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1. Article title: Deficiency in riboflavin biosynthesis affects tetrapyrrole biosynthesis in etiolated Arabidopsis tissue Journal: Plant Molecular Biology Authors:Boris Hedtke, Ali Alawady, Alfonso Albacete, Koichi Kobayashi, Michael Melzer, Thomas Roitsch, Tatsuru Masuda, Bernhard Grimm • Corresponding author: • Bernhard GrimmHumboldt University, Institute of Biology/Plant Physiology, Philippstr.13, Building 12, 10115 Berlin, Germany Email: bernhard.grimm@rz.hu-berlin.de

2. A: B: Supplemental Figure S1: Partial alignments of Arabidopsis RibA genes with A: GTP cyclohydrolase II from E. coli (EC_GCHII, acc. nr. BAA 14831, 196 aa) and B:3,4-dihydroxy-2-butanone-4-phosphate synthase from Methanocaldococcus jannaschii (MJ_DHBPS, NP 247019, 227 aa). Amino acids identical in all 4 sequences are shaded, residue numbers are given at the right margin. Herz et al. (2000) demonstrated both enzymatic activities for recombinant AtRIBA1 protein. Important residues and domains for both enzymes have been identified previously (highlighted in yellow, Fischer et al., 2002; Kaiser et al., 2002; Steinbacher et al., 2003; Ren et al., 2005). Substitutions and deletions in the catalytic or binding domains of RIBA2 and RIBA3 (marked in red) allow the prediction that only RIBA1 and RIBA3 are able to catalyse the GTP cyclohydrolase II reaction, while only RIBA1 and RIBA2 function as 3,4-dihydroxy-2-butanone-4-phosphate synthase. A similar difference in enzyme activity was found between the Bacillus subtilisRibD encoded dual-functional protein with pyrimidine deaminase and reductase activity and the homologous A. thaliana gene At4g20960, which encodes a protein of similar size with deamination reaction only (Fischer et al., 2004).

3. Supplemental Table S1: Differentially expressed genes showing at least two-fold reduction in rfd1 compared to wild type. All candidate genes show a logged expression coefficient (coef.) smaller than -1 and a probability (p-) value smaller or equal 0.01. Gene names (AGI ID) numbers and and a description based on the most recent TAIR annotation are given. LEA proteins are numbered according to Hundertmark and Hincha (2007, numbers in italics)

4. Supplemental Table S1, continued

5. Supplemental Table S2: Differential expression of tetrapyrrole biosynthesis genes in etiolated rfd1 seedlings. The expression coefficient (coef.) indicates logged expression differences in rfd1 compared to wild type. Genes with an coefficient > 0.3 are given in bold.

6. Supplemental Table S3: Chloroplast encoded genes accumulating more than two-fold in etiolated rfd1 seedlings. Selection criteria are, as described for Table 1, a logged expression coefficient bigger than 1 and a p- value smaller 0.01.

7. Supplemental Table S4: Oligonucleotides used in semi-quantitative and quantitative RT-PCR experiments. Supplemental References: Fischer M, Romisch W, Schiffmann S, Kelly M, Oschkinat H, Steinbacher S, Huber R, Eisenreich W, Richter G, Bacher A (2002) Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii. J Biol Chem 277 :41410-41416 Hundertmark M and Hincha DK (2008) LEA (late embryogenesis abundant) proteins and their encoding genes in Arabidopsis thaliana. BMC Genomics, 9, 118-141. Kaiser J, Schramek N, Eberhardt S, Püttmer S, Schuster M and Bacher A (2002) Biosynthesis of vitamin B2. Eur J Biochem 269:5264-70 Ren J, Kotaka M, Lockyer M, Lamb HK, Hawkins AR, Stammers DK (2005) GTP cyclohydrolase II structure and mechanism. J Biol Chem 280:36912-36919 Steinbacher S, Schiffmann S, Richter G, Huber R, Bacher A, Fischer M (2003) Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism. J Biol Chem 278:42256-42265