Nuclear Instruments and Methods in Physics Research B xxx (2010) xxx–xxx

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Contents lists available at ScienceDirect

Nuclear Instruments and Methods in Physics Research B

journal homepage: www.elsevier .com/locate /n imb

Cd induced redistribution of elements within leaves of the Cd/Zn hyperaccumulatorThlaspi praecox as revealed by micro-PIXE

Paula Pongrac a,b, Katarina Vogel-Mikuš b, Primoz Vavpetic a, Janja Tratnik b,1, Marjana Regvar b,Jurij Simcic a,2, Nataša Grlj a, Primoz Pelicon a,*

a Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Sloveniab Department of Biology, Biotechnical Faculty, Vecna pot 111, SI-1000 Ljubljana, Slovenia

a r t i c l e i n f o a b s t r a c t

Article history:Available online xxxx

Keywords:CadmiumMineral nutrientsThlaspi praecoxTrue X-ray microanalysisHydroponics

0168-583X/$ - see front matter � 2010 Elsevier B.V.doi:10.1016/j.nimb.2010.02.089

* Corresponding author. Tel.: +386 1 588 5294; faxE-mail address: primoz.pelicon@ijs.si (P. Pelicon).

1 Present address: Jozef Stefan Institute, Jamova 39,2 Present address: Jet Propulsion Laboratory, M/S 12

Pasadena, CA 91109, USA.

Please cite this article in press as: P. Pongrac etrevealed by micro-PIXE, Nucl. Instr. and Meth.

A detailed localisation of elements within leaf tissues of hydroponically grown Cd/Zn hyperaccumulatorThlaspi praecox (Brassicaceae) was determined by micro-PIXE at Jozef Stefan Institute (Ljubljana, Slove-nia) in order to study accumulation patterns of Cd and other elements in the case of a single metal (Cd)pollution. Plants were treated with increasing concentrations of Cd in the solution (0 (control), 1, 10and 100 lM). As expected, concentration of Cd in the leaves gradually increased with Cd concentrationin the solution. In order to reveal the main Cd storage compartment space within the leaves a relativeelement distribution (pool) was calculated based on concentrations of elements in specific leaf tissuesand their weight portions. Where present at detectable levels, Cd accumulated in the epidermal tissues(at 10 lM), but the contribution of epidermal pool decreased with increasing Cd concentration in solu-tion (at 100 lM). The opposite was observed for the mesophyll pool. In addition, in Cd treated plants, asignificant decrease in mesophyll Fe pool and an increase in the epidermal Fe pool were observed. Sim-ilar effect was seen for Mn pool at 100 lM Cd treatment accompanied by increasing Zn epidermal poolwith increasing Cd in nutrient solution. Altogether these results indicate repartitioning of essentialmesophyll cation pools (e.g., Fe, Mn and possibly Zn) when increasing Cd contents, that are insteadmore intensively stored in the epidermal cells. These results confirmed micro-PIXE as effective andpowerful technique providing essential information on metal localisation, repartitioning and major ele-mental stores in plants on the tissue levels that were not accessible using classical analytical tech-niques and thus complementing our current understanding of plant metal tolerance mechanisms asa whole.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

Hyperaccumulation of Cd (concentrations above 100 lg g�1 Cdin the aboveground dry biomass) is a rare phenomenon that hasto date been reported only in few plant species. It is still not clearhow these plants cope with such high, normally toxic concentra-tions of the metal in their tissues, therefore the mechanisms em-ployed are of great interest of modern biotechnology andphytoremediation. The reliable and detailed information on thespatial element distribution within plant tissues provides basicknowledge for understanding the metal tolerance mechanisms.Among the techniques providing this kind of information, micro-particle induced X-ray emission (micro-PIXE) has been successfully

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SI-1000 Ljubljana, Slovenia.1-104, 4800 Oak Grove Drive,

al., Cd induced redistribution oB (2010), doi:10.1016/j.nimb.20

applied to different plant parts such as seeds [1–3], leaves [4–7],stalks [5,8] and roots [9–11] of both non- and hyperaccumulatingplants. In the micro-PIXE localisation study of field collected leavesof a Cd/Zn hyperaccumulating plant species Thlaspi praecox [12] itwas demonstrated, that the highest concentrations of Zn, Cd, Ni,Cu, Pb, Fe and Mn were found in the upper/lower epidermis. How-ever, taking into account the contribution of the weight of specificleaf tissue it became clear, that the most prominent Zn pool was inepidermal tissue, most of accumulated Cd and Mn were stored inthe mesophyll, whereas Fe pool was predominant in both epider-mis and mesophyll [6,7]. Additionally, intense vascular Cd localisa-tion indicated high Cd mobility in the phloem [6] furthersupporting its hyperaccumulation in seeds and flowering stalksof T. praecox [1,13].

