Plant Physiol. (1999) 120: 675-684
EAF1 Regulates Vegetative-Phase Change and
Flowering Time in Arabidopsis1
Derek B. Scott,
Wei Jin,
Heidi K. Ledford,
Hou-Sung Jung, and
Mary
A. Honma*
Developmental, Cell, and Molecular Biology Group/Department of
Botany, Duke University, Durham, North Carolina 27708-1000
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ABSTRACT |
We have identified a new locus that
regulates vegetative phase change and flowering time in Arabidopsis. An
early-flowering mutant, eaf1
(early
flowering
1) was isolated and characterized.
eaf1 plants flowered earlier than the wild type under
either short-day or long-day conditions, and showed a reduction in the
juvenile and adult vegetative phases. When grown under short-day
conditions, eaf1 plants were slightly pale green and had
elongated petioles, phenotypes that are observed in mutants altered in
either phytochrome or the gibberellin (GA) response.
eaf1 seed showed increased resistance to the GA
biosynthesis inhibitor paclobutrazol, suggesting that GA metabolism
and/or response had been altered. Comparison of eaf1 to
other early-flowering mutants revealed that eaf1 shifts
to the adult phase early and flowers early, similarly to the
phyB
(phytochrome
B) and spy
(spindly)
mutants. eaf1 maps to chromosome 2, but defines a locus
distinct from phyB, clf
(curly leaf),
and elf3
(early-flowering 3). These results demonstrate that
eaf1 defines a new locus involved in an autonomous
pathway and may affect GA regulation of flowering.
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INTRODUCTION |
The transition from vegetative to reproductive development is of
vital importance for the survival of virtually all flowering plants.
The vegetative phase of Arabidopsis can be subdivided into juvenile and
adult phases that can be distinguished by physiological and
morphological markers (Martinez-Zapater et al., 1995
; Chien and Sussex,
1996; Telfer et al., 1997). Juvenile leaves are small and round in
shape, with adaxial trichomes and no abaxial trichomes (Telfer et al.,
1997). As development continues, emerging adult leaves are more
elongated and lanceolate in shape, and trichomes begin to appear on the
abaxial surfaces (Chien and Sussex, 1996; Telfer et al., 1997).
Application of the hormone GA3 will induce abaxial trichomes on leaves where they are not normally present (Chien
and Sussex, 1996; Telfer et al., 1997), although the earliest arising
leaves do not respond to this induction. At the appropriate stage in
development and in response to specific cues, the shoot apical meristem
becomes reprogrammed and generates a reproductive shoot that carries
either an inflorescence or a single flower. Daylength is a key
regulator of flowering in many plant species, and in Arabidopsis
flowering is hastened under LD conditions (Koornneef et al., 1998).
Vernalization accelerates flowering in some Arabidopsis ecotypes, and
the shoot meristem itself is thought to be the site of perception of
the vernalization signal (Dennis et al., 1996; Wilson and Dean, 1996).
Genetic studies with Arabidopsis and pea
have identified a large number of genes that regulate flowering (Reid
et al., 1996; Koornneef et al., 1998). In Arabidopsis at least two
flowering pathways are thought to exist, a photoperiod-sensitive
pathway and an autonomous pathway. GA may function as part
of the autonomous pathway or could define a third pathway. Each of the
flowering pathways includes both activator and repressor genes, and
epistasis tests and physiological experiments have led to the idea that these pathways function in parallel. In Arabidopsis several genes that
serve as promoters of flowering have been recently cloned, and the
sequences of the proteins encoded as well as their expression patterns
have suggested possible functions for these genes. For example, the
genes CO (Putterill et al., 1995) and LD (Lee et al., 1994) both appear to encode transcription factors, whereas FCA possibly affects RNA metabolism (Macknight et al.,
1997), suggesting that a cascade of gene activation events likely
controls flowering.
Genes that function to repress the transition to adult and/or
reproductive phases have been identified in Arabidopsis (Koornneef et
al., 1998). elf3
(early-flowering 3) (Hicks et al., 1996; Zagotta et al., 1996)
and lhy (long
hypocotyl) (Schaffer et al., 1998)
are photoperiod-insensitive mutants that show alterations in circadian clock function and appear to be involved in the repression of flowering
in the LD pathway. The HST
(HASTY) gene is thought to promote a juvenile pattern of development, and
loss-of-function mutations in this gene result in early transition to
the adult phase and early flowering (Telfer and Poethig, 1998). Arabidopsis mutants defective in phytochrome synthesis (Reed et al.,
1994; Koornneef et al., 1995) or phytochrome function (Ahmad and
Cashmore, 1996) flower early, indicating that this light receptor is
involved in repression of floral initiation. At least some of the
effects of phytochrome may be mediated by GA, because recent studies
(Reed et al., 1996) show that the hypocotyl tissue of phyB
(phytochrome
B) mutant seedlings is more responsive to
exogenous GA than wild-type seedlings. The tfl
(terminal flower) mutant flowers early and
produces a determinate inflorescence, often generating only one or a
few terminal flowers.
