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Physiological Mechanisms of Glyphosate Resistance

Published online by Cambridge University Press:  20 January 2017

Wendy Pline-Srnic*
Affiliation:
Biology and Logistics, Syngenta, Jealotts Hill International Research Centre, Bracknell, Berkshire RG42 6EY, U.K
*
Corresponding author's E-mail: wendy.pline-srnic@syngenta.com

Abstract

Glyphosate, a nonselective herbicide and also the world's most widely used herbicide, inhibits 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme in the aromatic amino acid biosynthetic pathway. Because of its broad-spectrum and potent weed control and favorable environmental characteristics, attempts to engineer glyphosate resistance have been intensive in the past few decades. The use of at least three different mechanisms has conferred glyphosate resistance in normally sensitive crop species. Early work focused on progressive adaptation of cultured plant cells to stepwise increases in glyphosate concentrations. The resulting cells were resistant to glyphosate because of EPSPS overexpression, EPSPS gene amplification, or increased enzyme stability. Further work aimed to achieve resistance by transforming plants with glyphosate metabolism genes. An enzyme from a soil microorganism, glyphosate oxidoreductase (GOX), cleaves the nitrogen– carbon bond in glyphosate yielding aminomethylphosphonic acid. Another metabolism gene, glyphosate N-acetyl transferase (gat), acetylates and deactivates glyphosate. A third mechanism, and the one found in all currently commercial glyphosate-resistant crops, is the insertion of a glyphosate-resistant form of the EPSPS enzyme. Several researchers have used site-directed mutagenesis or amino acid substitutions of EPSPS. However, the most glyphosate-resistant EPSPS enzyme to date has been isolated from Agrobacterium spp. strain CP4 and gives high levels of resistance in planta. Weeds resistant to glyphosate have offered further physiological mechanisms for glyphosate resistance. Resistant field bindweed had higher levels of 3-deoxy-d-arbino-heptulosonate 7-phosphate synthase, the first enzyme in the shikimate pathway, suggesting that increased carbon flow through the shikimate pathway can provide glyphosate resistance. Resistant goosegrass has reduced translocation of glyphosate out of the treated area. Although glyphosate resistance has been achieved by numerous mechanisms, currently the only independent physiological mechanism to give adequate and stable resistance to glyphosate for commercialization of glyphosate-resistant crops has been glyphosate-resistant forms of EPSPS.

Type
Research Article
Copyright
Copyright © Weed Science Society of America 

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References

Literature Cited

Amrhein, N., Johanning, D., Schab, J., and Schulz, A. 1983. Biochemical basis for glyphosate tolerance in a bacterium and a plant tissue culture. FEBS Lett. 157:191196.CrossRefGoogle Scholar
Anonymous. 2003a. January 16. Summary Information Format. Roundup Ready Sugar Beet. According to Council Regulation 2002/812/EC: Web page: http://gmoinfo.jrc.it/csnifs/C-BE-99-01.pdf. Accessed: January 23, 2004.Google Scholar
Anonymous. 2003b. June 30. Acreage-Biotechnology Varieties. National Agricultural Statistics Service, Cr. Pr. 2–5: Web page: http://usda.mannlib.cornell.edu/reports/nassr/field/pcp-bba/acrg0603.pdf. Accessed: January 21, 2004.Google Scholar
