Engineering vitamin E content: from Arabidopsis mutant to soy oil

We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of delta-tocopherol and decreased gamma-tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed delta-tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (gamma-tocopherol methyltransferase) in soybean, the seed accumulated to >95% alpha-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of alpha-tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.     

气象因子对油菜种子中油分积累的影响

选取2个甘蓝型油菜(Brassica napus)高油材料XZ37(含油量45.29%)和XZ366(含油量43.48%), 分别种植在南京、拉萨和西宁, 探讨种子发育过程中油分积累的差异, 并分析南京、拉萨和西宁3个地理生态环境下油菜花–角果期间的主要气象因子与种子油分之间的相关性及其对种子油分积累的影响。结果表明, 不同生态地区间种子中油分积累差异显著。在西宁种子油分快速积累始于开花后19天, 持续时长15天; 在南京种子油分快速积累始于开花后24天, 持续时长15天; 在拉萨种子油分快速积累始于开花后29天, 持续时间长达20天。研究显示, 日均温度、日均温差、日均降水量是影响甘蓝型油菜种子发育过程中油分积累的主要气候因子。不同地理生态地区, 影响油菜种子中油分积累的主要气候因子不同。日均温度是影响南京地区种子发育过程中油分积累的主要气候因子。该地区油菜开花后, 气温由低到高呈上升趋势, 成熟后期温度偏高, 不利于种子中油分积累。日均温差和日均降水量是影响拉萨和西宁两地种子油分积累的主要气候因子。两地种子发育过程中日均温差大, 种子中油分积累量大, 但由于拉萨日均降水量高于西宁, 日均温度偏低, 种子油分积累量低于西宁。因此, 在油菜种子发育过程中, 适宜的温度、较大的温差和较少的降水有利于种子积累油分, 并形成较高的含油量。     

Serum and tissue levels of tocopherols in cultured turbot (Scophthalmus maximus L.) fed vitamin E enriched diets

Turbot (Scophthalmus maximus) of approximate 110 g mean wet weight were fed three different diets supplemented with DL-alpha-tocopherol (1.4 g and 2.45 g · kg^-1 basal diet) and with non-alphatocopherols (0.29 g beta-, 1.29 g gamma-and 0.68 g delta-tocopherol kg^-1 basal diet). High dosages of alpha-tocopherol caused a linear increase of liver tocopherol (ninefold to controls). Spleen and blood serum accumulated also tocopherol. The level in muscle tissue was only poorly influenced by high vitamin E dosage. Non-alpha-tocopherols which normally do nct occur in blood and tissue have been resorbed from experiment diets and deposed in tissues in the same manner as alpha-tocopherol. The distribution of various tocopherols in the diets was reflected in muscle, spleen and serum of the fishes in the experiment. In the liver the distribution pattern of tocopherols was different to that of the diet. Conversion of alpha-tocopherol to non-alpha-tocopherpls in-vivo has not been found.     

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.     

Physical and chemical scavenging of singlet molecular oxygen by tocopherols

Singlet molecular oxygen (1O2) arising from the thermal decomposition of the endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate was used to assess the effectiveness of alpha-, beta-, gamma-, and delta-tocopherol in the physical quenching as well as the chemical reaction of 1O2. The relative physical quenching efficiencies of the tocopherol homologs were found to decrease in the order of alpha greater than or equal to beta greater than gamma greater than delta-tocopherol. The ability of physical quenching depends on a free hydroxyl group in position 6 of the chromane ring. Chemical reactivity of the tocopherol homologs with 1O2 was low, accounting for 0.1-1.5% of physical quenching with beta-tocopherol showing particularly low reactivity, resulting in the sequence alpha greater than gamma greater than delta greater than beta-tocopherol. Tocopheryl quinones were products of all tocopherol homologs, and in addition a quinone epoxide was a major product from gamma-tocopherol. This quinone epoxide was not cleaved by rat liver microsomal epoxide hydrolase; however, it reacted further with 1O2. It is concluded that methylation in position 5 of the chromane ring enhances physical quenching of 1O2, whereas chemical reactivity is favored by a methylated position 7. In view of the fact that beta-tocopherol is as effective as alpha-tocopherol in physical quenching of 1O2 but shows very low chemical reactivity, this tocopherol homolog might be particularly suitable for biological conditions in which an accumulation of oxidation products might weaken the antioxidant defense.     

