Effect of photoperiod on vitamin E and carotenoid biosynthesis in mung bean (Vigna radiata) sprouts.

Light affects the accumulation of vitamin E and carotenoids in many crops. This study investigated the impact of photoperiods on the metabolic regulation of vitamin E and carotenoids in mung bean sprouts considering their dietary health benefits. Mung beans were germinated under three different photoperiods: constant light, semilight and constant dark. Results revealed that the semilight photoperiod was optimum for vitamin E and carotenoid accumulation in mung bean sprouts. DXS was activated in the constant dark and was inhibited by constant light. GGPPS and HPT were sensitive to semilight photoperiod in the vitamin E biosynthetic pathway, playing dominant roles in vitamin E accumulation. The PSY, LCYE, LUT5, LUT1 and ZE genes, which are associated with carotenoid biosynthesis, were activated under semilight treatment and significantly regulated the accumulation of carotenoids. This knowledge improves knowledge on light-mediated regulation of vitamin E and carotenoids in mung bean sprouts.

Establishing reference intervals for vitamins A and E in Chinese elderly people using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Vitamins A and E play important roles in sustaining life activities and maintaining a good physical condition. However, most people, particularly the elderly, experience micronutrient deficiencies. This study aimed to establish reference intervals (RIs) for vitamins A and E in Chinese elderly people using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. METHODS: A total of 356 apparently healthy individuals aged ≥64 years who underwent health checkups were randomly selected for the study. Vitamin A and E levels were measured using LC-MS/MS. The effect of sex on vitamin A and E levels was evaluated, and RIs were established using a parametric method. RESULTS: Females showed significantly higher levels of vitamin E than males (p < 0.05). However, no significant sex-specific difference was observed with vitamin A levels. The RI for vitamin A in the elderly was 0.283-0.730 mg/L. For vitamin E, the RIs were 4.39-15.63, 4.51-16.14, and 4.41-14.67 mg/L for the total, female, and male participants, respectively. In multiple linear regression, alanine aminotransferase, glutamyl transpeptidase, urea, glucose, and uric acid levels increased with increasing vitamin A levels (p < 0.05), and total cholesterol and low-density lipoprotein cholesterol levels increased with increasing vitamin E levels (p < 0.05). Direct bilirubin levels decreased with increasing vitamin E levels (p < 0.05). CONCLUSIONS: This study established RIs for vitamins A and E in Chinese elderly individuals using an LC-MS/MS method. We also found that females had significantly higher vitamin E levels than males. The findings could provide a scientific basis for interpreting vitamin status in the elderly.

Antioxidant action of vitamin E in vivo as assessed from its reaction products with multiple biological oxidants.

Vitamin E acts as essential antioxidant against detrimental oxidation of biological molecules induced by multiple reactive species. To gain more insight into the physiological role of vitamin E, the levels of its oxidation products in humans under normal and pathological conditions were compared. α-Tocopherol quinone (α-TQ) and 5-nitro-γ-tocopherol (5-NgT) were focused. α-TQ is produced by multiple oxidants including oxygen radicals, peroxynitrite, hypochlorite, singlet oxygen, and ozone, while 5-NgT is produced by nitrogen dioxide radical derived from peroxynitrite and the reaction of nitrite and hypochlorite. The reported concentrations of α-TQ and 5-NgT in healthy human plasma are highly variable ranging from 15 to 360 and 4 to 170 nM, respectively. In general, the molar ratio 5-NgT/γ-tocopherol was higher than the ratio α-TQ/α-tocopherol. Both absolute concentrations of α-TQ and 5-NgT and the molar ratios to the parent tocopherols were elevated significantly in the plasma of patients with various diseases compared with healthy subjects except neurological diseases. The molar ratios of the products to the respective parent compounds decreased in the order of 5-NgT/γ-tocopherol > α-TQ/α-tocopherol > hydroxyoctadecadienoate/linoleate > 3-nitrotyrosine/tyrosine > isoprostane/arachidonate. The molar ratios of nitrated products to the respective parent compounds in human plasma are approximately 10-2 for 5-NgT and 10-5 for 3-nitrotyrosine, nitro-oleic acid, and 8-nitroguaine. These data indicate that vitamin E acts as an important physiological antioxidant and that α-TQ and 5-NgT represent biomarker for oxidative stress and nitrative stress respectively.

