Roles of Circadian Clock in Developmental Controls and Stress

Responses in Arabidopsis: Exploring a Link for Three

Components of Clock Function in Arabidopsis

RIM NEFISSI,* YU NATSUI,* KANA MIYATA,*

ABDELWAHED GHORBEL{ AND TSUYOSHI MIZOGUCHI*,1

*Gene Research Center, University of Tsukuba, Tsukuba,

Ibaraki, Japan
{
Biotechnology Center, Borj Cedria Science and Technology Park,
Route Touristique Borj Cédria-Soliman, Hammam-Lif, Tunisia

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
II. ELF3, a Novel Protein without Significant Similarity to any
Known Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
A. ELF3 as a Novel Nuclear Protein ........................................... 380
B. ELF3-Like Genes in Non-Arabidopsis Plant Species ..................... 380
C. Polyglutamine Tracts in ELF3 ............................................... 383
III. Roles of ELF3 in Light Signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
A. ELF3 in the light input pathway to the clock .............................. 384
B. ELF3 Interaction with PHYB ................................................ 385
C. PHYB-Independent Roles of ELF3 ......................................... 385
IV. Roles of ELF3 in the Control of Flowering Time . . . . . . . . . . . . . . . . . . . . . . . . . . 387
A. Current View of Photoperiodic Control of Flowering Time Linked
to the Circadian Clock in Arabidopsis....................................... 387
B. Co-Independent Role of ELF3 in the Control of Flowering Time ..... 388
C. Potential Role of ELF3 as a Scaffold for the Input, Oscillator and
Output of the Arabidopsis Clock ............................................. 388
D. Role of ELF3 as an Adaptor for COP1 and GI, Possibly in the
CRY2-Dependent Flowering Pathway...................................... 389

1
Corresponding author: E-mail: mizoguchi@gene.tsukuba.ac.jp

Advances in Botanical Research, Vol. 57 0065-2296/11 $35.00

Copyright 2011, Elsevier Ltd. All rights reserved. DOI: 10.1016/B978-0-12-387692-8.00011-4
378 R. NEFISSI ET AL.

E. Attempts to Identify Suppressors and Enhancers of

ELF3 Phenotypes............................................................... 389
V. Roles of ELF3 and Other Clock Proteins in Stress Tolerance. . . . . . . . . . . . . . 391
A. Ambient Temperature Stress ................................................. 391
B. Viability Under Very SD Regime ............................................ 391
C. Microarray Analysis ........................................................... 392
D. Gated induction of DREB/CBF by Low Temperature ................... 392
E. Roles of PRR9, PRR7 and PRR5 in the Cold-Stress Response ........ 393
F. PIF7 as a Possible Mediator between the Circadian Clock and
DREB1/CBF in Stress Signalling ............................................ 393
G. TOC1 as a Molecular Switch Connecting the Clock and Responses to
ABA and Drought ............................................................. 394
H. Release of Seed Dormancy.................................................... 394
VI. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397

ABSTRACT
Genetic analysis of the early flowering 3 (elf3) mutant of Arabidopsis thaliana indicates
that ELF3 plays key roles in the regulation of plant morphology, flowering time and
stress response, all of which are controlled by circadian clock. Although ELF3
appears to have multiple functions and has been shown to interact physically with
the photoreceptor phyB, its ability to regulate several distinct signalling pathways has
not been elucidated. This lack of information is attributable in part to the uniqueness
of the ELF3 gene, which encodes a novel nuclear protein with no significant sequence
similarity to any characterized protein in the existing public databases. Further, little
is known about direct protein–protein interactions of ELF3, or about mutations that
suppress elf3, phenotypes. Therefore, it is difficult to hypothesize about potential
factors downstream of ELF3. In this chapter, we summarize recent progress on the
characterization of ELF3 and discuss potential roles of ELF3 in plants. Several
reports have demonstrated that a circadian clock affects stress responses in Arabi-
dopsis and that DREB1A/CBF3 mediates between the clock and cold-inducible gene
expression. Therefore, possible roles of clock genes such as ELF3, PRRs, LHY and
CCA1 in the environmental stress responses of Arabidopsis are also discussed.

I. INTRODUCTION

Developmental transitions in plants are strongly affected by light quality,

intensity and duration. Arabidopsis is a facultative long-day (LD) plant, as it
flowers earlier under LD; (e.g. 16 h light/8 h dark) than under short-day (SD;
e.g. 8 h light/16 h dark) conditions (Simpson et al., 1999). In addition,
exposure to blue or far-red light promotes flowering in Arabidopsis.
Many genes are involved in the photoperiodic regulation of flowering in
Arabidopsis. In many prokaryotic and eukaryotic organisms, including
plants, biological clocks mediate the response of several physiological and
molecular processes to diurnal changes in environmental conditions such as
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 379

light quality and quantity, temperature and humidity. Circadian rhythms

persist with a period close to 24 h in the absence of any environmental time
cues and are generated by an endogenous timing mechanism. The basic
principles of circadian clocks are known for many organisms such as cyano-
bacteria, Neurospora, Arabidopsis, mice and humans. The clock consists of
oscillating molecules that form a negative, autoregulatory feedback loop
(Bell-Pedersen et al., 2005; Young and Kay, 2001).
Photoperiodic flowering is largely affected by the circadian clock. Genetic
approaches have identified more than two dozen key genes for clock func-
tions in Arabidopsis. Although phenotypic characterization of clock mutants
and identification of the corresponding genes have been performed, the
biochemical functions of most clock proteins are still largely unknown. For
example, EARLY FLOWERING 3 (ELF3; Hicks et al., 2001; Zagotta et al.,
1992, 1996), GIGANTEA (GI; Fowler et al., 1999) and EARLY FLOWER-
ING 4 (ELF4; Doyle et al., 2002; Kikis et al., 2005) are proposed to perform
key clock functions in Arabidopsis, and the genes for these clock proteins
have been known for nearly a decade. However, the novel proteins encoded
by the ELF3, GI and ELF4 genes have no significant sequence similarity to
any characterized proteins in the existing public databases, providing few
clues regarding their biochemical roles. It has recently been shown that GI
interacts with FKF1 and ZEITLUPE (ZTL; Somers et al., 2000) and that
these interactions are involved in the degradation of the proteins CDF1 and
TIMING OF CAB EXPRESSION 1 (TOC1; Imaizumi et al., 2005; Kim
et al, 2007; Más et al., 2003; Sawa et al., 2007).
The ELF3 gene of Arabidopsis is involved in the regulation of a wide variety
of processes, including plant morphology, flowering time and circadian rhythm
(Carré, 2002). The elf3-1 mutation was the first loss-of-function allele of elf3 to
be identified and was found in a screening for early flowering under an SD
regime (Zagotta et al., 1996). Mutations in ELF3 result in the loss of both
photoperiod sensitivity and circadian regulation, making ELF3 a candidate for
linking circadian clock function and photoperiodic induction of flowering
(Hicks et al., 2001). The elf3 mutant plants flower earlier than wild type under
both SD and LD conditions (Zagotta et al., 1996). The mutant plants exhibit
pale-green leaves and elongated hypocotyls and petioles, which are phenotypes
associated with a defect in the perception or transduction of light signals
(Coupland, 1997). Moreover, elf3-1 plants grow poorly compared with wild
type under both LD and SD conditions, suggesting that they are more sensitive
to other stresses in addition to light duration (Green et al., 2002). Taken
together, these observations suggest that ELF3 is a multifunctional protein.
Various approaches have been taken to uncover the mechanism by which
the mysterious ELF3 protein regulates several important biological processes
380 R. NEFISSI ET AL.

