Plant response to heavy metals and organic pollutants in cell culture and at whole plant level

R e v i e w A r t i c l e s P l a n t R e s p o n s e

Plant Response to Heavy Metals and Organic Pollutants in Cell Culture and at Whole Plant Level Avi Golan-Goldhirsh 1~, Oz Barazani 1, Ales Nepovim 2, Petr Soudek 2, Stanislav Smrcek 3 Lenka Dufkova 3, Sarka Krenkova 3, Kim Yrjala 4, Peter Schr6der s and Tomas Vanek 2

1Ben-Gurion University of The Negev, The Jacob Blaustein Institute for Desert Research, Albert Katz Department of Dryland Biotechnologies, Desert Plant Biotechnology Laboratory, Sede Boqer Campus, 84990, Israel

2Institute of Organic Chemistry and Biochemistry, AS CR, 166 10 Praha 6, Czech Republic 3 Faculty of Natural Sciences, Charles University, Hlavova 2030, Praha 2, Czech Republic 4 University of Helsinki, Department of Biosciences, Division of General Microbiology, P.O.B. 56, FIN-00014 Helsinki, Finland s Institute for Soil Ecology, GSF National Research Center for Environment and Health, Neuherberg, Ingolstaedter Landstrasse 1,

D-85758 Oberschleissheim, Germany

*Corresponding author ([email protected])

1 I n t r o d u c t i o n

Phytoremediation has become a growing endeavour for many research groups from various scientific disciplines, it therefore creates many opportunities for interdisciplinary cooperation in remediating of man-made environmental contamination. Phytoremediation, as the word itself suggests (from the Greek gro~6, plant), is a green, plant-based remediation. It can be used to clean-up soil and water from heavy metals and organic pollutants (xenobiotics). In most cases, these pollutants are not required by the plant for its regular metabolism. These compounds induce general tolerance mechanisms, degradation of xenobiotics or hyperaccumulation of heavy metals, which are quite effective in certain plant species.

Remediation of xenobiotics is different from that of heavy metal contaminants. In phytoremediation of hydrocarbons, or of nitrogenated and chlorinated organic compounds, the primary goal is to get biodegradation of the pollutants to harm- less substances such as CO 2, nitrate, chlorine and ammonia [7]. Remediation of heavy metal contaminants is based on removal of the contaminant from the soil, but there is no op- tion of degradation, since these substances are essentially im- mutable. The plant mechanisms involved in xenobiotic remediation are extraction, phytodegradation, rhizodegradation, sequestration and evaporation [25]. The possible plant mechanisms involved in heavy metals remediation are adsorption, transport, translocation, sequestration into vacuoles, hyperaccumulation and, in some cases, volatilization [20].

One obvious realization of recent European Cooperation in the field of Scientific and Technical Research (COST) meet- ings appeared to be the gap between the very fast accumu- lating data from in vitro, small scale experiments either in cell culture, hydroponics, or other simulated media, and in vivo, whole plant experiments under field conditions.

The use of in vitro systems enabled dissection of the complex system of plant, soil, and microbial interaction in order to evaluate the effect of stress factors on metabolism, specific enzymes and metabolites involved in the plant response

JSS - J Soils & Sediments 4 (2) 133 - 140 (2004) �9 ecomed publishers, D-86899 Landsberg, Germany and Ft. Worth/TX * Tokyo �9 Mumbai o Seoul �9 Melbourne o Paris

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Plant Response Review Articles

t o the pollutants. There is an advantage in testing the effects of a contaminant on a well-defined xenic cell culture under controlled conditions [10]. Cell cultures are also a useful system for metabolic engineering and for obtaining rapid evidence of the ecotoxicological behaviour of chemicals and heaw metals in plants with less analytical expense. Screening of plant species would be done much easier and faster under controlled conditions and independently of the external cli- matic conditions. However, extrapolation to the whole plant system under field conditions is not fully straightforward.

In this position paper, we put together a brief overview and description of current research run in our laboratories, which encompasses a range of experiments from in vitro tissue culture experiments all the way to the whole plant, in situ experiments. We hereby present some of our recent experi- mental results for the different levels of analysis of the interaction between the plant and pollutants, i.e. heavy metals and xenobiotics, in cell, tissue culture and whole plant experiments in soil.

