phosphatidylinositol 3-phosphate binding protein atph1 ...phosphatidylinositol 3-phosphate–binding...

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Phosphatidylinositol 3-phosphatebinding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis Astrid Agorio a,1 , Jérôme Giraudat a , Michele Wolfe Bianchi a,b , Jessica Marion a , Christelle Espagne a , Loren Castaings c , Françoise Lelièvre a , Catherine Curie c , Sébastien Thomine a,2 , and Sylvain Merlot a,2 a Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université ParisSud, Université Paris-Saclay, 91198, Gif-surYvette cedex, France; b Unité de Formation et de Recherche Sciences et Technologie, Université Paris-Est Créteil Val de Marne, 94010 Créteil, France; and c Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS, UMR 5004, Institut de Biologie Intégrative des Plantes, Montpellier, France Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved March 13, 2017 (received for review February 23, 2017) Too much of a good thingperfectly describes the dilemma that living organisms face with metals. The tight control of metal ho- meostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings re- quire the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient pheno- type of nramp3nramp4. Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 func- tion leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain pro- tein as a regulator of plant metal transporter localization, provid- ing evidence that PH domain proteins may be effectors of PI3P for protein sorting. metal transport | NRAMP | vacuole | late endosome | phosphatidylinositol 3-phosphate T he ability of iron (Fe) to switch between different oxidation states in cells is used in all biological kingdoms to catalyze essential biochemical reactions. Fe is found in the form of the Fe-S cluster, Heme, or Oxo di-iron as a cofactor in the catalytic center of a plethora of proteins involved in oxygen binding, electron transport chains, or DNA metabolism (1). As a conse- quence, Fe deficiency leads to severe symptoms in living or- ganisms including anemia in humans or chlorosis and growth inhibition in plants. On the other hand, the excess of Fe is del- eterious for cells because it may lead to the production of highly reactive oxygen species through the Fenton reaction. Therefore all organisms need to regulate Fe uptake and storage tightly according to their requirements. Fe homeostasis has been studied extensively in plants because of its strong impact on photosyn- thesis and therefore on biomass production and also because Fe biofortification of food crops is an important challenge to coun- teract Fe deficiency affecting billions of human beings (2, 3). In dicotyledonous plants, the ferric iron (Fe 3+ ) available as soluble chelates in soils is reduced to ferrous iron (Fe 2+ ) at the root surface by a plasma membrane-bound ferric-chelate re- ductase known as AtFRO2in Arabidopsis thaliana (4). Fe 2+ then is transported into root cells by the high-affinity iron- regulated transporter 1 (IRT1) from the ZIP/IRT family that is expressed in root epidermal cells in response to Fe starvation (5, 6). The natural resistance-associated macrophage protein1 from A. thaliana, AtNRAMP1, a homolog of the human DMT1 transporter, is able to transport Fe and manganese (Mn) when expressed in yeast (7, 8). In Arabidopsis, AtNRAMP1 was shown to mediate high-affinity Mn uptake (9) as well low-affinity Fe uptake (10). In photosynthetic cells, most of the cellular Fe is found in chloroplasts, within components of the photosynthetic electron transport chain, or bound to ferritin (2, 11). The vacuole is also an important site for Fe compartmentalization. In the A. thaliana embryo, Fe is stored in the vacuole of endodermal cells under the action of the VIT1 transporter of the CCC1 family (1214). The Fe pool stored in the vacuole of endodermal cells is remo- bilized during germination by two homologous vacuolar metal transporters, AtNRAMP3 and AtNRAMP4. The nramp3nramp4 double mutant is unable to develop into photosynthetic seedlings when germinated under Fe-limiting conditions because seed Fe is trapped in the vacuole of endodermal cells (13, 15). The nramp3nramp4 mutant is also hypersensitive to Cd and Zn for root growth, likely as a consequence of a globally unbalanced metal homeostasis (1618). Genetic analyses in yeast revealed that the regulation of metal homeostasis depends mainly on metal transporters and on the vacuolar protein-sorting pathway regulating the cellular traf- ficking of these transporters (1921). The main Mn transporter in yeast, Smf1p, a homolog of AtNRAMP1, localizes on both the plasma membrane and endosomes under Mn-sufficient condi- tions. The endosomal fraction of Smf1p is targeted to the vacuole Significance Metal homeostasis is essential for living organisms. Metal transporters play key roles in metal uptake and compartmen- talization. The tight regulation of metal homeostasis thus de- pends on accurate targeting of these metal transporters. Although the main metal transporters in plants have been identified, the mechanisms involved in their trafficking are still poorly understood. This study reveals that AtPH1, a pleckstrin homology (PH) domain containing protein binding to phos- phatidylinositol 3-phosphate (PI3P), controls the subcellular localization of the iron and manganese transporter AtNRAMP1. Our results further indicate that, in addition to proteins con- taining the FYVE and PHOX domains, proteins containing the PH domain can decode the PI3P signal in endosomal function. Author contributions: A.A., S.T., and S.M. designed research; A.A., J.G., M.W.B., J.M., C.E., L.C., F.L., S.T., and S.M. performed research; A.A., M.W.B., J.M., C.C., S.T., and S.M. ana- lyzed data; and M.W.B., S.T., and S.M. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 Present address: Department of Molecular Biology, Clemente Estable Biological Research Institute, CP 11600 Montevideo, Uruguay. 2 To whom correspondence may be addressed. Email: [email protected] or [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1702975114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1702975114 PNAS Early Edition | 1 of 10 PLANT BIOLOGY PNAS PLUS Downloaded by guest on March 16, 2020

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Page 1: Phosphatidylinositol 3-phosphate binding protein AtPH1 ...Phosphatidylinositol 3-phosphate–binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis

Phosphatidylinositol 3-phosphate–binding proteinAtPH1 controls the localization of the metaltransporter NRAMP1 in ArabidopsisAstrid Agorioa,1, Jérôme Giraudata, Michele Wolfe Bianchia,b, Jessica Mariona, Christelle Espagnea, Loren Castaingsc,Françoise Lelièvrea, Catherine Curiec, Sébastien Thominea,2, and Sylvain Merlota,2

aInstitute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris‐Sud, Université Paris-Saclay, 91198, Gif-sur‐Yvette cedex, France; bUnité deFormation et de Recherche Sciences et Technologie, Université Paris-Est Créteil Val de Marne, 94010 Créteil, France; and cLaboratoire de Biochimie etPhysiologie Moléculaire des Plantes, CNRS, UMR 5004, Institut de Biologie Intégrative des Plantes, Montpellier, France

