At the end of the last lecture I described one of the first approaches that was tried to develop plants with resistance to glyphosate, the active ingredient in the herbicide Roundup. This involved expressing a normal EPSP synthase (the target enzyme that is inhibited by glyphosate) at a higher level using the strategy outlined below:
A second approach that was tried was to make plants use the same mechanism as bacteria to become resistant to glyphosate. Bacteria synthesize aromatic amino acids using the same pathway as plants, the shikimic acid pathway. These bacteria also have EPSP synthase and their enzyme is inhibited by glyphosate so that bacteria are normally unable to grow in medium that contains glyphosate. However, mutants that are resistant to glyphosate can be selected quite easily, and this has been done with Salmonella and other species. In many cases, the bacteria have become resistant by producing an EPSP synthase that is no longer inhibited by glyphosate. The change of one single amino acid made the bacterial enzyme insensitive to glyphosate. This amino acid substitution resulted in the enzyme having a much lower affinity for glyphosate. See the overhead.
How was this used to make glyphosate-resistant plants? The following strategy was used:
Both of the approaches outlined above were successful in that the transgenic plants were more tolerant of glyphosate than the untransformed plants (see Nature [1985] 317, p.741; Science [1986] 233, 478). However, the level of tolerance was not sufficient for this glyphosate resistance gene to be used in crops. Transgenic plants that were sprayed with glyphosate exhibited symptoms of injury that would be unacceptable in field use. Simple overexpression of an EPSP synthase that was still inhibited by the herbicide was insufficient. The bacterial EPSP synthase was not targeted to the chloroplast, where EPSP synthase is normally located, and this likely accounted for its inability to provide "field resistance".
The next strategy was to combine the following features:
While transgenic plants with this transgene did exhibit good tolerance to glyphosate, they were still not sufficiently resistant for field use. One reason for this failure was that the mutation that reduced affinity for glyphosate also reduced the affinity of the enzyme for one of its substrates, PEP. This lowered the efficiency of the enzyme and made it difficult to sustain synthesis of aromatic amino acids after treatment with glyphosate. This led to a search for an isozyme (a form) of EPSP synthase with the following properties:
A number of approaches could be taken to identify an EPSP synthase with these properties:
The last approach is the one that turned out to be successful. In screening through bacterial collections, a strain of Agrobacterium (CP4) was identified that contained a suitable EPSP synthase. The gene was cloned and sequenced. From this a chimeric gene was assembled from the following components:
Transgenic plants carrying this gene had the desired level of glyphosate tolerance that was sufficient to allow development of crop plants with "field resistance" to glyphosate. This gene was then used to transform soybeans. A single transformed line forms the basis for the Roundup Ready soybeans that are on the market today. Once this gene was introduced into soybean, using the particle bombardment method, it was then incorporated into elite varieties by conventional plant breeding methods. Other versions of this gene, likely with the same protein but with different promoters, have been used to develop Roundup Ready corn and other crops.
A detailed description of the research leading to this development can be found in: Herbicide-Resistant Crops, ed.S.O. Duke, published by CRC Press (1996), pages 53-84. The same book contains a number of excellent chapters on development of crops with resistance to other herbicides, the subject of the remainder of today's lecture.
As you might imagine, Monsanto was not the only company to realise the potential of using biotechnology to add single genes to crop plants that would provide new resistance to herbicides. I will now outline the strategies that have been used to develop resistance genes for other herbicides.
Sulfonylureas and imidazolinones are herbicides that inhibit a central enzyme in the synthesis of another group of amino acids, the branched chain amino acids including leucine, isoleucine and valine. The target enzyme that is inhibited by these herbicides is called acetohydroxy acid synthase (AHAS). It is also known, rather confusingly, as acetolactate synthase (ALS). AHAS and ALS are the same enzyme.
It is quite straightforward to select plants with resistance to these herbicides. In fact one of the problems with these herbicides is that a single amino acid substitution in AHAS can give rise to a form of the enzyme that is no longer inhibited by these herbicides. So it s quite easy to select herbicide resistant weeds as well. One strategy to produce crops with resistance to these herbicides is as follows:
For a number of reasons, crop plants with resistance to these herbicides have not been developed using this approach.
