Transgenic Research

, Volume 22, Issue 2, pp 391–402 | Cite as

Improvement of vitamin E quality and quantity in tobacco and lettuce by chloroplast genetic engineering

  • Yukinori Yabuta
  • Hiroyuki Tanaka
  • Sahoko Yoshimura
  • Akiko Suzuki
  • Masahiro Tamoi
  • Takanori Maruta
  • Shigeru Shigeoka
Original Paper


Vitamin E (tocopherol: Toc) is an important lipid-soluble antioxidant synthesized in chloroplasts. Among the 8 isoforms of vitamin E, α-Toc has the highest activity in humans. To generate transgenic plants with enhanced vitamin E activity, we applied a chloroplast transformation technique. Three types of the transplastomic tobacco plants (pTTC, pTTMT and pTTC-TMT) carrying the Toc cyclase (TC) or γ-Toc methyltransferase (γ-TMT) gene and the TC plus γ-TMT genes as an operon in the plastid genome, respectively, were generated. There was a significant increase in total levels of Toc due to an increase in γ-Toc in the pTTC plants. Compared to the wild-type plants, Toc composition was altered in the pTTMT plants. In the pTTC-TMT plants, total Toc levels increased and α-Toc was a major Toc isoform. Furthermore, to use chloroplast transformation to produce α-Toc-rich vegetable, TC-overexpressing transplastomic lettuce plants (pLTC) were generated. Total Toc levels and vitamin E activity increased in the pLTC plants compared with the wild-type lettuce plants. These findings indicated that chloroplast genetic engineering is useful to improve vitamin E quality and quantity in plants.


