Characterization of an Arabidopsis mutant deficient in γ-tocopherol methyltransferase

Alpha-tocopherol (vitamin E) is synthesized from gamma-tocopherol in chloroplasts by gamma-tocopherol methyltransferase (gamma-TMT; VTE4). Leaves of many plant species including Arabidopsis contain high levels of alpha-tocopherol, but are low in gamma-tocopherol. To unravel the function of different forms of tocopherol in plants, an Arabidopsis plant (vte4-1) carrying a functional null mutation in the gene gamma-TMT was isolated by screening a mutant population via thin-layer chromatography. A second mutant allele (vte4-2) carrying a T-DNA insertion in the coding sequence of gamma-TMT was identified in a T-DNA tagged mutant population. In vte4-1 and vte4-2 leaves, high levels of gamma-tocopherol accumulated, whereas alpha-tocopherol was absent indicating that, presumably, these two mutants represents null alleles. Over-expression of the gamma-TMT cDNA in vte4-1 restored wild-type tocopherol composition. Mutant plants were very similar to wild type. During oxidative stress (high light, high temperature, cold treatment) the amounts of alpha-tocopherol and gamma-tocopherol increased in wild type, and gamma-tocopherol in vte4-1. However, chlorophyll content and photosynthetic quantum yield were very similar in wild type and vte4-1, suggesting that alpha-tocopherol can be replaced by gamma-tocopherol in vte4-1 to protect the photosynthetic apparatus against oxidative stress. Fatty acid and lipid composition were very similar in WT, vte4-1 and vte1, an Arabidopsis mutant previously isolated which is completely devoid of tocopherol. Therefore, a shift in tocopherol composition or the absence of tocopherol has no major impact on the amounts of specific fatty acids or on lipid hydrolysis.     

Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis

Tocopherols are essential components of the human diet and are synthesized exclusively by photosynthetic organisms. These lipophilic antioxidants consist of a chromanol ring and a 15-carbon tail derived from homogentisate (HGA) and phytyl diphosphate, respectively. Condensation of HGA and phytyl diphosphate, the committed step in tocopherol biosynthesis, is catalyzed by HGA phytyltransferase (HPT). To investigate whether HPT activity is limiting for tocopherol synthesis in plants, the gene encoding Arabidopsis HPT, HPT1, was constitutively overexpressed in Arabidopsis. In leaves, HPT1 overexpression resulted in a 10-fold increase in HPT specific activity and a 4.4-fold increase in total tocopherol content relative to wild type. In seeds, HPT1 overexpression resulted in a 4-fold increase in HPT specific activity and a total seed tocopherol content that was 40% higher than wild type, primarily because of an increase in gamma-tocopherol content. This enlarged pool of gamma-tocopherol was almost entirely converted to alpha-tocopherol by crossing HPT1 overexpressing plants with lines constitutively overexpressing gamma-tocopherol methyltransferase. Seed of the resulting double overexpressing lines had a 12-fold increase in vitamin E activity relative to wild type. These results indicate that HPT activity is limiting in various Arabidopsis tissues and that total tocopherol levels and vitamin E activity can be elevated in leaves and seeds by combined overexpression of the HPT1 and gamma-tocopherol methyltransferase genes.     

The role of homogentisate phytyltransferase and other tocopherol pathway enzymes in the regulation of tocopherol synthesis during abiotic stress

