Chapter 10: The Genetic Manipulation of Herbicide Tolerance

1. The use of herbicides in modern agriculture a. Biotic stress : weed, pest, disease b. Weeds affect yield & quality of crops compete for light, nutrients, contamination of harvested crop, weed harbour pest & disease

c. Broad spectrum herbicides are active against a wide range of weed. However, these can only be used at times when the crop is not itself vulnerable to herbicide action d. The development of herbicide-tolerant crops offers the chance to spray crops at the most effective time to kill weed sp w/o damage the crop e. Some crops are naturally resistant to certain herbicides and may appear through mutation & NS f. Herbicide more toxic to plants than animals generally affect plant-specific biological processes Herbicides are inhibitors of plant-specific processes inhibition of a single enzyme/protein Many plant-specific pathways are located in the plastids Belong to a wide range of different chemical families; with about 15 broad classes of mode of action Most herbicides only have 1 mode of action Certain enzyme seems to be relatively vulnerable to herbicide activity Eg, 5 chemical families of herbicide target the enzyme acetolactate synthase (ALS; or more correctly, acetohydroxy-acid synthase or AHAS) ALS catalyses the 1st reaction in biosynthetic pathways of branched-chain amino acid (Fig 5.6) 2 common classes of herbicide with widely differing chemical structures (Sulphonylureas & imidazolinones) that target this enzyme (table 5.3, pg 110) environmental persistence, biodegradability), mode of action Herbicide can also be grouped by Site of uptake into the plant (root or shoot) Degree of translocation within the plant (systemic or contact) Time of application (preplanting, pre-emergence, postemergence, or preharvesting)
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2. Strategies for engineering herbicide tolerant crop Strategy for Glyphosate tolerance: 1. Overexpression of the target protein Involves titrating the herbicide out by overproduction of the target protein. Eg, if the herbicide is a specific inhibitor of one particular enzyme, production of sufficient excess enzyme will partially overcome the inhibition. Overexpression achieved by the integration of multiple copies of the gene and/or the use of a strong promoter plus translational enhancer to drive expression of the gene. This strategy is to protect plants from round-up (glyphosate).

1. 2. Overexpression of a plant EPSPS gene a. Overexpression of a plant EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene was facilitated by the isolation of petunia cDNA from glyphosate tolerance tissue culture. b. Isolation of culture with high level of EPSPS enzyme which was found to be result of gene amplification rather than increased expression of EPSPS gene There were multiple (up to 20) copies of the EPSPS gene c. EPSPS - enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants, parasites, bacteria and fungi. d. EPSPS enzyme is not mutated itself. The tolerance was simply due to the high level of enzyme and become tolerance to glyphosate. e. Because of high level of EPSPS mRNA made it easier to isolate cDNA for this gene and to be re-introduced into the plant.
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Done by introducing the sensitive gene into plants, but with much stronger gene promoter. It would elevate the mRNA level, hence would increase EPSPS protein levels. By fusion of EPSPS cDNA to the cauliflower mosaic virus 35S promoter, plant had elevated levels of EPSPS & moderate tolerance to glyphosate. Result: 40x increase in EPSPS activity in transgenic plants & a tolerance to glyphosate aprayed in the field at a dose 2 to 4 X higher than that required to kill wild-type plants

f. The use of plant gene avoid protein targeting. g. No further manipulation of the cDNA was required to target this protein to the plastid site of activity, since the plant EPSPS cDNA sequence contains its own transit peptide to plastid. h. Disadvantage: insufficient to warrant commercialization. Transit Peptide : A peptide sequence, often at the N-terminus of a precursor protein, that directs a gene product to its specific cellular destination. Effect of the overexpression of EPSPS gene in transgenic petunia was tested on one of the earliest experiment on the engineering of herbicide resistance. The EPSPS cDNA was fused to the cauliflower mosaic virus 35S promoter and a nos promotor sequence in the vector pMON 546 and transformed into petunia using Agrobacterium. 1.3. Constructs for engineering glyphosate tolerance. 1. Direct overexpression of wild-type petunia (PET) EPSPS gene 2. Resistant E. coli gene fused to N-terminal of petunia gene 3. Resistant EPSPS gene (from A. tumefaciens) with enhanced CaMV 35S promoter (E35S) currently used to generate Roundup Ready crops (eg soybean, coton) *CTP chloroplast transit peptide 3 nos

CaMV 35S

Pet CTP

Petunia EPSP synthase cDNA

CaMV 35S

Pet CTP

Pet N

E. coli mutant EPSPS

3 nos

CaMV E35S

CTP4

A. tumafaciens CP4 mutant EPSPS

3 nos

2. Mutation of target protein ~Mutant EPSPS genes Steps:

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1. Find a modified/mutated target protein Has same function as native protein Resistant to inhibition by herbicide

2. Incorporate resistant target protein gene into plant genome Require knowledge of the mode of action of herbicide Eg. Glyphosate tolerance using mutant EPSPS gene

