Supporting Information legends Figure S1. Characterisation of the size of the T-DNA insert, sequence of produced transcript in myb103-2, F5H expression and S- and G-lignin content in the F1 generation resulting from allelic complementation of the two myb103 mutants. a) Genotyping of WT, myb103-1 and myb103-2. The data shows that both mutants are homozygous for the T-DNA insert. 1 kb, 1 Kb Plus DNA Ladder (Invitrogen); the 1000 bp band is marked out. 1, primers LP1 and RP1; 2, primers LB-GABI and RP1; 3, primers LP2 and RP2; 4, primers LB2-SAIL and RP2 (see Table S2 for primer details and Figure S1f for primer positions). b) Amplification of genomic DNA from WT and myb103-2 using primers LP2 and RP2 (see Table S2 for primer details and Figure S1f for primer positions), which bind to genomic DNA flanking the T-DNA insert in myb103-2. A ca 3 kbp fragment could be amplified from myb103-2 when a prolonged extension time (5 min) was used. The fragment amplified in WT was about 1 kbps, indicating that the size of the T-DNA insert was about 2 kbps. c) 5’ RACE PCR of MYB103 transcripts from WT and myb103-2 T-DNA mutant. WT produced a fragment of the expected size, but no fragment could be produced from myb103-2, probably because the transcript also contained a large portion of the T-DNA and, therefore, was too large for amplification. When a primer binding to the T-DNA was used, a fragment larger than that from WT was amplified from myb103-2. This shows that myb103-2 produces a transcript that also contains a part of the T-DNA. 5, primers GR5’P and RGSP; 6, primers LB2-SAIL and RGSP (see Table S2 for primer details and Figure S1f for primer positions). d) Sequencing of the PCR product obtained from myb103-2. Genomic sequence of the locus for MYB103 (MYB103_At1g63910) was aligned with sequences from the myb103-2 fragment obtained in (c), amplified with primer LB2-SAIL (myb103-2_LB2-SAIL) and with primer RGSP (myb103-2_RGSP). The TDNA is inserted 14 bps upstream of the 3’ end of the promoter. A start codon was located in the T-DNA 201 bps upstream of the WT start codon, and several stop codons were located downstream of this new start codon. This sequence should not result in the production of MYB103. Stop codons were found in all three reading frames. Cyan, promoter; yellow, 5’ UTR; green, start codon; magenta, alternative start codon; red, stop codon. e) Allelic complementation of myb103-1 and myb103-2. Two independent (silique 1 and 2) reciprocal crosses between myb103-1 and myb103-2 were performed and genotyping of the F1 plants showed the presence of each mutant allele in the offspring. 1, primers LP1 and RP1; 2, primers LB-GABI and RP1; 3, primers LP2 and RP2; 4, primers LB2-SAIL and RP2 (see Table S2 for primer details and Figure S1f for primer positions). f) Indication of positions for primers used. g) qPCR analysis of FERULATE-5-HYDROXYLASE (F5H) expression in basal stem segments of WT, myb103-1, myb103-2, myb103-1/myb103-2 and myb1032/myb103-1 plants. The heterozygous F1 plants of the allelic complemented lines showed similarly reduced F5H levels as the homozygous parental lines. This strongly indicates that both myb103-1 and myb103-2 are null mutants in MYB103. Mean ± SD, n = 3 biological replicates, each consisting of mRNA derived from 3 pooled basal stem segments from plants derived from both siliques 1 and 2. ** = p < 0.01, *** = p < 0.001, Student’s t-test comparison with WT. h) Syringyl lignin units of basal stems from WT, myb103-1, myb103-2, myb1031/myb103-2 and myb103-2/myb103-1 plants were estimated with Py-GC/MS and expressed as % of total ion counts. Mean ± SD, n = 3 biological replicates, each consisting of 3 pooled basal stem segments from plants derived from both siliques 1 and 2. *** = p < 0.001, Student’s t-test comparison with WT. i) Guaiacyl lignin units of basal stems from WT, myb103-1, myb103-2, myb1031/myb103-2 and myb103-2/myb103-1 plants were estimated with Py-GC/MS and expressed as % of total ion counts. Mean ± SD, n = 3 biological replicates, each consisting of 3 pooled basal stem segments from plants derived from both siliques 1 and 2. * = p < 0.05, ** = p < 0.01, Student’s t-test comparison with WT. Figure S2. Plant morphology and stem anatomy of WT, myb103-1 and myb103-2. (a) 6-week-old plants. (b) 4-week-old plants. (c) Transverse section of basal stems from 35 cm high plants stained with safranin / alcian blue. Scale bars = 200 µm. Figure S3. Py-GC/MS analysis of basal stems from WT, myb103-1 and myb103-2 plants. OPLS-DA scores plot showing the separation between WT and mutants. Each symbol represents an individual plant. Model details: 1 predictive + 2 orthogonal components, R2Y(cum) = 0.99, Q2(cum) = 0.93. Figure S4. 