32

1.5

1

0.5

Supplemental Figure 1. Characterization of an At5g32470 T-DNA Insertional Mutant.

(A) The position of the T-DNA insertion in the homozygous SAIL_572_G1 line determined bysequencing PCR products amplified from genomic DNA. The gene model is drawn to scale; exonsare black boxes, introns are black lines, and 5′- and 3′-UTRs are gray boxes. The positions of twopotential start codons flanking the insertion site are indicated in red.(B) Genotyping the SAIL_572_G11 line (SAIL). Genomic DNA from this line or the correspondingwild-type (Col-0) was used as a template for PCR with the primer pairs shown above. Ampliconswere analyzed by agarose-gel electrophoresis.(C) Evidence from RT-PCR for the presence of a truncated At5g32470 transcript in the SAIL line.Total RNA was used for first-strand cDNA synthesis, followed by PCR with primers designed toamplify the open reading frame from the first or second potential start codon (Full length orTruncated ORF, respectively), or a fragment of the Actin-2 transcript. Amplicons were analyzed byagarose-gel electrophoresis. The pairs of tracks for each sample represent replicates correspondingto two individual plants. L, DNA ladder.

A

B

100 bp1 2

3

Primer for genotyping Coding sequence UTR

At5g32470

T-DNA insertion positionSAIL_572_G11

Met1 Met2

kb

1

0.5

1.52

3

Col-0 SAIL

Primers: 1–2DNAladder

1

0.5

1.523

kbCol-0 SAIL

Primers: 3-2DNAladder

CCol-0 SAIL L Col-0 SAIL

kb

Col-0 SAIL

Actin-2 Full length ORF Truncated ORF

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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Supplemental Figure 2. The At5g32470 T-DNA Insertional Mutant Has Mild Growth Defects.

(A) Root growth of Col-0 (WT) and SAIL_572_G11 (CS824383) T-DNA insertion (KO) plants afterseven days growth from seed (stratified for five days) on half-strength MS medium containing 1%sucrose. Root length was determined with ImageJ software. Data are means and SE for 34 (WT) or 21(KO) replicate plants. Representative images of typical plants are shown. There was a significantdifference (Student’s t-test) between the length of WT and KO roots, P<0.00001.(B) Mass of the aerial parts of Col-0 (WT) and SAIL_572_G11 (CS824383) T-DNA insertion (KO) plantsafter 21 days growth from seed (stratified for five days) on half-strength MS medium containing 1%sucrose. Individual plants were cut at the base of the root and weighed immediately. Data are meansand SE for 36 (WT) or 48 (KO) replicate plants. There was a significant difference (Student’s t-test)between the mass of WT and KO plants, P=0.0014.

0

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5

Roo

t len

gth

(cm

)

WT KO WT KO0

4

8

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16

20

Aer

ial m

ass

(mg)

WT KO

BA

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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WT

KO

1 2 3 4 5 6 7 8 9 19 20 21 22 23 24 25 26 27

WT

KO

10 11 12 13 14 15 16 17 18

WT

KO

1 2 3

4 5 6

78 9

10 11 12

1314

15

1617 18

19 20 21

2223 24

25 2627

Supplemental Figure 3. The Phenotype of the At4g29530 th2-1 Double Mutant.

F2 seeds from a cross between the At4g29530 knockout (KO) and the th2-1 mutant were germinated on ½MS medium with no thiamin supplement, and plantlets showing the th2-1 phenotype (i.e., homozygous forthe th2-1 mutant allele) were transplanted to fresh medium. The th2-1 homozygotes were PCR screened toidentify double mutant homozygotes (lower panel). No obvious exacerbation of the th2-1 phenotype wasobserved for homozygous double mutants (indicated with red arrows) as compared to th2-1 single mutants.Plants were arbitrarily numbered 1 though 27 before genotyping.

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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Supplemental Figure 4. Levels of Thiamin and Its Phosphates in E. coli Cells Overexpressing Wild-Type or D317A Mutant At5g32470, or Maize Orthologs GRMZM2G148896 or GRMZM078283.

E. coli strain BL21-CodonPlus (DE3)-RIPL was transformed with pET28B alone (Vector) or harbor-ing a fragment encoding At5g32470, its D317A mutant, GRMZM2G148896, or GRMZM2G078283,all hexahistidine-tagged and minus their predicted targeting sequences. Cells were grown in LBmedium plus 50 μg/ml kanamycin. Isopropyl β-D-1-thiogalactopyranoside (final concentration 0.5mM) was added when OD600 reached 0.4, and cells were harvested when OD600 reached 1.0.Washed pellets from 10 ml of culture were taken for analysis. Data are means and SE for threereplicate cultures. Note that the values shown for ThDP are multiplied × 1/10.

