matricaria

7/28/2019 Matricaria

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

Jozef Kovcik a,*, Borivoj Klejdus b, Jir Grz c, Silvia Malcovsk a, Josef Hedbavny b

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

a r t i c l e i n f o

Article history:Received 11 February 2010Accepted 5 May 2010

Keywords:AntioxidantsHeavy metalsOxidative stressSalicylic acid

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 60lM solutions of individual metals over 7 days was stud-ied. Diploid plants accumulated higher amount of Cd in both shoots and roots compared to tetraploidplants while Ni accumulation was ploidy-independent. Cd presence caused higher accumulation of totalsoluble phenols and flavonoids and higher phenylalanine ammonia-lyase and guaiacol-peroxidase activ-ities in diploid cultivar in comparison with tetraploid but phenolic acids did not show direct correlationwith metal accumulation and even decreased in the leaves of Ni-exposed plants. Lignin content was pref-erentially elevatedin theroots of diploid cultivar. Among17 free amino acids, their sumincreased mainlyin the leaves of Cd-exposed plants (owing to increase in serine, alanine and proline). Potassium decreasein both cultivars in response to Cd was ploidy-independent and Ca, Mg and Fe accumulation were almostunaffected. It is concluded that Cd accumulation in chamomile may be mediated by the accumulation ofphenols but they have no active role in shoot Ni accumulation. Present findings in the context of our pre-vious studies and limited available literature about ploidy effect on metal accumulation are discussed.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Increasing industrial production leads to elevated release ofheavy metals into the environment, thus increasing risk for humanhealth if accumulated in crop plants. Among others, nickel (Ni) andcadmium (Cd) are potentially dangerous because of their toxic ef-fects and their accumulation in the food chain. Ni abundance insoils worldwide is >5 kg ha1 and in terms of plant metabolism,Ni is an essential ultramicronutrient, found to be the active centreof urease and the cofactor of one superoxide dismutase isoform(Kpper and Kroneck, 2007). In contrast, Cd has no known physio-logical function in plants. Both these metals are divalent and areunable to catalyze the generation of reactive oxygen species

(ROS) via FentonHaberWeiss reactions (Stohs and Bagchi,1995). Notwithstanding this, they have different effects on plantssince Ni shows lower toxicity in comparison with other metals(Kpper et al., 1996) and this observation has also been confirmedin chamomile (Matricaria chamomilla L.; Kovcik et al., 2006,2009b).

Besides antioxidative enzymes aimed to reduce the impact ofoxidative stress usually generated by excess of metals, plants alsosynthesize low molecular antioxidants such as ascorbate, glutathi-one and phenols. Phenolic metabolites are widely distributed com-

pounds involved also in plant defence (Dixon and Paiva, 1995).They may scavenge ROS directly or through enzymatic reactionsand may chelate metals in order to reduce the level of free metalions (Rice-Evans et al., 1996; Vasconcelos et al., 1999). Stimulationof phenolic metabolism in response to Cd excess has also beenfound in chamomile (Kovcik and Klejdus, 2008) while Ni showeda less-pronounced effect (Kovcik et al., 2009d).

Oxygen ligands such as organic acids are known to chelate met-als in hyperaccumulator plants (Kpper et al., 2004) or tobe a root-to-shoot translocation form (Bhatia et al., 2005), but little is knownabout the role of phenols in this process. Our previous study usingsalicylic acid-induced changes to phenolic metabolites has showncorrelation between soluble phenols and quantitative changes of

Cd in chamomile plants (Kovcik et al., 2009b).Polyploidisation is often used to increase the productivity of

plants through higher organ size but no corresponding increasein the content of active compounds has been observed in chamo-mile (Repck and Krausov, 2009). Besides, no clear correlation be-tween ploidy level and accumulation of phenols was found (Kimet al., 2009). There exist only a limited number of studies aboutthe role of ploidy in Cd uptake (Kraljevic-Balalic et al., 2009), andto our knowledge, there are no information about this phenome-non in terms of Ni accumulation. It was therefore the main aimof the present study to evaluate Cd and Ni uptake by two chamo-mile cultivars with different ploidy level (diploid vs. tetraploid).Selected biochemical parameters with special emphasis on

0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2010.05.012

* Corresponding author. Tel.: +421 905 678861; fax: +421 55 6337353.E-mail address: [email protected] (J. Kovcik).