Application of GA accelerates the onset of the adult phase, and induces
early flowering and the production of larger, slightly pale-green
leaves in wild-type Arabidopsis (Jacobsen and Olszewski, 1993; M. Honma, unpublished data). The GA-deficient mutant ga1 of
Arabidopsis flowers later when grown in LD and does not flower in SD
conditions, indicating that GA is required for the
photoperiod-insensitive (autonomous) flowering pathway (Wilson et al.,
1992). GA levels and response to the hormone are sensitive to changes
in photoperiod (Pharis et al., 1987; Zeevaart and Gage, 1993),
suggesting a role for GA in photoperiodic induction of flowering.
Suppressors of the ga1 mutant have been identified and shown
to suppress the late-flowering phenotype of ga1. These
suppressors known as spy (spindly)
(Jacobsen and Olszewski, 1993) and rga
(repressor of
ga1-3) (Silverstone et
al., 1997) are thought to have partially activated the GA response. spy flowers early, and spy mutant seed show
increased resistance to the GA biosynthesis inhibitor paclobutrazol.
The double mutant rga ga1 flowers earlier than
ga1 alone. The phenotypes of spy and
rga suggest that activating the GA response can cause
earlier flowering.
We have identified a new gene, eaf1
(early
flowering 1),
that appears to function in the autonomous pathway to repress the
transition from juvenile to adult development. eaf1 mutant
plants exhibit truncated juvenile and adult phases, resulting in early
flowering. eaf1 mutant plants exhibit phenotypes similar to
the GA response mutants spy and rga, and
eaf1 seed show increased resistance to paclobutrazol. Our
results are consistent with the notion that the eaf1 mutant
is altered in either GA biosynthesis or response to the hormone, and
that this change is responsible for the alteration in flowering time.
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MATERIALS AND METHODS |
Arabidopsis Seed Stocks
Seed stocks were obtained from the Arabidopsis Biological Resource
Center at Ohio State University, Columbus, or from individual researchers (Drs. D.R. Meeks-Wagner, J. Reed, G. Coupland, T.-p. Sun,
and R. Amasino). Acst and Ds
transgenic lines of ecotype Nossen used to generate the eaf1
mutant line were described previously (Honma et al., 1993). The
35S-Acst and
rbcS-Acst lines express the Ac
transposase under the control of either the cauliflower mosaic virus
35S or the Arabidopsis rbcS-1A promoters. The
DsALS construct carries the Arabidopsis
acetolactate synthase gene that encodes resistance to the herbicide
chlorsulfuron. The DsALS element resides
within the untranslated leader of a kanamycin resistance gene that
serves as marker for excision of Ds. The eaf1
mutant was originally identified in a mutant screen of a population
carrying transposed Ds elements (Honma et al., 1993; M. Honma, unpublished data). The eaf1 mutant line contained
three transposed Ds insertions: Ds-1,
Ds-2, and Ds-3.
Ds-1 and Ds-2 are tightly linked
to the eaf1 mutation, and most of the characterization described in this paper was done using a line that carried both of
these insertions.
Introgression of the eaf1 mutation into the Landsberg
background was accomplished by crossing eaf1 plants carrying
Ds-1 and Ds-2 to wild-type
Landsberg plants. Backcross progeny were screened using chlorsulfuron
to select for the presence of either Ds-1 or
Ds-2, both of which are linked to the
eaf1 mutation. These chlorsulfuron-resistant plants were
then backcrossed four more times to the Landsberg parent. No selection
for the early-flowering trait was done during the introgression, to
prevent selective maintenance of additional loci in the Nossen ecotype
that may condition earlier flowering. F1 plants
from the fifth backcross were self-fertilized and early-flowering
F2 progeny were identified.
Growth Conditions
Plants were grown either under sterile conditions on germination
medium (Valvekens et al., 1988) supplemented with appropriate antibiotics, as described previously (Honma et al., 1993), or in soil
under LD (16 h of light/8 h of dark) or SD (8 h of light/16 h of dark)
conditions at 18°C to 20°C during the dark period and at 20°C to
24°C during the light period. For most of the flowering-time experiments, seeds were either allowed to imbibe in water at 4°C for
4 d (in the dark or dim light) before transfer to soil or planted
directly in moist soil and cold treated. After the cold treatment,
seeds were transferred to growth rooms. Light intensity was 440 µE
m
2 s1 for SD and 240 µE m2 s1 for LD
conditions. Lighting was supplied by a 3:1 mixture of cool-white:Wide
Spectrum bulbs (General Electric). Plants were grown individually in
divided flats (Hummert, St. Louis, MO) at a density of 60 to 96 plants/1290 cm2 flat. In the screen used to
isolate the eaf1 mutant, seedlings were grown for 2 weeks on
germination medium plates, followed by transfer to soil at a density of
150 plants/1290 cm2 flat. Chlorsulfuron was a
gift from DuPont.
