Environmental Pollution 147 (2007) 50e59www.elsevier.com/locate/envpol

Localisation and quantification of elements within seedsof Cd/Zn hyperaccumulator Thlaspi praecox by micro-PIXE

Katarina Vogel-Mikus a, Paula Pongrac a, Peter Kump b, Marijan Ne�cemer b,Jure Sim�ci�c b, Primo�z Pelicon b, Milos Budnar b, Bogdan Povh c, Marjana Regvar a,*

a Department of Biology, Biotechnical Faculty, University of Ljubljana, Ve�cna pot 111, SI-1000 Ljubljana, Sloveniab Jo�zef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia

c Max-Planck-Institut fur Kernphysik, P.O. Box 103980, 69029 Heidelberg, Germany

Received 16 June 2006; received in revised form 24 August 2006; accepted 27 August 2006

Hyperaccumulation of Cd with partitioning in the epidermis of cotyledons was observed in T. praecox embryos.

Abstract

Cd, Zn and Pb accumulation, spatial distribution within seeds and germinating seedlings, and seeds fitness of metal hyperaccumulatingThlaspi praecox were investigated in order to gain more knowledge on plant reproductive success at metal polluted sites. The seeds containedup to 1351 mg g�1 (dry weight) of Cd, 121 mg g�1 of Zn and 17 mg g�1 of Pb. Seed fitness was negatively influenced by seed Cd hyperaccu-mulation. Nevertheless, the viability of seeds was decreased by maximally 20%, indicating very efficient tolerance of the plant embryos toCd. Localisation by micro-PIXE revealed preferential storage of most elements in the embryonic axis. Cd and Zn were preferentially localisedin the epidermis of cotyledons. The restriction of seed Pb and Zn uptake and hyperaccumulation of Cd, accompanied by partitioning of Cd in theepidermal tissues of cotyledons, may enable the survival of T. praecox embryos and seedlings in Cd polluted environments.� 2006 Elsevier Ltd. All rights reserved.

Keywords: Thlaspi praecox; Heavy metal; Zinc; Lead; Cadmium; Metal hyperaccumulation; TXRF; micro-PIXE

1. Introduction

Significant efforts towards understanding of the mechanismsinvolved in metal uptake strategies, transport, accumulation andsequestration in tissues of metal hyperaccumulating plants weremade in the last decade (Kupper et al., 1999, 2004; Lombi et al.,2001; Baker and Whiting, 2002). However, most studies havefocused on roots and leaves, while reports on metal accumula-tion and localisation in fruits and seeds are scarce (Mesjasz-

Abbreviations: AAS, atomic absorption spectrometry; MIC, Microanalyti-

cal Centre of the Jo�zef Stefan Institute; micro-PIXE, micro-proton induced

X-ray emission; STIM, scanning transmission ion microscopy; TXRF, total

reflection X-ray fluorescence; XRF, X-ray fluorescence.

* Corresponding author. Tel.: þ386 1 423 3388, fax: þ386 1 257 3390.

E-mail address: marjana.regvar@bf.uni-lj.si (M. Regvar).

0269-7491/$ - see front matter � 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2006.08.026

Przyby1owicz et al., 1999, 2001; Psaras and Manetas, 2001;Bhatia et al., 2003).

Nutrients for the seed formation are mainly transportedfrom the leaves via phloem, which may also incidentallydeliver other materials (e.g. certain metals) to the seeds(Patrick, 1997; Patrick and Offler, 2001; Grusak, 1994; Pear-son et al., 1995). Clearly, cellular sequestration of metals inthe vacuoles of root and shoot tissues of metal tolerant plantscan restrict the movement of the free metals in the symplast(Lasat et al., 1998; Wojcik et al., 2005). Therefore metalconcentrations within the seeds of metal tolerant plant eco-types (e.g. Thlaspi caerulescens, Silene vulgaris, Biscutellalaevigata, Thlaspi pindicum) are lower than of any other plantpart (Brooks, 1998a; Ernst, 1996; Mesjasz-Przyby1owicz et al.,1999, 2001; Ernst et al., 2000; Psaras and Manetas, 2001).Once loaded into the phloem, symplastic translocation ofnutrients and metals was found to stop at the funiculus or in

51K. Vogel-Mikus et al. / Environmental Pollution 147 (2007) 50e59

the seed coat, and the final translocation into the embryooccurs apoplastically (Patrick, 1997; Patrick and Offler,2001). The megasporangium, the placenta and/or the endo-sperm were suggested to act as barriers for metal translocationto the embryos (Ernst et al., 1992; Mesjasz-Przyby1owiczet al., 2001). Because of the inability of metals to crossthe seed apoplastic barrier (Bhatia et al., 2003), the exclusionof metals from embryonic tissues was frequently proposed as astrategy to maintain the reproductive success of hyper-accumulating plants on metal enriched soils (Ernst et al.,1992; Mesjasz-Przyby1owicz et al., 1999, 2001; Bhatia et al.,2003).

