3.2 Herbicide tolerance
As you discovered from Activity 1, herbicide tolerance is the trait most commonly incorporated into commercial GM plants. A crop can be made tolerant to herbicide by inserting a gene that causes plants to become unresponsive to the toxic chemical. Before considering how the genetic manipulation can be achieved, it is useful to understand a little about how herbicides act.
Many herbicides work by inhibiting a key plant enzyme necessary for growth (if you're not exactly sure what this means, see Box 2, below). The herbicide glyphosate (also known as Roundup™) is the world's largest-selling herbicide. It is a broad-spectrum herbicide which can kill a wide variety of monocot and dicot weeds. It is particularly effective because it is transported downwards in plants and so has the advantage of killing the roots of perennial weeds.
Glyphosate inhibits EPSP synthase, an enzyme that is involved in the shikimic acid pathway (see Figure 6). The enzyme catalyses the conversion of 3-phosphoshikimate to the compound EPSP. (If you are interested, this stands for 5-enolpyruvylshikimate-3-phosphate, but this level of detail doesn't really concern us here!). EPSP is converted, via a series of biochemical reactions, into essential aromatic amino acids like phenylalanine, tyrosine and tryptophan. Glyphosate acts by binding with EPSP synthase, and in doing so, prevents the enzyme from catalysing the reaction. If the shikimic acid pathway is blocked in this way, the plant is deprived of these essential amino acids and cannot make the proteins it requires. The plant weakens and eventually dies.
Box 2: Reading biochemical pathways
You may have come across biochemical pathways in your earlier studies. A key feature of all biochemical processes is that they take place in stages. Substances are made or broken down by an orderly sequence of linked chemical reactions called a metabolic pathway. Each chemical reaction in the pathway is catalysed by an enzyme. If the enzyme is not present, the rate of the reaction will usually be negligible. The precise mechanisms of these individual reactions form a fascinating area of study, but for our current purposes you do not need to have anything more than an outline.
To illustrate how these metabolic pathways are represented, we will look at an imaginary sequence of reactions, in which a substance A is converted into substance E by a sequence of four reactions:
Note that it is usual to represent the chemical transformation with a simple arrow, and to write the name of the enzyme catalysing the transformation beside the arrow. Such sequences usually focus on the most important chemical substances involved, and the less interesting participants are not included in the scheme. For example, many reactions will involve the gain or loss of phosphate groups or water molecules, but these are often omitted.
If for some reason, we wanted to block this pathway, we might try to prevent the action of one or more of these enzymes, a process known as inhibition. If, for example, we were able to effectively inhibit enzyme 2 in our sequence, we could slow or stop the conversion of substance B into substance C. This might cause the build up of substance B, and also prevent the production of our end product, substance E. The disruption of pathways in this way often severely damages an organism, and can kill it.
If crops can be made resistant to glyphosate, then the herbicide can be applied during the active growing phase without fear of damage to the crop. In the early 1980s, the biotechnology company Monsanto set about introducing glyphosate tolerance using a strategy that could be termed 'overproduction'. Petunia plants were selected that were expressing high levels of the enzyme EPSP synthase. The mRNA corresponding to the EPSP synthase gene was isolated and cDNA prepared. The cDNA was incorporated in an appropriate Ti plasmid, and the 'gene' was then used to produce transgenic plants using A. tumefaciens mediation. These showed 40- to 80-fold enhanced levels of EPSP synthase. The idea was that when glyphosate was applied, a proportion of the EPSP synthase would be inhibited, but sufficient quantities of enzyme would be produced to allow the shikimic acid pathway to function normally. Although the modified plants did show increased tolerance to glyphosate, the level was insufficient for commercial use and many of the plants showed growth retardation following glyphosate application.
Towards the middle of the 1980s, a different approach was explored. The idea was to discover an organism whose EPSP synthase had a reduced affinity for glyphosate but still had normal enzyme activity, so that the shikimic acid pathway could still operate normally. Although glyphosate is very effective in killing plants, some bacteria are able to tolerate it and these bacteria were potential sources of a gene coding for a glyphosate-tolerant EPSP synthase. One such gene was introduced into maize, using microprojectile bombardment (biolistic) transformation (Box 1). The novel EPSP synthase gene allowed the transgenic plants to continue producing aromatic amino acids in the presence of glyphosate, and conferred high levels of tolerance to the herbicide.