Phase 1 Xenobiotic Metabolic Systems in PlantsTamas Komives" and Gábor GullnerPlant Protection Institute of the Hungarian Academy of Sciences, H-1022 Budapest,Herman Otto u. 15, Hungary. E-mail: tkom@nki.hu

* Author for correspondence and reprint requests

z. Naturforsch. 60c, 179-185 (2005)

Phytoremediation uses living higher plants for the removal and biochemical decompositionof environmental pollutants. In this paper Phase 1 metabolic pathways in the biotransforma-tion reactions of organic pollutants in plants are reviewed. These reactions result in theintroduction of functional groups in the xenobiotic molecule or the exposure of preexistingfunctional groups and lead to the formation of more polar, more water-soluble, chemicallymore reactive and sometimes biologically more active derivatives. Phase 1 type reactions aremost important in the phytoremediation of hydrophobic, chemically stable organic pollutants,such as polycyclic aromatic hydrocarbons and (poly)chlorinated aliphatic and aromatic hy-drocarbons. Although Phase 1 reactions involve a wide range of chemical transformationsfrom hydrolysis to reduction, oxidative processes catalyzed by cytochrome P450 containingmonooxygenases are the most important. Transgenic plants with tailored Phase 1 enzymaticactivities may play major roles in the removal of environmentally stable organic pollutantsfrom contaminated fields.

Key words: Xenobiotics, Metabolism, Phytoremediation

Introduction

Fate of pollutants in the plant-soil system is de-termined by a highly complica ted set of chemical,biochemical, physical and biophysical reactions,elements which may play significant roles in deter-mining the ultimate success of the solution of aparticular pollution problem by phytoremediation.Thus, the rate and efficiency of phytoremediationof polluted soils depends on the physical andchemical properties of the pollutant and those ofthe soil: pollutants may be bound to soil partic1es,or taken up by microorganisms, plants, and ani-mals. In the process of phytoremediation the avail-ability of the pollutant for uptake by microorga-nisms of the rhizosphere and the plant root systemis of cruci al importance. Once in the plant tissuethe pollutant may be transformed by a wide vari-ety of chemical/biochemical metabolic reactions.Only those plants having the appropriate bio-chemical pathways are effective for phytoremedia-tion purposes. Complete mineralization of persis-tent pollutants may be very slow; intermediatesmay rem ain for substantial periods of time. Basicresearch in the field of plant physiology, biochem-istry and molecular biology of these processespaved the way for the development of phyto-remediation (Komives and Gullner, 2000).

Role of Phase 1 Transformations in theMetabolism of Xenobiotics in Plants

The "green liver" concept of Sandermann(1994) describes the fate of organic contaminantsin plan ts. Aceording to this concept, plants, as au-totrophic organisms, do not need organic com-pounds synthesized by other organisms as carbon,energy, and nitro gen source. Therefore, metabo-lism of foreign compounds (so-called xenobiotics)in plan ts serves detoxification purposes. Metabo-lism of xenobiotics in plants usually occurs in threephases (Fig.1). In Phase 1 xenobiotic moleculestypically undergo hydrolytic or oxidative trans-

Xenobiolic (hydrophobic)

1 Phase I

Funclionalized derivalive (Iess hydrophobic)

1 Phase 1/

Conjugale (hydrophilic)

1 Phase 11/

Tenninal melabolile (compartmentized)

Fig. 1. Scheme of degradation of xenobiotics in plants.

0939-5075/2005/0300-0179 $ 06.00 © 2005 Verlag der Zeitschrift für Naturforschung, Tübingen· http://www.znaturforsch.com .D

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formations. Phase 1 enzymes are also involved ina number of reductive reactions, gene rally underoxygen-deficiency condition. Usually, these reac-tions result in the introduction of functionalgroups in the molecule or the exposure of preex-isting functional groups and lead to the formationof more pol ar, more water-sol ub le, and usuallychemically and biologically more active deriva-tives. Therefore, Phase 1is often called the bioacti-vation phase of metabolism. In Phase II the actualdetoxification happens: the product of the Phase 1transformation is bound to an endogenous sugaror peptide molecule. Phase II reactions are calledconjugation processes and their products are theglycosyl, amino acid, or peptide conjugates of thexenobiotic compound. Conjugates are significantlyless toxic th an the original molecule or its Phase 1derivatives (Hatzios and Burgos, 2004). Plants -with the exclusion of aquatic plan ts - lack theexcretion system of animals. In plant cells toxicmetabolites and pollutants are sequestered intothe storage compartments. This Phase III type pro-cess is an active one and is catalyzed by mem-brane-bound ATP-driven pumps. Thus, in plan tsPhase III represents the final rem oval and decom-position of these conjugates via exporting theminto the cell vacuole, and/or incorporating th eminto biopolymers, such as lignin (Sandermann,1994).

