First published online 4 July 2007
doi: 10.1242/dev.02866
Development 134, 2841-2850 (2007)
Published by The Company of Biologists 2007
Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering
Malgorzata A. Domagalska1,
Fritz M. Schomburg2,
Richard M. Amasino2,
Richard D. Vierstra3,
Ferenc Nagy4 and
Seth J. Davis1,*
1 Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg
10, 50829 Cologne, Germany.
2 Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
53706, USA.
3 Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706,
USA.
4 Institute of Plant Biology, Biological Research Centre, Hungarian Academy of
Science, Szeged, Hungary.
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Fig. 1. bri1 is an enhancer of the autonomous mutant ld.
(A,B) Flowering time under long days (16 hours light/8 hours
darkness) of the wild-type control (Ws), the ld-3 single mutant and
the two double bri1 ld mutants isolated in the enhancer screen of
ld-3, measured as (A) days to flower or (B) rosette leaf number at
bolting. (C) Schematic representation of the effect of the
bri1-201 and bri1-202 mutations at the gene and protein
level. (D) Flowering time of various bri1 ld double-mutant
combinations under long days. A representative experiment is shown. Flowering
time was measured as the total number of rosette leaves formed when the bolt
was approximately 1 cm. The leaf number values are averages from 10-18 plants
per genotype. Error bars represent s.e.m. (E) Photographs at bolting of
bri1 ld double mutants compared with Ws and the single mutants
ld-2 and ld-3. Scale bar: 1 cm.
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Fig. 2. bri1 is a strong enhancer of FRI and of autonomous
mutants. (A) Photographs at bolting of double mutants between the
bri1-201 mutant and different flowering-time mutants: autonomous
(ld-3, fca-11), photoperiod (gi-11), gibberellin
(ga1-3), and dominant FRI in the Ws background. Each single
mutant is also shown. Plants were grown under long days. (B) Flowering
time under long days (16 hours light/8 hours darkness). (C) Flowering
time of the bri1-201, ld-3 and gi-11 single mutants and the
respective bri1 double mutants under short days (10 hours light/14
hours darkness). Representative experiments are presented. Flowering time was
measured as in Fig. 1. Scale
bar: 1 cm.
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Fig. 3. bri1 mutation leads to an elevation of FLC mRNA levels
in FRI and in autonomous mutants. Absolute (A) and
relative (B) FLC expression in bri1-201 ld-3 double
mutants, compared to the respective single mutants, under long-day
photoperiods. Samples were taken at the time indicated (number of days of
growth), until the flower buds were visible. (C) FLC
expression in bri1-201 ld-3 and bri1-202 ld-3 double mutants
and in the respective single mutants after 14 and 30 days of growth under
short days. (D) FLC mRNA levels in 30-day-old bri1 ld
double-mutant combinations, and (E) in the double-mutant bri1 fca,
bri1 FRI and bri1 gi lines under long days. FLC mRNA
abundance was monitored by RNA-blot analysis. (A-E) The ACTIN 1
(ACT1) gene was used as a loading control. FLC levels were
quantified, values were normalized to the levels of ACTIN 1, and the
highest value in each separate experiment was set to be 1.
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Fig. 4. Reduction of FLC expression in bri1 ld, bri1
fca and bri1 FRI double mutants efficiently accelerates
flowering. (A) The effect of vernalization treatment on flowering
time in the following mutants: bri1-201, ld-3, fca-11, FRI in the Ws
background, bri1-201 ld-3, bri1-201 fca-11 and bri1-201 FRI.
Germinated seedlings were vernalized for 6 weeks under short days (8 hours
light/16 hours darkness) at 4°C, after which they were grown under the
same long-day conditions as control non-vernalized plants. White bars,
non-vernalized controls; black bars, vernalization treatments. Absolute
(B) and relative (C) FLC mRNA levels before (-; white
bars) and after (+; black bars) vernalization. (D) Flowering phenotypes
of four independent FLC-RNAi transgenic lines in the bri1-201
ld-3 double-mutant background, compared to two independent lines of
bri1-201 ld-3 double mutants transformed with a control vector.
7-week-old plants are shown. (E) Flowering-time analysis of
FLC-RNAi bri1 ld mutants grown under a long-day photoperiod.
A representative result is shown. (F) RNA-blot analysis of FLC
mRNA levels in 20-day-old FLC-RNAi bri1 ld mutants compared
to the control bri1 ld lines. The ACT1 gene was used as a
loading control.
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Fig. 5. Combining the BR-deficient mutant cpd with ld delays
flowering and enhances FLC expression. (A,B)
Flowering time, under long days, of cpd-3939 ld-3 compared to
bri1-201 ld-3 mutants and to the respective single mutants, measured
as either days to bolt (A) or rosette leaf number at bolting (B). A
representative result is shown. (C,D) FLC expression
(C) and the abundance of the floral pathway integrators FT and
SOC1 (D) in the 30-day-old plants described in A. FLC mRNA
abundance was monitored as in Fig.
1. FT and SOC1 mRNA levels were measured by
reverse transcriptase (RT)-PCR and UBQ10 was used as a control.
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Fig. 6. Histone acetylation at the FLC locus is increased in bri1
ld mutants. (A) Regions of the FLC locus examined by
ChIP. Coding regions are indicated with vertical lines, and the nine fragments
amplified with PCR are depicted and numbered from I to IX. (B) ChIP
with antibodies against trimethylated histone 3 at lysine 4 (triMeH3K4).
21-day-old Ws, bri1-201, ld-3 and bri1-201 ld-3 mutants
grown under long days were analyzed, and region IV was examined. (C)
ChIP with antibodies against acetylated histone 3 (H3Ac). Samples are as
described in B. (B,C) Input, denotes DNA amplified from each chromatin sample
before immunoprecipitation. -, denotes samples immunoprecipitated with
antibodies against anti-rat-IgG (negative control). Anti-H3Ac refers to
fragments amplified from chromatin samples immunoprecipitated with antibodies
against H3Ac, whereas Anti-triMeH3K4 refers to samples immunoprecipitated with
antibodies against trimethylated H3K4. UBQ10 served as an internal
ChIP control. Representative ChIPs are illustrated. Fold enrichment calculated
for a biological replicate is the mean of averaged enrichment ratios from two
independent immunoprecipitations. Fold enrichment in Ws was arbitrarily set as
1.
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© The Company of Biologists Ltd 2007
