- Email: [email protected]
Engineered plants with elevated vitamin E: a nutraceutical success story
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migration of inner centromere protein (INCENP) [8,9], a reliable molecular indicator of cytokinesis. An accumulation of INCENP protein was found at the furrow indicating a non-essential role of the bipolar spindle in the migration of chromosome passenger-proteins. Having addressed the main question of spindle polarity versus furrow formation, the authors then turned their attention to the behavior of microtubules in monopolar spindles during cytokinesis. Kymographic analysis (a video digital image analysis technique) showed that microtubules extending to the cortex past the chromosomes were four times more stable than astral microtubules that extended away from the chromosome towards the polar cortical regions. Furthermore, in normal cells with bipolar spindles the polar microtubules were unstable even if they reached the cell cortex at the site of furrow formation. Thus the present study clearly shows an asymmetric nature of microtubule dynamics during cytokinesis, with furrows forming near highly populated and stable microtubule ends independent of the influence of spindle polarity. Conclusion The paper by Canman et al. [1] provides the first experimental evidence for uncoupling the traditional concept of spindle polarity and cell-furrow formation. However, an interesting deviation is found in Caenorhabditis elegans [10] in which a reduced population of microtubules triggers cytokinesis instead. This raises several questions: (i) does a directional microtubule gradient determine the onset and placement of the cell furrow? (ii) what is the critical threshold of cortical microtubule density that sets off furrow formation? (iii) given the immense diversity in cytokinesis regulation, can a generic model explain global features of cell-furrow regulation and (iv) what degree of microtubule overlap is necessary to induce cytokinesis? Although the contribution of polar versus equatorial microtubules is fairly clear for PtK1 cells, it would be interesting to study the differential contribution of kinetochore microtubules and overlapping microtubules in determining the cell furrows in various organisms. Furthermore, the role of overlapping microtubules in preventing ectopic furrowing (the process by which a cell cleaves itself away from the equatorial plane resulting in morphologically dissimilar daughter cells) merits greater attention in the future. The paper by Julie Canman and her colleagues [1] offers a fresh insight into
Vol.22 No.3 March 2004
post-mitotic events and indicates that there is more to cellequator formation than merely the physical distance between two poles. Future applications A possible future extension of the present study could be its application in the field of electronics. Miniaturization of electronic circuits to the nanoscale level has created a need for precisely understanding the physicochemical basis of microtubule organization and dynamics. Understanding the electrical and stress-sharing properties of overlapping microtubules could help in developing robust nanoscale systems. Microtubule dynamics also has potential applications in the drug industry, for example in directing the intracellular transport of microtubule-binding drugs, such as taxol, to a specific cellular address. Microtubule research has reached an exciting stage from which further advances are likely to drive both biotechnological and bioengineering applications. References 1 Canman, J.C. et al. (2003) Determining the position of the cell division plane. Nature 424, 1074– 1078 2 Walen, K.H. and Brown, S.W. (1962) Chromosomes in a marsupial (Potorous tridactylis) tissue culture. Nature 194, 406 3 Mayer, T.U. et al. (1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–974 4 Guenette, S. et al. (1995) Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes. J. Cell Sci. 108, 1195 – 1204 5 Wheatley, S.P. et al. (2001) INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow. Exp. Cell Res. 262, 122– 127 6 Canman, J.C. et al. (2002) Anaphase onset does not require the microtubule-dependent depletion of kinetochore and centromerebinding proteins. J. Cell Sci. 115, 3787– 3795 7 Murata-Hori, M. and Wang, Y.L. (2002) Both midzone and astral microtubules are involved in the delivery of cytokinesis signals: insights from the mobility of aurora B. J. Cell Biol. 159, 45 – 53 8 Cooke, C.A. et al. (1987) The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J. Cell Biol. 105, 2053 – 2067 9 Parra, M.T. et al. (2003) Dynamic relocalization of the chromosomal passenger complex proteins inner centromere protein (INCENP) and aurora-Bkinaseduringmalemousemeiosis.J.Cell Sci.116, 961–974 10 Dechant, R. and Glotzer, M. (2003) Centrosome separation and central spindle assembly act in redundant pathways that regulate microtubule density and trigger cleavage furrow formation. Dev. Cell 4, 333–344 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.01.001
Engineered plants with elevated vitamin E: a nutraceutical success story Imad Ajjawi and David Shintani Department of Biochemistry MS200, University of Nevada, Reno, NV 89557, USA
Vitamin E has been touted as a panacea for age-related diseases, including cardiovascular disease and Alzheimer’s disease and, thus, the demand for this nutraceutical has increased dramatically in recent years. Corresponding author: David Shintani ([email protected]). www.sciencedirect.com
This demand has, in turn, driven research to increase vitamin E production from plant sources. We have summarized the cumulative work of several groups in this area, describing the current status of efforts to bioengineer plants for elevated vitamin E content.
