转基因水稻中过表达OsHGGT基因提高种子三烯生育酚含量的研究

旨在提高稻米中三烯生育酚的含量,将来源于日本晴尿黑酸牻牛儿基牛儿基牻转移酶(homogentisic acid gerany-lgeranyl transferase,HGGT)基因导入粳稻品种武育粳3号过量表达。经PCR和RT-PCR分析证明外源基因已导入水稻中并能够在水稻胚乳中表达。HPLC测定结果表明,过表达HGGT后,转基因水稻种子糠层及胚乳中γ-三烯生育酚和总三烯生育酚的含量分别是未转化对照的1.52和1.67倍,且三烯生育酚的积累并未导致总生育酚含量的降低,最终糠层及胚乳中总三烯生育酚与总生育酚的比值分别提高到0.82和1.82,极显著高于(P<0.01)未转化对照(分别为0.54和1.27)。     

Vitamin E biosynthesis: functional characterization of the monocot homogentisate geranylgeranyl transferase

The biosynthesis of the tocotrienol and tocopherol forms of vitamin E is initiated by prenylation of homogentisate. Geranylgeranyl diphosphate (GGDP) is the prenyl donor for tocotrienol synthesis, whereas phytyl diphosphate (PDP) is the prenyl donor for tocopherol synthesis. We have previously shown that tocotrienol synthesis is initiated in monocot seeds by homogentisate geranylgeranyl transferase (HGGT). This enzyme is related to homogentisate phytyltransferase (HPT), which catalyzes the prenylation step in tocopherol synthesis. Here we show that monocot HGGT is localized in the plastid and expressed primarily in seed endosperm. Despite the close structural relationship of monocot HGGT and HPT, these enzymes were found to have distinct substrate specificities. Barley (Hordeum vulgare cv. Morex) HGGT expressed in insect cells was six times more active with GGDP than with PDP, whereas the Arabidopsis HPT was nine times more active with PDP than with GGDP. However, only small differences were detected in the apparent Km values of barley HGGT for GGDP and PDP. Consistent with its in vitro substrate properties, barley HGGT generated a mixture of tocotrienols and tocopherols when expressed in the vitamin E-null vte2-1 mutant lacking a functional HPT. Relative levels of tocotrienols and tocopherols produced in vte2-1 differed between organs and growth stages, reflective of the composition of plastidic pools of GGDP and PDP. In addition, HGGT was able to functionally substitute for HPT to rescue vte2-1-associated phenotypes, including reduced seed viability and increased fatty acid oxidation of seed lipids. Overall, we show that monocot HGGT is biochemically distinct from HPT, but can replace HPT in important vitamin E-related physiological processes.     

Tocotrienols inhibit human glutathione S-transferase P1-1

Tocopherols and tocotrienols are food ingredients that are believed to have a positive effect on health. The most studied property of both groups of compounds is their antioxidant action. Previously, we found that tocopherols and diverse tocopherol derivatives can inhibit the activity of human glutathione S-transferase P1-1 (GST P1-1). In this study we found that GST P1-1 is also inhibited, in a concentration-dependent manner, by alpha- and gamma-tocotrienol. The concentration giving 50% inhibition of GST P1-1 is 1.8 +/- 0.1 microM for alpha-tocotrienol and 0.7 +/- 0.1 microM for gamma-tocotrienol. This inhibition of GST P1-1 is noncompetitive with respect to both substrates CDNB and GSH. We also examined the 3D structure of GST P1-1 for a possible tocopherol/tocotrienol binding site. The enzyme contains a very hydrophobic pit-like structure where the phytyl tail of tocopherols and tocotrienols could fit in. Binding of tocopherol and tocotrienol to this hydrophobic region might lead to bending of the 3D structure. In this way tocopherols and tocotrienols can inhibit the activity of the enzyme; this inhibition can have far-reaching implications for humans.     

