Changes in elemental uptake and arbuscular mycorrhizal colonisation during the life cycle of Thlaspi praecox Wulfen

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Chemosphere 69 (2007) 1602–1609 www.elsevier.com/locate/chemosphere

Changes in elemental uptake and arbuscular mycorrhizal colonisation during the life cycle of Thlaspi praecox Wulfen Paula Pongrac a, Katarina Vogel-Mikusˇ a, Peter Kump b, Marijan Necˇemer b, Roser Tolra` c, Charlotte Poschenrieder c, Juan Barcelo´ c, Marjana Regvar a,* a

c

Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecˇna pot 111, 1000 SI-Ljubljana, Slovenia b Institute Jozef Stefan, Jamova 37, 1000 SI-Ljubljana, Slovenia Laboratorio de Fisiologı´a Vegetal, Facultad de Ciencias, Universidad Auto´noma de Barcelona, E-08193 Bellaterra, Spain Received 19 January 2007; received in revised form 16 May 2007; accepted 16 May 2007 Available online 5 July 2007

Abstract Elemental uptake and arbuscular mycorrhizal (AM) colonisation were studied during the life cycle of field collected Cd/Zn hyperaccumulating Thlaspi praecox (Brassicaceae). Plant biomass and tissue concentrations of Cd, Pb, Zn, Fe and Ni were found to vary during development, while no variation in P, K, Ca, Mn and Cu tissue concentrations were observed. The lowest Cd bioaccumulation in rosette leaves (BAFRL) observed during seeding was partially attributed to lower translocation from roots to rosette leaves and partially to high translocation to stalks, indicating a high Cd mobility to reproductive tissues, in line with our previous studies. The highest intensity of AM colonisation (M%) was observed in the flowering phase and was accompanied by increased root Cd, Zn, Pb and Fe contents. In addition, a positive correlation between AM colonisation and Fe contents in rosette leaves was found. The results indicate developmental dependence of AM formation, accompanied by selective changes in nutrient acquisition in T. praecox that are related to increased plant needs, and the protective role of AM colonisation on metal polluted sites during the reproductive period. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Development; Elemental uptake; Mycorrhiza; Thlaspi praecox

1. Introduction Numerous factors influence elemental uptake by plants, including soil element concentrations, soil characteristics (pH, organic matter, temperature, etc.) and plant genotype (Marschner, 1995). Plant nutrient requirements considerably change during the life cycle, the reproductive period being the most demanding phase (Obeso, 2002). Changes in uptake, mobility and transport capacity of nutrients reflect such changes in demand. Plants have the ability to alter elemental uptake to meet their requirements, either by morphological or biochemical changes in the root system, such as acidification and release of root exudates (Marschner and Ro¨mheld, 1996). In addition, arbuscular *

Corresponding author. Tel.: +386 01 4233388; fax: +386 01 2573390. E-mail address: [email protected] (M. Regvar).

0045-6535/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2007.05.046

mycorrhizal (AM) fungi represent a notable component providing a direct link between soil and roots (Chen et al., 2005; Marques et al., 2006; Trotta et al., 2006). It has been shown that Thlaspi praecox, from generally believed non-mycorrhizal Brassicaceae, forms AM symbiosis under field conditions (Regvar et al., 2003). A T. praecox population from metal polluted site in Slovenia is able to hyperaccumulate up to 1.5% Zn and 0.6% Cd without showing any metal toxicity symptoms (Vogel-Mikusˇ et al., 2005). In a greenhouse experiment, AM colonisation of T. praecox was only observed during the reproductive period of the plants and this colonisation contributed to changes in elemental uptake (Vogel-Mikusˇ et al., 2006). Similarly, the field grown pseudometallophyte, another Brassicaceae, Biscutella laevigata formed AM in the flowering stage and prior to seeding (Orłowska et al., 2002), suggesting that plant development affects AM formation.