Update
TRENDS in Biotechnology
The recent interest in phytochemical-based nutraceuticals has driven a surge in industrial and academic research in vitamin E biosynthesis in plants. Studies suggesting that vitamin E decreases the onset or occurrence of several degenerative diseases and cancers [1] have piqued the interest of the public and increased demand for vitamin E dietary supplements. Although the efficacy of vitamin E as a therapeutic agent has not been proven conclusively [1], because of its very low toxicity, many physicians still recommend its use to combat several ailments, including prostate cancer and Alzheimer’s disease. The growing demand for vitamin E has motivated several groups to develop higher yielding vitamin E crops through directed breeding and genetic engineering [2]. What is vitamin E? Vitamin E comprises a group of potent antioxidant compounds known as tocols, which have a common structure consisting of a polar chromanol head group and a nonpolar prenyl tail. The two major classes of tocols, tocopherols and tocotrienols, are structurally similar, with the exception that their prenyl tails are saturated and unsaturated, respectively. Tocols can be classified further based on the number and position of methyl groups on the chromanol head group. Tocols containing one methyl group are referred to as d-tocols. Tocols containing two methyl groups are known as g- or b-tocols. Fully methylsubstituted tocols containing three methyl groups are a-tocols (Figure 1). Animals and humans possess a tocopherol-binding protein (TBP) that is highly specific for a-tocopherol and ensures the preferential absorption and distribution of a-tocopherol throughout the body. For this reason, a-tocopherol is the most potent form of vitamin E, possessing vitamin E activity 2 – 33 times that of all other tocol species [1]. TBP is also stereospecific for the (R,R,R)-atocopherol stereoisomer, making natural a-tocopherol more potent than synthetic a-tocopherol, which is sold as a racemic mixture [1]. Plant sources of vitamin E The primary sources of dietary vitamin E are derived from plants, in which the quantity and composition of tocols varies between tissues and species. Oilseeds are the richest source of vitamin E, having total tocol levels ranging from 330 to 2,000 mg per gram of oil [2]. Unfortunately, many plant oils accumulate g-tocopherol, which has onetenth the vitamin E activity of a-tocopherol. Interestingly, another good source of vitamin E, green vegetables, has low tocol yields but high proportions of a-tocopherol. Although green plant tissues produce only between 20 and 50 mg of total tocols per gram of tissue, the tocol pools comprise almost entirely a-tocopherol [2]. From these observations, two complementary strategies for increasing the vitamin E content in plant foods emerge: (i) increase the flux through the tocopherol biosynthetic pathway to produce elevated levels of total tocols, and (ii) make a-tocopherol the predominant form of vitamin E. Increasing pathway flux would be especially useful in increasing the vitamin E content of green vegetable crops, and altering the tocol composition in favor of a-tocopherol would www.sciencedirect.com
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significantly increase the vitamin E content of oilseeds such as soybean. Flux control points in tocopherol biosynthesis Examination of the tocopherol biosynthetic pathway reveals that the pathway enzymes can be divided into upstream reactions that are important for flux regulation, and downstream reactions that control tocol composition. The first potential flux control points in tocopherol synthesis involve the enzymes hydroxyphenyl pyruvate dioxygenase (HPPD) and geranylgeranyl diphosphate (GGDP) reductase (GGDPR), which synthesize the aromatic headgroup precursor homogentisic acid (HGA) and the prenyl tail precursor phytyl-diphosphate (PDP), respectively (Figure 1). HPPD and GGDPR were selected as candidate regulatory enzymes based on in vivo observations showing that HGA and PDP might be limiting factors in tocopherol biosynthesis. Tissue culture studies have shown that exogenously supplied HGA and PDP both caused significant increases in tocopherol biosynthesis [3]. Furthermore, when PDP accumulates upon dark-induced senescence because of chlorophyll breakdown, large increases in tocopherol biosynthesis are observed [4]. To test the hypothesis that HGA levels limit tocopherol biosynthesis, the gene encoding HPPD was overexpressed in the leaves and seeds of both Arabidopsis and tobacco [5,6]. Although transgenic lines overexpressing the gene encoding HPPD showed large increases in enzyme activity compared with control lines, only small increases in tocopherol content were observed (10% in leaves and 30% in seeds) [5,6], suggesting that HPPD alone is not sufficient to increase tocopherol biosynthetic flux. Although similar experiments have not yet been performed to test directly the effect of increasing PDP