Post on 22-Jul-2020
S26
S98 S106
T143
Supplemental Figure 1. Protein sequence alignment of OsbZIP23, OsbZIP46, and TRAB1.
The red rectangles represent the Ser/Thr in the conserved RQXS/T motif, and the blue rectangle indicates the conserved Ser102 of TRAB1. The numbers represent the Ser/Thr location sites in
OsbZIP23.
OsbZIP23 357 AAs
S26 S98 S106 T143Ala, A
Point Mutation
S26A S98A S106A T143A
CaMV35S Ω NOSGAL4BD
CaMV35S Ω NOSGAL4BD OsbZIP23
GAL4BD
OsbZIP23-GAL4BD
CaMV35S Ω NOSSAPK2SAPK2-None
CaMV35S 5XGAL4-TATA NOSFirefly LUC35S-GAL4-fLUC
AtUbi3 NOSRenilla LUCAtUbi3-rLUC
Effector
Reporter
Internal Control
OsbZIP23S26A-GAL4BDOsbZIP23S98A-GAL4BDOsbZIP23S106A-GAL4BDOsbZIP23T143A-GAL4BD
OsbZIP23S26AS98A-GAL4BDOsbZIP23S26AS98AS106A-GAL4BDOsbZIP23S26AS98AT143A-GAL4BDOsbZIP23S26AS98AS106AT143A-GAL4BD
A
B
C
DRelative Fold Enrichment
0 1 2 3 4 5 6 7
GAL4BD/35S-GAL4-fLUC
OsbZIP23-GAL4BD/35S-GAL4-fLUC
OsbZIP23S26A-GAL4BD/35S-GAL4-fLUC
OsbZIP23S98A-GAL4BD/35S-GAL4-fLUC
OsbZIP23S106A-GAL4BD/35S-GAL4-fLUC
OsbZIP23T143A-GAL4BD/35S-GAL4-fLUC
OsbZIP23S26AS98A-GAL4BD/35S-GAL4-fLUC
OsbZIP23S26AS98AS106A-GAL4BD/35S-GAL4-fLUC
OsbZIP23S26AS98AT143A-GAL4BD/35S-GAL4-fLUC
OsbZIP23S26AS98AS106AT143A-GAL4BD/35S-GAL4-fLUC
**
Relative Induction Levels
0 1 2 3 4 5 6 7
SAPK2-None/OsbZIP23-GAL4BD/35S-GAL4-fLUC
SAPK2-None/OsbZIP23S26AS98A-GAL4BD/35S-GAL4-fLUC
SAPK2-None/OsbZIP23S26AS98AS106A-GAL4BD/35S-GAL4-fLUC
SAPK2-None/OsbZIP23S26AS98AT143A-GAL4BD/35S-GAL4-fLUC
SAPK2-None/OsbZIP23S26AS98AS106AT143A-GAL4BD/35S-GAL4-fLUC
**
Supplemental Figure 2. Effects of Ser/Thr substitution on SAPK2 mediated transcriptional activity activation of OsbZIP23. A, Schematic digram of amino acid substitution of OsbZIP23.
B, Scheme of the constructs used in the rice protoplast co-transfection assay. C, Ser/Thr
substitution significantly decrease the transcriptional activation activity of OsbZIP23 under ABA treatment. The relative fold enrichment represents the luciferase activity changes between
normal and ABA treatment conditions. D, Ser/Thr substitution significantly decreases the
transcriptional activation caused by SAPK2 under normal condition. The values in each column are the mean of three independent replicates and error bars indicate SD. The asterisks represent
a significantly difference determined by the Student’s t test. Two asterisks represents t< 0.01.
Supplemental Figure 3. Immunoblotting detects the specificity of the OsbZIP23 antibody. Wild
type (ZH11), osbzip23 and over expressed OsbZIP23 (OX-OsbZIP23) seedlings under both normal and drought stress conditions were used for nuclear protein extraction. OsbZIP23
antibody was made by Abmart (Shanghai, China) Company. After transformation, the PVDF membrane was blocked by non-fat milk and then incubated with OsbZIP23 antibody (1:5000).
Finally, HRP conjugated goat anti-Rabbit 2nd antibody were used. The signal was detected by
X-ray film (FUJIFILM).
anti-OsbZIP23
ZH11D
osbzip23N DN DN
OX-OsbZIP23
40kD
40kD
55kD Coommassie Blue G-250
Supplemental Figure 4. Statistical analysis of the length of the OsbZIP23 bound regions.
Sample Peak Numbers Average Length
ZH11N 1738 1413
Distribution of ZH11N Peaks Distribution of ZH11D Peaks
Sample Peak Numbers Average Length
ZH11D 4260 1548
Distribution of OX-OsbZIP23N Peaks
Sample Peak Numbers Average Length
OX-OsbZIP23 11611 1338
A B
C
Supplemental Figure 5. Functional categories of OsbZIP23 bound genes in drought stress.