P. Pongrac et al. / Chemosphere 69 (2007) 1602–1609

AM fungi are known to alleviate biotic and abiotic stress in plants through their contribution to enhanced plant nutritional and water status (Smith and Read, 1997). Inoculation with mycorrhizal fungi either enhances (e.g., Weissenhorn et al., 1995; Turnau and Mesjasz-Przybylowicz, 2003; Citterio et al., 2005; Chen et al., 2006; Marques et al., 2006) or decreases (Gildon and Tinker, 1983; Leyval et al., 1997; Hildebrandt et al., 1999; Andrade et al., 2004) plant metal uptake in cases of toxic soil metal concentrations. Being an integral part of terrestrial ecosystems, mycorrhizae contribute to their stability and productivity (van der Heijden et al., 1998) but the complexity of the effects of AM fungi on elemental uptake in plants makes the elucidation of general mycorrhizal effects extremely difficult. Thus, the present study was designed to elucidate the dynamics of elemental uptake and AM colonisation in relation to the life cycle of T. praecox plants growing on metal polluted soil under field conditions. 2. Materials and methods 2.1. Study site and sample collection Plants were collected from April to September 2006 in Zˇerjav, Northern Slovenia on a plot already described as plot P3 by Regvar et al. (2006). The plot is located on the rim of the valley, about 500 m from the main source of pollution, with closed vegetation and Sesleria caerulea L. and Thlaspi praecox as dominant plant species. The location is heavily polluted with Cd, Zn and Pb, a result of centuries of lead mining and smelting activities (Regvar et al., 2006). Five developmental phases during the plants’ life cycle were distinguished from April to September: (1) vegetative phase (VP) in the rosette stage; (2) flower induction phase (FI) with flowering stalks emerging from the rosette; (3) flowering phase (FP) with opened flowers collected in May; (4) seeding phase (SP) with yellowing silique collected in June and (5) senescence phase (SC) with dried, empty silique collected in September. Plants were sampled randomly and soil samples were taken from the rhizosphere of each plant. Only the soil closely attached to the root system was analysed. Five plants per plot were sampled and analysed in each phase, with the exception of the vegetative and flower induction phases where in total nine and six plants, respectively, had to be pooled into three composite samples per plot due to a small plant size. 2.2. Soil analyses Total soil Cd, Zn and Pb concentrations were determined by X-ray fluorescence spectrometry (XRF) as described in Vogel-Mikusˇ et al. (2006), concentration of available soil Cd, Zn and Pb fractions were analysed after extraction in ammonium acetate (NH4Ac) (Baker et al., 1994) and soil pH was determined in the water fraction after diluting 1 g of dried soil in 20 ml of MiliQ water ¨ hlinger, 1995). The and shaking vigorously for 2 h (O

1603

organic matter content was determined after Kandeler (1995) and plant available phosphorus in 5 g of soil sam¨ NORM L 1087 (1993). ples following the protocol of O 2.3. Plant analyses After a careful wash, the rosette leaves, roots and stalks (the latter only in the flowering and the seeding phase) of collected plants were separated, oven-dried (60 °C) for 48 h and weighed. Plant element concentrations were determined using total reflection X-ray fluorescence spectroscopy (TXRF) in 100 mg of dried plant material after grinding and homogenisation. 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 atomic absorption spectrometer (AAS) using an Aanalyst 100 instrument (Perkin–Elmer) and only these data were included in the results. Standard reference materials NIST SRM 2711, Montana Soil and BCR-CRM-101, spruce needles with certified concentrations of minerals were used for quality assurance of elemental analyses (Vogel-Mikusˇ et al., 2006). Plant elemental contents were calculated by multiplying concentrations by plant dry biomass. AM colonisation was determined by vital staining of fresh root fragments using the nitro blue tetrazolium chloride (NBT)-succinate method with deposition of dark bluepurple formazan as the viability indicator and counterstaining with acid fuchsin, clearly revealing both vital (dark blue-purple depositions) and non-vital (pink) intraradical fungal structures (Schaffer and Peterson, 1993). Roots were mounted in lactoglycerol and examined immediately. The portions of vital and total AM colonisation were estimated on 30 root fragments per plant specimen, at each developmental stage, according to Trouvelot et al. (1986). Mycorrhizal frequency (F% – frequency of root fragments with fungus) and mycorrhizal intensity (M% – global intensity of mycorrhizal colonisation in the root system) were determined. Total colonisation represents the fractions of vital and non-vital colonisation observed in root fragments, whereas, vital colonisation represents only the colonisation in which formazan deposition was observed. 2.4. Statistical analyses Bioaccumulation factors of rosette leaves (BAFRL = Crosette leaves/Ctotal soil) and stalks (BAFST = Cstalks/Ctotal soil) and translocation factors of rosette leaves (TFRL= Crosette leaves/Croots) and stalks (TFST = Cstalks/Crosette leaves) were calculated to quantify the accumulation and translocation of elements into and within the plant. To test the overall effect of the developmental stage on the studied parameters, the non-parametric Kruskal–Wallis test was applied and when significant, Mann–Whitney U test was used to determine the significance of differences between developmental phases at p < 0.05 (shown are means ±

