Engineering plant shikimate pathway for production of tocotrienol and improving herbicide resistance

Tocochromanols (tocopherols and tocotrienols), collectively known as vitamin E, are essential antioxidant components of both human and animal diets. Because of their potential health benefits, there is a considerable interest in plants with increased or customized vitamin E content. Here, we have explored a new strategy to reach this goal. In plants, phenylalanine is the precursor of a myriad of secondary compounds termed phenylpropanoids. In contrast, much less carbon is incorporated into tyrosine that provides p-hydroxyphenylpyruvate and homogentisate, the aromatic precursors of vitamin E. Therefore, we intended to increase the flux of these two compounds by deriving their synthesis directly at the level of prephenate. This was achieved by the expression of the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene in tobacco (Nicotiana tabacum) plants that already overexpress the Arabidopsis p-hydroxyphenylpyruvate dioxygenase coding sequence. A massive accumulation of tocotrienols was observed in leaves. These molecules, which were undetectable in wild-type leaves, became the major forms of vitamin E in the leaves of the transgenic lines. An increased resistance of the transgenic plants toward the herbicidal p-hydroxyphenylpyruvate dioxygenase inhibitor diketonitril was also observed. This work demonstrates that the synthesis of p-hydroxyphenylpyruvate is a limiting step for the accumulation of vitamin E in plants.     

Identification of a 3.2 kb 5'-flanking region of the murine keratocan gene that directs beta-galactosidase expression in the adult corneal stroma of transgenic mice

The mouse keratocan gene (Ktcn) expression tracks the corneal morphogenesis during eye development and becomes restricted to keratocytes of the adult, implicating a cornea-specific gene regulation of the mouse Ktcn [J. Biol. Chem., 273 (1998) 22 584–22 588]. To examine the functionality of the mouse Ktcn promoter, we have cloned and sequenced a 3.2 kb genomic DNA fragment 5′ of the mouse Ktcn gene, which was used to prepare a reporter gene construct that contained the 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, and β-geo hybrid reporter gene. The β-galactosidase (βGal) activity was assayed in tissues of two of five transgenic mouse lines obtained via microinjection. In adult transgenic mice, βGal activity was detected only in cornea, not in other tissues (e.g. lens, retina, sclera, lung, heart, liver, diaphragm, kidney, and brain). During ocular development, the spatial–temporal expression patterns of the βGal recapitulated that of endogenous Ktcn in transgenic mice. Using XGal staining, strong βGal activity was first detected in periocular tissues of E13.5 embryos, and restricted to corneal keratocytes at E14.5 and thereafter. Interestingly, in addition to cornea, βGal activity was transiently found in some non-ocular tissues, i.e. ears, snout, and limbs of embryos of E13.5 and E14.5 but was no longer detected in those tissues of E16.5 embryos. The transient expression of endogenous keratocan in non-ocular tissues during embryonic development was confirmed by in situ hybridization. Taken together, our results suggest that the 3.2 kb Ktcn promoter contains sufficient cis-regulatory elements to drive heterologous minigene expression in cells expressing keratocan. The identification of keratocyte-specific expression of βGal reporter gene in the adult transgenic mice is an important first step in characterizing the Ktcn promoter in order to use it to drive a foreign gene expression in corneal stroma.     

Generation and analyses of the transgenic potatoes expressing heterologous thermostable β-amylase

