Plant Growth Regul (2006) 50:239–247 DOI 10.



Matricaria chamomilla is not a hyperaccumulator, but tolerant to cadmium stress
Jozef Kovacik Æ Jaroslav Tomko Æ ´ˇ Martin Backor Æ Miroslav Repcak ˇ ˇ´

Received: 27 March 2006 / Accepted: 1 June 2006 / Published online: 20 October 2006 Ó Springer Science+Business Media B.V. 2006

Abstract The influence of low (3 lM) and high (60 and 120 lM) cadmium (Cd) concentrations were studied on selected aspects of metabolism in 4-week-old chamomile (Matricaria chamomilla L.) plants. After 10 days’ exposure, dry mass accumulation and nitrogen content were not significantly altered under any of the levels of Cd. However, there was a significant decline in chlorophyll and water content in the leaves. Among coumarin-related compounds, herniarin was not affected by Cd, while its precursors (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) increased significantly at all the levels of Cd tested. Cd did not have any effect on umbelliferone, a stress metabolite of chamomile. Lipid peroxidation was also not affected by even 120 lM Cd. Cd accumulation was approximately seven- (60 lM Cd treatment) to eleven- (120 lM Cd treatment) fold higher in the roots than that in the leaves. At high concentrations, it stimulated potassium leakage from the roots, while at the

lowest concentration it could stimulate potassium uptake. The results supported the hypothesis that metabolism was altered only slightly under high Cd stress, indicating that chamomile is tolerant to this metal. Preferential Cd accumulation in the roots indicated that chamomile could not be classified as a hyperaccumulator and, therefore, it is unsuitable for phytoremediation. Keywords Cadmium Æ Coumarins Æ Malondialdehyde Æ Matricaria chamomilla Æ Lipid peroxidation Æ Potassium leakage Abbreviations GMCAs (Z)- and (E)-2-b-D-glucopyranosyloxy4-methoxycinnamic acids MDA Malondialdehyde

´ˇ ˇ ˇ´ J. Kovacik Æ M. Backor Æ M. Repcak (&) Department of Botany, Institute of Biology and ˇ ´ Ecology, Faculty of Science, P. J. Safarik University, ´ ˇ Manesova 23, 041 67 Kosice, Slovak Republic e-mail: J. Tomko Department of Non-Ferrous Metals and Waste Treatment, Faculty of Metallurgy, Technical ´ ˇ University, Letna 9, 042 00 Kosice, Slovak Republic

Cadmium (Cd) pollution is a global concern as this metal not only persists in living organisms, but it is also transferred through the food chain to humans (Wagner 1993). In plants, it is taken up by the roots via calcium channels or Zn and Mn transporters (Zhao et al. 2002). After being taken up by the roots and partial translocation to the



Plant Growth Regul (2006) 50:239–247

shoots, it affects different aspects of plant metabolism. One of the symptoms of Cd toxicity is the perturbation of the plant–water relationship since it permeates through calcium channels in the leaves (Perfus-Barbeoch et al. 2002). In many plants, this results in the closure of stomata followed by a decrease in transpiration and inhibition of photosynthesis through an adverse effect on chlorophyll metabolism (Sandalio et al. 2001; Chaffei et al. 2004; Hsu and Kao 2004). This causes growth inhibition and imbalances in nutrient levels (Sandalio et al. 2001; Chaffei et al. 2004). The heavy metals cause oxidative stress resulting from the generation of reactive oxygen species (ROS). ROS cause damage to biomolecules, such as lipids (Shah et al. 2001), and can induce Ca2+ influx and K+ efflux in root cell protoplasts (Demidchik et al. 2003). Cd also induces changes in the membrane functionality by altering the lipid composition (Hernandez and Coke 1997), even though it is a non-redox metal, and is unable to produce ROS directly on its own ´ (Sanita di Toppi and Gabbrielli 1999). In plants, lipid peroxidation is usually quantified using malondialdehyde (MDA), which forms a coloured product with thiobarbituric acid (TBA), the so-called thiobarbituric acid reactive substances (TBARS). Because MDA is a result of lipid peroxidation that leads to the loss of membrane integrity, the leakage of ions is usually observed after exposure to heavy metals, e.g. Cr or Pb (Malkowski et al. 2002; Rai et al. 2004). The ability of Cd to induce MDA production is lower than that of Fe or Cu in both vascular ˇ plants and algae (Gallego et al. 1996; Backor unpublished results). On the other hand, high lipid peroxidation and strong oxidative stress occurred following exposure of Ceratophyllum demersum to 10 lM Cd (Aravind and Prasad 2003). In Phaseolus vulgaris, 94-h exposure to 5 lM Cd led to a significantly higher amount of MDA in all the plant organs investigated (Chaoui et al. 1997). Therefore, the sensibility of plants to metals is species-specific, and depends also on the concentrations tested and the exposure time. Exposure to Cd and other heavy metals has led to the evolution of various strategies for metal tolerance among the plant species. One of the