P. Pongrac et al. / Chemosphere 69 (2007) 1602–1609

standard error). Pearson’s correlation coefficient (r) was used when calculating correlations between mycorrhizal colonisation parameters and between mycorrhizal and plant parameters. Statistical tests were performed using Statistica StatsoftÒ (version 6.0) software. 3. Results 3.1. Soil properties

a

roots

c 0. 6

b b

a b

a VP

FI

b FP

b c

F%

c bc

60

ab b

40

b

b

a

20 0 VP

FI

FP

a

a

SP

SC

Developmental stage

c

tota l 10 8

M%

vital

b b

6

b c

4 2

a

b

b

a VP

Concentration of availalbe fraction (µg g-1)

SC

vita l

total 80

0 100000

SP

Developmental stage

3.2. Biomass and elemental concentrations in T. praecox As expected, the biomass of roots and rosette leaves progressively increased during the vegetative and the flower induction phases, reaching a maximum in the flowering and the seeding phase, with a decrease in the senescence phase (Fig. 2a). The lowest concentrations of Cd, Zn, Pb, Fe and Ni in the roots and of Cd, Pb and Fe in the rosette leaves were observed in the seeding phase (Table 1). In contrast, the highest concentrations of Cd and Fe in rosette leaves were found in the vegetative and of Ni in the senescence phase,

c

0. 3

0. 0

The rhizosphere soil of T. praecox was highly enriched in total Cd, Zn and Pb (84.3 ± 7.6, 1249 ± 141 and 16 225 ± 1865 mg kg 1, respectively). It contained 1.9 ± 0.3 mg kg 1 of available P, 18.4 ± 2.3% of organic matter and was of neutral pH (6.9 ± 0.1). Available metal fractions represented on average 28% of total soil Cd, 6% of total soil Zn and 10% of total soil Pb concentrations. Available soil Pb and P concentrations significantly increased during the growing season (Fig. 1). Similar trends were observed in available soil Cd and Zn, whereas, total soil concentrations did not differ significantly.

rosette leaves

0. 9

Dry biomass (g)

1604

FI

FP

a a

SP

SC

Developmental stage

1000 Pb Zn

Fig. 2. Plant biomass and mycorrhizal colonisation during the life cycle of field collected T. praecox: (a) plant biomass (g dry weight basis), (b) total and vital mycorrhizal frequency (F%), (c) total and vital mycorrhizal intensity (M%) (mean ± SE, n = 3–5); VP – vegetative phase, FI – flower induction phase, FP – flowering phase, SP – seeding phase, SC – senescence phase. Different letters beside the boxes indicate statistical differences, where significant (Mann–Whitney U test, p < 0.05).

