STREP CORYN IMPL2 VTC4 IMPL1 -------------------------------------------------------------------------------------------------------------------------MLAQSHFFSKSFDLIPPQSPALRSANPSLRISSSYSNSRLSFLSSSAIAVPVSRRRF 57 -----------------------------------------------------------MGRSLIFSGNMSLRISHLPRSSLPLQNPISGRTVNRTFRYRCTRILSN--SFKSTTRLQT

STREP CORYN IMPL2 VTC4 IMPL1 -------------------------------------------------------------------------------------------------------------------------MLAQSHFFSKSFDLIPPQSPALRSANPSLRISSSYSNSRLSFLSSSAIAVPVSRRRF 57 -----------------------------------------------------------MGRSLIFSGNMSLRISHLPRSSLPLQNPISGRTVNRTFRYRCTRILSN--SFKSTTRLQT

STREP CORYN IMPL2 VTC4 IMPL1 -------------------------------------------------------------------------------------------------------------------------MLAQSHFFSKSFDLIPPQSPALRSANPSLRISSSYSNSRLSFLSSSAIAVPVSRRRF 57 -----------------------------------------------------------MGRSLIFSGNMSLRISHLPRSSLPLQNPISGRTVNRTFRYRCTRILSN--SFKSTTRLQT 58 STREP CORYN IMPL2 VTC4 IMPL1 ---MPDYLDDLRLAHVLADAADAATMDRFKAL------------------DLKVETKPDM ---MSKYADDLALALELAELADSITLDRFEAS------------------DLEVSSKPDM CLTMASNSKRPNISNESPSELSDTELDRFAAVGNALADASGEVIRKYFRKKFDIVDKDDM ---MADNDSLDQFLAAAIDAAKKAGQIIRKGFYET----------------KHVEHKGQV KAVLSEVSDQTRYPRIGAKTTGTISPAHLLEVVELAAKTGAEVVMEAVNKPRNITYKGLS 39 39 117 41 118 STREP CORYN IMPL2 VTC4 IMPL1 TPVSEADKAAEELIRGHLSRARPRDSVHGEEFG-----VAGTGPRRWVIDPIDGTKNYVR TPVSDADLATEEALREKIATARPADSILGEEFGG-D--VEFSG-RQWIIDPIDGTKNYVR SPVTIADQMAEEAMVSIIFQNLPSHAIYGEEKGWRC--KEESADYVWVLDPIDGTKSFIT DLVTETDKGCEELVFNHLKQLFPNHKFIGEETTAAFGVTELTDEPTWIVDPLDGTTNFVH DLVTDTDKASEAAILEVVKKNFSDHLILGEEGGIIG---DSSSDYLWCIDPLDGTTNFAH 94 95 175 101 175 STREP CORYN IMPL2 VTC4 IMPL1 GVPVWATLIALMEAKEGGYQPVVGLVSAPALG--RRWWAVEDHGAFTGRSLTSAHRLHVS GVPVWATLIALLDNG----KPVAGVISAPALA--RRWWASEGAGAWRTFNGSSPRKLSVS GKPVFGTLIALLYKG----KPILGLIDQPILK--ERWIGMNGR-----RTKLNGEDISTR GFPFVCVSIGLTIGK----VPVVGVVYNPIME--ELFTGVQGKG-----AFLNGKRIKVS GYPSFAVSVGVLYRGNPAAASVVEFVGGPMCWNTRTFSATAGGG-----ALCNGQKIHVS 152 149 224 150 230 STREP CORYN IMPL2 VTC4 IMPL1 QVSTLSDASFAYSS---LSGWEEQGRLDGFLDLTREVWRTRAYG-DFWPYMMVAEGSVDL QVSKLDDASLSFSS---LSGWAERDLRDQFVSLTDTTWRLRGYG-DFFSYCLVAEGAVDI SCPKLSQAYLYTTS---PHLFSEEA--EKAYSRVRDKVKVPLYGCDCYAYALLASGFVDL AQSELLTALLVTEAGTKRDKATLDDTTNRINSLLTKVRSLRMSGSCALDLCGVACGRVDI KTDAVERALLITGFG-YEHDDAWSTNMELFKEFTDVSRGVRRLGAAAVDMCHVALGIAES 208 205 279 210 289 STREP CORYN IMPL2 VTC4 IMPL1 CAEPELS-LWDMAANAIIVTEAGGTFTGLDGR----------PGPHSGNAAASNGRLHDE AAEPEVS-LWDLAPLSILVTEAGGKFTSLAGV----------DGPHGGDAVATNGILHDE VIESGLK-PYDFLALVPVIEGAGGTITDWTGKRFLWEASSSAVATSFNVVAAGDSDIHQQ