3. Mutant EPSPS genes can be obtained by Isolating from glyphosate-resistant bacteria A single base substitution from C to T change amino acid from proline to serine in EPSPS - The glyphosate then cannot bind to mutant EPSPS enzyme. Thus, it provide resistance and the biosynthetic pathways of aromatic amino acids can take place

4. Case 1: a. A mutated aroA gene (from Salmonella typhimurium) insert between the promoter & terminator sequence of ocs gene (Of A. tumafaciens Ti plasmid) b. Only a moderate increase in herbicide tolerance. Why? enzyme was not transported into the chloroplast 5. Case 2: a. Construction of hybrid EPSPS gene (fusion of C-terminal end of mutated aroA gene (from E. coli) to N-terminal end of petunia EPSPS cDNA sequence containing transit peptide sequence b. Expression of chimaeric enzyme increased glyphosate tolerance in transgenic tobacco from 0.01 to 1.2 mmol/L glyphosate c. However, this method can bring problems: A mutant enzyme (EPSPS) with reduced affinity for a competitive inhibitor (glyphosate) may have a lower affinity (increased Km, the Michaelis-Menten constant) for substrate d. Solved by: A gene from herbicide-resistant A. tumafaciens strain CP4 encodes an glyphosateresistant EPSPS with low Km for PEP. This gene, in conjunction with an enhanced CaMV 35S promoter & a chloroplast transit peptide sequence from Arabidopsis or petunia is
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incorporated into the current range of Monsantos major monocotyledonous Roundup Ready crops (soybean, cotton, oilseed rape) Roundup Ready maize contains a resistant EPSPS gene (from maize, isolated after mutagenesis & selection in tissue culture), fused to a rice promoter & maize chloroplast transit peptide sequence

3. Detoxification of the herbicide a. Using a single gene from a foreign source. b. Detoxification is a mean of converting the herbicide to a less toxic form and/or removing it from the system. c. This strategy can be contrasted with the previous two because it does not require a detailed knowledge of the site of the action. d. An alternative strategy has been developed for engineering glyphosate tolerance based upon the specific detoxification mechanism by heterologous genes. In soil microorganisms, glyphosate can be degraded by cleavage by C-N bonds by enzyme (oxidoreductase) to form aminomethylphosphonic acid (AMPA) & glyoxylate. Gene encoding the enzyme glyphosate oxidase (GOX) has been isolated from a soil organism (Ochrobactrum anthropi strain LBAA), & modified by addition of transit peptide. Transgenic crops transformed with this gene show very good glyphosate resistance in the field. But, this strategy is not generally used in isolation.

e. Monsanto now employ a dual strategy for oilseed rape (canola) in which both the resistant Agrobacterium CP4 EPSPS gene and the GOX gene are expressed. enhanced glyphosate tolerance avoid accumulation of herbicide in plant (glyphosate is broken down into relatively harmless products) (glyoxylate anormal plant metabolite, AMPA can be converted to glycine)

f. accumulate to higher levels than normally found in that crops g. Strategy 3 : detoxification destruction of herbicide, or accumulation of a conjugate less harmful than the original compound Recently, a glyphosate acetylation detoxification system has been developed by molecular evolution (Box 5.5)

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4. Enhanced plant detoxification a. The aim here is to improve the natural plant defenses against toxic compounds. b. This requires detailed information about endogenous plant detoxification pathways and the mechanisms by which compounds are recognized and targeted for detoxification by the plant. c. Many xenobiotic (foreign) compounds are detoxified in plants but the pathways may involve more than one step such as hydroxylation, conjugation and transport stagesSo it may prove difficult to identify single-gene mechanisms to engineer tolerance. d. The hydroxylation of compounds involves enzymes such as the cytochrome P450 monooxygneases, which from a large gene family. e. For example, The analysis of weeds resistant to the herbicide bromoxynil revealed that the bromoxynil was being detoxified by an endogenous cytochrome P450 monooxygense. Plant detoxification pathways involve conjugation to glutathione by glutathione Stransferse (GST) activity, and specific transport of the conjugate into the vacuole. GST also comprise a large gene family, some members of which are known to be involved in endogenous metabolic reactions. In some cases, the hydroxylation and conjugation pathways operate in concert. The conjugation of atrazine to glutathione. The detoxification of atrazine is a two-stage process involving a 2-hydroxylation step (removing the chlorine residue) prior to the addition of glutathione. Glutathione is a tripeptide in which the key residue is the middle cysteine. The SH group is involved in a number of redox and conjugation reactions.

3. The development of super-weeds a. The herbicide-tolerant crop itself appears as a volunteer weed in fields where rotational crops are grown. This volunteer population could reproduce outside of cultivation and form a selfsustaining weed population.

b. Pollen from the herbicide-tolerant crop fertilizes weedy relatives of the crop plant, producing herbicide-tolerant hybrids. Subsequent backcrossing of these hybrids with the weed species could lead to introgression of the herbicide-tolerant trait into the weed population.

c. There is horizontal gene transfer by other mechanisms, such as viruses, that spread the herbicide-tolerant trait into a much wider range of plant species.

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