A Maize Gene Regulatory Network For Phenolic Metabolism · A Maize Gene Regulatory Network For...

A Maize Gene Regulatory Network For Phenolic Metabolism

Fan Yanga,b, Wei Lib,c, Nan Jianga,b, Haidong Yua,b, Kengo Morohashia,b, Wilberforce

Zachary Oumaa,b,d, Daniel E Morales-Mantillab,c,e, Fabio Gomez-Canoa,b, Eric Mukundia,b,

Luis Daniel Prada-Salcedob,c, Roberto Alers Velazquezb,c,e, Jasmin Valentinb,c,e, Maria

Katherine Mejía-Guerraa,b, John Grayf, Andrea I Doseffb,c, Erich Grotewolda,b

aCenter for Applied Sciences (CAPS), The Ohio State University, Columbus, OH 43210; bDepartment of Molecular Genetics, The Ohio State University, Columbus, OH 43210; cDepartment of Physiology and Cell Biology, Heart and Lung Research Institute, The

Ohio State University, Columbus, OH 43210; dMolecular, Cellular, and Developmental Biology (MCDB) Graduate Program, The Ohio

State University, Columbus, OH 43210. eSuccess in Graduate Education (SiGuE) Program, The Ohio State University, Columbus,

OH 43210. fDepartment of Biological Sciences, University of Toledo, Toledo, OH 43560.

A gene-centered approach based on the yeast one-hybrid assay revealed ~1,100

transcription factor-DNA interactions associated with the regulation of maize phenolic

compound biosynthesis. Results were validated by a combination of chromatin

immunoprecipitation and transient regulation. Several transcription factors recognize the

promoters of 10 or more genes, suggesting a role in coordinating phenolic metabolism.

1To whom correspondence should be addressed. E-mail, [email protected]; FAX :

(614) 292-8037; TEL (614) 292-2483

918 bps

E1 PAL3: GRMZM2G160541

-801 +116 +119 -144

-855 +122 +134

978 bps


-415 -792

+123 958 bps


-841 +116

903 bps

E1

PAL5: GRMZM2G081582

-763 +139 +178 +587 +2177

E2 ?

Cloned region

CAGE tags

TSS CAGE

TSS Annotation

LINEs

Dust

MuDR

Copia

hAT

TRF

ADN/EnSpm

Unknown

ADN/hAT

Gypsy

Helitron

RTE

DNA-TA

DNA/Harbinger

919 bps


-816 +102 +167 -549

-889 +93

C4H1: GRMZM2G147245

+111 982 bps

E1 -237 -472 -747

+906

904 bps


-870 +33 +100 -42 -187 -459 -645

941 bps

E1 C4H2: GRMZM2G139874

-938 +2 -39 +61 -174 -501 +187 -588

P1 P2

-402 -519

-886 +34 921 bps


-151 +109 -449 -683

P1 P2

-568 -648

P1 P2

-456 -535

P1 P2

-324 -414

Supplemental Figure 1

953 bps


-794 +158 -552 -6 -53 +159

P1 P2

-289 -398

951 bps


-874 +76 -345 +113 -392 -618 -298

P1 P2

-374

+126 961 bps

E1 4CL2: GRMZM2G174574

-934 +126

-517

500 bps


-490 +9 +206 -101 -262

902 bps

E1 HCT5: GRMZM2G158083

-679 +221 +247 -164 -410

942 bps


-787 +156 -199 -275 -504 +213

+84 957 bps


-875 +84

ATG

+208 -431 -516 -656 -716

921 bps

HCT11: GRMZM2G156296

-932 -12

+82 -56 -217 -408 E2 E1

+512 +9124 +7682

500 bps


-794 -296 +59

+43 1811 bps

E1 +480

+566

HCT13: GRMZM2G129266

-1244

922 bps

E1 CCoAOMT1: GRMZM2G033952

-890 +31 +82 -234

922 bps

E1 4CL1:GRMZM2G055320

-996 -76 +143 -339

ATG ATG

+243 +906 -407

500 bps


-550 -51 +145 -396

P1 P2

-338 -216

P1 P2

-352 -492

P1 P2

-220 -308

P1 P2

-245 -343

Supplemental Figure 1 (cont.)