FYELGFGGPWDIAAGIVIVKEAGGLIFDPSGK---------DLDITSQRIAASNASLKEL YWEYRLK-PWDMAAGVLIVEEAGGAVTRMDGG---------KFSVFDRSVLVSNGVLHPK 257 254 338 261 339 STREP CORYN IMPL2 VTC4 IMPL1 LLGYLNQRY----------------------TLDRLK-------------------------ALESLEWH-----------------------FAEALRLTGA---------------------LLERIAPATENLKSKGIDFSLWFKPEDYHTEL 266 260 346 271 371 Supplemental Figure S1. Alignment of the Arabidopsis proteins IMPL1, IMPL2 and VTC4 with known histidinol phosphate phosphatases from Streptomyces coelicolor (NP_629355) and Corynebacterium glutamicum (YP_225091). Identical residues are shaded in black, conserved residues in grey. The putative plastid transit peptide predicted by ChloroP analysis (Emanuelsson et al., 1999) in IMPL1 and IMPL2 is shown in green. The Gly residue previously shown to be required for HPP activity in the S. coelicolor enzyme is highlighted in red. Amino acid sequences were aligned using ClustalW. sequence:At4g39120 MASNSKRPNISNESPSELSDTELDRFAAVGNALADASGEVIRKYFRKKFDIV--DKDDMSPVTIADQMAEEAMVS IIFQNLPSHAIYGEEKGW---RCKEESADYVWVLDPIDGTKSFITGKPVFGTLIALLYKGKPILGLIDQPILKER WIGMNGRRTKLNGEDISTRSCPKLSQAYLYTTS--PHLF---SEEAEKAYSRVRDKV-KVPLYGCDCYAYALLAS GFVDLVIESGLKPYDFLALVPVIEGAGGTITDWTGKRFLWEASSSAVATSFNVVAAGDSDIHQQALESLEWH------* structure:2qfl ----------------------MHPMLNIAVRAARKAGNLIAKNYETPDAVEASQKGSNDFVTNVDKAAEAVIID TIRKSYPQHTIITEES---GELEGT-DQDVQWVIDPLDGTTNFIKRLPHFAVSIAVRIKGRTEVAVVYDPMRNEL FTATRGQGAQLNGYRLRGSTARDLDGTILATGF--PFKAKQYATTYINIVGKLFNECADFRATGSAALDLAYVAA GRVDGFFEIGLRPWDFAAGELLVREAGGIVSDFTGGHNYML--------TGNIVAGNPRVVKAMLANMRD-ELS----* structure:2bji --------------------DPWQECMDYAVTLAGQAGEVVREALKNEMNIMVKSSPA-DLVTATDQKVEKMLIT SIKEKYPSHSFIGEESVAAGEKSIL-TDNPTWIIDPIDGTTNFVHGFPFVAVSIGFVVNKKMEFGIVYSCLEDKM YTGRKGKGAFCNGQKLQVSHQEDITKSLLVTELGSSRTPETVRIILSNIERLLCLPIHGIRGVGTAALNMCLVAA GAADAYYEMGIHCWDVAGAGIIVTEAGGVLLDVTGGPFDLM--------SRRVIASSNKTLAERIAKEIQIIPLQ RDDE* Supplemental Figure S2. pGenTHREADER alignment used to generate homology model of IMPL2. A Mean signal intensity ( SE) 1200 1000 800 600 400 200 0 s yl af af ed on le le ot se c d t e l le il d u po ty en be ad hy v

bi co ju im st em n o e ip ot ue ry lle on ro tt iq b l o z l o i p s n ra ro em te t io a la g on el B Mean signal intensity ( SE) 1200 1000 800 600 400 200 0 ed at n i rm ge ed se g tte tt e in dl se se e o o r r se ng ed u p lo yo ve e d r r s s

g we we ue ue t in l o o q q l l i i o f f l l b si si g d e & un pe r o u o l rs y at ve m we o de fl C 3500 Mean signal intensity ( SE) 3000 2500 2000 VTC4 IMPL1 IMPL2 1500 1000 500 0 pollen ovary embryo imbibed seed Supplemental Figure S3. Expression patterns of VTC4, IMPL1 and VTC4 in Arabidopsis. (A) IMPL2 expression in tissue types, (B) IMPL2 expression at different developmental stages and (C) IMPL1, IMPL2 and VTC4 expression in pollen, ovary, embryo and imbibed seed. Values shown are mean signal intensities SE, and were obtained from analysis of publically available microarray datasets using Genevestigator V3. Rank r-value p-value e-value GeneID Annotation N'-5'-phosphoribosyl-formimino-5-aminoimidazole-4carboxamide ribonucleotide isomerase Expressed protein

1 2 0.702498 0.671816 0.00E+00 1.30E-43 7.00E-45 At2g36230 2.80E-39 At2g37860 3 4 0.667804 0.650538 6.20E-43 4.00E-40 1.30E-38 At4g33680 8.70E-36 At5g63890 5 0.637023 4.70E-38 1.00E-33 At2g22230 6 7 0.629804 0.628289 5.50E-37 9.10E-37 1.20E-32 At2g44040 2.00E-32 At3g57610 8 0.627427 1.20E-36 2.70E-32 At3g14390 Aminotransferase class I and II family protein low similarity to Aromatic Aminotransferase from Pyrococcus horikoshii Histidinol dehydrogenase Similar to beta-hydroxyacyl-ACP dehydratase from Toxoplasma gondii Dihydrodipicolinate reductase family protein, weak similarity to Dihydrodipicolinate reductase from Pseudomonas syringae Adenylosuccinate synthetase Diaminopimelate decarboxylase putative, similar to diaminopimelate decarboxylase Arabidopsis thaliana 9 0.625218 2.50E-36 5.50E-32 At1g15710 Prephenate dehydrogenase family protein 10 0.615464 6.00E-35 1.30E-30 At5g17660 Expressed protein contains Pfam profile PF02390: Putative methyltransferase Supplementary Table SI. Top 10 genes showing the strongest positive correlation with IMPL2 expression across 322 publically available microarray datasets. The list was generated using the Arabidopsis Coexpression Data Mining tool. Genes highlighted in grey are known to encode enzymes of the His biosynthetic pathway.

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