In planta studies on the biosynthesis of pyrrolizidine alkaloids and the involvement of copper-containing amine oxidases

Many plants especially from the families of Asteraceae, Boraginaceae, Heliotropiaceae, Fabaceae and Orchidaceae produce pyrrolizidine alkaloids (PAs) that are often toxic to fend off herbivores. Till this work only the first step of PA biosynthesis was elucidated. The entry-step of the PA biosynthesis is performed by an enzyme called homospermidinesynthase (HSS) which transfers an aminobutyl moiety of spermidine onto putrescine and creates the symmetric triamine, homospermidine. This enzyme, resulting from a duplication of the gene encoding the enzyme deoxyhypusine synthase (DHS) was shown to have developed several times independently. Using common comfrey (Symphytum officinale, Boraginaceae) as a model, different aspects of PA biosynthesis have been studied. It was shown that PA biosynthesis, though constitutive, is dynamic with respect to its regulation. A second site of PA biosynthesis was identified in planta within leaves subtending the reproductive parts (inflorescence) of the plants. Furthermore the work showed that this variability is also found on the intraspecific and intraindividual level by analyzing PA distribution and amounts in common comfrey. This result was validated also in another model system the Indian heliotrope (Heliotropium indicum, Heliotropiaceae) in respect to the variability of PA levels. This variability of PA levels was mirrored by the expression levels of HSS, an observation that was also valid for a copper-containing amine oxidase (CuAO). This CuAO was regarded as a candidate for a further step in PA biosynthesis, i.e. the oxidation of homospermidine. The sequence of the CuAO candidate was used to design virus-induced gene silencing (VIGS) experiments to knock down the candidate CuAO. Successful knock-downs showed reduced PA levels confirming the hypothesis of this CuAO is involved in PA biosynthesis. Chemical elucidation of the homospermidine transformation by the candidate CuAO resulted in the identification of a double oxidized and already double cyclized PA precursor. This result was further validated by tracer-feeding experiments to further support the involvement of the candidate CuAO, now named homospermidine oxidase in PA biosynthesis