OsbZIP23
Abiotic Stress
OsPIP2;6OsAPx8DWA1
OsWRKY45OsGPX3
OsMSRA4.1OsAPX1/APXa
SNAC1OsMSD
OsNAC5SQS
OsFER2OsFER1
OsARF16OsSRO1c
GF14c
OsbZIP23OsLti6bOsOAT
OsDREB1COsP5CSOsTZF1OSBZ8OsDhn1
OsGRAS23OsHsp70CP1
OsADC1Oshox22
OsClpB-cytOsMAPK6OsbZIP72OsZIP-2a
OCPI1OsRCI2-5OsLEA3-2OsLEA25
Rab16A/RAB21EFA27
OsHsfC1bAP59/OsBIERF3
DSM1DSM2
OsSEC24OsMAPK5OsETOL1OsCam1-1
RERJ1OsLti6a
Environmental Responses
GF14bOsWRKY45
OsCBPOsRING-1OsLecRK1ONAC131Sci1/PR6OsSSI2
AP59/OsBIERF3OsPHGPXOsBWMK1
HSP90OsNDPK1OsSDF2-1
Biotic StressDevelopment
Embroy/SeedOsAPL1/OsAGPL3
PDIL1TRAB1
OsALDH7RDD1AlaATVP1
OsVPE1GIC
OsISA2OsGRAS-32/D62/GS6
WxOsMAPK6SBE1/BEI
LP2ZN
NAL1/qFLW4NOL
NYC1SLAC7
OsYABBY4FCP1/OSCLE402
GLR1OsSIG5WSL1
OsCAO1
Leaf
Flowersped1-DOsCO3
OsCCT11OsBIP116bOsMADS18
GF14c
Shoot
OsGLU1RSG
Root
NAL1/qFLW4OsGPX3
OsKTN80c
ABA
MHZ4ZEBRA2/MHZ5
PSY1OsNCED4OsLCYe
OsPP2C09OsPP2C06OsPP2C68
DSM2SAPK3
Hormone ResponsesEthylene
OsACS2OsACS5
AP59/OsBIERF3OsETOL1OsBIERF1OsRTH2
GAOsGRAS-32/D62/GS6
OsRR9OsHK5
Cytokinin
OsBZR1 BR
AuxinOsARF16OsGH3-2OsCYP2
RERJ1 JA
Cellular ProcessesTransporters
COPT1OsMST4
KOB1OsFRDL1
OsSPX-MFS3OsSUT2
Metabolism Process
CYP32G2OsAPL3OsDXROsIsu1sps1
OsALSOsRad6OsUAM2OsSIZ1OsPAA2
OsRINGC2-2
ER Stress
OsERdj3APDIL1
Chromatin
HMGB1OsDDM1b
Supplemental Figure 6. OsbZIP23BGDs with known functions in various cellular processes and responses (Supplemental File 1). References for each gene can be found in RicENcode, http://
ricencode.ncpgr.cn.
A
B
0
0.005
0.01
0.015
0.02
0.025
0.03
LOC_Os06g01250P1 LOC_Os06g01250P2
% In
put�
ZH11NZH11DOX-OsbZIP23N
LOC_Os06g01250
OX-OsbZIP23N
ZH11D
ZH11N
P1 P2
0
0.005
0.01
0.015
0.02
0.025
0.03
LOC_Os01g55940P1 LOC_Os01g55940P2
% In
put�
ZH11NZH11DOX-OsbZIP23N
OX-OsbZIP23N
ZH11D
ZH11N
LOC_Os01g55940
P2P1
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
LOC_Os11g09280P1 LOC_Os11g09280P2
% In
put�
ZH11NZH11DOX-OsbZIP23N
LOC_Os11g09280
OX-OsbZIP23N
ZH11D
ZH11N
P1 P2
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
LOC_Os03g19290P1 LOC_Os03g19290P2
% In
put�
ZH11NZH11DOX-OsbZIP23N
OX-OsbZIP23N
ZH11D
ZH11N
LOC_Os03g19290
P1P2
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
LOC_Os06g50920 LOC_Os01g43650 LOC_Os03g60080 LOC_Os01g66120 LOC_Os02g50970 LOC_Os06g10880 LOC_Os08g36790 LOC_Os09g28310 LOC_Os05g46480
% In
put�
ZH11N
ZH11D
OX-OsbZIP23N
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
Actin LOC_Os03g57240P1 LOC_Os03g57240P2 LOC_Os03g57240C
% In
put�
ZH11NZH11DOX-OsbZIP23N
OX-OsbZIP23N
ZH11D
ZH11N
LOC_Os03g57240
C P1 P2
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
0.02
LOC_Os01g42860P2 LOC_Os01g42860P1 LOC_Os01g42860C
% In
put�
ZH11N
ZH11D
OX-OsbZIP23N
OX-OsbZIP23N
ZH11D
ZH11N
P2 P1 C
LOC_Os01g42860
Supplemental Figure 7. ChIP-qPCR verification of the ChIP-Seq results. A, Enrichment of 7 OsbZIP23 bound and 2 unbound genes (LOC_Os06g50920 and LOC_Os01g43650) were
examined by ChIP-qPCR. B, ChIP-qPCR verification of the OsbZIP23 bound genes. The
OsbZIP23 binding profiles in the promoter of these six genes were arranged below the histogram. The primers used in the qPCR assay are represented with black lines in the promoter region. The
values in each column are the means of three independent replicates and error bars represent the
SD. The primers used in this experiment are listed in Supplemental Table S4.