Edited by Natasha V. Raikhel, Center for Plant Cell Biology, Riverside, CA, and approved March 13, 2017 (received for review February 23, 2017)

“Too much of a good thing” perfectly describes the dilemma thatliving organisms face with metals. The tight control of metal ho-meostasis in cells depends on the trafficking of metal transportersbetween membranes of different compartments. However, themechanisms regulating the location of transport proteins are stilllargely unknown. Developing Arabidopsis thaliana seedlings re-quire the natural resistance-associated macrophage proteins(NRAMP3 and NRAMP4) transporters to remobilize iron from seedvacuolar stores and thereby acquire photosynthetic competence.Here, we report that mutations in the pleckstrin homology (PH)domain-containing protein AtPH1 rescue the iron-deficient pheno-type of nramp3nramp4. Our results indicate that AtPH1 bindsphosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the lateendosome compartment. We further show that loss of AtPH1 func-tion leads to the mislocalization of the metal uptake transporterNRAMP1 to the vacuole, providing a rationale for the reversion ofnramp3nramp4 phenotypes. This work identifies a PH domain pro-tein as a regulator of plant metal transporter localization, provid-ing evidence that PH domain proteins may be effectors of PI3P forprotein sorting.

metal transport | NRAMP | vacuole | late endosome | phosphatidylinositol3-phosphate

The ability of iron (Fe) to switch between different oxidationstates in cells is used in all biological kingdoms to catalyze

essential biochemical reactions. Fe is found in the form of theFe-S cluster, Heme, or Oxo di-iron as a cofactor in the catalyticcenter of a plethora of proteins involved in oxygen binding,electron transport chains, or DNA metabolism (1). As a conse-quence, Fe deficiency leads to severe symptoms in living or-ganisms including anemia in humans or chlorosis and growthinhibition in plants. On the other hand, the excess of Fe is del-eterious for cells because it may lead to the production of highlyreactive oxygen species through the Fenton reaction. Thereforeall organisms need to regulate Fe uptake and storage tightlyaccording to their requirements. Fe homeostasis has been studiedextensively in plants because of its strong impact on photosyn-thesis and therefore on biomass production and also because Febiofortification of food crops is an important challenge to coun-teract Fe deficiency affecting billions of human beings (2, 3).In dicotyledonous plants, the ferric iron (Fe3+) available as

soluble chelates in soils is reduced to ferrous iron (Fe2+) at theroot surface by a plasma membrane-bound ferric-chelate re-ductase known as “AtFRO2” in Arabidopsis thaliana (4). Fe2+

then is transported into root cells by the high-affinity iron-regulated transporter 1 (IRT1) from the ZIP/IRT family that isexpressed in root epidermal cells in response to Fe starvation(5, 6). The natural resistance-associated macrophage protein1from A. thaliana, AtNRAMP1, a homolog of the humanDMT1 transporter, is able to transport Fe and manganese (Mn)when expressed in yeast (7, 8). In Arabidopsis, AtNRAMP1 was

shown to mediate high-affinity Mn uptake (9) as well low-affinityFe uptake (10).In photosynthetic cells, most of the cellular Fe is found in

chloroplasts, within components of the photosynthetic electrontransport chain, or bound to ferritin (2, 11). The vacuole is alsoan important site for Fe compartmentalization. In the A. thalianaembryo, Fe is stored in the vacuole of endodermal cells underthe action of the VIT1 transporter of the CCC1 family (12–14).The Fe pool stored in the vacuole of endodermal cells is remo-bilized during germination by two homologous vacuolar metaltransporters, AtNRAMP3 and AtNRAMP4. The nramp3nramp4double mutant is unable to develop into photosynthetic seedlingswhen germinated under Fe-limiting conditions because seed Feis trapped in the vacuole of endodermal cells (13, 15). Thenramp3nramp4 mutant is also hypersensitive to Cd and Zn forroot growth, likely as a consequence of a globally unbalancedmetal homeostasis (16–18).Genetic analyses in yeast revealed that the regulation of metal

homeostasis depends mainly on metal transporters and on thevacuolar protein-sorting pathway regulating the cellular traf-ficking of these transporters (19–21). The main Mn transporterin yeast, Smf1p, a homolog of AtNRAMP1, localizes on both theplasma membrane and endosomes under Mn-sufficient condi-tions. The endosomal fraction of Smf1p is targeted to the vacuole

Significance

Metal homeostasis is essential for living organisms. Metaltransporters play key roles in metal uptake and compartmen-talization. The tight regulation of metal homeostasis thus de-pends on accurate targeting of these metal transporters.Although the main metal transporters in plants have beenidentified, the mechanisms involved in their trafficking are stillpoorly understood. This study reveals that AtPH1, a pleckstrinhomology (PH) domain containing protein binding to phos-phatidylinositol 3-phosphate (PI3P), controls the subcellularlocalization of the iron and manganese transporter AtNRAMP1.Our results further indicate that, in addition to proteins con-taining the FYVE and PHOX domains, proteins containing thePH domain can decode the PI3P signal in endosomal function.

Author contributions: A.A., S.T., and S.M. designed research; A.A., J.G., M.W.B., J.M., C.E.,L.C., F.L., S.T., and S.M. performed research; A.A., M.W.B., J.M., C.C., S.T., and S.M. ana-lyzed data; and M.W.B., S.T., and S.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Present address: Department of Molecular Biology, Clemente Estable Biological ResearchInstitute, CP 11600 Montevideo, Uruguay.