β-Amylase hydrolyzes the -1,4-glycosidic linkages of starch resulting in the release of maltose. This reaction is of industrial importance for maltose production and for the preparation process of fermented foods and alcoholic beverages. A demand for an acceleration of the rate of enzymatic cleavage of the starch macro-molecule is a prerequisite for large-scale and highly efficient production. Increasing the temperature up to the optimum of approximately 60 °C can significantly speed up the reaction. However, at higher temperatures, the effect on protein denaturation becomes dominant, and the conversion rate decreases. The primary objective of this study was to generate transgenic plants of the “Kennebec” potato variety for production of thermostable β-amylase using Agrobacterium-mediated transformation. Four chimeric genes encoding the β-amylase with or without signal peptide sequences for targeting expression in cytoplasm, amyloplasts, or vacuoles were constructed and driven by high tuber expression promoter from Sucrose synthetase gene Sus4. Forty-two transgenic lines were selected for this study. Transgenic lines with various β-amylase constructs were verified for the existence and expression of the transgenes by PCR approaches. The expression level of the introduced β-amylase protein was estimated by immunoblot analyses using polyclonal antibodies. Recombinant β-amylase was successfully expressed in Escherichia coli B21 (DE3), and temperature ranges of these inducible recombinant proteins were found to be between 40 and 90 °C. This enzymatic complex produced in the in vitro cultured microtubers and field-grown tubers from transgenic potatoes were proved to be stable and active at 60 °C. The relative activities of β-amylase in tubers of field-grown potatoes were compared, and the maximum increase was found with transgenic line #6A of the pSUS4-AMY construct which has an 11-fold greater increase than the untransformed “Kennebec”. Variations of the chemical compositions were found in the selected transgenic lines. Results of this study suggest the feasibility of utilizing thermostable β-amylase in transgenic potatoes for the starch-processing industries.     

Tocotrienols in health and disease: the other half of the natural vitamin E family

Tocochromanols encompass a group of compounds with vitamin E activity essential for human nutrition. Structurally, natural vitamin E includes eight chemically distinct molecules: -, β-, γ- and δ-tocopherol; and -, β-, γ- and δ-tocotrienol. Symptoms caused by -tocopherol deficiency can be alleviated by tocotrienols. Thus, tocotrienols may be viewed as being members of the natural vitamin E family not only structurally but also functionally. Palm oil and rice bran oil represent two major nutritional sources of natural tocotrienol. Taken orally, tocotrienols are bioavailable to all vital organs. The tocotrienol forms of natural vitamin E possesses powerful hypocholesterolemic, anti-cancer and neuroprotective properties that are often not exhibited by tocopherols. Oral tocotrienol protects against stroke-associated brain damage in vivo. Disappointments with outcomes-based clinical studies testing the efficacy of -tocopherol need to be handled with caution and prudence recognizing the untapped opportunities offered by the other forms of natural vitamin E. Although tocotrienols represent half of the natural vitamin E family, work on tocotrienols account for roughly 1% of the total literature on vitamin E. The current state of knowledge warrants strategic investment into investigating the lesser known forms of vitamin E.     

Over-expression of strawberry d-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance

Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed transgenic potato plants (Solanum tuberosum L. cv. Taedong Valley) over-expressing strawberry GalUR gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the GalUR gene in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid (AsA) levels in transgenic tubers were determined by high-performance liquid chromatography (HPLC). The over-expression of GalUR resulted in 1.6–2-fold increase in AsA in transgenic potato and the levels of AsA were positively correlated with increased GalUR activity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen (MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of GalUR gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control.     

Vitamin E supplementation increases the attractiveness of males' scent for female European green lizards

Background

In spite that chemoreception is important in sexual selection for many animals, such as reptiles, the mechanisms that confer reliability to chemical signals are relatively unknown. European green lizards (Lacerta viridis) have substantial amounts of α-tocopherol ( = vitamin E) in their femoral secretions. Because vitamin E is metabolically important and can only be attained from the diet, its secretion is assumed to be costly. However, its role in intraspecific communication is unknown.

Methodology/Principal Findings

Here, we experimentally show that male European green lizards that received a dietary supplement of vitamin E increased proportions of vitamin E in their femoral secretions. Furthermore, our experiments revealed that females preferred to use areas scent marked by males with experimentally increased vitamin E levels in their secretions. Finally, female preferences were stronger when vitamin E differences between a pair of males'' secretions were larger.

Conclusions/Significance

Our results demonstrate that female green lizards are able to discriminate between males based on the vitamin E content of the males'' femoral secretions. We suggest that the possible cost of allocating vitamin E to secretions, which might be dependent on male quality, may be a mechanism that confers reliability to scent marks of green lizards and allows their evolution as sexual signals.     

