Balancing of B6 Vitamers Is Essential for PlantDevelopment and Metabolism in Arabidopsis

Maite Colinas,a Marion Eisenhut,b Takayuki Tohge,c Marta Pesquera,a Alisdair R. Fernie,c Andreas P.M. Weber,b

and Teresa B. Fitzpatricka,1

a Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerlandb Institute of Plant Biochemistry, Cluster of Excellence on Plant Science, Heinrich-Heine-University, 40225 Düsseldorf, GermanycMax-Planck-Institute for Molecular Plant Physiology, 14476 Potsdam-Golm, Germany

ORCID IDs: 0000-0001-7053-2983 (M.C.); 0000-0002-2743-8630 (M.E.); 0000-0003-1584-8402 (M.P.); 0000-0003-0970-4672 (A.P.M.W.);0000-0001-7694-5631 (T.B.F.)

Vitamin B6 comprises a family of compounds that is essential for all organisms, most notable among which is the cofactorpyridoxal 59-phosphate (PLP). Other forms of vitamin B6 include pyridoxamine 59-phosphate (PMP), pyridoxine 59-phosphate(PNP), and the corresponding nonphosphorylated derivatives. While plants can biosynthesize PLP de novo, they also havesalvage pathways that serve to interconvert the different vitamers. The selective contribution of these various pathways tocellular vitamin B6 homeostasis in plants is not fully understood. Although biosynthesis de novo has been extensivelycharacterized, the salvage pathways have received comparatively little attention in plants. Here, we show that the PMP/PNPoxidase PDX3 is essential for balancing B6 vitamer levels in Arabidopsis thaliana. In the absence of PDX3, growth anddevelopment are impaired and the metabolite profile is altered. Surprisingly, RNA sequencing reveals strong induction ofstress-related genes in pdx3, particularly those associated with biotic stress that coincides with an increase in salicylic acidlevels. Intriguingly, exogenous ammonium rescues the growth and developmental phenotype in line with a severe reduction innitrate reductase activity that may be due to the overaccumulation of PMP in pdx3. Our analyses demonstrate an importantlink between vitamin B6 homeostasis and nitrogen metabolism.

INTRODUCTION

Vitamin B6 is a water-soluble vitamin essential for all living or-ganisms. The term vitamin B6 collectively refers to six differentvitamers that have apyridine ring in commonbut differ in their 4ʹaswell as their 5ʹ moieties, comprising the alcohol pyridoxine (PN),the amine pyridoxamine (PM), the aldehyde pyridoxal (PL), andtheir 59 phosphorylated forms (pyridoxine 59-phosphate [PNP],pyridoxamine 59-phosphate [PMP], and pyridoxal 59-phosphate[PLP]) (Fitzpatrick et al., 2007). The PLP vitamer acts as a cofactorin numerous (over 140) enzymatic reactions involved in a variety ofprocesses (http://bioinformatics.unipr.it/cgi-bin/bioinformatics/B6db/home.pl). In addition, vitamin B6 has been shown to be anefficient reactive oxygen species quencher in vitro and is con-sidered to act in the same capacity in vivo (Bilski et al., 2000;Havaux et al., 2009). Furthermore, it was recently suggested thatPLP is important for proper folding of certain PLP-dependentenzymes, fulfilling a chaperone role (Cellini et al., 2014).

Biosynthesis denovoofPLP takesplace inbacteria, plants, andfungi, but not in animals, making it an essential compound in thediet of the latter. Two different pathways have been described forbiosynthesis de novo: the deoxyxylulose 5-phosphate (DXP)-dependent and the DXP-independent pathway (Fitzpatrick et al.,2007). The DXP-dependent pathway takes place in Escherichia

coli as well as some other members of the g-division of proteo-bacteria, uses DXP as an intermediate, and requires sevenenzymes to form PLP (Fitzpatrick et al., 2007). By contrast, theDXP-independent pathway involves the action of only twoenzymes,PDX1 and PDX2 (Tambasco-Studart et al., 2005), and takes placein the majority of bacteria, fungi, and plants (Ehrenshaft et al.,1999; Mittenhuber, 2001). PLP can also be generated from otherB6vitamers insalvagepathways,whicharepresent inall kingdomsof life including those thatdonotproducevitaminB6denovo, suchas animals (Ruiz et al., 2008). In the salvage pathways of the plantmodel Arabidopsis thaliana, PN, PM, and PL can be phosphor-ylatedbyakinase (namedSALTOVERLYSENSITIVE4 [SOS4]; Shiet al., 2002; Shi and Zhu, 2002), and the phosphorylated forms,PNP and PMP, can be converted into PLP by the action of theoxidase PDX3 (González et al., 2007; Sang et al., 2007, 2011)(Figure 1). Interestingly, the PDX3 enzyme consists of two do-mains; the PMP/PNP oxidase domain is located at the C terminusand is fused to an N-terminal epimerase that has recently beenimplicated in nicotinamide nucleotide repair (annotated NNRE)(Marbaix et al., 2011; Colinas et al., 2014; Niehaus et al., 2014). Inaddition, a pyridoxal reductase converting PL into PN, found origi-nally in yeast (Morita et al., 2004), has more recently been charac-terized in Arabidopsis (Herrero et al., 2011). Activities of (possiblyunspecific)phosphatases involved invitaminB6 interconversionhavebeen suggested to occur in plants also, but genes encoding theseenzymes have not been identified to date (Huang et al., 2011). Theselectivecontributionof thesevariouspathways tocellular vitaminB6

homeostasis in plants is not fully understood.Several Arabidopsis mutants completely or partly impaired in

vitamin B6 metabolism have been described. A complete loss of

1 Address correspondence to teresa.fitzpatrick@unige.ch.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: Teresa B. Fitzpatrick(teresa.fitzpatrick@unige.ch).www.plantcell.org/cgi/doi/10.1105/tpc.15.01033

The Plant Cell, Vol. 28: 439–453, February 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

vitamin B6 biosynthesis de novo is embryo lethal (Tambasco-Studart et al., 2005; Titiz et al., 2006), whilemutants in either oneofthe two catalyticPDX1genes (PDX1.1orPDX1.3) have short rootsand are stress hypersensitive (Chen and Xiong, 2005; Titiz et al.,2006; Wagner et al., 2006; Boycheva et al., 2015). The latterphenotypes are remarkably similar to mutants in the salvagepathway kinaseSOS4 (Rueschhoff et al., 2013), originally isolateddue to their salt sensitivity (Shi et al., 2002). Recently, the phe-notypes associated with pdx1.1 and pdx1.3 have been linkedprimarily to a perturbation in ethylene and auxin homeostasis(Boycheva et al., 2015), two phytohormones that require PLP asa cofactor for their biosynthesis. By contrast, mutants partiallyimpaired in the expression of the salvage pathway enzymePDX3were reported to bemorphologically indistinguishable fromwild-type plants when grown on soil under normal conditionsbut hypersensitive to high sucrose levels, high light, and chilling(González et al., 2007). An independent study employingknockdown lines using small interfering RNA constructs alsoreported no significant visible phenotype but hypersensitivity to

high light was confirmed (Sang et al., 2011). It was thus suggestedthat a minimum level of expression of PDX3 is sufficient for ho-meostasis,but theeffectof itscompleteabolishment isnotknown.In eukaryotes, PLP is needed as a cofactor in numerous sub-cellular compartments most notably associated with enzymesinvolved in amino acid metabolism, as well as phytohormonebiosynthesis, photorespiration, and others (Mooney andHellmann,2010). Biosynthesis of PLP de novo takes place in the cytosol,whereas the PLP salvage enzyme SOS4 has been reported tobe plastidic (Rueschhoff et al., 2013). An early study reporteda plastidic location for PDX3 (Sang et al., 2011), but it was morerecently demonstrated to localize to mitochondria (Colinas et al.,2014; Niehaus et al., 2014). Taken together, the contribution ofPDX3 in provisioning PLP within the subcellular pool and its role ingrowth and development remain to be elucidated.To further our understanding, we report here on the functional

relevance of PDX3 and demonstrate that it is essential for normalgrowth and development in Arabidopsis. Significantly, pdx3knockout mutants have an imbalance in B6 vitamer levels and

Figure 1. Scheme of B6 Vitamer Metabolism in Arabidopsis.

PLP can be biosynthesized de novo from ribose 5-phosphate (R5P), glyceraldehyde 3-phosphate (G3P), and glutamine by the catalytic action of PDX1 andPDX2 (orangepanel). A salvage pathwayalso operates (blue panel) inwhichPLPmay also beproduced fromeither PMPorPNP through the action of PDX3.The kinaseSOS4 is alsopart of this pathwayandcanphosphorylatePM,PN, orPL.Phosphatases (probably unspecific) perform the reverse reactions to thelatter. PLmay also be reduced to PN through the action of pyridoxal reductase (PLR1). PN and PLmay also be glycosylated, but the enzymes carrying outthese reactionsarenot known inplants. Inall cases, thecofactor formPLPmaybe transferred toconfer catalyticactivity toPLP-dependent enzymessuchastransaminases and decarboxylases. Modified from González et al. (2007).

