1 John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
2 Max Planck Institute for Plant Breeding, Carl Von Linne Weg 10, D-50829
Cologne, Germany
Author for correspondence at address2 (e-mail:
coupland{at}mpiz-koeln.mpg.de)
Accepted 26 August 2002
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SUMMARY |
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Key words: Flowering, Arabidopsis thaliana, Photoperiod, Vernalization, ESD4
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INTRODUCTION |
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A systematic genetic approach to identifying genes that regulate flowering
time has been taken in Arabidopsis (reviewed by
Araki, 2001;
Mouradov et al., 2002
;
Simpson and Dean, 2002
). Many
mutations have been identified that delay flowering, and genetic and
physiological analysis has placed these mutations in at least three
independent pathways that promote flowering
(Koornneef et al., 1998
).
These are the long day pathway, the autonomous pathway and the gibberellic
acid (GA)-dependent pathway. Mutations affecting the long day pathway (co,
fd, fe, sha, ft, fwa, gi and lhy) delay flowering under long day
conditions, whereas those affecting the autonomous pathway (fca, fpa, fve,
fy and ld) delay flowering under all photoperiods. Mutations
that strongly reduce GA biosynthesis delay flowering under long days, and
almost abolish flowering under short days
(Wilson et al., 1992
). The
existence of these pathways is supported by the phenotypes of double mutants
(Koornneef et al., 1991
;
Koornneef et al., 1998
).
Partial redundancy between the long day, autonomous and GA pathways probably
explains why no single mutation has been identified that prevents flowering.
However a triple mutant, in which all three flowering pathways are impaired
does not flower under long or short days, indicating that these pathways are
absolutely required for flowering under these conditions
(Reeves and Coupland,
2001
).
The cloning of several flowering-time genes, and analysis of their
expression in wild-type and mutant backgrounds, have led to detailed models of
the mechanisms underlying the flowering response
(Lee et al., 1994;
Putterill et al., 1995
;
Macknight et al., 1997
;
Schaffer et al., 1998
;
Michaels and Amasino, 1999a
;
Sheldon et al., 1999
;
Kardailsky et al., 1999
;
Kobayashi et al., 1999
;
Samach et al., 2000
;
Lee et al., 2000
;
Suárez-López et al.,
2001
; El-Assal et al.,
2001
; Gendall et al., 2002). These models are supported by the
phenotypes of transgenic plants in which flowering-time genes are
overexpressed (Kardailsky et al.,
1999
; Kobayashi et al.,
1999
; Onouchi et al.,
2000
; Lee et al.,
2000
). An endogenous circadian clock acts to control the
expression patterns of genes within the long day pathway, enabling the
promotion of flowering under appropriate day lengths
(Schaffer et al., 1998
;
Fowler et al., 1999
;
Park et al., 1999
;
Suárez-López et al.,
2001
). The autonomous pathway appears to promote flowering by
reducing the expression of the FLC gene that encodes a repressor of
flowering (Michaels and Amasino,
1999a
; Sheldon et al.,
1999
; Michaels and Amasino,
2001
). More recently, it has become apparent that the long day,
autonomous, and GA pathways converge on a common set of target genes to
regulate flowering time and flower development. For example, all three
pathways are involved in the regulation of expression of LEAFY
(Simon et al., 1996
;
Blázquez et al., 1998
;
Nilsson et al., 1998
;
Blázquez and Weigel,
2000
), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS
(SOC1)/AGAMOUSLIKE 20 (AGL20)
(Samach et al., 2000
;
Lee et al., 2000
;
Borner et al., 2000
;
Michaels and Amasino, 2001
)
and FT (Kardailsky et al.,
1999
; Kobayashi et al.,
1999
; Samach et al.,
2000
;
Suárez-López et al.,
2001
; Ohto et al.,
2001
).
Additional flowering pathways promote flowering specifically in response to
vernalization. The vernalization response shares common targets with the
autonomous pathway, and acts to repress FLC mRNA abundance
(Michaels and Amasino, 1999a;
Sheldon et al., 1999
).
However, vernalization is not mediated by any of the three pathways described
above, because the vernalization response is not abolished in mutants
representative of each pathway (Koornneef
et al., 1991
; Chandler et al.,
2000
; Michaels and Amasino,
1999b
), nor in a co-2 fca-1 gal-3 triple mutant in which
all three pathways are impaired (Reeves
and Coupland, 2001
). The FRI gene confers a vernalization
response on naturally occurring varieties of Arabidopsis
(Johanson et al., 2000
), and
is involved in the promotion of FLC mRNA levels
(Michaels and Amasino, 1999a
;
Sheldon et al., 1999
;
Johanson et al., 2000
). Stable
repression of FLC by vernalization requires VERNALIZATION 2, which
was proposed to act within a protein complex similar to Polycomb group
complexes of Drosophila (Gendall
et al., 2001
). Both the promotion of flowering by the autonomous
pathway and the delay in flowering by strong FRI alleles are
absolutely dependent on active FLC, although the vernalization
response also has an FLC independent component
(Michaels and Amasino,
2001
).
