Genome-Wide Identification and Characterization of Isoflavone Synthase (IFS) Gene Family, and Analysis of GgARF4-GgIFS9 Regulatory Module in Glycyrrhiza glabra

Isoflavone synthase (IFS) is the key enzyme in isoflavonoid biosynthesis and has been functionally characterized in numerous plant species. <i>Glycyrrhiza</i> species, valued for their medicinal properties, accumulate flavonoids with significant physiological activities. Among these, isoflavones play crucial roles in plant growth, development and stress responses. However, the <i>IFS</i> gene family in <i>Glycyrrhiza</i> remains poorly understood. In this study, we identified 10, 9 and 9 <i>IFS</i> genes in <i>G. uralensis</i>, <i>G. inflata</i> and <i>G. glabra</i>, respectively. Phylogenetic analysis classified these genes into four distinct clades (Clade A-D). Further characterization included chromosomal localization, gene structure, conserved motifs, <i>cis</i>-acting elements and synteny analysis. Using yeast one-hybrid (Y1H) screening, dual-luciferase assays and an electrophoretic mobility shift assay (EMSA), these results revealed that auxin response factor 4 (GgARF4) directly binds to the isoflavone synthase 9 <i>(GgIFS9)</i> promoter and activates its expression. Following indole-3-acetic acid (IAA) treatment, RNA-seq revealed that in the differentially expressed genes (DEGs), the genes involved in isoflavonoid and flavonoid biosynthesis pathways were significantly enriched. The result of quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that <i>GgIFS9</i> was strongly induced by IAA. β-Glucuronidase (GUS) assays confirmed that IAA activates the expression of the <i>GgIFS9</i> promoter in <i>Nicotiana tabacum</i>. Our findings reveal that, through GgARF4 and its downstream-activated gene <i>GgIFS9,</i> IAA may promote flavonoid synthesis in <i>G. glabra</i>. This study provides novel insights into the auxin-mediated regulation of secondary metabolism in <i>Glycyrrhiza</i> species.