05.06.2008 Doctoral dissertation: Metabolic engineering and metabolite profiling of strawberry

In the PhD work of Kati Hanhineva, MSc, garden strawberry (Fragaria × ananassa) was genetically modified in order to enhance resistance to the grey mould fungus Botrytis cinerea. The public examination of the doctoral dissertation is on 5th June 2008 at the Faculty of Natural and Environmental Sciences, University of Kuopio.

A gene encoding stilbene synthase from frost grape Vitis riparia (NS-Vitis3) was introduced in strawberry to create biosynthetic pathway for resveratrol, a phenolic phytoalexin compound. Several genes encoding stilbene synthase have been used widely in genetic modification of plants, resulting in a succesful synthesis of resveratrol derivatives and providing enhanced resistance to pathogens including B. cinerea. The coding sequence of the gene NS-Vitis3 was introduced in strawberry under the control of two different promoters, i.e., a flower-specific (fil1) and general (cauliflower mosaic virus 35S) promoter. In the leaf tissue of the 35S:NS-Vitis3 line the integrated transgene was shown to cause down-regulation of an endogenous gene, chalcone synthase, resulting in the depletion of flavonoids and accumulation of phenylpropanoid compounds, as revealed by metabolite profiling by UPLC-qTOF-MS.

An in vitro leaf test suggested decreased resistance to grey mould, most likely resulting from the significant alteration of the metabolite composition. A detailed metabolite profiling was crucial for the understanding of the unexpected consequences of the genetic modification. The modification did not have clear impact on the floral organs, but the analysis of flowers allowed the identification of several metabolites and metabolite groups that differed between the different floral organs. Furthermore, NMR identification of a set of metabolites that showed unexpectedly high accumulation in the leaves of the modified plants led to the discovery of a metabolite class previously uncharacterized in strawberry, i.e., phenylpropanoid glycosides. The phenylpropanoid glycosides bring an interesting new aspect in the research of strawberry phytochemicals, since they are highly bioactive metabolites that have been studied in the context of Asian folk medicine, and have been found to contribute to the therapeutic activities of several plant species.

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