Chemical Papers 62 (4) 358–363 (2008)DOI: 10.2478/s11696-008-0036-9

ORIGINAL PAPER

Effect of different Fe(III) compounds on photosyntheticelectron transport in spinach chloroplasts and on iron accumulation

in maize plants

aKatarína Kráľová*, aElena Masarovičová, aFrantišek Šeršeň,bIveta Ondrejkovičová

aInstitute of Chemistry, Faculty of Natural Sciences, Comenius University, SK-842 15 Bratislava, Slovakia

bDepartment of Inorganic Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology,

SK-812 37 Bratislava, Slovakia

Received 26 October 2007; Revised 21 January 2008; Accepted 23 January 2008

Dedicated to Professor Milan Melník on the occasion of his 70th birthday.

Synthesis and spectral characteristics of [Fe(nia)3Cl3] and [Fe(nia)3(H2O)2](ClO4)3 are described.The effect of these compounds as well as of FeCl3 · 6H2O on photosynthetic electron transport inspinach chloroplasts was investigated using EPR spectroscopy. It was found that due to the in-teraction of these compounds with tyrosine radicals situated at the 161st position in D1 (TyrZ)and D2 (TyrD) proteins located at the donor side of photosystem (PS) II, electron transport be-tween the photosynthetic centres PS II and PS I was interrupted. In addition, the treatment with[Fe(nia)3(H2O)2](ClO4)3 resulted in a release of Mn(II) from the oxygen evolving complex situatedon the donor side of PS II. Moreover, the effect of the Fe(III) compounds studied on some productioncharacteristics of hydroponically cultivated maize plants and on Fe accumulation in plant organswas investigated. In general, the production characteristic most inhibited by the presence of Fe(III)compounds was the leaf dry mass and [Fe(nia)3(H2O)2](ClO4)3 was found to be the most effec-tive compound. The highest Fe amount was accumulated in the roots, and the leaves treated withFe(III) compounds contained more Fe than the stems. The treatment with FeCl3 · 6H2O caused themost effective translocation of Fe into the shoots. Comparing the effect of nicotinamide complexes,[Fe(nia)3(H2O)2](ClO4)3 was found to facilitate the translocation of Fe into the shoots more effec-tively than [Fe(nia)3Cl3]. This could be connected with the different structure of these complexes.[Fe(nia)3(H2O)2](ClO4)3 has ionic structure and, in addition, coordinated H2O molecules can beeasily substituted by other ligands.c© 2008 Institute of Chemistry, Slovak Academy of Sciences

Keywords: EPR spectroscopy, translocation, iron–nicotinamide complexes, maize plants, bioaccu-mulation

Introduction

Iron is an essential element for plants and it is in-dispensable for a variety of cellular functions. Thiselement is important because of its physico-chemicalproperties: coordinated at metalloprotein active sites,

it participates in most of the basic redox reactions re-quired in both the production and the consumption ofoxygen. Iron is also involved in many vital enzymaticreactions required for nitrogen fixation, DNA synthe-sis, and hormone synthesis. Plants require approxi-mately 10−8 mol dm−3 Fe, but in calcareous soils to-

*Corresponding author, e-mail: kralova@fns.uniba.sk

K. Kráľová et al./Chemical Papers 62 (4) 358–363 (2008) 359

Table 1. Elemental analysis of Fe(III) compounds prepared

wi(calc.)/%wi(found)/%

Compound Formula MrC H N Cl Fe

[Fe(nia)3Cl3] FeC18H18N6O3Cl3 528.59 40.90 3.43 15.90 20.12 10.5741.40 3.73 16.03 19.95 9.99

