Glyphosate is a broad spectrum herbicide that was introduced in the 1970s for management of annual, perennial and biennial herbaceous grasses, sedges, and broadleaves, as well as woody brush and trees (Franz et al., 1996). 

Glyphosate controls plants by inhibiting the enzyme EPSPS (5-enolpyruvylshikimate-3-phosphate synthase). EPSPS is an essential enzyme in the shikimate pathway that ultimately leads to the production of aromatic amino acids (tryptophan, tyrosine, and phenylalanine). The shikimate pathway for synthesizing aromatic amino acids, and therefore the enzyme EPSPS, is found in plants, bacteria and fungi, but not animals.

The structure of glyphosate resembles the structure of the substrate of EPSPS, which is phosoenolpyruvate (PEP). Therefore, glyphosate competes with PEP for the enzyme’s active site and prevents conversion of PEP to the precursor that is required in the synthesis of aromatic amino acids. Aromatic amino acids are essential for many plant processes, such as protein synthesis, cell wall formation, pathogen defense and hormone production.  By preventing the production of aromatic amino acids, glyphosate is toxic to virtually all plants.

There is no significant endogenous tolerance to glyphosate in crop plants or their weedy relatives. Consequently, attempts to conventionally breed most crops for glyphosate resistance have not succeeded (Padgette et al., 1995). Resistance was found in tissue cultures derived from 10 species. However, the resistance had no commercial value because in some cases it was not genetically based; in others, it was not stably inherited.

It is difficult to find glyphosate-resistant EPSPS molecules that still possess sufficient catalytic activity to provide adequate functioning of the shikimate pathway (Kishore et al., 1992; Padgette et al., 1996b). A naturally-occurring multiple mutation in corn EPSPS was discovered and has been utilized to generate glyphosate resistance (Lebrun et al., 1997).  Chemically mutating corn callus also led to an EPSPS with two amino acid changes that provided resistance to glyphosate without impairing EPSPS function (Dill, 2005).   However, multiple copies (amplification) of this double mutant are needed to provide a level of glyphosate tolerance that is acceptable to farmers.

In 1983 scientists discovered a bacterial gene, CP4, which encodes for a highly efficient, glyphosate-resistant form of EPSPS (Padgette et al., 1996a). Using the new, gene-based methods of biotechnology, crop developers were able to deliver the Agrobacterium CP4 gene into crop plants. As a result, biotech crops contain both the glyphosate-susceptible form of EPSPS, encoded by crop genes, and the glyphosate-resistant EPSPS encoded by the microbial CP4 gene. Having the bacterial form of EPSPS allows crops to continue to synthesize aromatic amino acids, even as the plant EPSPS enzyme is inactivated by glyphosate competitive inhibition, because glyphosate does not bind to bacterial EPSPS. The bacterial CP4 gene provides the basis of glyphosate resistance in most biotech crops that are resistant to the herbicide.

Biotech crop developers have utilized two strategies to enhance glyphosate resistance: increasing access of bacterial EPSPS to glyphosate and bolstering EPSPS insensitivity-based resistance with a new molecular mechanism, herbicide deactivation.

• Increased access
  EPSPS is found in chloroplasts. Enhancing the efficiency of the choloroplast transfer proteins (CTP) that are necessary for moving EPSPS into chloroplast has increased the chloroplast concentration of EPSPS molecules that are resistant to glyphosate. This allows the insensitive EPSPS molecule to out compete the plant’s endogenous EPSPS molecules for access to glyphosate.
• Deactivation
  Resistance to glyphosate encoded by the CP4 gene can be enhanced with other novel genes to improve their level of resistance. For example, glyphosate resistant canola contains, in addition to CP4, a bacterial gene that encodes the enzyme glyphosate oxidoreductase (GOX). The microorganism Ochrobactrum anthropi, the source of the GOX gene,  uses the   enzyme to degrade glyphosate to glyoxylate and aminomethylphosphonate (AMPA), two compounds that are non-toxic to plants. This mechanism is most likely the dominant glyphosate degradation pathway that soil microorganisms evolved in response to glyphosate exposure, because they, too, rely on the shikimate pathway to synthesize aromatic amino acids. So, glyphosate resistant canola plants rely on two molecular mechanisms: herbicide deactivation and target insensitivity.

Recently, a new mechanism to detoxify glyphosate in planta, deactivation by acetylation, was developed (Castle et al., 2004). The enzyme, glyphosate acetyltransferase, converts glyphosate into the non-toxic molecule acetylglyphosate, before glyphosate can reach and inhibit the the EPSPS enzyme. The transferase gene was derived from a naturally occurring soil bacterium (Bacillus licheniformis). Researchers used a technology, known as gene shuffling, to optimize both the enzyme’s acetylation efficiency and its specificity before incorporating the gene into plants (Castle et al., 2004; Siehl et al., 2005).

In summary, the mechanisms that have been used to provide glyphosate resistance to crop plants are:

1. Target insensitivity - EPSPS molecules that do not bind glyphosate;
2. Target insensitivity plus amplification to increase the number of insensitive sites;
3. Herbicide deactivation - GOX and glyphosate acetyltransferase;
4. Improved transport of insensitive targets to increase access to glyphosate (CTP).