Cd P 10

0 VP

FI

FP

SP

whereas, the concentrations of P, K, Ca, Mn and Cu did not differ significantly during the plants’ life cycle (Table 1). A negative correlation between available soil Cd concentrations and Fe concentrations in rosette leaves and positive correlations between Cd and Fe concentrations in both the roots and rosette leaves were found (Table 2).

SC

Developmental stage Fig. 1. Concentrations of available soil Pb, Cd, Zn, and P fractions (lg g 1) during the life cycle of field collected T. praecox (mean ± SE, n = 3–5); VP – vegetative phase, FI – flower induction phase, FP – flowering phase, SP – seeding phase, SC – senescence phase.

3.3. Bioaccumulation and translocation of cadmium The highest bioaccumulation of Cd from soil to rosette leaves, expressed by the bioaccumulation factor (BAFRL = Crosette leaves/Ctotal soil), was observed in the vegetative and

P. Pongrac et al. / Chemosphere 69 (2007) 1602–1609

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Table 1 Plant elemental concentrations (lg g 1) in roots, rosette leaves and stalks, Cd bioaccumulation (BAF) and translocation factors (TF) of rosette leaves and stalks during the life cycle of field collected T. praecox (mean ± SE, n = 3–5) Developmental stages 1

VP

Fl

FP

SP

SC

Cd (lg g )

Roots Rosette leaves Stalks

891 ± 47b 1536 ± 309d –

636 ± 37a 729 ± 55bc –

867 ± 41b 405 ± 61ab 597 ± 114

508 ± 104a 153 ± 15a 370 ± 63

875 ± 89b 1067 ± 45cd –

Pb (lg g 1)

Roots Rosette leaves Stalks

1576 ± 457b 845 ± 255b –

1260 ± 280b 1411 ± 649b –

2987 ± 485c 393 ± 103b 182 ± 113

515 ± 17a 179 ± 47a 116 ± 26

848 ± 164ab 221 ± 46a –

Zn (lg g 1)

Roots Rosette leaves Stalks

847 ± 72b 3605 ± 680 –

844 ± 62ab 3692 ± 99 –

1099 ± 117c 4134 ± 605 2447 ± 332

660 ± 63a 2630 ± 325 2448 ± 626

1093 ± 43c 3798 ± 313 –

Fe (lg g 1)

Roots Rosette leaves Stalks

597 ± 218 1505 ± 781c –

376 ± 84 623 ± 198b –

560 ± 103 410 ± 63b 124 ± 35

164 ± 16 226 ± 39a 118 ± 25

461 ± 23 411 ± 20b –

Ni (lg g 1)

Roots Rosette leaves Stalks

8.1 ± 1.5a 18.6 ± 1.6b –

7.2 ± 1.6a 12.0 ± 1.3a –

8.0 ± 1.3a 15.3 ± 3.1abc 14.3 ± 4.4

5.3 ± 1.2a 13.3 ± 2.5a 10.1 ± 1.4

59 ± 11.4b 65 ± 4.6c –

P (lg g 1)

Roots Rosette leaves Stalks

1753 ± 18 2810 ± 113 –

1632 ± 97 3338 ± 141 –

2408 ± 363 3039 ± 148 3158 ± 284

2507 ± 469 2830 ± 510 1861 ± 377

2039 ± 144 3349 ± 397 –

K (lg g 1)

Roots Rosette leaves Stalks

6688 ± 460 6458 ± 618 –

6788 ± 1466 7797 ± 702 –

6000 ± 522 10 702 ± 3075 11 858 ± 2545

6437 ± 730 8287 ± 1208 4613 ± 1340

7785 ± 407 8929 ± 59 –

Ca (lg g 1)

Roots Rosette leaves Stalks

6978 ± 1916 22 900 ± 6543 –

5118 ± 837 17 767 ± 780 –

4831 ± 427 16 650 ± 2317 13 671 ± 2264

3529 ± 338 18 032 ± 1580 15 050 ± 3280

3659 ± 308 20 230 ± 2747 –

Mn (lg g 1)

