Food and Chemical Toxicology 48 (2010) 21092114

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Food and Chemical Toxicology

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Role of ploidy in cadmium and nickel uptake by Matricaria chamomilla plants

ik a,*, Bor ivoj Klejdus b, Jir Grz c, Silvia Malc ovsk a, Josef Hedbavny b Jozef Kovc
a

Department of Botany, Institute of Biology and Ecology, Faculty of Science, P.J. afrik University, Mnesova 23, 041 67 Koice, Slovak Republic de lsk 1, 613 00 Brno, Czech Republic Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry Brno, Zeme c University and Institute of Experimental Botany ASCR, lechtitelu 11, 783 71 Olomouc, Czech Republic Laboratory of Growth Regulators, Palacky
b

a r t i c l e

i n f o

a b s t r a c t
Cadmium and nickel uptake by diploid and tetraploid chamomile (Matricaria chamomilla L.) cultivars (Novbona and Lutea, respectively) exposed to 60 lM solutions of individual metals over 7 days was studied. Diploid plants accumulated higher amount of Cd in both shoots and roots compared to tetraploid plants while Ni accumulation was ploidy-independent. Cd presence caused higher accumulation of total soluble phenols and avonoids and higher phenylalanine ammonia-lyase and guaiacol-peroxidase activities in diploid cultivar in comparison with tetraploid but phenolic acids did not show direct correlation with metal accumulation and even decreased in the leaves of Ni-exposed plants. Lignin content was preferentially elevated in the roots of diploid cultivar. Among 17 free amino acids, their sum increased mainly in the leaves of Cd-exposed plants (owing to increase in serine, alanine and proline). Potassium decrease in both cultivars in response to Cd was ploidy-independent and Ca, Mg and Fe accumulation were almost unaffected. It is concluded that Cd accumulation in chamomile may be mediated by the accumulation of phenols but they have no active role in shoot Ni accumulation. Present ndings in the context of our previous studies and limited available literature about ploidy effect on metal accumulation are discussed. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 11 February 2010 Accepted 5 May 2010

Keywords: Antioxidants Heavy metals Oxidative stress Salicylic acid

1. Introduction Increasing industrial production leads to elevated release of heavy metals into the environment, thus increasing risk for human health if accumulated in crop plants. Among others, nickel (Ni) and cadmium (Cd) are potentially dangerous because of their toxic effects and their accumulation in the food chain. Ni abundance in soils worldwide is >5 kg ha1 and in terms of plant metabolism, Ni is an essential ultramicronutrient, found to be the active centre of urease and the cofactor of one superoxide dismutase isoform (Kpper and Kroneck, 2007). In contrast, Cd has no known physiological function in plants. Both these metals are divalent and are unable to catalyze the generation of reactive oxygen species (ROS) via FentonHaberWeiss reactions (Stohs and Bagchi, 1995). Notwithstanding this, they have different effects on plants since Ni shows lower toxicity in comparison with other metals (Kpper et al., 1996) and this observation has also been conrmed ik et al., 2006, in chamomile (Matricaria chamomilla L.; Kovc 2009b). Besides antioxidative enzymes aimed to reduce the impact of oxidative stress usually generated by excess of metals, plants also synthesize low molecular antioxidants such as ascorbate, glutathione and phenols. Phenolic metabolites are widely distributed com* Corresponding author. Tel.: +421 905 678861; fax: +421 55 6337353. ik). E-mail address: jozkovacik@yahoo.com (J. Kovc 0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.05.012

pounds involved also in plant defence (Dixon and Paiva, 1995). They may scavenge ROS directly or through enzymatic reactions and may chelate metals in order to reduce the level of free metal ions (Rice-Evans et al., 1996; Vasconcelos et al., 1999). Stimulation of phenolic metabolism in response to Cd excess has also been ik and Klejdus, 2008) while Ni showed found in chamomile (Kovc ik et al., 2009d). a less-pronounced effect (Kovc Oxygen ligands such as organic acids are known to chelate metals in hyperaccumulator plants (Kpper et al., 2004) or to be a rootto-shoot translocation form (Bhatia et al., 2005), but little is known about the role of phenols in this process. Our previous study using salicylic acid-induced changes to phenolic metabolites has shown correlation between soluble phenols and quantitative changes of ik et al., 2009b). Cd in chamomile plants (Kovc Polyploidisation is often used to increase the productivity of plants through higher organ size but no corresponding increase in the content of active compounds has been observed in chamo k and Krausov, 2009). Besides, no clear correlation bemile (Repc tween ploidy level and accumulation of phenols was found (Kim et al., 2009). There exist only a limited number of studies about the role of ploidy in Cd uptake (Kraljevic-Balalic et al., 2009), and to our knowledge, there are no information about this phenomenon in terms of Ni accumulation. It was therefore the main aim of the present study to evaluate Cd and Ni uptake by two chamomile cultivars with different ploidy level (diploid vs. tetraploid). Selected biochemical parameters with special emphasis on