The majority of metal uptake studies in plants employ bulkatomic absorption spectrometry (AAS) and inductively cou-pled plasma atomic emission spectrometry (ICP-AES),whereas studies employing X-ray fluorescence techniques inanalysing plant samples are rare (Brooks, 1998b; Vargaet al., 2000; Vogel-Mikus et al., 2006). The limits of detection(LOD) of standard X-ray fluorescence (XRF) technique formedium and high atomic number elements can reach a fewmg g�1 of plant tissue, whereas the sensitivity of the total re-flection X-ray fluorescence (TXRF) technique is by two ordersof magnitude higher and thus sufficient for the analysis oftrace elements in plant tissues (Gunther et al., 1995; Kumpet al., 1996; Varga et al., 1999; Golob et al., 2005). Moreover,compared to AAS and ICP-AES, TXRF proved to be fasterand more economical in analysing a larger number of samples(Gunther et al., 1995; Kump et al., 1996; Golob et al., 2005). Areliable technique for providing unique information on thespatial element distribution is micro-Proton-Induced X-rayEmission (micro-PIXE). With its list mode analysis it offersthe highest sensitivity and accuracy in performing elementalmapping and quantification of the data extracted from arbi-trarily selected micro-areas of selected tissues. It was thereforeemployed in elemental localisation within fruits, seeds andvegetative tissues of metal hyperaccumulating plants (Mes-jasz-Przyby1owicz et al., 1997, 1999, 2001; Bhatia et al.,2003, 2004; Przyby1owicz et al., 2004).

Thlaspi praecox Wulf., successfully colonising Pb, Cd andZn polluted area in Slovenia (Regvar et al., 2006), was re-cently reported to hyperaccumulate up to 1.5% Zn, 0.6% Cdand 0.4% Pb in shoots (dry weight) (Vogel-Mikus et al.,2005). Because of the potential of metal hyperaccumulatingThlaspi species for phytoremediation of contaminated soils,the strategies enabling the reproductive success of T. praecoxin highly polluted environments could be of interest forbroader scientific community.

The main aims of this study were therefore to (i) determineheavy metal accumulation in T. praecox seeds using TXRFand AAS; (ii) investigate the spatial distribution of metalswithin the seeds (seed coat, embryonic tissues) and germinatedseedlings (cotyledons, hypocotyl and radicle) using micro-PIXE at the Microanalytical Centre (MIC) of the Jo�zef StefanInstitute, Ljubljana, Slovenia; and (iii) relate the accumulationand partitioning of Cd, Zn and Pb in seeds and germinatingseedlings to the mechanisms enabling their survival at themetal polluted sites.

2. Materials and methods

2.1. Plant and soil material

Mature (dry) seeding stalks with siliquae were collected in July 2002 and

2005 from approx. 35 plants at three differentially polluted plots (P1eP3) se-

lected at different distances from a lead mine and smelter in Zerjav (N:

46�280, E: 14�510), southern limestone Alps, Slovenia. Sampling site P1

was selected in the nearest vicinity of the smelter’s chimneys (burdened by

dust deposits). The soil is scarcely covered by vegetation, with Minuartia ger-

ardii (Wild.) Hayek as the predominant plant species (Regvar et al., 2006). P2

was selected near the abandoned lead mine entrances (burdened by ore de-

posits) on a 40� slope with patches of vegetation dominated by grasses, espe-

cially by Sesleria caerulea (L.) Ard. Both P1 and P2 were located in a closed

valley, whereas P3 was located on the rim of the valley, about 500 m from the

smelter, with closed vegetation and T. praecox, Erysimum sylvestre Scop.,

Thymus serphyllum (L.) agg. and Sesleria caerulea (L.) Ard. as the predomi-

nant plant species (Regvar et al., 2006). The bedrock of all plots is composed

mainly of Triassic limestone and metalliferous dolomite. The soils are stony

of rendzina type with pH of 6.0e7.4 and various humus layers (2.5e44%

of organic matter) polluted with heavy metals, particularly Pb, Cd and Zn

(Table 1). Ammonium acetate extractable metals represented 9.6e40%,

16.5e44.5% and 8.5e27.6% of total soil Pb, Cd and Zn concentrations, re-

spectively (Vogel-Mikus et al., 2005).

A non-polluted reference site (C) was selected in Zaplana (N: 45�580, E:

14�140) near Vrhnika, Slovenia in a meadow near a local road, harbouring

a calcareous grassland community. The soil is of rendzina type with pH of

7.7 and 8.6% of organic matter (Vogel-Mikus et al., 2005).

Samples for soil analyses were collected from T. praecox rhizosphere in

May 2002. Five plants per plot were carefully dug from the substrate and

the majority of the bulk soil was manually removed from the roots. The sub-

strate closely attached to the root system was combined into two composite

samples per plot for analyses.

2.2. Seed biomass, germination, viability anddormancy tests

The seed batches collected at each of the corresponding plots were mixed,

divided into sub-samples and stored at room temperature for 6e8 weeks be-

fore germination. Seed moisture content was 5% as determined after drying

at 50 �C for 3 days.

Prior to germination, the seeds were surface sterilised in 10% H2O2 for

10 min. Seed germination potential was determined by plating the seeds on

Petri-dishes in deionised water on a filter paper (n ¼ 100, No. of repli-

cates ¼ 4 plot�1). Seeds were germinated in a growth chamber (23 �C;

100% relative humidity; 16 h photoperiod; 200 mmol m�2 s�1) for 8 days

and only the seeds with clearly emerged radicle were considered germinated.