Lipid characterization of a wrinkled sunflower mutant

As part of a sunflower mutagenesis program carried out to obtain lines with fatty acid profiles in their oils, the half-palmitic CAS-7 line, with ca. 14% palmitic acid content, was isolated. Attempts to obtain a homozygotic line proved to be futile due to the lack of growth of the seedlings 10-12 days after germination. At this age, the seedlings stop growing, displayed a lack of chlorophyll and poor linolenic acid content, a fatty acid intimately linked to photosynthetic membranes. Accordingly, this line has only been maintained through heterozygotic seeds. Likewise, the cotyledons of seeds from this line with medium levels of palmitic acid present a characteristic wrinkled phenotype. In the oil of these seeds, the triacylglycerol content displayed a reduction of approximately 57% with respect to the control line, although a similar reduction was not observed in the polar lipids. Furthermore this mutant has 40.0% of trilinolein, the higher content found until today in sunflower seeds. These data indicate that the CAS-7 mutant possesses a multiple phenotype having a reduced triacylglycerol seed content, a modified intraplastidial fatty acid synthesis, together with a seedling blocked growth and poor green colour and reduced chloroplast development.     

Analysis of sequential accumulation of individual pigeonpea protease inhibitors during seed development

Seeds of pigeonpea are known to accumulate protease inhibitors (PIs), belonging to the Bowman-Burk inhibitor family. PIs are important for natural defense against phytophagous insect pests. Most insects attack crops at the early stages of seed development. Accumulation patterns of individual PIs and their relationship with each other were studied in developing seeds of 76 pigeonpea lines derived from BDN2 cultivar by ethyl methane sulfonate induced chemical mutagenesis. PIs extracted from these lines, collected between 10 and 40 days after flowering (DAF) and from mature seeds of BDN2 cultivar, were detected by using gel X-ray film contact print method. A total of 9 trypsin–chymotrypsin inhibitors were detected in mature seeds. All the nine PIs were capable of inhibiting proteases. Appearance of detectable levels of individual PIs started around 10 DAF. The PI-3 appeared early and was the most stable. It was accumulated in all the studied lines and was also detected in 84 % of the samples collected 10 DAF. In particular, 14 DAF, the individual PIs were accumulated and accumulation sequence was observed (PI-3, PI-2, PI-5, PI-7, PI-6, PI-4, PI-8, PI-9 and PI-1). Accumulation continued up to 40 DAF when seeds started hardening. Chemical mutagenesis could not produce any variation in the profile of individual PIs in the 76 studied lines in mature seeds. The process of accumulation of inhibitors is sturdy and mutagenesis fails to alter it. The robust mechanism is responsible for early appearance of PI-3. Early accumulated PIs in this study need further exploration for strengthening natural defense of pigeonpea against pests.     

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.     

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.     

Alterations in tocopherol cyclase activity in transgenic and mutant plants of Arabidopsis affect tocopherol content, tocopherol composition, and oxidative stress

Tocopherol belongs to the Vitamin E class of lipid soluble antioxidants that are essential for human nutrition. In plants, tocopherol is synthesized in plastids where it protects membranes from oxidative degradation by reactive oxygen species. Tocopherol cyclase (VTE1) catalyzes the penultimate step of tocopherol synthesis, and an Arabidopsis (Arabidopsis thaliana) mutant deficient in VTE1 (vte1) is totally devoid of tocopherol. Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from alpha-tocopherol to gamma-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves. Tocopherol deficiency in vte1 resulted in the increase in ascorbate and glutathione, whereas accumulation of tocopherol in VTE1 overexpressing plants led to a decrease in ascorbate and glutathione. Deficiency in one antioxidant in vte1, vtc1 (ascorbate deficient), or cad2 (glutathione deficient) led to increased oxidative stress and to the concomitant increase in alternative antioxidants. Double mutants of vte1 were generated with vtc1 and cad2. Whereas growth, chlorophyll content, and photosynthetic quantum yield were very similar to wild type in vte1, vtc1, cad2, or vte1vtc1, they were reduced in vte1cad2, indicating that the simultaneous loss of tocopherol and glutathione results in moderate oxidative stress that affects the stability and the efficiency of the photosynthetic apparatus.     

Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin

Carboxychroman metabolites of the major dietary tocopherols are excreted in human urine, but the mechanism of their synthesis is unknown. We employed well-characterized inhibitors of specific cytochrome P-450 (CYP) enzymes to determine which form was likely involved in tocopherol side chain oxidation. Ketoconozole (1.0 microM), a potent and selective inhibitor of CYP3A, substantially inhibited metabolism of gamma- and alpha-tocopherol in rat primary hepatocytes, and metabolism of gamma- and delta-tocopherol in HepG2/C3A cells. Sulphaphenazole and cyclosporin, inhibitors of CYP2C and CYP27, respectively, were without effect. Sesamin, a sesame lignan that causes elevation of tissue tocopherol concentration in rats, strongly inhibited tocopherol metabolism by HepG2/C3A cells at 1.0 microM. These results support a CYP3A-dependent mechanism of side chain metabolism of tocopherols to water-soluble carboxychromans, and provide the first evidence of a specific enzyme involved in vitamin E metabolism. The data further suggest that sesamin increases tissue tocopherol concentration by inhibiting tocopherol catabolism.     

