correlations in gene expression – part i · igmt1 at1g21100 igmt2 at1g21120 at1g21130 at1g76790...

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

Affymetrix Arabidopsis ATH1 Microarrays (N = 1436)

Correlations in Gene Expression – Part I

>0.6>0.5>0.4

>0.9

>0.7>0.8

Supplemental Figure 1. Correlations in gene expression – Part I.

Correlations in the expression between CYP81F and O-MT genes were calculated based on 1436 Affymetrix Arabidopsis ATH1 microarray experiments, available from TAIR (www.arabidopsis.org).Shown are correlations for CYP81F1, 2, 3, and 4, confirmed O-MT genes IGMT1 and IGMT2, and putative indole glucosinolate O-methyltransferase genes At1g21110, At1g21130 and At1g76790. All correlations (P < 0.00001) remain significant after Bonferroni correction. Colour codes indicate the strength of the correlation coefficient r.

Supplemental Data. Pfalz et al. (2011). Plant Cell 10.1105/tpc.110.081711

1

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

IGMT1At1g21100

IGMT2At1g21120

At1g21130

At1g76790

CYP81F3At4g37400

CYP81F4At4g37410

CYP81F1At4g37430

CYP81F2At5g57220

At1g21110

Abiotic Stress (N = 149) Pathogens (N = 36)

Development (N = 79) Hormones (N = 115)

>0.6>0.5>0.4

>0.9

>0.7>0.8

Correlations in Gene Expression – Part II


2

Supplemental Figure 2. Correlations in gene expression – Part II.

Correlations in CYP81F and O-MT gene expressions were calculated for microarray experiments focusing on plant organs at different developmental stages (N = 79), after hormone treatments (N =115), exposure to abiotic stress (N = 149) or to pathogens (N = 36). Information regarding experimental details is available from AtGenExpress (www.weigelworld.org/resources/microarray/AtGenExpress/).Colour codes indicate the strength of the correlation coefficient r. All correlations are significant after Bonferroni correction, except for correlations with r < 0.5 in the data set entitled ‘Pathogens’. The dashed line in ‘Abiotic Stress’ indicates a negative correlation.


3

0

100

200 IGMT1(At1g21100)

0

15

30 IGMT2At1g21120

0

500

1000 At1g76790

0

250

500 At1g21130

0

50

100

150CYP81F2

(At5g57220)

0

500

1000

1500

CYP81F1(At4g37430)

0

50

100

150CYP81F3

(At4g37400)

1 Dry seed

2 Imbibed seed, 24 h

3 1st Node

4 Flower Stage 12, Stamens

8 Entire Rosette AfterTransition To Flowering

5 Cauline Leaf

7 Root

6 Cotyledon

9 Flower Stage 9

10 Flower Stage 10/11

11 Flower Stage 12

12 Flower Stage 15

13 Flower Stage 12, Carpels

14 Flower Stage 12, Petals

15 Flower Stage 12, Sepals

16 Flower Stage 15, Carpels

17 Flower Stage 15, Petals

18 Flower Stage 15, Sepals

19 Flower Stage 15, Stamen

20 Flowers Stage 15, Pedicels

21 Leaf 1 + 2

22 Leaf 7, Petiole

23 Leaf 7, Distal Half

24 Leaf 7, Proximal Half

25 Hypocotyl

26 Root

27 Rosette Leaf 2

28 Rosette Leaf 4

29 Rosette Leaf 6

30 Rosette Leaf 8

31 Rosette Leaf 10

32 Rosette Leaf 12

33 Senescing Leaf

34 Shoot Apex, Inflorescence

35 Shoot Apex, Transition

36 Shoot Apex, Vegetative

37 Stem, 2nd Internode

38 Mature Pollen

39 Seeds Stage 3 w/ Siliques



42 Seeds Stage 6 w/o Siliques





47 Vegetative Rosette

1 2 4 7 9 11 14 16 18 21 23 25 27 29 31 33 34 35 36 37 38 39 40 41 42 44433 5 6 8 10 1213 20 22 24 26 28 30 3215 17 19 45 46 47

0

1000

2000 CYP81F4(At4g37410)

0

25

50 At1g21110

Abso

lute

Exp

ress

ion

Valu

e


4

Supplemental Figure 3. In silico analysis of CYP81F and O-MT gene expression patterns.

