A B - The Plant Cell · 10/06/2010 · Supplemental Data. Je et al. (2010). Plant Cell...
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Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 . IV III II I IV III II I p p sheath blade collar age stem A B D C C L S 0 20 40 60 80 100 120 whole Panicle I II III IV ratio (%) ravl1-1 WT Supplemental Figure 1. Expression patterns and phenotypes of RAVL1 plants. (A) RAVL1 expression in lateral roots and their primordia was detected using GUS staining (top left panel) and in situ hybridization (lower left panel). Immature leaves folded inside stems showed strong GUS staining (top right panel) and in situ hybridization signal (lower right panel). (B) As leaves and stems become mature, the overall expression of GUS was restricted to lamina joints and nodes. (C) Strong expression was detected in coleoptiles (left panel) and stigmas and lodicules of flowers (right panel). C, S, and L indicate coleoptiles, stigma, and lodicules, respectively. (D) Internode length (left panel) and ratio of internode length of mutants were compared with those of wild-type plants (right panel). Error bars are standard errors (SE) of the means of 10 plants.
Transcript of A B - The Plant Cell · 10/06/2010 · Supplemental Data. Je et al. (2010). Plant Cell...
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
IV
III
II
I
IV
III
II
Ip
p
sheath
blade
collar
age
stem
A B
D
CC
L
S
0
20
40
60
80
100
120
whole Panicle I II III IV
ratio
(%)
ravl1-1 WT
Supplemental Figure 1. Expression patterns and phenotypes of RAVL1 plants.
(A) RAVL1 expression in lateral roots and their primordia was detected using GUS
staining (top left panel) and in situ hybridization (lower left panel). Immature leaves
folded inside stems showed strong GUS staining (top right panel) and in situ
hybridization signal (lower right panel).
(B) As leaves and stems become mature, the overall expression of GUS was restricted to
lamina joints and nodes.
(C) Strong expression was detected in coleoptiles (left panel) and stigmas and lodicules
of flowers (right panel). C, S, and L indicate coleoptiles, stigma, and lodicules,
respectively.
(D) Internode length (left panel) and ratio of internode length of mutants were compared
with those of wild-type plants (right panel). Error bars are standard errors (SE) of the
means of 10 plants.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
0
1
2
3
4
5
6
7
0M 1nM 10nM 100nM 1µM
NAA
Roo
t len
gth
(cm
)
WTbri1 d61-1ravl1-1
0
1
2
3
4
5
6
7
0M 1nM 10nM 100nM 1µM
GA (0.01% DMSO)
Roo
t len
gth
(cm
)
WTbri1 d61-1ravl1-1
0
1
2
3
4
5
6
7
0M 1nM 10nM 100nM 1µM
ABA (0.01% ethanol)
Roo
t len
gth
(cm
)
WTbri1 d61-1ravl1-1
0
1
2
3
4
5
6
7
0M 1nM 10nM 100nM 1µM
ACC
Roo
t len
gth
(cm
)
WTbri1 d61-1ravl1-1
Supplemental Figure 2. Growth responses of seminal roots to ACC, ABA, GA, and
NAA treatment.
Germinated seeds were grown for 3 days in 1 mM CaCl B2 B solution with the indicated
concentrations of ACC, ABA, GA, or NAA. Error bars are SE of the means of 9 plants.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 . A
WT
bri1 d61-1
ravl1-10 1p 10p 100p 1n 10n 100n 1µM
Concentration of epiBL
0
10
20
30
40
50
60
70
0M 1pM 10pM 100pM 1nM 10nM 100nM 1µM
epiBL (0.01% DMSO)
Roo
t gro
wth
(mm
)
WTbri1 d61-1ravl1-1
B
C
10n 100n 1µM 10n 100n 1µM 10n 100n 1µM
Concentration of epiBL
WT bri1 d61-1 ravl1-1
Supplemental Figure 3. Growth and root curling responses of seminal roots to epiBL.
(A) Root growth responses to epiBL. Germinated seeds were grown for 3 days in 1 mM
CaCl B2 B solution with the indicated concentrations of epiBL.
