|
Plant Physiol. (1999) 120: 1025-1032
Independent Regulation of Flowering by Phytochrome B and
Gibberellins in Arabidopsis1
Miguel A. Blázquez and
Detlef Weigel*
Plant Biology Laboratory, The Salk Institute for Biological
Studies, 10010 North Torrey Pines Road, La Jolla, California 92037
 |
ABSTRACT |
Phytochromes and gibberellins (GAs)
coordinately regulate multiple aspects of Arabidopsis development.
Phytochrome B (PHYB) promotes seed germination by increasing GA
biosynthesis, but inhibits hypocotyl elongation by decreasing the
responsiveness to GAs. Later in the life cycle of the plant, PHYB and
GAs have opposite effects on flowering. PHYB delays flowering, while
GAs promote flowering, particularly under noninductive photoperiods. To
learn how PHYB and GAs interact in the control of flowering, we have analyzed the effect of a phyB mutation on flowering time
and on the expression of the floral meristem-identity gene
LFY
(LEAFY). We show that the early flowering caused by phyB
correlated with an increase in LFY expression, which
complements our previous finding that GAs are required for activation
of LFY under noninductive photoperiods (M.A.
Blázquez, R. Green, O. Nilsson, M.R. Sussman, D. Weigel [1998]
Plant Cell 10: 791 800). Since phyB did not change the
GA responsiveness of the LFY promoter and suppressed the
lack of flowering of severe GA-deficient mutants under short days, we
propose that PHYB modulates flowering time at least partially through a
GA-independent pathway. Interestingly, the effects of PHYB on flowering
do not seem to be mediated by transcriptional up-regulation of genes
such as CO (CONSTANS) and
FT (Flowering locus
T), which are known to mediate the effects
of the photoperiod-dependent floral-induction pathway.
| |
INTRODUCTION |
The control of plant development by light is exerted through the
activity of photoreceptors. Among these, phytochromes mediate the
responses to red and far-red light (Fankhauser and Chory, 1997 ). In
Arabidopsis, phytochromes are encoded by five different genes,
PHYA through PHYE (Sharrock and Quail, 1989;
Quail et al., 1995). While each phytochrome seems to have specific
roles, there is also considerable overlap in the function of individual
phytochromes (Reed et al., 1994; Aukerman et al., 1997; Devlin et al.,
1998). For example, PHYA seems to have a primary role in germination and in the regulation of seedling morphogenesis by far-red light (Nagatani et al., 1993; Parks and Quail, 1993; Whitelam et al., 1993).
Later in development, PHYA is involved in sensing photoperiod, which is
reflected in the insensitivity of phyA mutants to night breaks (Reed et al., 1994). PHYB, on the other hand, is an essential component of the shade-avoidance mechanism, and modulates the expression of genes in response to red light. Null mutations in PHYB cause increased elongation of hypocotyls, leaf
petioles, and stems, as well as decreased chlorophyll accumulation and
earlier flowering under both long and short photoperiods (Reed et al., 1993).
Phytochromes interact with plant hormones of the GA class to regulate
certain aspects of plant development (Chory and Li, 1997). The
phenotype of mutants defective in GA biosynthesis has confirmed that
GAs regulate processes such as seed germination, cell expansion, and
flowering, all of which are also under the control of phytochromes. For
example, the Arabidopsis spy (spindly) mutant has
a slender stature, is pale green, and flowers early, thus resembling
phyB mutants, or wild-type plants treated with GA3 (Jacobsen and Olszewski, 1993). An opposite
phenotype is seen in GA-deficient mutants of Arabidopsis that are
dark-green dwarves that flower late (Koornneef and van der Veen, 1980).
This phenotype is particularly severe in the
ga1-3 mutant, which is blocked in a very early
step of GA biosynthesis, and never flowers under short days (Wilson et
al., 1992). Reduced expression of LFY (LEAFY), a
floral meristem-identity gene, seems to be a main cause of the flowering defect in ga1-3 mutants, since
constitutive expression of LFY from a transgene is
sufficient to restore flowering of ga1-3 mutants
under short days (Blázquez et al., 1998).
There are at least two possible mechanisms by which phytochromes and
GAs may interact. The finding of elevated GA levels in the
phyB-deficient mutants ein of Brassica
rapa and ma3R of
sorghum has suggested that PHYB regulates GA biosynthesis in certain
species (Rood et al., 1990; Beall et al., 1991). However, the
relationship between GA biosynthesis and PHYB activity in sorghum is
complex, as GA biosynthesis follows a circadian regulation, and the
ma3R mutant shows a phase
shift in GA accumulation (Lee et al., 1998). An interaction between
phytochromes and GA biosynthesis is also supported by the finding that
GA-biosynthetic genes in Arabidopsis and spinach are regulated by
light. In both species, GA 20-oxidase mRNA levels are higher under long
than under short photoperiods (Xu et al., 1995; Wu et al., 1996). It
has been suggested that GA 20-oxidase activity is limiting for stem
elongation in long days, and that long photoperiods raise the
concentration of active GAs above a certain threshold (Talón et
al., 1991).
