Plant Soil (2007) 299:43–53 DOI 10.1007/s11104-007-9359-5
REGULAR ARTICLE
Arabidopsis arenosa (Brassicaceae) from a lead–zinc waste heap in southern Poland – a plant with high tolerance to heavy metals E. Przedpełska & M. Wierzbicka
Received: 13 December 2006 / Accepted: 16 July 2007 / Published online: 19 September 2007 # Springer Science + Business Media B.V. 2007
Abstract The morphological traits and tolerance to heavy metals (zinc, cadmium and lead) of two populations of Arabidopsis arenosa (Brassicaceae) were compared. One population was from a zinc–lead waste heap in Bolesław near Olkusz (southern Poland), the other one from the Kampinoski National Park (central Poland). Biometric measurements were done in the field and repeated after cultivation under controlled conditions (garden soil, phytotron chamber). Significant heritable morphological differences between the two populations were found. The plants from the waste-heap are smaller in comparison with the reference population, and their leaves are narrower, thicker with fewer trichomes, indicating. genetic adaptation to xerothermic conditions. The level of tolerance to heavy metals (zinc, cadmium, and lead) was compared by the root test. Very high tolerance to the three metals tested was found in the waste-heap population. Its tolerance exceeded that of four other predominant plant species populations growing on the same waste heap that had previously Responsible Editor: SE: Henk Schat. E. Przedpełska : M. Wierzbicka (*) Department of Ecotoxicology, Institute of Experimental Plant Biology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland e-mail:
[email protected] E. Przedpełska e-mail:
[email protected]
been tested in our laboratory. We consider the wasteheap population of A. arenosa to be a very suitable ecotype for the study of heavy metal tolerance mechanisms in plants. Keywords Arabidopsis arenosa (synonym: Cardaminopsis arenosa) . Zinc–lead waste heap . Morphological traits . Tolerance index
Introduction Strong anthropogenic pressure is the cause of environmental pollution which is endangering the natural environment and human health. Heavy metals are among the most dangerous pollutants. Removing them from soil is a difficult, lengthy, and very expensive process. A chance to resolve this problem lies in phytoremediation. This is a process whereby plants growing on a polluted site remove the contaminations. Some plant species have developed protective mechanisms that enable them to survive under extreme environmental conditions. Plant species (varieties or ecotypes) growing in areas naturally rich in heavy metals have evolved genetically fixed mechanisms of tolerance to them. Members of the Brassicaceae family have attracted much interest. This family has the largest number of identified heavy metal hyperaccumulators – 87 (Peer et al. 2003; Prasad and de Freitas 2003). Arabidopsis arenosa (synonym: Cardaminopsis arenosa) also belongs to this family. A. arenosa is found throughout
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of Europe in disturbed habitats at forest margins, roadsides, railroad tracks, river banks and in grassy and sandy areas, from near sea level to 2,000 m (Al-Shebaz and O’Kane 2002). This species has often been described as commonly occurring on heavy-metal polluted sites. In Poland, it is the dominating species on all calamine (zinc–lead) sites (Jędrzejczyk 2004). A. arenosa is also a common species on some waste heaps in Austria (Kudejka 2002). Grodzińska et al. (2001) have reported that A. arenosa is the common species in the vegetation, seed rain and seed bank of old as well as new waste heaps in the Olkusz region (southern Poland). Silver and lead ores have been mined and smelted in the vicinity of Olkusz in Poland since the thirteenth century, and in the eighteenth century, zinc was added to the metals mined and processed there. There are numerous zinc–lead waste heaps (calamine waste heaps) in this area, the oldest ones being there for more than hundred years. The study described in this paper was conducted on the oldest waste heap. It is located in Bolesław near Olkusz, is about 130 years old and has since long been the site of numerous botanical studies (Wójcicki 1913; Pax 1918; Dobrzańska 1955; Grodzińska et al. 2001). The reference site used for comparison has a very low level of heavy metal pollution and is located in the Central Mazovian Lowland of Poland. On the basis of the literature it can be said that A. arenosa is a nonmycorrhizal species (Pawlowska et al. 1996) commonly occurring in Central Europe (Hultén and Fries 1986; Hegi 1919). We did not find any reports about its tolerance to heavy metals, although A. arenosa is frequently found on metalliferous soils (Jędrzejczyk 2004; Kudejka 2002). A. arenosa is closely related to hyperaccumulators of zinc and cadmium, such as Arabidopsis halleri and Thlaspi caerulescens, as well as to A. thaliana, which is why A. arenosa may be a very interesting species, or even a model organism, for studying heavy metal tolerance mechanisms in plants (Clauss and Koch 2006). The objective of this study was to compare the population of A. arenosa plants dominating the zinc– lead (calamine) waste heap in Bolesław near Olkusz with the population occurring in an area with a low level of heavy-metal pollution, the Kampinos National Park. We were particularly interested in determining the level of heavy metal tolerance and in characterizing the adaptations responsible for the
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significant reproductive success of this species in areas highly polluted by heavy metals.