910 bps

E1 CCR1: GRMZM2G099420

-827 +82 +113 -585

910 bps


-735 +173 +196

937 bps

E1 CAD1: GRMZM2G046070

-827 +109 -133 +123 -63

907 bps

E1 CAD3 (bm1): GRMZM5G844562

-838 +68 +142 -154 -417 -628 -683 -765

924 bps

E1 CCoAOMT2: GRMZM2G099363

-844 +79 +104 -614

941 bps

ALDH5: GRMZM2G097706

-846 +94

-391 +111 -712 E2 E1

901 bps

E1 F5H1: AC210173.4_FG005

-840 +60 +103 -164 -511

953 bps

E1 COMT2: GRMZM2G082007

-936 +13 +359 -419 -263 -340 +15

P1 P2

-377 -452

P1 P2

-230 -329


921 bps


-654 +266 +100 -237 -19 +151

E2 +324

ATG ATG

-324 -420 -566

P1 P2

-128 -216

901 bps

E1

ALDH1 (rf2): GRMZM2G058675

-735 +165 +190 -163 +2770

E2 +290

P1 P2

-242 -344

812 bps

E1

COMT1 (bm3): AC196475.3_FG004

-810 +1 +114 -83 -392 -725

P1 P2

-460 -558

WHP1: GRMZM2G151227

941 bps

E1

-787 +51 -472 +119 -498 -667

E2

+63 1095 bps

E1 -358 -775 -1004

CHI1: GRMZM2G155329

-1080

929 bps

E1 F3H1 (fht1): GRMZM2G062396

-866 +62 +115 -788

869 bps

E1 FNS1 (fnsi1): GRMZM2G099467

-770 +98 +110 -164

927 bps

E1 F3’H (pr1): GRMZM2G025832

-933 -4 +59

982 bps -982 -1

E1 F3’H3: GRMZM2G313750

-184 -283 +37

FLS2: GRMZM2G069298

+34 1500 bps

E1 -491 -709 -1326

-1464 +33

+36 969 bps

E1

FLS1: GRMZM2G152801

-934 +35

-530 -872

P1 P2

-532 -652

915 bps

E1 F3’H2: GRMZM2G160763

-851 +63 +66 -189 -327 -488

P1 P2

-97 -197

-256 -380 794 bps

E1 C2: GRMZM2G422750

-781 +12 +725 -673

P1 P2


+23 988 bps

E1 F3H2: GRMZM2G146234

-976

A1: GRMZM2G026930

+87 1866 bps -776

E1 E2 +220 +315 +497

-1370

-135 -763

+496

A2: GRMZM2G345717

-1186 +4

1122 bps

E1 -167 -554

438 bps

E1 A4: GRMZM2G013726

-329 +106 +43 -101 -262

911 bps

E1 ANR1: GRMZM2G097841

-738 +173 +241 -699

500 bps

E1 Bz1: GRMZM2G165390

-491 +8 +81 -344

500 bps

E1 Bz2: GRMZM2G016241

-582 -83 +7 -136 -337

+1134 992 bps

E1

-28 -537

RHM1 (sm1): GRMZM2G031311

-1018 -217 -293

937 bps

E1 RHM2: GRMZM2G072911

-887 +49 -233 +112 -63 -360

+890 997 bps

E1

-48 -967

RHM4: GRMZM2G166767

-1039

-245 -161

P1 P2

P1 P2

-46 -558


+953 1000 bps

E1 +1 +502 +88

RHM3: GRMZM2G044281

ATG

+825 -46

+551 +599 +843

ATG

+952

Supplemental Figure 1. Graphic representation of the gene promoters used as baits in the Y1H screens. Representation of the promoters of the 54 genes in the phenolic biosynthetic pathway used in this study. Gene name and model are shown in the top. Transcription start sites (TSSs; indicated by +1) were obtained from CAGE data (red arrows) (Mejia-Guerra et al., 2015), or from the latest genome annotation (V3.0, http://www.maizegdb.org/) (black arrows), respectively. Repetitive elements (RE) present in the cloned regions used in the Y1H assays (represented by a blue line) are in accordance with the annotation available in MaizeGDB and are represented by the colored boxes following the criteria shown in the legend box. Exons (E; gray rectangles) are linked with UTRs (thinner gray rectangles). P1 and P2 represent the primers used for ChIP-qPCR experiments.

Num

ber o

f pla

smid

s pe

r ng

1.0E+00

1.0E+02

1.0E+06

1.0E+04

ALF

18O

FP41

MY

B11

8H

AG

15M

YB

19

GR

AS

70

YH

AB

5bH

LH14

5LB

D9

HS

F3W

RK

Y82

OR

PH

AN

314

ZIP

84

GB

P17

ZIM

32

bHLH

10M

YB

108

C3H

21

Supplemental Figure 2. Representation of TFs in the AD-TF library.Number of plasmid molecules present per ng of AD-TF library, as determined by qPCR using specific standards, are shown in blue. Number of promoters to which each TF was found to bind are shown in red. Values represent means±SE of three replicates.

