biologia plantarum

International journal on Plant Life established by Bohumil Němec in 1959

Biologia plantarum 57:635-645, 2013 | DOI: 10.1007/s10535-013-0344-y

Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

M. Pičmanová1, D. Reňák1, J. Feciková1, P. Růžička1, P. Mikšátková2, O. Lapčík2, D. Honys1,3,*
1 Laboratory of Pollen Biology, Institute of Experimental Botany of the ASCR, Prague, Czech Republic
2 Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical Technology, Institute of Chemical Technology in Prague, Prague, Czech Republic
3 Faculty of Science, Charles University in Prague, Prague, Czech Republic

Isoflavone synthase (IFS; CYP93C) plays a key role in the biosynthesis of phenolic secondary metabolites, isoflavonoids. These compounds, which are well-known for their benefits to human health and plant defence, are produced mostly in legumes. However, more than 200 of them have been described in 59 other plant families without any knowledge of their respective IFS orthologue genes (with the sole exception of sugar beet). In this study, we selected IFS from Pisum sativum L. (CYP93C18) for functional expression. CYP93C18 was isolated, cloned, and introduced into Arabidopsis thaliana. The presence of the gene was shown by Southern blot analysis and its expression in the transgenic Arabidopsis was proven by RT-PCR and Western blots. The functional activity of the heterologous IFS was verified by HPLC-MS analysis of the metabolite levels: the isoflavone genistein and its derivatives tectorigenin and biochanin A were detected in the overexpressing lines. In addition, 35S::CYP93C18::GFP fused proteins were transiently expressed in the leaves of Nicotiana benthamiana and the localization of the GFP signal was observed on the endoplasmic reticulum using confocal microscopy which is consistent with the data from the literature and with our in silico predictions. The putative mode of attachment of IFS to the endoplasmic reticulum membrane is suggested. The undemanding methodology presented in this paper is applicable to the functional analysis of newly-identified isoflavone synthase genes from various species.

Keywords: Arabidopsis thaliana; cytochrome P450; endoplasmic reticulum; isoflavonoids; Nicotiana benthamiana; Pisum sativum
Subjects: isoflavon synthase; isoflavonoids; cytochrome; endoplasmatic reticulum; amino acid sequences; Southern blot; 3-D structure model; tobacco; pea

Received: November 13, 2012; Accepted: April 3, 2013; Published: December 1, 2013  Show citation

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Pičmanová, M., Reňák, D., Feciková, J., Růžička, P., Mikšátková, P., Lapčík, O., & Honys, D. (2013). Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18. Biologia plantarum, 57(4), 635-645. doi: 10.1007/s10535-013-0344-y
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    References

