Radicle emergence (%)

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A MDCA (µm) Radicle emergence (%) 99.65 5 99.32 75 98.96 1 99.39 125 97.3 15 98.2 175 97.78 2 98.

(A) Radicle emergence (%) of seeds 2 DAG grown on.

1 A MDCA (µm) Radicle emergence (%) B 5 µm 75 µm 1 µm 125 µm 15 µm 175 µm 2 µm Figure S1. Effect of MDCA on germination of Arabidopsis. (A) Radicle emergence (%) of seeds 2 DAG grown on.5xms-medium supplemented with different concentrations of MDCA (n>25) (B) Phenotype of seedlings (12 DAG) grown on.5xms-medium supplemented with different concentrations of MDCA (n>25) (scale bar:.5 cm).

2 Compound Top-1 Retention time (min) 1 µm MDCA Average St. Dev. Average St. Dev. Fold change MDCA/ 1. Cinnamoyl aspartate 1 2. MDCA glutamate 1 3. MDCA aspartate 1 4. Cinnamoyl aspartate 2 5. MDCA aspartate 2 6. MDCA glutamate 2 7. Unknown 8. Cinnamoyl glutamate 9. Cinnamoyl aspartate 1 (fragment) 1. MDCA aspartate 2 (heterodimer) Compound Top-1 DWN 1. 4-methylthio-N-butyl-glucosinolate 2. 1-methoxyindol-3-ylmethyl-glucosinolate 3. G(8--4)ferulic acid + hexose + hexose 4. G(8--4)ferulic acid + hexose + hexose 5. 8-methylsulfinyloctyl-glucosinolate 6. Kaempferol + hexose + deoxyhexose 7. 5-methylthio-N-pentyl-glucosinolate 8. Unknown 9. Quercetin + hexose + deoxyhexose 1. 8-methylthio-N-octyl-glucosinolate (dimer) Figure S2. Metabolites with an altered abundance in 1 μm MDCA-treated Arabidopsis seedlings. An overview of the top-1 significantly differential compounds with at least a 1-fold increase () or decrease (DWN) in 1 μm MDCA-treated seedlings as compared to -treated seedlings (n=6). The top-1 defined as those compounds with the highest normalized peak area in MDCA-treated seedlings, whereas the top-1 DWN were defined as those compounds with the highest normalized peak area in -treated seedlings. The retention time is expressed in minutes. For each compound, normalized average peak areas (unitless) of - and 1 μm MDCA-treated seedlings are given. Peak areas are normalized relative to the dry weight of the pellet remaining after methanol extraction. The term implies that a peak could only be detected in 1 μm MDCA-treated seedlings and not in -treated seedlings.

3 A PA + hexose (formic acid adduct) (5.92 min, ) 1 intensity (%) ; in source fragment, [M-H + -dehyrated hexose] - = [PA-H + ] theoretical [M+ formic acid - H + ] - = retention time (min) B MS/MS @ 5.92 min (in-source fragment of PA + hexose) [PA-H + -C 2 -CH 2 ] hexose H H intensity (%) [PA-H + -C 2 ] [PA-H + ] C PA + malate (9.71 min, ) 1 intensity (%) ; in source fragment, [M-H + -dehyrated malate] - = [PA-H + ] theoretical [M -H + ] - = retention time (min) D MS/MS @ 9.71 min (in-source fragment of PA + malate) [PA-H + -C 2 -CH 2 ] [PA-H + -C 2 ] H H intensity (%) [PA-H + ] 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 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 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 eluting at 9.71 min showed characteristic fragmentation pattern of PA.

4 Compound Retention time (min) 1 µm MDCA Average St. Dev. Average St. Dev. Fold change MDCA/ Cinnamic acid derivatives Cinnamoyl hexose (formic acid adduct) Cinnamoyl malate 1 Cinnamoyl malate *** p-coumaric acid derivatives p-coumaric acid 4--hexoside p-coumaroyl hexose 1 p-coumaroyl hexose 2 Dihydro-p-coumaric acid + hexose *.1516***.4744*** Caffeic acid derivatives Caffeic acid 1 Caffeic acid 2 Caffeic acid 3/4--hexoside 3 Caffeoyl hexose 1 Caffeoyl hexose 3/4--hexoside 1 Caffeoyl malate 1 Caffeoyl malate *** ***

