Annurev-Arplant-043014-115619 The Intracellular Dynamics of Circadian Clocks Reach For The Light of Ecology and Evolution

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Circadian Clocks Reach for the
Light of Ecology and Evolution
Annu. Rev. Plant Biol. 2016.67:595-618. Downloaded from www.annualreviews.org
Andrew J. Millar
Access provided by 190.236.203.234 on 08/02/23. For personal use only.
SynthSys and School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF,
Scotland, United Kingdom; email: andrew.millar@ed.ac.uk
Annu. Rev. Plant Biol. 2016. 67:595–618 Keywords

First published online as a Review in Advance on circadian rhythms, biological clocks, plant photoperiodism, flowering,
November 19, 2015
Arabidopsis thaliana, systems biology
The Annual Review of Plant Biology is online at
plant.annualreviews.org Abstract
This article’s doi: A major challenge for biology is to extend our understanding of molecu-
10.1146/annurev-arplant-043014-115619
lar regulation from the simplified conditions of the laboratory to ecologi-
Copyright c 2016 by Annual Reviews. cally relevant environments. Tractable examples are essential to make these
All rights reserved
connections for complex, pleiotropic regulators and, to go further, to link
relevant genome sequences to field traits. Here, I review the case for the bio-
logical clock in higher plants. The gene network of the circadian clock drives
pervasive, 24-hour rhythms in metabolism, behavior, and physiology across
the eukaryotes and in some prokaryotes. In plants, the scope of chronobiol-
ogy is now extending from the most tractable, intracellular readouts to the
clock’s many effects at the whole-organism level and across the life cycle,
including biomass and flowering. I discuss five research areas where recent
progress might be integrated in the future, to understand not only circadian
functions in natural conditions but also the evolution of the clock’s molecular
mechanisms.
595
Contents
1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596
2. PERVASIVE DAILY RHYTHMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
2.1. Clock-Controlled Gene Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
2.2. Canonical Outputs from the Plant Clock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
2.3. A Broader Set of Clock Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
3. THE CLOCK’S TIMING MECHANISMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
3.1. The Transcriptional-Translational Feedback Loops . . . . . . . . . . . . . . . . . . . . . . . . . . 601
3.2. Abstracting to Understand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
3.3. Environmental Inputs and Modifying Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
3.4. The Ecology of Oscillators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604
4. VARIANTS IN CLOCK GENES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
4.1. Crop Varieties and Breeding Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605

4.2. Natural Genetic Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606

5. PHYSIOLOGICAL IMPORTANCE AND ENVIRONMENTAL
CONDITIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
5.1. Advantages of Circadian Timing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606
5.2. Clocking the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
6. UNDERSTANDING THE RHYTHMIC PHENOTYPE . . . . . . . . . . . . . . . . . . . . . . . 608
6.1. Plant Rhythms in the Field: Which Rhythms Are Selected? . . . . . . . . . . . . . . . . . . . 608
6.2. A Community Model to Study Complex Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
Behavior (in an
organism):
the genome’s way of 1. INTRODUCTION
coping with the Understanding how a particular molecular regulation contributes to the physiology, behavior,
environment; it
and selective advantage of a complex organism in a relevant environment is extremely difficult.
centers on movement
in animals but on Convincingly linking molecular regulation from the genotype to a whole-organism phenotype is
biochemistry and hard enough. The progress in five areas of research suggests that, in the case of the plant circadian
development in plants clock, we can extend our understanding of molecular regulation from the simplified conditions
Behavior (in a of the laboratory to environments that are ecologically relevant, and from the most tractable
dynamical system): intracellular readouts to the clock’s many effects across the plant life cycle. We might then also
the form of a system’s understand how the environment controls the plant clock on a much longer timescale, through
change over time, such
as oscillation
the evolution of the clock’s molecular regulation, and consequently how rational manipulation
might fine-tune biological timing in the future.
Circadian clock: in a
narrow sense, a small
Biological clocks are thought to have evolved in response to the Earth’s 24-hour rotation,
or minimal set of which drives the predictable day-night cycle of light and temperature in the environments of
components that almost all organisms. Photosynthetic organisms face a particular challenge because sunlight—the
generate circadian energy supply for their metabolic systems—is predictably unavailable for half of that cycle. Adding
rhythms; the term can complication, the duration of daylight within the cycle can vary widely across the seasons. The
also overlap with
circadian system
seasonal variation in photoperiod (day length) depends on the organism’s latitude, changing by
only minutes at the equator but from constant light to constant darkness at the poles.
Photoperiod:
the duration of light
This circuit of understanding, from genotype to phenotype and back, crosses all the life science
within the day-night disciplines, from molecular and cell biology to physiology, ecology, evolution, and population
cycle genetics. What makes chronobiology, the study of biological timing, a useful thread to connect
these disparate fields? First, the clock is a pervasive regulator. It drives so many 24-hour rhythms
596 Millar
that it affects many aspects of plant physiology, including important crop traits (Section 2). Second,
clock function depends intimately on environmental signals, which ensure that the endogenous
biological rhythms are set appropriately to local time. The circuit of gene regulation that creates
Clock-regulated
the circadian clock (Section 3) is therefore likely to be affected by present and future climate processes:
change, which will in turn affect all the downstream clock-regulated processes. Third, the plant conceptually, pure
clock system has recently been tested for natural genetic variation (Section 4). Clock gene variants outputs of the clock
have also been selected by plant breeders in the past to expand the geographical range for particular that do not feed back
to alter circadian
crops, allowing the plants to thrive in new environments. Fourth, the circadian clock has evolved to
timing; in practice,
address biological challenges that are ubiquitous, affecting most organisms in most environments clock outputs might
(Section 5). Circadian biologists therefore routinely compare results among species and expect that feed back indirectly
the concepts learned in one species will apply broadly to many others. The comparisons are made Mathematical model:
easier because our focus is on a single dimension (time) with an obvious and universal coordinate the description of a
system (the day-night cycle). This broad view is one of the major attractions of chronobiology, biological system or
process formalized in a
justifying the inclusion here of examples from other taxa, especially where we lack comparable
mathematical
results from plants. Finally, chronobiology has routinely included formal mathematical models,
representation, such as
giving this area a head start on the multiscale models that are now required to link intracellular ordinary differential
dynamics to the whole-organism scale and beyond (Section 6). equations
I refer below to a few of the recent reviews on each specific area. Broader treatments are included Laboratory model
in a retrospective from Colin Pittendrigh, a British American founder of the field (113), and in species: a species for
a comprehensive textbook (40). An overview of the historical consensus on transcriptional clock which experimental
mechanisms in laboratory model species (155) also presents a comparison across all taxonomic protocols, tools, and
resources have been
groups, which is now rare.
developed to facilitate
laboratory studies
2. PERVASIVE DAILY RHYTHMS Circadian rhythm:

a self-sustaining
Early observations on plants first identified endogenous circadian rhythms (24). Research on biological rhythm with
many plant species has contributed to the field and continues to do so (88). The most noticeable a temperature-
rhythms in plants included rhythmic movements of leaves and petals (38), rhythmic emission of compensated period
close to 24 hours,
floral fragrance (48), and rhythmicity in photosynthesis (34) and in the responses to day length
which is normally
(133). A shared vocabulary describes the properties of circadian rhythms in all organisms (Figure 1; entrained to the
see also sidebar Rhythmic Properties). day-night cycle
2.1. Clock-Controlled Gene Expression

The diversity of plant rhythms suggests that the clock mechanism must control many different
biochemical or molecular processes. The potential breadth of rhythmicity was most dramatically
revealed by the first large-scale study of the rhythmic transcriptome in any eukaryote (60), which
is also among several advances from research on the laboratory model species Arabidopsis thaliana
(mouse ear cress). The rhythmic genes functioned in a huge variety of metabolic, developmental,
and stress response pathways, which were apparently identified by reading several hundred papers,
because the Arabidopsis genome sequence, published in the same year, had only sparse annotation.
Genes that functioned together were often expressed at the same phase, suggesting many more
biological processes that might be rhythmically controlled. What proportion (and which) of these
RNA rhythms are physiologically relevant remains largely an open question.
More recent analyses have confirmed this temporal clustering, with at least 30% of transcripts
showing circadian clock control in Arabidopsis under constant conditions and up to 90% of tran-
scripts scored as rhythmic under diel light and/or temperature conditions (23, 89). Similar results
are now available from ten plant species and two green algae (reviewed in 54, 109) (see sidebar
www.annualreviews.org • Biological Timing in Higher Plants 597

a Phases, ϕ Amplitude Period, τ b 20 6L:18D

12L:12D
18L:6D
GI mRNA level (A.U.)

