Advertisement

Overexpression of the maize γ-tocopherol methyltransferase gene (ZmTMT) increases α-tocopherol content in transgenic Arabidopsis and maize seeds

  • Lan Zhang
  • Yanzhong Luo
  • Bin Liu
  • Liang Zhang
  • Wei Zhang
  • Rumei Chen
  • Lei WangEmail author
Original Paper
  • 49 Downloads

Abstract

The vitamin E family includes tocopherols and tocotrienols, which are essential lipid-soluble antioxidants necessary for human and livestock health. The seeds of many plant species, including maize, have high gamma (γ)-tocopherol but low alpha (α)-tocopherol contents; however, α-tocopherol is the most effective antioxidant. Therefore, it is necessary to optimize the tocopherol composition in plants. α-Tocopherol is synthesized from γ-tocopherol by γ-tocopherol methyltransferase (γ-TMT, VTE4) in the final step of the tocopherol biosynthetic pathway. In the present study, the full-length coding sequence (CDS) of γ-TMT was isolated from Zea mays, named ZmTMT. The ZmTMT CDS was 1059 bp in size, encoding 352 amino acids. Recombinant ZmTMT was expressed in Escherichia coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. A comparison of enzyme activities showed that the activity of ZmTMT was higher than that of GmTMT2a (Glycine max) and AtTMT (Arabidopsis thaliana). Overexpression of ZmTMT increased the α-tocopherol content 4–5-fold in transgenic Arabidopsis and around 6.5-fold in transgenic maize kernels, and increased the α-/γ-tocopherol ratio to approximately 15 and 17, respectively. These results show that it is feasible to overexpress ZmTMT to optimize the tocopherol composition in maize; such a corn product might be useful in the feed industry in the near future.

Keywords

α-Tocopherol α-/γ-Tocopherol ratio γ-Tocopherol methyltransferase (γ-TMT) Zea mays 

Notes

Acknowledgements

We thank Dr. Ling Jiang at Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, for manuscript revise and valuable discussions. This work was supported by the National Special Program for GMO Development of China (Grant Number 2016ZX08003-002).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11248_2019_180_MOESM1_ESM.docx (134 kb)
Supplementary material 1 (DOCX 134 kb)

