ABA-related GRN BRC1-dependent

A Bud dormancy genes Upregulated genes

B
Gene ID Name Description
At2g18550 HB21 HD-ZIP Homeobox protein 21
At4g36740 HB40 HD-ZIP Homeobox protein 40
At5g66700 HB53 HD-ZIP Homeobox protein 53
At1g18330 RVE7 MYB protein
At4g11360 RHA1B RING-H2 finger A1B
At4g34000 ABF3/DPBF5 bZIP ABA responsive elements-binding factor 3
At2g46270 GBF3 bZIP G-box binding factor 3
At1g69490 NAP NAC domain containing protein activated by AP3/PI
At5g39610 ORE1/NAC6 NAC domain containing protein 6
At3g29575 AFP3 ABI five binding protein 3
At1g75490 ERF/AP2 DREB subfamily A-2 of ERF/AP2 protein
At2g36080 ABS2/NGAL1 AP2/B3-like transcriptional factor family protein
At2g18050 HIS1-3 Histone 1.3
At3g14440 NCED3 Nine-cis-epoxycarotenoid dioxygenase 3
At4g14930 Survival protein SurE-like phosphatase/nucleotidase
At2g47770 TSPO Outer membrane tryptophan-rich sensory protein
At1g79520 Cation efflux family protein
At4g17550 G3Pp4 Major facilitator superfamily protein
At1g73480 Alpha/beta-Hydrolases superfamily protein
At3g17520 LEA Late embryogenesis abundant protein
At2g35950 EDA12 Embryo sac development arrest 12
At1g79270 ECT8 Evolutionarily conserved C-terminal region 8
At1g11210 Protein of unknown function
At2g28400 Protein of unknown function
At2g36220 Protein of unknown function
At3g03870 Protein of unknown function

Fig. S1. A, ABA-related bud dormancy genes from (1) were compared with BRC1-dependent
genes induced in wild-type but not in brc1 W+FR-treated buds (FDR>0.06, (2)) using Venny (http://
bioinfogp.cnb.csic.es/tools/venny/) B, Gene list showing the 26 BRC1-dependent genes belonging
to the ABA-related GRN. HB21, HB40, HB53 are highlighted in blue. Additional genes analyzed in
this work are highlighted in green.
Fig. S2. Phylogenetic tree of Arabidopsis thaliana Class I HD-Zip genes. Class II HD-Zip HAT2
was included as an outgroup. HD-Zip domain aminoacid sequences were aligned with Clustal
Omega (3) and a phylogenetic tree was obtained with MrBayes (4). Numbers in branches indicate
the probability supporting the branch after generation of 100000 trees. The HB21, HB40, HB53
subclade is highlighted.
A  

B  

Fig. S3. HB21, HB40 and HB53 mRNA levels correlate with BRC1 levels and with the state of
bud activity. mRNA levels of BRC1, HB21, HB40 and HB53 analysed by qPCR in axillary buds. A,
buds 24 h after decapitation compared with buds of intact plants or plants in which 50 µM NAA was
applied to the decapitated stem. B, buds 8 h after application of 400 µM sucrose or 400 µM
mannitol (control). Error bars are SEM of three biological replicates. Asterisks are significant
differences (T-student, P<0.05) between control and treated plants. Letters denote significant
differences (one-way anova, P<0.05) among means.
 Time after estradiol induction
A
75 KDa
α-HA
50 KDa
Ponceau

B
0   2 4   6   8  

10   12 14   16   18  

C
100

80
% GFP signal

60

40

20

0
0 2 4 6 8 10 12 14 16 18 20 22
Hours after induction

Fig. S4. BRC1 protein accumulation in estradiol-inducible lines. A, HA:BRC1 protein

accumulation analyzed by immunoblot using α-HA antibody in estradiol-inducible HA:BRC1ind
plants. Protein extracts (15 µg) from 7-day-old seedlings treated with 10 µM estradiol for 0-24 h
were separated by 10% SDS-PAGE and HA:BRC1 was identifed as a 60 KDa band. B, Images of
a time-lapse study of an induction experiment similar to that in A, performed on BRC1:GFPind
plants. BRC1:GFP accumulation was visualized as a GFP signal detected by confocal microscopy.
C, Quantification of GFP signal was carried out with ImageJ (5). Error bars are SEM of four
biological replicates.
 Mock
Estradiol

1.4  

1.2  

1  
Relative mRNA levels

0.8  

0.6  

0.4  

0.2  

0  
HB21 HB40 HB53

Fig. S5. HB21, HB40 and HB53 are not upregulated in estradiol-inducible GUS (GUSind)
control lines after an estradiol treatment. This is the negative control of Figure 1d. Seven-day-
old seedlings carrying an estradiol-inducible construct LEXA:GUS (GUSind). mRNA levels of HB21,
HB40 and HB53 analyzed by qPCR 8 h after a 10 µM estradiol treatment. Error bars are SEM of
three biological replicates.
Fig. S6. Conservation of BRC1 binding motifs in Brasicaceae HB genes. HB21, HB40 and HB53
orthologs in Arabidopsis lyrata, Thellungiella halophila, Caspela rubella and Brassica rapa were
obtained from phytozome (http://phytozome.jgi.doe.gov/) using the full protein sequence from
Arabidopsis thaliana as a query. AlHB21, Alyrata343486; ThHB21, Thhalv10022814; CrHB21,
Carubv10014603; BrHB21, Bra040941; AlHB40, Alyrata490960; ThHB40, Thhalv10026207;
CrHB40, Carubv10006436; BrHB40a, Bra017780; BrHB40b, Bra010573; AlHB53, Alyrata332898;
ThHB53, Thhalv10005612; CtHB53, Carubv10027040; BrHB53, Bra012093. Search for conserved
BRC1 binding sites was performed as in Figure 1f.
Fig. S7. Conservation of BRC1 binding motifs in Brasicaceae HB genes. AlHB21, Alyrata343486;
ThHB21, Thhalv10022814; CrHB21, Carubv10014603; BrHB21, Bra040941; AlHB40,
Alyrata490960; ThHB40, Thhalv10026207; CrHB40, Carubv10006436; BrHB40a, Bra017780;
BrHB40b, Bra010573; AlHB53, Alyrata332898; ThHB53, Thhalv10005612; CtHB53,
Carubv10027040; BrHB53, Bra012093. Alignment of the conserved motifs included in sites 1-6
analyzed by ChIP (Figs. 1f and 1g).
 HA:BRC1ind BRC1:GFPind HA:BRC1ind
BRC1:GFPind