The Arabidopsis eFP-Browser (http://bar.utoronto.ca) was used to visualize the expression of CYP81F and O-MT genes in different tissues during plant development (Winter et al., 2007). Panels show absolute expression values obtained from Affymetrix Arabidopsis ATH1 microarrays for CYP81F1, 2, 3, and 4, for confirmed O-MT genes IGMT1 and IGMT2, and for putative indole glucosinolate O-methyltransferase genes At1g21110, At1g21130 and At1g76790 in different organs during various stages of development. These organs/developmental stages are numbered from 1– 47, and are listed on the left of the panels. The CYP81F1 antisense gene, At4g37432, is not represented on the ATH1 microarrays and could thus not be evaluated.

CYP81F1 is expressed throughout all developmental stages and tissues but expression is particularly strong in sepals. CYP81F2 is highly expressed in imbibed seeds, and in all vegetative tissues of seedlings and mature plants. However, expression in roots is less pronounced than in leaves. No or little expression is visible in flowers and during most stages of seed development. CYP81F3 is expressed throughout all developmental stages and tissues, most strongly in roots. CYP81F4 has very high expression in the roots of seedlings, and in the roots and the hypocotyl of mature plants, but is also expressed in leaves although at a comparatively low level.

Four of the O-MT genes, IGMT1, At1g21110, IGMT2, and At1g21130, have very similar expression patterns. They are expressed in seedlings, during all leaf stages and in the hypocotyl but show little expression in flowers or during seed development. IGMT1, At1g21110 and IGMT2 but not At1g21130 are also strongly expressed in roots. Interestingly, none of these four genes has pronounced expression in imbibed seeds where CYP81F2 is strongly expressed. At1g76790 is strongly expressed during this developmental stage, but also in the roots of seedlings and of older plants, and in the hypocotyl.


5

0

8

4x108

0

8

4x108

8

4x108

I3M

0

4

2x107 4OH-I3M standard

8

0

4x108

0

CYP81F1

4OH-I3M

8

4x108

CYP81F3

0

8

4x108

CYP81F4

6 8time [min]

CYP81F2

Negative control

I3M biosynthesis

D

E

F

G

C

A

B369.1

391.1

300 400 500

6

8

2

0

4

x108

384.9

464.9

406.9

300 400 500

34

10

2

x107

369.1

469.2

391.1

300 400 500

6

2

0

4

x108

8

305.1 353.2385.1

407.1

465.1 493.1

300 400 500

3

5

10

2

x107

4

369.1

391.1

300 400 500

6

20

4

x108

8

359.3

385.1

407.1

421.3 465.0502.

300 400 500

6

20

4

x107

8

0

300

100

200

300200 250 [nm]

[mAU]

0

3000

1000

2000

300200 250 [nm]

[mAU]

200

100

0

300200 250 [nm]

[mAU]m/z m/z

H

I

J

K

L

M

N

O

P

Supplemental Figure 4. Engineering of CYP81F genes in Nicotiana benthamiana – MS data.

Shown are the same LC-MS runs as in Figure 7 (A – G), and corresponding MS data for I3M (K – M) and for 4OH-I3M (H – J). Shown are also UV-spectra for I3M (N) and 4OH-I3M (O, P). UV- spectra for I3M are from I3M produced by heterologous expression of the indole glucosinolate core pathway in Nicotiana benthamiana (B), and UV-spectra for 4OH-I3M from an authentic standard (C), isolated from Isatis tinctorea (Dyer’s woad) as described in Pfalz et al. (2009), and from co- expression of CYP81F2 with glucosinolate core pathway genes (D) for comparison.


