Supporting InformationSugawara et al. 10.1073/pnas.0811226106SI Materials and MethodsGeneration of cyp79b2 cyp79b3 Double Mutants. The T-DNA mu-tants were genotyped as described in Zhao et al. (1). Primer79B2–5P (5�-ATGAACACTTTTACCTCAAAC-3�) and LBa1primer (5�-TGGTTCACGTAGTGGGCCATCG-3�) were usedto identify the T-DNA insertion at 925 bp (cyp79b2–2) after theATG of the CYP79B2 gene. Primer 79B3–2-5P (5�-ATG-GATACTTTAGCTTCAAAC-3�) and pAC161-LB primer (5�-GGGCTACACTGAATTGGTAGCTC-3�) were used to iden-tify the T-DNA insertion at 1781 bp (cyp79b3–2) after the startcodon of the CYP79B3 gene. The primer 79B3–3-5P (5�-TCACTTCACCATTGGGTAAAGG-3�) and pAC161-LBprimer were used to identify the T-DNA insertion at 1962 bp(cyp79b3–3) after the start codon of the CYP79B3 gene. Theinsertions were confirmed by DNA sequencing of the PCRfragments generated with LBa1 primer, pAC161-LB primer, andgene-specific primers. cyp79b2–2 cyp79b3–2 and cyp79b2–1cyp79b3–3 double mutants were generated by crossing thecorresponding single mutants.

Synthesis of Chemicals

General Experimental Conditions. 1H and 13C NMR spectra wererecorded on a JNM Lambda 400 NMR spectrometer (JEOL).Chemical shifts are shown as �-values from TMS as the internalreference. Peak multiplicities are quoted in Hertz (Hz). Massspectra were measured on a JMS-700 spectrometer (JEOL).Column chromatography was carried out on columns of silica gel60 (230�400 mesh, Merck). All chemicals were purchased fromSigma-Aldrich, unless otherwise stated.

Synthesis of IAOx, D5-IAOx, and 13C6-IAOx.Oximes were synthesized as described by Rausch et al. (2) and

Glawischnig et al. (3).

Indole-3-ethanol. IAA (100 mg, 0.57 mmol) was added to asuspension of lithium aluminum hydride (LAH, 50 mg, 1.32mmol) in dry tetrahydrofuran (10 mL) under N2 at roomtemperature. After stirring for 2 h, excess LAH was quenched byaddition of 1N-HCl solution. The whole mixture was extractedwith ethyl acetate and the organic layer was washed with H2Oand brine, dried (MgSO4) and evaporated to dryness in vacuo.The crude product was subjected to silica gel column chroma-tography eluted with hexane-EtOAc (1:1) to afford indole-3-ethanol (86 mg, 94%) as a white solid.

1H-NMR (CDCl3, 400 MHz) � ppm: 1.60 (1H, bs), 3.03 (2H,t, J � 6.3 Hz), 3.90 (2H, t, J � 6.3 Hz), 7.05 (1H, d, J � 2.2 Hz),7.13 (1H, bt, J � 7.4 Hz), 7.21 (1H, bt, J � 7.5 Hz), 7.35 (1H, bd,J � 8.0 Hz), 7.62 (1H, bd, J � 7.6 Hz), and 8.08 (1H, bs).13C-NMR (CDCl3, 400 MHz) � ppm: 28.7, 62.6, 111.2, 112.2,118.8, 119.4, 122.2, 122.5, 127.4, and 136.4.

IAAld and IAOx. Sulfur trioxide pyridine complex (SO3/Pyr, 1.1 g,6.9 mmol) was added to a stirred solution of indole-3-ethanol(370 mg, 2.3 mmol), DMSO (3.2 mL) and triethylamine (Et3N)(1.6 ml, 11.5 mmol) in dry dichloroethane (DCE, 12 mL) at 0 °Cunder N2. After stirring at 0 °C for 30 min, the reaction mixturewas directly subjected to a short column of silica gel chroma-tography to afford crude IAAld as a yellow-brown oil, which wasused for the next step without further purification. Hydroxyl-amine hydrochloride (319 mg, 4.6 mmol) and sodium acetate(377 mg, 4.6 mmol) were added to a stirred solution of the crudeIAAld in ethanol (15 mL) at room temperature. After stirring

overnight, the reaction mixture was directly purified by columnchromatography eluted with hexane-EtOAc (3:2) to give IAOx(230 mg, mixture of cis/trans-form, 58%, 2 steps) as a yellow solid.The NMR spectral data were consistent with those reported inthe literature (3).