(B) Primary root lengths. Error bars are SE of the means of 20–30 plants.
(C) Distinct curling of wild-type roots was detected at epiBL concentrations less than
10 nM. ravl1-1 mutants did not show any distortion in roots. bri1 d61-1 plants exhibited
weak curling in the presence of 100 nM of epiBL.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
WT ravl1-1 OX1 OX2 OX3 OX4 OX5 OX6 OX7
very weak weak middle strong
A
WT OX1 OX3 OX4 OX6
EtBr
RAVL1
B
28±2(90±5)
88±5(150±15)
68±4(120±10)
73±4(115±10)
cell number in 700µm
(total)
OXbri1 d61-1
ravl1-1WTgenotype
WT ravl1-1 bri1 d61-1 OX
C
D
Supplemental Figure 4. Phenotypes of RAVL1 overexpressor.
(A) RAVL1 overexpressors (OX) lines were arranged and numbered according to the
severity of lamina inclination.
(B) RNA gel blot was performed to measure the levels of RAVL1 mRNA.
(C) Perpendicular sections of the adaxial cell layer of the lamina joint. Bar indicates 50
μm.
(D) Cell density in the adaxial side of the lamina joints. The number of cells was counted in the 2P
ndP layer of the adaxial sides. Error bars are SE of the means ofU 3-5U plants.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
35S RAVL1 GFP
1 400B3
86 174
85 194
35S:RAVL1-GFP
B3193 360
35S:RAVL1(ΔB3)-GFP
35S:RAVL1(Δ3’)-GFP
A
35S:RAVL1-GFPWT
35S:RAVL1(ΔB3)-GFP
B
35S:RAVL1(Δ3’)-GFP
Supplemental Figure 5. Nuclear localization of RAVL1-GFP in transgenic plant roots.
(A) GFP fusion constructs with full length RAVL1 and partial deletions of the B3
domain (ΔB3) or the C terminal domain (Δ3’). The numbers indicate OsRAVL1 amino
acid positions.
(B) GFP is detected in nuclei of RAVL1-GFP (top right panel) and URAVL1(ΔB3)-GFP U
(lower left panel) cells. However, GFP fluorescence is not detected in nuclei of wild
type (top left panel) and RAVL1(Δ3’)-GFP (lower right panel) cells. GFP fusion
proteins were observed in lateral roots using a confocal laser scanning microscope.
Figures are overlay images of the GFP and PI (propidium iodide) images. PI was used
to stain nuclei and cell walls).
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
probe
- + - - - - - - - - -antibodyOs RAVL1 - + +
R-B3
Os RAVL1
Os RAVL1+antibody
- + - + - + - + R-AP2 R-B3 m1 m2
A
RAV1-AP2: GAATTCTGGGAAGAACAACAGTAGTGGTAAGCTTRAV1-B3 : GAATTCTGGCCGAAACACCTGAGTTCGTAAGCTTRAV1-B3-m1: GAATTCTGGCCGAAACACCTAAGTTCGTAAGCTTRAV1-B3-m2: GAATTCTGGCCGAAACACCAGAGTTCGTAAGCTT
Probe AT AC CA GA AA AG GC TA CG
Os RAVL1 - + - + - + - + - + - + - + - + - +
B
probe : GAATTCTGGCCGAAACANNTGAGTTCGTAAGCTT
Supplemental Figure 6. EMSA to test binding target sequences of Os RAVL1.
(A) The left panel shows the super shift of the DNA-protein-antibody complex and the
right panel shows the binding affinity of Os RAVL1 to the target element of the B3
domain of At RAV1. The arrow indicates shifted or super shifted DNA-protein
complexes. The sequences of the probes used are shown at the bottom of the figure.
