Arabidopsis TH2 Encodes the Orphan Enzyme ThiaminMonophosphate PhosphataseOPEN

Manaki Mimura,a,1 Rémi Zallot,b,1 Thomas D. Niehaus,a,1 Ghulam Hasnain,a,1 Satinder K. Gidda,c

Thuy N.D. Nguyen,c Erin M. Anderson,c Robert T. Mullen,c Greg Brown,d Alexander F. Yakunin,d

Valérie de Crécy-Lagard,b Jesse F. Gregory III,e Donald R. McCarty,a,2 and Andrew D. Hansona,2

a Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611bMicrobiology and Cell Science Department, University of Florida, Gainesville, Florida 32611cDepartment of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, CanadadDepartment of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canadae Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida 32611

ORCID IDs: 0000-0002-1263-6343 (M.M.); 0000-0002-7317-1578 (R.Z.); 0000-0002-6915-7407 (R.T.M.); 0000-0001-8694-5117 (D.R.M.);0000-0003-2585-9340 (A.D.H.)

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) tothiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold,implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic andtranscriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) familyphosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470.Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and itsmaize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activityresides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivoexperiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also tomitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDPdepletion can produce severe deficiency symptoms.

INTRODUCTION

In all forms of life, thiamin diphosphate (ThDP) is an essentialcofactor for central metabolic enzymes such as a-keto acid de-hydrogenases, transketolase, pyruvate decarboxylase, andacetolactate synthase (Goyer, 2010). ThDP issynthesizeddenovoby prokaryotes, fungi, and plants but not by humans and otheranimals, which can only convert preformed thiamin (vitamin B1)to ThDP (Jurgenson et al., 2009). Plants are the main source ofthiamin in humandiets, and there is consequently active interest inmetabolic engineeringof theplantpathway to thiaminandThDP tocombat thiamin deficiency among populations whose diet con-sists largely of low-thiamin foods (Pourcel et al., 2013; Dong et al.,2015). Aseconddriver for interest in engineering thiaminsynthesisis its potential to increase plant resistance to biotic and abioticstresses (Tunc-Ozdemir et al., 2009; Dong et al., 2015).

Rational metabolic engineering of the thiamin pathway usingtransgenic technology or natural variation rests on thorough

knowledge of the steps in the pathway and of the correspondinggenes and enzymes (Martin, 2013). Fortunately, the steps in theplant thiaminpathwayand their subcellular locationsarenow fairlywell understood (Figure 1A) (Gerdes et al., 2012; Pourcel et al.,2013; Dong et al., 2015). In brief, the pyrimidine and thiazolemoieties of thiamin are made separately and coupled together inplastids by thiamin-phosphate diphosphorylase (TH1) to formthiaminmonophosphate (ThMP). ThMP is then dephosphorylatedto give thiamin, either in the plastid or after export to the cytosol.Finally, thiamin is converted to ThDP by cytosolic thiamin di-phosphokinases TPK1 and 2. Genes and enzymes are known forall thesteps in theplantpathwayexcept for adeadenylationstep inthiazole synthesis and for ThMPdephosphorylation (Gerdes et al.,2012). This is also basically the case for the yeast pathway(Nosaka, 2006; Begley et al., 2012; Kuznetsova et al., 2015).That ThMP phosphatase is still an “orphan” enzyme and gene

(i.e., without known sequences; Hanson et al., 2009) has led tothe general belief that ThMP is dephosphorylated by nonspecifichydrolases such as the broad-spectrum acid phosphatase iso-lated from maize (Zea mays) (Rapala-Kozik et al., 2009; Goyer,2010; Pourcel et al., 2013). However, this belief (that no dedicatedThMP phosphatase exists) has recently been overturned, forbacteria at least (Hasnain et al., 2016). Bacteria that synthesizeThDP via a plant-typepathwaywere found to havegenes encodingHAD (haloacid dehalogenase) family phosphatases that occur inpredicted operons with, or as fusions to, thiamin synthesis genes,

1 These authors contributed equally to this work.2 Address correspondence to adha@ufl.edu or drm@ufl.edu.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Donald R. McCarty (drm@ufl.edu).OPENArticles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.16.00600

The Plant Cell, Vol. 28: 2683–2696, October 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

and these HAD enzymes were found to strongly prefer ThMP toawiderangeofotherphosphoesters.Thesamestudyalso identifieda homologous enzyme in Arabidopsis thaliana (At4g29530) thatshoweda similarly strongpreference for ThMP.However, knockingout the At4g29530 gene did not affect growth or the levels of thi-amin, ThMP, or ThDP, indicating that At4g29530 is not the majorArabidopsis ThMP phosphatase (Hasnain et al., 2016).Apossiblecandidate for themissingmajorThMPphosphatase in

Arabidopsis is the product of the classical genetic locus TH2 onchromosome5; this locus isoneofseveral thiaminbiosynthesis locifor which auxotrophic mutations can be recovered (Li and Rédei,1969; Meinke et al., 2009), which contrasts with other vitaminbiosynthesis pathways in plants (Li et al., 1967). Mutants at thislocus can be rescued by supplying thiamin but not its thiazole orpyrimidine precursors, which places th2 lesions late in the thiaminpathway (Li and Rédei, 1969), potentially at the ThMP de-phosphorylation step (Figure 1A). Accordingly, in this studywe firstmeasured the levelsof thiaminand itsphosphates in th2-1mutants.As this analysis revealed ThMP accumulation, we used genomic,biochemical, and cell biology approaches to clone theTH2gene, todemonstrate that its product is themain ThMPphosphatase and todetermine where this enzyme localizes within cells. To confirm thatour findings apply to other plant species, we biochemically char-acterizedmaizeTH2orthologs.Wealso found that the th2-1mutantsheds light on a little studied area: thiamin deficiency in plants.

RESULTS

The Arabidopsis th2-1 Mutant Accumulates ThMP

The th2-1mutant plants grow slowly and have variegated yellow-green leaves unless given small doses of thiamin (Langridge,1965; Li and Rédei, 1969). To probe the nature of the th2-1 lesion,we compared the thiamin, ThMP, and ThDP levels in seedlings ofthemutant and the corresponding wild type (Ler-0) when culturedaxenically on sucrose-containing medium. Relative to the wildtype, ThDP levels in th2-1 seedlings fell by half (from 1.18 to0.65 nmol g21 fresh weight) and ThMP levels rose 5-fold (Figure1B). This mutant phenotype could be accounted for by a defect inThMP export from plastids or by a defect in ThMP dephosphor-ylation in plastids or cytosol (Figure 1A).

The th2-1 Mutation Maps to the At5g32470 Gene

The ThMP-ThDPmetabolic phenotype of the th2-1mutant led usto map the mutation by combining genome sequencing andtranscriptomic data. The th2 locus in the centromere region ofchromosome 5 is defined by a single allele induced by x-raymutagenesis (Langridge, 1955) of a wild-type strain identified as‘Estland’ (Est) (Langridge, 1965). The th2-1 mutant stock dis-tributed by the Arabidopsis Resource Center is the product ofintrogression into a Landsberg erecta (Ler) background (StockCS80; www.arabidopsis.org). Given the wealth of haplotype dataavailable for hundreds of Arabidopsis ecotypes (Cao et al., 2011;Schmitz et al., 2013), we reasoned that in principle candidates forTH2 could be identifiedusing awhole-genome sequencing (WGS)approach. To this end, we generated 12.2 Gb of WGS data fromth2-1 using the Illumina platform (150-base unpaired reads). In

Figure 1. The Arabidopsis ThDP Synthesis Pathway and the ThiaminPhenotype of the th2-1 Mutant.