2 Cell and Plant Culture Experiments: Some enzymes related to xenobiotic degradation

Plant biotransformation activity is preferably studied by using plant cell cultures or in vitro cultivated whole plants. Ex- periments under field conditions involve many environmental variables, which are less controllable. Furthermore, the transformation reactions catalysed by microorganisms, light or other abiotic factors cannot be distinguished from specific plant reactions [28]. The advantages of plant tissue cultures can thus be summarized as follows: 1. Constant reproducible growth during long periods 2. Cultures are sterile 3. Physiological activities can be controlled by environmental conditions 4. Large cell surface area increases the rate of penetration of tested

compounds into the ceils 5. Simple isolation and analysis of metabolites

Cell culture therefore offers a useful tool for investigation of metabolic t ransformation of organic compounds and xenobiotics. However, cell cultures consist of organ-undif- ferentiated cells and therefore are not appropriate for the study of uptake and transport processes. Organ root culture is therefore often used for the study of transport mechanism of contaminants in roots with a focus on the interface between root tip, root hairs and the rhizosphere.

2.1 Effect of nitroaromatics on horseradish root culture glutathione S-transferase and peroxidase

A great deal of research in phytoremediation of xenobiotics is directed in recent years to nitroaromatic compounds. Syn- thetic nitroaromatics are of considerable industrial impor- tance [11] and some of these compounds such as nitrobenzene, nitroglycerine and/or 2,4,6-trinitrotoluene (TNT) are produced on a large scale. T N T belongs to a group of nitroaromatic compounds, which contaminate large areas around ammunition factories and their storage sites all over the world. Due to its high toxicity to living organisms [38], and persistency in the soil, nitroaromatics represent a seri- ous environmental problem [34].

Recent evidence suggested that herbicides and other xenobiotics are recognized, transported, and metabolised by plants in similar ways as the natural metabolites [26]. It was therefore assumed that nitroaromatic compounds might be removed from the soil by plants. In this study, the effect of TNT and its derivatives on enzymatic activity of glutathione S-transferase (GST) and peroxidase (POX) was tested in 'hairy root' cultures of horseradish (Arrnoracia sp.).

TNT and its aminoderivates, 4-aminodinitrotoluene (ADNT), a mixture of isomers of diaminonitrotoluenes (DANTs) and 2,4-dinitrotoluene (DNT), were dissolved in 0.1% dimethyl sulfoxide (DMSO) and added to the growth medium for 6 and 27 hours at concentrations of 0.1 m M and 1 mM. The activity of GST was measured with the following substrates: 1,2-dichloro-4-nitrobenzene {DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB). Peroxidase activity was measured by guaiacol/hydrogen peroxide assay [33].

The GST activity assayed by DCNB was induced in all treated root cultures (Fig. la). Different time of exposure and concentration of ni t roaromatic compounds (NACs) caused changes in activity when compared to the control (DMSO) treated roots. When CDNB was used as substrate a smaller effect on enzyme activity was measured (Fig. lb). An induction effect was measured after 6 hours treatment with T N T and ADNT at both concentrations and by DANT (0.1 raM). A slight inhibition effect was determined in roots treated by ADNT (0.1 mM) for 6 hours, DANT (0.1 mM) and DNT (0.1 mM) for 27 hours. POX was inhibited after 6 hours by T N T and ADNT (Fig. lc). However, there was a partial recovery or induction of POX activity in ADNT after 27 hours of treatment (Fig. lc), but it was still inhibited in case of TNT. Longer exposition to DANT and DNT caused induction of activity, compared to 6 hours treatments.

In general, treatment of roots by nitroaromatics was accompanied by a decrease in the oxidized (GSSG) and reduced forms of glutathione (GSH) (Fig. 2a and b), which might be related to oxidation by reactive oxygen species. Nevertheless, high concentration of GSH was measured after 6 hours of treatment with TNT and DANT both at 0.1 mM (Fig. 2b), while the content of GSSG was increased after 27 h of treatment with DANT (1 raM) and DNT (0.1 mM) (Fig. 2a). This might point to oxidative stress and the involvement of antioxidants in the detoxification of nitroaromatics in horseradish.