in Arabidopsis. The purpose of this chapter is to summarize recent advances

in our understanding of the roles played by ELF3 and other clock proteins in
the molecular mechanisms underlying the regulation of flowering time, organ
elongation, and environmental stress responses in Arabidopsis. In the first
section, we discuss the general characteristics of the amino acid sequence of
ELF3 and ELF3-like proteins in plants. This is followed by an examination
of the roles of ELF3 in light signalling (Section II) and in photoperiodic
flowering (Section III). Finally, stress responses affected by clock genes,
including ELF3, are summarized. For reference, all the genes discussed in
this chapter are listed in Table I.

II. ELF3, A NOVEL PROTEIN WITHOUT SIGNIFICANT

SIMILARITY TO ANY KNOWN PROTEINS

Although the amino acid sequence of ELF3 shows no similarity to those of

other proteins reported in the public databases, ELF3 and ELF3-like pro-
teins in Arabidopsis and other plants share several conserved domains.
Among these, the polyglutamine-repeat tracts (Q repeats) are thought to be
responsible for several quantitative trait loci involved in responses to light,
hypocotyl elongation and circadian rhythms. The possible functions of the Q
repeats in ELF3 are discussed in more detail in this first section.

A. ELF3 AS A NOVEL NUCLEAR PROTEIN

ELF3 is a novel protein of 695 amino acids, with little similarity to previously
characterized proteins of Arabidopsis (Hicks et al., 2001). ELF3 was pro-
posed to function as a transcription factor, as it is particularly rich in serine,
proline and glutamine/threonine regions similar to those found in some
transcriptional regulators (Carré, 2002). As it contains a large number of
potential phosphorylation sites, phosphorylation may be involved in ELF3
regulation (Hicks et al., 2001).

B. ELF3-LIKE GENES IN NON-ARABIDOPSIS PLANT SPECIES

Genes similar to Arabidopsis ELF3 are found in other plant species such as
rice, Lemna and Mesembryanthemum crystallinum (common ice plant). Rice
has two genes similar to ELF3 (Izawa et al., 2003), and these two OsELF3-
like amino acid sequences show considerable similarities with the Arabidopsis
ELF3 sequence. However, their expression profiles do not reflect the rhyth-
mic expression pattern of Arabidopsis ELF3, whose expression is under
 TABLE I
Arabidopsis flowering time genes and their description

Gene name Accession no. Description

ELF3: EARLY FLOWERING 3 AT2G25930 A novel protein that is expressed rhythmically and interacts with phyB to control
plant development, flowering and circadian rhythms
GI: GIGANTEA AT1G22770 A protein that regulates several developmental processes, including photoperiodic
flowering, phyB signaling, circadian rhythms, carbohydrate metabolism and
cold stress response
ELF4: EARLY FLOWERING 4 AT2G40080 A protein necessary for light-induced expression of both CCA1 and LHY
FKF1: FLAVIN-BINDING, AT1G68050 A protein containing a PAS domain, kelch repeats and an F-box domain, involved
KELCH REPEAT, F-BOX 1 in protein degradation
ZTL: ZEITLUPE AT5G57360 A protein containing a PAS domain, kelch repeats and an F-box domain, involved
in protein degradation
CDF1: CYCLING DOF FACTOR 1 AT5G62430 A transcription factor with a Dof-type zinc-finger motif involved in repression of
the expression of CO
TOC1/PRR1: TIMING OF CAB AT5G61380 A pseudo-response regulator involved in the control of circadian rhythms
EXPRESSION 1/PSEUDO-
RESPONSE REGULATOR 1
LHY: LATE ELONGATED AT1G01060 A myb-related transcription factor involved in the control of circadian rhythms
HYPOCOTYL along with CCA1
CCA1: CIRCADIAN CLOCK AT2G46830 A myb-related transcription factor involved in the control of circadian rhythms
ASSOCIATED 1 along with LHY
SVP: SHORT VEGETATIVE AT2G22540 A MADS-box protein that acts as a floral repressor
PHASE
PHYB: PHYTOCHROME B AT2G18790 A red/far-red photoreceptor involved in the regulation of de-etiolation, light
promotion of seed germination and shade avoidance response
TIC: TIME FOR COFFEE AT3G22380 A plant-specific clock regulator working close to the central oscillator and
affecting the circadian gating of light responses
FHY3: FAR-RED ELONGATED AT3G22170 A component of the PHYA signaling network that mediates the FR-HIR response
HYPOCOTYLS 3 to far-red light

(continues)
 TABLE I (continued )