In the present study micro-PIXE analysis was applied to cryo-fixed and freeze-dried specimens for simultaneous elementallocalisation, detection of elemental redistributions and determina-tion of the sites of preferential accumulation of Cd and essentialelements (pools) within leaf cross-sections of T. praecox, treated

f elements within leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as10.02.089

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with increasing Cd concentrations in hydroponic solution undercontrolled experimental conditions.

Fig. 2. PIXE spectrum measured in a section of a leaf of Thlaspi praecox grown inhydroponic solution; (A) 100 lM Cd and (B) control. For Cd detection, the Cd Ka lineat 23 keV was used for evaluation, as the Cd L lines overlap with the strong K Kaline.

2. Experimental

2.1. Plant material and hydroponic experiment

Seeds of T. praecox Wulfen were obtained from the heavy-metalpolluted site in Zerjav in Northern Slovenia [12] and were surfacesterilized in 10% H2O2 for 10 min and germinated on a mixture ofvermiculite/perlite (1/1, v/v). The seedlings were then transferredto a hydroponic solution containing 3 mM KNO3, 1 mM NH4H2PO4,2 mM Ca(NO3)2, 0.5 mM MgSO4, 66 lM FeEDTA, 50 lM KCl, 46 lMH3BO3, 9 lM MnSO4, 1.5 lM ZnSO4, 1.5 lM CuSO4 and 0.14 lM(NH4)Mo7O24 [14]. The nutrient solution was aerated continuouslyand renewed each week. After a two-week adaptation period, Cd(as CdSO4) was added in final concentrations (0-control, 1, 10and 100 lM) in solution. The plants were grown in a controlledgrowth chamber with a 16 h day period, a light intensity of160 lmol m�2 s�1, 18 �C/16 �C day/night temperature and 50–60%relative humidity. Plants were treated with Cd for six weeks.

2.2. Sample preparation for micro-PIXE analysis

Leaf cross-sections were taken at the upper part of the leaf, onthe right hand side of the main vein of the leaf (Fig. 1A). Fullydeveloped leaves were selected for the analysis, detached from in-tact plants, quickly washed in deionised water and sectioned witha razor blade to smaller pieces of approx. 2 � 5 mm2. The pieceswere immediately inserted in a 2 mm stainless steel needle anddipped into liquid propane cooled by liquid nitrogen [15,6,7]. Leafpieces were sectioned with a Leica CM3050 cryotome (Leica, Bens-heim, Germany). The temperature of the microtome head and thechamber were in the range from �25 to �20 �C and the sectionthickness was 60 lm. The sections were placed in pre-cooled Alholders and transferred to an Alpha 2–4 Christ freeze dryer usinga cryo-transfer-assembly cooled by liquid nitrogen and thenfreeze-dried at �30 �C and a pressure of 0.4 mbar for three days.Dry leaf cross-sections were mounted on the Al sample holder be-tween two thin layers of Pioloform foil [15].

2.3. Micro-PIXE analysis

For the detection of the X-ray energies from 1 keV up to 25 keVhigh-purity germanium X-ray detector with an active area of 95mm2 and 25 lm-thick beryllium window positioned at the angleof 135� with respect to the beam direction was used. The protondose was determined by rotating in-beam chopper [2]. Measure-

Sectioned part of the

leaf

A B

Fig. 1. In cryo-fixed and freeze-dried leaf cross-section of Thlaspi praecox leavesexcised from a marked region (A) the following anatomical areas were distin-guished (B) UE, upper epidermis; PM, palisade mesophyll; VB, vascular bundle; SM,spongy mesophyll; LE, lower epidermis. Photographed with a digital camera(AxioCam) mounted on ZEISS Axioskop 2 (Carl Zeiss, Göttingen, Germany) lightmicroscope using AxioVision 3.1 programme. Bar represents 100 lm.