References

  1. Arita M, Sato Y, Miyata A, Tanabe T, Takahashi E, Kayden HJ, Arai H, Inoue K (1995) Human α-tocopherol transfer protein: cDNA cloning, expression and chromosomal localization. Biochem J 306(2):437–443CrossRefGoogle Scholar
  2. Arun M, Subramanyam K, Theboral J, Sivanandhan G, Rajesh M, Kapil Dev G, Jaganath B, Manickavasagam M, Girija S, Ganapathi A (2014) Transfer and targeted overexpression of gamma-tocopherol methyltransferase (gamma-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars. Appl Biochem Biotechnol 172(4):1763–1776.  https://doi.org/10.1007/s12010-013-0645-9 CrossRefPubMedGoogle Scholar
  3. Bergmüller E, Porfirova S, Dörmann P (2003) Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase. Plant Mol Biol 52:1181–1190CrossRefGoogle Scholar
  4. Chander S, Guo Y, Yang X, Yan J, Zhang YR, Song T, Li J (2008) Genetic dissection of tocopherol content and composition in maize grain using quantitative trait loci analysis and the candidate gene approach. Mol Breeding 22(3):353–365.  https://doi.org/10.1007/s11032-008-9180-8 CrossRefGoogle Scholar
  5. Chen R, Xue G, Chen P, Yao B, Yang W, Ma Q, Fan Y, Zhao Z, Tarczynski MC, Shi J (2008) Transgenic maize plants expressing a fungal phytase gene. Transgenic Res 17(4):633–643.  https://doi.org/10.1007/s11248-007-9138-3 CrossRefPubMedGoogle Scholar
  6. Chinese feed industry association (2008) Chinese feed industry yearbook 2006/2007. Chinese Commercial Press, Shanghai, pp 144–145Google Scholar
  7. Cho EA, Lee CA, Kim YS, Baek SH, Reyes BG, Yun SJ (2005) Expression of γ-tocopherol methyltransferase transgene improves tocopherol composition in lettuce (Latuca sativa L.). Mol Cells 19(1):16–22PubMedGoogle Scholar
  8. Chung YK, Mahan DC, Lepine AJ (1992) Efficacy of dietary D-a-tocopherol and DL-a-tocopheryl acetate for weanling pigs. J Anim Sci 70:2485–2492CrossRefGoogle Scholar
  9. Clough S, Bent A (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743CrossRefGoogle Scholar
  10. Collakova E, DellaPenna D (2003) Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol 131(2):632–642.  https://doi.org/10.1104/pp.015222 CrossRefPubMedPubMedCentralGoogle Scholar
  11. DellaPenna D (2005) Progress in the dissection and manipulation of vitamin E synthesis. Trends Plant Sci 10(12):574–579.  https://doi.org/10.1016/j.tplants.2005.10.007 CrossRefPubMedGoogle Scholar
  12. D’Harlingue A, Camara B (1985) Plastid enzymes of terpenoid biosynthesis. J Biol Chem 260(28):15200–15203PubMedGoogle Scholar
  13. Diepenbrock CH, Kandianis CB, Lipka AE, Magallanes-Lundback M, Vaillancourt B, Gongora-Castillo E, Wallace JG, Cepela J, Mesberg A, Bradbury PJ, Ilut DC, Mateos-Hernandez M, Hamilton J, Owens BF, Tiede T, Buckler ES, Rocheford T, Buell CR, Gore MA, DellaPenna D (2017) Novel loci underlie natural variation in vitamin E levels in Maize Grain. Plant Cell 29(10):2374–2392.  https://doi.org/10.1105/tpc.17.00475 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Evans H, Bishop K (1922) On the existence of a hitherto unrecognized dietary factor essential for reproduction. Science 56:650–651CrossRefGoogle Scholar
  15. Falk J, Andersen G, Kernebeck B, Krupinska K (2003) Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of the vitamin E content in seeds but not in leaves1. FEBS Lett 540(1–3):35–40.  https://doi.org/10.1016/s0014-5793(03)00166-2 CrossRefPubMedGoogle Scholar
  16. Grusak M, DellaPenna D (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu Rev Plant Physiol Plant Mol Biol 50:133–161CrossRefGoogle Scholar
  17. Herbers K (2003) Vitamin production in transgenic plants. J Plant Physiol 160(7):821–829.  https://doi.org/10.1078/0176-1617-01024 CrossRefPubMedGoogle Scholar
  18. Hunter SC, Cahoon EB (2007) Enhancing vitamin E in oilseeds: unraveling tocopherol and tocotrienol biosynthesis. Lipids 42(2):97–108.  https://doi.org/10.1007/s11745-007-3028-6 CrossRefPubMedGoogle Scholar
  19. Karunanandaa B, Qi Q, Hao M, Baszis SR, Jensen PK, Wong YH, Jiang J, Venkatramesh M, Gruys KJ, Moshiri F, Post-Beittenmiller D, Weiss JD, Valentin HE (2005) Metabolically engineered oilseed crops with enhanced seed tocopherol. Metab Eng 7(5–6):384–400.  https://doi.org/10.1016/j.ymben.2005.05.005 CrossRefPubMedGoogle Scholar
  20. Kim Y, Seo H, Park T, Baek S, Shin W, Kim H, Kim J, Choi Y, Yun S (2005) Enhanced biosynthesis of α-tocopherol in transgenic soybean by introducing γ-TMT gene. Journal of Plant Biotechnology 7(3):1–7Google Scholar
  21. 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 Organ Cult 92(1):47–54.  https://doi.org/10.1007/s11240-007-9301-9 CrossRefGoogle Scholar
  22. Li Q, Yang X, Xu S, Cai Y, Zhang D, Han Y, Li L, Zhang Z, Gao S, Li J, Yan J (2012) Genome-wide association studies identified three independent polymorphisms associated with alpha-tocopherol content in maize kernels. PLoS ONE 7(5):e36807.  https://doi.org/10.1371/journal.pone.0036807 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Lipka AE, Gore MA, Magallanes-Lundback M, Mesberg A, Lin H, Tiede T, Chen C, Buell CR, Buckler ES, Rocheford T, DellaPenna D (2013) Genome-wide association study and pathway-level analysis of tocochromanol levels in maize grain. G3 (Bethesda) 3(8):1287–1299.  https://doi.org/10.1534/g3.113.006148 CrossRefGoogle Scholar