440 The Plant Cell

a severe aberrant leaf phenotypeandshowdelayeddevelopment.Genetic complementation with PDX3 reverts the developmentalphenotype of pdx3mutants and restores vitamin B6 homeostasis.The B6 vitamer imbalance in pdx3 mutants results in grosschanges in themetabolite profile, notably toward steady stateamino acid levels. Most surprisingly, RNA sequencing revealsstrong induction of stress-related genes, particularly those as-sociated with biotic stress. This coincides with an increase insalicylic acid levels, implying that thepdx3mutant is constitutivelyactivated for defense. Intriguingly, exogenous ammonia rescuesthe pdx3 growth and developmental phenotype, as nitrate re-ductase activity is severely reduced. Overall, our analyses dem-onstrate an important link betweenB6 vitamer status and nitrogenmetabolism. In particular, the effect of the ratio of PMP to PLPcontent on the perceived nitrogen status of the plant is discussed.

RESULTS

PDX3 Is Required for Plant Vegetative Growthand Development

To provide insight into the functional relevance of PDX3, twoindependent T-DNA insertion lines (SALK_054167C and GK-260E03) were obtained from the available collections isolated tohomozygosity. PCR analysis established insertion of the T-DNAin intron 7 of SALK_054167C and in exon 4 of GK-260E03(Figure 2A). Analysis of the transcript level of PDX3 expressionby quantitative real-time RT-PCR (qPCR) with independentprimer pairs confirmed severely reduced expression (Figure 2B),although residual expression downstream and upstream of theT-DNA insertion site can be observed in GK-260E03 and

Figure 2. PDX3 Is Essential for Vegetative Growth and Development inArabidopsis.

(A)Genemodel ofPDX3withexonsdepictedasblackbars, intronsas lines,and the untranslated regions as gray bars. The regions corresponding to

the NNRE and PMP/PNP oxidase (POX) domains are as indicated. Thelocations of the T-DNA insertion in SALK_054167C (pdx3-3) and GK-260E03 (pdx3-4) are as depicted and were confirmed by genotyping andsequencing. Primer pairs indicated (Supplemental Table 2) were used forqPCR.(B)Quantitative analysis ofPDX3expression inmutant lines versus thewildtype (Col-0). Two sets of primer pairs were used (see [A]). Some residualexpression downstream of the insertion site can be observed in pdx3-4.The data are the average of three independent biological replicates withbars representing SE.(C) Immunochemical analysis of PDX3 levels indicates that the protein isnot detectable in pdx3 mutant lines compared with the wild type. Ex-pression is restored in transgenic pdx3 lines expressing PDX3 under thecontrol of the CaMV 35S promoter. Immunochemical analysis of ACTIN-2was used as a loading control.(D) Photographs of 21-d-old pdx3 lines compared with the wild type(Col-0), showing gross morphological defects during leaf development.Leaves display reduced leaf blade area, enhanced serration at leafmargins, and asymmetric leaf expansion. Notably, the convex, slightlyepinastic curvature typically observed in wild-type plants grown underthe same conditions is lost. Complementation of the phenotype isachieved in transgenic pdx3-3 and pdx3-4 expressing PDX3.(E) Photographs of the individual leaves of the pdx3 lines and wild-typeplants shown in (D). The severe curling of the leaves and asymmetric leafexpansion can be observed in pdx3-3 in particular. Plants were grownunder a 16-h photoperiod (120 mmol photons m22 s21) at 22°C and 8 h ofdarkness at 18°C.

B6 Vitamer Balance Is Required in Plants 441

SALK_054167C, respectively. However, no or extremely low levelsof protein could be detected in either mutant line by immuno-chemical analysis using an antibody specifically raised againstArabidopsis PDX3 (Figure 2C). By contrast, a strongprotein bandof;55 kD could be detected in wild-type protein extracts consistentwith theexpectedmolecularmassofmaturePDX3(52.2kD) (Colinaset al., 2014; Niehaus et al., 2014). Therefore, these two lines can beconsideredasstrongpdx3mutants.Given that twopartial knockoutorweakmutantsofpdx3havebeendescribedpreviously (Gonzálezet al., 2007) and annotated as pdx3-1 (SALK_060749C) and pdx3-2(SALK_149382C), we annotateSALK_054167Caspdx3-3andGK-260E03 as pdx3-4.

Interestingly, both pdx3-3 and pdx3-4 showed gross mor-phological defects during leaf development (Figures 2D and 2E).

Leaveshad reduced leaf bladearea andenhancedserration at leafmargins anddisplay asymmetric leaf expansion. Thus, the convexslightly epinastic curvature typically observed in wild-type plantsgrown under the same conditions was lost (Figure 2D). Moreover,severe curling of the leaves was observed at maturity (Figure 2E).These phenotypes were more pronounced in pdx3-3 comparedwith pdx3-4, consistent with a higher level of PDX3 transcriptthat would encode the PNP/PMP oxidase domain in the latter(Figure 2B) and were not observed in either pdx3-1 or pdx3-2(Supplemental Figure 1A), corroborating only partial knockdownstatus.Notably, duringearly development (up to the emergenceofthe second pair of true leaves), pdx3-3 and pdx3-4mutants wereindistinguishable from the wild type. Photoperiod affectedthe severity of the developmental phenotype, whichwasmost

Figure 3. PDX3 Is Essential for Timely Reproductive Development in Arabidopsis.

(A) Representative photographs of 27-d-old plants illustrating the early bolting phenotype of pdx3 mutant lines compared with the wild type (Col-0).Complementation of the phenotype is observed in transgenicpdx3 lines expressingPDX3under control of theCaMV35Spromoter (pdx3-3/35S-PDX3 andpdx3-4/35S-PDX3, respectively).(B)Bolting timeof the same lines as shown in (A). Thebolting timeofpdx3-3, in particular, is earlier than thewild type (Col-0), as indicatedby theasterisk, butrecovers in pdx3-3/35S-PDX3.(C) and (D)Timeof opening of the first flower andnumber of leaves at this stage in the same lines as shown in (A). The flowering time andnumber of leaves ofpdx3-3, in particular, are statistically different from the wild type (Col-0), as indicated by the asterisks, but recover in pdx3-3/35S-PDX3.(E) Reduced apical dominance is observed in pdx3-3, in particular, compared with the wild type (Col-0).(F) Increased number of emerging primary stems (arrows) observed in pdx3-3 compared with the wild type (Col-0).(G)Seedyield for all linesas in (A)measuredasweightof total seedsperplant, shownasboth freshweightanddryweight. Theyield is significantlydecreasedin pdx3-3.Plantsweregrownunder a 16-hphotoperiod (120µmol photonsm22 s21) at 22°Cand8hof darkness at 18°C.Statistical differences from thewild typewerecalculated by a two-tailed Student’s t test and indicated by an asterisk for P < 0.01. In all cases, error bars represent SE.

442 The Plant Cell

pronounced under continuous light or long days (16 h light) andless severe under a short photoperiod (8 h light) (SupplementalFigures 1B and 1C). To confirm that the phenotypic observationsof pdx3-3 and pdx3-4 were a consequence of PDX3 impairment,we transformed the latter mutants with a construct designed toexpress PDX3 under the control of the CaMV 35S promoter.Several lines inwhich theexpressionofPDX3was restored towild-type protein levels were isolated, examples of which are shown(Figure 2C, pdx3-3/35S-PDX3 and pdx3-4/35S-PDX3). In all ca-ses, the aberrant vegetative growth andmorphological defects inleaf development observed in both pdx3-3 and pdx3-4 were fullyrescued (Figure 2D). We therefore conclude that the loss of PDX3is responsible for the vegetative developmental defects observedin the pdx3-3 and pdx3-4 mutant lines.

PDX3 Is Also Required for Reproductive Development

In addition to the vegetative developmental phenotype, thetransition to the reproductive stage was earlier in pdx3-3 in par-ticular (Figure 3A), as judged from bolting time 23 d after germi-nation (DAG) under long-day conditions, comparedwithwild-typeplants (26 DAG) (Figure 3B). A weak early-bolting phenotype wasalso observed in pdx3-4 (25 DAG). Opening of the first flower inpdx3-3 (29 DAG) was 2 d earlier than in the wild type (31 DAG),while that of pdx3-4 was similar to the wild type under the sameconditions (Figure 3C). Consequently, the number of leavesin pdx3-3 was less than in the wild type at the equivalentdevelopmental stage (Figure 3D). Later during development,

reduced apical dominance was observed in pdx3-3 in particular,compared with wild-type plants (Figure 3E), consistent with anincreased number of primary stems (Figure 3F). There were fewersiliques overall; thus, there was a severe reduction in seed yield inpdx3-3 (by 60%) compared with the wild type (Figure 3G). Thepdx3-4 mutant also had reduced seed yield, but this was lesspronounced than that of pdx3-3 compared with the wild type.Furthermore, we performed reciprocal test crosses of pdx3-3 andpdx3-4, the T1 progeny of which displayed the weaker pdx3-4phenotypes, demonstrating that the mutants are allelic and thatthepdx3phenotype is recessive (Supplemental Figure2).Notably,these amalgamated pleiotropic phenotypes have not been de-scribed for a vitamin B6 mutant previously. Importantly, all of thereproductive phenotypes were fully restored to those of the wildtype in the genetically complemented pdx3-3 and pdx3-4 lines(Figure 3). Taken together, it must therefore be concluded thatPDX3 is needed for both vegetative and reproductive growth anddevelopment in Arabidopsis.

