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Expression of tobacco tocopherol cyclase in rice regulates antioxidative defense and drought tolerance


Tocopherols (α-, β-, γ-, and δ-tocopherol) represent a group of lipophilic antioxidants that are synthesized only by photosynthetic organisms. It is widely believed that the main functions of tocopherols are protection of pigments and proteins of photosystem and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species. In the present study, we report on the cloning and characterization of NtTC, which is a tocopherol cyclase (TC) ortholog isolated from tobacco. To enhance tocopherol contents, we generated independent transgenic rice events in expressing NtTC or NtTC along with Perilla γ-tocopherol methyltransferase genes. The transgenic TC line significantly increased α-, total tocopherol, total glutathione, and total antioxidant status activity levels compared with the wild type. Furthermore, TC rice plants showed higher tolerance to drought than wild-type rice plants. On the basis of these studies, we concluded that overexpression of NtTC could increase the tolerance to drought stress and that the increase in tocopherol affects cellular signaling and antioxidant defense of plants in response to drought.

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Reactive oxygen species


Tocopherol cyclase




Total antioxidant status


Reverse transcription


Gas chromatography


Time-of-flight mass spectrometry


Internal standard


Open reading frame


γ-Tocopherol methyltransferase


Arabidopsis thaliana vitamin E biosynthetic gene


  1. Abbasi AR, Hajirezaei M, Hofius D, Sonnewald U, Voll LM (2007) Specific roles of α- and γ-tocopherol in abiotic stress responses of transgenic tobacco. Plant Physiol 143:1720–1738

  2. Alscher RG, Erturk N, Heath LS (2002) Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. J Exp Bot 53:1331–1341

  3. Azzi A (2007) Molecular mechanism of alpha-tocopherol action. Free Radic Biol Med 43:16–21

  4. Bergmüller E, Porfirova S, Dörmann P (2003) Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase. Plant Mol Biol 52:1181–1190

  5. Bramley PM, Elmadfa I, Kafatos A, Kelly FJ, Manios Y, Roxborough HE, Schuch W, Sheehy PJA, Wagner KH (2000) Vitamin E. J Sci Food Agric 80:913–938

  6. Collakova E, DellaPenna D (2003) The role of homogentisate phytyltransferase and other tocopherol pathway enzymes in the regulation of tocopherol synthesis during abiotic stress. Plant Physiol 133:930–940

  7. d’Harlingue A, Camara B (1985) Plastid enzymes of terpenoid biosynthesis. Purification and characterization of gamma-tocopherol methyltransferase from Capsicum chromoplasts. J Biol Chem 260:15200–15203

  8. Foyer CH, Noctor G (2003) Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plant 119:355–364

  9. Grusak MA, DellaPenna D (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Ann Rev Plant Physiol Plant Mol Biol 50:133–161

  10. Havaux M, Eymery F, Porfirova S, Rey P, Dörmann P (2005) Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana. Plant Cell 17:3451–3469

  11. Hofius D, Sonnewald U (2003) Vitamin E biosynthesis: biochemistry meets cell biology. Trends Plant Sci 8:6–8

  12. Jang IC, Choi WB, Lee KH, Song SI, Nahm BH, Kim JK (2002) High-level and ubiquitous expression of the rice cytochrome c gene OsCc1 and its promoter activity in transgenic plants provides a useful promoter for transgenesis of monocots. Plant Physiol 129:1473–1481

  13. Jeong SC, Pack IS, Cho EY, Youk ES, Park S, Yoon WK, Kim CG, Choi YD, Kim JK, Kim HM (2007) Molecular analysis and quantitative detection of a transgenic rice line expressing a bifunctional fusion TPSP. Food Control 18:1434–1442

  14. Jiang Q, Elson-Schwab I, Courtemanche C, Ames BN (2000) Gamma-Tocopherol and its major metabolite, in contrast to alpha-tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells. Proc Natl Acad Sci USA 97:11494–11499

  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 137:713–723

  16. Kempna P, Reiter E, Arock M, Azzi A, Zingg JM (2004) Inhibition of HMC-1 mast cell proliferation by vitamin E: Involvement of the protein kinase B pathway. J Biol Chem 279:50700–50709

  17. Koch M, Lemke R, Heise KP, Mock HP (2003) Characterization of γ-tocopherol methyltransferases from Capsicum annum L and Arabidopsis thaliana. Eur J Biochem 270:84–92

  18. Ledford HK, Niyogi KK (2005) Singlet oxygen and photo-oxidative stress management in plants and algae. Plant Cell Environ 28:1037–1045

  19. 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 Tiss Organ Cult 92:47–54

  20. Lee K, Yi BY, Kim KH, Kim JB, Suh SC, Woo HJ, Shin KS, Kweon SJ (2011) Development of efficient transformation protocol for soybean (Glycine max L.) and characterization of transgene expression after Agrobacterium-mediated gene transfer. J Korean Soc Appl Biol Chem 54:37–45