Seed viability was tested using the tetrazolium test (Cottrell, 1947; Inter-

national Seed Testing Association, 1999). After soaking the seeds (n ¼ 100;

No. of replicates ¼ 4 plot�1) in deionised water overnight, the seed coats

were carefully removed and the embryos were soaked in a 1% solution of

2,3,5-triphenyl tetrazolium chloride (Sigma) in the dark at 20 �C. After 4 h

only homogeneously red stained embryos were counted as viable. Seed dor-

mancy was calculated as the difference between seed viability and germination

potential.

2.3. Elemental analyses

2.3.1. XRF analysis of soil samplesTwo composite soil samples, each collected from the rhizosphere of 5

plants per plot, were dried at 30 �C for 1 week, sieved (<2 mm), homogenised

and ground by a mechanical pulveriser (Fritsch, Idar-Oberstein, Germany), us-

ing a tungsten carbide mortar. Pellets were pressed from about 0.5 g of pow-

dered sample material using a pellet die and hydraulic press.

For fluorescence excitation, annular radioisotope sources from Isotope

Products Laboratories (USA) were utilised. Cd-109 (300 MBq, 22 keV) was

used for the measurement of Zn and Pb, and Am-241 (750 MBq, 60 keV)

52 K. Vogel-Mikus et al. / Environmental Pollution 147 (2007) 50e59

Table 1

Cd, Zn and Pb total soil (XRF) and T. praecox seed concentrations (TXRF), seed bioaccumulation factors (BAF), biomass, germination potential, viability and

dormancy from polluted (P1eP3) and non-polluted (C) sites in Slovenia

Polluted site Non-polluted site

P1 P2 P3 C

Total soil Cd (mg g�1) 212 � 0 a 109 � 1 b 77 � 7 c 4 � 1 d

Zn (mg g�1) 3980 � 130 a 3730 � 150 a 1500 � 290 b 237 � 3 c

Pb (mg g�1) 67200 � 950 a 57000 � 800 b 29900 � 100 c 114 � 25 d

Seed Cd (mg g�1)a 1060 � 180 ab 1350 � 160 a 623 � 78 b 18 � 3 c

Zn (mg g�1) 116 � 15 a 121 � 39 a 103 � 23 a 27 � 12 b

Pb (mg g�1) 6.2 � 1.2 a 16.6 � 5.8 a 15.3 � 7.9 a n.d.

Cd BAF 5.0 � 0.9 a 12.3 � 1.5 b 8.1 � 1.1 a 4.4 � 0.5 a

Zn BAF 0.029 � 0.004 a 0.033 � 0.010 a 0.069 � 0.015 ab 0.114 � 0.036 b

Pb BAF 0.0001 � 0.0001 a 0.0003 � 0.0001 a 0.0005 � 0.0003 a n.d.

Biomass (mg seed�1) 0.42 � 0.01 a 0.41 � 0.03 a 0.45 � 0.01 a 0.58 � 0.02 b

Germination potential (%) 48.5 � 1.8 a 49.2 � 1.1 a 76.0 � 2.1 b 91.0 � 4.0 c

Viability (%) 83.2 � 0.3 a 79.5 � 3.5 a 87.0 � 0.4 b 96 � 1.0 c

Dormancy (%) 34.8 � 2.0 a 31.0 � 2.0 a 11.0 � 2.3 b 5.0 � 3.0 b

Different letters in a row indicate statistically significant differences (LSD test, p < 0.05). Total soil Cd, Zn and Pb concentrations (mean � SE; n ¼ 2 composite

samples plot�1); seed Cd, Zn and Pb concentrations and biomass (mean � SE; n ¼ 4 composite samples plot�1); seed germination potential and viability (mean �SE; n ¼ 100 seeds, No. of replicates ¼ 4 plot�1). XRF, X-ray fluorescence; TXRF, total reflection X-ray fluorescence; AAS, atomic absorption spectrometry; SE,

Standard Error; n.d., not detected.a Seed Cd concentrations were determined by AAS.

for the determination of Cd. An X-ray spectrometer, based on a Si (Li) detec-

tor (EG and G ORTEC, USA) with a 25 mm thick Be window was used. The

energy resolution of the spectrometer at count rates below 1000 c s�1 was

175 eV at 5.9 keV. XRF measurements were performed in air and the samples

were irradiated for 3000 s. The measured complex X-ray spectra were ana-

lysed for pure element fluorescence intensities by the AXIL program (van Es-

pen and Janssens, 1993), included in the QXAS (Vekemans et al., 1994)

software package. The fundamental parameter program QAES (Quantitative

Analysis of Environmental Samples) developed in the XRF laboratory at the

Jo�zef Stefan Institute was used for quantification of the measured spectra.

The quantification was validated by NIST SRM 2711 Montana soil as certified

reference material.

2.3.2. TXRF analyses of minerals and AAS analysesof Cd in seeds

The seeds (n ¼ 4 composite samples of 100 mg seeds plot�1) were homo-

genised by grinding in a mortar with liquid N2, soaked overnight in 5 ml of an

ultra-pure mixture of HNO3/HClO4 ¼ 7:1 (Merck) and mineralised by gradual

heating up to 270 �C in an Al block until the acid evaporated. After digestion

the residues were dissolved with 5 ml of 0.2% HNO3. One ml of diluted

(20 mg ml�1) digested sample was spiked with 10 ml of Ga standard solution

of 1000 mg g�1 as an internal standard. Ten microlitres of this solution was de-

posited onto a quartz sample carrier plate and dried under an infrared lamp.