D5-IAOx. [indole-D5]IAOx (56 mg, cis/trans � 1.8:1, 97% D5-labeled) was synthesized from [indole-D5]IAA (100 mg, 97%D5-labeled, Cambridge Isotope Laboratories) as describedabove for IAOx. 1H-NMR (CD3OD, 400 MHz) � ppm: cis form,3.59 (2H, d, J � 6.5 Hz), 7.48 (1H, t, J � 6.5 Hz); trans form, 3.79(2H, d, J � 5.4 Hz), 6.79 (1H, t, J � 5.4 Hz).

13C6-IAOx. [phenyl-13C6]IAOx (4 mg, 98% 13C6-labeled) was pre-pared from [phenyl-13C6]IAA (20 mg, 98% 13C6-labeled, Cam-bridge Isotope Laboratories) as described above for IAOx.1H-NMR (CD3OD, 400 MHz) � ppm: cis form, 3.60 (2H, dd, J �6.4, 3.9 Hz), 6.7–7.8 (6H, m); trans form, 3.79 (2h, d, J � 5.2, 4.1Hz), 6.7–7.8 (6H, m).

Synthesis of 13C6-IAM. [phenyl-13C6]IAA (10 mg, 0.06 mmol, 98%13C6-labeled), N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinone(EEDQ, 15.5 mg, 0.06 mmol) (4), and ammonium hydrogencar-bonate (NH4HCO3, 13.5 mg, 0.17 mmol) were suspended inCHCl3 (4 mL), and the mixture was stirred overnight at roomtemperature. The mixture was then subjected to silica gel columnchromatography eluted with EtOAc-acetone (20:1) to afford13C6-IAM (8 mg, 80% yield, 98% 13C6-labeled) as a white solid.1H-NMR (CD3OD, 400 MHz) � ppm: 3.76 (2H, d, J � 4.4 Hz),5.39 (1H, bs), 5.64 (1H, bs), 6.9–7.9 (5H, m), 8.22 (1H, bs).

Synthesis of D2-IAN. 1.8-Diazabicyclo[5.4.0]undec-7-ene wasadded to a stirred solution of IAN in 50% DMF in D2O (vol/vol,2 mL, 98% D-labeled) at room temperature under argon. Themixture was heated to 80 °C for 3 h. After cooling, saturatedNaCl solution was added and the resulting mixture was extractedwith ether. The ether layer was dried over MgSO4. Removal ofthe solvent in vacuo afforded a residue, which was purified bycolumn chromatography eluted with hexane-EtOAc (3:1), togive [2�-D2]IAN (202 mg, 95% D2-labeled) as yellow oil.

1H-NMR (CDCl3, 400 MHz) � ppm: 7.1–7.3 (3H, m), 7.38 (1H,d, J � 8.0 Hz), 7.58 (1H, d, J � 8.0 Hz), and 8.21 (1H, bs).

Synthesis of 15N2-TAM. [15N2]TAM (60 mg, 98% 15N2-labeled) wassynthesized as described by Barton et al. (5) using [15N2]TRP(100 mg, 98% 15N2-labeled, Cambridge Isotope Laboratories).The structure of [15N2]TAM was confirmed as described above,LC-ESI-MS/MS analysis of TAM.

LC-ESI-MS/MS and GC-MS analysis of IAA and IAA inter-mediates

Analysis of IAOx. For analysis of IAOx, �0.5 g of fresh Arabidopsistissue were weighed quickly, frozen with liquid N2 and lyophi-lized overnight. The dried plant material was pulverized withliquid N2-chilled ceramic beads (10 mm) using a vortex for 2 minfollowed by extraction with 80% acetone (2.5 mL) containing 0.5ng of D5-IAOx (97% D5-labeled) at 4 °C for 30 min with gradualshaking. The extracts were centrifuged at 3,000 � g for 10 min,and the supernatant was removed. The extraction was repeatedwith 80% acetone and the supernatants were pooled and evap-orated with a Speed Vac (Thermo) at 35 °C. The dried extractwas resuspended with 200 �L of DMSO, and centrifuged at

