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Craig Schenck 11/12/09

Increasing the vitamin E content in plants by overexpressing the γ-tocopherol methyltransferase gene. Craig Schenck 11/12/09. Vitamin E. Collective term for α -, β -, γ -, δ -tocopherols and four corresponding derivatives α -, β -, γ -, δ -tocotrienols

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Craig Schenck 11/12/09

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  1. Increasing the vitamin E content in plants by overexpressing the γ-tocopherol methyltransferase gene Craig Schenck 11/12/09

  2. Vitamin E • Collective term for α-,β-,γ-,δ-tocopherols and four corresponding derivatives α-,β-,γ-,δ-tocotrienols • α-tocopherol is the most active form of vitamin E and is considered the most important for human health • Tocopherols are synthesized exclusively in photosynthetic organisms

  3. Health Benefits for Humans • Vitamin E is a strong antioxidant • Vitamin E may also be implicated in the prevention of heart disease, cancer, loss of memory and eye disorders • Deficiencies in vitamin E cause poor transmission of nerve impulses, muscle weakness and vision degradation

  4. Sources of Vitamin E • Egg, whole, fresh .................................... 1 large ........................................0.88 ......................................5.8 • Almond oil ..........................................1 tablespoon ....................................5.3 ......................................35.3 • Corn oil (Mazola) ................................1 tablespoon ..................................... 3 .......................................... 5 • Cottonseed oil .....................................1 tablespoon ....................................4.8 ....................................... 32 • Olive oil ..............................................1 tablespoon ....................................1.6 ......................................10.6 • Peanut oil ............................................1 tablespoon ....................................1.6 ......................................10.6 • Soybean oil ..........................................1 tablespoon ....................................1.5 ....................................... 10 • Vegetable-oil spray .......................... 2.5 second spray ................................0.51 ......................................3.4 • Wheat-germ oil ...................................1 tablespoon ...................................20.3 ....................................135.3 • Apple with skin .....................................1 medium .....................................0.81 ......................................5.4 • Spaghetti pasta, enriched ......................... 1 cup .........................................1.03 ......................................6.8 • Almonds, dried ....................................... 1 ounce .......................................6.72 .....................................44.8 • Hazelnuts, dried ..................................... 1 ounce ........................................6.7 ......................................44.6 • Peanut butter (Skippy) ........................1 tablespoon ..................................... 3 .......................................... 5 • Peanuts, dried ......................................... 1 ounce .......................................2.56 ...................................... 17 • Margarine (Mazola) ............................1 tablespoon ..................................... 8 ........................................53.3 • Mayonnaise (Hellmann’s) ...................1 tablespoon .................................... 11 .......................................73.3 • Spinach, raw ............................................1/2 cup .......................................0.53 ......................................3.5 • Sweet potato ..........................................1 medium .....................................5.93 .....................................39.5 • Tomato, red, raw ....................................1 tomato ......................................0.42 ......................................2.8 Food Serving Milligrams % RDA

  5. Tocopherol biosynthesis The tocopherol biosynthetic pathway and mutants in Arabidopsis thaliana and Synechocystis sp. PCC6803. Enzymes are indicated by black boxes and mutations by red letters and lines. Bold arrows show the primary biosynthetic route in wild-type Arabidopsis leaves and Synechocystis. vte1, vte2, vte3, vte4 and pds1 are mutants that have defects in tocopherol cyclase (TC), homogentisic acid (HGA) phytyltransferase (HPT), 2-methyl-6-phytyl-1,4-benzoquinol (MPBQ) methyltransferase (MT), g-tocopherol methyltransferase (g-TMT) and hydroxyphenylpyruvate (HPP) dioxygenase (HPPD) in Arabidopsis, respectively. slr1737, slr1736, sll0418, slr0089 and slr0090 are the corresponding mutants in Synechocystis. Null alleles of Arabidopsis pds1 and vte3 are lethal because of plastoquinone (PQ) deficiency [28,41], whereas the disruption of slr0090 or sll0418 has no impact on PQ and viability in Synechocystis [28,42]. DMPBQ, 2,3-dimethyl-6-phytyl-1,4-benzoquinol; PDP, phytyl-diphosphate; SDP, solanesyl-diphosphate.