A diverse group of mutations causes early flowering in Arabidopsis
(Alvarez et al., 1992;
Zagotta et al., 1996
;
Hicks et al., 1996
;
Goodrich et al., 1997
;
Telfer and Poethig, 1998
;
Somers et al., 1998
;
Soppe et al., 1999
;
Scott et al., 1999
;
Hartmann et al., 2000
;
Michaels and Amasino, 2001
;
Gomez-Mena et al., 2001
). For
example, the phyB mutation disrupts the gene encoding the red/far-red
light receptor PHYTOCHROME B (PHYB) and causes early flowering under both long
and short days (Reed et al.,
1993
; Koornneef et al.,
1995
). The elf3 mutation causes early flowering under
short days so that mutants flower at the same time irrespective of daylength
(Hicks et al., 1996
). Under
continuous light, elf3 also disrupts the rhythmic expression of the
circadian clock-regulated gene CAB2, and the rhythmic movement of
leaves. ELF3 is proposed to act by gating light signalling to the
circadian clock (McWatters et al.,
2000
). The early-flowering mutant, hasty, forms adult
leaves earlier in vegetative development than wild type
(Telfer and Poethig, 1998
).
The apical meristem of the hasty mutant can also respond more rapidly
to expression of the floral meristem-identity gene LEAFY, suggesting
that HASTY reduces the competence of the shoot to respond to
flowering signals. EMBRYONIC FLOWER mutations cause extreme early
flowering, probably by inactivating transcriptional repression complexes that
in wild-type plants repress the expression of floral identity genes such as
APETALA1 (Chen et al.,
1997
; Kinoshita et al.,
2001
).
Combining mutations causing early or late flowering can provide information
on how the functions of the affected genes are inter-related
(Yang et al., 1995;
Koornneef et al., 1995
;
Koornneef et al., 1998a; Weller et al.,
1997
; Soppe et al.,
1999
; Michaels and Amasino,
2001
). For example, in Arabidopsis, the efs gene
was suggested to act within the autonomous pathway on the basis of double
mutant analysis (Soppe et al.,
1999
). However, in general, little information is available on how
mutations causing early or late flowering interact in Arabidopsis and
the molecular basis for such interactions is even less clear.
Here we describe a novel mutation, early in short days 4 (esd4), that causes an extreme early-flowering phenotype in Arabidopsis. The analysis demonstrates genetic and molecular relationships between ESD4 and genes in the autonomous flowering pathway.
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MATERIALS AND METHODS |
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Mutant seed stocks were all in Ler and were provided by the following individuals: fca-1, fve-1, co-2, fwa-1, ft-1 M. Koornneef (University of Wageningen), ag-3 J. Goodrich (University of Edinburgh), gai-1, ga1-3, (Nottingham Stock Centre).
Growth conditions and measurement of flowering time
Flowering time was measured under controlled conditions as described
previously (Reeves and Coupland,
2001). Short days consisted of a photoperiod of 10 hours light,
whereas long days consisted of 10 hours light with a 6 hour daylength
extension supplied by incandescent bulbs. At least 15 plants were used to
examine the flowering time of each genotype. For analysis of FT, SOC1
and CO mRNA expression patterns, plants were grown in cabinets under
true long days of 16 hours light with no daylength extension.
Mapping
The map position of ESD4 was defined using the CAPS markers 326
(5'-GTGACGTACTCGGTGAAG, 5'-CTCTACTACACACCACAC; StyI) and
SC5 (5'-TCGACGACTCTCAAGAACCC, 5'-CACAAGCTATACGATGCTCACC;
AccI).
Cryo-scanning electron microscopy
The sample was mounted on an aluminium stub which was then plunged into
liquid nitrogen slush and transferred onto the cryostage of a CT1000 Hexland
cryo-transfer system at -85°C (Oxford Instruments, Oxford) fitted to a
CamScan Mark IV scanning electron microscope (Gresham-CamScan, Cambridge). The
sample was transferred to the pre-chamber, placed on a stage at -195°C and
sputter coated with gold for 6 minutes at 2 mA. It was returned to the main
cryo-stage of the microscope and viewed at 16 kV.
Construction and analysis of double mutants
Information on the construction of double mutants can be obtained from the
authors.
To examine the flowering times of esd4 gal-3 and gal-3
plants seeds were germinated without applying exogenous GA as described
previously (Reeves and Coupland,
2001).
Northern analysis of mRNA abundance
RNA extraction, northern blotting and hybridisation was as described by
Suárez-López et al.
(Suárez-López et al.,
2001). The FLC probe was provided by A. Gendall and C.
