Identification and Characterization of R2R3-MYB and bHLH Transcription Factors Regulating Anthocyanin Biosynthesis in Gentian Flowers

Abstract

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.

bHLH05 Is an Interaction Partner of MYB51 and a Novel Regulator of Glucosinolate Biosynthesis in Arabidopsis

“…Furthermore, the MYB-bHLH protein complexes involved in the regulation of flavonoid genes in Arabidopsis and also in other species such as rice (Oryza sativa), maize (Zea mays), cotton (Gossypium hirsutum), apple (Malus domestica), strawberry (Fragaria spp. ), snapdragon (Antirrhinum majus), beefsteakplant (P. frutescens), gerbera (Gerbera hybrida), and clustered gentian (Gentiana triflora; Martin et al, 1991;Grotewold et al, 2000;Hu et al, 2000;Aharoni et al, 2001;Sompornpailin et al, 2002;Elomaa et al, 2003;Humphries et al, 2005;Schwinn et al, 2006;Sweeney et al, 2006;Espley et al, 2007;Furukawa et al, 2007;Nakatsuka et al, 2008;Brueggemann et al, 2010;LinWang et al, 2010) have been rigorously characterized.…”

Production of picotee-type flowers in Japanese gentian by CRES-T

“…Our own and Suntory Holdings' research groups have isolated and characterized most of the flavonoid biosynthetic genes responsible for flower pigmentation in gentian (Fujiwara et al 1998;Fukuchi-Mizutani et al 2003;Nakatsuka et al 2005aNakatsuka et al , 2008aTanaka et al 1996). In addition, we also revealed that the pigmentation of gentian flowers was regulated by two transcription factors, R2R3-MYB and basic Helix-LoopHelix (bHLH), designated GtMYB3 and GtbHLH1, respectively (Nakatsuka et al 2008b). It was demonstrated that in gentian flowers the GtMYB3/ GtbHLH1 transcription factor complex regulated the expressions of anthocyanin biosynthetic genes (Nakatsuka et al 2008b).…”

“…In addition, we also revealed that the pigmentation of gentian flowers was regulated by two transcription factors, R2R3-MYB and basic Helix-LoopHelix (bHLH), designated GtMYB3 and GtbHLH1, respectively (Nakatsuka et al 2008b). It was demonstrated that in gentian flowers the GtMYB3/ GtbHLH1 transcription factor complex regulated the expressions of anthocyanin biosynthetic genes (Nakatsuka et al 2008b). …”

Transcriptional regulators of flavonoid biosynthesis and their application to flower color modification in Japanese gentians

“…On the other hand, G. triflora WDR ortholog 1 (GtWDR1) is expressed throughout the entire plant (our unpublished data). A yeast two-hybrid analysis has actually confirmed a protein-protein interaction between GtMYB3 and GtbHLH1, while a transient expression assay has demonstrated that cotransformation of GtMYB3 and GtbHLH1 can enhance the promoter activities of the late anthocyanin biosynthetic genes GtA5/3′AT and GtF3′5′H (Nakatsuka et al 2008a). In addition, the white-flowered cultivar 'Polarno-White' has been characterized as a GtMYB3-deficient mutant derived from the insertion of a transposable element into the coding region of the GtMYB3 gene.…”

“…In gentian, temporal expressions of GtMYB3 and GtbHLH1 are well correlated with those of late biosynthetic genes (Nakatsuka et al 2008a). On the other hand, G. triflora WDR ortholog 1 (GtWDR1) is expressed throughout the entire plant (our unpublished data).…”