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37
Response of plant development to environment: control of
flowering by daylength and temperature
Paul H Reeves* and George Coupland†
The transition from vegetative growth to flowering is often
controlled by environmental conditions and influenced by the age
of the plant. Intensive genetic analysis has identified pathways
that regulate flowering time of Arabidopsis in response to
daylength or low temperature (vernalization). These pathways are
proposed to converge to regulate the expression of genes that
act within the floral primordium and promote floral development.
In the past year, genes that confer the responses to daylength or
vernalization have been cloned and have enabled aspects of the
genetic models to be tested at the molecular level.
Addresses
John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
*e-mail: [email protected]
†e-mail: [email protected]
Current Opinion in Plant Biology 2000, 3:37–42
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
AG
AGAMOUS
AP
APETALA
CAB2
CHLOROPHYLL A/B BINDING PROTEIN 2
CCA1
CIRCADIAN CLOCK ASSOCIATED 1
CRY2
CRYPTOCHROME 2
CVI
Cape Verde Islands
EDI
EARLY DAYLENGTH INSENSITIVE
elf3
early flowering 3
FLC
FLOWERING LOCUS C
FRI
FRIGIDA
GI
GIGANTEA
ld
luminidependens
LFY
LEAFY
lhy
late elongated hypocotyl
vrn
vernalization
Introduction
The time of year at which plants flower varies widely
and is closely adapted to the environment in which they
grow. Temperature and daylength are the principal environmental conditions that synchronise flowering to the
changing seasons. Many papers published from the
1920s onward described variations in the flowering
response of varieties of the same species isolated from
different latitudes and, in some cases, identified genetic
loci that confer these responses. The use of genetic
approaches to compare the flowering responses of naturally occurring varieties of Arabidopsis and to isolate
induced mutations that disrupt these responses has
enabled the isolation of genes that underlie the control
of flowering by photoperiod and vernalization. Models
that were initially based on genetic interactions are
being confirmed or adapted on the basis of the analysis
of gene expression in different genetic backgrounds. In
this short review we concentrate on progress that has
been made in understanding the control of flowering by
both photoperiod and vernalization since publication of
the most recent longer reviews [1–3].
Vernalization
Many Arabidopsis ecotypes collected at high latitudes or
from mountainous regions are winter annuals that flower in
the spring after exposure to winter conditions. In the laboratory, these strains flower very late but will flower much
earlier if exposed to low temperatures for several weeks.
This process, called vernalization, prevents flowering late
in the summer when seed maturation may be curtailed by
onset of winter conditions.
Genetic analysis has identified two classes of late-flowering Arabidopsis plants that will respond to vernalization.
First, naturally occurring late-flowering varieties collected
from many locations across Europe carry dominant alleles
at the FRIGIDA (FRI) locus that delay flowering, and
these plants will flower early if given low temperature
treatments [4–6]. Second, a group of induced recessive
mutations delay flowering in early flowering varieties such
as Landsberg erecta and Columbia and this phenotype can
be corrected by low temperature treatments [7,8]. These
mutations were assigned to the autonomous flowering
pathway, because they delay flowering in all photoperiods.
The observation that these mutants respond to low temperature treatment suggests that vernalization can
overcome the requirement for the autonomous pathway.
During the past year it has become clear that the flowering-time gene FLOWERING LOCUS C (FLC) plays a
central role in the vernalization response (Figure 1). The
FLC gene was identified genetically because the widely
used laboratory strain Landsberg erecta carries an inactive
FLC allele. This was recognised when dominant FRI alleles or the luminidependens (ld) mutation that affects the
autonomous pathway were shown not to delay flowering in
Landsberg erecta, because for FRI and ld to cause late flowering an active FLC allele is required [9,10]. On the basis
of these genetic interactions it was proposed that FLC
inhibits flowering and that this inhibition is enhanced by
FRI and repressed by LD. These interpretations have
been supported by the molecular analysis of FLC.
The FLC gene was cloned independently by activation TDNA tagging [11••] and chromosome walking [12••]. Both
experiments identified the same MADS-box containing
gene, and proposed that it acts to repress flowering.
Analysis of FLC expression provided a means to test genetic models for the control of vernalization. Late-flowering
plants that can be induced to flower early if given a low
temperature treatment were shown to have high FLC
mRNA levels, mainly in roots and at the shoot apex, that
38
Growth and development
(vrn) mutations are likely to define genes that are required
for the vernalization process and at least the vrn2 mutation
prevents the reduction of FLC mRNA abundance that is
observed on low temperature treatment [11••].
Figure 1
Vernalization
Autonomous
pathway
LD
Photoperiod response
FCA
FRI
VRN2
FLC
Vegetative growth
Flowering
Current Opinion in Plant Biology
Genetic and molecular interactions involved in vernalization in
Arabidopsis. The FLC gene encodes a MADS-box transcription factor
that represses the transition from vegetative growth to flowering. The
abundance of FLC mRNA is reduced by low temperature treatment
and is proposed to play a central role in vernalization. The FRI, FCA,
LD and VRN2 genes regulate the abundance of FLC mRNA
[11••,12••]. FCA acts within the autonomous flowering pathway and
other genes within this pathway have similar effects to those of FCA.
The FRI gene increases FLC expression. The VRN2 gene acts during
vernalization to reduce FLC expression in response to low temperature
treatments. The FCA and LD genes reduce FLC mRNA abundance, so
that fca and ld mutants are late flowering but this can be corrected by
low temperature treatments.
are reduced on vernalization [11••,12••]. Dominant alleles
at the FRI locus, ld mutations and other mutations that
affect the autonomous pathway increase FLC mRNA abundance (Figure 1). Thus genotypes with a vernalization
requirement have high FLC mRNA levels and vernalization acts to reduce FLC mRNA abundance [11••,12••].