strategies employs active accumulation of metals in the shoots to exceptionally high concentrations, even though the metals are not available at high concentrations in the soil (Pollard et al. 2002). This phenomenon is known as hyperaccumulation. For Cd, the threshold level conventionally used to define this ability is 100 mg kg–1 of shoot dry mass (Baker and Walker 1990; Pollard et al. 2002). Besides, the metal-hyperaccumulating plants are endemic to soils that have high metal content; therefore, they are usually termed as strict metallophytes (Pollard et al. 2002). A stimulatory effect on growth is also a distinctive characteristic of hyperaccumulators (Pollard et al. 2002; Roosens et al. 2003). So far, only Thlaspi caerulescens has been identified as a Cdhyperaccumulator with some of its population accumulating up to 10,000 mg kg–1 shoot dry mass without suffering phytotoxicity in hydroponic conditions (Zhao et al. 2002). As reported by Roosens et al. (2003), the Cd content in the shoots of T. caerulescens from different localities including Cd non-contaminated areas was always much higher than that in the roots; the shoot:root ratio was 2.0–6.5 when the plant was cultivated in the presence of 3 and 30 lM Cd. Many of metaltolerant plants are facultative metallophytes, of which metal excluders are the most frequent. These plants can grown on both normal and metalliferous substrates and preferentially accumulate metals in the roots (Pollard et al. 2002). Chamomile (Matricaria chamomilla) is reported to accumulate high amounts of Cd preferentially in ˇ ´ the roots and also in anthodia (Grejtovsky and Pirc ´ ´ ˇ ´ 2000; Kral’ova and Masarovicova 2003; Chizzola and Mitteregger 2005), indicating that it belongs to the group of facultative metallophytes or metal excluders. However, detailed physiological studies on the impact of Cd on this medicinal plant are not ´ available. Grejtovsky et al. (1998) studied the effects of Cd on secondary metabolites of chamomile, and did not observe any changes in apigenin7-glucoside and other derivatives in anthodia. On the other hand, the quantities of two coumarins in the leaves, herniarin and umbelliferone, as well as herniarin glucosidic precursors (Z)- and (E)-2b-D-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs), were affected by foliar application of ˇ´ Cu2+ ions and biotic stress (Repcak et al. 2001;


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ˇ ´ Eliasova et al. 2004). These two stress factors resulted in a decrease in GMCAs, but an increase in herniarin as well as umbelliferone as compared to the control. However, nutritional starvation, such as nitrogen deficiency, did not cause this pattern of coumarins dynamics, indicating the presence of other mechanisms governing their ´ˇ accumulation (Kovacik et al. 2006). The main aim of our hydroponic experiment was to study the influence of low (3 lM) and high (60 and 120 lM) Cd levels on selected physiological aspects of 4-week-old plants of M. chamomilla. After 10 days’ exposure, plant growth, nitrogen, chlorophyll and coumarin contents were measured. Cd accumulation in the leaves and roots was determined in order to indicate whether this medicinal plant was a Cd-hyperaccumulator or metal excluders. The extent of lipid peroxidation (determined as MDA production) and potassium leakage from the roots were also investigated.