6

7 8time [min]

0

0

0

10

5

x107

0

0

10

5

x107

0

10

5

x106

10

5

x107

0

10

5

x107

10

5

x107

10

5

x107

I3M4MO-I3M

1MO-I3M

B

C

D

E

F

G

A

Control

IGMT2

CYP81F1IGMT1

CYP81F2IGMT1

CYP81F3IGMT2

CYP81F4IGMT1

Col-0

6

2

0

4

x106

1301.1

333.2481.3

407.2

300 400 500

0

5

12

x106

43

307.1 369.2421.2 449.1 497.3

391.1

300 400 500

0

1

x106

1.5

0.5301.2

346.2

378.2399.2

436.2

452.2

479.1

421.2

300 400 500

0

1

x107

1.5

0.5399.1

479.1

421.1

300 400 500

0

1

x107

0.5

331.2

407.2

465.1

301.2

300 400 5000

1

x108

1.5

0.5369.1

391.1

300 400 500

1.5

0.5

0

1

x108

369.1

391.1

300 400 5000

0.5

1

x107

1.5

1.2320.2

407.2

465.1

301.1

300 400 500

0.5

0

1x108

369.1

391.1

300 400 500

0

1

2

x106

3

4

301.1

320.1359.2

407.2

421.2

455.1

307.1

300 400 5000

2

4

x107

6

8

369.1449.1

391.1

300 400 500

0.5

0

1

x108

369.1

391.1

300 400 5000

0.5

x107

301.2

331.2 359.1 439.3

481.3

407.2

300 400 500

4MO-I3M

Contaminant (7.4 min)

1MO-I3M

I3M

Col-0

Col-0

Col-0

Control

IGMT2

CYP81F2IGMT1

CYP81F4IGMT1

CYP81F3IGMT2

IGMT2

CYP81F1IGMT1

CYP81F1IGMT1

CYP81F2IGMT1

CYP81F3IGMT2

m/zm/z

H

I

J

K

L

M

N

O

Q

R

S

T

P

Supplemental Figure 5. Engineering of CYP81F and O-MT genes in Nicotiana benthamiana – MS data I.

Shown are the same extracted ion chromatograms as in the left panels of Figure 8 (A – G), and corresponding MS data for I3M (K – O) and for a contaminant eluting at 7.4 min with m/z 407 (P – T). MS data of 4MO-I3M (H), I3M (I) and 1MO-I3M (J) extracted from the seedlings of Arabidopsis Col-0 are shown for comparison.


7

91.2

367.2

421.1

479.1

399.1

300 400 500

0

1.5

0.5

1

x107

0

2.5

0.51

x107

1.52

389.3

421.3

479.2

300 400 500

0

4

1

2

x106

3

301.2 367.2

399.2

479.1

421.1

300 400 5000

5

1

3

x107

2

4

1.2

399.2

479.1

421.1

300 400 500

0

2

4

x107

6

1.2

399.1

479.1

421.1

300 400 500012

x106

543

301.2 367.2

421.1

451.2 479.1

399.2

300 400 500

0

4

1

2

x106

3

305.3 343.2 367.2

399.2

479.1

421.1

300 400 500

0

5

12

x106

43

307.1 369.2421.2 449.1 497.3

391.1

300 400 500

0

1

x106

1.5

0.5301.2

346.2

378.2399.2

436.2

452.2

479.1

421.2

300 400 500

0

1

x107

1.5

0.5399.1

479.1

421.1

300 400 500

4MO-I3M

1MO-I3M

I3M

Col-0

Col-0

Col-0

7 8time [min]

0

0

0

10

5

x107

0

0

10

5

x107

0

10

5

x106

10

5

x107

0

10

5

x107

10

5

x107

10

5

x107

I3M4MO-I3M

1MO-I3M

B

C

D

E

F

G

A

Control

IGMT2

CYP81F1IGMT1

CYP81F2IGMT1

CYP81F3IGMT2

CYP81F4IGMT1

Col-0

CYP81F4IGMT1

CYP81F1IGMT1

CYP81F2IGMT1

CYP81F3IGMT2

CYP81F1IGMT1

CYP81F2IGMT1

CYP81F3IGMT2

m/z

m/z

H

I

J

K

L

M

N

O

Q

P

0

150

50

100

300200 250 [nm]

[mAU]

300200 250 [nm]0

1000

500

[mAU]R

S

Supplemental Figure 6. Engineering of CYP81F and O-MT genes in Nicotiana benthamiana – MS data II.

Shown are the same extracted ion chromatograms as in the left panels of Figure 8 (A – G), and corresponding MS data for 4MO-I3M (K – M) and for 1MO-I3M (N – Q). MS data of 4MO-I3M (H), I3M (I) and 1MO-I3M (J) extracted from the seedlings of Arabidopsis Col-0 are shown for comparison. Shown are also the UV-spectra of 4MO-I3M (R) and 1MO-I3M (S).