The analysis of FLC has extended the genetic models and
more clearly demonstrated a link between the autonomous
pathway and vernalization. The mechanism by which low
temperatures reduce FLC mRNA levels now becomes a
key question. The reduced level of FLC mRNA caused by
treating seedlings with low temperatures is maintained
throughout the development of the treated plant but their
progeny again show high levels of FLC mRNA and the
acceleration of flowering caused by low temperatures is
progressive with a six week exposure having a more severe
effect than a two week exposure [7,11••,12••]. There are
similarities between this and the regulation of gene
expression by methylation, and some early flowering
plants in which methyl transferase activity was reduced
using anti-sense technology show reduced levels of FLC
mRNA, suggesting that methylation might play a role in
FLC regulation [11••]. However, genes required for vernalization have been identified genetically by the isolation of
mutations that reduce the acceleration of flowering caused
by low temperature treatments [13]. These vernalization
Flowering of Arabidopsis is also regulated by daylength.
Flowering occurs much earlier under long days of 16 hours
light than under short days of 8 hours, and daylengths
between these two extremes give an intermediate
response. Mutations that disrupt the effect of photoperiod
on flowering have been described, and one gene has been
identified in an analysis of allelic differences between ecotypes. The daylength-insensitive mutants fall into two
classes: late-flowering mutants, which compared to wildtype flower late under long days but are unaffected under
short days, and early-flowering mutants, which flower
much earlier under short days. The late-flowering mutants
are believed to affect a single genetic pathway that promotes flowering in response to long days [10,14••]. In
addition, an extensive analysis of the allelic differences in
flowering time genes between the Cape Verde Islands
(CVI) and Landsberg erecta ecotypes identified a locus
EARLY DAYLENGTH INSENSITIVE (EDI) [15••]. When
introgressed into Landsberg erecta the dominant CVI allele
of EDI caused early flowering and these plants were
almost daylength insensitive. No induced dominant mutation has been isolated that causes early flowering,
highlighting the benefits of studying natural variation in
flowering time.
Classic experiments implicated the circadian clock as the
timer in daylength measurement, and analysis of daylengthinsensitive flowering-time mutants has provided genetic
support for this. The first of these was early flowering 3 (elf3),
which in addition to causing early flowering that is insensitive to daylength also shows alterations in circadian clock
regulation [16,17]. In elf3 plants grown under light/dark
cycles and then shifted to continuous light, the regulation of
genes such as CHLOROPHYLL A/B BINDING PROTEIN 2
(CAB2) by the circadian clock is disrupted as is the rhythm
in leaf movements [17]; the effect of the elf3 mutation is,
however, conditional and does not affect circadian clock
function under continuous darkness.
Two other genotypes were recently described that have
dramatic effects on flowering time and disrupt circadian
clock function [18••,19••]. One of these is the late elongated
hypocotyl (lhy) mutation, which was identified by activation
tagging and falls into the group of late-flowering mutations
causing delays in flowering only under long days [18••].
The LHY gene encodes a protein containing a single MYB
repeat and its expression is regulated by the circadian clock
with a peak in expression just after dawn. In the mutant,
the LHY gene is expressed at an elevated and constant
level. In entrained mutant plants shifted to either continuous dark or continuous light, all circadian clock controlled
rhythms that have been tested are disrupted. This pheno-
Control of flowering by daylength and temperature Reeves and Coupland
type together with the daylength-insensitive flowering
time of the mutant suggests a relationship between
daylength responses and circadian clock function [18••].
Transgenic plants in which the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene is expressed from the cauliflower
mosaic virus 35S promoter show a similar phenotype to lhy
mutants [19••]. CCA1 was initially identified because its
protein product binds to the CAB promoter [20]. The
CCA1 protein is 43% identical to LHY overall, and 87%
identical in the DNA-binding domain [18••]. The initial
publications on the effects of CCA1 and LHY on circadianclock regulation relied on the phenotypes of plants in
which the genes were overexpressed. The recent observation that inactivation of the CCA1 gene also affects
circadian-clock control of gene expression was therefore an
important step in establishing the importance of these
genes in circadian-clock regulation [21••]. A T-DNA insertion within an intron of the CCA1 gene prevents synthesis
of CCA1 protein, and causes the circadian clock to run
approximately three hours faster than in wild-type plants
under continuous light [21••]. The CCA1 knockout allele
may have a less severe effect on circadian-clock control
than CCA1 overexpression, because of the presence of
genes such as LHY that may be able to partially compensate for the loss of CCA1 function. No effect of the cca1
mutation on flowering time was reported; another mutation that shortens the period of the circadian clock, toc 1,
was however, previously shown to cause early flowering
under short days in the Landsberg erecta ecotype, but not
in the C24 ecotype [22,23••]. It is possible, therefore, that
introgression of cca1 into other ecotypes may be required
to observe any effect on flowering time and that allelic differences between ecotypes can mask the effects of period
mutations on flowering time.
