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REVIEW: PART OF A SPECIAL ISSUE ON FLOWER DEVELOPMENT
Molecular control of seasonal flowering in rice, arabidopsis and temperate cereals
Roshi Shrestha, Jorge Go
´mez-Ariza, Vittoria Brambilla and Fabio Fornara*
University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milan, Italy
* For correspondence. E-mail fabio.fornara@unimi.it
Received: 1 November 2013 Returned for revision: 11 December 2013 Accepted: 4 February 2014
†Background Rice (Oryza sativa) and Arabidopsis thaliana have been widely used as model systems to understand
how plants controlflowering time in response to photoperiod and cold exposure. Extensive research has resulted in the
isolation of several regulatory genes involved in flowering and for them to be organized into a molecular network
responsive to environmental cues. When plants are exposed to favourable conditions, the network activates expres-
sion of florigenic proteins that are transported to the shoot apical meristem where they drive developmental repro-
gramming of a population of meristematic cells. Several regulatory factors are evolutionarily conserved between
rice and arabidopsis. However, other pathways have evolved independently and confer specific characteristics to
flowering responses.
†Scope This review summarizes recent knowledge on the molecular mechanisms regulating daylength perception
and flowering time control in arabidopsis and rice. Similarities and differences are discussed between the regulatory
networks of the two species and they are compared with the regulatory networks of temperate cereals, which are evo-
lutionarily more similar to rice but have evolved in regions where exposure to low temperatures is crucial to confer
competence to flower. Finally,the role of flowering time genes in expansion of rice cultivation to Northern latitudes is
discussed.
†Conclusions Understanding the mechanisms involved in photoperiodic flowering and comparing the regulatory
networks of dicots and monocots has revealed how plants respond to environmental cues and adapt to seasonal
changes. The molecular architecture of such regulation shows striking similarities across diverse species.
However, integration of specific pathways on a basal scheme is essential for adaptation to different environments.
Artificial manipulation of flowering time by means of natural genetic resources is essential for expanding the culti-
vation of cereals across different environments.
Key words: Oryza sativa, rice, Arabidopsis thaliana, cereals, photoperiodic flowering, vernalization, florigen,
flower development.
INTRODUCTION
Floral initiation is a major physiological change that sets the
switch from vegetative to reproductive development in most
plant species. The transition from a vegetative (production of
stem and leaves) to a reproductive stage (production of inflores-
cences and flowers) determines the time of flowering (or heading
date in cereals) and is one of the most important developmental
switches in the life cycle of plants. To maximize reproductive
success and guarantee sufficient seed production for propagation
of the species, flowering time should be tightly regulated through
the integration of environmental inputs (daylength, temperature,
light quality, water and nutrient availability) with endogenous
cues (juvenility, stage of development). Depending on their re-
quirement for daylength, plants can be classified into three cat-
egories. Long-day (LD) plants flower when the photoperiod
exceeds a critical daylength, short-day (SD) plants flower
when the photoperiod is shorter than a critical daylength and day-
neutral plants flower regardless of daylength. The critical day-
length for floral induction is specific to each species but often
varies between accessions of the same species. In many plant
species, flowering can also be stimulated by exposure to low non-
freezing temperatures for several weeks. This process, known as
vernalization, occurs in temperate zones during winter and pre-
pares the plant to switch to reproductive growth only after the
cold season, when temperatures become favourable again.
Molecular genetic studies on model plants such as arabidopsis
and rice (Oryza sativa) have allowed identification of genes con-
trolling responses to environmental inputs and many of those
have been shown to be conserved between the two model
species, despite 150 million years of divergent evolution
(Chaw et al., 2004). However, during evolution, other pathways
have evolved and several factors have been recruited to novel
functions, increasing the diversity of flowering behaviours and
adapting the species to grow across broader areas. Although ara-
bidopsis and rice have been crucial to establish the basal architec-
ture of floral regulatory networks, studies conducted on other
species, including temperate cereals (e.g. wheat and barley)
greatly contributed to our understanding of the molecular
mechanisms controlling flowering.
In this review we will focus on rice as a model for SD plants
and discuss the pathways involved in daylength measurement
and flowering, mainly in comparison with arabidopsis. We will
discuss similarities of the core regulatory pathways controlling
photoperiodic flowering but will also address the pathways that
have evolved specifically in rice, which are not present in arabi-
dopsis. Finally, we will contrast regulatory networks active in
temperate cereals with those of arabidopsis and rice. We will
take into account the spatial separation of functions in leaves
and at the shoot apex.
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Besides its importance as a model system, rice is a crop and
staple food for most parts of the world. It was first domesticated
in Southern China and has evolved to adapt to a range of geo-
graphical regions over time. Currently, rice is cultivated over a
wide range of latitudes from 558Nto368S(Khush, 1997). This
has been possible through the diversification of flowering time.
In fact, flowering of rice is accelerated under SD conditions;
however, artificial selection has led to successful cultivation
even under LD conditions. Some varieties of rice with weak
or no photoperiod sensitivity flower very early at Northern
latitudes, particularly in Europe, Northern China and Japan.
We will discuss the genetic factors that have allowed such expan-
sion and that represent an increasing list of molecular tools
that plant breeders can use to expand further rice cultivation
worldwide.
THE PHOTOPERIODIC PATHWAY IN
ARABIDOPSIS AND RICE
Facultative LD species, such as arabidopsis, accelerate flowering
as days become longer, and several genes have been isolated that
control responses to photoperiodic cues (Andre
´s and Coupland,
2012). The corresponding mutants flower later when exposed to
long inductive days, typical of spring or early summer, but do not
affect flowering time when plants are grown under SDs (Turck
et al., 2008). Central to the photoperiod pathway in arabidopsis
is CONSTANS (CO), a zinc finger transcription factor that inte-
grates environmental and endogenous information to trigger
flowering at the appropriate time of the year. In the vascular
tissue of leaves, the CO protein directly activates expression of
FLOWERING LOCUS T (FT), which encodes a florigenic
protein promoting flowering (An et al., 2004;Tiwari et al.,
2010). Expression of CO is regulated by the circadian clock
that sets its rhythmic cycling to reach a peak at the end of the
day under LDs. Cycling of CO mRNA depends on the activity
of a protein complex formed by GIGANTEA (GI) and
FLAVIN BINDING, KELCH REPEAT, F-BOX 1 (FKF1), a
protein containing an F-box and blue light photoreceptor
domains. The interaction between GI and FKF1 requires light,
and the presence of GI protein is necessary to confer stability
to the FKF1 protein (Sawa et al., 2008; Fornara et al., 2009).
Upon interaction with GI, FKF1 targets a group of DOF tran-
scription factors, collectively known as CYCLING DOF
FACTOR (CDF) genes, for degradation (Fornara et al., 2009).
CDF proteins directly bind to the promoters of CO and FT,to
prevent their expression when plants are exposed to short photo-
periods (Imaizumi et al., 2005;Y.H.Song et al., 2012).
Degradation of the CDFs occurs at the DNA of target loci and
results in de-repression of CO and FT, allowing flowering to
occur (Fig. 1). Therefore, genotypes in which the activity of
the CDF genes is strongly reduced are insensitive to daylength,
and flower early under any photoperiodic condition (Fornara
et al., 2009;Go
´mez-Ariza and Fornara, 2012).
CONSTANS is also tightly regulated at the post-
transcriptional level. Despite its transcription being high
during the night under both SDs and LDs, the protein does not
accumulate in the dark because it is quickly directed to the
proteasome through the activity of an E3 ubiquitin ligase
encoded by CONSTITUTIVE PHOTOMORPHOGENIC 1
(COP1)(Jang et al., 2008;Liu et al., 2008). Other proteins,
including phytochromes and ubiquitin ligases, influence the sta-
bility of CO protein at different times of day, to ensure it accumu-
lates only when the day is sufficiently long (Valverde et al., 2004;
Pineiro et al., 2012;Y.H.Song et al., 2012).