GFP:BRC1ind
BRC1ind

BRC1ind
GFP:BRC1ind

GUSind GUSind

Mock 10 µM estradiol

Fig. S8. Phenotype of estradiol-inducible GUSind, BRC1ind, HA:BRC1ind, GFP:BRC1ind and

BRC1:GFPind lines grown for 10 days on mock (left) or 10 µM estradiol (right) MS, 1% sucrose
plates. Plants of the GUSind line are unaffected by estradiol. Growth arrest is strongest in the
untagged BRC1ind but it is comparable in HA:BRC1ind, GFP:BRC1ind and BRC1:GFPind lines.
 A B C

HB40p:GUS

D D E

HB53p:GUS

F G H

HB53p:GUS

Fig. S9. GUS staining of HB40p:GUS and HB53:GUS lines. A, HB40p:GUS flower showing GUS
staining. Close-up of stained HB40p:GUS sepal stomata (B) and pollen grains (C). D, GUS staining
of HB53p:GUS flower pedicel. E, Close-up of stained HB53p:GUS flower pedicel stomata. F to H,
GUS staining of HB53p:GUS lateral roots. GUS expression was noticeable from stages II to VI of
lateral root initiation.
 A

B
HB21 1.6 HB40 1.2 HB53 1.4 BRC1
1.4
Relative mRNA levels

1.4 1.2
1
1.2
1.2
1
1 0.8
1
0.8
0.8
0.8 0.6
0.6 0.6
0.6
0.4
0.4 0.4
0.4
0.2 0.2
0.2 0.2
0 * 0
* 0 * * 0

C HB21 HB40 HB53 HB53

Relative mRNA levels

* *

* * * *
* * *
Primer pair: 1 2 3 1 2 3 1 2 3 1 2 3

Fig. S10. HB21, HB40 and HB53 insertional mutants. A, Schematic representation of the
genomic structure of HB21, HB40 and HB53 and T-DNA insertions. Red triangles indicate the
insertion site for each mutation. Rectangles indicate exons; lines, introns. Primers used in qPCR
experiments shown in B and C are indicated. B, mRNA levels of BRC1, HB21, HB40 and HB53
analyzed by qPCR in axillary buds of mutants shown in A. Primer pair 2 was used for HB genes. C,
mRNA levels of BRC1, HB21, HB40 and HB53 analyzed by qPCR in axillary buds of mutants using
primer pairs indicated in A. Error bars are SEM of three biological replicates. Asterisks are
significant differences (T-student, P<0.05) between wild-type and mutant plants.
 0.55 0.56 0.67
3 W  
A a 0.54 a
0.63
a a
W+FR  
Number of RI branches
2.5 ac

2 bc
b
b b
1.5 b

0.5

0
wildwt
type hb21 hb40 hb53-1 hb53-2

0.62 0.78 0.94

W  
B 3
ab ab
b
b
W+FR  
Number of CII branches

ac
2 c

0
wildwt
type hb21
hb214053-1 hb21
hb214053-2
hb40 hb40
hb53-1 hb53-2
C wild type hb21hb40hb53-1
10 10
Number  of  individuals  

Reproductive

8 8 Vegetative 3

6 6 Vegetative 2

4 4 Vegetative 1

2 2 Meristem

Empty
0 0
c1 c2 L1 L2 L3 L4 L5 L6 L7 L8 L9 L10 c1 c2 L1 L2 L3 L4 L5 L6 L7 L8 L9 L10

Fig. S11. Shoot branching phenotype of hb21, hb40 and hb53 mutants. A, Number of primary
rosette branches (RI) of wild type and single hb mutants B, Number of secondary cauline branches
(CII) of wild type, hb21 hb40 hb53-1 and hb21hb40hb53-2 mutants. In A and B plants were grown
for 2 weeks after bolting in W or W+FR (N=28). Error bars are SEM. Letters denote significant
differences (one-way anova, P<0.05) among means. Rosette leaf number was undistinguishable in
the mutants and the wild type. C, Developmental stages of buds in the axils of cotyledons (c1 and
c2) and rosette leaves (L1 to L12) of wild-type (left) and hb21hb40hb53-1 (right) individuals
(N=10). Plants were grown in W light and long-day conditions after bolting. Developmental stages
are as defined in (6).  
A 4000 HB21
*
300 HB40
*
500 HB53
*
Relative mRNA levels

3500
Control 250 400
3000 Estradiol
200
2500 300
2000 150
1500 200
100
1000
50 100
500
0 0 0
GUSind HB21indHB40indHB53ind GUSind HB21ind HB40ind HB53ind GUSind HB21ind HB40ind HB53ind

B HA:HB21ind HA:HB40ind HA:HB53ind

37 KDa
α-HA

Ponceau

Mock Estradiol Mock Estradiol Mock Estradiol

Fig. S12. Estradiol-Inducible HB lines. A, mRNA levels of HB21, HB40 and HB53 analyzed by
qPCR in 7-day-old HBind seedlings 8 h after a 10 µM estradiol treatment. Error bar are SEM of
three biological replicates. Asterisks are significant differences (T-student, P<0.05) between control
and treated plants. B, HA:HB protein accumulation analyzed by immunoblot using α-HA antibody
in estradiol-inducible HA:HBsind plants. Protein extracts (15 µg) from 7-day-old seedlings after an 8
h treatment with mock or 10 µM estradiol were separated by 10% SDS-PAGE. HA:HBs were
identified as a 30 KD band. Membranes were probed with α-HA-HRP antibody (1:1000; Roche).
 A B
HB21
HB21