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15,000 � g for 5 min. The supernatant was applied to a 5-�m,4.6 � 150-mm, Symmetry shield C18 column (Waters) coupledto a 5-�m, 4.6 � 10-mm, C18 guard column (Senshu Pak)connected to a HPLC system equipped with a Waters 2475 multi�-f luorescence detector. The samples were eluted at a flow rateof 1 mL/min with 0.01 M ammonium acetate (solvent A) and100% methanol (solvent B) using 10% solvent B for 1 min anda gradient from 10 to 50% solvent B over 30 min. During elution,the samples were monitored continuously using a fluorescentdetector (280 nm excitation and 355 nm emission). Fractionseluting at the retention time of trans and cis forms of IAOx (28.6and 31.1 min) were collected and evaporated immediately usinga Speed Vac. The dried IAOx fraction was redissolved with H2O(2 mL) and applied to an Oasis HLB column (Waters). Thecolumn was washed with H2O (3 mL) before eluting the IAOxwith methanol (3 mL). The methanolic eluate was then evapo-rated to dryness using a Speed Vac.

IAOx was analyzed by an ACQUITY Ultra PerformanceLiquid Chromatography (UPLC)-MS/MS Q-Tof-premier (Wa-ters). The chromatography was performed on an ACQUITYUPLC BEH C18 1.7-�m, 2.1 � 50-mm column (Waters). TheIAOx fraction was redissolved in 50% methanol/H2O containing1% acetic acid (20 �L) and injected to UPLC. Elution of thesamples was carried out with H2O (solvent A) and acetonitrilewith 0.05% acetic acid (solvent B) using 3% solvent B for 0.1 min,and a gradient from 3 to 30% solvent B over 10 min, at a flowrate of 0.2 mL/min. The retention time (trans/cis form) of IAOxand D5-IAOx were 8.84/9.39 min and 8.82/9.38 min, respectively.MS/MS conditions were as follows: capillary, 2.8 kV; sourcetemperature, 80 °C; desolvation temperature, 400 °C; cone gasflow, 0 L/h; desolvation gas flow, 500 L/h; collision energy, 12;and MS/MS transition (m/z): 175.2/158.1 for unlabeled IAOx and180.2/163.1 for D5-IAOx. Quantification was carried out usingthe extracted ion chromatogram of IAOx and D5-IAOx. Thelinear dynamic range was determined by injecting a standardmixture of IAOx and D5-IAOx in a wide concentration range (10pg/�L to 200 pg/�L). The detection limit of our IAOx analysis:10 pg.

For analysis of IAOx in rice coleoptiles (�0.2 g), maizecoleoptiles (�50 mg), and tobacco apexes (�0.5 g), each samplewas extracted with 80% acetone (0.25–2.5 mL) containing 0.3 ngof D5-IAOx, purified with HPLC and a HLB column, andanalyzed by LC-ESI-MS/MS as described above.

Analysis of IAM. For analysis of IAM, �0.1–0.2 g of fresh planttissues were quickly weighed, chilled with liquid N2, and freeze-dried overnight. Plant materials were homogenized in 80%acetone/H2O (0.5 mL) containing 0.3–1 ng of 13C6-IAM withceramic beads (3 mm) using Tissue Lyser (QIAGEN) for 3 min.The extracts were centrifuged at 15,000 � g for 5 min, and thesupernatant was transferred to a new glass tube. This extractionwas repeated without 13C6-IAM and the supernatants werecombined and evaporated using a Speed Vac. The IAM fractionwas collected from HPLC (retention time, 19.2 min) as describedabove and evaporated with a Speed Vac. The IAM fraction wasredissolved in 5% ammonium solution (1–2 mL) and subjectedto Oasis HLB column. The column was washed with 5%ammonium solution (2 mL), and IAM eluted with 50% meth-anol/H2O containing 2% formic acid (3 mL) and evaporated todryness using a Speed Vac. The IAM fraction was redissolved inmethanol (2 mL) and applied to successive Oasis MAX andMCX columns. The flow-through was collected and the solventswere evaporated to give the IAM fraction. IAM was analyzed byLC-ESI-MS/MS. Elution of the samples was carried out withH2O (solvent A) and acetonitrile with 0.05% acetic acid (solventB) using 3% solvent B for 0.1 min, and a gradient from 3 to 30%solvent B over 10 min, at a flow rate of 0.2 mL/min. The retentiontime of IAM and 13C6-IAM was 5.25 min. MS/MS conditions

were as follows: collision energy, 9 and MS/MS transition (m/z):175.1/130.1 for unlabeled IAM and 181.1/136.1 for 13C6-IAM,respectively. Quantification was carried out using the extractedion chromatogram of IAM (m/z 130.1) and 13C6-IAM (m/z136.1). The linear dynamic range was determined by injecting astandard mixture of IAM and 13C6-IAM in a wide concentrationrange (1 pg/�L to 100 pg/�L).

