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Plant Physiology and Biochemistry 52 (2012) 91e97

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Plant Physiology and Biochemistry

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Research article

Uptake and incorporation of iron in sugar beet chloroplasts

Ádám Solti a, Krisztina Kovács b, Brigitta Basa a, Attila Vértes b, Éva Sárvári a, Ferenc Fodor a,*aDepartment of Plant Physiology and Molecular Plant Biology, Institute of Biology, Eötvös Loránd University, Pázmány P. lane 1/C, Budapest 1117, HungarybDepartment of Analytical Chemistry, Institute of Chemistry, Eötvös Loránd University, Pázmány P. lane 1/A, Budapest 1117, Hungary

a r t i c l e i n f o

Article history:Received 13 October 2011Accepted 29 November 2011Available online 8 December 2011

Keywords:ChloroplastIron uptakeBeta vulgaris (sugar beet)Ferric citrateBathophenantroline disulphonateMössbauer spectroscopy

* Corresponding author. Tel./fax: þ36 1381 2164.E-mail address: ffodor@ludens.elte.hu (F. Fodor).

0981-9428/$ e see front matter � 2011 Elsevier Masdoi:10.1016/j.plaphy.2011.11.010

a b s t r a c t

Chloroplasts contain 80e90% of iron taken up by plant cells. Though some iron transport-related envelopeproteins were identified recently, the mechanism of iron uptake into chloroplasts remained unresolved. Toshed more light on the process of chloroplast iron uptake, trials were performed with isolated intactchloroplasts of sugar beet (Beta vulgaris). Iron uptake was followed by measuring the iron content ofchloroplasts in the form of ferrous-bathophenantrolineedisulphonate complex after solubilising the chlo-roplasts in reducing environment. Ferric citratewas preferred to ferrous citrate as substrate for chloroplasts.Strong dependency of ferric citrate uptake on photosynthetic electron transport activity suggests that ferricchelate reductase usesNADPH, and is localised in the inner envelopemembrane. TheKm for ironuptake fromferric-citrate poolwas 14.65� 3.13 mMFe(III)-citrate. The relatively fast incorporation of 57Fe isotope into Fe-Sclusters/heme, detected byMössbauer spectroscopy, showed the efficiency of the biosyntheticmachinery ofthese cofactors in isolated chloroplasts. The negative correlation between the chloroplast iron concentrationand the rate of iron uptake refers to a strong feedback regulation of the uptake.

� 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction

Under natural conditions, iron can be present as di- or trivalentcation depending on the chemical environment, what makes ita good cofactor for oxidoreductase type enzymes. Nevertheless,free ferrous ions are dangerous for living organisms as they cancatalyze Fenton-reactions, and produce reactive radicals [1]. Inshoot tissues of plants, 80e90% of iron can be found in chloroplasts[2,3], where thylakoidmembranes themselves contain about 60% ofleaf iron [4]. In chloroplasts, the major iron sinks are proteinscontaining single iron ions, Fe-S clusters and heme cofactors, aswell as ferritins, the iron storage proteins [5,6]. In chloroplasts,iron-sulphur proteins are the enzymes of N- and S-metabolism(nitrite reductase, sulphite reductase), photosystem (PS) I, ferre-doxins, Rieske-like proteins, protein import machinery (Transloconof Inner Chloroplast envelope 55, TIC55), and chlorophyll a oxy-genase [5,7]. Heme groups are present e.g. in cytochromes andperoxidases such as ascorbate peroxidase [6,8]. In addition, chlo-roplasts are the major source of heme groups present in plant cells,they synthesize and export heme into the cytoplasm [6,9]. Asmaller portion of chloroplast iron content is represented by singleions as protein cofactors, e.g. in PSII reaction centres and ironsuperoxide dismutase (FeSOD) [10,11]. Iron may also be stored in

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chloroplasts. Ferritins are eukaryotic iron storage proteins encodedin the nuclear genome [12]. However, they were shown to accu-mulate in undifferentiated plastids of young leaves, and can befound in chloroplasts only under stress conditions [13].

Iron is essential for both development and function of plants. Itsabsence induces strong deficiency symptoms, the so-called ironchlorosis which is connected to the iron dependence of chlorophyllsynthesis [14]. Thus, the biosynthesis of chlorophylls and iron-sulphur clusters as well as the assembly of pigmenteproteincomplexes in thylakoidmembranes are strongly retarded under irondeficiency [15,16], which leads to a decrease in the photosyntheticcapacity and the productivity of plants. As iron is required to produceiron-containing enzymes involved in protection against oxidativestress, antioxidative defence alsogets damaged in the absence of iron.

