Supporting InformationChen et al. 10.1073/pnas.1120992109SI Materials and MethodsProcessing of Plant Material. Mature vanilla beans were separatedinto the seed and pod (residue left after seed isolation), and werecut into 2-cm sections and soaked with chloroform:methanol (2:1vol/vol) overnight to facilitate the isolation of seeds. Solubleextractives were removed by three successive extractions withchloroform:methanol (2:1 vol/vol), methanol, and finally water,all at room temperature, and the samples were freeze-dried andthen ground to powder with a freezer mill (SPEX SamplePrep)under liquid nitrogen.

Preparation of Vanilla planifolia Whole-Tissue Samples for NMR.Preparation of vanilla tissue NMR samples was via methodslargely described previously (1, 2). Briefly, isolated vanilla seedcoat, pod (residue remaining after seed coat isolation) and stemwere pre-extracted with 80% aqueous ethanol (sonication, 30min, three times). Pre-extracted vanilla tissues (2.5 g) were ball-milled (41 × 5 min, 5-min cooling cycle) by using a Retsch PM100ball-mill vibrating at 600 rpm with ZrO2 vessels containing ZrO2ball bearings. For gel-state NMR experiments (2), ∼50 mg of theball-milled vanilla materials were directly transferred into 5-mmNMR tubes and were swelled in 600 μL of DMSO-d6/pyridine-d5(4:1, vol/vol). For solution-state NMR of acetylated whole seedcoat (1), the ball-milled seed coat (92 mg) was dissolved inDMSO/N-methylimidazole (NMI) (2:1, vol/vol, 3 mL) at roomtemperature, acetic anhydride (1 mL) was added, and then stirredfor 2 h at room temperature. The mixture was poured into dis-tilled water (1,000 mL). The resultant precipitate was recoveredby filtration, washed with ultrapure water (1,000 mL) and thenlyophilized to yield acetylated whole seed coat (120 mg).

Isolations of Soluble Lignins from V. planifolia Tissues. The ball-milled vanilla seed coat and stem (2.4 g) were placed in centrifugetubes and digested at 30 °C with crude cellulases (Cellulysin;Calbiochem; lot no. D00074989; 30 mg/g of sample, in pH 5.0acetate buffer; three times over 2 d; fresh buffer and enzymeadded each time), leaving all of the phenolic polymers and re-sidual polysaccharides totaling 2.00 g (83%, seed coat), 805 mg(34%, pod), and 563 mg (24%, stem) (3). The cellulase-treatedvanilla seed coat (1.00 g) and stem (300 mg) were suspended indioxane-water (96:4, vol/vol; 50 mL/g) and stirred at 30 °C for 6 h(4, 5). The mixture was centrifuged (10,000 × g, 15 min) and thesupernatant was collected. These operations were repeated threetimes. The combined supernatant was concentrated to about5 mL with a rotary evaporator and then precipitated in 200 mLof 0.01 M aqueous HCl. The precipitated phenolic polymerswere recovered by centrifugation, reprecipitated into diethylether (100 mL) from methanol-dichloromethane (1:4, vol/vol,3 mL), and recovered by centrifugation to yield purified dioxane/water-soluble lignins (157.3 mg, 20% from cellulose-treated tis-sues, seed coat; 67.8 mg, 23%, stem). The cellulase-treated seedcoat (200 mg) was also extracted with DMSO in the samemanner described for the extraction with dioxane-water, yieldinga DMSO-soluble seed coat lignin (58.8 mg, 29% from the cel-lulose-treated seed coat). Acetylation of the isolated lignins wasvia acetic anhydride and pyridine: 10–50 mg of isolated ligninswere dissolved in acetic anhydride/pyridine (1:1, vol/vol, 2 mL).After stirring at room temperature overnight, the mixture waspoured into distilled water (200 mL). The resultant precipitatewas recovered by filtration, washed with ultrapure water (200mL) and then lyophilized to yield acetylated phenolic polymers(weight yield typically 115–122%).

