the chloroplast permease pic1 regulates plant growth and

The Chloroplast Permease PIC1 Regulates Plant Growthand Development by Directing Homeostasis andTransport of Iron1[W]

Daniela Duy, Roland Stube, Gerhard Wanner, and Katrin Philippar*

Biochemie und Physiologie der Pflanzen (D.D., R.S., K.P.) and Ultrastrukturforschung (G.W.), DepartmentBiologie I-Botanik, Ludwig-Maximilians-Universitat Munchen, D–82152 Planegg-Martinsried, Germany

The membrane-spanning protein PIC1 (for permease in chloroplasts 1) in Arabidopsis (Arabidopsis thaliana) was previouslydescribed to mediate iron transport across the inner envelope membrane of chloroplasts. The albino phenotype of pic1knockout mutants was reminiscent of iron-deficiency symptoms and characterized by severely impaired plastid developmentand plant growth. In addition, plants lacking PIC1 showed a striking increase in chloroplast ferritin clusters, which function inprotection from oxidative stress by sequestering highly reactive free iron in their spherical protein shell. In contrast, PIC1-overexpressing lines (PIC1ox) in this study rather resembled ferritin loss-of-function plants. PIC1ox plants suffered fromoxidative stress and leaf chlorosis, most likely originating from iron overload in chloroplasts. Later during growth, plants werecharacterized by reduced biomass as well as severely defective flower and seed development. As a result of PIC1 proteinincrease in the inner envelope membrane of plastids, flower tissue showed elevated levels of iron, while the content of othertransition metals (copper, zinc, manganese) remained unchanged. Seeds, however, specifically revealed iron deficiency,suggesting that PIC1 overexpression sequestered iron in flower plastids, thereby becoming unavailable for seed iron loading.In addition, expression of genes associated with metal transport and homeostasis as well as photosynthesis was deregulated inPIC1ox plants. Thus, PIC1 function in plastid iron transport is closely linked to ferritin and plastid iron homeostasis. Inconsequence, PIC1 is crucial for balancing plant iron metabolism in general, thereby regulating plant growth and in particularfruit development.

Chloroplasts originated about three billion yearsago from endosymbiosis of an ancestor of today’scyanobacteria with a mitochondria-containing host cell(Moreira et al., 2000; Palmer, 2000). During evolution,chloroplasts in higher plants established as the site ofphotosynthesis and thus became the basis for all lifedependent on oxygen and carbohydrate supply. Tofulfill this task, plastid organelles are loaded with thetransition metal iron, which due to its potential forvalency changes is essential for photosynthetic electrontransport (Raven et al., 1999). In consequence, chloro-plasts represent the iron-richest system in plant cells(Terry and Low, 1982). However, the improvement ofoxygenic photosynthesis in turn required adaptation ofprocesses related to iron transport and homeostasis. (1)Oxidation of soluble ferrous iron (Fe2+) to almost insol-

uble ferric iron (Fe3+) turned the most abundant metalin the earth’s crust into a limiting factor (Morel andPrice, 2003). Thus, higher plants developed certainstrategies for iron acquisition from the surroundingsoil (for overview, see Palmer and Guerinot, 2009).(2) Metal-catalyzed generation of reactive oxygen spe-cies (ROS; Halliwell and Gutteridge, 1992) causes oxi-dative damage and requires tight control of transport,storage, and cofactor assembly of metal ions (Kosman,2010). Studies on plants with abnormally high ironlevels, therefore, show increased ROS levels and thepromotion of oxidative stress responses (Kampfenkelet al., 1995; Caro and Puntarulo, 1996; Pekker et al.,2002). This is most acute in iron-rich chloroplasts,where radicals and transition metals are side by sideand production of ROS-like hydrogen peroxide (H2O2)is a usual feature of photosynthetic electron trans-port (Asada, 1999; Mubarakshina et al., 2010). Thus, onthe one hand, when bound in FeS cluster or hemeproteins, chloroplast-intrinsic iron is a prerequisite forphotoautotrophic life, but on the other hand, it is toxicwhen present in its highly reactive, hydroxyl radical-generating, free ionic forms. In consequence, plastidiron homeostasis and transport has to be tightly con-trolled and is crucial throughout plant developmentand growth.

Molecular mechanisms of iron uptake into cells ofdicotyledonous plants are best studied in the plasmamembrane of Arabidopsis (Arabidopsis thaliana) roots,

1 This work was supported by the Deutsche Forschungsgemein-schaft (grant no. PH73/3–2 to K.P.) and in the framework of theTransnational Cooperation (Germany, France, Spain) within thePLANT-KBBE Initiative funded by the Bundesministerium furBildung und Forschung (grant no. FKZ:0315458A to K.P., frameworkof the GABI initiative).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Katrin Philippar ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.110.170233

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where the ferric-chelate reductase oxidase FRO2(Robinson et al., 1999) reduces soil ferric iron prior toFe2+ uptake into epidermal cells by the divalent metaltransporter IRT1 (for iron-regulated transporter1; Vertet al., 2002, for overview, see Palmer and Guerinot,2009). In contrast, current knowledge on iron transportacross the two envelope membranes of plastids is stillscarce. Uptake experiments with isolated chloroplastsand purified inner membrane vesicles described plas-tid Fe(III)-chelate transport (Bughio et al., 1997) andFe2+ uptake across the inner envelope (Shingles et al.,2001, 2002). Localization of FRO7 (for ferric-chelatereductase oxidase7) in envelope membranes (Jeonget al., 2008) identified one component of the generaliron transport pathway. Because fro7 loss-of-functionmutants have less iron in chloroplasts and showchlorosis under iron-limiting conditions, FRO7 mostlikely plays a role in plastid iron uptake. Only recently,MAR1/IREG3 (for multiple antibiotic resistance1/iron-regulated protein3) was described as a plastid memberof the ferroportin/IREG transporter family (Conteet al., 2009). Although MAR1/IREG3 specifically im-ports aminoglycoside antibiotics, due to sequencesimilarity to the metal transporters IREG1 and IREG2(Schaaf et al., 2006; Morrissey et al., 2009) and the factthat MAR1 overexpression is causing leaf chlorosisthat can be rescued by iron, the authors suggest thatMAR1/IREG3 functions naturally in the uptake ofthe iron-chelating polyamine nicotianamine (NA)into chloroplasts (Conte et al., 2009; Conte and Lloyd,2010).

With PIC1 (for permease in chloroplasts 1), wepreviously identified the first molecular componentof the plastid iron-transport pathway in the innerenvelope membrane (Duy et al., 2007). PIC1 was ableto complement the growth of iron uptake-deficientyeast by mediating iron accumulation within cells.Besides their severe dwarf, albino phenotype thatresembled iron-deficiency symptoms in plants, themost striking feature of pic1 knockout mutants wasthe accumulation of ferritin protein clusters. Plastid-localized ferritins represent the central component forbalancing iron homeostasis, because they precipitateand store free iron in their oligomeric protein shell andthus withdraw this highly reactive metal from intra-cellular generation of ROS (for overview, see Briatet al., 2010). By generating plant lines overexpressingthe iron permease PIC1, we here provide additionalevidence that the function of PIC1 in the inner envel-ope of chloroplasts (iron transport) is tightly linked toferritin (iron storage) and iron homeostasis. Thus, bothplastid proteins represent key players in regulating irontransport and metabolism as well as metal-generatedoxidative stress. Furthermore, PIC1 overproductionraised iron levels in chloroplasts and specificallychanged iron content in flowers (increase) and seeds(decrease). Therefore, by mediating iron uptake andsequestering processes in plastids, PIC1 is crucial forplant growth and development in general and inparticular for fruit maturation.

RESULTS

Generation of Transgenic Arabidopsis PlantsOverexpressing PIC1

To obtain stable plant lines overexpressing PIC1, thecoding sequence of the chloroplast-targeted PIC1 pre-cursor protein (Duy et al., 2007) under the control ofthe cauliflower mosaic virus 35S promoter (35S::PIC1)was introduced into ecotype Columbia (Col-0) wild-type Arabidopsis plants. Eight independent T1 linesthat inherited the 35S::PIC1 construct and producedviable seeds in the T2 generation were generated. Twoof these lines (lines 2 and 4) showed identical pheno-types that segregated in a Mendelian fashion (Fig. 1).About 25% of the progeny was of wild-type appear-ance, 25% of the seedlings were dwarfed and albino,and the remaining 50% showed yellow-green, chlo-rotic leaves, visible after 2 to 3 weeks of growth. PCRgenotyping confirmed that the green plants of lines 2and 4 were wild type for PIC1, while the albinoseedlings and the chlorotic plantlets both containedthe 35S::PIC1 construct. Because the yellow-green prog-eny in all subsequent generations showed this pheno-typic segregation pattern, we conclude that these plantsare heterozygous for 35S::PIC1. In consequence, thedwarfed albino seedlings represent homozygous allelesfor the 35S::PIC1 construct. Since all albino seedlingswere nonviable, experiments were performed on theheterozygous chlorotic progeny of the independentlyderived 35S::PIC1 lines 2 and 4.

