Genetically engineered herbicide tolerance to glyphosate

Glyphosate is a non-selective herbicide that kills plants by inhibiting their ability to syn-thesise the aromatic amino acids phenylalanine, tyrosine and tryptophan (see Chapter 9 section 9.4). It accomplishes this by blocking the action of the enzyme EPSP synthase, the penultimate step in the shikimate pathway, vital in the biosynthesis of these three amino acids. Interestingly, glyphosate competes with one substrate (phosphoenol pyruvate, PEP)

Figure 13.2 An overview of the steps taken in the production of a genetically modified plant. From Bruce and Bruce, 1988.

Shikimate-3-phosphate

CP4

Plant

EPSPS

Activity inhibited by glyphosate

Aromatic amino acids

Figure 13.3 Biosynthesis of aromatic amino acids indicating how CP4 EPSPS allows synthesis in the presence of glyphosate. EPSP, 5-enoylpyruvyl shikimic acid 3-phosphate; EPSPS, EPSP synthase.

but forms a very stable herbicide-enzyme complex with the other, resulting in a 'full stop' to the metabolic pathway. This also subsequently reduces the ability of the plant to synthe-sise a number of vital metabolites including hormones, flavonoids and lignins. GM crops that are tolerant to glyphosate are produced by the insertion of a gene coding for a glypho-sate-insensitive EPSP synthase that is obtained from a soil bacterium Agrobacterium CP4. In this way, plant EPSP synthase is still inhibited, but the bacterial EPSP synthase is unaffected, allowing the plant to still synthesise aromatic amino acids and the other essential metabolites resulting from the shikimate pathway (Figure 13.3) (for an example of this process, see Vande Berg et al., 2008). The kinetic properties of plant and bacterial wild-type and mutated EPSP synthases are shown in Table 13.3.

Table 13.3 Kinetic properties for selected EPSP synthases (from Dill, 2005). K ( Km (PEP) is a measure of the selectivity of EPSPS for PEP over glyphosate. A higher value indicates a greater tolerance to glyphosate while the enzyme still possesses EPSPS activity.

Enzyme source

Km (PEP) (|M)

Ki (glyphosate) (| M)

Ki / Km

Petunia (wild type)

5.0

0.4

0.08

G101A

210

2000

9.5

T102I/P106S

10.6

58

5.5

P106S

17

1

0.06

Agrobacterium sp. CP4

12

2720

227

Figure 13.4 The major GM traits that are being commercially exploited (2006). Figures represent the % of total commercial GM plantings worldwide in 2006. HT = herbicide tolerance; Bt = Bacillus thuringiensis-mediated insect resistance.

Crops that have been modified by insertion of the EPSP synthase CP4 gene are commercialised under the trade name Roundup-Ready™ and represent the largest proportion of the GM-HT crop market worldwide. In 2006, 68% of GM crops grown were herbicide tolerant (Figure 13.4) and the vast majority of these were Roundup-Ready™. CP4 is a mutant form of EPSP synthase that has an amino acid replacement (Gly100 ^ Ala) which results in extremely high tolerance to glyphosate (Padgette et al., 1996). The low mammalian toxicity, fast degradation in soil and translocation within plants make glyphosate a popular and effective herbicide. The use of glyphosate-tolerant crops has reduced the number of herbicide applications necessary for a number of crops in addition to reducing fuel costs and land compaction due to reduced machinery use. In addition, these GM crops increase the flexibility of timing of weed control, as glyphosate can be used on weeds at growth stages in excess of those that conventional herbicides can effectively control. Such practices have allowed for the increase in no- t illage and conservation tillage practices alongside GM-HT crops in the USA and Canada.

The effectiveness of glyphosate as a herbicide means that weed control through cultivation is not always necessary. The high level of weed control achieved, however, will

OH HO Glyphosate

H OH

Glyoxylate

Figure 13.5 The detoxification of glyphosate by glyphosate oxidoreductase (GOX).

Aminomethyl phosphonic acid (AMPA)

clearly impact on biodiversity, as indicated in some of the data collected in the UK during the three-year Farm- Scale Evaluations (see section 13.13) . In addition, the overuse of glyphosate as the sole method of weed control may create an unacceptable selection pressure for the survival of naturally occurring populations of glyphosate-resistant weeds. This is a very real concern for the future. As such, GM glyphosate-)olerant crops should be considered alongside GM crops that are tolerant to other herbicides and non-GM crops in rotation in cropping systems. Studies such as those by Westra et al. (2008) will also prove important in assessing the effects of changing land management practices under glyphosate-tolerant cropping systems on weed population dynamics.

An alternative method for conferring tolerance to glyphosate involves the insertion of the gox gene from Ochrobactrum anthropi into crop plants (CaJacob et al., 2004; Reddy et al., 2004). Glyphosate oxidoreductase (GOX) is an enzyme that catalyses the breakdown of glyphosate to AMPA (aminomethylphosphonic acid) and glyoxylic acid (Figure 13.5).

In some oilseed rape lines, both the CP4 EPSP synthase and gox genes are inserted and expressed, giving the resultant plant two methods of avoiding damage by glyphosate. The reasons for this gene-stacking have not been made public and the build-up of AMPA (due to the presence of GOX) has been implicated in phytotoxic symptoms in some GM oilseed rape lines (Reddy et al., 2004).

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