levels on tocopherol yields through overexpression of GGDP reductase, it has been shown that increasing the total flux through the nonmevalonate pathway by overexpressing the gene encoding deoxyxylulose phosphate synthase caused a 40% increase in leaf tocopherol content [7]. Another important flux-regulating enzyme is homogentisate phytyltransferase (HPT). This enzyme catalyzes the committed step in tocopherol biosynthesis in which HGA and PDP are condensed to form the first prenylquinone intermediate methylphytylbenzoquinone (MPBQ). In overexpression studies, a 10-fold increase in HPT activity translated to a 4.4-fold increase in leaf tocopherol levels, relative to wild-type plants [8]. Similar results were obtained in seeds but the magnitude of the tocopherol increase was lower, ranging from 0.4-fold to twofold compared with wild-type levels [8,9]. Recently, Cahoon et al. [10] identified a HPT variant from barley, the sequence of which diverged significantly from previously characterized HPTs. Functional analyses of this protein revealed that it possessed homogentisate geranylgeranyltransferases (HGGT) activity. HGGT catalyzes an analogous reaction to HPT, but instead of using PDP as its prenyl substrate, it is highly specific for GGDP (Figure 1). As such, HGGT catalyzes the first committed step in tocotrienol synthesis and is, therefore, likely to play a role in regulating pathway flux. This was shown to be the case in experiments in which the maize HGGT gene
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Non-mevalonate pathway Glyceraldehyde-3-P and pyruvate
Tyrosine catabolism (shikimate Pathway)
4-Hydroxyphenylpyruvate O2 HPPD CO2
DXP synthase 1-Deoxy-D-xylulose-5-P
O OH
GGDP reductase HGA
Geranylgeranyl-DP
OH
OPP
OPP OH
Phytyl-DP
Homogentisic acid
HGGT
HPT MPBQMT
Tocotrienols
β-Carotene Abscisic acid Gibberellins
Chlorophylls Plastoquinones
DMPBQ
MPBQ Cyclase
Cyclase
HO
HO H O
H
δ-T
CH3
γ-TMT
O
H 3C
3
γ-T
CH3
Tocopherols
CH3
3
γ-TMT CH3
HO
HO H O CH3
3
β-T
H O
H3C CH3
3
α-T
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Figure 1. Tocopherol biosynthesis in plants. Enzymes that are responsible for flux regulation of the pathway are highlighted in red, whereas those responsible for an overall compositional change in tocol content are highlighted in blue. Broken lines represent alternative pathways that compete for tocopherol precursors. Abbreviations: DMPBQ, dimethylphytylbenzoquinone; DXP, 1-deoxy-D-xylulose-5-phosphate synthase; GGDP, geranylgeranyl diphosphate; HGGT, homogentisate geranylgeranyl transferase; HPPD, p-hydroxyphenyl-pyruvate dioxygenase; HPT, homogentisate phytyltransferase; MPBQ, methylphytylbenzoquinone; MT, methyltransferase; T, tocopherol; g-TMT, g-tocopherol methyltransferase; P, phosphate.
was overexpressed in maize seeds, leading to a 20-fold increase in tocotrienol levels, which translated to an eightfold increase in total tocols (tocopherols and tocotrienols) [10]. This result is the largest increase in tocol production ever observed in plants, and significantly increases the antioxidant potential of corn. Unfortunately, because dietary tocotrienols are not absorbed as well as a-tocopherol, the large increase in tocotrienol levels observed in the HGGT-overexpressing maize seed did not add much to the vitamin E nutritional value of these plants. However, because tocotrienols have superior in vitro antioxidant activity [11], transgenic plants with elevated tocotrienol levels could be used as sources of chemical antioxidants for industrial applications, such as oxidative stabilizers for paints, coatings and other lipophilic products. Furthermore, it has been reported that tocotrienols might have a therapeutic role in decreasing cholesterol levels in humans [12]. Interestingly, when Cahoon et al. [10] overexpressed the barley HGGT gene in Arabidopsis leaves, tocotrienols, which are not usually synthesized by dicot species, accumulated to very high levels, whereas tocopherol levels were not affected. This indicates that HGGT and HPT are highly specific for their prenyl substrates (GGDP and PDP, respectively) and must compete with one another for HGA. Although overproduction of tocotrienols clearly shows that www.sciencedirect.com
GGDP and HGA are abundantly available, the fact that HGGT overexpression has no effect on tocopherol production indicates that PDP must be limiting. Therefore, overexpression of GGDP reductase could increase PDP availability significantly and, thus, increase pathway flux. Regulation of tocopherol composition The enzymes catalyzing the later steps of the tocopherol biosynthetic pathway, specifically the MPBQ methyltransferase (MPBQMT), the tocopherol cyclase (TC) and the g-tocopherol methyltransferase (g-TMT), are important in determining tocopherol composition (Figure 1). In a recent report by Van Eenennaam et al. [13], the genes encoding g-TMT and MPBQMT were overexpressed in soybean seeds to improve this important dietary source of vitamin E. Like many other oilseeds, soybean seeds do not accumulate a-tocopherol, the most potent form of vitamin E, but instead accumulate precursor tocopherol species (i.e. dand g-tocopherol; Figure 1) with much lower vitamin E activity. The overexpression of the two tocopherol methyltransferases resulted in a 95% conversion of these lesser forms of vitamin E to a-tocopherol, which translated to a fivefold increase in vitamin E activity. To put this into a real-world perspective, although four tablespoons of soybean oil from wild-type plants contains only 13 international units (IU) of vitamin E, the same volume of oil