Similarly, GA 3 -hydroxylation is promoted by pulses of far-red light
in cowpea (Martínez-García and
García-Martínez, 1992) and by red light in Arabidopsis
seeds (Yamaguchi et al., 1998), indicating that the synthesis of active
GA species is under phytochrome control. In different situations,
however, phytochromes have been shown to affect the responsiveness to
GAs, rather than their biosynthesis. Putative phyB mutants
of pea and cucumber, lv and lh, as well as the
phyB mutant of Arabidopsis have wild-type levels of
endogenous GAs but show enhanced hypocotyl elongation in response to
exogenous GAs (Weller et al., 1994; López-Juez et al., 1995; Reed
et al., 1996). At least in Arabidopsis, PHYB thus appears to control
GA-dependent hypocotyl elongation by modulating GA sensitivity as
opposed to regulating GA biosynthesis.
In this study, we have addressed the question of whether PHYB and GAs
interact in the regulation of flowering in a way similar to what is
observed for other responses, such as hypocotyl elongation, in
Arabidopsis. Using promoter activity of the floral regulator LFY as an indicator, we show that PHYB and GAs regulate
LFY expression independently. This finding is corroborated
by the observation that the loss of PHYB function allowed GA-deficient
mutants to flower under short days.
| |
MATERIALS AND METHODS |
Plant Material
LFY::GUS lines (DW150-304 and 304G1) in the
Landsberg erecta background of Arabidopsis have been
previously described (Blázquez et al., 1997, 1998; Hempel et al.,
1997). Lines 304B5 (phyB-5 LFY::GUS) and 304G1B5
(ga1-3 phyB-5 LFY::GUS) were
constructed by crossing line 304G1 (ga1-3
LFY::GUS) to plants carrying the phyB-5 mutations, a null allele in the Landsberg
erecta background (Reed et al., 1993). Transgenic plants
homozygous for ga1-3 were initially identified by
their short stature and dark-green color, and were then confirmed by
PCR (Silverstone et al., 1997). The presence of the
phyB-5 allele was also monitored by PCR using a
dCAPS marker (Neff et al., 1998). Lines homozygous for the transgene were identified by testing F3 progeny.
Growth Conditions
For experiments on soil, seeds were stratified for 2 to 3 d
at 4°C before sowing. Plants were grown at 23°C in long (16 h of
light and 8 h of dark) or short days (9 h of light and 15 h of dark) under a mixture of 3:1 cool-white and Gro-Lux fluorescent lights (Osram Sylvania, Danvers, MA). The spectral quality of the light
received by the plants under these conditions was determined with a
portable spectroradiometer (model LI-1800, LI-COR) and is shown in
Figure 1. ga1-3
mutants required exogenous GAs to germinate (Koornneef and van der
Veen, 1980) and were incubated with 50 µM
GA3 (Sigma) during stratification. Seeds were
rinsed thoroughly with water before sowing. Vegetatively growing plants were sprayed twice weekly with a solution of 100 µM GA3 and 0.02% (v/v) Tween 20 (Bio-Rad).
View larger version (24K):
[in this window]
[in a new window]
| Figure 1.
Light spectrum inside the growth chambers used in
this work. The solid black line represents the spectrum provided by a
3:1 mixture of cool-white to fluorescent light, while the dotted line
represents the spectrum provided by cool-white light alone.
|
|
The dose-response experiments with GA3 or
paclobutrazol (Zeneca Ag Products, Wilmington, DE) were performed with
seedlings growing on MS plates (Murashige and Skoog, 1962) without
Suc under the light conditions described above. The fluence rate was
around 66 µmol m 2 s1.
In experiments with soil-grown plants, paclobutrazol was applied by
watering with a 37 mg/L solution.
Hypocotyl and GUS Activity Measurements
Hypocotyls were measured using a digitized image of 12 to 18 seedlings that had been placed between transparent acetate sheets (Neff
and Chory, 1998). The image was analyzed with the public domain NIH
Image program (developed at the United States National Institutes of
Health and available on the Internet at
http://rsb.info.nih.gov/nih-image). For quantitative measurements of
GUS activity using
4-methylumbelliferyl--D-glucopyranoside as a substrate,
samples of plants grown on soil or MS plates were collected and treated
as previously described (Blázquez et al., 1997).
RNA Extraction and Analysis
Total RNA was extracted with TRIzol reagent as indicated by the
manufacturer (GIBCO-BRL). RT-PCR was conducted on 1 µg of total RNA.
cDNA synthesis was performed with a reverse transcription kit
(Promega). A fragment of the CO (CONSTANS)
gene was amplified using oligonucleotides 5 -ACG CCA TCA GCG
AGT TCC-3 and 5-AAA TGT ATG CGT TAT GGT TAA TGG-3 as primers (P. Reeves and G. Coupland, personal communication). FT was
amplified using 5-ACT ATA TAG GCA TCA TCA CCG TTC GTT ACT CG-3 and
5-ACA ACT GGA ACA ACC TTT GGC AAT G-3 (J.H. Ahn and D. Weigel,
unpublished data). As a control we used oligos 5-GAT CTT TGC CGG AAA
ACA ATT GGA GGA TGG T-3 and 5-CGA CTT GTC ATT AGA AAG AAA GAG ATA ACA
GG-3, which amplify two polyubiquitin gene fragments in the Landsberg erecta ecotype (Callis et al., 1995). The amplified
fragments were separated on an agarose gel, blotted onto a membrane,
and hybridized with radiolabeled CO, FT, and
UBQ10 probes. Signal intensities were determined with a
phosphor imager (Molecular Dynamics), and the values in the exponential
range of amplification were compared.