2.2 Influence of pentachlorophenol on selected plant enzymes

For many years, pentachlorophenol (PCP) was the most prevalent wood preservative and its salts were widely used as fungicide, herbicide, insecticide, and moluscicide [6]. It was shown that PCP can also be generated in the environment during water chlorination, degradation of other pesticides, or burning of PCP treated biomass [1,36]. Although the direct use of PCP is restricted at present, a low-level of PCP residues is widely found in nature. Microbial degradation and the effect of environmental parameters on processes of PCP degradation were reported in detail [6,19], information on phytoremediation potential, however, is scarce. These facts were conducive to the trials presented here.

134 JSS - J Soils & Sediments 4 (2) 2004

Review Articles Plant Response

Fig. 2" Effect of nitroaromatic compounds on the concentration of GSSG (A) and GSH (B) in root tissue cultures after 6 (m) and 27 h (r-I) of treatment

Fig. 1: Activity of glutathione S-transferase after 6 and 27 h of treatment with 0.1 mM (m) and 1.0 mM (r-I) of nitroaromatic compounds determined with the following substrates: 1,2-dichloro-4-nitrobenzene - DCNB (A) and 1-chloro-2,4-dinitrobenzene - CDNB (B) and peroxidase with guaiacol/H202 substrates (C). Enzyme fraction used, ammonium sulfate precipitate at 40% to 80% saturation. The level of activity in control (not containing nitroaromatics nor solvent) and DMSO solvent control is shown on the right hand side

Zea mays plants were cultivated in aseptic Murashige and Skoog (MS) liquid medium supplemented with PCP at 14, 18 and 22 mg/l. During growth, the following parameters were evaluated: phytoextraction potential (Fig. 3), content of chlorophyll A and B, and enzymatic activity of peroxidase, [3,glucosidase and phosphoenolpyruvate carboxylase (PEPCase) (Fig. 4). Peroxidase activity was measured by the guaiacol/hydrogen peroxide assay [29]. [3-Glucosidase activity was measured spectrophotometrically using p-nitro- phenyl-13-D-glucopyranoside as a substrate [17]. PEPCase was measured spectrophotometrically at 340 nm by the decrease of NADH concentration in a coupled reaction with malate dehydrogenase [24]. Chlorophyll content was determined spec- trophotometricaUy in acetone extracts, and was calculated using the McKinney-Arnon equations [2].

Fig. 3: Removal of PCP by Zea mays from aseptic liquid growth medium. The initial concentration of PCP was: 22 mg/I (T), 18 mg/l (�9 and 14 mg/I (O). Aliquots were removed at time intervals for analysis of PCP

Zea mays tolerated PCP up to 22 mg/l (data not shown). The concentration of PCP declined by approximately 90% within 10 days of treatment (Fig. 3). The fate of PCP, whether it is degraded or accumulated in plant cells needs further

JSS - J Soils & Sediments 4 (2) 2004 135


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investigation. PCP concentrations up to 15 mg/1 stimulated peroxidase activity. The activity ranged between 0.31 lamol/ min in the control, 0.60 lamol/min at 9.2 mg/1 of PCP and reached a maximal value of 0.64 lamol/min at 14.2 mg/l of PCP, then decreased to approx. 0.25 lamol/min at 25 mg/l of PCP (Fig. 4). 13-Glucosidase activity in untreated plants was 63.4 lamol/min, and it declined under increasing concentrations of PCP (Fig. 4). The situation in a soil environment is different, since microbial retransformation of glucoside will render the pollutant less toxic, which would lead to more uptake of PCP-glucosides by the plant. Direct inhibition of ~-glucosidase by free PCP would enable further degradation of glucoside-bound PCP [16,27].

PEPCase activity increased up to a maximal level of 108.4 IJmol/ min at 15.7 mg/1PCP, while a decrease of its activity was measured at higher concentrations (Fig. 4). The highest enzyme activity measured was about 3.5 fold higher than that measured in control plants, indicating elevated metabolic activity, which can contribute to phytoremediation efficiency. The decrease in chlorophyll content with the increase of PCP concentration in the medium was from 67.2 mg/g fresh weight (FW) in control plants to 15.6 mg/g FW at 19 mg/l of PCP (Fig. 4).

