:: agris arabidopsis thaliana web page:: - interaction between … · 2013. 2. 18. · articles...

ART I C L E S

Interaction between BZR1 and PIF4 integratesbrassinosteroid and environmental responsesEunkyoo Oh1, Jia-Ying Zhu1 and Zhi-Yong Wang1,2

Plant growth is coordinately regulated by environmental and hormonal signals. Brassinosteroid (BR) plays essential roles in growthregulation by light and temperature, but the interactions between BR and these environmental signals remain poorly understood atthe molecular level. Here, we show that direct interaction between the dark- and heat-activated transcription factorphytochrome-interacting factor 4 (PIF4) and the BR-activated transcription factor BZR1 integrates the hormonal andenvironmental signals. BZR1 and PIF4 interact with each other in vitro and in vivo, bind to nearly 2,000 common target genes,and synergistically regulate many of these target genes, including the PRE family helix–loop–helix factors required for promotingcell elongation. Genetic analysis indicates that BZR1 and PIFs are interdependent in promoting cell elongation in response to BR,darkness or heat. These results show that the BZR1–PIF4 interaction controls a core transcription network, enabling plant growthco-regulation by the steroid and environmental signals.

In plants, cell elongation and seedling morphogenesis are controlledby multiple environmental factors and endogenous hormones,including light, temperature, brassinosteroid (BR), gibberellin (GA)and auxin. How these signals regulate largely overlapping cellular andphysiological responses through distinct signalling pathways remainsan outstanding question in plant biology1,2. In particular, BR is requiredfor hypocotyl elongation responses to dark, shade and high temperaturein Arabidopsis3–7. How BR mediates or modulates the environmentalresponses is poorly understood at themolecular level.Light switches the developmental programme of seedlings from

skotomorphogenesis to photomorphogenesis, causing inhibition ofhypocotyl elongation, cotyledon opening and expansion, chloroplastdevelopment and a switch from heterotrophic to photoautotrophicgrowth. Light acts through a suite of photoreceptors, among whichthe red light-activated photoreceptor phytochromes directly interactwith the basic helix–loop–helix (bHLH) transcription factors namedphytochrome interacting factors (PIFs; refs 8,9). PIFs accumulate inthe dark to promote skotomorphogenesis, but are phosphorylated anddegraded on light activation of phytochromes9–12. The activities ofPIFs are also regulated by GA (refs 13,14), the circadian clock andtemperature7,15–17. Thus, PIFs are considered key transcription factorsthat integratemultiple hormonal and environmental signals.BR is known to play a key role in light and temperature regulation

of plant development, as the BR mutants, such as de-etiolated 2 (det2),

1Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, USA.2Correspondence should be addressed to Z-Y.W. (e-mail: [email protected])

Received 13 January 2012; accepted 13 June 2012 ; published online 22 July 2012; DOI: 10.1038/ncb2545

show light-grown morphology and express light-induced genes inthe dark3,4,18,19, and are defective in hypocotyl elongation responsesto high temperature and shade5–7,20. BR signalling is mediated by theBRI1 receptor kinase and its downstream signal transduction cascadethat leads to dephosphorylation and activation of the BZR1 familytranscription factors21,22. Constitutive active forms of BZR1, due toeither increased dephosphorylation by protein phosphatase 2A (PP2A)(bzr1-1D) or reduced binding by the 14-3-3 proteins (BZR1-S173A),suppress the photomorphogenesis-in-the-dark phenotypes and mostof the gene expression changes of the BR-deficient or BR-insensitivemutants, indicating that BR promotes skotomorphogenesis throughBZR1 (refs 19,22–24).The crosstalk between the light and BR pathways is believed to

occur at or downstream of BZR1, because light does not significantlyaffect the levels of BR and BZR1 (ref. 25). Indeed, BZR1 modulatesthe expression levels of many light-signalling components19,25,26. Inaddition, genome-wide protein–DNA interaction analysis revealedBZR1 binding to the promoters of a significant portion of light-regulated genes19, suggesting that BR and light signals converge atthe promoters of common target genes through direct interactionbetween BZR1 and some light-signalling transcription factors. Here,we demonstrate that PIF4 directly interacts with BZR1, and togetherthey programme a central transcriptional network that controls cellelongation and seedling photomorphogenesis. Our genetic evidence

802 NATURE CELL BIOLOGY VOLUME 14 | NUMBER 8 | AUGUST 2012© 2012 Macmillan Publishers Limited. All rights reserved.

mailto:[email protected]://www.nature.com/doifinder/10.1038/ncb2545

ART I C L E S

indicates that this interaction is a key mechanism for BR-dependentgrowth responses to light and temperature.

RESULTSBZR1 interacts with PIF4To test if BZR1 interacts with any light-signalling transcription factors,we carried out transient bimolecular fluorescence complementation(BiFC) assays in tobacco (Nicotiana benthamiana), by co-expressing aBZR1 fused to the carboxy-terminal half of yellow florescent protein(BZR1–cYFP) and light-signalling transcription factors fused to theamino-terminal half of YFP (nYFP). As shown in Fig. 1a, a strong YFPfluorescence signal was observed in the nucleus when BZR1–cYFP wasco-transformed with PIF4–nYFP and PIF1–nYFP, indicating specificBZR1 interactions with PIF4 and PIF1. Co-immunoprecipitationassays using transgenic plants expressing BZR1–CFP (cyan fluorescentprotein) and PIF4–Myc from their native promoters confirmed theirinteraction in vivo (Fig. 1b). In vitro pulldown assays showed thatmaltose-binding protein (MBP)–BZR1 interacted with glutathioneS-transferase (GST)–PIF4 and GST–PIF1 but not GST alone (Fig. 1c),and GST–PIF4 interacted with full length MBP–BZR1, MBP–BZR2and the N-terminal DNA-binding domain but not the C-terminalfragment of BZR1 (Fig. 1d,e,g), whereas MBP–BZR1N interactedwith both the N-terminal fragment and bHLH domain but not theC-terminal fragment of PIF4 (Fig. 1f,h). The results indicate thatBZR1 and PIF4 interact through their DNA-binding domains andthe N-terminal domain of PIF4.

BZR1 and PIFs act interdependently in promoting hypocotylelongationWe examined whether BR response requires PIFs. Members of thePIF family play overlapping roles in promoting skotomorphogenesisand cell elongation. The quadruple mutant (pifq) lacking fourPIFs (PIF1, also known as PIL5, PIF3, PIF4 and PIF5, alsoknown as PIL6) exhibits a constitutive de-etiolation phenotype inthe dark, characterized by short hypocotyl and open cotyledons,similar to the BR-deficient or BR-insensitive mutants11,12. Thepifq mutant was less sensitive to exogenous brassinolide (BL, themost active form of BR) and more sensitive to BR biosynthesisinhibitor brassinazole (BRZ; Fig. 2a,b), suggesting that the loss ofPIFs compromises BR response. However, the pif4 single mutantresponded to BRZ similarly to wild type (Fig. 2b), indicating redundantfunctions of PIFs with regard to BR response. The bzr1-1D gain-of-function mutation causes constitutive dephosphorylation of BZR1by PP2A (ref. 22) and a BRZ-resistant phenotype23. However,the pifq;bzr1-1D quintuple mutant had similar short hypocotylsas pifq grown on the medium with or without BRZ (Fig. 2c,d),suggesting that PIFs are required for the BZR1 promotion of hypocotylelongation in the dark.In contrast to the etiolation-promoting effect observed in the

dark-grown bzr1-1D seedlings, the light-grown bzr1-1Dmutant plantsare weak dwarfs, with shorter hypocotyls and petioles thanwild type23,27

(Fig. 2e,f). It seems that a light-inactivated factor, possibly PIFs, isrequired for BZR1 promotion of cell elongation. Indeed bzr1-1Dincreased hypocotyl elongation in the PIF4-overexpression (PIF4-OX)background under light (Fig. 2e), suggesting that BZR1’s function ofpromoting cell elongation requires PIF4.