PIC1 RNA and Chloroplast-Intrinsic Protein AreIncreased in 35S::PIC1 Lines

The 35S::PIC1 transgenic lines were analyzed fortheir PIC1 transcript and protein content by quantita-

Figure 1. Phenotypic segregation of 35S::PIC1 plants. Plants of the35S::PIC1 lines 2 and 4 were grown for 3 weeks on plates. Both linessegregated in aMendelian fashion, producing progenywith greenwild-type(left), chlorotic (middle), and albino (right) phenotypes. A, Of 123 plantsanalyzed in the T2 generation (35S::PIC1 line 2), 29% were of green wild-type appearance, 43% showedyellow-green leaves, and 28%were dwarfedalbinos. B, From the 35S::PIC1 line 4, 97 plants in the T2 generation splitinto progeny of 32% green, 45% chlorotic, and 23% albino phenotype.

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tive real-time reverse transcription (RT)-PCR and im-munoblot analysis, respectively (Fig. 2). Thereby, itbecame evident that 2-week-old, chlorotic seedlings ofthe heterozygous 35S::PIC1 lines 2 and 4 containedabout 26 times more PIC1 mRNA than Col-0 (Fig. 2A;Supplemental Table S1). In all segregating wild-typeplantlets as well as in seedlings of other 35S::PIC1lines generated, PIC1 mRNA content was not signif-icantly elevated. In contrast, in the lethal albino seed-

lings from lines 2 and 4, identified as homozygous for35S::PIC1, 5- to 10-fold more PIC1 transcripts than inheterozygous chlorotic seedlings of the same motherplant were detected (data not shown), indicating agene dosage-dependent effect of 35S::PIC1 overexpres-sion. Since progeny of line 3 contained the 35S::PIC1construct as verified by PCR genotyping but showedneither a yellow-green leaf phenotype nor a strongincrease in PIC1 transcripts (2-fold; Fig. 2A; Supple-mental Table S1), this line was used as a wild-type-likecontrol in the following.

To analyze PIC1 increase on the protein level, wesubjected proteins from 5-week-old rosette leaves ofthe heterozygous 35S::PIC1 lines 2 and 4 to immuno-blot analysis (Fig. 2B). In the extract from chloroticleaves, the PIC1 antibody detected a strong signal atabout 26.5 kD, corresponding to the predicted size ofthe overexpressed mature PIC1 construct. Note thatdue to the presence of a C-terminal tag of 4 kD, theoverexpressed PIC1 protein is bigger than the endog-enous mature form of PIC1 (22.5 kD; compare Duyet al., 2007). Because the same construct was able tocomplement the strong pic1 knockout phenotype, wededuce that the introduced PIC1 preprotein (constructof 36.2 kD) in the 35S::PIC1 lines 2 and 4 was properlyprocessed by the chloroplast stromal peptidase andcorrectly inserted into the inner envelope membrane.Chloroplast-intrinsic PIC1 proteins were confirmed byimmunoblotting on mature leaves, flowers, and puri-fied chloroplasts (Fig. 2C). Again the PIC1 antibodyspecifically labeled proteins only in tissue of the het-erozygous 35S::PIC1 line 2 but not in the correspond-ing wild types. Since PIC1-overexpressing (PIC1ox)signals in chloroplasts, leaf, and flower tissue were ofthe same size and comparable strong intensity, weconclude that the majority of the overexpressed PIC1is present in the inner envelope membrane of chlo-roplasts. Thus, the overproduced PIC1 proteins rep-resent functional permeases. On proteins of leaves,flowers, and chloroplasts of Col-0 and the wild-typeprogeny, segregating from lines 2 and 4, however,a-PIC1 failed to recognize the endogenous maturePIC1 (Fig. 2, B and C). In general, to detect endogenousPIC1 signals via antiserum in wild-type plants, en-velope preparations, which are strongly enriched forPIC1 proteins, have to be used (compare Duy et al.,2007). In summary, we conclude that the chloroticphenotype of the independently derived heterozy-gous 35S::PIC1 lines 2 and 4 is due to highly increasedlevels of the iron-permease PIC1 in the inner envelopeof chloroplasts. Therefore, we designate these lines asPIC1ox 2 and PIC1ox 4.

pic1 knockout mutants were characterized by in-creased transcript levels for FER1 and FER4 (for ferri-tin1 and 4), two isoforms of the iron-storage proteinferritin, resulting in the accumulation of ferritin clus-ters in plastids (Duy et al., 2007). To check whether wecan detect a reciprocal behavior of ferritin gene ex-pression in PIC1ox plants, we performed quantitativereal-time RT-PCR analysis on 2-week-old seedlings of

Figure 2. 35S::PIC1 lines overproduce PIC1 in chloroplasts and differ-entially express plastid iron homeostasis genes. A, Quantification ofPIC1 transcripts by real-time RT-PCR in 14-d-old plantlets of Col-0 and35S::PIC1 lines 2, 3, and 4. PIC1 mRNA (arbitrary units) was calculatedrelative to 10 molecules of actin 2/8 (n = 3 6 SD; for line 3, n = 2). Forlines 2 and 4, only the chlorotic progeny heterozygous for 35S::PIC1was used, while PIC1 mRNA in line 3 (wild-type-like control) wasdetermined in all seedlings containing the 35::PIC1 construct. B,Immunoblot analysis of PIC1 in protein extracts (100 mg per lane)from 5-week-old rosette leaves of PIC1ox lines 2 and 4. The PIC1-specific signal at 26.5 kD was detectable only in the chlorotic progeny,heterozygous for 35S::PIC1 (he; arrowheads). In the segregating wild-type alleles (wt), a-PIC1 stained only background signal at about 24 kD.C, Immunoblot analysis of PIC1 in protein extracts from mature leaves,flowers, and isolated chloroplasts of PIC1ox line 2 and the Col-0 wildtype. For leaves and flowers, 100 mg, and for chloroplasts, 12.5 mg, ofprotein was loaded per lane. Note that, in contrast to the blot in B, 4 M

urea was used during gel separation, which shifted PIC1-specificsignals in overexpressing plants of PIC1ox 2 he (arrowheads) to a sizeof about 24 kD and background in all tissues to 28 kD. D, Quantifi-cation of FER1 (black bars) and FER4 (white bars) transcripts by real-time RT-PCR as described in A. FER1 and FER4 mRNA (arbitrary units)was calculated relative to actin 2/8 and normalized to the amount inCol-0, which was set to 1.0. Asterisks indicate a significant reduction ofFER1 transcripts (two-sample Student’s t test, P , 0.005) in PIC1ox 2and 4 when compared with lines with PIC1 wild-type content (Col-0and 35S::PIC1 line 3). E, Immunoblot analysis of the plastid-localizediron-superoxide dismutase FSD1 in protein extracts (12.5 mg per lane)from mature leaves of PIC1ox line 2 (he and wt) and the Col-0 wildtype. Mature FSD1 proteins migrate at 22 kD.

Plastid PIC1 Regulates Fe Homeostasis and Plant Growth

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PIC1ox lines 2 and 4 as well as on PIC1 wild-typecontrols Col-0 and 35S::PIC1 line 3 (Fig. 2D; Supple-mental Table S1). Thereby, we could show that FER1but not FER4 transcripts were decreased by about60% in PIC1ox lines, showing indeed down-regulationof the major leaf ferritin FER1 (Briat et al., 2010) inseedlings overexpressing PIC1. Furthermore, protein ofthe chloroplast-localized FSD1 (for Fe-dependent super-oxide dismutase) increased in PIC1ox leaves (Fig. 2E),again showing a reciprocal behavior as in pic1 knockoutplants (down-regulation; Duy et al., 2007). Thus, inPIC1ox plants, a noticeable regulation of plastid ironhomeostasis genes is apparent (see “Discussion”).

The Phenotype of PIC1ox Plants Closely ResemblesMutants Lacking Ferritin and Is Most Likely Caused byIron Overload in Chloroplasts

During further growth and development of PIC1oxplants, the yellow-green leaf phenotype became more

pronounced. Chlorosis was first visible at the centralvein, then spread over the entire surface in matureleaves (Fig. 3A). In 4-week-old leaves, this led to adecrease in chlorophyll a and b by 50% when com-pared with wild-type plants (Fig. 3B; SupplementalTable S1). Staining of 6-week-old rosette leaves (Fig.3C) revealed that chlorosis was accompanied by theproduction of H2O2, most likely originating from chlo-roplasts (Mubarakshina et al., 2010). Underlining theproposed function of PIC1 in iron uptake (Duy et al.,2007), we further found that mature chloroplasts ofPIC1ox line 2 contained about 3-fold more iron thancorresponding wild-type organelles (Fig. 3D; Supple-mental Table S1).