Gene name Accession no. Description

PHYA: PHYTOCHROME A AT1G09570 A red/far-red light photoreceptor involved in the regulation of de-etiolation,
gravitropism, flowering and phototropism
CO: CONSTANS AT5G15840 A protein with zinc-finger motif involved in regulation of photoperiodic flowering
under long days
FT: FLOWERING LOCUS T AT1G65480 FT, together with LFY and SOC1, promotes flowering and is antagonistic with its
homologous protein, TFL1
FLC: FLOWERING LOCUS C AT5G10140 A MADS-box protein that functions as a repressor of floral transition and
contributes to temperature compensation of the circadian clock
SOC1: SUPPRESSOR OF AT2G45660 A MADS-box protein that functions as activator of floral transition
OVEREXPRESSION OF CO 1
CRY2: CRYPTOCHROME 2 AT1G04400 A blue-light receptor mediating blue-light-regulated cotyledon expansion,
hypocotyl elongation and flowering time
CIB1: CRYPTOCHROME- AT4G34530 A transcription factor that interacts with CRY2 and acts together with additional
INTERACTING BASIC- CIB1-related proteins to promote CRY2-dependent floral initiation
HELIX-LOOP-HELIX 1
TFL1: TERMINAL FLOWER 1 AT5G03840 A protein required for the control of inflorescence meristem identity and involved
in the floral initiation process
PIF7: PHYTOCHROME- AT5G61270 A transcription factor with a basic helix–loop–helix (bHLH) motif that negatively
INTERACTING FACTOR 7 regulates the phyB-mediated seedling de-etiolation
DREB1A/CBF3: DEHYDRATION AT4G25480 A member of the DREB subfamily A-1 of the ERF/AP2 transcription factor
RESPONSE ELEMENT family involved in response to low temperature, dehydration and ABA
BINDING 1A/C-REPEAT
BINDING FACTOR 3
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 383

robust circadian control (Murakami et al., 2007). In contrast, the mRNA

expression profile of LgELF3H1, an ELF3-like gene from Lemna species, is
similar to that of Arabidopsis ELF3 under light/dark conditions (Miwa et al.,
2006). Serikawa et al. (2008) showed that the role of LgELF3H1 in the
circadian clock is comparable to that of ELF3 in Arabidopsis. In fact, the
RNAi suppression of LgELF3H1 under constant light (LL) reduced the
rhythmic expression amplitude of CCA1 and PRR1, and over-expression of
LgELF3H1 lengthened the period of the circadian rhythm (Serikawa et al.,
2008). These phenotypes appear similar to the phenotypes induced by elf3
mutation and ELF3 over-expression in Arabidopsis. McELF3, an ELF3-like
gene in M. crystallinum (Boxall et al., 2005), displays an expression pattern
similar to that of Arabidopsis ELF3, with transcription under circadian
control and peaking in the evening.

C. POLYGLUTAMINE TRACTS IN ELF3

Unlike other clock proteins in Arabidopsis, ELF3 possesses a motif known as Q

repeats. Based on a comparison of ELF3 sequences reported from 60 different
wild-type Arabidopsis plants, the number of Q-repeats in the C-terminal region
is a polymorphic trait (Tajima et al., 2007). There was significant correlation
between the number of Q repeats and two circadian markers, amplitude and
period length, in Arabidopsis, suggesting that the length of the polyglutamine
tracts may affect the function of ELF3 in the control of circadian rhythm
(Tajima et al., 2007). The Q repeats are conserved in Arabidopsis lyrata subsp.
lyrata (Q  19; NCBI Accession No. CAL85633) and subsp. petraea (Q  19,
20 and 21; CAL85631, CAL85630 and CAL85632), but not in M. crystallinum
(AAQ73529), Vitis vinifera (CAO43769), Triticum aestivum (ABL11477),
Oryza sativa (NP_001056770), Lemna gibba (BAD97872), Lemna paucicostata
(BAD97868) or Pisum sativum (ABP81864).
A far-red pulse at the end of day is a light signal that stimulates plants
living in natural shade. Recent genetic mapping of natural variations in the
shade-avoidance response (Coluccio et al., 2011) identified EODINDEX1 as
the most significant quantitative trait locus involved in the response to the
end-of-day signal. ELF3 was proposed as the most likely candidate gene
underlying the natural variation in EODINDEX1.

III. ROLES OF ELF3 IN LIGHT SIGNALLING

The circadian clock is composed of three components: the input pathway, a

central oscillator and the output pathway (Fig. 1). Photoreceptors such as
phytochromes and cryptochromes play major roles in the input pathway.
384 R. NEFISSI ET AL.

Input Output
pathways Oscillator pathways

LHY
CCA1

PHYB Gl CO FT

ELF3 TOC1
ELF4 Flowering

Fig. 1. A model showing the molecular hierarchy that controls flowering time in
Arabidopsis in response to photoperiod and the proposed interlocking feedback loops.
ELF3 expression enables the oscillator to control the light sensitivity of both the
resetting of the clock and the induction of circadian outputs. This suggests that ELF3
may mediate among the input, the oscillator and one of the outputs of the circadian
clock in Arabidopsis.

Clock proteins are believed to function in the input pathway or as central

oscillators, or both. In this section, we examine the possible roles of ELF3 in
general light signalling and in the light input pathway. We also discuss the
protein–protein interactions between ELF3 and phyB, and phyB-dependent
and -independent roles of ELF3 in light signalling.

A. ELF3 IN THE LIGHT INPUT PATHWAY TO THE CLOCK

An elf3 loss-of-function mutation (elf3-1) in Arabidopsis conferred arrhyth-

mia for all rhythms tested under LL. However, when transferred to constant
darkness (DD), elf3-1 plants retained the circadian rhythm. This indicates
that ELF3 has little, if any, role in regulating circadian clock functions in the
dark (Jeong and Clark, 2005; Liu et al., 2001; McWatters et al., 2000). Thus,
instead of being an essential component of the clock itself, ELF3 may
mediate an interaction between phototransduction and the circadian clock.
This would be consistent with the light-dependent arrhythmia in plants with
defective ELF3 (McWatters et al., 2000; Reed et al., 2000). The ELF3 gene
product was proposed to function in a light input pathway to the circadian
oscillator, and the absence of ELF3 was hypothesized to alter the coordina-
tion between the light and circadian regulatory pathways, resulting in the
altered flowering time and photoperiodic intensity observed in elf3 mutants
(Barak et al., 2000; Hicks et al., 2001; Searle and Coupland, 2004).
In Arabidopsis, there are two major types of photoreceptors, the red-light-
absorbing phytochromes and blue-light-absorbing cryptochromes (Covington
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 385

