Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data....

11
Ponceau -CUL3 * * * -GFP Col0 wri1-3 175 kDa 80 kDa 58 kDa 46 kDa 30 kDa -CUL3 Ponceau Ponceau -WRI1 B C D E A Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 1 : Verification of α-CUL3 and α-WRI1 antibodies. (A) Upper sequence: partial alignment of Arabidopsis CUL3a and CUL3b sequences. Lower sequence: Arabidopsis WRI1. Marked in gray are the peptide sequences used for antibody generation. (B) Western blot analysis in WT, cul3a, cul3b and cul3 hyp backgrounds with α-CUL3. (C) The specificity of α-CUL3 was confirmed by western blot analysis in transient expression assays with a GFP:CUL3a construct in tobacco. (D) PCR identification of homozygous T-DNA mutant wri1-3.A WRI1 gene-specific product was amplified in WT but not in wri1-3, while one T-DNA specific product was amplified in wri1-3 but not in WT. (E) WRI1 is detectable in WT but not in wri1-3.

Transcript of Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data....

Page 1: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

Ponceau

-CUL3* * *

-GFP

Col0

wri1-3

175 kDa80 kDa58 kDa46 kDa

30 kDa

-CUL3 Ponceau

Ponceau

-WRI1

B C

D E

ASupplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 1 : Verification of α-CUL3 and α-WRI1 antibodies. (A) Upper sequence:partial alignment of Arabidopsis CUL3a and CUL3b sequences. Lower sequence: Arabidopsis WRI1.Marked in gray are the peptide sequences used for antibody generation. (B) Western blot analysis inWT, cul3a, cul3b and cul3hyp backgrounds with α-CUL3. (C) The specificity of α-CUL3 wasconfirmed by western blot analysis in transient expression assays with a GFP:CUL3a construct intobacco. (D) PCR identification of homozygous T-DNA mutant wri1-3. A WRI1 gene-specific productwas amplified in WT but not in wri1-3, while one T-DNA specific product was amplified in wri1-3but not in WT. (E) WRI1 is detectable in WT but not in wri1-3.

Page 2: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

-WRI1

-CUL3

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 28 29 3031 32 33 34 3527*75 kDa

*43 kDa

*440 kDa

WT

GFP:WRI1 DAPI GFP:WRI1

DAPI

A

B

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 2: FPLC analysis and subcellular localization of WRI1. (A) Western blot analysis of FPLC fractions from two-week-old ArabidopsisWT plantlets tested with either α-WRI1 or α-CUL3, showed co-migration of the two proteins in fractions between 70 and 150 kDa. WRI1 is also present infractions corresponding to proteins of around 40-50 kDa, and in fractions higher than 150 kDa, indicating that the protein also assembles in complexes distinctfrom CUL3. (B) Transient expression analysis of GFP:WRI1 in tobacco shows that the fusion protein is located in the nucleus (green fluorescence is derivedfrom GFP; blue fluorescence from DAPI staining).

Page 3: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

02468

1012

WRI

1Ex

pres

sion

(F

old

Cha

nge)

*

*B

0100200300400500600700

IAA5

Expr

essi

on(F

old

Cha

nge)

*

*C

-WRI1

Ponceau

CHX100M

6h

CHX50M

6hMock

A MG13210M

6hMock

Supplemental Figure 3: CHX treatment does not reduce the protein level ofWRI1, but it induces the transcription level of WRI1. (A) WRI1 protein levelsdo not decline 6h after CHX treatment (50 µM or 100 µM). (B) CHX but notMG132 treatment up-regulates WRI1 expression (p< 0.01; indicated by asterisk).(C) IAA5 (auxin-responsive protein IAA5) was used as positive control for CHXtreatment. The expression of IAA5 was strongly induced by CHX treatment (p<0.01; indicated by asterisk). In all cases, two-week-old seedlings were used.Error bars indicate standard error.

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Page 4: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

BPM1MATH:NLS

BPM1MATH

A

B

C

expr

essi

on le

vel (

fold

cha

nge)

0

20

40

60

80

100

120

140

160

180

200

WRI1

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 4: Sub-cellular localization of GFP:BPM1MATH and GFP:BPM1MATH:NLS. (A) Transient expression analysis ofGFP:BPM1MATH in tobacco demonstrates that the fusion protein is present in the cytosol and nucleus (bar=5 µm). (B) GFP:BPM1MATH:NLS

fusion protein is strictly localized to the nucleus (bar=10 µm). Arrows indicate nuclei. (C) The expression levels of WRI1 in WT and twoindividual lines of GFP:BPM1MATH , GFP:BPM1MATH:NLS.

Page 5: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

WT

BPM1MATH #1

BPM1MATH #4

BPM1MATH:NLS #1

6xamiBPM #1

BPM1MATH:NLS #3

6xamiBPM #2

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 5: Rosette leaf phenotype on the time of primaryinflorescence. 6xamiBPM and MATH-overexpressing plants develop fewer, shorterleaves than WT. Leaves of transgenic plants frequently develop wider blades (scalebar=1cm).

Page 6: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

WT 6xamiBPM #1

6xamiBPM #2

-WRI1

Ponceau

DMSO ActD2+CHX-WRI1

Ponceau

A

B

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 6: Stability assays of WRI1 in WT, 6×amiBPM, andMATH overexpressing lines. In comparison to WT, WRI1 protein is stabilized inboth 6×amiBPM and MATH overexpressing plants.

