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Towards engineering glucosinolates into non-cruciferous plants

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Abstract

Glucosinolates are amino acid-derived secondary metabolites present in cruciferous plants. Glucosinolates and their hydrolysis products are involved in defence against insects and pathogens, but are also known for their characteristic flavor and their cancer-preventive and antibacterial properties. This wide range of bioactivities has prompted a desire to engineer glucosinolates into non-cruciferous plants. We report the one-step transfer of the last three steps of the benzylglucosinolate pathway (comprising the C–S lyase, glycosyltransferase and sulfotransferase) from Arabidopsis to tobacco. This was achieved using an expression construct consisting of a single 2A polycistronic open reading frame, which allowed the expression of the three coding-sequences from a single promoter. When compared to wildtype plants, transgenic tobacco lines showed increased ability to convert the intermediate phenylacetothiohydroxamate to benzylglucosinolate upon in vivo feeding. Enzymatic assays using plant extracts demonstrated that the individual activities required for this conversion were enhanced in the transgenic plants. The relatively high conversion by wildtype plants in feeding assays supports the hypothesis that the last part of the glucosinolate pathway was recruited from existing detoxification reactions. Immunoblots confirmed that individual proteins were being successfully produced from the 2A polycistronic open reading frame, albeit fusion proteins could also be detected. In summary, we transferred the last three steps of the benzylglucosinolate pathway to tobacco as a first step towards engineering glucosinolates into non-cruciferous plants.

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Abbreviations

ORF:

Open reading frame

BGLS:

Benzylglucosinolate

dBGLS:

Desulfobenzylglucosinolate

PATH:

Phenylacetothiohydroxamate

ST:

Sulfotransferase

GT:

Glucosyltransferase

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Acknowledgments

The authors would like to thank Douglas Grubb, Brandon Zipp (University of California, Davis, USA) and Leszek Doszczak (Universität Würzburg, Germany) for advice on the synthesis of PATH; Claire Halpin (University of Dundee, Scotland) for advice on the 2A system and Bodil Jørgensen (University of Copenhagen, Denmark) for advice on transformation of tobacco. Niels Agerbirk (University of Copenhagen, Denmark) is acknowledged for the kind gift of benzylglucosinolate. This work was supported by the Danish International Developmental Agency (DANIDA, project 91175) and by the Villum Kann Rassmusen Fond through its support to the VKR Research Centre Pro-Active Plants.

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Authors and Affiliations

  1. Plant Biochemistry Laboratory, VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen, 40 Thorvaldsensvej, 1871, Frederiksberg C, Denmark

    Fernando Geu-Flores & Barbara Ann Halkier

  2. Department of Natural Sciences, Faculty of Life Sciences, University of Copenhagen, 40 Thorvaldsensvej, 1871, Frederiksberg C, Denmark

    Carl Erik Olsen

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  1. Fernando Geu-Flores
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  2. Carl Erik Olsen
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  3. Barbara Ann Halkier
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Correspondence to Barbara Ann Halkier.

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Geu-Flores, F., Olsen, C.E. & Halkier, B.A. Towards engineering glucosinolates into non-cruciferous plants. Planta 229, 261–270 (2009). https://doi.org/10.1007/s00425-008-0825-y

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