et al., 2001). The hypocotyl elongation response under blue light in plants over-
expressing ELF3 (ELF3-OX) is similar to that in wild-type plants, suggesting
that ELF3 is not involved in the blue-light inhibition mediated by crypto-
chromes. Under continuous red light, ELF3-OX plants do not exhibit the
long hypocotyl phenotype that is seen in phyB mutants (Liu et al., 2001). This
indicates that the ELF3 gene product is a negative regulator of phyB-mediated
induction of circadian outputs (Covington et al., 2001; Kikis et al., 2005;
Salomé and McClung, 2005). Further, ELF3 must affect some downstream
aspect of phyB signalling or a phyB-independent regulatory pathway (Reed
et al., 2000).
Other genes have been shown to be involved in the light input pathway to
the clock. The mutation of TIME FOR COFFEE (TIC) also causes a defective
gating of light, but at a different time of the cycle. TIC acts at middle to late
night (Ding et al., 2007; Hall et al., 2003), whereas ELF3 acts with maximal
effect during early night (Hicks et al., 1996). The double mutant tic;elf3
showed additive morphological, rhythmic and gene expression phenotypes,
indicating that ELF3 and TIC have independent functions (Hall et al., 2003).
Recently, another component of the light gating pathway, FAR-RED
ELONGATED HYPOCOTYL 3 (FHY3), was identified in Arabidopsis
(Allen et al., 2006). FHY3 regulates light signalling during the day; fhy3
mutants conferred arrhythmia of central oscillator genes and disrupted reset-
ting of the clock under continuous red light (Allen et al., 2006).

B. ELF3 INTERACTION WITH PHYB

The N-terminal region of ELF3 was shown to physically interact with phyB,
suggesting that ELF3 may function as a direct modulator of signal informa-
tion from phyB via this interaction (Fig. 2; Liu et al., 2001). Yeast two-hybrid
system experiments demonstrated that ELF3 interacts with the C-terminal
domain of phyB, but not that of phyA, suggesting that its role is specific to
the regulation of phyB (Carré, 2002; Liu et al., 2001). The ELF3–phyB
complex may regulate multiple signalling pathways as well as gene expres-
sion. A phyB-mediated modification of ELF3 may allow interactions with
other proteins to control gene transcription (Liu et al., 2001).

C. PHYB-INDEPENDENT ROLES OF ELF3

In constant red light, elf3-1;phyB-9 double mutants had longer hypocotyls,

more elongated petioles and earlier flowering compared with single mutants.
These additive phenotypes suggest that ELF3 and phyB may act indepen-
dently of each other, at least in part (Kim et al., 2005; Reed et al., 2000).
386 R. NEFISSI ET AL.

Hypocotyl elongation
3 3
1 1 4
DREB1/CBF
PhyB PIF/PILs
1 Cold stress
2
CK2A CK2B response
2 2 PRR9

PRR7 ABA signaling

1 2 5
2 2 PRR5
ELF3 CCA1 LHY 5
8 TOC1/PRR1
LHY/CCA1family Drought stress
4 4
response
6 TOC1/PRR family
8 ZTL
6
8 GI 4 6
FLC SVP 6
FKF1 SKPs
8 8 6 7
6
CDFs
7 7 7 7
CO LKP2 26S Proteasome
Protein
FT ZTL/FKF degradation
Flowering family
time

Fig. 2. Functional interactions between various clock proteins and input/output

pathways for the regulation of flowering time, organ elongation, protein degradation
and stress responses. (1) Ni et al. (1998) and Liu et al. (2001). (2) Barak et al. (2000),
Mizoguchi et al. (2002) and Mizoguchi et al. (2006). (3) Kurup et al. (2000). (4) Más
et al. (2003) and Yamashino et al. (2003). (5) Kidokoro et al. (2009). (6) Imaizumi
et al. (2005), Nakasako et al. (2005), Kim et al. (2007), Rubio and Deng (2007) and
Sawa et al. (2007). (7) Calderon-Villalobos et al. (2007). (8) Yoshida et al. (2009).

The elf3 mutations eliminated circadian rhythm outputs in LL, whereas the
phyB mutations had quantitative effects on the amplitude and period without
eliminating rhythmicity. This suggests that ELF3 cannot be considered as
simply a unique phy-signalling component (Reed et al., 2000). In LL, elf3
mutations abolished the rhythmic expression of clock-controlled genes such
as LHY, CCA1, TOC1, GI, CO, FT and CAB, which suggests that these
genes may be downstream targets of transcriptional regulation by ELF3
(Barak et al., 2000; Hall et al., 2003; Hazen et al., 2005; Hicks et al., 2001).
As physiological studies have previously related circadian dysfunction to
chlorosis, the pale-green phenotype of elf3 may be due to the loss of circadian
control over chloroplast functions, in addition to abnormal gene regulation
(Dowson-Day and Millar, 1999).
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 387

IV. ROLES OF ELF3 IN THE CONTROL OF

FLOWERING TIME

Photoperiodic flowering is one of the clock output pathways and is arguably the
most studied developmental process under circadian control in plants. Loss-
and gain-of-function of ELF3 accelerates and delays flowering time in Arabi-
dopsis, respectively, indicating the importance of ELF3 in this process. A short
history and the current state of research on photoperiodic flowering are sum-
marized in this section. We discuss the roles of ELF3 in both CO-dependent
and -independent pathways and the potential role of ELF3 as a scaffold or
adaptor protein. Finally, our recent attempts to identify new proteins that may
function in tandem with ELF3 in the control of flowering are discussed.

