Induction of isoflavonoid pathway in the model legume Lotus japonicus: molecular characterization of enzymes involved in phytoalexin biosynthesis.

Treatment of the seedlings of Lotus japonicus, a model legume for molecular genetic studies, with reduced glutathione (GSH) resulted in the accumulation of an isoflavan phytoalexin, vestitol. Using PCR strategies based on the conserved amino acid sequences, full length P450 cDNAs were obtained from GSH-treated seedling roots. When the clones, LjCYP-1 (CYP93C family) and LjCYP-2 (CYP81E family), were heterologously expressed in yeast, the proteins exhibited 2-hydroxyisoflavanone synthase (IFS) and isoflavone 2'-hydroxylase (I2'H) activities, respectively. The transcription levels of LjCYP-1, LjCYP-2 and isoflavone reductase, which are all involved in vestitol biosynthesis, coordinately increased upon elicitation. Genomic Southern blot analysis indicated that the IFS gene forms a small gene family and a single copy of the I2'H gene is present in the L. japonicus genome. Molecular biological aspects of P450s involved in the isoflavonoid pathway and the genomic approach to flavonoid metabolism in this unique plant are discussed.

Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice.

Isoflavonoids are distributed predominantly in leguminous plants and play critical roles in plant physiology. A cytochrome P450 (P450), 2-hydroxyisoflavanone synthase, is the key enzyme in their biosynthesis. In cultured licorice (Glycyrrhiza echinata L., Fabaceae) cells, the production of both an isoflavonoid-derived phytoalexin (medicarpin) and a retrochalcone (echinatin) is rapidly induced upon elicitation. In this study, we obtained a full-length P450 cDNA, CYP Ge-8 (CYP93C2), from the cDNA library of elicited G. echinata cells. When the flavanones liquiritigenin and naringenin were incubated with the recombinant yeast microsome expressing CYP93C2, major products emerged and were readily converted to the isoflavones daidzein and genistein by acid treatment. The chemical structures of the products from liquiritigenin (2-hydroxyisoflavanone and isoflavone) were confirmed by mass spectrometry. CYP93C2 was thus shown to encode 2-hydroxyisoflavanone synthase, which catalyzes the hydroxylation associated with 1,2-aryl migration of flavanones. Northern-blot analysis revealed that transcripts of CYP93C2, in addition to those of other P450s involved in phenylpropanoid/flavonoid pathways, transiently accumulate upon elicitation.