Synthetic Model Dimers. Compound 1 (Fig. S4), a benzodioxanedehydrodimer of caffeyl alcohol, was synthesized from radicalcoupling reactions of caffeyl alcohol via silver carbonate(Ag2CO3) oxidation (6, 7): caffeyl alcohol (2.1 g, 0.0125 mol) wasdissolved in acetone-toluene (30 mL, 1:2, vol/vol) and Ag2CO3(4.1 g, 0.015 mol) was added at room temperature. After stirringat room temperature for 12 h, the solid was filtered off and theorganic solvents were evaporated under reduced pressure to givea solid residue. Purification by silica-gel chromatography yieldedcompound 1 as a colorless solid, 1.3 g, 64% yield. This productwas a mixture of cis- and trans-isomers of compound 1 (cis-1:trans-1, 5:95, by 1H-NMR). trans-1: NMR (DMSO-d6): δH =3.28–3.34 (1H, m, A′γ), 3.47–3.54 (1H, m, A′γ), 3.99–4.04 (1H,m, A′β), 4.06 (2H, t, J = 4.70 Hz, B′γ), 4.80 (1H, d, J = 7.80 Hz,A′α), 6.20 (1H, dt, J = 15.90 and 4.70 Hz, B′β), 6.41 (1H, d, J =15.90 Hz, B′α), 6.9 (1H, br. d, J = 8.10 Hz, A′6), 6.74 (1H, br. d,J = 8.10 Hz, A′5), 6.79 (1H, s, A′2), 6.86 (br. d, J = 8.30 Hz, B′5),6.92 (br. d, J = 8.45 Hz, B′6), 6.95 (1H, s, B′2); δC = 60.19 (A′γ),61.60 (B′γ), 75.66 (A′α), 78.32 (A′β), 114.25 (B′2), 114.94 (A′2),115.48 (A′5), 116.78 (B′5), 118.88 (A′6), 119.41 (B′6), 127.59 (A′1), 128.14 (B′α), 128.84 (B′β), 130.33 (B′1), 142.70 (B′4), 143.65(B′3), 145.25 (A′3), 145.80 (A′4). cis-1: NMR (DMSO-d6): δH =4.32 (m, Hβ), 5.17 (d, J = 2.35 Hz, A′α); δC = 58.52 (A′γ), 75.09(A′α), 77.31 (A′β). HR-MS (ESI) calculated for C18H18O6[M+]: 330.1098; found: 330.1096.Compound 2 (Fig. S4) was synthesized by methylation of

compound 1 with methyl iodide (MeI) and potassium carbonate(K2CO3): compound 1 (590 mg, 0.0018 mol) was dissolved inacetone (20 mL), MeI (1 mL, 0.018 mol) and K2CO3 (740 mg,0.0054 mol) was added and then refluxed for 2 h. After cooling toroom temperature, the solid was filtered off and the organic sol-vents were evaporated under reduced pressure to give an oil,which was purified by silica-gel chromatography to yield com-pound 2 as a colorless solid, 1.3 g, 64% yield. trans-2: NMR(DMSO-d6): δH = 3.28–3.33 (1H, m, Aγ), 3.49–3.76 (1H, m, Aγ),3.75 (3H, s, OMe), 3.76 (3H, s, OMe), 4.06 (2H, t, J = 4.47 Hz,Bγ), 4.15–4.18 (1H, m, Aβ), 4.93 (1H, d, J = 7.85 Hz, Aα), 6.21(1H, dt, J=15.95 and 4.48Hz, Bβ), 6.42 (1H, d, J=15.95Hz, Bα),6.88 (1H, br. d, J=8.25Hz, B5), 6.93 (1H, dd, J=8.25 and 1.85Hz,B5), 6.94–6.99 (3H, overlapped, A5, A6, and B2), 7.03 (1H, s, A2).δC = 55.50, 55.54 (OMe), 60.14 (Aγ), 61.58 (Bγ), 75.68 (Aα),78.01 (Aβ), 111.08 (A2), 111.48 (A5), 114.28 (B2), 116.79 (B5),119.47 (B6), 120.27 (A6), 128.09 (Bα), 128.86 (Bβ), 129.04 (A1),130.34 (B1), 142.71 (B4), 143.56 (B3), 148.71 (A3), 149.05 (A4).cis-2: NMR (DMSO-d6): δH = 4.44 (m, Aβ), 5.29 (d, J= 2.60 Hz,Aα); δC = 58.20 (Aγ), 74.92 (Aα), 77.20 (Cβ). HR-MS (ESI)calculated for C20H22O6 [M+]: 358.1411; found: 358.1421.Acetylations of compounds 1 and 2 were via acetic anhydride