| |
RESULTS |
Enhanced LFY Up-Regulation in phyB Mutants
Mutations at the PHYB locus cause early flowering,
especially under short photoperiods (Goto et al., 1991; Reed et al.,
1993) (Table I). Since several other
mutations that affect flowering time also change the expression level
of LFY (Blázquez et al., 1998; Nilsson et al., 1998),
we investigated the effect of the phyB-5 null
mutation on the activity of the LFY promoter using a fusion
of the LFY promoter to the GUS reporter, which
faithfully reflects endogenous LFY expression
(Blázquez et al., 1997). Plants homozygous for the
phyB-5 mutation and a
LFY::GUS transgene (304B5) and isogenic
PHYB+ plants (DW150-304) were grown on soil
under long and short photoperiods, and GUS activity in the apices was
determined at different ages during the vegetative phase. As shown in
Figure 2, the
phyB-5 mutation caused an increase in the
expression of LFY::GUS under both photoperiods,
although this effect was more pronounced under short days. This result
paralleled the acceleration of flowering observed in these plants
(Table I), and indicates that PHYB represses LFY expression.
View this table:
[in this window]
[in a new window]
|
Table I.
Flowering time of the ga1-3 and phyB-5 lines used in
this study
All lines are in the Landsberg erecta background and
homozygous for a LFY::GUS transgene. RL, Rosette
leaves; CL, cauline leaves; TL, total number of leaves. Values are the
means ± 2 SE (i.e. with a 95% confidence interval).
n  12 plants.
|
|
View larger version (19K):
[in this window]
[in a new window]
| Figure 2.
LFY::GUS expression during vegetative
growth of phyB-5 mutants. Plants
homozygous for the LFY::GUS transgene either
in a PHYB+ (DW150-304; white symbols) or
phyB-5 background (307B5; black symbols)
were grown in long days (squares) or short days (circles) until flower
buds were visible to the naked eye. Values are expressed as means ± 2 SE (n = 12). Time represents days
after sowing. Error bars that are not visible are contained within the
symbol. MUG, 4-Methylumbelliferyl--D-glucopyranoside.
|
|
Application of GA3 from germination on
accelerated flowering of wild-type plants, but not of
phyB-5 mutants under short days (Table I).
Although the number of rosette leaves was lower in GA3-treated phyB-5 plants
than in untreated plants, the total number of leaves, including cauline
leaves, was not significantly different between these populations
(Table I). Consistent with the absence of an effect on flowering time,
we observed no further increase in LFY::GUS
expression when phyB-5 plants were treated with
GA3 (results not shown).
Suppression of the Flowering Defect of ga1 Mutants
by phyB
The increased expression level of LFY::GUS in
phyB-5 mutants resembles what is seen upon
application of GAs to wild-type plants (Blázquez et al., 1997) or
in mutants with enhanced GA-signaling, such as spy
(Blázquez et al., 1998). Since several other effects of PHYB
signaling, such as hypocotyl elongation and seed germination, appear to
be mediated by GAs, we wanted to know whether the early flowering of
phyB mutants depended on the activity of GAs. Therefore, we
constructed ga1-3 phyB-5 mutant plants
and cultivated them under long and short photoperiods. Under long days,
the double mutants were similar in size to ga1-3
plants, and much smaller than phyB-5 or wild-type
plants, as previously described (Peng and Harberd, 1997). While
flowering time of ga1-3 phyB-5 mutants under long days was not different from that of
ga1-3 plants, the phyB-5
mutation suppressed the flowering defect of ga1-3
mutants under short days (Table I; Fig.
3). When plants were grown in a mixture
of fluorescent and cool-white light, suppression was observed in over
90% of the double mutants after 7 weeks. Among these plants, the
number of leaves produced before flowering did not deviate much from
the mean, and the value was intermediate between that of wild-type and
phyB-5 plants (Table I).
View larger version (81K):
[in this window]
[in a new window]
| Figure 3.
Suppression of the
ga1-3 flowering defect in short days by
phyB-5. Representative plants were
photographed 50 d after sowing. Arrowhead indicates flowers.
|
|
Decreased LFY expression appears to be a main cause of the
late-flowering phenotype of ga1 mutants under long days, and
of the inability of ga1-3 mutants to flower at
all under short days (Blázquez et al., 1998). To determine
whether phyB suppressed the ga1 mutant flowering
defect by restoring more normal levels of LFY promoter
activity, we examined LFY::GUS expression in
ga1-3 phyB-5 double mutants. As shown
in Figure 4A,
LFY::GUS expression under long days followed a
similar pattern in both ga1-3 and
ga1-3 phyB-5 mutants. The expression
levels were very low during the first 15 d of growth, and although
LFY::GUS expression eventually increased, it never
reached the levels seen in wild-type plants. Application of
GA3 restored the expression pattern seen in
wild-type plants and phyB-5 single mutants. In
contrast to long days, LFY::GUS expression
remained very low in short-day-grown ga1-3 single
mutants during the entire experimental period (Fig. 4B) (Blázquez
et al., 1998). Although LFY::GUS expression in
ga1-3 phyB-5 double mutants increased
shortly before flowering occurred, it was not different from the
ga1-3 single mutant during the first 4 weeks. Upon application of GA3,
ga1-3 and ga1-3
phyB-5 mutants flowered as early as wild-type and
phyB-5 plants treated with
GA3 (Table I), which was paralleled by a similar
increase in LFY::GUS expression (Fig. 4B).