Activity
ZT (h) 12 0 12 0 12 0 12 24 36 48 60 0.2 6L:18D

12L:12D
LD LL
18L:6D
0.02
c 0 12 24 36 48 60 72
8L:16D 16L:8D Time (h)
CO CO d
Phase of mRNA midpoint (h)

24
mRNA level
CO fall
18
CO rise
12 GI fall
FT FT GI rise
6 CCA1 fall
CCA1 rise
0
–6
0 8 16 24 0 8 16 24 6 12 18
Time (h) Time (h) Photoperiod (h)
Figure 1
Circadian rhythms in Arabidopsis. For additional discussion of these concepts, see the Rhythmic Properties sidebar. (a) Basic properties
of circadian rhythms under light:dark cycles (LD) and constant light (LL). The activity of a rhythmic, biological process is plotted on
the y axis. The Zeitgeber time (ZT) on the x axis is measured in hours from the last onset of light. The white bars represent light, the
black bars represent dark, and the gray bars represent predicted darkness during LL. (b) Rhythmic GI mRNA [in arbitrary units (A.U.)]
from quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays of seedlings under 6L:18D, 12L:12D, and 18L:6D
followed by LL. The error bars show the ranges of biological duplicates. Data replotted from Reference 41 on a logarithmic scale, to
show the high amplitude of rhythmic regulation in LD and the lower amplitude in LL. (c) Graphs showing that an external coincidence
between light and rhythmic CO mRNA (shaded area) induces FT expression and flowering under long photoperiods. The darker
shading and thick arrow denote greater induction in the late day, when FKF1 (not shown) is expressed. (d ) Graph showing that the
midrise and midfall phases of mRNA waveforms under LD cycles change with photoperiod. The white and black areas show the light
and dark intervals, respectively. A dawn-tracking rhythm would follow the horizontal line of dawn (dusk sensitivity = 0); a dusk-
tracking rhythm would follow the diagonal of dusk (dusk sensitivity = 1). The various dusk sensitivities of these RNA markers form
part of the Arabidopsis clock’s dynatype (26). Data replotted from Reference 41; the phases for GI were taken from plots of the data in
panel b, on a linear scale.
Online Resources for Plant Chronobiology). Promoter sequences that are enriched among genes
expressed at the same phase include the Evening Element, which is bound by the clock proteins
LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1
(CCA1) (see Section 3). The rhythmic transcriptional regulators of the clock mechanism provide
one mechanism for pervasive rhythmic control, as chromatin immunoprecipitation studies show
that these proteins individually interact with hundreds to thousands of downstream genes (69).
We still need to understand how these rhythmic regulators interact on their target promoter
sequences in order to explain the earlier observations that several regions of each promoter can
support rhythmic expression (1, 60). Designing synthetic promoters that target expression to any
desired phase will then validate that understanding.
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RHYTHMIC PROPERTIES
Our vocabulary for biological rhythms supplements the mathematical description of oscillations with properties
that are shared by the circadian rhythms of all organisms. Figure 1a illustrates the basic properties. Time is usually
measured from the last onset of light, defined as Zeitgeber time 0 (ZT0). A Zeitgeber (“time-giver”) is an entraining
signal. The absolute amplitude is measured as peak level minus midpoint, as shown in the figure, but for assays that
return arbitrary units, relative amplitude (peak divided by trough) is more often cited. Phase refers to a point on
the waveform and is measured from a reference point, usually ZT0 under a light:dark cycle (LD, a diel cycle). The
12-hour photoperiod illustrated in Figure 1a is denoted 12L:12D. Period refers to the time between successive
occurrences of the same phase. For a stably entrained clock, as in LD, the clock’s period is by definition the same
as T, the period of the entraining diel cycle. The endogenous period τ is measured under constant conditions, such
as constant light (LL). This period is rarely exactly 24 hours, so the phase progressively changes with respect to the
predicted times of dawn and dusk (compare with the dashed lines at ZT0 and ZT48 in Figure 1a).
The data in Figure 1b illustrate how light directly affects clock components to entrain the clock, in this case
increasing mRNA expression of GI in Arabidopsis. The logarithmic scale shows the >100-fold relative amplitude of
regulation in LD, which progressively decreases in LL. The fall in relative amplitude is referred to as damping.
Figure 1c illustrates the concept of external coincidence in the photoperiod response pathway (reviewed in 133).
Under short days of 8L:16D, little CO mRNA is present during the light interval. The shading indicates that the
CO protein is also unstable in the early day, so little expression of CO’s target, the flowering gene FT, is activated.
Under long days of 16L:8D, CO mRNA is abundant in the extended day and the CO protein is also stabilized,
inducing high FT levels (and hence rapid flowering).
Figure 1b and Figure 1d show that the waveform of the clock components also reflects particular entraining
signals, in this case 6L:18D, 12L:12D, or 18L:6D. The effect on the clock’s phase is detectable in subsequent LL, for
example, in the GI peak at approximately 54 hours in Figure 1b. The change in phase under different photoperiods
is termed dusk sensitivity (41). The observed change in phase under LL is small compared with the change in
photoperiod, so the Arabidopsis clock is termed dawn-dominant; a larger phase change was observed in constant
darkness (DD) (41). The waveforms under LD combine direct (acute) environmental responses and feedback from
other clock components (see Figure 2, below), such that the midrise and midfall phases are delayed by several hours
under long photoperiods (Figure 1d ). These dynamic behaviors of the clock system can be referred to collectively
as the system’s dynatype, by analogy with the phenotype (26).
New approaches to studying the global transcriptional network are now contributing to our
understanding of the output network downstream of the clock in Arabidopsis. First, systematic
analyses of DNA binding site preferences have gone beyond focused studies on specific clock
proteins (55, 63) or promoter sequences (120). Clock-relevant proteins have been analyzed in
broad-scale studies using protein-binding microarrays, including for CCA1 and REVEILLE 1
(RVE1) among over 30 Arabidopsis proteins (51) and for Arabidopsis LUX ARHYTHMO [LUX,
also known as PHYTOCLOCK 1 (PCL1)] and Ostreococcus tauri transcription factors among more
than 1,000 other eukaryotic transcription factors (147). Second, nuclease-sensitivity assays cou-
pled with high-throughput sequencing (134) are now extending, genome-wide, the footprinting
methods first used to identify protein binding on the early examples of clock-regulated promoters
(1). Importantly, neither of these approaches requires specific antibodies against the clock proteins
or epitope-tagged proteins in transgenic plants, which are time-consuming to produce. Instead,
rhythmically changing nuclease sensitivity at a particular promoter sequence may implicate a spe-
cific set of potential DNA-binding factors, each with an associated probability. The mutant lines
available in Arabidopsis for these binding factors, or inducible misexpression constructs, can then

ONLINE RESOURCES FOR PLANT CHRONOBIOLOGY
Reference 49 describes publicly accessible time-series data for clock gene RNAs in multiple conditions and
genotypes.
Diurnal (http://diurnal.mocklerlab.org) is a database of transcriptome time-series data for several plant species,
scored for RNA expression phase and waveform (94).
Plant Systems Biology Modelling (PlaSMo; http://www.plasmo.ed.ac.uk) is a repository of models for plant
growth and systems biology, including circadian clock models (28).
The Biological Data Repository (BioDare; http://www.biodare.ed.ac.uk) is a portal for time-series data other
than transcriptomes, with tools to document, analyze, and visualize rhythmic data (49, 95).
The Arabidopsis Information Portal (AraPort; http://www.araport.org) and The Arabidopsis Information
Resource (TAIR; http://www.arabidopsis.org) are databases for the model organism Arabidopsis thaliana that
also link to many related resources.

