0

50

75

100

125

150

175

200

Radicle emergence (%)

99.65

99.32

98.96

99.39

97.03

98.02

97.78

98.10

MDCA (µM)

A

B

mock 50 µM 75 µM 100 µM

125 µM 175 µM 200 µM150 µMFigure S1. Effect of MDCA on germination of Arabidopsis.

(A) Radicle emergence (%) of seeds 2 DAG grown on 0.5xMS-medium supplemented with different concentrations of MDCA (n>250) (B) Phenotype of seedlings (12 DAG) grown on 0.5xMS-medium supplemented with different concentrations of MDCA (n>250) (scale bar: 0.5 cm).

1. Cinnamoyl aspartate 12. MDCA glutamate 13. MDCA aspartate 14. Cinnamoyl aspartate 25. MDCA aspartate 26. MDCA glutamate 27. Unknown8. Cinnamoyl glutamate9. Cinnamoyl aspartate 1 (fragment) 10. MDCA aspartate 2 (heterodimer)

mock 10 µM MDCARetentiontime (min)

m/z

10.218.938.307.4910.0910.767.1710.9610.228.32

262.07320.07306.06262.07306.06320.07319.09276.08218.08900.23

00821125800000

16801114708713072976376640364930928905277282733123111

0049280600000

3002027639349931749318981794210706312054745081

UPUP1596.799852.950.9UPUPUPUPUP

Fold change MDCA/mockAverage St. Dev. Average St. Dev.

CompoundTop-10 UP

CompoundTop-10 DOWN

1. 4-methylthio-N-butyl-glucosinolate2. 1-methoxyindol-3-ylmethyl-glucosinolate3. G(8-O-4)ferulic acid + hexose + hexose4. G(8-O-4)ferulic acid + hexose + hexose5. 8-methylsulfinyloctyl-glucosinolate 6. Kaempferol + hexose + deoxyhexose7. 5-methylthio-N-pentyl-glucosinolate8. Unknown9. Quercetin + hexose + deoxyhexose10. 8-methylthio-N-octyl-glucosinolate (dimer)

3.438.013.103.2212.9210.005.442.178.6314.10

420.04955.15713.23713.23492.10593.15434.06338.08609.15953.24

298229287062357721304207741862817369127121139610038

246332325132312028453881188575350184

191900171014733427212097367011795298626706169

22996360433121811602651413259353375

0.08260.08100.05610.05640.04070.02080.06840.04530.03070.0184

Figure S2. Metabolites with an altered abundance in 10 μM MDCA-treated Arabidopsis seedlings.

An overview of the top-10 significantly differential compounds with at least a 10-fold increase (UP) or decrease (DOWN) in 10 μM MDCA-treated seedlings as compared to mock-treated seedlings (n=6). The top-10 UP defined as those compounds with the highest normalized peak area in MDCA-treated seedlings, whereas the top-10 DOWN were defined as those compounds with the highest normalized peak area in mock-treated seedlings. The retention time is expressed in minutes. For each compound, normalized average peak areas (unitless) of mock- and 10 μM MDCA-treated seedlings are given. Peak areas are normalized relative to the dry weight of the pellet remaining after methanol extraction. The term UP implies that a peak could only be detected in 10 μM MDCA-treated seedlings and not in mock-treated seedlings.

Figure S3. UHPLC-MS based detection of PA + hexose and PA + malate in MDCA treated Arabidopsis seedlings.

Structural identification of the compounds was done via accurate m/z match and MS/MS fragmentation spectra. (A) PA + hexose was detected at 5.92 min as formate adduct and as an in-source fragment. (B) The MS/MS of the in-source fragment of PA + hexose with m/z 165.015 eluting at 5.92 min showed characteristic fragmentation pattern of PA. (C) PA + malate was detected at 9.71 min as molecular ion and as an in-source fragment. (D) The MS/MS of the in-source fragment of PA + malate with m/z 165.015 eluting at 9.71 min showed characteristic fragmentation pattern of PA.