  6. Soybean and perilla Soybean Perilla

  7. Methodology • Soybean transformation • Particle bombardment via gene gun • Shot gold/DNA coated microprojectiles into embryogenic cultures • Cell culture work, lasting approximately 7 months Schematic representation of the binary vector constructed for soybean transformation. The T-DNA region of the p1304PfTMT binary vector showing the assembly of γ -TMT expression cassette (Vicilin P:γ -TMT:OCS T), hygromycin expression cassette (35S P:HYG:35Spoly A) and GFP–GUS reporter fusion gene expression cassette (35SP:GFP–GUS:Nos poly A). γ -TMT: γ -tocopherol methyltransferase;OCS T: Agrobacterium tumefaciens octopine synthase poly A; 35SP: CaMV 35S promoter; 35S poly A: CaMV 35S poly A; HYG: hygromycin;GFP–GUS: green fluorescent protein – β-glucronidase fusiongene; Nos poly A: Agrobacterium tumefaciens nopaline synthasepoly A

  8. Methodology • Perilla transformation • Similar construct to soybean transformation however Agrobaterium-mediated transformation was used pBKI Figure 1. Schematic representation of T-DNA region of binary vectors pIG121Hm (top) and pBK I (bottom). LB, left border; RB, right border; NOS PRO, nopaline synthase promoter; NPT II, neomycin phosphotransferase II; NOS TER, nopaline synthase gene terminator; 35S PRO, cauliflower mosaic virus 35S promoter; GUS, b-glucuronidase gene; HPT, hygromycin phosphotransferase; 35S poly A, 35S terminator; BAR, gene encoding phosphinothricin acetyl transferase; Vic PRO, vicillin promoter; OCS, octopinesynthase terminator.

  9. Identifying transgenic lines and gene expression of soybeans Northern blot of RNA isolated from different tissues of wild type and T134 plant Southern blot probed with full length GUS gene

  10. Transgenic perilla lines (b) PCR amplification of transformed γ-TMT gene. M, molecular size marker P, plasmid (pBK I) as a positive control; Y, Yeupsil as a negative control; P1–P4, transgenic T0 plants recovered from different transformation events; P1-1, a T1 progeny of P1 that does not carry the transgene due to segregation; P1-2 and P1-3, T1 progenies of P1 carrying the transgene. Southern blot of T2 plants

  11. Analyzing progeny and expression pattern • Northern blot analysis confirms • seed specific expression in both • T0 and T1 generation - γ-TMTgene is heritable Southern and northern blot analysis of T1 progeny probed with full length γ-TMTgene

  12. Expression pattern of γ-TMT gene • γ-TMT gene is expressed only in the • immature seed • The gene is heritable through multiple • generations Fig. 2 Northern blot analysis of transformed c-TMT gene transcript. Upper panel: Northern blot. lane 1, Yeupsil leaf; lane 2, T0 plant (P2 leaf); lane 3, Yeupsil (immature seed) 10 days after flowering; lane 4–7, Immature seeds of P1-2, P1- 3, P2-1, and P2-2, respectively, 10 days after flowering. Lower panel: ethidium-bromide gel staining of RNA samples as loading control

  13. Tocopherol content in seeds of soybeans

  14. Tocopherol content in seeds of perilla

  15. Conclusion • Successfully transformed γ-TMT gene in both cases • The trait is heritable across multiple generations • α-tocoperol content increased 10-20 • Manipulation of vitamin E synthesis did not significantly impact total lipid contents and fatty acid compositions of the seeds. • Agrobaterium-mediated transformation more efficient than particle bombardment?

  16. References • Tavva VK, Kim YH, Kagan IA, Dinkins RD, Kim KH, Collins GB (2007) Increased α-tocopherol content in soybean seed overexpressing the Perilla frutescens γ-tocopherol methyltransferase gene. Plant Cell Rep 26:61–70. • Lee BK, Kim SL, Kim KH, Yu SH, Lee SC, Zhang Z, Kim MS, Park HM, Lee JY (2008) Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (Perilla frutescens). Plant Cell Tissue and Organ Culture 92:47-54. • Maeda H, DellaPenna D (2007) Tocopherol functions in photosynthetic organisms. Current Opinion in Plant biology 10:260-265. • Department of Human Nutrition, "Vitamin E." November 2004.http://ohioline.osu.edu/hyg-fact/5000/5554.html (accessed 11/8/09). • Office of Dietary Supplements, "Vitamin E Fact Sheet." October 4, 2007.http://ods.od.nih.gov/factsheets/vitamine.asp#en4 (accessed 11/8/09).

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