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Plant and Cell Physiology Advance Access published online on October 30, 2008
Plant and Cell Physiology, doi:10.1093/pcp/pcn163
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Identification and characterization of R2R3-MYB and bHLH transcription factors regulating anthocyanin biosynthesis in gentian flowers

Takashi Nakatsuka1, Katia Sanae Haruta1,2, Chetsadaporn Pitaksutheepong3, Yoshiko Abe1, Yuko Kakizaki1, Kazuo Yamamoto2, Norimoto Shimada1, Saburo Yamamura1 and Masahiro Nishihara1

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: Dr. Masahiro Nishihara, Address: Iwate Biotechnology Research Center, 22-174-4, Narita, Kitakami, Iwate, 024-0003, Japan., Telephone number: +81-197-68-2911, Fax number: +81-197-68-3881, E-mail address: mnishiha{at}ibrc.or.jp


  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 RACE. Phylogenic 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 phylogenic 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

(Received October 1, 2008; Accepted October 26, 2008)
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