InputAnti-Myc

Anti-GFP

IP: anti-GFP

a

c d

b

Input Pulldown

nYFP

At3g60390–nYFP

Inpu

tP

ulld

own

BZR1–cYFP+

Inpu

tP

ulld

own

e

BZR1C

DNA binding domain

bHLH domain

gf

h

PIF4–nYFPPIF1–nYFP

PIF4

p::PI

F4–m

yc

PIF4

p::PI

F4–m

yc;

BZR

1p::B

ZR1–

CFP

MBP

BZR1

N

BZR1

C

BZR1

MBP

BZR1

BZR2

GST

GSTGS

T–PI

F4

GST–

PIF4

C1

PIF1

PIF4

GST

PIF1

PIF4

1 109 334BZR1

BZR1N 1 109

110 334

1 254 313 430PIF4

PIF4N1

PIF4N2

PIF4C1

PIF4C2

PIF4C3

1 313

1 253

254 430

430301

430314

Pul

ldow

nIn

put

GST–

PIF4

C3

GST–

PIF4

C2

GST–

PIF4

N2

GST–

PIF4

N1

Figure 1 BZR1 interacts with PIF4. (a) BiFC assay shows PIFs’ interactionwith BZR1 in tobacco leaf cells. The images show overlays of fluorescenceand light views. (b) Co-immunoprecipitation assay of BZR1 with PIF4. Plantsexpressing BZR1–CFP from native BZR1 promoter and PIF4–Myc fromnative PIF4 promoter or plants expressing only PIF4–Myc were incubated at28 ◦C for 8 h to accumulate PIF4 protein and treated with 100nM BL for1.5 h. BZR1–CFP was immunoprecipitated using anti-GFP (green fluorescentprotein) antibody and immunoblotted using anti-Myc or anti-GFP antibody.(c) PIFs directly interact with BZR1 in vitro. GST–PIF1 and GST–PIF4 werepulled down by MBP–BZR1 immobilized on maltose agarose beads andeluted and analysed by immunoblotting using anti-GST antibody. (d) PIF4interacts with both BZR1 and BZR2 in vitro. MBP–BZR1 and MBP–BZR2were pulled down by GST–PIF4 immobilized on glutathione–agarose beadsand then eluted and analysed by immunoblotting using anti-MBP antibody.(e) The N-terminal DNA-binding domain of BZR1 interacts with PIF4 in vitro.Various fragments of BZR1 fused to MBP were pulled down by GST–PIF4and then eluted and analysed by immunoblotting using anti-MBP antibody.(f) Both N-terminal and bHLH domains of PIF4 interact with BZR1 in vitro.Various fragments of PIF4 fused to GST were pulled down by MBP–BZR1Nand then eluted and analysed by immunoblotting using anti-GST antibody.(g,h) Box diagrams of the various fragments of BZR1 (g) and PIF4 (h) usedin the pulldown assays in e and f. The black boxes indicate the DNA-bindingdomains. The numbers indicate the amino acids. Uncropped images ofblots/gels are shown in Supplementary Fig. S7.

To test whether PIF4 promotion of cell elongation also requiresBR signalling and active BZR1, we introduced PIF4-OX into thebri1-116 single mutant, in which BZR1 is phosphorylated and inactive,

NATURE CELL BIOLOGY VOLUME 14 | NUMBER 8 | AUGUST 2012 803© 2012 Macmillan Publishers Limited. All rights reserved.

ART I C L E S

0.8∗∗

1.2∗∗

Hyp

ocot

yl le

ngth

(mm

)

BL (nM)

∗∗ ∗∗

∗∗

∗∗

∗∗ ∗∗

BRZ (µM)

Hyp

ocot

yl le

ngth

(%)

Hyp

ocot

yl le

ngth

(%)a

e f g

Hyp

ocot

yl le

ngth

(mm

)

1.25∗∗

2.93∗∗0.87∗∗

1.06∗∗+BRZ–BRZ

1.3∗∗

Col-0 pifq

0 1 10 1000

100

200

300

020406080

100120

0.0 0.5 1.0 2.0

+BRZ–BRZ

1.3∗∗

2.2∗∗

BRZM0

5

10

15

20

25 Dark Light

1.3∗∗

12.1∗∗

0

5

10

15

20

Hyp

ocot

yl le

ngth

(mm

)

Col-0

Col-0br

i1

bri1;

bzr1

-1D

bzr1

-1D

PIF4

-OX

PIF4

-OXbr

i1

bri1;

bzr1

-1D

bri1;

PIF4

-OX

bri1;

bzr1

-1D;

PIF4

-OX

Col-0

bzr1

-1D

bri1

bri1;

bzr1

-1D

bri1;

PIF4

-OX

bri1;

bzr1

-1D;

PIF4

-OX

Col-0

bzr1

-1D

pifq

pifq;

bzr1

-1DCo

l-0

bzr1

-1D

pifq

pifq;

bzr1

-1D

Col-0

bzr1

-1D

pifq;

bzr1

-1D

pifq

Col-0

bzr1

-1D

pifq

pifq;

bzr1

-1D

0

5

10

15

20

25b c d

Col-0 pif4 pifq

Col-0 bzr1-1D PIF4-OX bzr1-1D;PIF4-OX

Figure 2 BZR1 and PIFs act interdependently in promoting hypocotylelongation. (a) The pifq mutant is hyposensitive to BL when comparedwith wild type (Col-0). Seedlings were grown on various concentrationsof BL under white light for seven days before hypocotyl lengths weremeasured. Error bars indicate s.d. (n=10 plants) and ∗∗P

ART I C L E S

AT4G38850

AT3G15540 AT3G1 AT

Non-PIF regulatedPIF regulated

Bin

din

g p

eaks

55% 52% 56%

CACGTGCACATGCTATAG

–1,000 0 +1,000

Distance from binding peak

Count

Peak position PIF4

Pea

k p

ositi

on B

ZR

1

Num

ber

of m

otifs

E

nric

hmen

t

ChIP–reChIP

1,5372,826 6,054

CisGenom

e

PRI-CA

TCom

mon

1,2351,800 1,286

Freq

uenc

y

UpDownAll

IAA19

SAUR15

All light regulated (4,919)

638

3,130

461690

0

1

2

Bits

1 2 3 4 5 6 7 8 910

PIF4

PIF4

BZR1

BZR1

171819

161413121110

98764321

End +1,000–5,000 Start–5,000

Start

End

+1,000

0

50

100

150

200

250

300

0

1,000

2,000

3,000

4,000

5,000

–5,000 Start End +1,0000

0.010.020.030.040.050.06

01020304050

PP2A

SAUR

15IAA

19AC

S5

a

e f g h

b c dPIF4 target

(4,363)PIF regulated (7,591)

PIF4 target (4,363)

BZR1 target (3,620)

