) were analyzed by the NIH image program. (B) The trajectory of one nutation, obtained from the
coordinates in (A). The duration of one nutation was estimated at
120 min in this case.
Some indexes can be influenced by multiple factors, because
circumnutation is a complicated integration of physiological
phenomena such as elongation and reversible cell volume variations (Lubkin 1994). ‘The duration of one nutation’ could be
independent of these complex phenomena, especially independent of the longitudinal elongation rate, because this index
would reflect the transfer speed of areas with changing cell volume around the bending zone in the circumnutating organ.
‘The duration of one nutation’ is referred to here in terms of
circumnutation ‘speed’ for the sake of simplicity.
Plants were grown under 12 h light/ 12 h dark cycles (LD)
before transferring to constant white light conditions (LL). The
movements of the inflorescence stems were monitored under
LL with digital video cameras, which recorded images of the
circumnutating stems at intervals of 5 min for several days. We
analyzed the modulation of circumnutation on the basis of these
images (Fig. 1A). In Arabidopsis C24 wild-type plants, circumnutation speed was not uniform. It fluctuated, with the phases
of highest and lowest speed at subjective dawn and dusk,
respectively (Fig. 2A). Free-running periods (FRPs) and the
best-fit curve were estimated from each of nine plants by biomathematical analysis. The fast Fourier transform-non-linear
least squares method (FFT-NLLS; Plautz et al. 1997) was
applied in order to estimate the FRPs. This revealed that circumnutation speed fluctuated rhythmically with a period close
Fig. 2 The circadian patterns of circumnutation speed in A. thaliana
inflorescence stem and of CCR2 expression. Plants were grown under
12 h light, 12 h dark cycles (LD) before each examination under continuous light (LL). Time in LL shows hours after transfer to LL. (A)
Circadian rhythms of circumnutation speed in A. thaliana C24 wild
type under constant light. We traced nine inflorescence stems, and this
figure shows two representative examples. (B) The expression of
CCR2, 72–144 h after transfer of the plants to LL. Exp, experiment.
to 24 h under LL in C24 wild-type plants (23.9 ± 3.0 h, mean ±
SD; n = 9).
To verify that the circadian clock was well entrained to the
employed conditions, we assayed CCR2 (COLD CIRCADIAN
RHYTHM RNA BINDING 2) expression under LL using realtime reverse transcription–PCR (RT–PCR). This gene shows a
robust circadian rhythm with peak expression at circadian time
(CT) 8–12 h after dawn (Kreps and Simon 1997). The plants
were grown under LD cycles and then transferred to LL, in the
same way as in the above circumnutation experiment. The
CCR2 expression had a circadian oscillation with the peak at
approximately CT 10 h, even 72–144 h after transfer of the
plants to LL as reported (Fig. 2B; Kreps and Simon 1997).
We then investigated the modulation of circumnutation
speed in two loss-of-function mutants, toc1 and elf3. The toc1
mutation shortens the period for all circadian processes analyzed (Somers et al. 1998). In toc1 and its parental line C24,
circumnutation speed fluctuated rhythmically with a period
length of 24.2 ± 3.0 h (n = 9) and 21.9 ± 5.12 h (n = 10),
respectively (Fig. 3). This suggests that the toc1 mutation
shortens the period of circumnutation speed, the same as in
other circadian outputs.
elf3 causes arrhythmic circadian outputs under LL (Hicks
et al. 1996). The nutation speed was almost constant in the elf3
Circadian rhythm of circumnutation in Arabidopsis
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Fig. 3 The shortened period in toc1. The modulation of circumnutation speed in toc1 (A) and C24 (B) in LL. We traced 11 (toc1) and nine
(C24) inflorescence stems, respectively. Two representative patterns
are shown. Time in LL shows hours after transfer of the plants to LL.
Exp, experiment.
mutant lines and the elf3 mutation seemed to abolish the
rhythm of modulation of circumnutation speed (Fig. 4A). In
contrast, its parental line, Col-0, showed a robust circadian
rhythm under LL (Fig. 4B). Fig. 4C and D shows the distribution of rhythmic periods within the 5–60 h range calculated by
FFT-NLLS. The periods were plotted against the associated relative amplitude error (RAE). RAE is used to determine the significance of a given rhythm, which ranges from 0 (perfect sine
wave) to 1 (rhythm not significant), and RAEs >0.7 represent
very altered circadian rhythms (Más et al. 2003). Col-0 plants
displayed clustered periods in the circadian range (15–35 h),
with low RAEs (Fig. 4D), which is indicative of circadian
rhythms. In contrast, the periods in elf3 had no tendency
toward any particular range, with high RAE (Fig. 4C). Moreover, only 30% (n = 10) of elf3 plants yielded periods in the
circadian range (15–35 h) with RAEs <0.7, a value that is very
low compared with that of Col-0 at 63.6% (n = 11). These
results suggest that the modulation of circumnutation speed is
arrhythmic in elf3 mutants, which is consistent with a previous
report in other rhythmic outputs (Hicks et al. 1996). The results
in toc1 and elf3 demonstrated genetically that the circadian
clock controls circumnutation speed, for the first time.
Fig. 4 Arrhythmic circumnutation in elf3. The modulation of circumnutation speed in elf3 (A) and Col-0 (B) was monitored in LL. We
traced 10 (elf3) and 11 (Col-0) inflorescence stems. Two representative patterns are shown. Time in LL shows hours after transfer of the
plants to LL. Rhythmic periods in the 5–60 h range were estimated by
FFT-NLLS and plotted against the associated relative amplitude error
(RAE) for elf3 (C) and Col-0 (D). RAE is used to determine the significance of a given rhythm, and RAEs >0.7 represent greatly altered circadian rhythms (Más et al. 2003). Exp, experiment.
