supplemental figure 1. altered leaf development in ... · ecori atg atg lb xve t3a kan nos t rb 35s...

Supplemental Figure 1. Altered leaf development in seedlings induced by heat-shock for cre/lox-mediated excision of RBR (RBRlox). (A) Two-week-old WT plant induced for 11 consecutive days. Box: analyzed region by

scanning electron miscroscope (SEM) shown in (C).

(B) Two-week-old RBRlox induced for 11 consecutive days. Box: analyzed region by

SEM shown in (D).

(C) Puzzle-shaped pavement cells on the adaxial side of the mock induced WT leaf

visualized by SEM.

Supplemental Data. Borghi et al. (2010). Plant Cell 10.1105/tpc.110.074591

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(D) Adaxial side of the induced RBRlox leaf visualized by SEM. Mutant cells did not

acquire the typical pavement cell shape.

(E) Mutant leaf sectors (GFP expression was activated after cre/lox-mediated excision

of RBR, see F) did not develop properly (notice the leaf lateral asymmetry).

(G) Six days after induction, young leaves initiated before RBR excision (arrowhead)

were fully GFP positive.

(H) Nine days after induction, new expanding leaves were GFP negative (arrowhead).

(I) Top view of the shoot apical meristem (SAM) of an induced cre/lox seedling

visualized by confocal laser scanning microscopy (CLSM). No GFP sectors, and thus no

RBR excision, were detectable in the central zone (asterisk).

Scale bars A, B, E, F, G, H = 1 mm; C, D, I = 100 µm.


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EcoRI

ATG

ATG

XVE T3A Kan nos T RBLB35S nos P

RBRhp T3A hpt nos T RBLBolexA nos P

A

B

Supplemental Figure 2. Design of the β-estradiol inducible RNAi against RBR

(RBRi). (A) Map of the hairpin (RBRhp) built to promote RNAi against RBR. A 1131-bp long

RBR genomic fragment was cloned upstream of the corresponding inverted RBR cDNA

using a native EcoRI site.

(B) RBRhp was inserted into the binary inducible system constructed by Brand et al.,

2006. The system driver expresses the chimeric transcription factor XVE under control

of the 35S promoter. The RBRhp target was inserted downstream of the OLexA

sequence, the binding site for XVE. After β-estradiol treatment, XVE can enter the cell

nucleus and bind (dashed arrow) the OLexA sequence, thus activating the RBRi

system.


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Supplemental Figure 3. Induction test of 35S:XVE; OLexA:GUS plants by β-

estradiol spraying or supply in MS plates.

(A) Before β-estradiol treatment, no blue patches were detected after GUS infiltration.

(B) Two days after induction by β-estradiol spraying, the GUS signal was observed in

every type of plantlet tissue. Five-day-old 35S:XVE; OLexA:GUS seedlings germinated

on MS medium plus β-estradiol were evenly blue stained (insert).

(C,D) Higher magnifications of 35S:XVE; OLexA:GUS leaf blades before and after β-

estradiol induction.

Scale bars A, B = 0.5 cm; C, D = 0.5 µm.


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Supplementary Figure 4 online. RBRpromoter:GUS expression patterns and RBR protein levels during Arabidopsis development. (A-G) RBR promoter activity was monitored in ProRBR:GUS seedlings. ProRBR:GUS

was constructed by cloning the 1600-bp promoter-region of RBR upstream of the GUS

gene in pCambia1300. GUS staining had a conspicuous maximum in cotyledons two

days after germination (A=1 d.a.g., B=2 d.a.g., C=5 d.a.g.). GUS staining was also

strong in dividing cells, such as root tips (F), emerging lateral roots (E), and the proximal

half of young cotyledons (C, D). As a control, a wild-type plant is displayed in G.

(H) Analysis of RBR protein levels. Proteins were analyzed by SDS-PAGE and

immunoblotted with an antibody raised against the N-terminal domain of RBR

(specificity of the antibody is shown in I). The migration of proteins of standard sizes

(kDa) is indicated. A Coomassie-stained gel with the same protein loading is shown as

control. Samples were collected from 3-, 14- or 30-day-old seedlings grown under long

day conditions.

(I) Specificity of α-RBR antibodies. Antibodies were raised in rabbit against the N-

terminal domain of RBR. In a soluble protein extract prepared from seven-day-old wt

seedlings, a band at around 115 kDa was recognized by the serum but not by the pre-

immune serum. The band disappeared when the blotting membrane was co-incubated

with the antigene. All experiments described in the manuscript were performed using

affinity-purified antibodies.

