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.     

Genetic and biochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production

Vitamin E tocotrienol synthesis in monocots requires homogentisate geranylgeranyl transferase (HGGT), which catalyzes the condensation of homogentisate and the unsaturated C20 isoprenoid geranylgeranyl diphosphate (GGDP). By contrast, vitamin E tocopherol synthesis is mediated by homogentisate phytyltransferase (HPT), which condenses homogentisate and the saturated C20 isoprenoid phytyl diphosphate (PDP). An HGGT‐independent pathway for tocotrienol synthesis has also been shown to occur by de‐regulation of homogentisate synthesis. In this paper, the basis for this pathway and its impact on vitamin E production when combined with HGGT are explored. An Arabidopsis line was initially developed that accumulates tocotrienols and homogentisate by co‐expression of Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) and Escherichia coli bi‐functional chorismate mutase/prephenate dehydrogenase (TyrA). When crossed into the vte2–1 HPT null mutant, tocotrienol production was lost, indicating that HPT catalyzes tocotrienol synthesis in HPPD/TyrA‐expressing plants by atypical use of GGDP as a substrate. Consistent with this, recombinant Arabidopsis HPT preferentially catalyzed in vitro production of the tocotrienol precursor geranylgeranyl benzoquinol only when presented with high molar ratios of GGDP:PDP. In addition, tocotrienol levels were highest in early growth stages in HPPD/TyrA lines, but decreased strongly relative to tocopherols during later growth stages when PDP is known to accumulate. Collectively, these results indicate that HPPD/TyrA‐induced tocotrienol production requires HPT and occurs upon enrichment of GGDP relative to PDP in prenyl diphosphate pools. Finally, combined expression of HPPD/TyrA and HGGT in Arabidopsis leaves and seeds resulted in large additive increases in vitamin E production, indicating that homogentisate concentrations limit HGGT‐catalyzed tocotrienol synthesis.     

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)。     

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.     

A review of characterization of tocotrienols from plant oils and foods

Tocotrienols, members of the vitamin E family, are natural compounds found in a number of vegetable oils, wheat germ, barley and certain types of nuts and grains. Vegetable oils provide the best sources of these vitamin E forms, particularly palm oil and rice bran oil contain higher amounts of tocotrienols. Other sources of tocotrienols include grape fruit seed oil, oats, hazelnuts, maize, olive oil, buckthorn berry, rye, flax seed oil, poppy seed oil and sunflower oil. Tocotrienols are of four types, viz. alpha (α), beta (β), gamma (γ) and delta (δ). Unlike tocopherols, tocotrienols are unsaturated and possess an isoprenoid side chain. A number of researchers have developed methods for the extraction, analysis, identification and quantification of different types of vitamin E compounds. This article constitutes an in-depth review of the chemistry and extraction of the unsaturated vitamin E derivatives, tocotrienols, from various sources using different methods. This review article lists the different techniques that are used in the characterization and purification of tocotrienols such as soxhlet and solid–liquid extractions, saponification method, chromatography (thin layer, column chromatography, gas chromatography, supercritical fluid, high performance), capillary electrochromatography and mass spectrometry. Some of the methods described were able to identify one form or type while others could analyse all the analogues of tocotrienol molecules. Hence, this article will be helpful in understanding the various methods used in the characterization of this lesser known vitamin E variant.     

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.     

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.     

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

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

Biosynthesis,regulation and functions of tocochromanols in plants

Tocopherols and tocotrienols have been originally identified as essential nutrients in mammals based on their vitamin E activity. These lipid-soluble compounds are potent antioxidants that protect polyunsaturated fatty acids from lipid peroxidation. The biosynthesis of tocopherols and tocotrienols occurs exclusively in photosynthetic organisms. The biosynthetic precursors and the different pathway intermediates have been identified by biochemical studies and the different vitamin E biosynthetic genes (VTE genes) have been isolated in several plants and cyanobacteria. The characterization of transgenic plants overexpressing one or multiple VTE genes combined with the study of vitamin E deficient mutants allows from now on understanding the regulation and the function of tocopherols and tocotrienols in plants.     

Multifaceted role of tocotrienols in cardioprotection supports their structure: function relation

Tocotrienols are a class of vitamin E which modulates several mechanisms associated with cardioprotection, anti-cancer, anti-diabetic, and neuroprotection. Unlike other Vitamin E-like compounds, tocotrienols possess inimitable properties. Quite a lot of studies have determined the cardioprotective abilities of tocotrienols and have been shown to possess novel hypocholesterolemic effects together with an ability to reduce the atherogenic apolipoprotein and lipoprotein plasma levels. In addition, tocotrienol has been suggested to have an antioxidant, anti-thrombotic, and anti-tumor effect indicating that tocotrienol may serve as an effective agent in the prevention and/or treatment of cardiovascular disease and cancer. The bioactivity exhibited is due to the structural characteristics of tocotrienols. Rich sources of tocotrienols which include rice bran, palm oil, and other edible oils exhibit protective effect against cardiovascular disorders. The conclusions drawn from the early literature that vitamin E group of compounds provides an inevitable role in cardioprotection is sustained in many more recent studies.     

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.     

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.     

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.     

