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)