3 2 3 2 3 SC4DM - The Plant Cell...Nov 20, 2013 · Merged images of GFP fluorescence and chloroplast endogenous autofluorescence are shown. Scale bars = 10 µm. GFP Chlorophyll$

CH3 COOH4HO

CSD

CO2

SMO1

O

HO

HO

HO

HO cycloeucalenol

lanosterol

HO HOergosterol cholesterol

squalene epoxide

cycloartenol

24-methylene-cycloartanol

HO obtusifoliol

HO 4-α-methylergosta-8,14,24(28)-trienol

HO

HO HO

HO

HO

HO

HO

HO stigmasterol

sitosterol

24-methylenelophenol

HO

HO

HO 24-methylenecholesterol

HO campesterol

OO

HO

HO

OH

OH

brassinolide

4-α-methylfecosterol

episterol

5-dehydroepisterol

24-ethylenelophenol

Δ7-avenasterol

5-dehydroavenasterol

isofucosterol

Brassinosteroid pathway

SC4DM

SC4DM

SC4DM

SC4DM

HOCH3 CH3

24-Methylene Cycloartanol

SMO1

HOCH3

Cycloeucalenol

4

4

HO

24-Methylenelophenol

HO HO

SMO2 SMO2

HOOHO

Episterol

CSD

CO2

SMO2

SKR

CH2OHCH3 CHO

COOH

4

4

4

4

4

4

HOCH3 CH2OH

SMO1

OCH3

SKR

4

4

HOCH3 CHO

4

Supplemental Figure 1. Sterol biosynthetic pathway showing the cryptic steps (solid-lined box) leading to the formation of reactive SBIs from the SC4DM multienzyme complex.

Supplemental Data. Mialoundama et al. (2013). Plant Cell 10.1105/tpc.113.115576

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Supplemental Figure 2. Multiple sequence alignment of Arabidopsis ERG28 with homologous proteins between different species. Identical amino acids are shown against a black to grey background. The abbreviations refer to At, Arabidopsis thaliana; Os, Oryza sativa; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae and Sp, Schizosaccharomyces pombe. Accession numbers : At_ERG28 (At1g10030), Os_ERG28, (NP_001067350), Hs_ERG28 (NP_009107), Sc_ERG28 (NP_010962), and Sp_ERG28 (O74820). The sequence alignment was performed with the Geneious 5.3 software (BIOMATTERS Ltd, New Zealand) using the BLOSUM62 matrix.

F!M! S!L!Q!D! I!V! T!T!


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RTNLB2-‐GFP

ERG28-‐GFP

SMO1-‐GFP

CSD-‐GFP

SKR-‐GFP

Supplemental Figure 3. ER localization of the Arabidopsis SC4DM complex components. Arabidopsis leaves were transfected using the ERG28-GFP, SMO1-GFP, CSD-GFP and SKR-GFP constructs. The reticulon like RTNLB2-GFP was used as a positive marker of the ER membranes. Merged images of GFP fluorescence and chloroplast endogenous autofluorescence are shown. Scale bars = 10 µm.

GFP Chlorophyll Merged


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Supplemental Figure 4. Characterization of Arabidopsis erg28RNAi (erg28R2-1 to erg28R2-6) and erg28T-DNA (erg28T2-1 to erg28T2-6) mutants. (A) erg28RNAi plants were named erg28R2-1 to erg28R2-6. RT-PCR (top) and qRT-PCR analyses (middle) of ERG28 transcript of erg28R2-1 to erg28R2-6 and wild-type (WT) plants fol lowed by immunoblot analysis (bottom) using anti-A r a b i d o p s i s E R G 2 8 . α - Tu b u l i n (At1g04820) was used as a positive control for the RT-PCR (27 cycles for α-Tubulin and 35 cycles for erg28R2-1 to erg28R2-6 plants). The results of the qRT-PCR analysis represent mean values (error bars are s.d.; n = 3). (B) erg28T-DNA plants derived from the SALK line (SALK_000240) bearing an insertion in the ERG28 gene (At1g10030) were designated erg28T2-1 to erg28T2-6 and characterized. Schematic diagram (top) shows the T-DNA insertion indicated by the open triangle (black boxes, thin lines and white boxes indicate respectively exons, introns and UTR; D1, R1 and R2 were used as primers), followed by RT-PCR (upper middle), qRT-PCR (lower middle) and immunoblot analyses (bottom) performed as in (A) compared to WT plants. The results of the qRT-PCR analysis represent mean values (error bars are s.d.; n = 3). The combinations of D1-R1 and D1-R2 give the same result. (C) RT-PCR characterization of erg28T-DNA knockout plants complemented with Arabidopsis ERG28 (com2) compared to wild-type (WT) plants. Experimental conditions were as shown in (A).

