Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18

0420942 - ÚEB 2014 RIV NL eng J - Článek v odborném periodiku
Pičmanová, Martina - Reňák, David - Feciková, Jana - Růžička, Petr - Mikšátková, P. - Lapčík, O. - Honys, David
Functional expression and subcellular localization of pea polymorphic isoflavone synthase CYP93C18.
Biologia Plantarum. Roč. 57, č. 4 (2013), s. 635-645. ISSN 0006-3134. E-ISSN 1573-8264
Grant CEP: GA ČR GA525/09/0994; GA MŠMT(CZ) OC10054
Výzkumný záměr: CEZ:AV0Z50380511
Klíčová slova: Arabidopsis thaliana * cytochrome P-450 * endoplasmic reticulum
Kód oboru RIV: EF - Botanika
Impakt faktor: 1.740, rok: 2013

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.
Trvalý link: http://hdl.handle.net/11104/0227411