Scale bars: A, D, E, F = 100 µm; B, C, G = 500 µm.


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Supplemental Figure 5. RBR protein recovery after β-estradiol withdrawal. Digital

quantification (integrated density measurement) via ImageJ® of RBR protein extracted

from 120 seedlings per genotype / treatment. COL-ms: WT Arabidopsis thaliana grown

on MS medium; RBRi-ms: RBRi seedlings grown on MS medium; COL-e: WT

Arabidopsis grown on β-estradiol-containing medium (mock). RBRi-e: RBRi seedlings

grown on β-estradiol-containing medium (induced); COL-ms-10: 10-day-old WT

seedlings grown on MS medium; RBRi-ms-10: 10-day-old RBRi seedlings grown on MS

medium; COL-rec: 10-day-old Arabidopsis grown on β-estradiol-containing medium

(mock); RBRi-rec: RBR recovery is visible five days after β-estradiol withdrawal

(compare RBRi-rec to RBRi-e). The Coomassie-blue stained gel is shown as a

quantitative control.


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Supplemental Figure 6. Cell number quantification on adaxial and abaxial sides of

WT and RBRi β-estradiol-treated seedlings.

(A,B) Scanning electron microscopy (SEM) images of adaxial sides of WT and RBRi

leaf blades, respectively. Each digitally-drawn yellow spot marks a single cell.

(C,D) SEM pictures of abaxial sides of WT and RBRi-induced leaf blades.

(E) Cell number counting on adaxial (light grey) and abaxial (dark grey) sides on a total

leaf surface of 145000 µm2 in WT (a total of 530 cells counted) and induced RBRi (a

total of 1460 cells counted) leaves.

Scale bars: 100 µm.


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Supplemental Figure 7. Cell number quantification in WT and β-estradiol-treated

RBRi shoot apical meristems (SAM). Cytoplasmic dense cells belonging to L1, L2

and L3 layers in the shoot apical meristem were counted. Five days after β-estradiol

treatment, the RBRi SAM contained a higher number of cells.


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Supplemental Figure 8. Quantification of not-puzzle-shaped, brick-like pavement

cells on abaxial sides of WT and β-estradiol-treated RBRi leaves.

(A-C) Proximal, middle and distal, respectively, representative areas of a WT leaf. The

area covered by small, not-puzzle-shaped pavement cells was slightly larger at the

proximal side of the leaf (G, light grey bars), where cell proliferation was still detected

(see Figure 4A).

(D-F) In induced RBRi seedlings, more than 50% of the pavement cells did not acquire

the typical puzzle shape in the proximal part of the leaf blade (G, dark grey bars), while

the distal part was phenotypically similar to WT.

Scale bars A-F = 100 µm.


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Supplemental Figure 9. Quantitative PCR analysis of genes involved in stomate

development in WT and β-estradiol-treated RBRi leaves.

(A) Expression levels of genes required for early (SDD1, TMM) and late (EPF1, FAMA,

MUTE) stomate development were analyzed before and after RBRi induction. SPCH,

target of a MAPK cascade in regulation of cell proliferation (Lampard et al., 2008), is

upregulated. Gene expression quantification is relative to Time0 (before β-estradiol

treatment).

(B-D) ProMUTE:MUTE-GFP signal in WT leaves five days after β-estradiol treatment.

(E-G) ProMUTE:MUTE-GFP signal in RBRi leaves five days after β-estradiol treatment.

Scale bars B-G = 40 µm.


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Supplemental Figure 10. Silique elongation and fertility in 30 day-old WT and

RBRi plants treated with β-estradiol for two consecutive weeks.

(A) Tip of a β-estradiol treated WT inflorescence (inset, full plant).

(B) Tip of an induced RBRi inflorescence (inset, full plant).

(C) Number of WT (green) and RBRi (red) siliques belonging to four different length

classes (aborted: length<0.2 cm).

(D) After β-estradiol treatment, the few siliques produced by RBRi plants contained only

aborted ovules.

Scale bars A,B = 1 cm; D = 120 µm.


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Supplemental Figure 11. Nuclear DNA ploidy analysis in WT and β-estradiol

treated RBRi leaves. (A) Ploidy of leaves (n=8) number 3,4 from 24-day-old plants.

(B) Ploidy of leaves (n=8) number 7,8 from 24-day-old plants.

(C) Ploidy of leaves (n=8) number 3,4 from 24-day-old β-estradiol-induced plants.

(D) Ploidy of leaves (n=8) number 7,8 from 24-day-old β-estradiol-induced plants.

