Plant and Cell Physiology Advance Access originally published online on October 30, 2008
Plant and Cell Physiology 2008 49(12):1818-1829; doi:10.1093/pcp/pcn163
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Identification and Characterization of R2R3-MYB and bHLH Transcription Factors Regulating Anthocyanin Biosynthesis in Gentian Flowers
1Iwate Biotechnology Research Center, 22-174-4, Narita, Kitakami, Iwate, 024-0003 Japan
2Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577 Japan
3National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
*Corresponding author: E-mail, mnishiha{at}ibrc.or.jp; Fax, +81-197-68-3881.
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Abstract |
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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.
Keywords: Anthocyanin biosynthesis - bHLH - Gentian - R2R3-MYB - Transcription factor - White flower
Abbreviations: 5AT, hydroxycinnamoyl-CoA:anthocyanin 5-O-acyltransferase; bHLH, basic helix–loop–helix; CHS, chalcone synthase; EBG, early biosynthetic gene; F3'5'H, flavonoid 3',5'-hydroxylase; GUS, β-glucuronidase; LBG, late biosynthetic gene; ORF, open reading frame; RACE, rapid amplification of cDNA ends; RT–PCR, reverse transcription–PCR; TIR, terminal inverted repeat; TSD, target site duplication; WDR, WD40 repeat
(Received October 1, 2008; Accepted October 26, 2008)
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