Succinate-ubiquinone reductase linked recycling of alpha-tocopherol in reconstituted systems and mitochondria: requirement for reduced ubiquinone.

Studies have demonstrated that accumulation of mitochondrial tocopheroxyl radical, the primary oxidation product of alpha-tocopherol, accompanies rapid consumption of tocopherol. Enzyme-linked electron flow lowers both the steady-state concentration of the radical and the consumption of tocopherol. Reduction of tocopheroxyl radical by a mitochondrial electron carrier(s) seems a likely mechanism of tocopherol recycling. Succinate-ubiquinone reductase (complex II) was incorporated into liposomes in the presence of tocopherol and ubiquinone-10. After inducing formation of tocopheroxyl radical, it was possible to show that reduced ubiquinone prevents radical accumulation and tocopherol consumption. There was no evidence of direct reduction of tocopheroxyl radical by succinate-reduced complex II. These reactions were also measured using ubiquinone-1 and alpha-C-6-chromanol (2,5,7,8-tetramethyl-2-(4'-methylpentyl)-6-chromanol) which are less hydrophobic analogues of ubiquinone-10 and alpha-tocopherol. Mitochondrial membranes were made deficient in ubiquinone but sufficient in alpha-tocopherol and were reconstituted with added quinone. With these membranes it was shown that mitochondrial enzyme-linked reduction of ubiquinone protects alpha-tocopherol from consumption, and there is a requirement for ubiquinone. This complements the observations made in liposomes and we propose that reduced mitochondrial ubiquinones have a role in alpha-tocopherol protection, presumably through efficient reduction of the tocopheroxyl radical.     

Chickpea Defensive Proteinase Inhibitors Can Be Inactivated by Podborer Gut Proteinases

Developing chickpea (Cicer arietinum L.) seeds 12 to 60 d after flowering (DAF) were analyzed for proteinase inhibitor (Pi) activity. In addition, the electrophoretic profiles of trypsin inhibitor (Ti) accumulation were determined using a gel-radiographic film-contact print method. There was a progressive increase in Pi activity throughout seed development, whereas the synthesis of other proteins was low from 12 to 36 DAF and increased from 36 to 60 DAF. Seven different Ti bands were present in seeds at 36 DAF, the time of maximum podborer (Helicoverpa armigera) attack. Chickpea Pis showed differential inhibitory activity against trypsin, chymotrypsin, H. armigera gut proteinases, and bacterial proteinase(s). In vitro proteolysis of chickpea Ti-1 with various proteinases generated Ti-5 as the major fragment, whereas Ti-6 and -7 were not produced. The amount of Pi activity increased severalfold when seeds were injured by H. armigera feeding. In vitro and in vivo proteolysis of the early- and late-stage-specific Tis indicated that the chickpea Pis were prone to proteolytic digestion by H. armigera gut proteinases. These data suggest that survival of H. armigera on chickpea may result from the production of inhibitor-insensitive proteinases and by secretion of proteinases that digest chickpea Pis.     

Influence of systemic insecticides on lipid biosynthesis in seeds of Indian mustard (Brassica juncea L.)

The systemic insecticides namely phorate (Thimet 10 G) oxydemeton methyl (metasystox 25 EC) and dimethoate (Rogor 30 EC) decreased oil content in the developing seeds of Indian mustard (Brassica juncea L.) but showed an increase in the mature seeds. The inhibitory effect in the developing seed was accompanied by an increase in soluble sugars and a corresponding decrease in malate dehydrogenase and G6P dehydrogenase activity. The relative proportion of triacylglycerols and glycolipids decreased significantly while that of phospholipids and free fatty acids increased in the developing seeds. In the mature seeds, the proportion of triacylglycerols did not change appreciably from that in control. The erucic acid synthesis which was less at 10 and 20 days after fertilization (DAF) increased at 30 DAF with oxydemeton methyl and dimethoate; phorate was ineffective. In mature seeds, the proportion of erucic acid increased at the cost of linoleic and linolenic acids. All the insecticides appreciably decreased the rate of [1-¹⁴C]acetate incorporation into lipids both in vivo and in vitro experiments. In the in vivo experiment, the synthesis of polar lipids was enhanced at 10 and 20 DAF, and the higher doses of oxydemeton methyl and dimethoate at 30 DAF. On the other hand, the ¹⁴C-incorporation into triacylglycerols showed an opposite trend to that of polar lipids. In the in vitro experiment, oxydemeton methyl and dimethoate enhanced the synthesis of polar lipids at 10 and 20 DAF while these inhibited it at 30 DAF. The synthesis of triacylglycerols was inhibited by the use of these insecticides.     