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ik et al. / Food and Chemical Toxicology 48 (2010) 21092114 J. Kovc Free amino acids were extracted with 80% aqueous ethanol and analyses were performed on an HP 1100 liquid chromatograph (Hewlett Packard, Waldbronn, Germany) with uorometric detector FLD HP 1100 and using precolumn derivatization ik et al., 2009c). with o-phtalaldehyde and 9-uorenylmethyl chloroformate (Kovc 2.5. Statistical analyses

phenolic metabolism not only due to their role in plant stress protection but also because they are potent antioxidants in human diet were evaluated. Accumulation of free amino acids and selected mineral nutrients were estimated as well.
2. Materials and methods 2.1. Cultivation of plants and experimental design Seeds of experimental plants originated from our own experimental eld (summer 2005, germination rate >90%) in the Botanical Garden of P.J. afrik University in Koice. Twenty-one day old seedlings of M. chamomilla L. (Asteraceae) of diploid (Novbona, 2n = 18) and tetraploid (Lutea, 2n = 36) cultivar germinated in sand (with 34 true leaves) were placed to slightly modied Hoagland solution routinely ik et al., 2009ae, in press). Uniform plants were culused in our laboratory (Kovc tivated in brown plastic 5 L boxes (25 plants per box) with continual aeration of the solutions. The experiment was performed in a growth chamber under controlled conditions: 12 h day (6.00 am6.00 pm); photon ux density was 210 lmol m2 s1 PAR at leaf level supplied by cool white uorescent tubes TLD 36 W/33 (Philips, France); 25/20 C day/night temperature; and relative humidity $60%. In these conditions, plants form basal leaf rosettes only. Solutions were renewed weekly to prevent nutrient depletion. Plants, which had been cultivated hydroponically for 6 weeks were used in the experiment and further cultured for 7 days in 60 lM Cd- or Ni-enriched solutions (added in the form of NiCl26H2O and CdCl22H2O, Lachema Brno, Czech Republic) and solutions were applied twice (at the start and after 3 days). Based on the results of earlier work, these concentrations were selected to avoid damaging of the plants and consequently causing misleading results. Controls did not contain any additional chemicals and pH was checked to be 6.0 0.1 in all variants. Fresh and dry masses (dried at 70 C to constant weight) were estimated in order to determine the plant water content [100 (dry mass 100/fresh mass)] allowing recalculation of parameters measured in fresh samples. These dried samples were further analyzed for free amino acids, phenolic acids, lignin and mineral nutrients including Ni and Cd. Plants for fresh massrequiring parameters were powdered using liquid N2 and extracted as described below. For all enzymes, randomly selected supernatants were boiled to destroy enzyme activity and to check that the observed reaction was enzymatic. Spectrophotometry was carried out with an Uvi Light XTD 2 (Secomam, ALES Cedex, France). 2.2. Quantication of Ni, Cd and selected mineral nutrients Samples for quantication of metals were prepared as described previously ik et al., 2009b,e, in press): dry material was kept overnight in HNO3 and (Kovc H2O2 mixture (10 ml + 10 ml, Suprapur, Merck) at laboratory temperature and next day evaporated to dryness at 90 C in a water bath (56 h). Dry residue was dissolved in 5% HNO3 and diluted to a nal volume of 10 ml. All measurements were carried out using an atomic absorption spectrometer AA30 (Varian Ltd., Mulgrave, Australia) and an air-acetylene ame. Samples for quantication of intra-root Ni and Cd were washed in 10 mM CaCl2 (one root system in 300 ml) at 4 C for ik et al., 30 min in order to remove metals adsorbed to the root surface (Kovc 2009e) and all other mineral nutrients were quantied in these samples. For quantication of total root Ni/Cd, samples were washed with deionized water only (and Ca content was determined in these samples). 2.3. Analyses of phenolic metabolism-related parameters Total soluble phenols were extracted with 80% methanol from fresh tissue and measured using FolinCiocalteu method with gallic acid as standard; avonoids were estimated in the same supernatant using AlCl3 procedure and quercetin as ik et al., 2009d). Root lignin content was estimated by the thioglystandard (Kovc ik and Klejdus, 2008). Total contents of individual phenocolic acid reaction (Kovc lic acids were quantied after acid hydrolysis of methanolic extracts with 2 M HCl. Extraction and quantication using UPLCMS/MS system was done as described in detail previously (Ayaz et al., 2005; Grz et al., 2008). Activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was determined as the production of trans-cinnamic acid from phenylalanine using the HPLC method ik et al., with homogenates prepared using sodium borate buffer, pH 8.7 (Kovc 2009c). To determine the activities of cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195), polyphenol oxidase (PPO, EC 1.10.3.2) and guaiacol-peroxidase (GPX, EC 1.11.1.7), samples were homogenized in 50 mM potassium phosphate buffer (pH 7.0) containing 5 mM polyvinyl polypyrrolidone at 4 C. Measurements and cal ik et al., 2009a,b). culations were done as described earlier (Kovc 2.4. Quantication of soluble proteins and free amino acids Proteins were quantied according to Bradford (1976) using 20 ll of supernatants and bovine serum albumin as standard.