Several models have been proposed to explain the circumnutation mechanism. The ‘internal oscillator model’, which
was created by Darwin and Darwin (1880), predicts the existence of oscillators driving nutation independent of external
stimuli. The ‘gravitropic shoot model’ views nutation as a continuing gravitropic response (Israelsson and Johnsson 1967).
Furthermore, a model that combined these two models has
been proposed; it predicts that the internal oscillator causes
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Circadian rhythm of circumnutation in Arabidopsis
plant organs to oscillate in the absence of gravity, but that the
activity of the circumnutation is amplified by a gravitropic
feedback system (Hatakeda et al. 2003). We demonstrated that
the modulation of circumnutation speed is regulated by a circadian clock. Our results point to the existence of an internal
oscillator that controls the speed of circumnutation, and this
oscillator is under the control of a circadian system containing
TOC1 and ELF3. Further analysis of mutations on both circadian rhythm and gravitropism will provide us with a much
clearer image of the mechanisms of how these affect circumnutation speed in Arabidopsis.
portray arrhythmicity (Plautz et al. 1997). The RAE is used to determine the significance of a given rhythm.
Materials and Methods
Alabadí, D., Yanovsky, M.J., Más, P., Harmer, S.L. and Kay, S.A. (2002) Critical role for CCA1 and LHY in maintaining circadian rhythmicity in
Arabidopsis. Curr. Biol. 12: 757–761.
Buda, A., Zawadzki, T., Krupa, M., Stolarz, M. and Okulski, W. (2003) Daily
and infradian rhythms of circumnutation intensity in Helianthus annuus.
Physiol. Plant. 119: 582–589.
Caré, A.F., Nefed’ev, L., Bonnet, B., Millet, B. and Badot, P.M. (1998) Cell
elongation and revolving movement in Phaseolus vulgaris L. twinning
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in a spinning world. Plant Physiol. 132: 732–738.
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Takahashi, H. (2003) Gravitropic response plays an important role in the
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circumnutating plants. Bull. Math. Biol. 56: 795–810.
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circadian rhythm in Arabidopsis thaliana. Plant Cell Physiol. 41: 791–803.
Más, P., Alabadí, D., Yanovsky, M.J., Oyama, T. and Kay, S.A. (2003) Dual role
of TOC1 in the control of circadian and photomorphogenesis responsesin
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McClung, C.R. and Kay, S.A. (1994) Circadian rhythms in Arabidopsis
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We used Arabidopsis thaliana C24 wild-type, toc1-1 mutant, the
toc1-1 parent line that is the transgenic CAB2::LUC line in C24 wildtype (referred to as C24; Millar et al. 1995), elf3-1mutant and elf3-1
parental line Columbia gl1 (referred to as Col-0). They were obtained
from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA).
Plant seeds were sown on commercial artificial soils (Jiffy-7,
Jiffy Products International AS, Kristiansand, Norway), and plants
were grown in a plant growth chamber at 22 ± 1°C under LD cycles of
cool white fluorescent light with a photon flux density of about
90 µmol m–2 s–1. The plants were analyzed at the age of approximately
1 month, when the inflorescence stems were about 40 (±10) mm long.
The movement of the inflorescence stems was monitored under
LL of cool white fluorescent light at 32–40 µmol m–2 s–1, from above
with a video camera (DCR-TRV30, Sony Corporation, Tokyo, Japan).
Plants were grown at 22 ± 1°C in a temperature-controlled growth
chamber. Data were recorded at 5 min intervals for several days.
On the basis of the monitored images, we analyzed the duration
of one circumnutation. One circumnutation was a movement, the
beginning and end of which were defined as maximal at an ordinate
value of the inflorescence tip (Fig. 1A, B; Buda et al. 2003). The
images were traced by plotting the coordinates of the inflorescence
stem tips by using the NIH image program (National Institutes of
Health, Bethesda, MD, USA). We monitored the circumnutation from
0 h after transfer to LL, but we did not trace the circumnutation when
the inflorescence stem was too short or had fallen because it had
become too long.
Total RNA was prepared using TRIzol Reagent (Invitrogen,
Carlsbad, CA, USA) from fresh C24 wild-type plants. Real-time RT–
PCR was performed with an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA), as described by
Lamb et al. (2002). The primers for the CCR2 were CCR2-5′ (5′GGAGGATGGTAATTCCTTTAATTAGGT-3′) and CCR2-3′ (5′-CACACAAAACCAAGTAGAAGCATAACA-3′). The probe for CCR2
was (5′-TGGGATTACCAATGAATGTTCTCTCTCTCGC-3′). The
probe for a control gene (UBIQUITIN 10; UBQ10) was (5′-CTCACCGGAAAGACAATCACCCTCGA-3′). The primers for UBQ10 were
the same as described previously (Wang and Tobin 1998). The RT–
PCR analysis was performed twice with independent RNA samples.
Similar results were obtained from these experiments.
The data on circumnutation speed and CCR2 expression were
analyzed by the FFT-NLLS program, as described previously (Plautz et
al. 1997). To estimate the mean circadian period, the results were
restricted to the period falling within the circadian range (15–35 h), as
described by Dowson-Day and Millar (1999). In addition, all period
estimates within the range 5–60 h were plotted against their RAEs to
Acknowledgments
We are grateful to the Arabidopsis Biological Resource Center
(ABRC) for providing the mutant and wild-type seeds. We thank our
colleagues for their support and helpful comments during our work,
especially Dr. Gouthu Satyanarayana for his valuable advice. We also
thank Mr. Coen Arts for improving the English of this manuscript.
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Circadian rhythm of circumnutation in Arabidopsis
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(Received April 7, 2005; Accepted May 11, 2005)