A. CURRENT VIEW OF PHOTOPERIODIC CONTROL OF FLOWERING TIME

LINKED TO THE CIRCADIAN CLOCK IN ARABIDOPSIS

Garner and Allard classified plants into different day-length response types
(Garner and Allard, 1920): LD plants (LDPs) require a shorter time to flower
when light exposure exceeds a critical day length, and SD plants (SDPs) flower
sooner when light exposure is shorter than a critical day length. Subsequent
experiments demonstrated that SDPs actually measure the length of the night,
which must exceed a critical length to induce flowering, and that these plants do
not flower when grown under LL. Photoperiodic control of flowering time is
tightly linked to the circadian clock, which acts as the time-keeping mechanism
that measures the durations of day and night (Más, 2005; Suárez-López et al.,
2001; Yanovsky and Kay, 2002). The circadian clock is an endogenous oscilla-
tor with an approximate period of 24 h and can be synchronized, or entrained,
to the exact period of daily oscillations in light and temperature (Dunlap, 1999).
This process phases the biological activities to the correct time of day. The
LDPs are classified into two types, those that only flower (an absolute LDP)
and those that flower most rapidly (a facultative LDP) when light exposure
exceeds a certain number of hours during a 24-h period (Thomas and Vince-
Prue, 1997). As a facultative LDP, Arabidopsis flowers much earlier with a daily
regime of a long light period and short dark period (e.g. 16 h light/8 h dark) than
with a short light period and long dark period (e.g. 8–10 h light/16–14 h dark).
In Arabidopsis, the regulatory MYB proteins LHY and CCA1, close relatives of
each other, are essential clock components with redundant functions in
controlling the rhythmic expression of flowering-time genes for photoperiodic
flowering (Carré and Kim, 2002; Mizoguchi et al., 2002, 2005). In particular,
LHY and CCA1 regulate a flowering pathway, composed of the genes GI, CO
388 R. NEFISSI ET AL.

and FT, in light/dark cycles (Más, 2005; Mizoguchi et al., 2002, 2005). FT gene
expression is activated under LDs primarily through a conserved pathway
consisting of GI and CO (Mizoguchi et al., 2005). Several other Arabidopsis
genes, whose mutation also delays or accelerates flowering, have been previ-
ously identified (Más, 2005). The relationship between flowering and day length
in Arabidopsis involves rhythmic, circadian clock-controlled expression of CO
mRNA. In this model, CO mRNA levels rise and fall over the course of a day,
producing unstable levels of the CO protein. If CO mRNA levels are high when
the plant is exposed to light, CO protein is stabilized and activates the expres-
sion of FT (Más, 2005; Suárez-López et al., 2001; Valverde et al., 2004).
Changing the timing of CO expression can alter the length of daylight that
triggers flowering (Böhlenius et al., 2006; Yanovsky and Kay, 2002), but does
not alter the photoperiodic response type of Arabidopsis from facultative LDP
to SDP. A comparative analysis of Arabidopsis and rice, an SDP plant, suggests
that functional differences between Arabidopsis CO and its rice orthologue
(Heading date1, Hd1) may be the basis for the reversal of response type
(Hayama and Coupland, 2004). In rice, Hd1 represses flowering under LD by
repressing the expression of the rice FT orthologue (Heading date3, Hd3a),
whereas in Arabidopsis, CO activates flowering by activating FT expression
(Hayama and Coupland, 2004). FT and Hd3a are candidates for the floral
hormone florigen (Corbesier et al., 2007; Tamaki et al., 2007).

B. CO-INDEPENDENT ROLE OF ELF3 IN THE CONTROL OF FLOWERING TIME

The expression of GI, CO and FT was increased in the elf3-1 mutant, indicating
that ELF3 negatively regulates the transcript levels of all three genes (Kim et al.,
2005). CO mediates between GI and FT to control flowering time (Mizoguchi
et al., 2005). CO loss-of-function, as in co-1 and co-2 (Kardailsky et al., 1999;
Kobayashi et al., 1999), decreased the FT expression level. Surprisingly, the
elf3-1;co-1 double mutant flowered much earlier than co-1 in LD, although FT
expression remained very low (Kim et al., 2005). These results indicate that
elf3-1 partially suppresses late flowering in co-1 through a CO-independent
mechanism. ELF3 may be involved in post-transcriptional regulation of FT
protein or the transcriptional regulation of a gene downstream of FT, such as
SOC1.

C. POTENTIAL ROLE OF ELF3 AS A SCAFFOLD FOR THE INPUT, OSCILLATOR

AND OUTPUT OF THE ARABIDOPSIS CLOCK

Recently, we reported that mutations in the circadian clock genes LHY and
CCA1 caused Arabidopsis to show characteristics of an SDP (Fujiwara et al.,
2008; Mizoguchi and Yoshida, 2009; Yoshida et al., 2009). The lhy;cca1
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 389

mutant flowered later under LL than under SD, a behaviour pattern exhib-
ited by SDPs. Characterization of suppressors of the late-flowering pheno-
type of lhy;cca1 under LL indicated that inversion of the response appeared to
involve enhanced activities of ELF3 and two floral repressors, SHORT
VEGETATIVE PHASE (SVP) and FLOWERING LOCUS C (FLC;
Fujiwara et al., 2008; Mizoguchi and Yoshida, 2009; Yoshida et al., 2009).
ELF3 directly interacted with both CCA1 and SVP, which are part of the
central oscillator and output pathway of the Arabidopsis circadian clock,
respectively (Fig. 2; Yoshida et al., 2009). As described in the previous section,
ELF3 interacts with the photoreceptor phyB (Liu et al., 2001; Reed et al.,
2000), which plays key roles in the input pathway. These findings suggest
that ELF3 may function as a scaffold to form a clock protein complex in
Arabidopsis. We have proposed that the circadian clock may control path-
ways that promote and repress flowering and that altering the balance among
these pathways can switch the photoperiodic response type of a single species.

D. ROLE OF ELF3 AS AN ADAPTOR FOR COP1 AND GI, POSSIBLY IN THE

CRY2-DEPENDENT FLOWERING PATHWAY

Yeast two-hybrid analysis and co-immunoprecipitation assays have demon-

strated molecular interactions between ELF3 and the E3 ubiquitin-ligase
CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), and between
ELF3 and GI (Yu et al., 2008). It was proposed that ELF3 acts as an adaptor
protein between COP1 and GI. The blue-light receptor CRY2 may negatively
regulate COP1 via a direct interaction between the two (Wang et al., 2001).
Genetic analysis under LD and SD regimes support this idea. For example,
the late-flowering phenotype of cry2 was suppressed by cop1 under LD,
suggesting that COP1 may be a downstream factor of CRY2 under various
light/dark cycles. Thus, blue-light-activated CRY may stabilize the CO pro-
tein in the GI-independent pathway by inhibiting COP1 (Valverde et al., 2004;
Yu et al., 2008). Indeed, Jang et al. (2008) and Liu et al. (2008a,b) have shown
that COP1 triggers degradation of the floral inducer CO. In this way, the cry2
loss-of-function mutation would cause increased accumulation of COP1,
thereby inhibiting the stabilization of CO.