GIGANTEA (GI) is also implicated in the control of flowering in response to daylength [8,24–26], and the gene was
isolated during the past year [27••,28••]. The characterisation of GI again emphasises the role of the circadian clock
in daylength responses. The gi mutation was first
described as causing late flowering under long days and
daylength insensitivity [8,24]. GI encodes a large protein
that is predicted to be located in the plasma membrane
and to contain at least five membrane-spanning domains in
the first 660 amino acid residues. The carboxy-terminal
region is hydrophilic and has no homology to any protein
of known function but is probably important for GI function because several mutant alleles affect this region of the
protein [27••]. The abundance of GI mRNA is also regulated by the circadian clock and peaks around 10 hours
after dawn. Furthermore, expression of GI does not show
its normal peak in expression in the lhy mutant nor in
plants overexpressing CCA1 [27••], which may be expected as the expression of all other circadian-clock regulated
genes tested is also disrupted in these genotypes. More
surprisingly, however, gi mutations also reduce the expression of LHY and CCA1 in long-day grown plants and in
39
Figure 2
Photoperiod
Vernalization
(a)
(c)
(b)
CO/GI
FLC
FT/FWA
(d)
?
(h)
(e)
(g)
AP1
(f)
LFY
(i)
AP3/PI
AP1/AP2
AG
Floral patterning
Current Opinion in Plant Biology
The activation of floral-meristem identity genes by floral promotion
pathways. Photoperiod and vernalization control the timing of floral
induction through independent genetic pathways [1,2]. Eventually these
pathways must converge to activate genes, such as LFY and AP1, that
confer floral identity upon the shoot apical meristem [34••]; however,
little is known about how signals from separate floral promotion
pathways become integrated. (a) The main response to photoperiod in
Arabidopsis is the promotion of flowering under long days. Both the
circadian clock and light receptors are involved in this response, which
is mediated through genes such as CO and GI [14••,26,27••,38].
(b) Genetic analysis suggests that the FT and FWA genes are also
involved in the promotion of flowering in response to long days [14••].
(c) One role of vernalization is to reduce the mRNA abundance of the
floral repressor FLC [11••,12••]. The autonomous floral promotion
pathway also interacts with FLC (see Figure 1). (d) The photoperiodic
and autonomous floral promotion pathways act additively to promote
flowering and are involved in the regulation of the same floral meristem
identity genes, but the stage at which these pathways converge is
unknown. (e) Genetic and molecular evidence illustrates that genes
from the photoperiodic and autonomous floral promotion pathways are
involved in the regulation of the LFY gene [37••,38,39,40•,41•].
(f,g) Although flowering time genes have been shown to be involved in
the regulation of LFY, they are also likely to regulate other floral
meristem identity genes [37••,38,40•,41•]. AP1 is a possible candidate,
but other floral meristem identity genes are also likely to be involved. For
example, ld ; ap1 ; cal triple mutants show a more severe shoot
phenotype than lfy; ap1; cal mutant plants, suggesting that LD could
act on floral meristem identity genes such as APETALA2 or UNUSUAL
FLORAL ORGANS [40•]. (h) The main function of FT and FWA is not
the transcriptional activation of LFY [37••]. One function of these genes
is likely to be the activation of AP1, as AP1 expression is abolished in
the floral structures formed on ft ; lfy and fwa ; lfy double mutants [39].
(i) Floral meristem identity genes establish and regulate the expression
patterns of floral homeotic genes [34••].
40
Growth and development
plants shifted to continuous light indicating that
LHY/CCA1 and GI do not act in a simple linear pathway
but that they affect each other’s expression [27••,28••]. But
gi does not, however, affect LHY expression in plants shifted to continuous darkness indicating that GI may play a
role in the control of expression of circadian-clock regulated genes in response to light [28 ••]. There are
complications in establishing a connection between the
effects of gi on flowering time and on the period of the circadian clock because two alleles that showed different
effects on the period of CAB2 expression had very similar
effects on flowering time [28••]. The delay in flowering
caused by gi may be due to a general reduction in the
amplitude of expression of circadian clock regulated genes,
as observed for LHY and CCA1 [27••,28••].
In order to detect and respond to daylength, light receptors
must act within the long-day pathway. Classically, phytochrome was implicated as the photoreceptor that controls
photoperiodic responses. This was supported by the
demonstration that mutations in the gene encoding phytochrome A abolish the photoperiodic control of flowering in
pea plants [29]. In Arabidopsis and other crucifers, however,
both far red and blue light have long been known to promote flowering [30], and mutations in the gene encoding
the blue light receptor CRYPTOCHROME (CRY) 2 both
delay flowering and reduce the photoperiodic response
[31••]. As described above, the circadian clock regulates the
expression of genes that act within the long-day pathway,
and the circadian clock is itself entrained (or synchronised)
to the daily cycle of light and dark by light. Perhaps surprisingly, however, cry2 mutations have a weak effect on
circadian clock entrainment. This has become clear from a
detailed analysis of the effect of mutations in different light
receptor genes on circadian clock controlled expression of
the CAB2 gene. Using a fusion of the CAB2 promoter to
luciferase it was shown that under high fluence light mutations in the PHYB or CRY1 genes lengthen the period of the
circadian clock but that mutations in PHYA and CRY2 only
do so under specialised conditions of low fluence light
[32••]. This indicates that the phyA and cry2 mutations do
not affect the photoperiodic response by affecting circadian
clock control but may do so by more directly affecting the
expression or function of genes that control flowering time.
This is supported by recent observations that the promotion
of flowering by CRY2 occurs through antagonising an
inhibitory effect on flowering that is mediated by PHYB,
and may act by increasing the expression of the flowering
time gene CONSTANS [31••,33•].
The interaction between flowering time and
floral meristem identity
Several of the genes that act during the early stages of flower
development to confer floral identity on the developing floral meristem have been isolated and their expression
analysed in detail. LEAFY (LFY) is the earliest acting of
these genes and encodes a transcription factor that acts within the developing primordium to promote the expression of
another floral meristem identity gene, APETALA (AP) 1, or
together with co-activators that are expressed within particular domains of the floral meristem to activate expression of
genes such as AP3 and AGAMOUS (AG) that specify the
identity of particular floral organs [34••–36••].