Despite there are plenty of data on iron uptake from the soil andits translocation [14], iron uptake of shoot tissues and that of cellorganelles is much less known. Leaf mesophyll cells are known totake up both ferrous and ferric iron [17]. However, at least a part ofthe ferric iron should undergo reduction mediated by FRO reduc-tases for an effective iron uptake [18]. In mesophyll symplast, ironcan form iron-citrate or ironenicotianamine complexes [19], buttheir importance is still not known. Iron uptake machinery oforganelles is even less known. Very recently, an iron transporter ofmitochondria was identified (MIT: Mitochondrial Iron Transporter)[20], in the absence of which mitochondrial Fe-S cluster biogenesisdoes notwork. Iron uptake of chloroplasts, the other endosymbioticorganelles of plant cells, may differ from that of eukaryotic cells as

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iron should cross both outer and inner envelope membranes. Thefirst protein found to be involved in chloroplast iron acquisition is thePermease In Chloroplast 1/Translocon of Inner Chloroplast Envelope21 (PIC1/TIC21) [21,22] that is localized in the inner envelope ofchloroplasts in Arabidopsis, and often thought to be a member of theinner envelope protein translocon machinery (TIC21). Based onrecent results obtained onPIC1 overexpressing lines, PIC1 also seemsto be a regulator of chloroplast iron metabolism [23]. An antibiotictransporter, Multiple Antibiotic Resistance 1 (MAR1), was alsopostulated to transport Fe(III)-nicotianamine complexes into thechloroplasts [24]. Bacterial type III secretion system protein-likeFDR3 was also suggested to be involved in chloroplast iron metabo-lism but it is localized in the thylakoidmembranes [25]. By this time,no IRT, NRAMP and YSL family transporters were identified in thechloroplast envelope [26]. A ferric chelate oxidoreductase-familyprotein, FRO7, localized in the chloroplasts in Arabidopsis, proved tohave an important role in chloroplast iron uptake, as in its absence,chloroplasts were not able to collect iron from the cytoplasm [18].However, precise localisation of FRO7 in the envelope membranesystemisnot known.Mechanismof ironuptake into chloroplastswasalso studied only in a few papers. Bughio et al. [27] showed thatbarley (Hordeumvulgare) chloroplasts were able to take up iron fromferric epihydroxy mugineic acid chelates. This uptake proved to bedirectly light/photosynthesis dependent as uptake could be inhibitedby adding 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSIIinhibitor. Isolated pea (Pisum sativum) inner envelope vesicles wereable to transport ferrous iron when a proton gradient existed acrossthe membrane, and direction of iron transport was ruled by thedirection of the gradient [28]. This transport could be inhibitedcompetitively by divalent transition metal ions. Therefore, thoughiron plays a crucial role in chloroplast structure and function, onlyfew pieces of information are available about protein components ofthe ironuptake systemand the invivomechanismsof ironacquisitionof chloroplasts.

In this paper, we studied iron uptake and incorporation intoisolated chloroplasts by using a bathophenantroline disulphonate(BPDS) method to follow changes in their iron content, and Möss-bauer spectroscopy to unravel the chemical forms of iron taken upby chloroplasts.

2. Materials and methods

2.1. Plant material

Hydroponically cultured sugar beet (Beta vulgaris L. cv. Orbis)plants were grown in climate chamber with 14/10 h light(120 mmol m�2 s�1 photosynthetic photon flux density, PPFD)/darkperiods, 24/22 �C and 70/75% relative humidity in modified ¼strength Hoagland solution (1.25 mM Ca(NO3)2, 1.25 mM KNO3,0.5 mM MgSO4, 0.25 mM KH2PO4, 0.25 mM NaCl, 11.56 mM H3BO3,4.6mMMnCl2, 0.19mMZnSO4, 0.12mMNa2MoO4, 0.08mMCuSO4)with10 mM Fe(III)-citrate (iron:citrate ¼ 1:1, Reanal Kft., Hungary) as ironsource. Seeds were germinated at moderate light, and planteddirectly into nutrient solution in plastic containers of 12 l volume.Seedlings were pre-cultivated up to four-leaf stage. Leaves emergedduring the next 3 weeks were used for chloroplast isolation. In thecase of plants grown forMössbauer spectroscopymeasurements, theiron source was replaced by 57Fe(III)-citrate. 57Fe(III)-citrate was madefrom enriched 57FeCl3 (ca. 90% 57Fe) according to Kovács et al. [29].