Investigation of ultrathin sections of leaves with trans-mission electron microscopy (Fig. 4) showed significantdifferences of chloroplast ultrastructure in PIC1ox lines2 and 4 compared with the wild type: small membranevesicles with an electron-translucent matrix (diameterabout 70 nm) were accumulated throughout the stroma

Figure 3. PIC1ox leaves are chlorotic and suffer from oxidative stress as well as iron overload in chloroplasts. A, A 3-week-oldPIC1ox plant (line 4) in comparison with Col-0. Images of both plants are depicted with the same settings for magnification andbrightness. At right is a closeup of PIC1ox leaves to highlight the development of chlorosis (arrowheads). B, Chlorophyll (Chl)content in rosette leaves of 4-week-old Col-0 and 35S::PIC1 lines 2, 3, and 4. Chlorophyll a (dark green) and chlorophyll b (lightgreen) concentrations were determined in leaf discs of identical size (mg mm22 leaf tissue; n = 5 6 SD). In the chlorotic PIC1oxlines 2 and 4 (he; yellow grid), chlorophyll a and chlorophyll b were about half of all PIC1 wild-type alleles (wt). C, DAB stain of6-week-old rosette leaves of Col-0 (left) and PIC1ox lines 2 and 4 (middle, segregating wild-type alleles; right, chlorotic plants,heterozygous for 35S::PIC1). D, Iron content (ng mg21 chlorophyll g21 fresh weight [FW]; n = 26 SD) in chloroplasts purified frommature leaves of Col-0 and PIC1ox line 2. In PIC1ox chloroplasts (he; red bar), the iron content was increased more than 3-foldwhen comparedwith chloroplasts of PIC1wild-type alleles (wt; white bars). Note that the depicted iron content per chloroplast isan estimate, assuming that isolated chlorophyll per leaf fresh weight represents intact chloroplasts in each preparation.Chloroplast isolation and determination of iron levels are described in detail in “Materials and Methods.”

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of PIC1ox mesophyll chloroplasts. Due to the presenceof numerous, significantly smaller vesicles in the vi-cinity of the inner plastid envelope, they most likelyoriginated by budding from this membrane. Thestructure of mitochondria and peroxisomes, however,was not altered when compared with wild-type cells.Although the developmental stage was similar to

that of Col-0, PIC1ox plants produced less biomass.When measured just before bolting in 39-d-old plants,the rosette dry weight of PIC1ox line 2 was only 28% ofthe corresponding wild types (Supplemental Table S1).In general, adult PIC1ox plants were smaller in sizeand of a bushy appearance, characterized by the absenceof a central inflorescence stalk (Fig. 5A). Bolting of flowersoccurred simultaneously with the wild type; however,further silique and seed development was severely im-paired. Either siliques did not reach their full size,producing small and shriveled seeds, or empty, crum-pled siliques appeared (Fig. 5, B and C). In consequence,these defects in fruit development of PIC1ox plants ledto a drastic reduction in seed yield and weight (Fig. 5, Dand E) as well as to a dramatically reduced seed germi-nation rate (between 0% and 15%). The latter was moresevere in PIC1ox line 4, resulting in almost no viable seedprogeny of this line. Therefore, subsequent analysescould only be performed on plants of the PIC1ox line 2.When we measured metals in dry PIC1ox seeds, we

found that iron content per seed grain was significantlyreduced (Fig. 6A), while the concentration of the transi-tion metals copper, zinc, and manganese did not change

(Supplemental Table S2), indicating a PIC1 impact onseed iron loading. To test whether iron deficiency oroverload might influence germination of PIC1ox lines,we found that addition of neither Fe(III)-EDTA (100 mM)nor of the iron-chelator ferrozine (300 mM) increased ordecreased the germination of PIC1ox lines 2 and 4 (datanot shown). In contrast to seeds, impaired flower de-velopment in PIC1ox plants, however, was accompaniedby an increase in iron concentration (Fig. 6B). Again,changes were iron specific, because copper, zinc, andmanganese were unchanged when compared with thewild type (Supplemental Table S2).

In summary, we conclude that the described phe-notypic defects of PIC1ox plants most likely originatedfrom chloroplast iron overload, which is mediated byoverexpressed PIC1 proteins.

PIC1 Overexpression Leads to Deregulated GeneExpression in Flowers

Because flowers of PIC1ox plants showed impaireddevelopment in combination with elevated iron levels,we monitored differential gene expression by micro-array analysis (Affymetrix ATH1 GeneChip) in theidentical samples that were used for metal determina-tion (floral stages as depicted in Fig. 5B). When wecompared transcript levels in flowers of PIC1ox line 2with those of wild-type plants, we found that 1,809genes could be classified as up-regulated, while ex-pression of 1,963 genes was down-regulated (Fig. 7;

Figure 4. PIC1 overexpression induces vesicle formation in chloroplasts. Transmission electron micrographs of ultrathin sectionsfrom mesophyll tissue of 17-d-old rosette leaves are shown. A, Col-0. B, PIC1ox line 2. Characteristic for chloroplasts of PIC1oxlines are numerous small membrane vesicles in the vicinity of the inner envelope membrane (circles). Lager vesicles are presentthroughout the stroma between thylakoid membranes (squares). M, Mitochondrion; Pe, peroxisome; S, starch grain.





Supplemental Table S3). Disposition into functionalcategories by the MAPMANArabidopsis pathway anal-ysis program revealed that besides those withstill unknown functions (512 up, 538 down), the largestfractions of differentially expressed genes were assignedto the categories protein (320 up, 224 down), RNA (197up, 177 down), miscellaneous (82 up, 132 down), trans-port (74 up, 120 down), and signaling (47 up, 106 down).

To further identify hotspots of regulation, wesearched for functional categories that showed a highratio between numbers of up- and down-regulatedgenes. The greatest differences pertained to photosyn-thesis, tetrapyrrole synthesis, mitochondrial electrontransport, metal handling, and polyamine metabolism(Fig. 7). In particular for photosynthesis-associatedgenes, divergence was striking. Whereas only fourgenes either with a function in the Calvin cycle orphotorespiration were down-regulated, transcripts of117 genes increased. The majority of these are codingfor proteins directly involved in light reactions of PSIand PSII (e.g. light-harvesting complexes or electrontransport chain; for details, see Supplemental TableS3). Accordingly, genes with a function in tetrapyrrolesynthesis only showed up-regulation. Thus, PIC1oxflowers are characterized by elevated iron levels and asimultaneous increase in transcripts for proteins witha function in photosynthesis as well as chlorophyll andheme biosynthesis.

Since PIC1 was previously described to function inmediating iron transport across the chloroplast inner

envelope membrane (Duy et al., 2007) and metalhandling was another focus for differential regulationof expression in PIC1ox flowers, we took a closer lookat metal transport and homeostasis. First, we couldconfirm that PIC1 transcripts (11-fold induction; TableI) and proteins (Fig. 2C) were constantly and stronglyincreased in PIC1ox flowers. Of the eight other genesrelated to metal transport and up-regulated in PIC1oxflowers, five are reported to function in plastid mem-branes: FRO7, MAR1/IREG3, NiCo, YSL6, and PAA1/HMA6 (for details, see “Discussion”). In contrast, noneof the down-regulatedmetal transporters, according tocurrent knowledge, is localized in plastids. Regulationof metal handling genes (Table II) revealed the samedistribution and plastid focus. On the one hand, pro-teins from seven of eight genes induced in PIC1oxflowers are plastid intrinsic (FSD1, CSD2, OSA1, FC2,DWARF27, ClpC1/HSP93-V, and CUTA). On the otherhand, among genes of this functional category thatshowed transcript decrease, none is in plastids (fordetails, see “Discussion”).

DISCUSSION

PIC1 and Ferritin Are Key Players in Balancing IronHomeostasis as Well as Plant Growth and Development

Previously, we described PIC1 to function in irontransport across the inner envelope membrane ofplastids and thus to control plastid and cellular metal

Figure 5. PIC1 increase causes im-paired flower and seed development.A, Mature Col-0 (left) in comparisonwith PIC1ox line 4 (right). B, Flowerand early fruit development in Col-0(left) and PIC1ox line 2 (right). Bracketsindicate developmental stages used formetal determination and microarrayanalysis. C, Seeds of Col-0 (left) andPIC1ox line 2 (right). D, Seed yieldper plant of the Col-0 wild-type and35S::PIC1 lines 2, 3, and 4 (n = 10 6SD). E, Weight of 100 seeds from Col-0and 35S::PIC1 lines 2, 3, and 4 (n =10 6 SD). White bars in D and E depictplants with wild-type PIC1 content(wt), and black bars represent linesoverexpressing PIC1 (heterozygous for35S::PIC1; he).

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homeostasis (Duy et al., 2007). In that earlier study, wefound that ferritin protein clusters accumulated inplastids of pic1 knockout mutants. Because in thesemutants most likely the major iron-uptake pathwayinto plastids was blocked, free iron accumulated in thecytosol, thereby causing severe oxidative damage. Inconsequence, the plant cell was up-regulating ferritinexpression (Duy et al., 2007). Interestingly, Ravet et al.

(2009) recently showed that in the ferritin knockoutmutant fer1,3,4, which is devoid of all ferritins in-volved in vegetative growth, PIC1 transcript levelswere increased in flowers when plants got excess iron.Thus, expression of PIC1 and ferritin is reciprocallyregulated: when PIC1 proteins are lacking, ferritinlevels rise, and when ferritin is missing, PIC1 isincreased. This observation was underlined by thefinding that in mesophyll chloroplasts from Zea mays,PIC1 belongs to the most abundant proteins identified,while ferritin was not detected. In contrast, in chloro-plasts of Pisum sativum, FER1 and FER4 proteins werefound, but PIC1 was absent (see Supplemental TableS3 in Brautigam et al., 2008).