Estimation of chlorophyll and nitrogen The total chlorophyll content was estimated according to the relationships proposed by Wellburn (1994). Samples were prepared by the extraction of whole fresh leaf rosettes in methanol (1 g of fresh mass in 100 ml) at ambient temperature (20°C). The nitrogen content was ´ˇ estimated by the Kjeldahl method (Kovacik et al. 2006) in five different plants for each Cd treatment. HPLC estimation of coumarins and CdCl2 application on the leaves The contents of (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxycinnamic acids, herniarin and umbelliferone were estimated by gradient HPLC ˇ´ (Repcak et al. 2001) in seven different plants for each Cd treatment including the control. Dried leaves were extracted with 80% methanol. The coumarin contents in the leaves sprayed with an aqueous solution of 1% CdCl2 Æ 2 1/2H2O were estimated in a parallel experiment, and measured at 24, 48 and 120 h after Cd application. Estimation of Cd content and potassium leakage The total Cd content in leaf rosettes and roots was estimated by flame atomic absorption spectrometry in five different plants for each treatment using Perkin Elmer 3030B spectrometer (Perkin–Elmer Corp., Norwalk, Connecticut) equipped with deuterium lamp for background correction and air/acetylene flame, wavelength 228.8 nm. Samples were prepared by digestion of 50 mg (dry mass) of leaf rosettes or roots in HNO3 and H2O2 (5:3, v/v) at 90°C to the whole acid evaporation. Dry residue was dissolved in HNO3 (1%; Suprapur, Merck, Darmstadt) and diluted to the final volume of 10 ml. After 10 days’ exposure to Cd, potassium leakage from the roots of Cd-treated plants was estimated by the same method as described above (wavelength 710 nm) in a parallel experiment. Leakage was determined as the difference in potassium concentrations in the spent liquid

Materials and methods Plant cultivation Twenty-one-day old plants of M. chamomilla L. (tetraploid ‘Lutea’; Asteraceae) germinated in sand were transplanted to slightly modified Hoa´ˇ gland hydroponic solution (Kovacik et al. 2006). Ten plants per litre were cultivated in plastic boxes with 7 l of solution (70 plants per box) with continual aeration under a 12-h photoperiod (cool white fluorescent tubes TLD 36 W/33 Philips, France; PPFD: 210 lmol m–2 s–1 at the leaf level). Plants cultivated in hydroponics for 4 weeks were used in the experiment. Cd was supplemented in the medium as CdCl2 Æ 2 1/2H2O to the final concentration of 0, 3, 60 and 120 lM. All the treatments were applied twice, at the start of the experiment and after 5 days. Control and Cd-treated plants were collected mid-day at the start of the experiment and after 10 days of exposure to Cd, and dried at 95°C. The roots were carefully dried on filter papers. Fresh and dry masses of both the leaf rosettes and roots were estimated in order to determine the relative water content [100 – (dry mass · 100/fresh mass)].



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media between the Cd-treatment and the control (145 mg l–1). The results were expressed in mg K+ g root dry mass–1. Membrane lipid peroxidation Lipid peroxidation was measured as the amount of MDA determined by the TBA reaction (Esterbauer and Cheeseman 1990). Fresh leaves or roots (0.2 g) were homogenized in 0.1% (w/v) trichloroacetic acid (TCA) in a final volume of 1.5 ml. The homogenate was centrifuged at 10,000g for 15 min. To 0.5 ml of supernatant, 1.5 ml of 20% TCA containing 0.5 % (w/v) of TBA was added, and the mixture was heated at 95°C in a water bath for 30 min followed by rapid cooling on ice. The mixture was centrifuged at 4,000g for 10 min again. The absorbance of supernatant was measured at 532 nm (using extinction coefficient of MDA–TBA complex, 155 mM–1 cm–1) and corrected for non-specific absorption at 600 nm. The results were expressed in lmol MDA g dry mass–1. Statistical analyses One-way analysis of variance and Tukey’s pairwise comparisons (MINITAB Release 11, Minitab Inc., State College, Pennsylvania) were used to evaluate the significance of differences in parameters shown in Figs. 1 and 4 (P 6 0.05). The mean data on coumarin-related compounds after exogenous application of 1% CdCl2 (Fig. 2) and MDA content (Fig. 3) were compared using Student’s t-test. One plastic box containing 70 plants represents one replication for each treatment. Plants in which coumarin-related compounds after foliar application of 1% CdCl2 were measured and plants for measurements of potassium leakage were cultivated using the same volume of nutrient solution and plants density in separate boxes.