Dean (JIC, Norwich), and consisted of a 403 bp PCR fragment corresponding to
nucleotides 298-700 of the FLC cDNA sequence. The FT probe
was described previously (Samach et al.,
2000
). The UBQ10 probe was as described by Wang et al.
(Wang et al., 1997
). The
SOC1 probe was a 459 bp PCR fragment, amplified from the
SOC1 cDNA with the primers 5'-AATATGCAAGATACCATAGATCG-3'
and 5'-TCTTGAAGAACAAGGTAACCCAAT-3'. The strength of hybridisation
signals was assessed using a Phosphorimager (Molecular Dynamics). The ratio of
signal intensity compared to the UBQ10 control was calculated for
each sample using ImageQuant software (Molecular Dynamics).
Analysis of CO mRNA abundance
RT-PCR analysis of CO mRNA abundance was as described by
Suárez-López et al.
(Suárez-López et al.,
2001).
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RESULTS |
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The esd4 mutant was back-crossed to Ler three times before further phenotypic analysis, and crossed to Columbia to enable its position to be determined relative to RFLP markers. For the mapping, DNA was extracted from 200 F2 plants that were homozygous for esd4 and analysed with several CAPS markers. Linkage was detected to markers on the lower arm of chromosome 4. For example, esd4 was located approximately 1.8 cM distal to marker 326, and 0.5 cM proximal to SC5. Relative to phenotypic markers, esd4 is therefore located around 2.3 cM distal to cop9 and 0.5 cM proximal to fca. The map position of esd4 excluded the possibility that it was an allele of any previously described mutation causing early flowering.
esd4 causes early flowering and has pleiotropic effects on
shoot development
Plants homozygous for esd4 were grown under long and short days,
and their flowering time compared with that of Ler
(Table 1;
Fig. 1). The early-flowering
phenotype was most dramatic under short days where wild-type plants flowered
after forming around 49 rosette and cauline leaves compared to only 10 for
esd4. The mutants also flowered slightly earlier than wild-type
plants under long days, forming only 5 leaves compared to 9 for Ler.
The esd4 mutation therefore causes early flowering under long and
short days, and the flowering time of the mutant is influenced by daylength,
although less strongly than that of wild-type plants
(Table 1).
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Arabidopsis plants exhibit heteroblasty, forming juvenile rosette
leaves that have trichomes only on their adaxial (upper) side; adult rosette
leaves that develop trichomes on their adaxial and abaxial (lower) sides, and
cauline leaves that form on the stem above the rosette and have trichomes on
both surfaces (Chien and Sussex,
1996; Telfer et al.,
1997
). The early-flowering hasty mutant forms fewer
juvenile rosette leaves than wild type but approximately the same numbers of
adult rosette and cauline leaves (Telfer
and Poethig, 1998
). As shown in
Fig. 2, esd4 showed a
reduction in all types of leaf. However, the most dramatic effect was on adult
rosette leaves, which were absent from esd4 mutants grown under long
days and dramatically reduced in number under short days.
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The main inflorescence of esd4 mutants showed several abnormalities. Internodes between the cauline leaves and solitary flowers are shorter, and the leaves are smaller than in wild-type plants (Fig. 1). Also at the nodes at which the last leaf or the first solitary flower form there are often alterations to phyllotaxy compared to wild-type plants (Fig. 1). For example, in approximately 13% of esd4 mutants, two solitary flowers develop at the first node after the last cauline leaf, while in another 10% of mutants the last cauline leaf to develop and the first solitary flower form at the same node on opposite sides of the stem. More complex abnormalities also occur at this point of transition from cauline leaves to solitary flowers (Fig. 1).
In addition, esd4 mutants formed fewer flowers than wild-type plants (Table 1). The main inflorescence of wild-type plants contained approximately 37 solitary flowers under long days, and around 59 under short days. However, the main inflorescence of esd4 mutants formed many fewer solitary flowers: approximately 19 and 34 under long and short days, respectively. This reduction in flower number was in part associated with the conversion of the shoot apical meristem into a carpelloid, pistil-like structure (Fig. 1) that does not occur in wild-type plants (Fig. 1). Under long days, the main inflorescence of approximately 80% of esd4 mutants terminated with this structure. Under short days, this phenotype was less severe, and was only observed in around 25% of plants.
After fertilisation, the gynoecium of Arabidopsis plants elongates
to form a silique that contains the developing seeds
(Bowman, 1993). The siliques of
esd4 mutants are shorter than those of wild type, and are broader at
the tip (Fig. 1). The siliques
of esd4 mutants contain 2 valves, as do those of wild-type plants
(Bowman, 1993
).
The esd4 mutation is recessive and behaves as a single
genetic locus
Plants heterozygous for esd4 were generated by back-crossing to
Ler. The shoot of the heterozygotes showed a wild-type phenotype, and
the heterozygous plants flowered at the same time as wild-type under long and
short days (Table 1). The
esd4 mutation is therefore recessive with respect to all aspects of
the mutant phenotype.