Regulation of photoperiodic flowering through CO has fea-
tures of the external coincidence model of photoperiodism, ori-
ginally proposed by Pittendrigh and Minis (1964). According to
this model, an endogenous oscillator sets the rhythmic phase of
expression of target molecules. Coincidence of a particular
phase of expression with an external factor, such as light, triggers
a developmental response. Several steps of the photoperiodic
cascade of arabidopsis represented in Fig. 1require light at the
appropriate time of a circadian cycle to induce flowering. The
GI–FKF1 regulatory complex is expressed and stabilized only
when the peak of mRNA expression of the two genes coincides
with light under long daylengths. This allows the corresponding
proteins to interact and the complex to be stabilized. Similarly,
CO protein accumulation takes place only at the end of an LD,
in the presence of light that stabilizes it. Only under these condi-
tions can the CO protein accumulate, activate FT expression and
induce flowering. Under SDs, GI and FKF1 proteins do not inter-
act, preventing CO mRNA from increasing at the end of the day.
Therefore, light is necessary at the appropriate time of day to
activate the pathway. In agreement with this model, in mutants
in which the diurnal waveform of CO mRNA is also displaced
towards the light phase under SDs, or in which the protein is
allowed to accumulate because of mutations in genes controlling
its post-transcriptional stability, FTexpression and flowering are
activated regardless of daylength (Yanovsky and Kay, 2002;
Valverde et al., 2004;Jang et al., 2008;Fornara et al., 2009).
This mechanism incorporates endogenous and environmental
information to synchronize flowering with seasons characterized
by LDs.
Rice shares a similar photoperiodic pathway, mediating day-
length responses. OsGI and Heading Date 1 (Hd1) have been
cloned and shown to encode homologues of arabidopsis GI and
CO, respectively (Yano et al., 2000;Hayama et al., 2003).
Several homologues of FT are encoded in the rice genome and
at least three of them can promote flowering when overexpressed,
i.e. Hd3a,RICE FLOWERING LOCUS T 1 (RFT1) and FT-like 1
(FTL1)(Izawa et al., 2002;Kojima et al., 2002;Ogiso-Tanaka
et al., 2013).
Similar to arabidopsis, control of Hd1 expression is crucial to
confer a photoperiodic response (Brambilla and Fornara, 2013).
Hd1 was originally identified as a major quantitative trait locus
(QTL) and was later cloned by map-based approaches (Yano
et al., 2000). Hd1 is highly homologous to CO and the Hd1
protein is thought to be involved in DNA binding. However,
direct interaction of Hd1 with the Hd3a promoter has not been
reported. Additionally, whereas CO promotes flowering under
LDs, Hd1 promotes flowering under SDs but represses flowering
under LDs. The bi-functionality of Hd1 became clear through the
analysis of plants carrying hd1 loss-of-function alleles that cause
late flowering under SDs and early flowering under LDs (Yano
et al., 2000;Izawa et al., 2002). The effect on flowering is corre-
lated with Hd3a mRNA levels that are reduced in hd1 mutants
grown under SDs and increased when hd1 mutants are grown
under LDs (Izawa et al., 2002). Therefore, Hd1 has opposite
effects on Hd3a expression and flowering that switch depending
on daylength.
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OsGI is a positive regulator ofHd 1 expression, under both SDs
and LDs. In arabidopsis, overexpression of GI induces CO tran-
scription and rapid flowering under any daylength. Also in rice,
overexpression of OsGI in transgenic plants triggers higher Hd1
expression. However, because of the bi-functional features of
Hd1, higher levels of OsGI and Hd1 lead to decreased Hd3a tran-
scription and inhibition of flowering under both SDs and LDs
(Hayama et al., 2003). The same inhibitory effect was observed
when the Hd1 gene itself was overexpressed under SDs, abolish-
ing its diurnal cycling and causing high levels of transcripts also
to accumulate during the light phase. These data suggested that
exposure of Hd1 protein to light converted it to a repressor of
flowering, even if the length of the light phasewas below the crit-
ical threshold necessary for flowering (Ishikawa et al., 2011).
The molecular mechanisms responsible for switching Hd1 func-
tion are currently unknown. In arabidopsis, CO acts as floral ac-
tivator only and, therefore, what is crucial in this species is the
shape of CO protein accumulation during the day, which needs
to be tightly controlled in order to reach a maximum at dusk
under LDs. Conversely, accumulation of Hd1 protein occurs
during both the light and dark phases, suggesting that a different
mechanism is responsible for the bi-functional activity of Hd1
(Ishikawa et al., 2011).
OsGI transcription is controlled by the circadian clock and
shows a rhythmic expression pattern which peaks at the end of
the light period, similarly to arabidopsis (Hayama et al., 2003).
Mutations in osgi affect a large set of rice genes, besides Hd1,
and about 75 % of rice transcripts show altered levels in the
mutant, during a diurnal time course (Izawa et al., 2011).
Many of these genes encode proteins probably involved in
circadian clock function, at least based on homology with
known arabidopsis clock regulators, and OsGI can affect expres-
sion of LATE ELONGATED HYPOCOTYL (LHY), and of several
PSEUDO RESPONSE REGULATOR (PRR) genes, including
PRR1,PRR59 and PRR95. Interestingly, Hd2/PRR37 expression
was not affected in the osgi mutant, possibly indicating inde-
pendent control of heading date by these factors (Fig. 1).
However, how OsGI protein activates expression of Hd1 has
not been clarified yet. A homologue of FKF1 exists in rice and
shows a diurnal expression pattern identical to that described
in arabidopsis (Murakami et al., 2007;Higgins et al., 2010).
DOF transcription factors are also present in the rice genome,
and one of them, OsDOF12, is implicated in photoperiodic flow-
ering (Li et al., 2008,2009). Transcription of OsDOF12 is high in
leaves and diurnally regulated with a trough during the night
(Izawa et al., 2011). Transgenic rice overexpressing OsDOF12
flowers early compared with wild-type plants under LD, but
not SD conditions, showing higher Hd3a expression compared
with non-transgenic controls (Li et al., 2009). However, no
altered levels of Hd1 mRNA were reported, suggesting a differ-
ent genetic route for OsDOF12 action on flowering (Li et al.,
2009).
Based on these observations, it was possible to conclude that
the external coincidence model can be applied to rice, albeit
with modifications from the scheme proposed for an LD plant
(Hayama et al., 2003). Short-day plants measure the duration
of the dark phase, during which expression of florigenic proteins
starts. Hd1 is likely to act as a sensor of night length, being con-
verted to a floral activator when exposed to darkness. Expression
of Hd1 peaks during the night phase and, when this is sufficiently
Arabidopsis Rice
GI + FKF1
miR172 CDFs
OsGI
Phy B
PPD1
FT/VRN3 VRN1
Flowering Cold
VRN2
Ghd7
Ghd8
Hd16
OsCOL4
OsMADS51
Long day
Short day
Circadian clock regulated
Vernalization pathway
Ehd1 inducers
Ehd1 repressors
Hd17
Ehd2
Ehd3
Ehd4
OsMADS50
Temperate cereals
CO
FRI
Hd1 Ehd1
FLC
Flowering
FT
Cold
Phy B Phy B
Hd6
PRR37
Hd3a RFT1
miR156
Flowering
LD
LD
LD
CO
SD
SD
SD
SD
SD
SD
LD LD
LD
LD
LD
LD
SD
FIG. 1 . Simplified regulatory networkscontrolling florigen production inleaves of different plant species. Networks of arabidopsis (left panel), rice (central panel)
and temperate cereals (right panel) are compared. Small white and black boxes indicate regulatory connections occurring primarily under LDs and SDs, respectively.
Arrows indicate transcriptional activation, whereas flat-ended arrows indicate transcriptional repression. The blue boxes in both the arabidopsis and the temperate
cereal models include genes involvedin vernalization responses. The green and red boxes in the rice model include positiveand negative regulators of Ehd1 expression,
respectively. A clock symbol close to a gene indicates that its transcription is controlled by the circadian clock.