The role flowering-time genes play in the activation of
these floral meristem identity genes is also being uncovered, although to date the greatest progress has been made
in assessing the interactions between flowering time genes
and LFY. One approach has been to analyse the effect of
mutations causing late flowering on both LFY gene expression and on early-flowering transgenic plants that
overexpress the LFY gene from the 35S promoter [37••]. In
general, two classes of flowering-time genes have been
identified. The first class of gene appears to be involved in
the transcriptional upregulation of LFY: mutations in
genes from this class cause relatively weak upregulation of
LFY promoter activity during development and do not
severely attenuate the early flowering of 35S::LFY plants.
Genes from several genetically distinct flowering time
pathways including FCA/FVE (autonomous pathway),
CO/GI (photoperiod pathway), and GIBBERELLIN
RESPONSIVE 1/GIBBERELLIN INSENSITIVE (gibberellin-dependent pathway) fall into this group,
suggesting that LFY is a common target for these flowering
pathways (Figure 2; [37••,38]).
The second group is mainly defined by a subgroup of
genes in the photoperiod pathway, namely FT and FWA.
This class of gene does not appear to be involved in the
transcriptional upregulation of LFY but is instead required
for the response to LFY activity: mutations in these genes
have only a small effect on the activity of the LFY promoter and the late-flowering phenotype of ft and fwa is
epistatic to the early flowering of 35S::LFY plants
(Figure 2; [37••]). It is likely that one function of this second group is the activation of the AP1 gene [39].
More detailed genetic analysis combining individual lateflowering mutants with a range of meristem-identity
mutants supports the conclusions reached from the analysis of the LFY gene [40•–42•]. These studies have also
revealed that although some genes are involved in the
transcriptional upregulation of LFY this is unlikely to be
their sole function. For example, double fca ; lfy mutants
show an enhancement of the lfy phenotype, suggesting
that FCA also acts to promote flowering and flower development in a pathway parallel to LFY [41•].
The analysis of the interaction of the FCA gene with
meristem-identity genes also highlights an overlooked role
of the AP1 gene in the regulation of flowering [41•].
Overexpression of the AP1 gene causes early flowering
[43], suggesting that one function of AP1 is to promote
flowering, and the appearance of AP1 gene expression in
primordia is generally considered to be a good marker for
the commitment to flowering [44]. However, ap1 mutant
Control of flowering by daylength and temperature Reeves and Coupland
plants also flower slightly earlier than wild-type plants,
suggesting that the AP1 gene may also repress flowering
[41•]. As the late flowering of the fca mutant is epistatic to
the early flowering of ap1 plants, AP1 may repress flowering by antagonising the action of the autonomous
promotion pathway [41•].
Conclusions
Flowering time is regulated by balancing the promotive or
inhibitory effects of different environmental conditions.
For example, the responses to vernalization and photoperiod are regulated by parallel genetic pathways that
promote flowering in Arabidopsis. Recent progress has
demonstrated the importance of the circadian clock in controlling the photoperiod pathway and shown that
vernalization acts at least in part to reduce the abundance
of the FLC mRNA. These two pathways somehow converge to regulate the expression of LFY — a gene that acts
within the floral primordium to promote the expression of
genes that specify floral organ identity (Figure 2). Future
experiments will describe the molecular mechanism of this
convergence and whether it occurs at the LFY gene itself
or at an earlier stage in the transition from vegetative
growth to flowering.
Acknowledgements
Our work on flowering time is supported by grants from the European
Commission and the Human Frontiers Science Program.
References and recommended reading
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have been highlighted as:
• of special interest
•• of outstanding interest
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Growth and development
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the AP1 promoter.
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The cloning of the GIGANTEA gene by T-DNA tagging is reported, and its
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LHY, and CCA1 genes in the regulation of GI expression is also examined.
28. Park DH, Somers DE, Kim YS, Choy YH, Lim HK, Soh MS, Kim HJ,
•• Kay SA, Nam HG: Control of circadian rhythms and photoperiodic
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1999, 285:1579-1581.
This paper describes the isolation of the GIGANTEA gene using a map-based
cloning strategy. Mutations in this gene are shown to affect the period of the
circadian clock, although the two gi alleles analysed have different effects. The
effect of light fluence rate on the clock period in the gi-1 mutant, and the
reduced effect gi mutations have on period length in continuous darkness,
suggests that GI may be involved in controlling light signaling to the clock.
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31. Guo H, Yang W, Mockler TC, Lin C: Regulation of flowering time by
•• Arabidopsis photoreceptors. Science 1998, 279:1360-1363.
The late flowering of the fha mutant, which affects the photoperiodic floral
promotion pathway, is shown to be due to a mutation in the blue light receptor CRYPTOCHROME 2.
32. Somers DE, Devlin PF, Kay SA: Phytochromes and cryptochromes
•• in the entrainment of the Arabidopsis circadian clock. Science
1998, 282:1488-1490.
The authors explore the effect of light fluence rate on the circadian period in
mutants that are deficient in specific phytochromes and cryptochromes. The
results show that several photoreceptors are involved in the regulation of the
circadian clock.
33. Mockler TC, Guo H, Yang H, Duong H, Lin C: Antagonistic actions
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of Arabidopsis cryptochromes and phytochrome B in the
regulation of flowering. Development 1999, 126:2073-2082.
Genetic analysis allows the authors to propose a detailed model for the interaction of cryptochromes and phytochrome B in the regulation of flowering
time. Light quality is shown to be most important during a developmental
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•• gene in Arabidopsis. Science 1999, 285:585-587.