View larger version (18K):
[in this window]
[in a new window]
| Figure 4.
LFY::GUS expression during vegetative
growth of ga1-3 and
ga1-3 phyB-5 mutants.
Plants homozygous for the LFY::GUS transgene
were grown in long (A) or short (B) days until flower buds were visible
to the naked eye, except in the case of the
ga1-3 mutant without GA3
treatment under short days ( ), which had not flowered at the end of
the experiment. 304G1 (ga1-3, white
symbols) and 304G1B5 (ga1-3
phyB-5, black symbols) were treated with
GA3 (circles) or left untreated (squares). Values are
expressed as means ± 2 SE (n = 12). Time represents days after sowing. Error bars that are not visible
are contained within the symbol. MUG,
4-Methylumbelliferyl--D-glucopyranoside.
|
|
Interaction between PHYB and GAs
The suppression by phyB-5 of the flowering
defect of ga1-3 mutants, along with the weak
up-regulation of LFY::GUS expression in the
ga1-3 phyB-5 double mutants are
compatible with the idea that PHYB modulates flowering and
LFY expression independently of GAs. However, the
ga1-3 mutation does not completely abolish GA
biosynthesis, and several GA species are still detectable in the
ga1-3 mutant (Zeevaart and Talón, 1992; A. Silverstone, and T.-P. Sun, personal communication). In addition, there
is the possibility that physiologically relevant levels of exogenous GAs are carried over from the parental generation or from the seed
treatment required for germination. Thus, PHYB could act by increasing
the low levels of GA biosynthesis or by enhancing the responsiveness
toward the small amount of GAs present in ga1-3 mutants. An effect of phyB on GA biosynthesis is unlikely,
since overall levels of several GA intermediates are unchanged in
phyB mutants compared with the wild type (Reed et al.,
1996). phyB mutants show enhanced responsiveness to GAs, as
monitored by the dose response of hypocotyl elongation (Reed et al.,
1996). We have found that continuous watering of plants with
paclobutrazol, an inhibitor of the early steps of GA biosynthesis
(Rademacher, 1991), did not prevent the flowering of ga1
phyB or phyB mutants under short days, while it
abolished flowering of wild-type plants (results not shown).
To resolve the question of whether PHYB affects flowering by regulating
GA biosynthesis or GA response, we determined whether increased
responsiveness to GAs could account for the increased LFY::GUS expression in phyB mutants.
ga1-3 and ga1-3
phyB-5 seedlings carrying a
LFY::GUS transgene were grown on plates containing increasing concentrations of GA3. The hypocotyl
length of each seedling was determined before measuring
LFY::GUS activity. As previously reported (Reed et al.,
1996), the hypocotyl of phyB mutants was longer than that of
PHYB+ plants at all
GA3 concentrations. More importantly,
phyB mutants were also more responsive to exogenous
GA3 than wild-type plants (Fig.
5A). In contrast, levels of
LFY::GUS activity in phyB mutants did not show
increased responsiveness to exogenous GA3 over
the range of concentrations tested (Fig. 5B). The results were the same
under long or short days.
View larger version (14K):
[in this window]
[in a new window]
| Figure 5.
Responses of ga1-3
and ga1-3 phyB-5 seedlings
to exogenous GA3. Seeds of the lines 304G1
(LFY::GUS ga1-3,  ) and
304G1B5 (LFY::GUS ga1-3
phyB-5,  ) were sown on MS plates containing
the indicated concentrations of GA3. Hypocotyl length (A)
and GUS activity (B) were determined 7 d after sowing. Values are
expressed as means ± 2 SE (n = 12). Time represents days after sowing. Error bars that are not visible
are contained within the symbol. MUG,
4-Methylumbelliferyl--D-glucopyranoside.
|
|
To confirm that the responsiveness to exogenous
GA3 reflects the behavior toward endogenous GAs,
we analyzed both hypocotyl length and LFY::GUS activity in
wild-type and phyB-5 seedlings growing on plates
with increasing concentrations of paclobutrazol. As expected,
paclobutrazol reduced the elongation of wild-type and phyB
hypocotyls starting at concentrations as low as 0.03 µM (Fig. 6A)
(Reed et al., 1996). At higher concentrations, paclobutrazol inhibited
hypocotyl elongation faster in phyB-5 mutants
than in wild-type plants. Although paclobutrazol reduced
LFY::GUS expression in both wild-type and
phyB-5 plants, there was no difference in responsiveness between the two lines (Fig. 6B).
View larger version (17K):
[in this window]
[in a new window]
| Figure 6.
Responses of ga1-3
and ga1-3 phyB-5 seedlings
to the GA-biosynthesis-inhibitor paclobutrazol. Seeds of the lines
DW150-304 (LFY::GUS, ) and 304B5
(LFY::GUS phyB-5, ) were
sown on MS plates containing the indicated concentrations of
paclobutrazol. Hypocotyl length (A) and GUS activity (B) were
determined 7 d after sowing. Values are expressed as means ± 2 SE (n = 12). Time represents days
after sowing. Error bars that are not visible are contained within the
symbol. MUG, 4-Methylumbelliferyl--D-glucopyranoside.
|
|
Effect of phyB Mutation on Expression of CO
and FT
GAs act redundantly with the long-day-dependent pathway of floral
induction, as GA deficiency has much weaker effects on flowering in
long than in short days (Wilson et al., 1992). In addition, double
mutants carrying both the ga1-3 mutation and a
mutation in CO, an essential element of the long-day
pathway, often do not flower at all in long days (Putterill et al.,
1995). Since all of our other data pointed to a GA-independent effect
of PHYB on flowering, we wanted to determine whether the early
flowering of phyB mutants was caused by an increase in the
expression of genes known to be involved in the photoperiod-dependent
pathway that promotes flowering. The expression of two genes in this
pathway has been shown to be limiting for flowering, since their
overexpression causes very early flowering under both long and short
days. These genes are CO (Simon et al., 1996) and
FT (I. Kardailsky and D. Weigel, unpublished data).