B erg28T2 (SALK_000240)

541

ATG Stop D1

R1 R2

WBR2

RTTUBR2

WBTD2

RTTUBTD2

RTTD2

ERG28

ERG28

ERG28

α-‐Tubulin

TUB COM2

A

C

Com2L2

RNAI3bis4

0.0

0.5

1.0

1.5

2.0

2.5

ERG28

Rel

ativ

e ex

pres

sion

of

ER

G28

0.0

0.5

1.0

1.5

2.0

2.5

Rel

ativ

e ex

pres

sion

of

ER

G28

ERG28

α-‐Tubulin

α-‐Tubulin


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Class 1 I J B A Class 5

erg28R2-1 erg28T2-1 erg28T2-5 erg28R2-5

Class 2

E F Class 3

Class 4 Control Com erg28R2-3 erg28T2-3

Supplemental Figure 5. Phenotypes of Arabidopsis erg28RNAi (erg28R2-1 to erg28R2-6) and erg28T-DNA (erg28T2-1 to erg28T2-6) mutants. Phenotypes resulting from the RNAi knockdown (erg28R2-1 to erg28R2-6) and knockout (erg28T2-1 to erg28T2-6) of ERG28 were categorized into six phenotypic classes according to their vegetative development and compared to wild-type (WT) and complemented (Com) plants. (A, B) Class 1, 45-day-old erg28R2-1 (A) and erg28T2-1 (B) plants. (C, D) Class 2, 35-day-old erg28R2-2 (C) and erg28T2-2 (D) plants. (E, F) Class 3, 35-day-old erg28R2-3 (E) and erg28T2-3 (F) plants. (G, H) Class 4, 40-day-old erg28R2-4 (G) and erg28T2-4 (H) plants. (I, J) Class 5, 40-day-old erg28R2-5 (I) and erg28T2-5 (J) plants showing leaf fusion phenotype (arrowhead, close-up view below). (K, L) Class 6, 50-day-old erg28R2-6 (K) and erg28T2-6 (L) plants showing pin-shaped phenotype (arrowhead, close-up view below). (M, N) 20-day-old wild-type (M) and erg28T-DNA knockout plants complemented with Arabidopsis ERG28 cDNA (N). Scale bars, 0.5 cm.

erg28R2-2 erg28T2-2

C D

H G

erg28T2-4 erg28R2-4

K L erg28R2-6

erg28T2-6

Class 6

M N

WT Com2


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A B C

WT erg28R1-5 erg28R1-5

Supplemental Figure 6. Characteristics of specific organs and tissues of class 5 and class 6 erg28 mutants. (A-C) Cleared and green adult leaves of erg28R1-5 (A, B) compared to wild-type (WT) adult leaves (C). (A, B) 40-day-old and (C) 20-day-old plants. Scale bars, 0.5 cm. (D, E) Defects in the differentiation of interfascicular fibers of the inflorescence stems of erg28R1-5 adult plants (Figure 3O) observed in toluidine blue stained cross sections (arrows) (D) compared to WT plants (E). (D) 90-day-old and (E) 50-day-old plants. Scale bars, 100 µm. (F, G) Xylem strands and root hairs of erg28R1-6 plants (Figure 3PQ) are formed close to the root apex (arrows) (F) compared to WT plants (G). (F) 50-day-old and (G) 20-day-old plants. Scale bars, 100 µm.

D E

erg28R1-5 WT

F G erg28R1-6 WT

Class 5

Class 6


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Supplemental Figure 7. Effect of phytohormones on Arabidopsis erg28 development. Class 1 (erg28R1-1and erg28T1-1) plants were grown for 45 days on Murashige and Skoog solid medium in the absence or presence of different hormones as indicated. Scale bars, 0.5 cm.

24-Epibrassinolide

Indole 3-acetic acid

Gibberellin A3

trans-Zeatin

1-Aminocyclopropane 1 carboxylic acid

1  2.5 1 2.5 µM

erg28R1-1 erg28T1-1

WT

erg28R1-1 erg28T1-1 Hormones (-)

Hormones (+)


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Supplemental Figure 8. Effect of IAA and NAA on primary root elongation of wild-type (WT) and erg28 plants. erg28R1-4, erg28R1-5, erg28T1-4, erg28T1-5 and WT plants were grown for 8 days on Murashige and Skoog solid medium containing different concentrations of IAA (A) and NAA (B) (error bars are s.d., n = 3).

B

0 250 500 750 1000 12500

25

50

75

100

1-NAA (nM)

erg28T1-4

WT

erg28T1-5

erg28R1-4 erg28R1-5

0 250 500 750 1000 12500

25

50

75

100R

elat

ive

prim

ary

ro

ot e

long

atio

n

(%)

A

erg28T1-4

WT

erg28T1-5

erg28R1-4 erg28R1-5

IAA (nM)

Rel

ativ

e pr

imar

y

root

elo

ngat

ion

(%

)


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Supplemental Figure 9. Expression of ERG28 in Arabidopsis tissues and organs. Analysis was performed using the Genvestigator database (https://www.genevestigator.com/gv/plant.jsp). Bars represent standard errors from different microarrays.