2To whom correspondence may be addressed. Email: [email protected] [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1702975114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1702975114 PNAS Early Edition | 1 of 10

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for degradation by the Trp/Bsd2p/Rsp5 ubiquitin-dependent path-way (22–26). However, under Mn-deficient conditions, the fractionof Smf1p present at the plasma membrane is increased, likely tostimulate Mn uptake. Recent studies revealed that the homeo-stasis of metals and minerals is regulated by the trafficking oftransporters in plants as well. The Fe transporter IRT1 cyclesbetween the trans-Golgi network (TGN)/early endosome (EE)compartment and the plasma membrane of root epidermal cells,but a fraction is targeted to the vacuole for degradation throughthe late endosome (LE)/multivesicular bodies (MVB) compart-ment (27). This pathway depends on the ubiquitination of IRT1and its direct binding to FYVE1/FREE1 acting as noncanonicalESCRT-0 in the endosomal-sorting complex required for trans-port (ESCRT) machinery in plants (28, 29). Similarly, the traf-ficking of the boron transporter BOR1 and the phosphatetransporter PHT1 between the plasma membrane and the vac-uole is regulated by ubiquitin-dependent pathways, although theproteins involved in the trafficking of these transporters are notyet known (30–32).To identify previously unknown elements regulating Fe ho-

meostasis in the model plant A. thaliana, we looked for rever-tants of the nramp3nramp4 double mutant. Here we report thatloss-of-function mutations in the pleckstrin homology (PH) domaincontaining protein AtPH1 alleviate the phenotypes of nramp3nramp4.We further show that the localization of AtPH1 in the LE/MVBcompartment depends on binding to phosphatidylinositol 3-phosphate(PI3P) and that AtPH1 is involved in the regulation of AtNRAMP1localization. Our results therefore reveal the involvement ofAtPH1 in metal homeostasis in plants as well as the unsuspectedrole of PH domain-containing proteins as effectors of PI3P inprotein sorting.

ResultsThe suppressor1 Mutation Mitigates nramp3nramp4 Growth DefectUnder Fe-Deficient Conditions. To identify genes involved in theregulation of metal homeostasis in plants, we screened for re-vertants of the Arabidopsis nramp3nramp4 mutant, which is hy-persensitive to Fe starvation and to the presence of cadmium(Cd) at the early developmental stage (13, 15, 16). The nns1(nramp3nramp4suppressor1) mutant was selected from a nramp3-1nramp4-1 ethyl methanesulfonate (EMS)-mutagenized M2 pop-ulation (Ws accession) on the basis of a significant reduction of Cdsensitivity for root growth, used as a proxy for Fe deficiency(Fig. 1 A and B). We then directly tested whether the suppressor1mutation also alleviates the hypersensitivity to Fe starvation ofnramp3nramp4 seedlings (Fig. 1 C and D). After 7 d of growth onan Fe-limited medium, the nns1 root length (17.1 ± 5.3 mm) wasintermediate between that of wild-type (31.8 ± 4.6 mm) andnramp3nramp4 (4.2 ± 2.6 mm) plants. This result indicated thatthe suppressor1 mutation partially reverts the Fe-deficient phe-notype of nramp3nramp4.

The suppressor1 Mutation Affects the PH Domain-Containing ProteinAtPH1. To analyze the heredity of the suppressor 1 mutation, thenns1mutant was backcrossed with the parental nramp3-1nramp4-1mutant, and the F2 progeny was tested for the reversion of thenramp3nramp4 phenotype. The progeny showed a Mendelian3:1 segregation ratio for the reversion of the nramp3nramp4phenotype, suggesting that suppressor1 was a single-locus recessivemutation. To identify the suppressor1mutation, we then generatedan F2 recombinant population by outcrossing the nns1 mutantwith the nramp3-2nramp4-2 mutant (Col accession). Using F2plants showing the nns1-like phenotype, we mapped the suppressor1mutation on chromosome 2 between the simple sequence-lengthpolymorphism (SSLP) markers MSAT2-4 and MSAT2-11 usingthe polymorphism between Ws and Col accessions. We selected 19F2 plants that had recombined between these two markers andsequenced the DNA of this subpopulation as a pool using next-

generation sequencing technology. The analysis of the assembly ofthe sequencing reads to the A. thaliana Col genome further re-stricted the localization of the suppressor1 mutation to a regionenriched in Ws-linked SNPs encompassing 580 kb on chromosome2 (nucleotides 12,272,151–12,852,172). To identify the suppressor1mutation, we searched for SNPs between the nns1-like populationand the nramp3-1nramp4-1 mutant in this region containing209 annotated genes. We identified a G-to-A transition covered by18 reads in the ORF of At2g29700.1 (Fig. 2A). This transition re-sults in the nonconservative substitution of Arg46 to His (R46H) inthe predicted protein (Fig. 2 B and C). The At2g29700 gene en-codes a PH domain-containing protein, originally called “AtPH1,”the biological function of which was still unknown (33, 34). Thisprotein was later named “PH2” (35), but we will use the originalname, AtPH1, in agreement with sequence databases.To demonstrate that the point mutation identified in AtPH1,

hereafter called “atph1-1,” is responsible for the reversion ofnramp3nramp4 phenotypes, we expressed AtPH1 fused to GFP

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Fig. 1. nns1 partially reverts nramp3nramp4 phenotypes. (A) Wild-type (Ws),nramp3-1nramp4-1, and nns1 plants were grown vertically for 9 d on ABISagar medium containing 30 μM CdCl2 (+Cd). (B) Root length of plants growingon ABIS medium (black bars) or supplemented with 30 μM CdCl2 (whitebars). Data are shown as mean ± SD; n = 27–34 roots. (C ) Wild-type (Ws),nramp3-1nramp4-1, and nns1 plants were grown vertically for 7 d on ABIS agarmedium without Fe (−Fe). (D) Root length of plants growing on ABIS mediumwithout Fe (white bars) or supplemented with 50 μM Fe-HBED (black bars).Data are shown as mean ± SD; n = 21–31. (Scale bars in A and C: 10 mm.)

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at the C terminus (AtPH1-GFP) under the control of the 2x35Spromoter in nns1. Transgenic nns1 plants expressing AtPH1-GFPgrew normally on a medium supplemented with Fe (Fig. S1) butshowed a reduction in root growth similar to that of nramp3-1nramp4-1 when grown on an Fe-limited medium (Fig. 2D), in-dicating that the expression of AtPH1-GFP complements thesuppressor1mutation. In addition, we isolated a T-DNA (transferDNA) insertion mutant of AtPH1 in the Col accession, which wascalled “atph1-2” (Fig. S2). Neither atph1-1 (Ws) nor atph1-2(Col) single mutants displayed a visible alteration of develop-ment or a change in sensitivity to Fe deficiency (Fig. S3). Likeatph1-1, the atph1-2 mutation partially reverted the phenotype ofnramp3-2nramp4-2 double mutant (Col) in the absence of Fe orin the presence of Cd (Fig. 2E), thus recapitulating the pheno-type of nns1. All these data demonstrate that the loss of functionof AtPH1 is responsible for the partial reversion of thenramp3nramp4 phenotypes. The fact that atph1 only partiallyreverts nramp3nramp4 phenotypes may be caused by the ex-pression of an AtPH1 homolog playing a redundant function. Toaddress this possibility, we isolated a knockout mutation in the

At5g05710 gene, hereafter called “AtPH2,” which is the closest ho-molog to AtPH1 (Fig. S2). However, the analysis of the effect of theatph2-1 mutation in the nramp3nramp4 and nramp3nramp4atph1mutant backgrounds indicated that AtPH2 does not play a re-dundant function with AtPH1 for the reversion of the nramp3nramp4Fe-deficiency phenotype in roots (Fig. S4).