The B6 Vitamer Profile Is Imbalanced in pdx3 Mutants

To obtain further insight into the molecular basis of the pdx3mutant phenotypes, we determined the B6 vitamer profile ofrosette leaves using HPLC. In this way, the six different B6

vitamers canbe separated andquantified (Szydlowski et al., 2013)(Supplemental Figure 3). The analysis revealed that there wasa considerable perturbation in the levels of certain B6 vitamers inpdx3-3 and pdx3-4 compared with the wild type (Figure 4A).

Figure 4. PDX3 Is Required for Balancing B6 Vitamer Content.

(A) Individual B6 vitamer contents of lines as indicated, determined by HPLC from rosette leaves of 21-d-old plants. Contents of PMP and PNP are higher,whereas PLP contents are slightly lower in pdx3 mutant lines compared with the wild type (Col-0), confirming the enzymatic activity of PDX3 in vivo.ReversionofB6 vitamers towardwild-type levels is observed in the transgenic linesexpressingPDX3under control of theCaMV35Spromoter (pdx3-3/35S-PDX3 and pdx3-4/35S-PDX3, respectively).(B) Total B6 vitamer content of lines as indicated, demonstrating that the overall B6 content remains similar in all lines examined.Different letters indicate statistically significant differences between means for each vitamer as determined by a two-way ANOVA (corrected with Tukey’smultiple comparisons test) for P < 0.05. Plants were 21 d old grown under a 16-h photoperiod (120 mmol photons m22 s21) at 22°C and 8 h of darkness at18°C.

B6 Vitamer Balance Is Required in Plants 443

Specifically, the levels of PMP and PNP were increased, whilethere was a tendency for a decrease in the levels of PLP. This is inagreement with the assumed in vitro biochemical function ofPDX3, in that the enzyme oxidizes PNP and PMP to produce PLP(Sangetal., 2007). Thus, the resultsprovide invivoevidence for thefunction of PDX3 in Arabidopsis. Notably, the correspondingnonphosphorylated vitamers were also perturbed in a similarmanner, i.e., PL contents were reduced, whereas PM contentswereslightly increased (Figure4A). This is likelydue to theactionofnonspecificcellular phosphatases. Therefore, the ratio of thePMPplus PM to PLP plus PL contents (PMP+PM:PLP+PL) was con-siderably higher in pdx3-3 (1.46) and pdx3-4 (1.28) comparedwiththe wild type (0.57). The distribution of the B6 vitamer content waspartially restored toward wild-type levels in the genetically com-plementedpdx3-3 andpdx3-4mutant lines (Figure 4A); in turn, thePMP+PM to PLP+PL ratio was reduced (being 1.02 and 0.85 forpdx3-3/35S-PDX3 and pdx3-4/35S-PDX3, respectively). The in-complete rescue in vitamer levels in the complemented lines towild-type levels may be due to the slightly reduced expression ofPDX3 in these lines compared with the wild type (Figure 2C).Supplementation with PM or PL did not serve to exaggerate orrescue, respectively, the developmental phenotypes in pdx3 butdid rescue the pale leaf and stunted phenotype of the de novobiosynthesis mutant pdx1.3 (Supplemental Figure 4), as wasshown previously with PL and PN (Chen and Xiong, 2005; Titizet al., 2006). However, we noted that PM supplementation did notlead to an accumulation of PMP in the wild type but did in pdx3(even though the corresponding kinase SOS4 is present in both).This suggests that PMP contents are kept in check through theaction of PDX3. Significantly, we also found that the total vitaminB6content (i.e., thesumofall vitamers)wasnotchangedbetweenthemutant lines and thewild type (Figure4B). Taken together, thedata demonstrate the in vivo function of PDX3 and that thegrowth and developmental phenotypes observed in pdx3-3 andpdx3-4 are not a consequence of a change in the total content ofvitamin B6 but rather are due to an imbalance in the B6 vitamerprofile.

Loss of PDX3 Causes Major Alterations in Profiles ofMetabolites, Particularly Amino Acids

Given the involvement of PLP in numerous metabolic pathwaysanddue to the remarkablephenotypeof thepdx3mutants,wenextdetermined themetabolic profile of pdx3-3 and pdx3-4 to provideinsight into the predominant pathways affected. Rosette leaves of21-d-old plants were chosen for the analysis, a developmentaltime point when the leaf phenotype of the pdx3 mutants is par-ticularly pronounced. From this analysis, we were able to identify66 compounds (Supplemental Table 1), out of which 41 weresignificantly changed compared with the wild type (Figure 5A).Among thechangedcompounds in themutants, aminoacidswereparticularly prominent. Seventeen of the detected compoundscould be directly related to vitamin B6 metabolism because theyare either substrates or products of PLP-dependent reactions(Figure 5B; Supplemental Data Set 1). Of these compounds, 13were significantly changed in the mutants (Figures 5A and 5B).Thus, 76% of the compounds that could be detected and thatare directly related to vitamin B6 metabolism based on known

PLP-dependent reactions were altered in the pdx3-3 and pdx3-4mutants. Interestingly, only 3 of the 41 altered compounds showedsignificantly lower levels, while the rest were all overaccumulated(Figure 5A).

Defense Responses Are Activated in pdx3 Mutants

To further investigate and relate changes in the metabolite profileofpdx3 to global changes in gene expression,weperformedhigh-throughput RNA-seq analysis. To generate RNA-seq data sets,cDNA libraries were prepared of three independent biologicalreplicates of 21-d-old rosette leaves of pdx3-3, pdx3-4, and thewild type (Col-0) and subjected to Illumina sequencing. The re-sulting reads were mapped onto the reference Arabidopsis ge-nome assembly (TAIR10 version) and evaluated for changes ingene expression. A global analysis of changes in gene expressionin pdx3-3 and pdx3-4 showed that 1184 genes were induced atleast 2.0-fold and 254 genes were repressed at least 0.5-fold inboth pdx3 alleles (P < 0.05, see Methods for P value calculation),respectively (Figure 6A; Supplemental Data Set 2), out of a total of17,721 expressed genes. Importantly, while there ismisregulationof a unique set of transcripts in RNA-seq data sets of both pdx3-3and pdx3-4, the vast majority of the misregulated transcriptsoverlap (Figure 6A). Given the biochemical function of PDX3 insupplying PLP, we first searched for alteration of genes associ-atedwithPLP-dependentpathways.However,ouranalysis revealedthat of the 133 genes annotated as encoding PLP-dependent en-zymes in the Arabidopsis genome (Supplemental Data Set 1), themajority was not significantly altered. Specifically, while the tran-scriptome data set covered 87% of the annotated PLP-dependentgenes, only 11% of this gene category was affected (Figure 6B).Notably, a previous study reported on the upregulation of genesassociated with vitamin B6 metabolism in the pdx3-1 and pdx3-2mutant lines (González et al., 2007), specificallySOS4 of the salvagepathway and PDX1.1/PDX1.2/PDX1.3 of the biosynthesis de novopathway. By contrast, the transcriptome analysis performed in thisstudy with pdx3-3 and pdx3-4 showed that neither of the salvagepathway genes (SOS4 and PLR1) nor PDX2 of the biosynthesis denovo pathway were significantly changed and only slight changes(<2.0-fold) were observed for PDX1.1-1.3 (Figure 6C). Importantly,reads corresponding to expression of PDX3 were virtually absentafter the location of the T-DNA insertion in pdx3-3; however, readscouldbeobserved inpdx3-4after the locationof theT-DNAinsertion,although at lower levels than those of the wild type (SupplementalFigure 5). Therefore, we also specifically examined the expression ofthese genes by qPCR in pdx3-3 and pdx3-4 comparedwith thewildtype,whichservedtoconfirmthesefindings (Figure6D).OnlyPDX1.2was slightly upregulated in pdx3-3 in particular, compared with thewild type,whereasall othergeneswerenot significantlyaltered in themutants.Intriguingly, an analysis by Gene Ontology (GOslim; https://

www.arabidopsis.org/tools/bulk/go/index.jsp) indicated thatamong the induced or repressed genes the categories “responseto stress” and “response to abiotic or biotic stimulus” wereoverrepresented (Supplemental Data Set 2). In particular, genesassociatedwith pathogen defensewere strongly enriched amongthe upregulated transcripts (Figure 6E). Moreover, the list of up-regulated genes was considerably enriched (20%) for several

444 The Plant Cell

process categories associated specifically with salicylic acid-mediated defense and immunity responses, as well as functionalcategories associatedwith the transmembrane receptor activities(Supplemental Data Set 2). This is interesting considering theoveraccumulation of salicylic acid observed in the metabo-lite analysis of pdx3 mutants compared with the wild type(Figure 5A) and suggests that pdx3 is hyperactivated/primed fordefense. Furthermore, we could demonstrate by qPCR that keygenes in the salicylic acid-mediated defense pathway, such asPATHOGENESIS RELATED1 (PR1), ENHANCED DISEASESUSCEPTIBILITY1 (EDS1), and SUPPRESSOR OF npr1-1,CONSTITUTIVE1 (SNC1; specific to Col), were upregulated inboth pdx3-3 and pdx3-4 (Figure 6F). Thus, the data show thatdefense responses are activated in pdx3. Notably, among themisregulated genes there is enrichment for those encoding pro-teins localized to the plasma membrane or extracellular spaceaccording to a GOrilla analysis (http://cbl-gorilla.cs.technion.ac.il/)(Edenetal.,2009;SupplementalDataSet2).This furthersupports theobservationthatpdx3displaysanactivateddefenseresponse,asthelatter is associated with major rearrangements of the cell wall andplasma membrane (Malinovsky et al., 2014).