(E) Ploidy of leaves (n=8) number 3,4 from 16-day-old plants.

(F) Ploidy of leaves (n=8) number 5,6 from 16-day-old plants.

(G) Ploidy of leaves (n=8) number 3,4 from 16-day-old β-estradiol-induced plants.

(H) Ploidy of leaves (n=8) number 5,6 from 16–day-old β-estradiol-induced plants.


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Supplemental Figure 12. Exemplary coexpression clusters of genes deregulated

in RBRi plants after β-estradiol treatment.

Coordinate expression changes in expression modules associated with general stress

response proteins WRKY33 (A) and STZ/ZAT10(B), DNA replication (BARD1, C) and

cell cycle checkpoints (WEE1, D) are shown. The co-expression modules were derived

from the ATTED II database and expression changes are depicted schematically by red

(upregulation upon RBRi induction) or green (downregulation) bars in the order (each

set of bars, from left to right) 3, 6, 12 and 24 h after induction, respectively. Empty bars

denote unchanged expression.


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Supplemental Figure 13. Quantitative PCR analysis of WRKY33 and WRKY40, two transcription factors involved in stress response, in WT and RBRi induced plants. WRKY33 and WRKY40 expression levels were strongly upregulated in RBRi plants

treated with β-estradiol (n=50 for each time point / genotype / treatment). Smaller gene

expression changes were detected in WT and mock spraying tests (n=50 for each time

point / genotype / treatment). The error bars represent the SE from two biological

replicas.


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Supplemental Figure 14. Quantitative PCR analysis of AGP12, 17 and 18

expression levels in WT and RBRi β-estradiol treated plants. AGP12, AGP17 and

AGP18 expression levels were reduced in induced RBRi plants (n=50 for each time

point / genotype / treatment), in comparison to WT (n=50 for each time point / genotype

/ treatment), 12 hours after β-estradiol treatment. The error bars represent the SE from

two biological replicas.


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0

1

2

3

4

5

6

RPA2

ATORC1

ATORC2

ATORC4

ATORC6

PCNA1

PCNA2

MCM

3

MCM

4

MCM

5

MCM

7

MCM

9

CYCA3;1

CYCA3;2

CDKB1;2

CDKB2;1

E2FC

H2A

H3

H5

MSI2

MSI3

VIM1

FAS2

DD

M1

DD

M2

Sign

al to

log

rati

o re

lati

ve to

Tim

e0 (b

efor

e in

duct

ion)

Selected genes 12 and 24 hours after induction of RBRi

12 hai 24 hai Supplemental Figure 15. Selection of DNA-replication related genes upregulated

12 (light grey) and 24 (dark grey) hours after β-estradiol treatment.


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Time points q m n k phyper3 - 6 h 118 189 22557 295 1.34E-1846 - 12 h 79 295 22451 335 3.90E-7912 -24 h 75 335 22411 367 2.46E-65

3 - 6 -12 h 55 118 22628 335 1.91E-716 -12 - 24 h 7 79 22667 367 4.09E-05

3 - 6 -12 -24 h 4 55 22691 367 1.91E-03 Supplemental Table 1. Coexpression analyses and validation via hyper geometrical

distribution of genes upregulated at 3-6-12-24 h after β-estradiol treatment of RBRi

seedlings. Strong coexpressions (phyper < E-05) were detected at 3-6-12 hrs after β-

estradiol treatments and 12-24 h after induction. q = common upregulated genes at

analyzed time points; m = genes upregulated at the first time point; n = total gene

number on ATH1 Affymetrix microarray subtracted by m; k = genes upregulated at the

second time point.

Time points q m n k phyper3 - 6 h 32 183 22563 124 2.58E-416 - 12 h 16 124 22622 265 9.11E-1412 -24 h 44 264 22482 401 4.73E-31

3 - 6 -12 h 4 32 22714 265 3.22E-056 -12 - 24 h 4 16 22730 401 6.18E-06

3 - 6 -12 -24 h 1 4 22742 401 1.82E-03 Supplemental Table 2. Coexpression analyses and validation via hyper geometrical

distribution of genes downregulated at 3-6-12-24 h after β-estradiol treatment of RBRi

seedlings. Strong coexpressions (phyper < E-05) were detected at 3-6-12 h after β-

estradiol treatments and 12-24 h after induction. Genes upregulated early (3-6 h) and

late (12-24 h) after RBR downregulation belong to different functional categories (see

Supplemental Table 3) q = common upregulated genes at analyzed time points; m =

genes upregulated at the first time point; n = total gene number on ATH1 Affymetrix

microarray subtracted by m; k = genes upregulated at the second time point.