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from the methyltransferase-overexpressing lines contain 65 IU of vitamin E. Because 100 IU is the recommended minimum therapeutic dose to decrease the risk of heart disease, the work of Van Eenennaam et al. has done much to increase the nutraceutical potential of plant-derived vitamin E. Until now, transgenic experiments have not been reported with the gene encoding TC. However, it is likely that similar degrees of success will be seen when the investigations are reported. The ability to manipulate the three tocopherol-composition-regulating enzymes should allow for the tailoring of plant tissues with novel tocopherol composition. Therefore, in addition to nutritional applications, plants can also be engineered to accumulate g-, b- and d-tocopherol, which all have superior in vitro antioxidant activities to a-tocopherol [11], for the development of antioxidants for food processing and industrial applications. Concluding remarks Through the combined efforts of several groups [1,2,4,13], a clear picture has emerged that identifies enzymatic steps regulating quantitative and qualitative changes in planttissue tocol pools. This information is now being exploited by plant breeders and genetic engineers to develop plants with elevated vitamin E content. Although fiscal gains have been one of the primary motivating forces behind these efforts, altruistic aims have also driven the research. Specifically, groups are interested in developing foods that have been biofortified with vitamin E. The rationale is that although most people can obtain sufficient amounts of vitamin E from a typical diet, current foods do not provide the therapeutic levels of vitamin E that would allow the public to enjoy the added health benefits of this vitamin. Biofortified plants would provide a sustainable alternative to a prescribed regimen of vitamin E supplementation that would be available to everyone, regardless of income or class.
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References 1 Institute of Medicine (2000) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, National Academies Press 2 Grusak, M.A. and Dellapenna, D. (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 133 – 161 3 Furuya, T. et al. (1987) Production of tocopherols in cell culture of safflower. Phytochemistry 26, 2741– 2747 4 Moshe, R. et al. (1989) Accumulation of a-tocopherol in senescing organs as related to chlorophyll degradation. Plant Physiol. 89, 1028– 1030 5 Tsegaye, Y. et al. (2002) Overexpression of the enzymes phydroxyphenylpyruvate dioxygenase in Arabidopsis and its relationship to tocopherol biosynthesis. Plant Physiol. Biochem. 40, 913 – 920 6 Falk, J. et al. (2003) Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of vitamin E content in seeds but not in leaves. FEBS Lett. 540, 35 – 40 7 Estevez, J.M. (2001) 1-deoxy-d-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J. Biol. Chem. 276, 22901 – 22909 8 Collakova, E. and Dellapenna, D. (2003) Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol. 131, 632 – 642 9 Savige, B. et al. (2002) Isolation and characterization of homogentisate phytyltransferase from synechocystis sp. Pcc 6803 and Arabidopsis. Plant Physiol. 129, 321 – 332 10 Cahoon, E.B. et al. (2003) Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat. Biotechnol. 21, 1082– 1087 11 Kamal-Eldin, A. and Appelqvist, L.A. (1996) The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 31, 671 – 701 12 Theriault, A. et al. (1999) Tocotrienol: a review of its therapeutic potential. Clin. Biochem. 32, 309 – 319 13 Van Eenennaam, A.L. et al. (2003) Engineering vitamin E content: from Arabidopsis mutant to soy oil. Plant Cell 15, 3007– 3019
0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.01.008
How can genetically modified foods be made publicly acceptable? Gene Rowe Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK
A recent study by Lusk suggests that consumers might voluntarily pay more for a genetically modified (GM) food than a non-GM equivalent if made aware of the possible health benefits. However, other research indicates that the acceptability of novel hazards is affected by a variety of factors, in addition to benefits, and that making agricultural biotechnology publicly acceptable will be more complex than indicated by the results from Lusk’s study.