Food and Chemical Toxicology 48 (2010) 21092114

Contents lists available at ScienceDirect

Food and Chemical Toxicology

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phenolic metabolism not only due to their role in plant stress pro-tection but also because they are potent antioxidants in humandiet were evaluated. Accumulation of free amino acids and selectedmineral nutrients were estimated as well.

2. Materials and methods

2.1. Cultivation of plants and experimental d esign

Seeds of experimental plants originated from our own experimental field (sum-mer 2005, germination rate >90%) in the Botanical Garden of P.J. afrik Universityin 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 placedto slightly modified Hoagland solution routinelyused in our laboratory (Kovcik et al., 2009ae, in press). Uniform plants were cul-tivated in brown plastic5 L boxes (25plants per box) with continual aeration of thesolutions. The experiment was performed in a growth chamber under controlledconditions: 12 h day(6.00 am6.00 pm); photonflux density was210lmol m2 s1

PAR at leaf level supplied by cool white fluorescent tubes TLD 36 W/33 (Philips,France); 25/20 C day/night temperature; and relative humidity$60%. In thesecon-ditions, plants form basal leaf rosettes only. Solutions were renewed weekly to pre-vent nutrient depletion. Plants, which had been cultivated hydroponically for6 weeks were used in the experiment and further cultured for 7 days in 60lMCd- 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 se-lectedto avoid damaging of the plants andconsequently causing misleading results.Controls did not contain any additional chemicals and pH was checked to be6.0 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 (drymass100/fresh mass)] allowing recalculation of parameters measured in freshsamples. These dried samples were further analyzed for free amino acids, phenolicacids, lignin and mineral nutrients including Ni and Cd. Plants for fresh mass-requiring parameters were powdered using liquidN2 andextractedas describedbe-low. For all enzymes, randomly selected supernatants were boiled to destroy en-zyme activity and to check that the observed reaction was enzymatic.Spectrophotometrywas carried out with an UviLight XTD2 (Secomam,ALES Cedex,France).

2.2. Quantification of Ni, Cd and selected mineral nutrients

Samples for quantification of metals were prepared as described previously

(Kovcik et al., 2009b,e, in press): dry material was kept overnight in HNO3 andH2O2 mixture (10 ml + 10 ml, Suprapur, Merck) at laboratory temperature and nextday evaporated to dryness at 90 C in a water bath (56 h). Dry residue was dis-solved in 5% HNO3 and diluted to a final volume of 10 ml. All measurements werecarried out using an atomic absorption spectrometer AA30 (Varian Ltd., Mulgrave,Australia) and an air-acetylene flame. Samples for quantification of intra-rootNi and Cd were washed in 10 mM CaCl2 (one root system in 300ml) at 4 C for30 min in order to remove metals adsorbed to the root surface (Kovcik et al.,2009e) and all other mineral nutrients were quantified in these samples. For quan-tification of total root Ni/Cd, samples were washed with deionized water only (andCa content was determined in these samples).

2.3. Analyses of phenolic metabolism-related p arameters

Total soluble phenols were extracted with 80% methanol from fresh tissue andmeasured using FolinCiocalteu method with gallic acid as standard; flavonoidswere estimated in the same supernatant using AlCl3 procedure and quercetin as

standard (Kovcik et al., 2009d). Root lignin content was estimated by the thiogly-colic acid reaction (Kovcik and Klejdus, 2008). Total contents of individual pheno-lic acids were quantified after acid hydrolysis of methanolic extracts with 2 M HCl.Extraction and quantification using UPLCMS/MS system was done as described indetail previously (Ayaz et al., 2005; Grz et al., 2008).

Activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was determined asthe production oftrans-cinnamic acid from phenylalanine using the HPLC methodwith homogenates prepared using sodium borate buffer, pH 8.7 (Kovcik et al.,2009c).

To determine the activities of cinnamyl alcohol dehydrogenase (CAD, EC1.1.1.195), 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(pH7.0) containing 5 mM polyvinylpolypyrrolidone at 4 C. Measurements andcal-culations were done as described earlier (Kovcik et al., 2009a,b).

2.4. Quantification of soluble proteins and free amino acids

Proteins were quantified according to Bradford (1976) using 20 ll of superna-tants and bovine serum albumin as standard.

Free amino acids were extracted with 80% aqueous ethanol and analyses wereperformedon an HP 1100 liquidchromatograph (Hewlett Packard, Waldbronn, Ger-many) with fluorometric detector FLD HP 1100 and using precolumn derivatizationwith o-phtalaldehyde and 9-fluorenylmethyl chloroformate (Kovcik et al., 2009c).