Results Throughout the experiments no visible symptoms of Cd toxicity were apparent in chamomile leaves,

but the roots exposed to 60 and 120 lM Cd turned brownish compared to the whitish roots of control plants and those cultivated in the presence of 3 lM Cd. After 10 days’ Cd exposure, there were no significant changes in dry mass of leaf rosettes and the roots (Fig. 1). Leaf water content decreased when Cd was tested at high concentrations, but at 3 lM level it did not significantly affect the leaf water content as compared to the control. There was a significant reduction in chlorophyll content due to Cd presence in the medium, while nitrogen content remained constant in leaf rosettes and roots at all the levels of Cd as compared to the control (Fig. 1). After 10 days, Cd at all the levels increased the GMCAs; however, the differences were not significant (Fig. 1). Interestingly, the herniarin content was not influenced by Cd. The concentrations of umbelliferone were 0.0029–0.0155 mg g dry mass–1 in the controls and 0.0025–0.0295 mg g dry mass–1 after exposure to various levels of Cd. The results also showed that the exogenous application of 1% CdCl2 on the leaves of chamomile plants did not alter the amount of either GMCAs or herniarin (Fig. 2), and umbelliferone content was similar to values as described above. The amount of Cd accumulated in the leaves and in the roots increased with increasing levels of Cd in the solution (Fig. 1). However, Cd content was always higher in the roots than that in the leaves at all the levels of exogenous Cd application. After 10 days’ exposure, its content increased as follows (leaves/roots, n-multiple of control): 13/19 (3 lM Cd), 95/124 (60 lM Cd), 103/196 (120 lM Cd). Cd content in control plants was 2.49 mg kg dry mass–1 and 14.61 mg kg dry mass–1 in the leaves and roots, respectively. The Cd shoot:root ratios were 0.11 (3 lM Cd), 0.13 (60 lM Cd) and 0.09 (120 lM Cd). The results showed that there was no significant alteration in MDA production in the leaf rosettes and roots exposed to 120 lM of Cd as compared to the control (Fig. 3). Potassium leakage from the roots occurred when the plants were exposed to higher Cd concentrations (60 and 120 lM). However, Cd at a low concentration (3 lM), stimulated potassium uptake by the roots (Fig. 4).


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310 a Dry mass (mg plant ) 248 b 186 124 a

93 a Leaves ab Roots Leaf water content (%) ab 92 91 90 89 88 87 86 80 Nitrogen content (mg g dry mass –1) a ab bc c c


a ab bc c






0 Total chlorophyll (mg g dry mass –1) 28

Leaves 60 a a 40 ab a b a b a



b a




18 GMCAs Herniarin

10000 Leaves Roots b a

Coumarins (mg g dry mass –1)


a ab

Cd content ( µg g dry mass )

a 12

1000 c 100 d 10 d d 1 C0 C10 3 60 120 d b a

bc c



a 0 C0

a C10 3






Cadmium (µM)
Fig. 1 Dry mass production (n = 15), leaf water content (n = 15), total chlorophyll (n = 5), nitrogen content (n = 5), coumarin accumulation (n = 7) and Cd content (logarithmic scale, n = 5) in various plant parts of M. chamomilla

Cadmium (µM)
under varying Cd stress. C0 and C10 represent the controls at the start and after 10 days’ Cd exposure, respectively. Means with common letters are not significantly different at P 6 0.05, according to Tukey’s test