To determine whether the esd4 phenotype was due to a mutation at a single locus, 78 F2 plants derived from a back-cross of esd4 to wild-type plants were examined. Nineteen plants flowered at a similar time to esd4 and showed all of the shoot phenotypes previously described for the esd4 mutant, while 59 plants flowered at a similar time to the Ler wild-type, and showed a wild-type shoot phenotype. The ratio of esd4-like plants to wild-type like plants was approximately 3:1, suggesting that the esd4 phenotype is caused by a mutation at a single genetic locus.
AGAMOUS is not required for esd4 to cause early
flowering
Ectopic expression of the floral-organ identity gene AGAMOUS was
previously shown to cause early flowering
(Mizukami and Ma, 1992;
Goodrich et al., 1997
). To
test whether AG was required for the early flowering of
esd4, double mutants carrying both esd4 and ag-3
were made. These plants flowered at the same time as esd4 under long
and short days. AG is therefore not required for the early flowering
of esd4 plants. However, ectopic expression of other genes that
encode MADS box containing proteins can also cause early flowering. We
therefore constructed the esd4 ap3, esd4 pi and esd4 ap1
double mutants and they all flowered at the same time as esd4,
indicating that AP3, PI and AP1 are also not required for early flowering of
esd4.
The interaction of esd4 with mutations affecting the long
day promotion pathway
To test the relationship between ESD4 and genes that promote
flowering, double mutants were made containing esd4 and mutations
causing late flowering. The co, ft and fwa mutations were
proposed to affect the long-day pathway
(Koornneef et al., 1998). The
double mutants esd4 co-2, esd4 ft-1 and esd4 fwa-1 were
constructed to test the effects of the mutations on the esd4
phenotype.
Under long days, all three double mutants flowered later than esd4 (Table 2; Fig. 3). The flowering times of the esd4 co-2, esd4 fwa-1 and esd4 ft-1 double mutants were similar to that of wild type. Under short days, the co-2, ft-1 and fwa-1 mutants do not show late flowering compared to wild type. However, these mutations delayed flowering of the esd4 mutant under these conditions (Table 2, Fig. 4). The ft-1 and fwa-1 mutations caused a more severe delay in the flowering time of esd4 than co-2, and under short days the esd4 ft-1 and esd4 fwa-1 plants flowered almost as late as wild-type and the late flowering parent. The latest flowering genotype was esd4 fwa-1 that formed around 23 rosette leaves under short days compared to approximately 9 for esd4 (Table 2).
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The pleiotropic effects of esd4 were also reduced in severity in the esd4 co-2, esd4 ft-1 and esd4 fwa-1 plants. More flowers were formed on the primary inflorescences of the double mutants, and the proportion of plants in which the primary inflorescence was determinate was reduced. For example, in long days esd4 co-2, esd4 ft-1 and esd4 fwa-1 produced over 35, 72 and 61 flowers, respectively, compared to the 20 flowers produced by esd4. Only around 37% of esd4 co-2 mutants showed a determinate main inflorescence, whereas no esd4 ft-1 or esd4 fwa-1 plants showed this phenotype. The frequency with which abnormalities in floral phyllotaxy were observed at the node representing the transition from cauline leaves to flowers (Fig. 1) was also reduced. These were visible in fewer than 8% of esd4 co-2, and 3% of esd4 ft-1 mutants. No esd4 fwa-1 mutants showed these abnormalities. The double mutants retained the altered silique shape of esd4, and remained slightly dwarfed with respect to wild type, with the exception of esd4 ft-1, which was taller than wild type.
The FCA and FVE genes are not required for
esd4 to cause early flowering
The fca and fve mutations affect the autonomous
flowering-time pathway (Koornneef et al.,
1998). The double mutants esd4 fve-1 and esd4
fca-1 were made and their flowering times scored under long and short
days (Table 2; Figs
4 and
5).
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Under long days, esd4 fca-1 and esd4 fve-1 double mutants
flowered earlier than wild type, and were earlier flowering than the esd4
co-2, esd4 ft-1 and esd4 fwa-1 plants described above. Some of
the double mutant plants were indistinguishable from esd4, although
on average esd4 fve-1 and esd4 fca-1 formed 1 or 2 rosette
leaves more than esd4 (Table
2). esd4 fve-1 double mutants flowered slightly earlier
than esd4 fca-1 double mutants, probably because fve-1
causes a weaker phenotype than fca-1
(Table 2) (Koornneef et al., 1991).