In the initial experiment to determine linkage of the Ds
elements to the early-flowering phenotype, seedlings were first grown on germination medium plates for 10 to 14 d before transfer to soil.
Plants were grown in a growth chamber (Conviron, Winnipeg, Manitoba,
Canada) with a mixture of cool-white and incandescent bulbs. The light
intensity was approximately 120 µE m2
s1 and the temperature was maintained at
22°C. In the GA experiment sterilized seeds were plated on
germination medium plates with or without
105 M
GA3 and allowed to imbibe at 4°C for 2 d
before growth under SD (220 µmol photons m2
s1) conditions in a growth chamber (model
CU32L, Percival Scientific, Boone, IA). After 11 d of growth the
seedlings were transferred to soil and grown under SD conditions, 440 µE m2 s1.
GA3 (100 µL of 105
M) was applied weekly to the base of each plant
and continued until the plants had flowered.
For vernalization treatment, seeds were allowed to imbibe by planting
in moist soil; they were grown at 4°C for 8 weeks under low-intensity
SD conditions (19 µE m2
s1) before transfer to standard SD conditions
(440 µE m2 s1,
22°C). Untreated control seeds were allowed to imbibe for 4 d at
4°C and transferred to the SD growth rooms on the same day as the
vernalized plants.
Morphological Analysis
Days to flowering was scored as the length of time between
germination and visible appearance of the first floral bud. The number
of rosette leaves was counted weekly, and cauline leaves were counted
after seed set. Juvenile-stage leaves were those true leaves present in
the rosette that lacked abaxial trichomes. The appearance of
abaxial trichomes was monitored using a stereomicroscope.
Hypocotyl elongation in response to red light was measured as described
previously by Nagatani et al. (1993).
Allelism Tests
eaf1 was crossed to the early-flowering mutants
clf (curly
leaf),
elf3, and hy3, and F1 and
F2 plants were scored for days to flowering and
leaf number under SD conditions. clf and
phyB(hy3) are alleles in the Landsberg ecotype
and elf3 is in the Columbia ecotype. The control crosses eaf1 × Landsberg, eaf1 × Columbia,
and Nossen × clf, elf3, or phyB(hy3) were included. Twenty to fifty plants
were scored in each experiment.
Molecular Analysis
DNA was isolated from leaf tissue using a modification of the
method described by Dellaporta et al. (1983). Southern blotting was by
reverse capillary transfer as described previously (Ausubel et al.,
1988) and Southern hybridizations were carried out according to the
method of Church and Gilbert (1984). DNA fragments used as probes were
a 1.5-kb fragment from the 5
end of Ac and the Ds-2 genomic flanking sequence (PCR product).
These probes were generated by random-prime labeling (Feinberg and
Vogelstein, 1983).
Genetic Mapping of Ds-2 Insertion and the
eaf1 Mutation
Inverse PCR was used to isolate genomic DNA sequences flanking the
Ds-2 insertion (Healy et al., 1993). Mapping of
this genomic sequence was done using recombinant inbred lines as
described previously by Osborne et al. (1995).
Wild-type Nossen, Columbia, and Landsberg ecotypes were compared using
restriction fragment-length polymorphism analysis to determine if the
region carrying eaf1 in Nossen showed polymorphisms with
respect to the Columbia or Landsberg ecotypes. Southern analysis with
restriction fragment-length polymorphism clones that map to the middle
of chromosome 2 (Lister and Dean, 1993) showed that the eaf1
region in Nossen appears to be polymorphic compared with the same
region in Columbia (data not shown). Thus, the Nossen × Columbia
cross was the most likely to yield polymorphisms that could be used for
mapping. A mapping population was constructed by crossing the
eaf1 mutant (Nossen background) carrying
Ds-1 and Ds-2 to Columbia
wild type, and the F1 plants were self-fertilized to generate F2 siblings. Tissue was collected for
molecular analysis from 596 early-flowering plants that arose from a
population of approximately 2500 F2 plants. The position of
the eaf1 mutation was determined using
cleaved-amplified polymorphic sequence markers positioned on chromosome
2 (Lister and Dean, 1993)
(http://genome-www.stanford.edu/Arabidopsis/ww/Aug98RImaps/index.html). Progeny from plants that showed recombination events between the eaf1 mutation and the marker tested were scored for their
flowering phenotype to confirm that they were homozygous for the
eaf1 mutation.
Germination Assays
Paclobutrazol resistance was determined as described previously
(Jacobsen and Olszewski, 1993) with minor modifications. For each
paclobutrazol treatment, 120 seed of each line were sterilized by
treatment with 0.1% Triton. After the seeds were rinsed with water,
they were washed with the respective paclobutrazol solutions and
allowed to imbibe in the same solutions for 4 d at 4°C. Seeds were suspended in a small volume of 0.1% agarose, plated on four stacked filter paper circles in small Petri dishes, and allowed to dry
(30 seeds/plate). Paclobutrazol (1.5 mL) in 0.01% Tween was applied to
the filter paper and the Petri dishes were sealed with parafilm and
incubated at 22°C for 7 d under a LD photoperiod. Germination
was scored under a stereomicroscope as emergence of the radicle.