AtPH1 Localization Depends on PI3P Binding in Vivo. The PH domainof AtPH1 contains a putative phosphatidylinositol-3,4,5-trisphosphate-binding motif (PPBM). However, AtPH1 was shown to bind PI3Pspecifically in vitro (33). The R46H substitution identified in nns1affects an invariant residue of the PPBM motif (Fig. 2C and Fig.S5), which is expected to interact directly with the 3-phosphategroup of the inositol ring (36–39). Mutations of the correspondingamino acid in the human proteins PKB/AKT and BTK were shownto affect binding to phosphatidylinositol-3,4,5-trisphosphate andcause X-linked agammaglobulinemia disease, respectively (39, 40).Accordingly, the R46H mutation abrogated AtPH1 binding to PI3Pin vitro (Fig. S5). In root cells, AtPH1-GFP was predominantlydetected in small, punctate structures in the cytoplasm and on

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Fig. 2. The suppressor1 mutation affects AtPH1. (A) Alignment of reads from the nns1 mutant to the Col-0 reference genome (TAIR10) at the AtPH1/At2g27900 locus. Paired reads are in blue; broken pairs are in red and green. The translation of the At2g27900.1 gene model is given under the nucleotidesequence. The asterisk denotes the mutation identified in nns1 converting Arg46 to His. (B) Amino acid sequence of AtPH1/At2g27900.1. The PH domain ishighlighted in gray, and the PPBM is highlighted in red. (C) Alignment of the AtPH1 PH domain N terminus showing conservation to other PH domain fromOryza sativa (XP_015638090.1), Physcomitrella patens (XP_001766896.1), Dictyostelium discoideum (XP_643886.1), A. thaliana DRP2A (NP_172500.1), Homosapiens PKB/AKT1 (NP_001014431.1) and DAPP1 (NP_055210.1), and Saccharomyces cerevisiae ScOSH2 (NP_010265.1). The background color reflects sequenceconservation. (D) Wild-type (Ws), nramp3-1nramp4-1, nns1, and homozygous T3 nns1 lines constitutively expressing AtPH1-GFP were grown vertically for 10 don ABIS medium without iron (−Fe). (E) Wild-type (Col), nramp3-2nramp4-2, and nramp3-2nramp4-2atph1-2 plants were grown vertically for 8 d on ABISmedium without iron (−Fe) or for 10 d on medium containing 30 μM CdCl2 (+Cd). (Scale bars: 10 mm.)

Agorio et al. PNAS Early Edition | 3 of 10

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the vacuolar membrane (Fig. 3A). In contrast, AtPH1 carryingthe R46H mutation (AtPH1R46H-GFP) was uniformly dis-tributed in the cytosol, suggesting that the cellular localizationof AtPH1 depends on binding to PI3P (Fig. 3A). Accordingly,AtPH1-GFP colocalized with the PI3P marker 2xFYVE-mCherry (Fig. 3B), and treatment of roots with the PI3K in-hibitor wortmannin led to uniform redistribution of AtPH1-GFP in the cytosol (Fig. 3C). Together, these data indicatethat AtPH1 cellular localization depends on binding to PI3P.The phenotype of the nns1 mutant further suggests that bindingto PI3P is essential for AtPH1 function.

AtPH1 Localizes in the LE/MVB Compartment. PI3P accumulates inthe membranes delimiting the LE/MVB compartment and thevacuole of plant cells (28, 41). To characterize the localization ofAtPH1 further, we outcrossed the nns1 line complemented byAtPH1-GFP with transgenic lines expressing cellular markersfused to monomeric RFP (mRFP) and analyzed F1 plants byconfocal imaging. AtPH1 colocalized with the LE/MVB markersARA7 and ARA6 but only very weakly with the TGN/EE markerSYP43 or the trans-Golgi marker ST (Fig. 4 and Fig. S6A). Thesedata indicate that AtPH1 mainly localizes in the LE/MVB inplant cells. Using electron transmission microscopy, we observeda minor reduction of the diameter of LE/MVB vesicles but nodifference in the number of intraluminal vesicles (ILV) in thenns1 mutant compared with the wild type (Fig. S7). These resultsindicate that AtPH1 does not play a major role in the biogenesisof LE/MVB and ILVs.

AtNRAMP1 Is Involved in the Reversion of nramp3nramp4 by atph1.Because AtPH1 localizes in the LE/MVB, we hypothesized thatatph1 might affect the trafficking of a metal influx transporter,leading to its localization on the vacuolar membrane where itcould compensate for the loss of AtNRAMP3 and AtNRAMP4activity. The metal transporter AtNRAMP1 acting at the plasmamembrane for the uptake of Mn and Fe from the external me-dium to the cytoplasm (9, 10) was considered as a candidate forthe reversion of nramp3nramp4 by atph1. We generated the

nramp3nramp4atph1nramp1 quadruple mutant to test the effectof AtNRAMP1 loss of function on the nramp3nramp4atph1phenotype. When grown on an Fe-limited medium, the rootlength of nramp3nramp4atph1nramp1 was intermediate betweenthat of nramp3nramp4 and nramp3nramp4atph1 (Fig. 5). UnderFe-replete conditions, all genotypes displayed similar root growth.Therefore, our genetic analysis indicates that AtNRAMP1 isrequired for the reversion of nramp3nramp4 by atph1. However,because nramp1 does not fully suppress the effect of atph1,we cannot exclude the possibility that other mechanisms maybe involved.

