A flavonol synthase gene GtFLS defines anther-specific flavonol accumulation in gentian

Flavonoids are important for male gametophyte development. Here we report on the flavonoid components and their biosynthetic regulation in gentian anthers. Among flavonoids, flavonols, including kaempferol, quercetin and isorhamnetin derivatives, accumulated abundantly in gentian anthers. However, flavones and anthocyanins, which are the main flavonoids accumulating in petals, were not detected. Northern blot analysis of nine flavonoid biosynthetic genes showed that the 'early' flavonoid biosynthetic genes were expressed in both anthers and petals, and that flavonol synthase (FLS) transcripts were restricted to anthers. In contrast, flavone synthase II (FNSII) and 'late' flavonoid biosynthetic genes were expressed specifically in gentian petals. To confirm anther-specific expression of FLS, the 5′-upstream region of FLS (GtFLSpro) was cloned by inverse PCR and fused to the uidA (GUS) reporter gene. Tobacco, Arabidopsis and gentian plants, transformed with the GtFLSproGUS construct, exhibited anther-specific GUS expression. Expression was observed in the tapetum and in pollen at late stages of anther development in transgenic plants. These results revealed that flavonol accumulation in gentian anthers was regulated by the spatial expression of GtFLS. Our results also suggest that anther-specific regulation of FLS is conserved among higher plants and the GtFLS promoter is useful for induction of specific gene expression in anthers.