2D NMR analysis of dissolved cell wall material from basal stems of WT, myb103-1 and myb103-2 with different cut-off values. The peaks shown in the loadings plot are the ones that contribute to separation of mutants from WT, but note that this only shows qualitative differences, and that the size of the peak in the loadings plot cannot be translated into quantitative differences. By increasing the cut-off filter, only the most important peaks that contribute to the model that separates the two samples are visualized. The intensity of a peak is determined both by the relative difference in peak area between the samples, and the within-sample variation for the actual peak (see experimental procedures). Black peaks have higher relative intensities in WT and green peaks have higher relative intensity in the myb103 samples. C, cellulose; G, guaiacyl lignin; M, mannose; Met, O-methyl (mainly from syringyl lignin); S, syringyl lignin; X, xylan. (a) Loadings plot of separating components for myb103-1 with a cut-off value of 0.5 showing, in addition to the S and G lignin peaks shown in Figure 2b, a decrease in cellulose and mannose and an increase in xylan. (b) Loadings plot of separating components for myb103-2 with a cut-off value of 0.5. In addition to the peaks observed for myb103-1, three peaks not assigned to any cell wall component appear for this line (marked with circles). (c) Loadings plot of separating components for myb103-2 with a cut-off value of 0.95. Figure S5. FT-IR microspectroscopic analysis of xylem cell types extracted from high spatial resolution, focal plane array images of stem cross-sections prepared from WT plants. OPLS-DA scores plots for pair-wise comparison between cell types. IF, interfascicular fibres; XF, xylem fibres; VE, vessel elements. Each symbol represents spectra from one cell. Spectra were collected from 3 independent plants and at least two different image positions in each section, with about 5 spectra from each image used in the analysis (10 spectra/plant). Model details: XF vs VE (a), 1 predictive + 2 orthogonal components, R2X(cum) = 0.82, R2Y(cum) = 0.85, Q2(cum) = 0.81; XF vs IF (b), 1 predictive + 2 orthogonal components, R2X(cum) = 0.83, R2Y(cum) = 0.87, Q2(cum) = 0.84; IF vs VE (c), 1 predictive + 6 orthogonal components, R2X(cum) = 0.92, R2Y(cum) = 0.97, Q2(cum) = 0.90. Figure S6. Levels of sinapoyl malate in basal stems of WT, myb103-1 and f5h1-4. Means SE, n = 8 biological replicates. *** = p < 0.001, Student’s t-test comparison with WT. Figure S7. Comparison of FERULATE-5-HYDROXYLASE (F5H) promoters from Arabidopsis thaliana (At) and Medicago truncatula (Mt), and their transactivation by AtSND1 and AtNST1. a) MYB1AT, a MYB binding promoter element, is present in six repeats on the Medicago MtF5H promoter (orange bands), which was previously used for assays of transactivation by Arabidopsis AtSND1 in Zhao et al. (2010b). The MYB1AT element is not present in the AtF5H1 promoter. Promoter sequences were analysed using the PLACE tool (Prestridge, 1991; Higo et al., 1999). b) Protoplast transactivation analysis of AtSND1 and AtNST1 (as effectors) acting on the AtF5H1 and MtF5H promoters (reporters). Reporter activities were normalized to the activities produced from a 35S-driven Renilla luciferase construct. The control value represents the reporter activity obtained in the absence of any effector, and has been set to 1. Mean SE, n = 8 replicates. *** = p < 0.001, Student’s t-test comparison between the corresponding TF and control on the same effector. Table S1. Comparison of Arabidopsis thaliana (At) and Medicago truncatula (Mt) FERULATE-5-HYDROXYLASE (F5H) promoter sequences. Cells in orange indicate the presence of MYB1AT and the number of repeats of this MYB binding promoter element. Promoter sequences were analysed using the PLACE tool (Prestridge, 1991; Higo et al., 1999). Table S2. Primers used for genotyping, 5’ RACE and sequencing of myb103 mutants. For insertion line myb103-1, LP1, RP1 and LB-GABI were combined, and for insertion line myb103-2, LP2, RP2, LB2-SAIL, GR5’P and RGSP were combined. Table S3. Primers used for qPCR analysis and cloning. Forward (FP) and reverse primers (RP) were used to amplify fragments from cDNA templates for qPCR, or cloning of open reading frames (ORFs) for over-expression (OE) of target genes. Methods S1. Supplementary information to materials and methods.