Metabolism of xenobiotics in humans has be enstudied extensively because of toxicological andpharmacological reasons. Recently, in order to ex-plain differential individual response to drug treat-ments of humans, a new discipline has evolved:pharmacogenetics aims to study these variations totailor therapy for improved response and reducedtoxicity (Bell et al., 2003). Most research so far hasfocused on single polymorphisms of key Phase 1metabolic enzym es. In general four phenotypeswere identified: poor metabolizers, who lack thefunctional metabolic enzyme; intermediary meta-bolizers, who are heterozygous for one differentallele or carry two alleles that cause reduced activ-ity; extensive metabolizers, who have two normalalleles; and ultrarapid metabolizers, who havemultiple gene copies (Ingelman-Sundberg, 2002).In addition, attempts have been made to correlatemetabolic rates of animals belonging to differentspecies and extrapolating the results to humans(Vermeulen, 2003).

In contrast to drug metabolism in animals andhumans, phytoremediation is a more complex pro-

T. Komives and G. Gullner . Phase 1 Systems in Plants

cess because it involves the uptake and the trans-location of the xenobiotic in the plant, as well asthe participation of soil microorganisms in theprocess. Therefore, in phytoremediation a morecomprehensive approach is necessary in order tosuccessfully predict and influence plant responseand the gene tic variation in entire biological path-ways needs to be considered (Campanella et al.,2002; Coleman et al., 2002). Efforts to constructpathways of genes that have phytoremediation rel-evance for individual pollutants are necessary. To-gether, these pathway strategies offer a higherlikelihood of achieving the promise of geneticallyguided phytoremediation.

Although not part of the xenobiotic metabolicphases enzymes participating in the antioxidativesystems often determine the ability of a plant totolerate environment al stress. Oxygen radicals areproduced at various electron transfer sites or viadifferent oxidation reactions in plant tissues. Un-der chemical stress conditions production of theseradicals is powerfully enhanced. For example,rnicromolar concentrations of mercury(II) ions in-duce lipid peroxidation reactions in leaf tissues ofmaize (Zea mays) plants (Komives et al., 1994).Plants are able to protect themselves against thedamaging effects of endogenous oxygen radicals.It has been shown that a critical balance exists be-tween oxyradical-generating factors and the activ-ity of the systems that protect the cell from theirharmful effects. Antioxidant defenses split intothree general classes including water-soluble re-ductants, e.g. compounds that contain thiol-groups[cysteine, glutathione (v-t-glutamyl-t-cysteinyl-glycine, GSH), etc.], ascorbate and catechols; lipid-soluble compounds, e.g. a-tocopherol and f3-caro-tene; and enzymatic antioxidants, e.g. GSH peroxi-dase (GP, E.C. 6.4.11.6), ascorbate peroxidase(E.C. 1.11.1.11), catalase (E.C. 1.11.1.6), and su-peroxide dismutase (E.C. 1.15.1.1) (Komives et al.,1997). Coordinated activities of these enzymescontribute to plant fitness and will thereby alsoinfluence phytoremediation efficiency.

Hydrolysis reactions

In plants hydrolytic transformations of xenobio-tics containing ester and amide bonds usually oc-cur with the participation of esterase (E.C.3.1.1.1), amidase (E.C. 3.5.1.4), or protease en-zymes (E.C. 3.4.-.-) (Cummins and Edwards,2004). The product of the hydrolysis reaction is the

T. Komives and G. Gullner . Phase 1 Systems in Plan ts

CH3

02NAN02

YN02

TNT

02N....N~N"' ..N02L....