Peroxidase has an important function in xenobiotic detoxification, its increased activity in PCP-treated plants is worth noting. The decrease of ~-glucosidase activity in PCP treatment could be related to the formation of water-soluble glycosides and their release from plant tissues to the environment. It can be concluded, that Zea mays can effectively transform PCP up to ca. 12 mg/1 of PCP in the medium, however this was accompanied by stress symptoms.

2.3 Biotransformation and degradation of dye precursors by callus cultures of $trombocactus disciforrnis and Epithelantha micromeris

Processes of transformation and/or degradation of organic compounds constitute a complex variety of reactions in plants and in symbiotic microorganisms. Plants are able to exude compounds to the environment, including release of enzymes, and small organic molecules [22], which are involved in these processes. Glycosides can complex organic and inorganic compounds in soil and serve as nutrients for soil microorganisms. Enzymes released to the rhizosphere by plants and

microorganisms may participate in detoxification of xenobiotics. In order to study this complex system, we have to dissect it in such a way as to understand better the role of each component before one can reconstitute the system and study its function as a whole. Therefore, in this study, in vitro cultures of cacti were used to test the effect of degradation and/or biotransformation of dye precursors by plants.

Cacti were shown to produce protective compounds, such as alkaloids, from a family of aromatic hydrocarbons (e.g. hordenine, tyramine, 3,4-dimethoxyphenethyleamine and others) [4]. Thus, it was hypothesized that the metabolic pathway of synthesis and degradation of xenobiotic aromat- ics would be more likely to be found in these plants. This represents a case of our overall strategy of searching ex- traordinary pathways for environmentally-friendly-removal of toxic compounds in selected plant species.

Cultures of E. micromeris and S. disciformis were grown on an MS medium supplemented with growth regulator (2,4-D) with the following dye precursors: 2-hydroxy-4-sulfo-6-nitro- 1-naphtalenediazonium (Compound I), 2-chloro-5-nitrobenzene-sulfonic acid (Compound II), 2-hydroxy-4-sulfo-1- naphtalenediazonium (Compound III) and 2-amino-4-nitrophenol (Compound IV), each at 1 mM. HPLC analysis was used to separate and determine the content of these compounds and their by-products in the medium. The growth curves of E. micromeris and S. disciformis in the presence of PCPs, are shown in Fig. 5 and 6, respectively.

Compound I was removed from the medium within 4 days by cells ofS. disciformis and 8 days by E. micromeris cells (Fig. 7). It was slowly transformed and a new product at a retention time of 10.1 min was detected on the chromatogram (data not shown). The latter product was exuded to the medium in 27% after 36 days, and 52% after 28 days for S. disciformis and E. micromeris cells, respectively (Fig. 7). In comparison with rhubarb cells [8,9], where two degradation products of compound 1 were found, only one intermediate was detected here.

After 40 days of incubation, compound II was removed from the medium by 60% and 40% for cells of S. disciformis and E. micromeris, respectively (Fig. 8). In both species, biotrans-

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Fig. 5: Growth of Epithelantha micromeris cells in the presence of 2- hydroxy-4-sulfo-6-nitro-l-naphtalenediazonium (e), 2-chloro-5-nitrobenzene-sulfonic acid (O), 2-hydroxy-4-sulfo-l-naphtalenediazonium (v), 2- amino-4-nitrophenol (V) in comparison to the control (~)



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Fig. 6: Growth of Strombocactus disciformis cells in the presence of 2- hydroxy-4-sulfo-6-nitro-l-naphtalenediazonium (e), 2-chloro-5-nitrobenzene-sulfonic acid (O), 2-hydroxy-4-sulfo-l-naphtalenediazonium (v) , 2- amino-4-nitrophenol (V) in comparison to the control (~1,)

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Fig. 7: Accumulation of 2-hydroxy-4-sulfo-6-nitro-l-naphtatenediazonium by callus culture of Epithelantha micromeres (e) and Strombocactus disciformis (�9 and its transformation to by-products by the two cultures (v and ~ , respectively)

formed products were not detected. The results for rhubarb cells showed a fast decrease of compound II, and by-products were not detected either [8,9]. The question as to whether compound II was modified and stored in the cacti cells still has to be clarified.