[Fe(nia)3(H2O)2](ClO4)3 FeC18H22N6O17Cl3 756.58 28.58 2.93 11.10 7.3828.79 2.98 11.38 7.30

tal soluble iron reaches no more than 10−10 mol dm−3.Therefore it is evident that without active mechanismsfor iron extraction from soil, most plants would ex-hibit iron-deficiency symptoms such as leaf interveinalchlorosis (Guerinot & Yi, 1994; Briat et al., 1995).Mobilization of Fe by plants is achieved by differ-

ent strategies, e.g. by secretion of plant-borne chela-tors or by reductive and proton-promoted processes.Plants utilizing the so-called Strategy I, i.e. non-graminaceous plants, can lower the pH in their rhi-zosphere. Lowering the pH values increases solubil-ity of Fe(III) and promotes its reduction to Fe(II).Dissolved Fe(III) is reduced by reducing proteins as-sociated with the cellular membranes to Fe(II) be-fore it crosses the cellular membrane. Plants utiliz-ing the so-called Strategy II, i.e. graminaceous plants(grasses, maize, etc.), excrete phytosiderophores, non-protein amino acids that dissolve Fe(III) ions andform an Fe-phytosiderophore complex (Guerinot &Yi, 1994; Romheld & Schaaf, 2004; Charlson & Shoe-maker, 2006). As mentioned above, iron is taken upas Fe(II) into the root symplast in plants utilizingStrategy I, and as an Fe(III) complex in the plantsemploying Strategy II. During symplasmic transport,the intracellular environment is protected against thereactive species of iron by handling iron in chelatedforms. Root cell vacuoles may compete with the trans-port stream forming an iron store. In the presenceof an iron excess, plants can escape the deleteriouseffects of free iron by depositing it in phytoferritinas a storage protein. A pool of metabolically avail-able iron is formed also by nicotianamine (Stephan,2002).The release of phytosiderophores is positively cor-

related with genotypical differences in the resistanceto iron chlorosis. Siderophores form high-spin, ki-netically labile chelates with a ferric ion, which arecharacterized by exceptional thermodynamic stabil-ity (Schwarzenbach & Schwarzenbach, 1963). Thesiderophore ligand can be said to be “virtually spe-cific” for Fe(III) among the naturally occurring metalions of abundance. The fact that the siderophore lig-and shows strong affinity only for the higher oxidationstate of iron sets this natural complexing agent apartfrom molecules, such as heme, which serve effectivelyas electron shuttles. With few exceptions, the “hard”acid ion, Fe(III), is linked to hard base atoms, such

as oxygen, which accounts for the preference for ferricions (Neilands, 1995).Pot experiments with lettuce (Lactuca sativa L.)

showed that biodegradable ligands are able to serveas chelators to sustain Fe availability in calcareous en-vironments (Ylivainio et al., 2006). Iron amino acidchelates, such as iron glycinate chelates, were pre-pared to be used as food fortificants and therapeuticagents in the prevention and treatment of iron defi-ciency anaemia (Hertrampf & Olivares, 2004).This study is aimed at the investigation of the

effect of three Fe(III) compounds on photosyntheticelectron transport in spinach chloroplasts, on dry massof plant organs of maize (Zea mays L.) as well as oniron accumulation in roots, stems, and leaves of maizeplants.

Experimental

FeCl3 · 6H2O of analytical purity was supplied byLachema (Brno, Czech Republic). Two Fe(III) com-pounds containing 3 nicotinamide molecules (nia) and3 anions (Cl− or ClO−

4 ) were synthesized accordingto the following procedure: the mixture of ethanolicsolutions of corresponding Fe(III) salts (FeCl3 ·6H2Oor Fe(ClO4)3 ·12H2O) and nicotinamide (mole ration(Fe) : n(nia) = 1 : 4) was refluxed for 2 h. Af-ter cooling, fine crystalline products [Fe(nia)3Cl3] and[Fe(nia)3(H2O)2](ClO4)3 were obtained.Composition of the products was confirmed by el-

emental analysis; Fe content was determined chelato-metrically with Chelaton 3 using sulfosalicylic acid(Table 1). Elemental analyses were carried out bymeans of a Flash EA 1112 analyzer.Characteristic vibrations of the Fe(III) complexes

studied with nicotinamide ligands in the IR spectraare shown in Table 2. Other bands, at 1113 cm−1, 934cm−1, 629 cm−1, and 457 cm−1, in the IR spectrumof [Fe(nia)3(H2O)2](ClO4)3 are typical for the ionicbonded ClO−

