FIGURE LEGENDS
Figure 1. BLH1 modulates ABA sensitivity during seed germination and early
seedling development.
(a) The expression of BLH1 in Arabidopsis tissues. Total RNAs were isolated from
the indicated tissues. Leaves and roots, and stems and flowers were obtained from
2- and 5-week-old plants, respectively. The transcript levels of BLH1 were
normalized to those of UBQ1, and the relative level of BLH1 transcript in the roots
was set to 1. (b) The expression of BLH1 was induced by ABA. Five-day-old wildtype (Ler), pyr1 pyl1 pyl2 pyl4 mutant, and abi1-1 mutant seedlings were grown in
the presence or absence of 100 M ABA for 3 h. Transcript levels of BLH1 were
normalized to those of UBQ1, and the relative level of BLH1 transcripts in mocktreated wild-type seedlings was set to 1. (c) BLH1 positively modulates the ABA
response during seed germination. The germination rates of Col-0, blh1, 35S:BLH1
and pyr1 pyl1 pyl2 pyl4 were recorded from 1 to 5 days after stratification in the
presence of 0.3 M ABA (top) or 100 mM NaCl (bottom). (d) BLH1 affects the
opening of cotyledons. Cotyledon opening of Col-0, blh1, and 35S:BLH1 in the
presence of 0.5 M ABA was determined and photographs were taken 7 days after
stratification. Numbers in brackets represent the number of seedlings with open
cotyledons per total number of seeds examined. (e, f) BLH1 is positively involved in
ABA response during primary root growth. Three-day-old Col-0, 35S:BLH1, and blh1
seedlings were transferred to growth media containing 0M, 5 M, or 15 M of ABA,
grown for another 14 days, and then photographed. Representative seedlings are
shown (e) and the primary root lengths are presented (f). The data represent
averages from at least 10 individual seedlings. Error bars denote S.E. (n=3 in (a) and
(b), n=150 in (c); *P<0.05, **P<0.01 for Student’s t-tests).
Figure 2. BLH1 regulates the expression of ABA-inducible genes.
The expression patterns of ABA signaling-related RD29A, RD29B, ABI3, and ABI5,
and embryogenesis-related Em1 and Em6 were determined in 1-week-old Col-0,
blh1, and 35S:BLH1 seedlings treated with 100 M ABA for 2 h. The transcript level
of each gene was normalized to that of UBQ1, and the relative level of each gene in
mock-treated Col-0 was set to 1. The inset graph presents the expression of the
indicated genes in the mock-treated seedlings. Error bars denote S.E. (n=4, *P<0.05
for Student’s t-tests).
Figure 3. KNAT3 regulates ABA response during seed germination.
(a) KNAT3 is positively involved in the ABA response during seed germination. The
germination rates of Col-0, knat3, knat5, and knat6 were recorded from 1 to 5 days
(ABA) or at 5 days (NaCl) after stratification in the presence of 0.3 M ABA or the
indicated concentration of NaCl. (b) KNAT3 affects cotyledon opening. Cotyledon
opening of Col-0, knat3, knat5, and knat6 seedlings in the presence of 0.3 M ABA
was determined and photographs were taken 5 days after stratification. Numbers in
brackets represent the number of seedlings with open cotyledons per the total
number of seeds. (c) The expression of KNAT3 was induced by ABA. Five-day-old
wild-type (Ler), pyr1 pyl1 pyl2 pyl4 mutant, and abi1-1 mutant seedlings were grown
in the presence or absence of 100 M ABA for 3 h. The transcript level of each gene
was normalized to that of UBQ1, and the relative level of each gene in mock-treated
wild-type seedlings was set to 1. (d) The hypersensitivity of 35S:BLH1 to ABA during
seed germination was partially resolved by a loss-of-function knat3 mutation. The
germination rates of Col-0, knat3, 35S:BLH1, and 35S:BLH1 knat3 were recorded
from 1 to 5 days after stratification in the presence of 0.3 M ABA. Error bars denote
S.E. (n=150 in (a) and (d); n=3 in (c); *P<0.05, **P<0.01 for Student’s t-tests).
Figure 4. BLH1 and KNAT3 synergistically increase the ABA response.
(a) Schematic diagram of KNAT3 deletion mutants. Full-length KNAT3 contains four
putative functional domains: KNOX1, KNOX2, ELK, and homeodomain (HD).