ERG28


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Supplemental Figure 10. Spatial expression pattern of ERG28 according the Arabidopsis electronic fluorescent pictograph Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). The color code indicates the absolute intensity of ERG28 expression (from low (yellow) to high (red)).


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Seedlin

g

Flower

Young Siliq

ueSte

m

Juve

nile le

af

Adult lea

f

Senes

cent l

eaf

Root0

50

100

150

Supplemental Figure 11. qRT-PCR analysis of ERG28 expression in different Arabidopsis tissues. TIP41-LIKE (At4g34270) and GAPDH (At1g13440) were used to normalize the qRT-PCR data. The results of the qRT-PCR analysis represent mean values (error bars are s.d.; n = 3).

Rel

ativ

e ex

pres

sion


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Rnai

Sitosterol

Stimagsterol

Campesterol

Cholesterol

0

50

100

150150 S

tero

l con

tent

(µg

g-1 F

W)

100

50

0

Ste

rol c

onte

nt (µ

g g-

1 FW

)

Sitosterol

Stimagsterol

Campesterol

Cholesterol

0

50

100

150150

100

50

0

Sitosterol Stigmasterol Campesterol Cholesterol

Sitosterol Stigmasterol Campesterol Cholesterol

Supplemental Figure 12. Regular sterol content of wild-type (WT), erg28R1-1 to erg28R1-6 and erg28T1-1 to erg28T1-6 plants. Plants were grown in vitro on MS for 35 days (error bars are s.d. ; n = 3).


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cycloartenol

24 methylene*

Cycloeucalenol

Obtusifolio

l

4 methyl e

rgoasta*0.0

0.1

0.2

0.3

0.4

0.5

cycloartenol

24 methylene*

Cycloeucalenol

Obtusifolio

l

4 methyl e

rgoasta*0.0

0.1

0.2

0.3

0.4

0.50.5

Ste

rol c

onte

nt (µ

g g-

1 FW

)

0.4

0.3

0

Ste

rol c

onte

nt (µ

g g-

1 FW

)

0.5

0.4

0.2

0

Cycloartenol 24-Methylene-

cycloartanol Cycloeucalenol Obtusifoliol

Supplemental Figure 13. Levels of upstream sterol intermediates of wild-type (WT), erg28R1-1 to erg28R1-6 and erg28T1-1 to erg28T1-6 plants. Plants were grown in vitro on MS for 35 days (error bars are s.d. ; n = 3).

4-α-Methylergosta- 8,14,24(28)-trienol

0.2

0.1

0.3

0.1

Cycloartenol 24-Methylene-

cycloartanol Cycloeucalenol Obtusifoliol 4-α-Methylergosta- 8,14,24(28)-trienol


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24 m

ethyle

nelophen

ol

epist

erol

24 m

ethyle

necholes

tero

l

24 et

hylenelo

phenol

aven

aste

rol

isofu

cost

erol

0.0

0.5

1.0

1.5

2.0

2.52.5

Ste

rol c

onte

nt (µ

g g-

1 FW

)

2.0

1.5

24-Methylene- lophenol Episterol Δ7-Avenasterol

1.0

0.5

Isofucosterol 24-Methylene-

cholesterol 24-Ethylene-

lophenol

24 m

ethyle

nelophen

ol

epist

erol

24 m

ethyle

necholes

tero

l

24 et

hylenelo

phenol

aven

aste

rol

isofu

cost

erol

0.0

0.5

1.0

1.5

2.0

2.5

0

Ste

rol c

onte

nt (µ

g g-

1 FW

)

2.5

2.0

1.0

1.5

0.5

0

Supplemental Figure 14. Levels of sterol intermediates of campesterol and sitosterol branches of wild-type (WT), erg28R1-1 to erg28R1-6 and erg28T1-1 to erg28T1-6 plants. Plants were grown in vitro on MS for 35 days (error bars are s.d. ; n = 3).

24-Methylene- lophenol Episterol Δ7-Avenasterol Isofucosterol

24-Methylene- cholesterol

24-Ethylene- lophenol


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Standard

Supplemental Figure 15. Mass fragmentation patterns of CMMC from erg28 plants and of standard CMMC by UPLC MS/MS-APPI+. 35-day-old erg28R1-1, erg28T1-2 and erg28R1-6 plants were used. The parent ion [M+ H - H2O] = 453 was selected. The cone voltage (25 V) and the medium collision energy (23 V) were used to generate the fragments. RI : relative intensity.