Under short days, esd4 fca-1 and esd4 fve-1 mutants again flowered earlier than wild-type Ler and the late-flowering parent (Table 2). The double mutant plants flowered later than the esd4 mutant, with esd4 fca-1 plants and esd4 fve-1 plants forming a total of 19 and 15 leaves, compared to 12 in the esd4 mutant. Furthermore, although the fca-1 and fve-1 mutants flowered much later than any of the long-day pathway mutants described in the previous section (Table 2; see above), the relative severity of their phenotypes was in general reversed in the presence of the esd4 mutation: the esd4 fca-1 and esd4 fve-1 double mutants flowered much earlier under short days than esd4 fwa-1 and esd4 ft-1.
Under both long and short days, the double mutants still showed the pleiotropic effects caused by the esd4 mutation; the siliques retained their club-like appearance, the shoot terminated in a carpelloid structure, and the phyllotaxy of flowers on the shoot was disrupted. The frequency with which an abnormality occurs at the node representing the transition from cauline leaves to flowers was reduced from 45% in esd4 to 19% in esd4 fca-1 and 15% in esd4 fve-1. The number of flowers formed on the main inflorescence was also affected: under long days, 29 flowers were formed by esd4 fca-1 plants and 21 by esd4 fve-1 plants compared to 20 for esd4. However, the fca-1 and fve-1 mutations reduced the severity of the pleiotropic effects of esd4 to a much lesser extent than the long-day pathway mutations did (see previous section).
The effect of mutations affecting synthesis or response to the growth
regulator gibberellin on the flowering time of esd4 mutants
Mutations that affect GA synthesis or signal transduction delay flowering
weakly under long days, and severely under short days
(Wilson et al., 1992). The
severe mutation ga1-3 disrupts an early step in GA biosynthesis, and
prevents flowering under short days (Sun
and Kamiya, 1994
; Wilson et
al., 1992
). The gai mutation affects GA signalling
(Peng et al., 1997
). To
determine whether GA synthesis and response pathways are required for the
early flowering caused by esd4, plants carrying esd4 ga1-3
and esd4 gai were constructed.
Under both long and short days the esd4 gai double mutants flowered earlier than wild type, particularly under short days, and at approximately the same time as esd4 mutants (Table 2). The gai mutation therefore has almost no effect on the early-flowering phenotype caused by esd4. The esd4 ga1-3 plants showed a flowering time intermediate between the esd4 and ga1-3 parents, indicating that GA synthesis is required for the extreme early-flowering phenotype caused by esd4. However, esd4 still has a dramatic effect on the flowering time of ga1-3 mutants, particularly under short days where ga1-3 flowered after forming approximately 80 leaves, while the esd4 gal-3 double mutants formed approximately 15 (Table 2; Fig. 4).
esd4 can promote flowering in a co-2 fca-1 ga1-3
background
A co-2 fca-1 ga1-3 triple mutant, in which all three flowering
time pathways are impaired, does not flower under long days
(Reeves and Coupland, 2001).
To determine whether esd4 promotes flowering in this triple mutant
background, esd4 co-2 fca-1 ga1-3 quadruple mutants were constructed
and their flowering time examined under long days. As previously shown, the
co-2 fca-1 ga1-3 control plants did not flower under long-day
conditions (Table 2)
(Reeves and Coupland, 2001
).
However, the esd4 co-2 fca-1 ga1-3 quadruple mutant flowered after
the production of 28 leaves.
The level of FLC mRNA is reduced in esd4
mutants
The esd4 mutation most effectively suppressed the late-flowering
phenotype of mutations that impair the autonomous pathway
(Fig. 3;
Table 2). Therefore,
esd4 might cause early flowering by increasing the activity of the
autonomous pathway downstream of FCA and FVE, or by
bypassing the requirement for the autonomous pathway. FLC, which
encodes a repressor of flowering, is a downstream target of both the
autonomous and the vernalization-dependent floral promotion pathways
(Michaels and Amasino, 1999a;
Michaels and Amasino, 2001
;
Sheldon et al., 1999
;
Sheldon et al., 2000
).
FLC mRNA abundance is increased in mutants impaired in the autonomous
pathway and this increase is responsible for their late-flowering phenotype.
Therefore, esd4 may suppress the effect of autonomous pathway
mutations by reducing FLC mRNA levels.
The abundance of the FLC mRNA was compared in wild-type, esd4,
fca-1, esd4 fca-1, fve-1 and esd4 fve-1 seedlings that were 7
days old and had been grown in long days
(Fig. 5A). FLC mRNA
abundance was higher in both fca-1 and fve-1 mutants than in
wild-type plants, as previously shown
(Sheldon et al., 1999;
Michaels and Amasino, 1999a
).
However, in esd4 fca-1 and esd4 fve-1 double mutants, the
level of FLC mRNA was reduced compared to the late flowering
fca-1 and fve-1 parents. Nevertheless, FLC mRNA
abundance was still higher than in wild-type plants, which flower later than
esd4 fca-1 and esd4 fve-1 mutants. FLC mRNA levels
were also compared between wild-type plants and esd4 mutants.