De Novo High-Titer Production of Delta-Tocotrienol in Recombinant Saccharomyces cerevisiae.

Delta-tocotrienol as a vitamin E isomer has received much attention because of its diverse biomedical applications. Microbial biosynthesis of delta-tocotrienol is a promising strategy for its economic and environmental advantages. Here, we accomplished complete biosynthesis of delta-tocotrienol in Saccharomyces cerevisiae from glucose. We first constructed and incorporated a heterologous pathway into the genome of S. cerevisiae by incorporating the genes hpd (from Pseudomonas putida KT2440), hpt (from Synechocystis sp. PCC 6803), and vte1 (from Arabidopsis thaliana) for the biosynthesis of delta-tocotrienol. We further enhanced the biosynthesis of the precursor geranylgeranyl diphosphate by overexpressing the thmg1 and ggppssa (from Sulfolobus acidocaldarius) genes, leading to a production titer of delta-tocotrienol of 1.39 ± 0.01 mg/L. Finally, we optimized the fermentation medium using the response surface methodology, enabling a high-titer production of delta-tocotrienol (3.56 ± 0.25 mg/L), ∼2.6-fold of that of the initial culture medium. Fed-batch fermentation in a 2 L fermenter was further used to enhance the production titer of delta-tocotrienol (4.10 ± 0.10 mg/L). To the best of our knowledge, this is the first report on the de novo biosynthesis of delta-tocotrienol in S. cerevisiae, and the highest titer obtained for microbial production of delta-tocotrienol.

Metabolic engineering for the production of fat-soluble vitamins: advances and perspectives.

Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based on biosynthetic pathways, considerable advances of α-tocopherol and ß-carotenes have been achieved in transgenic plants and microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and ß-carotenes, is attracting considerable attention because it is an environment friendly process. In this review, we address the function and applications of fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants, microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins by biotechnological routes.

Identifying Vitamin E Biosynthesis Genes in Elaeis guineensis by Genome-Wide Association Study.

Elaeis guineensis is a tropical oil crop and has the highest oil yield per unit area. Palm oil has high palmitic acid content and is also rich in vitamins, including vitamin E. We conducted genome-wide association studies in a diversity panel of 161 E. guineensis accessions to identify single-nucleotide polymorphisms (SNPs) linked with vitamin E and validated candidate genes in these marker-associated intervals. Based on the SNPs reported in our previous research, 47 SNP markers were detected to be significantly associated with the variation of tocopherol and tocotrienol content at a cutoff P value of 6.3 × 10-7. A total of 656 candidate genes in the flanking regions of the 47 SNPs were identified, followed by pathway enrichment analysis. Of these candidate genes, EgHGGT (homogentisate geranylgeranyl transferase) involved in the biosynthesis of tocotrienols had a higher expression level in the mesocarp compared to other tissues. Expression of the EgHGGT gene was positively correlated with the variation in α-tocotrienol content. Induced overexpression of the gene in Arabidopsis caused a significant increase in vitamin E content and production of α-tocotrienols compared to wild Arabidopsis.

Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm.

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and ß-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3Δ9,12,15 or GLA, 18:3Δ6,9,12), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.

De novo transcriptome of Gymnema sylvestre identified putative lncRNA and genes regulating terpenoid biosynthesis pathway.