Photoprotective potential of lycopene, β-carotene, vitamin E, vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, β-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10–15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low μM concentrations of vitamin E, vitamin C, or carnosic acid but not with β-carotene or lycopene. Indeed, in the presence of 0.5–1.0 μM β-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5–2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, β-carotene or lycopene (0.5–1.0 μM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and β-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Genetic Transformation of Tobacco with the Trehalose Synthase Gene from Grifola frondosa Fr. Enhances the Resistance to Drought and Salt in Tobacco

Trehalose is a non-reducing disaccharide of glucose that functions as a protectant in the stabilization of biological structures and enhances the tolerance of organisms to abiotic stress. In the present study, we report on the expression of the Grifolafrondosa Fr. trehalose synthase (TSase) gene for manipulating abiotic stress tolerance in tobacco (Nicotiana tabaccum L.). The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and was transferred into tobacco by Agrobacterium tumefaciens EHA105. Compared with non-transgenic plants, transgenic plants were able to accumulate high levels of products of trehalose, which were increased up to 2.126-2.556 mg/g FW, although levels were undetectable in non-transgenic plants. This level of trehalose in transgenic plants was 400-fold higher than that of transgenic tobacco plants cotransformed with Escherichia coli TPS and TPP on independent expression cassettes, twofold higher than that of transgenic rice plants transformed with a bifunctional fusion gene (TPSP) of the trehalose-6-phosphate (T-6-P) synthase (TPS) and T-6-P phosphatase (TPP) of E. coli, and 12-fold higher than that of transgenic tobacco plants transformed the yeast TPS1 gene.It has been reported that transgenic plants with E. coli TPS and/or TPP were severely stunted and had morphological alterations of their roots. Interestingly, our transgenic plants have obvious morphological changes, including thick and deep-coloured leaves, but show no growth inhibition; moreover, these morphological changes can restore to normal type in T2 progenies. Trehalose accumulation in 35S-35S:TSase plants resulted in increased tolerance to drought and salt, as shown by the results of tests on drought, salt tolerance, and drought physiological indices, such as water content in excised leaves, malondialdehyde content, chlorophyll a and b contents, and the activity of superoxide dismutase and peroxidase in excised leaves. These results suggest that transgenic plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought and salt.     

Studies on the protective role of vitamin C and E against polychlorinated biphenyl (Aroclor 1254)--induced oxidative damage in Leydig cells

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. Polychlorinated biphenyls (PCBs) are global environmental contaminants that cause disruption of the endocrine system in human and animals. The present study was conducted to elucidate the protective role of vitamin C and E against Aroclor 1254-induced changes in Leydig cell steroidogenesis and antioxidant system. Adult male rats were dosed for 30 days with daily intraperitoneal (ip) injection of 2 mg/kg Aroclor or vehicle (corn oil). One group of rats was treated with vitamin C (100 mg/kg bw/day) while the other group was treated with vitamin E (50 mg/kg bw/day) orally, simultaneously with Aroclor 1254 for 30 days. One day after the last treatment, animals were euthanized and blood was collected for the assay of serum hormones such as luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin (PRL), triiodothyronine (T₃), thyroxine (T₄), testosterone and estradiol. Testes were quickly removed and Leydig cells were isolated in aseptic condition. Purity of Leydig cells was determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were used for quantification of cell surface LH receptors and steroidogenic enzymes such as cytochrome P₄₅₀ side chain cleavage enzyme (P₄₅₀scc), 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β- HSD). Leydig cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), γ-glutamyl transpeptidase (γ-GT), glutathione-S-transferase (GST) and non-enzymatic antioxidants such as vitamin C and E were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. Aroclor 1254 treatment significantly reduced the serum LH, TSH, PRL, T₃, T₄, testosterone and estradiol. In addition to this, Leydig cell surface LH receptors, activities of the steroidogenic enzymes such as cytochrome P₄₅₀scc, 3β-HSD, 17β-HSD, antioxidant enzymes SOD, CAT, GPX, GR, γ-GT, GST and non-enzymatic antioxidants such as vitamin C and E were significantly diminished whereas, LPO and ROS were markedly elevated. However, the simultaneous administration of vitamin C and E in Aroclor 1254 exposed rats resulted a significant restoration of all the above-mentioned parameters to the control level. These observations suggest that vitamin C and E have ameliorative role against adverse effects of PCB on Leydig cell steroidogenesis.     