This paper describes a LEAFY (LFY) responsive enhancer contained within
the second intron of the AGAMOUS (AG) gene, and shows that LFY is an
activator of AG.
36. Wagner D, Sablowski, RWM, Meyerowitz EM: Transcriptional
•• activation of APETALA1 by LEAFY. Science 1999, 285:582-584.
The authors demonstrate that AP1 is an immediate target of transcriptional
activation by LFY using steroid-inducible LFY activity.
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Nilsson O, Lee I, Blázquez MA, Weigel D: Flowering-time genes
modulate the response to LEAFY activity. Genetics 1998,
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A detailed analysis of the effect of late-flowering mutations on the expression
of the LFY gene, and the phenotype of transgenic 35S::LFY plants. The separation of genes into groups that affect LFY transcription and those that
affect the response to LFY, does not correspond to the classical groupings
that were made on the basis of physiological responses. A model for how
the different floral promotion pathways interact is also proposed.
38. Simon R, Igeño MI, Coupland G: Activation of floral meristem
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flowering-time gene LUMINIDEPENDENS is expressed primarily
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This report describes the expression pattern of the LD gene and the nuclear
localization of the LD protein. In addition, the interaction of LD with floral
meristem identity genes is also examined and indicates that, although LD is
involved in the activation of LFY, it is also likely to be required for the activation of other meristem identity genes.
41. Page T, Macknight R, Yang C, Dean C: Genetic interactions of FCA,
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an Arabidopsis gene controlling flowering time, with genes
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mutants and 35S::LFY plants indicates that FCA is likely to promote flowering in multiple pathways.
42. Melzer S, Kampmann G, Chandler J, Apel K: FPF1 modulates the
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competence to flowering in Arabidopsis. Plant J 1999,
18:395-405.
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suggests that FPF1 acts to promote flowering in a parallel pathway to LFY.
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Arabidopsis. Nature 1995, 377:522-524.
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Feldman LJ, Yanofsky MF: Floral determination and expression of
floral regulatory genes in Arabidopsis. Development 1997,
124:3845-3853.
Response of plant development to environment: control of
flowering by daylength and temperature
Paul H Reeves* and George Coupland†
The transition from vegetative growth to flowering is often
controlled by environmental conditions and influenced by the age
of the plant. Intensive genetic analysis has identified pathways
that regulate flowering time of Arabidopsis in response to
daylength or low temperature (vernalization). These pathways are
proposed to converge to regulate the expression of genes that
act within the floral primordium and promote floral development.
In the past year, genes that confer the responses to daylength or
vernalization have been cloned and have enabled aspects of the
genetic models to be tested at the molecular level.
Addresses
John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
*e-mail: [email protected]
†e-mail: [email protected]
Current Opinion in Plant Biology 2000, 3:37–42
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
AG
AGAMOUS
AP
APETALA
CAB2
CHLOROPHYLL A/B BINDING PROTEIN 2
CCA1
CIRCADIAN CLOCK ASSOCIATED 1
CRY2
CRYPTOCHROME 2
CVI
Cape Verde Islands
EDI
EARLY DAYLENGTH INSENSITIVE
elf3
early flowering 3
FLC
FLOWERING LOCUS C
FRI
FRIGIDA
GI
GIGANTEA
ld
luminidependens
LFY
LEAFY
lhy
late elongated hypocotyl
vrn
vernalization
Introduction
The time of year at which plants flower varies widely
and is closely adapted to the environment in which they
grow. Temperature and daylength are the principal environmental conditions that synchronise flowering to the
changing seasons. Many papers published from the
1920s onward described variations in the flowering
response of varieties of the same species isolated from
different latitudes and, in some cases, identified genetic
loci that confer these responses. The use of genetic
approaches to compare the flowering responses of naturally occurring varieties of Arabidopsis and to isolate
induced mutations that disrupt these responses has
enabled the isolation of genes that underlie the control
of flowering by photoperiod and vernalization. Models
that were initially based on genetic interactions are
being confirmed or adapted on the basis of the analysis
of gene expression in different genetic backgrounds. In
this short review we concentrate on progress that has
been made in understanding the control of flowering by
both photoperiod and vernalization since publication of
the most recent longer reviews [1–3].
Vernalization
Many Arabidopsis ecotypes collected at high latitudes or
from mountainous regions are winter annuals that flower in
the spring after exposure to winter conditions. In the laboratory, these strains flower very late but will flower much
earlier if exposed to low temperatures for several weeks.
This process, called vernalization, prevents flowering late
in the summer when seed maturation may be curtailed by
onset of winter conditions.
Genetic analysis has identified two classes of late-flowering Arabidopsis plants that will respond to vernalization.
First, naturally occurring late-flowering varieties collected
from many locations across Europe carry dominant alleles
at the FRIGIDA (FRI) locus that delay flowering, and
these plants will flower early if given low temperature
treatments [4–6]. Second, a group of induced recessive
mutations delay flowering in early flowering varieties such
as Landsberg erecta and Columbia and this phenotype can
be corrected by low temperature treatments [7,8]. These
mutations were assigned to the autonomous flowering
pathway, because they delay flowering in all photoperiods.
The observation that these mutants respond to low temperature treatment suggests that vernalization can
overcome the requirement for the autonomous pathway.
During the past year it has become clear that the flowering-time gene FLOWERING LOCUS C (FLC) plays a
central role in the vernalization response (Figure 1). The
FLC gene was identified genetically because the widely
used laboratory strain Landsberg erecta carries an inactive
FLC allele. This was recognised when dominant FRI alleles or the luminidependens (ld) mutation that affects the
autonomous pathway were shown not to delay flowering in
Landsberg erecta, because for FRI and ld to cause late flowering an active FLC allele is required [9,10]. On the basis
of these genetic interactions it was proposed that FLC
inhibits flowering and that this inhibition is enhanced by
FRI and repressed by LD. These interpretations have
been supported by the molecular analysis of FLC.