Baerson, S. R., Rodriguez, D. J., Tran, M., Feng, Y., Best, N. A., and Dill, G. M. 2002. Glyphosate resistant goosegrass. Identification of a mutation in the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol. 129:12651275.CrossRefGoogle ScholarPubMed
Barry, G., Kishore, G., Padgette, M., Kolacz, K., Weldon, M., Re, D., Eichholtz, D., Fincher, K., and Hallas, L. 1992. Inhibitors of amino acid biosynthesis: strategies for imparting glyphosate tolerance to crop plants. in Singh, B. K., Flores, H. E., and Shannon, J. C., eds. Biosynthesis and Molecular Regulation of Amino Acids in Plants. Rockville, MD: American Society of Plant Physiologists. Pp. 139145.Google Scholar
Bradshaw, L. D., Padgette, S. R., Kimball, S. L., and Wells, B. H. 1997. Perspectives on glyphosate resistance. Weed Technol. 11:189198.Google Scholar
Castle, L. A., Siehl, D., Giver, L. J., Minshull, J., Ivy, C., Chen, Y. H., and Duck, N. B. inventors; Maxygen, Inc. and Pioneer Hi-Bred International, Inc., assignees. 2002. Novel glyphosate N-acetyltransferase (GAT) genes. WO 02/36782. May 10, 2002.Google Scholar
Castle, L. A., Siehl, D. L., and Gorton, R. et al. 2004. Discovery and directed evolution of a glyphosate tolerance gene. Science 304:11511154.Google Scholar
Comai, L., Sen, L., and Stalker, D. M. 1983. An altered aroA gene product confers resistance to the herbicide glyphosate. Science 221:370371.Google Scholar
Comai, L., Facciotti, D. D., Hiatt, W. R., Thompson, G., Rose, R. E., and Stalker, D. M. 1985. Expression in plants of a mutant aroA gene from Salmonella typhimurium confers tolerance to glyphosate. Nature 317:741744.Google Scholar
Cresswell, R. C., Fowler, M. W., and Scragg, A. H. 1988. Glyphosate tolerance in Catharanthus roseus . Plant Sci. 54:5563.CrossRefGoogle Scholar
Dams, T. R., Anderson, P. C., Daines, R. J., Gordon-Kamm, W. J., Kausch, A. P., Mackey, C. J., Orozco, E. M., Orr, P. M., and Stephens, M. A. inventors; Dekalb Genetics Corporation, assignee. 1995. Fertile, transgenic maize plants and methods for their production. World patent WO 95/06128. March 2, 1995.Google Scholar
Delannay, X., Bauman, T. T., and Beighley, D. H. et al. 1995. Yield evaluation of a glyphosate-tolerant soybean line after treatment with glyphosate. Crop Sci. 35:14611467.Google Scholar
Dröge, W., Bröer, I., and Puhler, A. 1992. Transgenic plants containing the phosphinothricin-N-acetyltransferase gene metabolize the herbicide L-phosphinothricin (glufosinate) differently from untransformed plants. Planta 187:142151.CrossRefGoogle ScholarPubMed
Duke, S. O., Rimando, A. M., Pace, P. F., Reddy, K. N., and Smeda, R. J. 2003. Isoflavone, glyphosate, and aminomethylphosphonic acid levels in seeds of glyphosate-treated, glyphosate-resistant soybean. J. Agric. Food Chem. 51:340344.Google Scholar
Dyer, W. E., Weller, S. C., Bressan, R. A., and Herrmann, K. M. 1988. Glyphosate tolerance in tobacco (Nicotiana tabacum L). Plant Physiol. 88:661666.CrossRefGoogle ScholarPubMed
Elmore, R. W., Roeth, F. W., Klein, R. N., Knezevic, S. Z., Martin, A., Nelson, L. A., and Shapiro, C. A. 2001. Glyphosate-resistant soybean cultivar response to glyphosate. Agron. J. 93:404407.CrossRefGoogle Scholar
Fillatti, J. J., Kiser, J., Rose, R., and Comai, L. 1987. Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Biotechnology 5:726730.Google Scholar
Forlani, G., Nielsen, B., and Racchi, M. L. 1992. A glyphosate-resistant 5-enol-pyruvyl-shikimate-3-phosphate synthase confers tolerance to a maize cell line. Plant Sci. 85:915.CrossRefGoogle Scholar
