title: 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.
authors.affiliation: Iwate Biotechnology Research Center, 22-174-4, Narita, Kitakami, Iwate, 024-0003 Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakatsuka
authors.forename: Katia Sanae
authors.initials: KS
authors.lastname: Haruta
authors.forename: Chetsadaporn
authors.initials: C
authors.lastname: Pitaksutheepong
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Abe
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kakizaki
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Norimoto
authors.initials: N
authors.lastname: Shimada
authors.forename: Saburo
authors.initials: S
authors.lastname: Yamamura
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nishihara
mesh_terms: D000872:Anthocyanins / Q000096:biosynthesis; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D001578:Benzopyrans; D003001:Cloning, Molecular; D035264:Flowers / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D030012:Gentiana / Q000235:genetics* / Q000378:metabolism; D005960:Glucosides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000872:Anthocyanins; D051792:Basic Helix-Loop-Helix Proteins; D001578:Benzopyrans; D005960:Glucosides; D000073718:Pancreatitis-Associated Proteins; D010940:Plant Proteins; D018749:RNA, Plant; D014157:Transcription Factors; C000612141:REG3A protein, human; C414932:gentiodelphin