Γ-tocopherol Methyltransferase Gene Research Articles

γ-tocopherol methyltransferase gene sequencing and SNP discovery associated with olive oil quality.

γ-tocophérol methyltransferase (GTMT), a key enzyme in the tocopherols biosynthesis pathway, is involved in the conversion of δ- and l'γ-tocophérol to β- et l'α-tocophérol, respectively. In fact, it plays an important role in the α-tocopherol composition and the quality of olive oil. A total of 14 olive tree cultivars (Olea Europaea L.) were chosen and used in this study. They were sampled from different regions of Tunisia. Four cultivars from four Mediterranean regions (Greece, Algeria, Morocco, and Spain) were included for comparison. For each variety, DNA was extracted from young leaves. The Vte4 gene was PCR amplified from the 14 olive varieties and verified by electrophoresis on a 2% agarose gel for each variety. DNA sequencing of the olive cultivars revealed several single-nucleotide polymorphisms (SNPs). Statistical and bioinformatics analysis draw attention to some associations between some of the SNPs, tocopherols contents and oleic acid content. In fact, two significant associations are obtained between SUBS24 and both Total-Tocopherols and Beta-Tocopherol. Moreover, dendrograms reveals that there is a correlation between genetic variability and chemical characteristics which make the Vte4 gene more interesting in terms of tocopherols levels.

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

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 1059bp 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.

Cloning and Bioinformatic Analysis of γ-Tocopherol Methyltransferase Gene (<I>γ-TMT</I>) in Peanut

Cloning and Bioinformatic Analysis of γ-Tocopherol Methyltransferase Gene (<I>γ-TMT</I>) in Peanut

Stability of PAT Protein Expression of Multiple Generations of Genetically Modified Soybean Developed in Korea

 Abstract—Genetically modified (GM) soybeans (Glycine max (L.) Merrill) expressing the γ-tocopherol methyltransferase gene (γ-TMT) and the selectable marker phosphinothricin acetyltransferase (PAT) were developed by the Rural Development Administration in Korea. In this study, we used Southern blot analysis to examine the stability of inserted genes in GM soybeans, and enzymelinked immunosorbent assay (ELISA) was conducted to analyze inherent PAT protein levels. PAT expression levels varied among different plant generations and plant organs isolated from GM soybean. PAT expression was the highest in the leaves of plants at the beginning of the pod stage (R3) (12.36 μg/g) and the lowest in pods collected from plants that were at the fully mature stage (R8) (1.43 μg/g). The PAT protein showed a decreasing pattern of expression during plant growth. As expected, the expression of PAT proteins was not detected in control soybean plants.

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

A cDNA encoding γ-tocopherol methyltransferase from Brassica napus (BnTMT) was overexpressed in soybean [Glycine max (L.) Merr.] under the control of seed-specific promoter of Arabidopsis fatty acid elongase 1 (FAE1) or soybean glycinin G1. Two and three transgenic plants were selected, respectively, after Agrobacterium-mediated transformation. Polymerase chain reaction (PCR) and Southern blots confirmed that BnTMT was single-copy integrated into the genome of transgenic plants. RT-PCR analysis showed that the expression of BnTMT was higher in the immature cotyledons than in the mature cotyledons, while no expression was detected in the leaves. Moreover, the expression level under the control of FAE1 was higher than that of G1. HPLC analysis indicated that the seed-specific expression of BnTMT resulted in 11.1-fold and 18.9-fold increase in α- and β-tocopherol content, respectively, in T2 seed. These results suggested that introducing BnTMT into soybean can be used to increase the vitamin E composition in seeds.

Genetic variation of γ-tocopherol methyltransferase gene contributes to elevated α-tocopherol content in soybean seeds

BackgroundImprovement of α-tocopherol content is an important breeding aim to increase the nutritional value of crops. Several efforts have been conducted to improve the α-tocopherol content in soybean [Glycine max (L.) Merr.] through transgenic technology by overexpressing genes related to α-tocopherol biosynthesis or through changes to crop management practices. Varieties with high α-tocopherol content have been identified in soybean germplasms. The heritability of this trait has been characterized in a cross between high α-tocopherol variety Keszthelyi Aproszemu Sarga (KAS) and low α-tocopherol variety Ichihime. In this study, the genetic mechanism of the high α-tocopherol content trait of KAS was elucidated.ResultsThrough QTL analysis and fine mapping in populations from a cross between KAS and a Japanese variety Ichihime, we identified γ-TMT3, which encodes γ-tocopherol methyltransferase, as a candidate gene responsible for high α-tocopherol concentration in KAS. Several nucleotide polymorphisms including two nonsynonymous mutations were found in the coding region of γ-TMT3 between Ichihime and KAS, but none of which was responsible for the difference in α-tocopherol concentration. Therefore, we focused on transcriptional regulation of γ-TMT3 in developing seeds and leaves. An F5 line that was heterozygous for the region containing γ-TMT3 was self-pollinated. From among the progeny, plants that were homozygous at the γ-TMT3 locus were chosen for further evaluation. The expression level of γ-TMT3 was higher both in developing seeds and leaves of plants homozygous for the γ-TMT3 allele from KAS. The higher expression level was closely correlated with high α-tocopherol content in developing seeds. We generated transgenic Arabidopsis plants harboring GUS gene under the control of γ-TMT3 promoter from KAS or Ichihime. The GUS activity assay showed that the activity of γ-TMT3 promoter from KAS was higher than that of Ichihime.ConclusionsThe genetic variation in γ-TMT3, which plays a major role in determining α-tocopherol concentration, provides significant information about the regulation of tocopherol biosynthesis in soybean seeds. This knowledge will help breeding programs to develop new soybean varieties with high α-tocopherol content.