Data were evaluated using ANOVA followed by a Tukeys test (MINITAB Release 11, Minitab Inc., State College, Pennsylvania) at P < 0.05. Number of replications (n) in tables/gures denotes individual plants measured for each parameter. One box containing 25 plants was used for each cultivar/treatment, thus the whole experiment included six boxes. Two independent repetitions of the whole experiment were performed in order to check reproducibility and representative values are shown.

3. Results 3.1. Distribution of Cd and Ni in chamomile plants Leaf Cd content was signicantly higher in diploid plants, while Ni accumulation did not differ if diploid and tetraploid ones are compared (Fig. 1). In the roots, Cd amount was higher in both diploid and tetraploid plants in comparison with Ni. Both total and intra-root Cd was found to be signicantly higher in diploid cultivar (Fig. 1). In the case of root Ni, total and intra-root Ni content was not different either in diploid or tetraploid cultivar. 3.2. Phenolic metabolites and enzymes Content of total soluble phenols and avonoids was similar in the leaf rosettes and roots of control plants of both cultivars (Fig. 2). Soluble phenols and avonoids were strongly enhanced by Cd in diploid plants while a less-pronounced effect was found in tetraploid plants. Ni application evoked comparable response of these two parameters independently on ploidy level (only root avonoids were more enhanced in tetraploid ones). Within 10 detected phenolic acids, six benzoic and four cinnamic acid derivates have been recorded in chamomile leaves (Table 3). Protocatechuic, caffeic and sinapic acids were not affected by any of the treatments. Vanillic and gentisic acids decreased in response to Ni while syringic and ferulic acids increased in response to Cd in both cultivars. Sum of phenolic acids was reduced by Ni treatment in both cultivars while Cd stimulated it in Cd-exposed tetraploid ones. After 7 days of exposure to metals, leaf PAL activity was elevated in both cultivars being expressed the most in diploid ones (Fig. 3). In the roots, Cd evoked the highest PAL activity in diploid roots. GPX activity was preferentially enhanced in diploid plants in comparison with tetraploid plants in response to both metals (Table 1). Activity of PPO was not altered in the leaves and in the roots it showed similar response to both metals without effect of ploidy (Table 1). Root CAD activity was enhanced by Ni in both cultivars and by Cd in Lutea. Root lignin content increased in response to Cd in both cultivars and in response to Ni in Novbona (Table 1). 3.3. Quantitative changes of free amino acids and proteins Within 17 detected free amino acids, the majority of them increased in leaves of Cd-exposed plants of both cultivars, leading to more expressive increase of their sum in diploid ones (Table 2). This was caused mainly by increase in serine, alanine and proline. Among aromatic amino acids, phenylalanine increased in both cultivars in response to Cd while tyrosine increased only in tetraploid plants. Proline content was elevated by Cd and Ni in diploid but only by Ni in tetraploid plants. Methionine was not affected by any of the treatments (Table 2).

 ik et al. / Food and Chemical Toxicology 48 (2010) 21092114 J. Kovc

2111

250

leaf Cd content ( g g DW)

-1

200

leaf Ni content ( g g DW)