2.5. Statistical analyses

Data were evaluated using ANOVA followed by a Tukeys test (MINITAB Release11, Minitab Inc., State College, Pennsylvania) at P< 0.05. Number of replications (n)in tables/figures denotes individual plants measured for each parameter. One boxcontaining 25 plants was used for each cultivar/treatment, thus the whole experi-ment included six boxes. Two independent repetitions of the whole experimentwere performed in order to check reproducibility and representative values areshown.

3. Results

3.1. Distribution of Cd and Ni in chamomile plants

Leaf Cd content was significantly higher in diploid plants, whileNi accumulation did not differ if diploid and tetraploid ones arecompared (Fig. 1).

In the roots, Cd amount was higher in both diploid and tetra-

ploid plants in comparison with Ni. Both total and intra-rootCd was found to be significantly higher in diploid cultivar(Fig. 1). In the case of root Ni, total and intra-root Ni contentwas not different either in diploid or tetraploid cultivar.

3.2. Phenolic metabolites and enzymes

Content of total soluble phenols and flavonoids was similar inthe leaf rosettes and roots of control plants of both cultivars(Fig. 2). Soluble phenols and flavonoids were strongly enhancedby Cd in diploid plants while a less-pronounced effect was foundin tetraploid plants. Ni application evoked comparable responseof these two parameters independently on ploidy level (only rootflavonoids were more enhanced in tetraploid ones).

Within 10 detected phenolic acids, six benzoic and four cin-namic acid derivates have been recorded in chamomile leaves(Table 3). Protocatechuic, caffeic and sinapic acids were notaffected by any of the treatments. Vanillic and gentisic acidsdecreased in response to Ni while syringic and ferulic acidsincreased in response to Cd in both cultivars. Sum of phenolic acidswas reduced by Ni treatment in both cultivars while Cd stimulatedit in Cd-exposed tetraploid ones.

After 7 days of exposure to metals, leaf PAL activity was ele-vated in both cultivars being expressed the most in diploid ones(Fig. 3). In the roots, Cd evoked the highest PAL activity in diploidroots. GPX activity was preferentially enhanced in diploid plants incomparison 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 ofploidy (Table 1). Root CAD activity was enhanced by Ni in both cul-tivars and by Cd in Lutea. Root lignin content increased in responseto 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 in-creased in leaves of Cd-exposed plants of both cultivars, leadingto more expressive increase of their sum in diploid ones (Table2). This was caused mainly by increase in serine, alanine and pro-line. Among aromatic amino acids, phenylalanine increased in bothcultivars in response to Cd while tyrosine increased only in tetra-ploid plants. Proline content was elevated by Cd and Ni in diploid

but only by Ni in tetraploid plants. Methionine was not affected byany of the treatments (Table 2).

2110 J. Kovcik et al. / Food and Chemical Toxicology 48 (2010) 21092114


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a

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leafCdcon

tent(gg

-1D

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rootCdconte

nt(gg

-1D

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-1D

W)

ab

a

cbc

0

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400

600

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diploid tetraploid diploid tetraploid

rootNiconten

t(gg

-1D

W)

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

dcd

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d d

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rootflavonoids(mgg

-1D

W)

Fig. 2. Accumulation of total soluble phenols and AlCl3-reactive flavonoids in the leaf rosettes and roots of diploid and tetraploidMatricaria chamomilla plants exposed to

60lM 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 significantly different according toTukeys test (P< 0.05).

J. Kovcik et al./Food and Chemical Toxicology 48 (2010) 21092114 2111


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Soluble proteins were not affected by any of the treatments inthe leaf rosettes (Table 1). In the roots, both Cd and Ni reduced pro-teins 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 intetraploid cultivar if metal-exposed and control plants are com-pared (Table 4). Fe accumulation was only elevated in the leavesof Cd-exposed diploid. Potassium decreased with similar extentin both cultivars and metals. Shoot Na content increased moreexpressively in tetraploid plants while root Na remained unaf-

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

4. Discussion

Accumulation of metals in crops including medicinal plantssuch as chamomile is a global problem owing to health risk for hu-man. Therefore selection of cultivars with reduced root-to-shootmetal translocation is of great importance (Ali et al., 2009; Wanget al., 2009).