HB40
HB40

HB53
HB53

C
-843 TTCCAATTATTGTGT
At3g14440.1  

-­‐973  
-­‐843  
-­‐2747  

-­‐1849  
-973 TCCGAATAATTGATA
-1849 ATTAAATTATTAGAC NCED3  
-2747 TCTATATTATTGCTC
-100
-226

D 100 bp

NCED3
TSS

E
*  
Fold enrichment

BRC1-no tag
BRC1-GFP

NCED3 ACT2

Fig. S13. HB21, HB40 and HB53 and BRC1 bind the NCED3 promoter. A. Sequence logos
representing the top motifs bound by HB21, HB40 and HB53 in the Arabidopsis genome, as
identified by DAP-seq (7). B. HB21, HB40 and HB53 binding in vitro to the 5´ genomic region of
NCED3 (At3g14440). Coordinates correspond to the distance from TSS to the middle position of
the peaks defined in (7). C. Sequence alignment of motifs found in the middle position of the peaks
shown in B. In red, the sequence corresponding to the logo consensus. D. NCED3 promoter region
analyzed. E. Relative enrichment of GFP:BRC1 binding to the NCED3 5´genomic region shown in
D. ACT2 was used as a negative control. Error bars are SEM. Asterisks are significant differences
(T-student, P<0.05) between untagged BRC1 ind and GFP:BRC1ind lines.
A  
Relative mRNA levels

B   3
Control
ABA *
Relative mRNA levels

2
* *
1

0
BRC1 HB21 HB40 HB53

C  
BRC1
Relative mRNA levels

0.5

0 wt hb21hb40hb53

wild type hb21

hb40
hb53-1

Fig. S14. Response of BRC1, HB21, HB40 and HB53 in nced3 mutants and after ABA
application in axillary buds. A, mRNA levels of BRC1, HB21, HB40 and HB53 analyzed by qPCR
in axillary buds of wild-type or nced3 plants treated with W or W+FR for 8 h. B, mRNA levels of
same genes analyzed by qPCR in axillary buds, 8 h after application of mock or 50 µM ABA. C,
mRNA levels of BRC1 analyzed by qPCR in axillary buds of wild-type and hb21 hb40 hb53-1
plants. Error bars are SEM of three biological replicates. Asterisks are significant differences (T-
student, P<0.05) between control and treated plants.
 A  
wild type brc1
Fold Fold
Gene ID AGI logSignal FDR logSignal FDR
Change Change
ABF3 At4g34000 1.66 10.26 0.0139446 1.03 9.37 0.99997021
AFP3 At3g29575 2.81 10.915 0.02748892 1.755 9.96 0.19698109
GBF3 At2g46270 1.56 9.19 0.04363698 1.44 9.01 0.21433049
NAP At1g69490 3.165 9.345 0.00703062 1.89 8.425 0.33826567

B  

C   D  

Fig. S15. Response of ABF3, AFP3, GBF3 and NAP. A, response to W+FR in wild-type and brc1
mutant buds. Data obtained from (2). Microarray was hybridized with mRNA from wild-type and
brc1 axillary buds treated for 8 h with W or W+FR. Data obtained from six biological replicates.
Fold change is W+FR vs W. logSignal, logarithm of signal intensity. FDR, false discovery rate. Fold
change of microarray data in this experiment was shown to underestimate the fold change
validated by q-RT-PCR (2). In brc1 mutants gene induction is not statistically significant,
FDR>0.05. B, response to W+FR in wild-type and nced3-2 mutant buds. Expression of GBF3 (C)
and AFP3 (D) in 7-day-old GUSind, HA:BRC1ind, HA:HB21ind, HA:HB40ind and HA:HB53ind seedlings
treated for 8 h with 10 µM estradiol. Error bars are SEM of three biological replicates. Asterisks are
significant differences (T-student, P<0.05) between control and treated plants. Letters denote
significant differences (one-way anova, P<0.05) among means.
 A
W
W+FR

GRAS (At1g63100) KIN12B (At3g23670) OASC (At3g59760) ROPGEF6 (At3g55660)

0.70 0.55 0.58 0.49 0.64 0.80 0.59 0.46

RelaGve  mRNA  levels  

2 2 1.5 2
a a b
a a ab
1.5 1.5 1.5
1
a a b ab
1 a a 1 1 a
b b a
0.5
0.5 0.5 0.5

0 wt hb21hb40hb53
0 wt hb21hb40hb53
0 wt hb21hb40hb53
0 wt hb21hb40hb53
wild type hb21 wild type hb21 wild type hb21 wild type hb21
hb40 hb40 hb40 hb40
hb53-1 hb53-1 hb53-1 hb53-1

B
Mock
Estradiol
GRAS (At1g63100) KIN12B (At3g23670)
1.4 1.4
RelaGve  mRNA  levels  

1.2 1.2
1 1
0.8 0.8
0.6 0.6
0.4 0.4
0.2 0.2
0 GUSind HB21ind HB40ind HB53ind
0 GUSind HB21ind HB40ind HB53ind

1.5 OASC (At3g59760) 2 ROPGEF6 (At3g55660)

RelaGve  mRNA  levels  

1.5
1
1
0.5
0.5

0 GUSind HB21ind HB40ind HB53ind

0 GUSind HB21ind HB40ind HB53ind

Fig. S16. HB21, HB40 and HB53 do not regulate other BRC1-dependent GRNs. A, mRNA
levels of bud activation genes (2) analyzed by qPCR in axillary buds of wild-type and hb21 hb40
hb53-1 plants treated with W or W+FR for 8 h. B, mRNA levels of same genes analyzed by qPCR
in 7-d-old seedlings of HBind lines treated for 8 hours with 10 µM estradiol. These genes were
downregulated in BRC1ind lines in the same conditions (2). Error bars are SEM of three biological
replicates. Asterisks are significant differences (T-student, P<0.05) between control and treated
plants. Letters denote significant differences (one-way anova, P<0.05) among means.
Fig. S17. Schematic representation of the proposed BRC1 function in the control of bud
dormancy. BRC1 represses (red) and activates (green) several GRNs. BRC1 effect on bud
dormancy would be the additive result of its efect on these GRNs. Those GRNs described in (2),
involved in cell cycle and protein synthesis, are included. In this paper we propose that BRC1
induces an ABA-related GRN at least in part via HB21, HB40 and HB53.
A  