Compound III was rapidly and almost completely taken-up by cacti cells within 4 days, but then 30% and 10% of the initial concentration was slowly exuded to the medium by S. disciformis and E. micromeris, respectively (Fig. 9). In addition to the parent compound, which appeared on the HPLC chromatogram at a retention time of 11.3 min., two additional peaks were detected in plant extract at 9.1 min. and 10.1 rain. (data not shown). Duc et al. [8,9] reported the same results with rhubarb cells, although the by-products were only detected in cell extracts.

The initial concentration of compound IV decreased to 10 % and 45% of the initial concentration within 40 and 12 days in E. micromeris and S. disciformis, respectively (Fig. 10). One intermediate product was detected at a concentration of 13 % (E. micromeris) and 7% (S. disciformis) within 28 and 32

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Fig. 9: Accumulation of 2-hydroxy-4-sulfo-l-naphtalenediazonium by callus culture of Epithelantha micromeres (0 ) and Strombocactus disciformis (�9 and its transformation into two by-products, 1 (x , II), and 2 (V,O) by the two cultures, respectively

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days of culture, respectively. The growth curves (Fig. 5 and 6) showed that the applied concentration of compound IV is lethally toxic to the cells for both cultures. Thus, the products may be stress related compounds and not necessarily degradation products of the dye.



3 Whole Plant Experiments

3.1 Growth of selected desert plants in soil from a solid waste contaminated site

There is still a great need for experiments that test the removal of pollutants from contaminated soil in whole plant- soil experiments, under controlled conditions and in field experiments, in a comprehensive way. Such approaches will enable better and more specific answers to practical phytoremediation questions. As much as the in vitro experiments can provide information about mechanisms of uptake, translocation, degradation of xenobiotics and cellular compartmen- tation, the ultimate solution to environmental soil remediation lays at the whole plant level. The finding that application of certain chelates to soil increased the translocation of heaw metals from soil into the plant shoot has opened a new range of possibilities to remediate heavy metal contaminated soil [3]. Ethylenediaminetetraacetic acid (EDTA) was added to the growth medium of plants containing Cd, Cu, Ni, Zn and Pb, and was shown to significantly improve uptake and translocation of the metals [3,14,23]. Also, addition of compost to growth medium was reported to improve metal uptake by plants by lowering soil pH and enhancing the bioavailability of metal ions [37]. Furthermore, compost is rich in inorganic ligands like NO3- , PO4 -2 and organic compounds, which may facilitate metal uptake by plants.

In the following experiments, the accumulation of heavy metals from contaminated soil from a solid waste disposal site (Fig. 11) in the Negev desert is presented.

Soil from the solid-waste-contaminated site in the Negev Desert, which was used in this study, is dominated by big shrubs of Nicotiana glauca (Fig. 11). The concentration of the heavy metals Cu and Zn in the soil was found to be more than an order of magnitude higher than in non-contaminated desert soil. The following species: Bassia indica, Mesembryanthemurn nodiflorum, Nicotiana ghtaca and Atriplex halimus, which were found to be heavy metal hyperaccumulators in hydroponic experiments [18,30], were tested in pot experiments. Plants were grown for four weeks in contaminated or uncontaminated soils with the addition

Fig. 11: A solid waste contaminated site in the Negev, Israel. The only big shrub growing on the site is Nicotiana glauca

Fig. 12: Effect of EDTA, compost and EDTA+compost on root and shoot dry weight of Nicotiana glauca. Capital letters indicate uncontaminated soil and lower case letters indicate contaminated soil. 'A' and 'a', 'B' and 'b', 'C' and 'c' and 'D' and 'd' represent control, EDTA, compost and EDTA+compost treatments, respectively

of EDTA, compost or a combination of EDTA and compost. Heavy metal accumulation was measured in plant dry matter by atomic absorption spectrophotometer (Perkin- Elmer 1100 B).