5 Compound Retention time (min) 1 µm MDCA Average St. Dev. Average St. Dev. Fold change MDCA/ Ferulic acid derivatives Ferulic acid Feruloyl hexose 1 Feruloyl hexose 2 Ferulic acid -4-hexoside Feruloyl malate 1 Feruloyl malate ***.68***.1733***.2158 Sinapic acid derivatives Sinapic acid Sinapoyl hexose 1 Sinapoyl hexose 2 Disinapoyl hexose 1 Disinapoyl hexose 2 Sinapoyl malate 1 Sinapoyl malate ** Varia Coniferin (formic acid adduct) Scopolin (formic acid adduct) ***.2442***

6 Compound Retention time (min) 1 µm MDCA Average St. Dev. Average St. Dev. Fold change MDCA/ Coniferyl alcohol - ferulic acid conjugates G(8--4) ferulic acid G(8--4) ferulic acid + hexose 1 G(8--4) ferulic acid + hexose 2 G(8--4) ferulic acid + hexose 3 G(8--4) ferulic acid + hexose 4 G(8--4) ferulic acid + hexose 5 G(8--4) ferulic acid + hexose 6 G(8--4) ferulic acid + malate 1 G(8--4) ferulic acid + malate 2 G(8--4) ferulic acid + malate 3 G(8--4) ferulic acid + malate 4 G(8--4) ferulic acid Da 1 G(8--4) ferulic acid Da *** 1.798*** ***.6178**.6748*.2275***.3841***.448***.542*** G(8-5) feruloyl hexose 1 G(8-5) feruloyl hexose 2 G(8-5) feruloyl hexose 3 G(8-5) feruloyl malate G(8-5) feruloyl malate -4-hexoside 1 G(8-5) feruloyl malate -4-hexoside ***.2451***.4154*.4891**.319***.5487

7 Compound Retention time (min) 1 µm MDCA Average St. Dev. Average St. Dev. Fold change MDCA/ Hexosylated oligolignols G(8-5)G + hexose G 4--hexoside(8-5)G G(red8-5)G + hexose G(red8-8)G 8/4--hexoside G(8-8)G hexoside 1 G(8-8)G hexoside 2 G(8-8)G + hexose + hexose G(8--4)G(red8-5)G + hexose 1 G(8--4)G(red8-5)G + hexose ***.1814***.2128***.542***.475***.17*** *** Flavonol glycosides Isorhamnetin + hexose + deoxyhexose Isorhamnetin-3--rhamnoside-7--glucoside Isorhamnetin + deoxyhexose + deoxyhexose Isorhamnetin + hexose + deoxyhexose ***.431***.87.37***

8 Compound Retention time (min) 1 µm MDCA Average St. Dev. Average St. Dev. Fold change MDCA/ Flavonol glycosides Kaempferol-3--rhamnosyl(1-->2)- glucoside -7--rhamnoside Kaempferol + deoxyhexose + deoxyhexose Kaempferol + hexose + hexose+ deoxyhexose Kaempferol-3--rhamnoside-7--glucoside Kaempferol + hexose + deoxyhexose + deoxyhexose Kaempferol-3--rhamnoside-7--rhamnoside Kaempferol + hexose + deoxyhexose Kaempferol + hexose Kaempferol + deoxyhexose Kaempferol + deoxyhexose *.786**.3747** ***.386***.338***.4919 Quercetin + hexose + deoxyhexose Quercetin-3--rhamnosyl(1-->2)- glucoside -7--rhamnoside Quercetin + hexose + hexose+ deoxyhexose Quercetin + hexose + deoxyhexose + deoxyhexose Quercetin-3--rhamnoside-7--glucoside Quercetin + hexose + deoxyhexose Quercetin + deoxyhexose **.6289**.683***.58**.6831*.37***.754*** Figure S4. Metabolites with an altered abundance in 1 μm MDCA-treated Arabidopsis seedlings. A targeted approach was used to investigate which of the identified compounds are altered in 1 μm MDCA-treated seedlings in comparison with -treated seedlings (n=6). For each compound, normalized average peak areas (unitless) of - and 1 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 1 μm MDCA-treated and -treated plants as determined by Dunnett s test. Dunnett s test P-values: *.1 P <.5, **.1 P <.1, *** P <.1. The term implies that the compound could only be detected in 1 μm MDCA-treated seedlings and not in -treated seedlings.

Number of endodermal cells 6 5 4 3 2 1 *** ** 1 MDCA (µm) no compl. compl. compl. MDCA 1 µm compl.+ MDCA 1 µm Figure S5. The impact of the MDCA-induced lignin reduction on the plant phenotype.