and pyridine, yielding acetylated compounds 1-Ac and 2-Ac (Fig.S4), respectively. Compound 1-Ac: trans-1-Ac: NMR (chloro-form-d): δH = 2.05, 2.09, 2.30, and 2.31 (12H, s, OAc), 3.99 (1H,dd, J = 12.35 and 4.20 Hz, A′γ), 4.18–4.23 (1H, m, A′β), 4.38(1H, dd, J = 12.35 and 3.55 Hz, A′γ), 4.69 (2H, d, J = 6.50 Hz,B′γ), 4.97 (1H, d, J = 7.80 Hz, A′α), 6.14 (1H, dt, J = 15.85 and6.50 Hz, A′β), 6.55 (1H, d, J = 15.85 Hz, A′α), 6.91 (1H, br. d,J = 8.30 Hz, B′5), 6.95 (1H, dd, J = 8.35 and 1.80 Hz, B′6), 7.00(1H, d, J = 1.80 Hz, B′2), 7.23–7.30 (overlapped, A′2, A′5, andA′6). 13C NMR (chloroform-d): δ= 20.64 and 20.66 (OAc), 62.48(A′γ), 65.12 (B′γ), 75.39 (A′β), 75.68 (A′α), 115.09 (B′6), 117.24(B′5), 120.55 (B′2), 121.84 (B′β), 122.53 (A′2), 123.91 (A′5),125.36 (A′6), 130.35 (B′1), 133.56 (B′α), 134.51 (A′1), 142.43and 142.64 (A′3 and A′4), 142.79 (B′4), 143.16 (B′3), 167.93,

Chen et al. www.pnas.org/cgi/content/short/1120992109 1 of 8

167.99, 170.42, and 170.87 (OAc). cis-1-Ac: NMR (chloroform-d): δH = 5.28 (d, J = 2.50 Hz, Hα); δC = 60.18 (A′γ), 74.10(A′β), 74.57 (A′α). HR-MS (ESI) calculated for C26H26NaO10[(M+Na)+]: 5212.1419; found: 521.1422. Compound 2-Ac: trans-2-Ac: NMR (chloroform-d): δH = 2.09 and 2.10 (6H, s, OAc), 3.90and 3.91 (6H, s, OMe), 3.96 (1H, dd, J = 11.90 and 4.43 Hz, Aγ),4.24–4.28 (1H, m, Aβ), 4.38 (1H, dd, J = 12.10 and 2.83 Hz, Aγ),4.70 (2H, d, J = 6.55 Hz, Bγ), 4.90 (1H, d, J = 7.80 Hz, Aα), 6.13(1H, dt, J = 15.85 and 6.58 Hz, Aβ), 6.56 (1H, d, J = 15.85 Hz,Aα), 6.88–6.98 (5H, overlapped, A2, A5, A6, B5, and B6), 7.03(1H, d, J = 1.60 Hz, B6). 13C NMR (chloroform-d): δ = 20.72,21.01 (OAc), 55.91 and 55.93 (OMe), 62.97 (Aγ), 65.14 (Bγ),75.69 (Aβ), 76.32 (Aα), 109.73 (A2), 11.18 (A5), 115.09 (B6),117.17 (B5), 120.10 (A6), 120.41 (B2), 121.69 (Bβ), 128.03 (A1),130.17 (B1), 133.65 (Bα), 143.00 (B4), 143.57 (B3), 149.42 (A3),149.78 (A4), 170.48, and 170.84 (OAc). cis-2-Ac: NMR (chlo-roform-d): δH = 4.67 (m, Aβ), 5.25 (d, J = 2.70 Hz, Aα); δC =60.69 (Aγ), 74.62 (Aβ), 75.19 (Aα). HR-MS (ESI) calculated forC24H26O8 [M+]: 442.1623; found: 442.1635.

Dehydrogenation Polymer from Caffeyl Alcohol. A dehydrogenationpolymer (C-DHP) from caffeyl alcohol was generated via HRP-catalyzed polymerization as previously described (3, 8, 9): caffeylalcohol (166 mg, 1 mmol) in 240 mL of acetone/sodium phos-phate buffer (0.1 M, pH 6.5) (1:9, vol/vol) and a separate solu-tion of hydrogen peroxide (1.2 mmol) in 240 mL of water wereadded by peristaltic pump over a 20-h period at 25 °C to 60 mLof buffer containing HRP (5 mg). The reaction mixture wasfurther stirred for 4 h and then the precipitate was collected bycentrifugation (10,000 × g, 15 min), washed with ultrapure water(100 mL × 3), and lyophilized to afford C-DHP as a brownishpowder (97.4 mg, 59%). Acetylation of C-DHP was via aceticanhydride and pyridine, yielding acetylated C-DHP.

