Estrogen-related receptor ␣ (ERR␣) was identified as a gene related to estrogen receptor ␣ (ER␣) and belongs to a class of nuclear orphan receptors. ERR␣ binds to estrogen responsive element(s) (ERE) and is considered to be involved in modulation of estrogenic actions. However, biological significance of ERR␣ remains largely unknown. Therefore, we examined the expression of ERR␣ in human breast carcinoma tissues using immunohistochemistry (n ‫؍‬ 102) and real-time reverse transcription-PCR (n ‫؍‬ 30). ERR␣ immunoreactivity was detected in the nuclei of carcinoma cells in 55% of breast cancers examined, and relative immunoreactivity of ERR␣ was significantly (P ‫؍‬ 0.0041) associated with the mRNA level. Significant associations were detected between ER␣ and ERE-containing estrogen-responsive genes, such as pS2 (P < 0.0001) and EBAG9/RCAS1 (P ‫؍‬ 0.0214), in breast carcinoma tissues. However, no significant association was detected between ER␣ and pS2 (P ‫؍‬ 0.1415) in the ERR␣-positive cases (n ‫؍‬ 56) or between ER␣ and EBAG9/RCAS1 (P ‫؍‬ 0.8271) in the ERR␣-negative group (n ‫؍‬ 46). ERR␣ immunoreactivity was significantly associated with an increased risk of recurrence and adverse clinical outcome by both uni-(P ‫؍‬ 0.0097 and P ‫؍‬ 0.0053, respectively) and multi-(P ‫؍‬ 0.0215 and P ‫؍‬ 0.0118, respectively) variate analyses. A similar tendency was also detected in the group of breast cancer patients who received tamoxifen therapy after surgery. Results from our study suggest that ERR␣ possibly modulates the expression of ERE-containing estrogen-responsive genes, and ERR␣ immunoreactivity is a potent prognostic factor in human breast carcinoma.

Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6Ј-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6Ј-deoxychalcone and 6Ј-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1-cCHI3) from a model legume, Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that four CHI genes (CHI1-3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed in Escherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae.

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.

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.