Future Perspectives in Plant Biology
Plant Biology in the Fourth Dimension1
Stacey Harmer*
Department of Plant Biology, College of Biological Sciences, University of California, Davis, California 95616
The nature of time has long been an obsession for
philosophers, scientists, and laymen. This nearly universal interest in time is encapsulated by the fact that
the quotation “Time is nature’s way of keeping everything from happening at once” has been attributed to
both Woody Allen and Albert Einstein! Although
some argue that time is an illusion, ongoing work
has revealed that most organisms possess an internal
oscillator, the circadian clock, which has a pervasive
influence on growth, development, and responses to
the environment. Studies of circadian rhythms in
plants reveal that because everything doesn’t happen
at once, plants have evolved sophisticated mechanisms to partition physiological processes so that they
occur at the most advantageous times, both on seasonal and daily scales.
A BRIEF HISTORY OF TIMING
It is apparent to even the most casual observer that
plant physiology is strongly influenced by time: for
example, plants generally fix carbon during the day
but are carbon consumers at night, and plant reproduction is strongly tied to the seasons. Although these
changes in physiology are clearly linked to changes in
the environment, they are also strongly influenced
by the plant circadian clock. Studies spanning the
past 300 years have revealed that many features of
plant physiology are affected by the circadian clock
(McClung, 2006). Clock-influenced processes range
from once-in-a-lifetime events such as germination
and the transition from vegetative to reproductive
growth, to annual events such as the onset of flowering
or winter dormancy, to daily processes such as rhythmic movements of petals and leaves and emission of
floral fragrance. Ongoing, intensive investigation into
clock-regulated processes are revealing that plants are
even more sophisticated time keepers than we previously thought, with most or all aspects of plant
physiology influenced by the circadian system. The
molecular nature of the plant oscillator is now becoming clear, and the development of mathematical
models describing this clock are raising hopes that
we will someday be able to predict how particular
1
This work was supported by the National Institutes of Health
(grant no. GM069418) and the National Science Foundation (grant
no. 0616179).
* E-mail [email protected].
www.plantphysiol.org/cgi/doi/10.1104/pp.110.161448
environmental conditions will interact with a given
genotype to shape plant growth and development in
the real world.
HOW DO PLANTS TELL TIME?
One hallmark of circadian rhythms is that these
roughly 24-h cycles persist in constant environmental
conditions; that is, although they can be reset by
external cues such as changes in light or temperature,
they do not depend upon these outside influences for
their rhythmicity. In all organisms examined thus far,
circadian rhythms are generated in a cell autonomous
fashion. In eukaryotes as disparate as fungi, animals,
and plants, the clock mechanism involves interlocked
transcriptional feedback loops. However, despite these
similarities, the clock genes involved in these transcriptional feedback loops are not conserved across
higher taxa (Harmer, 2009). Extensive functional studies, carried out primarily in Arabidopsis (Arabidopsis
thaliana), have begun to reveal the architecture of the
circadian system in higher plants. The central transcriptional feedback loop consists of the Myb-like
transcription factors CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL
(LHY) and the pseudo-response regulator protein
TIMING OF CAB EXPRESSION1 (TOC1; Fig. 1, loop
A). CCA1 and LHY repress TOC1 expression by binding to its promoter. TOC1 in turn indirectly promotes
expression of CCA1 and LHY; in the case of CCA1, this
is accomplished in part by inhibition of the transcription factor CHE (Fig. 1, loop D). Additional regulatory
loops are also vital for clock function in Arabidopsis.
The TOC1 homologs PSEUDO-RESPONSE REGULATOR5 (PRR5), PRR7, and PRR9, inhibit expression of
LHY and CCA1. CCA1 and LHY in turn promote expression of PRR7 and PRR9, forming yet another
negative feedback loop (Fig. 1, loop B). An additional
transcriptional feedback loop has been proposed
based on mathematical modeling, although the molecular identities of its components are currently
unknown (Fig. 1, loop C). Additional genes such
as EARLY FLOWERING3 (ELF3), ELF4, and LUX
ARRYTHMO/PHYTOCLOCK1 (LUX/PCL1) are known
to function in the circadian network but have not yet
been assigned specific functions.
Posttranscriptional regulation is also essential for
proper clock function. The F-box protein ZEITLUPE
(ZTL) targets TOC1 for degradation in a manner modulated by PRR3. ZTL protein stability is in turn regulated by its interactions with GIGANTEA (GI; Fig. 1).