  24. Ministry of agriculture, PRC (2004a) Agricultural industry standards, PRC NY/T 33-2004 Feeding standard of chicken. Chinese Agricultural Press, Beijing, pp 3–8Google Scholar
  25. Ministry of agriculture, PRC (2004b) Agricultural industry standards, PRC NY/T 65-2004 Feeding standard of swine. Chinese Agricultural Press, Beijing, pp 4–5Google Scholar
  26. Munne-Bosch S, Falk J (2004) New insights into the function of tocopherols in plants. Planta 218(3):323–326.  https://doi.org/10.1007/s00425-003-1126-0 CrossRefPubMedGoogle Scholar
  27. Naqvi S, Farre G, Zhu C, Sandmann G, Capell T, Christou P (2011) Simultaneous expression of Arabidopsis rho-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content. Transgenic Res 20(1):177–181.  https://doi.org/10.1007/s11248-010-9393-6 CrossRefPubMedGoogle Scholar
  28. Roquet J, Nockels CF, Papas AM (1992) Cattle blood plasma and red blood cell a-tocopherol levels in response to different chemical forms and routes of administration of vitamin E. J Anim Sci 70:2542–2550CrossRefGoogle Scholar
  29. 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. Phytochemistry 72(4–5):321–329.  https://doi.org/10.1016/j.phytochem.2010.12.013 CrossRefPubMedGoogle Scholar
  30. Shintani D, DellaPenna D (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282:2098–2111CrossRefGoogle Scholar
  31. Soil J, Schultz G (1979) Comparison of geranylgeranyl and phytyl substituted methylquinols in the tocopherol synthesis of spinach chloroplasts. Biochem Biophys Res Commun 91(3):715–720CrossRefGoogle Scholar
  32. Streatfield SJ, Bray J, Love RT, Horn ME, Lane JR, Drees CF, Egelkrout EM, Howard JA (2010) Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production. GM Crops 1(3):162–172.  https://doi.org/10.4161/gmcr.1.3.12816 CrossRefPubMedGoogle Scholar
  33. Tavva V, Kim Y, Kagan I, Dinkins R, Kim K, Collins G (2007) Increased alpha-tocopherol content in soybean seed overexpressing the Perilla frutescens gamma-tocopherol methyltransferase gene. Plant Cell Rep 26(1):61–70.  https://doi.org/10.1007/s00299-006-0218-2 CrossRefPubMedGoogle Scholar
  34. Tomes D, Ross M, Songstad D (1995) Direct DNA transfer into intact plant cells via microprojectile Bombardment. In: Gamborg O, Phillips G (eds) Plant cell, tissue and organ culture. Springer Lab Manual. Springer, Berlin, Heidelberg, pp 197–213CrossRefGoogle Scholar
  35. Van Eenennaam AL, Lincoln K, Durrett TP, Valentin HE, Shewmaker CK, Thorne GM, Jiang J, Baszis SR, Levering CK, Aasen ED, Hao M, Stein JC, Norris SR, Last RL (2003) Engineering vitamin E content: from Arabidopsis mutant to soy oil. Plant Cell 15(12):3007–3019.  https://doi.org/10.1105/tpc.015875 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Wang H, Xu S, Fan Y, Liu N, Zhan W, Liu H, Xiao Y, Li K, Pan Q, Li W, Deng M, Liu J, Jin M, Yang X, Li J, Li Q, Yan J (2018) Beyond pathways: genetic dissection of tocopherol content in maize kernels by combining linkage and association analyses. Plant Biotechnol J 16(8):1464–1475.  https://doi.org/10.1111/pbi.12889 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Wong J, Lambert R, Tadmor Y, Rocheford T (2003) QTLs associated with accumulation of tocopherols in maize. Crop Sci 43:2257–2266CrossRefGoogle Scholar
  38. Xu S, Zhang D, Cai Y, Zhou Y, Trushar S, Farhan A, Li Q, Li Z, Wang W, Li J, Yang X, Yan J (2012) Dissecting tocopherols content in maize (Zea mays L.), using two segregating populations and high-density single nucleotide polymorphism markers. BMC Plant Biol 12:201–214CrossRefGoogle Scholar
  39. Yoshida H, Yusin M, Kuhlenkamp J, Kaplowitz N (1990) The purification and characterization of tocopherol binding protein tbp in rat liver. Hepatology 12(4 PART 2):930Google Scholar
  40. Zhang L, Wang X, Bi Y, Zhang C, Fan Y, Wang L (2008) Isolation and functional analysis of transcription factor GmWRKY57B from soybean. Sci Bull 53(22):3538–3545.  https://doi.org/10.1007/s11434-008-0483-2 CrossRefGoogle Scholar
  41. Zhang C, Cahoon RE, Hunter SC, Chen M, Han J, Cahoon EB (2013a) Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production. Plant J 73(4):628–639.  https://doi.org/10.1111/tpj.12067 CrossRefPubMedGoogle Scholar
  42. Zhang L, Luo Y, Zhu Y, Zhang L, Zhang W, Chen R, Xu M, Fan Y, Wang L (2013b) GmTMT2a from soybean elevates the alpha-tocopherol content in corn and Arabidopsis. Transgenic Res 22(5):1021–1028.  https://doi.org/10.1007/s11248-013-9713-8 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.National Key Facility of Crop Gene Resources and Genetic Improvement, Biotechnology Research InstituteChinese Academy of Agricultural SciencesBeijingChina

Personalised recommendations