Roots Rosette leaves Stalks

20 ± 9.4 39 ± 18.9 –

12 ± 3.1 34 ± 9.4 –

20 ± 3.8 27 ± 3.3 16.5 ± 1.2

11 ± 0.7 31 ± 5.5 19.4 ± 5.8

24 ± 6.3 35 ± 6.7 –

Cu (lg g 1)

Roots Rosette leaves Stalks

14 ± 2.6 19 ± 5.2 –

11 ± 2.3 15 ± 0.6 –

11.1 ± 1.3 12.1 ± 2.0 7.9 ± 1.1

7.0 ± 0.6 9.7 ± 1.6 9.3 ± 1.6

14 ± 1.9 18 ± 2.7 –

Cd BAF Cd TF

BAFRL BAFST TFRL TFST

21.6 ± 3.6c – 1.7 ± 0.3b –

11.9 ± 1.4b – 1.1 ± 0.04b –

6.8 ± 2.6b 8.7 ± 2.2b 0.4 ± 0.05a 1.5 ± 0.20

2.7 ± 1.5a 2.4 ± 0.7a 0.4 ± 0.07a 2.1 ± 0.7

13.2 ± 1.9b – 1.3 ± 0.10b –

VP – vegetative phase, Fl – flower induction phase, FP – flowering phase, SP – seeding phase, SC – senescence phase. BAFRL = Crosette BAFST = Cstalks/Ctotal soil. TFRL = Crosette leaves/Croots; TFST = Cstalks/Crosette leaves. Different letters beside the numbers indicate statistically significant differences, where significant (Mann–Whitney U test, p < 0.05).

the lowest in the seeding phase (Table 1). Bioaccumulation of Cd in stalks, expressed by bioaccumulation factors (BAFST = Cstalks/Ctotal soil) above 1, along with high Cd concentrations in stalks (over 100 lg g 1 dry weight) indicate hyperaccumulation of Cd in stalks during flowering and seeding. In addition, intense translocation of Cd from the rosette leaves to stalks was observed, as expressed by translocation factors (TFST = Cstalks/Crosette leaves) above 1, when present (Table 1). 3.4. Mycorrhizal colonisation Significant variation in mycorrhizal colonisation frequency (F%) and intensity (M%) during the life cycle of

leaves/Ctotal soil;

T. praecox was observed (Fig. 2b and c), with clear peaks in total and vital M% while flowering. Rare vital arbuscules were observed only from the vegetative to the flowering phase. Both biomass (Fig. 2a) and mineral concentrations (Table 1) were significantly affected by plant developmental stage; therefore, elemental contents of roots and rosette leaves were calculated. Contents of P, Cd, Zn, Pb, Fe and Ni in roots and of Cd and Ni in the rosette leaves were found to vary during the plants life cycle (Figs. 3 and 4). In addition, significant positive correlations of vital mycorrhizal intensity (M% vital) with Cd, Zn, Pb and Fe contents in roots were observed, accompanied by significant positive correlation of M% total and Fe contents in rosette leaves (Table 2).

P. Pongrac et al. / Chemosphere 69 (2007) 1602–1609

Fe roots (lg g 1)

Fe shoots (lg g 1)

Cd soil available Cd roots Cd rosette leaves

ns – 0.69***

0.58* 0.55* –

Element content (lg)

M% vital

M% total

Cd roots Zn roots Pb roots Fe roots Fe rosette leaves

0.56* 0.44** 0.65** 0.45** 0.44*

ns ns ns ns ns

ns: Not significant. * p < 0.05. ** p < 0.01. *** p < 0.001.