Our present study has revealed higher content of Cd in bothshoots and roots of diploid plants in comparison with tetraploidones. This is in accordance with a previous report using pot cultureand flowering plants (Grejtovsky and Pirc, 2000). Despite manyinvestigations focused on Cd accumulation in different genotypes(e.g. Wang et al., 2009), relation of ploidy to Cd accumulation re-mains unclear. Higher Cd accumulation in tetraploid wheat geno-types (in comparison with hexaploid) has been described(Kraljevic-Balalic et al., 2009), supporting our findings. It may beassumed that an increase in ploidy level reduces Cd accumulationin plants and exact features of this phenomenon need to be eluci-

cc

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PAL

activity

control Cd Ni

cdc

b

a

d d

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diploid tetraploid

roo

tPALactivity

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

Table 1

Selected physiological/biochemical parameters in the leaf rosettes and roots of diploid and tetraploid Matricaria chamomilla plants exposed to 60l

M 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 significantly 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 Tetraploid

Control Cd Ni Control Cd Ni

Leaf rosettesTissue water content (%; n = 12) 92.26a 90.73c 91.29bc 92.74a 91.09c 92.30abSoluble proteins (mg g1 DW; n = 4) 91.2a 83.5a 86.4a 96.8a 82.7a 94.9aGPX activity (n = 4) 0.21d 0.58a 0.63a 0.18d 0.29c 0.36bPPO activity (n = 4) 1.68a 1.63a 1.76a 1.52a 1.47a 1.72a

RootsTissue water content (%; n = 12) 93.82a 92.23c 93.99a 94.11a 93.23b 94.22aSoluble proteins (mg g1 DW; n = 4) 55.7a 37.6b 30.1bc 50.2a 24.8c 27.3cGPX activity (n = 4) 1.78d 6.71a 3.31c 1.85d 5.54b 2.01dPPO activity (n = 4) 0.89c 2.72a 1.26b 1.03bc 2.81a 1.19bcLignin content (mg g1 DW; n = 4) 25.9c 47.9a 33.1b 26.2c 37.1b 27.2c

CAD activity (n = 4) 55.1c 57.2c 150.1a 60.2c 137.1a 100.2b

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 significantly different according to Tukeys test

(P < 0.05).

Diploid Tetraploid


ASP 0.325d 0.724a 0.489c 0.277d 0.633b 0.532cGLU 0.279b 0.607a 0.318b 0.132c 0.303b 0.620aSER 1.572c 4.125a 3.740a 1.705c 3.378b 3.144bHIS 0.022c 0.025bc 0.017c 0.032ab 0.034a 0.038aGLY 0.266c 0.538a 0.398b 0.252c 0.358bc 0.309cTHR 0.359c 0.663a 0.111d 0.327c 0.414b 0.334bcARG 0.634a 0.514b 0.294c 0.559b 0.360c 0.179dALA 3.704cd 5.810a 3.939bc 3.300d 4.338b 3.371cdTYR 0.246ab 0.304a 0.177b 0.161b 0.273a 0.189bCYS 0.446c 0.720a 0.463c 0.325d 0.563b 0.353dVAL 0.555bc 0.933a 0.587b 0.361d 0.585b 0.470cMET 0.011a 0.017a 0.013a 0.014a 0.016a 0.015aPHE 0.296b 0.389a 0.277b 0.156c 0.326ab 0.169cILE 0.520c 0.874a 0.516c 0.280e 0.646b 0.382dLEU 0.428b 0.771a 0.333c 0.287c 0.449b 0.315cLYS 0.131c 0.216b 0.135c 0.126c 0.236b 0.307a

PRO 3.601c 5.375b 6.229b 6.262b 6.189b 8.994aSum 13.40d 22.66a 18.04c 14.55d 19.10bc 19.72b



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dated. No data are available about the comparison of Ni content inchamomile or any other plant cultivars with different ploidy. Non-significant difference in Ni content and its lower content in com-parison with Cd found in both cultivars (Fig. 1) suggest substan-tially different dynamics of Cd and Ni accumulation. Wetherefore studied several biochemical parameters in order to findany possible correlation with accumulation of metals.