Analysis of IAA. For analysis of IAA, �0.1–0.2 g of fresh planttissue were homogenized in 80% methanol/H2O (0.1–0.25 mL)containing 1–2 ng of 13C6-IAA, purified with HPLC (retentiontime of IAA, 10.7 min) as described above, and evaporated in aSpeed Vac. The IAA fraction was redissolved in 1% aceticacid/H2O (1–2 mL) and subjected to an Oasis HLB column. Thecolumn was washed with H2O (3 mL), and IAA eluted withmethanol (3 mL) and evaporated to dryness in vacuo to give theIAA fraction. IAA was analyzed by LC-ESI-MS/MS. The IAAfraction was redissolved in 50% methanol/H2O containing 1%acetic acid (20 �L) and injected to UPLC. Elution of the sampleswas carried out with isocratic solutions 85% A2 (H2O with 0.05%acetic acid) and 15% B (acetonitrile with 0.05% acetic acid). Theretention time of IAA and 13C6-IAA was 6.69 min. MS/MSconditions were as follows: collision energy, 10 and MS/MStransition (m/z): 176.2/130.1 for unlabeled IAA and 182.2/136.1for 13C6-IAA, respectively. Quantification was carried out usingthe extracted ion chromatogram of IAA (m/z 130.1) and 13C6-IAA (m/z 136.1). The linear dynamic range was determined byinjecting a standard mixture of IAA and 13C6-IAA in a wideconcentration range (10 pg/�L to 200 pg/�L).

For analysis of IAA in small amounts of plant material, 20–30mg of fresh plant tissue were quickly weighed and homogenizedin 80% methanol/H2O (0.1–0.25 mL) containing 0.2–0.5 ng ofD2-IAA (97% D2-labeled, Sigma-Aldrich) with ceramic beads (3mm) using Tissue Lyser for 3 min. The extracts were centrifugedat 15,000 � g for 5 min, and the supernatant was transferred toa new 1.5-mL tube. This extraction was repeated without D2-IAA and the supernatants were combined and evaporated usinga Speed Vac at 35 °C. The residue was resuspended in 1 mL ofH2O and adjusted to pH 2–3 with 1N HCl, and IAA absorbedto 100–200 mg of Sigma-Aldrich XAD7 (Amberlite XAD7HP)for 30 min at 4 °C. The aqueous solution was removed, and theXAD7 was washed with 1% acetic acid/H2O (1 mL � 3). Afterremoval of 1% acetic acid/H2O, IAA was extracted with dichlo-romethane (0.5 mL � 2) and concentrated with a Speed Vac.IAA was analyzed by LC-ESI-MS/MS as described above. Elu-tion of the samples was carried out with gradient solutions A2and B of 0–3 min 3% B and 0.1–10 min 3–98% B, at a flow rateof 0.2 mL/min. The retention times of IAA and D2-IAA were4.28 min and 4.26 min, respectively. MS/MS conditions were asfollows: collision energy, 10 and MS/MS transition (m/z): 178.2/132.1 for D2-IAA. Quantification was carried out using theextracted ion chromatogram of IAA (m/z 130.1) and D2-IAA(m/z 132.1). The linear dynamic range was determined byinjecting a standard mixture of IAA and D2-IAA in a wideconcentration range (10 pg/�L to 200 pg/�L).

