Session 2 Plant uptake and metabolism of organic pollutants: progress and bottlenecks

Chair: John Palmer (GB)

Session 2 (Oral presentations) ..........................................................................................................................1 Uptake, Metabolism and Persistence of Xenobiotics in Different Plant Systems .............................................................................................................................. 2 Oxidative enzymes involved in organic xenobiotic metabolism .................................. 5 Conjugating enzymes for the bioremediation of organic xenobiotics in plants ................................................................................................................. 9 Cellular transport and compartmentation of xenobiotics .......................................... 10 The role of plant associated rhizosphere bacteria for phytoremediation of organic pollutants ........................................................................... 12 Integration of organic xenobiotic metabolism................................................................ 15

Session 2 (Posters) .................................................................................................................................. 17 Phytoremediation of persistent organic pollutants in soils : study of the absorption mechanism in two cucurbitacean species and future applications .................................................................................................................. 17 The role of extracellular esterases in xenobiotic metabolism .................................... 19 The use of plant peroxidases for the in situ phytoremediation of soils and waste waters contaminated with phenolic compounds .............................. 20 Uptake of sewage sludge associated organic contaminants in crop plants ........................................................................................................................................... 21 Functional genomics of Arabidopsis glycosyl tranferases............................................ 22 “In vitro” plant biodegradation of pentachlorophenol ................................................ 24 Enzymes involved in a degradation of explosives......................................................... 25 Cytochromes P450s................................................................................................................. 28 The role of plant associated rhizosphere bacteria for phytoremediation of organic pollutants ........................................................................... 30

Uptake, Metabolism and Persistence of Xenobiotics in Different Plant Systems

Hans Harms, Maria Bokern, Marit Kolb and Claudia Bock

Institute of Plant Nutrition and Soil Science, Federal Agricultural Research Centre (FAL), Bundesallee 50, D-38116 Braunschweig, Germany

Phytoremediation evolves to become a major technology for clean-up of

contaminated sites. Among the mechanisms involved in the phytoremediation of organic pollutants the direct uptake and accumulation of contaminants and metabolism in plant tissue is one of the frequently used technologies. In order to use this technique most efficiently the metabolic potential of suitable plant species for transforming and degrading xenobiotics have to be evaluated. In order to satisfy these requirements, standardized test methods have been developed. Besides tests with intact plants under septic and aseptic conditions, in vitro techniques, such as plant cell suspension cultures or differentiated root cultures, have been used for studying the phytotoxicity, metabolic fate and persistence of xenobiotics in plants.

Especially plant cell and tissue cultures and recently organ cultures have become a major tool in the study of an increasing number of fundamental and applied problems in plant sciences. Thus, these in vitro systems contributed a lot to better understand the action of agrochemicals and the fate of xenobiotics.

Using these standardised in vitro plant cell test systems the metabolic fate of various xenobiotic compounds like polychlorinated biphenyls (PCBs), polycyclic aromatic hydro-carbons (PAHs), phenolic compounds, and surfactants have been tested.

Polychlorinated Biphenyls are industrial compounds, which have been detected in almost every compartment of the global ecosystem. Studies on the metabolism of 10 different PCB congeners in cell cultures of 12 different plant species revealed that the decrease in PCBs, which can be attributed to metabolism, is dependent on the plant species tested and on the tested PCB congener. Some plant species metabolise a variety of different congeners while others metabolise only one or two congeners but these to a high extent. With lower chlorination grade a higher possibility for metabo-lism is observed. The penta- and the hexachlorinated PCBs were not metabolized by any of the tested cell cultures. Also is a higher water solubility related to better metabolism. There seems to be a relationship between the structural and physical properties of PCBs. Those PCBs which possess a free ortho-meta and meta-para position are metabolised best, while those with free ortho-meta or meta - para positions are metabolized by only some of the cultures to a less extent. If these positions are substituted by chloro atoms, no metabolism is detectable. Investigations on the metabolism of 14C-labelled 2-chlorobiphenyl (PCB 1) and 2,2’5,5’-tetrachlorobiphenyl (PCB 52) as well as 3,3'4,4'-ztetrachlorobiphenyl (PCB 77) show that these congeners were metabolized to hydroxylated compounds. The

main metabolite of PCB 1 has been identified as 2'-chloro-4-biphenylol, the others as a 2'-chloro-3-biphyenylol while the very polar one was 2'-chloro-3,4-biphenyldiol. For PCB 52 the 2,2',5,5'-tetrachloro-4-bipennylol was the main metabolite, but also further monohydroxylated and dihydroxylated biphenyls could be detected. The main metabolites of PCB 77 could be identified as 2-Hydroxy-3,3'4,4'-tetrachlorobiphenyl and 5-Hydroxy-3,3'4,4'-tetrachlorobiphenyl. Furthermore a 6-Hydroxy-3,3'4,4'-tetrachlorobiphenyl has been identified in rose cultures.

Fluoranthene is one of the most abundant PAHs and has been detected in air, water, soils, sediments and even in biota including man. Cell suspension cultures of various plant species have been incubated with this chemical. The data demonstrate that most of the applied activity is still present as parent compound. Only the cultures of salad, tomato, wheat and the rose culture revealed remarkable turnover rates. For identification the metabolites were analyzed by GC-MS (EI) and HPLC-DAD and could be attributed to monohydroxylated fluoranthene isomers. The comparison of the UV/VIS spectra of these isomers with reference compounds confirm that the metabolites being formed correspond to 8-hydroxyfluoranthene, 1-hydroxy-fluoranthene and 3-hydroxyfluoranthene, respectively.

Phenolic Compounds and Aromatic Amines: Pentachlorophenol and its salts have been used as wood protectants and as a herbicide in rice. 4-Chloroaniline is a known degradation pro-duct of a variety of substituted phenylurea herbicides. A common feature of the metabolism of these compounds in soil and plants is a large proportion of nonextractable residues bound to high molecular weight compounds such as humic substances in soil or lignin in plants. 41 % of the radiolabel of PCP was converted (via the conjugate fraction) into the non-extractable residue fraction. It was bound mainly to lignin and to a high molecular weight hemicellulose fraction. In cell cultures, 72 % of 4-chloro-aniline was detected in the bound residue fraction. Further studies showed that this high proportion of 14C-label was mainly associated to the pectin and lignin fractions of the cell wall.

Surfactants: 4-Nonylphenol (4-NP) is one of the persistent products of the degradation of alkylphenolpolyethoxylates (non-ionic surfactants). The compound has been of public concern since recently new discussions have been triggered by the finding that nonylphenols are weak estrogenics. Its metabolism in cell cultures of wheat was examined according to a standardized method. Four major radioactively labelled fractions were detected and isolated by chromatographic procedures. By enzymatic hydrolysis, HPLC, GC-MS and ESI-MS/MS the chemical structures were elucidated and shown to be 4-(hydroxy)- and 4-(dihydroxy)-nonylphenols which were glucosylated at the phenolic OH-group and further glucosylated, glucuronylated, and acylated with acetic acid or malonic acid. Plants grown in nutrient media containing 14C-4-n-NP incorporated the compound and metabolized it. The amount taken up differed enormously depending on the plant species. No mineralisation to 14CO2 took place. The presence of radioactivity in shoots proved that 4-n-NP or its metabolites were transported from the root to the shoot. Thin layer chromatography gave a similar pattern of metabolites as being found in cell cultures. In soil-plant transfer studies plants took up 4-n-NP. However, over a period of 21 days, the amount did not exceed

1.5 % of the applied amount. Neverthe-less it is noteworthy that higher concentrations lead to a higher uptake rates and further that radioactivity is transported into the shoot. Uptake rates strongly differed between plant species, which has been shown also for cell cultures with 4-n-NP and other chemicals.