Chemical Analyses. Klason lignin analysis was according to the lit-erature method (10). The distribution of amorphous sugars(hemicelluloses and pectins) and crystalline glucan (cellulose) wascalculated by treating the plant material with a weak acid, tri-fluoroacetic acid, and analyzing the amorphous sugars, as alditolacetates, by gas chromatography (GC)-MS with inositol as internalstandard (11). The residue was washed with theUpdegraff reagent(12), stripped of further hemicelluloses and amorphous glucan,totally hydrolyzed with sulfuric acid (13), and glucose quantified bythe anthrone assay. Crude protein content was determined fromthe N content (Kjeldahl method) using a 6.25 factor (14). Proan-thocyanidins in vanilla seeds weremeasured using the butanol-HClassay described previously (15). Thioacidolysis analysis was ac-cording to the method described previously (16, 17). Briefly, ∼30mg of extractive-free samples were reacted with 3 mL of 0.2 MBF3-etherate in an 8.75:1 dioxane/ethanethiol mixture at 100 °Cfor 4 h.After adding 4mLof water, themixture was extracted using4 mL of dichloromethane (three times). The organic layer wasseparated and dried under a stream of nitrogen. After de-rivatization with BSTFA + 1% TMCS (Pierce Biotechnology),lignin-derived monomers were identified by GC-MS and quanti-fied by GC as their trimethylsilyl derivatives. GC-MS was per-formed on a Hewlett-Packard 5890 series II gas chromatographwith a 5971 series mass-selective detector (column: HP-1; 60 m ×0.25 mm; 0.25-μm film thickness), and mass spectra were recordedin electron impactmode (70 eV) with a 50–650m/z scanning range.

NMR Spectroscopy. The NMR spectra were acquired on a BrukerBiospin AVANCE 500 MHz spectrometer fitted with a cryogeni-cally cooled 5-mm TCI gradient probe with inverse geometry(proton coils closest to the sample) and spectral processing usedBruker’s Topspin 3.1 (Mac) software. ForNMRexperiments, ball-

milledwhole vanilla tissueswere swelled inDMSO-d6/Pyridine-d5,unacetylated samples of isolated lignins and C-DHP were dis-solved in DMSO-d6, and acetylated samples of isolated lignins, C-DHP and ball-milled whole vanilla seed coat were dissolved inchloroform-d; the central solvent peaks were used as internalreference (δC/δH: DMSO, 39.5/2.49; chloroform, 77.0/7.26 ppm).Standard Bruker implementations of the traditional suite of one-dimensional and 2D [gradient-selected, 1H-detected; for example,correlation spectroscopy (COSY), 13C–1H correlation (HSQC),heteronuclear multiple bond correlation (HMBC)] NMR ex-periments were used for structural elucidation and assignmentauthentication for monomer and dimers. Adiabatic 2D-HSQC(“hsqcetgpsisp2.2”) experiments for ball-milled vanilla tissues ina gel-state (2), and acetylated ball-milled seed coat, isolated lig-nins and DHP samples in a solution-state (3, 8), were carried outusing the parameters described previously. Processing used typicalmatched Gaussian apodization in F2 (LB = −0.1, GB = 0.001)and squared cosine-bell and one level of linear prediction (32coefficients) in F1. For quantification of aromatic distributions,only the carbon-2 correlations fromG and C units and the carbon-2/6 correlation from S units were used, and the S integrals werelogically halved; linear prediction was not used for spectra beingintegrated. For an estimation of the various interunit linkagetypes, the well-resolved Cα-Hα contours (Iα, IIα, IIIα, IVα, and Vα)(Fig. 2) were integrated; no correction factors were used (3).HMBC experiments (“hmbcgplpndqf”) for isolated lignins andDHP samples had the following parameters. Spectra were ac-quired from 10 to 0 ppm in F2 (1H) using 4-k datapoints (acqui-sition time 410 ms), 200–0 ppm in F1 (13C) with 400 increments(F1 acquisition time 8.0 ms) of 64 scans with a 1-s interscan delayand a 80-ms long-range coupling delay. Processing to a finalmatrixof 2 k by 1-k datapoints used typical matched Gaussian apodiza-tion in F2 (LB, −30; GB, 0.122), squared sine-bell in F1, and onelevel of linear prediction in F1 (32 coefficients).