191

205 191

149 109 123

369

453

109

123 149 205 369

453

453

369 205 191 149 123

109

109 123

149

205

191

453

100

0

100

RI %

100  120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 m//z

m//z

m//z

m//z

100  120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100  120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

100  120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 0

100

RI %

0

100

RI %

0

RI %

erg28R1-1

erg28T1-2

erg28R1-6


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Standard

MRM transition m/z 453 > 109 6.30e3

MRM transition m/z 453 >109 1.67e4


MRM transition m/z 453 >109 1.98e5

MRM transition m/z 453 > 109 587

m/z = 109

0

WT

Retention time (min) 12 14 16 18 20 22 24

100

RI %

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24 1

100

RI %

100

RI %

100

RI %

100

RI %

0

4

1

0

Supplemental Figure 16. Selected reaction monitoring (MRM) chromatogram of CMMC (453/109, 453/123 m/z). (A) MRM of CMMC (arrowhead) from lipid extracts of 35-day-old erg28R1-1, erg28T1-2 and erg28R1-6 and WT plants compared to authentic CMMC standard (arrow) using the transition (453/109 m/z). RI refers to relative intensity. (B) MRM of CMMC from lipid extracts (A) using the transition (453/123 m/z) .

HO

OOH

erg28R1-1

erg28T1-2

erg28R1-6






m/z = 123

Retention time (min)

Standard

WT

v

v

v

V

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

100

RI %

2

1

2

17

1

100

RI %

100

RI %

100

RI %

100

RI %

HO

OOH

erg28R1-1

erg28T1-2

erg28R1-6

A

B


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m/z= 149

Standard

WT

v

v

v

v

100


RI %

2

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24 2

100

RI %

100

RI %

100

RI %

100

RI %

6

11

1

2

Supplemental Figure 17. Selected reaction monitoring (MRM) chromatogram of CMMC (453/149, 453/191 m/z). (A) MRM of CMMC (arrowhead) from lipid extracts of 35-day-old erg28R1-1, erg28T1-2 and erg28R1-6 and WT plants compared to authentic CMMC standard (arrow) using the transition (453/149 m/z). (B) MRM of CMMC from lipid extracts (A) using the transition (453/191 m/z).

HO

OOH

erg28R1-1

erg28T1-2

erg28R1-6

MRM transition m/z 453.5 > 191 1.02e3

MRM transition m/z 453.5 > 191 1.77e3

MRM transition m/z 453.5 > 191 721



m/z = 191


Standard

WT

v

v

v

V

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

100

RI %

8

5

9

21

18

100

RI %

100

RI %

100

RI %

100

RI %

HO

OOH

erg28R1-1

erg28T1-2

erg28R1-6

A

B


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Standard

WT

v

v

v

V

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

100

RI %

7

4

5

11

0

100

RI %

100

RI %

100

RI %

100

RI %

Supplemental Figure 18. Selected reaction monitoring (MRM) chromatogram of CMMC (453/205, 453/369 m/z). (A) MRM of CMMC (arrowhead) from lipid extracts of 35-day-old erg28R1-1, erg28T1-2 and erg28R1-6 and WT plants compared to authentic CMMC standard (arrow) using the transition (453/205 m/z). (B) MRM of CMMC from lipid extracts (A) using the transition (453/369 m/z).

HO

OOH

HO

OOH

m/z= 205 erg28R1-1

erg28T1-2

erg28R1-6






m/z = 369


Standard

WT

v

v

v

V

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24

12 14 16 18 20 22 24 7

3

4

11

0

100

RI %

100

RI %

100

RI %

100

RI %

100

RI %

HO

OOH

erg28R1-1

erg28T1-2

erg28R1-6

B

A


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ABCB19

ABCB1

Supplemental Figure 19. CMMC competes with NPA binding-sites and docks into the nucleotide binding site of homology-modeled Arabidopsis ABCB19 and ABCB1 auxin transporters. (A) Microsomal membranes from rosette leaves of 35-day-old wild-type rosette leaves were incubated with 20 nM [3H]NPA, in the absence (black bars) or presence (white bars) of 20 nM CMMC (error bars are s.d. ; n = 3). Surface (B, F) and ribbon (C, G) representation of the docked complex of ABCB19 and ABCB1 auxin efflux transporters with CMMC (cyan), cholestanoic acid (magenta), NPA (yellow) and ATP (white) shown as stick structures. The proposed nucleotide-binding domains (NBDs) are shown in a close-up view for the NBD1 (D, H) and NBD2 (E, I) of ABCB19 and ABCB1 in a dimeric representation. Scale bars, 10Å in (B, C, F, G) and 1.5 Å in (D, E, H,I).