The Nottingham Arabidopsis Stock Centre (NASC; http://www.arabidopsis.info) is the European Arabidopsis
seed and DNA stock center.
more easily be tested for misexpression of the target genes in order to validate the prediction.
Hit-and-run regulators that only transiently bind to a promoter should still be identified by this
approach (110).
Two challenges will remain. First, the levels of the relevant transcription factors are as impor-
tant as their relative binding affinities, but the absolute protein abundance (protein copy number
per cell) is unknown for these proteins, with only indirect estimates in the alga O. tauri (141). Sec-
ond, resolving the contributions of multiple, overlapping, or redundant DNA-binding proteins—
for example, by using multiply-mutant plants (67), multifunctional artificial microRNAs (125), or
model species with smaller genomes (22)—remains laborious. Nonetheless, there is now a realistic
prospect of connecting clock-controlled genes to a genome-wide network for higher plant tran-
scription. The gene network could then be linked down to mechanistic clock models that explain
rhythmic dynamics (see Section 3), while coregulated genes in putative functional pathways are
linked up to physiological processes in the whole plant (see Section 5).
2.2. Canonical Outputs from the Plant Clock

The first clock-controlled genes studied in plants were highly expressed, encoded abundant pro-
teins, and were initially identified for their strong regulation, for example, by light [LIGHT-
HARVESTING COMPLEX (LHC) and RUBISCO ACTIVASE (RCA)] or cold [COLD AND
CIRCADIAN-REGULATED (CCR), also known as GLYCINE-RICH PROTEIN (AtGRP)]. Most
interest now focuses on the genes that regulate key aspects of clock-controlled physiology, where
the canonical examples include cold signaling (54), the daily control of hypocotyl growth and light-
regulated gene expression (136), and the regulation of flowering by day length (photoperiod) (133).
In each case, the clock controls the expression of intermediate transcription factors that integrate
abiotic and biotic environmental signals with temporal information from the clock (Figure 1c).
In the flowering pathway, intermediate transcription factors of the B-box and DNA-binding with
one finger (DOF) families, notably CONSTANS (CO) and the CYCLING DOF FACTOR (CDF)
genes, converge to regulate FLOWERING LOCUS T (FT ) (Figure 1c). In hypocotyl elongation,
basic helix-loop-helix factors of the PHYTOCHROME INTERACTING FACTOR (PIF) family are
most strongly implicated, along with basic leucine zipper proteins such as LONG HYPOCOTYL
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5 (HY5) (136). In both the output pathways and the clock mechanism (see Section 3), light input
is mediated by transcriptional regulation and by posttranscriptional mechanisms, notably protein
degradation via the CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1) ubiquitination
Circadian system:
complex (119). These mechanisms, together with the interaction of the PIF and CO/CDF path- the biological
ways downstream of the clock, were recently represented in an integrated mathematical model that components that
included both canonical and genome-wide clock targets (127). Environmental regulation of RNA mediate circadian
processing is one of the modifying mechanisms that should be integrated in the future (103, 130). rhythms; it is
conceptually divided
into input, oscillator,
and output
2.3. A Broader Set of Clock Targets
components, although
Circadian control of plant metabolism has long been documented but is now known to extend in practice a
well beyond photosynthesis (34), including secondary metabolites such as floral fragrance and component may
contribute to multiple
defense compounds. In several cases, this regulation is likely mediated by rhythmic transcription
functions
of the relevant enzymes (48, 56, 77). A fascinating example is the clock-mediated prediction
Oscillator: the
of the time of dawn, which sets the degradation rate of transient starch in the chloroplast at
biological components
dusk (58). The mechanism of starch regulation in the chloroplast might be posttranslational, that mediate self-
although this does not preclude transcriptional clock regulation of the posttranslational modifier sustaining rhythms,
(reviewed in 126). Intriguingly, circadian regulation has been discovered in fundamental cellular entrained by the input
systems such as ribosome biogenesis and translation (76), in addition to the cell cycle (see components and
regulating the output
Section 3). The clock’s relationship with central and ancient aspects of cellular function and
components of the
possible nontranscriptional clock outputs are both gaining new relevance as our view of the circadian system
circadian clockwork itself expands to consider potentially ancient nontranscriptional mechanisms.
Pseudo-response
regulator: a protein
with homology to the
3. THE CLOCK’S TIMING MECHANISMS receiver domain of
The circadian system of each organism includes a phylum-specific gene regulatory network that bacterial
histidine-aspartate
is required for most rhythmicity and is intimately connected to the environmental day-night cycle
phosphorelay proteins
(155), as well as one or more nontranscriptional oscillators that are less well characterized in but lacking the
eukaryotes. phospho-accepting
aspartate
3.1. The Transcriptional-Translational Feedback Loops

In plants, the clock gene network has been best studied in Arabidopsis thaliana, revealing a com-
plicated circuit of highly connected negative regulators (Figure 2) as well as positive regulators
that mediate light input to the clock and, more recently, rhythmic activators (reviewed in 68).
Expression of the MYB-related transcription factors LHY, CCA1, and RVE8 begins rising at
midnight and peaks at dawn [Zeitgeber time 0 (ZT0); see sidebar Rhythmic Properties], when
LHY and CCA1 inhibit the expression of later-expressed genes, including PSEUDO-RESPONSE
REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1, also called PRR1). The
biochemical mechanism of inhibition includes interaction with general TOPLESS repressor pro-
teins (145), DE-ETIOLATED 1 (DET1) (82), and chromatin modification at the target pro-
moters (reviewed in 4). The DNA-binding pseudo-response regulator genes PRR9 and PRR7 are
expressed after dawn (ZT2–6). LHY and CCA1 bind to the promoters of these two genes, but
the genetic evidence that this results in activation of PRR9 and PRR7 (46, 123) has not yet been
reconciled with the fact that LHY and CCA1 are repressors for other well-studied targets. Ex-
pression of PRR9 and PRR7 and then of PRR5 and TOC1 ends the morning phase. The PRR
proteins bind to and inhibit LHY and CCA1 expression, as demonstrated by experiments (55, 69,
101) and predicted by modeling (115, 116). Moreover, the data suggest that later-expressed PRR