8

x 107

x 106

x 107

x 107

x 107

x 107

x 107

x 106

x 106

2

0

4

2

0

4

20

64

20

64

20

64

20

64

0.5

0

1

0.5

0

1

20

64

CYP81F1IGMT1

Col-0

CYP81F1IGMT2

CYP81F2IGMT1

CYP81F2IGMT2

CYP81F3IGMT1

CYP81F3IGMT2

CYP81F4IGMT1

CYP81F4IGMT2

I3M

4MO-I3M

1MO-I3MA

B

C

D

E

F

G

H

I

0.5

1

0

1

0

0.5

0.5

1

0

6 8 10

x 107

x 107

x 107

IGMT1

IGMT2

Control

J

K

L

a

b

a

a

b

b

b

b

b


9

Supplemental Figure 7. Co-expression of CYP81F and IGMT genes in Nicotiana benthamiana.

Shown are extracted ion chromatograms for m/z 391 and m/z 421 for all combinations of CYP81F and IGMT1 or IGMT2, coexpressed with the genes for the indole glucosinolate core pathway in N.benthamiana (B – I), and extracted ion chromatograms for m/z 391, m/z 407 and m/z 421 for IGMT1 or IGMT2 expressed without CYP81Fs (J, K) and for a control where Agrobacterium without expression constructs was infiltrated into leaves of N. benthamiana (L). Glucosinolate profiles from Arabidopsis Col-0 seedlings are shown for comparison (A), and I3M, 4MO-I3M and 1MO-I3M peaks are labelled. The peak corresponding to 1MO-I3M is indicated in panels B – H (black arrows). No 4MO-I3M peak is visible in assays with CYP81F4 (H, I). Experimental artefacts (a, b) are indicated.


10

FLAG_140B06

10.0

3.04.05.0

2.0

BamHI

M M M W W

SALK_024438

10.0

3.04.05.0

2.0

HindIII

M M M W WW

SALK_031939

10.0

3.04.0

5.0

2.0 BamHI

W W W W MM

Supplemental Figure 8. DNA blots of T-DNA insertion lines.

Shown are the results of a non-radioactive hybridization with a T-DNA-specific probe (see: METHODS for experimental details). The hybridization pattern for FLAG_140B06 is compatible with a single insertion in mutants (M), while SALK_024438 and SALK_031939 mutants contain multiple insertions. Wildtypes (W) have no T-DNA insertions.

[kb][kb]

[kb]


11

Purpose Primer name Sequence (5' � 3')

Verification of T-DNA insertions 031939F CATACAACCACACAACCGTTG for SALK_031939 T-DNA insertion line 031939R CCTCCCAAATCTCTGGATCTC 024438F TGGGTCGAGTCTTACACAAGG for SALK_024438 T-DNA insertion line 024438R GGCTTAATGCTGGTAACGAATC 140B06F AACCCTCATGATACACGTATTTG for FLAG_140B06 T-DNA insertion line 140B06R TCTCGGCCCATTATGAGTAAG 123882-f TCGCCAAGTCGTAGTGATCTC for SALK_123882 T-DNA insertion line 123882-r TGCCTTTTTCAACACTTCAGG 005861-f GACTTTTCGGTTGTGTCCAG for SALK_005861 T-DNA insertion line 005861-r GTTCTTTCAGGCATTTCATCG

primer for left border of FLAG T-DNA FLAG-LB1 CGGCTATTGGTAATAGGACACTGG primer for left border of SALK T-DNA LBb1 AGTTGCAGCAAGCGGTCCACGC

Amplification of a T-DNA fragment as a probe for hybridization

FLAG-F GATGCGATGTTTCGCTTGGTG for FLAG T-DNA FLAG-R CTGGGCACAACAGACAATCG T-DNA_L1F TCCAGTTTGGAACAAGAGTCCA for SALK T-DNA T-DNA-L1R CCTGGCGTTACCCAACTTAATC

qRT-PCR with T-DNA insertion lines

RT-CYP400F GCCTAATGCTGGGCATGATG for CYP81F3 (At4g37400) RT-CYP400R CTTTAATATCCTCTGTTGGTG 37410seq2 CCCTGAGTACTACACTGAC for CYP81F4 (At4g37410) RT-410-(09)R CAAAACTTTTGGGTGGTTCAAC