NBD1 NBD2 NPA

NPA + MCCM

0

50

100

150

Control CMMC

150

100

50

NPA

bin

ding

(f

mol

mg-

1 pr

otei

n)

0

A

Surface Ribbon B

F

C

G

D

H

E

I


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Supplemental Table 1. In gel digestion and Nano LC-MS/MS of Arabidopsis ERG28 interactors in the SC4DM multienzyme complex.

Proteins were separated by SDS-PAGE. Bands (P1, P2 and P3) shown in the Figure 1D were digested and used for protein identification by liquid chromatography coupled to tandem mass spectrometry. The protein accession numbers : gi|15234416, gi|18401656 and gi|30686710 correspond respectively to SMO1 (At4g12110),CSD (At1g47290) and SKR (At5g18210).

Gel Band Enzyme MS/MS sample name Protein name

Protein accession numbers

Protein molecular

weight (Da)

Number of unique

peptides

Percentage sequence coverage

Peptide sequence Previous amino acid

Next amino acid

Best Mascot Ion score

Best Mascot Identity score

Mascot Ion - Identity score

Number of enzymatic

termini

Calculated +1H Peptide Mass (AMU)

Peptide start index

Peptide stop index Assigned

Chymotrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% ATVEEASIALGRNL Y T 45,7 29,3 16,4 2 1443,7802 5 18 trueChymotrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% DFPWSPTKY Y I 34,9 31 3,9 2 1140,5361 222 230 trueAspN Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% DFPWSPTKYIPFYGGAEYH Y D 35 28,2 6,8 2 2275,0496 222 240 trueChymotrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% ILVVGPL F Q 25,5 25 0,5 2 710,4812 97 103 trueTrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% LETLWFDYSATK R S 73,6 28,1 45,5 2 1473,7261 21 32 trueTrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% QMEAIETHSGYDFPWSPTK R Y 26,2 25 1,2 2 2239,9967 211 229 trueTrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% SASGLFNR R Y 72,3 29,3 43,00 2 851,4369 64 71 trueChymotrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% SLVPLPL F V 36,7 25 11,7 2 738,4761 50 56 trueTrypsin Mudpit_X00661 SMO1-1 (STEROL-4ALPHA-METHYL OXIDASE 1-1); 4,4-dimethyl-9beta,19-cyclopropylste gi|15234416,gi 34644,4 9 29,50% VNYSLSDMFK K C 61,9 27,3 34,6 2 1219,5664 78 87 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% AAGQAYFITNMEPIK K F 98,9 28,5 70,4 2 1653,8306 252 266 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% AEGEALILK K A 57 28,3 28,7 2 943,546 166 174 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% ALASGGEVCAK R A 68,1 30,1 38,00 2 1062,5249 241 251 trueChymotrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% ASGGEVCAKAAGQAY L F 38,6 28,4 10,2 2 1439,6584 243 257 trueAspN Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% DESLPYPPKHN A D 37,5 31,1 6,4 2 1296,622 148 158 trueAspN Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% DFTYVENVVHAHVCA Y E 89,3 28,9 60,4 1 1760,806 224 238 trueAspN Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% DGVHGTLNA F D 37,8 28,9 8,9 2 883,4269 139 147 trueChymotrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% DGVHGTLNADESLPYPPKHNDSY F S 46,7 25 21,7 2 2526,1534 139 161 trueAspN Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% DSYSATKAEG N E 33,6 31,5 2,1 1 1028,4531 159 168 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% FWEFMSQLLEGLGYERPSIK K I 52,7 26,7 26,00 2 2446,2114 267 286 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% IPASLMMPIAYLVELAYK K L 91,4 27,1 64,3 2 2055,0907 287 304 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% LLGPYGMK K V 49,8 30,7 19,1 2 894,4756 305 312 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% LMVPSLVTAAR K A 51,2 28,2 23,00 2 1157,6711 198 208 trueChymotrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% MVPSLVTAARAGKSKF L I 32,9 26,8 6,1 2 1678,931 199 214 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% PSSIFGPGDK R L 66,9 30,2 36,7 1 1004,5049 188 197 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% PSSIFGPGDKLMVPSLVTAAR R A 40,2 26,3 13,9 1 2159,1532 188 208 trueChymotrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% SATKAEGEAL Y I 32,6 31,8 0,8 2 976,4946 162 171 trueChymotrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% SATKAEGEALIL Y K 43,4 30,7 12,7 2 1202,6628 162 173 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% SGLLTCCIR R P 60,3 30,8 29,5 1 1079,5338 179 187 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% SGLLTCCIRPSSIFGPGDK R L 47,4 28 19,4 2 2065,0209 179 197 trueChymotrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% TCCIRPSSIF L G 40,5 30,2 10,3 2 1240,5814 183 192 trueTrypsin Mudpit_X00661 AT3BETAHSD/D1 (3BETA-HYDROXYSTEROID-DEHYDROGENASE/DECARBOXYLASE gi|18401656,gi 48107,2 22 38,00% VPVLTPSR K V 48 25 23,00 2 868,5251 313 320 trueTrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% AAVEAMVK K I 56,7 31,3 25,4 2 834,439 173 180 trueAspN Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% DGKSEETVMNII F E 42,3 31,7 10,6 1 1335,6462 208 219 trueChymotrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% DGKSEETVMNIIERSPF F G 64,2 28 36,2 2 1951,9431 208 224 trueTrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% GLGITANCVSPGPVATEMFFDGK K S 131 26,6 104,4 2 2384,1264 188 210 trueTrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% GSFLCCK R E 37 25 12,00 2 871,3802 130 136 trueTrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% IILLTSSLTEALIPGQGAYTASK R A 116 25 91,00 2 2347,3122 150 172 trueChymotrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% LCCKEAAKRL F K 50,4 32,1 18,3 2 1248,6553 133 142 trueTrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% SEETVMNIIER K S 82,9 28,6 54,3 2 1336,6414 211 221 trueChymotrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% TASKAAVEAMVKIL Y A 39,9 27,7 12,2 2 1447,819 169 182 trueChymotrypsin Mudpit_X00660 short-chain dehydrogenase/reductase (SDR) family protein [Arabidopsis thaliana] gi|30686710,gi 27248,1 10 33,30% TASKAAVEAMVKILAKEL Y K 46,1 25 21,1 2 1873,0828 169 186 true