Differential distribution of tocopherols and tocotrienols in photosynthetic and non-photosynthetic tissues

Tocopherols and tocotrienols are vitamin E compounds, differing only in the saturation state of the isoprenoid side chain. Tocopherol biosynthesis, physiology and distribution have been studied in detail. Tocopherols have been found in many different plant species, and plant tissues. In contrast, comparatively little is known about the physiology and distribution of tocotrienols. These compounds appear to be considerably less widespread in the plant kingdom. In this study 80 different plant species were analysed for the presence of tocotrienols. Twenty-four species were found to contain significant amounts of tocotrienols. No taxonomic relation was apparent among the 16 dicotyledonous species that were found to contain tocotrienol. Monocotyledonous species (eight species) belonged either to the Poaceae (six species) or the Aracaceae (two species). A more detailed analysis of tocotrienol accumulation revealed the presence of tocotrienols in several non-photosynthetic tissues and organs, i.e. seeds, fruits and in latex, in concentrations up to 2000 ppm. No tocotrienols could be detected in mature photosynthetic tissues. However, we found the transient accumulation of low levels of tocotrienols in the young coleoptiles of plant species whose seeds contained tocotrienols. No measurable tocotrienol biosynthesis was apparent in coleoptiles, or in chloroplasts isolated from such coleoptiles. In line with these results, we found that tocotrienol accumulation in coleoptiles was not associated with chloroplasts. Based on our data, we conclude that tocotrienols may be transiently present in photosynthetically active tissues, however, it remains to be proven whether the tocotrienols are biosynthesised in such tissues, or imported from elsewhere in the plant.     

Vitamin E sensitive genes in the developing rat fetal brain: a high-density oligonucleotide microarray analysis

Vitamin E (tocopherols and tocotrienols) is essential for normal neurological function. Recently we have reported that the neuroprotective properties of tocotrienols are much more potent than that of the widely studied tocopherols (Sen, C.K., Khanna, S., Roy, S. and Parker, L. (2000) J. Biol. Chem. 275, 13049–13055). The objective of this study was to evaluate whether (i) oral supplementation of tocotrienols during pregnancy is bioavailable to fetal and mother brains; (ii) short-term change in dietary vitamin E levels of pregnant rats influences gene expression profile of developing fetal brains. We report that dietary tocotrienol is bioavailable to both mother and fetal brains. The enrichment is more in fetal brain tissue. Using a GeneChip microarray expression profiling approach we have identified a specific set of vitamin E sensitive genes in the developing rat fetal brain.     

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.     

Tocopherol and tocotrienol accumulation during development of caryopses from barley (Hordeum vulgare L.)

Tocotrienols are lipophilic antioxidants belonging to the tocochromanols, better known as vitamin E. Although present in cereal grains in high quantities not much is known about their function in plants. In a detailed study the temporal and spatial accumulation of tocotrienols and tocopherols during grain development in two barley cultivars was analyzed. Tocochromanols and lipids accumulated in parallel until 80% of the final dry weight of the kernels was reached. Later on the tocochromanol content did not change while the lipid content decreased. Generally, only about 13% of the tocochromanols were found in the germ fraction, whereas the pericarp fraction contained about 50% and the endosperm fraction about 37% of the tocochromanols. Altogether, about 85% of the tocochromanols were tocotrienols in both cultivars. In case of the tocopherols about 80% were found in the germ fraction and the remaining 20% in the pericarp fraction. Tocotrienols were almost equally present in the pericarp and the endosperm fraction. Individual forms of tocopherols and tocotrienols accumulated with different kinetics during barley grain development. The differences in distribution and accumulation indicate different functions of the individual tocochromanols during grain development.     

Microbiological production of tocopherols: current state and prospects

Tocopherols are antioxidants that prevent various diseases caused by oxidative stress. Tocochromanols comprise four isoforms of tocopherols and four isoforms of tocotrienols but α-tocopherol is the most abundant and active isoform in human and animal tissues. Tocopherols are used as dietary supplements for human, as food preservatives, in manufacture of cosmetics, and for fortification of animal feed. Only photosynthetic cells are known to accumulate detectable concentrations of tocopherols. Tocopherols can be extracted and purified or concentrated from vegetable oils and other higher plant materials. However, the concentrations in these higher plant materials are very low and there are high proportions of the less-active homologues of tocopherols. Among the many strains of photosynthetic microorganisms known to accumulate tocopherols, Euglena gracilis is promising for commercial production of α-tocopherol. The growth rate and α-tocopherol contents are relatively high and α-tocopherol comprise more than 97% of all the tocopherols accumulated by Euglena gracilis. Although a lot of work has been done to increase the contents and composition of tocopherols in higher plants through genetic and metabolic engineering, work on genetic modification of microorganisms for increased tocopherol accumulation is scarce. Many cultivation systems have been investigated for efficient production of tocopherol by Euglena gracilis. However, those that involve heterotrophic metabolism are more promising. Bubble columns and flat-plate photobioreactors are more suitable for commercial production of tocopherols, than the tubular, internally illuminated, and open-air photobioreactors.     

Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.     

Tocotrienols in Vellozia gigantea leaves: occurrence and modulation by seasonal and plant size effects

Vitamin E occurs in all photosynthetic organisms examined to date. Tocopherols predominate in photosynthetic tissues (α-tocopherol being the major form), while either tocopherols or tocotrienols (or both) are present in seeds. Tocotrienols have not been described in photosynthetic tissues thus far. Here, we report on the presence of tocotrienols in leaves of higher plants. Both tocopherols and tocotrienols accumulated in leaves of Vellozia gigantea, an endemic plant found in the rupestrian fields of Serra do Cipó, Brazil. Increased plant size had a remarkable effect on the vitamin E composition of leaves, α-tocopherol and β-tocotrienol levels being highest in the largest individuals, but only during the dry season. Vitamin E levels positively correlated with lipid hydroxyperoxide levels, which also increased in the largest individuals during the dry season. However, the maximum efficiency of PSII photochemistry (F _v/F _m ratio) kept above 0.75 throughout the experiment, thus indicating absence of photoinhibitory damage to the photosynthetic apparatus. It is concluded that higher plants, such as V. gigantea, can accumulate tocotrienols in leaves, aside from tocopherols, and that the levels of both tocopherols and tocotrienols in the leaves of this species are strongly modulated by seasonal and plant size effects.     

Vitamin E and drug metabolism

Tocopherols and tocotrienols are metabolized by side chain degradation initiated by cytochrome P450 (CYP)-catalyzed omega-hydroxylation followed by beta-oxidation. Whereas alpha-tocopherol is only poorly metabolized, high amounts of the final products, carboxyethyl hydroxychroman (CEHC), are found from other tocols in HepG2 cells and in human urine. CYP3A4 and CYP4F2 were suggested to be involved in tocopherol degradation. CYP3A4 metabolizes most of the drugs and is induced by many of its substrates via the activation of the pregnane X receptor (PXR). Also tocopherols and in particular tocotrienols induce the expression of a PXR-driven reporter gene and the expression of endogenous CYP3A4 and CYP3A5 which is supported by sporadic publications spread over the last 30 years. The potential interference of vitamin E with drug metabolism is discussed in the light of related complications evoked by herbal remedies.     

Functional diversity of tocochromanols in plants

Tocochromanols encompass a group of compounds with vitamin E activity essential for human nutrition. They accumulate in photooxidative organisms, e.g. in some algae and in plants, where they localize to thylakoid membranes and plastoglobules of chloroplasts. Tocochromanols contain a polar chromanol head group with a long isoprenoid side chain. Depending on the nature of the isoprenoid chain, tocopherols (containing a phytyl chain) or tocotrienols (geranylgeranyl chain) can be distinguished in plants. The tocochromanol biosynthetic pathway has been studied in Arabidopsis and Synechocystis in recent years, and the respective mutants and genes were isolated. Mutant characterization revealed that tocopherol protects lipids in photosynthetic membranes and in seeds against oxidative stress. In addition to its antioxidant characteristics, tocopherol was shown be involved in non-antioxidant functions such as primary carbohydrate metabolism. A considerable proportion of tocopherol is synthesized from free phytol suggesting that excess amounts of phytol released from chlorophyll breakdown during stress or senescence might be deposited in the form of tocopherol in chloroplasts.     

Composition of fatty acids triacylglycerols and unsaponifiable matter in Calophyllum calaba L. oil from Guadeloupe

The composition of the kernel oils of two Calophyllum species (Calophyllum calaba L. and Calophyllum inophyllum L.) was investigated. The physico-chemical properties and fatty acid composition of the kernel oils were examined. In two species, oleic acid C18:1 (39.1-50%) is the dominating fatty acid followed by linoleic acid C18:2 (21.7-31.1%) as the second major fatty acid. Stearic C18:0 (13.4-14.3%) and palmitic C16:0 (11-13.7%) acids are the major saturates. The oils contains an appreciable amount of unsaturated fatty acids (70.8-73.10%). Most of the fatty acids are present as triacylglycerol (76.7-84%), twenty one triacylglycerols are detected with predominantly unsaturated triacylglycerols. The total unsaponifiable content, its general composition and the identity of the components of the sterol and tocopherol fractions are presented. In both species, analysis of the unsaponifiable fractions revealed the preponderance of phytosterols, mainly stigmasterol (35.8-45.1%) and beta-sitosterol (41.1-43.1%). Among the eight tocopherols and tocotrienols present in two species, variations exist; alpha-tocopherol (183 mg/kg) is the main tocopherol in Calophyllum calaba L. and Delta-tocotrienol (236 mg/kg) is the dominant tocotrienol in Calophyllum inophyllum L.     