Fig. S18. Potential cross-regulations within the GRN. A. Summary of Dap-seq and ChIP-seq
data (7, 8) for the 26 BRC1-dependent co-regulated genes of Fig. S1B. A large proportion of these
genes are bound in vitro and/or in vivo (blue cells) by TFs of the list. No data is available for BRC1,
At1g75490 and At2g36080. B. Number and percentage of genes bound (7, 8) by BRC1-dependent
TFs of Fig. S1B: i) in the Arabidopsis genome, ii) in the 26 genes of Fig. S1B (Core) and iii) in their
co-regulated genes (GRN, Dataset S1). *,** Other binding motifs are found in (8). Most values are
significantly higher than those expected in a random gene list (Pearson X2 test).
 Supplemental Materials and Methods

Plant material

Wild-type Arabidopsis thaliana plants were of the Columbia-0 (Col-0) ecotype.

The brc1-2 mutant was described (6). The hb21-1 (WiscDsLox468G4), hb40-1

(SALK_115125C), hb53-1 (GK-085A06-011845) and hb53-2 (GK-312E06-015817)

mutants were obtained from NASC (arabidopsis.info). Mutants backcrossed with

wild-type Col-0 were studied. In all experiments wild-type, single, double and

triple mutant siblings segregating from the same families were compared. The

nced3-2 mutants (9) were provided by Dr. K. Shinozaki (Riken, Japan).

Plant growth conditions

Plant were grown in soil or in vitro as previously described3 in a 16 h light/8

h dark photoperiod at 22°C in white light (PAR 120 µmol m−2 s−1). For short day

experiments, plants were grown in the same conditions, but with 8h light/16h dark.

Light sources

Light sources were like those described previously (2). W was provided by

tubes of white light-emitting diodes (LEDs) T8-160CM-DW (CCT 4000-4500;

Prosolda) or cool-white 20-W F20T12/CW tubes (Phillips). Supplementary FR light

was provided by lamp tubes carrying FR 735-nm LEDs (L735-03AU; Epitex). All

light measurements were monitored with a JAZ spectroradiometer (Ocean Optics).

Plant treatments

The W vs W+FR treatments were done on plants grown in W and transferred

to the light treatment for 8 h. In the decapitation assays, soil-grown plants with 1-2

cm-long main inflorescences were decapitated and the cut stump was inserted into

an inverted 0.5 mL tube with 500 µL of 0.6% (w/v) agarose and 50 µM NAA or
0.005% NaOH 1N (control), samples were collected 24 h after decapitation. For

sugar treatments, 500 µL of 400 mM sucrose or mannitol (control) and 0.1% (v/v)

Tween-20 were added to rosette axillary buds, and samples were collected 8 h

after beginning of the treatment. For estradiol induction experiments, seedlings

were grown in horizontal plates for 7 days, until estradiol treatment (2 mL of 10 µM

estradiol in DMSO or 0.0005% (v/v) DMSO (control) per plate). They were

collected 8 h after the treatment. For branching responses to ABA, after bolting,

400 µL of 50 µM ABA (SIGMA, A1049), 0.1% (v/v) Tween-20 were added directly

to the base of the stem, covering all the axillary buds, every day and branches were

measured 2 weeks after beginning of the treatment.

Phenotypic analyses

Wild-type, brc1 and hb mutant plants were either maintained in W or

transferred to W+FR after bolting. Two weeks after the main inflorescence became

visible, RI branches (shoots longer than 0.5 cm) were counted. For bud

development analyses in long days, 21 days after germination, 10 individuals of

each genotype were dissected and the developmental stage of each axillary bud

was determined with a stereoscopic microscope as described (6). For bud

development analyses in short days, plants were grown for 8 weeks and rosette

leaves were removed. The number of plants with more than one axillary bud with

leaves longer than 1 mm was counted. The number of rosette leaves (that could

affect the number of buds and branches) was undistinguishable in all the mutants

analysed.
RNA preparation and qPCR analysis

Rosettes leaves with their petioles, main inflorescence, and roots were carefully

dissected out, leaving a tissue highly enriched in axillary buds and subtending

tissue. Material was harvested from at least eight individuals, and three biological

replicates per genotype/treatment and was stored in N2(l). For the estradiol

induction experiment in seedlings, 30 seedlings were collected per biological

replicate. RNA preparation and quantitative RT PCR were performed as described

(2). Three technical replicates (reactions) were done for each biological replicate.

Pairs of primers described in S2 dataset were used. SAND was used as a reference

gene (10). Relative fold expression changes were calculated using qBASE software

(11).

Generation of binary constructs

Plasmids generated in this work are listed in S2 dataset. HB21, HB40 and

HB53 coding sequences (CDS) were obtained from axillary bud RNA using

Superscript III (Thermo Fischer), amplified by PCR with Ampli-Taq Gold

polymerase (Roche) using ATG and STOP primers for each gene (S2 dataset) and

cloned in pGEM-T easy (Promega). For Gateway (Invitrogen) plasmids, PCRs were

performed with primers described in S2 dataset using Phusion Taq polymerase

(New England Biolabs). PCR fragments were cloned in pDONR207 using Gateway

BP Clonase (Invitrogen) and these plasmids were used as entry vectors to generate

binary vectors.
Generation of transgenic lines

Transgenic arabidopsis plants were generated by agroinfiltration using the

floral dip method (12). At least 6 T3 homozygous lines were generated and

analysed for each construct.

Protein binding matrix

A MBP:BRC1 plasmid was transformed into a BL21 E. coli strain. 50 mL of

LB culture were induced with 1 mM IPTG, grown at 37°C for 4 h and then

centrifuged at 20000 g and frozen in N2(l). MBP:BRC1 protein extraction and PBM

binding was performed as described (13). The top 10 motifs with the best score

were aligned and a frequency matrix was generated. WebLogo was used to create

the logo for the BRC1 binding site. This frequency matrix was used in Rsat Pattern

Matching, Matrix-scan tool (14). Sites with a score >1 and P<0.05 were

represented.

