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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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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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Correspondence to Shelly Praveen or T. Vinutha.

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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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Keywords

  • α-Tocopherol
  • Glycine max
  • γ-TMT3
  • γ-TMT1
  • Sub-cellular localization
  • Confocal microscopy