PUB - Publikationen an der Universität Bielefeld

The Japanese Hydrangea serrata Oamacha is a traditional medical plant and known for its sweet tasting tea leaves and bioactive specialised metabolites hydrangenol and phyllodulcin. A variety of health improving bioactivities are owned by these two 3,4-dihydroisocoumarins and their glycosides. The detailed understanding of their biosynthesis is of special interest. A similar biocatalysis mechanism is well known from the chalcone synthase superfamily, a group of plant specific Type III polyketide synthases. Chalcone synthases and their homologues, the stilbene synthases produce a huge amount of specialised plant metabolites including phenylpropanoids. The dihydroisocoumarin biosynthesis is assumed to be enhanced by elicitors and mediated by polyketide synthases, like in cases of phenylpropanoid derived phytoalexins. This work includes the treatment of H. serrata Oamacha with different stressors, yeast-elicitor, jasmonic acid, aluminium chloride or drought. None of the applied stressors enhanced the hydrangenol or phyllodulcin accumulation in leaves or roots. Other studies on stressor treatments, resulted in similar outcomes. The purpose of 3,4-dihydroisocoumarins from H. serrata as possible phytoalexins was shown in one recent study on methyl jasmonic acid. The abiotic stressor aluminium chloride is toxic to plants, induces stilbenoid biosynthesis and changes the gene expression according to other studies. The effect on H. serrata Oamacha was analysed in this work on the level of metabolite contents and differential gene expression. A de-novo transcriptome assembly of leaves and roots alongside with functional annotation was generated and examined in two ways. The aim of this work was to isolate new polyketide synthase homologues from H. serrata with prospective of heterologous expression in the bacterial host Pseudomonas putida KT2440. The major findings of this work are summarized in the following:
• Many aspects about the main mechanisms of aluminium detoxification which were described in other studies on hydrangeas, were observed in this work as well. Transporter and cell wall stability (monolignol biosynthesis) related genes were differentially expressed like it was shown in a study on H. macrophylla. Interestingly, H. serrata in contrast to Hydrangea macrophylla showed changes in the terpenoid biosynthesis related genes in response to aluminium chloride. To fully explore the differential gene expression in terms of dihydroisocoumarin biosynthesis, the functional annotation based on orthologues from A. thaliana was not sufficient. To overcome this limitation, a comparison of homology to other polyketide synthases was made on amino acid level. Amino acid motifs were worked out from p-coumaroyltriacetic acid synthase and stilbenecarboxylate synthase and compared to H. serrata transcriptome derived peptide sequences. It was possible to isolate new stilbene synthase homologues.
• A LC-MS analysis was basis for leaf and root profiles of dihydroisocoumarins. A hydrangenol isomer, putatively hydrangeic acid, was quantified in roots. Moreover, glycosidic versions including diglycosides were quantified in the two organs.
• The chalcone synthase from H. macrophylla was successfully expressed in P. putida KT2440, as a blueprint for the use of the new isolated polyketide synthase homologue. A pathway which supplies p-coumaroyl-CoA as precursor was assembled therefore and extended by the chalcone synthase as well as the new H. serrata polyketide synthase homologue. The result of the cultivation of the respective KT2440 strain was a product which was detected in LC-MS but was different to dihydroisocoumarins and needs to be characterised.