LUC activity measurements

Seven-day-old seedlings were transferred to a 96-well microtiter plate filled

with 170 µL MS medium, and 15 µL D-luciferin substrate (20 mg/mL, Molecular

Probes) were added. 4 h later, 15 µL of MS with estradiol or DMSO (control), were

added to a final concentration of 10 µM estradiol or 0.0005% (v/v) DMSO. LUC

activity was quantified with an LB 960 Microplate Luminometer Center (Berthold)

using MikroWin software. 8 plants per construction/treatment were measured. LUC

levels were made relative to the first measurement after estradiol/DMSO addition.
GUS staining

GUS staining was performed as described (15) in buffers containing 10 mM

ferri- and ferrocyanide salts. Histological plastic sections were performed as

described in (16).

Chromatin immunoprecipitation

Ten-day-old GFP:BRC1ind seedlings grown in 125 mm, MS, 0.7 (w/v) % agar

plates were induced with 5 mL of 10 µM estradiol per plate. 4 h later, 1.5 g of

tissue were collected per sample. ChIP was performed as described (17), using

µMACS Anti-GFP (Miltenyi). Primers used for qPCR are in Dataset S2. Regions of

ACT2 and HSF1 genes without BRC1 binding sites were used as negative controls.

HSF1 was used as a normalizer for DNA quantity and ACT2 as a background

control for immunoprecipitated DNA. The same results were obtained when ACT2

was used as a normalizer and HSF1 as a background control.

ABA measurements

200 mg (fresh weight) of plant tissue were suspended in 80% methanol-1%

acetic acid containing the deuterium-labeled dABA as internal standard and

mixed by shaking during one hour at 4ºC. The extract was kept a -20ºC overnight

and then centrifuged and the supernatant dried in a vacuum evaporator. The dry

residue was dissolved in 1% acetic acid and passed through an Oasis HLB

(reverse phase) column. The dried eluate was dissolved in 5% acetonitrile-1%

acetic acid, separated through a reverse phase UHPLC chromatography (2.6 µm

Accucore RP-MS column, 50 mm length x 2.1 mm i.d.; ThermoFisher Scientific)

and was analysed with a Q-Exactive mass spectrometer (Orbitrap detector;

ThermoFisher Scientific) by targeted Selected Ion Monitoring (SIM).

Concentrations in the extracts were determined using embedded calibration

curves and the Xcalibur 2.2 SP1 build 48 and TraceFinder programs.

Analysis and representation of Dap-seq data    

DAP-Seq raw data in FASTQ format was obtained from GEO and mapped to

the TAIR10 genome sequence using Bowtie2 (18) with default parameters. Read

coverage across the genome was computed in 10 bp bins using deepTools (19)

and visualized under Integrated Genome Browser (IGB; http://bioviz.org/igb).

Coordinates correspond to the distance from TSS to the middle position of the

peaks defined in (7). Frequency matrices were downloaded from PlantCistromeDB

(http://neomorph.salk.edu/PlantCistromeDB) and used to obtain the logos with

enoLOGOS (20).
 Supplemental Dataset 1

BRC1-dependent (2): ABA GRN (1): Genes BRC1-dep

383 genes induced in wild-type 25 genes induced in dormant in the ABA GRN:
but not in brc1 buds buds (vs active buds) in three 26 genes common in list A and
after an 8 h W+FR treatment different treatments (blue), and B (blue) and their 201 co-
(FDR<0.06) their 195 coregulated genes regulated genes (white, ATTED-
(white, ATTED-II, (20)) II, (20))