Gymnema sylvestre is a highly valuable medicinal plant in traditional Indian system of medicine and used in many polyherbal formulations especially in treating diabetes. However, the lack of genomic resources has impeded its research at molecular level. The present study investigated functional gene profile of G. sylvestre via RNA sequencing technology. The de novo assembly of 88.9 million high quality reads yielded 23,126 unigenes, of which 18116 were annotated against databases such as NCBI nr database, gene ontology (GO), KEGG, Pfam, CDD, PlantTFcat, UniProt & GreeNC. Total 808 unigenes mapped to 78 different Transcription Factor families, whereas 39 unigenes assigned to CYP450 and 111 unigenes coding for enzymes involved in the biosynthesis of terpenoids including transcripts for synthesis of important compounds like Vitamin E, beta-amyrin and squalene. Among them, presence of six important enzyme coding transcripts were validated using qRT-PCR, which showed high expression of enzymes involved in methyl-erythritol phosphate (MEP) pathway. This study also revealed 1428 simple sequence repeats (SSRs), which may aid in molecular breeding studies. Besides this, 8 putative long non-coding RNAs (lncRNAs) were predicted from un-annotated sequences, which may hold key role in regulation of essential biological processes in G. sylvestre. The study provides an opportunity for future functional genomic studies and to uncover functions of the lncRNAs in G. sylvestre.

Genotype-dependent regulation of vitamin E biosynthesis in olive fruits as revealed through metabolic and transcriptional profiles.

Vitamin E is a general term used to describe a group of eight lipophilic compounds known as tocochromanols. These vitamin E variants are chemically categorised into two classes formed by α-, ß-, γ- and δ- tocopherols and tocotrienols isoforms, respectively. The present study describes the concurrent regulation of genes and metabolites orchestrating vitamin E biosynthesis in olive drupes of five distinctive Greek olive cultivars. A combination of analytical, biochemical and molecular approaches was employed in order to carry out comparative analyses, including real-time RT-qPCR for gene expression levels and HPLC analysis of metabolite content. Findings indicated that tocochromanol levels and composition, oil content, gene expression levels as well as total antioxidant activity were highly dependent on cultivar and, to a lesser extent, on fruit developmental stage. Specifically, cultivars 'Kalokairida' and 'Lianolia Kerkyras' demonstrated the highest vitamin E content. The latter possessed high tocochromanol content combined with highest overall antioxidant activity in all developmental stages, concomitant with the up-regulation expression profile of HPPD. The genotypic imprint versus the temporal contribution to vitamin E levels, as well as the potential link to lipid peroxidation amelioration, are discussed.

Transcriptional Regulation of Vitamin E Biosynthesis during Germination of Dwarf Fan Palm Seeds.

Vitamin E, a potent antioxidant either presents in the form of tocopherols and/or tocotrienols depending on the plant species, tissue and developmental stage, plays a major role in protecting lipids from oxidation in seeds. Unlike tocopherols, which have a more universal distribution, the occurrence of tocotrienols is limited primarily to monocot seeds. Dwarf fan palm (Chamaerops humilis var. humilis) seeds accumulate tocotrienols in quiescent and dormant seeds, while tocopherols are de novo synthesized during germination. Here, we aimed to elucidate whether tocopherol biosynthesis is regulated at the transcriptional level during germination in this species. We identified and quantified the expression levels of five genes involved in vitamin E biosynthesis, including TYROSINE AMINOTRANSFERASE (ChTAT), HOMOGENTISATE PHYTYLTRANSFERASE (ChHPT), HOMOGENTISATE GERANYLGERANYL TRANSFERASE (ChHGGT), TOCOPHEROL CYCLASE (ChTC) and TOCOPHEROL γ-METHYLTRANSFERASE (Chγ-TMT). Furthermore, we evaluated to what extent variations in the endogenous contents of hormones and hydrogen peroxide (H2O2) correlated with transcriptional regulation. Results showed an increase of ChTAT and ChHPT levels during seed germination, which correlated with an increase of jasmonic acid (JA), gibberellin4 (GA4), and H2O2 contents, while ChHGGT and Chγ-TMT expression levels decreased, thus clearly indicating vitamin E biosynthesis is diverted to tocopherols rather than to tocotrienols. Exogenous application of jasmonic acid increased tocopherol, but not tocotrienol content, thus confirming its regulatory role in vitamin E biosynthesis during seed germination. It is concluded that the biosynthesis of vitamin E is regulated at the transcriptional level during germination in dwarf fan palm seeds, with ChHPT playing a key role in the diversion of the vitamin E pathway towards tocopherols instead of tocotrienols.