Analysis of TAM. For analysis of TAM, �0.2–0.5 g of fresh planttissue were quickly weighed, chilled with liquid N2, and freeze-dried overnight. Plant material was homogenized in 80% ace-tone/H2O (0.5 mL) containing 0.1–0.2 ng of 15N2-TAM asdescribed above. The TAM fraction was collected by HPLC(retention time of TAM, 15.9 min) and evaporated using a SpeedVac. The TAM fraction was redissolved in 2% formic acid/H2O(1–2 mL) and subjected to a MCX column. The column waswashed with 2% formic acid/H2O (2 mL), methanol (2 mL), 2%formic acid/H2O (2 mL), and TAM eluted with methanolcontaining 5% ammonium solution (3 mL) and evaporated todryness with a Speed Vac. TAM was analyzed by LC-ESI-MS/

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MS. Elution of the samples was carried out with gradientsolutions A2 and B of 0–3 min 3% B and 0.1–10 min 3–30% B,at a flow rate of 0.2 mL/min. The retention times of TAM and15N2-TAM were 3.67 min and 3.69 min, respectively. MS/MSconditions were as follows: collision energy, 6 and MS/MStransition (m/z): 161.2/144.1 for TAM and 163.2/145.1 for 15N2-TAM. Quantification was carried out using the extracted ionchromatogram of TAM (m/z 144.1) and 15N2-TAM (m/z 145.1).The linear dynamic range was determined by injecting a standardmixture of TAM and 15N2-TAM in a wide concentration range(2 pg/�L to 100 pg/�L).

GC-MS Analysis of IAN. For analysis of IAN in cyp79b2 cyp79b3plants, �1 g of plant material was extracted with 80% acetone/H2O (5 mL) containing 5 ng of D2-IAN as described above. TheIAN fraction was collected by HPLC (retention time of IAN �30.5 min) and evaporated using a Speed Vac. The IAN fractionwas redissolved with methanol (2 mL) and applied to an OasisMCX column. The flow-through was collected and the solventswere dried with a Speed Vac. The IAN fraction was redissolvedin 20 �L of 1% trimethylchlorosilane/N-methyl-N-trimethylsi-lyltrif luoroacetamide (MSTFA) solution (PIERCE) and heatedat 70 °C for 10 min. IAN was analyzed as trimethylsilyl (TMS)-IAN by GC-MS with an Agilent 6890 series GC system con-nected to a JEOL GC-mate II mass spectrometer (sourceconditions: electric ionization mode and 70 eV at 250 °C) witha He gas flow rate of 1 mL/min. A DB-1 capillary column (J &W Scientific, 0.25 mm � 15 m, 0.25 �m film thickness) was used.The oven temperature program was 80 °C for 1 min, 80–245 °Cat 30 °C/min, 245–280 °C at 5 °C/min, and 280 °C for 1 min. Theretention times of TMS-IAN and TMS-D2-IAN were 5.22 and5.23 min, respectively. The molecular ions for TMS-IAN andTMS-D2-IAN were m/z 228 and 230, respectively. Quantificationwas carried out using the extracted ion chromatogram of TMS-IAN (m/z 228) and TMS-D2-IAN (m/z 230). The linear dynamicrange was determined by injecting a standard mixture of TMS-IAN and TMS-D2-IAN in a wide concentration range (100pg/�L to 2 ng/�L).

For analysis of IAN in WT and sur1 mutants, �10 mg of plantmaterials were homogenized in 80% acetone/H2O (200 �L)containing 100 ng D2-IAN with ceramic beads (3 mm) usingTissue Lyser for 3 min. The extracts were centrifuged at 15,000 �g for 5 min, and the supernatant was transferred to a new 1.5 mLtube. This extraction was repeated without D2-IAN and thesupernatants were combined and evaporated using a Speed Vacat 40 °C. The residue was redissolved with methanol (2 mL) andapplied to an Oasis MCX column. The flow-through was col-lected and the aliquot (200 �L) was dried using a Speed Vac.TMS-derivatization of IAN and GC-MS analysis of TMS-IANwere performed as described above.

For analysis of IAN in rice coleoptiles (�20 mg), maizecoleoptiles (�20 mg), and apexes of tobacco (�50 mg), each

plant material was extracted in 80% acetone/H2O (200 �L)containing 2 ng D2-IAN, separated with HPLC (retention time,30.5 min) and purified with an Oasis MCX column as describedabove. Each IAN fraction was redissolved in 10 �L of MSTFAsolution, heated at 70 °C (10 min), and injected (5 �L) toGC-MS. The detection limit of our IAN analysis: 100 pg.

