An Integrated-Omics/Chemistry Approach Unravels Enzymatic and Spontaneous Steps to Form Flavoalkaloidal Nudicaulin Pigments in Flowers of Papaver nudicaule L.

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins in Papaver nudicaule L. (Iceland poppy) are unique due to their rarity and unparalleled flavoalkaloid structure. Nudicaulins are derived from pelargonidin glycoside and indole, products of the flavonoid and indole/tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, differential gel electrophoresis (DIGE)-based proteome, and ultra-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS)-based metabolome data of P. nudicaule petals with chemical investigations. We identified candidate genes and proteins for all biosynthetic steps as well as some key metabolites across five stages of petal development. Candidate genes of amino acid biosynthesis showed a relatively stable expression throughout petal development, whereas most candidate genes of flavonoid biosynthesis showed increasing expression during development followed by downregulation in the final stage. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were co-expressed with flavonoid biosynthesis genes, and are probably providing free indole. The fusion of indole with pelargonidin glycosides was retraced synthetically and promoted by high precursor concentrations, an excess of indole, and a specific glycosylation pattern of pelargonidin. Thus, nudicaulin biosynthesis combines the enzymatic steps of two different pathways with a spontaneous fusion of indole and pelargonidin glycoside under precisely tuned reaction conditions.

Formation of Nudicaulins In Vivo and In Vitro and the Biomimetic Synthesis and Bioactivity of O-Methylated Nudicaulin Derivatives.

Nudicaulins are yellow flower pigments accounting for the color of the petals of Papaver nudicaule (Papaveraceae). These glucosidic compounds belong to the small group of indole/flavonoid hybrid alkaloids. Here we describe in vivo and in vitro experiments which substantiate the strongly pH-dependent conversion of pelargonidin glucosides to nudicaulins as the final biosynthetic step of these alkaloids. Furthermore, we report the first synthesis of nudicaulin aglycon derivatives, starting with quercetin and ending up at the biomimetic fusion of a permethylated anthocyanidin with indole. A small library of nudicaulin derivatives with differently substituted indole units was prepared, and the antimicrobial, antiproliferative and cell toxicity data of the new compounds were determined. The synthetic procedure is considered suitable for preparing nudicaulin derivatives which are structurally modified in the indole and/or the polyphenolic part of the molecule and may have optimized pharmacological activities.

Biosynthetic and Functional Color-Scent Associations in Flowers of Papaver nudicaule and Their Impact on Pollinators.

Despite increasing evidence for biosynthetic connections between flower pigments and volatile compounds, examples of such relationships in polymorphic plant species remains limited. Herein, color-scent associations in flowers from Papaver nudicaule (Papaveraceae) have been investigated. The spectral reflectance and scent composition of flowers of four color cultivars was determined. We found that pigments and volatiles occur in specific combinations in flowers of P. nudicaule. The presence of indole in the bouquets is strongly associated with the occurrence of yellow pigments called nudicaulins, for which indole is one of the final biosynthetic precursors. Whereas yellow flowers emit an excess of indole, orange flowers consume it during nudicaulin production and lack the substance in their bouquet. By using the honeybee, Apis mellifera, evaluations were made on how color and scent affect the discrimination of these flowers by pollinators. Honeybees were able to discriminate artificial odor mixtures resembling those of the natural flower odors. Bees trained with stimuli combining colors and odors showed an improved discrimination performance. The results indicate that the indole moiety of nudicaulins and emitted indole might be products of the same biochemical pathway. We propose that conserved pathways account for the evolution of color-scent associations in P. nudicaule and that these associations positively affect flower constancy of pollinators.

The Occurrence of Flavonoids and Related Compounds in Flower Sections of Papaver nudicaule.

Flavonoids play an important role in the pigmentation of flowers; in addition, they protect petals and other flower parts from UV irradiation and oxidative stress. Nudicaulins, flavonoid-derived indole alkaloids, along with pelargonidin, kaempferol, and gossypetin glycosides, are responsible for the color of white, red, orange, and yellow petals of different Papaver nudicaule cultivars. The color of the petals is essential to attract pollinators. We investigated the occurrence of flavonoids in basal and apical petal areas, stamens, and capsules of four differently colored P. nudicaule cultivars by means of chromatographic and spectroscopic methods. The results reveal the specific occurrence of gossypetin glycosides in the basal spot of all cultivars and demonstrate that kaempferol glycosides are the major secondary metabolites in the capsules. Unlike previous reports, the yellow-colored stamens of all four P. nudicaule cultivars are shown to contain not nudicaulins but carotenoids. In addition, the presence of nudicaulins, pelargonidin, and kaempferol glycosides in the apical petal area was confirmed. The flavonoids and related compounds in the investigated flower parts and cultivars of P. nudicaule are profiled, and their potential ecological role is discussed.

Unprecedented Utilization of Pelargonidin and Indole for the Biosynthesis of Plant Indole Alkaloids.

Nudicaulins are a group of indole alkaloid glycosides responsible for the color of yellow petals of Papaver nudicaule (Iceland poppy). The unique aglycone scaffold of these alkaloids attracted our interest as one of the most unusual flavonoid-indole hybrid structures that occur in nature. Stable isotope labeling experiments with sliced petals identified free indole, but not tryptamine or l-tryptophan, as one of the two key biosynthetic precursors of the nudicaulin aglycone. Pelargonidin was identified as the second key precursor, contributing the polyphenolic unit to the nudicaulin molecule. This finding was inferred from the temporary accumulation of pelargonidin glycosides in the petals during flower bud development and a drop at the point in time when nudicaulin levels start to increase. The precursor-directed incorporation of cyanidin into a new 3'-hydroxynudicaulin strongly supports the hypothesis that anthocyanins are involved in the biosynthesis of nudicaulins.