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

Expression of γ-tocopherol methyltransferase gene from Brassica napus increased α-tocopherol content in soybean seed

Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a fluorescence measurements

Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the γ-tocopherol methyl transferase (γ-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of α-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the γ-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial “O” level to the “P” (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that α-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.

Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (Perilla frutescens)

Increasing vitamin E activity in economically important oil crops such as perilla will enhance the nutritional value of these crops. Perilla (Perilla frutescens Britt) represents an important oil crop in Asian countries, including Korea. Using Agrobacterium-mediated transformation, we have engineered perilla with the γ-tocopherol methyltransferase (γ-TMT) gene under the control of seed-specific vicillin promoter. Molecular characterization including PCR, Southern and Northern blots confirmed that the γ-TMT transgene was successfully inherited to and expressed in the progeny plants. The γ -TMT transgene was specifically expressed in immature seeds of transgenic plants, leading to efficient conversion of γ-tocopherol to α-tocopherol and dramatic increase in seed α-tocopherol content, as detected by high performance liquid chromatography analysis. We also showed that such a high α-tocopherol content phenotype was transmitted to the progeny plants. In addition, there was no significant change in fatty acid composition in transgenic seeds as compared with untransformed control Yeupsil cultivar, suggesting the lack of interplay between the fatty acid and tocopherol biosynthesis pathways. This was the first report on over expression of the γ-TMT gene in transgenic perilla displaying desirable high α-tocopherol content phenotype. Since α-tocopherol has the highest vitamin E activity, the transgenic perilla with high α-tocopherol content in seeds developed in this study will benefit both human and animal health.

Increased α-tocopherol content in soybean seed overexpressing the Perilla frutescens γ-tocopherol methyltransferase gene

Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In soybean, gamma-tocopherol, the biosynthetic precursor to alpha-tocopherol, is the predominant form found in the seed, whereas alpha-tocopherol is the most bioactive component. This suggests that the final step of the alpha-tocopherol biosynthetic pathway catalyzed by gamma-tocopherol methyltransferase (gamma-TMT) is limiting in soybean seed. Soybean oil is the major edible vegetable oil consumed, so manipulating the tocopherol biosynthetic pathway in soybean seed to convert tocopherols into more active alpha-tocopherol form could have significant health benefits. In order to increase the soybean seed alpha-tocopherol content, the gamma-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter. One transgenic plant was recovered and the progeny was analyzed for two generations. Our results demonstrated that the seed-specific expression of the P. frutescens gamma-TMT gene resulted in a 10.4-fold increase in the alpha-tocopherol content and a 14.9-fold increase in the beta-tocopherol content in T2 seed. Given the relative contributions of different tocopherols to vitamin E activity, the activity in T2 seed was calculated to be 4.8-fold higher than in wild-type seed. In addition, the data obtained on lipid peroxidation indicates that alpha-tocopherol may have a role in preventing oxidative damage to lipid components during seed storage and seed germination. The increase in the alpha-tocopherol content in the soybean seed could have a potential to significantly increase the dietary intake of vitamin E.

Cloning and analysis of a γ-tocopherol methyltransferase gene from Brassica oleracea and the function of its recombinant protein*

Abstract The full-length cDNA (BoTMT) of γ-TMT is obtained from Brassica oleracea by 3′- and 5′-RACE methods. The 1265 bp cDNA contains an open reading frame of 1044 bp, which encodes a protein of 347 arnino acids with a predicted chloroplast transit peptide and two S-adenosylmethionine (SAM)-binding domains. Sequence analysis shows that the deduced protein shares 41.8% – 86.5% similarity to known γ-TMTs. Semi-quantitative RT-PCR reveals that BoTMT is expressed preferentially in flowers and leaves of B. oleracea. The recombinant γ-TMT protein is obtained by cloning its encoding region into the prokaryotic expression vector pET30a. The protein expressed in E. coli accounts for 22% of total bacterial protein. The enzyme activity assay indicates that the recombinant protein has relatively high activity to convert γ-tocopherol to α-tocopherol in vitro.

Enhancement of Vitamin E Levels in Corn

Vitamin E is the common name that describes eight naturally occurring compounds possessing α-tocopherol activity. These eight vitamin E compounds are collectively termed tocols, and all have antioxidant activity. There is natural variation among different corn breeding lines for levels of tocols. The two predominant isomers present in corn grain are γ-tocopherol and α-tocopherol. α-tocopherol is considered more desirable for human and animal consumption because it has higher biological activity than γ-tocopherol. Most corn breeding lines naturally have much more γ-tocopherol than α-tocopherol. Therefore a breeding goal is to increase levels of α-tocopherol relative to γ-tocopherol. However, recent research suggests that γ-tocopherol and compounds metabolized from it have properties important to human health that are unique from properties of α-tocopherol. Therefore it may be desirable to not only increase levels of α-tocopherol in corn grain, but also levels of γ-tocopherol. Determination of levels of tocopherols in corn grain is very laborious, requires HPLC analysis and is too time consuming for use in routine commercial corn breeding programs. Therefore we are performing biotechnology enabled molecular marker mapping of chromosomal regions with genes that control levels and ratios of α- and γ-tocopherol. Breeders can use molecular markers we have identified to expediently select for desirable alleles of genes that will improve levels of α- and γ-tocopherol in corn grain, without having to perform laborious HPLC assays. Another biotechnology strategy we have initiated is genetic transformation of corn with the γ-tocopherol methyl transferase gene to enhance conversion of γ-tocopherol to α-tocopherol and thus increase levels of α-tocopherol. This transgenic strategy has been demonstrated in the model plant Arabidopsis, and we are now applying this approach to corn.