Identification of novel C-glycosylflavones and their contribution to flower colour of the Dutch iris cultivars.

Seventeen C-glycosylflavones including four novel ones, were isolated from the flowers of Dutch iris (Iris hollandica Hort. ex Todd.) cultivar 'Blue Diamond'. Four new C-glycosylflavones were identified as isovitexin 2″-O-(4″'-acetylrhamnoside) (F1), swertisin 2″-O-(4″'-acetylrhamnoside) (F2), isovitexin 2″-O-(4″'-acetylrhamnoside)-4'-O-glucoside (F3) and swertisin 2″-O-(4″'-acetylrhamnoside)-4'-O-glucoside (F4) by UV spectra, LC-MS and (1)H and (13)C NMR. Furthermore, to understand the contribution of flavones to flower colour, the relationships with flower colours of three Dutch iris cultivars and flavonoid components were examined. The degree of blueness in the bluish cultivars 'Blue Diamond' and 'Blue Magic' were higher than that of the violet cultivar, 'Yesterday', and it was suggested that the flower colour expression from violet to blue colour of Dutch iris cultivars depend on the high ratio of total flavone contents/total delphinidin contents (F/A ratio). In addition, in vitro examination was carried out by the isolated anthocyanin and flavone. The mixture solutions were prepared in respective F/A ratio of three Dutch iris cultivars and could essentially reconstruct their visible absorption spectra of flowers. In conclusion, it was confirmed that isolated flavones contribute to blueness due to intermolecular co-pigmentation with anthocyanins.

Molecular cloning and characterization of O-methyltransferases from the flower buds of Iris hollandica.

In plants, O-methyltransferases (OMTs) play an important role in methylation of secondary metabolites, especially flavonoids and other phenylpropanoids, and two cDNA clones, IhOMT1 and IhOMT2 (Iris hollandica OMT), encoding OMTs were successfully isolated from a cDNA library of flower buds of I. hollandica. IhOMT1 encodes an open reading frame (ORF) of 365 amino acids with calculated molecular mass of 40,193Da and isoelectric point (pI) of 5.54, while IhOMT2, which shares 31.5% amino acid sequence identity with IhOMT1, encodes 369 amino acids with calculated molecular mass of 40,385Da and pI of 5.50. In addition, the molecular masses of both recombinant IhOMT1 and IhOMT2 proteins were estimated to be about 40kDa by protein gel blot analysis. Characterization of the enzymatic properties using the recombinant IhOMT1 protein confirmed that IhOMT1 cDNA encodes a S-adenosyl-L-methionine (SAM)-dependent caffeic acid 3-OMT, which catalyzes the transfer of the methyl moiety from SAM to caffeic acid to form ferulic acid. Its optimum activity was observed at pH 7.5-8.0 and at 35 degrees C. This is the first report of the isolation and characterization of a COMT cDNA clone involved in the phenylpropanoid biosynthesis of Iridaceae plants. In contrast, IhOMT2 showed no activity in SAM-dependent assays for various phenylpropanoids.