Corresponding author: Gene Rowe ([email protected]). www.sciencedirect.com
A recent paper by Lusk indicates that consumers could be willing to pay extra for a genetically modified (GM) food than a non-GM equivalent if told of the potential health benefits they might receive from eating it [1]. Indeed, as Lusk states in a Purdue newsletter (http://news.uns. purdue.edu/html4ever/031024.Lusk.rice.html), ‘This study is one of the first to show that people are willing to pay a premium for a food that’s been improved using biotechnology.’ Lusk attributes this result – which is contrary to past findings – to the emphasis of his study being on the potential benefits of GM food from a consumer’s, rather
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migration of inner centromere protein (INCENP) [8,9], a reliable molecular indicator of cytokinesis. An accumulation of INCENP protein was found at the furrow indicating a non-essential role of the bipolar spindle in the migration of chromosome passenger-proteins. Having addressed the main question of spindle polarity versus furrow formation, the authors then turned their attention to the behavior of microtubules in monopolar spindles during cytokinesis. Kymographic analysis (a video digital image analysis technique) showed that microtubules extending to the cortex past the chromosomes were four times more stable than astral microtubules that extended away from the chromosome towards the polar cortical regions. Furthermore, in normal cells with bipolar spindles the polar microtubules were unstable even if they reached the cell cortex at the site of furrow formation. Thus the present study clearly shows an asymmetric nature of microtubule dynamics during cytokinesis, with furrows forming near highly populated and stable microtubule ends independent of the influence of spindle polarity. Conclusion The paper by Canman et al. [1] provides the first experimental evidence for uncoupling the traditional concept of spindle polarity and cell-furrow formation. However, an interesting deviation is found in Caenorhabditis elegans [10] in which a reduced population of microtubules triggers cytokinesis instead. This raises several questions: (i) does a directional microtubule gradient determine the onset and placement of the cell furrow? (ii) what is the critical threshold of cortical microtubule density that sets off furrow formation? (iii) given the immense diversity in cytokinesis regulation, can a generic model explain global features of cell-furrow regulation and (iv) what degree of microtubule overlap is necessary to induce cytokinesis? Although the contribution of polar versus equatorial microtubules is fairly clear for PtK1 cells, it would be interesting to study the differential contribution of kinetochore microtubules and overlapping microtubules in determining the cell furrows in various organisms. Furthermore, the role of overlapping microtubules in preventing ectopic furrowing (the process by which a cell cleaves itself away from the equatorial plane resulting in morphologically dissimilar daughter cells) merits greater attention in the future. The paper by Julie Canman and her colleagues [1] offers a fresh insight into
Vol.22 No.3 March 2004
post-mitotic events and indicates that there is more to cellequator formation than merely the physical distance between two poles. Future applications A possible future extension of the present study could be its application in the field of electronics. Miniaturization of electronic circuits to the nanoscale level has created a need for precisely understanding the physicochemical basis of microtubule organization and dynamics. Understanding the electrical and stress-sharing properties of overlapping microtubules could help in developing robust nanoscale systems. Microtubule dynamics also has potential applications in the drug industry, for example in directing the intracellular transport of microtubule-binding drugs, such as taxol, to a specific cellular address. Microtubule research has reached an exciting stage from which further advances are likely to drive both biotechnological and bioengineering applications. References 1 Canman, J.C. et al. (2003) Determining the position of the cell division plane. Nature 424, 1074– 1078 2 Walen, K.H. and Brown, S.W. (1962) Chromosomes in a marsupial (Potorous tridactylis) tissue culture. Nature 194, 406 3 Mayer, T.U. et al. (1999) Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–974 4 Guenette, S. et al. (1995) Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes. J. Cell Sci. 108, 1195 – 1204 5 Wheatley, S.P. et al. (2001) INCENP binds directly to tubulin and requires dynamic microtubules to target to the cleavage furrow. Exp. Cell Res. 262, 122– 127 6 Canman, J.C. et al. (2002) Anaphase onset does not require the microtubule-dependent depletion of kinetochore and centromerebinding proteins. J. Cell Sci. 115, 3787– 3795 7 Murata-Hori, M. and Wang, Y.L. (2002) Both midzone and astral microtubules are involved in the delivery of cytokinesis signals: insights from the mobility of aurora B. J. Cell Biol. 159, 45 – 53 8 Cooke, C.A. et al. (1987) The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J. Cell Biol. 105, 2053 – 2067 9 Parra, M.T. et al. (2003) Dynamic relocalization of the chromosomal passenger complex proteins inner centromere protein (INCENP) and aurora-Bkinaseduringmalemousemeiosis.J.Cell Sci.116, 961–974 10 Dechant, R. and Glotzer, M. (2003) Centrosome separation and central spindle assembly act in redundant pathways that regulate microtubule density and trigger cleavage furrow formation. Dev. Cell 4, 333–344 0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.01.001
Engineered plants with elevated vitamin E: a nutraceutical success story Imad Ajjawi and David Shintani Department of Biochemistry MS200, University of Nevada, Reno, NV 89557, USA
Vitamin E has been touted as a panacea for age-related diseases, including cardiovascular disease and Alzheimer’s disease and, thus, the demand for this nutraceutical has increased dramatically in recent years. Corresponding author: David Shintani ([email protected]). www.sciencedirect.com
This demand has, in turn, driven research to increase vitamin E production from plant sources. We have summarized the cumulative work of several groups in this area, describing the current status of efforts to bioengineer plants for elevated vitamin E content.