Materials and methods Field studies The field studies on A. arenosa plants were conducted: (1) in an area heavily polluted by heavy metals – a calamine (zinc–lead) waste heap in Bolesław near Olkusz, Poland. The waste heap in Bolesław is situated on the western edge of the Cracow and Cze$stochowa Jura, about 43 km west of Krakow. The concentrations of heavy metal in the soil are 40,000 mg/kg zinc, 1,650 mg/kg lead and 170 mg/kg cadmium and the pH is 7.3 (Godzik 1984). (2) In an area with little heavy metal pollution – the Kampinos National Park, in the Central Mazovian Lowland of Poland. One of the sites where A. arenosa occurs in this area is the village of Laski in the borough of Izabelin. This village is located in the buffer zone of the Kampinos National Park Biosphere Reserve. The terrain here is mainly sandy and covered by pine forests, which are part of the buffer zone of the Kampinos National Park. The Kampinos National Park Biosphere Reserve is known as “Warsaw’s green lungs” and lies 12 km from the Warsaw city center. The biometric measurements in the field were done in 30 individuals per population. The plants for measurements were randomly drawn from the flowering individuals. Nine traits were measured: the number of leaves per plant, the length and width of five leaves of each plant, the height of the plants, the number of reproductive shoots and flowers per plant, the length of five siliques per plant, the total number of seeds per silique and the weight of the seeds. Laboratory studies A total of 110 plants per population were cultivated from seeds collected in the field. The plants were grown in a phytotron chamber (16/8 h photoperiod, temperature 24±1°C). The complete vegetative period of the plants lasted 2.5 months. The biometric measurements were done in the phytotron chamber after the plants had entered the reproductive phase (i.e., when the first bud blossomed), which was determined individually for each plant. The biometric measurements were carried on
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until the end of the growing season under the phytotron chamber conditions. The plants for measurements were randomly drawn from the flowering individuals. In this way, the plants were of the same biological age when the measurements were taken. Twenty one morphological traits were measured: the number of leaves per rosette, leaf length, width, thickness, leaf water content, number of stomata on both sides of leaves, number of trichomes on both sides of leaves, plant height, number of reproductive shoots, degree of branching of the main reproductive shoot, diameter of the main reproductive shoot, fresh and dry weights, number of inflorescences, number of flowers per inflorescence, number of flowers per plant, seed weight, the length of siliques, the total seed numbers per silique and the rate of entering into the reproductive phase. Leaf length and width were measured with an accuracy of ±1 mm on eight randomly selected leaves from 65 randomly chosen individuals from each population. In total, 1,040 leaves were measured. The number of leaves per rosette was determined in 65 randomly selected plants from each population. Leaf thickness was measured using a micrometric microscope eyepiece on manually prepared crosssections of 50 randomly selected leaves from each population. The number of stomata was determined on an area of 426 μm2. A thin layer of nail polish was applied to upper and lower leaf surfaces. After drying, the imprint of epidermal cells was carefully removed and the number of stomata determined under a microscope with the aid of LUCIA software. The number of trichomes on both sides of the leaves was determined under a binocular microscope on a surface of 640 μm2 of five randomly selected leaves from 65 randomly selected individuals from each population. A total of 650 leaves was analyzed. Moreover, ten randomly selected individuals were used to measure (with an accuracy of ±1 mm): plant height, number of generative shoots, degree of branching of the main generative shoot, and diameter of the main generative shoot. Moreover, the rate at which the plants entered into the generative phase, number of inflorescences, and total number of flowers were determined for 110 plants per population. The number of flowers in 33 inflorescences from ten randomly chosen plants per population was counted. The seeds of each population (10×50) were weighed.