N

)

IN02

RDX

~'O'

02NO ON02

GTN PETN

Fig. 2. Chemical structures of the environmental pollu-tant explosives TNT, RDX, GTN, PETN.

free carboxylic acid that is subsequently conju-gated to a sugar molecule (usually glucose) in aPhase II process (Van Eerd et al., 2004). Hydroly-sis of carbamic acid esters liberates the unstablecarbamic acids that decompose to the amine andcarbon dioxide. However, hydrolytic phytoremedi-ation of carbamate-polluted environment is com-plica ted by the fact that these chernicals areknown to inhibit hydrolytic enzymes (Newmanet al., 2001).

Soil and groundwater contamination due toexplosives (Fig. 2) such as glycerol trinitrate(GTN, nitroglycerin), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), pentaerythritol tetranitrate(PETN), and 2,4,6-trinitrotoluene (TNT) i~. aproblem at many ammunition plants. The abilityof plants to metabolize GTN was examined usingcultured sugar beet (Beta vulgaris) plant cells andplant cell extracts. Intact ceIls rapidly degr~d~dGTN with the initial formation of glycerol dinit-rate (GDN) and the later formation of glycerolmononitrate. A material balance analysis of theseintermediates indicated little, if any, formation ofreduced, conjugated or cell-bound carbonaceousmetabolites. Cell extracts were shown to be capa-ble of degrading GTN with the simultaneous for-mation of GDN in stoichiometric amounts (Goelet al., 1997).

Thiolysis reactions

Thiol groups as compared to hydroxyl groupsare characterized with significantly higher chemi-cal reactivity. Endogenous thiol group-containingpeptides and proteins are known to play majorroles in Phase II detoxification reactions. Themechanism of an interesting Phase 1 type thiol-

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catalyzed isomerization has been described byJablonkai et al. (1999). The re action results in bio-activation of a thiadiazole compound to a herbici-dal thiadiazolone derivative.

Oxidation reactions

The most common Phase 1 reactions are oxida-tive process es that involve cytochrome P450 en-zymes (CYP, E.C. 1.14.-.-). These enzymes s~p-port the oxidative, peroxidative and redu~tl~emetabolism of both endogenous and xenobioticsubstrates. They comprise a superfamily of heme-thiolate proteins present in every class of organ-isms, including Archaea, and in humans they areresponsible for 70-80% of all Phase I-dependentmetabolism of clinically used drugs (Danielson,2002; Ingelman-Sundberg, 2002). In plants there .isa surprisingly high number of CYP genes: 246 mArabidopsis (representing approximately 1% ofthe plant's gene complement) compared with lessthan a 100 in humans (Nelson et al., 2004). CYPenzymes are characterized by the high diversity ofreactions that they catalyze and the high range oftheir chernically divergent substrates. Increasingemphasis on functional genornic approaches toCYP research recently has greatly advanced ourunderstanding of CYP-mediated reactions inplants (Nelson et al., 2004; Schuler and Werck-Reichhardt, 2003). Selective inducibility of CYPisozymes plays important roles in increasing cropplant tolerance to phytotoxic compounds (Komi-ves and Dutka, 1989).

The halogenated organic aliphatic solven ts, suchas car bon tetrachloride, chloroform, and trichloro-ethylene (TCE) are among the most commontoxic substances found at hazardous waste sites.Oxidative transformation of these chemicals inplants is of high importance during phytoremedia-tion. Thus, axenic cultures of poplar (Populus spp.)tissues were shown to convert TCE to trichloro-ethanol, trichloroacetic acid, dichloroacetic acidand a small amount of CO2. At the whole plantlevel TCE was not toxic to the poplar trees at con-centrations much higher than levels usually foundat hazardous waste sites. The trees did not releasesignificant amounts of TCE into the air, becausethey not only took up the compound, but also me-tabolized it within their tissues to a far greater ex-tent as was found in the laboratory studies. Similarresults were obtained with carbon tetrachloride(CCI4), i.e. about 90% of the material was re-

182

-:CI CI>=< --CI H <,

CI OHH+--{

CI O

CI HCI-1-f-H

CI OH

HO O

HO OH. /"

CI OH»:CinCI O

Fig. 3. Metabolism of TCE in plants.

moved from the water at 20 ppm and no CC14

could be detect ed in the transpirate. Thus, intissues of poplar trees chlorinated aliphatic hy-drocarbons can undergo oxidation and dechlorina-tion process es, and finally, complete mineraliza-tion to CO2 (Gordon et al., 1998).