a P2011
Rice ef7, Hd17
b Repressilator c Quadripressilator
Barley Ppd-H1 Pea LATE Barley eam10 Pea Barley Mat-a (= eam8)
Wheat Ppd-D1a Brassica rapa GI Einkorn Eps3a DNE Pea HR
PRR9 PRR5
PRRs PRR7 TOC1
CK2, CK1: COP1
rice Hd6,
Hd16
LHY/ PRR9 PRR7 PRR5 TOC1 GI LUX ELF4 ELF3 EC
CCA1 LHY/
EC LHY/CCA1
CCA1 EC
ZTL RVE8
Activation Inhibition
Figure 2
Simplified schemas of the Arabidopsis clock gene circuit. (a) The clock genes (rectangles) as they were connected in the P2011 model
(115). LHY and CCA1 are combined into one component; yellow flashes and dots denote transcriptional and posttranscriptional light
inputs, respectively; and dashed connections denote posttranslational regulation. Such circuits and their associated biochemical
parameter values have been termed the clock’s chemotype (26). Note that inter-regulation of the PRRs was modeled as a forward,
activation cascade, for want of experimental data. Current results support a reverse, repression cascade, shown in simplified form in
panel c. Homologous genes that control crop flowering are shown above the schema. Einkorn refers to Triticum monococcum. (b) The
clock circuit of the P2011 model, oversimplified as a repressilator (115). The red arrow indicates the proposed activation of early PRR
genes by LHY and CCA1, previously termed the morning loop; negative autoregulation of the Evening Complex (EC) forms an
evening loop independent of LHY and CCA1. (c) A four-component quadripressilator circuit, abstracted from current understanding
(see Sections 3.1 and 3.2). This circuit retains the ring of repressors, with RVE8 as an activator (red ); the inhibition of PRR9 and PRR7
by LHY and CCA1 remains to be demonstrated (dashed line) (50). In panels b and c, the few activating links are highlighted in red.
proteins in general inhibit the earlier-expressed PRR genes (reviewed in 15, 50, 115), providing a
mechanism to explain the distinct timing of each PRR gene expression and hence to explain the
temporal wave of PRR expression within the day.
Falling LHY and CCA1 levels derepress expression of EARLY FLOWERING 3 (ELF3),
ELF4, and LUX, allowing these transcripts to peak at approximately ZT10. RVE8 [also called
LHY/CCA1-LIKE 5 (LCL5)] protein also accumulates (several hours after its peak RNA abun-
dance, owing to an enigmatic delay) and activates evening-expressed genes (67), interacting
with NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) proteins (112).
GIGANTEA (GI), a large plant-specific protein, is rhythmically expressed under LHY/CCA1
Waveform: the control but functions at a posttranslational level through, for example, stabilization of the TOC1-
temporal profile of a
degradation factor ZEITLUPE (ZTL) (79). The protein products of ELF3, ELF4, and LUX or
biological rhythm over
one cycle; it includes its homolog BROTHER OF LUX ARHYTHMO (BOA, also called NOX) interact to form another
phase, period, and repressor, the Evening Complex, at ZT12–14 (32, 63, 64). The Evening Complex ends the day
amplitude information phase, as it binds to and inhibits expression of PRR9 and PRR7. ELF4 and LUX are also targets
and the more of the Evening Complex, forming an autoregulatory feedback loop that ends ELF4 and LUX
complicated properties
expression several hours before the rise of their other major repressors, LHY and CCA1. The
of wave shape
resulting rhythmic waveforms of the clock genes are robust across plant age, growth conditions,
Parameter: a usually
and multiple laboratories (49). A range of mathematical models have helped elucidate how the
constant value in a
mathematical model clock’s dynamic behavior, or dynatype, arises from this complicated circuit (reviewed in 11, 50)
that affects the and from its parameter values (see below), or chemotype (26).
dynamics of the model
components, such as
the maximum rates and 3.2. Abstracting to Understand
binding affinities in
biochemical reactions The known clock circuit does not obviously divide into modules (50, 67). However, simplification
provides an intellectual framework to organize detailed results, and in the future should allow
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the abstraction of simpler mathematical models from our current, fine-grained versions. Starting
from the observation that later-expressed components often inhibit earlier-expressed compo-
nents, Figure 2 shows two simpler networks. A three-loop structure was previously described
(84) in which a morning loop and an evening loop were coupled around a repressilator (100, 115)
(Figure 2b). The repressilator is a ring of three inhibitors in which the expression of each gene
inhibits the earlier-expressed gene, as noted above. The circuit was implemented in Escherichia coli
in a canonical example of synthetic biology (reviewed in 118). However, James Locke and David
Rand (personal communication) have pointed out that, in contrast to the repressilator, the plant
clock has sufficient components to separate the control of the rising and falling phases of each
gene expression peak.
Slightly extending the scheme suggests a unidirectional ring of four inhibitors surrounding
two diagonal interactions (Figure 2c). Each group of genes is regulated by two or three inhibitory
interactions, allowing separate control of rising and falling phases. LHY/CCA1 and the late PRR
genes (PRR5 and TOC1) are mutual inhibitors across one diagonal, and the Evening Complex
inhibits the early PRR genes (PRR9 and PRR7) across the other diagonal. The four-inhibitor
ring or the two-member mutual inhibition motif would naturally have toggle-switch equilibria
rather than oscillations (140), suggesting that the combination of both, along with the asymmetric
elements, may be important for oscillation. It will be fascinating to discover how these dynamics
are altered in larger genomes, for example, in crop plants (see Section 4). In the minimal genome of
O. tauri, where homologs of LHY, TOC1, and a LIGHT, OXYGEN, AND VOLTAGE (LOV)
domain–histidine kinase are the proposed clock components, either a simple negative feedback
model (96, 137) or a repressilator (105) has been proposed to account for the data. The closely
related chlorophyte Chlamydomonas reinhardtii includes clock genes that differ from the plant
sequences (104). The growing resources in further algal and diatom species will likely reveal much
more diversity among clocks in the green lineage (reviewed in 104). In addition, several processes
and components identified in Arabidopsis remain to be fully integrated into our view of the circuit.
3.3. Environmental Inputs and Modifying Kinetics

Posttranscriptional, translational, and posttranslational regulation, often modified by protein in-
teractions, modulate the clock’s dynamics by changing the rate or strength of the connections
in the transcriptional-translational feedback loop (reviewed in 80, 112, 128). These quantitative
dynamics are critical in a timing system. Except in the Kai oscillator of cyanobacteria (see below),
it is unclear what biochemistry allows the slow, 24-hour circadian timescale to emerge from the
normally faster dynamics of the molecular processes involved. Measuring the absolute values of
the relevant biochemical parameters for the plant clock (as in 108) has taken more time than in
animal systems, where a larger field and a context of pharmaceutical interest maintains a greater
focus on biochemistry.
Environmental control of these parameter values allows both entrainment under diel cycles
(Figure 1) and the buffering of circadian timing over a range of constant temperatures (tem-
perature compensation). Multiple effects of light and temperature have now been documented
(reviewed in 68). Mathematical modeling is essential to integrate their joint effects and to under-
stand how they allow particular behaviors, such as dusk sensitivity (Figure 1) and temperature
compensation (57). Further work remains to test the models’ proposals. For example, the most
recent detailed clock models (50, 115) include light-dependent degradation of the Evening Com-
plex based on a model of hypothetical COP1-containing protein complexes (117), which was also
adopted by leading researchers of COP1 function (129). Further aspects of that model might
now be validated as dynamic regulation of the Evening Complex components is identified (144)

and linked to the relevant phytochrome, cryptochrome, and LOV-domain photoreceptors (135).
These environmental responses of the clock genes and proteins link the intracellular regulation
of all the clock-regulated processes to the anticipated changes in the global environment. The
challenge now is to measure their quantitative contributions to plant performance (see Section 6).
3.4. The Ecology of Oscillators