But what happens when PIC1 protein is increased inArabidopsis chloroplasts? By generating plant lineswith stable overexpression of PIC1 in this study, wecould show that, again, expression of the major leafferritin FER1 in 2-week-old seedlings behaved con-trarily to PIC1. However, even more remarkable wasthe high similarity of phenotypes between fer1,3,4knockout mutants and PIC1ox lines. When fer1,3,4plants are challenged with excess iron, they showreduced biomass, oxidative stress, as well as impairedflower development and decreased seed yield (Ravetet al., 2009). All these defects were observed in PIC1oxlines as well, although no additional iron treatmentwas necessary, indicating that PIC1-transport functionby itself induced a rise in plastid-intrinsic iron levels(Fig. 3D). The highest correlation between fer1,3,4 andPIC1ox phenotypes was found in flowers. Here, bothplant lines have elevated levels of iron, resulting instress and in turn impaired fruit development. Due tothe loss of ferritins in fer1,3,4 flower tissue, plastids areincapable of buffering excess free iron by the ferritinprotein shell, which in turn is causing massive oxida-tive stress. Interestingly, PIC1 mRNA was increased

Figure 7. Gene expression in PIC1oxflowers is deregulated. The distributionof differentially expressed genes intofunctional categories in PIC1ox versuswild-type flowers is shown. Differentialexpression was calculated by micro-array analysis as described in “Mate-rials and Methods.” Classification ofgenes was performed by MAPMAN(http://gabi.rzpd.de/projects/MapMan,version 3.1.1 [April 2, 2010]). In total,1,809 genes were assigned to be up-regulated (left), while 1,963 genes wereclassified as down-regulated (right).Note that numbers and definitions ofcolor codes apply only to regulatedgenes in functional categories, whichare mentioned in the text (for a com-plete list, see Supplemental Table S3).

Figure 6. PIC1ox seeds contain low iron and flowers have elevatediron. A, Iron content (ng per seed grain; n = 5 6 SD; for Col-0, n = 3)in seeds of Col-0 and PIC1ox lines 2 and 4. B, Iron concentration(mg g21 dry weight [DW]; n = 36 SD) in flowers of Col-0 and PIC1oxline 2. Asterisks indicate significantly different iron levels (two-sample Student’s t test; ** P , 0.005, * P , 0.05) in PIC1 wild-typealleles (wt; white bars) when compared with PIC1ox lines (he; blackbars).





concomitantly with floral iron accumulation (about7-fold in Col-0 and 14-fold in fer1,3,4; Ravet et al.,2009). PIC1ox flowers in our study showed a strongincrease in PIC1 transcripts, as well as proteins andhad to deal with iron overload, too. Thus, as shown forleaves, an increase in PIC1 proteins in this case mostlikely resulted in elevated iron inside flower plastidsas well, which was due to enhanced iron transport andnot reduced iron storage capacity, as for ferritin knock-outs. Hence, in flowers, more PIC1 protein causes thesame effect as the loss of ferritin, resulting in oxidativedamage followed by impaired fruit development aswell as a dramatic decrease in seed yield and germi-nation. About mechanisms of the opposite regulationof PIC1 and ferritin expression, however, we canonly speculate. In the case of pic1 knockout lines, themassive induction of ferritin expression is a well-described reaction upon oxidative stress and increaseof cytosolic iron levels (Duy et al., 2007; Briat et al.,2010). In PIC1ox lines, in contrast, the plant responseto the regulation of ferritin expression is only slight,but as described above, mutant phenotypes and pro-posed roles of both proteins during plant developmentcan be closely linked. Therefore, we conclude that inplastids, the functions of PIC1 (iron transport) andferritin (iron storage) correlate reciprocally and that

both proteins are crucial for balancing iron homeosta-sis throughout plant growth and development.

PIC1-Induced Oxidative Damage Is Iron Specific andOriginates from Chloroplasts

Oxidative stress of PIC1ox plants most likely origi-nated in chloroplasts, as documented by the productionof H2O2, plastid intrinsic vesicle formation, decrease inchlorophyll, and iron overload in leaf chloroplasts.During the life cycle, stress became detrimental forthe entire plant, resulting in leaf chlorosis and reducedbiomass. In particular, iron homeostasis was impairedin flowers and seed tissue, causing severe defectsduring fruit development. In PIC1ox as well as pic1knockout (Duy et al., 2007) plants, expression of theiron- and copper-dependent superoxide dismutasesFSD1 and CSD2, which detoxify ROS in the chloroplaststroma (Kliebenstein et al., 1998; Abdel-Ghany et al.,2005), was differentially regulated. In particular, thepronounced up-regulation of FSD1 in PIC1ox (oppositeto the down-regulation in pic1 knockouts), togetherwith the rise of iron levels in PIC1ox flowers and leafchloroplasts, indicate that oxidative stress is due tohigh iron in plastids. Although described to be cyto-solic (Myouga et al., 2008), recent proteomic (Ferro

Table I. Differentially expressed metal transport genes in PIC1ox versus the wild type

Microarry analysis was performed on flowers of the wild type (wt) and PIC1ox line 2 (PIC1ox). Names and Arabidopsis Genome Initiative (AGI)codes for regulated metal transport genes are listed. Respective membranes (loc) of corresponding proteins are designated as follows: C-env,chloroplast envelope; C-ie, chloroplast inner envelope; ER, endoplasmic reticulum; G, Golgi; PM, plasma membrane; V, tonoplast; ?, unknown. Ifsubcellular localizations are clearly predicted but not yet experimentally verified, abbreviations are given in parentheses. The average scaled signals(n = 5 for PIC1ox, n = 6 for the wild type), the fold change (FCH), and the corresponding P values of the analysis are shown. Signals for FRO6 andFRO7 are detected by the same ATH1 probe (bracket). Annotation of genes and proteins was done according to publications and ARAMEMNON(Schwacke et al., 2003) as well as TAIR (Swarbreck et al., 2008) databases.

Name AGI loc wt PIC1ox FCH P

UpPIC1 At2g15290 C-ie 956.52 10,421.83 10.90 1.17E-11FRO6 At5g49730 PM 523.90 2070.95 3.95 1.37E-05FRO7 At5g49740 C-envMAR1/IREG3 At5g26820 C-env 480.93 673.53 1.40 6.10E-04YSL6 At3g27020 (C-env) 1,062.36 1,227.80 1.16 2.45E-02NiCo At2g16800 (C-env) 492.37 621.87 1.26 4.43E-03PAA1/HMA6 At4g33520 C-ie 264.79 343.00 1.30 4.09E-03COPT1 At5g59030 PM 1,098.02 1,384.59 1.26 4.14E-03MTP1/ZAT1 At2g46800 V 1,041.03 1,355.03 1.30 3.02E-03MTP11 At2g39450 ER/G 915.42 1,107.12 1.21 3.12E-02

DownFRO8 At5g50160 (PM) 315.73 210.84 0.67 5.06E-04IRT1 At4g19690 PM 143.10 90.13 0.63 3.92E-02ZIP1 At3g12750 (PM) 472.92 292.99 0.62 9.12E-04ZIP6 At2g30080 (PM) 249.96 191.71 0.77 5.71E-03ZIFL1 At5g13750 (PM) 206.87 134.09 0.65 1.16E-03ZTP29 At3g20870 ER 932.62 757.43 0.81 6.30E-03PCR11 At1g68610 (PM) 334.11 149.63 0.45 1.18E-02YSL1 At4g24120 (PM) 1,185.67 863.06 0.73 4.08E-02YSL8 At1g48370 ? 299.81 220.04 0.73 1.04E-02NRAMP2 At1g47240 ? 341.41 270.11 0.79 2.59E-03NRAMP6 At1g15960 ? 120.70 71.81 0.59 7.61E-03MGT9/MRS2-2 At5g64560 ? 427.85 325.68 0.76 5.45E-03

Duy et al.




et al., 2010) and GFP-targeting as well as proteinimport studies (W.-L. Chang and B. Bolter, unpub-lished data) clearly demonstrate plastid targeting ofFSD1 without a classical signal peptide. The plastidorigin of PIC1-controlled oxidative stress also is dem-onstrated by increased transcripts of the chloroplastenvelope protein OSA1 in PIC1ox flowers. OSA1(again contrarily regulated in pic1 knockouts) hasbeen shown to play an essential role in balancingoxidative stress generated by heavy metals, H2O2, andlight (Jasinski et al., 2008).Furthermore, proteins for all other metal-handling

genes with elevated expression in PIC1ox, except one,are in plastids. At the same time, three of four of theseproteins are involved in iron homeostasis: ferrochela-tase (FC2) assembles iron into protoporphyrin IXduring heme synthesis in chloroplasts; DWARF27, aplastid-localized iron-containing protein, functions inthe biosynthesis of strigolactones (Lin et al., 2009);ClpC1, originally described as HSP93-V chaperone forplastid protein import, was recently shown to be in-volved in iron homeostasis, since chlorosis of ClpC1loss-of-function mutants could be rescued by iron ad-dition (Wu et al., 2010). In contrast, down-regulation ofmetal homeostasis genes is neither plastid nor ironspecific but rather linked to other metals. Decreasedtranscripts in PIC1ox flowers include those for cytosoliccopper-superoxide dismutase CSD1 (plastid CSD2 isup-regulated) and copper chaperones (CCH and ATX1),which deliver copper to heavy metal P-type ATPases

and are linked to senescence and oxidative stress(Himelblau et al., 1998; Mira et al., 2001; Puig et al.,2007). HMA domains (for heavy metal transport/detoxification) can be found in proteins of four otherdown-regulated genes that belong to a class of isopre-nylated proteins binding transition metals like copper,nickel, or zinc (Dykema et al., 1999; Barth et al., 2009).The decreased transcripts MT1c, MT2b, and MT3 codefor small plant metallothioneins, which function inmetal homeostasis, in particular for copper, cadmium,or zinc tolerance (van Hoof et al., 2001; Zimeri et al.,2005; Guo et al., 2008; Gao et al., 2009; Hassinen et al.,2009). In this list, ARD1 (for acireductase dioxygenase1), as part of the Met salvage pathway (Burstenbinderet al., 2007), is the only down-regulated protein thatrequires iron. In summary, increased expression ofmetal-handling genes in PIC1ox flowers is clearlyiron and plastid specific, indicating iron overload inchloroplasts. In contrast, down-regulated genes re-flect a disturbance of cellular metal homeostasis ingeneral.