A PA + hexose (formic acid adduct) (5.92 min, m/z 373.075)

5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.40

100

50

75

25inte

nsity

(%)

m/z 373.069-373.089

m/z 165.009-165.029; in source fragment,[M-H+-dehyrated hexose]- = [PA-H+]-

m/ztheoretical [M+ formic acid - H+]- = 373.0776

O O

hexose

inte

nsity

(%)

B MS/MS 165.015@ 5.92 min (in-source fragment of PA + hexose)

C PA + malate (9.71 min, m/z 281.027)

D MS/MS 165.015@ 9.71 min (in-source fragment of PA + malate)

9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 11.00

100

50

75

25inte

nsity

(%)

m/z 281.023-281.043

m/z 165.009-165.029; in source fragment,[M-H+-dehyrated malate]- = [PA-H+]-

m/ztheoretical [M -H+]- = 281.0303

O OO

O

HO

HO

91.015

121.028

165.020

70 100 130 160 190 220 250 280 310 3400

25

50

75

100

[PA-H+]-

[PA-H+-CO2]-

[PA-H+-CO2-CH2O]-

91.012

152.996

165.021[PA-H+]-

121.027[PA-H+-CO2]-

[PA-H+-CO2-CH2O]-

70 100 130 160 190 220 250 280 310 3400

25

50

75

100

inte

nsity

(%)

retention time (min)

retention time (min)

m/z

m/z

O

HOH

OO

OO

Cinnamic acid derivativesCinnamoyl hexose (formic acid adduct)Cinnamoyl malate 1Cinnamoyl malate 2

p-Coumaric acid derivativesp-Coumaric acid 4-O-hexosidep-Coumaroyl hexose 1p-Coumaroyl hexose 2Dihydro-p-coumaric acid + hexose

Caffeic acid derivativesCaffeic acid 1Caffeic acid 2Caffeic acid 3/4-O-hexoside 3Caffeoyl hexose 1Caffeoyl hexose 3/4-O-hexoside 1Caffeoyl malate 1Caffeoyl malate 2

mock 10 µM MDCARetentiontime (min)

m/z

9.8312.6614.05

3.024.675.244.03

4.905.805.643.453.125.655.91

355.10147.04147.04

325.09325.09325.09327.10

179.03179.03341.09341.09503.14295.05295.05

001

138519190942437

0077889511500

10194905813

204296913791156

00471131951900

002

6315361944617

001446806900

961222197

911543177155

009429212400

UPUP1018.7***

1.47130.5720*0.1516***0.4744***

000.6058***1.47324.5233***00

Fold changeMDCA/mock Average St. Dev. Average St. Dev.

Compound

Ferulic acid derivativesFerulic acidFeruloyl hexose 1Feruloyl hexose 2Ferulic acid O-4-hexosideFeruloyl malate 1Feruloyl malate 2

Sinapic acid derivativesSinapic acidSinapoyl hexose 1Sinapoyl hexose 2Disinapoyl hexose 1Disinapoyl hexose 2Sinapoyl malate 1Sinapoyl malate 2

VariaConiferin (formic acid adduct)Scopolin (formic acid adduct)

mock 10 µM MDCARetentiontime (min)

m/z

9.705.496.003.859.068.50

8.415.686.2415.4512.009.219.50

3.964.62

193.05355.10355.10355.10309.06309.06

223.06385.11385.11591.17591.17339.07339.07

387.13399.09

0188716110231220

383113076297975457503349682548218

7898730358

095010177260

2211270223733356278687210629147620

376177413

0284682881260

180281917438122110779118455230

157398264

02711970410

152272898502155020067201527285

49112109

00.5034***0.0608***0.1733***0.21580

0.57721.12331.25291.03111.7259**1.09780.9876

0.4762***0.2442***

Fold change MDCA/mockAverage St. Dev. Average St. Dev.

