Gentian plants have vivid blue-colored flowers, caused by accumulation of a polyacylated anthocyanin 'gentiodelphin'. We previously performed expression analysis of gentiodelphin biosynthetic genes, and hypothesized that the white-flowered gentian cultivar 'Polarno White' might have resulted from the mutation of certain regulatory factors responsible for anthocyanin biosynthesis in flower petals. In this study, we isolated 26 R2R3-MYB gene fragments including four full-length cDNAs (GtMYB2a, GtMYB2b, GtMYB3 and GtMYB4) and one basic helix-loop-helix (bHLH) gene (GtbHLH1) from blue-flowered gentian by degenerate PCR and rapid amplification of cDNA ends (RACE). Phylogenetic tree analysis showed that GtMYB3 was categorized into a clade involved in anthocyanin biosynthesis including petunia AN2 and Arabidopsis PAP1. On the other hand, GtbHLH1 exhibited high identity with petunia AN1 based on both phylogenetic and genomic structural analyses. Temporal profiles of GtMYB3 and GtbHLH1 transcript levels corresponded well with those of gentiodelphin accumulation and their biosynthetic genes in petals. Yeast two-hybrid analysis showed that GtbHLH1 interacted with GtMYB3. Moreover, transient expression analysis indicated that the co-expression of GtMYB3 and GtbHLH1 could enhance the promoter activities of late anthocyanin biosynthetic genes in tobacco BY2 cells. We also revealed that in cv. 'Polarno White' the GtMYB3 genes were mutated by insertions of transposable elements or uncharacterized sequences, indicating that the white coloration was caused by GtMYB3 mutation. These results strongly suggested that GtMYB3 and GtbHLH1 are involved in the regulation of gentiodelphin biosynthesis in gentian flowers.

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3'-hydroxylase [F3'H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully.

Flavonoids are one of the major plant pigments for flower colour. Not only coloured anthocyanins, but also co-pigment flavones or flavonols, accumulate in flowers. To study the regulation of early flavonoid biosynthesis, two R2R3-MYB transcription factors, GtMYBP3 and GtMYBP4, were identified from the petals of Japanese gentian (Gentiana triflora). Phylogenetic analysis showed that these two proteins belong to the subgroup 7 clade (flavonol-specific MYB), which includes Arabidopsis AtMYB12, grapevine VvMYBF1, and tomato SlMYB12. Gt MYBP3 and Gt MYBP4 transcripts were detected specifically in young petals and correlated with the profiles of flavone accumulation. Transient expression assays showed that GtMYBP3 and GtMYBP4 enhanced the promoter activities of early biosynthetic genes, including flavone synthase II (FNSII) and flavonoid 3′-hydroxylase (F3′H), but not the late biosynthetic gene, flavonoid 3′,5′-hydroxylase (F3′5′H). GtMYBP3 also enhanced the promoter activity of the chalcone synthase (CHS) gene. In transgenic Arabidopsis, overexpression of Gt MYBP3 and Gt MYBP4 activated the expression of endogenous flavonol biosynthesis genes and led to increased flavonol accumulation in seedlings. In transgenic tobacco petals, overexpression of Gt MYBP3 and Gt MYBP4 caused decreased anthocyanin levels, resulting in pale flower colours. Gt MYBP4-expressing transgenic tobacco flowers also showed increased flavonols. As far as is known, this is the first functional characterization of R2R3-MYB transcription factors regulating early flavonoid biosynthesis in petals.

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