In addition to nontranscriptional modulators of the canonical clock gene circuit, there is steadily
accumulating evidence for 24-hour rhythms that are independent of rhythmic gene expression
(121). The cyanobacterial KaiABC oscillator is the canonical example of such timing (reviewed
in 73), although convincing reports came decades earlier from measurements of rhythmic photo-
synthesis in giant algal cells from which the nucleus had been removed (for references, see 150).
Recent results point to rhythms involving calcium levels in several organisms, the calcium-release
factor cyclic ADP-ribose in plants, and cyclic AMP in animal cells (reviewed in 142). The most di-
rect evidence for the nontranscriptional oscillator in eukaryotes, including Arabidopsis, now comes
from rhythms in the oxidation of peroxiredoxin, a highly conserved, redox-sensitive enzyme that
detoxifies hydrogen peroxide. This posttranslational modification was shown to be rhythmic in
nontranscribing cells, using human erythrocytes and dark-adapted O. tauri (106, 107, 141). The
key result has now been replicated independently in each system (10, 20). Redox regulation has also
gained further prominence, for example, interacting with the clock gene network during salicylic
acid–mediated pathogen defense (158).
There has consequently been renewed interest in the multiple links between circadian
rhythms, metabolism more generally (reviewed in 62, 66), and other rhythmic processes. The
best-established link between the clock and another oscillator is with the cell cycle in eukaryotes
and cyanobacteria, based on the observation that many types of cells divide at a characteristic
circadian phase. This regulation was often described as the clock creating a permissive phase for
cell division, or gating the cell cycle, for example, in the green algae C. reinhardtii (102) and O. tauri
(97). Recent mathematical analysis based on imaging single cells, by contrast, has shown that the
mammalian circadian clock and the cell cycle are phase locked (47). Neither oscillator waits for, or
gates, the other; rather, they progress together as a coupled system. These observations support
earlier results showing that canonical cell-cycle genes such as that encoding the protein kinase
Wee1 not only are clock regulated but also affect the pace of the clock as much as the canonical
clock genes (157). Such clock coupling to the cell cycle has not been so clearly documented
in higher plants and seems to be quite different in root primordia. The root apical meristem
maintains a dawn phase in constant darkness, leaving its progeny cells to progress later in phase
(52). As multiple cell divisions form a new meristem in lateral roots, the clock is dramatically reset
(143).
The evolutionary breadth of the discussion is slowly extending to species that lack obvious
circadian rhythms, such as the yeast Saccharomyces cerevisiae. Yeast chemostat cultures show that a
well-documented metabolic cycle, which separates oxidative from reductive metabolism, controls
cell division and the expression of all yeast genes. Manipulation of the chemostat conditions
suggests that its period might extend from less than 6 hours (in the initial reports) to as long as 24
hours (44). A recent study showed that the peroxiredoxin oxidation state changes with the yeast
metabolic cycle and, intriguingly, that inhibition or deletion of the homologs of the mammalian
clock-related protein kinases Casein Kinase 1 (CK1) and Glycogen Synthase Kinase 3 (GSK3)
alters the yeast cycle’s period (16). Although the effects could result from conserved metabolic
regulation, they might also reflect an ancestral timing mechanism that linked these cellular
systems, which are now most often studied separately.
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4. VARIANTS IN CLOCK GENES

Notwithstanding tantalizing speculation on the roles of nontranscriptional oscillators, the demon-
strated physiological roles of the clock so far relate to its gene circuits. Clock mutants in Arabidopsis
have been important in identifying many plant clock genes (see Section 3) and now have further
importance in understanding the genetic variation in natural and breeding populations.
4.1. Crop Varieties and Breeding Lines

Bünning’s pioneering work of the 1930s in Phaseolus multiflorus was perhaps only one generation
away from identifying the first genetic variant in a clock-affecting plant gene (data reanalyzed in
91). The genetic variation in the breeding germplasm of many crop species also includes many
clock-affecting loci, as Figure 3a illustrates for vegetable brassicas (122). A subset of flowering-
time variation in crops is due to allelic variation in clock gene homologs—for example, in pea (148),
sorghum (98), rice (72), and wheat (53, 92)—although many other crop flowering traits are clearly
due to alleles of downstream, clock-regulated genes, such as the B-BOX 19 (BBX19) homolog
that controls FT floral regulators in beet (25). The northward progress of barley cultivation over
several millennia, from the Fertile Crescent to Scotland and Scandinavia, was accompanied by
selection for mutants in the homologs of PRR7, ELF3, and LUX (7, 156), which are also known
as single-gene mutations that alter flowering time (13). The PRR7 homolog Photoperiod H1 (Ppd-
H1) (12), however, did not affect clock gene expression rhythms in barley leaf RNA samples,
although it did affect expression of CO and FT homologs (14). Ppd-H1 is therefore specialized
either for a nonclock flowering function (139) or at most for the clock of a minor cell type. The
vascular phloem companion cells provide the critical timing signal for photoperiodic flowering
in Arabidopsis (21, 45), so a specialized clock in these cells remains a possibility, as proposed in
Ipomoea (61).
a b c
N×G Cvi × Ler
27 8
recombinant lines
20
6
Number of
Period (h)
26
4
10
25
2
24 0 0
N G A12 GD33 B M H S 23 24 25 26 27 23 24 25 26 27 28
Brassica oleracea line Period (h) Period (h)
Figure 3
Genetic variation in the plant clock. (a) Circadian period of leaf movement under constant light in Brassica oleracea lines (blue) (122).
Genetic variation among the lines alters the circadian period by almost 3 hours, as much as the strong, single-gene clock mutants in
Arabidopsis. The parental lines N (var. botrytis, cauliflower) and G (var. gemmifera, Brussels sprout) are shown in dark blue. (b,c) Graphs
showing the distribution of circadian period among recombinant progeny lines, compared with the periods of their parental varieties
(arrows). The wider, transgressive variation in the progeny compared with the parents indicates that the parents carried allelic variants
at multiple clock-affecting loci. Segregation of these alleles leads to longer and shorter periods among the progeny lines. Transgressive
variation is modest in the N × G population of B. oleracea (122) (panel b; blue) but large in the Cape Verde Islands (Cvi) × Landsberg
erecta (Ler) Arabidopsis population tested at 12◦ C (43) (panel c; green). Although the parent Arabidopsis lines had nearly identical periods,
the progeny segregated for a major clock-affecting quantitative trait locus; the underlying gene or genes remain to be identified.

4.2. Natural Genetic Variation

The many accessions of Arabidopsis thaliana distributed from community seed stock centers (see
sidebar Online Resources for Plant Chronobiology) with known variation in flowering time pre-
sented a potential gold mine of genetic variation to identify new clock genes (reviewed in 3).
Indeed, crossing different accessions resulted in progeny lines with very wide variation in circa-
dian properties, indicating that the rhythms in each parent line were due to a combination of
clock-lengthening and clock-shortening alleles. The first circadian quantitative trait loci (QTLs)
were located within five years after the first induced clock mutants were created. However, the
difficulty of identifying the causal gene for any subtle, quantitative trait was compounded by the
relatively laborious assays for circadian rhythms [by using leaf movement (43, 122) or whole-plant
movement (151) or by transforming many lines with firefly luciferase (LUC) reporters (9, 27,
81)]. Only two such genes were identified in the following 15 years [ELF3 (2) and FLOWERING
LOCUS C (FLC)], along with a suggested metabolic input (77) (see below). Natural variation at
FLC is well studied (83); its effects on the clock circuit include slightly altered LUX expression
(42) and genetic interaction with LHY and CCA1 (154). Very recently, natural variation in Brassica
rapa was mapped to an allele of GI (151). A study of Arabidopsis transformed 77 accessions with
a GI:LUC reporter and performed QTL analysis in a Liepowiez 0 (Lip-0) × Columbia (Col) re-
combinant breeding population. The photoreceptor PHYTOCHROME B (PHYB) was identified
as the causative gene for an altered expression phase under long photoperiods (30), similar to the
result of an earlier mutant screen (124). The QTL did not affect circadian period in constant
conditions but did alter light induction of GI transcription, emphasizing the clock genes’ function
as integrators of environmental signals.
Thus, the three most prominent nuggets so far from the gold mine turned out to be known
regulators. Each study has also revealed several other QTLs that remain to be identified (Figure 3c)
and might indeed be additional clock components. Streamlined phenotypic assays will be important
to tap this genetic resource, perhaps including the single-time-point transcriptome approach to
estimate phase, which suggested that the glucosinolate biosynthetic pathway could alter the clock
(77). The second hope in the studies of natural variation was that the accessions’ habitat of origin
would yield new insight into the ecological opportunities and constraints for circadian timing.
Understanding the role of circadian timing in nature, however, depends on background knowledge
of circadian physiology and of ecological contexts.
5. PHYSIOLOGICAL IMPORTANCE AND ENVIRONMENTAL