2.2. Chloroplast isolation and determination of intactness

Sugar beet leaves were homogenized in isolation buffer (50 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-KOH,pH 7.0, 330 mM sorbitol, 2 mM ethylenediaminetetraacetic acid

(EDTA), 2 mM MgCl2, 0.1% (w/v) bovine serum albumine (BSA),0.1% (w/v) Na-ascorbate) by Waring Blendor for 5 s. Homogenatewas filtered on 4 layers gauze and 2 layers Miracloth (Calbiochem-Novabiochem), and centrifuged at 1500� g for 5 min. All centri-fugation steps were carried out in a swing-out rotor. The pelletwas resuspended in washing buffer (50 mM HEPES-KOH, pH 7.0,330 mM sorbitol, 2 mM MgCl2), layered on the top of a stepwisesucrose gradient (50 mM HEPES-KOH, pH 7.0, 20/45/60% sucrose,2 mM EDTA, 2 mM MgCl2), and centrifuged at 2000� g for 15 min.Intact chloroplasts were collected from the 45/60% sucroseinterface. After five-time dilution with washing buffer, plastidfraction was centrifuged at 2500� g for 5 min. The pellet wasresuspended in washing buffer. Number of chloroplasts wascounted in a Bürker chamber in Nikon Optiphot-2 microscope(Zeiss Apochromatic 40/0.95 160/0.17 objective) equipped withNikon D70 DSLR camera.

To test cross-contamination of chloroplast suspensions, westernblotting was performed against mitochondrial inner membranealternative oxidase (AOX). Chloroplasts were solubilised in 62.5 mMtris(hydroxymethyl)aminomethane (Tris)eHCl (pH 6.8), 2% sodiumdodecyl sulfate (SDS), 2% dithiothreitol (DTT), 10% glycerol and0.001% bromophenol blue and proteins were separated on 10e18%gradient SDS polyacrilamide gel electrophoresis [30] containing 10%glycerol. Proteins were transferred to nitrocellulose membranes(SigmaeAldrich) in a 25 mM TriseHCl (pH 8.3), 192 mM glicine, 10%(v/v) methanol and 0.02% (m/v) SDS buffer. Membranes weredecorated with rabbit polyclonal antibody against mitochondrialalternative oxidase (AOX) (Agrisera AG, Vännäs, Sweden) followingthe manufacturer’s instruction. Horseradish peroxidase- (HRP-)conjugated goat-anti-rabbit IgG (BioRad, Inc.) was used to detectbands following the manufacturer’s instructions.

To quantify the intactness of isolated chloroplasts, both intactleaves and purified chloroplasts were solubilised, and proteinswere separated and blotted to nitrocellulose membranes as above.Membranes were decorated with rabbit polyclonal antibodyagainst aporotein of light harvesting complex II (apoLHCII) (a giftfrom Dr. Udo Johanningmeier, Bohum Universität, Germany) andrabbit polyclonal antibody against chloroplast triose phosphatetranslocator (cTPT) with a known cross-reaction to ribulose-1,5-bisphosphate carboxylase oxygenase large subunit (RbcL) (ob-tained from Agrisera AG, Vännäs, Sweden), and dissolved in 20 mMTriseHCl (pH 7.5), 0.15 M NaCl, 1% gelatine following the manu-facturers’ instructions. Bands were detected by HRP reaction asabove. Densities of bands were evaluated by Phoretics v. 4.01software (Phoretix International, Newcastle upon Tyne, UK). Chlo-roplast integrity was calculated by comparing RbcL/apoLHCII ratioin intact leaf and chloroplast samples [26].