0

1

2

3

4

5

6

Vector At5g32470 At5g32470-D317A GRMZM2G148896 GRMZM2G078283

nmol

mg-

1F

resh

wei

ght

Thiamin ThMP ThDP x 1/10

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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Supplemental Figure 5. Isolation of Recombinant At5g32470, GRMZM2G148896, and GRMZM-2G078283 Proteins and Estimation of Native Molecular Mass.

(A) Proteins were purified by Ni2+-affinity chromatography and gel filtration. Purified proteins (5 μg)were analyzed by SDS-PAGE with Coomassie staining. The positions of molecular mass markersare 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 monomermolecular mass values.

72

kDa

56

43

34

95

130 170

A

B

Protein Calculated monomer mass (kDa)

Estimated native mass (kDa)

At5g32470 64.97 66.6

GRMZM2G148896 64.62 64.6

GRMZM2G078283 64.43 64.4

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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Supplemental Figure 6. Alignment of the N-terminal Regions of the At5g32470, GRMZM2G148896, andGRMZM078283 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 highlightedin black; similar residues are highlighted in gray. The putative second start site in each sequence is highlightedin 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 At5g32470 that were fused toGFP for in vivo subcellular localization experiments (refer to Figure 7). Mitochondrial targeting prediction scoresfrom TargetP (Emanuelsson et al., 2000) and Predotar (Small et al., 2004) are shown to the right of eachsequence. These scores exceeded plastidial and endoplasmic reticulum scores in all cases except for Aquilegiaand Populus trichocarpa (where TargetP gave higher plastidial scores) and Triticum aestivum (where Predotargave a higher endoplasmic reticulum score).

.

At5g32470 MRFLF...PTRLINNSSLGLLRSPHTTAPIRSLWFRTKSPVFRSATTPIMTAVAFSSSLSIPP....TS.EEALPGKLWIKF.NRECLFSIYSPFAVCLAAGNL 0.926 0.47Brassica_napus MRILNNS.LALLRSPRAAAPIRSLLFGSKK......SSVSRSAAAFSSAMSIPPPSISTS.EEALAGRLWIKF.NRECLFSMYSPFAVSLAAGNL 0.818 0.67Aquilegia_hybrid MRFLI.RNPSFRAISSSICGFEALNLTSLRKSLRSSFSARFYSKSCSSSNFNLKSSKSISTKMGVVAVSDEEGIARRFWIKF.KRETVFAMYTPFVVCLASGNL 0.502 0.64Populus_trichocarpa MRLLLFTSPNPIKTSSSLYFLNSLRSNLTKRTLPTRRS..FIPARMAIPPRSIASAPSCTTTSGRSNINIEEGLASKFWIKF.RRESVFAMYTPFVISLASGTL 0.676 0.49Phaseolus_vulgaris MRMRWFLRSPIKNPLNLNLSPSRSFSLRALVPQWPSPASPISLPRSSIPMAAIHNHSNS..EAGLARRFWIKF.TRQSIFAMYTPFAIALASGNL 0.824 0.88Vigna_unguiculata MRMRWFLRSPIQSPLNLNLSPRRSFSLRALVPHWPSPASRRSLPRSSIPMAAIHNNSNS..EAGLARRFWIKF.TRQSIFAMYTPFAIALASGNL 0.833 0.89Glycine_max MRMRWFLRSPIIKTSLLNLSPPISFR.....PHW....ARRTFTSSRLSMAAIHNHSNSNSETGLARRFWIKF.TRESIFAMYTPFAIALASGNL 0.848 0.89Helianthus_ciliaris MRLLSTKFATTIR....SAISNTNPSRNFKFLTISSMATTPKPNEEGTAKRFWDKF.SKESILTLYTPFVVSLASGNL 0.499 0.50Parthenium_argentatum MPSFFTKFTIPIRPIIRSAIFNTNPSLKIKFLTLSSMAN...PTEEGTAKRFWDKF.CKESILTLYTPFVFSLASGNL 0.253 0.46GRMZM2G148896 MLVLRRLRF..PLPRPLLVSS..SLAPLS.PSTSSSS.CWSSTGEGRRAMASSPSSASAAVVAEGSAARRFWIAASTREAAFAAYTPFLLSLAAGNL 0.752 0.84GRMZM2G078283 MLVLRRLRLRLPLPRPLLVSSFSSTSPSSSPSTSSSSSCWSSTGESRRAMASSPSPDSAAVVAEGSAARRFWIAASTREAAFAAYTPFLLSLAAGNL 0.790 0.89Panicum_virgatum MLVLRRLRL..PLPRPLPVSS...........SSSSSWWWSSTSQRRPAMASS...SSAAVVAEGSAARRFWIAASTREAAFAAYTPFLLSLAAGNL 0.784 0.90Oryza_sativa MRGLLRRVYLRLP...PFPPAT.....SLYYWSRTR...PAAAGPNHPIPRRMSTSSTAAAVVAEGSAARRFWIAAASREAAFAAYTPFLVSLAAGAL 0.960 0.45Triticum_aestivum MRLLRLRLRLPLPALLPSPNPT.....TKSFFSLSSCWPGVTRTSPTSHHEQMSTTSSSAAAVAEGSAARRFWIAASSREAAFATYTPFLLSLAAGSL 0.813 0.61Hordeum_vulgare MRLLCLRLRLPLPALRPNPNQT.....TKSFFSPSSYWLRVTRTSPTNHQKQMSSASSSAAAVAEGSAARRFWIAASSREAAFATYTPFLLSLAAGSL 0.877 0.89