Tocopherols are amphipathic antioxidants synthesized exclusively by photosynthetic organisms. Tocopherol levels change significantly during plant growth and development and in response to stress, likely as a consequence of the altered expression of pathway-related genes. Homogentisate phytyltransferase (HPT) is a key enzyme limiting tocopherol biosynthesis in unstressed Arabidopsis leaves (E. Collakova, D. DellaPenna [2003] Plant Physiol 131: 632-642). Wild-type and transgenic Arabidopsis plants constitutively overexpressing HPT (35S::HPT1) were subjected to a combination of abiotic stresses for up to 15 d and tocopherol levels, composition, and expression of several tocopherol pathway-related genes were determined. Abiotic stress resulted in an 18- and 8-fold increase in total tocopherol content in wild-type and 35S::HPT1 leaves, respectively, with tocopherol levels in 35S::HPT1 being 2- to 4-fold higher than wild type at all experimental time points. Increased total tocopherol levels correlated with elevated HPT mRNA levels and HPT specific activity in 35S::HPT1 and wild-type leaves, suggesting that HPT activity limits total tocopherol synthesis during abiotic stress. In addition, substrate availability and expression of pathway enzymes before HPT also contribute to increased tocopherol synthesis during stress. The accumulation of high levels of beta-, gamma-, and delta-tocopherols in stressed tissues suggested that the methylation of phytylquinol and tocopherol intermediates limit alpha-tocopherol synthesis. Overexpression of gamma-tocopherol methyltransferase in the 35S::HPT1 background resulted in nearly complete conversion of gamma- and delta-tocopherols to alpha- and beta-tocopherols, respectively, indicating that gamma-tocopherol methyltransferase activity limits alpha-tocopherol synthesis in stressed leaves.     

Inhibition of oxidative metabolism of tocopherols with omega-N-heterocyclic derivatives of vitamin E

The oxidative metabolism of tocopherols and tocotrienols by monooxygenases is a key factor in the plasma and tissue clearance of forms of vitamin E other than alpha-tocopherol. It is well known that a commonly ingested form of vitamin E, gamma-tocopherol, has greatly reduced plasma half-life (faster clearance) than alpha-tocopherol. The tocotrienols are metabolized even faster than gamma-tocopherol. Both gamma-tocopherol and alpha- and delta-tocotrienol possess intriguing biological activities that are different from alpha-tocopherol, making them potentially of interest for therapeutic use. Unfortunately, the fast clearance of non-alpha-tocopherols from animal tissues is a significant hurdle to maximizing their effect(s) as dietary supplements. We report here the design and synthesis of N-heterocycle-containing analogues of alpha-tocopherol that act as inhibitors of Cyp4F2, the key monooxygenase responsible for omega-hydroxylation of the side chain of tocols. In particular, an omega-imidazole containing compound, 1, [(R)-2-(9-(1H-imidazol-1-yl)nonyl)-2,5,7,8-tetramethylchroman-6-ol] had an ED(50) for inhibition of gamma-CEHC production from gamma-tocopherol of approximately 1 nM when tested in HepG2 cells in culture. Furthermore, feeding of 1 to mice along with rapidly metabolized delta-tocopherol, resulted in a doubling of the delta-tocopherol/alpha-tocopherol ratio in liver (P<0.05). Thus, 1 may be a useful adjuvant to the therapeutic use of non-alpha-tocopherols.     

Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status

Postabsorptive elimination of the various forms of vitamin E appears to play a key role in regulation of tissue tocopherol concentrations, but mechanisms of tocopherol metabolism have not been elucidated. Here we describe a pathway involving cytochrome P450-mediated omega-hydroxylation of the tocopherol phytyl side chain followed by stepwise removal of two- or three-carbon moieties, ultimately yielding the 3'-carboxychromanol metabolite that is excreted in urine. All key intermediates of gamma-tocopherol metabolism via this pathway were identified in hepatocyte cultures using gas chromatography-mass spectrometry. NADPH-dependent synthesis of the initial gamma- and alpha-tocopherol 13'-hydroxy and -carboxy metabolites was demonstrated in rat and human liver microsomes. Functional analysis of several recombinant human liver P450 enzymes revealed that tocopherol-omega-hydroxylase activity was associated only with CYP4F2, which also catalyzes omega-hydroxylation of leukotriene B(4) and arachidonic acid. Tocopherol-omega-hydroxylase exhibited similar binding affinities but markedly higher catalytic activities for gamma-tocopherol than alpha-tocopherol, suggesting a role for this pathway in the preferential physiological retention of alpha-tocopherol and elimination of gamma-tocopherol. Sesamin potently inhibited tocopherol-omega-hydroxylase activity exhibited by CYP4F2 and rat or human liver microsomes. Since dietary sesamin also results in elevated tocopherol levels in vivo, this pathway appears to represent a functionally significant means of regulating vitamin E status.     

