Fig. S1 Mass spectra analysis of XXT4 reaction products demonstrating xylosyltransferase activity towards cellohexaose substrate. Predominant peaks represent cellohexaose (6G), celloheptaose (7G) and cellooctaose (8G) substrates. Note that oligosaccharides in the assay reaction were partly acetylated (Ac) during purification and these present in separated peaks; 6G+Ac, 7G+Ac and 8G+Ac. Mono- and di-xylosylated cellohexaose (6G+X or 6G+X+Ac and 6G+2X or 6G+2X+Ac) masses corresponding to the reaction prodcuts are underlined. 6G 6G+Ac 7G 7G+Ac 8G 8G+Ac 6G+X 6G+X+Ac 6G+2X6G+2X+Ac % Intensity Mass (m/z)

Fig. S2 Illustration of T-DNA insertion positions in the xxt mutants used in this study. Long black bars represent exons, and thin lines within the genes represent introns. Thin lines in front represent mRNA 5’ untranslated regions. The arrows on triangles indicate the direction of the left border primer of inserted T-DNAs. Black arrows on gene structures indicate primer positions for allele identifications, with primer names respected to each mutant allele. Dashed block arrows indicate primer positions for RT-PCR amplification. Primer sequences can be found in Tables S4 and S5. 3’ 5’ xxt1-2 5’ xxt2-1 5’ xxt3 XXT1 XXT2 XXT3 5’ 3’ xxt4 5’ xxt5 XXT4 XXT5 xxt2-2 xxt F 1-2F 1-3R 1-2R 2-1F 2-2F 2-2R2-1R 5F 5R 3F 3R 4F 4R

GABI_142A08 (xxt1-3) SALK_ (xxt2-2) SALK_ (xxt2-1) SALK_ (xxt1-2) M WT M WT cDNA WTgDNA WT cDNA xxt1-2 cDNA WTgDNA WT cDNA xxt1-3 cDNA WTgDNA WT cDNA xxt2-1 cDNA WTgDNA WT cDNA xxt2-2 GABI_411G05 (xxt5) SALK_ (xxt3) SALK_ (xxt4) M WT M WT M WT cDNA WTgDNA WT cDNA xxt3 cDNA WT gDNA WT cDNA xxt4 cDNA WTgDNA WT cDNA xxt5 a b cd e f g M WT Genotyping RT-PCR Genotyping RT-PCR xxt5xxt3xxt4 WT 18s rRNA control WT xxt1-2 18s rRNA control xxt2-1 xxt1-3 xxt2-2 h

Fig. S3 Identification of homozygous T-DNA insertion lines of the Arabidopsis GT34s by PCR amplification. PCR identification of each line is displayed as upper and lower panels. The upper panel is amplifications in an order of wild type (wt) and mutant line (M). Homozygosity of the mutant indicated by the absence of PCR product from gene specific primer, and the presence of PCR products from left border and reverse primers. The lower panels show the disruption of gene expression in the homozygous T-DNA lines by RT-PCR. Gels in a-g show the genomic DNA amplification of the line indicated below where a homozygous T-DNA line (M) is compared to the wild type (WT) using gene specific primers in lanes 1 and 2, and left border with reverse primers in lanes 3 and 4. RT-PCR results from each line are presented in the following order: cDNA of homozygous T-DNA lines (lane 1), WT cDNA (lane 2) and WT genomic DNA (lane 3). Amplification of 18s rRNA from cDNAs from each of the insertion lines was used as a control to indicate the quality of the RNA samples (h).

xxt2-1 xxt4 M WT M WT xxt2-1 xxt5 M WT M WT 2-1F + 2-1R 2-1R + LBb1 5F + 5R 5R + GABI left border 4F + 4R 4R + LBb1 2-1F + 2-1R 2-1R + LBb1 xxt4 xxt5 xxt1-2 xxt5 1-2F + 1-2R 1-2R + LBb1 4R + LBb14F + 4R 5R + GABI left border 5F + 5R 5R + GABI left border 5F + 5R Fig. S4 Identification of double knock-out lines. Homozygosity is indicated by the absence of PCR products amplified using gene specific primers and the presence of PCR products using border and reverse primers. M indicates insertion double mutant gDNA and WT indicates wild type gDNA.