Evaluation of Biosynthesis, Accumulation and Antioxidant Activityof Vitamin E in Sweet Corn (Zea mays L.) during Kernel Development.

Sweet corn kernels were used in this research to study the dynamics of vitamin E, by evaluatingthe expression levels of genes involved in vitamin E synthesis, the accumulation of vitamin E, and the antioxidant activity during the different stage of kernel development. Results showed that expression levels of ZmHPT and ZmTC genes increased, whereas ZmTMT gene dramatically decreased during kernel development. The contents of all the types of vitamin E in sweet corn had a significant upward increase during kernel development, and reached the highest level at 30 days after pollination (DAP). Amongst the eight isomers of vitamin E, the content of γ-tocotrienol was the highest, and increased by 14.9 folds, followed by α-tocopherolwith an increase of 22 folds, and thecontents of isomers γ-tocopherol, α-tocotrienol, δ-tocopherol,δ-tocotrienol, and ß-tocopherol were also followed during kernel development. The antioxidant activity of sweet corn during kernel development was increased, and was up to 101.8 ± 22.3 µmol of α-tocopherol equivlent/100 g in fresh weight (FW) at 30 DAP. There was a positive correlation between vitamin E contents and antioxidant activity in sweet corn during the kernel development, and a negative correlation between the expressions of ZmTMT gene and vitamin E contents. These results revealed the relations amongst the content of vitamin E isomers and the gene expression, vitamin E accumulation, and antioxidant activity. The study can provide a harvesting strategy for vitamin E bio-fortification in sweet corn.

Advances in Genetic Improvement for Tocotrienol Production: A Review.

Tocotrienols are forms of vitamin E that are present in several important food crops. Compared to tocopherols, less research has been conducted on these compounds because of their low bioavailability and distribution in plant tissues. Both tocotrienols and tocopherols are known for their antioxidant and anticancer activities, which are beneficial for both humans and animals. Moreover, tocotrienols possess certain properties which are not found in tocopherols, such as neuroprotective and cholesterol-lowering activities. The contents of tocotrienols in plants vary. Tocotrienols constitute more than 70% and tocopherols less than 30% of the total vitamin E content in palm oil, which is the best source of vitamin E. Accumulation of tocotrienols also occurs in non-photosynthetic tissues, such as the seeds, fruits and latex of some monocotyledonous and dicotyledonous plant species. The use of biotechnological techniques to increase the tocotrienol content in plants, their biological functions, and benefits to human health are discussed in this review.

Plasmodium falciparum uses vitamin E to avoid oxidative stress.

BACKGROUND: Plasmodium falciparum is sensitive to oxidative stress in vitro and in vivo, and many drugs such as artemisinin, chloroquine and cercosporin interfere in the parasite's redox system. To minimize the damage caused by reactive radicals, antioxidant enzymes and their substrates found in parasites and in erythrocytes must be functionally active. It was shown that P. falciparum synthesizes vitamin E and that usnic acid acts as an inhibitor of its biosynthesis. Vitamin E is a potent antioxidant that protects polyunsaturated fatty acids from lipid peroxidation, and this activity can be measured by detecting its oxidized product and by evaluating reactive oxygen species (ROS) levels. RESULTS: Here, we demonstrated that ROS levels increased in P. falciparum when vitamin E biosynthesis was inhibited by usnic acid treatment and decreased to basal levels if exogenous vitamin E was added. Furthermore, we used metabolic labelling to demonstrate that vitamin E biosynthesized by the parasite acts as an antioxidant since we could detect its radiolabeled oxidized product. The treatment with chloroquine or cercosporin of the parasites increased the ratio between α-tocopherolquinone and α-tocopherol. CONCLUSIONS: Our findings demonstrate that vitamin E produced endogenously by P. falciparum is active as an antioxidant, probably protecting the parasite from the radicals generated by drugs.

The manipulation of gene expression and the biosynthesis of Vitamin C, E and folate in light-and dark-germination of sweet corn seeds.

