Investigations of metabolic transgressions after synthetic allopolyploidisation of tobacco species

Allopolyploidy describes the combination of genomes from different species, resulting in a new organism with a combined and multiply increased set of chromosomes. Plant allopolyploids typically exhibit heterosis effects. The terms "transgression" and "heterosis" in this context describe phenotypic characteristics of the new organisms that lie outside the parental spectrum. A special method for allopolyploidisation is a grafting-based technology that has allowed the asexual creation of synthetic allopolyploid tobacco species from Nicotiana tabacum and N. glauca or N. benthamiana through horizontal genome transfer. The new species were named "N. tabauca" and "N. benthacum", respectively. The aim of this study was to characterise the effects of synthetic allopolyploidisation on the metabolome of the new species. For this purpose, the primary and secondary metabolome of the first generations of the synthetic allopolyploid species were analysed using gas chromatography and liquid chromatography coupled with mass spectrometry. Transgression was observed almost exclusively in polar secondary metabolites, but not in primary metabolites. Some metabolic transgressions also occurred in a lipophilic metabolite fraction. The most pronounced positive transgressions were observed in both new species in the class of "nicotianosides", i.e., the 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). These specialised metabolites are known to protect plants against herbivorous insects. To understand the changes in the HGL-DTG profile, the occurrence of the individual HGL-DTGs was analysed in detail in the progenitor species, specifically with regard to their biosynthesis and underlying enzymatic steps that may be responsible for HGL-DTG accumulation in the allopolyploid species. Enzyme candidates of a putative geranyllinalool synthase as well as rhamnosyl- and glucosyltransferases from the HGL-DTG synthesis pathway were cloned into an Agrobacterium tumefaciens vector and transiently overexpressed in N. benthamiana leaves, thereby determining the cytosolic cellular localisation of the enzymes. This study of synthetically and asexually generated allopolyploid plant species is consistent with previous reports on sexually generated allopolyploid plant species and demonstrates the emergence of metabolic transgressions already in the first generation after their formation. It provides rare insights into changes in specialised metabolism following allopolyploidisation events. Furthermore, it provides opportunities to mechanistically investigate the observed transgressions based on detailed knowledge of the corresponding specialised metabolite class and by transient expression of selected candidate enzymes.