2.3. Measurement of iron uptake by BPDS method

Chloroplast suspension was diluted by washing buffer to 100 mgchlorophyll (Chl) ml�1, equal to about 76000 � 9500 chloroplastml�1. Chl concentration was determined in 80% (v/v) acetone bya UVeVIS spectrophotometer (Shimadzu, Japan) using theabsorption coefficients of Porra et al. [31]. Uptake medium con-tained 0.5 ml chloroplast suspension and an iron source of either100 mM Fe(III)-citrate (Fe3þ:citrate 1:1, Reanal Kft., Hungary) or100 mM Fe(II)-citrate (100 mM Fe(III)-citrate reduced into ferrousform by 100 mM ascorbic acid). 10 mM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was used to inhibit the photosyntheticelectron transport. Plastids were kept on ice in darkness, and werewarmed up to room temperature in darkness for 5 min before usefor iron uptake experiments, which were carried out either indarkness or in the light. Iron uptake in the light was initiated by120 mmol m�2 s�1 PPFD (light source: mercury lamp), and

Fig. 1. Dependence of chloroplast ferric iron uptake on the external (A) and internal (B)concentration of iron (A). Iron uptake was measured on light for 30 min. Error barsshow SD values, n ¼ 9.

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terminated by placing the samples on ice in darkness. Sampleswere centrifuged promptly at 2500� g in swing-out rotor for 5 min.Pelleted chloroplasts were washed in 0.25 ml washing buffer with2 mM (v/v) EDTA. Chloroplasts were resuspended in 0.25 ml ofwashing buffer and solubilised in 1% SDS, 1% DTT solution at roomtemperature for 30 min. Non-solubilised material (starch) wasremoved by centrifugation of samples at 10000� g for 5 min. Ironcontent of solubilised chloroplast material was measured photo-metrically as [Fe(BPDS)3]4� complex at 535 nm by UVeVIS spec-trophotometer (Shimadzu, Japan) after the addition of 100 mMascorbic acid to reduce the total iron content of the samples intoFe2þ and 300 mM BPDS disodium salt (Sigma). Steady-state absor-bance was reached after 60 min incubation in darkness at roomtemperature. An absorption coefficient of E ¼ 22.14 mM�1 cm�1

given by Smith et al. [32] was used to calculate iron content ofchloroplasts before and after iron uptake.

Amount of iron reduced by light in a Fe(III)-citrate solution wasalso determined by the BPDS method, but without the addition ofascorbic acid.

2.4. Mössbauer spectroscopy

To identify iron forms present in the chloroplasts before theuptake, chloroplasts were isolated from 57Fe(III)-citrate-grownplants. To follow the forms of iron taken up by chloroplasts duringan uptake period of 30min, chloroplasts isolated from Fe(III)-citrate-grown plants were used but the uptake was carried out ina medium containing 100 mM 57Fe(III)-citrate as iron source. Afteriron uptake, chloroplasts were washed as before, and concentratedchloroplast suspensions frozen in liquid nitrogen were used assamples for Mössbauer spectroscopy. In order to study the ironspecies present in 57Fe(III)-citrate containing iron uptake medium,Mössbauer spectrum of 0.01 M stock 57Fe(III)-citrate solution wasalso measured in frozen state [33]. Samples were measured bya conventional constant acceleration type Mössbauer spectrometer(Wissel) in a liquid nitrogen bath cryostat at 80 K. A 57Co(Rh) sourceof w109 Bq activity was used, and the spectrometer was calibratedwith a-iron at room temperature. Evaluation of spectra was carriedout using the MOSSWIN code [34]. The Mössbauer parameterscalculated for the spectral components were: isomer shift (d, mms�1), quadrupole splitting (D, mm s�1) and partial resonantabsorption areas (Sr, %). These parameters provide information onthe electron densities at the Mössbauer nuclei (indicating thevalence state) and on the magnitude of any electric field gradients(indicating the coordination number of the resonant atom). Thequantitative analytical information for the species can be obtainedfrom the relative spectral areas [35].

2.5. Statistical analysis

Iron uptake measurements were performed in 3 technicalrepetitions in each of the 3-4 biological repetitions. To comparemultiple treatments, ANOVA was performed with TukeyeKramermultiple comparison post hoc test by InStat v. 3.00 (GraphPadSoftware, Inc.). Origin v. 6.01 (Origin Lab, Co.) was used to fitmathematical functions on data points.

3. Results

3.1. Chloroplast purity and integrity

Based onwestern blotting against mitochondrial inner envelopeAOX, no mitochondrial contamination was found in purified chlo-roplast suspensions. By comparing the RbcL/apoLHCII ratios in totalleaf extracts with those of chloroplast suspensions used in uptake

experiments, the integrity of chloroplast suspensions was>85%. Nosignificant change was found in their intactness under uptakeconditions for 30 min. However, a slow, continuous decline inchloroplast intactness was observed when external iron concen-tration exceeded 200 mM.