80

60

b
150 100 50 0 4000

-1

40

20

800

root Cd content ( g g DW)

total

"intra-root"
root Ni content ( g g DW)

a ab
600
-1

3000

b c

bc

-1

2000

400

d
1000

200

diploid

tetraploid

diploid

tetraploid

Fig. 1. Accumulation of Cd and Ni in the leaf rosettes and roots of diploid and tetraploid Matricaria chamomilla plants exposed to 60 lM Cd or Ni over 7 days (n = 4). Data are means, bars indicate SDs. Values within each graph, followed by the same letter(s), are not signicantly different according to Tukeys test (P < 0.05). Intra-root metals were measured after removal of adhered metals by CaCl2 solution (see Section 2).

25

control

Cd b

Ni leaf flavonoids (mg g DW) bc

-1

leaf phenols (mg g DW)

20

-1

15

cd cd d

b b

10

0 9

a root flavonoids (mg g DW)

1.2

a b
0.8

ab

root phenols (mg g DW)

bc

bc

-1

-1

0.4

0 diploid tetraploid

0 diploid tetraploid

Fig. 2. Accumulation of total soluble phenols and AlCl3-reactive avonoids in the leaf rosettes and roots of diploid and tetraploid Matricaria chamomilla plants exposed to 60 lM Cd or Ni over 7 days (n = 4). Data are means, bars indicate SDs. Values within each graph, followed by the same letter(s), are not signicantly different according to Tukeys test (P < 0.05).

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ik et al. / Food and Chemical Toxicology 48 (2010) 21092114 J. Kovc

0.9

a a leaf PAL activity

0.6

control

Cd

Ni

Table 2 Accumulation of free amino acids (lmol g1 DW; n = 4) in the leaf rosettes of diploid and tetraploid Matricaria chamomilla plants exposed to 60 lM Cd or Ni over 7 days. Data are means (for the lucidity of table, SDs are not shown). Values within rows, followed by the same letter(s), are not signicantly different according to Tukeys test (P < 0.05). Diploid Control Cd 0.724a 0.607a 4.125a 0.025bc 0.538a 0.663a 0.514b 5.810a 0.304a 0.720a 0.933a 0.017a 0.389a 0.874a 0.771a 0.216b 5.375b 22.66a Ni 0.489c 0.318b 3.740a 0.017c 0.398b 0.111d 0.294c 3.939bc 0.177b 0.463c 0.587b 0.013a 0.277b 0.516c 0.333c 0.135c 6.229b 18.04c Tetraploid Control 0.277d 0.132c 1.705c 0.032ab 0.252c 0.327c 0.559b 3.300d 0.161b 0.325d 0.361d 0.014a 0.156c 0.280e 0.287c 0.126c 6.262b 14.55d Cd 0.633b 0.303b 3.378b 0.034a 0.358bc 0.414b 0.360c 4.338b 0.273a 0.563b 0.585b 0.016a 0.326ab 0.646b 0.449b 0.236b 6.189b 19.10bc Ni 0.532c 0.620a 3.144b 0.038a 0.309c 0.334bc 0.179d 3.371cd 0.189b 0.353d 0.470c 0.015a 0.169c 0.382d 0.315c 0.307a 8.994a 19.72b

0.3

0 2

a
1.5

root PAL activity

0.5

cd

ASP GLU SER HIS GLY THR ARG ALA TYR CYS VAL MET PHE ILE LEU LYS PRO Sum

0.325d 0.279b 1.572c 0.022c 0.266c 0.359c 0.634a 3.704cd 0.246ab 0.446c 0.555bc 0.011a 0.296b 0.520c 0.428b 0.131c 3.601c 13.40d

0 diploid tetraploid

fected. Changes of Cu content were found preferentially in the roots (Table 4).

Fig. 3. Activity of phenylalanine ammonia-lyase (PAL; nmol min1 mg1 protein) in the leaf rosettes and roots of diploid and tetraploid Matricaria chamomilla plants exposed to 60 lM Cd or Ni over 7 days (n = 4). Other details as in Fig. 2.