Franz, J. E., Mao, M. K., and Sikorski, J. A. 1997. Uptake, transport and metabolism of glyphosate in plants. in Franz, J. E., Mao, M. K., and Sikorski, J. A., eds. Glyphosate: A Unique Global Herbicide. ACS Monogr 189:143181.Google Scholar
Gasser, C. S., Winter, J. A., Hironaka, C. M., and Shah, D. M. 1988. Structure, expression, and evolution of the 5-enolpyruvylshikimate-3-phosphate synthase genes of petunia and tomato. J. Biol. Chem. 263:42804289.Google Scholar
Geiger, D. R. and Bestman, H. D. 1990. Self-limitation of herbicide mobility by phytotoxic action. Weed Sci. 38:324329.Google Scholar
Goldsbrough, P. B., Hatch, E. M., Huang, B., Kosinsky, W. G., Dyer, W. E., Herrmann, K. M., and Weller, S. C. 1990. Gene amplification in glyphosate tolerant tobacco cells. Plant Sci. 72:5362.Google Scholar
Gorlach, J., Schmid, J., and Amrhein, N. 1994. Abundance of transcripts specific for genes encoding enzymes of the prechorismate pathway in different organs of tomato (Lycopersion esculentum L.) plants. Planta 193:216223.Google Scholar
Gougler, J. A. and Geiger, D. R. 1981. Uptake and distribution of N-phosphonomethylglycine in sugarbeet plants. Plant Physiol. 68:668672.Google Scholar
Gower, S. A., Loux, M. M., Cardina, J., and Harrison, S. K. 2002. Effect of planting date, residual herbicide, and postemergence application timing on weed control and grain yield in glyphosate-tolerant corn (Zea mays). Weed Technol. 16:488494.CrossRefGoogle Scholar
Gower, S. A., Loux, M. M., and Cardina, J. et al. 2003. Effect of postemergence glyphosate application timing on weed control and grain yield in glyphosate-resistant corn: results of a 2-yr multistate study. Weed Technol. 17:821828.Google Scholar
Hauptman, P. M., della-Cioppa, G., Smith, A. G., Kishore, G. M., and Widholm, J. M. 1988. Expression of glyphosate resistance in carrot somatic hybrid cells through the transfer of an amplified 5-enolpyruvylshikimate-3-phosphate synthase gene. Mol. Gen. Genet. 211:357363.Google Scholar
He, M., Nie, Y. F., and Xu, P. 2003. A T42M substitution in bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) generates enzymes with increased resistance to glyphosate. Biosci. Biotechnol. Biochem. 67:14051409.Google Scholar
He, M., Yang, Z. Y., Nie, Y. F., Wang, J., and Xu, P. 2001. A new type of class I bacterial 5-enopyruvylshikimate-3-phosphate synthase mutants with enhanced tolerance to glyphosate. Biochim. Biophys. Acta. 1568:16.Google Scholar
Hoagland, R. E. 1980. Effects of glyphosate on metabolism of phenolic compounds: VI. Effects of glyphosine and glyphosate metabolites on phenylalanine ammonia-lyase activity, growth, and protein, chlorophyll, and anthocyanin levels in soybean (Glycine max) seedlings. Weed Sci. 28:393400.CrossRefGoogle Scholar
Holländer-Czytko, H., Johanning, D., Meyer, H. E., and Amrhein, N. 1988. Molecular basis for the overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant cell suspension culture of Corydalis sempervirens . Plant Mol. Biol. 11:215220.Google Scholar
Holländer-Czytko, H., Sommer, I., and Amrhein, N. 1992. Glyphosate tolerance of cultured Corydalis sempervirens cells is acquired by an increased rate of transcription of 5-enolpyruvylshikimate 3-phosphate synthase as well as a reduced turnover of the enzyme. Plant Mol. Biol. 20:10291036.Google Scholar
Jones, M. A. and Snipes, C. E. 1999. Tolerance of transgenic cotton to topical application of glyphosate. J. Cotton Sci. 3:1926.Google Scholar