The Lack of PDX3 Renders the Plant with an Ammonium-Dependent Phenotype

Wenoted thatpdx3was indistinguishable from thewild typewhengrown in culture onMS saltmedium (Murashige andSkoog, 1962)and that the strong developmental phenotypes (Figures 2 and 3)became apparent only when pdx3was grown on soil, suggestinga nutrient-dependent response that was alleviated on MS saltmedium. Notably, MS salt medium contains both ammonium andnitrate as nitrogen sources, whereas nitrate is the predominantsource of nitrogen in soil due to microbial nitrification (Bloom,2015). In this context, it has previously been shown that there is aninteraction between defense responses and nitrogenmetabolism(Park et al., 2011; Wang et al., 2013). Specifically, the de-velopmental phenotypes observed in these aforementionedstudies were caused by a low ammonium-to-nitrate ratio andcould be repressed by simple ammonium supplementation. Wetherefore grew pdx3 on modified MS salt medium in which am-monium was omitted and indeed observed the aberrant leafphenotype, in particular with pdx3-3, previously seen only on soil(Figure 7A). As this suggested that a low ammonium-to-nitrateratio was responsible for the pdx3 phenotype on soil, we thenwatered soil-grown plants with different salts (ammonium nitrate,potassium nitrate, or ammonium chloride). When ammoniumwaspart of the salt mixture, pdx3 could not be distinguished from the

Figure 5. Metabolite Profiling Reveals Perturbations in pdx3.

(A) List of changed metabolites in pdx3-3 (white) and pdx3-4 (gray)compared with the wild type (Col-0). Compounds underlined are directlyrelated to PLP-dependent reactions.(B) Proportional Venn diagram of metabolites detected as a function ofvitamin B6 metabolism. Of the 66 compounds detected, 41 were changed

in abundance in pdx3 mutants, 13 of which could be directly related toPLP-dependentenzymaticreactions, theremaining28beingunrelated.Fourof the detected compounds that could be associated with PLP-dependentreactions were unchanged in the pdx3 mutants relative to the wild type.Notably, another71compounds related toPLP-dependent reactionscouldbe expected to be found in Arabidopsis (written in gray) but were notdetected in our analysis.Statistically significant changes compared with the wild type were cal-culated by a two-tailed Student’s t test for P < 0.05 in (A) (SupplementalTable1)andare indicatedbyanasterisk. Inall cases, errorbars represent SE.

B6 Vitamer Balance Is Required in Plants 445

Figure 6. RNA-Seq Analysis Reveals That Defense Responses Are Activated in pdx3-3 and pdx3-4.

(A) Venndiagrams demonstrating that themajority of transcriptsmisregulated in pdx3-3 and pdx3-4 overlap, according to the RNA-seq transcriptome analysis.(B) Pie charts demonstrating the percentage of genes altered in pdx3-3 and pdx3-4 that encode PLP-dependent enzymes. Note that the filtered tran-scriptome set contained 115 genes out of 133 genes that encode or are predicted to encode for PLP-dependent enzymes in the Arabidopsis genome.(C) Expression of genes encoding enzymes involved in vitamin B6 metabolism as depicted by the RNA-seq transcriptome analysis of pdx3-3 and pdx3-4.Only slight changes (<2.0-fold) were observed for PDX1.1-1.3, whereas expression of the other genes was not statistically significantly altered comparedwith Col-0, with the exception of PDX3, which was reduced (P < 0.05 calculated as described for the transcriptome analysis).(D)Expressionof thesamesetof vitaminB6metabolismgenesasshown in (B)measuredbyqPCRwithgene-specificprimers. Thedataare representativeofthreebiological replicatesand two technical repeatswith errorbars representing SE.Only theexpressionofPDX1.2wasstatistically significantly upregulated(P < 0.05, two-tailed Student’s t test) in both pdx3 mutants.(E) Gene Ontology terms related to pathogen defense and immune response are strongly overrepresented in the list of 2-fold upregulated genes in bothpdx3-3 and pdx3-4. A list of Gene Ontology terms and the associated genes found in each list and taken into account for this graph can be found inSupplemental Data Set 2, as well as a GOslim analysis.(F)Expression analysis of selected key genes involved in salicylic acid-mediated defense by qPCRanalysis. The data are presented asmeans6 SE for threebiological and three technical replicates. Statistically significant changes compared with the wild type were calculated with a two-tailed Student’s t test forP < 0.05 and are indicated by an asterisk.In all cases, the analyses were done on rosette leaves of 21-d-old plants grown under a 16-h photoperiod (120 mmol photons m22 s21) at 22°C and 8 h ofdarkness at 18°C.

446 The Plant Cell

wild type,whereas in theabsenceof ammoniumsupplementation,the developmental phenotypes could be clearly observed (Figure7B). That the response is specific to ammonium and not nitrate ishighlighted by exclusive rescue of the pdx3 phenotype uponwatering with ammonium chloride or ammonium nitrate but notwith potassium nitrate (Figure 7B).

To investigate a potential interaction between nitrogen me-tabolism and vitamin B6, the profile of B6 vitamers was analyzedin the ammonium-supplemented rescued lines. We found thatammonium supplementation led to considerable accumulation ofthe PMP vitamer, in both the wild type and the pdx3mutant lineseither when grown in culture or on soil (Figures 8A and 8B). Thissuggests that elevated PMP contents signify ammonium avail-ability. As a corollary, ammoniumavailabilitymaybeconstitutivelyperceived in pdx3 mutant lines (due to the abundance of PMP inthese mutants even in the absence of ammonium). This wouldserve to explain the dependence of pdx3mutants on ammoniumwhen grown on soil. Furthermore, PMP has been reported toinhibit nitrate reductase activity in vitro (Sims et al., 1968).Therefore, we measured this activity in the pdx3 lines and thewild type. Indeed, there was a considerable decrease in nitratereductase activity in both pdx3-3 and pdx3-4 (reduced by 60 to80% of that in the wild type) (Figure 8C). This would serve tocompound the dependence of pdx3 on ammonium. Takentogether, it can be concluded that pdx3 is an ammonium-dependent mutant, which possibly results from its constitutiveoveraccumulation of PMP.

DISCUSSION

In nature, the success of plants is determined by their abilityto withstand adverse environmental conditions. Consequently,plants have developed an arsenal of mechanisms to sense un-favorable situations and respond through a variety of signalingpathways that mediate a response. Inevitably, the launch of suchresponses requires resources and the plant often needs to makea trade-off that can affect yield (Todesco et al., 2010; Kerwin et al.,2015). In this study, our initial observations suggested that pdx3mutants were activated for defense and suffered a yield penalty.However, further investigation allowed us to demonstratethat these mutants were ammonium dependent and provided anunanticipated link between vitamin B6 homeostasis and nitrogenmetabolism.Specifically,perturbationof thebalanceofB6vitamersin the cell, in this case through disruption of the salvage pathwayenzyme PDX3, leads to accumulation of PMP and compromisesnitrogen metabolism, such that the plant becomes dependent onammonium. In the absence of the latter nutrient, the plant suffersstrong developmental impairments.

PDX3 Is Vital for Balancing B6 Vitamer Content

The functional significance of PDX3 becomes apparent in thisstudy, as loss-of-function mutants display severe impairments invegetative and reproductive development in Arabidopsis. Theseimpairments are more pronounced in the stronger allele pdx3-3than in theweakerpdx3-4 allele. Complementation of the aberrantphenotypes upon introduction of the PDX3 transgene validatesthe dependence of these phenotypes in the pdx3 mutant alleles.