Figure 3 BZR1 and PIF4 share a large number of genomic targets. (a) TotalPIF4-binding peaks identified by ChIP-seq analyses using two statisticalmethods. Numbers indicate the percentages of binding peaks associatedwith at least one PIF-regulated gene. (b) The Venn diagram shows significantoverlap between PIF4-binding target genes and PIF-regulated genesidentified by microarray analysis of pif4;pif5 or pifq and RNA-seq analysisof pifq. (c) Distribution of PIF4-binding peaks (frequency) relative to genestructure (−5 kb to +1 kb downstream of 3� end). (d) Frequency of showncis elements around the PIF4-binding sites. The sequence logo shows themost enriched motifs in the PIF4-binding regions. (e) Venn diagrams ofPIF4-target genes, BZR1-target genes identified by previous ChIP-chipassay and all light-regulated genes from the light-treatment microarrayassays25. (f) Spatial distribution of PIF4- and BZR1-binding peaks along

the promoter (−5,000 bp to start), coding (start-end, grey) and 3� (end to+1,000bp) regions of common target genes. The frequency of peak pairsis represented with a colour scale. Counts are numbers of peak pairs ina certain area. (g) Representative PIF4- and BZR1-binding peaks in thepromoters of common target genes (IAA19 and SAUR15 ). (h) ChIP–reChIPanalysis shows that BZR1 and PIF4 co-occupy common targets. Thechromatin of 35s-BZR1–myc;35s-PIF4–YFP double transgenic plants wasfirst immunoprecipitated using anti-Myc antibody, and then using anti-GFPantibody, and the precipitated DNA was quantified by qPCR. Enrichment ofDNA was calculated as the ratio between BZR1–myc;PIF4–YFP and wildtype control, normalized to that of the PP2A coding region as an internalcontrol. Error bars indicate the s.d. of three independent experiments(n=3).

also enriched but to a lesser degree, whereas the frequency of theCTATAG motif as the negative control was not increased near thePIF4-binding peaks (Fig. 3d and Supplementary Fig. S2c). Similarcis-element enrichment was found in PIF-activated or repressed genes(Supplementary Fig. S2c).

The target genes of PIF4 include over half (51.7%) of the BZR1-targetgenes identified by ChIP microarray19 (Fig. 3e). These target genesof both BZR1 and PIF4 contain a higher portion of light-regulatedgenes than the target genes of either BZR1 or PIF4 alone (Fig. 3e).When plotted along the gene structure, most of the binding peaksof BZR1 and PIF4 were clustered along the diagonal line (Fig. 3f),suggesting that they bind to nearby or the same cis elements (Fig. 3g).Motif analysis showed that the G-box (CACGTG), which containstwo inverted repeats of the core of the BZR1-binding site (CGTG;ref. 27), was the most enriched motif in the regions bound by bothBZR1 and PIF4 (Supplementary Fig. S2d,e). To test if BZR1 and PIF4bind to the same promoter elements at the same time or exclusivelyof each other, chromatin from transgenic Arabidopsis expressing bothBZR1–Myc and PIF4–YFP was immunoprecipitated sequentially usinganti-Myc and anti-YFP antibodies, and then analysed by quantitativePCR (qPCR). The results show a high enrichment of the BZR1 and PIF4common target promoters, indicating that BZR1 and PIF4 co-occupythese promoters in vivo (Fig. 3h). These results, together with in vitrodata showing direct interaction between their DNA-binding domains,suggest that BZR1 and PIF4 form a heterodimer to bind to promotersof the common target genes (Fig. 1).

BZR1 and PIF4 co-regulate light- and hormone-responsive genesOf the 2,879 BZR1-regulated genes, 857 (335 expected randomly)were regulated by PIFs (Fig. 4a). Whereas 80% (682) of the BZR1-and PIF-co-regulated genes were also regulated by light, only 18%of BZR1-only-regulated and 69% of PIF-only-regulated genes wereregulated by light, indicating that the co-regulated genes are morelikely to be light responsive than genes regulated by only BZR1 or PIF4(Fig. 4a). BZR1 and PIF4 regulated most genes in a similar directionand their effects were opposite to that of light (Fig. 4b), consistent withtheir roles as negative regulators of photomorphogenesis.Consistent with previous reports of PIF4 functions in the envi-

ronmental and hormonal responses such as light, high temperature,circadian rhythm, shade avoidance, auxin and GA responses9,13–15,17,32,gene ontology (GO) analyses showed that all these PIF4-mediatedresponses are highly represented by the PIF4-target genes (Supplemen-tary Fig. S3), indicating that PIF4 directly regulates these responses.In particular, genes involved in cell size regulation and responses toauxin and GA showed the highest enrichment in genes that wereactivated directly by BZR1 and PIF4 (Fig. 4c). In contrast, genesencoding photosynthetic and chloroplast proteins were most enrichedamong the genes that are repressed by BZR1 and PIFs but are notdirect targets (Fig. 4c). These results indicate that the BZR1–PIF4heterodimer tends to directly regulate the genes involved in auxinand GA responses and cell elongation, but indirectly regulate thegenes encoding photosynthetic and chloroplast proteins throughdownstream transcription factors.


ART I C L E S

a

e f g

b c d

Rel

ativ

e ex

pres

sion

s

Rel

ativ

e ex

pres

sion

s Col-0 bzr1-1D PIF4-OX PIF4-OX;bzr1-1D Col-0 PIF4-OX Col-0 (BRZ) PIF4-OX (BRZ) bzr1-1D (BRZ) PIF4-OX;bzr1-1D (BRZ)

682 361

1,661

1,015

531 175

1,178

BZR1 regulated (2,879)

PIF regulated (2,566)

Red light regulated (3,236)

4 2 3 1 0 3

18

6 8 2 0 2 1 0 0 0

12

49

2 0 2 0 2

22

1 0 1 0 0 7

Per

cent

age

of g

enes

BZR1-up and PIF-up (all) BZR1-up and PIF-up (BZR1, PIF4 target) BZR1-down and PIF-down (all) BZR1-down and PIF-down (BZR1, PIF4 target) Total

–10

0

10

0

bzr1

-1D

/Col

-0

bzr1-1D/pifq;bzr1-1D

R = 0.87

bzr1-1D/Col-0 (2,151)

bzr1-1D/pifq;bzr1-1D (3,176)

261611

1,062

558 217

1,603

pifq;bzr1-1D/pifq(1,330)

294

0

10

0

bzr1-1D/Col-0

pifq

;bzr

1-1D

/pifq

y = x

h i

y = 0.92x + 0.28 y = 0.25x + 0.05

BZR1 PIF Light

0

10

20

30

40

50

60

Respon

se

to auxin

Respon

se

to GA

Regulat

ion

of cell s

izeCel

l wall

modifica

tion

Photosy

nthesis

Chloro

plast

–10 10

–10

–10 10

Col-0 bzr1-1D pifq pifq;bzr1-1D

SAUR

15IAA

19AC

S5PR

E1PR

E5PR

E6FA

D5GE

R1 CPD

BR6O

X2DW

F40

5

10

15

20

SAUR

15IAA

19AC

S5PR

E1PR

E5PR

E6GE

R1DW

F4

SAUR

15IAA

19AC

S5PR

E1PR

E5PR

E60

5

10

15

20

25

Rel

ativ

e ex

pres

sion

s

0

5

10

15

20

25

–3 30

Figure 4 BZR1 and PIF4 regulate different genes interdependently andindependently. (a) The Venn diagram shows significant overlap betweenBZR1-regulated and PIF-regulated and red light-regulated genes. (b) Heatmap of 682 BZR1-, PIF- and light-co-regulated genes. The scale barshows fold changes (log 2 value). (c) GO analyses of BZR1 and PIF4targets, and BZR1-regulated, PIF4-regulated genes. Numbers indicate thepercentages of genes belonging to each GO category. Total: Arabidopsistotal genes. (d) Comparison of BZR1-regulated genes (bzr1-1D versusCol-0 and pifq;bzr1-1D versus pifq) and PIF-regulated genes (bzr1-1Dversus pifq;bzr1-1D ) identified by RNA-seq analysis. Differentiallyexpressed genes were defined by a twofold difference between sampleswith P -value >0.01. (e) Scatter plot of log 2-fold change values in

bzr1-1D/Col-0 or bzr1-1D/pifq;bzr1-1D RNA-seq data for 1,279 BZR1-and PIF-co-regulated genes. (f) Scatter plot of log 2-fold change valuesof 2,151 BZR1-regulated genes in the wild-type versus pifq mutantbackground. (g,h) qRT–PCR analysis of the expression levels of BZR1- andPIF-co-regulated genes and BR-biosynthesis genes. Seedlings were grownon 2 µM BRZ medium in the dark (g) or under red light (h) for five days.(i) PIF4 activation of target gene expression requires BR signalling andBZR1. qRT–PCR analysis of seedlings grown on media without or with 2 µMBRZ (BRZ) under red light for five days. All gene expression levels werenormalized to that of PP2A and are shown relative to the expression levelsin wild type (Col-0). All error bars indicate the s.d. of three independentexperiments (n=3).

