An integrated –omics/ chemistry approach unravels enzymatic and spontaneous steps of nudicaulin biosynthesis in Papaver nudicaule L. flowers

Flower colour is an important trait for plants to attract pollinators and ensure their reproductive success. Among yellow flower pigments, the nudicaulins present in Papaver nudicaule L. flowers occupy a special position due to their rarity and unique flavoalkaloid structure. Nudicaulins are de-rived from the direct precursors pelargonidin glycoside and indole, which are products of the flavo-noid and indole/ tryptophan biosynthetic pathway, respectively. To gain insight into the molecular and chemical basis of nudicaulin biosynthesis, we combined transcriptome, DIGE-based proteome, and UPLC-HRMS-based metabolome data of P. nudicaule petals with chemical investigations of the precursor's fusion reaction. 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 towards developmental stage 4 followed by downregulation in stage 5. Notably, gene candidates of indole-3-glycerol-phosphate lyase (IGL), sharing characteristic sequence motifs with known plant IGL genes, were coexpressed with flavonoid biosynthesis genes, and are probably providing free indole. The final step, fusion of indole with pelargonidin glycosides (pelargonidin 3-O-ß-D-sophoroside and orientalin), was retraced synthetically in parallel experiments. The reaction was promoted by high precursor concentrations, excess of indole, as well as a second glycosylation at position 7 of the pelargonidin aglycon. 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.