Update
TRENDS in Biotechnology
The recent interest in phytochemical-based nutraceuticals has driven a surge in industrial and academic research in vitamin E biosynthesis in plants. Studies suggesting that vitamin E decreases the onset or occurrence of several degenerative diseases and cancers [1] have piqued the interest of the public and increased demand for vitamin E dietary supplements. Although the efficacy of vitamin E as a therapeutic agent has not been proven conclusively [1], because of its very low toxicity, many physicians still recommend its use to combat several ailments, including prostate cancer and Alzheimer’s disease. The growing demand for vitamin E has motivated several groups to develop higher yielding vitamin E crops through directed breeding and genetic engineering [2]. What is vitamin E? Vitamin E comprises a group of potent antioxidant compounds known as tocols, which have a common structure consisting of a polar chromanol head group and a nonpolar prenyl tail. The two major classes of tocols, tocopherols and tocotrienols, are structurally similar, with the exception that their prenyl tails are saturated and unsaturated, respectively. Tocols can be classified further based on the number and position of methyl groups on the chromanol head group. Tocols containing one methyl group are referred to as d-tocols. Tocols containing two methyl groups are known as g- or b-tocols. Fully methylsubstituted tocols containing three methyl groups are a-tocols (Figure 1). Animals and humans possess a tocopherol-binding protein (TBP) that is highly specific for a-tocopherol and ensures the preferential absorption and distribution of a-tocopherol throughout the body. For this reason, a-tocopherol is the most potent form of vitamin E, possessing vitamin E activity 2 – 33 times that of all other tocol species [1]. TBP is also stereospecific for the (R,R,R)-atocopherol stereoisomer, making natural a-tocopherol more potent than synthetic a-tocopherol, which is sold as a racemic mixture [1]. Plant sources of vitamin E The primary sources of dietary vitamin E are derived from plants, in which the quantity and composition of tocols varies between tissues and species. Oilseeds are the richest source of vitamin E, having total tocol levels ranging from 330 to 2,000 mg per gram of oil [2]. Unfortunately, many plant oils accumulate g-tocopherol, which has onetenth the vitamin E activity of a-tocopherol. Interestingly, another good source of vitamin E, green vegetables, has low tocol yields but high proportions of a-tocopherol. Although green plant tissues produce only between 20 and 50 mg of total tocols per gram of tissue, the tocol pools comprise almost entirely a-tocopherol [2]. From these observations, two complementary strategies for increasing the vitamin E content in plant foods emerge: (i) increase the flux through the tocopherol biosynthetic pathway to produce elevated levels of total tocols, and (ii) make a-tocopherol the predominant form of vitamin E. Increasing pathway flux would be especially useful in increasing the vitamin E content of green vegetable crops, and altering the tocol composition in favor of a-tocopherol would www.sciencedirect.com
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significantly increase the vitamin E content of oilseeds such as soybean. Flux control points in tocopherol biosynthesis Examination of the tocopherol biosynthetic pathway reveals that the pathway enzymes can be divided into upstream reactions that are important for flux regulation, and downstream reactions that control tocol composition. The first potential flux control points in tocopherol synthesis involve the enzymes hydroxyphenyl pyruvate dioxygenase (HPPD) and geranylgeranyl diphosphate (GGDP) reductase (GGDPR), which synthesize the aromatic headgroup precursor homogentisic acid (HGA) and the prenyl tail precursor phytyl-diphosphate (PDP), respectively (Figure 1). HPPD and GGDPR were selected as candidate regulatory enzymes based on in vivo observations showing that HGA and PDP might be limiting factors in tocopherol biosynthesis. Tissue culture studies have shown that exogenously supplied HGA and PDP both caused significant increases in tocopherol biosynthesis [3]. Furthermore, when PDP accumulates upon dark-induced senescence because of chlorophyll breakdown, large increases in tocopherol biosynthesis are observed [4]. To test the hypothesis that HGA levels limit tocopherol biosynthesis, the gene encoding HPPD was overexpressed in the leaves and seeds of both Arabidopsis and tobacco [5,6]. Although transgenic lines overexpressing the gene encoding HPPD showed large increases in enzyme activity compared with control lines, only small increases in tocopherol content were observed (10% in leaves and 30% in seeds) [5,6], suggesting that HPPD alone is not sufficient to increase tocopherol biosynthetic flux. Although similar experiments have not yet been performed to test directly the effect of increasing PDP levels on tocopherol yields through overexpression of GGDP reductase, it has been shown that increasing the total flux through the nonmevalonate pathway by overexpressing the gene encoding deoxyxylulose phosphate synthase caused a 40% increase in leaf tocopherol content [7]. Another