atph1 Affects the Localization of AtNRAMP1. To study the locali-zation of AtNRAMP1 in planta, we first expressed NRAMP1-GFP under the control of the endogenous NRAMP1 promoter inthe nramp1-1 mutant. However, the fluorescent GFP signal de-tected in roots was too weak to analyze NRAMP1-GFP locali-zation in these lines by confocal microscopy. We thereforeexpressed NRAMP1-GFP under the control of the UBIQUITIN10promoter in the nramp1-1 mutant. In four independent stabletransgenic lines tested, the expression of AtNRAMP1-GFP com-plemented the Mn-deficient phenotype of nramp1-1, indicatingthat NRAMP1-GFP is functional (Fig. S8). We observed thatNRAMP1-GFP localized mainly in cytoplasmic granular struc-tures in epidermal cells and also frequently on the plasma mem-brane, notably in young epidermal cells (Fig. 6A). Colocalizationanalyses indicated that NRAMP1-GFP is distributed across thetrans-Golgi, TGN/EE, and LE/MVB compartments (Fig. 6Band Fig. S6B).The localization of NRAMP1-GFP was not visibly affected in

the nramp3-1nramp4-1 mutant (Fig. 7A). However, in the nns1(nramp3-1nramp4-1atph1-1) mutant, we observed that a signifi-cant fraction of NRAMP1-GFP colocalized with the vacuolarmembrane marker TagRFP-SYP22 (Fig. 7 B and C). Whennramp3-1nramp4-1 and nns1 plants expressing NRAMP1-GFPwere placed in the dark, a condition reducing vacuolar proteoly-sis of GFP (42, 43), we observed the appearance of a GFP signal inthe lumen of vacuoles, indicating that a fraction of NRAMP1-GFP

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Fig. 3. AtPH1 localization depends on binding to PI3P. (A) Confocal images of the root elongation zone of nns1 plants expressing AtPH1-GFP or AtPH1R46H-GFP under the control of the 2x35S promoter. The boxed area in the left panel is enlarged at right. Cell walls stained by PI are in magenta. The white arrowindicates the vacuolar membrane detaching from the cell periphery. (B) nns1 plants expressing AtPH1-GFP were crossed with plants expressing the PI3Pmarker 2xFYVE-mCherry. Colocalization of both fluorescent markers was analyzed by confocal microscopy on roots of F1 plants. On the merged picture theoverlap of GFP (green) and mCherry (magenta) channels appears in white. Cell contours are represented by dashed lines. Pearson (rp) and Spearman (rs)correlation coefficients as well as M1 (GFP) and M2 (mCherry) Manders overlap coefficients above threshold were calculated. (C) The roots of nns1 plantsexpressing AtPH1-GFP were treated by 30 μM wortmannin or 0.1% DMSO for 3 h. (Scale bars: 10 μm.)

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is targeted in the vacuole for degradation in both genetic back-grounds (Fig. 7D). Treatment of seedlings with Concanamycin A,an inhibitor of vacuolar ATPase (V-ATPase), which also led to areduction of vacuolar protease activity (27, 43), confirmed theseobservations (Fig. S9). Together, these results suggest thatNRAMP1 traffics through the endosomal system en route to itsdegradation in the vacuolar compartment and that the atph1 mu-tation leads to the mislocalization of a fraction of NRAMP1 on thevacuolar membrane.

DiscussionThe aim of this work was to identify elements involved in theregulation of metal homeostasis in plants using a forward geneticscreen to isolate revertants of the nramp3nramp4 mutant. Thisscreen identified a loss-of-function mutation in the gene codingfor the PH domain-containing protein AtPH1 (Fig. 2), whosecellular localization and biological function were unknown. Wefurther showed that the atph1 mutation reverts the defects of thenramp3nramp4 mutant by an original mechanism of suppression:atph1 leads to mislocalization of at least one metal transporter,AtNRAMP1, to the vacuolar membrane, where it substitutesfor AtNRAMP3 and AtNRAMP4 function. More generally, ourresults uncover the involvement of AtPH1 in the regulation of

ST-mRFP mRFP-SYP43 mRFP-ARA7

rp=0.70 rs=0.76M1=0.58 M2=0.51

rp=0.36 rs=0.37M1=0.30 M2=0.16

rp=0.22 rs=0.25M1=0.24 M2=0.15

Fig. 4. AtPH1 colocalizes with a marker of the LE/MVB. nns1 plantsexpressing AtPH1-GFP were crossed with plants expressing the trans-Golgimarker ST-mRFP, the TGN/EE marker mRFP-SYP43, or the LE/MVB markermRFP-ARA7. Colocalization of fluorescent markers was analyzed by confocalmicroscopy on root epidermal cells of F1 plants. On merged pictures theoverlap of GFP (green) and mRFP (magenta) channels appears white. Cellcontours are represented by dashed lines. The regions within the whiteoutline are enlarged in the lower panels (magnification: 3×). Pearson (rp) andSpearman (rs) correlation coefficients as well as M1 (GFP) and M2 (mRFP) Man-ders overlap coefficients above threshold were calculated. (Scale bars: 10 μm.)

nramp3nramp4 nramp3nramp4nramp1

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Fig. 5. NRAMP1 loss of function partially reverts the effect of atph1. (A) Themultiple mutants nramp3-2nramp4-2, nramp3-2nramp4-2atph1-2, nramp3-2nramp4-2nramp1-1atph1-2, and nramp3-2nramp4-2nramp1-1 (Col) weregrown vertically for 10 d on Hoagland-agar medium containing a limitedamount of Fe (0.1 μM Fe-HBED) or sufficient Fe (10 μM Fe-HBED). Repre-sentative plants from four independent culture plates were realigned for thepictures. (Scale bars: 10 mm.) (B) Quantification of root lengths shown inA. Results are shown as mean value ± SD; n = 21–27, four independent plates;letters indicate significant differences according to a Kruskal–Wallis testcorrected by a Dunn’s multiple comparison test; P < 0.01.

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plant metal homeostasis and highlight the role of a PH domainprotein specifically binding PI3P in intracellular protein sorting.

