No abstract

Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or 'metabolons', which represent the molecular level of organization. Here, we have confirmed the existence of the long-postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4-hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non-consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.

Soybean chalcone isomerase (CHI) family contains twelve members with unique evolutionary background, expression patterns and is compartmentalized to specific subcellular locations. The phenylpropanoid pathway produces a diverse array of plant natural products. A key branch-point enzyme, chalcone isomerase, catalyzes the reaction producing flavanones, the backbone for many downstream metabolites such as flavonoids and isoflavonoids. We have identified twelve soybean GmCHIs that fall into four subfamilies. The study of this family in soybean in the context of various CHIs and CHI-like proteins, across divisions in the plant kingdom and beyond, shows an evolutionary journey from fatty acid-binding proteins (FAPs) to sterically restricted folds that gave rise to the chalcone-to-flavanone isomerase. There are four GmCHIs with this functionality, three of which belong to a legume-specific clade known as 'type II' CHIs. Tissue-specific expression of eight core members of the soybean CHI family showed differential temporal and spatial expression, pointing to the potential function of GmCHI1A in seed isoflavonoid production. Promoter analysis of the GmCHIs described the minutiae of sub-organ expression patterns. Subcellular localization of the family was conducted to investigate the possibility of pathway-specific compartmentalization. Subfamilies 1, 2 and 4 localized to the nucleus and cytoplasm, with nuclear localization of CHIs raising questions about alternate function. GmCHI3 isoforms localized to the chloroplast, which, in conjunction with their position on the phylogenetic tree and expression patterns, closely associates them with the FAPs. This study provides the first comprehensive look at soybean CHIs, a family of unique evolutionary background and biochemical function, with the catalytically active members producing the backbone substrate in an important plant metabolic pathway.