important flux-regulating enzyme is homogentisate phytyltransferase (HPT). This enzyme catalyzes the committed step in tocopherol biosynthesis in which HGA and PDP are condensed to form the first prenylquinone intermediate methylphytylbenzoquinone (MPBQ). In overexpression studies, a 10-fold increase in HPT activity translated to a 4.4-fold increase in leaf tocopherol levels, relative to wild-type plants [8]. Similar results were obtained in seeds but the magnitude of the tocopherol increase was lower, ranging from 0.4-fold to twofold compared with wild-type levels [8,9]. Recently, Cahoon et al. [10] identified a HPT variant from barley, the sequence of which diverged significantly from previously characterized HPTs. Functional analyses of this protein revealed that it possessed homogentisate geranylgeranyltransferases (HGGT) activity. HGGT catalyzes an analogous reaction to HPT, but instead of using PDP as its prenyl substrate, it is highly specific for GGDP (Figure 1). As such, HGGT catalyzes the first committed step in tocotrienol synthesis and is, therefore, likely to play a role in regulating pathway flux. This was shown to be the case in experiments in which the maize HGGT gene
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Non-mevalonate pathway Glyceraldehyde-3-P and pyruvate
Tyrosine catabolism (shikimate Pathway)
4-Hydroxyphenylpyruvate O2 HPPD CO2
DXP synthase 1-Deoxy-D-xylulose-5-P
O OH
GGDP reductase HGA
Geranylgeranyl-DP
OH
OPP
OPP OH
Phytyl-DP
Homogentisic acid
HGGT
HPT MPBQMT
Tocotrienols
β-Carotene Abscisic acid Gibberellins
Chlorophylls Plastoquinones
DMPBQ
MPBQ Cyclase
Cyclase
HO
HO H O
H
δ-T
CH3
γ-TMT
O
H 3C
3
γ-T
CH3
Tocopherols
CH3
3
γ-TMT CH3
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Figure 1. Tocopherol biosynthesis in plants. Enzymes that are responsible for flux regulation of the pathway are highlighted in red, whereas those responsible for an overall compositional change in tocol content are highlighted in blue. Broken lines represent alternative pathways that compete for tocopherol precursors. Abbreviations: DMPBQ, dimethylphytylbenzoquinone; DXP, 1-deoxy-D-xylulose-5-phosphate synthase; GGDP, geranylgeranyl diphosphate; HGGT, homogentisate geranylgeranyl transferase; HPPD, p-hydroxyphenyl-pyruvate dioxygenase; HPT, homogentisate phytyltransferase; MPBQ, methylphytylbenzoquinone; MT, methyltransferase; T, tocopherol; g-TMT, g-tocopherol methyltransferase; P, phosphate.
was overexpressed in maize seeds, leading to a 20-fold increase in tocotrienol levels, which translated to an eightfold increase in total tocols (tocopherols and tocotrienols) [10]. This result is the largest increase in tocol production ever observed in plants, and significantly increases the antioxidant potential of corn. Unfortunately, because dietary tocotrienols are not absorbed as well as a-tocopherol, the large increase in tocotrienol levels observed in the HGGT-overexpressing maize seed did not add much to the vitamin E nutritional value of these plants. However, because tocotrienols have superior in vitro antioxidant activity [11], transgenic plants with elevated tocotrienol levels could be used as sources of chemical antioxidants for industrial applications, such as oxidative stabilizers for paints, coatings and other lipophilic products. Furthermore, it has been reported that tocotrienols might have a therapeutic role in decreasing cholesterol levels in humans [12]. Interestingly, when Cahoon et al. [10] overexpressed the barley HGGT gene in Arabidopsis leaves, tocotrienols, which are not usually synthesized by dicot species, accumulated to very high levels, whereas tocopherol levels were not affected. This indicates that HGGT and HPT are highly specific for their prenyl substrates (GGDP and PDP, respectively) and must compete with one another for HGA. Although overproduction of tocotrienols clearly shows that www.sciencedirect.com
GGDP and HGA are abundantly available, the fact that HGGT overexpression has no effect on tocopherol production indicates that PDP must be limiting. Therefore, overexpression of GGDP reductase could increase PDP availability significantly and, thus, increase pathway flux. Regulation of tocopherol composition The enzymes catalyzing the later steps of the tocopherol biosynthetic pathway, specifically the MPBQ methyltransferase (MPBQMT), the tocopherol cyclase (TC) and the g-tocopherol methyltransferase (g-TMT), are important in determining tocopherol composition (Figure 1). In a recent report by Van Eenennaam et al. [13], the genes encoding g-TMT and MPBQMT were overexpressed in soybean seeds to improve this important dietary source of vitamin E. Like many other oilseeds, soybean seeds do not accumulate a-tocopherol, the most potent form of vitamin E, but instead accumulate precursor tocopherol species (i.e. dand g-tocopherol; Figure 1) with much lower vitamin E activity. The overexpression of the two tocopherol methyltransferases resulted in a 95% conversion of these lesser forms of vitamin E to a-tocopherol, which translated to a fivefold increase in vitamin E activity. To put this into a real-world perspective, although four tablespoons of soybean oil from wild-type plants contains only 13 international units (IU) of vitamin E, the same volume of oil