This study investigates the potential interrelationship between gene expression and biosynthesis of vitamin C, E and folate in sweet corn sprouts. Germination of sweet corn kernels was conducted in light and dark environments to determine if this relationship was regulated by photo-illumination. Results indicated that light and dark environments affected the DHAR, TMT and GTPCH expression and that these genes were the predominant genes of vitamin C, E and folate biosynthesis pathways respectively during the germination. Levels of vitamin C and folate increased during the germination of sweet corn seeds while vitamin E had a declining manner. Sweet corn sprouts had higher vitamin C and E levels as well as relevant gene expression levels in light environment while illumination had little influence on the folate contents and the gene expression levels during the germination. These results indicate that there might be a collaborative relationship between vitamin C and folate regulation during sweet corn seed germination, while an inhibitive regulation might exist between vitamin C and E.

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination.

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of ß-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling.

Vitamin E - Occurrence, Biosynthesis by Plants and Functions in Human Nutrition.

OBJECTIVE: This review examines various aspects of vitamin E, both in plant metabolism and with regard to its importance for human health. Vitamin E is the collective name of a group of lipidsoluble compounds, chromanols, which are widely distributed in the plant kingdom. Their biosynthetic pathway, intracellular distribution and antioxidant function in plants are well recognized, although their other functions are also considered. CONCLUSION: Analytical methods for the determination of vitamin E are discussed in detail. Furthermore, the vitamin E metabolism and its antioxidant action in humans are described. Other nonantioxidant functions of vitamin E are also presented, such as its anti-inflammatory effects, role in the prevention of cardiovascular diseases and cancer, as well as its protective functions against neurodegenerative and other diseases.

Genetic variation of carotenoids, vitamin E and phenolic compounds in Provitamin A biofortified maize.

BACKGROUND: Biofortified maize is not only a good vehicle for provitamin A carotenoids for vitamin A deficient populations in developing countries but also a source of vitamin E, tocochromanols and phenolic compounds, which have antioxidant properties. Using high-performance liquid chromatography and a total antioxidant performance assay, the present study analyzed the antioxidant variation and antioxidant activity of 36 provitamin A improved maize hybrids and one common yellow maize hybrid. RESULTS: The ranges of major carotenoids in provitamin A carotenoids biofortified maize were zeaxanthin [1.2-13.2 µg g-1 dry weight (DW)], ß-cryptoxanthin (1.3-8.8 µg g-1 DW) and ß-carotene (1.3-8.0 µg g-1 DW). The ranges of vitamin E compounds identified in provitamin A carotenoids biofortified maize were α-tocopherol (3.4-34.3 µg g-1 DW), γ-tocopherol (5.9-54.4 µg g-1 DW), α-tocotrienol (2.6-19.5 µg g-1 DW) and γ-tocotrienol (45.4 µg g-1 DW). The ranges of phenolic compounds were γ-oryzanol (0.0-0.8 mg g-1 DW), ferulic acid (0.4-3.6 mg g-1 DW) and p-coumaric acid (0.1-0.45 mg g-1 DW). There was significant correlation between α-tocopherol and cis isomers of ß-carotene (P < 0.01). Tocotrienols were correlated with α-tocopherol and γ-oryzanol (P < 0.01). CONCLUSION: Genotype was significant in determining the variation in ß-cryptoxanthin, ß-carotene, α-tocopherol and γ-tocopherol contents (P < 0.01). A genotype × environment interaction was observed for γ-tocopherol content (P < 0.01). © 2016 Society of Chemical Industry.

Genetically Programmed Changes in Photosynthetic Cofactor Metabolism in Copper-deficient Chlamydomonas.