From amolecular point of view, there is perturbation of vitamin B6

homeostasis in the pdx3 alleles examined. In particular, thePMP-to-PLP ratio is imbalanced in thesepdx3mutants (with PMPcontentsmarkedly increasedandPLPcontentsdecreased), asarethe ratios of the corresponding nonphosphorylated derivatives,although the total vitamin B6 content remains unchanged. No-tably, this observation validates the presumed in vivo biochemicalfunction of PDX3, which is the oxidation of PMP (or PNP) to thecofactor form PLP (Sang et al., 2007). In line with this, metaboliteprofiling revealed that a high proportion of detected compoundsaltered in the pdx3 mutants examined here could be directly re-lated to vitamin B6 metabolism. The perturbation of amino acidswas particularly prominent and may be due to the prevalence ofPLP-dependent enzymes for their metabolism, suggesting thatPDX3 is important for the supply of PLP to certain enzymes. In-triguingly, most of the metabolite compounds detected were in-creased rather than decreased. This is similar to what has beenobserved in other vitamin B6 biosynthesis mutants with the ex-ception of the amino acid glycine, which has been observed todecrease in other mutants of this pathway (Wagner et al., 2006;Raschke et al., 2011), although glycine accumulation has beenreported upon overexpression of the de novo biosynthesis genePDX1.3 (Leuendorf et al., 2010). Interestingly, we did not observesubstantial changes in the expression of the majority of the othergenes involved in the biosynthesis of PLP, such as PDX1.1/PDX1.3 and PDX2 involved in biosynthesis de novo (Tambasco-Studart et al., 2005)or thesalvagepathwaykinaseSOS4 (Shi et al.,2002; Sang et al., 2007). Thus, in contrast to a previous studybased on partial knockdown mutants of pdx3 (pdx3-1 and pdx3-2),we did not see evidence of a compensatory mechanism for thelack of PLP provision by PDX3. In other words, PDX3 playsaspecific role inArabidopsis that cannotbe fulfilledbyothergenesinvolved in vitaminB6metabolism.Notably in this context, PDX3 isthe only enzyme known in vitamin B6 metabolism that is able torecycle PLP from PMP or PNP. Significantly, it must be taken intoaccount that the PDX3 protein was below detection in the pdx3-3and pdx3-4 mutants described in this study, although pdx3-4carries a transcript that would include the PNP/PMP oxidasedomain, which despite not being detected at the protein level mayaccount for the weaker phenotype in this allele. Nonetheless, weconclude that pdx3-3 and pdx3-4 represent stronger mutantalleles than the promoter T-DNA insertion mutants pdx3-1 andpdx3-2, which were shown to still express PDX3 and have PNP/PMPoxidaseactivity (Gonzálezetal., 2007).Thismaysuggest thateven reduced amounts of PDX3 in pdx3-1 and pdx3-2 can sensePLP levels (by an as yet unknown mechanism) and transmit im-balances in homeostasis to the other vitamin B6 metabolic genesallowing them to compensate for a decrease in PDX3 activity andtherebymaintainhomeostasis.Thus, the functional significanceofthe gene only becomes apparent upon analyzing loss-of-functionalleles and is exemplified in this study through the growth anddevelopmental consequences described in the absence of PDX3.Despite the fact that PDX3 is necessary for vitamin B6 homeo-stasis, we did not observe changes in the expression of the vastmajority of PLP-dependent enzymes at the transcriptional level,suggesting thatPDX3effectsaremediatedat theposttranscriptionallevel. In this context, a recent study has highlighted that thestability and abundance of certain PLP-dependent enzymes may

B6 Vitamer Balance Is Required in Plants 447

be directly affected by a low PLP content (de la Torre et al., 2006;Cellini et al., 2014).

PDX3 Plays an Important Role in Nitrogen Metabolism

Although vitamin B6 has been previously implicated in defense(Denslow et al., 2005), the upregulation of specific pathogen re-sponse genes and increased salicylic acid content of pdx3 wasunanticipated and initially suggested that PDX3 contributes to

repression of pathogen responses. In fact, the stunted shootgrowth, early flowering, dwarfism, and reduced seed yield of pdx3are hallmarks of mutants that show constitutive defense re-sponses in the absence of pathogen infection, i.e., autoimmunemutants (Todesco et al., 2010; Alcázar and Parker, 2011).However, a key finding in this study was the demonstration thatwhereas the pdx3 mutants show strong developmental im-pairments on soil, they are phenotypically normal when grown inculture on MS salt medium (Murashige and Skoog, 1962). Animportant difference between these two growth conditions isthat the essential macronutrient nitrogen is provided in the formof nitrate and ammonium in MS salt medium but is presentmainly as nitrate in soil due to microbial nitrification (Bloom,2015). As ammonium supplementation completely abrogatedthe developmental impairments of pdx3 on soil, we thereforeconclude that the primary defect in this mutant is due toammonium insufficiency triggered by the abnormal accu-mulation of PMP and that the morphological irregularitiesand constitutive defense response are consequential to thisdefect.Given the biochemical function of PDX3 as a PMP oxidase, it is

interesting that several decades ago the ratio of PMP to PLP washypothesized to reflect the nitrogen state of a plant (Teixeira andDavies, 1974; Matsumoto et al., 1975). These studies have beenlargely overlooked since, but the ammonium transporter mutantamt1.1has recently been reported to havealterations in vitaminB6

content, although it was not clear which vitamer(s) were changing(Pastor et al., 2014). In a normal cell, the equilibriumbetweenPMPand PLP levels must be maintained. An excess of PMP may in-terfere with PLP-dependent transaminase activity (the formerbeing an intermediate in such reactions), and excess PLP can betoxic as it is a very reactive aldehyde that readily interacts withamines and thiols in the cell. From the data in this study, we canconclude that PDX3 plays an important role in this process; in itsabsence, PMP accumulates and PLP diminishes. In the vitamerprofiles, part of this misregulation is likely to be reflected in thelevels of the corresponding nonphosphorylated vitamers pro-duced through the action of nonspecific phosphatases, suchthat the ratio of PMP+PN contents to PLP+PL contents isconsiderably perturbed (i.e., elevated) in pdx3 compared withthe wild type. In this study, we also show that exogenous am-monium,but notnitrate, leads toaccumulationofPMP (Figure8),which allows us to propose a working model (Figure 9) on thepossible mechanism underlying our observations. First, wesuggest that the level of PMP is a chemical sign (either directly orindirectly) of the cellular ammonium status. Accumulation ofPMP indicates that ammonium availability is sufficient andconcomitantly inhibits nitrate reductase activity, preventingfurther reduction of nitrate to ammonium, which is energeticallywasteful and could in turn lead to ammonia toxicity. Understandard soil conditions, ammonium is derived from nitrate, asthe predominant nitrogen source due to microbial activity. Anyimbalance in PMP contents is offset by the activity of PDX3,whose job is to maintain the PMP-to-PLP equilibrium, thusensuring metabolic processes such as amino acid metabo-lism do not get perturbed. This is corroborated by the increasein PL contents (possibly derived from PLP) in the wild typebut not in pdx3 in the presence of an ammonium source

Figure 7. The pdx3 Mutants Are Dependent on Ammonium.

(A) On standard MS salt medium (Murashige and Skoog 1962), pdx3mutant leaves are phenotypically indistinguishable from the wild type.When grown on a version of this medium with ammonium omitted, thenarrowcurly leafdevelopmental phenotypebecomesvisible.Pictureswerecaptured of 21-d-old plants grown under a 16-h photoperiod (120 mmolphotons m22 s21) at 22°C and 8 h of darkness at 18°C.(B) Plants were germinated and grown on soil with water supplementedwith 50 mM of the indicated compounds or with water only as a controlevery nine days. Pictures were captured of 21-day-old plants grown undera 16-h photoperiod (120 mmol photons.m22 s21) at 22°C and 8 h ofdarkness at 18°C. Note that supplementation with either ammoniumchloride or ammonium nitrate complements the phenotype, whereassupplementation with potassium nitrate does not.

448 The Plant Cell

(Figures 8A and 8B). However, in the pdx3 mutants, theoveraccumulation of PMP may inadvertently signal ammo-nium availability constitutively to the plant, leading to consis-tent inhibition of nitrate reductase activity. Therefore, pdx3mutants grown on soil suffer ammonium insufficiency with theconsequential negative effect on growth and developmentand the inappropriate defense response. These defects can bealleviated by ammonium supplementation. Taken together, ourdata indicate that PMP levels reflect the ammonium status ofthe plant and that PDX3 is important for maintaining PMP andPLP homeostasis.

METHODS

General Plant Material and Growth Conditions

Arabidopsis thaliana (Columbiaecotype),pdx3-1 (SALK_060749C,N681169),pdx3-2 (SALK_149382C, N669245), and pdx3-3 (SALK_054167C,N659883)were obtained from the European Arabidopsis Stock Centre, whereas thepdx3-4 mutant (GK-260E03) was obtained from the GABI-KAT collection(Kleinboelting et al., 2012). The mutant lines pdx3-3 and pdx3-4 werebackcrossed towild-typeColumbia and reisolated basedon cosegregationofgenotypeandphenotype,whileensuring thepresenceofasingle insertionand used for all experiments described. Plants homozygous for pdx3-3 and

Figure 8. Interaction between Nitrogen Metabolism and Vitamin B6 Homeostasis.

(A)B6vitamerprofilesofplantsgrownonsoilwateredwitheither ammoniumchloride (NH4Cl), ammoniumnitrate (NH4NO3), orpotassiumnitrate (KNO3), or inthe absence of supplementation (H2O) as indicated. The analysis was performed on rosette leaves of 21-d-old plants grown under a 16-h photoperiod(120mmol photonsm22 s21) at 22°C and 8 h of darkness at 18°C. A considerable increase in PMP levels, in particular, is observed in both the wild type andpdx3 mutants upon ammonium supplementation. Different letters indicate statistically significant differences between means for each vitamer asdetermined by a two-way ANOVA (corrected with Tukey’s multiple comparisons test) for P < 0.05.(B)B6vitamerprofilesofplantsgrownonMSmediummodified tobe in theabsenceorpresenceofammonium (NH4

+). Theanalysiswasperformedon rosetteleaves of 10-d-old plants grown under a 16-h photoperiod (120 mmol photons m22 s21) at 22°C and 8 h of darkness at 18°C. Different letters indicatestatistically significant differences betweenmeans for each vitamer asdeterminedbya two-wayANOVA (correctedwith Tukey’smultiple comparisons test)for P < 0.05.(C)Nitrate reductase activity in extracts of rosette leavesof 19-d-oldplants grownonsoil under a16-hphotoperiod (120mmol photonsm22 s21) at 22°Cand8hofdarknessat18°C.Error bars represent SEof 18biological replicates.Activitywasstrongly reduced inpdx3mutants, as indicatedby theasterisks,whichrepresent statistically significant changes compared with the wild type calculated by a two-tailed Student’s t test for P < 0.01.

