A multifaceted approach to identify unknown enzymes of pyrrolizidine alkaloid biosynthesis
Pyrrolizidine alkaloids (PAs) are anti-herbivorous toxins that are found in several angiosperm lineages. The first pathway-specific enzyme, the homospermidine synthase (HSS), evolved repeatedly by gene duplication from the primary metabolism gene deoxyhypusine synthase in different lineages. Remarkably, although the independent evolution resulted in very similar PA structures, the integration into the plant’s pathway was different in the various lineages. In this thesis, a further facet was added to the versatile regulation of the PA pathway. Previous studies revealed that comfrey (Symphytum officinale L.) produces PAs in the root. This study presents evidence that Symphytum activates a second site of PA biosynthesis in young leaves to boost defense of inflorescences against herbivores during flowering. The finding that Symphytum has two sites where PAs are produced, was used to identify further enzymes of the pathway. For a substractive approach, the transcriptomes of three different tissues were sequenced (PA-producing leaves, non-PA-producing leaves, and PA-producing roots). The transcript abundance was measured in silico and transcripts that were up-regulated in PA-producing tissues were enriched. By searching the enriched transcripts, diamine oxidases (DAOs) were identified that are postulated to catalyze the second step of the PA biosynthesis. These DAO coding transcripts were further analyzed on their occurrence in various tissues and cell types and their enzyme activity with the postulated substrate homospermidine was determined. The results indicate that two different DAOs are involved in PA biosynthesis in different PA-producing tissues. To test DAOs and further candidate genes in planta, RNAi knockdown in Symphytum hairy roots was established. In a proof-of-concept experiment, HSS was used as a target for RNAi-mediated knockdown and it was shown that a reduced level of HSS transcripts leads to a reduced level of PAs.