Whereas pic1 knockout mutants were characterizedby a dramatic decrease of the majority of photosyn-thesis proteins (Duy et al., 2007), the opposite occurredin PIC1ox plants. Here, transcript levels of most genesinvolved in primary photosynthesis and tetrapyrrolesynthesis were higher than in the wild type. However,PIC1ox plants were suffering from chlorosis, reducedbiomass, and a leaf chlorophyll decrease by 50%. Vesicleformation in plastids further indicates the accumulation

Table II. Differential expression of genes related to metal homeostasis in PIC1ox versus the wild type

Microarry analysis was performed on flowers of the wild type (wt) and PIC1ox line 2 (PIC1ox). Names and Arabidopsis Genome Initiative (AGI)codes of regulated genes are given. Localization (loc) of corresponding proteins is designated as follows: C, chloroplast; C-env, envelope membrane;C-ims, intermembrane space; C-str, stroma; cyt, cytosol; nuc, nucleus; ?, unknown. If subcellular destinations are clearly predicted but not yetexperimentally verified, abbreviations are in parentheses. The average scaled signals (n = 5 for PIC1ox, n = 6 for the wild type), the fold change(FCH), and the corresponding P values of the analysis are listed. Annotation of genes and proteins was done according to publications andARAMEMNON (Schwacke et al., 2003) as well as TAIR (Swarbreck et al., 2008) databases.

Name AGI loc wt PIC1ox FCH P

UpFSD1 At4g25100 C-str 212.56 910.94 4.29 1.32E-02CSD2 At2g28190 C-str 3,187.82 4,009.61 1.26 2.39E-03OSA1 At5g64940 C-env 973.65 1,234.68 1.27 4.72E-03FC2 At2g30390 C-str 762.15 881.59 1.16 3.41E-02DWARF27 family At1g64680 (C) 216.31 361.31 1.67 1.24E-04ClpC1/HSP93-V At5g50920 C-str 3,266.86 4,492.04 1.38 7.76E-04CUTA At2g33740 C-(ims) 482.76 564.29 1.17 2.58E-02HMA domain At5g03380 ? 329.71 470.26 1.43 1.15E-02

DownCSD1 At1g08830 cyt 7,134.23 5,578.74 0.78 5.71E-03CCH At3g56240 cyt 4,898.91 3,034.82 0.62 6.96E-04ATX1 At1g66240 cyt 1,186.55 995.26 0.84 1.55E-02HMA domain At5g17450 ? 498.20 185.93 0.37 2.29E-04HMA domain At5g50740 ? 139.20 87.95 0.63 3.49E-02FP3 (HMA domain) At5g63530 ? 646.98 546.59 0.84 2.21E-02FP6/HIPP26 (HMA domain) At4g38580 nuc 1,166.19 913.89 0.78 9.05E-03MT1c At1g07610 ? 1,925.47 1,287.33 0.67 2.44E-04MTb2 At5g02380 ? 17,090.44 14,107.21 0.83 2.59E-03MT3 At3g15353 ? 12,402.44 10,648.57 0.86 2.02E-02ARD1 At4g14716 ? 3,163.84 2,481.71 0.78 9.77E-04





of ROS, in particular H2O2, as documented by leafstaining. As in ferritin knockouts challenged with ad-ditional iron (Ravet et al., 2009), in PIC1ox flowers, theusually beneficial effects of elevated iron levels (e.g.increased biomass upon iron fertilization) becamedeleterious. Therefore, oxidative damage, most likelycaused by iron and H2O2 accumulation in plastids,prevented efficient photosynthesis.

According to the regulation of metal homeostasisand photosynthesis genes in PIC1ox and the describedphenotypical characteristics, we propose that PIC1-induced oxidative damage is iron specific and origi-nates from plastids.

PIC1 Most Likely Functions in the Uptake andSequestration of Iron in Plastids

The metal transport category showed a similar iron-and plastid-focused gene regulation pattern as dis-cussed for metal homeostasis genes. In this case, fiveof eight genes with increased transcripts in PIC1oxflowers are reported to function in plastid membranes,while none of the down-regulated genes, accordingto current knowledge, is plastid localized. The mostpronounced increase was for mRNA of the ferric-chelate reductases FRO6 and FRO7, which both aredetected by identical probes on the ATH1 chip.WhereasFRO6 is localized in the plasma membrane, FRO7 isplastid intrinsic and might reduce ferric iron prior touptake into chloroplasts (Jeong et al., 2008). Sinceacross the plastid inner envelope membrane, ironmostlikely is transported as ferrous ion (Shingles et al.,2001, 2002), PIC1 could represent the so far unknownpermease acting in concert with FRO7. Interestingly,FRO8 (down-regulated by cytosolic iron deficiency;Mukherjee et al., 2006) and nonplastid representativesof the ZIP (for ZRT/IRT-like protein) transportersare decreased in PIC1ox, again indicating plastidincrease and cytosolic shortage of iron. Furthermore,down-regulation of IRT1, ZIP6, YSL1, and YSL6 (foryellow-stripe like1 and 6; see below) is also found iniron-challenged fer1,3,4 flowers (Ravet et al., 2009) thatsuffer from elevated free iron in plastids. Since, inaddition, we could document that PIC1ox chloroplastscontained increased iron levels, we conclude that PIC1overexpression results in plastid iron overload as well,closely linking PIC1 function to iron uptake.

Furthermore, transcripts of the chloroplast-localizedMAR1/IREG3 (Conte et al., 2009) are increased inPIC1ox flowers. Because overexpression of MAR1/IREG3 causes leaf chlorosis that can be rescued byiron, it was suggested that this aminoglycoside trans-porter might function naturally in uptake of the iron-chelating polyamine NA (Conte and Lloyd, 2010).Interestingly, leaf chlorosis induced by MAR1/IREG3and PIC1 overexpression showed similar progressionalong the midveins, while chlorosis of NA synthase-less mutants is described to be interveinal (Ling et al.,1999; Klatte et al., 2009). Conte and coworkers (Conteet al., 2009; Conte and Lloyd, 2010) argue that the

unusual MAR1/IREG3-mediated midvein chlorosismight be due to redistribution of the cytoplasmic NApool to plastids. In consequence, iron would be se-questered within the organelles, and NA function inlong-distance transport of metals, which for iron mostlikely is in phloem loading/unloading processes (forreview, see Curie et al., 2009), would be restricted.Therefore, it seems likely that PIC1-mediated veinalchlorosis might be due to increased and immobilizedplastid iron and in turn disturbed iron and NA ho-meostasis in the phloem.

Another link to iron-NA transport in PIC1ox flowersis provided by specific down-regulation of polyaminebiosynthesis genes connected to NA synthesis and up-regulation of the potential iron-NA transporter YSL6(for overview, see Curie et al., 2009). Only recently,YSL6 was reported to be integral to chloroplast envel-ope membranes as well; however, the direction andsubstrates of transport have not yet been clarified (C.Curie, personal communication). In contrast to YSL6,transcripts of YSL1 and YSL8 are decreased in PIC1oxflowers. YSL1, most likely in the plasma membrane,fulfills a function in the delivery of iron-NA chelatesto seeds, as demonstrated by the decreased levels ofiron andNA in ysl1 knockout seeds (Le Jean et al., 2005).Because PIC1ox seeds too suffered from iron shortage,it is tempting to speculate that increased PIC1 functionaccumulates iron inside plastids, thereby disturbingNA distribution as well and subsequently influencingfruit development and in particular seed iron loading.

In conclusion, the majority of effects caused by PIC1overexpression, in particular the significant accumu-lation in PIC1ox chloroplasts, together with the factthat PIC1 mediated iron uptake into yeast cells and thestrong iron-deficient phenotype of pic1 knockouts(Duy et al., 2007) favor a function of PIC1 in ironinflux into plastids.