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long, can promote enough Hd3a protein expression and initiate
flowering. In this modified model, the coincidence of darkness
with peak expression of Hd1 is crucial to trigger a developmental
response.
THE EHD1–GHD7 PATHWAY ISUNIQUE TO RICE
Flowering in rice does not strictly require the OsGI – Hd1 – Hd3a
signalling pathway, and activation of florigens can be achieved
through independent mechanisms. Natural variation between
rice species and cultivars allowed cloning of EARLY
HEADING DATE 1 (Ehd1) which encodes a B-type response
regulator that does not have orthologues in arabidopsis and
defines a unique floral activation pathway in rice (Doi et al.,
2004). Ehd1 promotes flowering particularly under SDs, in par-
allel to Hd1, but can also promote flowering under LDs, when
Hd1 acts as a repressor (Doi et al., 2004). The Ehd1 protein con-
sists of a receiver domain at its N-terminus and a GARP
DNA-binding motif (Riechmann et al., 2000). It induces flower-
ing under SDs and LDs by upregulating Hd3a or RFT1 independ-
ently of Hd1, demonstrating the potential of Ehd1 and Hd1 to act
redundantly on separate pathways (Doi et al., 2004).
Regulation of Ehd1 is crucial for correct flowering time in rice,
and a large group of activatorsand repressors has been cloned and
shown to modulate its expression (Fig. 1). Repressors of Ehd1
have a central role in the photoperiodic network and, among
them, Grain number, plant height and heading date 7 (Ghd7)
is particularly important to shape Ehd1 diurnal and seasonal tran-
scription. Ghd7 encodes a CCT domain protein and is expressed
at higher levels under LDs, correlating with limited expression
and activity of Ehd1 under non-inductive photoperiods (Xue
et al., 2008). Besides its effect on flowering, Ghd7 controls
other traits in rice including grain number and plant height, indi-
cating pleiotropic roles for the protein in other processes (Xue
et al., 2008).
Analysis of Ghd7 and Ehd1 expression in response to light has
defined a novel coincidence mechanism and a double gating
system that sets critical daylength recognition for Hd3a expres-
sion under specific photoperiods (Itoh et al., 2010). Rice plants
exposed to photoperiods shorter than 13.5 h induce Hd3a and
Ehd1 expression, while reducing Ghd7 expression. Such inter-
play is achieved by a double regulatory mechanism dependent
on OsGI and phytochromes. Induction of Ehd1 expression in
the morning requires functional OsGI, which sets a gate (a sensi-
tive phase set by the circadian clock in response to light) around
dawn, which is independent of daylength, because it occurs at the
same time under both LDs and SDs. The gate is sensitive to blue
light in the morning and, when open, Ehd1 expression increases
and Hd3a is activated. OsGI protein accumulation reaches its
trough at dawn and therefore its effect on the gate is likely to
be indirect. Under LDs, Ghd7 expression is high and its induci-
bility is gated at the same time as the OsGI and blue light-
dependent gate. Under these conditions, induction of Ghd7 in
the morning is sufficient to repress Ehd1 transcription and
delay flowering. However, as daylength decreases under a critical
threshold, maximum inducibility of Ghd7 is gated during the
night, resulting in reduced expression the following morning
and de-repression of Ehd1 (Itoh et al., 2010). Expression of
Ghd7 requires functional phytochromes and it is abolished in
the PHOTOPERIOD INSENSITIVITY 5 (SE5) mutant, which
encodes a haem oxygenase very similar to LONG
HYPOCOTYL 1 (HY1) of arabidopsis and is impaired in phyto-
chrome chromophore biosynthesis (Izawa et al., 2000). Plants
in which SE5 is mutated are insensitive to photoperiod and
flower early under both SDs and LDs (Izawa et al., 2000).
Expression of Ghd7 is also strongly reduced in plants where a
long night is interrupted by a short red light pulse (a ‘night
break’) that converts phytochromes to the inactive form. These
data suggested that functional phytochromes are required for
correct expression of Ghd7 and floral repression, and that red
light signals are integrated in the photoperiodic flowering
network through Ghd7. Rice has three phytochrome genes
(PhyA–PhyC) but se5 single mutants lack all functional
forms. Therefore, single and double phytochrome mutants
have been useful tools to understand how correct Ghd7 expres-
sion is determined (Osugi et al., 2011). The results suggest that
phytochromes are not required to set the light-sensitive phase
for Ghd7 expression. However, PhyA homodimers and PhyB –
PhyC heterodimers are independently sufficient to trigger
Ghd7 transcription, whereas PhyB can repress it. The action of
phytochromes on Ghd7 expression is therefore more complex
than previously anticipated by the analysis of SE5.
Neither such a double gating mechanism nor the existence of
homologues of Ehd1 and Ghd7 has been observed in arabidopsis.
Expression of FT, as opposed to Hd3a expression, showed no
critical daylength threshold in mathematical modelling experi-
ments based on biological data (Salazar et al., 2009).
Additionally, the flowering time of arabidopsis accessions
grown under several SD and LD photoperiods indicated that
most ecotypes could discriminate variations of 2 h under
several SDs and LDs, and no critical photoperiod could be deter-
mined (Giakountis et al., 2010). Despite the fact that broad
genetic variation could also be expected among rice varieties,
these data point to a crucial difference in the way photoperiod
is perceived in an LD and SD plant to promote flowering, prob-
ably reflecting the different adaptation to their environment.
Regulators of Ghd7
The double gating mechanism based on the interplay between
Ehd1 and Ghd7 is crucial for daylength measurement, and proper
regulation of Ghd7 expression and activity sets the sensitivity of
the measure. Consistent with Ghd7 being central to the pathway,
extensive natural genetic variation was reported at the Ghd7
locus and at loci regulating its activity. Such variation has
allowed the cloning of a number of additional QTLs involved
in flowering.
Map-based cloning of Heading date 16 (Hd16) revealed that
this gene encodes a caseine kinase-I protein (Hori et al., 2013;
Kwon et al., 2014). The Hd16 protein directly interacts with
and phosphorylates Ghd7, thus converting it to an active repres-
sor of flowering. Natural allelic variants of Hd16 showing
reduced functionality de-repress expression of Ehd1 and the
florigens, leading to accelerated flowering particularly under
LD conditions. Natural variation at the Ehd3 locus allowed
cloning of a repressor of Ghd7. Ehd3 encodes a nuclear protein
with two PHD-finger motifs, and Matsubara et al. (2011) demon-
strated that it is a repressor of Ghd7 and activator of Ehd1 expres-
sion particularly under LDs. The two functions can be
genetically separated, indicating that activation of Ehd1 by
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Ehd3 can also be achieved independently of Ghd7 repression
(Matsubara et al., 2011).
Finally, cloning of Hd17 revealed that it encodes a homologue
of EARLY FLOWERING 3 (ELF3) which represses Ghd7 expres-
sion under SDs and LDs, resulting in increased levels of Ehd1.
The effect on flowering is probably indirect and due to the influ-
ence of OsELF3 on circadian clock function (Saito et al., 2012;
Zhao et al., 2012;Y.Yang et al., 2013).
Activators of Ehd1 expression
Several genes have been cloned whose activity eventually
converges on Ehd1 transcriptional regulation.
Early heading date 2 (Ehd2) encodes a putative transcription
factor with zinc finger motifs, which is an orthologue to
INDETERMINATE1 (ID1) of maize (Matsubara et al., 2008a).
Similarly to ID1,Ehd2 is expressed mainly in leaves
(Colasanti et al., 2006). Plants mutated in Ehd2 prevent upregu-
lation of Ehd1, show delayed flowering under SDs and cannot
flower under LDs, indicating that Ehd2 is a fundamental gene
for the LD promotion of flowering (Matsubara et al., 2008a;
Wu et al., 2008).