Transgenic tobacco (Nicotiana tabacum) plantsexpressing the mammalian cytochrome P450 2E1also metabolized TCE at an elevated rate: up to640-fold as compared with null vector controlplants. Metabolism led to the formation of trichlo-roethanol glycoside and oxalic acid (Fig.3). Thetransgenic plants also showed an increased uptakeand debromination of ethylene dibromide (Dotyet al., 2000; Shang et al., 2001). Tobacco plantsoverexpressing the soybean (Glycine max) enzymeCYP71A10 transforrned the herbicide chlorto-luron to a nontoxic hydroxymethyl compound ascompared to the wild-type plants where the me-tabolism lead to the formation of the still-toxic N-demethylated derivative (Fig.4; Siminszky et al.,1999).

Recalcitrant organic pollutants are resistant toattack by microbial and plant enzymes. Rationalprotein engineering of bacterial and mammalianCYP enzymes led to mutants with dramatically

CI-p-~ NH CH}--

I 3- N\

H,C j Cyp o CH,

CypCly-~ NH CH

}--I 3

- N\

HO O CH3

CI-p-~ NH CHLI 3

- I/NH3C O LOH

(decomp.)CI-p-~ NH CH

LI 3- rNHH3C O

Fig. 4. CYP-mediated metabolism of chlorotoluron inplants.

T. Komives and G. Gullner . Phase 1 Systems in Plants

increased ability to oxidize polyaromatic hydro-carbons (pyrene, benzo[ a]pyrene, and fluor-anthrene) and chlorinated aromatics (chloroben-zenes and 2,3,7,8-tetrachlorodibenzodioxin)(Carmichael and Wong, 2001; Bell et al., 2003;Shinkyo et al., 2003). Plants transforrned to pro-duce the engineered CYP isozymes may be capa-ble to phytoremediate recalcitrant environmentalcontaminants.

Reduction reactions

Phytotoxicity of metals and metalloids can bedecreased in plants via chemical reduction of theelement followed by its incorporation into organiccompounds. For example, selenium is volatilizedby plants in the form of dimethylselenide that isseveral hundred times less toxic than inorganicforms of this element (de Souza et al., 1998).Transgenic Arabidopsis plants containing and ex-pressing the bacterial gene merB convert thehighly toxic methylmercury to mercury(II), andshow high resistance against toxic levels of methyl-mercuric chloride and phenylmercuric acetate(Bizily et al., 1999). Similarly, the processes leadingto the reduction of chromium(VI) to chromi-um(III), mercury(II) to mercury(O), cadmium(II)to cadmium(O) and perchlorate to chlorite andchloride are important detoxification mechanismsin plants (Dhankher et al., 2003; Rugh et al., 1996;Susarla et al., 2000). The enzyme pentaerythritoltetranitrate reductase (E.C. 1.7.99.-) of the explo-sive-degrading bacterium Enterobacter cloacae en-ables it to decompose organic nitrate esters andseveral nitroaromatic explosives (Khan et al.,2004). Seeds of transgenic tobacco plants express-ing this enzyme were able to germinate and growin the presence of GTN and TNT at concentra-tions that inhibited germination and growth ofwild-type seeds. The transgenic seedlings alsoshowed more rapid and complete denitration ofGTN than wild-type seedlings (Freneh et al., 1999).CYP enzymes are usually oxidases but under con-ditions of low O2 tension they may also participatein reductive reactions (Guengerich, 2001). Thus,they may catalyze reductive dehalogenation re ac-tions and the reduction of many nitrogen com-pounds. For example, recent work shows that ex-plosives, such as RDX may be rapidly decomposedby membrane associated bacterial and mammalianCYP or flavin adenine dinucleotide-containing en-zymes (Bhushan et al., 2003, 2004).

