Col-0 SAIL Col-0 SAIL L Col-0 SAIL

A Met Met At5g470 00 bp Primer for genotyping T-DNA insertion position SAIL_57_G Coding sequence UTR B kb DNA ladder Primers: Col-0 SAIL kb DNA ladder Primers: - Col-0 SAIL.5.5 0.5 0.5 C kb Actin- Full length ORF Truncated ORF Col-0 SAIL Col-0 SAIL L Col-0 SAIL.5 0.5 Supplemental Figure. Characterization of an At5g470 T-DNA Insertional Mutant. (A) The position of the T-DNA insertion in the homozygous SAIL_57_G line determined by sequencing PCR products amplified from genomic DNA. The gene model is drawn to scale; exons are black boxes, introns are black lines, and 5 - and -UTRs are gray boxes. The positions of two potential start codons flanking the insertion site are indicated in red. (B) Genotyping the SAIL_57_G line (SAIL). Genomic DNA from this line or the corresponding wild-type (Col-0) was used as a template for PCR with the primer pairs shown above. Amplicons were analyzed by agarose-gel electrophoresis. (C) Evidence from RT-PCR for the presence of a truncated At5g470 transcript in the SAIL line. Total RNA was used for first-strand cdna synthesis, followed by PCR with primers designed to amplify the open reading frame from the first or second potential start codon (Full length or Truncated ORF, respectively), or a fragment of the Actin- transcript. Amplicons were analyzed by agarose-gel electrophoresis. The pairs of tracks for each sample represent replicates corresponding to two individual plants. L, DNA ladder.

A 5 B 0 Root length (cm) 4 Aerial mass (mg) 6 8 4 0 WT KO WT KO 0 WT KO Supplemental Figure. The At5g470 T-DNA Insertional Mutant Has Mild Growth Defects. (A) Root growth of Col-0 (WT) and SAIL_57_G (CS848) T-DNA insertion (KO) plants after seven days growth from seed (stratified for five days) on half-strength MS medium containing % sucrose. Root length was determined with ImageJ software. Data are means and SE for 4 (WT) or (KO) replicate plants. Representative images of typical plants are shown. There was a significant difference (Student s t-test) between the length of WT and KO roots, P<0.0000. (B) Mass of the aerial parts of Col-0 (WT) and SAIL_57_G (CS848) T-DNA insertion (KO) plants after days growth from seed (stratified for five days) on half-strength MS medium containing % sucrose. Individual plants were cut at the base of the root and weighed immediately. Data are means and SE for 6 (WT) or 48 (KO) replicate plants. There was a significant difference (Student s t-test) between the mass of WT and KO plants, P=0.004.

0 9 0 4 5 6 4 5 4 7 8 9 6 7 8 5 6 7 WT KO 4 5 6 7 8 9 0 4 5 6 7 8 9 0 4 5 6 7 WT KO WT KO Supplemental Figure. The Phenotype of the At4g950 th- Double Mutant. F seeds from a cross between the At4g950 knockout (KO) and the th- mutant were germinated on ½ MS medium with no thiamin supplement, and plantlets showing the th- phenotype (i.e., homozygous for the th- mutant allele) were transplanted to fresh medium. The th- homozygotes were PCR screened to identify double mutant homozygotes (lower panel). No obvious exacerbation of the th- phenotype was observed for homozygous double mutants (indicated with red arrows) as compared to th- single mutants. Plants were arbitrarily numbered though 7 before genotyping.

6 Thiamin ThMP ThDP x /0 5 nmol mg - Fresh weight 4 0 Vector At5g470 At5g470-D7A GRMZMG48896 GRMZMG0788 Supplemental Figure 4. Levels of Thiamin and Its Phosphates in E. coli Cells Overexpressing Wild- Type or D7A Mutant At5g470, or Maize Orthologs GRMZMG48896 or GRMZM0788. E. coli strain BL-CodonPlus (DE)-RIPL was transformed with pet8b alone (Vector) or harboring a fragment encoding At5g470, its D7A mutant, GRMZMG48896, or GRMZMG0788, all hexahistidine-tagged and minus their predicted targeting sequences. Cells were grown in LB medium plus 50 μg/ml kanamycin. Isopropyl β-d--thiogalactopyranoside (final concentration 0.5 mm) was added when OD 600 reached 0.4, and cells were harvested when OD 600 reached.0. Washed pellets from 0 ml of culture were taken for analysis. Data are means and SE for three replicate cultures. Note that the values shown for ThDP are multiplied /0. 4

