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JXB Advance Access published online on August 8, 2005
Journal of Experimental Botany, doi:10.1093/jxb/eri251
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© The Author [2005]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
Received February 21, 2005
Accepted June 21, 2005

RESEARCH PAPER

A comprehensive analysis of six dihydroflavonol 4-reductases encoded by a gene cluster of the Lotus japonicus genome

Norimoto Shimada 1 *, Ryohsuke Sasaki 1 *, Shusei Sato 2, Takakazu Kaneko 2, Satoshi Tabata 2, Toshio Aoki 1, and Shin-ichi Ayabe 1*

1 Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
2 Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan

* To whom correspondence should be addressed.
Shin-ichi Ayabe, E-mail: ayabe{at}brs.nihon-u.ac.jp


  Abstract

Dihydroflavonol 4-reductase (DFR) is the first committed enzyme of the anthocyanin and condensed tannin pathways. Several DFR cDNAs have been cloned, and different specificities of DFR isozymes in the substrate hydroxylation patterns have been reported, but only fragmentary knowledge of DFR gene organization is available. Reported here is a comprehensive analysis of DFRs of a model legume, Lotus japonicus. A total of five DFR genes were found to form a cluster within a 38 kb region in the L. japonicus genome, whereas six cDNAs, including two splicing variants resulting from a transversion at a splicing acceptor site, were cloned. All the genes were expressed, with different organ specificities, in the mature plant. Three of the DFR proteins heterologously expressed in Escherichia coli showed catalytic activity, and their substrate preferences agreed with the variation of a specific active site residue (Asp or Asn) reported to control the specificity. The hydroxylation patterns of anthocyanidins and condensed tannin units in the stems did not reflect the substrate specificity of the expressed isozymes, implying complex regulation mechanisms in the biosynthesis. The two splicing variants and one DFR with Ser at the specificity-controlling position failed to show the activity, but a revertant protein replacing the unusual splicing restored the activity. The phylogenetic tree, constructed with known DFR sequences, showed evolutionary divergence of some of the DFR genes prior to the plant speciation. This work affords the basis for genetic and biochemical studies on the diversity of DFR and the flavonoid products.

Keywords: Anthocyanin; condensed tannin; dihydroflavonol 4-reductase; flavanone 4-reductase; gene duplication; Lotus japonicus.

*These authors contributed equally to this paper.


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