TXRF analysis was assembled at the Jo�zef Stefan Institute (Kump et al.,

1996; Golob et al., 2005; Vogel-Mikus et al., 2006). A focused X-ray beam

from a fine focus X-ray tube (Seifert, Germany) with a Mo anode operating

at 40 kV and 30 mA and monochromatised using a carbon-tungsten (C/W)

multilayer to the energy of the Mo Ka line (17.4 keV) was used for excitation.

The sample deposit on the optically flat quartz carrier plate was irradiated for

300 s in air at an incident angle lower than the total reflection critical angle of

the substrate material (�1.8 mrad). X ray spectra were detected by a Si (Li)

detector (EG and G ORTEC) with a 25 mm thick Be window. The energy res-

olution of the spectrometer at count rates below 1000 c s�1 was 140 eV at

5.9 keV. The quantification procedure was based on Ga as an internal standard

(Kump et al., 1996; Golob et al., 2005; Vogel-Mikus et al., 2006). Analysis of

Cd by TXRF was possible by measurement of the L-series X-rays (3.13 keV),

which in our case were subjected to interference by the much more intense

lines of potassium K-series X-rays (3.34 keV). Therefore Cd was analysed

by AAS using an Aanalyst 100 instrument (Perkin Elmer) and only these

data were included in the results. The quantification was validated by BCR

CRM 129 Hay powder as certified reference material.

2.3.3. Elemental localisation within seeds and

germinating seedlings by micro-PIXESeeds were cut in half under a stereomicroscope using a sharp razor blade.

The seed halves and seedlings (germinated in deionised water for 8 days) were

rapidly frozen in propane cooled by liquid N2 in order to prevent elemental

losses or redistribution during freeze drying (Schneider et al., 1999; 2002).

Frozen plant material was carefully transferred to a freeze drier (Alpha 2-4,

Christ) via a cryo-transfer-assembly cooled by liquid N2 and freeze-dried at

�50 �C and 0.04 mbar for 3 days. The seed samples were then individually

mounted between two thin layers of Pioloform foil (1 g Pioloform diluted in

75 ml chloroform) on a plastic frame.

Micro-PIXE analysis was performed at the Microanalytical centre of Jo�zef

Stefan Institute (MIC), Ljubljana, Slovenia. Dissected seeds and germinating

seedlings were raster scanned with a 1 � 1 mm2 proton micro-beam with an en-

ergy of 2 or 3 MeV and a current of approx. 50 pA, over areas from approx.

1750 � 1750 mm2 down to 150 � 150 mm2. The samples were attached to a 5-

axes motorised vacuum goniometer alongside 125, 25 and 12.5 mm Cu mesh

and a quartz visualiser, which were used to optimise the size and profile of the

proton beam. To enable the positioning of the sample in the focal plane of the

quadrupole triplet lens system, a high magnification optical microscope with

a depth of field of 100 mm was applied. The irradiation of the sample with the

proton beam induced the emission of characteristic X-rays. The X-ray energy al-

lowed identification of the elements in the samples irrespective of their chemical

bonding, whereas the absolute element concentration in mg g�1 was calculated

from the number of induced X-rays, the sample area density and the total charge

(Johansson and Campbell, 1988). A rough estimate of the thickness of the freeze-

dried seed or seedling was approx. 1 mm on average. The range of 2 or 3 MeV

protons in pure cellulose (C6H11O5), chosen as the matrix of the measured sam-

ples, was 7.5 mg cm�2. Assuming the density of cellulose to be 0.8 g cm�3, the

proton range was estimated to be approx. 80 mm. The X-ray spectra were de-

tected with a high purity Ge detector (resolution 150 eV at 5.9 keV) with

a 25 mm thick Be window, placed in a vacuum at 45� relative to the beam direc-

tion at a working distance of 30 mm. No additional absorbers were used. The

lowest energy in the spectrum was at approx. 1.2 keV. Beam current normalisa-

tion was carried out with a graphite rotating vane covered with a 3 mm thick gold

foil, which intersected the proton beam approx. 20 times s�1. The spectrum of

back-scattered (RBS) protons from the gold foil, measured with the particle de-

tector, served for estimation of the number of protons irradiating the sample dur-

ing the measurement. A particle detector in the geometry of scanning

transmission ion microscopy (STIM), positioned at zero degrees, measured the

thickness of the Pioloform foil, and was used in estimating its corresponding

X-ray attenuation.

53K. Vogel-Mikus et al. / Environmental Pollution 147 (2007) 50e59

Fig. 1. Morphological structures of T. praecox seed; light microscopy coupled with UV light source. (a) Whole seed, (b) seed coatdcotyledon area. C, cotyledons;

SC, seed coat; T, testa; A, aleurone with hyaline layer; R, radicle; EC, epidermis of cotyledons; MC, mesophyll of cotyledons.