4 anion, which is usual for iron complexes(Melník et al., 1997). Another important vibration fre-quency is ν (Fe—Cl) at 250 cm−1 for the coordinatedCl− anion in [Fe(nia)3Cl3].The infrared spectra (4000–100 cm−1) were record-

ed on a Nicolet Magna 750 FTIR spectrophotometerusing the solid-state KBr technique.Chloroplasts were obtained from market spinach

360 K. Kráľová et al./Chemical Papers 62 (4) 358–363 (2008)

Table 2. Selected data from IR spectra of nicotinamide and Fe(III)–nicotinamide complexes

Vibration (cm−1) νas(NH2)* νs(NH2)* ν (CO) ν (Fe—N)

nia 3357 3150 1678 –[Fe(nia)3Cl3] 3341 3245, 3150 1705, 1684 230, 225[Fe(nia)3(H2O)2](ClO4)3 3335 3220, 3192 1687, 1673 229, 221

*νas, νs asymmetric and symmetric vibrations, respectively.

332 334 336 338 340

Inte

nsity

/a.u

.

Magnetic induction/mT

g = 2.0026A

332 334 336 338 340

Magnetic induction/mT

g = 2.0026B

Inte

nsity

/a.u

.

Fig. 1. EPR spectra of untreated spinach chloroplasts (A) and chloroplasts treated with 0.02 mol dm−3 [Fe(nia)3(H2O)2](ClO4)3(B). Spectra recorded in dark (solid line) and under light exposure (dotted line). Spectra A were recorded at doublesensitivity compared to spectra B.

employing the procedure of Walker (1980) partly mod-ified by Šeršeň et al. (1990) using the TRIS buffer (20mmol dm−3, pH = 7.0) containing 0.4 mol dm−3 sac-charose and 20 mmol dm−3 MgCl2.EPR measurements were carried out on an ERS

230 instrument (ZWG, AdW, Berlin, Germany) op-erating in the X-band. The EPR spectra of spinachchloroplasts were recorded at 5 mW of microwavepower with a 0.5 mT amplitude modulation at 25◦C.The samples containing 3.9 g of chloroplasts in dm3

were measured in a flat quartz cell and their irradi-ation (about 400 µE m−2 s−1 PAR) was carried outdirectly in the resonance cavity with a 250 W halogenlamp from a 0.5 m distance employing a 5 cm waterfilter.The seeds of maize (Zea mays L., c.v. Lucia) were

soaked in distilled water for 24 h, then germinated infilter paper moisturized with distilled water for 3 days,and consequently hydroponically cultivated in Knopnutrient solution under controlled conditions (7 days;photoperiod 16 h day/8 h night; irradiance 80 µmolm−2 s−1 PAR; pH = 5.5; mean air temperature (25± 1)◦C). The concentration of the Fe(III) compoundsapplied was 0.25 mmol dm−3, 0.50 mmol dm−3, and1.00 mmol dm−3, respectively. For each concentration,15 seedlings were used. After 7 days of cultivation, drymass of roots and shoots were estimated. The resultswere statistically evaluated using ANOVA (P ≤ 0.05)after a preceding verification of normality and homo-geneity of the variance. The multiple comparison ofmeans was based on the method of the Tukey-contrast.For the determination of metal accumulation in in-

dividual parts of maize plants (root, stem, leaf) theatomic absorption spectrometry method (AAS, PerkinElmer, Model 1100) was used.