(A) ThDP synthesis pathway. The pyrimidine (2-methyl-4-amino-5-hydroxymethylpyrimidine diphosphate [HMP-PP]) and thiazole [4-methyl-5-(2-phosphoethyl)-thiazole (THZ-P)] moieties of thiamin are made in plastidsand coupled together by TH1 to give ThMP. ThMP is then hydrolyzed tothiamin, which is converted to ThDP in the cytosol by thiamin diphospho-kinases TPK1 and 2. It is not knownwhether ThMP is exported fromplastidsand dephosphorylated in the cytosol or dephosphorylated in plastids fol-lowed by thiamin export. These alternatives are shown as red and bluedashed arrows, respectively. AIR, aminoimidazole ribotide; P, phosphategroup; ThMPase, ThMP phosphatase.(B) Levels of thiamin and its phosphates in wild-type and th2-1 seedlings.Wild-type Ler-0 and th2-1 homozygote plants were cultured for 3 weeks onagar medium. Whole plants were harvested for thiamin analysis. Data aremeans and SE for three biological replicates, each from different seedlings.

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parallel, we performed a replicated RNA-seq analysis of aerialorgans of Ler and th2-1 seedlings to identify genes that are ex-pressed differentially between these genotypes.

To identify candidate genes, we used a bottom-up approachbased on k-mer frequency analysis to identify genes on chro-mosome 5 of the Col reference genome that were polymorphic(i.e., contained at least one single nucleotide polymorphism) inth2-1. JELLYFISH (Marçais and Kingsford, 2011) was used tocompare frequencies of all 22-mer sequences included in codingsequencesof the representative gene set for chromosome5 in theCol reference genome (TAIR10) and the th2-1 WGS data set.Overall, the average frequency of single-copy Col k-mers in theth2-1 data set was 91 counts, consistent with the expected ge-nome coverage of the WGS data. We then analyzed the distri-bution of Col single copy k-mers that were absent (<5 counts) inth2-1 WGS data to identify genes that were either polymorphicor missing from the th2-1 genome. A single nucleotide poly-morphism in a single-copy gene sequence typically eliminatescounts for 22 consecutive k-mers; by this measure, at least 2375 of6414 genes in the representative gene set for chromosome5werepolymorphic. This set included 29 Col genes for which >85% ofk-mers were absent in the th2-1 data set (Figure 2A). The latterwere of particular interest because the x-ray-induced th2-1 mu-tation (Langridge, 1955)hadastrong likelihoodofbeingadeletion.

To further narrow the list of candidates for TH2, the k-merprofiles were overlaid with the RNA-seq data to identify poly-morphic genes that also showed a qualitative difference in

expression in th2-1 seedlings compared with the wild type. Eightof the 29 putative missing genes had significant FPKM values intriplicate wild-type samples but no detectable expression in th2-1(Figure 2B). This set included four genes (At5g32440,At5g32450,At5g32460, and At5g32470) in a contiguous block located nearthe expected position of th2 based on the classical genetic map(Meinke et al., 2009). Further analysis of single-copy k-mers fromthe surrounding region of the Est genome sequence (Schmitzet al., 2013) resolved a 13.9-kb deletion in th2-1 between nu-cleotides 12,075,450 and 12,089,360 of the chromosome 5 ref-erence sequence. Notably, one of the four genes in this interval,At5g32470, encodesaprotein thathaspreviouslybeen implicatedin thiamin metabolism (Zallot et al., 2014); this protein, which isconserved throughout land plants, comprises anN-terminal TenAdomain andaC-terminalHADdomain (Figure 2A). Bycontrast, theannotations of At5g32440, At5g32450, and At5g32460 (Figure2B) did not suggest obvious connections to thiamin.

Complementation and Insertional Mutation ConfirmAt5g32470 as the TH2 Locus

We first used complementation to confirm that deletion ofAt5g32470 is indeed responsible for the th2 phenotype. An 8-kbfragment of Ler-0 genomicDNAcontaining theAt5g32470 codingsequence and 2.2-kb of the 59 sequence was cloned into thepromoterless pCAMBIA1300 vector and introduced into the ho-mozygous th2-1mutant.Primary transformantsweresupplemented

Figure 2. The th2-1 Mutation Is Linked to a 13.9-kb Deletion on Chromosome 5.

(A) Physical map positions of 29missing reference genes (diamonds) identified by profiling frequencies of 22-mers inWGSdata from th2-1. Locations of eightgenes in thisset that alsoshowedaqualitativeexpressiondifferencebetweenLerand th2-1 (B)are colored red.Thebold lineabove thechromosomeshows theapproximatephysicalpositionof the th2 locusdeterminedby integrationofphysicalandclassicalgeneticmaps (Meinkeetal., 2009).A13.9-kbdeletion (triangle)in the vicinity of the th2 locus includes four genemodels. Nucleotide coordinates of the deletion breakpoints are indicated on the left and right of the triangle.The TenA and HAD domains of the protein encoded by At5g32470 are shown below the gene model. TP, predicted N-terminal targeting peptide.(B)RNA-seqdata for eight genes in the set of 29missinggenes (reddiamonds in [A]) that showedsignificant expression inwild-typeLerplants, but not in theth2-1 mutant. The differentially expressed genes include the four deleted genes diagrammed in (A). The At5g32470 candidate gene is in red font.

Thiamin Monophosphate Phosphatase 2685

with thiamin and allowed to self-pollinate, and three independentT1 progenies were cultured without thiamin, along with emptyvector and wild-type controls (Figure 3A). Each T1 progenysegregated 3:1 for the wild-type phenotype versus the th2-1phenotype, indicating that thewild-typeAt5g32470genewasableto complement the lesion in the th2-1 mutant. Consistent withcomplementation, the phenotypically normal transformantprogeny had wild-type levels of ThMP and ThDP (Figure 3B),whereas empty-vector control plants showed the expected ThMPaccumulation and ThDP depletion to below the level (0.65 nmolg21 fresh weight) associated with severe symptoms (Figure 1B).The wild-type At5g32470 gene is thus sufficient to fully revert thegrowth and vitamin phenotypes of the th2-1 mutant.Further support for correspondence between At5g32470 and

theTH2 locuswasobtained fromaT-DNAmutantwith an insertion32 codons after the first potential start codon of the At5g32470coding sequence (Supplemental Figures 1A and 1B). The positionof the insert made this mutant a priori likely to be leaky and,consistent with leakiness, homozygotes had detectable levelsof a truncated but potentially functional At5g32470 transcript(Supplemental Figure 1C) and showed modest reductions in rootlength (37%) and shoot growth (17%) (Supplemental Figure 2).Analysis of thiamin and its phosphates in the T-DNA mutantshowed a pattern of ThMP accumulation and ThDP depletion likethat in the th2-1mutant (Figure 1B), but less drastic inasmuch asthe ThDP level was well above the 0.65 nmol g21 level associatedwith severe symptoms (Figure 3C). Partial insertional inactivationofAt5g32470 thus results in a ThDP synthesis lesion similar to theth2-1 mutation but less severe.

Inactivating HAD Phosphatase At4g29530 Does Not Affectthe Phenotype of the th2-1 Mutant

Although the At5g32470 gene is deleted in the th2-1mutant, thismutant clearly retains some ThMP phosphatase activity becauseThDP synthesis is not completely eliminated (Figure 1B). Ourprevious work raised the possibility that the At4g29530 HAD en-zyme makes a minor contribution to total ThMP phosphataseactivity invivo (Hasnainetal., 2016).WethereforecheckedwhetherAt4g29530activity isasourceof the residualThMPphosphatase inth2-1mutant plantsbycrossing theAt4g29530 knockout (Hasnainet al., 2016) with th2-1 plants to obtain the double homozygousmutant. Double mutant individuals did not show a more seriousgrowth defect than th2-1 single mutants (Supplemental Figure 3),indicating that At4g29530 does not contribute significantly to theresidual ThMP phosphatase activity in the th2-1 mutant.

The At5g32470 HAD Domain and Its Maize Orthologs AreThMP-Selective Phosphatases

To corroborate the genetic evidence that the At5g32470 geneproduct is a ThMPphosphatase, and toconfirm that this is also true

Figure 3. Biochemical-Genetic Evidence That the At5g32470 GeneCorresponds to the TH2 Locus.