T. Komives and G. Gullner . Phase 1 Systems in Plants

Future Perspectives

Full potential of the Phase 1 plant detoxificationsystem can be exploited only if the uptake, thePhase II conjugation, and metabolite disposal pro-cesses (Phase Ill) are efficient, too. For example,transgenic plants with increased ability to secretemetal-chelating products are more efficient in ta-king up and accumulation of metals in aeri al tis-sues (Tesfaye et al., 2001). Transgenic tobaccoplants expressing a calmodulin-binding protein(involved in metal uptake across the plasma mem-brane) showed increased accumulation of lead(Arazi et al., 1999) and overexpression of Al-in-duced genes in Arabidopsis was confirmed to ame-liorate Al toxicity (Ezaki et al., 2004). Phase IIconjugative enzymes are of crucial importance totransform bioactivated hydrophobic pollutantsinto chemically less reactive and less toxic deriva-tives. Engineering transgenic plants to express cer-tain glutathione S-transferase isoenzymes is alsoimportant for crop breeding with useful traits in-cluding phytoremediation. Recent studies showedthe existence of a Phase III system that is involvedin the elimination of potentially toxic pollutantsfrom the cytosol. Tobacco plants expressing anArabidopsis calcium exchanger antiporter accu-mulate more Ca2+, Cd2+, and Mn2+ and are moretolerant to elevated Mn2+ levels (Hirschi et al.,2000) and Arabidopsis plants expressing the yeastvacuolar transporter YCF1 are more tolerant toCd2+ and Pb2+ (Tong et al., 2004).

Conclusions

Phytoremediation research has made significantstrides in re cent years by taking advantage of the

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spectacular developments in molecular biologyand gene tic engineering. As a result, our knowl-edge of the factors that influence phytoremedia-tion efficacy has expanded greatly. It became evi-dent that phytoremediation efficacy is stronglyinfluenced by the ability of the plant to escapedeleterious concentrations of the toxic form ofthe pollutant and the active oxygen species thatmight be generated in the treated tissue. The keyrole of the Phase 1 metabolic system in thebiotransformation of organic pollutants in sometolerant plants has been clearly established. How-ever, much is yet to be learned about this systemin plan ts, especially, with respect to its specificityand the mechanisms of its induction. In vivo andin vitro studies to follow pollutant-inducedchanges in plant biochemistry, biophysics andmolecular biology provide us with intriguingchallenges for further research. Attention is nowbeing focused on the development and use oftransgenic plants specifically tailored for the bior-emediation of persistent organic pollutants andheavy metals. Given the existing advances in plantmolecular biology, the use of such plants in thedaily practice of phytoremediation could soon be-come a reality.

Acknowledgements

The financial aid of the Organization of Eco-nomic, Cooperation and Development (OECD)and grants from the Hungarian Scientific ResearchFund (OTKA T 43476), the Hungarian NationalOffice for Research and Technology and theHungarian Ministry of Economy and Transport(GVOP-2004-3.1.1) are gratefully acknowledged.

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Arazi T., Sunkar R., Kaplan B., and Fromm H. (1999),A tobacco plasma membrane calmodulin-bindingtransporter confers Ni2+ tolerance and Pb2+ hyper-sensitivity in transgenic plants. Plant J. 20, 171-182.

Bell S. G., Chen x., Xu F., Rao Z., and Wong L. (2003),Engineering substrate recognition in catalysis bycytochrome P450cam. Biochem. Soc. Trans. 31, 558-562.

Bhushan B., Paquet L., Spain J. c., and Hawari J. (2003),lnitial reaction(s) in biotransformation of CL-20 iscatalyzed by salicylate 1-monooxygenase from Pseu-domonas sp. strain ATCC 29352. Appl. Environ.Microbiol. 70, 4040-4047.

Bhushan B., Trott S., Spain J. c., Halasz A., Paquet L.,and Hawari J. (2004), Biotransformation of hexahy-dro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbitliver cytochrome P450: insight into the mechanism ofRDX biodegradation by Rhodococcus sp. strainDN22. Appl. Environ. Microbiol. 69, 1347 -1351.

Bizily S. P., Rugh C. L., Summers A. 0., and Meagher,R. B. (1999), Phytoremediation of methylmercury pol-lution: merB expression in Arabidopsis thaliana con-fers resistance to organomercurials. Proc. NatI. Acad.Sci. USA 96, 6808-6813.

Campanella B., Bock c., and Schröder P. (2002), Phyto-remediation to increase the degradation of PCBs andPCDDlFs. Environ. Sci. Pollut. Res. 9, 73-85.