Please cite this article in press as: P. Pongrac et al., Cd induced redistribution orevealed by micro-PIXE, Nucl. Instr. and Meth. B (2010), doi:10.1016/j.nimb.20

ment of micro-PIXE and data evaluation for the biological samplesof intermediate thickness at the micro-PIXE setup at JSI has beenpreviously described in detail [1,2,6,11]. After final sample posi-tioning and scan size selection, the object slits were nearly closedand zero-degree scanning transmission ion microscope (STIM)maps were measured in the list mode. This was followed by ahigh-current mode with opened object slits during which PIXEmaps of the same region were measured in the list mode over aperiod of at least ten hours. After that the STIM map was againmeasured on the same area in the list mode to check for sampleconsistency, thinning and possible shrinking.

The X-ray and STIM spectra corresponding to distinct morpho-logical structures (upper/lower epidermis, palisade/spongy meso-phyll and vascular bundles) were identified (an example isshown in Fig. 2) and extracted from the selected regions (markedregions are shown in upper left panel of Fig. 3). Assuming a cellu-lose matrix, the average thickness of the selected area was calcu-lated from the STIM spectrum and used for matrix corrections inthe GUPIXWIN programme used for fitting of obtained PIXE spectra[16]. For the evaluation of Cd concentration, the Cd Ka line at23 keV was used, as the Cd L lines overlaps with the pronouncedKa line of potassium. This circumstance strongly increases re-quired time to reach sufficient statistics in the Cd Ka line acquiredspectra [7]. The calibration of the PIXE method was verified by theanalysis of the multi-elemental standard reference materials NISTSRM 1573a (tomato leaves, homogenized powder, analysed in aform of pressed pellet), NIST SRM 1107 (Naval Brass B, alloy) andNIST SRM 620 (Soda-Lime Flat Glass). The inter-calibration of PIXEand STIM was verified by thin mono-elemental metallic foils.

Resulting qualitative elemental maps (Fig. 3) were formed fromthe existing list mode results by Dynamic Analysis method [17].The imaging with Dynamic Analysis is able to resolve overlappingX-ray lines and exclude strong spectral background, which is notthe case for the simple discriminating method integrated in theOM_DAQ software [18]. The integration of the X-ray detectors effi-ciency and proton dose normalisation for proper quantificationwith the Dynamic Analysis method is currently under way in JSIlaboratory.

3. Results

In leaves of hydroponically grown T. praecox five distinct ana-tomical areas were distinguished, namely upper and lower epider-

f elements within leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as10.02.089

Fig. 3. Scanning Transmission Ion Microscopy (STIM) image (upper left panel) with marked regions from which PIXE spectra were extracted (green, epidermises; red,mesophyll and blue, vascular bundles) and qualitative elemental maps of P, S, K, Ca, Zn in leaf cross-section of Thlaspi praecox treated with 100 lM Cd in hydroponic solutionobtained by Dynamic Analysis method [17]. Scan size is 1090 � 870 lm2. (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

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mis, palisade and spongy mesophyll, and vascular bundles (Fig. 1B)in which essential elements and metals (P, S, Cl, K, Ca, Mn, Fe, Zn,and Cd) were detected by micro-PIXE. Some of the correspondingelemental maps with sufficient count statistics are presented inFig. 3, showing highly inhomogeneous element distribution withinleaf cross-section as well as within particular tissues (e.g. Zn in epi-dermis). The concentrations of elements in particular tissues werecalculated using GUPIXWIN software after extracting spectra fromencircled regions corresponding to epidermis (upper and lower),mesophyll (palisade and spongy) and vascular bundles (Table 1).Concentrations of Cd in the leaves of controls and 1 lM treatedplants were below the limit of detection, while in 10 and 100 lMtreated plants the concentration increased with increasing Cd con-centration in the solution.

Due to highly inhomogeneous element distribution within par-ticular tissues and because it was impossible to map exactly thesame leaf regions comprising the same proportion of specific tis-sues and/or cells in each Cd treatment, obtained element tissue

Please cite this article in press as: P. Pongrac et al., Cd induced redistribution orevealed by micro-PIXE, Nucl. Instr. and Meth. B (2010), doi:10.1016/j.nimb.20

concentrations were highly variable (Table 1). Therefore the rela-tive elemental distributions (%) within leaf cross-sections (pools)were calculated on the basis of the relative weight portions of par-ticular tissues in T. praecox leaves determined in our previousstudy, namely vascular bundle 18.3%, mesophyll 74.8% and epider-mis 6.9% [6] and concentration of the element in particular tissue.Changes in relative distribution due to the Cd treatment were ob-served for Cd and essential plant micronutrients Fe, Mn and Zn(Fig. 4A–D) but not for other elements that were mostly locatedin the mesophyll (data not shown).