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Tolerance to lead, cadmium and zinc Seeds collected from the field were germinated in Petri dishes on filter paper moistened with diluted Knop nutrient solution supplemented with A–Z trace elements (Strebeyko 1967; diluted 1:8), in the light at a temperature of 24±1°C. Next, seedlings having about 10 mm long roots were transferred to glass stands and placed in 25-l pots containing 1:8 Knop’s medium (200 g/l Ca(NO3)2 ×4H2O; 71.5 g/l KNO3; 35.5 g/l KCl; 71.5 g/l MgSO4; 28 g/l EDTA–Fe; pH=6) with trace elements. The medium did not contain PO3 4 ions to avoid precipitation of heavy metals. All pots were continuously aerated. The plants were grown in phytotron chambers (photoperiod 16/8 h, temperature 24±1°C). After 24 h of cultivation, one of the listed metal salts was added to achieve the following concentrations: 1. 2. 3. 4. 5. 6. 7.
5.5 mg dm−3 (85 μM) Zn2+ from Zn(NO3)2 11 mg dm−3 (170 μM) Zn2+ from Zn(NO3)2 2 mg dm−3 (18 μM) Cd2+ from Cd(NO3)2 4 mg dm−3 (36 μM) Cd2+ from Cd(NO3)2 3 mg dm−3 (15 μM) Pb2+ from Pb(NO3)2 6 mg dm−3 (30 μM) Pb2+ from Pb(NO3)2 Control
The metal concentrations were chosen on the basis of preliminary experiments. Concentrations that caused reversible effects and did not excessively inhibit root growth were chosen. Root length (in mm) was measured every 48 h for 8 days. The index of tolerance, IT (Wilkins 1957, 1978) to these metals was calculated using the formula:
IT ¼
Length of roots exposed to metal 100% Length of control roots
Lead tolerance was tested on 65 plants per concentration, and cadmium and zinc tolerance on 46 plants per combination. A total of 314 plants were used to assess tolerance. Chromosome number Cytogenetic studies were“ performed in order to determine the chromosome number in the somatic cells of plants from both populations. Root tips were incubated in a 2% orcein stain in 45% acetic acid for 1
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Plant Soil (2007) 299:43–53
Fig. 1 Overall habit of 2month-old Arabidopsis arenosa plants. a Reference population – tall plant with one reproductive shoot. b Waste-heap population – short plant with numerous reproductive shoots
hour. The stained meristematic region of the root was macerated and examined under a light microscope at 600×. The number of chromosomes was counted in cells being in the metaphase stages of mitosis. Statistical analysis The results were subjected to statistical analysis by Student t test (α=0.05). All of the results present on the graphs and in the tables are arithmetic averages and standard errors (SE) is marked.
Results Morphological differences between the populations in the field On the Kampinos National Park stand, A. arenosa plants had two times bigger size than those from the waste-heap stand. They were higher, had three times more reproductive shoots, and consequently four times more flowers per individual (Fig. 1a,b; Table 1). They also had two times longer siliques which contained two times more seeds. Hence the total seeds number per plant was higher in reference population than in the waste-heap population. Only for seed weight the trait level value was higher in the waste heap population than in the reference one. Morphological differences between the populations under identical controlled phytotron conditions The marked differences observed in the field (described above) between A. arenosa plants occurring in the waste-heap and Kampinos stands may have been caused by differences in the conditions prevailing in these stands. To determine if there are any genetically
established differences between these two populations, a comparative culture of these plants on normal garden soil was conducted under identical conditions (phytotron chamber). The results are presented in Table 2. Significant differences were found between the plants in both populations (Figs. 1a,b; 2a,b; 3a,b; 4a, b; 5; and 6). The reference plants were on average 1.7 times higher than those from the waste-heap population (Fig. 1a, b; Table 2). They had 1.5 times broader leaves than those from the waste-heap stand. But the waste-heap plants had two times more leaves per rosette and 1.4 times thicker leaves than those from Kampinos National Park (Table 2). The waste heap plants had about ten reproductive shoots, whereas those in the reference population had an average of two more strongly branched reproductive shoots (Fig. 1a,b). This did not affect the final number of flowers per plant, however, which averaged 118 in Table 1 Comparison of morphological traits of Arabidopsis arenosa plants from the waste heap in Bolesław near Olkusz and from The Kampinos National Park, Laski near Warsaw Stand: Bolesław Stand: Kampinos waste heap National Park Plant height (mm) Number of leaves per rosette Leaf width (mm) Leaf length (mm) Number of reproductive shoots Number of flowers Seed weight (μg) Silique length (mm) Number of seed per one silique
125.5±5.6* 16.0±0.9
244.9±13.5 16.9±1.3
5.0±0.2* 15.8±0.5 2.5±0.5*
11.7±0.5 35.8±1.1 7.9±0.6
5.0±0.5* 91±6.0* 13.9±0.4* 11.9±1.5*
20.4±2.6 52±4.2 23.7±0.5 22.5±1.8
Values are mean±SE. Asterisks indicate significant differences at P