At1g01520 At1g08570 At1g11210

At1g02470 At1g11210 At1g18330
At1g02930 At1g15330 At1g69490
At1g03457 At1g22890 At1g73480
At1g04310 At1g32700 At1g75490
At1g04610 At1g49750 At1g79270
At1g04770 At1g52890 At1g79520
At1g05440 At1g69260 At2g18050
At1g05710 At1g69490 At2g18550
At1g06360 At1g73480 At2g28400
At1g06420 At2g15830 At2g35950
At1g06980 At2g18050 At2g36080
At1g08520 At2g18550 At2g36220
At1g08560 At2g28400 At2g46270
At1g09140 At3g14440 At2g47770
At1g09250 At3g17520 At3g03870
At1g10140 At3g29575 At3g14440
At1g10657 At4g26080 At3g17520
At1g11210 At4g27410 At3g29575
At1g11580 At4g34000 At4g11360
At1g12240 At4g38060 At4g14930
At1g13245 At5g01520 At4g17550
At1g13990 At5g22270 At4g34000
At1g14890 At5g39610 At4g36740
At1g15080 At5g66700 At5g39610
At1g18100 At4g11880 At5g66700
At1g18330 At2g31945 At1g01190
At1g18590 At3g19200 At1g01240
At1g19050 At1g23710 At1g02190
At1g19940 At2g35680 At1g02390
At1g21525 At4g28140 At1g03790
At1g21680 At1g64110 At1g03890
At1g21830 At4g35690 At1g06040
At1g22190 At2g04240 At1g06570
At1g22250 At4g14060 At1g07040
At1g22400 At4g11900 At1g07430
At1g22530 At1g73390 At1g10140
At1g22910 At4g11360 At1g13740
At1g23050 At3g16120 At1g13990
At1g23390 At3g15500 At1g15740
At1g26260 At2g27830 At1g16850
At1g26570 At2g39400 At1g16950
At1g26800 At3g27210 At1g17020
At1g28330 At1g48000 At1g19700
At1g29195 At3g47500 At1g20070
At1g29420 At5g19430 At1g21000
At1g29465 At2g44010 At1g21410
At1g29490 At1g18330 At1g22370
At1g30040 At1g02190 At1g22770
At1g31230 At5g05220 At1g23710
At1g31690 At4g01280 At1g24600
At1g32520 At2g21820 At1g27630
At1g36060 At4g22950 At1g27730
At1g36940 At4g19860 At1g29680
At1g44830 At4g36740 At1g30500
At1g45010 At3g50410 At1g32560
At1g47210 At3g47080 At1g48000
At1g49660 At3g46450 At1g49720
At1g51805 At2g23110 At1g51140
At1g52400 At5g53970 At1g52690
At1g53180 At4g27530 At1g52820
At1g53390 At1g30500 At1g52890
At1g55950 At2g35300 At1g53170
At1g58270 At1g74840 At1g56300
At1g58340 At5g52300 At1g56600
At1g60600 At4g20930 At1g60190
At1g61667 At3g15670 At1g63440
At1g62770 At3g11580 At1g63800
At1g63100 At5g44620 At1g64110
At1g64640 At3g58450 At1g67970
At1g64660 At2g15695 At1g68500
At1g65490 At2g20880 At1g69260
At1g66050 At4g14930 At1g69270
At1g66080 At1g53170 At1g69310
At1g66540 At1g07040 At1g69480
At1g67180 At5g40690 At1g70420
At1g67830 At4g23880 At1g71130
At1g68620 At3g61890 At1g72770
At1g68780 At1g01190 At1g73390
At1g69490 At3g45300 At1g76590
At1g69610 At2g41090 At1g76600
At1g69850 At4g12000 At1g77680
At1g70000 At2g36220 At1g78070
At1g70070 At1g80110 At1g78600
At1g70985 At1g22770 At1g79160
At1g71110 At4g00870 At1g80110
At1g71720 At1g20070 At1g80160
At1g72050 At1g80840 At1g80840
At1g72230 At5g63190 At2g15830
At1g72670 At5g18630 At2g15890
At1g73480 At3g05640 At2g16365
At1g73830 At3g63060 At2g17840
At1g74000 At1g79160 At2g19900
At1g74470 At1g05000 At2g20880
At1g75440 At1g16260 At2g21820
At1g75490 At5g52660 At2g22240
At1g75590 At1g77680 At2g22590
At1g77450 At1g52820 At2g27830
At1g79270 At5g04000 At2g29300
At1g79520 At2g44210 At2g29380
At2g01010 At1g56600 At2g29650
At2g01570 At3g24520 At2g31945
At2g01830 At5g18130 At2g33380
At2g04100 At1g06570 At2g35300
At2g04270 At4g21440 At2g38820
At2g07699 At3g04070 At2g39000
At2g07726 At2g29300 At2g41190
At2g10940 At2g16365 At2g43010
At2g14890 At1g22640 At2g45210
At2g16586 At3g03870 At2g46680
At2g16660 At5g19250 At2g47260
At2g16720 At1g63440 At2g47460
At2g17560 At4g11350 At3g02310
At2g18010 At5g43840 At3g02480
At2g18050 At4g33905 At3g04070
At2g18550 At4g17840 At3g05200
At2g19310 At5g07330 At3g05640
At2g19580 At5g64170 At3g11410
At2g21210 At3g54340 At3g11580
At2g21220 At1g79270 At3g11670
At2g22430 At5g01880 At3g13310
At2g22540 At5g45310 At3g15210
At2g23010 At1g27730 At3g15500
At2g24600 At4g17550 At3g15670
At2g26400 At1g60190 At3g15780
At2g27500 At5g01300 At3g17800
At2g28150 At5g44630 At3g19200
At2g28210 At1g80160 At3g19580
At2g28250 At1g49720 At3g23920
At2g28400 At2g46270 At3g24520
At2g28570 At4g38810 At3g26890
At2g29090 At5g66110 At3g27210
At2g29890 At3g04060 At3g27880
At2g30695 At2g46680 At3g46450
At2g30860 At2g19900 At3g47080
At2g31110 At1g76590 At3g47160
At2g32230 At5g13330 At3g47500
At2g33793 At5g18270 At3g50410
At2g33830 At1g78600 At3g50970