Non-extractable (bound) Residues: After application of radiolabelled compounds to cell cultures, and especially to intact plants, large amounts of the radioactivity are often found associated with insoluble plant components. These bound residues were assigned to defined cell wall fractions using a sequential fractionation procedure. The pattern of binding of the different xenobiotics depended on the plant species, and on the physical and chemical properties of the compound. The data for phytotoxicity and the amount of nonextractable residues of the respective cell cultures suggest that the formation of nonextractable residues may be associated with a higher tolerance to 4-n-NP. Among the plant species tested, the members of the Fabaceae exhibited high capacities for residue formation and were at the same time quite tolerant to 4-n-NP whereas the tested Chenopodiaceae species formed only limited amounts of bound residues and were shown to be relatively sensitive.

The results of this study demonstrate that all compounds, even non-polar ones, are assimilated by intact plants and different in vitro systems. Uptake depended on the plant species and on the physico-chemical properties of the chemicals. The main metabolites being formed are polar conjugates with carbohydrates and amino acids. Polycyclic aromatic hydrocarbons (PAHs) are partly converted to oxygenated derivatives which are known to be even more toxic. Depending on the plant species partly large amounts of the chemicals and/or their metabolites, are frequently incorporated into non-extractable (bound) residues. The association, and type of binding to cell wall components, enable conclusions to be made about the bioavailability of these bound residues.

Oxidative enzymes involved in organic xenobiotic metabolism

Patricia J. Harvey1 , Daniele Werck-Reichhart2 , Mohammad Qasim Chaudhry 3 , Cait Coyle4 , Thomas Vanek5

1 School of Chemical & Life Sciences, University of Greenwich, Wellington St., London SE18

6PF. p.j.harvey@gre.ac.uk 2Institute of Plant Molecular biology, CNRS-UPR 406, 28, Rue Goethe, F-67000 Strasbourg,

France. Daniele.werck@ibmp-ulp.u-strasbg.fr 3Central Science Laboratory, MAFF, Sand Hutton, GB-Y041 1LZ, York United Kingdom.

q.chaudhry@csl.gov.uk 4Department of Chemical & Life Sciences, Waterford Institute of Technology, Cork Rd.,

Waterford, Co. Waterford, Ireland. Caitcoyle@ireland.com 5Institute of Organic Chemistry & Biochemistry, AS CR, Flemingovo n. 2, 166 10 Praha 6, Czech

Republic. Vanek@uochb.cas.cz Introduction Phytoremediation requires that a plant overcomes the physical and chemical

properties of organics and man-made chemicals that tend to make them non-utilisable and often toxic to most organisms. The main problems that organics pose include their relative insolubility in water and their chemical unreactivity. Certain micro-organisms have evolved mechanisms for conversion of hydrocarbons into chemical constituents of the cell, but this ability appears to be extremely limited in plants. A goal of phytoremediation is to enhance the capability of plants to degrade or assimilate organic pollutants.

Hydrocarbons are more highly reduced than cellular molecules and need to be

oxidised by the addition of an oxygen atom before they can be incorporated into cellular material. The addition of oxygen increases both their solubility and chemical reactivity and can be achieved either by dioxygenases; mono-oxygenases (Cytochromes P450); and peroxidases.

Cytochromes P450 Cytochromes P450 (P450s) are haem-thiolate proteins involved in the

metabolism of a wide variety of both exogenous and endogenous compounds and are vital for the biosynthesis of numerous compounds (hormones, lipids, and secondary metabolites). They constitute the largest family of enzymatic proteins in higher plants: about 300 P450 genes are expected in the diminutive genome of Arabidopsis. P450s use electrons from NADPH to activate molecular oxygen. The CYP (cytochromes P450) gene superfamily is composed of at least 51 families and 104 subfamilies. Over 400 plant CYP genes have now been reported, which belong to either CYP51 (also present in mammals), or CYP71 to CYP99 and CYP701 to CYP721.

The reaction they catalyse is usually a mono-oxygenation, with formation of a molecule of water and an oxygenated product (1), but other more atypical activities, like dealkylations, dimerisations, isomerisations, dehydrations, reductions have also been reported.

(1) R-H + O2+ NADPH + H+ ----> R-OH + H2O + NADP+

Electrons from NADPH are transferred to P450s via flavoproteins called

cytochrome P450-reductases. Both plant P450s and their reductases are usually bound via their N-terminus to the cytoplasmic surface of the endoplasmic reticulum. P450 proteins have molecular masses ranging from 45-62 kDa, and may have as little as 16 % amino acid identity. However, their overall three-dimensional structure is conserved, as are a few residues on both sides of the heme.

The role of P450s in the metabolism of industrial pollutants has not yet been

investigated. However, an impressive metabolic capacity has been demonstrated with agrochemicals and model compounds. Through selection, or genetic modification, it is hoped to develop plants with enhanced P450 activity against a given class of pollutants. Certain chemicals can also induce higher P450 activity in plants, which may lead to the co-metabolism of certain pollutants.

In the last 5 years an increasing number of P450 genes have become available,

expressed in recombinant systems. Enzymes primarily involved in major plant pathways, such as biosynthesis of lignin, have been investigated for their ability metabolise exogenous compounds. The first genes of P450s that very actively metabolised herbicides were isolated only recently. Both CYP76B1 and CYP71A10 expressed in yeast converted herbicides of the class of phenylurea to non-phytotoxic metabolites and conferred herbicide resistance to transgenic plants.

Peroxidases Peroxidases of both fungal and plant origin reduce H2O2 to water at the expense

of aromatic reductants, for which they appear to be relatively non-specific. Located both intracellularly as well as in extracellular compartments, they have been found in both membrane-bound and soluble fractions; there are several isoforms and both constitutive as well inducible forms are known.

Peroxidase reactions do not typically lead to the incorporation of oxygen into

the hydrocarbon, although oxygen may be incorporated by non-enzymatic means via the radical products of single-electron oxidation. Plant peroxidases are noted for coupling reactions that lead to lignin synthesis and formation of humic residues, whereas fungal peroxidases are noted for their role in catalysing the oxidative depolymerisation and aromatic ring-opening of lignin. In recent years the degradative capacity of fungal peroxidases has been intensively investigated and now includes a

broad range of xenobiotics (phenanthrene; benzo(a)pyrene; pentachlorophenol; mono- and dichlorobenzenes; TNT; and polyaromatic azo dyes).

Plant peroxidases Peroxidase-catalysed oxidation of phenolic precursors in the plant cell wall

yields radicals that couple randomly to form polymeric lignin. Polymerisation continues so long as there are activated precursors and space in the wall; the growing polymer therefore tends to displace other macromolecules as well as water, resulting in a very strong hydrophobic meshwork surrounding the other wall components. Organic xenobiotic degradative products have been found in the extracellular cell wall compartment of plants, however little is known about the control mechanisms involved. Recognising their ability to catalyse coupling reactions, current research in phytoremediation aims to reduce the bioavailability of phenolic xenobiotics by forming bound residues in the soil using peroxidase liberated from horseradish (Armoracia rusticana) roots. The successful decontamination of wastewaters and soils has already been achieved using this approach. Guaiacol peroxidases have also been found in sterile root exudates and shown to oxidise Mn2+ to form Mn3+ chelates that are capable of decolourising aromatic dyes.