CONDITIONS
5.1. Advantages of Circadian Timing

The physiological benefit of circadian timing initially seems to be a mystery. Plants are necessarily
exposed to light:dark cycles (dormant seeds, tubers, and polar residents excepted) and have multiple
photoreceptors. One might ask, if it were advantageous to carry out some biological process
in the daytime rather than at night, couldn’t the photoreceptors activate that process directly?
If a delayed rather than an immediate response were required, couldn’t that be achieved with
simpler biochemistry than a full-blown oscillator that is self-sustaining even in constant light?
Timing a process to the daylight hours is an example of external coordination, one of the two
conceptual benefits that have long been proposed for circadian timing, which is discussed in
more detail below. Internal coordination, by contrast, is simply providing a temporal sequence
to control internal events, but direct evidence to validate this benefit is limited. The sequence
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could avoid conflicts, such as separating oxygen-sensitive nitrogen fixation in the subjective night
from oxygenic photosynthesis in a unicellular cyanobacterium (75), and gain synergies, such as
the coregulation of multiple steps in a biosynthetic pathway (see Section 2). Internal coordination
Simulation:
by the circadian clock is therefore an alternative to the direct regulation of one biological process in this context, a
by another, to avoid (or promote) co-occurrence. The yeast metabolic cycle (see Section 3.2) is mathematical
presumably an example of pervasive internal coordination. The cycle seems to occur in individual, representation of a
unsynchronized yeast cells as well as in chemostat cultures (131), so this type of regulation may biological process as a
result of numerically
be more general than was first thought. However, an experimental test for internal coordination
solving a mathematical
by the circadian clock in the cyanobacterium Synechococcus elongatus found little benefit, as an model of that process
arrhythmic kaiC mutant suffered no competitive disadvantage under constant light (149). Most in a given set of
plant biologists therefore focus on the clock’s advantages for external coordination. conditions
The clock offers three benefits compared with a simple, direct environmental response, namely
multiple phases, the integration of multiple signals, and, crucially, history. First, clock components
are expressed throughout the day and night (see Section 3.1) and can directly regulate output
processes at many different phases, not only following the light:dark transitions. The output
processes can, of course, be diverse, including the rhythmic gating of other signaling pathways
at particular phases. Second, the clock is sensitive to changing conditions (e.g., temperature and
metabolism) even though circadian timing is maintained with little alteration in a range of constant
conditions (reviewed in 74). Most important, circadian rhythms continue to predict the time of day
even if such environmental inputs are altered, because the clock is self-sustaining. That prediction
is modulated by past environmental inputs, such as photoperiod, and updated by present inputs,
through the inputs’ entraining effects on the clock components (Figure 1b,d). The clock’s history
dependence and network structure can reduce the effects of environmental variability, such as
variation in the daily light intensity (36). Temperature history also affects circadian rhythms in
Arabidopsis (9).
The extent of the history effect varies among clock systems and was recently tested using LUC
reporter genes in the transition from the short-day to the long-day state. The expression profiles
of an indicator clock component readjusted over three transient cycles in the complicated clock of
Arabidopsis (31, 33), whereas the markers available for O. tauri adjusted on the second cycle (31).
These results agreed with theoretical expectations, which show at the extreme that the simplest
timers have no history. The level of sand in a running hourglass determines when the timer will
run out, for example, irrespective of the events that led to that sand level. By combining history and
multiple phase outputs, clocks can time events in sophisticated ways, for example, by controlling
phase with intermediate dusk sensitivity rather than dawn or dusk tracking (Figure 1d ). The
result is that clocks offer robust but evolvable timing across many conditions and seasons (114).
5.2. Clocking the Environment

These conceptual advantages of circadian timing lead to an evolutionary perspective, because
they predict how the clock mechanism will eventually evolve to reflect the particular timing
challenges (and opportunities) of the organism in its ecological context. For example, the slow re-
entrainment of an intricate clock gene circuit might reflect a selective advantage that was conferred
by a prolonged effect of the clock’s history. Simulations can readily show these effects. Model gene
circuits that were evolved in silico to anticipate the time of dusk in simple light:dark conditions
had very simple circuits that functioned as sand timers: No clock was required. Self-sustaining
clocks with complicated feedback circuits evolved only when the time of dusk varied unpredictably
and the photoperiod also changed, simulating weather and seasons, or when they were evolved

to match a year of measured natural weather and seasons (138). Extreme circadian evolution has
led to loss of rhythmicity in some animals that temporarily (reindeer in the Arctic summer) or
permanently (Mexican cave fish) lose daily environmental Zeitgeber signals (5, 85), although not
in others (75). In plants, loss of rhythmicity has been documented in Norway spruce (59) and in
chestnut and Arabidopsis at low temperature (6). Most evidence for the physiological importance
of plant rhythms, however, has been derived during ongoing environmental cycles.
Experimental settings can simulate extreme conditions, such as life on a different planet with
a non-24-hour rotation, known as a T-cycle experiment. Classic studies of particularly sensitive
species, such as tomato, showed many-fold more growth in 24-hour cycles compared with 12-
or 48-hour cycles (65). In Arabidopsis, comparisons of 20-, 24-, and 28-hour cycles showed that
wild-type plants favored 24-hour cycles, whereas toc1 and ztl mutants with 20- and 28-hour periods
grew better in their resonant T-cycle than in the opposite T-cycle (35); a similar study detected an
altered allele ratio in a subsequent generation, indicative of a selective advantage (152). A straight-
forward mechanism was suggested because a resonant T-cycle restores normal phase relationships,
relative to the environmental cycle, in such period mutants. While replicating the earlier results,
subsequent work also showed that both 20- and 28-hour mutants grew better in 24-hour days than
in either altered T-cycle (58), indicating that another mechanism in addition to circadian phase
was involved (see Section 6).
The most persuasive data from altered natural environments involve latitudinal and altitudinal
clines, where the input signals change progressively. The signatures of evolution can then be
detected in common-garden studies in a single condition. Early collections of Arabidopsis were
likely too disparate in origin to reveal such changes in circadian properties. Even an extensive study
of 150 accessions revealed only a correlation of circadian period with midsummer day length (90),
a nonlinear transformation of the latitude of origin that has since been mis-cited as a latitudinal
cline. A study of 27 accessions across three temperatures found no obvious relationship with
latitude (43). Now that many more Arabidopsis accessions have been collected with more detailed
habitat information, a second limitation has come into play, namely the laborious assays required
to test circadian phenotypes. The phase of CO expression in European aspen (Populus tremula)
trees was shown strikingly to delay with higher latitudes of origin in the handful of varieties tested
(8), where the photoperiodic induction of cold-hardy buds is a crucial overwintering adaptation.
Several studies have implicated PHYB and the clock genes LHY1 and LHY2 in photoperiodic bud
set in Populus, with some evidence for clinal variation (70, 86). Bud set in Norway spruce and
Siberian spruce has been linked to clinal variation in clock gene homologs such as PRR3 as well
as in FT-related genes (17, 18). A key challenge now is to understand the dynamics of the clock
network of any plant species in ecologically relevant conditions, preferably in species where we
also find tractable genetic variation and ecological understanding.
6. UNDERSTANDING THE RHYTHMIC PHENOTYPE
6.1. Plant Rhythms in the Field: Which Rhythms Are Selected?

