Genetic, biochemical and molecular biological studies of flavone formation in Gerbera hybrids

Within the naturally occuring flavonoid compounds, the flavones represent one of the most abundant and important class of secondary metabolites in nature. Besides their occurrence in flowers, they were also found in many other parts of higher plants with many different functions and are also abundant in plant-derived foods (e.g. vegetables and fruits). Furthermore, their benefits as bioactive substances and drugs in animal and human diseases are well known. In flower extracts of chemicogentically defined genotypes of Gerbera hybrids an enzyme activity was demonstrated which catalyses the formation of flavones. Further characterisation proved a correlation between the gene Fns and this enzyme activity, identified as flavone synthase II (FNS II) - a microsomal NADPH-dependent cytochrome P450 monooxygenase - and also the monogen inheritance of flavone formation in Gerbera. Further genetic experiments were performed to get hinds on the contribution of flavones on flower colour in Gerbera hybrids. Based on these data PCR techniques were used to clone full length cDNA (CYP93B2) of this gene. Northern blot analysis of total RNA of different Gerbera lines indicated that the CYP93B2 gene was only transcribed in lines with the dominant allele fns⁺ and that the transcription level during flower development is in agreement with measured enzyme activity of FNS II and flavone accumulation. Microsomes from yeast cells expressing CYP93B2 catalysed the direct formation of [¹⁴C]flavones from the respective [ ¹⁴C]flavanones. Thus, CYP93B2 was shown to encode FNS II. This new clone enables the control and specific manipulation of a step in flavonoid biosynthesis that is located at an important junction of this pathway leading to different flavonoids, such as flavones, isoflavones, flavonols, proanthocyanidins, catechins and anthocyanidins.