FLF, Alm.del - 2010-11 (1. samling) - Endeligt svar på spørgsmål 47: Spm. om oversendelse dokumentationen for, at restindholdet af spirotetramat i sojsbønner og bomuldsfrø m.v., til fødevareministeren

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Potential influence of the genetically modified EPSPS expressing genes andintroduction of the pat or bar genes on the metabolism of spirotetramat incotton and soybean compared to the wild-types

Leo Buerkle, Ingeborg Andersch

Bayer CropScience AGD-40789 MonheimGermany

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Request from EFSA:“...EFSA notes that some residue trials on soya and cotton have been performed with geneticallymodified crop. The applicant should therefore provide evidence that the genetically modification doesnot alter metabolism in the modified crop. If in the genetically modified crop a gene is inserted thatconveys active ingredient resistance due to pesticide metabolism, additional studies are requiredelucidating the metabolism in genetically modified crops.”

Some of the supervised residue trials in cotton and soybean were conducted with genetically modifiedplants being resistant towards the herbicide “Roundup”, which contains the active ingredient glyphosate(Roundup-Ready plants). Some cotton trials were conducted with glufosinate-resistant plants Liberty-Link plants.In this statement, the mode of action of the herbicides and mechanism of herbicide resistance isexplained, followed by the consideration about an influence of genetic modification on the metabolismof spirotetramat.Glyphosate resistant plantsGlyphosate is a non-selective herbicide. Its primary mode of action is the inhibition of the plant enzyme5-enolpyruvyl-3-shikimate-phosphate synthase (EPSPS), an enzyme in the shikimate pathway. Thismetabolic pathway produces aromatic amino acids in plants (i.e. phenylalanine, tyrosine, andtryptophan). These aromatic acids serve as precursors for numerous secondary plant products such asanthocyans, lignin, growth promoters, growth inhibitors, and phenolics, as well as proteins. The EPSPSenzyme converts phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to 5-enolpyruvyl-3-shikimate phosphate (EPSP) (see Figure 1). The EPSPS enzyme is encoded in the nuclear genome andis localized in the plastids.Glyphosate competitively inhibits the enzyme with respect to phosphoenolpyruvate with a Ki of 1 �M(Duke, 1988). Glyphosate structurally resembles the PEP oxonium ion with the exception that thephosphor in its phosphonate moiety is not attached to oxygen as in PEP but to a carbon atom. Thisallows glyphosate to bind very efficiently to the enzyme, but it cannot take part in the enzymaticreaction since the carbon-phosphor bond lacks the high energy of the oxygen-phosphor bond (P-C513.4 kJ mol-1, P-O 596.6 kJ mol-1, CRC, 1986-1987). The glyphosate molecule locks the activecomplex of the EPSPS enzyme and its substrate, and the glyphosate inhibitor remains bound to theenzyme (Schönbrunn et al., 2001). In this inhibitory mechanism the enzymatic reaction is completelyblocked and neither the S3P substrate nor the inhibitor is further processed by the enzyme.Blockage of the EPSPS enzyme results in a disruption of the shikimate pathway and consequently in amassive accumulation of shikimate in affected plant tissue. The in vivo activity of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, an earlier enzyme in the shikimate pathway, is also increased as aresult of glyphosate inhibition of EPSPS (Pinto et al., 1988). Glyphosate is the only compound knownto inhibit EPSPS sufficiently to be a valuable herbicide.

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Inhibition of the EPSPS mediated reaction by the glyphosate moleculeEPSPsynthase

Shikimate-3-phosphate(S3P)

Phoshoenolpyruvate(PEP)

5-Enolpyruvyl-3-shikimate phosphate(EPSP)

COO-PPOOHShikimate-3-phosphate(S3P)Glyphosate

POOH5-Enolpyruvyl-3-shikimate phosphate(EPSP)

Glyphosate tolerant crop varieties have now been developed by introduction of an alternative EPSPSexpressing gene into the plant’s genome. The resulting EPSPS enzymes, i.e. a CP4 EPSPS version inRoundup-Ready plants from Monsanto, or a 2mEPSPS version from BCS, have a significantly loweraffinity to glyphosate, but no altered functionality in synthesizing aromatic amino acids when comparedto the wild-type crops.In an alternative approach to render the plant tolerant to the glyphosate, a copy of an enzyme thatmetabolizes this herbicide is introduced. Two different activities are currently used in this strategy: theglyphosate oxidoreductase (GOX) that is degrading glyphosate to non-active AMPA (aminomethyl-phosphonic acid) and the glyphosate acetyl transferase (GAT) that is catalyzing the acetylation of theherbicide and thus inactivating the phytotoxic effect (Franz et al., 1997).In Roundup-Ready plants production of the genetically modified EPSP synthase is implementedresulting in glyphosate tolerance by production of an EPSPS enzyme that is not inhibited by glyphosatewhile its enzymatic activity remained virtually unchanged. As this modified enzyme is not involved inthe co-metabolism of xenobiotica the metabolic transformations of other herbicides, e.g. Spirotetramat,remains unchanged compared to the wild-type species.