E. ATTEMPTS TO IDENTIFY SUPPRESSORS AND ENHANCERS OF

ELF3 PHENOTYPES

To understand the roles of ELF3 as a multi-functional protein, a reasonable

approach is to isolate mutations that suppress or enhance all or some of the
phenotypes seen in elf3 mutants under different growth conditions. Using
390 R. NEFISSI ET AL.

this general approach, we looked for mutations that delayed flowering under
LL, but not LD or SD, in genetic backgrounds lacking functional ELF3, in
order to identify genes required for inversion of the photoperiodic flowering
type in lhy;cca1 mutants of the ELF3–SVP/FLC-independent pathway
(Fujiwara et al., 2008; Mizoguchi and Yoshida, 2009; Yoshida et al., 2009).
We isolated seven elf3-suppressors (sel1, sel3, sel5, sel7, sel14, sel15 and
sel20) and one enhancer (E#1) by mutagenesis of elf3-1 (Col accession)
with a heavy-ion beam (Natsui et al., 2010; Neffisi et al., unpublished).
Two additional suppressors were obtained with EMS mutagenesis of elf3-
101 (Ler accession) (Natsui et al., 2010). One mutant, the elf3-1;sel20 plants,
exhibited accelerated floral transition under LD and SD regimes but re-
pressed flowering under LL. This photoperiodic flowering response of elf3-
1;sel20 was similar to that of lhy;cca1 and completely different from that of
wild type. The sel20 mutation was determined to be a new deletion allele
consisting of a mutation in the gene for the blue-light receptor CRY2 (Neffisi
et al., unpublished). CRY2 has been shown to interact with and negatively
regulate COP1, thereby accelerating flowering time via CO degradation (Liu
et al., 2008a,b). Delay of flowering in cry1;cry2 was largely suppressed
by cop1, and co mutation repressed the acceleration of flowering time caused
by cop1.
CRY2 was proposed to regulate the COP1–ELF3–GI pathway for the
control of flowering time, as explained in Section IV.D. We believe this
pathway is unlikely to play a major role in the delay of flowering in lhy;
cca1 under LL; the downregulation of GI activity would decrease CO ex-
pression (Mizoguchi et al., 2005; Suárez-López et al., 2001), but there was no
significant difference in the CO expression level between lhy;cca1 and wild-
type plants under LL (Fujiwara et al., 2008). The sel20/cry2 mutation also
did not affect the CO expression level in elf3-1 under LL. These results
suggest that late flowering of both lhy;cca1 and elf3;cry2 under LL was
unlikely to depend on decreased GI activity. An additive effect of lhy;cca1
and gi on late flowering under LL supports this idea. We prefer a model
based on the function of CIB1 (Liu et al., 2008a,b) to explain LL-specific
delay of flowering in the elf3;cry2 double mutant. According to this model,
CIB1 directly interacts with CRY2. CIB1 activity requires CRY2 and is
increased by blue light. CIB1 binds to the FT gene and activates flowering
via the control of FT expression. The increase of FT expression in CIB1 over-
expressing plants was much more pronounced under the LL regime, but not
under the LD regime. Without CRY2, the effect of the loss-of-function of
CIB1 would be stronger under constant white light than under LD. It would
be interesting to test whether downregulation of CRY2 and CIB1 can
explain, at least in part, the delay of flowering in lhy;cca1 under LL.
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 391

V. ROLES OF ELF3 AND OTHER CLOCK PROTEINS IN

STRESS TOLERANCE

Photoperiodic flowering and organ elongation are well-characterized devel-

opmental processes controlled by a circadian clock in plants. Signal transdu-
cers and transcription factors that mediate between the clock and these
developmental processes have been identified, and molecular mechanisms
underlying these processes are beginning to be understood (see Section II).
After a microarray analysis demonstrated that the expression of a certain set of
stress-inducible genes was under the control of a circadian clock (Harmer and
Kay, 2000), our knowledge of the circadian clock control of plant stress
responses rapidly advanced. In this section, we summarize the reported roles
of clock genes, including ELF3, in responses to environmental stresses,
including ambient temperature, very short days, low temperature and drought.

A. AMBIENT TEMPERATURE STRESS

Sub-optimal, but not near-freezing, temperatures affect flowering through

the thermosensory pathway, which overlaps with the autonomous pathway
(Blázquez et al., 2003). Two distinct pathways regulate ambient temperature-
dependent flowering (Strasser et al., 2009). One pathway requires ELF3,
while the other requires TFL1. Delayed flowering at lower temperatures
was partially suppressed in single elf3 and tfl1 mutant plants. Further, elf3;
tfl1 double mutants were insensitive to temperature, suggesting that both
ELF3 and TFL1 are important in the control of flowering under ambient
temperature stress.

B. VIABILITY UNDER VERY SD REGIME

The over-expression of either CCA1 or LHY in Arabidopsis (CCA1-ox and

lhy-1) resulted in a loss of circadian rhythmicity under constant conditions
such as LL or DD. A loss-of-function mutation in ELF3 (elf3) or in
both CCA1 and LHY (lhy;cca1) also produced an arrhythmic phenotype
under LL. However, all of the CCA1-ox, lhy-1, lhy;cca1 and elf3 plants
retained the ability to respond to diurnal changes in light (Green et al.,
2002; Hicks et al., 2001; Song and Carré, 2005; Yoshida et al., 2009). The
transcript levels of clock-controlled genes oscillated robustly in these plants,
even under light/dark cycles.
In wild-type plants, the expression of clock-controlled genes changes in
anticipation of light/dark transitions. However, the CCA1-ox, lhy-1, lhy;cca1
and elf3 plants lost the ability to anticipate this daily change in their
392 R. NEFISSI ET AL.

environment. Green and co-workers examined the effects of loss of circadian

regulation on the fitness of Arabidopsis under different photoperiodic condi-
tions: 16 h light/8 h dark (16L8D), 8L16D and 4L20D. The elf3, CCA1-ox
and lhy-1 plants were less viable under very SD (4L20D) conditions. It would
be interesting to test the viability of lhy;cca1 and other clock mutants
under a very SD regime. Photoperiods with different T-cycles should also
be used.