Fungal peroxidases Lignin peroxidase (LiP), the most noteworthy among fungal peroxidases has

been crystallised and is structurally similar to plant peroxidases. It also shares a similar catalytic cycle. At present, however, there seems little immediate prospect of engineering plant peroxidases such as horseradish peroxidase (HRP) to behave as LiP. LiP uses a redox mediator (3,4-dimethoxybenzyl alcohol (veratryl alcohol, VA) in catalysis, which confers non-specificity toward electron donor. HRP, however, appears unable to use the oxidising power of H2O2 to oxidise VA, which has a redox potential of +1.42V. A Cβ-hydroxylated tryptophan (Trp171) has been identified in LiP, which is absent in HRP; this residue may be important in the pathway of electron flux from substrate to oxidised haem, particularly under conditions of low VA concentration. The pH optimum of LiP is acidic, (pH 3.0), in contrast to that of plant peroxidases (pH 7.0), which may be important in redox mediation with VA. LiP has no tyrosine residues, which may safeguard against auto-oxidation of the protein by substrate radical cations and promote their release to catalyse the oxidation of secondary substrates. Alternatives to clone LiP into the plant genome may be more immediately promising, if alternative redox cofactors of plant origin can be consigned to function as non-specific redox mediators as alternatives to VA. In this context, 2,5-dimethoxybenzyl alcohol shows better properties for redox mediation than the fungal cofactor, 3,4-dimethoxybenzyl alcohol. Current research is aimed at cultivating white-rot fungi to achieve soil bioremediation in combination with plant-based technologies.

Oxidases and TNT degradation TNT degradation has been described by poplar Populus sp. 2-amino-4,6-

dinitrotoluene, 4-amino-2,6-dinitrotoluene and a number of unidentified products more polar than TNT have been found, which may be incorporated into the cell wall. Little is known about the mechanisms involved although two redox enzymes

NADPH: thioredoxin reductase (TR, EC 1.6.4.5) and xanthine oxidase (XOD) may be involved in initial reductive reactions.

Conjugating enzymes for the bioremediation of organic xenobiotics in plants

Peter Schröder1 , Burkhard Messner2 and Tony Schäffner2

GSF-National Research Center of Environment and Health, D-85764 Neuherberg, FRG, Institute

for Soil Ecology1 and Institute for Biochemical Plant Pathology2.

Organic xenobiotics are subject to several biotransformation reactions in plants,

among them reduction, hydrolysis, oxidation, cleavage and conjugation. Whereas most of the mentioned reactions are reversible or render the xenobiotics more toxic by activation, true detoxification is only reached by conjugating enzymes. Conjugation of biomolecules to xenobiotics may include glutathione, sugars and amino acids. Several enzyme classes are known to catalyze these reactions, and glucosyl transferases and glutathione transferases are the most abundant enzymes found in all organs of plants.

Current research on GST has elucidated numerous isoforms with broad substrate specificities, depending on gene class and subunit combination. Heterodimer formation is not well understood. Specifically inducing heterodimer formation could be a means to strengthen GST mediated detoxification for remediation projects. Information on GTs stems mainly from investigations in A.thaliana, where substrate specifictities of recombinant enzymes are assayed, expression patterns and inducibilties through xenobiotics will be checked in parallel, and changes in metabolism can be studied via genetic manipulation of the expression levels.

Transport and catabolism of GS- as well as sugar conjugates leads to excretion of xenobiotic residues as mercapturic acids or glucuronyl compounds in animals, the vacuole is discussed to act as final storage organelle for xenobiotic and natural conjugates in plants. However, on the background of the available metabolism data, it seems logical that xenobiotic conjugates are intermediates rather than end products of the detoxifying pathway. Transport and storage phenomena may be central events in detoxification, but they are followed by catabolic reactions. Elaborate investigations of herbicide metabolism in several plant species have elucidated that GS-conjugates have only short lifetimes, and that they are rapidly further metabolized. Interestingly, a shortcut exists between the glutathione and the sugar derived detoxification pathways, leading to secondary conjugation and final excretion to the cell wall.

For phytoremediation the conjugating enzymes of plants are of special interest because they transform foreign compounds irreversibly to metabolites that are less toxic and more water soluble than parent compounds. Further studies must include inducibility, genetic engineering of isoforms and molecular modelling of active sites to increase the activity for practical use.

Cellular transport and compartmentation of xenobiotics

Julian Coleman1 and Enrico Martinoia2

1Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire,

AL5 2JQ, UK. 2Laboratoire de Physiologie Végétale, Université de Neuchâtel, 2007 Neuchâtel, Switzerland

Uptake, metabolism and compartmentation determine the fate of xenobiotics in plant cells. Furthermore, the presence and expression of these cellular systems depend on the plant species and is the basis for differences in rates of detoxification and tolerance to pollutants.

In general, metabolism of an organic pollutant requires it's penetration into a

living plant cell and this necessarily involves the passage across the plasma membrane. Occasionally a xenobiotic may be transported by a specific membrane protein that exists for the transport of an endogenous metabolite or an essential nutrient, but often the uptake of xenobiotics relies on passive transport. The rate of passage across the membrane via passive transport is critically dependent on the solute charge and the partition coefficient or log P. These physico-chemial properties are related in a general principle the "pH partition principle"; that is, for a molecule to pass unaided through a biological membrane it should be uncharged. For the uptake of weak acids and bases, their pKa values should be such as to allow a good proportion of the compound to be unionized at the pH of the external aqueous/ membrane interphase. The unionized fraction will partition into the membrane at a rate dependent on its log P, and likewise will partition out of the membrane on the cytosolic side. In the apoplastic compartment there are enzymes that catalyse chemical changes which can affect uptake and subsequent metabolism of compound by revealing charges and altering the log P. For example apoplastic carboxylesterases catalyse the hydrolysis of non-ionised esters to carboxylic acid anions with reduced log Ps (i.e. lower lipophilicity).

When an organic compound enters a plant cell it can either be transported unmodified to the vacuole or metabolised (chemically transformed) and then the products are transported to the vacuolar compartment. Two types of reaction bring about chemical modifications: Phase I (oxidation, reduction or hydrolysis) and Phase II (conjugation), by which a product of Phase I or the original compound is detoxified by conjugation to an endogenous metabolite such as glucose, malonate or glutathione. The conjugates formed are more water-soluble than the parent compound and are transported to the vacuole by specific solute transporters in the tonoplast. Vacuolar compartmentation is a critical step in the detoxification of organic metabolites because it removes conjugated products from vulnerable sites of the cytosol and also because further processing of the conjugates may take place in the vacuolar matrix. How xenobiotics and xenobiotic conjugates are sequestered in the vacuole has

recently been resolved with the discovery that specific transporters belonging to the ATP-binding cassette (ABC) transporter superfamily mediate their transport. Plant homologues of a human ABC transporter the multidrug resistance associated protein (MRP), are responsible for the transport of conjugates across the tonoplast. Searches of the Arabidopsis thaliana genome databases reveal that there are eight members of the AtMRP family. So far only three of these genes (At MRP1, AtMRP2 and AtMRP3) and the proteins they encode have been characterised in any detail. Heterologous expression in yeast (Saccharomyces cerevisae) has shown that all three proteins catalyse the transport of glutathione conjugates, two (AtMRP2 and AtMRP3) exhibit transport of malonylated chlorophyll catabolites that are produced during leaf senescence and one (AtMRP3) confers cadmium resistance.