The Function of PIC1

The protein NiCo, whose corresponding transcriptsare increased in PIC1ox flowers, is targeted to chloro-plast envelopes and predicted to function in the trans-port of nickel and/or cobalt (Fig. 8A; The ArabidopsisInformation Resource [TAIR] gene model at www.arabidopsis.org [June 15, 2009]). Only recently, weidentified NiCo in a yeast-based screen for PIC1-interacting proteins (R. Stube and K. Philippar, un-published data). Furthermore, the orthologous proteinin Nicotiana benthamiana is annotated as “chloroplastzebra-necrosis protein” (GenBank accession no.ACA03877), suggesting chlorotic NiCo mutant pheno-types similar to PIC1ox or knockout plants. With ageneral low specificity of plant transporters for diva-lent metal ions (compare for IRT1; Korshunova et al.,1999), it thus is tempting to speculate that PIC1 andNiComight function together in plastid iron transport,although interaction so far was shown in heterozygousyeast only. Presumably, PIC1 in this potential chloro-plast metal translocon (Fig. 8B) is not directly trans-

Duy et al.




porting iron but rather regulating or modulating aNiCo-like transporter.Because vesicles in PIC1ox seem to bud from the

inner plastid membrane, a function of PIC1 in ironhandover to either ferritin proteins and/or the FeScluster biogenesis machinery (for overview, see Yeet al., 2006; Rouhier et al., 2010) might be possible aswell (Fig. 8B). In consequence, PIC1-induced vesiclesin plastids would be overloaded with accumulatingproteins for photosynthetic light harvesting and elec-

tron transport, in particular FeS cluster or heme pro-teins, which subsequently could not be integrated intofunctional complexes in thylakoids. This hypothesis issupported by the up-regulation of genes for plastidFeS cluster biogenesis (CpSufE, HCF101), heme syn-thesis (ferrochelatase), and FeS cluster proteins (GLU1,CAO, TIC55) in PIC1ox (for details, see SupplementalTable S3). Thus, the hypothetical model depicted inFigure 8B could explain VIPP1-like (for vesicle-inducingprotein in plastids 1) vesicle budding in PIC1ox plastids(Kroll et al., 2001) as well as potential beneficiary effectson protein import. (1) VIPP1 (also up-regulated inPIC1ox) is functioning in thylakoid membrane biogen-esis/maintenance by inducing vesicle formation fromthe inner plastid membrane and thereby most likelychanneling imported preproteins to their thylakoid des-tination (for review, see Vothknecht and Soll, 2005;Jouhet and Gray, 2009). (2) In parallel to its function iniron transport, PIC1 was described as TIC21, being partof the preprotein translocon in the inner envelopemembrane of chloroplasts (Teng et al., 2006; Kikuchiet al., 2009). Thus, PIC1 in loose association with theactual protein translocon channels TIC20 and/or TIC110could deliver iron to the FeS cluster biogenesis machin-ery and thereby drive the assembly of FeS clusters intofreshly imported proteins as well as subsequent VIPP1-mediated vesicle budding.

CONCLUSION

In summary, our data strongly suggest a functionof PIC1 in mediating iron transport across the innerenvelope membrane of chloroplasts. Whether this roleis in direct transport or more oblique in regulatingother transporters or the handover of iron still re-mains to be clarified. Although we cannot fully ex-clude an export function of PIC1, according to thePIC1-mediated iron accumulation in chloroplasts (thisstudy) and in complemented yeast cells (Duy et al.,2007) as well as due to the described phenotypes ofpic1 knockout (iron-deficiency symptoms) and PIC1oxplants (iron-overload appearance), iron uptake is mostlikely. Together with ferritin, therefore, PIC1 repre-sents a crucial element for the integration of plastidiron homeostasis into plant growth and development.

MATERIALS AND METHODS

Plant Material and Growth

All experiments were performed on Arabidopsis (Arabidopsis thaliana)

ecotype Col-0 (Lehle Seeds) and the respective 35S::PIC1 lines. Before sowing,

seeds were surface sterilized and kept at 4�C for 3 d. If not indicated

otherwise, seedlings were grown for 2 weeks on plates (0.3% Gelrite medium

[Roth], 1% D-Suc, and 0.53Murashige and Skoog salts, pH 5.7) and afterward

transferred to soil. Plant growth occurred in chambers with a 16-h-light (21�C;photon flux density of 100 mmol m22 s21)/8-h-dark (16�C) cycle.

To generate plants overexpressing PIC1 under the control of the 35S

promoter, the 888-bp-long coding sequence of PIC1 (Duy et al., 2007) was

subcloned into the plasmid vector pH2GW7 (Karimi et al., 2002). PIC1/

pH2GW7was transformed into Agrobacterium tumefaciens GV3101 cells, which

Figure 8. Model of a potential metal translocon in the inner envelopemembrane of chloroplasts. A, PIC1 and NiCo insert into the innerenvelope membrane of chloroplasts (IE), most likely with four and sixa-helical domains, respectively. The potential metal-binding amino acidmotif HX5DH defines NiCo as a member of the Ni2+-Co2+ transporterfamily (Transporter Classification Database; www.tcdb.org). The firsthelix and the soluble N terminus of PIC1 (green box) are predicted tocontain a TonB-dependent receptor protein signature (Prosite predic-tion; www.expasy.ch). In a yeast split ubiquitin assay, this domain wasnecessary and sufficient to mediate the interaction with NiCo (R. Stubeand K. Philippar unpublished data). The bottom panels show in vivotargeting of PIC1-GFP and NiCo-GFP fusion proteins performed asdescribed by Duy et al. (2007). Green represents GFP signals and redrepresents chlorophyll autofluorescence. B, The hypothetical metaltranslocon, consisting of PIC1 and NiCo subunits (PIC-NIC), is shuttlingiron (Fe) across the inner envelope of chloroplasts (IE; black line). Inproximity, the TIC protein translocon imports proteins (for details, seeJouhet and Gray, 2009). The FeS protein biogenesis machinery (FS) in thestroma generates FeS cluster proteins, which are subsequently targeted totheir destination (e.g. inserted into thylakoid membranes [thy]). Excessiron can be buffered by ferritin. At right, overproduction of PIC1 mightcause VIPP1-mediated formation of vesicles, which contain elevated ironlevels and are destined to fuse with thylakoid membranes.





subsequently were used to transfect Col-0 plants as described by Duy et al.

(2007). T1 lines were selected by hygromycin (30 mg mL21), and stable insertion

of 35S::PIC1 was verified by PCR genotyping (T1 and T2 generations).

Immunoblot Analysis

Total leaf and flower proteins were extracted for 30 min on ice in 50 mM

Tris-HCl, pH 8.0, 50 mM EDTA, 2% lithium dodecyl sulfate, 10 mM dithio-

threitol, and 100 mM phenylmethylsulfonyl fluoride. Cell debris was pelleted

for 15 min at 14,000g and 4�C. Proteins were separated by SDS-PAGE and

transferred to an Immobilon-P polyvinyl difluoride membrane (Millipore).

Polyclonal antiserum against recombinant PIC1 protein (Duy et al., 2007) was

raised in rabbit (Pineda Antibody Service) and used in a 1:1,000 dilution (0.1 M

Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.2% Tween 20). Secondary horseradish

peroxidase-coupled antibodies (Sigma-Aldrich) were diluted 1:8,000. Non-

specific signals were blocked by 1% skim milk powder and 0.03% bovine

serum albumin. Blots were stained with 100 mM Tris-HCl, pH 8.5, 0.5% (w/v)

luminol, 0.22% (w/v) coumaric acid, and 0.009% (v/v) H2O2. Luminescence

was detected byKodak BiomaxMRfilms (Perkin-Elmer). FSD1 immunostaining

was performed as described by Duy et al. (2007).

Chlorophyll Extraction

For chlorophyll extraction, a defined leaf area of 0.64 cm2 was punched and

quickly ground in 1 mL of 80% acetone. After 10 min of dark incubation, cell

debris was pelleted by centrifugation at 16,000g for 10 min. Chlorophyll a

and b contents in the supernatant were measured in a spectrophotometer (750,

663, and 645 nm) and calculated as described (Arnon, 1949).

Detection of H2O2

H2O2 was visually detected by using 3,3-diaminobenzidine (DAB) ac-

cording to Thordal-Christensen et al. (1997). Leaves were vacuum infiltrated

with 1 mg mL21 DAB (pH 3.8) for 20 min, followed by DAB incubation for 8 h

at 25�C and subsequent washing in distilled, deionized water. The reaction

was stopped by 10 min of immersion in boiling ethanol. Residual chlorophyll

was extracted with fresh ethanol.

Electron Microscopy

For electron microscopy, plantlets were grown for 17 d on plates. Fixation

and embedding of leaf tissue as well as preparation of ultrathin sections were

performed as described (Duy et al., 2007). Micrographs were taken with an EM

912 electron microscope (Zeiss) equipped with an integrated energy filter

operated in the zero loss mode.

Chloroplast Isolation and Determination ofMetal Content

To provide independent biological replicates, chloroplasts were isolated

twice from mature leaf tissue of PIC1ox line 2 and the Col-0 wild type as

described by Philippar et al. (2007). Subsequently, iron concentration (see

below) was measured in 100 mL of lyophilized chloroplast suspension. To

estimate the iron content per chloroplast, we determined the yield of chloro-

phyll per fresh weight of leaf tissue initially used for chloroplast preparation.