Although FLC is expressed at a low level in Ler wild-type
plants, this was further reduced in esd4 mutant plants. Similar
reductions in the level of FLC mRNA were also observed under short
days (data not shown).
esd4 can partially suppress the effect of fca-1 on
FT and SOC1 mRNA levels
The reduction in FLC mRNA levels in esd4 may contribute
to the early-flowering phenotype of the mutant. The repression of flowering by
FLC is probably caused in part by reduced expression of the flowering-time
genes SOC1 and FT (P.Suárez-López and
G.Coupland, unpublished results) (Michaels
and Amasino, 2001; Ohto et
al., 2001
). Therefore, whether the early flowering and reduced
expression of FLC in genotypes containing esd4 was also
associated with increased expression of SOC1 and FT was
tested. Over a 24-hour long day cycle, FT and SOC1 mRNA
abundance was compared between wild-type and esd4 plants, and between
fca-1 and esd4 fca-1 plants.
In wild-type plants, FT showed the expected pattern of expression,
with the main peak in mRNA abundance occurring between 12 and 16 hours after
dawn (Fig. 5B)
(Suárez-López et al.,
2001). In esd4 single mutants, FT mRNA abundance
was slightly higher than in wild-type plants
(Fig. 5B). The fca-1
mutation caused a reduction in the level of FT mRNA, so that a lower
level peak was detectable at the same time as in wild-type plants. This
reduction in FT expression is probably caused by increased
FLC expression in fca-1 mutants
(Michaels and Amasino, 2001
),
and was partially suppressed by the esd4 mutation so that FT
mRNA levels were increased in the esd4 fca-1 double mutant compared
to fca-1, and similar to those of wild-type plants
(Fig. 5B).
Similar observations were made with SOC1. In wild-type plants
SOC1 mRNA peaked around 8 hours after dawn, although the peak was of
lower amplitude than that of FT
(Fig. 5C)
(Samach et al., 2000). In
esd4 single mutants, SOC1 expression was increased, and the
fca-1 mutation caused a severe reduction in the level of
SOC1 mRNA, although a low level peak was still detectable at the same
time as in wild-type plants (Fig.
5C). As for FT, the esd4 mutation partially
suppressed the effect of fca-1 on SOC1 expression
(Fig. 5C), so that the level of
SOC1 mRNA in the double mutant was similar to that of wild-type
plants.
The increases in FT and SOC1 expression in esd4 fca-1 plants compared to fca-1 mutants are consistent with the proposal that esd4 causes earlier flowering by reducing FLC expression and thereby increasing the expression of genes that are repressed by FLC.
esd4 does not affect the expression pattern of CONSTANS
To test at the molecular level whether esd4 influenced the
activity of the long-day pathway, CO mRNA abundance was compared in
wild-type and esd4 plants. CO acts downstream of many of the
other long-day pathway genes and is not repressed by FLC, although its
expression is increased in several early-flowering mutants or transgenic
plants that affect the long-day pathway
(Onouchi et al., 2000;
Suárez-López et al.,
2001
). CO shows a diurnal pattern of expression, with the
main peak of mRNA abundance occurring around 16 hours after dawn in long-day
grown plants (Suárez-López
et al., 2001
). In wild-type plants, CO mRNA levels showed
the expected pattern (Fig. 5D)
(Suárez-López et al.,
2001
). No significant difference in either the diurnal expression
pattern or the amplitude of expression of CO was observed in the
esd4 mutant.
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DISCUSSION |
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Pleiotropy of the esd4 mutation suggests ESD4 plays a broad
role in plant development
The esd4 mutation has pleiotropic effects on the architecture of
the shoot and on silique shape (Fig.
1). These pleiotropic effects of esd4 may be related to
the early flowering of the mutant, perhaps due to ectopic expression of
flowering time or floral meristem identity genes. Some of the previously
described early flowering mutants show pleiotropic effects
(Goodrich et al., 1997;
Soppe et al., 1999
;
Gomez-Mena et al., 2001
)
whereas others do not (Scott et al.,
1999
; Michaels and Amasino,
2001
). Moreover, transgenes causing ectopic expression of
flowering time genes can also result in developmental defects
(Kardailsky et al., 1999
;
Kobayashi et al., 1999
;
Onouchi et al., 2000
). Some of
the mutations causing late flowering largely suppressed the effects of
esd4 on flowering time, shoot determinacy and floral phyllotaxy, but
none of these mutations abolished the effect of esd4 on plant height
or silique shape. It is therefore unlikely that all the pleiotropic effects of
esd4 can be explained by altered expression of flowering time genes,
and therefore ESD4 probably plays a broader role in the regulation of
plant development.
Genetic interactions between esd4 and mutations causing late
flowering
Models for the genetic control of flowering time in Arabidopsis
propose that three independent genetic pathways promote flowering under long
photoperiods (see Introduction) (reviewed by
Araki, 2001;
Mouradov et al., 2002
;
Simpson and Dean, 2002
).