In addition to the MRP family other ABC transporters are likely to play an

important role in the distribution, compartmentation and detoxification of organic pollutants. A total of 60 open reading frames (ORF,s) encoding ABC proteins can be identified in the Arabidopsis genome databases (50% of the genome has so far been sequenced). At least 49 of these putative proteins are ABC transporters. These observations suggest that the number of ABC transporter systems in plants is significantly larger than found in S. cerevisae. This difference may reflect the capacity of plants for synthesising and sequestering large numbers of organic (secondary) compounds. This capability is a useful asset for phytoremediation. Therefore functional characterisation of ABC proteins will offer a better understanding of the detoxification and compartmentation of organic pollutants by plants.

Suggested reading

1. Kreuz, K., R. Tommasini, R. and Martinoia, E. (1996) Old enzymes for a new job: How cells dispose of herbicides. Plant Physiol.111, 349-353.

2. Rea, P.A., Martinoia, E., Li, Z-S., Lu, Y-P. and Drozdowicz, Y.M. (1998) From vacuolar GS-X pumps to multispecific ABC transporters. Annual Review of Plant Physiology and Plant Molecular Biology 49, 727-760

3. Rea, P.A. (1999) MRP subfamily ABC transporters from plants and yeasts. Journal of Experimental Botany 50 (special issue), 895-913.

4. Davies, T.G.E. and Coleman, J.O.D. (2000) The Arabidopsis thaliana ATP-binding cassette proteins ñ an emerging superfamily. Plant, Cell and Environment (In press)

The role of plant associated rhizosphere bacteria for phytoremediation of organic pollutants

Daniel van der Lelie1 , Zita Snellinx1 , 2 , Safieh Taghavi1 , Jaco Vangronsveld2 , Frank Laturnus3 , and Tomas Vanek4

1Vlaamse Instelling voor Technologisch Onderzoek (VITO), Environmental Technology Centre,

2400 Mol, Belgium 2 Limburgs Universitair Centrum (LUC), Universitaire Campus, Diepenbeek, Belgium

3 Department Plant Biology and Biogeochemistry, Risoe National Laboratory, Roskilde, Denmark 4 Department Plant Cell Cultures, Institute of Organic Chemistry and Biochemistry, Academy of

Sciences of the Czech Republic, Praha, Czech Republic Introduction The use of biological techniques can strongly reduce the cost for remediating

sites loaded with pollutants like heavy metals and organic xenobiotics. For large contaminated sites, bioremediation is the only alternative economically and socially acceptable. Therefore, phytoremediation, one of the soft bioremediation techniques, became an acceptable alternative for the treatment of sites and wastewater contaminated with organic xenobiotics. Phytoremediation is based on the combined action between plants and their associated microorganisms, such as mycorrhiza and bacteria. In this paper, the possibilities to use plant-associated bacteria to improve the efficiency of phytoremediation of organic xenobiotics are discussed.

Conditions for phytoremediation The development of an efficient strategy for phytoremediation of organic

xenobiotics requires several conditions to be fulfilled: the pollutant must be accessible for removal; the bioavailable concentration should not exceed toxicity thresholds of the plant; an uptake of the contaminants may be desirable; the contamination should not exceed areas accessible by the root system of the plants.

Bioavailability of contaminants The availability of a pollutant for degradation or uptake is one of the limiting

factors for efficient phytoremediation. Especially hydrophobic pollutants like e.g. PAHs are difficult to mobilise due to their low watersolubility. Both, plants and their associated rhizosphere bacteria can release surfactants. These compounds can increase the solubility, and subsequently the mobility and bioavailability of hydrophobic contaminants, which may lead to an increase in degradation of the pollutant by the plant rhizosphere system. Additionally, beside a direct degradation an uptake of the contaminants by the plants through the roots may also be possible. In general, lipophylic pollutants are difficult to take up by the root system. Only those contaminants are easily accessible by the root system with an octanol-water partition coefficient log Kow between 1 and 3. Fortunately, this is the case for many pollutants

of environmental concern, such as BTEX, PCBs, PAHs and explosives. However, by choosing plants with a high lipid content also compounds with a higher log Kow are available for plant-uptake, as these plants are considered highly capable of accumulation of hydrophobic contaminants.

Phytotoxicity of organic xenobiotics decreased by rhizosphere bacteria The possibilities to use phytoremediation as a cleanup strategy can be hindered

by the toxicity of the organic xenobiotics. Suitable rhizosphere bacteria can decrease the phytotoxicity of organic xenobiotics due to two processes. First, the bacteria can have general beneficial effects on plants, allowing them to tolerate higher toxicity levels. Secondly, bacteria can degrade the organic xenobiotic to a level below phytotoxicity that allow efficient phytoremediation. This clearly shows that both plants and their associated rhizosphere bacteria have an important contribution in the degradation process, which is based on a clear interaction between plant and associated bacteria. By secreting specific root exudates plants can stimulate the bacterial degradation of specific xenobiotics. Most bacterial degradation pathways only become induced when the concentration of a contaminant arrives above a threshold value. In case the bioavailable concentration of the contaminant is below this value, no degradation by the bacteria will occur. Under such conditions plants may excrete root exudates, which qua structure strongly resemble the contaminant, that are able to specifically induce the desired degradation pathway. These root exudates are plant specific and their production depends on many environmental factors. However, the suitable combination of plant and its associated rhizosphere bacteria is a prerequisite for the well functioning of the system.

Also less specific interactions exist by which plant can stimulate the degradation potential of its rhizosphere. An example is the secretion of sugars or phenolic compounds, which is independent of the presence of a specific pollutant. The secretion of phenolic compounds can, due to cometabolism, stimulate the degradation of TCE, e.g. by toluene monooxygenases of rhizosphere bacteria. The secretion of sugars will result in a general stimulation of the activity of the rhizosphere, which will speed up the degradation processes.

Plant growth stimulate by rhizosphere bacteria An increased root biomass or root activity in the soil will result in an improved

degradation activity in the root zone and better uptake of pollutants by plants. By using bacterial inocula, which stimulate plant growth, the efficiency of phytoremediation can be improved. This principle was demonstrated for the degradation of 2-CBA by wheat, where seeds were inoculated with the plant growth stimulating rhizosphere strain Pseudomonas aeruginosa R75. This strain is unable to degrade 2-CBA, but its presence strongly stimulated the overall degradation processes. In other words, it is possible to use a suitable bacterial inoculum to improve root growth and activity and subsequently the rhizosphere, resulting in better decontamination.

Conclusions

By playing with the interactions between plants and their associated microorganisms it is possible to improve the rhizosphere degradation and phytodegradation of organic xenobiotics. However, these are complex interactions that need to be well studied before they can be manipulated in order to improve the overall efficiency of phytoremediation. This counts both for heavy metals and organic xenobiotics.

Integration of organic xenobiotic metabolism

Michel Tissut1 and John Palmer2

1UMR Ecosystèmes et Changements Environnementaux, UJF, BP 53 X, 38041 Grenoble, France 2Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2

AZ, England The objective of phytoremediation is to use plants to remove organic

xenobiotics from the environment and concentrate them into harmless products. To achieve this, the plants must be able to (a) accumulate the material from the environment, (b) translocate the material to the site of metabolism (c) to metabolize the xenobiotic into harmless products.

Entry into the plant is normally by passive diffusion down a concentration gradient. There is evidence that some enzymatic transformation by esterases may occur in the apoplast before entry. Efficient removal in the transpiration stream or active metabolization at the root level will maintain this concentration gradient and maximise the rate of entry into the plant. Several molecules such as TNT or atrazine may bind to the cell wall polymers (cellulose or lignin) whilst lipophilic molecules will partition into the lipid phases of the cell (membranes). Such immobilisation of the xenobiotics will lower translocation and excessive accumulation could eventually disrupt normal metabolism and poise a threat to the natural food chain. Relatively hydrophilic molecules such as RDX are readily translocated to the leaves in the transpiration stream and can be accumulated there in amounts, exceeding the safety limit and can cause damage to the tissue.

