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Study of subcellular localization of Glycine max γ-tocopherol methyl transferase isoforms in N. benthamiana

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Abstract

Gamma-tocopherol methyltransferase (γ-TMT) converts γ-toc to α-toc—the rate limiting step in toc biosynthesis. Sequencing results revealed that the coding regions of γ-TMT1 and γ-TMT3 were strongly similar to each other (93% at amino acid level). Based on the differences in the N-terminal amino acids, Glycine max-γ-TMT proteins are categorized into three isoforms: γ-TMT1, 2 and 3. In silico structural analysis revealed the presence of chloroplast transit peptide (cTP) in γ-TMT1 and γ-TMT3 protein. However, other properties of transit peptide like presence of hydrophobic amino acids at the first three positions of N-terminal end and lower level of acidic amino acids were revealed only in γ-TMT3 protein. Subcellular localization of GFP fused γ-TMT1 and γ-TMT3 under 35S promoter was studied in Nicotiana benthamiana using confocal microscopy. Results showed that γ-TMT1 was found in the cytosol and γ-TMT3 was found to be localized both in cytosol and chloroplast. Further the presence γ-TMT3 in chloroplast was validated by quantifying α-tocopherol through UPLC. Thus the present study of cytosolic localization of the both γ-TMT1 and γ-TMT3 proteins and chloroplastic localization of γ-TMT3 will help to reveal the importance of γ-TMT encoded α-toc in protecting both chloroplastic and cell membrane from plant oxidative stress.

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References

  • Arun M, Subramanyam K, Theboral J, Sivanandhan G, Rajesh M, Dev GK, Ganapathi A (2014) Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars. Appl Biochem Biotechnol 172(4):1763–1776

    CAS  PubMed  Google Scholar 

  • Azzi A, Stocker A (2000) Vitamin E: non-antioxidant roles. Prog Lipid Res 39(3):231–255

    CAS  PubMed  Google Scholar 

  • Bergmüller E, Porfirova S, Dörmann P (2003) Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase. Plant Mol Biol 52(6):1181–1190

    PubMed  Google Scholar 

  • Botha FC, Black KG (2000) Sucrose phosphate synthase and sucrose synthase activity during maturation of internodal tissue in sugarcane. Funct Plant Biol 27(1):81–85

    CAS  Google Scholar 

  • Bruce BD (2000) Chloroplast transit peptides: structure, function and evolution. Trends Cell Biol 10(14):40–447

    Google Scholar 

  • Chen DF, Zhang M, Wang YQ, Chen XW (2012) Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed. Biol Plant 56(1):131–134

    CAS  Google Scholar 

  • 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

    CAS  PubMed  Google Scholar 

  • DellaPenna D, Pogson BJ (2006) Vitamin synthesis in plants: tocopherols and carotenoids. Annu Rev Plant Biol 57:711–738

    CAS  PubMed  Google Scholar 

  • DellaPenna D (2005) A decade of progress in understanding vitamin E synthesis in plants. J Plant Physiol 162:729–737

    CAS  PubMed  Google Scholar 

  • Drevon CA (1991) Absorption, transport and metabolism of vitamin E. Free Radic Res Commun 14(4):229–246

    CAS  PubMed  Google Scholar 

  • Dwiyanti MS, Yamada T, Sato M, Abe J, Kitamura K (2011) Genetic variation of γ-tocopherol methyltransferase gene contributes to elevated α-tocopherol content in soybean seeds. BMC Plant Biol 11(1):152

    CAS  PubMed  PubMed Central  Google Scholar 

  • Frigerio L, de Virgilio M, Prada A, Faoro F, Vitale A (1998) Sorting of phaseolin to the vacuole is saturable and requires a short C-terminal peptide. Plant Cell 10(6):1031–1042

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fryer MJ (1992) The antioxidant effects of thylakoid vitamin E (α-tocopherol). Plant Cell Environ 15(4):381–392

    CAS  Google Scholar 

  • Ghimire BK, Seong ES, Lee CO, Lim JD, Lee JG, Yoo JH, Chung IM, Kim NY, Yu CY (2011) Enhancement of alpha-tocopherol content in transgenic Perilla frutescens containing the gamma-TMT gene. Afr J Biotechnol 10:2430–2439

    CAS  Google Scholar 

  • Guo J (2006) Overexpression of VTE1 from Arabidopsis resulting in high vitamin E accumulation and salt stress tolerance increase in tobacco plant. Chin J Appl Environ Biol 12(4):468

    CAS  Google Scholar 

  • Hass CG, Tang S, Leonard S, Traber MG, Miller JF, Knapp SJ (2006) Three non-allelic epistatically interacting methyltransferase mutations produce novel tocopherol (vitamin E) profiles in sunflower. Theor Appl Genet 113(5):767–782

    CAS  PubMed  Google Scholar 

  • Horvath G, Wessjohann L, Bigirimana J, Jansen M, Guisez Y, Caubergs R, Horemans N (2006) Differential distribution of tocopherols and tocotrienols in photosynthetic and non-photosynthetic tissues. Phytochemistry 67(12):1185–1195