12 Control GMCAs (mg g dry mass–1) ns 8 1% Cd ns ns

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1200 Malondialdehyde (µmol g dry mass –1) Control 120 µM Cd 900 ns 600 ns Leaf rosettes ns ns



0 1.2 Herniarin (mg g dry mass–1)

0 350 Malondialdehyde (µmol g dry mass –1) ns 280 ns 210 ns Roots ns

ns 0.8





0 24 48 Time (h) 120

0 1 2 6 10

Time (days)

Fig. 2 Effect of exogenous application of 1% CdCl2 Æ 2 1/2 H2O aqueous solution on (Z)- and (E)-2-b-D-glucopyranosyloxy-4-methoxycinnamic acids (GMCAs) and herniarin contents in the leaves of Matricaria chamomilla. ns = significantly not different from the control at P 6 0.05, according to Student’s t-test

Fig. 3 MDA production in the leaf rosettes and roots of M. chamomilla following exposure to 120 lM Cd for varying periods. ns = significantly not different from the control at P 6 0.05, according to Student’s t-test

Discussion The brownish colour of the roots at high Cd concentrations, the only visible symptom of Cd presence in hydroponic culture, has earlier been described in other plants, e.g. Beta vulgaris (Larbi et al. 2002), and is apparently induced by the accumulation of phenolics and other compounds in the roots (Ederli et al. 2004). However, a marked reduction in growth and chlorophyll content due to 10–50 lM Cd as observed in Lycopersicon esculentum (Chaffei et al. 2004) or Pisum sativum (Sandalio et al. 2001) is in contrast with our results. We did not observe any significant

growth inhibition in chamomile when the plants were exposed to even comparatively higher Cd concentrations (60 and 120 lM). On the other ˇ ´ hand, Grejtovsky and Pirc (2000) reported a decline in shoot biomass of chamomile (12–15%) when cultivated in Cd-enriched soil (30 mg kg–1). We found that the reduction in chlorophyll content was relatively small (about 16%) as compared to the control when the plants were exposed to the highest cadmium level. A significant decline in water content of the plants at higher Cd concentrations supports the hypothesis that Cd affects the water status more than gas exchange in chamomile. This is in agreement with the results obtained by Costa and Morel (1994) in Lactuca sativa cultivated in the presence of 100 lM Cd.


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50 Potassium leakage (mg g root dry mass –1) a 25 a


0 3 –25 b –50 Cadmium (µM) 60 120

Fig. 4 Potassium leakage from the roots of M. chamomilla after 10 days’ exposure to varying Cd stress. Means with common letters are not significantly different at P 6 0.05, according to Tukey’s test

In the present study, we did not observe any significant changes in nitrogen content of the leaves and roots due to Cd presence, although its distribution in plants has been reported as strongly influenced by a concentration as low as 10 lM (Sandalio et al. 2001). In a long-term experiment, no significant changes in nitrogen content could be observed when the same chamomile cultivar was cultivated in the soil in the presence of 30 mg ˇ ´ Cd kg–1 (Grejtovsky and Pirc 2000). The coumarin contents in the leaves of chamomile were affected by foliar CuCl2 application with an increment in herniarin content, but a decline in ˇ ´ ˇ´ GMCAs (Eliasova et al. 2004). Similarly, Repcak et al. (2001) also observed this trend in chamomile in response to biotic stress. However, in contradiction to these results, we observed that Cd stress significantly increased the GMCAs in the present study. Increased production of GMCAs in nitrogen-deficient chamomile plants was also observed ´ˇ in our earlier study (Kovacik et al. 2006), and it is apparently related to phenylalanine ammonialyase (PAL) activity. This enzyme is essential for the formation of cinnamic acid and related structures, which are required as precursors for compounds, such as coumarins (Harborne 1980); with respect to active centre structure, PAL can functions in stressed plant tissue (Ritter and Schulz 2004). Interestingly, in the present study, the herniarin content was not influenced by Cd stress.