B6 Vitamer Balance Is Required in Plants 449

pdx3-4 were reciprocally crossed to generate the double pdx3-3 pdx3-4insertion mutants. The T1 progeny heterozygous for each of the pdx3insertions were analyzed for the purpose of a test cross. Unless statedotherwise,plantsweregrownunder long-daygrowthconditions (60%relativehumidity, 100 to 150 mmol photonsm22 s21 generated by fluorescent lamps[Philips Master T-D Super 80 18W/180] and 22°C for 16 h followed by 8 hof darkness at 18°C) and ambient CO2. Plant lines carrying the pdx3-3 andpdx3-4 T-DNA insertionswere verified byPCR analysis of genomic DNA (seeSupplemental Table 2 for oligonucleotides used). The absence of expressionof PDX3 in the respective lines was verified by qPCR and immunochemicalanalyses (see below).

Construction of Transgenic Plant Lines

Full-length PDX3 including the region encoding the transit peptide butexcluding the stop codon was amplified from cDNA of 10-d-old seedlingsusing a proofreading polymerase (Stratagene) and specific oligonucleo-tides (Supplemental Table 2). The amplified products were cloned intothe pENTR/D-TOPO vector using the pENTR/D-TOPO cloning kit (LifeTechnologies) according to the manufacturer’s instructions and se-quenced. A stop codon was introduced by site-directed mutagenesisusing specific oligonucleotides (Supplemental Table 2). Subsequently,the PDX3 insert was cloned into the Gateway destination vectorpB7YWG2 (Karimi et al., 2002) using LR clonase enzyme mix II (LifeTechnologies). The construct was introduced into Agrobacteriumtumefaciens strain C58 and used to transform pdx3-3 and pdx3-4Arabidopsis mutant plants by the floral dip method (Clough and Bent,1998). As the respective constructs contain the BAR gene, trans-formants were selected by resistance to BASTA and phenotypiccomplementation. Resistant plants were allowed to self-fertilize, andhomozygous lines were selected from the T3 generation according totheir segregation ratio for BASTA resistance.

Metabolite Profiling

Ten experimental replicates of Col-0, pdx3-3, and pdx3-4 were grownunder long-day conditions and harvested when they were 21 d old. Thematerial of four to six plantswas pooled and ground in liquid nitrogen usinga mortar and pestle. The resulting powder was split for standard gaschromatography-mass spectrometry and fatty acid analysis; the exactweight was recorded and the material was kept at280°C until extraction.Gas chromatography-mass spectrometry was performed exactly as de-scribed (Lisec et al., 2006) with peak annotation based on libraries ofauthentic standards (Kopka et al., 2005), while fatty acid analysis wasperformed as detailed by Raschke et al. (2011).

Transcriptome Analysis by RNA Sequencing

RNA was extracted from 21-d-old Col-0, pdx3-3, and pdx3-4 soil-grownplants under long-day conditions (three biological replicates) using thePureLink RNA Mini kit (Life Technologies) and poly(A)-selected using theTruSeq Stranded mRNA sample preparation LS kit (Illumina). The ninesamples were pooled and sequenced on an Illumina HiSeq 2000 se-quencing platform. The quality control of the resulting readswas donewithFastQC and the reads mapped to the Arabidopsis TAIR10 Ensembl ge-nome with the TopHat v.2 software. The number of reads and mappedreads per sample is given in Supplemental Data Set 2. For differentialexpression analysis, the gene features were counted with HTSeq v0.6p1(htseq-count) on the Ensembl TAIR 10 gene annotation. The statisticalanalysis was performed using the R/Bioconductor package EdgeR v3.4.2.The counts were normalized according to the library size and genes withless than one count per million were filtered out. The filtered data setcontained17,721genes. Thedifferentially expressedgene testsweredonewith a general linear model using a negative binomial distribution. TheP values of the differentially expressed genes were corrected for multipletesting with a 5% false discovery rate using Benjamini-Hochberg

Figure 9. Proposed Working Model for the Interaction of Vitamin B6 Homeostasis and Nitrogen Metabolism.

Growth on standard soil conditions, where nitrate (NO32) is the predominant nitrogen source and ammonium (NH4

+) is usually not abundant and freelyavailable. Left panel: NO3

2 is taken up and reduced to NH4+ through the actions of nitrate/nitrite (NO2

2) reductase (NR and NIR, respectively). The ac-cumulation of NH4

+ causes PMP levels to rise (as is observed in this study by NH4+ supplementation), which in turn inhibits NR activity regulating nitrate

reductionand thuspreventsaccumulationof toxic levelsofNH4+.PMPandPLP levels are inequilibriumduringvitaminB6metabolismandconstantly turning

over through theactionof PLP-dependent enzymes that arepredominantly involved in aminoacidmetabolism.Maintenanceof thisB6 vitamer homeostasisincludes the action of PDX3, which ensures the PMP and PLP equilibrium such that amino acidmetabolism is not perturbed. Right panel: In the absence ofPDX3, the PMP and PLP equilibrium is perturbed, manifested as the accumulation of PMP and diminishing PLP. The excess PMP inhibits NR, whichinadvertently leads to NH4

+ insufficiency in pdx3 disturbing metabolic homeostasis, in particular amino acid metabolism. Thus, these defects can bebypassed by feeding with NH4

+.

450 The Plant Cell

correction. Gene Ontology terms of the resulting gene set were retrievedfromTAIR. TheGOrilla analysiswasdoneusing thewhole transcriptomeasa background set.

Gene Expression Analysis by qPCR

Tissue sampleswere collected from21-d-old soil grownplants under long-day conditions. RNA was extracted using the RNA NucleoSpin Plant kit(Machery-Nagel) according to the manufacturer’s instructions. DNA wasremoved by an on-columnDNase digest during the RNA extraction and byan additional in-solution digest using DNase RQ1 (Promega) for 20 minaccording to the manufacturer’s instructions. Reverse transcription wasperformedusing1mg totalRNAasa templateandSuperscript II (Invitrogen)according to the instructions with the following modifications: oligo(dT)20primers stock concentration was 50 ng/mL and 0.5 mL Superscript II en-zymewasusedper reaction. qPCRwasperformed in 384-well plates on an7900HT Fast real-time PCR system (Applied Biosystems) using PowerSYBR Green master mix (Applied Biosystems) and the following amplifi-cation program: 10min denaturation at 95°C followedby 40 cycles of 95°Cfor 15 s and 60°C for 1min. The data were analyzed using the comparativecycle threshold method (22DCT) normalized to three reference genes,UBC21, SAND, and PP2A, as described (Vandesompele et al., 2002).Primers used are listed in Supplemental Table 2. Each experiment wasperformed with three biological and three technical replicates.

Immunochemical Analyses

Plant material was ground either using a tissue lyser (Qiagen) or a micro-pestle on liquid nitrogen. One volume of extraction buffer (50 mM sodiumphosphate buffer, pH 7.0, containing 5 mM b-mercaptoethanol, 10 mMEDTA, 0.5%Triton X-100 [v/v], 0.1mMPMSF, and 1% [v/v] complete plantprotease inhibitor cocktail [Sigma-Aldrich]) was added immediately to theground material and homogenized briefly, and samples were then kept onice. After centrifugation for 15 min at 16,000g at 4°C, the supernatant wasdecanted and the protein concentration determined by the Bradford assaykit (Bio-Rad) (Bradford, 1976). The samples were then separated by 10%SDS-PAGE loading30mgof total proteinper lane. Immunoblot analyses fordetection of PDX3 were performed with a-PDX3 (Colinas et al., 2014)(custom antibody raised against recombinant PDX3 in rabbits) at a 1:5000dilution as well as a peroxidase-conjugated goat anti-rabbit secondaryantibody (Bio-Rad) at a 1:5000 dilution, using the iBlot system (Invitrogen)and the SNAP i.d. 2.0 system (Millipore) as described previously (Colinaset al., 2014). As a loading control, a monoclonal antibody against ACTIN-2(Sigma-Aldrich; catalog number A0480, lot 065K4793) was used incombination with the peroxidase-conjugated goat anti-rabbit secondaryantibody (Bio-Rad; catalog number 1721019, lot 350003011), both ata 1:10,000 dilution. Chemiluminescence was detected using WesternBright ECL (Advansta) and captured using an ImageQuant LAS 4000system (GE Healthcare).

Vitamin B6 Analysis by HPLC

Vitamin B6 was extracted from 21-d-old soil-grown plants under long-dayconditions. Six rosettes from each line and experimental replicate wereharvested separately andgroundusingglass beadsanda tissue lyser. Twovolumes of 50 mM ammonium-acetate (pH 4) were added, the sampleswere vortexed vigorously for 10 min, centrifuged at 20,238g for 15 min,heated to 99°C for 3 min, and centrifuged as before. The resulting su-pernatant was decanted and used for analysis in two runs, using 10- and50-mL injection volumes. Separation anddetectionofB6 vitamers byHPLCwas performed as described previously (Szydlowski et al., 2013). Allstandards except PNP were purchased from Sigma-Aldrich. PNP is notcommercially available and was produced enzymatically using PN asa substrate and recombinant Escherichia coli PdxK (Park et al., 2004).