1.0E+01

1.0E+05

1.0E+03

0

20

15

10

5

25

30

Num

ber o

f pro

mot

ers

in

whi

ch T

F w

as id

entif

ied

bHLH

1

LBD

1LI

M6

LBD

20LB

D35

BA

F60.

21bH

LH17

7C

PP

8M

YB

31M

YB

40

OR

PH

AN

249

HB

112

MY

B3

Number of plasmids per ng

Random selected TFs TFs of interest

Number of promoters in which TF was identified

PAL5

PAL1

CO

MT2

F5H

1

CC

R4

CC

R3

CAD

3 (b

m1)

ALD

H5

CC

R1

CO

MT1

(bm

3)

ALD

H1

(rf2)

CAD

1

CC

oAO

MT2

CC

oAO

MT1

HC

T13

PAL2

4CL3

HC

T12

HC

T11

HC

T10

HC

T6H

CT5

4CL4

4CL2

4CL1

C4H

1PA

L9PA

L8PA

L7PA

L6

PAL4

PAL3

F3’H

2F3

’H (p

r1)

FNS1

(fns

i1)

F3H

2 F3

H1

(fht1

)C

HI1

WH

P1C

2

A4A1FLS2

FLS1

F3’H

3

RH

M3

RH

M4

RH

M2

RH

M1

(sm

1)Bz

2Bz

1AN

R1

A2C4H

2

Promoter TestedGeneral

Lignins Flavonoids Transcription Factors

Phenylpropanoids

Frequency

Direct Interactions

0

0.4

0.2

0.1

0.3

Supplemental Figure 3. Heat map representing PDIs identified by Y1H. Genes encoding phenolic pathway enzymes are shown on top. The 568 TFs identified as recognizing one or more of the promoters indicated in the columns are listed on the right. The brown-yellow gradient represents PDIs identified from the Y1H screenings, with the color corresponding to the frequency by which a particular TF was identified. Blue represent PDIs identified from Y1H directed assays. TFs are ordered from most (top) to least (bottom) interactions. All the data used to generate this heat map is available as Supplemental Table 2.

*

*

*

0 2015105

*

*

0 252015105

*

0

*

54321A B

SD-His-Ura-Leu+ 3AT (mM)

Fold enrichment over input

p35S::GFP-JMJ13p35S::GFP

p35S::GFP-MBF1.3p35S::GFP

p35S::GFP-GBP20p35S::GFP

Copia

ALDH1

4CL3

C2

Copia

4CL3

PAL5

Bz2

Copia

FLS1

CCoAOMT2

C2

*

*

*

0 12963 15

p35S::GFP-DOF28p35S::GFPCopia

4Cl3

CCR3

HCT10

C2

2

567

8

43

1

HCT10 CCR3

C2 4CL3

1,3,5,7: EV2,4,6,8: AD-DOF28

21

56 3

4

1,3,5: EV2,4,6: AD-GBP20

FLS1CCoAOMT2

C2

21

5

Bz2

4CL3 PAL5

21

5

C2

ALDH1 4CL3

1,3,5: EV2,4,6: AD-JMJ13

1,3,5: EV2,4,6: AD-MBF1.3

6 34

6 3

4

60

80 60

60 20

100 60

40

40

60 60

SD-His-Ura-Leu

60

120

Supplemental Figure 4. Comparison of ChIP and Y1H validation of select PDIs(A) Plates show the growth of various promoter bait yeast strains transformed with AD-JMJ13, AD-MBF1.3, AD-GBP20 and AD-DOF28 in non-selective (SD-His-Ura-Leu) and selective (SD-His-Ura-Leu +3-AT) media. AD empty vector (EV) was used as a negative control. (B) Validation of Y1H-identified PDIs in maize protoplasts transformed with p35S::GFP-TF by ChIP-qPCR using αGFP antibodies. Fold enrichment was calculated using the Copia genomic regions as control, and normalizing to the respective input controls (see Methods). Values represent the mean and SE of three independent biological replicates (represented by transformation of three different protoplast preparations from seedlings grown at different times). Statistically (two-sided Student t-test) significant values are indicated with an asterisk (p<0.05).