Therefore, the observed changes of GMCAs and herniarin are not ‘typical’ answer of these compounds to Cd stress in comparison with the results ˇ´ obtained in previous studies (Repcak et al. 2001; ˇ ova et al. 2004). This assumption is also sup´ Elias ported by the presence of a low amount of umbelliferone, which is known as a stress metabolite of ˇ´ chamomile (Repcak et al. 2001). Also non-stress pattern of GMCA-herniarin relation following Cd application on the leaves is in contrast with the ˇ´ reported effects of Cu (Repcak et al. 2001; ˇ ´ Eliasova et al. 2004), indicating substantial differences between the effects of these two heavy metals on chamomile. This can be explained in part by the lack of redox ability of Cd in contrast to Cu ´ (Sanita di Toppi and Gabbrielli 1999). Our results presented in this study support the previous results that in chamomile Cd is mainly ˇ ´ accumulated in the roots (Grejtovsky and Pirc ´ ´ ˇ ´ 2000; Kral’ova and Masarovicova 2003), and, therefore, should be considered as a facultative metallophyte or metal excluder. However, the conventional minimal content of 0.01% Cd in the leaf rosettes (dry mass) defined for hyperaccumulating plants was recorded at 60 and 120 lM Cd (approximately twice higher than the limit). This conventional minimal limit cannot be considered as a unique criterion to determine the hyperaccumulating capacity of a plant, because it is based on observations in nature, not in hydroponic conditions. Additionally, since the translocation of ions to the shoots in hyperaccumulating plants probably occur at the xylem-loading stage (Pollard et al. 2002 and the references therein), the higher Cd content at 60 and 120 lM exogenous Cd supply can indicate damage to ionspecific transporters, thus accounting for its unspecific increase in the shoot. The assumption that chamomile is not a hyperaccumulator can also be supported by the fact that only 9.52% increase of Cd content in the shoots occurred with an increase in exogenous Cd supply from 60 to 120 lM. Therefore, we conclude that chamomile is tolerant to Cd stress under tested conditions, and should be considered as a metal excluder. Cd has been reported to enhance lipid peroxidation in many plant species, e.g., Helianthus annuus (Gallego et al. 1996), Oryza sativa (Hsu and Kao 2004) and P. sativum (Sandalio



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et al. 2001). However, in the present study, we observed that Cd even at 120 lM did not cause any significant increase in MDA production. This indicated an indirect or undetectable Cd effect on the plasma membrane of chamomile cells. Earlier ˇ ´ Grejtovsky and Pirc (2000) showed that potassium content in chamomile shoots and roots was not affected by Cd in a long-term soil experiment. In P. sativum, a decrease in potassium content occurred when the plants were cultivated in the presence of 10–50 lM Cd in hydroponics, indicating potassium leakage into the culture medium (Sandalio et al. 2001). In agreement with these findings, we also observed potassium leakage from chamomile roots into the nutrient solution supplemented with 60 and 120 lM Cd. This indicates that even though membrane damage could not be detected, Cd perhaps directly affects K+-channels resulting in K+-efflux from root cells as described earlier (Demidchik et al. 2003). However, Cd at 3 lM had a negative effect on potassium efflux, indicating a stimulatory effect on its uptake. In conclusion, chamomile appears to be tolerant to Cd since overall metabolism was only slightly altered by high concentrations of this metal (60 and 120 lM). However, as it has been confirmed that chamomile has the ability to accumulate Cd, the soils used for the production of chamomile drug (flos chamomillae) is required to be checked. Preferential Cd accumulation in the roots and fine root system did not support the classification of chamomile as a hyperaccumulator and, therefore, it is not suitable for phytoremediation.
Acknowledgements This work was supported by the grant of the Slovak Grant Agency VEGA (1/3260/06). The ´ authors thank Prof. Dianne Fahselt and Prof. Luigi Sanita di Toppi for constructive comments on the manuscript. ˇ ´ ´ Mrs. Anna Michalcova and Mrs. Margita Buzinkaiova are also acknowledged for their excellent technical assistance.

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