Transgenic plants- Abiotic stress tolerance
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The document discusses the development of transgenic plants that are engineered for herbicide and abiotic stress tolerance, including methods for conferring herbicide resistance through gene modification and detoxification strategies. It highlights specific herbicides like glyphosate and phosphinothricin, their mechanisms of action, and successful genetic modifications made to enhance crop resilience. Additionally, the document addresses environmental impacts, challenges such as super weeds, and advancements in water deficit stress tolerance through osmoprotectant production and ice-nucleating bacteria resistance.
Transgenic plants- Abiotic stress tolerance
- 2. Introduction • The different types of external stresses that influence the plant growth and development. • These stresses are grouped based on their characters Biotic Abiotic • Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress to plants. • This damages the cellular constituents of plants which is associated with a reduction in plant yield.
- 3. Herbicide Resistance: • Weeds (wild herbs) are unwanted and useless plants that grow along with the crop plants. • Weeds compete with traps for light and nutrients, besides harbouring various pathogens. • It is estimated that the world’s crop yield is reduced by 10-15% due to the presence of weeds.
- 4. • A good or an ideal herbicide is expected to possess the following characteristics: i. Capable of killing weeds without affecting crop plants. ii. Not toxic to animals and microorganisms. iii. Rapidly trans-located within the target plant. iv. Rapidly degraded in the soil. • None of the commercially available herbicides fulfills all the above criteria. For this reason, the crops are also affected by herbicides, hence the need to develop herbicide-resistant plants.
- 5. Strategies for engineering herbicide resistance: 1. Overexpression of the target protein: The target protein, being acted by the herbicide can be produced in large quantities so that the affect of the herbicide becomes insignificant. Overexpression can be achieved by integrating multiple copies of the genes and/or by using a strong promoter.
- 6. 2. Improved plant detoxification: The plants do possess natural defense systems against toxic compounds (herbicides). Detoxification involves the conversion of toxic herbicide to non-toxic or less toxic compound. By enhancing the plant detoxification system, the impact of the herbicide can be reduced. 3. Detoxification of herbicide by using a foreign gene: By introducing a foreign gene into the crop plant, the herbicide can be effectively detoxified.
- 7. 4. Mutation of the target protein: • The target protein which is being affected by the herbicide can be suitably modified. • The changed protein should be capable of discharging the functions of the native protein but is resistant to inhibition by the herbicide. • Once the resistant target protein gene is identified, it can be introduced into the plant genomes, and thus herbicide-resistant plants can be developed.
- 8. Some of the developments made in the herbicide resistance of plant • Glyphosate Resistance: • Glyphosate, is a glycine derivative. • It acts as a broad-spectrum herbicide and is effective against 76 of the world’s worst 78 weeds. • Glyphosate is less toxic to animals and is rapidly degraded by microorganisms.
- 9. Mechanism of action of glyphosate: • Glyphosate is rapidly transported to the growing points of plants. • It is capable of killing the plants even at a low concentration. • Glyphosate acts as a competitive inhibitor of the enzyme 5-enoylpyruvylshikimate 3- phosphate synthase (EPSPS). • This is a key enzyme in shikimic acid pathway that results in the formation of aromatic amino acids (tryptophan, phenylalanine and tyrosine), phenols and certain secondary metabolites
- 10. • Glyphosate has some structural similarly with the substrate phosphoenol pyruvate. • Glyphosate binds more tightly with EPSPS and blocks the normal shikimic acid pathway. • Thus, the herbicide glyphosate inhibits the biosynthesis of aromatic amino acids and other important products. • This results in inhibition of protein biosynthesis (due to lack of aromatic amino acids). As a consequence, cell division and plant growth are blocked. • Glyphosate is non-toxic to animals (including humans), since they do not possess shikimate pathway.
- 11. Strategies for glyphosate resistance: 1. Overexpression of crop plant EPSPS gene: • An overexpressing gene of EPSPS was detected in Petunia. • This expression was found to be due to gene amplification rather than an increased expression of the gene. • EPSPS gene from Petunia was isolated and introduced into other plants. • The increased synthesis of EPSPS (by about 40 fold) in transgenic plants provides resistance to glyphosate. • These plants can tolerate glyphosate at a dose of 2-4 times higher than that required to kill wild-type plants.
- 12. 2. Use of mutant EPSPS genes: • An EPSPS mutant gene that conferred resistance to glyphosate was first detected in the bacterium Salmonella typhimurium. • It was found that a single base substitution (C to 7) resulted in the change of an amino acid from proline to serine in EPSPS. • This modified enzyme cannot bind to glyphosate, and thus provides resistance. • The mutant EPSPS gene was introduced into tobacco plants using Agrobacterium Ti plasmid vectors.
- 13. • The transgene produced high quantities of the enzyme EPSPS. • It was later known that the shikimate pathway occurs in the chloroplasts while the glyphosate resistant EPSPS was produced only in the cytoplasm. • In later years, the mutant EPSPS gene was tagged with a chloroplast- specific transit peptide sequence. By this approach, the glyphosate- resistant EPSPS enzyme was directed to freely enter chloroplast and confer resistance against the herbicide.
- 14. 3. Detoxification of glyphosate: • The soil microorganisms possess the enzyme glyphosate oxidase that converts glyphosate to glyoxylate and aminomethylphosponic acid. • The gene encoding glyphosate oxidase has been isolated from a soil organism Ochrobactrum anthropi. • With suitable modifications, this gene was introduced into crop plants e.g. oilseed rape. The transgenic plants were found to exhibit very good glyphosate resistance in the field.