4. Discussion Numerous reports indicate that the phenological rhythms of plants cannot be ruled out as factors affecting seasonal variation of soil characteristics, arbuscular mycorrhizal (AM) colonisation and elemental uptake (Ruotsalainen et al., 2002; Bohrer et al., 2004; Deram et al., 2006). Previous reports on the presence of AM colonisation in B. laevigata and T. praecox during the flowering phase or in the phase prior to seeding (Orłowska et al., 2002; Vogel-Mikusˇ et al., 2006) indicated plant developmental effects on AM development. In T. praecox, it was related to the extended vernalization period and higher soil metal concentrations, presumably due to enhanced nutrient demands during the reproductive period (Vogel-Mikusˇ et al., 2006). The present study confirmed developmental variation of both elemental uptake and AM colonisation in field collected T. praecox. Differing nutrient requirements during plant developmental phases (Jime´nez et al., 1996) are believed to be met by enhanced root exudation, directly affecting the solubility of metal ions and indirectly affecting microbial activity (Adriano, 2001). AM colonisation development is highly related to low soil P levels (Smith and Read, 1997; Li et al., 2006). This, however, does not explain the peak in AM development in T. praecox in this study because P availability was higher in the flowering than in the vegetative phase (Fig. 1). Seasonal effects with a peak in P availability at the end of July found in this study are in line with a report from low-alpine meadows (Ruotsalainen et al., 2002). Our result supports the view that the increased elemental demand during flowering favours mycorrhizal colonisation, thus providing adequate concentrations of most of the essential elements under study (P, K, Ca, Zn, Mn and Cu) in rosette leaves, despite their increased biomass (Table 1, Fig. 2a). The lowest Cd content in shoots during seed production was previously observed in field collected metallicolous

roots

2400

P content (µg)

Element concentration (lg g 1)

a

rosette leaves

1800

c

1200

c 600

b

ab a

0 VP

FI

FP

SP

SC

Developmental stage

b

rosette leaves

roots

800

Cd content (µg)

Table 2 Pearson’s correlation coefficients (r) between Cd and Fe concentrations (lg g 1) and between vital and total mycorrhizal intensity (M%) and plant element content (lg) found in field collected T. praecox

c 600

b

400

c b

a

a

200 0

a

a

ab VP

FI

b FP

SP

SC

Developmental stage

c Zn content (µg)

1606

rosette leaves

roots

2400 1800 1200

d cd

600

a

bc

b

0 VP

FI

FP

SP

SC

Developmental stage Fig. 3. Plant elemental contents (lg) during the life cycle of field collected T. praecox: (a) P, (b) Cd, and (c) Zn (mean ± SE, n = 3–5); VP – vegetative phase, FI – flower induction phase, FP – flowering phase, SP – seeding phase, SC – senescence phase. Different letters beside the boxes indicate statistical differences, where significant (Mann–Whitney U test, p < 0.05).

populations of Arrhenaterum elatius L. (Deram et al., 2006). In T. praecox it was accompanied by the lowest bioaccumulation factors (BAFRL), partially due to lower transfer from soil to the rosette leaves as expressed by the transfer factor (TFRL), and partially due to intensive translocation of Cd to the flowering/seeding stalks, confirmed by stalk translocation factors (TFST) (Table 1). Cadmium concentrations in the flowering/seeding stalks, reached values above the hyperaccumulation criteria set at 100 lg g 1 dry weight (Brooks, 1998). This indicates the extremely high mobility of Cd in T. praecox tissues and may help to explain seed Cd hyperaccumulation in this species as found in a previous study (Vogel-Mikusˇ et al., 2007).

P. Pongrac et al. / Chemosphere 69 (2007) 1602–1609

a

roots

Fe content (µg)

600

rosette leaves

d 400

c

200

0

ab

a VP

FI

bc FP

SP

SC

Developmental stage

b

roots

Ni content (µg)

28

rosette leaves

d

21 14

c bc b

7

d

a a

0

ab VP

FI

c

bc

FP

SP

SC

Developmental stage

c

roots

Pb content (µg)

3000

rosette leaves

c

2400 1800 1200 600

b b a

b

0 VP

FI

FP

SP

SC

Developmental stage Fig. 4. Plant elemental contents (lg) during the life cycle of field collected T. praecox: (a) Fe, (b) Ni, and (c) Pb (mean ± SE, n = 3–5); VP – vegetative phase, FI – flower induction phase, FP – flowering phase, SP – seeding phase, SC – senescence phase. Different letters beside the boxes indicate statistical differences, where significant (Mann–Whitney U test, p < 0.05).

