Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18
- 348 Downloads
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.
Additional key wordsArabidopsis thaliana cytochrome P450 endoplasmic reticulum isoflavonoids Nicotiana benthamiana Pisum sativum
keyhole limpet hemocyanin
green fluorescent protein
Unable to display preview. Download preview PDF.
- 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.2010.11.012Google Scholar
- 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.PubMedCrossRefGoogle Scholar
- Hofmann, K., Stoffel, W.: TMbase — a database of membrane spanning proteins segments — Biol. Chem. Hoppe-Seyler 374: 166, 1993.Google Scholar
- Jaganath, I.B.: Dietary Flavonoids: Bioavailabilty and Biosynthesis. — PhD Thesis, University of Glasgow, Glasgow 2005.Google Scholar
- 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.PubMedCrossRefGoogle Scholar
- 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.PubMedCrossRefGoogle Scholar
- Weigel, D., Glazebrook, J. (ed.): Arabidopsis. A Laboratory Handbook. — Cold Spring Harbor Laboratory Press, Cold Spring Harbor — New York 2002.Google Scholar
- 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.Google Scholar