-10
-5
0
5
10
15
20
-10 -5 0 5 10 15 20RN
A-S
eq Z
H11
D/Z
H11
N (L
og2)�
qPCR ZH11D/ZH11N (Log2)�
R² = 0.87709
-2
0
2
4
6
8
10
12
-4 -2 0 2 4 6 8 10 12 14
RNA-
Seq
OX-
Osb
ZIP2
3N/Z
H11N
(Log
2)�
qPCR OX-OsbZIP23N/ZH11N (Log2)�
R² = 0.92072
-10
-8
-6
-4
-2
0
2
4
6
-8 -6 -4 -2 0 2 4 6
RN
A-S
eq o
sbzi
p23N
/ZH
11N
(Log
2)�
qPCR osbzip23N/ZH11N (Log2)�
R² = 0.77169
-6
-4
-2
0
2
4
6
8
10
-4 -2 0 2 4 6 8 10
RNA-
Seq
OX-
Osb
ZIP2
3D/Z
H11D
(Log
2)�
qPCR OX-OsbZIP23D/ZH11D (Log2)�
R² = 0.90885
-6
-4
-2
0
2
4
6
8
-4 -3 -2 -1 0 1 2 3 4 5 6
RN
A-S
eq o
sbzi
p23D
/ZH
11D
(Log
2)�
qPCR osbzip23D/ZH11D (Log2)�
R² = 0.45404
A B
C D
E
Supplemental Figure 8. Correlation analysis between the RT-qPCR and the RNA-Seq data. A to
E, 29 genes were used in the validation of the RNA-Seq results by RT-qPCR. The primers used in this experiment are listed in Supplemental Table S4.
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
ospp2c49-10 ZH11 ospp2c49-23(CK)
Plan
t Hei
ght (
cm)�
2µM ABA
0.0
5.0
10.0
15.0
20.0
25.0
ospp2c49-10 ZH11 ospp2c49-23(CK)
Plan
t Hei
ght (
cm)�
Normal
Nor
mal
2µM
AB
A
ospp2c49-10 ZH11 ospp2c49-23 (CK)
GGGCTCGACCGCCGTTGTCGCGG-TCGTCGGTCC980
GGGCTCGACCGCCGTTGTCGCGGTTCGTCGGTCC
OsPP2C49
ospp2c49-10
A
B
Supplemental Figure 9. The ospp2c49 showed similar ABA sensitivity with the wild-type. A,
Mutation sites of ospp2c49. The underlined sequences indicate the sgRNA and the dash represents the insertion site. B, Performance of ospp2c49-10, ZH11 and control line (ospp2c49-23) in 1/2
strength MS medium containing 2µM ABA. Plant height was measured after growth for 14d. Bar represent the SD (n>6).
0
0.005
0.01
0.015
0.02
0.025
0.03
ZH11
N
ZH11
D
OX-
Osb
ZIP2
3N
ZH11
N
ZH11
D
OX-
Osb
ZIP2
3N
anti-IgG anti-OsbZIP23
% In
put�
DSMP1DSMP2
OX-OsbZIP23N
ZH11D
ZH11NChI
P-Se
qDSM2
P1 P2
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.018
ZH11
N
ZH11
D
OX-
Osb
ZIP2
3N
ZH11
N
ZH11
D
OX-
Osb
ZIP2
3N
anti-IgG anti-OsbZIP23
% In
put�
PSY1P1PSY1P2
PSY1
P1 P2
OX-OsbZIP23N
ZH11D
ZH11NChI
P-Se
q
Shift
Free probe
++ +-
- - -+
OsbZIP23-GST
GST
Non-Labeled Probe
-
+
PSY1 LOC_Os06g51290
++ +-
- - -+
OsbZIP23-GST
GST
Non-Labeled Probe
-
+
Shift
Free probe
LCYe LOC_Os01g39960
++ +-
- - -+
OsbZIP23-GST
GST
Non-Labeled Probe
-
+
Shift
Free probe
DSM2 LOC_Os03g03370
LCYe
P2P1ChI
P-Se
q
OX-OsbZIP23N
ZH11D
ZH11N
0
0.02
0.04
0.06
0.08
0.1
0.12
ZH11
N
ZH11
D
OX-
Osb
ZIP2
3N
ZH11
N
ZH11
D
OX-
Osb
ZIP2
3N
anti-IgG anti-OsbZIP23
% In
put�
LCYeP1LCYeP2
A B
C D
E F
Supplemental Figure 10. OsbZIP23 directly binds to the pomoters of the ABA biosynthesis genes.
