- Email: [email protected]
Response to Kutchan: Genetic engineering, natural variation and substantial equivalence
Update
TRENDS in Biotechnology
4 Sweetlove, L.J. et al. (2003) Predictive metabolic engineering: a goal for systems biology. Plant Physiol. 132, 420–425 5 Chen, F. et al. (2003) Profiling phenolic metabolites in transgenic alfalfa modified in lignin biosynthesis. Phytochemistry 64, 1013–1021 6 Hirai, M.Y. et al. (2004) Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 101, 10205–10210 7 Ye, X. et al. (2000) Engineering the provitamin A (b-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305 8 Ralley, L. et al. (2004) Metabolic engineering of ketocarotenoid formation in higher plants. Plant J. 39, 477–486 9 Muir, S.R. et al. (2001) Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nat. Biotechnol. 19, 470–474
Vol.23 No.8 August 2005
383
10 Niggeweg, R. et al. (2004) Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol. 22, 746–754 11 Mahmoud, S.S. and Croteau, R. (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc. Natl. Acad. Sci. U. S. A. 98, 8915–8920 12 Lu¨cker, J. et al. (2004) Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon. Plant Physiol. 134, 510–519 13 Li, L. et al. (2003) Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc. Natl. Acad. Sci. U. S. A. 100, 4939–4944 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.05.005
Research Focus Response
Response to Kutchan: Genetic engineering, natural variation and substantial equivalence Birger Lindberg Møller and Søren Bak Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology (PlaCe), Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark
In the Research Focus article by Toni Kutchan [1], published in this issue of Trends in Biotechnology, Kutchan concludes that genetic engineering can be used to introduce new natural products in a plant with only marginal inadvertent effects. A prerequisite for a successful approach is thorough biochemical understanding of the metabolic pathway to be altered or introduced. We fully agree. But how do these marginal inadvertent effects in a plant compare with natural variation? The rapid experimental developments within transcriptomics, proteomics and metabolomics provide the tools to answer the question of how marginal inadvertent effects of genetic engineering in a plant compare with natural variation, and to identify new plant cultivars that strictly adhere to the principle of substantial equivalence [2]. It also enables us to assess the magnitude of natural variation within the same species [3,4]. Such variation might, for example, reflect: (i) evolutionary differences between ecotypes associated with adaptation to the prevailing environmental conditions in geographically distinct regions and different habitats; (ii) differences in climatic conditions; (iii) differences in natural product content owing to plant responses to herbivore and pathogen attack; (iv) classical breeding to enhance a desired trait or to remove an unwanted property; (v) diurnal changes frozen-in by the time point at which the plant was harvested; or (vi) harvesting of the plant at different developmental stages [5,6]. The differences in the transcriptome and metabolome observed by sulfur starvation of Arabidopsis thaliana plants [7], differences in the transcriptome observed between the A. thaliana ecotypes Columbia and Corresponding author: Møller, B.L. ([email protected]). Available online 20 June 2005 www.sciencedirect.com
Landsberg erecta [8] and the differences in the metabolome of wild and cultivated strawberry with respect to fruit flavour compounds [9] (Figure 1) are significant. The differences also exceed the marginal and minute inadvertent differences in the transcriptome and metabolome of A. thaliana observed upon introduction of the dhurrin pathway by genetic engineering (Figures 2 and 3 in [10]). The differences that sometimes are reported between organically and traditionally grown plant products (e.g. with respect to the content of heavy metals [11] and flavonoids [12]) fall into the same category and are likewise marginal. Such marginal differences between organic, conventional or genetically modified foods have no impact on food quality and human health. To improve public health, it is important to secure access to a varied and balanced diet and to educate people in industrialized countries who have the opportunity but do not take advantage of the offer. Plants are the organic chemists par excellence in nature and constantly adjust their content of natural products as a response to biotic and abiotic challenges. Metabolic crosstalk contributes to the diversity of the responses, but is controlled by metabolon formation [13]. Introduction of a new biosynthetic pathway in the form of an enzyme metabolon would be predicted to reduce greatly the undesired impact on other metabolic pathways [10]. Unravelling of the mechanisms that control metabolon formation in natural product synthesis will thus dramatically expand the possibilities of engineering crop plants with improved content of pro-vitamins, flavours, fragrances, pharmaceuticals and other valuable fine chemicals that strictly adhere to the criteria of substantial equivalence.