Dry weight of shoots of N. glauca grown in uncontaminated soil were higher than those grown in contaminated soil (Fig. 12). Whereas, the dry matter content of B. indica and M. nodiflorum in each of the two soil types were not significantly different among the treatments (data not shown). Dry weight of A. halimus grown in uncontaminated soil amended with compost was significantly higher (p<0.05) than that grown in uncontaminated soil without compost (Fig. 13). Metal analysis showed, in EDTA and EDTA+compost treatments, that Zn and Cu concentrations in shoot of N. glauca and A. halimus were significantly higher (p<0.05) than in control and compost treatments (Figs. 14 and 15). There was a positive relationship between the increased solubilization, uptake and translocation to the shoot of Cu and Zn from soils amended with EDTA. While addition of compost to the soil did not affect metal solubilization, uptake and translocation. This is in contrast to a report that compost enhanced the bioavailability of metals by lowering soil pH [37]. Although EDTA enhanced the bioavailability of metals, none of the tested plants accumulated Zn or Cu at hyperaccumulator levels during the 4 weeks of the experiment. Thus, the species tested can be regarded as tolerant to Zn and Cu, but longer exposure



Fig. 15: Effect of EDTA, compost and EDTA+compost on Zu (left) and Zn (right) concentration in root (up) and shoot (down) of Atriplex halimus. Letter designation as in Fig. 12

Fig. 13: Effect of EDTA, compost and EDTA+compost on root and shoot dry weight of Atriplex halimus. Letter designation as in Fig. 12

Fig. 14: Effect of EDTA, compost and EDTA+compost on Cu (left) and Zn (right) concentration in root (up) and shoot (down) of Nicotiana glauca. Letter designation as in Fig. 12

may be required for hyperaccumulation. Among all plants tested, N. glauca accumulated the highest concentrations of heavy metals (Fig 14). It appears as a potential species for further studies.

3.2 Phytoremediation with tree species

Poplar (Populus spp.) and willow (Salix spp.) are often used for phytoremediation. They are fast growing trees with a high capacity of water uptake. The extensive root system of poplar holds stream banks in place, the tree is effective in shoreline

protection, and revegetating eroded stream channels [12]. Willows have commercial uses as firewood (e.g.S. viminalis). The major advantages of trees in phytoremediation are: 1. Perennial growth and accumulat ion with no need for annual

harvest 2. Well developed root system in mature trees 3. Effective water uptake, which can be used to stabilize the pol-

luted site 4. Production of litter that provides nutrients to poor soils after de-

composition 5. Some potential tree species for phytoremediation are also of eco-

nomical value

Great effort has been generated in the use of woody species for phytoremediation of soils and groundwater from organic pollutants [e.g. 5,13,21]; aromatic hydrocarbons [35], and halogenated compounds [12]. Mycorrhizal fungi infect trees, and the effect of the symbiotic association on uptake of pollutants was studied [e.g. 31,32].

In search for suitable plants for the phytoremediation of polluted sites, the use of trees is therefore one possible alter- native. Some tree species can be seen as the first 'immigrants' on such sites [15].

4 Conclusion

In this review, we have described a wide spectrum of phytoremediation experiments of xenobiotics and heavy metals from in vitro cultures to whole plant level. A picture emerges, in which the in vitro experiments add insight on mechanisms of detoxification, uptake of contaminants and their metabolism. However, they do not necessarily predict the practical potential of these plants and enzyme systems in the phytoremediation of contaminated soil in a straightforward way. Therefore, we suggest that studies on phytoremediation be approached by multidisciplinary teams, consisting of cell biologists, biochemists and agronomists, testing mechanisms of uptake, translocation, metabolism, and testing perform- ance of the same species in soil and field experiments in parallel. Such a comprehensive approach may lead to under- standing the mechanisms of phytoremediation as well as solving actual problems of soil contamination.



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Received: September 8th, 2003 Accepted: March 3rd, 2004

OnlineFirst: March 5th, 2004


Plant response to heavy metals and organic pollutants in cell culture and at whole plant level

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