The metabolic pathways involved in phytoremediation are not fully understood and vary depending on the type of substrate and species of plant. Some of them are induced by safeners. The metabolic details are only known for a few xenobiotics, mainly herbicides. The initial step (phase 1) involves an activation of the molecule, this creates a reactive chemical in the xenobiotic molecule. The activation involves hydrolysis by esterases, oxidation by cytochrome P 450 or peroxidases, reduction or hydroxylation. Once activated, the molecule can be metabolized directly or become conjugated to a "carrier" molecule during Phase 2 of metabolism. Conjugation to glutathione by the enzymes glutathione-S-transferases is the best characterised reaction of Phase 2. Conjugation to sugar derivatives such as glucose or galactose can also occur.Transfer of the activated xenobiotic to a malonyl derivative is also possible. These conjugated derivatives now enter the final Phase 3 of metabolism. This can occur within the cytosol where the conjugate is formed or they can be transported across biological membranes using an ATPase, translocation is normally into the vacuole. The fate of the conjugate once in the vacuole has not been resolved. There is evidence that the glutathione

conjugates are hydrolysed by peptidases. Further work is needed to describe the subsequent fate of the xenobiotic moiety. It is undesirable that the xenobiotics should simply be stored in the vacuole, it would limit the scope of plants in the process of phytoremediation and the excessive accumulation would exceed the safe levels and could be detrimental to the normal metabolic processes in the plant. Many of the metabolic processes characteristic or Phase 1 and Phase 2 of metabolism are closely associated with the redox chemistry of the plant and may expose the plant to environmental stresses not experienced under normal growth conditions.

Session 2 (Posters)

Phytoremediation of persistent organic pollutants in soils : study of the absorption mechanism in two cucurbitacean species and future applications

B. Campanella and R. Paul

Laboratoire de Toxicologie Environnementale, Faculté universitaire des Sciences Agronomiques, Gembloux, Belgium

In the wake of the observation by Hülster and Marschner (Hülster A., Müller J.F. and Marschner H. 1994) that zucchini (Cucurbita pepo L. var. Diamant) absorbs large quantities of polychlorodibenzo-p-dioxins and polychlorodi-benzofurans (PCDD/Fs) through its roots, our research is a contribution to the description of the mechanism involved in this process. We use, for this purpose, 14C-labelled tetrachlorodibenzo-p-dioxin (TCDD) as a model. Some important results have been obtained by Neumann’s group at Stuttgart Plant Nutrition Institute (Neumann G., Hülster A. and Marschner H. 1997).

PCDD/Fs are very hydrophobic (log Kow = 5 to 8) soil contaminants and therefore have low bioavailability. Their stability leads these pollutants to accumulate in soil and food chains. Many efforts have been made since the 80’s to reduce dioxin emissions (OMS, 1998), but some soils or sludges remain very concentrated. These high concentrations are limiting factors for the use of sewage or dredging sludges in agriculture. A low cost decontamination technique would be helpful to avoid the accumulation of these sludges. Unfortunately, as other persistent organic pollutants, dioxins are recalcitrant to bio- or phytoremediation. The very low microbial degradation rate is, for a large part, due to the poor bioavailability of these molecules. A better understanding of how cucurbits are able to absorb dioxins could have some applications in soils and sediments decontamination.

Our early trials confirmed both root absorption and further translocation in all plant parts in three zucchini varieties (Diamant, Tarmino and Ronde de Nice). Bioconcentration factor (BCF) reached 19 for Diamant stems, and 58 for Ronde de Nice leaves. Plants were cultivated only on glass marbles and nutrient solution. One can expect lower BCF values in soil conditions. Cucumber (Cucumis sativus L.) has been used as a control species.

According to the current hypothesis, a molecule produced by zucchini and released into its root exudates complexes with the pollutant. This complexed form present a greater water solubility and so, an enhanced mobility in comparison with TCDD. The transfer in soil and in root apoplast is increased. After its diffusion across the plasma membrane, TCDD is complexed again by molecules present in plant vascular systems. Meanwhile, complexation is also reversible, and dioxin can be adsorbed on root epidermis or cell walls. At equilibrium, the TCDD mobility in a

medium is fonction of both complexation and adsorption kinetics. This would explain the presence of 14C-TCDD in the leaves, but also its huge accumulation in stems.

We attempted to isolate one or several binding molecules in other members of the Cucurbitaceae family. These trials showed that such a molecule exists in melon. Extracts of melon root exudates and leaves increased the TCDD apparent solubility 3.5 and 26.1 times, respectively. To measure this increasing in apparent solubility, one realised a liquid-liquid extraction with toluene on an aqueous phase containing complexing molecule and labelled with 14C-TCDD. The more the aqueous phase activity was high, the more the apparent solubility increased. This would be linked to the presence of complexing molecules.

We are now trying to isolate and identify such a molecule. Some previous results suggested a proteinic nature. The step-wise precipitation showed that there are at least two groups of molecules with different hydratation properties (and perhaps different molecular weights) in leaf extracts. Separation on Sephadex G-25 showed that the complexing substances contained in melon leaves have a molecular weight above 5 kD. Further identification will be realised by electrophoretic separation and sequencing. References 1. Hülster A. Müller J.F. and Marschner H. (1994): “Soil-plant transfer of PCDD/F to

vegetables of the Cucumber family (Cucurbitaceae).” Environ. Sci. Technol. 28(6): 1110-1115.

2. Neumann G. Hülster A. and Marschner H. (1997). Identifizierung PCDD/F-mobilisierender verbindungen in wurzelexsudaten von Zucchini. Rhizosphärenprozesse, umweltstress und ökosystemstabilität. M. W.: 167-175.

3. OMS (1998). Assessment of the health risk of dioxins: re-evaluation of the Tolerable Daily Intake (TDI). Geneva, Switzerland, WHO European Centre for Environment and Health International Programme on Chemical Safety.

The role of extracellular esterases in xenobiotic metabolism

Richard Haslam1 , 2 , David Cole2 and Julian Coleman1

1Biochemistry and Physiology Department, IACR-Rothamsted, UK 2Aventis CropScience UK Limited

Plants encounter a wide range of xenobiotic organic compounds such as

herbicides, which may or may not have a deleterious effect. To have a herbicidal effect, a compound must enter plant cells by penetrating the hydrophobic layers at the plant surface. Therefore herbicides that are active as carboxylic acid anions are often formulated as uncharged carboxylesters, in order to enhance uptake. The localization of specific esterases in the apoplast, (one of the first points of contact between the plant and a xenobiotic) capable of de-esterifying herbicides will consequently influence the whole plant uptake and activity of the compound. Apoplastic proteins were extracted by vacuum infiltration, and analyzed by 2D electrophoresis, with isoelectric focusing in the first dimension and native separation in the second dimension. By activity staining fourteen esterases were identified in apoplastic extracts from seven-day-old wheat seedlings. Reverse HPLC analysis has shown that these extracts were capable of hydrolysing herbicide esters such as fenoxaprop-ethyl and diclofop-methyl to the active acid form. Using the model ester substrate 2-nitrophenyl acetate, the induction of wheat esterases in response treatment with fenoxaprop-ethyl and the safener fenchlorazole-ethyl was investigated. The implications of esterases on biodelivery and metabolism of herbicides and other xenobiotics will be presented.

