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Biologia Plantarum

, Volume 57, Issue 4, pp 635–645 | Cite as

Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

  • M. Pičmanová
  • D. Reňák
  • J. Feciková
  • P. Růžička
  • P. Mikšátková
  • O. Lapčík
  • D. Honys
Original Papers

Abstract

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 words

Arabidopsis thaliana cytochrome P450 endoplasmic reticulum isoflavonoids Nicotiana benthamiana Pisum sativum 

Abbreviations

IFS

isoflavone synthase

KLH

keyhole limpet hemocyanin

P450

cytochrome P450

GFP

green fluorescent protein

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • M. Pičmanová
    • 1
  • D. Reňák
    • 1
  • J. Feciková
    • 1
  • P. Růžička
    • 1
  • P. Mikšátková
    • 2
  • O. Lapčík
    • 2
  • D. Honys
    • 1
    • 3
  1. 1.Laboratory of Pollen BiologyInstitute of Experimental Botany of the ASCRPragueCzech Republic
  2. 2.Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical TechnologyInstitute of Chemical Technology in PraguePragueCzech Republic
  3. 3.Faculty of ScienceCharles University in PraguePragueCzech Republic

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