Identification of protein-protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.).

Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis.

Transcription analyses of GmICHG, a gene coding for a ß-glucosidase that catalyzes the specific hydrolysis of isoflavone conjugates in Glycine max (L.) Merr.

Isoflavone conjugate-hydrolyzing ß-glucosidase (GmICHG) of soybeans [Glycine max (L.) Merr.] catalyzes the specific hydrolysis of isoflavone conjugates (ß-7-O-(malonyl)glucosides of isoflavones) to produce free isoflavones. In this study, changes in the transcription levels of GmICHG in the individual organs of soybean seedlings (cv. Enrei) in response to microbial infection and abiotic stresses were analyzed and compared with those of genes coding for 2-hydroxyisoflavanone synthase (GmIFS) and isoflavone 7-O-glucosyltransferase (GmIF7GT). GmICHG was originally expressed in abundance only in the roots and at low levels only in the other organs. The transcription of GmICHG in the roots and other organs was suppressed upon infection of the roots by Phytophthora sojae. Upon wounding of the cotyledon, a transient long-distance up-regulation of GmICHG transcription in the roots was observed; upon fungal infection in the cotyledon, however, a delayed elevation of GmICHG transcription took place in the roots with the maximum at 10 h after the infection. Such long-distance up-regulation patterns were not observed with either GmIFS or GmIF7GT. The transcription levels of GmICHG remained essentially unchanged upon treatment of the roots with Bradyrhizobium japonicum. The transcription of GmICHG in the roots was also sensitive to a variety of stresses on the roots, such as flooding, elicitation with yeast extract, drought, and treatment with plant hormones such as abscisic, salicylic, and jasmonic acids and ethylene.