Antioxidant Plastid transformation Tocopherol Tobacco Lettuce 





Homogentisic acid


Homogentisate phytyltransferase




p-hydroxyphenylpyruvate dioxygenase


Methylerythritol phosphate




2-methyl-6-phytylbenzoquinone methyltransferase




Tocopherol cyclase


γ-tocopherol methyltransferase


  1. Adachi N, Migita M, Ohta T, Higashi A, Matsuda I (1997) Depressed natural killer cell activity due to decreased natural killer cell population in a vitamin E-deficient patient with Shwachman syndrome: reversible natural killer cell abnormality by α-tocopherol supplementation. Eur J Pediatr 156:444–448PubMedCrossRefGoogle Scholar
  2. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  3. Cahoon EB, Hall SE, Ripp KG, Ganzke TS, Hitz WD, Coughlan SJ (2003) Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat Biotech 21:1082–1087CrossRefGoogle Scholar
  4. Cheng Z, Sattler S, Maeda H, Sakuragi Y, Bryant DA, DellaPenna D (2003) Highly divergent methyltransferases catalyze a conserved reaction in tocopherol and plastoquinone synthesis in Cyanobacteria and photosynthetic eukaryotes. Plant Cell 15:2343–2356PubMedCrossRefGoogle Scholar
  5. Cho EA, Lee CA, Kim YS, Baek SH, de los Reyes BG, Yun SJ (2005) Expression of γ-tocopherol methyltransferase transgene improves tocopherol composition inlettuce (Latuca sativa L.). Mol Cells 19:16–22PubMedGoogle Scholar
  6. Collakova E, DellaPenna D (2001) Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol 127:1113–1124PubMedCrossRefGoogle Scholar
  7. Collakova E, DellaPenna D (2003) Homogentisate phytyl transferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol 131:632–642PubMedCrossRefGoogle Scholar
  8. Farré G, Sudhakar D, Naqvi S, Sandmann G, Christou P, Capell T, Zhu C (2012) Transgenic rice grains expressing a heterologous p-hydroxyphenylpyruvate dioxygenase shift tocopherol synthesis from the γ to the α isoform without increasing absolute tocopherol levels. Transgenic Res. doi:10.1007/s11248-012-9601-7 PubMedGoogle Scholar
  9. Golds T, Maliga P, Koop H-U (1993) Stable plastid transformation in PEG-treated protoplasts of Nicotiana tabacum. Bio/Technology 11:95–97CrossRefGoogle Scholar
  10. Grusak MA, DellaPenna D (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu Rev Plant Physiol Plant Mol Biol 50:133–161PubMedCrossRefGoogle Scholar
  11. Hunter SC, Cahoon EB (2007) Enhancing Vitamin E in Oilseeds: unraveling Tocopherol and Tocotrienol Biosynthesis. Lipids 42:97–108PubMedCrossRefGoogle Scholar
  12. Ichikawa Y, Tamoi M, Sakuyama H, Maruta T, Ashida H, Yokota A, Shigeoka S (2010) Generation of transplastomic lettuce with enhanced growth and high yield. GM Crops 1:322–326PubMedCrossRefGoogle Scholar
  13. Kamal-Eldin A, Appelqvist LA (1996) The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 31:671–701PubMedCrossRefGoogle Scholar
  14. Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa K (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15:205–217PubMedCrossRefGoogle Scholar
  15. Kanwischer M, Porfirova S, Bergmüller E, Dörmann P (2005) Alterations in tocopherol cyclase activity in transgenic and mutant plants of Arabidopsis affect tocopherol content, tocopherol composition, and oxidative stress. Plant Physiol 137:713–723PubMedCrossRefGoogle Scholar
  16. Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P (1988) Stable genetic transformation of intact Nicotiana cells by the particle bombardment process. Proc Natl Acad Sci USA 85:8502–8505PubMedCrossRefGoogle Scholar
  17. Kumar S, Dhingra A, Daniell H (2004) Stable transformation of the cotton plastid genome and maternal inheritance of transgenes. Plant Mol Biol 56:203–216PubMedCrossRefGoogle Scholar
  18. Lee K, Lee SM, Park SR, Jung J, Moon JK, Cheong JJ, Kim M (2007) Overexpression of Arabidopsis homogentisate phytyltransferase or tocopherol cyclase elevates vitamin E content by increasing γ-tocopherol level in lettuce (Lactuca sativa L.) Mol. Cells 24:301–306Google Scholar
  19. Lim S, Ashida H, Watanabe R, Inai K, Kim YS, Mukougawa K, Fukuda H, Tomizawa K, Ushiyama K, Asao H, Tamoi M, Masutani H, Shigeoka S, Yodoi J, Yokota A (2011) Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts. Plant Mol Biol 76:335–344PubMedCrossRefGoogle Scholar
  20. Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313PubMedCrossRefGoogle Scholar
  21. Mangialasche F, Kivipelto M, Mecocci P, Rizzuto D, Palmer K, Winblad B, Fratiglioni L (2010) High plasma levels of vitamin E forms and reduced Alzheimer’s disease risk in advanced age. J Alzheimers Dis 20:1029–1037PubMedGoogle Scholar
  22. Naqvi S, Farré G, Zhu C, Sandmann G, Capell T, Christou P (2011) Simultaneous expression of Arabidopsis p-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content. Transgenic Res 20:177–181PubMedCrossRefGoogle Scholar
  23. National Research Council (NRC) (1989) Recommended dietary allowances, 10th edn. National Academy Press, WashingtonGoogle Scholar