AtPH1 Loss of Function Partially Reverts the nramp3nramp4 Fe-Deficiency Phenotype. Mutations affecting AtPH1 partially revertthe Fe-deficient phenotype of the nramp3nramp4 mutant (Fig.1). Genetic evidence further suggests that the metal transporterNRAMP1 is required for the reversion of nramp3nramp4 phe-notypes by atph1 (Fig. 5). NRAMP1 was previously shown tolocalize on the plasma membrane of A. thaliana root hair cells(9). Although we also observed NRAMP1 at the plasma mem-brane of root epidermal cells, the main pool of NRAMP1 islocalized in the endosomal system (Fig. 6), suggesting thatNRAMP1 localization depends on culture conditions and celltypes. Indeed, we observed that NRAMP1 is mistargeted to thevacuolar membrane in the presence of the atph1 mutation (Fig.7). Therefore, we propose that the fraction of NRAMP1 presenton the vacuolar membrane in the nns1 mutant can compensatefor the lack of NRAMP3 and NRAMP4 activities in the remo-bilization of Fe from vacuolar stores (Fig. 8). Because atph1mutations partially rescue nramp3nramp4 phenotypes (Figs. 1and 2E), it could be argued that the effect of the atph1 mutationis partially masked by genes playing redundant functions. TheA. thaliana genome encodes 50 PH domain-containing proteins(35). Our genetic analysis, however, indicates that AtPH1 and itsclosest homolog, AtPH2/At5g05710.1, do not have redundantfunctions in the reversion of the nramp3nramp4 Fe-deficiencyphenotype in roots (Fig. S4). This result does not exclude thepossibility that these two proteins might have redundant functions

in other pathways or different tissues, nor can we completely ex-clude the possibility that more distantly related PH domain pro-teins might play redundant roles with AtPH1. The partial reversionof nramp3nramp4 could be the consequence of insufficient ex-pression or activity of NRAMP1 and possibly other Fe transportersthat are targeted on the vacuolar membrane in root cells in atph1.Although it was previously shown that NRAMP1 is expressed invascular tissues, as are NRAMP3 and NRAMP4 (9), this trans-porter has a lower affinity for Fe than NRAMP3 and NRAMP4 (8,10), thus providing a rationale for the partial reversion ofnramp3nramp4. Interestingly, the identification of AtNRAMP1,which is the high-affinity Mn transporter in Arabidopsis, as a targetof AtPH1 opens the possibility that this PI3P-binding proteinmight have a role in the regulation of Mn homeostasis.

Possible Modes of Action of AtPH1. Our results indicate that themutation of AtPH1 leads to an accumulation of NRAMP1 on themembrane of the vacuole, thus compensating for the lack ofvacuolar Fe export activity in the nramp3nramp4 mutant back-ground (Fig. 8). Several hypotheses may account for the mis-localization of NRAMP1. First, the accumulation of NRAMP1on the vacuolar membrane could result from its stabilizationon the outer membrane of LE/MVB, which subsequently fuseswith the vacuole (Fig. 8). A similar mislocalization of the auxintransporter PIN2 on the vacuolar membrane was observed in thefyve1/free1-knockout mutant that does not produce ILVs (29).The PI3P-binding protein FYVE1/FREE1 plays an essential roleas the ESCRT-0 component of the plant ESCRT machinery thatregulates the trafficking and the degradation in the vacuole of

ST-mRFP mRFP-ARA7mRFP-SYP43

rp=0.73 rs=0.58M1=0.70 M2=0.73

rp=0.84 rs=0.76M1=0.67 M2=0.89

rp=0.77 rs=0.57M1=0.57 M2=0.89

A BNRAMP1-GFP DIC

Fig. 6. NRAMP1 is present in the endosomal pathway and on the plasma membrane. Root epidermal cells of nramp1 plants complemented by NRAMP1-GFPwere imaged by confocal microscopy. (A) The vacuolar plane of elongating cells (Upper) and cortical plane of dividing cells (Lower) showing that NRAMP1-GFPwas present mostly in cytoplasmic vesicles but also was detected on the plasma membrane (white arrow), more clearly in younger cells. Transmitted-light(differential interference contrast, DIC) images are shown also. (B, Upper) NRAMP1-GFP plants were crossed with plants expressing the trans-Golgi markerST-mRFP, the TGN/EE marker mRFP-SYP43, and the LE/MVB marker mRFP-ARA7. Colocalization of fluorescent markers was analyzed in root epidermal cells ofF1 plants. On the merged pictures the overlap of GFP (green) and mRFP (magenta) channels appears white. Cell contours are represented by dashed lines.(Lower) Enlarged view of the region marked by the white outline in the upper row (magnification: 3×). Pearson (rp) and Spearman (rs) correlation coefficientsas well as M1 (GFP) and M2 (mRFP) Manders overlap coefficients above threshold were calculated. (Scale bars: 10 μm.)

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several ubiquitinated proteins, including PIN2, IRT1, and theABA receptor PYL4 (28, 29, 44). As a consequence, the fyve1/free1-knockout mutant is strongly affected in LE/MVB functionsand in the biogenesis of vacuoles, resulting in growth arrest at avery early stage of seedling development (28, 29, 45). In contrast,the atph1-knockout mutant is not lethal, does not display a visibledevelopmental phenotype, and is not affected in the biogenesis ofILVs (Figs. S1 and S6). These observations suggest that AtPH1might play a more specific role than FYVE1/FREE1 in LE/MVB,possibly targeting a limited number of cargo proteins, including

NRAMP1, to ILVs for their degradation in the vacuole. Furtherexperiments will be required to confirm the role of AtPH1 in thispathway and consequently to determine if AtPH1 acts indepen-dently or in concert with FYVE1/FREE1. Alternatively, theaccumulation of NRAMP1 on the outer membrane of LE/MVB could be the consequence of a defect in the recycling ofNRAMP1 from LE/MVB back to the plasma membrane in thenns1 mutant (Fig. 8). Although still debated in the literature(46), the existence of this recycling pathway in plants is supportedby the function of the plant-specific GTPase of the Rab5 family,

nns1

NRAMP1-GFP

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Fig. 7. NRAMP1 localizes on the vacuole in the nns1 mutant. (A and B) NRAMP1-GFP and TagRFP-SYP22 were expressed in nramp3-1nramp4-1 (A) andnramp3-1nramp4-1atph1-1 (nns1) (B) mutants. Root epidermal cells in the elongation zone were imaged on a spinning disk confocal microscope for GPF andtagRFP. (Scale bars: 10 μm.) (C) NRAMP1 localization on the vacuole was quantified in single-plane images corresponding to individual cells of nramp3nramp4(n = 48) and nramp3nramp4atph1 (n = 42) as a normalized fraction of NRAMP1-GFP signal colocalizing with the TagRFP-SYP22 signal. Note that because ofthe close proximity of cytoplasmic NRAMP1 structures with the vacuole, the value of the vacuolar membrane localization index is never null. In the graph,boxes include the two central quartiles, separated by the median. The whiskers extend to the 5th and 95th percentiles, and outliers are represented by a dot.P < 0.0001; Mann–Whitney test. (D) Root epidermal cells of nramp3nramp4 and nns1 mutants expressing NRAMP1-GFP were imaged across vacuolar planesusing spinning disk confocal imaging. GPF fluorescence and transmitted-light (DIC) images are displayed. The asterisks denote the position of vacuoles. Plantswere placed in darkness for 14 h (Dark) before imaging to reduce GFP degradation in the vacuoles. The contrast of the images was adjusted for the visu-alization of GFP in vacuoles. (Scale bar: 10 μm.)