BZR1 and PIF4 regulate different genes interdependentlyand independentlyWe carried out RNA-seq analysis to determine whether genes arecontrolled independently or interdependently by PIFs and BZR1. Wild-type, bzr1-1D, pifq and pifq;bzr1-1D plants were grown on the mediumcontaining 2 µM BRZ (to inactivate wild-type BZR1) in the dark forfive days. RNA-seq analyses identified 2,151 genes affected more thantwofold by bzr1-1Dmutation when compared with wild type (BZR1-regulated genes) (Fig. 4d and Supplementary Table S4), and 3,176 genesaffected by the pifqmutation in the bzr1-1D background (pifq;bzr1-1Dversus bzr1-1D, PIF-regulated genes; Fig. 4d and Supplementary TableS5). About 59% (1,279) of the BZR1-regulated genes are also regulatedby PIFs, mostly in the same direction (correlation coefficient= 0.87;Fig. 4e). There are more genes activated than repressed by BZR1 andPIFs, and this is particularly true for the co-target genes of BZR1 and

PIF4 (Supplementary Fig. S4). The overall effects of bzr1-1D on the geneexpression are significantly diminished in the pifq background whencompared with the wild-type background (Fig. 4f), with differentialexpression of about 78% of the 2,151 bzr1-1D-affected genes abolishedor reversed in the pifq background (Fig. 4d,f). Among the 3,176genes affected by pifq in the bzr1-1D background, 2,039 genes (64%)were unaffected or affected in opposite ways by pifq in the wild-typebackground, which lacks BZR1 activity when grown on the BRZmedium (Supplementary Fig. S5). These results demonstrate that BZR1and PIFs regulate a large portion of downstream genes interdependentlyon each other but also regulate some genes independently.We carried out quantitative PCR with reverse transcription

(qRT–PCR) of several BZR1–PIF4 common target genes in thebzr1-1D, pifq and bzr1-1D;pifqmutants to confirm their independent orinterdependent regulation by BZR1 and PIF4. As shown in Fig. 4g, the


ART I C L E S

Light intensity (µmol m–2 s–1)

Col-0 pre-amiR

Hyp

ocot

yl le

ngth

(mm

)

pifq;PRE1-OX

Hyp

ocot

yl le

ngth

(mm

)

Hyp

ocot

yl le

ngth

(%)

PR

E5p:

Luc

b c d

BL (nM)

Col-0 pre-amiR

Hyp

ocot

yl le

ngth

(%)

∗∗

∗∗

∗∗

Col-0 Rel

ativ

e ex

pres

sion

s

PIF4BZR1 – + – +

– – + +

15

0

5

10Col-0 pre-amiR

00.51.01.52.02.5

PRE6

PRE1

PRE2

PRE3

PRE5

pre-am

iR det2

bril1-

116

0 10 100 10000

100200300400500600 M BRZ

0

5

10

15

20

Col-0

pre-

amiR

bzr1

-1D

bzr1

-1D;

pre-

amiR

M BL M BL

Col-0 pre-amiR

1 2Col-0 pifq0

2

4

6

8

10

∗∗∗∗

∗∗

0 7 17 400

20

40

60

80

100

120

a

e f g h

Figure 5 PREs promote hypocotyl elongation downstream of BZR1 and PIF4.(a) Transient gene expression assays of co-regulation of PRE5 expression byBZR1 and PIF4. The PRE5 promoter (2 kb) fused to the firefly luciferasereporter gene was co-transfected with 35S::BZR1, 35S::PIF4 or bothinto Arabidopsis mesophyll protoplast. The firefly luciferase activities werenormalized by Renilla luciferase as an internal control. Error bars indicate thes.d. of three independent experiments (n=3). (b) Expression levels of PRE1to PRE6 in the pre-amiR plants. PRE4 was not detected in this condition.Similar results were obtained in two independent experiments. (c) Thepre-amiR plants show dwarfism. Four-week-old plants were photographed.(d,e) The pre-amiR plants had reduced sensitivity to BL. Seedlings were

grown on various concentration of BL medium under light for five days.Representative seedlings grown on either mock (M) or 100nM BL (BL)media are shown in d. Error bars indicate the s.d. (n = 10 plants) and∗∗P

ART I C L E S

–PPZ

Col-0 pifq pifq; bzr1-1D

bzr1-1D

+PPZ

20 °C

28 °C

20 °C 28 °C

ba

c

d

Hyp

ocot

yl le

ngth

(mm

)

–PPZ +PPZe

f

20 28 20 2028 28

PIF4–Myc

BZR1–CFP

20 28 20 2028 28

Hyp

ocot

yl le

ngth

(mm

)

3.54.6

1.3 1.2 1.3 1.31.2

3.2

Rel

ativ

e ex

pres

sion

s

SAUR15 IAA19

Col-0 pifq pifq; bzr1-1D

bzr1-1D

1 h 4 h 24 h

1 h 4 h 24 h

Col-0 bzr1-1D pifq Col-0 bzr1-1D pifqpifq; bzr1-1D

pifq; bzr1-1D

pre-amiRCol-00

2

4

6

8

10

PRE1 PRE50

2

4

6

8

10

121820

Col-0 (20) Col-0 (28) bri1 (20) bri1 (28) bri1; bzr1-1D (20) bri1; bzr1-1D (28) pifq (20) pifq (28)

0

2

4

6

8

10

12

20 28 20 20 2028 20 20 20 2028 28 28 28 2828

Figure 6 High temperature promotion of hypocotyl elongation requires bothBZR1 and PIF4. (a,b) Both BZR1 and PIF4 are required for high-temperaturepromotion of hypocotyl elongation. Seedlings were grown either on mock(−PPZ) or 2 µM PPZ (+PPZ) at 20 ◦C or 28 ◦C for seven days. Numbersindicate the ratios of hypocotyl lengths of seedlings at 28–20 ◦C. Errorbars indicate the s.d. (n = 10 plants). (c) PIF4 protein accumulatesat high temperature. Seedlings expressing PIF4–Myc from native PIF4promoter were grown at 20 ◦C for five days and transferred to 28 ◦Cfor 1, 4 and 24h. (d) BZR1 accumulation and phosphorylation statusare not significantly affected at high temperature. Seedlings expressing

BZR1–CFP from native BZR1 promoter were grown at 20 ◦C for five daysand transferred to 28 ◦C for 1, 4 and 24h. (e) Both BZR1 and PIF4 arerequired for high-temperature-induced gene expressions. Seedlings weregrown at 20 ◦C for four days and transferred to 28 ◦C for 24h (28) or keptat 20 ◦C (20). Relative gene expression levels were normalized to thatof PP2A. Similar results were obtained in two independent experiments.(f) The pre-amiR plants are defective in the high-temperature promotion ofhypocotyl elongation. Seedlings were grown at 20 ◦C or 28 ◦C for seven days.Error bars indicate the s.d. (n=10 plants). Uncropped images of blots/gelsare shown in Supplementary Fig. S7.

lengths of pre-amiR plants showed greatly reduced response to hightemperature (Fig. 6f), supporting a key role of PREs in hypocotylelongation downstream of BZR1 and PIF4.