3.2. Characteristics of chloroplast iron uptake

Iron uptake of chloroplast was followed by measuring thechanges in their iron content with the BPDS method after solubil-ising washed chloroplasts and reducing all ferric ions present in thesolution into ferrous form. Light-induced iron uptake of chloro-plasts from ferric iron pool was strongly dependent on ferric ironconcentration in the uptake medium: either iron efflux or influxwas observed after 30 min of incubation (Fig. 1A). Over5.03 � 0.20 mM external Fe(III) iron concentration iron uptake wasmeasured, which followed saturation kinetics up to about 100 mMFe(III). The rate of iron uptake at the saturation point was33.51�1.44 amol Fe min�1 plastid�1 (equal to 25.47� 1.09 pmol Femin�1 mg�1 Chl). Km value of iron acquisition from ferric iron poolwas 14.65 � 3.13 mM Fe(III)-citrate. Optimal external iron concen-tration for chloroplast iron uptake was found to be 100 mM Fe(III).Above 100 mM external iron concentration, iron uptake capacitystarted to decrease, and turned into iron release gradually. At400 mM external iron, the release was 12.2 � 2.1 amol Fe min�1

chloroplast�1. However, the intactness of chloroplasts incubatedwith 400 mM iron in the light for 30 min decreased drastically to20.6 � 3.8%.

Fig. 3. Iron uptake rate of chloroplasts from ferric and ferrous iron pools in thepresence or absence of DCMU at light (total) and in darkness (grey) for 30 min. Errorbars show SD values, n ¼ 9.

Á. Solti et al. / Plant Physiology and Biochemistry 52 (2012) 91e9794

The rate of chloroplast iron uptake at 100 mM external ironconcentration was negatively affected by higher internal ironconcentration. The actual rate (vactual) for iron uptakewas abolishedafter reaching about 4.54 � 0.10 mM internal iron concentration ofchloroplasts (Fig. 1B). In parallel to the increase of internal ironconcentration, a decrease in the initial rate (vmax) for iron uptakewas observed in those not-typical chloroplasts which showedelevated iron concentration originally (Fig. 1B). Nevertheless, nochloroplast population of iron supplied sugar beet plants was iso-lated containing less than 1.63 � 0.21 mM internal ironconcentration.

Iron uptake of chloroplasts at 100 mM Fe(III)-citrate external ironconcentration in the light showed saturation kinetics in time(Fig. 2). No lag period was observed, and the process was saturatedafter 30 min incubation. The initial rate of iron uptake was42.78 � 3.52 amol Fe min�1 chloroplast�1. Iron content of chloro-plasts was 204.5 � 16.4 amol plastid�1 (equal to 155.4 � 12.5 pmolFe mg�1 Chl or 2.03 � 0.16 mM internal iron concentration inchloroplasts) before the uptake measurements. During the 30 minincubation period, chloroplast iron content rose up to440.0 � 12.7 amol plastid�1, whereas the iron concentration in theuptake medium decreased from 100 mM to 62.02 � 8.46 mM.

In order to test the role of photosynthetic electron transport andthe valence state preference in the iron uptake machinery, chlo-roplasts were incubated in an uptake medium containing 100 mMferric or ferrous iron in form of citrate complex either in darkness orunder light for 30 min. Ferrous iron was produced by addingequimolar amount of ascorbate. The Fe(III)-citrate pool also getsreduced chemically to some extent on light. After 30 min incuba-tion at 120 mmol m�2 s�2 PPFD, nearly 10% of ferric iron content gotreduced into ferrous form in the ferric-citrate containing medium,which means a very moderate abiotic ferrous iron formation.Therefore, the effective ferric-complex concentration decreasedduring the experimental time, and some ferrous iron (possibly inform of ferrous-citrate) became available in the uptake medium. Indarkness, only moderate iron uptake was detected: chloroplastswere able to take up both ferric and ferrous iron, but the uptake offerric ironwas about three times higher than that of the ferrous one(Fig. 3). Iron uptake from ferrous chelate pool did not differsignificantly in darkness and in the light, whereas iron uptake fromferric citrate was markedly enhanced by light. 10 mM DCMU, whichtotally inhibits electron transport activity, caused no significantchange in ferrous iron uptake whereas the light-dependent ironuptake from ferric iron pool was strongly inhibited (Fig. 3).