The use of plant peroxidases for the in situ phytoremediation of soils and waste waters contaminated with phenolic compounds

Cait Coyle

Department of Chemical and Life Sciences, Waterford Institute of Technology, Cork rd., Waterford, Co. Waterford, Ireland

e-mail:caitcoyle@ireland.com

The proposed phytoremediation technique is based on the exploitation and

optimisation of a naturally occurring process known as oxidative coupling. Oxidative coupling, mediated abiotically or biotically, results in the formation of bound residues via covalent linkages.

The process is mediated biotically using peroxidase contained in horseradish. Peroxidase is an ubiquitous enzyme in plants but especially high activities are

found in horseradish (Armoracia rusticana) and fig tree roots. This group of enzymes may be divided into (1) monophenol monoxygenases and (2) peroxidases. Oxidative coulpling is involved in the synthesis of humic substances in soil. The structural resemblance of many xenobiotics to humic acid building blocks means that their bioavailability may be reduced using peroxidase via a two step mechanism, i.e.phytodegradation and phytostabilization due to bound residue formation. The successful decontamination of waste waters (Dec and Bollag, 1994; Klibanov, Tsu-Man, and Scott, 1983; Roper, Dec and Bollag, 1996) and soils (Berry and Boyd, 1985; Shannon and Bartha, 1988) has already being achieved using this approach.

References 1. Dec, J. and Bollag, J.-M.1994. Use of plant materials for the decontamination of

water polluted with phenols.Biotechnol.Bioeng.44, 1132-1139 2. Klibanov, A., Tsu-Man, T. and Scott, K.P. 1983. Peroxidase-catalysed removal of

phenols from coal-conversion waste waters. Science 221, 259-261. 3. Roper, J.C., Dec, J., and Bollag, J.-M. 1996. Using minced horseradish roots for the

treatment of polluted waters. J.Environ.Qual. 25, 1242-1247 4. Berry, D.F. and Boyd, S.A. 1985. Decontamination of soil through enhanced

formation of bound residues. Environ. Sci. Technol. 19, 1132-1133 5. Shannon, M. J. R. and Bartha, R. 1988. Immobilization of leachable toxic soil

pollutants by using oxidative enzymes. Appl. Environ. Microbiol. 54, 1719-1723

Uptake of sewage sludge associated organic contaminants in crop plants

Liv Kure, Gerda Mortensen, Frank Laturnus and Per Ambus

Department of Plant Biology and Biogeochemistry, Risoe National Laboratory. P.O. Box 49, DK-4000 Roskilde, Denmark. e-mail: liv.kure@risoe.dk

Recycling of nutrients and organic matter from organic wastes is important in

the development of a sustainable agricultural production. However, a number of organic contaminants - of which some are known to act as endocrine disrupters - accumulate in sewage sludge, that is applied to agricultural soils. Uptake of these compounds in crop plants and possible transfer to humans has been investigated in our laboratory.

Selected representative 14C-labelled compounds (DEHP, LAS, Nonylphenol

(NP), Nonylphenoxy-ethoxy-ethanol (NPEO2) and Pyrene) were used in hydroponics using different crop species (barley, rape, carrot and tomato) to investigate the potential for these plants to accumulate and translocate contaminants from roots to shoots. The physical-chemical properties of the compounds do to some extent determine adsorption to roots and translocation into shoots, but species differences in root physiology and morphology leads to ten fold differences in root accumulation factors (RCF) and transpiration stream concentration factors (TSCF). The bioavailability of the investigated contaminants in soil is, however, very low, and the results from laboratory experiments could not be verified in greenhouse or field experiments where levels of contaminants in plants were close to or below detection limits.

Recent results indicate that roots of at least rape and carrot metabolise the

contaminants used in the study to polar metabolites very rapidly. In experiments with LAS, NP or NPEO2 we found that after 24 hours less than 10 % of the 14C-activity in the media in which plants were grown were still present as the original compound.

Functional genomics of Arabidopsis glycosyl tranferases

Burkhard Messner, Heinrich Sandermann, Anton Schaeffner, Oliver Thulke, Sigrun Wegener

Institute of Biochemical Plant Pathology, GSF Research Center forEnvironment and Health, D-

85764 Neuherberg / Muenchen, Germany (schaeffner@gsf.de)

Plant secondary metabolite glycosyl transferases comprise a huge family ofenzymes involved in glycosylation of both endogenous metabolites andxenobiotic substances. In xenobiotic detoxification schemes glucosyltransferase activities have been classified as phase II enzymes taking part in conjugation reactions. However, thereis only little information on the molecular level on the expression andsubstrate specificities of individual enzymes as well as on their roles in different endogenous and defense or detoxification reactions. The genomesequencing project of the model plant Arabidopsis thaliana now offers the unique possibility that all genes of a single plant specieswill be accessible. Currently more than 80% of the whole genome have beensequenced. Based on this information we know more than 80 putativesecondary metabolite glycosyl transferases suggesting about 100 sequences in total.

In a functional genomics approach we want to investigate the role of this family of enzymes. In order to study their spatial and developmental expressionpatterns as well as their inducibility by xenobiotic substances we designedshort PCR fragments that will serve as specific probes on DNA microarrays.This will allow parallel expression analyses via reverse Northern hybridization.

A second line of evidence on the function of individual isozymes will bederived from the analyses of recombinantly expressed enzymes. These enzymeswill be challenged with a series of potential endogenous substrates like kaemperol, anthocyanidines, hormones, or salicylic acidand xenobiotic substances like e.g. chlorophenols, herbicide metabolites ormicrobial toxins. Preliminary results based on a few enzymes indicate arather broad substrate specificity for both endogenous and xenobiotic substances. Interestingly, xenobioticsubstrates like trichlorophenols or mycotoxins tend to be good substrateseven when using crude enzyme preparations from A. thaliana.

A third, complementary tool is the identification and characterization ofinsertion mutants that lead to specific gene disruption. These lines offerthe possibility to

investigate the role of glycosyl transferases inplanta, e.g. by studying alterations of metabolic profiles of endogenous orexogenous substrates and products.

Finally, these combined informations will also reveal insight intointeractions between different endogenous pathways and detoxification ofxenobiotics.

“In vitro” plant biodegradation of pentachlorophenol

Stanislav Smrcek Charles University in Prague, Department of Organic and Nuclear Chemistry, Hlavova 2030, 128

40 Prague 2, Czech Republic. e-mail: smrcek@natur.cuni.cz

There are known basically three ways for the pentachlorophenol (PCP)

degradation in the environment. The first is abiotic method based on the photochemical decomposition. The interaction of the specific compound with UV radiation is limited by the presence of the free phenolic hydroxyl in a molecule. The second one is the microbial degradation that was intensively studied for many microbial species. The third - plant biodegradation method could play the important role in the remediation processes. The use of the specific plants for this aim is limited by the toxicity of the chlorophenols for plants. This fact need not be so important, because the contaminated soils usually do not contain too high xenobiotic concentrations and the plants with the relatively high resistance toward chlorinated phenols can be found.

The growth ability of the commonly cultivated plants Beta vulgaris, Zea mays, Medicago sativa, Trifolium repens, Trifolium incarnatum, Pisum sativum, Sinapis alba, Helianthus annuus, and Phacelia tanacetifolia on media containing various concentrations PCP were tested. The preliminary results showed the possibility of the tested plants cultivation in the presence of pentachlorophenol. The maximal toxic concentration levels are different for the each plant species and ranges in the region 5 – 40 mg/l.