(A)Visualphenotypesof theT1progenyof th2-1mutantplants transformedwith the Ler-0At5g32470geneorwith empty vector;wild-type Ler-0plantsare included for comparison. Plants were cultured on thiamin-freemediumfor 2 weeks. Note the presence of approximately one-quarter transgene-free segregants, as expected.(B) Levels of thiamin and thiamin phosphates in the T1progenyof the th2-1mutant transformed with At5g32470 or empty vector. Only th2-1 trans-formants with a normal phenotype were analyzed. Wild-type Ler-0 plantswere included for comparison. Aerial parts were analyzed. Data aremeans and SE for three biological replicates, each from different seed-lings, which for T1 plants represented independent transformationevents. The dashed red line shows the ThDP level in the mutant th2-1plants of Figure 1B.

(C)Levelsof thiaminand itsphosphates in leavesofwild-typeCol-0and thehomozygousAt5g32470T-DNAmutant.Plantswereculturedon thiamin-freemedium for 3 weeks. Data are means and SE for three biological replicates,each from different seedlings. The dashed red line is the same as above.

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oforthologousproteins indivergentplant species,we turnedfirst tofunctionalcomplementationofanEscherichiacolistrainengineeredto require an active ThMP phosphatase for growth by deleting thiLand expressing mouse thiamin diphosphokinase from a plasmid(Hasnain et al., 2016). Expression in this strain of the At5g32470protein or either of its maize orthologs (GRMZM2G148896 andGRMZM2G078283) enabled growth on minimal medium (Figure4A), indicating that these proteins act as efficient ThMP phos-phatases in E. coli. Consistent with ThMP phosphatase activity,E. coli cells expressing these proteins had smaller ThMPpools andup to 20-fold larger thiamin pools than empty vector controls(Supplemental Figure 4). The complementation assay was alsoused to show that the ThMP phosphatase activity resides in theHAD domain of the At5g32470 protein: Substituting alanine forthe catalytically essential active site aspartate nucleophile (D317)of the HAD domain (Burroughs et al., 2006) abolished com-plementing activity (Figure 4A). Consistent with complete activityloss, cells overexpressing the D317A mutant protein had normalThMP and thiamin levels (Supplemental Figure 4).To obtain direct evidence that the At5g32470 protein and its

maize orthologs are ThMP phosphatases, they were expressed inE. coli in histidine-tagged form and purified by Ni2+ affinity chro-matography followed by gel filtration (Supplemental Figure 5A),which indicated that they exist asmonomers (Supplemental Figure5B). The purified proteins were then screened for activity againsta panel of 95 diverse phosphate esters (Supplemental Table 1). Allthree proteins showed their highest activity against ThMP; they alsoshowed substantial activity against flavin mononucleotide andThDP, as well as somewhat less against inorganic pyrophosphate,CTP, and dATP (Figure 4B). The activities against ThDP, pyro-phosphate, and nucleoside triphosphates, i.e., phosphoanhydridehydrolase activities, are quite unusual for HADphosphatases,mostof which act only on monophosphates (Burroughs et al., 2006;Kuznetsova et al., 2006, 2015). For all three enzymes, activitiesagainst the other 89 substrates tested were very low (<4% of thatwith ThMP) or undetectable. Kinetic characterization of all threeenzymes with ThMP as substrate gave Km values in 1.9 to 3.6 mMrange,andkcatvaluesof8.2 to16.9s

21 (Table1).TheseKmvaluesaretwo to three orders of magnitude lower than those for recombinantbacterial ThMP phosphatases (0.58 to 1.38 mM) or ArabidopsisAt4g29530 (5.1 mM) (Hasnain et al., 2016). Taken with the com-plementation data, these findings confirm that the HAD domains ofAt5g32470and itsorthologsareThMP-selectivephosphatasesandare highly catalytically efficient (Bar-Even et al., 2011).

The At5g32470 TenA Domain Has Thiamin SalvageHydrolase Activity

The TenA domains of At5g32470 and itsmaize orthologs belong tothe subfamily of TenA proteins (TenA_C) that have an active-site

Figure4. PhosphataseActivities of theAt5g32470TenA-HADProtein andIts Maize Orthologs.

(A) Evidence that the TenA-HAD proteins have in vivo ThMP phosphataseactivity in E. coli and that the activity resides in the HAD domain. Threeindependent isolatesof anE.coliDthiLstrain transformedwith vector alone(V) or harboring mouse TPK only (T), or mouse TPK plus native At5g32470(1), the At5g32470 D317A mutant (2), maize GRMZM2G148896 (3), ormaize GRMZM078283 (4) were plated on M9-glucose medium with orwithout 3.5 mMThDP. The DthiL strain transformed with E. coli thiL (L) wasincluded as apositive control. Plates contained50mg/mLchloramphenicoland 1 mg/mL anhydrotetracycline inducer. Images were captured afterincubation for 24 h at 37°C. The predicted targeting sequences were re-moved from all the TenA-HAD proteins.

(B) Substrate profiles of recombinant TenA-HAD proteins, truncated toremove predicted targeting sequences. The panel of 95 substratesscreened, and their abbreviations, are listed in Supplemental Table 1.Assays contained 0.25 mM substrate and 10 mM Mg2+. Data are meansand SE for four independent replicate assays. Activities against substratesnot shown were <4% of that against ThMP for each enzyme.

Thiamin Monophosphate Phosphatase 2687

cysteine (Zallot et al., 2014). TenA_C proteins from yeast (Onozukaet al., 2008) and bacteria (Jenkins et al., 2007; Barison et al., 2009)participate in salvage of the thiamin pyrimidine moiety; theyhydrolyze the breakdown product 4-amino-5-aminomethyl-2-methylpyrimidine (amino-HMP) to the thiamin precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) (Figure 5A) and can insome cases also slowly hydrolyze thiamin itself. TenA_C proteinslack activity against theN-formyl derivative of amino-HMP (anotherthiamin breakdown product; Figure 5A) (Jenkins et al., 2007; Zallotet al., 2014). We therefore tested the At5g32470 protein for activityagainst the breakdown products amino-HMP, N-formylamino-HMP, anddesthiothiamin, aswell as thiamin, ThMP, and ThDP.Wedetected activity only against amino-HMP (Figure 5B). This activitywas quite low, 5.4 nmol min21 mg21 protein, i.e., one to two ordersof magnitude less than standalone bacterial TenA_C proteins(Jenkins et al., 2007; Barison et al., 2009). Mutating the active-sitecysteine (Cys-213) to alanine abolished the activity (Figure 5C),confirming that it resides in the TenA domain.

The At5g32470 Protein Localizes to the Cytosol andPotentially to Mitochondria

The At5g32470, GRMZM2G148896, and GRMZM2G078283proteins and their orthologs from other plants have N-terminalextensions that TargetP (Emanuelsson et al., 2000) and Predotar(Small et al., 2004) predict to be mitochondrial matrix targetingpeptides (Supplemental Figure6).However, aproteomic study (Itoet al., 2011) found the At5g32470 protein in a cytosolic fraction.Promptedby this latterfinding,wediscovered thatAt5g32470andall of the orthologous sequences examined include a methionineresidue near the end of the targeting peptide (residue 47 inAt5g32470) that could potentially serve as an alternative trans-lation start site (Supplemental Figure 6), as reported for other plantproteins (Daras et al., 2014; Niehaus et al., 2014; Bohrer et al.,2015; Ellens et al., 2015). If used, this second translation start sitewould eliminate the putative mitochondrial targeting peptide.