The FLC gene was cloned independently by activation TDNA tagging [11••] and chromosome walking [12••]. Both
experiments identified the same MADS-box containing
gene, and proposed that it acts to repress flowering.
Analysis of FLC expression provided a means to test genetic models for the control of vernalization. Late-flowering
plants that can be induced to flower early if given a low
temperature treatment were shown to have high FLC
mRNA levels, mainly in roots and at the shoot apex, that
38
Growth and development
(vrn) mutations are likely to define genes that are required
for the vernalization process and at least the vrn2 mutation
prevents the reduction of FLC mRNA abundance that is
observed on low temperature treatment [11••].
Figure 1
Vernalization
Autonomous
pathway
LD
Photoperiod response
FCA
FRI
VRN2
FLC
Vegetative growth
Flowering
Current Opinion in Plant Biology
Genetic and molecular interactions involved in vernalization in
Arabidopsis. The FLC gene encodes a MADS-box transcription factor
that represses the transition from vegetative growth to flowering. The
abundance of FLC mRNA is reduced by low temperature treatment
and is proposed to play a central role in vernalization. The FRI, FCA,
LD and VRN2 genes regulate the abundance of FLC mRNA
[11••,12••]. FCA acts within the autonomous flowering pathway and
other genes within this pathway have similar effects to those of FCA.
The FRI gene increases FLC expression. The VRN2 gene acts during
vernalization to reduce FLC expression in response to low temperature
treatments. The FCA and LD genes reduce FLC mRNA abundance, so
that fca and ld mutants are late flowering but this can be corrected by
low temperature treatments.
are reduced on vernalization [11••,12••]. Dominant alleles
at the FRI locus, ld mutations and other mutations that
affect the autonomous pathway increase FLC mRNA abundance (Figure 1). Thus genotypes with a vernalization
requirement have high FLC mRNA levels and vernalization acts to reduce FLC mRNA abundance [11••,12••].
The analysis of FLC has extended the genetic models and
more clearly demonstrated a link between the autonomous
pathway and vernalization. The mechanism by which low
temperatures reduce FLC mRNA levels now becomes a
key question. The reduced level of FLC mRNA caused by
treating seedlings with low temperatures is maintained
throughout the development of the treated plant but their
progeny again show high levels of FLC mRNA and the
acceleration of flowering caused by low temperatures is
progressive with a six week exposure having a more severe
effect than a two week exposure [7,11••,12••]. There are
similarities between this and the regulation of gene
expression by methylation, and some early flowering
plants in which methyl transferase activity was reduced
using anti-sense technology show reduced levels of FLC
mRNA, suggesting that methylation might play a role in
FLC regulation [11••]. However, genes required for vernalization have been identified genetically by the isolation of
mutations that reduce the acceleration of flowering caused
by low temperature treatments [13]. These vernalization
Flowering of Arabidopsis is also regulated by daylength.
Flowering occurs much earlier under long days of 16 hours
light than under short days of 8 hours, and daylengths
between these two extremes give an intermediate
response. Mutations that disrupt the effect of photoperiod
on flowering have been described, and one gene has been
identified in an analysis of allelic differences between ecotypes. The daylength-insensitive mutants fall into two
classes: late-flowering mutants, which compared to wildtype flower late under long days but are unaffected under
short days, and early-flowering mutants, which flower
much earlier under short days. The late-flowering mutants
are believed to affect a single genetic pathway that promotes flowering in response to long days [10,14••]. In
addition, an extensive analysis of the allelic differences in
flowering time genes between the Cape Verde Islands
(CVI) and Landsberg erecta ecotypes identified a locus
EARLY DAYLENGTH INSENSITIVE (EDI) [15••]. When
introgressed into Landsberg erecta the dominant CVI allele
of EDI caused early flowering and these plants were
almost daylength insensitive. No induced dominant mutation has been isolated that causes early flowering,
highlighting the benefits of studying natural variation in
flowering time.
Classic experiments implicated the circadian clock as the
timer in daylength measurement, and analysis of daylengthinsensitive flowering-time mutants has provided genetic
support for this. The first of these was early flowering 3 (elf3),
which in addition to causing early flowering that is insensitive to daylength also shows alterations in circadian clock
regulation [16,17]. In elf3 plants grown under light/dark
cycles and then shifted to continuous light, the regulation of
genes such as CHLOROPHYLL A/B BINDING PROTEIN 2
(CAB2) by the circadian clock is disrupted as is the rhythm
in leaf movements [17]; the effect of the elf3 mutation is,
however, conditional and does not affect circadian clock
function under continuous darkness.
Two other genotypes were recently described that have
dramatic effects on flowering time and disrupt circadian
clock function [18••,19••]. One of these is the late elongated
hypocotyl (lhy) mutation, which was identified by activation
tagging and falls into the group of late-flowering mutations
causing delays in flowering only under long days [18••].
The LHY gene encodes a protein containing a single MYB
repeat and its expression is regulated by the circadian clock
with a peak in expression just after dawn. In the mutant,
the LHY gene is expressed at an elevated and constant
level. In entrained mutant plants shifted to either continuous dark or continuous light, all circadian clock controlled
rhythms that have been tested are disrupted. This pheno-
Control of flowering by daylength and temperature Reeves and Coupland
type together with the daylength-insensitive flowering
time of the mutant suggests a relationship between
daylength responses and circadian clock function [18••].