In addition to Ehd2,OsMADS51, a type I MADS-box tran-
scription factor, promotes flowering upstream of Ehd1 under
SD conditions (Kim et al., 2007). Plants in which expression
of OsMADS51 is artificially increased or decreased display
altered heading dates that can be observed only under SD, but
not under LD conditions. Expression of OsMADS51 depends
on OsGI, and Kim et al. (2007) demonstrated that reduction of
OsGI transcription by RNA interference (RNAi) is correlated
with lower expression of OsMADS51 and Ehd1. This study
showed how an OsGI-dependent signalling cascade can activate
Hd3a under SDs, independently of Hd1, through Ehd1 and
OsMADS51.
The Ehd4 locus encodes a nuclear-localized CCCH-type zinc
finger protein unique to the Oryza genus (Gao et al., 2013).
Loss-of-function mutants flower late under any condition, but
particularly under LDs. Delayed flowering is associated with
low levels of Ehd1 and the florigens, but not of Hd1, indicating
that Ehd4 specifically targets the Ehd1 pathway (Fig. 1).
Repressors of Ehd1 expression
Several repressors of Ehd1 expression have been cloned that
can function in a range of photoperiods. Hd5/DAYS TO
HEADING 8 (DTH8)/Ghd8 (from here on Ghd8) encodes a pu-
tative HEME ACTIVATOR PROTEIN 3 (HAP3) subunit of the
CCAAT-box-binding transcription factor complex. It acts as
floral repressor under LD conditions and delays flowering by
downregulating the expression of Ehd1,Hd3a and RFT1 (Wei
et al., 2010). Under SDs, Ghd8 was reported to induce expression
of these floral regulators, promoting flowering and showing some
degree of bi-functionality, similarly to Hd1 (Yan et al., 2011).
Expression of Ghd8 is not influenced by Ghd7 and Hd1, two
major LD repressors, indicating a distinct genetic pathway for
flowering time control (Wei et al., 2010). However, in arabidop-
sis, HAP3 and HAP5 proteins have been shown to interact phys-
ically with CO protein, forming a CCAAT-box-binding complex
directly controlling FT expression (Wenkel et al., 2006;Cai
et al., 2007;Kumimoto et al., 2010). If such a mechanism
were operating in rice, HAP/Ghd8 proteins would act in the
same genetic pathway as Hd1 to control Hd3a expression,
perhaps directly. It will be interesting to determine whether
Ghd8 and Hd1 proteins can physically interact to control expres-
sion of Ehd1 or the florigens and influence photoperiodic flower-
ing responses. Genetic data indicate that when overexpressed in
arabidopsis, Ghd8 triggers early flowering under LDs, and
causes no alteration of flowering time under SDs, similarly to
overexpression of other HAP subunits of arabidopsis, providing
evidences that the function of this class of proteins is conserved
between monocots and dicots (Kumimoto et al., 2010;Yan et al.,
2011).
OsLFL1 (Oryza sativa LEC2 and FUSCA3 Like 1) is a B3 tran-
scription factor that can delay flowering upon overexpression, by
repressing Ehd1 and its downstream targets (Peng et al., 2008).
Repression of Ehd1 mediated by OsLFL1 is probably direct, as
demonstrated by chromatin immunoprecipitation and gel shift
assays (Peng et al., 2007). Binding of OsLFL1 protein is
mediated by RY motifs present in the promoter region of Ehd1.
Such motifs can also mediate transactivation of a reporter gene
in yeast when OsLFL1 protein is expressed from an effector
plasmid. OsLFL1 is the only direct regulator of Ehd1 reported
to date. Its transcriptional control is mediated by chromatin mod-
ifications that require O. sativa VERNALIZATION INSENSITIVE
LIKE 2 (OsVIL2) and O. sativa EMBRYONIC FLOWER 2b
(OsEMF2b), the former encoding a PHD finger histone-binding
protein, and the latter encoding a component of Polycomb
Repressor Complex 2(PRC2) (J. Yang et al., 2013). A protein
complex containing OsVIL2 and OsEMF2b can associate with
the OsLFL1 promoter and enrich histones with H3K27me3
marks, leading to silencing of the locus. Consistently, osvil2
and osemf2b mutants are late flowering and show decreased ex-
pression of Ehd1 and the florigens (J. Yang et al., 2013). These
mechanisms highlight the importance of epigenetic regulation
of gene expression to fine-tune environmental responses. The
fundamental nature of these processes also accounts for its occur-
rence across divergent plant groups (Sung et al., 2006;Oliver
et al., 2009;J.Yang et al., 2013).
OsCO-Like 4 (OsCOL4) is a member of the CONSTANS-LIKE
(COL) family in rice. It is a constitutive flowering repressor that
functions under both SD and LD conditions (Lee et al., 2010).
The OsCOL4 mutant plants showed early flowering under
both SDs and LDs, while the overexpressing transgenic lines
showed a late flowering phenotype (Lee et al., 2010). The expres-
sion of Ehd1 and Hd3a was higher in OsCOL4 mutants, suggest-
ing that it functions upstream of these floral regulators. Neither
the overexpressors nor the mutant plants had altered transcription
levels of Hd1 or OsGI, indicating that OsCOL4 is specific to the
Ehd1 pathway.
DIFFERENTIAL REGULATION OF FLORIGEN
EXPRESSION UNDER LONG AND SHORT DAYS
Plants exposed to inductive daylengths activate expression of
florigenic proteins in the vasculature of leaves. In arabidopsis,
FT and TWIN SISTER OF FT (TSF) proteins are expressed in
the phloem of leaves and act as mobile, long-distance signals
to trigger developmental reprogramming at the shoot apical
meristem (SAM) (An et al., 2004;Yamaguchi et al., 2005;
Corbesier et al., 2007;Jaeger and Wigge, 2007;Mathieu et al.,
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2007). Hd3 and RFT1 encode two rice florigens and, similarly to
their arabidopsis orthologues, are expressed in the rice phloem
and move to the SAM (Tamaki et al., 2007;Komiya et al.,
2009). Plants suppressing both Hd3a and RFT1 mRNA expres-
sion by RNAi did not flower after 300 d under SDs. RNAi sup-
pression of Hd3a only delayed flowering under SDs, whereas
suppression of RFT1 expression delayed flowering under LDs
but not under SDs, indicating that the two florigens have distinct
effects on floral promotion depending on the photoperiod.
Additionally, RFT1 is redundant to Hd3a under SDs (Komiya
et al., 2008,2009). Critical daylength recognition does not
affect RFT1 expression as severely as Hd3a expression, and
RFT1 is essentially less influenced by LD repression as com-
pared with Hd3a (Itoh et al., 2010). These data have contributed
to build a model by which RFT1 and Hd3a encode LD- and
SD-specific florigens, respectively. The diverse impact of these
two florigens on photoperiodic flowering raises interesting
observations, as they are very similar in structure and located
physically close to each other (11.5 kb), suggesting a common
origin through tandem duplication, but also indicating the exist-
ence of distinct mechanisms of transcriptional regulation
(Komiya et al., 2008). Accumulating evidence indicates that
changes in the chromatin state can influence florigen expression,
in both rice and arabidopsis, and possibly cause differential regu-
lation of florigens in rice (Komiya et al., 2008;Adrian et al.,
2010;Gu et al., 2013). Periodic deacetylation of histones at the
FT locus in arabidopsis are associated with transcriptional
repression of FT. Components of a histone deacetylase complex
(HDAC) associate with the FT locus to limit its expression at
the end of the day (Gu et al., 2013). Plants mutated in such com-
ponents show enrichment of acetylated histones at FT, increase
FT expression and cause earlier flowering compared with wild-
type controls. Interestingly, in such mutants, FT induction still
requires functional CO,suggesting that chromatin modifications
leading to de-repression of FT still need the presence of a tran-
scriptional activator. Although acetylation of histones at the FT
locus has been associated with increased transcription of FT,
these chromatin modifications were reported to bea consequence
of FT activation, rather than its cause, suggesting that the timing
of these changes needs to be carefully monitored in order to reach
a conclusion about a causal relationship (Adrian et al., 2010). In
rice, accumulation of RFT1 mRNA in Hd3a RNAi-suppressed
plants was associated with increased H3K9 acetylation at the
RFT1 locus, indicating a possible relationship between histone
modifications and transcriptional activity at florigenic loci
(Komiya et al.,2008). Whether such modifications also involve
the Hd3a locus and precede transcriptional activation remains
to be determined. A mutantdefective ina histone methyltransfer-
ase, SDG724, demonstrated delayed flowering and reduced levels
of Hd3a and RFT1 (Sun et al., 2012). Interestingly, the
OsMADS50 and RFT1 loci, but not the Hd3a locus, showed en-
richment of H3K36me2/me3 chromatin marks, associated with
transcriptionally active chromatin. These data indicate differen-
tial regulation of the LD flowering pathway by histone methyla-
tion and provide an example of how RFT1 and Hd3a could be
differentially controlled by the dynamics of chromatin states.