Paclobutrazol was a gift from Zeneca (Wilmington, DE).
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RESULTS |
Isolation of an Early-Flowering Mutant,
eaf1
A mutant that flowered earlier than the wild type was identified
in a Ds-mutagenized population of plants of the Nossen
ecotype grown under LD conditions. This early-flowering mutant was
designated eaf1. The eaf1 mutant was
characterized with regard to flowering time, appearance, and overall
growth and development. Under LD conditions the mutant plants flowered
2 d before the wild type and generated three fewer leaves (Table
I). Under SD conditions the
early-flowering phenotype became much more extreme, and eaf1 flowered 20 d earlier with 27 fewer leaves than the wild type. The
eaf1 mutant flowered earlier under LD than under SD
conditions, indicating that it remains responsive
to changes in the photoperiod. This is in contrast to the phenotype of
other flowering-time mutants, elf3, co
(constans), and gi
(gigantea), that flower at the
same time under either SD or LD conditions and appear to be photoperiod
insensitive.
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Table I.
Days to flowering and rosette leaf no. of wild-type
Nossen and the eaf1 mutant in response to photoperiod
Days to flowering was measured as the no. of days from germination to
appearance of the floral bud. Leaf no. is the no. of rosette leaves
produced before flowering. Each value represents the mean ± 2 SE. Unless otherwise noted, plants from within each group
were significantly different from the wild-type (WT) controls (P < 0.05, Student's t test).
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Vernalization accelerates flowering in some Arabidopsis ecotypes
(Napp-Zinn, 1985), and it is possible that eaf1 has an
activated vernalization response. eaf1 and Nossen wild-type
plants were tested for a response to vernalization. Seedlings were
vernalized under SD conditions and scored for flowering time. As shown
in Table II, both Nossen wild type and
eaf1 respond to vernalization, flowering earlier with fewer
rosette leaves. This result suggests that response to vernalization has
not been altered in eaf1 or that this response has not been
saturated.
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Table II.
Days to flowering and rosette leaf no. of wild-type
Nossen and the eaf1 mutant in response to vernalization
Days to flowering was measured as the no. of days from germination to
appearance of the floral bud. Leaf no. is the no. of rosette leaves
produced before flowering. Each value represents the mean ± 2 SE. Unless otherwise noted, plants from within each group
were significantly different from the wild-type (WT) controls (P < 0.05, Student's t test). vern, Vernalization.
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eaf1 Is Recessive and Not Allelic to Other
Early-Flowering Mutants
Genetic analysis showed that eaf1 is a recessive
mutation. Homozygous eaf1 plants were crossed to wild-type
Nossen, and the resulting eaf1/+ F1
plants from two independent crosses (a and b) were scored for flowering
(Table III). Plants that were
heterozygous for eaf1 flowered similarly to wild-type
control plants, showing no statistical difference in terms of days to
flowering. Although the eaf1/+ plants flowered at the same
time as wild-type plants, the numbers of rosette leaves appeared to be
slightly reduced. One possibility is that the heterozygous plants have
a slightly reduced rate of leaf initiation, and if so, this would
indicate that the eaf1 mutation is not completely recessive
for this phenotype. Two F1 plants were
self-fertilized, and the F2 progeny were scored for flowering phenotype under SD conditions. The two
F2 populations segregated mutant:wild-type plants
in the ratios of 1:3, indicating that eaf1 is recessive and
that the early-flowering phenotype was due to a mutation at a single
locus (data not shown).
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Table III.
Days to flowering and rosette leaf no. of the eaf1
mutant in the Nossen background
Days to flowering was measured as the no. of days from germination to
appearance of the floral bud. Leaf no. is the no. of rosette leaves
produced before flowering. Each value represents the mean ± 2 SE. Unless otherwise noted, plants from within each group
were significantly different from the wild-type (WT) controls (P < 0.05, Student's t test).
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The original eaf1 mutant line carried three Ds
insertions, Ds-1, Ds-2, and
Ds-3. Backcross of eaf1 to wild-type
Nossen generated a set of lines carrying different combinations of the
Ds insertions. Lines that were hemizygous for
Ds-1 and Ds-2 or
Ds-2 and Ds-3 produced 25%
mutant progeny. Plants carrying only Ds-3
flowered similarly to the wild type. These results indicated that
Ds-2 is most closely linked to the
eaf1 mutation, with Ds-1 and
Ds-3 not responsible for causing the
early-flowering phenotype. The Ds-1 insertion is
approximately 2 centimorgans from eaf1, with the
Ds-3 insertion loosely linked on the same
chromosome (data not shown). The genomic sequence flanking
Ds-2 was isolated by inverse PCR (see
``Materials and Methods'') and used in Southern hybridization
experiments (data not shown). Analysis of 50 early-flowering
F2 progeny showed that all were homozygous for
the Ds-2 insertion, indicating that eaf1 was <1 centimorgan from Ds-2.