Results and discussion

Chloroplasts of vascular plants exhibit EPR sig-nals (so called signal I and signal II) in the region offree radicals (g ≈ 2.00) (Hoff, 1979) belonging to bothphotosystems. Fig. 1 shows the EPR spectra of thecontrol suspension of spinach chloroplasts (Fig. 1A)and of the chloroplast suspension treated with 0.02mol dm−3 [Fe(nia)3(H2O)2](ClO4)3 (Fig. 1B) takenin the dark (full line) and in the light (dotted line).EPR signal II, of untreated chloroplasts, is composedof two parts, the so-called signal IIslow (correspondingto the full line in Fig. 1A) and IIveryfast (correspondingto the difference between the EPR signals registered inthe light and in the dark; Fig. 1A). The spectroscopicparameters of both signals are g = 2.0046 and ∆Bpp =1.9 mT. In the presence of [Fe(nia)3(H2O)2](ClO4)3,both slow and fast components of the EPR signals,i.e. signal IIslow (Fig. 1B, full line) and signal IIveryfast(Fig. 1B, dotted line) decreased (almost disappeared).Signals IIslow and IIveryfast belong to intermediates Z+

/D+ , i.e. to tyrosine radicals situated at the 161st

position in D1 (TyrZ) and D2 (TyrD) proteins whichare located at the donor side of photosystem II (PSII) (Svensson et al., 1991). EPR signal I (g = 2.0026and ∆Bpp = 0.8 mT) belongs to an oxidized dimmerof chlorophyll in P700+ of PS I (Hoff, 1979) and itcan be observed usually in the EPR spectra of chloro-

K. Kráľová et al./Chemical Papers 62 (4) 358–363 (2008) 361

280 300 320 340 360 380 400

Inte

nsity

/a.u

.

Magnetic induction/mT

g = 2.0026

Fig. 2. EPR spectra of Mn(II) ions in spinach chloroplaststreated with 0.02 mol dm−3 [Fe(nia)3(H2O)2](ClO4)3.

plasts with damaged PS II under light. It was foundthat also [Fe(nia)3(H2O)2](ClO4)3 alone oxidizes thereaction centre of PS I (P700), which is documentedby the registered signal I in the EPR spectrum ofthe treated chloroplasts (Fig. 1B, full line). Due tothe interaction of [Fe(nia)3(H2O)2](ClO4)3 with TyrZand TyrD, electron transport between the photosyn-thetic centres PS II and PS I was interrupted whichwas manifested by the intensity increase of signal Irecorded in the light (Fig. 1B, dotted line). Similar ef-fects as [Fe(nia)3(H2O)2](ClO4)3 were also exhibitedby [Fe(nia)3Cl3] and FeCl3 ·6H2O.Applying [Fe(nia)3(H2O)2](ClO4)3 in the EPR

spectra of spinach chloroplasts, new signal con-sisting of six lines belonging to free Mn(II) ionswas observed (Fig. 2). Thus, it can be concludedthat [Fe(nia)3(H2O)2](ClO4)3 interacts with the man-ganese cluster which is situated in the oxygen evolv-ing complex on the donor side of PS II. Due to thisinteraction, Mn(II) ions from the manganese clusterare released into the interior of the thylakoid mem-brane. It was not possible to record the signal of man-ganese in the EPR spectra of control chloroplasts atroom temperature due to mutual interactions of man-ganese ions occuring in the manganese cluster contain-ing different oxidation states of this element. However,due to the interaction with [Fe(nia)3(H2O)2](ClO4)3,the released manganese ions occur only in the form ofMn(II) ions detectable by EPR spectroscopy alreadyat room temperature (Fig. 2).An adverse effect of iron stress on the pho-

tosynthetic electron transport was observed previ-ously by several researchers (e.g. Mallic & Rai, 1992;Kampfenkel et al., 1995). Mallic and Rai (1992) inves-tigated metal induced (Cu, Ni, and Fe) inhibition ofphotosynthesis and photosynthetic electron transportchain of Anabaena doliolum and Chlorella vulgaris andfound that the mode of inhibition of photosyntheticelectron transport chain of both the algae was simi-lar; however, PS II showed greater sensitivity to thetested metals. Kampfenkel et al. (1995) observed thatiron excess caused a 40 % decrease of the photosyn-