The relative epidermal pool of Cd decreased with increasingCd concentration in solution, whereas the opposite was observedin the mesophyll, where Cd pool gradually increased withincreasing Cd concentration in solution (Fig. 4A). The concentra-tions of Cd detected in the vascular bundles were relativelyconstant.

In Cd treated plants there was a slight decrease in mesophyll Fepool while an increase in epidermal Fe pool was observed (Fig. 4B).

f elements within leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as10.02.089

Table 1Micro-PIXE localization of elements within leaf cross-section of Thlaspi praecox grown in hydroponics for six weeks. The GUPIXWIN programme [18] was used for the evaluationof the elemental concentrations given in micrograms per gram of dry tissue.

0 lM Cd (510 � 510 lm2) 1 lM Cd (1220 � 1220 lm2) 10 lM Cd (1020 � 1020 lm2) 100 lM Cd (1220 � 1220 lm2)

lg g�1 Stat. err % LOD lg g�1 Stat. err % LOD lg g�1 Stat. err % LOD lg g�1 Stat. err % LOD

Epidermis P 6753 10.8 753 1082 13.2 158 1674 44.8 804 2271 15.2 384S 7642 4.23 436 1381 5.8 84 3426 12.4 456 3444 4.5 215Cl 3396 4.74 263 685 6.23 48 6058 4.3 271 2072 3.7 123K 62832 0.61 175 12204 0.68 63 76907 0.38 219 28928 0.39 73Ca 29576 1.04 356 16908 0.6 45 51346 0.71 327 14356 1.14 143Mn 304 17.8 40 107 14.4 10 388 8.39 32 232 7.6 22Fe 223 31.7 47 106 11.7 8 378 17.5 46 435 5.53 21Zn 2105 7.12 44 235 8.8 9 3222 3.3 49 339 9.3 25Cd n.d. / / n.d. / / 6506 34 2082 8029 18 1529

Mesophyll P 4280 3.99 188 1268 8.95 132 1636 43.5 804 1716 8.2 116S 8805 1.22 101 1493 4.23 68 6662 6.81 485 2794 2.4 57Cl 1109 5.05 67 642 5.17 40 8548 3.11 278 421 7.71 35K 31093 0.26 42 12254 0.48 36 143494 0.24 194 10874 0.48 31Ca 18965 0.65 99 13356 0.52 46 47482 1.12 428 13668 0.46 37Mn 133 6.2 8 46 13.5 5 186 18.4 30.1 38 16.7 5.4Fe 347 3.5 10 68 9.99 6 166 28.5 37.2 232 3.9 4.5Zn 158 6.8 7 100 10.4 5 235 25.7 25.5 22 31.1 4.9Cd n.d. / / n.d. / / 1518 68.4 2417 1805 29.8 463

Vascular bundle P 6973 2.16 279 2064 3.4 137 16988 1.79 562 8325 2.3 307S 14875 0.63 136 4002 1.04 67 7493 2.19 309 7734 1.38 153Cl 813 5.32 90 655 2.98 40 1853 5.08 201 597 7.81 100K 38177 0.18 60 14768 0.23 33 99745 0.14 93 28985 0.3 50Ca 17379 0.46 113 11599 0.34 47 8444 1.98 310 6210 1.24 118Mn 239 3.9 13 44.3 8.17 5.9 360 4.2 20 80.9 13.2 15Fe 326 3.5 16 65.3 6.13 6.3 139 12.1 30 361 4.5 15Zn 122 7.32 8 84.1 7.05 4.8 257 7.8 20 54.2 26 19Cd n.d. / / n.d. / / 2502 43.7 2095 2900 35.03 1219

Stat. err (%), error of the measurement; LOD, minimum limit of detection of the measurement (lg g�1); Micro-PIXE, micro-proton induced X-ray emission; n.d., not detected.