Growth under Different Ammonium Conditions

For culture in vitro, seedswere surface-sterilizedwith ethanol and sownonplates containing modified MSmedium (Murashige and Skoog 1962)(Sigma-Aldrich; containing no ammonium nitrate) and 0.55% agar(Duchefa). For growth in vitro including ammonium, modified MS sup-plementedwith 10.3mMammoniumchloridewasused. For growthonsoil,seeds were sown on soil soaked with water or water supplemented witheither ammonium chloride, ammoniumnitrate, or potassium nitrate (50mM)and subsequently watered with the described solutions every 10 d.

Measurement of Nitrate Reductase Activity

Nitrate reductaseassayswereperformedasdescribedpreviously (Yuetal.,1998; Park et al., 2011). Whole shoots from 19-d-old plants grown on soilunder long-day conditions were harvested in liquid nitrogen and groundwith glass beads on a tissue lyser (Qiagen). Then, 50 mL extraction buffer(250mMTris-HCl, pH 8.0, containing 1mMEDTA, 5 µMFAD, 1 µMsodiummolybdate, 3 mM DTT, 0.1% BSA, 12 mM b-mercaptoethanol, and 1%plant protease inhibitor cocktail [Sigma-Aldrich]) was added per 25 mgtissue. Thesampleswerekept on ice andcentrifugedat 13,000g for 15min.Fifteenmicroliters of the supernatant wasmixedwith 85mL reaction buffer(100 mM sodium phosphate buffer, pH 7.4, containing 40 mM sodiumnitrate and 10 mM NADH) and incubated in the dark at 25°C. After 2 h, thereaction was stopped by the addition of 25 mL 1% (w/v) sulfanilamide and25 mL 0.05% (w/v) napthylethylenediamine. As a background control,samples were mixed as above but the reaction was stopped immediately.The absorbance at 540 nm was then measured in a 96-well plate reader(BioTek Synergy 2).

B6 Vitamer Supplementation Experiments

SupplementationexperimentswithPMandPLwereperformedbywateringplants on soil with a solution containing 500mMof the respective vitamers.

Accession Numbers

Sequence data from this article can be found in the EMBL/GenBank/TAIRdata libraries under the following Arabidopsis Genome Initiative locusidentifiers: PDX3, At5g49970; SOS4, At5g37850; PLR1, At5g53580;PDX1.1, At2g38230;PDX1.2, At3g16050;PDX1.3, At5g01410;PDX2,At5g60540; PR1, At2g14610; EDS1, At3g48090; SNC1, At4g16890;UBC21, At5g25760; SAND, At2g28390; and PP2AA3, At1g13320. AllRNA-seq datagenerated in thisstudyweredeposited in theGeneExpressionOmnibus under accession number GSE77428.

Supplemental Data

Supplemental Figure 1. Photoperiod affects the severity of the pdx3developmental phenotype.

Supplemental Figure 2. Reciprocal test crosses of pdx3-3 and pdx3-4.

Supplemental Figure 3. B6 vitamer profile of Arabidopsis extracts.

Supplemental Figure 4. Supplementation experiments with PM or PL.

Supplemental Figure 5. RNA sequencing reads corresponding toPDX3 expression in pdx3-3 and pdx3-4.

Supplemental Table 1. List of metabolites detected in Arabidopsis lines.

Supplemental Table 2. Oligonucleotide primers used in this study.

Supplemental Data Set 1. Arabidopsis genes predicted to encodePLP-dependent enzymes.

Supplemental Data Set 2. RNA sequencing analysis of pdx3-3 andpdx3-4 compared with the wild type.

B6 Vitamer Balance Is Required in Plants 451

ACKNOWLEDGMENTS

We thank Mylène Docquier and Natacha Civic from the iGE3 GenomicsPlatform (University of Geneva) for performing the RNA-seq. Financialsupport is gratefully acknowledged from the Swiss National Science Founda-tion (Grant 31003A-141117/1) to T.B.F. as well as the University of Geneva.We also thank the Ernest Boninchi Foundation and the Ernst and LucieSchmidheiny Foundation for financial contributions. Additional financial sup-port is gratefully acknowledged from the European Molecular Biology Orga-nization (ASTF485-2014) toM.C.M.E.andA.P.M.W.appreciatesupportby theDeutsche Forschungsgemeinschaft (WE2231/8-2 and EXC 1028). We thanktheEuropeanArabidopsisStockCentre for seedsofSALK_060749C (pdx3-1),SALK_149382C (pdx3-2), and SALK_054167C (pdx3-3), GABI-KAT forGK-260E03 (pdx3-4), and Svetlana Boycheva for critical reading of the article.

AUTHOR CONTRIBUTIONS

T.B.F. and M.C. designed the research. M.C., M.E., T.T., and M.P. per-formed the research. M.C., M.E., T.T., M.P., A.R.F., A.P.M.W., and T.B.F.analyzed data. M.C. and T.B.F. wrote the article with input from all otherauthors.

Received December 10, 2015; revised January 19, 2016; accepted Feb-ruary 2, 2016; published February 8, 2016.

REFERENCES

Alcázar, R., and Parker, J.E. (2011). The impact of temperature onbalancing immune responsiveness and growth in Arabidopsis.Trends Plant Sci. 16: 666–675.

Bilski, P., Li, M.Y., Ehrenshaft, M., Daub, M.E., and Chignell, C.F.(2000). Vitamin B6 (pyridoxine) and its derivatives are efficient sin-glet oxygen quenchers and potential fungal antioxidants. Photo-chem. Photobiol. 71: 129–134.

Bloom, A.J. (2015). The increasing importance of distinguishingamong plant nitrogen sources. Curr. Opin. Plant Biol. 25: 10–16.

Boycheva, S., Dominguez, A., Rolcik, J., Boller, T., and Fitzpatrick,T.B. (2015). Consequences of a deficit in vitamin B6 biosynthesis denovo for hormone homeostasis and root development in Arabi-dopsis. Plant Physiol. 167: 102–117.

Bradford, M.M. (1976). A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principle ofprotein-dye binding. Anal. Biochem. 72: 248–254.

Cellini, B., Montioli, R., Oppici, E., Astegno, A., and Voltattorni,C.B. (2014). The chaperone role of the pyridoxal 59-phosphate andits implications for rare diseases involving B6-dependent enzymes.Clin. Biochem. 47: 158–165.

Chen, H., and Xiong, L. (2005). Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxida-tive stresses. Plant J. 44: 396–408.

Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation of Arabidopsis thaliana.Plant J. 16: 735–743.

Colinas, M., Shaw, H.V., Loubéry, S., Kaufmann, M., Moulin, M.,and Fitzpatrick, T.B. (2014). A pathway for repair of NAD(P)H inplants. J. Biol. Chem. 289: 14692–14706.

de la Torre, F., De Santis, L., Suárez, M.F., Crespillo, R., andCánovas, F.M. (2006). Identification and functional analysis ofa prokaryotic-type aspartate aminotransferase: implications forplant amino acid metabolism. Plant J. 46: 414–425.

Denslow, S.A., Walls, A.A., and Daub, M.E. (2005). Regulation ofbiosynthetic genes and antioxidant properties of vitamin B6vitamers during plant defense responses. Physiol. Mol. PlantPathol. 66: 244–255.

Eden, E., Navon, R., Steinfeld, I., Lipson, D., and Yakhini, Z. (2009).GOrilla: a tool for discovery and visualization of enriched GO termsin ranked gene lists. BMC Bioinformatics 10: 48.

Ehrenshaft, M., Bilski, P., Li, M.Y., Chignell, C.F., and Daub, M.E.(1999). A highly conserved sequence is a novel gene involved inde novo vitamin B6 biosynthesis. Proc. Natl. Acad. Sci. USA 96:9374–9378.

Fitzpatrick, T.B., Amrhein, N., Kappes, B., Macheroux, P., Tews, I.,and Raschle, T. (2007). Two independent routes of de novovitamin B6 biosynthesis: not that different after all. Biochem. J.407: 1–13.

González, E., Danehower, D., and Daub, M.E. (2007). Vitamer levels,stress response, enzyme activity, and gene regulation of Arabidopsislines mutant in the pyridoxine/pyridoxamine 59-phosphate oxidase(PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6salvage pathway. Plant Physiol. 145: 985–996.

Havaux, M., Ksas, B., Szewczyk, A., Rumeau, D., Franck, F.,Caffarri, S., and Triantaphylidès, C. (2009). Vitamin B6 deficientplants display increased sensitivity to high light and photo-oxidativestress. BMC Plant Biol. 9: 130.

Herrero, S., González, E., Gillikin, J.W., Vélëz, H., and Daub, M.E.(2011). Identification and characterization of a pyridoxal reductaseinvolved in the vitamin B6 salvage pathway in Arabidopsis. PlantMol. Biol. 76: 157–169.

Huang, S., Zeng, H., Zhang, J., Wei, S., and Huang, L. (2011).Characterization of enzymes involved in the interconversions ofdifferent forms of vitamin B(6) in tobacco leaves. Plant Physiol.Biochem. 49: 1299–1305.