Compound

Coniferyl alcohol - ferulic acid conjugates

G(8-O-4) ferulic acid G(8-O-4) ferulic acid + hexose 1G(8-O-4) ferulic acid + hexose 2G(8-O-4) ferulic acid + hexose 3G(8-O-4) ferulic acid + hexose 4G(8-O-4) ferulic acid + hexose 5G(8-O-4) ferulic acid + hexose 6G(8-O-4) ferulic acid + malate 1G(8-O-4) ferulic acid + malate 2G(8-O-4) ferulic acid + malate 3G(8-O-4) ferulic acid + malate 4G(8-O-4) ferulic acid + 224 Da 1G(8-O-4) ferulic acid + 224 Da 2

G(8-5) feruloyl hexose 1G(8-5) feruloyl hexose 2G(8-5) feruloyl hexose 3G(8-5) feruloyl malateG(8-5) feruloyl malate O-4-hexoside 1G(8-5) feruloyl malate O-4-hexoside 2

mock 10 µM MDCARetentiontime (min)

m/z

8.966.096.216.596.867.147.499.589.8310.4310.6311.9512.11

9.7910.3211.1114.0110.369.99

389.12551.18551.18551.18551.18551.18551.18505.14505.13505.13505.13775.25775.25

533.17533.17533.17487.12649.18649.17

22621540135013461380233133421257819671084149734342661

1229402455828653118119

20601345335230143080343276015931328247575154144

100986232140199565

4723612052904965670602304309234209581519

30273118264031046

56926512124093666392014294331631878892

962558735834161

0.91060.87290.2479***1.7098***0.81011.03590.2221***0.6178**0.6748*0.2275***0.3841***0.0448***0.0542***0.0814***0.2451***0.4154*0.4891**0.3190***0.5487

Fold changeMDCA/mockAverage St. Dev. Average St. Dev.

Compound

Hexosylated oligolignolsG(8-5)G + hexoseG 4-O-hexoside(8-5)GG(red8-5)G + hexoseG(red8-8)G 8/4-O-hexosideG(8-8)G hexoside 1G(8-8)G hexoside 2G(8-8)G + hexose + hexoseG(8-O-4)G(red8-5)G + hexose 1G(8-O-4)G(red8-5)G + hexose 2

Flavonol glycosidesIsorhamnetin + hexose + deoxyhexoseIsorhamnetin-3-O-rhamnoside-7-O-glucosideIsorhamnetin + deoxyhexose + deoxyhexoseIsorhamnetin + hexose + deoxyhexose

mock 10 µM MDCARetentiontime (min)

m/z

9.3610.948.419.2610.6010.737.2610.209.90

7.118.6810.0010.37

519.19519.19521.20521.20519.19519.19681.24717.28717.28

623.16623.16607.17623.16

261829144768177956541620261325502916

11133805315462872

3615291015962304326400225

4416368124811

514430616362886326429317263

2805356309792

1581012274429074870109

49315740626

0.1380***0.1814***0.2128***0.0542***0.4075***0.0017***1.01020.00000.0772***

0.0394***0.4301***0.80700.0037***

Fold change MDCA/mockAverage St. Dev. Average St. Dev.

Compound

Flavonol glycosides

Kaempferol-3-O-rhamnosyl(1-->2)- glucoside -7-O-rhamnosideKaempferol + deoxyhexose + deoxyhexoseKaempferol + hexose + hexose+ deoxyhexoseKaempferol-3-O-rhamnoside-7-O-glucosideKaempferol + hexose + deoxyhexose + deoxyhexoseKaempferol-3-O-rhamnoside-7-O-rhamnosideKaempferol + hexose + deoxyhexoseKaempferol + hexoseKaempferol + deoxyhexoseKaempferol + deoxyhexose

Quercetin + hexose + deoxyhexoseQuercetin-3-O-rhamnosyl(1-->2)- glucoside -7-O-rhamnosideQuercetin + hexose + hexose+ deoxyhexoseQuercetin + hexose + deoxyhexose + deoxyhexoseQuercetin-3-O-rhamnoside-7-O-glucosideQuercetin + hexose + deoxyhexoseQuercetin + deoxyhexose

mock 10 µM MDCARetentiontime (min)

m/z

6.40

6.727.788.358.93

9.6510.0110.4112.2014.97

5.657.72

6.547.177.238.6312.72

739.21

593.15755.21593.15739.21

577.16593.15447.09431.10431.10

609.15755.21

771.20755.21609.15609.15447.09

87791

111451447929121038

8435718628931318483917

450923472

3301970264027113962578

90604

9680201665835389

90086388360621927

180514762

226492943735350194

19898

202231314139130

1012636702054351721

9473643

6021770130752670666

19866

275546716130323

2083026569983966

9403284

14212748181353226

1.0320

0.86851.3932*0.7086**0.3747**1.06790.0208***0.0386***0.0338***0.4919

0.4003**0.6289**0.0683***0.5080**0.6831*0.0307***0.0754***

Fold changeMDCA/mockAverage St. Dev. Average St. Dev.