Recent cloning of Hd2 showed that the gene underlying the
QTL is encoded by OsPRR37, a homologue of PRR7 of arabi-
dopsis and PPD1 of wheat and barley (Koo et al., 2013).
Flowering of plants carrying loss-of-function alleles of
OsPRR37 is accelerated under any daylength, but is particularly
enhanced under LDs. Under such conditions, OsPRR37 sup-
presses Hd3a but not RFT1 expression, indicating differential
sensitivity of the florigens to the presence of this floral regulator.
The molecular mechanisms that allow OsPRR37 to discriminate
between Hd3a and RFT1 are unclear, but provide another layer of
control that fine-tunes photoperiodic responses (Fig. 1).
A GENE NETWORK AT THE SHOOT
APICAL MERISTEM INTEGRATES
ENVIRONMENTAL CUES
In previous sections, we have described how daylength affects
flowering and how light duration is monitored through a regula-
tory network. The products of such a network are florigenic pro-
teins which are highly expressed in response to inductive
photoperiods and encode long-distance transmissible signals.
Florigens have been isolated from several plant species and
shown to control floral induction. In arabidopsis and rice, FT,
Hd3a and RFT1 proteins are produced in leaves and transported
to the SAM upon perception of the appropriate photoperiods, ini-
tiating panicle development (Corbesier et al., 2007;Tamaki
et al., 2007). A complex network of regulatory proteins controls
perception of florigenic signals at the apex and drives down-
stream developmental events.
Proteins that interact with Hd3a at the shoot apical meristem
Florigens cannot directly function as transcriptional regula-
tors in meristematic cells and thus interaction with other tran-
scription factors is essential for their functioning. In several
plant species, basic leucine zipper (bZIP) transcription factors
have been described as FT-interacting proteins required for flori-
gen activity at the apical meristem (Abe et al., 2005;Wigge et al.,
2005;Muszynski et al., 2006;Li and Dubcovsky 2008;Taoka
et al., 2011;Dong et al., 2012). Arabidopsis FD and rice
OsFD1 encode bZIP transcription factors required for florigen
function. According to current models, the heterodimer formed
by FT and FD in arabidopsis is a molecular hub integrating envir-
onmental cues and spatial information at the apical meristem
(Abe et al., 2005;Wigge et al., 2005;Jaeger et al., 2013). The
rice homologue of FD, OsFD1, interacts with Hd3a and the
dimer fulfils similar roles to the FT– FD unit. Direct interaction
between OsFD1 and Hd3a could not be demonstrated, but
contact between the two proteins was shown to be mediated by
14-3-3 proteins, now considered to be receptors of florigens
(Taoka et al., 2011,2013). Initial studies performed in different
species and aimed at identifying interactors of florigens wereper-
formed in yeast that contains proteins probably mediating the
interaction between FD homologues and florigens (Pnueli
et al., 2001;Abe et al., 2005;Wigge et al., 2005;Li and
Dubcovsky, 2008). The use of yeast as a heterologous system
to test protein– protein interactions might have thus hidden the
nature of the florigen receptorcomplex (or FAC, florigen activa-
tion complex). Structural and in vivo analyses in rice have
demonstrated that the FAC unit is actually a heterohexamer
formed by two molecules each of Hd3a, OsFD1 and a 14-3-3
protein that bridges the interaction between the florigen and the
bZIP transcription factor. A similar structure for the FAC
might apply to other plant species (Fig. 2). Mutagenesis of key
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residues at the interaction surface between Hd3a and 14-3-3 and
between 14-3-3 and OsFD1 has uncovered several features of the
complex and contributed to build the current model of florigen
action at the SAM (Taoka et al., 2013). Florigen is first received
by a 14-3-3 protein in the cytosol of a meristematic cell, from
which it is translocated to the nucleus where it binds OsFD1.
Phosphorylation of OsFD1 is required for binding to 14-3-3
receptors, and to activate target genes (Taoka et al., 2011). The
presence of the bZIP transcription factor is a prerequisite for con-
tacting DNA at the promoters of target genes, because florigens
or 14-3-3 proteins have no DNA binding property. The full extent
of potential targets of the FAC is currently unknown; however, it
is becoming clear that assembly of the FAC is to a certain extent
combinatorial, and bZIPs homologous to OsFD1 can replace it to
generate complexes controlling processes other than flowering
(Tsuji et al., 2013). The OsFD2 transcription factor is one such
example of a bZIP protein capable of forming a FAC, but control-
ling leaf development rather than floral transition. Plant architec-
ture is altered when OsFD2 is overexpressed, but not when a
mutated version unable to bind to 14-3-3 receptors is overex-
pressed. Since 14-3-3 proteins are ubiquitously expressed and
florigens are detected in the entire meristem, bZIP proteins are
probably restricting different FAC complexes to different cell
types. The broad extent of florigen– bZIP interactions and the po-
tential role of FACs in rice development are still to be fully
explored. Additionally, the role of RFT1 in FAC formation has
not been addressed yet, which might suggest additional com-
binatorial possibilities. These aspects are opening up novel pos-
sibilities for dissecting the full range of florigen functions in rice.
Molecular events occurring at the shoot apical meristem
in response to photoperiodic induction
Inductive photoperiods trigger florigen expression and move-
ment to the apical meristem, affecting the regulation of genes that
are involved in inflorescence formation. In arabidopsis apices the
FT–FD complex is recruited to the promoter of APETALA1
(AP1), encoding a MADS-box transcription factor necessary
for flower development, and triggers its activation (Wigge
et al., 2005). Early events occurring during the floral transition
also include upregulation of other related MADS-box transcrip-
tion factors, including SUPPRESSOR OF OVEREXPRESSION
OF CONSTANS 1 (SOC1) and FRUITFULL (FUL), which are
required for the promotion of flowering by FT (Fig. 2). In rice
protoplasts, co-expression of Hd3a and OsFD1 proteins is
required to induce expression of OsMADS15, a homologue of
AP1 (Taoka et al., 2011). Mutagenized variants of Hd3a
unable to bind to 14-3-3 proteins cannot activate OsMADS15,
and an RNAi mutant simultaneously silencing four isoforms of
14-3-3 proteins also fails to induce OsMADS15 to the levels
observed in wild-type plants (Taoka et al., 2011). A homologue
of SOC1, encoded by OsMADS50, has been isolated in rice and
shown to be required for flowering (Lee et al., 2004). It is unclear
if OsMADS50 participates in the network regulating flowering at
the SAM, but its transcription can be detected at the apex, sug-
gesting that it shares features of its arabidopsis homologue
(Kobayashi et al., 2012). Other MADS-box genes, including
OsMADS14,OsMADS18 and OsMADS34, are upregulated at
the apex in response to reproductive transition and are necessary
for correct inflorescence development (Kobayashi et al., 2012).
These data indicate the existence of a conserved mechanism for
floral induction at the apical meristem of plants, whereby flori-
gens, interacting with FD-like transcription factors, activate
expression of a set of MADS-box genes at the early stages of in-
florescence development (Fig. 2). Further regulatory layers are
connected with this basic developmental plan to fine-tune and
stabilize floral transition from environmental noise (Fornara
et al., 2009;Jaeger et al., 2013). Additionally, partial or complete
redundancy between FD-like or MADS-box genes probably con-
tributes to co-ordinate and stabilize inflorescence meristem spe-
cification downstream of florigenic signals (Kobayashi et al.,
2012;Torti and Fornara, 2012;Jaeger et al., 2013).