Carrnichae1 A. B. and Wong L. L. (2001), Protein engi-neering of Bacillus megaterium CYP102 - The oxida-tion of polycyc1ic aromatic hydrocarbons. Eur. J. Bio-chem. 268, 3117-3125.

Coleman J. O., Frova c., Schröder P., and Tissut M.(2002), Exploiting plant metabolism for the phytore-mediation of persistent herbicid es. Environ. Sci. Pol-lut. Res. Int. 9, 18-28.

Cummins I. and Edwards R. (2004), Purification andcloning of an esterase from the weed black-grass (Alo-pecurus myosuroides), which bioactivates aryl-oxypbenoxypropionate herbicides. Plant J. 39, 894-904.

Danielson P. B. (2002), The cytochrome P450 superfam-ily: biochemistry, evolution and drug metabolism inhumans. Current Drug Metab. 3, 561-597.

de Souza M. P., Pilon-Smits E. A., Lytie C. M., HwangS., Tai J., Honma T. S., Yeh L., and Terry N. (1998),Rate-limiting steps in selenium assimilation and vola-tilization by Indian mustard. Plant Physiol. 117,1487-1494.

Dhankher O. P., Shasti N. A., Rosen B. P., Fuhrmann M.,and Meagher R. B. (2003), Increased cadmium toler-ance and accumulation by plants expressing bacterialarsenate reductase. New Phytologist 159, 431-441.

Doty S. L., Shang T. Q., Wilson A. M., Tangen J., West-ergreen A. D., Newman L. A., Strand S. E., and Gor-don M. P. (2000), Enhanced metabolism of haloge-nated hydrocarbons in transgenic plants containingmammalian cytochrome P450 2E1. Proc. NatI. Acad.Sci. USA 97, 6287 -6291.

Ezaki B., Suzuki M., Motoda H., Kawamura M., Nakas-hima S., and Matsumoto H. (2004), Mechanism ofgene expression of Arabidopsis glutathione S-trans-ferase, AtGSTl, and AtGSTll in response to alumi-num stress. Plant Physiol. 134, 1672-1682.

T. Komives and G. Gullner . Phase 1 Systems in Plants

Freneh C. E., Rosser S. J., Davies G. J., Nicklin S., andBruce N. C. (1999), Biodegradation of explosives bytransgenic plants expressing pentaerythritol tetrani-trate reductase. Nature Biotechnol. 17, 491-494.

Goel A., Kumar G., Payne G. F., and Dube S. K. (1997),Plant cell biodegradation ofaxenobiotic nitrate ester,nitroglycerin. Nature Biotechnol. 15, 174-177.

Gordon M., Choe N., Duffy J., Ekuan G., Heilman P.,Muiznieks I., Ruszaj M., Shurtleff B. B., Strand S.,Wilmoth J., and Newman L. A. (1998), Phytoremedia-tion of trichloroethylene with hybrid poplars. Environ.Health Perspect. 106, 1001-1004.

Guengerich F. P. (2001), Uncommon P450-catalyzed re-actions. Curr. Drug Metab. 2, 93-115.

Hatzios K. K. and Burgos N. (2004), Metabolism-basedherbicide resistance: regulation by safeners. Weed Sci.52, 454-467.

Hirschi K. D., Korenkov V. D., Wilganowski N. L., andWagner G. J. (2000), Expression of Arabidopsis CAX2in tobacco. Altered metal accumulation and increasedmanganese tolerance. Plant Physiol. U4, 125 -133.

Ingelman-Sundberg M. (2002), Polymorphism of cyto-chrome P450 and xenobiotic toxicity. Toxicology 181-182, 447 -452.

Jablonkai 1., Komives T., Böger P., Sato Y., and Waka-bayashi K. (1999), Chemical catalysis of the isomerisa-tion of peroxidising herbicid al thiadiazolidines. Pesti-cide Sci. SS, 657 -658.

Khan H., Barna T., Harris R. J., Bruce N. c., BarsukovI., Munro A. W., Moody P. C. E., and Scrutton N. S.(2004), Atomic resolution structures and solution be-havior of enzyme-substrate comp1exes of Enterobactercloacae PB2 pentaerythritol tetranitrate reductase. J.Biol. Chem. 279, 30563-30572.