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467
Harmer
Figure 1. The plant clock is composed of multiple interacting transcriptional feedback loops (A–D). Posttranslational regulation is also
key to clock function (pink circle). Figure adapted from Harmer (2009).
Additional posttranscriptional mechanisms are also
key (Somers et al., 2007).
How will unknown components of the plant clock
(e.g. X and Y in Fig. 1) be identified? Recent genetic
screens have identified multiple alleles of known genes,
suggesting they are approaching saturation. However,
high-throughput genomic approaches may reveal
members of gene families with partially redundant
functions in the circadian system (Pruneda-Paz et al.,
2009). Careful genetic studies on higher-order mutants
will then be required to determine their respective
contributions to clock function.
Recent studies of the plant circadian clock indicate
that its molecular composition can vary between different cell and organ types. For example, PRR3 appears to modulate TOC1 stability in plant vasculature
but not in other cell types (Para et al., 2007). CCA1 and
LHY are expressed but not able to inhibit TOC1
expression in dark-grown roots (James et al., 2008). It
seems likely that as we continue to study cell- and
organ-specific clock function we will find many additional examples of specializations in clock architecture
in distinct tissues. The underlying reasons for these
specializations may become apparent as we delve
more deeply into the mechanisms connecting the
circadian clock and plant physiology.
Recent studies have also shown that the clock is
more thoroughly integrated with other signaling pathways than previously suspected. At least one-third of
the plant genome is under circadian regulation. In
addition, genes regulated by any one of the major
plant hormones are more likely to be clock regulated
than expected by chance, suggesting specific clock
involvement in these pathways (Covington et al.,
2008). Indeed, functional studies have shown that the
circadian clock modulates plant sensitivity to exogenous auxin: even when plants are maintained in
constant environmental conditions, responsiveness to
auxin varies at different times of day (Fig. 2; Covington
and Harmer, 2007). The clock also regulates responsiveness to abscisic acid (Legnaioli et al., 2009), and it
seems likely that the clock will modulate other hormone pathways as well. Clock control of multiple
signaling pathways may help to coordinate plant
growth (Nozue et al., 2007; Michael et al., 2008). Intriguingly, exogenous hormone treatment can also
influence circadian clock function (Robertson et al.,
2009), suggesting that not only can the clock influence
hormone signaling, but that hormone signaling may
also influence the clock.
The circadian clock can also affect plant sensitivity
to a variety of environmental stimuli. For example,
chilling-sensitive plants show differential damage
when the same cold treatment is applied at different
circadian times (Rikin et al., 1993). Similarly, plant
sensitivity to light or to drought stress varies in a
circadian manner (Millar and Kay, 1996; Legnaioli
et al., 2009). Responsiveness to biotic stimuli may also
be influenced by the clock. For example, many genes
regulated by mechanical wounding are also clock
regulated, suggesting that plant defense responses
may be modulated by the circadian system (Walley
et al., 2007).
We are just beginning to appreciate the extensive
connections between the circadian clock and plant
metabolism. Clock genes influence nitrogen assimilation, tricarboxylic acid cycle intermediates, and gen-
The Circadian Clock and Physiology—Complex
Interacting Networks
Early representations of the circadian system consisted of three tidy categories: inputs, the core oscillator, and output pathways. Work in many model
organisms has led to the disintegration of these boundaries, since it is now abundantly clear that key clock
genes can play roles in input to the clock and that
outputs can feed back to regulate clock function
(Harmer, 2009).
Figure 2. Clock gating of auxin response. Plants expressing the auxinsensitive reporter eDR5::LUC were treated with auxin at 4-h intervals.
The black line indicates untreated control plants while the colored lines
indicate plants treated at different circadian times. Data are from
Covington and Harmer (2007). cts, counts; LL, constant light.
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Plant Biology in the Fourth Dimension
eral signaling molecules (Dodd et al., 2007; Gutierrez
et al., 2008; Fukushima et al., 2009). The clock has long
been known to influence photosynthesis, and recent
work has demonstrated that the clock also controls the
rate at which starch is utilized during the night (Graf
et al., 2010). It now seems most appropriate to think of
the circadian system as a network that is functionally
interconnected with many (or perhaps all) signaling
and metabolic networks within plant cells.