- 15. Phosphinothricin (glufosinate)Resistance • Phosphinothricin (or glufosinate) is also a broad spectrum herbicide like glyphosate. • Phosphinothricin is more effective against broad-leafed weeds but least effective against perennials. • Phosphinothricin is an unusual herbicide, being a derivative of a natural product namely bialaphos. Certain species of Streptomyces produce bialaphos
- 16. Mechanism of action of phosphinothricin: • Phosphinothricin acts as a competitive inhibitor of the enzyme glutamine synthase. • This is possible since phosphinothricin has some structural similarity with the substrate glutamate. • As a consequence of the inhibition of glutamine synthase, ammonia accumulates and kills the plant cells. • Further, disturbance in glutamine synthesis also inhibits photosynthesis.
- 17. • The gene responsible for coding phosphinothricin acetyl transferase (bar gene) has been identified in Streptomyces hygroscopicus. • Some success has been reported in developing transgenic maize and oilseed rape by introducing bar gene. These plants were found to provide resistance to phosphinothricin.
- 18. Sulfonylureas and Imidazolinones Resistance: • Sulfonylureas and imidazolinones inhibit the enzyme acetolactate synthase (ALS), a key enzyme in the synthesis of branched chain amino acids namely isoleucine, leucine and valine. • Mutant forms of this enzyme and the corresponding genes have been isolated, identified and characterized. • Transgenic plants with the mutant genes of ALS were found to be resistant to sulfonylureas and imidazolinones e.g. maize, tomato, sugar beet.
- 19. Resistance to other herbicides:
- 20. • Environmental Impact of Herbicide-Resistant Crops: • The development genetically modified (GM) herbicide-resistant crops has undoubtedly contributed to increase in the yield of crops. • farmers particularly in the developed countries (e.g. USA) have started using these GM crops. • Eg: Herbicide resistant soybean plants grown in USA increased from 17% in 1997 to 68% in 2001.
- 21. Disadvantage: Herbicide-resistant transgenic plants are at field-trial stage. Due to environmental concern, a few of these plants are withdrawn e.g. atrazine- resistant crops. • Disturbance in biodiversity due to elimination of weeds. • Rapid development of herbicide-resistance weeds that may finally lead to the production of super weeds.
- 22. Tolerance to Water Deficit Stresses: • The environmental conditions such as temperature (heat, freezing, chilling), water availability (shortage due to drought), and salinity influence the plant growth, development and yield. • The abiotic stresses due to temperature, drought and salinity are collectively regarded as water deficit stresses
- 23. Causes of water deficit: • Reduced soil water potential. • Increased water evaporation (in dry, hot and windy conditions). • High salt concentration in the soil (decrease soil water potential). • Low temperature resulting in the formation of ice crystals.
- 24. Effects of water deficit: • Results in osmotic stress. • Inhibits photosynthesis. • Increases the concentration of toxic ions (reactive oxygen species) within the cells. • Loss of water from the cell causing plasmolysis and finally cell death.
- 25. • Osmoprotectants are non-toxic compatible solutes, Plants produce osmoprotectants or osmolytes to overcome osmotic stresses. Divided into two groups. • 1. Sugar and sugar alcohols e.g. mannitol, sorbitol, pinitol, ononitol, trehalose, fructans. • 2. Zwitterionic compounds: These osmoprotectants carry positive and negative charges e.g. proline, glycine betaine. • The production of a given osmoprotectant is species dependent. The formation of mannitol, proline and glycine betaine are more closely linked to osmotic tolerance.
- 26. Transgenic plants with glycine toetaine production: • Glycine betaine is a quaternary ammonium compound and is electrically neutral. • Functioning as a cellular osmolytes, glycine betaine stabilizes proteins and membrane structures. • Some of the key enzymes for the production of glycine betaine have been identified e.g. choline mono-oxygenase, choline dehydrogenase, betaine aldehyde dehydrogenase. • The genes coding these enzymes were transferred to develop transgenic plants.
- 27. Resistance against Ice-Nucleating Bacteria: • Formation of ice on the plant cells (outer membrane) is a complex chemical process. The importance of ice-nucleating bacteria. The ice nucleating bacteria synthesize proteins, which coalesce with water molecules to form ice crystals at temperature around 32°F. As the ice crystals grow, they can pierce the plant cells and severely damage the plants. • Chemical treatment of plants to protect from ice formation:Copper & Urea
- 28. Ice-minus bacteria to resist plants from cold temperatures: • The bacterium Pseudomonas syringae is one of the highly prevalent ice-forming organisms in nature. • The gene that directs the synthesis of ice-related bacterial proteins in P. syringae was removed. • newly developed bacteria are referred to as ice-minus bacteria.
- 29. • The researchers proposed to spray the transgenic ice-minus bacteria on to young plants. • The intention was that these bacteria would give frost tolerance to the plants; and thus increase the crop yield. • The bacterial mutants may create some health complication in humans. • It was in 1987, ice-minus bacteria were sprayed on to the field of potato plants and strawberry plants. • P. syringae commercially labeled, as Frostban was later developed and used in crop fields. • Ice-minus bacteria of P. syringae were the first transgenic bacteria that were used outside the laboratory.
- 31. Arabidopsis with cold- tolerant genes: • Scientists were successful in developing cold- tolerant genes (around 20) in Arabidopsis when this plant was gradually exposed to slowly declining temperatures. • They also identified a coordinating gene that encodes a protein, which acts as a transcription factor for regulating the expression of cold- tolerant genes.More work is in progress in this direction.
- 32. Reference • U. Satyanarayana-Biotechnology • https://www.biologydiscussion.com/plants/transgenic- plants/applications-of-transgenic-plants-6-applications/10838