LBD35 LBD24 LBD20

DOF28 MYB65CPP8

NLP14 bZIP63 MYB19

C3H42 MBF1.3

General Phenylpropanoids

Flavonoids

Lignins

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM

Guaiacyl lignin Syringyl ligninp-Hydroxyphenyl

C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR

Stablization and vacuolar sequestration

ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

RHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM

Guaiacyl lignin Syringyl linginp-Hydroxyphenyl

C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

PAL C4H

4CL

CHS

CHI

F3’H

DFR

FNSF3HFLS

FLS FNSF3H

DFR

F3’H

LDOX/ANS

UGT

F2H

Phlobaphene Maysin

CGTUGTRHM


C3H CSE

HCT

CAD CADCADCAD

4CLCCoAOMT

CCRF5H COMT

F5H COMT

CCR

GST

HCT

lignin

ANR


ALDHALDH

Supplemental Figure 5. Distribution of candidate target genes for those TFs binding the largest number of promoters.Representation of the phenolic pathway shown in Figure 1 indicating in red which of the biosynthesis genes are targeted by each of the indicated TFs.

Supplemental Table 1. Yeast bait strains representing genes encoding main enzymes of the phenolic pathway.

Bait strain name

Promoter gene name

Promoter gene identifier

[3-AT] used in

Y1H screen (mM)

Number of transformants

screened

Number of TFs

validated

Y346 PAL1 GRMZM2G074604 100 94 10 Y062 PAL2 GRMZM2G441347 20 94 1 Y171 PAL3 GRMZM2G160541 100 94 2 Y153 PAL4 GRMZM2G063917 80 94 3 Y347 PAL5 GRMZM2G081582 80 94 3 Y349 PAL6 GRMZM2G118345 40 94 0 Y096 PAL7 GRMZM2G170692 40 94 2 Y185 PAL8 GRMZM2G334660 80 94 1 Y108 PAL9 GRMZM2G029048 60 94 4 Y258 C4H1 GRMZM2G147245 80 144 4 Y221 C4H2 GRMZM2G139874 80 113 10 Y143 4CL1 GRMZM2G055320 40 96 17 Y092 4CL2 GRMZM2G174574 20 96 10 Y050 4CL3 GRMZM2G096020 60 96 10 Y028 4CL4 GRMZM2G122787 40 96 7 Y114 HCT5 GRMZM2G158083 60 96 17 Y285 HCT6 GRMZM2G035584 80 96 31 Y149 HCT10 GRMZM2G034360 80 96 34 Y140 HCT11 GRMZM2G156296 80 192 22 Y033 HCT12 GRMZM2G179703 40 96 14 Y269 HCT13 GRMZM2G129266 100 96 11 Y330 CCoAOMT1 GRMZM2G033952 100 192 2 Y335 CCoAOMT2 GRMZM2G099363 100 96 21 Y159 CCR1 GRMZM2G099420 40 96 2 Y279 CCR3 GRMZM2G131205 40 306 36 Y214 CCR4 GRMZM2G131836 20 144 8 Y112 CAD1 GRMZM2G046070 100 96 13 Y178 CAD3 (bm1) GRMZM5G844562 40 96 7 Y188 ALDH1 (rf2) GRMZM2G058675 60 96 28 Y319 ALDH5 GRMZM2G097706 80 96 26 Y239 F5H1 AC210173.4_FG005 80 408 22 Y332 COMT1 (bm3) AC196475.3_FG004 80 1670 159 Y302 COMT2 GRMZM2G082007 100 288 8 Y203 C2 GRMZM2G422750 100 290 50 Y255 WHP1 GRMZM2G151227 80 138 2 Y316 CHI1 GRMZM2G155329 40 94 10 Y081 F3H1 (fht1) GRMZM2G062396 40 94 2 Y183 F3H2 GRMZM2G146234 40 92 4 Y161 FNS1 (fnsi1) GRMZM2G009467 80 94 13 Y296 F3'H (pr1) GRMZM2G025832 80 94 5 Y206 F3'H2 GRMZM2G160763 40 138 5

Y150 F3'H3 GRMZM2G313750 100 92 11 Y323 FLS1 GRMZM2G152801 100 94 5 Y271 FLS2 GRMZM2G069298 40 94 12 Y230 A1 GRMZM2G026930 80 559 83 Y098 A4 GRMZM2G013726 80 192 7 Y341 A2 GRMZM2G345717 40 94 2 Y326 ANR GRMZM2G097841 60 94 2 Y339 Bz1 GRMZM2G165390 100 461 129 Y350 Bz2 GRMZM2G016241 80 94 27 Y077 RHM1 (sm1) GRMZM2G031311 60 164 11 Y227 RHM2 GRMZM2G072911 80 96 14 Y344 RHM3 GRMZM2G044281 100 94 2 Y337 RHM4 GRMZM2G166767 40 94 3

TOTAL 8999 944

A Maize Gene Regulatory Network For Phenolic Metabolism · A Maize Gene Regulatory Network For...

Documents

Transcript of A Maize Gene Regulatory Network For Phenolic Metabolism · A Maize Gene Regulatory Network For...