Two lines hemizygous for Ds-2 alone have been
identified; when self-fertilized, both lines produced mutant:wild-type
progeny in the ratio of 1:3 (18 mutant:60 wild type and 20 mutant:59
wild type), confirming that Ds-1 and
Ds-3 were not responsible for causing the
early-flowering phenotype and that the eaf1 mutation was
tightly linked to the Ds-2 insertion. However,
meiotic mapping experiments using cleaved-amplified polymorphic sequence markers have more precisely localized the
Ds-2 insertion site to 0.35 ± 0.2 centimorgans away from the eaf1 mutation; thus Ds-2 is tightly linked but not inserted into the
eaf1 gene (W. Jin and M. Honma, unpublished data). Moreover,
sequence analysis of the genomic region flanking
Ds-2 indicates that the element is not inserted
within an open reading frame (W. Jin and M. Honma, unpublished data).
Assignment of an initial map position to the eaf1 mutation
was determined with the aid of the Ds-2
insertion, which lies 0.35 centimorgans away. The genomic sequence
flanking Ds-2 was isolated by inverse PCR (see
``Materials and Methods'') and this genomic sequence was mapped using a recombinant inbred population (Lister and Dean, 1993). The
Ds-2 insertion resides on chromosome 2, near
mi238. A mapping population was generated (as described in ``Materials and Methods'') by crossing eaf1 to wild-type Columbia
plants, and the early-flowering phenotype was mapped using
cleaved-amplified polymorphic sequence markers located in the middle of
chromosome 2 (Lister and Dean, 1993)
(http://genome-www.stanford.edu/Arabidopsis/ww/Aug98RImaps/index.html). Our results placed the eaf1 mutation between
mi139 and mi238. The early-flowering mutations
phyB(hy3), clf, and elf3
map within this region, but at locations different from eaf1
(Fig. 1). eaf1 lies 1.9 centimorgans south of phyB (hy3) and is >1.7
centimorgans north of clf. elf3 is 6.6 centimorgans south of
eaf1, which is close to the marker GPA1 (Zagotta et
al., 1996; K.A. Hicks, T.M. Albertson, and D.R. Meeks-Wagner, personal
communication). To confirm that eaf1 was not allelic to
phyB (hy3), clf, or elf3, complementation tests (described in ``Materials and Methods'') were
done. eaf1 (in Nossen) was crossed to phyB
(hy3) (in Landsberg), clf (in Landsberg), elf3 (in Columbia), wild-type Landsberg, or wild-type
Columbia. phyB(hy3), clf, and
elf3 were each crossed to wild-type Nossen. All
F1 plants flowered at the same time, and after
self-fertilization they produced both early-flowering and wild-type
F2 progeny (data not shown). These results
demonstrate that eaf1 is not allelic to
phyB(hy3), clf, and elf3.
Therefore, eaf1 defines a new locus on chromosome 2 that
affects flowering time, in addition to the previously known
phyB(hy3), clf, and elf3
loci.
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| Figure 1.
Map location of eaf1 and other
early-flowering mutants on chromosome 2. Distance shown with a bracket
indicates number of centimorgans between the marker and the
early-flowering phenotype. Distances shown with arrows were derived
from the recombinant inbred lines (Lister and Dean, 1995).
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eaf1 Regulates Vegetative-Phase Transition
eaf1 mutant and wild-type plants were analyzed for the
appearance of abaxial trichomes, a marker associated with the shift from the juvenile to the adult phase (Chien and Sussex, 1996; Telfer et
al., 1997). The first rosette leaf bearing abaxial trichomes is counted
as the first adult leaf. The number of juvenile, adult, and
reproductive (cauline) leaves was determined, and the results are
presented in Figure 2. Abaxial trichomes
first appeared on leaf 8 in mutant plants as compared with leaf 13 in
wild-type plants grown under SD
conditions. Under LD
conditions abaxial trichomes appeared on leaf 5 in mutant plants and
leaf 6 in wild-type plants. These results show that the juvenile phase
has been shortened in the mutant plants when grown in either SD or LD
conditions. The adult phase in the mutant was also affected, because
only nine adult leaves with abaxial trichomes were produced when grown under SD, as compared with 30 adult leaves in the wild type.
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| Figure 2.
Effect of daylength on number and type of leaves
produced in wild-type Nossen and eaf1 mutant plants (A)
grown under LD or SD conditions. Wild-type Landsberg and
tfl, spy,
phyB(hy3), and eaf1 mutant
plants all in the Landsberg background grown under LD (B) or SD (C)
conditions. Twenty to fifty plants of each line were tested and error
bars represent ±2 SE.
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eaf1 Mutant Plants Resemble Phytochrome and GA Signal
Transduction Mutants
eaf1 mutant plants had elongated petioles, were lighter
green compared with wild-type plants (Fig.