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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Supplemental Table 1. The Panel of 95 Phosphoesters Used in Activity Screens Substrate Abbreviation Substrate Abbreviation 2',5'-Adenosine diphosphate 2',5'-ADP Pyridoxal 5'-phosphate PLP

2'-Adenosine monophosphate 2'AMP Phosphono-acetate Po-acetate

2'-Cytidine monophosphate 2'CMP Phosphono-formate Po-formate

2-Phospho-ascorbate 2P-Ascorbate N-(Phosphonomethyl)glycine Po-methyl-Gly

2-Deoxyribose 5-phosphate dRib-5P Phosphorylethanolamine P-ethanolamine

3'-Adenosine monophosphate 3'AMP Phytic acid Phytate

3'-Cytidine monophosphate 3'CMP Polyphosphate Poly-P

3-Phosphoglyceraldehyde 3-PGA Inorganic pyrophosphate PPi

3-Phosphoglycerate 3P-Glycerate Ribose 5-phosphate Rib-5P

6-Phosphogluconate 6P-Gluc Ribulose-1,5-diphosphate Ru-1,5-P2

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-1,6-diphosphate Fru-1,6 diP Xanthosine monophosphate XMP

Fructose 1-phosphate Fru-1P α-D-Glucosamine 1-phosphate a-D-Glcn 1-P

Fructose 6-phosphate Fru-6P α-Glucose 1-phosphate a-Glc-1P

Guanosine diphosphate GDP β-Glucose 1-phosphate b-Glc-1P

Guanosine monophosphate GMP α-Glucose 6-phosphate a-Glc-6P

Guanosine triphosphate GTP β-Glucose 6-phosphate b-Glc-6P

Galactose 1-phosphate Gal-1P Deoxyadenosine diphosphate dADP

Glucosamine 6-phosphate Glcn-6P Deoxyadenosine monophosphate dAMP

Glucose-1,6-diphosphate Glucose-1,6P2 Deoxyadenosine triphosphate dATP

Glycerol 1-phosphate Glyc-1P Deoxycytidine diphosphate dCDP

Glycerol 2-phosphate Glyc-2P Deoxycytidine monophosphate dCMP

Glycerol 3-phosphate Glyc-3P 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-2-Phosphoglycerate 2P-Gly Deoxyinosine monophosphate dIMP

Lactose 1-phosphate Lactose-1P Deoxyinosine triphosphate dITP

Mannose 1-phosphate Man-1P Deoxythymidine monophosphate dTMP

Mannose 6-phosphate Man-6P Deoxyuridine monophosphate dUMP

N-Acetyl-α-D-glucosamine-1-phosphate NAGlcn-1P 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 3',5'-diphosphate PAP Phospho-tyrosine P-Tyr

Adenosine 3'-phosphate 5'-phosphosulfate PAPS p-Nitrophenylphosphate pNPP

Phospho(enol)pyruvate PEP

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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Supplemental Table 2. Oligonucleotide Primers Used in This Study

Primer Name SequenceSAIL_572_G11 Genotyping

1 LP At5g32470 F gttgttcaatggatccattttac 2 RP At5G32470 R cgagaagagaacttacgcgtg 3 BP SAIL LB3 tagcatctgaatttcataaccaatctcgatacac