Update
384
TRENDS in Biotechnology
Vol.23 No.8 August 2005
Cultivated: Fragaria ananassa OH
Abundance
OPP
Nerolidol
50000
TPS
40000
Mg2+, Mn2+
30000
Linalool
20000 10000 0
S-Linalool
Geranyl diphosphate 6.00
8.00
10.00
12.00
14.00
16.00
18.00
Time
Myrtenyl acetate
Abundance
28000
OH
Wild: Fragaria vesca
OPP
24000
TPS
20000 16000 12000
Mg2+, Mn2+
β-Myrcene
8000 4000 α-Pinene
β-Phellandrene α-Terpineol
6.00
8.00
10.00
(3S)-(E)-Nerolidol
Farnesyl diphosphate
0 12.00
14.00
16.00
18.00
Time
CH2OH
CH2OCOCH3
OPP
Geranyl diphosphate
TPS
Cytochrome P450
AAT
Mg2+, Mn2+
NADPH + O2
Acetyl-CoA
α-Pinene
Myrtenol
Myrtenyl acetate
Figure 1. Terpenoid production in cultivated and wild strawberry. The terpenoids were detected by gas chromatographic–mass spectrometric (GC–MS) headspace analysis of ripe fruits. Reactions catalysed by terpene synthases (TPS) and acetyltransferases (AAT), forming linalool, nerolidol, a-pinene, myrtenol and myrtenol acetate, are shown. Reproduced, with permission, from [9].
Acknowledgements Support is gratefully acknowledged from the Danish National Research Foundation and the Danish Research Council for Technology and Production.
References 1 Kutchan, T.M. (2005) Predictive metabolic engineering in plants: still full of surprises. Trends Biotechnol. 23. doi: 10.1016/j.tibtech. 2005.05. 005 2 WHO (2000) Safety aspects of genetically modified foods of plant origin. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology, WHO, Geneva 3 Koornneef, M. et al. (2004) Naturally occurring genetic variation in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55, 141–172 4 Borevitz, J.O. and Nordborg, M. (2003) The impact of genomics on the study of natural variation in Arabidopsis. Plant Physiol. 132, 718–725 5 Urbanczyk-Wochniak, E. et al. (2003) Parallel analysis of transcript and metabolic profiles: a new approach in systems biology. EMBO Rep. 4, 989–993 6 Urbanczyk-Wochniak, E. et al. Profiling of diurnal patterns of metabolite and transcript abundance in potato (Solanum tuberosum) leaves. Planta (in press) 7 Nikiforova, V.J. et al. (2004) Towards dissecting nutrient metabolism in plants: a systems biology case study on sulphur metabolism. J. Exp. Bot. 55, 1861–1871
www.sciencedirect.com
8 Borevitz, J.O. et al. (2003) Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res. 13, 513–523 9 Aharoni, A. et al. (2004) Gain and loss of fruit flavour compounds produced by wild and cultivated strawberry species. Plant Cell 16, 3110–3131 10 Kristensen, C. et al. (2005) Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome. Proc. Natl. Acad. Sci. U. S. A. 102, 1779–1784 11 Gundersen, V. et al. (2000) Comparative investigation of concentrations of major and trace elements in organic and conventional Danish agricultural crops. 1. Onions (Allium cepa Hysam) and peas (Pisum sativum Ping Pong). J. Agric. Food Chem. 48, 6094–6102 12 Grinder-Pedersen, L. et al. (2003) Effect of diets based on foods from conventional versus organic production on intake and excretion of flavonoids and markers of antioxidative defense in humans. J. Agric. Food Chem. 51, 5671–5676 13 Jørgensen, K. et al. (2005) Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr. Opin. Plant Biol. 8, 280–291
0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.06.004
TRENDS in Biotechnology
4 Sweetlove, L.J. et al. (2003) Predictive metabolic engineering: a goal for systems biology. Plant Physiol. 132, 420–425 5 Chen, F. et al. (2003) Profiling phenolic metabolites in transgenic alfalfa modified in lignin biosynthesis. Phytochemistry 64, 1013–1021 6 Hirai, M.Y. et al. (2004) Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U. S. A. 101, 10205–10210 7 Ye, X. et al. (2000) Engineering the provitamin A (b-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305 8 Ralley, L. et al. (2004) Metabolic engineering of ketocarotenoid formation in higher plants. Plant J. 39, 477–486 9 Muir, S.R. et al. (2001) Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols. Nat. Biotechnol. 19, 470–474
Vol.23 No.8 August 2005
383