Seed morphology structures of T. praecox were determined using light mi-

croscopy in accordance with (Vaughan and Whitehouse, 1971) and Garnock-

Jones (1991) (Fig. 1). The X-ray spectra corresponding to distinct morphological

structures of seeds or seedlings (Tables 2e4) were extracted from the encircled

regions on the basis of morphology of the structures revealed by light microscopy

and in seeds also on the basis of micro-PIXE qualitative elemental maps. The

complex X-ray spectra were deconvoluted by GUPIX software (Maxwell et al.,

1989, 1995; Campbell et al., 2000). The elemental concentrations corresponding

to different morphological structures were calculated as average concentrations

of two measured representative samples, taking into account the uncertainties

Table 2

Micro-PIXE localisation of elements within seed structures of T. praecox, with TXRF (AAS for Cd) bulk sample analysis

Micro-PIXE: Scanned area 1750 � 1750 mm2

TXRF Bulk sample

El. mg g�1 LOD mg g�1 Error (%) LOD mg g�1 Error (%) LOD mg g�1 Error (%) LOD mg g�1 Error (%) LOD

P 3491 � 823 503 5870 0.6 30.9 1077 4.1 55.9 11813 0.6 57.3 10099 2.1 141.1

S 6633 � 1070 280 5504 0.2 6.5 4155 0.5 12.4 11256 0.3 12.4 9818 1.2 32.7

K 4560 � 530 60.2 3707 0.1 3.0 4712 0.2 3.6 4824 0.2 5.6 5139 1.2 11.7

Ca 5900 � 727 39.6 3586 0.2 7.1 5736 0.2 11.3 5717 0.2 9.0 2823 1.3 16.3

Cda 623 � 77.5 n.d. 374 10.5 34.6 157 31.8 41.6 575 11.1 41.1 549 36.0 161.7

Zn 103 � 22.9 3.4 72.7 1.3 0.2 32.6 3.6 0.6 110 1.6 0.4 207 3.7 1.9

Fe 109 � 32.4 6.4 62.5 1.1 0.6 50.0 2.5 1.4 87.0 1.4 1.0 126 3.3 2.5

Mn 11.3 � 1.19 8.2 16.0 2.9 0.5 25.0 3.6 1.1 15.5 5.5 1.0 13.8 19.1 2.4

Cu 13.9 � 1.63 3.7 15.3 3.0 0.4 12.6 6.1 0.7 18.3 4.5 0.7 34.3 9.0 2.6

Pb 15.3 � 7.94 6.7 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

TXRF of bulk sample (mean � SE; n ¼ 4 composite samples). Figures represent a longitudinally cut seed scanned by the 3 MeV proton-beam. The X-ray spectra

corresponding to the different morphological seed structures were extracted from the encircled regions and analysed using GUPIX software. The concentrations of

elements corresponding to different morphological seed structures represent an average concentration of two representative measured samples, considering the

error of the measurement. LOD, minimum limit of detection of the measurements (mg g�1); TXRF, total reflection X-ray fluorescence; micro-PIXE, micro-proton

induced X-ray emission; n.d., not detected.a Cd analysis of bulk samples was performed using AAS; micro-PIXE Cd concentrations were calculated using CdeK line.

54 K. Vogel-Mikus et al. / Environmental Pollution 147 (2007) 50e59

Table 3

A detailed tissue localisation micro-PIXE scan (150 � 150 mm2) of seed tissues (testa, aleurone, epidermis, mesophyll) of longitudinally cut seed of T. praecox

Micro-PIXE: Scanned area 150 � 150 mm2

Seed coat Cotyledon

El. mg g�1 Error (%) LOD mg g�1 Error (%) LOD mg g�1 Error (%) LOD mg g�1 Error (%) LOD

P 1350 2.5 55.9 5146 0.8 60.3 16114 0.3 55.9 21786 0.3 61.7

S 6321 0.2 12.0 13988 0.3 12.7 15047 0.1 11.3 15643 0.1 13.1

K 14992 0.1 4.2 6002 0.2 4.2 6400 0.1 12.3 6531 0.1 8.2

Ca 9049 0.1 26.0 11294 0.2 11.8 8284 0.1 12.9 8377 0.1 13.0

Cd 47.8 64.6 33.1 444 9.9 32.7 1797 2.6 23.3 784 5.6 19.5

Zn 33.1 2.5 0.8 59.4 1.7 0.5 236 0.6 0.6 191 0.8 0.8

Fe 66.2 1.9 2.2 22.0 3.8 1.7 19.0 4.3 1.4 168.4 0.9 1.6

Mn 42.2 2.1 1.5 24.1 3.3 1.4 46.7 1.5 1.1 30.8 2.6 1.2

Cu 17.8 3.1 0.7 13.5 3.8 0.7 6.8 6.3 0.7 11.8 5.3 0.7

Pb 6.7 42.2 4.0 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Figures represent part of a longitudinally cut seed scanned by the 3 MeV proton-beam. The X-ray spectra corresponding to the different morphological seed struc-

tures were extracted from the encircled regions and analysed using GUPIX software. The concentrations of elements corresponding to different morphological seed

structures represent an average concentration of two measured representative samples, considering the error of the measurement. LOD, minimum limit of detection

of the measurements (mg g�1); micro-PIXE, micro-proton induced X-ray emission; n.d., not detected.

