Journal Article

Expression and functional characterization of a white clover isoflavone synthase in tobacco

Benjamin K. Franzmayr, Susanne Rasmussen, Karl M. Fraser and Paula E. Jameson

in Annals of Botany

Published on behalf of The Annals of Botany Company

Volume 110, issue 6, pages 1291-1301
Published in print November 2012 | ISSN: 0305-7364
Published online August 2012 | e-ISSN: 1095-8290 | DOI:

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  • Ecology and Conservation
  • Evolutionary Biology
  • Plant Sciences and Forestry


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Background and Aims

Trifolium repens (white clover) is a valuable component of pastures due to its ability to fix nitrogen. Productivity of T. repens is sometimes threatened by insect pests, and it has been suggested that phenylpropanoid-derived isoflavonoids such as formononetin can protect white clover from insect damage. The aim of this study was to isolate and functionally characterize an isoflavone synthase (IFS2_12) from T. repens by expressing it in Nicotiana tabacum (tobacco), a plant which does not naturally produce isoflavonoids.


To induce anthocyanin production and increase isoflavonoid precursors in tobacco, the tomato R2R3 MYB transcription factor ANT1 was expressed in tobacco (Nt-ANT1 plants). IFS2_12 was heterologously expressed in tobacco both transiently and stably, and isoflavonoids in leaf extracts were analysed by liquid chromatography (LC) coupled to mass spectrometry (MSn). As a positive control, a double construct of soybean IFS and alfalfa chalcone isomerase (IFS/CHI), which had been previously shown to induce isoflavonoid production in tobacco, was also expressed. Stable transformants expressing IFS2_12, soybean/alfalfa IFS/CHI and ANT1 were crossed and the resulting plants were analysed for isoflavonoid production.

Key results

Leaves of tobacco plants expressing ANT1 had a range of phenotypes from mainly green to uniformly bronze coloured. Both transient and stable expression of the IFS2_12 or IFS/CHI constructs resulted in the production of the isoflavonoid genistein and its conjugates. The highest levels (up to 19·2 mg g−1 d. wt) accumulated in a progeny of a cross between a purple ANT1 and a IFS/ CHI transformant, while the second highest concentration was found in a plant derived from a selfed IFS2-12 transformant.


It is concluded that the gene IFS2_12 isolated from T. repens encodes an isoflavone synthase. This study paves the way for engineering white clover plants with higher levels of isoflavonoids than naturally found in this species for sufficient insect protection.

Keywords: Trifolium repens; Nicotiana tabacum; isoflavone synthase; isoflavonoid biosynthesis; transient gene expression; liquid chromatography–mass spectrometry; genistein

Journal Article.  6168 words.  Illustrated.

Subjects: Ecology and Conservation ; Evolutionary Biology ; Plant Sciences and Forestry

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