A kda 70 0 95 7 56 4 4 B Protein Calculated monomer mass (kda) Estimated native mass (kda) At5g470 64.97 66.6 GRMZMG48896 64.6 64.6 GRMZMG0788 64.4 64.4 Supplemental Figure 5. Isolation of Recombinant At5g470, GRMZMG48896, and GRMZM- G0788 Proteins and Estimation of Native Molecular Mass. (A) Proteins were purified by Ni + -affinity chromatography and gel filtration. Purified proteins (5 μg) were analyzed by SDS-PAGE with Coomassie staining. The positions of molecular mass markers are indicated. The preparations used for enzyme assays were judged to be 90% homogeneous. (B) Native molecular mass values estimated by gel filtration compared to the calculated monomer molecular mass values. 5

. At5g470 MRFLF...PTRLINNSSLGLLRSPHTTAPIRSLWFRTKSPVFRSATTPIMTAVAFSSSLSIPP...TS.EEALPGKLWIKF.NRECLFSIYSPFAVCLAAGNL 0.96 0.47 Brassica_napus MRILNNS.LALLRSPRAAAPIRSLLFGSKK...SSVSRSAAAFSSAMSIPPPSISTS.EEALAGRLWIKF.NRECLFSMYSPFAVSLAAGNL 0.88 0.67 Aquilegia_hybrid MRFLI.RNPSFRAISSSICGFEALNLTSLRKSLRSSFSARFYSKSCSSSNFNLKSSKSISTKMGVVAVSDEEGIARRFWIKF.KRETVFAMYTPFVVCLASGNL 0.50 0.64 Populus_trichocarpa MRLLLFTSPNPIKTSSSLYFLNSLRSNLTKRTLPTRRS..FIPARMAIPPRSIASAPSCTTTSGRSNINIEEGLASKFWIKF.RRESVFAMYTPFVISLASGTL 0.676 0.49 Phaseolus_vulgaris MRMRWFLRSPIKNPLNLNLSPSRSFSLRALVPQWPSPASPISLPRSSIPMAAIHNHSNS..EAGLARRFWIKF.TRQSIFAMYTPFAIALASGNL 0.84 0.88 Vigna_unguiculata MRMRWFLRSPIQSPLNLNLSPRRSFSLRALVPHWPSPASRRSLPRSSIPMAAIHNNSNS..EAGLARRFWIKF.TRQSIFAMYTPFAIALASGNL 0.8 0.89 Glycine_max MRMRWFLRSPIIKTSLLNLSPPISFR...PHW...ARRTFTSSRLSMAAIHNHSNSNSETGLARRFWIKF.TRESIFAMYTPFAIALASGNL 0.848 0.89 Helianthus_ciliaris MRLLSTKFATTIR...SAISNTNPSRNFKFLTISSMATTPKPNEEGTAKRFWDKF.SKESILTLYTPFVVSLASGNL 0.499 0.50 Parthenium_argentatum MPSFFTKFTIPIRPIIRSAIFNTNPSLKIKFLTLSSMAN...PTEEGTAKRFWDKF.CKESILTLYTPFVFSLASGNL 0.5 0.46 GRMZMG48896 MLVLRRLRF..PLPRPLLVSS..SLAPLS.PSTSSSS.CWSSTGEGRRAMASSPSSASAAVVAEGSAARRFWIAASTREAAFAAYTPFLLSLAAGNL 0.75 0.84 GRMZMG0788 MLVLRRLRLRLPLPRPLLVSSFSSTSPSSSPSTSSSSSCWSSTGESRRAMASSPSPDSAAVVAEGSAARRFWIAASTREAAFAAYTPFLLSLAAGNL 0.790 0.89 Panicum_virgatum MLVLRRLRL..PLPRPLPVSS...SSSSSWWWSSTSQRRPAMASS...SSAAVVAEGSAARRFWIAASTREAAFAAYTPFLLSLAAGNL 0.784 0.90 Oryza_sativa MRGLLRRVYLRLP...PFPPAT...SLYYWSRTR...PAAAGPNHPIPRRMSTSSTAAAVVAEGSAARRFWIAAASREAAFAAYTPFLVSLAAGAL 0.960 0.45 Triticum_aestivum MRLLRLRLRLPLPALLPSPNPT...TKSFFSLSSCWPGVTRTSPTSHHEQMSTTSSSAAAVAEGSAARRFWIAASSREAAFATYTPFLLSLAAGSL 0.8 0.6 Hordeum_vulgare MRLLCLRLRLPLPALRPNPNQT...TKSFFSPSSYWLRVTRTSPTNHQKQMSSASSSAAAVAEGSAARRFWIAASSREAAFATYTPFLLSLAAGSL 0.877 0.89 Supplemental Figure 6. Alignment of the N-terminal Regions of the At5g470, GRMZMG48896, and GRMZM0788 Proteins and their Orthologs, and Mitochondrial Targeting Prediction Scores. Protein sequences deduced from cdnas or ESTs were aligned in Multalin (http://multalin.toulouse.inra.fr/multalin/) and printed with BoxShade (http://www.ch.embnet.org/software/box_form.html). Identical residues are highlighted in black; similar residues are highlighted in gray. The putative second start site in each sequence is highlighted in green. The red triangle indicates approximately where the N termini of homologous bacterial proteins align. Blue triangles mark the last residue in each of the N-terminal sequences from At5g470 that were fused to GFP for in vivo subcellular localization experiments (refer to Figure 7). Mitochondrial targeting prediction scores from TargetP (Emanuelsson et al., 000) and Predotar (Small et al., 004) are shown to the right of each sequence. These scores exceeded plastidial and endoplasmic reticulum scores in all cases except for Aquilegia and Populus trichocarpa (where TargetP gave higher plastidial scores) and Triticum aestivum (where Predotar gave a higher endoplasmic reticulum score). 6

