The following points highlight the five novel transgenic approaches for herbicide tolerance. The approaches are: 1. Glyphosate Resistance 2. Glyphosate Resistance by over expression of EPSPS Gene 3. Cytochrome p450 Mediated Tolerance 4. Engineering Herbicide Tolerance by p450 5. Overexpression of At PgP, or Apyrase.
Approach # 1. Glyphosate Resistance:
Majority of the weeds in the world can be destroyed by using broad-spectrum herbicide- glyphosate. This potential herbicide able to kill the weeds by acting as competitive inhibitor of the enzyme 5-enol pyruvyl shikimate-3 phosphate synthase (EPSPS). The glycine derivative glyphosate binds tightly to the EPSPS shikimate-3 phosphate complex.
The enzyme EPSPS play a key role in the biosynthetic pathways of the aromatic amino acids phenyalanine, tryosine and tryptophan. Glyphosate inflicts damage on the plant by inhibiting the biosynthetic process of aromatic amino acids and other products of shikimate pathway (Fig. 20.10).
Shikimate is one of the important intermediate compounds formed in shikimic acid pathway. Once shikimate is formed it is then phosphorylated to produce shikimate-3-phosphate. This acts as substrate for EPSPS. The EPSPS binds the enoipyruvyi side chain to shikimate-3-phosphate to form EPSP.
In the subsequent step, chorismate is formed by the elimination of phosphate from EPSP. Chorismate acts as a precursor of the phenolic and indole rings of the aromatic amino acids. Therefore, inhibition of this enzyme results in the death of weeds due to the deficiency of aromatic amino acids in the plant.
Approach # 2. Glyphosate Resistance by over Expression of EPSPS Gene:
Overexpression of plant EPSPsynthase gene for enhancing herbicide tolerance was earliest and effective one. This was evidenced in petunia cells growing on different levels of glyphosate. Herbicide tolerant petunia cells were found to have high level of EPSPS enzyme.
In a transgenic technique, EPSPS cDNA was fused to the CaM 35 promoter and a nos terminator sequence transformed into petunia using Agrobacterium. Since plant EPSPS gene contains its own transit peptide signal, there is no requirement of outsourcing transit signal peptide as this was practical when microbial aro A gene was used (Fig. 20.10).
Approach # 3. Cytochrome p450 Mediated Tolerance:
There have been number of reports on the herbicide resistant among weed population. Sometimes even multiple or cross-resistance to herbicides with different target sites. Development of resistance mechanism is due to mutation of target sites. Recent studies however, revealed that resistance is due to the involvement of cytochromes p450, which increases the rate of metabolism.
This has been confirmed by invivo experiments using inhibitors. An increase in metabolism is due to the involvement of p450 was first demonstrated in herbicide tolerant plant Lolium rigidum biotypes from Australia and phenyl urea resistant Phalaris minor in Asia.
p450 are heme proteins distributed among wide array of organism such as bacteria, plant and animals. It has been shown that several hundreds of p450 proteins are probably encoded by most plant species. Cytochromes p450 constitute the largest family of enzymatic proteins in higher plants.
p450 are monooxygenase have molecular weight ranging from 45 to 62 kDa that insert one atom of oxygen into inert hydrophobic molecules to transform them into more reactive p450, help plant to combat harmful chemicals such as pesticides, herbicides and industrial pollutants, convert them into less phytotoxic.
Plants 450 S and p450 reductase bonded to endoplasmic reticulum via N-terminal region. p450 reductase channeled electron from NADH to p450 protein by transferring them from FAD to FMN (Fig. 20.11).
A comparative study on the involvement of p450 in detoxification of herbicides has shown that most classes of herbicides are metabolised by p450 by alkyl-hydroxylated, dealkylation product formation. Dealkylation takes place due to the oxygenation of a carbon to hetero atom, followed by hydrolytic cleavage and elimination of aldehyde.
For example, in the detoxification of herbicides like prosulfuran, diclotop and chlortoluran can be converted by p450 into several metabolites (Fig. 20.12). Detoxification of phenyl urea, chlortoluran, and other herbicide is accomplished either via hydroxylation of the methyl ring or demethylation. The demethylated products are phytotoxic.
Approach # 4. Engineering Herbicide Tolerance by p450:
Demonstration of p450 in the detoxification of herbicide leads to the isolation of the gene for their control of herbicide tolerance in crops and weeds. The first genes SV1 and SV2 encoding herbicide metabolizing p450 were isolated from bacteria Streptomyces greseolus. SV1 (cyP105 A1) gene was fused to chloroplast transit peptide signal targeted to chloroplast stroma due to the availability to adequate redox molecule like ferrodoxin.