1 2 3 4

0

20

40

60

80

100

120

0

Sam

ple

to c

ontr

ol d

ry m

ass

ratio

/%

c/(mmol dm-3)0.25 0.50 1.00

g g g

fe

e

d d

c

ab

bc

Fig. 3. Dependence of the leaf dry mass of maize plantson the concentration of FeCl3 · 6H2O (black stripes),[Fe(nia)3Cl3] (white stripes), [Fe(nia)3(H2O)2](ClO4)3(grey stripes). The values indicated by the same let-ter do not differ statistically (P ≤ 0.05), n = 15, barsindicating standard error.

thetic rate in Nicotiana plumbaginifolia plants within12 h and the inhibition of photosynthesis was accom-panied by increased reduction of PS II.Maize (Zea mays L.) is a suitable model plant for

ecotoxicological assessment of metal contaminants be-cause its root system responds very sensitively to thepresence of toxic metals in the environment. In gen-eral, in the presence of high concentration of Fe(III)compounds, the inhibition of primary root growth ofmaize was much greater than that of shoot. At thehighest concentration of Fe(III) compounds applied,the growth of adventitious roots as well as root hairswas suppressed. Dry mass of roots and shoots wasless affected by the studied compounds than the rootand shoot growth; however, at higher Fe(III) concen-trations, higher shoot dry mass reduction was ob-served. On the other hand, in plants cultivated inthe presence of the highest Fe(III) concentration (1mmol dm−3), significant reduction of leaf dry mass oc-curred whereby the most toxic effect was exhibited by[Fe(nia)3(H2O)2](ClO4)3 (Fig. 3). In the [Fe(nia)3Cl3]complex, the Cl− anions are coordinated to the Featom; in [Fe(nia)3(H2O)2](ClO4)3, the ClO

2−4 anions

are not bound to the Fe atom by a coordination bondand it can be assumed that they exert toxic effect.Moreover, in the coordination sphere of this complexthere are water molecules which can easily be substi-tuted by another ligand (“bioactive ligand” such asresidues of amino acids in proteins). On the otherhand, the toxicity of [Fe(nia)3Cl3] was comparablewith that of FeCl3 · 6H2O.Fe concentrations in dry mass of plant organs

(roots, stems, and leaves) of maize treated with thestudied compounds and the corresponding bioaccu-mulation (BAF) and translocation factors (TF) areshown in Table 3.

362 K. Kráľová et al./Chemical Papers 62 (4) 358–363 (2008)

Table 3. Fe concentrations in dry mass of roots, stems, and leaves of maize treated with the compounds studied and the corre-sponding bioaccumulation (BAF) and translocation factors (TF)

w(Fe)/(mg kg−1)BAF

Compound c/(µmol dm−3) TF

Root Stem Leaf

Control 0 1949 65.6 137.7 0.223

FeCl3 · 6H2O 0.25 1889135.3

84.46.1

158.611.4

0.277

0.50 257492.2

128.68.1

223.58.0

0.265

1.00 6023107.8

450.48.1

240.04.3

0.143

[Fe(nia)3Cl3] 0.25 12398888.1

106.57.6

197.014.1

0.046

0.50 15862567.9

149.65.4

332.011.9

0.038

1.00 15571278.8

423.07.6

539.09.7

0.066

[Fe(nia)3(H2O)2](ClO4)3 0.25 3443246.6

146.010.5

245.017.6

0.142

0.50 277899.5

159.55.7

303.010.9

0.189

1.00 327758.7

162.82.9

518.09.3

0.137

The bioaccumulation factor (BAF) expresses theratio of the metal concentration in the biological ma-terial (in mmol or mg per kg of dry mass) to the metalconcentration in external solution in (mmol or mg perdm3) (Brooks & Robinson, 1998). The translocationfactor (TF) corresponds to the ratio of accumulated Feamount in shoots and roots and, thus, it depends alsoon the actual dry mass of these plant organs. HigherTF values reflect more effective mobility of Fe withinplants. From the values presented in Table 3 it is ev-ident that the highest Fe-accumulating capacity wasexhibited by maize roots and the lowest one by itsstems.Based on the comparison of the three studied