Although contents of total soluble phenols and flavonoids in

control plants of diploid and tetraploid cultivars were similar, theywere enhanced more expressively in both shoots and roots of dip-loid plants in response to Cd. In combination with comparable ex-tent of increase in Cd accumulation in the shoot of diploid, it maybe assumed that soluble phenols including flavonoids are involvedin this process. This assumption may be supported by at least threefacts: (i) flavonoids (mainly flavonols which are the main flavo-noid-like compounds giving reaction in the assay we used; Dengand van Berel, 1998) are long-distance transported molecules(Buer et al., 2007), (ii) salicylic acid-induced decrease in shoot Cdcontent was correlated with decrease in soluble phenols in tetra-ploid chamomile (Kovcik et al., 2009b) and (iii) Ni content didnot differ in diploid and tetraploid plants, as also soluble phenolsand leaf flavonoids did (Figs. 1 and 2). Further studies will be con-

ducted in order to verify the role of flavonoids in metal uptake bychamomile. 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-creases plant height and organ size also in chamomile (Repckand Krausov, 2009), this reduces the expression of selected genesincluding phenolic metabolism-related parameters (Zagoskinaet al., 1997). It is also visible that although leaf PAL activity wasstimulated by Ni in diploid, this was not correlated with eitherinduction of soluble phenols or flavonoids, suggesting differenttime dynamics in comparison with Cd. In fact, phenolic metabo-lism showed only low induction in tetraploid plants (Kovciket al., 2009d). We have also measured the quantity of selected phe-nolic acids, among which, e.g. protocatechuic acid is a phenol witha high chelating strength and could be involved in Cd tolerance inBrassica juncea shoots (Irtelli and Navari-Izzo, 2006). We found noclear correlation between the majority of individual phenolic acidsand metal accumulation (Table 2) and sum of these compoundseven decreased in the leaves of Ni-exposed cultivars. Accumulationof salicylic acid, an important plant signalling molecule, was lowerin diploid plants and it showed more expressive quantitativechanges in tetraploid leaves with opposite trend if Cd and Ni arecompared (Table 3). This may also confirm different effect of Cdand Ni on chamomile tissue and different relation of SA to metalaccumulation (Kovcik et al., 2009b). Gentisic acid was accumu-lated to a high extent in Cd-exposed plants while it decreased inNi treated plants. Gentisic acid followed the same pattern of accu-mulation as salicylic acid which is in accordance with our previousstudy (Kovcik et al., 2009b). Significance of higher root flavonoidsin Ni-exposed tetraploid plants remains unclear but it was foundthat more resistant lentil cultivars contained more UV-absorbingcompounds mainly in vacuoles and cell-wall which was correlatedwith lower Cu accumulation (Janas et al., 2010). It is also knownthat phenols may be exuded into the culture medium aimed to re-duce metal uptake (Jung et al., 2003).

Althoughdiploid plants had higher root lignin content (Table 1),this was not an effective barrier against Cd translocation into theabove-ground organs. This paradox may sufficiently be explained

by rapid shoot Cd uptake (Kovcik and Backor, 2008) but lowerdynamics of lignin accumulation in comparison, e.g. with Cu(Kovcik and Klejdus, 2008). In the process of lignification, partic-ipation of CAD (dos Santos et al., 2006) was also visible (Table 1).Partial discrepancy between CAD activity and lignin content mayagain be explained by different time dynamics (sufficient/higheramount of lignin does not need elevated CAD activity). Moreexpressive induction of GPX in diploid plants is another indicationof higher lignin content. Although we measured soluble fractionwhich serves as ROS-scavenger in connection with phenols (Saki-hama et al., 2002), cell-wall bound GPX has direct correlation withlignification and both soluble and bound GPX were found to showsimilar trend being correlated with increase in lignin deposition(Zanardo et al., 2009). This exclusive role of peroxidase-mediated

lignification may be confirmed by unaltered PPO activity (if diploidand tetraploid roots from each variant are compared). Mainly Cuwas found to be effective inductor of PPO activity in chamomile(Kovcik et al., 2009e).

We have also studied profile of free amino acids in the leaf ro-settes since, e.g. histidine and cysteine have been suggested as a li-gand for Ni accumulation in hyperaccumulator plants (Bhatia et al.,2005; Ali et al., 2009). Sum of free amino acids was preferentiallyelevated in Cd-exposed plants but also by Ni in both cultivars (incontradiction to majority of phenols) and this may be caused, themost probably, by different quantity of Cd and Ni in the leaf ro-settes (Fig. 1). Within individual compounds, we did not find in-crease in histidine and cysteine therefore their role in Nitranslocation in chamomile could be negligible. Metals also stimu-

lated increase in serine in both cultivars, an observation which wasfound in the xylem sap of Ni-exposed Brassica napus cultivars

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 significantly different according to Tukeys test

(P< 0.05).

