Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Franzmayr, Benjamin K., Rasmussen, Susanne, Fraser, Karl M., Jameson, Paula E.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478045/
https://www.ncbi.nlm.nih.gov/pubmed/22915577
http://dx.doi.org/10.1093/aob/mcs168
_version_ 1782247269300436992
author Franzmayr, Benjamin K.
Rasmussen, Susanne
Fraser, Karl M.
Jameson, Paula E.
author_facet Franzmayr, Benjamin K.
Rasmussen, Susanne
Fraser, Karl M.
Jameson, Paula E.
author_sort Franzmayr, Benjamin K.
collection PubMed
description 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. METHODS: 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 (MS(n)). 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. CONCLUSIONS: 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.
format Online
Article
Text
id pubmed-3478045
institution National Center for Biotechnology Information
language English
publishDate 2012
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-34780452012-10-22 Expression and functional characterization of a white clover isoflavone synthase in tobacco Franzmayr, Benjamin K. Rasmussen, Susanne Fraser, Karl M. Jameson, Paula E. Ann Bot Breeding Strategies for Forage and Grass Improvement 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. METHODS: 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 (MS(n)). 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. CONCLUSIONS: 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. Oxford University Press 2012-11 2012-08-22 /pmc/articles/PMC3478045/ /pubmed/22915577 http://dx.doi.org/10.1093/aob/mcs168 Text en © The Author 2012. Published by Oxford University Press on behalf of the Annals of Botany Company. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Breeding Strategies for Forage and Grass Improvement
Franzmayr, Benjamin K.
Rasmussen, Susanne
Fraser, Karl M.
Jameson, Paula E.
Expression and functional characterization of a white clover isoflavone synthase in tobacco
title Expression and functional characterization of a white clover isoflavone synthase in tobacco
title_full Expression and functional characterization of a white clover isoflavone synthase in tobacco
title_fullStr Expression and functional characterization of a white clover isoflavone synthase in tobacco
title_full_unstemmed Expression and functional characterization of a white clover isoflavone synthase in tobacco
title_short Expression and functional characterization of a white clover isoflavone synthase in tobacco
title_sort expression and functional characterization of a white clover isoflavone synthase in tobacco
topic Breeding Strategies for Forage and Grass Improvement
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478045/
https://www.ncbi.nlm.nih.gov/pubmed/22915577
http://dx.doi.org/10.1093/aob/mcs168
work_keys_str_mv AT franzmayrbenjamink expressionandfunctionalcharacterizationofawhitecloverisoflavonesynthaseintobacco
AT rasmussensusanne expressionandfunctionalcharacterizationofawhitecloverisoflavonesynthaseintobacco
AT fraserkarlm expressionandfunctionalcharacterizationofawhitecloverisoflavonesynthaseintobacco
AT jamesonpaulae expressionandfunctionalcharacterizationofawhitecloverisoflavonesynthaseintobacco