Chapter 6: Plant Biotechnology

1. Chapter 6: Plant Biotechnology Plant genetic engineering: methodology Plant genetic engineering: applications Crop improvement (herbicide, insect and virus resistance, altered oil content, delayed fruit ripening, and pollen control) Genetically engineered food (soybean, corn) Nutritionally enhanced plants (golden rice) Molecular farming (edible vaccines, biopolymers) Safety issues

2. Genetic Engineering of Plants: Methodology Plant transformation with the Ti plasmid of Agrobacterium tumefaciens Ti plasmid derived vector systems Physical methods of transferring genes to plants Microprojectile bombardment Use of reporter genes in transformed plant cells

3. To improve the agricultural, horticultural or ornamental value of a crop plant To serve as a living bioreactor for the production of economically important proteins or metabolites To provide a powerful means for studying the action of genes (and gene products) during development and other biological processes Why genetically engineer plants?

4. A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumors Tumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T-DNA (transferred DNA) The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A. tumefaciens Plant transformation with the Ti plasmid of Agrobacterium tumefaciens

5. The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the plant nuclear genome

6. The Ti plasmid of Agrobacterium tumafaciens and itsT-DNA region containing eukaryotic genes for auxin, cytokinin, and opine production.

7. Fig. 28-27 Crown Gall on Tobacco Infection of a plant with A. tumefaciens and formation of crown galls

8. The binary Ti plasmid system involves using a small T-DNA plasmid (A) and a disarmed Ti plasmid in A. tumefaciens (B) A. The small T-DNA plasmid (~10 kb) B. The disarmed Ti plasmid; i.e., the Ti plasmid lacking the T-DNA

9. (disarmed) Plant genetic engineering with the binary Ti plasmid system Clone YFG (your favorite gene) or the target gene in the small T-DNA plasmid in E. coli, isolate the plasmid and use it to transform the disarmed A. tumefaciens as shown. Transgenic plant

10. Plant cell DNA-delivery methods * Most commonly used methods

11. Microprojectile bombardment or biolistic-mediated DNA transfection equipment(a) lab version(b) portable version * When the helium pressure builds to a certain point, the plastic rupture disk bursts, and the released gas accelerates the flying disk* with the DNA-coated gold particles on its lower side. The gold particles pass the stopping screen, which holds back the flying disk, and penetrate the cells of the plant.

12. Some plant cell reporter and selectable marker gene systems

13. Reporter Genes • For how reporter genes work, see: http://bcs.whfreeman.com/lodish5e/pages/bcs-main.asp?v=category&s=00010&n=15000&i=15010.01&o=|00510|00610|00520|00530|00540|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1322 • GFP Researchers Win Nobel Prize (October 8, 2008) Osamu Shimomura, Martin Chalfie, and Roger Tsien won the Nobel Prize in chemistry for their work on green flourescent protein, a tool that has become ubiquitous in modern biology as a tag and molecular highlighter, vastly improving our ability to understand what goes on inside cells. • Perhaps you may even want to see a 10 minute YouTube video on GFP; if so please see http://www.youtube.com/watch?v=Sl2PRHGpYuU

14. Strong, constitutive promoters (35S Cauliflower mosaic virus promoter or 35S CaMV or 35S) Organ and tissue specific promoter (e.g., the leaf-specific promoter for the small subunit of the photosynthetic enzyme ribulosebisphosphate carboxylase or rbc) Promoterless reporter gene constructs to find new organ- and tissue-specific promoter Inducible promoters Secretion of transgene products by inclusion of a signal peptide sequence between a root promoter and YFG and growing the transgenic plant hydroponically (YFG product will be secreted) Manipulation of gene expression in plants

15. Genetic Engineering of Plants: Applications Insect-, pathogen-, and herbicide-resistant plants Stress- and senescence-tolerant plants Genetic manipulation of flower pigmentation Modification of plant nutritional content Modification of plant food taste and appearance Plant as bioreactors Edible vaccines Renewable energy crops Plant yield