Fe(III) compounds, it is evident that a markedlyhigher Fe concentration in plant roots was observedfor the treatment with [Fe(nia)3Cl3]. Thus, it couldbe assumed that this Fe(III) complex uptaken byroots will be mostly stored in unchanged form inthe root cell vacuoles. It was found previously thatthe amounts of Fe accumulated in the cells of algaScenedesmus quadricauda were 2.7-19.6 times higherfor Fe supplied in the form of complexes comparedto the inorganic salt (FeCl3 · 6H2O) (Fargašová et al.,2000). On the other hand, both Fe(III) complexes,([Fe(nia)3Cl3] and [Fe(nia)3(H2O)2](ClO4)3), as wellas FeCl3 · 6H2O reached higher Fe concentrations inthe leaves than in the stems. It is presumable thatFeCl3 · 6H2O and [Fe(nia)3(H2O)2](ClO4)3 will substi-

tute their H2O ligands and form complexes with or-ganic amino acids occurring in the cell. Consequently,these complexes will secure the mobility of Fe withinthe plant. This assumption is also supported by theTF values which decreased in the following order:FeCl3 ·6H2O, [Fe(nia)3(H2O)2](ClO4)3, [Fe(nia)3Cl3].Monocotyledonous plants, such as maize, release

siderophores which are able to dissolve external Fe(III)and the plants take up the formed complex Fe(III)-siderophore (Schmidt, 1999). The plants producemany ligands for metals. Citrate is usually present inexcess compared to the Fe content in xyleme exudates(Tiffin, 1966a, 1966b). Clark et al. (1973) confirmedthe presence of Fe-citrate associates in the exudatesof maize stems and increased production of organicacids was observed firstly in plants with insufficientFe supply (Abadía et al., 2002).Physiological effects of iron bound in the com-

plex on root nutrition depend on the conditions ofmineral nutrition as well as on the concentration,stability, and absorption properties of the Fe com-plex. In plant cells, dissociation of the complex dueto ligand exchange (residues of amino acids in pro-teins) or eventually other mechanisms could occur.Moreover, the excess of Fe in the cell can gener-ate formation of toxic oxygen radicals which im-pair physiological processes in the cell (Kampfenkel,1995). Using Mossbauer spectrometry it was con-firmed that the majority of Fe in vascular plants

K. Kráľová et al./Chemical Papers 62 (4) 358–363 (2008) 363

occurs in the form of Fe(III) (Terri & Abadía,1986).The main chelator in vascular plants is a non pro-

tein amino acid nicotianamine forming chelates notonly with Fe but also with other metals (von Wirén etal., 1999). In general, it can be concluded that the Fereactivity depends not only on its ligands but also onpH and the presence of chelators. Chelates with dif-ferent molecules should be compared for their efficacyconsidering their ability to maintain Fe in solution andtheir capacity to release iron to the roots. Acceptingthe turnover hypothesis, their efficacy is also depen-dent on the ability of the chelating agent to form thechelate using native iron from the soil. The ability ofchelates to maintain Fe in solution and to utilize nativeions from the soil is related to their chemical stabil-ity, while plants make better use of iron from the lessstable chelates. Plant response is the ultimate eval-uation method to compare commercial products withthe same chelating agent or different chelates (Lucena,1969). Inside the cells, generation of highly toxic hy-droxyl radicals by iron redox reactions is avoided byintricate chelation mechanisms. Organic acids, mostnotably nicotianamine, and specialized proteins bindiron before it can be inserted into target moleculesfor biological function. Uptake and trafficking of ironthroughout the plant is therefore a highly integratedprocess of membrane transport and reduction, traffick-ing between chelator species, whole-plant allocation,and genetic regulation (Hell & Stephan, 2003).

Acknowledgements. This study was supported by grants

VEGA of the Ministry of Education of the Slovak Republic and

the Slovak Academy of Science, No. 1/3489/06 and 1/4454/07.

The authors thank Dr. Jana Kubová from the Institute of Ge-

ology, Faculty of Natural Sciences, Comenius University in

Bratislava for determination of the Fe content in plants.

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