Kishore, G. M., Brundage, L., Kolk, K., Padgette, S. R., Rochester, D., Huynh, K., and della-Cioppa, G. 1986. Isolation, purification and characterization of a glyphosate tolerant mutant E. coli EPSP synthase. Fed. Proc. 45:1506.Google Scholar
Kishore, G. M. and Jacob, G. S. 1987. Degradation of glyphosate by Pseudomonas sp. PG2982 via a sarcosine intermediate. J. Biol. Chem. 262:1216412168.Google Scholar
Klee, H. J., Muskopf, Y. M., and Gasser, C. S. 1987. Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants. Mol. Gen. Genet. 210:437442.Google Scholar
Komossa, D., Gennity, I., and Sandermann, H. 1992. Plant metabolism of herbicides with C-P bonds: glyphosate. Pestic. Biochem. Physiol. 43:8594.Google Scholar
Lebrun, M., Sailland, A., and Freyssinet, G. inventors; Rhone-Poulenc Agrochimie, assignee. 1997. Mutated 5-enol pyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene. World patent application WO9704103. February 6, 1997.Google Scholar
Lebrun, M., Sailland, A., Freyssinet, G., and Degryse, E. inventors; Bayer Crop Science S.A., assignee. 2003. Mutated 5-enolpyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene. U.S. patent 6,566,587. May 20, 2003.Google Scholar
Light, G. G., Bauman, T. A., Dotray, P. A., Keeling, J. W., and Wester, D. B. 2003. Yield of glyphosate-tolerant cotton as affected by topical glyphosate applications on the Texas High Plains and Rolling Plains. J. Cotton Sci. 7:231235.Google Scholar
Malik, J., Barry, G., and Kishore, G. 1989. The herbicide glyphosate. Biofactors 2:1725.Google Scholar
Mannerlöf, M., Tuvesson, S., Steen, P., and Tenning, P. 1997. Transgenic sugar beet tolerant to glyphosate. Euphytica 94:8391.Google Scholar
McAllister, R. S. and Haderlie, L. C. 1985. Translocation of 14C-glyphosate and 14CO2-labeled photoassimilates in Canada thistle (Cirsium arvense). Weed Sci. 33:153159.Google Scholar
Moore, J. K., Braymer, H. D., and Larson, A. D. 1983. Isolation of a Pseudomonas sp. which utilizes the phosphonate herbicide glyphosate. Appl. Environ. Microbiol. 46:316320.Google Scholar
Murata, M., Ryu, J-H., Caretto, S., Rao, D., Song, H-S., and Widholm, J. M. 1998. Stability and culture limitations of gene amplification in glyphosate resistant carrot cell lines. J. Plant Physiol. 152:112117.Google Scholar
Nafziger, E. D., Widholm, J. M., Steinrucken, H. C., and Killmer, J. L. 1984. Selection and characterization of a carrot cell line tolerant to glyphosate. Plant Physiol. 76:571574.CrossRefGoogle ScholarPubMed
Nap, J. P., Metz, P. L. J., Escaler, M., and Conner, A. J. 2003. The release of genetically modified crops into the environment. Part I. Overview of current status and regulations. Plant J. 33:118.CrossRefGoogle ScholarPubMed
Nida, D. L., Kolacz, K. H., and Buehler, R. E. et al. 1996. Glyphosate-tolerant cotton: genetic characterization and protein expression. J. Agric. Food Chem. 44:19601966.Google Scholar
Odell, J. T., Nagy, F., and Chua, N-H. 1985. Identification of DNA sequences required for activity of the Cauliflower Mosaic Virus 35S promoter. Nature 313:810812.Google Scholar
Padgette, S. R., Kolacz, K. H., and Delannay, X. et al. 1995. Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci. 35:14511461.Google Scholar
Padgette, S. R., Re, D. B., Barry, G. F., Eichholtz, D. E., Delannay, X., Fuchs, R. L., Kishore, G. M., and Fraley, R. T. 1996. New weed control opportunities: development of soybeans with a Roundup Ready gene. in Duke, S. O., ed. Herbicide-Resistant Crops: Agricultural, Economic, Environmental, Regulatory, and Technological Aspects. Boca Raton, FL: CRC. Pp. 5384.Google Scholar