转TPSP融合基因小麦的耐旱相关特性

在获得转TPSP基因小麦纯合株系的基础上,对3个转基因株系的耐旱相关生理特性进行了分析。脯氨酸含量测定显示,干旱胁迫过程中小麦叶片中脯氨酸含量逐渐增加,且3个转基因株系叶片中脯氨酸的积累速度和积累量均显著高于非转基因对照;叶绿素荧光参数测定显示,3个转基因株系的Fv/Fm值在胁迫过程中均略高于非转基因对照,转基因株系4-4-4的Fv/Fo值显著高于非转基因对照,表明转基因株系在水分胁迫条件下光合系统II（PSII）的光合效率有所增强;转基因小麦耐旱性鉴定显示：模拟干旱胁迫100h时对照小麦叶片几乎全部萎蔫,而3个转基因株系均表现出较强的耐旱性;复水24h后转基因株系4-9-1、4-4-4和30-1-2的叶片黄化率分别为25.2%、23.3%和27.6%,显著低于非转基因对照（48.8%）。上述研究结果表明转TPSP基因小麦具有较强的耐旱能力,为转基因材料进一步应用于小麦抗旱育种提供了依据。     

Content of fat-soluble vitamins in human blood serum during the use of food additives with vitamin E and carotene of plant origin]

Vitamin E, A and carotene levels in blood serum of human that were affected by the definite irradiation doses accepting the admixtures of vitamin E and carotene preparations were estimated. The consuming of vitamin E increases vitamin E and A content in blood serum. While obtaining carotene significant increase of vitamin E and carotene content is observed, but it does not influence to vitamin A content. Simultaneous application of both preparations leads to vitamin E level growth being similar to persons, receiving only vitamin E; in this case the vitamin A and carotene content is increasing more actively and produces as early as in the month the carotene accumulation in blood serum. Along side with this the vitamin A content increases only in 3 month, and vitamin E practically does not change. Among estimated persons 45 years older revealed content of vitamin E increase in 3 month of both application of preparation. Application of vitamin E and carotene preparations have expressive positive change of fat-soluble vitamin status in blood serum.     

Tocotrienols, the unsaturated forms of vitamin E, can function as antioxidants and lipid protectors in tobacco leaves

Vitamin E is a generic term for a group of lipid-soluble antioxidant compounds, the tocopherols and tocotrienols. While tocotrienols are considered as important vitamin E components in humans, with functions in health and disease, the protective functions of tocotrienols have never been investigated in plants, contrary to tocopherols. We took advantage of the strong accumulation of tocotrienols in leaves of double transgenic tobacco (Nicotiana tabacum) plants that coexpressed the yeast (Saccharomyces cerevisiae) prephenate dehydrogenase gene (PDH) and the Arabidopsis (Arabidopsis thaliana) hydroxyphenylpyruvate dioxygenase gene (HPPD) to study the antioxidant function of those compounds in vivo. In young leaves of wild-type and transgenic tobacco plants, the majority of vitamin E was stored in thylakoid membranes, while plastoglobules contained mainly delta-tocopherol, a very minor component of vitamin E in tobacco. However, the vitamin E composition of plastoglobules was observed to change substantially during leaf aging, with alpha-tocopherol becoming the major form. Tocotrienol accumulation in young transgenic HPPD-PDH leaves occurred without any significant perturbation of photosynthetic electron transport. Tocotrienols noticeably reinforced the tolerance of HPPD-PDH leaves to high light stress at chilling temperature, with photosystem II photoinhibition and lipid peroxidation being maintained at low levels relative to wild-type leaves. Very young leaves of wild-type tobacco plants turned yellow during chilling stress, because of the strongly reduced levels of chlorophylls and carotenoids, and this phenomenon was attenuated in transgenic HPPD-PDH plants. While sugars accumulated similarly in young wild-type and HPPD-PDH leaves exposed to chilling stress in high light, a substantial decrease in tocotrienols was observed in the transgenic leaves only, suggesting vitamin E consumption during oxygen radical scavenging. Our results demonstrate that tocotrienols can function in vivo as efficient antioxidants protecting membrane lipids from peroxidation.     

