Transgenic Research

, Volume 21, Issue 4, pp 757–771

Coloring genetically modified soybean grains with anthocyanins by suppression of the proanthocyanidin genes ANR1 and ANR2

  • Nik Kovinich
  • Ammar Saleem
  • Tara L. Rintoul
  • Daniel C. W. Brown
  • John T. Arnason
  • Brian Miki
Original Paper

Abstract

Detection and quantification of the levels of adventitious presence of genetically modified (GM) soybeans in non-GM grain shipments currently requires sophisticated tests that can have issues with their reproducibility. We show here that pigment biosynthesis in the soybean seed coat can be manipulated to provide a distinct color that would enable the simple visible detection of the GM soybean grain. We observed that a distinct red-brown grain color could be engineered by the simultaneous suppression of two proanthocyanidin (PA) genes, ANTHOCYANIDINREDUCTASE1 (ANR1) and ANR2. Multiple reaction monitoring by liquid chromatography tandem mass spectrometry was used to quantify differentially accumulated seed coat metabolites, and revealed the redirection of metabolic flux into the anthocyanin pigment pathway and unexpectedly the flavonol-3-O-glucoside pathway. The upregulations of anthocyanin isogenes (DFR1 and GST26) and the anthocyanin/flavonol-3-O-glycosyltransferase (UGT78K2) were identified by quantitative RT-PCR to be endogenous feedback and feedforward responses to overaccumulation of upstream flavonoid intermediates resulting from ANR1 and ANR2 suppressions. These results suggested the transcription of flavonoid genes to be a key component of the mechanism responsible for the redirection of metabolite flux. This report identifies the suppression of PA genes to be a novel approach for engineering pigmentation in soybean grains.

Keywords

Metabolic engineering Visual marker Soybean seed coat color Anthocyanin Proanthocyanidin ANTHOCYANIDIN REDUCTASE 

Supplementary material

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Supplementary material 1 (XLSX 12 kb)
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Supplementary material 2 (XLSX 10 kb)
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Supplementary material 3 (XLS 25 kb)
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Supplementary material 4 (XLS 23 kb)