The data that directly measure rhythms in field conditions are limited but now growing. Ecologi-
cally relevant species have been tested; for example, gas exchange rhythms in tropical tree species
have been evaluated under constant conditions (39). Opening of stomatal pores in anticipation of
dawn can be clock regulated (e.g., 35). The resulting nighttime transpiration in field conditions in
cotton and bean, tested using large transpiration balances, is claimed to be of similar magnitude
to the expected change in transpiration due to climate change (29). Linking these responses with
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clock gene circuit dynamics, however, will depend on transcriptional profiling. Following early
transcriptomes that mapped the seasonal responses of free-living poplar (132), a landmark study
has used hundreds of RNA microarrays to test the rice transcriptome over two growing seasons,
with variable time resolution, in a field site close to a national meteorological station (99). The
many rhythmic gene profiles were then tested for correlation to weather data, first for the best
single correlating factor and then for multiple meteorological factors (87). Rhythmically mod-
ulated responses to temperature were a widespread explanatory factor, although circadian clock
genes such as OsLHY were strikingly temperature insensitive. We likely have much still to learn
from these data sets. Nicotiana attenuata, by contrast, offers exceptional ecological background
information on plant-insect interactions in field research sites and is now being developed as a
circadian system (78, 153). Circadian regulation of plant defense can clearly alter insect herbivory
(56). It is possible that the clock evolves to optimize such defenses and, if so, that adaptations of
circadian timing will be identifiable in N. attenuata. As in the trees and crop species, the circadian
regulation of photoperiodic flowering time and possibly of seed germination (111) also very likely
mediate part of the clock’s contribution to fitness in natural settings.
a b Wild type Mutant

Inputs Carbon Functional-
dynamic structural
Light
CO2
Photo- Plant
Temperature synthesis structure
Photo- Sugar- Organ

period starch growth
c
Environment
Clock Genotype Chemotype Dynatype Phenotype
Field traits
Genome
Flowering
Vernalization
FT RNA Fitness
Thermal time Selection Variation Yield
Flower Switch?
Management
Figure 4
A community model to understand plant circadian systems. Panel a shows a highly simplified schema of the first Arabidopsis Framework
Model, which predicted the biomass and area of each leaf (panel b) in a mature wild-type rosette under standard conditions, to within
experimental error (19). The model combined four earlier models of various types (blue dashed outlines), representing the processes
depicted in shaded rectangles, with links among models (red arrows) and from environmental inputs (black arrows). One model
component (in green) represented the rosette and root structure. The clock model only regulated photoperiod-responsive flowering,
although the clock controls many processes in the plant. The present challenge is to understand (explain and predict) complex,
whole-plant phenotypes such as the pleiotropic effects of circadian clock mutants, quantitatively and based on the underlying
mechanisms. Such understanding would link the plant’s genotype, via chemotype and dynatype, to its phenotype in a given
environment (panel c). The multiscale Framework Model promises to make this link. For example, the right-hand plant model in
panel b shows the predicted growth defect caused by a simulated mild defect in starch mobilization, compared with the wild-type plant
model in the center (Y.H. Chew, A. Zardilis & A.J. Millar, unpublished results). If the clock model also controlled starch degradation
(see Section 2.3), then the model might predict the growth defect of a simulated clock mutant. Our ambition is to go further, starting
from the genome sequence and extending to relevant field traits (panel c). It should then be possible to link the field traits to yield, for a
given approach to agricultural management, or to fitness in natural conditions. Combined with an understanding of (natural or
breeding) genetic variation, we may then hope to understand the selection that shaped the sequences and interconnections of the plant
clock genes (Figure 2).

6.2. A Community Model to Study Complex Traits

The flowering and bud set traits discussed above are among the physiological processes that
contribute to phenology, the temporal sequence of plant development, and thus to the plant’s life
history. Phenological traits are significant at a global scale because they affect how the vegetation
component of the biosphere contributes to the Earth system. Indeed, Earth system models include
simple phenology models for plants in various functional groups. Will it be possible to build on
our molecular understanding of the circadian system to understand, for example, how the clocks
of particular deciduous trees will alter the timing of bud set under future climates? Mathematical
models will be crucial to cross the necessary scales of biological organization from the intracellular
to the ecological (Figure 4). One recent example is the Arabidopsis Framework Model (Figure 4a),
which combined simple models of carbon metabolism, plant structure, flowering time, and a
clock-driven photoperiod sensor (19). This model predicted whole-plant and leaf-level biomass
and area based on existing models with minimal reparameterization, suggesting that mechanistic
models of additional processes could be incorporated in a community approach, to which multiple

groups can contribute. Similar optimism is shared among marine biologists (93) and in the fields of
ecology and evolution, such that one might hope to understand the evolutionary feedback from the
macroscopic, whole-organism scale of the phenotype back to the molecular scale of the genome
(Figure 4c). A recent review noted that “no. . .model has incorporated all of the components
discussed [in the review], but such integration is possible and would be a powerful approach for
studying ecological and evolutionary outcomes” (37, p. 75). It seems entirely possible that we will
develop noninvasive, time-resolved assays to understand clock effects in the field, together with
the cognate models, in order to link our molecular understanding of biological rhythms up to
larger scales in the coming decade.
SUMMARY POINTS
1. The biological clock in higher plants is a tractable example of a complex, pleiotropic
regulator that affects many processes across the plant life cycle.
2. Clock-regulated transcription has been studied in many individual plant genes; in Ara-
bidopsis, rhythmic regulation might next be integrated into a genome-wide transcriptional
regulatory network.
3. The mechanism of the clock gene circuit in Arabidopsis comprises a set of interlocked
feedback loops; I outline a simplified schema named the quadripressilator.
4. Nontranscriptional, 24-hour rhythms have been demonstrated in eukaryotes, as well as in
cyanobacteria, as part of the cell’s ecology of oscillators; the mechanisms and contribution
of nontranscriptional oscillators to daily timing in eukaryotes await further study.
5. Homologs of the Arabidopsis clock genes control photoperiodic, seasonal rhythms in
crops and trees. Genetic variation in these genes can contribute to crop domestication,
and latitudinal variation has been described in some species.
6. Circadian rhythms are sensitive to the environment, and plant rhythms are now being
measured in detail under natural conditions. A current challenge is to understand the
link from circadian timing to physiological traits in the field.
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7. Mathematical models are essential to define and test the quantitative, mechanistic links
from molecular regulation to field traits. The Arabidopsis Framework Model suggests a
modular, community-based approach to their construction.
FUTURE ISSUES
1. If synthetic promoters can direct plant gene expression to predictable phases—or, better,
to predictable waveforms in diel cycles—then can synthetic genomics reengineer the
temporal expression of all genes for a metabolic or signaling pathway in order to test the
contribution of their rhythmic control to plant growth?
2. Why are the known gene circuits of plant clocks more complicated than the clocks
of animals with comparable genome sizes (fungal and cyanobacterial clocks might be
genome limited)? Does this result from an evolutionary requirement, an experimental

artifact (confounding multiple tissue-specific clocks, for example), or a preference of the
scientists involved? Our genetic screens aspired to identify all discoverable Arabidopsis
clock genes, as in cyanobacteria. By contrast, Konopka’s rule, based on his discovery
of the fly period mutants in an earlier era, advises, “If you don’t find it in the first two
hundred, then quit” (146).
3. How and when does a cell’s “ecology of oscillators,” including the nontranscriptional
oscillator that controls peroxiredoxin, support or supplant rhythmic regulation by the
canonical clock gene circuit?
4. What new mathematical methods will allow us to analyze and understand the dynamic
coupling of multiple oscillators to biological processes, without abstracting so much that
we lose contact with measurable biochemical entities?
DISCLOSURE STATEMENT
The author is not aware of any affiliations, memberships, funding, or financial holdings that might
be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
I am grateful to past and present lab members and collaborators, especially to David Rand and
James Locke for discussion of repressilators that informed Figure 2c. Funding was provided by
European Union FP7 collaborative project TiMet (245143) and by Biotechnology and Biologi-
cal Sciences Research Council (BBSRC) awards BB/J009423/1 and BB/F005237/1. SynthSys is
supported in part by Synthetic Biology Research Center award BB/M018040/1 from the BBSRC,
the Engineering and Physical Sciences Research Council (EPSRC), and the Medical Research
Council (MRC).
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618 Millar
PP67-FrontMatter ARI 12 March 2016 14:40
Annual Review of
Plant Biology
Contents Volume 67, 2016
The Path to Thioredoxin and Redox Regulation in Chloroplasts

Bob B. Buchanan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Learning the Languages of the Chloroplast: Retrograde Signaling
and Beyond
Kai Xun Chan, Su Yin Phua, Peter Crisp, Ryan McQuinn, and Barry J. Pogson p p p p p p p p25
NDH-1 and NDH-2 Plastoquinone Reductases in Oxygenic