β-Carotene, vitamin C and vitamin E content of the marine microalga Dunaliella tertiolecta cultured with different nitrogen sources

Variations in the β-carotene, vitamin C and vitamin E content of D. tertiolecta have been shown to result from the nitrogen source used in the culture medium. Differences of 101%, 38% and 69% have been found in β-carotene, ascorbic acid and tocopherol content in mg/g of dry matter, respectively, and differences of 147%, 63% and 37% occurred in β-carotene, vitamin C and E concentrations in mg/litre of culture, respectively. Considering the β-carotene, vitamin C and vitamin E content in mg/g of chlorophyll a, maximum variations occurred in β-carotene content, with differences of 145% among the different nitrogen sources. Maximum β-carotene and vitamin C values were found in urea cultures, whereas urea cultures showed the minimum values for vitamin E.     

Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance

Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is part of the biosynthetic pathway leading to plastoquinone and vitamin E. This enzyme is also the molecular target of various new bleaching herbicides for which genetically engineered tolerant crops are being developed. We have expressed a sensitive bacterial hppd gene from Pseudomonas fluorescens in plastid transformants of tobacco and soybean and characterized in detail the recombinant lines. HPPD accumulates to approximately 5% of total soluble protein in transgenic chloroplasts of both species. As a result, the soybean and tobacco plastid transformants acquire a strong herbicide tolerance, performing better than nuclear transformants. In contrast, the over-expression of HPPD has no significant impact on the vitamin E content of leaves or seeds, quantitatively or qualitatively. A new strategy is presented and exemplified in tobacco which allows the rapid generation of antibiotic marker-free plastid transformants containing the herbicide tolerance gene only. This work reports, for the first time, the plastome engineering for herbicide tolerance in a major agronomic crop, and a technology leading to marker-free lines for this trait.     

Effects of tocopherols and 2,2'-carboxyethyl hydroxychromans on phorbol-ester-stimulated neutrophils

Tocopherol vitamers [e.g., alpha-, gamma- and delta-tocopherol (-TOC, γ-TOC and δ-TOC, respectively)] and their water-soluble 2,2′-carboxyethyl hydroxychroman metabolites (e.g., -, γ- and δ-CEHC) all possess antioxidant properties. As a consequence, and similarly to other natural antioxidants, vitamin E compounds may be useful in preventing inflammatory and oxidative-stress-mediated diseases. In this study, we investigated the concentration-dependent effect of tocopherols and water-soluble metabolites on a key event in oxidative stress, for example, the oxidative burst in neutrophils. It was found that not only -TOC but also γ-TOC and δ-TOC as well as -, γ- and δ-CEHC at physiological concentrations inhibit superoxide anion (O₂^•−) production in phorbol-ester-stimulated neutrophils. This effect was mediated by the inhibition of the translocation and activation of protein kinase C (PKC) enzyme, which is the key event in the phorbol-ester signaling. Importantly, CEHCs were stronger inhibitors of PKC as compared with the vitamer precursors, and the gamma forms of both tocopherol and CEHC showed the highest inhibitory activities. Tocopherols, but not CEHCs, directly inhibit the fully activated nicotine–adenine–dinucleotide phosphate (NADPH) oxidase. However, none of the test compounds was able to directly scavenge O₂^•− when tested in a cell-free system. In conclusion, vitamin E compounds can control the neutrophil oxidative burst through the negative modulation of PKC-related signaling and NADPH oxidase activity. As an original finding, we observed that CEHC metabolites might contribute to regulate PKC activity in these cells. These results may have important implications in the anti-inflammatory and antioxidant role of vitamin E compounds.     