  21. Liebler DC, Burr JA (1992) Oxidation of vitamin E during iron-catalyzed lipid peroxidation: evidence for electron-transfer reactions of the tocopheroxyl radical. Biochemistry 31:8278–8284

  22. Liebler DC, Kling DS, Reed DJ (1986) Antioxidant protection of phospholipid bilayers by α-tocopherol. Control of α-tocopherol status and lipid peroxidation by ascorbic acid and glutathione. J Biol Chem 261:12114–12119

  23. Machlin LJ, Filipski R, Nelson J, Horn LR, Brin M (1977) Effects of a prolonged vitamin E deficiency in rat. J Nutr 107:1200–1208

  24. Miller JN, Rice-Evans C, Davies MJ, Gopinathan V, Milner A (1993) A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci 84:404–412

  25. Munné-Bosch S (2005) The role of α-tocopherol in plant stress tolerance. J Plant Physiol 162:743–748

  26. Oh SJ, Song SI, Kim YS, Jang HJ, Kim SY, Kim M, Kim YK, Nahm BH, Kim JK (2005) Arabidopsis CBF3/DREB1A and ABF3 in transgenic rice increased tolerance to abiotic stress without stunting growth. Plant Physiol 138:341–351

  27. Park SY, Lee SM, Lee JH, Ko HS, Kweon SJ, Suh SC, Shin KS, Kim JK (2012) Compositional comparative analysis between insect-resistant rice (Oryza sativa L.) with a synthetic cry1Ac gene and its non-transgenic counterpart. Plant Biotechnol Rep 6:29–37

  28. Porfirova S, Bergmuller E, Tropf S, Lemke R, Doermann 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 99:12495–12500

  29. Ricciarelli R, Tasinato A, Clement S, Ozer NK, Boscoboinik D, Azzi A (1998) Alpha-Tocopherol specifically inactivates cellular protein kinase C alpha by changing its phosphorylation state. Biochem J 334:243–249

  30. Rimbach G, Minihane AM, Majewicz J, Fischer A, Pallauf J, Virgli F, Weinberg PD (2002) Regulation of cell signalling by vitamin E. Proc Nutr Soc 61:415–425

  31. Schneider C (2005) Chemistry and biology of vitamin E. Mol Nutr Food Res 49:7–30

  32. Shin KS, Lee JH, Lim MH, Woo HJ, Qin Y, Suh SC, Kweon SJ, Cho HS (2013) Qualitative and quantitative PCR detection of insect-resistant genetically modified rice Agb0101 developed in korea. J Plant Biotechnol 40:18–26

  33. Shintani D DellaPenna, DellaPenna D (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282:2098–2100

  34. Sohn SI, Kim YH, Cho JH, Kim JK, Lee JY (2006) An efficient selection scheme for Agrobacterium-mediated co-transformation of rice using two selective marker genes hpt and bar. Korean J Breed 38:173–179

  35. Tavva VS, Kim YH, Kagan IA, Dinkins RD, Kim KH, Collins GB (2007) Increased alpha-tocopherol content in soybean seed overexpressing the Perilla frutescens gamma-tocopherol methyltransferase gene. Plant Cell Rep 26:61–70

  36. Wefers H, Sies H (1988) The protection of ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin E. Eur J Biochem 174:353–357

  37. Xu X, Xie G, He L, Zhang J, Xu X, Qian R, Liang G, Liu JH (2013) Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco. Plant Cell Tiss Organ Cult 114:161–169

  38. Yabuta Y, Tanaka H, Yoshimura S, Suzuki A, Tamoi M, Maruta T, Shigeoka S (2013) Improvement of vitamin E quality and quantity in tobacco and lettuce by chloroplast genetic engineering. Transgenic Res 22:391–402

  39. Yokota T, Igarashi K, Uchihara T, Jishage K, Tomita H, Inaba A, Li Y, Arita M, Suzuki H, Mizusawa H, Arai H (2001) Delayed-onset ataxia in mice lacking alpha-tocopherol transfer protein: Model for neuronal degeneration caused by chronic oxidative stress. Proc Natl Acad Sci USA 98:15185–15190

  40. Yusuf MA, Sarin NB (2007) Antioxidant value addition in human diets: genetic transformation transformation of Brassica juncea with gamma-TMT gene for increased alpha-tocopherol content. Transgenic Res 16:109–113

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We gratefully thank Kyung-Hwan Kim and Kijong Lee for kindly providing us the pC3301-TMT plasmid containing the γ-TMT gene. This study was supported by a grant from Research Program for Agricultural Science & Technology Development (Project No. PJ008545), National Academy of Agricultural Science and the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ008021), Rural Development Administration, Republic of Korea.

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Correspondence to Hee-Jong Woo.

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All authors contributed equally to this work.

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Woo, H., Sohn, S., Shin, K. et al. Expression of tobacco tocopherol cyclase in rice regulates antioxidative defense and drought tolerance. Plant Cell Tiss Organ Cult 119, 257–267 (2014).

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  • Transgenic rice
  • Tocopherols
  • Drought tolerance
  • Tocopherol cyclase
  • Antioxidant