At2g35150 At5g59220 At3g50980
At2g35950 At5g62490 At3g54340
At2g36080 At5g49690 At3g54510
At2g36220 At4g30960 At3g57540
At2g36270 At2g41190 At3g59060
At2g36630 At5g04340 At3g61890
At2g37170 At5g59700 At3g62090
At2g37260 At5g06760 At3g63060
At2g37450 At1g56300 At4g00870
At2g37640 At1g01360 At4g02280
At2g37660 At1g72770 At4g03510
At2g37750 At1g32560 At4g10020
At2g38530 At1g24600 At4g11350
At2g39010 At2g29995 At4g11880
At2g39980 At5g50760 At4g12000
At2g40000 At1g75490 At4g13530
At2g40610 At5g66400 At4g14060
At2g40900 At3g17800 At4g14270
At2g41010 At1g64380 At4g15480
At2g41230 At3g05200 At4g15610
At2g41760 At1g71130 At4g16520
At2g42220 At4g13530 At4g19230
At2g42620 At1g78070 At4g19860
At2g42690 At5g12840 At4g19960
At2g42900 At1g13740 At4g20930
At2g43060 At4g19960 At4g21440
At2g43375 At1g67970 At4g22950
At2g43710 At2g36080 At4g23880
At2g44080 At5g24080 At4g24960
At2g45480 At5g57550 At4g26080
At2g45960 At4g28240 At4g27460
At2g46270 At4g19230 At4g28140
At2g46610 At1g69270 At4g35190
At2g46650 At3g02310 At4g35690
At2g47240 At3g57540 At4g38060
At2g47770 At5g44580 At5g01200
At2g47930 At2g47770 At5g01300
At3g01470 At3g23920 At5g02020
At3g02450 At1g21410 At5g02810
At3g02910 At1g07430 At5g04340
At3g03270 At3g50980 At5g06760
At3g03820 At2g33380 At5g06980
At3g03830 At4g02280 At5g09930
At3g03870 At5g66780 At5g10100
At3g04010 At2g47260 At5g12330
At3g04630 At3g53040 At5g13330
At3g05150 At5g15800 At5g13800
At3g06030 At5g59820 At5g15190
At3g06590 At5g24470 At5g15450
At3g07350 At5g01200 At5g15800
At3g08030 At1g17020 At5g18130
At3g08970 At5g15190 At5g18140
At3g10040 At1g58180 At5g18630
At3g11690 At4g25580 At5g19430
At3g11930 At1g21000 At5g19875
At3g12050 At1g69480 At5g22270
At3g12170 At4g05100 At5g24080
At3g14190 At1g02390 At5g24470
At3g14200 At5g54080 At5g26040
At3g14440 At1g15740 At5g35970
At3g14540 At1g05100 At5g43840
At3g14920 At2g35950 At5g44620
At3g15770 At3g15780 At5g44630
At3g16050 At3g11410 At5g45310
At3g16290 At4g16520 At5g45830
At3g17100 At3g61900 At5g46180
At3g17160 At3g19580 At5g46710
At3g17520 At1g79520 At5g47640
At3g17580 At2g17840 At5g49690
At3g18170 At5g12330 At5g51070
At3g18390 At1g76600 At5g52300
At3g18550 At1g01240 At5g52310
At3g18900 At4g35190 At5g54080
At3g20230 At1g22370 At5g57050
At3g20270 At1g03400 At5g57550
At3g22740 At3g10250 At5g57660
At3g23080 At5g46710 At5g59220
At3g23150 At1g66970 At5g59480
At3g23170 At1g16950 At5g59550
At3g23580 At1g06040 At5g59820
At3g23670 At5g57050 At5g62490
At3g26510 At5g63190
At3g28460 At5g64170
At3g28857 At5g66110
At3g29575 At5g66400
At3g43430 At5g66780
At3g44890 At5g66880
At3g44970 At5g67030
At3g45980
At3g46940
At3g47340
At3g48280
At3g48500
At3g48990
At3g50560
At3g51895
At3g52920
At3g53232
At3g53420
At3g53650
At3g53830
At3g54420
At3g54510
At3g54960
At3g55660
At3g57010
At3g57400
At3g59040
At3g59760
At3g59900
At3g61680
At3g62650
At4g01310
At4g04610
At4g04620
At4g05070
At4g09040
At4g10040
At4g10060
At4g10150
At4g10310
At4g11360
At4g12510
At4g12520
At4g12730
At4g13395
At4g14120
At4g14130
At4g14270
At4g14930
At4g15550
At4g15800
At4g17030
At4g17340
At4g17500
At4g17550
At4g17560
At4g18480
At4g19460
At4g20130
At4g21320
At4g21705
At4g21930
At4g22545
At4g22880
At4g23250
At4g24040
At4g25080
At4g25260
At4g27260
At4g27280
At4g27440
At4g28720
At4g29060
At4g30270
At4g30290
At4g30650
At4g31351
At4g31610
At4g31800
At4g32480
At4g34000
At4g34480
At4g34770
At4g35720
At4g36648
At4g36740
At4g36990
At4g37550
At4g38400
At4g38860
At5g01740
At5g02020
At5g02230
At5g02760
At5g03210
At5g03790
At5g04970
At5g05860
At5g07000
At5g07010
At5g08030
At5g10770
At5g12440
At5g12940
At5g13500
At5g14060
At5g14260
At5g14690
At5g14780
At5g17780
At5g17800
At5g18030
At5g19120
At5g23660
At5g23870
At5g25190
At5g25350
At5g25590
At5g26742
At5g27320
At5g28287
At5g38970
At5g39610
At5g41315
At5g43450
At5g44260
At5g44973
At5g47190
At5g47370
At5g47420
At5g47560
At5g47730
At5g48150
At5g49170
At5g49700
At5g49740
At5g50335
At5g50360
At5g51210
At5g51440
At5g51670
At5g53210
At5g53980
At5g54190
At5g54240
At5g54490
At5g55520
At5g57660
At5g57760
At5g59690
At5g59845
At5g59970
At5g60840
At5g60850
At5g62790
At5g63087
At5g64850
At5g66590
At5g66690
At5g66700
At5g67420
AtMg00210
AtMg01170
 Supplemental Data Set 2a. Primers used in qPCR experiments