P2

P1

P3


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Supplemental Table 2. Rescue of yeast erg28 null mutant by Arabidopsis ERG28. Sterol profile of yeast erg28 null mutant transformed with the pVT vector alone (pVT, Control) and pVT containing the full-length Arabidopsis ERG28 cDNA (pVT-ERG28).

Data shown are the mean + s.d. of four different experiments. apercent of total free sterols, bpercent of total sterol, nd : not detectable.

Sterols (acetates)

erg28 + pVT

erg28 + pVT-ERG28

% % Ergosta-5,7,22,28(28)tetraen-3βol 1.5a + 0.3 2.5 + 0.3 Ergosterol 26.6 + 5.5 75.7 + 2.2 Ergosta-7,24(28)-dien-3βol 3.2 + 0.8 nd Lanosterol 10.1 + 2.8 11.2 + 1.2 4α, 14α-Dimethyl-cholesta-8,24-dien-3βol nd 3.9 + 0.5 Zymostenone 1.1 + 0.4 nd 4α-Methyl-zymostenone 0.8 + 0.3 nd Fecosterone 3.9 + 1.9 nd Ergosta-7,24(28)-dien-3-one 4.0 + 1.4 5.6 + 1.5 4α-Methyl-fecosterone 3.3 + 0.9 nd 4α-carboxy-zymosterol 6.3 + 1.8 0.5 + 0.1 4α-carboxy-fecosterol 8.9 + 1.7 0.2 + 0.05 4β-methyl,4α-carboxy-zymosterol 30.3 + 4.4 0.5 + 0.1 Total 3β-hydroxy-sterols 41 + 9 93 + 1 Total 3-keto-steroids 13 + 5 6 + 2 Total 4α-carboxy-sterols 46 + 10 1.2 + 0.4 Total free sterols 100 100 Total free sterols 90b + 4 68 + 1 Total esterified sterols 10 + 4 32 + 1 Total sterols 100 100


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Supplemental Table 3. Binding free energy prediction of CMMC within Arabidopsis ABCB1 (At2g36910) and ABCB19 (At3g28860) nucleotide-binding domains.

EB ABCB1 ABCB19 (kcal mol-1)

Ligand Site 1 Site 2 Site 1 Site 2

NPA - 8.7 - 8.1 - 9.4 - 9.0 CMMC - 9.4 - 8.8 - 8.9 - 9.2 CA - 10.0 - 8.2 - 8.8 - 9.6

EB : predicted free energy; NPA (naphthylphthalamic acid), CMMC (4-carboxy-4-methyl-24-methylenecycloartanol) and compound CA (cholestanoic acid).


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Supplemental Table 4. Primer sequences used for cloning, genotyping and expression studies.