Padgette, S. R., Re, D. B., and Gasser, C. S. et al. 1991. Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site. J. Biol. Chem. 266:2236422369.CrossRefGoogle ScholarPubMed
Papanikou, E., Brotherton, J. E., and Widholm, J. M. 2004. Length of time in tissue culture can affect the selected glyphosate resistance mechanism. Planta 218:589598.Google Scholar
Pline, W. A., Price, A. J., Wilcut, J. W., Edmisten, K. L., and Wells, R. 2001. Absorption and translocation of glyphosate in glyphosate-resistant cotton as influenced by application method and growth stage. Weed Sci. 49:460467.Google Scholar
Pline, W. A., Viator, R., Wilcut, J. W., Edmisten, K. L., Thomas, J., and Wells, R. 2002. Reproductive abnormalities in glyphosate-resistant cotton caused by lower CP4-EPSPS levels in the male reproductive tissue. Weed Sci. 50:438447.Google Scholar
Reddy, K. N., Rimando, A. M., and Duke, S. O. 2004. Is aminomethylphosphonic acid, a metabolite of glyphosate, causing injury in glyphosate-treated glyphosate-resistant soybean? WSSA Abstr. 44:116.Google Scholar
Reinbothe, S., Nelles, A., and Partbier, B. 1991. N-(phosphonomethyl)glycine (glyphosate) tolerance in Euglena gracilis acquired by either overproduced or resistant 5-enolpyruvyl shikimate-3-phosphate synthase. Eur. J. Biochem. 198:365373.Google Scholar
Rubin, J. L., Gaines, C. G., and Jensen, R. A. 1984. Glyphosate inhibition of 5-enolpyruvylshikimate 3-phosphate synthase from suspension-cultured cells of Nicotiana silvestris . Plant Physiol. 75:839845.Google Scholar
Rueppel, M. L., Brightwell, B. B., Schaefer, J., and Marvel, J. T. 1977. Metabolism and degradation of glyphosate in soil and water. J. Agric. Food Chem. 25:517528.CrossRefGoogle Scholar
Ruff, T., Eichholtz, D., Re, D., Padgette, S., and Kishore, G. 1991. Effects of amino acid substitutions on glyphosate tolerance and activity of EPSPS. Plant Physiol. 96: (Suppl.). 94.Google Scholar
Sandberg, C. L., Meggitt, W. F., and Penner, D. 1980. Absorption, translocation and metabolism of 14C-glyphosate in several weed species. Weed Res. 20:195200.Google Scholar
Sellin, C., Forlani, G., Dubois, J., Nielsen, E., and Vasseur, J. 1992. Glyphosate tolerance in Cichorium intybus L. var. Magdebourg. Plant Sci. 85:223231.Google Scholar
Shah, D. M., Horsch, R. B., and Klee, H. J. et al. 1986. Engineering herbicide tolerance in transgenic plants. Science 233:478481.Google Scholar
Shyr, Y. Y. J., Hepburn, A. G., and Widholm, J. M. 1992. Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells. Mol. Gen. Genet. 232:377382.Google Scholar
Singer, S. R. and McDaniel, C. N. 1985. Selection of glyphosate-tolerant tobacco calli and the expression of this tolerance in regenerated plants. Plant Physiol. 78:411416.Google Scholar
Smart, C. C., Johanning, D., Muller, G., and Amrhein, N. 1985. Selective overproduction of 5-enol-pyruvylshikimic acid 3-phosphate synthase in a plant cell culture which tolerates high doses of the herbicide glyphosate. J. Biol. Chem. 260:1633816346.CrossRefGoogle Scholar
Smith, C. M., Pratt, D., and Thompson, G. A. 1986. Increased 5-enolpyruvylshikimic acid 3-phosphate synthase activity in a glyphosate-tolerant variant strain of tomato cells. Plant Cell Rep. 5:298301.Google Scholar