Compound

Figure S4. Metabolites with an altered abundance in 10 μM MDCA-treated Arabidopsis seedlings.

A targeted approach was used to investigate which of the identified compounds are altered in 10 μM MDCA-treated seedlings in comparison with mock-treated seedlings (n=6). For each compound, normalized average peak areas (unitless) of mock- and 10 μM MDCA-treated seedlings are given. Peak areas are normalized relative to the dry weight of the pellet remaining after methanol extraction. The retention time is expressed in minutes. Asterisks represent significant differences between 10 μM MDCA-treated and mock-treated plants as determined by Dunnett’s test. Dunnett’s test P-values: *0.001 ≤ P < 0.05, **0.0001 ≤ P < 0.001, *** P <0.0001. The term UP implies that the compound could only be detected in 10 μM MDCA-treated seedlings and not in mock-treated seedlings.

0 10

Num

ber o

f end

oder

mal

cel

ls

0

10

20

30

40

50

60

***

**

compl.no compl.

mock

MDCA 10 µM

compl.

compl.

+

MDCA 10 µM

MDCA (µM)

Figure S5. The impact of the MDCA-induced lignin reduction on the plant phenotype.

Visualization of the effect of MDCA treatment on the Casparian strip formation (white; no compl.) in Arabidopsis seedlings 5 DAG and complementation by exogenous application of two monolignols (grey; compl.): 50 μM of each coniferyl alcohol and sinapyl alcohol, which allows for the formation of a functional Casparian strip (n=10). See manuscript for additional explanation on this experiment. Error bars represent standard deviations and asterisks were used to indicate statistically significant differences compared to the corresponding mock-treated control sample as determined by Dunnett's test P-values: *P < 0.05, **P < 0.001, *** P <0.0001. (B)

Peak

are

a/dr

y w

eigh

t (m

g-1)

0

2

4

6

8

10

mock MDCA10 µM

*

Figure S6. Detection of salicylic acid in 10 μM MDCA-treated Arabidopsis seedlings.

The average peak area of salicylic acid (SA) in mock- and 10 μM MDCA-treated seedlings 12 DAG (n=10). The unitless peak areas were normalized to the dry weight of the pellet remaining after methanol extraction (in mg). Error bars represent standard deviations. The asterisk represents significant difference in SA levels between 10 μM MDCA-treated and mock-treated plants (0.01 < P-value < 0.05) as determined by Dunnett's test.

A

IAA MDCA

x

y

z

z

y

x

B

MM

pKa

cLogP([octanol]/[water]) partition

#H-acceptors

#H-donors

Total surface area

Polar surface area

Rotable bonds

Aromatic rings

175.186

4.66

1.1822

3

2

166

53.09

2

2

IAA MDCA

192.17

3.55

1.5853

4

1

168

55.76

2

1

Physiochemical Properties

D

C

E

O

O

O

OH

O

N

H

OH

Figure S7. Chemical and physical characteristics and docking of MDCA for TIR1.

(A) The molecular structure of IAA and MDCA. (B) Top view of energy minimized structures represented as sticks with bicyclic ring structures planar. The IAA carboxylic acid group does not position in the plane of the aromatic ring, whilst for MDCA, the carboxylic acid is fixed planar to the ring due to the trans-alkene bond. (C) Physiochemical properties of IAA and MDCA. (D) The binding pocket of TIR1 shown as a surface representation in relation to the whole structure whereby the binding region is defined as an 18Å x 18Å x 18Å box. Docking of IAA from the crystal structure (purple) with that of the docked result (grey) showed almost identical and superimposable results. (E) The best possible pose for MDCA in the lower region of the TIR1 pocket.