    1. Adams, N.R.: Detection of the effects of phytoestrogens on sheep and cattle. - J. anim. Sci. 73: 1509-1515, 1995. Go to original source...
    2. Akashi, T., Aoki, T., Ayabe, S.: Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice. - Plant Physiol. 137: 821-828, 1999. Go to original source...
    3. Akashi, T., Aoki, T., Ayabe, S.: Molecular and biochemical characterization of 2-hydroxyisoflavanone dehydratase. Involvement of carboxylesterase-like proteins in leguminous isoflavone biosynthesis. - Plant Physiol. 137: 882-891, 2005. Go to original source...
    4. Baker, N.A., Sept, D., Joseph, S., Holst, M.J., McCammon, J.A.: Electrostatics of nanosystems: application to microtubules and the ribosome. - Proc. nat. Acad. Sci. USA 98: 10037-10041, 2001. Go to original source...
    5. Baudry, J., Rupasinghe, S., Schuler, M.A.: Class-dependent sequence alignment strategy improves the structural and functional modeling of P450s. - Protein Engn. Design Select. 19: 345-353, 2006. Go to original source...
    6. Bendtsen, J.D., Nielsen, H., Von Heijne, G., Brunak, S.: Improved prediction of signal peptides: SignalP3.0. - J. mol. Biol. 340: 783-795, 2004. Go to original source...
    7. Bryson, K., McGuffin, L.J., Marsden, R.L., Ward, J.J., Sodhi, J.S., Jones, D.T.: Protein structure prediction servers at University College London. - Nucl. Acids Res. 33(Suppl.): W36-38, 2005. Go to original source...
    8. Chang, Z., Wang, X., Wei, R., Liu, Z., Shan, H., Fan, G., Hu, H.: Functional expression and purification of CYP93C20, a plant membrane-associated cytochrome P450 from Medicago truncatula. - Protein Expres. Purif. http://dx.doi.org/10.1016/j.pep.2010.11.012
    9. Clough, S.J., Bent, A.F.: Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. - Plant J. 16: 735-743, 1998. Go to original source...
    10. Cooper, L.D., Doss, R.P., Price, R., Peterson, K., Olivern, J.E.: Application of Bruchin B to pea pods results in the upregulation of CYP93C18, a putative isoflavone synthase gene, and an increase in the level of pisatin, an isoflavone phytoalexin. - J. exp. Bot. 56: 1229-1237, 2005. Go to original source...
    11. Cornwell, T., Cohick, W., Raskin, I.: Dietary phytoestrogens and health. - Phytochemistry 65: 995-1016, 2004. Go to original source...
    12. Crozier, A., Jaganath, I.B., Clifford, M.N.: Phenols, polyphenols and tannins: an overview. - In: Crozier, A., Clifford, M.N., Ashihara, H. (ed.): Plant Secondary Metabolites: Occurrence, Structure and Role in the Human Diet. Pp. 1-24. Blackwell Publishing, Oxford 2006. Go to original source...
    13. Dai, R., Pincus, M.R., Friedman, F.K.: Molecular modeling of cytochromes P450 2B1: mode of membrane insertion and substrate specifity. - J. Protein Chem. 17: 121-129, 1998. Go to original source...
    14. Dixon, R.A., Pasinetti, G.M.: Flavonoids and isoflavonoids: from plant biology to agriculture and neuroscience. - Plant Physiol. 154: 453-457, 2010. Go to original source...
    15. Franzmayr, B.K., Rasmussen, S., Fraser, K.M., Jameson, P.E.: Expression and functional characterization of a white clover isoflavone synthase in tobacco. - Ann. Bot. 110: 1291-1301, 2012. Go to original source...
    16. Haseloff, J., Siemering, K.R., Prasher, D.C., Hodge, S.: Removal of a cryptic intron and subcellular localisation of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. - Proc. nat. Acad. Sci. USA 94: 2122-2127, 1997. Go to original source...
    17. He, X., Blount, J.W., Ge, S., Tang, Y., Dixon, R.A.: A genomic approach to isoflavone biosynthesis in kudzu (Pueraria lobata). - Planta 233: 843-855, 2011. Go to original source...
    18. Hofmann, K., Stoffel, W.: TMbase - a database of membrane spanning proteins segments - Biol. Chem. Hoppe-Seyler 374: 166, 1993.
    19. Jaganath, I.B.: Dietary Flavonoids: Bioavailabilty and Biosynthesis. - PhD Thesis, University of Glasgow, Glasgow 2005.
    20. Jung, W., Yu, O., Lau, S.C., O'Keefe, D., Odell, J., Fader, G., McGonigle, B.: Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes. - Nat. Biotechnol. 18: 208-212, 2000. Go to original source...
    21. Lapčík, O.: Isoflavonoids in non-leguminous taxa: a rarity or rule? - Phytochemistry 68: 2909-2916, 2007. Go to original source...
    22. Lapčík, O., Honys, D., Koblovská, R., Macková, Z., Vítková, M., Klejdus, B.: Isoflavonoids are present in Arabidopsis thaliana despite the absence of any homologue to known isoflavonoid synthases. - Plant Physiol. Biochem. 44: 106-114, 2006. Go to original source...