Double mutants carrying esd4 and mutations previously shown to affect
each of these three pathways were constructed to determine whether
esd4 promotes early flowering by acting through one or more of these
pathways. No true epistatic relationships were identified between
esd4 and mutations causing late flowering, although epistatic
relationships have previously been reported between Arabidopsis
mutations causing early and late flowering
(Yang et al., 1995
;
Soppe et al., 1999
). However,
mutations that affect the autonomous pathway, such as fca and
fve, had only weak effects on the esd4 phenotype under long
days. One interpretation of these results is that ESD4 acts within
the autonomous pathway downstream of FCA and FVE
(Fig. 6A). In this model FCA
and FVE promote flowering by repressing the activity of ESD4, as was proposed
previously for efs (Soppe et al.,
1999
).
|
Construction of double mutants demonstrated that under both long and short
days mutations in the long-day pathway (co, ft and fwa)
caused a severe delay in flowering of esd4 mutants in comparison to
the effect of autonomous pathway mutations. A similar effect was observed for
GA pathway mutations under long days. ESD4 could therefore act in the long-day
pathway before the CO, FT and FWA genes to reduce their
expression, or within the GA pathway to reduce GA signalling or synthesis.
This is consistent with previous observations that overexpression of the
long-day pathway gene CO from the 35S promoter causes early flowering
and largely suppresses the late-flowering caused by fca
(Onouchi et al., 2000), and
that mutations in suppressors of GA signalling cause early flowering
(Jacobsen and Olszewski,
1993
). However, no increase in CO mRNA abundance was
detected in an esd4 mutant, and these mutant plants are not resistant
to the GA biosynthesis inhibitor paclobutrazol (data not shown) nor do they
show elongated internodes, which are effects characteristic of mutations such
as spindly that cause GA-signal transduction independently of GA
(Jacobsen and Olszewski,
1993
).
An alternative explanation for the partial suppression of mutations in the
long-day and GA pathways by esd4 is that ESD4 acts
predominantly in pathways related to the autonomous pathway
(Fig. 6), and that increased
activity of these pathways in esd4 mutants can partially suppress the
effect of mutations in the long-day or GA pathways. The esd4 mutation
would then be proposed to activate the autonomous pathway downstream of
FCA and FVE, because of the early flowering of esd4
fca and fve esd4 double mutants, or would somehow bypass the
effect of these mutations on the autonomous pathway. The autonomous pathway
appears to facilitate the action of the long day and GA pathways, but on its
own to have only a weak floral promotion activity
(Nilsson et al., 1998;
Reeves and Coupland, 2001
).
Thus, if esd4 acts predominantly through the autonomous pathway
(Fig. 6A), then mutations
within other floral promotion pathways would be expected to reduce the
severity of the esd4 phenotype. This reduction in severity of
esd4 by mutations in the long-day and GA pathways did occur, but not
for ga1 under short days, suggesting that under these conditions
there may be a closer relationship between esd4 and the GA pathway.
Nevertheless, we propose that the effect of ESD4 on flowering time is most
closely associated with the autonomous pathway.
Response of esd4 mutants to daylength
The esd4 mutant flowers later under short than long days, and is
therefore still responsive to daylength. In wild-type plants the daylength
response is conferred by the long-day pathway. Therefore, esd4
mutants that are mainly affected in the autonomous pathway would be expected
to retain a response to daylength. Such an argument can also explain why
esd4 suppresses mutations that impair the autonomous pathway much
less effectively under short-day conditions, because the activity of the
long-day pathway would be reduced under these conditions and this would
further delay flowering of esd4 fca-1 plants. The esd4 co-2
double mutant is only slightly later flowering under short days compared to
long days, consistent with the long-day pathway not having a strongly
promotive effect on flowering under short days even in an esd4
mutant. The co mutation had a similarly weak effect on the flowering
time of the efs mutant under short days
(Soppe et al., 1999).
The role of ESD4 in the regulation of FLC and its
downstream targets
Mutations within the autonomous pathway cause an increase in the expression
of the floral repressor FLC (Michaels and Amasino, 1999;
Sheldon et al., 1999). To test
the genetic model of ESD4 function, the effect of esd4 on the
regulation of FLC mRNA was examined. FLC mRNA abundance was
reduced in esd4 compared to wild-type plants, and in esd4
fca-1 and esd4 fve-1 double mutants compared to the late
flowering mutants. This suggests that ESD4 may act to delay flowering
by increasing the abundance of FLC mRNA. However, FLC mRNA
levels are still higher in esd4 fca-1 double mutants than in
wild-type plants, although the esd4 fca-1 plants flowered earlier
than wild type. This indicates that ESD4 is unlikely to act solely
through FLC. However, we cannot rule out the possibility that
esd4 may reduce FLC mRNA abundance to a very low level in a
small subset of cells that are critical for the regulation of flowering or
that ESD4 may have an additional post-transcriptional effect on FLC protein.