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Glufosinate resistant plantsGlufosinate is a non-selective post-emergence herbicide that controls weed by irreversibly inhibiting theenzyme glutamine synthetase which plays a primary role in the plant metabolism. This enzymeincorporates glutamate and ammonia to form the amino acid glutamine as shown in the followingschema (Figure 2).As glufosinate is a structurally analogous compound to glutamate it competes during the glutaminesynthetase reaction.GlufosinateHOOOHOPCH3NH2HOONH2GlutamateHOO

Inhibition of the glutamine synthetase by glufosinateGlutamateHOOO

ATPNADPHGlutamateSynthaseGlutamineSynthetaseO

HOalpha-ketoglutaratealpha-ketoglutamate

Glutamine synthetase contained in leaves and roots having different isoforms that show differentsensitivities to glufosinate. The inhibition of glutamine synthetase is manifested by ammoniaaccumulation, inhibition of amino acid synthesis and inhibition of photosynthesis.

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Glufosinate tolerance is conferred to plants by incorporation of either the pat (phosphinothricin acetyltransferase) or the bar (bialaphos resistance) gene, both of which code for enzymes that inactivateglufosinate by acetylation (Liberty Link plants). Both, the pat and bar enzymes are similar in theirability to selectively acetylate glufosinate but not other amino acids (Figure 3).Figure 3: Acetylation of glufosinateHOOOOHOPCH3GlufosinateNH2HOO

N-Acetyl-Glufosinate

The acetylation of glufosinate by pat and bar-gene-expressed acetyl transferase enzymes is very specificto glufosinate. Xenobiotic molecules with a different structure, e.g. spirotetramat, are metabolically notchanged if compared to the wild-type species.

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Influence of a genetic modifications of the EPSPS expressing and pat or bar genes onspirotetramat metabolismThe enzymes responsible for the glyphosate and glufosinate tolerance in plants exhibit very specificreactions that do not influence the metabolism of other xenobiotica.EPSP synthase exclusively appears in the shikimate pathway. A production of the genetically modifiedversion of this enzyme in Roundup-Ready plants results in the same biochemical synthesis reactions asobserved in the wild-type species and does not have any influence on spirotetramat metabolism.The acetylation of glufosinate by genetically modified bar and pat gene-expressing enzymes in Liberty-Link plants is also considered as a very specific process. Other amino acids are not acetylated(Wehrmann et al., 1996). As spirotetramat and its metabolites in plants are structurally completelydifferent from glufosinate (Figure 4), the enzyme acetylating glufosinate would not alter metabolism ofspirotetramat.Considering the specifity of the enzymes and the structure of their substrates, there is no evidence thatthe genetical modification in Roundup-Ready or Liberty-Link soybean or cotton plants could have aninfluence on the metabolic transformation of spirotetramat.Figure 4: Structures of spirotetramat and its plant metabolites:SpirotetramatH3COCH3ONHOCH3HOO CH3

Spirotetramat-enolHHOCH3O CH3

Spirotetramat-enol-glucosideHOHOOHOHO CH3

Spirotetramat-ketohydroxyHOCH3OHCH3O CH3

Spirotetramat-monohydroxyHeHOCH3OCH3

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ReferencesNo123Author(s), year, title, source, edition, pagesCRC Handbook of Chemistry and Physics, 67th ed., 1986-1987, pp. F-168, F-171Duke, S. 0. (1988) Glyphosate. In: Herbicides-Chemistry, Degradation and Mode of Action,Vol. 3 (P. C. Kearney and D. D. Kaufman, Eds.), Marcel Dekker, Inc., New York.Pinto, J. E. B. P., Dyer, W. E., Weller, S. C. and Hermann, K. M. (1988) Glyphosate induces3-deoxy-D-arabino-heptulosinate 7-phosphate synthase in potato (Solanum tuberosum L.)cells grown in suspension culture. Plant Physiol. 87: 891-893Schönbrunn, E., Eschenburg, S., Shuttleworth, W.A;, Schloss, J. V., Amrhein, N., Evans, J.N.and Kabsch, W. (2001) Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phospahte synthase in atomic detail, Proc. Natl. Acad. Sci; U.S.A. 98,1376-1380.Franz J. E; Mao, M. K. and Sikorski, J. A. (1997) Glyphosate: A unique global herbicide.ACS Monograph 189, American Chemical Society, Washington , DCWehrmann, A., A. Van Vliet, C. Opsomer, J. Botterman, and A. Schulz. 1996. The similaritiesofbarandpatgene products make them equally applicable for plant engineers. Nat.Biotechnology. 14:1274-1278.