4. Discussion Accumulation of metals in crops including medicinal plants such as chamomile is a global problem owing to health risk for human. Therefore selection of cultivars with reduced root-to-shoot metal translocation is of great importance (Ali et al., 2009; Wang et al., 2009). Our present study has revealed higher content of Cd in both shoots and roots of diploid plants in comparison with tetraploid ones. This is in accordance with a previous report using pot culture , 2000). Despite many and Pirc and owering plants (Grejtovsky investigations focused on Cd accumulation in different genotypes (e.g. Wang et al., 2009), relation of ploidy to Cd accumulation remains unclear. Higher Cd accumulation in tetraploid wheat genotypes (in comparison with hexaploid) has been described (Kraljevic-Balalic et al., 2009), supporting our ndings. It may be assumed that an increase in ploidy level reduces Cd accumulation in plants and exact features of this phenomenon need to be eluci-

Soluble proteins were not affected by any of the treatments in the leaf rosettes (Table 1). In the roots, both Cd and Ni reduced proteins in diploid plants and the same, but with higher intensity, was found in tetraploid plants (Table 1).

3.4. Accumulation of selected mineral nutrients Amounts of Ca and Mg were affected neither in diploid nor in tetraploid cultivar if metal-exposed and control plants are compared (Table 4). Fe accumulation was only elevated in the leaves of Cd-exposed diploid. Potassium decreased with similar extent in both cultivars and metals. Shoot Na content increased more expressively in tetraploid plants while root Na remained unaf-

Table 1 Selected physiological/biochemical parameters in the leaf rosettes and roots of diploid and tetraploid Matricaria chamomilla plants exposed to 60 lM Cd or Ni over 7 days. Data are means (for the lucidity of table, SDs are not shown). Values within rows, followed by the same letter(s), are not signicantly different according to Tukeys test (P < 0.05). Units are lmol min1 mg1 protein, DA min1 mg1 protein and nmol min1 mg1 protein for GPX, PPO and CAD activity, respectively. Diploid Control Leaf rosettes Tissue water content (%; n = 12) Soluble proteins (mg g1 DW; n = 4) GPX activity (n = 4) PPO activity (n = 4) Roots Tissue water content (%; n = 12) Soluble proteins (mg g1 DW; n = 4) GPX activity (n = 4) PPO activity (n = 4) Lignin content (mg g1 DW; n = 4) CAD activity (n = 4) 92.26a 91.2a 0.21d 1.68a 93.82a 55.7a 1.78d 0.89c 25.9c 55.1c Cd 90.73c 83.5a 0.58a 1.63a 92.23c 37.6b 6.71a 2.72a 47.9a 57.2c Ni 91.29bc 86.4a 0.63a 1.76a 93.99a 30.1bc 3.31c 1.26b 33.1b 150.1a Tetraploid Control 92.74a 96.8a 0.18d 1.52a 94.11a 50.2a 1.85d 1.03bc 26.2c 60.2c Cd 91.09c 82.7a 0.29c 1.47a 93.23b 24.8c 5.54b 2.81a 37.1b 137.1a Ni 92.30ab 94.9a 0.36b 1.72a 94.22a 27.3c 2.01d 1.19bc 27.2c 100.2b

 ik et al. / Food and Chemical Toxicology 48 (2010) 21092114 J. Kovc Table 3 Accumulation of phenolic acids (lg g1 DW; n = 4) in the leaf rosettes of diploid and tetraploid Matricaria chamomilla plants exposed to 60 lM Cd or Ni over 7 days. Data are means (for the lucidity of table, SDs are not shown). Values within rows, followed by the same letter(s), are not signicantly different according to Tukeys test (P < 0.05). Diploid Control Protocatechuic p-OH benzoic Vanillic Syringic Salicylic Gentisic Caffeic p-Coumaric Ferulic Sinapic Sum 7.48b 5.34a 96.3a 6.41c 3.34cd 55.4d 14.7a 8.21a 1.65c 9.09a 208.1d Cd 7.83b 5.27a 93.3a 15.3a 4.77c 66.2d 13.2a 7.50a 3.30a 8.51a 225.3d Ni 6.85b 3.10b 66.1b 5.48c 1.38d 25.0e 12.9a 5.25b 1.55c 8.61a 136.3e Tetraploid Control 12.5a 5.06a 72.5b 8.04bc 8.41b 318.9b 11.9a 5.80b 1.44c 9.48a 454.1b Cd 12.1a 5.14a 70.8b 19.5a 17.2a 422.4a 12.1a 5.74b 2.55b 9.07a 576.7a Ni 11.6a 5.20a 36.4c 10.7b 4.73c 230.8c 12.9a 3.60c 1.46c 8.40a 325.9c

