Supplemental Figures and Tables - Plant Physiology · Supplemental Figure 3. Immunoblotting detects the specificity of the OsbZIP23 antibody. Wild type (ZH11), osbzip23 and over expressed

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



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


ZH11D 4260 1548

Distribution of OX-OsbZIP23N Peaks


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


% In

put�


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


% In

put�


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


% In

put�


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�


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


% 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


% 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


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


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



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

Documents

Supplemental Figures and Tables - Plant Physiology · Supplemental Figure 3. Immunoblotting detects the specificity of the OsbZIP23 antibody. Wild type (ZH11), osbzip23 and over expressed