In Vivo Labeling Experiments. For 15N2-TAM feeding experiments,7-day-old WT seedlings were transferred to MS liquid media (15mL) containing 100 �M of 15N2-TAM, and incubated asepticallyfor 10 days with shaking at 100 rpm. Total IAOx and IAA werepurified from 15N2-TAM fed plants (ca 0.5 g) and analyzed byLC-ESI-MS/MS as described above except for the addition ofinternal standards. For 15N2-IAOx analysis, MS/MS transitionwas set to m/z 177.2/160.2. The 15N-incorporation rate into IAOxwas calculated using the extracted ion chromatogram of IAOx(m/z 158.2) and 15N2-IAOx (m/z 160.2). For 15N-IAA analysis,MS/MS transition was set to m/z 177.2/131.1. 15N-Incorporationrate into IAA was calculated using the extracted ion chromato-gram of IAA (m/z 130.1) and 15N-IAA (m/z 131.1). Total IANwas purified from seedlings (�0.05 g) and analyzed by GC-MSas described above without addition of the internal standard. The15N2-incorporation rate into IAN was calculated using theextracted ion chromatogram of IAN (m/z 228) and 15N2-IAN(m/z 230).

For the feeding experiment with 13C6-IAOx, 10-day-oldcyp79b2 cyp79b3 and WT seedlings were transferred to MS agarmedia containing 13C6-IAOx (3 �M and 10 �M) and incubatedaseptically for 24 h. For analysis of 13C6-incorporation intoproducts, total IAM, IAA, and IAN were purified from seed-lings (�0.1 g) and analyzed by LC-ESI-MS/MS and GC-MS,respectively, as described above (without addition of internalstandards). The 13C6-incorporation rate into IAM was calculatedusing the extracted ion chromatogram of IAM (m/z 130.1) and13C6-IAM (m/z 136.1). Similarly, the 13C6-incorporation rate intoIAA was calculated using the extracted ion chromatogram ofIAA (m/z 130.1) and 13C6-IAA (m/z 136.1). The 13C6-incorporation rate into IAN was calculated using the extractedion chromatogram of TMS-IAN (m/z 228) and TMS-13C6-IAN(m/z 234), respectively.

For feeding of D2-IAN to WT plants, 7-day-old seedlings weretransferred to MS agar media containing D2-IAN (30 �M) andincubated aseptically for 4 days. Total IAM and IAA werepurified from seedlings (�0.1 g) using HPLC as described abovewithout addition of the internal standard and analyzed byLC-ESI-MS/MS. For D2-IAM analysis, MS/MS transition wasset to m/z 177.1/132.1. The D2-incorporation rate was calculatedusing the extracted ion chromatogram of IAM (m/z 130.1) andD2-IAM (m/z 132.1). For D2-IAA analysis, MS/MS transitionwas set to m/z 178.1/132.1. The D2-incorporation rate wascalculated using the extracted ion chromatogram of IAA (m/z130.1) and D2-IAA (m/z 132.1).

1. Zhao Y, et al. (2002) Trp-dependent auxin biosynthesis in Arabidopsis: involvement ofcytochrome P450s CYP79B2 and CYP79B3. Genes Dev 16:3100–3112.

2. Rausch T, Helmlinger J, Hilgenberg W (1985) High-performance liquid chromato-graphic separation and some properties of (E)- and (Z)-3-indoleacetaldoxime. J Chro-matog 318:95–102.

3. Glawischnig E, Hansen BG, Olsen CE, Halkier BA (2004) Camalexin is synthesized fromindole-3-acetaldoxime, a key branching point between primary and secondary me-tabolism in Arabidopsis. Proc Natl Acad Sci USA 101:8245–8250.

4. Nozaki S, Muramatsu I (1988) Convenient synthesis of N-protected amino acid amides.Bull Chem Soc Jpn 61:2647–2648.

5. Barton DHR, Kirby GW, Prager RH, Wilson EM (1965) On the origin of the C-1 fragmentin indole alkaloids. J Chem Soc 3990–3994.

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Time (min) Fig. S1. LC-ESI-MS/MS analysis of IAOx. (A and B) The MS/MS chromatogram ([M � H � 17]�) for trans and cis IAOx (m/z 158.1, 9.05 and 9.60 min) and transand cis D5-IAOx (m/z 163.1, 8.95 and 9.55 min) in WT seedlings. (C and D) The MS/MS chromatogram ([M � H � 17]�) for the IAOx fraction (m/z 158.1) and transand cis D5-IAOx (m/z 163.1, 8.95 and 9.55 min) in cyp79b2 cyp79b3 seedlings.

