Two different mutations are involved in the formation of white-flowered gentian plants

Japanese cultivated gentian plants have naturally blue flowers, but some white-flowered cultivars are being bred through the utilization of spontaneous mutants. To determine the molecular basis of white coloration in gentian flowers, we compared two white-flowered cultivars Homoi and Polano White to a blue-flowered cultivar Maciry using biochemical and molecular approaches. High performance liquid chromatography (HPLC) analyses showed that flavone levels in cv. Polano White were only about one-half the amounts measured in the other two cultivars, while anthocyanins were absent in the two white-flowered cultivars compared to cv. Maciry in which high levels accumulated. Northern blot analysis of 10 flavonoid biosynthetic structural genes, previously reported to be temporally regulated in cv. Maciry [1] showed that cv. Homoi lacked transcripts for the anthocyanidin synthase (ANS) gene while cv. Polano White had decreased expressions for ANS as well as for chalcone synthase (CHS), flavanone 3-hydroxylase (F3H), flavonoid 3′,5′-hydroxylase (F3′,5′H), dihydroflavonol 4-reductase (DFR), UDP-glucose:flavonoid 3-glucosyltransferase (3GT) and anthocyanin 5-aromatic acyltransferase (5AT). Southern blot analysis confirmed the deficiency of one of two ANS loci in cv. Homoi. Transient expression of ANS in flower petals also strongly suggested that white flowers of cv. Homoi were derived from ANS mutation. Furthermore, analysis of stress-induced flower pigmentation suggested that rather than mutations in multiple structural genes being the cause, a defect in one or more regulatory factors controlling the later steps of flavonoid biosynthesis is responsible for white coloration in cv. Polano White.