Identification and Functional Characterization of <i>Isoflavone synthase</i> Gene Family in Pea ( <scp> <i>Pisum sativum</i> </scp> ): The Entry Point to Pisatin Biosynthesis

ABSTRACT Isoflavone synthase (IFS), a cytochrome P450 monooxygenase of the CYP93C subfamily, catalyzes the conversion of flavanones into isoflavones, the first committed step in the biosynthesis of isoflavonoid phytoalexins. In pea ( Pisum sativum L .), the phytoalexin pisatin plays a pivotal role in defense against pathogens. However, the molecular basis underlying IFS function in pea remains poorly understood. In this study, we performed a comprehensive genome‐wide identification and characterization of IFS genes in pea. Three IFS candidates, PsIFS7A , PsIFS7B , and PsIFS7C , were identified that reside on chromosome 7, each harboring all conserved cytochrome P450 signature motifs. PsIFS genes exhibited predominant expression in root tissue, with transcript levels induced rapidly upon Aphanomyces euteiches infection. Enzymatic assays confirmed their catalytic activity in converting the flavanones naringenin and liquiritigenin into the isoflavones genistein and daidzein, respectively, both in vitro and in planta systems. Furthermore, all three PsIFS genes were found in close proximity to quantitative trait loci (QTL) associated with Aphanomyces root rot resistance. Together, these findings provide novel insights into the IFS gene family in pea and lay a foundation for metabolic engineering or molecular breeding strategies to enhance disease resistance through targeted modulation of pisatin biosynthesis.