10 Niggeweg, R. et al. (2004) Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat. Biotechnol. 22, 746–754 11 Mahmoud, S.S. and Croteau, R. (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc. Natl. Acad. Sci. U. S. A. 98, 8915–8920 12 Lu¨cker, J. et al. (2004) Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon. Plant Physiol. 134, 510–519 13 Li, L. et al. (2003) Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc. Natl. Acad. Sci. U. S. A. 100, 4939–4944 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.05.005
Research Focus Response
Response to Kutchan: Genetic engineering, natural variation and substantial equivalence Birger Lindberg Møller and Søren Bak Plant Biochemistry Laboratory, Department of Plant Biology, Center for Molecular Plant Physiology (PlaCe), Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark
In the Research Focus article by Toni Kutchan [1], published in this issue of Trends in Biotechnology, Kutchan concludes that genetic engineering can be used to introduce new natural products in a plant with only marginal inadvertent effects. A prerequisite for a successful approach is thorough biochemical understanding of the metabolic pathway to be altered or introduced. We fully agree. But how do these marginal inadvertent effects in a plant compare with natural variation? The rapid experimental developments within transcriptomics, proteomics and metabolomics provide the tools to answer the question of how marginal inadvertent effects of genetic engineering in a plant compare with natural variation, and to identify new plant cultivars that strictly adhere to the principle of substantial equivalence [2]. It also enables us to assess the magnitude of natural variation within the same species [3,4]. Such variation might, for example, reflect: (i) evolutionary differences between ecotypes associated with adaptation to the prevailing environmental conditions in geographically distinct regions and different habitats; (ii) differences in climatic conditions; (iii) differences in natural product content owing to plant responses to herbivore and pathogen attack; (iv) classical breeding to enhance a desired trait or to remove an unwanted property; (v) diurnal changes frozen-in by the time point at which the plant was harvested; or (vi) harvesting of the plant at different developmental stages [5,6]. The differences in the transcriptome and metabolome observed by sulfur starvation of Arabidopsis thaliana plants [7], differences in the transcriptome observed between the A. thaliana ecotypes Columbia and Corresponding author: Møller, B.L. ([email protected]). Available online 20 June 2005 www.sciencedirect.com
Landsberg erecta [8] and the differences in the metabolome of wild and cultivated strawberry with respect to fruit flavour compounds [9] (Figure 1) are significant. The differences also exceed the marginal and minute inadvertent differences in the transcriptome and metabolome of A. thaliana observed upon introduction of the dhurrin pathway by genetic engineering (Figures 2 and 3 in [10]). The differences that sometimes are reported between organically and traditionally grown plant products (e.g. with respect to the content of heavy metals [11] and flavonoids [12]) fall into the same category and are likewise marginal. Such marginal differences between organic, conventional or genetically modified foods have no impact on food quality and human health. To improve public health, it is important to secure access to a varied and balanced diet and to educate people in industrialized countries who have the opportunity but do not take advantage of the offer. Plants are the organic chemists par excellence in nature and constantly adjust their content of natural products as a response to biotic and abiotic challenges. Metabolic crosstalk contributes to the diversity of the responses, but is controlled by metabolon formation [13]. Introduction of a new biosynthetic pathway in the form of an enzyme metabolon would be predicted to reduce greatly the undesired impact on other metabolic pathways [10]. Unravelling of the mechanisms that control metabolon formation in natural product synthesis will thus dramatically expand the possibilities of engineering crop plants with improved content of pro-vitamins, flavours, fragrances, pharmaceuticals and other valuable fine chemicals that strictly adhere to the criteria of substantial equivalence.