Table 4

Average concentrations of elements within few days old germinating seedling of T. praecox as estimated using micro-PIXE analysis of two representative seedlings

Micro-PIXE: Scanned area: 1000 � 1000 mm2

El. mg g�1 Error (%) LOD mg g�1 Error (%) LOD mg g�1 Error (%) LOD

P 14764 0.16 18.6 19141 0.10 17.9 15093 0.21 14.6

S 26678 0.12 6.9 28474 0.04 6.9 17964 0.19 6.9

K 15037 0.13 24.7 15849 0.03 32.3 20153 0.20 10.9

Ca 6598 0.14 32.6 5457 0.05 38.3 2436 0.21 45.1

Cd 4079 2.02 70.8 4421 2.17 103.6 1199 4.92 85.6

Zn 330.9 0.44 0.6 309.0 0.50 0.6 168.0 0.72 0.6

Fe 112.9 0.56 1.2 146.7 0.54 1.6 133.3 0.65 1.3

Mn 32.5 1.22 1.0 34.7 1.44 1.3 15.7 2.82 1.3

Cu 18.2 2.12 0.6 20.8 2.36 0.6 33.8 1.63 0.5

Pb n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Figures represent a germinating seedling scanned by the 2 MeV proton-beam. The X-ray spectra corresponding to the whole seedling, radicle, hypocotyl and cotyledons

were extracted from the encircled regions and analysed using GUPIX software. The cotyledons ruptured during specimen preparation, thus the rupture is not encircled.

The concentrations of elements corresponding to different morphological seed structures represent an average concentration of two measured samples, considering the

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

55K. Vogel-Mikus et al. / Environmental Pollution 147 (2007) 50e59

of the measurements. The quantification was validated by BCR-CRM 101 Spruce

needles as certified reference material. A pellet was prepared from approx. 0.5 g

of powdered sample material using a pellet die and hydraulic press and measured

by micro PIXE as described above. The samples were thick in both cases there-

fore the validation was acceptable. The measurements by micro-PIXE were also

compared to the bulk seed analysis (using TXRF and AAS for Cd).

2.4. Data analysis

The differences in total soil and seed metal concentrations, seed biomass, ger-

mination potential, viability and dormancy among the plots (P1eP3 and C) were

analysed by the ANOVA LSD test (p < 0.05). The data on viability (%) and ger-

mination potential (%) were transformed ( y ¼ arc sin x) prior to statistical anal-

ysis. Bioaccumulation factors were calculated as the ratios between average seed

metal and average total soil metal concentrations according to Baker et al. (1994).

Pearson’s correlation coefficients (r) were calculated to reveal correlations be-

tween soil and seed metal concentrations. The effects of seed Cd, Zn and Pb con-

centrations on the seed biomass, germination potential, viability and dormancy

were tested using multiple regression analysis (Statistica Statsoft� software).

3. Results

3.1. The morphological structures of T. praecox seedsand germinating seedlings

The seeds of T. praecox are campylotropous, comprised ofa seed coat and an embryo with cotyledons filling the majorityof the seed (Fig. 1, Table 2). The approx. 70e80 mm thick seedcoat is comprised of three-layered testa (including epidermis,palisade and a pigment layer) and an endosperm remnantdthealeurone (intimately connected with the testa)din line withthe description of Brassicaceae seed morphology (Vaughanand Whitehouse, 1971; Garnock-Jones, 1991). The storage pa-renchyma cells of the cotyledons were ensheathed by a 30e40 mm thick dermal cell layer (epidermis) (Fig. 1b, Table 3)consistent with the description of Patrick and Offler (2001).After germination the hypocotyl elongated, the cotyledons ex-panded and resembled leaves, whereas the epicotyl did notshow significant development at an early germinating seedlingstage (Table 4).

3.2. Seed Cd, Zn and Pb accumulation

Total soil elemental analysis by XRF revealed high heavymetal concentrations at the polluted site with maximum con-centrations of 212 mg g�1 Cd, 3980 mg g�1 Zn and67,200 mg g�1 Pb (Table 1). Seed concentrations determinedby TXRF were 17 mg g�1 for Pb and 121 mg g�1 for Zn withseed bioaccumulation factors (BAF) << 1 (Table 1). In con-trast, hyperaccumulation of Cd with up to 1350 mg g�1 de-termined by AAS was accompanied by high BAF (up to12) (Table 1). Seed Cd concentrations positively correlatedwith total soil Cd concentrations (r ¼ 0.585, p < 0.05),while no such correlation was observed for Zn and Pb. Sig-nificantly lower soil and seed metal concentrations werefound at the non-polluted reference site with seed Pb con-centrations under detection limits. Still, a relatively highBAF for Cd (of 4) was observed at the non-polluted site(Table 1).

3.3. Seed biomass, viability, germination potential anddormancy

Seeds from the polluted site showed significantly lowerseed biomass, germination potential and viability when com-pared to the non-polluted reference sites that were accompa-nied with increased seed dormancy (Table 1). Negativeeffects of the seed Cd concentrations on seed biomass(b ¼ �0.734; p < 0.01), seed germination potential(b ¼ �0.904; p < 0.01) and seed viability (b ¼ �0.752;p < 0.05), and a positive effect on seed dormancy (b ¼ 0.79;p < 0.05) were revealed by multiple regression analysis.

3.4. Micro-PIXE elemental analysis of seeds

The elemental concentrations obtained by micro-PIXEanalysis of whole T. praecox seed cross sections were in gen-eral within the range of concentrations found by the bulkTXRF (AAS for Cd) analyses, with the exception of the con-centrations of Pb, which were under the micro-PIXE detectionlimit (Table 2).

Most elements were preferentially stored in the embryonictissues, but high concentrations of Ca and Mn were also foundin the seed coat. Elemental distribution within the embryonicaxis revealed an even distribution of Cd between cotyledons(575 mg g�1) and radicle (549 mg g�1), whereas Zn(207 mg g�1), Fe (126 mg g�1) and Cu (34 mg g�1) were pref-erentially localised in the radicle (Table 2).