Transgenic plants in which the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) gene is expressed from the cauliflower
mosaic virus 35S promoter show a similar phenotype to lhy
mutants [19••]. CCA1 was initially identified because its
protein product binds to the CAB promoter [20]. The
CCA1 protein is 43% identical to LHY overall, and 87%
identical in the DNA-binding domain [18••]. The initial
publications on the effects of CCA1 and LHY on circadianclock regulation relied on the phenotypes of plants in
which the genes were overexpressed. The recent observation that inactivation of the CCA1 gene also affects
circadian-clock control of gene expression was therefore an
important step in establishing the importance of these
genes in circadian-clock regulation [21••]. A T-DNA insertion within an intron of the CCA1 gene prevents synthesis
of CCA1 protein, and causes the circadian clock to run
approximately three hours faster than in wild-type plants
under continuous light [21••]. The CCA1 knockout allele
may have a less severe effect on circadian-clock control
than CCA1 overexpression, because of the presence of
genes such as LHY that may be able to partially compensate for the loss of CCA1 function. No effect of the cca1
mutation on flowering time was reported; another mutation that shortens the period of the circadian clock, toc 1,
was however, previously shown to cause early flowering
under short days in the Landsberg erecta ecotype, but not
in the C24 ecotype [22,23••]. It is possible, therefore, that
introgression of cca1 into other ecotypes may be required
to observe any effect on flowering time and that allelic differences between ecotypes can mask the effects of period
mutations on flowering time.
GIGANTEA (GI) is also implicated in the control of flowering in response to daylength [8,24–26], and the gene was
isolated during the past year [27••,28••]. The characterisation of GI again emphasises the role of the circadian clock
in daylength responses. The gi mutation was first
described as causing late flowering under long days and
daylength insensitivity [8,24]. GI encodes a large protein
that is predicted to be located in the plasma membrane
and to contain at least five membrane-spanning domains in
the first 660 amino acid residues. The carboxy-terminal
region is hydrophilic and has no homology to any protein
of known function but is probably important for GI function because several mutant alleles affect this region of the
protein [27••]. The abundance of GI mRNA is also regulated by the circadian clock and peaks around 10 hours
after dawn. Furthermore, expression of GI does not show
its normal peak in expression in the lhy mutant nor in
plants overexpressing CCA1 [27••], which may be expected as the expression of all other circadian-clock regulated
genes tested is also disrupted in these genotypes. More
surprisingly, however, gi mutations also reduce the expression of LHY and CCA1 in long-day grown plants and in
39
Figure 2
Photoperiod
Vernalization
(a)
(c)
(b)
CO/GI
FLC
FT/FWA
(d)
?
(h)
(e)
(g)
AP1
(f)
LFY
(i)
AP3/PI
AP1/AP2
AG
Floral patterning
Current Opinion in Plant Biology
The activation of floral-meristem identity genes by floral promotion
pathways. Photoperiod and vernalization control the timing of floral
induction through independent genetic pathways [1,2]. Eventually these
pathways must converge to activate genes, such as LFY and AP1, that
confer floral identity upon the shoot apical meristem [34••]; however,
little is known about how signals from separate floral promotion
pathways become integrated. (a) The main response to photoperiod in
Arabidopsis is the promotion of flowering under long days. Both the
circadian clock and light receptors are involved in this response, which
is mediated through genes such as CO and GI [14••,26,27••,38].
(b) Genetic analysis suggests that the FT and FWA genes are also
involved in the promotion of flowering in response to long days [14••].
(c) One role of vernalization is to reduce the mRNA abundance of the
floral repressor FLC [11••,12••]. The autonomous floral promotion
pathway also interacts with FLC (see Figure 1). (d) The photoperiodic
and autonomous floral promotion pathways act additively to promote
flowering and are involved in the regulation of the same floral meristem
identity genes, but the stage at which these pathways converge is
unknown. (e) Genetic and molecular evidence illustrates that genes
from the photoperiodic and autonomous floral promotion pathways are
involved in the regulation of the LFY gene [37••,38,39,40•,41•].
(f,g) Although flowering time genes have been shown to be involved in
the regulation of LFY, they are also likely to regulate other floral
meristem identity genes [37••,38,40•,41•]. AP1 is a possible candidate,
but other floral meristem identity genes are also likely to be involved. For
example, ld ; ap1 ; cal triple mutants show a more severe shoot
phenotype than lfy; ap1; cal mutant plants, suggesting that LD could
act on floral meristem identity genes such as APETALA2 or UNUSUAL
FLORAL ORGANS [40•]. (h) The main function of FT and FWA is not
the transcriptional activation of LFY [37••]. One function of these genes
is likely to be the activation of AP1, as AP1 expression is abolished in
the floral structures formed on ft ; lfy and fwa ; lfy double mutants [39].
(i) Floral meristem identity genes establish and regulate the expression
patterns of floral homeotic genes [34••].
40
Growth and development
plants shifted to continuous light indicating that
LHY/CCA1 and GI do not act in a simple linear pathway
but that they affect each other’s expression [27••,28••]. But
gi does not, however, affect LHY expression in plants shifted to continuous darkness indicating that GI may play a
role in the control of expression of circadian-clock regulated genes in response to light [28 ••]. There are
complications in establishing a connection between the
effects of gi on flowering time and on the period of the circadian clock because two alleles that showed different
effects on the period of CAB2 expression had very similar
effects on flowering time [28••]. The delay in flowering
caused by gi may be due to a general reduction in the
amplitude of expression of circadian clock regulated genes,
as observed for LHY and CCA1 [27••,28••].