Constitutive E2F expression in tobacco plants exhibits altered cell cycle control and morphological change in a cell type-specific manner

The E2F family plays a pivotal role in cell cycle control and is conserved among plants and animals, but not in fungi. This provides for the possibility that the E2F family was integrated during the development of higher organisms, but little is known about this. We examined the effect of E2F ectopically expressed in transgenic tobacco (Nicotiana tabacum) plants on growth and development using E2Fa (AtE2F3) and DPa from Arabidopsis. E2Fa-DPa double transgenic lines exhibited altered phenotypes with curled leaves, round shaped petals, and shortened pistils. In mature but not immature leaves of the double transgenic lines, there were enlarged nuclei with increasing ploidy levels accompanied by the ectopic expression of S phase- but not M phase-specific genes. This indicates that a high expression of E2F promotes endoreduplication by accelerating S phase entry in terminally differentiated cells with limited mitotic activity. Furthermore, mature leaves of the transgenic plants contained increased numbers of small cells, especially on the palisade (adaxial) side of the outer region toward the edge, and the leaf strips exhibited hormone-independent callus formation when cultured in vitro. These observations suggest that an enhanced E2F activity modulates cell cycle in a cell type-specific manner and affects plant morphology depending on a balance between activities for committing to S phase and M phase, which likely differ between organs or tissues.     

天山雪莲SiSAD基因与拟南芥AtFAB2基因转化 烟草的抗寒性分析

通过转基因烟草(Nicotiana tabacum)验证天山雪莲(Saussurea involucrata) Δ9硬脂酰-ACP脱饱和酶基因SiSAD与拟南芥(Arabidopsis thaliana)中同源基因AtFAB2的抗寒性功能。利用农杆菌介导法将植物表达载体P_SiSAD:AtFAB2和P_SiSAD:SiSAD导入烟草, 然后将2种转基因和野生型烟草分别置于20°C、10°C、5°C、0°C及-2°C下处理2小时, 检测其相对电导率、丙二醛(MDA)含量、叶绿素荧光参数(F_v/F_m)及脂肪酸含量。将-2°C处理2小时后的植株置于25°C培养1周进行生长恢复实验。结果表明, 生长恢复实验中转SiSAD基因烟草的恢复效果显著优于转AtFAB2基因和野生型烟草。在0°C和-2°C处理2小时后, 转SiSAD、AtFAB2基因型和野生型烟草的相对电导率和丙二醛含量呈现显著递增趋势; 转SiSAD、AtFAB2基因型烟草的F_v/F_m显著高于野生型烟草, 其中, 转SiSAD基因烟草的F_v/F_m显著高于转AtFAB2基因烟草。转AtFAB2基因型和野生型烟草的油酸(C18:1)含量随着温度的降低逐渐升高后降低并在0°C时达到最高值; 而转SiSAD基因型烟草C18:1含量持续升高, 并在-2°C时达到最高值, 其含量分别是转AtFAB2基因型和野生型烟草的1.58倍和1.7倍。以上结果表明, 天山雪莲Δ9硬脂酰-ACP脱饱和酶基因SiSAD与拟南芥中同源基因AtFAB2均可以显著增强非低温驯化烟草的抗寒性, 但是SiSAD基因效果显著优于AtFAB2。     

Serum α-tocopherol and γ-tocopherol concentrations and prostate cancer risk in the PLCO Screening Trial: a nested case-control study

Background

Vitamin E compounds exhibit prostate cancer preventive properties experimentally, but serologic investigations of tocopherols, and randomized controlled trials of supplementation in particular, have been inconsistent. Many studies suggest protective effects among smokers and for aggressive prostate cancer, however.

Methods

We conducted a nested case-control study of serum α-tocopherol and γ-tocopherol and prostate cancer risk in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, with 680 prostate cancer cases and 824 frequency-matched controls. Multivariate-adjusted, conditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CIs) for tocopherol quintiles.