When we analyzed the expression of CO and FT by
RT-PCR, we found that the expression levels of CO and
FT did not differ dramatically between wild-type and
phyB-5 plants (Fig. 7),
suggesting that the early-flowering phenotype of phyB
mutants under short days is not caused by overexpression of genes in
the long-day pathway.
View larger version (22K):
[in this window]
[in a new window]
| Figure 7.
CO and FT RNA
expression in phyB mutants. Seedlings of the lines
DW150-304 (wild type [WT], white bars) and 304B5
(phyB-5, black bars) were grown on MS plates under short
days (SD), and harvested during the 8th h of light on the indicated
days. Expression was analyzed by RT-PCR (bottom panel) and the signals
quantified and normalized using UBQ expression as a control
(top panel, arbitrary units).
|
|
| |
DISCUSSION |
The level of LFY expression is an important determinant
for the identity of the primordia that arise at the flanks of the shoot
apical meristem during the transition to flowering (Blázquez et
al., 1997). This idea has been corroborated by the observation that
certain mutations that delay flowering, such as co or
gi (gigantea), also reduce the level
of LFY expression during the time that the transition to
flowering occurs in wild type (Nilsson et al., 1998). In a similar way,
the acceleration of flowering caused by mutations such as
spy is paralleled by increased LFY expression
(Blázquez et al., 1998). Hence, it is not surprising that PHYB,
which represses flowering, functions as a negative regulator of the
LFY promoter, although it has been difficult to assess
whether phyB mutants flower early exclusively because of
increased LFY expression, or also because of an increased
response to LFY activity (Nilsson et al., 1998).
In our growth conditions (Fig. 1), PHYB affected flowering time mainly
under short photoperiods. Although the total number of leaves produced
was similar under long and short days (Table I), the number of days
needed to produce the first flower was higher under short days, as
previously reported (Koornneef et al., 1995). Accordingly, the increase
of LFY promoter activity caused by the phyB
mutation was more clearly observed under short than under long days. It
has been previously observed that the contribution of different
photoreceptors to the control of flowering time changes with
photoperiod (Bagnall et al., 1995). For example, a specific role for
the blue/UVA photoreceptor encoded by CRY2 is the promotion
of flowering under long days (Guo et al., 1998; Lin et al., 1998).
The observation that a phyB mutation has a more pronounced
effect on LFY expression under short days suggests an
interaction with GAs, since GAs are essential for flowering under
noninductive conditions (Martínez-Zapater et al., 1994).
However, we present evidence that PHYB and GAs regulate LFY
expression through independent pathways, since a phyB
mutation did not enhance the response of the LFY promoter to
GAs. An independent action was confirmed by the observation that
phyB suppressed the flowering defect of ga1 mutants under short days, even when any GA1-independent
synthesis of ent-kaurene was inhibited by paclobutrazol
treatment. The simplest scenario for this suppression would be
activation of the long-day-dependent pathway of flowering. However, the
observation that the levels of RNA expression of CO and
FT, the two genes believed to act downstream in the long-day
pathway (Simon et al., 1996; Koornneef et al., 1998), are not
dramatically changed in phyB mutants suggests that this
activation would not occur at the transcriptional level. Alternatively,
the suppression could take place through the FCA-dependent autonomous pathway (Macknight et al., 1997).
An important conclusion from our study and previous studies is that the
relationship between PHYB and GAs is complex. For certain responses,
such as germination, the GA-deficient ga1 mutant is
completely epistatic over phyB. However, most other
characteristics of ga1 phyB double-mutant seedlings are
intermediate between those observed for the single mutant parents (Peng
and Harberd, 1997; this study). Finally, while PHYB regulates hypocotyl
elongation by modulating responsiveness to GAs (Reed et al., 1996),
PHYB acts independently of GAs in the control of flowering time (this study). That genes do not always interact in the same fashion, even
when controlling the same targets, is not uncommon in development. For
instance, PHYA and PHYB regulate common responses to light, but they do
so differently depending on the particular response. While both
phytochromes have an inhibitory effect on hypocotyl elongation and
promote seed germination, flowering is repressed by PHYB but promoted
by PHYA.
It is not easy to imagine the molecular mechanism that integrates the
different interactions between GAs and PHYB. Based on the effects of
application of different GAs to ryegrass, it has been proposed that
certain GA species possess low florigenic activity but promote stem
elongation very efficiently and vice versa (Evans et al., 1990). For
instance, 3-hydroxylation is required for the promotion of stem
elongation, but not of flowering in this plant (Evans et al., 1994). If
these findings reflect the presence of different receptors for the
various active GA species, PHYB might regulate receptors specific for
GA species involved in hypocotyl and stem elongation but not the ones
involved in flowering.
| |
FOOTNOTES |
1
This work was supported by grants from the
National Science Foundation (no. MCB-9723823 to D.W.) and the Human
Frontiers Science Program Organization (no. RG 303/97 to D.W.). M.A.B.
received fellowships from the Spanish Ministry of Education and the
Human Frontiers Science Program Organization. D.W. is a National
Science Foundation Young Investigator and receives support from
Agritope (Oregon).