0

20

40

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80

Concentration of Cd in nutrient solution (µM)

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D

Fig. 4. Relative element distribution (%) of (A) Cd, (B) Fe, (C) Mn and (D) Zn considering relative amounts (lg) of corresponding tissues in the leaf cross-section ofhydroponically grown Thlaspi praecox. Relative weight proportions (%) were adopted from [7] and were for mesophyll 74.8% and for epidermis 18.3%. n.d., not detected.

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Similarly at 100 lM Cd treatment there was a significant decreasein the mesophyll Mn pool whereas the epidermal Mn pool in-creased (Fig. 4C). Zn was most abundant in the epidermises and

Please cite this article in press as: P. Pongrac et al., Cd induced redistribution orevealed by micro-PIXE, Nucl. Instr. and Meth. B (2010), doi:10.1016/j.nimb.20

the epidermal pools slightly increased with Cd treatment, whilethere was a decrease in mesophyll Zn pool. Zn pools in the vascularbundles were relatively constant (Fig. 4D).

f elements within leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as10.02.089

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4. Discussion

The physiological mechanisms of metal tolerance in plants takeplace at the cellular, tissue and whole plant level [19]. Nowadays,intense research focuses on elucidating tolerance mechanisms atthe cellular level (recently reviewed by [20]). For the studies on tis-sue level however, micro-PIXE is very suitable technique providingdata on simultaneous tissue localisation of several elements, theirconcentrations and repartitioning, essential for the studies of metaltolerance strategies in biological systems. Therefore, we employedmicro-PIXE to study the distribution fingerprint of Cd and otherelements in leaves of hydroponically grown T. praecox treated withCd under controlled experimental conditions. T. praecox was re-cently reported to hyperaccumulate up to 0.6% Cd in shoots underfield conditions [12] and up to 0.3% Cd in hydroponics [21]. Gener-ally Cd is very toxic to plants and concentration of above 1 lg g�1

on dry weight biomass typically leads to visible toxicity symptomsthat are result of Cd interference with plant metabolism [22]. Asexpected, increasing Cd concentration in the leaves with increasingexternal Cd concentration was observed (Table 1) but no visibletoxicity symptoms were seen in T. praecox which is in line withthe results obtained in the pot experiment [23]. It is generally be-lieved that Cd has no known function in plant metabolism how-ever, growth enhancement by Cd was repeatedly observed in Cdhyperaccumulators [24,25,23] therefore a physiological role of Cdwas proposed [26]. In leaves of field collected T. praecox the highestconcentration of Cd was seen in the epidermis but if the relativevolume of certain tissue was taken into account it was obvious thatthe majority of Cd is localised in the mesophyll [6,7]. This was alsoobserved in T. caerulescens [27] and Arabidopsis halleri [28]. Differ-ential contribution of particular leaf tissue in particular sectionsand the heterogeneous distribution of the elements within leaf tis-sues resulted in very variable element tissues concentrations ob-tained, for example if more large vacuolated cells wereencountered in the scan, higher Zn concentrations were detected(Fig. 3, Table 1) due to specific Zn accumulation pattern reportedfor T. praecox [6,7]. The relative element distribution (%), encoun-tering element concentrations and the weight of the tissue [6,7],was thus introduced and only those results will be discussed inthe following text. A gradual accumulation of Cd in mesophyll poolwas observed at the expense of the epidermal pool with increasingCd concentrations in solution (Fig. 4A). The observed pattern mayreflect the limited accumulation capacity or saturation of tolerantmechanisms in the epidermises leading to allocation of Cd to themesophyll as seen also in A. halleri [28], posing specific threatsfor plant metabolic processes, in particular photosynthesis [29].

Simultaneously, a slight decrease in mesophyll Fe pool was ob-served, that may help in explaining a decreased chlorophyll con-centration in leaves of T. praecox treated with increasing Cdconcentration in a pot experiment [23]. Iron is crucial for properphotosynthetic activity and is thus an essential nutrient for opti-mal plant productivity [30]. It is highly reactive with organic li-gands, however, as internal Cd to Fe ratio increased due to Cdtreatment the replacement of Fe by Cd may have taken place inour experiment. Stimulatory effect of Fe deficiency on Cd uptakein a Cd hyperaccumulator T. caerulescens Ganges ecotype wasattributed to an up-regulation in the expression of genes encodingfor Fe2+ uptake [31]. More detailed analyses using techniques withhigher lateral resolution and those that enable the identification ofFe and Cd speciation and coordination will be needed to confirm Fereplacement by Cd.