Estrogen receptor beta. Potential functional significance of a variety of mRNA isoforms

A putative 2-methyl-6-phytylbenzoquinone (MPBQ) methyltransferase gene, SLL0418, was identified from the Synechocystis PCC6803 genome based on its homology to previously characterized gamma-tocopherol methyltransferases. Genetic and biochemical evidence confirmed open reading frame (ORF) SLL0418 encodes a MPBQ methyltransferase. An SLL0418 partial knockout mutant accumulated beta-tocopherol with no effect in the overall tocopherol content of the cell. In vitro assays of the SLL0418 gene expressed in Escherichia coli showed the enzyme efficiently catalyzes methylation of ring carbon 3 of MPBQ. In addition, the enzyme also catalyzes the methylation of ring carbon 3 of 2-methyl-6-solanylbenzoquinol in the terminal step of plastoquinone biosynthesis.     

拟南芥2-甲基-6-叶绿基-1,4-苯醌甲基转移酶启动子的分离及表达特性分析

维生素E是一类人体必需的脂溶性抗氧化剂, 具有重要的生理功能。2-甲基-6-叶绿基-1,4-苯醌甲基转移酶(MPBQ MT)是天然维生素E合成途径中的关键酶之一, 催化MPBQ甲基化, 生成DMPBQ。从拟南芥分离了MPBQ MT基因1018bp的启动子序列, 构建了含该启动子和GUS报告基因的植物表达载体, 通过农杆菌介导转化拟南芥, 获得了转基因植株。GUS组织化学染色结果表明, 在MPBQ MT启动子驱动下, 报告基因GUS在拟南芥的茎、叶、花萼、雄蕊、种荚均有表达, 且在茎、叶、种荚中表达量较高, 而在根、花瓣和种子中则没有观察到GUS基因的表达, 表明MPBQ MT基因可能仅在拟南芥幼嫩茎、叶、种荚等绿色组织中特异性高表达。     

Vitamin E: function and metabolism.

Although vitamin E has been known as an essential nutrient for reproduction since 1922, we are far from understanding the mechanisms of its physiological functions. Vitamin E is the term for a group of tocopherols and tocotrienols, of which alpha-tocopherol has the highest biological activity. Due to the potent antioxidant properties of tocopherols, the impact of alpha-tocopherol in the prevention of chronic diseases believed to be associated with oxidative stress has often been studied, and beneficial effects have been demonstrated. Recent observations that the alpha-tocopherol transfer protein in the liver specifically sorts out RRR-alpha-tocopherol from all incoming tocopherols for incorporation into plasma lipoproteins, and that alpha-tocopherol has signaling functions in vascular smooth muscle cells that cannot be exerted by other forms of tocopherol with similar antioxidative properties, have raised interest in the roles of vitamin E beyond its antioxidative function. Also, gamma-tocopherol might have functions apart from being an antioxidant. It is a nucleophile able to trap electrophilic mutagens in lipophilic compartments and generates a metabolite that facilitates natriuresis. The metabolism of vitamin E is equally unclear. Excess alpha-tocopherol is converted into alpha-CEHC and excreted in the urine. Other tocopherols, like gamma- and delta-tocopherol, are almost quantitatively degraded and excreted in the urine as the corresponding CEHCs. All rac alpha-tocopherol compared to RRR-alpha-tocopherol is preferentially degraded to alpha-CEHC. Thus, there must be a specific, molecular role of RRR-alpha-tocopherol that is regulated by a system that sorts, distributes, and degrades the different forms of vitamin E, but has not yet been identified. In this article we try to summarize current knowledge on the function of vitamin E, with emphasis on its antioxidant vs. other properties, the preference of the organism for RRR-alpha-tocopherol, and its metabolism to CEHCs.     

