Dihydro-p-coumaroyl-CoA dehydrogenase

This project studies the key step in the biosynthesis of phloridzin, which is the prevalent polyphenolic compound in apple. Phloridzin represents more than 90% of the soluble phenolic compounds in apple leaves. The presence of such high amounts of phloridzin makes apple unique since other species accumulate only very low amounts and many closely related species like pear are not able to form phloretin or its glucosylated relative phloridzin. The last decade has seen an explosion of research on the beneficial effects of phloretin and phloridzin for human health but the physiological relevance for apple is still unclear. A possible involvement in disease resistance is discussed. Previously we have shown with apple leaf extracts that phloridzin formation is based on three biosynthetic steps: (1) the formation of dihydro-p-coumaroyl-CoA from p-coumaroyl-CoA by a dehydrogenase, (2) further formation of phloretin by the common chalcone synthase and (3) the glucosylation of phloretin in position 2. Whereas the last two steps were already intensively studied, the knowledge of the first step is limited. The enzyme is crucial, because it seems to be the key point making the phloridzin-hoarding apple unique in comparison to other plants. In our previous FWF project (P25399-B16) we successfully completed a challenging purification process and were able to purify for the first time a candidate enzyme from apple leaves, which exhibits strong enzyme activity with p-coumaroyl-CoA to form dihydro-p-coumaroyl-CoA. The planned follow-up project will now target the detailed characterization of this important enzyme from apple leaves for the first time. Structural studies will resolve the enzymatic mechanism, such as protein crystallization and effects of substrates, inhibitors/effectors or other factors. The DNA sequence of the dehydrogenase will be isolated from apple and transferred into bacteria to produce large amounts of the enzyme for detailed characterization. It will be tested, in which tissue and developmental stage the dehydrogenase gene is switched on or off. Functional activity of the gene products will be tested with genetically modified plants where phloretin formation will be enabled by the dehydrogenase in thale cress (Arabidopsis) or disabled in apple. Comparison of the DNA and protein sequence of the dehydrogenase from different plant species will give insight to structure-activity relationship of the enzyme on the molecular level. The project members consist of three teams which provide complementary know-how and resources: One of the Austrian teams offers knowledge in phloridzin biosynthesis, molecular biology and enzymatic evaluation, the other Austrian team has profound experience in protein characterization and crystallisation, whereas the German team provides the infrastructure and long-term experience in the creation of transgenic plants. An external team from New Zealand, which is funded by own sources will additionally contribute its know-how on pear transformation.

Phloridzin is present in large quantities in apples, but the biosynthesis pathway has not yet been fully elucidated. The crucial step is the conversion of the phloridzin precursor p-coumaroyl-CoA to dihydro-p-coumaroyl-CoA by an enzyme (dehydrogenase). In a previous FWF project, we were able to isolate this enzyme from apple leaves for the first time in a complex purification process, while in the current project a corresponding characterization was carried out at different levels: Firstly, the production of the dehydrogenase in bioreactors with bacterial cells was optimized, the enzyme was isolated and used for activity tests. Biochemical characteristics such as optimal reaction conditions, specificity with different substrates, or thermal and temporal stability, etc. were determined. Furthermore, it was investigated how strongly the gene variants for the enzyme are switched on in different apple varieties and tissues (leaves, individual flower organs, shoot tips). So-called transcriptome analyses were also used, which made it possible to analyse individual variants of the gene and their expression in apples and other plant species. In order to assess the physiological relevance of the dehydrogenase, genetically modified plants were produced. To do this, the dehydrogenase was integrated into the genome of the model plant thale cress (Arabidopsis sp.) (overexpression experiments). Compared to the wild type, the transgenic plants produced new ingredients that can be attributed to the activity of the dehydrogenase, which also confirmed the activity of the enzyme in the plant. Furthermore, transgenic apple plants were produced in which the gene for the dehydrogenase was switched off (silencing experiments). The evaluation of the transgenic line is not yet complete. Overall, the results of the project led to a better understanding of the biosynthesis of phloridzin in the apple, in particular of the dehydrogenase involved, on several levels.