STREP ------------------------------------------------------------ CORYN ------------------------------------------------------------ IMPL2 ---MLAQSHFFSKSFDLIPPQSPALRSANPSLRISSSYSNSRLSFLSSSAIAVPVSRRRF 57 VTC4 ------------------------------------------------------------ IMPL1 MGRSLIFSGNMSLRISHLPRSSLPLQNPISGRTVNRTFRYRCTRILSN--SFKSTTRLQT 58 STREP ---MPDYLDDLRLAHVLADAADAATMDRFKAL------------------DLKVETKPDM 39 CORYN ---MSKYADDLALALELAELADSITLDRFEAS------------------DLEVSSKPDM 39 IMPL2 CLTMASNSKRPNISNESPSELSDTELDRFAAVGNALADASGEVIRKYFRKKFDIVDKDDM 117 VTC4 ---MADNDSLDQFLAAAIDAAKKAGQIIRKGFYET----------------KHVEHKGQV 41 IMPL1 KAVLSEVSDQTRYPRIGAKTTGTISPAHLLEVVELAAKTGAEVVMEAVNKPRNITYKGLS 118 STREP TPVSEADKAAEELIRGHLSRARPRDSVHGEEFG-----VAGTGPRRWVIDPIDGTKNYVR 94 CORYN TPVSDADLATEEALREKIATARPADSILGEEFGG-D--VEFSG-RQWIIDPIDGTKNYVR 95 IMPL2 SPVTIADQMAEEAMVSIIFQNLPSHAIYGEEKGWRC--KEESADYVWVLDPIDGTKSFIT 175 VTC4 DLVTETDKGCEELVFNHLKQLFPNHKFIGEETTAAFGVTELTDEPTWIVDPLDGTTNFVH 101 IMPL1 DLVTDTDKASEAAILEVVKKNFSDHLILGEEGGIIG---DSSSDYLWCIDPLDGTTNFAH 175 STREP GVPVWATLIALMEAKEGGYQPVVGLVSAPALG--RRWWAVEDHGAFTGRSLTSAHRLHVS 152 CORYN GVPVWATLIALLDNG----KPVAGVISAPALA--RRWWASEGAGAWRTFNGSSPRKLSVS 149 IMPL2 GKPVFGTLIALLYKG----KPILGLIDQPILK--ERWIGMNGR-----RTKLNGEDISTR 224 VTC4 GFPFVCVSIGLTIGK----VPVVGVVYNPIME--ELFTGVQGKG-----AFLNGKRIKVS 150 IMPL1 GYPSFAVSVGVLYRGNPAAASVVEFVGGPMCWNTRTFSATAGGG-----ALCNGQKIHVS 230 STREP QVSTLSDASFAYSS---LSGWEEQGRLDGFLDLTREVWRTRAYG-DFWPYMMVAEGSVDL 208 CORYN QVSKLDDASLSFSS---LSGWAERDLRDQFVSLTDTTWRLRGYG-DFFSYCLVAEGAVDI 205 IMPL2 SCPKLSQAYLYTTS---PHLFSEEA--EKAYSRVRDKVKVPLYGCDCYAYALLASGFVDL 279 VTC4 AQSELLTALLVTEAGTKRDKATLDDTTNRINSLLTKVRSLRMSGSCALDLCGVACGRVDI 210 IMPL1 KTDAVERALLITGFG-YEHDDAWSTNMELFKEFTDVSRGVRRLGAAAVDMCHVALGIAES 289 STREP CAEPELS-LWDMAANAIIVTEAGGTFTGLDGR----------PGPHSGNAAASNGRLHDE 257 CORYN AAEPEVS-LWDLAPLSILVTEAGGKFTSLAGV----------DGPHGGDAVATNGILHDE 254 IMPL2 VIESGLK-PYDFLALVPVIEGAGGTITDWTGKRFLWEASSSAVATSFNVVAAGDSDIHQQ 338 VTC4 FYELGFGGPWDIAAGIVIVKEAGGLIFDPSGK---------DLDITSQRIAASNASLKEL 261 IMPL1 YWEYRLK-PWDMAAGVLIVEEAGGAVTRMDGG---------KFSVFDRSVLVSNGVLHPK 339 STREP LLGYLNQRY----------------------- 266 CORYN TLDRLK-------------------------- 260 IMPL2 ALESLEWH------------------------ 346 VTC4 FAEALRLTGA---------------------- 271 IMPL1 LLERIAPATENLKSKGIDFSLWFKPEDYHTEL 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.

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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

1 0.702498 0.00E+00 7.00E-45 At2g36230N'-5'-phosphoribosyl-formimino-5-aminoimidazole-4-carboxamide ribonucleotide isomerase

2 0.671816 1.30E-43 2.80E-39 At2g37860 Expressed protein

3 0.667804 6.20E-43 1.30E-38 At4g33680Aminotransferase class I and II family protein low similarity to Aromatic Aminotransferase from Pyrococcus horikoshii

4 0.650538 4.00E-40 8.70E-36 At5g63890 Histidinol dehydrogenase

5 0.637023 4.70E-38 1.00E-33 At2g22230Similar to beta-hydroxyacyl-ACP dehydratase from Toxoplasma gondii

6 0.629804 5.50E-37 1.20E-32 At2g44040Dihydrodipicolinate reductase family protein, weak similarity to Dihydrodipicolinate reductase from Pseudomonas syringae

7 0.628289 9.10E-37 2.00E-32 At3g57610 Adenylosuccinate synthetase

8 0.627427 1.20E-36 2.70E-32 At3g14390Diaminopimelate 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 At5g17660Expressed 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.