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Fig. S2. In vivo labeling of IAOx and TAM from [13C8,15N]indole in Arabidopsis. The TRP-auxotroph trp1–1 homozygotes were selected from the progeny ofTRP1–1/trp1–1 plants after incubation on MS agar plates for 7 days. Five trp1–1 seedlings were transferred to MS liquid media (15 mL) containing [13C8,15N]indole(IND) (100 �M) in a 100-mL flask. Seedlings were incubated for 10 days at 100 rpm until the phenotype of the trp1–1 seedlings were nearly identical to WT. IAOxand TAM were purified from plants and analyzed by LC-ESI-MS/MS. (A and B) WT and trp1–1 seedlings (3-week-old). (C) Scheme for the in vivo labeling experimentusing trp1–1 mutants. (D) The MS/MS chromatogram ([M � H � 17]�) for IAOx and [13C8,15N]IAOx. The chromatograms for IAOx and [13C8,15N]IAOx are shownby blue and red lines, respectively. (E) The MS/MS chromatogram ([M � H � 17]�) for TAM and [13C8, 15N]TAM. The chromatogram for TAM and [13C8,15N]TAMare shown by blue and red lines, respectively.

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0 800400 1200 20001600 2400bps

CYP79B2/At4g39950

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SALK_113348 (cyp79b2-2) SALK_130570 (cyp79b2-1)

GABI_198F06 (cyp79b3-2) GABI_142B08 (cyp79b3-3)

WT cyp79b2-1 cyp79b3-3cyp79b2-1 cyp79b3-1 cyp79b2-2 cyp79b3-2

A

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Fig. S3. Analysis of cyp79b2 cyp79b3 double mutants. (A) Identification of T-DNA insertion mutants for the CYP79B2 and CYP79B3 genes. The T-DNA insertionsites are schematically indicated and the exact insertion sites shown as the distance from the ATG codon in the genomic sequence. (B) Two-week-old seedlingsgrown at 21 °C. The T-DNA insertion sites for cyp79b2–1 cyp79b3–1 mutants were previously reported by Zhao et al. (1). (Scale bar, 2 cm.)

1. Zhao Y, et al. (2002) Trp-dependent auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes Dev 16:3100–3112.

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yuc1 yuc2 yuc4 yuc6WTFig. S4. Phenotype of 4-week-old yuc1 yuc2 yuc4 yuc6 quadruple mutants. Seeds were germinated and grown on MS agar plates for 2 weeks, then transferredto soil and grown for another 2 weeks. Plants were collected before bolting for LC-ESI-MS/MS analysis. (Scale bar, 1 cm.)

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Rel

ativ

e ab

unda

nce

(%) 100

0

100

05.00 6.00 4.00

5.00 6.00 4.00

Rel

ativ

e ab

unda

nce

(%)

m/z 130.1

m/z 136.1

Time (min)

Time (min)

Fig. S5. LC-ESI-MS/MS analysis of IAM. The MS/MS chromatogram ([M � H � 45]�) for IAM (m/z 130.1, 5.25 min) and 13C6-IAM (m/z 136.1, 5.25 min) from WTseedlings.

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WT axr1-3

Mock IAA IAM Mock IAA IAM

Fig. S6. Effect of IAM on WT and axr1–3 seedlings. Seeds of WT and axr1–3 were germinated and grown for 5 days on MS agar media containing IAA (1 �M)or IAM (10 �M). (Scale bar, 4 mm.)

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IAA

TAM

TRP

N

COOH

N

COOH

NH2

N

NH2

N

N

S Glu

OSO3-

IGHTAMN

NHOH

YUC

SUR2

SUR1N

O

COOH

IPA

CYP71A13

IAM

AMI

Arabidopsis

IAAldN

CHON

NH2

O

CL

TAA1

N

NS

IAOxN

NOH

CYP79B2CYP79B3

IAMN

NH2

ONIT

N

C N

IAN

NIT

N

C N

IAN

Fig. S7. Alternative model for IAOx-dependent IAA biosynthesis in Arabidopsis. Dotted square indicates the IAOx metabolic pathway in Arabidopsis. Thealternative pathway for IAOx-dependent IAA biosynthesis is shown in the gray square. Cloned genes that potentially encode enzymes for IAA, CL, and IGbiosyntheses are shown in italics.

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