Elicitor-Induced Association of Isoflavone O-Methyltransferase with Endomembranes Prevents the Formation and 7-O-Methylation of Daidzein during Isoflavonoid Phytoalexin Biosynthesis

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Abstract
The bioactive isoflavonoids of the Leguminosae often are methylated on the 4 � -position of their B-rings. Paradoxically, reverse genetic evidence implicates alfalfa isoflavone O -methyltransferase (IOMT) in the biosynthesis of 4 � - O -methyl- ated isoflavonoids such as the phytoalexin medicarpin in vivo, whereas biochemical studies indicate that IOMT has strict specificity for methylation of the A-ring 7-hydroxyl of daidzein, the presumed substrate for O -methylation, in vitro. Radiolabeling and isotope dilution studies now confirm that daidzein is not an intermediate in isoflavonoid phytoalexin biosynthesis in alfalfa. Furthermore, protein gel blot analysis and confocal microscopy of a transiently expressed IOMT-green fluorescent protein fusion in alfalfa leaves show that the operationally soluble IOMT localizes to en- domembranes after elicitation of the isoflavonoid pathway. We propose that IOMT colocalizes with the endoplasmic reticulum-associated isoflavone synthase cytochrome P450 to ensure rapid B-ring methylation of the unstable 2,4 � ,7-trihydroxyisoflavanone product of isoflavone synthase, thereby preventing its dehydration to daidzein and sub- sequent A-ring methylation by free IOMT. In this way, metabolic channeling at the entry point into isoflavonoid phytoal- exin biosynthesis protects an unstable intermediate from an unproductive metabolic conversion.

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