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m q values for phyper RBRi 3 h pvalue RBRi 6 h pvalue RBRi 12 h pvalue RBRi 24 h pvalue

574 E2Fa/DP ox 78 2.23E-50 114 9.05E-9420 WEE1 ATTED 7 2.20E-10 7 4.53E-1020 BARD1 ATTED 9 6.81E-14 17 6.68E-3120 STZ/ZAT10 ATTED 7 2.26E-12 8 1.40E-12 11 9.70E-1820 WRKY33 ATTED 7 2.26E-12 11 2.08E-18 7 2.20E-1020 WRKY40 ATTED 14 5.28E-28 14 5.06E-25 14 3.53E-24

k=189 295 335 367

Supplemental Table 3. Coexpression analyses and validation via hyper geometrical

distribution between genes de-regulated at 3-6-12-24 hrs after β-estradiol treatment of

RBRi seedlings and E2F/DP over expression (ox) clusters or ATTED clusters. Stress

related ATTED clusters (STZ/ZAT10, WRKY33 and WRKY40) strongly coexpress with

genes early de-regulated after RBRi induction. E2F/DP ox, WEE1 and BARD1

coexpress with genes later de-regulated by β-estradiol inductions. q = common

upregulated genes at analyzed time points; m = genes upregulated at the first time

point; n = total gene number on ATH1 Affymetrix microarray subtracted by m; k = genes

upregulated at the second time point.


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Supplemental Methods

Primer sequences

Gene Forward primer (5'-3') Reverse primer (5'-3')

AGP12 GCT CAT AGT GGT GTT GAT GG CAG CAT CGG AAG TAG GACAGP17 ACC AAG CGA TGC TTT CTC AAC AAG TAG AGA CCA TGC GAAGP18 CCG CTA CAT TCC TCA ACA C CAC CAC CGA CTG AAT CTCANT CTT GTG GAG ACA AGT GTT GG GAA GCA CCC CTA GAG AAA CCBARD1 TAG GGT ATC TGA GGC CAC TGG CGC TAT GGA CAT GGA TAA CAT TGGCLV3 CCG GTC CAG TTC AAC AAC TG CCA ACG GTA CAT TGC TTT GGA GCYCB1;1 AAA ATC CCA CGC TTC TTG TGG A CAT ATG GGA ACT TGG CGG AGA G EPF1 GCT GCT CTT GAA GAT TTG GCT CAA CCT AGA CCC AGC CAC CTFAMA TAA CAA GTG AAA CGA GGT TTA CGG GTT AGA GCT TCC AGA TAT GTT GGTKNAT1 GCC AAA AGA TTG GAG TCC AC GCT ACC TTC TCT GAC TCA GAMUTE CGT TGT TAA GAT AGG ATT GGA GTG TC GGT AGA GAC GAT CAC TTC ATC AGA CPP2A CAA GTG AAC CAG GTT ATT GGG A ATA GCC AGA CGT ACT CTC CAGProRBR CCC CAG CTG AAA ACA TGC AAA ATC AAG C CCG GTA CCA CTA GGA ATT TGC CTC GTG TCRBR AGT GAA GAG GCT TAG TGT GAG TCC ACA ATA TTT AGC TTC AGA GCCSDD1 TCG TGA ATA CAT TTC CGC AAG AG CAT TGC CAA GAA CAT TCG CCSPCH GCT GCT CTT GAA GAT TTG GCT CAC TCA ATT CCA ATC TTG ATG GTGSTM AAT AGT GAT GGT CCG ATG TG TGG AGA GCT CTT GCT CAT ACTMM GGA CGT GAA GCA TCT AAG CGA TCA GCT TTC TCC TCA TCC TCCWRKY33 TGC AGA CAA CGA GTG ATA TTG AC TTG TAG TAG CTT CTT GGA TTT GGGWRKY40 GAA GAT CCA CCG ACA AGT GC CCT CAA GAC GTT GTA GTT GTC ACWUS ACA GCA TCA GCA TCA TCA TC TTG GCC ATA CTT CCA GAT GG

Supplemental references

Brand, L., Horler, M., Nuesch, E., Vassalli, S., Barrell, P., Yang, W., Jefferson, R.A., Grossniklaus, U., and Curtis, M.D. (2006). A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis. Plant Physiol 141, 1194-1204.

Lampard, G.R., Macalister, C.A., and Bergmann, D.C. (2008). Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 322, 1113-1116.


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