Photosynthesis
Gilles Peltier, Eva-Mari Aro, and Toshiharu Shikanai p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p55
Physiological Functions of Cyclic Electron Transport Around
Photosystem I in Sustaining Photosynthesis and Plant Growth
Wataru Yamori and Toshiharu Shikanai p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p81
The Costs of Photorespiration to Food Production Now
and in the Future
Berkley J. Walker, Andy VanLoocke, Carl J. Bernacchi, and Donald R. Ort p p p p p p p p p p p 107
Metabolite Damage and Metabolite Damage Control in Plants
Andrew D. Hanson, Christopher S. Henry, Oliver Fiehn,
and Valérie de Crécy-Lagard p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 131
The Regulation of Essential Amino Acid Synthesis and Accumulation
in Plants
Gad Galili, Rachel Amir, and Alisdair R. Fernie p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 153
Triacylglycerol Metabolism, Function, and Accumulation in Plant
Vegetative Tissues
Changcheng Xu and John Shanklin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 179
The Plant Polyester Cutin: Biosynthesis, Structure, and Biological
Roles
Eric A. Fich, Nicholas A. Segerson, and Jocelyn K.C. Rose p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 207
Biosynthesis of the Plant Cell Wall Matrix Polysaccharide Xyloglucan
Markus Pauly and Kenneth Keegstra p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 235
TOR Signaling and Nutrient Sensing
Thomas Dobrenel, Camila Caldana, Johannes Hanson, Christophe Robaglia,
Michel Vincentz, Bruce Veit, and Christian Meyer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 261
v
Rapid, Long-Distance Electrical and Calcium Signaling in Plants

Won-Gyu Choi, Richard Hilleary, Sarah J. Swanson, Su-Hwa Kim,
and Simon Gilroy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 287
Endocytosis and Endosomal Trafficking in Plants
Julio Paez Valencia, Kaija Goodman, and Marisa S. Otegui p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 309
Staying Tight: Plasmodesmal Membrane Contact Sites and the
Control of Cell-to-Cell Connectivity in Plants
Jens Tilsner, William Nicolas, Abel Rosado, and Emmanuelle M. Bayer p p p p p p p p p p p p p p p p 337
Pre-Meiotic Anther Development: Cell Fate Specification and
Differentiation
Virginia Walbot and Rachel L. Egger p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 365
Plant Sex Chromosomes

Deborah Charlesworth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 397

Haploidization via Chromosome Elimination: Means and Mechanisms
Takayoshi Ishii, Raheleh Karimi-Ashtiyani, and Andreas Houben p p p p p p p p p p p p p p p p p p p p p p p 421
Mechanisms Used by Plants to Cope with DNA Damage
Zhubing Hu, Toon Cools, and Lieven De Veylder p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 439
The Conservation and Function of RNA Secondary Structure in Plants
Lee E. Vandivier, Stephen J. Anderson, Shawn W. Foley, and Brian D. Gregory p p p p p p 463
Toxic Heavy Metal and Metalloid Accumulation in Crop Plants
and Foods
Stephan Clemens and Jian Feng Ma p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 489
Light-Mediated Hormonal Regulation of Plant Growth
and Development
Mieke de Wit, Vinicius Costa Galvão, and Christian Fankhauser p p p p p p p p p p p p p p p p p p p p p p p 513
Transcriptional Responses to the Auxin Hormone
Dolf Weijers and Doris Wagner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 539
Mapping Transcriptional Networks in Plants: Data-Driven Discovery
of Novel Biological Mechanisms
Allison Gaudinier and Siobhan M. Brady p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 575
The Intracellular Dynamics of Circadian Clocks Reach for the Light
of Ecology and Evolution
Andrew J. Millar p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 595
Environmental Control of Root System Biology
Rubén Rellán-Álvarez, Guillaume Lobet, and José R. Dinneny p p p p p p p p p p p p p p p p p p p p p p p p p p 619
The Haustorium, a Specialized Invasive Organ in Parasitic Plants
Satoko Yoshida, Songkui Cui, Yasunori Ichihashi, and Ken Shirasu p p p p p p p p p p p p p p p p p p p p p 643
vi Contents
Antibody Production in Plants and Green Algae

Vidadi Yusibov, Natasha Kushnir, and Stephen J. Streatfield p p p p p p p p p p p p p p p p p p p p p p p p p p p p 669
Perennial Grain and Oilseed Crops
Michael B. Kantar, Catrin E. Tyl, Kevin M. Dorn, Xiaofei Zhang,
Jacob M. Jungers, Joe M. Kaser, Rachel R. Schendel, James O. Eckberg,
Bryan C. Runck, Mirko Bunzel, Nick R. Jordan, Robert M. Stupar,
M. David Marks, James A. Anderson, Gregg A. Johnson, Craig C. Sheaffer,
Tonya C. Schoenfuss, Baraem Ismail, George E. Heimpel,
and Donald L. Wyse p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 703
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://www.annualreviews.org/errata/arplant
Contents vii

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PP67CH24-Millar ARI 14 March 2016 12:46

Annu. Rev. Plant Biol. 2016. 67:595–618 Keywords

4.1. Crop Varieties and Breeding Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605

4.2. Natural Genetic Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606

2. PERVASIVE DAILY RHYTHMS Circadian rhythm:

2.1. Clock-Controlled Gene Expression

www.annualreviews.org • Biological Timing in Higher Plants 597

a Phases, ϕ Amplitude Period, τ b 20 6L:18D

GI mRNA level (A.U.)

ZT (h) 12 0 12 0 12 0 12 24 36 48 60 0.2 6L:18D

Phase of mRNA midpoint (h)

www.annualreviews.org • Biological Timing in Higher Plants 599

ONLINE RESOURCES FOR PLANT CHRONOBIOLOGY

also link to many related resources.

seed and DNA stock center.

2.2. Canonical Outputs from the Plant Clock

3.1. The Transcriptional-Translational Feedback Loops

www.annualreviews.org • Biological Timing in Higher Plants 601

3.3. Environmental Inputs and Modifying Kinetics

www.annualreviews.org • Biological Timing in Higher Plants 603

3.4. The Ecology of Oscillators

4. VARIANTS IN CLOCK GENES

4.1. Crop Varieties and Breeding Lines

www.annualreviews.org • Biological Timing in Higher Plants 605

4.2. Natural Genetic Variation

5. PHYSIOLOGICAL IMPORTANCE AND ENVIRONMENTAL

5.1. Advantages of Circadian Timing

5.2. Clocking the Environment

www.annualreviews.org • Biological Timing in Higher Plants 607

6. UNDERSTANDING THE RHYTHMIC PHENOTYPE

6.1. Plant Rhythms in the Field: Which Rhythms Are Selected?

a b Wild type Mutant

Photo- Sugar- Organ

www.annualreviews.org • Biological Timing in Higher Plants 609

6.2. A Community Model to Study Complex Traits

models of additional processes could be incorporated in a community approach, to which multiple

genome limited)? Does this result from an evolutionary requirement, an experimental

www.annualreviews.org • Biological Timing in Higher Plants 611

24. Daan S. 2010. A history of chronobiological concepts. Protein Rev. 12:1–35

www.annualreviews.org • Biological Timing in Higher Plants 613

clock protein, TOC1, is a DNA-binding transcription factor. PNAS 109:3167–72

jasmonate-mediated defense with insect circadian behavior. PNAS 109:4674–77

www.annualreviews.org • Biological Timing in Higher Plants 615

without transcription in a eukaryote. Nature 469:554–58

response. Genome Biol. 15:R45

www.annualreviews.org • Biological Timing in Higher Plants 617

thaliana. New Phytol. 182:838–50

The Path to Thioredoxin and Redox Regulation in Chloroplasts

NDH-1 and NDH-2 Plastoquinone Reductases in Oxygenic

Rapid, Long-Distance Electrical and Calcium Signaling in Plants

Plant Sex Chromosomes

Deborah Charlesworth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 397

Antibody Production in Plants and Green Algae

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