Gel Permeation Chromatography.Gel permeation chromatography(GPC) was performed on a Shimadzu LC-20A LC system (Shi-madzu) equipped with a photodiode array (PDA) detector (SPD-M20A; Shimadzu) using the following conditions: column: TSKgel α-M + α-2500 (Tosoh); eluent: dimethylformamide con-taining 0.1 M lithium bromide; flow rate: 0.5 mL min−1; columnoven temperature: 40 °C; sample detection: PDA response at280 nm. The molecular weight calibration was via polystyrenestandards. The data acquisition and computation used LCsolu-tion version 1.25 software (Shimadzu).

Chiral HPLCy. Analytical and preparative chiral HPLC for enan-tiomeric separation of benzodioxane dimer 1 was performed ona Shimadzu LC-20A LC system equipped with a PDA detector(SPD-M20A; Shimadzu) and a fraction collector (FRC-10A;Shimadzu) using the following conditions: column: Lux Cellu-lose-1 (Phenomenex); eluent: hexane/isopropanol/formic acid(82.5/17.5/0.2, vol/vol/v); flow rate: 2 mL min−1; column oventemperature: 40 °C; sample detection: PDA response at 280 nm.The data acquisition and computation used LCsolution version1.25 software (Shimadzu).

CD Spectroscopy. CD spectra were run on an Model 202SF CDspectrophotometer (Aviv Biomedical) operated by IGOR-Prosoftware (Wavemetrics). All of the samples were dissolved in90:10 acetonitrile/water at a concentration of 50 μg/mL andplaced in a 1-cm quartz cuvettete holding a sample volume of300 μL. Spectra were obtained at 25 ± 0.1 °C using 2-s averagingin 1-nm steps over the range of 235–350 nm, baseline-corrected(using a scan of the blank solvent), and smoothed (splinesmoothing function; SF, 1; SD, 0.2).

Chen et al. www.pnas.org/cgi/content/short/1120992109 2 of 8

1. Lu F, Ralph J (2003) Non-degradative dissolution and acetylation of ball-milled plantcell walls: High-resolution solution-state NMR. Plant J 35:535–544.

2. Kim H, Ralph J (2010) Solution-state 2D NMR of ball-milled plant cell wall gels inDMSO-d(6)/pyridine-d(5). Org Biomol Chem 8:576–591.

3. Wagner A, et al. (2011) CCoAOMT suppression modifies lignin composition in Pinusradiata. Plant J 67:119–129.

4. Ralph J, et al. (2006) Effects of coumarate 3-hydroxylase down-regulation on ligninstructure. J Biol Chem 281:8843–8853.

5. Stewart JJ, Akiyama T, Chapple CCS, Ralph J,Mansfield SD (2009) The effects on lignin struc-ture of overexpression of ferulate 5-hydroxylase in hybrid poplar. Plant Physiol 150:621–635.

6. Ralph J, et al. (2001) NMR evidence for benzodioxane structures resulting fromincorporation of 5-hydroxyconiferyl alcohol into Lignins of O-methyltransferase-deficient poplars. J Agric Food Chem 49:86–91.

7. Lu F, et al. (2010) Sequencing around 5-hydroxyconiferyl alcohol-derived units incaffeic acid O-methyltransferase-deficient poplar lignins. Plant Physiol 153:569–579.

8. Tobimatsu Y, Davidson CL, Grabber JH, Ralph J (2011) Fluorescence-tagged monolignols:Synthesis, and application to studying in vitro lignification. Biomacromolecules 12:1752–1761.

9. Tobimatsu Y, Takano T, Kamitakahara H, Nakatsubo F (2008) Studies on the dehydroge-native polymerizations of monolignol β-glycosides. Part 3: Horseradish peroxidase-catalyzedpolymerizationsof triandrinand isosyringin. JWoodChemTechnol28(2):69–83.

10. Hatfield RD, Jung HG, Ralph J, Buxton DR, Weimer PJ (1994) A comparison of theinsoluble residues produced by the Klason lignin and acid detergent ligninprocedures. J Sci Food Agric 65(1):51–58.