Indole-3-actetic acid (IAA)

Precursors

Anthranilate

Tryptophan

Tryptamine

Indole-3-acetamide

Indole-3-acetonitrile

Indole-3-acetaldoxime

Indole-3-acetaldehyde

Indole-3-pyruvic acid

Conjugates, catabolites

2-oxindole-3-acetic acid

IAA-glutamate

IAA-aspartate

mock MDCA 5 µM MDCA 10 µM

15.5 (2.50)

26.50 (6.4)

2170.90 (508.90)

0.18 (0.05)

1.13 (0.26)

388.30 (79.50)

12.50 (2.40)

95.30 (24.60)

129.50 (15.30)

109.17 (26.10)

0.94 (0.16)

0.85 (0.20)

20.60 (2.30) *

36.50 (7.80)

2202.90 (389.10)

0.28 (0.07) *1.86 (0.37) **592.70 (126.70) *16.90 (3.00) *126.00 (20.80)

121.50 (22.60)

113.80 (20.22)

1.66 (0.36) **1.70 (0.37) **

27.80 (2.60) ***

38.10 (9.30)

3038.50 (648.90)

0.30 (0.08) *2.71 (0.61) ***1009.80 (189.10) ***20.20 (3.40) **160.00 (43.10) *149.00 (39.00)

149.36 (26.82) *2.24 (0.28) ***2.66 (0.62) ***

Figure S8. Concentration of auxin and auxin metabolites after MDCA treatment of Arabidopsis seedlings.

The concentration of free IAA, IAA-precursors, IAA-amino acid conjugates and catabolites in seedlings (12 DAG) grown on 0.5xMS-medium supplemented with MDCA (5 μM and 10 μM). Each biological replicate represents ten seedlings that were pooled and analyzed (n=6). Standard deviations are mentioned inbetween brackets and asterisks represent statistically significant differences between MDCA-treated and mock-treated plants as determined by Dunnett's test. P-values: *P < 0.05, **P < 0.001, *** P <0.0001.

Indole-3-actetic acid (IAA)

Precursors

Anthranilate

Tryptophan

Tryptamine

Indole-3-acetamide

Indole-3-acetonitrile

Indole-3-acetaldoxime

Indole-3-acetaldehyde

Indole-3-pyruvic acid

Conjugates, catabolites

2-oxindole-3-acetic acid

IAA-glutamate

IAA-aspartate

WT ref3-2 mutant

9.1 (0.7)

26.8 (7.4)

5251.3 (1083.3)

1.0 (0.3)

1.2 (0.3)

333.1 (117.8)

7.5 (1.4)

69.8 (10.7)

218 (42)

65.2 (15.6)

2.5 (0.6)

6.5 (1.8)

13.3 (1.9) ***

15.6 (2.6) **4300.4 (1037.8)

1.1 (0.2)

2.2 (0.5) **578.2 (71.1) **4.0 (0.9) ***126.7 (42.7) * 344 (87) **

93.4 (20.0) *9.5 (2.1) ***72.8 (13.9) ***

Figure S9. Concentration of auxin and auxin metabolites in the c4h mutant ref3-2.

The concentration of free IAA, IAA-precursors, IAA-amino acid conjugates and catabolites in the leaves of 2 months old ref3-2 plants. Each biological replicate represents one plant (n=6). Standard deviations are mentioned inbetween brackets and asterisks represent statistically significant differences between wild type (WT) and c4h mutant ref3-2 plants as determined by Dunnett's test. P-values: *P < 0.05, **P < 0.001, *** P <0.0001.

A

B

Figure S10. The impact of MDCA on auxin transport in suspension-grown Arabidopsis T87 cells.

(A-B) Effect of MDCA on the net accumulation of (A) [3H]-NAA or (B) [3H]-2,4-D in four-day old suspension-grown Arabidopsis cells (20 minute uptake period). Arrows point at time of application of MDCA. Error bars in (A-B) represent standard deviations (n=4).

Time (min)

Accu

mul

atio

n of

[3 H]-2

,4-D

(p

mol

per

10

mg

FW)

mockMDCA 50 µM

0 4 8 12 160

0.05

0.10

0.15

0.20

0.25

Accu

mul

atio

n of

[3 H]-N

AA

(pm

ol p

er 1

0 m

g FW

)

0 4 8 12 160

0.05

0.10

0.15

0.20

0.25

mockMDCA 50 µM

Time (min)