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Table 4 Accumulation of selected mineral nutrients (n = 4) in the leaf rosettes and roots of diploid and tetraploid Matricaria chamomilla plants exposed to 60 lM Cd or Ni over 7 days. Data are means (for the lucidity of table, SDs are not shown). Values within rows, followed by the same letter(s), are not signicantly different according to Tukeys test (P < 0.05). Diploid Control Leaf rosettes K (mg g1 DW) Na (mg g1 DW) Ca (mg g1 DW) Mg (mg g1 DW) Fe (mg g1 DW) Cu (lg g1 DW) Roots K (mg g1 DW) Na (mg g1 DW) Ca (mg g1 DW) Mg (mg g1 DW) Fe (mg g1 DW) Cu (lg g1 DW) 92.2b 4.35d 12.1b 3.65a 0.18b 12.5ab 87.0a 4.37b 11.8a 1.63a 7.25a 35.4d Cd 76.3c 5.22bc 11.7b 3.52a 0.23a 13.7ab 66.9b 4.64b 11.4a 1.55a 7.50a 44.8c Ni 87.9b 4.76cd 12.3b 3.40a 0.21ab 13.1ab 86.1a 4.54b 11.8a 1.41a 7.17a 42.7cd Tetraploid Control 102.9a 5.64b 14.8a 3.63a 0.19ab 16.2a 88.7a 5.75a 13.3a 1.61a 7.55a 35.2d Cd 78.1c 6.64a 14.7a 3.67a 0.17b 11.7b 66.0b 5.47a 13.0a 1.50a 6.93a 65.1a Ni 89.2b 6.45a 15.8a 3.59a 0.19ab 15.5ab 88.9a 5.18a 11.5a 1.42a 7.51a 53.4b

dated. No data are available about the comparison of Ni content in chamomile or any other plant cultivars with different ploidy. Nonsignicant difference in Ni content and its lower content in comparison with Cd found in both cultivars (Fig. 1) suggest substantially different dynamics of Cd and Ni accumulation. We therefore studied several biochemical parameters in order to nd any possible correlation with accumulation of metals. Although contents of total soluble phenols and avonoids in control plants of diploid and tetraploid cultivars were similar, they were enhanced more expressively in both shoots and roots of diploid plants in response to Cd. In combination with comparable extent of increase in Cd accumulation in the shoot of diploid, it may be assumed that soluble phenols including avonoids are involved in this process. This assumption may be supported by at least three facts: (i) avonoids (mainly avonols which are the main avonoid-like compounds giving reaction in the assay we used; Deng and van Berel, 1998) are long-distance transported molecules (Buer et al., 2007), (ii) salicylic acid-induced decrease in shoot Cd content was correlated with decrease in soluble phenols in tetra ik et al., 2009b) and (iii) Ni content did ploid chamomile (Kovc not differ in diploid and tetraploid plants, as also soluble phenols and leaf avonoids did (Figs. 1 and 2). Further studies will be conducted in order to verify the role of avonoids in metal uptake by chamomile. Higher induction of phenols in diploid plants was

