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.
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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):437–443
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
Bergmüller E, Porfirova S, Dörmann P (2003) Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase. Plant Mol Biol 52:1181–1190
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
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
Chinese feed industry association (2008) Chinese feed industry yearbook 2006/2007. Chinese Commercial Press, Shanghai, pp 144–145
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):16–22
Chung YK, Mahan DC, Lepine AJ (1992) Efficacy of dietary D-a-tocopherol and DL-a-tocopheryl acetate for weanling pigs. J Anim Sci 70:2485–2492
Clough S, Bent A (1998) Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743
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
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
D’Harlingue A, Camara B (1985) Plastid enzymes of terpenoid biosynthesis. J Biol Chem 260(28):15200–15203
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
Evans H, Bishop K (1922) On the existence of a hitherto unrecognized dietary factor essential for reproduction. Science 56:650–651
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
Grusak M, DellaPenna D (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu Rev Plant Physiol Plant Mol Biol 50:133–161
Herbers K (2003) Vitamin production in transgenic plants. J Plant Physiol 160(7):821–829. https://doi.org/10.1078/0176-1617-01024
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
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
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):1–7
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
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
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
Ministry of agriculture, PRC (2004a) Agricultural industry standards, PRC NY/T 33-2004 Feeding standard of chicken. Chinese Agricultural Press, Beijing, pp 3–8
Ministry of agriculture, PRC (2004b) Agricultural industry standards, PRC NY/T 65-2004 Feeding standard of swine. Chinese Agricultural Press, Beijing, pp 4–5
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
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
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 70:2542–2550
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
Shintani D, DellaPenna D (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282:2098–2111
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):715–720
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
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
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 197–213
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
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
Wong J, Lambert R, Tadmor Y, Rocheford T (2003) QTLs associated with accumulation of tocopherols in maize. Crop Sci 43:2257–2266
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 12:201–214
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):930
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
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
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
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).
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Zhang, L., Luo, Y., Liu, B. et al. Overexpression of the maize γ-tocopherol methyltransferase gene (ZmTMT) increases α-tocopherol content in transgenic Arabidopsis and maize seeds. Transgenic Res 29, 95–104 (2020). https://doi.org/10.1007/s11248-019-00180-z
- α-/γ-Tocopherol ratio
- γ-Tocopherol methyltransferase (γ-TMT)
- Zea mays