Results

Serum α-tocopherol and γ-tocopherol were inversely correlated (r = −0.24, p<0.0001). Higher serum α-tocopherol was associated with significantly lower prostate cancer risk (OR for the highest vs. lowest quintile = 0.63, 95% CI 0.44–0.92, p-trend 0.05). By contrast, risk was non-significantly elevated among men with higher γ-tocopherol concentrations (OR for the highest vs. lowest quintile = 1.35, 95% CI 0.92–1.97, p-trend 0.41). The inverse association between prostate cancer and α-tocopherol was restricted to current and recently former smokers, but was only slightly stronger for aggressive disease. By contrast, the increased risk for higher γ-tocopherol was more pronounced for less aggressive cancers.

Conclusions

Our findings indicate higher α-tocopherol status is associated with decreased risk of developing prostate cancer, particularly among smokers. Although two recent controlled trials did not substantiate an earlier finding of lower prostate cancer incidence and mortality in response to supplementation with a relatively low dose of α-tocopherol, higher α-tocopherol status may be beneficial with respect to prostate cancer risk among smokers. Determining what stage of prostate cancer development is impacted by vitamin E, the underlying mechanisms, and how smoking modifies the association, is needed for a more complete understanding of the vitamin E-prostate cancer relation.     

Chloroplast membrane photostability in chlP transgenic tobacco plants deficient in tocopherols

The phototolerance of three chlP transgenic tobacco (Nicotiana tabacum) lines, affected in geranylgeranyl reductase and, hence, deficient in tocopherols (vitamin E), was estimated by in vivo luminescence and fluorescence measurements and was compared with that of the wild type (WT). Exposure of leaf discs to high light (1 mmol photon m(-2) s(-1)) and low temperature (10 degrees C) led to a rapid inhibition of photosystem II (PSII) photochemistry that showed little dependence on the tocopherol level. PSII photo-inhibition was followed by lipid peroxidation with a time delay of about 4 h, and this phenomenon was exacerbated in the tocopherol-deficient leaves. A linear correlation was observed in these short-term experiments between resistance to photooxidation and tocopherol content. When whole plants were exposed to the same treatment, PSII was severely photo-inhibited in mature leaves of all genotypes. Lipid peroxidation was also observed in all plants, but it occurred much more rapidly in tocopherol-deficient transgenic plants relative to WT plants. The time at which extensive lipid peroxidation occurred was correlated with the tocopherol content of the leaves. The present results show that tocopherols protect thylakoid membranes against photodestruction through lipid peroxidation. However, tocopherol deficiency was compensated in young, developing leaves that were able to photo-acclimate in the long term and did not suffer from photooxidative damage. Soluble antioxidants (glutathione and ascorbate) did not accumulate in photo-acclimated chlP transgenic leaves relative to WT leaves. In contrast, a selective accumulation of xanthophyll cycle pigments was observed in young transgenic leaves, and this could represent a compensatory mechanism for tocopherol deficiency.     

Cytoprotective effects of the antioxidant phytochemical indicaxanthin in beta-thalassemia red blood cells

Antioxidant phytochemicals are investigated as novel treatments for supportive therapy in β-thalassemia. The dietary indicaxanthin was assessed for its protective effects on human β-thalassemic RBCs submitted in vitro to oxidative haemolysis by cumene hydroperoxide. Indicaxanthin at 1.0-10 μM enhanced the resistance to haemolysis dose-dependently. In addition, it prevented lipid and haemoglobin (Hb) oxidation, and retarded vitamin E and GSH depletion. After ex vivo spiking of blood from thalassemia patients with indicaxanthin, the phytochemical was recovered in the soluble cell compartment of the RBCs. A spectrophotometric study showed that indicaxanthin can reduce perferryl-Hb generated in solution from met-Hb and hydrogen peroxide (H₂O₂), more effectively than either Trolox or vitamin C.

Collectively our results demonstrate that indicaxanthin can be incorporated into the redox machinery of β-thalassemic RBC and defend the cell from oxidation, possibly interfering with perferryl-Hb, a reactive intermediate in the hydroperoxide-dependent Hb degradation. Opportunities of therapeutic interest for β-thalassemia may be considered.