(B) All possible E-box motifs were examined by EMSA. The two-base sequences listed
as “probe” are the possible combinations for 'NN' in the probe sequences shown at the
bottom of the figure.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
Labeled P1 1 1 1 1 P2 1 1 1 1
RAVL1 + + + + + + + +
Unlabeled P2 0 1 5 25 P1 0 1 5 25
※ 1=40ng
A
Labeled D2-A 1 1 1 1 BRD1-A 1 1 1 1
RAVL1 + + + + + + + +
Unlabeled BRD1-A 0 1 5 25 D2-A 0 1 5 25
B
Labeled D11-A 1 1 1 1 BRD1-A 1 1 1 1
RAVL1 + + + + + + + +
Unlabeled BRD1-A 0 1 5 25 D11-A 0 1 5 25C
Supplemental Figure 7. Binding competition assays of RAVL1 target sequences.
A series of concentrations of an unlabeled probe were added to the binding reaction of
RAVL1 with a labeled probe.
(A) The comparison of the strength of the binding of the P1 and P2 probes to RAVL1
revealed that P1 binding is stronger than P2 binding.
(B) BRD1-A binding is stronger than D2-A binding.
(C) BRD1-A showed a binding affinity that is similar to that of D11-A.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
A
WT d2-2 d2-2;OX OX
D
E
WT OX d2-2 d2-2;OX
F
WT OX d11-1 d11-1;OX
WT d11-1 d11-1;OX OX
BWT OX bri1 d61-1 bri1d61-1;OX
0
20
40
60
WT d d;OX OX
Hei
ght (
cm)
d61-1 x OXd2-2 x OXd11-1 x OX
C
Supplemental Figure 8. Phenotypes of RAVL1 overexpression in BR mutants.
(A) Double genetic combinations of d2-2 and RAVL1 overexpressor (OX) were shown
along with wild type, d2-2, and OX. Plants were grown for 3 weeks in the greenhouse.
(B) Double genetic combinations of d11-1and RAVL1 overexpressor (OX) were shown
along with wild type, d11-1, and OX.
(C) The heights of the four genotypes from each genetic combination. Error bars are SE
of the means of 5-10 plants.
(D-F) Skotomorphogenesis phenotypes of double combinations of bri1 d61-1 (D), d2-2
(E), and d11-1 (F) with RAVL1 overexpressors (OX).
Plants were grown in soil for two weeks in the dark. The arrows indicate nodes.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
0 1 10 100 1000
d2-2
WT
d2-2;OX
OX
A
d11-1;OX
d11-1
WT
OX
0 1 10 100 1000
B
C
0
1
2
3
4
5
BRI1
Rel
ativ
e ra
tio
WTravl1-1bri1-D;ravl1-1bri1-D
0
50
100
150
200
0 1 10 100 1000
Ang
le(°
)
WTd2-2d2-2;OXOX
0
50
100
150
200
0 1 10 100 1000
Ang
le (°
)
WTd11-1d11-1;OXOX
Supplemental Figure 9. Lamina inclination in OX;d2-2 and OX;d11-1 plants exposed
to epiBL, and expression of BRI1 in bri1-D and bri1-D;ravl1-1 plants.
(A and B) The lamina inclination assay was performed with OX;d2-2 (A) and OX;d11-1
(B). The indicated amounts of epiBL in 1 μl of ethanol were spotted on the tip of the
second lamina of 6-day-old seedlings (top panels). The graph shows measurements of
lamina inclinations in response to epiBL. Error bars are SE of the means of 10 plants
(bottom panels).
(C) The expression levels of BRI1 were examined in wild type, Uravl1-1U, bri1-D, and
bri1-DU;ravl1-1 Uplants of the F2 generation which was the result of a genetic cross
between Uravl1-1U and bri1-D. 25S rRNA was used control to normalize the expression
data. Error bars are SE of the means of three qPCR replicates.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
Precursors
Intermediates
Brassinolide
Feedback Regulation
BRI1Signal Activation
OO
H
OH
OH
HO
HO
RAVL1Basal Activation
P450
P450
Supplemental Figure 10. Functional model of RAVL1 in BR homeostasis.
RAVL1 ensures the basal activity of the BR signaling and biosynthetic pathways by
activating BRI1 and the biosynthetic genes that participate in the BR antagonistic
relationship. However, it appears that RAVL1 might be not involved in the feedback
suppression of these genes.