Gene AGI ID 5' primer 3' primer

SAND AT2G28390 AACTCTATGCAGCATTTGATCCACT TGATTGCATATCTTTATCGCCATC

OASC AT3G59760 TGCTGATGGTTCGGAACGA AGGTCCAGTCTCGCGCTTAA
GRAS AT1G63100 GACGCGATCAGATCAAGAAACA CACCGGTTCTTGCAATGAAA
ROPGEF6 AT3G55660 GGTTCAGGCTCTCTAAGGGAGAA GTGCCTGAGAATGACACAGAGAA
KIN12B AT3G23670 CAGTTTCTCCCTCGCAAAGTTG GCACGCTGAGCAAATCTCAA
BRC1 AT3G18550 TTCCCAGTGATTAACCACCAT TCCGTAAACTGATGCTGCTC
HB21 pair 1* AT2G18550 TCGCCGTCTGGTTCCAA CATCCTCGACTCGTTTGTTCTTC
HB21 pair 2 AT2G18550 GATCCAACGGCTTGCAAAAA CAATGGTCACAGAGGATGAGATAGG
HB21 pair 3 AT2G18550 GCCAATCATACGACACCGTTT CGTCAGCCTCACCGTCAAA
HB40 pair 1 AT4G36740 TTTATCTCTCAACTGTACCCTGATGTCT GCTTCGGCTGCTTCACTTCT
HB40 pair 2* AT4G36740 GGAGGAGGAAGAAGACCAAAGG ACAAACCGTTGCCTCCATCT
HB40 pair 3 AT4G36740 AAGTTGCCGTTTGGTTTCAGA TTCCTCGAGCCTTTTGTTCTTC
HB53 pair 1* AT5G66700 GGAGTCAGGGAGGAAGGAGAA CCACCTGTCTCGGGTCAAGA
HB53 pair 2 AT5G66700 ACGTCGTACTCGGCCAATG GAGCTTCACTCAATTGTTCTGTTAGTTT
HB53 pair 3 AT5G66700 GAAGAAATGCCAACCAACAGTTC TGGTGCATTGTTGGCTTCA
NCED3 AT3G14440 TCGTCGTACCTGACCAGCAA CCCACCGCGGATCATCT
AFP3 AT3G29575 GCCCTTCAAGTGCCCAATC GTTTTGGCTCGCTTCTTTGC
NRT1.2 AT1G69850 GGCAAATGACGTCACCAATTT GGAAACCACCGAGGAGAGCTA
GBF3 AT2G46270 AAACAGGCCGAGACAGAAGAAC GCCATGTTTTCGGCTGTCA
ABF3 AT4G34000 CCAAAGAGCGCCCTGGAT TTTTCTCACTCGCCCAAACAT
ChIP Site 1 AT2G18550 TTATCTTTCCGTACAAACACCCTTT AAGAGAGACCCAATGAGTCAATGAC
ChIP Site 2 AT4G36740 GCCCACATCAGGAAAATGACA GGGAAGAGGAAGACCTATGATTTATAAG
ChIP Site 3 AT4G36740 TCAATGGGCTCAGTTCAACAGT GCAAGCATCTGATTACACTTAAATGG
ChIP Site 4 AT5G66700 TGGTCATCTTCGGGATCCA AATAAGGTGGCTGACGACAATTC
ChIP Site 5 AT5G66700 GGGAAAGACGACCCAAGAATAA AGGTTGAGTCCTATGAGATGCTTTTT
ChIP Site 6 AT5G66700 AAAAAAGTTCCGTCCGTACAATCT AATGAGTGGGCAACTGTAAATGG
ChIP ACT2 AT5G09810 CCCTCGTAGATTGGCACAGT GGCCGTTCTTTCTCTCTATGC
ChIP HSF1 AT4G17750 GCTATCCACAGGTTAGATAAAGGAG GAGAAAGATTGTGTGAGAATGAAA
ChIP NCED3 AT3G14440 TGTGAAAGGTCTCAAGTCTCAAC AGAGGGAATGGAGTGAGTGG

* Primers used in all the experiments unless otherwise stated

 Supplemental Data Set 2b. Other primers used in this work

Name Sequence

attB1-BRC1 ggggacaagtttgtacaaaaaagcaggctTAATGAACAACAACATTTTCA
attB2-BRC1 ggggaccactttgtacaagaaagctgggtTCAATACATGTTTTGATAGTTGTGCATGAGGTC
Xba1-BRC1 GGGGGTCTAGAATGAACAACAACATTTTCAGTACTACTACCACC
BRC1-BamH1 GTGTGGGATCCTCAATACATGTTTTGATAGTTGTGCATGAGGTC
attB1-HA ggggacaagtttgtacaaaaaagcaggctATGGCATACCCATACGACGTTCCG
attB2-BRC1NOSTOP ggggaccactttgtacaagaaagctgggtAATACATGTTTTGATAGTTGTGCATG
attB1-HB21p1k ggggacaagtttgtacaaaaaagcaggctTCCTCTAATCCTCTCATCACA
attB2-HB21p ggggaccactttgtacaagaaagctgggtGTTTCTTCTTTTCGTTCTCT
attB1-HB40p1k ggggacaagtttgtacaaaaaagcaggctATGTATATATATCATTGAATT
attB2-HB40p ggggaccactttgtacaagaaagctgggtGTTCTTGGATCTCTCTCTGTC
attB1-HB53p2k ggggacaagtttgtacaaaaaagcaggctGTTCGTTGCCCACACATTACT
attB2-HB53p ggggaccactttgtacaagaaagctgggtTTTCTCTCTCTAGTTTTTCAG
attB1-HB53p1k ggggacaagtttgtacaaaaaagcaggctCTCGGGAAGATTCGTGGTTAT
attB2-HB21NOSTOP ggggaccactttgtacaagaaagctgggtTCATAAATTGGCTCATCCAGT
attB2-HB40NOSTOP ggggaccactttgtacaagaaagctgggtTTATGTATAGACTCATCCATT
attB2-HB53NOSTOP ggggaccactttgtacaagaaagctgggtTTACATACAAACCATCCCAAT
HB21-ATG ATGAATAACCAGAATGTAGATGA
HB21-STOP TTACATAAATTGGCTCATCC
HB40-ATG ATGAACTACACGGTGGATGAT
HB40-STOP TTATATGTATAGACTCATCCATT
HB53-ATG ATGGATCATGGTAGGTTAATG
HB53-STOP TTATACATACAAACCATCCCA
XhoI-GFP CCGCTCGAGatggtgagcaagggcgaggagctg
BRC1-PacI ccccTTAATTAAtcAATACATGTTTTGATAG
XhoI-BRC1 CCGCTCGAGatgaacaacaacattttcagtactactaccacc
GFP-PacI ccccTTAATTAAttacttgtacagctc
XbaI-HB21 TGCtctagaATGAATAACCAGAATGTAGATGA
HB21-BamHI GCGggatccTTACATAAATTGGCTCATCC
XbaI-HB40 TGCtctagaATGAACTACACGGTGGATGAT
HB40-BglII TCCagatctTTATATGTATAGACTCATCCATT
XbaI-HB53 TGCtctagaATGGATCATGGTAGGTTA
HB53-BamHI GCGggatccTTATACATACAAACCATCCCAAT
HB53-3' AATTTCACTTTGAGCTTCACTC
HB53-5' ACGTCGTACTCGGCCAATG
HB40-3' GGTTCTGAAACCAAACGGCA
HB21 genot5 TGGACACCAAGTGAAACCATCA
HB21 genot3 GGGTCCAAACTTGACCTCCAT
p745 AACGTCCGCAATGTGTTATTAAGTTGTC
LBa1 tggttcacgtagtgggccatcg
o8409 ATATTGACCATCATACTCATTGC
attB1 GUS ggggacaagtttgtacaaaaaagcaggcttaATGTTACGTCCTGTAGAAA
attB2 GUS ggggaccactttgtacaagaaagctgggtTCATTGTTTGCCTCCCTGCT
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