C. MICROARRAY ANALYSIS

The possible involvement of the circadian clock system in stress tolerance

was suggested by microarray analysis (Covington et al., 2008; Harmer and
Kay, 2000, Kant et al., 2008, Kreps et al., 2002). Many stress-inducible genes,
as well as genes for key regulators of stress signalling pathways, were shown
to be affected by a circadian clock.
The cis-element required for induction by dehydration stress was identified
and named the dehydration responsive element (DRE; Yamaguchi-
Shinozaki and Shinozaki, 1994). The core sequence of the DRE is AGCC-
GAC. The DRE was shown to be required for induction by low temperature.
A transcription factor that specifically binds to the DRE was identified in a
yeast one-hybrid system and named DRE BINDING 1 (DREB1; Liu et al.,
1998). Sakuma et al. (2002) grouped 145 Arabidopsis DREB/ERF-related
proteins into five subfamilies (AP-2, RAV, DREB, ERF and others) based
on amino acid sequence similarities among the AP2/ERF domains. DREB1-
related genes were classified into three subgroups (DREB1, 2 and 3). DRE
and DREB were also identified as C-box and CBF, respectively (Stockinger
et al., 1997). The clock-controlled gene DREB1A/CBF3 was elucidated by
Harmer and Kay (2000).

D. GATED INDUCTION OF DREB/CBF BY LOW TEMPERATURE

The expression levels of CBF3 and certain CBF-regulated genes exhibit

circadian cycling under unstressed conditions (Harmer and Kay, 2000).
Fowler et al. (2005) found that the cold induction of CBF/DREB was gated
by a circadian clock, suggesting that the expression of genes for key regula-
tors of cold-stress responses may be fine-tuned by several layers of regula-
tion. The extent to which the CBF/DREB mRNA level increased in response
to cold stress depended on the time of day that the plants were exposed to
cold, with the highest and lowest levels at 4 and 16 h after subjective dawn,
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 393

respectively. Constitutive over-expression of CCA1 abolished gating of CBF/

DREB induction by the circadian clock in response to cold stress.

E. ROLES OF PRR9, PRR7 AND PRR5 IN THE COLD-STRESS RESPONSE

In a comparative microarray analysis between the triple mutant prr9-11;prr7-

10;prr5-10 (d975) and wild-type plants, Nakamichi and co-workers found
significant overlap between the set of upregulated genes in d975 and the set of
cold-responsive genes (Nakamichi et al., 2009). The expression level of
DREB1/CBF was higher in d975 than in wild-type plants. Consistent with
this, d975 plants were more tolerant to cold, high salinity, and drought
compared with wild-type plants. Raffinose and L-proline, which are usually
increased under stress conditions, accumulated at higher levels in d975, even
under normal conditions. Based on these results, Nakamichi et al. have
proposed that PRR9, PRR7 and PRR5 may be involved in a mechanism
that anticipates diurnal cold stress and initiates a stress response by mediat-
ing cyclic expression of stress response genes, including DREB1/CBF.

F. PIF7 AS A POSSIBLE MEDIATOR BETWEEN THE CIRCADIAN CLOCK AND

DREB1/CBF IN STRESS SIGNALLING

Although several research groups have independently shown clock-controlled

expression of DREB1/CBF, the mechanism by which clock proteins affect
DREB1/CBF expression has not been elucidated. There are six conserved
motifs, boxes I–VI, in the promoter sequences of DREB1s. Kidokoro et al.
(2009) demonstrated that box V with the G-box sequence negatively regulated
the clock-controlled expression of DREB1C. Using yeast one-hybrid screens,
they identified phytochrome-interacting factor 7 (PIF7) as a factor that
specifically binds to the G-box of the DREB1C promoter (Fig. 2). Transacti-
vation experiments with Arabidopsis protoplasts indicated that PIF7 func-
tions as a repressor for DREB1C expression. PIF7 interacts with the clock
protein TOC1/PRR1 and the red-light photoreceptor phyB (Kidokoro et al.,
2009; Leivar et al., 2008; Yamashino et al., 2003), and the activity of PIF7 is
enhanced by co-expression of TOC1 or phyB (Kidokoro et al., 2009). In a
PIF7 loss-of-function mutant, the circadian rhythms of DREB1B and
DREB1C expression were disrupted under LL; the transcript levels of both
were elevated, and the amplitudes of the rhythmic expression were much
lower in pif7, compared with wild-type plants. Kidokoro et al. (2009) pro-
posed that the negative regulation of DREB1 expression may be important for
394 R. NEFISSI ET AL.

avoiding growth retardation of plants owing to an accumulation of DREB1

protein under unstressed conditions.

G. TOC1 AS A MOLECULAR SWITCH CONNECTING THE CLOCK AND

RESPONSES TO ABA AND DROUGHT

The expression of genes for the biosynthesis and signalling of plant hormones
such as ABA, auxin, ethylene, cytokinine, GA and brassinosteroid are con-
trolled by a circadian clock (Hanano et al., 2006). TOC1/PRR1 is a critical
component of the circadian clock system in Arabidopsis. Recently, Legnaioli
et al. (2009) demonstrated that TOC1/PRR1 protein was bound to the
promoter of the ABA-related gene (ABAR/CHLH/GUN5) and controlled
its circadian expression (Fig. 2). TOC1/PRR1 expression was acutely induced
by ABA, before TOC1/PRR1 binding to the ABAR promoter. The circadian
control of ABAR expression was modulated by ABA. The gated induction of
TOC1/PRR1 by ABA was suppressed in ABAR RNAi plants, suggesting that
reciprocal regulation between TOC1 and ABAR was important for ABA
signalling. The over-expression of TOC1/PRR1 significantly reduced the
plant tolerance to drought, whereas plants with loss-of-function of TOC1
(toc1-2 and TOC1 RNAi) responded to water-deficient conditions better than
wild-type plants. ABA INSENSITIVE 3 (ABI3), a transcription factor with
a B3 domain, functions as a major regulator of ABA signalling (Giraudat
et al., 1992). TOC1/PRR1 was identified as the ABI3-interacting protein 1
(AIP1; Kurup et al., 2000). These results suggest that clock-dependent gating
of plant hormone functions is vital for cellular homeostasis under various
environmental conditions.

