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

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.

Analysis of γ-Tocopherol methyl transferase3 promoter activity and study of methylation patterns of the promoter and its gene body.

Soybeans are known for its good source of protein (40%), oil (20%) and also serve as a source of nutraceutical compounds including tocopherols (toc). To know the molecular basis of differential α-toc accumulation in two contrasting soybean genotypes: DS74 (low α-toc - 1.36 µg/g and total-toc -29.72 µg/g) and Bragg (high α-toc - 10.48 µg/g and total-toc 178.91 µg/g), the analysis of γ-TMT3 promoter activity and its methylation patterns were carried out. The sequencing results revealed nucleotide variation between Bragg:γ-TMT3-P and DS74:γ-TMT3-P, however none of the variations were found in core-promoter region or in cis-elements. The histochemical GUS assay revealed higher promoter activity of Bragg:γ-TMT3-P than that of DS74:γ-TMT3-P and correlated with significantly higher and lower (P < 0.05) expression of γ-TMT3 gene respectively. To know the molecular basis of differential accumulation of α-toc in these contrasting soybean genotypes, the DNA methylation pattern of γ-TMT3 gene body and its promoter was studied in both varieties. The results showed higher percentage (62.5%) of methylation in DS74:γ-TMT3-P than in Bragg:γ-TMT3-P (50%). Out of all the methylation sites in the promoter region, one of methylation site was found at CAAT box (-190 bp) of DS74:γ-TMT3-P. Further gene body methylation patterns revealed lowest % (40%) of CG methylation in DS74:γ-TMT3 gene as compared to Bragg:γ-TMT3 (64.2%). Thus our study revealed that, expression of γ-TMT3 gene was influenced by its promoter activity and methylation patterns in cis-elements of γ-TMT3 promoter and gene body. This study will help us to understand the possible role of methylation and promoter activity in determining the α-toc content in soybean seeds.