Diploid Tetraploid


Protocatechuic 7.48b 7.83b 6.85b 12.5a 12.1a 11.6ap-OH benzoic 5.34a 5.27a 3.10b 5.06a 5.14a 5.20aVanillic 96.3a 93.3a 66.1b 72.5b 70.8b 36.4cSyringic 6.41c 15.3a 5.48c 8.04bc 19.5a 10.7bSalicylic 3.34cd 4.77c 1.38d 8.41b 17.2a 4.73cGentisic 55.4d 66.2d 25.0e 318.9b 422.4a 230.8cCaffeic 14.7a 13.2a 12.9a 11.9a 12.1a 12.9ap-Coumaric 8.21a 7.50a 5.25b 5.80b 5.74b 3.60cFerulic 1.65c 3.30a 1.55c 1.44c 2.55b 1.46cSinapic 9.09a 8.51a 8.61a 9.48a 9.07a 8.40aSum 208.1d 225.3d 136.3e 4 54.1b 576.7a 3 25.9c

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 withinrows, followed by the same letter(s), are not significantly different according to

Tukeys test (P< 0.05).

Diploid Tetraploid


Leaf rosettesK (mg g1 DW) 92.2b 76.3c 87.9b 102.9a 78.1c 89.2bNa (mg g1 DW) 4.35d 5.22bc 4.76cd 5.64b 6.64a 6.45aCa (mg g1 DW) 12.1b 11.7b 12.3b 14.8a 14.7a 15.8aMg (mg g1 DW) 3.65a 3.52a 3.40a 3.63a 3.67a 3.59aFe (mg g1 DW) 0.18b 0.23a 0.21ab 0.19ab 0.17b 0.19abCu (lg g1 DW) 12.5ab 13.7ab 13.1ab 16.2a 11.7b 15.5ab

RootsK (mg g1 DW) 87.0a 66.9b 86.1a 88.7a 66.0b 88.9aNa (mg g1 DW) 4.37b 4.64b 4.54b 5.75a 5.47a 5.18a

Ca (mg g1

DW) 11.8a 11.4a 11.8a 13.3a 13.0a 11.5aMg (mg g1 DW) 1.63a 1.55a 1.41a 1.61a 1.50a 1.42aFe (mg g1 DW) 7.25a 7.50a 7.17a 7.55a 6.93a 7.51aCu (lg g1 DW) 35.4d 44.8c 42.7cd 35.2d 65.1a 53.4b

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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(Kovcik et al., in press). Within aromatic amino acids, higher con-tent of free phenylalanine may serve as a pool for enhanced PALactivity which we recorded. Proline accumulation was elevatedin the leaves of both cultivars. Gajewska and Skodowska (2005)observed a similar trend in Ni-exposed pea plants and suggestedthat it may be involved in the osmoregulation.

Decrease in potassium content is one of the symptoms of heavymetals toxicity also in chamomile as judged from its increase in thecultivation medium (Kovcik et al., 2006, 2008). Our present studyrevealed that this depletion is not ploidy-dependent and Cd ismore toxic for this parameter in comparison with Ni (Table 4). Al-most unaffected Fe and Mg contents are in accordance with ab-sence of visible chlorosis or necrosis. Overall, mineral nutrients,with the exception of K+, were relatively slightly affected, confirm-ing chamomile tolerance to Ni and Cd excess in the given experi-mental conditions.

Present study adds new evidence to our earlier hypothesis thatsoluble phenols and possibly flavonoids may by involved in Cdtranslocation in chamomile plants but shoot Ni accumulation isphenol-independent. Besides, lower ploidy level allows higheraccumulation of phenolic metabolites under stress conditions, de-spite the fact that polyploidisation may lead to higher biomass ofplants. In terms of practical cultivation, it may be concluded thatalthough diploid plants contained higher amounts of active com-pounds (such as flavonoid-like apigenin derivatives) in flowers(Repck and Krausov, 2009), it is less suitable for cultivation onCd-contaminated soil owing to higher Cd accumulation.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Grant of Academy of Sciencesof the Czech Republic (KAN 200380801), Czech Ministry of Educa-tion(MSM 6198959216), Grant Agency of the Czech Republic(525/07/0338) and partially by the Grant of P.J. afrik University Rectorfor Young Scientists (to J.K., No. VVGS 1/09-10).

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