We therefore used a coupled transcription-wheat germ trans-lation system to test which translation start sites in the At5g32470and GRMZM2G148896 sequences are functional in vitro. Whenpreceded by a strong Kozak translation initiation sequence(ACCATG. . .) (Kozak, 1981), both full-length sequences yieldedtwo protein products, whereas sequences truncated up to thesecond start site yielded only the smaller product (Figure 6A). Theproducts’ masses, as estimated from the gel (;70 and ;65 kD),agreewith the calculatedmasses of the proteins initiated from the

first andsecondstart sites, and forbothArabidopsisandmaize thesmaller product predominates (Figure 6A). These results establishthat the TenA-HADproteins have alternative translation start sitesand that the second site is preferred in vitro. Dual import assayswith pea (Pisum sativum) chloroplasts and mitochondria (Rudheet al., 2002) showed that translation from the first start site in theArabidopsis or maize sequence generates a protein that is tar-geted to and processed by mitochondria, but not chloroplasts(Figure 6B). A rabbit reticulocyte system was used in these ex-periments as it privileges the first start site (Figure 6B) and is thestandard system for dual import assays (Rudhe et al., 2002).We next assessed intracellular localization in vivo by transiently

expressing various At5g32470-C-terminal GFP fusions in Agro-bacterium tumefaciens-transformed tobacco leaf epidermal cells,a well-characterized system for studying protein localization

Table 1. Kinetic Parameters of the At5g32470, GRMZM2G148896, andGRMZM2G078283 TenA-HAD Proteins with ThMP as Substrate

Enzyme Km (mM) kcat (s21) kcat/Km (s21 M21)

At5g32470 1.86 6 0.37 13.5 6 0.8 7.25 3 106

GRMZM2G148896 3.61 6 0.68 16.9 6 1.3 4.68 3 106

GRMZM2G078283 3.20 6 1.04 8.2 6 1.0 2.57 3 106

All proteins were truncated to remove predicted targeting sequences.Assays contained 50 mM Tris-HCl buffer, pH 7.5, 10 mM MgCl2, 10%glycerol, and 1 mg/mL BSA. Data are means and SE for three in-dependent determinations.

Figure 5. Amino-HMP Aminohydrolase Activity of the At5g32470 TenA-HAD Protein.

(A) Thiamin breakdown and salvage reactions, showing the two salvagereactions that are mediated by typical TenA_C proteins, i.e., the majoramino-HMP aminohydrolase activity (solid arrow, black highlighting) andthe minor thiaminase II activity (dashed arrow, gray highlighting).(B) Chromatographic evidence that recombinant Arabidopsis At5g32470protein converts amino-HMP (1) to HMP (2). Reactions (50 mL) containing1mMamino-HMPand60mgofproteinwere incubatedat30°C for the timesindicated and analyzed by HPLC with UV detection. The small amount ofHMP present in the zero time (t0) reaction was formed during sampleprocessing.(C)Elimination of theaminohydrolaseactivityofArabidopsisAt5g32470bythe Cys213Ala mutation. Assay conditions were as in (B).

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(Sparkes et al., 2006). In total, eight fusion constructs were ex-amined with respect to their intracellular localization based ondifferences in translational initiation from the two potential startsitesand thepredictedN-terminalmitochondrial targetingpeptide(see schematic illustrations in Figure 7 and the corresponding

legend for details). When the first translation start site inAt5g32470 was preceded by its native Kozak translation initiationsequence, the full-length native fusion construct (FL Kozak-N)appeared to localize exclusively to the cytosol, as judged by itsdiffusefluorescencepatterndistributed throughout thecell (Figure7, row 1, left). When preceded by the strong Kozak sequence,however, the full-length construct (FL Kozak-S) exhibited a dis-tinct punctate fluorescence pattern that coincided with that at-tributable to themitochondrial marker protein, Cherry-At1g55450(Marty et al., 2014) coexpressed in the same cell (Figure 7, row 3,left). Similarly, the constructs in which the predicted N-terminaltargeting peptide (residues 1 to 44 or 1 to 63), preceded by thestrong Kozak sequence, was fused directly to GFP, also localizedto Cherry-At1g55450-containing mitochondria (TP44 Kozak-Sand TP63 Kozak-S; Figure 7, rows 1 and 2, right). Two N-terminalpeptides were tested because the cleavage site of the processingpeptidase could not be precisely predicted (Supplemental Figure6). Mitochondrial localization was also observed for both full-length M47L fusion constructs, wherein the second methioninecodon (codon47)waschanged to leucine (M47L)andprecededbythe native Kozak sequence (FL M47L Kozak-N) or a strong Kozaksequence (FL M47L Kozak-S) (Figure 7, rows 3 and 4, right). Fi-nally, as expected, the N-terminal-truncated constructs lackingthe predicted targeting peptide (Tr Kozak-N and Tr Kozak-S) bothlocalized solely to the cytosol (Figure 7, rows 2 and 4, left).Overall, these results indicate that, at least in tobacco leaf

epidermal cells, the native At5g32470 sequence is translatedprimarily from the second potential start site, resulting in theprotein residing predominantly in the cytosol. However, whenpreceded by a strong Kozak sequence, the first start codon inAt5g32470 can also be used in vivo, as it can in vitro (Figure 6A),so that the protein is localized predominantly to mitochondria.Whether or not the latter results are a result of capturing a normalprocess exaggerated by overexpression, it is clear that theAt5g32470 protein has the potential for dual targeting to thecytosol and mitochondria.

DISCUSSION

Identification of the TH2 Gene Product as a ThMP-SelectiveHAD Phosphatase

Our results provide genomic, genetic, and biochemical evidenceestablishing that the classical Arabidopsis TH2 locus (Li andRédei, 1969) corresponds to theAt5g32470gene encoding ThMPphosphatase, a hitherto “orphan” enzyme of ThDP biosynthesiswhose existence as a specific enzyme was in doubt. With theidentification of the ThMP phosphatase, the set of plant thiaminand ThDP synthesis enzymes and genes is now almost complete;only the enzyme mediating a deadenylation step in thiazolesynthesis remains unidentified in plants and other organisms(Gerdes et al., 2012). Theway is thus nowclear to engineering verynearly all the enzymatic steps in the thiamin pathway.That the ThMP phosphatases encoded by the TH2/At5g32470

geneand itsmaizeorthologsbelong to theHADfamily conforms toan established pattern, for this family includes other pathway-specific phosphatases as well as nonspecific phosphatases

Figure 6. In Vitro Evidence for Dual Translational Start Sites and PotentialMitochondrial Targeting of Arabidopsis and Maize TenA-HAD Proteins.

(A) In vitro-coupled transcription-translation identifies the first and secondmethionine codons as alternative start sites. Full-length (FL) At5g32470 andGRMZM2G148896cDNAsandcorresponding truncated (Tr) cDNAsstartingat the second methionine codon (codon 47 and codon 44, respectively;Supplemental Figure 6) were transcribed and translated (in a wheat germsystem) in thepresenceof [3H]leucine.Translationproductswere resolvedbySDS-PAGE; radioactive bandswere visualized by fluorography. Positions ofmolecular mass markers are indicated. All sequences were preceded bya strong Kozak translation initiation sequence (ACCATG. . .).(B)The full-lengthAt5g32470andGRMZM2G148896proteinsare importedinto and processed by mitochondria, but not chloroplasts. At5g32470 andGRMZM2G148896full-lengthcDNAsprecededbynativeKozaksequencesweretranscribed invitroandtranslated (ina rabbit reticulocytesystem) in thepresence of [3H]leucine. The translation products (TP)were incubated in thelight with mixed pea mitochondria (M) and chloroplasts (C). The organelleswere mock treated (2) or thermolysin (TL) treated (+) to remove adsorbedproteins and then reisolated on a Percoll gradient. Proteins were separatedbySDS-PAGEand visualized by fluorography. Sampleswere loadedon thebasisofequal chlorophyll ormitochondrial proteincontentnext toanaliquotof the translation product. Exposure times were adjusted to give compa-rable band intensities in all tracks. Positions of molecular mass markers (inkD) are shown. Note that the reticulocyte translation system, unlike thewheatgermsystem(Figure6A),privileges thefirst translationstart site, doesnot recognize the second, and apparently recognizes one or more minor,artifactual start sites within the sequence of the mature protein.