Together with the determination of the maximal toxic concentration level the decrease of pentachlorophenol concentrations in media was proved. Using HPLC analysis of pentachlorophenol content in media the time dependence and the efficiency of phytoextraction were determined. The plant tissues were analysed and neither free starting compound nor volatile phenolic derivatives have been found. From these results follows that chlorinated phenols or their metabolites were extracted into plant organisms and stored there in the form of the conjugates with compounds present in the plant metabolism.

Enzymes involved in a degradation of explosives

T. Vanek and A. Nepovím

Institute of Organic Chemistry and Biochemistry AS CR, Flemingovo n. 2, 166 10 Praha 6, Czech Republic

In the recent years considerable attention has been paid to the nitrocompounds degradation by microorganisms (bioremediation) and by tissue cultures of higher plants (phytoremediation), especially to the elucidation of the degradation pathways of these compounds as well as the identification of individual intermediates (Fig. 1) (Hughes, 1997; Montpas, 1997; Fiorella, 1997). Several reports focused on the study of the mechanism of their degradation have been published recently, too. Degradation of nitrocompounds may be catalyzed by two types of enzymes. The first group includes enzymes catalysing transfer of two electrons onto nitro-group in one step which are insensitive towards the presence of molecular oxygen. The second group of enzymes is characteristic by transfer of only one electron and by sensitivity towards the molecular oxygen. After transfer of one electron under aerobic conditions back-oxidation of nitroanion radical to the coresponding nitrocompound occurs, which is connected with the formation of superoxide anion radical from molecular oxygen. This cycle takes place as long as the oxygen is available. The group of enzymes insensitive to the towards the presence of molecular oxygen includes flavoprotein isolated from Escherichia coli (Anlezark, 1992). The group of enzymes sensitive to molecular oxygen contains xanthin oxidase (Kutcher, 1984), glutathion reductase (Carlberg, 1986), cytochrom c reductase (Moreno, 1984), ferredoxin: NADP reductase and nitrobenzen nitroreductase (Shah, 1997).

NR

O

O

NR

O

O 2

2 e -

NR

H

O H

NR

H

H

e -

e -

NR

O

O-

O 2-

2 e -

2 e -

u z a v øe n ? c y k lu s

Figure. 1 Reductive metabolism of nitrocompounds. - R = aryl, furan, imidazol (Bryant, 1991). Metabolism of nitrocompound degradation intermediates - aminocompounds -

has been studied mostly in case of fungi and bacteria, where mineralization has been detected (Scheibner, 1997; Samson, 1998; Nishino, 1993; Boopathy, 1994). In case of plants TNT degradation has been described in case of poplar Populus sp. In this species 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene and a number of unidentified products more polar than TNT were found (Thompson, 1998). Vanderford (1997) suppose that products of nitrocompounds degradation might be in plants incorporated into the cell wall. Until now the mechanism of this part of degradation in plants has not been reported.

To the enzymes exhibiting nitroreductase activity belongs flavoenzyme NADPH: thioredoxin reductase (TR, EC 1.6.4.5), present in both prokaryotes and eukaryotes (Holmgren, 1989). In plants TR is localized in cytosol. Thioredoxin (redox protein reducing disulfide bounds) is involved in the regulation of the light control of enzymes of oxidative photosynthesis and germination (Florencio, 1988; Buchanan, 1994). Miškinine et al. (1998) found that TR isolated from Arabidopsis thaliana reduced nitroaromatic compounds via one-elektron transfer. They also found that TNT together with tetryl increased the fluorescence intensity of FAD at ? =518 nm.

Studying the reduction of p-nitrobenzen by xanthin oxidase both mechanisms of electron transfer were found: one-, two- and more elektron. NADH was used as an electron donor and the ratio of one-electron transfer to the whole electron flow was dependent on medium pH, however, not on the concentration of nitrocompounds (Tatsumi, 1981).

References

1. Anlezark G.M., Melton R.G., Sherwood R.F., Coles B., Friedlos F., Knox R.J. (1992) Biochem. Pharmacol. 44: 2289-2295.

2. Boopathy R., Kulpa C.F., Manning J., Montemagno C.D. (1994) Bioresour. Technol. 47: 205-208.

3. Bryant Ch., DeLuca M. (1991) J. Biol. Chem. 266: 4119-4125. 4. Buchanan, B., Schurmann P., Decottignies P., Lozano R.M. (1994) Arch.

Biochem. Biophys. 314: 257-260. 5. Carlberg I., Mannervik B. (1986) J. Biol. Chem. 261: 1629-1635. 6. Fiorella P.D., Spain J.C. (1997) Appl. Environ. Microbiol. 63: 2007-2015. 7. Florencio F.J., Yee B.C., Johnson T.C:, Buchanan B.B. (1988) Arch. Biochem.

Biophys. 266: 496-507. 8. Holmgren A., J.Biol. Chem. 264: 13693-13696 (1989). 9. Hughes J.B:, Shanks J., Vanderford M:, Lauritzen J., Bhadra R. (1997)

Environ. Sci. Technol. 31: 266-271. 10. Kutcher W.W., McCalla D.R. (1984) Biochem. Pharmacol. 33: 799-805. 11. Mishkiniene V., Sharlauskas J. Jacquot J.P., Chenas N. (1998) Biochim.

Biophys. Acta 1366: 275-283. 12. Montpas S., Samson J., Langlois E., Lei J., Piché Y., Chenevert R. (1997)

Biotechnol. Lett. 19: 291-294. 13. Moreno S.N.J., Mason R.P., Decampo R. (1984) J. Biol. Chem. 259: 6298-

6305. 14. Nishino S.F., Spain J.C. (1993) Appl. Environ. Microbiol. 59: 2520-2525. 15. Samson J., Langlois E., Lei J., Piché Y., Chenevert R. (1998) Biotechnol. Lett.

20: 355-358. 16. Shah M.M., Campbell J.A. (1997) Biochem. Biophys. Res. Commun.241: 794-

796. 17. Tatsumi K., Inoue A., Yoshimura H. (1981) J. Pharmacobiodyn. 4: 101-108. 18. Thompson P.L., Ramer L.A., Schnor J.L. (1998) Environ. Sci. Technol. 32:

975-980. 19. Vanderford M., Shanks J.V., Hughes J.B. (1997) Biotechnol. Lett. 19: 277-

280.

Cytochromes P450s

Daniele Werck-Reichhart

Intitute of Plant Molecular Biology CNRS-UPR 406, 28, rue Goethe

F-67000 Strasbourg France Daniele.werck@ibmp-ulp.u-strasbg.fr

Cytochromes P450 (P450s) constitute the largest family of enzymatic proteins

in higher plants: about 300 P450 genes are expected in the diminutive genome of Arabidopsis. P450s are heme proteins using electrons from NADPH to activate molecular oxygen. The reaction they catalyze is usually a monooxygenation, with formation of a molecule of water and an oxygenated product (1), but other more atypical activities, like dealkylations, dimerisations, isomerisations, dehydrations, reductions have also been reported.

(1) R-H + O2+ NADPH + H+ ----> R-OH + H2O + NADP+

Electrons from NADPH are transferred to P450s via flavoproteins called cytochrome P450-reductases. Both plant P450s and their reductases are usually bound via their N-terminus to the cytoplasmic surface of the endoplasmic reticulum. P450 proteins have molecular masses ranging from 45 to 62 kDa, and may have as little as 16 % amino acid identity. However, their overall tridimensional structure is conserved, as are a few residues on both sides of the heme.