3), and produced hypocotyls that were
approximately 20% longer than the wild type (data not shown). The
elongated petiole and pale-green phenotype has also been observed with
other early-flowering mutants, phyB, spy, and elf3 (Jacobsen and Olszewski, 1993; Reed et al., 1993;
Zagotta et al., 1996) and the late-flowering mutant lhy
(Schaffer et al., 1998). In addition, elf3 produces long
hypocotyls when grown under SD conditions (Zagotta et al., 1992), and
phyB and lhy mutant seedlings produce long
hypocotyls when grown under LD conditions (Koornneef et al., 1980;
Schaffer et al., 1998).
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| Figure 3.
Wild-type and eaf1 mutant plants
(Landsberg ecotype) grown under SD conditions for approximately 8 weeks. eaf1 plants had flowered 3 weeks before this
photograph was taken and wild-type plants had not yet flowered.
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Although we had previously shown that eaf1 was not allelic
to phyB, it is still possible that eaf1 is
defective in some other phytochrome or phytochrome-related process.
eaf1 mutant and Nossen wild-type seedlings were tested for
inhibition of hypocotyl elongation in response to red light, a
phytochrome-mediated response. Both wild-type and eaf1
mutant seedlings responded by producing shorter hypocotyls at higher
fluences of red light, indicating that the eaf1 mutation was
not affected in this aspect of phytochrome function. (M. Honma and J. Reed, unpublished data).
GA has long been known to be involved in flowering, and application of
GA accelerates the transition to the adult phase and flowering in
Arabidopsis (Jacobsen and Olszewski, 1993). Some of the phenotypic
characteristics of eaf1 mutant plants, such as early
flowering, pale color, and elongated petioles, are similar to
GA-treated plants or mutants altered in GA response (Jacobsen and
Olszewski, 1993). Therefore, GA levels might be elevated or GA response
may have been increased in the eaf1 mutant. To test whether
eaf1 is able to respond to exogenous GA, an experiment was
performed by applying GA3 and measuring flowering
time. If the GA signal transduction pathway has been activated such
that its response is saturated, then increased levels of exogenous GA
will not have an effect. Our results showed that eaf1 was
still able to respond to applied GA, and treated plants exhibited more elongated petioles, were paler green (data not shown), and were earlier
flowering (Table IV). This suggests that,
even if GA synthesis or signaling has been activated, the response to
the hormone had not been saturated.
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Table IV.
Days to flowering and rosette leaf no. of wild-type
Nossen and the eaf1 mutant in response to GA
Days to flowering was measured as the no. of days from germination to
appearance of the floral bud. Leaf no. is the no. of rosette leaves
produced before flowering. Each value represents the mean ± 2 SE. Unless otherwise noted, plants from within each group
were significantly different from the wild-type controls (P < 0.05, Student's t test). ND, Not determined.
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Quantitative measurements of response to GA or GA inhibitors on
soil-grown plants are extremely difficult. To address the question of
whether eaf1 is altered in some aspect of GA function, seed
germination in response to paclobutrazol was examined. Germination requires GA, and paclobutrazol interferes with GA biosynthesis such
that germination of wild-type seeds is inhibited. Germination of
wild-type Nossen and eaf1 mutant seeds was measured in the presence of varying levels of paclobutrazol (Fig.
4). eaf1 showed increased
resistance to paclobutrazol compared with wild-type Nossen. Between 0 and 3 µM paclobutrazol, both the wild type and eaf1 showed similar high levels of germination. Germination
of wild-type Nossen dropped to below 75% on 10 µM paclobutrazol, whereas eaf1
germination remained at 95%. Increasing levels of paclobutrazol showed
a decreasing level of germination for the wild type, with only 15%
germination at 300 µM. In contrast, germination of eaf1 seed remained high at 100 µM, and even at 300 µM
paclobutrazol, 35% of the seed still germinated. Thus, the
eaf1 mutant shows increased resistance to paclobutrazol,
similarly to the spy mutant (Jacobsen and Olszewski, 1993).
This resistance could be the result of elevated levels of GA, such that
higher levels of paclobutrazol are required to have an effect, or the
result of an altered response to GA. The pale pigmentation,
early-flowering, elongated petiole, and paclobutrazol-resistant
phenotypes of eaf1 mutant plants are all consistent with the
notion that GA levels or responses have been altered.
View larger version (27K):
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| Figure 4.
Response of eaf1 to paclobutrazol.
Percentage germination of Nossen wild-type and eaf1
mutant seed in the presence of exogenous paclobutrazol. Open bars
represent eaf1; striped bars represent wild type. Error
bars represent ±1 SE.
|
|
Comparison of eaf1 and Other Early-Flowering Mutants
To determine whether all early-flowering mutants show premature
appearance of abaxial trichomes, flowering time and abaxial trichome
formation were examined in a variety of early-flowering mutants grown
under different photoperiods (Table V;
Fig. 2). eaf1 was compared with the early-flowering mutants
phyB(hy3), spy, and tfl, of
which alleles exist in the Landsberg ecotype. Because wild-type
Landsberg and Nossen differ in flowering time and other
characteristics, the eaf1 mutation was introgressed into the
Landsberg background for this comparison. Plants of each mutant line
were grown in either LD or SD conditions, and the appearance of abaxial trichomes and time of floral bud emergence were
noted. Two experiments were done and similar results were obtained.