Accumulation dynamics of seed tocopherols in sunflower lines with modified tocopherol levels

Sunflower (Helianthus annuus L.) seeds have a tocopherol profile dominated by alpha-tocopherol. The objective of this research was to study the dynamics of tocopherol accumulation in sunflower lines with altered total tocopherol content or tocopherol profile. Developing seeds were sampled at regular intervals in two lines with reduced and increased total tocopherol content, respectively, and six lines with modified tocopherol profiles. The line with reduced tocopherol content showed a tocopherol accumulation rate reduced by half, whereas the line with increased tocopherol content showed a tocopherol accumulation rate twofold higher than the control. In the three cases, alpha-tocopherol followed a sigmoid accumulation pattern. Modified tocopherol profiles were expressed at early stages of tocopherol accumulation. In most lines with modified profiles, tocopherol accumulation pattern differed from the alpha-tocopherol lines, with maximum tocopherol content at 18 or 21 days after flowering (DAF) that was reduced to reach a plateau from 33 or 36 DAF onward. Such a reduction was caused by continued dry matter accumulation after tocopherol accumulation ceased or slowed down. In lines with increased levels of beta-tocopherol or both gamma- and delta-tocopherol, the synthesis of beta- and delta-tocopherol started and stopped earlier than the synthesis of alpha- and gamma-tocopherol, respectively.     

Simultaneous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis> ρ-hydroxyphenylpyruvate dioxygenase and MPBQ methyltransferase in transgenic corn kernels triples the tocopherol content

The quantity and composition of tocopherols (compounds with vitamin E activity) vary widely among different plant species reflecting the expression, activity and substrate specificity of enzymes in the corresponding metabolic pathway. Two Arabidopsis cDNA clones corresponding to ρ-hydroxyphenylpyruvate dioxygenase (HPPD) and 2-methyl-6-phytylplastoquinol methyltransferase (MPBQ MT) were constitutively expressed in corn to further characterize the pathway and increase the kernel tocopherol content. Transgenic kernels contained up to 3 times as much γ-tocopherol as their wild type counterparts whereas other tocopherol isomers remained undetectable. Biofortification by metabolic engineering offers a sustainable alternative to vitamin E supplementation for the improvement of human health.     

Tocopherol and fatty acid composition of twenty-five species of Onagraceae Juss.

Chemotaxonomic relationships in Onagraceae have been investigated at the level of leaf and flower compounds but not for seed traits. The objective of this study was to characterize a set of 26 accessions (25 species, 7 genera) of this family for seed oil content, fatty acid composition, tocopherol content and composition, and to evaluate the chemotaxonomic implications of the results. The accessions showed a large variation for all the traits. Gamma-linolenic acid was exclusively found in Oenothera spp. where two groups, showing different concentrations of gamma-linolenic acid and alpha-tocopherol, were observed. The species of Clarkia Pursh were markedly different to the rest of species, showing low alpha-linolenic acid and low gamma-tocopherol concentration. Circaea lutetiana L. had a characteristic tocopherol profile, with high levels (13%) of beta-tocopherol. Variation for fatty acid and tocopherol composition in Epilobium spp. suggested some phylogenetic relationships. The annual species E. pankulatum Nutt. showed a characteristic composition very similar to the closely related Boisduvalia densiflora (Lindl.) S. Watson. The accessions of the phylogenetically related E. fleischeri Hochst. and E. dodonaei Vill., also showed similar fatty acid and tocopherol composition. The results of this study suggest a potential chemotaxonomic value of seed fatty acids and tocopherols in Onagraceae.     

Isolation and properties of gamma-tocopherol methyltransferase in Euglena gracilis.

Gamma-Tocopherol methyltransferase (EC2.1.1.-), which catalyzes the conversion of gamma-tocopherol into alpha-tocopherol, was present in a cell homogenate of Euglena gracilis. The enzyme was loosely bonded to the outer membrane of chloroplasts and solubilized from chloroplast membranes by a detergent, followed by partial purification in a three-step procedure. The methyltransferase showed a pH optimum of 7.5 and a temperature optimum of 35 degrees C and had an M(r) of 150,000. The activity was about 1.4-fold higher with gamma-tocopherol than with beta-tocopherol as substrate. The enzyme was specific for S-adenosylmethionine as a methyl donor, with a Km value of 50 microM. The addition of homogentisate, L-tyrosine and L-phenylalanine into a suspension of Euglena cells increased the relative pool sizes of alpha- and gamma-tocopherol, but not those of beta- and delta-tocopherol. The contents of alpha- and gamma-tocopherol in a chloroplast fraction of Euglena were always higher than those of any other fraction after any period of incubation with homogentisate. Based on the results of the present experiments, we propose a biosynthetic pathway of alpha-tocopherol in Euglena gracilis.     