16. Are we eating genetically engineered plants now? You bettcha! 80 genetically engineered plants approved in the US Your query has returned 80 records. For further information on a particular event, click on the appropriate links under the Event column in the following table. Creeping Bentgrass   Sugar Beet   Argentine Canola Papaya   Chicory   Melon   Squash   Soybean   Cotton   Flax, Linseed   Tomato   Alfalfa   Tobacco   Rice   Plum   Potato   Wheat   Maize  132 genetically engineered plants approved in the world Your query has returned 132 records. For further information on a particular event, click on the appropriate links under the Event column in the following table. Creeping Bentgrass   Sugar Beet   Argentine Canola   Polish Canola Papaya   Chicory   Melon   Squash   Carnation   Soybean   Cotton   Sunflower   Lentil   Flax Linseed   Tomato   Alfalfa   Tobacco   Rice   Plum   Potato   Wheat   Maize -See http://www.agbios.com/dbase.php for details

17. Genetic engineering of insect resistant plants or Bt-plants • Bacillus thuringiensisis a soil bacterium that produces a toxin (Bt toxin) which kills certain insects • Expression of truncated Bt genes encoding the N-terminal portion of Bt increase effectiveness • Effectiveness enhanced by site-directed mutagenesis increasing transcription/translation • Effectiveness further enhanced by making codon bias changes (bacterial to plant) • 35S CaMV and rbcS promoters used • Integration and expression of the Bt gene directly in chloroplasts • Note that Lepidopteran insects like corn rootworm, cotton bollworm, tobacco budworm, etc., cause combined damages of over $7 Billion dollars yearly in the US

18. A binary T-DNA plasmid for delivering the Bt gene to plants (NPT or kanr) (35S-Bt gene-tNOS) (Spcr)

19. For a visual look at the effectiveness of Bt-plants: • You can download a quicktime movie clip on “Insect resistance with Bt” from Dr. Goldberg’s web site http://www.mcdb.ucla.edu/Research/Goldberg/research/movie_trailers-index.htm • Or you can see a video embedded at this web site http://www.crop.cri.nz/home/research/plants/brassica-faqs.php

20. Procedure for putting CuMV coat protein into plants Virus-resistant plants • Overexpression of the virus coat protein (e.g. cucumber mosaic virus in cucumber and tobacco, papaya ringspot virus in papaya and tobacco, tobacco mosiac virus in tobacco and tomato, etc.) • Expression of a dsRNase (RNaseIII) • Expression of antiviral proteins (pokeweed)

21. B (pNOS-NPT-tNOS) (pNOS-NPT-tNOS) (35S-CuMV sense-tRBC) (35S-CuMV antisense-tRBC) (Spcr) (Spcr) Binary cloning vector carrying the protein-producing sense (A) or antisense RNA-producing (B) orientation of the cucumber mosaic virus coat protein (CuMV) cDNA

22. Genetically engineered Papaya to resist the Papaya Ringspot-Virus by overexpression of the virus coat protein

23. Herbicides and herbicide-resistant plants • Herbicides are generally non-selective (killing both weeds and crop plants) and must be applied before the crop plants germinate • Four potential ways to engineer herbicide resistant plants • Inhibit uptake of the herbicide • Overproduce the herbicide-sensitive target protein • Reduce the ability of the herbicide-sensitive target to bind to the herbicide • Give plants the ability to inactivate the herbicide

24. Herbicide-resistant plants:Giving plants the ability to inactivate the herbicide • Herbicide: Bromoxynil • Resistance to bromoxynil (a photosytem II inhibitor) was obtained by expressing a bacterial (Klebsiella ozaenae) nitrilase gene that encodes an enzyme that degrades this herbicide

25. Herbicide-resistant plants:Reducing the ability of the herbicide-sensitive target to bind to the herbicide • Herbicide: Glyphosate (better known as Roundup) • Resistance to Roundup (an inhibitor of the enzyme EPSP involved in aromatic amino acid biosynthesis) was obtained by finding a mutant version of EPSP from E. coli that does not bind Roundup and expressing it in plants (soybean, tobacco, petunia, tomato, potato, and cotton) • 5-enolpyruvylshikimate-3-phosphate synthase (EPSP) is a chloroplast enzyme in the shikimate pathway and plays a key role in the synthesis of aromatic amino acids such as tyrosine and phenylalanine • This is a big money maker for Monsanto!