KNAT3KNOX1 and KNAT3KNOX2 indicate the deletion of the KNOX1 and KNOX2
domain, respectively. Amino acid numbers are indicated above the diagram. (b)
BLH1 interacted with KNAT3, but not with KNAT3KNOX1 and KNAT3KNOX2. BLH1-HA
was co-transfected with KNAT3-FLAG, KNAT3KNOX1-FLAG, or KNAT3KNOX2-FLAG
into mesophyll protoplasts. Co-immunoprecipitation assays were performed with
monoclonal anti-FLAG antibodies in total protein extracts from protoplasts, and
proteins
were
detected
with
anti-HA or anti-FLAG antibodies.
(c) BLH1
synergistically activates ProRD29B with KNAT3. Protoplasts were co-transfected
with 10 g of ProRD29B:LUC as a reporter and the indicated effector plasmids, and
treated with 100 M ABA for 1 h. The amount of DNA used in each experiment was
10 (+) or 20 g (++), as indicated. Renilla luciferase (Rluc) was used as an internal
control. The relative luciferase activity in mER7-GFP-transfected cells not treated
with ABA was set to 1. Error bars denote S.E. (n=3; **P<0.01 for Student’s t-tests).
Figure 5. BLH1 promotes the nuclear localization of KNAT3.
(a) Amino acid sequence alignment of KNOX1 domains from multiple KNOX proteins
in Arabidopsis. The conserved sequences of the nuclear export signal (NES) in the
KNOX1 domain are indicated by boxes. The NES consensus sequence is shown
below. (b) Localization of BLH1-GFP and KNAT3-RFP. Top row: protoplasts
expressing BLH1-GFP or KNAT3-RFP. Middle row: protoplasts expressing KNAT3GFP in the presence of leptomycin B or KNAT3L173S-RFP. Samples were treated with
leptomycin B, an exportin inhibitor, for 2 h. Bottom row: protoplasts expressing
KNAT3KNOX1-RFP or KNAT3KNOX2-RFP. Bright field (left) and fluorescence (right)
images were taken 6 h after transfection. (c) KNOX1 and KNOX2 domains are
necessary for the BLH1-mediated nuclear retention of KNAT3. Top row: protoplasts
expressing both BLH1-GFP and KNAT3-RFP. Middle row: protoplasts expressing
both BLH1-GFP and KNAT3KNOX1-RFP. Bottom row: protoplasts expressing both
BLH1-GFP and KNAT3KNOX2-RFP. Bright field, GFP, RFP, and merge (left to right)
images were taken 6 h after transfection. Scale bars: 10 m in (b) and (c).
Figure 6. The BLH1/KNAT3 complex increases the activity of the ABI3 promoter.
(a) BLH1 strongly binds to the ABI3 promoter in the presence of KNAT3. Upper
panel: schematic diagram of the ABI3 promoter. BLH1-binding TGGA motifs (white
circles) and the regions (R1-R4) analyzed by ChIP. Lower panel: protoplasts were
transfected with 40 g of BLH1-HA (black bars), 40 g of KNAT3-HA (light gray bars),
or 20 g of BLH1-HA and 20 g of KNAT3-HA (dark grey bars). The chromatin
associated with BLH1, KNAT3, or BLH1 and KNAT3 was immunoprecipitated with
anti-HA antibodies and the regions of the ABI3 promoter indicated in the upper panel
were amplified by quantitative RT-PCR using the specific primers. The fold
enrichment was calculated and normalized to the values of the protoplasts without
antibodies. Error bars denote S.E. (n=5~6; *P<0.05 for Student’s t-tests). (b) BLH1
synergistically activates the ABI3 promoter with KNAT3. Protoplasts were cotransfected with 5 g of ProABI3:LUC as a reporter plasmid and 5 g of each
effector plasmid as indicated. Renilla luciferase was used as an internal control. The
relative luciferase activity in mER7-GFP-transfected cells without ABA was set to 1.
(c) Hypothetic model of BLH1/KNAT3 action during ABA signaling. Under low
concentrations of ABA, low levels of BLH1 and KNAT3 proteins are produced.
KNAT3 monomers are translocated to the cytosol via exportin, and thus BLH1
weakly induces ABI3 expression. Under stress conditions (e.g., high levels of ABA),
the expression of BLH1 and KNAT3 is enhanced. Increased amounts of BLH1
proteins promote the retention of KNAT3 in the nucleus by masking NES. The
resulting BLH1/KNAT3 complex strongly binds to the ABI3 promoter and enhances
the ABA responses.