Specific roles of alpha- and gamma-tocopherol in abiotic stress responses of transgenic tobacco

Tocopherols are lipophilic antioxidants that are synthesized exclusively in photosynthetic organisms. In most higher plants, alpha- and gamma-tocopherol are predominant with their ratio being under spatial and temporal control. While alpha-tocopherol accumulates predominantly in photosynthetic tissue, seeds are rich in gamma-tocopherol. To date, little is known about the specific roles of alpha- and gamma-tocopherol in different plant tissues. To study the impact of tocopherol composition and content on stress tolerance, transgenic tobacco (Nicotiana tabacum) plants constitutively silenced for homogentisate phytyltransferase (HPT) and gamma-tocopherol methyltransferase (gamma-TMT) activity were created. Silencing of HPT lead to an up to 98% reduction of total tocopherol accumulation compared to wild type. Knockdown of gamma-TMT resulted in an up to 95% reduction of alpha-tocopherol in leaves of the transgenics, which was almost quantitatively compensated for by an increase in gamma-tocopherol. The response of HPT and gamma-TMT transgenics to salt and sorbitol stress and methyl viologen treatments in comparison to wild type was studied. Each stress condition imposes oxidative stress along with additional challenges like perturbing ion homeostasis, desiccation, or disturbing photochemistry, respectively. Decreased total tocopherol content increased the sensitivity of HPT:RNAi transgenics toward all tested stress conditions, whereas gamma-TMT-silenced plants showed an improved performance when challenged with sorbitol or methyl viologen. However, salt tolerance of gamma-TMT transgenics was strongly decreased. Membrane damage in gamma-TMT transgenic plants was reduced after sorbitol and methyl viologen-mediated stress, as evident by less lipid peroxidation and/or electrolyte leakage. Therefore, our results suggest specific roles for alpha- and gamma-tocopherol in vivo.     

Less than adequate vitamin E status observed in a group of preschool boys and girls living in the United States

In that data were not available on the vitamin E status of young children, the aim of the study was to evaluate the vitamin E status of preschool children by three commonly used criteria: vitamin E intakes, plasma alpha-tocopherol concentrations and plasma alpha-tocopherol/total lipid ratios. Twenty-two ethnically diverse preschool children (13 males and 9 females), aged 2 to 5 years, living in Lincoln, NE, served as subjects. The subjects were in two groups: 2-3 and 4-5 years old. Energy, fat, and alpha- and gamma-tocopherol intakes of the subjects were estimated utilizing two 24-h food recalls. Plasma alpha- and gamma-tocopherol and total lipid concentrations were ascertained. No significant differences by age grouping or gender were observed for vitamin E intakes, plasma alpha-tocopherol concentrations, plasma gamma-tocopherol concentrations and plasma alpha-tocopherol/total lipid ratios of subjects. Plasma alpha-tocopherol concentrations indicative of less than adequate status (<12 micromol/L) were observed in 91% of the children, and values <7 micromol/L (proposed cutoff for pediatric populations) in 68%. Sixty-eight percent of the subjects had plasma alpha-tocopherol/total lipid values <0.8 mg/g. The majority of the 2- to 5-year-old children included in the study had less than adequate vitamin E status.     

Improving alpha-tocopherol production in plant cell cultures

Suspension cell cultures of Helianthus annuus L. were previously established for the production of the most active component of vitamin E, alpha-tocopherol, by optimizing medium composition and culture conditions. In the present work, the possibility of enhancing alpha-tocopherol production by the addition of jasmonic acid to the culture medium was investigated both in sunflower and Arabidopsis cell cultures. A considerable increase (49% and 66%, respectively) of alpha-tocopherol production was obtained in both, after a 72-h treatment with 5 microM jasmonic acid. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.