  24. Norris SR, Shen X, DellaPenna D (1998) Complementation of the Arabidopsis pds1 mutation with the gene encoding p-hydroxy-phenylpyruvate dioxygenase. Plant Physiol 117:1317–1323PubMedCrossRefGoogle Scholar
  25. Porfirova S, Bergmuller E, Tropf S, Lemke R, Dormann P (2002) Isolation of an Arabidopsis mutant lacking vitamin E and identification of a cyclase essential for all tocopherol biosynthesis. Proc Natl Acad Sci USA 99:12495–12500PubMedCrossRefGoogle Scholar
  26. Prasad KN, Kumar A, Kochupillai V, Cole WC (1999) High doses of multiple antioxidant vitamins: essential ingredients in improving the efficacy of standard cancer therapy. J Am College Nutr 18:13–25Google Scholar
  27. Pryor WA (2000) Vitamin E and heart disease: basic science to clinical intervention trials. Free Radic Biol Med 28:141–164PubMedCrossRefGoogle Scholar
  28. Sano M, Ernesto C, Thomas RG, Klauber MR, Schafer K, Grundman M, Woodbury P, Growdon J, Cotman CW, Pfeiffer E, Schneider LS, Thal LJ (1997) A controlled trial of selegiline, alpha-tocopherol, or both as treatment for Alzheimer’s disease. The Alzheimer’s Disease Cooperative Study. N Engl J Med 336:1216–1222PubMedCrossRefGoogle Scholar
  29. Sattler SE, Cheng Z, DellaPenna D (2004a) From Arabidopsis to agriculture: engineering improved vitamin E content in soybean. Trends Plant Sci 9:365–367PubMedCrossRefGoogle Scholar
  30. Sattler SE, Gilliland LU, Magallanes-Lundback M, Pollard M, DellaPenna D (2004b) Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination. Plant Cell 16:1419–1432PubMedCrossRefGoogle Scholar
  31. Savidge B, Weiss JD, Wong Y-HH, Lassner MW, Mitsky TA, Shewmaker CK, Post-Beittenmiller D, Valentin HE (2002) Isolation and characterization of homogentisate phytyltransferase genes from Synechocystis sp. PCC 6803 and Arabidopsis. Plant Physiol 129:321–332PubMedCrossRefGoogle Scholar
  32. Schledz M, Seidler A, Beyer P, Neuhaus G (2001) A novel phytyltransferase from Synechocystis sp. PCC 6803 involved in tocopherol biosynthesis. FEBS Lett 499:15–20PubMedCrossRefGoogle Scholar
  33. Seo YS, Kim SJ, Harn CH, Kim WT (2011) Ectopic expression of apple fruit homogentisate phytyltransferase gene (MdHPT1) increases tocopherol in transgenic tomato (Solanum lycopersicum cv. Micro-Tom) leaves and fruits. Phytochem 72:321–329CrossRefGoogle Scholar
  34. Shigeoka S, Ishiko H, Nakano Y, Mitsunaga T (1992) Isolation and properties of γ-tocopherol methyltransferase in Euglena gracilis. Biochem Biophys Acta 1128:220–226PubMedCrossRefGoogle Scholar
  35. Shintani D, DellaPenna D (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282:2098–2100PubMedCrossRefGoogle Scholar
  36. Shintani DK, Cheng Z, DellaPenna D (2002) The role of 2- methyl-6-phytylbenzoquinone methyltransferase in determining tocopherol composition in Synechocystis sp. PCC6803. FEBS Lett 511:1–5PubMedCrossRefGoogle Scholar
  37. Staub JM, Maliga P (1995) Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids. Plant J 7:845–848PubMedCrossRefGoogle Scholar
  38. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917PubMedCrossRefGoogle Scholar
  39. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530PubMedCrossRefGoogle Scholar
  40. Traber MG, Sies H (1996) Vitamin E in humans: demand and delivery. Ann Rev Nutr 16:321–347CrossRefGoogle Scholar
  41. Tsegaye Y, Shintani DK, DellaPenna D (2002) Overexpression of the enzyme p-hydroxyphenylpyruvate dioxygenase in Arabidopsis and its relation to tocopherol biosynthesis. Plant Physiol Biochem 40:913–920CrossRefGoogle Scholar
  42. Yabuta Y, Motoki T, Yoshimura K, Takeda T, Ishikawa T, Shigeoka S (2002) Thylakoid-membrane bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress. Plant J 32:915–925PubMedCrossRefGoogle Scholar
  43. Yabuta Y, Tamoi M, Yamamoto K, Tomizawa K, Yokota A, Shigeoka S (2008) Molecular design of photosynthesis-elevated chloroplasts for mass accumulation of a foreign protein. Plant Cell Physiol 49:375–385PubMedCrossRefGoogle Scholar
  44. Zhang GY, Liu RR, Xu G, Zhang P, Li Y, Tang KX, Liang GH, Liu QQ (2012) Increased α-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis γ-tocopherol methyltransferase. Transgenic Res. doi:10.1007/s11248-012-9630-2 Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Yukinori Yabuta
    • 1
  • Hiroyuki Tanaka
    • 2
  • Sahoko Yoshimura
    • 2
  • Akiko Suzuki
    • 2
  • Masahiro Tamoi
    • 2
  • Takanori Maruta
    • 2
    • 3
  • Shigeru Shigeoka
    • 2
  1. 1.School of Agricultural, Biological, and Environmental Sciences, Faculty of AgricultureTottori UniversityTottoriJapan
  2. 2.Department of Advanced Bioscience, Faculty of AgricultureKinki UniversityNaraJapan
  3. 3.Faculty of Life and Environmental ScienceShimane UniversityShimaneJapan

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