A more detailed localisation of the elements within theseeds by micro-beam scanning (150 � 150 mm2) of particulartissues (testa, aleurone, epidermis of cotyledons and meso-phyll) confirmed the preferential localisation of Ca in the aleu-rone and Mn in the testa (Table 3).

Within the cotyledonal tissues (epidermis/mesophyll) thehighest concentrations of Cd, Zn and Mn were observed inthe epidermal cells, whereas P and Fe were accumulated inthe mesophyll (Table 3).

3.5. Micro-PIXE elemental analysis of germinatedseedlings

Seedlings (germinated in deionised water for 8 days) werescanned by the micro-beam to reveal the distribution of ele-ments at the organ level of the seedlings. The elemental con-centrations in cotyledons and hypocotyl were comparable(Table 4) and are therefore not commented on separately.Most of S, Cd, Zn and Mn were found in the cotyledons/hypo-cotyl and most Cu was detected in the radicle of the seedlings.The concentrations of Cd (up to 4079 mg g�1 dry weight)found in the cotyledons confirmed hyperaccumulation. Thecontents of P, Ca, Fe and Cu in cotyledons of seedlings andseeds were comparable, whereas the concentrations of S, K,Cd, Zn and Mn found in seedlings were substantially increased(Tables 2 and 4), presumably as a consequence of the in-creased metal levels in the parent seeds.

56 K. Vogel-Mikus et al. / Environmental Pollution 147 (2007) 50e59

4. Discussion

4.1. Accumulation of Cd, Zn and Pb in T. praecox seeds

Field collected T. praecox plants were shown to accumulateup to 14,590 mg g�1 Zn, 5960 mg g�1 Cd and 3500 mg g�1 Pbin shoot dry weight (DW), mainly depending on total soilmetal concentrations, pH and organic matter levels (Vogel-Mikus et al., 2005). T. praecox rhizosphere soil concentrationsmeasured in this experiment are consistent with the rhizo-sphere contents found in Vogel-Mikus et al., 2005 that are,however, significantly higher than the average metal concen-trations measured according to DIN ISO 10381-1 (1996) onthe same plots (Regvar et al., 2006). These results indicatethat hyperaccumulating T. praecox generates hot spots ofmetals in its rhizosphere as previously observed for T. caeru-lescens (Perronet et al., 2000).

Field collected seeds contained up to 17 mg g�1 of Pb and121 mg g�1 of Zn with seed bioaccumulation factors(BAF) << 1, revealing the restriction of Pb and Zn uptakeinto the seeds, that may be mainly attributed to the metal de-toxification mechanisms in vegetative plant parts, since mostof Pb usually remains bound to the cell walls and vacuolesof the roots (Tung and Temple, 1996; Seregin and Ivanov,2001), whereas most of Zn is sequestered in the vacuoles ofleaf epidermal cells in Zn hyperaccumulating Thlaspi species(Frey et al., 2000), thus lowering symplastic metal concentra-tions (Lasat et al., 1998; Kupper et al., 1999; Wojcik at al.,2005).

In contrast, seed hyperaccumulation of Cd (1351 mg g�1)with seed BAF of up to 12 was observed in T. praecox seedsthat may be connected to a less effective Cd compartmentationin the mother plant, resulting in higher Cd mobility throughoutthe plant tissues and possibly enhanced phloem loading duringseed development, as previously observed in non-tolerant mus-tard, sunflower, wheat, peanut and potato plants (Gaur andGupta, 1994; Aggarwal et al., 1995; Popelka et al., 1996; Cak-mak et al., 2000; Reid et al., 2003). In the sieve tubes the metalsshould be transported in complex form, due to their physiochem-ical properties and prevalent phloem sap pH ranging between 7and 8 (Kruger et al., 2002; Graham and Strangoulis, 2003).However, data concerning the chemical form and transportmechanisms of Cd movement, especially in phloem, are notavailable. Nicotianamine was proposed to bind metal micronu-trients such as, Cu, Mn and Zn in phloem (Schmidke et al., 1999;Kruger et al., 2002), whereas Cd has greater affinity for formingcomplexes with sulphydryl groups (e.g. proteins) and Cl, whichare usually abundant in phloem (Reid et al., 2003).

To our knowledge this is the first report on hyperaccumula-tion of Cd in seeds. The observed results indicate that the up-take mechanism(s) of Cd in seeds differ substantially fromthose of Pb and Zn.

4.2. The effects of metal accumulation on seed fitness

Enhanced exposure to metals affects their uptake into seedsand thus burdens the next generation, frequently affecting seed

biomass or resulting in decreased seed germination potential(Ernst and Nelissen, 2000; Ernst et al., 2000). The significantlyreduced T. praecox seed biomass accompanied by reducedgermination and increased dormancy of the seeds collectedat the most polluted sites was attributed mainly to the en-hanced Cd accumulation within the seeds. However, the via-bility of seeds containing the highest Cd concentrations wasreduced maximally by 20%, thus pointing to an extremelyhigh tolerance of T. praecox embryos to accumulated Cd. Asignificant positive correlation of the seed Cd concentrationswith increased seed dormancy was observed, possibly contrib-uting to plant fitness as a mechanism of survival strategy at thepolluted sites, as it increases the likelihood of the offspring’ssurvival in years less suitable for seed germination (Anderssonand Mildberg, 1998). However, the effects of other environ-mental conditions during seed maturation (Fenner, 1991; An-dersson and Mildberg, 1998) may also have contributed to theincreased seed dormancy observed.