This value was used as a measure for intact chloroplasts and to normalize iron

levels in all preparations (for data, see Supplemental Table S1). The observa-

tion that we could isolate significantly less intact chloroplasts per g fresh

weight from PIC1ox leaves was underlined by the fact that equal leaf areas

contained decreased chlorophyll as well (Fig. 3B; Supplemental Table S1).

Note that the calculated iron content per chloroplast is an estimate, assuming

that chlorophyll per fresh weight represents intact chloroplasts.

For concentration measurements of copper, iron, manganese, and zinc,

100 mg of fresh flower tissue and 20 mg of seeds were sampled from at least

10 individual plants. Metals in all tissue samples were determined by induc-

tively coupled plasma atomic emission spectrometry analysis after pressure

digestion (Central Inorganic Analysis Service, Helmholtz Zentrum Munchen).

Transcript Level Profiling

Total RNA was isolated using the Plant RNeasy extraction kit (Qiagen).

Quantitative real-time RT-PCR was performed as described by Duy et al.

(2007). To decrease biological variation, three samples each of 10 seedlings

were harvested and at least two technical replicates were accomplished.

For microarray analysis, flowers (Fig. 5B) of mature heterozygous PIC1ox

line 2 and wild-type plants were used. To provide biological replicates, five

PIC1ox and six wild-type samples were harvested from 10 individual plants.

RNA (5 mg) was processed and hybridized to Affymetrix GeneChip Arabi-

dopsis ATH1 Genome Arrays using the Affymetrix One-Cycle Labeling and

Control (Target) kit according to the manufacturer’s instructions (Affymetrix).

Raw signal intensity values (CEL files) from PIC1ox (n = 5) and the wild type

(n = 6) were computed from the scanned array images using the Affymetrix

GeneChip Command Console 3.0. For quality check and normalization, the

raw intensity values were processed with Robin software (Lohse et al., 2010)

default settings. Specifically, for background correction, the robust multiarray

average normalization method (Irizarry et al., 2003) was performed across all

arrays (between-array method). Statistical analysis of differential gene ex-

pression of PIC1ox versus wild-type flowers was carried out using the linear

model-based approach developed by Smyth (2004). The obtained P values

were corrected for multiple testing using the nestedF procedure, applying a

significance threshold of 0.05 in combination with the Benjamini and Hochberg

(1995) false-discovery rate control. Genes that showed a signal difference be-

tween PIC1ox and the wild type greater than 30 (arbitrary units) were consid-

ered to be differentially expressed.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table S1. Parameters determined during phenotype anal-

ysis of PIC1ox plants.

Supplemental Table S2. Metal concentration in flowers and seeds of

PIC1ox and the wild type.

Supplemental Table S3. Differentially expressed genes in PIC1ox versus

wild-type flowers.

ACKNOWLEDGMENTS

We thank Julia Neumann and Silvia Dobler for technical assistance and

Ann-Christine Konig for intensive work on PIC1ox seeds (all from Ludwig-

Maximilians-Universitat Munchen, Planegg-Martinsried, Germany). Antiserum

against FSD1 was donated by Marinus Pilon (Colorado State University, Fort

Collins).

Received November 30, 2010; accepted February 18, 2011; published Feb-

ruary 22, 2011.

LITERATURE CITED

Abdel-Ghany SE, Muller-Moule P, Niyogi KK, Pilon M, Shikanai T

(2005) Two P-type ATPases are required for copper delivery in Arabi-

dopsis thaliana chloroplasts. Plant Cell 17: 1233–1251

Arnon DI (1949) Copper enzymes in isolated chloroplasts: polyphenoloxi-

dase in Beta vulgaris. Plant Physiol 24: 1–15

Asada K (1999) The water-water cycle in chloroplasts: scavenging of active

oxygen and dissipation of excess photons. Annu Rev Plant Physiol Plant

Mol Biol 50: 601–639

Barth O, Vogt S, Uhlemann R, Zschiesche W, Humbeck K (2009) Stress

induced and nuclear localized HIPP26 from Arabidopsis thaliana inter-

acts via its heavy metal associated domain with the drought stress

related zinc finger transcription factor ATHB29. Plant Mol Biol 69:

213–226

Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a

practical and powerful approach to multiple testing. J R Stat Soc B 57:

289–300

Brautigam A, Hoffmann-Benning S, Weber APM (2008) Comparative

proteomics of chloroplast envelopes from C3 and C4 plants reveals

specific adaptations of the plastid envelope to C4 photosynthesis and

candidate proteins required for maintaining C4 metabolite fluxes. Plant

Physiol 148: 568–579

Briat JF, Ravet K, Arnaud N, Duc C, Boucherez J, Touraine B, Cellier F,

Gaymard F (2010) New insights into ferritin synthesis and function

Duy et al.




highlight a link between iron homeostasis and oxidative stress in plants.

Ann Bot (Lond) 105: 811–822

Bughio N, Takahashi M, Yoshimura E, Nishizawa NK, Mori S (1997)

Light-dependent iron transport into isolated barley chloroplasts. Plant

Cell Physiol 38: 101–105

Burstenbinder K, Rzewuski G, Wirtz M, Hell R, Sauter M (2007) The

role of methionine recycling for ethylene synthesis in Arabidopsis. Plant J

49: 238–249

Caro A, Puntarulo S (1996) Effect of in vivo iron supplementation on

oxygen radical production by soybean roots. Biochim Biophys Acta

1291: 245–251

Conte S, Stevenson D, Furner I, Lloyd A (2009) Multiple antibiotic

resistance in Arabidopsis is conferred by mutations in a chloroplast-

localized transport protein. Plant Physiol 151: 559–573

Conte SS, Lloyd AM (2010) The MAR1 transporter is an opportunistic

entry point for antibiotics. Plant Signal Behav 5: 49–52

Curie C, Cassin G, Couch D, Divol F, Higuchi K, Le Jean M, Misson J,

Schikora A, Czernic P, Mari S (2009) Metal movement within the plant:

contribution of nicotianamine and yellow stripe 1-like transporters. Ann

Bot (Lond) 103: 1–11

Duy D, Wanner G, Meda AR, von Wiren N, Soll J, Philippar K (2007)

PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron

transport. Plant Cell 19: 986–1006

Dykema PE, Sipes PR, Marie A, Biermann BJ, Crowell DN, Randall SK

(1999) A new class of proteins capable of binding transition metals.

Plant Mol Biol 41: 139–150

Ferro M, Brugiere S, Salvi D, Seigneurin-Berny D, Court M, Moyet L,

Ramus C, Miras S, Mellal M, Le Gall S, et al (2010) AT_CHLORO, a

comprehensive chloroplast proteome database with subplastidial local-

ization and curated information on envelope proteins. Mol Cell Proteo-

mics 9: 1063–1084

Gao W, Xiao S, Li H-Y, Tsao S-W, Chye M-L (2009) Arabidopsis thaliana

acyl-CoA-binding protein ACBP2 interacts with heavy-metal-binding

farnesylated protein AtFP6. New Phytol 181: 89–102

Guo WJ, Meetam M, Goldsbrough PB (2008) Examining the specific

contributions of individual Arabidopsis metallothioneins to copper

distribution and metal tolerance. Plant Physiol 146: 1697–1706

Halliwell B, Gutteridge JMC (1992) Biologically relevant metal ion-

dependent hydroxyl radical generation: an update. FEBS Lett 307:

108–112

Hassinen VH, Tuomainen M, Peraniemi S, Schat H, Karenlampi SO,

Tervahauta AI (2009) Metallothioneins 2 and 3 contribute to the metal-

adapted phenotype but are not directly linked to Zn accumulation in the

metal hyperaccumulator, Thlaspi caerulescens. J Exp Bot 60: 187–196

Himelblau E, Mira H, Lin SJ, Culotta VC, Penarrubia L, Amasino RM

(1998) Identification of a functional homolog of the yeast copper ho-

meostasis gene ATX1 from Arabidopsis. Plant Physiol 117: 1227–1234

Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf

U, Speed TP (2003) Exploration, normalization, and summaries of high

density oligonucleotide array probe level data. Biostatistics 4: 249–264

Jasinski M, Sudre D, Schansker G, Schellenberg M, Constant S,

Martinoia E, Bovet L (2008) AtOSA1, a member of the Abc1-like family,

as a new factor in cadmium and oxidative stress response. Plant Physiol

147: 719–731

Jeong J, Cohu C, Kerkeb L, Pilon M, Connolly EL, Guerinot ML (2008)

Chloroplast Fe(III) chelate reductase activity is essential for seedling

viability under iron limiting conditions. Proc Natl Acad Sci USA 105:

10619–10624

Jouhet J, Gray JC (2009) Is chloroplast import of photosynthesis proteins

facilitated by an actin-TOC-TIC-VIPP1 complex? Plant Signal Behav 4:

986–988

Kampfenkel K, Van Montagu M, Inze D (1995) Effects of iron excess on

Nicotiana plumbaginifolia plants. Plant Physiol 107: 725–735

Karimi M, Inze D, Depicker A (2002) Gateway vectors for Agrobacterium-

mediated plant transformation. Trends Plant Sci 7: 193–195

Kikuchi S, Oishi M, Hirabayashi Y, Lee DW, Hwang I, Nakai M (2009) A

1-megadalton translocation complex containing Tic20 and Tic21 medi-

ates chloroplast protein import at the inner envelope membrane. Plant

Cell 21: 1781–1797

Klatte M, Schuler M, Wirtz M, Fink-Straube C, Hell R, Bauer P (2009) The

analysis of Arabidopsis nicotianamine synthase mutants reveals func-

tions for nicotianamine in seed iron loading and iron deficiency re-

sponses. Plant Physiol 150: 257–271

Kliebenstein DJ, Monde RA, Last RL (1998) Superoxide dismutase in

Arabidopsis: an eclectic enzyme family with disparate regulation and

protein localization. Plant Physiol 118: 637–650

Korshunova YO, Eide D, Clark WG, Guerinot ML, Pakrasi HB (1999) The

IRT1 protein from Arabidopsis thaliana is a metal transporter with a

broad substrate range. Plant Mol Biol 40: 37–44

Kosman DJ (2010) Redox cycling in iron uptake, efflux, and trafficking.