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ARA6/AtRABF1 (47–49), that colocalizes with AtPH1 (Fig. S6A).Finally, the mislocalization of NRAMP1 in the absence of func-tional AtPH1 may result from other processes, e.g., a defect ofNRAMP1 degradation on the vacuole by invagination of the vac-uolar membrane. This process, called “microphagy,” has beenproposed to regulate the turnover of the vacuolar potassiumchannel TPK1 (50, 51). The identification of AtPH1-interactingproteins and the analysis of the trafficking of other cargo pro-teins in the atph1mutant should help in discriminating between thedifferent hypotheses regarding the mode of action of AtPH1.

AtPH1 as an Effector of PI3P in the Endosomal Pathway. In-dependently of its mode of action, our results demonstrate thatthe function of AtPH1 depends on PI3P binding in vivo. Using aphospholipid overlay assay, it was previously shown that AtPH1binds specifically PI3P in vitro (33). We confirmed these data(Fig. S5) and, more importantly, showed that the localization ofAtPH1 in the LE/MVB compartment of root cells depends on itsability to bind PI3P in vivo (Figs. 3 and 4). The loss-of-functionmutation identified in the nns1 mutant affects the highly con-served residue Arg46 in the PH domain of AtPH1, which ispredicted to bind the 3-phosphate group of the inositol ring ofphosphoinositides (36–39). Accordingly, the R46H mutationabrogates both AtPH1 binding to PI3P in vitro (Fig. S5) and itslocalization in the LE/MVB compartment (Fig. 3A). These re-sults strongly suggest that the direct binding of AtPH1 to PI3P isnecessary for AtPH1 function in the endosomal pathway. Moregenerally, this work provides genetic evidence supporting a rolefor a PH domain protein directly binding PI3P in endosomalfunctions. Indeed, the involvement of PH domain proteins inendosomal functions has largely been overlooked compared withproteins containing FYVE or PHOX domains, because the bindingspecificity and affinity of PH domains for PI3P are usually lowerthan those of FYVE and PHOX domains (52–54). AtPH1 has noobvious orthologs outside the plant kingdom, but several PHdomain-containing proteins specifically binding PI3P in vitro havebeen identified from yeast and human genomes (33, 55–57). Futuregenetic and functional studies might uncover a role for some ofthese proteins in the fine-tuning of endosomal trafficking.

Materials and MethodsA. thaliana Genotypes. The A. thaliana mutants nramp3-1nramp4-1 (Ws ac-cession), nramp3-2nramp4-2 (Col accession), and nramp1-1 (Col) were de-

scribed previously (9, 13, 16). The atph1-2 allele (Col) isolated in this studycorresponds to the GABI-Kat line GK-310H12 (58). The single atph1-1mutantwas isolated after outcrossing the nns1 mutant with the wild-type (Ws ac-cession) strain. The multiple mutants nramp3-2nramp4-2atph1-2 (Col),nramp1-1nramp3-2nramp4-2 (Col), and nramp1-1nramp3-2nramp4-2atph1-2(Col) were obtained by outcrossing available mutants. Homozygous mutantsof the expected genotypes were selected by PCR using gene- and T-DNA–specific primers or a derived cleaved amplified polymorphic sequence (dCAPS)approach (SI Materials and Methods and Table S1).

Arabidopsis nramp3nramp4 Suppressor Screening. Seeds of the Arabidopsisthaliana nramp3-1nramp4-1 (Ws accession) were mutagenized by EMS aspreviously described (15). Approximately 36,000 seeds corresponding to theprogeny of 1,800 mutagenized M1 seeds were sown on ABIS-Agar platessupplemented with 20 μM CdCl2 and were grown vertically for 14 d at 21 °Cwith 16 h light/d. Plantlets showing longer roots than nramp3-1nramp4-1were selected as nramp3nramp4 suppressors (nns). The nns1 mutant wasbackcrossed twice with nramp3-1nramp4-1 to perform physiological studies.

Culture of Plants for Root-Growth Assays. Plants were grown vertically at 21 °Cwith 16 h of light for 5–9 d on ABIS or Hoagland-Agar medium (16, 59).When indicated, the medium was supplemented with Fe-HBED [N,N′-di(2-hydroxybenzyl) ethylenediamine-N,N′diacetic acid monochloride hydrate](Strem Chemicals) and CdCl2 (Sigma-Aldrich). For the Mn-depleted condition,MnCl2 was omitted from the Hoagland medium, and metal-free agar wasprepared as previously described (60).

Identification of the nns1Mutation. The nns1mutant was outcrossed with thenramp3-2nramp4-2 mutant (Col accession). The initial mapping of the sup-pressor1 mutation in the F2 population was performed using the MSATcollection of SSLP markers (www7.inra.fr/vast/msat.php) (61). The genomicDNA was purified from selected F2 plants showing an nns1-like phenotypeand from the parental nramp3-1nramp4-1 mutant. The two pools of geno-mic DNA were sequenced using Illumina 100-bp paired-end reads technol-ogy (Fasteris SA). We obtained 70.4 × 106 reads for the nns1-like pool and88.2 × 106 reads for nramp3-1nramp4-1. The reads were mapped to theA. thaliana Columbia-0 reference genome (TAIR10) using CLCbio GenomicWorkbench 5.5.1 (QIAGEN). The genomic region linked to the nns1 pheno-type was mapped by analysis of SNP distribution between Ws and Col ac-cessions using the variant detection tool. To identify the suppressor1 EMSmutation, we restricted the SNP analysis to the genomic region linked to thenns1 phenotype, selected SNPs corresponding to G-to-A and C-to-T transi-tions, filtered out SNPs present in parental nramp3-1nramp4-1, and focusedon SNPs affecting the coding sequence of annotated genes.