Page 7: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

0

5

10

15

20

25

30

35

40

45

50

14:0 16:0 16:1 16:2 18:0 16:3 18:1 18:2 20:0 18:3 20:1 22:0 22:1 24:0

Col0wri1-36xamiBPM #16xamiBPM #3

fatt

y ac

ids c

ompo

sitio

n in

µg/

30 se

eds

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

rela

tive

met

abol

ite a

bund

ance

sugars/sugar alcoholsfatty acids

0

0.005

0.01

0.015

0.02

0.025

rela

tive

met

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ite a

bund

ance

amino acids

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0.005

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0.015

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0.03

0.035

0.04organic acids

rela

tive

met

abol

ite a

bund

ance

WT

6xamiBPM #2

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 7: Fatty acid profile and metabolic profile in seeds of WT, wri1-3, and two 6xamiBPM lines. All extractions and measurements were done from mature and desiccated seeds from WT, wri1-3, and 6xamiBPM lines. In thegraph for fatty acids, numbers indicate the type of fatty acid (carbon chain length and number of double bonds). All values are based on five independent samples. Error bar indicates the standard deviation.

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ECol06xamiBPM #3

*

0.0

0.5

1.0

1.5

2.0

2.5

3.0Fo

ld c

hang

e /W

T (s

eeds

)

B

D

0

0.5

1

1.5

2

2.5

3

3.5

Expr

essi

on le

vel

( fol

d ch

ange

rela

tvie

to A

CTI

N) BCCP1 ATGLB1

Col0

6xamiBPM #3

A

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

seed

s dry

wei

ght i

n m

g (2

0 se

eds)

WT 6xamiBPM #3

WT6xamiBPM #3

-WRI1

Ponceau

C

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 8: Seed phenotype analysis for 6xamiBPM #3. (A) WRI1 protein level is elevated in the mutantline in comparison to WT, which (B,C) correlates with seed size and weight, and (D) increased expression of BCCP1and GLB1. Scale bar represents 1 mm. (E) Fatty acid levels are significantly increased in 6xamiBPM #3 in comparisonto WT (p<0.05, T-test).

GLB1

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BPM2

BPM5

BPM6

BPM4

BPM3

BPM1

Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292

Supplemental Figure 9: Predicted target sites for artificial microRNA (in red) on the different BPM genes. Numbersindicate base pairs.

Page 10: Supplemental Data. Chen et al. (2013). Plant Cell 10.1105 ... · 6/12/2013  · Supplemental Data. Chen et al. (2013). Plant Cell 10.1105/tpc.112.107292 Supplemental Figure 2: FPLC

Supplemental Table 1: Primers used for different PCR-based approaches Cloning of constructs Name of Primer Sequence of Primer (5'-3') T7BPM1MATHFW TAATACGACTCACTATAGGGAGAATGTTCAAGATCTGTGGGTAC BPM1MATHRW CTACATTTCTAGACTGGACCTCCTG BPM1-MATH-RW-NLS TCGTCCTCAGTGGACGCTTAGAGAGCACTTCTAGACTGGACCTCCTG NLS-1RW ACGCTTACGCTCAGATGGCTCACCGTCGTCGTCCTCACGTGGACGCTTAG NLS-RW2 CACGACCGTCCTTAGAACGCTCGTCACGCTCACGCTTACGCTCAGATGGC Attb2stop-NLS AGAAAGCTGGGTCACGACGGTTACCACCACGACCGTCC WRI1-FW ATGAAGAAGCGCTTAACCAC WRI-RW TCAGACCAAATAGTTACAAG qRT-PCR Name of Primer Sequence of Primer (5'-3') ACTIN2-qRT FW CCTGCCATGTATGTTGCCATT ACTIN2-qRT RW AATCGAGCACAATACCGGTTGT BPM1-qRT-FW ATTGGCGTCTACTCTTGT BPM1-qRT-RW AATGATGCTGCTCTGCTA BPM2-qRT-FW TAATCGGCACAGACTTGA BPM2-qRT-RW ACTCGCATATTGTTCTAAGC BPM3-qRT-FW CACCAGTTCACGATTCAAG BPM3-qRT-RW CCACCAACGGAGAAGATAT BPM4-qRT-FW TCCTGATGGCAAGAATCC BPM4-qRT-RW CGAAGTGGCTATGAACCT BPM5-qRT-FW TTAGGCTCAGGTTGTTGT BPM5-qRT-RW TCATCCTTCATCTGTTGGTA BPM6-qRT-FW GCATAAGGTTCATAGCCATT BPM6-qRT-RW AGATGTCTCAAGCAAGGA WRI1-qRT-FW GAGCAACAAGAAGCAGAG WRI1-qRT-RW CCACAACGATCCATTTCC BCCP1-qRT-FW CAGCCAAATCGTCACT BCCP1-qRT-RW GTTCCGGTATGGTCAG AtGLB1-qRT-FW CTTTCACCGTCTTAGGAACAAACAG AtGLB1-qRT-RW TAGGAACAGAGTTTCGATGTCTGAGAAC BPM1-MATH-qRTFW CGGAGGATAACTCGTCTT BPM1-MATH-qRTRW AATGGCTATGAACCTTATGC

 

 

 

 

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Supplementary Table 1 (continued): Primers used for different PCR-based approaches ChIP-qPCR Name of Primer Sequence of Primer (5'-3') EF1-qRT-FW CTGGAGGTTTTGAGGCTGGTTA EF1-qRT-RW CCAAGGGTGAAAGCAAGAAGA ProBCCP1-qRT-FW AAGTGAACTGTTGTTGTT ProBCCP1-qRT-RW CGTCTTCTTATTGTTATTGG Pro-GLB1-qRT-FW TTCCAATAATTACCTCCTT Pro-GLB1-qRT-RW TTTAACACAACTTTCAAAG