26. How to make a Roundup Ready Plant

27. Development of stress- and senescence-tolerant plants: genetic engineering of salt-resistant plants • Overexpression of the gene encoding a Na+/H+ antiport protein which transports Na+ into the plant cell vacuole • This has been done in Arabidopsis and tomato plants allowing them to survive on 200 mM salt (NaCl)

28. Flavor development pathway Red Green Fruit softening pathway polygalacturonase Soft Hard Development of stress- and senescence-tolerant plants: genetic engineering of flavorful tomatoes • Fruit ripening is a natural aging or senescence process that involves two independent pathways, flavor development and fruit softening. • Typically, tomatoes are picked when they are not very ripe (i.e., hard and green) to allow for safe shipping of the fruit. • Polygalacturonase is a plant enzyme that degrades pectins in plant cell walls and contribute to fruit softening. • In order to allow tomatoes to ripen on the vine and still be hard enough for safe shipping of the fruit, polygalacturonase gene expression was inhibited by introduction of an antisense polygalacturonase gene and created the first commercial genetically engineered plant called the FLAVR SAVR tomato. antisense polygalacturonase

29. Modification of plant nutritional content: increasing the vitamin E (a-tocopherol) content of plants • Plants make very little a-tocopherol but do make g-tocopherol; they do not produce enough of the methyltransferase (MT) • The MT gene was identified and cloned in Synechocystis and then in Arabidopsis • The Arabidopsis MT gene was expressed under the control of a seed-specific carrot promoter and found to produce 80 times more vitamin E in the seeds Dean DellaPenna, Michigan State Univ. Professor B.S. 1984, Ohio University

30. Modification of plant nutritional content: increasing the vitamin A content of plants GGPP Phytoene Lycopene b-carotene Vitamin A • 124 million children worldwide are deficient in vitamin A, which leads to death and blindness • Mammals make vitamin A from b-carotene, a common carotenoid pigment normally found in plant photosynthetic membranes • Here, the idea was to engineer the b-carotene pathway into rice • The transgenic rice is yellow or golden in color and is called “golden rice” Daffodil phytoene synthase gene Bacterial phytoene desaturase gene Daffodil lycopene b-cyclase gene Endogenous human gene

32. Plants as bioreactors: production of pharmaceutical human proteins in plant systems Growth hormone Serum albumin Epidermal growth factor a-interferon Erythropoietin Hemoglobin Interleukins 2 and 4 a1-Antitrypsin

33. Production of (edible) vaccines in plants Herpes virus B surface antigen Rabies glycoprotein Norwark virus coat protein Foot and mouth virus VP1 Cholera toxin B subunit Human cytomegalovirus glycoprotein B

34. Renewable Energy/Biofuels: Cellulosic Ethanol Review Nature Reviews Genetics 9, 433-443 (June 2008) | doi:10.1038/nrg2336 Focus on: Global Challenges Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol Mariam B. Sticklen1About the author Top of pageAbstract Biofuels provide a potential route to avoiding the global political instability and environmental issues that arise from reliance on petroleum. Currently, most biofuel is in the form of ethanol generated from starch or sugar, but this can meet only a limited fraction of global fuel requirements. Conversion of cellulosic biomass, which is both abundant and renewable, is a promising alternative. However, the cellulases and pretreatment processes involved are very expensive. Genetically engineering plants to produce cellulases and hemicellulases, and to reduce the need for pretreatment processes through lignin modification, are promising paths to solving this problem, together with other strategies, such as increasing plant polysaccharide content and overall biomass.

35. The Plant Cell Wall a | Cell wall containing cellulose microfibrils, hemicellulose, pectin, lignin and soluble proteins.b | Cellulose synthase enzymes are in rosette complexes, which float in the plasma membrane.c | Lignification occurs in the S1, S2 and S3 layers of the cell wall.

36. Cellulosic Ethanol Production and Research Challenges This figure depicts some key processing steps in a future large-scale facility for transforming cellulosic biomass (plant fibers) into biofuels. Three areas where focused biological research can lead to much lower costs and increased productivity include developing crops dedicated to biofuel production (see step 1), engineering enzymes that deconstruct cellulosic biomass (see steps 2 and 3), and engineering microbes and developing new microbial enzyme systems for industrial-scale conversion of biomass sugars into ethanol and other biofuels or bioproducts (see step 4). Biological research challenges associated with each production step are summarized in the right portion of the figure.

37. Potential Bioenergy Crops