Thiamin Monophosphate Phosphatase 2689

(Burroughs et al., 2006). The Arabidopsis and maize proteins arehighly selective for ThMPand show lowKm and high kcat values forthis substrate, which is consistent with their functioning in vivo asThMP phosphatases.

Our genome sequence data demonstrate that the th2-1 mu-tation is a full deletion. That thismutation is not lethal (Li andRédei,1969) implies that another phosphatase or phosphatases canpartly substitute for At5g32470when ThMPbuilds up to abnormallevels. The At4g29530 phosphatase (Hasnain et al., 2016) most

likely does not play such a substitutionary role because thephenotype of the th2-1 At4g29530 double mutant is no moresevere than that of the th2-1 single mutant. More generally, thepartial redundancy of ThMP phosphatase contrasts with thenonredundancy of other biosynthetic phosphatases in plants,notably those acting on phosphoserine (Cascales-Miñana et al.,2013) and histidinol (Petersen et al., 2010). However, there arecases of partial redundancy of biosynthetic phosphatases inbacteria (Klaus et al., 2005; Lu et al., 2011).

Figure 7. In Vivo Evidence for Targeting of the Arabidopsis TH2 Protein to Cytosol and Mitochondria.

Representative confocal laser scanning micrographs of tobacco leaf epidermal cells transiently expressing various At5g32470-C-terminal-GFP fusions,including, asshown inschematic illustrationsat the top,GFP fused toeither the full-lengthAt5g32470sequence (FL)beginningatmethioninecodon1 (Met1)(which can potentially be translated to yield proteins with or without the predicted N-terminal mitochondrial targeting peptide); the truncated At5g32470sequence (Tr) beginning at methionine codon 47 (Met-47) (which yields a protein that lacks the predicted N-terminal targeting peptide); the full-lengthAt5g32470 sequencewithmethionine codon 47 changed to leucine (FLM47L) (which yields a protein that contains theN-terminal targeting peptide); or thepredictedN-terminalmitochondrial targeting peptide inAt5g32470 corresponding to either first 44 residues (TP44) or 63 residues (TP63) of theprotein (refertoSupplemental Figure 6). All coding sequenceswereprecededeither by thenativeKozak sequence (Kozak-N) immediately upstreamof the first translationstart site in At5g32470 (TTTTATG. . . for full-length sequences; AATAATG. . . for truncated sequences) or by a strong Kozak translation initiation sequence(Kozak-S) (ACCATG. . .). Cells shown were coagroinfiltrated with binary plasmids encoding each fusion construct (diagrammed above the images) alongwith a Cherry-tagged mitochondrial marker protein (the mitochondrial outer membrane protein At1g55450; Marty et al., 2014). Each row of imagescorresponds to thefluorescenceattributable to (as indicatedby labels) thecandidate fusionprotein (GFP)and themitochondrialmarker (Mito) (greenandred,respectively) and the corresponding merged image. Note that the FL M47L Kozak-N construct displayed an atypically low transformation efficiency andweak fluorescence signal relative to the other constructs, suggesting it was poorly expressed. Bar = 20 mm.

2690 The Plant Cell

The TenA Domain of TH2 Proteins

The TH2/At5g32470 protein and its orthologs in other plants arefusions between the HAD phosphatase domain and a domainbelonging to a family (TenA) that is associated with thiamin me-tabolism (Jenkins et al., 2007; French et al., 2011; Hasnain et al.,2016). The TenA domain belongs to the TenA_C subfamily, othermembers of which have amino-HMP aminohydrolase activity(Figure 5A) (Jenkins et al., 2007; French et al., 2011). We dem-onstrated that the At5g32470 TenA domain likewise has amino-HMP aminohydrolase activity. That this activity was low could bedue to instability during isolation because activity appeared not tosurvive beyond the Ni2+-affinity step in purification. The low ac-tivity could also be because amino-HMP is not the physiologicalsubstrate, this substrate being a breakdown product upstreamof amino-HMP (Figure 5A), as proposed for Helicobacter pyloriTenA_C (Barison et al., 2009). If so, the TenA-HAD fusion could berationalized as follows. Amino-HMP is the last in a series of ill-characterized thiamin or ThDP breakdown products containingsuccessively less of the thiazole moiety (Jenkins et al., 2007).At5g32470 could attack an early breakdown product that retainsthe thiazole side chain with an attached diphosphate group. Thisgroup blocks the action of other TenA_C proteins (Murata, 1982;Müller et al., 2009). The HAD domain of At5g32470 and its or-thologs, via their di- and monophosphatase activities, could re-move both phosphate groups from ThDP breakdown products,allowing the TenA_C domain to act.

Intracellular Localization of ThMP Phosphatase andIts Implications

Weobtainedstrongbioinformatic andexperimental evidence that,as proteome data (Ito et al., 2011), suggest the At5g32470 proteinis localized to the cytosol as a consequence of translational ini-tiation from the second of two potential start sites and that thesame likely applies to an orthologous maize protein. That ThMPphosphatase is cytosolic disambiguates the substrate specificityof the still missing transporter that exports a thiamin moiety fromplastids (Gerdes et al., 2012), i.e., if ThMP hydrolysis occurs in thecytosol, the plastidial transporter must act on ThMP, not thiamin(Figure 1A).

Given the presence of a conserved upstream start site(Supplemental Figure 6) whose use (when directed by a strongKozak sequence) results in the At5g32470 protein being tar-geted to mitochondria both in vitro (Figure 6B) and in vivo(Figure 7), this protein clearly has the potential for dual local-ization to cytosol and mitochondria. However, whether thispotential is realized and, if so, what its functional significancemay be remain to be investigated. Nevertheless, one possiblefunction for a mitochondrially located TenA-HAD protein re-lates to the two-step salvage role proposed above. ThreemajorThDP-dependent enzymes (the pyruvate, 2-oxoglutarate, andbranched-chain 2-oxoacid dehydrogenase complexes) are inmitochondria, which are hotspots for reactive oxygen speciesdamage (Noctor et al., 2007). The thiazole moiety of ThDP ishighly vulnerable to such damage (Sümegi and Alkonyi, 1983;Bunik et al., 2007). A mitochondrial TenA-HAD protein mighttherefore mediate HMP salvage from oxidatively damaged

ThDP, while its cytosolic counterpart functions mainly in ThDPsynthesis.

Thiamin Deficiency in Plants

Afinal inference fromourdata is thataseveredeficiencysyndromesets in when the ThDP level in Arabidopsis falls from the normalrange (1.2 to 2.0 nmol g21) to 0.65 nmol g21 or below, i.e., by;50to 70%. This inference is broadly consistent with results forArabidopsis tpk1 (thiamin diphosphokinase) mutant seedlings,which grew normally with modestly depleted ThDP levels (Ajjawiet al., 2007). The threshold for severe ThDP deficiency may behigher in maize, in which the thi2-blk thiamin synthesis mutantshowed grossly defective leaf blade development and meristemmaintenance although its ThDP levelwasonly;30% less than thewild type (Woodward et al., 2010). However, caution is needed incomparing thresholds in this way (1) because ThDP levels in bulktissue (asmeasured in all these cases) may not reflect levels in thedividingandexpandingcells thatdrivegrowth, and (2) because, asjust noted, the “normal” level is a range, not a constant referencevalue that can be set at 100%.The ThMP that accumulates in the th2-1 mutant can be dis-

counted as a cause of its severe chlorosis and growth symptomsbecause (1) ThMP levels are similarly elevated in the At5g32470insertional mutant, which does not display severe symptoms, and(2) ThMP is not known to substantially inhibit any plant enzyme(Chang et al., 2015).The consequences of particular levels of ThDP deficiency in

plants cannot be compared directly with those in animals.However, data for young rats (Rains et al., 1997) indicate thatweight gain totally ceased when liver ThDP level fell by ;60%,implying that smaller ThDP losses must substantially impactgrowth. Thus, there is some indication that animals and plantsalike develop severe deficiency symptoms when ThDP levelsdrop by about half, i.e., that neither group has a large marginof safety.If plants normally operate quite close to ThDP deficiency, this

could help explain two sets of classical observations. First, itcould help account for the large number of reported thiaminmutants (Boynton, 1966; Li et al., 1967; Kumar and Sharma,1986); high susceptibility to ThDP deficiency would make de-ficiency easy to attain via hypomorphic as well as null alleles ofbiosynthesis genes. Second, normal operation not far abovethe threshold of ThDP deficiency could explain reports thatplants grown in stress conditions benefit from thiamin sup-plementation, whereas controls in nonstressed conditions donot (Bonner, 1943; Tunc-Ozdemir et al., 2009). Perhaps stress-related decreases in ThDP production or increases in itsbreakdown drive ThDP levels down to the deficiency threshold.