View this table:
[in this window]
[in a new window]
|
Table V.
Days to flowering and rosette leaf no. of wild-type
Landsberg and various early-flowering mutants
Days to flowering was measured as the no. of days from germination to
appearance of the floral bud. Leaf no. is the no. of rosette leaves
produced before flowering. Each value represents the mean ± 2 SE. Unless otherwise noted, plants from within each group
were significantly different from the wild-type controls (P < 0.05, Student's t test).
|
|
One set of experiments is shown in Table V and Figure 2. In the
Landsberg background, eaf1 exhibited a shorter juvenile
phase and flowered early in LD conditions. spy also produced
fewer juvenile leaves and both eaf1 and spy
produced the normal number of adult leaves.
phyB(hy3) and tfl had slightly
shortened juvenile phases, had markedly reduced adult phases, and
flowered early. The phenotype of tfl1-2 in the LD
condition is similar to what has been previously reported for Columbia
alleles of tfl (Telfer and Poethig, 1998). When grown under
SD conditions, tfl appeared to be very
similar to wild-type Landsberg. The reason for no observable phenotype in the SD condition is not clear, but alleles of tfl in the
Landsberg ecotype have been previously reported to show a milder
phenotype than those in the Columbia ecotype (Alvarez et al., 1992).
Perhaps the light intensity, light quality, or temperatures used in our experiments were unable to reveal such a phenotype.
phyB(hy3) flowered early under SD
conditions, but the length of the adult phase
appeared to be less affected than either spy or
eaf1. When grown under the LD condition, the adult phase of
phyB(hy3) appeared to have been shortened
compared with the wild type. spy and eaf1 behaved
very similarly when grown in SD conditions, with reduced juvenile and
greatly reduced adult phases and early flowering. Thus, by comparison,
eaf1 is similar phenotypically to spy under both
photoperiod regimes tested.
| |
DISCUSSION |
The EAF1 gene that controls vegetative-phase change and
flowering time has been identified by mutational analysis and shown to
reside on chromosome 2. Both the juvenile and adult phases of
eaf1 mutant plants are shortened, resulting in an early
transition to reproductive development. eaf1 appears
primarily to affect the length of the adult phase, with a less dramatic
alteration of the juvenile phase. The eaf1 allele behaves as
a recessive mutation, and if this mutation is due to loss-of-function
of the eaf1 gene, then the EAF1 gene product may
function to repress flowering by delaying adult development.
Alternatively, eaf1 could be a recessive neomorph, in which
case the wildtype product may not normally function to regulate
flowering. Additional alleles of the eaf1 gene will provide
important information regarding the role of EAF1 in
control of flowering.
Seed of eaf1 show increased resistance to paclobutrazol
compared with Nossen wild type, a phenotype also seen with the
spy mutant. Increased resistance to paclobutrazol suggests
that eaf1 is involved in regulation of GA levels or response
to the hormone. An increase in bioactive GAs could be the result of
increased biosynthesis, decreased catabolism or inactivation, or loss
of feedback regulation on the biosynthetic pathway (Chiang et al., 1995). Elongation of the inflorescence stem (bolt) after flowering is a
GA-regulated process, and paclobutrazol treatment will inhibit elongation. Preliminary results indicate that eaf1 plants
are more resistant to paclobutrazol than wild-type Nossen in terms of
bolt elongation, demonstrating that early developmental stages such as
germination and late stages such as bolting are both altered in
eaf1. These results support the idea that alteration in GA metabolism or signaling in eaf1 is responsible for the
early-flowering phenotype. Levels of paclobutrazol resistance observed
with the Nossen wild type are higher than has been seen previously with wild-type seeds of Landsberg or Columbia ecotypes (D. Scott and M. Honma, unpublished data). Thus, the Nossen ecotype may produce more
bioactive GA than other ecotypes or have an altered response to the
hormone. Plants of the Nossen ecotype have leaves that are paler green
with longer petioles than Landsberg or Columbia plants, phenotypes
consistent with increased GA levels or GA signaling. Identification of
loci that differ between Nossen and Landsberg responsible for
resistance to paclobutrazol is currently in progress.