Manganese distribution in mesophyll of T. praecox leaves inhydroponics is in agreement with the distribution observed inleaves of field collected plants [7]. At 100 lM Cd treatment how-ever, the Mn distribution changed drastically and most of the Mnpool was directed to epidermises (Fig. 4C). Manganese is an essen-

Please cite this article in press as: P. Pongrac et al., Cd induced redistribution orevealed by micro-PIXE, Nucl. Instr. and Meth. B (2010), doi:10.1016/j.nimb.20

tial micronutrient with low phloem mobility that plays a majorrole in photosynthesis, respiration and activation of several en-zymes [32]. In Mn accumulator Phytolacca americana the interac-tions between Mn and Cd in uptake indicated that Mn and Cdions might use the same transport proteins for metal uptake[33]. In a hydroponic experiment with increasing Cd concentra-tions a decrease of Mn and Fe concentration in shoots of A. halleriwas seen [28]. The interaction between Cd and Mn was also re-ported in lettuce [34] and an increase of Zn, Mn and Ca in thehydroponic solution was shown to decrease Cd uptake in T. cae-rulescens Prayon ecotype, further supporting significant interac-tions of Cd and the plant essential micronutrients mentioned[35]. As for Fe it seems that increased Cd in solution led to thereplacement of Mn by Cd in mesophyll seen at the highest Cd treat-ment of T. praecox plants.

Thlaspi praecox was reported also to hyperaccumulate Zn [12]meaning that Zn concentration in shoots of field collected plantsexceeded 1% of dry biomass. Zn is an essential nutrient for plantsand plays a catalytic and structural role in enzyme reactions. Con-centrations of up to 100 mg Zn kg�1 are usually found in plants[32]. Zn was demonstrated to be accumulated in the large vacuo-lated epidermal cells in field collected leaves of T. praecox [6,7].Binding capacity and removal of Zn from the cytosol in Zn tolerantplants is very fast and may be one of the reasons for higher Znrequirement in these plant species [36]. This may help in explain-ing the observed Zn redistribution by increasing external Cd con-centration in our experiment and confirm the existence ofinteractions between Cd and Zn in T. praecox as previously reportedfor T. caerulescens Ganges ecotype [37,38]. Similar interactionswere also observed by bulk sample analyses of greenhouse grownT. praecox plants [40].

The homeostasis of the essential transition metals Fe, Mn andZn requires balanced activities of transporters that mediate importinto the cell, distribution to organelles and export from the cell[39]. These processes in plants are dynamic, changing between tis-sues, sub-cellular compartments and across developmental stages[41] and may be attributed to changes in the activity of metaltransporter(s). Some of them have been shown to simultaneouslytransport Fe, Zn, Mn and Cd [41]. Based on our results we concludethat increasing Cd interferes with the homeostasis of Mn and Fe inT. praecox, a Cd tolerant hyperaccumulating plant species.

In conclusion, micro-PIXE enabled us to study the elemental fin-gerprints in Cd/Zn hyperaccumulating T. praecox. Besides elemen-tal concentrations, it also enabled us to determine majorelemental pools in different plant tissues and follow the impactof increasing Cd concentration in solution on the elemental redis-tributions within leaves under controlled experimental conditions.Gradual replacement of plant essential micronutrients (Fe, Mn, Zn)by building up Cd pools within plant tissues indicate specific met-abolic consequences for plants. Thus micro-PIXE is a valuable tech-nique providing crucial information for studies of plant metaltolerance mechanisms on tissue level thus complementing theexisting knowledge on metal hyperaccumulating plants that maypotentially be used in phytoremediation of heavy-metal pollutedsoils.

Acknowledgements

Authors would like to thank G. Kukec for his help with softwarefor Dynamic Analysis. The work was supported by the followingprograms and projects of Slovenian Research Agency: P1-0212,P1-0112, J7-0352, Fellowships for ‘‘Young researchers”, Supportof Accelerator Laboratory in the frame of Research Infrastructure.Part of work was done within EU Projects COST 859 and EU FP7Project No. 2270112 ‘‘SPIRIT”. A scholarship from the WorldFederation of Scientists and a national fellowship awarded by

f elements within leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as10.02.089

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L’OREAL-UNESCO-The Slovenian Science Foundation to P. Pongracis gratefully acknowledged.

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f elements within leaves of the Cd/Zn hyperaccumulator Thlaspi praecox as10.02.089