SAIL_572_G11 RT-PCR 4 FL AT5G32470 F atgcgcttcctcttccc 5 FL AT5G32470 R ttactcccatccaagagcga 6 Met2 AT5G32470 F atgacggcggtcgct 7 At3g18780 Actin2 F atggctgaggctgatgatat 8 At3g18780 Actin2 R aggtctcaaacatgatttgagtc

In vitro transcription-translation 9 FL At5g32470 SacI F cagtcagagctcaccatgcgcttcctcttccc 10 At5g32470 XbaI R cagtcatctagattactcccatccaagagcga 11 Met2 AT5G32470 SacI F cagtcagagctcaccatgacggcggtcgct 12 FL GRMZM2G148896 EcoRI F cagtcagaattcaccatgcttgttctccgccgc 13 GRMZM2G148896 SphI R cagtcagcatgctcagctgaaatcgcttccc 14 Met2 GRMZM2G148896 EcoRI F cagtcagaattcaccatggcgtcatctccgtct

GFP fusions 13 TraB (At1g05270) BamH1 F ccggccggatccacgatagagccgacgcaatc 14 TraB (At1g05270) XbaI R ccggcctctagactactttcttcttgaaagaagtatac 15 FL At5g32470 XbaI native kozak F ctagtctagattttatgcgcttcctcttccc 16 FL At5g32470 XbaI strong kozak F ctagtctagaaccatgcgcttcctcttccc 17 At5g32470 BamHI R cgcggatccctcccatccaagagcgaat 18 Met2 At5g32470 XbaI native kozak F ctagtctagaaataatgacggcggtcgct 19 Met2 At5g32470 XbaI strong kozak F ctagtctagaaccatgacggcggtcgct 20 At5g32470 BamHI 15 aa after met2 R cgcggatccttccgaggtagggggaatc 21 At5g32470 BamHI 2 aa before met2 R cgcggatccagtagtcgccgatcggaag

GFP fusions PDONR207 22 Gateway R (GFP R) ggggaccactttgtacaagaaagctgggtcttacttgtacagctcgtccatgccg 23 FL At5g32470 GFP native kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagattttatgcgcttcctcttccc 24 FL At5g32470 GFP strong kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatgcgcttcctcttccc 25 Met2 At5g32470 GFP native kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaataatgacggcggtcgct 26 Met2 At5g32470 GFP strong kozak F ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatgacggcggtcgct

Quickchange 27 At5g32470 Quickchange Met2 to Leu F gactactccaatattgacggcggtcgct 28 At5g32470 Quickchange Met2 to Leu R agcgaccgccgtcaatattggagtagtc 29 AtTenAHAD-D317A-F ctggtgatcttttctgcttttgatctgacttgcaccgttg 30 AtTenAHADD317A-R caacggtgcaagtcagatcaaaagcagaaaagatcaccag 31 AtTenAC213A-F caacggtgcaagtcagatcaaaagcagaaaagatcaccag 32 AtTenAC213A-R ttacgagaaa ggcatacaac cgcatggcag gtgtcacagca

Functional complementation 33 AtTenAHAD-F acaaggatccgaaggagatataccatgacggcggtcgctttctc 34 AtTenAHAD-R aacctgcagg ttactcccatccaagagcgaat 35 ZMTenAHAD1-F acaaggatccgaaggagatataccatggcgtcatctccgtcttc 36 ZMTenAHAD1-R aacctgcagg tcagctgaaatcgcttcccag 37 ZmTenAHAD2-F acaaggatccgaaggagatataccatggcgtcatctccttctcc 38 ZmTenAHAD2-R aacctgcaggttagctgaaatcacttcccagtacg

Protein expression 39 At5g32470 truncated cloning pET28b NcoI atgcatccatggcggcggtcgctttc 40 Rev At5g32470 truncated cloning pET28b NotI atgcatgcggccgcctcccatccaagagcga 41 GRMZM2G148896 truncated cloning pET28b NcoI atgcatccatggcgtcatctccgtct 42 Rev GRMZM2G148896 truncated cloning pET28b NotI atgcatgcggccgcgctgaaatcgcttccca 43 ZmTenHAD2-p28-F gatcgccatggcgtcatctccttctcc 44 ZmTenHAD2-p28-R acaactcgaggctgaaatcacttcccagtac

Plant transformation 45 TENA_C_inf_XbaI_F ccggggatcctctagatcgggtttgggtattatgcctagg 46 TENA_C_inf_SalI_R atgcctgcaggtcgaccttctctattccacgtttcagttctcg

 

Supplemental Data. Mimura et al. (2016). Plant Cell 10.1105/tpc.16.00600

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