H. RELEASE OF SEED DORMANCY

The dormant stage of seeds is highly tolerant to stress conditions, and seed
dormancy and germination are controlled by plant hormones such as ABA
and GA. Recently, Penfield and Hall demonstrated that clock genes such as
LHY, CCA1, GI and TOC1 play key roles in responses to the signals that
break seed dormancy in Arabidopsis (Penfield and Hall, 2009).

VI. PERSPECTIVES

Loss-of-function of the ELF3 gene produced pleiotropic phenotypes, sug-

gesting that ELF3 may have several functions in more than two signalling
pathways. A protein–protein interaction between ELF3 and phyB was
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 395

Clock components MAPK cascades

PhyA/ Ssk2/
PhyB FKF Ste11 AtMEKK1 MAPKKK
PhyB Ssk22

LHY/
ELF3

Ste5
PRRs GI AtMEK/
CCA1 Ste7 Pbs2
AtMKK2
MAPKK

SVP/ PIFs/
CDFs Kss1/
FLC PILs
Fus3
Hog1 AtMPK4 MAPK

Hypocotyl Osmotic Multiple

Flowering Flowering Mating stresses
elongation stress

Fig. 3. Analogy between clock components and MAPK cascades, showing a

possible role of ELF3 as a scaffold protein. For details, see the text.

reported. Recently, two groups demonstrated several additional protein–

protein interactions of ELF3 (Fig. 3; Yoshida et al., 2009; Yu et al., 2008).
Mitogen-activated protein kinases (MAPKs) play central roles in many
distinct signal transduction pathways (Mizoguchi et al., 1997) and
are activated by environmental stimuli (e.g. high or low osmotic stress,
dehydration, high or low temperature), growth substances (e.g. growth hor-
mones, pheromones) and biotic stresses (e.g. infection by bacteria or viruses,
attack by insects or nematodes). MAPKs are good research models for
studying both specificity and diversity of signalling pathways. For activation,
MAPKs require phosphorylation of both the threonine and tyrosine residues
in the conserved TXY motif, by their upstream factors called MAPK kinases
(MAPKKs). MAPKKs themselves are also phosphorylated and activated by
their upstream factors, MAPKK kinases (MAPKKKs). In Saccharomyces
cerevisiae, three types of MAPKs participate in three different signalling
pathways (Schwartz and Madhani, 2004). Fus3/Kss1, Hog1 and Mpk1 func-
tion in the responses to mating pheromone, osmotic stress and cell wall
biosynthesis, respectively. These three MAPKs are activated by three differ-
ent MAPKKs and MAPKKKs. To achieve this kind of specificity, a scaffold
protein interacts with all three kinases. For example, Ste5 is a scaffold
protein for the MAPK cascade of the mating-pheromone pathway (Seeliger
and Kuriyan, 2009) and Ste11 (MAPK), Ste7 (MAPKK) and Fus3/Kss1
396 R. NEFISSI ET AL.

(MAPKs) all interact with Ste5 (Fig. 3). There is no epistatic relationship
between Ste5 and any other cascade of protein kinases. In the Ssk2/Ssk22–
Pbs2–Hog1 pathway of S. cerevisiae and in the AtMEKK1–AtMEK/
AtMKK2–AtMPK4 pathway, Pbs2 and AtMEKK1, respectively, appear
to function as scaffold proteins (Fig. 3; Ichimura et al., 1998; Maeda et al.,
1995; Mizoguchi et al., 1998; Posas and Saito, 1997). In Arabidopsis, there are
20 MAPKs, 10 MAPKKs and more than 50 MAPKKKs (MAPK Group,
2002). Specificity for the interaction and activation in each MAPK cascade
may be controlled by unidentified scaffold proteins.
Many of the clock proteins in Arabidopsis interact with other proteins and
are proposed to function in signalling complexes (Fig. 2). These putative
complexes include photoreceptors (phyA, phyB, CRY2, FKF1 and ZTL),
F-box proteins involved in protein degradation (FKF1 and ZTL) and tran-
scription factors (SVP, FLC, CDFs and PIFs/PILs). We have recently identi-
fied phyB mutations as suppressors of the late-flowering phenotype of lhy;
cca1 under LL (Miyata and Mizoguchi, unpublished). In a manner similar to
Ste5, ELF3 may function as a scaffold protein for phyB, LHY/CCA1 and
SVP/FLC in the control of flowering time (Figs. 2 and 3; Mizoguchi and
Yoshida, 2009). ELF3 was proposed to function as an adaptor protein for
COP1 and GI (see Section IV.D), and CRY2 directly interacts with ELF3 and
COP1. Thus, ELF3 appears to function as a scaffold protein for different sets
of partners in distinct signalling pathways, which may explain the pleiotropic
phenotype of elf3 mutants. FKF1 and ZTL, closely related F-box proteins
(Baudry et al., 2010), interact with and are regulated by GI. Molecular targets
for protein degradation of FKF1 and ZTL are CDFs, which control CO
expression, and TOC1, which is a central oscillator, respectively (Kim et al,
2007; Sawa et al., 2007). CDFs and TOC1 bind to ZTL and FKF1, and
therefore, these two F-box proteins appear to function as scaffold proteins.
In the phyA/phyB–PRRs–PIFs/PILs pathway, the PIFs/PILs probably func-
tion as scaffold proteins.
The expression of many genes, including stress-inducible genes, is under
the control of a circadian clock (Covington et al., 2008; Harmer and Kay,
2000, Kant et al., 2008, Kreps et al., 2002). The molecular mechanism
underlying the clock-controlled response to low temperature is starting to
be understood; however, the machinery behind the responses to other stres-
sors such as desiccation, high temperature, UV and high/low osmolarity
remains unknown. Transcriptional as well as post-transcriptional and post-
translational regulation may play key roles in clock-controlled responses to
environmental stress. The identification of enzymatic activities that are posi-
tively or negatively regulated by a circadian clock is the next important
challenge.
 DEVELOPMENTAL CONTROLS AND STRESS RESPONSES 397

ACKNOWLEDGEMENTS

This work was supported in part by the Bilateral Joint-Lab Project between
Japan and France of the Ministry of Education, Culture, Sports, Science and
Technology (MEXT; to T. M.).

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