    CAS  PubMed  Google Scholar 

  • Hosomi A, Arita M, Sato Y, Kiyose C, Ueda T, Igarashi O, Inoue K (1997) Affinity for α-tocopherol transfer protein as a determinant of the biological activities of vitamin E analogs. FEBS Lett 409(1):105–108

    CAS  PubMed  Google Scholar 

  • Hofius D, Hajirezaei MR, Geiger M, Tschiersch H, Melzer M, Sonnewald U (2004) RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants. Plant Physiol 135:1256–1268

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jin S, Daniell H (2014) Expression of γ-tocopherol methyltransferase in chloroplasts results in massive proliferation of the inner envelope membrane and decreases susceptibility to salt and metal-induced oxidative stresses by reducing reactive oxygen species. Plant Biotechnol J 12(9):1274–1285

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lee DW, Lee S, Lee GJ, Lee KH, Kim S, Cheong GW, Hwang I (2006) Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco. Plant Physiol 140(2):466–483

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lehtimäki N, Koskela MM, Dahlström KM, Pakula E, LintalaM SM, Hippler M, Hanke GT, RokkaA BN, Salminen TA (2014) Posttranslational modifications of FERREDOXIN-NADP+ OXIDOREDUCTASE in Arabidopsis chloroplasts. Plant Physiol 166(4):1764–1776

    PubMed  PubMed Central  Google Scholar 

  • Leister D (2003) Chloroplast research in the genomic age. Trends Genet 19(1):47–56

    CAS  PubMed  Google Scholar 

  • Lichtenthaler HK, Buschmann C, Döll M, Fietz HJ, Bach T, Kozel U, Rahmsdorf U (1981) Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves. Photosynth Res 2(2):115–141

    CAS  PubMed  Google Scholar 

  • López-Juez E (2006) Plastid biogenesis, between light and shadows. J Exp Bot 58(1):11–26

    PubMed  Google Scholar 

  • Mène-Saffrané L, Jones AD, DellaPenna D (2010) Plastochromanol-8 and tocopherols are essential lipid-soluble antioxidants during seed desiccation and quiescence in Arabidopsis. Proc Natl Acad Sci 107(41):17815–17820

    PubMed  Google Scholar 

  • Millar AH, Whelan J, Small I (2006) Recent surprises in protein targeting to mitochondria and plastids. Curr Opin Plant Biol 9(6):610–615

    CAS  PubMed  Google Scholar 

  • Miras S, Salvi D, Piette L, Seigneurin BD, Grunwald D, Reinbothe C, Joyard J, Reinbothe S, Rolland N (2007) Toc159-and Toc75-independent import of a transit sequence-less precursor into the inner envelope of chloroplasts. J Biol Chem 282(40):29482–29492

    CAS  PubMed  Google Scholar 

  • Munné-Bosch S (2005) The role of α-tocopherol in plant stress tolerance. J Plant Physiol 162(7):743–748

    PubMed  Google Scholar 

  • Munné-Bosch S, Alegre L (2002a) The function of tocopherols and tocotrienols in plants. Crit Rev Plant Sci 21(1):31–57

    Google Scholar 

  • Munné-Bosch S, Alegre L (2002b) Plant aging increases oxidative stress in chloroplasts. Planta 214(4):608–615

    PubMed  Google Scholar 

  • Munné-Bosch S, Schwarz K, Alegre L (1999) Enhanced formation of α-tocopherol and highly oxidized abietane diterpenes in water-stressed rosemary plants. Plant Physiol 121(3):1047–1052

    PubMed  PubMed Central  Google Scholar 

  • Munné-Bosch S, Weiler EW, Alegre L, Müller M, Düchting P, Falk J (2007) α-Tocopherol may influence cellular signaling by modulating jasmonic acid levels in plants. Planta 225(3):681–691

    PubMed  Google Scholar 

  • Patron NJ, Waller RF (2007) Transit peptide diversity and divergence: a global analysis of plastid targeting signals. BioEssays 29(10):1048–1058

    CAS  PubMed  Google Scholar 

  • Richly E, Leister D (2004) An improved prediction of chloroplast proteins reveals diversities and commonalities in the chloroplast proteomes of Arabidopsis and rice. Gene 329:11–16

    CAS  PubMed  Google Scholar 

  • Russin WA, Evert RF, Vanderveer PJ, Sharkey TD, Briggs SP (1996) Modification of a specific class of plasmodesmata and loss of sucrose export ability in the sucrose export defective1 maize mutant. Plant Cell 8(4):645–658

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sakamoto W, Miyagishima SY, Jarvis P (2008) Chloroplast biogenesis: control of plastid development, protein import, division and inheritance. Arabidopsis book 6, e0110

  • Sattler SE, Gilliland LU, Magallanes LM, Pollard M, DellaPenna D (2004) Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination. Plant Cell 16(6):1419–1432

    CAS  PubMed  PubMed Central  Google Scholar 

  • Schneider C (2005) Chemistry and biology of vitamin E. Mol Nutr Food Res 49(1):7–30

    CAS  PubMed  Google Scholar 

  • Shintani D, DellaPenna D (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282(5396):2098–2100