In order to detect and respond to daylength, light receptors
must act within the long-day pathway. Classically, phytochrome was implicated as the photoreceptor that controls
photoperiodic responses. This was supported by the
demonstration that mutations in the gene encoding phytochrome A abolish the photoperiodic control of flowering in
pea plants [29]. In Arabidopsis and other crucifers, however,
both far red and blue light have long been known to promote flowering [30], and mutations in the gene encoding
the blue light receptor CRYPTOCHROME (CRY) 2 both
delay flowering and reduce the photoperiodic response
[31••]. As described above, the circadian clock regulates the
expression of genes that act within the long-day pathway,
and the circadian clock is itself entrained (or synchronised)
to the daily cycle of light and dark by light. Perhaps surprisingly, however, cry2 mutations have a weak effect on
circadian clock entrainment. This has become clear from a
detailed analysis of the effect of mutations in different light
receptor genes on circadian clock controlled expression of
the CAB2 gene. Using a fusion of the CAB2 promoter to
luciferase it was shown that under high fluence light mutations in the PHYB or CRY1 genes lengthen the period of the
circadian clock but that mutations in PHYA and CRY2 only
do so under specialised conditions of low fluence light
[32••]. This indicates that the phyA and cry2 mutations do
not affect the photoperiodic response by affecting circadian
clock control but may do so by more directly affecting the
expression or function of genes that control flowering time.
This is supported by recent observations that the promotion
of flowering by CRY2 occurs through antagonising an
inhibitory effect on flowering that is mediated by PHYB,
and may act by increasing the expression of the flowering
time gene CONSTANS [31••,33•].
The interaction between flowering time and
floral meristem identity
Several of the genes that act during the early stages of flower
development to confer floral identity on the developing floral meristem have been isolated and their expression
analysed in detail. LEAFY (LFY) is the earliest acting of
these genes and encodes a transcription factor that acts within the developing primordium to promote the expression of
another floral meristem identity gene, APETALA (AP) 1, or
together with co-activators that are expressed within particular domains of the floral meristem to activate expression of
genes such as AP3 and AGAMOUS (AG) that specify the
identity of particular floral organs [34••–36••].
The role flowering-time genes play in the activation of
these floral meristem identity genes is also being uncovered, although to date the greatest progress has been made
in assessing the interactions between flowering time genes
and LFY. One approach has been to analyse the effect of
mutations causing late flowering on both LFY gene expression and on early-flowering transgenic plants that
overexpress the LFY gene from the 35S promoter [37••]. In
general, two classes of flowering-time genes have been
identified. The first class of gene appears to be involved in
the transcriptional upregulation of LFY: mutations in
genes from this class cause relatively weak upregulation of
LFY promoter activity during development and do not
severely attenuate the early flowering of 35S::LFY plants.
Genes from several genetically distinct flowering time
pathways including FCA/FVE (autonomous pathway),
CO/GI (photoperiod pathway), and GIBBERELLIN
RESPONSIVE 1/GIBBERELLIN INSENSITIVE (gibberellin-dependent pathway) fall into this group,
suggesting that LFY is a common target for these flowering
pathways (Figure 2; [37••,38]).
The second group is mainly defined by a subgroup of
genes in the photoperiod pathway, namely FT and FWA.
This class of gene does not appear to be involved in the
transcriptional upregulation of LFY but is instead required
for the response to LFY activity: mutations in these genes
have only a small effect on the activity of the LFY promoter and the late-flowering phenotype of ft and fwa is
epistatic to the early flowering of 35S::LFY plants
(Figure 2; [37••]). It is likely that one function of this second group is the activation of the AP1 gene [39].
More detailed genetic analysis combining individual lateflowering mutants with a range of meristem-identity
mutants supports the conclusions reached from the analysis of the LFY gene [40•–42•]. These studies have also
revealed that although some genes are involved in the
transcriptional upregulation of LFY this is unlikely to be
their sole function. For example, double fca ; lfy mutants
show an enhancement of the lfy phenotype, suggesting
that FCA also acts to promote flowering and flower development in a pathway parallel to LFY [41•].
The analysis of the interaction of the FCA gene with
meristem-identity genes also highlights an overlooked role
of the AP1 gene in the regulation of flowering [41•].
Overexpression of the AP1 gene causes early flowering
[43], suggesting that one function of AP1 is to promote
flowering, and the appearance of AP1 gene expression in
primordia is generally considered to be a good marker for
the commitment to flowering [44]. However, ap1 mutant
Control of flowering by daylength and temperature Reeves and Coupland
plants also flower slightly earlier than wild-type plants,
suggesting that the AP1 gene may also repress flowering
[41•]. As the late flowering of the fca mutant is epistatic to
the early flowering of ap1 plants, AP1 may repress flowering by antagonising the action of the autonomous
promotion pathway [41•].
Conclusions
Flowering time is regulated by balancing the promotive or
inhibitory effects of different environmental conditions.
For example, the responses to vernalization and photoperiod are regulated by parallel genetic pathways that
promote flowering in Arabidopsis. Recent progress has
demonstrated the importance of the circadian clock in controlling the photoperiod pathway and shown that
vernalization acts at least in part to reduce the abundance
of the FLC mRNA. These two pathways somehow converge to regulate the expression of LFY — a gene that acts
within the floral primordium to promote the expression of
genes that specify floral organ identity (Figure 2). Future
experiments will describe the molecular mechanism of this
convergence and whether it occurs at the LFY gene itself
or at an earlier stage in the transition from vegetative
growth to flowering.
Acknowledgements
Our work on flowering time is supported by grants from the European
Commission and the Human Frontiers Science Program.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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