When this gene was expressed in tobacco targeted to the chloroplast due to the availability of electron from Fd it activates the harmless sulforylurea and convert this into highly phytotoxic herbicides. The plant dies due to the expression of SV1 in whole plant. However, when this gene was expressed using tapetum specific promoter herbicide treatment of immature flower result in male sterility.
Another bacterial p450 gene from Rhodococcus strain has the ability to detoxify the herbicide S-ethyl dipropyl carbo methioate (EPTC) and exert a bio-safening activity. This is being used currently protecting maize from herbicide injury by its bio-safening activity. Certain Mammalian p450 genes known to degrade drugs and other xenobiotics are used to produce transgenic plants for herbicide degradation.
This strategy has been continued by using rat cyP A, to produce transgenic herbicide tolerant tobacco and potato plants. This gene product is an enzyme assigned to degrade various xenobiotics, including atrazine, chlortoluran. In one of the strategies cyP A, gene fused with the yeast p450 reductase gene and consequent expression in plants.
In another study, fusion protein is targeted to the chloroplast in order to improve coupling of p450 reduction with photosynthetic electron transfer. One of the drawbacks of the process is the transfer of mammalian gene into plant as it is known to activate polyaromatic hydrocarbon into procarcinogens.
Since good proportion of p450 genes occurs in plants and considering their diversity in the different plant spices, p450 can provide adequate source of detoxyfying genes for engineering parameter and herbicide tolerant by plant transformation. The plant gene cyP 76B, was isolated from Jerusalem artichoke (Helianthes tuberosus), expressed in yeast fused with Arabidopsis p450 reductase.
The cyP 76 B, protein convert xenobiotic including penyl urea into non-toxic product. Another gene cyP 71A 10 was isolated from soyabean and expressed in yeast proved that is convert chlortoluran to demethylated non-toxic compound. Metabolization of chlortoluran is also aided by cyp 81 B2 and cyp 71A11, isolated from tobacco.
Efforts have also been made in the isolation of p450 genes from monocot crops and weeds as it was shown in the isolation of single herbicide inducible p450 from wheat. Overexpression of these two genes i.e., cyp 761A10 and cyp All in tobacco confer increased tolerance to phenyl urea. Recently identified cyp 72 from tolerant line capable of metabolises both bentazon and chlortoluran.
Approach # 5. Overexpression of At PgP, or Apyrase:
Overexpression of AtPgP1 or apyrase gene in Arabidopsis thaliana was able to show resistance to cyclohexamide and other commercial herbicides studies indicated that herbicide resistance was achieved by removal of toxin from plant cells and resistance involve action of apyrase.
Their finding revealed that both AtPgP, and apyrase proteins are involved in multidrug resistance (MDR) mechanism in plants. Multi-drug resistance is observed in bacteria, human cancers and recently in plants. This phenomenon is often related to ABCB, i.e., p-glycoprotein, a member of the ATP binding cassette (ABC).
Expression of these ABC transporters is responsible for MDR in prokaryotes and eukaryotes. Arabidopsis contains 130 numbers of ABC transporter families. This p-glycoprotein mediated resistance is correlated to the efflux of herbicide to release ATP. Over expression of ATPg in A. thaliana results in increasing accumulation of ATP outside the cell and efflux of ATP correlated with toxin resistance.
Therefore, it can be correlated removal of toxin from the cell is effected by co-symport of the toxin and ATP through p-glycoprotein. Maintenance of extra-cellular ATP by apyrase is necessary for continuous removal of xenobiotics. Dinitroaniline resistance has also been successfully generated in transgenic tobacco through overexpression of mutant α and β-tubilin gene.
Transgenic tobacco exhibit complete resistance to dinitroaniline. To engineer a multi herbicide resistance and obtained high level of resistance. Several options are enforced like expression of multiple copies of the genes involved and selection of modified p-glycoprotein or related ABC transporter herbicide.
Herbicide resistance transgenic Panax-ginseng were produced using the PAT (phosphinothricin) gene. PAT gene is obtained from Streptomyces virido chromogene encode for the enzyme PAT, which catalyses free ammonium groups of phosphinothricin. The active ingredient of herbicide bialophos and biolophos is non-toxic to transgenic plants expressing PAT gene.