DISCUSSIONSignalling crosstalk is considered important for cellular decision-making, but few examples of direct crosstalk between signallingpathways have been elucidated in plants, although plants are expectedto have more complex signalling system than animals2. Genetic andphysiological studies have suggested a close relationship between BR-and light-signalling pathways, yet evidence for direct crosstalk has beenelusive. Here we show that light-regulated PIF4 interacts directly withBR-regulated BZR1. Together they bind to thousands of common targetgenes in the genome, and interdependently control gene expression andseedlingmorphogenesis. The functional interdependence between PIF4and BZR1 explains why BR is required for cell elongation responses tomultiple environmental signals that activate PIFs, including darkness,shade and high temperature3,5–8,17. This relation is also consistent withthe light-dependent phenotype of bzr1-1D (ref. 27). The interactionseems conserved among further members of the BZR1 and PIFfamilies. Although bes1-D was reported to show a light-independentlong-hypocotyl phenotype37, the difference could be due to differentgenetic background or a possibly light-independent activity of BES1.Our strong genetic and molecular evidence indicates that BZR1–PIF4interaction is a key mechanism for coordination of growth regulationby BR, light and temperature.

Most of the BZR1–PIF4-co-regulated target genes were positivelyregulated by both BZR1 and PIFs, suggesting that the BZR1–PIF4heterodimer functions as a transcription activator. However, theinteractions between BZR1 and PIF4 seem to be promoter specific, andBZR1 and PIF4 each also controls large numbers of unique targets. Thispotentially enables differential regulation of various processes by BRand environmental signals. The genome-wide analysis confirmed thatthe major functions of BZR1–PIF4 are activating genes involved in cellelongation while repressing the transcription pathways for chloroplastdevelopment. In addition to cell-wall-related genes, BZR1–PIF4promotion of cell elongation seems to also require the downstream PREfamily ofHLH factors, which are homologous to the human Inhibitor ofDNA-binding (Id) protein35,36. Previous studies have shown the majorfunction of the PREs in promoting growth by antagonizing other bHLHfactors including IBH1, AIF1 and PAR1 (refs 35,36,38). We show herethat PREs are co-activated by BZR1 and PIF4, and they are required forthe BZR1/PIF4-mediated hypocotyl elongation responses to BR, dark-ness and high temperature. A recent study showed that PAR1 interactswith PIF4 (ref. 39); therefore, the transcription activation of PREs mayalso activate a feedback loop, further activating PIF4. BZR1 and PIFsseem to act at the centre of a complex transcriptional network thatcontrols cellular growth and development of photosynthetic apparatus.Interestingly, BZR1 and PIF4 tend to regulate photosyn-

thetic/chloroplast genes indirectly through their target transcriptionfactors such as GLK1 and GLK2, which are key transcription factorsregulating expression of photosynthetic apparatus40–42. GLK1 is a


ART I C L E S

target gene of both BZR1 and PIF4, whereas GLK2 is a PIF4 target;both are repressed by BZR1, BZR2 (also known as BES1) and PIFs(refs 12,19,43). Of the 120 GLK1/2-activated genes identified bymicroarray analysis, 55 genes (46%) were repressed by BZR1 and/orPIFs (Supplementary Table S6), consistent with the repression ofGLK1/2 by BZR1 and PIFs.Previous studies have shown that BZR1 inhibits photomorphogen-

esis by transcriptional repression of light-signalling components19,including phytochrome B as well as GATA2 and BZS1 transcriptionfactors19,25,26. As such, BZR1 not only directly cooperates with thenegative regulators (PIFs) but also transcriptionally represses thepositive regulators of photomorphogenesis, providing two levels ofcontrol. These interactions at multiple levels support the importanceof steroid regulation of light sensitivity. In addition to light andtemperature, circadian rhythm and GA also control the activitiesof PIF factors13–15, and thus the responses to these signals may alsodepend on BR/BZR1. The BZR1–PIF4 module therefore seems to bethe core of a complex network that integrates multiple endogenousand environmental signals. �

METHODSMethods and any associated references are available in the onlineversion of the paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSWe thank the Stanford Center for Genomics and Personalized Medicine (SCGPM)service centre led by M. Snyder and A. Sidow for the sequencing service, Z. Wen forcarrying out the sequencing, Y. Bai for help with genomic data analysis and Y. Haofor experimental assistance. Research was primarily supported by a grant from theNIH (R01GM066258).

AUTHOR CONTRIBUTIONSE.O. and J-Y.Z. carried out experiments. E.O. analysed data and wrote themanuscript. Z-Y.W. designed experiments, analysed data and wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at www.nature.com/doifinder/10.1038/ncb2545Reprints and permissions information is available online at www.nature.com/reprints

1. Depuydt, S. & Hardtke, C. S. Hormone signalling crosstalk in plant growth regulation.Curr. Biol. 21, R365–R373 (2011).

2. Vert, G. & Chory, J. Crosstalk in cellular signaling: background noise or the real thing?Dev. Cell 21, 985–991 (2011).

3. Li, J., Nagpal, P., Vitart, V., McMorris, T. C. & Chory, J. A role for brassinosteroids inlight-dependent development of Arabidopsis. Science 272, 398–401 (1996).

4. Szekeres, M. et al. Brassinosteroids rescue the deficiency of CYP90, a cytochromeP450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85,171–182 (1996).

5. Nemhauser, J. L., Mockler, T. C. & Chory, J. Interdependency of brassinosteroid andauxin signaling in Arabidopsis. PLoS Biol. 2, E258 (2004).

6. Kozuka, T. et al. Involvement of auxin and brassinosteroid in the regulation of petioleelongation under the shade. Plant Physiol. 153, 1608–1618 (2010).

7. Stavang, J. A. et al. Hormonal regulation of temperature-induced growth inArabidopsis. Plant J. 60, 589–601 (2009).

8. Leivar, P. & Quail, P. H. PIFs: pivotal components in a cellular signaling hub. TrendsPlant Sci. 16, 19–28 (2011).

9. Chen, M. & Chory, J. Phytochrome signaling mechanisms and the control of plantdevelopment. Trends Cell Biol. 21, 664–671 (2011).

10. Al-Sady, B., Ni, W., Kircher, S., Schafer, E. & Quail, P. H. Photoactivatedphytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediateddegradation. Mol. Cell 23, 439–446 (2006).

11. Shin, J. et al. Phytochromes promote seedling light responses by inhibiting fournegatively-acting phytochrome-interacting factors. Proc. Natl Acad. Sci. USA 106,7660–7665 (2009).

12. Leivar, P. et al. Definition of early transcriptional circuitry involved in light-inducedreversal of PIF-imposed repression of photomorphogenesis in young Arabidopsisseedlings. Plant Cell. 21, 3535–3553 (2009).

13. Feng, S. et al. Coordinated regulation of Arabidopsis thaliana development by lightand gibberellins. Nature 451, 475–479 (2008).

14. De Lucas, M. et al. A molecular framework for light and gibberellin control of cellelongation. Nature 451, 480–484 (2008).

15. Nozue, K. et al. Rhythmic growth explained by coincidence between internal andexternal cues. Nature 448, 358–361 (2007).

16. Foreman, J. et al. Light receptor action is critical for maintaining plant biomass atwarm ambient temperatures. Plant J. 65, 441–452 (2011).

17. Koini, M. A. et al. High temperature-mediated adaptations in plant architecturerequire the bHLH transcription factor PIF4. Curr. Biol. 19, 408–413 (2009).

18. Song, L. et al. Genome-wide analysis revealed the complex regulatory network ofbrassinosteroid effects in photomorphogenesis. Mol. Plant 2, 755–772 (2009).

19. Sun, Y. et al. Integration of brassinosteroid signal transduction with the transcriptionnetwork for plant growth regulation in Arabidopsis. Dev. Cell 19, 765–777 (2010).

20. Gray, W. M., Ostin, A., Sandberg, G., Romano, C. P. & Estelle, M. High temperaturepromotes auxin-mediated hypocotyl elongation in Arabidopsis. Proc. Natl Acad. Sci.USA 95, 7197–7202 (1998).