Control of macaw palm seed germination by the gibberellin/abscisic acid balance

The hormonal mechanisms involved in palm seed germination are not fully understood. To better understand how germination is regulated in Arecaceae, we used macaw palm (Acrocomia aculeata (Jacq.) Lodd. Ex Mart.) seed as a model. Endogenous hormone concentrations, tocopherol and tocotrienol and lipid peroxidation during germination were studied separately in the embryo and endosperm. Evaluations were performed in dry (D), imbibed (I), germinated (G) and non‐germinated (NG) seeds treated (+GA₃) or not treated (control) with gibberellins (GA). With GA₃ treatment, seeds germinated faster and to a higher percentage than control seeds. The +GA₃ treatment increased total bioactive GA in the embryo during germination relative to the control. Abscisic acid (ABA) concentrations decreased gradually from D to G in both tissues. Embryos of G seeds had a lower ABA content than NG seeds in both treatments. The GA/ABA ratio in the embryo was significantly higher in G than NG seeds. The +GA₃ treatment did not significantly affect the GA/ABA ratio in either treatment. Cytokinin content increased from dry to germinated seeds. Jasmonic acid (JA) increased and 1‐aminocyclopropane‐1‐carboylic acid (ACC) decreased after imbibition. In addition, α‐tocopherol and α‐tocotrienol decreased, while lipid peroxidation increased in the embryo during germination. We conclude that germination in macaw palm seed involves reductions in ABA content and, consequently, increased GA/ABA in the embryo. Furthermore, the imbibition process generates oxidative stress (as observed by changes in vitamin E and MDA).     

A survey of sesamin and composition of tocopherol variability from seeds of eleven diverse sesame (Sesamum indicum L.) genotypes using HPLC-PAD-ECD

The objective of this study was to determine the composition and content of sesamin and desmethyl tocopherols such as alpha-tocopherol (alphaT), delta-tocopherol (deltaT) and gamma-tocopherol (gammaT) in seeds of sesame (Sesamum indicum L.) for 11 genotypes conserved in the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Plant Genetic Resources Conservation Unit (PGRCU) in Griffin, Georgia, USA. Seed accessions studied were collections from eight countries worldwide, including one landrace from Thailand and two cultivars from Texas, USA. Novel methodologies and analytical techniques described herein consisted of reverse-phase high-performance liquid chromatography (HPLC) connected in series with two detection systems specific for each analyte class. Photodiode array detection was employed for sesamin analysis and electrochemical array detection was used in the determination of tocopherols. A preliminary study was conducted to assess sesamin levels in 2003 and tocopherol levels in 2004 from sesame seed samples conserved at the USDA, ARS and PGRCU. In 2005, sesame seed samples were grown, harvested and evaluated for sesamin as well as tocopherol levels. The overall results (n = 3) showed that sesamin, alphaT, deltaT and gammaT levels were 0.67-6.35 mg/g, 0.034-0.175 microg/g, 0.44-3.05 microg/g and 56.9-99.3 microg/g respectively, indicating that the sesame seed accessions contained higher levels of sesamin and gammaT compared with alphaT and deltaT. Statistical analysis was conducted and significant differences were observed among the 11 different sesame genotypes. This suggests that genetic, environmental and geographical factors influence sesamin and desmethyl tocopherol content.     