SUPPLEMENTARY LEGENDS
Figure S1. Characterization of the T-DNA insertion mutants and overexpressing
lines, blh1, knat3, knat5, knat6, 35S:BLH1, and 35S:BLH1 knat3 line. RT-PCR
analysis of BLH1 expression in Col-0, blh1, 35S:BLH1, and 35S:BLH1 knat3 lines,
and of KNOX expression in Col-0 and the indicated KNOX mutants. UBQ1 was used
as an RT-PCR control.
Figure S2. BLH1 is positively involved in stress responses during seed germination.
(a) Germination rates of Col-0, blh1, and 35S:BLH1 were recorded from 1 to 5 days
after stratification in the presence of the indicated concentrations of ABA or NaCl. (b)
Cotyledon opening rates of Col-0, 35S:BLH1, and blh1 were similar under normal
growth conditions. The photographs were taken 5 days after stratification. Error bars
denote S.E. (n=150).
Figure S3. BLH1 interacts with KNAT3, KNAT5, and KNAT6. BLH1-HA was cotransfected with KNAT3-FLAG, KNAT5-FLAG, or KNAT6-FLAG into mesophyll
protoplasts. Co-immunoprecipitation assays were performed with monoclonal antiFLAG antibodies in total protein extracts from protoplasts, and proteins were
detected with anti-HA or anti-FLAG antibodies.
Figure S4. KNAT3 is positively involved in the ABA response during seed
germination. Germination rates of Col-0, knat3, knat5, and knat6 were recorded from
1 to 5 days after stratification in the presence of the indicated concentrations of ABA.
Error bars denote S.E. (n=150).
Figure S5. The hypersensitivity of 35S:BLH1 to ABA during seed germination was
partially compromised by a loss-of-function mutation of KNAT3. The germination
rates of Col-0, knat3, 35S:BLH1, and 35S:BLH1 knat3 were recorded from 1 to 5
days after stratification in the presence of the indicated concentrations of ABA. Error
bars denote S.E. (n=150).
Figure S6. ABA-dependent induction of ABA signaling-related genes in 35S:BLH1
was partly suppressed by a loss-of-function knat3 mutation. The expression patterns
of RD29A, RD29B, and ABI3 were determined in 1-week-old Col-0, 35S:BLH1, and
35S:BLH1 knat3 seedlings treated with 100 M ABA for 3 h. The transcript level of
each gene was normalized to that of UBQ1, and the relative level of each gene in
mock-treated Col-0 was set to 1. The inset graph presents the expression of the
indicated genes in the mock-treated seedlings. Error bars denote S.E. (n=3, *P<0.05
for Student’s t-tests).
Figure S7. The reduced expression of KNAT3 could reinforce the ABA insensitivity of
blh1. Protoplasts isolated from each line were transfected with 30 g of control
plasmid or KNAT3 RNAi plasmid as indicated. The transcript level of each gene was
normalized to that of UBQ1, and the relative level of each gene in mock-treated Col0 was set to 1. The inset graph presents the expression of RD29B in the mocktreated protoplasts. Error bars denote S.E. (n=3, *P<0.05 for Student’s t-tests).
Figure S8. KNAT3L173S activates ProRD29B more strongly than does KNAT3.
Protoplasts were co-transfected with 10 g of ProRD29B:LUC as a reporter and 10
g of the indicated effector plasmids, and treated with 100 M ABA for 1 h. Rluc was
used as an internal control. The relative luciferase activity in mER7-GFP-transfected
cells without ABA was set to 1. Error bars denote S.E. (n=3; *P<0.05 for Student’s ttests).
Figure S9. Localization of KNAT5-RFP and KNAT6-RFP. Top row: protoplasts
expressing KNAT5-RFP or KNAT6-RFP. Middle row: protoplasts expressing both
BLH1-GFP and KNAT5-RFP. Bottom row: protoplasts expressing both BLH1-GFP
and KNAT6-RFP. Brightfield (left) and fluorescence (right) images were taken 6 h
after transfection. Scale bars: 10 m.
Figure S10. The ABA hypersensitivity of BLH1 overexpression was inhibited in the
abi3-8 mutant. Col-0 and abi3-8 protoplasts were transfected with 30 g of mER7GFP (control) or BLH1-HA. The transcript level of each gene was normalized to that
of UBQ1, and the relative level of each gene in mER7-GFP-transfected cells not
treated with ABA was set to 1. Error bars denote S.E. (n=3).
Table S1. List of primers used in this study.