21. Kim, T. W. & Wang, Z. Y. Brassinosteroid signal transduction from receptor kinasesto transcription factors. Annu. Rev. Plant Biol. 61, 681–704 (2010).

22. Tang, W. et al. PP2A activates brassinosteroid-responsive gene expression and plantgrowth by dephosphorylating BZR1. Nat. Cell Biol. 13, 124–131 (2011).

23. Wang, Z. Y. et al. Nuclear-localized BZR1 mediates brassinosteroid-inducedgrowth and feedback suppression of brassinosteroid biosynthesis. Dev. Cell 2,505–513 (2002).

24. Gampala, S. S. et al. An essential role for 14-3-3 proteins in brassinosteroid signaltransduction in Arabidopsis. Dev. Cell 13, 177–189 (2007).

25. Luo, X-M. et al. Integration of light and brassinosteroid signaling pathways by a GATAtranscription factor in Arabidopsis. Dev. Cell 19, 872–883 (2010).

26. Fan, X. Y. et al. BZS1, a B-box protein, promotes photomorphogenesis downstreamof both brassinosteroid and light signaling pathways. Mol. Plant 5, 65–74 (2012).

27. He, J-X. et al. BZR1 is a transcriptional repressor with dual roles in brassinosteroidhomeostasis and growth responses. Science 307, 1634–1638 (2005).

28. Ji, H., Jiang, H., Ma, W. & Wong, W. H. Using CisGenome to analyze ChIP-chip andChIP-seq data. Curr. Protoc. Bioinformatics 33, 2.13.1–2.13.45 (2011).

29. Muino, J. M., Hoogstraat, M., van Ham, R. C. & van Dijk, A. D. PRI-CAT: a web-toolfor the analysis, storage and visualization of plant ChIP-seq experiments. NucleicAcids Res. 39, W524–W527 (2011).

30. Richter, R., Behringer, C., Muller, I. K. & Schwechheimer, C. The GATA-typetranscription factors GNC and GNL/CGA1 repress gibberellin signaling downstreamfrom DELLA proteins and PHYTOCHROME-INTERACTING FACTORS. Genes Dev. 24,2093–2104 (2010).

31. Lorrain, S., Trevisan, M., Pradervand, S. & Fankhauser, C. Phytochrome interactingfactors 4 and 5 redundantly limit seedling de-etiolation in continuous far-red light.Plant J. 60, 449–461 (2009).

32. Nozue, K., Harmer, S. L. & Maloof, J. N. Genomic analysis of circadian clock-, light-,and growth-correlated genes reveals PHYTOCHROME-INTERACTING FACTOR5 as amodulator of auxin signaling in Arabidopsis. Plant Physiol. 156, 357–372 (2011).

33. Martinez-Garcia, J. F., Huq, E. & Quail, P. H. Direct targeting of light signals to apromoter element-bound transcription factor. Science 288, 859–863 (2000).

34. Oh, E. et al. Genome-wide analysis of genes targeted by PHYTOCHROMEINTERACTING FACTOR 3-LIKE5 during seed germination in Arabidopsis. Plant Cell.21, 403–419 (2009).

35. Lee, S. et al. Overexpression of PRE1 and its homologous genes activatesGibberellin-dependent responses in Arabidopsis thaliana. Plant Cell Physiol. 47,591–600 (2006).

36. Zhang, L. Y. et al. Antagonistic HLH/bHLH transcription factors mediatebrassinosteroid regulation of cell elongation and plant development in rice andArabidopsis. Plant Cell. 21, 3767–3780 (2009).

37. Yin, Y. et al. BES1 accumulates in the nucleus in response to brassinosteroids toregulate gene expression and promote stem elongation. Cell 109, 181–191 (2002).

38. Wang, H., Zhu, Y., Fujioka, S., Asami, T. & Li, J. Regulation of Arabidopsisbrassinosteroid signaling by atypical basic helix-loop-helix proteins. Plant Cell. 21,3781–3791 (2009).

39. Hao, Y., Oh, E., Choi, G., Liang, Z. & Wang, Z. Y. Interactions between HLH and bHLHfactors modulate light-regulated plant development.Mol. Plant 5, 162–171 (2012).

40. Waters, M. T., Moylan, E. C. & Langdale, J. A. GLK transcription factorsregulate chloroplast development in a cell-autonomous manner. Plant J. 56,432–444 (2008).

41. Waters, M. T. et al. GLK transcription factors coordinate expression of thephotosynthetic apparatus in Arabidopsis. Plant Cell. 21, 1109–1128 (2009).

42. Fitter, D. W., Martin, D. J., Copley, M. J., Scotland, R. W. & Langdale, J. A. GLKgene pairs regulate chloroplast development in diverse plant species. Plant J. 31,713–727 (2002).

43. Yu, X. et al. A brassinosteroid transcriptional network revealed by genome-wide identification of BESI target genes in Arabidopsis thaliana. Plant J. 65,634–646 (2011).


http://www.nature.com/doifinder/10.1038/ncb2545http://www.nature.com/doifinder/10.1038/ncb2545http://www.nature.com/doifinder/10.1038/ncb2545http://www.nature.com/doifinder/10.1038/ncb2545http://www.nature.com/reprints

METHODS DOI: 10.1038/ncb2545

METHODSPlant materials and growth conditions. A. thaliana plants were grown in agreenhouse with a 16-h light/8-h dark cycle at 22–24 ◦C for general growth andseed harvesting. All the plants were in Col-0 ecotype background. To generatePIF4-overexpressing transgenic plants, the full length of the PIF4 coding sequencewas cloned into gateway compatible pEarleyGate 101 (ref. 44) and transformedinto Col-0. To generate PRE knockdown mutants (pre-amiR), we used WMD3(Web MicroRNA Designer 3; ref. 45) to design an artificial microRNA sequence(5�-TTATGTAGTTGCATGTCTCGT-3�) targeting PRE1, PRE2, PRE5 and PRE6and transformed the artificial microRNA driven by the 35s promoter into Col-0.The pifq mutant11 was provided by G. Choi (Korea Advanced Institute of Scienceand Technology).

Hypocotyl length measurements. Seeds sterilized by 70% (v/v) ethanol and0.01% (v/v) Triton X-100 were grown on half-strengthMSmedium (PhytoTechnol-ogy Laboratories) supplementedwith 0.8%phytoagar. After three days of incubationat 4 ◦C, seedlings were irradiated by white light for 6 h to promote germinationand then incubated in specific light conditions for five days. For high-temperatureexperiments, seedlings were incubated at 20 ◦C for one day and incubated at28 ◦C for five to six days. Seedlings were photocopied and hypocotyl lengths weremeasured by using ImageJ software (http://rsb.info.nih.gov/ij).

Transient gene expression assays. Protoplast isolation and PEG transformationwas carried out as described previously (tape method)46. Plasmid DNAs wereextracted using the Qiagen Plasmid Maxi Kit according to the manufacturer’sinstructions. 1× 104 isolated mesophyll protoplasts were transfected with a totalof 10 µg of DNA (35s::PIF4–GFP, 35s::BZR1–GFP, 35s::Renilla luciferase and PRE5promoter::firefly luciferase) and incubated overnight. Protoplasts were harvested bycentrifugation and lysed in 50 µl of passive lysis buffer (Promega). Firefly andRenilla(as internal standard) luciferase activities were measured using a dual-luciferasereporter kit (Promega).

