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JXB Advance Access published online on March 28, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern031
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© The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Cloning and characterization of the UDP-glucose:anthocyanin 5-O-glucosyltransferase gene from blue-flowered gentian

Takashi Nakatsuka1 *, Kei Sato2 *, Hideyuki Takahashi1, Saburo Yamamura1 and Masahiro Nishihara1,{dagger}

1Iwate Biotechnology Research Center, 22-174-4, Narita, Kitakami, Iwate 024-0003, Japan
2Graduate School of Agriculture, Iwate University, 3-18-8, Ueda, Morioka, Iwate 020-8550, Japan

{dagger} To whom correspondence should be addressed. E-mail address: mnishiha{at}ibrc.or.jp

Blue-flowered gentian (Gentiana triflora) is known to accumulate gentiodelphin, a unique polyacylated delphinidin-type anthocyanin, in the petals. Almost all of the structural genes involved in gentiodelphin biosynthesis have been isolated, but an important gene encoding UDP-glucose:anthocyanin 5-O-glucosyltransferase (5GT) remained to be identified. In this study, an attempt was made to isolate and characterize gentian 5GT, which is responsible for glucosylation of anthocyanidin 3-glucoside. A PCR-based cloning strategy identified seven 5GT candidates from gentian flowers. Among them, the deduced amino acid sequence of the 5GT gene from gentian petal cDNA, designated Gt5GT7, exhibited 36.0–41.7% identities with those of 5GTs from other plant species, and phylogenic analysis also suggested that Gt5GT7 belongs to the 5GT subfamily. The expression analysis showed that Gt5GT7 transcripts were detected predominantly in petals and weakly in filaments but not in leaves, stems, and other floral organs. In addition, increased levels of Gt5GT7 transcripts in petals coincided with flower development, a pattern identical to that of 5GT enzymatic activity as determined by in vitro assay using petal crude proteins. The substrate specificity of Gt5GT7 was analysed in vitro using the recombinant enzyme produced by Escherichia coli. Gt5GT7 could transfer a glucosyl moiety to anthocyanidin 3-glycosides but not to other flavonoid compounds. Delphinidin 3-glucoside, the precursor of gentiodelphin, was the best substrate among several anthocyanidin 3-glycosides tested. Heterologous expression of Gt5GT7 in tobacco plants led to additional accumulation of cyanidin 3-rutinoside-5-glucoside, confirming that Gt5GT7 has a valid enzymatic activity in planta.

Key words: Anthocynian 5-O-glucosyltransferase, flower colour, gentian, gentiodelphin, 5GT, transgenic tobacco

* These authors contributed equally to this work.

Received 29 November 2007; Revised 11 January 2008 Accepted 21 January 2008


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