A, Validation of OsbZIP23 direct binding sites in the DSM2 promoter by ChIP-qPCR analysis. B,
EMSA showing that OsbZIP23 could directly bind to the promoter of DSM2. C, Validation of OsbZIP23 direct binding sites in the LCYe promoter by ChIP-qPCR analysis. D, EMSA showing
that OsbZIP23 could directly bind to the promoter of LCYe. E, Validation of OsbZIP23 direct
binding sites in the PSY1 promoter by ChIP-qPCR analysis. F, EMSA showing that OsbZIP23 could directly bind to the promoter of PSY1. In the ChIP-qPCR assay, the Rabbit IgG was used as a
control and the primers used in the qPCR assay were represented by black lines in the promoter
region of each gene. In the EMSA assay, 5-, 10- and 30- fold excess non-labeled probe was used for competition.
Supplemental Table S1. Summary statistics for ChIP-Seq library alignment.
Sample Total Reads Mapped Reads Unique Mapped Reads
ZH11N 22269610 9773237 (43.89%) 7797925 (35.02%)
ZH11D 20707873 13053956 (63.04%) 9927059 (47.94%)
OX-OsbZIP23N 20756525 8457039 (40.74%) 6963151 (33.55%)
ChIP-Seq Data RNA-Seq Data
Gene Symbol MSU ID 7.0 ZH11N Peak ZH11D PeakOX-OsbZIP23N
PeakZH11N/OX-OsbZIP23N ZH11N/ZH11D
OsPP2C06 LOC_Os01g40094 N Y Y N/A 3.107146928OsPP2C08 LOC_Os01g46760 N Y Y N/A N/A
OsPP2C09 LOC_Os01g62760 Y Y Y N/A 2.096307912
OsPP2C30 LOC_Os03g16170 Y Y Y N/A 5.2083152
OsPP2C37 LOC_Os04g08560 N N N N/A N/A
OsPP2C49 LOC_Os05g38290 Y Y Y 2.819251871 5.409323663
OsPP2C50 LOC_Os05g46040 N N Y N/A 4.241217471
OsPP2C51 LOC_Os05g49730 N Y Y 2.725393679 9.823846266
OsPP2C53 LOC_Os05g51510 N Y Y N/A N/A
OsPP2C68 LOC_Os09g15670 Y Y Y N/A 6.627762714
Supplemental Table S2. OsbZIP23 directly binds to the promoter of clade A PP2Cs in rice.
Supplemental Table S3. OsPYLs and SAPKs in OsbZIP23BGDs.
ChIP-Seq Data RNA-Seq DataGene Symbol MSU ID 7.0 ZH11N Peak ZH11D Peak OX-OsbZIP23N
PeakZH11N/ZH11D
OsPYLs
OsPYL1 LOC_Os10g42280 N N N N
OsPYL2 LOC_Os06g36670 N N N N
OsPYL3 LOC_Os02g13330 N N N N
OsPYL4 LOC_Os01g61210 N N Y NOsPYL5 LOC_Os05g39580 N N N NOsPYL6 LOC_Os03g18600 N N N -4.019988316
OsPYL7 LOC_Os06g33480 N N N NOsPYL8 LOC_Os06g33640 N N N NOsPYL9 LOC_Os06g33690 N N N NOsPYL10 LOC_Os02g15640 N N N NOsPYL11 LOC_Os05g12260 N N N NOsPYL12 LOC_Os02g15620 N N N N
SAPKSSAPK1 LOC_Os03g27280 N N Y 3.755917517SAPK2 LOC_Os07g42940 N N N NSAPK3 LOC_Os10g41490 Y Y Y NSAPK4 LOC_Os01g64970 N N Y 3.8734785
SAPK5 LOC_Os04g59450 N N Y NSAPK6 LOC_Os02g34600 N N Y 2.06026931SAPK7 LOC_Os04g35240 N N N -2.513927433SAPK8 LOC_Os03g55600 N N N NSAPK9 LOC_Os12g39630 N N N 2.506073579SAPK10 LOC_Os03g41460 N N Y 2.021317306