Karimi, M., Inzé, D., and Depicker, A. (2002). GATEWAY vectors forAgrobacterium-mediated plant transformation. Trends Plant Sci. 7:193–195.

Kerwin, R., et al. (2015). Natural genetic variation in Arabidopsisthaliana defense metabolism genes modulates field fitness. eLife 4:10.7554/eLife.05604.

Kleinboelting, N., Huep, G., Kloetgen, A., Viehoever, P., andWeisshaar, B. (2012). GABI-Kat SimpleSearch: new features ofthe Arabidopsis thaliana T-DNA mutant database. Nucleic AcidsRes. 40: D1211–D1215.

Kopka, J., et al. (2005). GMD@CSB.DB: The Golm MetabolomeDatabase. Bioinformatics 21: 1635–1638.

Leuendorf, J.E., Osorio, S., Szewczyk, A., Fernie, A.R., andHellmann, H. (2010). Complex assembly and metabolic profilingof Arabidopsis thaliana plants overexpressing vitamin B₆ biosynthesisproteins. Mol. Plant 3: 890–903.

Lisec, J., Schauer, N., Kopka, J., Willmitzer, L., and Fernie, A.R.(2006). Gas chromatography mass spectrometry-based metaboliteprofiling in plants. Nat. Protoc. 1: 387–396.

Malinovsky, F.G., Fangel, J.U., and Willats, W.G. (2014). The role ofthe cell wall in plant immunity. Front. Plant Sci. 5: 178.

Marbaix, A.Y., Noël, G., Detroux, A.M., Vertommen, D., VanSchaftingen, E., and Linster, C.L. (2011). Extremely conservedATP- or ADP-dependent enzymatic system for nicotinamidenucleotide repair. J. Biol. Chem. 286: 41246–41252.

Matsumoto, H., Hirasawa, E., Kawano, S., and Takahashi, E.(1975). Some regulative properties of glutamine synthetase incucumber leaves. Soil Sci. Plant Nutr. 21: 379–389.

Mittenhuber, G. (2001). Phylogenetic analyses and comparativegenomics of vitamin B6 (pyridoxine) and pyridoxal phosphatebiosynthesis pathways. J. Mol. Microbiol. Biotechnol. 3: 1–20.

452 The Plant Cell

Mooney, S., and Hellmann, H. (2010). Vitamin B6: Killing two birdswith one stone? Phytochemistry 71: 495–501.

Morita, T., Takegawa, K., and Yagi, T. (2004). Disruption of the plr1+gene encoding pyridoxal reductase of Schizosaccharomycespombe. J. Biochem. 135: 225–230.

Murashige, T., and Skoog, F. (1962). A revised medium for rapid growthof bioassays with tobacco tissue culture. Physiol. Plant. 15: 473–497.

Niehaus, T.D., Richardson, L.G., Gidda, S.K., ElBadawi-Sidhu, M.,Meissen, J.K., Mullen, R.T., Fiehn, O., and Hanson, A.D. (2014).Plants utilize a highly conserved system for repair of NADH andNADPH hydrates. Plant Physiol. 165: 52–61.

Park, B.S., Song, J.T., and Seo, H.S. (2011). Arabidopsis nitrate re-ductase activity is stimulated by the E3 SUMO ligase AtSIZ1. Nat.Commun. 2: 400.

Park, J.-H., Burns, K., Kinsland, C., and Begley, T.P. (2004). Char-acterization of two kinases involved in thiamine pyrophosphate andpyridoxal phosphate biosynthesis in Bacillus subtilis: 4-amino-5-hydroxymethyl-2methylpyrimidine kinase and pyridoxal kinase.J. Bacteriol. 186: 1571–1573.

Pastor, V., Gamir, J., Camañes, G., Cerezo, M., Sánchez-Bel, P., andFlors, V. (2014). Disruption of the ammonium transporter AMT1.1 altersbasal defenses generating resistance against Pseudomonas syringaeand Plectosphaerella cucumerina. Front. Plant Sci. 5: 231.

Raschke, M., Boycheva, S., Crèvecoeur, M., Nunes-Nesi, A., Witt, S.,Fernie, A.R., Amrhein, N., and Fitzpatrick, T.B. (2011). Enhancedlevels of vitamin B(6) increase aerial organ size and positively affectstress tolerance in Arabidopsis. Plant J. 66: 414–432.

Rueschhoff, E.E., Gillikin, J.W., Sederoff, H.W., and Daub, M.E.(2013). The SOS4 pyridoxal kinase is required for maintenance ofvitamin B6-mediated processes in chloroplasts. Plant Physiol. Bio-chem. 63: 281–291.

Ruiz, A., García-Villoria, J., Ormazabal, A., Zschocke, J., Fiol, M.,Navarro-Sastre, A., Artuch, R., Vilaseca, M.A., and Ribes, A.(2008). A new fatal case of pyridox(am)ine 59-phosphate oxidase(PNPO) deficiency. Mol. Genet. Metab. 93: 216–218.

Sang, Y., Barbosa, J.M., Wu, H., Locy, R.D., and Singh, N.K. (2007).Identification of a pyridoxine (pyridoxamine) 59-phosphate oxidasefrom Arabidopsis thaliana. FEBS Lett. 581: 344–348.

Sang, Y., Locy, R.D., Goertzen, L.R., Rashotte, A.M., Si, Y., Kang, K.,and Singh, N.K. (2011). Expression, in vivo localization and phyloge-netic analysis of a pyridoxine 59-phosphate oxidase in Arabidopsisthaliana. Plant Physiol. Biochem. 49: 88–95.

Shi, H., Xiong, L., Stevenson, B., Lu, T., and Zhu, J.-K. (2002). TheArabidopsis salt overly sensitive 4 mutants uncover a critical role forvitamin B6 in plant salt tolerance. Plant Cell 14: 575–588.

Shi, H., and Zhu, J.-K. (2002). SOS4, a pyridoxal kinase gene, is requiredfor root hair development in Arabidopsis. Plant Physiol. 129: 585–593.

Sims, A.P., Folkes, B.F., and Bussey, A.H. (1968). Mechanisms in-volved in the regulation of nitrogen assimilation in micro-organismsand plants. In Recent Aspects of Nitrogen Metabolism in Plants, E.J.Hewitt and C.V. Cutting, eds (New York: Academic Press), pp. 15–63.

Szydlowski, N., Bürkle, L., Pourcel, L., Moulin, M., Stolz, J., andFitzpatrick, T.B. (2013). Recycling of pyridoxine (vitamin B6) byPUP1 in Arabidopsis. Plant J. 75: 40–52.

Tambasco-Studart, M., Titiz, O., Raschle, T., Forster, G., Amrhein,N., and Fitzpatrick, T.B. (2005). Vitamin B6 biosynthesis in higherplants. Proc. Natl. Acad. Sci. USA 102: 13687–13692.

Teixeira, A.R.N., and Davies, D.D. (1974). The control of plant glu-tamate dehydrogenase by pyridoxal-59-phosphate. Phytochemistry13: 2071–2079.

Titiz, O., Tambasco-Studart, M., Warzych, E., Apel, K., Amrhein,N., Laloi, C., and Fitzpatrick, T.B. (2006). PDX1 is essential forvitamin B6 biosynthesis, development and stress tolerance inArabidopsis. Plant J. 48: 933–946.

Todesco, M., et al. (2010). Natural allelic variation underlying a majorfitness trade-off in Arabidopsis thaliana. Nature 465: 632–636.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N.,De Paepe, A., and Speleman, F. (2002). Accurate normalization ofreal-time quantitative RT-PCR data by geometric averaging ofmultiple internal control genes. Genome Biol. 3: H0034.

Wagner, S., Bernhardt, A., Leuendorf, J.E., Drewke, C.,Lytovchenko, A., Mujahed, N., Gurgui, C., Frommer, W.B.,Leistner, E., Fernie, A.R., and Hellmann, H. (2006). Analysis ofthe Arabidopsis rsr4-1/pdx1-3 mutant reveals the critical function ofthe PDX1 protein family in metabolism, development, and vitaminB6 biosynthesis. Plant Cell 18: 1722–1735.

Wang, H., Lu, Y., Liu, P., Wen, W., Zhang, J., Ge, X., and Xia, Y.(2013). The ammonium/nitrate ratio is an input signal in the tem-perature-modulated, SNC1-mediated and EDS1-dependent auto-immunity of nudt6-2 nudt7. Plant J. 73: 262–275.

Yu, X., Sukumaran, S., and Mrton, L. (1998). Differential expressionof the Arabidopsis nia1 and nia2 genes. cytokinin-induced nitratereductase activity is correlated with increased nia1 transcriptionand mRNA levels. Plant Physiol. 116: 1091–1096.

B6 Vitamer Balance Is Required in Plants 453

DOI 10.1105/tpc.15.01033; originally published online February 8, 2016; 2016;28;439-453Plant Cell

Weber and Teresa B. FitzpatrickMaite Colinas, Marion Eisenhut, Takayuki Tohge, Marta Pesquera, Alisdair R. Fernie, Andreas P.M.

Vitamers Is Essential for Plant Development and Metabolism in Arabidopsis6Balancing of B

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