11. Albersheim P, Nevins DJ, English PD, Karr A (1967) A method for the analysis of sugarsin plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydr Res 5:340–345.

12. Updegraff DM (1969) Semimicro determination of cellulose in biological materials.Anal Biochem 32:420–424.

13. Selvendran RR, O’Neill MA (1987) Isolation and analysis of cell walls from plantmaterial. Methods Biochem Anal 32:25–153.

14. Darvill A, McNeil M, Albersheim P, Delmer D (1980) The primary cell wall of floweringplants. The Biochemistry of Plants, ed Tolbert NE (Academic Press, New York), Vol 1,pp 91–162.

15. Peel GJ, Pang Y, Modolo LV, Dixon RA (2009) The LAP1 MYB transcription factororchestrates anthocyanidin biosynthesis and glycosylation in Medicago. Plant J 59:136–149.

16. Lapierre C, Monties B, Rolando C (1985) Thioacidolysis of lignin: Comparison withacidolysis. J Wood Chem Technol 5:277–292.

17. Lapierre C, Monties B, Rolando C (1986) Thioacidolysis of poplar lignins: identificationof monomeric syringyl products and characterization of guaiacyl-syringyl ligninfractions. Holzforschung 40(2):113–118.

OHOH

OHOH

OH

OHOMe

OH

OHOH

OHOH

OMe

OH

MeO

MeO

OO

O OOMe

OO

OOMe

MeO

MeO

MH MG MS

MC M5-OH-G

H G S

C 5-OH-G

PH PG PS

PC P5-OH-G

Fig. S1. A schematic version of the monolignol pathway, showing the successive hydroxylation and O-methylation reactions. Primary lignin monomers, themonolignols, p-coumaryl (MH), coniferyl (MG), and sinapyl (MS) alcohols, are synthesized by a pathway that involves sequential aromatic ring hydroxylation andO-methylation. The final monolignols undergo radical coupling reactions during lignification resulting in generic lignin polymer units, p-hydroxyphenyl (H),guaiacyl (G), and syringyl (S) units, respectively. Lignification with hydroxycinnamyl alcohols that may be regarded as unconventional (incompletely methyl-ated) monolignols, caffeyl (MC) and 5-hydroxyconiferyl (M5-OH-G) alcohols, generating novel catechyl (C) and 5-hydroxy-guaiacyl (5-OH-G) lignin units, has beenidentified in transgenic plants in which genes encoding O-methyltransferases in the monolignol biosynthetic pathway are substantially down-regulated.

Chen et al. www.pnas.org/cgi/content/short/1120992109 3 of 8

A B

6 we

eks

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eeks

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eeks

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ease

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ease

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ioac

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ysis

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omer

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ol /g

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R)

Fig. S2. (A and B) Time-course study for C-lignin accumulation during V. planifolia seed development. Flowers were hand pollinated, and immediately taggedwith the time and date of pollination. Developing pods were then harvested at the times indicated, and seed coats dissected and analyzed by thioacidolysis.

ppm13C

1H5.5

95

5.0 4.56.0

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100

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95

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(1 4)- -D-Manp

podseed coat stem

-L-Araf

2-O-Ac- -D-Xylp

(1 4)- -D-Xylp

3-O-Ac- -D-Xylp

(1 4)- -D-Glcp

(1 6)- -D-Glcp

(1 4)- -D-Galp

-D-Xylp (R) +-D-Glclp (R)

-D-Xylp (R) +-D-Glclp (R)

4-O-Me-Glucuronate

2,3-di-O-Ac- -D-Xylp

-L-Araf

2-O-Ac- -D-Xylp

(1 4)- -D-Xylp

3-O-Ac- -D-Xylp

(1 4)- -D-Glcp

(1 6)- -D-Glcp

(1 4)- -D-Galp

???

-D-Xylp (R) +-D-Glclp (R)

4-O-Me-Glucuronate

2,3-di-O-Ac- -D-Xylp

(1 4)- -D-Manp

(1 4)- -D-Glcp

-L-Araf

-D-Glclp (R)

-D-Glclp (R)

???

B A C

Fig. S3. (A–C) Partial gel-state short-range 13C–1H (HSQC) spectra of vanilla tissues: polysaccharide anomeric regions. Tentative assignments were according toliterature data (1–3).