RT-CYP430F GAACCGCTCTTATCTGATTG RT-CYP430R GGAAATAGCCGGAATGTTTC for CYP81F1 (At4g37430) and antisense

(At4g37432), respectively RT-CYP430-1F GCAGCGTTTGTTAGATGAATG Cyt2F GTCACAGGGAGACGCTACTAC for CYP81F2 (At5g57220) Cyt2R CACCACTGTTGTCATTGATGTC

EIF4A-f TGAACACCAGGCGTAAGGTTGA control gene EIF4A1 (At3g13920) EIF4A-r CCTTTCTCCCGAACCTTCCACT

Generation of expression constructs for Sf9 insect cell lines 400_Ins-Exp-KOZ-F GTGATGGTTTACTATGTGATTGTCC for CYP81F3 (At3g37400) 400_Ins-R ACCTTTTAAATGGGCCTGAAG 410_Ins-Exp-KOZ-F GTGATGGTTAACTATGTGATTATTCTTC for CYP81F4 (At3g37410) 410_Ins-R AACTTTCGTGTAGGCCGGG

Supplemental Table 1. Primer Sequences Used in This Work.

Supplemental Table 1. Primer Sequences Used in This Work.


12

Purpose Primer name Sequence (5' � 3')

Generation of expression constructs for Nicotiana benthamiana CYP79B2f GGCTTAAUATGAACACTTTTACCTCAAACTCTTCG for CYP79B2 CYP79B2r GGTTTAAUTCACTTCACCGTCGGGTAGA CYP83B1f GGCTTAAUATGGATCTCTTATTGATTATAGCCGGT for CYP83B1 CYP83B1r GGCTTAAUTCAGATGTGTTTCGTTGGTGC

cAt1g21100F ATGGGATACCTTTTTCAAGAAAC amplification of full-length coding sequence from IGMT1 (At1g21100) cAt1g21100R TTATTTACAGAATTCAATAATCCAGC

cAt1g21120F ATGGGATACCTTTTCGAAGAAAC amplification of full-length coding sequence from IGMT2 (At1g21120) cAt1g21120R TTATTTACAGAACTCAATAATCCAGC

c400_F ATGTTTTACTATGTGATTGTCC amplification of full-length coding sequence from CYP81F3 c400_R CTAACCTTTTAAATGGGCCTG

c410_F ATGTTTAACTATGTGATTATTCTTCC amplification of full-length coding sequence from CYP81F4 c410_R CTAAACTTTCGTGTAGGCCG

c430_F ATGTTATACTTCATCCTTCTCC amplification of full-length coding sequence from CYP81F1 c430_R TTAGAGTAAAAGCTTTGACATAA

c220_F ATGGATTACGTTTTGATTGTTTTGC amplification of full-length coding sequence from CYP81F5 c220_R TTAAGCCAAGAGATTAGTCATAATG

21100_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGGGATACCTTTTTCAAGAAAC 21100/21120_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTG TTATTTACAGAAYTCAATAATCCAGC GATEWAY adaptor primer for IGMT1 and

IGMT2, respectively 21120_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGGGATACCTTTTCGAAGAAAC 400_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGTTTTACTATGTGATTGTCC GATEWAY adaptor primer for CYP81F3 400_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTG CTAACCTTTTAAATGGGCC 410_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGTTTAACTATGTGATTATTCTTC GATEWAY adaptor primer for CYP81F4 410_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTG CTAAACTTTCGTGTAGGCC 430_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGTTATACTTCATCCTTCTCC GATEWAY adaptor primer for CYP81F1 430_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTG TTAGAGTAAAAGCTTTGACATA 220_attB1F GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGGATTACGTTTTGATTGTTTTGC GATEWAY adaptor primer for CYP81F2 220_attB2R GGGGACCACTTTGTACAAGAAAGCTGGGTG TTAAGCCAAGAGATTAGTCATAATG

Supplemental Table 1, cont.


13

correlations in gene expression – part i · igmt1 at1g21100 igmt2 at1g21120 at1g21130 at1g76790...

Documents