References

  1. Albert S, Delseny M, Devic M (1997) BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat. Plant J 11:289–299PubMedCrossRefGoogle Scholar
  2. Basaran P, Rodriguez-Cerezo E (2008) Plant molecular farming: opportunities and challenges. Crit Rev Biotechnol 28:153–172PubMedCrossRefGoogle Scholar
  3. Besseau S, Hoffmann L, Geoffroy P, Lapierre C, Pollet B, Legrand M (2007) Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell 19:148–162PubMedCrossRefGoogle Scholar
  4. Blount JW, Korth KL, Masoud SA, Rasmussen S, Lamb C, Dixon RA (2000) Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway. Plant Physiol 122:107–116PubMedCrossRefGoogle Scholar
  5. Bolwell G, Cramer C, Lamb C, Schuch W, Dixon R (1986) l-Phenylalanine ammonia-lyase from Phaseolus vulgaris: modulation of the levels of active enzyme by trans-cinnamic acid. Planta 149:97–107CrossRefGoogle Scholar
  6. Demeke T, Perry D, Scowcroft W (2006) Adventitious presence of GMOs: scientific overview for Canadian grains. Can J Plant Sci 86:1–23CrossRefGoogle Scholar
  7. Devic M, Guilleminot J, Debeaujon I, Bechtold N, Bensaude E, Koornneef M, Pelletier G, Delseny M (1999) The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Plant J 19:387–398PubMedCrossRefGoogle Scholar
  8. Finer JJ, McMullen MD (1991) Transformation of soybean via particle bombardment of embryogenic suspension culture tissue. In Vitro Cell Dev Biol Plant 27:175–182CrossRefGoogle Scholar
  9. Geekiyanage S (2009) Potential of VlmybAl-2 as a candidate marker for visual identification of transgenic plants: induced anthocyanin production in Arabidopsis and tobacco. Trop Agric Res Ext 12:35–41Google Scholar
  10. Hahlbrock K, Knobloch K, Kreuzaler F, Potts J, Wellmann E (1976) Coordinated induction and subsequent activity changes of two groups of metabolically interrelated enzymes. Light-induced synthesis of flavonoid glycosides in cell suspension cultures of Petroselinum hortense. Eur J Biochem 61:199–206PubMedCrossRefGoogle Scholar
  11. Hartmann U, Sagasser M, Mehrtens F, Stracke R, Weisshaar B (2005) Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes. Plant Mol Biol 57:155–171PubMedCrossRefGoogle Scholar
  12. Holton TA (1995) Modification of flower colour via manipulation of P450 gene expression in transgenic plants. Drug Metabol Drug Interact 12:359–368PubMedCrossRefGoogle Scholar
  13. Holton TA, Brugliera F, Tanaka Y (1993) Cloning and expression of flavonol synthase from Petunia hybrida. Plant J 4:1003–1010PubMedCrossRefGoogle Scholar
  14. Karg SR, Kallio PT (2009) The production of biopharmaceuticals in plant systems. Biotechnol Adv 27:879–894PubMedCrossRefGoogle Scholar
  15. Kovinich N, Saleem A, Arnason JT, Miki B (2010) Functional characterization of a UDP-glucose:flavonoid 3-O-glucosyltransferase from the seed coat of black soybean (Glycine max (L.) Merr.). Phytochemistry 71:1253–1263PubMedCrossRefGoogle Scholar
  16. Kovinich N, Arnason JT, De Luca V, Miki B (2011a) Coloring soybeans with anthocyanins? In: Gang DR (ed) Recent advances in phytochemistry. Springer, New York, pp 47–57Google Scholar
  17. Kovinich N, Saleem A, Arnason JT, Miki B (2011b) Combined analysis of transcriptome and metabolite data reveals extensive differences between black and brown nearly-isogenic soybean (Glycine max) seed coats enabling the identification of pigment isogenes. BMC Genomics 12:381PubMedCrossRefGoogle Scholar
  18. Kovinich N, Saleem A, Arnason JT, Miki B (Submitted) Identification of two anthocyanidin reductase genes from the seed coat of brown soybean and three red-brown soybean accessions that have reduced ANTHOCYANIDIN REDUCTASE1 mRNA, activity, and seed coat proanthocyanidin amounts. J Agric Food ChemGoogle Scholar
  19. Krueger R, Le Buanec B (2008) Action needed to harmonize regulation of low-level presence of biotech traits. Nat Biotechnol 26:161–162PubMedCrossRefGoogle Scholar
  20. Lee Y, Yoon H, Paik YS, Liu JR, Chung W-I, Choi G (2005) Reciprocal regulation of Arabidopsis UGT78D2 and BANYULS is critical for regulation of the metabolic flux of anthocyanidins to condensed tannins in developing seed coats. J Plant Biol 48:356–370CrossRefGoogle Scholar
  21. Lemaux PG (2008) Genetically engineered plants and foods: a scientist’s analysis of the issues (part I). Annu Rev Plant Biol 59:771–812PubMedCrossRefGoogle Scholar
  22. Lepiniec L, Debeaujon I, Routaboul JM, Baudry A, Pourcel L, Nesi N, Caboche M (2006) Genetics and biochemistry of seed flavonoids. Annu Rev Plant Biol 57:405–430PubMedCrossRefGoogle Scholar
  23. Loake GJ, Choudhary AD, Harrison MJ, Mavandad M, Lamb CJ, Dixon RA (1991) Phenylpropanoid pathway intermediates regulate transient expression of a chalcone synthase gene promoter. Plant Cell 3:829–840PubMedGoogle Scholar
  24. McCallum JA, Walker JRL (1990) Proanthocyanidins in wheat bran. Cereal Chem 67:282–285Google Scholar
  25. Meyer P, Heidmann I, Forkmann G, Saedler H (1987) A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature 330:677–678PubMedCrossRefGoogle Scholar
  26. Miki D, Shimamoto K (2004) Simple RNAi vectors for stable and transient suppression of gene function in rice. Plant Cell Physiol 45:490–495PubMedCrossRefGoogle Scholar
  27. Mol J, Grotewold E, Koesa R (1998) How genes paint flowers and seeds. Trends Plant Sci 3:212–217CrossRefGoogle Scholar
  28. Morisset D, Demsar T, Gruden K, Vojvoda J, Stebih D, Zel J (2009) Detection of genetically modified organisms-closing the gaps. Nat Biotechnol 27:700–701PubMedCrossRefGoogle Scholar