Nevertheless, the observation that flc null mutants do not flower as
early as esd4 mutants under short days
(Michaels and Amasino, 2001
),
supports our view that the regulation of FLC is not the only role for
ESD4 in the regulation of flowering time.
Although the reduction in FLC mRNA levels is not the only cause of
the esd4 phenotype, it is likely to contribute to the early flowering
of esd4 mutants and the suppression of the late flowering phenotype
of autonomous pathway mutations. Therefore we examined the effect of
esd4 on FT and SOC1, two flowering time genes whose
expression levels are repressed by high levels of FLC (P.
Suárez-López and G. Coupland, unpublished results)
(Michaels and Amasino, 2001;
Ohto et al., 2001
). In
esd4 plants, an increase in FT and SOC1 mRNA levels
relative to wild-type was observed, suggesting that these genes may contribute
to the early flowering of esd4. esd4 partially suppressed the
reduction in FT and SOC1 mRNA caused by the fca-1
mutation, consistent with the partial reduction in FLC mRNA levels
observed in the esd4 fca-1 genotype. In esd4 fca-1 plants
the levels of FT and SOC1 were similar to those of wild-type
plants. This suggests that it is unlikely that the increase in FT and
SOC1 expression alone explains the effect of esd4 on
flowering time, as esd4 fca-1 plants flower earlier than wild-type
despite having similar levels of FT and SOC1. Furthermore,
it is unlikely that the effects on FT and SOC1 are mediated
solely through FLC as esd4 fca-1 have higher levels of
FLC mRNA compared to wild type. Thus, the genetic and molecular data
indicate that ESD4 has a role in increasing FLC, and
consequently decreasing FT and SOC1 expression, but also
that it has additional functions in the control of flowering.
Other genes have previously been shown to increase the level of
FLC. For example, the FRIGIDA gene, which is responsible for
the vernalization requirement of many naturally occurring winter varieties of
Arabidopsis promotes FLC expression
(Michaels and Amasino, 1999a;
Sheldon et al., 1999
;
Johanson et al., 2000
).
ESD4 may therefore act together with FRI to promote
FLC. However, esd4 was isolated in the Landsberg
erecta ecotype, which lacks an active FRI allele
(Johanson et al., 2000
).
Therefore, it is unlikely that the early flowering of esd4 is due to
effects on FRI. The vernalization response also acts through
FLC and mutations that impair vernalization cause increased
FLC mRNA levels (Chandler et al.,
1996
; Sheldon et al.,
1999
). Moreover, vernalization does not act solely through
FLC (Michaels and Amasino,
2001
). ESD4 may therefore act to antagonize the activity
of genes that are required to promote flowering in response to vernalization,
such as the VRN genes (Chandler et
al., 1996
; Gendall et al.,
2001
). In this case loss of esd4 would result in
upregulation of their activity, in effect resulting in esd4 behaving
as a constitutively vernalized mutant as has been proposed for the early
flowering hos1 mutant (Lee et
al., 2001
). However, vernalization appears more effective in
repressing FLC than loss of ESD4 function
(Michaels and Amasino, 1999a
;
Sheldon et al., 1999
). The
identification and cloning of additional genes that control the vernalization
response should enable the interaction of ESD4 with the vernalization
pathway to be examined in more detail.
A model for ESD4 in the control of flowering time
We propose that ESD4 is closely associated with the regulation of
FLC within the autonomous pathway. Mutations in ESD4 promote
flowering at least partly by decreasing FLC mRNA levels and thereby
increasing expression of downstream flowering-time genes such as FT
and SOC1. Increases in FT and SOC1 expression would
be expected to lead to earlier expression of floral meristem identity genes,
such as LEAFY and APETALA1
(Ruiz-Garcia et al., 1997;
Kardailsky et al., 1999
;
Kobayashi et al., 1999
).
However, the late flowering phenotype of autonomous pathway mutations such as
fca and fve is completely dependent on functional
FLC: fca and fve mutants do not flower any later
than wild type in a flc null mutant background
(Michaels and Amasino, 2001
),
whereas the early flowering of esd4 cannot be explained solely by its
effects on FLC. We therefore propose that ESD4 has an additional role
in a pathway that by-passes the requirement for the autonomous pathway
(Fig. 6B). In this model,
ESD4 has two roles. The first is to ensure high FLC
expression that leads to repression of flowering through repressing
FT and SOC1, and probably other genes (represented by X in
Fig. 6B). The second is to
regulate flowering-time genes independently of FLC (represented by Y in
Fig. 6B). This model is
consistent with the analysis of gene expression in esd4 mutants as
well as with the single and double mutant phenotypes under long and short
days.
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ACKNOWLEDGMENTS |
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Footnotes |
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