Soybean (Glycine max) 5-deoxyisoflavonoids (daidzein and its conjugates) are precursors of glyceollin phytoalexins. They are also converted to equol by microbes in the human intestine, resulting in health benefits. 5-Deoxyisoflavonoids accumulate in the roots (93% mol/mol of the total root isoflavonoids) and seeds of unstressed soybean plants. Chalcone reductase (CHR) is a key enzyme mediating 5-deoxyisoflavonoid biosynthesis because it catalyzes the production of 6′-deoxychalcone through its effects on the chalcone synthase (CHS)-catalyzed reaction. The soybean genome encodes at least 11 CHR-related homologs, but it is unclear which ones are functionally important for daidzein accumulation in unstressed plants. Among the CHR homologs, the temporal and spatial expression patterns of GmCHR5 were the most correlated with the distribution patterns of 5-deoxyisoflavonoids. The CHR activity of GmCHR5 was confirmed in vitro and in planta. In the in vitro assays, the ratio of CHR products (6′-deoxychalcone) to total CHS products (R value) was dependent on GmCHR5 and CHS concentrations, with higher concentrations resulting in higher R values (i.e. approaching 90%). Subcellular localization analyses revealed that GmCHR5 was present in the cytoplasm and nucleus. Protein–protein interaction assays indicated that GmCHR5, but not GmCHR1 and GmCHR6, interacted with 2-hydroxyisoflavanone synthase (IFS) isozymes. The CHS isozymes also interacted with IFS isozymes but not with GmCHR5. The proposed micro-compartmentalization of isoflavone biosynthesis through the formation of an IFS-mediated metabolon is probably involved in positioning GmCHR5 close to CHS, resulting in an R value that is high enough for the accumulation of abundant 5-deoxyisoflavonoids in soybean roots.
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