HPLC analysis of vitamin E isoforms in human epidermis: correlation with minimal erythema dose and free radical scavenging activity

The content and composition of different vitamin E isoforms was analyzed in normal human skin. Interestingly the epidermis contained 1% alpha-tocotrienol, 3% gamma-tocotrienol, 87% alpha-tocopherol, and 9% gamma-tocopherol. Although the levels of tocotrienol in human epidermis appear to be considerably lower than reported in the hairless mouse, the presence of significant amounts of tocotrienol levels leads to speculation about the physiological function of tocotrienols in skin. Besides antioxidant activity and photoprotection, tocotrienols may have skin barrier and growth-modulating properties. A good correlation was found for epidermal alpha-tocopherol (r = 0.7909, p <.0003), gamma-tocopherol (r = 0.556, p <.025), and the total vitamin E content (r = 0.831, p <.0001) with the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging in epidermis, as assessed by electron paramagnetic resonance (EPR) spectroscopy. In human epidermis, alpha-tocopherol is quantitatively the most important vitamin E isoform present and comprises the bulk of first line free radical defense in the lipid compartment. Epidermal tocotrienol levels were not correlated with DPPH scavenging activity. The minimal erythema dose (MED), an individual measure for sun sensitivity and a crude indicator for skin cancer susceptibility, did not correlate with the epidermal content of the vitamin E isoforms. Hence it is concluded that vitamin E alone is not a determinant of individual photosensitivity in humans.     

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.     

植物维生素E生物强化研究进展

维生素E是一种只能在光合组织中合成的脂溶性小分子有机化合物,是人体和动物营养不可缺少的重要维生素。由于植物中维生素E含量较低,人类大多处于慢性缺乏维生素E--“隐性饥饿”的状态,而动物饲料中则需要添加外源合成的维生素E以满足其营养需求。因此,提高植物中维生素E的含量是改善维生素E缺乏的重要途径之一。从维生素E的合成途径入手,详细地综述了维生素E合成关键酶基因的表达变化以及前体物质的含量变化对维生素E合成的影响,发现三烯生育酚和α-生育酚的生物强化效果较好,而生育酚总量提高受限;进而从遗传的角度探讨了维生素E合成受限的原因以及遗传上可能影响维生素E合成的其他代谢途径;最后结合可能影响维生素E合成的调控因子以及其前体物质的转运等方面为今后维生素E的生物强化提出了新的思路。     

Overexpression of the maize γ-tocopherol methyltransferase gene (ZmTMT) increases α-tocopherol content in transgenic Arabidopsis and maize seeds

Transgenic Research - The vitamin E family includes tocopherols and tocotrienols, which are essential lipid-soluble antioxidants necessary for human and livestock health. The seeds of many plant...     

Increases in Both Acute and Chronic Temperature Potentiate Tocotrienol Concentrations in Wild Barley at ‘Evolution Canyon’

Biosynthesis of tocols (vitamin E isoforms) is linked to response to temperature in plants. ‘Evolution Canyon’, an ecogeographical microcosm extending over an average of 200 meters (range 100–400) wide area in the Carmel Mountains of northern Israel, has been suggested as a model for studying global warming. Both domestic (Hordeum vulgare) and wild (Hordeum spontaneum) barley compared with wheat, oat, corn, rice, and rye show high tocotrienol/tocopherol ratios. Therefore, we hypothesized that tocol distribution might change in response to global warming. α‐, β‐, γ‐, and δ‐tocopherol, and α‐, β‐, γ‐, and δ‐tocotrienol concentrations were measured in wild barley (H. spontaneum) seeds harvested from the xeric (African) and mesic (European) slopes of Evolution Canyon over a six‐year period from 2005–2011. Additionally, we examined seeds from areas contiguous to and distant from the part of the Canyon severely burned during the Carmel Fire of December 2010. Increased α‐tocotrienol (p<0.01) was correlated with 1) temperature increases, 2) to the hotter ‘African’ slope in contrast to the cooler ‘European’ slope, and 3) to propinquity to the fire. The study illustrates the role of α‐tocotrienol in both chronic and acute temperature adaptation in wild barley and suggests future research into thermoregulatory mechanisms in plants.     

Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study

With the aim to enhance the plant vitamin E content, the barley gene encoding 4-hydroxyphenylpyruvate dioxygenase was overexpressed in tobacco plants under control of the 35S promoter. Transgenic lines have a higher capacity for homogentisate biosynthesis as evident by a more than 10-fold higher resistance towards the bleaching herbicide sulcotrione. Seeds from transgenic lines have an up to two-fold enhanced level of vitamin E without a change in the ratio of γ-tocopherol and γ-tocotrienol. While the vitamin E content is not affected in leaves, the level of plastoquinone is enhanced in leaves of transgenic lines during leaf senescence.     

三烯生育酚生物学功能及合成调控研究进展

介绍了三烯生育酚的生物合成途径,重点综述了三烯生育酚在神经保护、抗癌、降低胆固醇以及抗氧化等方面的优越生物学功能,以及利用关键酶的高效表达和前体物质水平的提高等植物代谢工程手段提高植物体内三烯生育酚生物合成水平的研究进展。