Cloning of AtPH1 and NRAMP1 Coding Sequences. The AtPH1 (AT2G29700.1)and NRAMP1 (AT1G80830.1) coding sequences without a stop codon were

TGN/EE LE/MVB

Plasma membrane

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Fig. 8. Possible hypotheses to explain the role of AtPH1 in the reversion of nramp3nramp4 through the regulation of NRAMP1 trafficking. (A) In wild-typeplants, NRAMP1 could cycle between the plasma membrane and TGN/EE. A fraction of NRAMP1 is directed to the vacuole, where it is degraded. AtPH1 thatlocalizes in LE/MVB and the vacuolar membrane could be involved in the targeting of NRAMP1 in ILVs (1), in the recycling of NRAMP1 from the LE/MVBcompartment to the plasma membrane (2), or in the degradation of NRAMP1 on the vacuolar membrane (3). (B) The absence of functional AtPH1 in the nns1mutant (nramp3nramp4atph1) leads to the accumulation of NRAMP1 on the membrane of the vacuole, thus complementing the activity of NRAMP3 andNRAMP4 in the remobilization of Fe from the vacuole.

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amplified by PCR using high-fidelity Phusion polymerase (Thermo Scientific)from genomic DNA and cDNA respectively. AtPH1R46H was obtained usingnns1 genomic DNA as template. Coding sequences were recombined usingGateway cloning (Invitrogen) into the pDONR207 vector and then in theexpression vectors pMDC83 (62) and pMUBI83 (63) to generate pMDC83-AtPH1, pMDC83-AtPH1R46H, and pMUBI83-NRAMP1. All constructs wereverified by Sanger sequencing (GATC Biotech).

Expression of AtPH1 and NRAMP1 in Transgenic Plants. A. thaliana nns1(nramp3-1nramp4-1atph1-1) plants were transformed with pMDC83-AtPH1 and pMDC83-AtPH1R46H by floral dipping using the agrobacteriumAGL0 strain to express AtPH1-GFP and AtPH1R46H-GFP fusion proteins, re-spectively. The nramp1-1, nramp3-1nramp4-1, and nns1 mutants were trans-formed with pMUBI83-NRAMP1 to express NRAMP1-GFP fusion protein.

Transgenic lines were selected in vitro on half-strength Murashige andSkoog (MS) agar medium supplemented with 50 μg/mL Carbenicillin and15 μg/mL Hygromycin B. Homozygous single-locus-insertion transgenic T3 lineswere selected for further experiments.

Confocal Imaging and Colocalization Analyses. For colocalization analyses, thehomozygous Arabidopsis transgenic lines nns1/pMDC83-AtPH1 and nramp1-1/pMUBI83-NRAMP1 were outcrossed with lines expressing mRFP-SYP43,ARA6-mRFP, and mRFP-ARA7 under their endogenous promoters, ST-mRFPunder the control of the 35S promoter (47, 64, 65), and 2xFYVEHRS-mCherryunder the control of the UBI10 promoter (28, 41). F1 plants were used forcolocalization analysis. Homozygous nramp3-1nramp4-1 and nns1 linesexpressing NRAMP1-GFP (pMUBI83-NRAMP1) were transformed withpBGW/pSYP22:tagRFP-SYP22 (66), and primary transformants were selectedon half-strength MS agar medium supplemented with 15 μg/mL Basta.T2 lines expressing both fluorescent markers were selected for furtherexperiments.

Seedlings were grown vertically for 5–9 d on Hoagland Agar mediumcontaining 1% sucrose and 50 μM Fe-HBED before imaging. When indicated,seedlings were treated for 3 h with 30 μM wortmannin (Sigma) or 0.1%DMSO as control and with 1% (wt/vol) propidium iodide (PI) for 5 min.Seedlings were mounted in culture medium, and confocal images of rootepidermal cells in the division and elongation zone were obtained on eithera Leica SP8 microscope to acquire GFP (λex= 490 nm, λem= 500–550 nm) andmCherry/mRFP/PI (λex= 590 nm, λem= 600–650 nm) fluorescence simul-taneously or on a Nipkow spinning disk confocal system by high-speed

(300 ms) sequential acquisition of the two channels (GFP and TagRFP) using aHamamatsu ORCA-Flash 4.0_LT or a Photometrics EMCCD Evolve camera (67).

Image processing (cropping, contrast adjustment, and background sub-traction) and analysis were performed with ImageJ (Wayne Rasband; Na-tional Institutes of Health). Colocalization coefficients were computed withthe JACoP plugin [rp, Pearson correlation coefficient; M1 and M2, Mandersoverlap coefficients above threshold (68)] or the Coloc2 plugin (rs, Spearmancorrelation coefficient) of the FiJi distribution of ImageJ (69). To evaluate thedifferential colocalization of NRAMP1-GFP with tagRFP-SYP22, an auto-mated procedure was used. First, the fraction of total GFP fluorescenceabove threshold (established by the Huang method of ImageJ) present onSYP22 regions (segmented by thresholding with the Isodata method) wascomputed. These values then were normalized to the extent (fraction oftotal cytoplasmic surface) of vacuolar membrane segmented in each image,to obtain a vacuolar membrane localization index. Such an index corre-sponds to a Manders overlap coefficient normalized to the relative extentof vacuolar membrane (SYP22 signal) in each image. The data shown inFig. 7C represent the pool of two independent experiments, performed ondifferent dates, which gave virtually identical results. In each experiment,20–27 epidermal cells from the root elongation zones of four to sevenseedlings were analyzed individually for each genotype. The quantificationwas conducted on single-plane images corresponding to the vacuolar plane.

Statistical Analyses. Statistical analyses were performed using GraphPad Prismversion 7.00 (GraphPad Software, www.graphpad.com).

Accession Numbers. Sequence data from this article can be found in theArabidopsis Genome Initiative or GenBank/NCBI databases under the fol-lowing accession numbers: AtPH1 (AT2G29700), AtPH2 (AT5G05710), NRAMP1(AT1G80830), NRAMP3 (AT2G23150), and NRAMP4 (AT5G67330).

ACKNOWLEDGMENTS. We thank Takashi Ueda, Emi Ito, and TomohiroUemura for kindly providing mRFP marker lines and the pBGW/pSYP22:tagRFP-SYP22 construct and Gregory Vert for critical reading of the manu-script. This work was supported by Grants ANR-11-BSV6-0004 (to S.T.) andANR-2011-ISV6-001-01 (to C.C.). A.A. was the recipient of a PostdoctoralFellowship from the Spanish Ministry of Science and Innovation. This workhas benefited from the core facilities of Imagerie-Gif (www.i2bc.paris-saclay.fr/spip.php?rubrique184), a member of Infrastructures en Biologie Santé etAgronomie (IBiSA) (www.ibisa.net), supported by France BioImaging GrantANR-10INBS-04-01 and the Saclay Plant Science Labex Grant ANR-11-IDEX-0003-02.

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