*
Corresponding author; e-mail weigel{at}salk.edu; fax 858-558-6379.
Received February 19, 1999;
accepted April 29, 1999.
| |
ABBREVIATIONS |
Abbreviation:
RT, reverse transcriptase.
| |
ACKNOWLEDGMENTS |
We thank Ove Nilsson, Michael Neff, and José Miguel
Martínez-Zapater for discussion and critical reading of the
manuscript; Trevor Phan for technical assistance with the hypocotyl
length measurements; Ji Hoon Ahn, Paul Reeves, and George Coupland for advice concerning the RT-PCR experiments; and Aron Silverstone for
communicating unpublished results. phyB-5 mutant
seeds were obtained from Joanne Chory.
| |
LITERATURE CITED |
Aukerman MJ,
Hirschfeld M,
Wester L,
Weaver M,
Clack T,
Amasino RM,
Sharrock RA
(1997)
A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing.
Plant Cell
9:
1317-1326
[Abstract]
Bagnall DJ,
King RW,
Whitelam GC,
Boylan MT,
Wagner D,
Quail PH
(1995)
Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.
Plant Physiol
108:
1495-1503
[Abstract]
Beall FD,
Morgan PW,
Mander LN,
Miller FR,
Babb KH
(1991)
Genetic regulation of development in Sorghum bicolor. V. The ma3R allele results in gibberellin enrichment.
Plant Physiol
95:
116-125
[Abstract/Free Full Text]
Blázquez MA,
Green R,
Nilsson O,
Sussman MR,
Weigel D
(1998)
Gibberellins promote flowering of Arabidopsis by activating the LEAFY promoter.
Plant Cell
10:
791-800
[Abstract/Free Full Text]
Blázquez MA,
Soowal L,
Lee I,
Weigel D
(1997)
LEAFY expression and flower initiation in Arabidopsis.
Development
124:
3835-3844
[Abstract]
Callis J,
Carpenter T,
Sun CW,
Vierstra RD
(1995)
Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia.
Genetics
139:
921-939
[Abstract]
Chory J,
Li H
(1997)
Gibberellins, brassinosteroids and light-regulated development.
Plant Cell Environ
20:
801-806
[CrossRef]
Devlin PF,
Patel SR,
Whitelam GC
(1998)
Phytochrome E influences internode elongation and flowering time in Arabidopsis.
Plant Cell
10:
1479-1487
[Abstract/Free Full Text]
Evans LT,
King RW,
Chu A,
Mander LN,
Pharis RP
(1990)
Gibberellin structure and florigenic activity in Lolium temulentum, a long-day plant.
Planta
182:
97-106
Evans LT,
King RW,
Mander LN,
Pharis RP
(1994)
The relative significance for stem elongation and flowering in Lolium temulentum of 3-hydroxylation of gibberellins.
Planta
192:
130-136
Fankhauser C,
Chory J
(1997)
Light control of plant development.
Annu Rev Cell Dev Biol
13:
203-229
[CrossRef][ISI][Medline]
Goto K,
Kumagai T,
Koornneef M
(1991)
Flowering responses to light breaks in photomorphogenic mutants of Arabidopsis thaliana, a long-day plant.
Physiol Plant
83:
209-215
[CrossRef]
Guo H,
Yang H,
Mockler TC,
Lin C
(1998)
Regulation of flowering time by Arabidopsis photoreceptors.
Science
279:
1360-1363
[Abstract/Free Full Text]
Hempel FD,
Weigel D,
Mandel MA,
Ditta G,
Zambryski P,
Feldman LJ,
Yanofsky MF
(1997)
Floral determination and expression of floral regulatory genes in Arabidopsis.
Development
124:
3845-3853
[Abstract]
Jacobsen SE,
Olszewski NE
(1993)
Mutations at the SPINDLY locus of Arabidopsis alter gibberellin signal transduction.
Plant Cell
5:
887-896
[Abstract/Free Full Text]
Koornneef M,
Alonso-Blanco C,
Peeters AJM,
Soppe W
(1998)
Genetic control of flowering in Arabidopsis.
Annu Rev Plant Physiol Plant Mol Biol
49:
345-370
[CrossRef][ISI]
Koornneef M,
Hanhart C,
van Loenen-Martinet P,
Blankestijn de Vries H
(1995)
The effect of daylength on the transition to flowering in phytochrome-deficient, late-flowering and double mutants of Arabidopsis thaliana.
Physiol Plant
95:
260-266
[CrossRef]
Koornneef M,
Rolff E,
Spruit CJP
(1980)
Genetic control of light-inhibited hypocotyl elongation in Arabidopsis thaliana (L.) Heynh.
Z Pflanzenphysiol
100:
147-160
Koornneef M,
van der Veen JH
(1980)
Induction and analysis of gibberellin sensitive mutants in Arabidopsis thaliana.
Theor Appl Genet
58:
257-263
[CrossRef][ISI]
Lee IJ,
Foster KR,
Morgan PW
(1998)
Photoperiod control of gibberellin levels and flowering in sorghum.