Sost, D. and Amrhein, N. 1990. Substitution of Gly-96 to Ala in the 5-enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae results in a greatly reduced affinity for the herbicide glyphosate. Arch. Biochem. Biophys 282:433436.CrossRefGoogle Scholar
Sost, D., Schulz, A., and Amrhein, N. 1984. Characterization of a glyphosate-insensitive 5-enolpyruvyl-shikimic acid-3-phosphate synthase. FEBS Lett. 173:238242.Google Scholar
Stalker, D. M., Hiatt, W. R., and Comai, L. 1985. A single amino acid substitution in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase confers resistance to the herbicide glyphosate. J. Biol. Chem. 260:47244728.Google Scholar
Steinrucken, H. C., Schulz, A., Amrhein, N., Porter, C. A., and Fraley, R. T. 1986. Overproduction of 5-enolpyruvyl-shikimate 3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line. Arch. Biochem. Biophys 244:169178.Google Scholar
Suh, H., Hepburn, A. G., Kriz, A. L., and Widholm, J. M. 1993. Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate resistant carrot cells. Plant Mol. Biol. 22:195205.Google Scholar
Tharp, B. E. and Kells, J. J. 1999. Influence of herbicide application rate, timing, and interrow cultivation on weed control and corn (Zea mays) yield in glufosinate-resistant and glyphosate-resistant corn. Weed Technol. 13:807813.Google Scholar
Thompson, C. J., Movva, R. N., Tizard, R., Crameri, R., Davies, J. E., Lauwereys, M., and Botterman, J. 1987a. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus . EMBO J. 9:25192523.Google Scholar
Thompson, G. A., Hiatt, W. R., Gacciotti, D., Stalker, D. M., and Comai, L. 1987b. Expression in plants of a bacterial gene coding for glyphosate resistance. Weed Sci. 35: (Suppl. 1). 1923.Google Scholar
Torstensson, L. 1985. Behavior of glyphosate in soils and its degradation. in Grossbard, E. and Atkinson, D., eds. The Herbicide Glyphosate. London: Butterworth. Pp. 137150.Google Scholar
Torstensson, N. T. L. and Aamisepp, A. 1977. Detoxification of glyphosate in soil. Weed Res. 17:209212.Google Scholar
Wang, H. Y., Li, Y. F., Xie, L. X., and Xu, P. 2003. Expression of a bacterial aroA mutant, aroA-M1, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants. J. Plant Res. 116:455460.Google Scholar
Wang, Y. X., Jones, J. D., Weller, S. C., and Goldsbrough, P. B. 1991. Expression and stability of amplified genes encoding 5-enolpyruvylshikimate-3-phosphate synthase in glyphosate-tolerant tobacco cells. Plant Mol Biol. 17:11271138.Google Scholar
Warner, S. A. J., Hawkes, T. R., and Andrews, C. J. inventors; Syngenta Limited, assignee. 2002. Herbicide Resistant Plants. WO 02/26995. April 4, 2002.Google Scholar
Weaver, L. M. and Herrmann, K. M. 1997. Dynamics of the shikimate pathway in plants. Trends Plant Sci. 2:346351.CrossRefGoogle Scholar
Westwood, J. H. and Weller, S. C. 1997. Cellular mechanisms influence differential glyphosate sensitivity in field bindweed (Convolvulus arvensis) biotypes. Weed Sci. 45:211.Google Scholar
Widholm, J. M., Chinnala, A. R., Ryu, J., Song, H., Eggetta, T., and Brothertona, J. E. 2001. Glyphosate selection of gene amplification in suspension cultures of 3 plant species. Physiol. Plant. 112:540545.CrossRefGoogle ScholarPubMed
Wyrill, J. B. and Burnside, O. C. 1976. Absorption, translocation, and metabolism of 2,4-D and glyphosate in common milkweed and hemp dogbane. Weed Sci. 24:557566.CrossRefGoogle Scholar
Zhou, H., Arrowsmith, J. W., and Fromm, M. E. et al. 1995. Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Rep. 15:159163.Google Scholar