Co-immunoprecipitation assays. Plants expressing both BZR1–CFP from nativeBZR1 promoter (BZR1p::BZR1–CFP) and PIF4–Myc from native PIF4 promoter(PIF4p::PIF4–myc) or plants expressing only PIF4–Myc were incubated at 28 ◦C for8 h to accumulate PIF4 protein and treated with 100 nM BL for 1.5 h. Harvestedtissues were ground in liquid nitrogen, homogenized in immunoprecipitationbuffer (50mM Tris-Cl at pH 7.5, 1mM EDTA, 75mM NaCl, 0.1% Triton X-100,5% glycerol, 1mM phenylmethylsulphonyl fluoride, 1× protease inhibitor) andsonicated four times to break the nuclei. After centrifugation at 20,000g for 10min,1ml of the supernatant was incubated overnight with anti-GFP (5 µg) immobilizedon protein A/G agarose beads. The beads were then washed three times with 1ml ofimmunoprecipitation buffer and eluted samples were analysed by immunoblot.

Pulldown assays. GST–PIF4, GST–PIF1, GST–PIF4N1, GST-PIFN2, GST–PIF4C1, GST–PIFC2, GST–PIFC3, GST, MBP–BZR1, MBP–BZR2, MBP–BZR1N,MBP–BZR1C and MBP proteins were expressed in BL21 codon plus Escherichiacoli cells and purified either using glutathione beads (GE Healthcare) or amyloseresin (NEB) according to the manufacturer’s protocol respectively. 1 µg ofMBP–BZR1-bound maltose agarose beads were incubated with either 1 µg ofGST or GST–PIF4 or GST–PIF1 in 1× TEN buffer (20mM Tris-HCl, pH 7.5,100mM NaCl, 1mM EDTA) at 4 ◦C for 1 h. The beads were then washed threetimes with NETN buffer (200mM NaCl, 1mM EDTA, 0.5% Nonidet P-40, 20mMTris-HCl, pH 8.0), and eluted samples were analysed by immunoblotting usinganti-GST antibody (GST (Z-5) antibody: sc-459; Santa Cruz) at 1:5,000 dilution.MBP–BZR1,MBP–BZR2,MBP–BZR1N,MBP–BZR1C andMBPwere pulled downby GST–PIF4 immobilized on glutathione agarose beads and then eluted andanalysed by immunoblotting using anti-MBP antibody (anti-MBP monoclonalantibody; NEB) at 1:5,000 dilution. GST–PIF4N1, GST-PIFN2, GST–PIF4C1,GST–PIFC2, GST–PIFC3 and GST were pulled down by MBP–BZR1N-terminalfragment immobilized on maltose agarose beads and then eluted and analysed byimmunoblotting using anti-GST antibody.

Bimolecular fluorescence complementation (BiFC) assays. BiFC assays werecarried out as described previously4. Briefly, agrobacterial suspensions containingPIF4–nYFP or PIF1–nYFP and BZR1–cYFP constructs were injected into the lowerepidermis of tobacco leaves. The transfected plants were kept in the greenhouse forat least 36 h at 22 ◦Cand fluorescence signals were visualized by using a spinning-discconfocal microscope (Leica Microsystems).

Gene expression analysis. Total RNA was extracted from five-day-old seedlingsusing the Spectrum Plant Total RNA kit (Sigma). M-MLV reverse transcriptase

(Fermentas) was used to synthesize complementary DNA from the RNA.Quantitative real-time PCR (qPCR) was carried out using LightCycler 480 (Roche)and the Bioline SYBRGreenMasterMix. PP2Awas used as an internal control. Genespecific primers are listed in Supplementary Table S7.

Chromatin immunoprecipitation (ChIP) assays. To generate PIF4 nativepromoter-driven PIF4–Myc (pPIF4::PIF4–myc) in the pifq mutant background, aPIF4 genomic fragment including 2 kb upstream of the transcription start site wascloned into the gateway compatible pGWB17 vector47 and transformed into thepifq mutant. A line showing the wild-type phenotype was selected and used forChIP assay. The pPIF4::PIF4–myc;pifq plants were grown under white light for twoweeks or in the dark for five days and cross-linked for 20min in 1% formaldehydeunder vacuum. The chromatin complex was isolated as previously described34 andsheared by sonication to reduce the average DNA fragment size to around 500 bp.The sonicated chromatin complex was immunoprecipitated by anti-Myc antibody(Myc-Tag (9B11) mousemAb; Cell Signaling). The beads were washed with low-saltbuffer (50mMTris-HCl at pH 8.0, 2mMEDTA, 150mMNaCl, 0.5%TritonX-100),high-salt buffer (50mM Tris-HCl at pH 8.0, 2mM EDTA, 500mM NaCl, 0.5%Triton X-100), LiCl buffer (10mM Tris-HCl at pH 8.0, 1mM EDTA, 0.25 M LiCl,0.5% NP-40, 0.5% deoxycholate) and TE buffer (10mM Tris-HCl at pH 8.0, 1mMEDTA) and eluted with elution buffer (1% SDS, 0.1M NaHCO3). After reversecross-linking, the DNA was purified with a PCR purification kit (Fermentas) andanalysed by ChIP–qPCR. Primers are listed in Supplementary Table S7.

ChIP-seq analysis. For ChIP-seq library construction, we used 10 ng of ChIPDNA pooled from three biological repeats to reduce sample variation. Blunting end,adapter ligation and amplification were carried out using Illumina Genomic DNASample PrepKits according to themanufacturer’s protocol with smallmodifications.We used diluted (1:10) adapter and 150–400 bp gel-purified amplified DNA.

Illumina Genome Analyser IIx was used for high-throughput sequencing of theChIP-seq library. The raw sequence data were processed using Illumina sequencedata analysis pipeline GAPipeline1.3.2. Sequences in Solexa FASTQ format weremapped to the Arabidopsis genome, TAIR9. Only unique mapping reads wereused for calling PIF4-binding peaks. Two-Sample-Peak-Calling in CisGenome withparameters Bin Size B=50, Max Gap=50, Min Peak Length=100,Win Stat CutoffC>= 2.5, Local Rate Cutoff= 1e-005 and PRI-CAT with default parameters andcutoff (FDR

DOI: 10.1038/ncb2545 METHODS

five-day-old seedlings using the Spectrum Plant Total RNA kit (Sigma). Librarieswere constructed using a TruSeq RNA sample preparation kit (Illumina) accordingto the manufacturer’s instruction. Total reads were mapped to the A. thalianagenome (TAIR9, www.arabidopsis.org) using the TopHat software50. Read countsfor every gene were generated using HTSeq with unionmode. Differential expressedgenes between samples were defined by DEseq (ref. 51), using fold change> 2 andP-value < 0.01.

GEO accession numbers. The ChIP-seq data used in this study may be viewedunder GSE35315.

The RNA-seq data used in this study may be viewed under GSE37160.

44. Earley, K. W. et al. Gateway-compatible vectors for plant functional genomics andproteomics. Plant J. 45, 616–629 (2006).

45. Schwab, R., Ossowski, S., Riester, M., Warthmann, N. & Weigel, D. Highlyspecific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell. 18,1121–1133 (2006).

46. Wu, F. H. et al. Tape-Arabidopsis Sandwich—a simpler Arabidopsis protoplast isola-tion method. Plant Methods http://dx.doi.org/10.1186/1746-4811-5-16 (2009).

47. Nakagawa, T. et al. Development of series of gateway binary vectors, pGWBs, forrealizing efficient construction of fusion genes for plant transformation. J. Biosci.Bioeng. 104, 34–41 (2007).

48. Machanick, P. & Bailey, T. L. MEME-ChIP: motif analysis of large DNA datasets.Bioinformatics 27, 1696–1697 (2011).

49. Furlan-Magaril, M., Rincon-Arano, H. & Recillas-Targa, F. Sequential chro-matin immunoprecipitation protocol: ChIP–reChIP. Methods Mol. Biol. 543,253–266 (2009).

50. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions withRNA-seq. Bioinformatics 25, 1105–1111 (2009).