Why has evolution led to these pervasive interconnections? Recent studies suggest that consonance
between the period of the circadian clock and environmental cycles enhances plant productivity and may
provide an adaptive advantage (Dodd et al., 2005;
Graf et al., 2010). That is, fitness is enhanced when
the circadian system is able to appropriately time
physiological processes so that they occur at the appropriate time in the day/night cycle. Modulation of clock
function may also play a role in hybrid vigor (Ni et al.,
2009).
The Beginning of Timing
An important outstanding question is how the plant
clock evolved. Comparative genomic and functional
studies suggest that the molecular nature of the plant
clock is similar across the angiosperms and in the moss
Physcomitrella patens (Okada et al., 2009; McClung,
2010). Studies in two green algae, Ostreococcus tauri
and Chlamydomonas reinhardtii, paint a more complex
picture, however. The O. tauri and C. reinhardtii genomes each contain single homologs of CCA1/LHY,
TOC1, and LUX. Functional studies demonstrated a
role for the CCA1/LHY-like and TOC1-like genes in the
O. tauri circadian clock (Corellou et al., 2009) and for the
CCA1/LHY-like and LUX-like genes in C. reinhardtii
(Matsuo et al., 2008). These functional similarities suggest that the central transcriptional feedback loop
between CCA1/LHY and TOC1 (Fig. 1, loop A) is conserved between green algae and land plants. But despite these similarities with angiosperm clocks, there
must also be fundamental differences. The O. tauri and
C. reinhardtii genomes lack additional PRR-like genes
and do not have recognizable ELF3, ELF4, GI, or ZTL
homologs (Matsuo et al., 2008; Corellou et al., 2009). In
addition, forward genetic studies in C. reinhardtii have
demonstrated that genes without obvious homologs in
higher plants are essential for normal clock function in
this species (Iliev et al., 2006; Matsuo et al., 2008). It will
be fascinating to learn the mechanistic similarities and
differences between the clocks of the green algae and
those of the land plants.
The similarity between the overall architecture of
circadian networks in higher eukaryotes coupled with
the lack of conservation of clock genes make for a
fascinating evolutionary puzzle. Are the similarities in
circadian organization in animals, plants, and fungi
due to convergent evolution, or evolution by descent?
One possibility is that the last common ancestor to
these eukaryotes possessed a circadian clock that
relied upon metabolic oscillations, and that the clock
genes that we now study evolved relatively recently to
make this primordial biochemical clock more robust
and more thoroughly integrated with environmental
input and physiological output pathways.
Clocks in the Real World
As sessile organisms, plants are exquisitely well
adapted to their local environments. However, as our
global climate undergoes rapid warming, plants are
changing their latitudinal ranges in an attempt to
offset changes in the environment (Parmesan and
Yohe, 2003). The resulting alterations in seasonal day
lengths will likely necessitate evolutionary changes in
circadian clock function for plants to maintain appropriate regulation of daily and seasonal events. Indeed,
a recent study revealed that Arabidopsis plants grown
in the field are acutely sensitive to seasonal influences,
with small differences in time of sowing resulting in
plants that either flower rapidly or overwinter before
undergoing the transition from vegetative to reproductive growth (Wilczek et al., 2009). A relatively
simple photothermal model of Arabidopsis development accurately predicted flowering time of multiple
genotypes in this study. Mathematical models describing the plant circadian clock are currently being developed (Locke et al., 2006; Zeilinger et al., 2006).
Future models may someday be used to inform breeding to optimize plant growth in diverse environments.
The above studies, and much research not discussed
due to space constraints, give us a better appreciation
of how plant physiology varies at different times of
day. Further investigation of the mechanisms underlying the plant clockwork and its integration with
pathways essential for growth and development will
help us understand how plants are able to thrive in the
ever-changing conditions of the real world.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: CCA1, At2g46830; ELF3,
At2g25930; ELF4, At2g40080; GI, At1g22770; LHY, At1g01060; LUX/PCL1,
At3g46640; PRR3, At5g60100; PRR5, At5g24470; PRR7, At5g02810; PRR9,
At2g46790; TOC1, At5g61380; and ZTL, At5g57360.
ACKNOWLEDGMENT
I would like to thank J.N. Maloof for helpful comments on the manuscript.
Received June 16, 2010; accepted July 2, 2010; published October 6, 2010.
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