In arabidopsis, the vegetative to reproductive phase change is
largely controlled by microRNAs, including miR156 and
miR172. Expression of miR156 decreases as plants age, and this
pattern is complementary to that shown by miR172,whose
Arabidopsis
FT Hd3a FT/VRN3
FT + (14-3-3) + FD
? ?
Hd3a + (14-3-3) + OsFDI FT/VRN3 + (14-3-3) + FDL
AP1 OsMADS15 VRN1
OsMADS14
OsMADS18
OsMADS34
SOC1
FUL
Rice Temperate cereals
FIG. 2. Molecular responses of the shoot apical meristem to florigenic proteins. Interaction of florigen with FD-like genes is required to promote expression of
MADS-box transcription factors, one of the first molecular events occurring upon floral transition. The requirement for 14-3-3 proteins has not been demonstrated
in arabidopsis and temperate cereals, and the linker protein is therefore indicated with a question mark on the top. Arrows indicate direct transcriptional activation.
Dashed arrows indicate indirect transcriptional activation.
Shrestha et al. — Seasonal flowering in rice, arabidopsis and temperate cereals Page 7 of 14
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expression increases during development. Overexpression of
miR156 delays the floral transition in arabidopsis by limiting ex-
pression of SQUAMOSA PROMOTER BINDING LIKE (SPL)
transcription factors (Wu and Poethig, 2006). Conversely,
miR172 accelerates flowering, by promoting FT expression in
leavesandbytargertingsixrelatedmembersoftheAPETALA2
(AP2) clade, that repress flowering (Jung et al., 2007;Yant et al.,
2010). The transcriptional dynamics of miR172 depend on some
of the SPLs targeted by miR156, generating a molecular loop
that allows progression of phase change as plants age (Wang
et al., 2009;Wuet al., 2009). Heading date in rice is only partially
dependent on these microRNAs. Rice plants overexpressing
miR172 showed altered flower development, including homeotic
convertions of floral organs and loss of determinacy (Zhu et al.,
2009). However, no altered heading date could be observed,
unlike arabidopsis plants overexpressing mir172 that showed
extremely early flowering (Yant et al., 2010). Overexpression of
rice miR156 influenced heading date, delaying flowering by
several days and was associated with a strong increase in tiller
number and dwarfism (Xie et al., 2006). Whether the levels of
mir156 and mir172 are reciprocally regulated in rice remains to
be established.
SEASONAL FLOWERING RESPONSES IN
TEMPERATE CEREALS
Flowering time pathways share a high degree of conservation
between monocots and dicots. However, homologous genes
could also be recruited to different functions during evolution,
and novel pathways could evolve in specific lineages, to allow
adaptation of species to different environments.
Most plants adapted to temperate climates, including arabi-
dopsis and temperate cereals such as wheat (Triticum spp.) and
barley (Hordeum vulgare), require prolonged exposure to cold
temperatures before flowering. This process is known as vernal-
ization. Since rice was domesticated in tropical regions it does
not show vernalization responses. However, this is a crucial
adaptation for species and varieties adapted to higher latitudes,
because it prevents flowering when temperature is unfavourable,
thus protecting the delicate inflorescence meristem from cold
damage. Arabidopsis has been used extensively to understand
the genetic and molecular bases of vernalization responses,
and two genes, FRIGIDA (FRI) and FLOWERING LOCUS C
(FLC), play a major role in preventing flowering before cold ex-
posure (Ream et al., 2012). Plants experiencing low temperatures
repress FLC expression and maintain its repression also when
returned to warm temperatures. Stable downregulation of FLC
expression is associated with epigenetic silencing of FLC chro-
matin that is converted from active to inactive (J. Song et al.,
2012). Until recently it was believed that no homologue of
FLC existed in monocots, but a recent report suggested that
this is not the case. Ruelens et al. (2013) showed that tandem
arrangements of MADS-box genes, including FLC, are evolu-
tionarily conserved across Angiosperms, and FLC homologues
can also be traced in monocot genomes (Ruelens et al., 2013).
These studies also suggest that OsMADS51 and OsMADS37
are the closest homologues of FLC in rice. Interestingly,
OsMADS51 has been shown to control heading date as anactiva-
tor of Ehd1 expression, indicating an opposite function in
regulation of florigenic proteins to that performed by FLC in ara-
bidopsis (Kim et al., 2007).
In temperate cereals, vernalization responses are controlled by
VERNALIZATION (VRN) loci (Ream et al., 2012). In wheat and
barley, broad genetic variation in the vernalization responses has
been reported, and many varieties are known to have strict or no
vernalization requirement. Varieties that need to be exposed to
cold are planted before winter and flower only during the subse-
quent spring, whereas vernalization-insensitive accessions can
be planted after winter. Such variation has been instrumental in
isolating genes controlling the vernalization processand in estab-
lishing regulatory connections between vernalization genes
(Fig. 1)(Yan et al., 2003,2004;Trevaskis et al., 2003;Karsai
et al., 2005;Hemming et al., 2008;Ream et al., 2012).
Temperate cereal varieties showing vernalization requirements
express VRN2 at high levels before vernalization. The VRN2
locus encodes a CCT-domain protein showing sequence similar-
ity to Ghd7 of rice, and acts as a potent floral repressor that has to
be downregulated during floral transition. Mutations in VRN2
cause insensitivity to vernalization and confer a spring habit
(Yan et al., 2004). Exposure to low temperatures increases
expression of VRN1, a floral promoter homologue of FUL
and AP1 of arabidopsis, and reduces expression of VRN2
(Trevaskis et al., 2006). Dominant allelic variants of VRN1 car-
rying mutations in its regulatory regions express VRN1 inde-
pendently of exposure to low temperatures, and confer a spring
growth habit (Loukoianov et al., 2005). High levels of VRN1 ex-
pression are associated with repression of VRN2 transcription,
which supported the idea that VRN1 acts as repressor of VRN2.
However, by loss-of-function vrn1 mutants, it became clear
that VRN1 induction is not necessary to initiate repression of
VRN2 during vernalization, but is required to maintain its repres-
sion after exposure to cold (Chen and Dubcovsky, 2012). These
data indicate that cold signals co-ordinately repress VRN2 and
activate VRN1 expression during vernalization, whereas after
vernalization VRN1 maintains the repressed state at the VRN2
locus (Fig. 1).
In barley, transcriptional dynamics of VRN1 mRNA are prob-
ably caused by changes in the chromatin state of the VRN1 locus,
in which cold promotes an active chromatin state that is later
maintained after plants are exposed to warm temperatures
(Oliver et al., 2009). Changes in the chromatin state were not
observed at the VRN2 locus, suggesting that VRN1 is the
primary target of chromatin remodelling complexes during
vernalization (Oliver et al., 2009).
As VRN2 levels decrease, the vernalization requirement is sat-
isfied and if plants are exposed to LDs, VRN3, a homologue of
Hd3a and FT, is transcribed and moves to the apical meristem
where it promotes flowering during spring (Yan et al.,2006;
Hemming et al.,2008). The molecular mechanisms through
which VRN3 promotes flowering at the apex are conserved
(Fig. 2). In wheat, VRN3/TaFT protein can interact with TaFDL
transcriptional regulators, homologues of FD and OsFD1,and
promote expression of VRN1 (Li and Dubcovsky, 2008).
Expression of VRN1 is directly controlled by the TaFT– TaFDL
heterodimer as at least one TaFDL protein can bind the promoter
of VRN1. Whether 14-3-3 proteins mediate the interaction
between TaFT and TaFDL is currently unclear (Fig. 2).