51. Anders, S. & Huber, W. Differential expression analysis for sequence count data.Genome Biol. 11, R106 (2010).

NATURE CELL BIOLOGY

© 2012 Macmillan Publishers Limited. All rights reserved.

www.arabidopsis.orghttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE35315&submit.x=25&submit.y=6http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE37160&submit.x=17&submit.y=9http://dx.doi.org/10.1186/1746-4811-5-16

S U P P L E M E N TA RY I N F O R M AT I O N

WWW.NATURE.COM/NATURECELLBIOLOGY 1

DOI: 10.1038/ncb2545

M BL M BL

WT pifq

p-BZR1

BZR1

Figure S1 BZR1 phosphorylation status is not affected in pifq mutant. Seedlings were grown on the medium containing 2 μM BRZ for 4 days in the dark and then treated with either mock (M) or 100 nM BL for 2 hr. Endogenous BZR1 was detected by anti-BZR1 antibody. p-BZR1 : phosphorylated BZR1, BZR1 : de-phosphorylated BZR1.


http://www.nature.com/doifinder/10.1038/ncbxxxx


2 WWW.NATURE.COM/NATURECELLBIOLOGY

0

1

2

3

4

5

CACGTG CACATG

Up Down All Genome

PIF4-myc

Control

HFR1

Down35.2% Up

58.2%

a b

PRPT, 1537

c

Num

ber o

f mot

if pe

r 1kb

Complex 6.6%

E-value : 4.5e-167

d e

0 1 2 3 4 5 6

CACGTG CACATG

PIF4_BZR1 common Genomic

Num

ber o

f mot

ifs p

er 1

kb

BZR1_PIF4 common

PIF4-myc

Control

PIL1

Figure S2 PIF4 ChIP-seq analyses. (a) Representative PIF4 binding peaks. Peaks in the promoters of HFR1 and PIL1 were detected in the PIF4-myc sample, but not detected in the wild type control sample. (b) 1537 PIF-regulated-PIF4-target (PRPT) genes. Up indicates PIF-activated gene; Down indicates PIF-repressed gene; Complex indicates gene differently regulated by PIF in the different data set. (c) Number of enriched motifs per 1 kb

in the PIF4 binding peaks. Enriched motifs frequency were not different between PIF-activated (up) and PIF-repressed (down) PIF4 target genes. Genomic indicates Arabidopsis total genome. (d) Sequence logo shows the most enriched motif in the BZR1 and PIF4 common binding regions. (e) Number of enriched motifs per 1 kb in the BZR1 and PIF4 common binding regions. Genomic indicates Arabidopsis total genome.




0 1 2 3 4 5 6 7 8 9

response to auxin stimulus

response to brassinosteroid stimulus

response to gibberellin stimulus

response to ethylene stimulus

response to abscisic acid stimulus

response to jasmonic acid stimulus

response to light stimulus

response to red or far red light

shade avoidance

response to temperature stimulus

circadian rhythm

regulation of cell size

regulation of transcription

Total

PRPT

3.9E-26

1.6E-3

5.6E-9

4.3E-4

3.4E-8

4.3E-4

9.8E-16

1.6E-15

2.6E-10

3.4E-6

7.7E-4

1.8E-5 1.4E-8

Figure S3 GO analyses of PIF-regulated PIF4 target genes (PRPT). Enriched GO (gene ontology) categories of PRPT compared with total Arabidopsis genes (Total). Blue numbers indicate p-value.




-7

0

7

-7 0 7

bzr1

-1D

/ C

ol-0

bzr1-1D / pifq;bzr1-1D

All BZR1,PIF4 co-target

Total 1279 103

co-activated 812 (63%) 74 (72%)

co-repressed 406 (32%) 16 (15%)

opposite 61 (5%) 13 (13%)

Figure S4 BZR1 and PIF4 co-targets are more likely to be activated by BZR1 and PIFs. Scatter plot of log2 fold change values in bzr1-1D/Col-0 or bzr1-1D/pifq;bzr1-1D RNA-Seq data for 103 BZR1 and PIF4 co-regulated

co-targets genes. In the table, “All” indicates BZR1 and PIF4 co-regulated genes (1279) and “BZR1,PIF4 co-targets” indicates BZR1 and PIF4 co-regulated co-target genes (103). Numbers indicate number of genes.




bzr1-1D / Col-0 (2151)

bzr1-1D / pifq;bzr1-1D (3176)

828 1069

970

1034

309

682

Col-0 / pifq (2361)

190

a

b

-10

0

10

-10 0 10

bzr1-1D / pifq;bzr1-1D

Col

-0 /

pifq

y=0.38x + 0.13 y=x

Figure S5 PIF effect on gene regulation is dependent on BZR1 activity. (a) Comparison of BZR1-regulated genes (bzr1-1D vs WT(Col-0)) and PIF-regulated genes (bzr1-1D vs pifq;bzr1-1D and WT vs pifq) identified by RNA-Seq analysis.

Differential expressed genes were defined by 2-fold difference between samples with p-value< 0.01. (b) Scatter plot of log2 fold change values of 3176 PIF-regulated genes in the bzr1-1D versus wild type background.




-3

2

7

12

PP2A PRE1 PRE5 PRE6

a

PRE1

PRE5

PRE6

b

0

2

4

6

8

10

PP2A PRE1 PRE5 PRE6

Enr

ichm

ent

PIF4 binding peaks

PIF4 binding

c BZR1 binding

Enr

ichm

ent

0

0.2

0.4

0.6

0.8

1

1.2

IAA19 PRE5 PRE6

WT pif4 pifq

Rel

ativ

e ex

pres

sion

s

d

Figure S6 PREs mediate BR and light crosstalk downstream of BZR1 and PIF4. (a) PIF4 binding peaks in the promoters of PRE1, PRE5 and PRE6. (b) ChIP-qPCR using PIF4p::PIF4-myc shows PIF4 binding to PREs promoters. Enrichment of pulled DNA was calculated as ratio between PIF4-myc and wild type, normalized to that of PP2A coding region as an internal control. Similar results were obtained in two independent experiments. (c) BZR1 binds to PREs promoters. 5 days dark-grown BZR1p::bzr1-1D-CFP

transgenic seedlings were used for ChIP assay. Enrichment of pulled DNA was calculated as ratio between bzr1-1D-CFP and wild type, normalized to that of PP2A coding region as an internal control. Error bars indicate s.d. of three independent experiments (n=3). (d) PREs are redundantly regulated by PIFs. Seedlings were grown in the dark for 4 days. Relative gene expression levels were normalized to that of PP2A. Error bars indicate s.d. of three independent experiments (n=3).




Figure S7 Full scan images of immunoblots.


Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responsesRESULTSBZR1 interacts with PIF4

BZR1 and PIFs act interdependently in promoting hypocotyl elongationBZR1 and PIF4 bind to overlapping genomic targetsBZR1 and PIF4 co-regulate light- and hormone-responsive genesBZR1 and PIF4 regulate different genes interdependently and independentlyPREs promote cell elongation downstream of BZR1 and PIF4High temperature promotion of hypocotyl elongation requires both BZR1 and PIF4DISCUSSIONMETHODSACKNOWLEDGEMENTSAUTHOR CONTRIBUTIONSCOMPETING FINANCIAL INTERESTSFigure 1 BZR1 interacts with PIF4.Figure 2 BZR1 and PIFs act interdependently in promoting hypocotyl elongation.

Figure 3 BZR1 and PIF4 share a large number of genomic targets.Figure 4 BZR1 and PIF4 regulate different genes interdependently and independently.Figure 5 PREs promote hypocotyl elongation downstream of BZR1 and PIF4.Figure 6 High temperature promotion of hypocotyl elongation requires both BZR1 and PIF4.

:: agris arabidopsis thaliana web page:: - interaction between … · 2013. 2. 18. · articles...

Documents