Fig. 2. Kinetics of light-dependent iron uptake of chloroplasts from ferric iron pool.

3.3. Iron forms in the chloroplasts

Mössbauer spectrum of the 57Fe(III)-citrate solution used in theiron uptake buffer for chloroplasts showed a quadrupole doublet(Fig. 4) with the parameters of d ¼ 0.48(1) mm s�1, D ¼ 0.62(1) mms�1. Mössbauer spectra of the intact chloroplasts of 57Fe(III)-citrate-grown plants can be evaluated with one symmetrical quadrupoledoublet (FeA component) with Mössbauer parameters ofd ¼ 0.43(1) mm s�1, D ¼ 1.05(2) mm s�1 (Fig. 5A). Chloroplastsisolated from Fe(III)-citrate-grown plants collected no measureable57Fe iron if they were removed promptly from the uptake buffercontaining 57Fe(III)-citrate. 30 min of 100 mM 57Fe(III)-citrate acqui-sition by these chloroplasts gave the possibility to study the formsof iron taken up during the iron acquisition period. The Mössbauerspectrum of these chloroplasts (Fig. 5B) differed from those isolatedfrom 57Fe(III)-citrate-grown plants. Namely it shows a broadenedquadrupole doublet which can be evaluated as a superposition oftwo doublets with the following hyperfine parameters:d ¼ 0.46(1) mm s�1, D ¼ 1.06(4) mm s�1 (FeA component) and

Fig. 4. 80 K Mössbauer spectrum of 0.01 M stock solution of 57Fe(III)-citrate used inferric iron uptake experiments.

Fig. 5. 80 K Mössbauer spectrum of intact chloroplasts isolated from 57Fe(III)-citrate-grown plants (A) and from plants grown on Fe(III)-citrate but taken up iron from100 mM 57Fe(III)-citrate for 30 min (B), the broadened quadrupole of which was eval-uated as a superposition of FeA and FeB components (parameters see: in text).

Á. Solti et al. / Plant Physiology and Biochemistry 52 (2012) 91e97 95

d ¼ 0.48(1) mm s�1, D ¼ 0.61(3) mm s�1 (FeB component). Therelative abundance of FeA component was approximately one thirdof the total iron present in the sample (35 � 10%).

4. Discussion

Despite iron plays a crucial role in chloroplasts [6], we have onlya few pieces of information on their iron acquisition machinery andthe iron exchange between cytoplasm and chloroplasts in barley[27], pea [28], and Arabidopsis [18,22]. Due to the high complexityof leaves and even mesophyll cells, chloroplast iron uptake cannotbe easily examined in leaves or their cells, while isolated chloro-plasts are more suitable for this purpose.

Extraction of the total iron content of chloroplasts by SDS-DTTsolubilisation was as effective as the acidic digestion for ICP-MSmeasurements [36]. Thus, BPDS method is a cheap and fast way todetermine iron content inplant tissues and to follow iron uptake intochloroplasts. According to Mössbauer spectroscopy, no measureable57Fe was found in chloroplasts if they were removed promptly fromthe uptake buffer containing 57Fe(III)-citrate. It means that EDTAwashing eliminated all the iron bound/precipitated, if any, on thesurface of chloroplast outer envelope or on damaged chloroplastmembranes. Therefore, the results on iron uptake are reasonable.

4.1. Mechanism of iron uptake into the chloroplast

According to our measurements, iron acquisition from ferriccitrate pool was preferred to that from ferrous citrate pool (Fig. 3).This was in accordancewith the results obtained by Jeong et al. [18],who showed that ferric chelate reductase activity was necessary forchloroplast iron acquisition under normal iron supply, and indi-cated that the substrates used preferentially in the iron acquisitionby chloroplasts are ferric chelates. Ferric-dicitrate complexes arealso the main iron source for Synechocystis [37], which is related tothe progenitors of chloroplasts. Fe(III)-chelates are known to bephotoreactive, a reaction in which blue and UV light are involved[38]. Formation of free ferrous iron in the uptakemedium, however,was much less than the iron uptake capacity of chloroplastsmeasured in Fe(III)-citrate containingmedia. Therefore, the decreasein Fe(III)-citrate concentration did not limit iron uptake, and

conversely, the iron uptake of chloroplasts was not closely corre-lated with the abiotic formation of Fe(II)-citrate.