Genetic and genomic studies indicate that copper deficiency triggers changes in the expression of genes encoding key enzymes in various chloroplast-localized lipid/pigment biosynthetic pathways. Among these are CGL78 involved in chlorophyll biosynthesis and HPPD1, encoding 4-hydroxyphenylpyruvate dioxygenase catalyzing the committed step of plastoquinone and tocopherol biosyntheses. Copper deficiency in wild-type cells does not change the chlorophyll content, but a survey of chlorophyll protein accumulation in this situation revealed increased accumulation of LHCSR3, which is blocked at the level of mRNA accumulation when either CGL78 expression is reduced or in the crd1 mutant, which has a copper-nutrition conditional defect at the same step in chlorophyll biosynthesis. Again, like copper-deficient crd1 strains, cgl78 knock-down lines also have reduced chlorophyll content concomitant with loss of PSI-LHCI super-complexes and reduced abundance of a chlorophyll binding subunit of PSI, PSAK, which connects LHCI to PSI. For HPPD1, increased mRNA results in increased abundance of the corresponding protein in copper-deficient cells concomitant with CRR1-dependent increased accumulation of γ-tocopherols, but not plastoquinone-9 nor total tocopherols. In crr1 mutants, where increased HPPD1 expression is blocked, plastochromanol-8, derived from plastoquinone-9 and purported to also have an antioxidant function, is found instead. Although not previously found in algae, this metabolite may occur only in stress conditions.

Overexpression of Medicago sativa TMT elevates the α-tocopherol content in Arabidopsis seeds, alfalfa leaves, and delays dark-induced leaf senescence.

Alfalfa (Medicago sativa L.) is a major forage legume for livestock and a target for improving their dietary quality. Vitamin E is an essential vitamin that animals must obtain from their diet for proper growth and development. γ-tocopherol methyltransferase (γ-TMT), which catalyzes the conversion of δ- and γ-tocopherols (or tocotrienols) to ß- and α-tocopherols (or tocotrienols), respectively, is the final enzyme involved in the vitamin E biosynthetic pathway. The overexpression of M. sativa L.'s γ-TMT (MsTMT) increased the α-tocopherol content 10-15 fold above that of wild type Arabidopsis seeds without altering the total content of vitamin E. Additionally, in response to osmotic stress, the biomass and the expression levels of several osmotic marker genes were significantly higher in the transgenic lines compared with wild type. Overexpression of MsTMT in alfalfa led to a modest, albeit significant, increase in α-tocopherol in leaves and was also responsible for a delayed leaf senescence phenotype. Additionally, the crude protein content was increased, while the acid and neutral detergent fiber contents were unchanged in these transgenic lines. Thus, increased α-tocopherol content occurred in transgenic alfalfa without compromising the nutritional qualities. The targeted metabolic engineering of vitamin E biosynthesis through MsTMT overexpression provides a promising approach to improve the α-tocopherol content of forage crops.

Subgenome-specific assembly of vitamin E biosynthesis genes and expression patterns during seed development provide insight into the evolution of oat genome.

Vitamin E is essential for humans and thus must be a component of a healthy diet. Among the cereal grains, hexaploid oats (Avena sativa L.) have high vitamin E content. To date, no gene sequences in the vitamin E biosynthesis pathway have been reported for oats. Using deep sequencing and orthology-guided assembly, coding sequences of genes for each step in vitamin E synthesis in oats were reconstructed, including resolution of the sequences of homeologs. Three homeologs, presumably representing each of the three oat subgenomes, were identified for the main steps of the pathway. Partial sequences, likely representing pseudogenes, were recovered in some instances as well. Pairwise comparisons among homeologs revealed that two of the three putative subgenome-specific homeologs are almost identical for each gene. Synonymous substitution rates indicate the time of divergence of the two more similar subgenomes from the distinct one at 7.9-8.7 MYA, and a divergence between the similar subgenomes from a common ancestor 1.1 MYA. A new proposed evolutionary model for hexaploid oat formation is discussed. Homeolog-specific gene expression was quantified during oat seed development and compared with vitamin E accumulation. Homeolog expression largely appears to be similar for most of genes; however, for some genes, homoeolog-specific transcriptional bias was observed. The expression of HPPD, as well as certain homoeologs of VTE2 and VTE4, is highly correlated with seed vitamin E accumulation. Our findings expand our understanding of oat genome evolution and will assist efforts to modify vitamin E content and composition in oats.