Gene Locus Sense Primer Reverse Primer Experiment ERG28 At1g10030 GCTTCTGTTTGGTTTGGTTTC GAGAGTGCAGGTCAAGAGTG qRT-PCR GAPDH At1g13440 TTGGTGACAACAGGTCAAGCA AACTTGTCGCTCAATGCAATC qRT-PCR TIP41 At4g34270 TGAAAACTGTTGGAGAGAAGCAA TCAACTGGATACCCTTTCGCA qRT-PCR Lipid transfer protein At2g37870 GTAACATCCGAAACCGCC AATGCTCATAAGATCATGGAACAA qRT-PCR Aminotransferase At3g08860 CCCCGCTCTTTTTCACTTCT AACCGTCGCTATTCCTCCA qRT-PCR Acid phosphatase type 5 At3g17790 CGGGAGATAACTTCTACGACAATG GGTTTCCCAAAACACTGTACCA qRT-PCR VPE At1g62710 GCTGGTTCTTCTGGATATGGA TTTCTTAGTATTTGATATGCGTGACA qRT-PCR - ERG28 SALK_025834 SALK_000240 - α-Tubulin

At1g10030 At1g04820

D1 :ATGAAGGCGTTGGGGTATTGGTTAATG D1 :ATGAAGGCGTTGGGGTATTGGTTAATG GAGATTGTTGACCTGTGCTTAGACCG

R1 : GAAAGTTTGGAGTGTGGTTGTTCAAGG R2 : CCTGCAAAGAAGCCCACAGTTGAGAG GGCTTTCTCTGCGGAGATGACTGGGGC

Genotyping

ERG28

At1g10030

Including BamHI restriction site CGCGGATCCATGGGCTCTTCGTCTC GCTGTCTTCTCTCAGAC Including BglII restriction site GAAGATCTGCTGGGCTCTTCGTCTC GCTGTCTTCTCTCAGAC GTATTGGTTAATGGTGGTTGGTTCACTG

Including EcoRI restriction site CGGAATTCAAATGATAGAAACGTAGC CAAGTATAATGGTTTG CACAGTTGAGAGATTCGCGATGGTC

RNAi lines RT-PCR on RNAi lines

ERG28

At1g10030

Including XbaI restriction site TGCTCTAGAGATGAAGGCGTTGGGGTA TTGGTTAATG

Including XbaI restriction site TGCTCTAGAGGAGAAAGTTTGGAGTGTG GTTGTTC

Complementation

SMO1 CSD SKR ERG28 RNLB2

At4g12110 At1g47290 At5g18210 At1g10030 At4g11220

Including PciI restriction site GCGACATGTGAATGATTCCTTACGCTACAG TCGAAGAAG Including PciI restriction site GCGACATGTGAATGGTGATGGAAGTTACAG AGACTGAG Including NcoI restriction site CATGCCATGGGAATGGCTTCCTCAGTCTC CTCTCTCGC Including NcoI restriction site CATGCCATGGGAATGAAGGCGTTGGGGTATT GTTAATG Including NcoI restriction site CATGCCATGGGAATGGCGGATGAACA TAAGCATGAAG

including PciI restriction site GCGACATGTCATCGGATTTTATTCCTC CGTTATGC including PciI restriction site GCGACATGTCGTCGATCTTCTTGCTCC CGAACACTTTC Including NcoI restriction site CATGCCATGGCTGAGCTTTTCCAGTTTTG CTTCTTG Including NcoI restriction site CATGCCATGGCAGAAAGTTTGGAGTGTGG TTGTTCAAG Including NcoI restriction site CATGCCATGGCATCCTTCTTCTTGTCTT TCAACGGTCC

GFP fusion


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Gene Locus Sense Primer Reverse Primer Experiment SMO1 CSD SKR ERG28

At4g12110 At1g47290 At5g18210 At1g10030

Including XbaI restriction site TGCTCTAGAGATGTGAATGATTCCTT ACGCTACAGTCGAAGAAG Including XbaI restriction site TGCTCTAGAGATGTGAATGGTGATGGA AGTTACAGAGACTGAG Including XbaI restriction site TGCTCTAGAGATGGGAATGGCTTCCT CAGTCTCCTCTCTCGC Including XbaI restriction site TGCTCTAGAGATGGGAATGAAGGCGTT GGGGTATTGGTTAATG

Including XbaI restriction site TGCTCTAGAGGTTTGTATAGTTCATCC ATGCCATGTGT Including XbaI restriction site TGCTCTAGAGGTTTGTATAGTTCATCC ATGCCATGTGT including XbaI restriction site TGCTCTAGAGGTTTGTATAGTTCATCC ATGCCATGTGT including XbaI restriction site TGCTCTAGAGGTTTGTATAGTTCATCC ATGCCATGTGT

Affinity interaction

SMO1 CSD SKR ERG28

At4g12110 At1g47290 At5g18210 At1g10030

ATGATTCCTTACGCTACAGTCGAAG CCGAGTGTTGTGTTTGACGGGGTC CTAGTTAACTCAGCTGGAATCCTC ATGAAGGCGTTGGGGTATTGGTTAATG

ATCGGATTTTATTCCTCCGTTATGC CCTAACCCTAGAAGGTGTTAGCAC CTCACCAAGCCTACCAAAAGGACTC AGAAAGTTTGGAGTGTGGTTGTTC

Protein expression


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Supplemental Methods

Plant growth and hormone treatments

Phenotypic rescue of erg28 mutants was tested using class 1 (erg28R1-1and erg28T1-1)

plants in which shoot and root development are dramatically altered. Plants were grown on