Supplemental Table. The Panel of 95 Phosphoesters Used in Activity Screens Substrate Abbreviation Substrate Abbreviation ',5'-Adenosine diphosphate ',5'-ADP Pyridoxal 5'-phosphate PLP '-Adenosine monophosphate 'AMP Phosphono-acetate Po-acetate '-Cytidine monophosphate 'CMP Phosphono-formate Po-formate -Phospho-ascorbate P-Ascorbate N-(Phosphonomethyl)glycine Po-methyl-Gly -Deoxyribose 5-phosphate drib-5p Phosphorylethanolamine P-ethanolamine '-Adenosine monophosphate 'AMP Phytic acid Phytate '-Cytidine monophosphate 'CMP Polyphosphate Poly-P -Phosphoglyceraldehyde -PGA Inorganic pyrophosphate PPi -Phosphoglycerate P-Glycerate Ribose 5-phosphate Rib-5P 6-Phosphogluconate 6P-Gluc Ribulose-,5-diphosphate Ru-,5-P Adenosine diphosphate ADP Sucrose 6-phosphate Sucrose-6P Adenosine monophosphate AMP Sorbitol 6-phosphate Sorb-6P Adenosine monophosphoramidate AMP-ram Thymidine monophosphate TMP Adenosine triphosphate ATP Thymidine diphosphate TDP Cytidine diphosphate CDP Thymidine triphosphate TTP Cytidine monophosphate CMP Thiamine monophosphate Thiamine-P Cytidine triphosphate CTP Thiamine pyrophosphate Thiamine-PP Coenzyme A CoA Trehalose 6-phosphate Trehalose-6P Dihydroxyacetone phosphate DHAP Uridine diphosphate UDP Erythrose 4-phosphate Eryth-4P Uridine monophosphate UMP Riboflavin 5'-monophosphate FMN Uridine triphosphate UTP Fructose-,6-diphosphate Fru-,6 dip Xanthosine monophosphate XMP Fructose -phosphate Fru-P α-d-glucosamine -phosphate a-d-glcn -P Fructose 6-phosphate Fru-6P α-glucose -phosphate a-glc-p Guanosine diphosphate GDP β-glucose -phosphate b-glc-p Guanosine monophosphate GMP α-glucose 6-phosphate a-glc-6p Guanosine triphosphate GTP β-glucose 6-phosphate b-glc-6p Galactose -phosphate Gal-P Deoxyadenosine diphosphate dadp Glucosamine 6-phosphate Glcn-6P Deoxyadenosine monophosphate damp Glucose-,6-diphosphate Glucose-,6P Deoxyadenosine triphosphate datp Glycerol -phosphate Glyc-P Deoxycytidine diphosphate dcdp Glycerol -phosphate Glyc-P Deoxycytidine monophosphate dcmp Glycerol -phosphate Glyc-P 5-Methyldeoxycytidine monophosphate Me-dCMP Glyphosate Glyphosate Deoxycytidine triphosphate dctp Inosine diphosphate IDP Deoxyguanidine diphosphate dgdp Inosine monophosphate IMP Deoxyguanidine monophosphate dgmp Inosine triphosphate ITP Deoxyguanidine triphosphate dgtp L--Phosphoglycerate P-Gly Deoxyinosine monophosphate dimp Lactose -phosphate Lactose-P Deoxyinosine triphosphate ditp Mannose -phosphate Man-P Deoxythymidine monophosphate dtmp Mannose 6-phosphate Man-6P Deoxyuridine monophosphate dump N-Acetyl-α-D-glucosamine--phosphate NAGlcn-P Deoxyuridine triphosphate dutp N-Acetyl-α-D-glucosamine-6-phosphate NAGlcn-6P Phosphorylcholine P-Cho β-nicotinamide mononucleotide NMN Phosphorylserine P-Ser β-nicotinamide adenine dinucleotide phosphate NADP Phospho-threonine P-Thr Adenosine ',5'-diphosphate PAP Phospho-tyrosine P-Tyr Adenosine '-phosphate 5'-phosphosulfate PAPS p-nitrophenylphosphate pnpp Phospho(enol)pyruvate PEP 7