J Biol Chem 285: 26729–26735

Kroll D, Meierhoff K, Bechtold N, Kinoshita M, Westphal S, Vothknecht

UC, Soll J, Westhoff P (2001) VIPP1, a nuclear gene of Arabidopsis

thaliana essential for thylakoid membrane formation. Proc Natl Acad

Sci USA 98: 4238–4242

Le Jean M, Schikora A, Mari S, Briat JF, Curie C (2005) A loss-of-function

mutation in AtYSL1 reveals its role in iron and nicotianamine seed

loading. Plant J 44: 769–782

Lin H, Wang R, Qian Q, Yan M, Meng X, Fu Z, Yan C, Jiang B, Su Z, Li J,

et al (2009) DWARF27, an iron-containing protein required for the

biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant

Cell 21: 1512–1525

Ling HQ, Koch G, Baumlein H, Ganal MW (1999) Map-based cloning of

chloronerva, a gene involved in iron uptake of higher plants encoding

nicotianamine synthase. Proc Natl Acad Sci USA 96: 7098–7103

Lohse M, Nunes-Nesi A, Kruger P, Nagel A, Hannemann J, Giorgi FM,

Childs L, Osorio S, Walther D, Selbig J, et al (2010) Robin: an intuitive

wizard application for R-based expression microarray quality assess-

ment and analysis. Plant Physiol 153: 642–651

Mira H, Martınez-Garcıa F, Penarrubia L (2001) Evidence for the plant-

specific intercellular transport of the Arabidopsis copper chaperone

CCH. Plant J 25: 521–528

Moreira D, Le Guyader H, Philippe H (2000) The origin of red algae and

the evolution of chloroplasts. Nature 405: 69–72

Morel FMM, Price NM (2003) The biogeochemical cycles of trace metals in

the oceans. Science 300: 944–947

Morrissey J, Baxter IR, Lee J, Li L, Lahner B, Grotz N, Kaplan J, Salt DE,

Guerinot ML (2009) The ferroportin metal efflux proteins function in

iron and cobalt homeostasis in Arabidopsis. Plant Cell 21: 3326–3338

Mubarakshina MM, Ivanov BN, Naydov IA, Hillier W, Badger MR,

Krieger-Liszkay A (2010) Production and diffusion of chloroplastic

H2O2 and its implication to signalling. J Exp Bot 61: 3577–3587

Mukherjee I, Campbell NH, Ash JS, Connolly EL (2006) Expression

profiling of the Arabidopsis ferric chelate reductase (FRO) gene family

reveals differential regulation by iron and copper. Planta 223: 1178–1190

Myouga F, Hosoda C, Umezawa T, Iizumi H, Kuromori T, Motohashi R,

Shono Y, Nagata N, Ikeuchi M, Shinozaki K (2008) A heterocomplex of

iron superoxide dismutases defends chloroplast nucleoids against ox-

idative stress and is essential for chloroplast development in Arabidopsis.

Plant Cell 20: 3148–3162

Palmer CM, Guerinot ML (2009) Facing the challenges of Cu, Fe and Zn

homeostasis in plants. Nat Chem Biol 5: 333–340

Palmer JD (2000) A single birth of all plastids? Nature 405: 32–33

Pekker I, Tel-Or E, Mittler R (2002) Reactive oxygen intermediates and

glutathione regulate the expression of cytosolic ascorbate peroxidase

during iron-mediated oxidative stress in bean. Plant Mol Biol 49:

429–438

Philippar K, Geis T, Ilkavets I, Oster U, Schwenkert S, Meurer J, Soll J

(2007) Chloroplast biogenesis: the use of mutants to study the etioplast-

chloroplast transition. Proc Natl Acad Sci USA 104: 678–683

Puig S, Mira H, Dorcey E, Sancenon V, Andres-Colas N, Garcia-Molina A,

Burkhead JL, Gogolin KA, Abdel-Ghany SE, Thiele DJ, et al (2007)

Higher plants possess two different types of ATX1-like copper chape-

rones. Biochem Biophys Res Commun 354: 385–390

Raven JA, Evans MCE, Korb RE (1999) The role of trace metals in

photosynthetic electron transport in O2 evolving organisms. Photosynth

Res 60: 111–149

Ravet K, Touraine B, Boucherez J, Briat JF, Gaymard F, Cellier F (2009)

Ferritins control interaction between iron homeostasis and oxidative

stress in Arabidopsis. Plant J 57: 400–412

Robinson NJ, Procter CM, Connolly EL, Guerinot ML (1999) A ferric-

chelate reductase for iron uptake from soils. Nature 397: 694–697

Rouhier N, Couturier J, Johnson MK, Jacquot JP (2010) Glutaredoxins:

roles in iron homeostasis. Trends Biochem Sci 35: 43–52

Schaaf G, Honsbein A, Meda AR, Kirchner S, Wipf D, von Wiren N

(2006) AtIREG2 encodes a tonoplast transport protein involved in iron-





dependent nickel detoxification in Arabidopsis thaliana roots. J Biol

Chem 281: 25532–25540

Schwacke R, Schneider A, van der Graaff E, Fischer K, Catoni E,

Desimone M, Frommer WB, Flugge UI, Kunze R (2003) ARAMEM-

NON, a novel database for Arabidopsis integral membrane proteins.

Plant Physiol 131: 16–26

Shingles R, North M, McCarty RE (2001) Direct measurement of ferrous

ion transport across membranes using a sensitive fluorometric assay.

Anal Biochem 296: 106–113

Shingles R, North M, McCarty RE (2002) Ferrous ion transport across

chloroplast inner envelope membranes. Plant Physiol 128: 1022–1030

Smyth GK (2004) Linear models and empirical Bayes methods for assess-

ing differential expression in microarray experiments. Stat Appl Genet

Mol Biol 3: Article 3

Swarbreck D, Wilks C, Lamesch P, Berardini TZ, Garcia-Hernandez M,

Foerster H, Li D, Meyer T, Muller R, Ploetz L, et al (2008) The

Arabidopsis Information Resource (TAIR): gene structure and function

annotation. Nucleic Acids Res 36: D1009–D1014

Teng YS, Su YS, Chen LJ, Lee YJ, Hwang I, Li HM (2006) Tic21 is an

essential translocon component for protein translocation across the

chloroplast inner envelope membrane. Plant Cell 18: 2247–2257

Terry N, Low G (1982) Leaf chlorophyll content and its relation to the

intracellular location of iron. J Plant Nutr 5: 301–310

Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular

localization of H2O2 in plants, H2O2 accumulation in papillae, and

hypersensitive response during the barley-powdery mildew interaction.

Plant J 11: 1187–1194

van Hoof NA, Hassinen VH, Hakvoort HW, Ballintijn KF, Schat H,

Verkleij JA, Ernst WH, Karenlampi SO, Tervahauta AI (2001) En-

hanced copper tolerance in Silene vulgaris (Moench) Garcke populations

from copper mines is associated with increased transcript levels of a 2b-

type metallothionein gene. Plant Physiol 126: 1519–1526

Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, Briat JF,

Curie C (2002) IRT1, an Arabidopsis transporter essential for iron uptake

from the soil and for plant growth. Plant Cell 14: 1223–1233

Vothknecht UC, Soll J (2005) The protein import pathway into chloro-

plasts: a single tune or variations on a common theme? In SG Moller, ed,

Plastids: Annual Plant Reviews, Vol 13. Blackwell Publishing, Oxford,

pp 157–179

Wu H, Ji Y, Du J, Kong D, Liang H, Ling HQ (2010) ClpC1, an ATP-

dependent Clp protease in plastids, is involved in iron homeostasis in

Arabidopsis leaves. Ann Bot (Lond) 105: 823–833

Ye H, Pilon M, Pilon-Smits EAH (2006) CpNifS-dependent iron-sulfur

cluster biogenesis in chloroplasts. New Phytol 171: 285–292

Zimeri AM, Dhankher OP, McCaig B, Meagher RB (2005) The plant

MT1 metallothioneins are stabilized by binding cadmiums and are

required for cadmium tolerance and accumulation. Plant Mol Biol 58:

839–855

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