METHODS

Chemicals

Amino-HMPandHMPwere fromTorontoResearchChemicals.N-Formyl-4-amino-5-aminomethyl-2-methylpyrimidine (formylamino-HMP) wasprepared from amino-HMP as described (Jenkins et al., 2007; Zallotet al., 2014). Thiamin hydrochloride, thiamin monophosphate chloridedihydrate, thiamin diphosphate, and other chemicals were obtained

Thiamin Monophosphate Phosphatase 2691

from Sigma-Aldrich. Desthiothiamin was prepared as described (Kurataet al., 1968). Briefly, thiamin-HCl (15.5 mmol) and NaOH (31.1 mmol) weredissolved in 10 mL water, and glycine (15.5 mmol) and NaOH (15.5 mmol)weredissolved in7.5mLwater.Thetwosolutionsweremixedandincubatedfor 3 d at 22°C with continuous stirring. Desthiothiamin crystals wereharvested by filtration and washed with ethanol. Oligonucleotide primerswere from Eurofins MWG Operon and are listed in Supplemental Table 2.

Plant Material and Growth Conditions

Seeds of the Arabidopsis thaliana th2-1 (CS80), Ler-0 (CS20), and Col-0(CS60000) lines, the At4g29530 SALK_081194.31.30.x T-DNA knockoutline, and the At5g32470 SAIL_572_G11 (CS824383) T-DNA knockout linewere from theArabidopsisBiological ResourceCenter. Seedswere surface-sterilized and plated on half-strength MS medium containing 1% (w/v)sucrose, 0.1%(w/v)MES,pH5.7, and0.6%(w/v)Phytagel; for th2-1, 10mMthiamin was added to the medium. Plates were held at 4°C for 3 d beforebeing placed under fluorescent lights (Sylvania F40/CWP 40W cool-whiteplus, photosynthetic photon flux 100 to 150 mmol photons m22 s21) ona 12-h-light/12-h-dark cycle at 22°C. For thiamin vitamer profiling ex-periments, 36 seedswere evenly spacedon9-cmsquareplates containingmedium with the indicated concentration of thiamin; aerial portions of theplants were harvested, immediately frozen in liquid N2, and stored at280°C. Togenerate seeds, plantswere transferred tosoil 2 to3weeksaftergermination; th2-1 plants were watered with 100 mM thiamin until seedswere harvested. Note that this procedure did not supply a controlledthiamin dose because plants were watered as needed (i.e., at irregularintervals based on water use) and thiamin is unstable in soil. Thus, theamount of thiamin available to developing siliques most likely varied be-tween plants and with time, causing differences in thiamin endowmentsamong seeds. Such differences could account for the observed hetero-geneity in the growth of th2-1 plants transformed with empty vector whencultured without thiamin (Figure 3A).

Genomic DNAwas isolated from young leaves of theAt5g32470SAILT-DNA line and from wild-type Col-0 plants; PCR reactions were per-formedwithprimerpairs for thewild-typeorT-DNA insertionalleles (1and2, or 3 and 2, respectively; Supplemental Figure 1). Amplicons wereanalyzed by agarose-gel electrophoresis. The amplicon obtained withthe T-DNA insertion allele primers was T/A cloned into pGEM-T Easy(Promega) and sequenced to confirm the insertion site. Total RNA wasisolated from homozygous plants using the RNeasy Plant Mini Kit(Qiagen) with on-column DNase treatment. First-strand cDNA wascreated with the Superscript III first-strand synthesis system (LifeTechnologies). Todetect transcripts, PCR reactionswereperformedwithprimers designed to amplify the full-length coding sequence of theAt5g32470 transcript (4 and 5), the full-length coding sequence startingfrom the secondpotential start codon (5 and6), or a fragment of the actin-2 transcript (7 and 8); amplicons were analyzed by agarose-gel elec-trophoresis. Genotyping the At4g29530 SALK T-DNA knockout line waspreviously described (Hasnain et al., 2016).

Nicotiana benthamiana (tobacco) plants used for Agrobacterium tu-mefaciens-mediated transient expression experiments were grown ina growth chamber in soil at 28°C under a mixture of fluorescent (Sylvania60W cool-white plus) and incandescent (Philips T8 bulbs [F96T8/TL841/H0/Plus/Alto) lighting (100 to 150 mmol photonsm22 s21) with a 16-h-light/8-h-dark cycle. Leaves of 4-week-old plants were infiltrated with Agro-bacterium (strain LBA4404) carrying selected binary vectors (see Sub-cellular Localization section below for details on vector construction).Agrobacterium transformed with the tomato bushy stunt virus gene P19was included in all infiltrations to enhance transgene expression (Petrieet al., 2010). Procedures for Agrobacterium growth, transformation, in-filtration, and processing of leaf material for microscopy (see below) havebeen described elsewhere (McCartney et al., 2005; Cai et al., 2015).

Genome and Transcriptome Analysis

For transcriptome analysis, th2-1 and Ler-0 seeds were plated onmediumlacking thiamin (36 seedsevenly spacedon9-cmsquare plates) andgrownfor 17dafter germination. Aerial portions of plantswere collected, frozen atonce in liquid N2, and stored at280°C. Total RNAwas isolated from plantscollected from three independent plates using the RNeasy Plant Mini Kitwith on-column DNase treatment. RNA-seq libraries were prepared usingthe TruSeq Stranded Total RNA Sample Prep Kit (Illumina) and sequencedwith the IlluminaNextSeq500platform.RNA-seqdatawere analyzed usingtheGalaxyWeb-basedplatformprovidedbyUniversity of FloridaResearchComputing. Briefly, the short reads from RNA-seq were mapped to theArabidopsis TAIR10 genome using TopHat2. Cufflinks was used to as-semble transcripts and estimate their abundance on TAIR10 genomeannotation downloaded from the Ensemble Plants website (http://plants.ensembl.org). Cuffdiff was then used to find genes differentially expressedin th2-1 and Ler-0.

For WGS analysis, th2-1 seeds were plated on medium supplementedwith 10 mMthiamin and grown for 23 d after germination. Entire plantswerecollected and DNAwas isolated with the DNeasy Plant Mini Kit (Qiagen). AWGS library was constructed with the NEXTflex DNA sequencing kit fol-lowing the manual. A unidirectional, mid-scale run on the Illumina Next-Seq500 instrument yielded 14 Gb of raw sequence. To detect thepolymorphisms in the th2-1 genome, k-mer frequency analysis was per-formed with JELLYFISH software (Marçais and Kingsford, 2011) usinga k-mer length of 22 bases. Frequencies of k-mers derived from geneslocated on chromosome 5 and included in the TAIR10 representative geneset (http://plants.ensembl.org) were profiled in the th2-1WGS data set toidentify genes that were either absent or highly polymorphic in the th2-1genome (genes for which a high percentage of k-mers had zero frequencyin the WGS data). The average frequency of k-mers that are single copy inthe Columbia genomewas;90, indicating 903 genome coverage overall.The analysis yielded a set of 29 genes fromchromosome5 forwhich >85%ofk-merswereabsent from theWGSdata. TheRNA-seqanalysis identifiedtwo genes (At5g32460 and At5g32470) in this set that were also signifi-cantly downregulated in th2-1 plants.