    CAS  PubMed  Google Scholar 

  • Sies H, Menck CF (1992) Singlet oxygen induced DNA damage. Mutat Res DNAging 275(3–6):367–375

    CAS  Google Scholar 

  • Skovsen E, Snyder JW, Lambert JD, Ogilby PR (2005) Lifetime and diffusion of singlet oxygen in a cell. J Phys Chem B 109(18):8570–8573

    CAS  PubMed  Google Scholar 

  • Soll J, Schultz G, Joyard J, Douce R, Block MA (1985) Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts. Arch Biochem Biophys 238(1):290–299

    CAS  PubMed  Google Scholar 

  • Stocker A, Azzi A (2000) Tocopherol-binding proteins: their function and physiological significance. Antioxid Redox Signal 2(3):397–404

    CAS  PubMed  Google Scholar 

  • Sun S, Zeng H, Robinson DB, Raoux S, Rice PM, Wang SX, Li G (2004) Monodisperse mfe2o4 (m = fe, co, mn) nanoparticles. J Am Chem Soc 126(1):273–279

    CAS  PubMed  Google Scholar 

  • Szymańska R, Kruk J (2008a) γ-Tocopherol dominates in young leaves of runner bean (Phaseolus coccineus) under a variety of growing conditions: the possible functions of γ-tocopherol. Phytochemistry 69:2142–2148

    PubMed  Google Scholar 

  • Szymańska R, Kruk J (2008b) Tocopherol content and isomers' composition in selected plant species. Plant Physiol Biochem 46(1):29–33

    PubMed  Google Scholar 

  • Tavva VS, 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(1):61–70

    CAS  PubMed  Google Scholar 

  • Taylor SL, Lamden MP, Tappel AL (1976) Sensitive fluorometric method for tissue tocopherol analysis. Lipids 11(7):530–538

    CAS  PubMed  Google Scholar 

  • Traber MG, Sies H (1996) Vitamin E in humans: demand and delivery. Annu Rev Nutr 16(1):321–347

    CAS  PubMed  Google Scholar 

  • Van Wijk KJ (2004) Plastid proteomics. Plant Physiol Biochem 42(12):963–977

    PubMed  Google Scholar 

  • Villarejo A, Burén S, Larsson S, Déjardin A, Monné M, Rudhe C, Von Heijne G (2005) Evidence for a protein transported through the secretory pathway en route to the higher plant chloroplast. Nat Cell Biol 7(12):1224

    PubMed  Google Scholar 

  • Vinutha T, Bansal N, Prashat GR, Krishnan V, Kumari S, Dahuja A, Sachdev A, Rai RD (2015) Three dimensional structure prediction and in silico functional analysis of gamma tocopherol methyl transferase from Glycine max. Int J Bioinform Res 6:289–299

    Google Scholar 

  • Vinutha T, Bansal N, Kumari K, Rama Prashat G, Sreevathsa R, Krishnan V, Kumari S, Dahuja A, Lal S, Sachdev A, Praveen S (2017) Comparative analysis of tocopherol biosynthesis genes and its transcriptional regulation in soybean seeds. J Agric Food Chem 65(50):11054–11064

    Google Scholar 

  • Von Heijne G, Steppuhn J, Hermann SG (1989) Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 80:535–545

    Google Scholar 

  • Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress. Plant Cell 6:251–264

    CAS  PubMed  PubMed Central  Google Scholar 

  • Yusuf MA, Sarin NB (2007) Antioxidant value addition in human diets: genetic transformation of Brassica juncea with γ-TMT gene for increased α-tocopherol content. Transgenic Res 16(1):109–113

    CAS  PubMed  Google Scholar 

  • Zybailov B, Rutschow H, Friso G, Rudella A, Emanuelsson O, Sun Q, van Wijk KJ (2008) Sorting signals, N-terminal modifications and abundance of the chloroplast proteome. PLoS One 3(4):1994

    Google Scholar 

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Acknowledgements

The authors are thankful for financial support mainly from DST-SERB, Receipt Number-No. SB/389 YS/LS-100/2013 and also thankful for great support from ICAR- IARI Fund.

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Authors and Affiliations

  1. Division of Biochemistry, ICAR-Indian Agricultural Research Institue, New Delhi, 110012, India

    Khushboo Kumari, Navita Bansal, Sweta Kumari, Anil Dahuja, Archana Sachdev, Shelly Praveen & T. Vinutha

  2. Amity University, Noida, Uttar Pradesh, 201313, India

    Khushboo Kumari, Monika Prakash Rai & Navita Bansal

  3. Division of Genetics, ICAR-Indian Agricultural Research Institute, New Delhi, 110012, India

    G. Rama Prashat

  4. ICAR-National Institute for Plant Biotechnology, New Delhi, 110012, India

    Rohini Srivathsa

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  1. Khushboo Kumari
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Kumari, K., Rai, M.P., Bansal, N. et al. Study of subcellular localization of Glycine max γ-tocopherol methyl transferase isoforms in N. benthamiana. 3 Biotech 10, 110 (2020). https://doi.org/10.1007/s13205-020-2086-9

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