As in arabidopsis, temperate cereals flower earlier if exposed to
LDs, whereas flowering is delayed under SDs. Photoperiodic
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control of flowering becomes relevant onlyafter the vernalization
requirement has been satisfied and plants become competent to
respond to daylength. The major gene controlling photoperiod
sensitivity in both wheat and barley is PHOTOPERIOD 1
(Ppd1), encoding a pseudoresponse regulator similar to
OsPRR37 of rice and PRR7 of arabidopsis (Turner et al.,2005;
Beales et al.,2007). Photoperiod-insensitive wheat plants carry
Ppd-D1a, a semi-dominant allele that bears a 2 kb deletion in its
upstream regulatory region and induces early flowering under
both LDs and SDs (Beales et al., 2007). The Ppd-D1a deletion
causes increased expression of Ppd-D1a also during the night
under SDs, when transcription of the wild-type gene is normally
repressed. Misexpression induces TaFT constitutive activation
and flowering, irrespective of daylength. In barley, the recessive
ppd-H1 loss-of-function allele cannot induce high expression of
HvFT when plants are exposed to LD conditions. This results in
delayed flowering and is advantageous for spring-sown varieties
that can prolong vegetative growth, producing more biomass
and eventually seeds (Turner et al.,2005).
Interestingly, the effect of homologues of OsPRR37 on flower-
ing depends on the species. In LD plants such as arabidopsis and
temperate cereals, functional alleles promote flowering through
transcriptional induction of florigens; conversely, in SD species
such as rice and sorghum, PRR genes repress expression and
flowering of florigens under LDs (Murphy et al., 2011;Koo
et al., 2013). The molecular mechanisms underlying PRR func-
tion will provide important clues to understand how information
on daylength is elaborated and the causes FT activation.
Homologues of CO and Hd1 have been cloned from temperate
cereals; however, they seem not to be crucial to confer a photo-
periodic response (Nemoto et al., 2003;Shimada et al., 2009).
Overexpression of HvCO1 in a ppd-H1-deficient accession
accelerates flowering under both LD and SD conditions, indicat-
ing that HvCO is independent of the PRR pathway. However,
plants overexpressing HvCO retained responsiveness to photo-
period, flowering later under SDs and indicating the existence
of additional factors with major effects on flowering. Indeed,
allelic variation at Ppd-H1 was shown to be a major determinant
of HvFT1 expression and flowering time, and acted independent-
ly of HvCO1 (Campoli et al., 2012). Similar conclusions were
suggested from studies in wheat (Shaw et al., 2012) and point
to a model whereby florigen expression is the convergence
point of independent pathways with limited cross-talk (Fig. 1).
In temperate cereals the responses to daylength and vernaliza-
tion are integrated. Aftera period of growth under LDs, exposure
to SDs accelerates flowering in wheat, and can largely substitute
for vernalization treatments (Dubcovskyet al., 2006). Flowering
is due to downregulation of VRN2 expression under SDs, an
effect also observed in barley and Brachypodium (Trevaskis
et al., 2006;Ream et al., 2012). Thus, VRN2 is a convergence
point integrating photoperiodic and temperature information,
and its correct expression is key for flowering at the most appro-
priate time of year.
RICE ADAPTATION TO LONG-DAY CONDITIONS
INVOLVED ALLELIC CHANGES AT HEADING
DATE LOCI
Rice domestication startedabout 10 000– 13 000years agoin the
surroundings of the Pearl River in Southern China (Huang et al.,
2012). During this process, the founder ecotypes, probably
belonging to the O. rufipogon progenitor, split into the five
groups of cultivated rice known to date. Over the centuries, the
area of rice cultivation expanded, first within tropical and sub-
tropical Asia and then to other regions of the world, reaching tem-
perate areas at higher latitudes. The success of rice adaptation
depended on the acquisition of cold tolerance traits and the
loss of photoperiod sensitivity. In temperate areas, seasonal var-
iations in temperatures limit the period of rice cultivation from
late spring to early autumn. Thus, rice flowering occurs during
summer days, which are warm but long, and varieties adapted
to temperate climates showreduced sensitivity to changes in day-
length, flowering under conditions normally non-inductive.
Several genetic studies have been carried out in order to
identify the molecular mechanisms that allow rice to flower
at high latitudes. Five major QTLs controlling heading date
in response to photoperiod were identified and described in
detail in previous sections (Yamamoto et al., 1998,2000).
All genes underlying these major QTLs have been cloned
and, interestingly, four of them (Hd1,Hd2/PRR37,Hd4/Ghd7
and Hd5/DTH8/Ghd8) were demonstrated to be repressors of
flowering under LD conditions, whereas Hd3a encodes the
major florigen normally targeted by the Hd1 –Hd4 repressors
(Yano et al., 2000;Kojima et al., 2002;Xue et al., 2008;
Wei et al., 2010;Yan et al., 2011;Fujino et al., 2012;Koo
et al., 2013). This genetic architecture probably reflects the
tropical origin of the species, and indicates that floral repression
is a default state that needs to be overcome in order for flower-
ing to occur. However, it also provides the substrate for artifi-
cial selection of varieties better adapted to regions where
daylength is not permissive.
Polymorphisms at loci encoding florigens exist and can partly
account for flowering diversity. In particular, variations at the
Hd3a promoter regions contribute to diversification of flowering
time of a rice core collection (Takahashi et al., 2009). A recent
study has demonstrated how single nucleotide polymorphisms
(SNPs) in the regulatory genomic region and an amino acid sub-
stitution in the protein sequence of RFT1 provide flowering time
divergence under LD conditions (Ogiso-Tanaka et al., 2013).
Rice varieties growing under natural LD conditions (where day-
length is longer than 13 h and latitude over 23.68N) use both the
RFT1- and Hd3a-dependent pathways to promote flowering,
whereas rice varieties growing at southern latitudes mostly
use the Hd3a pathway (Fig. 3A) (Ogiso-Tanaka et al., 2013).
However, since florigens are highly conserved across rice var-
ieties and species, flowering diversification has mainly resulted
from the regulation of florigen expression levels that are highly
correlated with flowering time.
Repressors (or suppressors) of flowering play a crucial role in
reducing florigen gene expression under LD conditions, leading
to a strong delay in heading date. Non-functional alleles of
repressors (or suppressors) of LD-dependent flowering have
been associated with loss of sensitivity to photoperiod.
Loss-of-function alleles of such genes cause an increase in flori-
gen gene expression to promote flowering under LDs. Thus, de-
fective alleles of repressors can be used by breeders to introduce
variations in flowering time in rice varieties that grow under LD
conditions. These alleles have been useful tools to introduce trop-
ical varieties into temperate areas and to increase the northern
limit of rice cultivation.
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Non-functional alleles at Hd1,PRR37,Ghd7 and Ghd8 loci
are generated by SNPs, insertions or deletions, leading to dramat-
ic changes in florigen expression and heading dates. Special at-
tention is required for Hd1, which acts as a repressor under
LDs but as an inducer under SDs. A high occurrence of natural
polymorphisms in Hd1 has been correlated with variation in
flowering time and Hd3a mRNA levels (Takahashi et al.,
2009). Rice cultivars with functional Hd1 alleles showed
50°N
A
B
hd16/el1
RFT1 pathway
Hd3a pathway
ghd 7
prr 37
dth8/ghd8
hd1
Hd3a pathway
Nipponbare_SNP (strong)
Koshihikari_SNP (weak)
Group A4 (strong)
Groups A1 & A2 (weak)
Hap_3 (strong)
Hap_2 (weak)
40°N
30°N
20°N
10°N
10°S
50°N
40°N
30°N
20°N
10°N
Hd17 DTH2 Ehd4
10°S
0°
0°
FIG. 3 . Distribution of alleles influencing heading date in rice. The maps show the distribution of loss-of-function alleles of floral repressors, including hd16,hd1,
prr37,ghd7 and dth8/ghd8 (A) or allelic variants of floralpromoters associated with weak or strongactivity, which include Hd17,DTH2 and Ehd4 (B). The colour of the
distribution matches that of the corresponding gene, which is indicated on the right-hand side of the map. The length of the coloured bars on the right covers the lati-
tudinal range acrosswhich varieties bearing the allelic variant are grown. Grey arrows(represented only in A) indicate the requirement forHd3a expression at southern
latitudes and for Hd3a and RFT1 expression at higher latitudes. The maps are based on data reported in Xue et al. (2008),Takahashi et al. (2009),Wei et al. (2010),
Fujino et al. (2012),Matsubara et al. (2012),Gao et al. (2013),Koo et al. (2013), Kwon et al. (2013) and Wu et al. (2013).
