Imaging Mass Spectrometry Analysis of Flavonoids in Blue Viola Petals and Their Enclosure Effects on Violanin during Color Expression.

The color expression of anthocyanin pigments in blue flowers is precisely controlled by their chemical and physical properties such as pH and the presence of metal ions or colorless copigments. Despite the large number of known blue flowers, their coloration mechanisms have not been examined in sufficient detail. In this work, the blue coloration of Viola cornuta petals was expressed via the copigmentation of various flavonol 3- O-glycosides. By using a combination of imaging mass spectrometry with liquid chromatography mass spectrometry, the structures and contents of flavonols colocalized with violanin in the discrete blue-colored regions of the petal were identified. The obtained data allowed the in vitro reconstruction of the color expression that was consistent with the visible spectrum of the viola petal. The results of visible spectral analysis indicated that neither the increase in the solution pH inside the vacuole cells nor the presence of metal ions affected the color development process. Ultimately, it was experimentally confirmed that the excess amounts of flavonol 3- O-glycosides complexed with violanin, which prevented violanin molecules from forming a levorotatory helical self-assembly during the blue color expression via copigmentation.