Increased ��-tocotrienol content in seeds of transgenic rice overexpressing Arabidopsis ��-tocopherol methyltransferase

Vitamin E comprises a group of eight lipid soluble antioxidant compounds that are an essential part of the human diet. The ??-isomers of both tocopherol and tocotrienol are generally considered to have the highest antioxidant activities. ??-tocopherol methyltransferase (??-TMT) catalyzes the final step in vitamin E biosynthesis, the methylation of ??- and ??-isomers to ??- and ??-isomers. In present study, the Arabidopsis ??-TMT (AtTMT) cDNA was overexpressed constitutively or in the endosperm of the elite japonica rice cultivar Wuyujing 3 (WY3) by Agrobacterium-mediated transformation. HPLC analysis showed that, in brown rice of the wild type or transgenic controls with empty vector, the ??-/??-tocotrienol ratio was only 0.7, much lower than that for tocopherol (~19.0). In transgenic rice overexpressing AtTMT driven by the constitutive Ubi promoter, most of the ??-isomers were converted to ??-isomers, especially the ??- and ??-tocotrienol levels were dramatically decreased. As a result, the ??-tocotrienol content was greatly increased in the transgenic seeds. Similarly, over-expression of AtTMT in the endosperm also resulted in an increase in the ??-tocotrienol content. The results showed that the ??-/??-tocopherol ratio also increased in the transgenic seeds, but there was no significant effect on ??-tocopherol level, which may reflect the fact that ??-tocopherol is present in very small amounts in wild type rice seeds. AtTMT overexpression had no effect on the absolute total content of either tocopherols or tocotrienols. Taken together, these results are the first demonstration that the overexpression of a foreign ??-TMT significantly shift the tocotrienol synthesis in rice, which is one of the world??s most important food crops.     

Tocopherol biosynthesis: chemistry, regulation and effects of environmental factors

Tocopherols are lipophilic antioxidants and together with tocotrienols belong to the vitamin-E family. The four forms of tocopherols (??-, ??-, ??- and ??-tocopherols) consist of a polar chromanol ring and lipophilic prenyl chain with differences in the position and number of methyl groups. The biosynthesis of tocopherols takes place mainly in plastids of higher plants from precursors derived from two metabolic pathways: homogentisic acid, an intermediate of degradation of aromatic amino acids, and phytyldiphosphate, which arises from methylerythritol phosphate pathway. The regulation of tocopherol biosynthesis in photosynthetic organisms occurs, at least partially, at the level of key enzymes as such including p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), homogentisate phytyltransferase (HPT, EC 2.5.1.-), tocopherol cyclase (TC, EC 5.4.99.-), and two methyltransferases. Tocopherol biosynthesis changes during plant development and in response toward different stresses induced by high-intensity light, drought, high salinity, heavy metals, and chilling. It is supposed that scavenging of lipid peroxy radicals and quenching of singlet oxygen are the main functions of tocopherols in photosynthetic organisms. The antioxidant action of tocopherols is related to the formation of tocopherol quinone and its following recycling or degradation. However, until now, the mechanisms of tocopherol degradation in plants have not been established in detail. This review focuses on mechanisms of tocopherols biosynthesis and its regulation in photosynthetic organisms. In addition, available information on tocopherol degradation is summarized.     

Seed size variation in the palm Euterpe edulis and the effects of seed predators on germination and seedling survival

Intraspecific variation in seed size is common in wild plant populations and has important consequences for the reproductive success of individual plants. Multiple, often conflicting evolutionary forces mediated by biotic as well as abiotic agents may maintain such a variation. In this paper we assessed seed size variation in a population of the threatened, commercially important palm Euterpe edulis in southeast Brazil. We investigated (i) how this variation affects the probability of attack by vertebrate and invertebrate post-dispersal seed predators, and (ii) if seed size influences the outcome of seeds damaged by beetles in terms of seed germination and early survival of seedlings. Euterpe edulis seeds varied in diameter from 8.3 to 14.1 mm. Neither insects nor rodents selected the seeds they preyed upon based on seed size. Seed germination and total, shoot and root biomasses of one-year seedlings were significantly and positively affected by seed size. Root biomass and seedling survival were negatively affected by seed damage caused by a scolytid beetle (Coccotrypes palmarum) whose adults bore into seeds to consume part of the endosperm, but do not oviposit on them. Seed size had a marginally significant effect on seedling survival. Therefore, if any advantage is accrued by E. edulis individuals producing large seeds, this is because of greater seed germination success and seedling vigor. If this is so, even a relatively narrow range of variation in seed size as observed in the E. edulis population studied may translate into differential success of individual plants.     

Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60(c-Src) kinase activation and death of HT4 neuronal cells

HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death. Tocotrienols were more effective than alpha-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of alpha-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60(c-Src) kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of alpha-tocotrienol, but not alpha-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. alpha-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4-10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.