    23. Lapčík, O., Hill, M., Černý, I., Lachman, J., Al-Maharik, N., Adlercreutz, H., Hampl R.: Immunoanalysis of isoflavonoids in Pisum sativum and Vigna radiata. - Plant Sci. 148: 111-119, 1999. Go to original source...
    24. Li, J.F., Park, E., Von Arnim, A.G., Nebenführ, A.: The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. - Plant Methods 5: 6, 2009. Go to original source...
    25. Liu, C.-J., Blount, J.W., Steele, C.L., Dixon, R.A.: Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis. - Proc. nat. Acad. Sci. USA 99: 14578-14583, 2002. Go to original source...
    26. Liu, C.-J., Dixon, R.A.: Elicitor-induced association of isoflavone O-methyltransferase with endomembranes prevents the formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesis. - Plant Cell 13: 2643-2658, 2001. Go to original source...
    27. Macková, Z., Koblovská, R., Lapčík, O.: Distribution of isoflavonoids in non-leguminous taxa - an update. - Phytochemistry 67: 849-855, 2006. Go to original source...
    28. Misra, P., Pandey, A., Tewari, S.K., Nath, P., Trivedi, P.K.: Characterization of isoflavone synthase gene from Psoralea corylifolia: a medicinal plant. - Plant Cell Rep. 29: 747-755, 2010. Go to original source...
    29. Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., Kimura, T.: Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. - J. Biosci. Bioeng. 104: 34-41, 2007. Go to original source...
    30. Ososki, A.L., Kennelly, E.J.: Phytoestrogens: a rewiev of present state of research. - Phytoterap. Res. 17: 845-869, 2003. Go to original source...
    31. Oulehlová, D., Hála, M., Potocký, M., Žárský, V., Cvrčková, F.: Plant antigens cross-react with rat polyclonal antibodies against KLH-conjugated peptides. - Cell Biol. Int. 33: 113-118, 2009. Go to original source...
    32. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., Ferrin, T.E.: UCSF chimera - a visualization system for exploratory research and analysis. - Comput. Chem. 25: 1605-1612, 2004. Go to original source...
    33. Sali, A., Blundell, T.L.: Comparative protein modelling by satisfaction of spatial restraints. - J. mol. Biol. 234: 779-815, 1993. Go to original source...
    34. Sawada, Y., Ayabe, S.: Multiple mutagenesis of P450 isoflavonoid synthase reveals a key active-site residue. - Biochem. biophys. Res. Commun. 330: 907-913, 2005. Go to original source...
    35. Sawada, Y., Kinoshita, K., Akashi, T., Aoki, T., Ayabe, S.: Key amino acid residues required for aryl migration catalysed by the cytochrome P450 2-hydroxyisoflavanone synthase. - Plant J. 31: 555-564, 2002. Go to original source...
    36. Steele, C.L., Gijzen, M., Qutob, D., Dixon, R.A.: Molecular characterization of the enzyme catalyzing the aryl migration reaction of isoflavonoid biosynthesis in soybean. - Arch. Biochem. Biophys. 367: 146-150, 1999. Go to original source...
    37. Thelen, P., Scharf, J.G., Burfeind, P., Hemmerlein, B., Wuttke, W., Spengler, B., Christoffel, V., Ringert, R.H., Seidlová-Wuttke, D.: Tectorigenin and other phytochemicals extracted from leopard lily Belamcanda chinensis affect new and established targets for therapies in prostate cancer. - Carcinogenesis 26: 1360-1367, 2005. Go to original source...
    38. Veitch, NC: Isoflavonoids of the leguminosae. - Nat. Prod. Rep. 24: 417-464, 2007. Go to original source...
    39. Wang, W., Vignani, R., Scali, M., Cresti, M.: A universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysis. - Electrophoresis 27: 2782-2786, 2006. Go to original source...
    40. Weigel, D., Glazebrook, J. (ed.): Arabidopsis. A Laboratory Handbook. - Cold Spring Harbor Laboratory Press, Cold Spring Harbor - New York 2002.
    41. Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F., McRee, D.E.: Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functionaldiversity. - Mol. Cells 5: 121-131, 2000. Go to original source...
    42. Wiriyaampaiwong, P., Thanonkeo, S., Thanonkeo, P.: Molecular characterization of isoflavone synthase gene from Pueraria candollei var. mirifica. - Afr. J. agr. Res. 7: 4489-4498, 2012. Go to original source...
    43. Yu, O., Jung, W., Shi, J., Croes, R.A., Fader, G.M., McGonigle, B., Odell, J.T.: Production of the isoflavones genistein and daidzein in non-legume dicot and monocot tissues. - Plant Physiol. 124: 781-794, 2000. Go to original source...
    44. Yu, O., McGonigle, B.: Metabolic engineering of isoflavone biosynthesis. - Adv. Agr. 86: 147-190, 2005. Go to original source...

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