Plant Physiol
116:
1003-1011
[Abstract/Free Full Text]
Lin C,
Yang H,
Guo H,
Mockler T,
Chen J,
Cashmore AR
(1998)
Enhancement of blue-light sensitivity of Arabidopsis seedlings by a blue light receptor cryptochrome 2.
Proc Natl Acad Sci USA
95:
2686-2690
[Abstract/Free Full Text]
López-Juez E,
Kobayashi M,
Sakurai A,
Kamiya Y,
Kendrick RE
(1995)
Phytochrome, gibberellins, and hypocotyl growth. A study using the cucumber (Cucumis sativus L.) long hypocotyl mutant.
Plant Physiol
107:
131-140
[Abstract]
Macknight R,
Bancroft I,
Lister C,
Page T,
Love K,
Schmidt R,
Westphal L,
Murphy G,
Sherson S,
Cobbett C
(1997)
FCA, a gene controlling flowering time in Arabidopsis, encodes a protein containing RNA-binding domains.
Cell
89:
737-745
[CrossRef][ISI][Medline]
Martínez-García JF,
García-Martínez JL
(1992)
Phytochrome modulation of gibberellin metabolism in cowpea epicotyl elongation.
In
CM Karssen,
LC van Loon,
D Vreugdenhil,
eds, Progress in Plant Growth Regulation.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 585-590
Martínez-Zapater JM, Coupland G, Dean C, Koornneef M
(1994) The transition to flowering in Arabidopsis. In EM
Meyerowitz, CR Somerville, eds, Arabidopsis. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, pp 403-433
Murashige T,
Skoog F
(1962)
A revised medium for rapid growth and bio-assay with tobacco tissue cultures.
Physiol Plant
15:
473-497
[CrossRef]
Nagatani A,
Reed JW,
Chory J
(1993)
Isolation and initial characterization of Arabidopsis mutants that are deficient in phytochrome A.
Plant Physiol
102:
269-277
[Abstract]
Neff MM,
Chory J
(1998)
Genetic interactions between phytochrome A, phytochrome B and cryptochrome 1 during Arabidopsis development.
Plant Physiol
118:
27-35
[Abstract/Free Full Text]
Neff MM,
Neff JD,
Chory J,
Pepper AE
(1998)
dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics.
Plant J
14:
387-392
[CrossRef][ISI][Medline]
Nilsson O,
Lee I,
Blázquez MA,
Weigel D
(1998)
Flowering-time genes modulate the response to LEAFY activity.
Genetics
150:
403-410
[Abstract/Free Full Text]
Parks BM,
Quail PH
(1993)
hy8, a new class of Arabidopsis long hypocotyl mutants deficient in functional phytochrome A.
Plant Cell
5:
39-48
[Abstract/Free Full Text]
Peng J,
Harberd NP
(1997)
Gibberellin deficiency and response mutations suppress the stem elongation phenotype of phytochrome-deficient mutants of Arabidopsis.
Plant Physiol
113:
1051-1058
[Abstract]
Putterill J,
Robson F,
Lee K,
Simon R,
Coupland G
(1995)
The CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors.
Cell
80:
847-857
[CrossRef][ISI][Medline]
Quail PH,
Boylan MT,
Parks BM,
Short TW,
Xu Y,
Wagner D
(1995)
Phytochromes: photosensory perception and signal transduction.
Science
268:
675-680
[Abstract/Free Full Text]
Rademacher W (1991) Biochemical effects of plant growth
retardants. In HW Gausman, ed, Plant Biochemical Regulators.
Marcel Dekker, New York, pp 169-200
Reed JW,
Foster KR,
Morgan PW,
Chory J
(1996)
Phytochrome B affects responsiveness to gibberellins in Arabidopsis.
Plant Physiol
112:
337-342
[Abstract]
Reed JW,
Nagatani A,
Elich TD,
Fagan M,
Chory J
(1994)
Phytochrome A and phytochrome B have overlapping but distinct functions in Arabidopsis development.
Plant Physiol
104:
1139-1149
[Abstract]
Reed JW,
Nagpal P,
Poole DS,
Furuya M,
Chory J
(1993)
Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development.
Plant Cell
5:
147-157
[Abstract]
Rood SB,
Williams PH,
Pearce D,
Murofushi N,
Mander LN,
Pharis RP
(1990)
A mutant gene that increases gibberellin production in Brassica.
Plant Physiol
93:
1168-1174
[Abstract/Free Full Text]
Sharrock RA,
Quail PH
(1989)
Novel phytochrome sequences in Arabidopsis thaliana: structure, evolution, and differential expression of a plant regulatory photoreceptor family.
Genes Dev
3:
1745-1757
[Abstract/Free Full Text]
Silverstone AL,
Mak PY,
Martinez EC,
Sun T-P
(1997)
The new RGA locus encodes a negative regulator of gibberellin response in Arabidopsis thaliana.
Genetics
146:
1087-1099
[Abstract]
Simon R,
Igeño MI,
Coupland G
(1996)
Activation of floral meristem identity genes in Arabidopsis.
Nature
382:
59-62
Talón M,
Zeevaart JAD,
Gage DA
(1991)
Identification of gibberellins in spinach and effects of light and darkness on their levels.
Plant Physiol
97:
1521-1526
[Abstract/Free Full Text]
Weller JL,
Ross JJ,
Reid JB
(1994)
Gibberellins and phytochrome regulation of stem elongation in pea.