Lipid biosynthesis in seeds of mustard (Brassica juncea) influenced by zinc and sulphur deficiency

In developing seeds of mustard ( Brassica juncea L. cv. RLM 198) the period between 20 and 30 days after fertilization (DAF) was identified as the period of active lipid biosynthesis, although dry matter continued to accumulate until maturity. The period of lipid synthesis was associated with a decrease in starch, soluble sugars and protein, thus, giving rise to precursors for the biosynthesis of lipids. Besides decreasing the dry matter content (on both % and seed basis), Zn and S deficiency caused a significant ( P > 0.05) reduction in oil content. As compared to control, the decrease in oil content was 11, 12 and 18% at 30 DAF and 4, 9 and 16% at maturity in Zn, S and (Zn+S) deficient treatments, respectively. Throughout the period of seed development, a significant decrease in starch and protein with a slight accumulation of soluble sugars was observed due to deficiency of Zn or S. The rate of [l-¹⁴C]-acetate incorporation into total lipids, which was maximal at 30 DAF, also displayed a significant decrease due to the abovementioned mineral deficiencies. Addition of Zn or S in vitro, enhanced the lipid synthesis at all stages of seed development. Under Zn and S deficiency, the phospholipids increased from 10 to 30 DAF and then declined until maturity. However, the proportion of glycolipids and free fatty acids increased, with a corresponding decrease in total glycerides. Further, in deficiency treatments, there was an increase in 22:1 with a corresponding decrease in 18:1, 18:2 and 18:3 in developing and mature mustard seeds.     

Modulation of proteasome activity by vitamin E in THP-1 monocytes

In THP-1 monocytes, cellular proteasome inhibition by ritonavir or ALLN is associated with increased production of oxidative stress. Both compounds produced comparable amounts of oxidative stress; however, normalization by alpha-tocopherol occurred solely after inhibition by ritonavir, and not by ALLN. Similar to that, alpha-tocopherol could normalize the reduced formation of 3-nitrotyrosine-modified proteins only after ritonavir treatment. In the absence of any proteasome inhibitor, intrinsic cellular proteasome activity was not modulated by alpha-, beta-, and gamma-tocopherols; however, delta-tocopherol, alpha-tocotrienol, and alpha-tocopheryl phosphate could significantly inhibit cellular proteasome activity and increased the level of p27(Kip1) and p53. Since oxidative stress was reduced by alpha-tocopherol only after proteasome inhibition by ritonavir and not by ALLN, it is concluded that, in this experimental system, alpha-tocopherol does not act as an antioxidant but interferes with the inhibitory effect of ritonavir.     

A new insight into application for barley chromosome addition lines of common wheat: achievement of stigmasterol accumulation

Barley (Hordeum vulgare) has a much higher content of bioactive substances than wheat (Triticum aestivum). In order to investigate additive and/or synergistic effect(s) on the phytosterol content of barley chromosomes, we used a series of barley chromosome addition lines of common wheat that were produced by normal crossing. In determining the plant sterol levels in 2-week-old seedlings and dry seeds, we found that the level of stigmasterol in the barley chromosome 3 addition (3H) line in the seedlings was 1.5-fold higher than that in the original wheat line and in the other barley chromosome addition lines, but not in the seeds. Simultaneously, we determined the overall expression pattern of genes related to plant sterol biosynthesis in the seedlings of wheat and each addition line to assess the relative expression of each gene in the sterol pathway. Since we elucidated the CYP710A8 (cytochrome P450 subfamily)-encoding sterol C-22 desaturase as a key characteristic for the higher level of stigmasterol, full-length cDNAs of wheat and barley CYP710A8 genes were isolated. These CYP710A8 genes were mapped on chromosome 3 in barley (3H) and wheat (3A, 3B, and 3D), and the expression of CYP710A8 genes increased in the 3H addition line, indicating that it is responsible for stigmasterol accumulation. Overexpression of the CYP710A8 genes in Arabidopsis increased the stigmasterol content but did not alter the total sterol level. Our results provide new insight into the accumulation of bioactive compounds in common wheat and a new approach for assessing plant metabolism profiles.