GA is known to promote vegetative-phase change and flowering in a
variety of plants, including Arabidopsis (Chien and Sussex, 1996;
Telfer et al., 1997). The SPY, RGA, and
GAI genes are negative regulators of GA signaling (Jacobsen
and Olszewski, 1993; Peng et al., 1997; Silverstone et al., 1997), and
loss-of-function alleles exhibit phenotypes indicating that these genes
act downstream of GA biosynthesis. The spy mutant of
Arabidopsis, which is altered in response to GA, exhibits phenotypic
modifications similar to GA-treated plants and is able to suppress most
of the ga1 phenotypic changes, including reduced
germination. Although eaf1 does possess some phenotypic
alterations in common with spy, it does not show increased
height or reduced fertility. However, because only one allele of
eaf1 is currently available, other mutant alleles may have
more severe phenotypes or eaf1 may regulate a different
subset of GA-controlled functions than spy. Both
rga and spy are able to suppress the
late-flowering defect of ga1 and accelerate the production
of adult leaves (Silverstone et al., 1997). It is thought that
rga functions downstream of GA biosynthesis, in a pathway independent of spy (Silverstone et al., 1997). Preliminary
characterization of an eaf1 ga1 double-mutant line suggests
that eaf1 is not able to suppress the germination defect of
ga1, similar to rga and gai mutations
(H. Ledford and M. Honma, unpublished data). If eaf1 is also
involved in GA response, this mutation will likely be able to suppress
the late-flowering alteration of ga1. Thus, we would expect
that ga1 eaf1 would flower earlier than ga1 under LD conditions. GAI also functions as a negative regulator of
GA response, and GA can release this repression (Peng et al., 1997). Recently, RGA and GAI have been shown to encode
proteins with similar amino acid sequences, suggesting that they may
have redundant functions in GA signaling (Peng et al., 1997;
Silverstone et al., 1998).
The role of GAs in promotion of flowering could be the consequence of
early transition to the adult phase, which then hastens transition to
reproductive development. Alternatively, regulation of transition to
the adult phase might be independent of transition to the reproductive
phase, but with components in common, one of which may be GAs. GAs
could contribute to generate a signal that promotes phase transition or
may function in making the meristem more competent to respond to such
factors. Thus, GAs may act by causing developmental changes that
eventually result in early flowering, rather than acting directly as a
floral inducer. Recently it was shown that expression of the floral
meristem identity gene LEAFY is regulated in response to GA
(Blázquez et al., 1998). Construction of double-mutant lines altered
in eaf1 and the GA and phytochrome genes will allow study of
genetic interactions between these genes. Such experiments are in
progress and will indicate whether eaf1 functions within the
GA or photoperiod pathways.
Flowering-time mutants can be grouped into classes based on duration of
juvenile and adult phases. To compare eaf1 with other early-flowering mutants, it was introgressed into the Landsberg background and the duration of juvenile and adult phases and flowering time of all genotypes compared. eaf1 appears to be most
similar to the spy mutant, which shows reduced juvenile and
adult phases. In contrast, the hst gene (Telfer and Poethig,
1998) appears to have a primary role during the juvenile phase.
hst mutants exhibit a shortened juvenile phase and a
normal-length adult phase, flower earlier than wild-type plants, and
appear to be pleiotropic (Telfer and Poethig, 1998). When grown under
LD conditions, we find that tfl mutant plants have a
slightly shorter juvenile phase, a greatly shortened adult phase, and
flower early, as has been previously reported (Shannon and
Meeks-Wagner, 1991). The existence of mutants that primarily affect one
phase but not the other would suggest that flowering and phase
transition are separate processes, which may share common regulatory
factors. eaf1 defines a new locus in Arabidopsis that
represses the shift to the adult phase. Future studies of
eaf1 in conjunction with GA response and flowering-time genes will further our understanding of the complex interactions that
control vegetative-phase change and reproductive development.
| |
FOOTNOTES |
1
This work was supported by grants from the
American Cancer Society (no. JFRA-607), the National Science Foundation
(no. IBN-9509229), and the North Carolina Biotechnology Center (no.
9513 ARG-0039) to M.A.H.
*
Corresponding author; e-mail honma{at}acpub.duke.edu; fax
1-919-613-8177.
Received November 30, 1998;
accepted April 2, 1999.
| |
ABBREVIATIONS |
Abbreviations:
LD, long-day.
SD, short-day.
| |
ACKNOWLEDGMENTS |
We thank Dr. J. Reed (University of North Carolina, Chapel Hill)
for collaboration on the hypocotyl elongation experiment, Dr. B. Osborne and C. Corr (Plant Gene Expression Center [PGEC]-U.S. Department of Agriculture [USDA], Albany, CA) for mapping the Ds-2 insertion, Dr. M. Anderson (Nottingham
Arabidopsis Stock Centre) for assistance with mapping of
phyB, and Dr. C. Waddell (PGEC-USDA) for collaborating on
the initial Ac/Ds mutant screen. M.H. gratefully
acknowledges support and encouragement from Dr. B. Baker (PGEC-USDA)
during the early stages of this work. We enjoyed interesting
discussions on GA with Drs. T.-P. Sun, S. Yamaguchi, and A. Silverstone; and Drs. T-P. Sun, X. Dong, B. Kohorn, J. Boynton, and J. Siedow provided critical comments on the manuscript.
| |
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Abstract
Introduction