1. Kim H, Ralph J (2010) Solution-state 2D NMR of ball-milled plant cell wall gels in DMSO-d(6)/pyridine-d(5). Org Biomol Chem 8:576–591.2. Jensen JK, et al. (2011) The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis. Plant J 66:387–400.3. Rencoret J, et al. (2011) Lignin composition and structure in young versus adult Eucalyptus globulus plants. Plant Physiol 155:667–682.

Chen et al. www.pnas.org/cgi/content/short/1120992109 4 of 8

Fig. S4. Partial short-range (HSQC) and long-range (HMBC) 13C–1H spectra of (A) benzodioxane dimers and (B) an in vitro polymer (C-DHP) synthesized fromcaffeyl alcohol, and (C and D) their acetylated samples.

Chen et al. www.pnas.org/cgi/content/short/1120992109 5 of 8

O

OH

OH

OHO

7.0 6.0 5.0 4.08.0not assigned herecarbohydrates,

solvent, etc.

Vα

C2, C5, C6

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Vbenzodioxane

( )

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OO

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26

OO

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5

26

OHOH

catechyl units(non-etherified)

Pcellulose

C

C

Fig. S5. Partial short-range (HSQC) 13C–1H spectra of acetylated whole V. planifolia seed coat.

}

A seed coat lignin B stem lignin

6.07.0 5.08.0 4.0

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S2/6

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III

III

I

I

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II

III III

III

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X

IIIresinol( )

O

O

IIphenylcoumaran

( -5)

OHO

5

I-aryl ether( -O-4)

OHO

HO

OMe

not assignedcarbohydrates,solvents, etc.

Xcinnamyl alcohol

end-units

OH

methoxyl

Vttrans-

benzodioxane

4

OO

HO

Vccis-

benzodioxane

4

OO

HO4

Vbenzodioxane

( -O-4)

4

OO

HO

5

26

OO

catechyl units

OMeO

5

26

guaiacyl units

OMeO

26

OMe

syringyl units

G S C

Fig. S6. Partial short-range (HSQC) 13C–1H spectra of isolated lignins (extracted with dioxane-water, 96:4, vol/vol) from V. planifolia (A) seed coat and (B) stem.

Chen et al. www.pnas.org/cgi/content/short/1120992109 6 of 8

Fig. S7. Partial long-range (HMBC) 13C–1H spectra of acetylated lignins isolated from V. planifolia (A) seed coat and (B) stem.

Table S1. Chemical composition of isolated V. planifolia tissues

Fraction Seed coat Pod Stem

Klason lignin (mg/g) 817 166 129DMSO-extracted lignin (mg/g) 242 n.d. n.d.Dioxane/water-extracted lignin (mg/g) 166 n.d. 55Crystalline glucan, cellulose (mg/g) 164 361 426Amorphous neutral sugars (mg/g) 15 142 165Rhamnose (%) 2.4 4.4 2.0Arabinose (%) 7.7 17.9 3.4Xylose (%) 8.7 35.5 76.9Mannose (%) 2.7 5.3 1.1Galactose (%) 27.8 18.1 4.2Glucose (%) 49.5 16.5 12.1Crude proteins (mg/g) 21 88 111

n.d., not determined.

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Table S2. Lignin properties from V. planifolia tissues

Samples Mn* Mw* Mw/Mn* DPn†

Whole seed coat‡ 6060 22000 3.63 29.4Seed coat lignin§ 3770 14100 3.74 18.3Seed coat lignin¶ 2700 7840 2.90 13.1Stem lignin¶ 5990 24500 4.09 20.7C-DHP 1670 4330 2.59 8.1

GPC-derived number-average (Mn) and weight-average (Mw) molecularweights, polydispersity index (Mw/Mn), and number-average degree of po-lymerization (DPn) data of acetylated samples of V. planifolia whole seedcoat, isolated seed coat lignins, and in vitro synthetic lignin from caffeylalcohol (C-DHP).*Molecular-weight calibration was via polystyrene standards.†Calculated based on NMR-derived aromatic composition ratio (Fig. 3) andthe molecular weights of guaiacyl β-aryl ether, syringyl β-aryl ether, andcatechyl benzodioxane intermonomeric structures.‡Whole seed-coat materials, ball-milled and acetylated via DMSO-N-methyl-imidazole method (see main text).§Isolated lignins extracted with DMSO.¶Isolated lignins extracted with dioxane-water (96:4, vol/vol).

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