Shrestha et al. — Seasonal flowering in rice, arabidopsis and temperate cerealsPage 10 of 14
at Biblioteca Biologica on March 24, 2014http://aob.oxfordjournals.org/Downloaded from
higher Hd3a expression levels and earlier flowering times under
SD conditions, whereas those with non-functional Hd1 alleles
showed lower Hd3a expression levels and later flowering times
(Takahashi et al., 2009). Since the presence of non-functional
alleles of Hd1 influences flowering in opposite ways depending
on daylength, rice varieties carrying natural hd1 mutants have
been found in a wide range of latitudes (Fig. 3A). Varieties
bearing non-functional Hd1 alleles grown under SDs will
delay flowering time, which could be important to elongate the
vegetative phase in order to increase grain production. The
effect of non-functional Hd1 alleles under SDs can be reinforced
by the presence of non-functional Ehd1 alleles, as observed in
some Taiwanese rice varieties (Doi et al., 2004). Conversely,
non-functional Hd1 alleles in varieties grown under LDs will an-
ticipate heading, contributing to cultivation at high latitudes
(Izawa, 2007). A recent study has revealed that Hd2/PRR37
downregulates Hd3a expression under LD conditions and has
demonstrated that natural variation at PRR37 in many Asian
rice cultivars has contributed to the expansion of rice cultivation
to temperate areas, similar to previous reports in sorghum
(Murphy et al., 2011;Koo et al., 2013). Non-functional PRR37
alleles (Fig. 3A) were detected in a wide range of latitudes, in-
cluding the northern limit of rice cultivation (Koo et al., 2013).
Genetic analysis revealed that the effect of PRR37 on heading
date is additive to that of Ghd7, and rice varieties carrying non-
functional PRR37 and Ghd7 showed extremely early flowering
under LDs (Koo et al., 2013). Ghd7 has been previously
described as a key component in the adaptation of rice to northern
latitudes because it downregulates the expression of Ehd1 and,
consequently, that of Hd3a and RFT1 under LDs. Natural ghd7
mutants (Fig. 3A) were found in early flowering rice varieties
grown in central and southern China and in varieties from the
Heilongjiang Province of North-eastern China, the latter being
characterized by cool summers and a short growing season
(Xue et al., 2008). Japonica cultivars with both Ghd7 and
PRR37 mutations were also found at high-latitude regions of
North-eastern Asia, including Northern Japan. This suggests
that naturally occurring mutations in PRR37 and Ghd7 play an
important role in rice adaptation from low to high latitudes
(Koo et al., 2013). However, Ghd7 acts on a separate genetic
pathway to that of PRR37 (Xue et al., 2008;Fujino et al.,
2012;Koo et al., 2013). This might indicate that pyramiding of
non-functional alleles in cultivated varieties has probably
allowed further expansion of the cultivation area, and artificial
construction of early flowering genotypes has been particularly
successful when independent repressor pathways were targeted
(Ebana et al., 2011). Polymorphisms in the DTH8/Ghd8 se-
quence that create non-functional alleles have been related to
loss of photoperiod sensitivity. Natural ghd8 mutants were
found in several provinces of China, the Philippines, Indonesia
and Northern Japan (Wei et al., 2010;Fujino et al., 2012)
(Fig. 3A). The mutant allele has been used in breeding pro-
grammes outside of Japan, its country of origin, and spread to
Europe, where it probably conferred an agronomic advantage
over functional alleles (Wei et al., 2010;Fujino et al., 2012).
Ghd8 expression does not affect Ghd7 or Hd1 expression, sug-
gesting that multiple targeting of repressor pathways has the po-
tential to accelerate flowering strongly. Combinations of
defective alleles generate stronger phenotypes, as demonstrated
with prr37 ghd7 cultivars under LDs (Koo et al., 2013),
suggesting that accumulation of additional Hd mutant alleles
could contribute to further reduction of photoperiodic sensitivity
and crop cycle.
In addition to these major loci, polymorphisms in the DNA se-
quence of other alleles have also contributed to the northern adap-
tation of rice. From additional QTL analyses, Hd6, a minor
heading date allele, was detected (Yamamoto et al., 2000). Hd6
enhances the repressive activity of Hd1 and is defective in some ja-
ponica cultivars (Takahashi et al., 2001;Ogiso et al., 2010;Ebana
et al., 2011). This reduces (but does not abolish) Hd1-mediated re-
pression under LDs, further contributing to diversification of flow-
ering time. Matsubara et al. (2008b)identified new QTLs related to
photoperiodic flowering. Among them, recent studies have shown
how naturally occurring variants of EL1/Hd16 alleles in japonica
cultivars influence Ghd7 activity (Matsubara et al., 2012;Hori
et al., 2013;Kwon et al., 2014). Hd16 acts as a suppressor of
LD-dependent flowering by phosphorylating Ghd7 (Hori et al.,
2013). Cultivars carrying non-functional EL1/Hd16 variants
(Fig. 3A) are closely associated with high latitudes, whereas the
cultivars carrying functional EL1/Hd16 variants are randomly dis-
tributed independently of latitude (Kwon et al., 2014).
Natural variation at loci encoding floral activators has recently
been shown to have an important role in adaptation to northern
latitudes. In contrast to all the genes described above, allelic var-
iants of Hd17, encoding an OsELF3-like protein, DTH2, which
encodes a CONSTANS-like protein, and Ehd4 do not create
loss-of-function alleles but rather genetic variants showing a
gradient of activity (Matsubara et al., 2012;Gao et al., 2013;
Wu et al., 2013). Genetic studies demonstrated that the
Nipponbare_SNP of Hd17, allele 4 (A4) of DTH2 and haplotype
3ofEhd4 have been fixed during the domestication of rice at high
latitudes (Fig. 3B). These showed a stronger effect as floral pro-
moters under natural LD conditions in comparison with other
alleles (Matsubara et al., 2012;Gao et al., 2013;Wu et al., 2013).
CONCLUSIONS AND PERSPECTIVES
Decades of research on flowering control have greatly expanded
our understanding of the molecular mechanisms that initiate
and drive reproductive phase transitions in different species.
Molecular control networks are becoming increasingly complex
as novel genes and regulatory mechanisms are described.
Research that takes advantage of arabidopsis as a model organ-
ism often leads the way and opens up the possibility of exploring
the function of orthologues from other species. However, arabi-
dopsis is not representative of all plant species, and several
examples discussed in this review indicate that several monocot-
specific (or even Oryza-specific) genes do not have functional
equivalents in dicots (Doi et al., 2004;Yan et al., 2004;Xue
et al., 2008;Matsubara et al., 2011; Wang et al., 2013; Wu
et al., 2013). Exploring genomes by DNA sequencing, QTL
and association mapping, transcriptome profiling or mutant
screens will keep providing new exciting insights into the way
genes allow plants to interface with the environment. Mining
genetic variation in crop species and their progenitors, and coup-
ling it with the enormous potential of next-generation sequen-
cing, will reach the dual objective of identifying novel
regulators, perhaps difficult to pinpoint with other tools, and to
exploit diversity to accelerate breeding programmes (Huang
et al., 2011;Zhao et al., 2011).
Shrestha et al. — Seasonal flowering in rice, arabidopsis and temperate cereals Page 11 of 14
at Biblioteca Biologica on March 24, 2014http://aob.oxfordjournals.org/Downloaded from
ACKNOWLEDGEMENTS
This work was supported by an ERC Starting Grant (#260963)
to F.F.
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