Update
384
TRENDS in Biotechnology
Vol.23 No.8 August 2005
Cultivated: Fragaria ananassa OH
Abundance
OPP
Nerolidol
50000
TPS
40000
Mg2+, Mn2+
30000
Linalool
20000 10000 0
S-Linalool
Geranyl diphosphate 6.00
8.00
10.00
12.00
14.00
16.00
18.00
Time
Myrtenyl acetate
Abundance
28000
OH
Wild: Fragaria vesca
OPP
24000
TPS
20000 16000 12000
Mg2+, Mn2+
β-Myrcene
8000 4000 α-Pinene
β-Phellandrene α-Terpineol
6.00
8.00
10.00
(3S)-(E)-Nerolidol
Farnesyl diphosphate
0 12.00
14.00
16.00
18.00
Time
CH2OH
CH2OCOCH3
OPP
Geranyl diphosphate
TPS
Cytochrome P450
AAT
Mg2+, Mn2+
NADPH + O2
Acetyl-CoA
α-Pinene
Myrtenol
Myrtenyl acetate
Figure 1. Terpenoid production in cultivated and wild strawberry. The terpenoids were detected by gas chromatographic–mass spectrometric (GC–MS) headspace analysis of ripe fruits. Reactions catalysed by terpene synthases (TPS) and acetyltransferases (AAT), forming linalool, nerolidol, a-pinene, myrtenol and myrtenol acetate, are shown. Reproduced, with permission, from [9].
Acknowledgements Support is gratefully acknowledged from the Danish National Research Foundation and the Danish Research Council for Technology and Production.
References 1 Kutchan, T.M. (2005) Predictive metabolic engineering in plants: still full of surprises. Trends Biotechnol. 23. doi: 10.1016/j.tibtech. 2005.05. 005 2 WHO (2000) Safety aspects of genetically modified foods of plant origin. Report of a Joint FAO/WHO Expert Consultation on Foods Derived from Biotechnology, WHO, Geneva 3 Koornneef, M. et al. (2004) Naturally occurring genetic variation in Arabidopsis thaliana. Annu. Rev. Plant Biol. 55, 141–172 4 Borevitz, J.O. and Nordborg, M. (2003) The impact of genomics on the study of natural variation in Arabidopsis. Plant Physiol. 132, 718–725 5 Urbanczyk-Wochniak, E. et al. (2003) Parallel analysis of transcript and metabolic profiles: a new approach in systems biology. EMBO Rep. 4, 989–993 6 Urbanczyk-Wochniak, E. et al. Profiling of diurnal patterns of metabolite and transcript abundance in potato (Solanum tuberosum) leaves. Planta (in press) 7 Nikiforova, V.J. et al. (2004) Towards dissecting nutrient metabolism in plants: a systems biology case study on sulphur metabolism. J. Exp. Bot. 55, 1861–1871
www.sciencedirect.com
8 Borevitz, J.O. et al. (2003) Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res. 13, 513–523 9 Aharoni, A. et al. (2004) Gain and loss of fruit flavour compounds produced by wild and cultivated strawberry species. Plant Cell 16, 3110–3131 10 Kristensen, C. et al. (2005) Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome. Proc. Natl. Acad. Sci. U. S. A. 102, 1779–1784 11 Gundersen, V. et al. (2000) Comparative investigation of concentrations of major and trace elements in organic and conventional Danish agricultural crops. 1. Onions (Allium cepa Hysam) and peas (Pisum sativum Ping Pong). J. Agric. Food Chem. 48, 6094–6102 12 Grinder-Pedersen, L. et al. (2003) Effect of diets based on foods from conventional versus organic production on intake and excretion of flavonoids and markers of antioxidative defense in humans. J. Agric. Food Chem. 51, 5671–5676 13 Jørgensen, K. et al. (2005) Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr. Opin. Plant Biol. 8, 280–291
0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.06.004