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 . Supplemental Table 1. The list of primers used for real-time RT-PCR Primer Primer sequence Gene name
OSR-F 5′– TCC TCA CCA ACT CCA CAT TAC GGT–3′
OSR-R 5′– CAG ATC GAG ATC CAA CGA GGA–3′
RAVL1
25SR-F 5′– AAG GCC GAA GAG GAG AAA GGT–3′
25SR-R 5′–CGT CCC TTA GGA TCG GCT TAC–3′
25SR-R1 5′– TTG GCG GGC CGT TAA GCA GAA AAG A –3′
25S Ribosomal RNA
D2-F 5′– ATG TGA TAA CAG AGA CGC TGC GGT –3′
D2-R 5′– TGG TGA CCA AGT GGT GAA GGA AGA –3′
D2
D11-F 5′– AGT GAA GAG GGA GCA TGA AGG CAT –3′
D11-R 5′– ATC TGC AGG GCT GAA ATT GTT GGG –3′
D11
BRD1-F 5′– GAG AAG AAC ATG GAA TCA CAT CCA –3′
BRD1-R 5′– TCA GTA ATC TTG AAC GCG GAT ATG –3′
BRD1
OsBRI1-F 5′–CAGCTACTTGGCTATCTTGAAGCTCAGC –3′
OsBRI1-R 5′–CCATTCTTGTTGAAGGTGTACTCCGTGC –3′
BRI1 (Shimada et al., 2006)
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 . Supplemental Table 2. The list of primers used for ChiP and EMSA Primer sequence Gene name
CON1-F 5′–CTG ATC AGG CGA TCA CTG CAC–3′
CON1-R 5′–CGA GCT AGA AAG GAC GCT TAG–3′
BRI1 promoter
BRI1P1-F 5′–CTC CTC CTC ACC CGG CTT AAG–3′
BRI1P1-R 5′–TTA AAT GTG CAT GAC ACA CCA G–3′
BRI1 promoter
BRI1P2-F 5′–ATT CTC CCG CGC CGC GCC GGA TTT T–3′
BRI1P2-R 5′–CCC AAA ATA TTA TAC CAT CT–3′
BRI1 promoter
ACTIN-F 5′– ACT ACA TAG ATA CAC ATA CTT AAC A –3′
ACTIN-R 5′– TAG CTC CCA CAC ACT AAC ATA ATT G –3′
ACTIN promter (Os03g0718100)
D11A-F 5′–GCG CAT AAG CTT CAT CAG ATT C–3′
D11A-R 5′–CAG AGT AGC TAG CAT CTA GGC TG–3′
D11 promoter
D2A-F 5′–GGT TGA AAT AAC GGG AAG CGT–3′
D2A-R 5′–GTT TAA AAC AGG CCC TAA ATC ATC–3′
D2 promoter
D2B-F 5′–TTA AAA CAT CAT CGA CCA ATA GTG–3′
D2B-R 5′–AAG TCT CAC CTC ACC TCC TCG TG–3′
D2 promoter
BRD1A-F 5′–ATA CAC ATT TGA AGT ATT AAA CGT–3′
BRD1A-R 5′–AAA ATC AAT TGT ACA GTT TGC A–3′
BRD1 promoter
Supplemental Data. Je et al. (2010). Plant Cell 10.1105/tpc.109.069575 .
Supplemental Table 3. The list of primers used for vector construction in transient assays.
Primer sequence Gene name
TEVR-F 5′–GGA GAT GGA GCA GGA GCA GGA TG–3′
TEVR-R 5′–TCA CAG ATC GAG ATC CAA CGA GG –3′
RAVL1
TBZR1-F 5′–GGA GCT GGT GGG TAT GAC GTC –3′
TBZR1-R 5′–ATG GCC GTC ATT TCG CGC CGA –3′
BZR1
TPBRI1-F 5′– GCG TAC ACA ATT CTT ACT GTC –3′
TPBRI1-R 5′– GTA CGA GCG AGC TCA CTG CCT –3′
BRI1 promoter
TBRD1-F 5′– CGA TTC ACG ACG AGC TTC ACC –3′
TBRD1-R 5′– CTT CTT CTT CCT CCT CTC TTG GCT A –3′
BRD1 promoter