Komives T. and Dutka F. (1989), Effects of herbicidesafeners on 1evels and activities of cytochrome P450and other enzymes of com. In: Herbicide Safeners:Development, Uses and Modes of Action (HatziosK. K. and Hoagland R. E., eds.). Academic Press,New York, pp. 129-151.

Komives T. and Gullner G. (2000), Phytoremediation.In: Piant-Environment Interactions (WilkinsonR. E., ed.). Marcel Dekker Inc., New York, pp. 437-452.

Komives T., Aioub A. A. A., and Gullner G. (1994), Ef-fects of mercuric chloride on the glutathione transfer-ase enzyme activity in com (Zea mays L.) plants. Ce-real Res. Commun. 22, 99-104.

Komives T., Gullner G., and Király Z. (1997), The ascor-bate-glutathione cyc1e and oxidative stresses in plants.In: Regulation of Enzymatic Systems DetoxifyingXenobiotics in Plants (Hatzios K. K., ed.). KluwerAcademic Publishers, Dordrecht, The Netherlands,pp. 85-96.

Nelson D. R., Schuler M. A., Paquette S. M., Werck-Reichhart D., and Bak S. (2004), Comparative geno-mics of rice and Arabidopsis. Analysis of 727 cyto-chrome P450 genes and pseudogenes from a monocotand a dicot. Plant Physiol. 135, 756-772.

Newman J. W., Denton D. L., Morisseau c., Koger C. S.,Wheelock C. E., Hinton D. E., and Hammock B. D.(2001), Evaluation of fish models of soluble epoxidehydrolase inhibition. Environ. Health Perspect. 109,61-66.

T. Komives and G. Gullner . Phase 1 Systems in Plan ts

Rugh C. L., Wilde H. D., Stack N. M., Thompson D. M.,Sumrners A. O., and Meagher R. B. (1996), Mercuricion reduction and resistance in transgenic Arabidopsisthaliana plants expressing a modified bacterial merAgene. Proc. Nat!. Acad. Sci. USA 93, 3182-3187.

Sandermann H. (1994), Higher plant metabolism ofxenobiotics: The 'green liver' concept. Pharmacoge-netics 4, 225-241.

Schuler M. A. and Werck-Reichhart D. (2003), Func-tional genomics of P450s. Annu. Rev. Plant Bio!. 54,629-667.

Shang T. Q., Doty S. L., Wilson A. M., Howald W. N.,and Gordon M. P. (2001), Trichloroethylene oxidativemetabolism in plan ts: the trichloroethanol pathway.Phytochemistry 58, 1055-1065.

Shinkyo R., Sakaki T., Takita T., Ohta M., and InouyeK. (2003) Generation of 2,3,7,8-TCDD-metabolizingenzyme by modifying rat CYP1A1 through site-di-rected muta gene sis. Biochem. Biophys. Res. Commun.308,511-517.

Siminszky B., Corbin F. T., Ward E. R., FleischmannT. J., and Dewey R. E. (1999), Expression of a soy-

185

bean cytochrome P450 monooxygenase cDNA inyeast and tobacco enhances the metabolism ofphenylurea herbicides. Proc. Nat!. Acad. Sci. USA 96,1750-1755.

Susaria S., Bacchus S., Harvey G., and McCutcheon S.(2000), Phytotransformation of perchlorate contami-nated waters. Environ. Techno!. 21, 1055-1065.

Tesfaye M., Stephen J., Temple S. J., Allan D. L., VanceC. P., and Samac D. A. (2001) Overexpression of ma-late dehydrogenase in transgenic alfalfa enhances or-ganic acid synthesis and confers tolerance to alumi-num. Plant Physio!. 127, 1836-1844.

Tong Y. P., Kneer R., and Zhu Y. G. (2004), Vacuolarcompartmentalization: a second-generation approachto engineering plants for phytoremediation. TrendsPlant Sci. 9, 7 -9.

Van Eerd L. L., Hoagland R. E., Zablotowicz R. M., andHall J. C. (2004), Pesticide metabolism in plants andmicroorganisms. Weed Sci. 51, 472-495.

Vermeulen N. P. E. (2003), Prediction of drug metabo-lism: the case of cytochrome P450 2D6. Curr. Top.Med. Chem. 3, 1227-1239.