Functional Complementation Tests of TH2 Genes

For functional complementation tests, theEscherichia coliBW25113DthiLstrain andplasmid pBY279.1 carrying themousemTPK1cDNAwere used,with theE.coli thiLgeneaspositivecontrol (Hasnainetal., 2016).To test theThMPase activity of TH2 proteins, artificial operons were constructed inpBY279.1 with the At5g32570, GRMZM2G148896, or GRMZM2G078283sequences, starting at the second methionine (Supplemental Figure 6),placed downstream from mTPK1. Amplifications used PfuTurbo DNApolymerase (Stratagene) andprimerswith restriction sites for cloning. Site-directed mutagenesis was performed on the pBY279.1-TPK/At5g32570construct using the Agilent QuikChange II Kit to change aspartate codon317 toalanine (GAT toGCT); themutatedconstructwassequence-verified.TheDthiL strainwas transformedwith pBY279.1 alone or containingE. colithiL, mTPK1, or mouse TPK1 plus At5g32570, At5g32570-D317A,GRMZM2G148896, or GRMZM2G078283. Transformants were selectedon LB medium containing 50 mg/mL kanamycin, 50 mg/mL chloram-phenicol, and 3.5 mM ThDP. Three transformants per construct were thenstreakedonM9-glucosemediumcontaining50mg/mLkanamycin,50mg/mLchloramphenicol, and 1 mM anhydrotetracycline, plus or minus 3.5 mMThDP. Plates were incubated at 37°C for 24 h.

Genetic Experiments

For functional complementation tests, an 8-kb Ler-0 genomic region in-cluding theAt5g32470 coding sequence and 2.2-kb promoter was clonedinto the pCAMBIA1300 vector. PCR was performed using primers

2692 The Plant Cell

containing 16 bases homologous to sequences in the pCAMBIA1300vector (Supplemental Table 2). pCAMBIA1300 was cut with XbaI and SalI,and the 8-kb genomic fragment was inserted using the In-Fusion HDCloning Kit (Clontech). The resulting plasmid was transformed intoAgrobacterium strain GV3101, which was used to transform the th2-1homozygousmutant (T0 generation) by the floral dip method (Zhang et al.,2006). Seeds were collected from T0 generation plants and T1 plants wereidentified by hygromycin-resistant and thiamin-independent growthphenotypes. Progeny of T1 plants (T2 generation) were grown for 2 weekson the half-strength MS medium described above without thiaminand used for thiamin vitamer analysis. To cross the At4g29530 SALK_081194.31.30.x T-DNA knockout line and th2-1, plants were grown in soil(th2-1plantswerewateredwith 10mMthiamin) andcrossedusingstandardprocedures. Briefly, pollen-producing anthers fromSALK_081194.31.30.xplants were used to pollinate the stigmas of prepared pistils from th2-1plants. Recovered F1 seeds were germinated on plates and transferred tosoil, genomic DNA was isolated from young leaves, and PCR reactionswere performed with wild-type allele primers for At4g29530 or T-DNAinsertion allele primers (Supplemental Table 2); amplicons were analyzedby agarose-gel electrophoresis. F2 seeds were collected from plantsshown to be heterozygous for the T-DNA insertion in At4g29530 andgerminatedonplates. About one-quarter of theF2plants showed the th2-1phenotype; these plants were genotyped as described above.

Expression and Isolation of Recombinant Proteins

mRNAwasextracted fromArabidopsis andmaize (Zeamays) tissues usingthe RNeasy Plant Mini Kit (Qiagen). First-strand cDNA was synthesizedusing 1 mg of total RNA, oligo(dT) primer, and SuperScript III reversetranscriptase (Invitrogen). The At5g32470 and GRMZM2G148896 codingsequenceswere amplifiedbyPCR from the leaf cDNAsusingPhusionDNApolymerase (New England Biolabs). The clones ZM_BFb0076H17 andZM_BFc0019H05 carrying GRMZM2G078283 cDNAs were ordered fromthe Arizona Genomics Institute and used as templates for further cloning.All constructs were verified by DNA sequencing. The At5g32570,GRMZM2G148896, or GRMZM2G078283 cDNAs (starting at the secondmethionine)werecloned intopET28b (Novagen), adding theC-terminalHistag encoded by the vector. Sequence-verified constructs were introducedinto E. coliBL21-CodonPlus (DE3)-RIPL cells. Cultures (1 liter) were grownat 37°C inTerrificBroth containing50mg/mLkanamycin and100mMThDP.When OD600 reached 0.8, IPTG was added (final concentration 1 mM)and incubation was continued for 24 h at 16°C. Cells were harvested bycentrifugation,washedwithPBS,and frozen in liquidN2.Subsequent stepswere at 4°C.Bacterial pelletswere suspended in 50mMNaH2PO4, 300mMNaCl, and 10 mM imidazole, pH 8.0, and sonicated. The extract wasclarified by centrifugation (10,000g, 30 min, 4°C), applied to a 0.5-mL Ni2+-nitrilotriacetic acid agarose column (Qiagen) equilibrated with the abovebuffer, and allowed to drain. After washing with 16 mL 50 mM NaH2PO4,300 mM NaCl, and 20 mM imidazole, pH 8.0, proteins were eluted with2.5 mL of this buffer containing 250 mM imidazole, desalted on PD-10columns (GE Healthcare) equilibrated in 50 mM Tris-HCl, pH 7.5, 10 mMMgCl2, 10% (v/v) glycerol, and concentrated to;1mg/mL inAmiconUltra-0.5mL 10K units. Proteins were further purified using an FLPCgel filtrationstep. A Superdex 200 (GE Healthcare Life Sciences) was equilibrated with50mMTris-HCl, pH7.0, and150mMNaCl. After sample injection, proteinswere eluted using 1 column volume (24 mL) of equilibration buffer witha constant flow rate of 0.5 mL/min, and 1-mL fractions were collected.Proteinswere detected by absorption at 280 nm.Nativemolecularmasseswere estimated using a calibration curve with a Sigma-Aldrich gel filtrationmolecularweightmarker kit (12,000 to200,000D). Fractionswereanalyzedby SDS-PAGE and Coomassie Brilliant Blue staining. Purified proteinswere desalted andconcentrated as above, frozen in liquidN2, andstored at280°C. For TenA activity assays, proteins were purified by Ni2+-affinity

chromatography only, as described (Zallot et al., 2014). The AgilentQuikChange II Kit was used as above to make a Cys213Ala mutation.Protein concentrations were estimated by dye binding (Bradford, 1976)with BSA as standard.

Enzyme Assays

PurifiedTH2proteinswerescreened forphosphataseactivityagainstapanelof 95 phosphorylated metabolites (Supplemental Table 1) using the Mala-chite Green reagent as previously described (Kuznetsova et al., 2006). Thescreeningassayswererun for15minat37°C; theycontained50mMHEPES-KOH, pH 7.5, 10mMMg2+, 0.25mMsubstrate, and 1mg of enzyme. Kineticstudies of ThMPase activity used triplicate 100-mL reaction mixtures con-taining 50mM Tris-HCl, pH 7.5, 10mMMgCl2, 10% (v/v) glycerol, 1 mg/mLBSA, the specified concentrations of ThMP, and 2 ng of enzyme. Assayswere run at 37°C for 2min and stopped and derivatized by adding 100mL ofoxidation reagent (3.35 M NaOH containing 12.14 mM potassium ferricya-nide), which converts thiamin vitamers to thiochrome derivatives. Standardswerediluted inassaybuffer andoxidized to thiochromesbyadding100mLofoxidation reagent.Samplesandstandardswere thenneutralizedwith100mLof 3 M phosphoric acid, followed by the addition of 15 mL methanol anddeproteinization using Amicon Ultra 0.5-mL 10K units. Aliquots (typically30 to150mL)of theflow-throughwereseparatedonanAltimaHPC18amidecolumn (15034.6mm;5mm;190Å;Alltech).The thiochromesweredetectedfluorometrically (excitation 367 nm, emission 435 nm). The mobile phase(1 mL/min) consisted of a gradient of potassium phosphate (140 mM, pH7.00)/12%methanol (A) to70%methanol inwater (B).Runsbeganwith100%A; the proportion of B was increased linearly to 50% during the first 10 min,then from50to70%during thenext2min,andfinally from70to100%during5 min. Values of kcat and Km were estimated by nonlinear fitting usingGraphPad Prism version 7.00.