Cd concentrations and contents in the rosette leaves increased again in the senescence phase, partially due to increased bioaccumulation (BAFRL) and partially due to increased Cd translocation from roots (TFRL). In the perennial T. praecox this may significantly contribute to rhizome detoxification after decay of the rosette in autumn, which is in line with the disposal hypothesis (Baker, 1981). Similarly, highest concentrations and contents of Ni found in the rosette leaves in the senescence phase coincide with increased translocation of Ni to the seeding stalks, as Ni is a phloem mobile element and is known to be easily translocated to the seeds (Adriano, 2001). In fact, up to 90 lg g 1 of Ni was measured in T. praecox seeds from the same plot (Vogel-Mikusˇ, personal communication).

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The disposal of Cd from the decayed rosette leaves enriches the surface soil beneath them with this metal, thus creating hotspots of Cd in the T. praecox rhizosphere as previously reported (Vogel-Mikusˇ et al., 2007) hence enhancing its competitiveness, as proposed in the interference hypothesis of Baker and Brooks (1989). In addition, increased Cd concentrations interfere with Fe uptake as previously demonstrated in T. caerulescens Ganges populations (Roosens et al., 2003), and therefore, the Cd hyperaccumulation system has been proposed as an inadvertent result of selection for the acquisition of essential nutrients, such as Fe (Lombi et al., 2002; Roosens et al., 2003). In line with the results found in T. caerulescens (Basic et al., 2006) we found a negative correlation between soil Cd concentrations and Fe concentrations in the rosette leaves of T. praecox and positive correlations between Cd and Fe concentrations in the roots and in the rosette leaves, further supporting this hypothesis. The uptake of Cd predominantly via a high-affinity uptake system for Fe demonstrated for a T. caerulescens Ganges population (Lombi et al., 2001), may have contributed to the observed results. Mycorrhizal symbiosis in contaminated sites is mostly cited in relation to the improvement of plant mineral status due to a shortage of available mineral nutrients on sites with excessive heavy metal concentrations (Shetty et al., 1995), whereas, its effects on plant elemental concentrations vary (e.g., Gildon and Tinker, 1983; Weissenhorn et al., 1995; Leyval et al., 1997; Hildebrandt et al., 1999; Turnau and Mesjasz-Przybylowicz, 2003; Andrade et al., 2004; Chen et al., 2005; Citterio et al., 2005; Vogel-Mikusˇ et al., 2006). The positive correlation between total AM colonisation intensity (M% total) and Fe contents (but not for Cd or P) in the rosette leaves of field collected T. praecox further supports the hypothesis of AM being an important factor determining the nutrient status of plants on metal polluted sites. Furthermore, the positive correlations between AM colonisation levels and root Cd, Zn, Pb and Fe contents in T. praecox indicate the interactions of AM colonisation with metal concentrations. Metal adsorption by the fungal biomass of the hyphal intracellular network, demonstrated for Cd and Zn, was proposed as one of the tolerance strategies in heavy metal polluted soil (Joner et al., 2000) and may contribute significantly to the protection of plants on metal polluted sites. Improved mineral status and reduced metal uptake in T. praecox was already reported from a greenhouse experiment, suggesting that inoculation with an indigenous AM fungal mixture induced changes in its metal tolerance strategies (VogelMikusˇ et al., 2006). The present study is the first report demonstrating this in T. praecox under field conditions. In conclusion, developmental effects on both elemental uptake and AM colonisation in field collected T. praecox presumably result from the increased elemental requirement during flowering/seeding periods, thus efficiently providing essential elements as well as simultaneous protection from excess metals, and therefore, significantly contributing to plant fitness on metal polluted sites.

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