A second trategy, not involving gene transfer but instead relying on selection and regeneration from tissue culture, has been used to develop a number of imidazolinone-resistant crops, including corn and canola. The approach here is as follows:
Imidazilonone resitant corn hybrids are now on the market, and programs to develop canola and wheat varieties with the same trait are well advanced. The same approach (selection in tissue culture of herbicide resistant cells followed by regeneration of plants) has been used to develop corn with resistance to sethoxydim, a herbicide that blocks synthesis of fatty acids. Sethoxydim inhiibits an enzyme called acetyl CoA carboxylase, ACCase. Most monocots are sensitive to this herbicide while dicots are not affected because the dicot ACCase is not inhibited by sethoxydim. Development of corn lines with resistance to this herbicide now gives another option for weed control in corn production.
This herbicide blocks this synthesis of glutamine. Because this enzymatic reaction utilizes ammonia, when the pathway is blocked by the herbicide it results in the accumulation of ammonia to toxic levels that kill the plant. This is another broad spectrum herbicide.
The strategy to develop plants with resistance to this herbicide was based on knowledge of where the herbicide was originally discovered. Glufosinate was discovered as an antibiotic, produced by a Streptomycete fungus. As with most antibiotics, the organism that produces the compound has a mechanism to protect itself from its toxic effects. In this case, the Streptomycete produces an enzyme that inactivates the antibiotic. This led to the following scheme which uses the metabolism strategy to impart herbicide resistance:
This has been successfully developed for many crops. The commercial name of the herbicide is Liberty, but it has also been sold as Ignite or Basta. Corn hybrids with this resistance mechanism are sold as Liberty Link seed. The company developing this resistance is AgrEvo. There have been some delays in getting this into the marketplace, in part I believe because of patent challenges and other legal actions.
This herbicide is an inhibitor of photosynthesis, acting on photosystem II. It is a "contact" herbicide that does not get transported throughout the plant, unlike the other herbicides that I have described. This is a selective herbicide, killing broadleaf weeds, and is registered for use in wheat, barley, oats and a number of other monocots. It is known that one mechanism that likely contributes to the tolerance of these crops is metabolism of the herbicide to an inactive form. However, plant genes encoding the enzyme(s) involved in this metabolic pathway have not been identified. Where could you find other genes encoding enzymes that would metabolize this compound? Well, you could look at any organism on the planet, but the easiest to study, grow and select for unusual metabolic properties are microbes. And where would you have a higher likelihood of finding microbes that can metabolise this compound? In soil that is contaminated with the herbicide. A survey of bacteria from bromoxynil-contaminated soil identified a strain of Klebsiella ozaenae that was able to grow on bromoxynil as its sole source of nitrogen and was obviously highly resistant to the herbicide. This microbe produced an enzyme that metabolized bromoxynil, and so a now familiar strategy was used:
As a result of this research, cotton plants with resistance to bromoxynil (BXN cotton) have been developed and are on the market. This will provide some alternative weed control strategies for cotton growers. It is likely that Roundup Ready cotton will also be introduced in the near future as well.
A similar strategy has been used to identify genes that can metabolise another herbicide, 2,4-D. In this case the gene for metabolism of 2,4-D was found in another bacterium, Alacligenes eutrophus. After cloning, modification and transfer into plants, herbicide tolerant plants were produced. It is less likely that this will be developed and incorporated into plant varieties because of concerns about the safety of 2,4-D.
These examples illustrate the potential of biotechnology to develop crop plants with new herbicide tolerances. One point that I want to emphasize from these examples is where the various genes were found that were used and manipulated to provide herbicide tolerance. In most cases these genes were not moved between different plants. Instead they were first identified in microbes with unusual properties, e.g. the ability to grow in the presence of high concentrations of a herbicide. This highlights the point that biotechnology can expand the pool of genes that is available for crop improvement. Such genes for herbicide tolerance in most cases could not have been identified in the conventional germplasm of these crops. An important element of biotechnology is the process of gene discovery, finding the genes that can carry out specific functions, from whatever sources are available.