  29. Naczk M, Nichols T, Pink D, Sosulski F (1994) Condensed tannins in Canola Hulls. J Agric Food Chem 42:2196–2200CrossRefGoogle Scholar
  30. Nagamatsu A, Masuta C, Matsuura H, Kitamura K, Abe J, Kanazawa A (2009) Down-regulation of flavonoid 3′-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence. J Plant Physiol 166:32–39PubMedCrossRefGoogle Scholar
  31. Nakatsuka T, Abe Y, Kakizaki Y, Yamamura S, Nishihara M (2007) Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes. Plant Cell Rep 26:1951–1959PubMedCrossRefGoogle Scholar
  32. Nishihara M, Nakatsuka T (2011) Genetic engineering of flavonoid pigments to modify flower color in floricultural plants. Biotechnol Lett 33:433–441PubMedCrossRefGoogle Scholar
  33. Pelletier MK, Murrell JR, Shirley BW (1997) Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis. Further evidence for differential regulation of “early” and “late” genes. Plant Physiol 113:1437–1445PubMedCrossRefGoogle Scholar
  34. Ramessar K, Capell T, Twyman RM, Christou P (2010) Going to ridiculous lengths—European coexistence regulations for GM crops. Nat Biotechnol 28:133–136PubMedCrossRefGoogle Scholar
  35. Routaboul JM, Kerhoas L, Debeaujon I, Pourcel L, Caboche M, Einhorn J, Lepiniec L (2006) Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana. Planta 224:96–107PubMedCrossRefGoogle Scholar
  36. Shen L, Petolino J (2006) Pigmented maize seed via tissue-specific expression of anthocyanin pathway gene transcription factors. Mol Breed 18:57–67CrossRefGoogle Scholar
  37. Smyth S, McHughen A (2008) Regulating innovative crop technologies in Canada: the case of regulating genetically modified crops. Plant Biotechnol J 6:213–225PubMedCrossRefGoogle Scholar
  38. Stewart CN Jr (2005) Monitoring the presence and expression of transgenes in living plants. Trends Plant Sci 10:390–396PubMedCrossRefGoogle Scholar
  39. Stewart CN Jr (2006) Go with the glow: fluorescent proteins to light transgenic organisms. Trends Biotechnol 24:155–162PubMedCrossRefGoogle Scholar
  40. Stracke R, Ishihara H, Huep G, Barsch A, Mehrtens F, Niehaus K, Weisshaar B (2007) Differential regulation of closely related R2R3-MYB transcription factors controls flavonol accumulation in different parts of the Arabidopsis thaliana seedling. Plant J 50:660–677PubMedCrossRefGoogle Scholar
  41. Stracke R, De Vos RC, Bartelniewoehner L, Ishihara H, Sagasser M, Martens S, Weisshaar B (2009) Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase. Planta 229:427–445PubMedCrossRefGoogle Scholar
  42. Sugimoto T, Kawasaki T, Kato T, Whittier RF, Shibata D, Kawamura Y (1992) cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean. Plant Mol Biol 20:743–747PubMedCrossRefGoogle Scholar
  43. Tanaka Y, Tsuda S, Kusumi T (1998) Metabolic engineering to modify flower color. Plant Cell Physiol 39:1119–1126CrossRefGoogle Scholar
  44. Tsuda S, Fukui Y, Nakamura N, Katsumoto Y, Yonekura-Sakakibara K, Fukuchi-Mizutani M, Ohira K, Ueyama Y, Ohkawa H, Holton TA, Kusumi T, Tanaka Y (2004) Flower color modification of Petunia hybrida commercial varieties by metabolic engineering. Plant Biotechnol 21:377–386CrossRefGoogle Scholar
  45. Tuteja JH, Clough SJ, Chan WC, Vodkin LO (2004) Tissue-specific gene silencing mediated by a naturally occurring chalcone synthase gene cluster in Glycine max. Plant Cell 16:819–835PubMedCrossRefGoogle Scholar
  46. Venglat P, Xiang D, Qiu S, Stone SL, Tibiche S, Cram D, Alting-Mees M, Nowak J, Cloutier S, Deyholos M, Bekkaoui F, Sharpe A, Wang E, Rowland G, Selvaraj G, Datla R (2011) Gene expression analysis of flax seed development. BMC Plant Biol 11:74PubMedCrossRefGoogle Scholar
  47. Wang C-S, Vodkin LO (1994) Extraction of RNA from tissues containing high levels of procyanidins that bind RNA. Plant Mol Biol Rep 12:132–145Google Scholar
  48. Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493PubMedCrossRefGoogle Scholar
  49. Xie DY, Sharma SB, Paiva NL, Ferreira D, Dixon RA (2003) Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 299:396–399PubMedCrossRefGoogle Scholar
  50. Yu O, Shi J, Hession AO, Maxwell CA, McGonigle B, Odell JT (2003) Metabolic engineering to increase isoflavone biosynthesis in soybean seed. Phytochemistry 63:753–763PubMedCrossRefGoogle Scholar
  51. Zhao J, Dixon RA (2010) The ‘ins’ and ‘outs’ of flavonoid transport. Trends Plant Sci 15:72–80PubMedCrossRefGoogle Scholar
  52. Zhao J, Huhman D, Shadle G, He XZ, Sumner LW, Tang Y, Dixon RA (2011) MATE2 mediates vacuolar sequestration of flavonoid glycosides and Glycoside Malonates in Medicago truncatula. Plant Cell 23:1536–1555PubMedCrossRefGoogle Scholar

Copyright information

© Her Majesty the Queen in Right of Canada 2011

Authors and Affiliations

  • Nik Kovinich
    • 1
    • 2
  • Ammar Saleem
    • 3
  • Tara L. Rintoul
    • 1
  • Daniel C. W. Brown
    • 4
  • John T. Arnason
    • 3
  • Brian Miki
    • 1
  1. 1.Bioproducts and Bioprocesses, Research BranchAgriculture and Agri-Food CanadaOttawaCanada
  2. 2.Department of Biology, Ottawa–Carleton Institute of BiologyCarleton UniversityOttawaCanada
  3. 3.Department of Biology and Center for Research in Biopharmaceuticals and BiotechnologyUniversity of OttawaOttawaCanada
  4. 4.Southern Crop Protection and Food Research CentreAgriculture and Agri-Food CanadaLondonCanada

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