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


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.
AgResearch Limited, Grasslands Research Centre, Tennent Drive, Palmerston North 4442, New Zealand.
Benjamin K
BK
Franzmayr
Susanne
S
Rasmussen
Karl M
KM
Fraser
Paula E
PE
Jameson
D053898:Biosynthetic Pathways; D003433:Crosses, Genetic; D015870:Gene Expression; D005818:Genetic Engineering; D019833:Genistein / Q000378:metabolism; D007529:Isoflavones / Q000302:isolation & purification / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000235:genetics; D009584:Nitrogen / Q000378:metabolism; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D018515:Plant Leaves / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D036226:Seedlings / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D029921:Trifolium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D007529:Isoflavones; D010940:Plant Proteins; D014157:Transcription Factors; D019833:Genistein; D010105:Oxygenases; C048489:isoflavone synthase; D009584:Nitrogen