Iron acquisition from ferric iron pool was strongly influenced bythe concentration of the supplied Fe(III)-citrate. At low external ironconcentration, a tendentious but not significant iron export wasmeasured from the chloroplasts (Fig. 1A). It may representa continuous export of some organic iron compounds from intactchloroplasts and refer to a heme/protoheme release, as the proto-porphyrin IX ferrochelatase, which synthesizes heme groups forthe whole plant cell, is only found in chloroplasts [9]. At higher ironconcentrations, this release can be masked by the mass uptake ofiron. In our experiments, the Km for ferric iron uptake was in similarrange as the values given in Singles et al. [28], and intermediatebetween the values characteristic to the high affinity transportersand channels [39]. Similarly to the results of Moreau et al. [40]obtained with isolated bacteroids of Glycine max plants, chloro-plast iron uptake decreased at higher than saturating concentrationof the external iron. In our case, the reason for it was the rupture ofchloroplasts, which was caused possibly by Fenton-reactions [41].

Iron acquisition from ferric citrate iron pool had a strongdependency on photosynthetic electron transport. It was inhibitedin darkness or in the light with DCMU as it was also shown in barleyby Bughio et al. [27] for the uptake of ferric epihydroxy mugineicacid chelates. Ferric chelate reductases (FRO family proteins) wereshown to contain NADPþ binding domain [42]. However, isolatedroot plasma membrane ferric chelate oxidoreductase used bothNADH and NADPH with similar efficiency [43]. In irradiated chlo-roplasts, NADPH is produced primarily. Dark and DCMU inhibitionof iron uptake by isolated chloroplasts indicates that chloroplastferric chelate reductases most probably use NADPH produced byphotosynthetic light reactions. As chloroplast inner envelope is notpermeable to NADPH, ferric chelate reductase should be located inthe chloroplast inner envelope membrane [44]. Dark uptake ofeither ferric or ferrous citrate was insensitive to DCMU, but uptakeof the former was more pronounced. It refers to the existence ofa common or a homologous mechanism for dark acquisition offerric and ferrous iron, which can be mediated by chloroplast outerenvelope transporter(s). Nevertheless, we have little informationon the outer envelope cation transport, yet [45,46].

Iron uptake of chloroplasts resembles to the bacterial mecha-nisms in some aspects. The free-living cyanobacterium Synecho-cystis has plasma membrane ferrous iron transporters, prefersferricedicitrate complexes, and uptake of ferrous iron is importantonly under iron-starvation conditions [37]. Marshall et al. [47]demonstrated that mutants of the Gram-negative bacteriumPseudomonas aeruginosa, deficient in cytoplasmic ferric chelatepermeases, can take up iron from ferric iron pool mediated byferrous ion transporters if ferrous iron was produced by ferric ironphotoreduction. This mechanism is analogous to that of chloro-plasts of Arabidopsis fro7 mutants [18], which may also use pho-toreduced ferric iron and can survive only at elevated iron supply.Isolated bacteroids of Glycine max plants also prefer to take upferrous iron produced by a ferric chelate reductase located in theperibacteroid membrane, which reduces iron using cytoplasmicNADH [40]. Concentration dependence of the bacteroid and chlo-roplast iron acquisition is similar indicating that the inner enve-lope/bacterial membrane ferrous iron transporter or the ferricchelate reductase may have similar kinetics. The above-mentionedsimilarities of cyanobacterial, bacterial and bacteroidal iron uptakemechanism and changes in iron uptake of fro7 mutant chloroplaststogether with our results suggest that chloroplasts may havea bacterial-type iron uptake system lacking inner envelope/bacte-rial plasma membrane ferric chelate permease but having a bacte-rial-type ferrous iron transporter and a eukaryotic type ferricchelate reductase.

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The only and essential chloroplast inner envelope iron trans-porter identified so far is the PIC1 iron permease, which is of cya-nobacterial origin [22]. Shingles et al. [28] measured ferrous ironmovement through chloroplast inner envelope vesicles which canrepresent the transport kinetics of PIC1. According to Mössbauerspectroscopy measurements, ferrous iron released by chloroplastferric chelate reductase does not accumulate in high spincomplexes (eg. [Fe(H2O)6]2þ or Fe(II)-carboxylates which have thetypical hyperfine parameters of dw1.2e1.4 mm s�1,Dw3.1e3.4 mm s�1 [33]) between the two envelope membranes asit does in roots after reduction by FRO2 [29]. The absence of theaccumulation of a ferrous-hexahydrate pool indicates that thereduced free ferrous iron possibly crosses promptly the chloroplastinner envelope membrane. Deductively, these results may lead tothe conclusion that chloroplast envelope ferric chelate reductaseand permease work in close cooperation.

