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

• 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

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

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

Tocopherol biosynthesis

The tocopherol biosynthetic pathway and mutants in Arabidopsis thaliana and Synechocystis sp. PCC6803. Enzymes are indicated by blackboxes 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 sll0418has 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.

Soybean and perilla

Perilla

Soybean

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

Methodology

• Perilla transformation– Similar construct to soybean transformation

however Agrobaterium-mediated transformation was used

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.

pBKI

Identifying transgenic lines and gene expression of soybeans

Southern blot probed with full length GUS gene

Northern blot of RNA isolated from different tissuesof wild type and T134 plant

Transgenic perilla lines

(b) PCR amplificationof transformed γ-TMT gene. M, molecular sizemarker P, plasmid (pBK I) as a positive control; Y, Yeupsil asa 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

Analyzing progeny and expression pattern

Southern and northern blot analysis of T1 progeny probed withfull length γ-TMT gene

- Northern blot analysis confirmsseed specific expression in bothT0 and T1 generation

- γ-TMT gene is heritable

Expression pattern of γ-TMT gene

Fig. 2 Northern blot analysis of transformed c-TMT genetranscript. 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. Lowerpanel: ethidium-bromide gel staining of RNA samples asloading control

-γ-TMT gene is expressed only in the immature seed

-The gene is heritable through multiple generations

Tocopherol content in seeds of soybeans

Tocopherol content in seeds of perilla

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?

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).