Assays for hydrolysis of amino-HMP, N-formylamino-HMP, desthio-thiamin, thiamin, and ThDP were made in 50-mL reaction mixtures con-taining 50mMTris-HCl, pH 7.5, 1mMDTT, 500mMglycine betaine, 1mMsubstrate, and 60 mg of enzyme protein. Assays were run at 30°C for 1 to4h, stoppedon ice,mixedwith250mLwater, and thendeproteinizedat4°Cusing Amicon Ultra 0.5-mL 10K units. Samples (typically 100 mL) of theflow-through were analyzed by HPLC with UV detection as described(Zallot et al., 2014) except that the elution buffer was 10 mM potassiumphosphate, pH 6.6. The detection limit in these assays was 6 pmol min21

mg21 protein.

Thiamin Vitamer Analysis

Plant samples (;300 mg) were ground to a fine powder in liquid N2. E. coliBL21-CodonPlus (DE3)-RIPL cells (Agilent) harboring pET28b alone orencoding At5g32570, At5g32570-D317A, GRMZM2G148896, orGRMZM2G078283, His-tagged and minus their predicted targeting se-quences as above, were cultured in 10mL of LBmedium containing 50 mg/mL kanamycin at 37°C, with continuous shaking at 220 rpm. When OD600

reached 0.4, IPTG (final concentration 0.5 mM) was added and incubationwas continued until OD600 reached 1.0. Cells were then harvested, washedwith PBS, and frozen in liquid N2. To analyze thiamin and its phosphates,plant and bacterial samples were extracted in 500 mL of 7.2% perchloricacid by sonicating for 30 min in a bath and holding on ice for 15 min withperiodic vortex-mixing, then cleared by centrifugation (14,000g, 10 min,22°C) and converted to thiochrome derivatives followed by HPLC withfluorometric detection (Fraccascia et al., 2007; Hasnain et al., 2016).

Subcellular Localization

Full-length or N-terminally truncated At5g32470 and GRMZM2G148896cDNAs were cloned into pGEM4Z (Promega), and the constructs were

Thiamin Monophosphate Phosphatase 2693

verifiedbyDNA sequencing. In vitro transcription-translationwasperformedusingaTnTSP6CoupledWheatGermExtractSystem(Promega), accordingto the manufacturer’s instructions, with 1 mg of plasmid and 35-40 mCi[3H]leucine (115.8 Ci/mmol; Perkin-Elmer) as label. Samples were re-solved bySDS-PAGE (10%gel), and the gelswere impregnatedwith 2,5-diphenyloxazole before the bands were visualized by fluorography.

Full-length orN-terminally truncatedAt5g32470cDNA, or thepredictedmitochondrial targetingsequence,werecloned into theNcoI siteofpUC18/NcoI-mGFP, immediately 59of the open reading frame formonomeric GFP(Clark et al., 2009) to create peptide-mGFP fusions. The targeting regionsthat were removed, or fused directly to GFP, are shown in Figure 7. TheAgilent QuikChange II Kit was used as above to change the ATG codoncorresponding to the second methionine to TTG (leucine). The generatedplasmids containing the peptide-GFP fusions obtained were used astemplates for PCR amplification followed by cloning of the correspondingsequences into Gateway plasmid pDONR207 (Life Technologies) usingGateway BP Clonase II Enzyme Mix (Life Technologies). The constructswere then transferred to the destination vector pMDC32, harboring a dual35SCaMVpromoter andaNOSterminator (CurtisandGrossniklaus, 2003),using Gateway LR Clonase II enzyme mix. Construction of the pMDC32-based binary plasmid encoding Cherry-At1g55450, which encodes themonomeric Cherry fluorescent protein fused to the N terminus ofAt1g55450, an S-adenosyl-L-methionine-dependent methyltransferaseprotein that is localized in the outer membrane of mitochondria, has beendescribed previously (Marty et al., 2014). Primers used for each constructare given in Supplemental Table 2.

Agrobacterium-infiltrated tobacco leaves were processed for confocallaser scanning microscopy imaging as described previously (Cai et al.,2015). Microscopy images of transiently cotransformed tobacco leaveswere acquired using a Leica DM RBE microscope with a Leica 633 PlanApochromat oil-immersion objective, a Leica TCSSP2scanning head, andthe Leica TCS NT software package (Leica). GFP and Cherry were excitedwitha488-nmanda543-nm laser, respectively, andemissionfluorescencesignals were collected at 500 to 540 nm for GFP and 590 to 640 nm forCherry. All fluorophore emissions were collected sequentially and single-labeling experiments revealed no detectable crossover at the settingsused for data collection. Images were acquired as individual single opticalsections and all fluorescence images of cells shown are representative ofat least two separate experiments, including at least 15 independenttransformations. All figure compositions were generated using AdobePhotoshop CS and Illustrator CS2 (Adobe Systems).

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for the genes mentioned inthis article are as follows: TH2, At5g32470; and HAD phosphatase,At4g29530.

Supplemental Data

Supplemental Figure 1. Characterization of an At5g32470 T-DNAInsertional Mutant.

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

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

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

Supplemental Figure 5. Isolation of Recombinant At5g32470,GRMZM2G148896, and GRMZM2G078283 Proteins and Estimationof Native Molecular Mass.

Supplemental Figure 6. Alignment of the N-Terminal Regions of theAt5g32470, GRMZM2G148896, and GRMZM078283 Proteins and TheirOrthologs, and Mitochondrial Targeting Prediction Scores.

Supplemental Table 1. The Panel of 95 Phosphoesters Used inActivity Screens.

Supplemental Table 2. Oligonucleotide Primers Used in This Study.

ACKNOWLEDGMENTS

This research was supported by U.S. National Science Foundation AwardIOS-1444202 (to A.D.H.), by National Sciences and Engineering ResearchCouncil ofCanadaAward217291 (toR.T.M.), byNSERCStrategicNetworkGrant IBN (to A.F.Y.), and by an endowment from the C.V. Griffin Sr.Foundation. R.T.M. holds aUniversity ofGuelphResearchChair.We thankK.N. Allen for advice on HAD enzyme catalysis, J. Pillet for help in vectorconstruction, and M. Ziemak for technical support.

AUTHOR CONTRIBUTIONS

D.R.M. and A.D.H. designed research. M.M., R.Z., T.D.N., G.H., V.d.C.-L.,G.B., A.F.Y., J.F.G., D.R.M., and A.D.H. performed biochemical and ge-netic research. G.B. andA.F.Y. ran phosphatase activity screening assays.S.K.G., T.N.D.N., E.M.A., and R.T.M. carried out in vivo protein localizationstudies. All authors analyzed data. D.R.M., R.T.M, and A.D.H. wrote thearticle.

ReceivedJuly29, 2016; revisedSeptember 20, 2016; acceptedSeptember26, 2016; published September 27, 2016.

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DOI 10.1105/tpc.16.00600; originally published online September 27, 2016; 2016;28;2683-2696Plant Cell

Crécy-Lagard, Jesse F. Gregory III, Donald R. McCarty and Andrew D. HansonNguyen, Erin M. Anderson, Robert T. Mullen, Greg Brown, Alexander F. Yakunin, Valérie de

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