4.2. Incorporation of iron into chloroplasts

57Fe Mössbauer spectroscopy is a non-destructive, uniquetechnique that gives information on the valence state and coordi-nation environment of the 57Fe nuclei and thus can help to revealthe types of iron compounds present in the sample [48,49]. Irontaken up by isolated chloroplasts from 57Fe(III)-citrate pool in thelight during the 30 min acquisition time was represented by twoforms (Fig. 5B). The major component (65%) was FeB with Möss-bauer parameters very typical of non-heme high spin Fe(III)

compounds in octahedral-O6 environment [35] such as Fe(III)-carboxylate complexes formed with organic acids [29,50]. Thequadrupole splitting of D ¼ 0.61(3) mm s�1 suggests the presenceof Fe(III)-citrate since the Mössbauer spectrum of 0.01 M 57Fe(III)-citrate solution at pH 6.0 gives a quadrupole doublet with the sameparameters (Fig. 4). Thorough EDTA washing and resuspension ofchloroplasts ensures that FeB may only represent ferric-citratecomplexes having crossed the outer but not the inner envelopemembrane. Therefore, FeB can represent the Fe(III)-citrate taken upfrom the iron uptake medium into the space between the twoenvelopes.

Free ferrous iron is transported preferentially across innerenvelope membrane [28]. Based on the Mössbauer parametersmeasured and calculated in intact chloroplasts isolated from57Fe(III)-citrate-grown plants, the second component can be iden-tified as FeA, which can be either Fe4S4 centres in accordance withthe data gained on Fe4S4 proteins found in plants [51], bacteria[52,53] and in isolated photosystems [48,54,55] or heme groups.However, low-spin heme Fe(II) compounds such as cytochromes,which probably give high contribution to the FeA component,cannot be separated by Mössbauer measurements made only at80 K [54]. This smaller portion of iron that is present as Fe4S4centres and/or heme groups represents the amount of iron incor-porated as cofactors in chloroplast proteins, possibly including PSI,cytrochrome b6f complex, and oxidoreductase-type defenceenzymes. The relatively fast incorporation of iron shows thateffective machineries exist for Fe-S cluster and heme biogenesis inintact chloroplasts [5e7]. No iron incorporated into ferritin-likeiron-storage proteins (dw0.5 mm s�1, Dw0.7e0.8 mm s�1 [49])was found in intact chloroplasts of 57Fe(III)-citrate-grown plants, inaccordance with that ferritin is formed in mature chloroplasts onlyunder high iron excess [13].

Chloroplast iron uptake was strictly regulated. It showed satu-ration in time (Fig. 2). The reason of decreasing iron uptake capacityseems to be the increase in the internal iron concentration ofchloroplasts, as isolated chloroplast populations with high ironcontent showed no or little iron uptake (Fig. 1B). This tendentiousdecline in iron uptake capacity as the function of increasing internal

iron concentration shows that some kind of sensing of internal ironcontent and a strong feedback regulation of iron uptake shouldexist. This sensor/regulator may be the chloroplast Fe-S clustersynthase (NAP) complex, which is regulated by iron excess throughthe iron binding to NAP1 [6,56].

4.3. Conclusion

Iron uptake machinery of intact chloroplasts seems to preferferric iron complexes to ferrous iron complexes as iron source.Chloroplast iron uptake is also strongly connected to the photo-synthetic performance of chloroplasts in a dicotyledonous modelplant (sugar beet). Increasing internal iron content of chloroplasts isa negative feedback regulator of iron uptake. Once iron has beencrossed the inner envelop of chloroplasts, it incorporates into Fe-S/heme cofactors.

Acknowledgement

We would like to thank to Zsuzsanna Ostorics for technicalassistance. This work was supported by the grants NN-74045(OTKA) and NN-84307 (ERA Chemistry e OTKA). Brigitta Basaalso acknowledges the financial support of the European Union andthe European Social Fund (TÁMOP 4.2.1./B-09/1/KMR-2010-0003).

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