MS agar and transferred at day 15 postgermination to MS agar in the absence or in the

presence of indole-3-acetic acid (IAA), 1-naphthalene acetic acid (NAA), gibberellin A3,

trans-zeatin, 24-epibrassinolide (from Kalys) or 1-aminocyclopropane 1 carboxylic acid

(ACC) (from Sigma) for 45 days. Primary root elongation was analyzed using wild-type and

erg28 (erg28R1-4, erg28R1-5, erg28T1-4 and erg28T1-5) grown for 8 days on vertical plates

as described (Jadid et al., 2011) to evaluate the effect of IAA and NAA.

Subcellular localization of Arabidopsis SC4DM complex protein components

Constructs encoding in frame GFP fusion proteins of the Arabidopsis SC4DM protein

complex (ERG28-GFP, SMO1-GFP, CSD-GFP, SKR-GFP) and RTNLB2-GFP used as an

ER marker (Nziengui et al., 2007) were generated using pCATs-GFP vector (Bouvier et al.,

2006). DNA sequences corresponding to ERG28 (At1g10030), SMO1 (At4g12110), CSD

(At1g47290), SKR (At5g18210) and RTNLB2 (At4g11220) were amplified using

oligonucleotide primers (Supplemental Table 4 online) and inserted in the NcoI site. The

resulting plasmids were used for transient expression in Arabidopsis leaves (Jadid et al.,

2011). Confocal laser scanning microscopy imaging was performed using a Zeiss LSM510

confocal laser scanning microscope (Bouvier et al., 2006).

Anatomical and histological analyses

Fused leaves were cleared after fixation as described previously (Leon-Kloosterziel et al.,

1994) and observed using a Microscope Zeiss AX10 Imager Z1. For interfascicular fiber

analysis, stem sections were fixed with 1% glutaraldehyde, embedded in LR white resin

(EMS, Fort Washington), before staining with 5% toluidine blue and observation using a

Leica DMRB microscope.

Yeast complementation

The yeast erg28 mutant (Gachotte et al., 2001) was complemented using Arabidopsis ERG28

cloned into the XbaI and XhoI sites of the pVT102U shuttle vector before transformation


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(Rahier et al., 2006). Yeast sterols were extracted and analyzed as described previously

(Gachotte et al., 2001; Rahier et al., 2006) using a GC-MS spectrometer (Agilent 5973N).

Gene expression analysis

In silico analyses of ERG28 expression were determined with GENEVESTIGATOR

(https://www.genevestigator.com/gv/plant.jsp) and BAR

(http://bar.utoronto.ca/welcome.html). STRING (http://string-db.org) was used for

coexpression and protein-protein interaction analyses and ATTEd-II (http://atted.jp/) was used

for coregulatory network analysis.

Inhibitor binding

Competitive displacement binding assays were performed as described (Jacobs and Rubery,

1988; Noh et al., 2001) using [2,3,4,5-3H]NPA, (40 Ci/mmol) obtained from Hartmann

Analytic GmbH, Germany. Microsomal membranes (equivalent to 100 µg protein) were

resuspended in 20 mM sodium citrate-buffer pH 5.3 containing 0.25 M sucrose and incubated

for 1 h at 4°C with 20 nM [3H]NPA or 20 nM [3H]NPA plus 20 µM non radioactive NPA (to

estimate the non specific binding). For competitive displacement binding experiments, 20 nM

CMMC was added in the reaction mixture.

Homology modelling and docking studies

For homology modelling we used the E. coli ABC-transporter haemolysin B structure solved

as a dimer with bound ATP (Schmitt et al., 2003). Models for PAT inhibitors binding to

Arabidopsis ABCB1 and ABCB19 were generated using Modeller (Sali and Blundell, 1993).

Ligand 3D coordinates were generated in PDB compatible format with the Dundee

GlycoBioChem PRODRG2 web Server (Schuttelkopf and van Aalten, 2004). AutoDockTools

(Sanner, 1999) was used to add non-polar hydrogens and to compute the Gasteiger charges

before running Autodock Vina (Trott and Olson, 2010) to carry out the docking computations.

The grid volume was set to cover a box of 40Åx40Åx40Å centered on X=38.8Å Y=103.5Å

Z=44.8Å for NBD1 and X=24.0Å Y=84.6Å Z=61.9Å for NBD2. Visual inspection of the

models and docked ligands were performed using the PyMOL, Molecular Graphics System.


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Documents

3 2 3 2 3 SC4DM - The Plant Cell...Nov 20, 2013 · Merged images of GFP fluorescence and chloroplast endogenous autofluorescence are shown. Scale bars = 10 µm. GFP Chlorophyll$