9 Number of endodermal cells *** ** 1 MDCA (µm) no compl. compl. compl. MDCA 1 µm compl.+ MDCA 1 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.): 5 μm of each coniferyl alcohol and sinapyl alcohol, which allows for the formation of a functional Casparian strip (n=1). 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 -treated control sample as determined by Dunnett's test P-values: *P <.5, **P <.1, *** P <.1. (B)

10 Peak area/dry weight (mg -1 ) * MDCA 1 µm Figure S6. Detection of salicylic acid in 1 μm MDCA-treated Arabidopsis seedlings. The average peak area of salicylic acid (SA) in - and 1 μm MDCA-treated seedlings 12 DAG (n=1). 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 1 μm MDCA-treated and -treated plants (.1 < P-value <.5) as determined by Dunnett's test.

A H N H B IAA MDCA x z H y z x y C Physiochemical Properties MM pka clogp([octanol]/[water]) partition #H-acceptors #H-donors Total surface area Polar surface area Rotable bonds Aromatic rings D IAA

(A) The molecular structure of IAA and MDCA. (B) Top view of energy minimized structures represented as sticks with bicyclic ring structures planar.

11 A H N H B IAA MDCA x z H y z x y C Physiochemical Properties MM pka clogp([octanol]/[water]) partition #H-acceptors #H-donors Total surface area Polar surface area Rotable bonds Aromatic rings D IAA MDCA E 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.

12 MDCA 5 µm MDCA 1 µm Indole-3-actetic acid (IAA) 15.5 (2.5) 2.6 (2.3) * 27.8 (2.6) *** Precursors Anthranilate 26.5 (6.4) 36.5 (7.8) 38.1 (9.3) Tryptophan Tryptamine Indole-3-acetamide Indole-3-acetonitrile Indole-3-acetaldoxime Indole-3-acetaldehyde (58.9).18 (.5) 1.13 (.26) (79.5) 12.5 (2.4) 95.3 (24.6) (389.1).28 (.7) * 1.86 (.37) ** (126.7) * 16.9 (3.) * 126. (2.8) (648.9).3 (.8) * 2.71 (.61) *** 19.8 (189.1) *** 2.2 (3.4) ** 16. (43.1) * Indole-3-pyruvic acid (15.3) (22.6) 149. (39.) Conjugates, catabolites 2-oxindole-3-acetic acid IAA-glutamate IAA-aspartate (26.1).94 (.16).85 (.2) (2.22) 1.66 (.36) ** 1.7 (.37) ** (26.82) * 2.24 (.28) *** 2.66 (.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.5xms-medium supplemented with MDCA (5 μm and 1 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 -treated plants as determined by Dunnett's test. P-values: *P <.5, **P <.1, *** P <.1.

13 Indole-3-actetic acid (IAA) WT 9.1 (.7) ref3-2 mutant 13.3 (1.9) *** Precursors Anthranilate Tryptophan Tryptamine Indole-3-acetamide Indole-3-acetonitrile Indole-3-acetaldoxime Indole-3-acetaldehyde Indole-3-pyruvic acid 26.8 (7.4) (183.3) 1. (.3) 1.2 (.3) (117.8) 7.5 (1.4) 69.8 (1.7) 218 (42) 15.6 (2.6) ** 43.4 (137.8) 1.1 (.2) 2.2 (.5) ** (71.1) ** 4. (.9) *** (42.7) * 344 (87) ** Conjugates, catabolites 2-oxindole-3-acetic acid IAA-glutamate IAA-aspartate 65.2 (15.6) 2.5 (.6) 6.5 (1.8) 93.4 (2.) * 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 <.5, **P <.1, *** P <.1.

14 A.25 Accumulation of [ 3 H]-NAA (pmol per 1 mg FW) B Accumulation of [ 3 H]-2,4-D (pmol per 1 mg FW) MDCA 5 µm Time (min) MDCA 5 µm Time (min) Figure S1. The impact of MDCA on auxin transport in suspension-grown Arabidopsis T87 cells. (A-B) Effect of MDCA on the net accumulation of (A) [ 3 H]-NAA or (B) [ 3 H]-2,4-D in four-day old suspension-grown Arabidopsis cells (2 minute uptake period). Arrows point at time of application of MDCA. Error bars in (A-B) represent standard deviations (n=4).

Figure 1. Identification of UGT74E2 as an IBA glycosyltransferase. (A) Relative conversion rates of different plant hormones to their glucosylated

Figure 1. Identification of UGT74E2 as an IBA glycosyltransferase. (A) Relative conversion rates of different plant hormones to their glucosylated form by recombinant UGT74E2. The naturally occurring auxin