Besides their physiological functions in the biosynthesis of hormones, lipids, and secondary metabolites, P450s help plant to cope with harmful exogenous chemicals making them less phytotoxic. Their role in the metabolism of industrial pollutants was not yet investigated, but an impressive metabolic capacity was demonstrated with agrochemicals and model compounds. First data were obtained from analysis of metabolites formed in vivo, or with preparations of microsomes from crop plants. In the last 5 years an increasing number of P450 genes have become available expressed in recombinant systems. It was thus possible to start investigating the ability of enzymes primarily involved in major plant pathways such as biosynthesis of lignin to metabolize exogenous compounds (1,2). The first genes of P450s very actively metabolizing herbicides were isolated only recently. Both CYP76B1 (3, 4) and CYP71A10 (5) expressed in yeast converted herbicides of the class of phenylurea to non-phytotoxic metabolites and conferred herbicide resistance to transgenic plants. For more information on plant P450s in metabolism of exogenous molecules refer to (6).

References 1. Schalk M., Batard Y., Seyer A., Nedelkina S., Durst F. and Werck-Reichhart

(1997) Design of fluorescent substrates and potent inhibitorsof CYP73As, P450s that catalyze 4-hydroxylation of cinnamic acid in higher plants. Biochemistry 36, 15252-15261.

2. Schalk, M., Pierrel, M.A., Zimmerlin, A., Batard, Y., Durst, F. and Werck-Reichhart, D. (1997) Xenobiotics: Substrates and inhibitors of the plant P450s. Env. Sci. Pollution Res. 4, 229-234.

3. Batard Y., LeRet M., Schalk M., Robineau T., Durst F. and Werck-Reichhart D. (1998) Molecular cloning and functional expression in yeast of CYP76B1, a xenobiotic-inducible 7-ethoxycoumarin O-deethylase from Helianthus tuberosus. Plant J. 14, 111-120.

4. Robineau T., Batard Y., Nedelkina S., Cabello-Hurtado F., LeRet M., Sorokine O., Didierjean L and Werck-Reichhart D. (1998) The chemically-inducible plant cytochrome P450 CYP76B1 actively metabolizes phenylureas and other xenobiotics. Plant Physiol. 118, 1049-1056.

5. Siminsky, B., Corbin, F. T., Ward, E. R., Fleischmann, T. J., and Dewey, R.E. (1999) Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides, Proc. Natl. Acad. Sci. USA 96, 1750-1755.

6. Werck-Reichhart, D., Hehn, A. and Didierjean, L. (2000) Cytochromes P450 for Engineering Herbicide Tolerance, Trends Plant Sci., March issue.

The role of plant associated rhizosphere bacteria for phytoremediation of organic pollutants

Daniel van der Lelie1 , Zita Snellinx1 , 2 , Safieh Taghavi1 , Jaco Vangronsveld2 , Frank Laturnus3 and Tomas Vanek4

1Vlaamse Instelling voor Technologisch Onderzoek (VITO), Environmental Technology Centre,

2400 Mol, Belgium 2Limburgs Universitair Centrum (LUC), Universitaire Campus, Diepenbeek, Belgium

3Department Plant Biology and Biogeochemistry, Risoe National Laboratory, Roskilde, Denmark 4Department Plant Cell Cultures, Institute of Organic Chemistry and Biochemistry, Academy of

Sciences of the Czech Republic, Praha, Czech Republic

Introduction The use of biological techniques can strongly reduce the cost for remediating

sites loaded with pollutants like heavy metals and organic xenobiotics. For large contaminated sites, bioremediation is the only alternative economically and socially acceptable. Therefore, phytoremediation, one of the soft bioremediation techniques, became an acceptable alternative for the treatment of sites and wastewater contaminated with organic xenobiotics. Phytoremediation is based on the combined action between plants and their associated microorganisms, such as mycorrhiza and bacteria. In this paper, the possibilities to use plant-associated bacteria to improve the efficiency of phytoremediation of organic xenobiotics are discussed.

Conditions for phytoremediation The development of an efficient strategy for phytoremediation of organic

xenobiotics requires several conditions to be fulfilled: the pollutant must be accessible for removal; the bioavailable concentration should not exceed toxicity thresholds of the plant; an uptake of the contaminants may be desirable; the contamination should not exceed areas accessible by the root system of the plants.

Bioavailability of contaminants The availability of a pollutant for degradation or uptake is one of the limiting

factors for efficient phytoremediation. Especially hydrophobic pollutants like e.g. PAHs are difficult to mobilise due to their low watersolubility. Both, plants and their associated rhizosphere bacteria can release surfactants. These compounds can increase the solubility, and subsequently the mobility and bioavailability of hydrophobic contaminants, which may lead to an increase in degradation of the pollutant by the plant rhizosphere system. Additionally, beside a direct degradation an uptake of the contaminants by the plants through the roots may also be possible. In general, lipophylic pollutants are difficult to take up by the root system. Only those contaminants are easily accessible by the root system with an octanol-water partition coefficient log Kow between 1 and 3. Fortunately, this is the case for many pollutants of environmental concern, such as BTEX, PCBs, PAHs and explosives. However, by choosing plants with a high lipid content also compounds with a higher log Kow are

available for plant-uptake, as these plants are considered highly capable of accumulation of hydrophobic contaminants.

Phytotoxicity of organic xenobiotics decreased by rhizosphere bacteria The possibilities to use phytoremediation as a cleanup strategy can be hindered

by the toxicity of the organic xenobiotics. Suitable rhizosphere bacteria can decrease the phytotoxicity of organic xenobiotics due to two processes. First, the bacteria can have general beneficial effects on plants, allowing them to tolerate higher toxicity levels. Secondly, bacteria can degrade the organic xenobiotic to a level below phytotoxicity that allow efficient phytoremediation. This clearly shows that both plants and their associated rhizosphere bacteria have an important contribution in the degradation process, which is based on a clear interaction between plant and associated bacteria. By secreting specific root exudates plants can stimulate the bacterial degradation of specific xenobiotics. Most bacterial degradation pathways only become induced when the concentration of a contaminant arrives above a threshold value. In case the bioavailable concentration of the contaminant is below this value, no degradation by the bacteria will occur. Under such conditions plants may excrete root exudates, which qua structure strongly resemble the contaminant, that are able to specifically induce the desired degradation pathway. These root exudates are plant specific and their production depends on many environmental factors. However, the suitable combination of plant and its associated rhizosphere bacteria is a prerequisite for the well functioning of the system.

Also less specific interactions exist by which plant can stimulate the degradation potential of its rhizosphere. An example is the secretion of sugars or phenolic compounds, which is independent of the presence of a specific pollutant. The secretion of phenolic compounds can, due to cometabolism, stimulate the degradation of TCE, e. g. by toluene monooxygenases of rhizosphere bacteria. The secretion of sugars will result in a general stimulation of the activity of the rhizosphere, which will speed up the degradation processes.

Plant growth stimulate by rhizosphere bacteria An increased root biomass or root activity in the soil will result in an improved

degradation activity in the root zone and better uptake of pollutants by plants. By using bacterial inocula, which stimulate plant growth, the efficiency of phytoremediation can be improved. This principle was demonstrated for the degradation of 2-CBA by wheat, where seeds were inoculated with the plant growth stimulating rhizosphere strain Pseudomonas aeruginosa R75. This strain is unable to degrade 2-CBA, but its presence strongly stimulated the overall degradation processes. In other words, it is possible to use a suitable bacterial inoculum to improve root growth and activity and subsequently the rhizosphere, resulting in better decontamination.

Conclusions By playing with the interactions between plants and their associated

microorganisms it is possible to improve the rhizosphere degradation and phytodegradation of organic xenobiotics. However, these are complex interactions that need to be well studied before they can be manipulated in order to improve the

overall efficiency of phytoremediation. This counts both for heavy metals and organic xenobiotics.