Planta
192:
489-496
Whitelam GC,
Johnson E,
Peng J,
Carol P,
Anderson ML,
Cowl JS,
Harberd NP
(1993)
Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light.
Plant Cell
5:
757-768
[Abstract/Free Full Text]
Wilson RN,
Heckman JW,
Somerville CR
(1992)
Gibberellin is required for flowering in Arabidopsis thaliana under short days.
Plant Physiol
100:
403-408
[Abstract/Free Full Text]
Wu KQ,
Li L,
Gage DA,
Zeevaart JAD
(1996)
Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.
Plant Physiol
110:
547-554
[Abstract]
Xu YL,
Li L,
Wu K,
Peeters AJ,
Gage DA,
Zeevaart JA
(1995)
The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.
Proc Natl Acad Sci USA
92:
6640-6644
[Abstract/Free Full Text]
Yamaguchi S,
Smith MW,
Brown RG,
Kamiya Y,
Sun T
(1998)
Phytochrome regulation and differential expression of gibberellin 3-hydroxylase genes in germinating Arabidopsis seeds.
Plant Cell
10:
2115-2126
[Abstract/Free Full Text]
Zeevaart JAD, Talón M (1992) Gibberellin mutants in
Arabidopsis thaliana. In CM Karssen, LC van Loon, D
Vreugdenhil, eds, Progress in Plant Growth Regulation. Kluwer Academic
Publishers, Dordrecht, The Netherlands, pp
34-42
This article has been cited by other articles:
|
|
|
|
 
A. C. Wollenberg, B. Strasser, P. D. Cerdan, and R. M. Amasino
Acceleration of Flowering during Shade Avoidance in Arabidopsis Alters the Balance between FLOWERING LOCUS C-Mediated Repression and Photoperiodic Induction of Flowering
Plant Physiology,
November 1, 2008;
148(3):
1681 - 1694.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. R. Andersson, C. A. Helliwell, D. J. Bagnall, T. P. Hughes, E. J. Finnegan, W. J. Peacock, and E. S. Dennis
The FLX Gene of Arabidopsis is Required for FRI-Dependent Activation of FLC Expression
Plant Cell Physiol.,
February 1, 2008;
49(2):
191 - 200.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. H. Kebrom and T. P. Brutnell
The molecular analysis of the shade avoidance syndrome in the grasses has begun
J. Exp. Bot.,
October 5, 2007;
(2007)
erm205v1.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Zhao, X. Yu, E. Foo, G. M. Symons, J. Lopez, K. T. Bendehakkalu, J. Xiang, J. L. Weller, X. Liu, J. B. Reid, et al.
A Study of Gibberellin Homeostasis and Cryptochrome-Mediated Blue Light Inhibition of Hypocotyl Elongation
Plant Physiology,
September 1, 2007;
145(1):
106 - 118.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Hanano, M. A. Domagalska, F. Nagy, and S. J. Davis
Multiple phytohormones influence distinct parameters of the plant circadian clock
Genes Cells,
December 1, 2006;
11(12):
1381 - 1392.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Laubinger, V. Marchal, J. Gentilhomme, S. Wenkel, J. Adrian, S. Jang, C. Kulajta, H. Braun, G. Coupland, and U. Hoecker
Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability
Development,
August 15, 2006;
133(16):
3213 - 3222.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. FRANKLIN and G. C. WHITELAM
Phytochromes and Shade-avoidance Responses in Plants
Ann. Bot.,
August 1, 2005;
96(2):
169 - 175.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Mizoguchi, L. Wright, S. Fujiwara, F. Cremer, K. Lee, H. Onouchi, A. Mouradov, S. Fowler, H. Kamada, J. Putterill, et al.
Distinct Roles of GIGANTEA in Promoting Flowering and Regulating Circadian Rhythms in Arabidopsis
PLANT CELL,
August 1, 2005;
17(8):
2255 - 2270.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Yamaguchi, Y. Kobayashi, K. Goto, M. Abe, and T. Araki
TWIN SISTER OF FT (TSF) Acts as a Floral Pathway Integrator Redundantly with FT
Plant Cell Physiol.,
August 1, 2005;
46(8):
1175 - 1189.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Jeong and S. E. Clark
Photoperiod Regulates Flower Meristem Development in Arabidopsis thaliana
Genetics,
February 1, 2005;
169(2):
907 - 915.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. An, C. Roussot, P. Suarez-Lopez, L. Corbesier, C. Vincent, M. Pineiro, S. Hepworth, A. Mouradov, S. Justin, C. Turnbull, et al.
CONSTANS acts in the phloem to regulate a systemic signal that induces photoperiodic flowering of Arabidopsis
Development,
August 1, 2004;
131(15):
3615 - 3626.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. R. Stinchcombe, C. Weinig, M. Ungerer, K. M. Olsen, C. Mays, S. S. Halldorsdottir, M. D. Purugganan, and J. Schmitt
A latitudinal cline in flowering time in Arabidopsis thaliana modulated by the flowering time gene FRIGIDA
PNAS,
March 30, 2004;
101(13):
4712 - 4717.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Pineiro, C. Gomez-Mena, R. Schaffer, J. M. Martinez-Zapater, and G. Coupland
EARLY BOLTING IN SHORT DAYS Is Related to Chromatin Remodeling Factors and Regulates Flowering in Arabidopsis by Repressing FT
PLANT CELL,
July 1, 2003;
15(7):
1552 - 1562.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