4.3. Localisation of elements within T. praecox seeds andseedlings

The localisation of Ca in the endosperm is in accordance withthe patterns observed in Ni hyperaccumulating Stackhousiatryonii and metal tolerant Biscutella laevigata (Mesjasz-Przy-by1owicz et al., 2001; Bhatia et al., 2003). The release of Cafrom the apoplast and/or intracellular stores of the aleurone cellsis a prerequisite for the release of storage carbohydrates for thedeveloping embryo (Lovegrove and Hooley, 2000).

The localisation of P within the embryonic tissues of T. prae-cox seeds may be connected to the deposition of phytic acid andproteins in protein storage vacuoles, providing nutrients to thegerminating embryo (Jiang et al., 2001), whereas the high S con-tents found in the aleurone, epidermis and mesophyll may beconnected to the synthesis of S-containing storage proteins(Shewry et al., 1995) and glucosinolates (Fahey et al., 2001),known to accumulate in the reproductive tissues of Brassicaceaeduring seed development as a consequence of the transport fromleaves and de novo synthesis in siliquae walls (Du and Halkier,1998; Chen et al., 2001).

The preferential localisation of Zn in Biscutella laevigata isthe endosperm where the highest metal concentrations werefound (Mesjasz-Przyby1owicz et al., 2001). The same effectwas not observed in T. praecox. In contrast, the preferential lo-calisation of Cd in the embryonic axis of T. praecox is the firstknown report of any metal localisation in a hyperaccumulatingplant. The existence of symplastic connections of the outer in-tegument with the funicular phloem, demonstrated in Arabidop-sis thaliana, another Brassicaceae species, during the globularand heart stage embryonic development (Stadler et al., 2005),may have at least partially contributed to the observed result.The lack of symplastic connections between the seed coat/endo-sperm and endosperm/embryo observed in mature A. thalianaseeds (Stadler et al., 2005), indicates the possibility of an apo-plastic movement of Cd presumably accompanied by a car-rier-mediated transport, which enables Cd transport to theembryonic tissues in the later-stage development of T. praecox

57K. Vogel-Mikus et al. / Environmental Pollution 147 (2007) 50e59

embryos, similar to the high affinity Cd transporters found in T.caerulescens (Lombi et al., 2001).

Some metals (e.g. Zn, Fe and Cu) showed preferential lo-calisation in the radicle of T. praecox embryos, whereas nopreferential distribution on the organ level (cotyledons/radicle)was found for Cd. Within the cotyledons, however, most of theCd, Zn and Mn were found in the epidermal tissues, presum-ably bound to the cell walls and compartmented into the vac-uoles (Vazquez et al., 1994; Cosio et al., 2004; Ma et al.,2005). The compartmentation of Cd and Zn away from the an-cestral tissues of the photosynthetic mesophyll may be attrib-uted to the deleterious effects of high Cd and Znconcentrations on the photosynthetic apparatus (Prasad andStrza1ka, 1999) and may therefore be viewed as a tolerancemechanism of T. praecox embryos to Zn and Cd, similar tothat observed in the leaves of T. caerulescens (Vazquezet al., 1994; Wojcik et al., 2005).

Seedlings of T. praecox (germinated in deionised water foreight days) showed extremely high Cd concentration (up to4421 mg g�1) that were partially attributed to their epidermaland sub-epidermal localisation (as a consequence of the esti-mated micro-beam range; see Section 2 for details), and par-tially to the high Cd levels in the originating seeds. Incontrast to the even distribution of Cd in the seed embryonicaxis, the preferential localisation of Cd in the seedlings wasthe cotyledons, where most of Zn and Mn were also found.Typical shoot localisation of Cd and Zn was also found in fieldcollected T. praecox plants (Vogel-Mikus et al., 2005). Cu, onthe other hand, was preferentially stored in the radicles of thegerminated seedlings, resembling the distribution already seenin the embryos.

5. Conclusions

This is the first report on Cd hyperaccumulation in theseeds of a metal hyperaccumulating plant. The mechanismsenabling survival of the embryos and germinating seedlingsof T. praecox at highly heavy metal polluted sites involve (i)the restriction of Pb and Zn uptake into the seeds, (ii) the ex-istence of (an) efficient tolerance mechanism(s) to Cd in em-bryos and germinated seedlings, (iii) the partitioning of Cdin the epidermis of cotyledons, away from the ancestral photo-synthetic tissues of the expanded cotyledons, and (iv) in-creased seed dormancy as a strategy of an increasedlikelihood of the offspring’s survival in years less suitablefor seed germination. Further research will be needed to revealthe physiology of Cd tolerance in T. praecox embryos at thecellular and molecular levels.

Acknowledgements

The authors are indebted to Dr Stefan Scheloske for sharinghis knowledge and his hints on technical details and to Assoc.Professor Dr Damjana Drobne for the access to the AAS forCd analysis. The work was supported by the following pro-jects: MSZS L1-5146-0481 Tolerance of Organisms inStressed Ecosystems and Potential for Remediation with the

sponsorship of Rudnik Me�zica MPI, �Crna na Koroskem andMobitel d.d.; MSZS PO-0212 Biology of Plants research pro-gram and EU COST 859 entitled ‘‘Phytotechnologies to Pro-mote Sustainable Land Use and Improve Food Safety’’.

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