clearly correlated with PAL activity at least in Cd-exposed plants (Fig. 3). It may be assumed that although polyploidisation in k creases plant height and organ size also in chamomile (Repc and Krausov, 2009), this reduces the expression of selected genes including phenolic metabolism-related parameters (Zagoskina et al., 1997). It is also visible that although leaf PAL activity was stimulated by Ni in diploid, this was not correlated with either induction of soluble phenols or avonoids, suggesting different time dynamics in comparison with Cd. In fact, phenolic metabo ik lism showed only low induction in tetraploid plants (Kovc et al., 2009d). We have also measured the quantity of selected phenolic acids, among which, e.g. protocatechuic acid is a phenol with a high chelating strength and could be involved in Cd tolerance in Brassica juncea shoots (Irtelli and Navari-Izzo, 2006). We found no clear correlation between the majority of individual phenolic acids and metal accumulation (Table 2) and sum of these compounds even decreased in the leaves of Ni-exposed cultivars. Accumulation of salicylic acid, an important plant signalling molecule, was lower in diploid plants and it showed more expressive quantitative changes in tetraploid leaves with opposite trend if Cd and Ni are compared (Table 3). This may also conrm different effect of Cd and Ni on chamomile tissue and different relation of SA to metal ik et al., 2009b). Gentisic acid was accumuaccumulation (Kovc lated to a high extent in Cd-exposed plants while it decreased in Ni treated plants. Gentisic acid followed the same pattern of accumulation as salicylic acid which is in accordance with our previous ik et al., 2009b). Signicance of higher root avonoids study (Kovc in Ni-exposed tetraploid plants remains unclear but it was found that more resistant lentil cultivars contained more UV-absorbing compounds mainly in vacuoles and cell-wall which was correlated with lower Cu accumulation (Janas et al., 2010). It is also known that phenols may be exuded into the culture medium aimed to reduce metal uptake (Jung et al., 2003). Although diploid plants had higher root lignin content (Table 1), this was not an effective barrier against Cd translocation into the above-ground organs. This paradox may sufciently be explained ik and Bac kor, 2008) but lower by rapid shoot Cd uptake (Kovc dynamics of lignin accumulation in comparison, e.g. with Cu ik and Klejdus, 2008). In the process of lignication, partic(Kovc ipation of CAD (dos Santos et al., 2006) was also visible (Table 1). Partial discrepancy between CAD activity and lignin content may again be explained by different time dynamics (sufcient/higher amount of lignin does not need elevated CAD activity). More expressive induction of GPX in diploid plants is another indication of higher lignin content. Although we measured soluble fraction which serves as ROS-scavenger in connection with phenols (Sakihama et al., 2002), cell-wall bound GPX has direct correlation with lignication and both soluble and bound GPX were found to show similar trend being correlated with increase in lignin deposition (Zanardo et al., 2009). This exclusive role of peroxidase-mediated lignication may be conrmed by unaltered PPO activity (if diploid and tetraploid roots from each variant are compared). Mainly Cu was found to be effective inductor of PPO activity in chamomile ik et al., 2009e). (Kovc We have also studied prole of free amino acids in the leaf rosettes since, e.g. histidine and cysteine have been suggested as a ligand for Ni accumulation in hyperaccumulator plants (Bhatia et al., 2005; Ali et al., 2009). Sum of free amino acids was preferentially elevated in Cd-exposed plants but also by Ni in both cultivars (in contradiction to majority of phenols) and this may be caused, the most probably, by different quantity of Cd and Ni in the leaf rosettes (Fig. 1). Within individual compounds, we did not nd increase in histidine and cysteine therefore their role in Ni translocation in chamomile could be negligible. Metals also stimulated increase in serine in both cultivars, an observation which was found in the xylem sap of Ni-exposed Brassica napus cultivars

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where increased with increasing endogenous Ni (Ali et al., 2009). Similar shoot serine increase was found in Cu-exposed Silene plants ik et al., in press). Within aromatic amino acids, higher con(Kovc tent of free phenylalanine may serve as a pool for enhanced PAL activity which we recorded. Proline accumulation was elevated in the leaves of both cultivars. Gajewska and Skodowska (2005) observed a similar trend in Ni-exposed pea plants and suggested that it may be involved in the osmoregulation. Decrease in potassium content is one of the symptoms of heavy metals toxicity also in chamomile as judged from its increase in the ik et al., 2006, 2008). Our present study cultivation medium (Kovc revealed that this depletion is not ploidy-dependent and Cd is more toxic for this parameter in comparison with Ni (Table 4). Almost unaffected Fe and Mg contents are in accordance with absence of visible chlorosis or necrosis. Overall, mineral nutrients, with the exception of K+, were relatively slightly affected, conrming chamomile tolerance to Ni and Cd excess in the given experimental conditions. Present study adds new evidence to our earlier hypothesis that soluble phenols and possibly avonoids may by involved in Cd translocation in chamomile plants but shoot Ni accumulation is phenol-independent. Besides, lower ploidy level allows higher accumulation of phenolic metabolites under stress conditions, despite the fact that polyploidisation may lead to higher biomass of plants. In terms of practical cultivation, it may be concluded that although diploid plants contained higher amounts of active compounds (such as avonoid-like apigenin derivatives) in owers k and Krausov, 2009), it is less suitable for cultivation on (Repc Cd-contaminated soil owing to higher Cd accumulation. Conict of Interest The authors declare that there are no conicts of interest. Acknowledgements This work was supported by the Grant of Academy of Sciences of the Czech Republic (KAN 200380801), Czech Ministry of Education (MSM 6198959216), Grant Agency of the Czech Republic (525/ 07/0338) and partially by the Grant of P.J. afrik University Rector for Young Scientists (to J.K., No. VVGS 1/09-10). References
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