Final Report Summary - BIOSYN-NUDICAUL (Biosynthetic studies of nudicaulins in laser-microdissected petal cell of Papaver nudicaule by the use of NMR spectroscopy and hyphenated technique LC-NMR) One of the most attracting natural phenomena is the colour diversity that is observed in plant kingdom and especially the broad colourful range of flowers. Usually the floral colours are conferred to pigments like flavonoids, carotenoids or betalains. However the colour of yellow Iceland poppy flowers is conferred to a group of pigments called nudicaulins. The recent identification of nudicaulins' chemical structure revealed a unique molecular structure consisting of an indole ring and a C6-C3-C6 moiety similar to that of flavonoids. The major objectives of project BIOSYN-NUDICAUL could be summarised to:- Elucidation of the nudicaulins biosynthesis- Distribution of nudicaulins in plant kingdom- Investigation of their ecological role and physiological function- Potential pharmacological use.To successfully achieve the objectives of the project the following tasks were performed:- Growing plants under controlled conditions and study their development- Establish conditions for in vitro cultures of Papaver nudicaule plants- Development of an analytical protocol for analysis of plant extract and isolation of target compounds by the combination of liquid chromatography (LC) and spectroscopic methods (NMR; MS)- Targeted analysis of nudicaulins in samples from other Papaver species- Incubation of Iceland poppy plants with supplemental UV-B radiation and phytochemical analysis of flower petals by HPLC- Metabolic profile of Papaver nudicaule plant organs during the development of plant- Hydrolysis of nudicaulins under basic and acidic conditions for removal of malonyl and glucosidic conjugations, respectively. Isolation of demalonylated nudicaulins and aglyca.- Bioassay of isolated nudicaulins for pharmacological activities- Administration of 13C labelled precursors to buds from Iceland poppy plants- Pulse/chase labelling experiments with 13CO2, followed by isolation of primary and targeted secondary metabolites.- Retrobiosynthetic analysis of NMR data- Comparative genomic and proteomic analysis (additional task).The microscopic examination of flower petals has shown a homogenous distribution of pigments. The analysis of flower extracts from other Papaver species revealed the presence of nudicaulins in flower petals of Welsh and Alpine poppy. Comparing the metabolic profile of white flowers from Iceland poppy plants with that of yellow flower, the only difference that was observed is the absence of nudicaulins. It seems that the metabolic pathway responsible for biosynthesis of nudicaulins is inactive in white petals, and the corresponding genes and enzymes are not expressed. The analysis of other plant organs (leaves, roots, stems) from Iceland poppy during the different development stages of plant demonstrates that nudicaulin pigments are located only in petals. Therefore, the role of those pigments seems to be high importance for the pollination mechanism.Processing and deconvolution of spectroscopic data from the feeding experiments reveals that the indole ring system comes from the indole/tryptophan pathway while the rest of the aglycon skeleton structure is originated from the chalcone/flavonoid pathway. According to our literature knowledge, there is no other example of natural products formed by joining precursors form the indole and from flavonoid pathway. Since most of the known indole alkaloids are formed by the fusion of precursors coming from indole pathway with metabolites derived from monoterpenoid pathway, the example of nudicaulins is unique. Thus, the present paradigm of alkaloids with flavonoid/indole hybrid structure reveals the presence of a novel pathway. This breakthrough in plant biology and biochemistry could lead to discovery of new enzymes or to novel activities of known enzymes. An integrated knowledge of nudicaulins biosynthetic pathways would be a powerful tool in genetic engineering in floriculture providing novel flower colours.