Supplemental Table. Oligonucleotide Primers Used in This Study Primer Name Sequence SAIL_57_G Genotyping LP At5g470 F gttgttcaatggatccattttac RP At5G470 R cgagaagagaacttacgcgtg BP SAIL LB tagcatctgaatttcataaccaatctcgatacac SAIL_57_G RT-PCR 4 FL AT5G470 F atgcgcttcctcttccc 5 FL AT5G470 R ttactcccatccaagagcga 6 Met AT5G470 F atgacggcggtcgct 7 Atg8780 Actin F atggctgaggctgatgatat 8 Atg8780 Actin R aggtctcaaacatgatttgagtc In vitro transcription-translation 9 FL At5g470 SacI F cagtcagagctcaccatgcgcttcctcttccc 0 At5g470 XbaI R cagtcatctagattactcccatccaagagcga Met AT5G470 SacI F cagtcagagctcaccatgacggcggtcgct FL GRMZMG48896 EcoRI F cagtcagaattcaccatgcttgttctccgccgc GRMZMG48896 SphI R cagtcagcatgctcagctgaaatcgcttccc 4 Met GRMZMG48896 EcoRI F cagtcagaattcaccatggcgtcatctccgtct GFP fusions TraB (Atg0570) BamH F ccggccggatccacgatagagccgacgcaatc 4 TraB (Atg0570) XbaI R ccggcctctagactactttcttcttgaaagaagtatac 5 FL At5g470 XbaI native kozak F ctagtctagattttatgcgcttcctcttccc 6 FL At5g470 XbaI strong kozak F ctagtctagaaccatgcgcttcctcttccc 7 At5g470 BamHI R cgcggatccctcccatccaagagcgaat 8 Met At5g470 XbaI native kozak F ctagtctagaaataatgacggcggtcgct 9 Met At5g470 XbaI strong kozak F ctagtctagaaccatgacggcggtcgct 0 At5g470 BamHI 5 aa after met R cgcggatccttccgaggtagggggaatc At5g470 BamHI aa before met R cgcggatccagtagtcgccgatcggaag GFP fusions PDONR07 Gateway R (GFP R) ggggaccactttgtacaagaaagctgggtcttacttgtacagctcgtccatgccg FL At5g470 GFP native kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagattttatgcgcttcctcttccc 4 FL At5g470 GFP strong kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatgcgcttcctcttccc 5 Met At5g470 GFP native kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaataatgacggcggtcgct 6 Met At5g470 GFP strong kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatgacggcggtcgct Quickchange 7 At5g470 Quickchange Met to Leu F gactactccaatattgacggcggtcgct 8 At5g470 Quickchange Met to Leu R agcgaccgccgtcaatattggagtagtc 9 AtTenAHAD-D7A-F ctggtgatcttttctgcttttgatctgacttgcaccgttg 0 AtTenAHADD7A-R caacggtgcaagtcagatcaaaagcagaaaagatcaccag AtTenACA-F caacggtgcaagtcagatcaaaagcagaaaagatcaccag AtTenACA-R ttacgagaaa ggcatacaac cgcatggcag gtgtcacagca Functional complementation AtTenAHAD-F acaaggatccgaaggagatataccatgacggcggtcgctttctc 4 AtTenAHAD-R aacctgcagg ttactcccatccaagagcgaat 5 ZMTenAHAD-F acaaggatccgaaggagatataccatggcgtcatctccgtcttc 6 ZMTenAHAD-R aacctgcagg tcagctgaaatcgcttcccag 7 ZmTenAHAD-F acaaggatccgaaggagatataccatggcgtcatctccttctcc 8 ZmTenAHAD-R aacctgcaggttagctgaaatcacttcccagtacg Protein expression 9 At5g470 truncated cloning pet8b NcoI atgcatccatggcggcggtcgctttc 40 Rev At5g470 truncated cloning pet8b NotI atgcatgcggccgcctcccatccaagagcga 4 GRMZMG48896 truncated cloning pet8b NcoI atgcatccatggcgtcatctccgtct 4 Rev GRMZMG48896 truncated cloning pet8b NotI atgcatgcggccgcgctgaaatcgcttccca 4 ZmTenHAD-p8-F gatcgccatggcgtcatctccttctcc 44 ZmTenHAD-p8-R acaactcgaggctgaaatcacttcccagtac Plant transformation 45 TENA_C_inf_XbaI_F ccggggatcctctagatcgggtttgggtattatgcctagg 46 TENA_C_inf_SalI_R atgcctgcaggtcgaccttctctattccacgtttcagttctcg 8