Formation of UV-honey guides in Rudbeckia hirta

Honey guides are part of the pigmentation patterning in flowers. Their physiological function is to guide pollinating insects to the center, where the sex organs and nectar are present. Often they are brightly colored spots, dots or lines as the black spots in the inner part of poppy flowers or the red dots of foxgloves. More frequently, they are invisible to the human eye, due to the accumulation of UV-absorbing pigments. However, for honey bees, who are able to detect ultraviolett light, they are clearly visible as color contrasts. Rudbeckia hirta, coneflower, is a well- known modell system for the formation of such UV-honey guides. To the human eye, their flower seems to be uniformly yellow, but in UV light two concentrical circles can be detected, the outer one UV-reflecting and bright, while the inner is dark-absorbing. It could be shown, that in the UV-reflecting outer parts only carotenoids are present, while in the inner parts flavonoids, in particular 6- hydroxyflavonols, are responsible for UV-absorption. The presence of UV-honey guides in Rudbeckia sp. has been demonstrated with numerous photographs and photometrical investigations. In this project, the underlying biochemical and molecular base will be investigated for the first time. First, the biosynthesis of the pigments responsible for the UV-absorption will be studied in detail. According to literature, 6-hydroxquercetin (quercetagetin) derivatives, which show different glucosylation and methylation patterns, are present. Although flavonoid biosynthesis has been extensively studied, only a few reports on higher hydroxylated flavonols are available. The studies will focus on the introduction of an additionalhydroxyl group in position 6, the introduction of glucosyl moieties in positions 3 and 7 and of methyl groups in positions 7 and 6. The enzymes involved could be demonstrated for the first time in Rudbeckia hirta in a preliminary experiment for this project and will be studied now in detail. Special emphasis will be placed on their substrate specificity. Since the presence of several methyltransferases and glucosyltransferases could be shown, purification of the enzymes will be necessary to enable biochemical studies. After the biochemical studies, the corresponding structural genes will be isolated and characterized in detail. The last part of the project will be dedicated to the elucidation of UV-honey guide formation on the biochemical and molecular level. Optimized enzyme assays and the isolated cDNA clones will be used in combination with HPLC-analysis to determine, which enzymes and structural genes are responsible for the local accumulation of UVabsorbing pigments in the petals of R. hirta.

Honey guides are part of the pigmentation patterning in flowers. Their physiological function is to guide pollinating insects to the center, where the sex organs and nectar are present. Often they are brightly colored spots, dots or lines as the black spots in the inner part of poppy flowers or the red dots of foxgloves. More frequently, they are invisible to the human eye, due to the accumulation of UV-absorbing pigments. However, for honey bees, which are able to detect ultraviolett light, they are clearly visible as color contrasts. Rudbeckia hirta, coneflower, is a well-known model system for the formation of such UV-honey guides. To the human eye, their flower seems to be uniformly yellow, but in UV light two concentrical circles can be detected, the outer one UV-reflecting and bright, while the inner is dark-absorbing. It could be shown, that in the UV-reflecting outer parts only carotenoids are present, while in the inner parts flavonoids, in particular 6-hydroxyflavonols, are responsible for UV-absorption. The presence of UV-honey guides in Rudbeckia sp. has been demonstrated with numerous photographs and photometrical investigations. In this project, the underlying biochemical and molecular base was investigated for the first time. First, the biosynthesis of the pigments responsible for the UV-absorption was studied in detail. This included the demonstration and characterization of five novel enzymes and their corresponding genes. We were able to show that gene expression and thus, the corresponding activities of the enzymes involved in the formation of the UV-absorbing 6-hydroxyflavonols was much higher in the inner part of the petals. Future studies will deal with the changes of gene expression and corresponding enzyme activities during flower development.