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from the methyltransferase-overexpressing lines contain 65 IU of vitamin E. Because 100 IU is the recommended minimum therapeutic dose to decrease the risk of heart disease, the work of Van Eenennaam et al. has done much to increase the nutraceutical potential of plant-derived vitamin E. Until now, transgenic experiments have not been reported with the gene encoding TC. However, it is likely that similar degrees of success will be seen when the investigations are reported. The ability to manipulate the three tocopherol-composition-regulating enzymes should allow for the tailoring of plant tissues with novel tocopherol composition. Therefore, in addition to nutritional applications, plants can also be engineered to accumulate g-, b- and d-tocopherol, which all have superior in vitro antioxidant activities to a-tocopherol [11], for the development of antioxidants for food processing and industrial applications. Concluding remarks Through the combined efforts of several groups [1,2,4,13], a clear picture has emerged that identifies enzymatic steps regulating quantitative and qualitative changes in planttissue tocol pools. This information is now being exploited by plant breeders and genetic engineers to develop plants with elevated vitamin E content. Although fiscal gains have been one of the primary motivating forces behind these efforts, altruistic aims have also driven the research. Specifically, groups are interested in developing foods that have been biofortified with vitamin E. The rationale is that although most people can obtain sufficient amounts of vitamin E from a typical diet, current foods do not provide the therapeutic levels of vitamin E that would allow the public to enjoy the added health benefits of this vitamin. Biofortified plants would provide a sustainable alternative to a prescribed regimen of vitamin E supplementation that would be available to everyone, regardless of income or class.
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References 1 Institute of Medicine (2000) Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, National Academies Press 2 Grusak, M.A. and Dellapenna, D. (1999) Improving the nutrient composition of plants to enhance human nutrition and health. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 133 – 161 3 Furuya, T. et al. (1987) Production of tocopherols in cell culture of safflower. Phytochemistry 26, 2741– 2747 4 Moshe, R. et al. (1989) Accumulation of a-tocopherol in senescing organs as related to chlorophyll degradation. Plant Physiol. 89, 1028– 1030 5 Tsegaye, Y. et al. (2002) Overexpression of the enzymes phydroxyphenylpyruvate dioxygenase in Arabidopsis and its relationship to tocopherol biosynthesis. Plant Physiol. Biochem. 40, 913 – 920 6 Falk, J. et al. (2003) Constitutive overexpression of barley 4-hydroxyphenylpyruvate dioxygenase in tobacco results in elevation of vitamin E content in seeds but not in leaves. FEBS Lett. 540, 35 – 40 7 Estevez, J.M. (2001) 1-deoxy-d-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J. Biol. Chem. 276, 22901 – 22909 8 Collakova, E. and Dellapenna, D. (2003) Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol. 131, 632 – 642 9 Savige, B. et al. (2002) Isolation and characterization of homogentisate phytyltransferase from synechocystis sp. Pcc 6803 and Arabidopsis. Plant Physiol. 129, 321 – 332 10 Cahoon, E.B. et al. (2003) Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content. Nat. Biotechnol. 21, 1082– 1087 11 Kamal-Eldin, A. and Appelqvist, L.A. (1996) The chemistry and antioxidant properties of tocopherols and tocotrienols. Lipids 31, 671 – 701 12 Theriault, A. et al. (1999) Tocotrienol: a review of its therapeutic potential. Clin. Biochem. 32, 309 – 319 13 Van Eenennaam, A.L. et al. (2003) Engineering vitamin E content: from Arabidopsis mutant to soy oil. Plant Cell 15, 3007– 3019
0167-7799/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2004.01.008
How can genetically modified foods be made publicly acceptable? Gene Rowe Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK
A recent study by Lusk suggests that consumers might voluntarily pay more for a genetically modified (GM) food than a non-GM equivalent if made aware of the possible health benefits. However, other research indicates that the acceptability of novel hazards is affected by a variety of factors, in addition to benefits, and that making agricultural biotechnology publicly acceptable will be more complex than indicated by the results from Lusk’s study.
Corresponding author: Gene Rowe ([email protected]). www.sciencedirect.com
A recent paper by Lusk indicates that consumers could be willing to pay extra for a genetically modified (GM) food than a non-GM equivalent if told of the potential health benefits they might receive from eating it [1]. Indeed, as Lusk states in a Purdue newsletter (http://news.uns. purdue.edu/html4ever/031024.Lusk.rice.html), ‘This study is one of the first to show that people are willing to pay a premium for a food that’s been improved using biotechnology.’ Lusk attributes this result – which is contrary to past findings – to the emphasis of his study being on the potential benefits of GM food from a consumer’s, rather